Low-Intensity Vibration Protects the Weight-Bearing Skeleton and Suppresses Fracture Incidence in Boys With Duchenne Muscular Dystrophy: A Prospective, Randomized, Double-Blind, Placebo-Controlled Clinical Trial.

The ability of low-intensity vibration (LIV) to combat skeletal decline in Duchenne Muscular Dystrophy (DMD) was evaluated in a randomized controlled trial. Twenty DMD boys were enrolled, all ambulant and treated with glucocorticoids (mean age 7.6, height-adjusted Z-scores [HAZ] of hip bone mineral density [BMD] -2.3). Ten DMD boys were assigned to stand for 10 min/d on an active LIV platform (0.4 g at 30 Hz), while 10 stood on a placebo device. Baseline and 14-month bone mineral content (BMC) and BMD of spine, hip, and total body were measured with DXA, and trabecular bone density (TBD) of tibia with quantitative computed tomography (QCT). All children tolerated the LIV intervention well, with daily compliance averaging 78%. At 14 months, TBD in the proximal and distal tibia remained unchanged in placebo subjects (-1.0% and -0.2%), while rising 3.5% and 4.6% in LIV subjects. HAZ for hip BMD and BMC in the placebo group declined 22% and 13%, respectively, contrasting with no change from baseline (0.9% and 1.4%) in the LIV group. Fat mass in the leg increased 32% in the placebo group, contrasting with 21% in LIV subjects. Across the 14-month study, there were four incident fractures in three placebo patients (30%), with no new fractures identified in LIV subjects. Despite these encouraging results, a major limitation of the study is-despite randomized enrollment-that there was a significant difference in age between the two cohorts, with the LIV group being 2.8y older, and thus at greater severity of disease. In sum, these data suggest that noninvasive LIV can help protect the skeleton of DMD children against the disease progression, the consequences of diminished load bearing, and the complications of chronic steroid use. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Dose-dependent effects of pharmaceutical treatments on bone matrix properties in ovariectomized rats.

As both anabolic and anti-catabolic osteoporosis drugs affect bone formation and resorption processes, they may contribute to bone's overall mechanical behavior by altering the quality of the bone matrix. We used an ovariectomized rat model and a novel fracture mechanics approach to investigate whether treatment with an anabolic (parathyroid hormone) or anti-catabolic (alendronate) osteoporosis drugs will alter the organic and mineral matrix components and consequently cortical bone fracture toughness. Ovariectomized (at 5 months age) rats were treated with either parathyroid hormone or alendronate at low and high doses for 6 months (age 6-12 months). Specifically, treatment groups included untreated ovariectomized controls (n = 9), high-dose alendronate (n = 10), low-dose alendronate (n = 9), high-dose parathyroid hormone (n = 10), and low-dose parathyroid hormone (n = 9). After euthanasia, cortical microbeams from the lateral quadrant were extracted, notched, and tested in 3-point bending to measure fracture toughness. Portions of the bone were used to measure changes in the 1) organic matrix through quantification of advanced glycation end-products (AGEs) and non-collagenous proteins, and 2) mineral matrix through assessment of mineral crystallinity. Compared to the ovariectomized group, rats treated with high doses of parathyroid hormone and alendronate had significantly increased cortical bone fracture toughness, which corresponded primarily to increased non-collagenous proteins while there was no change in AGEs. Additionally, low-dose PTH treatment increased matrix crystallinity and decreased AGE levels. In summary, ovariectomized rats treated with pharmaceutical drugs had increased non-collagenous matrix proteins and improved fracture toughness compared to controls. Further investigation is required for different doses and longer treatment periods.

Reporting Guidelines for Whole-Body Vibration Studies in Humans, Animals and Cell Cultures: A Consensus Statement from an International Group of Experts.

