Three-Dimensional Microtissue Spheroids of Dental Pulp Stem Cells and Endothelial Cells in Vascularised Pulp Regeneration

Abstract

Objectives: To fabricate the microtissue spheroids of dental pulp stem cells (DPSCs) and human umbilical vein endothelial cells (HUVECs); and to examine the potential of these three-dimensional spheroids in vascularised pulp regeneration. Methods: Three-dimensional microtissue-spheroids of DPSC-alone and DPSC-HUVEC cocultures were fabricated using 12-series micro-molds (MicroTissues Inc.). Cellular organization within the spheroids (CellTracker dyes) and cell 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 slices and implanted into the subcutaneous space on the back of 6-8-week-old female severe combined immunodeficient (SCID) mice. Four weeks after the transplantation, the mice were euthanized and the tooth fragments were removed for histological (Haematoxylin and eosin) and immunohistochemical (human mitochondria, human CD31, Human Nestin antibodies) analysis. Experiments were conducted in triplicate using DPSCs from three different donors and statistically analysed (ANOVA). Results: DPSCs and HUVECs were self-aggregated into spheroids with no evidence of cell death at the centre. HUVECs were organized into a dense-network of tubular-like structures throughout the DPSC:HUVEC co-cultured microtissues. Elevated levels of ALP, BSP and RUNX2 (p < 0.05) in DPSC:HUVEC microtissues compared to DPSC-alone microtissues confirmed that HUVECs enhanced osteo/ odontogenic differentiation. Both DPSC-alone and DPSC: HUVEC groups showed vascularized pulp-like tissue with an odontoblast-like cell layer adjacent to the dentin after transplantation in SCID mice. DPSC-HUVEC 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. DPSCs and HUVECs in three-dimensional microtissue spheroids synergistically enhance osteo/odontogenic differentiation and angiogenesis in-vitro. These prevascularized microtissue spheroids can successfully regenerate vascularised pulplike tissue in-vivo.