Session VI - Fracture Repair

Do Fractures Heal by the Re-Induction of Genes Expressed during Bone Development?

Theodore Miclau, MD; Eytan Alpern, DDS, PhD; C. Ferguson; Jill Helms, MD, University of California, San Francisco, San Francisco, CA

Introduction: Despite advances in implant design and surgical techniques, delayed union and nonunion persist. Biologically based therapeutic strategies including the application of single growth factors, such as Bmps, continue to gain great clinical interest. Potential limitations of these strategies, however, include timing of application, lack of associated factors, and ineffective targeting of "upstream" factors. While the molecular mechanisms mediating bone formation remain largely unknown, histological similarities between fetal bone formation and adult fracture repair suggest that the processes may be related. In addition, given that bone heals with bone rather than scar tissue, the molecular processes mediating fracture healing may recapitulate those expressed during fetal bone development. The purpose of this study is to evaluate the expression of cbfa1, a transcription factor felt critical for fetal bone formation, and the genes in the indian hedgehog pathway (indian hedgehog (ihh) and bone morphogenic protein 6 (bmp6)), felt to regulate chondrogenic differentiation during fetal skeletogenesis, in a mouse tibia fracture model. The indian hedgehog gene is not expressed in adult mouse bone.

Materials and Methods: Adult 10-12 week Swiss Webster mice underwent closed, standardized unstabilized fractures produced via three-point bending. Fractures were confirmed via radiography. Callus tissues were collected at post-fracture days 3, 6, 8, 10, and 14 (n=7). Callus tissues were decalcified, dehydrated, and embedded. Tissue was analyzed histologically using Safranin O-Fast Green staining. In-situ hybridizations, sensitive methods for visualizing gene expression patterns, were performed using cDNAs of the mouse genes cbfa1, ihh, and bmp6. This technique was also performed for other markers for bone and cartilage: osteocalcin (oc), collagen type II (col2), and collagen type X (col10).

Results: Three days post-fracture, a cellular callus had developed that lacked cartilage by proteoglycan staining. Compared to the normal periosteum, the periosteum near the fracture site was thicker and expressed both cbfa1 and oc. Ihh and col10 were not detected, suggesting that chondrocytes had not yet undergone maturation or hypertrophy. Six days post-fracture, the callus exhibited increased cellularity and contained areas of cartilage that had begun to hypertrophy and express col10. Cbfa1 was pronounced in the mesenchymal cells within the central callus and within the hypertrophic cartilage. In addition, the periosteum continued to express cbfa1 and oc. By 8 and 10 days post-fracture, histological signs of new bone were present.

Cbfa1 expression was most pronounced within these areas of neo-ossification. Cbfa1 continued to be expressed within the hypertrophic cartilage. Ihh was seen in maturing and hypertrophic chondrocytes. At 14 days post-fracture, cbfa1 was present throughout the fracture, but was no longer detected within the periosteum. Ihh was still expressed in the cartilagenous domains.

Conclusion: The temporal and spatial expression patterns of cbfa1, ihh , and the genes associated with these two pathways are similar to those seen during fetal limb development. These findings support the hypothesis that embryonic genes are re-induced in adult fracture repair. These fetally expressed genes may represent targets for future therapeutic intervention.