NOTCH signaling in skeletal progenitors is critical for fracture repair
Cuicui Wang, … , Hani A. Awad, Matthew J. Hilton
Cuicui Wang, … , Hani A. Awad, Matthew J. Hilton
Published March 7, 2016
Citation Information: J Clin Invest. 2016;126(4):1471-1481. https://doi.org/10.1172/JCI80672.
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Research Article Bone biology

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.

Authors

Cuicui Wang, Jason A. Inzana, Anthony J. Mirando, Yinshi Ren, Zhaoyang Liu, Jie Shen, Regis J. O’Keefe, Hani A. Awad, Matthew J. Hilton

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Figure 4

Fracture nonunions observed in RBPjκPrx1 mutants are likely due to the significant reduction of BMSC numbers and altered differentiation status.

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Fracture nonunions observed in RBPjκPrx1 mutants are likely due to the s...
(A) CFU-F assays on BMSCs isolated from WT and RBPjκPrx1 mutant fractures at 42 dpf. Representative images for crystal violet staining of CFU-Fs and ALP staining of CFU-OBs are both shown. (B) RBPjκPrx1 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.