Experimental Study Of Bone Marrow Stromal Cells Seeded On Demineralized Bone Matrix For Articular Cartilage Repair Of Rabbits

[Objective] To observe the potential of using Demineralized bone matrix as a scaffold for cartilage tissue engineering, determine the optimal co-culture time for implantation, and the feasibility of using bone marrow stromal cells seeded on demineralized bone matrix for articular cartilage repair of rabbits.[Methods] 1.Demineralized bone matrix was prepared according to Urist's method with different demineralizing time of 6 hours, 12 hours and 24 hours. The Ultra structure of different DBM was observed with scanning electron microscope (SEM) . Bone marrow stromal cells were cultured with different DBM, and MTT growth curve was used to analyze the effect of different DBM on Bone marrow stromal cells. The growth of Bone marrow stromal cells on different DBM was also observed with SEM.2.Bone marrow was obtained from the iliac bone of rabbits respectively. BMSCs were purified and ampilified in number to the second passage. The third passage BMSCs labeled by Brdu were seeded into DBM in vitro. The DBM / BMSCs complex was cultured and transplanted into full thickness defects on intercondylar notch of both knee joints. Twenty-seven healthy New Zealand rabbits were divided into three groups randomly, and there were 9 rabbits in each group. The cartilage defects were repaired with DBM / BMSCs complex in group A (experimental group), repaired with only DBM in group B (controlled experimental group), and no repair in group C (controlled group). Three rabbits were killed at 4th, 8th and 12th weeks after the operation in each group, and the reparative tissue samples were evaluated grossly, histologically, immunohistochemically and evaluate the repairing effect by Wakitani's score system. SPSS 11.5 software was applied to proceed the statistic alanalysis. The sources of the reparative tissue were detected also.[Results] 1.The appearance of the DBM scaffold was white cancellous feature. SEM showed the pores were connected each other ,the pore and porosity of DBM with different demineralized time (6 hours, 12 hours and 24 hours) had no difference. BMSCs co-cultured with the DBM ampilified until the eighth day, DBM could promote the expansion of BMSCs, and the DBM with a demineralizing time of 6 hours had the most effect.2. Defective regions were repaired by hyaline cartilage at 4 weeks after transplantation and were barely recognizable with natural articular cartilage at 12 weeks. Neither mononuclear cell infiltration nor vascular invasion could be seen in or around the transplanted tissue. Transplanted BMSCs marked by Brdu remain viable and contribute to repair in the host cartilage defects at 4 week after surgery.[Conclusion] 1. The DBM scaffold had a structural feature of tissue engineering scaffold. DBM could promote the proliferation of BMSCs. and the DBM with a demineralizing time of 6 hours had the most effect. So it was a kind of ideal scaffold in cartilage tissue engineering.2. DBM /BMSCs complex could be an efficient alternative for articular cartilage defects repair. Transplanted cells remain viable and contribute to repair in the host cartilage defects at 4 week after transplantation.