Precision of bone mechanoregulation assessment in humans using longitudinal high-resolution peripheral quantitative computed tomography in vivo.

Local mechanical stimuli in the bone microenvironment are essential for the homeostasis and adaptation of the skeleton, with evidence suggesting that disruption of the mechanically-driven bone remodelling process may lead to bone loss. Longitudinal clinical studies have shown the combined use of high-resolution peripheral quantitative computed tomography (HR-pQCT) and micro-finite element analysis can be used to measure load-driven bone remodelling in vivo; however, quantitative markers of bone mechanoregulation and the precision of these analyses methods have not been validated in human subjects. Therefore, this study utilised participants from two cohorts. A same-day cohort (n = 33) was used to develop a filtering strategy to minimise false detections of bone remodelling sites caused by noise and motion artefacts present in HR-pQCT scans. A longitudinal cohort (n = 19) was used to develop bone imaging markers of trabecular bone mechanoregulation and characterise the precision for detecting longitudinal changes in subjects. Specifically, we described local load-driven formation and resorption sites independently using patient-specific odds ratios (OR) and 99 % confidence intervals. Conditional probability curves were computed to link the mechanical environment to the remodelling events detected on the bone surface. To quantify overall mechanoregulation, we calculated a correct classification rate measuring the fraction of remodelling events correctly identified by the mechanical signal. Precision was calculated as root-mean-squared averages of the coefficient of variation (RMS-SD) of repeated measurements using scan-rescan pairs at baseline combined with a one-year follow-up scan. We found no significant mean difference (p < 0.01) between scan-rescan conditional probabilities. RMS-SD was 10.5 % for resorption odds, 6.3 % for formation odds, and 1.3 % for correct classification rates. Bone was most likely to be formed in high-strain and resorbed in low-strain regions for all participants, indicating a consistent, regulated response to mechanical stimuli. For each percent increase in strain, the likelihood of bone resorption decreased by 2.0 ± 0.2 %, and the likelihood of bone formation increased by 1.9 ± 0.2 %, totalling 38.3 ± 1.1 % of strain-driven remodelling events across the entire trabecular compartment. This work provides novel robust bone mechanoregulation markers and their precision for designing future clinical studies

Evaluating the theory of bone mechanoregulation in the physiological loading scenario

In this paper, the theory of bone mechanoregulation under physiological loading was evaluated. The entire right tibiae of wild type (WT, N=5) and parathyroid hormone (PTH, N=5) treated C57BL/6J female mice were scanned using an in vivo μCT imaging system at 14, 16, 17, 18, 19, 20, 21, and 22 weeks. The PTH intervention started from week 18 until week 22. Subject-specific finite element (FE) models were created from the μCT images and physiological loading condition was defined in the FE models. The rates of changes in bone mineral content (BMC), bone mineral density (BMD), and bone tissue density (TMD) were quantified over 40 anatomical compartments across the entire mouse tibia. The resulting values were then correlated to the average 1st principal tensile strain (ε1) and the strain energy density (SED) for every compartment at weeks 18, 20, and 22. It was found that: in both groups, ε1 had a minimal effect on the variability of ΔBMC (p>0.01); SED had a significant effect on the variability of ΔBMC only in the WT group (p0.01). These results are the first to reveal the mechanism of bone mechanoregulation in the physiological loading scenario

Atherosusceptible Shear Stress Activates Endoplasmic Reticulum Stress to Promote Endothelial Inflammation.

Atherosclerosis impacts arteries where disturbed blood flow renders the endothelium susceptible to inflammation. Cytokine activation of endothelial cells (EC) upregulates VCAM-1 receptors that target monocyte recruitment to atherosusceptible regions. Endoplasmic reticulum (ER) stress elicits EC dysregulation in metabolic syndrome. We hypothesized that ER plays a central role in mechanosensing of atherosusceptible shear stress (SS) by signaling enhanced inflammation. Aortic EC were stimulated with low-dose TNFα (0.3 ng/ml) in a microfluidic channel that produced a linear SS gradient over a 20mm field ranging from 0-16 dynes/cm2. High-resolution imaging of immunofluorescence along the monolayer provided a continuous spatial metric of EC orientation, markers of ER stress, VCAM-1 and ICAM-1 expression, and monocyte recruitment. VCAM-1 peaked at 2 dynes/cm2 and decreased to below static TNFα-stimulated levels at atheroprotective-SS of 12 dynes/cm2, whereas ICAM-1 rose to a maximum in parallel with SS. ER expansion and activation of the unfolded protein response also peaked at 2 dynes/cm2, where IRF-1-regulated VCAM-1 expression and monocyte recruitment also rose to a maximum. Silencing of PECAM-1 or key ER stress genes abrogated SS regulation of VCAM-1 transcription and monocyte recruitment. We report a novel role for ER stress in mechanoregulation at arterial regions of atherosusceptible-SS inflamed by low-dose TNFα

