Nicotine induced proliferation and cytokine release in osteoblastic cells.

Smoking has deleterious effects on osteoporosis and periodontitis both characterized by bone loss. Smoking also interferes with the protective effect that hormone replacement therapy (HRT) has on bone loss. Our study investigated two mechanisms by which smoking may affect bone metabolism: nicotine-induced proliferation and nicotine-induced cytokine secretion in osteoblasts. Two osteoblastic cell models were used: mouse osteoblasts derived from mouse calvaria and human osteoblasts. Thymidine incorporation and immunoassays were used to evaluate proliferation, interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-alpha) secretion. Parametric and nonparametric statistical analyses were used for comparisons. The results showed that nicotine induced stimulation and inhibition of proliferation in both osteoblastic cell models. In human osteoblasts, the proliferative and inhibitory effects were also donor dependent. Il-6 secretion showed different patterns in mouse and human osteoblasts. In mouse osteoblasts, nicotine significantly increased IL-6 secretion and estradiol significantly inhibited the nicotine-induced IL-6 release. In human osteoblasts, cells derived from one subject did not respond to nicotine. However, in the second sample, nicotine increased secretion of Il-6 but estradiol did not oppose this effect. In human osteoblasts, nicotine also induced an increase in the TNF-alpha secretion and estradiol opposed this increase. These results suggest that nicotine affects bone metabolism by modulating proliferation, and Il-6 and TNF-alpha secretion. These studies provide a possible explanation for differences in bone loss among subjects who smoke and offer a possible mechanism for the oppositional effect of smoking on HRT in subjects with bone loss.

Alveolar bone osteoblast differentiation and Runx2/Cbfa1 expression.

Alveolar bone cells have a unique origin and functionality, but may resemble skeletal osteoblasts. Osteoblast differentiation and gene expression are regulated by the Runx2/Cbfa1 transcription factor. However, most studies on Runx2/Cbfa1 expression have been on rodent cells and the few studies on human osteoblasts have had differing results. The purpose of this study was to characterize Runx2/Cbfa1 expression in primary cell cultures derived from human alveolar bone. An alveolar bone chip was incubated in alpha-minimum essential medium (alpha-MEM) supplemented with fetal calf serum (10% FCS). Explant cultures were harvested after 3-4 weeks of outgrowth and grown in alpha-MEM with FCS. This media was supplemented with ascorbate, beta-glycerophosphate and dexamethasone to promote osteoblast differentiation over 14 days. RT-PCR analysis and Western blots showed a rapid increase in Runx2/Cbfa1 mRNA (2.1-fold) and protein (2.3-fold) levels in 3 days, followed by a slight decline. There was also a rapid increase in bone sialoprotein expression (2.9-fold) in 3 days, followed by a further increase (3.6-fold) at 14 days. There was a slower increase in alkaline phosphatase expression (1.6-fold) and activity (3.1-fold) over 7 days, followed by a gradual decline. In contrast, collagen mRNA levels showed little change over 14 days. These findings attest to the osteogenic potential of primary cell cultures derived from human alveolar bone. Osteoblastic differentiation in human alveolar bone involves an increase in Runx2/Cbfa1 expression that may be an important component of the differentiation process.