NOTCH signaling in skeletal progenitors is critical for fracture repair

Abstract

Fracture nonunions develop in 10%-20% of patients with fractures, resulting in prolonged disability. Current data suggest that bone union during fracture repair is achieved via proliferation and differentiation of skeletal progenitors within periosteal and soft tissues surrounding bone, while bone marrow stromal/stem cells (BMSCs) and other skeletal progenitors may also contribute. The NOTCH signaling pathway is a critical maintenance factor for BMSCs during skeletal development, although the precise role for NOTCH and the requisite nature of BMSCs following fracture is unknown. Here, we evaluated whether NOTCH and/or BMSCs are required for fracture repair by performing nonstabilized and stabilized fractures on NOTCH-deficient mice with targeted deletion of RBPjk in skeletal progenitors, maturing osteoblasts, and committed chondrocytes. We determined that removal of NOTCH signaling in BMSCs and subsequent depletion of this population result in fracture nonunion, as the fracture repair process was normal in animals harboring either osteoblast- or chondrocyte-specific deletion of RBPjk. Together, this work provides a genetic model of a fracture nonunion and demonstrates the requirement for NOTCH and BMSCs in fracture repair, irrespective of fracture stability and vascularity.

Figure 1. Loss of NOTCH signaling in MSCs results in fracture nonunion.

(A) A real-time radiographic comparison of 2 representative nonstabilized tibia fractures from WT and RBPjκ^Prx1 mutant mice at 0, 14, 28, and 42 dpf revealed persistent fracture lines (yellow arrows) at 42 dpf, suggesting an established fracture nonunion in RBPjκ^Prx1 mutants. n = 12 mice per genotype per time point. (B) μCT analyses of 14-, 28-, and 42-day-old WT and RBPjκ^Prx1 mutant fractures revealed substantial periosteal external callus formation by 14 dpf and beyond, but apparent radiolucent space (yellow arrows) between broken cortices at 42 dpf in RBPjκ^Prx1 mutants. n = 7 mice per genotype per time point. (C) Reconstruction of μCT data reflected the normal and robust periosteal response in RBPjκ^Prx1 mutants; however, the new bone remodeling was delayed in these animals. n = 7 mice per genotype per time point. *P < 0.05 compared with WT by 2-way ANOVA followed by Dunnett’s post hoc test. Results are expressed as mean ± SD.

Figure 2. Loss of NOTCH signaling in MSCs results in histological changes consistent with the pathology of fracture nonunion.

(A) ABH/OG-stained callus sections from WT and RBPjκ^Prx1 mutants at 7, 14, 28, and 42 dpf showed prominent external callus formation (blue dotted line area) and persistent mesenchymal tissue (yellow dotted line area) within the internal callus area, which was ultimately filled with mesenchymal fibrous tissue (green arrows) in RBPjκ^Prx1 mutant fractures. Mesenchymal/fibrotic callus regions are shown at high magnification in blue boxes. n = 5 mice per genotype per time point. (B) IHC for COL3A1 on callus sections from WT and RBPjκ^Prx1 mutant fractures at 14, 28, and 42 dpf confirmed the formation of mesenchymal-like fibrous tissue in the fracture gap of RBPjκ^Prx1 mutants. n = 5 mice per genotype per time point. Original magnification, ×5. Red arrows indicate the expression of COL3A1.

Figure 3. RBPjκ^Prx1 mutant fractures have altered callus composition with remarkably inferior biomechanical properties.

(A) Histomorphometric quantifications of the cartilage, bone, and mesenchymal areas (Ar.) from ABH/OG-stained sections show robust cartilage and bone formation in the external callus regions and the progression of fracture nonunion in RBPjκ^Prx1 mutant fractures. n = 5 mice per genotype per time point. *P < 0.05 compared with WT by 2-way ANOVA followed by Dunnett’s post hoc test. Results are expressed as mean ± SD. (B) Biomechanical torsion testing of WT and RBPjκ^Prx1 mutant fractures at 42 dpf. All biomechanical parameters, including the maximum torque, torsional rigidity, and energy to maximum, which represent the bone strength, bone stiffness, and bone toughness, respectively, were markedly lower in RBPjκ^Prx1 mutant repaired tibia than those in the WT controls. n = 7 mice per genotype. *P < 0.05 compared with WT by 2-tailed, unpaired Student’s t test. Results are expressed as mean ± SD.

Figure 4. Fracture nonunions observed in RBPjκ^Prx1 mutants are likely due to the significant reduction of BMSC numbers and altered differentiation status.

