Low Density Extracorporeal Shock Wave And Low Dose Pulsed RhPTH1-34's Effect To Rat Osteoblast And Its Intracellular Signal Transduction

It has been widely known that many physical and chemical factors contribute to bone formation. Various effects of different stimulates are shown according to different periods and different organs they take effect. Detailing of these mechanisms are of vital importance to medical research.As a kind of sound wave, ESW can produce tremendous pressure variation in an extremely short period of time. When ESW traveling through medias of different densities, the relief of energy will result in kinds of biochemical effect to stress sensitive cells, such as osteoblast. In vivo, changes of local micro environment and fluid shearing stress(FSS) caused by ESW can cause series of bio-effect to these cells at molecular level. Many research have confirmed that osteoblast can be activated by ESW stimulate to contribute to bone formation. Therefore, the conception of stress construction in bone tissue engineering arises in recent years.The balance of bone formation and bone absorption is modulated by many kinds of chemicals, including PTH. In vivo, the secretion model of PTH are of 2 kinds. First, the quantity of secreted PTH is frequently altered, which contribute to the maintenance of constant calcium concentration in serum. Second, the amount and period of PTH secretion are constant, which contribute to bone formation. Thus, different patterns of PTH stimulates may cause different result to bone formation. Some researches show that continual high dose rhPTH1-34 inhibit bone formation, while low dose impulse rhPTH1-34 stimulate enhance it. But the mechanism is still unclear.ESW has been proved to be effective to enhance bone formation. But, as a kind of stress stimulate, its mechanism is still unclear. Many aspects of its clinical application, such as suitable energy density, the frequency, etc, are all left to be identified. The same as ESW, PTH's mechanism of enhancing bone formation is not clear either. Exploring of these mechanisms, especially the intracellular signal transduction, are of key importance to Bone Tissue Engineering research and clinical application.1,Primary culture and identification of rat osteoblast.1.1 Aims: to establish reasonable and effective new method and process of in vitro osteoblast culture and identification.1.2 Methods: modified isolation method of osteoblast by type I collagenase was adopted to get rice's calvarial bone cell. The cells were cultured invitro and identified. The growth line was drew to observe the proliferation of cells. Cell morphology was observed and recorded by microscope. Cells'Alkaline Phoshatase (ALP) and type I collagen expression was detected by method of Immunohistochemical staining.1.3 Results: by modified isolation method, purified bone cells were obtained. Growth line shows excellent proliferation rate of the cells. Immunohistochemical staining of ALP shows brown and yellow region exist in plasma of positive cells and the positive percentage is 77.3%. Type I collagen's immunohistochemical staining shows the positive cells'plasma turn into brown and yellow and the positive rate is 77.3%. Calcic node was identified in the matrix of osteoblast had been cultured invitro for longer than 3 weeks.1.4 Conclusions: Modified isolation by type I collagenase is an excellent osteoblast primary culture method.2,Low Density Extracorporeal Shock Wave and Low Dose Pulsed rhPTH1-34's Effect to proliferation and differentiation of Rat Osteoblast2.1 objective to investigate low density ESW and low dose pulse rhPTH1-34 stimulation's effect on proliferation and differentiation of ROB. 2.2 method After stimulations of different times of 0.18mJ/mm2ESW, different pattern of rhPTH1-34 and ESW+pulse rhPTH1-34 (10-11M), ROB cells were collected to observe cell proliferation by cell counting, MTT and DNA cycle analysis and to observe cell differentiation by measuring ALP activity and type I collagen expression.2.3 result 0.18mJ/mm2ESW(60-150 times), pulse rhPTH1-34 (10-11and 10-10M) and ESW(60-150 times)+pulse rhPTH1-34 (10-11M) stimulation