Animal experiments

All animal experiments were performed in accordance with protocols approved by the Animal Care and Use Committee of Showa Medical University (approval numbers: 224056, 224066). C57BL/6J male mice were purchased from Japan SLC (Shizuoka, Japan) and maintained under a specific pathogen-free (SPF) condition at the animal facility of Showa Medical University. They were housed under a 12-hour light/dark cycle with controlled temperature and humidity, and provided access to food and water ad libitum. For tissue collection, mice were euthanized by cervical dislocation performed by trained personnel, in accordance with institutional guidelines. Because all subsequent experiments were conducted in vitro using isolated cells, no anesthesia or analgesia was administered. To alleviate animal suffering, mice were handled gently, monitored daily, and euthanized promptly when tissues were required, minimizing the duration of handling and stress.

Isolation of osteoclast progenitor cells

Bone marrow cells were isolated from femurs and tibias of 5- to 8-week-old male C57BL/6J mice on Day 0, and cultured in α-minimum essential medium (α-MEM; Fujifilm Wako, Osaka, Japan) supplemented with 20 ng/mL M-CSF (R&D Systems, Minneapolis, USA) at 37°C in a 5% CO₂ atmosphere. After 3 days of culture, non-adherent cells were removed, and adherent bone marrow-derived macrophages (BMMs) were used as osteoclast progenitors in the assays described below.

Medium and reagents

The base medium was α-MEM or Leibovitz’s L-15 (Fujifilm Wako, Osaka, Japan), while supplements included 10% fetal bovine serum (FBS; Gibco, Grand Island, USA) and penicillin-streptomycin mixed solution (Nacalai Tesque, Kyoto, Japan). Differentiation factors used were M-CSF at 20 ng/mL and RANKL at 100 ng/mL (R&D Systems, Minneapolis, USA). Vitamin C (ascorbic acid; Fujifilm Wako, Osaka, Japan) at 50 mg/L was added only in pit assays performed under atmospheric CO₂ conditions (0.04%), because Leibovitz’s L-15 medium, which is suitable for CO₂-independent culture, does not contain Vitamin C, unlike α-MEM. Medium was replaced every 3–4 days (specific schedule indicated in each experimental section).

Hypergravity devices and calibration

Hypergravity cultures were performed using Zeromo CL-5100 (Kitagawa, Hiroshima, Japan) and LIX-140SP (Tomy Seiko, Tokyo, Japan) devices. CL-5100 is able to generate different gravity levels depending on the culture vessel, with 209 rpm producing 3G for 24-well plates (Falcon, Corning, USA) and 5G for 25-mL flasks (Falcon, Corning, USA). The LIX-140SP generates 30G at 390 rpm. Hypergravity exposure was performed at 37°C, with CO₂ concentrations specified for each experiment. Because the LIX-140SP does not allow CO₂ control during centrifugation, experiments conducted using this device were performed under atmospheric CO₂ conditions (0.04%) with CO₂-independent Leibovitz’s L-15 medium, whereas experiments using the CL-5100 were conducted in a standard 5% CO₂ environment. Importantly, within each experimental setup, both the 1G control and corresponding hypergravity groups were cultured under identical CO₂ conditions, and gravity-dependent comparisons were therefore not confounded by differences in CO₂ concentration.

Pit assay: Bone marrow macrophage monocultures (1G vs 30G)

Osteoclast resorption activity on dentin slices was evaluated. On Day 3, BMMs were detached and switched to Leibovitz’s L-15 medium containing M-CSF (20 ng/mL), RANKL (100 ng/mL), and vitamin C (50 mg/L). Dentin slices (diameter 5 mm) were pre-fixed to the bottom of 96-well plates (Thermo Fisher Scientific, Waltham, USA) using Medical Adhesive Silicone Type A (Dow Corning, Midland, USA), then BMMs were seeded at 2.0 × 10⁴ cells/well. The cultures were maintained at 37°C under 0.04% CO₂ at 1G or 30G until Day 16, with medium replaced every 3 days. On Day 16, cells were fixed with 4% paraformaldehyde (PFA; Muto Pure Chemicals, Tokyo, Japan).

Pit assay: BMM and primary osteoblast co-cultures (1G vs 30G)

Primary osteoblasts were isolated from calvaria of neonatal C57BL/6J mice by treatment with 0.1% collagenase and 0.2% dispase. Isolated osteoblasts were cryopreserved in liquid nitrogen until use. 4 days before BMM seeding (designated Day −4, relative to BMM isolation), osteoblasts were thawed and cultured in α-MEM with medium changes every 3 days. Cells were expanded by passaging as needed. On Day 3 of BMMs culture, osteoblasts were detached using 0.25% Trypsin-EDTA (Fujifilm Wako, Osaka, Japan). and co-seeded together with BMMs onto dentin slices pre-fixed to the bottom of 96-well plates at a total density of 2.0 × 10⁴ cells/well. Leibovitz’s L-15 medium supplemented with 100 ng/mL of prostaglandin E₂ (Sigma-Aldrich, St. Louis, USA), 100 ng/mL of 1α,25(OH)₂D₃ (Fujifilm Wako, Osaka, Japan), and 50 mg/L of vitamin C was used, and cultures were maintained at 37°C with 0.04% CO₂ at 1G or 30G until Day 14, with medium replaced every 3 days. On Day 14, cells were fixed with 4% PFA.

Pit assay: RepCell-derived osteoclasts (1G vs 3G)

BMMs were seeded onto RepCell (CellSeed, Tokyo, Japan) culture dishes on Day 3 and differentiated in α-MEM containing M-CSF (20 ng/mL) and RANKL (100 ng/mL) until Day 14. Cells were then detached at 4°C for 10 minutes and seeded onto dentin slices (diameter 5 mm) in 24-well plates at 2.0 × 10⁴ cells/well. Pit assays were performed under 1G or 3G at 37°C with 5% CO₂ until Day 20, with medium replaced every 4 days. On Day 20, cells were fixed with 4% PFA.

