Low intensity vibration with zoledronate reduces musculoskeletal weakness and adiposity in estrogen deprived female mice

Domain: biology
Published: 2026-01-21
Editorial outcome: major_revision

Abstract
Aromatase inhibitors are widely used in the treatment of hormone-sensitive breast cancer, but their suppression of estrogen production accelerates bone loss, increases fracture risk, and negatively impacts muscle and fat metabolism. Here, we demonstrate that daily low intensity vibration, serving as an on-drug mimetic for exercise, protects musculoskeletal health in skeletally immature, female mice under complete estrogen deprivation. Subsequent improvements in vertebral bone density are paralleled by greater and leaner skeletal muscle mass and functional once considered reduced fat accretion and circulating metabolites. In mature, estrogen deprived mice, vibration enhances weekly bisphosphonate treatment, improving bone density, cortical thickness, and mechanical resistance to fracture. These findings support the proposed hypothesis that low intensity vibration reduces musculoskeletal frailty in estrogen deprived mice, with stronger effects observed in younger cohorts, while in skeletally mature mice combination therapy with anti-resorptive treatment is necessary to suppress cancer-treatment induced musculoskeletal degradation.

Introduction
Breast cancer is among the most prevalent malignancies afflicting individuals in the United States. Approximately 80% of individuals with breast cancer have estrogen (E2) receptor-positive disease, with the majority of cases occurring in early-to postmenopausal women. Untreated E2-receptor-positive breast cancer exhibits a high affinity to metastasize to bone, liver, and lung. The skeleton is a favorable site of metastasis due to the high concentration and co-localization of growth factors; some of which are derived from the bone matrix, such as transforming growth factor-β (TGF-β), and others are directly secreted by the tumor, including parathyroid hormone-related peptide. By virtue of proximity, these growth factors are locally available to drive tumor progression. Suppressing tumor advancement through pharmacologic intervention and/or radiologic modalities is integral to prolonging patient lives. Clinical strategies to inhibit breast cancer progression include selective E2-receptor (ER) modulators (e.g., tamoxifen) for pre-menopausal patients and hormone deprivation therapies (e.g., aromatase inhibitors; AI) for post-menopausal patients. Third-generation AIs (e.g., letrozole) curtail peripheral estradiol synthesis from adrenal tissue by inhibiting aromatase production. These anti-E2 therapies are highly effective in decreasing tumor-associated mortality and increasing disease-free survival.

While the inhibition of estradiol synthesis by aromatase inhibitors (AIs) confers therapeutic efficacy in postmenopausal patients, this intervention concurrently promotes osteoclast-mediated bone resorption, thereby exacerbating skeletal degeneration and increasing the risk of secondary osteoporotic complications. Such bone loss not only fuels tumor growth but leads to increased release of TGF-β, which exacerbates muscle weakness through dysregulation of ryanodine receptor-1 (RyR-1), a critical Ca2+-dependent Ca2+ release channel within the sarcoplasmic reticulum. Denosumab, an antibody recognizing receptor-activator of nuclear-factor Kappa-β ligand (RANKL), is the first line of treatment prescribed to inhibit osteoclast-mediated bone resorption in postmenopausal women, as well as in patients with hormone receptor-positive breast cancer treated with AIs. Including bisphosphonates [e.g., zoledronic acid (ZA)] in the treatment regimen for breast cancer also effectively inhibits osteoclast activity and reduces cancer recurrence, improving survival in postmenopausal ER+ breast cancer patients. Bisphosphonates prevent adhesion of metastatic cells to bone and induce osteoclast apoptosis in vivo and in vitro. Thus, clinical challenges persist following treatment with AIs, including muscle weakness, progressive adiposity, and imbalanced bone remodeling. As these toxicities reduce quality of life, lead to poor patient adherence, and result in treatment discontinuation, innovative approaches are needed to reduce the musculoskeletal burden on these individuals to ultimately maintain adherence to AI-mediated therapies and to increase survival.

Daily physical activity is universally recommended as an alternative or supplemental therapy in standards-of-care to prevent bone and muscle loss, including in clinical cancer-treatment guidelines. Exercise is known to have an anabolic effects on bone and muscle while suppressing fat formation. Additionally, exercise reduces inflammation and regulates glucose metabolism. Despite its well-recognized benefits and patient willingness, breast cancer patients often find it difficult to participate in typical exercise regimens due to decreased energy and muscle strength. Cancer patients also experience heightened pain and frailty; thus, incorporating strenuous loads, such as those induced by exercise, may elevate fracture risk, therein precipitating the fractures that the exercise was intended to prevent.

As an alternative to the high-intensity loads generated by daily locomotion let alone physical activity or exercise regimens, low-intensity vibration (LIV) provides a low-magnitude (<1g acceleration, where 1g = Earth’s gravitational field or 9.8 m/s2) mechanical signal delivered through vertical (uniaxial) acceleration at a physiologically high frequency (~20–100 Hz) but low-magnitude force that is anabolic to bone in both translational and clinical studies. LIV does not require strenuous engagement by the patient, providing a fraction of the dynamic mechanical energy used in postural muscle contraction to maintain upright balance, which is itself a significant contributor to bone homeostasis and wanes with age or as a result of dystrophy or disease. We previously showed that LIV safely preserves bone and reduces tumor burden in murine ovarian and myeloma cancer models. Additionally, LIV inhibits adiposity in mice fed a high-fat diet and increases the density of muscle-bound satellite cells in ovariectomized mice. In mesenchymal stem cells, LIV induces osteogenic differentiation while attenuating adipogenesis. We’ve also shown that the timing of LIV dosing affects outcomes, where delivering multiple bouts of LIV separated by a rest period results in beneficial effects in both bone tissue and cells. We recently showed that direct application of LIV to MDA-MB-231 breast cancer cells resulted in decreased invasion and impaired the ability to secrete paracrine factors that promote osteoclast differentiation. In the clinic, LIV improved bone formation in young women with low bone mineral density and in adolescent patients with anorexia nervosa and cerebral palsy, as well as increased t-scores of childhood cancer survivors after 1 year of LIV treatment when compared with placebo-treated patients.

In the current study, we utilize mouse models to simulate the extreme bone loss, muscle weakness, and increased adiposity experienced by patients undergoing AI treatment to induce complete E2 deprivation. As LIV exhibits protective effects on bone and muscle in preclinical studies of high-fat diet and cancer models and in patients with cancer remission and suppressed E2 production, we hypothesized that daily administration of LIV would preserve critical bone and muscle endpoints in the context of treatment-induced E2 deprivation. LIV protects bone and muscle function from degradation and adverse accumulation of adiposity and short-chain fatty acids in younger mice. Coupling LIV with a standard anti-resorptive agent enhances bone strength more than either treatment alone and partially mitigates muscle fatigue in skeletally mature mice.

Results
Experiment I
The following experiment was designed to determine the effects of LIV in young, skeletally immature E2-deprived mice. Results from this experiment are detailed below.

LIV increased trabecular bone microarchitecture and histomorphometric markers. Trabecular bone volume fraction (Tb.BV/TV) and trabecular connectivity density (Tb.Conn.D) in L5 vertebrae of OVX/AI+LIV(y) mice were greater (p=0.048, p=0.017, respectively) than in OVX/AI(y) mice (Fig. 1a). (Data from micro-computed tomography (µCT) analyses are presented in their entirety in Supplementary Table S2). The number of osteoclasts (N.OC/BS) and osteoclast surface area (OC.S/BS) in OVX/AI+LIV(y) L5 vertebrae were lower (p=0.02, p=0.002, respectively) than in OVX/AI mice (Fig. 1b). The number of osteoblasts (OB/BS) in OVX/AI+LIV(y) mice was greater (p=0.01) than in OVX/AI(y) mice (Fig. 1b). Dynamic histomorphometric analysis of vertebral trabecular bone indicated that bone formation rate (BFR/BS) was greater (p=0.01) in OVX/AI+LIV(y) mice relative to OVX/AI(y) mice (Fig. 1c). Dynamic histomorphometry of femoral trabecular bone showed an increase (p=0.001) in bone formation rate (BFR/BS) and in mineral apposition rate (MAR) (p=0.003) of OVX/AI+LIV(y) mice relative to that of OVX/AI(y) mice (Fig. 1c). No differences were observed in N.OC/BS or OC.S/BS across the femoral metaphysis.

LIV increased muscle strength and function following E2 deprivation. From 8 weeks of age (after OVX) until euthanasia (32-week-old), longitudinal change in forelimb grip strength measurements relative to baseline readings were significantly greater (p=0.05) in OVX/AI+LIV(y) mice than in OVX/AI(y) mice (Fig. 1d). By 26 weeks after OVX, OVX/AI+LIV(y) mice exhibited greater (p=0.03) grip strength than OVX/AI(y) mice (Fig. 1d). Throughout the study, significant effects were observed as a result of treatment with LIV (p=0.05), time (p=0.0001), and between subjects (p=0.0001), and an interaction effect (p=0.0001) was also observed (Fig. 1d). Longitudinal DXA scans showed that OVX/AI+LIV(y) mice had greater (p=0.03) lean mass relative to OVX/AI(y) mice by week 28 (Fig. 1e). Muscle from LIV-treated mice had increased myofiber size with decreased spacing between individual fibers (Fig. 1f). Histological sections of OVX/AI+LIV(y) quadratus femoris showed increased myofiber cross-sectional area (CSA; p=0.034) by 24 weeks compared with OVX/AI(y) mice (Fig. 1f). No differences in the number of myofibers were observed between the two groups (Fig. 1f). Tetanic stimulation of the EDL at euthanasia exhibited an interaction (p=0.01), time (p=0.001), and subject-related (p=0.001) effect on muscle fatigue between OVX/AI(y) and OVX/AI+LIV(y) mice (Supplementary Fig. 2). Although there were time and subject-related effects, contractility of the EDL did not exhibit any differences in muscle-specific force between OVX/AI(y) and OVX/AI+LIV(y) mice (Supplementary Fig. 2).

