Chemically-Induced Hypoxic Response Affects Osteogenic Differentiation of Adipose Tissue-Derived Mesenchymal Stem Cells

Aim: Insufficient vascularization is among the major obstacles hindering the clinical translation of bone engineering strategies for reconstructing large bone defects. Limitations for the local delivery of vascular endothelial growth factor (VEGF) include its short half-life and off-target effects. Delivery of Prolylhydroxylase inhibitors (PHIs) is an interesting approach to upregulate VEGF by stabilizing hypoxia inducible factor-1α (HIF-1α) and inducing a hypoxic response. It is, however, necessary to assess the direct influence of the PHIs on osteogenesis. This study aims to compare two PHIs: dimethyloxalylglycine (DMOG) and Baicalein for their effects on HIF-1α stabilization and osteogenic potential of human adipose tissue-derived mesenchymal stem cells (AT-MSCs).
Material, methods and results: Immunofluorescent detection and western blots revealed the ability of both tested drugs to stabilize HIF-1α in a dose-dependent manner. DMOG showed a more sensitive HIF-1α stabilization response, which declined rapidly over time. Cellular proliferation assays showed that Baicalein and DMOG have evident antiproliferative effects, which were dose- and time-dependent. At a non-cytotoxic concentration, DMOG enhanced VEGFA relative gene expression with and without osteogenic induction. Addition of DMOG almost abolished the effect of osteogenic induction on the expression of osteogenic marker genes RUNX2, ALPL, and COL1A1. Addition of DMOG to osteogenic induction medium also suppressed the formation of a mineralized matrix, as measured by alkaline phosphatase activity, alizarin red s stain, and hydroxyproline assays. The reduced osteogenic response of AT-MSCs could not be attributed to the reduced collagen in the extracellular matrix. Baicalein showed similar effects on matrix formation as DMOG. Baicalein did, however, upregulate the expression of osteogenic marker genes BMP2 and SPP1, and osteogenic cytokines as revealed by human cytokine antibody array analysis.
Conclusion: Although both PHIs suppress the direct osteogenic response of AT-MSCs, Baicalein holds, however, more pro-osteogenic potential than DMOG on both genetic and paracrine levels for targeting angiogenesis-osteogenesis coupling in bone engineering applications.