Mechanobiology

Breast and prostate cancers preferentially metastasise to bone tissue, with metastatic lesions
forming in the skeletons of most patients. On arriving in bone tissue, disseminated tumour cells
enter a mechanical microenvironment that is substantially different to that of the primary tumour
and is largely regulated by bone cells. Osteocytes, the most ubiquitous bone cell type, orchestrate
healthy bone remodelling in response to physical exercise. However, the effects of mechanical
loading of osteocytes on cancer cell behaviour is still poorly understood. The aim of this study
was to characterise the effects of osteocyte mechanical stimulation on the behaviour of breast and
prostate cancer cells. To replicate an osteocyte-controlled environment, this study treated breast
(MDA-MB-231 and MCF-7) and prostate (PC-3 and LNCaP) cancer cell lines with conditioned media
from MLO-Y4 osteocyte-like cells exposed to mechanical stimulation in the form of fluid shear stress.
We found that osteocyte paracrine signalling acted to inhibit metastatic breast and prostate tumour
growth, characterised by reduced proliferation and invasion and increased migration. In breast
cancer cells, these effects were largely reversed by mechanical stimulation of osteocytes. In contrast,
conditioned media from mechanically stimulated osteocytes had no effect on prostate cancer cells.
To further investigate these interactions, we developed a microfluidic organ-chip model using the
Emulate platform. This new organ-chip model enabled analysis of cancer cell migration, proliferation
and invasion in the presence of mechanical stimulation of osteocytes by fluid shear stress, resulting
in increased invasion of breast and prostate cancer cells. These findings demonstrate the importance
of osteocytes and mechanical loading in regulating cancer cell behaviour and the need to incorporate
these factors into predictive in vitro models of bone metastasis.

"Biological cells have the amazing ability to sense a wide variety of stimuli. Recent work suggests cells are also able to sense light, touch and even, to a certain extent, smell. But can cells hear?

This is a surprisingly complex subject. A number of experiments have shown that sound can have an impact on cellular activity, but explanations are elusive. Studies have found mechanical interactions and oscillations in cellular systems, which could plausibly be modified by sound. However, research is at a relatively early stage and it is unclear how sound-based mechanisms would work.
Crucially, we currently do not understand how sound affects molecules. The subject has even been dubbed a ‘black art’4, so how are we to understand the role of sound in the complexity of biology? The confusion seems rooted in the gap between our quantum mechanical and classical understanding of sound; we simply do not know how the two relate. However this is proving a fertile area for insightful new studies."

The osteocyte is believed to act as the primary sensor of mechanical stimulus in bone, controlling signalling for bone growth and resorption in response to changes in the mechanical demands placed on bones throughout life. Alterations in local bone tissue composition and structure arising during osteoporosis likely disrupt the local mechanical environment of these mechanosensitive bone cells, and may thereby initiate a mechanobiological response. However, due to the difficulties in directly characterising the mechanical environment of bone cells in vivo, the mechanical stimuli experienced by osteoporotic bone cells are not known. The global aim of this thesis is to discern the in vivo mechanical environment of the osteocyte, both in healthy bone tissue and during the disease of osteoporosis.
The first study of this thesis involved the development of 3D finite element models of osteocytes, including their cell body and the surrounding pericellular matrix (PCM) and extracellular matrix (ECM), using confocal images of the lacunar-canalicular network. These anatomically representative models demonstrated the significance of geometry for strain amplification within the osteocyte mechanical environment. A second study employed fluid-structure interaction (FSI) modelling to investigate the complex multi-physics environment of osteocytes in vivo. These models built upon the anatomically representative models developed in the first study, and FSI methods were used to simulate loading-induced interstitial fluid flow through the lacunar-canalicular network. Interestingly, the in vivo mechanical stimuli (strain and shear stress) predicted using these computational approaches were above thresholds known to elicit osteogenic responses from osteoblastic cells in vitro, and thereby provide a novel insight into the complex multi-physics mechanical environment of osteocytes in vivo.
The third study of this thesis sought to experimentally characterise the strain environment of osteoblasts and osteocytes under physiological loading conditions in healthy and osteoporotic bone, using a rat model of osteoporosis. A custom-designed loading device compatible with a confocal microscope was constructed to apply strains to fluorescently stained femur samples from normal and ovariectomised rats. Confocal imaging was performed simultaneously during loading and digital image
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correlation techniques were used to characterise cellular strains from the images acquired. These results suggested that the mechanical environment of osteoblasts and osteocytes is altered during early-stage osteoporosis, and it is proposed that a mechanobiological response restores the homeostatic mechanical environment by late-stage osteoporosis. A final study applied these results as inputs for the developed computational models to investigate whether changes in tissue properties, lacunar-canalicular architecture or amplification mechanisms during osteoporosis could explain the altered mechanical stimulation of osteocytes observed. The findings of this study shed new light on the osteocyte mechanical environment in both healthy and osteoporotic bone, elucidating a possible mechanobiological relationship between increases in strain stimulation of the osteocyte and subsequent increases in mineralisation of bone tissue as key events in the progression of osteoporosis.
Together, the studies in this thesis provide a novel insight into the closed mechanical environment of the osteocyte. Using both computational and experimental methods, the mechanical stimuli that osteocytes experience under physiological loading in vivo, in both healthy and osteoporotic bone, were elucidated. In particular, the research in this thesis provides a missing mechanobiological link in the temporal development of post-menopausal osteoporosis, and the information gained from this body of work may inform future treatments for osteoporosis.

