Collagen chitosan porous scaffolds with improved biostability for skin tissue engineering

ARTICLE IN PRESS Biomaterials 24 (2003) 4833–4841 Collagen/chitosan porous scaffolds with improved biostability for skin tissue engineering Lie Maa, Changyou Gaoa,*, Zhengwei Maoa, Jie Zhoua, Jiacong Shena, Xueqing Hub, Chunmao Hanb a Department of Polymer Science and Engineering, Zhejiang University, Hangzhou 310027, China b Faculty of Burn, Second Affiliated Hospital of Zhejiang University, Hangzhou 310027, China Received November 2002; accepted 13 May 2003 Abstract Porous scaffolds for skin tissue engineering were fabricated by freeze-drying the mixture of collagen and chitosan solutions Glutaraldehyde (GA) was used to treat the scaffolds to improve their biostability Confocal laser scanning microscopy observation confirmed the even distribution of these two constituent materials in the scaffold The GA concentrations have a slight effect on the cross-section morphology and the swelling ratios of the cross-linked scaffolds The collagenase digestion test proved that the presence of chitosan can obviously improve the biostability of the collagen/chitosan scaffold under the GA treatment, where chitosan might function as a cross-linking bridge A detail investigation found that a steady increase of the biostability of the collagen/chitosan scaffold was achieved when GA concentration was lower than 0.1%, then was less influenced at a still higher GA concentration up to 0.25% In vitro culture of human dermal fibroblasts proved that the GA-treated scaffold could retain the original good cytocompatibility of collagen to effectively accelerate cell infiltration and proliferation In vivo animal tests further revealed that the scaffold could sufficiently support and accelerate the fibroblasts infiltration from the surrounding tissue Immunohistochemistry analysis of the scaffold embedded for 28 days indicated that the biodegradation of the 0.25% GA-treated scaffold is a long-term process All these results suggest that collagen/chitosan scaffold cross-linked by GA is a potential candidate for dermal equivalent with enhanced biostability and good biocompatibility r 2003 Elsevier Ltd All rights reserved Keywords: Collagen; Chitosan; Biostability; Cross-link; Tissue engineering Introduction The skin loss is one of the oldest and still not totally resolved problems in surgical field Due to the spontaneous healing of the dermal defects would not occur, the scar formation for the full thickness skin loss would be inevitable unless some skin substitutes are used In the past decades, many skin substitutes such as xenografts, allografts and autografts have been employed for wound healing However, because of the antigenicity or the limitation of donor sites, the skin substitutes mentioned above cannot accomplish the purpose of the skin recovery and yet not be used widely [1–5] Therefore, many studies are turning toward the tissue engineering *Corresponding author Tel.: +86-571-87951108; fax: +86-57187951948 E-mail address: cygao@mail.hz.zj.cn (C Gao) 0142-9612/03/$ - see front matter r 2003 Elsevier Ltd All rights reserved doi:10.1016/S0142-9612(03)00374-0 approach, which utilizes both engineering and life science discipline to promote organ or tissue regeneration and to sustain, recover their functions [6–9] One crucial factor in skin tissue engineering is the construction of a scaffold A three-dimensional scaffold provides an extra cellular matrix analog which functions as a necessary template for host infiltration and a physical support to guide the differentiation and proliferation of cells into the targeted functional tissue or organ [10,11] An ideal scaffold used for skin tissue engineering should possess the characteristics of excellent biocompatibility, suitable microstructure such as 100–200 mm mean pore size and porosity above 90%, controllable biodegradability and suitable mechanical property [12–15] Collagen is known to be the most promising materials and have been found diverse applications in tissue engineering for their excellent biocompatibility and biodegradability However, the fast biodegrading rate ARTICLE IN PRESS L Ma et al / Biomaterials 24 (2003) 4833–4841 4834 scaffolds owing to the large number of amino groups in its molecular chain (Fig 1) Hence, one can expect that less GA could be used in the presence of chitosan and the potential cytotoxicity of GA might be decreased Herein we describe the fabrication of collagen porous scaffold in the presence of 10 wt% chitosan, which functions as a cross-linking bridge in the further treatment of GA cross-linkage The microstructure, the swelling capacity, as well as the degradability both in vivo and in vitro of the collagen/chitosan scaffold were investigated