Antioxidant Strategy to Prevent Simulated Microgravity-Induced Effects on Bone Osteoblasts

The effects induced by microgravity on human body functions have been widely described, in particular those on skeletal muscle and bone tissues. This study aims to implement information on the possible countermeasures necessary to neutralize the oxidative imbalance induced by microgravity on osteoblastic cells. Using the model of murine MC3T3-E1 osteoblast cells, cellular morphology, proliferation, and metabolism were investigated during exposure to simulated microgravity on a random positioning machine in the absence or presence of an antioxidant—the 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox). Our results confirm that simulated microgravity-induced morphological and metabolic alterations characterized by increased levels of reactive oxygen species and a slowdown of the proliferative rate. Interestingly, the use of Trolox inhibited the simulated microgravity-induced effects. Indeed, the antioxidant-neutralizing oxidants preserved cell cytoskeletal architecture and restored cell proliferation rate and metabolism. The use of appropriate antioxidant countermeasures could prevent the modifications and damage induced by microgravity on osteoblastic cells and consequently on bone homeostasis.


Introduction
Bone physiology is featured by a complex crosstalk between different cell phenotypes and their interaction with organic matrix and inorganic matter. In this scenario, biophysical interactions and mechanical loading have a key role in regulating bone tissue metabolism and functional balance of bone remodeling [1]. On Earth, body weight by 1 g-gravity and muscle activities are the main forces that drive bone functional adaptation. This is impaired in microgravity conditions where body weight and muscle activities are deeply modified [2,3]. Considering the increasing interest in space colonization and the number of human missions to explore the deep-space environment, the importance of preserving bone health is becoming a crucial task. The main macroscopic effect induced by microgravity exposure on bone tissue is the decrease of bone mass. In particular, bone mass loss is more evident in bones of legs and the lower back, possibly due both to the unloading and body fluid shifting, and consequently, the decreased blood perfusion [4][5][6].
These effects are due to microgravity-induced modifications at cellular levels that in bone tissue may result in an impairment in the activities and signaling of osteoclasts, osteoblasts, and osteocytes.
Trolox can neutralize the harmful effects of oxidative stress thus counteracting morphological changes and intracellular triggered signals (Ca 2+ and ROS levels) evoked by s-microgravity exposure.

Cell Morphology and Proliferation under s-Microgravity Conditions
The MC3T3-E1 cells were cultured up to 96 h under s-microgravity (exposed cells, RPM) or at 1 g (control cells, Ctr) conditions. At selected times (24,48,72, and 96 h) morphological parameters, cell viability, and expression levels of main cytoskeleton proteins were assayed. Figure 1 shows representative images (XZ projection-or single optical section-images) of control and s-microgravity-exposed cells (see also z-stack reconstruction-images in Supplementary Figure S1). Herewith, by using the model of murine MC3T3-E1 osteoblast cells exposed to RPM-generated s-microgravity, we focused our attention on 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), a well-known water-soluble analogue of vitamin E acting as a scavenger of free radicals. Trolox can neutralize the harmful effects of oxidative stress thus counteracting morphological changes and intracellular triggered signals (Ca 2+ and ROS levels) evoked by s-microgravity exposure.

