The involvement of dental pulp stem cells in inflammation and regeneration

Abstract

Following infection or trauma, dental pulp tissue maintains, defends and repairs itself; furthermore, this tissue tries to regenerate the damaged/lost dentin/pulp complex. Under these circumstances, dental pulp stem cells (DPSCs), a subpopulation of pulpal cells, have the potential to proliferate and differentiate into odontogenic lineage during repair and regeneration processes. Furthermore, inflammation causes accumulation of DPSCs at the inflamed sites by upregulation of proinflammatory cytokines, such as tumor necrosis factor alpha (TNF-α) and interleukin-1beta (IL-1β). On the other hand, various growth factors and cytokines regulate the recruitment and differentiation of DPSCs during angiogenesis under inflammatory stimuli, such as vascular endothelial growth factor (VEGF) or stromal derived factor 1 alpha (SDF-1α). This study aimed to investigate the potential roles of TNF-α, VEGF and SDF-1α in regulating DPSCs during reactionary/reparative dentin formation and pulp regeneration. TNF-α is expressed in gradient concentrations in dental pulp tissue during the pulpitis process, which may promote stem cells’ migration to injured site and odontogenesis in an appropriate concentration. Once the signaling of EphB/ephrin-B is activated, DPSCs may be driven from the perivascular niche of pulp tissue, and subsequently migrate to the injury area. However, the effects of TNF-α on DPSCs in dentin biomineralization and repair remained unclear. Therefore, the present study investigated whether TNF-α could regulate EphB2/ephrin-B1 signaling in DPSCs, and how this process may operate at a molecular level. DPSCs could upregulate the mRNA and protein expression of EphB2/ephrin-B1 by stimulating with TNF-α. Both TNF receptor 1 (TNFR1) and TNF receptor 2 (TNFR2) were involved in regulating the cellular mRNA activities partially at protein level. TNF-α induced EphB2/ephrin-B1 mRNA expression was c-Jun N-terminal kinases (JNK)-dependent signaling. It is known that cytokines also plays an active role in mediating the pro-angiogenesis of DPSCs. Pulp tissue could be regenerated by combining DPSCs, scaffolds and growth factors if a sufficient blood supply into root canals is secured. Therefore, vascularization is vital for the survival of cells after implantation in pulp regeneration in vivo. However, it is not clear how the angiogenic factors, VEGF and/or SDF-1α act on regenerative potential in DPSCs. In this study, the vascularization potential of DPSCs transfected with VEGF or SDF-1α was investigated in vitro and in vivo. After gene modification, reverse transcription polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA) and western blot analysis verified stable gene and protein overexpression. Transfected DPSCs have higher cell proliferation and enhance HUVECs migration and tube formation in vitro. The root segments mixed with gene-modified DPSCs and PuraMatrix were implanted in severe combined immunodeficient (SCID) mice and retrieved after four weeks. The histology and immunohistochemistry results demonstrated that the mixture of DPSCs transfected with VEGF and SDF-1α enhance the volume of newly vascularized pulp-like tissue in pulp regeneration in vivo. In conclusion, the current findings suggest that upregulation of TNF-α-stimulated EphB2/ephrin-B1 expression in DPSCs through the JNK-dependent signaling pathway. The combination of VEGF-transfected and SDF-1α-transfected DPSCs can enhance the sizable formation of pulp-like tissues. This study enhances our understanding on the molecular mechanism of pulp regeneration.