In vitro culture of human dermal fibroblasts and in vivo animal tests demonstrated that the scaffolds showed good cytocompatibility and could effectively guide the infiltration and growth of fibroblasts and the low mechanical strength of the untreated collagen scaffold are the crucial problems that limit the further use of this material Cross-linking of the collagen-based scaffolds is an effective method to modify the biodegrading rate and to optimize the mechanical property For this reason, the cross-linking treatment to collagen has become one of the most important issues for the collagen-based scaffolds Currently, there are two different kinds of cross-linking methods employed in improving the properties of the collagen-based scaffolds: chemical methods and physical methods The latter include the use of photooxidation, dehydrothermal treatments (DHT) and ultraviolet irradiation, which could avoid introducing potential cytotoxic chemical residuals and sustain the excellent biocompatibility of the collagen materials [16] However, most of the physical treatments cannot yield high enough crosslinking degree to satisfy the demand of skin tissue engineering Therefore, the treatments by chemical methods are still necessary in almost all cases The reagents used in the cross-linking treatment recently involve traditional glutaraldehyde (GA), 1-ethyl-3-(3dimethylaminopropyl)-carbodiimide (EDAC), polyglycidyl ether and polyepoxidic resins, etc [17–21] GA is a kind of bifunctional cross-linking reagents that can bridge amino groups between two adjacent polypeptide chains and has become the predominant choice in skin tissue engineering because of its water solubility, high cross-linking efficiency and low cost [22] Chitosan is another biomaterials used in a variety of biomedical fields such as drug delivery carriers, surgical thread, and wound healing materials [23] Due to its many advantages for wound healing such as hemostasis, accelerating the tissue regeneration and the fibroblast synthesis of collagen, many applications of chitosan in skin tissue engineering have been reported [24–27] In addition, chitosan can function as a bridge to increase the cross-linking efficiency of GA in the collagen-based Materials and methods 2.1 Materials Chitosan (viscosity average molecular weight MZ: 1.0  105–1.7  105, 75–85% deacetylation degree), collagenase I (278 U/mg), rhodamine B isothiocyanate, fluorescein isothiocyanate (FITC) and fluorescein diacetate (FDA) were purchased from Sigma Trypsin (250 U/mg) was a commercial product from Amresco Glutaraldehyde (GA), 25% water solution, was purchased from Shanghai Pharm Co (China) All other reagents and solvents are of analytical grade and used as received Collagen type I was isolated from fresh bovine tendon by trypsin digestion and acetic acid dissolution method Briefly, after removed the fat and muscle impurity substances the bovine tendon was cut into pieces as thin as possible and digested in trypsin solution (0.25%) at 37 C for 24 h The tendon pieces were then incubated in 0.5 m acetic acid (HAc) at 4 C for 48 h A tissue triturator was employed to agitate the swollen tendon COOH COOH (Collagen) NH2 NH2 R CH N GA NH2 Chitosan H2N NH2 NH2 COOH R NH2 H2N CH N HC N CH NH2 Chitosan H2N HC N R N N CH COOH Fig Schematic presentation of collagen cross-linked with glutaraldehyde in the presence of chitosan ARTICLE IN PRESS L Ma et al / Biomaterials 24 (2003) 4833–4841 pieces violently so that the collagen fibers could be well dispersed The collagen solution was then centrifuged to get rid of insoluble impurities The supernatant was precipitated by wt% NaCl solution The precipitate was re-dissolved in 0.5 m HAc to repeat the same process for purification Finally the collagen extraction was dialyzed with double distilled water for 72 h, changing the water every 12 h, and then was lyophilized The composition and purity of the collagen type I was characterized and confirmed by UV spectroscopy, IR spectroscopy and amino acid analysis Rhodamine labeled collagen (Rd-Col) and FITC labeled chitosan (FITC-Chi) were prepared by mixing 0.2 mg/ml rhodamine B isothiocyanate or FITC into 0.5% (w/v) biomacromolecule solutions at 4 C for 48 h, respectively The free dyes were dialyzed off in 0.05 m acetic acid solution for weeks 2.2 Preparation of collagen/chitosan scaffold Collagen or chitosan was dissolved in 0.5 m HAc solution to prepare a 0.5% (w/v) solution, respectively The chitosan solution was slowly dropped into collagen suspension in the ratio of 9:1 (collagen:chitosan) and homogenized to obtain collagen/chitosan blend After deaerated under vacuum to remove entrapped airbubbles, the collagen/chitosan blend was injected into a home-made mould (diameter: 16 mm, depth: mm), frozen