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In the human molar, multirooted tooth formation occurs through the formation of dentin islands, termed subpulpal lobus, in the dental papilla. However, the mechanisms underlying subpulpal lobus formation currently remain unclear. Thus, using the rat molar, in which multirooted teeth formed through subpulpal lobus formation, similar to that in humans, we observed the process of subpulpal lobus formation over multirooted tooth formation histologically and immunohistologically, and discussed accessory root canal formation in the furcation area. The upper second molar (M2) was examined from rats 8 to 18 days after birth, stained with hematoxylin, and observed under a stereoscopic microscope. The maxilla including M2 was dissected out en bloc and decalcified, and paraffin-embedded sections were prepared following the standard method. Sections were subjected to hematoxylin-eosin double staining and immunohistochemical staining with anti-pan-keratin (PK), anti-heat shock protein 27 (HSP27), and anti-sonic hedgehog (Shh) antibodies. After the completion of tooth crown formation, a part of Hertwig’s epithelial root sheath (HERS) in the cervical region extended and formed an epithelial projection (EP) that was reactive with the anti-PK antibody. The EP induced the differentiation of dental papilla cells facing it into anti-Hsp27 antibody-reactive odontoblasts and formed the subpulpal lobus. EP and subpulpal lobus-forming odontoblasts reacted with the anti-Shh antibody, suggesting that cell differentiation in subpulpal lobus formation occurs through the epithelial-mesenchymal interaction. While forming subpulpal lobus, the EP extended without making contact with the opposing EP or HERS in the cervical region. The furcation area was formed by fusion between the dentin projection (DP) formed in the cervical region and subpulpal lobus and between subpulpal lobus. HERS in the cervical region and EP formed the furcation area without making contact with each other, suggesting that the accessory root canal is formed where DP and the subpulpal lobus do not fuse.

Histological and Immunohistochemical Observation of the Furcation Area Formation with the Subpulpal Lobus of Rat Molar