Chitosan-gelatin Scaffolds Research Articles

Characterization of chitosan-gelatin scaffolds for dermal tissue engineering

Porous scaffolds for dermal tissue engineering were fabricated by freeze-drying a mixture of chitosan and gelatin (CG) solutions. Different crosslinking agents including glutaraldehyde, 1-(3-dimethylaminopropyl)-3-ethyl-carbodimide hydrochloride (EDC), and genipin were used to crosslink the scaffolds and improve their biostability. The porous structure and mechanical properties were determined for the scaffolds. The proliferation of human fibroblasts in the scaffolds was analyzed. It was found that EDC crosslinked scaffolds had the greatest amount of cells after four days. EDC crosslinked CG scaffolds had tensile modulus in a dry state and compressive modulus in a wet state similar to commercial collagen wound dressing. They also showed appropriate pore size, high water absorption, and good dimensional stability during cell culture. When human fibroblasts were seeded on acellular porcine dermis (APD), acellular human dermis (AHD), and CG scaffolds for 3D cell culture, they were well-distributed in the centre of the CG scaffolds but stayed only on the superficial layer of APD or AHD after seven days. A gelatin-based bioglue was applied to the CG scaffolds where the keratinocytes were seeded to mimic epidermal structure. After 14 days, the bioglue degraded and keratinocytes grew to form monolayers on the scaffolds. This study showed that CG scaffolds crosslinked by EDC and seeded with human fibroblasts could serve as dermal constructs, while the bioglue coating seeded with keratinocytes could serve as an epidermal construct. Such a combination could help regenerate skin with integrated dermal and epidermal layers and a have potential use in tissue-engineered skin.

Compatibility between chitosan-gelatin scaffold and synovium-derived mesenchymal stem cells

Objective To investigate the compatibility between chitosan-gelatin scaffold and synovium-derived mesenchymal stem cells (SMSCs).Methods The SMSCs were obtained with passage isolation, and then seeded on chitosan-gelatin scaffold. Cytocompatibility was detected by using scanning electron microscopy (SEM) and methyl thiazol tetrazolium (MTT) assay at day 3, 7 and 10 after seeding.Results SEM image of scaffold showed that SMSCs spread well and homogenously distributed throughout the entire scaffold. MTT assay revealed that the cell viability in 3D scaffolds was as follows: (1.43±0.11) at day 10≥ (1.35±0.08) at day 7 > (0.43±0.08) at day 3, and at day 7 and 10, it was significantly higher than that in controls (day 7: 0.64±0.15, and day 10: 0.59±0.13;P＜0.05).Conclusion The chitosan-gelatin scaffold is compatible with SMSCs. Key words: Synovium-derived mesenchymal stem cells; Chitosan-gelatin scaffold; Tissue engineering; Articular cartilage

Incorporation of PLGA nanoparticles into porous chitosan–gelatin scaffolds: Influence on the physical properties and cell behavior

Bone regeneration can be accelerated by localized delivery of appropriate growth factors/biomolecules. Localized delivery can be achieved by a 2-level system: (i) incorporation of biomolecules within biodegradable particulate carriers (nanoparticles), and (ii) inclusion of such particulate carriers (nanoparticles) into suitable porous scaffolds. In this study, freeze-dried porous chitosan–gelatin scaffolds (CH–G: 1:2 ratio by weight) were embedded with various amounts of poly(lactide-co-glycolide) (PLGA) nanoparticles, precisely 16.6%, 33.3% and 66.6% (respect to CH–G weight). Scaffolds loaded with PLGA nanoparticles were subjected to physico-mechanical and biological characterizations including morphological analysis, swelling and dissolution tests, mechanical compression tests and cell viability tests. Results showed that incorporation of PLGA nanoparticles into porous crosslinked CH–G scaffolds: (i) changed the micro-architecture of the scaffolds in terms of mean pore diameter and pore size distribution, (ii) reduced the dissolution degree of the scaffolds, and (iii) increased the compressive modulus. On the other hand, the water uptake behavior of CH–G scaffolds containing PLGA nanoparticles significantly decreased. The incorporation of PLGA nanoparticles did not affect the biocompatibility of CH–G scaffolds.

