Impairment of 7F2 osteoblast function by simulated partial gravity in a Random Positioning Machine

Justin Braveboy-Wagner; Peter I. Lelkes

doi:10.1038/s41526-022-00202-x

Impairment of 7F2 osteoblast function by simulated partial gravity in a Random Positioning Machine

Braveboy-Wagner, Justin; Lelkes, Peter I. 2022-06-07 00:00:00 www.nature.com/npjmgrav ARTICLE OPEN Impairment of 7F2 osteoblast function by simulated partial gravity in a Random Positioning Machine 1 1✉ Justin Braveboy-Wagner and Peter I. Lelkes The multifaceted adverse effects of reduced gravity pose a signiﬁcant challenge to human spaceﬂight. Previous studies have shown that bone formation by osteoblasts decreases under microgravity conditions, both real and simulated. However, the effects of partial gravity on osteoblasts’ function are less well understood. Utilizing the software-driven newer version of the Random SW Positioning Machine (RPM ), we simulated levels of partial gravity relevant to future manned space missions: Mars (0.38 G), Moon −3 (0.16 G), and microgravity (Micro, ~10 G). Short-term (6 days) culture yielded a dose-dependent reduction in proliferation and the enzymatic activity of alkaline phosphatase (ALP), while long-term studies (21 days) showed a distinct dose-dependent inhibition of mineralization. By contrast, expression levels of key osteogenic genes (Alkaline phosphatase, Runt-related Transcription Factor 2, Sparc/osteonectin) exhibited a threshold behavior: gene expression was signiﬁcantly inhibited when the cells were exposed to Mars-simulating partial gravity, and this was not reduced further when the cells were cultured under simulated Moon or microgravity conditions. Our data suggest that impairment of cell function with decreasing simulated gravity levels is graded and that the threshold proﬁle observed for reduced gene expression is distinct from the dose dependence observed for cell proliferation, ALP activity, and mineral deposition. Our study is of relevance, given the dearth of research into the effects of Lunar and Martian gravity for forthcoming space exploration. npj Microgravity (2022) 8:20 ; https://doi.org/10.1038/s41526-022-00202-x INTRODUCTION bioreactors and clinostats) that recapitulate certain aspects of microgravity. These experiments demonstrated that simulated Loss of structural skeletal mineral is a health complication facing −3 12,13 microgravity (Micro) conditions (equivalent to ~10 G ), any human presence in space or, to a yet unknown degree, on 12,14 suppress osteoblast differentiation and alter osteoclast func- other terrestrial bodies with reduced gravity, like the Moon tion and survival , similar to the effects observed in orbital (~0.16 G) or Mars (~0.38 G). The detrimental effects of microgravity microgravity, making these venues cost-effective substitutes for on the musculoskeletal system have been known, albeit not fully gravity modeling and experimentation. understood, since the onset of manned spaceﬂight. The effect was While alternatives like parabolic ﬂights exist for short-term conﬁrmed as astronauts and cosmonauts began to spend studies, the experimental gold standards for simulating micro- increasingly more time in space . In studies conducted on the gravity on Earth are rotating clinostats or microgravity- International Space Station (ISS), signiﬁcant trabecular volumetric 12,17 simulating bioreactors . There is ample evidence in the bone mineral density (vBMD) losses in the spine and hip were literature suggesting that the results of ground-based modeled noted with femoral vBMD showing an average loss of 2.7%/month 18–20 microgravity studies mimic real space conditions . In com- and losses at the trabecular hip of 2.3%/month . If not mitigated parative studies, similar degrees of inhibition of proliferation and by proper countermeasures, loss of bone mass poses an increased differentiation have been observed both in osteoblast-like cells risk of fracture in the workplace (space) and negatively impacts cultured in true-microgravity in space as well as in experiments quality of life on return to Earth. carried out in simulated microgravity using clinostats . The The primary cause of bone loss in microgravity (and presumably Random Positioning Machine (RPM), a 3D clinostat, randomizes also under the partial-gravity conditions found on Mars or Moon) orientation with respect to Earth’s gravitation ﬁeld so that a is believed to be the inhibition of osteoblast and osteocyte sample’s gravity vector averages out to close to zero, providing a activity, resulting in decreased bone mineralization , concomitant state of simulated microgravity . Using outer and inner frames, with elevated osteoclast resorption. The upset equilibrium results ﬁxed to two separate axes, the RPM can rotate independently in in a substantial loss of bone mass over time, as mentioned above. three dimensions and simulate partial gravities beyond micro- The effects of microgravity on osteoblasts have been previously gravity, such as Moon and Mars. This is an advantage the RPM has explored: in experiments carried out in orbital microgravity (at −4 −6 4 over the two-dimensional rotation of the rotating wall vessel ~10 –10 G) osteoblast proliferation was inhibited, osteogenic (RWV) or 2D clinostat. In vitro studies carried out in the RPM have differentiation was delayed, and the expression of genes 5–7 yielded results similar to those performed in actual (orbital) controlling bone differentiation was reduced , while the bone 8,9 microgravity in bone-marrow-derived mesenchymal stem cells resorption by osteoclasts increased aggressively . The result is an 23 24 overall loss of bone mineral density, wherein load-bearing bones (MSCs) and 2T3 preosteoblasts . An alternative approach to the 10 SW operation of an RPM (RPM ) is to control the range of clinostat are prone to atrophy , although one study reports a small motion through the use of speciﬁc path ﬁles that move the arms increase in skull bone mineral density . These effects have been replicated when osteoblasts and preosteoblasts were cultured on of the device through pre-determined paths. These paths are not earth in microgravity analogs (rotating wall vessel (RWV) random, rather they are set for every iteration of the experiment, Department of Bioengineering, College of Engineering, Temple University, Philadelphia, PA, USA. email: pilelkes@temple.edu Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA 1234567890():,; J. Braveboy-Wagner and P.I. Lelkes allowing for the simulation of distinct partial gravities: in our case the 3D printed mounting cage for loading and unloading ﬂasks simulating the reduced gravity levels encountered for Mars, Moon, and the perfusion system used to eliminate bubbles and 25–27 and Microgravity . change media. For the sake of scientiﬁc rigor and within the context of this paper, the term Simulated Partial Gravity (SPG) or Simulated Random Positioning Machine Microgravity (SMG) refers speciﬁcally to the respective reduction To simulate reduced gravity, all experiments were carried out in in the net gravitational vector as generated by the motion- the second generation of the software-driven Random Positioning averaged movement of the 3D clinostat or RPM. The use of the SW Machine (RPM 2.0) (originally DutchSpace Airbus, Leiden, terms “Mars” and “Moon” does not indicate that we actually Netherlands, now Yuri GmbH, Meckenbeuren, Germany). The recreated the respective extraterrestrial partial-gravity conditions. Mode of operation of this RPM differs from that of the earlier Rather, operating the RPM in the speciﬁc path ﬁles generates generation, which was developed for generating completely earth-based analog environments that simulate many of the random paths, thus creating a simulated microgravity condition, environmental conditions expected at 0.38 G (Mars) and 0.16 G 18–20 and used for years in research . The “mode of operation” of the (Moon). SW RPM for simulating micro and partial gravity (see Supplemen- In this study, we cultured murine preosteoblasts (7F2 cells) in tary Fig. 3) was described by Benavides Damm et al. and was −3 the RPM under conditions that simulate microgravity (10 G), and previously used to test the effects of modeled Mars gravity in the partial gravities on Moon (0.16 G), and Mars (0.38 G). 25,31 plants . To simulate partial gravity the RPM utilizes pre- Speciﬁcally, we mostly focused on the effects of altered simulated determined path ﬁles, developed, and validated by the manu- gravity conditions on several distinct stages of initial osteoblastic facturer (Dutch Airbus), in which the software directs the motion cell functions, such as proliferation and osteogenic differentia- of the RPM arms in a non-random fashion. These paths will have a tion . In addition, we also evaluated the effects of these degree of preference along the Earth gravity vector, and the result simulated partial-gravity conditions on later-stage matrix miner- is a net positive gravity that is greater than net-zero but <1 G alization. We hypothesized that the inhibition of these stage- normal. The motion of the RPM in random mode over time can be speciﬁc osteoblast functions would depend on the simulated visualized as a sphere while the motion over time of a path ﬁle can partial-gravity levels. In testing this hypothesis, we observed that be visualized as a prolate spheroid. In the center of this spheroid, a the inhibition of two of these osteoblast functions, proliferation, sample is not weightless, as with a net-zero sphere, but instead and maturation (ALP-enzymatic activity and mineral deposition) experiences partial gravity, where the larger the eccentricity of the was dose-dependent. By contrast, we found a distinct threshold spheroid the higher the level of gravity. Additional validation of proﬁle for the inhibition of osteogenic marker gene expression. the simulation of partial gravity via vector averaging was achieved through maintenance software of the RPM, which converts feedback from the frames into a real-time vector average MATERIALS AND METHODS (Supplementary Fig. 4). Materials Alpha-minimum essential medium (a-MEM) and Fetal Bovine Alkaline phosphatase (ALP) activity assay Serum (FBS) were purchased from Gibco Life Technologies Alkaline phosphatase (ALP) enzymatic activity was used as a (Carlsbad, CA, USA). L-ascorbic acid (AA), β–glycerophosphate marker for osteoblastic differentiation and quantitated spectro- (β-GP), para-Nitrophenylphosphate (pNPP), Alizarin Red, and Tri photometrically. Following a room-temperature PBS wash, cell Reagent® for processing tissues were purchased from Sigma- monolayers were scraped in 250 µl PBS and transferred into 1 ml Aldrich (St. Louis, MO, USA). Quant-iT™ PicoGreen™ dsDNA Assay microcentrifuge tubes. The cells were then lysed with 250 µl 0.2% Kit was purchased from Invitrogen Molecular Probes (Eugene, OR, Triton in PBS, to a ﬁnal concentration of 0.1% (v/v) Triton X-100 USA) via Thermo Fisher Scientiﬁc. TaqMan Fast Universal PCR (500 µl), followed by one freeze-thawing cycle (−80 °C/RT). After Master Mix (2X) and Taqman primers were purchased through thawing and centrifugation (2000 × g, 1 min), the supernatants Applied Biosystems (Foster City, CA, USA). RNeasy Protect Mini Kits were used to determine ALP activity according to the protocol of were purchased from Qiagen (Hilden, GER). Lin et al. with some modiﬁcations: the buffer used was 10 mM MgCl , 0.5 M AMP (2-Amino-2-Methyl-1-Propanol), supplemented Cell culture techniques with 9 mM of the ALP substrate, p-nitrophenylphosphate (pNPP). 