Diminished osteogenic capacity in mature periodontal ligament stem cells via RELB downregulation

Although periodontal ligament stem cells (PDLSCs) are pivotal for periodontal tissue regeneration, their regenerative capacity diminishes with the PDL maturity. This study aims to elucidate the mechanisms underlying this phenomenon.Single-cell RNA sequencing (scRNA-seq) was conducted 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.Our 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).This study demonstrates 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 findings provide a theoretical basis for strategies aimed at optimizing the bone regeneration capacity of PDLSCs. For orthodontic reasons, human premolars without endodontic or periodontal disease were extracted at the Stomatology Hospital of Xi’an Jiaotong University and used for PDL isolation. All donors signed informed consent forms. According to the root developmental stage and donor age, the obtained teeth were divided into 2 groups, one group with 1/2-2/3 root development was called immature group, and the other group with complete root development was named mature group. PDL tissues from the root surface were collected for PDLSCs culture and scRNA-seq.After enzymatic digestion and lysis of red blood cells, the cell suspension of PDL tissues were filtered and counted. Libraries were constructed using Single-Cell 3′ Reagent Kit v3.1 (10× Genomics) according to the manufacturer’s protocol. Indexed libraries were pooled and sequenced on an Illumina sequencer using paired-end 26×98 bp read length.After non-linear dimensional reduction using uniform manifold approximation and projection (UMAP), clustering was run using the FindNeighbors and FindClusters functions with the SLM algorithm and 27 interactions. Clustering was performed at varying resolution values with a value of 1.5 used as the initial stage. Clusters were assigned preliminary identities based on the expression of combinations of known marker genes for major cell classes and types. Subclustering was performed on each major cell type data subset to resolve additional cell types and subtypes.