(A) CFU-F assays on BMSCs isolated from WT and RBPjκ^Prx1 mutant fractures at 42 dpf. Representative images for crystal violet staining of CFU-Fs and ALP staining of CFU-OBs are both shown. (B) RBPjκ^Prx1 mutant fractures are associated with significantly reduced type 1 colonies (CFU-Fs) and ALP-positive colonies (CFU-OBs), but an increased ratio of CFU-OB to CFU-F. n = 6 mice per genotype. *P < 0.05 compared with WT by 2-tailed, unpaired Student’s t test. Results are expressed as mean ± SD. (C) CFU-F assays for BMSCs isolated from WT mice at 2 months of age and treated with either DMSO or DAPT for 14 days beginning on their third day in culture. Representative images for crystal violet staining of CFU-Fs are shown. (D) Quantification of DMSO- and DAPT-treated BMSC cultures show significantly reduced type 1 colonies (CFU-Fs). *P < 0.05 compared with DMSO control by 2-tailed, unpaired Student’s t test. Results are expressed as mean ± SD of 4 independent experiments. (E) Relative gene expression for Hes1, Lepr, Col1a1, Alp, and Oc in DAPT-treated BMSCs as compared with DMSO-treated control. *P < 0.05, compared with DMSO control by 2-tailed, unpaired Student’s t test. Results are expressed as mean ± SD of 3 independent experiments.

Figure 5. Loss of NOTCH signaling in mature osteoblasts does not lead to fracture nonunion.

(A) A real-time radiographic comparison of 2 representative nonstabilized tibia fractures from WT and RBPjκ^Col1 mutant mice at 0, 14, 28, and 42 dpf revealed normal fracture repair in RBPjκ^Col1 mutants. n = 7 mice per genotype. (B) Representative μCT images of fracture calluses from WT and RBPjκ^Col1 mutants at 42 dpf. n = 7 mice per genotype. *P < 0.05 compared with WT by 2-tailed, unpaired Student’s t test. Results are expressed as mean ± SD. (C) Reconstruction of μCT data revealed a similar amount of mineralized calluses between WT and RBPjκ^Col1 mutants at 42 dpf. (D) ABH/OG-stained callus sections from RBPjκ^Col1 mutants and controls at 42 dpf. Original magnification, ×2.5. (E and F) Histomorphometric analyses of ABH/OG-stained callus sections indicated no significant differences in bone and mesenchyme area between WT and RBPjκ^Col1 mutant fractures. n = 7 mice per genotype. *P < 0.05 compared with WT by 2-tailed, unpaired Student’s t test. Results are expressed as mean ± SD.

Figure 6. Loss of NOTCH signaling in fracture callus chondrocytes does not result in fracture nonunion.

(A) A real-time radiographic comparison of 2 representative nonstabilized tibia fractures from WT and RBPjκ^AcanTM mutant mice at 0, 14, and 28 dpf, revealed normal fracture repair in RBPjκ^AcanTM mutants. n = 5 mice per genotype per time point. (B) IHC- and ABH/OG-stained callus sections from RBPjκ^AcanTM mutants and controls at 10 and 28 dpf. IHC analyses for RBPjκ shows an extremely efficient removal of RBPjk protein in RBPjκ^AcanTM mutant cartilage calluses. ABH/OG-stained callus sections indicate no identifiable tissue or cellular alterations in fracture repair between WT and RBPjκ^AcanTM mutant fractures. n = 5 mice per genotype per time point. Original magnification, ×20 (IHC); ×5 (ABH/OG).

Figure 7. Insufficient fracture stabilization is not absolutely required for the fracture nonunion observed in RBPjκ^Prx1 mutants.

(A) A real-time radiographic comparison of 1.2-mm osteotomies in WT and RBPjκ^Prx1 mutants. n = 6 mice per genotype. (B) Representative μCT images of 1.2-mm osteotomies in WT and RBPjκ^Col1 mutants at 42 dpf. n = 6 mice per genotype. (C and D) Amira analyses of μCT data revealed significantly lower bone volume and minimum PMOI in defect zone. n = 6 mice per genotype. *P < 0.05 compared with WT by 2-tailed, unpaired Student’s t test. Results are expressed as mean ± SD. (E) ABH/OG staining and IHC for COL3A1 staining on femur fracture sections (1.2-mm osteotomy) from WT and RBPjκ^Prx1 mutants at 42 dpf revealed the formation of mesenchymal-like fibrous tissue (red arrows) in the 1.2-mm gap. n = 6 mice per genotype. Original magnification, ×5.