The NATO project: nanoparticle-based countermeasures for microgravity-induced osteoporosis

Recent advances in nanotechnology applied to medicine and regenerative medicine have an enormous and unexploited potential for future space and terrestrial medical applications. The Nanoparticles and Osteoporosis (NATO) project aimed to develop innovative countermeasures for secondary osteoporosis affecting astronauts after prolonged periods in space microgravity. Calcium- and Strontium-containing hydroxyapatite nanoparticles (nCa-HAP and nSr-HAP, respectively) were previously developed and chemically characterized. This study constitutes the first investigation of the effect of the exogenous addition of nCa-HAP and nSr-HAP on bone remodeling in gravity (1 g), Random Positioning Machine (RPM) and onboard International Space Station (ISS) using human bone marrow mesenchymal stem cells (hBMMSCs). In 1 g conditions, nSr-HAP accelerated and improved the commitment of cells to differentiate towards osteoblasts, as shown by the augmented alkaline phosphatase (ALP) activity and the up-regulation of the expression of bone marker genes, supporting the increased extracellular bone matrix deposition and mineralization. The nSr-HAP treatment exerted a protective effect on the microgravity-induced reduction of ALP activity in RPM samples, and a promoting effect on the deposition of hydroxyapatite crystals in either ISS or 1 g samples. The results indicate the exogenous addition of nSr-HAP could be potentially used to deliver Sr to bone tissue and promote its regeneration, as component of bone substitute synthetic materials and additive for pharmaceutical preparation or food supplementary for systemic distribution.

In healthy adults there are two coordinated processes, predominantly dependent upon the activity of bone-resorbing osteoclasts and bone-forming osteoblasts, which are responsible for continuously remodelling bone tissue. The interrelated processes of bone formation and bone resorption are tightly regulated by both local and systemic signals and by external stimuli. Imbalance in the regulation of these processes may result in osteoporosis, a systemic skeletal condition that is characterized by low bone mass, reduced bone strength and loss of the bone microarchitecture/mineralization. These conditions affect the mechanical properties of bone tissue, leading to an increased risk of fracture due to the decreased density and increased bone fragility 1 .
Conventionally, osteoporosis is defined as primary osteoporosis when associated with sex hormone deficiency and ageing, and as secondary osteoporosis when associated with chronic diseases or disuse. It is known that sedentary lifestyles and immobilization can increase the risk of bone loss, leading to secondary osteoporosis 2 . Additionally, genetic factors, inadequate calcium intake, alcohol and tobacco abuse can increase the risk of osteoporosis 3,4 .
Current clinical therapies include (a) the use of calcium and vitamin D dietary supplements, combined with specific treatments with anti-resorptive or anti-catabolic agents that suppress the activity of osteoclasts (e.g. oestrogen, bisphosphonates, calcitonin, or selective oestrogen receptor modulators), (b) anabolic or bone-forming drugs that stimulate the osteoblast function (e.g. the parathyroid hormone) and (c) anabolic-anti-catabolic The replacement of Ca 2+ with Sr 2+ in the strontium-substituted hydroxyapatite nanopowder allowed the complete solubility of such alkaline ion in the HAP crystalline lattice, making it suitable as Sr 2+ ions vector for bone tissue regeneration 19 . The previously biocompatibility studies performed by adding exogenous nanoparticles suspensions to the culture of osteosarcoma cell line SAOS-2 showed the best performance in term of viability and proliferation using the nSr-HAP suspensions with the highest load of Sr 2+ .
The effect of nanoparticles in suspension on hBM-MSCs cultures in 1 g conditions. Cell viability, proliferation, apoptosis and morphological studies. In a previous study 19 , various suspensions of nanoparticles with different concentrations (6.25 μg/ml, 62.5 μg/ml, 625 μg/ml) and time incubations (1, 3 and 7 days) were biologically validated using the osteosarcoma cell line SAOS-2. The present study evaluated the effect of the addition of nanoparticles to the hBM-MSCs cultures on cell viability and proliferation. Furthermore, apoptosis, cell morphology and intracellular uptake of nanoparticles were assessed at 24 hr by CLSM observations. We assessed the dose-and time-dependence effects of exogenous addition of both types of nanoparticles on hBM-MSCs proliferation, by treating cells with 5 different concentrations of nCa-HAP and nSr-HAP for 1, 3 and 7 days in comparison with untreated cells (Fig. 1). The results showed all tested nCa-HAP concentrations did not significantly decrease cell viability in samples treated for 1 and 3 days compared to untreated samples (Fig. 1, panels a The presented data are the average ± standard deviations of three measurements in two separate experiments. Statistically significant values are indicated as ***p < 0.001. Panels (d-g) Apoptosis evaluation performed at 24 h by PSVue 480 staining on untreated cells (d), treated with 100 mM hydrogen peroxide (H 2 O 2 ) (e), 62.5 µg/ mL of nCa-HAP (f) or nSr-HAP (g) nanoparticles for 24 h. Panels, (h-j) Cell morphology was evaluated by staining β-tubulin (green fluorescence) and F-actin (red fluorescence) filaments of either untreated (h), nCa-HAP (i) or nSr-HAP (j) treated hBM-MSCs at 24 h. Panels, (k-l) Representative CLSM images and orthogonal projections of hBM-MSCs incubated for 24 h with FITC conjugated nCa-HAP (k) or nSr-HAP (l). Each scale bar represents 50 μm. and b). The substitution of Ca 2+ with Sr 2+ did not compromise cell viability, rather longer incubation times (up to 7 days) promoted an increase of cell proliferation (20-50%) in a concentration-dependent manner up to 62.5 μg/ mL (Fig. 1, panel c). In this context, the 62.5 μg/mL was the safest and most efficient concentration of nSr-HAP to be used for hBM-MSCs cultures, as it has been previously observed in the SAOS-2 cell line 19 .