Cell Morphology and Proliferation under s-Microgravity Conditions
The MC3T3-E1 cells were cultured up to 96 h under s-microgravity (exposed cells, RPM) or at 1 g (control cells, Ctr) conditions. At selected times (24,48,72, and 96 h) morphological parameters, cell viability, and expression lSupplementaryevels of main cytoskeleton proteins were assayed. Figure 1 shows representative images (XZ projection-or single optical section-images) of control and s-microgravity-exposed cells (see also z-stack reconstruction-images in Supplementary Figure S1). Morphological assessment of MC3T3-E1 cells exposed to s-microgravity conditions. (A) Representative orthogonal projections (XZ) obtained from the z-stack images from control cells at 1g (Ctr) or cells exposed to s-microgravity (RPM) at different times (24,48,72, and 96 h), stained with Alexa Fluor 546 Phalloidin (for f-actin) and DAPI (for nuclei). (B) The graph shows quantitative analyses of cell height. This was calculated as described in Materials and Methods and as shown in the example image (h = height). (C) Representative single optical section-images of Ctr or RPM cells, stained with Alexa Fluor 546 Phalloidin (for f-actin) and DAPI (for nuclei), and the formula used to assay nuclei roundness. (D-F) Quantitative analyses of nucleus area, nucleus roundness, and the mean length of actin filaments of Ctr or RPM cells at different exposure times (24-96 h). The data in the graphs are presented as the means ± SEM from three independent experiments. * p < 0.05 vs. Ctr; ** p < 0.01 vs. Ctr, *** p < 0.001 vs. Ctr. Figure 1. Morphological assessment of MC3T3-E1 cells exposed to s-microgravity conditions. (A) Representative orthogonal projections (XZ) obtained from the z-stack images from control cells at 1g (Ctr) or cells exposed to s-microgravity (RPM) at different times (24,48,72, and 96 h), stained with Alexa Fluor 546 Phalloidin (for f-actin) and DAPI (for nuclei). (B) The graph shows quantitative analyses of cell height. This was calculated as described in Materials and Methods and as shown in the example image (h = height). (C) Representative single optical section-images of Ctr or RPM cells, stained with Alexa Fluor 546 Phalloidin (for f-actin) and DAPI (for nuclei), and the formula used to assay nuclei roundness. (D-F) Quantitative analyses of nucleus area, nucleus roundness, and the mean length of actin filaments of Ctr or RPM cells at different exposure times (24-96 h). The data in the graphs are presented as the means ± SEM from three independent experiments. * p < 0.05 vs. Ctr; ** p < 0.01 vs. Ctr, *** p < 0.001 vs. Ctr.
The quantitative analyses revealed that the 48h-exposure time was a key time for morphological changes. In comparison to control cells, there were the following findings: (1) a small but significant increase of cell height in 48h-exposed cells followed by a decreased height after 72 and 96h-exposure ( Figure 1B); (2) a bigger nuclear area in 48h-exposed cells ( Figure 1C,D); (3) a significant increase in roundness values of nuclei starting from 48h-exposure ( Figure 1C,E); (4) an increased mean length of actin filaments starting from 48h-exposure ( Figure 1F). The effects observed at 48h-exposure were probably dependent on the higher expression levels of β actin and β tubulin (even if this latter one did not reach the statistical significance), main components of the cytoskeleton, in 24h-exposed cells in comparison to control cells ( Figure 2A). Furthermore, in 24h-exposed cells, an increased expression levels of β1 integrin, a cell adhesion molecule responsible of cell-matrix contacts, was observed ( Figure 2B).
Starting from 48h-exposure to s-microgravity, MC3T3-E1 cells significantly decreased the proliferative rate in comparison to control cells ( Figure 2C). The following 72h-and 96h-exposures did not affect the proliferative trend ( Figure 2C), in addition, the trypan blue-exclusion test revealed a very low percentage of blue-stained cells at any time both in controls and s-microgravity-exposed cells (<5%). The quantitative analyses revealed that the 48h-exposure time was a key time for morphological changes. In comparison to control cells, there were the following findings: (1) a small but significant increase of cell height in 48h-exposed cells followed by a decreased height after 72 and 96h-exposure (Figure 1 B); (2) a bigger nuclear area in 48h-exposed cells (Figure 1 C,D); (3) a significant increase in roundness values of nuclei starting from 48h-exposure (Figure 1 C,E); (4) an increased mean length of actin filaments starting from 48h-exposure (Figure 1 F). The effects observed at 48h-exposure were probably dependent on the higher expression levels of β actin and β tubulin (even if this latter one did not reach the statistical significance), main components of the cytoskeleton, in 24h-exposed cells in comparison to control cells (Figure 2 A). Furthermore, in 24h-exposed cells, an increased expression levels of β1 integrin, a cell adhesion molecule responsible of cell-matrix contacts, was observed (Figure 2 B).
Starting from 48h-exposure to s-microgravity, MC3T3-E1 cells significantly decreased the proliferative rate in comparison to control cells (Figure 2 C). The following 72h-and 96h-exposures did not affect the proliferative trend (Figure 2 C), in addition, the trypan blue-exclusion test revealed a very low percentage of blue-stained cells at any time both in controls and s-microgravity-exposed cells (< 5%). Representative immunoblots of β actin, β tubulin, and β1 integrin expression levels in extracts from control cells at 1g (Ctr) or cells exposed to s-microgravity (RPM) at different exposure times (24-96 h). The densitometric analyses are plotted as the relative expression calculated as a ratio between the optical density (OD) × mm 2 of each band and OD × mm 2 of the corresponding GAPDH band, used as loading control. (C) Cell proliferation tested on Ctr and RPM-exposed cells at different exposure times (24-96 h). The data are presented as the means ± SEM from three independent experiments. *p< 0.05 vs Ctr.

