Experimental Study On Constructing The Tissue Engineering Cartilage In Vitro

Objective: To study the effect of cell density on the cell proliferation and matrix synthesis of chondrocytes embedded in fibrin gel in vitro, on the volum change of fibrin gel. To discuss the best appropriate cell density using to construct the tissue engineering cartilage in vitro.Methods: Chondrocytes were isolated from harvested articular cartilage of the 3-week-old Japanese white rabbits using sequential trypsin and collagenase digestion, and were cultured. Third passage chondrocytes were collected and embedded in fibrin gel at a density of 1×10~6/ml (A group), 10 × 10~6/ml (B group) and 50×10~6/ml (C group) , respectively. The morphology , structure , cell density and composition of chondrocyte-fibrin gels was observed and semi-measured by histochemical stain , immunohistochemical stain and scan electron microscope. The content of glycosaminoglycans(GAG) was measured by DMMB spectrometry. The number of chondrocytes in chondrocyte-fibrin gels were counted by trypan-blue exclusion test in order to calculate the cell proliferation. Results: The chondrocytes could been resuspended uniformly in fibrin gelsand could keep live during preparing the chondrocyte-fibrin gels, and the mixture formed a solid with desired shape and did not break down. The number of chondrocytes in gel samples from group A and B were increased during the 3-week culture in vitro, and reached the peak on the 3rd week, 4.532 fold and 2.097 fold increase respectively. The number of chondrocytes in gel samples from group C reached the acme on the 3rd day and increased 1.147 fold, then decreased to the lowerest on the 3rd week, 0.907 fold. In contrast, most chondrocytes in the C group showed increased capability to produce GAG and type II collagen, however, this made the gel contracted seriously in the early stage.Conclusion: The proliferation, and GAG and typeII collagen sythesis of chondrocyte influenced by the cell density. The 50 X 106/ml group(C group) inhibited the proliferation, but the 1X 106/ml group(A group) promoted the proliferation. The high cell density promoted the matrix synthesis. Under ours experimental conditions, 50 X 106/ml group is the most appropriate cell density.Experimental TwoAllogeneic acellular extracellular matrix as a scaffold for cartilage reconstruction in vitroObjective: To study the feasibility of applying the small intestinal submucosa(SIS) and allogenous acellular cartilaginous matrix(ACM) as the scaffold in constructing tissue engineering cartilage in vitro. Methods: We processed the bovine cartilaginous matrix by combination of lyophilization (freeze dry) and repeated freeze-thaw(12 cycles) and enzyme digestion in order to remove the cell component. Small intestine submucosawas obtained from the small intestine of specific pathogen-free pigs. The tunica submucosa layer was isolated from the mucosal, muscular, and serosal layers by gentle mechanic abrasion. The SIS was made acellular by combination of detergent and enzyme digestion. The 3rd passage chondrocytes were seeded onto the SIS and ACCM , and had been cultured for 3 weeks. The cell growth, attachment and distribution were detected by histochemical stain ^ immunohistochemical stain and scan electron microscope.Results: The chondrocytes could adhere and grow well on the matrix surface, and synthesize a large of the GAG and type II collagen. However, the chondrocytes grew only on the surface and superficial layer of the scaffold, they did not move into the inner part of the scaffold.Conclusion: Both SIS and ACM have good cellular compatibility without cytotoxicity and provide temporary substrate to which these anchorage-dependent cells can adhere, and stimulate the chondrocytes anchored on the scaffold to proliferate and keep differentiated phenotype. It needs to study furtherly to promote the ability of chondrocyte chemotaxis in order to distribute the chondrocytes into the whole scaffold uniformly.Experimental ThreeThe Effect of Centrifugal Force on Constructing TissueEngineering Cartilage in vitroObjective: To study the effect of the centrifugal force on the vitality and the functional expression of chondrocytes, and learn the role of centrifugal culture in tissue engineering cartilage. Methods: The first series of centrifugation sought to determine the conditionsof pressure on the cells that would damage their vitality. The flasks which carried the 3rd passage chondrocytes was put into a centrifuge with horizontal micropalte rotor. Several speed duration experimentals were then conducted and the results monitored using the trypan-blue exclusion test for cell vitality. We adopted histochemistrical stain and immunohistochemistrical stain to observe the structure of tissue engineering cartilage and the level of II collagen expression, and measured the content of glycosamin-oglycans(GAG) by DMMB spectrometry.Results: The centrifugation speed of 2000 rev/min and duration of 60 min caused >10% cell death. The centrifugation speed of 500 rev/min and duration of 120 min caused <10% cell death. During the same culture periods in vitro, the amounts of II collagen stained by immunohistochemistrical stain in the static culture group was less than that in the centrifugal culture group. The differences of GAG sythesis and proliferation between the centrifugal culture group and the static culture group had obvious statistic significance on2> 4> 6 weeks of culture in vitro(P<0.05).Conclusion: The centrifugal force (528g, 2000rev/min) will do harm to the chondrocyte vitality, so it cannot been used to apply in construct tissue engineering cartilage. The centrifugal force (33g, 2000rev/min) takes little effect on the chondrocyte vitality. The centrifugal force (132g,1000rev/min) on the chondrocytes promotes chondrocytes to secrete the GAG and type II collagen, and affects the structural arrange of the tissue engineering cartilage. The technique of centrifugal culture is a alternative means to construct tissue engineering cartilage.