IADR Abstract Archives
HIF-1α Stabilization Enhances Odonto/Osteogenic Properties of SHED in-Vitro and in-Vivo
Objectives: To explore the odonto/osteogenic potential of hypoxia inducible factor-1α (HIF-1α) stabilized stem cells from human exfoliated deciduous teeth (SHED).
Methods: HIF-1α stabilization in SHED was achieved by knocking down prolyl-hydroxylase domain-containing protein-2 (PHD2) using target-specific shRNA lentiviral particles. Knockdown efficiency was measured by real-time PCR and western blotting. Subsequently, odontogenic and osteogenic marker genes DSPP, DMP-1, ALP, and Runx2 expression in SHED were examined by real-time PCR and western blotting after odonto/osteogenic induction for 14 days. ALP and Alizarin red staining were performed 7 and 21 days after odonto/osteogenic induction to evaluate mineralization capacity. Furthermore, full-length human tooth roots were used in an ectopic pulp regeneration model in severe combined immunodeficient (SCID) mice to examine the capacity of HIF-1α stabilized SHED in regenerating pulp-dentin complex. Tetracycline (20 mg/kg/5 days, s.c.) was injected as a probe to detect new dentin formation. After 4 weeks of implantation, the samples were retrieved and examined for histology by hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC) for DSPP.
Results: PHD2 was knocked-down in SHED with a 90-95% efficiency and in turn, HIF-1α was stabilized under normoxia. PCR and western blotting results showed a significantly increased expression of odonto/osteogenic genes including DSPP, DMP1, ALP, and Runx2 in HIF-1α stabilized SHED (P<0.05) compared with the control-SHED. In addition, ALP and Alizarin red staining also demonstrated that HIF-1α stabilization resulted in higher mineralization capacity in SHED after odonto/osteogenic induction (P<0.05). In vivo, examination for the auto-fluorescence from tetracycline showed that HIF-1α stabilized SHED group had more dentin-like tissue formation on the interface between the tooth dentin and pulp-like tissue. H&E staining and IHC for DSPP indicated that more odontoblast-like cells could be observed along the dentin wall in HIF-1α stabilized group (P<0.05).
Conclusions: HIF-1α stabilization enhances the odonto/osteogenic potential of SHED both in-vitro and in-vivo.
Methods: HIF-1α stabilization in SHED was achieved by knocking down prolyl-hydroxylase domain-containing protein-2 (PHD2) using target-specific shRNA lentiviral particles. Knockdown efficiency was measured by real-time PCR and western blotting. Subsequently, odontogenic and osteogenic marker genes DSPP, DMP-1, ALP, and Runx2 expression in SHED were examined by real-time PCR and western blotting after odonto/osteogenic induction for 14 days. ALP and Alizarin red staining were performed 7 and 21 days after odonto/osteogenic induction to evaluate mineralization capacity. Furthermore, full-length human tooth roots were used in an ectopic pulp regeneration model in severe combined immunodeficient (SCID) mice to examine the capacity of HIF-1α stabilized SHED in regenerating pulp-dentin complex. Tetracycline (20 mg/kg/5 days, s.c.) was injected as a probe to detect new dentin formation. After 4 weeks of implantation, the samples were retrieved and examined for histology by hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC) for DSPP.
Results: PHD2 was knocked-down in SHED with a 90-95% efficiency and in turn, HIF-1α was stabilized under normoxia. PCR and western blotting results showed a significantly increased expression of odonto/osteogenic genes including DSPP, DMP1, ALP, and Runx2 in HIF-1α stabilized SHED (P<0.05) compared with the control-SHED. In addition, ALP and Alizarin red staining also demonstrated that HIF-1α stabilization resulted in higher mineralization capacity in SHED after odonto/osteogenic induction (P<0.05). In vivo, examination for the auto-fluorescence from tetracycline showed that HIF-1α stabilized SHED group had more dentin-like tissue formation on the interface between the tooth dentin and pulp-like tissue. H&E staining and IHC for DSPP indicated that more odontoblast-like cells could be observed along the dentin wall in HIF-1α stabilized group (P<0.05).
Conclusions: HIF-1α stabilization enhances the odonto/osteogenic potential of SHED both in-vitro and in-vivo.