Research Paper Volume 16, Issue 6 pp 4948—4964

Figure 2. Effects of MB and MitoQ on osteogenesis in vitro. BM cells from 6-7 months old UM-HET3 mice were seeded in 24 well plate at a density of 1.5 10⁶ cells/well. Adherent cells were subjected for osteoblast differentiation treating with osteogenic factors in the presence or absence of MB or Mito Q at different concentrations (0, 0.125, 025 and 0.5 μM). (A) On day 18 in culture, cells were stained for Alkaline-phosphatase (Alk-Phos). The area of Alk-Phos positive colonies treated with (B) MB or (C) MitoQ were quantified in the wells were measured. (D) Non-adherent BM mononuclear cells were separated by Ficoll-Paque density gradient and seeded on 96 wells plate and induced for osteoclast differentiation with the supplementation of RANKL, M-CSF in the presence or absence of (E) MB or (F) MitoQ (0, 0.125, 025 and 0.5 μM). Multinucleated osteoclasts (>3 nuclei/cell) formed after 5 days in culture were visualized using TRAP staining kit and counted. Differentiated osteoblasts (14 days in culture) were treated with (G) MB or (H) MitoQ for 48 hours and oxygen consumption rate (OCR) was measured using mitochondrial stress assay kit. (I) Basal OCR and (J) maximal OCR were recorded along the assay. Data presented as mean ± SEM. We used n = 6 mice for assays in A-C, n = 4 for assays in D–F, n = 6 for mitochondrial stress assay in G–J. Data tested by multivariate ANOVA. Significance accepted at p < 0.05 (^*p < 0.05, ^**p < 0.01, ^***p < 0.001, ^****p < 0.0001).