HIF-1α Stabilization Promotes Post-Implantation Cell Survival During Dental Pulp Regeneration

Abstract

Objectives: To promote dental pulp regeneration by ameliorating post-implantation survival of stem cells from human exfoliated deciduous teeth (SHED) through hypoxia-inducible factor-1alpha (HIF-1α) stabilization. Methods: HIF-1α was stabilized in SHED by knocking-down prolyl-hydroxylase domain-containing protein-2 (PHD2) using target-specific shRNA lentiviral particles in-vitro. Subsequently, HIF-1α-stabilized-SHED (H1αSS) cell viability under normoxia and hypoxia (1%O2) was assessed by CCK-8 assay, apoptosis assay, and ki67 staining. Cellular and mitochondrial ROS levels were evaluated by CM-H2DCFDA and MitoSOX staining. HIF-1α downstream genes involved in ROS homeostasis and glycolysis were examined by western-blotting and RT-PCR. In-vivo experiment, H1αSS and Control-SHED (CS) were encapsulated in 0.5% PuraMatrixTM hydrogel, injected into root canals of tooth fragments, and implanted subcutaneously in SCID mice. After 7 and 28 days, implants were retrieved, and histological and immunofluorescent staining were performed to evaluate cell viability and dental pulp-like tissue. Results: HIF-1α was successfully stabilized in SHED after knockdown of PHD2 with a 90-95% efficiency. HIF-1α stabilization increased viability of SHED under hypoxia (P<0.05), which could be attributed to reduced cell apoptosis and ROS levels (P<0.05). RT-PCR and western-blotting results showed a significantly increased expression of genes [PDK1, HK2, and Glut1 (P<0.05)] involved in ROS maintenance and glycolysis in H1αSS compared to CS. In-vivo, at 7 days post-implantation, staining for DNA damage and ki67 demonstrated significantly lower cell apoptosis and higher cell proliferation in H1αSS (P<0.05), respectively. Histological analysis revealed that H1αSS regenerated a pulp-like tissue with significantly higher number of vessels with larger vascular lumens (P<0.05). Immunofluorescent staining demonstrated that HIF-1α stabilization contributed to endothelial differentiation (human-CD31 and human-vWF) along with host vessel recruitment (mouse-CD31) and odonto-/osteogenic differentiation (DSPP) of SHED resulting in augmentation of dental pulp-like tissue and dentin, respectively (P<0.05). Conclusions: HIF-1α stabilization in SHED promotes dental pulp regeneration by enhancing post-implantation cell survival through modulating cell metabolism.