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10. 7759/cureus. 58564

Cureus

Dry Mouth Dilemma: A Comprehensive Review of Xerostomia in Complete Denture Wearers

Xerostomia, commonly known as dry mouth, presents a significant challenge for individuals wearing complete dentures, affecting their oral health and quality of life. This review explores the relationship between saliva and complete dentures, highlighting the varied management strategies for xerostomia. Saliva plays a critical role in denture retention, lubrication, and oral environment buffering. Complete denture wearers often experience reduced salivary flow, aggravating symptoms of xerostomia. Various management approaches are discussed, including general measures such as hydration and salivary stimulation techniques which aim to boost saliva production naturally. The use of salivary substitutes provides artificial lubrication and moisture to alleviate dry mouth discomfort. Oral lubricating devices, such as sprays, gels, and lozenges, offer relief by mimicking saliva's lubricating properties, thereby improving denture stability and comfort. This review addresses the etiology of xerostomia in complete denture wearers and explores preventive measures to reduce its impact. A comprehensive approach has been discussed for the management of xerostomia which will help to improve the oral health and well-being of complete denture wearers experiencing dry mouth.

Introduction and background The presence of optimal salivary flow, consistency, and composition holds importance, particularly in completely edentulous patients. It is imperative for the prosthodontist to meticulously consider these salivary characteristics throughout the stages of denture fabrication from initial assessment to post-insertion care. Salivary flow is crucial for maintaining oral health and comfort. The average daily secretion of saliva typically ranges from 500 ml to 1500 ml (approximately 50. 72 oz), underscoring its significance in oral function and comfort [ 1 ]. Xerostomia, characterized by dryness of the oral cavity, arises due to a reduction or absence of salivary flow. It is a symptomatic manifestation of multifactorial conditions such as radiation exposure or a side effect of certain medications [ 2 ]. Oral mucosal changes in xerostomia commonly occur with alterations in the appearance of the oral mucosa. The mucosa may exhibit a cobblestoned or erythematous appearance, resembling a pebbled texture due to reduced lubrication and moisture. In cases of chronic xerostomia, the filiform papillae on the dorsal surface of the tongue may undergo atrophy. This can result in a smooth, glossy appearance of the tongue surface, known as a "bald tongue" or "smooth tongue. " Xerostomia can contribute to the development of fissures or cracks on the surface of the tongue (fissured tongue) which can be associated with discomfort and pain [ 2 ]. The oral mucosal surfaces may exhibit increased adherence or stickiness upon light palpation. This can occur due to insufficient lubrication provided by saliva, leading to a sensation of dryness and discomfort in the oral cavity [ 3 ]. Diagnostic tests such as cotton wool drying followed by salivary gland milking may reveal delayed or absent salivary secretion from the duct orifices. This delay in salivary flow further confirms the presence of xerostomia and underscores the need for prompt intervention and management [ 4 ]. These clinical signs collectively contribute to the diagnosis and assessment of xerostomia in patients. The etiology of xerostomia The etiology of xerostomia encompasses a broad spectrum of factors, primarily categorized into two groups, water or metabolic loss and salivary gland damage, along with interference with neural transmission. These causes have significant implications for the management and treatment of xerostomia. Water or metabolic loss may occur due to dehydration arising from various conditions such as pyrexia, burns, hyperhidrosis, hemorrhage, vomiting, diarrhea, and renal dysfunction causing excessive urination or osmotic polyuria [ 5 ]. Protein-calorie malnutrition can exacerbate fluid and electrolyte loss, further contributing to xerostomia. Salivary gland impairment plays a crucial role in decreasing saliva production due to therapeutic radiation targeting the head, neck, and face, which can lead to salivary gland dysfunction. Diseases affecting the salivary glands are autoimmune disorders like Sjögren's syndrome, endocrine conditions such as type I and II diabetes mellitus, thyroid disorders, and adrenal gland disorders, which affect their function and reduce saliva production [ 6 ]. Systemic diseases like liver and kidney diseases, including hepatitis C infection, as well as viral infections, such as human immunodeficiency virus (HIV) and human T-lymphotropic virus-1 (HTLV-1), can indirectly affect salivary gland function, contributing to xerostomia [ 7 ]. Medications interfering with the neural control of salivary glands, such as cytotoxic drugs, anticholinergic drugs, proton pump inhibitors, psychoactive agents, sympathomimetic drugs, antihypertensives, and diuretics, are associated with dry mouth as a side effect [ 8 ]. Other factors contributing to xerostomia include age-related medication usage. Tobacco use, whether through chewing or smoking, increases the risk of dry mouth by affecting saliva production and quality [ 9 ]. Dehydration, caused by insufficient fluid intake, can also contribute to dry mouth. Physical exertion or exposure to heat may lead to temporary dry mouth due to dehydration and prioritization of fluid distribution away from the salivary glands [ 10 ]. Various health conditions and habits can also contribute to xerostomia, including anxiety disorders, depression, Parkinson's disease, poorly managed diabetes, Sjögren's syndrome, and certain sleep habits like mouth breathing or snoring [ 11 ]. Stroke and Alzheimer's disease may cause a perception of dry mouth, even when salivary gland function is intact [ 12 ]. For management and treatment strategies specific to address the underlying causes and alleviate symptoms effectively, understanding the multifactorial etiology of xerostomia is essential. Signs and symptoms Dry mouth, or xerostomia, presents a range of signs and symptoms that can significantly impact oral health and quality of life. One of the primary symptoms associated with dry mouth is halitosis or bad breath which arises due to decreased salivary flow and the subsequent build-up of bacteria in the oral cavity [ 13 ]. Cheilitis, characterized by inflammation, splitting, and cracking of the lips, may also occur due to inadequate moisture and lubrication provided by saliva. Cracking and splitting of the oral mucosa in the cheeks and lips can lead to discomfort and pain, as the skin at the corners of the mouth is susceptible to soreness and ulceration [ 14 ]. Dry mouth often results in dysgeusia or taste disorders, affecting the perception of flavors and altered taste sensations. Reduced saliva flow predisposes individuals to fungal infections in the mouth, such as oral thrush, which can manifest as white patches or lesions on the tongue, inner cheeks, or palate [ 15 ]. Glossodynia may also occur due to dryness and irritation of the oral mucosa. Inflammation of the tongue or the development of tongue ulcers may cause oral discomfort and compromise oral function. Dry mouth is associated with a higher incidence of periodontal disease, as saliva plays a crucial role in maintaining oral hygiene and buffering acidic pH levels in the mouth. Increased tooth decay and plaque accumulation are also common as saliva helps remineralize tooth enamel and neutralize acids produced by bacteria [ 16 ]. Practical difficulties in oral function are prevalent among individuals with dry mouth, including problems in speaking, swallowing, and chewing. Individuals wearing dentures may experience issues with denture retention, development of denture sores, and adhesion of the tongue to the roof of the mouth due to reduced saliva lubrication [ 17 ]. In severe cases of xerostomia, complications such as sialadenitis, a salivary gland infection, may arise, leading to localized pain, swelling, and inflammation of the affected gland [ 18 ]. Sore throat, sticky or stringy saliva, and compromised oral hygiene further contribute to the multifaceted nature of xerostomia's clinical presentation. Drugs linked with dry mouth Several classes of pharmaceuticals have been linked with xerostomia. Phenothiazines and benzodiazepines, commonly used for various psychiatric and neurological conditions, have been reported to cause dry mouth as a side effect [ 19 ]. Anticholinergic agents such as atropine and hyoscine, which block the action of acetylcholine in the body, can inhibit saliva secretion and contribute to xerostomia [ 20 ]. Opioids, including medications like morphine and codeine, are known to suppress saliva production through their central nervous system effects [ 21 ]. Certain cytotoxic drugs used in chemotherapy, which are intended to target rapidly dividing cells, can also damage salivary glands and impair saliva production [ 22 ]. Medications that influence the sympathetic nervous system can indirectly contribute to dry mouth by altering salivary gland function [ 23 ]. Acid pump inhibitors, including pantoprazole, commonly used to treat gastroesophageal reflux disease (GERD) and peptic ulcers, have also been associated with xerostomia as a potential adverse effect [ 24 ]. Alpha-1 antagonists like doxazosin, beta blockers like carvedilol, and alpha-2 agonists such as clonidine have all been reported to induce xerostomia. Diuretics subsequently reduce fluid levels in the body and can lead to dehydration and dry mouth [ 25 ]. Centrally acting psychoactive drugs, including certain antidepressants such as tricyclic compounds, have anticholinergic effects that can interfere with saliva production [ 26 ]. Consequences and complications Xerostomia can have wide-ranging consequences and complications that impact oral health, comfort, and overall well-being. Oropharyngeal dysfunction, including dysphagia, can significantly impact an individual's ability to eat and drink comfortably [ 27 ]. Dysarthria can affect communication and social interaction [ 28 ]. It can also cause mucosal trauma, ulcerations, and pharyngoesophageal pain [ 29 ]. Reduced saliva flow contributes to mucus accumulation in the mouth and difficulty in clearing the throat. Food residues may also accumulate in the oral cavity due to inadequate saliva for lubrication and cleansing, increasing the risk of plaque formation and dental caries. Hyposalivation compromises the mouth's natural defense mechanisms against bacterial overgrowth, resulting in alterations in the oral microflora and an increased susceptibility to oral infections such as candidiasis [ 30 ]. Nocturnal oral discomfort due to dryness can disrupt sleep patterns and reduce overall quality of life [ 31 ]. Review Saliva and complete denture Saliva also plays a very important role in preserving denture integrity by keeping the denture surfaces clean and in maintaining oral hygiene. It physically washes away food and other debris. The lubrication provided by saliva is important in the edentulous as this makes the surface of the dentures more compatible with the movements of the lips, cheek, and tongue during speech, mastication, and swallowing. Salivary β-glycoproteins facilitate the movement of soft tissues during oral functions and contribute to denture retention. Successful rehabilitation of edentulous patients with complete dentures has largely contributed to satisfactory denture retention [ 32 ]. The physical factors contribute to denture retention, including adhesion, cohesion, interfacial surface tension, atmospheric pressure, capillary attraction, and viscosity of saliva. Principal factors that aid denture retention are the adhesive action of the thin film between the saliva and denture base and underlying soft tissues. Such adhesive action of the saliva is achieved through ionic forces between charged salivary glycoproteins and surface epithelium on one side and denture base acrylic resin on the other. It also provides a cushioning effect and lubrication between the denture base and oral tissues and tends to reduce friction [ 33 ]. The quantity, quality, consistency, and flow rate affect the stability of the denture. A copious amount of thin viscosity saliva lubricates the mucosa and assists in denture retention. Dry mouth affects the retention of the denture and also leads to soft tissue trauma due to enhanced friction between the mucosa and denture. The presence of thick ropy saliva leads to dislodgement of the maxillary denture due to the negative hydrostatic force that is created in the anterior to posterior palatal seal. While taking impressions, tissue details are poorly recorded due to thick saliva. In excessive salivation, impression-making becomes difficult and may cause pits and voids. Management of xerostomia General Measures The management of xerostomia and hyposalivation depends upon its etiology. A multidisciplinary approach allows for comprehensive care addressing both the oral and systemic aspects of the condition. Medication-related xerostomia can be reduced by substituting it with other medication with fewer side effects affecting salivary flow. Adjusting the timing or dosing schedule of medications can also help minimize dry mouth symptoms while maintaining therapeutic efficacy [ 2 ]. Hydration is essential, especially for individuals on low-salt diets or those using antidiuretic drugs, which can exacerbate dehydration. Patients are advised to consume at least 2 liters of fluid daily to ensure proper hydration and support salivary gland function. Multidisciplinary management of systemic conditions such as Sjögren's syndrome and other diseases like rheumatoid arthritis, HIV infection, and diabetes is necessary, which can contribute to xerostomia [ 34 ]. Salivary Stimulation Salivary stimulation therapies offer effective options for managing xerostomia by enhancing saliva production and improving oral hydration. Stimulation methods can be achieved through mechanical or chemical means, often involving lifestyle modifications or pharmacological interventions. Mechanical stimulation involves strategies such as consuming more frequent meals and chewing sugar-free gum. Chewing gum stimulates saliva production by mechanically activating the salivary glands, promoting the release of saliva into the oral cavity. Xylitol, a sugar alcohol commonly used in chewing gum, has been shown to enhance saliva production and improve oral hydration [ 35 ]. Chemical stimulation, on the other hand, involves the use of sialagogues-medications that directly stimulate the salivary glands to increase saliva production. Several pharmacological agents have been identified for this purpose. Pilocarpine: It is a cholinergic agonist that stimulates muscarinic receptors on the salivary gland cells, leading to increased saliva secretion. Pilocarpine has been extensively studied and shown to be effective in improving salivary flow rates and relieving symptoms of xerostomia [ 36 ]. Bromhexine: This mucolytic agent has been found to possess sialogogue properties by stimulating the secretion of saliva. It acts by enhancing the production and secretion of mucus in the salivary glands, thereby promoting saliva flow [ 37 ]. Cevimeline: Another cholinergic agonist that selectively activates muscarinic receptors in the salivary glands, resulting in increased saliva production. Cevimeline has been approved for the treatment of xerostomia associated with Sjögren's syndrome and has demonstrated efficacy in improving salivary flow rates and relieving dry mouth symptoms [ 2 ]. Anethole trithione: A compound with sialogogue properties, anethole trithione acts by stimulating saliva secretion through its cholinergic activity. It has been used in the treatment of xerostomia, although further research is needed to fully elucidate its efficacy and safety profile [ 38 ]. The duration of action for these sialagogues typically ranges from two to three hours, providing temporary relief from dry mouth symptoms. However, it is important to note that these medications may have side effects and contraindications and their use should be carefully monitored [ 2 ]. Use of Salivary Substitutes The main drawback of artificial saliva is that patients need to manually introduce it into the oral cavity at regular intervals. The use of salivary substitutes is a key component of the treatment approach for xerostomia, aiming to reduce dry mouth symptoms and maintain oral health. Salivary substitutes can be categorized into various types, each with its own advantages and considerations. Glycerin and lemon-based substitutes: These substitutes are relatively simple and readily available. They may have drawbacks such as potential erosion of natural teeth due to acidity and astringent properties and possible discomfort or stinging sensation in soft tissues due to glycerin content. Carboxymethyl cellulose (CMC)-based substitutes: CMC-based substitutes offer a more complex formulation, providing improved lubrication and moisture retention compared to glycerin-based options. These substitutes can effectively coat oral surfaces and provide relief from dry mouth symptoms [ 2 ]. Mucin-based substitutes: Mucin is a glycoprotein present in natural saliva and contributes to its lubricating and moisturizing effects and protective qualities. Mucin-based substitutes that closely resemble the composition of natural saliva are considered to have superior properties in terms of mimicking natural saliva. In addition to conventional artificial saliva products, milk can also serve as a natural salivary substitute due to its chemical and physical properties. Milk not only moisturizes and lubricates the oral mucosa but also helps buffer oral acidity, decrease enamel solubility, and promote enamel remineralization [ 39 ]. Despite the benefits of salivary substitutes, one drawback is the need for manual introduction into the oral cavity at regular intervals, as the effects are not sustained over extended periods. Patients may need to frequently administer these substitutes to maintain oral comfort and prevent complications. Use of Oral Lubricating Devices The utilization of oral lubricating devices presents an innovative approach to the treatment of xerostomia, offering a convenient and effective means of delivering saliva substitutes to reduce dry mouth symptoms and enhance oral comfort. These devices are designed to meet specific requirements to ensure optimal performance and patient satisfaction. Reservoir Dentures Reservoir dentures feature compartments or reservoirs within the denture base that can hold saliva substitutes or lubricants. These reservoirs gradually release the solution throughout the day, providing continuous relief from dry mouth symptoms [ 2 ]. Designing reservoirs in dentures involves a meticulous process aimed at integrating specialized compartments within the denture base to store and dispense salivary substitutes. A comprehensive assessment of the patient's oral health status, including the severity of xerostomia, oral tissue condition, and denture requirements, is crucial before designing reservoirs in dentures [ 40 ]. Patient evaluation should encompass factors such as the etiology of xerostomia, salivary gland function, and individual treatment objectives. Salivary gland function tests are diagnostic procedures used to assess the production and secretion of saliva by the salivary glands such as salivary flow rate measurement, sialometry, salivary pH test, salivary composition analysis, salivary gland imaging, and salivary gland biopsy. The placement and configuration of reservoirs within the denture base are important aspects of the designing process [ 41 ] for the optimal distribution and retention of saliva substitutes. The most common locations for reservoirs include the palate and the lingual aspect of the denture base. Through the electronic search of the keyword "salivary reservoir denture, " 22 case series were found from 1986 to 2024 [ 42, 43 ]. The location for reservoir preference seemed almost equal among both the arches, out of which, maxillary reservoirs were placed in the palate. Mandibular reservoirs were designed in the lingual flange or split denture technique with inter-compartments (Figure 1 and Figure 2 ). Figure 1 Maxillary denture with salivary reservoir Figure 2 Mandibular denture with salivary reservoir Reservoirs in these areas ensure that saliva substitutes are evenly dispersed across the oral cavity, providing maximum relief from dry mouth symptoms. Reservoirs are designed to accommodate 2-5 ml volume of saliva substitutes; the duration of flow ranges from two to five hours while maintaining the integrity and aesthetics of the denture [ 44 ]. The shape of reservoirs may vary depending on the patient's oral anatomy and the desired distribution of saliva substitutes. Reservoirs may be elongated to cover a larger surface area of the palate or lingual tissues. The orientation of reservoirs is adjusted to optimize the release of saliva substitutes during mastication and speaking. The choice of materials for reservoir fabrication should prioritize durability, flexibility, and biocompatibility with saliva substitutes. Medical-grade polymers, such as silicone elastomers, are commonly used [ 45 ]. These materials ensure that reservoirs remain comfortable and safe for long-term use, minimizing the risk of irritation or allergic reactions in the oral cavity. Saliva substitutes, such as artificial saliva or lubricating agents, are introduced into the reservoirs to provide moisture and lubrication to the oral mucosa [ 46 ]. Careful calibration of the reservoirs ensures the controlled release of saliva substitutes, preventing over-hydration or under-hydration of the oral tissues. Following the fabrication of dentures with reservoirs, thorough evaluation and patient feedback are essential to assess fit, comfort, and efficacy [ 47 ]. Any necessary adjustments to the reservoir design or placement are made based on patient response and clinical outcomes, ensuring optimal performance and patient satisfaction. Other Oral Lubricating Devices These devices are designed to administer saliva substitutes or lubricants, commonly induced by medication side effects, systemic conditions, or radiation therapy. Developed for sustained release, they maintain optimal moisture levels over time, ensuring enduring comfort for patients. With user-friendly designs, they allow for effortless application and administration, promoting patient compliance and convenience. Seamlessly integrating into the oral cavity, these devices do not interfere with normal oral function, enabling patients to speak, chew, and swallow comfortably while benefiting from their use. Saliva sprays: Saliva sprays are portable and convenient devices that deliver a fine mist of artificial saliva directly into the mouth. These sprays offer quick relief, are suitable for on-the-go use, and offer immediate moisture to the oral cavity [ 36 ]. Oral gels: Oral gels are viscous formulations applied directly to the oral mucosa. They coat the tissues, providing lubrication and moisture to alleviate dryness and discomfort. These gels often contain ingredients such as glycerin, xylitol, or cellulose derivatives which help to retain moisture and soothe dry mouth symptoms [ 48 ]. Lozenges: Lozenges are solid formulations that dissolve slowly in the mouth, releasing active ingredients to moisturize and lubricate the oral tissues. They offer prolonged relief and are available in various flavors [ 49 ]. Mouth rinses: Some mouth rinses are specifically formulated to combat dry mouth symptoms. These rinses contain moisturizing agents such as glycerin or xylitol, which help to hydrate the oral tissues and alleviate dryness. Additionally, mouth rinses may have antimicrobial properties to promote oral health and prevent complications associated with dry mouth [ 50 ]. Oral lubricating devices work through various mechanisms to provide relief from dry mouth symptoms. The primary function of these devices is to replenish moisture in the oral cavity. Ingredients such as water, glycerin, or xylitol mimic the hydrating properties of saliva, helping to moisten the oral mucosa and alleviate dryness. Lubricating agents in oral gels, sprays, and lozenges coat the oral tissues, reducing friction and discomfort associated with dry mouth. This lubrication enhances oral comfort during speaking, chewing, and swallowing. Some oral lubricating devices contain ingredients that can stimulate salivary gland function, promoting the production of natural saliva. For example, xylitol-based products may stimulate saliva secretion by activating salivary reflexes. Certain formulations are designed to maintain the pH balance of the oral cavity, which is essential for oral health. By buffering acidic conditions, these devices help prevent oral mucosal irritation and protect against dental erosion. Oral lubricating devices contain antimicrobial agents that help reduce the risk of oral infections. These agents inhibit the growth of bacteria and fungi in the mouth, promoting oral hygiene and preventing complications associated with dry mouth. Denture design modifications Modified denture designs for patients with xerostomia involve specific alterations in both the design and fabrication process to address the challenges associated with reduced salivary flow and dry mouth symptoms. These modifications aim to enhance comfort, retention, and oral health for individuals experiencing xerostomia. One key aspect of modified denture design is the incorporation of reservoirs or compartments within the denture base. Softer and more flexible materials may be preferred to minimize friction and irritation on the oral mucosa. These materials offer greater comfort and reduce the risk of tissue trauma, especially in individuals with sensitive oral tissues [ 51 ]. Liquid-supported denture is a viable surface modification for xerostomic patients. The technique involves the incorporation of a polyurethane sheet in the denture to create space which was further filled with glycerin or any other lubricating liquid. This will help in reducing occlusal pressure on the ridge and soft tissues by the distribution of stresses through the liquid. This has the advantages of both denture relining material and soft liners which increases comfortability over long-term usage of the denture [ 52 ]. Surface modifications are another consideration in modified denture design for xerostomia patients. Special coatings or treatments such as soft liners may be applied to the denture surface to reduce friction and pressure spots on soft tissue. Soft liners acrylic or silicone type can be used depending on the shelf-life of the material. These surface modifications help improve the overall comfort and adaptability of the dentures, particularly in individuals with compromised salivary flow [ 53 ]. The contour and fit of the dentures are customized to accommodate changes in oral anatomy resulting from xerostomia. Prosthodontists pay close attention to the fit of the denture borders and ensure optimal retention and stability. This meticulous adjustment of the denture contours helps prevent displacement and discomfort during speaking and eating. Advanced techniques such as digital denture design and computer-aided design/computer-aided manufacturing (CAD/CAM) may be utilized to achieve accurate and predictable results [ 54 ]. Denture adhesives It is seen that the wetting mechanism of saliva is very important for the retention of the complete denture. Denture adhesives have shown quite impressive results in the case of dry mouth. Denture adhesives come in various forms, including pastes, powders, and strips. The most common components of denture adhesives include polymers such as CMC or methyl cellulose, along with mineral oil, petrolatum, or other petroleum-based compounds. These ingredients work together to enhance adhesion between the denture and oral tissues. Polymer-based adhesives, such as those containing CMC, form a gel-like layer when hydrated, which adheres to both the denture and the oral tissues. This adhesive layer fills in gaps and irregularities, improving the fit and stability of the denture. Mineral oil and petrolatum act as lubricants, reducing friction between the denture and the oral mucosa, thereby minimizing irritation and discomfort. Some denture adhesives may contain antimicrobial agents, such as zinc or fluoride, which help maintain oral hygiene by inhibiting the growth of bacteria and fungi on the denture surface. These antimicrobial properties contribute to overall oral health and may prevent oral infections associated with prolonged denture wear [ 55 ]. When selecting a denture adhesive for patients with xerostomia, it is essential to consider factors such as product compatibility, ease of application, and patient preference. Patients should be instructed to use an adequate amount of denture adhesives as per the manufacturer's instructions. Dentures with adhesives need to be wetted before use to maximize their benefits [ 56 ]. Maximum 12-14 hours denture adhesives can be used. Dentures need to be cleaned thoroughly before re-application to remove any remnants of old adhesive, which can cause bacterial and fungal growth. Preventive measures For the prevention of oral complications such as reduced salivary output, additional measures should be implemented, for example, dental evaluations should be carried out once every 4-6 months. Annual radiographs are recommended to assess for potential dental caries, bone loss, or other pathology [ 56 ]. To prevent dental caries, especially in patients wearing single complete dentures or overdentures, maintaining a low-sugar diet is advisable. Proper oral hygiene practices, including regular brushing and flossing, are essential. Furthermore, the use of topical fluoride treatments is recommended to strengthen tooth enamel and prevent decay. In cases of rampant caries due to hyposalivation, neutral pH sodium fluoride is effective [ 57 ]. Dentifrices, varnishes, oral gels, and mouth rinses containing fluorides and remineralizing solutions can be used with or without applicator trays. The specific fluoride regimen should be decided on the individual patient's needs, the magnitude of hypofunction of the salivary gland, and the rate of caries [ 58 ]. For patients undergoing radiation therapy, strategies to mitigate radiation-induced salivary dysfunction without compromising oncologic treatment are crucial. These may include sparing the parotid glands from radiation, using tissue protectants, and surgical transfer of salivary glands. These approaches aim to preserve salivary gland function and minimize the impact of radiation therapy on saliva production [ 59 ]. In denture-wearing patients, it is advised to wet dentures before placing them into the mouth and spraying prostheses with artificial saliva before applying denture adhesives as this will help in reducing the discomfort. Additionally, increasing fluid intake during meals and wetting dentures before eating can aid in mastication and swallowing, facilitating a more comfortable eating experience [ 2 ]. Advanced treatment for xerostomia Electrical Stimulation Electrical stimulation of salivary glands is a promising therapeutic approach for xerostomia. This technique involves the application of electrical currents to the salivary glands to stimulate saliva production. Electrical stimulation can enhance salivary gland function by increasing blood flow, promoting glandular cell proliferation, and stimulating neurotransmitter release [ 60 ]. Several studies have demonstrated the efficacy of electrical stimulation in improving saliva flow rates and relieving xerostomia symptoms in patients with various underlying conditions, including Sjögren's syndrome, radiation-induced xerostomia, and salivary gland hypofunction [ 61, 62 ]. Electrical stimulation devices, such as intraoral electrostimulators, have shown promising results in clinical trials, providing a non-invasive and potentially effective treatment option for xerostomia [ 63 ]. These devices can be easily incorporated into the dentures or mouth guards to effectively and locally provide salivary stimulation. Tissue Engineering Tissue engineering approaches hold great promise for regenerating damaged or dysfunctional salivary glands in patients with xerostomia. Tissue engineering involves the construction of artificial tissues or organs using a combination of cells, biomaterials, and bioactive factors. In the context of xerostomia treatment, tissue engineering strategies aim to create functional salivary gland tissues capable of producing saliva [ 64 ]. Researchers have explored various approaches to tissue engineering for salivary glands, including the use of stem cells, bioengineered scaffolds, growth factors, and tissue culture techniques. By harnessing the regenerative potential of stem cells and the biocompatibility of biomaterials, tissue engineering holds the potential to restore salivary gland function and alleviate xerostomia symptoms [ 65 ]. Clinical studies evaluating tissue-engineered salivary gland constructs have shown promising results in preclinical models, demonstrating the feasibility of this approach for xerostomia treatment. However, further research is needed to optimize tissue engineering strategies and translate them into clinically viable treatments for xerostomia [ 66 ]. Gene Therapy Gene therapy offers another innovative approach for treating xerostomia by targeting the underlying genetic or molecular causes of salivary gland dysfunction. Gene therapy involves the delivery of therapeutic genes to target cells or tissues to correct genetic defects, enhance cell function, or modulate gene expression [ 67 ]. In the context of xerostomia, gene therapy can be used to promote salivary gland regeneration, enhance saliva production, or protect salivary gland tissues from damage. Researchers have investigated various gene therapy approaches for xerostomia, including the delivery of growth factors, anti-inflammatory cytokines, or transcription factors to promote salivary gland function and repair. Preclinical studies using animal models have shown promising results for gene therapy in restoring salivary gland function and alleviating xerostomia symptoms. However, the clinical translation of gene therapy for xerostomia remains in the early stages, with further research needed to optimize gene delivery methods, ensure safety and efficacy, and address potential regulatory challenges [ 62 ]. Conclusions Xerostomia is a condition which affects the overall health and quality of life. The above review summarises the challenges faced by a xerostomic denture patient and measures which can be introduced to overcome them. The xerostomic patient faces not only difficulties due to decreased salivary flow but also increased chances of trauma to mucosal surfaces. The review has briefed about xerostomia in detail including etiology, characteristics, and management. The ways to reduce the impact of dry mouth include general measures along with systemic drugs used at times in conjunction with salivary stimulation. Various denture surface modifications have been incorporated to lessen the struggle in adaptation to dentures. Salivary substitutes also play a vital role in the efficient management of xerostomia. Similarly, tissue engineering, gene therapy, and electric stimulation represent an innovative approach to enhance salivary gland function. However, further research and clinical trials are needed to validate their efficacy, safety, and long-term outcomes in human patients.

10. 7759/cureus. 58572

Cureus

Exploring the Horizons of Four-Dimensional Printing Technology in Dentistry

In dentistry, the integration of additive manufacturing, particularly 3D printing, has marked significant progress. However, the emergence of 4D printing, which allows materials to change shape dynamically in response to stimuli, opens up new avenues for innovation. This review sheds light on recent advancements and potential applications of 4D printing in dentistry, delving into the fundamental principles and materials involved. It emphasizes the versatility of shape-changing polymers and composites, highlighting their ability to adapt dynamically. Furthermore, the review explores the challenges and opportunities in integrating 4D printing into dental practice, including the customization of dental prosthetics, orthodontic devices, and drug delivery systems and also probing into the potential benefits of utilizing stimuli-responsive materials to improve patient comfort, treatment outcomes, and overall efficiency and the review discusses current limitations and future directions, emphasizing the importance of standardized fabrication techniques, biocompatible materials, and regulatory considerations. Owing to its diverse applications and advantages, 4D printing technology is poised to transform multiple facets of dental practice, thereby fostering the development of healthcare solutions that are more tailored, effective, and centered around patient needs.

