Long-term spaceflights induce bone loss as a result of profound modifications of bone remodeling, the modalities of which remain unknown in humans. We measured intact parathyroid hormone (PTH) and serum calcium; for bone formation, serum concentrations of bone alkaline phosphatase (BAP), intact osteocalcin (iBGP), and type 1 procollagen propeptide (PICP); for resorption, urinary concentrations (normalized by creatinine) of procollagen C-telopeptide (CTX), free and bound deoxypyridinoline (F and B D-Pyr), and Pyr in a 36-year-old cosmonaut (RTO), before (days −180, −60, and −15), during (from days 10 to 178, n = 12), and after (days +7, +15, +25, and +90) a 180-day spaceflight, in another cosmonaut (ASW) before and after the flight. Flight PTH tended to decrease by 48% and postflight PTH increased by 98%. During the flight, BAP, iBGP, and PICP decreased by 27%, 38%, and 28% respectively in CM1, and increased by 54%, 35%, and 78% after the flight. F D-Pyr and CTX increased by 54% and 78% during the flight and decreased by 29% and 40% after the flight, respectively. We showed for the first time in humans that microgravity induced an uncoupling of bone remodeling between formation and resorption that could account for bone loss.
Background: Microgravity induces bone loss by mechanism(s) that remain largely unknown. Methods: We measured biochemical markers related to bone remodeling in two cosmonauts before, during, and after 21- and 180-day space flights, respectively. Results: During both flights, type I procollagen propeptide and bone alkaline phosphatase decreased as early as 8 days after launch. Undercarboxylated osteocalcin percentage increased early and remained high during both flights. Vitamin K supplementation restored carboxylation of osteocalcin during the long-term flight. Urinary and serum C-telopeptide of type I collagen (CTX) increased as early as day 8 of the flights; the increase was greater in serum than in urine. Pyridinoline, free deoxypyridinoline, and N-telopeptide increased less than CTX during the short-term space flight. The circadian rhythm of bone resorption assessed by urine CTX and free deoxypyridinoline was not altered by microgravity. Conclusion: Vitamin K metabolism or action and bone remodeling may be altered in cosmonauts.
In this study we found that weight gain, related to refeeding only, reversed the anorexia nervosa-induced uncoupling of bone remodelling and restored circadian variation of a bone resorption marker.
Six days of microgravity (Bion10 mission) induced dramatic shape changes in ROS 17/2.8 osteoblasts (7). During the Foton 11 and 12 space flights, we studied the kinetics (0-4 days) of ROS 17/2.8 morphology and adhesion, the relationships between adhesion and cell cycle progression after 4 days in space, and osteoblastic growth and activity after 6 days in space. Quantitative analysis of high-resolution adhesion [focal adhesion area imaged by total interference reflection fluorescent microscopy (TIRFM)] and integrin-dependent adhesion (imaged on confocal microscope by vinculin and phosphotyrosine staining) as well as cell cycle phase classification [Ki-67 staining, S-G2, mitotic cells and G1 (postmitotic cells)] were performed using programs validated in parabolic flight and clinostat. We observed disorganization of the cytoskeleton associated with disassembling of vinculin spots and phosphorylated proteins within focal contacts with no major change in TIRFM adhesion after 2 and 4 days of microgravity. Postmitotic cells, alone, accounted for the differences observed in the whole population. They are characterized by immature peripheral contacts with complete loss of central spots and decreased spreading. Osteocalcin, P1CP and alkaline phosphatase, and proliferation were similar in flight cells and 1 g centrifuge and ground controls after 6 days. In conclusion, microgravity substantially affected osteoblastic integrin-mediated cell adhesion. ROS17/2.8 cells responded differently, whether or not they were cycling by reorganizing adhesion plaque topography or morphology. In ROS 17/2.8, this reorganization did not impair osteoblastic phenotype.