7F2 murine preosteoblasts (American Type Culture Collection, The lysate was diluted 10×. Color development was read in situ in Manassas, VA, USA, CRL-12557) were cultured in α-MEM media an Inﬁnite 200 PRO multimode plate reader (Tecan Group Ltd., supplemented with 10 mM HEPES, 10% FBS, 1% streptomycin and Switzerland) every 2 min for 14 min at 405 nm. Readings were penicillin, and maintained in a humidiﬁed, 37 °C, 5% CO /air converted to concentration (nM) with a standard curve based on incubator (maintenance medium). For osteogenic induction, the 4-Nitrophenol. ALP results are presented as enzyme activity over cells were cultured in osteogenic media containing the above time (the rate of p-nitrophenol production from the complete alpha-MEM culture medium supplemented with 10 mM p-nitrophenylphosphate substrate) and normalized to cell number β-glycerophosphate and 10 µg/ml ascorbic acid (differentiation as calculated from DNA content, using PICO green (as below). The medium). The media was changed every 3 days. In addition to normalized results are expressed as the amount of substrate frequent equilibrated media changes, oxygenation was main- converted (ng) over time per number of cells (ng/min/10k cells). tained by using modiﬁed culture ﬂasks utilizing silicon caps in place of ventilated caps, and shear stress was minimized by PICO-green assay for cell proliferation having the ﬂask completely ﬁlled with culture medium and devoid of air bubbles, which are notoriously detrimental to maintaining PicoGreen dsDNA Quantitation Reagent (Invitrogen, Eugene OR, the slow-shear, simulated microgravity conditions , thus main- USA) was supplied as a 1-ml concentrated dye solution in taining the concept of near-solid body (“zero headspace”). Cells anhydrous dimethylsulfoxide (DMSO) and used following the were cultured as 2D monolayers in T-12.5 Falcon™ Tissue Culture manufacturer’s protocol, with precedent in osteoblast studies . Treated Flasks (Fisher Scientiﬁc, Waltham, MA, USA) retroﬁtted 100 µl of the 0.1% Triton monolayer lysate supernatant (see with Fischer-brand Silicone Recessed Septum Stoppers (internal above) was removed, diluted 400×, and added to a 96-well plate. diameter: 14.5–15.5 mm) for enhanced gas exchange. The 100 µl of the combined PicoGreen Reagent (1:200 PicoGreen accessories used are shown in Supplementary Fig. 1, including diluted in the TE buffer supplied with the kit) was added to each npj Microgravity (2022) 20 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA 1234567890():,; J. Braveboy-Wagner and P.I. Lelkes sample. After mixing and incubation for 5 min at room tempera- RESULTS ture, protected from light, ﬂuorescence was measured on an Inhibition of 7F2 cell proliferation in simulated micro and Inﬁnite 200 PRO multimode plate reader (Tecan Group Ltd., partial gravity −3 12 Switzerland) at 485 nm excitation, 535 nm emission. Standard Simulated microgravity, such as in the RPM (~10 ×G) , inhibits curves were constructed from known cell numbers (counted in cell proliferation in numerous cell types, including in bone- triplicate). marrow-derived adult stem cells (BMSCs) . The proliferation of i7F2 preosteoblasts was signiﬁcantly inhibited in a dose- dependent manner on days 4 and 6 for the simulated partial Alizarin Red and dissolved TECO assay for mineralization gravity (SPG) of Moon and Mars and simulated microgravity (Micro) (Fig. 1a). The time between days 2 and 4 represented a Mineralization of the cultures was assessed qualitatively by window in which the cells actively proliferated under all gravity Alizarin Red staining, essentially as previously described .In conditions; between days 4 and 6 the cells actively proliferated brief, following washing with PBS and ﬁxation with 10% neutral under Mars and Moon conditions but reached conﬂuence in Earth buffered formalin for 15 min, the cultures were stained using 0.5% gravity. Cell seeding was weighed between being too low, Alizarin Red S (pH 4.2) Digital images of the stained calciﬁed prompting cell senescence and inhibiting layer formation, and nodules were evaluated using ImageJ software (National Institutes being too high (rapid conﬂuence). During the 2–4 day window for of Health, NIH). Mineralization was also quantiﬁed using a 7F2s in these conditions, we observed statistically signiﬁcant commercially available, colorimetric calcium quantiﬁcation kit differences in the population doubling times (PDTs) between (Teco Diagnostics, Anaheim CA), following destructive decalciﬁca- Earth, Mars/Moon, and Micro (Fig. 1b). In Micro, the PDTs, speciﬁc tion of the cultures in 0.6 N HCl and analyzing the supernatant for that 2–4 day time period, increased three-fold over Earth according to the manufacturer’s instructions. Separate standard controls (2.95 ± 0.16 days (Micro) vs 0.96 ± 0.15 days (Earth)). The curves were established for each experiment. PDTs for Mars and Moon conditions were 1.4 ± 0.08 days and 1.5 ± 0.30 days, respectively (Fig. 1b). Compared to the speciﬁc PDT values of cells cultured in Earth gravity, exposure to Micro slowed RNA extraction and real-time PCR for osteogenic marker gene proliferation and resulted in a relative increase in the doubling expression (ALPL, RUN, ON) time by 307% (P < 0.001), by 156% in Moon (P < 0.001), and by The expression of select osteogenic marker genes was determined 146% for Mars (P < 0.001) (MAE). A semilogarithmic plot of PDTs by quantitative PCR (qPCR) essentially as previously described, against nominal gravity values on Earth, Mars, Moon, and 34 2 with some minor modiﬁcations . In brief, total RNA was extracted microgravity showed a very good ﬁt(R = 0.9919) (Fig. 1c). from 7F2 cells using a modiﬁed version of a hybrid Tri Reagent (Sigma-Aldrich, USA)/RNEasy® protocol. RNA was quantiﬁed using Alkaline phosphatase enzymatic activity is inhibited by partial a NanoDrop Spectrophotometer (Thermo Scientiﬁc, Waltham, MA), gravity and concentrations were brought to a uniform level using Alkaline phosphatase is a widely used marker for osteoblast additional RNase-free water. RNA was reverse transcribed to cDNA maturation and an important regulator of osteoblast mineraliza- 37–40 using the high-capacity cDNA reverse transcription kit (Applied tion . To quantify the impairment of osteogenic differentiation Biosystems, Foster City, CA) according to the manufacturer’s by SPG, we measured ALP activity in 7F2 preosteoblasts, cultured instructions. The cDNA was ampliﬁed in TaqMan Fast universal for up to 6 days in osteogenic media in Earth (control) and SPG PCR master mix with TaqMan assay primers and probes according conditions (Mars, Moon, Micro). ALP activity was normalized to cell to the manufacturer’s instruction. Genes of interest were: ALPL numbers as determined via the PICO-green assay. As seen in Fig. 2a, all simulated non-Earth gravities resulted in a time- and (Mm00475834_m1), RUNx2 (Mm00501584_m1) and Sparc/osteo- dose-dependent reduction in ALP activity in comparison to the nectin/BM40 (Mm00486332_m1). Quantitative PCR (qPCR) was Earth control. For example, on day 6, ALP activity was reduced by performed in a RealPlex Real-Time PCR System (Eppendorf, 27 ± 7% (p < 0.0001), 40 ± 5% (p < 0.0001) and 58 ± 9% (p < 0.0001) Enﬁeld, CT) with fast thermal cycling as described by Taqman for Mars, Moon and Micro, respectively (Fig. 2b) (Tukey post hoc). (Applied Biosystems). The level of expression of each gene was Plotting ALP activity on day 6, the time point of maximal signal, normalized to the level of expression of a common standard against altered gravity levels gives a linear trend (Fig. 2c) with a housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase correlation coefﬁcient of 0.967. (GAPDH) to determine the fold change in up/downregulation of the genes of interest using the comparative CT method 35 Simulated partial gravity downregulates the expression of (2-ΔΔCT) . osteogenic genes Next, we evaluated the effects of the three SPG conditions (Mars, Moon, Micro) on the early expression of select osteogenic marker Statistical analysis genes, Runt-related Transcription Factor 2 (RUN), Alkaline Phos- Throughout the text, unless otherwise speciﬁed, statistical phatase (ALPL), and Osteonectin (ON), using quantitative RT-PCR. differences between the samples were assessed by ANOVA and Expression levels of these osteogenic markers were measured post hoc analysis using Tukey’s HSD (honestly signiﬁcant every 2 days for 10 days. Qualitatively, the pattern of expression of difference). Mean absolute error (MAE) was used to determine all these genes followed a similar time- and dose/gravity- the difference between modeled projections. Results were plotted dependent proﬁle. For example, the transient expression of ALPL using Excel or JMP Pro. Data are presented as means ± standard peaked at day 6 under 1 G conditions (Fig. 3a). At its peak, ALPL deviation. P < 0.05 was considered signiﬁcant and noted as “*”, P < expression was downregulated under all levels of SPG, with no 0.01 and P < 0.001 were noted as “**” and “***”, respectively. signiﬁcant difference between Mars, Moon, or microgravity (Fig. 3b). By comparison to their expression levels on earth, all marker genes studied were suppressed by SPG. Speciﬁcally, on Reporting summary day 6, ALPL gene levels were reduced 3.86-fold in simulated Further information on research design is available in the Nature microgravity, 3.58-fold under Moon conditions, and 3.63-fold Research Reporting Summary linked to this article. under Mars conditions. Similarly, the expression of the RUN was Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2022) 20 J. Braveboy-Wagner and P.I. Lelkes Fig. 1 Inhibition of 7F2 cell proliferation in simulated partial gravity. a Increase in cell numbers on days 2 and 4 of culture in 1 G (Earth) and under the various altered simulated partial gravity conditions (Mars, Moon, Micro) (N = 3). b Speciﬁc partial population doubling times between days 2 and 4 and taking into account the mean absolute error (MAE). The average population doubling times were calculated as 0.96 ± 0.14 days for Earth (1 G), 1.4 ± 0.08 days for Mars, 1.5 ± 0.30 days for Moon, and 2.95 ± 0.17 days for simulated microgravity (Micro). c Semilogarithmic plot of the speciﬁc population doubling times vs. simulated partial gravity (R = 0.9919) for that 48-h window of time. Data are presented as means ± standard deviation. Asterisk (*) shows p < 0.05, (**) shows p < 0.01, (***) p < 0.001 as determined by Tukey’s post hoc analysis (panels (a) and (c)) or mean absolute error (MAE) (panel (b)). reduced 2.93-fold (simulated microgravity), 3.3-fold (Moon), 2.65- Moon and Micro (P < 0.001, Tukey post hoc), while Moon and fold (Mars), respectively, and ON by 3.43-fold (microgravity), 3.224- Micro were not signiﬁcantly different from each other. fold (Moon), 3.226-fold (Mars) (p < 0.0001) for all stated values) Percentage inhibition was calculated with 1 G (Earth) as the (Tukey post hoc). The expression levels of the individual genes at control. Five models were constructed to best ﬁt the data for days 2, 6, 8, and 14 can be found in Supplementary Fig. 2 in the various days of SPG (plus Earth), and also for an aggregate of all Appendix. days (R = 0.8972). SPG inhibited osteoblast mineralization along a linear gradient, with mineralization under Mars conditions being signiﬁcantly different from that under Moon or Micro conditions, Simulated partial-gravity conditions reduce long-term mineralization as illustrated in (Fig. 4c). The effects of simulated altered gravity over time on mineralization are shown in a three-dimensional