Untreated cells (Fig. 1, panel d) and H 2 O 2 treated cells (positive CTRL, Fig. 1, panel e), incubated with a 62.5 μg/mL nCa-HAP ( Fig. 1, panel f) or nSr-HAP ( Fig. 1, panel g) were stained with PSVue 480 to assess apoptosis after a 24 h incubation, as indicated in the materials and methods section. No differences were observed with other nanoparticle concentrations (data not shown). As expected, apoptosis was observed in the sample treated with H 2 O 2 , showing an intense green fluorescence, whereas no staining was detected in the treated and untreated samples with nanoparticles.
Untreated and treated samples with 62.5 μg/mL nCa-HAP or nSr-HAP suspensions were observed by CLSM after proper staining, in order to evaluate cell morphology at 24 h. No significant differences in the cytoskeleton were observed between the untreated cells ( Fig. 1, panel h) and the cells treated with nCa-HAP ( Fig. 1, panel i) or nSr-HAP (Fig. 1, panel j) exhibiting the typical fibroblast-like morphology of hBM-MSCs. In addition, the FITC conjugated nanoparticles (FITC-nPS) uptake was evaluated at 24 h (Fig. 1, panels k,l) by CLSM analysis. Interestingly, both types of FITC-NPs were detected inside the cells with no significant differences between the nCa-HAP (Fig. 1, panel k) or nSr-HAP (Fig. 1, panel l) treatments. Further studies are needed to evaluate the uptake mechanism and its effect on cell differentiation.
The observations made by using an Olympus camera connected to an optical microscope of the hBM-MSCs at 24 h (Fig. S1, panels b and c) and 72 h (Fig. S1, panels e and f) did not reveal any differences in growth, demonstrating that the exogenous addition of nanoparticles, when compared to untreated cells (Fig. S1, panels a and d), did not hamper the proliferation or physiological adhesion processes. The results obtained at 72 h were also confirmed by TEM images (Fig. S1, panels g-i).
Differentiation studies. Since the in vitro stem cells differentiation into osteoblasts is completed in approximately 28 days in the presence of an osteogenic medium (OM), we also evaluated the effect of the addition of both types of nanoparticles to hBM-MSCs cultured for 28 days in proliferative medium (PM, without osteogenic factors) in 1 g conditions. The rationale for this experiment was to clarify the role of the nanoparticles as potential osteogenic factors for stem cell differentiation to osteoblasts. To this end, the specificity of Sr 2+ on the spatiotemporal evolution of differentiation towards osteoblasts was tested by evaluating ALP activity at 2 time points (8 and 28 days). Moreover, the quantitative bone gene marker expression (qRT-PCR), and the calcified bone matrix deposited (alizarine red staining, ELISA assay and CLSM), either in PM or OM conditions, were detected after 28 days of treatment.
In OM culture conditions, a significant increase in ALP activity in the samples treated with either 31.25 or 62.5 μg/mL nSr-HAP (p < 0.01) were observed in comparison with untreated samples, while no differences were found in cells treated with nCa-HAP (p > 0.05) (Fig. S2). At higher nSr-HAP concentrations, a significant decrease of ALP activity and a higher number of adipocytes were revealed. In particular, adipocytes were recognized after 28 days of treatment of hBM-MSCs with nSr-HAP at concentrations of 312.5 and 625 μg/mL: in fact, these treated cells presented large lipid droplets in the cytoplasm confirmed by specific staining with an Oil red O dye (manuscript in preparation). ALP activity was not modified by the addition of nCa-HAP and nSr-HAP (p > 0.05) in PM conditions (Fig. S2).