Pit assay: RepCell-derived osteoclasts under inverted gravity conditions (1G, −1G, and −3G)

BMMs were seeded onto RepCell culture dishes on Day 3 and differentiated in α-MEM containing M-CSF (20 ng/mL) and RANKL (100 ng/mL). On Day 13, mature osteoclasts were detached from the RepCell dishes and seeded onto dentin slices (diameter 5 mm) pre-fixed to the bottom of 24-well plates at 2.0 × 10⁴ cells/well (cell replating). To ensure firm attachment of osteoclasts to the dentin surface and prevent detachment during subsequent inverted culture or centrifugation, cells were incubated overnight under standard upright 1G conditions at 37°C in 5% CO₂ prior to gravity loading. On Day 14, cultures were subjected to conventional upright culture (1G), inverted static culture (−1G), or inverted centrifugation culture (−3G) for 3 days at 37°C in 5% CO₂. The medium was replaced every 3 days during the culture period. At the end of the experiment, cells were fixed with 4% PFA for subsequent staining and analysis.

Short-term (30 minutes) hypergravity exposure (1G vs 30G, 1G vs 3G, 1G vs 5G)

BMMs were detached on Day 3 and reseeded in medium containing M-CSF (20 ng/mL) and RANKL (100 ng/mL). The medium was replaced on Day 6.

For 1G vs 30G exposure, BMMs were seeded into 48-well plates at 7.0 × 10⁴ cells/well and cultured under 0.04% CO₂ conditions. On Day 7, cells were exposed to 30G for 30 minutes at 37°C using the LIX-140SP hypergravity device. Control groups were maintained under identical culture conditions and exposed to 1G for 30 minutes. For 1G vs 3G exposure, BMMs were seeded into 24-well plates at 2.0 × 10⁴ cells/well and cultured under 5% CO₂ conditions. On Day 7, cells were exposed to 3G for 30 minutes at 37°C using the CL-5100 device. Control groups were maintained under identical culture conditions and exposed to 1G for 30 minutes. Cells were fixed with 4% PFA immediately after exposure. For exploratory experiments (S4 Fig., S5 Fig.), BMMs were seeded into 25-mL flasks at 2.0 × 10⁶ cells/flask on Day 3. Due to the larger medium volume and accelerated differentiation, hypergravity exposure was performed on Day 6. Cells were exposed to 5G for 30 minutes at 37°C under 5% CO₂. In some experiments, cells exposed to 5G for 30 minutes were subsequently returned to 1G conditions for an additional 30 minutes before fixation.

Rationale for seeding density and plate format

Seeding densities and plate formats were selected based on the requirements of each experimental setup and device configuration. Importantly, the aim of this study was not to compare absolute osteoclast differentiation efficiency across different plate formats or cell densities, but to evaluate the relative effects of different gravity conditions under identical culture conditions. Therefore, all gravity-dependent comparisons were performed strictly within the same plate format and seeding density (e.g., 1G vs. 30G, 1G vs. 3G, or 1G vs. 5G), and no direct comparisons were made across different well sizes or culture formats. Similarly, fixation time points were determined individually for each experimental system based on osteoclast maturation and assay requirements, such as differences in differentiation kinetics and the time required to achieve stable osteoclast morphology and resorption activity in each culture system. Gravity-dependent comparisons were always performed within the same system and time point, and no direct comparisons were made across different fixation time points.

Short-term (30 minutes) inverted gravity exposure (1G, −1G, and −3G)

BMMs were detached on Day 3 and reseeded into 24-well plates at 2.0 × 10⁴ cells/well in α-MEM containing M-CSF (20 ng/mL) and RANKL (100 ng/mL). The medium was replaced on Day 6. On Day 7, osteoclasts were subjected to conventional upright culture (1G), inverted static culture (−1G), or inverted centrifugation culture (−3G) for 30 minutes at 37°C in 5% CO₂ using the CL-5100 device. For the −3G condition, culture plates were mounted in an inverted orientation so that centrifugal force was directed away from the culture substrate. Control cells (1G) and inverted static controls (−1G) were maintained under otherwise identical environmental conditions. Immediately after exposure, cells were fixed with 4% PFA and processed for subsequent morphological analyses, including Rhodamine-phalloidin staining and TRAP staining.

Phosphoproteomic analysis (1G vs 5G)

BMMs were detached on Day 3 and seeded at 3.0 × 10⁶ cells/25-mL flask (first experiment) in medium containing M-CSF and RANKL. On Day 6, cells were exposed to 5G in the CL-5100 at 37°C with an atmosphere containing 5% CO₂ for 30 minutes, while the control cells were maintained at 1G. Cells were washed with PBS (-) (Shimadzu Diagnostics, Tokyo, Japan) and inactivated with 0.1 vol% trifluoroacetic acid-acetonitrile (ACN-0.1% TFA, Kanto Kagaku, Tokyo, Japan), then scraped and placed in conical tubes. Samples were then obtained and frozen in liquid nitrogen, and stored at −80°C. In a second independent phosphoproteomic experiment, BMMs were seeded at 1.59 × 10⁷ cells per 25-mL flask, and phosphoproteomic analysis was performed using three independent flasks per condition (n = 3). All experimental procedures, including culture conditions, gravity exposure, sample preparation, and fixation, were identical to those used in the first experiment. The first experiment (n = 1 per condition) was conducted as an exploratory screening to identify gravity-responsive phosphorylation events, while the second experiment (n = 3 per condition) was performed to confirm the reproducibility of phosphorylation changes observed in the screening analysis. The analyses were conducted by Kazusa Genome Technologies (Kisarazu, Japan).

Rhodamine-phalloidin staining

F-actin structures were visualized using Rhodamine-phalloidin staining. Cells were fixed with 4% PFA, washed with PBS (-), permeabilized with 0.1% Triton X-100 (MP Biomedicals, Santa Ana, USA) for 15 minutes, and blocked with 1% bovine serum albumin (BSA; Sigma-Aldrich, St. Louis, USA) for 30 minutes. Phalloidin, Rhodamine X conjugated (Fujifilm Wako, Osaka, Japan) was applied for 2 hours at room temperature. After washing, fluorescence images were acquired with a BZ-X810 fluorescence microscope (Keyence, Osaka, Japan). All images used for qualitative and quantitative comparisons were acquired within the same experiment under identical acquisition settings. Image contrast and intensity were not arbitrarily adjusted during image acquisition or subsequent analysis.