Intracellular Cell Responses to s-Microgravity
The 24h-and 48h-exposure to s-microgravity were also critical for biochemical cell behaviour. Starting from 24h-exposure, the MC3T3-E1 cells cultured on RPM showed increased intracellular Ca 2+ and ROS levels, in comparison to control cells (Figure 3 A,B). Intracellular Ca 2+ levels regained control values at 72h-and 96h-exposure, while ROS levels were found to be increased up to 96h-exposure (Figure 3 B). The cells appeared to react to this metabolic status because mitochondrial membrane potential decreased at 48h-or 72h-exposure, showing signs of mitochondria suffering (Figure 2 D). Representative immunoblots of β actin, β tubulin, and β1 integrin expression levels in extracts from control cells at 1g (Ctr) or cells exposed to s-microgravity (RPM) at different exposure times (24-96 h). The densitometric analyses are plotted as the relative expression calculated as a ratio between the optical density (OD) × mm 2 of each band and OD × mm 2 of the corresponding GAPDH band, used as loading control. (C) Cell proliferation tested on Ctr and RPM-exposed cells at different exposure times (24-96 h). The data are presented as the means ± SEM from three independent experiments. * p < 0.05 vs. Ctr.

Intracellular Cell Responses to s-Microgravity
The 24h-and 48h-exposure to s-microgravity were also critical for biochemical cell behaviour. Starting from 24h-exposure, the MC3T3-E1 cells cultured on RPM showed increased intracellular Ca 2+ and ROS levels, in comparison to control cells ( Figure 3A,B). Intracellular Ca 2+ levels regained control values at 72h-and 96h-exposure, while ROS levels were found to be increased up to 96h-exposure ( Figure 3B). The cells appeared to react to this metabolic status because mitochondrial membrane potential decreased at 48h-or 72h-exposure, showing signs of mitochondria suffering ( Figure 2D). The inference of s-microgravity conditions on the metabolic status of MC3T3-E1 cells was also evident considering the increased levels of glucose and lactate in the culture media during all exposure times (from 24 up to 96 h) ( Figure 3D,E).
The inference of s-microgravity conditions on the metabolic status of MC3T3-E1 cells was also evident considering the increased levels of glucose and lactate in the culture media during all exposure times (from 24 up to 96 h) (Figure 3 D,E). Glucose and lactate levels measured in the growth medium of the Ctr and RPM-exposed cells. The data in the graphs are presented as the means ± SEM from three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 vs Ctr.

Counter-Measures to s-Microgravity
The effects on cell metabolism described above suggested that s-microgravity induced an altered oxidative status in exposed cells. To prevent oxidative unbalance, MC3T3-E1 cells were exposed to smicrogravity in the presence of Trolox to test if this antioxidant could neutralize the morphological and protein expression changes showed in Figures 1 and 2. The data showed in Figures 4-6 point out that the presence of the antioxidant counteracted the s-microgravity-induced effects. In particular, starting from 24h-incubation, in RPM-exposed cells, Trolox restored intracellular ROS and Ca 2+ levels, the mitochondrial membrane potential, as well as glucose and lactate levels in the medium ( Figure  4A-E). This evidence was supported by the complete data sets and statistical analyses reported in Supplementary Tables. Glucose and lactate levels measured in the growth medium of the Ctr and RPM-exposed cells. The data in the graphs are presented as the means ± SEM from three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. Ctr.

