USE OF GENETICALLY ENGINEERED BONE MARROW STEM CELLS TO TREAT OSTEONECROSIS: AN EXPERIMENTAL STUDY

Abstract

Introduction: Treatment of osteonecrosis continues to be a challenging problem in orthopedic practice. Arthroplasty is generally successful but long-term results are inferior especially in young adults. Alternative treatments such as core decompression and trap-door procedures provide only temporary benefits and need much improvement. The replacement of necrotic bone to promote osteogenesis and angiogenesis and healing subchondral bone are future approaches. Autogenous cancellous bone is the preferred graft material but its supply is limited. Allografts are useful but not as desirable as autografts. Substitutes for bone grafts have been actively researched but few are available currently. In this study, we have attempted to use genetically engineered bone marrow stem cells in order to enhance the healing of a bone defect in a mouse model.

Materials and Methods: A bone marrow stem cell was cloned from Balb/c mice and transfected with LacZ and neomycin resistance genes. The cells were cultured for 7 to 10 days and both the osteoblastic and angiogenic properties of the cells were examined using Northern blots to detect osteocalcin and VEGF gene expression. The cells were also analyzed for alkaline phosphatase activity to demonstrate the osteoblastic phenotype of the cells. A suspension containing 2 x 10⁷ cells/ml phosphate buffered solution was prepared for cell transplantation. A total of forty-eight, 8-week old Balb/c mice were used in this study. A 1.2 mm defect was created bilaterally with an electric drill in the femurs of 24 mice to mimic the core decompression and trap-door procedures. 2 x 10⁶ cells were transplanted into each defect of the right femur while the left femur served as a control trap-door defect which was injected with PBS but without cells. An equal number of cells were injected either at subcutaneous sites, in the hindquarter muscles, or into the renal capsule (8 mice in each site) to evaluate ossification at ectopic sites. Animals were sacrificed at 2, 4, 6 and 8 weeks. Defect repair was evaluated radiographically and the contribution to osteogenesis by transplanted cells was studied histomorphometrically using tissue sections stained with X-gal as well as biochemically on DNA extracts using primers for the neomycin resistance gene.

Results: Radiopaque tissue appeared two weeks after the cells were transplanted into bone defects, muscle, subcutaneous sites, and the renal capsule. Histological analysis demonstrated that these tissues consist of newly formed bone from transplanted cells that stained positively with X-gal and contained neo DNA. The repair tissue did not contain cartilaginous areas indicating that ossification surrounding the D1-BAG cells was not through the endochondral process. At four weeks, 4 of 6 femora showed a defect that was filled with new bone. At 6 weeks, all of the defects (6 of 6) contained fully restored bone. However, in the control side that was injected with PBS (no cells) only 2 of 6 at 4 weeks, 3 of 6 at 6 weeks, and 5 of 6 at 8 weeks showed complete repair. All histological sections of bone defects (n = 24) were examined histomorphometrically using a computerized image analysis system. Transplantation of marrow stem cells into bone defects produced more bone at an earlier time point than controls and, the process of enhanced ossification continued throughout the healing process.

Discussion: The cloned bone marrow stem cell can directly form bone after transplantation into bone defects and into ectopic sites, indicating that the in vitro expanded bone marrow stem cells can serve as a grafting material to enhance healing of bone defects and the treatment of osteonecrosis. In addition, this study demonstrates that genetic labelling is a useful tool in studies of cell differentiation in vivo and that bone marrow stem cells may be useful as a carrier of genetically-engineered factors in the treatment of skeletal diseases.