in 70% ethanol bath at À20 C for h and then lyophilized for 24 h to obtain a porous collagen/chitosan scaffold 2.3 Cross-linking treatment To improve the biostability, the collagen/chitosan scaffolds were treated with GA All scaffolds were rehydrated in 0.05 m HAc solutions for 15 firstly, and then were cross-linked in the GA solutions (doubledistilled water, pH 5.6) with different concentrations (0.05–0.25%) at 4 C for 24 h [21,28] After washed with double-distilled water (10  times), the scaffolds were freeze-dried again to obtain the GA treated collagen/chitosan scaffolds 2.4 Microstructure observation The microstructure of the scaffolds was observed under scanning electron microscopy (SEM, Cambridge stereoscan 260) and confocal laser scanning microscopy (CLSM, Biorad 2100) Rd-Col and FITC-Chi were used for CLSM detection with double channels’ mode 2.5 Swelling test The collagen/chitosan scaffolds were placed into distilled water at room temperature and the wet weight 4835 (w) of the scaffold was determined after incubated for 24 h The swelling ratio of the scaffolds was defined as the ratio of weight increase (wÀw0) to the initial weight (w0) Each value was averaged from three parallel measurements 2.6 In vitro collagenase degradation In vitro biodegradation test of the collagen/chitosan scaffolds cross-linked by GA with different concentrations (0–0.25%) was performed by collagenase digestion Each kind of scaffolds was immersed in phosphate buffered saline (PBS, pH 7.4) containing 100 mg/ml (28 units) collagenase (type I, Sigma) at 37 C for 4, 16, 24 and 48 h The degradation was discontinued at the desired time interval by incubating the assay mixture in an ice bath immediately Following centrifugation at 1500 rpm for 10 min, the clear supernatant was hydrolyzed with m HCl at 120 C for 12 h The content of hydroxyproline released from the scaffold was measured with ultraviolet spectroscopy [29] The biodegradation degree is defined as the percentage of the released hydroxyproline from the scaffolds at different time to the completely degraded one with same composition and same weight 2.7 Cell culture Fibroblasts used in this study were isolated from human dermis by collagenase digestion Briefly, the epidermis and subcutaneous tissue of human skin were removed by the scalpel The residual dermis was diced into 0.5–1 mm3 sized tissues, and washed with phosphate buffer saline (PBS, pH 7.4) supplemented with penicillin (100 U/ml) and streptomycin (100 U/ml) times Then these dermis pieces were placed in a spinner flask containing 10 ml of mg/ml collagenase (type I, Sigma) in Dulbecco’s modified Eagle medium (DMEM) supplemented with penicillin (100 U/ml) and streptomycin (100 U/ml) After digested in an incubator (37 C, 5% CO2) for h, the digestion was discontinued by adding DMEM supplemented with penicillin (100 U/ml), streptomycin (100 U/ml) and 10% FBS (complete medium) The digesting solution was filtered through a copper mesh (cell strainer, 200 meshes) and then was centrifuged at 1000 rpm for 10 The cell suspension were cultured at 37 C, 5% CO2, and 95% humidity in the complete medium The culture medium was changed every days Cells were passaged at confluence and the 4–8th passage fibroblasts were used for the seeding The 0.25% GA treated collagen/chitosan scaffold (both rhodamine-labeled) was immersed in 75% ethanol for 12 h for sterilization, followed with solvent exchange by PBS for times The scaffold was then placed on a 24-well polystyrene plate and seeded with 200 ml human dermal fibroblast suspension at a density of  106 cells/ ml After incubation for h, ml complete medium was ARTICLE IN PRESS L Ma et al / Biomaterials 24 (2003) 4833–4841 4836 added and cultured in a 5% CO2 incubator at 37 C for days After washed with PBS for times, the fibroblasts were stained with mg/ml FDA solution in the incubator for 15 Following with removal of the unreacted FDA with double washing in PBS, ml complete medium was then added The live fibroblasts can metabolize FDA to form a fluorescence product Hence, the fibroblasts existed in the scaffolds are distinct from the rhodamine labeled scaffolds (red color) by the generation of green color under CLSM incubated for 12 h at 4 C with mouse anti-bovine type I collagen IgG (diluted 1:100) and washed with PBS (pH 7.4) (3 times, each for min) Subsequently, the sections were incubated for 30 at 37 C with biotinylated goat anti-mouse IgG (diluted 1:300) and washed with PBS (pH 7.4) The slides were then reacted with avidinconjugated peroxidase (diluted 1:30) at 37 C for 30 Finally, the sections were displayed with DAB and embedded by paraffin to yield a positive stain Sections were observed under light-microscope 2.8 In vivo animal evaluation Results and