In Vitro and In Vivo Biological Assessments of 3D-Bioprinted Scaffolds for Dental Applications.

Three-dimensional (3D) bioprinting is a unique combination of technological advances in 3D printing and tissue engineering. It has emerged as a promising approach to address the dilemma in current dental treatments faced by clinicians in order to repair or replace injured and diseased tissues. The exploration of 3D bioprinting technology provides high reproducibility and precise control of the bioink containing the desired cells and biomaterial over the architectural and dimensional features of the scaffolds in fabricating functional tissue constructs that are specific to the patient treatment need. In recent years, the dental applications of different 3D bioprinting techniques, types of novel bioinks, and the types of cells used have been extensively explored. Most of the findings noted significant challenges compared to the non-biological 3D printing approach in constructing the bioscaffolds that mimic native tissues. Hence, this review focuses solely on the implementation of 3D bioprinting techniques and strategies based on cell-laden bioinks. It discusses the in vitro applications of 3D-bioprinted scaffolds on cell viabilities, cell functionalities, differentiation ability, and expression of the markers as well as the in vivo evaluations of the implanted bioscaffolds on the animal models for bone, periodontal, dentin, and pulp tissue regeneration. Finally, it outlines some perspectives for future developments in dental applications.

Role of Biomaterials in the Development of Epithelial Support in 3D In Vitro Airway Epithelium Development: A Systematic Review.

Respiratory diseases have a major impact on global health. The airway epithelium, which acts as a frontline defence, is one of the most common targets for inhaled allergens, irritants, or micro-organisms to enter the respiratory system. In the tissue engineering field, biomaterials play a crucial role. Due to the continuing high impact of respiratory diseases on society and the emergence of new respiratory viruses, in vitro airway epithelial models with high microphysiological similarities that are also easily adjustable to replicate disease models are urgently needed to better understand those diseases. Thus, the development of biomaterial scaffolds for the airway epithelium is important due to their function as a cell-support device in which cells are seeded in vitro and then are encouraged to lay down a matrix to form the foundations of a tissue for transplantation. Studies conducted in in vitro models are necessary because they accelerate the development of new treatments. Moreover, in comparatively controlled conditions, in vitro models allow for the stimulation of complex interactions between cells, scaffolds, and growth factors. Based on recent studies, the biomaterial scaffolds that have been tested in in vitro models appear to be viable options for repairing the airway epithelium and avoiding any complications. This review discusses the role of biomaterial scaffolds in in vitro airway epithelium models. The effects of scaffold, physicochemical, and mechanical properties in recent studies were also discussed.

A Three-Dimensional Xeno-Free Culture Condition for Wharton's Jelly-Mesenchymal Stem Cells: The Pros and Cons.

Xeno-free three-dimensional cultures are gaining attention for mesenchymal stem cell (MSCs) expansion in clinical applications. We investigated the potential of xeno-free serum alternatives, human serum and human platelet lysate, to replace the current conventional use of foetal bovine serum for subsequent MSCs microcarrier cultures. In this study, Wharton's Jelly MSCs were cultured in nine different media combinations to identify the best xeno-free culture media for MSCs culture. Cell proliferation and viability were identified, and the cultured MSCs were characterised in accordance with the minimal criteria for defining multipotent mesenchymal stromal cells by the International Society for Cellular Therapy (ISCT). The selected culture media was then used in the microcarrier culture of MSCs to determine the potential of a three-dimensional culture system in the expansion of MSCs for future clinical applications, and to identify the immunomodulatory potential of cultured MSCs. Low Glucose DMEM (LG) + Human Platelet (HPL) lysate media appeared to be good candidates for replacing conventional MSCs culture media in our monolayer culture system. MSCs cultured in LG-HPL achieved high cell yield, with characteristics that remained as described by ISCT, although the overall mitochondrial activity of the cells was lower than the control and the subsequent effects remained unknown. MSC microcarrier culture, on the other hand, showed comparable cell characteristics with monolayer culture, yet had stagnated cell proliferation, which is potentially due to the inactivation of FAK. Nonetheless, both the MSCs monolayer culture and the microcarrier culture showed high suppressive activity on TNF-α, and only the MSC microcarrier culture has a better suppression of IL-1 secretion. In conclusion, LG-HPL was identified as a good xeno-free media for WJMSCs culture, and although further mechanistic research is needed, the results show that the xeno-free three-dimensional culture maintained MSC characteristics and improved immunomodulatory activities, suggesting the potential of translating the monolayer culture into this culture system in MSC expansion for future clinical application.