Antibody staining

Cells were fixed and permeabilized as detailed above, blocked with 1% BSA, and incubated with the following primary antibodies: α-Tubulin (Alexa Fluor 488, clone DM1A; Abcam, Cambridge, UK), Vinculin (Alexa Fluor 488, clone EPR8185; Abcam, Cambridge, UK), c-Src (Alexa Fluor 488, clone B-12; Santa Cruz Biotechnology, Dallas, USA), and Integrin β3 (Rabbit polyclonal; StressMarq Biosciences, Victoria, Canada), Alexa Fluor 488-conjugated antibodies were used directly, while other primary antibodies were detected using donkey anti-rabbit IgG (Alexa Fluor 488; Abcam, Cambridge, UK). Nuclei were stained with DAPI (Fujifilm Wako, Osaka, Japan). Microscopic images were acquired using a BZ-X810 fluorescence microscope. Immunofluorescence images represent independent single-color staining experiments performed with individual antibodies. Each antibody was applied and imaged separately, and these experiments were not designed for multiplex imaging or co-localization analysis.

TRAP staining

TRAP staining was performed to assess osteoclast differentiation and activity. Cells were fixed with ethanol/acetone for 1 minute, then washed and air-dried. Fast Red Violet LB Salt (Sigma-Aldrich, St. Louis, USA) and Naphthol AS-MX phosphate (Sigma-Aldrich, St. Louis, USA) in 0.1 M acetate buffer (pH 5.0) with 1% N,N-dimethylformamide (Fujifilm Wako, Osaka, Japan) were applied at 37°C for 20 minutes. Stained cells were imaged using a BZ-X810 fluorescence microscope. For TRAP staining, quantitative evaluation was restricted to comparisons between the 1G and hypergravity groups within the same culture system (monoculture or co-culture). TRAP staining intensity was not quantitatively compared across different culture systems.

Toluidine blue staining

Following the pit assay, dentin slices were stained with 1% Toluidine blue O (Polysciences, Warrington, USA) for 5 minutes and imaged using a VHX-6000 digital microscope (Keyence, Osaka, Japan).

Quantification and image analysis

Image analysis was performed using ImageJ, ver. 1.54g (National Institutes of Health, Bethesda, USA). Osteoclast number, Rhodamine-phalloidin-positive area, TRAP-positive area, and resorption pit area were quantified using thresholding, particle analysis, and macros. Consistent image acquisition and analysis settings were applied within each experiment, with threshold values adjusted between experiments according to staining intensity and background levels. Analyses were performed using 8-bit images. All images used for quantitative and qualitative comparisons were acquired within the same experiment using fixed image acquisition settings, including exposure time, illumination intensity, and detector gain. No arbitrary contrast or brightness adjustments were applied during image acquisition or analysis. The mean fluorescence intensity of the actin ring was quantified from Rhodamine-phalloidin-stained images using raw, unadjusted images, without thresholding or binarization. For some images, osteoclast and nuclei numbers were quantified by manual counting.

Statistical analysis

Statistical analyses were performed using JMP Pro, ver. 17.0.0 (Statistical Discovery, Durham, USA). Data are presented as mean ± standard deviation (SD), unless otherwise specified. Comparisons between two groups were made using a two-tailed Student’s t-test, whereas comparisons among three groups were conducted using one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test. Statistical significance was set at p < 0.05. In the figures, significance is indicated as *p < 0.05, **p < 0.01, or ***p < 0.001, and comparisons showing no significant differences are indicated as n.s.

Osteoclast bone resorption attenuated under severe hypergravity at 30G

To evaluate the effects of severe hypergravity on osteoclast function, BMMs were cultured either alone or in cocultures with osteoblasts under a 1G or 30G condition. TRAP staining of cell culture plates was performed to assess osteoclast differentiation, while pit assays of dentin slices were conducted to evaluate bone resorption (Fig 1A). For osteoclast monoculture experiments, bone marrow cells were isolated on Day 0 and cultured with M-CSF until Day 3, followed by culture with M-CSF and RANKL on cell culture plates or dentin slices until fixation on Day 16. For osteoclast-osteoblast coculture experiments, osteoblasts were prepared four days in advance and maintained with medium replacement until Day 3. BMMs were then added, and cocultures were maintained in the presence of prostaglandin E2 and 1α,25(OH)₂D₃ until fixation on Day 14. The LIX-140SP hypergravity device was set to 390 rpm and provided continuous 30G (Fig 1B, C). Under the hypergravity condition, osteoclasts in both monocultures and cocultures displayed no morphological or TRAP activity changes, indicating that hypergravity has no effect on osteoclast differentiation (Fig 1D-I). In addition, the number of TRAP-positive osteoclasts (≥100 µm) was quantified in monocultures and cocultures (Fig 1G, I). In monocultures, all osteoclasts contained three or more nuclei, confirming successful fusion. In cocultures, counting nuclei for fusion index was difficult due to overlapping cells; therefore, the number of TRAP-positive osteoclasts ≥100 µm was used instead. In pit assays, osteoclasts monocultured under 30G exhibited no significant change in pit area or pit depth (Fig 1J, L, M). In cocultures, resorption area showed no significant change, whereas pit depth was significantly reduced under 30G (Fig 1K, N, O). These findings indicate that severe hypergravity of 30G attenuates bone resorption, particularly when osteoclasts interact with osteoblasts, and that this effect is specific to pit depth rather than resorption area.

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Fig 1. Severe hypergravity (30G) attenuates osteoclastic bone resorption on dentin.

