Hyperbaric Oxygen Therapy Improves the Osteogenic and Vasculogenic Properties of Mesenchymal Stem Cells in the Presence of Inflammation In Vitro

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.

Keywords: adipose-derived stem cells; bone regeneration; hyperbaric oxygen therapy; inflammation; osteogenic differentiation; vasculogenic differentiation.

Figure 1

Metabolic activity and proliferation of human Adipose-Derived Stem Cells (hADSCs) after hyperbaric oxygen (HBO) treatment. (A) MTT assay and (B) quantification of DNA content in hADSCs 1 day (d1), 7 days (d7), 14 days (d14), and 21 days (d21) after osteogenic differentiation in Osteogenic Differentiation Medium (ODM) and hyperbaric oxygen (HBO), hyperbarism (HB), or hyperoxia (HO) treatment. The control condition (CTRL) is represented by cells grown under normoxia and normobarism. Data are shown as mean ± standard deviation (SD). * p < 0.05, *** and $$$ p < 0.001 indicate statistically significant difference between the HBO- or HO-treated cells and the control group, respectively.

Figure 2

Metabolic activity and proliferation of hADSCs after HBO treatment in the presence of inflammation. (A) MTT assay and (B) quantification of DNA content in hADSCs 1, 7, 14, and 21 days after osteogenic differentiation in the presence of 10 ng/mL Tumor Necrosis factor-α (TNF-α) and HBO, HB, or HO treatment. Data are shown as mean ± SD. In both graphs, # and $ p < 0.05 indicate statistically significant difference between the HB- or HO-treated cells and control. ** p < 0.01 and *** p < 0.001 indicate statistically significant difference between the HBO-treated cells and the control group; ### p < 0.001 indicates statistically significant difference between the HB-treated cells and the control group.

Figure 3

Matrix mineralization in hADSCs after HBO treatment. (A) Alizarin Red S (ARS) staining and (B) quantification of calcium deposits in hADSCs after d1, d7, d14, and d21 of culture in ODM and HBO, HB, or HO treatment. Scale bars 100 µm. Data are shown as mean ± SD. ** p < 0.01 and *** p < 0.001 indicate statistically significant difference between the HBO-treated cells and the control group, respectively. ### and $$$ p < 0.001 indicate statistically significant difference between HB- and HO-treated cells and control, respectively.

Figure 4

Matrix mineralization in hADSCs after HBO treatment in the presence of inflammation. (A) ARS staining and (B) quantification of calcium deposits in hADSCs after d1, d7, d14, and d21 of culture in ODM supplemented with 10 ng/mL TNF-α and HBO, HB, or HO treatment. Scale bars 100 µm. Data are shown as mean ± SD. ** p < 0.01 and *** p < 0.001 indicate statistically significant difference between the HBO-treated cells and the control group, respectively. ### p < 0.001 indicate statistically significant difference between the HB-treated cells and control.

Figure 5

Metabolic activity and matrix mineralization of pre-committed hADSCs. (A) MTT assay of hADSCs 14 and 21 days after osteogenic differentiation in ODM and HBO, HB, or HO treatment. Data are shown as mean ± SD (n = 3). (B) ARS staining and (C), quantification of calcium deposits in hADSCs after d14 and d21 of culture in ODM and HBO, HB, or HO treatment. Scale bars 200 µm. Data are shown as mean ± SD (n = 3).

Figure 6

Gene expression profile of osteogenic and vasculogenic markers in hADSCs after HBO treatment. Expression of the osteoblast lineage genes (A) RUNX2; (B) OSX; (C) ALP; (D) OCN; (E) OPN; (F) RANKL; (G) VEGFA; and (H) KDR measured by real-time PCR. Data are expressed as mean ± SD of the target gene versus reference gene (transferrin receptor, TFRC) ratio and represented by 2deltaCt. *, $ p < 0.05, ##, ** p < 0.01, and ###, $$$, ***p < 0.001 mark significant changes in gene expression level compared to the control within the same measurement day.

Figure 7

Gene expression profile of osteogenic and vasculogenic markers in hADSCs after HBO treatment in the presence of inflammation. Expression of (A) RUNX2; (B) OSX; (C) ALP; (D) OCN; (E) OPN; (F) RANKL; (G) VEGFA; and (H) KDR measured by real-time PCR. Data are expressed as mean ± SD of the target gene versus reference gene (TFRC) ratio and represented by 2deltaCt. * p < 0.05, **, $$ p < 0.01, and ***, ### p < 0.001 mark significant changes in gene expression level compared to the control within the same measurement day.

Figure 8

Schematic experimental design of treatments starting simultaneously with the osteogenic differentiation of hADSCs. (A) Image of hyperbaric culture chamber used for HBO exposures. (B) hADSCs are grown in ODM and contextually subjected to one of the following treatments: HBO, HB, or HO. The control condition is represented by cells grown under normoxia and normobarism. Each treatment is performed daily for a total of 21 days. (C) hADSCs are treated as described above but in the presence of 10 ng/mL TNF-α. Tests for evaluating cell metabolic activity and osteogenic differentiation, as evidenced in the legend, are performed at d1, d7, d14, and d21.

Figure 9

Schematic experimental design of treatments starting after osteogenic pre-commitment of hADSCs. hADSCs are pre-differentiated in ODM for 10 days (d10) before treatment with HBO, HB, or HO. Each treatment is performed daily for 60 min from the tenth day to d21 of culture. Tests for evaluating cell metabolic activity and osteogenic differentiation, as evidenced in the legend, are performed at d14 and d21.