Bone strength and composition in spacefaring rodents: systematic review and meta-analysis

Matthew Goldsmith; Sequoia D. Crooks; Sean F. Condon; Bettina M. Willie; Svetlana V. Komarova

doi:10.1038/s41526-022-00195-7

Bone strength and composition in spacefaring rodents: systematic review and meta-analysis

Goldsmith, Matthew; Crooks, Sequoia D.; Condon, Sean F.; Willie, Bettina M.; Komarova, Svetlana V. 2022-04-13 00:00:00 www.nature.com/npjmgrav ARTICLE OPEN Bone strength and composition in spacefaring rodents: systematic review and meta-analysis 1,2 1 1 1,3 1,2✉ Matthew Goldsmith , Sequoia D. Crooks , Sean F. Condon , Bettina M. Willie and Svetlana V. Komarova Studying the effects of space travel on bone of experimental animals provides unique advantages, including the ability to perform post-mortem analysis and mechanical testing. To synthesize the available data to assess how much and how consistently bone strength and composition parameters are affected by spaceﬂight, we systematically identiﬁed studies reporting bone health in spacefaring animals from Medline, Embase, Web of Science, BIOSIS, and NASA Technical reports. Previously, we reported the effect of spaceﬂight on bone architecture and turnover in rodents and primates. For this study, we selected 28 articles reporting bone strength and composition in 60 rats and 60 mice from 17 space missions ranging from 7 to 33 days in duration. Whole bone mechanical indices were signiﬁcantly decreased in spaceﬂight rodents, with the percent difference between spaceﬂight and ground control animals for maximum load of −15.24% [Conﬁdence interval: −22.32, −8.17]. Bone mineral density and calcium content were signiﬁcantly decreased in spaceﬂight rodents by −3.13% [−4.96, −1.29] and −1.75% [−2.97, −0.52] respectively. Thus, large deﬁcits in bone architecture (6% loss in cortical area identiﬁed in a previous study) as well as changes in bone mass and tissue composition likely lead to bone strength reduction in spaceﬂight animals. npj Microgravity (2022) 8:10 ; https://doi.org/10.1038/s41526-022-00195-7 INTRODUCTION which are tremendously expensive and have small sample size, making improved statistical power with meta-analysis very Long-duration spaceﬂight is now ﬁrmly on the agenda for important. Moreover, summarizing all the missions that occurred humanity . Currently, with plans for a human-manned mission in different crafts that ﬂew to space over 40–50 years, allows to to the Martian surface within the next two decades and plans for separate the common effects of spaceﬂight from hazards and the construction of a lunar outpost to facilitate deep-space potential mishaps occurring within individual missions. The exploration , we can expect that in the coming century human current study serves as a continuation of our team’s series of spaceﬂights will increase in frequency and duration. Longer space systematic reviews and meta-analyses regarding spaceﬂight- missions pose greater risk to human health, potentially augment- 6 13 induced changes to bone in humans and animals . Previously, ing the known spaceﬂight related physiological changes including 4–6 we demonstrated a signiﬁcant deterioration of both cortical and bone loss . Although countermeasures have been implemented trabecular bone architecture in spaceﬂight rodents and found to help mitigate microgravity-induced bone loss – primarily 4,5 5,7 bone turnover to be signiﬁcantly affected . Here, we analyzed the exercise & diet – they have not been completely effective . data reporting changes to bone mechanical properties, bone To enable development of countermeasures that prevent mass, characterized by bone tissue mineral density (BMD) and microgravity-induced bone loss, comprehensive understanding 4,5 bone composition in spaceﬂight animals. The goals of the present of the underlying phenomena is necesary . study were to (i) to systematically identify all available literature Animals have long been used as a model to study and concerning the mechanical properties, BMD and composition of understand physiological changes that result from various stimuli bone in animals sent to space; (ii) to quantitatively characterize in humans. Speciﬁcally in regards to microgravity and bone, the degree and consistency of change in bone strength and animal studies have the beneﬁt of post-mortem analysis, which composition parameters using a meta-analytic approach, and (iii) enabled bone mechanical testing to be performed on spaceﬂight identify confounding variables associated with observed changes subjects. This allows for direct measurement of bone strength, and to the included bone parameters. Analyzing how bone strength thus more accurate assessment of fracture risk. Bone strength is and composition are affected by spaceﬂight will provide further determined by various contributors including bone geometry, 8–10 insights into the underlying causes and the functional risks bone mass, and the properties of the constituent tissue .In microgravity can pose to humans. humans, direct measurement of bone strength is not possible, and one must rely on surrogate measures such as bone mineral density measured through clinical imaging (i.e., dual-energy X-ray RESULTS absorptiometry, DEXA, or peripheral quantitative computed Identiﬁed articles tomography, pQCT) or estimation of strength using ﬁnite element 9,11] analyses to predict fracture risk . Thus, animal experiments can The systematic searches were performed on Medline, Embase, PubMed, BIOSIS Previews, and Web of Science using the search be used to better understand changes occurring in humans 12 13 strategy reported in Fu, Goldsmith et al. . Additionally, 9 articles during long-duration missions . Meta-analysis is an important approach for quantitative were identiﬁed from other sources including the NASA Technical synthesis of prior work, especially for spaceﬂight experiments, Reporting Service and articles referenced in the compendium of 1 2 Research Centre, Shriners Hospital for Children – Canada, Montréal, QC, Canada. Faculty of Dental Medicine and Oral Health Sciences, McGill University, Montréal, QC, Canada. Department of Pediatric Surgery, McGill University, Montréal, QC, Canada. email: svetlana.komarova@mcgill.ca Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA 1234567890():,; M. Goldsmith et al. Fig. 1 Systematic review information ﬂow. Prisma diagram indicated the numbers of records assessed duringdifferent steps of the systematic review. NASA’s animal and cell spaceﬂight experiments compiled by Overview of included bone parameters & control groups in Ronca et al . Original search was performed on November 2, the study 2017, a full update was performed on November 1, 2019 and For each parameter, a minimum of 3 mission level outcomes were again on September 13, 2021. In total, 15,977 candidate non- required to be included in this study. Mechanical properties duplicate articles were identiﬁed (Fig. 1). The Preferred Reporting included in meta-analysis consisted of 6 whole-bone mechanical Items for Systematic Reviews and Meta-Analysis (PRISMA) checklist properties: max load, yield load, failure load, stiffness, work to max is provided in the Supplementary Table 1. Following title and load, and work to failure load (Supplementary Fig. 1a); and 2 abstract screening, 1159 were determined to be concerning tissue-level mechanical properties: elastic modulus and yield animals sent to space. Previously, we identiﬁed that a majority of stress. All included measures of bone strength were from either bone health-focused animal studies reported ﬁndings in mice, rats torsional tests or 3-point bending tests (3PBT) conducted on long and primates (348 articles) . In this study, we performed the full bones: tibia, femur, and humerus. For bone mineral density we 14–66 text screening of the these articles and identiﬁed 54 articles included measurements from the following techniques: mercury that contained quantitative measures of bone strength, bone porosimetry, dual-energy x-ray absorptiometry (DEXA), microcom- mineral density (BMD) and composition (included parameters are puted tomography (μCT), peripheral quantitative computed presented in Table 1 and Supplementary Table 2). Twenty-six tomography (pQCT) and calculated density obtained by authors 42–66 articles were excluded at this level with reasons described in by dividing the weight of cortical bone segment by its estimated 42,55,58,59 37–40 Supplementary Table 3. Of note, 4 articles presented volume. It is worth noting that 4 articles indicated that they relevant bone measures in primates but were excluded due to measured tissue mineral density (TMD) rather than bone mineral insufﬁcient quantity of any single measure of interest for density (BMD). However, we treated TMD and BMD identically, 14–41 quantitative synthesis. In the ﬁnal meta-analysis, 28 articles since the voxel size used in these μCT studies included were included, 20 regarding rats, and 8 regarding mice, ﬂown on a contributions from porosity . In addition, the polychromatic total of 17 spaceﬂight missions, with a total of 60 rats and 60 mice beam used in lab-based μCT leads to beam hardening effects, being described (overview of included article is in Table 2). which further limits the accuracy of tissue mineralization npj Microgravity (2022) 10 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA 1234567890():,; M. Goldsmith et al. Table 1. Included bone properties for meta-analysis. Parameter Description Unit(s) Measures of whole bone mechanical properties 1. Maximum load Load at which internal structure begins to fail , peak load on the load- cm-dyne or N displacement curve 2. Yield load Load beyond which permanent deformation occurs , the proportional limit on the N load-displacement curve 3. Failure load Load at which bone failure/fracture occurs. N 4. Stiffness Measure of whole bone resistance during elastic deformation , slope of the initial dyne/rad or N/mm linear portion of the load-displacement curve 5. Work to maximum load Energy required to reach max load, area under the load-displacement curve until Nmm or mJ max load 6. Work to failure load Energy required to reach failure load, area under the load-displacement curve until cm-dyne rad or Nmm failure load Measures of tissue-level mechanical properties 1. Elastic modulus Measure of tissue-level resistance to deformation, tissue-level stiffness GPa or MPa 2. Yield stress Measure of stress at the yield point MPa Bone density measure 3 3 3 1. Bone mineral density Mass of bone per unit volume or mass of bone per unit area mg/mm , g/cm , mg/cm ,or mg/cm Bone composition measures 1. Calcium content Amount of calcium per mass of dry bone g/100 g, mg/g, μg/mg, %dry weight, or mol/kg 2. Phosphorus content Amount of phosphorus per mass of dry bone g/100 g, mg/g, μg/mg, or mol/kg 3. Hydroxyproline content Mass of hydroxyproline per mass of dry bone mg/g or μg/mg 4. Osteocalcin content Mass of osteocalcin per mass of dry bone μg/mg, mg/g, or ng/mg measurements . Bone composition data for 4 compounds demonstrated that increased quality score was associated with present in bone, calcium, phosphorus, hydroxyproline, and decreased effect size magnitude for BMD and stiffness (Supple- osteocalcin, were included as the weight of the compound mentary Fig. 1e, h). This association was however confounded by compared to the overall dry bone weight. The speciﬁc measure- higher quality scores of newer articles, which also are describing ments present in each study are presented in Supplementary mouse studies. Quality score was not associated with BMD article- Table 4 and study characteristics used for covariate analysis in level standard error (Supplementary Fig. 1f). Supplementary Table 5. For the purposes of analysis, two types of control animal groups Long bone mechanical properties were considered; a vivarium control group (VC) comprised of We ﬁrst examined the effect of spaceﬂight on the bone strength animals housed in standard laboratory habitats, and a ground parameters yield load, max load, and failure load obtained using control group (GC) where some or all aspects of spaceﬂight other 3-point bending (3PBT) or torsional tests conducted on long than microgravity, including habitat, light/dark cycle, diet and bones (Fig. 2). Spaceﬂight signiﬁcantly reduced the max load in forces of liftoff and re-entry were simulated. To assess the hindlimb long bones (Fig. 2a, Supplementary Table 6) with the inﬂuence of microgravity, we calculated the normalized difference calculated effect size representing the normalized difference in between SF and GC. To determine the possible effect of max load between SF and GC of −15.42% with a 95% CI of conditions associated with spaceﬂight other than microgravity [−23.88, −6.96] in the femur, and –17.27% [−27.20, −7.34] in the on bone strength, we calculated the normalized difference tibia. The change in the forelimb long bones (humerus), was between GC and VC. negative but not signiﬁcant −12.66% [−27.05, 1.73]. For all the long bones, spaceﬂight signiﬁcantly reduced the max load with Heterogeneity, bias, and quality the calculated effect size of −15.24% [−22.32,−8.17] (Fig. 2a left). Among the 13 included parameters, statistical heterogeneity was In the femur was there a signiﬁcant difference between GC and high (I > 75%) for 3 datasets; stiffness, yield stress, and bone VC, with an increase of 15.52% [4.29, 26.75], however in other long density. Heterogeneity was moderate (55% > I > 40%) for 3 bones and overall max load in GC and VC was not signiﬁcantly datasets: max load; work to max load; and elastic modulus. The different (Fig. 2a right). Subgroup analysis of effect of measure- remaining 7 datasets showed low (I < 25%) heterogeneity. The ment technique on SF-induced changes to max load demon- largest and most heterogeneous dataset, BMD, was used to assess strated no signiﬁcant difference in outcomes resulting from global bias. From single study exclusion analysis, no single mission torsional test and 3PBT (Fig. 2b). Among measures of max load signiﬁcantly affected global heterogeneity or outcome (Supple- derived from 3PBT machinery, neither loading rate nor span mentary Fig. 1b). From cumulative