Since the treatment of hBM-MSCs with 62.5 μg/mL nanoparticles determined the best performance in terms of cell proliferation and ALP activity during differentiation, this concentration was used in our subsequent studies in OM culture conditions. Figure 2, panel a reports the ALP values of the untreated and nCa-Hap or nSr-HAP treated hBM-MSCs differentiated for 8 and 28 days in OM culture conditions. Data at 28 days confirmed the previous observations, whereas no significant differences were observed at day 8, indicating that the early differentiation process is probably unaffected by the addition of exogenous nanoparticles. ALP immunolocalization  Cell viability during the differentiation phase was monitored at 8 and 28 days using a 62.5 μg/mL concentration for both types of nanoparticles. We observed increased proliferation in cells treated with nSr-HAP in OM culture conditions when compared to the untreated or nCa-HAP treated samples (data not shown).
We further evaluated the effect of nCa-HAP or nSr-HAP on the levels of calcium deposited after 28 days of treatment in PM or OM culture conditions by Alizarin Red staining and its quantification after cetylpyridinium dissolution (by optical density reading at λ562), as shown in Fig. 3. In contrast to PM conditions (Fig. 3, panels a-c), the Alizarin red staining was more intense in the samples cultured in OM culture conditions (Fig. 3, panels d-f). Moreover, in the nCa-HAP and nSr-HAP treated hBM-MSCs, we observed a statistically higher number of calcium deposits in comparison with the untreated cells; this data was supported by an absorbance reading performed at 562 nm after cetylpyridinium dissolution (Fig. 3, panel g) (p < 0.001). These data suggest that nanoparticles, and in particular nSr-HAP, have a positive effect on bone matrix mineralization.
To get deeper insights into the ability of nCa-HAP or nSr-HAP to induce differentiation of hBM-MSCs into osteoblasts, we also tested the regulatory mechanisms underlying this process by analyzing the expression profile of marker genes in PM and OM conditions for 28 days. Increased expression levels of the COL1A1 gene, encoding the main protein occurring in connective tissues and bone matrix, was detected in PM conditions in the presence of both type of nanoparticles (p < 0.001). Furthermore, a positive correlation between gene expression and enzyme activity was revealed concerning the ALP gene (Fig. 4, panel a). Moreover, in the same conditions, the expression of the marker gene COL3A1, IBSP, OCN DCN, and RUNX2 did not show any significant difference in untreated and nCa-HAP or nSr-HAP treated samples (data not shown). On the other hand, in OM culture conditions, we detected an increase in gene expression of COL3A1, IBSP, and OCN in both treated samples although ALP, DCN and RUNX2 showed a significant fold increase only for samples treated with nSr-HAP (Fig. 4, panel b) (p < 0.001). All these genes codify proteins involved in the osteogenic differentiation process. Runx2 is a transcription factor required for osteoblast differentiation. It is interesting to note that Runx2-null mice are completely deprived of osteoblasts 27 . After Runx2 expression, osteoblast progenitors acquire bone-specific ALP properties, responsible for a high concentration of phosphate at the mineral deposition site. The late stage of osteoblast differentiation is characterized by high expression of proteins involved in bone matrix deposition, such as type-I and -III collagen (COL1A1 and COL3A1, respectively), decorin (DCN), osteocalcin (OCN), which is almost exclusively expressed in bone, and by the production of non-collagenous proteins such as bone sialo protein (IBSP) 20,28,29 .
Finally, deposits of specific bone matrix proteins were evaluated after 28 days in untreated and nCa-HAP or nSr-HAP treated hBM-MSCs cultured in PM and OM culture conditions by ELISA immunoassay (Fig. 5). Results indicated the expression levels of all tested proteins increased in OM culture conditions in nSr-HAP treated samples compared to the untreated samples (Fig. 5A). On the contrary, no differences were observed in PM culture conditions (Fig. 5A). Furthermore, Type-I collagen and osteocalcin were also analyzed by immunofluorescence The analyzed ECM proteins are organic components of bone matrix and impact on bone remodeling and formation. Type-I and type-III collagen are the major components of bone ECM and, through their organization in fibrils and fibers, they constitute the supporting structure of the bone matrix. OPN and FN are known to play important role in cell attachment and proliferation 30 , whereas ONT, OCN and DCN interact with mineral deposits inside the matrix 31 . The increased deposition of these proteins by the hBM-MSCs treated with nSr-HAP indicates the positive impact that these nanostructures have on the organic portion of the bone matrix as well as on the mineral part, as shown previously.
