The Effects Of The Mechanical Signal In Different Fluid Shear Stresses On The Biological Effect Of Osteoblasts

ObjectiveTo observe and study the effects of the mechanical signal in different fluid shear stresses on the biological effect of osteoblasts .Methods1. The cell culture: the osteoblasts were cultured in Dulbecco's modified Eagle medium (DMEM) as usual ,when the cells reached confluence ,they were trypsinized and removed from the flask , then they were made into cell suspension and cell counting on inverted phase contrast microscope. They were subcultured into a glass plate at a plating density of 10~4/ml. The medium were changed every 48h.2. Performance Evaluation for the osteoblasts(1) Cells morphology on phase-contrast microscope(2) ALP staining (Gomori Ca-Co method)(3) BGP identification on immunohistochemistry(4) Mineralization nodule staining(Alizarin Bordeaux staining method and Von kossa)3. The shear force on the slides with osteoblasts were exposed to shear stress in a special apparatus used previously to assess cell response to short exposures to fluid flow, in a sterile state .the culture medium is continuously recirculated through the flow chamber by a peristaltic pump, peristaltic pump can regulation the flow rate.4. To determine the effects of the mechanical signal in the fluid shear stress (7dynes/cm~2 or 12dynes/cm~2) on the morphology and the proliferation activity of the osteoblasts in different times (15min,30min,60min,120min) respectively. Beside that, this study initially assays the positive expression of BGP in osteoblasts in different shear forces.(1) To show the cell morphology on phase-contrast microscope.(2) To assay the proliferation activity of osteoblasts by MTT.(3) Semi quantitative analysis on the Area of cells and the immunohistochemistry results by IPP, a precise image analysis software.Results1. The effects of the fluid shear stress (7dynes/cm~2 or 12dynes/cm~2 ) on the Morphology of osteoblasts:(1) To observe and assay the Morphology of osteoblasts exposed to the shear stress of 7dynes/cm~2:Compared with control group, the Morphology of cells in all the treatment groups have significantly changed respectively, almost all of the long axis of cells keep the same direction with the shear stress ; Through the assay results of the average area of cells in this experiment, compared with the control group ,the groups (30min,60min,120min) showed significant differences (P<0.05) ,whereas no difference was found between the 15min group and the control group (P>0.05).(2) To observe and assay the Morphology of osteoblasts exposed to the shear stress of 12dynes/cm~2 :Compared with control group ,the Morphology of cells in the treatment groups (15min,30min,60min) have significantly changed respectively, almost all of the long axis of cells keep the same direction with the shear stress ;surprisely ,in the 120min group, the Morphology of cells have changed anormaly ,we have observed a few of cell nucleus desintegrated or apoptosis in this group . Through the assay results of the average area of cells in this experiment, compared with the control group ,the groups (60min,120min) showed significant differences (P<0.05) ,whereas no difference was found among the groups (15min,30min) and the control group (P>0.05).(3) The difference between the effects on the Morphology of osteoblasts exposed to the two kind of fluid shear stresses (of 7dynes/cm~2 and 12dynes/cm~2):By the assay results of the average areas of cells in this experiment, we can found that the effects on the average areas of the cells ,which exposed to the two kind shear forces respectively showed significant differences(P<0.05).2. The effect of the fluid shear stress of 7dynes/cm~2 or 12dynes/cm~2 on the proliferation activity of osteoblasts.(1) The effect of the fluid shear stress of 7dynes/cm~2 on the proliferation activity of osteoblasts:Compared with control group,the proliferation activity of the osteoblasts were increased in all the treatment groups with different exposed times (15min, 30min, 60min) (P<0.05) ; significantly higher levels of the proliferation activity was observed in the 60min group in this experiment.(2) The effect of the fluid shear stress of 12dynes/cm~2 on the proliferation activity of osteoblasts:Compared with control group,the proliferation activity of the osteoblasts were increased in all the treatment groups with different exposed times (15min, 30min, 60min) (P<0.05) ; significantly higher levels of the proliferation activity was observed in the 60min group in this experiment.(3) The difference between effects on the proliferation activity of the osteoblasts exposed to the two kind of fluid shear stresses ( 7dynes/cm~2 and 12dynes/cm~2):By the assay results of the proliferation activity of the osteoblasts in this experiment, we can found that the effects on the proliferation activity of the cells ,which exposed to the two kind shear forces respectively showed significant differences(P<0.05); Besides that, the proliferation activity of the cells exposed to the fluid shear stress of 12dynes/cm~2 for 60min was significantly higher than others in this experiment.3. The effect of the fluid shear stress of 12dynes/cm~2 on the positive expression of BGP in osteoblasts:Compared with control group(0min),just the positive expression of BGP in the osteoblasts, which exposed to the fluid shear stress of 12dynes/cm~2 for 60min, was significantly higher (P<0.05) ;Besides that, the positive expression of BGP in this group had more higher level than other treatment groups in this experiment (P<0.05 );ConclusionsIt showed that the mechanical signal in the fluid shear stresses of 7dynes/cm~2 and 12dynes/cm~2 could influence the biological effect of osteoblasts. In this experiment, the mechanical signal could influence the morphology and the proliferation activity of osteoblasts ; Besides that, the proliferation activity of the osteoblasts exposed to the fluid shear stress of 12dynes/cm~2 for 60min was significantly higher than others in this experiment. The experiment will be the foundation of the following study ,and the results will lay a foundation for the study on signal transduction and the critical protein on the surface of implants.