Bio-Medical Materials and Engineering 24 (2014) 633–641 DOI 10.3233/BME-130851 IOS Press
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Fabrication of Photo-crosslinked ChitosanGelatin Scaffold in Sodium Alginate Hydrogel for Chondrocyte Culture Peng Zhao a*§, Cuijun Deng a §, Hongzhen Xu b, Xing Tang a, Hailong He c, Chao Lin a*, and Jiansheng Su b* a
The Institute for Biomedical Engineering and Nanoscience, School of Medicine, Tongji University, Shanghai, 200092, P.R. China. b Laboratory of Oral Biomedical Science and Translational Medicine, School of Stomatology, Tongji University, Shanghai, 200072, P.R. China c Department of Orthopedics, Changzheng Hospital of Second Military Medical University, Shanghai 200003, P.R..China
Abstract. Photo-crosslinked chitosan-gelatin scaffolds were fabricated and applied for chondrocyte culture in vitro. Photocurable methacryloyl chitosan was synthesized and characterized by FTIR and 1H NMR, respectively. Microstructure and mechanical properties of the chitosan-gelatin scaffold treated with or without EDC as crosslinking agent were analyzed by scanning electronic microscopy (SEM), compression and viscoelastic measurement. It is demonstrated that EDC-treated chitosan-gelatin scaffold possesses better porous structure and improved mechanical properties. Photo-crosslinked chitosangelatin scaffold could be further integrated in sodium alginate hydrogel using calcium chloride to support proliferation of chondrocytes for over 21 days and maintain spherical phenotype, as evaluated by AlamarBlue assay and SEM, respectively, implying that the chitosan-gelatin-hydrogel system exhibits great cyto-biocompatibility. Results of this study show that photo-crosslinked chitosan-gelatin scaffold in sodium alginate hydrogel is suited as a scaffold candidate for cartilage tissue engineering. Keywords: chitosan, gelatin, chondrocyte, hydrogel, cartilage tissue engineering
1. Introduction Tissue engineering represents a novel strategy for injured cartilage therapy [1, 2]. For cartilage tissue regeneration, scaffold is a critical component which provides spatial frame for cell proliferation and promotes cell proliferation, migration, and differentiation [3]. A few types of synthetic polymers have been studied extensively for the preparation of scaffolds in cartilage tissue engineering due to the low-toxicity and degradability [4, 5]. For example, both poly(glycolic acid) and poly (lactide) exhibit excellent plasticity and mechanical strength, and can be modified to meet different demands in tissue *
Corresponding author. E-mail: [email protected] (PZ), [email protected] (CL), [email protected] (JS) The two authors contribute equally as co-first author. Tel.: +86 21 6598 8029; Fax: +86 21 6598 3706-0.
§
0959-2989/14/$27.50 © 2014 – IOS Press and the authors. All rights reserved
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P. Zhao et al. / Fabrication of photo-crosslinked chitosan-gelatin scaffold in sodium alginate hydrogel
engineering, but the inherent lack of biological signaling nature and inflammation caused by acidic degradation products significantly impede further clinical application [6, 7]. Opposed to synthetic polymers, biopolymers including collagen, gelatin, chitosan and fibrin possess superior cellular adhesive nature, biological signaling, and biodegradability in vivo [8, 9]. Moreover, these biopolymers can be fabricated to form scaffolds through hydrogen bonding, ionic interactions or chemical cross-linking. An ideal scaffold for cartilage tissue engineering is normally composed of two components-porous scaffold (sponges, fibrous meshes, nanofibers) and hydrogel [10]. Porous scaffolds have the advantage of high mechanical strength, but the pore size is hard to be regulated, and a small pore size may inhibit cells smoothly penetrating into the interior of scaffold, while a large one could lead to the leakage of cells from scaffolds. Alternatively, hydrogels consist of crosslinked polymer network containing a large amount of water, similar to natural cartilage matrix environment [11, 12]. This property endows hydrogel to be an ideal scaffold for chondrocyte culture and cartilage regeneration [13]. Moreover, the cells could be facilely and homogeneously integrated into hydrogels. However, hydrogels generally possess poor mechanical strength compared to porous scaffolds. For these reasons, combining