The Effects Of The Fluid Shear Stress On Osteoclasts And The Level Of TRAP Of Osteoclasts And The Expression Of MRNA Of RANKL In Vitro

Bone is a complex tissue consists of many different kinds cells, organics and minerals. The normal state of bone is sustained by continuing absorbing nutrients under certain pressure environment. After a tooth is extracted, due to the lack of occlusive stimulation, the absorption of the remains of alveolar ridge shows an irreversible process, due to the imbalance of the bone formation. Bones formation and absorption are always very highly adjusted procedure; many factors from the whole body and the local area all have effects on it. My research into signal transduction of the osteoclast and its biologic behavior under pressure may lead to a way of solving the problem mentions above.To find out more about the signal transduction of the osteoclast under mechanostimulus is a much-researched area overseas. There are two main points of view on the mechanisms of mechanostimulus induced osteoclast bone absorption differentiations: â‘ Stress has effects on the osteoclasts and its surrounding tissues, releasing many signal factors and therefore able to adjust osteoclasts function. â‘¡Stress works on osteoclast itself and changes its innerstructure; therefore adjust its function. Until now, the mechanism to control osteoclast division, growth, move and function and death is still not clear.Among all the signal factors that adjust the functions of osteoclast, ODF is considered the ultimate controlling factor. Many experiments have shown that ODF controls the growth of osteoclast precursor cell to maturity. It is through the connection between ODF/RANKL and the RANK in osteoclast, that signals can be transferred into the precursor of the osteoclast, which in turn induced a chain of reactions, that osteoclast starts dividing and coming back to life. This makes RANKL a very important factor for researchers to find out the signal transduction mechanism of osteoclast under the mechanostimulus.My research uses 1, 25 (OH) 2D3 to grow SD rat bone marrow osteoclast in vitro, at the same time using cell mechanostimulus and enzyme chemical method and testing method from molecular biology, observed the effects of the fluid shear stress on the growth of osteoclast in vitro and the changes of tartrate-resistant acid phosphatase (TRAP). Also researched is the effect of fluid shear stress on osteoclasts differentiation factor ODF/RANKL and discussed to molecular mechanism of the effects on the osteoclast division under mechanostimulus.The major work done under my research is: 1. The cultivation of osteoclast from the bone marrows of SD rat in vitroObjective: Using la, 25 (OH) ₂D₃ to grow osteoclast from the bone marrows of SD mouse in vitro, the process can help to establish reliable cell cultivate method and finding the best timing to observe the cell by providing biologic effects of osteoclasts. Method: Taking the thigh bone of SD rat under asepsis, cutting off both end, using a-MEM to flush out its bone marrow cells. Then culture it in cell culture plates. Changing liquid every three hours. Observe thegrowth and splitting of the cells under microscope. Using tartrate-resistant acid phosphatase (TRAP) to dye, and then observe under SEM, counting the cells with multiple cell nucleoli. Result and Conclusion: Using 10⁸mol/L, la, 25 (OH) 2D3 to grow osteoclast from the bone marrows of SD mouse in vitro, the osteoclast grown from it shows very specific characteristic with positive result from TRAP dye. The ox bone plate shows very clear absorption lacuna. The curve of osteoclast in vitro shows that we can get most number of cells on the seventh and eighth day. It is also the best timing to do research and observation on the osteoclast.2. The effects of the fluid shear stress on osteoclasts and the level of tartrate-resistant acid phosphatase (TRAP)Objective: Though the growth in vitro of osteoclast from bone marrow of SD mouse, observe the effect of different level of fluid shear stress on the growth of osteoclasts in vitro and the level of tartrate-resistant acid phosphatase (TRAP). Method: Using stress system added on cells in vitro. There are four groups of experiments: Group A is for comparison purpose, there is no added pressure on it Group B has 0.1dyne/cm2 stress added on for the period of 0.5 hours. Group C has 0.3dyne/cm2 stress added on for the period of 0.5 hours. Group D has 3.0dyne/cm2 for the period of 0.5 hours. There are six lots in every group. The pressure is added for two days and the observation results are acquired on the seventh day. The multiple nucleoli osteoclasts are observed and taken record of under microscope using TRAP dye. Result and Conclusion: Osteoclasts shrank under pressure, the numbers of typical multiple nucleoli osteoclast also have dropped. The colour under TRAP dye is lighter. After two days of added pressure, the osteoclasts are fuller in shape and the numbers have grown. It is very clear thatfluid shear stress has suppressive effects on the split, growth, and the metabolism enzyme of osteoclast, and the result shows the effects are relative to the level of stress between 0.3 to 3.0 dyne/cm².3. The effects of the fluid shear stress on osteoclasts differentiation factor RANKLmRNAObjective: Through the observation of the effects of the fluid shear stress on osteoclasts differentiation factor RANKLmRNA, discuss the molecule mechanism of fluid shear stress on the effect of suppress the formations of osteoclasts. Method: Add different level of fluid shear stress (0.1 dyne/cm², 0.3dyne/cm² and 3.0dyne/cm²) to bone marrow cells for the period of 0.5 hours, and add 0.3 dyne/cm² stress to bone marrow cells under different period of time (15. min, 30 min, 60 min, 240 min). Result and Conclusion: Fluid shear stress has suppressive effects on the RANKLmRNA in bone marrow cells. Apart from 0.1 dyne/cm , 30 min and 0.3 dyne/cm , 15 min groups don't have statistical meaning, all the other groups with added stress have prominent difference from the comparison group. Fluid shear stress has suppressive effects on the bone marrow osteoclasts in vitro through the early stage suppression of RANKL.4. The concerted effects of signal transduction ligand inhibitor and fluid shear stress on the osteoclastsObjective: Through the observation of the effects of the signal transduction ligand inhibitor and fluid shear stress on osteoclasts, search the distinct signal transduction pathway. Method: Add fluid shear stress of 0.3dyne/cm² and 0.5 hours and KT5720 Genestein separately,observe the osteoclast number, mean density of TRAP picture, and RANKL level. Result: Apart from RANKL level of Genestein group don't have statistical meaning, all the other groups with addedstress have prominent difference from the comparison group. Conclusion: The suppressive effects of fluid shear stress on the bone marrow osteoclasts in vitro attribute to cAMP-PKA pathway but not MAPK one.