Figure 5. Osteogenic potential of human NHO-MDSCs in response to proinflammatory stimuli.

(A) Neurogenic heterotopic ossification muscle-derived stromal cells (NHO-MDSCs) were isolated, cultured, and then subsequently induced to differentiate into 3 mesenchymal lineages using specific media. Differentiation into osteoblasts, adipocytes, and chondrocytes was evaluated by Alizarin Red S, Oil Red O, and Alcian blue staining, respectively. Original magnification, ×10. (B) NHO-MDSCs express classical mesenchymal markers, as shown by flow cytometry. (C) NHO-MDSCs were cultured in control medium (CT) or osteogenic medium alone (OB) or were supplemented with LPS (100 ng/ml) (OB + LPS) or TNF-α (100 ng/ml) (OB + TNF-α) for 3 weeks. Cells were then stained with Alizarin Red S. Calcium mineralization was quantified and expressed as mean ± SEM (n = 4). For statistical analysis, 1-way ANOVA followed by Dunnett’s post-hoc test were used (*P ≤ 0.05, between experimental conditions). (D) Runx2 and BSPII protein expression by Western blot of NHO-MDSC cell lysates with (OB) or without (control [CT]) osteoblastic differentiation medium (day 3 and day 21, respectively). Ratios correspond to RUNX2/actin or BSPII/actin.