study exclusion, 20% of studies length of the supports were signiﬁcantly associated with a change were excluded prior to the dataset reaching homogeneity, and the in outcome (Fig. 2c, d). Yield load and failure load decreased in SF outcome of the homogeneous dataset was similar to the complete compared to GC with a percent difference of −18.95% [−27.24, dataset (Supplementary Fig. 1c). The funnel plot demonstrated −10.66] and −10.41% [−21.99, 1.16] respectively, with only the uneven distribution; however, the presence of a speciﬁc bias was change to yield load being statistically signiﬁcant (Supplementary difﬁcult to ascertain (Supplementary Fig. 1d). Regression analysis Tables 7 and 8). When yield load, max load, and failure load were of article-level effect size as a function of quality score normalized to weight of respective animal group at the time of Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2022) 10 M. Goldsmith et al. Table 2. Overview of articles included in meta-analysis. Articles Mission Year Days Species n Type of control Bone(s) analyzed QS (/18) SF 14 a Morey-Holton 1978a Cosmos 782 1975 19.5 Rats 4 GC, VC Humerus 16 15 a Morey-Holton 1978b Cosmos 936 1977 18.5 Rats 4 GC, VC Femur 14 Prokhonchukov 1982 Cosmos 1129 1979 18.5 Rats 5 GC, VC Scapula 7 Rodgacheva 1984 6 Femur 11 Patterson-Buckendahl 1985 Spacelab 3 1985 7 Rats 6 GC Humerus 10 Patterson-Buckendahl 1987 6 Vertebrae (L3), Humerus 11 Shaw 1988 6 Humerus, Tibia 11 Simmons 1986 6 Vertebrae (T), Femur 11 22 a Cann 1990 Cosmos 1887 1987 12.5 Rats 5 GC, VC Vertebrae (L4) 7.5 Simmons 1990a 6 Calvarium, Vertebrae (L5) 15 Vailas 1990a 4 Humerus 17 Arnaud 1992 Cosmos 2044 1989 14 Rats 5 GC, VC Femur 13 26 a Cann 1994 5 Vertebrae (L5) 9 Vailas 1992 5 Humerus 16 28 a Vailas 1994 5 Vertebrae (L5) 8 Lafage-Proust 1998 STS-58 1993 14 Rats 5 GC Parietal bone, Vertebrae (T), Humerus, Tibia 14 Chapes 1999 STS-60 1994 8 Rats 6 GC, VC Femur, Tibia 15 STS-63 1995 8 Rats 6 GC, VC Femur, Tibia 15 Bateman 1998 STS-77 1996 10 Rats 6 VC Humerus, Femur 14 Vajda 2001 STS-78 1996 17 Rats 6 GC, VC Femur 14 Zerath 2000 6 Pelvic bone 14 Lloyd 2015 STS-108 2001 12 Mice 12 GC Vertebrae (L5), Femur, Tibia 14 Ortega 2013 STS-118 2007 13 Mice 12 GC Femur 14 Coulombe 2021 Femur, Tibia 15 Zhang 2013 STS-131 2010 15 Mice 7 GC Calvariae 14 Gerbaix 2017 Bion M1 2013 30 Mice 5 GC, VC Vertebrae (T12 & L3), Femur 15 Gerbaix 2018 5 Calcaneus, Navicular, Talus 15 Macaulay 2017 6 GC Calvariae 15 Coulombe 2021 SpaceX-4 2014 21 Mice 10 GC Femur, Tibia 14 Lee 2020 SpaceX-19 2019 33 Mice 8 GC Femur, Vertebrae (L2,3,5) 12 Days mission duration (days), n sample size of spaceﬂight animal group. Control groups: GC ground control, VC vivarium control. For vertebrae region: L SF lumber, T thoracic, C caudal. QS quality score calculated according to Supplementary note 1. Indicates articles sourced from NASA Final Reports of Soviet missions. sacriﬁce, we found the overall decrease in these parameters in SF failure load were not signiﬁcantly affected when normalized to animals compared to GC to be very similar: yield load −12.24% weight at sacriﬁce (Supplementary Tables 14–16). [−20.52, −3.95], max load −12.65% [−21.11, −4.18], failure load Given that included measures of bone strength were exclusively −11.36% [−21.47, −1.26], all of which were statistically signiﬁcant from torsional tests and 3PBT, reported tissue-level mechanical (Supplementary Tables 9–11). properties, elastic modulus and yield stress, were derived using Next, we assessed the effect of spaceﬂight on stiffness, work to engineering beam theory equations. In spaceﬂight rats, elastic max load, and work to failure load. Stiffness (Fig. 3, Supplementary modulus 1.64% [−19.98, 23.26] and yield stress 4.96 [−26.04, Table 12) decreased in the hindlimbs of SF animals by −15.40% 35.97] exhibited no signiﬁcant change from GC and had moderate [−23.38, −7.42] in the femur and by −16.09% [−23.48, −8.69] in to high heterogeneity (I ≥ 52%). Interestingly, elastic modulus the tibia, while the change in humerus stiffness was not demonstrated an overall signiﬁcant difference between the statistically signiﬁcant, −3.85[−26.54, 18.84]. When all long bones ground control and vivarium control with the decrease in GC by were combined, the spaceﬂight-induced change to stiffness was −21.61% [−35.02, −8.19] compared to VC (Supplementary Tables not statistically signiﬁcant (Fig. 3a left). There was no signiﬁcant 17 and 18). There were no available data reported for mice. The difference in long bones stiffness between GC and VC (Fig. 3a outcomes of elastic modulus and yield stress were unaffected right). The effect of spaceﬂight on bone stiffness in long bones did when normalized to weight (Supplementary Tables 19 and 20). not differ when sub-grouped by the measurement technique (Fig. 3b), and did not depend on loading rate (Fig. 3c) nor span length Bone Mineral Density (Fig. 3d) in 3PBT. The data for work to max load and work to failure BMD was signiﬁcantly lower in SF rats, −4.51% [−8.32, −0.70], and load were only available for rats. Both measures decreased in SF mice, −2.09 [−3.74, −0.44], compared to GC, with the overall animals by −16.41% [−47.85, 15.03] and −39.53% [−67.14, −11.92] respectively, with the considerably larger and statistically effect for rodents of −3.13% [−4.96, −1.29] and high hetero- signiﬁcant decrease for work to failure load (Fig. 4, Supplementary geneity (I = 83.4%) (Fig. 5a left). GC and VC were not dfferent (Fig. Tables 13). Outcomes for stiffness, work to max load, and work to 5a right). When stratiﬁed by the measurement technique, no npj Microgravity (2022) 10 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA M. Goldsmith et al. Fig. 2 Spaceﬂight-induced changes in max load of bone in rodent. a Forest plot of changes in max load in humerus, femur and tibia in spaceﬂight animals (SF) compared to ground control (GC) (Left); and GC compared to vivarium control animals (VC) (Right). Missions are ordered by mission year; mission name, duration (Days), SF and GC sample sizes (n /n ) are shown. Circle/line: effect size (%) and 95% CI, the SF GC size of the circle is proportional to the mission’s weight. Black diamonds: overall effect size and 95% CI for rats; color diamonds: overall effect size 2 2 and 95% CI for rodents. I and H are for rodents. *indicates missions where there was no GC, and SF was compared to VC. b Subgroup analysis of measured max load by mechanical test: torsional test (Torsional) and 3-point bending tests (3PBT). Square/line: effect size (%) and 95% CI. N : number of mission level outcomes. Meta-regression analysis of max load measures obtained by 3PBT as a function of loading rate (c) and span length (d) of the 3PBT machinery. Linear regression line (dark blue), its 95% CI (light blue area) and R are shown. signiﬁcant difference was found between measurements obtained not signiﬁcant decrease of −1.32% [−3.18, 0.54] (Fig. 6 middle). using mercury porosimetry, calculated from cross-sections, DEXA, Hydroxyproline content increased in the bone of spaceﬂight rats and computed tomography (Fig. 5b). Spaceﬂight-induced by 8.20% [−7.42, 23.83], although this change was not signiﬁcant decrease in BMD were statistically signiﬁcant in the hindlimb (Fig. 6 right). GC to VC comparisons for bone composition bones, femur and tibia, but not the humerus of the forelimb (Fig. parameters were not signiﬁcantly different (Supplementary Tables 5c). BMD measured from samples of bone that contained only 21–23). Osteocalcin content in bone was not affected by the cortical bone and samples that contained both cortical and spaceﬂight (Supplementary Table 24). trabecular bone demonstrated no signiﬁcant difference in SF to GC outcomes (Fig. 5d). When only measures from long bones were Covariate analysis considered, spaceﬂight-induced BMD deﬁcits were greater in We assessed the inﬂuence of covariates using subgroup and meta- regions containing both cortical and trabecular bone (metaphyses regression analyses on the 4 parameters with 6 or more mission- and epiphyses) with a decrease of −9.8% [−11.7, −7.8] compared level outcomes: max load, stiffness, BMD and calcium content. to regions containing only cortical bone (diaphyses) with a Animal related covariates included age at launch, age at sacriﬁce, decrease of −3.0% [−5.7, −0.4] (Fig. 5e). strain, sex, source or dealer, weight of spaceﬂight animals at recovery or sacriﬁce and the difference in weight between the Bone composition spaceﬂight and ground control animal groups (Δweight SF and The data for speciﬁc mineral and organic components of bone GC). Linear regression analysis identiﬁed a weak association were only available for rats, and included homogeneous (I = 0%) between age at launch and change in calcium content and no datasets for bone calcium, phosphorus, hydroxyproline and association with max load, stiffness, nor bone density (Fig. 7a). osteocalcin. Spaceﬂight rats demonstrated a signiﬁcant decrease Similarly, only change in calcium content was weakly associated in bone calcium content of −1.75% [−2.97, −0.52] (Fig. 6 left). with animal age at sacriﬁce (Supplementary Fig. 2a). All spaceﬂight Phosphorus content in spaceﬂight rats demonstrated a similar but mice were of C57BL/6 strains, therefore subgroup analysis on Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2022) 10 M. Goldsmith et al. Fig. 3 Spaceﬂight-induced changes in bone stiffness in rodents. a Forest plot of changes in stiffness in humerus, femur and tibia in spaceﬂight animals (SF) compared to ground control (GC) (Left); and GC compared to vivarium control animals (VC) (Right). Missions are ordered by mission year; mission name, duration (Days), SF and GC sample sizes (n /n ) are shown. Circle/line: effect size (%) and 95% CI, the SF GC size of the circle is proportional to the mission’s weight. Black diamonds: overall effect size and 95% CI for rats; color diamonds: overall effect size 2 2 and 95% CI for rodents. I and H are for rodents. *indicates missions where there was no GC, and SF was compared to VC. b Subgroup analysis of measured bone stiffness by mechanical test: torsional test (Torsional) and 3-point bending tests (3PBT). Square/line: effect size (%) and 95% CI. N number of mission level outcomes. Meta-regression analysis of stiffness measures obtained by 3PBT as a function of loading i: rate (c) and span length (d) of the 3PBT machinery. Linear regression line (dark blue), its 95% CI (light blue area) and R are shown. Fig. 4 Spaceﬂight-induced changes in work to failure load in rats. Forest plot of changes in work to failure load in spaceﬂight animals (SF) compared to ground control (GC) (Left); and GC compared to vivarium control animals (VC) (Right). Missions are ordered by mission year; mission name, duration (Days), SF and GC sample sizes (n /n ) are shown. Circle/line: effect size (%) and 95% CI, the size of the circle is SF GC proportional to the mission’s weight. Orange diamonds: overall effect size and 95% CI for rats. animal strain was only applied to rats, in which the decreases to Institute of Experimental Endocrinology of Czeckolslovakia, max load and stiffness were only signiﬁcant in Sprague-Dawley Taconic Farms (Germantown, NY or afﬁliated facilities), or Jackson rats, and not in Wistar rats, while density and calcium content Laboratory (Bar Harbor, ME). Subgroup analysis of mission level changes were similar for both strains (Supplementary Fig. 2b). All outcomes by source of animal did not affect the outcomes spaceﬂight rats were male, therefore subgroup analysis for animal (Supplementary Fig. 2c). Weight at time of sacriﬁce, or Δweight SF sex was only applied to mice. Comparing outcomes of BMD by sex and GC did not signiﬁcantly affected spaceﬂight outcomes in mice demonstrated a signiﬁcant decrease in female but not in (Supplementary Fig. 2d, e). male mice, although the number of datasets for male mice was Mission-related covariates included mission duration, SF hous- limited to 2 (Fig. 7b). Animals were obtained primarily from ing - either single or grouped, and year of mission launch. npj Microgravity (2022) 10 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA M. Goldsmith et al. Fig. 5 Spaceﬂight-induced changes in bone mineral density (BMD) in rodents. a Forest plot of changes in BMD in spaceﬂight animals (SF) compared to ground control (GC) (Left); and GC compared to vivarium control animals (VC) (Right). Missions are stratiﬁed by rodent species, and within each stratum, missions are ordered by mission year. Mission name, duration (Days), SF and GC sample sizes (n /n ) are shown. SF GC Circle/line: effect size (%) and 95% CI, the size of the circle is proportional to the mission’s weight. Black diamonds: overall effect size and 95% 2 2 CI for mice and rats; color diamonds: overall effect size and 95% CI for rodents. I and H are for rodents. b Subgroup analysis of changes in BMD by measurement technique, which included density derived from weight of cortical cross-section sample divided by volume determined either from mercury displacement (Mercury Porosimetry) or from geometric estimates (Cortical Cross-Sectional), as well as BMD obtained from DEXA, or pQCT/μCT.c Subgroup analysis of long bone BMD by the forelimb and hindlimb bones. d Subgroup analysis of all BMD outcomes by the bone type. e Subgroup analysis of long bone BMD by bone region. Square/line: effect size (%) and 95% CI. N number of mission level outcomes. N number of measurement level outcomes. Fig. 6 Spaceﬂight-induced changes to bone mineral composition in rats. Forest plot of changes in bone calcium (left), phosphorus (middle), and hydroxyproline (right) content of spaceﬂight animals (SF) compared to ground control (GC). Missions are ordered by mission year (old to recent). Mission name, duration (Days), SF and GC sample sizes (n /n ) are shown. Circle/line: effect size (%) and 95% CI, the size of the circle SF GC is proportional to the mission’s weight. Orange diamonds: overall effect size and 95% CI for rats. *indicates missions where there was no GC, and SF was compared to VC. Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2022) 10 M. Goldsmith et al. Fig. 7 Covariate analysis of spaceﬂight-induced changes in bone strength and composition. a Meta-regression analysis of max load, stiffness, BMD, and calcium content as a function of age at launch of SF animals. Linear regression line (dark color), its 95% CI (light color area) and R are shown. Subgroup analysis of BMD by animal sex (b) and by short (14 days or less) and long (greater than 14 days) mission duration (c). Square/line: effect size (%) and 95% CI. N number of mission level outcomes. d Meta-regression analysis of max load, stiffness, BMD, and i: calcium content as a function of mission duration. Linear regression line (dark color), its 95% CI (light color area) and R are shown. Subgroup analysis of BMD by single vs. grouped rat housing (e) and by how closely GC mimics SF conditions (f). For f: Group 1: GC housed in same habitat as the SF; Group 2: GC housed in same habitat as SF, the force of liftoff and/or re-entry were mimicked; Group 3: GC was mimicked by in-ﬂight centrifuge. Square/line: effect size (%) and 95% CI. N number of mission level outcomes. i: Subgroup analysis for short (<14 days) and long (≥14 days) DISCUSSION duration mission demonstrated no signiﬁcant difference between The objective of this study was to systematically review and mission duration subgroups for any parameter (Fig. 7c, Supple- quantitatively synthesize data regarding changes to bone strength mentary Fig. 3a); however, the decrease in stiffness compared to and bone composition in rodents sent to space. We demonstrate GC was only signiﬁcant in short durations missions, while the that whole bone mechanical properties in spaceﬂight rodents decrease in calcium was only signiﬁcant in long-duration missions were signiﬁcantly decreased in their hindlimbs but not in the (Supplementary Fig. 3a). Longer mission duration was weakly forelimbs. BMD was signiﬁcantly decreased in spaceﬂight rodents. associated with lower deﬁcits in BMD in linear regression analysis In spaceﬂight rats, bone calcium content was signiﬁcantly lower, (Fig. 7d). The max load, stiffness and BMD demonstrated greater with a decrease in phosphorus and an increase in hydroxyproline deﬁcits when rats were housed alone, although the difference that were not statistically signiﬁcant. We were able to perform a between groups was not statistically signiﬁcant (Fig. 7e, Supple- limited analysis of the effect of some covariates on the SF-induced mentary Fig. 3b). There was no association between launch year changes in bone strength and composition parameters. and outcome (Supplementary Fig. 3c). Spaceﬂight-induced deﬁcits in BMD were signiﬁcant in female Study related covariates included sacriﬁce delay and the degree mice, but not in male mice; decreases to bone strength to which GC animals mimic the conditions of SF animals (GC parameters were only signiﬁcant in Sprague-Dawley rats, and condition). Sacriﬁce delay was weakly associated with decreased not in Wistar rats; bone strength and density were affected more magnitude of max load, but did not affect other parameters in single-housed rats than group housed. However, the interac- (Supplementary Fig. 4a). BMD and calcium content outcomes were tions between multiple confounding factors, for example age and similar across all GC conditions (Fig. 7f, Supplementary Fig. 4b). sex, was not possible due to data paucity. Importantly, whole bone While max load and stiffness appeared to be affected in some GC mechanical, BMD, and mineral composition properties were not conditions, there were no consistent pattern (Supplementary signiﬁcantly different between the ground control and vivarium Fig. 4b). npj Microgravity (2022) 10 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA M. Goldsmith et al. 11,70,75 animal groups, suggesting that microgravity is the primary factor porosity also may have signiﬁcant effects on bone strength. causing these changes. Although the effect of spaceﬂight on HA crystallinity , and Our analysis only included measures of whole bone strength cortical porosity were measured in Bion M1 mission, we lacked derived from 3-point bending tests or torsional tests. The relative sufﬁcient data of these outcome measures for meta-analysis. change to bone strength from these two loading modes are While many common methods used today to measure mineral considered to be comparable as they both depend on the and matrix properties such as quantitative backscattered electron underlying geometric and material properties of the tested imaging, nanoindentation, small angle x-ray scattering, Fourier region which is composed of cortical bone in long bone diaphysis. transform infrared spectroscopy, and Raman spectroscopy , they We found that yield load and max load were signiﬁcantly lower in were not performed in enough studies to include in our analyses. SF with estimated decreases of −18.95% [−27.24, −10.66] and Thus, our study identiﬁes a signiﬁcant gap in our knowledge of the −15.24% [−22.32,−8.17] respectively, while a decrease in bone degree to which bone tissue level properties are affected by stiffness of −9.47 [−20.44,1.49] was not signiﬁcant. We found that microgravity. work to failure load, which represents the area under the force- Where it was possible, we investigated the effects of covariates displacement curve until failure , was the most affected on bone strength, density and composition outcomes. Similar to parameter in spaceﬂight animals with a decrease of −39.53% our previous ﬁndings , neither mission duration, nor age at [−67.14,−11.92], indicating a signiﬁcant decrease to bone launch were associated with signiﬁcant changes in measured toughness, although toughness is also deﬁned by fracture parameter, likely due to the relatively short mission durations, up mechanics parameters . Given that changes to stiffness, yield, to 33 days, as well as the younger age of included animals. We max and failure load were all estimated to be less than half of the conﬁrmed that housing type had a signiﬁcant effect on SF- work to failure load magnitude of change, we can deduce that induced changes. In rodents housed individually during space- post-yield displacement (PYD), a measure of bone ductility , may ﬂight, a greater decrease in bone stiffness, max load, and BMD was have been lower, potentially indicating increased bone brittleness observed compared to animals housed in groups. A hindlimb in spaceﬂight animals. This is supported by two pieces of unloading study that directly compared the effect of unloading on evidence. First, it has been reported that PYD has the greatest single-housed mice and those housed in pairs demonstrated that inﬂuence on work-to-fracture load . Second, in two included several immune and hypothalamic-pituitary-adrenal axis studies, Patterson-Buckendahl et al. report of SpaceLab3 and responses were signiﬁcantly different in these groups, suggestion Vailas et al. report of Cosmos 2044, max load and failure load strong contribution of social isolation to physiological responses occurred simultaneous. Tissue-level mechanical properties, elastic to unloading . However, in vivo mouse tibial loading studies modulus and yield stress determined from engineering beam performed on Earth have shown that the response to loading in theory equations did not change in spaceﬂight animals. However, male mice was reduced when mice were group housed, compared one must also consider the limitations of calculating tissue level to individually housed mice, likely due to increased mechanical properties from these equations, which has been reported to strains engendered in the tibiae during group-housed ﬁghting provide values that are greatly underestimated, with inconsistent activities that masked the bone (re)modeling response to and even inverse relative differences between experimental loading . We have also identiﬁed a potentially important groups compared to the relative differences reported by nano- difference between the responses to spaceﬂight in male and 72,73 indentation measurements . Therefore, our reported changes female mice, where only in female mice the spaceﬂight-induced in tissue-level mechanical properties should be interpreted with deﬁcits in BMD were signiﬁcant. However, low number of studies caution. Thus, whole bone mechanical properties are signiﬁcantly with male mice and no studies with female rats presented a major reduced in spacefaring rodents. limitation for further analysis. It has been reported that the whole bone mechanical properties We have found signiﬁcant regional differences in the bone depend on its mass, geometry and material compositional response to spaceﬂight. The change in BMD in the metaphyses of 8–10 properties . We demonstrated a signiﬁcant decrease in BMD long bones was greater than the change in the diaphysis. This of cortical bone diaphysis: −3.0% [−5.7, −0.4]. Comparing the trend is consistent with our previous report examining bone change in BMD to the changes in bone strength support the architecture, where a greater reduction in trabecular bone notion that changes to BMD alone may not explain the changes to compared to cortical bone was observed . We have found that 9,11 bone strength . We previously reported that in SF animals spaceﬂight-induced deﬁcits in maximum load, stiffness and BMD cortical bone area decreased signiﬁcantly by −5.9% [−8.0, −3.8] were higher in the hindlimb bones compared to the forelimb and cortical thickness decreased by −4.7% [−13.7,4.4] while there bone, supporting a region dependent changes in bone health due was no signiﬁcant change to marrow area . Thus, cortical bone to SF, which was similar to humans, for which the magnitude of mass decreased during spaceﬂight with no increase in total cross- bone loss was the highest in the legs, while arms were sectional area, which otherwise may have increased bone unaffected . Previously, we reported a trend to higher trabecular 10,70 13 strength . We also previously reported signiﬁcant reductions bone deﬁcits in distal skeletal regions compared to axial regions . in histomorphometric cortical bone formation indices only on the When we speciﬁcally analyzed the changes in humerus, femur and periosteal surface . These SF-induced alterations in cortical tibia, we found that spaceﬂight-induced changes in trabecular microstructure due to imbalanced bone (re)modeling are con- bone volume fraction (Tb.BV/TV) were −15.3% [−21.0, −9.7] in sistent with the reduction of bone strength in SF animals. humerus, −29.0% [−33.5, −24.5] for femur and −24% [−30.5, Our study suggests that alterations in bone composition −17.5] for tibia. This is also conﬁrmed by in ﬂight measurements properties due to SF also contributed to the altered bone of BMD using DEXA reported for SpaceX-19 mission, which strength. In the current study, we have demonstrated that bone reported that after 28 days of spaceﬂight decrease in BMD was calcium content signiﬁcantly decreased in SF rats compared to GC, observed in the femur and not the humerus . Analysis of with a trend of a decrease in phosphorus content, and a relative movement of mice sent to the International Space Station, noted increase in the organic component of bone quantiﬁed by the forelimb ambulation during the ﬁrst half of the mission as key in- increase in hydroxyproline, an amino acid unique to collagen is ﬂight activity . These data suggests that the increased use of the used as a relative measure of collagen content. There was no forelimbs may help to preserve bone health in this region. available data regarding calcium, phosphorus or hydroxyproline The limitations of this study included, i) variations in experi- content in SF mice, and thus possible species differences could mental designs between missions, ii) inconsistent reporting, iii) 10,69,70 not be determined. Other factors including HA crystallinity , variations in measures of BMD, and iv) use of skeletally immature, presence of microcracks , and changes in cortical bone growing animals. Limitations i and ii have been explored in detail Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2022) 10 M. Goldsmith et al. in Fu & Goldsmith et al. . In brief, mission designs and associated mechanical properties in spacefaring rodents. We demonstrated experiments have changed over time, and the included control signiﬁcant deterioration in bone health, including decreased group varied in terms of degree in which they mimic spaceﬂight- measures of bone architecture, strength and composition, and associated stressors. It was quite noticeable that reporting of altered bone turnover. Our analysis is important in providing solid certain parameters changes with time. For example, measures of quantitative estimates of the effect sizes with measures of whole bone mechanical properties were reported for all, but one variance, and in identifying gaps and directions for informing spaceﬂight mission involving rats. In contrast, only 2 of the 6 future spaceﬂight experiments. In addition to the need for more included studies on spaceﬂight missions involving mice report inﬂight measurements of bone mass and architecture, standardiz- whole bone mechanical properties. Similarly measures of bone ing measurement techniques, expanding the studies of animal calcium and phosphorus concentrations were only reported in rat sex, strain, age and spaceﬂight duration is critically important for missions, with no available data for mice. When grouping mission obtaining a clear picture on how bone is changed in microgravity SF to GC outcomes by degree to which control group mimic and how these changes can be prevented. spaceﬂight conditions, no clear association was observed suggest- ing that the microgravity is the main driver of the changes. METHODS Secondly, we observed that reported animal treatment was not consistent across publications. One example of this inconsistency This study was conducted in compliance with the Preferred is the great variation in reported sacriﬁce delays of SF animals Reporting Items for Systematic Reviews and Meta-Analysis among articles describing identical missions (Supplementary Table (PRISMA) statement. For the PRISMA Checklist, refer to Supple- 5). The third set of limitations was related to the use of several mentary Table 1. different measurement