Mechanical Regulation of Endocytosis: New Insights and Recent Advances

Endocytosis is a mechanosensitive process. It involves remodeling of the plasma membrane from a flat shape to a budded morphology, often at the sub‐micrometer scale. This remodeling process is energy‐intensive and is influenced by mechanical factors such as membrane tension, membrane rigidity, and physical properties of cargo and extracellular surroundings. The cellular responses to a variety of mechanical factors by distinct endocytic pathways are important for cells to counteract rapid and extreme disruptions in the mechanohomeostasis of cells. Recent advances in microscopy and mechanical manipulation at the cellular scale have led to new discoveries of mechanoregulation of endocytosis by the aforementioned factors. While factors such as membrane tension and membrane rigidity are generally shown to inhibit endocytosis, other mechanical stimuli have complex relationships with endocytic pathways. At this juncture, it is now possible to utilize experimental techniques to interrogate theoretical predictions on mechanoregulation of endocytosis in cells and even living organisms.Endocytosis involves extensive remodeling of the plasma membrane. Physical forces such as membrane tension, membrane rigidity, substrate stiffness, and physical properties of cargo play a role in this remodeling process. Recent advances in microscopy and mechanical manipulation at the cellular level have led to new insights into the mechanoregulation of endocytosis. These experimental techniques are being used to interrogate existing models to study the mechanoregulation of endocytosis.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/155473/1/adbi201900278.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/155473/2/adbi201900278_am.pd

Identification of Mechanosensitive Genes during Embryonic Bone Formation

Although it is known that mechanical forces are needed for normal bone development, the current understanding of how biophysical stimuli are interpreted by and integrated with genetic regulatory mechanisms is limited. Mechanical forces are thought to be mediated in cells by “mechanosensitive” genes, but it is a challenge to demonstrate that the genetic regulation of the biological system is dependant on particular mechanical forces in vivo. We propose a new means of selecting candidate mechanosensitive genes by comparing in vivo gene expression patterns with patterns of biophysical stimuli, computed using finite element analysis. In this study, finite element analyses of the avian embryonic limb were performed using anatomically realistic rudiment and muscle morphologies, and patterns of biophysical stimuli were compared with the expression patterns of four candidate mechanosensitive genes integral to bone development. The expression patterns of two genes, Collagen X (ColX) and Indian hedgehog (Ihh), were shown to colocalise with biophysical stimuli induced by embryonic muscle contractions, identifying them as potentially being involved in the mechanoregulation of bone formation. An altered mechanical environment was induced in the embryonic chick, where a neuromuscular blocking agent was administered in ovo to modify skeletal muscle contractions. Finite element analyses predicted dramatic changes in levels and patterns of biophysical stimuli, and a number of immobilised specimens exhibited differences in ColX and Ihh expression. The results obtained indicate that computationally derived patterns of biophysical stimuli can be used to inform a directed search for genes that may play a mechanoregulatory role in particular in vivo events or processes. Furthermore, the experimental data demonstrate that ColX and Ihh are involved in mechanoregulatory pathways and may be key mediators in translating information from the mechanical environment to the molecular regulation of bone formation in the embryo

Effect of dynamic mechanical compression on actin cytoskeleton network of human mesenchymal stem cells (hMSCs) in three dimensional collagen constructs

Actin filament, one type of cytoskeletons, plays a central role in mediating cellular in responses to mechanical loading. Many mechanorgulation studies are restricted to 2D models using isolated cells or monolayer cultures, even though it is know that cells behave differently in term of cell morphology, cell matrix adhesion composition and matrix mediated force transmission when they are in 3D configuration. This current study investigates the temporal change of actin network of hMSCs entrapped in 3D collagen construct upon cyclic compression. Human bone marrow mesenchymal stem cells were encapsulated in cylindrical collagen construct. A micromanipulator based loading device coupled to fluorescent microscope was used to deliver compression loading to the construct with 10% strain at 1Hz for different period of time. Rhodamine phalloidin was used to stain for the actin filament network to hMSC in the construct at different time points postcompression. An optimized loading protocol with 5hrs of continuous loading was delivered. Actin network concentrated at the cell periphery of cells exhibiting round morphology was observed immediately while elongated and polarized actin network was found after 24 hours. Detailed characterization of actin filament organization and their association with cell-matrix interaction molecules are warrented before the mechanisms of compression-induced hMSC alignment can be delineated © 2010 IEEE.published_or_final_versionThe IEEE 4th International Conference on Nano/Molecular Medicine and Engineering (NANOMED 2010), Hong Kong/Macau, China, 5-9 December 2010. In Proceedings of the IEEE International Conference on NANOMED, 2010, p. 136-13