Simultaneous regeneration of articular cartilage and subchondral bone in vivo using MSCs induced by a spatially controlled gene delivery system in bilayered integrated scaffolds

Engineering complex tissues is important but difficult to achieve in tissue regeneration. Osteochondral tissue engineering for the repair of osteochondral defect, involving simultaneous regeneration of bone and cartilage, has attracted considerable attention and also serves as an optimal model system for developing effective strategies aimed at regenerating complex tissues. In the present study, we formulated a bilayered gene-activated osteochondral scaffold consisting of plasmid TGF-β1-activated chitosan-gelatin scaffold for chondrogenic layer and plasmid BMP-2-activated hydroxyapatite/chitosan-gelatin scaffold for osteogenic layer. Mesenchymal stem cells seeded in each layer of the bilayered gene- activated osteochondral scaffold showed significant cell proliferation, high expression of TGF-β1 protein and BMP-2 protein respectively. The results showed that spatially controlled and localized gene delivery system in the bilayered integrated scaffolds could induce the mesenchymal stem cells in different layers to differentiate into chondrocytes and osteoblasts in vitro, respectively, and simultaneously support the articular cartilage and subchondral bone regeneration in the rabbit knee ostochondral defect model. This study gives the evidence that multi-tissue regeneration through the combination of biomimetic and multi-phasic scaffold design, spatially controlled and localized gene delivery system and multi-lineage differentiation of a single stem cell population represents a promising strategy for facilitating the development of complex tissue or organ systems.

Human PDL Fibroblasts Proliferation in Scaffolds on Bioactive Glass Modified Ceramics

Scaffolds are commonly used as cell’s vehicles in order to promote cell development. The aim of this study was to investigate the proliferation of human periodontal ligament fibroblasts (PDLF) in composite ceramic scaffolds. Chitin (CHN), chitosan (CHS) and chitosangelatin (CH-G) scaffolds were fabricated on the surface of ceramic disks, coated with a mixture of a bioactive glass modified ceramic. Cell proliferation was evaluated with the MTS method. All composite ceramic scaffolds were successfully loaded with PDLF cells. However, only in CH-G composite ceramicscaffolds cell population progressively increased until day 7 and presented a sudden drop at day 10 before being stabilized for the rest of the testing period. In the case of CHN and CHS the cell number was progressively decreasing. The successful observed cellular response in CH-G composite ceramic scaffolds could support the development of a protocol for tissue engineering on the materials used.

Preparation and evaluation of chitosan-gelatin composite scaffolds modified with chondroitin-6-sulphate

Abstract The objective of the present study was to investigate the properties of porous chitosan–gelatin scaffolds. The modification of samples was done by incorporation of chondroitin-6-sulphate in bulk phase through cross linking with glutaraldehyde. Scaffolds were prepared using the freeze-drying method. An in vitro cell culture of fibroblasts L-929 was employed to study the growth and attachment of cells to the scaffolds. Optical microscopy showed high cell density for the sample supplemented with chondroitin-6-sulphate. Scanning electron microscopy was used to analyze the growth and attachment of cells. Results of the tests revealed that the sample with a 65: 35 ratio of gelatin and chitosan was suitable for both cell attachment and proliferation in fibroblast cultures. The cell viability 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromide (MTT) assay with human mesenchymal stem cells on the previous sample showed about 80 % increase in cell viability. To analyze the mechanical properties of scaffolds, the tensile strengths of the samples were measured and all samples showed stress at break above 0.1 MPa. Optimum substratum composition for porous scaffolds would involve a compromise between the desired attributes of biocompatibility, promotion of cell growth and mechanical strength.

Porous Chitsan-Gelatin Scaffolds Embedded with PLGA Nanoparticles for Bone Repair

AbstractBone regeneration can be accelerated by localized delivery of appropriate growth factors/bio active molecules. Localized delivery can be achieved by incorporating bioactive molecules within biodegradable particulate carrier system followed by embed them in a suitable porous scaffolds. These carrier system facilitates the impregnated growth factor(s) to release at a desirable rate and concentration, and to linger at injury sites for a sufficient time to recruit progenitors and stimulate tissue healing processes. In this study, an attempt has been made to engraft the porous chitosan-gelatin scaffolds with PLGA nanoparticles for localized delivery of bioactive components. Scaffolds loaded with PLGA nanoparticles were subjected to physical and mechanical characterizations such as microarchitecture analysis, swelling, porosity, mechanical properties, dissolution studies.