Cellular Interaction of Human Skin Cells towards Natural Bioink via 3D-Bioprinting Technologies for Chronic Wound: A Comprehensive Review.

Skin substitutes can provide a temporary or permanent treatment option for chronic wounds. The selection of skin substitutes depends on several factors, including the type of wound and its severity. Full-thickness skin grafts (SGs) require a well-vascularised bed and sometimes will lead to contraction and scarring formation. Besides, donor sites for full-thickness skin grafts are very limited if the wound area is big, and it has been proven to have the lowest survival rate compared to thick- and thin-split thickness. Tissue engineering technology has introduced new advanced strategies since the last decades to fabricate the composite scaffold via the 3D-bioprinting approach as a tissue replacement strategy. Considering the current global donor shortage for autologous split-thickness skin graft (ASSG), skin 3D-bioprinting has emerged as a potential alternative to replace the ASSG treatment. The three-dimensional (3D)-bioprinting technique yields scaffold fabrication with the combination of biomaterials and cells to form bioinks. Thus, the essential key factor for success in 3D-bioprinting is selecting and developing suitable bioinks to maintain the mechanisms of cellular activity. This crucial stage is vital to mimic the native extracellular matrix (ECM) for the sustainability of cell viability before tissue regeneration. This comprehensive review outlined the application of the 3D-bioprinting technique to develop skin tissue regeneration. The cell viability of human skin cells, dermal fibroblasts (DFs), and keratinocytes (KCs) during in vitro testing has been further discussed prior to in vivo application. It is essential to ensure the printed tissue/organ constantly allows cellular activities, including cell proliferation rate and migration capacity. Therefore, 3D-bioprinting plays a vital role in developing a complex skin tissue structure for tissue replacement approach in future precision medicine.

Gelatin Hydrogels Loaded with Lactoferrin-Functionalized Bio-Nanosilver as a Potential Antibacterial and Anti-Biofilm Dressing for Infected Wounds: Synthesis, Characterization, and Deciphering of Cytotoxicity.

Gelatin hydrogels are attractive for wound applications owing to their well-defined structural, physical, and chemical properties as well as good cell adhesion and biocompatibility. This study aimed to develop gelatin hydrogels incorporated with bio-nanosilver functionalized with lactoferrin (Ag-LTF) as a dual-antimicrobial action dressing, to be used in treating infected wounds. The hydrogels were cross-linked using genipin prior to loading with Ag-LTF and characterized for their physical and swelling properties, rheology, polymer and actives interactions, and in vitro release of the actives. The hydrogel's anti-biofilm and antibacterial performances against S. aureus and P. aeruginosa as well as their cytotoxicity effects were assessed in vitro, including primary wound healing gene expression of human dermal fibroblasts (HDFs). The formulated hydrogels showed adequate release of AgNPs and LTF, with promising antimicrobial effects against both bacterial strains. The Ag-LTF-loaded hydrogel did not significantly interfere with the normal cellular functions as no alteration was detected for cell viability, migration rate, and expression of the target genes, suggesting the nontoxicity of Ag-LTF as well as the hydrogels. In conclusion, Ag-LTF-loaded genipin-cross-linked gelatin hydrogel was successfully synthesized as a new approach for fighting biofilms in infected wounds, which may be applied to accelerate healing of chronic wounds.

The Role of Calcium in Wound Healing.