Counter-Measures to s-Microgravity
The effects on cell metabolism described above suggested that s-microgravity induced an altered oxidative status in exposed cells. To prevent oxidative unbalance, MC3T3-E1 cells were exposed to s-microgravity in the presence of Trolox to test if this antioxidant could neutralize the morphological and protein expression changes showed in Figures 1 and 2. The data showed in Figures 4-6 point out that the presence of the antioxidant counteracted the s-microgravity-induced effects. In particular, starting from 24h-incubation, in RPM-exposed cells, Trolox restored intracellular ROS and Ca 2+ levels, the mitochondrial membrane potential, as well as glucose and lactate levels in the medium ( Figure 4A-E). This evidence was supported by the complete data sets and statistical analyses reported in Supplementary Tables.  (Tables S1-S5).
We observed that under ROS-evoked stress, not only both cytoskeleton architecture and cell shape are altered, but a wide range of essential biological cell processes, including cell adhesion, proliferation, and survival, are influenced [27,28]. We suggest that increase in ROS levels can likely trigger these changes. Indeed, the presence of Trolox in the medium of cells cultured on RPM almost completely abolished the effects induced by s-microgravity on cell height, the mean length of actin and the expression levels of β actin and β1 integrin ( Figure 5 A, B, C and E). However, antioxidant addition did not succeed in completely counteracting the s-microgravity-induced effects on the nucleus shape, as nucleus roundness was unaffected by treatment ( Figure 5 D).
The antioxidant activity of Trolox was also able to restore the proliferation rate when the molecule was present in the medium of cells exposed to s-microgravity. In addition, the trypan blueexclusion test revealed a very low percentage (< 5%) of blue-stained cells at any times in all tested cell populations (controls and s-microgravity-exposed cells also in the presence of Trolox). These percentages were not significantly different in controls and s-microgravity-exposed cells. The trypan blue-exclusion test is a non-specific assay and can reveal early-necrotic or late-apoptotic cells and, even if we did not perform further investigations regarding this aspect, possible apoptotic or autophagic mechanisms could be involved.  (Tables S1-S5).
We observed that under ROS-evoked stress, not only both cytoskeleton architecture and cell shape are altered, but a wide range of essential biological cell processes, including cell adhesion, proliferation, and survival, are influenced [27,28]. We suggest that increase in ROS levels can likely trigger these changes. Indeed, the presence of Trolox in the medium of cells cultured on RPM almost completely abolished the effects induced by s-microgravity on cell height, the mean length of actin and the expression levels of β actin and β1 integrin ( Figure 5A-C,E). However, antioxidant addition did not succeed in completely counteracting the s-microgravity-induced effects on the nucleus shape, as nucleus roundness was unaffected by treatment ( Figure 5D).
The antioxidant activity of Trolox was also able to restore the proliferation rate when the molecule was present in the medium of cells exposed to s-microgravity. In addition, the trypan blue-exclusion test revealed a very low percentage (<5%) of blue-stained cells at any times in all tested cell populations (controls and s-microgravity-exposed cells also in the presence of Trolox). These percentages were not significantly different in controls and s-microgravity-exposed cells. The trypan blue-exclusion test is a non-specific assay and can reveal early-necrotic or late-apoptotic cells and, even if we did not perform further investigations regarding this aspect, possible apoptotic or autophagic mechanisms could be involved. , and s-microgravity-exposed cells without (RPM) or with (RPM + Trolox) 100 μM Trolox up to 24 h of cell culture. The densitometric analyses are plotted as the relative expression calculated as the ratio between the optical density (OD) × mm 2 of each band and OD × mm 2 of the corresponding anti-glyceraldehyde-3-phosphatedehydrogenase (GAPDH) band, used as loading control. In all graphs, the data are presented as the means ± SEM from three independent experiments. * p < 0.05, ** p < 0.01 vs respective Controls; # p < 0.05, ## p < 0.01 vs RPM+DMSO. See the complete data sets and detailed statistical analyses, reported in Supplementary Tables (Tables S6-S10 and Western blot analysis). , and s-microgravity-exposed cells without (RPM) or with (RPM + Trolox) 100 µM Trolox up to 24 h of cell culture. The densitometric analyses are plotted as the relative expression calculated as the ratio between the optical density (OD) × mm 2 of each band and OD × mm 2 of the corresponding anti-glyceraldehyde-3-phosphatedehydrogenase (GAPDH) band, used as loading control. In all graphs, the data are presented as the means ± SEM from three independent experiments. * p < 0.05, ** p < 0.01 vs. respective Controls; # p < 0.05, ## p < 0.01 vs. RPM+DMSO. See the complete data sets and detailed statistical analyses, reported in Supplementary Tables (Tables S6-S10 and Western blot analysis).  (Table S11).