Introduction and background Exploring the dimensions: an introduction to 4D printing technology 4D printing denotes an additive manufacturing modality characterized by the fabrication of objects utilizing materials endowed with the capacity for self-metamorphosis or morphological adjustment in reaction to external stimuli during temporal progression. These stimuli encompass a spectrum of environmental variables, such as temperature, luminosity, moisture, and additional ambient factors [ 1 ]. The designation "4D" connotes the standard triad of spatial dimensions-length, width, and height-augmented by the temporal dimension. This temporal component facilitates the programmed transformation of printed artifacts, enabling predetermined alterations in shape or structural configuration post-fabrication. The prospective applications of this technology span diverse sectors, including aerospace, healthcare, architectural design, and consumer goods, indicating its substantial promise and versatility [ 2 ]. Evolution and milestones of 4D printing technology Additive manufacturing has transformed the landscape of the manufacturing industry, offering the capability to produce intricate geometries with unprecedented ease. The emergence of 4D printing technology represents a novel advancement beyond traditional 3D printing, introducing the temporal dimension. This innovative extension enables materials to autonomously self-assemble, alter shape, or undergo transformation into new configurations over time. In circa 2013, Skylar Tibbits, an innovative scholar affiliated with the Massachusetts Institute of Technology (MIT), introduced the groundbreaking notion of "4D printing, " envisioning a departure from the constraints of traditional 3D printing. Tibbits conceived of a manufacturing approach that integrates materials endowed with the unique ability for self-assembly or dynamic shape alteration over time, marking a pivotal moment in fabrication technology. The early part of the 2010s saw a surge in global exploration of 4D printing potentialities by various research entities, with MIT's Self-Assembly Lab, led by Tibbits, at the forefront of this movement [ 3 ]. Through the demonstration of proof-of-concept prototypes featuring self-folding structures and materials responsive to external stimuli, MIT's efforts showcased the transformative capabilities of 4D printing [ 4 ]. Despite challenges such as scalability and precise control over shape modulation, the extraordinary scientific potential of 4D printing suggests promising applications in fields like biomedical, medical, and dental sciences. Review Beyond three dimensions: the rise of the fourth dimension of printing technology 4D printing presents numerous advantages over conventional 3D printing by integrating the temporal dimension into the manufacturing process and employing dynamic materials capable of undergoing shape alterations or transformations in reaction to external stimuli [ 5 ]. An elaborate discourse delineating the ways in which 4D printing outperforms 3D printing across several domains is mentioned in Figure 1. Figure 1 A depiction of the distinguishing features that differentiate 4D printing technology from 3D printing technology Credit: Image created by the author Distinguishing Features Adaptive functionality: A key benefit of 4D printing lies in its capacity to fabricate objects endowed with adaptive functionality. In contrast to the static nature of objects manufactured through 3D printing, those created through 4D printing exhibit the capability to undergo changes in shape, properties, or functionality over time, prompted by external stimuli such as fluctuations in temperature, exposure to light, variations in humidity levels, or other environmental influences. This inherent adaptability renders 4D-printed objects well-suited for diverse applications wherein dynamic responses are imperative [ 6 ]. For instance, dental implants fabricated through 4D printing can autonomously conform to temperature fluctuations, ensuring a consistently accurate and comfortable fit amidst changing environmental factors within the oral cavity. Furthermore, adaptive 4D-printed orthodontic appliances can incrementally alter alignment in alignment with patient progress, thereby enhancing treatment efficacy and precision beyond what static 3D-printed counterparts can achieve [ 7 ]. Complex geometries: Although 3D printing facilitates the production of intricate geometries with considerable convenience, 4D printing elevates this capability by permitting the generation of objects with heightened intricacy and dynamicity. Through leveraging the shape-altering attributes of dynamic materials, 4D printing can yield objects boasting intricate internal architectures or mobile components that would present challenges or feasibility issues when employing conventional manufacturing techniques. As an illustration, dental models fabricated via 4D printing can integrate responsive attributes, enabling the development of deeply individualized prosthetic solutions. Furthermore, the intrinsic pliability of 4D-printed substrates permits the fabrication of intricate configurations, including bespoke implants tailored to accommodate shifting oral dynamics, thus guaranteeing superior performance and durability [ 8 ]. Self-assembly and self-repair: 4D printing facilitates the production of objects capable of self-assembling or self-repairing devoid of external intervention. For instance, structures created through 4D printing could autonomously fold or unfold into predefined configurations or mend damage incurred from wear and tear over time. This innate ability for self-assembly and self-repair holds considerable ramifications for domains including robotics, infrastructure upkeep, and healthcare [ 9 ]. For instance, dental implants crafted through 4D printing may incorporate self-healing materials, enabling automated repair of minor impairments. This feature not only extends the longevity of the implants and diminishes the necessity for frequent interventions but also elevates patient comfort while mitigating the potential hazards associated with compromised prostheses. This attribute underscores a distinct advantage over conventional 3D-printed counterparts. Material efficiency: A further benefit of 4D printing lies in its capacity for enhancing material efficiency. By harnessing the programmable shape-altering properties of dynamic materials, 4D printing can optimize the utilization of materials and diminish waste throughout the fabrication process. This attribute proves particularly advantageous in contexts where lightweight and resource-efficient structures are sought after, such as within the aerospace or automotive sectors. As an example, dental structures produced through 4D printing exhibit the capability to adaptively alter their shape and characteristics, consequently diminishing the requirement for surplus material consumption in the manufacturing process [ 10 ]. Enhanced functionality: 4D printing presents an opportunity to augment the functionality of printed objects beyond the capabilities achievable through 3D printing alone. Through the integration of sensors, actuators, or other functional elements into dynamic materials, 4D-printed objects can demonstrate advanced functionalities such as sensing, actuation, or communication. This broadens the horizon for the development of intelligent and interactive objects for diverse applications, encompassing realms such as healthcare, consumer electronics, and wearable technology [ 11 ]. Versatility and customization: 4D printing offers enhanced versatility and customization capabilities in contrast to 3D printing. By enabling precise control over the timing and extent of shape changes or transformations, 4D printing facilitates the production of tailor-made objects tailored to precise requirements or preferences. This inherent versatility and customization potential render 4D printing well-suited for a diverse array of applications spanning various industries, ranging from personalized medical implants to customizable consumer goods. For instance, dental appliances fabricated through 4D printing possess the capability to adaptively modify their characteristics in response to individual patient requirements and evolving oral circumstances, facilitating the creation of meticulously customized solutions [ 12 ]. General principles of 4D printing technology 4D printing technology constitutes a notable progression beyond conventional 3D printing methodologies, incorporating the temporal aspect into the manufacturing process. Fundamentally, the principles underpinning 4D printing hinge upon the utilization of dynamic materials capable of undergoing alterations in shape or form in reaction to external stimuli [ 13 ]. An intricate elucidation of the underlying principles guiding 4D printing is mentioned in Figure 2. Figure 2 A depiction of the general principles of 4D printing technology Credit: Image created by the author Principles Dynamic materials: Central to 4D printing is the utilization of dynamic materials endowed with the capability to undergo alterations in shape, properties, or functionality in response to particular external stimuli. Such materials encompass a variety of options, including shape-memory polymers, hydrogels, smart alloys, and other responsive substances [ 14, 15 ]. Responsive design: The design process in 4D printing is meticulously crafted to capitalize on the distinctive characteristics of dynamic materials. Objects are meticulously engineered with predetermined shape-altering functionalities that are triggered by external stimuli such as fluctuations in temperature, exposure to light, variations in moisture levels, or magnetic fields. Material response mechanism: Various dynamic materials demonstrate diverse response mechanisms to external stimuli. For instance, shape-memory polymers can undergo reversible alterations in shape upon heating or cooling, whereas hydrogels may expand or contract in reaction to fluctuations in humidity levels. Profound comprehension and effective utilization of these material response mechanisms are imperative in the realm of 4D printing [ 16 ]. Temporal dimensions: In contrast to conventional 3D printing, which yields static objects, 4D printing integrates the temporal dimension into the fabrication process. Printed objects are meticulously designed to undergo pre-defined shape alterations or transformations over time subsequent to fabrication, thereby incorporating the fourth dimension, time, into the process, hence the appellation "4D" (comprising three spatial dimensions alongside time) [ 17 ]. Precision control: Attaining meticulous control over the timing and extent of shape alterations or transformations holds paramount importance in the realm of 4D printing. This necessitates thorough deliberation of factors encompassing material characteristics, stimulus triggers, the geometry of the printed object, and environmental parameters to guarantee the attainment of the intended functionality [ 18 ]. Applications exploration: The potential applications of 4D printing are extensive and varied. For instance, in healthcare, implants fabricated through 4D printing could dynamically alter their shape to conform to precise anatomical structures. Ongoing research and development endeavors are continuously exploring and unveiling novel opportunities for this burgeoning technology across diverse industries [ 19 ]. The equations utilized or created in the realm of 4D printing to model the shape-morphing behaviors can be categorized into five distinct groups which are mentioned in Figure 3. Figure 3 A depiction of the shape-modifying behaviors Credit: Image created by the author Laws of 4D printing technology The prognostication of the time-evolving characteristics, which constitute the fourth dimension, in any structure produced through 4D printing is imperative. Through an exhaustive and methodical exploration of 4D printing and its associated domains, we are able to discern three overarching principles that dictate the shape-altering tendencies of nearly all 4D structures, notwithstanding the diversity of materials and stimuli involved. These principles serve a dual purpose: first, to gain comprehension, and second, to formulate models and projections regarding the fourth dimension [ 20 ]. The laws of 4D printing technology are mentioned in Figure 4. Figure 4 A depiction of the laws of 4D printing technology Credit: Image created by the author First Law The vast array of shape-altering tendencies are observed in multi-material 4D-printed structures, including those influenced by thermo-responsive stimuli, electrochemical and thermal, ultrasound, enzymes, hydro, photothermal, and photochemical solvents. These factors can be attributed to a fundamental phenomenon known as relative expansion between active and passive materials. This relative expansion serves as the underlying mechanism for the intricate shape-morphing behaviors exhibited in 4D printing, such as twisting, coiling, and curling. These behaviors are made possible through the incorporation of various forms of anisotropy between active and passive materials and the fabrication of diverse heterogeneous structures. Second Law The shape-altering characteristics exhibited by the majority of multi-material 4D-printed structures are governed by four distinct physical mechanisms which include mass diffusion, thermal expansion, molecular transformation, and organic growth. These mechanisms are thoroughly examined, quantified, and integrated into the subsequent sections. Each of these mechanisms contributes to the relative expansion between active and passive materials, resulting in the ensuing shape-morphing behaviors when subjected to stimuli. While these stimuli typically originate externally, they can also arise internally. Third Law The time-evolving shape-altering characteristics observed in the majority of multi-material 4D-printed structures are regulated by two distinct "categories" of time constants. In the simplest scenario, where a multi-material 4D-printed structure comprises one active and one passive layer, these constants play a pivotal role. The pictorial representation of 4D printing technology is mentioned in Figure 5. Figure 5 A depiction of the representation of 4D printing technology Credit: Image created by the author Material considerations for 4D printing technology and its application in the field of dentistry Material selection in 4D printing entails a meticulous evaluation of various factors to ensure the chosen material effectively accomplishes the desired shape-changing behavior. The process involves several key steps which include identifying the specific shape transformations required for the application and selecting a material that can respond appropriately to stimuli like heat, light, or moisture. Pinpointing the external stimulus that will trigger the shape change and ensuring the material is responsive under controlled conditions. Considering the mechanical, thermal, chemical, and biocompatibility properties of the material to ensure alignment with application requirements. Evaluating compatibility with the 3D printing process, including factors like printability and post-processing needs. Assessing cost-effectiveness and availability, while also conducting experimental validation to confirm the material's performance. Accounting for regulatory and safety considerations to ensure compliance with relevant standards and guidelines [ 21 ]. Shape Memory Polymers Shape memory polymers (SMPs) represent a category of intelligent materials endowed with the capacity to revert from a deformed state to their original, predetermined form under specific external stimuli, such as changes in temperature, light exposure, or alterations in pH levels [ 22 ]. This capability of shape recovery arises from the temporary fixation of the polymer's macromolecular chains into an interim configuration, which can be prompted to return to its permanent state upon activation [ 23 ]. SMPs demonstrate attributes akin to both elasticity and plasticity, enabling them to undergo reversible deformation and recuperation multiple times without enduring lasting damage. These materials find diverse applications across sectors such as medicine, aerospace, textiles, and robotics, where their shape-altering characteristics are harnessed for purposes like actuation, sensing, and the creation of adaptable structures [ 24 ]. The utilization of SMPs in the field of dentistry is mentioned in Table 1. Table 1 An illustration of the utilization of shape memory polymers in the field of dentistry PCL: Polycaprolactone; SMP: Smart memory polymers; PEG: Polyethylene glycol Shape memory polymer Inference Applications in dentistry Polycaprolactone (PCL) Biodegradable polyester that exhibits shape memory behavior [ 25 ]. Dental implants, temporary crowns, and orthodontic devices can conform to the patient's anatomy and then revert to their original shape once implanted. Polyurethane-based SMPs Polyurethane-based shape memory polymers offer flexibility and biocompatibility, making them suitable for various dental applications. Dental splints, aligners, and prosthetic devices that can adjust to the patient's mouth shape over time. Methacrylate-based SMPs Methacrylate-based shape memory polymers are commonly used in dental materials due to their excellent mechanical properties and biocompatibility. Temporary bridges, denture bases, and orthodontic brackets. Polyethylene glycol (PEG) based SMPs PEG-based shape memory polymers are hydrophilic and can swell in the presence of water, making them suitable for dental applications where moisture responsiveness is required. Dental adhesives, drug delivery systems, and oral tissue scaffolds that can adapt to the oral environment. Hydrogels Hydrogels employed in 4D printing represent interconnected networks of hydrophilic polymers with the ability to absorb and retain substantial quantities of water or biological fluids while preserving their structural integrity. These materials possess an inherent affinity for water, enabling them to expand when immersed in aqueous environments and undergo notable changes in volume in response to various environmental cues, including alterations in pH, temperature, light exposure, or electrical signals. Within the realm of 4D printing, hydrogels serve as essential components, functioning as both printing inks and supportive substrates to produce objects endowed with dynamic shape-shifting capabilities [ 26 ]. Through the integration of hydrogels into the printing process, intricate structures can be crafted that exhibit controlled deformation, bending, folding, or swelling over time. Such advancements hold promise for a myriad of applications across disciplines such as biomedicine, soft robotics, and tissue engineering [ 27 ]. The utilization of hydrogels in the field of dentistry is mentioned in Table 2. Table 2 An illustration of the utilization of hydrogels in the field of dentistry PEG: Polyethylene glycol; GelMA: Gelatin methacrylate Hydrogels Inference Applications in dentistry Alginate hydrogels Biocompatible, easy to handle. Dental impressions and temporary restorations of custom-fitted dental devices and implants Polyethylene glycol (PEG) hydrogels Commonly used in tissue engineering and drug delivery due to their biocompatibility and tunable properties. Dental scaffolds, oral drug delivery systems, or biocompatible coatings for dental implants. Methacrylate-based hydrogels Excellent mechanical properties and compatibility with dental restorative materials. Fabrication of dental prostheses, orthodontic appliances, and oral tissue scaffolds. Chitosan hydrogels Possess antimicrobial properties and biocompatibility, making them suitable for dental applications such as wound healing and tissue regeneration. Fabrication of dental implants, periodontal membranes, or bioactive dental materials. Gelatin methacrylate (GelMA) hydrogels Derived from gelatin and has been extensively studied for tissue engineering and regenerative medicine applications. Dental scaffolds, gingival tissue substitutes, or bioprinted constructs for tooth regeneration. Smart Elastomers Smart elastomers utilized in 4D printing encompass a category of materials that amalgamate the characteristics of elastomers with sensitivity to external triggers. This amalgamation enables them to undergo deliberate alterations in shape over time. These materials demonstrate elastic properties, permitting reversible deformation when subjected to mechanical forces, while also possessing the capability to react to particular stimuli such as changes in temperature, light exposure, or pH levels. Within the realm of 4D printing, smart elastomers serve as key components in generating objects endowed with dynamic shape-shifting abilities [ 28 ]. During the printing process, the desired shape changes are encoded into the material's structure. Through the integration of smart elastomers into 4D printing, intricate structures can be crafted with the ability to adapt, deform, or reconfigure in response to environmental cues. This advancement holds promise for a wide array of applications in fields such as soft robotics, wearable technology, and biomedical implants [ 29 ]. The utilization of smart elastomers in the field of dentistry is mentioned in Table 3. Table 3 An illustration of the utilization of smart elastomers in the field of dentistry UV: Ultraviolet Smart elastomers Inference Applications in dentistry Thermoresponsive elastomers Change their shape or properties in response to temperature variations. Dental devices that adapt to the oral environment, such as orthodontic aligners or customized mouthguards. Light-responsive elastomers Elastomers that respond to light stimuli, such as UV light, can be utilized in 4D printing for dental applications. Light-responsive elastomers enable precise control over shape changes during printing and can be used to fabricate dental prostheses, temporary crowns, or oral drug delivery systems. pH-sensitive elastomers Elastomers that exhibit changes in shape or properties in response to variations in pH levels. Oral drug delivery or tissue engineering. pH-sensitive elastomers enable targeted release of therapeutic agents or modulation of cellular responses within the oral cavity. Stimuli-responsive hybrid elastomers Hybrid elastomers that combine multiple responsiveness mechanisms, such as temperature and pH sensitivity, offer enhanced control over shape changes in 4D printing. Tailored for specific dental applications, including personalized orthodontic devices, periodontal membranes, or dental adhesives. Composites Composites employed in 4D printing technology involve the integration of multiple components with diverse properties to craft structures capable of dynamic shape alterations over time. These composite materials typically comprise a base material fortified with embedded elements like fibers, particles, or additives, which confer specific functionalities or response capabilities [ 30 ]. In the realm of 4D printing, these composites are applied to manufacture objects endowed with shape transformations that can be programmed strategically by incorporating materials exhibiting varied reactions to external stimuli, such as temperature fluctuations, light exposure, moisture changes, or mechanical forces. Through leveraging the synergistic attributes of their constituent elements, composites facilitate the production of intricate structures capable of adapting, deforming, or reconfiguring in response to environmental stimuli. This attribute renders them versatile and functional across a broad spectrum of applications spanning aerospace, engineering, biomedical, and consumer product domains [ 31 ]. The utilization of composites in the field of dentistry is mentioned in Table 4. Table 4 An illustration of the utilization of composites in the field of dentistry PLA: Polylactic acid; PGA: Polyglycolic acid Composites Inference Applications in dentistry Bioactive composites incorporate bioactive materials, such as calcium phosphates or bioactive glasses, into a polymer matrix. Bioactive composites promote remineralization and integration with natural tooth structures, making them suitable for dental restorations, fillings, or implants [ 32 ]. Antimicrobial composites Composites containing antimicrobial agents, such as silver nanoparticles or quaternary ammonium compounds, help prevent bacterial colonization and reduce the risk of dental infections. Dental prosthetics, orthodontic appliances, or periodontal membranes [ 33 ]. Biodegradable composites composed of polymers such as polylactic acid (PLA) or polyglycolic acid (PGA) degrade over time and are absorbed by the body, making them suitable for temporary dental devices or drug delivery systems Dental splints, sutures, and bone graft substitutes [ 34 ]. Hybrid composites combine different types of materials, such as ceramics, metals, or polymers, to achieve specific mechanical, aesthetic, or biocompatible properties [ 35, 36 ]. Dental crowns, bridges, or veneers that require enhanced strength and durability. Thermoresponsive composites containing thermoresponsive polymers undergo reversible shape changes in response to temperature variations. Thermoresponsive composites are employed in 4D printing for dental appliances that adapt to oral temperature changes, such as denture bases or orthodontic aligners. Conductive Materials Conductive materials utilized in 4D printing technology are substances endowed with the capacity to conduct electricity. These materials are integrated into printed objects to confer electrical conductivity or responsiveness to external electrical stimuli. Typically, these materials comprise conductive particles such as graphene, carbon nanotubes, or metal nanoparticles dispersed within a polymer matrix or ink. In the realm of 4D printing, conductive materials serve to fashion objects with functional electrical properties, including sensors, actuators, or electronic circuits, which can be activated or regulated via electrical signals [ 37 ]. By integrating conductive materials into the printing process, it becomes feasible to manufacture intricate structures capable of detecting, transmitting, or modulating electrical signals, thus offering potential applications in fields such as electronics, wearable technology, and smart devices. The utilization of conductive materials in the field of dentistry is mentioned in Table 5. Table 5 An illustration of the utilization of conductive materials in the field of dentistry CNT: Carbon nanotubes Conductive materials Inference Applications in dentistry Graphene A single layer of carbon atoms arranged in a hexagonal lattice is known for its excellent electrical conductivity. In dentistry, graphene-based conductive inks can be used to create dental sensors for monitoring parameters such as temperature, pH levels, or bacterial presence in the oral cavity [ 38 ]. Carbon nanotubes (CNTs) Carbon nanotubes exhibit exceptional electrical conductivity and mechanical strength. CNTs can be incorporated into dental materials to enhance their electrical properties or used to fabricate conductive pathways for electronic devices embedded within dental prostheses or orthodontic appliances [ 39 ]. Silver nanoparticles Possess high electrical conductivity and antimicrobial properties. Conductive dental adhesives, fillings, or coatings for dental implants with antibacterial functionalities. polyaniline or polypyrrole Electrical conductivity and flexibility, make them suitable for flexible electronic devices in dentistry. Fabrication of flexible sensors or electrodes for intraoral diagnostic applications [ 40 ]. Metal nanoparticles Metals such as gold or copper nanoparticles, exhibit good electrical conductivity and biocompatibility. Metal nanoparticle-based inks can be used to create conductive patterns for electronic circuits or implantable sensors for monitoring dental health parameters. Responsive Inks Responsive inks utilized in 4D printing technology are specialized printing materials with dynamic characteristics that react to external stimuli. These inks are engineered to undergo specific alterations, such as changes in color, shape, conductivity, or mechanical attributes, in response to environmental cues like fluctuations in temperature, exposure to light, variations in humidity, or chemical interactions [ 41 ]. Typically composed of functional components such as intelligent polymers, nanoparticles, or reactive dyes dispersed within a carrier medium such as a polymer solution or solvent, responsive inks enable the production of objects with programmable behaviors that evolve over time. In the realm of 4D printing, these inks facilitate the fabrication of intricate structures whose responsive properties are predetermined within the ink formulation and activated by external triggers during the printing process [ 42 ]. By leveraging the capabilities of responsive inks, 4D printing technology enables the creation of complex structures capable of adapting, evolving, or self-assembling in response to changing environmental conditions, opening new avenues for innovation in fields including materials science, biomedicine, and consumer electronics. The utilization of responsive inks in the field of dentistry is mentioned in Table 6. Table 6 An illustration of the utilization of responsive inks in the field of dentistry Responsive inks Inference Applications in dentistry Thermochromic inks These inks change color in response to temperature variations, allowing for the creation of dental devices that indicate temperature changes in the oral cavity. It can be incorporated into dental appliances like mouthguards or orthodontic aligners to alert users to potential issues such as temperature-sensitive dental conditions or improper fit. Hydrochromic inks Hydrochromic inks alter their color or transparency when exposed to moisture, making them suitable for dental applications where moisture levels play a role, such as detecting saliva or plaque accumulation. Fabrication of dental devices that provide visual feedback on oral hygiene or moisture levels in the mouth. pH-sensitive inks Change color in response to variations in pH levels, allowing for the creation of dental devices that monitor oral pH balance. These inks can be integrated into dental materials such as dental adhesives, restorations, or remineralization agents to indicate changes in oral acidity or alkalinity, aiding in the prevention of dental caries or enamel erosion [ 43 ]. Antimicrobial inks Antimicrobial inks contain agents that inhibit bacterial growth or promote oral health, making them suitable for dental applications where bacterial colonization is a concern. Fabrication of dental devices with built-in antimicrobial properties, such as dental implants, prosthetics, or orthodontic appliances, to prevent infections or promote healing in the oral cavity. Smart polymer inks They undergo reversible changes in shape, stiffness, or conductivity in response to external stimuli such as temperature, light, or moisture. Fabrication of dental devices with dynamic properties, such as shape-changing orthodontic appliances or self-adjusting dental materials that adapt to changes in the oral environment [ 44 ]. Biodegradable Polymers Biodegradable polymers employed in 4D printing technology are organic-based materials engineered to naturally decompose into harmless substances when exposed to environmental factors like moisture, heat, or microbial activity. These polymers are specially designed to undergo regulated degradation, resulting in the gradual breakdown of printed items into environmentally safe components [ 45 ]. In 4D printing, these polymers serve as printing substrates to create objects with shape-changing capabilities over time, where the degradation process influences the objects' temporal behaviors. By integrating biodegradable polymers into 4D printing processes, it becomes feasible to produce eco-friendly structures capable of adjusting, evolving, or disintegrating over time. Such advancements hold promise for applications in various sectors, including biomedical implants, environmental monitoring devices, and disposable consumer goods. The utilization of biodegradable polymers in the field of dentistry is mentioned in Table 7. Table 7 An illustration of the utilization of biodegradable polymers in the field of dentistry PLA: Polylactic acid; PGA: Polyglycolic acid; PCL: Polycaprolactone; PHA: Polyhydroxyalkanoates Biodegradable polymers Inference Applications in dentistry Polylactic acid (PLA) Biodegradable polymer derived from renewable resources such as corn starch or sugarcane. It is widely used in 4D printing for dental applications due to its biocompatibility, mechanical strength, and ability to degrade into non-toxic lactic acid under natural environmental conditions [ 46 ]. Fabrication of dental implants, temporary crowns, or orthodontic devices [ 47, 48 ]. Polyglycolic acid (PGA) Biodegradable polymers are commonly used in tissue engineering and drug delivery applications. To create biodegradable scaffolds for periodontal tissue regeneration or controlled-release systems for dental therapeutics [ 49 ]. Polycaprolactone (PCL) Biodegradable polyester with a slow degradation rate, making it suitable for long-term dental applications. To fabricate dental splints, bone graft substitutes, or customized dental implants that gradually degrade and integrate with surrounding tissues over time [ 50 ]. Polyhydroxyalkanoates (PHA) A group of biodegradable polymers produced by bacterial fermentation of renewable feedstocks. They exhibit biocompatibility and versatility in 4D printing for dental applications. Fabrication of biodegradable sutures, drug delivery systems, or tissue scaffolds for oral tissue regeneration. Chitosan Biodegradable polysaccharide derived from chitin, a natural polymer found in the exoskeleton of crustaceans. Create biodegradable films, membranes, or drug-delivery vehicles for periodontal therapy or oral wound healing [ 51 ]. Advantages of 4D printing technology 4D printing technology, with its unique capability to fabricate objects capable of altering shape or functionality in response to external stimuli, presents numerous distinct advantages within the domain of dentistry. Customization A paramount benefit of integrating 4D printing into dentistry lies in its unparalleled capacity for customization. This technology empowers dental practitioners to precisely tailor structures and appliances to harmonize with the singular anatomy and requirements of individual patients, thereby enhancing fit, comfort, and functionality. Intricate Geometries 4D printing facilitates the production of dental devices featuring intricate geometries that would pose considerable challenges or be unattainable through conventional manufacturing methods. Such capabilities enable the creation of highly intricate dental implants, prosthetics, and orthodontic appliances, elevating the sophistication of treatment options. Patient-Centric Solutions By harnessing 4D printing, dental professionals can devise treatment solutions meticulously tailored to the precise needs and oral conditions of each patient. This personalized approach not only augments treatment efficacy and patient satisfaction but also mitigates the likelihood of complications arising during dental interventions. Material Efficiency In contrast to traditional subtractive manufacturing approaches notorious for generating substantial material waste, 4D printing operates on an additive principle, utilizing only the requisite amount of material. Consequently, this methodological shift contributes to diminished material waste and reduced environmental impact in dental manufacturing processes [ 52 ]. Enhanced Efficiency and Time Management Despite inherent processing time constraints, the integration of 4D printing into dental workflows has the potential to streamline operations by consolidating multiple manufacturing stages into a cohesive process. This heightened efficiency translates into time savings throughout the fabrication and delivery of dental devices, benefiting both patients and dental practitioners. Cutting-Edge Materials Progressions in 4D printing technology have spurred the development of novel materials tailored specifically for dental applications. These advanced materials boast superior biocompatibility, durability, and flexibility, thereby augmenting the performance and longevity of dental devices. Minimally Invasive Techniques 4D-printed dental devices facilitate minimally invasive treatment modalities by conforming precisely to the patient's anatomical contours, thereby minimizing the necessity for extensive surgical interventions. Consequently, patients experience reduced trauma, accelerated recovery periods, and overall enhanced treatment experiences. Research and Development Opportunities The versatility inherent in 4D printing technology serves as a catalyst for innovation within dental research and development endeavors. Researchers are afforded the opportunity to explore avant-garde designs, materials, and functionalities, thereby addressing unmet clinical needs and propelling the evolution of dental science. Collectively, the advantages conferred by 4D printing in dentistry encapsulate a spectrum of benefits including personalized customization, geometric intricacy, operational efficiency, environmental sustainability, and pioneering innovation, all of which converge to elevate patient care standards and treatment outcomes within the dental sphere. Limitations of 4D printing technology 4D printing technology, which entails the fabrication of objects capable of morphing in shape or behavior over time in response to external stimuli, holds considerable promise across diverse fields, including dentistry. Nonetheless, akin to any nascent technological innovation, it is not devoid of limitations, particularly within the realm of dental applications. Several constraints specific to 4D printing in dentistry are discernible. Material Selection The materials utilized in 4D printing must conform to stringent criteria regarding biocompatibility, durability, and flexibility to be deemed suitable for dental purposes. The restricted availability of such materials may impede the advancement of intricate dental structures. Resolution and Accuracy Attaining optimal resolution and precision in 4D printing processes assumes paramount importance in crafting exacting dental implants, prosthetics, or orthodontic apparatuses. Existing constraints in printing resolution could potentially compromise the fit, functionality, and enduring efficacy of dental constructs. Processing Time The procedural duration encompassing both printing and activation phases in 4D printing can be notably protracted. Dental establishments necessitate streamlined workflows to ensure the expeditious delivery of bespoke dental solutions, a task rendered challenging by prolonged fabrication durations. Cost 4D printing technology remains relatively costly compared to conventional manufacturing methodologies prevalent in dentistry. The elevated expenses associated with equipment procurement and material acquisition might curtail widespread adoption, particularly among smaller dental practices or in developing regions. Complexity of Design and Fabrication The intricate geometries and multifaceted functionality inherent in designing and fabricating 4D-printed dental devices mandate specialized expertise and knowledge. Dentists and dental technicians may necessitate supplementary training to effectively harness the full potential of this technology [ 1 ]. Regulatory Approval and Standardization Negotiating the labyrinthine regulatory approval processes pertinent to 4D-printed dental products can prove intricate and time-intensive. Establishing standardized protocols governing manufacturing, quality control, and safety assessment assumes paramount importance in ensuring compliance with regulatory requisites [ 53 ]. Long-Term Performance A comprehensive evaluation of the enduring performance and biocompatibility of 4D-printed dental devices is imperative. Delving into the mechanical properties, degradation kinetics, and tissue response of 4D-printed materials is indispensable for appraising their suitability for prolonged clinical utilization. Notwithstanding these constraints, ongoing research endeavors and technological strides are progressively tackling many of these impediments, thereby augmenting the capabilities and applications of 4D printing in dentistry. Through further refinement and advancement, 4D printing stands poised to revolutionize diverse facets of dental care, proffering tailored and functional solutions to patients. Future directions The future potential of 4D printing in dentistry is vast, spanning multiple critical areas of advancement. This includes the transformation of dental prosthetics, where dynamic devices could adapt to oral changes, enhancing patient comfort and functionality [ 54 ]. Orthodontic treatments may become more efficient and personalized through customizable appliances adjusting to tooth alignment. Moreover, 4D printing's integration with biofabrication techniques offers possibilities for tissue regeneration, addressing conditions like periodontal disease. Additionally, 4D-printed dental devices could deliver localized therapeutic agents, improving treatment precision while minimizing systemic effects. Precision surgical guides and implant components could be tailored to individual anatomy, enhancing procedure accuracy and patient outcomes. In education, 4D printing enables immersive learning experiences, while advancements in digital dentistry could facilitate remote care delivery, expanding access to high-quality dental solutions. This trajectory of innovation underscores the transformative potential of 4D printing in dentistry, requiring continued research, collaboration, and technological refinement to realize its full impact. Conclusions The emergence of 4D printing technology in dentistry stands poised to transform the landscape of dental healthcare delivery and patient experience. This cutting-edge technology presents a wide array of opportunities, ranging from the creation of dynamic dental prosthetics to tailored orthodontic interventions and novel approaches to tissue regeneration. Through its capacity to drive innovation, enhance treatment precision, and widen the availability of sophisticated dental solutions, 4D printing heralds a significant advancement in the field. Ongoing research, collaboration, and refinement of this technology will be crucial in fully unlocking its potential to reshape the future of dental care.

10. 7759/cureus. 58664

Cureus

Synthesis and Analysis of Novel Hyaluronic Acid-Based Dual Photocrosslinkable Tissue Adhesive: An In Vitro Study

Background Tissue adhesives are mainly used for aiding in the attachment of adjacent tissues or to nearby hard tissue surfaces. They promote the natural healing processes of the tissues, especially for less painful closure, simple application, no need for sutures following surgery, and localized drug release. This study aimed to synthesize and assess the properties of hyaluronic acid (HA)-based, dual photocrosslinkable tissue adhesive. Materials and methodology N-hydroxysuccinimide (NHS), 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), HA, and polymethylmethacrylate, which served as a photoinitiator, were combined to synthesize a tissue adhesive. The prepared formulation was characterized, and its biocompatibility was assessed. Results Surface morphology, mechanical properties, and biological properties of the HA adhesive were comparable to those of conventional fibrin glue. Scanning electron microscopy (SEM) analysis showed the average size of the molecules, 10-25 mm in diameter, and also showed a smooth and nonporous surface. The specimens experienced maximum compressive stress of 0. 06 ± 0. 02 MPa, compressive strain of 3. 07 ± 2. 02, and a compressive displacement at break of 3. 04 ± 1. 23 mm, with a maximum force of 2. 33 ± 0. 07 N at break. The cytotoxicity assay results for HA and fibrin glue are almost equal. Conclusion HA-based photocrosslinkable tissue adhesive could be a potential biomaterial in various applications in the field of medicine, especially in soft tissue management.

Introduction Tissue adhesives are one of the most rapidly developing medical advances in recent times. It is one of the medical innovations that has developed most quickly in recent years [ 1 ]. Tissue adhesive, also known as surgical adhesive or medical glue, is a type of adhesive used in medicine and surgery to close wounds, incisions, or lacerations. It is designed to bond biological tissues together and promote healing without the need for traditional sutures or staples [ 2 - 5 ]. Tissue adhesives are typically made from biocompatible materials that are safe for internal use and can be absorbed or broken down by the body over time. The most common types of tissue adhesives include cyanoacrylates, fibrin sealants, and collagen-based adhesives [ 6 ]. Cyanoacrylate adhesives, often referred to as "superglues, " are widely used in medical applications. They polymerize quickly in the presence of moisture and form a strong bond between tissue surfaces. These adhesives are often used for closing small superficial wounds, such as lacerations on the skin, but are known to have an exothermic reaction, which might harm the tissues [ 7 ]. Fibrin sealants are composed of fibrinogen and thrombin, two proteins involved in the blood clotting process. When combined, these proteins form a clot-like substance that adheres to the wound, sealing it and promoting healing. Fibrin sealants are often used in surgeries involving blood vessels or other delicate tissues [ 8 ]. Collagen-based adhesives are derived from animal sources and contain collagen proteins. These adhesives mimic the natural extracellular matrix found in tissues and promote cell migration and tissue regeneration. They are commonly used in ophthalmic and neurological surgeries [ 9 ]. Tissue adhesives have several advantages over traditional sutures or staples. They can be applied quickly, reducing procedure time and potentially leading to improved patient comfort. Adhesives also provide a barrier against bacteria, reducing the risk of infection. Additionally, they eliminate the need for suture removal, as most tissue adhesives are absorbed or sloughed off naturally as the wound heals [ 10 ]. Hydrogels are another type of tissue adhesive that consists of water-absorbing polymers. These adhesives are biocompatible and can adhere to wet surfaces, making them suitable for use in moist environments, such as the gastrointestinal tract or mucosal surfaces. Hydrogels are used by various medical fraternities for wound closure, drug delivery systems, and tissue engineering [ 11 ]. Hyaluronic acid (HA) is a naturally found compound present in the human body in connective tissues and fluids such as synovial fluid. It is a glycosaminoglycan, which means it is a long chain of repeating sugar molecules. HA has also gained limelight in the field of medicine and cosmetics due to its unique properties, including its ability to retain water and provide lubrication [ 12 ]. The goal of this study was to develop a dual photocrosslinkable HA-based tissue adhesive, analyze its mechanism, describe its unique properties, discuss the benefits and drawbacks of using it in clinical settings, and find ideas for future research projects aimed at creating the next generation of tissue adhesives. Materials and methods Preparation of adhesive Initially, 10× phosphate-buffered saline (PBS) was formulated by adding 10 mL of PBS with 90 mL of distilled water, which was later mixed with HA. In a separate container, 120 mg of N-hydroxysuccinimide (NHS) was added to 55 mg of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC). The latter was mixed with activated HA. This product was kept at -80°C to ensure that the residue became sufficiently sedimented to be separated on its own. After that, ethanol was used to wash the residue four or five times. The supernatant solution was removed. Afterward, the pellets were stored at -80°C. Then, the moisture was extracted by vacuum-drying. Next, a 300-mL beaker containing 0. 1 L of deionized water and 1 g of HA was combined. After that, 50 mL of deionized water was mixed with 10 g of sodium hydroxide in a different beaker. This was followed by the transfer of 2 mL of NaOH to a 100 mL beaker. Finally, 7. 8 mL of poly(methyl methacrylate), a photoinitiator, was added to this mixture. Characterization Visual Observation Scanning electron microscopy (SEM) analysis was done to analyze the structural characteristics of the prepared adhesive sample (Japan Electron Optics Laboratory (JEOL) JSM-IT800, JEOL (Germany) GmbH, Freising, Germany, platinum sputter). The adhesive samples were split into multiple pieces using a razor blade. Before SEM analysis, the specimen was sputtered by platinum in a vacuum (JEOL, JFC-1600, Tokyo, Japan). The glue was characterized by Fourier-transform infrared spectroscopy (FTIR) with the aid of attenuated total reflectance (ATR) mode. The wave number ranged from 500 to 5, 000 cm -1 at a resolution of 1 cm -1, with an average of 64 scans. The equipment used was Bruker Alpha II - compact Fourier transform infrared spectrometer with platinum ATR. In a 1:5 ratio, freeze-dried adhesive was powdered and mixed with potassium bromide. Strength Test Compressive strength was evaluated with the help of a dynamic universal testing machine (UTM) (Instron Electroplus E3000), as shown in Figure 1. Specimen dimensions were 150 × 10 mm 2 and a distance of 100 mm between the grips initially. This was in accordance with the American Society for Testing Materials (ASTM) standard. One end of the specimen (fixed) was retained in the upper cross-head of the machine. The specimen was gradually loaded by the machine's loading unit while its other end was attached to the movable, adjustable cross-head. Figure 1 Compressive strength analysis using a universal testing machine Cell Viability Analysis A LIVE/DEAD fluorescence assay kit (produced by Molecular Probes) (Sigma Aldrich, Burlington, USA) was used to assess the viability test. In a qualitative biocompatibility test, Living Dead® (Sigma Aldrich, Burlington, USA) is utilized. The cells were seeded (3 × 106 cells/mL) in a 96-well plate and cultivated for 24 hours at 37°C in Dulbecco’s Modified Eagle Medium - Low Glucose (DMEM-LG) with 10% FBS. The cells were then cultured for 24 hours using the poly-l-lactic acid (PLLA) scaffolds, which measure 2 mm by 5 mm. Following a 24-hour period, the cells were cleaned with 200 L of PBS and then treated with the calcein acetoxymethyl ester (calcein-AM) and ethidium homodimer-1 solution per the manufacturer's instructions. Following a half-hour incubation at 37°C, the cells were cleaned and stored in PBS. The cells were analyzed using the Nikon E800's inverted fluorescence microscopy program (Image Pro-Plus software, Media Cybernetics, Rockville, USA). Results SEM analysis Microstructural morphological characteristics of the HA-based photocrosslinkable tissue adhesive is shown in Figure 2. SEM analysis verified that the average size of the molecules was 10-25 mm in diameter, showing a smooth and nonporous surface. Figure 2 SEM image of HA tissue adhesive BD, beam deceleration; FOV, field of view; HA, hyaluronic acid; SEM, scanning electron microscopy; UHD, ultra high definition; WD, working distance FTIR analysis FTIR analysis reveals the photocross linkage between HA and photoinitiator. The peak wave was seen at 3, 297. 17 and 1, 077. 5 cm -1, which indicates O-H and C-O structure, respectively (Figure 3 ). Figure 3 FTIR image of HA tissue adhesive FTIR, Fourier-transform infrared spectroscopy; HA, hyaluronic acid Compressive strength The test specimen's compressive strength was assessed with the aid of the Instron Universal Testing Machine (Instron, Norwood, USA). As seen in Figure 4, the displacement for the specimen was 3 mm at 0 kN. The specimens experienced maximum compressive stress of 0. 06 ± 0. 02 MPa, compressive strain of 3. 07 ± 2. 02, and compressive displacement at break of 3. 04 ± 1. 23 mm at maximum force of 2. 33 ± 0. 07 N at break. The compressive strength of the specimen was depicted graphically in Figure 4. The average compressive strength analysis values of the samples are tabulated in Table 1. Figure 4 Graphical representation of compressive strength of the specimen Table 1 Compressive strength analysis of the HA-based tissue adhesive Maximum force (N) Compressive displacement at break (standard) (mm) Compressive strain (displacement) at break (standard) (mm) Compressive stress at break (standard) (MPa) 2. 33 ±0. 07 3. 04 ± 1. 23 3. 07 ± 2. 02 0. 06 ± 0. 02 Viability by LIVE/DEAD® The polymer's biocompatibility with cells is qualitatively demonstrated by the LIVE/DEAD® experiment. The cells were grown using the biomaterial. Living cells interacted with the fluorescent marker SYTO® 9 (Thermo Fisher Scientific Inc. , Waltham, USA), which resulted in the green staining of healthy cells. Conversely, unviable cells were dyed red, signifying that they were dead cells. The LIVE/DEAD® technique is illustrated in Figure 5 and Figure 6 using the results of inverted fluorescence microscopy. Our findings from the cytotoxicity test revealed that while the fibrin glue increased the number of visible dead cells, the HA-based tissue adhesive enhanced the growth of L929 cells than the former. This demonstrates the efficacy and biocompatibility of our artificial HA-based tissue adhesive. Figure 5 Cell viability of fibrin glue-based tissue adhesive Figure 6 Cell viability of HA tissue adhesive HA, hyaluronic acid Discussion Biomaterials, which are used as tissue adhesives, have a crucial role in cell behavior, such as cell adhesion and gene expression, mainly because of their consistency. Thus, maintaining the mechanical properties of the materials would be advantageous [ 13 ]. Our study data showed that the stiffness of the HA-polyethylene glycol (PEG) hydrogels may be changed by adjusting the pH of a solution containing PEG-(NH2)6 prior to gelation. Two competing reactions - hydrolysis and reactivity with primary amines - with the NHS-activated carboxyl groups of HA occur simultaneously [ 14 ]. Carboxyl groups are produced through hydrolysis, while amide bonds are produced by reaction with primary amines. Typically, both reactions take place more frequently at higher pHs, with amide production increasing the most. Yet as the reaction progresses in this instance, the molecular weight of both polymers rises [ 15 ]. This causes the polymers containing carboxyl groups with NHS activation and primary amines to diffuse at slower rates, which lengthens the time it takes for the reaction between two functional groups [ 16 ]. Due to the slower diffusion rate, it is more likely that hydrolysis occurs first, before the reaction between two reactive groups. As a result, the reaction kinetics slow down when the pH is lowered, giving primary amines more time to travel to NHS-activated carboxyl groups and engage in reaction to generate amide bonds. As the number of amide bonds increases, the hydrogel becomes more crosslinked. This results in enhanced stiffness, as indicated by the greater compression modulus. This stiffness is managed with the help of altering the pH. When the density of cross-linkage and modulus rises, with the same composition in hydrogels synthesized from the same polymer, the equilibrium swelling ratio decline is predicted. The measured ratio of swelling was reduced when pH declined, as was predicted, which is in line with the compression modulus results. The variable HA content is responsible for these variabilities in the values [ 17 ]. Tissue integration has been found to be enhanced in terms of adherence to the underlying tissue. Here, we demonstrate that HA gels have a 10 times higher adherence to cartilage tissue compared to fibrin glue. Apart from creating mechanical crosslinking to the adjacent tissues, similar to fibrin glue, HA-based gel can initiate covalent crosslinks by building amide bonds with the primary amine groups located on collagen in the extracellular matrix [ 18 ]. Thus, the increased covalent crosslinks are responsible for the material's improved adhesion properties [ 19 ]. These findings are similar to those of previous studies done with HA as a tissue adhesive. Ming Li et al. explored the possibility of a novel HA hydrogel adhesive, which is based on phenylboronic acid-diol ester linkages. HA-3-(acrylamido)phenylboronic acid (HA-3APBA) hydrogels, formed through phenylboronic acid (PBA) and diols complexation, exhibit injectability, suitable mechanical properties, and a functional molecule for tissue binding. In vivo studies confirm the hydrogel's effectiveness in biocompatibility, wound exudate absorption, controlling bleeding, and accelerating wound closure. It outperforms commercial fibrin glue in adhesion tests [ 20 ]. Another study by Koivusalo et al. proved that dopamine-modified HA hydrogel enhanced tissue adhesion, enabled protein conjugation, and supported stem cell culture. It improved cell viability and mechanical properties and reduced swelling in cell culture [ 21 ]. The study by Milne et al. evaluated HA methacrylate aldehyde dual crosslinked network (HA-MA-CHO-DCN) hydrogels' adhesive performance through lap-shear and burst pressure tests, comparing them to bovine serum albumin glutaraldehyde (BSAG) glue hydrogels commonly used in surgery. The HA-MA-CHO-DCN hydrogels showed greater burst pressure in comparison to the control sealant, meeting requirements for various human tissue applications [ 22 ]. HA was combined with dopamine (HA-DN) along with thiol end-capped Pluronic F127 copolymer (Plu-SH), creating an HA/Pluronic composite gel, and the resulting hydrogels showed phase transitions that can vary depending on the temperature, with rapid sol-gel behavior, which is reversible. These hydrogels could be injected as a sol at room temperature and would quickly gel at body temperature, demonstrating excellent tissue adhesion and stability for potential drug and cell delivery applications [ 23 ]. An adhesive hydrogel made of MeHA and ELP developed by Shirzaei Sani et al. with antimicrobial properties and elastic behavior can be quickly photocrosslinked for tissue healing. MeHA/ELP hydrogels showed no significant inflammatory response, were effectively biodegradable, and facilitated the integration of new autologous tissue [ 24 ]. All the previous studies have discussed the physical and biochemical properties of the HA and the photocrosslinking property, but most of them are used in other specialities and not in dentistry. The results of these study validates the use of HA in tissue adhesives because of favorable compressive strength and biocompatibility like fibrin glue. Limitations Although the prepared HA-based tissue adhesive exhibited better mechanical and biological properties, clinical studies are further needed to substantiate these findings. The rate of degradation in the oral environment should also be analyzed in future studies. Conclusions HA-based tissue adhesives are promising candidates for a wide range of procedures especially in soft tissue management. The strength of this bio adhesive is comparable to the commercially available adhesive fibrin glue. Therefore, the HA-based photocrosslinkable adhesive could be a potential biomaterial in various applications in the field of medicine including dentistry for procedures like flap and mucogingival surgeries.

10. 7759/cureus. 58701

Cureus

Exploration of Whitlockite Nanostructures for Hemostatic Applications

Background Calcium magnesium phosphate (CMP)-based whitlockite is a promising biomaterial for hemostasis and regenerative applications. Regenerative approaches aim to advance tissue repair and recovery in different clinical scenarios. Whitlockite is a biocompatible and biodegradable mineral that has garnered impressive consideration for its interesting properties, making it an appealing candidate for therapeutic applications. Aim This study aimed to evaluate the hemostatic behavior of synthesized whitlockite nanoparticles. Materials and methods Coprecipitation and hydrothermal methods were used to synthesize whitlockite nanoparticles. Calcium nitrate, magnesium nitrate, and diammonium hydrogen phosphate were used as precursors to prepare this material. Results Crystalline phases of whitlockite (Ca 3 Mg) 3 (PO 4 ) and calcium magnesium phosphate Ca 7 Mg 2 P 6 O 2 were observed through X-ray diffraction (XRD) patterns, along with relevant properties of the phosphate functional group detected through Raman spectra. This study explores the hemostatic adequacy of CMP-based whitlockite using different methodologies. The capacity of the materials to actuate platelet conglomeration and encourage clot arrangement is assessed using in vitro experiments. Moreover, this study investigates the regenerative potential of CMP-based whitlockite in tissue-building applications. Conclusion The structural and morphological parameters provide crucial insights into the proper formation of the material, and the hemoclot assessment aids in understanding its coagulation behavior. Future investigations and clinical trials will be instrumental in fully harnessing the potential of CMP-based whitlockite for advancing hemostasis and regenerative medicine.

Introduction Mineral apatite is a crystalline form of calcium phosphate and is a naturally occurring hydroxyapatite. It is the primary mineral in mammalian bones and teeth, giving those structures their strength and rigidity. Ca 5 (PO 4 ) 3 (OH) is the chemical formula for hydroxyapatite, which is made up of atoms of calcium, phosphorus, oxygen, and hydrogen arranged in a particular crystal structure [ 1 ]. Due to its biocompatibility and capacity to integrate with natural tissues, hydroxyapatite is also utilized in several biomedical applications, such as tissue engineering scaffolds, drug delivery systems, and dental implants [ 2 ]. To be used in these applications, it can also be synthesized in a lab [ 3 ]. Hydroxyapatite is a renowned material for its capacity to support tissue regeneration and biocompatibility [ 4 ]. Hydroxyapatite is frequently used as a scaffold material in the field of tissue engineering to support the growth of new tissue [ 5 ]. Therefore, cells can adhere and multiply on the surface of the scaffold due to the structural resemblance between hydroxyapatite and natural bone [ 6 ]. New tissue can be created due to the cell's ability to add extracellular matrix to the scaffold gradually. Additionally, hydroxyapatite has been applied to dentistry [ 7 ]. Tricalcium phosphate and calcium magnesium phosphate are two essential minerals that are essential for bone and tissue regeneration [ 8 ]. The calcium that makes up a large portion of these structures is responsible for maintaining the strength and rigidity of the bones and teeth [ 9 ]. Calcium is essential for the development of new bone tissue when tissue regeneration is taking place. The proliferation and differentiation of osteoblasts and the cells responsible for bone formation are two cellular processes that depend on calcium ions and are crucial for bone growth and healing [ 10 ]. Another mineral that is critical for tissue regeneration is magnesium. It participates in various cellular functions, such as cell division, protein synthesis, and DNA synthesis [ 11 ]. Another mineral required for bone and tissue regeneration is phosphate. It contributes significantly to the mineral hydroxyapatite, which constitutes the majority of bone tissue. Tricalcium phosphate can be resorbed over time as new bone tissue develops due to its biocompatibility, which means it does not trigger an immune reaction or exhibit toxicity within the body [ 12 ]. In conclusion, crucial minerals for bone and tissue regeneration include calcium, magnesium, phosphate, and tricalcium phosphate. They can be used in various regenerative medicine applications to support the growth of new bone by playing critical roles in cellular processes vital for bone growth and healing [ 13 ]. Due to its distinct characteristics, whitlockite (WH), a rare calcium magnesium phosphate mineral, has recently attracted interest in the field of biomedical research. WH has the potential to be used in a variety of biomedical processes, such as cancer treatment, drug delivery, and bone tissue engineering [ 14 ]. WH biocompatibility prevents an immune reaction or toxicity in the body, which is one of its key characteristics. It is, therefore, a material that shows promise for use in biomedical applications [ 15 ]. WH is a desirable substance for bone tissue engineering applications because of its similar composition to the mineral phase found in natural bone. The current approaches for the development of WH for biomedical applications include its synthesis using different techniques, modification of its surface properties to enhance its interaction with biological molecules and cells, and investigation of its potential use as a biomaterial for bone tissue engineering and regeneration as well as hemostatic applications [ 16 ]. Selecting a synthesis methodology is critical when designing WH for biomedical applications. It is essential to optimize the synthesis parameters to achieve the desired properties, such as particle size, crystal structure, purity, and surface properties, for the intended biomedical application. Materials and methods The precipitation method was employed to synthesize the WH nanoparticles. Calcium nitrate (0. 5 M/25 mL) was mixed with magnesium nitrate (0. 5 M/3. 5 mL) to form a calcium magnesium solution. After mixing the solution, diammonium hydrogen phosphate (0. 5 M/20 mL) was added dropwise, and the pH of the solution was maintained at 6 using an ammonia solution. Then, the precipitated particles were collected and dried using a hot air oven (100 °C for 12 hours), along with the co-precipitated particles. For hydrothermally derived particles, the precipitates were placed in a hydrothermal setup and kept in a hot air oven for 15 minutes at a temperature of 200 °C to obtain WH powder. After cooling down the temperature, the obtained particles were washed with deionized water, followed by ethanol, and then dried in a hot air oven at 100 °C overnight to collect the nanoparticles, as depicted in Figure 1. Figure 1 Synthesis of whitlockite. The scheme represents the synthesis of whitlockite using the hydrothermal method. Without the hydrothermal setup, the dried powder is obtained through the coprecipitation method. Figure credits: Chitra Shivalingam. Characterization techniques To investigate the characteristics of the materials and evaluate the crystalline phases, X-ray diffraction (XRD) was used with the Cu Kα wavelength (Bruker D8 advance, Billerica, Massachusetts). Raman spectroscopy (WITEC ALPHA300 RA-Confocal Raman-AFM Microscope, Ulm, Germany) was used to examine the characteristics of functional groups. JEOL (JSM-IT 800, Tokyo, Japan) and Oxford Instruments (Abingdon, England) were utilized to analyze the morphological and elemental analysis. Results XRD patterns The XRD diffractogram of WH depicted the well-matched crystalline phases of WH (Ca, Mg) 3 (PO 4 ) 2 (ICDD:00-013-0404) and calcium magnesium phosphate Ca 7 Mg 2 P 6 O 24 (ICDD:00-020-0348) (Figure 2 ). Tricalcium phosphate (TCP) and magnesium-substituted tricalcium phosphate (TCMP) possess very close crystalline properties to WH. Magnesium is deposited in the cite of calcium in the TCP structure; however, in the case of WH, Mg 2+ can also be incorporated in HPO 4 2- [ 17 ]. Most literature reported Ca(OH) 2 and Mg(OH) 2 crystalline phases for WH. However, this study found calcium magnesium phosphate crystalline phases [ 18 ]. Similarly, Ca 18 Mg 2 (HPO 4 ) 2 (PO 4 ) 12 is the predominant crystalline phase for WH, with a polygonal structure [ 19 ]. Current results showed the dominance of (Ca, Mg) 3 (PO 4 ) with nanoparticles. Figure 2 XRD patterns of whitlockite. XRD patterns of whitlockite by the coprecipitation and hydrothermal methods. XRD, X-ray diffraction. Raman spectra The Raman spectroscopic analysis of the WH showed specific peaks at 967 and 409 cm −1 (Figure 3 ), which are the characteristic peaks of υ1 and υ4 PO 4 3− ions, while the peak at 409 cm −1 is a characteristic peak of υ2 PO 4 3−. This authenticates the prominent presence of phosphate and further reconfirms the presence of WH. The characteristic peaks of bone mineral apatite were observed at 959-975 cm -1 with sharp vibrations. The obtained peak broadening indicates the formation of WH [ 20 ]. The shoulder peak before 967 cm -1 indicates the proper formation of WH [ 21 ]. Hence, the obtained results indicate the formation of WH. Figure 3 Raman spectra of whitlockite. Raman spectra of whitlockite by the coprecipitation and hydrothermal methods. Field emission scanning electron microscopy (FE-SEM) and elemental analysis The WH nanoparticles synthesized using the hydrothermal method showed an elongated, spherical shape; similarly, the WH obtained from coprecipitation showed a small spherical morphology (Figure 4 ). Uniform temperature and pressure are crucial for promoting the homogeneous growth of elongated, spherical structures in the hydrothermal method. Generally, WH tends to grow in a distinctive polygonal shape [ 22 ]; however, in this case, a spherical morphology was distinctly formed. Rod-like, hexagonal, and polygonal shapes are the most commonly observed structures for WH [ 17, 19 ]; however, in this case, a spherical morphology was predominant. The Zn-infused WH showed highly agglomerated hexagonal particles measuring approximately 80 nm in size. The current results indicated that the particles are nearly homogeneous in size, measuring approximately 40 nm [ 23 ]. Figure 4 Morphology of whitlockite by field emission scanning electron microscopy. Morphology of whitlockite using field emission scanning electron microscopy by coprecipitation and hydrothermal methods. (A) WH - Hydrothermal and (B) WH - Coprecip. The elemental composition was confirmed through energy dispersive spectroscopy (EDS) and mapping spectra, revealing the presence of O, Ca, C, P, and Mg elements in both materials (Figures 5 - 7 ). Ca and P were consistently distributed throughout; Mg was also evenly dispersed on the nanoparticles. Based on these results, it can be concluded that nanoparticles contain the WH phase. Figure 5 The elemental composition of the prepared material was confirmed by EDS and elemental mapping. The elemental composition of the prepared material (by coprecipitation and hydrothermal methods) was confirmed by energy dispersive spectroscopy (EDS) and elemental mapping: (A) hydrothermal and (B) coprecipitation. Figure 6 Elemental mapping of the synthesized particles using the hydrothermal method. The elemental mapping data of whitlockite synthesized using the hydrothermal method: (A) a mixture of all the elements, (B) carbon, (C) phosphate, (D) calcium, (E) oxygen, and (F) magnesium. Figure 7 Elemental mapping of the synthesized particles using the coprecipitation method. The elemental mapping data of whitlockite synthesized using the coprecipitation method: (A) a mixture of all the elements, (B) calcium, (C) phosphate, (D) magnesium, (E) oxygen, and (F) carbon. Hemostatic assay through FE-SEM The normal hemostasis has no control over death during significant medical procedures and injuries. Subsequently, external hemostatic agents are utilized to help typical coagulation pathways and to control bleeding. These external specialists are monetarily accessible in different forms. The most ordinarily utilized hemostatic specialists are comprised of chitosan [ 24 ]. The hemostatic behavior of the blood was evaluated with the treatment of hydrothermally synthesized WH compared to control; rapid clotting behavior was observed in treated blood samples (Figure 6 ). The osteo-conductive and osteo-inductive properties of calcium phosphate have been widely used in bone regeneration applications [ 25 ]. In order to promote bone regeneration, calcium and phosphorus ions are released, which controls how osteoblasts and osteoclasts are activated, as shown in Figure 8 [ 26 ]. The control of calcium phosphate surface characteristics and porosity affects cell/protein adhesion and growth and manages the production of bone minerals [ 27 ]. Due to the differences in ion release, solubility, stability, and mechanical strength, different types of calcium phosphate, including hydroxyapatite and TCP, have different properties affecting their bioactivity that can be used in various biomedical applications [ 28 ]. Figure 8 Hemostatic property of whitlockite. The hemostatic behavior of the blood was evaluated under two conditions: (A) control and (B) treatment with hydrothermally synthesized whitlockite. Discussion CMP-based WH altogether advances cell expansion, separation, and extracellular framework blend, demonstrating its capacity to support tissue regeneration. In addition, the biocompatibility and biodegradability of CMP-based WH are broadly examined to guarantee its safety and long-term use in the human body [ 29 ]. Cytotoxicity studies, histological assessments, and contamination examinations demonstrate that CMP-based WH shows favorable tissue regeneration [ 30 ]. In conclusion, this study presents CMP-based WH as a flexible biomaterial with dual benefits: promoting successful hemostatic properties and advancing tissue recovery [ 16 ]. It is also a promising candidate for various restorative applications, including wound administration, surgical strategies, and tissue regeneration procedures. CMP-based WH is highly effective for hemostasis and regenerative potential. They have versatile applications in regenerative medicine applications, aiding in the growth of new bone by performing essential functions in cellular processes that are vital for bone growth and healing. The purity, crystal structure, morphology, particle size, and surface characteristics of WH can be used for regenerative applications [ 25 ]. Bio-ceramics have been extensively employed for treating bone defects. Magnesium and hydrogen combined are important elements found in bone WH. WH is an essential part of biological hard tissues, such as the teeth and bones, in children. Synthesizing WH crystal is essential for simulating tissue composition and structure, making it easier to build bioinspired materials for drug delivery systems, scaffolds for tissue engineering, and medical implants. Magnesium WH is created when heterogeneous species are nucleated by the phospholipids in the bone [ 13 ]. Phosphatidylserine and phosphatidylinositol facilitate the mineralization of hydroxyapatite in bones. In vitro, the process explicates the role of WH and upregulates the genes involved in cell differentiation. Magnesium is one of the significant components that stimulate osteogenic and neurological properties [ 23 ]. This WH continually releases magnesium ions that influence mineralization, cell proliferation, and alkaline phosphatase (ALP) activity and are directly affected by magnesium ions. The higher concentration of calcium and magnesium ions results in a greater amount of protein adsorption due to the negative surface charge of WH, which is associated with the material's improved capacity for bone repair. It increases the synthesis of alkaline phosphate, which is essential for bone growth and mineralization [ 30 ]. Therefore, it possesses a significant value as a material for bone tissue engineering, drug delivery, and other biomedical applications. Limitations of the study WH receives less attention compared to hydroxyapatite, tricalcium phosphate, and other calcium phosphate minerals. Hence, the lack of comprehensive reports may hinder its properties and efficiency in biomedical applications. The synthesis of WH in the appropriate phase is not adequately explored due to the limited understanding of the material. This study provided an explanation of the structural, morphological, and hemostatic properties of WH. A thorough comprehension of biocompatibility characteristics, specifically in terms of blood and cellular compatibility, is necessary to evaluate the material's potential in physiological environments. Furthermore, in order to comprehensively examine the effects of the biological environment on medical advancements, it is imperative to conduct animal studies to analyze both the positive and negative aspects. Conclusions An analysis was conducted on the impact of fabrication methods on the characteristics of WH. Through an XRD pattern, it was found that WH and calcium magnesium phosphate crystalline phases were present. The presence of phosphate peaks in Raman spectroscopy indicates the functional group properties of the materials. Small spherical morphologies were found through FE-SEM. The elemental composition of WH, such as calcium, magnesium, and phosphorus, was observed through EDS and elemental mapping techniques. The morphological analysis revealed a rapid hemostatic response. The hydrothermal synthesis of WH showed sharp crystalline indexing and distinct Raman spectra, which indicates enhanced WH features compared to coprecipitation methods for hemostatic and regenerative applications.

