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Therapeutic potential of HERS spheroids in tooth regeneration.

ABSTRACT: Hertwig's epithelial root sheath (HERS) plays indispensable roles in tooth root development, including controlling the shape and number of roots, dentin formation, and helping generate the cementum. Based on these characteristics, HERS cell is a potential seed cell type for tooth-related tissue regeneration. However, the application is severely limited by a lack of appropriate culture methods and small cell numbers.Methods: Here, we constructed a 3D culture method to expand functional HERS cells into spheroids, and investigated characteristics and application of dental tissue regeneration of these spheroids. HERS spheroids and HERS cells (2D monolayer culture) were compared in terms of biological characteristics (such as proliferation, self-renewal capacity, and stemness) in vitro and functions (including differentiation potential and inductive ability of dentin formation) both in vitro and in vivo. Further, transcriptome analysis was utilized to reveal the molecular mechanisms of their obvious differences.Results: HERS spheroids showed obvious superiority in biological characteristics and functions compared to 2D monolayers of HERS cells in vitro. In vivo, HERS spheroids generated more mineralized tissue; when combined with dental papilla cells (DPCs), HERS spheroids contributed to dentin-like tissue formation. Moreover, the generation and expansion of HERS spheroids rely to some degree on the HIF-1 pathway.Conclusion: HERS spheroid generation is beneficial for functional HERS cell expansion and can provide a useful cell source for further tooth regeneration and mechanistic research. Notably, HIF-1 pathway plays a critical role in HERS spheroid formation and function.

SUBMITTER: Duan Y

PROVIDER: S-EPMC7330840 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Project description:Whole tooth regeneration approaches currently are limited by our inability to bioengineer full-sized, living replacement teeth. Recently, decellularized organ scaffolds have shown promise for applications in regenerative medicine by providing a natural extracellular matrix environment that promotes cell attachment and tissue-specific differentiation leading to full-sized organ regeneration. We hypothesize that decellularized tooth buds (dTBs) created from unerupted porcine tooth buds (TBs) can be used to guide reseeded dental cell differentiation to form whole bioengineered teeth, thereby providing a potential off-the-shelf scaffold for whole tooth regeneration. Porcine TBs were harvested from discarded 6-mo-old pig jaws, and decellularized by successive sodium dodecyl sulfate/Triton-X cycles. Four types of replicate implants were used in this study: 1) acellular dTBs; 2) recellularized dTBs seeded with porcine dental epithelial cells, human dental pulp cells, and human umbilical vein endothelial cells (recell-dTBs); 3) dTBs seeded with bone morphogenetic protein (BMP)-2 (dTB-BMPs); and 4) freshly isolated nondecellularized natural TBs (nTBs). Replicate samples were implanted into the mandibles of host Yucatan mini-pigs and grown for 3 or 6 mo. Harvested mandibles with implanted TB constructs were fixed in formalin, decalcified, embedded in paraffin, sectioned, and analyzed via histological methods. Micro-computed tomography (CT) analysis was performed on harvested 6-mo samples prior to decalcification. All harvested constructs exhibited a high degree of cellularity. Significant production of organized dentin and enamel-like tissues was observed in dTB-recell and nTB implants, but not in dTB or dTB-BMP implants. Micro-CT analyses of 6-mo implants showed the formation of organized, bioengineered teeth of comparable size to natural teeth. To our knowledge, these results are the first to describe the potential use of dTBs for functional whole tooth regeneration.

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Therapeutic potential of HERS spheroids in tooth regeneration.

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