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. Author manuscript; available in PMC: 2026 Aug 7.
Published in final edited form as: Bone. 2026 May 6;210:117924. doi: 10.1016/j.bone.2026.117924

Age-Dependent Musculoskeletal Changes During Mechanical Unloading with Bisphosphonate Treatment

Sophie Orr 1, Natalie K Gilmore 2, Suraj Pathak 2, Hodo Ali Edan 2, Henning Langer 2,3, Keith Baar 2, Blaine A Christiansen 1
PMCID: PMC13445739  NIHMSID: NIHMS2189493  PMID: 42103043

Abstract

Mechanical unloading (disuse) leads to reductions in bone and muscle mass. Bone and muscle adaptation are often studied together in terms of mechanical stimulus, but non-mechanical (biological) crosstalk during periods of disuse and how this is affected by age and bone-preserving pharmaceuticals has not been assessed. This study aimed to determine how mechanical unloading and concurrent bisphosphonate treatment affect bone and muscle structure and function in young, middle-aged, and old mice. We hypothesized that unloading would cause bone loss in untreated mice, but bisphosphonate treatment would prevent this loss. Additionally, we expected that unloading would result in muscle atrophy and reduced contraction force, but we hypothesized that these reductions would be partially mitigated by bisphosphonate treatment due to decreased release of osteokines, and that this mitigation would decrease with age. To investigate these hypotheses, young (3-mo, n=40), middle-aged (12-mo, n=40), and old (20-m, n=40) male C57BL/6J mice received biweekly subcutaneous bisphosphonate injections (0.03 mg alendronate/mouse) or vehicle injections starting one week before unloading. Mice underwent hindlimb unloading (HLU) via tail suspension for 14 days. Maximum force production of the hind limb was measured after 14 days of unloading. Femurs were imaged with micro-computed tomography (μCT 35, SCANCO Medical AG); cortical bone was analyzed at the mid-diaphysis, and trabecular bone was analyzed at the distal femur to determine bone microstructural outcomes. Muscle fiber cross-sectional area (CSA) and fiber type were analyzed via IHC. Muscle myostatin and serum TGF-β1 levels were measured via ELISA. Achilles tendon mechanical properties were also assessed using tensile testing. We found that HLU decreased the mass of the triceps surae muscles, and this loss was not recovered during bisphosphonate treatment. Muscle mass in old mice decreased during HLU with bisphosphate treatment. Maximum hindlimb force production and respective force to muscle mass ratio differed between all age groups and did not correlate with bone or muscle changes. Muscle myostatin concentrations increased with age (p=0.040), bisphosphonate treatment (p=0.003), and unloading (p=0.002), as well as due to the interaction of treatment and unloading (p=0.0239). Serum levels of TGF-β1 increased with age (p<0.0001) and unloading (p=0.012), as well as with interactions between both age and treatment (p=0.006) and age and unloading (p=0.013). Age impacted muscle, tendon, and bone responses to unloading and/or bisphosphonate treatment. Bone and muscle adaptation to unloading are different across the lifespan, as are the effects of bisphosphonate treatment. Characterizing these changes is essential for understanding clinical outcomes related to periods of disuse and clinical bone and muscle preserving treatments during bedrest, immobilization, or even spaceflight across different age groups.

1. Introduction

Osteoporosis & sarcopenia are debilitating age-related diseases with high morbidity and mortality risks that impact millions. Osteoporosis is a degenerative disease characterized by reduced bone mass, leading to bone weakness and frailty [1], that impacts millions annually in the United States [2]. The incidence of osteoporosis increases with age in a gender-dependent manner, with women being 4-times more likely to develop osteoporosis than men [3]. Osteoporosis is often closely associated with sarcopenia, the wasting of skeletal muscle that occurs later in life, with both conditions leading to increased frailty, fracture risk, and mortality rates [4, 5].

Bone and muscle atrophy are accelerated during periods of mechanical unloading (disuse) [6, 7], especially in situations such as bed rest and spaceflight [8–11]. Mechanical loading (i.e., exercise) can theoretically be used to regenerate atrophied tissue [12]. However, with age, people’s ability to perform weight-bearing exercise is diminished, and bone and muscle tissue are less responsive to mechanical stimuli [13]. This is important because elderly patients make up a disproportionately large number of hospitalizations, and their length of stay for hospitalizations is longer on average than that of younger adults [14, 15]. Preventing musculoskeletal atrophy during unloading periods could benefit multiple at-risk populations, but these treatments will most likely vary as a function of age.

