COPYRIGHT © 2006 BY THE JOURNAL OF BONE AND JOINT SURGERY, INCORPORATED  Use of Genetically Engineered Bone-Marrow Stem Cells to Treat Femoral Defects: An Experimental Study BY QUANJUN CUI, MD, ZENGMING XIAO, MD, XUDONG LI, MD, PHD, KHALED J. SALEH, MD, MSC, FRCSC, AND GARY BALIAN, PHD Background: Treatment of osteonecrosis continues to be a challenging problem. The replacement of necrotic bone with graft materials that promote osteogenesis and angiogenesis may provide better outcomes for early stage dis- ease. In this study, genetically engineered bone-marrow stem cells were used to enhance repair of a defect in the dis- tal aspect of the femur. Methods: Cloned bone-marrow stem cells were transfected with traceable genes. Osteoblastic and angiogenic prop- erties of the cells were analyzed. A defect was created bilaterally in the distal portion of the femur of twenty-four mice to mimic a core decompression procedure. The cloned cells were transplanted into each defect of the right femur while the left femur served as control. Bone formation was evaluated radiographically and histomorphometrically. In addition, in twenty-four additional mice, the cells were injected into subcutaneous sites, muscles, and into the renal capsule (eight mice in each group) to evaluate ectopic osteogenesis. Results: Radiopaque tissue appeared two weeks after the cells were transplanted into bone defects and at ectopic sites. Histologic analysis demonstrated that these tissues consisted of newly formed bone from transplanted cells that expressed traceable genes. Four of six bone defects that received cell transplantation were filled with new bone at four weeks, and all of the defects (n = 6) demonstrated complete healing at six weeks. On the control side, com- plete repair was seen in only two of six bone defects at four weeks and in three of six defects at six weeks. Histomor- phometric analysis showed that transplantation of marrow stem cells into bone defects produced more bone at an earlier time-point than occurred in the controls. Conclusions: This study demonstrated that cloned bone-marrow stem cells can directly form bone after transplanta- tion into bone defects and at ectopic sites, indicating that the in vitro expanded bone-marrow stem cells can serve as a graft material to enhance bone repair and to treat osteonecrosis. Clinical Relevance: As an alternative graft material, bone-marrow stem cells may provide new and as yet technologically unachievable solutions to many clinical problems in the areas of musculoskeletal reconstruction and tissue regeneration. reatment of osteonecrosis of the femoral head contin- ues to be a challenging problem 1-4 . Hip arthroplasty is generally successful, but long-term results may be less than optimal, especially in active young adults 2,5 . Alternative treatments such as core decompression and bone-grafting procedures provide some benefits, but long-term success is not always reliable 6-12 . Replacement of necrotic bone at an early stage of the disease to promote osteogenesis and angiogenesis as well as to heal subchondral bone lesions may provide better outcomes for patients with the disease 13-15 . Autogenous cancel- lous bone is currently the gold-standard graft material, but its supply is limited and, in addition, donor-site complications can occur. Allografts are useful but not as desirable as au- tografts because of the problem of immunogenicity and the potential to transmit disease 16-19 . Growth and differentiation factors, delivered in the form of proteins and genes, have been widely tested both in animal studies and clinically for their potential to enhance bone repair, including the treatment of osteonecrosis, but no long-term results are available 1,13,15,20-26 . Autologous marrow transplantation has been used as a bone- graft substitute in the treatment of fracture nonunions and has been shown to be effective. Successful use of bone-marrow transplantation in the treatment of osseous defects is based on the osteogenic property of these cells 27,28 . However, the preva- lence of stem cells in a marrow preparation is low (approxi- mately one in 100,000 cells) which means that cell expansion will be needed if these cells are to be used for the treatment of bone defects 29 . Since few ideal substitutes for or alternatives to T