Diminished RELB impairs osteogenesis in mature periodontal ligament stem cells

Although periodontal ligament stem cells (PDLSCs) are pivotal for periodontal tissue regeneration, their regenerative capacity diminishes with the PDL maturity. To elucidate the mechanisms underlying this phenomenon, we performed single-cell RNA sequencing (scRNA-seq) on immature and mature human PDL tissues to characterize the heterogeneity and predict critical regulators affecting osteogenic capacity of PDLSCs. Subsequently, the predicted role of RELB was experimentally validated both in vitro and in vivo using small interfering RNA. The scRNA-seq analysis revealed significant heterogeneity within the PDLSC population. Notably, PDLSC-5 subtype, which exhibited osteogenic potential and positioned at the terminus of the pseudotime trajectory, was markedly reduced in mature PDL tissues. RELB was specifically overexpressed in PDLSC-5 and identified as a key transcription factor (TF) for this subcluster. Knockdown of RELB significantly inhibited the osteogenic differentiation capacity of PDLSCs in vitro and bone formation potential in vivo at both developmental stages. This inhibitory effect appears to be mediated, at least in part, through the downregulation of its target gene, superoxide dismutase 2 (SOD2). Our findings demonstrate that the proportion of PDLSCs with osteogenic ability significantly declines with maturity, accounting for the diminished regeneration capacity of mature periodontal tissue. Furthermore, RELB was identified as a crucial TF for maintaining the osteogenic potential of PDLSCs. These results provide a theoretical basis for strategies aimed at optimizing the bone regeneration capacity of PDLSCs.