Whole-body vibration (WBV) is an exercise modality or treatment/prophylaxis method in which subjects (humans, animals, or cells) are exposed to mechanical vibrations through a vibrating platform or device. The vibrations are defined by their direction, frequency, magnitude, duration, and the number of daily bouts. Subjects can be exposed while performing exercises, hold postures, sitting, or lying down. Worldwide, WBV has attracted significant attention, and the number of studies is rising. To interpret, compare, and aggregate studies, the correct, complete, and consistent reporting of WBV-specific data (WBV parameters) is critical. Specific reporting guidelines aid in accomplishing this goal. There was a need to expand existing guidelines because of continuous developments in the field of WBV research, including but not limited to new outcome measures regarding brain function and cognition, modified designs of WBV platforms and attachments (e.g., mounting a chair on a platform), and comparisons of animal and cell culture studies with human studies. Based on Delphi studies among experts and using EQUATOR recommendations, we have developed extended reporting guidelines with checklists for human and animal/cell culture research, including information on devices, vibrations, administration, general protocol, and subjects. In addition, we provide explanations and examples of how to report. These new reporting guidelines are specific to WBV variables and do not target research designs in general. Researchers are encouraged to use the new WBV guidelines in addition to general design-specific guidelines.

Modeling stem cell nucleus mechanics using confocal microscopy.

Nuclear mechanics is emerging as a key component of stem cell function and differentiation. While changes in nuclear structure can be visually imaged with confocal microscopy, mechanical characterization of the nucleus and its sub-cellular components require specialized testing equipment. A computational model permitting cell-specific mechanical information directly from confocal and atomic force microscopy of cell nuclei would be of great value. Here, we developed a computational framework for generating finite element models of isolated cell nuclei from multiple confocal microscopy scans and simple atomic force microscopy (AFM) tests. Confocal imaging stacks of isolated mesenchymal stem cells were converted into finite element models and siRNA-mediated Lamin A/C depletion isolated chromatin and Lamin A/C structures. Using AFM-measured experimental stiffness values, a set of conversion factors were determined for both chromatin and Lamin A/C to map the voxel intensity of the original images to the element stiffness, allowing the prediction of nuclear stiffness in an additional set of other nuclei. The developed computational framework will identify the contribution of a multitude of sub-nuclear structures and predict global nuclear stiffness of multiple nuclei based on simple nuclear isolation protocols, confocal images and AFM tests.

Towards reporting guidelines of research using whole-body vibration as training or treatment regimen in human subjects-A Delphi consensus study.

BACKGROUND: Whole-body vibration (WBV) is a method utilizing vibrating platforms to expose individuals to mechanical vibration. In its various applications, it has been linked to improved muscular, skeletal, metabolic, or cognitive functioning, quality of life, and physiological parameters such as blood pressure. Most evidence concerning WBV is inconclusive and meta-analytical reviews may not readily produce insights since the research has a risk of misunderstandings of vibration parameters and incomplete reporting occurs. This study aims at laying an empirical foundation for reporting guidelines for human WBV studies to improve the quality of reporting and the currently limited comparability between studies. METHOD: The Delphi methodology is employed to exploit the integrated knowledge of WBV experts to distil the specific aspects of WBV methodology that should be included in such guidelines. Over three rounds of completing online questionnaires, the expert panel (round 1/2/3: 51/40/37 experts respectively from 17 countries with an average of 19.4 years of WBV research experience) rated candidate items. RESULTS: A 40-item list was established based on the ratings of the individual items from the expert panel with a large final consensus (94.6%). CONCLUSION: The final consensus indicates comprehensiveness and valuableness of the list. The results are in line with previous guidelines but expand these extensively. The present results may therefore serve as a foundation for updated guidelines for reporting human WBV studies in order to improve the quality of reporting of WBV studies, improve comparability of studies and facilitate the development of WBV study designs.

Two-dimensional graphene oxide-reinforced porous biodegradable polymeric nanocomposites for bone tissue engineering.