Mineral nodule formation was initially assessed qualitatively by surface plot and heatmap (Fig. 4d). The surface plot was derived visual inspection following alizarin red staining (Fig. 4a). Quanti- from Equation (1), where (t) is time (days), (g) is gravity, and tative analysis of the differences in mineralization between the various simulated partial-gravity culture conditions (Mars, Moon, (mineral) is mineralization in µg/cm . Micro) was assessed using the TECO mineralization assay. Differences in calcium deposition between the various simulated ðmineralÞ¼ðÞ 3:87þ 0:34 ðÞ t þ 1:63 ðÞ g þðÞ ðÞ t 0:38525 gravities became ﬁrst noticeable by day 14 of RPM culture and ðÞ ðÞ ðÞ g 17:25 0:14 were studied through day 21 (Fig. 4b). Relative to the 1 G controls, mineralization was signiﬁcantly decreased by all SPG conditions. (1) Inhibition due to Mars gravity was signiﬁcantly different from npj Microgravity (2022) 20 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA J. Braveboy-Wagner and P.I. Lelkes Fig. 2 Simulated partial gravity inhibits osteogenic differentiation in 7F2 osteoblasts. a ALP activity as a result of exposure to different simulated partial-gravity conditions at different time points (N= 3). b ALP activity as a function of time between day 2 and day 6. c ALP activity as a function of altered simulated partial gravities at day 6. Data are presented as means ± standard deviation. Asterisk (*) shows p < 0.05, (**) shows p < 0.01, (***) p < 0.001 as determined by Tukey’s post hoc analysis. DISCUSSION paper, and also illustrated in the Appendix (Supplementary Figs. 3 SW and 4). To the best of our knowledge only the RPM (and similar The most signiﬁcant and novel ﬁnding in this work is the devices), or a centrifuge in orbit can provide long-term simulated uncovering of differential partial-gravity dependencies of the partial gravity of this quality and duration. parameters studied: dose dependence versus step-function. While The similarities between our results and those of others using most ground-based studies using the RWV or the RPM bioreactors clinostats as earth-based analogs for simulating microgravity and have focused on the effects of “simulated microgravity” (Micro, −3 25,31 partial-gravity conditions and results observed in space-ﬂown ~10 G), there are a few studies that compare the effects of sample in real microgravity, suggest that many of the reported Micro and non-terrestrial simulated partial gravities (Moon, Mars). effects might primarily be due to alterations in the modeled Given the preparations for upcoming manned spaceﬂights to reduced gravity conditions. However, for the sake of scientiﬁc Moon and Mars, there is a need for increased studies of partial- rigor, and as the mechanisms of gravisensing in SMG or SPG are as gravity research. Amongst the research priorities between 2018 yet unclear we would like to acknowledge the possibility that and 2024, recent NASA workshops and international committees some of the phenomena observed in clinostats and the RPM could have emphasized studies of “G-dose response in cell cultures also be (in part) due to indirect effects in the environment (e.g., using RPM” on Earth, as well as ISS-based counter-experiments . 44–47 changes in ﬂuid shear or mass transfer) . Moreover, while In the past, earth-based simulation of partial gravity has been challenging. Some of the previous approaches for generating differences in vibration, ﬂuid shear, or mass transfer may occur SW between different path ﬁles in the RPM , these have yet to be micro and/or partial gravity, such as drop towers (seconds of 25,31 quantiﬁed or identiﬁed as signiﬁcant . weightlessness), parabolic ﬂights (~10–20 s), sounding rockets The gravity-dependent, graded inhibition of cell proliferation (3–8 min in free-fall phase), and commercial sub-orbital rockets (minutes to hours) have signiﬁcant drawbacks and pitfalls, such as and ALP activity (Figs. 1 and 2) can be taken as an indirect short duration and ﬂuctuation of the gravitational load and validation of the ability of our system to simulate different magnitudes of partial gravity. Our identiﬁcation of graded, step, intrinsic experimental challenges . The system we used to and threshold inhibition due to partial gravitational unloading has simulate the extraterrestrial partial gravities of Moon and Mars in addition to simulating microgravity, the new software-driven some precedence: for example, using the RPM to simulate partial SW 25,31,43 SW RPM , has been described previously The RPM is the gravity, Kamal et al. observed a gradient in morphofunctional advanced version of the original “Random Positioning Machine”, types of nucleoli between Earth, Mars, and Micro after 24 h as well 19,20 31 as in Histone H4 acetylation . Manzano et al. reported that cell which has been widely used to simulate microgravity . However, instead of leveraging the random motion of the original proliferation of Arabidopsis thaliana plants at simulated Mars SW RPM, the RPM employs speciﬁc RPM path ﬁles, as described in gravity was essentially identical to that on Earth, while it was great detail in the Supplementary Information of Manzano et al., signiﬁcantly accelerated under Micro conditions, with Moon brieﬂy explained in the “Materials and methods” section of this inhibition being between Mars and Micro . The authors speculate Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2022) 20 J. Braveboy-Wagner and P.I. Lelkes that there is a likely threshold to gravity sensitivity intermediate of the expression of the three osteogenic marker genes studied between Moon and Mars. Swift et al. used a weight-bearing exhibited a threshold effect (Pattern III, Fig. 3): gene expression suspension device to investigate musculoskeletal losses following levels in cells exposed to Mars, Moon, and Micro were all similar to each other but signiﬁcantly different from gene expression in 1 G. full and partial hindlimb unloading, akin to modeled partial gravity ALP activity illustrates the contrast: inhibition of ALP-enzymatic on Moon, Mars, and in simulated microgravity . Close inspection activity was greater in Micro than on Mars, while expression of the of the data indicated different graded responses depending on ALPL gene was similar between Mars, Moon, and Micro (Fig. 3b). the anatomical location of the individual plantar-ﬂexor muscles. To the best of our knowledge, this study provides a ﬁrst For example, loss of vBMD (bone mineral density) of the proximal comparative analysis of the effects of non-terrestrial gravities tibia was similar for Moon and Mars, just as we found for our data (Mars, Moon) versus Earth and microgravity conditions on cultured on ALP activity and osteoblast proliferation (Figs. 1 and 2). While mammalian cells. Based on the predictive character of our different from our system in the details, the results of these studies mineralization model (Fig. 4e), we can make predictions about do show both gradients and thresholds in terms of gravity conditions based on other gravity wells, like Europa (0.134 G). dependence, just like our data. Our in vitro results are in line with previous space-based studies Our ﬁndings conﬁrm the results of previous studies indicating in orbital microgravity. For example, using the osteosarcoma MG- 12,17,33,36,49–51 the inhibitory effect of SMG on osteoblast function . 63 cell line, Carmeliet et al. examined the effects of “real” In expanding those studies, our data indicate a signiﬁcant microgravity in space on matrix formation and maturation, both at inhibitory effect of simulated partial gravity (SPG) on both the protein and mRNA levels. Cells were cultured for 9 days under proliferation, enzymatic activity, and gene expression in the orbital microgravity conditions aboard a Foton-10 satellite. Alka- short-term experiments (6 days), as well as on mineralization in line phosphatase activity in microgravity increased by only a factor the long-term studies (21 days). From our results we found three of 1.8 over the culture period, compared to the 3.8-fold increase in different patterns of inhibition: (Pattern I) in this pattern, inhibition the ground-based 1 g controls (p < 0.01). Similarly, gene expres- of proliferation between days 2 and 4 and enzymatic activity by sion for ALPL in microgravity was decreased, producing a fold Moon and Mars gravity levels were statistically indistinguishable change of 0.6 (p < 0.02) . These results are qualitatively similar to (Figs. 1b and 2a). A different gradient was observed in inhibition of our results in simulated microgravity: we observed a 2.5-fold mineralization (Pattern II) that displayed similarity between Moon reduction of ALP-enzymatic activity between Micro and Earth and and Micro (Fig. 4c). By contrast to the functional studies, inhibition a fold change of 0.8 in ALPL gene expression (Fig. 3). While ALP activity plays a key role in the mineralization and maintenance of bone, the exact mechanisms of action are unclear . Sugawara et al. demonstrated that the enzymatic activity of ALP is necessary for mineralization in MC3T3 cells, but does not require anchoring of ALP to the external surfaces of plasma membranes, as ALP is released by osteoblasts and steadily accumulates in the media . Elevated levels of ALP do not necessarily correlate with increased mineralization; for example, mechanical stimulation of osteoblasts via pulsatile ﬂow elicited an increase in ALP activity but did not result in a signiﬁcant difference in matrix mineralization . The decrease in the expression and activity of alkaline phosphatase and the inhibition of genes related to matrix proteins, like osteopontin and osteocalcin, which are characteristic of mature mineralizing osteoblasts, can indicate that osteogenic cells are sensitive to mechanical stimuli—in our case partial gravity—in both their mature and immature phases. While one study found no effects of SMG on the proliferation of 2T3 cells cultured in the RPM , most others have observed a signiﬁcant inhibition of cell proliferation. For example, Dai et al. reported that the proliferation of rat bone-marrow mesenchymal stem cells was inhibited by SMG, with cells arrested in the G0/G1 phase of the cell cycle . Similarly, skeletal unloading, a ground- based analog to SMG, has been shown to decrease the proliferation of osteoblast precursor cells . Our study suggests, for the ﬁrst time, a dose-dependent reduction of proliferation in osteoblasts by simulated partial gravity (Fig. 1). A plot of the calculated doubling times vs the distinct gravity levels suggests a logarithmic relationship (Fig. 1c). By contrast, the gravity Fig. 3 Effects of simulated partial gravities on osteogenic marker dependence of the inhibition of enzymatic ALP activity (Fig. 2) gene expression. a Time course of ALPL gene expression for or matrix mineralization (Fig. 4) appears to be linear. multiple simulated gravity conditions, following exposure of There is a consensus that ALPL mRNA levels are inhibited by conﬂuent 7F2 osteoblasts to osteogenic media. Relative expression was analyzed by real-time PCR and normalized to the levels of a microgravity over time, though there have been exceptions housekeeping gene (GAPDH), with 1 G conﬂuent cell culture (prior observed. Many studies have observed downregulation of ALPL in to osteogenic induction) as control. The relative expression of each both real and simulated microgravity versus Earth con- gene to control is presented as a fold-change expression for each 24,33,55,56 19 trols , for example in 2T3 cells after 3 days in the RPM . transcript. b