LIV suppressed body weight gain, adiposity, and increased glucose metabolism associated with E2 deprivation. Longitudinal measurements of body weight in young mice in experiment I showed no differences between the OVX/AI(y) and OVX/AI+LIV(y) groups (Fig. 1g). However, linear regression analysis from week 14 to week 28 after OVX showed a significant difference between slopes, with OVX/AI+LIV(y) mice gaining weight at half the rate of OVX/AI(y) mice (Fig. 1g). OVX/AI+LIV(y) mice had lower (p=0.0001) fat mass relative to OVX/AI(y) mice by week 28 (Fig. 1h). The weight of the fat pad harvested from the perigonadal depots at euthanasia was significantly (p=0.05) lower in OVX/AI+LIV(y) mice than in OVX/AI(y) mice after 28 weeks of treatment with LIV (Fig. 1i). Serum leptin exhibited a decreasing trend in OVX/AI+LIV(y) mice at 24 weeks post-OVX that, when compared with total fat mass at euthanasia, resulted in a significant correlation (R2=0.827, p=0.001; Fig. 1j). Marrow adipose tissue quantified from the tibiae of younger mice was 6.5% lower in OVX/AI+LIV(y) mice than in OVX/AI(y) mice when normalized to medullary cavity volume, but this was not significantly different (Fig. 1k). Blood glucose readings in young mice were recorded using a glucometer at 0, 15, 30, 60, and 120 min from blood draw at the tail vein. OVX/AI+LIV(y) mice exhibited a trend of lower serum blood glucose (p=0.09) than OVX/AI(y) mice, as quantified by the area under the curve (Fig. 1l).

Analysis of serum fatty acid oxidation intermediates (acylcarnitines) in young mice prior to and 24 weeks after commencing E2 deprivation identified significant increases in short-chain and medium-chain acylcarnitines. Specifically, 24 weeks of E2 deprivation induced 61% greater (p=0.0002) circulating short-chain acylcarnitine (2–7 carbon chains; C2–C7) concentrations in OVX/AI(y) compared to baseline (Supplementary Fig. 3c), which increased by just 26% (p=0.025) from baseline mice when compared to OVX/AI+LIV(y). Among the C2–C7 acylcarnitine groups, acetylcarnitine (C2), propionylcarnitine (C3), butyrylcarnitine/isobutyrylcarnitine (C4/Ci4), and hexanoylcarnitine (C6) contributed to the increased concentration in OVX/AI observed at 24 weeks post-E2 deprivation relative to baseline in experiment I (Supplementary Fig. 3c). C2 species in BL mice significantly (p=0.0005) increased in OVX/AI(y) but did not differ as compared to OVX/AI+LIV(y), while OVX/AI(y) decreased by 24% (p=0.0154) in OVX/AI+LIV(y) (Supplementary Fig. 3c). C3 species significantly (p=0.0013) increased in OVX/AI+LIV(y) mice relative to baseline mice but did not differ compared to OVX/AI(y) mice (Supplementary Fig. 3c). Both OVX/AI(y) and OVX/AI+LIV(y) mice increased (p=0.00005, p=0.000067, respectively) C4/Ci4 species as compared to baseline mice (Supplementary Fig. 3c). C6 species exhibited 9x (p=0.00004) and 5.5x increases (p=0.00117) in OVX/AI(y) and OVX/AI+LIV(y), respectively, as compared to baseline mice (Supplementary Fig. 3c). by Medium-chain (C8–C14) acylcarnitines significantly increased (p=0.006) after 28 weeks of E2 deprivation relative to baseline mice, but these levels did not decrease in OVX/AI+LIV(y) relative to baseline nor OVX/AI(y) mice (Supplementary Fig. 3d). Relative to baseline mice, C14:2 (tetradecadienoylcarnitine) significantly increased in OVX/AI(y) and OVX/AI+LIV(y) (p=0.017, p=0.002, respectively), while no differences were observed between either group at 28 w. No differences in long-chain (C16–C22) species were observed (Supplementary Fig. 3e).

E2 deprivation confirmed from uterine weights. Complete E2 deprivation was performed by ovariectomy surgery and maintained by an aromatase inhibitor. At euthanasia, uterine weights from either group, both E2-deprived, were not statistically different and were comparable to our reported prior data (Supplementary Fig. 1).

Experiment II
This experiment was designed to determine the effects of LIV in combination with zoledronic acid in skeletally mature E2-deprived mice. Results from this experiment are detailed below.

Bone mineral density and cortical and trabecular microarchitecture are enhanced when LIV is administered in combination with ZA. Data from µCT analyses are presented in their entirety in Supplementary Table S2. DXA-generated whole-body BMD in the older mice subject to E2 deprivation (OVX/AI) was decreased (p<0.00001) compared to SH mice (Fig. 2a). OVX/AI+LIV mice exhibited no differences relative to OVX/AI mice (Fig. 2a). OVX/AI+ZA mice exhibited increased (p<0.00001) whole-body BMD relative to OVX/AI mice but lower whole-body BMD (p=0.0029) than SH mice (Fig. 2a). However, whole-body BMD was increased (p<0.00001) in OVX/AI+LIV+ZA mice compared with OVX/AI mice, an increase when compared with SH mice (Fig. 2a). BMD analysis of the lumbar spine in the skeletally mature mice using DXA exhibited time, group, and interaction effects (p=0.0001; Fig. 2b). OVX/AI and OVX/AI+LIV mice exhibited a reduction (p=0.0001) in lumbar BMD relative to SH mice as early as 4 weeks post-OVX and this effect continued throughout the study (Fig. 2b). However, at 4 weeks post-OVX, lumbar BMD in OVX/AI+ZA mice were lower (p=0.01) than in SH mice (Fig. 2b). Lumbar BMD in OVX/AI+ZA mice was greater compared to OVX/AI and OVX/AI+LIV mice (Fig. 2b). Additionally, OVX/AI+LIV+ZA mice exhibited no differences in lumbar BMD 4 weeks post-OVX relative to SH mice, which was greater (p=0.0001) than the difference observed between OVX/AI and OVX/AI+LIV mice (Fig. 2b). By 15 weeks after OVX, BMD of the lumbar vertebrae in OVX-AI+ZA mice were higher (p=0.000039) than in OVX/AI mice but only mildly greater (p=0.01) than in OVX/AI+LIV mice (Fig. 2b). However, lumbar BMD of OVX/AI+LIV+ZA mice were greater (p<0.00001) than in OVX/AI mice at 15 weeks post-OVX (Fig. 2b).

L5 vertebrae in OVX/AI and OVX/AI+LIV mice exhibited a significant (p=0.0034, p=0.0039, respectively) decrease in Tb.BV/TV compared with those of SH mice by 15 weeks post-OVX (Fig. 2c). A trend of increased BV/TV was observed in OVX/AI+LIV+ZA mice relative to SH. In OVX/AI+ZA and OVX/AI+LIV+ZA mice, Tb.BV/TV increased (p=0.0001, p<0.000001, respectively) compared with untreated OVX/AI mice (Fig. 2c). Interaction (p=0.0001) and time (p=0.01) effects were found between groups throughout the study as a function of percentage change in L5 Tb.BV/TV (Fig. 2c). Untreated OVX/AI mice had decreased Tb.BV/TV (p=0.0001) relative to SH mice (Fig. 2c). OVX/AI+ZA mice increased (p=0.0001) in Tb.BV/TV relative to OVX/AI mice, and in OVX/AI+LIV mice, Tb.BV/TV was greater (p=0.0001) than in untreated OVX/AI mice; a similar effect was also observed (p=0.01) in OVX/AI+LIV+ZA mice (p<0.000001) (Fig. 2c). These findings were paralleled by similar differences in Tb.N and Tb.Th, each showing decreases (p=0.05, p=0.01, respectively) in OVX/AI mice relative to SH mice and increases (p=0.05, p=0.001, respectively) in mice treated with ZA (Supplementary Table S2). OVX/AI+LIV+ZA mice exhibited increased Tb.N and Tb.Th (p=0.001, p=0.0001, respectively) compared with OVX/AI mice, as well as increases relative to OVX/AI+ZA mice (p=0.05, p=0.05, respectively; Supplementary Table S2).

Femoral Ct.BA/TA exhibited significant interaction, time, and treatment (ZA and LIV)-related effects (p=0.0001) in older mice (Supplementary Table S2). Ct.BA/TA decreased (p=0.0001) in OVX/AI mice compared to SH mice by 15 weeks after OVX (Supplementary Table S2). Although OVX/AI+LIV mice did not show improved Ct.BA/TA, Ct.BA/TA was increased (p=0.001) in OVX/AI+ZA mice (Supplementary Table S2). OVX/AI+ZA mice had increased (p=0.0001) Ct.BA/TA, and OVX/AI+LIV mice had increased (p=0.0001) Ct.BA/TA as well (Supplementary Table S2). Ct.BA/TA in OVX/AI+LIV+ZA mice increased (p=0.0001) compared with that of OVX/AI mice, thereby improving on treatment with ZA alone (Supplementary Table S2). Corresponding decreases in Ct.Th (p=0.0001) and Ct.pMOI (p=0.05) were observed in OVX/AI mice compared with SH mice (Supplementary Table S2). OVX/AI+ZA mice showed increased (p=0.001) Ct.Th relative to OVX/AI mice, and Ct.Th increased further (p=0.0001) in OVX/AI+LIV+ZA mice (Supplementary Table S2). Ct.pMOI was not statistically different in OVX/AI+ZA or OVX/AI+LIV+ZA mice relative to OVX/AI or SH mice (Supplementary Table S2).