The results of our cell differentiation studies in 1 g conditions allowed us to set up the Simulated Microgravity and Space Flight experiments by adding nCa-HAP or nSr-HAP at 62.5 μg/mL in the osteogenic culture medium (OM). Furthermore, since the maximum time provided for the NATO payload experiment on the ISS to perform the cell differentiation studies in the absence and presence of each type of nanoparticles was 88 h, an identical period of incubation was used for the Simulated Microgravity (with RPM) experiments.

nca-and nSr-HAp as potential countermeasures for bone loss induced by microgravity. Pre-flight experiment in simulated microgravity with RPM. STROMA Hardware (HW) Experimental
Units (EU) were used to perform the NATO experiments onboard the ISS (Fig. 6, panel a) 32 . This type of hardware was developed for the STROMA experiment that investigated mice BMSCs and was launched in 2003 on the STS-107 Shuttle. The same type of HW was also used in 2006 (Soyuz 12 S), 2007 (Foton) and 2010 (Progress 40 P). In general, the EU are equipped with reservoirs to be filled with chemicals (culture medium, washing buffer, fixatives) and a culture chamber allowing for the growth of adherent cells on Thermanox. This system is capable of performing automatic 2D cell culturing in microgravity thanks to a microfluidic system (Fig. 6, panel d). Each STROMA EU used by the NATO experiment was accommodated into the KIC (in blue, Fig. 6, panels b and c) which contains programmable electronics, allowing for a predefined timeline of the experimental protocol. At the end of the experiment, the EU can be stowed in flight at controlled temperatures, down to −80 °C. After stowage and re-entry to Earth, the 2D cell cultures of the STROMA EU can be analyzed by microscopy techniques and by molecular biology-based approaches for genomic, transcriptomic and proteomic studies.
Preparations of the NATO Space Flight Experiments included an Experimental Sequence Test (EST) to assess the assembly development of the STROMA EUs, the loading of the reservoirs and the hBM-MSCs culture in simulated microgravity conditions using the Random Positioning Machine (RPM) 33 (Fig. 7, panel a).
Preliminary experiments were then carried out to determine the variables induced by both the microgravity conditions and by the STROMA EU culture system (Fig. S3). Once the exposure to simulated microgravity was over, Thermanox coverslips were removed from the culture chambers and the expression of bone gene markers was analyzed (Fig. S3). After 88 h of exposure to simulated microgravity, and as expected, the expression of osteoblastic gene markers was significantly reduced in the RPM samples, as compared to the GC samples (Fig. S3). Further experiments were performed on RPM samples in OM culture conditions with/without nCa-HAP and nSr-HAP to evaluate cell viability (data not shown) and ALP activity (Fig. 7, panel b). Cell viability was not reduced by the addition of both types of nanoparticles and the nSr-HAP treatment was able to counteract the effects of microgravity on ALP activity by greatly promoting to the same level of GC untreated samples (Fig. 7,  panel b).  (Fig. 8A). The cells were cultured in PM conditions and this yielded a 70% increase in cell number over a three-day period before the launch, reaching the required number of cells to be used for the ISS experiment. These cells, used for the experiment onboard the ISS, were inserted in six STROMA EUs (the EU numbers assigned to treatments were: #124 and #125, CTRL; #126 and #127, +nCa-HAP; # 128 and #129, +nSr-HAP) assembled and integrated into the KICs, and stored at 37 °C until their transfer to a BIOKIT ( Table 1). The BIOKIT is made of certified materials with good insulation and thermal properties. BIOKITs have predominantly been used to support flight HW transportation since their first use in 2003, for the STROMA mission loading on Dragon X (the orbital transport capsule for the material destined for the ISS). The space launch of the samples took place on April 14 th , 2015, in the context of the sixth SpaceX commercial space mission CRS-6. The transfer of samples to the ISS took place two days later, while the beginning of the in-flight experiment started on April 18 th 2015 with the loading of samples into the KUBIK incubator. When the KICs were inserted in the KUBIK the first medium change from reservoir #2 took place. Cells were cultured for 88 hours and then washed two times with PBS from reservoirs #3 and #4 and, finally, the RNA Cell Protector was inserted in the culture chamber to stabilize the RNA samples (Fig. 6, panel d). The pistons of the reservoirs were activated in series, allowing for medium changes. Cells fixed by RNA cell protector injection, were stored on board ISS initially at www.nature.com/scientificreports www.nature.com/scientificreports/ 4 °C. The KICs were then transferred at −95 °C into the MELFI until the return to Earth and handed over to our lab. Once back from the ISS, data relating to times and temperatures occurring during the experiment on board the ISS were recovered to allow for the reproduction of the entire experiment (Fig. S4). Temperature recordings  www.nature.com/scientificreports www.nature.com/scientificreports/ showed that the temperature never dropped below 26 °C during upload and that the incubation in the KUBIK was carried out at 37 °C as requested (Fig. S4, panel a). At the end of the incubation period, the temperature of the samples frozen in MELFI were less than −96 °C (Fig. S4, panel b).