Inhibition by simulated partial gravity of the three A smaller number of studies have reported ALPL upregulation in osteogenic marker genes is best discernable at the peak of their microgravity versus Earth gravity, for example in cultured expression at day-6 Alkaline phosphatase (ALPL), Runt-related 14,57,58 osteoblasts recovered after 10 days in space or in rats that Transcription Factor 2 (RUN), Sparc/osteonectin (ON). Data are were ﬂown in space for 6 days . Explaining this upregulation, presented as means ± standard deviation. Values are means ± SD of Kapitonova et al., speculated that this unexpected upregulation three independent cultures. Asterisk (*) shows p < 0.05, (**) shows p < 0.01, (***) p < 0.001 as determined by Tukey’s post hoc analysis. may be due to the change in the window of matrix maturation as npj Microgravity (2022) 20 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA J. Braveboy-Wagner and P.I. Lelkes Fig. 4 Effect of simulated partial gravity on long-term mineralization. a Alizarin red-stained mineralized nodules after 18 days on Earth (1 G) and in the RPM exposed to simulated partial -gravities of Moon and Mars. Images are representative micrographs of 1 × 1 cm areas from the bottom of T-12.5 Falcon™ Tissue Culture Treated Flasks. b Long-term mineralization under variable simulated gravity conditions, quantiﬁed as micrograms of calcium per square centimeter (μg/cm ). c Percentage inhibition of mineralization normalized versus Earth controls with modeled trendlines for various days: 14 days (R² = 0.8708), 16 days (R² = 0.92), 18 days (R² = 0.9735), 21 days (R² = 0.9308). d Three- dimensional surface plot and heatmap of the interplay between simulated gravity levels and mineralization over time. Data are presented as means ± standard deviation (N = 3). Asterisk (*) shows p < 0.05, (**) shows p < 0.01, (***) p < 0.001 as determined by Tukey’s post hoc analysis. Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2022) 20 J. Braveboy-Wagner and P.I. Lelkes microgravity inhibits osteoblasts. As noted by Landis et al. The threshold between Earth and Mars in terms of modulation of preosteoblasts cultured in space experienced a delayed progres- gene expression by SPG suggests a sensitivity of gene expression sion into a mature mineralizing state, as microgravity reduced the to the initial changes in gravitational load not shared by other expression of osteocalcin and type-I collagen . Finally, at least processes. one study that lasted one day only showed no difference in ALPL There is an intense interest in space biology regarding 16 42 expression . Our studies conﬁrm and build on the majority of thresholding, particularly for gravity sensing and for biological prior publications indicating that microgravity inhibited the impairment. Studies at simulated partial gravity can contribute to initiation of mineralization in osteoblasts and downregulated the determination of thresholds for various biological responses to genes related to osteoblast mineralization (Fig. 3). Another gravitational alterations. For example, the effects of partial gravity example for a similar outcome is MC3T3-E1 cells in Hu et al. . on critical physiological factors like the cell cycle are under- In addition to ALPL, our study also focused on Runx2 and studied . It is important to determine how exposure to distinct osteonectin. Runx2 (or RUN), a member of the runt homology magnitudes of gravity interacts with time (effects after 6 h, 24 h, domain transcription factor family, is a transcription factor weeks). Other papers, like Kamal et al. have, in plants, observed essential for osteoblast differentiation and mineralization , and threshold effects related to exposure time and, for example, for regulating osteocalcin expression . Decreased expression of ribosome biogenesis, where the effects are similarly inhibited by ALPL and RUN is consistently observed in most orbital experi- simulated Mars gravity and microgravity in comparison to Earth 17,24,60 31 ments and SMG cultures using calvaria, MSCs, and cell lines . gravity, suggesting a threshold between 1 g and 0.38 g , similar Peak RUN expression occurs at the end of the proliferative phase to our ﬁnding for the inhibition of gene expression in 7F2 and the beginning of mature mineralization and matrix deposi- preosteoblasts (Fig. 3b). tion. ALPL and RUN are often proﬁled in parallel. For example, While there are several hypotheses for gravity sensing Pardo et al., while ﬁnding no change in 2T3 cell proliferation, mechanisms, epigenetic triggers, and potential feedback loops reported that after 3 days of culture in the RPM under Micro in mammalian cells, that exacerbate inhibition over time , the conditions, ALPL gene expression was reduced ﬁve-fold while RUN dependence of these mechanisms across a range of simulated was reduced 1.88-fold when compared to 1 G Earth controls .Hu gravities has not yet been elucidated and warrants further et al. observed a signiﬁcant (~2-fold) reduction in the expression investigation in future spaceﬂights to Moon and Mars. In the of the genes for ALPL and RUN in 7-day differentiated MC3T3 cells meantime, a reasonable validation of these results, and other after 24 h in SMG . By comparison, the expression of the partial-gravity simulations on Earth, could also be done in orbital osteogenic genes in our experiments was downregulated (day 6, microgravity on the ISS, using a variable counterweight centri- Micro) as follows: ALPL was inhibited 3.85-fold and RUN 2.93-fold. fuge . In the past, centrifuges have been tested in space to In vitro conﬂuence and osteogenic differentiation resulted in simulate Earth gravity, though challenges persist in replicating the downregulation of proliferation and the transient expression methodologies and carrying out cell culture experiments that of ALPL and RunX2. Both these genes are associated with require intensive intervention or attention from astronauts. osteogenic differentiation, reaching a maximal expression on Taken together, exposure of 7F2 osteoblasts to various levels of −3 day 6, similar to the expression proﬁle in Choi et al., using simulated partial gravity (equivalent to ~0.6–10 G), resulted in 61 58 periodontal ligament cells , or Bikle et al. in hindlimb elevation , signiﬁcant, gravity-dependent inhibition of ALP activity in the or Guillot et al. in adult bone-marrow MSCs . The maximal short-term (6 days) and of mineralization in the long term expression followed by a decline may indicate the initiation of (21 days). Proliferation was also inhibited, with decreasing gravity differentiation and the transition of a majority of the cells from a signiﬁcantly lengthening population doubling times. Gradients for proliferating to a more mature matrix-mineralizing phenotype, inhibition exhibited slightly different patterns for the various making this time point (or transition point) of particular interest parameters studied: the pattern of inhibition of cell proliferation when investigating either the inhibition or delay of differentiation. and ALP activity was 1 G > Mars=Moon>Micro, while the pattern Stein et al. refer to this pattern as “stage-speciﬁc” or transient of inhibition of matrix mineralization was 1 G > Mars>Moon=Mi- expression . cro. Modulation of gene expression by reductions in simulated Osteonectin (ON), also known as secreted protein acidic and rich gravity levels exhibited a threshold-like behavior, with all partial- in cysteine (SPARC), is one of the most abundant non-collagenous gravity states and Micro resulting in similar levels of down- bone proteins . The effects of (simulated) microgravity on ON regulation. Describing, for the ﬁrst time, dose-dependent osteo- expression are controversial. While some studies show a signiﬁcant genic responses to reductions in simulated partial gravity, our reduction of ON expression in cells exposed to Micro conditions , comparative study is timely and relevant for forthcoming space just as we have demonstrated in this work (Fig. 3b), others have explorations, speciﬁcally for predicting biological effects of the seen no signiﬁcant reduction in expression due to micrograv- reduced gravity on Moon and Mars. 14,57 ity . By contrast, Kumei et al. reported a small increase in osteonectin under microgravity (space shuttle) conditions , while DATA AVAILABILITY Kapitonova et al. reported a non-signiﬁcant difference . Our The data of this study are available from the authors upon reasonable request. results (Fig. 3a) demonstrate a transient peak expression of osteogenic genes (e.g., ALPL, RANKL, BMP-4, Collagen-I, osteocal- cin), a proﬁle seen previously in other mineralizing cells, like Received: 14 May 2021; Accepted: 10 May 2022; periodontal cells differentiating into osteoblasts . Buravkova et al. have speculated that these mechanically sensitive markers of osteoblast differentiation are most vulnerable to changes in the gravitational ﬁeld at the peak of expression . Importantly, we did not observe statistically signiﬁcant differences REFERENCES in gene expression between the simulated partial gravities: Mars, 1. LeBlanc, A. D., Spector, E. R., Evans, H. J. & Sibonga, J. D. Skeletal responses to Moon, and microgravity. Thus, we presume that the inhibition of space ﬂight and the bed rest analog: a review. J. Musculoskelet. 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Microgravity signal ensnarls cell adhesion, cytoskeleton, and reprints matrix proteins of rat osteoblasts: osteopontin, CD44, osteonectin, and alpha- tubulin. Ann. N. Y Acad. Sci. 1090, 311–317 (2006). Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims 66. Takahashi, K. et al. Gravity sensing in plant and animal cells. NPJ Microgravity 7,2 in published maps and institutional afﬁliations. (2021). ACKNOWLEDGEMENTS Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, This study was supported in part by NASA grant 80NSSC18K1480 (P.I.L.) and a Temple adaptation, distribution and reproduction in any medium or format, as long as you give University Dissertation completion grant (J.B.W.). appropriate credit to the original author(s) and the source, provide a link to the Creative 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 AUTHOR CONTRIBUTIONS indicated otherwise in a credit line to the material. If material is not included in the All experiments were carried out by J.B.W. in partial fulﬁllment of a doctoral thesis. article’s Creative Commons license and your intended use is not permitted by statutory P.I.L. and J.B.W. shared in the experimental design, analysis of the data, and writing regulation or exceeds the permitted use, you will need to obtain permission directly the manuscript. from the copyright holder. To view a copy of this license, visit http://creativecommons. org/licenses/by/4.0/. COMPETING INTERESTS The authors declare no competing interests. © The Author(s) 2022 npj Microgravity (2022) 20 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/impairment-of-7f2-osteoblast-function-by-simulated-partial-gravity-in-uHMjhffjpf

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Publisher: Springer Journals
Copyright: Copyright © The Author(s) 2022
eISSN: 2373-8065
DOI: 10.1038/s41526-022-00202-x
Publisher site: See Article on Publisher Site

Abstract