Tras un período prolongado de recuperación, aproximadamente un año, el ligamento cicatrizado no alcanza las propiedades mecánicas ni las cualidades del ligamento normal, convirtiéndolo en un tejido susceptible de esguinces crónicos. Este hecho se asocia a la baja producción de colágeno y a la nueva orientación aleatoria de las fibras, lo cual ocasiona una distribución anormal de las cargas. Actualmente, es aceptado que la carga mecánica tiene efectos benéficos en la reparación del tejido, estimula la proliferación celular y producción de colágeno. Por ello, para entender cómo el tejido en reparación responde a los estímulos mecánicos, se recurre a la mecanobiología, un enfoque que describe los procesos de mecanostransducción en el tejido. Por tanto, el objetivo de este artículo es proveer una revisión sobre la mecanobiología y los factores que influyen en el proceso de reparación del ligamento tras sufrir una lesión.

The physical properties of cells are promising biomarkers for cancer diagnosis and prognosis. Here we determine the physical phenotypes that best distinguish human cancer cell lines, and their relationship to cell invasion. We use the high throughput, single-cell microfluidic method, quantitative deformability cytometry (q-DC), to measure six physical phenotypes including elastic modulus, cell fluidity, transit time, entry time, cell size, and maximum strain at rates of 10 2 cells per second. By training a k-nearest neighbor machine learning algorithm, we demonstrate that multiparameter analysis of physical phenotypes enhances the accuracy of classifying cancer cell lines compared to single parameters alone. We also discover a set of four physical phenotypes that predict invasion; using these four parameters, we generate the physical phenotype model of invasion by training a multiple linear regression model with experimental data from a set of human ovarian cancer cells that overexpress a panel of tumor suppressor microRNAs. We validate the model by predicting invasion based on measured physical phenotypes of breast and ovarian human cancer cell lines that are subject to genetic or pharmacologic perturbations. Taken together, our results highlight how physical phenotypes of single cells provide a biomarker to predict the invasion of cancer cells. Understanding how the physical phenotypes of cells – such as their deformability and size – are associated with cancer cell invasion would provide insights into mechanisms of invasion, and could also enable physical phenotypes to be used as a label-free biomarker for invasion. However, physical phenotyping measurements have been limited due to challenges in lack of measurement standardization and throughput. Here, we use the quantitative deformability cytometry (q-DC) method that we recently developed to rapidly obtain calibrated measurements of cellular physical phenotypes of across 19 distinct samples of human breast, ovarian, and pancreatic cancer cell lines. Using the data to train a machine learning algorithm, we develop the physical phenotyping model for invasion, which enables us to predict the invasion of cancer cell lines. More broadly, this methodology provides a framework for predicting functional behavior of cells based on physical phenotypes.

