Effects Of Mechanical Stretch On Osteogenic Differentiation Of MSCs

Msenchymal stem cells (MSCs) are a heterogenous cell population which can differentiate into various cell types in ectodermal, mesodermal and intradermal tissues including osteoblasts and chondroblasts both in vitro and in vivo. As primitive stem cells with multidifFerentiation potential, MSCs have been widely used as vector cells in tissue engineering and cell engineering. Therefore the applications of MSCs in these areas have drawn great attention and have been widely explored in clinical and preclinical studies.Bone regeneration and modeling involve complicate force stimuliti among which mechanical stimulus is one of the most important factors. Adequate mechanical stimulus is critical for osteoregeneration in many orthopaedic procedures especially during distraction osteogenesis. Various cells, including MSCs, OBs, osteoclasts, endothelial cells and fibroblast, participate in this process among which MSCs are mong the most important. Bone regeneration during DO is characterized by a series of cellular processes and complex molecular events that under appropriate mechanical stimulus result in chemataxic, proliferation and differentiation of uncommitted mesenchymal cells into bone-forming cells and eventually the formation of bone callus in the distraction zone. Despite recent advances in response of osteoblasts to mechanical stretchand its relating gene expressions, the mechanism of MSCs undergoing above osteogenic commitment under mechanical stimuli, the molecular cascades involved and the changes of cellular structure and functions, remain unknown yet.Based on above knowledge, the changes of cell structure and cell functions in MSCs under mechanical stretch, and expression profiles of bone-relating and apoptosis relating genes were investigated in this study. The results of this study will provide some valuable information to elucidate the molecular mechanism of MSCs participating in DO and orthodontic treatments during which mechanical force is usually employed.To establish an in vitro isolation and culture system of rat bone marrow stem cells, firstly, density gradient centrifugation and plastic adherence methods were used for isolation of bone marrow MSCs from adult SD rats. The purity and the proliferation ability of MSCs was compared by MTT examination.Then the MSC were also undergone osteogenic, neurogenic and myogenic induction and cytoch-emical and immunocytochemical staining were performed to verify their mutipo-tential. Based on above study, a four-point bending apparatus is utilized to perform a series of various mechanical strain on rat bone marrow MSCs, bone-relating and apoptosis relating genes were analyzed by SyberGreen Real-time PCR The expression patterns of these genes in mechanically strained MSCs are explored. To imitate once activation of clinical distractor, the MSCs were loaded by four-point bending system with tension stress (2000ue,40min) ,bone-relating genes were analyzed by Real-time PCR At same time ,to study the function of F-actin on mechanotransduction, rat bone marrow MSCs and calvarial osteoblast-like cells were exposed to cyclic mechanical stretch (4000ue) with a four-point bend-ing apparatus. Then the cells were checked under LSCM with same scanning condition. TUNEL was performed to detect and quantify the apoptotic cells with a in situ cell death detection kit. Results:1. Gradient centrifugation is a better method for isolation and purification of MSCs from bone marrow and the cells showed perfect proliferation ability.2. MSCs can trans-differentiate into neuron-like cells, osteoblasts and myoblasts under regulated in vitro culture conditions and were verified their pluripotential.3. The mechanical stimulus(400~3OOOu.fi) at physiologic magnitude could induce the larger expression of bone-relating genes The high-mechanical stimulus (>3000uï¿¡) at pathologic magnitude could induce the largest expression of apoptosis-relating gene.4. The mechanical stimulus(2000uï¿¡) is a cardinal driving factor for expression of bone-relating genes in MSCs and the expression of these genes will in turn result in osteogenic differentiation of MSCs. and expression of most of genes is peak value at 6h. The distraction protocol of 2 to 4 times per day is reasonable for clinical distraction osteogenesis.5. Mechanical stretch of 4000ustrain resulted in disruption of F-actin network and apoptotic changes in the stretched cells in a time-dependent manner and these changes were more obvious in MSCs than in OB-like cells.