Decreased bone formation and mineralization in E2-deprived mice due to ZA is attenuated in LIV-treated mice. dLS was reduced in OVX/AI+LIV and OVX/AI+ZA mice (p=0.03, p=0.0018, respectively) compared to SH mice (Fig. 2d); however, dLS of OVX/AI+LIV+ZA mice, although not statistically different from SH mice, was 2 times greater than that observed in OVX/AI+ZA mice (Fig. 2d). BFR/BS was lower (p=0.0062) in the L5 of OVX/AI+ZA mice than in SH mice, while OVX/AI+LIV+ZA mice exhibited trends of lower BFR/BS than SH mice.

Mineralizing surface area (MS/BS) was significantly reduced (p=0.002) in OVX/AI+ZA mice relative to SH mice, while OVX/AI+LIV+ZA mice exhibited trends of lower MS/BS compared to SH mice (Fig. 2d). OB/BS was significantly decreased (p=0.008) in OVX/AI+ZA mice compared with SH mice and was also lower (p=0.027) compared to OVX/AI+LIV mice (Fig. 2d). No differences were observed in Ir.L.Wi., OC.S/BS, or N.OC/BS (Fig. 2d). Sections stained with tartrate-resistant acid phosphatase exhibited no differences in the number of osteoclasts or osteoclast surface area between groups; however, there were fewer osteoclasts in OVX/AI+LIV mice at a similar proportion to that observed with OVX/AI+ZA mice relative to untreated OVX/AI mice.

The mechanical properties of vertebral and diaphyseal bone degraded by E2 deprivation are normalized with ZA but improved further when combined with LIV. Mechanical testing of bone data are presented in their entirety in Supplementary Table S3. Compression testing of L5 vertebrae of OVX/AI and OVX/AI+LIV mice exhibited decreased (p=0.0022, p=0.011, respectively) ultimate force compared to SH mice (Fig. 2e). Ultimate force increased in OVX/AI+ZA mice L5 vertebrae (p=0.0001) compared to untreated OVX/AI mice (Fig. 2e). Ultimate force of L5 in OVX/AI+LIV+ZA mice improved (p<0.00001) relative to OVX/AI mice, a 43% increase compared with OVX/AI+ZA (Fig. 2e). In comparison to OVX/AI+LIV mice, OVX/AI+ZA and OVX/AI+LIV+ZA mice showed greater ultimate force (p=0.0009, p=0.00007, respectively).

As measured by 3-point bending, femora of OVX/AI mice exhibited a reduction (p=0.038) in yield force compared to SH mice, but which was increased (p=0.001) in OVX/AI+ZA and OVX/AI+LIV+ZA mice relative to OVX/AI mice (p=0.043, p=0.0038, respectively) (Fig. 2f). Yield force in OVX/AI+LIV+ZA mice improved (p=0.012) relative to OVX/AI mice (Fig. 2f). Similarly, stiffness was also reduced (p=0.002) in OVX/AI mice relative to SH mice, but no significant differences were observed in OVX/AI+LIV, OVX/AI+ZA, or OVX/AI+LIV+ZA mice relative to either SH or OVX/AI mice (Fig. 2f). Tissue-level properties indicated similar decreases in yield stress, ultimate stress, resilience, and toughness in OVX/AI mice compared with SH mice (Fig. 2f). Improvements (p=0.007) were observed in OVX/AI+ZA mice compared with OVX/AI mice, while nonsignificant increases were observed in resilience (47%), toughness (38%), and yield stress (18%) in OVX/AI+LIV mice relative to untreated OVX/AI mice (Fig. 2e and Supplementary Table S2). OVX/AI+LIV+ZA mice exhibited increased resilience (p=0.0001), toughness (p=0.036), yield stress (p=0.04), and ultimate stress (p=0.02) compared with OVX/AI mice (Fig. 2e and Supplementary Table S2).

Muscle function improved following either LIV or ZA treatment. Peak percentage change in grip strength was observed 4 weeks following sham-OVX in the SH mice (Fig. 3a). Although all groups exhibited increased grip strength throughout the initial 2 weeks after surgery (OVX), OVX/AI+LIV and OVX/AI+LIV+ZA mice exhibited the sharpest increase among the groups, peaking at an average 125 g of force (Fig. 3a), but these data were not significant. At the initial 2 weeks post-surgery, OVX/AI+LIV mice had greater grip strength (p=0.05) than OVX/AI mice, and OVX/AI+LIV+ZA mice trended toward increased grip strength (p=0.06) relative to OVX/AI mice (Fig. 3a). At 5 weeks post-OVX, grip strength began to decline in all groups (Fig. 3a). By week 9, OVX/AI+LIV mice exhibited greater grip strength (p=0.05) than did OVX/AI mice (Fig. 3a). This margin increased (p=0.007) by week 11, at which point OVX/AI+LIV+ZA mice also exhibited increased grip strength (p=0.03) relative to OVX/AI mice (Fig. 3a). Grip strength of the OVX/AI+LIV mice remained consistently higher than all groups throughout the course of the study following E2 deprivation (Fig. 3a). Throughout the post-OVX period, an effect (p=0.01) was observed between groups (Fig. 3a).

Percentage decrease from baseline to the first tetanus (30 Hz stimulation) during ex vivo muscle contractility of the EDL yielded significant improvement in fatigue in OVX/AI+LIV mice (p=0.028) and OVX/AI+LIV+ZA mice (p=0.041) relative to SH mice (Fig. 3b). At the next frequency (40 Hz) in the stimulation protocol, OVX/AI+LIV and OVX/AI+LIV+ZA mice exhibited less muscle fatigue (p=0.0065, p=0.0017, respectively) than SH mice (Fig. 3b). Additionally, OVX/AI+ZA mice had reduced muscle fatigue (p=0.045) in contrast to SH mice; however, none of the groups differed from OVX/AI mice (Fig. 3b). At 50 Hz stimulation, treatment with LIV and LIV+ZA in OVX/AI mice both led to reduced muscle fatigue (p=0.0071, p=0.0032, respectively) than that seen in SH mice, whereas OVX/AI+ZA mice had 10% more muscle fatigue (p=0.00004) than that observed in SH mice (Fig. 3b). At this stimulation frequency, OVX/AI+ZA mice had less muscle fatigue (p=0.012) than did untreated OVX/AI mice (Fig. 3b). No differences were observed in muscle-specific force at any of the frequencies used to stimulate the muscle (Fig. 3c). Untreated OVX/AI mice showed lower myofiber CSA compared with SH mice (Fig. 3d); however, no significant differences in myofiber CSA or number of myofibers were observed in the skeletally mature mice (Fig. 3e, f, respectively).

E2 deprivation increased perigonadal and bone marrow adipose accrual, whereas either LIV or ZA alone reduced body weight and decreased adiposity. DXA analysis indicated significant increases in % fat mass of OVX/AI mice (p=0.01) and OVX/AI+ZA mice (p=0.02) relative to SH mice at 9 weeks post-OVX (Fig. 4a). By 14 weeks post-OVX, all E2-deprived mice had greater (p=0.0001) percent fat mass relative to SH mice (Fig. 4a). Neither LIV treatment nor ZA alone or in combination significantly altered percent fat mass compared to E2-deprived mice (Fig. 4a). No differences were observed in percent lean mass between groups. A significant effect (p=0.0001) was found in all groups with respect to time over the course of the study (Fig. 4a). Throughout experiment II (older cohort), body weight increased the least in SH mice, and OVX/AI mice had significantly greater (p=0.01) weight than SH mice (Fig. 4b). OVX/AI+LIV and OVX/AI+LIV+ZA mice exhibited greater (p=0.0001) body weight than SH mice by 20 weeks post-OVX (Fig. 4b). Body weight of OVX/AI+ZA mice was lower (p=0.05) than that of OVX/AI+LIV+ZA mice by the same endpoint (Fig. 4b). Taken together, these results indicated that young mice treated with LIV had a significantly slower rate of weight and fat gain compared with their untreated counterparts, but older E2-deprived mice gained significantly more weight than did their E2-replete, counterparts.

At euthanasia, both OVX/AI and OVX/AI+LIV+ZA mice had significantly more gonadal fat pad tissue (p=0.02) than did SH mice (Fig. 4e). OVX/AI+LIV mice had more gonadal fat pad tissue than SH mice, but fat pad tissue did not differ between OVX/AI-LIV mice and untreated OVX/AI mice (Fig. 4c). None of the treatment groups in experiment II significantly differed from OVX/AI mice in terms of gonadal fat pad tissue weight (Fig. 4c).

Marrow adipose tissue normalized to medullary cavity volume, as quantified by µCT, was greater (p=0.0001) in OVX/AI mice than in SH mice (Fig. 4d). OVX/AI+LIV mouse tibiae were not significantly different from those of OVX/AI mice, nor were those of OVX/AI+ZA mice (Fig. 4d). Marrow adipose tissue decreased in OVX/AI+LIV+ZA mice relative to OVX/AI mice, by 36% compared with SH mice, but this was not significant (Fig. 4d).