Effect of bovine pituitary extract on the formation of neocartilage in chitosan/gelatin scaffolds

This study investigates the influence of protein concentration in bovine pituitary extract (BPE) on the production of cartilaginous components in chitosan/gelatin (CG) scaffolds. Bovine knee chondrocytes (BKCs) in genipin-crosslinked CG scaffolds were cultured over 28 days for assessing the constructs. Scanning electron micrographs revealed that the pore structure of CG scaffolds was appropriate for cartilage tissue engineering. Also, an addition of BPE into culture medium stimulated the growth of BKCs on the pore surfaces of CG scaffolds. An increase in the protein concentration in BPE enhanced the amounts of BKCs, glycosaminoglycans and collagen. At a constant protein concentration in BPE, a scaffold prepared with a high concentration of biomaterial gels also favored the proliferation of BKCs and secretion of extracellular matrices. The histological and immunochemical staining showed chondrogenesis in the culture of BKCs using CG scaffolds and BPE-containing medium. In addition, type II collagen was synthesized in the constructs, demonstrating the chondrocytic phenotype of proliferated BKCs. Hence, combining CG scaffolds with BPE can be effectual in the regeneration of neocartilage.

Chitosan–gelatin scaffolds for tissue engineering: Physico-chemical properties and biological response of buffalo embryonic stem cells and transfectant of GFP–buffalo embryonic stem cells

The favorable cellular response of newly developed cell line, buffalo embryonic stem (ES) cells to three-dimensional biodegradable chitosan–gelatin composite scaffolds with regard to stem-cell-based tissue engineering is described. Chitosan–gelatin composites were characterized by a highly porous structure with interconnected pores, and the mechanical properties were significantly enhanced. Furthermore, X-ray diffraction study indicated increased amorphous content in the scaffold on the addition of gelatin to chitosan. To develop a transfectant of green fluorescence protein (GFP)–buffalo ES cell, transfection of GFP plasmid to the cell was carried out via the electroporation procedure. In comparison with pure chitosan, cell spreading and proliferation were greater in highly visualized GFP-expressing cell–chitosan–gelatin scaffold constructs. The relative comparison of biological response involving cell proliferation and viability on the scaffolds suggests that blending of gelatin in chitosan improved cellular efficiency. Studies involving scanning electron and fluorescence microscopy, histological observations and flow cytometer analysis of the constructs implied that the polygonal cells attached to and penetrated the pores, and proliferated well, while maintaining their pluripotency during the culture period for 28 days. Chitosan–gelatin scaffolds were cytocompatible with respect to buffalo ES cells. The study underscores for the first time that chitosan–gelatin scaffolds are promising candidates for ES-cell-based tissue engineering.

Resveratrol protects bone marrow mesenchymal stem cell derived chondrocytes cultured on chitosan-gelatin scaffolds from the inhibitory effect of interleukin-1β

To investigate the effects of resveratrol on interleukin-1beta (IL-1beta) induced catabolism in bone marrow mesenchymal stem cell (MSC) derived chondrocytes cultured on chitosan-gelatin scaffolds (CGS). The chondrogenesis of alginate-encapsulated MSCs was evaluated by toluidine blue staining, RT-PCR, and immunostaing. MSC-derived chondrocyte morphology cultured on CGS was evaluated by a scanning electron microscope (SEM) and a laser confocal microscope (LCM). When these cells on CGS were pre-stimulated with IL-1beta or co-treated with IL-1beta and resveratrol in the absence and presence of the specific beta1-integrin blocking antibody, collagen type II, aggrecan, matrix metalloproteinase-13 (MMP-13) expression, and the translocation of nuclear factor kappaB (NF-kappaB) were analyzed by Western blot analysis. Transforming growth factor beta 3 (TGF-beta3) combined with insulin-like growth factor I (IGF-I) induced the cartilage-specific collagen type II, aggrecan expression and extracellular matrix (ECM) accumulation at the end of a 3-week culture. CGS supported those differentiated chondrocytes'attachment, proliferation, migration, and ECM formation. When those cells cultured on CGS were stimulated with IL-beta1 alone, collagen type II and aggrecan expression was inhibited. However, MMP-13 expression increased. Resveratrol reversed the catabolic effects by reducing the translocation of NF-kappaB. A specific beta1-integrin blocking antibody abrogated the effects of resveratrol on IL-1beta stimulated MSC-derived chondrocytes. These results indicated that resveratrol acts as a NF-kappaB inhibitor to protect MSC-derived chondrocytes on the CGS from the IL-1beta catabolism and these effects are mediated by beta1-integrin.

