title: Osteogenic differentiation of mesenchymal stem cells from osteopenic rats subjected to physical activity with and without nitric oxide synthase inhibition.
abstract: Physical activity has potent and complex effects on bones. We hypothesized that physical activity has a positive effect upon osteopenic rat bones because it stimulates osteogenic differentiation of bone marrow mesenchymal stem cells (MSCs). We also postulated that local nitric oxide concentrations mediate the effects of physical activity on bones. The objective of this study was to investigate the osteogenic differentiation in vitro of MSCs from osteopenic female rats subjected to physical activity with and without nitric oxide synthase inhibition. We used MSCs from the femurs of Wistar female rats divided into six groups: Group 1, sham-operated (control); Group 2, sedentary osteopenic; Group 3, active osteopenic; Group 4, sham-operated with L-NAME; Group 5, sedentary osteopenic with L-NAME; and Group 6, active osteopenic with L-NAME. The cells were cultured at 37 degrees C and 5% CO2. Cells were phenotypically characterized with anti-CD45, anti-CD90, anti-CD73, and anti-CD54 using a FACScan cytometer. MSCs were cultured in osteogenic medium for 7, 14 and 21 days. Alkaline phosphatase activity, the capacity of dimethylthiazol conversion in formazan crystals, collagen synthesis and the number of mineralized nodules were analyzed. The means of all of the variables were compared using the SNK test. MSCs did not express CD45 in 96.94% of the cells, but there was expression of CD73, CD54 and CD90 in 93.99%, 95.10% and 86.77% of the cells, respectively. MSCs from osteopenic rats showed less osteogenic differentiation. Surprisingly, physical activity increased the osteogenic differentiation of MSCs in osteopenic rats. Inhibition of nitric oxide synthase in vivo had a negative effect upon the osteogenic potential of MSCs from normal rats and from osteopenic rats subjected to physical activity. Our results suggest that nitric oxide stimulates MSCs osteogenic differentiation and that nitric oxide mediates the beneficial effects of physical activity upon MSCs osteogenic differentiation.
authors.affiliation: Departamento de Clínica e Cirurgia Veterinárias, Escola de Veterinária da Universidade Federal de Minas Gerais, Avenida Presidente Antônio Carlos, 6627-CEP, 30.161-970, Belo Horizonte, Minas Gerais, Brazil.
authors.forename: N M
authors.initials: NM
authors.lastname: Ocarino
authors.forename: J N
authors.initials: JN
authors.lastname: Boeloni
authors.forename: A M
authors.initials: AM
authors.lastname: Goes
authors.forename: J F
authors.initials: JF
authors.lastname: Silva
authors.forename: U
authors.initials: U
authors.lastname: Marubayashi
authors.forename: R
authors.initials: R
authors.lastname: Serakides
mesh_terms: D000469:Alkaline Phosphatase / Q000378:metabolism; D000704:Analysis of Variance; D000818:Animals; D015703:Antigens, CD / Q000378:metabolism; D001851:Bone Diseases, Metabolic / Q000503:physiopathology*; D002113:Calcification, Physiologic / Q000187:drug effects; D002454:Cell Differentiation / Q000187:drug effects; D003094:Collagen / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D005562:Formazans / Q000378:metabolism; D016130:Immunophenotyping; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology*; D009569:Nitric Oxide / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000378:metabolism; D010012:Osteogenesis / Q000187:drug effects; D010052:Ovariectomy; D010805:Physical Conditioning, Animal; D011897:Random Allocation; D051381:Rats; D017208:Rats, Wistar; D013778:Tetrazolium Salts / Q000378:metabolism
chemical_list: D015703:Antigens, CD; D004791:Enzyme Inhibitors; D005562:Formazans; D013778:Tetrazolium Salts; C079782:MTT formazan; D009569:Nitric Oxide; D003094:Collagen; D019001:Nitric Oxide Synthase; D000469:Alkaline Phosphatase; D019331:NG-Nitroarginine Methyl Ester