Low intensity mechanical signals promote proliferation in a cell-specific manner: Tailoring a non-drug strategy to enhance biomanufacturing yields.

Biomanufacturing relies on living cells to produce biotechnology-based therapeutics, tissue engineering constructs, vaccines, and a vast range of agricultural and industrial products. With the escalating demand for these bio-based products, any process that could improve yields and shorten outcome timelines by accelerating cell proliferation would have a significant impact across the discipline. While these goals are primarily achieved using biological or chemical strategies, harnessing cell mechanosensitivity represents a promising - albeit less studied - physical pathway to promote bioprocessing endpoints, yet identifying which mechanical parameters influence cell activities has remained elusive. We tested the hypothesis that mechanical signals, delivered non-invasively using low-intensity vibration (LIV; <1g, 10-500Hz), will enhance cell expansion, and determined that any unique signal configuration was not equally influential across a range of cell types. Varying frequency, intensity, duration, refractory period, and daily doses of LIV increased proliferation in CHO-adherent cells (+79% in 96h) using a particular set of LIV parameters (0.2g, 500Hz, 3x30 min/d, 2h refractory period), yet this same mechanical input suppressed proliferation in CHO-suspension cells (-13%). Exposing these same CHO-suspension cells to distinct LIV parameters (30Hz, 0.7g, 2x60 min/d, 2h refractory period) increased proliferation by 210%. Particle image velocimetry combined with finite element modeling showed high transmissibility of these signals across fluids (>90%), and LIV effectively scaled up to T75 flasks. Ultimately, when LIV is tailored to the target cell population, its highly efficient transmission across media represents a means to non-invasively augment biomanufacturing endpoints for both adherent and suspended cells, and holds immediate applications, ranging from small-scale, patient-specific personalized medicine to large-scale commercial bio-centric production challenges.

Low-Magnitude Mechanical Signals Combined with Zoledronic Acid Reduce Musculoskeletal Weakness and Adiposity in Estrogen-Deprived Mice.

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 (E 2 )-deprivation in young and skeletally mature mice. Complete E 2 -deprivation (surgical-ovariectomy (OVX) and daily injection of aromatase inhibitor (AI) letrozole) were performed on 8-week-old C57BL/6 female mice for 4 weeks following commencement of LIV administration or control (no LIV), for 28 weeks. Additionally, 16-week-old C57BL/6 female E 2 -deprived mice were administered ±LIV twice daily and supplemented with ±ZA (2.5 ng/kg/week). By week 28, lean tissue mass quantified by dual-energy X-ray absorptiometry was increased in younger OVX/AI+LIV(y) mice, with increased myofiber cross-sectional area of quadratus femorii. Grip strength was greater in OVX/AI+LIV(y) mice than OVX/AI(y) mice. Fat mass remained lower in OVX/AI+LIV(y) mice throughout the experiment compared with OVX/AI(y) mice. OVX/AI+LIV(y) mice exhibited increased glucose tolerance and reduced leptin and free fatty acids than OVX/AI(y) mice. Trabecular bone volume fraction and connectivity density increased in the vertebrae of OVX/AI+LIV(y) mice compared to OVX/AI(y) mice; however, this effect was attenuated in the older cohort of E 2 -deprived mice, specifically in OVX/AI+ZA mice, requiring combined LIV with ZA to increase trabecular bone volume and strength. Similar improvements in cortical bone thickness and cross-sectional area of the femoral mid-diaphysis were observed in OVX/AI+LIV+ZA mice, resulting in greater fracture resistance. Our findings demonstrate that the combination of mechanical signals in the form of LIV and anti-resorptive therapy via ZA improve vertebral trabecular bone and femoral cortical bone, increase lean mass, and reduce adiposity in mice undergoing complete E 2 -deprivation. One Sentence Summary: Low-magnitude mechanical signals with zoledronic acid suppressed bone and muscle loss and adiposity in mice undergoing complete estrogen deprivation. Translational Relevance: Postmenopausal patients with estrogen receptor-positive breast cancer treated with aromatase inhibitors to reduce tumor progression experience deleterious effects to bone and muscle subsequently develop muscle weakness, bone fragility, and adipose tissue accrual. Bisphosphonates (i.e., zoledronic acid) prescribed to inhibit osteoclast-mediated bone resorption are effective in preventing bone loss but may not address the non-skeletal effects of muscle weakness and fat accumulation that contribute to patient morbidity. Mechanical signals, typically delivered to the musculoskeletal system during exercise/physical activity, are integral for maintaining bone and muscle health; however, patients undergoing treatments for breast cancer often experience decreased physical activity which further accelerates musculoskeletal degeneration. Low-magnitude mechanical signals, in the form of low-intensity vibrations, generate dynamic loading forces similar to those derived from skeletal muscle contractility. As an adjuvant to existing treatment strategies, low-intensity vibrations may preserve or rescue diminished bone and muscle degraded by breast cancer treatment.

