Fig 2: Intrinsic cellular inflammation induces Rbpjk via downregulation of Dnmt3b, leading to progenitor differentiation impairments and fracture repair defects.(A) Schematic demonstration of previously established NF-κB/Dnmt3b/Rbpjk axis in regulating fracture repair. Dashed line: Relationship established by in vitro experiments. Solid line: Relationship established in mice. Dashed circle: Role of the gene established in vitro. Solid circle: Role of the gene established in mice. (B) Chondrogenic pellet and osteogenic differentiation assays were performed within control and Ikk2ca PPCs in the presence or absence of Lenti-Dnmt3b (Dnmt3b GOF) viral infection. Alcian blue and alizarin red staining of chondrogenic pellet sections and osteogenic cultures on day 28 and day 21, respectively (n = 3). (C) ABH/OG staining of fracture callus sections from Ikk2cafl/+ (control), Prx1CreERT2;Ikk2cafl/+ (Ikk2caPrx1), Prx1CreERT2;Rosa-rtTAfl/+;Dnmt3b-Tg (Dnmt3b-tgPrx1), and Prx1CreERT2;Ikk2cafl/+;Rosa-rtTAfl/+;Dnmt3b-Tg (Ikk2ca;Dnmt3b-tgPrx1) mice at 7 and 10 dpf (n = 5). Scale bars: 100 mm. (D) ABH/OG staining of fracture callus sections from Dnmt3bfl/fl (control), Prx1CreERT2;Dnmt3bfl/fl (Dnmt3bPrx1), Prx1CreERT2;Rbpjkfl/fl (RbpjkPrx1), and Prx1CreERT2; Dnmt3bfl/fl;Rbpjkfl/fl (Dnmt3b;RbpjkPrx1) mice at 7 and 10 dpf (n = 5). Scale bars: 100 mm.

Fig 3: Rbpjk ablation abrogates fracture nonunion of RA mice.(A) ABH/OG staining of fracture callus sections from Rbpjkfl/fl (control) and Prx1CreERT2;Rbpjkfl/fl (RbpjkPrx1) RA mice at 7, 10, and 14 dpf (n = 5). Scale bars: 100 mm. (B) Histomorphometric measures of mesenchyme, cartilage, and bone areas based on ABH/OG staining (n = 5). (C) Micro-CT assessment of mineralized bony calluses of control and RbpjkPrx1 RA mice at 14 dpf (n = 5). Scale bars: 0.5 mm. (D) Bony callus volume and BV/TV quantifications on micro-CT analyses of control and RbpjkPrx1 RA fractures at 14 dpf (n = 5). (E) Biomechanical torsion testing of control and RbpjkPrx1 RA fractures at 28 dpf. Maximum torque and displacement at maximum torque were recorded during testing (n = 5). Data presented as mean ± SD. *P < 0.05 by 2-tailed Student’s t test.

Fig 4: Inflammation induces Rbpjk expression through Dnmt3b-mediated DNA methylation reduction.(A) Methylation qPCR for the Rbpjk promoter region in Ikk2cafl/+ PPCs following Ad-GFP (control) or Ad-Cre (Ikk2ca) transfection (n = 3). (B) IHC for Dnmt3b on callus sections from Ikk2cafl/+ (control) and Prx1CreERT2;Ikk2cafl/+ (Ikk2caPrx1) mice at 10 dpf (n = 5). Scale bars: 100 mm. (C) Real-time qPCR analyses of gene expression for Dnmt1, Dnmt3a, and Dnmt3b in control and Ikk2ca PPCs (n = 3). (D) Real-time qPCR analyses for Rbpjk within control and Ikk2ca PPCs in the presence or absence of Lenti-Dnmt3b (Dnmt3b GOF) viral infection (n = 3). (E) Schematic representing the selection of C3H10T1/2 cell lines modified by the dCas9-Dnmt3a epigenetic editing system specifically targeting CpG islands of the Rbpjk gene. (F) Western blot analyses for Rbpjk in protein extracts from dCas9-Dnmt3a-scramble gRNA (control) and 12 individual dCas9-Dnmt3a-Rbpjk gRNA-engineered C3H10T1/2 cell lines (n = 3). Lane C: dCas9-Dnmt3a-scramble gRNA. Lanes 1–10: dCas9-Dnmt3a-Rbpjk1-1-10 gRNAs targeting CpG island 1 of the Rbpjk gene. Lanes 11 and 12: dCas9-Dnmt3a-Rbpjk2-1-2 gRNAs targeting CpG island 2 of the Rbpjk gene. (G) Chondrogenic pellet and osteogenic differentiation assays were performed within dCas9-Dnmt3a-scramble (control) and dCas9-Dnmt3a-Rbpjk1-9 C3H10T1/2 cell lines in the presence or absence of IL-1β. Alcian blue and alizarin red staining of chondrogenic and osteogenic cultures on day 28 and day 21, respectively (n = 3). Data expressed as mean ± SD. *P < 0.05 determined by 2-tailed Student’s t test (A and C) or by 2-way ANOVA followed by Tukey’s test (D).

Fig 5: Rbpjk inhibition by epigenetic modification using CRISPR/dCas9/Dnmt3a editing system rescues fracture nonunion in RA mice.(A) PCL scaffold with dCas9-Dnmt3a–engineered C3H10T1/2 cells (blue arrow) was applied to the fractured bone in RA mice. (B) ABH/OG staining of callus sections from 14-dpf wild-type RA fractures grafted with PCL scaffolds that were fabricated with dCas9-Dnmt3a-scramble (control) or dCas9-Dnmt3a-Rbpjk1-9 C3H10T1/2 cells (Rbpjk epigenetically modified) (n = 5). Scale bars: 100 mm. (C) Histomorphometric quantifications of bone area based on ABH/OG staining (n = 5). (D) Micro-CT analyses of newly formed bony callus from 14-dpf wild-type RA fractures grafted with PCL scaffolds that were fabricated with control or Rbpjk epigenetically modified C3H10T1/2 cells (n = 5). Scale bars: 0.5 mm. (E) Bony callus volume and BV/TV measures of 14-dpf wild-type RA fractures grafted with PCL scaffolds fabricated with control or Rbpjk epigenetically modified C3H10T1/2 cells (n = 5). (F) Biomechanical torsion testing of 28-dpf wild-type RA fractures grafted with PCL scaffolds fabricated with control or Rbpjk epigenetically modified C3H10T1/2 cells. Maximum torque and displacement at maximum torque were recorded during testing (n = 8). Data presented as mean ± SD. *P < 0.05 by 2-tailed Student’s t test.