Bone and muscle atrophy are often researched together in the context of mechanical loading or unloading due to their roles in force transmission. However, any non-mechanical (e.g., biological crosstalk) interactions between these tissues have been poorly characterized. Cytokines are proteins released from cells that can trigger action in the secretory cell itself, other cells in proximity, or distant tissues within an organism. These proteins are often categorized by their tissue of origin; for example, osteokines originate in bone, while myokines originate in muscle. Biological crosstalk between bone and muscle involves osteokines and myokines regulating different aspects of muscle and bone activity, respectively [16, 17]. During disuse, osteoclasts break down existing bone tissue, releasing proteins stored within the calcified matrix that can affect muscle. One of the primary osteokines that affects muscle mass is transforming growth factor beta (TGF-β), which drives muscle atrophy or produces myopathy by signaling in target muscles [18, 19]. Similarly, the myokine myostatin (GDF-8) limits myogenesis and reduces muscle mass and strength [20–22], but also affects bone. Myostatin can regulate osteoblast differentiation, osteoclast development, bone morphology, and induce atrophy in normal and unloaded conditions [16, 17, 21–24]. Therefore, these and other cytokines play essential function, and crosstalk may contribute to musculoskeletal health, especially during periods of unloading. Understanding the role of crosstalk between bone and muscle tissues at different ages during mechanical unloading may help define novel pathways to improve bone and muscle health.

Bisphosphonates are bone-targeting drugs commonly prescribed to osteoporotic patients that maintain bone mass by interfering with normal osteoclast function and inhibiting bone resorption [25–27]. Bisphosphonates could also potentially be used in aerospace applications to prevent bone loss during long-duration spaceflight missions [28]. By blocking bone resorption, bisphosphonates could also prevent the release of proteins from the calcified matrix of bone and limit muscle atrophy. This study investigated the effect of bisphosphonate (alendronate) treatment on bone-muscle crosstalk and musculoskeletal atrophy during disuse at different ages. We hypothesized that the release of osteokines during unloading-induced bone atrophy contributes to muscle atrophy. We further hypothesized that preventing bone resorption during unloading would mitigate muscle atrophy by inhibiting the release of catabolic osteokines, and that this inhibition would diminish with age.

2. Methods

2.1. Animals

All animals were maintained and used in accordance with National Institutes of Health guidelines on the care and use of laboratory animals. All procedures were approved by the UC Davis Institutional Animal Care and Use Committee. Young (3-mo), middle-aged (12-mo), and old (20-mo) male C57BL/6J mice (n = 10 per age/treatment/HLU condition for a total of n=120) were purchased from Jackson Laboratory (Sacramento, CA). Mice were allowed to acclimatize to their cage settings for 7 days prior to beginning the study. Mice were treated with alendronate (0.003mg/mouse, in Phosphate Buffered Saline, PBS) or vehicle (PBS) biweekly starting 7 days before mechanical unloading. Mice were mechanically unloaded for 14 days via hindlimb unloading, at which point they were euthanized. Mice in each age group were randomly assigned to one of four groups (n=10 per group): Unloaded Control (CTR), Unloaded Bisphosphonate Treated (CTR+B), Hindlimb Unloaded Control (HLU), or Hindlimb Unloaded Bisphosphonate Treated (HLU+B). All mice were supplied with water, bedding, and chow available ad libitum (chow # 2918, Teklad Global, West Lafayette, Indiana, USA). Unloaded mice were group-housed in standard mouse cages. Hindlimb-unloaded mice were single-housed in Hindlimb unloading cages described previously [29], also with ad libitum access to food and water. All mice were maintained in a facility set to a thermoneutral temperature of 90°F to reduce the negative thermal effects of HLU positioning [30].

2.1. Hindlimb Unloading (HLU)

Mice were single-housed and suspended by their tails at approximately a 30-degree head-down angle so that their hindlimbs could not touch the cage floor, as previously described [31]. Tail suspension fixtures were attached to a swivel and slider on a bar across the top of the cage, allowing the mice the ability to move freely throughout the entire cage.

2.2. Bisphosphonate Treatment:

Alendronate sodium trihydrate, 97% (Thermo Scientific, Waltham, MA), was administered at a dose of 1.0 mg/kg body weight in phosphate-buffered saline (PBS) subcutaneously biweekly [32] starting 7 days before the beginning of HLU. PBS was administered to control groups at a dose of 0.1 mL subcutaneously on the same schedule [32].