Ad.Ar./Ma.Ar. in the tibial metaphysis was increased (p=0.01) in OVX/AI mice relative to SH mice, and these levels were decreased by 15% in OVX/AI+LIV mice (Fig. 4e). Ad.Ar./Ma.Ar. was decreased in OVX/AI+ZA mice by 10% relative to OVX/AI mice, and a nonsignificant reduction was also observed in OVX/AI+LIV+ZA mice (p=0.08; Fig. 4e). N.Ad./Ma.Ar. was increased (p=0.04) in OVX/AI mice relative to SH mice (Fig. 4f). Treatment with LIV resulted in a 33% reduction compared with OVX/AI mice, while N.Ad./Ma.Ar. in OVX/AI+ZA mice decreased by 26% (Fig. 4f). However, a 62% reduction was observed in OVX/AI+LIV+ZA mice as compared to OVX/AI mice (Fig. 4f).

E2 deprivation confirmed from uterine weights. Complete E2 deprivation was performed by ovariectomy surgery. Uterine weights measured at euthanasia in all OVX/AI experimental groups weighed significantly (p≤0.0001) less than SH mice (Supplementary Fig. 1) and were not statistically different from uterine weights from Experiment I.

Methods
All experimental procedures were approved by the Institutional Animal Care and Use Committee guidelines at Indiana University and complied with all relevant ethical regulations in strict accordance with the guidelines of the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals and the ARRIVE guidelines.

Study design
The overarching objective of this study was intended to assess the suitability and impact of low-magnitude mechanical signals, administered using low-intensity vibrations (LIV), either alone or in combination with an anti-resorptive agent, on musculoskeletal parameters and systemic or regional adiposity in a murine model of complete E2 deprivation. Our initial aim was to contrast the effects of daily exposure to LIV on mice experiencing aggressive bone and muscle degeneration and accretion of adiposity in skeletally immature mice while addressing effects on the quantity and functionality of these tissues. Following this aim, complete E2 deprivation was modeled in skeletally mature mice. All C57BL/6 mice underwent complete E2 deprivation through surgical ovariectomy and were maintained through daily letrozole injection to ensure inhibition of both ovarian and adrenal androgen sources, as this model had been previously validated by our group and translates clinically. µCT analysis and 3-point and compressive mechanical testing of femora and vertebrae were performed to quantify bone microarchitecture and resistance to fracture as a measure of bone quantity and quality. Body composition was measured from DXA scans. Histological analyses were performed to visualize and measure bone histomorphometry and muscle morphology. Longitudinal in vivo measurements of bone mineral density (BMD, g/cm2), % whole-body fat mass (grams), and % whole-body lean mass (grams) were collected via DXA scanning (Piximus2, Lunar Corporation, Madison, WI, USA) to determine body composition. Prior to each scan, mice were anesthetized with an intraperitoneal injection of a ketamine/xylene cocktail (100 mg/kg + 10 mg/kg). Whole-body regions of interest excluded the head and tail. DXA analysis of the lumbar spine was performed using a region of interest situated above the sacral spine and extending superior to L1.

Animal model
C57BL/6 mice (Envigo, Indianapolis, IN, USA) were monitored daily and maintained in accordance with the Institutional Animal Care and Use Committee guidelines at Indiana University and the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. Sample sizes were determined based on prior studies and power analyses (80% power, α=0.05), an n=10 was calculated for the preliminary experiment (I) a total of 20 mice per group was allocated to account for various downstream assays. Mice were housed five per cage at standard room conditions of 22 °C and 40–60% humidity and allowed ad libitum access to standard diet-chow (catalog #2018SX, Envigo) and water. All mice were monitored daily for signs of ill health, and humane endpoints were applied in accordance with institutional guidelines. A period of 1 week was allotted at the beginning of the study to acclimatize the mice to the handling procedures and LIV and mock-LIV treatment protocol. Group designations are detailed in Supplementary Table S1. For experiment I, 4-week-old female C57BL/6 mice were divided into two groups (n=10/group): a. ovariectomized (OVX) and treated with AI only [OVX/AI(y)] and b. OVX and treated with AI and LIV [OVX/AI+LIV(y)]. For experiment II, 15-week-old female C57BL/6 mice were divided into five groups (n=20/group): a. sham-ovariectomized age-matched controls given saline vehicle (SH); b. OVX and treated with AI only (OVX/AI); c. OVX and treated with AI and LIV (OVX/AI+LIV); d. OVX and treated with AI and ZA (OVX/AI+ZA); and e. OVX and treated with AI, LIV, and ZA (OVX/AI+LIV+ZA).

Complete E2 deprivation
Sham-surgery and vehicle (phosphate-buffered saline) injection were performed on SH mice, and all other treatment groups underwent OVX supplemented with subcutaneous injection of AI letrozole (Novartis International AG, Basel, Switzerland) administered at a dose of 5 µg/kg per day, 5 times per week. Serum β-estradiol was significantly decreased (p=0.004) in 28-week-old OVX/AI(y) mice and (p=0.0002) in 28-week-old OVX/AI+LIV(y) mice relative to 4-week-old mice at baseline. No differences in serum β-estradiol were detected between OVX/AI(y) and OVX/AI+LIV(y) mice at 28 weeks of age. This model, characterized in experiment I, was used in a similar fashion for experiment II.

Bisphosphonate administration
Low-dose (2.5 ng/kg) ZA (Zometa, Novartis) was subcutaneously injected in the skeletally mature mice (OVX/AI+ZA and OVX/AI+LIV+ZA groups) from experiment II once per week commencing the day following OVX surgery.

Mechanical loading protocol
Mechanical loading (LIV and mock-LIV) regimens commenced at 4 weeks of age for the younger cohort (experiment I) and at 16 weeks of age for the older cohort (experiment II). Mice assigned to the mechanical loading regimen were subjected to LIV (0.7±0.025g, 90 Hz sine wave) for 20 min per treatment, administered once per day, 5 days per week over 28 weeks (experiment I), or twice per day, 5 days per week over 24 weeks (experiment II). OVX/AI(y) mice underwent identical handling and loading protocols as OVX/AI+LIV(y) mice but without activation of the platform (mock-LIV). Daily loading consisted of placing mice into individual compartments within a larger container placed on a vertically oscillating platform (modified from Marodyne Medical, LLC) to administer the LIV signal. Displacements required to produce accelerations at 90 Hz induce membrane deformations of approximately 5 microstrain. The lead investigator was not blinded to the experimental groups during the LIV treatment windows.

Tissue harvest and preservation
At the termination of the study, each mouse was anesthetized using isoflurane inhalation. Euthanasia was confirmed by cervical dislocation. Whole blood collected via cardiac puncture was stored in serum-separation tubes, with serum aliquots stored at −80 °C. Bones for histologic processing, including the right femora and tibiae, were stripped of muscle and residual soft tissue and fixed in 10% neutral buffered formalin, which was replaced at 48 h with 70% ethanol. At euthanasia, lumbar vertebrae and femora harvested for mechanical testing were wrapped in saline-soaked gauze and frozen at −20 °C. The quadratus femoris muscle was formalin-fixed for 48 h following euthanasia and then transferred to 70% ethanol and preserved for downstream processing.

Micro-computed tomography (µCT)
In vivo and ex vivo scans were performed to quantify bone microarchitecture and visualize reconstructed 3-dimensional representations. Distal femora and L5 vertebrae were measured and evaluated ex vivo using X-ray µCT (μCT40, OpenVMS, Scanco Medical, Wayne, PA, USA). X-ray parameters were set to the following: in vivo source voltage E=55 kVp, current=145 µA, integration time=300 ms, and voxel size=19 µm; ex vivo source voltage E=55 kVp, current=145 µA, integration time=300 ms, and voxel size=10 µm. Cancellous (trabecular) bone parameters were identified using the following nomenclature: Tb.BV/TV, Tb.Conn.D, Tb.N, trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), trabecular BMD (Tb.BMD), and trabecular structure model index (Tb.SMI). Cortical bone parameters were identified using the following nomenclature: cortical bone area fraction (Ct.BA/TA), cortical thickness (Ct.Th), and cortical polar moment of inertia (Ct.pMOI). Starting 700 µm proximal to the femoral growth plate, 1000 µm of metaphyseal trabecular bone was evaluated. A 500-µm uniform length of trabecular bone (excluding the growth plate) was evaluated across the L5 vertebral bodies; 500 µm of cortical bone was quantified about the femoral mid-diaphysis.

Mechanical testing of bones
Mechanical testing was performed using a 3-point bending jig to quantify both structural and material-level cortical bone properties at the femoral mid-diaphysis, and vertical compression jigs were used to quantify the structural properties resisting fracture in the lumbar vertebrae. Force and displacement data were recorded (R controller, Test Resources, Shakopee, MN, USA), and structural and material properties were derived from force curves using a customized MATLAB (MathWorks, Natick, MA, USA) script.

Spines were thawed, then the L5 vertebrae were isolated using a scalpel and cleaned of intervertebral disks and cartilaginous tissue from proximal and distal ends. Pedicles and spinous processes were removed, leaving only the vertebral body. Sample vertebrae were loaded along the vertical axis with 2 N of preload force between platens using 11 lb-f load cells. Position-controlled rate of compression was applied until failure. Samples that slipped during compression were omitted from the dataset.

Femora were thawed, excess soft tissue was removed, and the femora were scanned using µCT to obtain the CSA. For 3-point mechanical testing of the femora, the distance between the lower supports (Δx=9 mm) was maintained throughout the analyses, while the upper support was centered between the bottom supports. 150 lb-f load cells were used for testing with 1 N of preload force, and the position-controlled rate was applied to each sample (femora were positioned anterior side facing up) until failure.

