Alpha Omega Alpha Honor Medical Society

2010 Research Abstract

The Differential Effects of Cellularly Engineered BMP-2, BMP-9 and Runx2 on Bone Healing in Murine Critical-Sized Calvarial Defects

Investigator: Deana Shenaq, University of Chicago Division of the Biological Sciences Pritzker School of Medicine
Mentor: Russell R. Reid, MD, PhD, University of Chicago Division of the Biological Sciences Pritzker School of Medicine

Introduction: Healing of critical-sized calvarial defects in the craniofacial skeleton occurs via intramembraneous ossification. This type of bony regeneration poses problems for patients older than two years, as the capacity for proliferation and differentiation of mesenchymal stem cells declines with age. Belonging to the TGF-beta superfamily, bone morphogenic proteins (BMPs) play a sentinel role in osteoblastic proliferation and differentiation, and their implementation into clinical practice can potentially revolutionize the management of cranial defects. Currently, only BMP-2 and BMP-7 are FDA approved for clinical use, yet it is unclear whether they play the most osteogenic role in the craniofacial skeleton. Our objective was to compare the differential effects of BMP-9, BMP-2 and RunX2 in primary calvarial cells in vitro and in an in vivo murine model of critical-sized calvarial defects.

Methods: Non-suture associated 4mm parietal defects were created in adult CD1 male mice (age >8 weeks, n=19). Adenoviral vectors encoding BMP-9 (n=6), BMP-2 (n=5), or GFP alone (control, n=5) were impregnated (0.5 x 10^6 pfu) into collagen sponges, which filled the defects (One group (n=3) was treated with collagen sponge alone. MicroCT scans of live subjects permitted serial defect survey (0, 3, 6, 12, 16-weeks post craniotomy). A second in vivo experiment was performed using calvarial derived bone marrow stromal cells, which were infected with adRunX2 and adBMP-9. This colony was expanded ex vivo and the infected cells were then transferred to defect sites as previously described. Defect closure and bony maturation were assessed in the form of defect bone intensity by creating 3D volumetric reconstructions at a threshold of 400 Hounsfield units. Calvaria were harvested at 20 weeks and de novo osseous regeneration was confirmed by histology. Mirroring in vivo experimentation, immortalized primary calvarial cells (iCALs) were also infected with adBMP-9, adBMP-2 and adGFP to assess for early and late osteogenic markers.

Results: MicroCT imaging showed increased bony regeneration in adBMP-9 and adBMP-2 groups by week 3. AdBMP-9 had a significant percent change in defect intensity from baseline (240.0% ± 65.0%) vs adGFP controls (63.0% ± 26%) by 6 weeks (p=0.04), whereas, a significant change was seen by 16 weeks in the adBMP- 2 group (336.7% ± 194.0% vs 72.0% ± 25.9%, p=0.03). The adBMP-9, adBMP-2, and adGFP groups all showed greater intensity changes than the sponge only control (p<0.05). No significant difference was seen between the adBMP groups at any time point. No bony regeneration was observed in the second in vivo experiment in either the BMP-9 or RunX2 groups. Significant elevations in alkaline phosphatase activity compared to GFP treated cells were observed in the adBMP-9 and adBMP-2 treated iCALs by days 3 and 9, and bone nodule formation was evident by days 14 and 21 respectively. Levels of osteocalcin mRNA were increased at day 8 in the adBMP-9 group and in the BMP-2 group by day 14.

Conclusions: In a reliable and reproducible model of critical-sized murine calvarial defects, we demonstrate that both BMP-9 and BMP-2 promote osseous healing. Moreover, both BMPs induce differentiation in calvarial-derived progenitor cells. Preliminary data supports that BMP-9 treated cells demonstrate an earlier and more robust osteogenic response. Further studies should be performed to comprehensively test the differing and possible synergistic effects of BMPs in the craniofacial skeleton.

Updated on June 8, 2011.

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