DNA methylation mediated Rbpjksuppression protects against fracture nonunion caused by systemic inflammation
Challenging skeletal repairs are frequently seen in patients experiencing systemic inflammation. Current therapies aimed at mitigating inflammation or enhancing stem cell function with osteogenic factors yield inconsistent clinical outcomes. To tackle the complexity and heterogeneity of fracture repair process, we performed single-cell RNA sequencing and revealed that progenitor cell was one of the major lineages responsive to elevated inflammation and this response adversely affected progenitor differentiation by upregulation ofRbpjkin fracture nonunion.We then validated the interplay between inflammation (via Ikk2ca) and Rbpjkspecifically in progenitors by using genetic animal models. Focusing on epigenetic regulation, we identifiedRbpjkas a direct target of Dnmt3b. Mechanistically, intrinsic inflammation decreased Dnmt3b expression in progenitor cells, consequently leading toRbpjkupregulation via hypomethylation within its promoter region. We also showed thatDnmt3bloss-of-function mice phenotypically recapitulated the fracture repair defects observed inIkk2camice, whereasDnmt3btransgenic mice alleviated fracture repair defects induced byIkk2ca. Moreover,Rbpjkablation restored fracture repair in bothIkk2camice andDnmt3bloss-of-function mice. Altogether, this work elucidates a common mechanism involving NF-kB/Dnmt3b/Rbpjkaxis within the context of inflamed bone regeneration. Building upon this novel mechanistic insight, we applied local treatment with epigenetically modified progenitor cells in RA mice and showed a functional restoration of bone regeneration under inflammatory condition through an increase in progenitor differentiation potential. These findings underscore the promise of epigenetic-based therapeutic interventions for addressing delayed fracture union and nonunion particularly associated with systemic inflammation. Overall design: Four fracture calluses were dissociated by scraping newly formed tissue along the periosteal surface from injured bone as a pool for each experiment. Callus samples were further dissociated in 1 mg/ml Collagenase P at room temperature for one hour. Then, cell suspension was filtered through a diameter of 30-mm strainer to generate a single-cell suspension. Four batches of single cell libraries for sequencing: callus cells from 4-dpf control fractures (control_4d, n=4), 7-dpf control fractures (control_7d, n=4), 4-dpf RA fractures (RA_4d, n=4), 7-dpf RA fractures (RA_7d, n=4). 10,000 cells for each sample were loaded in aim of preparing single-cell mRNA libraries for each group, barcoded, purified using Chromium Single Cell 3' kit (v3.1 Chemistry, 10x Genomics Inc), and sequenced using 2 x 150 bp pair-end configuration on an Illumina NovaSeq platform.