Bone histology/histomorphometry
The lengths of long bones were measured ex vivo using digital calipers. Formalin-fixed bone specimens for paraffin embedding and osmium tetroxide staining were decalcified in ethylenediaminetetraacetic acid (10%; pH=7.32) for 2 weeks. 5 µm paraffin-embedded sagittal cross sections were stained with hematoxylin and eosin. PMMA-embedded intact lumbar vertebrae were sectioned (4.5 µm) and were subsequently either left unstained (i.e., dynamic histomorphometry) or stained with tartrate-resistant acid phosphatase to quantify osteoclasts or with von Kossa-McNeil to quantify osteoblasts. Bone analysis software (v.18.1, BIOQUANT Osteo, Bioquant Image Analysis Corporation, Nashville, TN, USA) was used to analyze the results.

Forelimb grip strength
Muscle function was quantified with the use of a grip strength meter (Bioseb, Pinellas Park, FL, USA) to report maximum applied force (grams). Prior to each pass, the researcher suspended mice individually by their tails, placing forelimbs within reaching distance of the front of the mesh grid. A constant horizontal pulling force was applied until the mice released their grip from the mesh. Triplicate readings per mouse were averaged, and longitudinal measurements were taken throughout the study.

Muscle histology
Paraffin-embedded histologic sections (4.5 µm) of the quadratus femoris were stained with hematoxylin and eosin. Dedicated software (v.18.1 BIOQUANT Osteo) was used to acquire 5 fields-of-view at 10× magnification and then used to quantify myofiber CSA by contouring each myofiber within the field of view, thresholding out the space between the fibers, and normalizing to the total tissue area.

Muscle-specific force/muscle fatigue
Ex vivo contractility of the extensordigitorum longus (EDL) muscle was performed via tetanic stimulation as previously described. Briefly, whole EDL muscles were dissected from hindlimbs and placed in Tyrode buffer to maintain Ca2+ potential. Muscles were mounted to force transducers (Aurora Scientific, Ontario, Canada) using stainless steel hooks tied to the proximal and distal tendons, while remaining hydrated in a stimulation chamber containing O2/CO2 (95/5%) bubbled into Tyrode buffer. The excised muscle was stimulated to contract using a supramaximal stimulus between two platinum electrodes. Force–frequency and fatigue–frequency relationships were determined by triggering contraction using incremental stimulation frequencies (specific force: 0.5 ms pulses at 1–150 Hz for 350 ms at maximal voltage; fatigue: 0.5 ms pulses at 1–50 Hz for 350 ms at maximal voltage). Data were collected using proprietary “Dynamic Muscle Control/Data Acquisition” and “Dynamic Muscle Control Data Analysis” programs (Aurora Scientific). Wet tissue weights and lengths of gastrocnemius, EDL, soleus, and tibialis anterior muscles were recorded with digital calipers at euthanasia. For quantification of the specific force and fatigue of the EDL, the absolute force was normalized to muscle size and calculated by dividing muscle weight by muscle length using a muscle density constant (1.056 kg/m−3). Investigators performing the measurements were blinded to the treatment groups.

Marrow adipose histology
Histologic sections (20×, hematoxylin and eosin) of the distal tibial metaphysis were analyzed starting from the growth plate for 2000 µm distally to quantify the total adipocyte volume encased within a given marrow volume (Ad.Ar./Ma.Ar.) and total number of adipocytes within this space (N.Ad./Ma.Ar.). Briefly, the region of interest across the marrow space was contoured, followed by binary thresholding of the marrow and adipocytes independently, which were each quantified using Osteo (v.19.2).

Osmium tetroxide staining of marrow adipose tissue
Marrow adipose tissue was quantified as described previously. Formalin-fixed tibiae were scanned via µCT. Bones were decalcified in ethylenediaminetetraacetic acid (pH=7.32) for 10 days, rinsed with tap water, and transferred to Eppendorf tubes. A 1:1 ratio of osmium tetroxide (OsO4) to sodium bicarbonate mixture was added to each sample, allowing the OsO4 to permeate the marrow for 48 h. After discarding the waste solution, bones were washed with tap water again and stored in phosphate-buffered saline at 4 °C. Samples were scanned using µCT imaging (E=55 kVa, I=72 µA, voxel size=12.5 µm). Values were produced by thresholding tissue that absorbed OsO4 and normalizing this value to the medullary cavity volume across 6000 µm from the epiphysis to the inferior tibiofibular joint.

Immunoassays
Serum was collected at the termination of the study and stored at −80 °C. Thawed samples were used to quantify leptin (Quantikine, catalog #MOB00, Minneapolis, MN, USA) as analyzed by enzyme-linked immunosorbent assay per manufacturer guidelines.

Serum metabolite analyses
Serum isolated from whole blood at baseline (n=10) and euthanasia (OVX/AI(y): n=5, OVX/AI+LIV(y): n=10) was used to quantify fatty acid metabolite intermediates (acylcarnitines). Acylcarnitines were analyzed by flow injection electrospray ionization tandem mass spectrometry and quantified by isotope or pseudo-isotope dilution using methods described previously. Tissue homogenates were prepared at 50 mg/ml in 50% acetonitrile/0.3% formic acid, and serum samples were spiked with a cocktail of heavy-isotope internal standards (Cambridge Isotope Laboratories, MA, USA; CDN Isotopes, Canada) and deproteinated with methanol. The supernatants were dried and esterified with acidified methanol. Mass spectra for acylcarnitines were obtained in the positive mode using a precursor ion scan of m/z 99. The spectra were acquired in a multi-channel analyzer (MCA) mode to improve signal-to-noise. The data were generated using a Waters Xevo TQD mass spectrometer equipped with an AcquityTM UPLC system and a data system controlled by MassLynx 4.1 operating system (Waters, Milford, MA). Ion ratios of analyte to respective internal standard computed from centroided spectra were converted to concentrations using calibrators constructed from authentic aliphatic acylcarnitines (Sigma, MO, USA; Larodan Sweden) and dialyzed fetal bovine serum (Sigma, MO, USA). Acylcarnitine concentrations were analyzed using Metaboanalyst (v.2.0, McGill, Quebec, Canada) and run in the statistical package R (v.2.14.0). Initial unsupervised evaluation using principal component analysis identified the mouse strain as the primary source of variance; therefore, the metabolites were mean-centered per strain after log transformation of concentrations, resulting in a Gaussian distribution. All metabolite analyses were performed blinded to group, experimental treatment, and all other identifying data. After acylcarnitine values were determined, metabolomics analysis of grouped data was performed with the user blinded to all group designations. All metabolomics data has been uploaded to an online repository, Metabolomics Workbench.

Glucose tolerance testing
Mice aged to 26 weeks were fasted overnight and intraperitoneally injected the following morning with a bolus of glucose (20% glucose solution). Subsequent blood glucose samples drawn from the tail vein were read using a glucometer at 0, 15, 30, 60, and 120 min intervals. Serum glucose was not measured in the older mice in order to minimize potential stress.