G protein-coupled receptor kinase 3 modulates mesenchymal stem cell proliferation and differentiation through sphingosine-1-phosphate receptor regulation.

BACKGROUND: The bone marrow niche supports hematopoietic cell development through intimate contact with multipotent stromal mesenchymal stem cells; however, the intracellular signaling, function, and regulation of such supportive niche cells are still being defined. Our study was designed to understand how G protein receptor kinase 3 (GRK3) affects bone marrow mesenchymal stem cell function by examining primary cells from GRK3-deficient mice, which we have previously published to have a hypercellular bone marrow and leukocytosis through negative regulation of CXCL12/CXCR4 signaling. METHODS: Murine GRK3-deficient bone marrow mesenchymal stromal cells were harvested and cultured to differentiate into three lineages (adipocyte, chondrocyte, and osteoblast) to confirm multipotency and compared to wild type cells. Immunoblotting, modified-TANGO experiments, and flow cytometry were used to further examine the effects of GRK3 deficiency on bone marrow mesenchymal stromal cell receptor signaling. Microcomputed tomography was used to determine trabecular and cortical bone composition of GRK3-deficient mice and standard ELISA to quantitate CXCL12 production from cellular cultures. RESULTS: GRK3-deficient, bone marrow-derived mesenchymal stem cells exhibit enhanced and earlier osteogenic differentiation in vitro. The addition of a sphingosine kinase inhibitor abrogated the osteogenic proliferation and differentiation, suggesting that sphingosine-1-phosphate receptor signaling was a putative G protein-coupled receptor regulated by GRK3. Immunoblotting showed prolonged ERK1/2 signaling after stimulation with sphingosine-1-phosphate in GRK3-deficient cells, and modified-TANGO assays suggested the involvement of ß-arrestin-2 in sphingosine-1-phosphate receptor internalization. CONCLUSIONS: Our work suggests that GRK3 regulates sphingosine-1-phosphate receptor signaling on bone marrow mesenchymal stem cells by recruiting ß-arrestin to the occupied GPCR to promote internalization, and lack of such regulation affects mesenchymal stem cell functionality.

Exercise to Mend Aged-tissue Crosstalk in Bone Targeting Osteoporosis & Osteoarthritis.

Aging induces alterations in bone structure and strength through a multitude of processes, exacerbating common aging- related diseases like osteoporosis and osteoarthritis. Cellular hallmarks of aging are examined, as related to bone and the marrow microenvironment, and ways in which these might contribute to a variety of age-related perturbations in osteoblasts, osteocytes, marrow adipocytes, chondrocytes, osteoclasts, and their respective progenitors. Cellular senescence, stem cell exhaustion, mitochondrial dysfunction, epigenetic and intracellular communication changes are central pathways and recognized as associated and potentially causal in aging. We focus on these in musculoskeletal system and highlight knowledge gaps in the literature regarding cellular and tissue crosstalk in bone, cartilage, and the bone marrow niche. While senolytics have been utilized to target aging pathways, here we propose non-pharmacologic, exercise-based interventions as prospective "senolytics" against aging effects on the skeleton. Increased bone mass and delayed onset or progression of osteoporosis and osteoarthritis are some of the recognized benefits of regular exercise across the lifespan. Further investigation is needed to delineate how cellular indicators of aging manifest in bone and the marrow niche and how altered cellular and tissue crosstalk impact disease progression, as well as consideration of exercise as a therapeutic modality, as a means to enhance discovery of bone-targeted therapies.

Mechanical suppression of breast cancer cell invasion and paracrine signaling to osteoclasts requires nucleo-cytoskeletal connectivity.

