Effects Of Continuously Compressive Pressure On Biological Characteristics And Intracellular And Extracellalur Ca～(2+) Concentration Of Rat Osteoblasts In Vitro

Objective: The mechanical environment of the skeleton is crucial to bone development and architecture. Mechanical loading plays an important role in regulating bone metabolism. Increased mechanical loading increases bone formation and decreases bone resorption. The absent or unsuitable mechanical stimulation may reduce bone formation, and increase bone resorption. Although the effects of mechanical stimulation on bone formation are well documented, the cytological basis of their action is still poorly understood. So we investigated the effects of continuously compressive pressure (CCP) on the biological characteristics and intracellular Ca2+ concentration of rat osteoblasts in vitro.Methods Isolated and purified osteoblasts from the alveolar bone of newborn SD rats were primarily cultured and subcultured to passage three and four. A self-made hydrostatic compressive device was used to apply CCP at 100KPa or 30KPa on the cells. Cell proliferation was detected by cell counting. Biochemical analysis andradio-immunological methods were used to study the effects of different CCP on the alkaline phosphatase (ALP) activity, osteocalcin (OCN) synthesis and intracellular and extracellular Ca2+ concentration at different interval (0d/ld/3d/5d) and different levels of CCP force. The cell were subjected to CCP(100KPa, 30KPa)for 0/1/3/5 h and laser scanning confocal microscopy was used to observe the changes of intracellular Ca2+ concentration induced by CCP. Before the observation, cells had been loaded with Fluo-3, a fluorescent intracellular chelator.Results: The cells in CCP treatment group proliferated slower than those in control group, and the doubling time in the four groups (100KPa test group, 100KPa control group, 30KPa test group and 30KPa control group) are 73.43h, 63.06h, 60.95h and 58.94h respectively. The ALP activitiy in all groups increased with time and was significant different between test and control groups over 24h after applying CCP. Under the same conditions as described above, the secretion of OCN also increased with time. Under 100KPa CCP applied, the secretion of OCN increased over 24h after applying CCP and was significantly difference between the two groups (p<0.01); however, under 30KPa CCP, there was no significant difference of secretion of OCN (p>0.05). Application of CCP (30KPa, 100KPa) to osteoblasts for 1h and 3h produced an increase in intracellular Ca2+ concentration, but increased with the time the intracellular Ca2+ concentration decreased.Conclusion: CCP may inhibit the growth of osteoblasts and promote the differentiation of the cells at the mean time. Long time CCP might cause the decrease of intracellular Ca2+ concentration and augment of extra cellular Ca2+ concentration in osteoblasts. As a result CCP may be not fitting force for growth of osteoblasts.