Notch signaling components are upregulated during both endochondral and intramembranous bone regeneration

Abstract

Previous studies have demonstrated that Notch signaling regulates endochondral and intramembranous bone formation by controlling cell proliferation and differentiation. Notch signaling has also been shown to regulate healing in a variety of tissues. The objective of this study was to characterize and compare activation of the Notch signaling pathway during endochondral and intramembranous bone healing using tibial fracture and calvarial defect injury models, respectively. Bilateral tibial fractures or bilateral 1.5 mm diameter calvarial defects were created in mice, and tissues were harvested at 0, 5, 10, and 20 days post-fracture. Gene expression of Notch signaling components was upregulated during both tibial fracture and calvarial defect healing, with expression generally higher during tibial fracture healing. The most highly expressed ligand and receptor during healing, Jag1 and Notch2 (specifically the activated receptor, known as NICD2), were similarly localized in mesenchymal cells during both modes of healing, with expression decreasing during chondrogenesis, but remaining present in osteoblasts at all stages of maturity. Results suggest that in addition to embryological bone development, Notch signaling regulates both endochondral and intramembranous bone healing.

Figure 1

Gene expression of Notch ligands (left), receptors (middle) and target genes (right) during TF (white bars) and CD (grey bars). # indicates a significant difference between TF vs. CD at a given time point (p<0.05). A common letter above any two bars indicates a significant difference between those time points during TF (a,b,c) or CD (x,y,z) (p<0.05). Data is presented as relative gene expression to the housekeeping genes, calculated using the formula 2^{− ΔC(t)} (arbitrary units).

Figure 2

Jag1 and NICD2 are expressed in identical cell populations that participate in endochondral bone repair during TF. Undifferentiated mesenchymal cells (A) are largely positive (brown staining, black arrows), but expression gradually decreases as cells differentiate into proliferative (B), pre-hypertrophic (C), and hypertrophic chondrocytes (D), and then is re-expressed in terminal hypertrophic chondrocytes (E). Alternative to chondrogenesis, osteogenic cells at various stages of maturity, located in osteoid (F), primary (G) and remodeled bone formation (H) are mostly positive. Note that varying amounts of Jag1 and NICD2 negative cells are present in distinct cell population (white arrows). H&E and SafO images acquired at 200X magnification. Jag1 and NICD2 images acquired at 600X magnification.

Figure 3

Jag1 and NICD2 are expressed in vascular endothelial cells invading the cartilage matrix, as well as terminal hypertrophic chondrocytes adjacent to the invading vasculature. Black arrows and brown staining indicate positive cells. White arrows indicate negative cells. SafO image acquired at 200X magnification. Jag1 and NICD2 images acquired at 600X magnification

Figure 4

Jag1 and NICD2 are expressed in identical cell populations that participate in intramembranous bone repair during CD. Undifferentiated mesenchymal cells located in the periosteum (A) and adjacent to the defect site (B) are largely positive (brown staining, black arrows). As osteogenesis progresses, cells at various stages of maturity continue to stain positive in areas of new (C) and remodeled bone (D). Osteocytes (D) are both positive and negative. Note that Jag1 and NICD2 negative cells (white arrows) are present in each area. H&E images acquired at 400X magnification. Jag1 and NICD2 images acquired at 600X magnification.