This study investigates the mechanical properties and in vitro cytotoxicity of two-dimensional (2D) graphene oxide nanoribbons and nanoplatelets (GONRs and GONPs) reinforced porous polymeric nanocomposites. Highly porous poly(propylene fumarate) (PPF) nanocomposites were prepared by dispersing 0.2 wt % single- and multiwalled SONRs (SWGONRs and MWGONRs) and GONPs. The mechanical properties of scaffolds were characterized using compression testing and in vitro cytocompatibility was assessed using QuantiFlour assay for cellularity and PrestoBlue assay for cell viability. Immunofluorescence was used to assess collagen-I expression and deposition in the extracellular matrix. Porous PPF scaffolds were used as a baseline control and porous single and multiwalled carbon nanotubes (SWCNTs and MWCNTs) reinforced nanocomposites were used as positive controls. Results show that incorporation of 2D graphene nanomaterials leads to an increase in the mechanical properties of porous PPF nanocomposites with following the trend: MWGONRs > GONPs > SWGONRs > MWCNTs > SWCNTs > PPF control. MWGONRs showed the best enhancement of compressive mechanical properties with increases of up to 26% in compressive modulus (i.e., Young's modulus), ~60% in yield strength, and ~24% in the ultimate compressive strength. Addition of 2D nanomaterials did not alter the cytocompatibility of porous PPF nanocomposites. Furthermore, PPF nanocomposites reinforced with SWGONRs, MWGONRs, and GONPs show an improvement in the adsorption of collagen-I compared to PPF baseline control. The results of this study show that 2D graphene nanomaterial reinforced porous PPF nanocomposites possess superior mechanical properties, cytocompatibility, and increased protein adsorption. The favorable cytocompatibility results opens avenues for in vivo safety and efficacy studies for bone tissue engineering applications. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 1143-1153, 2019.

Differential Efficacy of 2 Vibrating Orthodontic Devices to Alter the Cellular Response in Osteoblasts, Fibroblasts, and Osteoclasts.

Modalities that increase the rate of tooth movement have received considerable attention, but direct comparisons between devices are rare. Here, we contrasted 2 mechanical vibratory devices designed to directly transfer vibrations into alveolar bone as a means to influence bone remodeling. To this end, 3 cells types intimately involved in modulating tooth movements-osteoblasts, periodontal ligament fibroblasts, and osteoclasts-were subjected to in vitro vibrations at bout durations prescribed by the manufacturers. As quantified by an accelerometer, vibration frequency and peak accelerations were 400% and 70% greater in the VPro5 (Propel Orthodontics) than in the AcceleDent (OrthoAccel Technologies) device. Both devices caused increased cell proliferation and gene expression in osteoblasts and fibroblasts, but the response to VPro5 treatment was greater than for the AcceleDent. In contrast, the ability to increase osteoclast activity was device independent. These data present an important first step in determining how specific cell types important for facilitating tooth movement respond to different vibration profiles. The device that engendered a higher vibration frequency and larger acceleration (VPro5) was superior in stimulating osteoblast and fibroblast cell proliferation/gene expression, although the duration of each treatment bout was 75% shorter than for the AcceleDent.

Low-Intensity Vibration Improves Muscle Healing in a Mouse Model of Laceration Injury.

Recovery from traumatic muscle injuries is typically prolonged and incomplete, leading to impaired muscle and joint function. We sought to determine whether mechanical stimulation via whole-body low-intensity vibration (LIV) could (1) improve muscle regeneration and (2) reduce muscle fibrosis following traumatic injury. C57BL/6J mice were subjected to a laceration of the gastrocnemius muscle and were treated with LIV (0.2 g at 90 Hz or 0.4 g at 45 Hz for 30 min/day) or non-LIV sham treatment (controls) for seven or 14 days. Muscle regeneration and fibrosis were assessed in hematoxylin and eosin or Masson's trichrome stained muscle cryosections, respectively. Compared to non-LIV control mice, the myofiber cross-sectional area was larger in mice treated with each LIV protocol after 14 days of treatment. Minimum fiber diameter was also larger in mice treated with LIV of 90 Hz/0.2 g after 14 days of treatment. There was also a trend toward a reduction in collagen deposition after 14 days of treatment with 45 Hz/0.4 g (p = 0.059). These findings suggest that LIV may improve muscle healing by enhancing myofiber growth and reducing fibrosis. The LIV-induced improvements in muscle healing suggest that LIV may represent a novel therapeutic approach for improving the healing of traumatic muscle injuries.

Absence of complement factor H alters bone architecture and dynamics.

