Fig. 5

NGF treatment results in matrix degeneration in OA cartilage. Cartilage explants from OA tissue were cultured in serum-free medium (control), or treated with NGF for 14 days, and then the explants and culture medium were analyzed separately. a Safranin O staining of cartilage explants isolated from a uniform region before and after 14 days of culture, with or without 10 ng/ml NGF. b Medium sGAG release, normalized to tissue wet weight (mg) (n = 4, biological replicates). Representative samples are presented: values are mean ± SE; NGF versus control, two-way ANOVA, p <0.0001). c MMP zymography analysis of medium MMP2 and MMP9 activity (n = 5; representative images shown). Zymography analysis of MMP2 release in medium at day 1 (n = 5 biological replicates; *p <0.05), semiquantified based on band intensity signal. d Western blot analysis of medium MMP3 release during culture; semiquantified based on band intensity (n = 3 biological replicates; values are mean ± SD; NGF versus control, two-way ANOVA, p <0.005; blots from same patient sample). e Medium MMP activity. Medium samples from day 1, days 5–7, and days 11–13 were assayed (n = 4, biological replicates; NGF versus control group, and NGF versus NGF + ab group at day 13; *p <0.05, values are mean ± SD). f Western blot analysis of medium TIMP1 release at days 1, 7, and 13 (n = 4, biological replicates; representative results shown). ab NGF neutralizing antibody (10 ng/ml), MMP matrix metalloproteinase, NGF nerve growth factor, sGAG sulfated glycosaminoglycan, TIMP1 tissue inhibitor of matrix metalloprotease-1