www.nature.com/npjmgrav ARTICLE OPEN Impairment of 7F2 osteoblast function by simulated partial gravity in a Random Positioning Machine 1 1✉ Justin Braveboy-Wagner and Peter I. Lelkes The multifaceted adverse effects of reduced gravity pose a signiﬁcant challenge to human spaceﬂight. Previous studies have shown that bone formation by osteoblasts decreases under microgravity conditions, both real and simulated. However, the effects of partial gravity on osteoblasts’ function are less well understood. Utilizing the software-driven newer version of the Random SW Positioning Machine (RPM ), we simulated levels of partial gravity relevant to future manned space missions: Mars (0.38 G), Moon −3 (0.16 G), and microgravity (Micro, ~10 G). Short-term (6 days) culture yielded a dose-dependent reduction in proliferation and the enzymatic activity of alkaline phosphatase (ALP), while long-term studies (21 days) showed a distinct dose-dependent inhibition of mineralization. By contrast, expression levels of key osteogenic genes (Alkaline phosphatase, Runt-related Transcription Factor 2, Sparc/osteonectin) exhibited a threshold behavior: gene expression was signiﬁcantly inhibited when the cells were exposed to Mars-simulating partial gravity, and this was not reduced further when the cells were cultured under simulated Moon or microgravity conditions. Our data suggest that impairment of cell function with decreasing simulated gravity levels is graded and that the threshold proﬁle observed for reduced gene expression is distinct from the dose dependence observed for cell proliferation, ALP activity, and mineral deposition. Our study is of relevance, given the dearth of research into the effects of Lunar and Martian gravity for forthcoming space exploration. npj Microgravity (2022) 8:20 ; https://doi.org/10.1038/s41526-022-00202-x INTRODUCTION bioreactors and clinostats) that recapitulate certain aspects of microgravity. These experiments demonstrated that simulated Loss of structural skeletal mineral is a health complication facing −3 12,13 microgravity (Micro) conditions (equivalent to ~10 G ), any human presence in space or, to a yet unknown degree, on 12,14 suppress osteoblast differentiation and alter osteoclast func- other terrestrial bodies with reduced gravity, like the Moon tion and survival , similar to the effects observed in orbital (~0.16 G) or Mars (~0.38 G). The detrimental effects of microgravity microgravity, making these venues cost-effective substitutes for on the musculoskeletal system have been known, albeit not fully gravity modeling and experimentation. understood, since the onset of manned spaceﬂight. The effect was While alternatives like parabolic ﬂights exist for short-term conﬁrmed as astronauts and cosmonauts began to spend studies, the experimental gold standards for simulating micro- increasingly more time in space . In studies conducted on the gravity on Earth are rotating clinostats or microgravity- International Space Station (ISS), signiﬁcant trabecular volumetric 12,17 simulating bioreactors . There is ample evidence in the bone mineral density (vBMD) losses in the spine and hip were literature suggesting that the results of ground-based modeled noted with femoral vBMD showing an average loss of 2.7%/month 18–20 microgravity studies mimic real space conditions . In com- and losses at the trabecular hip of 2.3%/month . If not mitigated parative studies, similar degrees of inhibition of proliferation and by proper countermeasures, loss of bone mass poses an increased differentiation have been observed both in osteoblast-like cells risk of fracture in the workplace (space) and negatively impacts cultured in true-microgravity in space as well as in experiments quality of life on return to Earth. carried out in simulated microgravity using clinostats . The The primary cause of bone loss in microgravity (and presumably Random Positioning Machine (RPM), a 3D clinostat, randomizes also under the partial-gravity conditions found on Mars or Moon) orientation with respect to Earth’s gravitation ﬁeld so that a is believed to be the inhibition of osteoblast and osteocyte sample’s gravity vector averages out to close to zero, providing a activity, resulting in decreased bone mineralization , concomitant state of simulated microgravity . Using outer and inner frames, with elevated osteoclast resorption. The upset equilibrium results ﬁxed to two separate axes, the RPM can rotate independently in in a substantial loss of bone mass over time, as mentioned above. three dimensions and simulate partial gravities beyond micro- The effects of microgravity on osteoblasts have been previously gravity, such as Moon and Mars. This is an advantage the RPM has explored: in experiments carried out in orbital microgravity (at −4 −6 4 over the two-dimensional rotation of the rotating wall vessel ~10 –10 G) osteoblast proliferation was inhibited, osteogenic (RWV) or 2D clinostat. In vitro studies carried out in the RPM have differentiation was delayed, and the expression of genes 5–7 yielded results similar to those performed in actual (orbital) controlling bone differentiation was reduced , while the bone 8,9 microgravity in bone-marrow-derived mesenchymal stem cells resorption by osteoclasts increased aggressively . The result is an 23 24 overall loss of bone mineral density, wherein load-bearing bones (MSCs) and 2T3 preosteoblasts . An alternative approach to the 10 SW operation of an RPM (RPM ) is to control the range of clinostat are prone to atrophy , although one study reports a small motion through the use of speciﬁc path ﬁles that move the arms increase in skull bone mineral density . These effects have been replicated when osteoblasts and preosteoblasts were cultured on of the device through pre-determined paths. These paths are not earth in microgravity analogs (rotating wall vessel (RWV) random, rather they are set for every iteration of the experiment, Department of Bioengineering, College of Engineering, Temple University, Philadelphia, PA, USA. email: pilelkes@temple.edu Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA 1234567890():,; J. Braveboy-Wagner and P.I. Lelkes allowing for the simulation of distinct partial gravities: in our case the 3D printed mounting cage for loading and unloading ﬂasks simulating the reduced gravity levels encountered for Mars, Moon, and the perfusion system used to eliminate bubbles and 25–27 and Microgravity . change media. For the sake of scientiﬁc rigor and within the context of this paper, the term Simulated Partial Gravity (SPG) or Simulated Random Positioning Machine Microgravity (SMG) refers speciﬁcally to the respective reduction To simulate reduced gravity, all experiments were carried out in in the net gravitational vector as generated by the motion- the second generation of the software-driven Random Positioning averaged movement of the 3D clinostat or RPM. The use of the SW Machine (RPM 2.0) (originally DutchSpace Airbus, Leiden, terms “Mars” and “Moon” does not indicate that we actually Netherlands, now Yuri GmbH, Meckenbeuren, Germany). The recreated the respective extraterrestrial partial-gravity conditions. Mode of operation of this RPM differs from that of the earlier Rather, operating the RPM in the speciﬁc path ﬁles generates generation, which was developed for generating completely earth-based analog environments that simulate many of the random paths, thus creating a simulated microgravity condition, environmental conditions expected at 0.38 G (Mars) and 0.16 G 18–20 and used for years in research . The “mode of operation” of the (Moon). SW RPM for simulating micro and partial gravity (see Supplemen- In this study, we cultured murine preosteoblasts (7F2 cells) in tary Fig. 3) was described by Benavides Damm et al. and was −3 the RPM under conditions that simulate microgravity (10 G), and previously used to test the effects of modeled Mars gravity in the partial gravities on Moon (0.16 G), and Mars (0.38 G). 25,31 plants . To simulate partial gravity the RPM utilizes pre- Speciﬁcally, we mostly focused on the effects of altered simulated determined path ﬁles, developed, and validated by the manu- gravity conditions on several distinct stages of initial osteoblastic facturer (Dutch Airbus), in which the software directs the motion cell functions, such as proliferation and osteogenic differentia- of the RPM arms in a non-random fashion. These paths will have a tion . In addition, we also evaluated the effects of these degree of preference along the Earth gravity vector, and the result simulated partial-gravity conditions on later-stage matrix miner- is a net positive gravity that is greater than net-zero but <1 G alization. We hypothesized that the inhibition of these stage- normal. The motion of the RPM in random mode over time can be speciﬁc osteoblast functions would depend on the simulated visualized as a sphere while the motion over time of a path ﬁle can partial-gravity levels. In testing this hypothesis, we observed that be visualized as a prolate spheroid. In the center of this spheroid, a the inhibition of two of these osteoblast functions, proliferation, sample is not weightless, as with a net-zero sphere, but instead and maturation (ALP-enzymatic activity and mineral deposition) experiences partial gravity, where the larger the eccentricity of the was dose-dependent. By contrast, we found a distinct threshold spheroid the higher the level of gravity. Additional validation of proﬁle for the inhibition of osteogenic marker gene expression. the simulation of partial gravity via vector averaging was achieved through maintenance software of the RPM, which converts feedback from the frames into a real-time vector average MATERIALS AND METHODS (Supplementary Fig. 4). Materials Alpha-minimum essential medium (a-MEM) and Fetal Bovine Alkaline phosphatase (ALP) activity assay Serum (FBS) were purchased from Gibco Life Technologies Alkaline phosphatase (ALP) enzymatic activity was used as a (Carlsbad, CA, USA). L-ascorbic acid (AA), β–glycerophosphate marker for osteoblastic differentiation and quantitated spectro- (β-GP), para-Nitrophenylphosphate (pNPP), Alizarin Red, and Tri photometrically. Following a room-temperature PBS wash, cell Reagent® for processing tissues were purchased from Sigma- monolayers were scraped in 250 µl PBS and transferred into 1 ml Aldrich (St. Louis, MO, USA). Quant-iT™ PicoGreen™ dsDNA Assay microcentrifuge tubes. The cells were then lysed with 250 µl 0.2% Kit was purchased from Invitrogen Molecular Probes (Eugene, OR, Triton in PBS, to a ﬁnal concentration of 0.1% (v/v) Triton X-100 USA) via Thermo Fisher Scientiﬁc. TaqMan Fast Universal PCR (500 µl), followed by one freeze-thawing cycle (−80 °C/RT). After Master Mix (2X) and Taqman primers were purchased through thawing and centrifugation (2000 × g, 1 min), the supernatants Applied Biosystems (Foster City, CA, USA). RNeasy Protect Mini Kits were used to determine ALP activity according to the protocol of were purchased from Qiagen (Hilden, GER). Lin et al. with some modiﬁcations: the buffer used was 10 mM MgCl , 0.5 M AMP (2-Amino-2-Methyl-1-Propanol), supplemented Cell culture techniques with 9 mM of the ALP substrate, p-nitrophenylphosphate (pNPP). 