Skin injury is quite common, and the wound healing is a complex process involving many types of cells, the extracellular matrix, and soluble mediators. Cell differentiation, migration, and proliferation are essential in restoring the integrity of the injured tissue. Despite the advances in science and technology, we have yet to find the ideal dressing that can support the healing of cutaneous wounds effectively, particularly for difficult-to-heal chronic wounds such as diabetic foot ulcers, bed sores, and venous ulcers. Hence, there is a need to identify and incorporate new ideas and methods to design a more effective dressing that not only can expedite wound healing but also can reduce scarring. Calcium has been identified to influence the wound healing process. This review explores the functions and roles of calcium in skin regeneration and reconstruction during would healing. Furthermore, this review also investigates the possibility of incorporating calcium into scaffolds and examines how it modulates cutaneous wound healing. In summary, the preliminary findings are promising. However, some challenges remain to be addressed before calcium can be used for cutaneous wound healing in clinical settings.

Evaluating Feasibility of Human Tissue Engineered Respiratory Epithelium Construct as a Potential Model for Tracheal Mucosal Reconstruction.

The normal function of the airway epithelium is vital for the host's well-being. Conditions that might compromise the structure and functionality of the airway epithelium include congenital tracheal anomalies, infection, trauma and post-intubation injuries. Recently, the onset of COVID-19 and its complications in managing respiratory failure further intensified the need for tracheal tissue replacement. Thus far, plenty of naturally derived, synthetic or allogeneic materials have been studied for their applicability in tracheal tissue replacement. However, a reliable tracheal replacement material is missing. Therefore, this study used a tissue engineering approach for constructing tracheal tissue. Human respiratory epithelial cells (RECs) were isolated from nasal turbinate, and the cells were incorporated into a calcium chloride-polymerized human blood plasma to form a human tissue respiratory epithelial construct (HTREC). The quality of HTREC in vitro, focusing on the cellular proliferation, differentiation and distribution of the RECs, was examined using histological, gene expression and immunocytochemical analysis. Histological analysis showed a homogenous distribution of RECs within the HTREC, with increased proliferation of the residing RECs within 4 days of investigation. Gene expression analysis revealed a significant increase (p < 0.05) in gene expression level of proliferative and respiratory epithelial-specific markers Ki67 and MUC5B, respectively, within 4 days of investigation. Immunohistochemical analysis also confirmed the expression of Ki67 and MUC5AC markers in residing RECs within the HTREC. The findings show that calcium chloride-polymerized human blood plasma is a suitable material, which supports viability, proliferation and mucin secreting phenotype of RECs, and this suggests that HTREC can be a potential candidate for respiratory epithelial tissue reconstruction.

3D Culture of MSCs on a Gelatin Microsphere in a Dynamic Culture System Enhances Chondrogenesis.

Recent advancement in cartilage tissue engineering has explored the potential of 3D culture to mimic the in vivo environment of human cartilaginous tissue. Three-dimensional culture using microspheres was described to play a role in driving the differentiation of mesenchymal stem cells to chondrocyte lineage. However, factors such as mechanical agitation on cell chondrogenesis during culture on the microspheres has yet to be elucidated. In this study, we compared the 2D and 3D culture of bone-marrow-derived mesenchymal stem cells (BMSCs) on gelatin microspheres (GMs) in terms of MSC stemness properties, immune-phenotype, multilineage differentiation properties, and proliferation rate. Then, to study the effect of mechanical agitation on chondrogenic differentiation in 3D culture, we cultured BMSCs on GM (BMSCs-GM) in either static or dynamic bioreactor system with two different mediums, i.e., F12: DMEM (1:1) + 10% FBS (FD) and chondrogenic induction medium (CIM). Our results show that BMSCs attached to the GM surface and remained viable in 3D culture. BMSCs-GM proliferated faster and displayed higher stemness properties than BMSCs on a tissue culture plate (BMSCs-TCP). GMs also enhanced the efficiency of in-vitro chondrogenesis of BMSCs, especially in a dynamic culture with higher cell proliferation, RNA expression, and protein expression compared to that in a static culture. To conclude, our results indicate that the 3D culture of BMSCs on gelatin microsphere was superior to 2D culture on a standard tissue culture plate. Furthermore, culturing BMSCs on GM in dynamic culture conditions enhanced their chondrogenic differentiation.

