A comparison of bone microarchitectural and transcriptomic changes in murine long bones in response to hindlimb unloading and aging.

Age- and disuse-related bone loss both result in decreases in bone mineral density, cortical thickness, and trabecular thickness and connectivity. Disuse induces changes in the balance of bone formation and bone resorption like those seen with aging. There is a need to experimentally compare these two mechanisms at a structural and transcriptomic level to better understand how they may be similar or different. Bone microarchitecture and biomechanical properties were compared between 6- and 22-month-old C57BL/6 J male control mice and 6-month-old mice that were hindlimb unloaded (HLU) for 3 weeks. Epiphyseal trabecular bone was the compartment most affected by HLU and demonstrated an intermediate bone phenotype between age-matched controls and aged controls. RNA extracted from whole-bone marrow-flushed tibiae was sequenced and analyzed. Differential gene expression analysis additionally included 4-month-old male mice unloaded for 3 weeks compared to age-matched controls. Gene ontology analysis demonstrated that there were age-dependent differences in differentially expressed genes in young adult mice. Genes related to downregulation of cellular processes were most affected in 4-month-old mice after disuse whereas those related to mitochondrial function were most affected in 6-month-old mice. Cell-cycle transition was downregulated with aging. A publicly available dataset (GSE169292) from 3-month female C57BL/6 N mice unloaded for 7 days was included in ingenuity pathway analysis (IPA) with the other datasets. IPA was used to identify the leading canonical pathways and upstream regulators in each HLU age group. IPA identified "Senescence Pathway" as the second leading canonical pathway enriched in mice exposed to HLU. HLU induced activation of the senescence pathway in 3-month and 4-month-old mice but inhibited it in 6-month-old mice. In conclusion, we demonstrate that hindlimb unloading and aging initiate similar changes in bone microarchitecture and gene expression. However, aging is responsible for more significant transcriptome and tissue-level changes compared to hindlimb unloading.

Hindlimb Unloading Induces Bone Microarchitectural and Transcriptomic Changes in Murine Long Bones in an Age-Dependent Manner.

Age and disuse-related bone loss both result in decreases in bone mineral density, cortical thickness, and trabecular thickness and connectivity. Disuse induces physiological changes in bone like those seen with aging. Bone microarchitecture and biomechanical properties were compared between 6- and 22-month-old C57BL/6J male control mice and 6-month-old mice that were hindlimb unloaded (HLU) for 3 weeks. Epiphyseal trabecular bone was the compartment most affected by HLU and demonstrated an intermediate bone phenotype between age-matched controls and aged controls. RNA extracted from whole-bone marrow-flushed tibiae was sequenced and analyzed. Differential gene expression analysis additionally included 4-month-old male mice unloaded for 3 weeks compared to age-matched controls. Gene ontology analysis demonstrated that there were age-dependent differences in differentially expressed genes. Genes related to downregulation of cellular processes were most affected in 4-month-old mice after disuse whereas those related to mitochondrial function were most affected in 6- month-old mice. Cell-cycle transition was downregulated with aging. A publicly available dataset (GSE169292) from 3-month female C57BL/6N mice unloaded for 7 days was included in ingenuity pathway analysis with the other datasets. IPA was used to identify the leading canonical pathways and upstream regulators in each HLU age group. IPA identified "Senescence Pathway" as the second leading canonical pathway enriched in mice exposed to HLU. HLU induced activation of the senescence pathway in 3- month and 4-month-old mice but inhibited it in 6-month-old mice. In conclusion, we demonstrate that hindlimb unloading and aging initiate similar changes in bone microarchitecture and gene expression. However, aging is responsible for more significant transcriptome and tissue-level changes compared to hindlimb unloading. Highlights: Epiphyseal trabecular bone is most susceptible to hindlimb unloading.Hindlimb unloaded limbs resemble an intermediate phenotype between age-matched and aged controls.Hindlimb unloading induces gene expression changes that are age dependent and may lead to inflammation and/or mitochondrial dysfunction depending on context.Younger mice (3-4 months) activate the senescence pathway upon hindlimb unloading, whereas skeletally mature (6 months) mice inhibit it.

Reambulation following hindlimb unloading attenuates disuse-induced changes in murine fracture healing.

