The Study On The Biological Effects And OPG/ODF Expression Of Rat Osteoblasts Under Flow-shear Stress

The mechanical response of Wistar rat osteoblasts to fluid shear stress was studied in flow chamber, which includes their proliferation, differentiation, mineralization and deformation. The regulating mechanism of the balance between bone resorption and bone formation by the mechanical stimulation was investigated through gene expression of osteoprotegerin(OPG) and osteoclast differentiation factor(ODF). The signal path of bone metabolic balance influenced by mechanical stimulation was investigated through the osteoclast formation induced by osteoblast. The main work and conclusions were as follows: Flow chamber was designed and used for investigating the effects of fluid shear stress on osteoblast. The shear stress of the flow chamber can be regulated by the flow rate of laminar flow fluid. The assembly and disassembly of the flow chamber is very easy, and about 10ml 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 purified by the difference of the adhibition time with other type of cells. It was identified by the morphology, alkaline phosphatase (ALP) activity and Von Kossa staining. The third transfer of the cultured osteoblasts was used in the mechanical experiment. The change of the cell morphology was observed by microscope and FITC-phalloidin staining. Cell cycle analysis was carried out by flow cytometry to determine the proliferation of osteoblasts. Osteoblasts were sheared with the stress of 5~30 dynes/cm~2 and 3~36 hours . The results showed that the proliferation index of osteoblasts increased respectively about 20.2% and 45.7% at 5 and 10 dynes/cm~2 after 12h , and the cells were elongated along the way of flow, and the cytoskeleton rearranged.Otherwise the proliferation index decreased respectively about 72.6% and 76.3% at 20 and 30dyn after 12h. The ALP activity and extracellular calcium deposition 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, 20 and 5 dynes/cm~2 was increased, and their effects were in turns of 10 dynes/cm~2, 20 dynes/cm2 and 5 dynes/cm~2. At the same time, 10 dynes/cm~2 and 20 dynes/cm~2 advanced the time of the peak value of ALP activity for 3 hours during the experiment period. While the shear stress of 30 dynes/cm~2 decreased the ALP activity. The extracellular calcium deposition of osteoblasts was increased by the shear stress of 5 dynes/cm2, 10 dynes/cm2, 20 dynes/cm2 and their turns were 10 dynes/cm2, 5 dynes/cm2 and 20 dynes/cm2, but it was decreased by the shear stress of 30 dynes/cm2. And the shear stress has no effect on the time of peak value, So it can be inferred that proper shear stress (here is 5~20 dynes/cm2) can increase the differentiation and mineralization of osteoblasts. The expression of OPG and ODF mRNA was detected by RT-PCR. The cells of static cultured for 24 hours were used in our tests. Invariable shear stress of 5,10 and 15 dynes/cm2 for 24 hours respectively, and step-wise increased shear stress from 5 dynes/cm2 to 10 dynes/cm2 and at last to 15 dynes/cm2 and each for 8 hours, and step-wise decreased shear stress from 15 dynes/cm2 to 10 dynes/cm2 and at last to 5 dynes/cm2 and each for 8 hours were applied.The results showed that compared with the static cells, the flow shear stress of 10 dynes/cm2 OPG was increased, but ODF was inhibited and its inhibition effect was increased with time. The shear stress of 10 and 15 dynes/cm2 induced more significant changes in OPG and ODF than 5 dynes/cm2. There was significant difference on the OPG and ODF between 10 and 15 dynes/cm2. No marked difference between the effects of step-wise increased shear stress and 15 dynes/cm2 after 24 hours, and between the effects of step-wise decreased shear stress and 5 dynes/cm2 after 24 hours were observed. So it can be inferred that the effects of the latter shear stress concealed the effects of the former shear stress, and the step-wise shear stress improved the response rate of shear stress on osteoblasts. In all, the balance of OPG/ODF mRNA was pushed towards more OPG by shear stress, which means that bone resorption was inhibited by the fluid flow. The bone marrow single nucleus cells were regarded as osteoclast precursor cell. the inducing effect of osteoblast on osteoclast was done in flow chamber. The results showed that the culture liquid medium of osteoblast can enhance the survival rate and growth state of bone marrow cells. The cell fusion of bone marrow cells was better in the static control than the sheared osteoblasts. That is to say the formation of osteoclasts was inhibited by shear stress. So it may be possible that this inhibition effect was related to the balance of bone metabolism in flow shear stress.