Degeneration of articular cartilage in osteoarthritis is a serious medical problem. We have isolated a population of cells from the connective tissue of mammals termed mesenchymal stem cells (MSCs) for their apparent unlimited growth potential and their ability to differentiate into several phenotypes of the mesodermal lineage, including cartilage and bone. These qualities make them ideal candidates for cartilage repair. We isolated MSCs from adult rabbit muscle and cultured them in vitro into porous polyglycolic acid polymer matrices. The matrices were implanted into 3-mm-diameter full thickness defects in rabbit knees with empty polymer matrices serving as the contralateral controls. The implants were harvested 6 and 12 weeks postop. At 6 weeks, the controls contained fibrocartilage while the experimentals seemed to contain undifferentiated cells. By 12 weeks postop, the controls contained limited fibrocartilage and extensive connective tissue, but no subchondral bone. In contrast, the implants containing MSCs had a surface layer of cartilage approximately the same thickness as normal articular cartilage and normal-appearing subchondral bone. There was good integration of the implant with the surrounding tissue. Implantation of MSCs into cartilage defects appears to effect repair of both the articular cartilage and subchondral bone. Studies are ongoing to further characterize the use of MSCs for cartilage repair.
We have previously shown a population of putative mesenchymal stem cells in the connective tissue surrounding embryonic avian skeletal muscle. These cells differentiate into at least five recognizable phenotypes in culture: fibroblasts, chondrocytes, myotubes, osteoblasts, and adipocytes. We have now isolated a similar population of cells from fetal and newborn rat skeletal muscle. Cells from rat leg muscle were dissected, minced, and then enzymatically digested with a collagenase-dispase solution. The dissociated cells were plated and allowed to differentiate into two recognizable populations: myotubes and stellate mononucleated cells. The cells were then trypsinized, filtered through a 20 microm filter to remove the myotubes, frozen at -80 degrees C, then thawed and replated. In culture the cells maintained their stellate structure. However, under treatment with dexamethasone, a nonspecific differentiating agent, seven morphologic conditions emerged: cells with refractile vesicles that stained with Sudan black B (adipocytes), multinucleated cells that spontaneously contracted in culture and stained with an antibody to myosin (myotubes), round cells whose extracellular matrix stained with Alcian blue, pH 1.0 (chondrocytes), polygonal cells whose extracellular matrix stained with Von Kossa's stain (osteoblasts), cells with filaments that stained with an antibody to smooth muscle a-actin (smooth muscle cells), cells that incorporated acetylated low density lipoprotein (endothelial cells), and spindle-shaped cells that grew in a swirl pattern (fibroblasts). The initial population is tentatively classified as putative mesenchymal stem cells. The presence of these cells point to the existence of stem cells in the postembryonic mammal that could provide a basis for tissue regeneration as opposed to scar tissue formation during wound healing.
Bone morphogenetic protein has previously been shown to induce the formation of cartilage and bone in vivo. We have isolated a population of mesenchymal stem cells from rat skeletal muscle capable of forming multiple mesodermal morphologies in vitro. These cells were treated with recombinant human bone morphogenetic proteins-2 and -4 to determine the differentiation-inducing activities of bone morphogenetic protein on these cells. The mesenchymal stem cells were cultured in medium with 10% preselected horse serum containing 0 to 100 ng/ml recombinant human bone morphogenetic proteins-2 or -4 for a maximum of 4 weeks. Control cultures maintained the stellate morphology of mesenchymal stem cells. Cultures treated with recombinant human bone morphogenetic protein-2 exhibited discrete cartilage nodules and mineralized bone nodules. The first increase in chondrogenesis was seen at 0.5 ng/ml. Cultures treated with recombinant human bone morphogenetic protein-4 also exhibited an increase in chondrogenesis at the higher concentration of 2 ng/ml. Skeletal myotubes and adipocytes also appeared in cultures treated with either bone morphogenetic protein. Mesenchymal stem cells do respond to inductive factors, but bone morphogenetic proteins-2 and -4 were not specific for the induction of cartilage and bone.
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