Effect of Negative Pressure on Human Bone Marrow Mesenchymal Stem Cells In Vitro

Abstract

The aim of this study was to determine how low-intensity intermittent negative pressure affects the differentiation and proliferation of human mesenchymal stem cells (MSCs), as well of OPG and OPGL mRNA expression in MSCs. MSCs were isolated from adult marrow using the density gradient separation method, passaged for three generations, and divided into the vacuum group, which was administrated at pressure of −50 kPa, for 30 min at a frequency of 2/d, and a control group. The differentiation of MSCs was examined through inverted phase contrast microscopy, measurement of alkaline phosphatase activity, alizarin-red staining, and immunohistochemistry for type I collagen, hypoxia-inducible factor-1α (HIF-1a), and vascular endothelial growth factor (VEGF). The MTT assay and flow cytometry were used to measure proliferation and apoptosis. Real-time PCR detected the expression of mRNA from OPG/OPGL. Compared to the control group, there was a decrease in the proliferation of cells in the vacuum group. The number of cells in S phase was reduced by 62.4%, while the rate of apoptosis, the activity of ALP, and calcium release all increased under vacuum conditions. Calcium nodes could be observed through alizarin-red staining, and the expression of collagen type I, VEGF, and HIF-1a were increased significantly. Expression of OPG mRNA was increased and the expression of OPGL mRNA decreased in the vacuum group relative to the control group. In conclusion, low-intensity intermittent negative pressure can inhibit the proliferation of human MSCs, induce differentiation to bone cells, promote the OPG mRNA expression, and reduce OPGL mRNA expression.