Scar tissue size following myocardial infarction is an independent predictor of cardiovascular outcomes, yet little is known about factors regulating scar size. We demonstrate that collagen V, a minor constituent of heart scars, regulates the size of heart scars after ischemic injury. Depletion of collagen V led to a paradoxical increase in post-infarction scar size with worsening of heart function. A systems genetics approach across 100 in-bred strains of mice demonstrated that collagen V is a critical driver of postinjury heart function. We show that collagen V deficiency alters the mechanical properties of scar tissue, and altered reciprocal feedback between matrix and cells induces expression of mechanosensitive integrins that drive fibroblast activation and increase scar size. Cilengitide, an inhibitor of specific integrins, rescues the phenotype of increased post-injury scarring in collagen-V-deficient mice. These observations demonstrate that collagen V regulates scar size in an integrin-dependent manner.

Mechanical forces play important roles in human embryonic stem cell (hESC) differentiation. To investigate the impact of dynamic mechanical forces on neural induction of hESCs, this study employs acoustic tweezing cytometry (ATC) to apply cyclic forces/strains to hESCs by actuating integrin-bound microbubbles using ultrasound pulses. Accelerated neural induction of hESCs is demonstrated as the result of combined action of ATC and neural induction medium (NIM). Specifically, application of ATC for 30 min followed by culture in NIM upregulates neuroecdoderm markers Pax6 and Sox1 as early as 6 h after ATC, and induces neural tube-like rosette formation at 48 h after ATC. In contrast, no changes are observed in hESCs cultured in NIM without ATC treatment. In the absence of NIM, ATC application decreases Oct4, but does not increase Pax6 and Sox1 expression, nor does it induce neural rossette formation. The effects of ATC are abolished by inhibition of FAK, myosin activity, and RhoA/ROCK signaling. Taken together, the results reveal a synergistic action of ATC and NIM as an integrated mechanobiology mechanism that requires both integrin-targeted cyclic forces and chemical factors for accelerated neural induction of hESCs.

Mechanobiology, the study of the influence of mechanical loads on biological processes through signaling to cells, is fundamental to the inherent ability of bone tissue to adapt its structure in response to mechanical stimulation. The immense contribution of computational modeling to the nascent field of bone mechanobiology is indisputable, having aided in the interpretation of experimental findings and identified new avenues of inquiry. Indeed, advances in computational modeling have spurred the development of this field, shedding new light on problems ranging from the mechanical response to loading by individual cells to tissue differentiation during events such as fracture healing. To date, in silico bone mechanobiology has generally taken a reductive approach in attempting to answer discrete biological research questions, with research in the field broadly separated into two streams: (1) mechanoregulation algorithms for predicting mechanobiological changes to bone tissue and (2) models investigating cell mechanobiology. Future models will likely take advantage of advances in computational power and techniques, allowing multiscale and multiphysics modeling to tie the many separate but related biological responses to loading together as part of a larger systems biology approach to shed further light on bone mechanobiology. Finally, although the ever-increasing complexity of computational mechanobiology models will inevitably move the field toward patient-specific models in the clinic, the determination of the context in which they can be used safely for clinical purpose will still require an extensive combination of computational and experimental techniques applied to in vitro and in vivo applications .