2.3. Micro-Computed Tomography (μCT) Analysis of Bone Microstructure

Femurs were collected and placed in 4% paraformaldehyde (PFA) on a rocker for 3 days at 4°C, after which the PFA was discarded, and the femurs were stored in 70% ethanol until scanning. Femurs were scanned using micro-computed tomography (SCANCO μCT 35, Brüttisellen, Switzerland) (SCANCO μCT 35, Brüttisellen, Switzerland) at X-ray tube potential 55 kVp, intensity 114 mA, integration time 900 ms, 6 μm isotropic nominal voxel size[33]. Trabecular bone was analyzed in the distal femoral metaphysis (200 slices per ROI starting at the end of the growth plate), and cortical bone was examined at the femoral diaphysis (100 slices per ROI).

2.4. Muscle Contractile Force Measurements

Immediately before euthanasia, mice were anesthetized with 2–4% inhaled isoflurane, and maximum muscle contractile force was measured via electrical stimulation of the hindlimb muscles using a dual-mode muscle lever and footplate system (305C-LR, Aroura Scientific, Aroura, ON, Canada) [34]. Briefly, the right hip, knee, and ankle were aligned at 90 degrees, and the foot was taped to the force transducer pedal. Needle electrodes were then inserted into gastrocnemius muscle. Maximum hindlimb force was elicited using three 150Hz (pulse width 0.2ms, duration 0.350s) stimuli with 20s of rest between contractions. Unfortunately, the force testing system malfunctioned on one collection day when many older animals were collected. The result is that force was only measured in 2 animals for the old HLU group. Torque traces were normalized to body weight, triceps surae weight, and tibialis anterior weight to investigate possible changes in muscle function. After euthanasia, the tibialis anterior, triceps surae (gastrocnemius, plantaris, and soleus muscles) were removed and weighed as a single unit.

2.5. Muscle Fiber Type Analysis

Following dissection and weighing, triceps surae from the right leg of each mouse were pinned to cork at a fixed length, and flash-frozen in LN2-cooled isopentane (2-methylbutane) before being stored at −80°C [35]. Muscles were then sectioned into 8-micron sections via cryotome and stored at −20°C. Immunohistology (IHC) staining was performed to identify Type I, IIa, and IIb fiber types. Unstained fibers were considered IIx. Briefly, tissue sections were blocked with Mouse on Mouse (MOM) and Normal Goat Serum in PBS with Tween (20%). The following Santa Cruz Biotechnology (Santa Cruz, CA, USA) secondary antibodies were used: IgG2B (blue, 350λ, type I), IgG1 (red, 555λ, type IIa), IgM (green, 488λ, type IIb), and RB 647λ (laminin). Changes in muscle fiber size and type were quantified using immunohistochemistry and analyzed using SMASH software [36].

2.6. Myostatin (GDF-8) and TGF-β1 Analysis

At the time of euthanasia, 0.5–1 mL of blood was collected via cardiac puncture. To extract serum, blood samples were centrifuged at 12,000g for 10 minutes at 4 C, and the top layer transferred to a new tube. Tibialis anterior and triceps surae muscles from the left leg of each mouse were collected and flash-frozen after dissection. Tissues were later powdered and the supernatants were analyzed via enzyme-linked immunosorbent assay (ELISA) for myostatin (GDF-8) following the manufacturer’s guidelines: Myostatin ELISA on Muscle supernatant (GDF8/Myostatin Quantikine ELISA kit, Cat #DGDF80, R&D Systems, Minneapolis, MN, USA). Serum samples were also analyzed via ELISA for circulating TGF-β following the manufacturer’s guidelines: (Human/Mouse/Rat/Porcine/Canine TGF-β1 Quantikine ELISA kit, Cat #DB100C, R&D Systems, Minneapolis, MN, USA).

