Two-dimensional co-culture systems to study epithelial-mesenchymal interactions during tooth development

Hypothesis. Tooth development is a complex process involving interactions between the oral epithelium and neural-crest derived mesenchyme. These interactions initiate a series of reciprocal and inductive signals that result in the determination and differentiation of odontogenic tissues. Organizational signals generated by the two cell types play a very critical role in initiation of cellular differentiation. Objectives. To design novel co-culture substrates mimicking the epithelial-mesenchymal interactions of a developing tooth to study mineralized matrix formation by differentiating cells as a result of organizational cues. Methods. Cell lines representing dental epithelial and mesenchymal cells, namely HAT-7 and RPC-C2A, were used to establish and characterize co-cultures on self-assembled monolayers. Immunohistochemical analysis was performed to observe morphological and organizational changes in the co-cultures. The co-cultures were subjected to mineralization inducing conditions and SEM and EDX analyses were performed to identify mineralized matrix deposition. Primary mesenchymal cells were extracted from wild type and GFP expressing developing mouse embryos at E14.5 and co-cultured with the epithelial cell line, HAT-7. These co-cultures were also treated with mineralization inducing media and analyzed for cellular differentiation using immunohistochemistry and SEM. Microfluidic devices were modeled and computer simulations were generated to predict molecular diffusion. Devices were fabricated using photolithography and HAT-7 and RPC-C2A were cultured in the microchannels. Results. Nodule formation and polarization of cells along the junction of epithelial-mesenchymal cell layers was observed. Histological evaluation of the co-cultures with alizarin red showed elongation and polarization of cells indicating differentiation. SEM analysis of co-cultures treated with mineralizing media showed calcium phosphate like crystal deposition. The presence of calcium and phosphate in these cultures was confirmed by elemental analysis using EDX. Condensation of primary mesenchymal cells near the epithelial cell layer resembling in-vivo organization was seen by histological staining of co-cultures containing HAT-7 and primary mesenchymal cells. SEM analysis of these cultures also showed deposition of calcium phosphate like crystals near the centers of cell nodules which is a sign of mineralized matrix deposition. The fabricated microfluidic devices demonstrated cell attachment and proliferation within the microchannels which was determined by staining the attached cells with actin. Conclusion. Self-assembled monolayers were characterized for establishment of co-cultures using epithelial and mesenchymal cells for different substrate patterns. All co-cultures showed cellular differentiation and mineralized matrix deposition validating the capability of this system to be used as a model for co-culture of two different cell types in any desired pattern. The designed microfluidic devices also proved effective in establishment of co-cultures in different patterns that have the potential to be employed to study cellular interactions.