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Combinatorial Gene Therapy with BMP2/7 Enhances Cranial Bone Regeneration
J.T. Koh1,2,
Z. Zhao1,
Z. Wang3,
I.S. Lewis1,
P.H. Krebsbach3 and
R.T. Franceschi1,4,*
1 Department of Periodontics and Oral Medicine and
3 Department of Biologic and Materials Sciences, School of Dentistry, and Center for Craniofacial Regeneration, University of Michigan, 1011 North University Ave., Ann Arbor, MI 48109-1078, USA;
2 BK21 Project for School of Dentistry and Dental Science Research Institution, Chonnam National University, Gwangju, 500-757, South Korea; and
4 Department of Biological Chemistry, School of Medicine, University of Michigan, Ann Arbor, MI 48109, USA
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Figure 1. Biological activity of conditioned medium (CM) from cells transduced with AdBMP2/7. (A) BMP content of conditioned medium. BLK fibroblasts were transduced with AdLacZ (vector control), AdBMP2 (moi 150), AdBMP7 (moi 60) alone, or the AdBMP2 plus AdBMP7 combination (same moi). Total viral titer was held constant at 210 moi by addition of the appropriate amount of AdLacZ. CM was collected after 24 hrs. A portion was used for measurement of BMP levels by ELISA, and the remainder was stored at –70°C for functional assays. As shown, for the specific lots of AdBMP2 and 7 used in this study, these viral titers directed the production of equivalent amounts of BMP2 and BMP7 in CM. (B) Alkaline phosphatase induction. C2C12 cells were plated in 24-well plates and incubated in a total volume of 300 µL containing up to 100 µL of each CM or combinations of CM. Volumes were equalized by the addition of the appropriate volume of CM from AdLacZ-treated cells. After 5 days, cells were harvested for measurement of ALP activity and DNA. Values are reported as means ± SD, N = 3. (C) SMAD and p38 phosphorylation. C2C12 cells were treated with BMP-containing CMs as described in (B). Cells were separately harvested at 2 hrs for Smad1/5/8 and at 30 min for p38. Phospho and total SMADs and p38 were measured on Western blots as described in MATERIALS & METHODS.
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Figure 3. Micro-computed tomography analysis of cranial defects. Representative three-dimensional images were rendered from µCT data. Dorsal (top) and ventral (bottom) three-dimensional renderings of calvarial bones are shown. These images were generated from calvarial samples shown in Fig. 2 (second column from left).
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Figure 4. Histology. Coronal sections through the midline of defects are shown. Margins of the original 7.0-mm trephine defect are shown (arrowheads). The same samples as shown in Fig. 3 were used for histology. Bar, 1 mm.
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Journal of Dental Research, Vol. 87, No. 9,
845-849 (2008)
DOI: 10.1177/154405910808700906
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