discussion Twelve health rabbits weighing about kg were obtained from the animal laboratory and were divided into four groups randomly The 0.25% GA treated collagen/chitosan (10 wt%) scaffolds were sterilized by immerged into 75% (v/v) ethanol for 30 and washing with PBS (pH 7.4) (5 times  min) Before implantation, the dorsal surface hairs of the rabbit ears were shaved Then all rabbits were anesthetized by intravenous administration of 20 mg/kg ketamine-HCl The ears of rabbits were sterilized with 5% PVP-I, on which subcutaneous pockets were made [30,31] In every group, four scaffolds (0.5  cm2) were embedded subcutaneously on the dorsal surface of rabbit ear Harvests were performed randomly in selected group at days, and 1, 2, weeks after implantation At harvest, the implantation sites were cut in a full thickness manner (including both sides of the ear skin and cartilage) Paraffin sections were stained with hematoxylin-eosin (HE) reagent for histological observations 2.9 Immunohistochemistry Sample of 0.25% GA treated collagen/chitosan scaffold after embedded for 28 days was fabricated into paraffin section After dewaxed and blocked with 3% (w/v) bovine serum albumin in PBS (pH 7.4) (BSA/PBS) for 20 at 20 C, sections of the scaffold were 50µm (a) 3.1 Distribution of collagen and chitosan One of the important purposes adding chitosan is providing additional amino groups which function as binding cites to increase the GA cross-linking efficiency Therefore, the interpenetration of collagen and chitosan in the scaffold is crucial Exploiting the sequential scanning mode of CLSM, the distribution of FITC-Chi (Fig 2a) and Rd-Col (Fig 2b) in their complex scaffold was separately measured at wet state A merged image is shown in Fig 2c The CLSM observations indicate that the scaffold was indeed composed with chitosan and collagen which were evenly dispersed through the scaffold In acidic solution, both collagen and chitosan are positively charged, either forming a real solution (for chitosan) or suspension (for collagen) [32] Their mixture in solution is stable and does not precipitate as that for collagen/chondroitin sulfate blend, where chondroitin sulfate is negatively charged [4,7] Therefore, sufficient mixing of these two hydrophilic biomacromolecules in sub-molecular level can be achieved 3.2 Morphology It is known that the microstructure such as pore size and its distribution, porosity as well as pore shape has 50µm (b) 50µm (c) Fig CLSM images of the distribution of chitosan (a) and collagen (b) in the Rd-Col/FITC-Chi porous scaffold; (c) is the merged image of (a) and (b)  400 ARTICLE IN PRESS L Ma et al / Biomaterials 24 (2003) 4833–4841 prominent influence on cell intrusion, proliferation and function in tissue engineering The cross-section morphologies of the collagen/chitosan scaffolds before and after GA treatment are shown in Fig The interconnected 3D porous structure of the scaffolds was retained after GA treatment; however, some other significant changes occurred with respect to pore size and morphology The mean pore size increased from À100 mm, the uncross-linked (Fig 3a), to >200 mm, the cross-linked scaffolds (Fig 3b–e) Accompanying with reduction of the fibers in between pores, more sheet-like structure appeared together with condensed walls No big difference between the cross-linked scaffolds was observed, except for which treated with highest GA concentration (Fig 3e), where elongated pores were existed The results indicate that the morphology difference is mainly caused by rehydration and relyophilization process in the GA cross-linking treatment This additional refreeze-drying can induce the collagen fibers to be combined again to form sheets, leading to the fusion of some smaller pores to generate larger ones It has to be noted that the slight collapse of the scaffold during this process should have an opposite effect to the pore fusion; i.e., reducing the pore size Hence, one can deduce from the above results that the fusion effect is more prominent than the collapse As a result, the pores are enlarged On the other hand, this collapse, if not (a) occurs homogeneously in 3D, will inevitably produce elongated pores as shown in Fig 3e 3.3 Swelling test The ability of a scaffold to preserve water is an important aspect to evaluate its property for skin tissue engineering The swelling ratios of various scaffolds are shown in Fig The swelling property of the uncross-linked scaffold was doubled than the GA treated scaffolds However, the cross-linked scaffolds did not show obvious difference regardless of the GA concentration The water-binding ability of the collagen/chitosan scaffold could be attributed to both of their hydrophilicity and the maintenance of their three-dimensional