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;dnMAMLf/-) 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.
Highlights
Bone fracture healing occurs through a series of carefully regulated spatiotemporal events
GFP was widely expressed in multiple cell populations present during fracture healing in dnMAML mice including undifferentiated mesenchymal cells, chondrocytes, osteoblasts, endothelial cells, hematopoietic cells, and inflammatory cells (Figure 1C), verifying that dnMAML was expressed during fracture healing
The Notch signaling pathway regulates embryological bone development [7,8,9,10,13,14,32,33], and because many aspects of development are recapitulated during repair [17,18,19], we set out to identify the role of Notch signaling during bone fracture healing
Summary
Bone fracture healing occurs through a series of carefully regulated spatiotemporal events. The vascular network mediates an influx of osteoprogenitor cells that differentiate to produce immature bone on top of the resorbing cartilage matrix. While the majority of fractures restore original structure and function in a scarless manner, some fractures result in delayed or non-union healing [2] This increases the cost of care, necessitates additional surgeries, and results in a prolonged period of convalescence, which is associated with increased mortality in an aged population [3]. The spatiotemporal progression of fracture healing is well-characterized [1], the signaling pathways that regulate events required for healing are not as well understood. Identifying and elucidating the roles of signaling pathways that regulate fracture healing will allow us to identify novel therapeutic targets for improved regeneration of bone
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