porous scaffold with hydrogel will provide an ideal scaffold with favorable mechanical and biological properties for cartilage regeneration. Chitosan, a deacetylated derivative of chitin, has been widely applied bio-medically due to its biocompatibility, biodegradability, and low immunogenicity [14]. Since N-acetyl-glucosamine residue of chitosan is also present in the glycosaminoglycans (GAGs) of cartilage matrix, chitosan is a promising biomaterial for promoting chondrocyte proliferation and maintaining its phenotype [15, 16]. However, chitosan is not conducive to cell adhesion. Alternatively, gelatin is a denatured collagen that can promote chondrocyte adhesion, migration, and differentiation [17-19]. As such, alginate-based scaffolds were investigated in this paper for chondrocyte culture due to its favorable cellular response [20], and photo-curable chitosan was synthesized on the theoretical basis of acylation chemistry to coupling benzoyl and methacryloyl group to local chitosan. Then, porous chitosan-gelatin porous scaffolds were fabricated by photo-crosslinking and subsequent particle-leaching method. Microstructure and mechanical properties of the scaffolds were characterized. Besides, chitosan-gelatin scaffold in sodium alginate hydrogel was constructed and its potential for chondrocyte culture was evaluated in vitro. It is indicated that photo-crosslinked, chitosan-gelatin scaffold in sodium alginate hydrogel is a potential material for cartilage tissue engineering. 2. Materials and Methods 2.1. Materials Chitosan with high molecular weight (Sigma-Aldrich) and Iragure 2959 (Ciba Specialty Chemicals) were purchased. Dimethyl sulfoxide (DMSO), methane sulfonic acid, benzoyl chloride, methacryloyl chloride and 1-ethyl-(3-3-dimethylaminopropyl carbodiimide hydrochloride) (EDC) were purchased from Aladdin. 2.2. Synthesis of light-curable chitosan Chemically modified light-curable chitosan was synthesized according to the method described previously [21]. Briefly, 2 g of chitosan was completely dissolved in 45 g of methane sulfonic acid by constantly stirring. Followed by dropwise adding the mixture of benzoyl chloride (2.2 g) and methac-
P. Zhao et al. / Fabrication of photo-crosslinked chitosan-gelatin scaffold in sodium alginate hydrogel
635
ryloyl chloride (2.45 g), the solution was stirred at room temperature for 3-4 hours. Precipitate was obtained in cold water by neutralization with aqueous ammonia. After repeated filtering and rinsing, the product was dried with P2O5 in vacuum for 2 days and stored at 4 ºC until required. Chemical structure of the light-curable chitosan was characterized using a FTIR–ATR (Research Series100, Madison Instrument, Madison, WI) and 1H NMR (Varian INOVA-400 (9.4 T), Varian, Walnut Creek, CA) operated at 400 MHz proton with d-DMSO as solvent. 2.3. Fabrication of 3D porous scaffolds 1 g of light-curable chitosan was dissolved in 20 g of DMSO containing 5 wt % of gelatin by stirring to obtain a 5 wt % gelatin-5 wt % photocurable chitosan-DMSO mixture. Then 10 mg of Iragure 2959 was added to the mixture. In order to obtain porous scaffolds, salt crystal (NaCl, 250-380 μm) was used as porogen. Firstly, the mold (8 mm in diameter and 8 mm in height) was filled with salt crystals. Then, added the solution dropwise into the salt pile and filled the interspaces between salt crystals with liquid, and then exposed the mixture to UV light until it was completely cured. Discs generated from mixture were immersed in DI water and washed for several times to remove the remaining solvent and salt. In order to improve mechanical strength of the photocured discs, one set of discs were further crosslinked after immersed into 1:4 (v/v) water-acetone containing 1% of EDC overnight and then washed with DI water. Morphology of the discs was determined by SEM. The photocured porous scaffolds were freezedried at -20 ºC, and then coated with gold before SEM analysis. Compressibility of the scaffolds with or without crosslinking gelatin with EDC was measured using a dynamic mechanical analyzer (DMA 8000). 