Effects Of Flow-Shear Stress On Physiological Activity And The Expression Of NOS Of Rat Osteoblasts

The effects of fluid shear stress on the physiological activity and gene expression of NOS of osteoblasts were studied through biomechanical, cell biological and molecular biology methods. The mechanism of bone resorption and bone formation by the mechanical stimulation was investigated through gene expression of eNOS and iNOS. The main work and conclusions were as follows:(1) 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.(2) 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.(3) Osteoblasts were sheared with the stress of 10 and 30 dynes/cm and 1-36 hours . Cell cycle analysis was carried out by flow cytometry to determine the proliferation of osteoblasts.The results showed that the proliferation index of osteoblasts increasedrespectively about 43.8% at 10 dynes/cm~2 on 12h. Otherwise the proliferation indexdecreased respectively at 30 dynes/cm~2 on 12h .(4) 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 dynes/cm~2 was increased. While the shear stress of 30 dynes/cm~2 decreased the ALP activity. The extracellular calciumdeposition of osteoblasts was increased by the shear stress of 10 dynes/cm~2 , but it wasdecreased by the shear stress of 30 dynes/cm~2 .(5) The change of NO deposition of osteoblasts were measured by colorimetry method. The results showed that the NO deposition of osteoblasts changed differentlysheared by 10 dynes/cm~2 and 30 dynes/cm~2 . The shear stress of 10 dynes/cm~2 increasedthe deposition of low level NO,while the shear stress of 30 dynes/cm~2 increased high level of NO in short time .(6) The expression of eNOS and iNOS mRNA was detected by RT-PCR. Osteoblasts were sheared with the stress of 10 and 30 dynes/cm~2 and 1-36 hours . The...