Studies On The Healing Of Bony Defects By Percutaneous Autologous MSC Grafting In Rabbits

Objective: The treatment of bony defects is a difficult problem for orthopedic surgeons. Open grafting with autologous bone, allograft and other artificial materials proved insufficient in lots of cases. Accompany with the development of bone tissue engineering and cell therapy, implantation of osteogenic precursor cell and growth factors became the focus of investigation. Bone marrow stromal cells(MSCs) could be extensively expanded in vitro and the proliferation and bone-forming capacity of MSCs could be enhanced by growth factors. The cell based approaches, implantation of MSCs to bony defects, which can increase the number of osteoblastic cell, have a promising future. In order to explore a simple, practical, minimally invasive method, we investigated whether the growth factors can control the proliferation and cpmrnitment of MSCs into cells of the osteogenic lineage in vitro and the effectiveness of percutaneous injection of autologous MSCs+BMP without matrix on healing of rabbits bony defect.Methods: (1) MSCs were obtained from 1ml aspirate from one tibia of New Zealand rabbits. G-CSF was intravenously injected at the dosage of 2.5ug/kg ?d for 5 days. Then MSCs were obtained from the other tibia of New Zealand rabbits. Cells were plated in tissue culture dishes and incubated at 37癈 with 5%humidified CO2. ALP activity and mineralized plagues were examined. A simple way of short period preservation of cells was established using ?-85癈 refrigerator. (2) The MSCs was stimulated with rhBMP-2, bFGF and rhG-CSF at a concentration of 50, lOOng/ml. The effects of different factors and a factor at different concentration combinated with BMP were compared by MTT. ALP activity was measured. (3) The monolayer cells were collected and injected with BMP into the established radial bone defect on one side in each rabbit, the other bone defect on the contralateral side was injected into fresh bone marrow, which was used as control.Results: (1) The adherent MSCs gave rise to colonies and grew quickly. The appearances of MSCs were not altered after several passages. Some colonies and cells locating in the center of colonies expressed significant ALP activity. Mineralized plagues were detected by von Kossa staining. Though G-CSF can raise the number of bone marrow nucleated cells, the number of colonies did not increased. (2) bFGF has the most active effect on the proliferation of MSCs and BMP dramatically increased the cellular ALP activity by promoting the cells differentiation. (3) Radiographic and histologic examination after the MSCs transplantation show that bone union with marrow was observed in 4-8 weeks on most of the experimental sides. There was a statistically significant difference with bone marrow grafting.Conclusion: This study indicated that: (l)G-CSF could not increase the number of MSC in bone marrow; (2)The proliferation and bone-forming capacity of MSCs could be enhanced by BMPand bFGF. MSCs could be used in bone tissue engineering. (3) Percutaneous injection of autologous MSCs+BMP could improve the repair of bony defects, and MSCs have a more powerful ability of bone formation than fresh bone marrow.