In vitro study of enhanced osteogenesis induced by HIF-1α-transduced bone marrow stem cells.
ABSTRACT Hypoxia-inducible factor 1α (HIF-1α) is a pivotal regulator of hypoxic and ischaemic vascular responses that drives transcriptional activation of hundreds of genes involved in vascular reactivity, angiogenesis and arteriogenesis. Previous reports based on gene knockout technology have demonstrated that HIF-1α can promote osteogenesis. However, this protein is easily degraded in a normoxic state, which makes in vitro studies of HIF-1α-induced mesenchymal stem cell (MSC) osteogenesis difficult. For better understanding of HIF-1α promoting osteogenesis, the role of HIF-1α-induced MSC osteogenesis in the normoxic state has been investigated here.
HIF-1α was made to overexpress using a lentiviral vector, and its effects on bone marrow-derived mesenchymal stem cell (BMSC) osteogenesis were investigated. Real-time quantitative and western blotting (to assess expression levels of angiogenic and osteogenic related genes regulated by Lenti-HIF-1α), alkaline phosphatase (ALP) and alizarin red-S staining analyses, were performed.
In HIF-1α gene-transfected BMSCs, expression levels of angiogenic, cartilaginous and osteogenic genes were all increased significantly compared to Lenti LacZ-transfected cells, at both mRNA and protein levels. ALP activity and alizarin red-S staining were significantly enhanced in HIF-1α transduced cells compared to control cells, on day 21.
These results indicate that Lenti-HIF-1α can induce BMSC overexpression levels of angiogenic and osteogenic genes in vitro in the normoxic state. Further study will be focused on whether HIF-1α can also improve bone repair in vivo.
- SourceAvailable from: plosone.org[show abstract] [hide abstract]
ABSTRACT: Although corticosteroid-induced osteonecrosis of the femoral head (ONFH) is common, the treatment for it remains limited and largely ineffective. We examined whether implantation of hypoxia inducible factor-1α (HIF-1α) transgenic bone marrow cells (BMCs) can promote the repair of the necrotic area of corticosteroid-induced ONFH. In this study, we confirmed that HIF-1α gene transfection could enhance mRNA expression of osteogenic genes in BMCs in vitro. Alkaline phosphatase activity assay and alizarin red-S staining indicated HIF-1α transgenic BMCs had enhanced osteogenic differentiation capacity in vitro. Furthermore, enzyme linked immunosorbent assay (ELISA) for VEGF revealed HIF-1α transgenic BMCs secreted more VEGF as compared to normal BMCs. An experimental rabbit model of early-stage corticosteroid-induced ONFH was established and used for an evaluation of cytotherapy. Transplantation of HIF-1α transgenic BMCs dramatically improved the bone regeneration of the necrotic area of the femoral head. The number and volume of blood vessel were significantly increased in the necrotic area of the femoral head compared to the control groups. These results support HIF-1α transgenic BMCs have enhanced osteogenic and angiogenic activity in vitro and in vivo. Transplantation of HIF-1α transgenic BMCs can potentially promote the repair of the necrotic area of corticosteroid-induced ONFH.PLoS ONE 01/2013; 8(5):e63628. · 3.73 Impact Factor