Complement system is an important arm of the immune system that promotes inflammation. Complement Factor H (FH) is a critical regulator of the alternative complement pathway. Its absence causes pathology in different organs resulting in diseases such as age related macular degeneration and dense deposit disease. Recent studies suggest that the complement system plays a role in bone development and homeostasis. To determine the role of FH in bone architecture, we studied the FH knockout (fh-/-) mice. 3D reconstructions of femur from 16 week old fh-/- mice reveal significant changes, such as decreased BV/TV (4.5%, p < 0.02), trabecular number (22%, p < 0.01), tissue mineral density (16%, p < 0.04), and increased marrow area (16% p < 0.01), compared to their wild type (WT) counterparts. Kidney function and histology remained normal indicating that bone changes occurred prior to kidney dysfunction. Next we examined cultured osteoblasts and osteoclasts isolated from bone marrow. FH is expressed ubiquitously in the osteoblasts and in the cytoplasm of osteoclasts. The changes caused by absence of FH include: increase in number of osteoblasts (362%) and osteoclasts (342%), increase in RNA (180%) and protein expression of cathepsin K and increased osteoclast function (pit formation, 233%). Actin rearrangement in both osteoblasts and osteoclasts was altered, with a loss of integrity of the F-actin ring at the periphery of the osteoclasts. For the first time our studies demonstrate a direct role of FH in the maintenance of bone structure and function and is highlighted as a promising therapeutic target in bone diseases.

Differences in bone structure and unloading-induced bone loss between C57BL/6N and C57BL/6J mice.

The C57BL/6 mouse, the most frequently utilized animal model in biomedical research, is in use as several substrains, all of which differ by a small array of genomic differences. Two of these substrains, C57BL/6J (B6J) and C57BL/6N (B6N), are commonly used but it is unclear how phenotypically similar or different they are. Here, we tested whether adolescent B6N mice have a bone phenotype and respond to the loss of weightbearing differently than B6J. At 9 weeks of age, normally ambulating B6N had lower trabecular bone volume fraction but greater bone formation rates and osteoblast surfaces than corresponding B6J. At 11 weeks of age, differences in trabecular indices persisted between the substrains but differences in cellular activity had ceased. Cortical bone indices were largely similar between the two substrains. Hindlimb unloading (HLU) induced similar degeneration of trabecular architecture and cellular activity in both substrains when comparing 11-week-old HLU mice to 11-week-old controls. However, unloaded B6N mice had smaller cortices than B6J. When comparing HLU to 9 weeks baseline control mice, deterioration in trabecular separation, osteoblast indices, and endocortical variables was significantly greater in B6N than B6J. These data indicate specific developmental differences in bone formation and morphology between B6N and B6J mice, giving rise to a differential response to mechanical unloading that may be modulated, in part, by the genes Herc2, Myo18b, and Acan. Our results emphasize that these substrains cannot be used interchangeably at least for investigations in which the phenotypic makeup and its response to extraneous stimuli are of interest.

Diets High in Fat or Fructose Differentially Modulate Bone Health and Lipid Metabolism.

Diets high in fat or carbohydrates can lead to obesity and diabetes, two interrelated conditions that have been associated with osteoporosis. Here, we contrasted the effects of a high fat (HF) versus fructose-enriched carbohydrate (CH) versus regular chow (SC) diet on bone morphology, fat content and metabolic balance in BALB/cByJ mice over a 15-week period. For 13 weeks, there were no differences in body mass between groups with small differences in the last 2 weeks. Even without the potentially confounding factor of altered body mass and levels of load bearing, HF consumption was detrimental to bone in the distal femur with lower trabecular bone volume fraction and thinner cortices than controls. These differences in bone were accompanied by twofold greater abdominal fat content and fourfold greater plasma leptin concentrations. High-fat feeding caused a decrease in de-novo lipid synthesis in the liver, kidney, white adipose and brown adipose tissue. In contrast to HF, the fructose diet did not significantly impact bone quantity or architecture. Fructose consumption also did not significantly alter leptin levels or de-novo lipid synthesis but reduced epididymal adipose tissue and increased brown adipose tissue. Cortical stiffness was lower in the CH than in HF mice. There were no differences in glucose or insulin levels between groups. Together, a diet high in fat had a negative influence on bone structure, adipose tissue deposition and lipid synthesis, changes that were largely avoided with a fructose-enriched diet.

