Abstract
Post-implantation cell survival and angio-/vasculogenesis are critical for the success of cell-based regenerative strategies. The current study aimed to overexpress B-cell lymphoma 2 (Bcl-2) gene in dental pulp stem cells (DPSCs) and examine the anti-apoptotic and angio-/vasculogenic effects both in-vitro and in-vivo. DPSCs were transduced with Bcl-2-green fluorescent protein (GFP) lentiviral particles and examined for cell proliferation and apoptosis. The cells were cultured under normoxic or hypoxic (0.5 mM CoCl2) conditions and examined for the expression of angiogenic factors and effects on endothelial cell proliferation, migration and vessel morphogenesis. Cells with or without hypoxic preconditioning were used in in-vivo Matrigel plug assay to study the post-implantation cell survival and angio-/vasculogenesis. Bcl-2-overexpressing-DPSCs showed significantly lower apoptosis than that of null-GFP-DPSCs under serum-free conditions. Under hypoxia, Bcl-2-overexpressing-DPSCs expressed significantly higher levels of vascular endothelial growth factor compared to that under normoxia and null-GFP-DPSCs. Consequently, Bcl-2-overexpressing-DPSCs significantly enhanced endothelial cell proliferation, migration and vascular tube formation on Matrigel. Immunohistological assessment of in-vivo transplanted Matrigel plugs showed significantly higher cell survival and vasculature in hypoxic preconditioned Bcl-2-overexpressing-DPSC group compared to null-GFP-DPSC group. In conclusion, Bcl-2 overexpression and hypoxic-preconditioning could be synergistically used to enhance post-implantation cell survival and angio-/vasculogenic properties of DPSCs.
Highlights
Dental pulp stem cells (DPSCs) are considered a promising population of stem cells in regenerative medicine for their ready availability and regenerative potential [1,2]
Western blotting assay confirmed the overexpression of B-cell lymphoma 2 (Bcl-2) in transduced DPSCs (Figure 1A) compared to that of null-green fluorescent protein (GFP) control and wild type groups, in which the expression was hardly detectable
No significant difference in cell proliferation was observed in Bcl-2-DPSCs in comparison to that of GFP-DPSCs (Figure 2A) as shown by the Cell Counting Kit-8 (CCK-8) assay
Summary
Dental pulp stem cells (DPSCs) are considered a promising population of stem cells in regenerative medicine for their ready availability and regenerative potential [1,2]. The survival and successful engraftment of engineered cellular/tissue constructs of DPSCs following implantation depends on adequate and timely supply of oxygen and nutrients and on the efficiency of removal of toxic waste [3]. The success of a tissue engineering strategy strictly relies on the rate of vascularization of the cellular/tissue construct mainly through angiogenesis. Cells have adapted to trigger an angiogenic response when they are faced with hypoxia or ischemia [5], which is mainly through secretion of vascular endothelial growth factor (VEGF) mediated by hypoxia inducible factors (HIFs) [6,7,8,9]. VEGF is one of the most potent angiogenic factors secreted in response to hypoxia [10,11,12]. It was shown that VEGF could induce the ingrowth of blood vessels into the ischemic tissues in both physiological and pathological conditions [9]
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