Discussion
Gravity, as well as many biophysical forces and constraints, plays a critical role in shaping bone structure and functions [29][30][31]. Namely, weightlessness promotes an imbalance between bone formation and resorption that can lead to bone loss [32][33][34]. The development of countermeasures capable of preventing this condition may ensure safe space missions for astronauts, while shedding light on a number of bone pathological ailments, akin to osteoporosis.
The osteoblasts, together with osteoclasts, are critically important for bone formation and remodeling in response to mechanical stimuli [35]. Numerous studies showed that weightlessness affects osteoblast morphology, proliferation, differentiation, gene expression, and oxidative status [22,30].
In the present study, we used the murine MC3T3-E1 cells as model, a widely accepted in vitro osteoblast-like phenotype, and the RPM device to simulate microgravity conditions. This choice was due to previously published reports that consider RPM as a useful, handy, and valid device able to simulate microgravity and to expose adherent cells [36,37]. The experiments were performed on proliferating MC3T3-E1 cells in the absence of any growth factor or differentiating stimulus in order to assess a mere relationship between cellular changes and variation of external forces.
In our experimental condition, the short-term exposure (24-48 h) of MC3T3-E1 cells at smicrogravity is critical for cell behavior, and the presence of an antioxidant preserves the cells. Our data revealed that s-microgravity conditions dramatically modify the cell and nucleus morphology of MC3T3-E1 osteoblasts. Early response includes cell increased height followed, after 72 h, by a more flattened shape, while nuclei adopt a more circular form as their roundness increased. It is well accepted that shape parameters are instrumental in allowing specific cell functions and cell fate commitment towards differentiation [38][39][40].
Several studies, carried out by adopting different weightlessness devices as well as on board the International Space Station, provide similar results. MC3T3-E1 cells exposed to weightlessness in high magnetic gradient environment changed into a flattened shape, their nucleus was enlarged and the cells appeared polygonal-shaped compared to those under 1 g condition [20]. The fractal analyses on MC3T3-E1 cells exposed to RPM revealed that these cells became larger and acquired a more  (Table S11).

