Dnmt3a-Mediated DNA Methylation Changes Regulate Osteogenic Differentiation of hMSCs Cultivated in the 3D Scaffolds under Oxidative Stress.

Liangping Li,Maik Stiehler,Xiaoying Chen,Yan Chen,Qihua Qi,Wenwu Dong,Hongxing Liao,Zemin Ling,Xuenong Zou,Michael Gelinsky,Hao Hu,Corina Vater

doi:10.1155/2019/4824209

Abstract

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 in vivo 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 μM H2O2 to simulate the microenvironment of osteogenesis after implantation. Our results showed that decreased methylation levels of ALP and RUNX2 were induced by H2O2 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 H2O2 treatment using the 3D in vitro model. The hypomethylation of ALP and RUNX2 induced by H2O2 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.

Highlights

Bone repair materials have developed rapidly and are widely used in the clinic, the development of a strategy for improving osteogenesis remains a big challenge in the field of orthopaedics
MTT staining indicated that the seeding method “small” was significantly better than the seeding method “big.” After only 24 h of cultivation, human mesenchymal stem cells (hMSCs) penetrated from the surface through less than 25% depth of the scaffolds in the “dmem+big” group, whereas hMSCs penetrated from the surface through more than a 50% depth of the scaffolds in the “dmem+small” group (Figure 1(a))
The methylation levels of alkaline phosphatase (ALP) and RUNX2 were determined after hMSCs were treated with 5-AZA and incubated with H2O2 and osteogenic medium (OM). 5-AZA led to decreased ALP methylation levels from 56.0% to 44.7% for non-OD hMSCs and from 42.3% to 21.9% for OD hMSCs, respectively (Figure 6(f)). 5-AZA led to a decrease in the levels of RUNX2 methylation from 83.6% to 66.0% for non-OD hMSCs and from 64.6% to 46.0% for OD hMSCs, respectively (Figure 6(g)). These results suggest that 5-AZA can enhance osteogenic differentiation of hMSCs seeded in the 3D scaffolds under oxidative stress (OS) probably through the hypomethylation of ALP and RUNX2 that is correlated with an increased expression of ALP and RUNX2

Summary

Introduction

Bone repair materials have developed rapidly and are widely used in the clinic, the development of a strategy for improving osteogenesis remains a big challenge in the field of orthopaedics. Bone formation involves the recruitment, commitment, proliferation, and osteogenic differentiation of mesenchymal stem cells (MSCs) [1]. In vivo bone formation after implantation of bone repair materials is a more complex process that is influenced by oxidative stress (OS), inflammation response, and vascularization [2]. MSCs initially migrate around the bone repair materials and subsequently undergo hypoxia stress, OS, and even endoplasmic reticulum stress after implantation. Thereafter, the minority of MSCs fail to maintain homeostasis and become apoptotic or even necrotic because these stress reactions are too dramatic. The majority of MSCs are capable of bringing about a series of adaptive reactions that enable them to survive, proliferate, differentiate, and achieve osteogenesis due to an appropriate stress intensity

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Conclusion