2.4. Biocompatibility and bioactivity in vitro 2.4.1. Chondrocytes culture and seeding Third-passage chondrocytes harvested from rabbit were used for cyto-biocompatibility testing [22]. The chondrocytes (2×105 cells) in sodium alginate solution were immersed in sterilized photocurable chitosan-gelatin scaffold (6 mm in diameter and 2.5 mm in thickness) and encapsulated in sodium alginate gel with calcium chloride in a 96-well plate. The plate was maintained in an incubator at 37 ºC for 1 h to allow cells infiltrating scaffold pores. High glucose DMEM (200 l) containing 10 wt % of FBS, 1 wt % of antibiotics and 1 wt % of ascorbic acid was renewed every 2 days. The chondrocytescaffold construct was incubated in the culture medium at 37 °C for 3 weeks. At different times, SEM and AlamarBlue assay were employed to determine the adhesion and phenotype of chondrocytes in the scaffold and evaluate cell proliferation. 2.4.2. SEM characterization Morphology of the complex consisting of chondrocytes and scaffolds was investigated with SEM (Hitachi S-2360N). All specimens were fixed by glutaraldehyde (2.5 wt %) and dehydrated with graded ethanol. Then these samples were dried at critical point and coated with gold before SEM. 2.4.3. Alamar blue assay The toxicity of scaffolds was evaluated via AlamarBlue assay to quantify the proliferation of chondrocytes. The cell-scaffold constructs were placed in 96-well plates with 200 ul of DMEM and 10 wt % of FBS per well. 10 v % of AlamarBlue solution was added in each well and incubated in hu-
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P. Zhao et al. / Fabrication of photo-crosslinked chitosan-gelatin scaffold in sodium alginate hydrogel
midified incubator at 37 ºC with 5 v % of CO2 for 4 h. The supernatants were collected and measured at 570 nm with a MK3 spectrophotometer (Thermo Fisher Scientific, USA). 2.5. Statistical Analysis All values were expressed as means ± SD (standard deviation). Student’s t test was conducted to compare differences between two groups using SPSS 14.0, which were considered to be significant when p
633
Fabrication of Photo-crosslinked ChitosanGelatin Scaffold in Sodium Alginate Hydrogel for Chondrocyte Culture Peng Zhao a*§, Cuijun Deng a §, Hongzhen Xu b, Xing Tang a, Hailong He c, Chao Lin a*, and Jiansheng Su b* a
The Institute for Biomedical Engineering and Nanoscience, School of Medicine, Tongji University, Shanghai, 200092, P.R. China. b Laboratory of Oral Biomedical Science and Translational Medicine, School of Stomatology, Tongji University, Shanghai, 200072, P.R. China c Department of Orthopedics, Changzheng Hospital of Second Military Medical University, Shanghai 200003, P.R..China
Abstract. Photo-crosslinked chitosan-gelatin scaffolds were fabricated and applied for chondrocyte culture in vitro. Photocurable methacryloyl chitosan was synthesized and characterized by FTIR and 1H NMR, respectively. Microstructure and mechanical properties of the chitosan-gelatin scaffold treated with or without EDC as crosslinking agent were analyzed by scanning electronic microscopy (SEM), compression and viscoelastic measurement. It is demonstrated that EDC-treated chitosan-gelatin scaffold possesses better porous structure and improved mechanical properties. Photo-crosslinked chitosangelatin scaffold could be further integrated in sodium alginate hydrogel using calcium chloride to support proliferation of chondrocytes for over 21 days and maintain spherical phenotype, as evaluated by AlamarBlue assay and SEM, respectively, implying that the chitosan-gelatin-hydrogel system exhibits great cyto-biocompatibility. Results of this study show that photo-crosslinked chitosan-gelatin scaffold in sodium alginate hydrogel is suited as a scaffold candidate for cartilage tissue engineering. Keywords: chitosan, gelatin, chondrocyte, hydrogel, cartilage tissue engineering
1. Introduction Tissue engineering represents a novel strategy for injured cartilage therapy [1, 2]. For cartilage tissue regeneration, scaffold is a critical component which provides spatial frame for cell proliferation and promotes cell proliferation, migration, and differentiation [3]. A few types of synthetic polymers have been studied extensively for the preparation of scaffolds in cartilage tissue engineering due to the low-toxicity and degradability [4, 5]. For example, both poly(glycolic acid) and poly (lactide) exhibit excellent plasticity and mechanical strength, and can be modified to meet different demands in tissue *
Corresponding author. E-mail: [email protected] (PZ), [email protected] (CL), [email protected] (JS) The two authors contribute equally as co-first author. Tel.: +86 21 6598 8029; Fax: +86 21 6598 3706-0.
