Osteoclast Calcium Fluid Shear Stress Response Characteristics And Mechanisms

Objective:Bone is a organ with evolutionary structure and its metabolism was performed under the mechanical environment. Bone is a porous tissue whose internal cavities are full of interstitial fluid. As bone cells are exposed to both vascular pressure and mechanical loading, the fluid may generate significant shear stresses on the osteoblasts and osteoclasts on the endosteal surface and osteocytes within the lacunae. Normal bone metabolism in human is maintained by the balance between osteoblasts and osteoclasts. Osteoblasts secret bone matrix to form new bone and osteoclasts play important roles during bone metabolism to resorbe the old bone matrix.A number of studies have demonstrated that the formation and differentiation of osteoclasts were closely related to calcium oscillation, however, there is still a lack of enough researches about the relationship between mechanical stimulation and the calcium oscillation of osteoclasts, which is useful for osteoclastic metabolism.In our research, we mainly focus on the following questions:If could fluid shear stress (FSS) induce calcium response in osteoclast? And if is the osteoclastic function influenced by this calcium response? Solving these problems can provide a reasonable guidance and scientific basis for the clinical regulation of mechanical stimulation on bone metabolism.Methods:The culture of osteoclasts. Three kinds of culture methods for osteoclasts were used in this study, i.e. the induction of primary hematopoietic stem cells with M-CSF and RANKL, the induction of RAW264.7cells with M-CSF and RANKL, and the induction of RAW264.7cells applying the condition medium (CM) from osteoblasts. The biological features of osteoclasts such as apoptosis and the relationship between nuclear number and cellular area, were observed and analyzed at different induction time (0day,4days and8days).The analyses on the features of calcium response of osteoclasts under FSS. We used Fluo-4AM to stain the intracellular calcium in osteoclasts, then mounted the coverslip with cells on a flow chamber. The fluid shear stress of1dyne/cm2or10dyne/cm2were applied on the cells and ended after10minutes. The time-lapsed fluorescent intensity of each cell was recorded and statistically analyzed.The analyses of the mechanisms of calcium response of osteoclasts induced by FSS. Seven pathways of calcium response were investigated, respectively, i.e. MSCC, PLC, L-VSCC, ATP, intracellular calcium stored in ER, gap junctions between cells as well as extracellular calcium. Comparing the different calcium response for these blocking groups with the normal group, we could conclude the mechanism of calcium response of osteoclasts induced by FSS.The expression of functional markers of osteoclasts under FSS stimulation. We detected the gene expression of c-Fos through Real-Time PCR technology.TRAP and NFATcl were the important markers for osteoclasts, and NFATcl was closely related with calcium response. We detected the protein expression of TRAP and NFATcl of osteoclasts under static condition and FSS stimulation through Western blot technology. Results:32pg/mL soluble RANKL(sRANKL) was detected in MC3T3-E1CM by ELISA. This CM was able to induce RAW264.7cells to fuse into osteoclasts. There is significant correlation between nuclear numbers and cellular areas of osteoclasts, therefore the nuclear number of an osteoclast can be estimated according to its spreading area.The results indicated a different property of calcium oscillation for the osteoclasts in different fusion stages, i.e., more calcium-responsive peaks appeared in small osteoclasts than those in the larger ones.Calcium response induced by FSS disappeared after blocking MSCC, PLC or removing intracellular calcium from ER, and there was only one calcium peaks when blocking ATP pathway or removing extracellular calcium. The calcium response was similar to control group after blocking gap junction and L-VSCC.Compared with the static condition, the FSS can significantly improve the c-Fos and NFATcl expression, but not TRAP expression. Conclusions:The condition medium from MC3T3-E1was able to promote the differentiation of RAW264.7cells into TRAP-positive, multinucleated osteoclasts.Fluid shear stress could induce calcium response in osteoclasts. It was concluded that FSS might first activate MSCC and PLC, then IP3, finally release the calcium ions stored in ER. In addition, extracellular calcium influx might be necessary for the subsequent calcium peaks after the first one. It was implied that the calcium response could significantly improve the expression of NFATcl.Fluid shear stress may be an independent factor playing the roles on the calcium response of osteoclasts similar to RANKL.