Cytoskeletal Configuration Modulates Mechanically Induced Changes in Mesenchymal Stem Cell Osteogenesis, Morphology, and Stiffness.

Mesenchymal stem cells (MSC) responding to mechanical cues generated by physical activity is critical for skeletal development and remodeling. Here, we utilized low intensity vibrations (LIV) as a physiologically relevant mechanical signal and hypothesized that the confined cytoskeletal configuration imposed by 2D culture will enable human bone marrow MSCs (hBMSC) to respond more robustly when LIV is applied in-plane (horizontal-LIV) rather than out-of-plane (vertical-LIV). All LIV signals enhanced hBMSC proliferation, osteogenic differentiation, and upregulated genes associated with cytoskeletal structure. The cellular response was more pronounced at higher frequencies (100 Hz vs 30 Hz) and when applied in the horizontal plane. Horizontal but not vertical LIV realigned the cell cytoskeleton, culminating in increased cell stiffness. Our results show that applying very small oscillatory motions within the primary cell attachment plane, rather than perpendicular to it, amplifies the cell's response to LIV, ostensibly facilitating a more effective transfer of intracellular forces. Transcriptional and structural changes in particular with horizontal LIV, together with the strong frequency dependency of the signal, emphasize the importance of intracellular cytoskeletal configuration in sensing and responding to high-frequency mechanical signals at low intensities.

Trabecular and Cortical Bone of Growing C3H Mice Is Highly Responsive to the Removal of Weightbearing.

Genetic make-up strongly influences the skeleton's susceptibility to the loss of weight bearing with some inbred mouse strains experiencing great amounts of bone loss while others lose bone at much smaller rates. At young adulthood, female inbred C3H/HeJ (C3H) mice are largely resistant to catabolic pressure induced by unloading. Here, we tested whether the depressed responsivity to unloading is inherent to the C3H genetic make-up or whether a younger age facilitates a robust skeletal response to unloading. Nine-week-old, skeletally immature, female C3H mice were subjected to 3wk of hindlimb unloading (HLU, n = 12) or served as normal baseline controls (BC, n = 10) or age-matched controls (AC, n = 12). In all mice, cortical and trabecular architecture of the femur, as well as levels of bone formation and resorption, were assessed with µCT, histomorphometry, and histology. Changes in bone marrow progenitor cell populations were determined with flow cytometry. Following 21d of unloading, HLU mice had 52% less trabecular bone in the distal femur than normal age-matched controls. Reflecting a loss of trabecular tissue compared to baseline controls, trabecular bone formation rates (BFR/BS) in HLU mice were 40% lower than in age-matched controls. Surfaces undergoing osteoclastic resorption were not significantly different between groups. In the mid-diaphysis, HLU inhibited cortical bone growth leading to 14% less bone area compared to age-matched controls. Compared to AC, BFR/BS of HLU mice were 53% lower at the endo-cortical surface and 49% lower at the periosteal surface of the mid-diaphysis. The enriched osteoprogenitor cell population (OPC) comprised 2% of the bone marrow stem cells in HLU mice, significantly different from 3% OPC in the AC group. These data show that bone tissue in actively growing C3H mice is lost rapidly, or fails to grow, during the removal of functional weight bearing-in contrast to the insignificant response previously demonstrated in female young adult C3H mice. Thus, the attributed low sensitivity of the C3H mouse strain to the loss of mechanical signals is not apparent at a young age and this trait therefore does not reflect a genetic regulation throughout the life span of this strain. These results highlight the significance of age in modulating the contribution of genetics in orchestrating bone's response to unloading and that the skeletal unresponsiveness of young adult C3H mice to the loss of weight bearing is not genetically hard-wired.

Low-intensity vibrations accelerate proliferation and alter macrophage phenotype in vitro.

