The Modification Of The Acellular Dermal Matrixcollagen Membrane Compounded With Multi-materials For Repairing The Articular Cartilage Defects

Objective: Natural collagen protein was a major component of connective tissue and it had good biocompatibility. But there had some disadvantages about the collagen, such as poor of flexibility, poor anti-tensile force. There's some difficults in meeting the needs of using it as a separate carrier material for the repair of cartilage defects. Cartilage matrix is rich in proteoglycan, glycosaminoglycan, type-Ⅱcollagen and other components. Articular cartilage relyed on a large number of autocrine factors in the maintenance of chondrocytes and normal phenotype matrix metabolism. Growth factors can regulate the proliferation, differentiation and metabolism of chondrocytes and participate in the physiological and pathological processes of articular cartilage. The objective of this experiment was using cattle dermis for the preparation of acellular dermal matrix collagen membrane (ADM). On this basis, further application of chondroitin sulfate (CS), sodium hyaluronate (HA), collagen typeⅡ(Col-Ⅱ), transforming growth factor -β(TGF-β) and insulin-like growth factor-Ⅰ(IGF-Ⅰ) were used for the modification of ADM. Simulating the greatest degree of chondrocytes growth environment, in order to promote the growth of chondrocytes.Methods: Using the Jishuitan Hospital Trauma Orthopedic Institute technical methods for the preparation of ADM. Disinfection and packaging.4 New Zealand fetal rabbit were sacrificed and the chondrocytes were gathered, then cultured and passed. Growth curve, HE and fluoresceindiacetate (FDA)-propidium iodide (PI) fluorescence staining were carried out.There's blank group, the CS group, HA group, Col-Ⅱgroup, TGF-βgroup, IGF-I group and mixed group. Prepare 0.5%(m/v)CS high glucose DMEM solution;1.0%(m/v)HA high glucose DMEM solution;0.5%(m/v)Col-Ⅱhigh glucose DMEM solution;1ng/ml TGF-βhigh glucose DMEM solution;10ng/ml IGF-I high glucose DMEM solution;Mixing the above five modified materials as 0.5%CS+1.0% HA+0.5%Col-Ⅱ+1ng/ml TGF-β+10ng/ml IGF-I high glucose DMEM mixture solution. Combine the total of six groups of solution with ADM.Chondrocytes were cultured in vitro to the third passage. Concentration of the chondrocytes suspension was 5×106/ml, and then mixed the chondrocytes suspension with the ADM. Scanning electron microscopy, embedding, HE staining, S100 immunohistochemical staining were carried out in 1, 3, 7, 14, 21days. After that, the positive cell count of immunohistochemical staining was also done. In 1, 3, 7 days, ADM were taken out and then analyzed with RT-PCR. BanScan4.3 gel image analysis software, analyzed the results.Made the model of articular cartilge defects of rabbits, divided in into three groups. A mixed group repair implants was a variety of composite materials modification ADM with chondrocytes. Pure collagen membrane group was the ADM for non-modified composite chondrocytes. The blank group did not composed with any material. Rbbits were sacrificed in 1, 2, 3 month. Then, the general observation, HE staining, Col-Ⅱimmunohistochemical staining and Wakitani for histologic score were analyzed.Results: ADM appears white and flexible structure. Electron microscope showed the porous structure of it. The porosity was suitable for the implantation and growth of chondrocytes.The acquired articular cartilage was digested by type-Ⅱcollagenase for 2 to 4 hours, and then removed the supernatant after centrifugation. Adding culture medium to the chondrocytes, and then could get more chondrocytes through the transferring in vitro. HE staining showed that cell morphology better, the FDA-PI fluorescence staining showed that cell survival rate was over 98%.Electron microscope showed the modification effect of all groups: Mixed bank> Col-Ⅱ> TGF-β>CS>HA and IGF-Ⅰ. HE and immunohistochemical staining showed the mixed bank had the best effect and the highest positive rate. Under the microscope, the numbers of positive cells were counted. The results show that: modification effects were similar to the result showed by the electron microscopy. Mixed bank had the best effect. The results of RT-PCR showed that on the first day, Col-Ⅱbank,TGF-βbank and mixed bank appeared obviously strap. The other banks could not see obviously strap. On the third and seventh days, Col-Ⅱbank and mixed bank had obviously strap. BanScan4.3 suggested that the Col-ⅡmRNA expression of the mixed bank was the highest.On the 1, 2, 3 month, general observation and HE staining showed that, the repaired organization of the mixed group was likely hyaline cartilage repair. Pure collagen membrane group and the blank group was the fiber restoration. The positive rate of Col-Ⅱimmunohistochemical staining showed that the mixed group>Simple materials group>control group. The result of Wakitani histologic score suggested that the mixed group had a better effect of restoration for the defects.Conclusion: ADM had better biocompatibility, suitable biodegradation, better cell adhesion surface activity, better plasticity and better three-dimensional porous structure.Mixed modification ADM had a better compatibility with chondrocytes.Mixed modification ADM appears as the most suitable scaffold for repairing of cartilage defects.