FOCAL ADHESION KINASE EXPRESSION DURING MANDIBULAR DISTRACTION OSTEOGENESIS

Participants: S. Buchman, M. Ignelzi, L. Tong, S. Rhee, D. Kayner, M. Stock

Keywords: distraction osteogenesis, focal adhesion kinase, rat mandible

Introduction

The use of distraction osteogenesis (DO) in mandibular reconstruction continues to grow rapidly. However, the cellular mechanisms underlying bone regeneration in this process remain poorly characterized.

We theorize that mechanical loading forces play a significant role in stimulating new bone formation during DO. In vitro studies have suggested that cells, including osteoblasts, react to mechanical strain via a number of cytoskeletal elements, including integrins, a major family of cell surface receptors. Integrins mediate a number of cell signaling pathways, which may lead to gene expression and cell differentiation critical to new bone formation. One key regulator of the integrin-mediated signal transduction pathway is focal adhesion kinase (FAK), a cytoskeletal-associated protein tyrosine kinase.

Using a unique rodent model of distraction osteogenesis, we were able to study the expression of FAK in vivo at multiple time points during DO. The goal was to identify FAK expression at a cellular level in an in vivo model of mechanical loading

Using immunohistochemistry techniques, we were able to demonstrate cellular FAK expression localized to areas associated with new bone formation where mechanical distraction forces are present during DO. We are also currently evaluating FAK expression in mandibles without distraction forces, but with new bone formation.

These data support our hypothesis that integrin-mediated signal transduction regulates gene expression and cell differentiation stimulated by mechanical loading forces during distraction osteogenesis. Our results also suggest that FAK may play an important role in the mechanism of mechanotransduction and biologic growth.

Materials and Methods

The custom-made distraction device consists of a frame-type bilateral distractor. Two posts secured to the right and left mandibular angle and ramus are attached at right angles to distraction rods situated parallel to the body of the rat mandible. These distraction rods are interlocked with a transversely threaded anterior post transfixed through the bone just behind the symphysis of the mandible, completing the rigid external mandibular frame. The distraction system utilizes a series of nuts and custom-milled titanium caps to secure the posts. This system allows controlled advancement of the mandible using the distraction rod. Locking the system in place at any advancement level along the distraction rod enables the distractor to function as a sturdy external fixator for the mandible with the stability of a three dimensional bilateral support system.

Animals were sacrificed at 4, 6, 9, 12 and 16 days post-operatively through CO2 chamber immersion. Mandibles were immediately harvested and fixed in 4% paraformaldehyde for 48h. Decalcification of the mandibles was then carried out with a sodium citrate/formic acid solution changed every two days, over 10 days. The specimens were subsequently embedded in paraffin, and 6 micron sagittal sections were mounted on glass slides.

Hematoxylin-eosin staining was performed to assess morphology. Immunohistochemistry was conducted with slides from both groups using polyclonal anti-FAK antibody (Santa Cruz), and visualized under fluorescent microscopy.

Progress

Hematoxylin-eosin staining of the distraction group mandibles demonstrated progressive stages of bony healing that appeared to undergo formation of a cartilaginous intermediate that was subsequently mineralized from the periphery. Immunohistochemical analysis revealed positive FAK staining in the distraction group at all time points during distraction. Initially, FAK was localized within gap tissues that were histologically undifferentiated.

After five days of distraction, mandibles revealed intense staining that appeared to be localized within cartilage cells in regions of new bone formation. At post-operative day 16 (after distraction was complete and four days of consolidation had elapsed), FAK staining was again expressed within cartilage cells in the central portion of the gap. In addition, FAK expression was seen at the periphery of the distraction tissues and was associated with cells that appeared to be osteoblasts.

We are currently examining a group of animals with acutely advanced mandibles. When the mandible is osteotomized and acutely advanced to 2mm, no distraction osteogenesis takes place; however, bone growth does occur at this gap size.

Results

Data accumulated from in vitro studies suggest FAK to be an important component of the integrin-mediated signal transduction cascade. We demonstrate that FAK is produced in vivo, during mandibular distraction osteogenesis. FAK is localized to cells associated with new bone formation in regions where mechanical distraction forces are present. These data support our hypothesis that mechanical forces produced by DO are transduced via the cytoskeleton through the integrin-mediated signal transduction pathway to regulate gene expression, cell differentiation, and new bone formation within the distraction gap. Furthermore, our results suggest that FAK may play an important role in the mechanism of mechanotransduction in vivo.