Male C57BL/6N mice were flown on the unmanned BION-M1 biosatellite at an altitude of 560–580 km for 30 days between April 19, and May 19, 2013. Joint tissues were acquired from 6 flight mice as part of NASA’s Biospecimen Sharing Program. The mice were specific pathogen-free and 19–20 weeks old at the time of launch and start of asynchronous control experiments. Joint issues were dissected from flight mice in Moscow 13–16.5 h after landing in Kazakhstan. In addition to the spaceflight mice (SF), there were 8 ‘flight’ vivarium male ground control mice (SFV), 7 asynchronous ground control males (GC), and 7 asynchronous vivarium ground control males (GCV) as determined by agreements between the Russian Space Agency and NASA. The asynchronous control mice were housed 2 months after the flight mission in the same flight habitats for 30 days under environmental conditions simulating the flight conditions including temperature, humidity and gas composition. Since the (asynchronous) control mice arm of the experiment was conducted 3 months after the flight arm of the experiment, the vivarium control cohorts were included concurrently with flight and asynchronous control experiments as reported by Andreev-Andrievskiy et al.
Dissection and tissue preparation
Each hind-limb was cut mid-femur and mid-tibia and the left limbs placed in 10% neutral-buffered formalin (NBF). The tissue surrounding the femoro-tibial and sternocostal joints were removed immediately and, following an additional 2 days fixation with 10% NBF, the joints were decalcified in 5% formalin containing 10% formic acid for 2 days at 4°C. The knees were bisected down the trochlear groove following decalcification using a razor blade. The medial halves were paraffin embedded, mounted in paraffin blocks and sections cut (10 mm) using standard methods. The remaining halves were placed in 1× phosphate-buffered saline (PBS) containing 0.01 M ethylenediaminetetraacetic acid (EDTA) at room temperature for 4–6 h and then stored in 70% ethanol. Joint samples were sectioned in the sagittal plane.
Following dewaxing, every second slide was stained concurrently for Safranin-O (Saf-O) and counterstained with hematoxylin in parallel to visualize the cartilage-subchondral bone boundary and bone marrow space using a protocol from the University of Rochester, Center for Musculoskeletal Research, (https://www.urmc.rochester.edu/musculoskeletal-research/core-services/histology/protocols.aspx). Images were acquired on a Nikon Eclipse TI microscope using a Media Cybernetics Evolution MP camera and ImagePro Plus software. All images are 2560×1920 pixels so at 10X magnification, the scale is approximately 0.11 mm2 per pixel.
The extent of subchondral bone was analyzed using ImageJ software and the relative proportions of bone and bone marrow space determined within the secondary ossification center of the femoral condyle and proximal tibia. The proportion of bone was calculated relative to the total area of the secondary ossification center and the number of adipocytes per field within the ossification center calculated.
For the femoro-tibial joint histological scoring analysis, measurements were taken from 6 GC, 7 GCV, 5 SF, and 6 SFV samples where good joint histology in the correct plane was obtained. Sections were from the same relative position through the joint centered in the medial femoral condyle. The Mann-Whitney U test was used to compare SF (n=5) bone fraction and adipocyte number with the combined control groups (GC, GCV, SFV) (n=19)(see Fig. 1C). First, omnibus Kruskal-Wallis P-values for multiple independent samples were calculated to determine whether differences exist between experimental groups (top right in each graph). Then, the Kruskal-Wallis test was used pairwise to determine whether differences exist between SF and each of the separate experimental groups (GC, GCV, SFV). Post-hoc testing by Conover of all pairwise combinations of experimental groups was used to determine which pairs were different for adipocyte number. P-values were adjusted according to the family-wise error rate of Holm and then by the Benjamini-Hochberg FDR method. Median, maximum score, minimum score, and 25th and 75th percentile for the bone area and adipocyte data plotted for each experimental group are shown in figure 1D–G.