Patients with bone and muscle loss from prolonged disuse have higher risk of falls and subsequent fragility fractures. In addition, fracture patients with continued disuse and/or delayed physical rehabilitation have worse clinical outcomes compared to individuals with immediate weight-bearing activity following diaphyseal fracture. However, the effects of prior disuse followed by physical reambulation on fracture healing cellular processes and adjacent bone and skeletal muscle recovery post-injury remains poorly defined. To bridge this knowledge gap and inform future treatment and rehabilitation strategies for fractures, a preclinical model of fracture healing with a history of prior unloading with and without reambulation was employed. First, skeletally mature male and female C57BL/6J mice (18 weeks) underwent hindlimb unloading by tail suspension (HLU) for 3 weeks to induce significant bone and muscle loss modeling enhanced bone fragility. Next, mice had their right femur fractured by open surgical dissection (stabilized with 24-gauge pin). Then, mice were randomly assigned to continued HLU or allowed normal weight-bearing reambulation (HLU + R). Mice given normal cage activity throughout the experiment served as healthy age-matched controls. All mice were sacrificed 4-days (DPF4) or 14-days (DPF14) following fracture to assess healing and uninjured hindlimb musculoskeletal properties (6-10 mice per treatment group/biological sex/timepoint). We found that continued disuse following fracture led to severely diminished uninjured hindlimb skeletal muscle mass (gastrocnemius and soleus) and femoral bone volume adjacent to the fracture site compared to healthy age-matched controls across mouse sexes. Furthermore, HLU led to significantly decreased periosteal expansion (DPF4) and osteochondral tissue formation by DPF14, and trends in increased osteoclastogenesis (DPF14) and decreased woven bone vascular area (DPF14). In contrast, immediate reambulation for 2 weeks after fracture, even following a period of prolonged disuse, was able to increase hindlimb skeletal tissue mass and increase osteochondral tissue formation, albeit not to healthy control levels, in both mouse sexes. Furthermore, reambulation attenuated osteoclast formation seen in woven bone tissue undergoing disuse. Our results suggest that weight-bearing skeletal loading in both sexes immediately following fracture may improve callus healing and prevent further fall risk by stimulating skeletal muscle anabolism and decreasing callus resorption compared to minimal or delayed rehabilitation regimens.

Global cannabinoid receptor 1 deficiency affects disuse-induced bone loss in a site-specific and sex-dependent manner.

Bone loss during mechanical unloading increases fracture risk and is a major concern for the general population and astronauts during spaceflight. The endocannabinoid system (ECS) plays an important role in bone metabolism. One of the main ECS receptors, cannabinoid receptor 1 (CB1), has been studied in regards to basic bone metabolism; however, little is known as to how CB1 and the ECS affect bone in different mechanical environments. In this study, we analyzed the influence of global CB1 deficiency and sex on mice during disuse caused by single limb immobilization. Female mice were more sensitive to disuse-induced BV/TV loss than males in both the femoral metaphysis and tibial epiphysis. Genotype also affected bone loss in a sex-dependent manner, with male mice deficient in CB1 receptors (CB1KO) and female wildtype (WT) mice experiencing increased bone loss in both the tibial metaphysis and femoral epiphysis. Genotype affected the response to disuse as CB1KO mice displayed greater changes in femoral ultimate force, along with lower tibial ultimate stress, compared to WT mice. Female mice had a significantly higher femoral, and lower tibial ultimate force compared to male mice. These results reveal that disuse-induced bone loss due to CB1 deficiency is sex-dependent. CB1 deficiency in male mice exacerbated bone loss, while in females CB1 deficiency appeared to protect against disuse-induced bone loss. Regardless of genotype, female mice were more sensitive than males to disuse. These results suggest that CB1 receptors may represent a potential therapeutic target for mitigation of disuse-induced bone loss.

Update on the effects of microgravity on the musculoskeletal system.

With the reignited push for manned spaceflight and the development of companies focused on commercializing spaceflight, increased human ventures into space are inevitable. However, this venture would not be without risk. The lower gravitational force, known as microgravity, that would be experienced during spaceflight significantly disrupts many physiological systems. One of the most notably affected systems is the musculoskeletal system, where exposure to microgravity causes both bone and skeletal muscle loss, both of which have significant clinical implications. In this review, we focus on recent advancements in our understanding of how exposure to microgravity affects the musculoskeletal system. We will focus on the catabolic effects microgravity exposure has on both bone and skeletal muscle cells, as well as their respective progenitor stem cells. Additionally, we report on the mechanisms that underlie bone and muscle tissue loss resulting from exposure to microgravity and then discuss current countermeasures being evaluated. We reveal the gaps in the current knowledge and expound upon how current research is filling these gaps while also identifying new avenues of study as we continue to pursue manned spaceflight.