目的:本研究旨在探讨机械刺激调节成骨分化的潜在机制。
方法:将成骨细胞暴露于压缩力(0-4g/cm2)1-3天或CGRP1或3天。受体活性修饰蛋白1(RAMP1)的表达,转录因子RUNX2,骨钙蛋白,通过蛋白质印迹分析p38和p-p38。通过茜素红应变分析钙矿化。
结果:使用压缩力处理,24小时的低压缩力(1和2g/cm2)可促进成骨细胞分化和矿物质沉积,而较高的压缩力(3和4g/cm2)不会产生成骨作用。通过蛋白质印迹分析,我们观察到受体活性修饰蛋白1(RAMP1)表达上调,在低强度压缩力促进成骨细胞分化过程中,p38丝裂原活化蛋白激酶(MAPK)被磷酸化。降钙素基因相关肽(CGRP)肽孵育的进一步研究,RAMP1的配体,表明CGRP在25和50ng/ml的浓度可以增加RUNX2和骨钙蛋白的表达水平,和矿化百分比,表明它的成骨潜力。此外,在相同的条件下,CGRP还显著上调RAMP1和磷酸化p38表达水平。此外,压缩力(1和2g/cm2)与50ng/mlCGRP的组合倾向于增加RAMP1表达,p38活性,和成骨标志物RUNX2水平,以及与单独的压缩力相比的矿化百分比。这表明RAMP1可能在成骨分化过程中充当p38信号传导的上游调节剂。
结论:这些研究结果表明,CGRP-RAMP1/p38MAPK信号传导与成骨细胞分化有关,以响应最佳大小的压缩力。这项研究有助于确定压缩刺激的潜在机制,也可能增强压缩刺激或CGRP肽的应用,作为在正畸治疗中加速牙齿移动的替代方法。
OBJECTIVE: The present study aimed to investigate the underlying mechanism of mechanical stimulation in regulating osteogenic differentiation.
METHODS: Osteoblasts were exposed to compressive force (0-4 g/cm2) for 1-3 days or CGRP for 1 or 3 days. Expression of receptor activity modifying protein 1 (RAMP1), the transcription factor RUNX2, osteocalcin, p38 and p-p38 were analyzed by western blotting. Calcium mineralization was analyzed by alizarin red straining.
RESULTS: Using compressive force treatments, low magnitudes (1 and 2 g/cm2) of compressive force for 24 h promoted osteoblast differentiation and mineral deposition whereas higher magnitudes (3 and 4 g/cm2) did not produce osteogenic effect. Through western blot assay, we observed that the receptor activity-modifying protein 1 (RAMP1) expression was upregulated, and p38 mitogen-activated protein kinase (MAPK) was phosphorylated during low magnitudes compressive force-promoted osteoblast differentiation. Further investigation of a calcitonin gene-related peptide (CGRP) peptide incubation, a ligand for RAMP1, showed that CGRP at concentration of 25 and 50 ng/ml could increase expression levels of RUNX2 and osteocalcin, and percentage of mineralization, suggesting its osteogenic potential. In addition, with the same conditions, CGRP also significantly upregulated RAMP1 and phosphorylated p38 expression levels. Also, the combination of compressive forces (1 and 2 g/cm2) with 50 ng/ml CGRP trended to increase RAMP1 expression, p38 activity, and osteogenic marker RUNX2 levels, as well as percentage of mineralization compared to compressive force alone. This suggest that RAMP1 possibly acts as an upstream regulator of p38 signaling during osteogenic differentiation.
CONCLUSIONS: These findings suggest that CGRP-RAMP1/p38MAPK signaling implicates in osteoblast differentiation in response to optimal magnitude of compressive force. This study helps to define the underlying mechanism of compressive stimulation and may also enhance the application of compressive stimulation or CGRP peptide as an alternative approach for accelerating tooth movement in orthodontic treatment.