Asymmetric Chitosan Membrane Containing Collagen I Nanospheres

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Biomacromolecules 2009, 10, 1642–1649

Asymmetric Chitosan Membrane Containing Collagen I Nanospheres for Skin Tissue Engineering Kuo-Yu Chen,† Wei-Ju Liao,‡ Shyh-Ming Kuo,§ Fuu-Jen Tsai,| Yueh-Sheng Chen,|,⊥,# Chih-Yang Huang,|,# and Chun-Hsu Yao*,|,⊥,# Department of Chemical and Materials Engineering, National Yunlin University of Science and Technology, Yunlin, 64002, Taiwan, Institute of Biomedical Engineering and Material Science, Central Taiwan University of Science and Technology, Taichung, 40601, Taiwan, Department of Biomedical Engineering, I-Shou University, Kaohsiung, 84001, Taiwan, and Graduate Institute of Chinese Medical Science, and Department of Biomedical Imaging and Radiological Science, China Medical University, Taichung, 40402, Taiwan Received February 25, 2009; Revised Manuscript Received April 15, 2009

A biodegradable chitosan membrane with an asymmetric structure, seeded with fibroblasts, was prepared as a novel skin substitute. Chitosan was cross-linked with genipin and then frozen and lyophilized to yield a porous asymmetric membrane (CG membrane). Nanoscale collagen I particles were injected into the CG membrane to form an asymmetric CGC membrane. The results reveal that the CG membrane treated with 0.125 wt % of genipin had a higher swelling ratio, porosity, and pore size. After 7 d of dynamic culture, many of the adhered cells exhibited a flat morphology and well spread on the surface of CGC membrane treated with 0.125 wt % of genipin. In animal studies, the CGC membrane seeded with fibroblasts and grown in vitro for 7 d was more effective than both gauze and commercial wound dressing, Suile, in healing wounds. An in vivo histological assessment indicated that covering the wound with the asymmetric CGC membrane resulted in its epithelialization and reconstruction. CGC membrane, thus, has great potential in skin tissue engineering.

Introduction Skin, the largest organ of the body, is commonly damaged by wounding or physical trauma.1 However, the problem of loss or damage of skin has not yet been fully solved in the surgical field. Large full-thickness skin defects can not heal spontaneously. Therefore, various skin substitutes such as autografts, allografts, and xenografts for covering fullthickness skin defects have been studied.2 However, these naturally derived skin substitutes cannot support skin regeneration due to a limited number of donor sites and antigenicity.3 Accordingly, several works4 have focused on skin tissue engineering to promote wound healing and to reduce scar formation. Chitosan, poly(1,4-D-glucosamine), is a cationic natural biopolymer that is formed by the alkaline N-deacetylation of chitin. Because it has numerous special characteristics and advantages, such as biocompatibility, biodegradability, hemostatic activity, antibacterial properties,5 accelerating of tissue regeneration, and collagen synthesis,6 various researchers7-9 have employed chitosan in skin tissue engineering in recent years. However, chitosan degrades rapidly, especially in an acid medium and in the presence of lysozyme,10 which is an enzyme present in the human body and which is also formed by macrophages during wound healing.9 It will reduce its * To whom correspondence should be addressed. Tel.: +886-4-22053366, ext. 7806. Fax: +886-4-2207-3220. E-mail: [email protected] and [email protected]. † National Yunlin University of Science and Technology. ‡ Central Taiwan University of Science and Technology. § I-Shou University. | Graduate Institute of Chinese Medical Science, China Medical University. ⊥ Department of Biomedical Imaging and Radiological Science, China Medical University. # These authors contributed equally to this work.

