Fig. 3.

Chronic cigarette smoke exposure inhibited bone mineralization and downregulated the gene expression of osteoblast differentiation markers. a Smoke exposure did not alter the number of osteoblasts on bone interface measured by histomorphometry in the protocol 1 mice model. b BMSCs were seeded at 5 × 10⁴ cells/cm² in 6-well plates and given differentiation medium for 10 days. Inorganic phosphate (5 mM) was added to the culture medium for another 4 days for mineralization assays. The capacity of bone mineralization was measured by von Kossa staining and alizarin red staining. c Gene expression of osteoblast differentiation markers including Runx2, OCN, Col I, OSX, BSP and OPN was determined by real-time RT-PCR. Levels of Runx2, OCN, Col I, OSX, BSP, and OPN mRNA were normalized to GAPDH. d ALP activity was normalized to total protein. e OPG and RANKL gene expression in the osteoblasts were examined by quantitative RT-PCR. The ratio of OPG to RANKL is illustrated. Data are representative of three independent experiments. Data are presented as the mean ± SD from triplicates. ^*P < 0.05 compared to non-smoke group; ^**P < 0.001 compared to non-smoke group