component of endochondral ossification. In the last decade, localization of neuronal nicotinic acetylcholine receptor (nAChR), a specific receptor of nicotine, has been widely detected in non- excitable cells [16]. Therefore, we hypothesized that nicotine affect growth plate chondrocytes directly and specifically through nAChR to delay skeletal growth. We here demonstrate that nicotine affected growth plate chondrocytes through alpha7 nAChR to decrease matrix synthesis and to suppress hypertrophic differentiation, thereby delaying skeletal growth. Results Detection and localization of nAChR in growth plate chondrocytes To date, many epidemiological [1–5] and experimental [13] studies suggested that endochondral ossification is affected by cigarette smoking, especially by its major component, nicotine [15]. We thus assumed that nicotine may directly affect chondrocytes, a key player in endochondral ossification. To investigate whether the impact of nicotine on chondrocytes is specific, we studied the expression pattern of the specific receptor, nAChR. For screening of the existing subunits of nAChR, RT- PCR was performed with primers for each subunit of nAChR. Human growth plate chondrocytes expressed alpha5, alpha7, beta1 and epsilon subunits of nAChR (Figure 1A). Among the detected subunits, only the alpha7 subunit can form a functional nAChR by forming a homopentameric receptor [17]. We thus tried to detect alpha7 subunit at a protein level. Western blot analysis revealed that chondrocytes produced alpha7 nAChR (Figure 1B). Immunocytochemical analysis also revealed that chondrocytes stained positive for alpha7 nAChR (Figure 1C). Moreover, the alpha7 subunit was detected at resting, proliferating and pre-hypertrophic chondrocytes of murine growth plate but not hypertrophic chondrocytes (Figure 1D). These results suggest that the growth plate chondrocytes in their non-hypertrophic stage express alpha7 homopentameric nAChR. Effect of nicotine on chondrocytes cultured in agarose gel To study the effect of nicotine on growth plate chondrocytes in vitro, two methods of suspension cultures, i.e., agarose gel culture and alginate bead culture, were employed. In agarose gel, the chondrocytes are initially embedded in the suspension layer solitarily. The chondrocytes then proliferate, differentiate, and aggregate to form a colony in the presence of ascorbic acid, and start to produce a matrix around themselves [18]. We applied the agarose gel culture to study the effect of nicotine on the proliferation and differentiation of growth plate chondrocytes in vitro. Nicotine was added to culture media for three weeks culture period. Nicotine decreased the percentage of colonies which produce matrix, as revealed by alcian blue (ALB) stains in a concentration-dependent manner (Figure 2A, upper panels). Similarly, nicotine suppressed Col X expression and enzyme activity of alkaline phosphatase (ALP) in a concentration-dependent manner (Figure 2A, middle and lower row panels). In contrast, nicotine did not affect colony density (Figure 2B, left panel) or the number of cells per colony (Figure 2B, right panel) which are indicators for cell proliferation. No nicotinic effect on cell proliferation was detected as assessed by immunohis- tochemistry using antibody to proliferating cell nuclear antigen (PCNA) (Figure S1). These results suggest that nicotine decreases the matrix synthesis and suppresses hypertrophic differentiation of growth plate chondrocytes, but has little effect on cell proliferation in vitro and vivo. To investigate if the nicotinic effect is mediated by alpha7 nAChR, we used MLA, the specific antagonist of alpha7 nAChR. MLA clearly reversed the effect, as assessed by ALB- Nicotine Delays Bone Growth stained colonies (Figure 2C), suggesting the involvement of alpha7 nAChR in the effect of nicotine on growth plate chondrocytes. Long-term (four months) effect of nicotine on growth plate chondrocytes in alginate beads Different from the case with agarose gel, human chondrocytes hardly proliferate in alginate beads, maintaining chondrocyte properties for more than eight months [19]. Moreover, molecular analysis can be done easily compared with that in agarose gel, since chondrocytes can be recovered from beads by chelation of divalent ions with ethylenediamine tetraacetic acid (EDTA) followed by centrifugation. We investigated the long-term effect of nicotine on growth plate chondrocytes by employing alginate bead culture. Chondrocytes encapsulated in alginate beads remained viable during the culture period (four months) in their lacunae. Nicotine did not affect viability of the chondrocytes at any indicated concentration. Nicotine dose-dependently suppressed ALB- and Safranin-O-stained areas at four months (Figure 3A). To investigate expression of chondrocyte-specific genes, we performed RT-PCR analysis on chondrocytes in alginate beads. Genes for collagen type II (Col II), Aggrecan, collagen type X (ColX), ALP, and indian hedgehog (Ihh) were up-regulated at three weeks after the start of alginate bead culture (Figure 3B). In contrast, genes for parathyroid hormone receptor type I (PTHR1), matrix metalloproteinase type 13 (MMP13), vascular endothelial growth factor (VEGF), and Sox9 were constitutionally expressed and their expression level remained unchanged. We then performed RT- PCR analysis to investigate the expression of chondrocyte-specific genes in chondrocytes treated by nicotine for four months. Nicotine dose-dependently decreased the expression of Col II, Aggrecan, Col X, ALP, and Ihh gene (Figure 3C). These findings suggest that nicotine suppresses matrix synthesis and hypertrophic maturation of chondrocytes in long-term culture using alginate beads. Functional calcium imaging To investigate the intracellular signals after nicotinic stimula- tion, we performed calcium imaging assay for primary chondro- cyte cultures, since alpha7 nAChR has large Ca2+ permeabilities and also induces elevated intracellular free calcium by releasing intracellular calcium stores [17]. Nicotine elicited a transient increase of intra-cellular calcium (Figure 4A) in a concentration- dependent manner (Figure 4B). MLA, the specific antagonist of alpha7 nAChR, inhibited the calcium signals in a concentration- dependent manner (Figure 4C), implying that the effect of nicotine on chondrocytes is mediated through the alpha7 nAChR. Maternal nicotine exposure in wild-type mice To study the effect of nicotine on endochondral ossification in vivo, ovulation-controlled pregnant C57BL/6J mice were given drinking water with or without nicotine during pregnancy, and skeletal growth of their fetuses was observed. At noon on gestational day 15, fetuses were surgicallly obtained and their legs were sectioned for measurement of the femur length (FL) and the length of the hypertrophic zone of the femur (HL) (Figure 5A). There were no significant differences of the amount of water consumed between nicotine-exposed group and control group. Maternal nicotine exposure significantly reduced the FL (Figure 5B) and HL/FL (Figure 5C) of mice at embryonic day 15.5 (E15.5), suggesting that nicotine delayed endochondral ossification. Maternal nicotine exposure in alpha7 nAChR-disrupted mice To clarify an involvement of alpha7 nAChR in nicotine- induced delayed skeletal growth in vivo, we investigated the effect PLoS ONE | www.plosone.org 2 December 2008 | Volume 3 | Issue 12 | e3945