Abstract
Treatments for congenital and acquired craniofacial (CF) bone abnormalities are limited and expensive. Current reconstructive methods include surgical correction of injuries, short-term bone stabilization, and long-term use of bone grafting solutions, including implantation of (i) allografts which are prone to implant failure or infection, (ii) autografts which are limited in supply. Current bone regenerative approaches have consistently relied on BMP2 application with or without addition of stem cells. BMP2 treatment can lead to severe bony overgrowth or uncontrolled inflammation, which can accelerate further bone loss. Bone marrow-derived mesenchymal stem cell-based treatments, which do not have the side effects of BMP2, are not currently FDA approved, and are time and resource intensive. There is a critical need for novel bone regenerative therapies to treat CF bone loss that have minimal side effects, are easily available, and are affordable. In this study we investigated novel bone regenerative therapies downstream of JAGGED1 (JAG1).
We previously demonstrated that JAG1 induces murine cranial neural crest (CNC) cells towards osteoblast commitment via a NOTCH non-canonical pathway involving JAK2-STAT5 (1) and that JAG1 delivery with CNC cells elicits bone regeneration in vivo. In this study, we hypothesize that delivery of JAG1 and induction of its downstream NOTCH non-canonical signaling in pediatric human osteoblasts constitute an effective bone regenerative treatment in an in vivo murine bone loss model of a critically-sized cranial defect. Using this CF defect model in vivo, we delivered JAG1 with pediatric human bone-derived osteoblast-like (HBO) cells to demonstrate the osteo-inductive properties of JAG1 in human cells and in vitro we utilized the HBO cells to identify the downstream non-canonical JAG1 signaling intermediates as effective bone regenerative treatments. In vitro, we identified an important mechanism by which JAG1 induces pediatric osteoblast commitment and bone formation involving the phosphorylation of p70 S6K. This discovery enables potential new treatment avenues involving the delivery of tethered JAG1 and the downstream activators of p70 S6K as powerful bone regenerative therapies in pediatric CF bone loss.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
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Changes were made for specificity and clarity of patient samples used. Figure 1 was revised to include transcriptional analysis of osteogenic genes. Figure 2 was revised for resolution and scale bars were added. Supplemental Figure 4 was added, showing the results of a pilot experiment that included an empty defect (no cells) condition. Supplemental Figure 5 was added with IHC staining of COL1A1 on calvarial defect tissues. Figure 5 was revised to focus specifically on p70 S6K inhibition and Supplemental Figures 6 and 7 were added to show mineralization results with NOTCH inhibition and gene expression with both inhibitors. Additionally, we have clarified our conclusions to reflect that p70 S6K is one of the non-canonical pathways that JAG1 may be activating in bone regeneration.
