Prevascularized Three-Dimensional DPSC Constructs in Dental Pulp Regeneration

Abstract

Objectives: To co-culture dental pulp stem cells (DPSCs) and human umbilical vein endothelial cells (HUVECs) in an injectable, peptide hydrogel scaffold: PuraMatrix while fabricating a vascular network in-vitro and to utilize this construct in pulp regeneration in-vivo. Methods: DPSCs and HUVECs were encapsulated in PuraMatrix (BD-Biosciences) as mono-cultures or co-cultures at different ratios (3:1, 1:1 1:3). Viability, morphology and three-dimensional organization of cells within PuraMatrix were assessed over 2-weeks under confocal microscopy. Cell-PHS constructs were induced for odonto/osteogenic differentiation (up to 21-days); and examined for alkaline phosphatase (ALP) activity and mineralization (von-Kossa staining). Cell-encapsulated PuraMatrix-constructs were injected into the canal space of full-length tooth-roots and implanted into the subcutaneous space of 6-8-week-old female severe combined immunodeficient mice. Two-to-four weeks after transplantation, mice were euthanized and tooth-roots were removed for histological (Haematoxylin and eosin) and immunohistochemical (human mitochondria, CD31) analysis. Experiments were conducted in triplicate using DPSCs from three different donors and analysed statistically (ANOVA).

Results: Results showed that both DPSCs and HUVECs survived well in co-cultures compared to monocultures. HUVECs, when co-cultured with DPSCs formed a vessel-like network throughout the PuraMatrix compared to HUVEC-monocultures where not only HUVECs failed to form any vessels but also underwent apoptosis. ELISA-assay revealed that DPSCs secrete VEGF in high amounts inhibiting apoptosis and promoting vessel formation by HUVECs. Higher ALP activity and mineralization were observed in co-cultures compared to monocultures (p<0.05). Both DPSC-monoculture and co-culture groups showed vascularised pulp-like tissue with patches of osteodentin after transplantation in mice. Co-cultured groups showed higher amounts of extracellular matrix, vascularisation and mineralization compared to DPSC-monocultures in-vivo. Immunohistochemistry for human mitochondria confirmed the contribution of transplanted cells in regenerated pulp-like tissue and vasculature.

Conclusion: DPSCs and HUVECs being encapsulated within PuraMatrix demonstrate synergistic effects in odontogenic differentiation and angiogenesis and have potential for engineering vascularised pulp tissues in-vivo.