Delivery of a Jagged1-PEG-MAL hydrogel with pediatric human bone cells regenerates critically sized craniofacial bone defects
- Department of Pediatric Otolaryngology, Emory University, United States
- Parker H. Petit Institute for Bioengineering and Biosciences, Georgia Institute of Technology, United States
- School of Chemistry and Biomolecular Engineering, Georgia Tech College of Engineering, United States
- Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology, United States
- Department of Orthopedics, Emory University, United States
- Neuroscience Program in College of Sciences, Georgia Institute of Technology, United States
- The Atlanta Veterans Affairs Medical Center Atlanta, United States
- Department of Surgery, Division of Oral and Maxillofacial Surgery, Emory University, United States
- Department of Cell Biology, Emory University, United States
- George W. Woodruff School of Mechanical Engineering, Georgia Tech College of Engineering, United States
- Department of Pediatric Otolaryngology, Children’s Healthcare of Atlanta, United States
Figures
Figure 1 with 2 supplements
JAGGED1-induced mineralization and gene expression in HBO cells: seven HBO cell lines were treated with growth media alone, osteogenic media alone or with Fc-Dynabeads (5.7 μM) or JAG1-Dynabeads (5.7 μM).
The cells were half-fed every 5 days. On day 21 cells were fixed with 50% ethanol and thereafter, stained with 1% Alizarin Red S. (A) Representative image of HBO2. (B) Alizarin Red S dye was …
Figure 1—figure supplement 1
Method of processing human bone samples to produce primary human bone-derived osteoblast-like cell lines.
Human bone-derived osteoblast-like cells (HBO) were isolated by collagenase digestion of pediatric healthy fibular bones. (A) Pediatric fibula specimen (B) Bone is mechanically segmented into small …
Figure 1—figure supplement 2
PEG-4MAL hydrogel-encapsulated JAGGED1-induced mineralization of HBO cells.
(A) HBO cells alone or in the presence of JAG1-Dynabeads complex (20 μM) were incorporated in 4% PEG-MAL hydrogels and grown in culture. The cells were half-fed every 5 days. On day 21 cells were …
Figure 2 with 3 supplements
JAG1 delivery in a PEG hydrogel stimulates bone regeneration in a critical-sized bone defect mouse model.
HBO cells alone or in the presence of JAG1-Dynabeads complex (20 μM) ± DAPT and bone morphogenetic protein 2 (BMP2; 2.5 µM) + Fc-Dynabeads were incorporated in 4% PEG-MAL hydrogels and implanted …
Figure 2—figure supplement 1
Visual depiction of calvarial defect studies.
(A) HBO cells alone or in the presence of other treatments were incorporated in 4% PEG-MAL hydrogels and (B) implanted into 4 mm critical-sized defects in the parietal bones of 6- to 8-week-old NOD …
Figure 2—figure supplement 2
Pilot study of JAG1-bds delivery in a PEG hydrogel stimulates bone regeneration in a critical-sized bone defect mouse model.
No cells (Empty Defect) or HBO cells alone or in the presence of JAG1-Dynabeads complex (20 μM) ± DAPT and bone morphogenetic protein 2 (BMP2; 2.5 µM) + Fc-Dynabeads were incorporated in 4% PEG-MAL …
Figure 2—figure supplement 3
Immunohistochemical staining of calvarial defect tissue for Collagen 1.
Formalin-fixed paraffin-embedded (FFPE) tissue from the calvarial defect experiment shown in Figure 2 was sectioned and stained with an antibody for COL1A1 (Cell Signaling #72026S) and …
Figure 3
Transcriptional profiling reveals genes and pathways stimulated by non-canonical NOTCH signaling.
(A) RNAseq reveals clusters of genes associated with the non-canonical NOTCH pathway (rows are z-scored, side color bar identifies up- and downregulated differentially expressed genes (DEGs) …
Figure 4
JAGGED1 induces a non-canonical NOTCH pathway in HBO cells.
HBO cells undergo mineralization through a non-canonical pathway. Luminex analysis of lysates obtained from three HBO cell lines untreated or treated with Dynabead-bound recombinant JAG1-Fc fragment …
Figure 5 with 2 supplements
p70 S6K is an essential target during JAGGED1-induced mineralization of HBO cells: HBO cells were treated with growth media alone, osteogenic media alone or with Fc-Dynabeads (5.7 μM), S6K-18 alone (a p70 S6K phosphorylation inhibitor) (50 μM), and JAG1-Dynabeads (5.7 μM) alone or in combination with S6K-18 (50 μM).
The cells were half-fed every 5 days. On day 21 cells are fixed with 50% ethanol and thereafter, stained with 1% Alizarin Red S.(A) Representative image of HBO7. (B) Alizarin Red S dye was extracted …
Figure 5—figure supplement 1
Mineralization assay with DAPT inhibition of the NOTCH canonical pathway.
HBO1 cells were treated with JAG1-Dynabeads (5.7 μM) alone or in combination with increasing concentrations of DAPT in a dose–response test (50 μM). The cells were half-fed every 5 days. On day 21 …
Figure 5—figure supplement 2
Inhibition of JAGGED1-induced mineralization of HBO cells with inhibitors of NOTCH and p70 S6K.
HBO cells were treated with JAG1-bds (5.7 μM) alone or in combination with DAPT (15 µM), S6K-18 (a p70 S6K phosphorylation inhibitor) (50 μM) or S6K-18 (50 μM) + DAPT (15 μM). The cells were …
Additional files
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Supplementary file 1
List in excel sheet attached shows differentially expressed genes (DEGs) in JAG1 and JAG1 + DAPT groups compared to no treatment.
Analysis revealed a total of 448 upregulated genes and 435 downregulated genes in the non-canonical pathway.
- https://cdn.elifesciences.org/articles/92925/elife-92925-supp1-v1.xlsx
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Supplementary file 2
Primer sequences used for qRT-PCR.
- https://cdn.elifesciences.org/articles/92925/elife-92925-supp2-v1.docx
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MDAR checklist
- https://cdn.elifesciences.org/articles/92925/elife-92925-mdarchecklist1-v1.pdf
