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. 2008 Dec 16;3(12):e3945. doi: 10.1371/journal.pone.0003945

Figure 2. Effect of nicotine on growth plate chondrocytes in agarose gel.

Figure 2

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 µm (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 µg/ml). The percentage of ALB-positive colonies exceeded 10% by using 500 nM MLA. *, statistically significant, P<0.02.