Lepr+ PDL Cells Are Functionally Compensated during Aging and Bone Repair

Periodontal ligament fibroblastic cells (PDLCs) harbor skeletal stem/progenitor cells (SSPCs), which are essential for maintaining periodontal hard tissues. PDLCs expressing leptin receptor (Lepr) are SSPCs capable of differentiating into osteoblasts and cementoblasts. However, the mechanisms underlying their differentiation and the long-term maintenance of their progenitor potential remain unclear. To investigate the dynamics of Lepr ⁺ PDLCs, we performed single-cell RNA sequencing and long-term lineage tracing in Lepr-CreER ; ROSA26-tdTomato mice. Lepr ⁺ PDLCs consisted of two distinct subpopulations: Runx2 ⁺ and Runx2⁻ cells. The Runx2 ⁻/ Lepr ⁺ cluster expressed Thy1 , a proposed SSPC marker, but was not enriched for other markers, including Axin2 , Gli1 , and Acta2 . Trajectory analysis revealed that Runx2 ⁻/ Lepr ⁺ PDLCs differentiate into osteoblasts and cementoblasts through a Runx2 ⁺ / Lepr ⁺ intermediate state. Long-term lineage tracing showed an increase in Lepr ⁺ cell numbers within the periodontal ligament over 1 year. However, their contribution to osteocytes and cementocytes peaked between 2 and 6 months and subsequently declined. In a tooth extraction model, Lepr ⁺ PDLCs contributed to bone regeneration by differentiating into osteocytes, although they accounted for fewer than 3% of regenerated osteocytes. The genetic ablation of Lepr ⁺ cells via a non-inducible Cre line had no significant impact on the volume of regenerated bone. These findings suggest that while Lepr ⁺ PDLCs are essential contributors to hard tissue formation during development and repair, their function diminishes with age and other progenitor populations compensate for their functional decline during both physiological and regenerative processes.