Abstract

Hyperbaric oxygen (HBO) therapy has been reported to be beneficial for treating many conditions of inflammation-associated bone loss. The aim of this work was to in vitro investigate the effect of HBO in the course of osteogenesis of human Mesenchymal Stem Cells (MSCs) grown in a simulated pro-inflammatory environment. Cells were cultured with osteogenic differentiation factors in the presence or not of the pro-inflammatory cytokine Tumor Necrosis Factor-α (TNF-α), and simultaneously exposed daily for 60 min, and up to 21 days, at 2,4 atmosphere absolute (ATA) and 100% O2. To elucidate osteogenic differentiation-dependent effects, cells were additionally pre-committed prior to treatments. Cell metabolic activity was evaluated by means of the MTT assay and DNA content quantification, whereas osteogenic and vasculogenic differentiation was assessed by quantification of extracellular calcium deposition and gene expression analysis. Metabolic activity and osteogenic properties of cells did not differ between HBO, high pressure (HB) alone, or high oxygen (HO) alone and control if cells were pre-differentiated to the osteogenic lineage. In contrast, when treatments started contextually to the osteogenic differentiation of the cells, a significant reduction in cell metabolic activity first, and in mineral deposition at later time points, were observed in the HBO-treated group. Interestingly, TNF-α supplementation determined a significant improvement in the osteogenic capacity of cells subjected to HBO, which was not observed in TNF-α-treated cells exposed to HB or HO alone. This study suggests that exposure of osteogenic-differentiating MSCs to HBO under in vitro simulated inflammatory conditions enhances differentiation towards the osteogenic phenotype, providing evidence of the potential application of HBO in all those processes requiring bone regeneration.

Highlights

  • Bone loss and subsequent repair are biological processes related to many pathological conditions affecting bones, including fractures, osteoporosis, osteoarthritis, osteomyelitis, osteonecrosis, and tumors [1]

  • The MTT assay and quantification of DNA content were performed at selected time points over a period of 21 days to compare the metabolic activity of human Adipose-Derived Stem Cells (hADSCs) exposed to different pressure and oxygen signals

  • Our results reveal that constant exposure to the pro-inflammatory cytokine Tumor Necrosis Factor-α (TNF-α) clearly inhibits hADSCs osteogenesis

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Summary

Introduction

Bone loss and subsequent repair are biological processes related to many pathological conditions affecting bones, including fractures, osteoporosis, osteoarthritis, osteomyelitis, osteonecrosis, and tumors [1]. Hyperbaric oxygen (HBO) has been proposed as an adjunctive therapy in the management of several conditions requiring bone healing [3,4,5,6]. Treatments usually involve pressurization between 1.5 and 3.0 ATA inside airtight chambers for periods between 60 and 120 min once or twice daily [7]. In this way, it is possible to deliver a greatly increased supply of oxygen to tissues, which is advantageous for fibroblasts proliferation, collagen fiber deposition, and formation of new blood vessels, accelerating tissue repair and healing, and easing pain [8]. High-pressure oxygen can enhance the viability and capacity of phagocytic cells, which are beneficial to the absorption and removal of necrotic tissues, and reduction of local inflammation [9]

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