10. 7759/cureus. 59202

Cureus

Green Synthesis and the Evaluation of Osteogenic Potential of Novel Europium-Doped-Monetite Calcium Phosphate by Cissus quadrangularis

Background The quest for an ideal bone grafting material has been ongoing for decades. Calcium phosphate, alone or in combination with other materials in natural bone, has been shown to aid in bone regeneration effectively. Monetite exhibits superior solubility and resorption rates among calcium phosphates, rendering it an optimal choice for bone regeneration applications. However, the degradation rate of the Monetite is much faster than that of all the other calcium phosphates. Hence, we have added Europium onto the matrix to alter the degradation profile and enhance the osteogenic ability of the prepared matrix. Materials and methods An exclusive Europium-Monetite composite was synthesized employing eco-friendly techniques involving Cissus quadrangularis. The osteogenic potential was gauged using the MG-63 cell line through a calcium mineralization assay employing an Alizarin Red solution, collagen estimation, and an alkaline phosphatase (ALP) assay. The composite's cytocompatibility was evaluated using the MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) assay across different concentrations ranging from 12. 5 µg to 100 µg. Results Scanning electron microscopy (SEM) analysis of the Europium-Monetite composite revealed a sheet-like arrangement in stacks, and the ATR-IR confirmed the presence of elements Ca, P, and Eu. The osteogenic potential, analyzed by ALP activity, calcium mineralization, and collagen staining, was 10% higher than that of the control (Monetite). Conclusion The prepared novel Europium-Monetite calcium phosphate complex can enhance the osteogenic potential and could be a promising material for bone regeneration/tissue engineering. The newly created Europium-Monetite calcium phosphate complex holds promise for various bone grafting applications, including integration into scaffolds and as a coating for implants.

Introduction Enormous progress has been made in bone regeneration to bring an alternative to autologous grafts, as autologous grafts are associated with disadvantages such as limited availability and morbidity to the patient. Numerous techniques have been used routinely with biomaterials such as allografts, xenografts, alloplastic, and growth factors. The gaps resulting from the utilization of alloplastic calcium phosphate grafts pose challenges including incomplete setting reactions leading to inflammation. Moreover, their lack of a macro-porous structure restricts cell adhesion speed, fluid exchange, and restoration capabilities. Additionally, they become brittle under tensile and shear stress [ 1 ]. Nevertheless, Monetite calcium phosphates used as bone grafts have demonstrated bone augmentation rates similar to those of autografts, which are considered the gold standard and sourced from the patient's own body [ 1 ]. They undergo conversion of the hydroxyapatite without any phase transformation. They also exhibit faster resorption and greater volume of newly formed bone [ 1 ]. However, the downside of using this Monetite as a bone augmentation material is its weak mechanical properties and efficacy in biological performance and degradation. Numerous studies have focused on improving the biological performance of Monetite structures by incorporating specific modifications into their matrix. Incorporating strontium into Monetite structures enhances the long-term viability of osteoblast-like cells, indicating potential applications in conditions characterized by excessive bone resorption [ 2, 3 ]. Rare earth nano-biomaterials are booming in bone tissue engineering and implant design [ 4 ]. Europium is a rare earth element of the lanthanide series used initially in medical applications for imaging due to its fluorescent properties [ 5 ]. These materials have been used effectively for bone regeneration due to their unique osteogenic, angiogenic, antimicrobial, and antioxidant properties and in vivo bone tissue imaging [ 5 ]. Numerous physical and chemical methods have been mediated for the synthesis of nanoparticles. Green nanoparticle synthesis using plants is more advantageous as it can stabilize the agents in the nanoparticles and are nontoxic, eco-friendly, and sustainable [ 6 - 8 ]. Cissus quadrangularis, a vining plant indigenous to India and Africa, belongs to the Vitaceae family and is also known as Vitis quadrangularis. It has been utilized for medicinal purposes for centuries. The stem and root of this plant exhibit antioxidant and antimicrobial properties. C. quadrangularis is rich in anabolic steroidal compounds and significant amounts of calcium and phosphorus. C. quadrangularis functioned as a reducing agent in producing silver, ZnO, and CaO nanoparticles [ 9 ]. Hence, to improve their osteogenic potential in the present study, we have incorporated Europium into the Monetite calcium phosphate. Therefore, we have fabricated an innovative and novel Monetite-structured green synthesis of Europium-Monetite calcium phosphate by C. quadrangularis, and the material's physiochemical characterization and osteogenic potential were assessed. Materials and methods The innovative Europium-Monetite composite was synthesized through green synthesis using C. quadrangularis, and its osteogenic potential was evaluated through calcium mineralization assay using Alizarin Red solution, collagen estimation, and alkaline phosphatase (ALP) assay. The ethical clearance has been successfully obtained from the institutional review board under reference SRB/SDC/PhD/PERIO-2312/23/TH-080. To prepare the Europium-Monetite composite via green synthesis using C. quadrangularis, 2 g of C. quadrangularis were dissolved in 100 mL of distilled water and incubated in a shaker overnight at 37°C as shown in Figure 1A. After filtration, the filtrate was combined with 0. 99 mol of calcium nitrate solution and 0. 01 mol of Europium, stirring for three to four hours. Then, 0. 67 mol of diammonium hydrogen phosphate was introduced to the stirred solution, which was further stirred for 24 hours until reaching a pH of 7. 0. The solution was subsequently dried as shown in Figure 1B yielding the sample for further analysis, including physiochemical characterization. Figure 1 A shows the green synthesis of Europium-Monetite by C. Quadrangularis, and B shows the Europium-Monetite composite SEM and EDX analysis Scanning electron microscopy (SEM) was utilized to assess the physical properties, while X-ray diffraction (XRD) analysis was employed to examine mineral phases and crystallinity. The surface morphology and topography were evaluated using SEM, conducted with a high-energy beam and electrons' backscattering, with X-rays' characteristics recorded and converted into images by electron detectors (FESEM, JOEL JSM IT800 (JEOL Ltd. , Tokyo, Japan)). Images were observed at various magnifications to analyze surface topography. Energy dispersive X-ray (EDX) analysis of the material was conducted using an EDX detector X-PLORE-30/C-SWIFT (Oxford Instruments, Wiesbaden, Germany) to determine the elemental composition correlated with the atomic number. This EDX analysis was coupled with SEM. XPS analysis X-ray photoelectron spectroscopy (XPS) analysis was performed using the Thermo Scientific instrument, Model NEXSA surface analysis. The instrument features a micro-focused monochromatic Al-Kα source (hν=1486. 6 eV), a hemispherical analyzer, and a 128-channel plate detector. This analysis aimed to characterize the surface properties of the novel Europium-doped-Monetite calcium phosphate. Raman spectroscopy Raman spectroscopy provides insights into the molecular composition of the Europium-doped-Monetite calcium phosphate complex. Analyzing the spectrum can reveal information about the various molecules present. If its components are present, relative peak intensities offer quantitative data regarding the composition of the mixture. The Raman spectroscopy of the innovative Europium-Monetite complex (Eu-MCaP) was carried out using WITEC ALPHA300 RA-Confocal Raman (AFM Microscope, Ulm, Germany). Cytotoxic assay Biocompatibility tests were conducted using human osteoblastic-like cells (MG63) cultured in Dulbecco's Modified Eagle Medium (DMEM, Sigma Aldrich). The metabolic activity of the cells was assessed using the MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) assay. Europium-doped-Monetite calcium phosphate samples at different concentrations were added to 96-well plates along with the cells, followed by a 24-hour incubation period. The assay was then measured using a microplate reader at a wavelength of 570 nm. Cell viability percentage was calculated using the formula: Cell viability (%) = (Test sample OD)_570 / (Control OD)_570 × 100 Calcium mineralization assay Using Alizarin Red solution, a dye that binds to calcium salts, the calcium generation in the control and experimental group Eu-mCaP was assessed at seven days. Calcium content was evaluated using the Alizarin Red S (ARS) treated cells, where the cells would be incubated at room temperature for 20-30 min with 1 mL of 40 mM ARS per well. The cells were incubated for three days, washed with phosphate-buffered saline (PBS) then fixed with 4% formaldehyde at room temperature for 15 min. The samples were viewed under the fluorescent microscope (Leica Stellaris) and analyzed after washing the cells. ARS-treated cells were mixed with 10% (v/v) acetic acid, agitated, and incubated for 30 min. The cells were then taken and placed in tubes, agitated for 30 sec, and incubated at 85°C for 10 min. Subsequently, it was placed for centrifugation for 15 min with 200 µL of supernatant and 10% NH 4 OH (v/v) of 22. 5 µL 0. 405 nm was used to measure the absorbance. Collagen estimation Osteoblasts produce the initial matrix consisting mostly of collagen after that the matrix is mineralized by the deposition of minerals. In this study, we evaluated the amount of collagen by staining the collagen using the histological method for control cells and cells incubated with Europium-Monetite calcium phosphate. Collagen estimation was performed by incubating the cells at 37°C for 48 hours with medium for both control and treated cells every 24 hours. After incubation, cells would be washed with saline, then harvested and fixed for 20 min with 4% formalin. Following fixation, the cells were rinsed with PBS solution three times and stained with 0. 1% Sirius Red (20 μL) at 37°C for 20 min. After this, the cells were treated with 10% acetic acid and washed with PBS solution. Then these cells were stained with Picro-Sirius Red for one hour. The samples were visualized under a fluorescent microscope after washing with acidified water and dehydrated with ethanol. Two blind investigators, working independently, meticulously conducted quantitative estimations of histochemical stainings. Each investigator thoroughly analyzed all tissue specimens, and their respective findings were harmoniously averaged and visually depicted according to established criteria. This approach ensures robustness and reliability in our data interpretation. ALP assay The activity of ALP serves as an indicator of the osteogenic differentiation process from mesenchymal stem cells to osteoblasts. This enzyme, expressed by osteoblasts, plays a crucial role in biomineralization by increasing the concentration of inorganic phosphate (Pi) through adenosine triphosphate (ATP) hydrolysis. The deposition of calcium and phosphate ions marks the initial stage of extracellular matrix mineralization during bone formation. ALP activity was assessed by measuring protein production. Cells were seeded with the sample and incubated for three days. Before incubation, cells were solubilized with Triton-X-100 and incubated for 1 min. Cell density was measured at 405 nm using an ELISA plate reader, and images were captured using a fluorescence microscope after adding 5-bromo-4-chloro-3-indolyl-phosphate (BCIP) and nitro blue tetrazolium (NBT) solution and incubating for 30 min. Protein content was determined using the Bradford assay. Samples were placed onto a new 96-well plate with Bradford reagent and AP buffer. Optical density was measured at each culture time by dividing the optical density by the cell count at 595 nm. Results The surface topography of Monetite exhibited a sheet-like configuration arranged in stacks. SEM micrographs of various magnifications of x1. 50k, x10. 0k, and x5. 0k are observed as shown in Figure 2. The composite featured a sheet-like arrangement with a thickness of 90 nm and a length of 1. 2 µm. In spectrum 1, the weight percentages of the elements were 25. 3%, 15. 2%, and 1. 4% for Ca, P, and Eu, and atomic percentages were 0. 2%, 0. 1%, and 0. 2% for Ca, P, and Eu, respectively, as illustrated in Figure 3. XPS spectra analysis revealed intensity peaks of various elements in the Europium-doped-Monetite phase of calcium phosphate. It visually represents these peaks, including 133. 9 for P2p, 351. 5 for Ca2p, 531. 7 for O1s, and 1135. 5, 1165. 2, and 1169. 4 for Eu 3d5 as shown in Figure 4. The XRD spectrum obtained for the novel Europium-doped-Monetite calcium phosphate matches with JCPDS No. 98-000-556. The structure of Monetite Ca(HPO 4 ) was determined to be anorthic and the peak between 20° and 30° depicts the Monetite as shown in Figure 5 and the thinner peak corresponds to the bigger crystal. Intensity peaks were observed by Raman spectroscopy at 954 cm21 corresponds to the n1 stretching of PO4 confirming the presence of carbonated apatite, 1050 cm -1 for HSO 4 -ions and 1116 cm -1 attributed to C-O stretching as shown in Figure 6 [ 8, 9 ]. Figure 2 SEM Images of the green synthesis of Europium-doped-Monetite phase of calcium phosphate at A) x1. 50k, B) x10. 0k, and C) x5. 00k SEM: scanning electron microscopy Figure 3 Elemental composition of the prepared compound by EDX analysis EDX: energy dispersive X-ray Figure 4 XPS spectra analysis for Europium-Monetite calcium phosphate XPS: X-ray photoelectron spectroscopy Figure 5 XRD spectrum of Europium-doped-Monetite phase of calcium phosphate XRD: X-ray diffraction Figure 6 Intensity peaks observed by Raman spectrum ALP assessment for cells incubated with Europium-Monetite calcium phosphate, compared to the control (cells without incubation), revealed significantly darker blue-violet staining. As confirmed through blue-violet staining, ALP-active cells exhibited higher levels of Europium-Monetite calcium phosphate than the control as shown in Figures 7A, 7B. The percentage of ALP activity in the control and Europium-Monetite calcium phosphate complex was tabulated in a bar graph as shown in Figure 8. Figure 7 A shows ALP activity observed in the microscopic staining panels for control, and B shows Eu-MCaP ALP: alkaline phosphatase; Eu-MCaP: Europium-Monetite complex Figure 8 Relative percentage of ALP activity in control and Eu-MCaP ALP: alkaline phosphate; Eu-MCaP: Europium-Monetite complex Calcium-rich deposition on cells after incubation with Eu-MCaP was quantified using ARS staining. The Eu-MCaP composition, featuring calcium phosphate, facilitated the formation of calcium nodules on cells. ARS staining showed dense red spots in the experimental group, indicating the presence of calcium nodules promoting bone regeneration as shown in Figures 9A, 9B [ 10, 11 ]. Eu-MCaP stimulated mature bone cells and precursors accelerated osteoblast activity through ERK1/2 and PI3K/Akt pathways and played a vital role in controlling osteoclast production and resorption [ 12, 13 ]. The relative percentage of calcium staining was higher in the Europium-Monetite calcium phosphate group than in the control as shown in Figure 10. Figure 9 A shows calcium staining observed microscopically in control, and B shows calcium staining observed microscopically when incubated in Eu-MCaP Eu-MCaP: Europium-Monetite complex Figure 10 Relative percentage of calcium staining in control and Eu-MCaP Eu-MCaP: Europium-Monetite complex Collagen matrix production, assessed by collagen staining during incubation, demonstrated higher quantities for Eu-MCaP than control cells, showcasing the material's efficacy in enhancing bone regeneration as shown in Figures 11A, 11B. The relative percentage of collagen staining was higher for the Eu-MCaP than the control and was tabulated in a bar graph as shown in Figure 12. Figure 11 A shows collagen staining observed microscopically in control, and B shows collagen staining observed microscopically when incubated with Eu-MCaP Eu-MCaP: Europium-Monetite complex Figure 12 Relative percentage of collagen content between control and Eu-mCaP Eu-mCaP: Europium-Monetite complex The material's safety and effectiveness were evaluated across concentrations (12. 5 µg to 100 µg), revealing increased cell viability and proliferation with higher percentages in the experimental group Eu-mCaP than the control group as shown in Figures 13A, 13B. Cells maintained their spindle shapes, emphasizing the excellent biocompatibility [ 13 ]. The relative percentage of cell viability observed between the control and Eu-mCaP across concentrations was tabulated in the bar graph as shown in Figure 14. Figure 13 A shows cell viability observed microscopically in control, and B shows cell viability observed microscopically at 100 µg Eu-MCaP Eu-MCaP: Europium-Monetite complex Figure 14 Relative percentage of cell viability across concentrations from 12. 5 µg to 100 µg of Eu-MCaP Eu-MCaP: Europium-Monetite complex Discussion The adoption of green synthesis in material production stands as a pivotal and sustainable method for crafting biomaterials, presenting substantial advantages such as cost-effectiveness, reduced raw material consumption, and minimized toxicity [ 14 ]. C. quadrangularis, chosen for its role as a climber, possesses a rich array of properties including antioxidant, antibacterial, anti-hemorrhoidal, antifungal, anti-inflammatory, analgesic, and bone fracture healing attributes [ 15 ]. Employed in green synthesis, C. quadrangularis contributes to bone regeneration by stimulating the growth and specialization of mesenchymal stem cells, its effectiveness varying with dosage [ 14 ]. Additionally, it promotes the mineralization of the extracellular matrix and exhibits anti-inflammatory properties, creating a conducive environment for bone healing and regeneration, including enhanced angiogenesis to facilitate optimal distribution of oxygen and nutrients [ 13, 16 ]. The incorporation of green synthesis in the production of C. quadrangularis may have played a role in shaping the Monetite structure, thereby augmenting the osteogenic activity of the innovative complex [ 16 ]. Europium, recognized as a soft and volatile rare earth element, is esteemed for its remarkable fluorescence capabilities, widely applied in cell imaging techniques. The novel Eu-MCaP, containing 0. 01% mol of Eu, retains the inherent luminescent property of Europium, allowing for the assessment of the material's degradation ability when utilized in scaffolds or as an implant coating. Bioactive materials like calcium polyphosphate scaffolds, Hydroxyapatite (HAp), and bioactive glasses (BGs) can enhance their osteogenic capabilities through the addition of signaling ions in suitable amounts [ 14 ]. While Eu shares a structure similar to Ca, it boasts a larger ionic potential [ 16, 17 ]. This enables it to regulate the expression of osteogenic markers such as ALP, COL1, OPN, and Runx2, influencing the release of Ca ions [ 18, 19 ]. This modulation affects the conformation of HAp during new bone formation and impacts genes associated with osteogenesis. Moreover, Eu increases H 2 O 2 synthesis, activating endothelial nitric oxide synthase (eNOS) through a PI3K-dependent pathway, ultimately promoting angiogenesis. The multifaceted properties of Eu encompass antibacterial efficacy, antitumor capabilities, and robust biocompatibility [ 20 ]. Monetite, classified as anhydrous dicalcium phosphate and a member of the calcium phosphate family, exhibits remarkable regenerative capacities, surpassing those of HAp-based graft materials in both volume generation and faster resorption, without converting to HAp [ 21, 19 ]. Recognized as a degradable matrix, Monetite serves as an effective vehicle for delivering drug conjugates and fostering bone regeneration [ 22 ]. Idowu et al. investigation into Monetite's osteoinductive potential as a scaffold under a non-conditioned medium revealed that human mesenchymal cells maintained their typical physiological, morphological, and proliferative characteristics. The scaffold exhibited intrinsic osteoinductive properties, akin to control HAp [ 23 ]. Numerous studies have explored the doping of Europium onto calcium phosphate for drug delivery and creatinine estimation [ 24 ]. This study stands as a pioneer in synthesizing the Eu-MCaP and evaluating its osteogenic potential for effective bone regeneration. Considering the study's limitations, it is crucial to evaluate both the degradation profile and antimicrobial efficacy of the material. Additionally, we conducted an in vitro analysis to examine the properties of this groundbreaking Eu-MCaP. Future research will aim to further assess its antimicrobial abilities, mechanical characteristics, and hemocompatibility, and explore potential applications like incorporating it into scaffolds, utilizing it as a drug delivery platform, and applying it as coatings for implants. Subsequent investigations will be done to involve animal studies and human trials to comprehensively evaluate its potential. Conclusions The innovative biomaterial, synthesized Europium-Monetite calcium phosphate complex, exhibits enhanced osteogenic potential with increased collagen and calcium staining, ALP activity, and robust biocompatibility. This biomaterial holds promise for various applications, including bone grafting, scaffold incorporation, and implant coatings. The novel Europium-Monetite calcium phosphate complex offers versatile applications in bone grafting, scaffold integration, and implant coatings, presenting a potential biomaterial with advanced osteogenic capabilities for effective bone regeneration. Future research should investigate the material's degradation profile, mechanical properties, antimicrobial properties, and broader applications in drug delivery and coating implants.

10. 7759/cureus. 5938

Cureus

Immunotherapy – Strategies for Expanding Its Role in the Treatment of All Major Tumor Sites

Immunotherapy is widely regarded to have the ability to transform the treatment of cancer, with immune checkpoint inhibitors already in use for cancers such as advanced melanoma and non-small cell lung cancer (NSCLC). However, despite its potential, the widespread adoption of immunotherapy for the treatment of other cancers has been largely limited. This can be partly attributed to additional immunosuppressive mechanisms in the tumor microenvironment that help promote and maintain a state of T cell exhaustion. As such, the exploration of combinatory immunotherapies is an active area of research and includes the combination of immune checkpoint inhibitors with cytotoxic therapies, cancer vaccines and monoclonal antibodies against other co-inhibitory and co-stimulatory receptors. Strategies are also being employed to improve the homing, extravasation and survival of chimeric antigen receptor (CAR)-T cells in the tumor microenvironment. Furthermore, the development of immunotherapies targeted to one or multiple neoantigens unique to a specific tumor may act to enhance anti-tumor immunity, as well as reduce immune-related adverse events (irAEs). As immunotherapy evolves to become a mainstay treatment for cancer, it is imperative that optimum treatment regimens that maximize efficacy and limit toxicity are developed. Foremost, appropriate biomarkers must be identified to help tailor combinatory immunotherapies to the individual patient and hence pave the way to a new era of personalized medicine.

Introduction and background The notion of harnessing the host’s immune system to eliminate cancer has been well-established for years, even though the field has only started taking off relatively recently. It stems from the knowledge that the immune system is a critical player in the prevention, as well as the development and progression of cancer. The prevention of tumorigenesis is achieved via numerous mechanisms, including protection against viral-induced tumors and suppression of tumor-promoting inflammatory environments. Arguably, the most important mechanism is that malignantly transformed cells often co-express ligands associated with DNA damage and tumor antigens, which can be recognized and targeted by the innate immune system and lymphocytes of the adaptive immune system, respectively, in a process termed immune surveillance [ 1 ]. The ability of the immune system to recognize antigens on malignant cells and target them for destruction forms the foundations of immunotherapy [ 2 ]. However, a distinguishing hallmark of cancer is its ability to evade immune destruction in a number of distinct ways. Notably, immune editing provides a selective pressure that gives rise to a less immunogenic population of neoplastic cells [ 2 - 3 ]. This is augmented by other immune-evading processes, including disruption of T cell function and signaling, defective antigen presentation, and the altered production of immune-suppressive mediators such as inhibitory cytokines and immunosuppressive cells. These processes ultimately lead to the proliferation of malignant cells, clinically manifesting as cancer [ 3 ]. Immunotherapy holds a lot of promise, not least because it avoids the many limitations of chemotherapy and radiotherapy - the current mainstay treatments for cancer. These limitations include systemic toxicities, lack of specificity for malignant cells, recurrence of drug-resistant tumors, and the inability to target and treat micrometastasis or subclinical disease [ 4 ]. The main forms of immunotherapy are immune checkpoint inhibitors, adoptive cell transfer, cytokine therapy, and cancer vaccines. As cancer immunotherapy effectively targets the immune system, in principle it should be able to treat a broad range of tumor types independent of the underlying histology or driver mutations. However, to date, immunotherapy has only demonstrated efficacy in a select group of cancers and usually in a minority of patients with those cancers, limiting its use as a treatment. Consequently, strategies for expanding its use is an active area of research and will be the focus of this review. Review Immune checkpoint inhibitors Immune checkpoint inhibitors have achieved notable clinical success as a novel class of immunotherapy, particularly in patients with advanced melanoma and non-small cell lung cancer (NSCLC) [ 4 ]. The primary effector cells of the adaptive immune response to cancer are T lymphocytes, including T helper cells and cytotoxic T lymphocytes (CTLs). These cells are primed and activated through interaction of their T cell receptors (TCRs) with tumor antigens presented on major histocompatibility complexes (MHCs) by antigen-presenting cells (APCs). Immune checkpoints essentially provide the co-stimulatory or co-inhibitory signals that regulate this process. Specifically, cytotoxic lymphocyte-associated protein 4 (CTLA-4) is a co-inhibitory receptor which, through binding to CD80 (B7. 1) and CD86 (B7. 2) ligands expressed on tumor cells and APCs, inhibits the process of T cell priming and activation. Normally, after priming and activation, CTLs migrate to the tumor where they exert their cytotoxic activity. This process is also controlled by immune checkpoints including programmed cell death protein 1 (PD-1) receptor, a co-inhibitory receptor present on activated T cells, regulatory T (Treg) cells, B cells, and natural killer (NK) cells. The ligands of this receptor, PD-L1 and PD-L2, are also expressed by both tumor cells and APCs, and once bound lead to T cell apoptosis and exhaustion, providing protection against CTL-mediated killing. As such, these inhibitory pathways are often found to be upregulated in cancer, as one of many mechanisms to evade immune surveillance. Given the crucial role of immune checkpoints in suppressing the anti-tumor immune response, the use of monoclonal antibodies (mAbs) as immune checkpoint inhibitors targeting CTLA-4 and PD-1/PD-L1 appears very promising, with their mechanism of action shown in Figure 1. Examples of approved immune checkpoint inhibitors include ipilimumab, an anti-CTLA-4 mAb, and nivolumab, an anti-PD-1 mAb. However, despite immune checkpoint inhibitors showing anti-tumor activity in a number of different malignancies, less than 25% of patients achieve any benefit [ 5 ]. This may in part be due to additional suppressive mechanisms that help contribute and maintain T cell exhaustion, preventing the immune system from mounting a sufficient response. For instance, targeting the PD-1 pathway alone does not result in complete restoration of T cell function and in fact expression of alternative co-inhibitory immune checkpoints has been associated with resistance to PD-1 blockade [ 6 ]. One such inhibitory receptor that is expressed on CTLs and Treg cells during exhaustion is lymphocyte activation gene 3 protein (LAG3), with efficacy studies demonstrating an enhanced response in melanoma patients treated with both nivolumab and relatlimab, an anti-LAG3 mAb [ 7 ]. Similarly, T cell immunoglobulin and mucin domain-containing 3 (TIM3) are also thought to maintain a state of T cell exhaustion and are found to be expressed on lymphocytes in a range of tumors. In fact, preclinical studies have already demonstrated a superior synergistic effect of combined TIM3 and PD-1 blockade in cancers, such as melanoma, gastric cancer, hepatocellular carcinoma (HCC), and acute myeloid leukemia (AML), which is superior to targeting either pathway alone [ 6, 8 ]. Figure 1 Mechanism of action of anti-CTLA-4 and anti-PD-L1 immune checkpoint inhibitors CTLA-4, cytotoxic lymphocyte-associated protein 4; MHC, major histocompatibility complex; PD-1, programmed cell death protein 1; TCR, T cell receptor; Treg cell, regulatory T cell The most studied immunotherapy combination is combined PD-1 and CTLA-4 blockade. This combination has been shown to be able to overcome T cell exhaustion and restore anti-tumor immunity in a wide range of cancers, with phase III trials demonstrating superiority of this approach in the treatment of melanoma, renal cell carcinoma, and NSCLC [ 9 - 11 ]. Other cancers being explored in early clinical trials include bladder cancer, ovarian cancer, colorectal cancer, breast cancer, HCC and pancreatic cancer, as shown in Table 1 [ 12 ]. This synergistic effect of dual blockade results from the alteration of different signaling pathways within T cells, with suppression of both Treg cells and inhibitory pathways within CTLs being thought of as the primary mechanism of action [ 12 ]. The action of killer inhibitory receptors (KIRs), which recognize MHC class I molecules and subsequently negatively regulate the cytotoxic activity of NK cells, is another mechanism of avoiding immune surveillance in tumor cells through retention of MHC class I expression [ 13 ]. Hence, KIR specific mAbs that are able to block this negative regulation may provide a further combinatory target. T cell exhaustion may also be overcome through tissue engineering approaches. For example, re-differentiation of pluripotent stem cells into the T cell lineage, followed by transduction with engineered TCRs or chimeric antigen receptors (CARs), has been shown to delay cancer progression in solid tumors [ 14 ]. However, it is currently unclear if this method overcomes the issue of T cell exhaustion. Interestingly, direct cell-cell contact between T cells and tumor cells has been shown to induce T cell defects after short-term incubation, suggesting that severing this contact may act to enhance T cell efficacy [ 15 ]. Table 1 A summary of significant combination immunotherapy regimens currently being explored in clinical trials Trial Tumor type Therapy Clinical outcome NCT03298451 Advanced hepatocellular carcinoma Durvalumab plus tremelimumab Initial data from phase I/II trial suggests improved objective response rate compared to durvalumab alone. NCT03434379 Advanced hepatocellular carcinoma Atezolizumab plus bevacizumab Initial data from phase Ib trial suggests improved response rate compared to monotherapy with either agent. NCT03713593 Advanced hepatocellular carcinoma Pembrolizumab plus lenvatinib Initial data from phase Ib trial suggests good anti-tumor response in unresectable tumors. NCT02425891 Triple-negative breast cancer Atezolizumab plus paclitaxel Improved overall survival and progression-free survival compared to monotherapy, particularly in PD-1 positive subgroup. NCT03036488 Triple-negative breast cancer Pembrolizumab plus chemotherapy regimen Improved pathological complete response and event-free survival compared to chemotherapy alone. NCT01844505 Advanced melanoma Nivolumab plus ipilimumab Improved overall survival compared to ipilimumab alone. NCT02231749 Advanced renal cell carcinoma Nivolumab plus ipilimumab Improved overall survival and objective response rate versus sunitinib. NCT02853331 Advanced renal cell carcinoma Pembrolizumab plus axitinib Improved overall survival, progression free survival and objective response rate versus sunitinib. NCT02684006 Advanced renal cell carcinoma Avelumab plus axitinib Improved progression free survival compared to sunitinib, though no improvement in overall survival. NCT02420821 Advanced renal cell carcinoma Atezolizumab plus bevacizumab Improved progression free survival compared to sunitinib. NCT02985957 Metastatic prostate cancer Nivolumab plus ipilimumab Initial data from phase II trial suggests improved objective response rate. NCT02039674 Advanced non-small cell lung cancer Pembrolizumab plus carboplatin plus paclitaxel Improved overall survival and progression free survival compared to carboplatin and paclitaxel alone. NCT01454102 Advanced non-small cell lung cancer Nivolumab plus ipilimumab Improved progression free survival compared to chemotherapy treatment. NCT02542293 Advanced non-small cell lung cancer Durvalumab plus tremelimumab No improvement in overall survival compared to chemotherapy. NCT02366143 Advanced non-small cell lung cancer Atezolizumab plus bevacizumab plus carboplatin plus paclitaxel Improved overall survival and progression-free survival compared to bevacizumab plus carboplatin plus paclitaxel. NCT03214250 Metastatic pancreatic cancer Nivolumab plus gemcitabine plus paclitaxel plus APX005M Initial data from phase Ib trial suggests promising anti-tumor response. NCT03036098 Metastatic bladder cancer Nivolumab plus ipilimumab Improved overall survival and progression free survival compared to chemotherapy. NCT02807636 Advanced or metastatic bladder cancer Atezolizumab plus platinum-based chemotherapy Improved progression free survival compared to atezolizumab alone. NCT02498600 Recurrent ovarian cancer Nivolumab plus ipilimumab Initial data from phase II trial demonstrates improved anti-tumor response compared to nivolumab alone. NCT02580058 Recurrent ovarian cancer Avelumab plus doxorubicin or platinum-based chemotherapy No improvement in overall survival or progression free survival compared to chemotherapy. NCT02788279 Metastatic colorectal cancer Atezolizumab plus cobimetinib No improvement in overall survival compared to regorafenib. NCT02060188 Metastatic colorectal cancer Nivolumab plus ipilimumab Initial data from phase II trial demonstrates promising objective response rate. Another reason for the limited efficacy of immune checkpoint inhibitors in certain cancers may relate to a poor Immunoscore, which is a scoring system that classifies cancers according to the level of immune cell infiltration. The Immunoscore ranges from I0 or ‘cold’ for poorly infiltrated tumors to I4 or ‘hot’ for well-infiltrated tumors. Hot tumors have been associated with improved response rates to checkpoint blockade [ 16 ]. Hence, combinatory treatments that are able to prime the immune system prior to checkpoint inhibitor therapy are another possible approach to expand the role of immunotherapy. For example, agonistic targeting of costimulatory receptors found on T cells, such as CD137 and OX40, enhances the anti-tumor immune response by promoting T cell proliferation and survival [ 17 - 18 ]. Alternatively, T cells can be primed and expanded through the use of neoantigen-based vaccines or by targeting cytokines, for example, dual IL-10 and PD-1 blockade is able to enhance the function of tumor-specific CTLs [ 19 ]. Conventional treatments such as chemotherapy or radiotherapy may also have a role in improving the response to immunotherapies, for example, as neoadjuvant therapies. They are also able to upregulate the production of chemokines and cytokines, increase the expression of MHC molecules, and facilitate tumor death, enabling the release of tumor-associated antigens [ 12 ]. This, in turn, leads to enhanced antigen presentation, and T cell recruitment and activation, which results in the upregulation of inhibitory immune checkpoints. Other agents also have the potential to upregulate immune checkpoints and enhance anti-tumor immunity. Such agents include 1) vascular endothelial growth factor (VEGF) inhibitors, which are able to increase lymphocyte infiltration into tumors and reduce expression of Treg cells, 2) adenosine (P1) receptor inhibitors, which increase APC activation and reduce Treg cell expression, and 3) mitogen-activated protein kinase (MAPK) inhibitors, which promote tumor cell death and the presentation of tumor-associated antigens by enhancing MHC class I expression [ 12, 20 ]. Another strategy to expand the use of immune checkpoint inhibitors is to target mechanisms of resistance. For example, indoleamine 2, 3-dioxygenase 1 (IDO1) has been implicated in resistance to both anti-CTLA-4 and anti-PD-1 mAbs, though as of yet phase III trials have not demonstrated any clinical improvements [ 21 ]. Mutations in immune effector signaling pathways are also capable of suppressing the activity of tumor-specific T cells and provide mechanisms of resistance to treatment. For example, mutations in Janus kinase 1 (JAK1) and Janus kinase 2 (JAK2) are associated with loss of interferon-gamma (IFNγ) responsiveness and antigen presentation. This has been shown to result in resistance to PD-1 blockade, which can be overcome by inhibition of JAK1/2 signaling [ 22 ]. Downregulation of antigen presentation may also be attributed to epigenetic changes, which may be overcome through the use of DNA methylation inhibitors and histone deacetylase inhibitors [ 23 ]. Given the role of the gut microbiome in regulating the mucosal immune system, it has also been shown to influence the response to immune checkpoint inhibitors [ 24 ]. Consequently, modulating the gut microbiome, for example, through fecal transplantation or simply by encouraging high-fiber diets in patients, may further increase the efficacy of immune checkpoint inhibitors [ 25 ]. Interestingly, however, recent evidence suggests that taking dietary supplements such as probiotics may hinder the response to checkpoint inhibitors by lowering the diversity of the gut microbiome, warranting further research in the area [ 25 ]. A summary of the various combinatory immunotherapies discussed and their corresponding synergistic effects on the immune system is provided in Figure 2. Figure 2 Combinatory immunotherapy approaches and their synergistic mechanisms of action CAR, chimeric antigen receptor; CTLA-4, cytotoxic lymphocyte-associated protein 4; IDO1, indoleamine 2, 3-dioxygenase 1; JAK, Janus kinase; LAG3, lymphocyte activation gene 3 protein; MAPK, mitogen-activated protein kinase; PD-1, programmed cell death protein 1; TIM3, T cell immunoglobulin and mucin domain-containing 3; Treg cells, regulatory T cells; VEGF, vascular endothelial growth factor. Adoptive cell transfer and vaccines Adoptive cell transfer (ACT) is another approach used in immunotherapy, where the patient’s own T lymphocytes with anti-tumor activity are identified, expanded in vitro and re-infused into the patient, often along with growth factors. CAR-T cell therapy is a particularly attractive strategy, where the patient’s T cells are genetically engineered to express modified CARs that target surface antigens whose epitope is unique to cancer cells. This approach is also attractive in that CARs are HLA-independent, eliminating the need for haplotype matching. However, while CAR-T therapy has had success in the treatment of hematological tumors, their application in solid tumors has been largely limited [ 26 ]. This can firstly be attributed to the lack of specific targetable antigens. For example, while CD19 has proven to be the most successful target antigen for CAR-T cell therapy due to its ubiquitous expression in almost all B cell malignancies, it is also expressed by non-malignant B cells [ 27 ]. However, fortunately, the B cell aplasia and hypogammaglobulinemia associated with this lack of specificity can be easily managed. Further barriers to CAR-T cell therapy include problems in T cell homing, infiltration, and subsequent survival in the tumor microenvironment. Infiltration may be improved through the engineering of CAR-T cells capable of degrading cellular components, such as αvβ6 integrin, VEGF receptor 2 and heparan sulphate proteoglycans (through heparanase release) [ 28 - 30 ]. To counteract the hostile and immunosuppressive nature of the tumor microenvironment, and hence improve T cell survival, several strategies can be employed. Such strategies include engineering CAR-T cells that 1) are resistant to TGF-β suppression via dominant-negative TGF-β receptor expression, 2) can counteract the action of reactive oxygen species (ROS) through catalase expression, reducing H 2 O 2, and 3) can convert IL-4’s suppressive effects to a stimulatory one through engineering chimeric receptors that express the IL-4 receptor ectodomain [ 31 - 33 ]. CAR-T cell therapy may also provide a selective pressure, facilitating the emergence of antigen loss variants with time [ 26, 34 ]. This can be overcome by enabling the CAR-T cells to target more than one antigen. For instance, through engineering the extracellular portion of the CD16-chimeric receptor to express an FcγR domain, it is able to bind to any therapeutic antibody directed against any tumor-associated antigen, triggering both a cellular immune response and antibody-dependent cellular cytotoxicity [ 35 ]. Alternatively, the two-component SUPRA CAR system has recently been developed, which is composed of a receptor expressed on T cells (zipCAR) and an antigen-binding component (zipFv). In this system zipCAR expressing T cells are activated once a zipFv component containing a matching leucine zipper is added. SUPRA CARs are universal in that multiple zipFv components expressing the same leucine zipper but different antigen-binding domains can be added, allowing the targeting of multiple antigens. Thus, throughout the course of therapy, antigen specificity can be altered depending on the patient response to improve treatment efficacy [ 36 ]. A further strategy is the dual recognition of tumor-associated antigens expressed by the same cell by two CARs [ 37, 38 ]. As well as enhancing T cell activation, this approach may also be able to reduce on-target/off-tumor toxicity by improving specificity to the target tumor, protecting normal tissues. Alternatively, toxicity can be reduced by combining CARs directed against target antigens with inhibitory CARs (iCARS). The idea is that target antigens for CAR-T cell therapy are also expressed by healthy tissues, albeit at lower levels compared to the tumor. iCARS are able to produce inhibitory signals that can override this low level of T cell activation against target antigens expressed by healthy cells, whilst enabling CAR-induced T cell activation against tumor cells [ 37 ]. For example, the inhibitory receptors CTLA-4 and PD-1 can be used in iCARs to negatively regulate the activation of T lymphocytes against normal tissue, thus reducing off-target toxicity [ 39 ]. Toxicity associated with CAR-T cells may also relate to the use of retroviral (RV) and lentiviral (LV) vectors, which may trigger immune and inflammatory responses [ 40 ]. Instead, alternative systems, such as the transposons piggyBac (PB) and Sleeping Beauty (SB) can be utilized, which also simplify and reduce the costs associated with transduction [ 26 ]. Furthermore, as they do not utilize reverse transcription, the likelihood of aberrant gene rearrangements is minimized. Most targeted antigens in immunotherapy are not tumor-selective and rather are just overexpressed in tumors [ 26 ]. However, neoantigens are not encoded by the normal genome and instead arise in tumors as a result of driver mutations and as by-products of increasing genetic instability (passenger mutations), often rendering the pattern of expression as highly unique to the individual [ 41 ]. Whilst this generally means neoantigens are not practical for CAR-T therapy, distinct neoepitopes have been identified. For example, MUC-1 targeting CAR-T cells have been shown to significantly delay tumor progression [ 42 ]. Furthermore, neoantigen-directed T cells from a patient or donor can also be identified and expanded in vitro for treatment, or alternatively T-cells can be genetically engineered to express neoantigen-specific TCRs [ 43 - 44 ]. Enhancement of antigen presentation through stimulation of the innate immune response and dendritic cell function, for instance by using type I IFN and toll-like receptor (TLR) ligands, may also promote the formation and presentation of neoantigens [ 45 ]. In turn, this may mount a more significant response by neoantigen-directed T cells. These methods combined overcome the problem of a lack of suitable neoantigens and alterations in antigen processing and/or presentation, which has been associated with impaired anti-tumor activity [ 46 ]. Vaccine based approaches targeting neoepitopes can also be utilized, typically employing synthetic peptides, DNA or RNA to encode the neoantigen. Whilst some neoantigens are shared between various tumors and patients, the repertoire is rather small [ 47 ]. Consequently, this largely limits the use of neoantigen-based vaccines for the treatment of cancer. However, with the advent of next generation sequencing technologies, mutations specific to an individual patient’s tumor can be identified, leading to the development of tailored neoantigen-based vaccines. Such vaccines exert their effect through numerous mechanisms, including priming the immune system and enhancing the response by CTLs [ 20, 48 ]. Furthermore, poly-neoantigen vaccines can be utilized to facilitate an augmented response. They may also be used to help overcome the issue of tumor heterogeneity and minimize the risk of clonal expansion of antigen loss variants of tumor cells, which can confer treatment resistance [ 47 ]. Through this effect, neoantigen-based vaccines are able to act as a crucial adjunct for both ACT therapy and treatment with immune checkpoint inhibitors [ 12, 20 ]. Challenges and future directions To facilitate the widespread adoption of immunotherapy for the treatment of cancer, a few barriers must be overcome first. Most importantly, toxicity resulting from enhanced activation of the immune system is an obstacle that prevents the regular use of immunotherapy, particularly for combinatory regimens. Such immune-related adverse events (irAEs) include acute episodes of autoimmune-like disease, as seen with immune checkpoint inhibitors, making efficacy and safety studies essential when considering such therapies. Furthermore, given the inherent complexity of tumors, preclinical models that accurately reflect the natural course of tumor development and the associated immunosuppressive microenvironment must be utilized, such as genetically engineered mouse models. Consideration also needs to be given to the route of delivery and other pharmacokinetic properties of immunotherapies in order to maximize bioavailability to the target tumor site, for example through the use of nanoparticle drug delivery systems. In addition, immunotherapy is often limited by the use of conventional chemotherapy as first-line treatment. As a result, by the time, immunotherapy is utilized the patient’s immune system may already be compromised due to advanced disease and/or previous therapy. It is therefore essential that appropriate treatment regimens that optimize the dose, schedule and duration of therapy are designed. Given the abundance of potential target molecules and the wide array of combinatory therapies, it is imperative that biomarkers are developed to help predict tumor responses. This will enable immunotherapy to be tailored to the individual patient, improving efficacy, and reducing toxicity. For instance, in addition to CAR-T cell therapy, neoantigens may be utilized as predictive biomarkers to identify tumors more amenable to checkpoint inhibitor therapy, due to the correlation between the number of mutations/neoantigens and the therapeutic response [ 49 ]. Similarly, tumor mutational burden and the presence of certain immune inhibitory molecules such as PD-L1, CTLA-4, and IDO1 can be used to predict response to checkpoint inhibitor blockade in a range of cancers, including NSCLC, renal cell carcinoma, bladder cancer, and melanoma [ 50 ]. To facilitate a shift to an era of more personalized cancer therapy, cost-effective and practical methods for identifying relevant biomarkers and/neoepitopes, and categorizing cancers according to the underlying immunosuppressive mechanism, must be developed. Conclusions While immunotherapy is still very much in its infancy, it has already shown huge promise and is well-aligned in becoming the mainstay treatment for cancer. In particular, the use of combination therapies and strategies to boost the immune response appear to be particularly attractive approaches, although careful consideration must be given to minimize the likelihood of irAEs. Tailoring immunotherapy to the individual patient through the use of neoantigens and predictive biomarkers should also be further explored in order to improve treatment efficacy, whilst minimizing toxicity. Given the heterogeneous nature of tumors, it is crucial that these different strategies are explored simultaneously and synergistically to ensure immunotherapy lives up to its potential in improving the treatment for the majority of cancers.