utility as a scaffold in skin regeneration. Various processes have been proposed to increase the structural stability of chitosan in acidic solutions using cross-linking treatments.9 Genipin is a naturally occurring and weakly cytotoxic crosslinking reagent, which can be obtained from its parent compound geniposide isolated from the fruits of Gardenia jasminoides Ellis.11 It has been adopted to fix biological tissues and crosslink native polymers because it is less cytotoxic than glutaraldehyde.12,13 Accordingly, this low-toxic cross-linking agent, genipin, is used in this work to cross-link the chitosan membrane. Collagen is a major protein component of bone, cartilage, skin, and connective tissue and also an important part of extracellular matrixes in animals. The role of collagen in wound healing has already been mentioned.14 Kratz et al.15 used a collagen scaffold as a dermal equivalent to induce fibroblast infiltration and dermal regeneration. Tsai et al.16 found that adding collagen as a cell-recognizing component facilitates the growth of human skin fibroblasts on the chitosan scaffolds. Various forms of collagen, such as films, sponges, and gels, have been developed for collagen-based dressing.2 However, no investigation has employed nanosized collagen particles to repair damaged skin. Mammalian cells evolve in vivo in close contact with the extracellular matrix, which is a substratum with nanoscale features.17 The interactions between cells and the extracellular matrix affect the behaviors of the cells. In this work, nanosized collagen particles were incorporated into a chitosam membrane to mimic the natural environment of cells. Given their huge surface areas, the nanosized collagen particles could promote the adhesion of fibroblasts to the scaffold. Few investigations have adopted porous scaffolds7,16,18-20 or nanofibrous membrane21 composed of chitosan and collagen to regenerate skin. No work has been published on the use of an

10.1021/bm900238b CCC: $40.75  2009 American Chemical Society Published on Web 05/06/2009

Chitosan Membrane Containing Collagen I Nanospheres

asymmetric chitosan membrane that contains nanosized collagen particles for skin tissue engineering. This study describes the preparation and characteristics of a novel asymmetric membrane that is seeded with fibroblasts as a tissue-engineered skin. The upper layer which faces to the air is designed to allow the drainage of wound exudates. The lower layer which directly contacts the wound and contains nanosized collagen I particles and fibroblasts is designed to absorb wound exudates and accelerate tissue regeneration. The degree of cross-linking, swelling ratios, porosity, and morphology of the asymmetric genipin-cross-linked chitosan membrane are determined. An in vivo experiment is conducted to examine wounds treated with the membrane in a rat model. The progress of wound repair toward the asymmetric membrane is evaluated histologically.

Experimental Section Materials. Chitosan with molecular weight of 7.0 × 104 was purchased from VA & G Bioscience Inc. (Taoyuan, Taiwan). The degree of deacetylation of the chitosan was approximately 85%. Genipin powder was obtained from Challenge Bioproducts Co. (Taichung, Taiwan). Nanosized collagen I particles were produced using a highvoltage electrostatic field system22,23 (kindly provided by Prof. ShyhMing Kuo, I-Shou University, Taiwan). All chemicals were used without further purification. Dulbecco’s modified Eagle medium (DMEM), horse serum, penicillin, and streptomycin were all purchased from Gibco (U.S.A.). 3-[4,5Dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) was purchased from USB, Amersham Life Science (Cleveland, OH). Glutaraldehyde was purchased from Acros (Geel, Belgium). Zoletil 50 was purchased from Virbac (France). Rompun solution was purchased from Bayer (Germany). Povidone-iodine solution was purchased from Chou Jen Pharmaceutical Co. (Nantou, Taiwan). Commercial wound dressings, Suile, were purchased from Swiss-American Products Inc. (Texas). Formalin and paraffin wax were purchased from Merck (Whitehouse Station, NJ). Hematoxylin-eosin (H&E) reagent and lysozyme (L-6876) were purchased from Sigma (St. Louis, MO). Cell culture flask was purchased from Costar (U.S.A.). Sprague-Dawley rats were purchased from the National Laboratory Animal Center, Taiwan. Preparation of Asymmetric CG and CGC Membranes. A 3 wt % chitosan solution was prepared by dissolving chitosan powder in 1% aqueous acetic acid. Genipin solution with a concentration of 20% was obtained by dissolving genipin powder in 60% ethanol. As the chitosan solution was heated to 35 °C, various volumes of 20 wt % genipin solutions were added to create a cross-linking reaction. Consequently, various weight percentages of genipin solutions from 0.125 to 0.5 wt % were present in the chitosan-genipin mixture. After 2 min of vigorous stirring, the mixture began to turn light bluish and became increasingly viscous. It was then poured on a glass dish of diameter 90 mm at 25 °C and subsequently quenched with liquid nitrogen for 10 s. The bottom of the dish was in direct contact with liquid nitrogen while being open to the air on top. The solidified samples were frozen at -70 °C for 24 h and freeze-dried for a further 24 h to yield a porous asymmetric structure. The dried samples were neutralized several times by adding aqueous 1 N sodium hydroxide and then flushed thoroughly using deionized water to wash off the alkali solution. Finally, these samples were frozen at -70 °C for 24 h and then lyophilized for 24 h to form porous asymmetric CG membranes. Nanosized collagen I particles were than seeded into the porous CG membrane by the static seeding method,24 which drove a particle suspension into a porous scaffold by gravity-driven diffusional processes. Briefly, a 0.2 mg/mL collagen solution that contained nanosized collagen I particles was prepared in phosphate buffer solution (PBS). A total of 250 µL of collagen solution was pipetted onto the top surface of lower layer of CG membrane (1.5 × 1.5 cm) and then dried in a Laminar-Flow Hood to yield the final CGC membrane.