2.7. Tendon Mechanical Properties

Achilles tendons were removed during dissection and frozen in PBS soaked gauze at −20° C. Tissues were thawed on the day of testing, and the mechanical and material properties were quantified using an Instron Model 68SC-1 single-column tensile tester as previously described [37, 38]. Briefly, the width and thickness of each tendon were determined with spectral-domain optical coherence tomography (OCT) using an OQ Labscope (Lumedica, Durham, NC). Cross-sectional area was calculated by measuring the width and thickness at the narrowest point and assuming the tendons have a rectangular shape. Tendons were stretched to failure using a Model 68SC-1 single-column tensile tester with a BioPuls Body Temperature Bath and BioPuls Submersible Pneumatic Side Action Grips (Instron, Norwood, MA) with a 100-Newton (N) load cell. During testing, tissues were submerged in 0.9% saline at 37°C. Grip tension was held at 80 lb/in2 (56245.57 kg/m2). The mechanical testing protocol included 10 preconditioning cycles (0.10–0.25 N) at a rate of 0.25 mm/s before loading to failure at a constant rate of 0.25 mm/s until the force dropped 80% from the maximum.

2.8. Statistical Analysis

Statistical analysis was performed with a 3-way analysis of variance (ANOVA) with age (3, 12, or 20 months old), treatment (saline or bisphosphonate), and loading condition (loaded or unloaded) as factors. Post hoc analysis was performed using Tukey’s Honest Significant Difference (HSD) test. All statistical tests were performed using Prism 10.4.1 for Mac (Graphpad Software, San Diego, CA). Statistically significant differences were identified at p<0.05; some non-significant trends were noted at p ≤0.10.

3. Results

3.1. MicroCT Analysis

Age impacts skeletal response to unloading and bisphosphonate treatment.

MicroCT analysis of cortical and trabecular bone of the femur demonstrated that two weeks of HLU significantly reduced cortical and trabecular bone morphology in 3-month-old mice, but not in 12-month-old mice. 20-month-old mice exhibited a reduced response to HLU. The selected alendronate dose successfully maintained bone volume lost during HLU in young animals and increased bone volume in middle-aged and old groups during HLU (Figs. 1 & 2, Supplemental Table 1). Several bone microarchitecture factors changed across the lifespan, including reduced metaphyseal bone volume, bone volume fraction (BV/TV), and trabecular number (Tb.N). Cortical bone area fraction (BA/TA) also decreased. HLU and alendronate interactions were noted in cortical (BA/TA) but no other properties. Trabecular thickness (Tb.Th) and trabecular separation (Tb.Sp) increased with age (Fig. 2 D & E). Age also altered the effect of HLU and alendronate treatment in trabecular bone, but not cortical bone (Fig. 2B–D).

Figure 1.

Figure 1.

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μCT analysis of cortical bone at the femoral mid-diaphysis during hindlimb unloading (HLU) and bisphosphonate (alendronate) treatment (CTR+B) in 3-,12-, and 20-month-old mice. Cortical bone microarchitecture: A) bone area (BA); B) bone volume fraction (BA/TA); C) cortical thickness (Ct.Th). Significant main effects and interactions were apparent in bone area (age: p < 0.0001; unloading: p < 0.0001; bisphosphonate: p = 0.025), BA/TA (age: p < 0.0001; unloading: p = 0.008; bisphosphonate: p < 0.0001; age x bisphosphonate: p = 0.045) and Ct.Th (age: p < 0.0001; bisphosphonate: p < 0.0001). Significance values of 3-way ANOVA with Tukey’s post-hoc analysis: * p-value < 0.05, ** p-value <0.01, *** p-value < 0.001, **** p-value < 0.0001.

Figure 2.

Figure 2.

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μCT analysis of trabecular bone af the distal femoral metaphysis during hindlimb unloading (HLU) and bisphosphonate (alendronate) treatment (CRT+B) in 3-,12-, and 20-month-old mice. Trabecular bone microarchitecture: A) bone volume (BV); B) bone volume fraction (BV/TV); C) trabecular number (Tb.N); D) trabecular thickness (Tb.Th); E) Trabecular Separation (Tb.Sp). Significant main effects and interactions were present in BV (age: p < 0.0001; HLU: p < 0.0001; unloading: p < 0.0001), BV/TV (age: p < 0.0001; HLU: p < 0.0001; unloading: p < 0.0001; age x HLU: p = 0.003), Tb.N (age: p < 0.0001; HLU: p = 0.004; unloading: p = 0.003; Age x HLU: p = 0.024; age x bisphosphonate: p = 0.046), Tb.Th (age: p < 0.0001; HLU: p < 0.0001; unloading: p < 0.0001; age x bisphosphonate: p = 0.017), and Tb.Sp (age: p < 0.0001). Significance values of 3-way ANOVA with Tukey’s post-hoc analysis: * p-value < 0.05, ** p-value <0.01, *** p-value < 0.001, **** p-value < 0.0001.