10. 7759/cureus. 59474

Cureus

Stem Cell Therapy for Myocardial Infarction and Heart Failure: A Comprehensive Systematic Review and Critical Analysis

In exploring therapeutic options for ischemic heart disease (IHD) and heart failure, cell-based cardiac repair has gained prominence. This systematic review delves into the current state of knowledge surrounding cell-based therapies for cardiac repair. Employing a comprehensive search across relevant databases, the study identifies 35 included studies with diverse cell types and methodologies. Encouragingly, these findings reveal the promise of cell-based therapies in cardiac repair, demonstrating significant enhancements in left ventricular ejection fraction (LVEF) across the studies. Mechanisms of action involve growth factors that stimulate angiogenesis, differentiation, and the survival of transplanted cells. Despite these positive outcomes, challenges persist, including low engraftment rates, limitations in cell differentiation, and variations in clinical reproducibility. The optimal dosage and frequency of cell administration remain subjects of debate, with potential benefits from repeated dosing. Additionally, the choice between autologous and allogeneic stem cell transplantation poses a critical decision. This systematic review underscores the potential of cell-based therapies for cardiac repair, bearing implications for innovative treatments in heart diseases. However, further research is imperative to optimize cell type selection, delivery techniques, and long-term efficacy, fostering a more comprehensive understanding of cell-based cardiac repair.

Introduction and background Ischemic heart disease (IHD) is a significant worldwide health problem, resulting in 17. 9 million fatalities per year [ 1, 2 ]. Despite recent improvements in treatment, a significant number of individuals still experience persistent ischemia and congestive heart failure (CHF). Despite the recent advances in the management of IHD in the form of medications or coronary revascularization, many patients are left with chronic ischemia and nearly one-third of them end up developing congestive heart failure (CHF) [ 3 ]. The available medical treatment for CHF can only address its symptoms without correcting the underlying pathology. Stem cell therapy, an innovative medical approach, has demonstrated encouraging outcomes in the regeneration of damaged heart muscle tissue and the restoration of its ability to contract. Cardiac stem cells (CSCs), mesenchymal stem cells (MSCs), and bone marrow-derived stem cells have demonstrated encouraging outcomes [ 4 ]. The suggested interventions for this therapy encompass anti-fibrotic properties, immunomodulatory effects, neovascularization, promotion of endogenous heart regeneration, and mitigation of unfavorable ventricular remodeling [ 5 ]. The primary emphasis of research investigations in the domain of cell-based therapy revolves around determining the most suitable cell type for delivery, the safest method of administration, the ideal cell dosage, the clinical feasibility, and addressing problems such as cell homing or survival [ 6 ]. The main aim of this systematic review is to shed light on the current status of stem cell therapy as a potential breakthrough in cardiac repair and the treatment of heart failure. We discuss the biology of several stem cell types, mechanisms of cardiac regeneration, translational findings in clinical settings, challenges with possible solutions, and different data from preclinical and clinical studies. Review Methods Search Strategy and Study Selection The Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) standards were adhered to during the process of preparing this systematic review. We conducted a systematic review of all published studies from January 2007 till December 2022 investigating stem cell therapy for heart failure and myocardial infarction (MI). The search for studies was conducted exclusively in the English language, utilizing databases such as Medline, PubMed, and Scopus. Additionally, gray literature sources such as the Centers for Disease Control and Prevention (CDC) and the World Health Organization (WHO) were investigated. The process of conducting a literature search and screening approach, selecting studies, extracting data, and assessing the risk of bias was carried out according to established inclusion criteria. These procedures were overseen by the senior author to ensure the maintenance of quality. The search strategy included keywords and Medical Subject Headings (MeSH) terms related to ischemic heart disease, heart failure, cell therapy, stem cells, and cardiac repair. The primary endpoint is left ventricular ejection fraction (LVEF), and the secondary endpoints include infarct size, ventricular wall thickness, end-systolic volume, and end-diastolic volume. The database search yielded several types of scholarly literature, including randomized control trials, observational studies, review articles, meta-analyses, and original publications, which focused on both animal models and human subjects. All studies were chosen based on their compliance with the inclusion criteria. Two independent authors then assessed the content of the studies to determine their eligibility. Further discrepancies were addressed through a collective deliberation, during which a choice was made to either incorporate or exclude a study based on pre-established criteria. The reviewer conducted a quality assessment to ascertain the presence of a well-defined research topic with precise outcomes, a clear delineation of inclusion and exclusion criteria, robust methodology, potential for generalizability, and acknowledgment of limitations. Inclusion​​​​ and Exclusion Criteria For the inclusion criteria, we will consider studies published from January 2007 to December 2022, in English, including randomized controlled trials, animal studies, systematic reviews, and meta-analyses. Exclusion criteria encompass studies published more than 16 years ago, studies in languages other than English, case studies, editorials, and studies not addressing LVEF as an endpoint. Data Extraction Two independent reviewers conducted a rigorous screening and data extraction process. Any discrepancies were resolved through discussion and consensus. Extracted data included study author, sample size, type of stem cells, mode of delivery, follow-up period, and outcomes. Statistical Analysis Due to the substantial heterogeneity among the included studies, a meta-analysis was not feasible. Therefore, a narrative synthesis of the findings focused on the main outcomes and trends observed across the selected studies. Results The study selection process was systematically conducted in adherence to PRISMA guidelines. The search initially yielded a total of 1457 records, which were deduplicated, resulting in 721 duplicate records being removed. Subsequently, the remaining 736 unique records underwent screening for relevance based on inclusion and exclusion criteria, leading to the exclusion of 652 records that did not meet these criteria. The remaining 84 records were further evaluated for eligibility, and 49 were excluded based on predefined criteria. Ultimately, 35 studies met the inclusion criteria and were included in this systematic review [ 7 - 41 ]. A PRISMA flow diagram (Figure 1 ) was used to visually represent the selection process, illustrating the number of records at each stage, from the initial search to the inclusion of studies. This approach ensured a transparent and systematic approach to study selection in line with PRISMA standards. Cell-based therapies appear to have promise for cardiac repair, with considerable improvements in LVEF found across investigations. The increase in LVEF indicates improved heart function following cell-based treatments. The characteristics and summary of the included studies are illustrated in Table 1. Figure 1 PRISMA flow diagram. PRISMA: Preferred Reporting Items for Systematic Reviews and Meta-Analyses. Table 1 Summary of the included studies. CSC: cardiac stem cell; MSC: mesenchymal stem cells; BM: bone marrow; LVEF: left ventricular ejection fraction; VEGF: vascular endothelial growth factor. Study No. of patients Type of stem cells Dose Mode of delivery Follow-up period Outcome Butler et al. , 2017 [ 7 ] Total = 22; Treated group = 10, Control group= 12 Ischemia-tolerant human donor allogeneic MSCs (itMSCs) 1. 5 × 10 6 cells/kg Intravenous 90 days -itMSC therapy was safe - Improvement in the health status and functional capacity - No significant differences in mortality, hospitalization, or serious adverse event Ulus et al. , 2020 [ 8 ] Total = 42; Treated group = 26; Control group = 16 Human umbilical cord-derived mesenchymal stromal cells or bone marrow-derived MSCs 21∼26 × 10 6 Intramyocardial 12 months There was a decline in NT-proBNP levels in the treated group. The HUC-MSC group specifically showed an increase in LVEF. Additionally, decreases in necrotic myocardium were observed. Bolli et al. , 2011 [ 9 ] (Stage A) Treated group = 9; Control group = 4. (Stage B) Treated group = 7; Control group = 3 Autologous CSCs Based on the number and location of the infarcts (not to exceed a total of 1 million cells) Intracoronary Four-month - Safe - Increase in LVEF - Decrease in infarct size Chin et al. , 2011 [ 10 ] Group A: Five had ischemic dilated cardiomyopathy (DCM) unlikely to benefit from revascularization alone. Group B: Two patients had previous revascularization and three had non-ischemic DCM Mesenchymal stromal cells Group A received 0. 5-1. 0 × 10 6 MSC/kg body weight - Group B received 2. 0-3. 0 × 10 6 MSC/kg body weight Group A: Intramyocardial Group B: Intracoronary Six-week, three-month, six-month, and 12-month - Safe and feasible - Increase in LVEF - Decrease in infarct size Chugh et al. , 2012 [ 11 ] Treated group = 20; Control group = 13 c-kit+ CSCs Eighteen treated patients received 1 × 10 6 cells and two received 5 × 10 5 cells Intracoronary Four- and 12-month - Improvement in both global and regional LV function. - Reduction in infarct size. - Persistent increase in viable tissue Gao et al. , 2015 [ 12 ] Total patients = 116; Treated group = 58; Control group = 58 Wharton’s jelly-derived mesenchymal stem cells (WJ-MSCs) 6 × 10 6 WJ-MSCs Intracoronary 18-month - Increase in the LVEF - Increase in the myocardial viability and perfusion within the infarcted region Guijarro et al. , 2016 [ 13 ] Ten patients MSCs 61. 5 × 10 6 cells per patient Intramyocardial One-month, 12-month, and two-year - Safe - Improvements in cardiac performance, LV remodeling, and patient functional status Hare et al. , 2017 [ 14 ] Thirty-seven patients in a 1:1 ratio MSCs 100 million MSCs Transendocardial Baseline, 30 days, and three-, six-, and 12-month - Allogeneic stem cell injection is safer and more effective than autologous stem cells for nonischemic DCM - Pivotal trials of allogenic MSCs are required according to these results Bartunek et al. , 2017 [ 15 ] Forty-eight patients Autologous bone marrow-derived and cardiopoietic MSCs 733 x 10 6 cells Endomyocardial - The primary endpoint (feasibility/safety) at two-year follow-up. Secondary endpoints (cardiac structure/function and clinical performance) six months post-therapy - Improved LVEF - Decrease in LV end-systolic volume - Improvement in exercise tolerance, quality of life, and physical performance Hare et al. , 2009 [ 16 ] Fifty-three patients BM-MSCs 0. 5, 1. 6, and five million cells/kg Intravenous Six-month - Safe and feasible - Increase in LVEF - Reversal of ventricular remodeling - Improved overall performance CONCERT-HF Trial. , 2021 [ 17 ] Total patients = 18; Treated group = 9; Control group = 9 MSCs + CSCs MSCs dose = 150×10 6 cells CSCs dose = 5×10 6 cells Intramyocardial Baseline, one-month, six-month, and 12-month N/A Li et al. , 2015 [ 18 ] Fifteen patients UC-MSCs Low-dose 3 x 10 6, mid-dose 4 x 10 6 and high-dose 5 x 10 6 groups Intracoronary 12-month and 24-month - Safe and feasible - Increase in LVEF and decrease in infarct size TAC-HFT randomized trial, 2014 [ 19 ] Total patients = 65 MSC; Treated group = 19; Control group = 11 BMC: Treated group = 19; Control group = 10 MSCs and bone marrow mononuclear cells (BMCs) N/A Transendocardial One-month and 12-month - Both are generally safe without serious adverse events following injection - Improved functional status with MSCs - Decrease in infarct size with MSCs - Increase in LVEF CADUCES Trial, 2014 [ 20 ] Treated group = 17 (plus 1 infused off-protocol 14 months post-MI), Control group = 8 Cardiosphere-derived cells (CDCs) 12. 5 to 25 × 10 6 Intracoronary One-year - Decrease in scar area - Increase in viable tissue - Improved LVEF and cardiac function - Safe Houtgraaf et al. , 2012 [ 21 ] Total = 14; Treated group = 10, Control group = 4 Adipose tissue-derived regenerative cells (ADRCs) 20 million ADRCs Intracoronary Six-month - Improvement in LVEF and cardiac function - Decrease in infarct area - Improvement in cardiac perfusion - Safe and feasible without serious adverse effects PRECISE Trial, 2014 [ 22 ] Total = 27; Treated group = 21, Control group = 6 Adipose-derived regenerative cells (ADRCs) 0. 4 x 10 6 ADRCs/kg, 0. 8 x 10 6 ADRCs/kg, and 1. 2 x 10 6 ADRCs/kg (escalating doses) Transendocardial One-, three-, six-, 12-, 18- up to 36-month - Safe and feasible - Improvement in total left ventricular mass and cardiac function - Improvement in cardiac perfusion - Improvement in exercise capacity MyStromalCell Trial, 2012 [ 23 ] Sixty patients AD-MSCs N/A Intramyocardial After four, 12, and 26 weeks and then after one, two, and three years - Decrease in infarct size in the LV size - Improvement in maximum oxygen consumption and exercise tolerance - Improvement in myocardial perfusion - Increase in LVEF - Improved symptoms Ascheim et al. , 2014 [ 24 ] Thirty patients were randomized in a ratio of 2:1 Allogeneic mesenchymal precursor cells (MPCs) 25 million MPCs Intramyocardial Twelve-month or until transplant, whichever came first - Safe with no difference in serious adverse events - There is a potential signal of efficacy that needs further evaluation Musialek et al. , 2015 [ 25 ] Ten patients WJ-MSCs 30 × 10 6 WJ-MSCs Intracoronary 12-month - Safe and feasible without major adverse effects RIMECARD Trial, 2017 [ 26 ] Treated group = 15; Control group = 15 UC-MSCs 1 × 10 6 cells/kg Intravenous Three-, six-, and 12-month - Increase in LVEF - Improvements of New York Heart Association functional class - Improvement in quality of life Zhao et al. , 2015 [ 27 ] Total patients = 59; Treated group = 30, Control group = 29 UC-MSCs N/A Intracoronary Baseline, six-month, and 12-month - Increase in six-minute walking distance of the treatment group - improvement of cardiac remodeling and cardiac function - Decrease in mortality rate Sürder et al. , 2016 [ 28 ] Total patients = 200; Treated group= 133, Control group = 67 Bone marrow-derived mononuclear cells (BM-MNC) N/A Intracoronary Baseline, and 12-month - Overall, one-year mortality was low - Among patients with AMI and LV dysfunction, treatment with BM-MNC either five to seven days or three to four weeks after AMI did not improve LV function at 12 months, compared with control SWISS-AMI, 2010 [ 29 ] Total patients = 192; Treated group = 128, Control group = 64 BM-MNCs Fifty milliliters of autologous bone marrow Intracoronary Baseline, four-month, 12-month, and 24-month - Improve remodeling of the left ventricle after acute MI TIME Randomized Trial, 2012 [ 30 ] Total patients = 120; Treated group = 79, Control group = 41 BM-MNCs 150 × 10 6 BMCs or placebo Intracoronary Baseline, and six-month - No complications were associated with intracoronary infusion. - No significant increase in LVEF TOPCARE-AMI, 2011 [ 31 ] Total patients = 55 CPC or BMC N/A Intracoronary Five-year follow-up Reduction in functional infarct size -Increment in LV-end diastolic volume Wöhrle et al. , 2010 [ 32 ] Total patients = 42; BMC group = 29, Placebo group = 13 Autologous bone marrow cells (BMCs) 381 x 10 6 mononuclear BMCs Intracoronary Baseline, one-month, and six-month No positive evidence of intracoronary BMC versus placebo therapy with respect to LV ejection fraction, LV volume indexes, or infarct size STAR-heart study, 2010 [ 33 ] Total patients = 391; Treated group = 191, Control group = 200 BMC (Progenitor or stem cells) 80–120 mL of BMC Intracoronary Baseline, three-month, 12-month, and 60-month -Significant improvement in hemodynamics (e. g. , LVEF, cardiac index, exercise capacity, oxygen consumption, and LV contractility - Decrease in long-term mortality in the BMC-treated patients compared with the control group) Stamm et al. , 2007 [ 34 ] Total patients = 55; Treated group = 35, Control group = 20 CD133+ bone marrow cell 2. 95 × 10 7 CD34 + cells Intramyocardial Baseline, and 12-month - Intramyocardial delivery of purified bone marrow stem cells together with CABG surgery is safe. - Improvement in LVEF REGENT Trial, 2009 [ 35 ] Total patients = 200; Treated group= 160, Control group = 40 Bone marrow-derived unselected mononuclear cells (UNSEL) and selected CD34(+)CXCR4(+) cells (SEL) N/A Intracoronary Baseline, and six-month - No significant improvement in LVEF -Use of selected CD34 + CXCR4 + cells in patients with significantly reduced LV function is safe, feasible, and needs further investigation RENEW Trial, 2016 [ 36 ] Total patients = 112; Treated group = 57, Control group = 55 Autologous CD34(+) cells (10 × 0. 2 ml) of 1 × 10 5 auto-CD34 + cell/kg up to 1 × 10 7 cells Intramyocardial Baseline, three-month, six-month, and 12-month - Autologous CD34(+) cell therapy is safe - Due to early termination, RENEW was an incomplete experiment IMPACT-CABG Trial, 2016 [ 37 ] Total patients = 40; Treated group = 20, Control group = 20 Autologous CD133 + stem cells 0. 5 × 10 6 -10 × 10 6 Intramyocardial Baseline, and six-month - Safe and feasible - No significant improvement in LVEF Qayyum et al. , 2017 [ 38 ] Total patients = 161; Treated group = 78, Control group = 83 Autologous VEGF-A 165 -stimulated adipose-derived stromal cells (ASCs) 10–15 injections of 0. 2 mL of ASCs Intracoronary Baseline, three-month, and six-month - Safe and feasible - Increase in exercise capacity in the ASCs group but not in the placebo group -Improvement in LVEF POSEIDON Randomized Trial, 2012 [ 39 ] Total patients = 31; Treated group = 31 MSCs 20 million, 100 million, or 200 million cells (five patients in each cell type per dose level) Transendocardial Baseline, six-month, 12-month, and 13-month - Improvement in the patient functional capacity, quality of life, and LV remodeling - Low-dose concentration MSCs (20 million cells) produced the greatest reductions in LV volumes and increased LVEF C-CURE Trial, 2013 [ 40 ] Total patients = 46; Treated group = 32, Control group = 15 MSCs N/A Intramyocardial Two-year follow-up -Cardiopoietic stem cell therapy was feasible and safe with signs of improvement in chronic heart failure - Improved six-minute walk distance Lee et al. , 2014 [ 41 ] Total patients = 80 MSCs 7. 2±0. 90 × 10 7 cells Intracoronary Baseline and six-month -Improved LVEF - Tolerable and safe - Modest improvement in LVEF at six-month follow-up Discussion Over the past few decades, animal and clinical studies have explored several types of stem cells to find the best one for cardiac repair and contractile function restoration [ 20, 21 ]. Stem cell types employed in cardiac cell-based therapy are briefly discussed below. MSCs MSCs are stem cells that can self-renew, clone, and differentiate into mesenchymal and non-mesenchymal lineages. They can differentiate into cardiomyocytes, blood vessel endothelial cells, and smooth muscles. MSCs can be obtained from bone marrow (BM), peripheral blood, adipose tissue, umbilical cord, or synovial tissue. BM-MSCs have several benefits, but their use in animal experiments is controversial due to potential risks. MSCs exhibit surface markers but lack MHC class II and other costimulatory markers [ 42 ]. Umbilical cord-derived mesenchymal stem cells (UC-MSCs) can be collected from discarded placenta and release angiogenic, anti-apoptotic, and growth factors. They are chemoattractive and produce pro-angiogenic molecules, such as VEGF, hepatocyte growth factor (HGF), and angiopoietin. AD-MSCs, another type of mesenchymal stem cell, are an ideal autologous source of therapeutic regenerative cells. They are multipotent, pro-angiogenic, and can differentiate into cardiomyocytes and endothelial cells without immunologic issues when employed for cell-based therapy after myocardial infarction. AD-MSCs are easy to isolate in high quantities by liposuction and are widely available [ 43 ]. CSCs Endogenous CSCs are an interesting discovery in heart regeneration medicine. This disproves the idea that the heart is terminally differentiated and that all cardiomyocytes cannot re-enter the cell cycle. However, the modest number of resident CSCs limits their therapeutic use. In addition, their endogenous regeneration potential cannot compensate for significant segmental myocardial loss in MI [ 44 ]. A mouse model using "mosaic analysis with double markers" showed that post-natal cardiomyocyte synthesis is rare and limited 50. CSCs include c-kit+, Sca-1+, Islet-1+, SP, Cs, and CDCs 11. Indeed, c-kit+ CSCs were the first heart CSC subtype identified, and they have shown promising effects after transplantation, reduction of ventricular remodeling, and cardiac function enhancement in animal investigations [ 45 ]. The SCIPIO trial, the first human phase 1, randomized controlled trial of autologous c-kit+ CSCs in heart failure patients, found that patients with ischemic heart failure who undergo coronary artery bypass grafting (CABG) can be treated with CSCs, which can be isolated from the heart during surgery [ 9 ]. In comparison to MSCs, CSCs differentiated into cardiomyocytes, had stronger angiogenic potential, improved engraftment, and balanced paracrine factor secretion. These superior qualities promote cardiac functioning more than extracardiac stem cells. Animal studies suggest dual stem cell transplantation is a promising cell-based therapy. CardioChimeras (CCs) generated by fusing CSCs and MSCs showed better cardiac healing and function than the injection of each cell type separately in an animal study by Pearl Quijada et al [ 46 ]. Other Low-Effective Stem Cell Types Induced pluripotent stem cells (iPSCs) and human embryonic stem cells (hESCs) provide an unlimited source of tissue-specific cell types, such as cardiomyocytes. Despite ethical and social issues surrounding ESCs, iPSCs can produce atrial, nodal, and ventricular cardiomyocytes. Genetic reprogramming from adult somatic cells creates pluripotent iPSCs, which can self-renew and differentiate into many cell types, including cardiomyocytes. However, iPSCs are vulnerable in disease modeling and personalized medical treatment due to their ability to maintain patient-specific genomic, transcriptomic, proteomic, and metabolomic information. Immature hiPSCs are the main challenge, and novel treatments such as transient reprogramming factors may overcome this barrier. Human amniotic fluid (hAF) cells can multiply and develop into all embryonic germ layers without becoming teratomas. A two-week human study found that cardiopoietic AF cells upregulated cardiac transcription factors and expressed multipotency markers [ 47 ]. Routes of Stem Cell Delivery Cell delivery is a crucial aspect of determining the safest and most effective way to deliver stem cells inside damaged cardiac muscle. There are several routes of cell delivery, including intracoronary, transendocardial, intravenous, and intramyocardial. The intracoronary route is an efficient method due to its safety, less invasiveness, and homogenous uniform distribution of transplanted stem cells into the targeted region of the heart. However, it has drawbacks such as the induction of further ischemia, the limitation of cell delivery in poorly perfused regions, and limited doses of stem cells [ 32, 33, 48 ]. The transendocardial route involves catheter-based injection of stem cells through the endocardium, which relies on electromechanical mapping (EMM) for precise identification and differentiation of infarcted and healthy myocardial tissue. It is a safe, highly effective, and less invasive method, but its efficacy is debatable in clinical trials [ 22, 39 ]. The transcoronary venous route is another potential method for stem cell delivery to the infarcted heart, but it has limitations such as variability of coronary venous circulation among patients and tortuosity of coronary veins leading to difficulty passing through them [ 49 ]. The intravenous route seems to be the safest, easiest, and least invasive, but its efficacy decreases due to the lack of proper homing and implantation into the targeted area of the heart. The pulmonary first-pass in the lungs is another concern that needs to be investigated [ 26 ]. A swine study suggested that intravenous injection of MSCs during the early post-infarct stage enhances regional myocardial perfusion and improves LVEF. However, clinical translation yielded conflicting results [ 50 ]. Intravenous injection of MSCs is generally safe and well-tolerated with some anti-inflammatory and immunomodulatory effects, but there was no significant difference in EF between treatment and placebo groups [ 50 ]. The surgical intramyocardial route, performed through thoracotomy during open-heart surgeries or as a separate procedure, offers the most direct and precise method for delivering stem cells into the infarcted region, with the highest rate of stem cell engraftment inside the determined region. However, the main disadvantage of the intramyocardial method is its highly invasive nature, which carries several safety concerns and can lead to complications such as cardiac perforation, arrhythmia induction, systemic embolization, and prolonged recovery time [ 36, 37, 40, 43 ]. Cell delivery for cardiac treatment can be accomplished through various methods, such as the intramyocardial route, the retrograde coronary venous infusion route, and myocardial tissue engineering. The intramyocardial route is the most direct, but it comes with risks such as cardiac perforation and arrhythmia. Retrograde coronary venous infusion is done through coronary sinus injection, beneficial during cardiac surgery, but has limitations like coronary vein tortuosity. Myocardial tissue engineering, specifically with bioactive scaffolds, exhibits the potential to address obstacles such as inadequate cell viability [ 7 - 41 ]. Cell sheet transplantation, a novel application of cardiac tissue engineering, requires further investigation in animal models. Mechanisms The role of paracrine factors in cell-based cardiac repair is a topic of controversy. Paracrine factors are bioactive molecules that diffuse along short distances to induce changes in surrounding cells, contributing to cardiac repair through various mechanisms. These mechanisms include cardiomyocyte proliferation, promotion of cardiomyocyte cell cycle re-entry, cytoprotection and anti-apoptosis actions, differentiation of resident CSCs, angiogenesis, enhancing cardiac metabolism and contractility, anti-fibrosis actions, and immunomodulatory effects [ 51 ]. Injecting exogenous CSCs into the infarcted area of the myocardium may promote the proliferation of endogenous CSCs in both infarcted and noninfarcted regions. CSCs generate growth factors such as hepatocyte growth factor (HGF) and insulin growth factor-1 (IGF-1) that induce the migration of additional CSCs via the cardiac interstitium, proliferation, and differentiation into cardiomyocytes and vascular elements. This activation of endogenous CSCs has been proposed to have a role in the positive effects of other cell types, including MSCs [ 52 ]. Angiogenesis is another mechanism of benefit for stem cells, as proangiogenic factors such as vascular endothelial growth factor (VEGF), HGF, IGF-1, and angiopoietin are secreted by stem cells to promote neovascularization. Stem cells can also inhibit ventricular remodeling by altering extracellular matrix elements, restricting infarct expansion, LV remodeling, and myocardial fibrosis. Preventing ventricular hypertrophy is another mechanism of benefit, with stem cell cardiac therapy in HF models resulting in inhibition of the hypertrophic response of surviving cardiomyocytes. Paracrine factors like IGF-1 released by stem cells have been shown to enhance cardiomyocytes' survival and inhibit their apoptosis [ 51, 52 ]. Cardiac regeneration involves the use of various genes, enzymes, and cytokines to enhance the functional recovery of the infarcted heart. C-Kit is a type III receptor tyrosine kinase involved in numerous intracellular signaling pathways, and it has been used as a marker to identify and enrich adult stem cells such as CSCs that are capable of differentiating into cardiomyocytes, endothelial cells, and smooth muscle cells in vitro and in vivo after myocardial infarction (MI). Pim-1 is another critical contributor to cardiac regeneration, expressed in stem cells, endothelial cells, and vascular smooth muscle cells. Genetic modification of CSCs and MSCs through increasing Pim-1 expression before injection into animal models has been associated with better cell engraftment and enhanced survival [ 53 ]. Combining growth factors with injected CSCs or MSCs is a novel approach to further enhance the functional recovery of the infarcted heart. Transforming growth factor (TGF-β) is a versatile and influential cytokine and growth factor that controls various cellular and metabolic reactions. It plays a role in reducing inflammation, modulating the immune system, promoting wound healing, stimulating the creation of new blood vessels, responding to cancer, and facilitating the regeneration of heart tissue. HIF-1 is a transcription factor responsible for the regulation of oxygen homeostasis, controlling cellular and systemic adaptive responses to hypoxia, and promoting angiogenesis, differentiation, survival, and protection of both transplanted and resident CSCs against the effects of hypoxia, inflammation, and reactive oxygen species in ischemic cardiac conditions [ 51 - 53 ]. Hepatocyte growth factor (HGF) and insulin-like growth factor-1 (IGF-1) are other examples of growth factors implemented in the process of cardiac regeneration. Combining HGF and IGF-1 with transplanted stem cells could be used as an adjuvant to cell injection for myocardial repair. The underlying mechanisms include promoting angiogenesis, differentiation, survival, and protection of both transplanted and resident CSCs against the effects of hypoxia, inflammation, and reactive oxygen species in ischemic cardiac conditions [ 54 ]. Heme oxygenase-1 (HO-1) is a Nrf2-regulated enzyme that protects against cellular injury, including heart damage. It catalyzes the breakdown of heme into CO, bilirubin, and ferrous ions. HO-1 increases VEGF, FGF, and IL-10 expression, while decreasing IL-1, IL-6, and TNF. It also generates angiogenic effects in MSCs and improves anti-oxidative and anti-apoptotic capacities [ 55 ]. Challenges Against Cell-Based Cardiac Repair and Possible Solutions Challenges in cell-based cardiac repair include issues with stem cell collection, ex-vivo expansion, intracardiac delivery methods, cell dosage, and achieving adequate engraftment, differentiation, and functional improvement in vivo. Only a small percentage of transplanted stem cells survive and integrate into the host myocardium due to factors like ischemia, reperfusion injury, and inflammation. Strategies such as prosurvival stimulation before injection, embedding cells in bioactive scaffolds, and guided cardiopoiesis aim to improve engraftment and differentiation [ 46, 52, 53, 56 ]. Clinical reproducibility poses a challenge, requiring preclinical studies in large animal models with physiological similarities to humans. Co-morbid conditions like hypertension, diabetes, and aging must be considered. Various clinical trials and meta-analyses have provided mixed results regarding the efficacy of stem cell therapy, with some showing benefits in terms of mortality reduction, improved cardiac function, and decreased hospital readmission, while others report no significant advantage [ 56 ]. Dosing and frequency of administration present uncertainties, as the relationship between cell dosage and therapeutic effects remains unclear. Studies suggest that dividing a given number of cells into multiple doses may be more effective than a single administration, emphasizing the importance of repeated dosing for maximum therapeutic efficacy. Further research is needed to establish the correlation between the number and frequency of cells delivered and their impact on heart function [ 57 ]. Possible Adverse Events Following Stem Cell Therapy Stem cell therapy, while promising in terms of safety and efficacy, has reported adverse events in some studies. Short-term mortality in patients who received stem cell therapy was linked to various causes, such as mediastinitis, cardiogenic shock, intestinal ischemia, ventricular fibrillation, congestive heart failure (CHF), and myocardial ischemia. Long-term mortality was associated with events like cerebrovascular hemorrhage, sepsis after a heart transplant, heart failure, sudden cardiac death, and lung malignancy [ 58 ]. Arrhythmia, particularly with intramyocardial injection, is a frequently reported adverse effect. Potential mechanisms include lack of electromechanical integration, graft automaticity, direct injury and edema, transplantation of non-cardiomyocyte contaminants, nerve sprouting, immunologic reactions, and expression of gap junctions affecting arrhythmogenic potential. Paracrine factors were suggested to play a protective role against arrhythmia [ 59 ]. Occurrences of nonfatal myocardial infarctions were documented throughout both short-term and long-term monitoring after stem cell transplantation. Rehospitalization due to heart failure was also noted, with no significant difference compared to control groups in terms of the risk of readmission [ 60 ]. Conclusions Cell-based therapy, a promising treatment option for IHD and CHF, has the potential to revolutionize the treatment of these conditions. However, the exact role of stem cells in treating these conditions remains unexplored. Despite early trials showing potential improvements in heart function, concerns about the mechanism of action, cell dose, timing, and delivery methods remain unresolved. Large-scale clinical studies and approved technologies are crucial for the widespread adoption of stem cell treatment.

10. 7759/cureus. 59848

Cureus

Fabrication of Periodontal Membrane From Nelumbo nucifera: A Novel Approach for Dental Applications

Background The periodontal membrane plays a crucial role in tooth support and maintenance. Natural materials with biocompatible and bioactive properties are of interest for periodontal membrane fabrication. Nelumbo nucifera, known for its therapeutic properties, presents a potential source for such materials. Aim This study aimed to fabricate a periodontal membrane from N. nucifera and evaluate its biocompatibility and potential for periodontal tissue regeneration. Materials and methods N. nucifera stems were collected dried, and aqueous extract was prepared. The extracted material was then processed into a membrane scaffold using a standardized fabrication method. The fabricated membrane was characterized by its physical and chemical properties. Biocompatibility was assessed using human periodontal ligament fibroblast (hPDLF) cells cultured on the membrane, followed by viability, proliferation, and anti-microbial assays. Results The fabricated N. nucifera membrane exhibited a porous structure with suitable mechanical properties for periodontal membrane application. The membrane supported the adhesion, viability, and proliferation of hPDLF cells in vitro. Conclusion The fabrication of a periodontal membrane from N. nucifera shows promise as a natural and biocompatible material for periodontal tissue regeneration. Further studies are warranted to explore its clinical potential in periodontal therapy.

Introduction Nelumbo nucifera, commonly referred to as lotus, is an aquatic perennial plant that falls within the Nelumbonaceae family. This plant serves as a valuable source of herbal medicine, exhibiting potent antipyretic, cooling, astringent, and demulcent properties. The seeds of the lotus plant are utilized in the preparation of various Ayurvedic remedies for conditions, such as tissue inflammation, cancer, diuretic effects, and skin diseases. Furthermore, these seeds are rich in compounds, particularly flavonoids, making them effective as an antidote for poisoning [ 1 ]. Previous studies have documented the capacity of lotus seeds to scavenge free radicals and protective effects against cytotoxicity and DNA damage generated by reactive nitrogen, sodium nitroprusside (SNP), peroxynitrite, and macrophage RAW 264. 7 cell lines. [ 2 ]. Various studies have used finite element analysis (FEA) to develop constitutive models for the periodontal ligament (PDL) and understand its mechanical behavior. These models have considered the anisotropic, inhomogeneous, and non-linear elastic nature of the PDL [ 3, 4 ]. Lotus seeds have been extensively studied for their antioxidant properties. Research has shown that different parts of the lotus plant, such as the seed epicarp, seed embryos, and rhizome knots, exhibit significant antioxidant activity due to their high phenolic content [ 5 ]. Lotus seed protein hydrolysate (LSPH) derived from lotus seeds has also demonstrated potent antioxidant abilities, with high scavenging activity against DPPH and H2O2 radicals [ 6 ]. Lotus root polysaccharide (LRP) and its carboxymethylated form have also been found to be natural antioxidants. The carboxymethylated form is better at getting rid of ferrous ions and hydroxyl radicals [ 7 ]. Furthermore, extracts from Ziziphus lotus L. seeds have exhibited antioxidant properties, with the methanol extract showing the highest DPPH radical scavenging activity [ 8 ]. These findings collectively highlight the free radical scavenging potential of lotus seeds and related plant parts. Barrier membranes used in PDL regeneration require specific biocompatibility requirements, such as high biocompatibility, low cellular permeability, tight tissue adhesion, moderate mechanical strength, storage stability, and handleability. Current limitations of these membranes include weak biocompatibility of nonabsorbable and absorbable synthetic polymer membranes [ 9 ]. Furthermore, it should be noted that natural collagen membranes demonstrating rapid degradation rates possess restricted rigidity and may not effectively sustain barrier functionality [ 10, 11 ]. To improve biocompatibility, possible solutions include using electrospinning techniques, nanofiber scaffolds, or developing functional gradient membranes [ 12 ]. Among them, clinicians currently use membranes made of poly-lactic acid (PLA) and poly-glycolic acid (PGA) that exhibit a diverse range of tensile strength, ranging from 40 to 140 Mpa. However, these membranes generate harmful oxidative species when they are broken down by polymorphonuclear leukocytes, and the material is directly proportional to the inflammatory response, thus leading to the failure of the treatment [ 13 ]. Hence, the aim of this research is to fabricate a PDL membrane/barrier membrane naturally from the stem of N. nucifera. Materials and methods Preparation of the PDL membrane from the stem extract N. nucifera is collected from local flower shops. Stems were removed, dried and crushed into powder, boiled in water, and filtered the extract, and the filtrate obtained is the aqueous extract (Figure 1A, 1B, 1C ). Five grams of the extract was mixed with 20 ml of sodium alginate and placed in a stirrer for five to 10 minutes. The polymer mixture was poured into the petri dish and kept overnight at -20 ºC after the complete mixture of seed extract in the alginate polymer. The membrane formed instantly was stored in the freezer, and 6% calcium chloride (a cross-linking solution that maintains the same solid state of the membrane formed out from the freezer) solution was added above the membrane and kept aside. Thus, the thin membrane was fabricated. The shelf life of a PDL membrane fabricated from plants can vary depending on the specific material and how it is processed and stored. If the membrane is properly processed, packaged, and stored, it can have a shelf life of around two to three years. Figure 1 Preparation of membrane from the stem of Nelumbo nucifera (A: stems were segregated, B: the extract preparation, C: membrane prepared from the extract) Membrane morphology Morphological analysis was done using a scanning electron microscope (JEOL-JSM-IT800 electron microscope, JEOL, Ltd. , Japan) at 1 Kv. The samples were dried up before SEM analysis with the help of a critical point dryer (Leica EM CPD300, Leica Microsystems GmbH, Germany). Hemocompatibility assessment The biocompatibility test was assessed by mixing 50 µl with 950 µl of double distilled water for the positive control and mixing 50 µl with 950 µl of phosphate-buffered saline (PBS) for the negative control. The solution was exposed to the membranes, and the rate of hemolysis was assessed using the positive and negative controls. Antimicrobial activity The antibacterial activity of the extract was evaluated against strains of Streptococcus mutans, Enterococcus faecalis, and Staphylococcus aureus using Muller-Hinton agar in order to ascertain the zone of inhibition. The agar medium was prepared, sterilized, and permitted to solidify for a duration of 16 minutes at a temperature of 121 °C. The test organisms were collected by swabbing them following the incision of the wells using a 9 mm sterile polystyrene tip. The sample was loaded with various concentrations (5, 10, 15, and 20 microliters) and the area of inhibition was assessed after incubating the plates at 37°C for 24 hours. The activity of Candida albicans is assessed through the application of the Agar Well Diffusion Assay, employing Rose Bengal Agar as the medium. Prior to the addition of varying volumes of the extract, specifically 5, 10, 15, and 20 microliters, the wells were subjected to a sterile medium that had been effectively swabbed with the test pathogen. The plates were raised at a temperature of 37 °C for a duration of 40-72 hours. Following the designated incubation period, the zone of inhibition was assessed. Confocal microscope The hPDLF cells were washed with PBS and fixed with formaldehyde. The cells were labeled with fluorescent dyes like acridine orange and proprium iodide targeting their attachments over the synthesized membrane. The labeled cells were imaged using a confocal laser scanning microscope. Results Morphological analysis of the membrane The alginate membrane exhibited a smooth surface characterised by few imperfections and irregularities, as depicted in Figure 2. The N. nucifera membrane exhibited surface roughness, resulting in an increased surface area. The findings indicate that the extended endurance of the material can be attributed to its robust crystalline structure. The incorporation of the stem extract has the potential to enhance the surface roughness of the membrane, hence facilitating the adhesion of cellular components to the hydrogel membrane. Figure 2 A and B show the smooth alginate membrane. C and D depict the membrane fabricated from Nelumbo nucifera displayed surface roughness. Hemocompatibility of the membrane The membrane's biocompatibility was evaluated using both positive and negative controls. Both solutions were applied to the membrane, and the rate of lysis was quantified using a UV spectrophotometer. The PDL membrane derived from N. nucifera exhibited a hemolysis rate of less than 5% (Figure 3 ). Figure 3 Hemolysis of the fabricated membrane with improved biocompatibility with increasing concentration PC: positive control, NC: negative control, S1: sample 1 (5 grams), S2: sample 2 (10 grams) Antimicrobial assessment of the membrane Although the membrane has good biocompatibility, the antibacterial activity against S. aureus and E. coli was very mild. The antimicrobial activity of the membrane against E. faecalis and Candida albicans were negative (Figure 4 ). Figure 4 Antimicrobial activity of the membrane fabricated from Nelumbo nucifera against Candida albicans (A), Staphylococcus aureus (B), Escherichia coli (C), Enterococcus faecalis (D) Confocal imaging of the cells in the membrane The adhesion, proliferation, and focal adhesion formation of the cultured human periodontal ligament fibroblast cells (hPDLF) cells were observed by confocal laser microscopy. The encapsulated hPDLF cells were homogenously distributed inside the matrix of the polymer membrane. After incubation for 24 hours, the cells were well attached to the lotus stem extract blended alginate membrane. The figure shows the uniform cytoskeleton of the hPDLF cell network interconnecting the polymer membrane. The phenomenon of cell spreading is evident, as the cells that are connected exhibit spindle-like processes (Figure 5 ). Figure 5 A and B show the attachment of human periodontal ligament fibroblast cells to the membrane with spindle-like processes over the fabricated membrane under a confocal microscope Discussion Plant-based PDL membranes are gaining significant attention in research due to their high biocompatibility [ 14, 15 ]. These membranes, derived from plant sources like olive leaves and Lythri herba, offer non-toxic, eco-friendly, and renewable alternatives for wound dressings and cell culture platforms [ 16, 17 ]. The use of plant-derived compounds in biomaterials enhances biocompatibility and accelerates tissue repair processes, making them ideal for applications in regenerative medicine [ 18 ]. In addition, decellularized plant-based scaffolds provide a natural and tunable substrate for 3D cell culture, promoting cell communication and differentiation. The versatility and effectiveness of plant-based PDL membranes highlight their potential as innovative solutions in biomedicine, offering promising avenues for further research and development. Our research is the first of its kind to fabricate a periodontal membrane from the stem of N. nucifera. A previous study evaluated the antibacterial activity of the scaffold with Ziziphus jujuba and found it to be microbicidal against S. aureus and E. coli [ 19 ]. A recent study in fabricating the GTR membrane using the mucilage of Chia seeds and lignin-mediated ZnO nanoparticles showed remarkable antibacterial properties against S. au reus and E. coli with good degradation properties [ 20 ]. These findings are consistent with our study and suggest that this new approach could be a promising solution for combating bacterial infections. In recent times, considerable research has been conducted on the composite scaffold, a biomaterial structure composed of two or more different materials, usually in the form of a matrix or framework, which are combined to create a scaffold with enhanced properties for tissue engineering and regenerative medicine applications. These scaffolds have proven to be highly effective in promoting cell adhesion, proliferation, and differentiation, as well as facilitating tissue regeneration in a variety of applications. One such composite scaffold synthesized from bacterial cellulose, chitosan, and hydroxyapatite was studied for the release of pomegranate peel extract. The study found that the release of the extract was dose-dependent and had a microbicidal effect with good wound-healing abilities [ 21, 22 ]. However, the precise control of concentration and its cost in fabrication, and interfacial bonding is still a problem. Hence, researchers need to consider the correlation between scaffold parameters and cell fate, as well as the effects of biochemical characteristics, structure architecture, biodegradability, and mechanical behavior of scaffold materials, in tissue engineering [ 23 ]. Hydroxyapatite was infused with the alginate membrane (HAp-Alg) and was found to have good biocompatibility with the alginate and pH-responsive degradability [ 24 ]. Another novel innovation for periodontal regeneration includes bacterial/plant-derived extracellular vesicles (BEVs/PEVs) has been shown to play a role in periodontal homeostasis and regeneration [ 25, 26 ]. These vesicles are secreted by bacteria and plants and contain biomolecules that mediate communication between cells. Studies have demonstrated that BEVs and PEVs have beneficial effects on periodontal regeneration, making them a potential alternative strategy for cell-based periodontal regeneration [ 27 ]. Therefore, there is a need for further in-depth research to understand the complex interactions between scaffold materials and cells. The development of advanced composite scaffolds that can mimic the natural extracellular matrix and provide an optimal microenvironment for cell growth and differentiation is crucial. The integration of multiple materials with different properties to create a composite scaffold with enhanced physical, biological, and mechanical properties is also an important area of focus. Limitations of the study The periodontal membrane fabricated from N. nucifera has demonstrated exceptional biocompatibility. However, the present study was limited to the evaluation of biocompatibility and potential for regeneration in vitro, which may not fully represent the complex in vivo environment of periodontal tissues. The biocompatibility was evaluated using hPDLF cells only. The interaction of the membrane with other cell types involved in periodontal regeneration, such as osteoblasts or immune cells, will be considered in the future. While the study suggests promise for clinical use, further research, including animal studies and clinical trials, is needed to validate its safety and efficacy in humans. Conclusions The fabrication of a periodontal membrane from N. nucifera demonstrates promising potential as a natural and biocompatible material for periodontal tissue regeneration. The membrane exhibited suitable physical and mechanical properties and supported the adhesion, viability, and proliferation of hPDLF cells in vitro. These findings suggest that N. nucifera could be a viable candidate for use in periodontal therapy. However, further studies, including in vivo investigations and clinical trials, are warranted to validate its efficacy and safety for clinical application.