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Evaluation of the Cross-Linking Index. A ninhydrin assay was employed to evaluate the degree of cross-linking of CG membranes.25 Ninhydrin (2,2-dihydroxy-1,3-indanedione) was used to determine the amount of free amino groups in each test sample. The test sample was ground and then swelled in distilled water. After it had been heated with a ninhydrin solution at 100 °C for 20 min, the optical absorbance was recorded using a spectrophotometer (Spectronic Unicam Genesys 10, NY) at 570 nm using various known concentrations of glycine as standard. The amounts of free amino groups in the test sample before (Ci) and after (Cf) cross-linking are proportional to the optical absorbance of the solution.26 The cross-linking index was calculated as cross-linking index (%) ) (Ci - Cf)/Ci × 100 (%). The values are given as mean ( standard deviation (n ) 6). Measuring Swelling Ratio. Each CG membrane (1.5 × 1.5 cm) was immersed in a sealed tube containing PBS. After soaking for 3, 6, 12, 24, and 48 h at 37 °C, the swollen sample was removed from the tube, gently blotted with filter paper to remove surface liquid, and immediately weighed (Wwet). The swollen sample then was lyophilized and weighed (Wdry). The swelling ratio (∆W%) of each CG membrane at each time point was calculated using the formula, ∆W (%) ) (Wwet - Wdry)/Wdry × 100 (%). Evaluating Porosity. The porosity (average void volume) of the CG membranes was determined using the Archimedes principle. The exterior volume (Vs) of each CG membrane (1.5 × 1.5 cm) was measured using vernier calipers. The sample was then cut into pieces and immersed in pycnometer that contained distilled water. The actual volume (Vm) of the sample was calculated using the formula, Vm ) (Ww - W0) - (Wt - Wp), where Ww is the weight of water and the pycnometer, W0 is the weight of the dry pycnometer, Wt is the weight of water, the pycnometer, and the sample fragments, and Wp is the weight of the dry pycnometer and dry sample fragments. The porosity was determined using the following formula: porosity (%) ) (Vs - Vm)/Vs × 100 (%). The values are given as mean ( standard deviation (n ) 6). Morphological Evaluations. Scanning electron microscopy (SEM, Hitachi S-3000N, Japan) was employed to observe the morphologies of the asymmetric CG membranes. The test samples were lyophilized and then immediately sputter-coated with gold for further SEM observation. The size and distribution of the collagen I particles in the CGC membrane were determined using a field-emission scanning electron microscope (FE-SEM, Hitachi S-4800, Japan). Determination of In Vitro Biodegradation Rate. The biodegradation rate of the CG membrane was tested under simulated physiological conditions. Each CG membrane (1.5 × 1.5 cm) was lyophilized and weighed (Wi). The sample then was immersed in 20 mL of PBS (pH 7.4) containing 4 mg/mL of lysozyme at the temperature of 37 °C. After soaking for 7, 14, 21, and 28 d, the sample was removed from the degradation medium, lyophilized, and weighed (Wf). The weight loss percentage (∆W%) at each time interval was calculated using the following formula: ∆W (%) ) (Wi - Wf)/Wi × 100 (%). The degradation rate for each CG membrane then was determined from the relationship between its weight loss percentage and the soaking time. Preparation of Extracts from CG Membranes. Following sterilization with 60Co gamma ray irradiation at a dose of 15 kGy, each CG membrane was placed in a sterilized tube that was filled with aseptic distilled water and incubated at 37 °C. After soaking for 1, 4, 7, 14, 21, and 28 d, the extracts were collected for residual analysis. The concentrations of residual genipin and chitosan in the extracts were determined using a spectrophotometer at 241 and 218 nm, respectively. Isolation of Rat Fibroblasts and Cell Culture. Rat fibroblasts adopted herein were isolated from the skin of 2-4-day-old SpragueDawley rats. Before the beginning of the study, the ethical committee for animal experiments at the Central Taiwan University of Science and Technology, Taichung, Taiwan, approved the protocols. Under sterile conditions, rat skin was excised, stripped of lipid membranes and blood vessels using DMEM, washed with PBS, and digested

