Hypoxia enhances osteoclastogenesis in periodontal ligament cells via expression of VEGF and RANKL

Introduction

Periodontal ligament (PDL) damage caused by dental trauma can lead to local circulatory disorders. The mechanisms through which PDL cells, once exposed to a transient hypoxic environment, contribute to tissue regeneration or resorption of pathological tooth roots after reoxygenation remain unclear. Therefore, we aimed to examine how changes in oxygen (O₂) concentration affect PDL healing.

Materials and methods

Human PDL stem cells (hPDL cells) were cultured under normoxic or hypoxic (20% or 1% O₂ concentration) conditions. Vascular endothelial growth factor (VEGF) and receptor activator of nuclear factor kappa-Β ligand (RANKL) expressions were measured using real-time quantitative polymerase chain reaction or Western blotting. Furthermore, a co-culture of hPDL and osteoclast precursor cells was used to demonstrate the effect of changes in O₂ concentration on osteoclast formation.

Results

VEGF expression considerably increased over time under hypoxia compared with normoxia. However, during reoxygenation (24 h hypoxia–24 h normoxia), expression markedly decreased under hypoxia. No significant difference in RANKL expression was observed in both conditions after 24 h; however, it remarkably increased under hypoxia compared with normoxia after 48 h. In the osteoclast formation assay, the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells considerably increased over time under hypoxia compared with normoxia. Notably, when VEGF expression was reduced using small interfering RNA, the number of TRAP-positive multinucleated cells decreased extensively.

Conclusion

Under hypoxic conditions, periodontal ligament cells produce VEGF to promote angiogenesis. However, excessive VEGF production, along with RANKL production, induces osteoclast formation. Osteoclast formation can be suppressed using rapid reoxygenation.