Chondrogenic Research Of Rabbit Allogenic Decalcified Bone Matrix Combined With Bone Marrow Mesenchymal Stem Cells Cultured In Rabbits' Knee Cavity

Objective To investigate the chondrosis of rabbit allogenic decalcified bone matrix combined with bone marrow mesenchymal stem cells cultured and activated by revulsant in rabbits'knee cavity。Methods Archiogeneration BMSC were isolated and purified by adhering to the culture glassware wall from neonatal New Zealand young rabbits, the second generation cells were identified by flow cytometry. the third generation BMSC induced to chondrocyte by transforming growth factor TGF-β1, insulin-like growth factor IGF-1 and VitC when the cells overspread 70 percent of the botton of the culture-flask. TypeⅡcollagen immunohistochemistry of the cells were carried out both the third generation BMSC and 14 days-induced. Allogenic DBM was made from rabbit's ilium and vertebral bone. 1 day later, the revulsive BMSC combined with DBM in vitro will be culture in rabbits'left knee cavities and culture homologous decalcified bone matrix in rabbits'right knee cavity. Every 4 weeks after cellular transplant, the specimens were observed and made in paraffin sections. All the specimens were carried out with HE stain, toluidine blue stain, Masson stain and typeⅡcollagen immunohistochemical staining with chromogen diaminobenzidine(DAB). Immunohistochemical optical density(OD) value were calculated by morphology analysis software. Measurement data were demonstrated with mean±standard, satistical analysis was performed by t test, using SPSS 17.0 software. Results The results of cells identification were CD34 negative and CD44 positive. There was no obvious rejection after complex implantation in New Zealand white rabbits'knee cavity, surgical incision was dry, no irritation, oozy and knee activities without obvious limited. At the 4th week, both the specimens were qualitative toughening and still retain the structure of DBM, but the pore was decreased. At the 8th week, part of the DBM of group A were absorbed and the structure was collapsed. Cancellous bone peculiar pores were almost disappeared. At the 12th week, the specimens'surface of group A form transparent chondroid substances and were similar to normal cartilage appearance, and observation of the specimen's cross section using scanning electron microscope shows that the inside was full of cartilage besides some hole in diameter of different size within 100um, but the specimens of group B were disappear. After cultivating for 12 weeks, H-E stain shows that the specimens maturated chondrocytes ,surrounded by a pond of cellular matrix infiltrate interior the scaffolds. The decalcified bone matrix were absorbed mostly, toluidine blue stain shows that cartilage cells secrete the acidic glass sample matrix was dyed red or purple, the structure of the bracket was dyed blue, Masson stain shows that cartilage cells, matrix aizen, according to certain stress direction alignment, Immunohistochemical identification ofⅡcollagen was positive, lots of brown-yellow stained particles could be discerned in extracellar matrix.Conclusion Tissue engineering cartilage can be cultured by cartilage induced rabbit BMSC combined with allograft DBM in rabbits'knee cavity .