Synergistic effects of dental pulp stem cells and endothelial cells in pulp regeneration

Abstract

Regeneration of the tissues to replace diseased, missing and traumatized dentin/pulp requires combining the recent progress in stem cell and tissue engineering research. Dental pulp stem cells (DPSCs) are considered as a promising population of cells in regenerative dentistry and shown to be able to produce dentin/pulp-like tissues following implantation in-vivo. Securing a good blood supply is critical in pulp regeneration, however, this is a challenging task due to the unique structure of the tooth, the anatomy of which permits only a microcirculatory system via a very small apical opening (<0.3-1mm). This limitation raises the need to develop novel methods to enhance angiogenesis during pulp regeneration. It was shown that DPSCs reside in the microvasculature region of the dental pulp and interact with perivascular cells. Therefore, endothelial cells could be a major source of modulators of pulp-dentin development and angiogenesis. If a pulp tissue substitute with pre-formed endothelial network could be engineered in-vitro, it would not only gain rapid anastomosis with host vasculature but also regulate DPSC function in pulp regeneration. In this study, for the first time, synergistic effects of DPSCs and human umbilical vein endothelial cells (HUVECs) on osteo/odontogenic differentiation and angiogenesis were investigated using two-dimensional and three-dimensional direct co-culture systems. Furthermore, the potential of three-dimensional DPSC constructs prevascularized with HUVECs in dental pulp regeneration in-vivo was exmined. HUVECs promoted odonto/osteogenic differentiation of DPSCs in direct two-dimensional co-cultures in-vitro. Further, addition of DPSCs stabilized the pre-existing vessel-like structures formed by HUVECs and increased the longevity of these structures on matrigel in-vitro. Using two different systems, scaffold-free self-assembling microtissue spheroids and peptide hydrogel scaffold, the interactions of DPSCs and HUVECs in three-dimensional cultures were investigated. The results demonstrated that DPSCs can self assemble into three-dimensional microtissue spheroids when cultured alone or with HUVECs. DPSCs promoted survival and vascular structure formation by HUVECs both in scaffold-free microtissue spheroids and peptide hydrogel scaffold. In contrast, HUVECs, when cultured alone, neither formed vascular structures nor survived in either of the 3D systems. The latter phenomenon was attributable to vascular endothelial growth factor secreted by DPSCs, a major factor responsible for endothelial function. Co-cultures also showed enhanced odonto/osteogenic differentiation in both three-dimensional microtissue spheroid and peptide hydrogel scaffold systems. Following implantation of tooth-root fragments filled with three-dimensional DPSC constructs into the subcutaneous space of immunodefficient mice, vascularised pulp-like tissue was regenerated within the root canals. Compared to DPSC-only group, DPSC/HUVEC co-culture groups showed higher vascularisation, extracellular matrix formation and mineralization in regenerated tissue. More importantly, HUVEC-lined vascular lumens were observed in regenerated tissues suggesting the successful integration of in-vitro formed pre-vascular structures to the host vasculature. In summary, the findings suggest that DPSCs and HUVECs display significant synergy during odonto/osteogenic differentiation and angiogenesis when co-cultured either in two-dimensional or three-dimensional culture systems. Unravelling these fundamental behavioural patterns of DPSCs provides novel insights into the process of pulp regeneration, leading to new avenues for more effective therapies in regenerative endodontics.