The Effects Of Cyclic Stretching On The Physiological Activity And Mechanical Property Of Rat Osteoblasts

The effects of cyclic stretching on the physiological activity and mechanical property of osteoblasts were studied through biomechanical ,cell biological and cell dynamical methods, as well as the mechanism by which osteoblasts respond to mechanical signal was investigated in the present paper. The main work and conclusions as follows: (1) Osteoblasts were delievered and cultured from the new-born Wistar rats calvaria by the digesting method. (2) In order to investigate the effects of cyclic stretching on the biochemical responses and mechanical property of osteoblasts at different strain magitude and loading time, a four-point bending apparatus was used to apply cyclic stretching on osteoblasts .The applied strain magnitudes were 500με,1000μεand 1500μεrespectively and working frequency was 0.4Hz. The results showed that:a) Cyclic strtching at 500με,1000μεand 1500μεwith the frequency of 0.4Hz affected the physiological activity of osteoblasts delivered from rat calvaria. Stretching at 500μεincreased cell proliferation, collagen type I secretion, extracellular calcium deposition, alkaline phosphatase (ALP) activity and osteocalcin production. On the contrary, stretching at 1000μεand 1500μεinhabited cells proliferation, collagen type I secretion , extracellular calcium deposition ,ALP activity and osteocalcin production. The inhabiting effects of steteching at 1500μεwere more significant than that of 1000με. The promoting or inhabiting effects of stretching on osteoblasts increased with the loading time. Furthermore, the reponses of osteoblasts to cyclic stimulation expressed accordant changes in prolliferation, matrix production, differentiation and mineralization.b)The micropipette aspiration technique was used to study the effects of cyclic stretching on the mechanical property of rat calvirial osteoblasts which were loaded at 500με,1000μεand 1500μεrespectively with the frequency of 0.4Hz. The stretching increased cells adhesive forces and spreading areas. Furthermore, cells adhesive forces and spreading areasincreased with the strain magnitude. Loading time had no significant effect on cells adhesive forces and spreading areas. (3) A new loading method, step by step increased stretching (step stretching in shortened form) was also designed and used in this article. Cells were stretched at 500μεfor 2h firstly, followed by stretching at 1000μεfor 2h. Then the strain level was raised to 1500με and cells were stretched for 2h. The step by step increased stretching methods was used to investigate osteoblasts response to the changing mechanical environment. The results showed that: a) When the stretching on osteoblasts were changed from an appropriate stimulation (500με) to an inhabiting stimulation (above 1000με), osteoblasts changed their physiological behaviors to adapt to the new mechanical environment. Cells proliferation, collagen typeⅠsecretion , extracellular calcium deposition ,ALP activity and osteocalcin production changed rapidly, suggesting that cells could distinguish different mechanical environment and adjust their biochemical responses accordingly.b) The effects of step loading on osteoblasts mechanical property had the same trends as physilogical activity. Cells modified their adhesive forces and spreading areas when the mechanical environment was changed.(4) The intracelluler calcium plays an important role in cell mechaotransduction. The fluorescence probe (fluo-3/AM) was used to investigate the changes of intracelluler calcium concentration when osteoblasts responded to stretching. The results showed that osteoblasts that were stretched increased their intracelluler calcium concentration. Cells were stretched after being treated with the panax notoginseng saponins, a Ca2+ channel inhibitor. The results showed that the rise of intracelluler calcium concentration was due to the influx of extracellular calcium and the release of intracellular calcium store, in which the influx of extracellular calcium play the main role.