Macrophages are essential for the efficient healing of various tissues. Although many biochemical signaling pathways have been well characterized in macrophages, their sensitivity to mechanical signals is largely unexplored. Here, we applied low-intensity vibrations (LIV) to macrophages to determine whether macrophages could directly transduce LIV signals into changes in the expression of genes and proteins involved in tissue repair. Two different LIV signal frequencies (30Hz or 100Hz) were combined with two acceleration magnitudes (0.15g or 1g) to generate four distinct LIV signals that were applied to cultured murine macrophages. All four LIV signals significantly increased macrophage number after 3 days of stimulation with the combination of the smallest acceleration and the highest frequency (0.15g at 100Hz) generating the largest response. Compared to non-LIV controls, gene expression of the pro-healing growth factors VEGF and TGF-ß increased with all four LIV signals (Day 1). LIV also decreased protein levels of the pro-inflammatory cytokines IL-6, IFN-γ, and TNF-α (Days 1 and 3). These data demonstrate the sensitivity of macrophages to high-frequency oscillations applied at low intensities and may suggest that the benefit of LIV for tissue repair may be based on reducing inflammation and promoting a pro-healing macrophage phenotype.

Osteocyte Apoptosis Caused by Hindlimb Unloading is Required to Trigger Osteocyte RANKL Production and Subsequent Resorption of Cortical and Trabecular Bone in Mice Femurs.

Osteocyte apoptosis is essential to activate bone remodeling in response to fatigue microdamage and estrogen withdrawal, such that apoptosis inhibition in vivo prevents the onset of osteoclastic resorption. Osteocyte apoptosis has also been spatially linked to bone resorption owing to disuse, but whether apoptosis plays a similar controlling role is unclear. We, therefore, 1) evaluated the spatial and temporal effects of disuse from hindlimb unloading (HLU) on osteocyte apoptosis, receptor activator of NF-κB ligand (RANKL) expression, bone resorption, and loss in mouse femora, and 2) tested whether osteocyte apoptosis was required to activate osteoclastic activity in cortical and trabecular bone by treating animals subjected to HLU with the pan-caspase apoptosis inhibitor, QVD (quinolyl-valyl-O-methylaspartyl-[-2,6-difluorophenoxy]-methylketone). Immunohistochemistry was used to identify apoptotic and RANKL-producing osteocytes in femoral diaphysis and distal trabecular bone, and µCT was used to determine the extent of trabecular bone loss owing to HLU. In both cortical and trabecular bone, 5 days of HLU increased osteocyte apoptosis significantly (3- and 4-fold, respectively, p < 0.05 versus Ctrl). At day 14, the apoptotic osteocyte number in femoral cortices declined to near control levels but remained elevated in trabeculae (3-fold versus Ctrl, p < 0.05). The number of osteocytes producing RANKL in both bone compartments was also significantly increased at day 5 of HLU (>1.5-fold versus Ctrl, p < 0.05) and further increased by day 14. Increases in osteocyte apoptosis and RANKL production preceded increases in bone resorption at both endocortical and trabecular surfaces. QVD completely inhibited not only the HLU-triggered increases in osteocyte apoptosis but also RANKL production and activation of bone resorption at both sites. Finally, µCT studies revealed that apoptosis inhibition completely prevented the trabecular bone loss caused by HLU. Together these data indicate that osteocyte apoptosis plays a central and controlling role in triggering osteocyte RANKL production and the activation of new resorption leading to bone loss in disuse. © 2016 American Society for Bone and Mineral Research.

Disc herniations in astronauts: What causes them, and what does it tell us about herniation on earth?

PURPOSE: Recent work showed an increased risk of cervical and lumbar intervertebral disc (IVD) herniations in astronauts. The European Space Agency asked the authors to advise on the underlying pathophysiology of this increased risk, to identify predisposing factors and possible interventions and to suggest research priorities. METHODS: The authors performed a narrative literature review of the possible mechanisms, and conducted a survey within the team to prioritize research and prevention approaches. RESULTS AND CONCLUSIONS: Based on literature review the most likely cause for lumbar IVD herniations was concluded to be swelling of the IVD in the unloaded condition during spaceflight. For the cervical IVDs, the knowledge base is too limited to postulate a likely mechanism or recommend approaches for prevention. Basic research on the impact of (un)loading on the cervical IVD and translational research is needed. The highest priority prevention approach for the lumbar spine was post-flight care avoiding activities involving spinal flexion, followed by passive spinal loading in spaceflight and exercises to reduce IVD hyper-hydration post-flight.

