Mapping the amelogenin protein expression during porcine molar crown development

Amelogenin (AMEL) plays critical roles during enamel and dentin matrix deposition and mineralization. Most studies focused on the expression patterns of AMEL through the bud, cap, and bell stages. The spatial-temporal expression of AMEL protein during different mineralization stages, especially from presence of crypts to crown completed stages, remains unknown. Thus, the distribution pattern of AMEL in tooth crown formation from Nolla Stage 1 to 6 was investigated. Porcine mandibular molar tooth germs from Nolla Stage 1 to 6 were obtained. The dynamic morphologic changes of tooth germs were examined by X-ray and surgical operating microscope. The AMEL protein expression was evaluated immunohistochemically, then analyzed semi-quantitatively, and further visualized via heat map. Tooth germs continuously increased in size from Nolla Stage 1 to 6. AMEL expression in the newly formed enamel kept negative, but presented intensively positive in the previously formed enamel from Stage 1 to 3. The adjacent enamel-dentin junction (EDJ) was strongly positive during the whole process. In predentin, AMEL was weakly seen at Stage 1 and then dramatically up-regulated from Stage 2 to Stage 3, then down-regulated but was still apparently seen in the whole process. AMEL expression in dentin was decreased during dentin matrix secretion and mineralization. This study identified the dynamic distribution of AMEL during porcine tooth crown formation. Semi-quantitative analysis and heat map emerged as reliable indicators in demonstrating AMEL distribution pattern.