§
0959-2989/14/$27.50 © 2014 – IOS Press and the authors. All rights reserved
634
P. Zhao et al. / Fabrication of photo-crosslinked chitosan-gelatin scaffold in sodium alginate hydrogel
engineering, but the inherent lack of biological signaling nature and inflammation caused by acidic degradation products significantly impede further clinical application [6, 7]. Opposed to synthetic polymers, biopolymers including collagen, gelatin, chitosan and fibrin possess superior cellular adhesive nature, biological signaling, and biodegradability in vivo [8, 9]. Moreover, these biopolymers can be fabricated to form scaffolds through hydrogen bonding, ionic interactions or chemical cross-linking. An ideal scaffold for cartilage tissue engineering is normally composed of two components-porous scaffold (sponges, fibrous meshes, nanofibers) and hydrogel [10]. Porous scaffolds have the advantage of high mechanical strength, but the pore size is hard to be regulated, and a small pore size may inhibit cells smoothly penetrating into the interior of scaffold, while a large one could lead to the leakage of cells from scaffolds. Alternatively, hydrogels consist of crosslinked polymer network containing a large amount of water, similar to natural cartilage matrix environment [11, 12]. This property endows hydrogel to be an ideal scaffold for chondrocyte culture and cartilage regeneration [13]. Moreover, the cells could be facilely and homogeneously integrated into hydrogels. However, hydrogels generally possess poor mechanical strength compared to porous scaffolds. For these reasons, combining porous scaffold with hydrogel will provide an ideal scaffold with favorable mechanical and biological properties for cartilage regeneration. Chitosan, a deacetylated derivative of chitin, has been widely applied bio-medically due to its biocompatibility, biodegradability, and low immunogenicity [14]. Since N-acetyl-glucosamine residue of chitosan is also present in the glycosaminoglycans (GAGs) of cartilage matrix, chitosan is a promising biomaterial for promoting chondrocyte proliferation and maintaining its phenotype [15, 16]. However, chitosan is not conducive to cell adhesion. Alternatively, gelatin is a denatured collagen that can promote chondrocyte adhesion, migration, and differentiation [17-19]. As such, alginate-based scaffolds were investigated in this paper for chondrocyte culture due to its favorable cellular response [20], and photo-curable chitosan was synthesized on the theoretical basis of acylation chemistry to coupling benzoyl and methacryloyl group to local chitosan. Then, porous chitosan-gelatin porous scaffolds were fabricated by photo-crosslinking and subsequent particle-leaching method. Microstructure and mechanical properties of the scaffolds were characterized. Besides, chitosan-gelatin scaffold in sodium alginate hydrogel was constructed and its potential for chondrocyte culture was evaluated in vitro. It is indicated that photo-crosslinked, chitosan-gelatin scaffold in sodium alginate hydrogel is a potential material for cartilage tissue engineering. 2. Materials and Methods 2.1. Materials Chitosan with high molecular weight (Sigma-Aldrich) and Iragure 2959 (Ciba Specialty Chemicals) were purchased. Dimethyl sulfoxide (DMSO), methane sulfonic acid, benzoyl chloride, methacryloyl chloride and 1-ethyl-(3-3-dimethylaminopropyl carbodiimide hydrochloride) (EDC) were purchased from Aladdin. 2.2. Synthesis of light-curable chitosan Chemically modified light-curable chitosan was synthesized according to the method described previously [21]. Briefly, 2 g of chitosan was completely dissolved in 45 g of methane sulfonic acid by constantly stirring. Followed by dropwise adding the mixture of benzoyl chloride (2.2 g) and methac-
P. Zhao et al. / Fabrication of photo-crosslinked chitosan-gelatin scaffold in sodium alginate hydrogel