Statistical analyses
All graphical data are represented by means±SEM. Repeated measures analysis of variance was performed for longitudinal measurements of body weight, grip strength, body composition, and bone mineral density as measured by DXA, muscle contractility, and µCT, followed by Tukey’s HSD procedure to adjust for multiple comparisons among groups (Prism, GraphPad Software v.7.0c, San Diego, CA, USA). Pearson correlations were performed to compare total adipose tissue mass with serum leptin and reported along with 95% confidence intervals. The student’s two-tailed t-test was performed on single-timepoint data to compare groups. Single-timepoint analyses of these groups were quantified by one-way ANOVA and Tukey’s post hoc procedure. A moderated t-statistic model (SAM) was used to identify metabolites that differed between groups at a false discovery rate <5% with Fisher’s least significant difference used for post-test comparisons.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this study the authors have tested if the addition of low-intensity vibrations (LIV) to zoledronic acid would prevent the bone and muscle loss and adiposity that comes with complete estrogen deprivation. This is clinically relevant considering how common complete estrogen deprivation is in women undergoing breast cancer treatment.\nIn young estrogen deprived mice not treated with zoledronic acid there seems to be a potent effect of LIV to improve bone, adipose and muscle parameters. In older mice however this effect is blunted and the addition of LIV to zoledronic acid did not significantly improve any or few of the measured parameters. However, many of the diagrams and tables are to tiny or blurry to interpret and the statistics is not clearly described, se comments below.\nThe techniques are relevant and improving the presentation of the data would make it easier for the reader to interpret the importance of the results.\nSpecific comments:\n1. Please show serum E2 levels in figure. Sometimes E2-levels in mice can be difficult to determine by ELISA as used by the authors and a more reliable indicator of E2-depletion would be uterus weight, please provide if possible.\n2. For the figures please add in each figure legend:\n-The number of animals (n)\n-The statistics used (if for example 2-way ANOVA, specify the factors tested and between which groups post-hoc test has been done).\n-Make graphs larger where needed, for example figure 2c and explain where the p-value comes from, is it comparison between the two groups at a certain time-point or something else?\n-Remove all personal comments on whether the data is increased or decreased especially if this is not supported by a significant difference and even if it is, I think these type of comments should be kept to a minimum in the figure legends.\n3.In the text it says that the p-value for figure 1A is 0.047 but in table 2 it says 0.048?\n4. Please refer to a figure or table when data is mentioned in the text or write data not shown (if this is accepted by the journal otherwise remove these sentences and only report data that is presented).\n5. The scores plot and the heat map in supplemental figure 1 and 2 is identical?", "reviewer_2": "Dear Authors\nCongratulations. The subject of your study is highly relevant. The findings obtained in your study bring important contributions to the field related to the effects of mechanical vibrations. Relevant references are cited, however, I am suggesting some additional references that could aid to improve the Introduction and Discussion sections. The methods that were used in the experimental assays are well defined.\nI am presenting some corrections to be done and they are in the attached file.", "reviewer_3": "This research builds upon a large mass of already previously published data showing that Low Magnitude Mechanical Signals (LIV) improve bone and muscle including in osteoporosis caused by low estrogen (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8734256/). This hypothesis is embedded to the extent that LIV is offered as a standard of care treatment to postmenopausal women to prevent bone loss and muscle weakness. The first part of the current study, shown in figure 1 confirms these previous data.\nIt is also well established that zoledronic acid inhibits osteoporosis induced by loss of estrogen in humans and mouse models, therefore the hypothesis tested in the current study that combining zoledronic acid with LIV may add further benefit does warrant testing. Unfortunately, however the data presented do not appear to support the title of the manuscript. Authors state that LIV combined with zoledronic acid reduce musculoskeletal weakness and adiposity in estrogen deprived mice, whilst this is not untrue the data presented show that giving zoledronic acid alone to estrogen deprived mice has the same effect as giving zoledronic acid and LIV. Given that the majority of osteoporotic patients will receive an anti-resorptive drug such as zoledronic acid as standard of care, this study suggests that there is no need to add LIV into their treatment schedule. I am therefore unsure how the data presented will lead to improved outcome for patients. For example:\nFigure 2 shows no difference in BMD, microarchitecture, mechanical or structural properties between estragen depleted mice treated with Zoledronic acid and those treated with zoledronic acid and LIV\nFigure 3 suggest no additional benefit of adding zoledronic acid to LIV. Figure 3B is impossible to interpret as there is no indication as to which groups are significant compared with which other groups. It would help to provide more clarity around how these analyses were performed and what the starts pertain to given that the lines appear to overlap.\nFigure 4 shows no additional benefit from adding zoledronic acid to LIV on fat metabolism and 4A has no legend making it impossible to interpret.\nThere are some experimental issues that the authors should consider:\nC57BL/6 mice gain bone mass much more rapidly following zoledronic acid treatment than other models. To ensure that data generated are not model specific experiments should be be performed in more than one model system, ideally in a second model from a different genetic background.\nAuthors should provide ELISA data showing estradiol levels in these mice to provide confidence in the model system and enable readers to interpret the data in relation to estardiol levels observed in women who have recieved endocrine treatments.\nThe way statistical significance is indicated in the various graphs are very difficult to interpret, please provide more detail in the figure legends."}, "author_response": "In general, we thank all of the reviewers for the time and commitment to reviewing and critiquing our manuscript and work. This opportunity has given us the chance to significantly improve the readability and clarity of our work to ensure its comprehension for a broader audience.\nReviewer #1 (Remarks to the Author):\nIn this study the authors have tested if the addition of low-intensity vibrations (LIV) to zoledronic acid would prevent the bone and muscle loss and adiposity that comes with complete estrogen deprivation. This is clinically relevant considering how common complete estrogen deprivation is in women undergoing breast cancer treatment. In young estrogen deprived mice not treated with zoledronic acid there seems to be a potent effect of LIV to improve bone, adipose and muscle parameters. In older mice however, this effect is blunted and the addition of LIV to zoledronic acid did not significantly improve any or few of the measured parameters. However, many of the diagrams and tables are to tiny or blurry to interpret and the statistics is not clearly described, see comments below. The techniques are relevant and improving the presentation of the data would make it easier for the reader to interpret the importance of the results.\nOur team would like to thank the Reviewer for their thoughtful and careful assessment of our manuscript highlighting the clinical relevance of the work, their time and support, as well as the opportunity and provided critique to improve the quality of the manuscript. Additionally, we appreciate the recognition of the observed LIV effects in young estrogen- deprived mice and the less pronounced differences in older cohorts, as anticipated. We acknowledge the concerns raised regarding data presentation and statistical clarity, and we have taken thorough steps to improve figure resolution and enhance the description of our statistical methods as detailed in the revised manuscript. The visual resolution of the figures has been drastically improved. We have also clarified the interpretation of data and statistical measurements from the figures, hoping these revisions improve the readability and convey the impact of our findings.\nSpecific Comments:\n1. Please show serum E levels in figure. Sometimes E -levels in mice can be difficult to 2 2 determine by ELISA as used by the authors and a more reliable indicator of E -depletion would 2 be uterus weight, please provide if possible.\nThank you for requesting confirmation of estradiol levels via uterine weights. Uterine weights are routinely measured at the conclusion of our studies. As such, we have provided uterine weights to demonstrate atrophy of the uterus following complete estrogen deprivation. These values have been provided in Supplemental Figure 1. (See File Supplemental Figures and Tables.pdf)\n2. For the figures please add in each figure legend:\n-The number of animals (n)\n-The statistics used (if for example 2-way ANOVA, specify the factors tested and between which groups post-hoc test has been done).\n-Make graphs larger where needed, for example figure 2c and explain where the p-value comes from, is it comparison between the two groups at a certain time-point or something else?\n-Remove all personal comments on whether the data is increased or decreased especially if this is not supported by a significant difference and even if it is, I think these type of comments should be kept to a minimum in the figure legends.\nThank you for these helpful remarks for guidance to improve clarity of the data reported, especially in the figures. Additionally, whilst consulting with our scientific editors we have clearly delineated the statistical comparisons, indicated the number of animals per study, and improved graphical clarity. Comments describing increased or decreased data have been kept to a minimum in the figure caption, unless these are trends, in which case they were omitted. Color was added to the graphs to aid in reading clarity. (See all Figures)\n3. In the text it says that the p-value for figure 1A is 0.047 but in table 2 it says 0.048?\nThank you for pointing this out. The full p-value for this is 0.0476. The text has now been adjusted to 0.048 to mirror the data in Table 2. (See page 7)\n4. Please refer to a figure or table when data is mentioned in the text or write data not shown (if this is accepted by the journal otherwise remove these sentences and only report data that is presented).\nWe thank you for this suggestion. Previously, we were unsure if adding multiple references to the same figure within the text body would be superfluous. We have made these additions throughout the manuscript to improve clarity when referencing data as pertains to their placement in the figures. (See throughout the Results Section)\n5. The scores plot and the heat map in supplemental figure 1 and 2 is identical?\nYes, the heat maps and plots in Supplemental Figures 1 and 2 were identical. Initially, the decision was made to separate the short-chain free fatty acids from the medium- and long- chain free fatty acids due to crowding in the figure. However, as with the assumption in the previous comment and considering the confusing presentation, we have decided to combine them into a single figure, Supplemental Figure 2. Thank you for a very helpful suggestion. (See File Supplemental Figures and Tables.pdf)\nReviewer #2 (Remarks to the Author):\nDear Authors,\nCongratulations. The subject of your study is highly relevant. The findings obtained in your study bring important contributions to the field related to the effects of mechanical vibrations. Relevant references are cited; however, I am suggesting some additional references that could aid to improve the Introduction and Discussion sections. The methods that were used in the experimental assays are well defined. I am presenting some corrections to be done, and they are in the attached file.\nWe would like to thank the author for their thoughtful feedback and enthusiastic support and validation of our work.