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The study of enamel formation of rats incisors by in situ hybridization
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Yoshishige Yamada, Yuzou Takagi, Hitoyata Shimokawa*,
Hiroshi Ono and Keiichi Ohya**

Department of Pedodontics, *Biochemistry and **Dental Pharmacology,
Faculty of Dentistry, Tokyo Medical and Dental University,
1-5-45 Yushima Bunkyo-ku,
Tokyo 113, Japan



Key word: In situ hybridization /enamel hypoplasia /bisphosphonate /amelogenin


Abstract:

The successive injection of 1-hydroxyethylidene-1, 1-bisphosphonate (HEBP) induced enamel hypoplasia in rat incisor (Jpn J Oral Biol, 33, 82-96, 1991). The present study was undertaken to investigate the amelogenin gene expression in ameloblast of enamel hypoplasia by in situ hybridization. Male Wistar rats, weighing 120-130g, were injected subcutaneously with HEBP at a dose of 8 mgP/kg/day. The control rats were injected with a physiological saline. They were sacrificed under anesthesia 24 hours after the seventh injection, and their maxillary incisors were used for in situ hybridization. In the light microscopic observation, the islets of enamel appeared along the incisor surface and enamel-free zone were observed between these islets. Contact microradiographs indicated that mineralization of the enamel had gradually developed in these islets of enamel, but several hypoplastic bands were observed in the enamel produced during the experimental period.
In situ hybridization using amelogenin RNA prove showed that amelogenin gene expression was uniformly observed in ameloblasts of the secretory stage in the control rat. In the HEBP injected rats, amelogenin gene expression was observed almost same level of the control rats and their expression in ameloblasts were not different between The region of enamel hypoplasia and islet enamel. These results indicated that the successive injection of HEBP did not alter the amelogenin gene expression in ameloblasts, but inhibited the process after gene transcription, resulting the enamel hypoplasia.


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Fig. 1
Contact microradiographies of the longitudinal ground surface of enamel in the ratmaxillary . (a) The HEBP 8 mgP/kg/day-injected group. Several islets of mineralized enamel are observed in the secretory stage. ( b) High power magnification of (a). The mineralization of dentin has occurred only under the islets of enamel. E : enamel, D : dentin.


Fig. 2
Light microscopic photographs of the longitudinal section of the rat maxillaryincisor.
(a) The control group. In situ hybridization showed the expression of amelogenin gene at the
secretory stage of ameloblasts. (b) -(d) The HEBP 8 mgP/kg/day-injected group. (b)Normal light
microscopic section. Several islets of enamel (*) appear along the dentin surfaceand enamel-free zones are observed between these islets. The dentin under the islets of enamel is more strongly stained with hematoxylin-eosin than the dentin under the enamel-free zone. The ameloblasts facing the enamel-free zone seem to be normal in form. The odontoblasts shows an unusual cell-arrangement. (c) Expression of amelogenin gene by in situ hybridization using anti-sense prove. Amelogenin gene expression are observed in the ameloblast between enamel-free area and islets of enamel. The level of expression is similar between enamel-free area and islets of enamel. (d) Expression of amelogenin gene using sense prove. The expression of amelogenin could not be observed in the ameloblast. E : enamel, D : dentin.