Studies performed at tissular (three-dimensional, 3-D) or cellular (two-dimensional, 2-D) levels showed that the loading pattern plays a crucial role in the osteoblastic physiology. In this study, we attempted to investigate the response of a 3-D osteoblastic culture submitted to either no external stress or static or dynamic stresses. Rat osteosarcoma cells (ROS 17/2.8) were embedded within collagen type I lattices and studied for 3 weeks. Entrapment and proliferation of cells within the hydrated collagen gel resulted in the generation of contractile forces, which led to contraction of the collagen gel. We used this ability to evaluate the influence of three modes of mechanical stresses on the cell proliferation and differentiation: (1) the freely retracted gels (FRG) were floating in the medium, (2) the tense gels (TG) were stretched statically and isometrically, with contraction prevented in the longitudinal axis, and (3) the dynamic gels (DG) were floating gels submitted to periodic stresses (50 or 25 rpm frequency). Gels showed maximum contraction at day 12 in 50 rpm DG, followed by 25 rpm DG, then FRG (88%, 81%, 70%, respectively) and at day 16 in TG (33%). The proliferation rate was greater in TG than in FRG (ϩ52%) but remained low in both DGs. Gel dimensions were related to the collagen concentration and on a minor extent to cell number. Cells in DG appeared rounder and larger than in other conditions. In TG, cells were elongated and oriented primarily along the tension axis. Scanning electron microscopy (SEM) showed that tension exerted by cells in TG led to reorientation of collagen fibers which, in turn, determined the spatial orientation and morphology of the cells. Transmission electron microscopy (TEM) performed at maximum proliferation showed a vast majority of cells with a distended well-developed RER filled with granular material and numerous mitochondria. Alkaline phosphatase activity peaked close to the proliferation peak in FRG, whereas in TG, a biphasic curve was observed with a small peak at day 4 and the main peak at day 16. In DG, this activity was lower than in the two other conditions. A similar time course was observed for alkaline phosphatase gene expression as assessed by Northern blots. Regardless of the conditions, osteocalcin level showed a triphasic pattern: a first increase at day 2, followed by a decrease from day 4 to 14, and a second increase above initial values at day 18. Microanalysis-x indicated that mineralization occurred after 14 days and TEM showed crystals within the matrix. We showed that static and dynamic mechanical stresses, in concert with 3-D collagen matrices, played a significant role on the phenotypic modulation of osteoblast-like cells. This experimental model provided a tool to investigate the significance and the mechanisms of mechanical activity of the 3-D cultured osteoblast-like cells. J. Cell. Biochem. 76:217-230, 1999. 1999 Key words: ROS 17/2.8; type I collagen; ultrastructure; proliferation; alkaline phosphatase; osteocalcin; mineralization...
Studies performed at tissular (three-dimensional, 3-D) or cellular (two-dimensional, 2-D) levels showed that the loading pattern plays a crucial role in the osteoblastic physiology. In this study, we attempted to investigate the response of a 3-D osteoblastic culture submitted to either no external stress or static or dynamic stresses. Rat osteosarcoma cells (ROS 17/2.8) were embedded within collagen type I lattices and studied for 3 weeks. Entrapment and proliferation of cells within the hydrated collagen gel resulted in the generation of contractile forces, which led to contraction of the collagen gel. We used this ability to evaluate the influence of three modes of mechanical stresses on the cell proliferation and differentiation: (1) the freely retracted gels (FRG) were floating in the medium, (2) the tense gels (TG) were stretched statically and isometrically, with contraction prevented in the longitudinal axis, and (3) the dynamic gels (DG) were floating gels submitted to periodic stresses (50 or 25 rpm frequency). Gels showed maximum contraction at day 12 in 50 rpm DG, followed by 25 rpm DG, then FRG (88%, 81%, 70%, respectively) and at day 16 in TG (33%). The proliferation rate was greater in TG than in FRG (+52%) but remained low in both DGs. Gel dimensions were related to the collagen concentration and on a minor extent to cell number. Cells in DG appeared rounder and larger than in other conditions. In TG, cells were elongated and oriented primarily along the tension axis. Scanning electron microscopy (SEM) showed that tension exerted by cells in TG led to reorientation of collagen fibers which, in turn, determined the spatial orientation and morphology of the cells. Transmission electron microscopy (TEM) performed at maximum proliferation showed a vast majority of cells with a distended well-developed RER filled with granular material and numerous mitochondria. Alkaline phosphatase activity peaked close to the proliferation peak in FRG, whereas in TG, a biphasic curve was observed with a small peak at day 4 and the main peak at day 16. In DG, this activity was lower than in the two other conditions. A similar time course was observed for alkaline phosphatase gene expression as assessed by Northern blots. Regardless of the conditions, osteocalcin level showed a triphasic pattern: a first increase at day 2, followed by a decrease from day 4 to 14, and a second increase above initial values at day 18. Microanalysis-x indicated that mineralization occurred after 14 days and TEM showed crystals within the matrix. We showed that static and dynamic mechanical stresses, in concert with 3-D collagen matrices, played a significant role on the phenotypic modulation of osteoblast-like cells. This experimental model provided a tool to investigate the significance and the mechanisms of mechanical activity of the 3-D cultured osteoblast-like cells.
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