Nicotine Delays Bone Growth Figure 3. Long-term (four months) effect of nicotine on growth plate chondrocytes in alginate beads. 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). doi:10.1371/journal.pone.0003945.g003 PLoS ONE | www.plosone.org 5 December 2008 | Volume 3 | Issue 12 | e3945