can all improve proliferation and differentiation of cultured ROB(p<0.05, compared with control). The effect of ESW+pulse rhPTH1-34 stimulation was stronger than ESW alone and pulse rhPTH1-34 (10-11M) alone(p<0.05).2.4 conclusion Suitable ESW stimulation and low dose pulse rhPTH1-34 stimulation can improve proliferation and differentiation of cultured ROB significantly. The effect of combining ESW with pulse rhPTH1-34 (10-11M) stimulation was the most strongest.3,The intracellular signal transduction of ROB stimulated by suitable ESW and Low Dose Pulsed rhPTH1-343.1 objective To have a study on the mechanism of ESW and low dose pulse rhPTH1-34's contribution to proliferation and differentiation of ROB, and to investigate the role of PKC and p38MAPK in the intracellular signal transduction path.3.2 Method Added with specific blocker of PKC or p38MAPK, proliferation of ROB are measured by Typan Blue Dye, MTT and DNA circle analysis by Flow Cytometry to observe the role of PKC and p38MAPK during the process. Added with specific blocker of PKC or p38MAPK, bone formation of ROB are observed by ALP activity detection and expression percentage of collagen I to observe the role of PKC and p38MAPK during the process. Added with specific blocker of PKC or p38MAPK, activation of p38MAPK is measured by Western Blot to define the role of PKC and p38MAPK in the related intracellular signal transduction path.3.3 Result 3.3.1 Cell proliferation and bone formation of ROB measured by methods mentioned above are significantly high in cells treated by low dose pulse rhPTH1-34, ESW and cooperation of both stimulates(P<0.05). The effect of cooperation of low dose pulse rhPTH1-34 and ESW is the strongest, and is significantly higher than low dose pulse rhPTH1-34 and ESW(P<0.05).3.3.2 Added with H7, the specific PKC blocker, proliferation and bone formation of ROB treated with low dose pulse rhPTH1-34 become significantly less than treated with rhPTH1-34 alone(P<0.05), which indicates that PKC may play a key role. But SB203580 can not affect proliferation and bone formation of ROB treated with low dose pulse rhPTH1-34(P>0.05), which indicates that p38MAPK don't take part in the process.3.3.3 Added with H7, the specific PKC blocker, proliferation and bone formation of ROB treated with ESW become significantly less than treated with ESW alone(P<0.05), which indicates that PKC may play a key role. SB203580, a specific p38MAPK blocker, can also affect proliferation and bone formation of ROB treated with ESW(P<0.05), which indicates that p38MAPK take part in the process too.3.3.4 Measured by Western Blot, P-p38MAPK expression of ROB treated with ESW alone and cooperation of ESW and rhPTH1-34 are higher than that of the control and treated with rhPTH1-34 alone(P<0.01), which confirms that p38MAPK can be activated by ESW, but not low dose pulse rhPTH1-34 stimulation. Activation of p38MAPK by ESW can be inhibited by H7(P<0.01), which shows that PKC take part in the activating. All these come together to indicate exist of the signal path: ESW→PKC→p38MAPK→ROB's proliferation and differentiation. While H7 can significantly inhibit the proliferation and bone formation of ROB treated with low dose pulse rhPTH1-34, p38MAPK can not be activated by rhPTH1-34, which indicate exist of another signal path: rhPTH1-34→PKC→ROB's proliferation and differentiation.3.4 Conclusion Suitable ESW stimulation and low dose pulse rhPTH1- 34 stimulation can improve proliferation and differentiation of cultured ROB significantly. The effect of cooperation of ESW and rhPTH1-34 stimulation was the most strongest. Proliferation and bone formation of ROB treated with ESW can be inhibited by H7 and SB203580, and activation of p38MAPK by ESW can be inhibited by H7, which indicate exist of the signal path: ESW→PKC(some isoforms)→p38MAPK→ROB's proliferation and differentiation. While H7 can significantly inhibit the proliferation and bone formation of ROB treated with low dose pulse rhPTH1-34, p38MAPK can not be activated by rhPTH1-34, which indicate exist of another signal path: rhPTH1-34→PKC(other isoforms)→ROB's proliferation and differentiation.