structure In general, the swelling ratio is decreased as the cross-linking degree is increased because of the decrease of the hydrophilic groups [33] The results in Fig indicate that the primary factor affected the swelling property is the procedure of the GA treatment other than the GA concentration (hence, the crosslinking degree) As mentioned above, the collapse during the refreeze-drying procedure will cause the reduction of the porosity, hence, the volume for water storage, leading to the decrease of the swelling capacity However, the absolute value is still over 80 times of its (b) (d) 4837 (c) (e) Fig The cross-section SEM images of collagen/chitosan scaffolds treated with different concentration of GA,  100 (a): control; (b): 0.05% GA; (c): 0.1% GA; (d): 0.2% GA; (e): 0.25% GA ARTICLE IN PRESS L Ma et al / Biomaterials 24 (2003) 4833–4841 4838 20 100 The biodegradation degree (%) 18 The swelling ratios (1000%) 16 14 12 10 80 60 40 20 0 0.05 0.1 0.2 col 0.25 GA concentrations (%) Fig The effect of GA concentrations on the swelling ratios of the collagen/chitosan scaffolds Values are mean 7S.D (n=3) col/chi-GA control 0.05% 0.1% 0.2% 0.25% 100 The biodegradation degree (%) The Fig compares the biodegradation degree of the pure collagen scaffold and the collagen/chitosan scaffold before and after GA treatment After incubated in collagenase solution for 12 h, the pure collagen scaffold (col) had been thoroughly biodegraded The addition of chitosan (col/chi) can somewhat increase the biostability, where slight lower biodegradation degree, 92.1%, was found After cross-linked with 0.25% GA, the biostability of the pure collagen scaffold (col-GA) was greatly enhanced, where only 12.8% was degraded in 12 h Owing to the expected larger cross-linking degree (Fig 1), the ability to resist collagenase degradation was further enhanced for the chitosan-combined scaffold These results reveal that both the addition of chitosan and GA cross-linking are indispensable for improving the scaffold biostability and the presence of chitosan can obviously improve the biostability of the collagen/ chitosan scaffold under the GA treatment, where chitosan might function as a cross-linking bridge The dynamic degradation of the collagen/chitosan scaffolds cross-linked by different concentrations of GA is illustrated in Fig The uncross-linked collagen/ chitosan scaffold was biodegraded so fast that its biodegradation degree had achieved to 41.5% just treated by the collagenase solution for h After biodegradation for 16 h, the uncross-linked scaffold had been dissolved in the collagenase solution thoroughly Fig shows that the biostability of the GA treated scaffolds were better than the uncross-linked one For example, even treated with the lowest GA concentration (0.05%), the biodegradation degree of the scaffold was only 6.3% in h When the GA col/chi Fig The biodegradation degree of the pure collagen scaffolds and the collagen/chitosan scaffolds (uncross-linked or GA treated) after incubated in 100 mg/ml (30 units) collagenase for 12 h Values are mean 7S.D (n=3) initial weight after GA treatment, which is high enough for skin tissue engineering 3.4 In vitro biodegradability col-GA 80 60 40 20 0 10 20 30 40 50 Biodegrading time (h) Fig The effect of GA concentrations on the biodegradability of the collagen/chitosan scaffolds (a): control; (b): 0.05% GA; (c): 0.1% GA; (d): 0.2% GA; (e): 0.25% GA concentration was up to 0.1%, the biodegradation degree increased very slowly with the degrading The highest biodegradation degree was just 26.1% after 48 h Fig shows also that with the GA concentration increase, the effect of GA concentration on the improvement of the biostability was slowed down 3.5 Cell culture Cell infiltration and proliferation are crucial for a scaffold to support and guide tissue regeneration Fig represents the CLSM images of the human fibroblasts cultured for days in the collagen/chitosan scaffold treated by 0.25% GA Exploiting the sequential scanning mode, a great number of fibroblasts (Fig 7a) were easily distinguished from the scaffold (Fig 7b) The merged image (Fig 7c) reveals that the fibroblasts were adhered on the walls of the scaffold tightly with typical ARTICLE IN PRESS L Ma et al / Biomaterials 24 (2003) 4833–4841 50µm (a) 50µm 4839 50µm (c) (b) Fig CLSM images of human dermal fibroblasts (a) cultured over the collagen/chitosan scaffold (b, rhodamine-labeled) for days; (c) is the merged image of (a) and (b)  400 (a) (b) (c) (d) Fig The histological response to the collagen/chitosan scaffolds treated with 0.25% GA, after embedded in rabbit ear for different time,  100 (a): days; (b): days; (c): 14 days; (d): 28 days Bar indicates 200 mm M: the implanted collagen/chitosan scaffold T: the