635
ryloyl chloride (2.45 g), the solution was stirred at room temperature for 3-4 hours. Precipitate was obtained in cold water by neutralization with aqueous ammonia. After repeated filtering and rinsing, the product was dried with P2O5 in vacuum for 2 days and stored at 4 ºC until required. Chemical structure of the light-curable chitosan was characterized using a FTIR–ATR (Research Series100, Madison Instrument, Madison, WI) and 1H NMR (Varian INOVA-400 (9.4 T), Varian, Walnut Creek, CA) operated at 400 MHz proton with d-DMSO as solvent. 2.3. Fabrication of 3D porous scaffolds 1 g of light-curable chitosan was dissolved in 20 g of DMSO containing 5 wt % of gelatin by stirring to obtain a 5 wt % gelatin-5 wt % photocurable chitosan-DMSO mixture. Then 10 mg of Iragure 2959 was added to the mixture. In order to obtain porous scaffolds, salt crystal (NaCl, 250-380 μm) was used as porogen. Firstly, the mold (8 mm in diameter and 8 mm in height) was filled with salt crystals. Then, added the solution dropwise into the salt pile and filled the interspaces between salt crystals with liquid, and then exposed the mixture to UV light until it was completely cured. Discs generated from mixture were immersed in DI water and washed for several times to remove the remaining solvent and salt. In order to improve mechanical strength of the photocured discs, one set of discs were further crosslinked after immersed into 1:4 (v/v) water-acetone containing 1% of EDC overnight and then washed with DI water. Morphology of the discs was determined by SEM. The photocured porous scaffolds were freezedried at -20 ºC, and then coated with gold before SEM analysis. Compressibility of the scaffolds with or without crosslinking gelatin with EDC was measured using a dynamic mechanical analyzer (DMA 8000). 2.4. Biocompatibility and bioactivity in vitro 2.4.1. Chondrocytes culture and seeding Third-passage chondrocytes harvested from rabbit were used for cyto-biocompatibility testing [22]. The chondrocytes (2×105 cells) in sodium alginate solution were immersed in sterilized photocurable chitosan-gelatin scaffold (6 mm in diameter and 2.5 mm in thickness) and encapsulated in sodium alginate gel with calcium chloride in a 96-well plate. The plate was maintained in an incubator at 37 ºC for 1 h to allow cells infiltrating scaffold pores. High glucose DMEM (200 l) containing 10 wt % of FBS, 1 wt % of antibiotics and 1 wt % of ascorbic acid was renewed every 2 days. The chondrocytescaffold construct was incubated in the culture medium at 37 °C for 3 weeks. At different times, SEM and AlamarBlue assay were employed to determine the adhesion and phenotype of chondrocytes in the scaffold and evaluate cell proliferation. 2.4.2. SEM characterization Morphology of the complex consisting of chondrocytes and scaffolds was investigated with SEM (Hitachi S-2360N). All specimens were fixed by glutaraldehyde (2.5 wt %) and dehydrated with graded ethanol. Then these samples were dried at critical point and coated with gold before SEM. 2.4.3. Alamar blue assay The toxicity of scaffolds was evaluated via AlamarBlue assay to quantify the proliferation of chondrocytes. The cell-scaffold constructs were placed in 96-well plates with 200 ul of DMEM and 10 wt % of FBS per well. 10 v % of AlamarBlue solution was added in each well and incubated in hu-
636
P. Zhao et al. / Fabrication of photo-crosslinked chitosan-gelatin scaffold in sodium alginate hydrogel
midified incubator at 37 ºC with 5 v % of CO2 for 4 h. The supernatants were collected and measured at 570 nm with a MK3 spectrophotometer (Thermo Fisher Scientific, USA). 2.5. Statistical Analysis All values were expressed as means ± SD (standard deviation). Student’s t test was conducted to compare differences between two groups using SPSS 14.0, which were considered to be significant when p
