Scaffold-free Three-dimensional Spheroids of DPSCs/ECs in Vascularised Pulp Regeneration

Abstract

Objectives: To determine the interactions between dental pulp stem cells (DPSCs) and endothelial cells (ECs) in a three-dimensional co-culture model; and to examine the potential of these cells in vascularised pulp regeneration. Methods: Three-dimensional microtissue-spheroids of DPSC-alone and DPSC-EC were fabricated using 12-series micro-molds (MicroTissues Inc.). Cells’ organization within the spheroids (CellTracker dyes) and viability (live/dead assay) were assessed at day-1, 7 and 14. Microtissue-spheroids 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 prevascularisation (Immunohistochemistry for CD31). Microtissues were inserted into the canal space of tooth-root fragments and implanted into the subcutaneous space on the back of 6- 8-week-old female severe combined immunodeficient (SCID) mice. 2-4 weeks after the transplantation, the mice were euthanized and the tooth fragments were removed for histological (Haematoxylin and eosin) and immunohistochemical (human mitochondria, CD31, DSP) analysis. Experiments were conducted in triplicate using DPSCs from three different donors and statistically analysed (ANOVA). Results: DPSCs and ECs were self-aggregated into spheroids with no evidence of cell death at the centre. ECs were organized into a dense-network of tubular-like structures throughout the DPSC:EC-microtissues. Elevated levels of ALP, BSP and RUNX2 (p < 0.05) in DPSC:EC-microtissues compared to DPSC-alone-microtissues confirmed that ECs enhanced osteo/odontogenic differentiation in 3D. Both DPSC-alone and DPSC:EC groups showed vascularised pulp-like tissue with an odontoblast-like cell layer adjacent to the dentin after transplantation in SCID mice. DPSC-EC-microtissue groups showed a significantly higher amount of extracellular matrix, vascularisation and mineralization compared to DPSC-alone-microtissues both in-vitro and in-vivo. Positive staining for antibodies against human mitochondria confirmed the contribution of transplanted microtissues in regenerated pulp-like tissue and vasculature. Conclusion: DPSCs and ECs synergistically enhance osteo/odontogenic differentiation and vascularisation in 3D in-vitro and regenerate vascularised pulp-like tissue in-vivo.