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Chen et al. Table 1. Cross-Linking Index of the CG Membranes as a Function of Genipin Concentration genipin (wt %)

cross-linking index (%)

0.125 0.25 0.375 0.5

16.2 ( 1.4 23.8 ( 2.3 24.4 ( 1.0 29.3 ( 0.7

Table 2. Porosity of the CG Membranes as a Function of Genipin Concentration

Figure 1. Effect of genipin concentrations on the swelling ratios of the CG membranes (n ) 3).

sequentially in collagenase solution. The isolated cells were rinsed and resuspended in a tissue culture medium (DMEM supplemented with 10% horse serum and 1% penicillin-streptomycin solution) before being plated into a 75 cm2 cell culture flask. Cell cultures were maintained in a humidified incubator with 5% CO2 at 37 °C. Culture medium was refreshed every 2 d. Cells were passaged at confluence. Rat fibroblasts at their second to third passage were used in all the experiments. Colorimetric MTT Assay for Cell Viability. Rat fibroblasts were used to determine the cytotoxicity of the composition of the CG membrane. A series of genipin and chitosan solutions in different concentrations were prepared to test the effects of genipin and chitosan on the growth of fibroblasts. The concentrations of genipin and chitosan solutions were in the ranges of 5-15 ppm and 5-20 ppm, respectively. The volume ratio of culture medium and the solutions to be evaluated was 1:1.27 In the control group, PBS was mixed with culture medium at 1:1 for cell cultures. A total of 100 µL of 5 × 104 cells/mL of cultured fibroblasts were seeded in individual wells of a 96-well tissue culture plate. The cells were cultured in a humidified atmosphere with 5% CO2 balance air incubator at 37 °C. After culturing for 2 d, the proliferation of fibroblasts was determined by MTT assay. The MTT assay is based on the reduction of MTT, a pale yellow soluble dye, by the mitochondrial succinate dehydrogenase, to form an insoluble dark-blue formazan product. Only viable cells with active mitochondria reduced substantial amount of MTT to formazan. After 2 d of culturing, the medium was replaced with 20 µL/well of MTT

genipin (wt %)

porosity (%)

0.125 0.25 0.375 0.5

57.7 ( 1.9 51.9 ( 3.6 48.8 ( 1.5 42.3 ( 3.3

solution (5 mg/mL) and 180 µL/well of culture medium. The plate was then incubated at 37 °C in a fully humidified atmosphere at 5% CO2 in air to enable the formation of formazan crystal. After 4 h of incubation, the solution was removed and 200 µL/well of acid isopropyl alcohol (0.04 M HCl in isopropyl alcohol) was added and mixed thoroughly to dissolve the formazan crystals. The dissolvable solution was jogged homogenously about 15 min at room temperature by the shaker. The optical density of the formazan solution was read on an ELISA plate reader (uQuant; Bio-Tek Instruments Inc.) at a wavelength of 570 nm and a reference wavelength of 650 nm. All experiments were repeated three times. Using the standard curve, the optical density values of the MTT assay were converted to cell numbers/well. Fibroblast Statically Cultured with the CG Membrane. The effects of cultured rat fibroblasts on the CG membrane were studied to evaluate directly the cytocompatibility of such a membrane. After it had been sterilized under 60Co gamma irradiation at a dose of 15 kGy, the CG membrane was placed in a 12 well culture dish, with each well seeded with 3 × 104 rat fibroblasts, immersed in 5 mL of culture medium, and then incubated at 37 °C in a 5% CO2 atmosphere for 2 d. For morphological observation, the cells on the CG membrane were washed with PBS three times to remove the nonadherent cells and then fixed using 2 vol % glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) for 48 h. After they had been thoroughly washed with PBS, the cells were dehydrated using graded ethanol (30, 50, 70, 80, 90, 95, and 100%) and then dried in a critical point drier (Hitachi HCP-2, Japan). The dried sample was immediately sputtered with gold. The morphology of the adhered fibroblasts was observed using SEM. Fibroblast Dynamically Cultured with the CGC Membrane. A spinner flask was used to perform the three-dimensional culture of

Figure 2. SEM micrographs of the CG membranes cross-linked with a different amount of genipin: (a) cross-section (0.125 wt %; ×30), (b) upper layer (0.125 wt %; ×100), (c) lower layer (0.125 wt %; ×100), (d) lower layer (0.125 wt %; ×800), (e) cross-section (0.25 wt %; ×30), (f) skin layer (0.25 wt %; ×100), (g) lower layer (0.25 wt %; ×100) and (h) lower layer (0.25 wt %; ×800).


