Gene transfer of Ephrin-B2 into dental pulp stem cells enhances angiogenic potential

Abstract

Objectives: Dental pulp stem cells (DPSCs) have shown great promise as cell-based therapy for promoting angiogenesis during dental pulp tissue repair and regeneration, but utilization of stem cells alone is limited due to the insufficient production of proangiogenic cytokines after cell transplantation. Recent focus has been put on enhancing inherent angiogenic capacity of stem cells through genetically modifying them with overexpression of angiogenic factors. Molecules of the Eph/ephrin family are expressed during tooth morphogenesis and development and Eprhin-B2 and its receptor Eph-B4 have been demonstrated as key regulators in postnatal neovascularization and vessel maturation. In this study, we therefore aimed to develop genetically modified, Ephrin-B2 high-expressing DPSCs for promotion of stem cell-mediated neovascularization. Methods: Human DPSCs were transfected with Ephrin-B2/GFP using lentiviral vectors and the transfection efficiency was examined by GFP expression in cells under fluorescent microscopy. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and western blot were performed to verify the increased expression of Ephrin-B2 in DPSCs transfected with Ephrin-B2 (DPSCs/Ephrin-B2). Proliferation of DPSCs/Ephrin-B2 and DPSCs transfected with GFP (DPSCs/GFP) was determined and compared by WST-8 assay. The effect of overexpression of Ephrin-B2 on DPSCs to form capillary-like structures was tested using an in vitro matrigel angiogenesis assay and cells grew on matrigel were harvested for detection of expression of endothelial markers (VEGFR-2, CD31 and vWF) by qRT-PCR and western blot. Results: qRT-PCR and western blot showed significantly enhanced expression of Ephrin-B2 in DPSCs/Ephrin-B2 compared to DPSCs/GFP. DPSCs/Ephrin-B2 showed lower proliferation rate than DPSCs/GFP, as demonstrated by WST-8 assay. In vitro matrigel assay showed more capillary-like structure formation in DPSCs/Ephrin-B2 than DPSCs/GFP under endothelial cell culture condition. Conclusions: DPSCs overexpressing Ephrin-B2 could be a potential cell source for promoting angiogenesis in dental pulp tissue engineering.