Indirect fabrication of microstructured chitosan-gelatin scaffolds using rapid prototyping

A porous chitosan-gelatin scaffold with specific geometry and complex internal microarchitectures was fabricated via a novel indirect rapid prototyping technique. A stereolithography (SL) technique was employed to build the resin mold, based on which a high-resolution poly-dimethylsilicone (PDMS) mold was microreplicated. The chitosan-gelatin hybrid solution was cast on the PDMS mold for pre-freezing and the monolayer scaffold with predefined features and highly porous structures was produced upon lyophilization. These monolayer structures along with the special interface layers can be stacked together to form a three-dimensional (3D) scaffold. The morphologies of the predefined features maintain well during the assembly process. Hepatocyte culture experiment indicates that the novel microstructured chitosan-gelatin porous scaffold could facilitate cells to migrate and proliferate into the deep layers of the scaffold in comparison with the traditional porous only scaffold. This approach provides a new way to fabricate a complex 3D scaffold using various natural biomaterials for vital organ engineering.

Preparation of chitosan–gelatin hybrid scaffolds with well-organized microstructures for hepatic tissue engineering

The structural organization of natural liver is instrumental in the multifunctionality of hepatocytes, and mimicking these specific architectures in tissue-engineered scaffold plays an important role in the engineering of an implantable liver equivalent in vitro. To achieve this goal, we have developed a novel fabrication process to create chitosan–gelatin hybrid scaffolds with well-organized architectures and highly porous structures by combining rapid prototyping, microreplication and freeze–drying techniques. The scaffolds obtained not only have analogous configurations of portal vein, central vein, flow-channel network and hepatic chambers, but also have high (>90%) porosity, with the mean pore size of 100 μm. Swelling and degradation studies showed that the scaffold has excellent properties of hydrophilicity and biodegradability. A hepatocyte culture experiment was conducted to evaluate the efficiency of the well-defined chitosan–gelatin scaffold in facilitating hepatocyte growth in the inner layer of the scaffold in vitro. Scanning electron microscopy and histological analysis showed that hepatocytes could form large colonies in the predefined hepatic chambers, and these cavities could the completely filled with hepatocytes during 7 day culture. Albumin secretion and urea synthesis further indicated that the well-organized scaffolds were more suitable for hepatocyte culture.

Preparation of chitosan–gelatin scaffold containing tetrandrine-loaded nano-aggregates and its controlled release behavior

A well-timed delivery of bioactive macromolecules from the porous scaffolds is very important in tissue engineering. Tetrandrine (Ted) is one of a large number of known plant derived bisbenzylisoquinoline alkaloids and is obtained from the roots of Stephania tetrandria. Ted can be used as a modifier to poly( l-lactic acid) scaffolds to promote chondrocyte differentiation and secrete type II collagen. But the effect of Ted on chondrocyte's behavior strongly depends on the concentration of Ted in the culture media. Here amphiphilic diblock copolymer (PLAE) composed of l-lactide and methoxy poly(ethylene glycol) (MePEG) was prepared and the Ted loaded copolymeric nanospheres were obtained by self-emulsification and then solvent evaporation. The mean sizes of core/shell type PLAE nanospheres and Ted-loaded nanospheres are about 60 and 100 nm, respectively. Chitosan–gelatin (Cs–Gel) porous scaffolds loaded with PLAE–Ted nanospheres were fabricated through freeze drying. Ted release behaviors from PLAE–Ted nanospheres and porous scaffolds were investigated. The result shows that the Ted-loaded nanospheres could be embedded within Cs–Gel scaffolds and no initial burst release could be observed in the release patterns. Here a room can be provided for the scaffolds to sustained release bioactive component in tissue engineering.