Effect of Multiple-Cycle Collections of Conditioned Media from Different Cell Sources towards Fibroblasts in In Vitro Wound Healing Model.

Conditioned media refers to a collection of the used cell culture media. The goal of this study was to evaluate the possible impacts of different conditioned media collected across a number of cycles on the fibroblast proliferation, migration, and profiles of protein release. Human dermal fibroblast (HDF) cells and Wharton jelly mesenchymal stem cells (WJMSC) were cultured and incubated for 3 days prior to being harvested as cycle-1 using the serum-free media F12:DMEM and DMEM, respectively. The procedures were repeatedly carried out until the fifth cycle of conditioned media collection. An in-vitro scratch assay was conducted to measure the effectiveness of wound healing. Collagen hydrogel was combined separately with both the Wharton jelly-conditioned medium (WJCM) and the dermal fibroblast-conditioned medium (DFCM) in order to evaluate the protein release profile. The conditioned medium from many cycles had a lower level of fibroblast attachment than the control (complete medium); however, the growth rate increased from 100 to 250 h-1, when supplemented with a conditioned medium collected from multiple cycles. The wound scratch assay showed that fibroblast cell migration was significantly increased by repeating cycles up to cycle-5 of DFCM, reaching 98.73 ± 1.11%. This was faster than the rate of migration observed in the cycle-5 of the WJCM group, which was 27.45 ± 5.55%. Collagen hydrogel from multiple cycles of DFCM and WJCM had a similar protein release profile. These findings demonstrate the potential for employing repeated cycles of DFCM- and WJCM-released proteins with collagen hydrogel for applications in wound healing.

Antibacterial polylysine-containing hydrogels for hemostatic and wound healing applications: preparation methods, current advances and future perspectives.

The treatment of various types of wounds such as dermal wounds, multidrug resistant bacteria-infected wounds, and chronic diabetic wounds is one of the critical challenges facing healthcare systems. Delayed wound healing can impose a remarkable burden on patients and health care professionals. In this case, given their unique three-dimensional porous structure, biocompatibility, high hydrophilicity, capability to provide a moist environment while absorbing wound exudate, permeability to both gas and oxygen, and tunable mechanical properties, hydrogels with antibacterial function are one of the most promising candidates for wound healing applications. Polylysine is a cationic polymer with the advantages of inherent antibacterial properties, biodegradability, and biocompatibility. Therefore, its utilization to engineer antibacterial hydrogels for accelerating wound healing is of great interest. In this review, we initially discuss polylysine properties, and then focus on the most recent advances in polylysine-containing hydrogels (since 2016) prepared using various chemical and physical crosslinking methods for hemostasis and wound healing applications. Finally, the challenges and future directions in the engineering of these antibacterial hydrogels for wound healing are discussed.

ECM-derived biomaterials for regulating tissue multicellularity and maturation.

Recent breakthroughs in developing human-relevant organotypic models led to the building of highly resemblant tissue constructs that hold immense potential for transplantation, drug screening, and disease modeling. Despite the progress in fine-tuning stem cell multilineage differentiation in highly controlled spatiotemporal conditions and hosting microenvironments, 3D models still experience naive and incomplete morphogenesis. In particular, existing systems and induction protocols fail to maintain stem cell long-term potency, induce high tissue-level multicellularity, or drive the maturity of stem cell-derived 3D models to levels seen in their in vivo counterparts. In this review, we highlight the use of extracellular matrix (ECM)-derived biomaterials in providing stem cell niche-mimicking microenvironment capable of preserving stem cell long-term potency and inducing spatial and region-specific differentiation. We also examine the maturation of different 3D models, including organoids, encapsulated in ECM biomaterials and provide looking-forward perspectives on employing ECM biomaterials in building more innovative, transplantable, and functional organs.

