Dental Pulp Stem Cell-Epithelial Interactions in Two Dimensional and Three Dimensional Co-culture

Epithelial mesenchymal interaction has been observed as the developmental basis of many organs. Hair, glands, lungs, kidneys and teeth develop through this interaction and understanding the cell-cell crosstalk may lead to advances in tissue engineering and regenerative medicine. Teeth serve as good models of this process because of their many stages of development and ease of accessibility. Additionally, understanding tooth formation can lead to advances in dentistry, leading to improved restoration techniques. Research has revealed the complex signaling that leads to odontogenesis. Communication between dental epithelium and dental mesenchyme are reciprocal and progressive. Many have replicated odontogenesis in vitro, and three dimensional cultures have been used to better replicate in vivo cell processes. Classic tissue recombinant studies have shown only one cell type of dental origin is required for tooth formation. Current in vitro research has shown mouse-mouse, or human-mouse epithelial mesenchymal co-cultures to exhibit dental characteristics, and can form tooth like structures when implanted in vivo. However, differences have been found between in human and mouse dental anatomy. This study was designed to observe the dental development in an allogeneic human mesenchymal epithelial co-culture. Two dental pulp stem cell lines and a foreskin epithelial cell line were co-cultured under osteogenic conditions. The epithelial cells were first cultured in low adhesion conditions to form spheroids, and then were co-cultured with a mesenchymal monoculture (3D/2D co-culture). 3D/2D co-cultures were compared to monocultures in both osteogenic and control conditions. Monocultures were also recombined with epithelial cells in single cell suspension (2D/2D co-culture). An additional mesenchymal cell of non-dental origin was also used in both 3D/2D and 2D/2D co-cultures. Cultures were assayed using Alizarin red staining, immunohistochemistry and qRT-PCR. Findings suggest a difference between monocultures, 2D/2D co-cultures and 3D/2D co-cultures. Dental pulp stem cells in 3D/2D co-cultures and osteogenic dental monocultures expressed markers of osteogenesis and early odontogenesis. However, variations between dental pulp stem cell lines were also observed, as one line had fewer markers of odontogenesis. Findings indicate the potential for allogeneic co-cultures to better represent human tooth formation. Further studies are required to better understand if allogeneic co-cultures can form tooth-like structures when implanted in animal models.