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

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Kawakita et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

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Growth plate chondrocytes in alginate beads were exposed to the indicated concentration of nicotine for four months. A: Microscopic view of chondrocytes in alginate beads after four-months cultivation. Upper panels: ALB stain, lower panels: Safranin-O stain. Chondrocytes were surrounded by matrix which they secreted. Nicotine decreased the area stained with ALB or Safranin-O in a concentration-dependent manner. *, statistically significant, P<0.02. B: RT-PCR analysis of chondrocyte-specific gene expression in the chondrocytes at the start of cultivation (lane 1: 0W) and three weeks (lane 2: 3W). From top to bottom: genes for Col II, Aggrecan, parathyroid hormone receptor type 1 (PTHR1), Col X, alkaline phosphatase (ALP), Indian hedgehog (Ihh), matrix metalloproteinase type13 (MMP13), vascular endothelial growth factor (VEGF), Sox9 and GAPDH. C: RT-PCR analysis of chondrocyte-specific gene expression in chondrocytes embedded in alginate beads exposed to the indicated concentration of nicotine for four months. Expression of early stage matrix-gene (Col II and Aggrecan) and markers of hypertrophic chondrocytes (Col X, ALP and Ihh) increased after three weeks of cultivation (B). Nicotine decreased the expression of these genes in a concentration-dependent manner, but had little effect for the expression of MMP13, VEGF, and control genes (Sox9 and GAPDH) (C).