(A) Experimental timeline for monoculture and coculture pit assays on dentin slices. (B) LIX-140SP hypergravity device with mounted plate. (C) Schematic diagram of gravity-generated mechanical loading under hypergravity applied to osteoclasts. (D, E) Representative osteoclasts on culture plates stained with TRAP under monoculture (D) and coculture (E) conditions. (F) Quantification of TRAP-positive area in osteoclast monocultures. (G) Number of TRAP-positive osteoclasts larger than 100 µm in monoculture. (H) Quantification of TRAP-positive area in coculture. (I) Number of TRAP-positive osteoclasts larger than 100 µm in coculture. (J, K) Representative resorption pits stained with Toluidine Blue and corresponding 3D reconstructions. Yellow lines indicate the positions used for cross-sectional analysis. (L) Relative pit area in monoculture. (M) Pit depth (µm) in monoculture. (N) Relative pit area in coculture. (O) Pit depth (µm) in coculture. Relative values are normalized to the 1G control group. For F-I, ImageJ threshold values were set to 50-170, and TRAP-positive osteoclasts with a diameter ≥100 µm were analyzed. For L and N, threshold values were set to 70-140, and resorption pits with a diameter ≥10 µm and an aspect ratio <1.5 were analyzed. Scale bar: 100 µm. Error bars indicate standard deviation (SD). Statistical comparisons were performed using a two-tailed unpaired Student’s t-test. p < 0.05. Data were obtained from three independent experiments. Each data point represents one well or one dentin slice (1G and 30G, n = 3 wells or dentin slices per condition), except for the 1G coculture condition (n = 2 dentin slices). For each well or dentin slice, quantitative analyses were performed using three randomly acquired images, and the averaged value per well or dentin slice was used for statistical analysis.

https://doi.org/10.1371/journal.pone.0351542.g001

Pronounced alterations in actin ring structure and nuclear repositioning induced by short-term 30G hypergravity

To investigate rapid cytoskeletal responses, osteoclasts were exposed to severe hypergravity at 30G for 30 minutes (Fig 2A). Representative microscopic images of osteoclasts immediately before and after hypergravity loading are provided (S1 Fig.). Immediately before gravity loading, actin ring organization was comparable between the 1G and 30G groups, with no apparent structural differences. In contrast, immediately after hypergravity exposure, localized alterations in the arrowhead region of the actin ring were observed in the 30G group, whereas the actin rings in the 1G group remained unchanged. Although immunofluorescence staining for cytoskeleton- and adhesion-related proteins, including Tubulin, Vinculin, c-Src, and Integrin β3 showed no obvious differences as compared with the 1G control (Fig 2B), Rhodamine-phalloidin staining indicated reduced fluorescence intensity and increased sealing zone (actin ring) width under 30G (Fig 2C-F). The percentage of regions with high actin ring fluorescence along the osteoclast circumference was significantly reduced in the 30G group compared with the 1G control (Fig 2D). In addition, the mean fluorescence intensity of the actin ring was significantly decreased under 30G conditions (Fig 2E). Consistent with these findings, cross-sectional analysis of F-actin fluorescence intensity along the green line in Fig 2C showed reduced F-actin accumulation at the sealing zone (actin ring) under 30G (Fig 2F). These changes were more pronounced than observed under 3G, which are described in the following section. Additionally, nuclear positioning was strongly affected by hypergravity, with the proportion of nuclei on actin rings increasing from 34.15% at 1G to 83.56% at 30G, though the total number of nuclei per cell remained unchanged (Fig 2G, H). Osteoclast diameter showed no significant difference between 1G and 30G following short-term exposure (Fig 2I). These results indicate that short-term strong hypergravity induces rapid cytoskeletal remodeling and nuclear repositioning in osteoclasts.

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Fig 2. Short-term (30 minutes) severe hypergravity (30G) alters F-actin organization and nuclei positioning in osteoclasts.

(A) Experimental timeline. Osteoclasts were exposed to 1G or 30G for 30 minutes. (B) Immunostaining for Tubulin, Vinculin, c-Src, and Integrin β3. Each marker was stained and imaged separately. (scale bar: 500 µm). (C) Rhodamine-phalloidin and DAPI staining, with merged images and 3D intensity maps. Yellow arrows indicate nuclei localized within actin rings, and red arrows indicate regions of high F-actin intensity. Scale bar: 100 µm. (D) Percentage of regions with high actin ring fluorescence. (E) Mean fluorescence intensity of actin rings. (F) Line-scan analysis of Rhodamine-phalloidin fluorescence intensity across actin ring cross-sections. (G, H) Nuclear positioning shown by representative images (G) and quantification of peripheral versus central nuclear localization (H). (I) Osteoclast diameter. Error bars indicate standard deviation (SD). Statistical comparisons were performed using a two-tailed unpaired Student’s t-test. ***p < 0.001. Data were obtained from two independent experiments. Panel E was analyzed using nine wells, with three osteoclasts imaged per well (n = 27 cells). Panels D and F-I were analyzed using 16 wells, with three osteoclasts imaged per well (n = 48 cells); however, in panel F, the numbers of analyzed cells differed between conditions (1G: n = 48 cells; 30G: n = 47 cells). In all analyses, individual osteoclasts were treated as data points (n = cell number).

https://doi.org/10.1371/journal.pone.0351542.g002

Effects of 3G hypergravity on osteoclast actin rings and bone resorption

Following the initial assessment at 30G, the effects of mild hypergravity (3G) were examined to evaluate potential impacts that may be more relevant to actual incidents of human exposure. To exclude any potential effects of hypergravity on the differentiation process, RepCell dishes were used to obtain premature osteoclasts that had already undergone differentiation prior to gravity loading. RepCell is a temperature-responsive culture system that allows intact osteoclasts to be detached without enzymatic treatment, ensuring preservation of cytoskeletal structures. BMM-derived osteoclasts were cultured on RepCell dishes, then transferred to dentin slices for pit assays at 1G or 3G for 6 days (Fig 3A) using a CL-5100 at 209 rpm (Fig 3B, C). When actin rings were exposed to 3G, the area of the sealing zone with fluorescence intensity above the predefined threshold was significantly reduced (Fig 3D, F), indicating that F-actin accumulation was decreased under hypergravity. In contrast, the total number of osteoclasts remained unchanged (Fig 3G). In resorption assays, neither the relative resorption area nor pit depth showed significant differences between the 1G and 3G groups (Fig 3E, H, I). In contrast, in osteoclast-osteoblast cocultures, pit depth was significantly reduced under 3G compared with 1G (S2 Fig.). These results suggest that modest 3G hypergravity induces significant alterations in sealing zone (actin ring) organization without affecting osteoclast number, and that attenuation of bone resorption, particularly in pit depth, becomes evident under osteoclast-osteoblast coculture conditions. To further distinguish the effects of hypergravity from those of force direction relative to the substrate, additional pit assays were performed under inverted loading conditions (1G, −1G, and −3G). Under these conditions, neither F-actin-positive area nor resorption pit depth differed significantly among the three groups (S3 Fig.), indicating that reversal of the gravity-generated force vector relative to the substrate did not impair osteoclast attachment or bone resorption on dentin slices.

