MARROW-DERIVED STROMAL CELL INJECTIONS INFLUENCE OSTEOGENESIS AFTER DISTRACTION

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

Keywords: distraction osteogenesis, mesenchymal stem cells, fracture repair

Introduction

Distraction osteogenesis is a process used for limb lengthening and generation of large volumes of bone to repair defects. The recent advent of cell-based therapies provides attractive options for facilitating bone regeneration after distraction. The osteo-chondrogenic potential of marrow-derived stromal cells (or mesenchymal stem cells, MSCs) in both ectopic sites and bone defects has been well established. The precise mechanism by which these cells are capable of stimulating bone regeneration and healing, however, is still unclear. In this study, a bilateral rat model of distraction osteogenesis was used to investigate the specific effects of injecting a large quantity of MSCs in either culture media or a collagen gel carrier into a distraction gap.

Materials and Methods

Surgery: Four threaded K-wires (0.067") were placed in the femora of adult male Fisher 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 6 until day 18, for a total of 6mm distraction.

Cell preparation: Whole femoral marrow plugs were obtained from syngeneic male Fisher rats, and dispersed by passage through 18 gauge needles. Cells were centrifuged, resuspended in DMEM + 10% fetal bovine serum, and incubated with a Di-I label prior to plating at a density of 1x106 cells per 75 mm flask. Cells were released by treatment with trypsin-EDTA for 6 min. at 37C, centrifuged, and resuspended in serum-free DMEM at the appropriate density. Collagen injections were prepared by mixing 0.25 ml of this cell suspension at a density of 10x106 cells/ml with 0.25 ml type I collagen gel (carrier), while culture media injections consisted of 0.5 ml of cell suspension at a density of 5x106.

Cell injection: On day 18, rats received cell+carrier injections in one random side, and injections of carrier alone into the contralateral limb. Injections were made directly into the distraction gap over a one minute period.

Tissue Analysis: Animals were sacrificed on day 21, both femora were harvested, and frozen in isopentane in a dry ice slurry for 3 minutes. The gaps were removed from the femora, and cryosectioned in the longitudinal direction. Analyses included histomorphometry, tissue staining and immunohistochemistry, and visualization of the Di-I labeled cells.

Progress

A total of 8 rats have received cell injections in either collagen gel (n=4) or culture media (n=4). Three rats in each group were sacrificed, their femurs dissected, and snap-frozen. The remaining specimens were sacrificed, their femurs dissected free, and then stored in 70% EtOH. After processing histologically, all specimens were sectioned longitudinally, and then stained with either Hemotoxylin and Eosin, Toluidine Blue, or Safranin O/ Fast Green. Protocols for cryosectioning, histology, and visualization of the cell labels are currently in progress.

Results

To date, little differences were seen in the healing of any treatment group. It is possible that the sacrifices are too early. In addition, the role of the injected cell in contrast to the carrier remains unclear.