Activation of the bone morphogenetic protein (BMP) signaling pathway. (a,b) Time course of the differential expression of BMP-2 mRNA in injured versus uninjured explants in (a) the presence or (b) the absence of fetal bovine serum (FBS) in the culture medium. Values are normalized for the housekeeping β actin gene and expressed as fold change of gene expression in the injured explants from paired uninjured controls. Diamonds indicate samples from preserved areas from joints affected by osteoarthritis; open squares indicate the sample pair from healthy cartilage. (c-g) Immunostaining for phosphorylated SMAD-1/-5 in: (c) freshly dissected normal cartilage; (g) the adjacent injured explant at day 1 after injury; (f) and the adjacent uninjured control at the same time-point. (d) Larger magnification of the area shown in the square in (c). In the freshly dissected sample, phosphorylated SMAD-1/-5-positive cells were detected predominantly in the intermediate layer indicated by the bracket in (c). (e) Image obtained by false coloring in red the image in (d) and superimposing it on the fluorescent image in the blue channel documenting the nuclear DAPI counterstain. The DAB precipitate in the phosphorylated SMAD-1/-5-positive cells quenched the DAPI fluorescence and, therefore, in this panel, phosphorylated SMAD-1/-5-positive cells appear red and the nuclei of negative cells appear blue. The top insets in (f,g) are large magnifications of the corresponding squared areas. (h) A graphic summary of the proportion of phospho-SMAD-1/-5-positive cells and the expression of BMP-2, FRZB, metalloproteinase (MMP)-3 and MMP-13 mRNAs in this experiment with normal adult human articular cartilage. Values are expressed as: percent of positive cells for phospho-SMAD-1/-5; relative gene expression normalized for the housekeeping β actin gene; percent of the day 6 time point for BMP-2, MMP-3 and MMP-13 mRNA; and percent of the freshly dissected cartilage for FRZB. *p < 0.05; **p < 0.01. D, day(s); H, hours; SF, serum free medium.