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Cui, Q., Azer, N.M., Saleh, K.J., Wang, G.J., Balian, G.
Department of Orthopaedic Surgery, University of Virginia, Box 800159, Charlottesville, Virginia 22908, USA. Corresponding Author: Quanjun Cui, MD. E-mail: qc4q@virginia.edu
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 bone defect in a mouse model.
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 48 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 injected it 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 labeling
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.