Scaffolds for Gingival Tissues: Model Synthetic Structures for In-Vitro Co-Culture

A significant challenge in tissue engineering is to achieve a successful integration of multiple types of cells and tissues into a physiologically correct architecture. In humans, gingival tissues mainly consist of dense fibrous connective tissues covered by stratified squamous epithelium. Objective: To assemble a composite matrix that can support the simultaneous proliferation, function, and layering of epithelial cells on the surface and fibroblasts within it. Methods: A RGD peptide impregnated Vinyl lysine urethane (VLU) scaffold was synthesized according to established protocols and bonded to a polycarbonate-urethane electro-spun nanofibre (130-890 nm) porous membrane using dimethyl acetamide to form a 2-phase composite matrix. HGF-1 cells were cultured onto the VLU scaffold for 2 weeks prior to the incubation of SSC-15 cells onto the nanofibre membrane side for 1 week. Live/Dead® Assay with confocal microscopy and histological staining with anti-CD40 and anti-EpCAM antibodies were performed at the end of each week to study cell migration and proliferation. Results: Migration of HGF-1 cells was observed by two-photon confocal microscopy to be 80µm and 250µm into the matrix for the 1st and 2nd weeks of culture respectively. The remained was isolated from the electro-spun membrane surface. Histological images revealed the presence of more cells inside the matrix at the end of 2nd week versus 1st week. Conclusion: While co-culture studies of HGF-1 and SCC-15 cells are still on-going, this work to-date was successful in developing a methodology for producing a dual phase tissue engineering matrix, establishing a protocol for carrying out the co-culture of epithelial and fibroblast cells and maintaining them in separate layers, and demonstrating that HGF cells can migrate several hundred microns within the scaffold, remaining viable and proliferating. Acknowledgements: Canadian Institutes of Health Research/NSERC of Canada.