Autoradiographic investigation of the effect of 1-hydroxyethylidene-1, 1-bisphosphonate on matrix protein synthesis and secretion by secretory ameloblasts in rat incisors.

Seven daily subcutaneous injections of 1-hydroxyethylidene-1, 1-bisphosphonate (HEBP) can induce enamel hypoplasia. Several enamel-free zones were observed along the crown-analogue side of rat incisors during the secretory stage of amelogenesis. Ameloblasts related to the enamel-free zones lay directly on the abnormally non-mineralized mantle dentine, whereas the adjacent ameloblasts, which were forming the enamel matrix layer, were associated with the region where mineralization of dentine was proceeding. The further purpose of this study was to investigate the synthetic and secretory activity of these two groups of ameloblasts and to trace the fate of the radioactively labelled proteins. [(3)H]-proline was administered to Wistar rats 12 h after the last injection of HEBP. Light-microscopic autoradiography was performed. Quantitative analysis indicated that the ameloblasts of the enamel-forming zones in the drug-treated group showed a distribution pattern of silver grains similar to that of the controls. The ameloblasts of the enamel-free zones also demonstrated incorporation of [(3)H]-proline at the same level. There was some labelling over the non-mineralized mantle dentine, which was supposed to indicate the penetration of ameloblast products. From these results, it is concluded that HEBP does not affect the ameloblast activity in protein synthesis. The complete failure of enamel-layer formation in some specific regions is probably due to the failure in protein secretion and protein deposition. This study provides additional evidence that the mineralization of dentine is an essential factor in successful enamel matrix secretion and deposition.

Gene expression and immunolocalization of amelogenin in enamel hypoplasia induced by successive injections of bisphosphonate in rat incisors.

Successive injections of 1-hydroxyethylidene-1, 1-bisphosphonate (HEBP) in rats induce enamel hypoplasia. To elucidate the pathogenesis of this hypoplasia, male Wistar rats were daily injected with HEBP or physiological saline for 7 days. After the last injection, they were killed under anaesthesia and their maxillary incisors were examined using an in situ hybridization technique and immunohistochemical staining to detect the gene expression and localization of amelogenin protein, respectively. In the HEBP-injected rats, several islets of partially mineralized enamel were present along crown-analogous surface of the incisor in the secretory stage of amelogenesis and enamel-free zones existed between these islets. In situ hybridization demonstrated amelogenin gene expression over the ameloblasts facing the islets of the matrix enamel as well as over those of the enamel-free zones. Immunohistochemical studies using rabbit antiamelogenin antibody revealed positive reaction both in the enamel matrix of the control group and in the islets of enamel matrix of the HEBP-injected group. Some small granules immunoreactive to amelogenin antibody were found in the distal portions of the ameloblasts in the HEBP-injected rats. The results indicate that HEBP does not alter amelogenin gene expression over ameloblasts, or the protein's existence in enamel matrix. There appeared to be some accumulation of amelogenin in the HEBP-treated ameloblasts. It is therefore suggested that the enamel hypoplasia in this experiment may not be due to a disturbance in amelogenin synthesis but to a disturbance in a later process, presumably of protein secretion.