Subcutaneous implantation of tooth germ stem cells over the masseter muscle in mice: An in vivo pilot study

Abstract

Objectives: This study aimed to explore the potential of tooth germ stem cells for regenerating tooth-like structures by subcutaneously implanting first molar tooth germ stem cells over the masseter muscle in mice. Methods: Five pairs of house mice, Mus musculus, were selected for mating. At gestational day 14 (E14), the fetuses were extracted, and the first molar tooth germ at the cap stage was isolated. Tooth germ stem cells were prepared into a suspension and seeded onto scaffolds, which were then implanted subcutaneously over the masseter muscle in male mice. The control group (n 1/4 5 male mice) received acellular scaffolds implanted at the same site. After 20 days, the regenerated tissues were resected and analyzed histologically using hematoxylin and eosin (H & E) staining, Masson's trichrome staining, and immunohistochemical (IHC) staining for cytokeratin (CK) and vimentin markers. Results: H & E staining showed the formation of integrated oval structures at the implant site in all samples. Masson's trichrome staining identified dispersed accumulations of cellular mineralized matrix within the connective tissue. IHC staining was positive for vimentin, confirming the mesenchymal origin of the loose tissue at the center, indicating future dental pulp development. Positive CK staining indicated the ectodermal origin of dense peripheral tissues, suggesting the future formation of inner enamel epithelium. The combined immunohistochemical results for vimentin and CK confirmed the ectomesenchymal origin of the regenerated tissue, which resembled a late bell-stage tooth germ observed around gestational days 17.5-18 and showed early indications of dentin formation (D0). Conclusion: The study indicates that tooth germ stem cells may have the potential to produce dense, tooth-like structures when implanted subcutaneously in mice. These findings provide preliminary insights into the possible applications of tooth germ stem cells in regenerative dental tissue engineering. (c) 2025 The Author(s). Published by Elsevier BV on behalf of The Japanese Society for Regenerative Medicine. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/ licenses/by-nc-nd/4.0/).