Fig. 1

Chronic cigarette smoke exposure caused bone loss in mouse model both in vivo and ex vivo. a microCT of the epiphyseal region of the fifth lumbar spine (L5). (Left) Three-dimensional images of microCT. (Right) The parameters of bone phenotype were determined by microCT, including bone volume vs tissue volume (BV/TV), trabecular numbers (Tb. N), trabecular thickness (Tb. Th), and trabecular separation (Tb. Sp). b BMD and trabecular BMD were measured by pQCT in the tibiae. (Upper panel) Slices were scanned by pQCT. The small box in the right panel indicates the tibia. The reference slice and the slices examined are indicated on a representative tibia. (Lower panel) Graph of the average bone density obtained by pQCT in tibiae from non-smoke and smoke groups. c Histological and histomorphometric analyses of the tibiae. (Left) TRAP staining on decalcified tibiae slides. (Right) Number of osteoclasts on bone surface as measured by bone histomorphometry analysis. d Ex vivo osteoclast formation and bone resorption assay were performed using the non-adherent bone morrow cells. Number of osteoclast-like multinucleated cells per well was quantified. Samples were evaluated in quadruplicate. Bone resorption area was determined microscopically with SPOT software. Samples were evaluated in triplicate. Results are reported as mean ± SD. e Gene expression of bone resorption markers including Cat K, TRAP, and CTR were examined by real-time RT-PCR. Specific mRNA expression was calculated relative to the expression of GAPDH using the ΔCT method. Bars are indicated mean ± SE. Data is representative of three independent experiments. ^*P < 0.05 compared to non-smoke group; ^**P < 0.001 compared to non-smoke group