Mobility of Indium-Labeled Mesenchymal Progenitor Cells During Augmentation of Bone Repair Following Distraction Osteogenesis

Participants: K.M. Kozloff, E.P. Frankenburg, L.A. Solchaga^*, E.A. Smith-Adaline, J.C. Taylor, D.P. Lennon^*, J.E. Dennis^*, J. Gao^*, M.R. Kilbourn^**, A.I. Caplan^*, S.A. Goldstein

* Skeletal Research Center, Case Western Reserve University

** Division of Nuclear Medicine, University of Michigan

Keywords: Distraction osteogenesis, mesenchymal stem cell, fracture healing, cell mobility, animal model

Introduction

Progenitor cells derived from bone marrow have been evaluated for their therapeutic repair potential in numerous musculoskeletal defects. Injection of marrow-derived progenitor cells into a fracture gap has been shown to enhance bone repair following distraction osteogenesis in the rat, however this response may have been due to genetic incompatibility between donor and recipient, not stem cell activity [1]. Furthermore, it is not known whether these cells remain localized to the fracture gap or whether they are mobilized toward other regions of repair or other organs. The purpose of this study was to track the mobility of marrow-derived progenitor cells upon injection into a consolidating distraction gap using Fisher rats as syngeneic donor and recipient animals.

Materials and Methods

Bilateral femoral osteotomies were created in adult male Fisher rats [1], and a custom, monolateral distraction fixator was applied. Six days of latency followed surgery, and beginning on day 7, femurs were distracted twice daily at 0.25 mm per distraction, for a total of 0.5 mm per day. The total distraction period lasted 12 days.

Mesenchymal progenitor cells (MPC) derived from adult male Fisher rats were obtained as previously described [2], and resuspended in serum free Dulbecco's modified Eagles medium at a concentration of 5 million cells per mL. Dermal fibroblasts (FIB) were obtained through a collagenase digestion, and resuspended at the same concentration. Indium[111] oxine, a commonly used radioactive marker, was added to each cell suspension and allowed to diffuse into the cells for 30 minutes, after which the suspensions were centrifuged to remove any free-floating indium from the supernatant. The cells were again resuspended at the desired concentrations of 5 million cells per mL [3]. Injection volumes of 0.5 mL were prepared and radiocounted prior to injection.

Following the distraction period (day 19), 2 rats received injections of radiolabeled MPC's in one femur and 3 rats received injections of radiolabeled dermal fibroblasts. All contralateral femurs received unlabeled media injections. The injection site was found by careful palpation of the distraction callus. A 21 gauge needle was inserted under fluoroscopy to visualize its placement within the center of the gap, and the 0.5 mL volume was injected over a period of approximately 70 seconds into the fracture site. Rats were allowed normal cage activity for 24 hours following the injection, then sacrificed, and the following tissues were dissected and analyzed for radiation dose: proximal femurs, distraction gaps, distal femurs, quadriceps, lung, spleen, heart, liver, brain, kidney, and testes.

A second group of animals received identical, non-radiolabeled injections of either MPC vs. media (n=2) or MPC vs. FIB (n=2) for long term study. These animals were sacrificed on day 35, 17 days post-injection, and analyzed for new bone volume by micro-ct, as well as precursor tissue composition by histology.

Results and Discussion

Radiocounts were normalized by the initial indium dose to obtain values of the percent injected dose per gram of tissue and percent injected dose per organ. Levels less than 1% were considered to be insignificant.

As shown in Figure 1, femoral activity is located predominantly in the distraction gap and in the quadriceps after injection of both progenitor cells and fibroblasts. Values of percent injected dose greater than 100 result from extremely high dose-to-tissue-weight measures. Low levels of radioactivity are seen in the proximal and distal femur, indicating that no significant amount of radiolabeled cells are in the bone or intramedullary space adjacent to the distraction gap. All contralateral distraction gaps, distal and proximal femurs, and quadriceps showed doses below background levels of 1 percent, indicating that no cells migrated to the contralateral side (data not shown).

Figure 1: % injected dose of In[111] per g of tissue a) MPC's b) FIB's

Figure 2: % injected dose of In[111] per organ a) MPC's b) FIB's

Figure 2 illustrates the injected cell population in the organs studied. Liver, kidney, and lung contain the highest amounts of cells, and in general, MPC's appear to be expressed in higher amounts in these organs than fibroblasts. This trend is most evident in the liver.

These results suggest that both fibroblasts and MPC's, when injected directly into a healing distraction gap, maintain high population levels at the injection site. However, MPC's appear to be more mobile in their abilities to reach organs such as the lung, liver, and kidney, than fibroblasts. Differences seen between rats in gap tissue and quadriceps radioactivity may reflect needle placement during injection. MPC rat 1 and FIB rat 2 have the highest expression of indium labeling in the gap, and low expression in the quadriceps. However, these animals show the greatest activity in other organs compared to their cell groups. This suggests that if the injection is localized directly in the distraction gap tissue, there is less uptake by the surrounding muscles, and the cells may be more easily mobilized to other distant organs. Conversely, MPC rat 2 and FIB rat 1 show high expression in the quadriceps, low expression in the gap, and the lowest expression in the other organs, suggesting that if a significant proportion of the injected volume enters the muscle, fewer cells are able to exit to other organs, and the cells may remain localized to the muscle tissue. An important result from this experiment shows that there is no significant migration of transplanted cells from one distraction gap to the contralateral side, suggesting that these cells are not being targeted to other healing tissues in the body.

Micro-ct analysis of new bone volume from the long term animals showed mixed results from both experimental groups. Subsequent analysis performed at Case Western Reserve University on subcultures of the injected cells indicated poor osteogenic potential measured by ingrowth in subcutaneously implanted ceramic cubes. Further investigation is required to determine whether the poor healing response from the progenitor cells was due to poor cell prep. Histology of these animals in currently in progress.

This work has been submitted as an abstract to the 2001 ASME Summer Bioengineering Conference.

References

[1] Richards et al, 1999, J. Orthop. Res, 17:900-908 [2] Dennis et al, 1993, J

Oral Implant, 19:106-137 [3] Gao et al, In Press 2000, Cells, Tissues, Organs