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      Thirty days of spaceflight does not alter murine calvariae structure despite increased Sost expression

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          Abstract

          Previously our laboratory documented increases in calvaria bone volume and thickness in mice exposed to 15 days of spaceflight aboard the NASA Shuttle mission STS-131. However, the tissues were not processed for gene expression studies to determine what bone formation pathways might contribute to these structural adaptations. Therefore, this study was designed to investigate both the structural and molecular changes in mice calvariae after a longer duration of spaceflight. The primary purpose was to determine the calvaria bone volume and thickness of mice exposed to 30 days of spaceflight using micro-computed tomography for comparison with our previous findings. Because sclerostin, the secreted glycoprotein of the Sost gene, is a potent inhibitor of bone formation, our second aim was to quantify Sost mRNA expression using quantitative PCR. Calvariae were obtained from six mice aboard the Russian 30-day Bion-M1 biosatellite and seven ground controls. In mice exposed to 30 days of spaceflight, calvaria bone structure was not significantly different from that of their controls (bone volume was about 5% lower in spaceflight mice, p = 0.534). However, Sost mRNA expression was 16-fold (16.4 ± 0.4, p < 0.001) greater in the spaceflight group than that in the ground control group. Therefore, bone formation may have been suppressed in mice exposed to 30 days of spaceflight. Genetic responsiveness (e.g. sex or strain of animals) or in-flight environmental conditions other than microgravity (e.g. pCO 2 levels) may have elicited different bone adaptations in STS-131 and Bion-M1 mice. Although structural results were not significant, this study provides biochemical evidence that calvaria mechanotransduction pathways may be altered during spaceflight, which could reflect vascular and interstitial fluid adaptations in non-weight bearing bones. Future studies are warranted to elucidate the processes that mediate these effects and the factors responsible for discordant calvaria bone adaptations between STS-131 and Bion-M1 mice.

          Highlights

          • Previously, 15 days of spaceflight augmented bone volume in mice calvariae.

          • In this study, calvaria bone structure was not altered after 30 days of spaceflight.

          • Sost mRNA expression was higher in murine calvariae after 30 days of spaceflight.

          • Longer duration, or other spaceflight factors, may negate short-term calvarial growth.

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          Most cited references46

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          Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method.

          The two most commonly used methods to analyze data from real-time, quantitative PCR experiments are absolute quantification and relative quantification. Absolute quantification determines the input copy number, usually by relating the PCR signal to a standard curve. Relative quantification relates the PCR signal of the target transcript in a treatment group to that of another sample such as an untreated control. The 2(-Delta Delta C(T)) method is a convenient way to analyze the relative changes in gene expression from real-time quantitative PCR experiments. The purpose of this report is to present the derivation, assumptions, and applications of the 2(-Delta Delta C(T)) method. In addition, we present the derivation and applications of two variations of the 2(-Delta Delta C(T)) method that may be useful in the analysis of real-time, quantitative PCR data. Copyright 2001 Elsevier Science (USA).
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            Guidelines for assessment of bone microstructure in rodents using micro-computed tomography.

            Use of high-resolution micro-computed tomography (microCT) imaging to assess trabecular and cortical bone morphology has grown immensely. There are several commercially available microCT systems, each with different approaches to image acquisition, evaluation, and reporting of outcomes. This lack of consistency makes it difficult to interpret reported results and to compare findings across different studies. This article addresses this critical need for standardized terminology and consistent reporting of parameters related to image acquisition and analysis, and key outcome assessments, particularly with respect to ex vivo analysis of rodent specimens. Thus the guidelines herein provide recommendations regarding (1) standardized terminology and units, (2) information to be included in describing the methods for a given experiment, and (3) a minimal set of outcome variables that should be reported. Whereas the specific research objective will determine the experimental design, these guidelines are intended to ensure accurate and consistent reporting of microCT-derived bone morphometry and density measurements. In particular, the methods section for papers that present microCT-based outcomes must include details of the following scan aspects: (1) image acquisition, including the scanning medium, X-ray tube potential, and voxel size, as well as clear descriptions of the size and location of the volume of interest and the method used to delineate trabecular and cortical bone regions, and (2) image processing, including the algorithms used for image filtration and the approach used for image segmentation. Morphometric analyses should be based on 3D algorithms that do not rely on assumptions about the underlying structure whenever possible. When reporting microCT results, the minimal set of variables that should be used to describe trabecular bone morphometry includes bone volume fraction and trabecular number, thickness, and separation. The minimal set of variables that should be used to describe cortical bone morphometry includes total cross-sectional area, cortical bone area, cortical bone area fraction, and cortical thickness. Other variables also may be appropriate depending on the research question and technical quality of the scan. Standard nomenclature, outlined in this article, should be followed for reporting of results. 2010 American Society for Bone and Mineral Research.
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              Biomechanical and molecular regulation of bone remodeling.

              Bone is a dynamic tissue that is constantly renewed. The cell populations that participate in this process--the osteoblasts and osteoclasts--are derived from different progenitor pools that are under distinct molecular control mechanisms. Together, these cells form temporary anatomical structures, called basic multicellular units, that execute bone remodeling. A number of stimuli affect bone turnover, including hormones, cytokines, and mechanical stimuli. All of these factors affect the amount and quality of the tissue produced. Mechanical loading is a particularly potent stimulus for bone cells, which improves bone strength and inhibits bone loss with age. Like other materials, bone accumulates damage from loading, but, unlike engineering materials, bone is capable of self-repair. The molecular mechanisms by which bone adapts to loading and repairs damage are starting to become clear. Many of these processes have implications for bone health, disease, and the feasibility of living in weightless environments (e.g., spaceflight).
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                Author and article information

                Contributors
                Journal
                Bone Rep
                Bone Rep
                Bone Reports
                Elsevier
                2352-1872
                18 August 2017
                December 2017
                18 August 2017
                : 7
                : 57-62
                Affiliations
                [a ]University of California, San Diego, UCSD Medical Center, Orthopaedic Surgery Department, 350 Dickinson Street, Suite 121, Mail Code 8894, San Diego, CA 92103-8894, USA
                [b ]University of California, San Diego, Altman Clinical and Translational Research Institute, Lower Level 2 West 417, 9452 Medical Center Drive, La Jolla, CA 92037, USA
                [c ]KBRwyle, 2400 NASA Parkway, Houston, TX 77058, USA
                Author notes
                [* ]Corresponding author at: University of Southern California, Division of Biokinesiology and Physical Therapy, 1540 E. Alcazar Street, CHP149, Los Angeles, CA 90089, USA.University of Southern CaliforniaDivision of Biokinesiology and Physical Therapy1540 E. Alcazar Street, CHP149Los AngelesCA90089USA tmacaula@ 123456usc.edu
                [1]

                Contribution: Data collection, data analysis, manuscript writing, final approval.

                [2]

                Senior authors.

                [3]

                Supported by NASA NNX09AP11G.

                [4]

                Supported by NSBRI NCC 9-58.

                Article
                S2352-1872(17)30025-6
                10.1016/j.bonr.2017.08.004
                5574818
                28875158
                ad51efa7-c3e2-463d-b874-7e7da033d8ed
                © 2017 The Authors

                This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

                Categories
                Article

                microgravity,mechanotransduction,hydrostatic pressure,osteocyte,weightlessness,skull

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