Hou Peng, Sun Yu, Yang Weichao, Wu Hongliu, Sun Luyuan, Xiu Xinjie, Xiu Chaoyang, Zhang Xiaonong, Zhang Wen
Orthopaedic Department, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, Shanghai, China.
State Key Laboratory of Metal Matrix Composites, School of Materials Science and Engineering, Shanghai Jiao Tong University, Shanghai, China.
J Biomed Mater Res B Appl Biomater. 2022 Jul;110(7):1594-1603. doi: 10.1002/jbm.b.35020. Epub 2022 Feb 2.
Magnesium (Mg) based alloy has been used as a biodegradable implant for fracture repair with considerable efficacy, and it has been proved that magnesium ion (Mg ), one of the degradation products, could stimulate osteogenesis. Here, we investigated the osteogenesis property of magnesium both in vitro and in vivo, and to identify the cellular and molecular mechanisms that mediate these effects. Results showed that magnesium exerts a dose-dependent increase in the proliferation of MC3T3 and MG63 cells, and in the expression of osteopontin (OPN), a promising biomarker of osteogenesis. Subsequently, the protein-protein interaction (PPI) network analysis showed the interactions between calmodulin (CaM) and calmodulin-dependent protein kinase (CaMK) and CREB1. The ratio of p-CaMKIV/CaMKIV and p-CREB1/CREB were increased at protein level in MC3T3 and MG63 cells after treatment with Mg . Dual-luciferase reporter gene assay showed that p-CREB1 could directly bind to OPN promoter and up-regulate the transcription of OPN after nuclear entry. Meanwhile, the expression of OPN and p-CREB1, which increased after Mg treatment, was down-regulated by sh-CaMKIV or sh-CREB1. Moreover, the mineralized deposit and expression of OPN were reduced after treatment with an inhibitor of CaMKIV, KN93. In addition, massive cavities in the cortical bone around the Mg screw were showed in vivo after injection of KN93. These data indicated that the osteogenic effect of Mg is related to the activation OPN through CaM/CaMKIV/CREB1 signaling pathway.
镁基合金已被用作具有显著疗效的骨折修复可生物降解植入物，并且已经证明，降解产物之一的镁离子（Mg²⁺）可以刺激骨生成。在此，我们研究了镁在体外和体内的成骨特性，并确定介导这些作用的细胞和分子机制。结果表明，镁对MC3T3和MG63细胞的增殖以及骨桥蛋白（OPN）的表达具有剂量依赖性增加，骨桥蛋白是一种有前景的成骨生物标志物。随后，蛋白质-蛋白质相互作用（PPI）网络分析显示钙调蛋白（CaM）与钙调蛋白依赖性蛋白激酶（CaMK）和CREB1之间的相互作用。用Mg²⁺处理后，MC3T3和MG63细胞中p-CaMKIV/CaMKIV和p-CREB1/CREB的比例在蛋白质水平上增加。双荧光素酶报告基因测定表明，p-CREB1可以直接结合到OPN启动子上，并在进入细胞核后上调OPN的转录。同时，Mg²⁺处理后增加的OPN和p-CREB1的表达被sh-CaMKIV或sh-CREB1下调。此外，用CaMKIV抑制剂KN93处理后，矿化沉积物和OPN的表达降低。另外，注射KN93后在体内显示Mg螺钉周围皮质骨中有大量空洞。这些数据表明，Mg的成骨作用与通过CaM/CaMKIV/CREB1信号通路激活OPN有关。