technique to assess BMD. Among these techniques, some measures were more precise such as using μCT, Search strategy, inclusion criteria and quality assessment others less so, such as estimating BMD by the weight of the bone The systematic search strategy used in this study was identical to sample, divided by the volume calculated as the cross-sectional that used in Fu & Goldsmith et al. 2021 . In brief a search strategy area of the sample multiplied by its thickness. Four studies using terms related to bone, space travel, and animals was 37–40 indicated that they report bone tissue mineral density , constructed and used to execute a search Medline, Embase, however the smallest voxel size used was 9 μm, while a resolution PubMed, BIOSIS Previews, and Web of Science on November 2nd, of 1 μm is required to distinguish cortical vasculature micro- 2017, with an updated search being performed on November 1st, architecture . For future studies, it would be valuable to also have 2019. An additional search of the NASA Technical Reporting analyses of bones using synchrotron-based tomography where Service (NTRS) and articles referenced in the compendium of smaller voxel sizes are possible and more accurate tissue mineral animal and cell spaceﬂight experiments compiled by Ronca et al. density can be determined without beam hardening artifacts that was performed manually. No language restrictions were applied to are present with lab-based computed tomography . The ﬁnal set considered articles. Title and abstract screening, performed of limitations was related to the use of skeletally immature independently by SDC & SFC for the primary search and by SVK rodents, particularly rats. Only one study included animals older for the update, selected articles describing any non-human than 6 months of age, and average age was ~ 11 weeks for rats, vertebrate sent to space. Studies that described humans, and 20 weeks for mice. C57BL/6 mice reach peak cancellous bone invertebrates or Earth-based spaceﬂight simulations were mass at 8–12 weeks of age. They achieve peak adult cortical bone excluded. Primary full text screening (conducted independently density in the femur by 16 weeks and whole bone strength in by SDC, SFC & MG for primary and MG for update) selected articles bending and torsion peaks by 20 weeks of age . Rats are describing the effects of spaceﬂight on bone health of mice, rats skeletally mature at 6–9 months of age . Since on average and primate. We included in the meta-analysis studies that included mice were closer to skeletal maturity, this may explain presented quantitative measures of strength, density and why the decrease to BMD was less severe to mice compared to SF composition of bones of the axial and appendicular skeleton in rats. However, one must keep in mind that age-related changes in mice and rats that were on normal diet, were not pregnant, and BMD and mechanical properties are genetic strain and sex did not have surgery other than sham. Only studies that presented 83 84 dependent in both mice and rats . It is clear from loading measures of bone strength resulting from three-point bending studies in rodents that young animals have a much greater bone tests (3PBT) or torsional tests were included as the relative 85,86 formation and resorptive response to mechanical loading .It changes in outcomes obtained using these loading modes were remains less clear how SF-induced bone (re)modeling changes are suggested to be comparable . Of studies reporting strength affected by age, but a recent study by Coulombe et al. showed 54 28 measures, only Zernicke et al. and Vailas et al. reported useable that mature 32-week-old female mice exposed to microgravity data derived from compression test machinery. Gerbaix et al. experienced greater bone loss than young 9-week-old mice with reported hardness and elastic modulus results using nanoindenta- net skeletal growth. However, aged mice similarly showed a tion, which precluded meta-analysis for these measures. Papers diminished recovery upon re-ambulation compared to adult included in meta-analysis were scored on an 18-point scale for mice . We were not able to perform extensive strain and sex reporting quality (Supplementary note 1). If the outcomes of two analysis, because of limited information. Subgroup analysis of separate missions were reported in a single article, quality score animal sex for BMD in mice demonstrated potential difference (QS) was assessed for each mission independently. between the responses in male and female mice, however only 2 groups of male mice were included both from the same Bion M1 Data extraction mission. Mechanical loading studies in mice have observed sex- 87,88 89 related differences in cortical bone , but not cancellous bone . The following data was extracted by MG and veriﬁed by SVK for all Genetic strain-speciﬁc differences in mechanoresponsive that studies included in meta-analysis: mission name and duration; have been reported between C57BL6, Balb/c, and C3H/HeJ animal species and sample size (n) of spaceﬂight, ground control, 90–92 mice . Future studies are needed to carefully examine how and vivarium control groups (when applicable); bone type and genetic strain, age and sex affect the mechano-adaptive bone region being measured; measurement technique; and mean response to SF. and median in the 13 bone parameters (Table 1); standard errors, The two meta-analytic studies (Fu & Goldsmith et al. and the standard deviations, and/or interquartile ranges; days when current study) quantitatively summarize previously reported measurements were taken. If the type of dispersion measure changes to bone architecture, turnover, composition and was not given, we assumed it to be a standard error to ensure a npj Microgravity (2022) 10 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA M. Goldsmith et al. conservative estimate. If a range of sample sizes was reported, the accordance with the RE model, global effect size, θ, was calculated smallest value was extracted. The following mission characteristics using mission-level outcomes θ and their associated weight w via i i were also extracted for covariate analysis: animal strain, age at Eq. (5), launch and sacriﬁce, weight at sacriﬁce or recovery, sex, source or ðÞ θ ´ w i i dealer of animals, year of mission, spaceﬂight group sacriﬁce θ ¼ P (5) delays, single vs grouped spaceﬂight habitat, and treatment i conditions of ground control group. Mission characteristics were where N is the number of combined mission-level outcomes. pooled from all applicable articles. If articles report differing values Equation (6) was used to calculate weight of mission-level for apparently identical samples, the data from the article with the outcomes w using mission-level standard error se(θ ) and the i i 2 2 higher quality score was included. If articles report conﬂicting DerSimonian-Laird interstudy variance estimator τ . τ was values for a single mission characteristic, the most frequently calculated using Eqs. (7), (8), and (9). reported was included if possible, otherwise, the value from the article with a higher quality score was included. If only an interval w ¼ i (6) seðÞ θ þτ of time was provided for age at launch the mean value was used, i if only an interval of time was provided for spaceﬂight animal Q ðN 1Þ sacriﬁce delay, the higher value was used. All alternate terms used (7) τ ¼ for included parameters are in Supplementary Table 2. ! ! P 2 seðÞ θ ´ θ i i 2 i Measurement-level outcomes (8) Q ¼ seðÞ θ ´ θ i i P seðÞ θ This study included relevant data of two control groups: the i vivarium control (VC) consisting of animals housed in standard laboratory habitats, and the ground control (GC) which modeled seðÞ θ X i (9) some or all aspects of spaceﬂight except for microgravity. Animals c ¼ seðÞ θ seðÞ θ sent to space and subjected to artiﬁcial gravity (AG) were i considered GC. When possible, GC was used as a comparison Standard error of global effect size was calculated using Eq. (10). group, in missions without GC, VC was used as the comparator for spaceﬂight (SF). For each bone measurement j, the mean SF value, 1 se θ ¼ qﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ μ , and the mean comparison control (CC) value, μ with their P (10) SFj CCj associated standard errors se(μ ), or standard deviations sd(μ ) were i j j recorded In instances where sd(μ ) was recorded, it was converted . j pﬃﬃﬃ 95% conﬁdence intervals (CI) was calculated as 95% to se(μ)as se μ ¼ sd μ = n, where n is n for spaceﬂight and ^ ^ ^ ^ j j j SF CI¼ θ±z ´ seðθÞ¼ θ ±1:96 ´ SEðθÞ. All the above analysis ð1α=2Þ n for the corresponding control. For median P and interquartile CC was repeated for GC to VC comparisons, replacing instances of SF range x − x , μ was calculated as μ ¼ðx þ P þ x Þ=3 upper lower j j upper lower pﬃﬃﬃ and GC with GC and VC respectively. with: se μ ¼ x x = n ´ 2:7: We calculated j upper lower measurement-level effect size as the normalized percent differ- Heterogeneity and publication bias analysis ence, θ , between μ and μ using Eq. (1). j SFj CCj 2 2 Heterogeneity of global outcomes were reported as H and I μ μ 2 SF CC 2 Q 2 H 1 j j which uses Cochran’s Q (Eq. (8)) as: H ¼ , and I ¼ .To θ ¼ ´ 100% 2 j (1) N1 H CC assess the contribution of individual missions to global outcome and heterogeneity, we performed single data exclusion analysis, The cumulative standard error in percentage, se(θ ), was calculated wherein one at a time each mission-level outcome was assuming the two groups were independent using Eq. (2). sequentially removed and heterogeneity statistics recalculated. vﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 0 12 0 12 In cumulative data exclusion analysis mission-level outcomes were se μ se μ u SF CC j j excluded sequentially starting with those that contributed the t@ A @ A (2) se θ ¼ þ ´ 100% highest heterogeneity. A funnel plot showing the distribution of μ μ SF CC j j seðÞ θ to θ was used to assess reporting bias. Independent of their i i contribution to heterogeneity or potential bias, we included all the studies in the ﬁnal analysis. Mission-level outcomes When measurement level outcomes of multiple unique b bones or Additional analysis bone regions were recorded for mission i, mission-level effect sizes The following 17 characteristics were used for covariate analysis: θ and standard error se(θ ) were calculated as unweighted means i i ﬂight duration, strain of rats, sex of mice, source or dealer of by Eqs. (3), (4) respectively. animals, age at launch & sacriﬁce, weight at sacriﬁce/recovery, change in weight between SF and CC group, launch year, SF (3) θ ¼ b sacriﬁce delay, single vs grouped housing condition, the degree to which GC group mimic the environmental conditions of SF (GC se θ j conditions), bone or bone region measured, measurement (4) seðÞ θ ¼ technique, span length & loading rate of 3PBT, and article quality score. Subgroup analysis was performed by combining mission- For a single mission, Bion M1, the data for two animal groups were level outcomes and standard error within each category for 38,39 reported separately . As a result, these two animal groups were categorical variables sex, strain, animal source, single vs grouped treated as independent missions. housing conditions, GC conditions, and measurement technique, as well as for short (<14 days) and long (≥14 days) duration Meta-analytic model and global outcome missions. Subgroup analysis for measurement-level outcomes was Considering that we combine data from two different rodent used for bone type or bone region analysis. Meta-regression species aboard spaceﬂight missions with highly heterogeneous analysis was performed on mission level outcomes for continuous methodologies, a random effects (RE) model was selected. In variables: ﬂight duration, launch year, age at launch & sacriﬁce, Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2022) 10 M. Goldsmith et al. weight at sacriﬁce or recovery, and change in weight between SF 8. van der Meulen, M. C. H., Jepsen, K. J. & Mikić, B. Understanding bone strength: size isn’t everything. Bone 29, 101–104 (2001). and CC group. Meta-regression analysis on measurement-level 9. Kim, G., Boskey, A. L., Baker, S. P. & van der Meulen, M. C. Improved prediction of outcomes was performed for span length & loading rate in 3PBT. rat cortical bone mechanical behavior using composite beam theory to integrate For quality score, missions reported in a single article were tissue level properties. J. Biomech. 45, 2784–2790 (2012). combined to create a paper-level score,θ and associated seðÞ θ p P 10. Wallace, J. M. in Basic and applied bone biology (eds David B. Burr & Matthew R. using Eqs. 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M.G. was supported by graduate scholarship from McGill Faculty of in published maps and institutional afﬁliations. Dentistry. AUTHOR CONTRIBUTIONS Open Access This article is licensed under a Creative Commons M.G., S.D.C., S.F.C., and S.V.K. performed screening; M.G. and S.D.C. performed data Attribution 4.0 International License, which permits use, sharing, extraction; M.G. performed meta-analysis; M.G., B.M.W., and S.V.K. performed critical adaptation, distribution and reproduction in any medium or format, as long as you give analysis of the data; M.G. and S.V.K. wrote the ﬁrst draft; all authors edited and appropriate credit to the original author(s) and the source, provide a link to the Creative approved the manuscript. Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the COMPETING INTERESTS article’s Creative Commons license and your intended use is not permitted by statutory The authors declare no competing interests. regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons. org/licenses/by/4.0/. ADDITIONAL INFORMATION Supplementary information The online version contains supplementary material © The Author(s) 2022 available at https://doi.org/10.1038/s41526-022-00195-7. Correspondence and requests for materials should be addressed to Svetlana V. Komarova. npj Microgravity (2022) 10 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png npj Microgravity Springer Journals http://www.deepdyve.com/lp/springer-journals/bone-strength-and-composition-in-spacefaring-rodents-systematic-review-uQJdDUNKLj

