ECs Enhance DPSC Properties And Therapeutic Potential In Three-dimensional Model

Abstract

Objectives: Dental pulp stem cells (DPSCs) are known to occupy a perivascular niche and interact with endothelial cells (ECs). This study, for the first time aimed to determine the interactions between DPSCs and ECs in a three-dimensional co-culture model, focusing on survival, angiogenesis and differentiation capacities to reflect cells’ in-vivo milieu more accurately. Methods: Three-dimensional microtissue-spheroids of DPSCs and ECs were fabricated using 12-series micro-molds (MicroTissues Inc.). CellTracker dyes were used to fluorescent label the cells and examined for the organization into spheroids. The cell viability was assessed with the live/dead viability assay kit at day-1, 7 and 14. 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 expression levels of osteo/odontogenic markers: alkaline phosphatase (ALP), bone sialoprotein (BSP) and RUNX2 (Real-time PCR), 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: DPSCs were aggregated to form spheroids when cultured with/without ECs in 3D conditions. The cell viability and turnover on day-14 remained equivalent to that of day-7 with no evidence of cell death in the centre of the spheroids. 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. Results confirmed that ECs enhanced osteo/odontogenic differentiation, on days-7 and 14, compared to DPSC only controls as shown by elevated ALP, BSP and RUNX2 levels (p < 0.05). DPSC-EC macrotissues showed a significantly higher amount of extracellular matrix and mineralization compared to DPSC-alone macrotissues in 3-D.

Conclusions: ECs regulate DPSC activity and their differentiation capacity in 3D, which may facilitate the maintenance of DPSC quiescence and osteo/odontogenic differentiation when exposed to induction stimuli.