Study On The Response Of Osteoblasts To Level-changed Shear Stress In Vitro

Cellullar experiments were carried out using osteoblasts in vitro here to figure out the influences of fluid shear stress on the bone metabolic and the most influential force or force process. The mechanical responses of Wistar rat osteoblasts to fluid shear stress were studied with flow chamber. The proliferation, differentiation and the regulation to osteoclasts via osteoprotegerin (OPG) and osteoclast differentiation factor (RANKL) were tested. The main work and conclusions were as follows:Flow chamber was designed and used in our study. It can provide laminar flow shear stress and the shear stress can be adjusted via the flow rate. The assembly and disassembly were very easy to do. About 30 ml of fluid was used in our experiments.The primary Wistar rats osteoblasts were cultured from the calvaria of new-born rats by the tissue piece culture method. The osteoblasts were identified by the morphology and Von Kossa staining. The third passage of osteoblasts was used in the mechanical experiment after they reached confluence. Five processes of shear stress were designed here, which were steady shear stress of 5, 10 and 15 dyn/cm2 for 12 h respectively, stepwise increase shear stress from 5 to 10 and to 15 dyn/cm2 and each level for 4 h, stepwise decrease shear stress from 15 to 10 and to 15 dyn/cm2 and each for 4 h.The expression of OPG and RANKL mRNA was detected by RT-PCR. The results showed that compared with the static cells, the flow shear stress of 5, 10 dyn/cm2 and stepwise changed shear stress have increased, while 15 dyn/cm2 has decreased the OPG expression The shear stress of 10 dyn/cm2 has the more significant increase effect. Shear stress of 5 and 10 dyn/cm2 have deceased the expression of RANKL. No marked different between the effects of decreased shear stress and increased shear stress were observed. In all, the balance of RANKL /OPG mRNA was pushed towards more OPG by shear stress of 5 and 10 dyn/cm2 and backwards by shear stress of 15 dyn/cm2, which means the bone resorption was inhibited by the fluid flow of 5 and 10 dyn/cm2 and promoted by 15 dyn/cm2.Cell cycle analysis was carried out by flow cytometry to determine the proliferation of osteoblast. The result showed that the proliferation of osteoblasts increased respectively at 5 and 10 dyn after 12h.The ALP activity and extracellular calcium secretion of osteoblasts were measured by colorimetry method. The results showed that compared with the static cells, the ALP activity of the osteoblasts sheared by 10 and 15 dyn/cm2 was increased, but no marked difference with 5 and stepwise changed shear stress. The extracelluar calcium secretion of osteoblasts was increased by all the shear stress, and the effects of steady shear stress were stronger than the stepwise changed shear stress.All the results showed the osteoblasts can response to the level-changed shear stress.