Synergistic Actions of DPSCs and ECs in Vascularising Bioengineered Pulp

Objectives:   To investigate the synergistic effects of dental pulp stem cells (DPSCs) and endothelial cells (ECs) on osteo/odontogenic differentiation and vasculogenesis in 2-dimensional and 3-dimensional culture systems in-vitro. Methods: Different ratios of DPSCs and ECs were cultured in direct-contact in optimized-medium and were induced for osteo/odontogenic differentiation up to 3-weeks. Alkaline phosphatase (ALP) activity, expression levels of ALP, bone sialoprotein (BSP), dentin sialophosphoprotein (DSPP) genes and Alizarin Red staining for mineralization at different time points were analyzed. Tubular network formation on Matrigel and gene expression levels of CD117, VEGF, CD34, Flk-1 were used to analyse vasculogenesis. Three-dimensional microtissue-spheroids of DPSCs and ECs were fabricated using 12-series micro-molds (MicroTissues Inc.). Microtissue-spheroids (1200) were transferred to a custom-designed, 3mm-diameter, agarose mold and cultivated for 4-days to self-assemble into macrotissue. The macrotissues were induced for odontogenic differentiation (21-days), examined for mineralization (Von-Kossa) and for vascularisation (Immunohistochemistry for CD31). Experiments were conducted in triplicate using DPSCs from three different donors and statistically analysed (ANOVA). Results: The quantification of ALP revealed significantly greater activity in DPSC:EC co-cultures compared to DPSC-alone cultures (p<0.05). DPSC:EC, 1:1 and 1:5 co-cultures had a greater amount of calcification under Alizarin Red staining compared to other cultures (p<0.01). The higher expression levels of ALP, BSP and DSPP genes further confirmed the greater osteo/odontogenic differentiation in co-cultures compared to those of DPSC-alone cultures. Matrigel assay showed that the addition of DPSCs stabilized pre-existing vessel-like structures formed by ECs and increased their longevity. DPSC-EC macrotissues showed a significantly higher amount of extracellular matrix and mineralization compared to DPSC-alone macrotissues in 3-D. In contrast to DPSC-alone macrotissues, a dense-network of ECs was found throughout the DPSC:EC macrotissues under immunohistochemical analysis for the EC-specific marker CD31. Conclusion: DPSCs and ECs synergistically enhance osteo/odontogenic differentiation and prevascularize pulp-like macrotissues in-vitro.