Dental cell sheet biomimetic tooth bud model

Abstract

Tissue engineering and regenerative medicine technologies offer promising therapies for both medicine and dentistry. Our long-term goal is to create functional biomimetic tooth buds for eventual tooth replacement in humans. Here, our objective was to create a biomimetic 3D tooth bud model consisting of dental epithelial (DE) – dental mesenchymal (DM) cell sheets (CSs) combined with biomimetic enamel organ and pulp organ layers created using GelMA hydrogels. Pig DE or DM cells seeded on temperature-responsive plates at various cell densities (0.02, 0.114 and 0.228 cells 106/cm2) and cultured for 7, 14 and 21 days were used to generate DE and DM cell sheets, respectively. Dental CSs were combined with GelMA encapsulated DE and DM cell layers to form bioengineered 3D tooth buds. Biomimetic 3D tooth bud constructs were cultured in vitro, or implanted in vivo for 3 weeks. Analyses were performed using micro-CT, H&E staining, polarized light (Pol) microscopy, immunofluorescent (IF) and immunohistochemical (IHC) analyses. H&E, IHC and IF analyses showed that in vitro cultured multilayered DE-DM CSs expressed appropriate tooth marker expression patterns including SHH, BMP2, RUNX2, tenascin and syndecan, which normally direct DE-DM interactions, DM cell condensation, and dental cell differentiation. In vivo implanted 3D tooth bud constructs exhibited mineralized tissue formation of specified size and shape, and SHH, BMP2 and RUNX2and dental cell differentiation marker expression. We propose our biomimetic 3D tooth buds as models to study optimized DE-DM cell interactions leading to functional biomimetic replacement tooth formation.

Three‐dimensional printing biotechnology for the regeneration of the tooth and tooth‐supporting tissues

“…These stem cell types are usually isolated from unerupted tooth buds extracted from infantile donor animals. The biomimetic tooth bud model was constructed through recombining DE and DM progenitor cells encapsulated within GelMA hydrogels (Monteiro et al, ; E. E. Smith et al, ). It has been well documented that the reaggregation of adult DE and DM cells is a crucial way to establish organ culture models of human tooth germ development in vitro.…”

“…A functional vasculature has been reported in 3D‐bioprinted thick human tissues (>1 cm; Kolesky, Homan, Skylar‐Scott, & Lewis, ). Additionally, certain light‐crosslinking hydrogels, encapsulated with HUVECs, have resulted in well‐organized neovasculature formation in the bioprinted dental pulp and tooth germ tissues (Khayat et al, ; Monteiro et al, ; E. E. Smith et al, ). Maintaining high activity and excellent angiogenesis, HUVECs are considered to be beneficial for bioengineered dental tissues.…”

“…Additionally, mimicking such an epithelial–mesenchymal interface that naturally occurs in the early stages of tooth formation has been proven to be the fundamental step in tooth germ regeneration (W. Zhang, Ahluwalia, & Yelick, ; W. Zhang, Vazquez, Oreadi, & Yelick, ). On the above foundation, Yelick focused on the controlled formation of cell aggregates in vitro through different methods (Monteiro et al, ; E. E. Smith et al, ; W. Zhang et al, ). For example, Monteiro et al encapsulated postnatal porcine DE (pDE), porcine DM (pDM) progenitor cells, and HUVECs within a two‐phase composite GelMA to simulate the tooth germ (E. E. Smith et al, ).…”

“…For example, Monteiro et al encapsulated postnatal porcine DE (pDE), porcine DM (pDM) progenitor cells, and HUVECs within a two‐phase composite GelMA to simulate the tooth germ (E. E. Smith et al, ). Additionally, Monteiro created a biomimetic 3D tooth bud model consisting of DE‐DM cell sheets combined with biomimetic enamel organ and pulp organ layers created using GelMA hydrogels (Monteiro et al, ). Notably, mineralized tissue of specified size and shape formed after in vivo implantation (Monteiro et al, ; E. E. Smith et al, ).…”

“…Based on the above findings, the construction of 3D‐printed scaffolds, where cells attach, migrate, and proliferate, has been used in the field of dental regeneration, especially in the regeneration of the periodontium complex (Lee et al, ; Park et al, ; Park et al, ; Vaquette et al, ). Additionally, although it remains in its early stages to print living cells and matrix materials together to produce dental tissue constructs depending on the existing research (Bottino, Pankajakshan, & Nor, ), 3D biofabrication utilizing photocrosslinkable polymers, such as gelatin methacryloyl (GelMA) hydrogels, has been proven to be promising for the dental pulp and whole‐tooth regeneration due to good biocompatibility and efficient revascularization (Monteiro et al, ; E. E. Smith et al, ).…”