Guangdong Provincial Key Laboratory of Orthopaedics and Traumatology, Orthopaedic Research Institute/Department of Spinal Surgery, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou 510080, China.
Centre for Translational Bone, Joint, and Soft Tissue Research, Medical Faculty and University Centre for Orthopaedics and Trauma Surgery, University Hospital Carl Gustav Carus at Technische Universität Dresden, Dresden 01307, Germany.
Oxid Med Cell Longev. 2019 Nov 15;2019:4824209. doi: 10.1155/2019/4824209. eCollection 2019.
Oxidative stress (OS) caused by multiple factors occurs after the implantation of bone repair materials. DNA methylation plays an important role in the regulation of osteogenic differentiation. Moreover, recent studies suggest that DNA methyltransferases (Dnmts) are involved in bone formation and resorption. However, the effect and mechanism of DNA methylation changes induced by OS on bone formation after implantation still remain unknown. Three-dimensional (3D) cell culture systems are much closer to the real situation than traditional monolayer cell culture systems in mimicking the microenvironment. We have developed porous 3D scaffolds composed of mineralized collagen type I, which mimics the composition of the extracellular matrix of human bone. Here, we first established a 3D culture model of human mesenchymal stem cells (hMSCs) seeded in the biomimetic scaffolds using 160 M HO to simulate the microenvironment of osteogenesis after implantation. Our results showed that decreased methylation levels of ALP and RUNX2 were induced by HO treatment in hMSCs cultivated in the 3D scaffolds. Furthermore, we found that Dnmt3a was significantly downregulated in a porcine anterior lumbar interbody fusion model and was confirmed to be reduced by HO treatment using the 3D model. The hypomethylation of ALP and RUNX2 induced by HO treatment was abolished by Dnmt3a overexpression. Moreover, our findings demonstrated that the Dnmt inhibitor 5-AZA can enhance osteogenic differentiation of hMSCs under OS, evidenced by the increased expression of ALP and RUNX2 accompanied by the decreased DNA methylation of ALP and RUNX2. Taken together, these results suggest that Dnmt3a-mediated DNA methylation changes regulate osteogenic differentiation and 5-AZA can enhance osteogenic differentiation via the hypomethylation of ALP and RUNX2 under OS. The biomimetic 3D scaffolds combined with 5-AZA and antioxidants may serve as a promising novel strategy to improve osteogenesis after implantation.
多种因素导致的氧化应激（OS）发生在骨修复材料植入后。DNA 甲基化在成骨分化的调节中发挥着重要作用。此外，最近的研究表明，DNA 甲基转移酶（Dnmts）参与骨形成和吸收。然而，OS 引起的 DNA 甲基化变化对植入后骨形成的影响和机制仍不清楚。三维（3D）细胞培养系统比传统的单层细胞培养系统更能模拟真实情况，更接近微环境。我们已经开发出由矿化 I 型胶原组成的多孔 3D 支架，该支架模拟了人骨细胞外基质的组成。在这里，我们首先使用 160µM 的 H2O2 建立了在仿生支架中接种人间充质干细胞（hMSCs）的 3D 培养模型，以模拟植入后成骨的微环境。我们的结果表明，HO 处理诱导 3D 支架中 hMSCs 的 ALP 和 RUNX2 甲基化水平降低。此外，我们发现 Dnmt3a 在猪前路腰椎间融合模型中显著下调，并通过 3D 模型证实 HO 处理使其减少。HO 处理诱导的 ALP 和 RUNX2 低甲基化被 Dnmt3a 过表达所消除。此外，我们的研究结果表明，Dnmt 抑制剂 5-AZA 可以在 OS 下增强 hMSCs 的成骨分化，这表现为 ALP 和 RUNX2 的表达增加，同时 ALP 和 RUNX2 的 DNA 甲基化减少。综上所述，这些结果表明，Dnmt3a 介导的 DNA 甲基化变化调节成骨分化，5-AZA 可以通过 OS 下 ALP 和 RUNX2 的低甲基化增强成骨分化。仿生 3D 支架结合 5-AZA 和抗氧化剂可能成为改善植入后骨形成的一种有前途的新策略。
