Chondrogenic Observation Of Rabbit Allogenic Decalcified Bone Matrix Combined With Bone Marrow Mesenchymal Stem Cells Cultured In Vitro

Objective 1. To investigate the methods of division, culturing, and identifying bone marrow mesenchymal stem cells(BMSC) in vitro, make a foundation for tissue engineered cartilage in knee articular cavity. 2. To investigate the chondrosis of allogenic decalcified bone matrix combined with bone marrow mesenchymal stem cells cultured and activated by revulsant in vitro。Methods 1. Archiogeneration BMSC were isolated and purified by adhering to the culture glassware wall from neonatal New Zealand young rabbits, some of the second generation BMSC were separated to do the identification of phaenotype, the third generation BMSC induce to chondrocyte by transforming growth factor TGF-β1, insulin-like growth factor IGF-1 and VitC. 14 days later, typeⅡcollagen immunohistochemical of the cells were carried out. 2. Following the method described by Urist, allogenic DBM was made. 3. To cultivate the revulsive BMSC combined with DBM in vitro. 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).Results 1. The morphological character of BMSC was clear that they all present fusiform shape. Confluence of BMSC reach to 90% 10-14 days afer primary culture or 6-7 days after subculture. CD44(+) and CD34(-) were detected by flow cytometry. Induced BMSC had characteristics of the chondrocyte. 2. Allogeneic DBM prepared was a three -dimensional highly-porous structure like sponge. 3. At 12th week, culture masses were observated, HE stain show that the chondrocytes have began to proliferate, caryon is blue. Toluidine blue stain show that the chondrocytes proliferated and have no order,surrounded by lots of extracellar matrix. Masson stain shows that chondrocytes are out of order; Immunohistochemical identification ofⅡcollagen was positive, brown-yellow stained particles could be discerned in extracellar matrix.Conclusion 1. BMSC can be separated and proliferated effectively by the whole bone marrow adherence culture. 2. BMSC can induced to chondrogenic differentiation in certain circumstances. 3. Tissue engineering cartilage can be cultured by BMSC combined with DBM in vitro.