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. 2013 Jul 3;8(7):e68726.
doi: 10.1371/journal.pone.0068726. Print 2013.

Systemic inhibition of canonical Notch signaling results in sustained callus inflammation and alters multiple phases of fracture healing

Michael I Dishowitz  1 , Patricia L Mutyaba, Joel D Takacs, Andrew M Barr, Julie B Engiles, Jaimo Ahn, Kurt D Hankenson
Affiliations

Systemic inhibition of canonical Notch signaling results in sustained callus inflammation and alters multiple phases of fracture healing

Michael I Dishowitz et al. PLoS One. .

Abstract

The Notch signaling pathway is an important regulator of embryological bone development, and many aspects of development are recapitulated during bone repair. We have previously reported that Notch signaling components are upregulated during bone fracture healing. However, the significance of the Notch pathway in bone regeneration has not been described. Therefore, the objective of this study was to determine the importance of Notch signaling in regulating bone fracture healing by using a temporally controlled inducible transgenic mouse model (Mx1-Cre;dnMAML(f/-)) to impair RBPjκ-mediated canonical Notch signaling. The Mx1 promoter was synthetically activated resulting in temporally regulated systemic dnMAML expression just prior to creation of bilateral tibial fractures. This allowed for mice to undergo unaltered embryological and post-natal skeletal development. Results showed that systemic Notch inhibition prolonged expression of inflammatory cytokines and neutrophil cell inflammation, and reduced the proportion of cartilage formation within the callus at 10 days-post-fracture (dpf) Notch inhibition did not affect early bone formation at 10dpf, but significantly altered bone maturation and remodeling at 20dpf. Increased bone volume fraction in dnMAML fractures, which was due to a moderate decrease in callus size with no change in bone mass, coincided with increased trabecular thickness but decreased connectivity density, indicating that patterning of bone was altered. Notch inhibition decreased total osteogenic cell density, which was comprised of more osteocytes rather than osteoblasts. dnMAML also decreased osteoclast density, suggesting that osteoclast activity may also be important for altered fracture healing. It is likely that systemic Notch inhibition had both direct effects within cell types as well as indirect effects initiated by temporally upstream events in the fracture healing cascade. Surprisingly, Notch inhibition did not alter cell proliferation. In conclusion, our results demonstrate that the Notch signaling pathway is required for the proper temporal progression of events required for successful bone fracture healing.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. GFP-dnMAML fusion protein is expressed in dnMAML mice and inhibits Notch signaling during fracture healing.
(A) GFP gene expression is upregulated in dnMAML fractures. (B) This correlates with a 30% reduction in Hes1 gene expression at 5dpf. (C) GFP is expressed in undifferentiated mesenchymal cells, chondrocytes, osteoblasts, endothelial cells, hematopoietic cells and inflammatory cells in dnMAML fractures. There is no expression in WT mice. GFP IHC images were acquired at 200x magnification. Gene expression data is presented as fold change to WT for each time point, calculated using the formula. 2-ΔΔC(t). *p<0.050 (dnMAML vs WT).
Figure 2
Figure 2. dnMAML decreases cartilage formation during fracture healing.
(A) Percent of cartilage area to total callus area (CA/TA) via SafO histomorphometric analysis is decreased in dnMAML fractures at 10dpf. (B) Col2a1 and (C) Sox9 gene expression are decreased in dnMAML fractures at 10dpf. (D) ColX gene expression is non-significantly decreased in dnMAML fractures at 10dpf. (E) There are no differences between WT and dnMAML fractures in percent of immature, mature or hypertrophic cartilage to cartilage area (CA) at 10dpf. (F) There are no differences in chondrocyte density within these areas at 10dpf. SafO images were acquired at 20x magnification. Gene expression data is presented as relative expression to β-actin, calculated using the formula. 2-ΔC(t). *p<0.050 tp<0.100 (dnMAML vs WT).
Figure 3
Figure 3. dnMAML increases the proportion of bone mass within the callus due to a moderate decrease in callus size.
(A) Bone volume fraction (BV/TV) via μCT analysis is increased in dnMAML fractures at 20dpf. (B) There is a non-significant increase in percent osseous bone tissue area to total callus area (BA/TA) via Masson’s Trichrome histomorphometric analysis. (C) Callus total volume is non-significantly decreased and (E) average callus total area (Avg TA) is decreased at 20dpf. (D) There are no differences in bone volume (BV) or (F) average osseous bone tissue area (Avg BA) at any time point. μCT images were acquired at a 21 µm voxel size. Masson’s Trichrome images were acquired at 20x magnification. *p<0.050 tp<0.100 (dnMAML vs WT).
Figure 4
Figure 4. dnMAML decreases osteogenic and osteoclast cell density.
(A) dnMAML expression decreases osteoblast density normalized by bone perimeter (Obl/BP) and (B) increases osteocyte density normalized by bone area (Ocy/BA) at 20dpf, (C) which results in an increased osteocyte-to-osteoblast ratio (Ocy:Obl). (D) However, dnMAML expression decreases overall osteogenic (osteoblast and osteocyte) cell density normalized by bone area (Osteo/BA) at 20dpf. (E) Osteocalcin (Ocn) gene expression is increased in dnMAML fractures at 20dpf. There are no differences in (F) osterix (Osx) or (G) collagen type I (Col1a1) gene expression. (H) dnMAML expression decreases osteoclast density normalized by bone area (Ocl/BA) and (I) TRAP gene expression at 20dpf. Masson’s Trichrome images were acquired at 200x and 400x magnification for cell-based histomorphometric analysis. Gene expression data is presented as relative expression to β-actin, calculated using the formula. 2-ΔC(t). *p<0.050 (dnMAML vs WT).
Figure 5
Figure 5. dnMAML prolongs inflammation during fracture healing.
(A) Percent void area to total callus area (Void/TA) via histomorphometric analysis is increased in dnMAML fractures at 10dpf. (B) Neutrophil inflammation via semi-quantitative analysis of H&E images is increased in dnMAML fractures at 10dpf. (C) There is no difference in mononuclear cell inflammation. (D) TNF-α and (E) IL-1β gene expression are increased in dnMAML fractures at 10dpf. IL-1β is decreased at 20dpf. Gene expression data is presented as relative expression to β-actin, calculated using the formula. 2-ΔC(t). *p<0.050 tp<0.100 (dnMAML vs WT).
Figure 6
Figure 6. dnMAML does not alter cell proliferation during fracture healing.
There are no differences in (A) PCNA and (B) Cyclin D1, gene expression during fracture healing. (C) PCNA IHC staining shows no differences in (D) % PCNA+ cells in undifferentiated mesenchymal or in pre-hypertrophic chondrocytes, and no differences in (E) PCNA+ area per bone perimeter (PCNA+ area/BP) in immature bone. PCNA IHC images were acquired at 400x magnification. Gene expression data is presented as relative expression to β-actin, calculated using the formula. 2-ΔC(t).
Figure 7
Figure 7. dnMAML inhibits osteoblast differentiation of mMSCs in culture.
(A) dnMAML does not alter cell number over time (24 hrs p=0.118; 96 hrs p=0.950). (B,C) dnMAML decreases calcified mineral deposition of differentiated mMSCs (p=0.045).
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