10. 7759/cureus. 59924

Cureus

Comparative Analysis of the Effectiveness of Four Distinct Remineralizing Agents in Artificial White Spot Lesions Following Chitosan Nanoparticle Pretreatment: An In Vitro Study

Aim The aim of the study was to compare the effectiveness of chitosan nanoparticle pretreatment with four different remineralizing agents in artificial white spot lesions. Methods A total of 100 human maxillary first premolars were selected and divided into five groups of 20 samples in each group. Artificial white spot lesions were created by immersing the samples in the demineralizing solution for 96 hours. Chitosan pretreatment was done for all samples followed by subjecting Group I samples to artificial saliva (control), Group II samples to 3M Clinpro, Group III samples to GC Tooth Mousse, Group IV samples to SHY-NM, and Group V samples with Aclaim using a cotton applicator tip. Each group was divided into two subgroups of 10 samples, which were subjected to hardness testing and mineral content analysis. Surface microhardness and the calcium phosphorous ratio were recorded using a Vickers microhardness tester and energy-dispersive X-ray (EDAX) analysis at three levels i. e. , baseline, after demineralization, and after remineralization and tabulated. Statistical analysis was conducted by analyzing data using ANOVA and post hoc followed by Dunnett's t-test using IBM SPSS Statistics for Windows, Version 16 (Released 2007; IBM Corp. , Armonk, New York, United States). Results Vickers surface hardness testing and EDAX analysis showed statistically significant values for all the groups. Among them, maximum remineralization potential was seen in samples treated with Chitosan and 3M Clinpro combination, and minimum remineralization potential was seen in Chitosan and artificial saliva combination. Conclusion The addition of chitosan nanoparticles with various remineralizing agents showed a significant synergistic effect on remineralization activity. Also, chitosan and Clinpro combination showed the maximum surface hardness and EDAX analysis values when compared to other groups.

Introduction Dental caries is one of the most common dental diseases and is recognized as a leading cause of tooth loss [ 1 ]. Fluoride has previously been used for caries treatment due to its cariostatic potential [ 2 ]. However, certain limitations (such as dental and skeletal fluorosis) have restricted its use. Research is being done to develop recent preventive agents that act as adjuncts to fluoride [ 1 ]. Clinpro 5000 (3M ESPE, Canada) contains 1. 1% NaF silica and a functionalized tricalcium 6 phosphate (fTCP) known as hydroxyapatite [ 2 ]. This has been shown to boost remineralization performance relative to fluoride-only systems. It controls calcium and phosphate delivery and prevents early interactions with fluoride [ 2 ]. CPP-ACP (GC Tooth Mousse) is developed from milk protein and acts as a reservoir of calcium and phosphate ions, resulting in the formation of hydroxyapatite or fluorapatite crystals, thereby promoting remineralization [ 3 ]. Novamine (SHY NM, Group Pharmaceuticals, India) is a bioactive glass that was originally used as a bone regenerative material [ 4 ]. It forms a hydroxycarbonate apatite (HCA) layer similar to naturally occurring biological apatite, thereby halting the demineralization and enhancing the remineralization process [ 1, 4 ]. Nanohydroxyapatite (Aclaim, Group Pharmaceuticals, India) toothpaste is morphologically similar to the apatite crystal of tooth enamel. It fills defects on demineralized teeth, continuously attracts calcium and phosphate ions to the defective surface, and promotes crystal integrity and growth [ 5 ]. Chitosan (Sigma Aldrich, Bangalore) is obtained from arthropod shells. It electrostatically attracts negatively charged microbial species, inhibiting their growth and the formation of dental plaque. Due to continuous adhesion provided by chitosan, biomineralization did not ease, even when the pH decreased below 6. 5 [ 6 ]. The Vickers hardness test measures the indentation hardness of thin sections of metals, ceramics, and other materials [ 7 ]. Energy-dispersive X-ray (EDAX) image analysis is the gold standard for mineral content estimation at the ultrastructural level. It is critical to select an appropriate material with high remineralizing potential, so this study compared the effectiveness of chitosan nanoparticle pretreatment with four remineralizing agents in artificial white spot lesions. Materials and methods A total of 100 human maxillary first premolars freshly extracted for orthodontic treatment with no caries were included. Exclusion criteria were teeth examined with extensive caries, restorations, enamel hypoplasia, and cracks. Each tooth was thoroughly cleaned and stored in a 10% formalin solution. The sample size was calculated using the following formula. n= 2S 2 (z 1 +z 2 ) 2 /(m 1 -m 2 ) 2 The teeth were decoronated 1 mm below the cementoenamel junction using a slow-speed diamond disc. The decoronated samples were stored in a 0. 1% thymol solution until the study was initiated. Custom plastic cylindrical molds were prepared and filled with chemical-cured resin. Each sample was placed in the resin mold with the buccal surface facing upward, parallel to the horizontal plane. In the middle of the sample, a 5 mm × 5 mm enamel window was created using adhesive tape. The window was made acid-resistant by applying nail varnish around it. After the samples were dried, the adhesive tape was removed using an explorer, exhibiting a rectangular area on the enamel surface. The surface microhardness and calcium phosphorous ratio were recorded using a Vickers microhardness tester and EDAX analysis, respectively, at three levels: baseline, after demineralization, and after remineralization. All the samples were divided into five groups (I, II, III, IV, and V) of 20 samples, which were further divided into two subgroups (a and b) of 10 samples. All the samples in subgroup a were subjected to Vickers hardness testing, and the samples in subgroup b were subjected to EDAX analysis. The demineralizing solution was prepared using 2. 2 mM potassium phosphate, 2. 2 mM calcium chloride, and 0. 05 M acetic acid, and the pH was maintained at 4. 4 with 1 M sodium hydroxide. The remineralizing solution was prepared using 0. 9 mM sodium phosphate, 1. 5 mM calcium chloride, and 0. 15 M potassium chloride, and the pH was maintained at 7. 0. Both demin/remin solutions were prepared in the department of biochemistry at Sree Mookambika Institute of Dental Sciences, Kulasekharam. All the samples were individually immersed in the demineralizing solution (20 ml) for 96 hours to produce artificial carious lesions in the enamel. A Vickers surface hardness test and an EDAX assessment were done for all the samples in each subgroup to record the values of artificially produced demineralized lesions. Then, 20 μl of chitosan (Sigma Aldrich, Bangalore) nanoparticle solution was applied on dried enamel surfaces using a cotton applicator twice daily for one minute. All the chitosan pretreated samples were subjected to four different remineralizing agents as mentioned in Table 1. Table 1 Treatment of samples with remineralizing agents Groups Remineralizing Agents Group I Artificial saliva (Apexion, Kozhikode) Group II Clinpro 5000 dentifrice (3M ESPE, Canada) Group III GC Tooth Mousse dentifrice (Group Pharmaceuticals, India) Group IV SHY NM dentifrice (Group Pharmaceuticals, India) Group V Aclaim dentifrice (Group Pharmaceuticals, India) A pH cycling model was created to mimic the changes in the oral environment. The remineralizing pastes were applied using applicator tips for two minutes, and samples were washed with deionized water. The samples were then individually immersed in 20 ml of demineralizing solution (pH 4. 4) for three hours and washed with deionized water. Then, the samples were treated again with the respective remineralizing agents for two minutes, which were then washed off with deionized water. All the enamel samples were then individually immersed in 20 ml of remineralizing solution (pH 7) for 17 hours. The pH cycling was carried out for 30 days. The remineralizing and demineralizing solutions were replaced every 48 hours and 5 days, respectively. All samples in each subgroup were individually subjected to Vickers microhardness and EDAX surface analyses to record the values of remineralization. The values were obtained at each of the three stages of the study: baseline, after demineralization, and after remineralization. Results Data were collected and statistical analysis was done using one-way ANOVA (post hoc) followed by Dunnett's t-test and tabulated. Table 2 denotes the mean load values of Vickers Hardness of different groups at baseline, after demineralization, and after one month of remineralization. Table 2 Vickers Hardness of different groups at baseline, after demineralization, and after one month of remineralization Groups Baseline (Mean±SD) Demineralization (Mean±SD) After one month (Mean±SD) Group I 239. 98±69. 43 196. 31±41. 38 205. 42±39. 84 Group II 251. 59±35. 24 235. 60±15. 63 246. 29±10. 78 Group III 259. 43±31. 05 219. 39±15. 98 225. 08±12. 42 Group IV 248. 91±18. 34 209. 25±46. 19 216. 73±44. 35 Group V 240. 53±49. 72 228. 64±29. 19 233. 61±18. 63 Table 3 denotes multiple comparisons of mean load values at baseline between the groups. Table 3 Intergroup comparison of baseline values Groups Baseline (Mean±SD) Comparison p-value Group I 239. 98±69. 43 I with II, III, IV, V 0. 78 Group II 251. 59±35. 24 II with I, II, III, IV, V 0. 31 Group III 259. 43±31. 05 III with I, II, IV, V 0. 42 Group IV 248. 91±18. 34 IV with I, II, III, V 0. 89 Group V 240. 53±49. 72 V with I, II, III, IV 0. 45 Table 4 denotes multiple comparisons of mean load values after demineralization between the groups. Table 4 Intergroup comparison of demineralization values Groups Demineralization (Mean±SD) Comparison p-value Group I 196. 31±41. 38 I with II, III, IV, V 0. 02 Group II 235. 60±15. 63 II with I, II, III, IV, V 0. 03 Group III 219. 39±15. 98 III with I, II, IV, V 0. 04 Group IV 209. 25±46. 19 IV with I, II, III, V 0. 02 Group V 228. 64±29. 19 V with I, II, III, IV 0. 04 Table 5 denotes multiple comparisons of mean load values after one month of remineralization between the groups. Table 5 Intergroup comparison of remineralization values Groups After one month (Mean±SD) Comparison p-value Group I 205. 42±39. 84 I with II, III, IV, V 0. 03 Group II 246. 29±10. 78 II with I, II, III, IV, V 0. 04 Group III 225. 08±12. 42 III with I, II, IV, V 0. 03 Group IV 216. 73±44. 35 IV with I, II, III, V 0. 04 Group V 233. 61±18. 63 V with I, II, III, IV 0. 04 Table 6 denotes mean load values of the calcium phosphorus ratio of different groups at baseline, after demineralization, and after one month of remineralization. Table 6 Intergroup comparison of calcium phosphorus ratio at baseline, after demineralization, and after remineralization Results obtained were statistically significant with p<0. 05 Groups Baseline (Mean±SD) After demineralization (Mean±SD) After remineralization (Mean±SD) Group I 2. 73±0. 15 2. 18±0. 06 2. 30±0. 15 Group II 2. 60±0. 14 2. 22±0. 16 2. 56±0. 15 Group III 2. 95±0. 14 2. 04±0. 15 2. 44±0. 15 Group IV 2. 93±0. 15 2. 83±0. 14 2. 41±0. 14 Group V 2. 69±0. 17 2. 25±0. 15 2. 51±0. 14 On comparing the values obtained, it was found that demineralized values were found to be lower than the baseline values in all the groups. Also, remineralized values were found to be higher than the demineralized values but always lower than the baseline values in all the groups. According to Vickers testing, Group II showed the maximum surface microhardness after one month of remineralization treatment followed by Group V, Group III, and Group IV and the least values were seen for Group I. According to EDAX analysis, Group II showed the maximum calcium phosphorous ratio after one month of remineralization treatment followed by Group V, Group III, and Group IV, and the least values were seen for Group I. The results obtained were statistically significant for all the groups indicating that the various agents used showed good remineralization properties and also inhibited caries formation. Discussion Dental caries is a prevalent noncommunicable oral disease. Clinically, it manifests as a white spot lesion in the early stage, which can be observed with the naked eye as a white, chalky lesion. Demineralization results from predominant diet variations, poor oral hygiene, or increased microbial activity. Saliva typically increases the buffering capacity, promoting remineralization [ 1 ]. Recently, the concept of a non-invasive approach has become popular. This approach includes detecting and treating the carious areas sooner, relying more on prevention than the traditional method [ 1 ]. Various remineralizing agents promote healing by replacing the minerals lost during demineralization [ 1 - 4 ]. Fluoride was recognized as the main remineralizing agent due to its cariostatic potential. Apart from halting caries progression, it has some limitations, such as skeletal and dental fluorosis [ 4 - 6 ]. Because of these limitations, other non-fluoride agents, such as CPP-ACP, Novamine, self-assembling peptides, and nanohydroxyapatite, were introduced as adjuncts to fluorides. In this study, all samples were pretreated with chitosan nanoparticles for one minute, in accordance with a study done by Zhang et al. [ 6 ], to avoid deep erosion or etching of the tooth surface. This was followed by treatments with different remineralizing agents for two minutes according to the ADA’s brushing time recommendations to obtain effective quantitative and therapeutic values. Chitosan (Sigma Aldrich, Bangalore) is a mucopolysaccharide present in crustaceans’ exoskeletons. It showed an inhibitory effect against streptococcus and enterococcus species with its electrostatic interaction. It functions as a bio-adhesive polymer with a prolonged retention time on the oral mucosa and synergistic antiplaque, antibacterial effect with chlorhexidine and other remineralizing agents [ 6, 8 ]. According to an in vitro study conducted by Ryge et al. [ 7 ], enamel treated with acid-soluble chitosan showed a higher Vickers surface microhardness than untreated samples. Nanoparticles range in size from 1 to 100 nm (1 nm = 10−9 m). Chitosan nanoparticles increase the surface area up to hundreds of times compared to microparticles (10−6 m) and increase the ability to bind to chemical groups promoting reactivity. Fluoride effectively increases the remineralization of early enamel lesions [ 1 ]. It is adsorbed into the enamel surface, resisting acid attacks, depositing minerals resulting in the formation of acid-resistant fluorapatite crystals, and acting as a bioreservoir of Ca, P, and F ions on adjacent porous enamel [ 9 - 12 ]. In this study, Clinpro (3M ESPE, Canada) dentifrice had high remineralization potential compared to other groups, which was in accordance with in-vitro studies conducted by Baysan et al. Clinpro contains sodium fluoride with functionalized tricalcium phosphate (f-TCP) as the main ingredient, which acts as a continuous reservoir of calcium and phosphate ions, providing optimal benefits even when delivered in a neutral pH environment. An important feature of this calcium phosphate system is that it is stable in an aqueous environment and does not affect the fluoride activity added to the dentifrice. f-TCP is a smart ingredient that controls calcium and phosphate delivery by surrounding them with a protective coating, preventing them from early interactions with fluoride until brushed on the tooth [ 13, 14 ]. Nanohydroxyapatite dentifrice is a biomimetic remineralizing paste that contains calcium and phosphate in the hydroxyapatite form as a key ingredient [ 1 ]. The small HAP nanoparticles resemble the features of biological apatite and self-assemble to form enamel-like structures [ 15 ], filling the fine pores to form a homogeneous apatite layer [ 16 ] and maintaining a high concentration of calcium and phosphate ions in the subsurface enamel. In this study, Aclaim (Group Pharmaceuticals, India) dentifrice was used, which showed a better remineralizing effect and synergistic effect with chitosan, in accordance with other studies. Though it possessed improved remineralizing potential, it was found to be less than fluoride dentifrice. In accordance with the study done by Kim et al. , nano-HAP-based dentifrice had comparable remineralizing potential to fluoride dentifrice and can be used as adjuvant to fluoride therapy [ 15 ]. Casein phosphopeptide (CPP) consists of multiphosphoseryl clusters that stabilize calcium phosphate by forming colloidal casein-phosphopeptide amorphous calcium phosphate (ACP) nanocomplexes, preventing the destruction of calcium and phosphate ions [ 1 ]. CPP-ACP nanocomplex can maintain high-concentration gradients of calcium and phosphate ions within the subsurface lesion, resulting in the formation of hydroxyapatite or fluorapatite crystals via crystal growth [ 17 - 20 ]. In this study, GC Tooth Mousse (Group Pharmaceuticals, India) showed superior hardness to Novamine technology but inferior to fluoride and nanohydroxyapatite toothpaste. The peculiar nature of CPP maintains a supersaturated state of calcium and phosphate ions through the reservoir of bound ACP that promotes diffusion into the lesion, thereby promoting remineralization [ 20, 21 ]. Novamine is a bioactive glass widely used in the fields of tissue engineering, bone regeneration, and dentin remineralization; it is also used as a desensitizing agent [ 22 - 24 ]. In this study, SHY-NM (Group Pharmaceuticals, India) contains a calcium sodium phosphosilicate that reacts with saliva, allowing sodium ions to exchange with hydrogen ions, thereby raising the pH to form a new acid-resistant HCA layer over demineralized areas, promoting remineralization [ 1, 24 ]. Chitosan pretreatment also improved remineralization when used with bioactive glass slurry through electrostatic interactions [ 8, 25 ]. In this study, a statistically significant decrease in calcium phosphorous ratio and microhardness values was found. In all groups, the samples had plugs that sealed demineralized pores and were capable of remineralization, but the bioactive glass had more potential to remineralize than CPP-ACP in a few studies. The clinical advantage is its cost-effectiveness and availability in the form of dentifrice, which may increase patient compliance [ 24, 25 ]. As in vitro values are variable, further research should be done to evaluate particular remineralizing agents in resisting demineralization and promoting remineralization. Conclusions All the remineralizing agents used in this study showed significant results in preventing ion loss and promoting remineralization. Vickers and EDAX analysis were highest for chitosan and Clinpro combination followed by Aclaim, GC Tooth Mousse, and SHY NM. Also, remineralized values were more than demineralized but always less than baseline values indicating that non-carious, non-hypoplastic enamel is strongly resistant to any mineral ions. Hence, we can conclude that chitosan can be used in optimal concentration on a daily basis as mouthwash followed by regular brushing with any of these remineralizing dentifrices to prevent caries formation at an early stage.

10. 7759/cureus. 59970

Cureus

The Use of Platelet-Rich Plasma and Stem Cell Injections in Musculoskeletal Injuries

Injuries to the musculoskeletal (MSK) system can have a significant impact on an individual’s activities of daily living, as this multifunctional unit is associated with physical movement. Treatment of MSK injuries often involves corticosteroid injections, supplements, pharmaceutical agents, and/or surgery. While these approaches have been shown to be effective for some patients over both the short and long term, they can be associated with limited relief, adverse effects, and/or decreases in activities of daily living. An unmet need exists to develop and/or implement more effective treatment approaches for MSK injuries. Treatment options being explored include platelet-rich plasma (PRP) and stem cell injections. This review outlines the current state of research evaluating PRP and stem cell injections in the treatment of various MSK injuries. A literature search was conducted using the PubMed database to identify the relevant published articles related to the use of PRP and/or stem cell injections for the treatment of MSK and cartilage injuries. PRP and stem cell injections have been shown to improve an individual’s quality of life (QOL) and are associated with fewer side effects as compared to invasive standards of care in multiple MSK injuries such as plantar fasciitis, Achilles tendinopathy, acute muscle and tendon tears, ligament injuries, chondral and medial collateral ligament (MCL) knee injuries and arthritis, rotator cuff lesions, and avascular femoral necrosis. Specifically, these studies on PRP and stem cell injections suggest that both approaches are associated with a quicker return to activities of daily living while providing longer lasting relief without significant adverse events. The studies reviewed demonstrated PRP and stem cell approaches to be effective and safe for the treatment of certain MSK injuries, but as standardized protocols were not utilized across studies in the discussion of similar injuries, it was therefore difficult to compare their efficacy and safety. As such, further research is warranted to establish standardized research protocols across MSK injury studies to gain further insight into the efficacy, safety, and durability of PRP and stem cell injections.

Introduction and background The musculoskeletal (MSK) system is a dynamic and multifunctional unit of the human body that is composed of bones, cartilage, ligaments, connective tissue, and tendons, which function to support the body and aid in physical movement. As such, injuries to the MSK system, including acute muscle tears, plantar fasciitis, lateral ankle sprain, Achilles tendinopathy, knee injuries, and rotator cuff lesions, can be associated with significant impacts on an individual’s activities of daily living. Current standards of treatment for MSK injuries include the use of pharmaceutical agents, corticosteroid injections, supplements, and/or surgery. While these approaches have been shown to be effective for some patients over both the short and long term, they can be associated with limited relief, adverse effects, lack of regenerative benefit, and/or decreases in the activities of daily living [ 1 ]. Some potential complications include but are not limited to opiate addiction, organ failure induced by nonsteroidal anti-inflammatory drug (NSAIDs), limited long-term benefits of corticosteroid injections, and bleeding/reinjury associated with surgery [ 1 ]. Corticosteroid injections are presently used to treat multiple MSK conditions. Based on current research, steroid injections only remain efficacious for an average of three months and have significant side effects such as hyperpigmentation, infection, and post-steroid syndrome: flushing of the skin [ 2 ]. Research on pharmaceutical agents such as aspirin, acetaminophen, oxycodone, and other pain-modulating substances is significant. For instance, NSAIDs increase the incidence of renal and hepatic toxicities, gastrointestinal bleeds, and cardiovascular problems whereas opioid medications lead to increased incidence of constipation, mental addiction disorder, tolerance, respiratory depression, and death [ 3, 4 ]. These side-effect profiles limit the use of these drugs in clinical practice and thus pose a limitation to the patient population that can be treated, therefore opening an avenue to alternative pain-modulating therapies. More effective approaches such as physical therapy and psychological treatments have been proven to be effective in providing long-term pain relief and improvements in overall quality of life (QOL) but may require longer time commitment [ 1 ]. As such, an unmet need exists to develop and/or implement more immediate treatment approaches for MSK injuries. Treatment options being explored include platelet-rich plasma (PRP) and stem cell injections. This review outlines the current state of research evaluating PRP and stem cell injections in the treatment of various MSK injuries, suggesting that both approaches improve an individual’s QOL, maintain long-term efficacy, and are associated with fewer side effects as compared to standards of care in multiple MSK injuries such as plantar fasciitis, Achilles tendinopathy, acute muscle and tendon tears, ligament injuries, chondral and medial collateral ligament (MCL) knee injuries and arthritis, rotator cuff lesions, and avascular femoral necrosis [ 5 ]. Specifically, PRP’s purported effectiveness is derived from its growth factors such as platelet-derived growth factor (PDGF), transforming growth factor beta (TGFB), and insulin-like growth factors, and ability to increase blood supply and nutrients to the affected area [ 6, 7 ]. The process of the preparation of PRP injections is seen in Figure 1. PRP provides a minimally invasive avenue to restore joint function, mobility, and rebalance the body's homeostatic mechanisms by increasing type II collagen synthesis and vascularity, restoring hyaluronic acid, and reducing inflammation [ 7 ]. PRP also offers a minimally invasive approach to treat injuries while decreasing the potential side effects that current invasive modalities present for patients. Figure 1 Scheme of platelet-rich plasma and stem cell injections For the stem cell injection, the isolated muscle stem cells were taken from the tissue or derived from the differentiation of induced pluripotent stem cells (iPSCs). Figure created using Canva and the data for the creation of the figure was taken from two articles [ 8, 9 ]. MSK: musculoskeletal Meanwhile, stem cells are undifferentiated cells that differentiate into a particular cell lineage with a specific function upon reception of a growth stimulus. In a broad analysis, there are two stem cell types: embryonic stem cells (ESCs) and adult stem cells (Figure 1 ). Since ESCs are challenging to obtain and raise large ethical issues, adult stem cells have become the primary field of study, as these cells are more easily obtainable and exist in nearly all tissues [ 10 ]. Within the adult stem cell category, there are many cellular regulators that decide the fate of the cells, such as extracellular matrix molecules, growth factors, cytokines, and matrix stiffness [ 10 ]. The stem cells considered to be the most applicable are bone marrow-derived mesenchymal stem cells (bmMSCs), as they can be induced in vitro and in vivo and differentiate into multiple cell lineages, depending on which regulators influence them. Invasive and pharmacologic treatments of MSK injuries have failed to provide lasting efficacy with minimal complication risk, demonstrating an unmet need for developing more effective management approaches [ 2 ]. Novel treatment modalities such as PRP and stem cell injections may result in an overall improvement of QOL, last longer than standard treatments, and present fewer adverse events with a potential for regeneration [ 5, 10 ]. This review aims to provide an insight into the current studies using PRP and stem cells in treating MSK-related injuries, as well as discuss the effects that stem cells and PRP injections have on different MSK injuries throughout the body. Review Study design The articles discussed in this paper were gathered, analyzed, and evaluated using previously peer-reviewed published articles on the PubMed database. The keywords used to narrow the search included “Platelet-Rich Plasma AND Cartilage injuries", “Platelet-Rich Plasma AND Musculoskeletal injuries", “Stem Cell AND Cartilage injuries", “Stem Cell AND Musculoskeletal injuries”, “Platelet-Rich Plasma AND Musculoskeletal injuries AND Effectiveness AND Management”, “Stem Cell AND Musculoskeletal injuries AND Effectiveness AND Management”, “Platelet-Rich Plasma AND Cartilage injuries AND Effectiveness AND Management”, and “Stem Cell AND Cartilage injuries AND Effectiveness AND Management”. A total of 3, 657 articles resulted, leading to a thorough review by applying the inclusion and exclusion criteria and removing any duplicates. The articles had to be primary articles, the time of their research within the last eight years, should have been published in English, should include cartilage injuries or MSK injuries using PRP and/or stem cell injections and exclude progenitor cells, platelet-rich fibrin, and any articles not including treatment of MSK and/or cartilage injuries with PRP and/or stem cells. Citation search was also included when conducting the review of PRP and stem cells. Therefore, a total of 25 articles resulted after the exclusion and inclusion criteria were applied. Eleven more articles were located on PubMed when conducting research on the background and discussion of MSK injuries and the current modalities used to treat these injuries other than PRP and stem cells as well as the creation of the figure. The information and clinical trials used in this review were limited to within the past eight years to better account for the newly emerging field of study and to provide the most accurate and up-to-date information. The identification of the articles was done and their eligibility for the review determined utilizing a flow diagram shown in Figure 2. Figure 2 Flow diagram: identification of studies via PubMed and other methods Platelet-rich plasma An unmet need exists in the management of musculoskeletal injuries to identify treatment options that are not only effective but associated with less adverse effects and faster return to activities of daily living and/or return to play. Currently, pharmacological therapies used for the treatment of musculoskeletal injuries have drawbacks and negative side effects including tolerance, addiction, and toxicity [ 1 ]. As such, alternative treatment approaches are much needed. PRP has demonstrated benefits without inducing the harmful side effects of currently available pharmaceutical agents and has been associated with a faster return to activities of daily living and/or playing sports. Specifically, PRP has been postulated to help increase tissue regeneration in a variety of injuries such as acute muscle tears, lumbar intradiscal disorders, plantar fasciitis, lateral ankle sprain, chronic midportion Achilles tendinopathy, acute Achilles tendon rupture, knee injuries, MCL injury, and rotator cuff lesions [ 11 - 20 ]. See Table 1 for platelet-rich-plasma-focused articles. Table 1 Summary points of platelet-rich plasma and stem cell articles PRP: platelet-rich plasma; QOL: quality of life; ROM: range of motion; bmMSCs: bone marrow mesenchymal stem cells; aMSCs: adipose-derived mesenchymal stem cells; MSCs: mesenchymal stem cells; OA: osteoarthritis; ON: osteonecrosis; BMAC-PRP: bone marrow aspirate concentrate-platelet-rich plasma [ 6, 7, 10, 11 - 32 ] Author(s), Year Title Sample Size Results Boesen et al. 2017 [ 6 ] Effect of high-volume injection, platelet-rich plasma, and sham treatment in chronic midportion Achilles tendinopathy 57, male, 18- to 59-year-olds PRP in combination with 12-week eccentric training allowed for a reduction in pain and tendon thickness, decreased intratendinous vascularity, and improved activity level. Zou et al. 2020 [ 7 ] Autologous platelet-rich plasma therapy for refractory pain after low-grade medial collateral ligament injury 52, male and female, 20- to 45-year-olds PRP allowed for a reduction of pain, decreased tenderness and stiffness, complete restoration of function, no edema as well as shortened healing time. Alleviation of pain is possibly due to anti-inflammatory properties of PRP. Li et al. 2015 [ 10 ] A study of autologous stem cell therapy assisted regeneration of cartilage in avascular bone necrosis 15, male and female, 26- to 55-year-olds Increased ROM and flexibility, restoration of femoral head cartilage, and decreased pain perception. However, the use of stem cell therapy is a subject of individual treatment. Rossi et al. 2017 [ 11 ] Does platelet-rich plasma decrease time to return to sports in acute muscle tear? A randomized controlled trial 72, male and female, 18- to 40-year-olds Use of PRP allowed for decreased off-time (faster return to play) and decreased pain severity scores. Recurrence rate was not statistically significant between PRP and control groups. Tuakli-Wosornu et al. 2016 [ 12 ] Lumbar intradiskal platelet-rich plasma injections: a prospective, double-blind, randomized controlled study 43, female, 45- to 65-year-olds PRP group showed improvement in function, pain, patient satisfaction, and duration of relief. No complications were reported post-injection. Peerbooms et al. 2019 [ 13 ] Positive effect of platelet-rich plasma on pain in plantar fasciitis: a double-blind multicenter randomized controlled trial 82, female, 40- to 60-year-olds PRP had a decrease in pain and disability scores. The PRP group continued to improve whereas the corticosteroid group initially improved and later declined. Blanco-Rivera et al. 2021 [ 14 ] Treatment of lateral ankle sprain with platelet-rich plasma: a randomized clinical study 21, female, 18 to 60-year-olds PRP injection allowed for faster recovery time, less pain, and better functionality. Kearney et al. 2021 [ 15 ] Effect of platelet-rich plasma injection versus sham injection on tendon dysfunction in patients with chronic midportion Achilles tendinopathy: a randomized clinical trial 221, male and female, 40- to 60-year-olds A single PRP injection did not yield a statistically significant difference compared to the sham group of dry needling. Swelling was a side effect but did not last long. Boesen et al. 2020 [ 16 ] Effect of platelet-rich plasma on nonsurgically treated acute Achilles tendon ruptures: a randomized, double-blinded prospective study 40, male, 18- to 60-year-olds Treatment with multiple PRP injections had no significant variation in tendon length, healing, or improved function between control and experimental groups. Therefore, no improvement in functional or clinical outcomes was seen, but different treatment regimens have yielded more successful outcomes. Alsousou et al. 2019 [ 17 ] Platelet-rich plasma injection for adults with acute Achilles tendon rupture: the PATH-2 RCT 216, male and female, 35- to 55-year-olds PRP showed no significant variation between the control and experimental groups. Danieli et al. 2021 [ 18 ] Leucocyte-poor-platelet-rich plasma intraoperative injection in chondral knee injuries improved patient outcomes: a prospective randomized trial 64, male and female, 18- to 50-year-olds Injection of PRP increased joint homeostasis and quality of synovial fluid as well as decreased synovial tissue inflammation. PRP can also slow down joint tissue degeneration. Sari et al. 2020 [ 19 ] Comparison of ultrasound-guided platelet-rich plasma, prolotherapy, and corticosteroid injections in rotator cuff lesions 120, male and female, 18- to 75-year-olds A single dose of PRP showed a significant decrease in pain score post 24 weeks and long-term improvement in rotator cuff lesions. Randelli et al. 2022 [ 20 ] Platelet-rich plasma in arthroscopic rotator cuff repair: clinical and radiological results of a prospective randomized controlled trial study at 10-year follow-up 53 male and female, 54 to 67-year-olds The PRP group reported less pain after two years; however, the difference between the PRP and control groups became negligible after 10 years. Alsousou et al. 2017 [ 21 ] Platelet-rich plasma in Achilles tendon healing 2 (PATH-2) trial: protocol for a multicenter, participant and assessor-blinded, parallel-group randomized clinical trial comparing platelet-rich plasma (PRP) injection versus placebo injection for Achilles tendon rupture 230, unspecified gender, 18-year-olds PRP showed no significant variation between the control and experimental groups. This paper was the protocol for the study with the results reported in 2019, see Ref. [ 17 ]. Hamid et al. 2014 [ 22 ] Platelet-rich plasma injections for the treatment of hamstring injuries: a randomized controlled trial 24, male and female, 17- to 49-year-olds Patients with PRP injection along with the rehabilitation program recovered quicker within a short-term interval and reduced pain. Ebert et al. 2017 [ 23 ] A midterm evaluation of postoperative platelet-rich plasma injections on arthroscopic supraspinatus repair: a randomized controlled trial 55, male, 29- to 74-year-olds Although no differences were observed between the PRP and control groups, it was noted that PRP did assist with improving pain-free abduction strength. Hurd et al. 2020 [ 24 ] Safety and efficacy of treating symptomatic, partial-thickness rotator cuff tears with fresh, uncultured, unmodified, autologous adipose-derived regenerative cells (UA-ADRCs) isolated at the point of care: a prospective, randomized, controlled first-in-human pilot study 15, male and female, 30- to 75-year-olds The use of stem cells was safe without adverse side effects and improved the function of the shoulder. Wyles et al. 2015 [ 25 ] Adipose-derived mesenchymal stem cells are phenotypically superior for regeneration in the setting of osteonecrosis of the femoral head 15, male and female, 28- to 58-year-olds aMSCs provided increased proliferation and bone differentiation efficiency compared to bmMSC in patients with osteonecrosis. Alentorn-Geli et al. 2019 [ 26 ] Effects of autologous adipose-derived regenerative stem cells administered at the time of anterior cruciate ligament reconstruction on knee function and graft healing 39, unspecified gender, 18- to 40-year-olds aMSCs allowed for improvement in pain, knee function, activity level, and graft maturation; however, it was not statistically significant compared to the group without aMSC injection post reconstruction. Kim et al. 2020 [ 27 ] Implantation of mesenchymal stem cells in combination with allogenic cartilage improves cartilage regeneration and clinical outcomes in patients with concomitant high tibial osteotomy 70, male and female, 42- to 68-year-olds Improvement in tibial/femoral axis deformity and in osteoarthritis grading. aMSC therapy provides symptomatic relief in cartilage-based repairs. Hernigou et al. 2018 [ 28 ] Subchondral stem cell therapy versus contralateral total knee arthroplasty for osteoarthritis following secondary osteonecrosis of the knee 30, male and female, 18- to 41-year-olds Subchondral bone marrow injection was more effective compared to total knee arthroplasty for the treatment of OA in patients with ON due to corticosteroid use. Toan et al. 2020 [ 29 ] The effectiveness of knee osteoarthritis treatment by arthroscopic microfracture technique in combination with autologous bone marrow stem cell transplantation 46, male and female, 46- to 69-year-olds Usage of autologous bone marrow stem cells was safe and effective in the treatment of OA due to a decrease in pain and improvement of knee function. Rodas et al. 2021 [ 30 ] Effect of autologous expanded bone marrow mesenchymal stem cells or leukocyte-poor platelet-rich plasma in chronic patellar tendinopathy (with gap >3 mm): preliminary outcomes after six months of a double-blind, randomized, prospective study 20, male, 18- to 48-year-olds The use of bmMSC injection along with rehabilitation resulted in a reduction of pain, improved activity, and patellar tendon structure compared to PRP. Kim et al. 2018 [ 31 ] Effects of bone marrow aspirate concentrate and platelet-rich plasma on patients with partial tear of the rotator cuff tendon 24, male and female, 47- to 66-year-olds BMAC-PRP injection improved shoulder function and pain. No significant difference in the decrease in tear size between the BMAC-PRP and control groups. Kim et al. 2017 [ 32 ] Effect of bone marrow aspirate concentrate-platelet-rich plasma on tendon-derived stem cells and rotator cuff tendon tear 1, female, 58-year-old BMAC and PRP assisted with the healing of tendon tears, increased growth, and migration of tendon-derived stem cells. Acute Muscle Tear with Platelet-Rich Plasma Injection Researchers have demonstrated that PRP can positively affect growth factors to decrease the time of tissue regeneration in a variety of injuries such as tendon rupture, cartilage injury, and ligament sprains. Rossi et al. aimed to study the effects of PRP on return to play after acute muscle injury from recreational and competitive sports in athletes two years post injury [ 11 ]. This study focused on lower extremity muscle strains of hamstrings, gastrocnemius, and quadriceps by measuring four factors: (a) time to return to play from the date of injury to the day the full range of motion was achieved, (b) improvement of strength and functional abilities without pain/stiffness, (c) overall pain severity, and (d) recurrence rate of injuries/strains at two, 12, and 24 months. The researchers found that PRP had a significant impact on shortening the time to return to sports where the control group had a mean time of 25 days versus the PRP group having a mean time of 21. 1 days. The recurrence rate at two years showed no statistical significance at 5. 7% and 10% for the PRP group and control group, respectively [ 11 ]. This study was limited by the size of the study population and lack of statistically significant difference in visual analog score (VAS) scores and recurrence rate [ 11 ]. Lumbar Intradiscal with Platelet-Rich Plasma Injection Low back pain is a significant cause of disability among Americans. Due to the intervertebral disc (IVD) being largely avascular, upon injury, healing can be problematic and may contribute to intervertebral disc syndrome (IDS). Given the failure of currently available pharmaceutical and other treatment modalities including surgery, PRP injections have been considered a potential treatment option for these patients, as PRP injections have been associated with increasing the amount of growth factors to promote the body’s natural healing processes [ 12 ]. PRP injections aim to offer a safer and more cost-effective approach as compared to surgery for patients with chronic lower back pain. In a study by Tuakli-Wosornu et al. , two participant groups received either PRP injections (n=29) or a contrast agent (control; n=18) at the midportion of the suspected disc levels and an additional injection at the disc level that presented with pain [ 12 ]. The aim of this study was to measure the improvement in pain (using the numeric rating scale (NRS)), function (using the functional rating index (FRI)), degree of satisfaction (using the North American Spine Society (NASS) Outcome Questionnaire), and side effects including increased pain, bleeding, infections, and neurologic deficits over an eight-week period [ 12 ]. The researchers found that PRP injection into patients with chronic discogenic lower back pain resulted in greater improvement of function, less pain, and better satisfaction after eight weeks compared to the control group. As measured by the internationally validated outcome surveys (FRI, NRS, SF-36, and the Modified NASS Outcomes Questionnaire), pain and functional improvements showed a statistically significant p-value of. 015 on the NRS scale for pain. In addition, these findings were sustained after one year. Further studies need to be performed to compare the control group with the experimental group after one year [ 12 ]. This study suggested that PRP therapy is promising for the long-term treatment of chronic low back pain with minimal side effects and warrants further investigation. Plantar Fasciitis with Platelet-Rich Plasma Injection Peerbooms et al. [ 13 ] utilized PRP injections on patients with chronic plantar fasciitis with the aim of decreasing pain and improving QOL. Two groups of patients were assessed in this study: one group in which patients received an injection of PRP and the other in which patients received an injection of corticosteroid. The Foot Function Index (FFI) pain score and World Health Organization Quality of Life Brief Version (WHOQOL-BREF) score were used at one, three, six, and 12 months post-injection therapy to measure the short-term and long-term effectiveness of these approaches as well as the presence of adverse events. After receipt of a PRP or corticosteroid injection, patients participated in stretching exercises prescribed by a physical therapist for two weeks, followed by strengthening exercises for two weeks [ 13 ]. At one year, PRP demonstrated improvement in patient’s QOL and decreased pain associated with chronic plantar fasciitis when compared to patients receiving corticosteroid injection [ 13 ]. This study suggests that PRP, in combination with directed stretching and strengthening exercises, is more effective than corticosteroid injections at one year due to the steady trend of pain relief, improvement of QOL, and decrease in risks associated with less repeat injections; however, future studies are warranted to determine long-term effects, adverse effects of the PRP injection, and comparison of PRP use alone without associated exercise. Lateral Ankle Sprain with Platelet-Rich Plasma Injection In 2019, a study by Blanco-Rivera et al. [ 14 ] evaluated the use of PRP in combination with short-term immobilization in patients diagnosed with acute lateral ankle sprains compared to a control group that underwent long-term immobilization. In this study, patients with lateral ankle sprains were injected with PRP followed by short-term immobilization for 10 days or only underwent long-term immobilization for three or more weeks. At eight-week follow-up, the PRP treatment group showed significant improvements in the VAS and the American Orthopedic Foot and Ankle Score (AOFAS) compared to the control group that underwent long-term immobilization [ 14 ]. Meanwhile, at 24 weeks follow-up, both groups reported comparable findings, VAS of 0. 1 and 0. 2 for the PRP and control groups, respectively (p-value=0. 493) [ 14 ]. Based on these results, study participants who were administered the PRP injection adjuvant therapy experienced a faster recovery of pain, which enabled them to get back into their daily lives and sports faster than the control group at eight weeks follow-up. While PRP had no greater improvement in long-term (24 weeks) symptoms relative to long-term immobilization alone, utilizing PRP and short-term immobilization provides improved symptom scores (VAS scores) and may be considered an early treatment option. Further studies are warranted due to the small sample size of 21 participants (10 control, 11 treatment). Chronic Midportion Achilles Tendinopathy with Platelet-Rich Plasma Injection Achilles tendinopathy is commonly treated with injections, exercise, orthotics, and electrotherapy [ 15 ]. The use of PRP therapy in Achilles tendinopathy is thought to facilitate the repair of the tendon by increasing growth factors such as PDGF, TGFB, and insulin-like growth factor [ 6 ]. Boesen et al. [ 6 ] sought to evaluate this theory in their study comparing the effect of sham saline injections (control group) to PRP injections with eccentric exercise - provided by a physical therapist - in those with chronic Achilles tendinopathy (three months or greater) and the control group. Both groups were assessed for improvements in pain intensity, activity levels, tendon thickness, and tendon vascularity. The study results showed that multiple injections of PRP followed by a 12-week eccentric training program resulted in a reduction of pain, increased activity level, reduction of tendon thickness, and decreased intratendinous vascularity [ 6 ]. These findings suggest that PRP with exercise results in improved outcomes relative to sham treatments. The findings of this study support the need for further research on the effectiveness of exercise combined with PRP on chronic tendinopathy in comparison to the standard of care due to the limiting factor of being unable to determine the exact concentration of platelets in the PRP injections and lack of exercise protocol details. Therefore, a consistent protocol of platelet concentration and therapeutic exercise protocol will assist with supporting the suggested positive results of PRP injection on chronic tendinopathy. Acute Achilles Tendon Rupture with Platelet-Rich Plasma Injection Boesen et al. focused on the effect of multiple PRP injections to treat nonsurgically treated Achilles tendon rupture (ATR) compared to placebo injections. The purpose of this experiment was to determine the effects of multiple injections since previous studies found no differences in function and clinical outcomes of ATR PRP injections upon a singular dose [ 16 ]. Following at-home exercises for both the PRP and placebo group, pain and function were measured as well as Achilles tendon length (p-value >. 05), calf circumference (p-value <. 001), and ankle dorsiflexion range of motion (p-value <. 001), all of which showed no significant variation between the control and experimental groups [ 16, 17, 21 ]. A standardized protocol of preparation of PRP injections is needed to better compare the results of single versus multiple injections across other similar studies in order to acquire more accurate data. Knee Injuries with Platelet-Rich Plasma Injection As chondral injuries have a decreased rate of healing and regeneration of fibrocartilage relative to hyaline cartilage, these injuries are difficult to treat and can result in decreased activity and early degeneration of the cartilage. The current treatment for chondral knee injuries is surgery, which leaves the cartilage more fragile, weaker, and with no prevention of degeneration due to the replacement of the cartilage with fibrocartilage [ 18 ]. PRP is currently being evaluated as an alternative treatment option. The regenerative properties of PRP including increased type II collagen synthesis, biological homeostatic regulator, hyaluronic acid restoration, reduction in inflammation, and induction of mesenchymal stem cell chondrogenesis can promote the healing process of chondral injuries and decrease pain from injuries such as tendinopathy and osteoarthritis [ 18 ]. Danieli et al. compared chondral knee injuries between two groups: a control group with no PRP injection and only surgery, and a treatment group with both surgery and PRP injection, by measuring improvement in function, joint homeostasis, synovial tissue inflammation, and quality of synovial fluid. The results of this study found that PRP treatment led to an improvement in function after anterior cruciate ligament (ACL) reconstruction and grade III chondral knee injury according to subjective International Knee Documentation Committee (IKDC) form, Knee Injury and Osteoarthritis Outcome Score (KOOS), and Tegner activity forms assessed before surgery and three, six, 12, and 24 months post-surgery [ 18 ]. PRP has the potential to improve joint homeostasis, decrease synovial tissue inflammation, and increase the quality of synovial fluid compared to surgery alone [ 18 ]. This is one of the few studies that suggest an improvement in the quality of synovial fluid with the use of PRP, but small participant size, lack of postoperative imaging, use of a single injection, and emphasis on small-medium size tears limit the study's generalizability. Acute Hamstring Injury with Platelet-Rich Plasma Injection Current treatment options most commonly used for acute hamstring injury include rest, ice, compression, and elevate (RICE), anti-inflammatory medications, rehabilitation programs, and electrotherapeutic treatments [ 22 ]. Hamid et al. evaluated the use of PRP for acute hamstring injury. Specifically, the researchers aimed to determine how a single PRP injection with a rehabilitation program would affect the return to play of athletes post hamstring injury compared to solely using a rehabilitation program. The measurements used were the number of days to return to play, pain severity, and pain intensity scores using Brief Pain Inventory - Short Form (BPI-SF), which also assisted with determining when the patient could return to the sport [ 22 ]. Patients also underwent an isokinetic strength assessment to determine hamstring strength. Patients with a single PRP injection along with a rehabilitation program recovered quicker than patients undergoing rehab alone without the use of PRP [ 22 ]. Hamid et al. focused on the short-term effects of PRP use in combination with a rehabilitation program, and whether the authors' findings remain true in the long term is an area for future study. Medial Collateral Ligament Injury with Platelet-Rich Plasma Injection One of the most common knee ligament injuries, MCL injury, has been shown to benefit from PRP. In one study, patients received three serial injections of PRP followed by a range of motion and progressive resistive weight-bearing exercises. The participants underwent follow-up at one, three, and six months, with comparisons made to their starting baseline IKDC reports [ 7 ]. The results suggest the use of PRP improved pain, stiffness, tenderness, gait stability, and walking with weight without pain as assessed by the IKDCb (p= 0. 00). These findings are noteworthy because it shows that intra-articular injection of PRP for intractable pain after low-grade MCL injury was able to improve subjective reports of function up to six months post-treatment [ 7 ]. It is important to note that because this study did not have a control group, direct claims about the efficacy of PRP therapy is not possible, and the results warrant further investigation. Rotator Cuff Lesions with Platelet-Rich Plasma Injection Rotator cuff repairs usually involve surgical treatment, which may be associated with retears due to incomplete healing or from reinjuries. In one study, Sari and Eroglu compared three types of injections, corticosteroids, PRP, and prolotherapy, to a control of lidocaine, which were all injected into rotator cuff lesions [ 19 ]. Due to the varying methods of each type of injection, the authors could not arrive at a consensus about which injection was the most successful in healing these lesions. All four injections - corticosteroids, PRP, prolotherapy, and lidocaine - proved to be beneficial treatments according to the VAS, American Shoulder and Elbow Surgeons (ASES), and Western Ontario Rotator Cuff Index (WORC) scores [ 19 ]. Further studies are needed that involve a larger study sample and multiple PRP injections as well as a longer follow-up than just 24 weeks. In another study, Ebert et al. conducted the longest follow-up of arthroscopic supraspinatus rotator cuff repair along with two PRP injections at seven and 14 days after surgery [ 23 ]. Using the Oxford Shoulder Score (OSS), Quick Disabilities of the Arm, Shoulder, and Hand (Quick-DASH) questionnaire, and VAS for pain, it was discovered that no significant differences were observed between the PRP as an adjuvant to surgery and the surgery-only group. In conclusion, the PRP injection did aid in attaining pain-free abduction; however, no evidence was found for the PRP injection leading to a robust tendon repair [ 23 ]. In contrast, Hurd et al. found that PRP injection as an adjuvant to arthroscopic surgery of a rotator cuff lesion was beneficial, as it improved outcomes including decreased pain and increased healing [ 24 ]. Further research needs to be conducted on the treatment of rotator cuff lesions with PRP injections as an adjuvant to arthroscopic repair. Arthroscopic Rotator Cuff Repair with Platelet-Rich Plasma Injection Randelli et al. compared PRP injection to a control group of no injection after arthroscopic rotator cuff repair with a 10-year follow-up. It was noted that at the two-year mark, the PRP group reported less pain compared to the control group, but this difference subsided after 10 years [ 20 ]. A lack of a standardized PRP composition protocol makes it difficult to compare studies due to the various methods of PRP preparations. As such, evaluating the concentration of growth factors, cytokines, and other components within the PRP mixture as well as the location and number of the injections could help standardize PRP use in research for comparison to one another. There are limited studies following patients over 10 years, and therefore future research is needed to validate the outcome of PRP injections observed in this study over the long term. Stem cells Musculoskeletal surgery is tied to increased risk of infection, use of anesthesia, graft rejection, medication addiction and tolerance, and increased functional off-time. Researchers and clinical practitioners have been investigating the effectiveness of MSCs as an alternative solution by decreasing immune-mediated host-graft rejection through the utilization of the patient's own human leukocyte antigen (HLA) immunological markers. The application of MSCs looks to utilize a less invasive approach to regenerate new hyaline cartilage and bone by differentiating them into osteogenic and chondrogenic cells. There are different types of stem cells: embryonic and adult stem cells. Embryonic being difficult to harvest because they differentiate into any type of cell whereas adult stem cells are easier to extract but have a more limited differentiation potential [ 10 ]. The most adequate stem cells are MSCs derived from either bone marrow or adipose tissue, which can differentiate into a variety of tissue types [ 10, 25 ]. Stem cells have become a focus of study for MSK injuries due to the less invasive approach with possible less side effects than the current modalities. See Table 1 for a compilation of the stem cell-focused articles. Avascular Necrosis of the Femoral Head with Stem Cell Injection Li et al. focused on the use of MSCs to treat avascular necrosis (AVN) of the femoral head. The current treatment for AVN includes the drilling of holes into the femoral head in order to allow an increase in blood flow to this area of interruption of blood supply or replacement arthroplasty for more severe AVN injuries [ 10 ]. The need for a less invasive approach led to the study of cells as a method of treatment such as the usage of stem cells in early AVN of the femoral head. Stem cells hold the ability to allow for bone regeneration and cartilage repair and therefore become a potential treatment option for AVN. Following bmMSC extraction and injection at a three-month follow-up time point, patients showed improvements in pain, range of motion, flexibility, and restoration of femoral head cartilage compared to pre-surgery [ 10 ]. Utilizing the patient's own bmMSCs to regenerate cartilage avoids the introduction of foreign material, significantly reduces the rate of immune rejection, and optimizes ROM of the joint. This potentially decreases the duration of postoperative care and rehabilitation. Stem cell therapy may offer an alternative approach to treating AVN. Bone Marrow MSCs Versus Adipose-derived MSCs with Stem Cell Injection Wyles et al. suggest mSCs are promising cellular candidates for regenerative orthopedics. The use of bmMSCs has demonstrated limited efficacy due to high physiologic stress to the surrounding tissues, high cell turnover, and loss of potency with age and disease [ 25 ]. Meanwhile, adipose-derived MSCs (aMSCs) may be a more effective approach to regeneration as they are more protective against these factors. In one study comparing bmMSCs and aMSCs for the repair of osteonecrosis of the femoral head, aMSCs had higher levels of proliferation capacity and better bone differentiation efficiency, suggesting a better regenerative therapeutic strategy compared to bmMSCs [ 25 ]. This is one of the few studies that allows a comparison between two lineages of stem cells. Although this study shows that aMSCs hold more potential, future studies are needed to demonstrate long-term efficacy and to elucidate adverse event profiles. Anterior Cruciate Ligament Tear with Stem Cell Injection ACL tears are common knee injuries in soccer and other sports. ACL tears are most commonly treated with arthroscopic-assisted reconstruction, but there is an increase in off-time and imperfect repair [ 26 ]. Stem cells may be a method to circumvent surgical procedures. ACL reconstruction with intraoperative administration of aMSCs was compared to intraoperative treatment alone. Patients who received intraoperative administration of aMSCs after ACL reconstruction had no significant advantage over those patients who received treatment without aMSCs [ 26 ]. It is suggestive that aMSCs do not improve healing or shorten recovery time in patients after ACL reconstruction [ 26 ]. Conflicting results exist in the literature. Wyles et al. [ 25 ] and Kim et al. [ 27 ] suggest that aMSC therapy provides symptomatic relief in some injuries - for example, cartilage-based repairs - to a better extent than others, such as ligament injuries. MSCs and Allogeneic Cartilage in Knee Osteoarthritis with Stem Cell Injection Medial compartmental knee osteoarthritis (OA) is caused by uneven joint load in the knees causing cartilage damage and is a common problem that affects athletes and the general population, as individuals age. Current treatment involves surgical approaches including high tibial osteotomy (HTO), which shifts the contact pressure of the cartilage [ 27 ]. However, long-term effects of this approach are questionable, as the cartilage is not repaired. This technique allows patients to walk without pain, but degeneration of the articular surfaces can occur despite the HTO correction [ 27 ]. Cell-based tissue engineering such as MSCs shows promise as its aim is to repair the cartilage by filling in the hyaline cartilage-like substances that will not deteriorate. In addition, cartilage regeneration is likely associated with more natural motion of the joints and less pain when compared to an artificial joint. The aim of this study was to determine whether MSC implantation or MSC plus allogeneic cartilage (AC) implantation is effective in treating patients with unresolved radiographically diagnosed grade III or IV medial compartmental knee OA with tibial and femoral axis deformity. To monitor long-term and short-term effects, patients were evaluated clinically and radiologically before surgery and after surgery at four weeks, three months, six months, and one year [ 27 ]. Although the mechanism behind the cartilage regeneration is uncertain, it is believed that it is due to the cell-to-cell interaction between MSCs and chondrocytes; the implantation of MSCs with the mega cartilage allows for the regeneration of the cartilage [ 27 ]. These results are possibly due to the synergistic nature of the AC when working with MSCs allowing for improved integration into human cartilage tissues. While the studies proved promising, future studies should contain a control group that has HTO alone, allowing for a better comparison of efficacy between the two groups. Osteoarthritis and Osteonecrosis of the Knee with Stem Cell Injection Although total knee arthroplasty (TKA) is one of the most commonly performed surgeries to treat osteonecrosis (ON) in patients who have OA, new techniques such as bone marrow concentrate (BMC) injections aim to provide an alternatively better and more therapeutic treatment option. By stimulating the proliferation of progenitor cells using BMC, the therapeutic window of efficacy may be increased from a “standard” window of three years to at least a decade, providing an approach that can be beneficial to younger populations [ 28 ]. One study aimed to evaluate patients who underwent bilateral TKA while injecting one side with a subchondral injection of BMC. The study consisted of 30 patients who had secondary bilateral ON of the knees related to corticosteroids. Radiographs and MRI confirmed the collapse and degradation of the cartilage surface with secondary OA [ 28 ]. Mesenchymal stem cells were aspirated from the bone marrow and were centrifuged to collect the concentrate. Knee arthroplasty was performed and then MSCs were injected into the subchondral medial and lateral femorotibial compartments of each knee. Patients were then evaluated clinically between four and six weeks, three months, one year, and then annually for about 12 years. The study showed that TKA and injection with subchondral bone marrow was more effective when compared to TKA alone in treating OA in patients with ON related to corticosteroids [ 28 ]. Although the subjects were young, they tended to have lower activity compared to other patients of the same age due to their condition. In addition, no biopsies were performed on the cartilage, leading to a weaker understanding of its composition. Similar findings were reported by Toan et al. with regards to combining bone marrow stem cells with arthroscopy in order to improve the VAS score KOOS points, which deals with knee function and symptoms [ 29 ]. In this study, a fibrin scaffold was introduced during the arthroscopy to increase growth factors and cytokines via stem cells, which aided in the repair of the knee [ 29 ]. The method of combining bone marrow stem cells with arthroscopy led to an invasive approach that was both safe and effective. Platelet-rich plasma and stem cells PRP and stem cells have become new approaches to treating a variety of musculoskeletal injuries. As these two harvesting procedures, PRP and stem cells, are derived from autogenous grafts, there is a substantially decreased probability of drug-mediated toxicity, tolerance, immunogenic rejection, immunosuppression, and bleeding that are often associated with current modalities such as surgery, thus offering a different avenue of treatment for various musculoskeletal injuries. Although still considered an emerging field, the combination of PRP and stem cell treatment has been used to treat injuries including chronic patellar tendinopathy and rotator cuff tendon tears. See Table 1 for a list of platelet-rich plasma and stem cell-focused articles. MSCs and PRP in Chronic Patellar Tendinopathy In one study, the researchers aimed to determine if PRP or bmMSC injections will help with pain, function, and tendon structure of a patellar tendinopathy along with a rehabilitation program at a six-month time point [ 30 ]. Two groups were evaluated: one group received bmMSC injections and the other group received PRP injections followed by both groups receiving the same supervised rehabilitation program. The results of this study indicated that following PRP or bmMSC injection therapy, there was a reduction of pain and improvement of activity in both groups; however, bmMSC showed greater improvement of tendon structure compared to the PRP [ 30 ]. Based on the results, the authors concluded that bmMSC offers a better therapeutic relief of patellar tendinopathy compared to PRP, leading to a faster recovery time and entry back into daily life. Further analysis and research are needed to determine the long-term benefits of PRP compared with bmMSC. Bone Marrow Aspirate Concentrate-PRP on Partial Tear of the Rotator Cuff Tendon Nonsurgical treatment for rotator cuff tendon tears includes exercise, medication, and corticosteroid injections. These approaches provide stable long-term outcomes, including but not limited to pain and symptomatic relief, increased QOL, and faster return to work. Regenerative bmMSC biological treatment and PRP were compared to a control group treated with physical therapy techniques (i. e. , scapular stretching, stabilization, and strengthening) to assess the improvement of symptoms in those with rotator cuff tears. Over the course of three months, the researchers found that pain and shoulder function improved after the injection of bone marrow aspirate concentrate (BMAC)-PRP compared to the control group [ 31 ]. In this study, the researchers did not perform individual tests on the effect of BMAC versus PRP due to the procedure being invasive and the desire to develop a treatment that demonstrated a maximal therapeutic effect. Tear size failed to improve, as it is dependent on surgical intervention [ 31 ]. From these results, decreased pain can be potentially attributed to the decreased inflammatory mediators as a result of treatment with PRP and BMAC [ 31 ]. Although this study only maximized therapeutic benefits such as pain reduction, PRP and BMAC should be further investigated as a treatment option using a broader range of participants to determine the presence of side effects and the total therapeutic benefit. BMAC-PRP on Tendon-Derived Stem Cells and Rotator Cuff Tendon Tear Kim et al. focused on the effects of combining PRP with BMAC to determine the effects of this combination on rotator cuff tendon repair. The results of this study show that dual therapy with PRP plus BMAC resulted in decreased partial tear size (initial: 30. 2 \begin{document}\pm\end{document} 24. 5 mm 2 to 22. 5 \begin{document}\pm\end{document} 18. 9 mm 2 at 3 months with a p>0. 05) and an increase in growth and migration of tendon-derived stem cells, while preventing aberrant chondrogenic and osteogenic differentiation [ 32 ]. A VAS score of 5. 8 \begin{document}\pm\end{document} 1. 9 in the PRP plus BMAC pretreatment decreased to 2. 8 \begin{document}\pm\end{document} 2. 3 at 3 months after treatment with PRP plus BMAC which demonstrated a reduction in clinical symptoms. It was shown that there is a specific stem cell differentiation induced with treating with a dual therapy increasing the accuracy and target of treatment; thus, dual therapy should be further considered for rotator cuff tendon repair over current arthroscopy and open surgery treatment, which hold the risk of fatal complications such as stroke, heart attack, pneumonia, or blood clots [ 32 ]. The dual therapy approach could offer a more advanced framework for treating the underlying pathology because the PRP provides a growth factor scaffold potentially aiding the regenerative potentials of BMAC [ 32 ]. Due to the limitations in this study of not having a control group to compare with the experimental group, dual therapy should be further considered, studied, and analyzed for long-term benefit and complications in comparison to both arthroscopic and open surgical approaches. Discussion Overall, multiple studies concluded the necessity for future research on the usage and effects of PRP and stem cells in the treatment of MSK-related injuries. Both types of injections allow for an increase in regeneration ability using endogenous material from each patient, a less invasive approach, lower side effects, and potential healing for the various parts of the MSK system. Some of the risks involved in PRP and stem cell injections include immune reactions such as inflammation and infections from the injections. But current studies have found very few side effects and the injections to be relatively safe [ 33 - 35 ]. However, further research is required on the risk factors that PRP and stem cell injections may pose. Another aspect to be considered is the cost of both injections. Both stem cells and PRP injections are not covered by many health insurance providers, leading to their high cost. According to Fuggle et al. , the cost for a single stem cell injection for OA could be on average $5, 156 and for a PRP injection about $714 [ 35 ]. Therefore, other treatment options such as steroid injections are favored due to the lower cost per injection. A common limitation throughout the studies was a lack of standardized protocol with creating the PRP and stem cell injections, which leads to the difficulty of comparing the effectiveness of multiple studies effectiveness between one another. No consistent standardized protocol creates a challenge of determining the effectiveness of each injection for the specific injuries acquired. Due to the varying results in the literature on a range of conditions, a broad generalization that PRP and/or stem cell injections are universally effective is not possible. Therefore, in the future, a standardized protocol for the creation of PRP and stem cell injections is needed in order to better compare the results of each study to the other. Conclusions This review outlined the current state of research evaluating PRP and stem cell injections in the treatment of various MSK injuries. Both approaches have shown the potential to improve an individual’s QOL, maintain long- term efficacy, and are associated with fewer side effects as compared to standards of care in multiple MSK injuries such as plantar fasciitis, Achilles tendinopathy, acute muscle and tendon tears, ligament injuries, chondral and MCL knee injuries and arthritis, rotator cuff lesions, and avascular femoral necrosis. PRP and stem cells can provide an alternative solution to common MSK-related injuries by decreasing the requirement for pain medication, decreasing adverse effects, and complications associated with more invasive procedures. Meanwhile, a limitation of both PRP and stem cell therapies is the lack of a standardized protocol to objectively compare the results of multiple studies together. It is important to note that current research is ongoing and new experiments, clinical studies, and results are continuously evolving to meet the demand for understanding this increasingly complex and popular therapy. As such, further research is still needed in order to explore their efficacy and safety in larger, randomized controlled trials.