3.2. Muscle Analysis

Alendronate treatment does not result in muscle mass maintenance.

Body weight was reduced in HLU animals with and without alendronate treatment compared to controls (Fig. 3A, Supplemental Fig. 1). Atrophy of the triceps surae and tibialis anterior muscles with unloading was apparent in all age groups (Fig. 3 C & E, Supplemental Fig. 2). 12-month-old mice were the only age group that did not demonstrate changes in the weight of tibialis anterior muscles between controls and HLU groups. 20-month-old mice had increased normalized force production after HLU with and without treatment, while 3-month and 12-month mice did not change significantly (Fig. 3 B, D, F).

Figure 3.

Figure 3.

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Mass and force production of triceps surae and tibialis anterior muscles during hindlimb unloading (HLU) and bisphosphonate (alendronate) treatment (CTR+B) in 3-,12-, and 20-month-old mice. A) mouse body weight; B) max force normalized to body weight; C triceps surae mass; D) muscle max force normalized to triceps surae mass; E) tibialis anterior mass; F) muscle max force normalized to tibialis anterior mass. Significant main effects and interactions were present in body weight (age: p < 0.0001; HLU: p < 0.0001; Age x HLU: p = 0.012; HLU x bisphosphonate: p = 0.012), Max Force: BW (age x HLU: p = 0.018; HLU x bisphosphonate: p = 0.019), triceps surae mass (age: p < 0.0001; HLU: p < 0.0001), Max Force: TS (HLU: p = 0.015; age x HLU: p = 0.018), tibialis anterior mass (age: p < 0.0001; HLU: p < 0.0001; age x bisphosphonate: p = 0.012), and Max Force: TA (age x HLU: p = 0.037). Significance values of 3-way ANOVA with Tukey’s post-hoc analysis: * p-value < 0.05, ** p-value <0.01, *** p-value < 0.001, **** p-value < 0.0001.

Age, HLU, and bisphosphonate treatment modified the average fiber type sizes in the soleus and gastrocnemius muscles.

The percentage of type I fibers in the soleus muscle differed as a function of age (Fig. 4, Supplemental Figure 3). Soleus type I fiber size (cross-sectional area, CSA) increased with bisphosphonate treatment (p<0.0001) and decreased with unloading (p=0.005). Age impacted the response to bisphosphonate treatment (p=0.027), with 12-month-old mice being less affected by these changes than 3-month-old and 20-month-old mice. Conversely, type I fibers in the gastrocnemius were reduced by bisphosphonate treatment (p=0.035) and unloading (p=0.002). Gastrocnemius type IIa fibers were also impacted by age (p=0.016) and unloading (p<0.0001), but not by bisphosphonate treatment. 3- and 20-month-old mice had significantly reduced gastrocnemius type IIa CSA between control mice and HLU bisphosphonate-treated mice.

Figure 4.

Figure 4.

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Muscle fiber types and sizes during hindlimb unloading (HLU) and bisphosphonate (alendronate) treatment (CTR+B) in 3-,12-, and 20-month-old mice. Muscle fiber type and cross-sectional area (CSA): A-B) soleus type I; C-D) gastrocnemius type I; E-F) gastrocnemius type IIa. Significant main effects and interactions were present in soleus type I fiber percentage (age: p = 0.044), soleus type I fiber size (age: p = 0.004; HLU: p = 0.005; bisphosphonate: p < 0.0001; age x bisphosphonate: p =0.027), gastrocnemius type I fiber percentage (age: p < 0.0001), gastrocnemius type I fiber size (age: p = 0.027; HLU: p = 0.002; bisphosphonate: p = 0.035), gastrocnemius type IIa fiber percentage (age: p = 0.001), and gastrocnemius type IIa fiber size (age: p = 0.016; HLU: p < 0.0001). Significance values of 3-way ANOVA with Tukey’s post-hoc analysis: * p-value < 0.05, ** p-value <0.01, *** p-value < 0.001, **** p-value < 0.0001.

3.3. ELISA Analysis

TGF-β1 and myostatin are not wholly responsible for the muscle response to mechanical unloading.

