ABSTRACT
Objective To investigate the effects of mechanical strain on Ca-calmodulin dependent kinase (CaMK)-cAMP response element binding protein (CREB) signal pathway and proliferation of osteoblasts.Methods Using a four-point bending device, MC3T3-E1 cells were exposed to mechanical tensile strains of 2500 µs and 5000 µs at 0.5 Hz respectively. The intracellular free Ca([Ca]i) concentration and calmodulin activity were assayed by fluorospectrophotometry, CaMK II β, CREB, and phosphorylated (activated) CREB (p-CREB) were assessed by Western blot, and cells proliferation was assayed with MTT. Pretreatment with verapamil was carried out to block Cachannel, and inhibitor U73122 was used to inhibit phospholipase C (PLC).Results Mechanical strains of 2500 µs and 5000 µs for 1 to 10 minutes both increased [Ca]i level of the cells. The 2500 µs strain, a periodicity of 1 h/d for 3 days, activated calmodulin, elevated protein levels of CaMK II β and p-CREB, and promoted cells proliferation, which were attenuated by pretreatment of verapamil or U73122. The effects of 5000 µs strain on calmodulin, CaMK II β, p-CREB and proliferation were contrary to 2500 µs strain.Conclusion The mechanical strain regulates osteoblasts proliferation through Ca-CaMK-CREB signal pathway via Cachannel and PLC/IPtransduction cascades.
ABSTRACT
Objective To investigate the effects of different vibration durations on expression level of osteogenesis-related proteins by loading low intensity mechanical vibration in the ovariectomized (OVX) rats. Methods Twenty-four 6-month old female Wistar rats were ovariectomized and then randomly divided into 8-week-control group (C8), 12-week-control group (C12), 8-week-vibration group (V8), and 12-week-vibration group (V12). Vibration treatment was started one week after all the rats were ovariectomized. Rats in both V8 and V12 groups were loaded with 35 Hz, 0.25 g low intensity mechanical vibration for 15 minutes per day. C8 and C12 groups served as control without any treatment. Rats were executed in batch at 8th and 12th week, respectively, to analyze expression level of osteogenesis-related proteins, including BMP-2, p-ERK, Runx2 and OCN. Results Low-intensity mechanical vibration enhanced the osteogenesis related protein expression in OVX rats (P<0.01). With the extension of vibration duration, the osteogenesis related proteins BMP-2、p-ERK、Runx2 and OCN in V12 group were increased by 22.61% (P<0.05), 27.96% (P<0.01), 25.85% (P<0.01), 27.05% (P<0.01), respectively, as compared with V8 group. But for the control groups, no significant differences were found in expression level of osteogenesis-related proteins. Conclusions The low intensity mechanical vibration could elevate expression level of osteogenesis-related proteins, and the osteogenesis was enhanced with the extension of vibration duration.
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Objective To investigate the effect of mechanical loading with different magnitudes on the proliferation, differentiation and activity of preosteoclasts and osteoclasts. Methods One group of RAW264.7 preosteoclastic cells cultured in osteoclast inductive medium were subjected to the cyclic tensile strain for three days, and then cultured for four days; the other group of RAW264.7 cells were induced in osteoclast inductive medium for four days to be osteoclasts, then subjected to the cyclic tensile strain for three days. Results Under the tensile strain at different magnitudes, the proliferation variations in two groups of RAW264.7 cells were approximately identical, but changes in the activities of tartrate-resistant acid phosphatage (TRAP) and numbers of TRAP-positive multinucleated cells (osteoclasts) in the two groups were significantly different. Under the moderate tensile strain (2 500 με), the reduction of TRAP activity and osteoclasts number were both the highest in the first group, and both the lowest in the second group. Conclusions The influence of different tensile strain on osteoclast differentiation and osteoclastic activity of preosteoclasts in early differentiation is different to that of the preosteoclasts already differentiated into osteoclasts.