Craniofacial distraction osteogenesis (DO) is an evolving reconstructive technique with expanding applications for the treatment of bony deficiencies of the facial skeleton. Mechanical force has been known to play a fundamental role in modulating Sustained osteogenic response and therefore is believed to function as a critical regulator of DO. We hypothesize that key clustering components of an integrin-mediated signaling pathway, including c-Sre (pp60(src)), are necessary for mediating the response to mechanical force. The specific aim of this Study is to demonstrate up-regulation of a key focal adhesion molecule, c-Src, selectively in new bone formation subject to the mechanical forces of distraction and to demonstrate a lack of that same up-regulation in new bone formation associated with simple fracture healing. An additional specific aim is to demonstrate colocalization of c-Src expression and bone rnorphogenetic protein (BMP 2/4) expression during mandibular DO. Using a rat model of mandibular DO, c-Src and BMP 2/4 expression were evaluated in critical size defects, subcritical size defects, and mandibles undergoing gradual distraction. Osseous regeneration was observed in the course of gradual distraction; this process was associated with increased expression of c-Src. Furthermore, the presence of BMP 2/4 closely approximated c-Src expression spatially and temporally, suggesting a link between cytoplasmic focal adhesion activation and the resultant nuclear regulation of osteogenic protein expression. In significant contradistinction, minimal c-Src expression was found in the subcritical-sized defects where the fractures healed secondarily but where no gradual distraction was performed. Instead, the new bone formation inherent in the secondarily heated subcritical-sized defects demonstrated expected BMP 2/4 expression but was devoid of an up-regulation of c-Src. Finally, as expected, minimal expression of both c-Src and BMP was found in fibrous nonunion specimens. C-src expression was observed during gradual distraction; furthermore, minimal c-Src expression was visualized during subacute and critical-size defect fracture healing. C-Src expression also closely approximated BMP expression during DO. These findings that c-Src expression is found primarily only during conditions of cyclic distraction forces strongly implicates that mechanical force during gradual distraction is associated with c-Src expression. These results provide in vivo support for previous in vitro evidence that mechanical force profoundly influences osseous regeneration during distraction osteogenesis by means of a c-Src dependent mechano-transduction pathway, resulting in increased expression of osteogenic proteins, including BMP 2/4.