TRPV4-A Missing Link Between Mechanosensation and Immunity

Transient receptor potential vanilloid-type 4 (TRPV4) cation channel is widely expressed in all tissues as well as in immune cells and its function as mechanosensitive Ca2+ channel seems to be conserved throughout all mammalian species. Of late, emerging evidence has implicated TRPV4 in the activation and differentiation of innate immune cells, especially in neutrophils, monocytes, and macrophages. As such, TRPV4 has been shown to mediate neutrophil adhesion and chemotaxis, as well as production of reactive oxygen species in response to pro-inflammatory stimuli. In macrophages, TRPV4 mediates formation of both reactive oxygen and nitrogen species, and regulates phagocytosis, thus facilitating bacterial clearance and resolution of infection. Importantly, TRPV4 may present a missing link between mechanical forces and immune responses. This connection has been exemplary highlighted by the demonstrated role of TRPV4 in macrophage activation and subsequent induction of lung injury following mechanical overventilation. Mechanosensation via TRPV4 is also expected to activate innate immune cells and establish a pro-inflammatory loop in fibrotic diseases with increased deposition of extracellular matrix (ECM) and substrate stiffness. Likewise, TRPV4 may be activated by cell migration through the endothelium or the extracellular matrix, or even by circulating immune cells squeezing through the narrow passages of the pulmonary or systemic capillary bed, a process that has recently been linked to neutrophil priming and depriming. Here, we provide an overview over the emerging role of TRPV4 in innate immune responses and highlight two distinct modes for the activation of TRPV4 by either mechanical forces (“mechanoTRPV4”) or by pathogens (“immunoTRPV4”)

Protrusive Push versus Enveloping Embrace: Computational Model of Phagocytosis Predicts Key Regulatory Role of Cytoskeletal Membrane Anchors

Encounters between human neutrophils and zymosan elicit an initially protrusive cell response that is distinct from the thin lamella embracing antibody-coated targets. Recent experiments have led us to hypothesize that this behavior has its mechanistic roots in the modulation of interactions between membrane and cytoskeleton. To test and refine this hypothesis, we confront our experimental results with predictions of a computer model of leukocyte mechanical behavior, and establish the minimum set of mechanistic variations of this computational framework that reproduces the differences between zymosan and antibody phagocytosis. We confirm that the structural linkages between the cytoskeleton and the membrane patch adherent to a target form the “switchboard” that controls the target specificity of a neutrophil's mechanical response. These linkages are presumably actin-binding protein complexes associating with the cytoplasmic domains of cell-surface receptors that are engaged in adhesion to zymosan and Fc-domains

Development of a micromanipulator-based loading device for mechanoregulation study of human mesenchymal stem cells in three-dimensional collagen constructs

Mechanical signal is important for regulating cellular activities, including proliferation, metabolism, matrix production, and orientation. Bioreactors with loading functions can be used to precondition cells in three-dimensional (3D) constructs so as to study the cellular responses to mechanical stimulation. However, full-scale bioreactor is not always an affordable option considering the high cost of equipments and the liter-sized medium with serum and growth factor supplements. In this study, a custom-built loading system was developed by coupling a conventional camera-equipped inverted research microscope with two micromanipulators. The system was programmed to deliver either cyclic compressive loading with different frequencies or static compressive loading for 1 week to investigate the cellular responses of human mesenchymal stem cells (hMSCs) entrapped in a 3D construct consists of reconstituted collagen fibers. Cellular properties, including their alignment, cytoskeleton, and cell metabolism, and properties of matrix molecules, such as collagen fiber alignment and glycosaminoglycan deposition, were evaluated. Using a MatLab-based image analysis program, reorientation of the entrapped cells from a random distribution to a preferred alignment along the loading direction in constructs with both static and cyclic compression has been demonstrated, but no such alignment was found in the free-floating controls. Fluorescent staining on filamentous actin cytoskeleton also confirmed the finding. Nevertheless, the collagen fiber meshwork entrapping the hMSCs remained randomly distributed, and no change in cellular metabolism and glycosaminoglycans production was noted. The current study provides a simple and affordable option toward setting up a mechanoregulation facility based on existing laboratory equipments and sheds new insights on the effect of mechanical loading on the alignment of hMSCs in 3D collagen constructs. Copyright © 2010, Mary Ann Liebert, Inc.published_or_final_versio