The ability of a eukaryotic cell to resist deformation, to transport intracellular cargo and to change shape during movement depends on the cytoskeleton, an interconnected network of filamentous polymers and regulatory proteins. Recent work has demonstrated that both internal and external physical forces can act through the cytoskeleton to affect local mechanical properties and cellular behaviour. Attention is now focused on how cytoskeletal networks generate, transmit and respond to mechanical signals over both short and long timescales. An important insight emerging from this work is that long-lived cytoskeletal structures may act as epigenetic determinants of cell shape, function and fate. In a 1960 lecture, cell and developmental biologist Paul A. Weiss encouraged his audience to think of the cell as an integrated whole " lest our necessary and highly successful preoccupation with cell fragments and fractions obscure the fact that the cell is not just an inert playground for a few almighty masterminding molecules, but is a system, a hierarchically ordered system, of mutually interdependent species of molecules, molecular groupings, and supramolecular entities; and that life, through cell life, depends on the order of their interactions " 1. This statement may be more relevant today than it was 50 years ago. Despite tremendous progress, fundamental gaps remain between our understanding of individual molecules and our understanding of how these molecules function collectively to form living cells. The sequencing of genomes outpaces characterization of the cellular components they encode and far exceeds our ability to reassemble these components into the types of complex system that can provide mecha-nistic insight into cellular behaviour. An even more difficult task is to connect the behaviour of cells in culture with that of more complex living tissues and organisms. Ever since muscle fibres were first examined under rudimentary microscopes in the seventeenth century, researchers have been motivated to understand how the process of self-organization generates dynamic, robust and elaborate structures that organize and 'animate' cells. The biological importance of establishing order over diverse length scales and timescales, as well as the challenges of understanding how systems of self-organizing molecules carry out cellular functions, is perhaps best illustrated by studies of the cytoskeleton. The cytoskeleton carries out three broad functions: it spatially organizes the contents of the cell; it connects the cell physically and biochemically to the external environment; and it generates coordinated forces that enable the cell to move and change shape. To achieve these functions, the cytoskeleton integrates the activity of a multitude of cytoplasmic proteins and organelles. Despite the connotations of the word 'skeleton' , the cytoskeleton is not a fixed structure whose function can be understood in isolation. Rather, it is a dynamic and adaptive structure whose component polymers and regulatory proteins are in constant flux. Many basic building blocks of the cytoskeleton have been identified and characterized extensively in vitro, and researchers are now using advanced light microscopy to determine, with great spatial and temporal precision, the locations and dynamics of these cytoskeletal proteins during processes such as cell division and motility. For example, more than 150 proteins have so far been found to contain binding domains for the protein actin, which polymerizes to form one of the key cytoskeletal filaments in cells 2. One set of actin regulators forms a macromolecular ensemble called the WAVE complex that promotes assembly of actin-filament networks at the leading edge of motile cells 3. High-resolution light microscopy of rapidly crawling leukocytes revealed that the WAVE complex forms highly coherent travelling waves whose movement correlates with cell protrusion 4. Such observations in living cells can stimulate the formation of detailed hypotheses for how molecules collaborate to form functional cytoskeletal structures, but to test these hypotheses definitively, the components must be isolated from cells and purified. Remarkably, experiments that combine a small number of purified proteins have demonstrated that many complex cytoskeletal structures observed in cells can be reconstituted in vitro from purified components. For example, only three proteins are required to actively track and transport cargo on the growing end of microtubules, which are formed by the polymerization of subunits consisting of αβ-tubulin heterodimers and are another key cytoskeletal filament in cells 5. Although the list of proteins associated with the cytoskeleton continues to grow, the ultimate goal remains — understanding how the interactions of the individual molecules of the cytoskeleton give rise to the large-scale cellular behaviours that depend on them. In this Review, we discuss recent progress towards an integrated understanding of the cytoskeleton. In particular, we focus on the mechanics of cytoskeletal networks and the roles that mechanics have in many cell biological processes. Rather than focusing on one cellular process or cytoskel-etal filament, we identify a set of basic concepts and link them to work in several cytoskeleton-related fields. We begin with a brief introduction to the major polymers that constitute the cytoskeleton and then shift focus from molecules to more complex structures, emphasizing three concepts that echo Weiss's 1960 challenge to view cells as an integrated whole. The first concept is that long-range order arises from the regulated self-assembly of components guided by spatial cues and physical constraints. The second is that beyond simply composition, it is the architecture of the cytoskeleton that controls the physical properties of the cell. And the third is that cytoskeletal links to the external microenvironment can mediate both short and long timescale changes in cellular behaviour. We finish by discussing the intriguing and under-appreciated question of whether long-lived cytoskeletal structures can function as a cellular 'memory' that integrates past interactions with the mechanical microenvironment and influences future cellular behaviour.

