Fig 1.
Schematic representation of the overall experimental design.
(A) Murine calvarial cells (CALs) were immortalized via retrovirally introduced SV40 large T antigen to produce iCALs. iCALs were then transduced with BMP9 via adenoviral vector and mixed with PPCN scaffolding material. (B) qPCR analysis demonstrating relative expression of BMP9 in iCALs infected with ad-BMP9 (blue bars) compared to control (ad-GFP)-infected cells (red bars). Gene transcript expression was normalized against GAPDH expression. (C) The mixture was subsequently tested in our murine craniofacial defect model. Four millimeter diameter full-thickness calvarial defects were created in the left parietal bone of 8-week-old male athymic (nu/nu) mice. The newly created empty defect reveals the underlying dura mater. PPCN alone or PPCN and adenovirally transduced immortalized calvarial cells were used to fill the defect site.
Fig 2.
Time-course microCT imaging of the calvarial defects.
At 24–48 hours postoperatively, baseline microCT imaging was performed and analyzed to determine defect volume. Follow-up imaging and analysis was performed at 2, 4, 6, 8, and 12 weeks postoperatively to quantify residual defect volume and new bone ossification. Representative images are shown.
Fig 3.
Quantitative analysis of the BMP9-induced calvarial defect repair.
(A, B, C) Average defect volumes (mm3) were calculated at 24–48 hours, 2, 4, 6, 8, and 12 weeks postoperatively using volumetric reconstructions generated in Amira®. Asterisks indicate a significant (p < 0.05) difference in average defect volume between test groups at the specified time point, as determined by one-way ANOVA. (D, E, F) The change in defect volume over time was used to deduce the percentage of baseline defect volume filled with new bone.
Fig 4.
Histologic evaluation of the BMP9-induced calvarial defect repair.
Histologic analysis of tissue microsections harvested 12 weeks post-treatment. Yellow arrows indicate the original defect borders. (A) The defect site of this AdGFP-treated mouse shows incomplete healing; there is some ingrowth of bone, but fibrous tissue fills part of the defect. (B) Conversely, the defect site of an AdBMP9-treated mouse has been completely bridged with new bone. All sections show no trace of PPCN material, indicating complete resorption.
Fig 5.
Assessment of the Trichrome histology.
Histologic analysis of tissue microsections harvested 12 weeks post-treatment. (A, B) Chondroid matrix and a smaller proportion of mature bone is shown in the defect site of a PPCN + iCAL + AdGFP-treated mouse. (C, D) A visibly higher proportion of mature bone can be appreciated in samples taken from the defect sites treated with PPCN + iCAL + AdBMP9.