Assessing the efficacy of curcumin-loaded alginate hydrogel on skin wound healing: A gene expression analysis.

Skin tissue engineering has gained significant attention as a promising alternative to traditional treatments for skin injuries. In this study, we developed 3D hydrogel-based scaffolds, Alginate, incorporating different concentrations of Curcumin and evaluated their properties, including morphology, swelling behavior, weight loss, as well as hemo- and cytocompatibility. Furthermore, we investigated the therapeutic potential of Alginate hydrogel containing different amounts of Curcumin using an in vitro wound healing model. The prepared hydrogels exhibited remarkable characteristics, SEM showed that the pore size of hydrogels was 134.64 µm with interconnected pores, making it conducive for cellular infiltration and nutrient exchange. Moreover, hydrogels demonstrated excellent biodegradability, losing 63.5% of its weight over 14 days. In addition, the prepared hydrogels had a stable release of curcumin for 3 days. The results also show the hemocompatibility of prepared hydrogels and a low amount of blood clotting. To assess the efficacy of the developed hydrogels, 3T3 fibroblast growth was examined during various incubation times. The results indicated that the inclusion of Curcumin at a concentration of 0.1 mg/mL positively influenced cellular behavior. The animal study showed that Alginate hydrogel containing 0.1 mg/mL curcumin had high wound closure(more than 80%) after 14 days. In addition, it showed up-regulation of essential wound healing genes, including TGFß1 and VEGF, promoting tissue repair and angiogenesis. Furthermore, the treated group exhibited down-regulation of MMP9 gene expression, indicating a reduction in matrix degradation and inflammation. The observed cellular responses and gene expression changes substantiate the therapeutic efficacy of prepared hydrogels. Consequently, our study showed the healing effect of alginate-based hydrogel containing Curcumin on skin injuries.

Multifunctionalised skin substitute of hybrid gelatin-polyvinyl alcohol bioinks for chronic wound: injectable vs. 3D bioprinting.

Chronic wounds are challenging to heal and increase global mortality. The effectiveness of skin graft is limited by rejection, fibrosis, and inadequate donor site. Multifunctionalised-hydrogel skin substitutes promoted higher wound healing by maintaining the moisture microenvironment and permit gas exchange/nourishment in prolong cell viability/activity. The purpose of this study was to evaluate a skin substitute using two strategies; via injectable and 3D bioprinting technique. New hydrogel formulations that composed of gelatin (GE) and polyvinyl-alcohol (PVA) were constructed using a pre-mix crosslinking approach with genipin (GNP) to generate the biodegradable and biocompatible skin substitute with reduced secondary traumatic wound. GPVA5_GNP (6% GE: 5% PVA crosslinked with GNP) was the most stable hydrogel for wound healing application with the longest enzymatic degradation and stable hydrogel for absorption of excess wound exudates. Primary human dermal fibroblasts (HDFs) migrated extensively through 3D bioprinted hydrogels with larger average pore sizes and interconnected pores than injectable hydrogels. Moreover, 3D bioprinted GPVA hydrogels were biocompatible with HDFs and demonstrated > 90% cell viability. HDFs maintained their phenotype and positively expressed collagen type-I, vinculin, short and dense F-actin, alpha-smooth muscle actin, and Ki67. Additionally, the presence of GNP demonstrated antioxidant capacity and high-ability of angiogenesis. The utilisation of the 3D bioprinting (layer-by-layer) approach did not compromise the HDFs' growth capacity and biocompatibility with selected bioinks. In conclusion, it allows the cell encapsulation sustainability in a hydrogel matrix for a longer period, in promoting tissue regeneration and accelerating healing capacity, especially for difficult or chronic wound.

PVA/PVP Nanofibres Incorporated with Ecklonia cava Phlorotannins Exhibit Excellent Cytocompatibility and Accelerate Hyperglycaemic Wound Healing.

