Search

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 10(6)/cm(2)) 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.

Physical forces drive cell shape across diverse processes such as cytokinesis, cell migration and tissue invasion. The actin cytoskeleton provides the framework for regulating cell shape in response to mechanical stress. In the social amoeba Dictyostelium discoideum, a myosin II-based mechanosensory system controls cellular contractility and cell shape. The IQGAP proteins bind to the actin crosslinker, cortexillin I, and regulate myosin accumulation under stress. To determine how these proteins interact to regulate contractility, we studied the protein dynamics using fluorescence recovery after photobleaching (FRAP). We observe mechanical stress-dependent reduction in the mobility and network release of these proteins from the cytoskeleton. These altered dynamics could reflect a new mechanism for the accumulation of the mechanosensory proteins under stress by local rearrangement of the actin cytoskeleton microstructure. Additionally, IQGAP2 regulates the dynamics of cortexillin I under stress, indicating a functional relevance for the biochemical interaction between these proteins. These studies will help determine how cell shape is regulated various mechanical contexts, which will be valuable for understanding processes such as metastasis and tissue remodeling.