Session V - Fracture Healing

Potentiation of Fracture Healing Using IGF-I Transfected Marrow Mesenchymal Cells

Francis H. Shen, MD; Jennifer M. Visger, BS; Shepard R. Hurwitz, MD; David R. Diduch, MD; Gary Balian, PhD, University of Virginia School of Medicine, Charlottesville, VA

Purpose: Consistent fracture healing continues to be an area of interest in orthopaedic trauma. Therapeutic measures directed at accelerating the stages of healing have included the use of bone marrow elements. Marrow elements, which include mesenchymal cells, growth and differentiation factors, and cell adhesion factors, play an important role in bone and musculoskeletal tissue repair. Pluripotent mesenchymal cells transfected with growth factors may have a role in potentiating fracture healing. The advantages of using these cells include both the primary osteogenic properties of the mesenchymal cells, coupled with secondary therapeutic benefits associated with the delivery of a gene product. Theoretically, the ability for transfected cells to produce increased levels of therapeutic growth hormone at fracture sites allows for elevated local levels while minimizing wider systemic side-effects. We hypothesized that transfected mesenchymal cells preferentially localize to a fracture site and potentiate fracture healing in a mouse model.

Method: A previously cloned pluripotential cell line derived from bone marrow stroma of balb/c mice was transfected with a gene for neomycin resistance (neo) for selection, and either insulin-like growth factor I (D1-IGF-I) for therapeutic purposes, or _-galactosidase (D1-BAG) as a marker to trace the cells by histochemical staining. Under anesthesia through a midline knee incision the right femur of each mouse underwent retrograde intramedullary stabilization with a 25-gauge needle. Closed transverse femur fractures (type 32-A3) were then produced in 108 mice. Mice were then divided into 3 groups receiving one million cells of either D1-IGF-I or D1-BAG by intravenous tail vein injection; mice injected with lactated Ringers (LR) solution served as control. At 2, 4, and 6 weeks, the mice were euthanized and femurs analyzed by DNA-PCR (using neo primer) to detect for the presence and location of transplanted cells. Histological sections were performed at equivalent time periods looking for _-galactosidase containing cells. Matrix mineralization and callus maturation were evaluated by histomorphometry.

Results: In the femurs examined at all time periods we were able to detect transfected cells by DNA-PCR for neo in D1-IGF-I and D1-BAG groups. We were also able to detect the presence of transfected cells histologically as an increased number of positively staining (blue) cells in the D1-BAG over the control group in the bone marrow and at the fracture site. As tissue healing progressed, the initial neo levels and number of blue cells in fracture calluses decreased with time. At all time periods, the neo signal and number of blue cells were consistently greater in the fractured compared with the non-fractured femur. Analysis of fracture calluses in the D1-IGF-I group demonstrated an earlier appearance and more rapid progression >from cartilaginous to osseous callus compared to the control group. By 4 weeks mice receiving D1-IGF-I cells had greater mineralized matrix and nearly completely ossified callus compared to the control group.

Discussion and Conclusions: The results indicate that after transfection, the cloned mesenchymal cells retain their ability to return to and repopulate the bone marrow. They appear to localize preferentially at the fracture site and decrease in number with time. The fracture healing process was accelerated in the D1-IGF-I group reflected by an increase in mineralized matrix and the earlier appearance of an osseous callus. The ability to combine a mesenchymal cell with the gene of a growth factor which is known to stimulate both chondrogenesis and osteogenesis in vivo may lead to the development of a cell-based gene delivery system for treatment of traumatic injuries. The results of our experiments suggest that using transfected marrow mesenchymal cells as such a delivery system could localize the delivery of the therapeutic gene to the injury site and potentiate fracture healing. Further studies include investigating the influence of these cells on biomechanical properties and strength testing.