BACKGROUND: Diabetic foot ulcer (DFU) is a major debilitating complication of diabetes. The lack of effective diabetic wound dressings has been a significant problem in DFU management. In this study, we aim to establish a phlorotannin-incorporated nanofibre system and determine its potential in accelerating hyperglycaemic wound healing. METHODS: The effective dose of Ecklonia cava phlorotannins (ECP) for hyperglycaemic wound healing was determined prior to phlorotannin nanofibre fabrication using polyvinyl-alcohol (PVA), polyvinylpyrrolidone (PVP), and ECP. Vapour glutaraldehyde was used for crosslinking of the PVA/PVP nanofibres. The phlorotannin nanofibres were characterised, and their safety and cytocompatibility were validated. Next, the wound healing effect of phlorotannin nanofibres was determined with 2D wound scratch assay, whereas immunofluorescence staining of Collagen-I (Col-I) and Cytokeratin-14 (CK-14) was performed in human dermal fibroblasts (HDF) and human epidermal keratinocytes (HEK), respectively. RESULTS: Our results demonstrated that 0.01 µg/mL ECP significantly improved hyperglycaemic wound healing without compromising cell viability and proliferation. Among all nanofibres, PVA/PVP/0.01 wt% ECP nanofibres exhibited the best hyperglycaemic wound healing effect. They displayed a diameter of 334.7 ± 10.1 nm, a porosity of 40.7 ± 3.3%, and a WVTR of 1718.1 ± 32.3 g/m2/day. Besides, the FTIR spectra and phlorotannin release profile validated the successful vapour glutaraldehyde crosslinking and ECP incorporation. We also demonstrated the potential of phlorotannin nanofibres as a non-cytotoxic wound dressing as they support the viability and proliferation of both HDF and HEK. Furthermore, phlorotannin nanofibres significantly ameliorated the impaired hyperglycaemic wound healing and restored the hyperglycaemic-induced Col-I reduction in HDF. CONCLUSION: Taken together, our findings show that phlorotannin nanofibres have the potential to be used as a diabetic wound dressing.

Progress in Wound-Healing Products Based on Natural Compounds, Stem Cells, and MicroRNA-Based Biopolymers in the European, USA, and Asian Markets: Opportunities, Barriers, and Regulatory Issues.

Wounds are breaks in the continuity of the skin and underlying tissues, resulting from external causes such as cuts, blows, impacts, or surgical interventions. Countless individuals suffer minor to severe injuries, with unfortunate cases even leading to death. In today's scenario, several commercial products are available to facilitate the healing process of wounds, although chronic wounds still present more challenges than acute wounds. Nevertheless, the huge demand for wound-care products within the healthcare sector has given rise to a rapidly growing market, fostering continuous research and development endeavors for innovative wound-healing solutions. Today, there are many commercially available products including those based on natural biopolymers, stem cells, and microRNAs that promote healing from wounds. This article explores the recent breakthroughs in wound-healing products that harness the potential of natural biopolymers, stem cells, and microRNAs. A comprehensive exploration is undertaken, covering not only commercially available products but also those still in the research phase. Additionally, we provide a thorough examination of the opportunities, obstacles, and regulatory considerations influencing the potential commercialization of wound-healing products across the diverse markets of Europe, America, and Asia.

Applications of drug delivery systems, organic, and inorganic nanomaterials in wound healing.

The skin is known to be the largest organ in the human body, while also being exposed to environmental elements. This indicates that skin is highly susceptible to physical infliction, as well as damage resulting from medical conditions such as obesity and diabetes. The wound management costs in hospitals and clinics are expected to rise globally over the coming years, which provides pressure for more wound healing aids readily available in the market. Recently, nanomaterials have been gaining traction for their potential applications in various fields, including wound healing. Here, we discuss various inorganic nanoparticles such as silver, titanium dioxide, copper oxide, cerium oxide, MXenes, PLGA, PEG, and silica nanoparticles with their respective roles in improving wound healing progression. In addition, organic nanomaterials for wound healing such as collagen, chitosan, curcumin, dendrimers, graphene and its derivative graphene oxide were also further discussed. Various forms of nanoparticle drug delivery systems like nanohydrogels, nanoliposomes, nanofilms, and nanoemulsions were discussed in their function to deliver therapeutic agents to wound sites in a controlled manner.

