Effect of Bone Morphogenetic Protein-2-Expressing Muscle-Derived Cells on Healing of Critical-Sized Bone Defects in Mice
Cells that express bone morphogenetic protein-2 (BMP-2) can now be prepared by transduction with adenovirus containing BMP-2 cDNA. Skeletal muscle tissue contains cells that differentiate into osteoblasts on stimulation with BMP-2. The objectives of this study were to prepare BMP-2-expressing muscle-derived cells by transduction of these cells with an adenovirus containing BMP-2 cDNA and to determine whether the BMP-2-expressing muscle-derived cells would elicit the healing of critical-sized bone defects in mice.
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 yen 10(5)) were implanted into a 5-mm-diameter critical-sized skull defect in female SCID (severe combined immunodeficiency strain) mice with use of a collagen sponge as a scaffold. Healing in the treatment and control groups was examined grossly and histologically at two and four weeks. Implanted cells were identified in vivo with use of the Y-chromosome-specific fluorescent in situ hybridization (FISH) technique, and their differentiation into osteogenic cells was demonstrated by osteocalcin immunohistochemistry.
Skull defects treated with muscle cells that had been genetically engineered to express BMP-2 had >85% closure within two weeks and 95% to 100% closure within four weeks. Control groups in which the defect was not treated (group 1), treated with collagen only (group 2), or treated with collagen and muscle cells without adBMP-2 (group 3) showed at most 30% to 40% closure of the defect by four weeks, and the majority of the skull defects in those groups showed no healing. Analysis of injected cells in group 4, with 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 immunohistochemically for osteocalcin, indicating that they had differentiated in vivo into osteogenic cells.
This study demonstrates that cells derived from muscle tissue that have been genetically engineered to express BMP-2 elicit the healing of critical-sized skull defects in mice. The cells derived from muscle tissue appear to enhance bone-healing by differentiating into osteoblasts in vivo. Clinical Relevance: Ex vivo gene therapy with muscle-derived cells that have been genetically engineered to express BMP-2 may be used to treat nonhealing bone defects. In addition, muscle-derived cells appear to include stem cells, which are easily obtained with muscle biopsy and could be used in gene therapy to deliver BMP-2.
Available from: Xing Liu
- "Athymic nude mice injected with BMP9- transduced C2C12 cells into the quadriceps muscles demonstrated significant orthotopic bone formation on both X-ray and histologic evaluation   . Several recent studies have suggested that skeletal muscle may harbor multipotent MSCs as well as osteoblastic progenitor cells   . When adenovirally-delivered BMP9 (AdBMP9) was directly injected into the quadriceps muscles of athymic nude mice, there was evidence of increased osteoid matrix production and formation of mature lamellar bone compared to BMP2-and 7-treated groups. "
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ABSTRACT: Bone morphogenetic proteins (BMPs) are members of the TGF-β superfamily and play a critical role in skeletal development, bone formation and stem cell differentiation. Disruptions in BMP signaling result in a variety of skeletal and extraskeletal anomalies. BMP9 is a poorly characterized member of the BMP family and is among the most osteogenic BMPs, promoting osteoblastic differentiation of mesenchymal stem cells (MSCs) both in vitro and in vivo. Recent findings from various in vivo and molecular studies strongly suggest that the mechanisms governing BMP9-mediated osteoinduction differ from other osteogenic BMPs. Many signaling pathways with diverse functions have been found to play a role in BMP9-mediated osteogenesis. Several of these pathways are also critical in the differentiation of other cell lineages, including adipocytes and chondrocytes. While BMP9 is known to be a potent osteogenic factor, it also influences several other pathways including cancer development, angiogenesis and myogenesis. Although BMP9 has been demonstrated as one of the most osteogenic BMPs, relatively little is known about the specific mechanisms responsible for these effects. BMP9 has demonstrated efficacy in promoting spinal fusion and bony non-union repair in animal models, demonstrating great translational promise. This review aims to summarize our current knowledge of BMP9-mediated osteogenesis by presenting recently completed work which may help us to further elucidate these pathways.