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Fig 3. Moderate hypergravity (3G) reduces F-actin accumulation in osteoclasts on dentin.

(A) Experimental timeline. BMM-derived osteoclasts differentiated on RepCell were transferred to dentin slices for pit assays. (B) CL-5100 hypergravity device with mounted 24-well plate. (C) Schematic diagram of gravity-generated mechanical loading under hypergravity applied to osteoclasts. (D) Representative osteoclasts on dentin slices stained with Rhodamine-phalloidin. Magnified views of the boxed regions are shown. (E) Representative resorption pits stained with Toluidine Blue, followed by 3D reconstruction. Yellow lines indicate cross-section positions. (F) Quantification of F-actin-positive area. ImageJ threshold values were set to 40-60, and cells with a diameter ≥20 µm were analyzed. (G) Osteoclast number was determined by manual counting of actin rings. (H) Quantification of pit area. Threshold values were set to 70-160, and pits with a diameter ≥10 µm and an aspect ratio <1.5 were analyzed. (I) Quantification of pit depth. Scale bar: 100 µm. Error bars indicate standard deviation (SD). Two-tailed unpaired t-test. *p < 0.05. Data were obtained from two independent experiments. Four dentin slices were analyzed per condition (n = 4 dentin slices). For image-based analyses, five fields per well were analyzed for panels F and G, and four fields per well for panels H and I.

https://doi.org/10.1371/journal.pone.0351542.g003

Short-term 3G hypergravity alters F-actin and nuclei distribution

To examine the immediate effects of mild hypergravity, osteoclasts were exposed to 3G for 30 minutes (Fig 4A). Decreased fluorescence intensity of actin rings was shown in localized regions by Rhodamine-phalloidin staining (Fig 4B), and quantitative analyses demonstrated a reduction in both the proportion of regions with high actin ring fluorescence and the mean fluorescence intensity of the actin ring under 3G conditions (Fig 4C, D). Additionally, nuclear positioning was altered, as the proportion of nuclei localized on the sealing zone increased to 39.96% at 3G compared with 20.78% at 1G. However, the number of nuclei per cell and osteoclast diameter remained unchanged (Fig 4E-G). These findings suggest that even a low level of hypergravity rapidly affects cytoskeletal dynamics. To examine whether similar rapid cytoskeletal responses were observed at an intermediate gravity level, osteoclasts were exposed to 5G for 30 minutes. Consistent with the 3G condition, Rhodamine-phalloidin staining revealed a significant reduction in actin ring fluorescence under 5G (S4 Fig.). Quantitative analyses confirmed a decrease in mean actin ring fluorescence intensity. In addition, nuclear positioning was similarly affected, with an increased proportion of nuclei localized on the sealing zone under 5G (35.15%) compared with 1G (22.08%) (S4 Fig.). To further characterize cytoskeletal responses after gravity loading, osteoclasts were exposed to 5G for 30 minutes followed by incubation at 1G for an additional 30 minutes. Under these conditions, no significant differences were detected in actin ring fluorescence intensity, nuclear distribution, or osteoclast morphology compared with cells analyzed immediately after 5G exposure (S5 Fig.). Together, these findings demonstrate that short-term hypergravity rapidly induces cytoskeletal remodeling and changes in nuclear positioning in osteoclasts, and that similar responses are observed across multiple gravity conditions examined in this study. To further examine the contribution of force direction, osteoclasts were subjected to short-term inverted loading conditions (1G, −1G, and −3G) for 30 minutes. Both inverted static culture (−1G) and inverted centrifugation culture (−3G) induced cytoskeletal changes comparable to those observed under conventional hypergravity, including a significant reduction in the proportion of regions with high actin ring fluorescence and an increase in actin ring width (S6 Fig.). Mean actin ring fluorescence intensity was significantly reduced under −3G, whereas the decrease under −1G did not reach statistical significance. In addition, both inverted conditions caused pronounced peripheral redistribution of nuclei without altering the total number of nuclei per osteoclast or overall cell diameter (S6 Fig.). These findings indicate that osteoclast actin ring organization and nuclear positioning respond rapidly not only to substrate-directed compressive loading under conventional hypergravity, but also to tensile loading generated when the net force is directed away from the substrate under inverted loading conditions.

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Fig 4. Mild effects of short-term (30 minutes) moderate hypergravity (3G) on F-actin and nuclei distribution.

(A) Experimental timeline. Osteoclasts were exposed to 1G or 3G for 30 minutes. (B) Rhodamine-phalloidin and DAPI staining shown with merged images, 3D fluorescence intensity maps, and TRAP staining. Yellow arrows indicate nuclei located within actin rings. (C) Percentage of regions with high actin ring fluorescence. (D) Mean fluorescence intensity of the actin ring. (E, F) Representative images showing nuclear positioning (E) and ratio of peripheral vs central localization (F). (G) Osteoclast diameter. Scale bar: 100 µm. Error bars indicate standard deviation (SD). Two-tailed unpaired t-test. *p < 0.05, ***p < 0.001. Data were obtained from two independent experiments. Panel D was analyzed using five wells, with three osteoclasts analyzed per well (n = 15 cells). Panels C and E-G were analyzed using eight wells, with three osteoclasts analyzed per well (n = 24 cells).

https://doi.org/10.1371/journal.pone.0351542.g004

Phosphoproteomic profiling of osteoclasts under hypergravity

To investigate early molecular events underlying hypergravity-induced sealing zone (actin ring) remodeling, osteoclasts were exposed to 5G hypergravity for 30 minutes using a CL-5100 centrifuge (Fig 5A-C). Phosphoproteomic analysis was performed with three independent biological replicates per condition (n = 3).