Discussion
Gravity, as well as many biophysical forces and constraints, plays a critical role in shaping bone structure and functions [29][30][31]. Namely, weightlessness promotes an imbalance between bone formation and resorption that can lead to bone loss [32][33][34]. The development of countermeasures capable of preventing this condition may ensure safe space missions for astronauts, while shedding light on a number of bone pathological ailments, akin to osteoporosis.
The osteoblasts, together with osteoclasts, are critically important for bone formation and remodeling in response to mechanical stimuli [35]. Numerous studies showed that weightlessness affects osteoblast morphology, proliferation, differentiation, gene expression, and oxidative status [22,30].
In the present study, we used the murine MC3T3-E1 cells as model, a widely accepted in vitro osteoblast-like phenotype, and the RPM device to simulate microgravity conditions. This choice was due to previously published reports that consider RPM as a useful, handy, and valid device able to simulate microgravity and to expose adherent cells [36,37]. The experiments were performed on proliferating MC3T3-E1 cells in the absence of any growth factor or differentiating stimulus in order to assess a mere relationship between cellular changes and variation of external forces.
In our experimental condition, the short-term exposure (24-48 h) of MC3T3-E1 cells at s-microgravity is critical for cell behavior, and the presence of an antioxidant preserves the cells. Our data revealed that s-microgravity conditions dramatically modify the cell and nucleus morphology of MC3T3-E1 osteoblasts. Early response includes cell increased height followed, after 72 h, by a more flattened shape, while nuclei adopt a more circular form as their roundness increased. It is well accepted that shape parameters are instrumental in allowing specific cell functions and cell fate commitment towards differentiation [38][39][40].
Several studies, carried out by adopting different weightlessness devices as well as on board the International Space Station, provide similar results. MC3T3-E1 cells exposed to weightlessness in high magnetic gradient environment changed into a flattened shape, their nucleus was enlarged and the cells appeared polygonal-shaped compared to those under 1 g condition [20]. The fractal analyses on MC3T3-E1 cells exposed to RPM revealed that these cells became larger and acquired a more rounded phenotype [12]. Also during parabolic flight shape changes were noticed even if microgravity occurred for very short-lasting time exposure (20 s) [41].
There are also growing evidence that the cell-microenvironment cross-talk can transduce mechanic stimuli through the integrin-cytoskeleton network, which, in turn, transmits physical stresses to nucleoskeleton, eventually leading to remodeling of chromatin structure and nuclear morphology [42][43][44].
In our model, after 24h-exposure to s-microgravity, there were transient increases in integrin and actin expression levels, whereas actin-filament mean length increased after 48h-exposure. Interestingly, this cytoskeleton reorganization is accompanied by the inhibition of cell proliferation, another macroscopic event that became significant only after 48-exposure time.
Testa and colleagues showed that the s-microgravity on RPM induced in murine osteoblasts a significant increase in the mean surface area and roundness, accompanied by a slow-down of cell proliferation, a concomitant increase in cell apoptosis and a reduction of expression level of β1-integrin [12]. In addition, studies conducted in MC3T3-E1 cells exposed to s-microgravity using 2/3D clinostats, showed cytoskeleton alterations accompanied by loss of stress fibers, decreased β-actin mRNA levels [19]. Some apparently contradictory data related to the time-course of the effects of microgravity on changes of cell shape and the different organization and expression of cytoskeleton-integrin networking could depend on several conditions: real or simulated microgravity environment, times of observation, and the different cellular models used (culture systems with specific supports, media, cell density etc).
In another cell model represented by TCam-2-an adherent seminoma cell line-after 24h-exposure to s-microgravity, we observed a modulation of cell shape, and cytoskeletal architecture associated with changes in cell metabolic status [45]. We hypothesized that also in adherent MC3T3-E1 cells there might be the same metabolic response. Indeed, after 24h-exposure to weightlessness, these cells showed reduced extracellular glucose uptake and increased lactate production, indicating an increase of anaerobic metabolic rate. This metabolic shift is accompanied by an increase in Ca 2+ and ROS levels. It is reported that increased ROS levels inhibit osteoblast functions suppressing osteoblast differentiation via phospholipase C-γ1/extracellular signal-regulated kinase 1/2/NF-KB signaling [46], and promote apoptosis in osteoblasts, that resulted protected by estrogenic treatment [47], therefore influencing the pathophysiology of bone loss [48]. It is well known that bone health status is the result of a balance among different bone cell phenotypes and their interaction with organic matrix and inorganic matter [1]. Consequently, the physiological dynamics of cell architecture and metabolic homeostasis are required to prevent bone alterations. In particular the former is important for cell compartmentalization, division as well as for cell-cell and cell-matrix interactions. The latter represents not only cell energy supply, but also the cell response to extracellular stimuli [35]. It has been noticed that microgravity increases the production of ROS and foster oxidative stress [22,45,49]. Conversely, some reports demonstrated the osteogenic benefits induced by the administration of several antioxidants acting against oxidative stress [50,51]. Based on this evidence, countermeasures have been adopted to restore physiological intracellular redox status [22]. In our experimental conditions, we used as antioxidant Trolox, a water-soluble analogue of vitamin E. The presence of Trolox in RPM-exposed cells counteracted the s-microgravity-induced increase of intracellular ROS levels, restoring both cell metabolic and proliferative rate, while promoting a normalization of their morphological main descriptors. Considering these results, we hypothesize that the alterations observed in RPM-exposed MC3T3-E1 osteoblasts may be the result of the increased ROS levels. ROS act as messengers in the transduction of certain cues (e.g., metabolic and environmental) that influence different signaling and functional pathways [52]. Calcium signaling can be regulated also by ROS production [53], and changes in Ca 2+ trafficking may affect, in turn, a number of cellular processes, including cell morphology, proliferation, and metabolism. From our results, we can speculate that the ROS increase, induced by s-microgravity, could affect the function (or even the expression) of membrane glucose transporters resulting in a decrease of glucose uptake. This, in turn, could alter cellular metabolic pathways leading to an increase of lactate production and a decrease of mitochondria functioning proven by the reduction of mitochondria membrane potential. Further experiments are need to elucidate this aspect and to detail the intracellular pathways triggered by the s-microgravity exposure. Our data open the way to identify those intracellular reactions that produce an increase of ROS levels in response to s-microgravity. In this scenario. some enzymes could be involved such as the NADPH oxidases present in different cell structures and compartments. Furthermore, considering the effect of s-microgravity on cell proliferation, also possible mechanisms related to this aspect should be explored such as cyclins' turnover, apoptosis, and autophagocytosis, all processes affected by the cell oxidative status. Other interesting aspects to be evaluated could be to monitor metabolic changes during osteogenesis in bone cells (also from human origin) during exposure to s-microgravity.
In our model, Trolox inhibited the increase in both ROS and Ca 2+ , ultimately "protecting" osteoblasts from the disruptive microgravity-related effects. Overall, these findings suggest that changes in ROS and Ca 2+ likely play a pivotal role in transducing weightlessness effects into cell physiology. Thereby, the use of appropriate antioxidant countermeasures could prevent the modifications and damage induced by microgravity on osteoblastic cells and so on bone homeostasis. Studies are warranted to deepen knowledge regarding the intracellular sources of increased ROS delivery in weightlessness to find pertinent protective strategies in support of astronaut's health.

