BACKGROUND:

Insufficient vascularization hampers bone tissue engineering strategies for reconstructing large bone defects. Delivery of prolyl-hydroxylase inhibitors (PHIs) is an interesting approach to upregulate vascular endothelial growth factor (VEGF) by mimicking hypoxic stabilization of hypoxia-inducible factor-1alpha (HIF-1α). This study assessed two PHIs: dimethyloxalylglycine (DMOG) and baicalein for their effects on human adipose tissue-derived mesenchymal 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-1α stabilization response. The optimized concentrations for long-term treatment were tested for their effects on the cell cycle, apoptosis, cytokine secretion, and osteogenic differentiation of AT-MSCs. Gene expression levels were evaluated for alkaline phosphatase (ALPL), bone morphogenetic protein 2 (BMP2), runt-related transcription factor 2 (RUNX2), vascular endothelial growth factor A (VEGFA), secreted phosphoprotein 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-1α in a dose-dependent manner and showed evident dose- and time dependent antiproliferative effects. With doses maintaining proliferation, DMOG and baicalein diminished the effect of osteogenic induction on the expression of RUNX2, ALPL, and COL1A1, and suppressed the formation of mineralized matrix. Suppressed osteogenic 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 stemness-related genes. PHIs proangiogenic potential should be weighed against their longterm direct inhibitory effects on the osteogenic differentiation of AT-MSCs.

中文翻译：

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促血管生成缺氧模拟剂减弱脂肪干/基质细胞的成骨潜力。

背景：

血管化不足阻碍了重建大骨缺损的骨组织工程策略。脯氨酰羟化酶抑制剂 (PHI) 的递送是一种有趣的方法，通过模拟缺氧诱导因子 1α (HIF-1α) 的缺氧稳定性来上调血管内皮生长因子 (VEGF)。本研究评估了两种 PHI：二甲基乙二酰甘氨酸 (DMOG) 和黄芩素对人类脂肪组织来源的间充质干/基质细胞 (AT-MSC) 的影响。

方法：

用 PHI 对分离的 AT-MSC 进行表征和处理，以评估细胞增殖反应。免疫染色和蛋白质印迹用于验证 HIF-1α 稳定反应。测试了长期治疗的优化浓度对 AT-MSCs 的细胞周期、细胞凋亡、细胞因子分泌和成骨分化的影响。评估碱性磷酸酶 ( ALPL )、骨形态发生蛋白 2 ( BMP2 )、矮小相关转录因子 2 ( RUNX2 )、血管内皮生长因子 A ( VEGFA )、分泌型磷蛋白 1 ( SPP1 ) 和 I 型胶原蛋白的基因表达水平阿尔法 1 ( COL1A1）。此外，还评估了干性相关基因 Kruppel 样因子 4 ( KLF4 )、Nanog 同源框 ( NANOG ) 和八聚体结合转录因子 4 ( OCT4 )。

结果：

PHI 以剂量依赖性方式稳定 HIF-1α，并显示出明显的剂量和时间依赖性抗增殖作用。在维持增殖的剂量下，DMOG和黄芩素降低了成骨诱导对RUNX2、ALPL和COL1A1表达的影响，并抑制了矿化基质的形成。抑制 AT-MSCs 的成骨反应伴随着干性相关基因的上调。

结论：

PHI 显着降低了 AT-MSCs 的成骨分化，而上调了干性相关基因。PHI 的促血管生成潜力应与其对 AT-MSCs 成骨分化的长期直接抑制作用进行权衡。