Performance Of Recombinant Human Collagen Ii Tissue Engineering Scaffolds And Biocompatibility

With the development of tissue engineering, biomaterial has attracted more and more attention. As a kind of natural biomaterial, collagen has low antigenicity, biodegradability, good biocompatibility and can promote cell migration and proliferation. Collagen has been widely used in tissue engineering field. RHLCⅡ(recombinant human-like collagenⅡ) as a kind of recombinant collagen was produced by high cell-density cultivation of recombinant Escherichia coli containing a partial cDNA that was reversed by mRNA from human collagen. Comparing with the collagen from animal tissues, RHLCⅡcan be free of virus and easily industrialized by large scale. The preparation of tissue engineering scaffold is the precondition of the application of RHLCⅡin tissue engineering field. In this paper, RHLCⅡwas crosslinked to form membranes and sponges with glutaraldehyde. We addressed on the study of the effect of various concentrations of glutaraldehyde on properties and biocompatibility of RHLCⅡscaffold.1. RHLCⅡwas crosslinked with various concentrations (0.01％, 0.025％, 0.05％, 0.075％, 0.1％) of glutaraldehyde to form RHLCⅡmembranes by natural drying. RHLCⅡmembranes was obtained when concentration of glutaraldehyde used was above 0.025％. Cytotoxicity of RHLCⅡmembranes was evaluated by BHK-21. The reaction scales was 1 level and cytotoxicity met the the requirement of standards. After being seeded on RHLCⅡmembranes, attachment of fibroblasts was observed with contrast and scanning electronic microscope. The proliferation of fibroblasts was tested by MTT and there was no significant difference on different RHLCⅡmembranes (P＞0.05). Furthermore, when the concentration of glutaraldehyde used was higher than 0.05％, tensile strength of RHLCⅡmembranes can reach to 11MPa.In conclusion, 0.05％was optimal concentration of glutaraldehyde to form RHLCⅡmembranes by natural drying.2. Crosslinking technics was improved to make the memebranes dry with sterile air. RHLCⅡwas crosslinked with various concentrations (0.05％and 0.1％) of glutaraldehyde to form RHLCⅡmembranes by new technics. Cytotoxicity of both RHLCⅡmembranes met the requirement of standards. The reaction scales was 0-1 level. Fibroblasts could proliferate and adhere to both RHLCⅡmembranes. Proliferation of fibroblasts on both RHLCⅡmembranes showed no significant difference(P＞0.05). Fibroblasts formed several confluent layers on both RHLCⅡmembranes by observation of scanning electronic microscope Furthermore, tensile strength of both RHLCⅡmembranes can reach to 11MPa. In conclusion, 0.05％concentration of glutaraldehyde was chosen when RHLCⅡmembranes was obtained with sterile air.3. RHLCⅡwas crosslinked with various concentrations (0.01％,0.05％,0.1％,0.15％,0.2％) of glutaraldehyde to form RHLCⅡsponges by freeze drying. RHLCⅡsponges could be obtained when the concentration of glutaraldehyde used was above 0.05％. Cytotoxicity of RHLCⅡsponges met the requirement of standards. The reaction scales was 0-1 level. The obvervation of contrast, scanning electronic and fluorescence microscope and the results of HE staining of frozen section showed good attachment and growth of fibroblasts on RHLCⅡsponges. The proliferation of the fibroblasts on RHLCⅡsponges with (0.1％-0.2％) concentrations of glutaraldehyde showed no significant difference(P＞0.05) and was significantly better than RHLCⅡsponges with 0.05％concentrations of glutaraldehyde(P＜0.05)by cell counting. RHLCⅡsponges had more than 90％porsity and tensile strength could reach to 0.11MPa when concentration of glutaraldehyde was above 0.1％. In conclusion, 0.1％was optimal concentration of glutaraldehyde to form RHLCⅡsponges.