collagen synthesis of human articular chondrocytes in vitro [21]. Cultured human growth plate chondrocytes derived from infant fingers serve as a good model for analyzing whether nicotine has direct action on growth plate chondrocytes. The present findings of nicotinic effect, i.e. decreasing matrix synthesis and suppressing hypertrophic differentiation but not proliferation on growth plate chondrocytes in vitro, indicate the direct effect of nicotine on growth plate chondrocytes. The findings are consistent with reports that maternal nicotine exposure has a negative effect on endochondral ossification in animals [13]. Besides, these findings are consistent, considering the fact that longitudinal skeletal growth is partly caused by matrix synthesis and hypertrophic differentiation of chondrocytes. Confirmation of the animal model using ‘‘human’’ chondrocytes is essential since certain chemicals, such as thalidomide, exhibit different effects in humans and rodents. Differences of expression levels of the genes for Col X, ALP, Ihh, MMP13, and VEGF in alginate beads culture (Figure 3B, C) may attribute to differential regulation among hypertrophic markers. Expression of the Ihh, Col X, and ALP genes were down-regulated by nicotine and the MMP13 and VEGF genes remained unaffected. Alternatively, the difference could be a result of chondrocyte culture, that is, artifactual induction ex vivo, and the MMP13 and VEGF genes were indeed expressed at the start of alginate bead culture with chondrocytes at passage 1 (Fig. 3B, lane 1: ‘‘0 W’’). In contrast, the Col X, ALP, and Ihh genes were appropriately regulated after three-dimensional culture (Figure 3B, lane 2: ‘‘3 W’’; Figure 3C, lane 1: without exposure to nicotine), as is the case with gene regulation in the growth plate. Involvement of alpha7 nAChR in delayed endochondral ossification The alpha7 nAChR-null mice exhibit normal development, including neural tissue, but alpha7 nAChR-null mice lack nicotinic currents in hippocampal neurons [22], and show abnormalities in late-stage keratinocyte development in the epidermis [23]. Lack of phenotypic abnormality in the femur of fetuses (Figure 5B) and adults indicates that ACh signaling through alpha7 nAChR has little involvement in the process of physiological skeletal growth. Results using MLA, the antagonist to alpha7 nAChR, strongly suggest the involvement of alpha7 nAChR in the nicotinic effect on chondrocytes. Such low-molecular weight substances may, however, have additional unclarified action in addition to any ‘‘specific’’ action. The proof of alpha7 nAChR involvement in delayed skeletal growth was strengthened by the in vivo experiments with alpha7 nAChR gene-disrupted mice. Especially so, considering the fact that maternal nicotine exposure caused delayed skeletal growth in only alpha7 nAChR +/+ fetuses compared with their alpha7 nAChR 2/2 littermates, fetal alpha7 nAChR but not maternal alpha7 nAChR is responsible for the mechanism of nicotine-induced delayed skeletal growth. Since nicotine exposure has been reported to be epidemiolog- ically and experimentally correlated with maternal effect, i.e., abnormal placental function and blood flow [10], the physiological and pathological function of alpha7 nAChR in growth plate was confirmed by comparing ‘‘littermates’’ of alpha7 nAChR (Figure 6B). This comparison confirms involvement of alpha7 nAChR on the fetus, and eliminates a possibility of maternal effect. Furthermore, decrease of relative femur length (Figure 6C, scatterplot and correlation, right panel, alpha7 nAChR +/+) and lack of nicotinic effect on body weight of alpha7 nAChR fetuses (Figure 6B, right panel, ‘‘BW’’) by maternal nicotine exposure indicate a specific effect of nicotine on bone growth rather than a systemic effect. Therefore, the effect of smoking during pregnancy Nicotine Delays Bone Growth on skeletal growth may be attributed to this direct action of nicotine on growth plate chondrocytes, at least in part. Our studies suggest that, from the large number of chemicals associated with cigarette smoking, nicotine may cause delayed skeletal growth and, indeed, amniotic fluid and breast milk both have higher concentrations of nicotine than maternal serum does [24]. In addition, metabolism of nicotine in the fetus and child is much slower than that in adults [25]. We therefore should pay close attention to the effect of smoking, regardless of being active or passive, on growth plate chondrocytes. This nicotinic effect may also extend to the delay of fracture repair or generation of non- union in adults, since the process of bone repair also partly depends on endochondral ossification. Materials and Methods Chondrocyte cultures Human chondrocytes were isolated from epiphysis of extra fingers, which were surgically excised from patients with polydactyly. Ethical approval for tissue collection was granted by the Institutional Review Board of the National Research Institute for Child Health and Development, Tokyo, Japan (#88). Minced tissue was incubated for 1 h at 37uC in 0.08% trypsin in PBS, then for 6 h at 37uC in 0.2% collagenase type 1 (Wako, Osaka, Japan) in Dulbecco’s Modified Eagle’s medium (DMEM). The released cells were washed and resuspended in DMEM containing 10% fetal bovine serum (FBS, Sanko Junyaku Co., Tokyo, Japan, lot number: 27110307) and plated at a density of 16106 cells per 100 mm dish for primary monolayer cultures, or 16106 cells per 35 mm dish for calcium influx assay and immunocytochemical assay of nAChR. In each experiment, we used one lot of cultured chondrocytes from extra fingers obtained from four patients. RT-PCR for detection of nAChR subunit Total RNA was prepared from epiphysis of extra fingers using Isogen (Nippon Gene) according to the manufacturer’s recom- mendations. DNase-treated RNA was reverse transcribed in 20 ml of RT-PCR mix (50 mM Tris, pH 8.3, 3 mM MgCl2, 75 mM KCl, 50 mM dNTPs, 2.5 mM oligo(dT)20, 5 mM DTT, 2 U RNaseOUT and 10 U SuperScriptIII (Invitrogen) at 50uC for 1 h. The PCR was performed in a final volume of 50 ml containing 1 ml of the single strand cDNA product, 25 mM TAPS (pH 9.3), 50 mM KCl, 2.0 mM MgCl2, 1 mM b-mercaptoethanol , 200 mM dNTPs, and AmpliTaq Gold (Applied Biosystems) and 20 pmol of each forward (59) and reverse (39) primers (Table S1). For each experiment the housekeeping gene GAPDH was amplified with 25–35 cycles to normalize the cDNA content of the samples. The amplification was performed for 30 cycles, with other conditions following polymerase-producing manufacturer’s rec- ommendations. Human brain and skeletal muscle RNAs were purchased from Ambion (Austin, TX). Western blot analysis for detection of nAChR subunit Total proteins were isolated from primary monolayer cultures using CelLyticTM-M Mammalian Cell Lysis/Extraction Reagent (Sigma). The proteins were separated by SDS-PAGE (Bio-Rad) in a 10% acrylamide gel, then blotted at 60 V for 2 h at 4uC onto a nitrocellulose membrane. Non-specific binding was blocked by incubation in TBS containing 10% BSA and 0.05% Tween-20. The membrane was subsequently incubated at 4uC overnight with the monoclonal antibody to nicotinic acetylcholine receptor, alpha7 subunit (Sigma, St-Louis, MO; product number: N 8158) diluted 1:3000. After rinsing, the membrane was incubated for 1 h at room temperature in horseradish peroxidase-conjugated rabbit PLoS ONE | www.plosone.org 8 December 2008 | Volume 3 | Issue 12 | e3945