Figure 3. FE-SEM micrographs of the CGC membrane containing nanosized collagen I particles.

Figure 4. Weight loss of the CG membranes cross-linked by 0.125 and 0.25 wt % of genipin during the soaking time (n ) 3).


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drier. The dry sample was sputtered with gold and observed using SEM. SEM examination was conducted to determine a suitable for the dynamically culturing fibroblasts for further animal studies. In Vivo Animal Evaluation. Adult male Sprague-Dawley rats weighing 300-350 g were used as experimental animals. The animals were housed in a manner consistent with national animal care guidelines. Before the beginning of the study, the ethical committee for animal experiments at the Central Taiwan University of Science and Technology, Taichung, Taiwan, approved the protocols. All animals were anaesthetized using Zoletil 50 and 2% Rompun solution (1:2 ratio, 1 mL/kg). The dorsal surface of each animal was shaved, sterilized with 10% povidone-iodine solution, and prepared for surgery, which was performed in an aseptic animal operation room. A full-thickness rectangular wound with a surface area of 2 × 2 cm was formed by dermoepidermic excisions on the back of each animal. Six rats were used to investigate the wound healing behavior. The wound was covered with the sterile CGC membrane, gauze, and a commercial wound dressing (Suile) of equal size. The lower layer of CGC membrane was placed on the wound. Then it was bound up with the sterile gauze. Wound repair was histologically evaluated. Anesthetized animals were killed 7 and 14 d postsurgery, respectively. At each transplantation time, three rats were operated on. The wounds and surrounding skin were excised and fixed in phosphate-buffered 10% formalin. Then they were washed with PBS and dehydrated using a graded series of ethanol solutions. The dehydrated samples were embedded in paraffin wax, thin-sectioned (10 µm), and stained with H&E reagent for histological observations. The reactions of tissue to the test samples were evaluated on the basis of the inflammation responses under an optical microscope (Olympus IX70, Japan). Statistical Analysis. All quantitative data were presented as mean ( standard deviation. Statistical differences among samples were evaluated by one-way analysis of variance followed by post hoc Fisher’s LSD multiple comparison test. Significance was regarded as probability p < 0.05.

Results and Discussion

Figure 5. Genipin and chitosan concentration released in the soaking solutions after the CG membrane cross-linked with 0.125 wt % of genipin had been soaked in distilled water.

fibroblasts in a CO2 incubator. A total of 50 mL of 2 × 102 cells/mL of cultured fibroblasts was statically seeded onto the biodegradable porous CGC membrane. After 5 mL of DMEM had been added, the cell-seeded scaffold was cultured at 37 °C in a 5% CO2 atmosphere for 1 d. At the end of incubation, the seed scaffold was placed in a spinner flask with stirring with a magnetic stirring bar at 70 rpm for 10 h and then at 50 rpm for 1, 7, and 14 d. The setup was placed at 37 °C in a humidified incubator that contained 5% CO2. The medium was replaced every 3 d. For morphological observation, each sample was washed three times with PBS and subsequently fixed using 2 vol % glutaraldehyde for 48 h. After that, the sample was washed with PBS, dehydrated through a series of graded ethanol solutions, and then dried in a critical point

Evaluation of the Cross-Linking Index. In this investigation, the chitosan was cross-linked with genipin at concentrations from 0.125 to 0.5 wt %. As stated in Table 1, the cross-linking index of CG membranes increased with the concentration of genipin from 0.125 to 0.5 wt % (p < 0.05), suggesting that the concentration of the genipin affected the degree of cross-linking of CG membranes. Measuring Swelling Ratio. Figure 1 plots the swelling ratios of CG membranes that were cross-linked with genipin at various concentrations. As shown, the swelling ratios decreased as the concentration of genipin increased before 12 h of soaking (p < 0.05), indicating that the swelling behaviors of the CG membranes seemed to depend on the number of hydrophilic free amino groups present. This result is consistent with earlier work. Jin et al.12 prepared chitosan-polyethylene oxide films that were cross-linked by genipin. The swelling ratio of the noncrosslinked film was roughly 10, and decreased approximately 4-fold with cross-linking. Furthermore, the attenuated swelling tendency of CG membranes was observed after 12 h of soaking, indicating that PBS had already saturated the CG membranes. After 48 h of soaking, the swelling ratios of CG membranes that had been cross-linked with 0.125, 0.25, and 0.375 wt % of genipin were in the range of 690-710% (p > 0.05). The high absorption of water by CG membranes was preferred for skin substitutes. Determination of Porosity. Table 2 reveals that the porosity of CG membranes was in the range of 46-58%. The CG membrane cross-linked with 0.125 wt % of genipin had the highest porosity of approximately 57.7% (p < 0.05) and that

