INFLUENCE OF MARROW ABLATION ON OSTEOGENESIS AFTER DISTRACTION

Participants: E.P. Frankenburg, K.M. Kozloff, E.A. Smith, T. Adachi, J.A. Baker, K.A. Sweet, B. Nolan, A.I. Caplan, S.A. Goldstein

Keywords: distraction osteogenesis, marrow ablation, fracture repair

Introduction

Distraction osteogenesis is a process used for limb lengthening and generation of large volumes of bone to repair defects. The clinical success of this technique depends greatly on the timing and rate of distraction, as well as fixator stiffness, which all relate to the specific mechanical environment created and its effects on the regenerating tissue. Animal models have been extremely useful tools for investigating these specific relationships, although the clinical difficulty of bone consolidation after distraction is often overlooked. Tibial marrow ablation has been shown to increase bone formation at distant sites (i.e. mandible), possibly by some circulating osteogenic peptide. It is therefore reasonable to hypothesize that performing marrow ablation after distraction may increase cytokine expression, which may then increase cell proliferation and subsequent bone formation within the distraction gap.

Materials and Methods

Four threaded K-wires (0.067") were placed in the femora of adult male Sprague-Dawley rats, and attached to a custom, external distraction fixator. A subperiosteal transverse osteotomy was performed in the mid-diaphysis. Both femora were distracted 0.25mm twice each day from day 5.5 until day 17, for a total of 5.75mm distraction. On day 17, a random tibia on each animal was vacuum marrow ablated and flushed with saline. Rats were sacrificed 6 days after marrow ablation (Day 23), both femora were harvested and frozen in isopentane for 3 minutes. The distraction gaps were removed from the bone and cryosectioned in the longitudinal direction. New bone formation will be assessed using histomorphometric techniques, and immunohistochemistry will be performed to analyze specific protein expression.

Progress

Eight animals have undergone surgery and distraction, then sacrificed and their femora harvested. All of the femoral specimens were snap-frozen, and prepared for cryosectioning. After sectioning, the specimens were stained using Hematoxylin and Eosin, Toluidine Blue, and Safranin-O/Fast Green on sequential sections. Point counting methods using the slides stained with Toluidine Blue were established and completed by three investigators. Protocols for digital color analysis and immunohistochemistry are also being finalized.