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Fig 5. Phosphoproteomic profiling of osteoclasts exposed to a higher level of moderate hypergravity (5G) for 30 minutes.

(A) Experimental timeline. (B) CL-5100 device with a mounted 25-mL flask. (C) Schematic diagram of gravity-generated mechanical loading under hypergravity applied to osteoclasts. (D) Volcano plot of phosphoproteomic analysis comparing 1G and 5G conditions (n = 3 per condition). Data were log2-transformed and imputed for missing values, followed by statistical testing using a two-sample t-test and permutation-based FDR correction (250 permutations; FDR = 0.05; S0 = 0.1). Red dots indicate significantly up-regulated phosphopeptides and blue dots indicate significantly down-regulated phosphopeptides (FDR < 0.05); gray dots indicate no significant change. Phosphopeptides with FDR < 0.01 are annotated by name. (E) Heatmap of differentially phosphorylated peptides identified in (D). Phosphopeptides up-regulated under 5G conditions constitute the up-regulated cluster in 5G, whereas down-regulated phosphopeptides constitute the down-regulated cluster in 5G. Phosphopeptides with FDR < 0.05 were Z-score-normalized and visualized. (F) Bar graph summarizing the number of up-regulated (59 peptides) and down-regulated (94 peptides) phosphorylation events identified in (D), with accompanying functional classification of the corresponding proteins.

https://doi.org/10.1371/journal.pone.0351542.g005

A total of 12,087 phosphopeptides were identified. Quantitative analysis revealed 59 phosphopeptides with significantly increased phosphorylation and 94 with decreased phosphorylation under hypergravity (Fig 5D; S1 Table). Differentially phosphorylated proteins were visualized by volcano plot analysis (Fig 5D) and hierarchical clustering heatmaps (Fig 5E), demonstrating clear separation between 1G and 5G conditions. Corresponding analyses based on unadjusted datasets are also shown (S7 Fig.).

Functional categorization of the differentially phosphorylated proteins showed that hypergravity predominantly affected six major cellular modules: (1) Cytoskeleton/ Cell adhesion/ Mechanotransduction, (2) Membrane trafficking/ Endocytosis/ Lipid signaling, (3) Kinase/ Phosphatase/ Signaling, (4) Nucleus/ Chromatin/ DNA repair, (5) RNA processing/ Translation, and (6) Mitochondria/ Metabolism/ Autophagy (Fig 5F). Among the upregulated phosphoproteins, substantial representation was observed in cytoskeleton/cell adhesion/mechanotransduction and kinase/phosphatase/signaling categories, together accounting for nearly half of the upregulated events, alongside a considerable fraction of nuclear and chromatin-associated proteins. This distribution suggests that hypergravity rapidly enhances phosphorylation events related to actin-membrane dynamics and intracellular signaling, while also engaging nuclear regulatory components. In contrast, downregulated phosphoproteins were more strongly biased toward nuclear/chromatin/DNA repair and RNA processing/translation categories, indicating preferential attenuation of phosphorylation within transcriptional and post-transcriptional regulatory pathways. Membrane trafficking-related proteins also constituted a notable fraction of downregulated phosphoproteins, suggesting functional rebalancing of vesicular and endocytic processes under hypergravity.

To assess the consistency between the exploratory and quantitative analyses, proteins commonly identified as differentially phosphorylated in both the dataset (n = 3) and the additional dataset (n = 1) were summarized (Table 1). An overview of the exploratory phosphoproteomic dataset is shown (S8 Fig.), and the complete list of identified phosphopeptides is provided (S2 Table). These overlapping proteins represent robust candidates for hypergravity-responsive phosphorylation events.

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Table 1. Commonly regulated phosphorylation sites identified in both quantitative (n = 3) and exploratory (n = 1) phosphoproteomic analyses under hypergravity-induced mechanical loading conditions.

https://doi.org/10.1371/journal.pone.0351542.t001

Together, these results demonstrate that short-term hypergravity induces a rapid and coordinated shift in phosphorylation states across cytoskeletal, membrane, and nuclear functional modules in osteoclasts.

Hypergravity attenuates osteoclast bone-resorptive activity

The present study was conducted to comprehensively analyze the effects of hypergravity-induced mechanical loading on osteoclast bone-resorptive function and cytoskeletal organization, as well as the molecular basis underlying these responses, based on phosphoproteomic profiling. Important findings included the following: (i) prolonged exposure to 3G or 30G altered sealing zone (actin ring) structures and was associated with suppression of bone resorption; (ii) even short-term exposure (30 minutes) induced actin ring structural changes, resulting in decreased F-actin fluorescence, ring broadening, and nuclear peripheral repositioning; (iii) hypergravity induced coordinated changes in phosphorylation across multiple functional modules, including cytoskeletal organization, membrane trafficking, intracellular signaling, and nuclear/chromatin-associated regulatory proteins; and (iv) similar cytoskeletal and nuclear changes were also observed under inverted loading conditions (−1G and −3G), indicating that osteoclasts respond rapidly to changes in the magnitude and direction of gravity-generated mechanical forces.

