Fabrication Of Allogenic Cartilage In Tube Shape Lining With Epithelial By A Novel Copolymer Scaffold

Objective:一. To improve a viable and convenient method for human nasoseptal chondrocyte culture in vitro and study cell's biological character.二. To study the biodegradation in vitro and in vivo of a chitosan nonwoven cloth and its biocompatibility in vivo and to discuss a possibility of its using in the cartilage tissue engineering.三. To fabricate a novel copolymer scaffold made by Poly (dl -lactide-co-glycolide) (PLGA) embedding with chitosan nonwoven cloth with good mechanical properties , compliance and three-dimensional microstructure, which can be used in cartilage repairing for tissue engineering, and to explore a novel method for developing polymeric scaffolds.四. To study the feasibility of the formation of tissue engineered cartilage in tube shape lining with epithelial by seeding allogenic chondrocytes onto a novel copolymer scaffold, which made by Poly (lactide-co-glycolide) (PLGA) embedding with chitosan nonwoven cloth.Methods:一. The chondrocytes were obtained by incomplete enzyme digestion of cartilage and were seeded with small piece of cartilage in 25 ml culture flasks coated with polylysine previously.This study determines the biologic synthesis of the chondrocytes by toluidine blue and type II collagen immunohistochemistry reaction. The proteoglycan synthesisefficiencies of cryopreservation on human nasoseptal chondrocytes cultured in vitro were measured by using 35S-Na2SO4 incorporation. We also compare the modified method with the conventional method in these synthesis.—. In vitro group, the chitosan nonwoven cloth was steeped in 1% or 0.1% bacteriolysin solution and bathed in a 37 °C vibrated water bath to observe the degradation rate of it in the time sequence. In vivo group, implant the chitosan nonwoven cloth into subcutaneous tissues of rabbits and histologilly investigated 2, 4, 8, 12 weeks after implantation to study the biodegradation and biocompatibility.JE￡. Double qualities of PLGA were lysised in chloroform and soaked the chitosan nonwoven cloth in the solutions and then bake to dry. Those to make them intersect steady.0. Allogenic chondrocytes were obtained from the ears of rabbits aged one month. After cultured in vitro for 3~4 generations, The cells were seeded onto the copolymer scaffold so as to form polymer graft, and then implanted to the rabbits. After 6, 12 and 18 weeks, the general and histological characteristics were investigated and the samples were stained with haematine-eosin, toluidine blue and Masson's trichrome.Result:—. The results suggest that human nasoseptal chondrocytes cultured by modified method still show the normal structure and morphology,and maintain the function of metabolism and selfduplication.The cell survival rate is higher and the quantity is more.—. In vitro group, the chitosan nonwoven cloth has been degraded 9.36% when steeped for 2 weeks. In vivo group, the chitosan nonwoven cloth has been degraded distinctly in the subcutaneous tissues.The chitosan nonwoven cloth remained its primary shape roughly and had the suitable degradation in vitro and in vivo and showed ideal biocompatibility by histologic analysis and general observation.H. The novel scaffolds made by this method had porosity of 82% to 86% respectively, their pore size ranging from 100 to 300 um.0. The matrix like cobweb was observed about 1 week after seeding chondrocytes onto the copolymer scaffold .After 6 weeks of implanting, the matrix was secreted and there were immature chondrocytes in the chondrocyte/ scaffold polymer. After 12weeks, the novel cartilage in tube shape lining with epithelial was fabricated, chondrocytes were nearly mature and formed cave. After 18 weeks, neocartilage was close to normal cartilage.Conclusion:—. The modified method is a viable and convenient method.—. The chitosan nonwoven cloth has different degradability in different media. The degradation rate is correspond to multiple factors in vivo. The chitosan nonwoven cloth had ideal characteristic of degradation and biocompatibility.H. The scaffolds made in this way possess stable three-dimensional microstructure with controllable parameters and have stable biomechanical performance, which can be applicable for tissue engineering.0. It is feasible to form neocartilage in tube shape lining with epithelial by seeding allogenic chondrocytes onto a novel copolymer scaffold , which made by Poly (lactide-co-glycolide) (PLGA) embedding with chitosan nonwoven cloth.