BACKGROUND: Insufficient vascularization hampers bone tissue engineering strategies for reconstructing large bonedefects. Delivery of prolyl-hydroxylase inhibitors (PHIs) is an interesting approach to upregulate vascular endothelialgrowth factor (VEGF) by mimicking hypoxic stabilization of hypoxia-inducible factor-1alpha (HIF-1a). This studyassessed two PHIs: dimethyloxalylglycine (DMOG) and baicalein for their effects on human adipose tissue-derivedmesenchymal stem/stromal cells (AT-MSCs). METHODS: Isolated AT-MSCs were characterized and treated with PHIs to assess the cellular proliferation response.Immunostaining and western-blots served to verify the HIF-1a stabilization response. The optimized concentrations forlong-term treatment were tested for their effects on the cell cycle, apoptosis, cytokine secretion, and osteogenic differentiationof AT-MSCs. Gene expression levels were evaluated for alkaline phosphatase (ALPL), bone morphogeneticprotein 2 (BMP2), runt-related transcription factor 2 (RUNX2), vascular endothelial growth factor A (VEGFA), secretedphosphoprotein 1 (SPP1), and collagen type I alpha 1 (COL1A1). In addition, stemness-related genes Kruppel-like factor 4(KLF4), Nanog homeobox (NANOG), and octamer-binding transcription factor 4 (OCT4) were assessed. RESULTS: PHIs stabilized HIF-1a in a dose-dependent manner and showed evident dose- and time dependent antiproliferativeeffects. With doses maintaining proliferation, DMOG and baicalein diminished the effect of osteogenic inductionon the expression of RUNX2, ALPL, and COL1A1, and suppressed the formation of mineralized matrix. Suppressedosteogenic response of AT-MSCs was accompanied by an upregulation of stemness-related genes. CONCLUSION PHIs significantly reduced the osteogenic differentiation of AT-MSCs and rather upregulated stemnessrelatedgenes. PHIs proangiogenic potential should be weighed against their longterm direct inhibitory effects on theosteogenic differentiation of AT-MSCs.

BACKGROUND: Insufficient vascularization hampers bone tissue engineering strategies for reconstructing large bonedefects. Delivery of prolyl-hydroxylase inhibitors (PHIs) is an interesting approach to upregulate vascular endothelialgrowth factor (VEGF) by mimicking hypoxic stabilization of hypoxia-inducible factor-1alpha (HIF-1a). This studyassessed two PHIs: dimethyloxalylglycine (DMOG) and baicalein for their effects on human adipose tissue-derivedmesenchymal stem/stromal cells (AT-MSCs). METHODS: Isolated AT-MSCs were characterized and treated with PHIs to assess the cellular proliferation response.Immunostaining and western-blots served to verify the HIF-1a stabilization response. The optimized concentrations forlong-term treatment were tested for their effects on the cell cycle, apoptosis, cytokine secretion, and osteogenic differentiationof AT-MSCs. Gene expression levels were evaluated for alkaline phosphatase (ALPL), bone morphogeneticprotein 2 (BMP2), runt-related transcription factor 2 (RUNX2), vascular endothelial growth factor A (VEGFA), secretedphosphoprotein 1 (SPP1), and collagen type I alpha 1 (COL1A1). In addition, stemness-related genes Kruppel-like factor 4(KLF4), Nanog homeobox (NANOG), and octamer-binding transcription factor 4 (OCT4) were assessed. RESULTS: PHIs stabilized HIF-1a in a dose-dependent manner and showed evident dose- and time dependent antiproliferativeeffects. With doses maintaining proliferation, DMOG and baicalein diminished the effect of osteogenic inductionon the expression of RUNX2, ALPL, and COL1A1, and suppressed the formation of mineralized matrix. Suppressedosteogenic response of AT-MSCs was accompanied by an upregulation of stemness-related genes. CONCLUSION PHIs significantly reduced the osteogenic differentiation of AT-MSCs and rather upregulated stemnessrelatedgenes. PHIs proangiogenic potential should be weighed against their longterm direct inhibitory effects on theosteogenic differentiation of AT-MSCs.