\nReviewer #3 (Remarks to the Author):\nThis research builds upon a large mass of already previously published data showing that Low Magnitude Mechanical Signals (LIV) improve bone and muscle including in osteoporosis caused by low estrogen (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8734256/). This hypothesis is embedded to the extent that LIV is offered as a standard of care treatment to postmenopausal women to prevent bone loss and muscle weakness. The first part of the current study, shown in figure 1 confirms these previous data.\nThank you, to the Reviewer, for acknowledgment of the work and relevance to clinical findings in postmenopausal women and its relationship to musculoskeletal degeneration as a corollary to complete estrogen deprivation therapy, and that LIV is a suitable agent to counter these adverse effects. We thank the reviewer for their highly-detailed and unbiased critique of the work we have presented. We have addressed other points of concern raised by the other Reviewer, which have strengthened the data, provided additional clarity to the figures.\nIt is also well established that zoledronic acid inhibits osteoporosis induced by loss of estrogen in humans and mouse models, therefore the hypothesis tested in the current study that combining zoledronic acid with LIV may add further benefit does warrant testing. Unfortunately, however the data presented do not appear to support the title of the manuscript. Authors state that LIV combined with zoledronic acid reduce musculoskeletal weakness and adiposity in estrogen deprived mice, whilst this is not untrue the data presented show that giving zoledronic acid alone to estrogen deprived mice has the same effect as giving zoledronic acid and LIV. Given that the majority of osteoporotic patients will receive an anti-resorptive drug such as zoledronic acid as standard of care, this study suggests that there is no need to add LIV into their treatment schedule. I am therefore unsure how the data presented will lead to improved outcome for patients. For example:\nWe agree with the Reviewer’s standpoint that the title may need to be more descriptive to reflect the findings in the manuscript. At the editor’s discretion we would be fine adjusting the title from “Low-Magnitude Mechanical Signals Combined with Zoledronic Acid Reduce Musculoskeletal Weakness and Adiposity in Estrogen-Deprived Mice” to either of the following as preferred:\na. “Bone-Protective Effects of Low-Magnitude Mechanical Signals and Zoledronic Acid with Partial Muscular and Marrow Adiposity Benefits in Estrogen-Deprived Mice”\nb. \"Summary of the Bone-Protective Effects of Low-Magnitude Mechanical Signals and Zoledronic Acid, with Partial Muscular and Marrow Adiposity Benefits in Estrogen-Deprived Mice\"\nFigure 2 shows no difference in BMD, microarchitecture, mechanical or structural properties between estragen depleted mice treated with Zoledronic acid and those treated with zoledronic acid and LIV.\nThank you for the analysis of the skeletal data within Figure 2. Indeed, LIV alone was unable to overcome the aggressive resorption imposed by complete estrogen deprivation. In fact, the whole-body BMD of the ZA alone and LIV+ZA group were not statistically different from one another at the final timepoint. Two points to note however, the slope in BMD of the LIV+ZA group is steeper than in the ZA alone group, inferring faster retention of bone between 4w and 14w. Some of the benefit of LIV to ZA treatment is likely diluted in the combined low-resolution and whole-body capture of the BMD data with the DXA machine. Isolating our region of interest to the lumbar spine, as it is predominantly trabecular bone, we note several differences between the ZA alone and LIV+ZA group. We invite the reviewer to note the statistical differences of each group in question as compared to the estrogen deprived control group; the LIV+ZA group has greater statistical significance as compared to the estrogen deprived control than the ZA alone group does. To provide further evidence of these differences, our microCT data, acquired at much higher resolution, demonstrates a clear advantage of the combined treatment of LIV+ZA in contrast to the ZA alone group. Histomorphometry (dynamic) demonstrates the suppressive nature of the ZA alone; the double-label surface (dLS), mineralizing surface (MS/BS), and bone formation rate (BFR/BS) is significantly suppressed in contrast to /Sham-controls (and is even lower than the estrogen deprived group), while the LIV+ZA group has a protective value that the ZA alone group does not, normalizing bone remodeling properties. From a bone quality aspect, the compression testing, ZA alone has a p<0.001 as compared to the estrogen deprived group where LIV+ZA has a p<0.0001 in comparison to the estrogen deprived group. Similar effects are evident from the 3-pt mechanical testing of the cortical bone in the femur. We hope these data, especially since they are now visually and descriptively clearer, provide the Reviewer with the rationale for our claim. (See pages 7-13 and Figure 2)\nFigure 3 suggest no additional benefit of adding zoledronic acid to LIV. Figure 3B is impossible to interpret as there is no indication as to which groups are significant compared with which other groups. It would help to provide more clarity around how these analyses were performed and what the starts pertain to given that the lines appear to overlap.\nThank you for posing this concern. With regards to Figure 3. Indeed, the longitudinal graphs associated with the muscle function assays (grip strength and fatiguability of the EDL) were not visually easy to interpret, and this has been addressed after refinement of the graph. Muscle fatigue was greatly reduced in both LIV-treated groups at 40 and 50 Hz stimulations compared to OVX/AI mice, as did OVX/AI+ZA at the 50Hz stimulation. In addition, OVX/AI+LIV+ZA mice retained the highest grip strength throughout the duration of the study. Histologically, OVX/AI+LIV mice generally had reduced myofiber cross-sectional area. Visually, empty spaces between muscle fibers were evident in untreated OVX/AI mice, and these were nearly absent in sections from the OVX/AI+LIV mice. Our Scientific Editor has added color and thinned the lines to provide distinction to each group. We have also indicated within the Results section the statistical comparisons that are being referenced. Closer inspection of the muscle function data indicates a partial benefit imparted on the muscle in mice administered with LIV and ZA. (See pages 13-14 and Figure 3)\nFigure 4 shows no additional benefit from adding zoledronic acid to LIV on fat metabolism and 4a has no legend making it impossible to interpret.\nThank you. We have clarified the visual interpretation in Figure 4A and have addressed its corresponding comparisons in the Figure 4 legend. With regards to the data presented as % fat mass in the Figure 4a, indeed the comparisons indicate that there is no derived benefit from LIV or ZA or the combined treatment as compared to the estrogen deprived counterparts sans treatment. However, it is also important to note that fat mass did not increase either, in response to these treatments. Visual examination of osmium-tetroxide- stained sections revealed a distinction between the tibiae of LIV-treated mice compared with vehicle-treated OVX/AI mice. Further, trends in the marrow adiposity as measured through histological evaluation indicated that at the cellular level, adipose cell area and the number of adipocytes in the OVX/AI+LIV+ZA mice were not significantly greater than SH nor were they significantly lower than OVX/AI alone, indicating a partial benefit in fat metabolism, which is now reflected in the title as suggested. (See pages 14-15 and Figure 4)\nThere are some experimental issues that the authors should consider:\nC57BL/6 mice gain bone mass much more rapidly following zoledronic acid treatment than other models. To ensure that data generated are not model specific experiments should be be performed in more than one model system, ideally in a second model from a different genetic background.\nThank you for the perspective and reminder of the variations in bone accrual across murine models. We have performed similar studies with balb/c athymic nude mice under complete estrogen deprivation (ovariectomy and aromatase inhibitor) and have also observed retention of bone following administration of zoledronic acid (Wright L, Oncotarget; 2016). We appreciate that the effects of zoledronic acid are strong, exhibiting a noticeable anti-resorptive effect. It should be noted that the dose of zoledronic acid administered was quite low and delivered subcutaneously only 1x/w. To this point, the purpose was to not occlude the effects of LIV for the purposes of bolstering the ZA effects.\nAuthors should provide ELISA data showing estradiol levels in these mice to provide confidence in the model system and enable readers to interpret the data in relation to estardiol levels observed in women who have recieved endocrine treatments.\nThank you, for reinforcing this suggestion, as Reviewer 1 has requested this as well. As Reviewer 1 has suggested, we have amended the supplemental figure (Supplemental Figure 1) to include uterine weights measured at euthanasia as a more reliable indicator of E - 2 depletion. Uterine weights are routinely measured at the conclusion of our studies and correlate to complete estrogen deprivation (Wright L, Oncotarget; 2016). As such, we have provided uterine weights to demonstrate atrophy of the uterus following complete estrogen deprivation. The corresponding values for Experiments 1 and 2 have been added to Supplemental Figure 1. (See pages 7 and 9 and Supplemental Figure 1)\nThe way statistical significance is indicated in the various graphs are very difficult to interpret, please provide more detail in the figure legends.\nOur group agrees with the Reviewers regarding the interpretation of the statistical measures in the graphs. A dedicated Scientific Editor was sought and has since assisted in a strong effort to improve the clarity of the plethora of graphs in the manuscript and updated the legends to reflect the statistical measures used in the analysis. Additionally, we have added references to each figure (i.e., Fig. 1) in the Results section. Figure legends now include statistical tests used to compare data within and between groups, as applicable. We thank this Reviewer for their perspective and appreciate the chance to improve the illustrations. (See Figures 1-4 and throughout the Results Section)"}, {"round": 2, "reviewer_comments": {"reviewer_1": "Revision 2, Reviewer 1.\nPlease find my comments to the authors below in bold:\nReviewer #1 (Remarks to the Author): In this study the authors have tested if the addition of low-intensity vibrations (LIV) to zoledronic acid would prevent the bone and muscle loss and adiposity that comes with complete estrogen deprivation. This is clinically relevant considering how common complete estrogen deprivation is in women undergoing breast cancer treatment. In young estrogen deprived mice not treated with zoledronic acid there seems to be a potent effect of LIV to improve bone, adipose and muscle parameters. In older mice however, this effect is blunted and the addition of LIV to zoledronic acid did not significantly improve any or few of the measured parameters. However, many of the diagrams and tables are to tiny or blurry to interpret and the statistics is not clearly described, see comments below. The techniques are relevant and improving the presentation of the data would make it easier for the reader to interpret the importance of the results.\nOur team would like to thank the Reviewer for their thoughtful and careful assessment of our manuscript highlighting the clinical relevance of the work, their time and support, as well as the opportunity and provided critique to improve the quality of the manuscript. Additionally, we appreciate the recognition of the observed LIV effects in young estrogen-deprived mice and the less pronounced differences in older cohorts, as anticipated. We acknowledge the concerns raised regarding data presentation and statistical clarity, and we have taken thorough steps to improve figure resolution and enhance the description of our statistical methods as detailed in the revised manuscript. The visual resolution of the figures has been drastically improved. We have also clarified the interpretation of data and statistical measurements from the figures, hoping these revisions improve the readability and convey the impact of our findings.