The reduction in osteoclast resorptive activity caused by hypergravity is notable. While microgravity encountered during spaceflight has been shown to accelerate bone resorption and contribute to bone loss [27,28], the present findings suggest that increased gravity-generated mechanical loading may suppress osteoclast function. In particular, exposure to severe hypergravity at 30G resulted in significantly decreased resorption pit depth, indicating direct inhibition of osteoclast-mediated bone matrix degradation. Similarly, prolonged exposure to moderate hypergravity (3G) also reduced resorptive activity, demonstrating that this inhibitory effect is not restricted to extreme loading conditions. The present in vitro observations are consistent with previous in vivo findings showing that hypergravity suppressed bone resorption in ovariectomized rats [25]. Thus, these results suggest that osteoclasts show a bidirectional response to altered gravitational loading, with microgravity enhancing and hypergravity attenuating bone resorption, which highlights the role of mechanical cues in skeletal homeostasis. Although microgravity and hypergravity exert opposite effects on osteoclast activity, both conditions represent deviations from the normal gravitational environment and are likely to challenge cellular mechanosensing systems. In this regard, cytoskeletal organization may constitute a gravity-sensitive structural target that responds to altered mechanical loading, while downstream functional outcomes diverge depending on the direction and magnitude of the gravitational perturbation.

Importantly, however, in our experimental system, the functional consequences of altered gravitational loading were not uniform. Hypergravity significantly suppressed osteoclast bone-resorptive activity under upright loading conditions (3G and 30G), whereas resorptive activity appeared to be preserved under inverted loading conditions (−1G and −3G). These findings indicate that osteoclast function is not impaired simply by changes in gravitational orientation. Rather, suppression of bone resorption appears to depend on increased gravity-generated mechanical loading. Thus, while cytoskeletal remodeling may represent a common gravity-sensitive response, its downstream functional consequences are determined by the magnitude and direction of the mechanical forces generated by gravity.

Cytoskeletal responses to altered gravity-generated forces

Structural changes occurring even after short-term hypergravity exposure indicate that osteoclasts have the capability to rapidly sense and respond to gravity-generated mechanical stress. It is likely that an altered F-actin assembly contributes to impaired adhesion and reduced resorptive activity under hypergravity-generated mechanical loading. The present pit assay results demonstrated that osteoclast migration was also suppressed by hypergravity conditions. The sealing zone (actin ring) has been shown to be closely associated with migration rate and resorption efficiency [29], thus their structural alterations observed in the present study may have a direct relationship with the reduced motility and functional capacity noted for osteoclasts.

Osteoclast activity is tightly regulated by osteoblast-derived cues, including RANKL/OPG signaling and contact-dependent interactions [30], which influence resorption efficiency as well as cytoskeletal organization. In the present study, suppression of resorption pit depth was observed in osteoclast-osteoblast cocultures under hypergravity conditions, suggesting that altered osteoblast-mediated signaling may contribute to the hypergravity-induced osteoclastic phenotype. At the same time, comparable alterations in actin ring organization were also detected in osteoclast monocultures, indicating that hypergravity directly affects osteoclasts in a cell-intrinsic manner, independent of osteoblast-derived regulation.

Another important finding is that nuclei were displaced onto actin rings under hypergravity. Such nuclear repositioning suggests modulation of intracellular tension, and potential involvement of the linker of nucleoskeleton and cytoskeleton (LINC) complex in cytoskeleton-nucleus coupling [31,32]. Similar findings have been reported for osteoblasts [33] and fibroblasts [34], but have rarely been described in osteoclasts.

Recent studies have begun to highlight the importance of intracellular spatial organization in multinucleated osteoclasts. In particular, proper clustering of centrosomes is required for polarized microtubule organization and efficient vesicular trafficking, which are essential for bone resorption [35]. Disruption of this organization leads to impaired cytoskeletal polarity and reduced resorptive activity. In addition, defective centrosome clustering has been shown to cause abnormal nuclear distribution and loss of polarized architecture in osteoclasts [36].

Actin filaments and podosome rings play critical roles in osteoclastogenesis and migration [37,38], and the present results further suggest that hypergravity-induced cytoskeletal remodeling may indirectly drive nuclear repositioning by altering intracellular force balance. Such aberrant nuclear localization could, in turn, interfere with actin-dependent processes by perturbing the spatial coordination required for sealing zone organization and polarized resorptive function.

Importantly, the inverted-loading experiments demonstrated that both inverted static culture (−1G) and inverted centrifugation (−3G) induced actin ring broadening and peripheral nuclear redistribution similar to those observed under conventional hypergravity. Osteoclasts remained firmly adherent to dentin slices under these inverted conditions and maintained bone-resorptive activity, with no significant differences in resorption pit depth among 1G, −1G, and −3G conditions. These findings further indicate that the observed cytoskeletal and nuclear responses are not primarily driven by substrate-directed compressive loading or hydrostatic pressure. If simple compression or medium-derived hydrostatic forces were the dominant determinants, opposite cellular responses would be expected under upright versus inverted orientations. Instead, the comparable phenotypes observed under both conditions support a model in which gravity-generated mechanical forces, despite opposite loading directions relative to the substrate, produce similar intracellular mechanical stresses and structural responses. Such effects may arise from differential displacement of intracellular components, including nuclei and organelles, thereby perturbing intracellular force balance throughout the multinucleated osteoclast. Although hydrostatic pressure may contribute as a secondary, uniformly distributed factor, it is unlikely to represent the principal driver of the observed structural changes.

Together, these findings point to a previously underappreciated aspect of osteoclast mechanotransduction, in which hypergravity perturbs both cytoskeletal organization and nucleo-cytoskeletal interactions, potentially leading to functional impairment. Importantly, the concurrent occurrence of actin remodeling and nuclear repositioning suggests that these structural responses are mechanically coupled rather than independent events. Although causality cannot be established in the present study, these findings suggest that cytoskeletal-nuclear reorganization may contribute to the functional impairment of osteoclasts under hypergravity-generated mechanical loading.

Molecular basis revealed by phosphoproteomics

In this study, phosphoproteomic analysis with biological replication (n = 3) revealed that hypergravity induces broad yet structured changes in phosphorylation across multiple cellular systems, rather than isolated modulation of individual proteins. Differentially phosphorylated proteins were distributed among defined functional modules, including cytoskeletal organization, membrane trafficking, intracellular signaling, nuclear/chromatin regulation, and RNA processing, indicating that gravity-generated mechanical stress elicits a system-level reorganization of mechanotransduction pathways in osteoclasts. This modular pattern is consistent with established models of mechanotransduction, in which mechanical cues are integrated across interconnected cellular compartments rather than conveyed through single linear signaling cascades [39,40].