Myostatin concentrations in the triceps surae increased with alendronate treatment in 3-month-old unloaded mice (HLU vs HLU+B) but not within other age groups (Fig. 5A). However, overall, unloading, alendronate treatment, and the interaction of unloading and treatment impacted myostatin concentrations. 20-month-old mice had lower concentrations of myostatin than 12-month-old mice. No significant interactions between age and HLU or age and alendronate treatment were observed. Serum TGF-β1 concentrations increased with age and unloading, though unloading results from old mice may skew this relationship (Fig. 5B). Only 20-month-old mice exhibited within-groups changes due to unloading and alendronate treatment. HLU increased TGF-β1 concentrations in this age group, and alendronate treatment reduced circulating TGF-β1. Age interacted with both HLU and alendronate treatment to increase circulating TGF-β1 concentrations. Simple linear regression analysis confirmed that muscle atrophy did not align with the levels of circulating TGF-β1 (all p > 0.05, Supplemental Fig. 4).

Figure 5.

Figure 5.

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ELISA cytokine analysis during hindlimb unloading (HLU) and bisphosphonate (alendronate) treatment (CTR+B) in 3-,12-, and 20-month-old mice. A) triceps surae muscle myostatin concentrations and B) serum (circulating) TGF-β1 concentrations. Significant main effects and interactions were apparent in both myostatin (age: p = 0.040; unloading: p = 0.002; bisphosphonate: p = 0.003; unloading x bisphosphonate: p=0.024) and TGF-β1 concentrations (age: p < 0.0001; unloading: p= 0.012; age x unloading: p= 0.013; age x bisphosphonate: p=0.006). Significance values of 3-way ANOVA with Tukey’s post-hoc analysis: * p-value < 0.05, ** p-value <0.01, *** p-value < 0.001, **** p-value < 0.0001.

3.4. Tendon Analysis

Tendon mechanical responses to unloading are most pronounced in older mice.

Force at maximum tensile load (MTL) was increased in 12-month-old mice undergoing alendronate treatment in both loaded and unloaded conditions (Fig. 6). Achilles tendons of 20-month-old mice had a higher modulus with alendronate treatment during HLU compared to control loaded and control HLU animals. Similarly, failure stress was reduced by alendronate treatment during HLU. Age and interactions between age and alendronate treatment significantly impacted tendon mechanical properties.

Figure 6.

Figure 6.

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Mechanical testing of Achilles tendon after hindlimb unloading (HLU) and bisphosphonate (alendronate) treatment (CTR+B) in 3-,12-, and 20-month-old mice. Achilles tendon mechanical properties: A) force at maximal tensile load; B) Young’s Modulus; C) tensile stress at ultimate tensile strength. Significance values of 3-way ANOVA with Tukey’s post-hoc analysis: * p-value < 0.05, ** p-value <0.01, *** p-value < 0.001, **** p-value < 0.0001.

4. Discussion

In this study, we investigated differences in bone, muscle, and tendon adaptation to mechanical unloading with bisphosphonate treatment in young, middle-aged, and old mice. Contrary to our hypothesis, muscle mass was not maintained with the use of bisphosphonates. We did, however, observe changes in musculoskeletal adaptation across the lifespan and in important crosstalk-related cytokine concentrations. Notably, this study demonstrates the significant effects of aging, mechanical unloading, and bisphosphonate treatment on musculoskeletal tissues osteokines.

Bone

Our results were partially consistent with our previous studies of bone loss during HLU in male mice[29]. The trabecular bone loss in young and old mice was consistent, with estimated reductions in metaphyseal trabecular bone between 30 and 45% (Supplemental Table 1). We noted a decrease in trabecular BV/TV between control and unloaded mice in young (−33.2%) and old (−33.6%), though the loss was not statistically significant in old animals. However, middle-aged mice had a less pronounced reduction in BV/TV than expected (−18.1%). We also observed less loss of cortical bone (BV/TV) than previously observed (10–15%) [29], especially in middle-aged mice (young: −9.1%, middle-aged: −2.6%, old: −5.1%). We noted similar changes to cortical thickness (young: −8.9%, middle-aged: −3.4 %, old: −6.2%). The differences between bone loss in middle-aged and young and old mice demonstrate an interesting resistance to unloading in middle-aged and older animals compared to their young counterparts. This is consistent with rat models, where older animals also demonstrate reduced response to unloading (e.g., nonsignificant loss) in trabecular bone during unloading compared to young animals [13, 39]. This underscores the importance of utilizing different age groups in unloading research with future clinical applications in mind.