Exercise benefits the musculoskeletal system and reduces the effects of cancer. The effects of exercise are multifactorial, where metabolic changes and tissue adaptation influence outcomes. Mechanical signals, a principal component of exercise, are anabolic to the musculoskeletal system and restrict cancer progression. We examined the mechanisms through which cancer cells sense and respond to low-magnitude mechanical signals introduced in the form of vibration. Low-magnitude, high-frequency vibration was applied to human breast cancer cells in the form of low-intensity vibration (LIV). LIV decreased matrix invasion and impaired secretion of osteolytic factors PTHLH, IL-11, and RANKL. Furthermore, paracrine signals from mechanically stimulated cancer cells, reduced osteoclast differentiation and resorptive capacity. Disconnecting the nucleus by knockdown of SUN1 and SUN2 impaired LIV-mediated suppression of invasion and osteolytic factor secretion. LIV increased cell stiffness; an effect dependent on the LINC complex. These data show that mechanical vibration reduces the metastatic potential of human breast cancer cells, where the nucleus serves as a mechanosensory apparatus to alter cell structure and intercellular signaling.

Postural Stability in Obese Preoperative Bariatric Patients Using Static and Dynamic Evaluation.

INTRODUCTION: Globally, 300 million adults have clinical obesity. Heightened adiposity and inadequate musculature secondary to obesity alter bipedal stance and gait, diminish musculoskeletal tissue quality, and compromise neuromuscular feedback; these physiological changes alter stability and increase injury risk from falls. Studies in the field focus on obese patients across a broad range of body mass indices (BMI >30 kg/m2) but without isolating the most morbidly obese subset (BMI ≥40 kg/m2). We investigated the impact of obesity in perturbing postural stability in morbidly obese subjects elected for bariatric intervention, harboring a higher-spectrum BMI. SUBJECTS AND METHODS: Traditional force plate measurements and stabilograms are gold standards employed when measuring center of pressure (COP) and postural sway. To quantify the extent of postural instability in subjects with obesity before bariatric surgery, we assessed 17 obese subjects with an average BMI of 40 kg/m2 in contrast to 13 nonobese subjects with an average BMI of 30 kg/m2. COP and postural sway were measured from static and dynamic tasks. Involuntary movements were measured when patients performed static stances, with eyes either opened or closed. Two additional voluntary movements were measured when subjects performed dynamic, upper torso tasks with eyes opened. RESULTS: Mean body weight was 85% (p < 0.001) greater in obese than nonobese subjects. Following static balance assessments, we observed greater sway displacement in the anteroposterior (AP) direction in obese subjects with eyes open (87%, p < 0.002) and eyes closed (76%, p = 0.04) versus nonobese subjects. Obese subjects also exhibited a higher COP velocity in static tests when subjects' eyes were open (47%, p = 0.04). Dynamic tests demonstrated no differences between groups in sway displacement in either direction; however, COP velocity in the mediolateral (ML) direction was reduced (31%, p < 0.02) in obese subjects while voluntarily swaying in the AP direction, but increased in the same cohort when swaying in the ML direction (40%, p < 0.04). DISCUSSION AND CONCLUSION: Importantly, these data highlight obesity's contribution towards increased postural instability. Obese subjects exhibited greater COP displacement at higher AP velocities versus nonobese subjects, suggesting that clinically obese individuals show greater instability than nonobese subjects. Identifying factors contributory to instability could encourage patient-specific physical therapies and presurgical measures to mitigate instability and monitor postsurgical balance improvements.

Quantitative ultrasound imaging monitoring progressive disuse osteopenia and mechanical stimulation mitigation in calcaneus region through a 90-day bed rest human study.