Maximizing Postoperative Recovery: The Role of Functional Biomaterials as Nasal Packs-A Comprehensive Systematic Review without Meta-Analysis (SWiM).

Numerous biomaterials have been developed over the years to enhance the outcomes of endoscopic sinus surgery (ESS) for patients with chronic rhinosinusitis. These products are specifically designed to prevent postoperative bleeding, optimize wound healing, and reduce inflammation. However, there is no singular material on the market that can be deemed the optimal material for the nasal pack. We systematically reviewed the available evidence to assess the functional biomaterial efficacy after ESS in prospective studies. The search was performed using predetermined inclusion and exclusion criteria, and 31 articles were identified in PubMed, Scopus, and Web of Science. The Cochrane risk-of-bias tool for randomized trials (RoB 2) was used to assess each study's risk of bias. The studies were critically analyzed and categorized into types of biomaterial and functional properties, according to synthesis without meta-analysis (SWiM) guidelines. Despite the heterogeneity between studies, it was observed that chitosan, gelatin, hyaluronic acid, and starch-derived materials exhibit better endoscopic scores and significant potential for use in nasal packing. The published data support the idea that applying a nasal pack after ESS improves wound healing and patient-reported outcomes.

Performance of hybrid gelatin-PVA bioinks integrated with genipin through extrusion-based 3D bioprinting: An in vitro evaluation using human dermal fibroblasts.

3D bioprinting technology is a well-established and promising advanced fabrication technique that utilizes potential biomaterials as bioinks to replace lost skin and promote new tissue regeneration. Cutaneous regenerative biomaterials are highly commended since they benefit patients with larger wound sizes and irregular wound shapes compared to the painstaking split-skin graft. This study aimed to fabricate biocompatible, biodegradable, and printable bioinks as a cutaneous substitute that leads to newly formed tissue post-transplantation. Briefly, gelatin (GE) and polyvinyl alcohol (PVA) bioinks were prepared in various concentrations (w/v); GE (6% GE: 0% PVA), GPVA3 (6% GE: 3% PVA), and GPVA5 (6% GE: 5% PVA), followed by 0.1% (w/v) genipin (GNP) crosslinking to achieve optimum printability. According to the results, GPVA5_GNP significantly presented at least 590.93 ± 164.7% of swelling ratio capacity and optimal water vapor transmission rate (WVTR), which is <1500 g/m2/h to maintain the moisture of the wound microenvironment. Besides, GPVA5_GNP is also more durable than other hydrogels with the slowest biodegradation rate of 0.018 ± 0.08 mg/h. The increasing amount of PVA improved the rheological properties of the hydrogels, leading the GPVA5_GNP to have the highest viscosity, around 3.0 ± 0.06 Pa.s. It allows a better performance of bioinks printability via extrusion technique. Moreover, the cross-section of the microstructure hydrogels showed the average pore sizes >100 µm with excellent interconnected porosity. X-ray diffraction (XRD) analysis showed that the hydrogels maintain their amorphous properties and were well-distributed through energy dispersive X-ray after crosslinking. Furthermore, there had no substantial functional group changes, as observed by Fourier transform infrared spectroscopy, after the addition of crosslinker. In addition, GPVA hydrogels were biocompatible to the cells, effectively demonstrating >90% of cell viability. In conclusion, GPVA hydrogels crosslinked with GNP, as prospective bioinks, exhibited the superior properties necessary for wound healing treatment.

Functionalised Hybrid Collagen-Elastin for Acellular Cutaneous Substitute Applications.

