Dentin-Pulp Complex Tissue Regeneration via Three-Dimensional Cell Sheet Layering

The dentin-pulp complex is a unique structure in teeth that contains both hard and soft tissues. Generally, deep caries and trauma cause damage to the dentin-pulp complex, and if left untreated, this damage will progress to irreversible pulpitis. The aim of this study was to fabricate a layered cell sheet composed of rat dental pulp (DP) cells and odontogenic differentiation of pulp (OD) cells and to investigate the ability to regenerate the dentin-pulp complex in a scaffold tooth. We fabricated two single cell sheets composed of DP cells (DP cell sheet) or OD cells (OD cell sheet) and a layered cell sheet made by layering both cells. The characteristics of the fabricated cell sheets were analyzed using light microscopy, scanning electron microscopy (SEM), hematoxylin-eosin (HE) staining, and immunohistochemistry (IHC). Furthermore, the cell sheets were transplanted into the subrenal capsule of immunocompromised mice for 8 weeks. Following this, the regenerative capacity to form dentin-like tissue was evaluated using micro-computed tomography (Micro-CT), HE staining, and IHC. The findings of SEM and IHC confirmed that layered cell sheets fabricated by stacking OD cells and DP cells maintained their cytological characteristics. Micro-CT of layered cell sheet transplants revealed a mineralized capping of the access cavity in the crown area, similar to that of natural dentin. In contrast, the OD cell sheet group demonstrated the formation of irregular fragments of mineralized tissue in the pulp cavity, and the DP cell sheet did not develop any hard tissue. Moreover, bone volume/tissue volume (BV/TV) showed a significant increase in hard tissue formation in the layered cell sheet group compared to that in the single cell sheet group (p<0.05). HE staining also showed a combination of soft and hard tissue formation in the layered cell sheet group. Furthermore, IHC confirmed that the dentin-like tissue generated from the layered cell sheet expressed characteristic markers of dentin but not bone equivalent to that of a natural tooth. In conclusion, this study demonstrates the feasibility of regenerating dentin-pulp complex using a bioengineered tissue designed to simulate the anatomical structure.

内容要旨・審査要旨・論文本文の公開
本論文は，著者Huijiao Yanの学位論文として提出され，学位審査・授与の対象となっている。

This is the accepted version of the following article: Dentin-Pulp Complex Tissue Regeneration via Three-Dimensional Cell Sheet Layering; Huijiao Yan, Masamitsu Oshima, Resmi Raju, Swarnalakshmi Raman, Kazumitsu Sekine, Arief Waskitho, Miho Inoue, Masahisa Inoue, Otto Baba, Tsuyoshi Morita, Mayu Miyagi, and Yoshizo Matsuka; Tissue Engineering Part C: Methods 2021 27:10, 559-570, which has now been formally published in final form at Tissue Engineering Part C: Methods at https://doi.org/10.1089/ten.tec.2021.0171. This original submission version of the article may be used for non-commercial purposes in accordance with the Mary Ann Liebert, Inc., publishers’ self-archiving terms and conditions.