Effects of Gel Concentration, Human Fibronectin, and Cation Supplement on the Tissue-Engineered Cartilage

Cultivation of bovine knee chondrocytes (BKCs) in various cationic additives was studied using chitosan-gelatin scaffolds, whose surfaces were modified by human fibronectin (HFN). Here, the genipin-crosslinked scaffolds were fabricated by the freezing/lyophilization method with various concentrations of the precursory gels. The experimental results indicated that a lower freezing temperature led to higher moisture content, porosity, and specific surface area of a scaffold. The higher the precursor concentration, the larger the moisture content of a scaffold. A fast biodegradation of scaffold matrix was generated by a high porosity with BKCs. A higher concentration of HFN coated on scaffold surfaces yielded a faster rate of BKC attachment from the culture medium. The amounts of BKCs, glycosaminoglycans, and collagen over 28-day cultivation increased with the scaffold porosity, the coating concentration of HFN, the seeding density of BKCs, and the calcium concentration in medium.

Effects of hydroxyapatite in 3-D chitosan–gelatin polymer network on human mesenchymal stem cell construct development

Human mesenchymal stem cells (hMSCs) have great potential in bone tissue engineering, and hydroxyapatite (HA), a natural component of human hard tissues, is believed to support hMSC growth and osteogenic differentiation. In this study, two types of biomimetic composite materials, chitosan–gelatin (CG) and hydroxyapatite/chitosan–gelatin (HCG), were fabricated and compared to examine the effects of HA on hMSC adhesion and 3-D construct development. The 2-D membranes were prepared to examine the influence of HA on adhesion efficiency of hMSCs, while 3-D porous scaffolds were produced to investigate the effects of HA on material adsorption properties and 3-D hMSC construct development. HA was found to promote protein and calcium ion adsorption of the 3-D porous scaffolds in the complete tissue culture media. HMSCs exhibited higher initial cell adhesion efficiency to 2-D HCG membranes, and maintained higher proliferation rates in the 3-D porous HCG than CG scaffolds with 3.3 times higher final DNA amount in HCG scaffolds over a 35-day period. Colony forming unit-fibroblast (CFU-F) assays showed that higher percentages of cells maintained their progenicity in the 3-D porous HCG scaffolds over the 35-day culture period. Differentiation assays indicated that the multi-lineage differentiation potential of the hMSCs was preserved in both 3-D porous scaffolds. However, higher alkaline phosphate activity was detected in the 3-D porous HCG scaffolds upon osteogenic induction indicating improved osteogenic differentiation potential. The results demonstrate that enhanced protein and calcium ion adsorption properties of HA in the CG polymer network improve initial cell adhesion and long-term growth, favor osteogenic differentiation upon induction, as well as maintain the progenicity of the 3-D hMSC constructs.

Porous chitosan-gelatin scaffold containing plasmid DNA encoding transforming growth factor- β1 for chondrocytes proliferation

Cartilage defects as a result of disease or injury have a very limited ability to heal spontaneously. Recently, tissue engineering and local therapeutic gene delivery systems have been paid much attention in the cartilage natural healing process. Gene-activated matrix (GAM) blends these two strategies, serving as local bioreactor with therapeutic agents expression and also providing a structural template to fill the lesion defects for cell adhesion, proliferation and synthesis of extracellular matrix (ECM). In the current study, we used chitosan-gelatin complex as biomaterials to fabricate three-dimensional scaffolds and plasmid DNA were entrapped in the scaffolds encoding transforming growth factor- β1 (TGF- β1), which has been proposed as a promoter of cartilage regeneration for its effect on the synthesis of matrix molecules and cell proliferation. The plasmid DNA incorporated in the scaffolds showed a burst release in the first week and a sustained release for the other 2 weeks. The gene transfectd into chondrocytes expresses TGF- β1 protein stably in 3 weeks. The histological and immunohistochemical results confirmed that the primary chondrocytes cultured into the chitosan-gelatin scaffold maintained round and owned characters of high secretion of specific ECM. From this study, it can be concluded that this gene-activated chitosan-gelatins matrix has a potential in the application of cartilage defects regeneration.

