On the developing periodontal ligament of rats, using a new specimen preparation method for SEM in comparison with histochemistry.

To visualize the cells and fibers of the developing periodontal ligament (PDL) by scanning electron microscope (SEM), we examined a new tissue preparation method including decalcification, sectioning by cryomicrotome, and chemical treatment for removal of cells or collagen fibers. The advantages of this method were as follows: (1) it was possible to expose the restricted area, (2) it caused no damage by heat or various embedding agents such as paraffin or resin, and (3) it was possible to make comparisons the SEM observation with histochemical or immunohistochemical observation using the neighboring sections. We could classify the development of PDL into three stages by alkaline phosphatase (ALPase) activity and observe each stage by this method. Stage I was the zone of dental follicle proper that showed negative ALPase activity. Stage II was the tissue surrounding the disrupted Hertwig's epithelial root sheath (HERS) which evinced intense ALPase activity, and stage III was the further advanced zone of differentiation that displayed moderate ALPase activity. Using this new method for SEM, cells with many processes and thin fibers were seen irregularly at stage II. On the other hand, at stage III, fibers were seen as interconnecting meshworks of thick bundles and cells that showed regularly arranged rows running obliquely to the surface of the root and alveolar bone. At the transition between stages II and III, the thickness and orientation of fibers changed abruptly.