Ephrin-B2 signaling regulates angiogenesis in dental pulp regeneration

Abstract

The success of dental pulp regeneration by transplantation of bioengineered pulp constructs largely depends upon i) the development of vascular endothelial tubule network (angiogenesis), and ii) penetration and connection of host vasculature with the bioengineered pulp replacement (anastomosis). Currently, a major challenge in dental pulp regeneration is the small apical opening below 1 mm in diameter that allows only a microcirculatory transportation system. Therefore, it is critical to develop novel approaches to achieve rapid anastomosis and enhance functional angiogenesis via integration of in vitro-bioengineered tissue and host vasculature. The critical molecules that participate in the formation of blood vessels include vascular endothelial growth factor (VEGF), angiopoietin-1 (Ang1), and ephrin-B2/EphB4, and therefore their genes are the therapeutic targets for enhancing the approaches to pulp regeneration. In this study, we aimed to identify the specific role and function of ephrin-B2 in angiogenesis, and develop a new approach to accelerating angiogenesis in pulp regeneration by modulating ephrin- B2 expression in stem cells from apical papilla (SCAPs). SCAPs were isolated from freshly extracted human third molars with immature roots (aged 18–25 years). Prior to using SCAPs for the experiments, freshly isolated cells were assessed for their “stemness” by flow cytometric analysis, indicating that SCAPs could strongly express CD73 and CD90, and weakly express CD45 and CD105. 26.35% of the cells expressed STRO-1. Multilineage differentiation assay further demonstrated that SCAPs enabled to differentiate into osteogenic, odontogenic, adipogenic and neurogenic cells. Tube formation assays showed that SCAPs cocultured with human umbilical vein endothelial cells (HUVECs) in Matrigel had increased tubule quantity, tubule length and branching points as compared with SCAPs or HUVECs alone. Notably, delayed addition of SCAPs to the established vessel-like structures of HUVECs could stabilize the immature neo-capillaries. In vitro experiments indicated that the VEGF secreted by SCAPs under hypoxia was metabolized by HUVECs to accelerate tube formation in the early stage of sprouting angiogenesis. In addition, ephrin-B2 expression in SCAPs induced endothelial cells (ECs) quiescence, and improved the stabilization and maturation of neo-capillaries. Interestingly, once SCAPs and HUVECs were co-seeded in Matrigel, phosphorylation of ephrin-B2 was activated instantly and peaked at 1 h. Silencing ephrin-B2 gene expression in SCAPs and HUVECs via siRNAs prevented the formation of vessel-like structures, suggesting that ephrin-B2 may play a critical role in angiogenesis. Our studies also found that overexpression of ephrin-B2 in SCAPs, by stable transfection with a lentiviral expression vector, significantly upregulated VEGF secretion. When cocultured with HUVECs, overexpression of ephrin-B2 in SCAPs resulted in early formation of vessel-like structures. Animal experiments confirmed that SCAPs co-transplanted with HUVECs enabled to generate greater amount of blood vessels than SCAPs alone. Furthermore, ephrin-B2- overexpressing SCAPs produced increased number of blood vessels with reference to GFP-SCAPs, and those co-transplanted with HUVECs generated vessels with larger tubule volumes. In conclusion, co-transplantation of SCAPs and ECs into root canals may accelerate vasculature formation. Overexpression of ephrin-B2 signaling in SCAPs generates functional neo-capillaries with greater vessel volumes. Ephrin- B2 signaling could be utilized to enhance dental pulp regeneration by virtue of accelerating angiogenesis.