\nRevision 2, Reviewer 1:\nThe presentation has improved but there are still some concerns regarding the figures and statistics used, please see below for details. In addition, please add a conclusion in the abstract where you state what you found and how it relates to your original hypothesis (“Combination treatment of Low-Intensity Vibration (LIV) with zoledronic acid (ZA) was hypothesized to preserve bone mass and muscle strength while reducing adipose tissue accrual associated with complete estrogen (E2) deprivation in young and skeletally-mature mice.”).\nSpecific Comments: 1. Please show serum E2 levels in figure. Sometimes E2-levels in mice can be difficult to determine by ELISA as used by the authors and a more reliable indicator of E2-depletion would be uterus weight, please provide if possible.\nThank you for requesting confirmation of estradiol levels via uterine weights. Uterine weights are routinely measured at the conclusion of our studies. As such, we have provided uterine weights to demonstrate atrophy of the uterus following complete estrogen deprivation. These values have been provided in Supplemental Figure 1. (See File Supplemental Figures and Tables.pdf)\nRevision 2, Reviewer 1:\nThank you!\n2. For the figures please add in each figure legend: -The number of animals (n) -The statistics used (if for example 2-way ANOVA, specify the factors tested and between which groups post-hoc test has been done). -Make graphs larger where needed, for example figure 2c and explain where the p-value comes from, is it comparison between the two groups at a certain time-point or something else? -Remove all personal comments on whether the data is increased or decreased especially if this is not supported by a significant difference and even if it is, I think these type of comments should be kept to a minimum in the figure legends.\nThank you for these helpful remarks for guidance to improve clarity of the data reported, especially in the figures. Additionally, whilst consulting with our scientific editors we have clearly delineated the statistical comparisons, indicated the number of animals per study, and improved graphical clarity. Comments describing increased or decreased data have been kept to a minimum in the figure caption, unless these are trends, in which case they were omitted. Color was added to the graphs to aid in reading clarity. (See all Figures)\nRevision 2, Reviewer 1:\nTo my knowledge two-way ANOVA is not an appropriate statistical test for the time-course measurements in Fig. 2a, b, c; Fig. 3a, b, c and Fig. 4a. Please consult with a statistician\n3. In the text it says that the p-value for figure 1A is 0.047 but in table 2 it says 0.048?\nThank you for pointing this out. The full p-value for this is 0.0476. The text has now been adjusted to 0.048 to mirror the data in Table 2. (See page 7)\nRevision 2, Reviewer 1:\nThank you!\n4. Please refer to a figure or table when data is mentioned in the text or write data not shown (if this is accepted by the journal otherwise remove these sentences and only report data that is presented).\nWe thank you for this suggestion. Previously, we were unsure if adding multiple references to the same figure within the text body would be superfluous. We have made these additions throughout the manuscript to improve clarity when referencing data as pertains to their placement in the figures. (See throughout the Results Section)\nRevision 2, Reviewer 1:\nThere are still mentioning of results for L4 but results are only presented for L5.\nIn Supplementary Figure 2, the authors sometimes indicate between which groups the post hoc tests are done and sometimes not.\nAll bar plots should be replaced by dot plots to make it easier for the reader to estimate the distribution of data.\n5. The scores plot and the heat map in supplemental figure 1 and 2 is identical?\nYes, the heat maps and plots in Supplemental Figures 1 and 2 were identical. Initially, the decision was made to separate the short-chain free fatty acids from the medium- and long-chain free fatty acids due to crowding in the figure. However, as with the assumption in the previous comment and considering the confusing presentation, we have decided to combine them into a single figure, Supplemental Figure 2. Thank you for a very helpful suggestion. (See File Supplemental Figures and Tables.pdf)\nRevision 2, Reviewer 1:\nOk", "reviewer_2": "Dear Authors.\nCongratulations. The manuscript was improved, but I suggest:\n-It is very important to add the reference that is a guideline to the use of whole-body vibration studies in humans, animals and cell cultures. This reference could be added in the Introduction section. The reference is: van Heuvelen MJG, Rittweger J, Judex S, Sañudo B, Seixas A, Fuermaier ABM, Tucha O, Nyakas C, Marín PJ, Taiar R, Stark C, Schoenau E, Sá-Caputo DC, Bernardo-Filho M, van der Zee EA. Reporting Guidelines for Whole-Body Vibration Studies in Humans, Animals and Cell Cultures: A Consensus Statement from an International Group of Experts. Biology (Basel). 2021 Sep 27;10(10):965. doi: 10.3390/biology10100965. PMID: 34681065; PMCID: PMC8533415.\n- Put all \"p\" related to statistical analysis in italic format throughout the manuscript.", "reviewer_3": "The authors have clarified key points and data analysis in this manuscript providing an informative, highly relevant study. I am satisfied that all previously expressed concerns have been addressed."}, "author_response": "Revision 2, Reviewer 1:\nResponse:\nThank you for the positive assessment and opportunity to further enhance the manuscript. We have added the following concluding remark to the Abstract in the manuscript (See page 3):\n“In summary, our findings support the proposed hypothesis, that LIV preserves bone and muscle mass while reducing fat accumulation in E -deprived mice, with stronger effects 2 observed in younger mice, in contrast to skeletally-mature mice where LIV combined with bisphosphonate (ZA) treatment significantly improved bone outcomes.”\n______________________________________________________________________________\nSpecific Comments:\n1. Please show serum E2 levels in figure. Sometimes E2-levels in mice can be difficult to determine by ELISA as used by the authors and a more reliable indicator of E2-depletion would be uterus weight, please provide if possible.\nThank you for requesting confirmation of estradiol levels via uterine weights. Uterine weights are routinely measured at the conclusion of our studies. As such, we have provided uterine weights to demonstrate atrophy of the uterus following complete estrogen deprivation. These values have been provided in Supplemental Figure 1. (See File Supplemental Figures and Tables.pdf)\nRevision 2, Reviewer 1:\nThank you!\nResponse:\nThank you.\n______________________________________________________________________________\n2. For the figures please add in each figure legend: -The number of animals (n) -The statistics used (if for example 2-way ANOVA, specify the factors tested and between which groups post-hoc test has been done). -Make graphs larger where needed, for example figure 2c and explain where the p-value comes from, is it comparison between the two groups at a certain time-point or something else? -Remove all personal comments on whether the data is increased or decreased especially if this is not supported by a significant difference and even if it is, I think these type of comments should be kept to a minimum in the figure legends.\nThank you for these helpful remarks for guidance to improve clarity of the data reported, especially in the figures. Additionally, whilst consulting with our scientific editors we have clearly delineated the statistical comparisons, indicated the number of animals per study, and improved graphical clarity. Comments describing increased or decreased data have been kept to a minimum in the figure caption, unless these are trends, in which case they were omitted. Color was added to the graphs to aid in reading clarity. (See all Figures)\nRevision 2, Reviewer 1:\nTo my knowledge two-way ANOVA is not an appropriate statistical test for the time-course measurements in Fig. 2a, b, c; Fig. 3a, b, c and Fig. 4a. Please consult with a statistician\nResponse:\nThank you for pointing this out. We consulted with a biostatistician to confirm the suitability of the test that was performed. Indeed, as you’ve mentioned, the nomenclature for the test reported (two-way ANOVA) was not accurate, though the actual test performed was correct (Repeated measures ANOVA). The statistical test being performed was Repeated measures ANOVA, as confirmed by the biostatistician (Roland Bassett, Jr) that we consulted with. Each of these Figure captions have been revised to reflect the correct notation. (See pages 32, 43, 45, and 47)\n______________________________________________________________________________\n3. In the text it says that the p-value for figure 1A is 0.047 but in table 2 it says 0.048?\nThank you for pointing this out. The full p-value for this is 0.0476. The text has now been adjusted to 0.048 to mirror the data in Table 2. (See page 7)\nRevision 2, Reviewer 1:\nThank you!\nResponse:\nThank you.\n______________________________________________________________________________\n4. Please refer to a figure or table when data is mentioned in the text or write data not shown (if this is accepted by the journal otherwise remove these sentences and only report data that is presented).\nWe thank you for this suggestion. Previously, we were unsure if adding multiple references to the same figure within the text body would be superfluous. We have made these additions throughout the manuscript to improve clarity when referencing data as pertains to their placement in the figures. (See throughout the Results Section)\nRevision 2, Reviewer 1:\nThere are still mentioning of results for L4 but results are only presented for L5.\nIn Supplementary Figure 2, the authors sometimes indicate between which groups the post hoc tests are done and sometimes not.\nAll bar plots should be replaced by dot plots to make it easier for the reader to estimate the distribution of data.\nResponse:\nThank you. We have removed all remaining instances of L4 in the body of the text.\nWe thank you for pointing out the post-hoc analysis data. These have been clearly indicated now, we have ensured the post-hoc analyses are indicated for their respective groups in Supplemental Figure 2.\nBar plots have been replaced by dot plots to maintain consistency in reporting across the manuscript and allow for easier visualization of the distribution of data for the reader.\n______________________________________________________________________________\n5. The scores plot and the heat map in supplemental figure 1 and 2 is identical?\nYes, the heat maps and plots in Supplemental Figures 1 and 2 were identical. Initially, the decision was made to separate the short-chain free fatty acids from the medium- and long-chain free fatty acids due to crowding in the figure. However, as with the assumption in the previous comment and considering the confusing presentation, we have decided to combine them into a single figure, Supplemental Figure 2. Thank you for a very helpful suggestion. (See File Supplemental Figures and Tables.pdf)\nRevision 2, Reviewer 1:\nOk\nResponse:\nThank you.\nReviewer #2 (Remarks to the Author):\nDear Authors.\nCongratulations. The manuscript was improved, but I suggest:\n-It is very important to add the reference that is a guideline to the use of whole-body vibration studies in humans, animals and cell cultures. This reference could be added in the Introduction section. The reference is: van Heuvelen MJG, Rittweger J, Judex S, Sañudo B, Seixas A, Fuermaier ABM, Tucha O, Nyakas C, Marín PJ, Taiar R, Stark C, Schoenau E, Sá-Caputo DC, Bernardo-Filho M, van der Zee EA. Reporting Guidelines for Whole-Body Vibration Studies in Humans, Animals and Cell Cultures: A Consensus Statement from an International Group of Experts. Biology (Basel). 2021 Sep 27;10(10):965. doi: 10.3390/biology10100965. PMID: 34681065; PMCID: PMC8533415.\n- Put all \"p\" related to statistical analysis in italic format throughout the manuscript.\nResponse:\nThank you for this suggestion. We have added this reference to the Introduction section as suggested. Please refer to reference #59. (See page 5).\nAdditionally, we have italicized all “p” related to statistical analyses throughout the manuscript.\nReviewer #3 (Remarks to the Author):\nThe authors have clarified key points and data analysis in this manuscript providing an informative, highly relevant study. I am satisfied that all previously expressed concerns have been addressed.\nWe thank you for your great suggestions that have served to improve the quality of the manuscript."}, {"round": 3, "reviewer_comments": {"reviewer_1": "Thank you, the authors have answered all my questions."}, "author_response": ""}]

Decision letter