Equipment and Cell Exposure Parameters
The s-microgravity was established using a desktop RPM connected to a control console through standard electrical cables (Dutch Space, Leiden, The Netherlands). The main characteristics of this apparatus were previously described [45,54,55] and the use parameters reported in the manufacturer's manual. Briefly, the RPM is a 3D clinostat whose running exerts continuous changes in orientation of the biological sample in order to minimize the gravitational force vector. The desktop RPM used in this study, works within a CO 2 incubator at 37 • C and 95% humidity.

Reagents and Materials
Unless indicated otherwise, reagents and materials were purchased as follows: cell culture medium, sera, and antibiotics from ThermoFisher Scientific (Monza, Italy), cell culture plastic ware from Becton Dickinson Falcon (Steroglass S.r.l., San Martino in Campo, Italy), and the chemicals and standards from Merck Life Science S.r.l. (Milan, Italy).

Cell Cultures
The murine MC3T3-E1 line (ATCC, CRL-2596TM, American type culture collection ATCC-LGC Standards, Teddington, UK), was cultured in growth medium (alpha minimal essential medium, a-MEM) supplemented with 10% foetal bovine serum, 200 mM L-glutamine, 100 IU/mL penicillin and 100 µg/mL streptomycin. After twenty-four hours from cell seeding (10 × 10 3 cells/cm 2 ), all experiments were performed on cells growing up to 96 h at 1 g or at s-microgravity on the RPM in the same incubator and culture conditions. MC3T3-E1 cells were plated as follows: in special-optic 96-well plates (Corning-Costar, Milan, Italy) for measurements of mitochondrial membrane potential and levels of intracellular Ca 2+ or ROS; in dishes (ø 3.5 cm) or 25 cm 2 flasks for trypan blue exclusion tests, Western blots analyses, measurements of glucose and lactate levels; on glass coverslips positioned in 3.5 cm-dishes for immunofluorescence staining. Where indicated, cells were grown at 1 g or on RPM in the presence of 100 µM Trolox. This agent was solubilized in dimethyl sulphoxide at concentration of 100 mM than diluted in cell medium at a final concentration of 100 µM. At the beginning of each experimental set, pilot experiments were performed using vehicle administration (1 µL dimethyl sulphoxide in 1 mL medium) to exclude its possible effect or toxicity on cells. All cell culture holders (microplates, flasks, etc.) were completely filled with culture medium to avoid air bubbles and to minimize liquid flow, and consequently buoyancy and shear stress during rotation.