Muscle

Whole body weight loss during HLU was consistent with the literature of similarly aged young and old mice (young: −14.4%, middle-aged: −14.9%, old: −11.2%, Supplemental Fig.1, Supplemental Table 2) [40]. However, our young and old mice lost more triceps surae muscle mass than the ~11% reported by other researchers [40]: we found a change of −19% in young mice and −18% in our old mice. Our middle-aged mice were more similar to their results, with a −12% loss in triceps surae mass. Loss of tibialis anterior mass was more consistent with their results (young: −12.9%, middle-aged: −7.5%, old: −13.1%). Loss of muscle mass in older animals indicates the risk of sarcopenia during unloading, which was not rescued by bisphosphonate use.

Muscle force production was significantly lower than observed in the literature, but activation methods differed (tibial nerve excitation [40] vs intramuscular gastrocnemius excitation). Our contractile force measurements demonstrated a loss of maximal plantar flexion force in young mice due to HLU, but no change in middle-aged mice and an increase in force production in old mice (young: −26.4%, middle-aged: +1%, old: +26.9%). However, several cohorts of our mice could not be tested due to equipment functionality. Thus, data collection for our old mice were incomplete limiting our ability to make strong conclusions.

The percentage of soleus Type I fibers decreased with age, but the size of fibers increased. One study by Chiu et al. has suggested that alendronate may prevent musculoskeletal atrophy during denervation through the SIRT3 pathway[41]. SIRT3 is a regulator of muscle metabolism which works by decreasing enzymatic activity through acetylation[42, 43]. Another group found that microgravity modulates SIRT3 expression in endothelial cells, pointing at an unloading effect. While we did not see a mass-based prevention of muscle atrophy, it is possible that alendronate increased muscle fiber type 1 size through this pathway. This would also explain the increase in muscle force seen in older animals with bisphosphonate treatment, as SIRT3 levels decrease with age[44]. In the gastrocnemius, type I and type IIa fiber cross-sectional area were reduced with age and unloading but not with alendronate treatment. This reduction in fiber type size does not explain the increase in muscle force production in the triceps surae in older mice. Additional mechanisms must be at play, such as SIRT3 regulation, increased specific force, or an increase in type IIb/x fiber area. Further exploration into possible mechanisms and SIRT3 regulation of muscle metabolism during unloading, aging and bisphosphonate use is warranted.

Bone-Muscle Crosstalk: Myostatin

Myostatin concentrations did not support our initial hypothesis that bone mass maintenance would influence changes in myokines during HLU. In young mice, myostatin concentrations increased with alendronate treatment during unloading, indicating increased myokine production during bone mass maintenance. No changes in myostatin concentration with unloading or treatment were apparent in middle-aged or old animals except for a trend with alendronate treatment in older animals undergoing unloading (Fig. 2.6). However, the interaction of age, loading, and alendronate treatment were all significant, as well as the interactions of with unloading and alendronate treatment. The small reduction of myostatin across the lifespan is consistent with the literature [45]. However, we did not see a decrease in muscle mass, and since the myostatin measures are made relative to muscle mass, not serum concentration, the claim that a reduction of muscle causes a loss of circulating myostatin does not directly apply to this observation.

Bone-Muscle Crosstalk: TGF-β1

Circulating TGF-β1 levels were age-dependent. Our results do not support the hypothesis that increased circulating TGF-β1 concentration caused by bone resorption impacts muscle atrophy in young or middle-aged mice. While unloading was implicated as an important main effect on circulating TGF-β1 concentrations (Fig. 2.6), alendronate treatment did not reduce circulating TGF-β1 in the young and middle-aged groups. The hypothesized effect was seen in older mice, where HLU increased circulating TGF-β, and alendronate treatment significantly reduced it. These results drive the main effects identified by ANOVA. These results do not support the current consensus in the literature that TGF-β1 is an important bone-to-muscle crosstalk agent in all animal ages [17, 19, 46, 47]. TGF-β1 release from bone may play a role in muscle atrophy; however, our data would suggest that this effect is only seen in old animals. This crosstalk does not account for the markedly large systemic increases seen in TGF-β1 during aging, as preventing its release from bone does not return circulating concentrations to that seen in younger animals.