Quantitative categorization demonstrated that upregulated phosphorylation events were particularly enriched in cytoskeleton/cell adhesion/mechanotransduction and kinase/phosphatase/signaling modules, together accounting for a substantial fraction of the hypergravity-induced phosphorylation response. In parallel, a notable proportion of upregulated phosphoproteins was also associated with nuclear and chromatin-related functions, suggesting that hypergravity rapidly engages both peripheral structural networks and nuclear regulatory components. The enrichment of cytoskeletal regulators, membrane-cytoskeleton linkers, actin-myosin-associated proteins, and vesicle trafficking factors supports the notion that hypergravity enhances phosphorylation-dependent reinforcement and remodeling of actin-membrane architectures. These molecular changes are consistent with the rapid sealing zone reorganization and altered osteoclast morphology observed at the cellular level in this study, as well as with established roles of cytoskeletal regulation in osteoclast function [41].

In contrast, downregulated phosphorylation events exhibited a stronger relative bias toward nuclear/chromatin/DNA repair and RNA processing/translation modules. The prominence of chromatin remodelers, histone-modifying enzymes, transcriptional regulators, and RNA processing factors among downregulated phosphoproteins suggests that hypergravity preferentially attenuates or reprograms phosphorylation-dependent nuclear regulatory pathways. Notably, membrane trafficking and endocytic components also constituted a considerable fraction of downregulated proteins, indicating that hypergravity does not simply activate peripheral pathways but induces a coordinated rebalancing of phosphorylation across multiple cellular systems.

The concurrent representation of nuclear and chromatin-associated proteins in both up- and downregulated datasets suggests that hypergravity does not uniformly suppress nuclear signaling. Instead, it likely induces selective phosphorylation remodeling within nuclear regulatory networks. The presence of proteins involved in nucleo-cytoskeletal coupling, centrosome organization, and chromatin architecture among the differentially phosphorylated proteins further supports a model in which hypergravity alters force transmission along the membrane-cytoskeleton-nucleus axis. Such modulation may influence nuclear positioning, chromatin accessibility, and transcriptional responsiveness through phosphorylation-dependent mechanisms, in line with emerging concepts of nuclear mechanotransduction [39,40,42].

Taken together, these phosphoproteomic data indicate that hypergravity signaling in osteoclasts is characterized not by a simple dichotomy between cytoskeletal activation and nuclear suppression, but by a coordinated, module-specific reprogramming of phosphorylation states. Enhanced phosphorylation of cytoskeletal and signaling components, coupled with selective attenuation of nuclear and RNA regulatory phosphorylation, provides a molecular framework linking mechanical stress to structural adaptation and altered cellular function. This coordinated response may underlie the impaired osteoclast activity observed under hypergravity-generated mechanical loading [39–41].

Notably, a limited subset of differentially phosphorylated proteins was consistently detected in both the exploratory phosphoproteomic analysis (n = 1) and the quantitative analysis with biological replication (n = 3). Although the overlap was modest, these shared candidates were distributed across cytoskeletal, membrane trafficking, and nuclear regulatory modules, providing additional support for the involvement of these functional systems in the hypergravity response [43]. The phosphoproteomic dataset generated in this study will serve as a valuable resource for future studies aimed at validating and functionally characterizing individual hypergravity-responsive phosphorylation‌‌ events.

Limitations and future directions

The present experiments were conducted in vitro and the effects of hypergravity on in vivo bone remodeling remain to be determined. In addition, it should be noted that the 3G and 30G experiments were performed using different hypergravity devices, which precludes direct quantitative comparison between these two conditions. Accordingly, the present study does not attempt to compare the magnitude of osteoclast responses between 3G and 30G. Nevertheless, qualitatively similar cytoskeletal changes, characterized by reduced F-actin fluorescence intensity and peripheral nuclear repositioning, were consistently observed across multiple hypergravity conditions, including 3G, 5G, and 30G. The reproducibility of these features across different gravity levels and experimental platforms suggests that they represent a common osteoclast response to gravity-generated mechanical loading, rather than device-specific effects. Future studies using a unified experimental system will be required to enable rigorous quantitative comparison across gravity magnitudes. Moreover, it remains unclear whether phosphorylation changes are causal for functional suppression or represent secondary responses. While disorganization of actin rings was induced by 30 minutes of hypergravity, the present data indicate that returning cells to 1G for 30 minutes was insufficient to restore actin ring fluorescence intensity to baseline levels (S5 Fig.). This suggests that recovery of cytoskeletal organization, if it occurs, may require longer periods under normal gravity. Further studies will be necessary to examine the time dependency and mechanisms underlying such potential reversibility, including targeted perturbation of candidate phosphoproteins, as well as extension of the analysis to in vivo models. Astronauts are instructed to perform exercise to mitigate microgravity-induced bone loss [44], underscoring the importance of mechanosensitive pathways in skeletal health.

A key consideration in the present experimental design is that osteoclasts cultured on dentin slices are exposed to distinct combinations of gravity-generated mechanical forces depending on sample orientation. Under conventional hypergravity, cells on the upper surface primarily experience substrate-directed compressive loading together with increased hydrostatic pressure from the overlying medium. In contrast, under inverted conditions, forces are directed away from the substrate, thereby imposing tensile loading while reducing compressive effects. During centrifugation, both upright and inverted samples are also subject to rotationally associated inertial forces, including minor shear-related components and Coriolis forces. However, these effects are absent under static inverted 1G (−1G) conditions. The observation that actin ring remodeling and nuclear redistribution occurred under both centrifuged and static inverted conditions indicates that these structural responses are not dependent on compressive loading or centrifugation-specific inertial forces alone. By contrast, the reduction in bone-resorptive activity observed only under conventional hypergravity suggests that compressive loading and/or increased hydrostatic pressure may be particularly important in suppressing osteoclast resorptive function. Future studies using systems that independently control compression, tension, hydrostatic pressure, and shear will be necessary to define the specific contributions of these individual mechanical stimuli.