Modulation of Notch1 signaling regulates bone fracture healing

Abstract

Fracture healing involves interactions of different cell types, driven by various growth factors, and signaling cascades. Periosteal mesenchymal progenitor cells give rise to the majority of osteoblasts and chondrocytes in a fracture callus. Notch signaling has emerged as an important regulator of skeletal cell proliferation and differentiation. We investigated the effects of Notch signaling during the fracture healing process. Increased Notch signaling in osteochondroprogenitor cells driven by overexpression of Notch1 intracellular domain (NICD1) (αSMACreERT2 mice crossed with Rosa-NICD1) during fracture resulted in less cartilage, more mineralized callus tissue, and stronger and stiffer bones after 3 weeks. Periosteal cells overexpressing NICD1 showed increased proliferation and migration in vitro. In vivo data confirmed that increased Notch1 signaling caused expansion of alpha-smooth muscle actin (αSMA)-positive cells and their progeny including αSMA-derived osteoblasts in the callus without affecting osteoclast numbers. In contrast, anti-NRR1 antibody treatment to inhibit Notch1 signaling resulted in increased callus cartilage area, reduced callus bone mass, and reduced biomechanical strength. Our study shows a positive effect of induced Notch1 signaling on the fracture healing process, suggesting that stimulating the Notch pathway could be beneficial for fracture repair.

Keywords: Notch signaling; alpha-smooth muscle actin; bone fracture; inducible Cre; osteoblast differentiation; periosteal progenitors.

Figure 1.. Validation of NICD1 overexpression.

A. Experimental design for Rosa-NICD1 fracture studies. Cre recombination was confirmed by DNA recombination in callus tissue 7 DPF, each lane represents a band from single animal callus tissue sample. B. Relative expression of Notch pathway target genes of male mice from primary periosteal cells derived from callus tissue digested 5 DPF and additionaly expanded for 3 days in cell culture, determined by qRT-PCR. Results are presented as fold change normalized to Cre⁻ expression, n=4–6, *p<0.05.

Figure 2.. Tracing the αSMA⁺ cells in fracture healing using αSMACreERT2/Rosa-NICD1 mice crossed with Ai9 reporter mice.

A. Experimental design for αSMA9/NICD1 lineage tracing during the fracture healing. Tamoxifen was injected on the day of the fracture, 2 and 4 DPF, and fractured femurs collected for histological analysis 5, 10 and 14 FPF and flow cytometry 5 DPF. B. Representative images show presence of TdTomato reporter in αSMA cells in controls (αSMA9/NICD1⁻) and with NICD1 overexpression (αSMA9/NICD1⁺) 5, 10 and 14 DPF. Dotted lines are surrounding the periosteal callus. Scale bar = 500 μm. PC-periosteal callus, CB-cortical bone, BM-bone marrow, PT-pin track, M-muscle. C. Representative dot plots and graph present αSMA9 cell frequency in αSMA9/NICD1⁻ and αSMA9/NICD1⁺ from digested periosteal callus tissue 5 DPF, *p<0.05. The results are presented as mean ± SD, (n=5 for Cre- and n=6 for Cre+). **p<0.01.

Figure 3.. NICD1 overexpression enhances fracture healing.

We produced a stabilized fracture on the right femur of male αSMACreNICD1 mice. A. Representative images of Safranin O (left) and von Kossa staining (right) are shown. a’ - d’ shows magnified cartilage area within the callus. Scale bar = 500 μm. Graphs indicate quantification of cartilage and mineralized area from the histological sections. (D7, n=4–7; D10 n=6–7: D14, n=16–18 (Cre+) and D7, n=3–7: D10, n=8–9; D14, n=13–14 for (Cre-)). B. μCT analysis at 21 DPF was used to calculate callus volume and bone mass. Torsion testing was then used to determine strength and stiffness (n=9). C. Reconstruction of representative calluses 21 DPF. Scale bar = 1 mm Results are presented as mean ± SD. * p<0.05, **p<0.01, ***p<0.001.

Figure 4.. NICD1 overexpression in αSMA osteoprogenitor cells increases proportion of αSMA and osteocalcin expressing cells.

Stabilized femur fracture was produced in male αSMACreERT2/Rosa-NICD1 mice crossed with Ai9 reporter mice and collected 10 DPF. A. Representative images of osteocalcin staining. Scale bar = 500 μm B. Stained cells were quantified as a proportion of DAPI-stained nuclei using image analysis. C. Osteoblasts positive for osteocalcin derived from αSMA cells were determined by counting double positive cells (positive for αSMA⁺ and OCN⁺) out of OCN⁺ cells. Arrows indicate tdTomato⁺ osteoblasts. n=6 for NICD⁻ and n=8 for NICD⁺. *p<0.05, **p<0.01.

Figure 5.. NICD1 overexpression in αSMA cells inhibits differentiation but enhances migration of periosteal cells in vitro.

Primary periosteal cells were isolated from 8-week-old male mice, and cultured as indicated in the diagrams. A. Alkaline phosphatase staining. B. Expression of indicated genes during differentiation, mean ± SD (n=3 for Cre-, and n=5 for cre+). C. EdU incorporation over 4h, (n=5 for Cre-, and n=6 for Cre+). D. Wound closure following a scratch assay, n=5.. * p<0.05. Hey1, Hairy/Enhancer-Of-Split Related with YRPW Motif Protein 1; BSP, bone sialoprotein; DOM, days in osteogenic medium; OCN, osteocalcin. Blue bars = Cre- and Red bars = Cre+.

Figure 6.. Notch1 inhibition impairs fracture healing.

A. Treatment scheme for aNRR1. B. Histology of femur fractures treated with aNRR1 or control antibody at 10 DPF. Cartilaginous and mineralized area within the callus were determined by Safranin O or von Kossa staining and images analyzed in ImageJ software. n=11 for control, and n=9 for aNRR1 treated group. C. Fractured femurs were analysed by μCT to determine callus volume and bone mass, and tested for biomechanical properties by torsion testing at 21 DPF. D. Representative images show reconstructed calluses 21 DPF in control and aNRR1 treated mice. Scale bar = 1 mm, n=7–9 for control, and n=10–11 for aNRR1 treated group. The results are presented as mean ± SD. * p<0.05, ***p<0.001.

Figure 7.. Proportion of αSMA cells in fracture healing during early Notch1 signaling inhibition.

A. Fractures were performed in αSMA9 mice, and tamoxifen and aNRR1 antibody were injected on 0, 2, and 4 DPF, n=5. B. The results are presented as mean ± SD. Representative dot plots are showing gating of (CD45/Ter119/CD31)⁻ cells and histograms of αSMA9 that are (CD45/Ter119/CD31)⁻ in two experimental groups. **p<0.01.