In this study, a novel freeze-gelation method instead of the conventional freeze-drying method was used to fabricate porous chitosan/collagen-based composite scaffolds for skin-related tissue engineering applications. To improve the performance of chitosan/collagen composite scaffolds, we added 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and amino acids (including alanine, glycine, and glutamic acid) in the fabrication procedure of the composite scaffolds, in which amino acid molecules act as crosslinking bridges to enhance the EDC-mediated crosslinking. This novel combination enhanced the tensile strength of the scaffolds from 0.70 N/g for uncrosslinked scaffolds to 2.2 N/g for crosslinked ones; the crosslinked scaffolds also exhibited slower degradation rates. The hydrophilicity of the scaffolds was also significantly enhanced by the addition of amino acids to the scaffolds. Cell compatibility was demonstrated by the in vitro culture of human skin fibroblasts on the scaffolds. The fibroblasts attached and proliferated well on the chitosan/collagen composite scaffolds, especially the one with glutamic acid molecules as crosslinking bridges, whereas cells did not grow on the chitosan scaffolds. Our results suggest that the collagen-modified chitosan scaffolds with glutamic acid molecules as crosslinking bridges are very promising biomaterials for skin-related tissue engineering applications because of their enhanced tensile strength and improved cell compatibility with skin fibroblasts.
The development of tissue engineering provides a novel approach to restore bodily functions by seeding cells onto various scaffolds. Although chitosan is a non-toxic biomaterial, its cytocompatibility still needs to be improved. In this study, gamma-poly(glutamic acid) (γ-PGA) was blended with chitosan to prepare both dense and porous γ-PGA/chitosan composite scaffolds using the freeze-gelation method. This method saves time and energy, and there is less residual solvent. SEM micrographs demonstrated that an interconnected porous structure with a pore size of 30-100 micrometer was present in the scaffolds. The hydrophilicity of the scaffolds was significantly improved by γ-PGA. Further, the tensile strength of the porous γ-PGA-modified chitosan scaffolds was about 50% higher than that of the unmodified chitosan scaffolds. The number of osteosarcoma cells cultured on the γ-PGA-modified scaffolds was about double that on the unmodified chitosan scaffolds on day 7. Thus, the γ-PGA/chitosan composite scaffolds, due to their better hydrophilicity, cytocompatibility, and mechanical strength, are very promising biomaterials for tissue engineering applications. We further demonstrated the use of glutamic acid to enhance the tensile strength of chitosan-based composite porous scaffolds. The tensile strength of the chitosan/collagen composite scaffolds was increased by more than 2 times with the addition of glutamic acids as cross-linking bridges. We found that the hepatocytes attached and proliferated well on these composite scaffolds, demonstrating that the glutamic acid modified-chitosan composite scaffolds are also potential tissue engineering biomaterials.
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