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Classical tooth development theory suggests that dental papilla cells (DPCs) are the precursor cells of odontoblasts, which are responsible for dentin development. However, our previous studies have indicated that dental follicle cells (DFCs) can differentiate into odontoblasts. To further our understanding of tooth development, and the differences in dentinogenesis between DFCs and DPCs, the odontogenic differentiation of DFCs and DPCs was characterized in vitro and in vivo. DFCs and DPCs were individually combined with treated dentin matrix (TDM) before they were subcutaneously implanted into the dorsum of mice for 8 weeks. Results showed that 12 proteins were significantly differential, and phosphoserine aminotransferase 1 (PSAT1), Isoform 2 of hypoxia-inducible factor 1-alpha (HIF1A) and Isoform 1 of annexin A2 (ANXA2), were the most significantly differential proteins. These proteins are related to regulation of bone balance, angiogenesis and cell survival in an anoxic environment. Both DFCs and DPCs express odontogenic, neurogenic and peridontogenic markers. Histological examination of the harvested grafts showed that both DFCs and DPCs form pulp-dentin/cementum-periodentium-like tissues in vivo. Hence, DFCs and DPCs have similar odontogenic differentiation potential in the presence of TDM. However, differences in glucose and amino acid metabolism signal transduction and protein synthesis were observed for the two cell types. This study expands our understanding on tooth development, and provides direct evidence for the use of alternative cell sources in tooth regeneration.
Three-dimensional (3D) bioprinting is an emerging method for tissue regeneration. However, promoting the epithelial-mesenchymal interaction (EMI), while maintaining the characteristics of epithelial cells has always been a challenge in tissue engineering. Since EMI acts as a critical factor in bone regeneration, this study aims to promote EMI by recombining epithelial and mesenchymal cells through 3D bioprinting. Hertwig's epithelial root sheath (HERS) is a transient structure appeared in the process of tooth root formation. Its epithelial characteristics are easy to attenuate under appropriate culture environment. We recombined HERS cells and dental papilla cells (DPCs) through 3D bioprinting to simulate the micro-environment of cell-cell interaction in vivo. HERS cells and DPCs were mixed with gelatin methacrylate (GelMA) separately to prepare bio-inks for bioprinting. The cells/GelMA constructs were transplanted into the alveolar socket of Sprague-Dawley rats and then observed for 8 weeks. Hematoxylin and eosin staining, Masson staining, and immunohistochemical analysis showed that dimensional cultural pattern provided ideal environment for HERS cells and DPCs to generate mineralization texture and promote alveolar bone regeneration through their interactions. 3D bioprinting technology provides a new way for the co-culture of HERS cells and DPCs and this study is inspiring for future research on EMI model.
Hertwig's epithelial root sheath (HERS) is important in guiding tooth root formation by differentiating into cementoblasts through epithelial-mesenchymal transition (EMT) and inducing odontoblastic differentiation of dental papilla through epithelial-mesenchymal interaction (EMI) during the tooth root development. Thus, HERS cells are critical for cementum and dentin formation and might be a potential cell source to achieve tooth root regeneration. However, limited availability and lifespan of primary HERS cells may represent an obstacle for biological investigation and therapeutic use of tooth tissue engineering. Therefore, we constructed, characterized, and tested the functionality of immortalized cell lines in order to produce a more readily available alternative to HERS cells.
Hertwig's epithelial root sheath (HERS) is critical for epithelial-mesenchymal interaction (EMI) during tooth root formation. However, the exact roles of HERS in odontogenic differentiation by EMI have not been well characterized, because primary HERS cells are difficult to obtain. Immortalized cell lines constitute crucial scientific tools, while there are few HERS cell lines available. Our previous study has successfully established immortalized HERS cell lines. Here, we confirmed the phenotype of our HERS-H1 by verifying its characteristics and functions in odontogenic differentiation through EMI. The HERS-H1-conditioned medium (CM-H1) effectively enhanced odontogenic differentiation of dental papilla cells (DPCs) in vitro. Furthermore, Smad4 and p-Smad1/5/8 were significantly activated in DPCs treated with CM-H1, and this activation was attenuated by noggin. In vivo, our implanted recombinants of HERS-H1 and DPCs exhibited mineralized tissue formation and expression of Smad4, p-Smad1/5/8, and odontogenic differentiation markers. Our results indicated that HERS-H1 promoted DPCs odontoblastic differentiation via bone morphogenetic protein/Smad signaling. HERS-H1 exhibits relevant key molecular characteristics and constitutes a new biological model for basic research on HERS and the dental EMI during root development and regeneration.
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