Comparative Study Of Rabbit Allogenic Decalcified Bone Matrix Combined With Bone Marrow Mesenchymal Stem Cells Cultured In Knee-joint With Cavity Cartilage

Objective To investigate the chondrosis of allogenic decalcified bone matrix combined with bone marrow mesenchymal stem cells cultured and activated by revulsant in vitro and in knee-joint。Methods 1. Archiogeneration BMSC were isolated and purified by adhering to the culture glassware wall from neonatal New Zealand young rabbits, the third generation BMSC induce to chondrocyte by transforming growth factor TGF-β1, insulin-like growth factor IGF-1 and VitC. 2. Following the method described by Urist, allogenic DBM was made and was observed with SEM. 3. To cultivate the revulsive BMSC combined with DBM in vitro(management A) and knee-joint(management B). Every 4 weeks after cellular transplant, the specimen and articular cartilage(management C) were observed with SEM and made in paraffin sections. All the specimen 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% 10-12 days afer primary culture or 6-7 days after subculture. 2. Induced BMSC had characteristics of the chondrocyte. 3. Allogeneic DBM prepared was a three -dimensional highly-porous structure like sponge. 4.At the 12th week, culture masses were observated with SEM, HE stain, Toluidine blue stain, Masson stain,and Immunohistochemical identification ofⅡcollagen. A:SEM: The surface of specimen is uneven, HE stain shows that the chondrocytes have began to proliferate but not much; Toluidine blue stain shows that there have little chondrocytes and extracellular matrix; Masson stain shows that chondrocytes are out of order; Immunohistochemical identification ofⅡcollagen shows that there have few brown-yellow stained particles.B:SEM: The surface of specimen is smooth, HE stain shows that the chondrocytes have began to proliferate, caryon is blue. Toluidine blue stain shows that the chondrocytes proliferated and cartilage lacunas surrounded by lots of extracellar matrix.Masson stain shows that cartilage cells, matrix aizen, according to certain stress direction alignment, Immunohistochemical identification ofⅡcollagen was positive, brown-yellow stained particles could be discerned in extracellar matrix.Conclusion Tissue engineering cartilage which cultured by BMSC combined with DBM in rabbits knee-joint close to normal cartilage.