BACKGROUND: Osteoporosis parallels aging and functional mechanical unloading (e.g., space flight and bed rest), jeopardizing mineral density, microstructure, and integrity of bone and leading to an increased risk of fracture. A way to combat this deterioration is to harness the sensitivity of bone to mechanical signals. OBJECTIVE: This study evaluates the longitudinal effect of a dynamic mechanical loading through the heel on human bone in vivo during 90-day bed rest, monitored by quantitative ultrasound (QUS) imaging and dual-energy X-ray absorptiometry (DXA) in localized regions of interests, i.e., calcaneus. METHODS: A total of 29 bed rest individuals were evaluated (11 control and 18 treatment) with a brief (10-minute) daily low-intensity (0.3g), high-frequency (30Hz) dynamic mechanical stimulation countermeasure through vibrational inhibition bone erosion (VIBE). Both QUS and DXA detected longitudinal bone density and quality changes. RESULTS: Ultrasound velocity (UV) decreased in the control group and increased in the group treated with low-intensity loading. The UV increased by 1.9% and 1.6% at 60- and 90-day bed rest (p=0.01) in VIBE over control groups. A trend was found in broadband ultrasound attenuation (BUA), with a VIBE benefit of 1.8% at day 60 and 0.5% at day 90 in comparison with control (p=0.5). Bone mineral density (BMD) assessed by DXA decreased -4.50% for control individuals and -2.18% for VIBE individuals, showing a moderate effect of the mechanical intervention (p=0.19). Significant correlations between QUS and DXA were observed, with a combined BUA and UV vs. BMD: r2=0.70. CONCLUSION: These results indicated that low-intensity, high-frequency loading has the potential to mitigate regional bone loss induced by long-term bed rest and that QUS imaging may be able to assess the subtle changes in bone alteration. TRANSLATIONAL POTENTIAL OF THIS ARTICLE: Quantitative ultrasound has shown the efficacy of noninvasively assessing bone mass and structural properties in cadaver and isolated trabecular bone samples. While its ability in measuring in vivo bone quality and density is still unclear, a scanning confocal ultrasound imaging is developed and can perform an instant assessment for the subtle changes of such bone loss. This ultrasound imaging modality can potentially be used in the clinical assessment of bone mass. Moreover, physical stimulation has shown the ability to prevent bone loss induced by functional disuse and estrogen deficiency in animal models. However, its treatment capability is unclear. This study has shown that low-magnitude mechanical signals, introduced using low-intensity vibration (LIV), can mitigate regional bone loss caused by functional disuse. Thus localized mechanical treatment, and the quantitative ultrasound imaging have shown translational potential to noninvasively attenuate bone loss, and assess bone mass in the clinic, e.g., in an extreme condition such as long-term space mission, and long-term bedrest such as in case of spinal cord injury.

Combating osteoporosis and obesity with exercise: leveraging cell mechanosensitivity.

Osteoporosis, a condition of skeletal decline that undermines quality of life, is treated with pharmacological interventions that are associated with poor adherence and adverse effects. Complicating efforts to improve clinical outcomes, the incidence of obesity is increasing, predisposing the population to a range of musculoskeletal complications and metabolic disorders. Pharmacological management of obesity has yet to deliver notable reductions in weight and debilitating complications are rarely avoided. By contrast, exercise shows promise as a non-invasive and non-pharmacological method of regulating both osteoporosis and obesity. The principal components of exercise - mechanical signals - promote bone and muscle anabolism while limiting formation and expansion of fat mass. Mechanical regulation of bone and marrow fat might be achieved by regulating functions of differentiated cells in the skeletal tissue while biasing lineage selection of their common progenitors - mesenchymal stem cells. An inverse relationship between adipocyte versus osteoblast fate selection from stem cells is implicated in clinical conditions such as childhood obesity and increased marrow adiposity in type 2 diabetes mellitus, as well as contributing to skeletal frailty. Understanding how exercise-induced mechanical signals can be used to improve bone quality while decreasing fat mass and metabolic dysfunction should lead to new strategies to treat chronic diseases such as osteoporosis and obesity.

Marrow Adiposity and Hematopoiesis in Aging and Obesity: Exercise as an Intervention.

PURPOSE OF REVIEW: Changes in the bone marrow microenvironment, which accompany aging and obesity, including increased marrow adiposity, can compromise hematopoiesis. Here, we review deleterious shifts in molecular, cellular, and tissue activity and consider the potential of exercise to slow degenerative changes associated with aging and obesity. RECENT FINDINGS: While bone marrow hematopoietic stem cells (HSC) are increased in frequency and myeloid-biased with age, the effect of obesity on HSC proliferation and differentiation remains controversial. HSC from both aged and obese environment have reduced hematopoietic reconstitution capacity following bone marrow transplant. Increased marrow adiposity affects HSC function, causing upregulation of myelopoiesis and downregulation of lymphopoiesis. Exercise, in contrast, can reduce marrow adiposity and restore hematopoiesis. The impact of marrow adiposity on hematopoiesis is determined mainly through correlations. Mechanistic studies are needed to determine a causative relationship between marrow adiposity and declines in hematopoiesis, which could aid in developing treatments for conditions that arise from disruptions in the marrow microenvironment.

Low-intensity vibration increases cartilage thickness in obese mice.