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Publisher: Springer Journals
Copyright: Copyright © The Author(s) 2022
eISSN: 2373-8065
DOI: 10.1038/s41526-022-00195-7
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Abstract

www.nature.com/npjmgrav ARTICLE OPEN Bone strength and composition in spacefaring rodents: systematic review and meta-analysis 1,2 1 1 1,3 1,2✉ Matthew Goldsmith , Sequoia D. Crooks , Sean F. Condon , Bettina M. Willie and Svetlana V. Komarova Studying the effects of space travel on bone of experimental animals provides unique advantages, including the ability to perform post-mortem analysis and mechanical testing. To synthesize the available data to assess how much and how consistently bone strength and composition parameters are affected by spaceﬂight, we systematically identiﬁed studies reporting bone health in spacefaring animals from Medline, Embase, Web of Science, BIOSIS, and NASA Technical reports. Previously, we reported the effect of spaceﬂight on bone architecture and turnover in rodents and primates. For this study, we selected 28 articles reporting bone strength and composition in 60 rats and 60 mice from 17 space missions ranging from 7 to 33 days in duration. Whole bone mechanical indices were signiﬁcantly decreased in spaceﬂight rodents, with the percent difference between spaceﬂight and ground control animals for maximum load of −15.24% [Conﬁdence interval: −22.32, −8.17]. Bone mineral density and calcium content were signiﬁcantly decreased in spaceﬂight rodents by −3.13% [−4.96, −1.29] and −1.75% [−2.97, −0.52] respectively. Thus, large deﬁcits in bone architecture (6% loss in cortical area identiﬁed in a previous study) as well as changes in bone mass and tissue composition likely lead to bone strength reduction in spaceﬂight animals. npj Microgravity (2022) 8:10 ; https://doi.org/10.1038/s41526-022-00195-7 INTRODUCTION which are tremendously expensive and have small sample size, making improved statistical power with meta-analysis very Long-duration spaceﬂight is now ﬁrmly on the agenda for important. Moreover, summarizing all the missions that occurred humanity . Currently, with plans for a human-manned mission in different crafts that ﬂew to space over 40–50 years, allows to to the Martian surface within the next two decades and plans for separate the common effects of spaceﬂight from hazards and the construction of a lunar outpost to facilitate deep-space potential mishaps occurring within individual missions. The exploration , we can expect that in the coming century human current study serves as a continuation of our team’s series of spaceﬂights will increase in frequency and duration. Longer space systematic reviews and meta-analyses regarding spaceﬂight- missions pose greater risk to human health, potentially augment- 6 13 induced changes to bone in humans and animals . Previously, ing the known spaceﬂight related physiological changes including 4–6 we demonstrated a signiﬁcant deterioration of both cortical and bone loss . Although countermeasures have been implemented trabecular bone architecture in spaceﬂight rodents and found to help mitigate microgravity-induced bone loss – primarily 4,5 5,7 bone turnover to be signiﬁcantly affected . Here, we analyzed the exercise & diet – they have not been completely effective . data reporting changes to bone mechanical properties, bone To enable development of countermeasures that prevent mass, characterized by bone tissue mineral density (BMD) and microgravity-induced bone loss, comprehensive understanding 4,5 bone composition in spaceﬂight animals. The goals of the present of the underlying phenomena is necesary . study were to (i) to systematically identify all available literature Animals have long been used as a model to study and concerning the mechanical properties, BMD and composition of understand physiological changes that result from various stimuli bone in animals sent to space; (ii) to quantitatively characterize in humans. Speciﬁcally in regards to microgravity and bone, the degree and consistency of change in bone strength and animal studies have the beneﬁt of post-mortem analysis, which composition parameters using a meta-analytic approach, and (iii) enabled bone mechanical testing to be performed on spaceﬂight identify confounding variables associated with observed changes subjects. This allows for direct measurement of bone strength, and to the included bone parameters. Analyzing how bone strength thus more accurate assessment of fracture risk. Bone strength is and composition are affected by spaceﬂight will provide further determined by various contributors including bone geometry, 8–10 insights into the underlying causes and the functional risks bone mass, and the properties of the constituent tissue .In microgravity can pose to humans. humans, direct measurement of bone strength is not possible, and one must rely on surrogate measures such as bone mineral density measured through clinical imaging (i.e., dual-energy X-ray RESULTS absorptiometry, DEXA, or peripheral quantitative computed Identiﬁed articles tomography, pQCT) or estimation of strength using ﬁnite element 9,11] analyses to predict fracture risk . Thus, animal experiments can The systematic searches were performed on Medline, Embase, PubMed, BIOSIS Previews, and Web of Science using the search be used to better understand changes occurring in humans 12 13 strategy reported in Fu, Goldsmith et al. . Additionally, 9 articles during long-duration missions . Meta-analysis is an important approach for quantitative were identiﬁed from other sources including the NASA Technical synthesis of prior work, especially for spaceﬂight experiments, Reporting Service and articles referenced in the compendium of 1 2 Research Centre, Shriners Hospital for Children – Canada, Montréal, QC, Canada. Faculty of Dental Medicine and Oral Health Sciences, McGill University, Montréal, QC, Canada. Department of Pediatric Surgery, McGill University, Montréal, QC, Canada. email: svetlana.komarova@mcgill.ca Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA 1234567890():,; M. Goldsmith et al. Fig. 1 Systematic review information ﬂow. Prisma diagram indicated the numbers of records assessed duringdifferent steps of the systematic review. NASA’s animal and cell spaceﬂight experiments compiled by Overview of included bone parameters & control groups in Ronca et al . Original search was performed on November 2, the study 2017, a full update was performed on November 1, 2019 and For each parameter, a minimum of 3 mission level outcomes were again on September 13, 2021. In total, 15,977 candidate non- required to be included in this study. Mechanical properties duplicate articles were identiﬁed (Fig. 1). The Preferred Reporting included in meta-analysis consisted of 6 whole-bone mechanical Items for Systematic Reviews and Meta-Analysis (PRISMA) checklist properties: max load, yield load, failure load, stiffness, work to max is provided in the Supplementary Table 1. Following title and load, and work to failure load (Supplementary Fig. 1a); and 2 abstract screening, 1159 were determined to be concerning tissue-level mechanical properties: elastic modulus and yield animals sent to space. Previously, we identiﬁed that a majority of stress. All included measures of bone strength were from either bone health-focused animal studies reported ﬁndings in mice, rats torsional tests or 3-point bending tests (3PBT) conducted on long and primates (348 articles) . In this study, we performed the full bones: tibia, femur, and humerus. For bone mineral density we 14–66 text screening of the these articles and identiﬁed 54 articles included measurements from the following techniques: mercury that contained quantitative measures of bone strength, bone porosimetry, dual-energy x-ray absorptiometry (DEXA), microcom- mineral density (BMD) and composition (included parameters are puted tomography (μCT), peripheral quantitative computed presented in Table 1 and Supplementary Table 2). Twenty-six tomography (pQCT) and calculated density obtained by authors 42–66 articles were excluded at this level with reasons described in by dividing the weight of cortical bone segment by its estimated 42,55,58,59 37–40 Supplementary Table 3. Of note, 4 articles presented volume. It is worth noting that 4 articles indicated that they relevant bone measures in primates but were excluded due to measured tissue mineral density (TMD) rather than bone mineral insufﬁcient quantity of any single measure of interest for density (BMD). However, we treated TMD and BMD identically, 14–41 quantitative synthesis. In the ﬁnal meta-analysis, 28 articles since the voxel size used in these μCT studies included were included, 20 regarding rats, and 8 regarding mice, ﬂown on a contributions from porosity . In addition, the polychromatic total of 17 spaceﬂight missions, with a total of 60 rats and 60 mice beam used in lab-based μCT leads to beam hardening effects, being described (overview of included article is in Table 2). which further limits the accuracy of tissue mineralization npj Microgravity (2022) 10 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA 1234567890():,; M. Goldsmith et al. Table 1. Included bone properties for meta-analysis. Parameter Description Unit(s) Measures of whole bone mechanical properties 1. Maximum load Load at which internal structure begins to fail , peak load on the load- cm-dyne or N displacement curve 2. Yield load Load beyond which permanent deformation occurs , the proportional limit on the N load-displacement curve 3. Failure load Load at which bone failure/fracture occurs. N 4. Stiffness Measure of whole bone resistance during elastic deformation , slope of the initial dyne/rad or N/mm linear portion of the load-displacement curve 5. Work to maximum load Energy required to reach max load, area under the load-displacement curve until Nmm or mJ max load 6. Work to failure load Energy required to reach failure load, area under the load-displacement curve until cm-dyne rad or Nmm failure load Measures of tissue-level mechanical properties 1. Elastic modulus Measure of tissue-level resistance to deformation, tissue-level stiffness GPa or MPa 2. Yield stress Measure of stress at the yield point MPa Bone density measure 3 3 3 1. Bone mineral density Mass of bone per unit volume or mass of bone per unit area mg/mm , g/cm , mg/cm ,or mg/cm Bone composition measures 1. Calcium content Amount of calcium per mass of dry bone g/100 g, mg/g, μg/mg, %dry weight, or mol/kg 2. Phosphorus content Amount of phosphorus per mass of dry bone g/100 g, mg/g, μg/mg, or mol/kg 3. Hydroxyproline content Mass of hydroxyproline per mass of dry bone mg/g or μg/mg 4. Osteocalcin content Mass of osteocalcin per mass of dry bone μg/mg, mg/g, or ng/mg measurements . Bone composition data for 4 compounds demonstrated that increased quality score was associated with present in bone, calcium, phosphorus, hydroxyproline, and decreased effect size magnitude for BMD and stiffness (Supple- osteocalcin, were included as the weight of the compound mentary Fig. 1e, h). This association was however confounded by compared to the overall dry bone weight. The speciﬁc measure- higher quality scores of newer articles, which also are describing ments present in each study are presented in Supplementary mouse studies. Quality score was not associated with BMD article- Table 4 and study characteristics used for covariate analysis in level standard error (Supplementary Fig. 1f). Supplementary Table 5. For the purposes of analysis, two types of control animal groups Long bone mechanical properties were considered; a vivarium control group (VC) comprised of We ﬁrst examined the effect of spaceﬂight on the bone strength animals housed in standard laboratory habitats, and a ground parameters yield load, max load, and failure load obtained using control group (GC) where some or all aspects of spaceﬂight other 3-point bending (3PBT) or torsional tests conducted on long than microgravity, including habitat, light/dark cycle, diet and bones (Fig. 2). Spaceﬂight signiﬁcantly reduced the max load in forces of liftoff and re-entry were simulated. To assess the hindlimb long bones (Fig. 2a, Supplementary Table 6) with the inﬂuence of microgravity, we calculated the normalized difference calculated effect size representing the normalized difference in between SF and GC. To determine the possible effect of max load between SF and GC of −15.42% with a 95% CI of conditions associated with spaceﬂight other than microgravity [−23.88, −6.96] in the femur, and –17.27% [−27.20, −7.34] in the on bone strength, we calculated the normalized difference tibia. The change in the forelimb long bones (humerus), was between GC and VC. negative but not signiﬁcant −12.66% [−27.05, 1.73]. For all the long bones, spaceﬂight signiﬁcantly reduced the max load with Heterogeneity, bias, and quality the calculated effect size of −15.24% [−22.32,−8.17] (Fig. 2a left). Among the 13 included parameters, statistical heterogeneity was In the femur was there a signiﬁcant difference between GC and high (I > 75%) for 3 datasets; stiffness, yield stress, and bone VC, with an increase of 15.52% [4.29, 26.75], however in other long density. Heterogeneity was moderate (55% > I > 40%) for 3 bones and overall max load in GC and VC was not signiﬁcantly datasets: max load; work to max load; and elastic modulus. The different (Fig. 2a right). Subgroup analysis of effect of measure- remaining 7 datasets showed low (I < 25%) heterogeneity. The ment technique on SF-induced changes to max load demon- largest and most heterogeneous dataset, BMD, was used to assess strated no signiﬁcant difference in outcomes resulting from global bias. From single study exclusion analysis, no single mission torsional test and 3PBT (Fig. 2b). Among measures of max load signiﬁcantly affected global heterogeneity or outcome (Supple- derived from 3PBT machinery, neither loading rate nor span mentary Fig. 1b). From cumulative study exclusion, 20% of studies length of the supports were signiﬁcantly associated with a change were excluded