subcutaneous connective tissue Arrowhead: the infiltrated fibroblasts shuttle-like morphology This result proves that the chitosan-combined and GA-treated scaffold preserves the original good cytocompatibility of collagen Potential cytotoxicity of GA residue was not evidenced This ensures the further study of the tissue response to the scaffolds in vivo 3.6 Histological examination The histological results of the 0.25% GA-treated scaffold embedded in the rabbit ear for different time are shown in Fig The pure collagen scaffold was easy to lose its contour structure and biodegraded quickly in days because of its low stability On the contrary, the structure of the 0.25% GA-treated scaffold was retained entirely and a few of fibroblasts and inflammatory cells could be observed in the scaffold after implanted for days (Fig 8a) After implanted for days, more fibroblasts were grown into the scaffold and the inflammatory cells were still in existence (Fig 8b) When the test had processed for 14 days, a large number of fibroblasts were infiltrated into the scaffold The morphology of the scaffold was similar to the surrounding dermal tissue and its structure could not be obviously observed (Fig 8c) After 28 days’ implantation, the scaffold had almost disappeared and the blood vessels could be observed (Fig 8d) These results demonstrate that the collagen/chitosan scaffolds can ARTICLE IN PRESS L Ma et al / Biomaterials 24 (2003) 4833–4841 4840 (a) (b) Fig The light microscopic (a) and immunostaining images (b) of the 0.25% GA-treated collagen/chitosan scaffold which embedded for 28 days  100 Bar indicates 200 mm Arrowhead: the un-degraded collagen scaffold effectively sufficiently support and accelerate the fibroblasts infiltration from the surrounding tissue All the in vitro and in vivo results have shown that the collagen/ chitosan scaffold treated with GA has a good biocompatibility 3.7 Immunohistochemistry To study the biodegradation behavior of the collagen/ chitosan scaffold in vivo, the image of the paraffin section of the 0.25% GA cross-linked scaffold after immunohistochemistry treatment is shown in Fig The GA cross-linked collagen/chitosan scaffold could not be distinguished from the new-formed collagen fiber under routine paraffin section with light microscope after 28 days’ implantation (Fig 9a) However, the immunohistochemistric assay shows that the bovine type I collagen had been partially preserved though the scaffold had macroscopically disappeared (Fig 9b) This result indicates that the biodegrading behavior of the GAtreated collagen/chitosan scaffold is a long-term process The long-term biodegradation of this kind scaffold in vivo should be studied further Conclusion Herein we have described the fabrication of porous collagen/chitosan scaffold by freeze-drying their mixture and the further cross-linking with GA Collagen and chitosan were evenly distributed in the scaffold The GA treatment had an influence on the morphology and the swelling property of the scaffold, while no significant differences were observed among the scaffolds treated with different concentration GA After addition of chitosan, the ability to resist the collagenase degradation was augmented obviously and can be controlled with the change of the GA concentration The cell culture and animal test prove that the GA-treated scaffold retained the original good biocompatibility and could induce the fibroblasts infiltration from the surrounding tissue successfully Immunohistochemistric assay indicates that the biodegrading behavior of the 0.25% GAtreated collagen/chitosan scaffold is a long-term period In conclusion, the GA-treated collagen/chitosan scaffold is a potential candidate for dermal equivalent with enhanced biostability and good biocompatibility Acknowledgements The authors thank Prof Yiyong Chen for his valuable discussion This work was supported by the Natural Science Foundation of China (50173024) and the Major State Basic Research Program of China (G1999054305) References [1] Boyce ST Design principles of composition and performance of cultured skin substitutes Burns 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À20 C for h and then lyophilized for 24 h to obtain a porous collagen/ chitosan scaffold 2.3 Cross-linking treatment To improve the biostability, the collagen/ chitosan scaffolds were treated with. .. composed with chitosan and collagen which were evenly dispersed through the scaffold In acidic solution, both collagen and chitosan are positively charged, either forming a real solution (for chitosan) ... function in tissue engineering The cross-section morphologies of the collagen/ chitosan scaffolds before and after GA treatment are shown in Fig The interconnected 3D porous structure of the scaffolds