Unfortunately, after ISS experiment, both EUs #124 and #126 evidenced pronounced levels of contamination. Nevertheless, the RNA cell protector was recovered from all of the culture chambers and RNAs and proteins were isolated. The quality of RNA was evaluated by Agilent BioAnalyzer 2100. The results, reported in Fig. S5, indicate that 4 out of 6 samples had a respectable level of RNA quality (with RNA integrity numbers between 7.5 and 8.6). Statistical significance values are indicated as *p < 0.05 vs CTRL, and # p < 0.05 vs nCa-HAP RPM. www.nature.com/scientificreports www.nature.com/scientificreports/ The RNA samples extracted from the two contaminated EUs were completely degraded. This contamination means that it is not possible to obtain statistically significant results from any RNA Sequence analysis and, as a result, has limited our ability to test the effects of nCa-HAP and nSr-HAP on hBM-MSCs cell differentiation in real microgravity conditions, onboard of the ISS. No study of RNA Seq were performed on GC.

NATO Payload Experiment
Interestingly, after the removal of the samples for the RNA Seq analysis, the Thermanox coverslips were immediately recovered and observed under the microscope (Fig. 8B). The images of the recovered Thermanox of both the ISS (Fig. 8B, panels a-c) and GC (Fig. 8B, panels d-f) samples and the results extrapolated by crystal size quantitative analysis (Fig. 8C, panels b) are also reported. The crystals of the ISS's untreated samples were smaller than those observed in GC conditions (p < 0.05) (Fig. 8C, panels b). In both culture conditions, ISS and GC, we observed an increment in crystal dimension for nSr-HAP treated samples when compared with nCa-HAP and untreated samples (p < 0.05) (Fig. 8C, panels b). It appears that nSr-HAP exerts a positive effect on extracellular matrix mineralization even in absence of gravity, as already demonstrated in 1 g condition (Fig. 3).
Collectively, these data, even if preliminary, shed a light on possibilities to mediate the physiological response of osteoblasts to nanoparticle treatments, allowing us to set up new on-ground experiments to unravel the molecular mechanisms underlying the osteoblast's activity.

conclusions and future perspectives
In our current work, stable suspensions of nSr-HAP were systematically synthesized and used to test their effects on hBM-MSCs cells during differentiation. These suspensions exhibited high levels of biocompatibility. This is confirmed by viability results and cell morphology evolution as well as a demonstrated capability to promote cell adhesion and proliferation. In vitro, these positive effects were enhanced by prolonged incubation periods and high Sr concentrations that induced cell apoptosis or death.
We observed an increased expression of bone marker genes during the differentiation phase, along with enhanced levels of mineralization and calcified matrix deposition in nSr-HAP-treated cells. This clearly indicates these nanoparticles have a role in driving a more rapid commitment of stem cells toward osteoblastic differentiation. In simulated microgravity conditions, the nSr-HAP treated cells were able to counteract microgravity-induced ALP reduction and, on ISS sample, stimulated the dimensions of crystals during hBM-MSCs differentiation. It was, unfortunately, not possible to test transcriptomic effects in real space condition due to sample contamination. Further research is now needed to understand the molecular mechanisms activated by nSr-HAP nanoparticles. The effect of both nCa-Hap and nSr-HAP suspensions on osteoclasts and osteocytes in vitro cultures is under investigation and work has been planned on osteoblast-osteoclast co-cultures. Moreover, taking into account the relevance of the Wnt/β-catenin pathways in skeletal biology and disease, additional studies have been planned in order to test the possible involvement of nCa-Hap, nSr-HAP and/or Sr 2+ in this signalling pathway, either in osteoblasts or in osteoclasts.
We may state, however, that the results achieved in 1 g and in simulated microgravity conditions clearly suggest that nSr-HAP could be used to delivery Sr 2+ to bone tissue and promote regeneration, as a component of a synthetic bone substitute material, via pharmaceutical preparations or as food supplements for systemic distribution.