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Figure 6. Effects of (a) genipin and (b) chitosan at various concentrations on the proliferation of fibroblasts, as determined by MTT assay (*p < 0.05).

Figure 7. SEM micrographs of rat fibroblasts cultured on the surface of (a) upper layer, (b) cross-section, and (c) lower layer of CG membrane cross-linked with 0.125 wt % of genipin after 2 d.

Figure 8. SEM pictures of rat fibroblasts attaching and adhering to the surface of lower layer of CGC membrane after dynamically culturing for (a) 1, (b) 7, and (c) 14 d.

cross-linked with 0.5 wt % of genipin had the lowest porosity of approximately 42.3% (p < 0.05), indicating that the porosity of the chitosan membranes decreased with cross-linking. Adekogbe et al.9 used dimethyl-3,3-dithio-bis′-propionimidate to cross-link chitosan as a scaffold in skin tissue engineering. They found that cross-linking significantly reduced the overall porosity of the scaffold. This observation is consistent with the results presented here. The porosity decreased as the concentrations of genipin increased for a given cross-linking period, because, as the cross-linking index increased, the CG membranes became less hydrophilic. When samples were less hydrophilic, the water was lost more rapidly, making the ice crystals smaller and fewer. A membrane with higher porosity had higher water adsorption capacity because of the increase of void to capillary-adsorbed water, which prevents the wound from accumulating fluid by adsorbing exudates.28 Morphological Evaluations. Figure 2 shows SEM micrographs of the cross-section, upper layer, and lower layer of CG membranes that were cross-linked with 0.125 and 0.25 wt % of genipin. The structure was not homogeneous. The pore size

of the lower layer that contacted the bottom of the glass dish was much smaller than that of the upper layer. Yao et al.29,30 prepared asymmetric chitosan-gelatin and chitosan-gelatinhyaluronic acid scaffolds by freezing and lyophilizing methods. They also found that the pore size of bottom layer directly contacting the cooling plate (-40 °C) was much smaller than that of the air contacting top layer. The difference in ice nucleation conditions probably contribute to the asymmetric structure.10 In this study, the bottom of the glass dish was in direct contact with liquid nitrogen. Therefore, the temperature of the bottom layer of the chitosan-genipin mixture was far below the water freezing point. It is generally accepted that, at a lower temperature, the number of crystal nuclei initially formed is larger than that it is at a higher freezing temperature.29 Thus, there are more ice crystal nuclei formed on the interface of chitosan-genipin mixture and glass dish, which results in smaller pores after lyophilizing in contrast to the air contacting upper layer of the mixture. In addition, a dense skin layer was obviously presented on the surface of CG membranes crosslinked with 0.25 wt % of genipin (Figure 2e), which could act


Figure 9. Macroscopic observations of the wounds treated with the (a)(b) CGC membrane, (c) Suile, and (d) gauze on the seventh postoperative day.