Obesity is associated with an elevated risk of osteoarthritis (OA). We examined here whether high fat diet administered in young mice, compromised the attainment of articular cartilage thickness. Further, we sought to determine if low-intensity vibration (LIV) could protect the retention of articular cartilage in a mouse model of diet-induced obesity. Five-week-old, male, C57BL/6 mice were separated into three groups (n = 10): Regular diet (RD), High fat diet (HF), and HF + LIV (HFv; 90 Hz, 0.2g, 30 min/d, 5 d/w) administered for 6 weeks. Additionally, an extended HF diet study was run for 6 months (LIV at 15 m/d). Articular cartilage and subchondral bone morphology, and sulfated GAG content were quantified using contrast agent enhanced µCT and histology. Gene expression within femoral condyles was quantified using real-time polymerase chain reaction. Contrary to our hypothesis, HF cartilage thickness was not statistically different from RD. However, LIV increased cartilage thickness compared to HF, and the elevated thickness was maintained when diet and LIV were extended into adulthood. RT-PCR analysis showed a reduction of aggrecan expression with high fat diet, while application of LIV reduced the expression of degradative MMP-13. Further, long-term HF diet resulted in subchondral bone thickening, compared to RD, providing early evidence of OA pathology-LIV suppressed the thickening, such that levels were not significantly different from RD. These data suggest that dynamic loading, via LIV, protected the retention of cartilage thickness, potentially resulting in joint surfaces better suited to endure the risks of elevated loading that parallel obesity. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:751-759, 2018.

Mechanical signals protect stem cell lineage selection, preserving the bone and muscle phenotypes in obesity.

The incidence of obesity is rapidly rising, increasing morbidity and mortality rates worldwide. Associated comorbidities include type 2 diabetes, heart disease, fatty liver disease, and cancer. The impact of excess fat on musculoskeletal health is still unclear, although it is associated with increased fracture risk and a decline in muscular function. The complexity of obesity makes understanding the etiology of bone and muscle abnormalities difficult. Exercise is an effective and commonly prescribed nonpharmacological treatment option, but it can be difficult or unsafe for the frail, elderly, and morbidly obese. Exercise alternatives, such as low-intensity vibration (LIV), have potential for improving musculoskeletal health, particularly in conditions with excess fat. LIV has been shown to influence bone marrow mesenchymal stem cell differentiation toward higher-order tissues (i.e., bone) and away from fat. While the exact mechanisms are not fully understood, recent studies utilizing LIV both at the bench and in the clinic have demonstrated some efficacy. Here, we discuss the current literature investigating the effects of obesity on bone, muscle, and bone marrow and how exercise and LIV can be used as effective treatments for combating the negative effects in the presence of excess fat.

Incorporating Refractory Period in Mechanical Stimulation Mitigates Obesity-Induced Adipose Tissue Dysfunction in Adult Mice.

OBJECTIVE: The aim of this study was to determine whether inclusion of a refractory period between bouts of low-magnitude mechanical stimulation (LMMS) can curb obesity-induced adipose tissue dysfunction and sequelae in adult mice. METHODS: A diet-induced obesity model that included a diet with 45% of kilocalories from fat was employed with intention to treat. C57BL/6J mice were weight matched into four groups: low-fat diet (LFD, n = 8), high-fat diet (HFD, n = 8), HFD with one bout of 30-minute LMMS (HFDv, n = 9), and HFD with two bouts of 15-minute LMMS with a 5-hour separation (refractory period, RHFDv, n = 9). Two weeks of diet was followed by 6 weeks of diet plus LMMS. RESULTS: HFD and HFDv mice continued gaining body weight and visceral adiposity throughout the experiment, which was mitigated in RHFDv mice. Compared with LFD mice, HFD and HFDv mice had increased rates of adipocyte hypertrophy, increased immune cell infiltration (B cells, T cells, and macrophages) into adipose tissue, increased adipose tissue inflammation (tumor necrosis factor alpha gene expression), and a decreased proportion of mesenchymal stem cells in adipose tissue, all of which were rescued in RHFDv mice. Glucose intolerance and insulin resistance were elevated in HFD and HFDv mice, but not in RHFDv mice, as compared with LFD mice. CONCLUSIONS: Incorporating a 5-hour refractory period between bouts of LMMS attenuates obesity-induced adipose tissue dysfunction and improves glucose metabolism.

The Efficacy of Low-intensity Vibration to Improve Bone Health in Patients with End-stage Renal Disease Is Highly Dependent on Compliance and Muscle Response.

