细胞外信号调节激酶ERK1/2在粗糙钛表面影响成骨细胞分化中的作用

Role of extracellular signal-regulated kinase in osteoblast differentiation on roughened titanium surfaces

目的:通过体外实验,研究喷砂酸蚀钛表面对成骨细胞分化行为的影响,并对喷砂酸蚀钛表面影响成骨细胞分化的信号通路进行研究,探讨粗糙钛表面调控成骨细胞分化的作用机制。方法:采用扫描电子显微镜(SEM)对光滑钛表面、普通喷砂酸蚀钛表面进行表征。将成骨细胞接种到各样本表面,采用实时荧光定量PCR检测成骨细胞在不同材料表面骨功能基因表达的差异。采用Western免疫印迹法研究普通喷砂酸蚀钛表面对成骨细胞内ERK1/2蛋白磷酸化水平的影响。采用SAS9.0软件包对数据进行统计学分析。结果:SEM结果显示,光滑钛表面的粗糙度只有0.2μm,普通喷砂酸蚀钛表面有不规则孔隙,其平均粗糙度约为3.2μm。与光滑钛表面相比,普通喷砂酸蚀钛表面能通过促进骨功能基因的表达显著促进成骨细胞分化(P<0.05)。普通喷砂酸蚀钛表面加入ERK1/2通路特异性抑制剂(PD98095),成骨细胞骨功能基因表达显著增强。Western印迹结果显示,与光滑钛表面相比,普通喷砂酸蚀钛表面组成骨细胞内ERK1/2蛋白的磷酸化水平降低。加入PD98095的光滑钛表面组和未加入该抑制剂的喷砂酸蚀钛表面组ERK1/2蛋白磷酸化水平均降低,但后者不如前者效应显著。结论:表面粗糙度是影响成骨细胞生物学行为的重要因素,粗糙钛表面能显著促进成骨细胞分化,并且其促进成骨细胞分化与抑制ERK1/2蛋白磷酸化有关。

PURPOSE： The purpose of this study was to evaluate the effect of sand-blasted and acid-etched titanium surface on MC3T3-E1 murine pre-osteoblast cell differentiation, and investigate the pathway of regulating osteogenic differentiation of MC3T3-E1 cells on sand-blasted and acid-etched titanium surface in order to elucidate the regulatory mechanisms of surface roughness on osteoblastie differentiation. METHODS： The characteristic of PT polished titanium （PT）, sand-blasted and acid-etched （SLA） titanium surface were examined by scanning electron microscopy （SEM）. Real- time PCR was applied to detect the expression of osteogenic genes including Runx2, OSX, OCN and OPN of the MC3T3- E1 cells cultured on the 2 groups of substrates.ERK1/2 activities in MC3T3-T1 cells were measured by Western blot on SLA surface. The data was analyzed using SAS9.0 software package. RESULTS： The result of SEM observation showed that the PT surface was turned titanium surfaces with the mean peak to valley roughness （Ra） of 0.2 I^m and the corresponding Ra value of SLA was 3.2 Ixm. The expression levels of Runx2, OSX, OPN and OCN were significantly higher and the cell proliferation was significantly lower on SLA surfaces than on PT surfaces （P〈0.05）. The expression levels of Runx2, OSX, OPN and OCN were up-regulated by the effect of SLA surface with PD98095. Compared with PT surface, ERK1/2 phosphorylation was continuously inhibited by SLA. Moreover, PT surfaces treated with PD98095 and SLA surfaces without PD98095 both demonstrated reduced ERK1/2 phosphorylation of the cells and the inhibitive effect of SLA surfaces was milder than that of PD98095. CONCLUSIONS： Surface roughness is an important factor that determines osteoblast behaviors. Surface roughness of titanium substrates seems to enhance the osteoblastic differentiation of MC3T3-E1 cells and the enhancing effect of surface roughness on cell differentiation may be mediated by suppressing the activity of ERK1/2 pathway.