10. 7759/cureus. 60201

Cureus

Enhancing Hip Arthroplasty Outcomes: The Multifaceted Advantages, Limitations, and Future Directions of 3D Printing Technology

In the evolving field of orthopedic surgery, the integration of three-dimensional printing (3D printing) has emerged as a transformative technology, particularly in addressing the rising incidence of degenerative joint diseases. The integration of 3D printing technology in hip arthroplasty offers substantial advantages throughout the surgical process. In preoperative planning, 3D models enable meticulous assessments, aiding in accurate implant selection and precise surgical strategies. Intraoperatively, the technology contributes to precise prosthesis design, reducing operation duration, X-ray exposures, and blood loss. Beyond surgery, 3D printing revolutionizes medical equipment production, imaging, and implant design, showcasing benefits such as enhanced osseointegration and reduced stress shielding with titanium cups. Challenges include a higher risk of postoperative infection due to the porous surfaces of 3D-printed implants, technical complexities in the printing process, and the need for skilled manpower. Despite these challenges, the evolving nature of 3D printing technologies underscores the importance of relying on existing orthopedic surgical practices while emphasizing the need for standardized guidelines to fully harness its potential in improving patient care.

Introduction and background In the dynamic landscape of modern medicine and surgery, the evolution of technology has propelled the field into new frontiers of innovation. One advancement is the application of three-dimensional printing (3DP) - a transformative technology with far-reaching implications, particularly in the realm of orthopedic surgery. The integration of 3D printing in medicine has revolutionized surgical approaches, providing unprecedented opportunities for customization and precision [ 1 - 4 ]. The incidence of degenerative joint diseases, including hip osteoarthritis and necrosis of the femoral head, has surged in recent decades, aligning with the aging global population. Total hip arthroplasty procedures stand as effective interventions for end-stage joint degenerative diseases, offering relief from pain and restoration of a functional range of motion [ 2, 5, 6 ]. As the number of primary arthroplasties has increased, complications necessitating revision surgery increase too, such as periprosthetic osteolysis, aseptic loosening, periprosthetic fractures, and periprosthetic infections [ 2, 7, 8 ]. This escalation in demand for joint reconstructions has underscored the critical need for innovative solutions that go beyond the limitations of traditional approaches [ 9 ]. Therefore, the emergence of 3D printing in the orthopedic landscape has been of importance. By leveraging advanced imaging techniques, such as computed tomography (CT) scans, surgeons can now obtain detailed three-dimensional reconstructions of the patient's hip joint [ 10 ]. Its ability to produce patient-specific implants has positioned 3D printing as a key-changer in addressing the unique anatomical variations among individuals [ 1, 11 - 13 ]. Recognizing the additive value of this technology, the present narrative review is conceptualized to explore the advancements, challenges, and future directions of 3D printing integration in the context of hip arthroplasty. Review Advantages related to the surgery The use of 3D models in total hip replacement surgeries provides several significant advantages. The use of 3D printing in orthopedic surgeries proved to be more beneficial preoperatively, intraoperatively, and even postoperatively when compared to surgeries done without the aid of 3D printing techniques. Preoperative 3D models serve as invaluable tools for preoperative planning, allowing surgeons to deeply analyze intricate cases. This detailed approach to planning allows the surgeon to simulate the fracture reduction process preoperatively, further refining the surgical strategy. Such meticulous preoperative assessments using 3D models not only facilitate accurate implant selection but also optimize the determination of the cup size, position, screw placement, and the need for reaming [ 9, 10, 14 ]. In the realm of complex revision hip arthroplasty, the employment of life-sized 3D models grants surgeons the ability for precise surgical simulations. This results in enhanced accuracy across multiple fronts including cup, augment, and buttress sizing, as well as cage templating and screw trajectory optimization, thereby reducing the risk of intraoperative neurovascular injury [ 10 ]. Furthermore, in the context of surgically applied anatomy, 3D models have proven to be more cost-effective compared to cadavers. These models enable trainers to illustrate the presence of pathology, an advantage not present in cadaveric training [ 15 ]. A study found that using 3D-printed models for preoperative examination of patient bone fractures offered a clearer understanding of the fracture patterns. This was in comparison to solely relying on 2D and 3D reconstructions viewed on a screen. The 3D models accurately represented joint fragmentations and the patterns of the articular surface, which assisted in the processes of reduction and fixation [ 16 ]. The intricate nature of 3D models plays a pivotal role in surgical readiness. It helps surgeons identify and classify bony deficiencies or fractures, presenting a clear advantage over traditional imaging techniques in understanding abnormal bone anatomy [ 1, 9, 17 - 19 ]. In their retrospective cohort study, Aprato and his team contrasted 3D CT scans with traditional CT scans to identify pelvic discontinuity in 56 patients who were receiving revision total hip replacements [ 20 ]. The initial surgical reports indicated pelvic discontinuity (PD) in nine of the 56 patients with type 3 acetabular bone defects. X-ray analysis by the first observer identified PD in 27 cases and the second observer in 21 cases. Standard CT scans showed PD in 25 cases for the first observer and 24 for the second. Using 3D models, both observers agreed on PD in 11 patients. Notably, the nine patients with PD reported in their initial surgical reports were confirmed to have PD in both CT scans and 3D models. Their findings showed that 3D models were more specific in detecting defects in the acetabulum, exhibiting flawless consistency among observers. They proposed that 3D printing technology could lead to more accurate diagnoses and better-informed management decisions compared to standard radiographic methods [ 20 ]. Moreover, Hughes et al. showed that the 3D printing technique enhances the estimation and treatment of complex pelvic deformities with greater precision. Models of actual size contributed to accurate operation simulations, facilitating improved preoperative planning and anatomical understanding. The accuracy and cost-effectiveness of this approach are likely to prove extremely valuable as a tool in clinical practice [ 15 ]. For patients, these models demystify complex acetabular fractures, making the consent process more comprehensible. This enhances the satisfaction of patients and their families [ 1, 2, 14, 21 ]. Childs conducted a comparison between standard human hip models and 3D-printed models during patient consultations for arthroscopic hip surgery treating femoroacetabular impingement (FAI). The study revealed that the use of 3D-printed models led to an improved understanding and retention of information among patients [ 22 ]. Intraoperative The value of 3D models extends beyond surgery preparation. They pinpoint the optimal surgical strategy, which then becomes the foundation of the surgeon's approach [ 21 ]. Such preparedness invariably cuts down on intraoperative decision-making time and ensures that placements are precise [ 23, 24 ]. Mistakes, especially those arising from inexperience or less-than-optimal surgical techniques, are substantially minimized [ 17, 24 ]. In a related study, using a full-size 3D-printed model was found to influence surgeons’ selection of preoperative locking plates, notably aiding less experienced surgeons in assessing complex fractures [ 25 ]. In addition, the success rates of surgeries have shown improvement when 3D-printed devices are utilized in the preoperative phase [ 21, 26 ]. The 3D models also aid in precise prosthesis design, enabling the manufacturing of individualized prosthetic implants [ 14 ]. Their effectiveness has been documented for procedures like periacetabular osteotomies in hip dysplasia and predicting outcomes of scoliosis corrective surgery [ 27 ]. At Mayo Clinic, a bilateral total hip replacement was successfully conducted on a dwarfism patient who was too small for standard implants. The surgeon created a 3D-printed model of the patient's hip, and based on this model, a custom-made implant was manufactured. This tailored implant was then utilized in the joint replacement procedure [ 28 ]. Despite being relatively new to the market with only short-term results and higher costs, custom-made 3D printed implants, particularly acetabular components, are becoming increasingly favored [ 29 ]. A study conducted in Belgium has demonstrated that the aMace 3D-printed implant was significantly cost-effective in the revision arthroplasty of Paprosky-type 3B acetabular defects, compared to custom three-flanged acetabular components [ 30 ]. On the educational front, 3D models improve the learning experience for surgeons, medical students, and physicians alike, elevating skill development in orthopedic surgery [ 14 ]. The use of 3D printing in orthopedics was emphasized in a study that underlined its comprehensive utility in understanding native anatomy through both visual and tactile stimuli. They noted its significant value in the education and training of students, trainees, and surgeons and also in patient education [ 1 ]. The intraoperative advantages of using 3D-printed prostheses and navigation templates are manifold, including reductions in operation duration, X-ray exposures, and minimizing potential damage to the femoral epiphysis [ 17, 21, 24, 26 ]. Furthermore, 3D models contribute to effective reductions in the neck shaft angle, trimming down surgery time, and limiting both intraoperative and postoperative blood losses [ 15, 23, 27 ]. Specific applications, like 3D-printed guide plates for core decompression, have shown better outcomes compared to traditional techniques, particularly in areas like reducing fluoroscopy time and intraoperative blood loss [ 10, 23, 27 ]. This was proved in a study that focused on assessing the effectiveness of 3D printing rapid prototyping (3DP-RP) in aiding percutaneous fixation for treating femoral intertrochanteric fractures using proximal femoral nail anti-rotation (PFNA). The study divided patients into two groups: 19 patients underwent PFNA with the assistance of 3DP-RP following computed tomography scanning, while another 20 patients received conventional PFNA treatment. The findings showed that the 3DP-RP group experienced significant reductions in surgery duration, intraoperative blood loss, and postoperative blood loss compared to the conventional surgery group [ 31 ]. 3D-printed implants, designed to align with a patient's unique anatomy, ensure a more accurate reconstruction of the hip center of rotation. This precise alignment significantly enhances the overall success and efficiency of the surgical process [ 8 ]. Postoperative The success stories of 3D printing in orthopedics are plentiful. From treating periacetabular malignant bone tumors with personalized 3D-printed hemi pelvic prostheses to the use of 3D-printed integrated prostheses for acetabular malignancies, the results are promising [ 23 ]. Postoperative results from surgeries that employed 3D printing show improved Harris Scores after total hip arthroplasty (THA) and reduced postoperative weight-bearing times [ 17, 19, 26 ]. This was evidently shown in a study that utilized 3D printing technology in THA for 74 patients, with a follow-up period of about 24 months. The outcomes, including Harris scores and the time taken for postoperative weight bearing, were better in the group that received 3D printing treatment compared to the conventional group [ 26 ]. One of the most notable advantages of 3D printing is its affordability, accessibility, and potential for distributed manufacturing, reasons for its wide-scale adoption in surgical settings [ 32 ]. Printing techniques Several 3D printing techniques are being employed in orthopedic surgery. Six main techniques have emerged as commonly utilized in this domain. Differential naming has emerged for the different techniques. For clarity, we will be using the American Society for Testing and Materials/ International Organization for Standardization (ISO/ASTM) standards for reference [ 33 ]. These include material extrusion (fused filament fabrication and direct ink writing), vat photopolymerization (stereolithography), powder bed fusion (selective laser sintering), binder jetting (particle binding), and material jetting (inkjet printing) [ 34 ]. Fused Filament Fabrication This falls under material extrusion and entails feeding a filament of the desired material (polymers or polymer/ceramic composite materials), melting it in a vessel through heat, and extruding it from the nozzle layer by layer to form a scaffold [ 35 ]. Vat Photopolymerization This utilizes a single-beam laser to polymerize or crosslink a photosensitive polymer, creating thin layers that are then stacked layer by layer to construct the final structure. This method presents an alternative to material extrusion. It offers precise control over the fabrication process [ 36 ]. Powder Bed Fusion This utilizes a high-power laser to sinter metal or ceramic powders [ 37 ]. The high-power laser irradiates the powders during the printing process, enabling their fusion into cohesive structures. Binder Jetting This presents an innovative approach to 3D printing. It deviates from the traditional laser-based methods. Particle binding employs a liquid binding solution to fuse particles within each layer, followed by a high-temperature sintering step to solidify the final 3D products [ 38 ]. This method provides an alternative means of achieving structural integrity. It offers versatility in material compatibility. Material Jetting This stands out for its ability to deposit minimal volumes of individual droplets onto a printing surface, aiming to form structures through post-printing solidification [ 39 ]. The precision afforded by inkjet printing makes it suitable for intricate structures and complex geometries. Direct Ink Writing This is classified under extrusion-based 3D printing. It involves the extrusion of viscous materials through nozzles by compressed gas, forming individual lines that solidify onto a build plate in a layer-by-layer fashion [ 40 ]. Fused deposition modeling and direct ink writing are methods that are easy to use and have been employed effectively for tissue engineering applications. Selective laser sintering and particle binding have been used to create devices for hard-tissue engineering applications. Inkjet printing has been used for tissue engineering (bioadhesives, scaffolds, and living cells) and pharmaceutical applications [ 34 ]. As for the disadvantages of these various techniques, they are mostly related to the resolution, the mechanical properties, and cost [ 41 ]. A major disadvantage is the fact that almost all of them require postprocessing [ 41 ]. Some of the main advantages and disadvantages of various 3D printing techniques are listed in Table 1. Table 1 Advantages and disadvantages of 3D printing techniques Technique Advantages Disadvantages Fused filament fabrication Adequate mechanical properties, usually does not require postprocessing, and is widely accessible and relatively inexpensive Lower resolution, needs previous filament fabrication, and needs a very high temperature for thermal degradation Vat photopolymerization Precise control over the fabrication process and has high resolution Possibility of material toxicity and relatively costly Powder bed fusion High-power laser that offers good control over internal microstructures and high resolution Requires a suitable particle size and relatively costly Binder and material jetting Fast and precise production, low temperatures are required, and relatively inexpensive Needs a suitable ink viscosity and not very adequate mechanical properties Direct ink writing High drug loading efficiency, low temperatures are required, and relatively inexpensive Lower resolution and high risk of nozzle clogging The diverse array of 3D printing techniques provides orthopedic surgeons with a range of options, each offering unique advantages. The choice of technique should align with the specific requirements of the intended application, considering factors such as material compatibility, structural integrity, and printing precision. Figure 1 depicts a flow diagram for the steps of 3D printing of a model. Figure 1 Steps of 3D printing of a model. Author credit: Wael Barakeh This image was developed by the author and not taken from anywhere else. Advantages beyond the surgery Prosthesis Materials and Design The materials and design of hip prostheses have been rapidly developing. There is a continuous strive to investigate novel materials and enhance technological modalities in implant manufacturing [ 42 ]. This includes various materials, such as metals, ceramics, polymers, composites, alloys, and hybrids. This review focuses on 3D-printed metal-based hip prosthetics. Currently used materials in hip implants, such as titanium-based alloys, cobalt-chrome alloys, and 316L stainless steel, are stiffer than bone. When a metal implant is inserted into the femur, a significant portion of the physiological load is transferred away from the more compliant surrounding bone. This altered load transmission induces underloading of the implanted femur, leading to bone remodeling. This is a process where living tissue undergoes resorption and loss of mass due to mechanotransduction sensitivity [ 43 ]. Yuan et al. suggested that increasing scaffold porosity improves biocompatibility. However, there may be a reduction in mechanical properties, such as yield strength and Young's modulus. Young's modulus is a material property that signifies its susceptibility to stretching and deformation. Porosity is viewed as inversely proportional to these properties and may result in wall thickness thinning [ 44 ]. The promise of 3D printing in orthopedic surgery extends to the development of fully porous femoral stems, with the potential to reduce stress shielding following THA and prolong implant longevity. Hip prostheses typically consist of two main components: the femoral component or stem and the acetabular component or Socket. Arabnejad et al. introduced a unique femoral stem design characterized by "tunable" mechanical properties, specifically in the form of a short stem taper-wedge design [ 45 ]. This innovative approach enables the microstructure of the implant to be fine-tuned to match the individual bone properties of the patient. The process involves a multi-level computerized program to adjust the stiffness of the implant in relation to the bone. The resultant specifications are then translated into a lattice mold, which is utilized in the creation of the implant through a technique referred to as "selective laser melting. " This intricate procedure optimizes the 3D-printed implant to minimize resorption [ 1, 45 ]. The study by Arabnejad et al. indicates that such a stem has the potential to reduce bone loss due to stress shielding by 75% [ 45 ]. These findings suggest that this innovative femoral stem design could become a crucial addition to the toolkit of orthopedic surgeons in future hip arthroplasties [ 1 ]. In addition, the ability to create customizable textured surfaces and regional stiffness can minimize irritation to overlying soft tissues, reduce stress concentration, and mitigate stress shielding [ 46 ]. A systematic review by Safavi et al. screened 2, 530 articles and included a total of 46 studies. The review examined the potential of additive manufacturing in reducing stress shielding by incorporating higher porosity levels achievable only through this manufacturing method [ 47 ]. Three porous design strategies were identified: uniform, graded, and optimized designs. Uniform porosity involves employing a single-unit cell design repeated throughout all porous areas of the implant. Functionally graded designs refer to the systematic incorporation of defined gradients, utilizing a specified number of distinct unit cell designs distributed rationally. These distributions are based on assumptions derived from analysis and literature. Optimized porosity involves utilizing optimization algorithms to achieve the most suitable design specification. Optimized designs based on patient-specific data were found to be the most promising [ 47 ]. Titanium Cups 3D-printed titanium cups offer distinct advantages in orthopedic applications. These cups feature a porous and rough surface, theoretically promoting local vascularization and osseointegration while mitigating stress shielding due to their low elastic modulus [ 48 ]. Unlike other highly porous titanium cups produced through conventional methods, 3D-printed cups exhibit larger pore sizes and higher porosity, thereby replicating a trabecular bone-like elastic modulus. This uniqueness is achieved in monoblock implants without the need for additional coatings [ 49 ]. Consequently, 3D-printed cups can offer distinct biological advantages, including enhanced osseointegration, reduced stress shielding, and unique failure modalities, such as cracking and ion release [ 49 ]. Trabecular titanium, a specialized material used in orthopedic applications, exhibits a structure that closely resembles the trabecular bone found in the human body. This unique material can only be reproduced using high-quality additive manufacturing technology [ 11 ]. 3D printing, which is one of the many additive manufacturing technologies, is particularly suited for custom-made implants, complex geometries, and diverse surfaces, even in serial production. By contrast, conventional manufacturing techniques are primarily geared toward mass production and off-the-shelf devices due to manufacturing constraints [ 50 ]. The ability of additive manufacturing to integrate surface porosity within a monoblock implant is key to improving osseointegration and potentially reducing stress shielding by mimicking the stiffness of the periprosthetic bone [ 50 ]. It was shown that 3D-printed cups maintain acceptable micromotion, even surpassing conventional cups with lower wall thickness [ 48 ]. The combination of wall thickness and a highly porous surface contributes to the stability of 3D cups. They allow for peripheral micromotion of the sockets while providing rigidity with thicker walls [ 51 ]. Castagnini et al. in 2019 reported the results of an eight-year comparison evaluating the survival rates and reasons for revisions between trabecular titanium cups and conventional cementless cups in THA [ 52 ]. The study found that trabecular titanium cups exhibited a statistically higher survival rate compared to the control group, which used conventional cementless cups. In addition, there was a statistically lower incidence of cup aseptic loosening observed in the trabecular titanium cup group. These findings suggest that trabecular titanium cups may offer improved long-term performance and a lower risk of cup loosening in hip arthroplasty procedures [ 52 ]. These findings underscore the significant advantages of 3D-printed titanium cups in orthopedic surgery, particularly in enhancing osseointegration, reducing stress shielding, and providing stability with unique design characteristics. Regeneration In addition to 3D-printed customized implants, there is a noteworthy advancement in the design of orthopedic implants. These optimized implants adopt a nonporous microstructure and are constructed using materials with high cell affinity. This innovative approach promotes the proliferation of osteoblasts within the artificial joint rather than solely on the implant surface, leading to a more extensive distribution compared to traditional joint replacements. Consequently, it results in a larger contact area and increased stress resistance [ 53 ]. Enhancing the stress resistance of artificial joints holds the potential to significantly improve their long-term survival rates. This technological advancement represents a promising development in orthopedic surgery that could lead to better outcomes for patients in need of joint replacements [ 2 ]. Infection Control In the management of artificial hip joint prosthesis infections, two-stage revisions are considered the gold standard for infection control. However, these reoperations may impose significant harm on patients due to their invasive nature. Therefore, the development of prosthetic materials with enhanced antibacterial properties can potentially reduce surgical trauma, decrease operation time, and expedite joint function recovery [ 24 ]. Zhu et al. recently employed a combination of 3D printing and antimicrobial nano-modification technology to produce zirconia ceramic implant materials with precise 3D structures and long-term wear resistance [ 54 ]. These hip prostheses precisely matched the affected area, exhibited good biocompatibility, and were sterilizable, offering the promise of improved antibacterial performance. In addition, Karaji et al. utilized electrophoretic deposition to create a silk fibroin protein solution that included calcium phosphate and vancomycin as a coating for porous titanium surfaces manufactured through additive methods [ 55 ]. This innovative approach resulted in implants with excellent antimicrobial properties and the ability to stimulate bone differentiation. Moreover, Kim et al. proposed the utilization of a 3D printed liner made from polylactic acid (PLA) as a potential solution to address the limitations of polymethylmethacrylate (PMMA) in orthopedic surgery [ 56 ]. In a comprehensive mechanical assessment, they established that PLA exhibited superior strength and ductility compared to PMMA. Furthermore, the study demonstrated the capability of PLA to facilitate controlled elution of antibiotics through the incorporation of reservoirs and microchannels [ 56 ]. Notably, the precise regulation of the liner's porosity was identified as a key factor in achieving sustained antibiotic release, a critical consideration in the context of arthroplasty-related infections. The integration of such advancements in implant materials holds the potential to improve patient outcomes by reducing infection-related complications and expediting recovery. Disadvantages and limitations While 3D printing technology has revolutionized many fields in orthopedics, including hip arthroplasty, it is not without its disadvantages. The utilization of 3D printing technology in orthopedics has indeed created new avenues for customized implant designs and enhanced surgical results, notwithstanding some of the limitations that can be inherent or manageable. The risk of infection is present due to the ubiquitous dispersal of microorganisms, and 3D-printed hip implants are not exempt from posing this risk to patients and healthcare professionals. When comparing 3D printing groups to common hip replacement groups, studies have shown that the rates of postoperative infection were higher in 3D printing groups [ 1, 19, 26 ]. This has to do with many underlying causes, primarily the high porosity and the often-rougher finish that 3D implants have compared to their counterparts, where such surfaces offer ideal environments for bacterial growth and proliferation, ultimately leading to biofilm formation, and potentially leading to severe infections [ 6, 57 ]. Periprosthetic hip infections have been estimated to occur between 0. 3% and 1. 7% of the time in the first two years following surgery [ 58 ]. Staphylococcus species are notorious for causing periprosthetic hip infections, and research is still ongoing to study fungal infections in orthopedic implants, namely, those caused by Candida and Aspergillus species [ 59 ]. Combating infections requires guidelines that enforce special sterilization techniques like gamma irradiation and autoclaving, proper preoperative skin preparation, sterile draping, the use of case-dependent antimicrobial prophylaxis, patient education, and other methods to ensure patient safety. 3D-printed hip arthroplasty also carries the burden of some technical challenges, whereby several factors like the printing time, the need for expert manpower, and the high cost that comes with it play a big role. According to Tserovski et al. , the 3D printing process requires advanced computer skills and additional training, and it is a lengthy process, whereby the completion of each model requires around 12 hours [ 17 ]. In fact, the printing and overall preparation time of these models is one of the most notable limitations [ 6, 14, 24, 60 ]. This is heavily attributed to the fact that the construction of the virtual models for the implants cannot be automated and must be handled by multidisciplinary teams that include surgeons, radiologists, and engineers [ 61 ]. After all, every patient has unique anatomical features that may prove challenging for automated algorithms to accurately capture. Another issue that can be considered a limitation is the fact that many insurance companies do not cover the use of additive manufacturing to create anatomical models [ 62, 63 ]. This may have to do with the fact that additional data supporting the need for 3D printing should be established. It has been suggested by Shah et al. that when this additional data support is gathered, insurance companies can then create a dedicated Current Procedural Terminology (CPT) code for 3D printing, thus allowing for a more widespread use of this technology [ 64 ]. As for the manpower, there is a necessity for skilled users, who can either be technicians or surgeons who would have to undergo extensive training in medical modeling software modalities, which adds to the cost of this process [ 27, 32, 65 ], especially since the very nature of medical 3D printing often requires customization, meaning skilled users must be able to convert clinical data into digital models tailored to each patient’s unique requirements. In addition, Kumar et al. argue that implant manufacturers and producers should embed the cleaning prerequisites in the planning stage given the geometric flexibility of the implants [ 66 ]. This proactive approach can help mitigate infection risk and ensure regulatory compliance, thereby enhancing patient safety. The lack of universal standards that guide the designing and manufacturing of 3D-printed models is an additional major limitation that needs to be addressed adequately. Guidelines Given that 3D printing technologies are evolving by the day, they can still improve and further revolutionize orthopedics in general and hip arthroplasty in specific. In the absence of universal guidelines, it becomes imperative that healthcare professionals working with 3D printing in hip arthroplasty must rely on a combination of existing orthopedic surgical guidelines and best practices tailored to the unique aspects of 3D printing technology. Guidelines can target the biocompatibility, mechanical properties, and durability of 3D-printed materials [ 67 ]. For example, the International Organization for Standardization (IOS) issued a set of guidelines for assessing the biocompatibility of medical equipment, including those made via additive manufacturing (ISO-10993) [ 68 ]. This organization has also commented on mandating adherence to Quality Management Systems (ISO-13485) [ 69 ]. Such systems monitor the adherence of medical devices to certain requirements and specifications [ 70 ]. In addition, ASTM International provided us with guidelines pertaining to radiopacity testing of medical devices (ASTM F640-12) [ 71 ], which is very crucial in the case of 3D-printed implants to ensure their proper visibility under medical imaging techniques. In addition, many guidelines have targeted labeling and traceability, post-marketing surveillance, and education and training. Moreover, Alexander et al. raise the issue of the importance of having standard general ISO/ASTM terminology for 3D printing [ 33 ]. Many organizations are working on improving the quality of guidelines pertaining to 3D printing technologies, including the International Electrotechnical Commission (IEC), Joint Technical Committee 1 (JTC1), International Medical Device Regulatory Forum (IMDRF), and other international standards and medical device regulatory organizations like the ISO and ASTM International [ 62 ]. However, the problem remains that 3D printing in orthopedics should have its own guidelines, given its current expansion and outreach, which raise questions about many ethical and legal considerations addressing patient consent, data privacy, and customized implants. Some potential ethical considerations include the sources of cells used in bioprinting (including human embryonic stem cells and xenogeneic cells) and the use of induced pluripotent stem cells (iPSCs) that raises concerns about tumorigenicity and genetic privacy [ 72 ]. In addition, confidentiality and privacy issues may arise due to the digitization of human anatomy for bioprinting purposes. As for legal concerns, the lack of a comprehensive regulatory framework for 3D bioprinting raises concerns regarding liability and quality control. Also, concerns regarding fair distribution and social stratification can arise from the possibility that access to bioprinting technologies could widen already-existing socioeconomic gaps [ 72, 73 ]. Most importantly, international harmonization is paramount when it comes to establishing specific guidelines to ensure consistency and interoperability and to further facilitate the global acceptance and market access for 3D-printed orthopedic devices. We propose the following insights as a starting point for future guidelines: the selection of biocompatible coatings that have demonstrated marked resistance to microbial colonization, the creation of implants with patient-specific textures and geometries to enhance osseointegration and lessen the chance of implant dislocation or loosening, and the employment of wearable sensors and remote monitoring to gather real-time data on patient mobility and implant stability. Given the patient-specific nature and the lack of long-term data for 3D printing technologies, the future development of universal guidelines thus becomes essential to promote standardization and efficiency and improve the quality of care. Conclusions While 3D printing in hip arthroplasty may present challenges, its profound advantages for both patients and surgeons cannot be understated. In the extensive exploration of 3D printing's applications in hip arthroplasty, a myriad of benefits emerges, ranging from meticulous preoperative planning and surgical readiness to enhanced prosthesis design and educational advancements. These advantages highlight why 3D-printed implants should be embraced. Success stories underscore its efficacy in treating complex cases, improving postoperative outcomes, and even contributing to the affordability and accessibility of orthopedic interventions on a global scale. Ultimately, these advancements are not just about innovation in surgical techniques; they are about patient safety and providing better care. Technological advancements, ongoing research, and a commitment to refining processes offer a pathway to overcome current limitations. Advancing orthopedic surgery with 3D printing technology necessitates conducting longitudinal studies to evaluate its efficacy against traditional methods, focusing on recovery, complications, and patient satisfaction. In the absence of universal guidelines, healthcare professionals engaging with 3D printing in hip arthroplasty must combine existing orthopedic surgical principles with tailored best practices. The imperative for future development of universal guidelines underscores the commitment to standardization, efficiency, and the continual improvement of care quality. While 3D printing in hip arthroplasty may present challenges, its profound advantages for both patients and surgeons cannot be understated. As the field embraces this transformative technology, the ongoing pursuit of innovation, refinement, and standardization ensures that 3D printing will play an indispensable role in shaping the future of orthopedic surgery.

