Experimental Study On The Effect Of Chitosan-Gelatin Hybrid Scaffold On Repairing Articular Cartilage Defects In Rabbits

Objective: To investigate the ability of repair of rabbit articular cartilage defects using hybrid scaffold of chitosan and gelatin in vivo by means of tissue engineering. Observe the influence of the hybrid scaffold on the chondrocytes regeneration and extracellular matrix secretion of rabbit articular. The advantage and disadvantage of Chitosan-Gelatin hybrid scaffold in tissue engineering are evaluated; the hybrid scaffold is also compared with the deminerized antigen-extradted cancellous bone which is also used to repair the rabbit articular cartilage defects as a kind of scaffold.Methods: A total of 48 healthy New Zealand rabbits, 6mm diameter, full-thick articular osteochondral defects were made in the femur trochlear groove of the rabbits, these rabbits were randomly divided into 3 groups according to the scaffolds which will be filled in these defects: A group (chitosan-gelatin hybrid scaffold), B group (deminerized antigen-extradted cancellous bone) and C group (void control group). The repair of defects was periodically examined by gross observation, histologically and gray scale value of safranin O staining after the operation of 4, 8, 12 and 16 weeks respectively.Results: After 4 weeks of the operation, gross observation: there are obvious dent on the surface of articular defects in A, B and C group, the boundary is identified clearly between the new tissue and normal cartilage around the defects. There is no statistical difference among the three groups (P>0.05) according to histological grade scale and gray scale value of safranin O staining. After 8 weeks of the operation, gross observation: The new tissue filled the entire area of cartilage defects in A and B groups. In A group, the new tissue is even higher than the normal cartilage, white, tenacious and tramosericeous. The boundary is not easily to see compared with 4 weeks. Histological observation: the new tissue filled the entire area of cartilage defects in A groups, the new tissue is composed mainly by hyaline-like cartilage. But the new cells in the defects are smaller than the normal chondrocytes and have a long and narrow shape. Their arrangements are also irregular. There are some isogenous groups in the defects. The staining of the cartilage-specific extracellular matrix was similar to that of normal cartilage around the defects. The boundary still existed. In B groups, there were not obvious dent on the surface of articular defects; we can see some pykno-fibrous tissue at the surface of the defects through the microscope. The arrangement of the fibers is regular. In the deep lamella of the new tissues, we also can see some hyaline-like chondrocytes, the cells are small, but lacunas are plenty. The staining of the cartilage-specific extracellular matrix is similar to that of normal cartilage around the defects and the boundary is not obvious any more. In the defects of C groups, we can still see obvious dent. The new tissue is composed mainly by fibrous tissue. Cells in the defects are smaller than the normal chondrocytes and have a long and narrow shape. Isogenous groups can not be founded. The staining of the cartilage-specific extracellular matrix is superficial and the boundary is still easily to see between the new tissue and normal cartilage around the defects. 12 weeks postoperatively, gross observation: the surface of articular defects is smooth in A and B groups. These defects were filled by hyaline-like tissue. The new tissue is tenacious. Histological observation: the new tissue filled the entire area of cartilage defects in A groups and composed mainly by hyaline-like cartilage. The surface of the defect is smooth, these new cells looks bigger than before, The staining of the cartilage-specific extracellular matrix was similar to that of normal cartilage around the defects. But the boundary is difficult to discriminate. In B groups, the surface of defects is smooth and also filled by hyaline-like chondrocytes. The densities of these new cells were lower than before and the number of the isogenous groups is much more than before. The staining of the cartilage-specific extracellular matrix is similar to that of normal cartilage around the defects. At the same time, in C groups, the defects are filled by fibrous tissue. Under the surface, there are some cartilage-like tissues; these cells are still small and slender. The staining of the cartilage-specific extracellular matrix is superficial and the boundary is still easily to see. After 16 weeks of the operation, the dent on the surface of articular defects in A and B groups disappeared totally. The new tissue is similar to that of the normal cartilage around the defects. Histological observation: hyaline-like cartilage filled the entire area of cartilage defects in A groups. These chondrocytes located in the lacunas and arrayed more regular than before. The staining of the cartilage-specific extracellular matrix was as the same as that of normal cartilage around the defects. The new tissue combined with normal cartilage firmly and the thickness is similar to that of normal cartilage. We can see the tidal line and subchondral-bone regenerated totally. In B groups the defects were almost as high as normal cartilage and filled by the hyaline-like cartilage. But number of these newly born chondrocytes is still higher than normal cartilage. Cells are small in the surface of the defects and bigger in the deep lamella of the new tissues. The staining of the cartilage-specific extracellular matrix was as the same as that of normal cartilage around the defects. The new tissue combined with normal cartilage firmly and the thickness is similar to that of normal cartilage. We can see the tidal line and subchondral-bone regenerated totally. In C groups, the defects were filled mainly by fibrous-like cartilage, the surfaces of which were uneven and the borders were identified clearly. Cells in the defects are smaller than the normal chondrocytes and have a long and narrow shape. These cells arrayed irregular. The staining of the extracellular matrix is superficial. The regeneration of the tidal line and subchondral-bone never happened. After 8, 12 and 16 weeks of the operation, there were statistical difference among the three groups (P<0.05) according to histological grade scale and gray scale value of safranin O staining, and there was no statistical difference between A and B groups (P<0.05).Conclusion: 1 Chitosan-Gelatin hybrid scaffold which was made by lyophilizing and some controlling technology is a kind of new tissue engineering scaffolds and have good biocompatibility and biodegradation. It can supply superordinary three dimensional structures for chondrocytes and is fit for defects of any shape and depth. Chitosan-Gelatin hybrid scaffold significantly accelerate the regeneration of chondrocytes and the secretion of ECM. It developed a new way for the selection of tissue engineering scaffold.2 Deminerized antigen-extradted cancellous-bones eliminate the shortages of bad biocompatibility and material source deficit. As a typical tissue engineering scaffold, it has three dimensional structures which is good for the adhesion and generation of chondrocytes and promoted the secretion of ECM. This study supports the potential use of Deminerized antigen-extradted cancellous-bones as a tissue engineering scaffold.3 In this study, there were not any cell or cytokine was adhered to the scaffolds, the volume and arrangement of chondrocytes are not ideal, the thickness of articular cartilage is still thinner than that of normal cartilage around the defects. There is no statistical difference among the three groups after 4 weeks of the operation, which is quite different compared with the conclusions of most researches whose scaffolds were integrated with some cells and cytokines, maybe these scaffolds supplied more assistance in accelerating the regeneration of chondrocytes and the secretion of ECM. Adhering some chondrocytes, MSCs and cytokines to Chitosan-Gelatin hybrid scaffold should be considered and the effect of repairing articular cartilage is anticipated.