Available from: Alexander Birbrair
- "Autologous cell transplantation circumvents the immunological and ethical concerns delaying cell therapy. Previous studies have discovered a population of multipotent skeletal muscle cells that differentiate into mesodermal lineages, including myogenic  , adipogenic , osteogenic , chondrogenic , endothelial , and under certain conditions, they can break germ-layer commitment and differentiate into ectodermal lineages, including neural cells [3,13–17]. "
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ABSTRACT: Reversing brain degeneration and trauma lesions will depend on cell therapy. Our previous work identified neural precursor cells derived from the skeletal muscle of Nestin-GFP transgenic mice, but their identity, origin, and potential survival in the brain are only vaguely understood. In this work, we show that Nestin-GFP+ progenitor cells share morphological and molecular markers with NG2-glia, including NG2, PDGFRα, O4, NGF receptor (p75), glutamate receptor-1(AMPA), and A2B5 expression. Although these cells exhibit NG2, they do not express other pericyte markers, such as α-SMA or connexin-43, and do not differentiate into the muscle lineage. Patch-clamp studies displayed outward potassium currents, probably carried through Kir6.1 channels. Given their potential therapeutic application, we compared their abundance in tissues and concluded that skeletal muscle is the richest source of predifferentiated neural precursor cells. We found that these cells migrate toward the neurogenic subventricular zone displaying their typical morphology and nestin-GFP expression two weeks after brain injection. For translational purposes, we sought to identify these neural progenitor cells in wild-type species by developing a DsRed expression vector under Nestin-Intron II control. This approach revealed them in nonhuman primates and aging rodents throughout the lifespan.
Available from: Yoshimasa Igarashi
- "The first challenge is to identify the source of cells for genetic engineering and transplantation . Bone marrow stromal cells (Riew et al., 1998; Musgrave et al., 2000; Cheng et al., 2001; Turgeman et al., 2001; Gysin et al., 2002; Partridge et al., 2002), periosteal cells (Breitbart et al., 1999), muscles (Riew et al., 1998; Lee et al., 2000, 2001, 2002; Cheng et al., 2001; Pelinkovic et al., 2001; Musgrave et al., 2002), and fibroblasts (Takayanagi et al., 1999; Franceschi et al., 2000; Krebsbach et al., 2000) have been successfully used as the source of transplanted cells in animal experiments. These cell types are strong candidates for clinical application, but these cause considerable challenge when applied in the dental clinics. "
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ABSTRACT: Several lines of evidence show that transplantation of osteoblastic cells or genetically engineered nonosteogenic cells expressing osteoblast-related genes into bone defects effectively promotes bone regeneration. To extend this possibility, we investigated whether oral mucosal fibroblasts are capable of differentiating into osteoblastic cells by conducting in vitro and in vivo experiments. We investigated the effects of bone morphogenetic protein-2 (BMP-2) on osteoblast differentiation of cultured fibroblasts isolated from canine buccal mucosa. We also transplanted green fluorescence protein (GFP)-expressing fibroblasts with gelatin/BMP-2 complexes into the subfascial regions of athymic mice, and investigated the localization of GFP-positive cells in the ectopically formed bones. The cultured canine buccal mucosal fibroblasts differentiated into osteoblastic cells by increasing their alkaline phosphatase (ALP) activity and Osteocalcin, Runx2, and Osterix mRNA expression levels in response to BMP-2. Transplantation experiments of GFP-expressing oral mucosal fibroblasts with gelatin/BMP-2 complexes revealed that 17.1% of the GFP-positive fibroblasts differentiated into ALP-positive cells, and these cells accounted for 6.2% of total ALP-positive cells in the ectopically formed bone. This study suggests that oral mucosal fibroblasts can differentiate into osteogenic cells in response to BMP-2. Thus, these cells are potential candidates for cell-mediated bone regeneration therapy in dentistry.
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