RATIONAL AND OBJECTIVES: Low intensity vibration (LIV) may represent a nondrug strategy to mitigate bone deficits in patients with end-stage renal disease. MATERIALS AND METHODS: Thirty end-stage renal patients on maintenance hemodialysis were randomized to stand for 20 minutes each day on either an active or placebo LIV device. Analysis at baseline and completion of 6-month intervention included magnetic resonance imaging (tibia and fibula stiffness; trabecular thickness, number, separation, bone volume fraction, plate-to-rod ratio; and cortical bone porosity), dual-energy X-ray absorptiometry (hip and spine bone mineral density [BMD]), and peripheral quantitative computed tomography (tibia trabecular and cortical BMD; calf muscle cross-sectional area). RESULTS: Intention-to-treat analysis did not show any significant changes in outcomes associated with LIV. Subjects using the active device and with greater than the median adherence (70%) demonstrated an increase in distal tibia stiffness (5.3%), trabecular number (1.7%), BMD (2.3%), and plate-to-rod ratio (6.5%), and a decrease in trabecular separation (-1.8%). Changes in calf muscle cross-sectional area were associated with changes in distal tibia stiffness (R = 0.85), trabecular bone volume/total volume (R = 0.91), number (R = 0.92), and separation (R = -0.94) in the active group but not in the placebo group. Baseline parathyroid hormone levels were positively associated with increased cortical bone porosity over the 6-month study period in the placebo group (R = 0.55) but not in the active group (R = 0.01). No changes were observed in the nondistal tibia locations for either group except a decrease in hip BMD in the placebo group (-1.7%). CONCLUSION: Outcomes and adherence thresholds identified from this pilot study could guide future longitudinal studies involving vibration therapy.

Exercise Decreases Marrow Adipose Tissue Through ß-Oxidation in Obese Running Mice.

The relationship between marrow adipose tissue (MAT) and bone health is poorly understood. We used running exercise to ask whether obesity-associated MAT can be attenuated via exercise and whether this correlates with gains in bone quantity and quality. C57BL/6 mice were divided into diet-induced obesity (DIO, n = 14) versus low-fat diet (LFD, n = 14). After 3 months, 16-week-old mice were allocated to an exercise intervention (LFD-E, DIO-E) or a control group (LFD, DIO) for 6 weeks (4 groups, n = 7/group). Marrow adipocyte area was 44% higher with obesity (p < 0.0001) and after exercise 33% lower in LFD (p < 0.0001) and 39% lower in DIO (p < 0.0001). In LFD, exercise did not affect adipocyte number; however, in DIO, the adipocyte number was 56% lower (p < 0.0001). MAT was 44% higher in DIO measured by osmium-µCT, whereas exercise associated with reduced MAT (-23% in LFD, -48% in DIO, p < 0.05). MAT was additionally quantified by 9.4TMRI, and correlated with osmium-µCT (r = 0.645; p < 0.01). Consistent with higher lipid beta oxidation, perilipin 3 (PLIN3) rose with exercise in tibial mRNA (+92% in LFD, +60% in DIO, p < 0.05). Tibial µCT-derived trabecular bone volume (BV/TV) was not influenced by DIO but responded to exercise with an increase of 19% (p < 0.001). DIO was associated with higher cortical periosteal and endosteal volumes of 15% (p = 0.012) and 35% (p < 0.01), respectively, but Ct.Ar/Tt.Ar was lower by 2.4% (p < 0.05). There was a trend for higher stiffness (N/m) in DIO, and exercise augmented this further. In conclusion, obesity associated with increases in marrow lipid-measured by osmium-µCT and MRI-and partially due to an increase in adipocyte size, suggesting increased lipid uptake into preexisting adipocytes. Exercise associated with smaller adipocytes and less bone lipid, likely invoking increased ß-oxidation and basal lipolysis as evidenced by higher levels of PLIN3. © 2017 American Society for Bone and Mineral Research.

Low intensity vibration mitigates tumor progression and protects bone quantity and quality in a murine model of myeloma.

