Effects Of Mechanical Stretching On The Physiological Activity And The Expression Of IGF-1 And MGF Of Rat Osteoblasts

The effects of mechanical stretching on the physiological activity and gene expression of osteoblasts were studied through biomechanical, cell biological and molecular biology methods. The mechanism by which osteoblasts respond to mechanical signal was investigated at gene expression level in the present paper. The main work and conclusion as follow: â‘ Osteoblasts were delivered and purified from the new-born Wistar rats calvaria by the digesting method. Then osteoblasts were cultured and identified. â‘¡In order to investigate the effects of mechanical stretching on the biochemical responses and gene expression of osteoblasts, a cyclic stretching unit and a static stretching unit were used to apply mechanical stretching on osteoblasts. The applied strain rate was 15% at all times and loading time were 6,12,24,36h respectively. 1) To determine the proliferative effect of mechanical stretching on osteoblasts, cell cycle analysis was carried out by flow cytometry. The results showed that the relative proliferative index(PIr) of osteoblasts was increased with the increase of loading time, which were subjected to 15% strain rate by cyclic stretching and static stretching. The PIr of cyclic stretched osteoblasts was bigger than static stretched osteoblasts, but they had same trend. Under the action of different loading time, the PIr of osteoblasts was the biggest of all when loading time was 6h. However, the PIr of osteoblasts decreased after loaded 12,24,36h, and they gradually tended toward 1. These results demonstrated that the mechanical stretching increased cell proliferation, and the action was related with loaded time. The PIr of osteoblasts was the biggest of all when initial loading stages (6h), and the increasing action of cyclic stretching was stronger than static stretching all the time. 2) Cyclic stretching or static stretching at 15% elongation affected the relative alkaline phosphatase(ALP) and relative extracellular calcium deposition of osteoblasts. The static stretching for 6h decreased relative ALP and relative extracellular calcium deposition of osteoblasts (<1). And the values increased with the increase of loading time(>1). The relative ALP peaked at 36h. The relative extracellular calcium deposition peaked at 24h and decreased at 36h(but >1). The relative ALP and relative extracellular calcium deposition were all less than 1 at every loaded time when osteoblasts were subjected to cyclic stretching. These results demonstrated that the static stretching inhibited differentiation and mineralization of osteoblasts at short loaded time, and increased at long loaded time. Then cyclic stretching inhibited differentiation and mineralization of osteoblasts at all loaded time. 3) The expression of IGF-1 and MGF mRNA was detected by semi-quantitive RT-PCR. The osteoblasts were subjected to cyclic stretching ('cs'), static stretching('ss') respectively, and the unstrained cells as control culture('cc'). The expression of IGF-1: 'cc', is low; 'cs'is significantly higher than 'ss'; but the expression of MGF: 'cc', is nothing; 'cs'is markedly higher than 'ss'. MGF mRNA expression peaked at 6h, and then later the IGF-1 mRNA expression peaked at 12h. These results demonstrated that the IGF-1 mRNA expression of osteoblasts increased markedly in response to mechanical stimulation, and MGF, the isoforms of IGF-1, was also expressed. It was certified that MGF expressed in bone too. The expression of two autocrine growth factor were affected by loading mode and loading time. The effect of IGF-1 and MGF mRNA expression cyclic stretching was stronger than static stretching, and the increase of mechanical stretching was the best at the short time. Following mechanical stretching stimulation, MGF was expressed quite rapidly and then largely expressed IGF-1, which was sustained for a considerably longer time.