Search

In this study, we attempted to localize the immunoreactivities of podoplanin/E11/gp38 and CD44, a counterpart possessing a high affinity to podoplanin/E11/gp38, as well as endomucin-immunoreactive blood vessels in the regions of odontoblast layers and the underlying sub-odontoblastic layers in murine tooth germs. Endomucin-reactive small blood vessels were scattered throughout the dental papillae of the tooth germs at postnatal day 1 but came to be localized close to the odontoblast/sub-odontoblastic layers until day 3. After postnatal day 5, small blood vessels were seen in odontoblast cell layers, while blood vessels with relatively larger diameters were seen forming in sub-odontoblastic layers. Immunoreactivities of podoplanin/E11/gp38 and CD44 were not detectable in the cells of dental papillae facing the inner enamel epithelium at postnatal day 1. However, at around postnatal days 3-5, podoplanin/E11/gp38 was localized in the odontoblast layer but not in the sub-odontoblastic layer, whereas CD44 was observed in the sub-odontoblastic layer but not in the odontoblast layer. The exclusive immunolocalization of podoplanin/E11/gp38 and CD44 in the odontoblast layers and sub-odontoblastic layers was seen after postnatal day 3 of the tooth germs, when the mesenchymal cells of dental papillae have already differentiated into mature odontoblasts at the cusp tip. Taken together, it seems likely that endomucin-reactive small blood vessels extended to the podoplanin/E11/gp38-positive odontoblast layers, whereas endomucin-reactive large blood vessels were already present in CD44-immmunopositive sub-odontoblastic layer, indicating the cellular regulation on the vascularization of endomucin-reactive endothelial cells during odontogenesis of the tooth germs.

Minodronate is highlighted for its marked and sustained effects on osteoporotic bones. To determine the duration of minodronate’s effects, we have assessed the localization of the drug in mouse bones through isotope microscopy, after labeling it with a stable nitrogen isotope ([15N]-minodronate). In addition, minodronate-treated bones were assessed by histochemistry and transmission electron microscopy (TEM). Eight-week-old male ICR mice received [15N]-minodronate (1 mg/kg) intravenously and were sacrificed after 3 hr, 24 hr, 1 week, and 1 month. Isotope microscopy showed that [15N]-minodronate was present mainly beneath osteoblasts rather than nearby osteoclasts. At 3 hr after minodronate administration, histochemistry and TEM showed osteoclasts with well-developed ruffled borders. However, osteoclasts were roughly attached to the bone surfaces and did not feature ruffled borders at 24 hr after minodronate administration. The numbers of tartrate-resistant acid phosphatase–positive osteoclasts and alkaline phosphatase–reactive osteoblastic area were not reduced suddenly, and apoptotic osteoclasts appeared in 1 week and 1 month after the injections. Von Kossa staining demonstrated that osteoclasts treated with minodronate did not incorporate mineralized bone matrix. Taken together, minodronate accumulates in bone underneath osteoblasts rather than under bone-resorbing osteoclasts; therefore, it is likely that the minodronate-coated bone matrix is resistant to osteoclastic resorption, which results in a long-lasting and bone-preserving effect.

Summary Matrix vesicle-mediated mineralization is an orchestrated sequence of ultrastructural and biochemical events that lead to crystal nucleation and growth. The influx of phosphate ions into the matrix vesicle is mediated by several proteins such as TNAP, ENPP1, Pit1, annexin and so forth. The catalytic activity of ENPP1 generates pyrophosphate (PPi) using extracellular ATPs as a substrate, and the resultant PPi prevents crystal overgrowth. However, TNAP hydrolyzes PPi into phosphate ion monomers, which are then transported into the matrix vesicle through Pit1. Accumulation of Ca2+ and PO43− inside matrix vesicles then induces crystalline nucleation, with calcium phosphate crystals budding off radially, puncturing the matrix vesicle’s membrane and finally growing out of it to form mineralized nodules.