nCa-HAP and nSr-HAP synthesis, suspension preparation and chemical-physical characteriza-
tion. The nCa-HAP and nSr-HAP were synthetized, suspended and characterized as previously reported 19 . cell culture. 1 g conditions. The Institutional Review Board of the University of Pavia (2011) approved this study. All methods were performed in accordance with the relevant guidelines and regulations. Informed consent was obtained from all participants and their legal guardians. The hBM-MSC samples were isolated and phenotypically analysed to assess their mesenchymal properties according to the International Society for Cellular Therapy, as previously described 34 . As reported in our previous studies 35,36 , hBM-MSCs were cultured at 37 °C in a humidified incubator with 5% CO 2 in a medium maintenance, low-glucose DMEM (Dulbecco's modified Eagle's medium) supplemented with 10% FBS, 1% glutamine, 50 μg/ml penicillin-streptomycin (P-S) and amphotericin B (Lonza Group Ltd.) (Proliferative medium, PM). hBM-MSCs were cultured in osteogenic medium (OM), α-MEM (Minimum Essential Medium) supplemented with 10% FBS, 50 μg/ml of P-S and the osteogenic mixture containing 100 nM dexamethasone, 5 mM β-glycerophosphate disodium and 50 mg/ml ascorbic acid (Sigma-Aldrich, S. Louis, MO, USA) to induce osteogenesis. The treatment lasted up to 28 days and the medium was changed every 3 days: for the samples cultured in PM or OM conditions the media was replaced with fresh culture media with/without nCa-HAP or nSr-HAP.
Random positioning machine (RPM). Microgravity conditions were simulated using the RPM (Dutch Space, Leiden, Netherlands) 37 . The RPM provides continuous random changes in orientation of the 1 g vector for an accommodated experiment. For the EST experiment, 150 × 10 3 hBM-MSCs were seeded on Thermanox supports (11 × 22 mm) and cultured in PM for 72 h before the samples were inserted into the STROMA EUs and then exposed to the RPM conditions. The STROMA EUs were supplemented with 12.5 mM HEPES (Sigma-Aldrich) to remove air bubbles and fixed on the RPM as close as possible to the centre of the platform, which was then rotated using the real random mode (random speed and random direction) of the machine. The RPM operated at 37 °C for 88 h, mirroring the duration of the ISS NATO space flight experiment. During the RPM experiment the samples cultured in OM, with and without nCa-HAP or Sr-HAP, were unexposed (Ground Control, GC) or exposed to RPM. The GC cultures, treated in parallel in identical equipment, were then placed on the RPM.
Immunofluorescence studies. Apoptosi. The hBM-MSCs were labelled using the PSVue480 ™ cell stain, according to the manufacturer's instructions (Molecular Targeting Technologies, Inc.), in order to determine the extent to which cell death resulted from the addition of the nanoparticle suspension from the culture medium.
PSVue480 ™ dye detects apoptosis by targeting the loss of phospholipid asymmetry in the plasma membrane, an early event in the apoptosis process irrespective of cell type, which results in the exposure of phosphatidylserine (PS) residues at the outer plasma membrane leaflet. The hBM-MSCs were seeded on glass coverslips (Thermo Scientific) with a density of 3 × 10 4 cells/cm 2 and incubated with H 2 O 2 (positive control; 100 mM), no addition (negative control) and with 62.5 μg/mL concentrations of nCa-HAP or nSr-HAP for 24 h. At the end of each culture, cells were stained with PSVue480 ™ solution. Samples were then counterstained with a Hoechst 33342 solution (2 μg/mL) to target the cellular nuclei and then observed under a fluorescence optical microscope (Nikon Eclipse 80i).
Morphological studies. The hBM-MSCs which were cultured in PM culture conditions for 24 h, either with or without the 62.5 μg/mL concentration of nCa-HAP or nSr-HAP, were fixed with a 4% (w/v) paraformaldehyde (PFA) solution in a 0.1 M phosphate buffer (pH 7.4) for 30 min at 4 °C and then washed with PBS three times. The hBM-MSCs were then made permeable with a 0.1% Triton X-100 solution for 1 h at room temperature (RT) and incubated with FITC conjugated antibody against β-tubulin (Invitrogen) for 90 min at RT. F-actin staining was performed using 10 μg/mL phalloidin, labelled with tetramethylrhodamine (TRITC) (Sigma) for 45 min. Lastly, nuclei were counterstained with Hoechst 33342 solution (2 μg/mL). Samples were examined using a Confocal Laser Scanning Microscope (CLSM) (Leica TCS SP2, Leica Instruments, Germany), acquiring images every 1.5 μm to a depth of 100 μm.
Bone extracellular matrix deposits. The hBM-MSCs cultured in OM culture conditions for 28 days, with or without the 62.5 μg/mL concentrations of nCa-HAP or nSr-HAP, were fixed with a 4% (w/v) paraformaldehyde solution in 0.1 M phosphate buffer (pH 7.4) for 30 min at 4 °C and washed with phosphate-buffered saline (PBS) three times. The cells were then blocked by incubating with PAT (PBS containing 1% [w/v] bovine serum albumin and 0.02% [v/v] Tween 20) for 2 h at room temperature and then washed. Cells were stained with anti-type-I collagen, anti-osteocalcin and anti-alkaline phosphatase (diluted to 1:500 in PAT) overnight at 4 °C. This was followed by an incubation with an Alexa-Fluor-488 goat anti-rabbit IgG (HþL; Invitrogen) at a dilution of 1:750 in PAT for 1 h at room temperature. At the end of the incubation period the samples were washed in PBS, counterstained with a Hoechst solution (2 μg/mL) to target the cellular nuclei and then washed. The images were taken by the Fluorescence microscope (Leica Microsystems, Bensheim, Germany) equipped with a digital image capture system at 40X magnification.
