Endothelial cells regulate the functions of dental pulp stem cells in stabilizing the newly formed blood vessels

Abstract

Timely re-establishment of functional blood circulation is critical for dental pulp regeneration. Prevascularization is one of the most promising approaches to accelerate vasculature formation within engineered tissues. Due to the multicellular structure of blood vessels, the strategies to co-transplant two or more types of cells, for example, the combination of endothelial cells (ECs) and mesenchymal stem cells (MSCs), stromal/mural cells, or fibroblasts, have raised more attention in the field of vascular tissue engineering. Dental pulp stem cells (DPSCs), which reside in the perivascular niche of the dental pulp tissue, can function as pericyte-like cells to extend the longevity of vessels when cocultured with ECs in vitro. Still, the mechanisms of DPSCs in stabilizing blood vessels are poorly understood. Therefore, in the first part of this study, DPSCs were treated with transforming growth factor beta 1 (TGF-β1) for 7 days (T-DPSCs), and the properties and functions of T-DPSCs were analyzed by functional contraction and 3D co-culture spheroidal sprouting assays. The results showed that T-DPSCs possess smooth muscle cell (SMC) properties and resemble pericyte-like cells to enclose human umbilical vein ECs (HUVECs) in the 3D sprouting model. Furthermore, T-DPSCs enhance the stabilization of blood vessels through Ang1/Tie2 and VEGF/VEGFR2 signaling pathways. Perivascular cells and ECs play critical roles in the angiogenic process. Therefore, a thorough understanding of their dynamic cell-cell interactions is vital for developing innovative strategies for vascular tissue engineering. In the second part of this study, the interactions of HUVECs and DPSCs in an in vitro coculture model were explored. The results suggested that HUVECs induce DPSC differentiation into SMCs in HUVEC+DPSC direct coculture. Furthermore, TGF-β1 was responsible for DPSC differentiation into SMCs in HUVEC+DPSC cocultures, and TGF-β1/ALK5 signaling pathway played a vital role in this process. In the third part of this study, the functions of E-DPSCs, which were DPSCs isolated from HUVEC+DPSC direct cocultures, and T-DPSCs on vascular stabilization were explored in vitro and in vivo. Functional contraction and 3D co-culture spheroid sprouting assays were conducted to assess the properties of E-DPSCs and T-DPSCs. Dental pulp angiogenesis in the SCID mouse model was used to explore the roles of E-DPSCs and T-DPSCs in vascularization in vivo. The results demonstrated that both E-DPSCs and T-DPSCs possess SMC properties, exhibiting the higher expression of SMC-specific markers, greater contractility, and the suppression of HUVEC sprouting. More importantly, E-DPSCs and T-DPSCs inhibited HUVEC sprouting through Ang1/Tie2/VE-cadherin and VEGF/VEGFR2 signaling pathways. In vivo study found more perfused and total blood vessels in the HUVEC+E-DPSC and HUVEC+T-DPSC groups, and the Angiopoietin1 (Ang1) and vascular endothelial protein tyrosine phosphatase (VE-PTP) inhibitor pretreatment groups. In summary, the current study demonstrated that HUVECs and TGF-β1 could induce DPSC differentiation into functional SMCs, and Ang1/Tie2/VE-cadherin and VEGF/VEGFR2 signaling pathways contributed to vascular stabilization induced by DPSCs. This study broadens our understanding of the angiogenic potential of DPSCs, which may open a new avenue for applying DPSCs in vascular tissue engineering for dental pulp regeneration.