Modulation of unloading-induced bone loss in mice with altered ERK signaling.

Genetic variations mediate skeletal responsiveness to mechanical unloading, with individual space travelers exhibiting large variations in the extent of bone loss. We previously identified genomic regions harboring several hundred genes that can modulate the magnitude of skeletal adaptation to mechanical unloading. Here, bioinformatic filters aided in shortlisting 30 genes with bone-related and mechanoregulatory roles. The genes CD44, FGF2, NOD2, and Fas, all associated with ERK signaling, were then functionally tested in hindlimb-unloaded (HLU) knockout (KO) mice. Compared to their respective normally ambulating wildtype (WT) controls, all KO strains, except Fas mice, had lower trabecular bone volume, bone volume fraction, and/or trabecular number. For cortical bone and compared to ambulatory WT mice, CD44(-/-) had impaired properties while FGF2(-/-) showed enhanced indices. NOD2(-/-) and Fas(-/-) did not have a cortical phenotype. In all KO and WT groups, HLU resulted in impaired trabecular and cortical indices, primarily due to trabecular tissue loss and mitigation of cortical bone growth. The difference in trabecular separation between HLU and ambulatory controls was significantly greater in CD44(-/-) and NOD2(-/-) mice than in WT mice. In cortical bone, differences in cortical thickness, total pore volume, and cortical porosity between HLU and controls were aggravated in CD44(-/-) mice. In contrast, deletion of NOD2 and Fas genes mitigated the differences in Po.V between HLU and control mice. Together, we narrowed a previous list of QTL-derived candidate genes from over 300 to 30, and showed that CD44, NOD2, and Fas have distinct functions in regulating changes in trabecular and cortical bone indices during unloading.

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.

Human evolution. Comment on "Human-like hand use in Australopithecus africanus".

Skinner and colleagues (Research Article, 23 January 2015, p. 395), based on metacarpal trabecular bone structure, argue that Australopithecus africanus employed human-like dexterity for stone tool making and use 3 million years ago. However, their evolutionary and biological assumptions are misinformed, failing to refute the previously existing hypothesis that human-like manipulation preceded systematized stone tool manufacture, as indicated by the fossil record.

Cell Mechanosensitivity to Extremely Low-Magnitude Signals Is Enabled by a LINCed Nucleus.

A cell's ability to recognize and adapt to the physical environment is central to its survival and function, but how mechanical cues are perceived and transduced into intracellular signals remains unclear. In mesenchymal stem cells (MSCs), high-magnitude substrate strain (HMS, ≥2%) effectively suppresses adipogenesis via induction of focal adhesion (FA) kinase (FAK)/mTORC2/Akt signaling generated at FAs. Physiologic systems also rely on a persistent barrage of low-level signals to regulate behavior. Exposing MSC to extremely low-magnitude mechanical signals (LMS) suppresses adipocyte formation despite the virtual absence of substrate strain (<0.001%), suggesting that LMS-induced dynamic accelerations can generate force within the cell. Here, we show that MSC response to LMS is enabled through mechanical coupling between the cytoskeleton and the nucleus, in turn activating FAK and Akt signaling followed by FAK-dependent induction of RhoA. While LMS and HMS synergistically regulated FAK activity at the FAs, LMS-induced actin remodeling was concentrated at the perinuclear domain. Preventing nuclear-actin cytoskeleton mechanocoupling by disrupting linker of nucleoskeleton and cytoskeleton (LINC) complexes inhibited these LMS-induced signals as well as prevented LMS repression of adipogenic differentiation, highlighting that LINC connections are critical for sensing LMS. In contrast, FAK activation by HMS was unaffected by LINC decoupling, consistent with signal initiation at the FA mechanosome. These results indicate that the MSC responds to its dynamic physical environment not only with "outside-in" signaling initiated by substrate strain, but vibratory signals enacted through the LINC complex enable matrix independent "inside-inside" signaling.