Myeloma facilitates destruction of bone and marrow. Since physical activity encourages musculoskeletal preservation we evaluated whether low-intensity vibration (LIV), a means to deliver mechanical signals, could protect bone and marrow during myeloma progression. Immunocompromised-mice (n=25) were injected with human-myeloma cells, while 8 (AC) were saline-injected. Myeloma-injected mice (LIV; n=13) were subjected to daily-mechanical loading (15min/d; 0.3g @ 90Hz) while 12 (MM) were sham-handled. At 8w, femurs had 86% less trabecular bone volume fraction (BV/TV) in MM than in AC, yet only a 21% decrease in LIV was observed in comparison to AC, reflecting a 76% increase versus MM. Cortical BV was 21% and 15% lower in MM and LIV, respectively, than in AC; LIV showing 30% improvement over MM. Similar outcomes were observed in the axial skeleton, showing a 35% loss in MM with a 27% improved retention of bone in the L5 of LIV-treated mice as compared to MM. Transcortical-perforations in the femur from myeloma-induced osteolysis were 9× higher in MM versus AC, reduced by 57% in LIV. Serum-TRACP5b, 61% greater in MM versus AC, rose by 33% in LIV compared to AC, a 45% reduction in activity when compared to MM. Histomorphometric analyses of femoral trabecular bone demonstrated a 70% elevation in eroded surfaces of MM versus AC, while measures in LIV were 58% below those in MM. 72% of marrow in the femur of MM mice contained tumor, contrasted by a 31% lower burden in LIV. MM mice (42%) presented advanced-stage necrosis of tibial marrow while present in just 8% of LIV. Myeloma infiltration inversely correlated to measures of bone quality, while LIV slowed the systemic, myeloma-associated decline in bone quality and inhibited tumor progression through the hindlimbs.

Cell Mechanosensitivity is Enabled by the LINC Nuclear Complex.

Mechanoresponses in mesenchymal stem cells (MSCs) guide both differentiation and function. In this review, we focus on advances in0 our understanding of how the cytoplasmic cytoskeleton, nuclear envelope and nucleoskeleton, which are connected via LINC (Linker of Nucleoskeleton and Cytoskeleton) complexes, are emerging as an integrated dynamic signaling platform to regulate MSC mechanobiology. This dynamic interconnectivity affects mechanical signaling and transfer of signals into the nucleus. In this way, nuclear and LINC-mediated cytoskeletal connectivity play a critical role in maintaining mechanical signaling that affects MSC fate by serving as both mechanosensory and mechanoresponsive structures. We review disease and age related compromises of LINC complexes and nucleoskeleton that contribute to the etiology of musculoskeletal diseases. Finally we invite the idea that acquired dysfunctions of LINC might be a contributing factor to conditions such as aging, microgravity and osteoporosis and discuss potential mechanical strategies to modulate LINC connectivity to combat these conditions.

Effect of Low-Magnitude Mechanical Stimuli on Bone Density and Structure in Pediatric Crohn's Disease: A Randomized Placebo-Controlled Trial.

Pediatric Crohn's Disease (CD) is associated with low trabecular bone mineral density (BMD), cortical area, and muscle mass. Low-magnitude mechanical stimulation (LMMS) may be anabolic. We conducted a 12-month randomized double-blind placebo-controlled trial of 10 minutes daily exposure to LMMS (30 Hz frequency, 0.3 g peak-to-peak acceleration). The primary outcomes were tibia trabecular BMD and cortical area by peripheral quantitative CT (pQCT) and vertebral trabecular BMD by QCT; additional outcomes included dual-energy X-ray absorptiometry (DXA) whole body, hip and spine BMD, and leg lean mass. Results were expressed as sex-specific Z-scores relative to age. CD participants, ages 8 to 21 years with tibia trabecular BMD <25th percentile for age, were eligible and received daily cholecalciferol (800 IU) and calcium (1000 mg). In total, 138 enrolled (48% male), and 121 (61 active, 60 placebo) completed the 12-month trial. Median adherence measured with an electronic monitor was 79% and did not differ between arms. By intention-to-treat analysis, LMMS had no significant effect on pQCT or DXA outcomes. The mean change in spine QCT trabecular BMD Z-score was +0.22 in the active arm and -0.02 in the placebo arm (difference in change 0.24 [95% CI 0.04, 0.44]; p = 0.02). Among those with >50% adherence, the effect was 0.38 (95% CI 0.17, 0.58, p < 0.0005). Within the active arm, each 10% greater adherence was associated with a 0.06 (95% CI 0.01, 1.17, p = 0.03) greater increase in spine QCT BMD Z-score. Treatment response did not vary according to baseline body mass index (BMI) Z-score, pubertal status, CD severity, or concurrent glucocorticoid or biologic medications. In all participants combined, height, pQCT trabecular BMD, and cortical area and DXA outcomes improved significantly. In conclusion, LMMS was associated with increases in vertebral trabecular BMD by QCT; however, no effects were observed at DXA or pQCT sites. © 2016 American Society for Bone and Mineral Research.