Wound contracture, which commonly happens after wound healing, may lead to physical distortion, including skin constriction. Therefore, the combination of collagen and elastin as the most abundant extracellular matrix (ECM) skin matrices may provide the best candidate biomaterials for cutaneous wound injury. This study aimed to develop a hybrid scaffold containing green natural resources (ovine tendon collagen type-I and poultry-based elastin) for skin tissue engineering. Briefly, freeze-drying was used to create the hybrid scaffolds, which were then crosslinked with 0.1% (w/v) genipin (GNP). Next, the physical characteristics (pore size, porosity, swelling ratio, biodegradability and mechanical strength) of the microstructure were assessed. Energy dispersive X-ray spectroscopy (EDX) and Fourier transform infrared (FTIR) spectrophotometry were used for the chemical analysis. The findings showed a uniform and interconnected porous structure with acceptable porosity (>60%) and high-water uptake capacity (>1200%), with pore sizes ranging between 127 ± 22 and 245 ± 35 µm. The biodegradation rate of the fabricated scaffold containing 5% elastin was lower (<0.043 mg/h) compared to the control scaffold (collagen only; 0.085 mg/h). Further analysis with EDX identified the main elements of the scaffold: it contained carbon (C) 59.06 ± 1.36-70.66 ± 2.89%, nitrogen (N) 6.02 ± 0.20-7.09 ± 0.69% and oxygen (O) 23.79 ± 0.65-32.93 ± 0.98%. FTIR analysis revealed that collagen and elastin remained in the scaffold and exhibited similar functional amides (amide A: 3316 cm-1, amide B: 2932 cm-1, amide I: 1649 cm-1, amide II: 1549 cm-1 and amide III: 1233 cm-1). The combination of elastin and collagen also produced a positive effect via increased Young's modulus values. No toxic effect was identified, and the hybrid scaffolds significantly supported human skin cell attachment and viability. In conclusion, the fabricated hybrid scaffolds demonstrated optimum physicochemical and mechanical properties and may potentially be used as an acellular skin substitute in wound management.

Human epidermal keratinocytes and human dermal fibroblasts interactions seeded on gelatin hydrogel for future application in skin in vitro 3-dimensional model.

Introduction: Plenty of biomaterials have been studied for their application in skin tissue engineering. Currently, gelatin-hydrogel is used to support three-dimensional (3D) skin in vitro models. However, mimicking the human body conditions and properties remains a challenge and gelatin-hydrogels have low mechanical properties and undergo rapid degradation rendering them not suitable for 3D in vitro cell culture. Nevertheless, changing the concentration of hydrogels could overcome this issue. Thus, we aim to investigate the potential of gelatin hydrogel with different concentrations crosslinked with genipin to promote human epidermal keratinocytes and human dermal fibroblasts culture to develop a 3D-in vitro skin model replacing animal models. Methods: Briefly, the composite gelatin hydrogels were fabricated using different concentrations as follows 3%, 5%, 8%, and 10% crosslinked with 0.1% genipin or non-crosslinked. Both physical and chemical properties were evaluated. Results and discussion: The crosslinked scaffolds showed better properties, including porosity and hydrophilicity, and genipin was found to enhance the physical properties. Furthermore, no alteration was prominent in both formulations of CL_GEL 5% and CL_GEL8% after genipin modification. The biocompatibility assays showed that all groups promoted cell attachment, cell viability, and cell migration except for the CL_GEL10% group. The CL_GEL5% and CL_GEL8% groups were selected to develop a bi-layer 3D-in vitro skin model. The immunohistochemistry (IHC) and hematoxylin and eosin staining (H&E) were performed on day 7, 14, and 21 to evaluate the reepithelization of the skin constructs. However, despite satisfactory biocompatibility properties, neither of the selected formulations, CL_GEL 5% and CL_GEL 8%, proved adequate for creating a bi-layer 3D in-vitro skin model. While this study provides valuable insights into the potential of gelatin hydrogels, further research is needed to address the challenges associated with their use in developing 3D skin models for testing and biomedical applications.