In vitro characterization of chitosan–gelatin scaffolds for tissue engineering

Recently, chitosan–gelatin scaffolds have gained much attention in various tissue engineering applications. However, the underlying cell–matrix interactions remain unclear in addition to the scaffold degradation and mechanical characteristics. In this study, we evaluated (i) the degradation kinetics of chitosan and chitosan–gelatin scaffolds in the presence of 10 mg/L of lysozyme for dimensional stability, weight loss, and pH changes for a period of 2 months, (ii) tensile and compressive properties of films and scaffolds in wet state at 37 °C, (iii) viability of fibroblasts and human umbilical vein endothelial cells (HUVECs) on scaffolds, and (iv) the alteration in cell spreading characteristics, cytoskeletal actin distribution, focal adhesion kinase (FAK) distribution and PECAM-1 expression of HUVECs under static and 4.5, 8.5, 13 and 18 dyn/cm 2 shear stress conditions. Degradation results showed that gelatin-containing chitosan scaffolds had faster degradation rate and significant loss of material than chitosan. Mechanical properties of chitosan are affected by the addition of gelatin although there was no clear trend. Three-dimensional chitosan and chitosan–gelatin scaffolds supported fibroblast viability equally. However, chitosan membranes decreased cell-spreading area, disrupted F-actin and localized FAK in the nucleus of HUVECs. Importantly, the lowest shear stress tested (4.5 dyn/cm 2) for 3 h washed away cells on chitosan suggesting weak cell adhesion. In the blends, effect of gelatin was dominant; actin and FAK distribution were comparable to gelatin in static culture. However, at higher shear stresses, presence of chitosan inhibited shear-induced increase in cell spreading and weakened cell adhesive strength. No significant differences were observed in PECAM-1 expression. In summary, these results showed significant influence of blending gelatin with chitosan on scaffold properties and cellular behavior.

Porous Poly (DL-Lactic Acid) Modified Chitosan-Gelatin Scaffolds for Tissue Engineering

Chitosan-gelatin (Cs-Gel) scaffolds are modified with poly (DL-lactic acid) (PDLLA) dichloromethane solution of different concentrations (0.1, 0.5, and 1.0%) and immersed in water after the evaporation of the solvent. The swollen scaffolds are freeze dried. The concentration of PDLLA has significant effects on both the physicomechanical properties and the cytocompatibility. Data reveal that only the 0.1% concentration could increase the tensile strength fourfold in comparison with the pristine Cs-Gel scaffold, while maintaining the human fibroblast adhesion, migration, and proliferation just like the Cs-Gel scaffold.

Tissue engineering of cartilage with the use of chitosan-gelatin complex scaffolds.

Chitosan has been shown to be a promising scaffold for various applications in tissue engineering. In this study, a chitosan-gelatin complex was fabricated as a scaffold by a freezing and lyophilizing technique. Chitosan's structure and characteristics are similar to those of glycosaminoglycan (GAG) and its analogs, and possesses various biological activities, whereas gelatin can serve as a substrate for cell adhesion, differentiation, and proliferation. With the use of autologous chondrocytes isolated from pig's auricular cartilage and seeded onto the chitosan-gelatin scaffold, elastic cartilages have been successfully engineered at the porcine abdomen subcutaneous tissue. After 16 weeks of implantation, the engineered elastic cartilages have acquired not only normal histological and biochemical, but also mechanical properties. The tissue sections of the engineered elastic cartilages showed that the chondrocytes were enclosed in the lacuna, similar to that of native cartilage. The presence of elastic fibers in the engineered cartilages was also demonstrated by Vehoeff's staining, and immunohistochemical staining confirmed the presence of type II collagen in the engineered cartilages. Quantitatively, the GAG in the engineered cartilages reached 90% of the concentration in native auricular cartilage. Furthermore, biomechanical analysis demonstrated that the extrinsic stiffness of the engineered cartilages reached 85% of the level in native auricular cartilage when it was harvested at 16 weeks. Thus, this study demonstrated that the chitosan-gelatin complex may serve as a suitable scaffold for cartilage tissue engineering.

A study on a chitosan-gelatin-hyaluronic acid scaffold as artificial skin in vitro and its tissue engineering applications

Chitosan-gelatin-hyaluronic acid scaffolds for tissue regeneration were fabricated by freezing and lyophilizing methods. The scaffolds showed a higher water uptake and retention abilities than chitosan-gelatin scaffolds did. Fibroblasts cultured in chitosan-gelatin-hyaluronic acid scaffolds grew and proliferated well, and they exhibited a strong viability. Keratinocytes were co-cultured with fibroblasts in chitosan-gelatin-hyaluronic acid scaffolds to construct an artificial bilayer skin in vitro . The artificial skin obtained was flexible and had good mechanical properties. The data from this study suggested that chitosan-gelatin-hyaluronic acid scaffolds are suitable for preparing a bilayer skin substitute.