Alkaline phosphatase (ALP) activity. The ALP activity of the hBM-MSCs, cultured in PM or OM culture conditions with or without the nCa-HAP or nSr-HAP suspensions, was determined using a colorimetric end point assay as previously described 39 . The assay measures the conversion of the colourless substrate p-nitrophenol phosphate (PNPP) by the enzyme ALP to the yellow product p-nitrophenol, in this process the rate of the colour change corresponds to the amount of enzyme present in the solution. In short, an aliquot (1 mL) of 0.3 M PNPP (dissolved in glycine buffer, pH 10.5) was added to each scaffold at 37 °C. After incubation, the reaction was stopped by the addition of 100 μL 5 M NaOH. The standards of PNPP, in concentrations ranging from 0 to 50 μM, were freshly prepared from dilutions of a 500 μM stock solution and incubated for 10 min with 7U of ALP (Sigma-Aldrich), previously dissolved in 500 μL of ddH 2 O. The absorbance reading was performed at 405 nm with a microplate reader (BioRad Laboratories) using 100 μL of standard or sample placed into individual wells on a 96-well plate. Samples were run in triplicate and compared against a calibration curve of p-nitrophenol standards. The enzyme activity was expressed as micromoles of p-nitrophenol produced per minute per milligram of enzyme (for analysis in 1 g culture conditions) and was normalized in relation to cell number seeded in the plate for analysis in STROAM EU on RPM culture conditions x 88 h.
Alizarin red staining. After 28 days, the hBM-MSCs cultured in PM or OM culture conditions, both the untreated samples and those treated with a 62.5 μg/mL concentration of nCa-HAP or nSr-HAP, were rinsed with PBS, fixed for 30 min at 4 °C with 4% PFA and stained for 10 min with 40 mM of Alizarin Red S (pH 4.2, Sigma-Aldrich) to analyse the Ca deposits. Samples were then washed three times with water and once with PBS for 10 min and observed under the microscope. Alizarin Red S staining was released from the cell matrix by incubation in a 10% cetylpyridinium chloride (Sigma-Aldrich) solution in 10 mM of sodium phosphate (pH 7.0) for 15 min and the absorbance was measured at 562 nm.
Gene expression. After 28 days, we extracted RNA from hBM-MSCs that have been cultured in PM or OM culture conditions and either untreated or treated with a 62.5 μg/mL solution of nCa-HAP or nSr-HAP. This was performed with the RNeasy Plus Mini Kit (Qiagen) and retro-transcribed into cDNA with the iScript cDNA Synthesis Kit (BioRad Laboratories), as previously reported 40 . A quantitative reverse-transcription polymerase chain reaction (qRT-PCR) analysis was performed in a 48-well optical reaction plate using a MiniOpticon Real-Time PCR System (BioRad Laboratories). The oligonucleotide primers were designed with gene sequences published in GenBank, indicated in Table S2. Reactions were performed in 20 μL with 2 μL of cDNA, 10 μL Brilliant SYBER Green qPCR Master Mix (Stratagene, La Jolla, CA), 0.4 μL of each primer, and 7.2 μL H 2 O. The PCR conditions were as follows: 3 min at 95 °C, 40 cycles of 5 sec at 95 °C, and 23 sec at 60 °C. Gene expression was normalized to the 18 S housekeeping gene expression. Each sample was analysed in triplicate and correlated against a standard curve. The reaction mixture, without cDNA, was used as a negative control in each run.
The same analysis was performed on samples cultured in the STROMA EU and exposed to RPM for 88 h.
Extraction of ECM proteins and enzyme-linked immunosorbent assay. After  A summary of the NATO experiment conducted onboard the ISS, one of the nine scientific research and technology projects selected for this mission by ASI, is reported in Table 1. NATO was designed to evaluate the influence of hydroxyapatite nanoparticles enriched with or without strontium on osteoblasts differentiation in real space environment by using the RNA Seq techniques 41,42 .
The hBM-MSCs were cultured in 6 STROMA EUs and then inserted in 6 KICs, and housed inside the KUBIK incubator for 3 days onboard of the ISS. The NATO experiment was prepared during the first two weeks of April 2015 at the Space Kennedy Center laboratories in Cape Canaveral (Florida, USA) before being transferred on the SpaceX CRS-6 cargo ship to the ISS. Six EUs were fully automated and managed by an internal microcomputer for the experiment onboard of the ISS, the other 6 EUs for GC were in a manual setting.