prior to the dataset reaching homogeneity, and the in outcome (Fig. 2c, d). Yield load and failure load decreased in SF outcome of the homogeneous dataset was similar to the complete compared to GC with a percent difference of −18.95% [−27.24, dataset (Supplementary Fig. 1c). The funnel plot demonstrated −10.66] and −10.41% [−21.99, 1.16] respectively, with only the uneven distribution; however, the presence of a speciﬁc bias was change to yield load being statistically signiﬁcant (Supplementary difﬁcult to ascertain (Supplementary Fig. 1d). Regression analysis Tables 7 and 8). When yield load, max load, and failure load were of article-level effect size as a function of quality score normalized to weight of respective animal group at the time of Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2022) 10 M. Goldsmith et al. Table 2. Overview of articles included in meta-analysis. Articles Mission Year Days Species n Type of control Bone(s) analyzed QS (/18) SF 14 a Morey-Holton 1978a Cosmos 782 1975 19.5 Rats 4 GC, VC Humerus 16 15 a Morey-Holton 1978b Cosmos 936 1977 18.5 Rats 4 GC, VC Femur 14 Prokhonchukov 1982 Cosmos 1129 1979 18.5 Rats 5 GC, VC Scapula 7 Rodgacheva 1984 6 Femur 11 Patterson-Buckendahl 1985 Spacelab 3 1985 7 Rats 6 GC Humerus 10 Patterson-Buckendahl 1987 6 Vertebrae (L3), Humerus 11 Shaw 1988 6 Humerus, Tibia 11 Simmons 1986 6 Vertebrae (T), Femur 11 22 a Cann 1990 Cosmos 1887 1987 12.5 Rats 5 GC, VC Vertebrae (L4) 7.5 Simmons 1990a 6 Calvarium, Vertebrae (L5) 15 Vailas 1990a 4 Humerus 17 Arnaud 1992 Cosmos 2044 1989 14 Rats 5 GC, VC Femur 13 26 a Cann 1994 5 Vertebrae (L5) 9 Vailas 1992 5 Humerus 16 28 a Vailas 1994 5 Vertebrae (L5) 8 Lafage-Proust 1998 STS-58 1993 14 Rats 5 GC Parietal bone, Vertebrae (T), Humerus, Tibia 14 Chapes 1999 STS-60 1994 8 Rats 6 GC, VC Femur, Tibia 15 STS-63 1995 8 Rats 6 GC, VC Femur, Tibia 15 Bateman 1998 STS-77 1996 10 Rats 6 VC Humerus, Femur 14 Vajda 2001 STS-78 1996 17 Rats 6 GC, VC Femur 14 Zerath 2000 6 Pelvic bone 14 Lloyd 2015 STS-108 2001 12 Mice 12 GC Vertebrae (L5), Femur, Tibia 14 Ortega 2013 STS-118 2007 13 Mice 12 GC Femur 14 Coulombe 2021 Femur, Tibia 15 Zhang 2013 STS-131 2010 15 Mice 7 GC Calvariae 14 Gerbaix 2017 Bion M1 2013 30 Mice 5 GC, VC Vertebrae (T12 & L3), Femur 15 Gerbaix 2018 5 Calcaneus, Navicular, Talus 15 Macaulay 2017 6 GC Calvariae 15 Coulombe 2021 SpaceX-4 2014 21 Mice 10 GC Femur, Tibia 14 Lee 2020 SpaceX-19 2019 33 Mice 8 GC Femur, Vertebrae (L2,3,5) 12 Days mission duration (days), n sample size of spaceﬂight animal group. Control groups: GC ground control, VC vivarium control. For vertebrae region: L SF lumber, T thoracic, C caudal. QS quality score calculated according to Supplementary note 1. Indicates articles sourced from NASA Final Reports of Soviet missions. sacriﬁce, we found the overall decrease in these parameters in SF failure load were not signiﬁcantly affected when normalized to animals compared to GC to be very similar: yield load −12.24% weight at sacriﬁce (Supplementary Tables 14–16). [−20.52, −3.95], max load −12.65% [−21.11, −4.18], failure load Given that included measures of bone strength were exclusively −11.36% [−21.47, −1.26], all of which were statistically signiﬁcant from torsional tests and 3PBT, reported tissue-level mechanical (Supplementary Tables 9–11). properties, elastic modulus and yield stress, were derived using Next, we assessed the effect of spaceﬂight on stiffness, work to engineering beam theory equations. In spaceﬂight rats, elastic max load, and work to failure load. Stiffness (Fig. 3, Supplementary modulus 1.64% [−19.98, 23.26] and yield stress 4.96 [−26.04, Table 12) decreased in the hindlimbs of SF animals by −15.40% 35.97] exhibited no signiﬁcant change from GC and had moderate [−23.38, −7.42] in the femur and by −16.09% [−23.48, −8.69] in to high heterogeneity (I ≥ 52%). Interestingly, elastic modulus the tibia, while the change in humerus stiffness was not demonstrated an overall signiﬁcant difference between the statistically signiﬁcant, −3.85[−26.54, 18.84]. When all long bones ground control and vivarium control with the decrease in GC by were combined, the spaceﬂight-induced change to stiffness was −21.61% [−35.02, −8.19] compared to VC (Supplementary Tables not statistically signiﬁcant (Fig. 3a left). There was no signiﬁcant 17 and 18). There were no available data reported for mice. The difference in long bones stiffness between GC and VC (Fig. 3a outcomes of elastic modulus and yield stress were unaffected right). The effect of spaceﬂight on bone stiffness in long bones did when normalized to weight (Supplementary Tables 19 and 20). not differ when sub-grouped by the measurement technique (Fig. 3b), and did not depend on loading rate (Fig. 3c) nor span length Bone Mineral Density (Fig. 3d) in 3PBT. The data for work to max load and work to failure BMD was signiﬁcantly lower in SF rats, −4.51% [−8.32, −0.70], and load were only available for rats. Both measures decreased in SF mice, −2.09 [−3.74, −0.44], compared to GC, with the overall animals by −16.41% [−47.85, 15.03] and −39.53% [−67.14, −11.92] respectively, with the considerably larger and statistically effect for rodents of −3.13% [−4.96, −1.29] and high hetero- signiﬁcant decrease for work to failure load (Fig. 4, Supplementary geneity (I = 83.4%) (Fig. 5a left). GC and VC were not dfferent (Fig. Tables 13). Outcomes for stiffness, work to max load, and work to 5a right). When stratiﬁed by the measurement technique, no npj Microgravity (2022) 10 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA M. Goldsmith et al. Fig. 2 Spaceﬂight-induced changes in max load of bone in rodent. a Forest plot of changes in max load in humerus, femur and tibia in spaceﬂight animals (SF) compared to ground control (GC) (Left); and GC compared to vivarium control animals (VC) (Right). Missions are ordered by mission year; mission name, duration (Days), SF and GC sample sizes (n /n ) are shown. Circle/line: effect size (%) and 95% CI, the SF GC size of the circle is proportional to the mission’s weight. Black diamonds: overall effect size and 95% CI for rats; color diamonds: overall effect size 2 2 and 95% CI for rodents. I and H are for rodents. *indicates missions where there was no GC, and SF was compared to VC. b Subgroup analysis of measured max load by mechanical test: torsional test (Torsional) and 3-point bending tests (3PBT). Square/line: effect size (%) and 95% CI. N : number of mission level outcomes. Meta-regression analysis of max load measures obtained by 3PBT as a function of loading rate (c) and span length (d) of the 3PBT machinery. Linear regression line (dark blue), its 95% CI (light blue area) and R are shown. signiﬁcant difference was found between measurements obtained not signiﬁcant decrease of −1.32% [−3.18, 0.54] (Fig. 6 middle). using mercury porosimetry, calculated from cross-sections, DEXA, Hydroxyproline content increased in the bone of spaceﬂight rats and computed tomography (Fig. 5b). Spaceﬂight-induced by 8.20% [−7.42, 23.83], although this change was not signiﬁcant decrease in BMD were statistically signiﬁcant in the hindlimb (Fig. 6 right). GC to VC comparisons for bone composition bones, femur and tibia, but not the humerus of the forelimb (Fig. parameters were not signiﬁcantly different (Supplementary Tables 5c). BMD measured from samples of bone that contained only 21–23). Osteocalcin content in bone was not affected by the cortical bone and samples that contained both cortical and spaceﬂight (Supplementary Table 24). trabecular bone demonstrated no signiﬁcant difference in SF to GC outcomes (Fig. 5d). When only measures from long bones were Covariate analysis considered, spaceﬂight-induced BMD deﬁcits were greater in We assessed the inﬂuence of covariates using subgroup and meta- regions containing both cortical and trabecular bone (metaphyses regression analyses on the 4 parameters with 6 or more mission- and epiphyses) with a decrease of −9.8% [−11.7, −7.8] compared level outcomes: max load, stiffness, BMD and calcium content. to regions containing only cortical bone (diaphyses) with a Animal related covariates included age at launch, age at sacriﬁce, decrease of −3.0% [−5.7, −0.4] (Fig. 5e). strain, sex, source or dealer, weight of spaceﬂight animals at recovery or sacriﬁce and the difference in weight between the Bone composition spaceﬂight and ground control animal groups (Δweight SF and The data for speciﬁc mineral and organic components of bone GC). Linear regression analysis identiﬁed a weak association were only available for rats, and included homogeneous (I = 0%) between age at launch and change in calcium content and no datasets for bone calcium, phosphorus, hydroxyproline and association with max load, stiffness, nor bone density (Fig. 7a). osteocalcin. Spaceﬂight rats demonstrated a signiﬁcant decrease Similarly, only change in calcium content was weakly associated in bone calcium content of −1.75% [−2.97, −0.52] (Fig. 6 left). with animal age at sacriﬁce (Supplementary Fig. 2a). All spaceﬂight Phosphorus content in spaceﬂight rats demonstrated a similar but mice were of C57BL/6 strains, therefore subgroup analysis on Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2022) 10 M. Goldsmith et al. Fig. 3 Spaceﬂight-induced changes in bone stiffness in rodents. a Forest plot of changes in stiffness in humerus, femur and tibia in spaceﬂight animals (SF) compared to ground control (GC) (Left); and GC compared to vivarium control animals (VC) (Right). Missions are ordered by mission year; mission name, duration (Days), SF and GC sample sizes (n /n ) are shown. Circle/line: effect size (%) and 95% CI, the SF GC size of the circle is proportional to the mission’s weight. Black diamonds: overall effect size and 95% CI for rats; color diamonds: overall effect size 2 2 and 95% CI for rodents. I and H are for rodents. *indicates missions where there was no GC, and SF was compared to VC. b Subgroup analysis of measured bone stiffness by mechanical test: torsional test (Torsional) and 3-point bending tests (3PBT). Square/line: effect size (%) and 95% CI. N number of mission level outcomes. Meta-regression analysis of stiffness measures obtained by 3PBT as a function of loading i: rate (c) and span length (d) of the 3PBT machinery. Linear regression line (dark blue), its 95% CI (light blue area) and R are shown. Fig. 4 Spaceﬂight-induced changes in work to failure load in rats. Forest plot of changes in work to failure load in spaceﬂight animals (SF) compared to ground control (GC) (Left); and GC compared to vivarium control animals (VC) (Right). Missions are ordered by mission year; mission name, duration (Days), SF and GC sample sizes (n /n ) are shown. Circle/line: effect size (%) and 95% CI, the size of the circle is SF GC proportional to the mission’s weight. Orange diamonds: overall effect size and 95% CI for rats. animal strain was only applied to rats, in which the decreases to Institute of Experimental Endocrinology of Czeckolslovakia, max load and stiffness were only signiﬁcant in Sprague-Dawley Taconic Farms (Germantown, NY or afﬁliated facilities), or Jackson rats, and not in Wistar rats, while density and calcium content Laboratory (Bar Harbor, ME). Subgroup analysis of mission level changes were similar for both strains (Supplementary Fig. 2b). All outcomes by source of animal did not affect the outcomes spaceﬂight rats were male, therefore subgroup analysis for animal (Supplementary Fig. 2c). Weight at time of sacriﬁce, or Δweight SF sex was only applied to mice. Comparing outcomes of BMD by sex and GC did not signiﬁcantly affected spaceﬂight outcomes in mice demonstrated a signiﬁcant decrease in female but not in (Supplementary Fig. 2d, e). male mice, although the number of datasets for male mice was Mission-related covariates included mission duration, SF hous- limited to 2 (Fig. 7b). Animals were obtained primarily from ing - either single or grouped, and year of mission launch. npj Microgravity (2022) 10 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA M. Goldsmith et al. Fig. 5 Spaceﬂight-induced changes in bone mineral density (BMD) in rodents. a Forest plot of changes in BMD in spaceﬂight animals (SF) compared to ground control (GC) (Left); and GC compared to vivarium control animals (VC) (Right). Missions are stratiﬁed by rodent species, and within each stratum, missions are ordered by mission year. Mission name, duration (Days), SF and GC sample sizes (n /n ) are shown. SF GC Circle/line: effect size (%) and 95% CI, the size of the circle is proportional to the mission’s weight. Black diamonds: overall effect size and 95% 2 2 CI for mice and rats; color diamonds: overall effect size and 95% CI for rodents. I and H are for rodents. b Subgroup analysis of changes in BMD by measurement technique, which included density derived from weight of cortical cross-section sample divided by volume determined either from mercury displacement (Mercury Porosimetry) or from geometric estimates (Cortical Cross-Sectional), as well as BMD obtained from DEXA, or pQCT/μCT.c Subgroup analysis of long bone BMD by the forelimb and hindlimb bones. d Subgroup analysis of all BMD outcomes by the bone type. e Subgroup analysis of long bone BMD by bone region. Square/line: effect size (%) and 95% CI. N number of mission level outcomes. N number of measurement level outcomes. Fig. 6 Spaceﬂight-induced changes to bone mineral composition in rats. Forest plot of changes in bone calcium (left), phosphorus (middle), and hydroxyproline (right) content of spaceﬂight animals (SF) compared to ground control (GC). Missions are ordered by mission year (old to recent). Mission name, duration (Days), SF and GC sample sizes (n /n ) are shown. Circle/line: effect size (%) and 95% CI, the size of the circle SF GC is proportional to the mission’s weight. Orange diamonds: overall effect size and 95% CI for rats. *indicates missions where there was no GC, and SF was compared to VC. Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2022) 10 M. Goldsmith et al. Fig. 7 Covariate analysis of spaceﬂight-induced changes in bone strength and composition. a Meta-regression analysis of max load, stiffness, BMD, and calcium content as a function of age at launch of SF animals. Linear regression line (dark color), its 95% CI (light color area) and R are shown. Subgroup analysis of BMD by animal sex (b) and by short (14 days or less) and long (greater than 14 days) mission duration (c). Square/line: effect size (%) and 95% CI. N number of mission level outcomes. d Meta-regression analysis of max load, stiffness, BMD, and i: calcium content as a function of mission duration. Linear regression line (dark color), its 95% CI (light color area) and R are shown. Subgroup analysis of BMD by single vs. grouped rat housing (e) and by how closely GC mimics SF conditions (f). For f: Group 1: GC housed in same habitat as the SF; Group 2: GC housed in same habitat as SF, the force of liftoff and/or re-entry were mimicked; Group 3: GC was mimicked by in-ﬂight centrifuge. Square/line: effect size (%) and 95% CI. N number of mission level outcomes. i: Subgroup analysis for short (<14 days) and long (≥14 days) DISCUSSION duration mission demonstrated no signiﬁcant difference between The objective of this study was to systematically review and mission duration subgroups for any parameter (Fig. 7c, Supple- quantitatively synthesize data regarding changes to bone strength mentary Fig. 3a); however, the decrease in stiffness compared to and bone composition in rodents sent to space. We demonstrate GC was only signiﬁcant in short durations missions, while the that whole bone mechanical properties in spaceﬂight rodents decrease in calcium was only signiﬁcant in long-duration missions were signiﬁcantly decreased in their hindlimbs but not in the (Supplementary Fig. 3a). Longer mission duration was weakly forelimbs. BMD was signiﬁcantly decreased in spaceﬂight rodents. associated with lower deﬁcits in BMD in linear regression analysis In spaceﬂight rats, bone calcium content was signiﬁcantly lower, (Fig. 7d). The max load, stiffness and BMD demonstrated greater with a decrease in phosphorus and an increase in hydroxyproline deﬁcits when rats were housed alone, although the difference that were not statistically signiﬁcant. We were able to perform a between groups was not statistically signiﬁcant (Fig. 7e, Supple- limited analysis of the effect of some covariates on the SF-induced mentary Fig. 3b). There was no association between launch year changes in bone strength and composition parameters. and outcome (Supplementary Fig. 3c). Spaceﬂight-induced deﬁcits in BMD were signiﬁcant in female Study related covariates included sacriﬁce delay and the degree mice, but not in male mice; decreases to bone strength to which GC animals mimic the conditions of SF animals (GC parameters were only signiﬁcant in Sprague-Dawley rats, and condition). Sacriﬁce delay was weakly associated with decreased not in Wistar rats; bone strength and density were affected more magnitude of max load, but did not affect other parameters in single-housed rats than group housed. However, the interac- (Supplementary Fig. 4a). BMD and calcium content outcomes were tions between multiple confounding factors, for example age and similar across all GC conditions (Fig. 7f, Supplementary Fig. 4b). sex, was not possible due to data paucity. Importantly, whole bone While max load and stiffness appeared to be affected in some GC mechanical, BMD, and mineral composition properties were not conditions, there were no consistent pattern (Supplementary signiﬁcantly different between the ground control and vivarium Fig. 4b). npj Microgravity (2022) 10 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA M. Goldsmith et al. 11,70,75 animal groups, suggesting that microgravity is the primary factor porosity also may have signiﬁcant effects on bone strength. causing these changes. Although the effect of spaceﬂight on HA crystallinity , and Our analysis only included measures of whole bone strength cortical porosity were measured in Bion M1 mission, we lacked derived from 3-point bending tests or torsional tests. The relative sufﬁcient data of these outcome measures for meta-analysis. change to bone strength from these two loading modes are While many common methods used today to measure mineral considered to be comparable as they both depend on the and matrix properties such as quantitative backscattered electron underlying geometric and material properties of the tested imaging, nanoindentation, small angle x-ray scattering, Fourier region which is composed of cortical bone in long bone diaphysis. transform infrared spectroscopy, and Raman spectroscopy , they We found that yield load and max load were signiﬁcantly lower in were not performed in enough studies to include in our analyses. SF with estimated decreases of −18.95% [−27.24, −10.66] and Thus, our study identiﬁes a signiﬁcant gap in our knowledge of the −15.24% [−22.32,−8.17] respectively, while a decrease in bone degree to which bone tissue level properties are affected by stiffness of −9.47 [−20.44,1.49] was not signiﬁcant. We found that microgravity. work to failure load, which represents the area under the force- Where it was possible, we investigated the effects of covariates displacement curve until failure , was the most affected on bone strength, density and composition outcomes. Similar to parameter in spaceﬂight animals with a decrease of −39.53% our previous ﬁndings , neither mission duration, nor age at [−67.14,−11.92], indicating a signiﬁcant decrease to bone launch were associated with signiﬁcant changes in measured toughness, although toughness is also deﬁned by fracture parameter, likely due to the relatively short mission durations, up mechanics parameters . Given that changes to stiffness, yield, to 33 days, as well as the younger age of included animals. We max and failure load were all estimated to be less than half of the conﬁrmed that housing type had a signiﬁcant effect on SF- work to failure load magnitude of change, we can deduce that induced changes. In rodents housed individually during space- post-yield displacement (PYD), a measure of bone ductility , may ﬂight, a greater decrease in bone stiffness, max load, and BMD was have been lower, potentially indicating increased bone brittleness observed compared to animals housed in groups. A hindlimb in spaceﬂight animals. This is supported by two pieces of unloading study that directly compared the effect of unloading on evidence. First, it has been reported that PYD has the greatest single-housed mice and those housed in pairs demonstrated that inﬂuence on work-to-fracture load . Second, in two included several immune and hypothalamic-pituitary-adrenal axis studies, Patterson-Buckendahl et al. report of SpaceLab3 and responses were signiﬁcantly different in these groups, suggestion Vailas et al. report of Cosmos 2044, max load and failure load strong contribution of social isolation to physiological responses occurred simultaneous. Tissue-level mechanical properties, elastic to unloading . However, in vivo mouse tibial loading studies modulus and yield stress determined from engineering beam performed on Earth have shown that the response to loading in theory equations did not change in spaceﬂight animals. However, male mice was reduced when mice were group housed, compared one must also consider the limitations of calculating tissue level to individually housed mice, likely due to increased mechanical properties from these equations, which has been reported to strains engendered in the tibiae during group-housed ﬁghting provide values that are greatly underestimated, with inconsistent activities that masked the bone (re)modeling response to and even inverse relative differences between experimental loading . We have also identiﬁed a potentially important groups compared to the relative differences reported by nano- difference between the responses to spaceﬂight in male and 72,73 indentation measurements . Therefore, our reported changes female mice, where only in female mice the spaceﬂight-induced in tissue-level mechanical properties should be interpreted with deﬁcits in BMD were signiﬁcant. However, low number of studies caution. Thus, whole bone mechanical properties are signiﬁcantly with male mice and no studies with female rats presented a major reduced in spacefaring rodents. limitation for further analysis. It has been reported that the whole bone mechanical properties We have found signiﬁcant regional differences in the bone depend on its mass, geometry and material compositional response to spaceﬂight. The change in BMD in the metaphyses of 8–10 properties . We demonstrated a signiﬁcant decrease in BMD long bones was greater than the change in the diaphysis. This of cortical bone diaphysis: −3.0% [−5.7, −0.4]. Comparing the trend is consistent with our previous report examining bone change in BMD to the changes in bone strength support the architecture, where a greater reduction in trabecular bone notion that changes to BMD alone may not explain the changes to compared to cortical bone was observed . We have found that 9,11 bone strength . We previously reported that in SF animals spaceﬂight-induced deﬁcits in maximum load, stiffness and BMD cortical bone area decreased signiﬁcantly by −5.9% [−8.0, −3.8] were higher in the hindlimb bones compared to the forelimb and cortical thickness decreased by −4.7% [−13.7,4.4] while there bone, supporting a region dependent changes in bone health due was no signiﬁcant change to marrow area . Thus, cortical bone to SF, which was similar to humans, for which the magnitude of mass decreased during spaceﬂight with no increase in total cross- bone loss was the highest in the legs, while arms were sectional area, which otherwise may have increased bone unaffected . Previously, we reported a trend to higher trabecular 10,70 13 strength . We also previously reported signiﬁcant reductions bone deﬁcits in distal skeletal regions compared to axial regions . in histomorphometric cortical bone formation indices only on the When we speciﬁcally analyzed the changes in humerus, femur and periosteal surface . These SF-induced alterations in cortical tibia, we found that spaceﬂight-induced changes in trabecular microstructure due to imbalanced bone (re)modeling are con- bone volume fraction (Tb.BV/TV) were −15.3% [−21.0, −9.7] in sistent with the reduction of bone strength in SF animals. humerus, −29.0% [−33.5, −24.5] for femur and −24% [−30.5, Our study suggests that alterations in bone composition −17.5] for tibia. This is also conﬁrmed by in ﬂight measurements properties due to SF also contributed to the altered bone of BMD using DEXA reported for SpaceX-19 mission, which strength. In the current study, we have demonstrated that bone reported that after 28 days of spaceﬂight decrease in BMD was calcium content signiﬁcantly decreased in SF rats compared to GC, observed in the femur and not the humerus . Analysis of with a trend of a decrease in phosphorus content, and a relative movement of mice sent to the International Space Station, noted increase in the organic component of bone quantiﬁed by the forelimb ambulation during the ﬁrst half of the mission as key in- increase in hydroxyproline, an amino acid unique to collagen is ﬂight activity . These data suggests that the increased use of the used as a relative measure of collagen content. There was no forelimbs may help to preserve bone health in this region. available data regarding calcium, phosphorus or hydroxyproline The limitations of this study included, i) variations in experi- content in SF mice, and thus possible species differences could mental designs between missions, ii) inconsistent reporting, iii) 10,69,70 not be determined. Other factors including HA crystallinity , variations in measures of BMD, and iv) use of skeletally immature, presence of microcracks , and changes in cortical bone growing animals. Limitations i and ii have been explored in detail Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2022) 10 M. Goldsmith et al. in Fu & Goldsmith et al. . In brief, mission designs and associated mechanical properties in spacefaring rodents. We demonstrated experiments have changed over time, and the included control signiﬁcant deterioration in bone health, including decreased group varied in terms of degree in which they mimic spaceﬂight- measures of bone architecture, strength and composition, and associated stressors. It was quite noticeable that reporting of altered bone turnover. Our analysis is important in providing solid certain parameters changes with time. For example, measures of quantitative estimates of the effect sizes with measures of whole bone mechanical properties were reported for all, but one variance, and in identifying gaps and directions for informing spaceﬂight mission involving rats. In contrast, only 2 of the 6 future spaceﬂight experiments. In addition to the need for more included studies on spaceﬂight missions involving mice report inﬂight measurements of bone mass and architecture, standardiz- whole bone mechanical properties. Similarly measures of bone ing measurement techniques, expanding the studies of animal calcium and phosphorus concentrations were only reported in rat sex, strain, age and spaceﬂight duration is critically important for missions, with no available data for mice. When grouping mission obtaining a clear picture on how bone is changed in microgravity SF to GC outcomes by degree to which control group mimic and how these changes can be prevented. spaceﬂight conditions, no clear association was observed suggest- ing that the microgravity is the main driver of the changes. METHODS Secondly, we observed that reported animal treatment was not consistent across publications. One example of this inconsistency This study was conducted in compliance with the Preferred is the great variation in reported sacriﬁce delays of SF animals Reporting Items for Systematic Reviews and Meta-Analysis among articles describing identical missions (Supplementary Table (PRISMA) statement. For the PRISMA Checklist, refer to Supple- 5). The third set of limitations was related to the use of several mentary Table 1. different measurement technique to assess BMD. Among these techniques, some measures were more precise such as using μCT, Search