10. 7759/cureus. 892

Cureus

Biological Treatment Approaches for Degenerative Disc Disease: A Review of Clinical Trials and Future Directions

Biologic-based treatment strategies for musculoskeletal diseases have gained traction over the past 20 years as alternatives to invasive, costly, and complicated surgical interventions. Spinal degenerative disc disease (DDD) is among the anatomic areas being investigated among this group, notably due to its high incidence and functional debilitation. In this review, we report the literature encompassing the use of biologic-based therapies for DDD. Articles published between January 1995 and November 2015 were reviewed, with a subset meeting the primary and secondary inclusion criteria of clinical trial results that could be sub-classified into bimolecular, cell-based, or gene therapies, as well as studies investigating the utility of allogeneic and tissue-engineered intervertebral discs. Ongoing clinical trials that have not yet published results are also mentioned to present the current state of the field. This exciting area has demonstrated positive and encouraging results across multiple strategies; thus, future bimolecular and regenerative techniques and understanding will likely lead to an increase in the number of human clinical trials assessing these therapies.

Introduction and background The radiographic findings of degenerative disc disease (DDD) can be found in 40% of individuals younger than 30 and in more than 90% of individuals older than 50 years of age [ 1 - 2 ]. While the majority of these imaging findings are part of the normal aging process, a subset of patients will present with symptomatic nerve root compression and chronic back pain ultimately requiring surgical intervention [ 3 - 4 ]. DDD can be treated pharmacologically with opiates, steroids, or non-steroidal anti-inflammatory drugs. Likewise, other conservative measures such as physical therapy and corticosteroid injections are frequently prescribed. However, these measures do not treat the underlying cause of the degenerative process and do not slow the natural progression of the disease. In progressively symptomatic patients not responsive to conservative measures, surgery is indicated. The type of intervention is based on the underlying pathology and symptomatology, ranging from discectomy to placement of an interbody graft for bony fusion. While controversial, reports of reherniation, pseudarthrosis, and adjacent segment disease can lead to recurrent symptoms and reoperations [ 5 - 6 ]. Prosthetic total disc replacement (TDR) devices are now being used in clinical practice as an alternative to fusion; however, multiple studies have shown that TDR devices also alter spine biomechanics significantly enough to lead to adjacent segment degeneration (ASD) [ 6 - 7 ]. Given the potential complications of these surgical interventions, attention to biologic-based therapies for DDD has gained traction. Trials of gene therapy, in addition to cellular- and acellular-based transplantations have been described in degenerative knee and metacarpophalangeal arthritis, with promising results [ 8 - 11 ]. Thus, translation to their spinal counterparts has been an intense area of research. However, the inherent multi-factorial nature of DDD presents a challenge for optimal treatment strategies. Biomechanical, immunologic, environmental, and genetic factors influence DDD, and their complex interactions are not well understood. Biomolecular therapies (e. g. genes and proteins), cell-based therapies, (e. g. stem cells and chondrocytes), and total disc replacement (allogeneic or tissue-engineered) are the broad categories of research in biologics for DDD. Depending on the stage of degeneration, different treatment strategies have been employed with varying degrees of success. In the present review, we present the published and unpublished clinical studies of biological disc repair and discuss future directions in this regenerative field. Review Materials and methods The PubMed, Google Scholar, Embase, ClinicalTrials. gov, and Cochrane Library databases were searched for relevant studies from January 1995 to November 2015. The following keywords were queried in combination with intervertebral disc or degenerative disc disease: gene therapy, cell therapy, molecular therapy, stem cell, mesenchymal stem cell, disc cell, nucleus pulposus cell, disc chondrocyte, disc regeneration, and tissue engineering. After the initial search, 213 studies were identified. These studies’ results were reviewed, duplicates were identified, and only relevant studies were included. The primary inclusion criterion was the presence of clinical results on disc regeneration. The secondary inclusion criterion was the ability to categorize into one of the following categories: biomolecular therapy, cell-based therapy, gene therapy, and tissue engineered intervertebral disc (IVD). These categories were generated based on the literature that is available on in vivo animal studies on biologic therapy for DDD. For each study, we identified the type of study, the questionnaire-based subjective assessment on pain, and radiologic outcome measures. Following this advanced filtering, a total of 24 studies were included for discussion. Pathophysiology of disc degeneration The IVD is composed of the nucleus pulposus (NP) surrounded by the annulus fibrosis (AF), sandwiched between cartilaginous endplates at the junction to the vertebral bodies located above and below the IVD. The NP is mainly composed of proteoglycans and type II collagen, which allows for the retention of water increasing the IVD’s ability to handle axial loading. The surrounding AF is more stiff and is primarily composed of type I collagen. With increasing age, the water content within the IVD decreases and results in the NP being less resilient to mechanical stressors. This progressive decrease in resiliency leads to NP fissures, which can extend into the AF. This marks the initial stages of degenerative destruction of the IVD, endplates, and associated vertebral bodies (Figure 1 ). Figure 1 Schematic for Degenerative Disc Disease and Biologic Therapies Biomolecular therapy for disc regeneration As previously stated, DDD is a multifactorial process including the progressive decline in NP hydration due to loss of proteoglycans and collagen. This decreased hydration results in loss of mechanical tension in the AF collagen fibers and results in abnormal spinal axial loading forces and segmental instability. These minor changes in stress forces on the spine can result in the development of neck or back pain and narrowing of the spinal canal over time. In early stage degeneration, the disc undergoes an imbalance of anabolic and catabolic factors that leads to extracellular matrix (ECM) degradation [ 12 ]. Specifically, the diseased state of decreased anabolism and increased catabolism can be modified by recombinant proteins and genes to regenerate expression of target molecules. The goal would be to facilitate ECM synthesis and promote NP rehydration and nutrition. The following section will review recent studies on biomolecules used to treat disc degeneration (Table 1 ). Table 1 Clinical Trials Using Biologic-based Therapies for Degenerative Disc Disease Primary Researcher Biologic Therapy Study Design Number of Patients Follow-Up (M) Findings Journal Meisel, et al. Autologous Disc Chondrocyte Transplantation (EuroDisc) Multicenter prospective, randomized, controlled, non-blinded study 28 24 Patients who received ADCT had lower pain scores as tabulated by the OPDQ than control. Patients who received ADCT had retention of better hydration of the disc than control, but no change in disc height EuroSpine J 2006, 2008 Orozco, et al. Autologous Bone Marrow Mesenchymal Cell Pilot Study/ Case Series 10 Improvement in pain, disability, and disc hydration Transplantation 2001 Yoshikawa, et al. Autologous Bone Marrow Mesenchymal Cell Pilot Study/Case Series 2 24 Both patients showed improvement in pain? And intensity of T2-weighted MRIs Spine 2010 Haufe SMW, et al. Hematopoietic Stem Cell Pilot Study/Case Series 10 12 No improvement in back pain Stem Cells Dev. 2006 Coric D, et al. Allogeneic Juvenile Chondrocytes (NuQu) Pilot Study/ Case Series 15 12 ODI, NRS SF-36 improvement from baseline with 89% of patients showing some improvement on MRI JNS 2013 Berlemann, et al. Injectable Biomimetic Nucleus Hydrogel Pilot Study/Case Series 14 24 Significant improvement in leg and back pain after micro-discectomy Euro Spine 2009 Ruan, et al. Total Disc Replacement with Allogeneic IVD Pilot Study/Case Series 5 60 The allograft engrafted the disc space without apparent immunoreaction; 4 out 5 implanted disc spaces preserved their range of motions after disc implantation Lancet 2007 Pettine, et al. Injection of Autologous Bone Marrow Concentrate Cells Pilot Study/Case Series 26 12 Improvement in pain scores prominently in patients with higher CFU-F concentrations. Rehydration of the discs observed (n=8) Stem Cells 2015 Recombinant Human Growth/Differentiation Factor-5 Growth/differentiation Factor-5 (GDF-5) is a member of the transforming growth factor-b (TGF-b) superfamily and the bone morphogenetic protein (BMP) subfamily and is known to influence the growth and differentiation of various tissues, including the intervertebral disc. In vitro and in vivo experiments have shown that human recombinant GDF-5 (rhGDF-5) can stimulate gene expression and synthesis of ECM proteins such as type II collagen and aggrecan [ 13 ]. In September 2014, a multicenter, randomized, double-blind, placebo-controlled clinical trial was conducted to evaluate the safety and tolerability of intradiscal rhGDF-5 in subjects with early lumbar disc degeneration [ 14 ]. Twenty-four subjects with persistent low back pain with at least three months of non-surgical therapy at one suspected symptomatic lumbar level (L3/4 to L5/S1) were included in the study. The subjects received a discogram to confirm that they had at least one symptomatic level attributable to DDD. Additionally, they all had an Oswestry Disability Index (ODI) for low back pain of 30 or greater and Visual Analog Scale (VAS) of four or greater. Subjects who had an abnormal neurological exam at baseline, radicular pain due to anatomic nerve root compression, extravasation of contrast during the discogram, or suspected symptomatic facet joints and/or severe facet joint degeneration were all excluded from the trial. The subjects were evaluated through a 12-month period followed by annual telephone contact at 24 and 36 months for subject health status follow-up. The secondary outcome was the preliminary effectiveness of intradiscal rhGDF-5 as compared to placebo at the same time frame. The results of this study have not been published yet. Platelet-rich Plasma Platelet-rich-plasma (PRP) is a fraction of plasma that can be produced by centrifugal separation of whole blood. A platelet contains the vast majority of biologically active molecules required for blood coagulation, such as adhesive proteins, coagulation factors, and protease inhibitors [ 15 ]. In addition to these factors, PRP also carries a number of factors that are known to increase collagen content, accelerate epithelial regeneration, promote angiogenesis, improve wound healing, and stimulate IVD metabolism [ 16 - 18 ]. More specifically, PRP includes growth factors such as TGF-b, platelet-derived growth factor (PDGF), epidermal growth factor (EGF), vascular endothelial growth factor, and insulin-like growth factor-1 (IGF-1). These factors have been shown to enhance cell viability, stimulate ECM metabolism, and stimulate proliferation of IVD cells [ 19 ]. The presence of these factors within PRP lead to the proposal to use it as an intradiscal therapy to stimulate regeneration or at least slow the progression of degeneration within a diseased IVD. A 2012 published abstract described the safety and feasibility of intradiscal PRP injection in reducing low back pain in patients with DDD [ 20 ]. This abstract included 12 individuals with one or more lumbar discs (L3/4 to L5/S1) with >3 months of low back pain without leg pain, degenerative changes on magnetic resonance imaging (MRI), and at least one symptomatic disc confirmed using a standardized provocative discography procedure. The participants were followed for six months with interval radiographic and clinical assessment. The PRP releasate solution, which was isolated from clotted PRP, was injected (2. 0 ml) into the center of the nucleus pulposus under fluoroscopic guidance. At one month, pain scores (as assessed by VAS and Japanese Orthopaedic Association (JOA)) showed a significant decrease and was sustained for 12 months after treatment. However, disc height indices were not significantly changed over the follow-up period (p=0. 12) and the mean T2 hydration assessments did not change significantly after treatment. Further evidence for PRP therapy in DDD was augmented in 2015 by a prospective, randomized controlled study assessing intradiscal PRP injections in discogenic-mediated low back pain. This study demonstrated improvements in pain and function in patients as early as eight weeks post-treatment and was sustained for up to one year [ 21 ]. Forty-seven adults with chronic (≥ 6 months) moderate to severe lumbar discogenic pain, unresponsive to conservative treatment were randomized to receive an intradiscal PRP injection or an intradiscal contrast agent after a provocative discography. Twenty-nine subjects were randomized to the treatment group and eighteen subjects were randomized to the control group. Data on pain, physical function, and overall satisfaction were collected at one week, four weeks, eight weeks, six months, and one year. The study had a 92% follow-up rate at eight-week time points or longer and found statistically significant improvement in participants who received intradiscal PRP injections with regard to pain, function, and overall satisfaction. Although this was a novel study with very promising results, there were a number of limitations in this study. One limitation of the study was the limited follow-up time of only eight weeks for the control group, thus limiting the assessment of how long the effects of the PRP injections are seen. In other words, if the PRP injected groups and control groups had no statistical difference in their pain and functionality scores at one year after the treatment, it could be stated that the PRP injection provided some short-term symptomatic relief but did not provide any longer-term symptomatic relief. Additionally, the participants in the study were not standardized by the degree of disc degeneration. Therefore, some participants had more disc degeneration and larger protrusions than others, which likely increased the variability of the responses. Lastly, there was no radiographic assessment of the degenerated disc. Thus this study did not show that injecting PRP affected the natural progression of the disc degeneration. This would have been critical information for the use of biomolecular therapy for DDD. Cell-based Therapy for Disc Regeneration Biomolecular therapy likely has limited efficacy in discs with higher grades of degeneration, as the number of cells responsive to injected genes and proteins decline with progression degeneration. Cell-based therapy is the optimal treatment strategy in mid-stage degeneration because it directly addresses the decreased number of viable chondrocytes and stem cells within the diseased disc space (Table 2 ). Table 2 Unpublished Clinical Trials Using Biologic-based Therapies for Degenerative Disc Disease PI/ Sponsor Title of Trial Biologic Therapy Study Design Number of Patients Follow-up (M) Status ISTO Technologies, Inc. A Study Comparing the Safety and Effectiveness of Cartilage Cell Injected Into the Lumbar Disc as Compared to a Placebo Allogeneic juvenile chondrocytes (NuQu) in fibrin carrier. Double-blind, Randomized, Phase 2 44 24 Phase II done Mesoblast, Ltd. Safety and Preliminary Efficacy Study of Mesenchymal Precursor Cells (MPCs, Mesoblast) in Subjects With Lumbar Back Pain 6 or 18 million MPCs (Mesoblast) in a hyaluronic acid carrier Double-blind, Randomized, Phase 2 100 36 Phase II done Red de Terapia Celular Treatment of Degenerative Disc Disease With Allogeneic Mesenchymal Stem Cells (MSV) (Disc_allo) 25 millions MSC in 2 ml of saline Double-blind, Randomized, Phase 1, 2 24 12 Ongoing K-Stemcell Co. , Ltd. Autologous Adipose Tissue Derived Mesenchymal Stem Cells Transplantation in Patient With Lumbar Intervertebral Disc Degeneration Autologous Adipose Tissue derived MSCs Non-randomized, Open label 8 6 Ongoing Bioheart, Inc. Adipose Cells for Degenerative Disc Disease Adipose tissue-derived stem cells suspended in platelet-rich plasma Non-randomized, Open label 100 12 Ongoing DePuy Spine Intradiscal rhGDF-5 (BMP14) for Early Stage Lumbar DDD rhGDF-5 Double-blind, Randomized, Phase 1, 2 38 36 Ongoing Mochida J, et al. Intradiscal rhGDF-5 (BMP14) for Early Stage Lumbar DDD Autologous NP cells from fusion, co-cultured with bone marrow MSCs Case Series 10 24 Ongoing Lutz, et al. HSS Lumbar Intradiscal PRP injections Single injection of PRP Double-blind, Randomized Controlled study 72 6 Complete Akeda, et al. Intradiscal Injection of PRP-releasate for the Treatment of Lumbar Disc Degeneration Injection of the soluble releasate isolated from clotted PRP Case-Series 6 6 Complete Autologous Disc Chondrocytes The use of autologous disc cells is an alternative approach to repair damaged or chronically inflamed tissue by addressing multiple propagators of degeneration at once. From 2002 to 2006, the first study of autologous disc chondrocyte transplantation (ADCT) in a large group enrolled patients in a multicenter prospective, randomized, controlled, non-blinded study to compare the safety and efficacy of ADCT. Known as the EuroDISC trial, this study compared ADCT plus discectomy to discectomy alone to evaluate if ADCT mitigated postoperative pain. In this study, 28 participants between the ages of 18 and 60 with a body mass index below 28 and 1-level lumbar canal stenosis requiring surgical intervention were included. Patients with multiple levels of stenosis requiring operative intervention, discs with sclerosis, edema, or modic changes of grade II or III, and/or focal spondylolisthesis were excluded. Twelve patients received percutaneous ADCT 12 weeks following discectomy and 16 patients received only discectomy. The patients were followed for two years and assessed using the Oswestry low back back pain disability questionnaire (OPDQ) as the primary criterion. Secondary criteria included MRI and X-ray evaluation. They found that the cell therapy group had continual improvement in their OPDQ after the initial surgery compared to no improvement in the control group, and this results persisted at the two-year follow-up [ 22 ]. Additionally, the analysis of the fluid content of the IVD as measured by T2 signal intensity on MRI showed 16% more retention of fluid within the nucleus pulposus. However, comparison of the mean IVD heights revealed no difference between the groups. These results were very promising; however, the final results and analysis have not been published yet. This ADCT system is currently manufactured and marketed in Germany but has not been approved by the FDA and thus is not available in the United States. Autologous Mesenchymal Stem Cells Mesenchymal stem cells (MSCs) derived from bone marrow are one of the most well-studied cell types in regenerative medicine due to their accessibility and expandability in ex vivo conditions. Additionally, the anti-inflammatory effects of MSCs have been demonstrated in numerous animal models of injury including myocardial infarction, renal ischemia, reperfusion injury, burn wounds, and osteoarthritis [ 23 - 24 ]. There have been multiple attempts to introduce mesenchymal precursor cells or MSC into the intradisc space to treat chronic low back pain. In 2010, Yoshikawa, et al. analyzed the regenerative ability of autologous MSCs in markedly degenerated IVDs of two patients with chronic low back pain, radiculopathy, and paresthesias [ 25 ]. MSCs isolated from bone marrow aspirate were coupled with collagen sponges and grafted percutaneously to the degenerated IVD following partial laminotomy. Two years after surgery, both patients had significant symptomatic relief as assessed by VAS, and T2-weighted MRIs showed high signal within the treated IVDs indicating high NP hydration without progressive degeneration. Although the study was limited by a very small sample size, it did serve as a proof of principle showing that autologous MSCs may play a role in the treatment of DDD. In a similar regard, Orozco, et al. published a study of 10 patients with chronic back pain diagnosed due to DDD with autologous MSCs injected directly into the NP [ 26 ]. These patients were followed for one year and pre- and post-treatment MRIs were obtained. Both lumbar pain and disability were strongly reduced at three months after MSC transplantation, followed by modest additional improvement at six and 12 months. The short form-36 (SF-36) life quality questionnaire revealed by the end of treatment a significant improvement of the physical component with no change of the mental component. They also found marginal improvement in disc hydration of the treated levels at one year but no significant change in disc height. Despite these results, the major challenge to MSCs therapy is the pain and cost associated with harvesting these cells. Since MSCs are found in adipose tissue, this poses an attractive source for harvesting and subsequent transplant due to the low-risk accessibility, and current trials evaluating their efficacy in intradiscal applications are ongoing [ 27 - 28 ]. Autologous Hematopoietic Stem Cells Hematopoietic stem cells are thought to be useful to treat disc degeneration due to their differentiation and proliferative capacities. However, as applies to most cells, the oxygen-poor environment poses a challenge for biologic therapies in spinal disc disease. In 2006, Haufe, et al. described a study utilizing hyperbaric oxygenation in 10 patients following percutaneous intradiscal injection of autologous hematopoietic precursor stem cells (HSCs) [ 29 ]. Ultimately, none of the 10 patients achieved any improvement of their discogenic pain after one year and eight out of the 10 patients underwent surgical treatment within one year study completion. Although disappointing, the study provided insight into the use of non-mesenchymal lineage cells in DDD. Autologous Bone Marrow Aspirate/Bone Marrow Concentrated Cells Harvesting, culturing, and expanding autologous chondrocytes or MSCs is an expensive process and requires the patient to undergo at least two procedures since the harvested cells must be cultured and expanded in a lab for weeks prior to transplantation (Figure 2 ). Figure 2 Preparation of Bone Marrow-Based Cell Therapy Bone marrow concentrated cells (BMCs) contain multiple stem and progenitor cells, including mesenchymal stem cells (MSCs) and can be autotransplanted at the time of surgery, precluding a second delayed intervention. Pettine, et al. evaluated the use of autologous, nonexpanded BMCs to treat moderate to severe discogenic low back pain [ 30 ]. The study included 26 participants who had 55 mL of bone marrow aspirate collected from the iliac crest and subsequently injected in the intradisc space of a diseased level. Significant improvement in ODI, VAS, and modified Pfirrmann score were reported at three, six, and 12 months posttreatment. Upon closer examination of the bone marrow aspirate, the authors discerned that patients who received >2, 000 colony-forming unit-fibroblasts (CFU-F) per milliliter of bone marrow aspirate had statistically significant improvement in pain scores compared to those patients with <2000 CFU-F/ml. The exact mechanism by which the increased concentration of CFU-F lead to improvement in discogenic back pain remains unclear and is currently the focus of follow-up studies. Allogeneic Mesenchymal Precursor Cells/Mesenchymal Stem Cells Allogeneic stem cells are another method of biologic treatment for DDD being investigated that is attractive due to the low costs of harvesting and preclusion of secondary procedures. Currently, two clinical trials are underway evaluating their effects. Mesoblast, a private cellular medicine company, is conducting a double-blinded, placebo-controlled Phase 2 clinical trial of 100 patients with DDD randomized to received intradisc injection of saline, hyaluronic acid, or allogeneic mesenchymal precursor cells (MPCs) of varying concentrations in a hyaluronic acid [ 31 ]. Similarly, a private group in Spain is conducting Phase 2 clinical trials for allogeneic MSCs in 12 patients compared with controls [ 32 ]. Clinical and radiographic endpoints will be collected and while results have not been published yet, the utility of allogeneic stem cells remain an intense area of investigation. Finally, biologic strategies for annular repair incorporating annulus fibrocytes and MSCs following surgical intervention may aid in slowing the progressive, degenerative nature of disc disease after manipulation (Figure 3 ). These strategies are still being developed in pre-clinical animal models, but promising preliminary data will likely see them transition to human clinical trials in the coming years. Figure 3 Injectable High-Density Collagen Gel Allogeneic Juvenile Disc Chondrocytes Similar to the previous reports of autologous disc chondrocyte transplantation, groups have assessed the efficacy of allogeneic juvenile chondrocytes into degenerated discs. In a prospective cohort study, Coric, et al. demonstrated that NuQu, an injectable percutaneous fibrin-based delivery of juvenile chondrocytes, helps improve low back pain refractory to conservative measures [ 33 ]. Fifteen patients, with a mean age of 40 years, were treated with a single percutaneous delivery of 1-2 mL of NuQu containing 10 7 juvenile chondrocyte cells/ml within a fibrin carrier. Ten of the 13 patients exhibited significant clinical and radiographic improvements, and interestingly eight of nine patients with the posterior annular tears had resolution of significant improvement in the degree of posterior annular tear, suggesting ongoing remodeling of the injured AF. Gene Therapy for DDD Injection of biologic molecules into the disc allows for direct delivery of protein products to address the causes of degenerative disc disease at the source. However, these biological molecules and many cell-based therapies have short half-lives which preclude their longevity following injection. Gene therapy has emerged as a potential solution to this, which comprises of delivery of recombinant genes into cells. The recombinant gene can then incorporate with the DNA of the host cell allowing for long-term expression of either a proanabolic or anticatabolic protein to facilitate healing of the degenerated disc space [ 34 ]. This strategy requires identification of relevant genes that play a role in the disc degeneration cascade and then the ability to deliver those potentially therapeutic cells to the disc space. Though the evidence generated from in vivo animal models has been promising, no clinical trials have been conducted using this technique in humans. Since viral carriers are often used for transfection, serious concerns involving unexpected mutations create challenges for Food and Drug Administration (FDA) approval and patient inclusion. Nonetheless, gene therapy has shown promising results in other neurologic and orthopedic diseases and thus remains an area of interest for biologic treatment strategies in DDD. Allogeneic and Tissue Engineered IVD Transplantation for DDD At present, both in vivo and in vitro experiments using tissue engineered IVD are in their preliminary states. One promising study published by Ruan, et al. included five patients with cervical spondylosis who underwent transplantation of fresh-frozen composite disc allografts following discectomy [ 35 ]. The disc allografts were harvested from 13 previously healthy organ donors aged 20–30 years. Within two hours of cardiac arrest, the cervical spine was removed en-bloc from C3 to T1 under sterile conditions. The patients were then implanted with the harvested allografts and were followed with serial flexion-extension X-rays and MRIs. Of note, these patients did not receive any immunosuppressive agents and were simply monitored with weekly measurements of erythrocyte sedimentation rate, C-reactive protein, and peripheral blood counts to assess for organ rejection. At the five-year follow-up, the motion and stability of the implanted spinal segment was preserved, but only two of the five implanted grafts showed signs of adequate NP hydration on T2-weighted MRI. All five patients reported improvement in symptoms at the five-year follow-up and none encountered immunoreaction. This proof-of-concept study has created a new alternative to biologic treatment of DDD; however, challenges are expected. First the supply of organ donors will need to be established and criteria for which donors are most suitable still need to be clearly defined. Furthermore, while none of these patients encountered immunologic reactions, the vast majority of transplantation recipients require some form of immunosuppression, which increases the risk of opportunistic infections and malignancies in a subset of patients. There have not been any human clinical trials for the implantation of tissue engineered IVD (TE-IVDs). However, there has been one human pilot study for using a biomimetic protein polymer that mimics the NP. Berlemann, et al. used NuCore® injectable nucleus (Spine Wave, Inc. , CT, USA), which is a protein polymer hydrogel that mimics the properties of the natural nucleus to treat post-discectomy patients [ 36 ]. The polymer chain is composed of silk and elastin components designed for both elasticity and toughness. This hydrogel is injected as a fluid through the annular defect as a replacement for nuclear tissue lost to herniation and microdiscectomy. Fourteen patients with single-level herniated discs that were unresponsive to conservative therapy had NuCore® hydrogel injected following microdiscectomy that was allowed to cure/harden over five minutes. Ultimately, the group found significant improvement in leg and back pain scores, and functional scores (as assessed by ODI) following the procedure. Postoperative MRI showed stable position of the implants, and radiographic measurements showed restoration of disc height. This study serves as the first of its kind to use an engineered polymer to replace a native biologic structure following surgical removal. Conclusions The information elucidated from various studies on biologics presents an exciting new area of research for DDD. Adaptations and modifications of similar modalities used in degenerative joint arthritis may one day be applicable to spinal disc disease as an upfront therapy. Future research into the use of viral vector gene therapy, RNA interference, and micro RNAs may provide fruitful alternatives to cell-based and whole IVD-based therapies, but significant challenges need to be addressed prior to translation to human clinical trials.

10. 7860/JCDR/2013/6915. 3638

Journal of Clinical and Diagnostic Research : JCDR

A Natural Meliorate: Revolutionary Tissue Engineering in Endodontics

Platelet-Rich Fibrin (PRF) was first described by Choukroun et al. , (2001). It has been referred to as a second-generation platelet concentrate, which has been shown to have several advantages over traditionally prepared platelet-rich plasma. PRF has a physiologic architecture that is very favourable to the healing process, obtained due to the slow polymerization process. The development of platelet concentrate as bioactive surgical additives that are applied locally to promote wound healing stems from the use of fibrin adhesive. Developments in the field of tissue engineering have made the generation of artificial substitutes in several areas of medicine. Various clinical applications in endodontics include Periapical surgeries, Revascularisationprocedures, Regenerative pulpotomy, Perforation repair. This article aims to discuss the various applications of PRF in the field of Endodontics with few case reports.

No full text available

10. 7860/JCDR/2014/7609. 3937

Journal of Clinical and Diagnostic Research : JCDR

Biomaterials in Tooth Tissue Engineering: A Review

Biomaterials play a crucial role in the field of tissue engineering. They are utilized for fabricating frameworks known as scaffolds, matrices or constructs which are interconnected porous structures that establish a cellular microenvironment required for optimal tissue regeneration. Several natural and synthetic biomaterials have been utilized for fabrication of tissue engineering scaffolds. Amongst different biomaterials, polymers are the most extensively experimented and employed materials. They can be tailored to provide good interconnected porosity, large surface area, adequate mechanical strengths, varying surface characterization and different geometries required for tissue regeneration. A single type of material may however not meet all the requirements. Selection of two or more biomaterials, optimization of their physical, chemical and mechanical properties and advanced fabrication techniques are required to obtain scaffold designs intended for their final application. Current focus is aimed at designing biomaterials such that they will replicate the local extra cellular environment of the native organ and enable cell-cell and cell-scaffold interactions at micro level required for functional tissue regeneration. This article provides an insight into the different biomaterials available and the emerging use of nano engineering principles for the construction of bioactive scaffolds in tooth regeneration.

No full text available

10. 7860/JCDR/2014/8257. 5034

Journal of Clinical and Diagnostic Research : JCDR

Regenerative Endodontics: A Road Less Travelled

Although traditional approaches like root canal therapy and apexification procedures have been successful in treating diseased or infected root canals, but these modalities fail to re-establish healthy pulp tissue in treated teeth. Regeneration-based approaches aims to offer high levels of success by replacing diseased or necrotic pulp tissues with healthy pulp tissue to revitalize teeth. The applications of regenerative approaches in dental clinics have potential to dramatically improve patients’ quality of life. This review article offers a detailed overview of present regenerative endodontic approaches aiming to revitalize teeth and also outlines the problems to be dealt before this emerging field contributes to clinical treatment protocols. It conjointly covers the basic trilogy elements of tissue engineering.

No full text available

10. 7860/JCDR/2015/13907. 6565

Journal of Clinical and Diagnostic Research : JCDR

Biomedical Biopolymers, their Origin and Evolution in Biomedical Sciences: A Systematic Review

Biopolymers provide a plethora of applications in the pharmaceutical and medical applications. A material that can be used for biomedical applications like wound healing, drug delivery and tissue engineering should possess certain properties like biocompatibility, biodegradation to non-toxic products, low antigenicity, high bio-activity, processability to complicated shapes with appropriate porosity, ability to support cell growth and proliferation and appropriate mechanical properties, as well as maintaining mechanical strength. This paper reviews biodegradable biopolymers focusing on their potential in biomedical applications. Biopolymers most commonly used and most abundantly available have been described with focus on the properties relevant to biomedical importance.

No full text available

10. 7860/JCDR/2015/9871. 5636

Journal of Clinical and Diagnostic Research : JCDR

SHED - Basic Structure for Stem Cell Research

The discovery that stem cells from dental pulp are capable of differentiating into endothelial cells raised the exciting possibility that these cells can be a single source of odontoblasts and vascular networks in dental tissue engineering. These so-called mesenchymal stem cell populations have been identified from human exfoliated deciduous teeth because of their ability to generate clonogenic adherent colonies when grown and expanded. In addition to these stem cells, other population of stem cells can be from adult human dental pulp and periodontal ligament. The identification and isolation of these stem cells in adult dental pulp was first reported by Gronthos and co-workers in 2000. These dental pulp stem cells have clonogenic abilities, rapid proliferative rates and the capacity to form mineralized tissues both in vitro and in vivo. The stem cells from human exfoliated deciduous teeth are distinct from dental pulp stem cells by virtue of their proliferation rate, increased cell population doublings and osteoinductive capacity in vivo. It is further demonstrated that human exfoliated deciduous teeth stem cells may not be a single-cell type, may well be a heterogenous population of cells from the pulp.

No full text available

10. 7860/JCDR/2016/16809. 7901

Journal of Clinical and Diagnostic Research : JCDR

Test Tube Tooth: The Next Big Thing

Unlike some vertebrates and fishes, humans do not have the capacity for tooth regeneration after the loss of permanent teeth. Although artificial replacement with removable dentures, fixed prosthesis and implants is possible through advances in the field of prosthetic dentistry, it would be ideal to recreate a third set of natural teeth to replace lost dentition. For many years now, researchers in the field of tissue engineering have been trying to bioengineer dental tissues as well as whole teeth. In order to attain a whole tooth through dental engineering, that has the same or nearly same biological, mechanical and physical properties of a natural tooth, it’s necessary to deal with all the cells and tissues which are concerned with the formation, maintenance and repair of the tooth. In this article we review the steps involved in odontogenesis or organogenesis of a tooth and progress in the bioengineering of a whole tooth.

No full text available

10. 9734/BBJ/2013/4309

British biotechnology journal

Constraining the Pluripotent Fate of Human Embryonic Stem Cells for Tissue Engineering and Cell Therapy – The Turning Point of Cell-Based Regenerative Medicine

To date, the lack of a clinically-suitable source of engraftable human stem/progenitor cells with adequate neurogenic potential has been the major setback in developing safe and effective cell-based therapies for regenerating the damaged or lost CNS structure and circuitry in a wide range of neurological disorders. Similarly, the lack of a clinically-suitable human cardiomyocyte source with adequate myocardium regenerative potential has been the major setback in regenerating the damaged human heart. Given the limited capacity of the CNS and heart for self-repair, there is a large unmet healthcare need to develop stem cell therapies to provide optimal regeneration and reconstruction treatment options to restore normal tissues and function. Derivation of human embryonic stem cells (hESCs) provides a powerful in vitro model system to investigate molecular controls in human embryogenesis as well as an unlimited source to generate the diversity of human somatic cell types for regenerative medicine. However, realizing the developmental and therapeutic potential of hESC derivatives has been hindered by the inefficiency and instability of generating clinically-relevant functional cells from pluripotent cells through conventional uncontrollable and incomplete multi-lineage differentiation. Recent advances and breakthroughs in hESC research have overcome some major obstacles in bringing hESC therapy derivatives towards clinical applications, including establishing defined culture systems for de novo derivation and maintenance of clinical-grade pluripotent hESCs and lineage-specific differentiation of pluripotent hESCs by small molecule induction. Retinoic acid was identified as sufficient to induce the specification of neuroectoderm direct from the pluripotent state of hESCs and trigger a cascade of neuronal lineage-specific progression to human neuronal progenitors and neurons of the developing CNS in high efficiency, purity, and neuronal lineage specificity by promoting nuclear translocation of the neuronal specific transcription factor Nurr-1. Similarly, nicotinamide was rendered sufficient to induce the specification of cardiomesoderm direct from the pluripotent state of hESCs by promoting the expression of the earliest cardiac-specific transcription factor Csx/Nkx2. 5 and triggering progression to cardiac precursors and beating cardiomyocytes with high efficiency. This technology breakthrough enables direct conversion of pluripotent hESCs into a large supply of high purity neuronal cells or heart muscle cells with adequate capacity to regenerate CNS neurons and contractile heart muscles for developing safe and effective stem cell therapies. Transforming pluripotent hESCs into fate-restricted therapy derivatives dramatically increases the clinical efficacy of graft-dependent repair and safety of hESC-derived cellular products. Such milestone advances and medical innovations in hESC research allow generation of a large supply of clinical-grade hESC therapy derivatives targeting for major health problems, bringing cell-based regenerative medicine to a turning point.

