Figure 5.

HN expression and protective effects of HN in cultured human growth plate cartilage and cultured human proliferative chondrocytes. A, B) Studies of endogenous levels of HN in human growth plate cartilage treated with Dexa (A; 1 µM) for 24 h and analyzed for changes in endogenous HN expression (brown staining; matrix stained blue with Alcian blue, n = 4) shown in R+P zone and hypertrophic H zone. Quantitative analysis of HN (B), expressed as fold change. C–F) Quantitative analysis of apoptosis and Bax levels in cultured human growth plate biopsies. Human growth plate cartilage was treated with Dexa (1 µM), HNG (100 nM), or both, for 24 h and analyzed for changes in apoptosis level (red staining, white arrows) by using TUNEL assay (C, D) and Bax expression (red staining, white arrows) using immunohistochemistry (E, F) (n = 4). G–I) HCS-2/8 chondrocytes were used to study cell death, proliferation, and changes in mitochondrial membrane potential (MMP). The cells were treated with Dexa (25 µM), HNG (1, 10, and 100 nM), or both, for 72 h. Apoptosis (G) was measured by Cell Death ELISA. MMP difference (H) by using a commercially available kit. Cell proliferation (I) by BrdU incorporation analyses applying a BrdU-ELISA (n = 4). All error bars indicate sd. For statistical analysis, 1-way ANOVA was performed. Original magnification (A, C, E), ×20. **P < 0.01, ***P < 0.001.