Abstract
Fracture repair is characterized by cytokine production and hypoxia. To better predict cytokine modulation of mesenchymal stem cell (MSC)‐aided bone healing, we investigated whether interleukin 4 (IL‐4), IL‐6, and their combination, affect osteogenic differentiation, vascular endothelial growth factor (VEGF) production, and/or mammalian target of rapamycin complex 1 (mTORC1) activation by MSCs under normoxia or hypoxia. Human adipose stem cells (hASCs) were cultured with IL‐4, IL‐6, or their combination for 3 days under normoxia (20% O 2) or hypoxia (1% O 2), followed by 11 days without cytokines under normoxia or hypoxia. Hypoxia did not alter IL‐4 or IL‐6‐modulated gene or protein expression by hASCs. IL‐4 alone decreased runt‐related transcription factor 2 (RUNX2) and collagen type 1 (COL1) gene expression, alkaline phosphatase (ALP) activity, and VEGF protein production by hASCs under normoxia and hypoxia, and decreased mineralization of hASCs under hypoxia. In contrast, IL‐6 increased mineralization of hASCs under normoxia, and enhanced RUNX2 gene expression under normoxia and hypoxia. Neither IL‐4 nor IL‐6 affected phosphorylation of the mTORC1 effector protein P70S6K. IL‐4 combined with IL‐6 diminished the inhibitory effect of IL‐4 on ALP activity, bone nodule formation, and VEGF production, and decreased RUNX2 and COL1 expression, similar to IL‐4 alone, under normoxia and hypoxia. In conclusion, IL‐4 alone, but not in combination with IL‐6, inhibits osteogenic differentiation and angiogenic stimulation potential of hASCs under normoxia and hypoxia, likely through pathways other than mTORC1. These results indicate that cytokines may differentially affect bone healing and regeneration when applied in isolation or in combination.
Highlights
Small bone injuries can heal spontaneously without intervention of an orthopedic surgeon
The mammalian target of rapamycin complex 1 signaling pathway is required for osteoblast proliferation and differentiation (Singha et al, 2008), and it plays an important role in the regulation of bone metabolism and skeletal development by regulating messenger RNA translation during preosteoblast differentiation (Bakker & Jaspers, 2015; Fitter et al, 2017)
We found that hypoxia inhibited Human adipose stem cells (hASCs) proliferation, observed by decreased DNA content in hASCs cultured without cytokines by 1.3–2.4‐fold at Day 2 and 7 (Figures 1a,b and 3a,b)
Summary
Small bone injuries can heal spontaneously without intervention of an orthopedic surgeon. Little is known about the conditions favouring the osteogenic potential of MSCs. Mimicking some aspects of the physiological conditions of bone healing will lead to the development of therapies to aid bone repair in large defects that are currently difficult to treat. The mammalian target of rapamycin complex 1 (mTORC1) signaling pathway is required for osteoblast proliferation and differentiation (Singha et al, 2008), and it plays an important role in the regulation of bone metabolism and skeletal development by regulating messenger RNA (mRNA) translation during preosteoblast differentiation (Bakker & Jaspers, 2015; Fitter et al, 2017). The aim of this study was to determine whether IL‐4, IL‐6, or the combination of both cytokines affects osteogenic differentiation and the angiogenic stimulation potential of MSCs under normoxia and hypoxia. The osteogenic and angiogenic effects exerted by IL‐4 and/or IL‐6 will be accompanied by mTORC1 activation in MSCs
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