7F2 murine preosteoblasts (American Type Culture Collection, The lysate was diluted 10×. Color development was read in situ in Manassas, VA, USA, CRL-12557) were cultured in α-MEM media an Inﬁnite 200 PRO multimode plate reader (Tecan Group Ltd., supplemented with 10 mM HEPES, 10% FBS, 1% streptomycin and Switzerland) every 2 min for 14 min at 405 nm. Readings were penicillin, and maintained in a humidiﬁed, 37 °C, 5% CO /air converted to concentration (nM) with a standard curve based on incubator (maintenance medium). For osteogenic induction, the 4-Nitrophenol. ALP results are presented as enzyme activity over cells were cultured in osteogenic media containing the above time (the rate of p-nitrophenol production from the complete alpha-MEM culture medium supplemented with 10 mM p-nitrophenylphosphate substrate) and normalized to cell number β-glycerophosphate and 10 µg/ml ascorbic acid (differentiation as calculated from DNA content, using PICO green (as below). The medium). The media was changed every 3 days. In addition to normalized results are expressed as the amount of substrate frequent equilibrated media changes, oxygenation was main- converted (ng) over time per number of cells (ng/min/10k cells). tained by using modiﬁed culture ﬂasks utilizing silicon caps in place of ventilated caps, and shear stress was minimized by PICO-green assay for cell proliferation having the ﬂask completely ﬁlled with culture medium and devoid of air bubbles, which are notoriously detrimental to maintaining PicoGreen dsDNA Quantitation Reagent (Invitrogen, Eugene OR, the slow-shear, simulated microgravity conditions , thus main- USA) was supplied as a 1-ml concentrated dye solution in taining the concept of near-solid body (“zero headspace”). Cells anhydrous dimethylsulfoxide (DMSO) and used following the were cultured as 2D monolayers in T-12.5 Falcon™ Tissue Culture manufacturer’s protocol, with precedent in osteoblast studies . Treated Flasks (Fisher Scientiﬁc, Waltham, MA, USA) retroﬁtted 100 µl of the 0.1% Triton monolayer lysate supernatant (see with Fischer-brand Silicone Recessed Septum Stoppers (internal above) was removed, diluted 400×, and added to a 96-well plate. diameter: 14.5–15.5 mm) for enhanced gas exchange. The 100 µl of the combined PicoGreen Reagent (1:200 PicoGreen accessories used are shown in Supplementary Fig. 1, including diluted in the TE buffer supplied with the kit) was added to each npj Microgravity (2022) 20 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA 1234567890():,; J. Braveboy-Wagner and P.I. Lelkes sample. After mixing and incubation for 5 min at room tempera- RESULTS ture, protected from light, ﬂuorescence was measured on an Inhibition of 7F2 cell proliferation in simulated micro and Inﬁnite 200 PRO multimode plate reader (Tecan Group Ltd., partial gravity −3 12 Switzerland) at 485 nm excitation, 535 nm emission. Standard Simulated microgravity, such as in the RPM (~10 ×G) , inhibits curves were constructed from known cell numbers (counted in cell proliferation in numerous cell types, including in bone- triplicate). marrow-derived adult stem cells (BMSCs) . The proliferation of i7F2 preosteoblasts was signiﬁcantly inhibited in a dose- dependent manner on days 4 and 6 for the simulated partial Alizarin Red and dissolved TECO assay for mineralization gravity (SPG) of Moon and Mars and simulated microgravity (Micro) (Fig. 1a). The time between days 2 and 4 represented a Mineralization of the cultures was assessed qualitatively by window in which the cells actively proliferated under all gravity Alizarin Red staining, essentially as previously described .In conditions; between days 4 and 6 the cells actively proliferated brief, following washing with PBS and ﬁxation with 10% neutral under Mars and Moon conditions but reached conﬂuence in Earth buffered formalin for 15 min, the cultures were stained using 0.5% gravity. Cell seeding was weighed between being too low, Alizarin Red S (pH 4.2) Digital images of the stained calciﬁed prompting cell senescence and inhibiting layer formation, and nodules were evaluated using ImageJ software (National Institutes being too high (rapid conﬂuence). During the 2–4 day window for of Health, NIH). Mineralization was also quantiﬁed using a 7F2s in these conditions, we observed statistically signiﬁcant commercially available, colorimetric calcium quantiﬁcation kit differences in the population doubling times (PDTs) between (Teco Diagnostics, Anaheim CA), following destructive decalciﬁca- Earth, Mars/Moon, and Micro (Fig. 1b). In Micro, the PDTs, speciﬁc tion of the cultures in 0.6 N HCl and analyzing the supernatant for that 2–4 day time period, increased three-fold over Earth according to the manufacturer’s instructions. Separate standard controls (2.95 ± 0.16 days (Micro) vs 0.96 ± 0.15 days (Earth)). The curves were established for each experiment. PDTs for Mars and Moon conditions were 1.4 ± 0.08 days and 1.5 ± 0.30 days, respectively (Fig. 1b). Compared to the speciﬁc PDT values of cells cultured in Earth gravity, exposure to Micro slowed RNA extraction and real-time PCR for osteogenic marker gene proliferation and resulted in a relative increase in the doubling expression (ALPL, RUN, ON) time by 307% (P < 0.001), by 156% in Moon (P < 0.001), and by The expression of select osteogenic marker genes was determined 146% for Mars (P < 0.001) (MAE). A semilogarithmic plot of PDTs by quantitative PCR (qPCR) essentially as previously described, against nominal gravity values on Earth, Mars, Moon, and 34 2 with some minor modiﬁcations . In brief, total RNA was extracted microgravity showed a very good ﬁt(R = 0.9919) (Fig. 1c). from 7F2 cells using a modiﬁed version of a hybrid Tri Reagent (Sigma-Aldrich, USA)/RNEasy® protocol. RNA was quantiﬁed using Alkaline phosphatase enzymatic activity is inhibited by partial a NanoDrop Spectrophotometer (Thermo Scientiﬁc, Waltham, MA), gravity and concentrations were brought to a uniform level using Alkaline phosphatase is a widely used marker for osteoblast additional RNase-free water. RNA was reverse transcribed to cDNA maturation and an important regulator of osteoblast mineraliza- 37–40 using the high-capacity cDNA reverse transcription kit (Applied tion . To quantify the impairment of osteogenic differentiation Biosystems, Foster City, CA) according to the manufacturer’s by SPG, we measured ALP activity in 7F2 preosteoblasts, cultured instructions. The cDNA was ampliﬁed in TaqMan Fast universal for up to 6 days in osteogenic media in Earth (control) and SPG PCR master mix with TaqMan assay primers and probes according conditions (Mars, Moon, Micro). ALP activity was normalized to cell to the manufacturer’s instruction. Genes of interest were: ALPL numbers as determined via the PICO-green assay. As seen in Fig. 2a, all simulated non-Earth gravities resulted in a time- and (Mm00475834_m1), RUNx2 (Mm00501584_m1) and Sparc/osteo- dose-dependent reduction in ALP activity in comparison to the nectin/BM40 (Mm00486332_m1). Quantitative PCR (qPCR) was Earth control. For example, on day 6, ALP activity was reduced by performed in a RealPlex Real-Time PCR System (Eppendorf, 27 ± 7% (p < 0.0001), 40 ± 5% (p < 0.0001) and 58 ± 9% (p < 0.0001) Enﬁeld, CT) with fast thermal cycling as described by Taqman for Mars, Moon and Micro, respectively (Fig. 2b) (Tukey post hoc). (Applied Biosystems). The level of expression of each gene was Plotting ALP activity on day 6, the time point of maximal signal, normalized to the level of expression of a common standard against altered gravity levels gives a linear trend (Fig. 2c) with a housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase correlation coefﬁcient of 0.967. (GAPDH) to determine the fold change in up/downregulation of the genes of interest using the comparative CT method 35 Simulated partial gravity downregulates the expression of (2-ΔΔCT) . osteogenic genes Next, we evaluated the effects of the three SPG conditions (Mars, Moon, Micro) on the early expression of select osteogenic marker Statistical analysis genes, Runt-related Transcription Factor 2 (RUN), Alkaline Phos- Throughout the text, unless otherwise speciﬁed, statistical phatase (ALPL), and Osteonectin (ON), using quantitative RT-PCR. differences between the samples were assessed by ANOVA and Expression levels of these osteogenic markers were measured post hoc analysis using Tukey’s HSD (honestly signiﬁcant every 2 days for 10 days. Qualitatively, the pattern of expression of difference). Mean absolute error (MAE) was used to determine all these genes followed a similar time- and dose/gravity- the difference between modeled projections. Results were plotted dependent proﬁle. For example, the transient expression of ALPL using Excel or JMP Pro. Data are presented as means ± standard peaked at day 6 under 1 G conditions (Fig. 3a). At its peak, ALPL deviation. P < 0.05 was considered signiﬁcant and noted as “*”, P < expression was downregulated under all levels of SPG, with no 0.01 and P < 0.001 were noted as “**” and “***”, respectively. signiﬁcant difference between Mars, Moon, or microgravity (Fig. 3b). By comparison to their expression levels on earth, all marker genes studied were suppressed by SPG. Speciﬁcally, on Reporting summary day 6, ALPL gene levels were reduced 3.86-fold in simulated Further information on research design is available in the Nature microgravity, 3.58-fold under Moon conditions, and 3.63-fold Research Reporting Summary linked to this article. under Mars conditions. Similarly, the expression of the RUN was Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2022) 20 J. Braveboy-Wagner and P.I. Lelkes Fig. 1 Inhibition of 7F2 cell proliferation in simulated partial gravity. a Increase in cell numbers on days 2 and 4 of culture in 1 G (Earth) and under the various altered simulated partial gravity conditions (Mars, Moon, Micro) (N = 3). b Speciﬁc partial population doubling times between days 2 and 4 and taking into account the mean absolute error (MAE). The average population doubling times were calculated as 0.96 ± 0.14 days for Earth (1 G), 1.4 ± 0.08 days for Mars, 1.5 ± 0.30 days for Moon, and 2.95 ± 0.17 days for simulated microgravity (Micro). c Semilogarithmic plot of the speciﬁc population doubling times vs. simulated partial gravity (R = 0.9919) for that 48-h window of time. Data are presented as means ± standard deviation. Asterisk (*) shows p < 0.05, (**) shows p < 0.01, (***) p < 0.001 as determined by Tukey’s post hoc analysis (panels (a) and (c)) or mean absolute error (MAE) (panel (b)). reduced 2.93-fold (simulated microgravity), 3.3-fold (Moon), 2.65- Moon and Micro (P < 0.001, Tukey post hoc), while Moon and fold (Mars), respectively, and ON by 3.43-fold (microgravity), 3.224- Micro were not signiﬁcantly different from each other. fold (Moon), 3.226-fold (Mars) (p < 0.0001) for all stated values) Percentage inhibition was calculated with 1 G (Earth) as the (Tukey post hoc). The expression levels of the individual genes at control. Five models were constructed to best ﬁt the data for days 2, 6, 8, and 14 can be found in Supplementary Fig. 2 in the various days of SPG (plus Earth), and also for an aggregate of all Appendix. days (R = 0.8972). SPG inhibited osteoblast mineralization along a linear gradient, with mineralization under Mars conditions being signiﬁcantly different from that under Moon or Micro conditions, Simulated partial-gravity conditions reduce long-term mineralization as illustrated in (Fig. 4c). The effects of simulated altered gravity over time on mineralization are shown in a three-dimensional Mineral nodule formation was initially assessed qualitatively by surface plot and heatmap (Fig. 4d). The surface plot was derived visual inspection following alizarin red staining (Fig. 4a). Quanti- from Equation (1), where (t) is time (days), (g) is gravity, and tative analysis of the differences in mineralization between the various simulated partial-gravity culture conditions (Mars, Moon, (mineral) is mineralization in µg/cm . Micro) was assessed using the TECO mineralization assay. Differences in calcium deposition between the various simulated ðmineralÞ¼ðÞ 3:87þ 0:34 ðÞ t þ 1:63 ðÞ g þðÞ ðÞ t 0:38525 gravities became ﬁrst noticeable by day 14 of RPM