Background: Anti-tumor necrosis factor a (anti-TNF-a) treatments are widely used in patients with rheumatoid arthritis (RA); however, the increased risk of infections is one of the most important side effects of anti-TNF-a agents. This study evaluated the differences between monoclonal antibodies and the soluble receptor for infections in patients with RA by direct comparison of observation studies.

The aim of the present study was to investigate the potential of bone mesenchymal stem cells (BMSCs) treated with a combination of vascular endothelial growth factor (VEGF) and bone morphogenetic protein-6 (BMP-6) genes for the treatment of avascular necrosis of the femoral head (ANFH). Rat BMSCs were isolated and purified using a density gradient centrifugation method. The purity and characteristics of the BMSCs were detected by cell surface antigens identification using flow cytometry. The experimental groups were administered with one of the following adeno-associated virus (AAV) vector constructs: AAV-green fluorescent protein (AAV-GFP), AAV-BMP-6, AAV-VEGF or AAV-VEGF-BMP-6. The expression of VEGF and BMP-6 was detected by reverse transcription-quantitative polymerase chain reaction, western blotting and ELISA assays. The effects of VEGF and BMP-6 on BMSCs were evaluated by angiogenic and osteogenic assays. The transfected BMSCs were combined with a biomimetic synthetic scaffold poly lactide-co-glycolide (PLAGA) and they were then subcutaneously implanted into nude mice. After four weeks, the implants were analyzed with histology and subsequent immunostaining to evaluate the effects of BMSCs on blood vessel and bone formation in vivo. In the AAV-VEGF-BMP-6 group, the expression levels of VEGF and BMP-6 were significantly increased and human umbilical vein endothelial cells tube formation was significantly enhanced compared with other groups. Capillaries and bone formation in the AAV-VEGF-BMP-6 group was significantly higher compared with the other groups. The results of the present study suggest that BMSCs expressing both VEGF and BMP-6 induce an increase in blood vessels and bone formation, which provides theoretical support for ANFH gene therapy.

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.

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