| Objective: In recent years, with the development of the tissue engineering, the use of tissue engineering treatment of articular cartilage defects has become a hotspot research. However, compared with those techniques, cartilage tissue engineering is a new strategy for cartilage repair emerging in recent years, and many studies about it are still ongoing. Because of its simple operation, little damage and few complications, the micro-fracture technique have become the first-line treatment of articular cartilage defects on clinical. In this study, we examined the feasibility of autologous uncultured BM-MNCs in combination with microfracture in a full-thickness articular cartilage defect model and attempted to provide experimental data, which can guide the clinical work well.Methods: The 40 rabbits were divided into group A ,B ,C and D randomly, 10 rabbits in each group. The group A and C were taken 5ml marrow from the left femur, then autologous BM-MNCs were separation and extraction. The full-thickness articular cartilage defects were made on femoral intercondylar fossa in right knees of rabbits. Group A: micro-fracture was made on the cartilage defect and then implanted autologous uncultured BM-MNCs - autologous fibrin gel complex. Group B: the same micro-fracture was made on the cartilage defect and implanted autologous fibrin gel. Group C: the cartilage defect was implanted autologous uncultured BM-MNCs - autologous fibrin gel complex. Group D: the cartilage defect was implanted autologous fibrin gel. Five rabbits were killed at 8 and 12 weeks after transplantation in each group, and the reparative tissue samples evaluated grossly, histologically and immunohistochemically were graded according to the gross and histological scale.Results:1 Gross observation: Eight weeks after transplantation, the defects were filled with milky white tissues in group A. The reparative tissues have a certain flexibility and the color is similar to the normal cartilage. The boundary of regenerated tissues was blurred. The group B and C looks similar. The reparative tissues was a little soft. The color of reparative tissues was light yellow and the boundary was clear. In group D, the defects were partially filled with fibrous tissues, there were still large holes. Twelve weeks after transplantation, the reparative tissue surface was smooth, flexibility; similar color with the surrounding cartilage, the boundary was blurred in group A. In group B and C, the reparative tissue was relatively smooth and the boundary was blurred. In group D, the defects were partially filled with milky white tissues, there were still obvious holes.2 Histological observation: Eight weeks after transplantation in group A, the reparative tissue was smooth. In the optical microscope, there were some cartilage capsules and cartilage lacunas and also the flat shape chondrocytes gradually become round, oval and bigger. But, compared with the normal cartilage, reparative tissue had more chondrocytes, the morphology and arrange in order of chondrocytes were little abnormal. As for the thickness, the reparative tissue is greater than the normal cartilage. In addition, the toluidine blue metachromasia of the repair tissue was obvious. In the optical microscope, the defects of the group B were filled with fibrous tissues and a large number of vacuoles. The cartilage cells arranged in disorder and the healing between the repair tissue and surrounding tissue was poor. In the optical microscope, the group C looks similar to the group B, but more regeneration cartilage cells. In group D, the defects were filled with a little fibrous tissue. The degree of integration with adjacent cartilage was poor. Twelve weeks after transplantation, defects were repaired by hyaline-like cartilage in group A. The degree of integration with adjacent cartilage was good. The cells showed columnar arrangement. The surface of repair tissue was smooth, toluidine blue staining of the repair tissue between the surrounding tissue was no significant difference. In group B and C, the surface of repair tissue was rough and chondrocytes was irregular. Defect was filled mainly by fibrous connective tissue in group D. There were still some defects.The statistical analysis on the histological gradings at 8 weeks and 12 weeks showed that group A was significantly better than group B, C and D (P <0.05), group B and C was better than group D (P <0.05), each group at 12 weeks was better than itself at 6 weeks (P <0.05).3 Immunohistochemistry staining of type-â…¡collagen: Eight weeks after transplantation in group A, some of the cartilage cells was positive in type-II collagen staining and the cytoplasm was brown. Twelve weeks after transplantation, the cartilage cells were round or oval and showed columnar arrangement. The cytoplasm and extracellular matrix was positive in type-II collagen staining. Compared with group B, type-II collagen staining was stronger in group C, but both were weak positive. In group D, the type-II collagen staining was negative after twelve weeks.Conclusion:1 Both of micro-fracture and transplantation of the uncultured autologous BM-MNCs combined autologous fiber gel can promote the repair of cartilage defects.2 Micro-fracture in combination with autologous uncultured BM-MNCs plays an effective role in articular cartilage regeneration, which provide the theoretical basis for clinical application.
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