Associations of Computed Tomography-Based Trunk Muscle Size and Density With Balance and Falls in Older Adults.

BACKGROUND: Deficits in balance and muscle function are important risk factors for falls in older adults. Aging is associated with significant declines in muscle size and density, but associations of trunk muscle size and density with balance and falls in older adults have not been previously examined. METHODS: Trunk muscle size (cross-sectional area) and attenuation (a measure of tissue density) were measured in computed tomography scans (at the L2 lumbar level) in a cohort of older adults (mean ± SD age of 81.9±6.4) residing in independent living communities. Outcome measures were postural sway measured during quiet standing and Short Physical Performance Battery (SPPB) at baseline, and falls reported by participants for up to 3 years after baseline measurements. RESULTS: Higher muscle density was associated with reduced postural sway, particularly sway velocities, in both men and women, and better Short Physical Performance Battery score in women, but was not associated with falls. Larger muscle size was associated with increased postural sway in men and women and with increased likelihood of falling in men. CONCLUSIONS: The results suggest that balance depends more on muscle quality than on the size of the muscle. The unexpected finding that larger muscle size was associated with increased postural sway and increased fall risk requires further investigation, but highlights the importance of factors besides muscle size in muscle function in older adults.

Focal enhancement of the skeleton to exercise correlates with responsivity of bone marrow mesenchymal stem cells rather than peak external forces.

Force magnitudes have been suggested to drive the structural response of bone to exercise. As importantly, the degree to which any given bone can adapt to functional challenges may be enabled, or constrained, by regional variation in the capacity of marrow progenitors to differentiate into bone-forming cells. Here, we investigate the relationship between bone adaptation and mesenchymal stem cell (MSC) responsivity in growing mice subject to exercise. First, using a force plate, we show that peak external forces generated by forelimbs during quadrupedal locomotion are significantly higher than hindlimb forces. Second, by subjecting mice to treadmill running and then measuring bone structure with µCT, we show that skeletal effects of exercise are site-specific but not defined by load magnitudes. Specifically, in the forelimb, where external forces generated by running were highest, exercise failed to augment diaphyseal structure in either the humerus or radius, nor did it affect humeral trabecular structure. In contrast, in the ulna, femur and tibia, exercise led to significant enhancements of diaphyseal bone areas and moments of area. Trabecular structure was also enhanced by running in the femur and tibia. Finally, using flow cytometry, we show that marrow-derived MSCs in the femur are more responsive to exercise-induced loads than humeral cells, such that running significantly lowered MSC populations only in the femur. Together, these data suggest that the ability of the progenitor population to differentiate toward osteoblastogenesis may correlate better with bone structural adaptation than peak external forces caused by exercise.

Diminished satellite cells and elevated adipogenic gene expression in muscle as caused by ovariectomy are averted by low-magnitude mechanical signals.

Age-related degeneration of the musculoskeletal system, accelerated by menopause, is further complicated by increased systemic and muscular adiposity. The purpose of this study was to identify at the molecular, cellular, and tissue levels the impact of ovariectomy on adiposity and satellite cell populations in mice and whether mechanical signals could influence any outcomes. Eight-week-old C57BL/6 mice were ovariectomized, with one half subjected to low-intensity vibration (LIV; 0.3 g/90 Hz, 15 min/day, 5 day/wk; n = 10) for 6 wk and the others sham vibrated (OVX; n = 10). Data are compared with age-matched, intact controls (AC; n = 10). In vivo µCT analysis showed that OVX mice gained 43% total (P < 0.001) and 125% visceral adiposity (P < 0.001) compared with their baseline after 6 wk, whereas LIV gained only 21% total (P = 0.01) and 70% visceral adiposity (P < 0.01). Relative to AC, expression of adipogenic genes (PPARγ, FABP4, PPARδ, and FoxO1) was upregulated in OVX muscle (P < 0.05), whereas LIV reduced these levels (P < 0.05). Adipogenic gene expression was inversely related to the percentage of total and reserve satellite cell populations in the muscle, with both declining in OVX compared with AC (-21 and -28%, respectively, P < 0.01). LIV mitigated these declines (-11 and -17%, respectively). These results provide further evidence of the negative consequences of estrogen depletion and demonstrate that mechanical signals have the potential to interrupt subsequent adipogenic gene expression and satellite cell suppression, emphasizing the importance of physical signals in protecting musculoskeletal integrity and slowing the fat phenotype.