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Bone Formation by BMP-7-transduced Human Gingival Keratinocytes

Center for Biorestoration of Oral Health, School of Dentistry, University of Michigan, 1011 N. University, Ann Arbor, MI 48109-1078; and
¹ Department of Oral Biology, School of Dentistry, University of Washington;

View larger version (106K): [in a new window]   Figure 1. BMP-7-transduced human oral keratinocytes (A) and fibroblasts (B) produce ossicles after subcutaneous implantation. 106 ex vivo BMP-7-transduced human oral (gingival) keratinocyte cells and gingival fibroblasts were harvested, suspended in a collagen thermoset hydrogel, and injected subcutaneously into immunocompromised mice. The ossicles were harvested after 3 wks in vivo and prepared for histological analyses. The overall bone mass is greater in the keratinocyte-produced ossicles, while bony trabeculae (arrow) and marrow (double arrow) are prominent in the fibroblast-produced ossicles. The keratinocyte-induced ossicles contained keratin-producing cells (C, arrow), while the gingival fibroblast ossicles did not (E). Developing the specimen with non-immune IgG instead of anti-keratin antibody demonstrates the specificity of the immuncytochemical assay for keratin production (D). The preparations in panels A and B were stained with H&E. Bar = 700 µm.

View larger version (108K): [in a new window]   Figure 2. BMP-7-transduced oral human keratinocyte (A,B) and gingival fibroblast produced ossicles (C,D) and secreted BMP-7 in vivo. Specimens were developed for immunohistochemical analyses with anti-BMP-7 (A,C), control non-immune IgG (B,D), and counterstained with hematoxylin. The ossicles were harvested and prepared for analysis 3 wks after implantation. Bar = 700 µm.

View larger version (77K): [in a new window]   Figure 3. BMP-7-transduced keratinocytes fail to form a bone-like matrix in diffusion chambers implanted for 6 wks into c57B/6 immunocompetent BC57B/6 mice. 105 BMP-7/HOKC (A), MT/HOKC (B), and BMP-7/HGF (D) were suspended in a thermoset collagen hydrogel and loaded into diffusion chambers as described (METHODS). As a control against the possibility of diffusion chamber leakage, some of the diffusion chambers were breached prior to implantation (C). The diffusion chambers were surgically implanted into subcutaneous tissue pouches in C57Bl6 mice, harvested after 6 wks, prepared for histological examination, and stained with H & E. Bar = 700 µm.