Continuous hypoxia regulates the osteogenic potential of mesenchymal stem cells in a time-dependent manner

The effects of hypoxia on the osteogenic potential of mesenchymal stem cells (MSCs) have been previously reported. From these studies, possible factors affecting the association between hypoxia and the osteogenic differentiation of MSCs have been suggested, including hypoxia severity, cell origin and methods of induction. The effect of the duration of hypoxia, however, remains poorly understood. The aim of the present study was to investigate the effect of continuous hypoxia on the induced osteogenesis of MSCs. Rat MSCs were isolated and cultured in vitro. Once the cells had been cultured to passage three, they were switched to 1% oxygen and cultured either with or without osteogenic medium, while cells in the control groups were cultured under normoxia in corresponding conditions. Four osteogenic differentiation biomarkers, runt-related transcription factor 2, osteopontin, osteocalcin and alkaline phosphatase, were analyzed by quantitative polymerase chain reaction and western blotting at defined intervals throughout the culture period. In addition, Alizarin Red staining was used to assess changes in mineralization. The results showed that 1% hypoxia was able to enhance and accelerate the osteogenic ability of the MSCs during the initial phases of differentiation, and the protein expression of certain associated biomarkers was upregulated. However, continuous hypoxia was shown to impair osteogenesis in the latter stages of differentiation. These findings suggest that hypoxia can regulate the osteogenesis of MSCs in a time-dependent manner.

缺氧对间充质干细胞（MSCs）成骨潜能的影响此前已有报道。从这些研究中，已经提出了可能影响缺氧与MSCs成骨分化之间关联的因素，包括缺氧程度、细胞来源和诱导方法。然而，缺氧持续时间的影响仍知之甚少。本研究的目的是探讨持续缺氧对MSCs诱导成骨的影响。大鼠MSCs在体外分离培养。当细胞培养到第3代时，将其置于1%氧气环境中，并在有或无成骨培养基的条件下培养，而对照组的细胞在常氧相应条件下培养。在整个培养期间的特定时间间隔，通过定量聚合酶链反应和蛋白质印迹法分析四种成骨分化生物标志物，即runt相关转录因子2、骨桥蛋白、骨钙素和碱性磷酸酶。此外，采用茜素红染色评估矿化变化。结果表明，1%缺氧能够在分化的初始阶段增强和加速MSCs的成骨能力，并且某些相关生物标志物的蛋白质表达上调。然而，持续缺氧在分化的后期阶段会损害成骨作用。这些发现表明，缺氧可以以时间依赖的方式调节MSCs的成骨作用。