EVALUATION OF REPAIR BLASTEMA CELLS IN THE HEALING PROCESS OF BONE

Participants: K.M. Kozloff, E.P. Frankenburg, E.A. Smith, B.T. Nolan, K. Lucas, R.L. Taylor, J.A. Baker, K.A. Sweet, S.A. Goldstein

Keywords: fracture healing, repair blastema, animal model

Introduction

It has been hypothesized that cortical size defects in bone might be induced to heal through the implantation of tissue engineering constructs composed of mesenchymal stem cells in a carrier matrix. The function of these pluropotential cells and their role in the repair, however, is unknown. The purpose of this study is to evaluate the effect of interposing a repair blastema involving these stem cells between diaphyseal vertical defect model.

Materials and Methods

The experimental model was designed to provide a reproducible surgical construct that would enable the investigators to statistically evaluate the effect of the repair blastema. The model utilized male Fisher rats, weighing approximately 400 grams. On one femur in each animal (randomly chosen), a four-pin external fixator was implanted utilizing a series of templates which ensured consistent percutaneous placement of the pins centralized on the femur. After placement of the external fixator, the mid-diaphysis of the femur was exposed through a one centimeter incision and subperiosteal dissection. Utilizing an oscillating microsaw with constant irrigation, a five millimeter gap was produced. Depending on the experimental group, the gap was filled with mesenchymal stem cells in a collagen carrier, fibroblasts in a collagen carrier, or nothing. The wound was then closed in layers and the skin stapled. A similar procedure was administered on the contralateral femur, where the addition of a blastema was dependent on the experimental group. The animals were maintained on anesthesia, and then were recovered and allowed to ambulate at will. After four weeks, the animals were sacrificed, and both femurs were excised and fixed in 70% EtOH for histologic analysis.

As noted above, each of the animals were randomly assigned to an experimental group, and the experimental procedures were randomly assigned to the right or left femur. Since this is a pilot study, three experimental groups will be studied, with four rats in each group. The specific treatment groups include mesenchymal stem cells vs. control dermal fibroblasts, mesenchymal stem cells vs. empty, fibroblasts vs. empty, and empty vs. empty. All animals were maintained on oral antibiotics, radiographed weekly, and sacrificed at four weeks post-operative.

Progress

A total of seventeen animals have undergone surgery, four animals in the MSC vs. fibroblast group, eight animals in the MSC vs. empty group, three animals in the fibroblast vs. empty group, and two animals in the empty vs. empty group. All of these animals were sacrificed at four weeks after surgery, and all but two animals had their femurs dissected and placed in 70% ethanol. The remaining two animals had their femurs dissected free, and the bones were subsequently snap frozen for histologic purposes.

All femoral specimens fixed in 70% EtOH were decalcified, embedded in paraffin and sectioned. Sequential sections were then subsequently stained with hematoxylin and eosin, Safranin O/ Fast green, or toluidine blue. The snap-frozen femurs were cryosectioned and similarly stained.

Results

With light microscopy, it was evident that the empty gaps contained little or no new bone formation, but contained mainly fibrous tissue. The gaps treated with fibroblasts or MSCs showed minimal new bone formation. Although one could conclude that MSCs loaded in a collagen gel do not help promote fracture healing, it appears that the collagen gel carrier might actually inhibit fracture healing due to its influence in causing contraction by the included cells. Studies are continuing with variations alternative matrices and to increase the statistical power.