1. INTRODUCTION Pluripotent human embryonic stem cells (hESCs) have both the unconstrained capacity for long-term stable undifferentiated growth in culture and the intrinsic potential for differentiation into all somatic cell types in the human body, holding tremendous potential for restoring human tissue and organ function [ 1 – 3 ]. Derivation of hESCs, essentially the in vitro representation of the pluripotent inner cell mass (ICM) or epiblast of the human blastocyst, provides not only a powerful in vitro model system for understanding human embryonic development, but also an unlimited source for in vitro derivation of a large supply of disease-targeted human somatic cells for tissue engineering and cell therapy. There is a large unmet healthcare need to develop hESC-based therapeutic solutions to provide optimal regeneration and reconstruction treatment options for normal tissue and function restoration in many devastating and life-threatening diseases and injuries. However, realizing the developmental and therapeutic potential of hESC derivatives has been hindered by conventional approaches for generating functional cells from pluripotent cells through uncontrollable, incomplete, and inefficient multi-lineage differentiation [ 2, 3 ]. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, which yields embryoid body (EB) consisting of a mixed population of cell types that may reside in three embryonic germ layers and results in inefficient, incomplete, and uncontrollable differentiation that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity [ 1 – 9 ]. Growing evidences indicate that incomplete lineage specification of pluripotent cells via multi-lineage differentiation often resulted in poor performance of such stem cell derivatives and/or tissue-engineering constructs following transplantation [ 2, 3, 10 ]. In order to generate a large supply of uniform functional cells for tissue engineering and cell therapy, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired lineage has been a major challenge for clinical translation. In addition, most currently available hESC lines were derived and maintained on animal feeder cells and proteins, therefore, such hESCs have been contaminated with animal biologics and unsuitable for clinical application [ 2, 3, 11 – 13 ]. Without a practical strategy to convert pluripotent cells direct into a specific lineage, previous studies and profiling of hESC differentiating multi-lineage aggregates have compromised their implications to molecular controls in human embryonic development. Developing novel strategies for well-controlled efficiently directing pluripotent hESCs exclusively and uniformly towards clinically-relevant cell types in a lineage-specific manner is not only crucial for unveiling the molecular and cellular cues that direct human embryogenesis, but also vital to harnessing the power of hESC biology for tissue engineering and cell-based therapies. To date, the lack of a clinically-suitable source of engraftable human stem/progenitor cells with adequate neurogenic potential has been the major setback in developing safe and effective cell-based therapies for regenerating the damaged or lost central nervous system (CNS) structure and circuitry in a wide range of neurological disorders. Similarly, the lack of a clinically-suitable human cardiomyocyte source with adequate myocardium regenerative potential has been the major setback in regenerating the damaged human heart. Given the limited capacity of the CNS and heart for self-repair, transplantation of hESC neuronal and heart cell therapy derivatives holds enormous potential in cell replacement therapy. Clinical applications of hESC therapy derivatives provide the right alternative for many incurable diseases and major health problems that the conventional mode of drugs and treatments cannot, such as heart disease and failure, diabetes, Parkinson’s diseases, ALS, Alzheimer disease, stroke, brain and spinal cord injuries. Each single one of those world-wide major health problems would cost the health care system more than $10 billion annually. Recent advances and breakthroughs in hESC research have overcome some major obstacles in bringing hESC therapy derivatives towards clinical applications, including establishing human stem cell technology platforms for defined culture systems for derivation and maintenance of clinical-grade pluripotent hESCs and lineage-specific differentiation of pluripotent hESCs by small signal molecule induction for direct conversion of pluripotent hESCs into a large supply of high purity neuronal cells or heart muscle cells with adequate capacity to regenerate neurons and contractile heart muscles for developing safe and effective stem cell therapies [ 3, 12 – 21 ]. bFGF, insulin, ascorbic acid, laminin, and activin-A were identified as the minimal essential elements for sustaining pluripotence of hESCs in a defined culture system ( Fig. 1 ), serving as a platform for de novo derivation of therapeutically-suitable pluripotent hESCs that can be directly converted into large supplies of safely engraftable neuronal or cardiac lineage-committed progenies for neural or cardiac repair in the clinical setting [ 3, 12 – 21 ]. Formulation of minimal essential defined conditions for hESCs renders pluripotent hESCs be uniformly converted into a specific neural or cardiac lineage by small signal molecule induction [ 3, 12 – 21 ] ( Figs. 2 – 4 ). Retinoic acid (RA) was identified as sufficient to induce the specification of neuroectoderm direct from the pluripotent state of hESCs and trigger a cascade of neuronal lineage-specific progression to human neuronal progenitors (hESC-I hNuP) and neurons (hESC-I hNu) of the developing CNS in high efficiency, purity, and neuronal lineage specificity by promoting nuclear translocation of the neuronal specific transcription factor Nurr-1 [ 3, 14, 16 – 21 ] ( Figs. 2, 3 ). Similarly, nicotinamide (NAM) was identified sufficient to induce the specification of cardiomesoderm direct from the pluripotent state of hESCs by promoting the expression of the earliest cardiac-specific transcription factor Csx/Nkx2. 5 and triggering progression to cardiac precursors and beating cardiomyocytes with high efficiency [ 3, 12 – 16 ] ( Figs. 2, 4 ). Such milestone advances and medical innovations in hESC research enable generation of a large supply of high purity clinical-grade hESC neuronal and heart cell therapy products for treating neurological and heart diseases and injuries. Currently, these hESC neuronal and cardiomyocyte therapy derivatives are the only available human cell sources with adequate capacity to regenerate neurons and contractile heart muscles, vital for CNS and heart repair in the clinical setting. The availability of human stem/progenitor/precursor cells in high purity and large quantity with adequate neurogenic or cardiogenic potential will greatly facilitate developing safe and effective cell-based regeneration and replacement therapies against CNS and heart disorders. Clinical translation of milestone advances and medical innovations in hESC research provides the only hope to many devastating and life-threatening diseases and injuries. Transforming pluripotent hESCs into fate-restricted therapy derivatives dramatically increases the clinical efficacy of graft-dependent repair and safety of hESC-derived cellular products, bringing cell-based regenerative medicine to a turning point. 2. Defined Platform for Well-Controlled Derivation, Maintenance, and Differentiation of Clinical-grade Pluripotent hESCs The hESCs have the capacity for long-term undifferentiated growth in culture and the theoretical potential for differentiation into all somatic cell types [ 1 ]. Pluripotent hESCs have been shown to generate teratomas in vivo as well as differentiate into many different lineages and cell types in vitro, including neural precursors, glia, neurons, cardiomyocytes, hematopoietic precursors, endodermal and endocrine cells, and skeletal myoblasts by allowing multi-lineage differentiation through aggregate formation in suspension or extended culture [ 22 – 31 ]. However, only a small fraction of those cells progresses to display targeted differentiation characteristics. Although procedures such as immunoselection for specific surface antigens, treating floating aggregates (embryoid body [EB]) with inducing molecules, coculturing with mouse stromal cells, or manipulating cell density or serum concentration appeared to enrich the populations of desired cell types, none has been able to produce a large population of uniform uncontaminated functional progenies from hESCs for therapeutic application [ 2 ]. In addition, the simultaneous emergence of substantial widely divergent uncharacterized cell types that may reside in all three embryonic germ-layers in the aggregates makes directing hESC differentiation en a particular route unpredictable and unreliable, compromising the therapeutic potential of hESCs [ 2 ]. Without a through understanding of the molecular and cellular cues that direct hESC differentiation programs, controlled differentiation of hESCs effectively into functional lineages has proven to be one of the daunting challenges for fulfilling the therapeutic promise of hESCs. Maintaining undifferentiated hESCs in a defined biologics-free culture system that allows faithful expansion and controllable direct differentiation is one of the keys to their therapeutic utility and potential, which requires a better understanding of the minimal essential components necessary for sustaining the pluripotent state and well-being of undifferentiated hESCs [ 2, 3 ]. The hESC lines initially were derived and maintained in co-culture with growth-arrested mouse embryonic fibroblasts (MEFs) [ 1 ]. Using this mouse-support system may compromise the therapeutic potential of these hESCs because of the risk of transmitting xenopathogens, altering genetic background, and promoting the expression of immunogenic proteins [ 3, 32 ]. In addition, the need for foreign biologics for derivation, maintenance, and differentiation of hESCs may make direct use of such cells and their derivatives in patients problematic [ 3 ]. To avoid those shortcomings, several human feeder, feeder-free, recombinant laminin, and artificially-formulated defined culture systems have been developed for derivation and maintenance of hESCs, though the elements for sustaining prolonged stable undifferentiated growth in those culture systems remain unsolved [ 33 – 39 ]. These exogenous feeder cells and molecules help maintain the long-term stable growth of undifferentiated hESCs while mask their ability to respond to differentiation inducing signals and molecules. Without an understanding of the essential developmental components for sustaining hESC pluripotence and self-renewal, such hESC lines are at risk for becoming unhealthy and unstable after prolonged culturing under artificially-formulated chemically-defined conditions [ 3 ]. Achieving effective differentiation of hESCs into a specific lineage, first and at least, requires a better understanding of the elements necessary and sufficient for sustaining the pluripotence of hESCs, a platform from which controlled differentiation can then directly proceed from the earliest developmental stage [ 3 ]. To overcome some of the major obstacles in basic biology and therapeutic application of hESCs, our recent studies have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of animal-free therapeutically-suitable hESCs and well-controlled efficient specification of such pluripotent cells exclusively and uniformly towards a particular lineage by small molecule induction [ 3, 12, 14 ] ( Fig. 1 ). bFGF (at an optimal concentration of 20 ng/ml), insulin (20 µg/ml), ascorbic acid (50 µg/ml), laminin, and activin-A (50 ng/ml) were identified as the minimal essential elements for sustaining pluripotence and self-renewal of clonal hESCs in a defined culture system, serving as a platform for de novo derivation of therapeutically-suitable pluripotent hESCs that can be directly induced by small molecules into large supplies of safely engraftable neuronal or cardiac lineage-committed progenies across the spectrum of developmental stages with adequate CNS or myocardial regenerative potential for neural or cardiovascular repair in the clinical setting [ 3, 12, 14 ]. Establishing defined platform for the long-tern stable maintenance of pluripotent hESCs has overcome some of the major obstacles in translational biology. Good manufacturing practice (GMP) quality, defined by both the European Medicine Agency (EMA) and the Food and Drug Administration (FDA), is a requirement for clinical-grade cells, offering optimal defined quality and safety in cell transplantation [ 11 ]. Resolving minimal essential requirements for the maintenance of pluripotent hESCs allows all poorly-characterized and unspecified biological additives, components, and substrates in the culture system, including those derived from animals, to be removed, substituted, or optimized with defined human alternatives for optimal production GMP-quality xeno-free hESC lines and their therapy derivatives [ 3, 12 ]. Maintaining pluripotent hESCs in a defined culture enables the spontaneous unfolding of early embryogenic processes in vitro that emulate the in vivo maintenance of the pluripotent epiblast [ 3, 12 ]. In early embryogenesis, the epiblast is composed of more progressed pluripotent cells developed from the ICM, serving as the most immediate precursors of the early somatic lineages [ 40 – 44 ]. We found that such defined conditions derived their efficacy from enabling the spontaneous unfolding of inherent early embryogenesis processes in vitro that emulated the maintenance of the pluripotent epiblast developed from the ICM in vivo. Therefore, such defined culture system not only rendered specification of clinically-relevant early lineages directly from the pluripotent state without an intervening multi-lineage germ-layer stage, but also allowed identify the signaling molecules necessary and sufficient for inducing the cascade of organogenesis in a process that might emulate the human embryonic development [ 3, 12 ]. The hESCs are not only pluripotent, but also incredibly stable and positive, as evident by that only the positive active chromatin remodeling factors, but not the negative repressive chromatin remodeling factors, can be found in the pluripotent epigenome of hESCs [ 14, 18, 20, 21 ]. In the last few years, pluripotency-inducing factors, most of which are known oncogenes, have been used to reprogram somatic cells to induced pluripotent stem cells (iPS cells) [ 44 – 47 ]. However, the extremely low efficiencies (< 0. 1%) of the iPS cell technique diminish its clinical implication. The scientific definition and proof for stem cells are that they have the intrinsic ability of both self-renewal and differentiation. Pluripotent hESCs can maintain prolonged normal stable growth or self-renewal in non-hostile growth environments containing the essential developmental components that sustain hESC pluripotence and self-renewal [ 3, 12 ]. However, so far, there is no evidence that pluripotent cells derived from sources harboring adult nuclei by somatic cell nuclear transfer or transcription-factor-based reprogramming or small-molecule-based reprogramming, such as iPS cells or ESC derived from cloned embryos, can maintain prolonged normal stable growth or self-renewal [ 44 – 50 ]. In addition, some techniques that those reports used for their analysis of pluripotent sticky cells, such as FACS for sorting non-sticky adult cells or western blot analysis for detecting weakly expressed molecules that cannot be detected by immunocytochemical analysis, would give false positive to a heterogeneous population or colony of cells that the majority of cells might be negative [ 44 – 50 ]. The normality and positivity of hESC open epigenome differentiate pluripotent hESCs from any other stem cells, such as pluripotent iPS cells reprogrammed from adult cells, ESC derived from cloned embryos, and tissue-resident stem cells [ 14, 18, 20, 21 ]. Although pluripotent, the iPS cells are made from adult cells, therefore, iPS cells carry many negative repressive chromatin remodeling factors and unerasable genetic imprints of adult cells that pluripotent hESCs do not have [ 21, 51, 52 ]. Somatic cell nuclear transfer and factor- or small-molecule-based reprogramming are incapable of restoring a correct epigenetic pattern of pluripotent hESCs [ 21, 51 – 54 ]. As an alternative approach to iPS cells, known neural-fate determining genes or chemicals were recently used to transdifferentiate or reprogram fibroblasts or tissues into induced adult neural cells by genetic engineering or induction with extremely low efficiencies [ 55 – 59 ]. Similarly, known cardiac-fate determining genes or chemicals were recently used to transdifferentiate or reprogram fibroblasts or tissues into induced adult cardiac progenitors and cardiomyocytes by genetic engineering or induction with extremely low efficiencies [ 60 – 63 ]. Reprogrammed somatic cells have historically been associated with abnormal gene expression, accelerated senescence, and immune-rejection following transplantation [ 21, 51 – 54 ]. These major drawbacks have severely impaired the utility of reprogrammed or deprogrammed or direct/trans-differentiated somatic cells as viable therapeutic approaches. Although small molecules used to induce hESC lineage-specific therapy derivatives are usually safe developmental signal molecules and morphogens, it should be cautious of the small molecules used in the reverse process to generate iPS cells or trans-differentiation, which are known toxic cancerogenic chemicals with too dangerous or even lethal side effects to be used for patients [ 3, 21, 48, 49, 63 ]. 3. TRANSFORM PLURIPOTENT HUMAN EMBRYONIC STEM CELLS INTO NEURONAL FATE-RESTRICTED THERAPY DERIVATIVES FOR CNS REGENERATION 3. 1 Direct Induction of a Cascade of Uniform Neuronal Lineage-Specific Progression from the Pluripotent State of hESCs Using Small Molecules The development of better differentiation strategies that permit to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to desired phenotypes is vital for realizing the therapeutic potential of hESCs. Conventional hESC differentiation procedures largely rely on the formation of multi-lineage aggregates that contain cells from all three embryonic germ layers, in part because it has been assumed that tissue and organ systems arise from the endo-, meso-, and ecto-derms. However, the nervous system and the heart are among the first tissue and organ systems formed from the cells of the ICM in embryogenesis. In fact, substantial neural and cardiac differentiation appears to occur at relatively early stages in embryonic stem cells cultivation under conditions that induce differentiation [ 22 – 26 ]. It is deducible that the specification of early embryonic neural and cardiac lineages may occur directly from the pluripotent hESCs, precede the germ layer formation, and subsequently influence lineage determination at later stages of the developmental continuum. Direct induction of pluripotent hESCs exclusively into a rich collection of neural- or cardiac-restricted progenies will open the door for investigating the molecular and cellular cues in directing hESC differentiation programs using effective in vitro model systems. These studies will permit control conditions to derive not only mature functional lineages but intermediate stem/progenitor/precursor cells for cell-based therapies. To achieve uniformly conversion of pluripotent hESCs to a lineage-specific fate, we have employed the defined culture system capable of insuring hESC proliferation to screen a variety of small molecules and growth factors on the pluripotent state of hESCs in our recent reports. We found that pluripotent hESCs maintained under the defined culture conditions can be uniformly converted into a specific neural or cardiac lineage by small molecule induction [ 3, 12 – 21 ]. RA was identified as sufficient to induce the specification of neuroectoderm direct from the pluripotent state of hESCs and trigger a cascade of neuronal lineage-specific progression to human neuronal progenitors (hESC-I hNuP) and neurons (hESC-I hNu) of the developing CNS in high efficiency, purity, and neuronal lineage specificity by promoting nuclear translocation of the neuronal specific transcription factor Nurr-1 [ 3, 14, 16 – 21 ] ( Figs. 2, 3 ). Upon exposure of undifferentiated hESCs maintained in the defined culture to RA, all the cells within the colony underwent morphology changes to large differentiated cells that ceased expressing pluripotence-associated markers (e. g. , Oct-4) and began expressing neuroectoderm-associated markers, but not markers associated with other lineages (Stage 1 – Human Neuroectodermal Cells) ( Figs. 2, 3 ) ( Table 1 ). These differentiating hESCs then formed neuroblasts that were uniformly positive for β-III -tubulin in suspension (Stage 2 – Human Neuronal Progenitor Cells [hESC-I hNuP]) ( Figs. 2, 3 ). After permitting the neuroblasts to attach, β-III-tubulin- and Map-2-expressing, exuberantly neurite-bearing cells and pigmented cells began to appear with a drastic increase in efficiency (> 90%) (Stage 3 – Human Neuronal Cells in the developing CNS [hESC-I hNu]) when compared to similarly cultured cells derived from untreated EBs (<5%) ( Figs. 2, 3 ). This technology breakthrough enables neuronal lineage-specific differentiation direct from the pluripotent state of hESCs with small molecule induction, providing a much-needed in vitro model system for investigating molecular controls in human CNS development in embryogenesis as well as a large supply of clinical-grade human neuronal cells across the spectrum of developmental stages for tissue engineering and cell therapy against CNS disorders. The traditional sources of engraftable human stem cells with neural potential for transplantation therapies have been multipotent human neural stem cells (hNSCs) isolated directly from the human fetal neuroectoderm or CNS [ 64 – 68 ]. These CNS-derived primary hNSCs are neuroepithelial-like cells that are nestin-positive and can spontaneously differentiate into a mixed population of cells containing undifferentiated hNSCs, neurons, astrocytes, and oligodendrocytes in vitro and in vivo [ 69 – 72 ]. These primary hNSCs can be maintained as stable cell lines that express neural stem cell markers (e. g. , nestin, Sox-2, musashi) in serum-free, mitogen-supplemented media [ 2, 67 ]. Upon removal of mitogens, 10–30% of these hNSCs spontaneously differentiate into cells that express the neuronal markers (e. g. , β-III-tubulin, Map-2) [ 73, 74 ]. The capacity of these primary hNSCs to respond to cues that might direct them towards a particular CNS neuronal subtype in vitro was best established in dopaminergic (DA) differentiation, producing as high as 5–10% tyrosine hydroxylase (TH)-positive ventral mesencephelon (VM) neuronal cells that expressed midbrain DA neuron markers (e. g. , Nurr1, Ptx3) [ 75, 76 ]. In animal models of DA dysfunction, as high as 5% of neuronal cells associated with a DA phenotype were observed [ 67, 70, 75, 76 ]. However, cell therapy based on CNS tissue-derived hNSCs has encountered supply restriction and difficulty to use in the clinical setting due to their declining plasticity with aging and limited expansion ability, making it difficult to maintain a large scale culture and potentially restricting the tissue-derived hNSC as an adequate source for graft material [ 2, 3 ]. Despite some beneficial outcomes, CNS-derived hNSCs appeared to exert their therapeutic effect primarily by their non-neuronal progenies through producing trophic and/or neuro-protective molecules to rescue endogenous host neurons, but not related to regeneration from the graft or host remyelination [ 2, 66, 67 ]. The small numbers of neuronal progenies generated from those engrafted hNSCs often fail to achieve the anticipated mechanism of direct reconstruction of the damaged CNS structure and circuitry [ 2, 66, 67 ]. So far, due to these major limitations, cell therapies based on CNS-derived hNSCs have not yielded the satisfactory results expected for clinical trials to move forward [ 77 ]. 3. 2 Neuroectoderm Specification Transforms Pluripotent hESCs into a More Neuronal Lineage-Specific Embryonic Neuronal Progenitor than the Prototypical Neuroepithelial-Like hNSCs Alternatively, the genetically stable pluripotent hESCs proffer cures for a wide range of neurological disorders by supplying the diversity of human neuronal cell types in the developing CNS for regeneration and repair [ 2, 3 ]. Therefore, pluripotent hESCs have been regarded as an ideal source to provide an unlimited supply of human neuronal cell types and subtypes for regenerating the damaged or lost nerve tissues in CNS disorders. Although neural lineages appear at a relatively early stage in differentiation, < 5% hESCs undergo spontaneous differentiation into neurons [ 2, 3 ]. RA does not induce neuronal differentiation of undifferentiated hESCs maintained on feeders [ 2, 3 ]. And unlike mouse ESCs, treating hESC-differentiating multi-lineage aggregates (EBs) only slightly increases the low yield of neurons [ 23, 24 ]. Under conventional protocols presently employed in the field, these neural grafts derived from pluripotent cells through multi-lineage differentiation yielded neurons at a low prevalence following engraftment, which were not only insufficient for regeneration or reconstruction of the damaged CNS, but also accompanied by unacceptably high incidents of teratoma and/or neoplasm formation [ 2, 3, 5 – 9 ]. Similar to CNS-derived hNSCs, these hESC-derived secondary hNSCs are neuroepithelial-like cells that are nestin-positive and can spontaneously differentiate into a mixed population of cells containing undifferentiated hNSCs, neurons (<10%), astrocytes, and oligodendrocytes in vitro and in vivo [ 25, 79 – 82 ]. Before further differentiation, those secondary hNSCs were tediously mechanically isolated or enriched from hESC-differentiating multi-lineage aggregates [ 2 ]. Previously, co-culturing with stromal cells or telomerase-immortalized midbrain astrocytes as well as exposing to FGF and sonic hedgehog (SHH) signaling have been used to improve the yield of β-III-tubulin- and TH-positive cells from hESC-derived hNSCs [ 83 – 85 ]. The signaling factors that operate along the rostrocaudal and dorsoventral axes of the neural tube to specify motor neuron fate in vivo have been used to direct hESCs differentiate into an early motor neuron phenotype through germ-layer induction in vitro, but with low efficiencies [ 86, 87 ]. Early study of these uncommitted hESC-derived hNSCs showed that the grafted cells not only yielded a small number of DA neurons (~ 0. 2%) in vivo following transplantation, but could not acquire a DA phenotype in the lesioned brain [ 88 ]. Transplanting DA neurons pre-differentiated from these hESC-derived hNSCs ex vivo did not increase the yield of DA neurons in the lesioned brain [ 89, 90 ]. Although a small number of motor neurons were observed following transplantation of the ESC-derived grafts into adult paralyzed rats, there was little evidence of improved behavior [ 86, 87, 91 ]. Similar to their CNS counterpart, the therapeutic effect of these hESC-derived hNSCs was mediated by neuroprotective or trophic mechanism to rescue dying host neurons, but not related to regeneration from the graft or host remyelination [ 2, 3 ]. Growing evidences indicate that these secondary hNSCs derived from hESCs via conventional multi-lineage differentiation in vitro appear to have increased risk of tumorigenicity but not improved neurogenic potential compared to primary hNSCs isolated from the CNS tissue in vivo, remaining insufficient for CNS regeneration. A recent report showed that further directed differentiation of those hESC-derived hNSCs into floor-plate precursors of the developing midbrain appeared to increase the efficiency of DA neuron engraftment in Parkinson’s disease models, further suggesting that the poor in vivo performance of those nestin-positive neuroepithelial-like hNSCs derived from hESCs in vitro was due to incomplete neuronal lineage specification [ 3, 10 ]. Development of a well-controlled strategy for efficiently committing hESCs into a more specific neuronal lineage in high purity and large quantity is vital to harnessing the therapeutic potential of pluripotent hESCs for CNS repair. Unlike the two prototypical hNSCs, hESC-I hNuPs, which have acquired a neuroectodermal identity through RA induction of pluripotent hESCs in vitro, did not express the canonical hNSC markers [e. g. , nestin], but assumed uniformly strong expression and nuclear localization of the neuronal specific transcriptional factor Nurr-1 [ 3, 17, 18 ]. Under neuronal differentiation conditions, hESC-I hNuPs yielded exclusively neurons that expressed neuronal markers with a drastic increase in efficiency (~ 95%) when compared to the yields of β-III-tubulin-positive neurons differentiated under similar conditions from hESC-derived hNSCs (~ 6%) or CNS-derived hNSCs (~ 13%) [ 3, 17 – 19 ]. These in vitro neuroectoderm-derived hESC-I hNuPs yielded neurons efficiently and exclusively, as they did not differentiate into other neural cell types such as glial cells, suggesting that they are a novel more neuronal lineage-specific embryonic neuronal progenitor than the prototypical neuroepithelial-like hNSCs. MiRNAs act as the governors of gene expression networks, thereby modify complex cellular phenotypes in development or disorders [ 16, 19 ]. MiRNAs play a key role in regulation of ESC identity and cell lineage in mouse and human ESCs [ 16, 19 ]. MiRNA expression profiling using microarrays is a powerful high-throughput tool capable of monitoring the regulatory networks of the entire genome and identifying functional elements in hESC development [ 16, 19 ]. Genome-scale profiling of microRNA (miRNA) differential expression showed that the expression of pluripotence-associated hsa-miR-302 family was silenced and the expression of Hox miRNA hsa-miR-10 family that regulates gene expression predominantly in neuroectoderm was induced to high levels in those hESC-derived neuronal progenitors [ 16, 19 ]. Following transplantation, they engrafted widely and yielded well-dispersed and well-integrated human neurons at a high prevalence within neurogenic regions of the brain [ 18, 19 ]. These studies suggest that these hESC neuronal derivatives have acquired a neuronal lineage-specific identity by silencing pluripotence-associated miRNAs and inducing the expression of miRNAs linked to regulating human CNS development to high levels, therefore, highly neurogenic in vitro and in vivo [ 3, 16 – 21 ]. Therefore, neuroectoderm specification of pluripotent hESCs produces an engraftable human embryonic neuronal progenitor in high purity and large supply with adequate neurogenic potential for scale-up CNS regeneration [ 3, 16 – 21 ] ( Fig. 2 ). Under protocols presently employed in the field, hESC-derived cellular products consist of a heterogeneous population of mixed cell types, including fully differentiated cells, high levels of various degrees of partially differentiated or uncommitted cells, and low levels of undifferentiated hESCs, posing a constant safety concern when administered to humans [ 2, 3 ]. Novel lineage-specific differentiation approach by small molecule induction of pluripotent hESCs not only provides a model system for investigating human embryogenesis, but also dramatically increases the clinical efficacy of graft-dependent repair and safety of hESC-derived cellular products. Thus, it offers an adequate human neurogenic cell source in high purity and large quantity for CNS tissue engineering and developing safe and effective stem cell therapy to restore the normal nerve tissue and function in a wide range of neurological disorders. 3. 3 Turning Pluriptoent hESCs into a Large Supply of Plastic CNS Derivatives for Modeling Human CNS Development, Tissue Engineering, and Cell Therapy Understanding the much more complex human embryonic development has been hindered by the restriction on human embryonic and fetal materials as well as the limited availability of human cell types and tissues for study. In particular, there is a fundamental gap in our knowledge regarding the molecular networks and pathways underlying the CNS and heart formation in human embryonic development. The enormous diversity of human somatic cell types and the highest order of complexity of human genomes, cells, tissues, and organs among all the eukaryotes pose a big challenge for characterizing, identifying, and validating functional elements in human embryonic development in a comprehensive manner. Derivation of hESCs provides a powerful in vitro model system to investigate the molecular controls in human embryonic development as well as an unlimited source to generate the diversity of human cell types and subtypes across the spectrum of development stages for repair. Development and utilization of hESC models of human embryonic development will facilitate rapid progress in identification of molecular and genetic therapeutic targets for the prevention and treatment of human diseases. After neuronal induction during early mammalian embryogenesis, neuroectodermal cells form the neural plate that develops into the neural tube. Subsequent development, together with vesiculation within the tube, gives rise to the brain and spinal cord of the CNS. The complexity in generating the enormous diversity of neuronal cell types is best illustrated in the development of mammalian telencephalon [ 92, 93 ]. The most rostral region of the neural tube, the prosencephalon, divides into the telencephalon and diencephalon. The dorsal region of the telencephalon gives rise to the cerebral cortex, which comprises the neucortex, piriform cortex and hippocampus, while the ventral telencephalon differentiates into the basal ganglia. The dorsoventral and rostrocaudal identities and subsequent specification of progenitors are established by diffusible morphogens, including Fgf, Shh, Bmp, Wnt, Nodal, and Notch proteins, through regional patterning and activation of transcription factors [ 94 ]. The key steps of neurogenesis include neuronal commitment of NSCs, the subtype specification of intermediate neuronal progenitors, postmitotic precursors, and mature neurons [ 92 – 94 ] ( Table 1 ). Developing multi-cellular models of human CNS development using hESCs will contribute tremendously to our knowledge regarding molecular neurogenesis in human embryonic development, and thereby, aid the formulation of more optimal cell-based therapeutic strategies for CNS repair. Recent development in innovative small molecule direct induction approach renders a cascade of neuronal lineage-specific progression directly from the pluripotent state of hESCs, providing much-needed in vitro model systems for investigating the human CNS development in embryogenesis [ 3, 16 – 21 ] ( Fig. 2, Fig. 3 ). This technology breakthrough not only opens the door for further identification of the developmental networks in human embryonic neurogenesis in a comprehensive manner, but also offers means for small-molecule-mediated direct control and modulation of the pluripotent fate of hESCs when deriving an unlimited supply of neuronal cell types and subtypes for regenerative medicine [ 3, 16 – 21 ]. Advances in large-scale profiling of developmental regulators in high-resolution have provided powerful genome-wide high-throughput approaches that lead to great advances in our understanding of the global phenomena of human embryogenesis. Studies to profile novel hESC models of human embryonic neurogenesis using genome-wide approaches, including employing chromatin/nucleosome-immunoprecipitation-coupled DNA microarray analysis (ChIP/NuIP-chip) and miRNA mapping, have revealed molecular controls and the underlying mechanisms in hESC neuronal lineage specification [ 16, 19 – 21 ]. Such genome-wide high-resolution maps will generate comprehensive knowledge of developmental regulators and networks underlying hESC neuronal cell type and subtype specification for systems biology approaches and network models of human embryogenesis. Unveiling developmental networks during human embryonic neurogenesis using novel hESC models will contribute tremendously to our knowledge regarding molecular embryogenesis in human development, thereby, reveal potential therapeutic targets and aid the development of more optimal stem-cell-mediated therapeutic strategies for the prevention and treatment of CNS disorders. The outcome of such research programs will potentially shift current research to create new scientific paradigms for developmental biology and stem cell research. Standard stem cell differentiation protocols involve cultivation in 2-dimensional (2D) settings, whereas in vivo organogenesis requires a 3-dimensional (3D) setting to provide the spatial and temporal controls of cell differentiation necessary for the formation of functional tissues [ 95 – 97 ]. The traditional methods of 2D culture often result in unpredictable stem cell function and behavior in vivo following transplantation [ 95 – 97 ]. Because of interspecies differences, conventional studies using animal models are often poor predictors of human efficacy and safety. Animal models are xeno-hosts for transplantation of human cells, not ideal for testing the safety and efficacy of therapeutic outcomes of human stem cells. Large primate models are very costly and often taken years to obtain results. In addition, the results of animal studies can be highly variable and difficult to reproduce, making them unreliable as benchmarks for decisions on human clinical trials. Developing strategies for complex 3D multi-cellular models of human embryogenesis and organogenesis will provide a powerful tool that enables analysis under conditions that are tightly regulated and authentically representing the in vivo spatial and temporal patterns. It will go beyond flat biology to increase the biological complexity of human-based in vitro models and assays to mimic the in vivo human organ systems and functions, which are controllable, reproducible, and scalable, and can be monitored and validated against responses on multiple hierarchical levels. Advancements in micro- and nano-fabrication techniques offer the possibility for highly reproducible mass-fabrication of systems with complex geometries and functionalities [ 96, 98 ]. Tissue specific extracellular matrix (ECM) gels can now create structures and surfaces with defined shapes that can be used to position cells and tissues, control cell shape and function, and create highly structured 3D culture microenvironments [ 96 – 98 ]. Hydrogels are excellent scaffolding materials for repairing and regenerating a variety of tissues because they can provide a highly swollen 3D environment similar to soft tissues [ 99, 100 ]. Bio-mimetic modification of hydrogels as tissue engineering scaffolds has emerged as an important strategy to modulate specific cellular responses for the incorporation of key biofunctions of natural ECMs to provide valuable insight into the regulation of cell function and developmental processes in tissue- and organ-specific differentiation and morphogenesis [ 101, 102 ]. In addition, the recently developed NanoCulture Plate has a precisely engineered pattern (microsquare or microhoneycomb) that promotes cells to form uniform spheroids that are highly reproducible. Realizing the developmental and therapeutic potential of hESCs has been hindered by conventional approaches for generating functional cells from pluripotent cells through multi-lineage differentiation in 2D culture, which is uncontrollable, inefficient, instable, highly variable, difficult to reproduce and scale-up [ 2, 3 ]. Development and utilization of multi-cellular 3D human embryonic models using hESCs will provide an authentic and reliable in vitro tool targeted for rapid and high fidelity safety and efficacy evaluation of human therapeutic candidates and products, and thus reduce the reliance on animal models to test potential therapeutic strategies and lead to advances in technologies used in the regulatory review. It will dramatically increase the overall turnover of investments in biomedical sciences and facilitate rapid progress in identification of therapeutic targets and approaches for the prevention and treatment of human diseases. Combining innovations in establishing highly efficient hESC neuronal lineage-specific differentiation protocol with the advancements in 3D culture microenvironments to develop the multi-cellular 3D models of the human CNS will provide much-needed in vitro tools for biomedical research. Under 3D neuronal subtype specification conditions, these hESC-derived neuronal cells by small molecule induction further proceeded to express subtype neuronal markers associated with ventrally-located neuronal populations, such as DA neurons and motor neurons, demonstrating their potential for neuron regeneration in vivo as stem cell therapy to be translated to patients in clinical trials [ 19 ]. Recent studies found that pluripotent hESCs maintained under the defined culture conditions can be uniformly converted into a specific neural or cardiac lineage by small molecule induction [ 3, 12 – 21 ] ( Fig. 2 ). This technology breakthrough enables well-controlled generation of a large supply of neuronal lineage-specific progenies across the spectrum of developmental stages direct from the pluripotent state of hESCs with signal molecules, providing unlimited source of engraftable hESC neuronal therapy derivatives in high purity, large scale, and neuronal-lineage specificity with adequate neurogenic capacity for regenerating the damaged or lost CNS structure and circuitry in a wide range of neurological disorders. Further assessment of their potential in disease models and 3D CNS models will offer critical insights into therapeutic strategies against CNS disorders as well as provide preclinical evidences of safety and efficacy for translating to patients in clinical trials. The availability of human neuronal progenitors and neuronal cells in high purity and large quantity with adequate neurogenic potential will facilitate CNS tissue-engineering and accelerate the development of safe and effective cell-based therapy against a wide range of neurological disorders that so far remain incurable, including Parkinson’s disease, Alzheimer disease, ALS, spinal muscular atrophy, stroke, brain and spinal cord injuries. 4. TRANSFORM PLURIPOTENT HUMAN EMBRYONIC STEM CELLS INTO CARDIAC FATE-RESTRICTED THERAPY DERIVATIVES FOR MYOCARDIUM REGENERATION 4. 1 A Well-Controlled Efficient Approach for hESC Cardiac Lineage-Specific Progression Direct from the Pluripotent Stage towards Beating Cardiomyocytes by Small-Molecule Induction Cardiovascular disease (CVD) is a major health problem and the leading cause of death in the Western World. So far, the lack of a clinically-suitable human cardiomyocyte source with adequate myocardium regenerative potential has been the major setback in regenerating the damaged human heart, either by endogenous cells or by cell-based transplantation or cardiac tissue engineering [ 3, 12, 13, 78, 103, 104 ]. In the adult heart, the mature contracting cardiac muscle cells, known as cardiomyocytes, are terminally differentiated and unable to regenerate. Damaged or diseased cardiomyocytes are removed largely by macrophages and replaced by scar tissue. Although cell populations expressing stem/progenitor cell markers have been identified in postnatal hearts, the minuscule quantities and growing evidences indicating that they are not genuine heart cells have caused skepticism if they can potentially be harnessed for cardiac repair [ 78, 105 – 110 ]. There is no evidence that stem/precursor/progenitor cells derived from other sources, such as bone marrow, cord blood, umbilical cord, fat tissue, or placenta, are able to give rise to the contractile heart muscle cells following transplantation into the heart [ 3, 78, 104 ]. The heart is the first organ formed from the cells of the ICM or epiblast of the blastocyst in early embryogenesis. During vertebrate embryogenesis, the major morphogenetic and regulative events that control myocardial progenitor cell differentiation into cardiomyocytes include four sequential but overlapping processes: specification of the cardiogenic mesoderm, determination of the bilaterally symmetric heart fields, patterning of the heart field, and finally cardiomyocyte differentiation and formation of the heart tube [ 111, 112 ] ( Table 2 ). In addition, endocardial and extracardiac cell populations, including smooth muscle cells, endothelial cells, and connective tissue elements, contribute to the fully functional mature heart. The development of the heart appears to be regulated by complex positive and negative signaling networks involving members of the Bmp, Shh, Fgf, Wnt, Nodal, and Notch proteins [ 108, 113 ]. The heart forms from two distinct progenitor cell populations or heart fields that segregate from a common progenitor at gastrulation [ 113, 114 ]. The primary and second heart fields can be distinguished by the expression of specific transcription factors and signaling molecules. For example, Tbx5 and Hand1 mark the primary heart field, whereas Hand2, Isl1, Tbx1, Foxh1, Mef2c, and Fgf8/10 mark the secondary heart field, although some cardiac regulatory genes, such as Nkx2. 5, are expressed in both heart fields [ 108, 113, 114 ]. Developing cellular models of human embryonic heart formation will reveal the biological pathways and molecular targets that control cardiogenesis in human embryonic development, thereby, aids identification of molecular and genetic therapeutic targets for the prevention and treatment of heart disease and failure [ 3, 115 ]. Due to the prevalence of CVD worldwide and acute shortage of donor organs or adequate human myocardial grafts, there is intense interest in developing hESC-based therapy for heart disease and failure. In hESC-differentiating multi-lineage aggregates (EBs), only a very small fraction of cells (< 4 %) spontaneously differentiate into cardiomyocytes [ 3, 26 ]. Immune-selection, co-culturing, and morphogens have been used to isolate and enrich populations of immature cardiomyocytes from hESC-differentiating EBs [ 26, 116 – 119 ]. Enriched hESC-derived cardiomyocytes could generate small grafts and function as the biological pacemaker in animal infarcted models [ 27 ]. Although such hESC-derived cardiomyocytes can partially remuscularize the injured heart and attenuate the progression of heart failure in animal models of acute myocardial infarction up to 12 weeks, equivalent to perhaps a few months in humans, the grafts generated by cell transplantation have been small and insufficient to restore heart function or to alter adverse remodeling of chronic infarcted models following transplantation [ 120 – 123 ]. Thus, developing novel strategies to channel the wide differentiation potential of pluripotent hESCs exclusively and predictably to a cardiac phenotype is vital to harnessing the power of hESC biology for cardiac repair. Recent studies found that formulation of minimal essential defined conditions for hESCs rendered small molecule NAM sufficient to induce the specification of cardiomesoderm direct from the pluripotent state of hESCs by promoting the expression of the earliest cardiac-specific transcription factor Csx/Nkx2. 5 and triggering progression to cardiac precursors and beating cardiomyocytes with high efficiency [ 3, 12 – 16, 21 ] ( Figs. 2, 4 ). Upon exposure of undifferentiated hESCs maintained in the defined culture to NAM, all the cells within the colony underwent morphology changes to large differentiated cells that down-regulated the expression of pluripotence-associated markers (e. g. , Oct-4, Sox-2) and began expressing the earliest marker for heart precursor (e. g. , Nkx2. 5, alpha-actinin), but not markers associated with other lineages (Stage 1 --- Human Cardiomesodermal Cells) ( Figs. 2, 4 ) ( Table 2 ). These differentiating hESCs then formed cardioblasts that uniformly expressed Nkx2. 5 in suspension (Stage 2 --- Human Cardiac Precursor Cells) ( Figs. 2, 4 ). After permitting the cardioblasts to attach and further treating them with NAM, beating cardiomyocytes began to appear after withdrawal of NAM with a drastic increase in efficiency when compared to similarly cultured cells derived from untreated EBs (Stage 3 --- Human Cardiomyocytes) ( Figs. 2, 4 ). Cells within the beating cardiospheres expressed markers characteristic of cardiomyocytes [ 3, 12, 15 ] ( Figs. 2, 4 ). Electrical profiles of the cardiomyocytes confirmed their contractions to be strong rhythmic impulses reminiscent of the p-QRS-T-complexes seen from body surface electrodes in clinical electrocardiograms [ 15 ]. This technology breakthrough enables cardiac lineage-specific differentiation direct from the pluripotent state of hESCs with small molecule induction, providing a much-needed in vitro hESC model system for investigating molecular controls in human embryonic heart formation as well as a large supply of clinical-grade human cardiomyocyte precursors and cardiomyocytes for myocardial tissue engineering and cell therapy against heart disease and failure. NAM appeared to induce global histone deacetylation, significant down-regulation of the expression of active chromatin remodeling factors associated with a pluripotent state, and nuclear translocation of the class III NAD-dependent histone deacetylase SIRT1 [ 16, 21 ]. This observation suggests that NAM triggers the activation of SIRT1 and NAD-dependent histone deacetylation that lead to global chromatin silencing yet selective activation of a subset of cardiac-specific genes, and subsequently cardiac fate determination of pluripotent hESCs [ 16, 21 ]. Further unveiling the neucleoprotein complex regulation in hESC cardiac lineage specific progression towards cardiomyocytes mediated by SIRT1 will provide critical understanding to the molecular mechanism underlying human embryonic cardiogenesis, thereby aid the development of more effective and safe stem cell-based therapeutic approaches in the heart field. 4. 2 Cardiac Lineage-Specific Differentiation of Pluripotent hESCs by Small Molecule Induction Opens the Door to Model the Human Heart Formation Advances in human miRNA expression microarrays, ChIP-chip, and chromatin-immunoprecipitation-combined second-generation high-throughput sequencing (ChIP-seq) have provided powerful genome-wide, high-throughput, and high resolution techniques that lead to great advances in our understanding of the global phenomena of human embryonic developmental processes using hESCs [ 16, 19 – 21, 124 – 126 ]. ChIP-seq is a most recently developed technique for genome-wide profiling of DNA-binding proteins, histone or nucleosome modifications using next-generation deep DNA sequencing technology [ 21, 125, 126 ]. ChIP-seq offers higher resolution, less noise and greater coverage than its array-based predecessor ChIP-chip, and has become an indispensable tool for studying gene regulation and epigenetic mechanisms in development [ 21, 125, 126 ]. However, without a practical strategy to convert pluripotent cells direct into a specific lineage, previous studies are limited to profiling of hESCs differentiating multi-lineage aggregates, such as EB that contain mixed cell types of endoderm, mesoderm, and ectoderm cells or a heterogeneous population of EB-derived cardiac or cardiovascular cells that contain mixed cell types of cardiomyocytes, smooth muscle cells, and endothelial cells [ 21, 124, 125 ]. Those previous reports have not achieved to utilize high-throughput approaches to profile one particular cell type differentiated from hESCs, such as neurons or cardiomyocytes [ 21, 124, 125 ]. Their findings have been limited to a small group of genes that have been identified previously in non-human systems, and thus, have not uncovered any new regulatory pathways unique to humans [ 21, 124, 125 ]. Due to the difficulty of conventional multi-lineage differentiation approaches in obtaining the large number of purified cells typically required for ChIP-seq experiments, studies to reveal the mechanism in hESC differentiation remain lacking [ 21 ], though genome-wide mapping of histone modifications and chromatin-associated proteins have already begun to reveal the mechanisms in mouse ESC differentiation [ 127 ]. Recent development in innovative small molecule direct induction approach renders a cascade of neuronal or cardiac lineage-specific progression directly from the pluripotent state of hESCs, providing much-needed in vitro model systems for investigating the human CNS development and heart formation in embryogenesis [ 3, 12 – 21 ] ( Figs. 2 – 4 ). Such in vitro hESC model systems enable direct generation of large numbers of high purity hESC neuronal or cardiomyocyte derivatives required for ChIP-seq analysis to reveal the mechanisms responsible for regulating the patterns of gene expression in hESC neuronal or cardiomyocyte specification [ 21 ]. This technology breakthrough not only opens the door for further identification of the developmental networks in human embryonic cardiogenesis in a comprehensive manner, but also offers means for small-molecule-mediated direct control and modulation of the pluripotent fate of hESCs when deriving an unlimited supply of human cardiac cells and cardiomyocytes for regenerative medicine. Profiling novel hESC models of human embryonic cardiogenesis using genome-wide approaches has begun to reveal molecular controls and the underlying mechanisms in hESC cardiac specification [ 16 ]. Such genome-wide high-resolution maps will generate comprehensive knowledge of developmental regulators and networks underlying hESC cardiomyocyte specification for systems biology approaches. Unveiling developmental networks during human embryonic heart formation using novel hESC models will contribute tremendously to our knowledge regarding molecular cardiogenesis in human embryonic development, thereby, reveal potential therapeutic targets and aid the development of more optimal stem-cell-mediated therapeutic strategies for the prevention and treatment of heart diseases. The outcome of such research program will potentially shift current research to create new scientific paradigms for developmental biology and stem cell research. Development and utilization of complex 3D multi-cellular hESC models of human embryonic heart formation will provide a powerful tool that enables analysis under conditions that are tightly regulated and authentically representing the in vivo spatial and temporal patterns of the heart, and thus reduce the reliance on animal models to test potential therapeutic strategies against CVD. Recent technology breakthrough enables well-controlled efficient generation of a large supply of cardiac lineage-specific progenies across the spectrum of developmental stages direct from the pluripotent state of hESCs for innovations to develop the multi-cellular 3D human embryonic model that can replicate the aspects of the human heart [ 3, 12 – 16, 21 ]. Heart formation requires cardiac cellular constituents and cardiovascular architectural support. The hESC-derived cardiac elements resemble the cardiac cells in human embryogenesis; therefore, they have the intrinsic potential to form human contractile heart muscle as well as the cardiovascular structure with 3D geometry and vasculature of the heart. The hESC-derived multi-cellular heart models will be able to represent cellular, functional and structural characteristics of the human heart to increase the biological complexity of human-based in vitro models and assays to mimic the in vivo structure, behavior, and function of the human organs. Such studies will provide a powerful tool targeted for rapid and high fidelity safety and efficacy evaluation of human therapeutic candidates and human cell therapy products against heart diseases, and thus lead to advances in technologies used in the regulatory review in the heart field. 4. 3. Human Embryonic Stem Cell Cardiomyocyte Derivatives for Heart Regeneration — the Vital Source for Myocardial Tissue Engineering and Myocardium Repair To date, the lack of a suitable human cardiomyocyte source with adequate myocardium regenerative potential has been the major setback for myocardial tissue engineering as well as for developing safe and effective cardiac cell therapy. Novel approach using small molecule direct induction of pluripotent cells into cardiac precursors and cardiomyocytes offers the benefits in efficiency, stability, safety, and scale-up production over existing conventional approaches [ 3, 12 – 16 ] ( Figs. 2, 4 ). Such technology breakthroughs in hESC research enable de novo derivation of clinically-suitable stable hESC lines from human blastocysts and direct conversion of such pluripotent hESCs into a large supply of clinical-grade functional human cardiomyocyte precursors and cardiomyocytes to be translated to patients for mending the damaged heart [ 3, 12, 13 ]. The availability of human cardiomyocyte derivatives in high purity and large quantity with adequate potential for myocardium regeneration will facilitate myocardial tissue-engineering and accelerate the development of safe and effective cell-based therapy for heart disease and failure that affect millions of survivors and so far have no cure [ 12, 13 ]. It makes heart disease and failure possible to be the first major health problem to be resolved by clinical translation of the advances of hESC research [ 12, 13 ]. Current cell delivery methods to the damaged heart, by injection of cells either directly into the infarcted region or via the coronary circulation, are inefficient [ 78, 103, 104 ]. In addition, arrhythmogenesis is a potential risk in cell-based cardiac repair [ 78, 103, 104 ]. So far, the need to regenerate or repair the damaged heart muscle (myocardium) has not been met by adult stem cell therapy, either endogenous or via cell delivery [ 78, 103, 104 ]. Heart transplantation with the donor organ has been the only definitive treatment for end-stage heart failure. For millions living with the damaged heart, there is no alternative definitive treatment available at present time. And there is an acute shortage of donor organs for patients who need the heart transplantation. For end-stage heart failure, stem/progenitor-cell-mediated cellular regenerative approach cannot be used as an alternative approach to heart transplantation. Those shortcomings provoke developing the technology of using hESC-derived cardiac elements to reconstitute the human hearts as the replacement organ in order to provide alternative treatment options to donor-based heart transplantation. In case of successful heart transplantation from suitable donor organ, it requires life-long immune suppression that is often associated with serious side effects. The hESCs and their derivatives are considerably less immunogenic than adult tissues [ 103, 104 ]. It is also possible to bank large numbers of human leukocyte antigen isotyped hESC lines so as to improve the likelihood of a close match [ 103, 104 ]. Therefore, making the heart from hESC-derived cardiac elements will not only provide a 3D human heart model that authentically represents the in vivo organ system and function for understanding human embryonic heart formation, but also will have tremendous potential to translate to clinical studies for organ-replacement therapy to meet some of the critical medical challenges resulted from shortage of donor organs and immune rejection in heart transplantation [ 12, 13 ]. Cardiomyocytes contribute to most of the structural volume of the heart. The relative simplicity in development and maturation of the embryonic heart makes it also possible to be the first organ to be reconstituted from hESCs, the in vitro representation of the ICM/epiblast. Establishing a controllable differentiation route to efficiently generate a large supply of human cardiac elements from hESCs will make it become feasible to reconstruct the human beating heart in 3D with appropriate cellular constituents and cardiovascular architecture [ 3, 12, 13, 15 ]. The hESC-derived cardiac elements in vitro may resemble the cardiac cells in human embryogenesis in vivo ; therefore, they have the potential to form a perfect match to the human beating heart [ 12, 13 ]. Using hESC cardiac derivatives to reconstitute the 3D human heart that reflects the biological complexity and microenvironment niche of the in vivo human heart and function will facilitate rapid progress in the identification of molecular and genetic therapeutic targets for prevention and treatment of CVD. Such studies will lead to reconstituting fully competent human hearts in 3D from hESC cardiac derivatives to meet the medical need of replacement organs for end-stage heart disease and failure, a major leap in regenerative medicine. It will provide groundbreaking technology platform for tissue and organ reconstitution from hESC-derived somatic elements, innovating in regenerative medicine that will have a tremendous impact on biomedical sciences and the healthcare industry. 5. FUTURE PROSPECTIVES Human stem cell therapy derivatives are extremely attractive for therapeutic development because they have direct pharmacologic utility in clinical applications, unlike any cells originated from animals and other lower organisms that are only useful as research materials. The human stem cell is emerging as a new type of pharmacologic agent of cellular entity in cell-based regenerative medicine, because human stem cell therapy derivatives have the potential for human tissue and function restoration that the conventional drug of molecular entity lacks. The ability of a human stem cell, by definition, to both self-renew and differentiation makes it a practically inexhaustible source of replacement cells for many devastating or fatal diseases that have been considered as incurable, such as neurodegenerative diseases and heart diseases. The pharmacologic activity of human stem cell therapy derivatives is measured by their extraordinary cellular ability to regenerate the tissue or organ that has been damaged or lost. In this regard, the pharmacologic utility of human stem cells cannot be satisfied only by their chaperone activity, if any, to produce trophic or protective molecules to rescue existing endogenous host cells that can simply be achieved by a small molecule or a drug of molecular entity. There is a large unmet healthcare need to develop hESC-based stem cell therapies to provide optimal regeneration and reconstruction treatment options to restore normal tissues and function. Clinical applications of hESC therapy derivatives provide the right alternative for many incurable diseases and major health problems that the conventional mode of drugs and treatments cannot. Recent advances and technology breakthroughs in hESC research have overcome some major obstacles in bringing hESC therapy derivatives towards clinical applications, including establishing defined culture systems for de novo derivation of clinically-suitable stable hESC lines from human blastocysts that have never been contaminated by animal cells and proteins, and direct conversion of such pluripotent hESCs into a large supply of clinical-grade functional human neuronal or cardiomyocyte therapy derivatives to be translated to patients for CNS or heart repair [ 3, 12 – 21 ]. Without an understanding of the essential developmental components for sustaining hESC pluripotence and self-renewal, hESC lines are at risk for becoming unhealthy and unstable after prolonged culturing under animal feeders, feeder-conditioned media, or artificially-formulated chemically-defined conditions [ 3, 128 ]. Resolving minimal essential requirements for sustaining embryonic pluripotence allows all poorly-characterized and unspecified biological additives, components, and substrates in the culture system, including those derived from animals, to be removed, substituted, or optimized with defined human alternatives for de novo derivation and long-term maintenance of GMP-quality xeno-free stable hESC lines and their human therapy derivatives [ 3, 12 ]. Formulation of minimal essential defined conditions renders pluripotent hESCs be directly and uniformly converted into a specific neural or cardiac lineage by small signal molecule induction [ 3, 12 – 21 ]. Such milestone advances and medical innovations in hESC research enable generation of a large supply of high purity clinical-grade hESC neuronal and heart muscle cell therapy products as powerful cellular medicines that can offer pharmacologic utility and capacity for CNS and heart regeneration that no conventional drug of molecular entity can. Currently, these hESC neuronal and cardiomyocyte therapy derivatives are the only available human cell sources with adequate capacity to regenerate neurons and contractile heart muscles, vital for CNS and heart repair in the clinical setting. The availability of human neuronal and cardiomyocyte therapy derivatives in high purity and large quantity with adequate potential for CNS and myocardium regeneration will facilitate CNS and myocardial tissue-engineering and accelerate the development of safe and effective cell-based therapy to resolve these major health problems. Further improving policy making and funding situation for hESC research would open up a new dimension of cell therapy-based future medicine to provide new medical treatments for many devastating and life-threatening diseases and injuries. Transforming pluripotent hESCs into fate-restricted therapy derivatives dramatically increases the clinical efficacy of graft-dependent repair and safety of hESC-derived cellular products, bringing cell-based regenerative medicine to a turning point.