We present a fluid-solid-growth model for the evolution of a saccular cerebral aneurysm on the internal carotid sinus artery. The model utilises a realistic constitutive model of the arterial wall that accounts for the structural arrangement of collagen fibres in the medial and adventitial layers, the natural reference configurations in which the collagen fibres are recruited to load bearing and the mass of the elastin and collagenous constituents. The structural model is integrated into a patient-specific geometry of the internal carotid artery. This enables growth and remodelling (G&R) of the aneurysmal section to be explicitly linked to physiologically realistic haemodynamic stimuli. In addition, a quasi-static approach is used to obtain the geometry of the aneurysmal section at systolic and diastolic pressures, enabling G&R to be explicitly linked to the magnitude of the cyclic stretches. To our knowledge, this is the first patient-specific model of cerebral aneurysm evolution that incorporates a realistic constitutive model of the arterial wall and explicitly links G&R to the pulsatile mechanical environment. It will provide the basis for further investigating and elucidating the aetiology of the disease.

As the primary structural protein of our bodies, fibrillar collagen and its organizational patterns determine the biomechanics and shape of tissues. While the molecular assembly of individual fibrils is well understood, the mechanisms determining the arrangement of fibers and thus the shape and form of tissues remain largely unknown. We have developed a cell culture model that successfully recapitulates early tissue development and the de novo deposition of collagen fibers to investigate the role of mechanical cues on collagen fiber alignment. The devices used a thin, collagencoated deformable PDMS membrane inside a tissue culture well built on microscope-grade coverslips. Deformations and strains in the PDMS membrane were quantified by tracking fluorescent bead displacement and through the use of a COMSOL model. Cyclical strains were applied to serumcultured rabbit corneal cells at 0.5 Hz for 24-48 h and showed a preferred alignment after 36 h of cyclical loading. Cells cultured with ascorbic acid under methylcellulose serum-free conditions deposited a collagenous matrix that was visible under live second harmonic generation microscopy at week 4. Our microfabricated tissue culture system allows for the controllable application of a wide range of stress profiles to cells, and for the observation and quantification of cells and de novo collagen formation in vitro. Future studies will involve the fabrication of models to study the formation and organization of collagen in ocular diseases.

The aim of this study was to explore how cell-matrix interactions and extrinsic mechanical signals interact to determine stem cell fate in response to transforming growth factor-β3 (TGF-β3). Bone marrow derived mesenchymal stem cells (MSCs) were seeded in agarose and fibrin hydrogels and subjected to dynamic compression in the presence of different concentrations of TGF-β3. Markers of chondrogenic, myogenic and endochondral differentiation were assessed. MSCs embedded within agarose hydrogels adopted a spherical cell morphology, while cells directly adhered to the fibrin matrix and took on a spread morphology. Free-swelling agarose constructs stained positively for chondrogenic markers, with MSCs appearing to progress towards terminal differentiation as indicated by mineral staining. MSC seeded fibrin constructs progressed along an alternative myogenic pathway in long-term free-swelling culture. Dynamic compression suppressed differentiation towards any investigated lineage in both fibrin and agarose hydrogels in the short-term. Given that fibrin clots have been shown to support a chondrogenic phenotype in vivo within mechanically loaded joint defect environments, we next explored the influence of long term (42 days) dynamic compression on MSC differentiation. Mechanical signals generated by this extrinsic loading ultimately governed MSC fate, directing MSCs along a chondrogenic pathway as opposed to the default myogenic phenotype supported within unloaded fibrin clots. In conclusion, this study demonstrates that external cues such as the mechanical environment can override the influence specific substrates, scaffolds or hydrogels have on determining mesenchymal stem cell fate. The temporal data presented in this study highlights the importance of considering how MSCs respond to extrinsic mechanical signals in the long term.

