Effect Of Fluid Shear Stress On The Bone Resorption Of Osteoclasts In Osteoporotic Rats

BackgroundOsteoporosis is characterised by low bone mass and microarchitectural deterioration of bone tissue, leading to enhanced bone fragility and consequent increase in fracture risk. It is a common condition affecting one in three women and one in 12 men, resulting in substantial morbidity, excess mortality, and health and social services expenditure. Therefore, it is important to develop strategies to prevent and treat osteoporosis in both men and women. Many factors can affect on osteoporosis, and one of them is mechanical loading. In vivo, mechanical loading has been shown to play a important role during bone development and can inhibit bone resorption and increase bone formation. Bone is a porous tissue that is continuously perfused by interstitial fluid. Fluid flow, driven by both vascular pressure and mechanical loading, may generate significant shear stresses through the canaliculi as well as along the bone lining at the endosteal surface. However, it is still unknown whether osteoclast is sensitive to shear stress and how osteoclasts transduce mechanical signals into biochemical response of osteoclasts. So, we hypothesized that osteoclasts and preosteoclasts may be mechanosensitive and integrinavp3 on osteoclasts may take part in mechanical signal transduction in osteoclasts.This purpose is to investigate the fluid shear stress effects on the bone resorption of osteoclasts derived from normal rats and ovariectomied rats, respectively and a probable molecular mechanism in the mechanical signal transduction pathways of the regulation of integrinα_vβ₃ on osteoclasts.Methods1. Establishment of animal model of osteroporosisAnimal model of osteoporosis were established for six month old Sprague-Dawley rats by ovariectomy. These animals were killed at 90 days after ovariectomy. Histopathological evaluation of the bone tissue was performed to indentify the animal model of osteoporosis.2. Osteoclast isolationOsteoclasts were obtained from lumber vertebra of normal rats and ovariectomied rats by the combination of induced differentiation of the bone marrow cells and mechanical separation process. Osteoclasts were identified by morphological observation, cytoskeleton marking, TRAP staining and formation of bone resorptive pit on bone slices, respectively. Cell couting and assay of TRAP enzyme activity were used to choice the optimal growth phase of osteoclast.3. Preparation of coverslip coated with hydroxyapatite ceramicCoverslips coated with hydroxyapatite(HA) ceramic were prepared with calcium nitrate and phosphoric anhydride by sol-gel process. Coverslips coated with HA were used to observe the resorption of the bone matrix, instead of bone slices.4. Fluid shear stress experimentsFluid shear stress at 5.97, 11.36, 16.08, 20.54 dyne/cm² was used for all experiments ,for 30 min. Perfuse solution of each group were collected for TRAP enzyme activity testing quickly by ultraviolet spectrophotometry after mechanical stimulation. Number and area of bone resorptive pits were calculated through image analysis by means of scaning electron micrography. According the above result, the fluid shear stress at 16.08 dyne/cm were loading on osteoclast from normal rats and ovariectomied rats for 30min,60min 90min 120min to observe the change of the expression of integrinα_vβ₃ on osteoclast and to evaluate the bone resorption of osteoclast.Results1. The bone mass decreased and the microartitecture of bone was impaired in osteoporotic rats at the 3^th month after ovariectomy.2. The number and TRAP activity of osteoclast were the optimal growth phase at 7^th of culture by bone marrow revulsion culture.in vitro3. Bone resorptive pits were formed on coverslips coated with hydroxyapati-te ceramic.4. In our research, FSS can increase the bone resorption of osteoclasts from normal rats and ovariectomied rats. This effect was the greatest in 16.08 dyne/cm² when FSS was loading in 30 min. But shear stress-induced increasing of bone resorption of osteoclast was higher in normal rats than ovariectomied rats.5. Expressinon of integrinα_vβ₃ of osteoclasts were increased with time prolong of FSS loading. This increase of expressinon of integrinα_vβ₃on osteoclast was at peak value at 60 min. At the same time, the number and area of bone absorptive pit were increased with expression of integrinα_vβ₃. But the change of bone resorption and expression of integrinα_vβ₃ on osteoclast were lower in ovx group than in control group.6. The increase of bone resorption of osteoclast induced by FSS partly decreased when integrinα_vβ₃ was blocked.Conclusion1. The osteoporotic rat modle can been wae established by ovariectomy.2. The optimal growth phase was at 7 of bone marrow revulsion culture in vitro.3. Coverslips coated with hydroxyapatite ceramic can simulate bone matrix and bone slice and the coverslip may use to examine the formation of bone resorption pit induced by osteoclast in vitro.4. The FSS selected can promote the bone resorption of osteoclast and increase the expression of integrinα_vβ₃ on osteoclast. And the increase of expression of integrinα_vβ₃ on osteoclast was time-dependent.5. The effect that FSS enhances the bone resorption of osteoclast was decreased when integrinα_vβ₃ on osteoclast was blocked. This indicates that integrinα_vβ₃ may have a important role in the mechanical signal transduction pathways in osteoclasts6. The sensitivity of osteoclast to fluid shear stress was lower in ovariectomied rats than normal rats.