Figure 4. Electron micrographs demonstrating various stages of calcification (a–d) and OPN localization (e,f) after 7 days. (a,b) At initial calcification sites, apatite crystals are observed within and outside the cells. (c,d) Calcification progresses to collagen fibrils (arrows in "c") from calcified nodules (CN). (e) Immunoreactivity for OPN is seen in the peripheral region of a calcified nodule. (f) The expanded collagen fibrils (asterisks) are immunopositive for OPN. Scale bar: 0.5 µm (d,e,f), 0.2 µm (b,c), 0.1 µm (a).