Nicotine Delays Bone Growth Figure 2. Effect of nicotine on growth plate chondrocytes in agarose gel. Growth plate chondrocytes were cultured in an agarose gel using the modified method previously described [28], and exposed to nicotine and MLA, a specific antagonist for alpha7 nAChR, at the indicated concentration. After three weeks of cultivation, suspension agarose was transferred to a glass slide and the following histological analyses were then performed. A: Microscopic appearance of chondrocyte colonies. From top to bottom: ALB (Alcian blue stain), Col X (immunocytochemistry by an anti- Col X antibody), ALP (enzyme cytochemistry of alkaline phosphatase). For ALB and Col X stain, the slides were counterstained with kernechtrot and hematoxylin, respectively. Percentage of ALB- stained, Col X- positive, and Alkaline phosphatase- positive colonies were counted (right panel, from top to bottom). All the ALP positive colonies in the panels are indicated by arrowheads. Nicotine concentration-dependently suppressed the percentage of the colonies stained with ALB, Col X, and ALP. *, statistically significant, P,0.02. B: Number of colonies and number of cells per colony. The number of colonies with a diameter greater than 50 mm (left panel) and cell number per colony (right panel) were counted on the ALB- stained agarose gel slides. C: Microscopic appearance of chondrocyte colonies stained with ALB. MLA reversed the decrease of ALB- positive matrix in a concentration-dependent manner under constant nicotine concentration (20 mg/ml). The percentage of ALB-positive colonies exceeded 10% by using 500 nM MLA. *, statistically significant, P,0.02. doi:10.1371/journal.pone.0003945.g002 PLoS ONE | www.plosone.org 4 December 2008 | Volume 3 | Issue 12 | e3945