as a barrier to prevent bacterial invasion. However, it would reduce the water vapor transmission rate and depress the permeation of oxygen. As shown, the pores in lower layer of the CG membrane that was cross-linked with 0.125 wt % of genipin (Figure 2c,d) were much larger and greater in number than those cross-linked with 0.25 wt % of genipin (Figure 2g,h). The larger pores will provide space for the migration of cells and improves the diffusion of nutrients and metabolites throughout the membrane. In this study, nanosized collagen I particles were injected into the pores of the CG membrane to yield a CGC membrane. The larger pores allowed more collagen particles into the lower layer of the membrane. Furthermore, the free amino groups of chitosan interacted with the carboxyl groups of collagen to form ionic bonds.31 As shown in Table 1, the CG membrane that was cross-linked with 0.125 wt % of genipin had more free amino groups, suggesting that the CG membrane cross-linked with 0.125 wt % of genipin was more suitable for growing cells. Based on the measurements of cross-linking index, swelling ratios, porosity and morphology, the optimal concentration of genipin for cross-linking the chitosan in CG membranes was 0.125 wt %. Therefore, the CG membrane cross-linked with 0.125 wt % of genipin was adopted as the best scaffold in an animal study and used after injection of nanosized collagen I particles and seeding with fibroblasts. Figure 3 shows that the collagen I particles were uniformly distributed throughout the CGC membrane and had good spherical shapes with diameters of 20-30 nm. Determination of In Vitro Degradation Rate. Figure 4 plots weight losses after various periods of soaking of CG membranes cross-linked with 0.125 and 0.25 wt % of genipin. Most of the noncross-linked chitosan molecules were dissolved and released during the first 7 d of soaking. The curve revealed a lower rate of degradation after 7 d of soaking, even after CG membranes had been soaked in PBS containing lysozyme for 28 d. These results suggest that the concentration of genipin was enough to delay the degradation of chitosan. The release patterns and cytoxicity of the major components of the CG membrane crosslinked by 0.125 wt % of genipin will be further investigated.


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Determination of the Amounts of Genipin and Chitosan Released from the CG Membrane. Figure 5 presents residual genipin and chitosan released from the CG membrane crosslinked with 0.125 wt % of genipin. As shown, significant release of genipin and chitosan was observed during the first 7 d of soaking (p < 0.05). After 7 d, the release of genipin and chitosan continuously increased. The concentrations of residual genipin and chitosan in the extract on the 28th day were approximately 10 and 4 ppm, respectively. The increased concentration of genipin could result from genipin trapped in the network of the chitosan--genipin structure, which was released as the CG membrane began degrading. This result demonstrates that uncross-linked genipin may have remained in the CG membrane as the material was under preparation. Evaluation of Cytotoxicity of Genipin and Chitosan. Because the residual genipin concentration in the CG membrane treated with 0.125 wt % of genipin in the soaking solution was below 15 ppm, the rat fibroblasts were separately cultured with media that contained 5, 10, and 15 ppm of genipin to carry out the cytotoxic test. After 2 d of culture, the MTT assay indicated that the fibroblasts proliferated typically in the medium (Figure 6a). The cell numbers of fibroblasts were significantly decreased and lower than those in the control group (p < 0.05) when genipin concentration was 15 ppm. However, the cell number of medium containing 15 ppm of genipin (7.5 × 103) was still larger than the original cell number (5 × 103). Moreover, the residual genipin concentration in the CG membrane in the soaking solution was lower than 15 ppm. Therefore, the CG membrane cross-linked by 0.125 wt % of genipin should not result in cell death. As illustrated in Figure 6b, it was found that the cell number of fibroblasts was significantly increased and higher than that in the control (p < 0.05). The number of cells in the chitosan solution proliferated from 9.3 × 103 to 9.8 × 103 when chitosan concentrations increased from 5 to 20 ppm. These results demonstrate that the chitosan molecules released from the CG membrane cross-linked by 0.125 wt % of genipin could promote proliferation of fibroblasts. Fibroblast Statically Cultured with the CG Membrane. When cells come into contact with materials, they undergo morphological changes to stabilize the cell-material interface. The morphology of cells on materials surfaces is typically used to determine the cytocompatibility of biomaterials. Figure 7a, b, and c present the morphology of rat fibroblasts that are attached to the surfaces of the upper layer, cross-section, and lower layer, respectively, of the CG membrane that was crosslinked with 0.125 wt % of genipin after 2 d of culture. As shown, numerous fibroblasts were attached to the surface and the interior pores of the CG membrane. These cells were flattened with effective spreading, indicating that the CG membrane had good cytocompatibility and the residues released from the CG membrane had no toxic effect on the fibroblasts. Moreover, SEM examination verified that the internal porous microstructure was maintained throughout the culture period. Boucard et al.32 prepared a bilayer physical hydrogel of chitosan for regenerating skin. It comprised of a rigid protective gel-layer and a soft gellayer. However, no cell was observed inside the gels during the 100 d of healing because the mean porosity of both the rigid and soft layers was between 100 and 500 nm. In this work, the proliferation of cells inside the membrane favored the regeneration of skin. Fibroblast Dynamically Cultured with the CGC Membrane. A dynamic culture system was employed to improve the exchange of nutrients and waste products between the

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Figure 10. Histological examinations of the wounds treated with (a) CGC membrane, (b) Suile, and (c) gauze removed on postoperative day 7 (F, fibroblast; C, collagen; I, inflammatory response; original magnification, ×200).