culture and ðÞ ðÞ ðÞ g 17:25 0:14 were studied through day 21 (Fig. 4b). Relative to the 1 G controls, mineralization was signiﬁcantly decreased by all SPG conditions. (1) Inhibition due to Mars gravity was signiﬁcantly different from npj Microgravity (2022) 20 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA J. Braveboy-Wagner and P.I. Lelkes Fig. 2 Simulated partial gravity inhibits osteogenic differentiation in 7F2 osteoblasts. a ALP activity as a result of exposure to different simulated partial-gravity conditions at different time points (N= 3). b ALP activity as a function of time between day 2 and day 6. c ALP activity as a function of altered simulated partial gravities at day 6. Data are presented as means ± standard deviation. Asterisk (*) shows p < 0.05, (**) shows p < 0.01, (***) p < 0.001 as determined by Tukey’s post hoc analysis. DISCUSSION paper, and also illustrated in the Appendix (Supplementary Figs. 3 SW and 4). To the best of our knowledge only the RPM (and similar The most signiﬁcant and novel ﬁnding in this work is the devices), or a centrifuge in orbit can provide long-term simulated uncovering of differential partial-gravity dependencies of the partial gravity of this quality and duration. parameters studied: dose dependence versus step-function. While The similarities between our results and those of others using most ground-based studies using the RWV or the RPM bioreactors clinostats as earth-based analogs for simulating microgravity and have focused on the effects of “simulated microgravity” (Micro, −3 25,31 partial-gravity conditions and results observed in space-ﬂown ~10 G), there are a few studies that compare the effects of sample in real microgravity, suggest that many of the reported Micro and non-terrestrial simulated partial gravities (Moon, Mars). effects might primarily be due to alterations in the modeled Given the preparations for upcoming manned spaceﬂights to reduced gravity conditions. However, for the sake of scientiﬁc Moon and Mars, there is a need for increased studies of partial- rigor, and as the mechanisms of gravisensing in SMG or SPG are as gravity research. Amongst the research priorities between 2018 yet unclear we would like to acknowledge the possibility that and 2024, recent NASA workshops and international committees some of the phenomena observed in clinostats and the RPM could have emphasized studies of “G-dose response in cell cultures also be (in part) due to indirect effects in the environment (e.g., using RPM” on Earth, as well as ISS-based counter-experiments . 44–47 changes in ﬂuid shear or mass transfer) . Moreover, while In the past, earth-based simulation of partial gravity has been challenging. Some of the previous approaches for generating differences in vibration, ﬂuid shear, or mass transfer may occur SW between different path ﬁles in the RPM , these have yet to be micro and/or partial gravity, such as drop towers (seconds of 25,31 quantiﬁed or identiﬁed as signiﬁcant . weightlessness), parabolic ﬂights (~10–20 s), sounding rockets The gravity-dependent, graded inhibition of cell proliferation (3–8 min in free-fall phase), and commercial sub-orbital rockets (minutes to hours) have signiﬁcant drawbacks and pitfalls, such as and ALP activity (Figs. 1 and 2) can be taken as an indirect short duration and ﬂuctuation of the gravitational load and validation of the ability of our system to simulate different magnitudes of partial gravity. Our identiﬁcation of graded, step, intrinsic experimental challenges . The system we used to and threshold inhibition due to partial gravitational unloading has simulate the extraterrestrial partial gravities of Moon and Mars in addition to simulating microgravity, the new software-driven some precedence: for example, using the RPM to simulate partial SW 25,31,43 SW RPM , has been described previously The RPM is the gravity, Kamal et al. observed a gradient in morphofunctional advanced version of the original “Random Positioning Machine”, types of nucleoli between Earth, Mars, and Micro after 24 h as well 19,20 31 as in Histone H4 acetylation . Manzano et al. reported that cell which has been widely used to simulate microgravity . However, instead of leveraging the random motion of the original proliferation of Arabidopsis thaliana plants at simulated Mars SW RPM, the RPM employs speciﬁc RPM path ﬁles, as described in gravity was essentially identical to that on Earth, while it was great detail in the Supplementary Information of Manzano et al., signiﬁcantly accelerated under Micro conditions, with Moon brieﬂy explained in the “Materials and methods” section of this inhibition being between Mars and Micro . The authors speculate Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2022) 20 J. Braveboy-Wagner and P.I. Lelkes that there is a likely threshold to gravity sensitivity intermediate of the expression of the three osteogenic marker genes studied between Moon and Mars. Swift et al. used a weight-bearing exhibited a threshold effect (Pattern III, Fig. 3): gene expression suspension device to investigate musculoskeletal losses following levels in cells exposed to Mars, Moon, and Micro were all similar to each other but signiﬁcantly different from gene expression in 1 G. full and partial hindlimb unloading, akin to modeled partial gravity ALP activity illustrates the contrast: inhibition of ALP-enzymatic on Moon, Mars, and in simulated microgravity . Close inspection activity was greater in Micro than on Mars, while expression of the of the data indicated different graded responses depending on ALPL gene was similar between Mars, Moon, and Micro (Fig. 3b). the anatomical location of the individual plantar-ﬂexor muscles. To the best of our knowledge, this study provides a ﬁrst For example, loss of vBMD (bone mineral density) of the proximal comparative analysis of the effects of non-terrestrial gravities tibia was similar for Moon and Mars, just as we found for our data (Mars, Moon) versus Earth and microgravity conditions on cultured on ALP activity and osteoblast proliferation (Figs. 1 and 2). While mammalian cells. Based on the predictive character of our different from our system in the details, the results of these studies mineralization model (Fig. 4e), we can make predictions about do show both gradients and thresholds in terms of gravity conditions based on other gravity wells, like Europa (0.134 G). dependence, just like our data. Our in vitro results are in line with previous space-based studies Our ﬁndings conﬁrm the results of previous studies indicating in orbital microgravity. For example, using the osteosarcoma MG- 12,17,33,36,49–51 the inhibitory effect of SMG on osteoblast function . 63 cell line, Carmeliet et al. examined the effects of “real” In expanding those studies, our data indicate a signiﬁcant microgravity in space on matrix formation and maturation, both at inhibitory effect of simulated partial gravity (SPG) on both the protein and mRNA levels. Cells were cultured for 9 days under proliferation, enzymatic activity, and gene expression in the orbital microgravity conditions aboard a Foton-10 satellite. Alka- short-term experiments (6 days), as well as on mineralization in line phosphatase activity in microgravity increased by only a factor the long-term studies (21 days). From our results we found three of 1.8 over the culture period, compared to the 3.8-fold increase in different patterns of inhibition: (Pattern I) in this pattern, inhibition the ground-based 1 g controls (p < 0.01). Similarly, gene expres- of proliferation between days 2 and 4 and enzymatic activity by sion for ALPL in microgravity was decreased, producing a fold Moon and Mars gravity levels were statistically indistinguishable change of 0.6 (p < 0.02) . These results are qualitatively similar to (Figs. 1b and 2a). A different gradient was observed in inhibition of our results in simulated microgravity: we observed a 2.5-fold mineralization (Pattern II) that displayed similarity between Moon reduction of ALP-enzymatic activity between Micro and Earth and and Micro (Fig. 4c). By contrast to the functional studies, inhibition a fold change of 0.8 in ALPL gene expression (Fig. 3). While ALP activity plays a key role in the mineralization and maintenance of bone, the exact mechanisms of action are unclear . Sugawara et al. demonstrated that the enzymatic activity of ALP is necessary for mineralization in MC3T3 cells, but does not require anchoring of ALP to the external surfaces of plasma membranes, as ALP is released by osteoblasts and steadily accumulates in the media . Elevated levels of ALP do not necessarily correlate with increased mineralization; for example, mechanical stimulation of osteoblasts via pulsatile ﬂow elicited an increase in ALP activity but did not result in a signiﬁcant difference in matrix mineralization . The decrease in the expression and activity of alkaline phosphatase and the inhibition of genes related to matrix proteins, like osteopontin and osteocalcin, which are characteristic of mature mineralizing osteoblasts, can indicate that osteogenic cells are sensitive to mechanical stimuli—in our case partial gravity—in both their mature and immature phases. While one study found no effects of SMG on the proliferation of 2T3 cells cultured in the RPM , most others have observed a signiﬁcant inhibition of cell proliferation. For example, Dai et al. reported that the proliferation of rat bone-marrow mesenchymal stem cells was inhibited by SMG, with cells arrested in the G0/G1 phase of the cell cycle . Similarly, skeletal unloading, a ground- based analog to SMG, has been shown to decrease the proliferation of osteoblast precursor cells . Our study suggests, for the ﬁrst time, a dose-dependent reduction of proliferation in osteoblasts by simulated partial gravity (Fig. 1). A plot of the calculated doubling times vs the distinct gravity levels suggests a logarithmic relationship (Fig. 1c). By contrast, the gravity Fig. 3 Effects of simulated partial gravities on osteogenic marker dependence of the inhibition of enzymatic ALP activity (Fig. 2) gene expression. a Time course of ALPL gene expression for or matrix mineralization (Fig. 4) appears to be linear. multiple simulated gravity conditions, following exposure of There is a consensus that ALPL mRNA levels are inhibited by conﬂuent 7F2 osteoblasts to osteogenic media. Relative expression was analyzed by real-time PCR and normalized to the levels of a microgravity over time, though there have been exceptions housekeeping gene (GAPDH), with 1 G conﬂuent cell culture (prior observed. Many studies have observed downregulation of ALPL in to osteogenic induction) as control. The relative expression of each both real and simulated microgravity versus Earth con- gene to control is presented as a fold-change expression for each 24,33,55,56 19 trols , for example in 2T3 cells after 3 days in the RPM . transcript. b Inhibition by simulated partial gravity of the three A smaller number of studies have reported ALPL upregulation in osteogenic marker genes is best discernable at the peak of their microgravity versus Earth gravity, for example in cultured expression at day-6 Alkaline phosphatase (ALPL), Runt-related 14,57,58 osteoblasts recovered after 10 days in space or in rats that Transcription Factor 2 (RUN), Sparc/osteonectin (ON). Data are were ﬂown in space for 6 days . Explaining this upregulation, presented as means ± standard deviation. Values are means ± SD of Kapitonova et al., speculated that this unexpected upregulation three independent cultures. Asterisk (*) shows p < 0.05, (**) shows p < 0.01, (***) p < 0.001 as determined by Tukey’s post hoc analysis. may be due to the change in the window of matrix maturation as npj Microgravity (2022) 20 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA J. Braveboy-Wagner and P.I. Lelkes Fig. 4 Effect of simulated partial gravity on long-term mineralization. a Alizarin red-stained mineralized nodules after 18 days on Earth (1 G) and in the RPM exposed to simulated partial -gravities of Moon and Mars. Images are representative micrographs of 1 × 1 cm areas from the bottom of T-12.5 Falcon™ Tissue Culture Treated Flasks. b Long-term mineralization under variable simulated gravity conditions, quantiﬁed as micrograms of calcium per square centimeter (μg/cm ). c Percentage inhibition of mineralization normalized versus Earth controls with modeled trendlines for various days: 14 days (R² = 0.8708), 16 days (R² = 0.92), 18 days (R² = 0.9735), 21 days (R² = 0.9308). d Three- dimensional surface plot and heatmap of the interplay between simulated gravity levels and mineralization over time. Data are presented as means ± standard deviation (N = 3). Asterisk (*) shows p < 0.05, (**) shows p < 0.01, (***) p < 0.001 as determined by Tukey’s post hoc analysis. Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2022) 20 J. Braveboy-Wagner and P.I. Lelkes microgravity inhibits osteoblasts. As noted by Landis et al. The threshold between Earth and Mars in terms of modulation of preosteoblasts cultured in space experienced a delayed progres- gene expression by SPG suggests a sensitivity of gene expression sion into a mature mineralizing state, as microgravity reduced the to the initial changes in gravitational load not shared by other expression of osteocalcin and type-I collagen . Finally, at least processes. one study that lasted one day only showed no difference in ALPL There is an intense interest in space biology regarding 16 42 expression . Our studies conﬁrm and build on the majority of thresholding, particularly for gravity sensing and for biological prior publications indicating that microgravity inhibited the impairment. Studies at simulated partial gravity can contribute to initiation of mineralization in osteoblasts and downregulated the determination of thresholds for various biological responses to genes related to osteoblast mineralization (Fig. 3). Another gravitational alterations. For example, the effects of partial gravity example for a similar outcome is MC3T3-E1 cells in Hu et al. . on critical physiological factors like the cell cycle are under- In addition to ALPL, our study also focused on Runx2 and studied . It is important to determine how exposure to distinct osteonectin. Runx2 (or RUN), a member of the runt homology magnitudes of gravity interacts with time (effects after 6 h, 24 h, domain transcription factor family, is a transcription factor weeks). Other papers, like Kamal et al. have, in plants, observed essential for osteoblast differentiation and mineralization , and threshold effects related to exposure time and, for example, for regulating osteocalcin expression . Decreased expression of ribosome biogenesis, where the effects are similarly inhibited by ALPL and RUN is consistently observed in most orbital experi- simulated Mars gravity and microgravity in comparison to Earth 17,24,60 31 ments and SMG cultures using calvaria, MSCs, and cell lines . gravity, suggesting a threshold between 1 g and 0.38 g , similar Peak RUN expression occurs at the end of the proliferative phase to our ﬁnding for the inhibition of gene expression in 7F2 and the beginning of mature mineralization and matrix deposi- preosteoblasts (Fig. 3b). tion. ALPL and RUN are often proﬁled in parallel. For example, While there are several hypotheses for gravity sensing Pardo et al., while ﬁnding no change in 2T3 cell proliferation, mechanisms, epigenetic triggers, and potential feedback loops reported that after 3 days of culture in the RPM under Micro in mammalian cells, that exacerbate inhibition over time , the conditions, ALPL gene expression was reduced ﬁve-fold while RUN dependence of these mechanisms across a range of simulated was reduced 1.88-fold when compared to 1 G Earth controls .Hu gravities has not yet been elucidated and warrants further et al. observed a signiﬁcant (~2-fold) reduction in the expression investigation in future spaceﬂights to Moon and Mars. In the of the genes for ALPL and RUN in 7-day differentiated MC3T3 cells meantime, a reasonable validation of these results, and other after 24 h in SMG . By comparison, the expression of the partial-gravity simulations on Earth, could also be done in orbital osteogenic genes in our experiments was downregulated (day 6, microgravity on the ISS, using a variable counterweight centri- Micro) as follows: ALPL was inhibited 3.85-fold and RUN 2.93-fold. fuge . In the past, centrifuges have been tested in space to In vitro conﬂuence and osteogenic differentiation resulted in simulate Earth gravity, though challenges persist in replicating the downregulation of proliferation and the transient expression methodologies and carrying out cell culture experiments that of ALPL and RunX2. Both these genes are associated with require intensive intervention or attention from astronauts. osteogenic differentiation, reaching a maximal expression on Taken together, exposure of 7F2 osteoblasts to various levels of −3 day 6, similar to the expression proﬁle in Choi et al., using simulated partial gravity (equivalent to ~0.6–10 G), resulted in 61 58 periodontal ligament cells , or Bikle et al. in hindlimb elevation , signiﬁcant, gravity-dependent inhibition of ALP activity in the or Guillot et al. in adult bone-marrow MSCs . The maximal short-term (6 days) and of mineralization in the long term expression followed by a decline may indicate the initiation of (21 days). Proliferation was also inhibited, with decreasing gravity differentiation and the transition of a majority of the cells from a signiﬁcantly lengthening population doubling times. Gradients for proliferating to a more mature matrix-mineralizing phenotype, inhibition exhibited slightly different patterns for the various making this time point (or transition point) of particular interest parameters studied: the pattern of inhibition of cell proliferation when investigating either the inhibition or delay of differentiation. and ALP activity was 1 G > Mars=Moon>Micro, while the pattern Stein et al. refer to this pattern as “stage-speciﬁc” or transient of inhibition of matrix mineralization was 1 G > Mars>Moon=Mi- expression . cro. Modulation of gene expression by reductions in simulated Osteonectin (ON), also known as secreted protein acidic and rich gravity levels exhibited a threshold-like behavior, with all partial- in cysteine (SPARC), is one of the most abundant non-collagenous gravity states and Micro resulting in similar levels of down- bone proteins . The effects of (simulated) microgravity on ON regulation. Describing, for the ﬁrst time, dose-dependent osteo- expression are controversial. While some studies show a signiﬁcant genic responses to reductions in simulated partial gravity, our reduction of ON expression in cells exposed to Micro conditions , comparative study is timely and relevant for forthcoming space just as we have demonstrated in this work (Fig. 3b), others have explorations, speciﬁcally for predicting biological effects of the seen no signiﬁcant reduction in expression due to micrograv- reduced gravity on Moon and Mars. 14,57 ity . By contrast, Kumei et al. reported a small increase in osteonectin under microgravity (space shuttle) conditions , while DATA AVAILABILITY Kapitonova et al. reported a non-signiﬁcant difference . Our The data of this study are available from the authors upon reasonable request. results (Fig. 3a) demonstrate a transient peak expression of osteogenic genes (e.g., ALPL, RANKL, BMP-4, Collagen-I, osteocal- cin), a proﬁle seen previously in other mineralizing cells, like Received: 14 May 2021; Accepted: 10 May 2022; periodontal cells differentiating into osteoblasts . Buravkova et al. have speculated that these mechanically sensitive markers of osteoblast differentiation are most vulnerable to changes in the gravitational ﬁeld at the peak of expression . Importantly, we did not observe statistically signiﬁcant differences REFERENCES in gene expression between the simulated partial gravities: Mars, 1. LeBlanc, A. D., Spector, E. R., Evans, H. J. & Sibonga, J. D. Skeletal responses to Moon, and microgravity. Thus, we presume that the inhibition of space ﬂight and the bed rest analog: a review. J. Musculoskelet. 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Braveboy-Wagner and P.I. Lelkes 62. Guillot, P. V. et al. Comparative osteogenic transcription proﬁling of various fetal ADDITIONAL INFORMATION and adult mesenchymal stem cell sources. Differentiation 76, 946–957 (2008). Supplementary information The online version contains supplementary material 63. Rosset, E. M. & Bradshaw, A. D. SPARC/osteonectin in mineralized tissue. Matrix available at https://doi.org/10.1038/s41526-022-00202-x. Biol. 52-54,78–87 (2016). 64. Zayzafoon, M., Gathings, W. E. & McDonald, J. M. Modeled microgravity inhibits Correspondence and requests for materials should be addressed to Peter I. Lelkes. osteogenic differentiation of human mesenchymal stem cells and increases adipogenesis. Endocrinology 145, 2421–2432 (2004). Reprints and permission information is available at http://www.nature.com/ 65. Kumei, Y. et al. Microgravity signal ensnarls cell adhesion, cytoskeleton, and reprints matrix proteins of rat osteoblasts: osteopontin, CD44, osteonectin, and alpha- tubulin. Ann. N. Y Acad. Sci. 1090, 311–317 (2006). Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims 66. Takahashi, K. et al. Gravity sensing in plant and animal cells. NPJ Microgravity 7,2 in published maps and institutional afﬁliations. (2021). ACKNOWLEDGEMENTS Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, This study was supported in part by NASA grant 80NSSC18K1480 (P.I.L.) and a Temple adaptation, distribution and reproduction in any medium or format, as long as you give University Dissertation completion grant (J.B.W.). appropriate credit to the original author(s) and the source, provide a link to the Creative 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 AUTHOR CONTRIBUTIONS indicated otherwise in a credit line to the material. If material is not included in the All experiments were carried out by J.B.W. in partial fulﬁllment of a doctoral thesis. article’s Creative Commons license and your intended use is not permitted by statutory P.I.L. and J.B.W. shared in the experimental design, analysis of the data, and writing regulation or exceeds the permitted use, you will need to obtain permission directly the manuscript. from the copyright holder. To view a copy of this license, visit http://creativecommons. org/licenses/by/4.0/. COMPETING INTERESTS The authors declare no competing interests. © The Author(s) 2022 npj Microgravity (2022) 20 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA

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Impairment of 7F2 osteoblast function by simulated partial gravity in a Random Positioning Machine

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Impairment of 7F2 osteoblast function by simulated partial gravity in a Random Positioning Machine

Impairment of 7F2 osteoblast function by simulated partial gravity in a Random Positioning Machine

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