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.