Tendon

Aging, disuse, and injury have been shown to reduce tendon modulus[48–50], and our results support this. Bisphosphonate treatment in older animals did recover and improve tendon stiffness after unloading. Counterintuitively, we did not observe a change in tendon force with bisphosphonate usage and saw a decrease in failure stress in the same animal groups. These findings are in line with spontaneous tendon ruptures seen in some patients taking bisphosphonates[51, 52]. Additional investigation into the biological mechanisms of these changes is warranted.

Future Investigations

Several other cytokines related to musculoskeletal maintenance should be investigated to further determine the importance of bone-muscle crosstalk during unloading. RANK, RANKL, osteocalcin, BAIBA, and IL6, 7, and 15 all have the potential to drive bone and muscle atrophy [16, 17, 53–65]. Additionally, alendronate treatment could mask crosstalk due to direct effects on muscle [66]. Alendronate has been linked with upregulation of SIRT3 in muscle, which can inhibit myostatin production, leading to muscle mass maintenance [41]. Our data refutes this hypothesis showing that alendronate increases muscle myostatin concentration while decreasing muscle fiber size in the gastrocnemius. Interetstingly, our data showed an increase in type I fiber CSA in the soleus muscle of young and old animals with alendronate. The mechanism underlying this adaptation should be investigated further. Whether myostatin inhibition is important in bone-muscle crosstalk should also be studied further. Myostatin inhibitors are being used clinically to systemically inhibit myostatin, a myokine that blocks muscle growth [67, 68], from binding to its receptor on muscle cells [69, 70]. While this treatment is currently successful in research models such as mice [71, 72], clinical therapies have not been successful in combating atrophic diseases such as muscular dystrophy [72]. It is presently unknown whether preserving muscle tissue with myostatin inhibitors affects bone tissue by limiting the effect of this myokine on bone, or whether bisphosphonate treatments prevent osteokine transfer to, and subsequent effects on, muscle function. Differences in cytokine transfer between these tissues across the lifespan are also poorly understood.

Limitations

Sex is one of the most important sources of biological variation, and the musculoskeletal system’s responses to stimuli are no exception[73]. However, we only utilized male mice for this study. Female C57Bl/6 mice exhibit significantly reduced trabecular bone in long bones as they age[74], making a comparison of femoral trabecular bone loss difficult in this aging study. Unloading and injury have been shown to affect bone differently in male and female mice[34, 54], and as such, bisphosphonate use during unloading should be investigated in female mice to identify how sex alters these responses. Additionally, an equipment malfunction impeded our ability to collect force data for some mice during this study, resulting in a lower sample size for old HLU mice during muscle force analysis (n=2).”

5. Conclusion

This study used young, middle-aged, and old mice treated with alendronate to identify key differences in musculoskeletal adaptation to unloading across the lifespan. Notably, we confirmed that alendronate increased bone mass across all age groups and prevented HLU - induced bone loss. Alendronate treatment also tended to reduce body and muscle mass loss across the lifespan. The potential crosstalk osteokine TGF-β was an effective indicator of bone loss in old animals, but not in young or middle-aged animals. Further, TGF-β did not correlate with improved muscle mass caused by alendronate treatment. Together, these results indicate the importance of considering patient age during clinical applications of alendronate treatment during mechanical unloading or where muscle loss is a concern. Additional monitoring of muscle mass and function may be warranted for patients and those who experience periods of unloading, such as astronauts, to prevent muscle wasting.

Supplementary Material

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Highlights.

  • Age, unloading, and bisphosphonates impact musculoskeletal health during unloading

  • Bisphosphonates did not prevent muscle atrophy during mechanical unloading

  • Circulating TGF- β1 was only increased in old mice during mechanical unloading

  • TGF- β1 bone-to-muscle crosstalk was not sufficient to cause muscle atrophy seen during unloading

Acknowledgements

This research was supported by the NIH NIAMS (R01 AR071459-01). Sophie V Orr was supported by a NIAMS funded training program in Musculoskeletal Health Research (T32 AR079099). Natalie Kori Gilmore was supported by the UC Davis T32 in Pharmacology (T32 GM144303).

Footnotes

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Declaration of interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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Supplementary Materials

1
NIHMS2189493-supplement-1.pdf (35.2KB, pdf)
2
NIHMS2189493-supplement-2.pdf (78.6KB, pdf)
3
NIHMS2189493-supplement-3.pdf (437.3KB, pdf)
4
NIHMS2189493-supplement-4.pdf (88.5KB, pdf)
5
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