The Effects Of Mechanical Environment On Recruitment And Differentiation Of MSCs At Fracture Site During Early Stage Of Fracture Healing

ObjectivesFracture healing is comprised by fracture, fusion of bone jointed and bone transplantation until natural bone is connected. Fracture healing is a complex and continuously repairing process and it is influenced by many environmental factors. It can stimulate the growth of the callus, accelerate the process of fracture healing when given fixed fractures at a suitable degree of micro-movement. The mechanism of the cell biology and molecular biology in mechanical environment of bone healing process is still unclear. Our group found in the clinical treatment that fixed completely of the healing fracture cannot accelerate the process, to give patients a certain appropriate activity can accelerate fracture healing. Therefore, the study of mechanics environment on the fracture healing process can not only make a better understanding of the mechanism of fracture healing, but also has an important guiding function on the treatment and rehabilitation of clinical fractures. The early stage of fracture healing is the most effective period for mechanical stimulation in promoting fracture healing. MSCs differentiate into a number of cell types, including osteoblasts and chondrocytes. Raising MSCs at fracture site plays an important role in bone repairment. It is an foundational prerequisite of raising a sufficient number of MSCs to fracture healing. We used flow cytometry, RT q PCR, Western blot and other technical means, to observe the different effect of different mechanical environment of bone end on recruitment and differentiation of MSCs. Methods1.We established femur fracture model in rats and fixed with external fixation frame of different activity coefficient.We set up the control group, high stiffness fixator group and low stiffness fixator group.2.After two weeks of the modeling, the bone callus and the fracture healing were observed on X-Ray film. The effects were evaluted by using gross observation,histopathology at 6 weeks respectively after operation.3.We get organization of fracture end respectively in 2 days, 4 days, 6 days, 10 days, separate nucleated cells and apply flow cytometry to detect proportion of CD29+CD90+ cells(MSCs).4.We get callus tissue of 2 days, 6 days, 10 days, 14 days after operation, Runx2, Osterix and Sox9 expression was measured by RT-q PCR and Runx2 and Sox9 was tested by Western-blot in order to determine impact of mechanics environment on differentiation of MSCs. Results1.X-ray showed that the amount of callus of high stiffness group is significantly less than low stiffness group. Histologic study shows, high stiffness group rats achieved the osseous links, but had less callus while low stiffness group rats achieved the cartilaginous links, cartilage part is very thin, but ultimately no ossification occured.2.Compareing three groups with each other respectively, we find that with the increase of healing time, the ratio of CD29 + CD90 +cells(MSCs) decreases constantly, 2 days > 4 days > 6 days>10 days, P < 0.05; the same day after Operation, low stiffness group > high stiffness group > the control group, difference between three groups were statistically significant, P < 0.05.3.The level of Runx2, Osterix increased within 14 days. The level of Sox9 reached the highest in control group and high stiffness group 10 days after treatment and declined in 14 days while low stiffness group has been rising within 14 days after surgery. The expression of Runx2, OSX and Sox9 in 14 days after operation was statistically significant difference between the three groups, low stiffness group > high stiffness group > the control group. ConclusionsDuring early stage of fracture healing in rats, the mechanical environmental do have effects on recruitment and differentiation of mesenchymal stem cells:1.When fixed with low intensity, activity coefficient is large, more MSCs can be recruited at fracture site.2.When given a lower fixation strength, level of osteogenesis related gene Runx2、 Osterix and cartilage related gene Sox9 relatively increased than the higher one.3.Micro motion can promote the differentiation to osteogenesis and cartilage of MSCs, but may not be able to obtain rigid healing over a certain range of it. In the process of clinical treatment of fracture, we should control the mechanical stimuli to promote fracture healing process and reach the final osseous healing.