Peptide Hydrogel Scaffold Promotes Angiogenesis And Osteo/Odontogenic Differentiation In-Vitro

Abstract

Objectives: To investigate cell viability, vascular network formation and differentiation of mono- or co-cultured human dental pulp stem cells (DPSCs) and human umbilical vein endothelial cells (HUVECs) within a three-dimensional peptide hydrogel scaffold (PHS). Methods: Monocultures and combinations of co-cultures (3:1, 1:1, 1:3) of DPSCs and HUVECs were encapsulated in different concentrations (0.5%, 0.25%, 0.15%) of PHS (BD-Biosciences, Bedford, MA). DPSCs and HUVECs were transfected with green fluorescent protein and red fluorescent protein respectively using pre-made lentiviral particles (GenTarget Inc, San Diego, CA) before encapsulation. Cellular morphologies and 3-dimensional organization of cultures within PHS were monitored using confocal microscopy. Cell viability was assessed using a live/dead viability assay kit at day 7 and 14. Cell cultures within PHS were induced for odonto/osteogenic differentiation (up to 21-days), examined for alkaline phosphatase (ALP) activity (ALP quantification assay) and mineralization (Von-Kossa staining). Experiments were conducted in triplicate using DPSCs from three different donors and statistically analysed (ANOVA).

Results: Live/dead assay revealed 0.15% as the optimum PHS concentration with a significantly high survival of HUVECs compared to 0.5% and 0.25% PHS. In monocultures, DPSCs survived and grew faster than HUVECs. In co-cultures both cells survived well and HUVECs formed an extensive vessel-like network throughout the PHS compared to HUVEC monocultures where it failed to form any vessel-like structures. This finding suggested that co-culture inhibits HUVECs apoptosis and secretes angiogenic factors to promote vessel-like network formation. At higher endothelial cell counts, the density of vessel-like structures was low and DPSC:HUVEC 3:1 was found as the optimum ratio. ALP activity of cells in co-culture was higher than that of DPSCs in monocultures (p<0.05). Despite the ratio, all the co-cultures showed higher amount of mineralization compared to monocultures.

Conclusion: These findings indicate that PHS promotes angiogenesis and osteo/odontogenic differentiation and has potential for engineering vascularised-pulp tissues.