The STROMA experimental units (EU) provided by Kayser Italia (Kayser Italia srl, http://www.kayser.it/) were used to cultivate cells in with and without nCa-HAP and nSr-HAP nanoparticles in order to study the effect of these nanoparticles on the levels of hBM-MSC differentiation to osteoblasts on the ISS: 6 STROMA EU were launched on ISS and 6 were cultured on Ground. 3 × 10 5 cells were seeded on 12 Thermanox coverslips (Nunc) and left to attach for 48 h. In addition, the medium was replaced with OM with and without nCa-HAP or nSr-HAP nanoparticles at the same concentration. Cell viability was examined with the Alamar Blu assay (Sigma-Aldrich), according to the manufacturer's instructions. After 8 days, but before the ISS experiments, a few samples were fixed for SEM observations, as previously described 39,40 . For the other samples, media were collected and kept at −80 °C and the Thermanox were built inside the culture chamber of the STROMA EU with a fresh medium with and without nanoparticles plus the addition of 12.5 mM HEPES (Sigma).
Filling the EU Reservoirs. Four of the five EU reservoirs were filled as follows: #2 with a fresh medium with or without nanoparticles (depending of the EU), #3 PBS (first washing), #4 PBS (second washing) and #5 RNA Cell Protector (Qiagen). 6 EUs inside 6 KICs (Kayser Italia srl, http://www.kayser.it/) were shipped inside a BIOKIT, ensuring a constant temperature of 37 °C during their transportation to the ISS through the space vector Falcon 9 of the SpaceX CRS6, launched from SLC-40 (Space Launch Complex 40) on April 14 th , 2015 from Kennedy Space Center -Cape Canaveral -Florida (USA). The payload reached the ISS on April 17 th , 2015 and was docked by Captain Samantha Cristoforetti who inserted the KICs inside the KUBIK, the incubator present on ISS. When the KICs were inserted in the KUBIK, the first medium change from reservoir #2 took place. Cells were cultivated for 88 hours and then washed two times with PBS from reservoirs #3 and #4 and, finally, the RNA Cell Protector was inserted into the culture chamber to stabilize RNA. The EUs were then transferred by Captain Samantha Cristoforetti from the KUBIC to the −95 °C MELFI. The 6 KICs of GC followed the same procedures, www.nature.com/scientificreports www.nature.com/scientificreports/ but manually performed. On May 21 th , 2015 the 6 ISS KICs frozen samples were carried to Earth by Dragon 6 vector, splashing down in the Pacific Ocean at 12.58 pm. The samples were then shipped to Italy.
As with the GC samples, the 6 manual EUs were assembled on the same day as the 6 EUs destined for the experiment on board of the ISS. The 6 GC EUs were kept at a temperature of 25 °C and sent to Italy. At this point they were transferred to an incubator at 26-28 °C until the beginning of the space flight experiment on board ISS, to simulate sample handling and temperature before ISS experiment. Eventually, the samples were activated and incubated at 37 °C for 88 h according to the flight experiment scheme, and at the end of incubation were stored at −80 °C until their opening with the flight samples.
ISS and GC samples analysis. The 12 KICs (6 ISS and 6 GC) were opened on June 9 th , 2015 and the RNA cell protector from the culture chambers, the Thermanox and culture media from washings were recovered. RNA (Fig. S4) and proteins ( Table S3, the subject of on-going studies) were extracted from RNA cell protector using RNeasy Mini Kit (Qiagen), according to the manufacturer's instructions, and stored at −80 °C. RNA was qualitatively analysed by Agilent BioAnalyzer 2100 in order to perform RNA Seq. For morphological analyses of deposited hydroxyapatite crystals, images of samples were acquired by a Nikon Eclipse Te200 equipped with 20x magnification and a Nikon Ds-Fi1 camera (sense: 5 MP CMOS) controlled by a Ds-U3camera controller and saved in TIFF format. Image analyses were performed under pre-calibrated ImageJ and the deposited hydroxyapatite crystals were manually segmented one by one in order to obtain crystal area.

Statistical analysis.
Quantitative results are expressed as the mean ± standard error of the mean (SEM).
One-way analysis of variance (ANOVA) with post hoc Bonferroni test was applied, with a significance level of 0.05 in order to compare results. For crystal images of deposited crystals, statistical analysis was performed by 1 Way ANOVA Holm-Sidak's multiple comparisons test (Graph Pad Software in., San Diego, USA) and p value < 0.05 was considered statistically different.

Data availability
The datasets generated during and/or analysed in the current study are included in this published article (and its Supplementary Information file).