strategy, inclusion criteria and quality assessment others less so, such as estimating BMD by the weight of the bone The systematic search strategy used in this study was identical to sample, divided by the volume calculated as the cross-sectional that used in Fu & Goldsmith et al. 2021 . In brief a search strategy area of the sample multiplied by its thickness. Four studies using terms related to bone, space travel, and animals was 37–40 indicated that they report bone tissue mineral density , constructed and used to execute a search Medline, Embase, however the smallest voxel size used was 9 μm, while a resolution PubMed, BIOSIS Previews, and Web of Science on November 2nd, of 1 μm is required to distinguish cortical vasculature micro- 2017, with an updated search being performed on November 1st, architecture . For future studies, it would be valuable to also have 2019. An additional search of the NASA Technical Reporting analyses of bones using synchrotron-based tomography where Service (NTRS) and articles referenced in the compendium of smaller voxel sizes are possible and more accurate tissue mineral animal and cell spaceﬂight experiments compiled by Ronca et al. density can be determined without beam hardening artifacts that was performed manually. No language restrictions were applied to are present with lab-based computed tomography . The ﬁnal set considered articles. Title and abstract screening, performed of limitations was related to the use of skeletally immature independently by SDC & SFC for the primary search and by SVK rodents, particularly rats. Only one study included animals older for the update, selected articles describing any non-human than 6 months of age, and average age was ~ 11 weeks for rats, vertebrate sent to space. Studies that described humans, and 20 weeks for mice. C57BL/6 mice reach peak cancellous bone invertebrates or Earth-based spaceﬂight simulations were mass at 8–12 weeks of age. They achieve peak adult cortical bone excluded. Primary full text screening (conducted independently density in the femur by 16 weeks and whole bone strength in by SDC, SFC & MG for primary and MG for update) selected articles bending and torsion peaks by 20 weeks of age . Rats are describing the effects of spaceﬂight on bone health of mice, rats skeletally mature at 6–9 months of age . Since on average and primate. We included in the meta-analysis studies that included mice were closer to skeletal maturity, this may explain presented quantitative measures of strength, density and why the decrease to BMD was less severe to mice compared to SF composition of bones of the axial and appendicular skeleton in rats. However, one must keep in mind that age-related changes in mice and rats that were on normal diet, were not pregnant, and BMD and mechanical properties are genetic strain and sex did not have surgery other than sham. Only studies that presented 83 84 dependent in both mice and rats . It is clear from loading measures of bone strength resulting from three-point bending studies in rodents that young animals have a much greater bone tests (3PBT) or torsional tests were included as the relative 85,86 formation and resorptive response to mechanical loading .It changes in outcomes obtained using these loading modes were remains less clear how SF-induced bone (re)modeling changes are suggested to be comparable . Of studies reporting strength affected by age, but a recent study by Coulombe et al. showed 54 28 measures, only Zernicke et al. and Vailas et al. reported useable that mature 32-week-old female mice exposed to microgravity data derived from compression test machinery. Gerbaix et al. experienced greater bone loss than young 9-week-old mice with reported hardness and elastic modulus results using nanoindenta- net skeletal growth. However, aged mice similarly showed a tion, which precluded meta-analysis for these measures. Papers diminished recovery upon re-ambulation compared to adult included in meta-analysis were scored on an 18-point scale for mice . We were not able to perform extensive strain and sex reporting quality (Supplementary note 1). If the outcomes of two analysis, because of limited information. Subgroup analysis of separate missions were reported in a single article, quality score animal sex for BMD in mice demonstrated potential difference (QS) was assessed for each mission independently. between the responses in male and female mice, however only 2 groups of male mice were included both from the same Bion M1 Data extraction mission. Mechanical loading studies in mice have observed sex- 87,88 89 related differences in cortical bone , but not cancellous bone . The following data was extracted by MG and veriﬁed by SVK for all Genetic strain-speciﬁc differences in mechanoresponsive that studies included in meta-analysis: mission name and duration; have been reported between C57BL6, Balb/c, and C3H/HeJ animal species and sample size (n) of spaceﬂight, ground control, 90–92 mice . Future studies are needed to carefully examine how and vivarium control groups (when applicable); bone type and genetic strain, age and sex affect the mechano-adaptive bone region being measured; measurement technique; and mean response to SF. and median in the 13 bone parameters (Table 1); standard errors, The two meta-analytic studies (Fu & Goldsmith et al. and the standard deviations, and/or interquartile ranges; days when current study) quantitatively summarize previously reported measurements were taken. If the type of dispersion measure changes to bone architecture, turnover, composition and was not given, we assumed it to be a standard error to ensure a npj Microgravity (2022) 10 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA M. Goldsmith et al. conservative estimate. If a range of sample sizes was reported, the accordance with the RE model, global effect size, θ, was calculated smallest value was extracted. The following mission characteristics using mission-level outcomes θ and their associated weight w via i i were also extracted for covariate analysis: animal strain, age at Eq. (5), launch and sacriﬁce, weight at sacriﬁce or recovery, sex, source or ðÞ θ ´ w i i dealer of animals, year of mission, spaceﬂight group sacriﬁce θ ¼ P (5) delays, single vs grouped spaceﬂight habitat, and treatment i conditions of ground control group. Mission characteristics were where N is the number of combined mission-level outcomes. pooled from all applicable articles. If articles report differing values Equation (6) was used to calculate weight of mission-level for apparently identical samples, the data from the article with the outcomes w using mission-level standard error se(θ ) and the i i 2 2 higher quality score was included. If articles report conﬂicting DerSimonian-Laird interstudy variance estimator τ . τ was values for a single mission characteristic, the most frequently calculated using Eqs. (7), (8), and (9). reported was included if possible, otherwise, the value from the article with a higher quality score was included. If only an interval w ¼ i (6) seðÞ θ þτ of time was provided for age at launch the mean value was used, i if only an interval of time was provided for spaceﬂight animal Q ðN 1Þ sacriﬁce delay, the higher value was used. All alternate terms used (7) τ ¼ for included parameters are in Supplementary Table 2. ! ! P 2 seðÞ θ ´ θ i i 2 i Measurement-level outcomes (8) Q ¼ seðÞ θ ´ θ i i P seðÞ θ This study included relevant data of two control groups: the i vivarium control (VC) consisting of animals housed in standard laboratory habitats, and the ground control (GC) which modeled seðÞ θ X i (9) some or all aspects of spaceﬂight except for microgravity. Animals c ¼ seðÞ θ seðÞ θ sent to space and subjected to artiﬁcial gravity (AG) were i considered GC. When possible, GC was used as a comparison Standard error of global effect size was calculated using Eq. (10). group, in missions without GC, VC was used as the comparator for spaceﬂight (SF). For each bone measurement j, the mean SF value, 1 se θ ¼ qﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ μ , and the mean comparison control (CC) value, μ with their P (10) SFj CCj associated standard errors se(μ ), or standard deviations sd(μ ) were i j j recorded In instances where sd(μ ) was recorded, it was converted . j pﬃﬃﬃ 95% conﬁdence intervals (CI) was calculated as 95% to se(μ)as se μ ¼ sd μ = n, where n is n for spaceﬂight and ^ ^ ^ ^ j j j SF CI¼ θ±z ´ seðθÞ¼ θ ±1:96 ´ SEðθÞ. All the above analysis ð1α=2Þ n for the corresponding control. For median P and interquartile CC was repeated for GC to VC comparisons, replacing instances of SF range x − x , μ was calculated as μ ¼ðx þ P þ x Þ=3 upper lower j j upper lower pﬃﬃﬃ and GC with GC and VC respectively. with: se μ ¼ x x = n ´ 2:7: We calculated j upper lower measurement-level effect size as the normalized percent differ- Heterogeneity and publication bias analysis ence, θ , between μ and μ using Eq. (1). j SFj CCj 2 2 Heterogeneity of global outcomes were reported as H and I μ μ 2 SF CC 2 Q 2 H 1 j j which uses Cochran’s Q (Eq. (8)) as: H ¼ , and I ¼ .To θ ¼ ´ 100% 2 j (1) N1 H CC assess the contribution of individual missions to global outcome and heterogeneity, we performed single data exclusion analysis, The cumulative standard error in percentage, se(θ ), was calculated wherein one at a time each mission-level outcome was assuming the two groups were independent using Eq. (2). sequentially removed and heterogeneity statistics recalculated. vﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 0 12 0 12 In cumulative data exclusion analysis mission-level outcomes were se μ se μ u SF CC j j excluded sequentially starting with those that contributed the t@ A @ A (2) se θ ¼ þ ´ 100% highest heterogeneity. A funnel plot showing the distribution of μ μ SF CC j j seðÞ θ to θ was used to assess reporting bias. Independent of their i i contribution to heterogeneity or potential bias, we included all the studies in the ﬁnal analysis. Mission-level outcomes When measurement level outcomes of multiple unique b bones or Additional analysis bone regions were recorded for mission i, mission-level effect sizes The following 17 characteristics were used for covariate analysis: θ and standard error se(θ ) were calculated as unweighted means i i ﬂight duration, strain of rats, sex of mice, source or dealer of by Eqs. (3), (4) respectively. animals, age at launch & sacriﬁce, weight at sacriﬁce/recovery, change in weight between SF and CC group, launch year, SF (3) θ ¼ b sacriﬁce delay, single vs grouped housing condition, the degree to which GC group mimic the environmental conditions of SF (GC se θ j conditions), bone or bone region measured, measurement (4) seðÞ θ ¼ technique, span length & loading rate of 3PBT, and article quality score. Subgroup analysis was performed by combining mission- For a single mission, Bion M1, the data for two animal groups were level outcomes and standard error within each category for 38,39 reported separately . As a result, these two animal groups were categorical variables sex, strain, animal source, single vs grouped treated as independent missions. housing conditions, GC conditions, and measurement technique, as well as for short (<14 days) and long (≥14 days) duration Meta-analytic model and global outcome missions. Subgroup analysis for measurement-level outcomes was Considering that we combine data from two different rodent used for bone type or bone region analysis. Meta-regression species aboard spaceﬂight missions with highly heterogeneous analysis was performed on mission level outcomes for continuous methodologies, a random effects (RE) model was selected. In variables: ﬂight duration, launch year, age at launch & sacriﬁce, Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2022) 10 M. Goldsmith et al. weight at sacriﬁce or recovery, and change in weight between SF 8. van der Meulen, M. C. H., Jepsen, K. J. & Mikić, B. Understanding bone strength: size isn’t everything. Bone 29, 101–104 (2001). and CC group. Meta-regression analysis on measurement-level 9. Kim, G., Boskey, A. L., Baker, S. P. & van der Meulen, M. C. Improved prediction of outcomes was performed for span length & loading rate in 3PBT. rat cortical bone mechanical behavior using composite beam theory to integrate For quality score, missions reported in a single article were tissue level properties. J. Biomech. 45, 2784–2790 (2012). combined to create a paper-level score,θ and associated seðÞ θ p P 10. Wallace, J. M. in Basic and applied bone biology (eds David B. Burr & Matthew R. using Eqs. 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M.G. was supported by graduate scholarship from McGill Faculty of in published maps and institutional afﬁliations. Dentistry. AUTHOR CONTRIBUTIONS Open Access This article is licensed under a Creative Commons M.G., S.D.C., S.F.C., and S.V.K. performed screening; M.G. and S.D.C. performed data Attribution 4.0 International License, which permits use, sharing, extraction; M.G. performed meta-analysis; M.G., B.M.W., and S.V.K. performed critical adaptation, distribution and reproduction in any medium or format, as long as you give analysis of the data; M.G. and S.V.K. wrote the ﬁrst draft; all authors edited and appropriate credit to the original author(s) and the source, provide a link to the Creative approved the manuscript. Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the COMPETING INTERESTS article’s Creative Commons license and your intended use is not permitted by statutory The authors declare no competing interests. regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons. org/licenses/by/4.0/. ADDITIONAL INFORMATION Supplementary information The online version contains supplementary material © The Author(s) 2022 available at https://doi.org/10.1038/s41526-022-00195-7. Correspondence and requests for materials should be addressed to Svetlana V. Komarova. npj Microgravity (2022) 10 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA

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Bone strength and composition in spacefaring rodents: systematic review and meta-analysis

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