Figure 11. Histological examinations of the wounds treated with the(a) CGC membrane, (b) Suile, and (c) gauze removed on postoperative day 14 (F, fibroblast; I, inflammatory response; original magnification, ×200).

interior and the exterior of the scaffold. Figure 8a, b, and c show the morphology of rat fibroblasts that were dynamically cultured on the lower layer surface of the CGC membrane for 1, 7, and 14 d, respectively. SEM observation reveals that, after culturing for 1 and 7 d, the attached cells were completely flattened and effectively spread. In contrast, cells exhibited aging and atrophy after 14 d of culture. Additionally, the SEM images seem to indicate that the number of cells after 7 d of culture exceeded that after 1 d of culture, suggesting that the optimal period for dynamic culture was 7 d. After 7 d of dynamic culture, the CGC membrane was adopted in the follow-up animal study. Animal Study. Figure 9 presents the macroscopic appearance of the wounds that were treated with the CGC membrane, Suile, and gauze on the seventh postoperative day. The CGC membrane dynamically cultured with fibroblasts for 7 d had adsorbed the wound exudates without any fluid accumulation, indicating that the CGC membrane effectively absorbed body fluids that exuded from the wounded area. The gauze was easily stripped from the CGC membrane surface (Figure 9a,b). In contrast, the wound exudates that leaked through the gauze (negative control) and the commercial wound dressing, Suile, were absorbed by the covering gauze, causing the gauze to adhere to the skin (Figure 9c,d), destroying newly regenerated tissue when it was removed, impairing the wound-healing process, and promoting scar formation in the healing area. Also, protein and electrolytes are thus lost from the wound surface. Figure 10 shows the histological results of the wounds that were covered with the CGC membrane, Suile, and gauze retrieved on postoperative day 7. As shown in Figure 10a, the wound treated with the CGC membrane exhibited a slight inflammatory reaction, the formation of fibroblasts, and the synthesis of collagen. Wound repair is a complex process. Fibroblast formation is the sine qua non of the healing process.33 During the initial inflammatory phase of wound healing, fibroblasts begin to enter the wound where they synthesis and later remodel new extracellular matrix material, of which

collagen is the main component.34 The wound covered with the gauze herein also formed fibroblasts, but a serious inflammatory response occurred (Figure 10c). A notable inflammatory reaction also occurred in the area that had been dressed with Suile (Figure 10b). At 14 d postsurgery, the regenerating epithelial tissue of the wound was present on the CGC membrane-covered wound. As displayed in Figure 11a, the epithelium was well-organized; the dermis had a considerable number of fibroblasts and collagen fibers. The newly synthesized collagen in the wound was well organized. These results all reveal perfect regeneration of the damaged tissue. However, no complete epidermis was formed when the wounds were treated with Suile and gauze (Figure 11b,c). Inflammatory responses with abundant polynuclear and macrophage inflammatory cells were still evident when the wound was treated with gauze, because bacteria easily came into contact with the wound surface. In contrast, almost no inflammatory response occurred when the CGC membrane was used, suggesting that CGC membrane can prevent bacterial invasion, and act as a body fluid controller to reduce the risk of wound dehydration, while allowing for drainage of wound exudates (Figure 9a,b). Animal test results demonstrate that the application of the CGC membrane promotes tissue reconstruction processes, indicating that this membrane is a potential skin substitute.

Conclusions In this investigation, a novel biodegradable membrane with a porous asymmetric structure, designed for use in skin tissue engineering, was prepared by freezing and lyophilizing method. Increasing the concentrations of genipin increases the degree of cross-linking of chitosan. When the concentration of genipin was 0.125 wt %, the membrane had an adequate swelling ratio, porosity, and pore size and favorable biocompatibility. Animal studies reveal that the asymmetric membrane promotes efficient


re-epithelialization of the wound, indicating perfect regeneration of damaged tissue. Results of this study establish the potential of the membrane as a scaffold in skin tissue regeneration. Acknowledgment. This work was financially supported by the National Science Council of the Republic of China, Taiwan (No. NSC93-2622-E-166-004-CC3).

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BM900238B

Asymmetric Chitosan Membrane Containing Collagen I Nanospheres

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