Trypan Blue Exclusion Test
The trypan blue exclusion assay tested cell viability. A trypan blue dye solution (0.5% in phosphate-buffered saline, PBS) stained the cells that were counted using a Bürker chamber. The non-viable MC3T3-E1 cells appeared completely blue-stained.

Fluorescence Staining
The MC3T3-E1 cells were processed at room temperature as follows: fixed with a 4% paraformaldehyde solution in PBS (15 min), washed in PBS, permeabilized with a 0.1% TritonX-100 solution (10 min), stained with Alexa Fluor 546 Phalloidin (1:100 dilution, cod. A22283, ThermoFisher Scientific, Monza, Italy) (1 h); the nuclei were counterstained with DAPI (10 µM, cod. D1306, ThermoFisher Scientific) (10 min). For each sample, images were acquired in 5 different randomly selected fields from 3 independent experiments, using a Zeiss LSM800 URGB confocal system equipped with an upright Axio Observer Z1 microscope, a 40X N.A. 1.3 oil immersion objective and the ZEN Blue 2.1 software (Carl Zeiss, Jena, D). Z scan acquisitions were performed at resolution of 512 × 512 pixel acquiring the focal planes every 0.420 µm using as starting-point the glass adherent side of the cells, and as end-point the plane where the cells vanished from the acquisition field. The actin filament lengths were analyzed using ImageJ software (image processing program, National Institute of Health, Bethesda, MD, USA).

Cell Height
Cellular height was calculated off-line using the ZEN Blue 2.1 software (Carl Zeiss Spa) from orthogonal projections (XZ) obtained from the Z-stack images acquisition. Measurements of each cell in the acquired field derived tracing the distance between the region of cell adhesion to the glass slide and the highest point of the same cell in proximity of the nuclear region. The quantitative analysis was carried out on at least 5 randomly selected fields from each experimental condition performed in triplicate.

Nuclear Morphometric Analysis
Quantitative analyses (nuclear area and roundness) were performed using FIJI distribution of ImageJ (ver1.52p) [56]. Nuclear area was calculated as the total number of pixels presented in a single nucleus and expressed in square micrometers (1 pixel = 0.156 µm 2 ). Nuclear roundness was calculated using the following formula: 4 × area/(π × major axis 2 ), in which the major axis was one of the best fitted ellipse to a given object. A high value for roundness indicates a round nucleus, instead a low value indicates a rather elongated nucleus [57]. The quantitative analysis was carried out on at least 5 randomly selected fields from each experimental condition performed in triplicate.

Actin Filament Length
This parameter was calculated using FIJI distribution of ImageJ (ver1.52p), optical sections deriving from equatorial plane were firstly processed to obtain a simplified morphological model binarizing and skeletonizing using the "make binary" and "skeletonize" functions respectively. On the obtained images the function "Analyze Skeleton (2D/3D)" calculated the averaged actin filament length in a cell expressed as an arbitrary unit (a.u.). The quantitative analysis of the actin filament length was carried out on at least 5 randomly selected fields from each experimental condition performed in triplicate.

Measurements of Glucose and Lactate Levels
The measurements of glucose and lactate levels in the growth media were assayed according to the manufacturer instructions, using a Free Style Optium glucometer (Abbot Laboratories, Rome, Italy) and a LactatePro Analyzer (Arkray Inc. Kyoto, Japan), respectively.

Spectrofluorometric Measurements
The MC3T3-E1 cells, after 40-min incubation at 37 • C, in NES (Normal External Solution: 140 mM NaCl, 2.8 mM KCl, 2 mM CaCl 2 , 2 mM MgCl 2 , 10 mM glucose and 10 mM HEPES, pH 7.3) containing one of the probes (ThermoFisher Scientific) reported in Table 1, were rinsed and fluorescence intensity was detected using a microplate reader (Synergy H1 multimode, Biotek, Bad Friedrichshall, Germany) [45,54]. The fluorescence values (from Fluo-4-or H 2 -DCFDA-loaded cells) were expressed as the means (±SEM) of fluorescence values recorded during 5-min acquisition. Fluorescence values of JC1-loaded cells were expressed as the means (±SEM) of the red/green fluorescence ratio, which depends on the probe chemical status in relation to the mitochondrial membrane potential. For each sample, three independent experiments were performed, each containing eight repetitions.