Effect of bone morphogenetic protein-2-expressing muscle-derived cells on healing of critical-sized bone defects in mice

Background: Cells that express bone morphogenetic protein-2 (BMP-2) can now be prepared by transduction with adenovirus containing BMP-2 cDNA. Skeleta l muscle tissue contains cells that differentiate into osteoblasts on stimu lation with BMP-2. The objectives of this study were to prepare BMP-2-expre ssing muscle-derived cells by transduction of these cells with an adenoviru s containing BMP-2 cDNA and to determine whether the BMP-2-expressing muscl e-derived cells would elicit the healing of critical-sized bone defects in mice. Methods: Primary cultures of muscle-derived cells from a normal male mouse were transduced with adenovirus encoding the recombinant human BMP-2 gene ( adBMP-2). These cells (5 x 10(5)) were implanted into a 5-mm-diameter criti cal-sized skull defect in female SCID (severe combined immunodeficiency str ain) mice with use of a collagen sponge as a scaffold. Healing in the treat ment and control groups was examined grossly and histologically at two and four weeks. Implanted cells were identified in vivo with use of the Y-chrom osome-specific fluorescent in situ hybridization (FISH) technique, and thei r differentiation into osteogenic cells was demonstrated by osteocalcin imm unohistochemistry. Results: Skull defects treated with muscle cells that had been genetically engineered to express BMP-2 had >85% closure within two weeks and 95% to 10 0% closure within four weeks. Control groups in which the defect was not tr eated (group 1), treated with collagen only (group 2), or treated with coll agen and muscle cells without adBMP-2 (group 3) showed at most 30% to 40% c losure of the defect by four weeks, and the majority of the skull defects i n those groups showed no healing. Analysis of injected cells in group 4, wi th the Y-chromosome-specific FISH technique showed that the majority of the transplanted cells were located on the surfaces of the newly formed bone, but a small fraction (approximately 5%) was identified within the osteocyte lacunae of the new bone. Implanted cells found in the new bone stained imm unohistochemically for osteocalcin, indicating that they had differentiated in vivo into osteogenic cells. Conclusions: This study demonstrates that cells derived from muscle tissue that have been genetically engineered to express BMP-2 elicit the healing o f critical-sized skull defects in mice. The cells derived from muscle tissu e appear to enhance bone-healing by differentiating into osteoblasts in viv o. Clinical Relevance: Ex vivo gene therapy with muscle-derived cells that hav e been genetically engineered to express BMP-2 may be used to treat nonheal ing bone defects. In addition, muscle-derived cells appear to include stem cells, which are easily obtained with muscle biopsy and could be used in ge ne therapy to deliver BMP-2.