The Roles Of BMSCs On Bone Healing And The Expression Of Chemokine In Fracture Microenvironment

Objectives:(1)To isolation,culture and identification of BMSCs carrying GFP gene.(2) To tagb and to evaluate the role of autologous BMSCs in bone healing.(3)To detect the expression of SDF-1, SCF, HGF, G-CSF, MCP-1, MMP-9 in the microenvironment.Find the most one,which is associate with the migration of BMSCs.Methods:(1)GFP positive BMSCs were harvested from bone marrow of GFP transgenic C57BL/6 mice by density centrifugation and identified through osteoblastic and adipogenic induction.Test CD44 and CD34 by flow cytometry.(2)Male GFP positive BMSCs and nonadherent cells of bone marrow from male C57BL/6 mice were injected into female C57BL/6 mice of 4-week-old through tail vein after 10 Gy X-ray irradiation to establish chimeric mice models.(3)Then the chimeric mices were used to make left shankbone injured models. At the 1st,3rd, 5th,7th,14th,21th day post fracture, the rate of GFP positive cells to all cells in the injured site and the rate of osteoblasts differentiated fome GFP positive cells to all the osteoblasts were detected. The levelof SDF-1, SCF, HGF, G-CSF, MCP-1, MMP-9 in the microenvironment were detected with immunohistochemistry technology.Results:(1)GFP positive BMSCs grew as clony-like manner, expressed calcium deposition after induction by osteoblastic culture medium and formed fat droplets in adipogenic culture medium.(2)After fracture,more and more BMSCs can migrate to injuried site markedly,half of them differentiate into osteoblasts.(3)At the each day post fracture,the expression of SDF-1, SCF, HGF, MCP-1, MMP-9 in the microenvironment are in each level,and the expression of G-CSF is negative. The expression of SDF-1 in the fracture microenvironment is associate with the migration of BMSCs.Conclusion:(1)GFP positive BMSCs can be harvested from bone marrow of GFP transgenic mice C57BL/6 by density centrifugation.(2)The autologous BMSCs take part in and take an important roles in bone healing.(3)SDF-1 may play an important roles in promoting BMSCs to migrate to the facture site and promoting bone healing.