The human pelvis has evolved over time into a remarkable structure, optimised into an intricate architecture that transfers the entire load of the upper body into the lower limbs, while also facilitating bipedal movement. The pelvic girdle is composed of two hip bones, os coxae, themselves each formed from the gradual fusion of the ischium, ilium and pubis bones. Unlike the development of the classical long bones, a complex timeline of events must occur in order for the pelvis to arise from the embryonic limb buds. An initial blastemal structure forms from the mesenchyme, with chondrification of this mass leading to the first recognisable elements of the pelvis. Primary ossification centres initiate in utero, followed post-natally by secondary ossification at a range of locations, with these processes not complete until adulthood. This cascade of events can vary between individuals, with recent evidence suggesting that fetal activity can affect the normal development of the pelvis. This review surveys the current literature on the ontogeny of the human pelvis.

Facial wrinkles are clear markers of the aging process, being chronological, photo-induced or reflecting repetitive facial expressions. Recent in silico results demonstrated the role of stratum corneum (SC) biomechanics on the skin folding. Cumulative and localized mechanical stresses, induced by facial expressions, could generate biomechanical fatigue in deep skin layers and contribute to create wrinkles. To such an issue, computational models theoretically alleviate that a long-lasting softening of the SC should decrease the severity of facial wrinkles. The objective of our study was to assess clinically this hypothesis. We have used the combination of clinical, instrumental in vivo data, digital methodology and proteomic data to give new insights to the biophysical and biological mechanisms involved. The long-term efficacy of a 20% glycerol based moisturizer (GBM) versus its vehicle was evaluated on crow's feet skin hydration, crow's feet wrinkles visibility and skin elasticity. A significant increase of hydration was measured at D56 and D112 for both formulae, but 2 times higher with GBM vs its vehicle. A significant increase of the skin elasticity was observed on the crow's feet site at D56 or D112 always in favor of GBM, versus its vehicle. There was a significant decrease in the severity of crow's feet wrinkles scoring, depth and volume at D112 for both formulae, albeit higher with the GBM. Proteomic data revealed an association between the effect of GBM and upregulation of four proteins among which three enzymes associated with the desquamation process, the cell-glycan extracellular interactions and the glycation/oxidation of proteins. Three functions closely related to tissue mechanical/adhesion properties. Our results provide an in vivo demonstration that a cumulative skin surface softening treatment might have an anti-ageing impact, both on crow's feet wrinkle and skin elasticity. The use of high concentration of glycerol should bring additional performance to other conventional cosmetic anti-ageing formulations.

Talins are cytoskeletal linker proteins that consist of an N-terminal head domain, a flexible neck region and a C-terminal rod domain made of 13 helical bundles. The head domain binds integrin β-subunit cytoplasmic tails, which triggers integrin conformational activation to increase affinity for extracellular matrix proteins. The rod domain links to actin filaments inside the cell to transmit mechanical loads and serves as a mechanosensitive signalling hub for the recruitment of many other proteins. The α-helical bundles function as force-dependent switches – proteins that interact with folded bundles are displaced when force induces unfolding, exposing previously cryptic binding sites for other ligands. This leads to the notion of a talin code. In this Cell Science at a Glance article and the accompanying poster, we propose that the multiple switches within the talin rod function to process and store time- and force-dependent mechanical and chemical information.

Because bone marrow-derived stromal cells (BMSCs) are able to generate many cell types, they are envisioned as source of regenerative cells to repair numerous tissues, including bone, cartilage, and ligaments. Success of BMSC-based therapies, however, relies on a number of methodological improvements, among which better understanding and control of the BMSC differentiation pathways. Since many years, the biochemical environment is known to govern BMSC differentiation, but more recent evidences show that the biomechanical environment is also directing cell functions. Using in vitro systems that aim to reproduce selected components of the in vivo mechanical environment, it was demonstrated that mechanical loadings can affect BMSC proliferation and improve the osteogenic, chondrogenic, or myogenic phenotype of BMSCs. These effects, however, seem to be modulated by parameters other than mechanics, such as substrate nature or soluble biochemical environment. This paper reviews and discusses recent experimental data showing that despite some knowledge limitation, mechanical stimulation already constitutes an additional and efficient tool to drive BMSC differentiation.

Experimental studies have shown that primary osteoporosis caused by oestrogen-deficiency results in localised alterations in bone tissue properties and mineral composition. Additionally, changes to the lacunar-canalicular architecture surrounding the mechanosensitive osteocyte have been observed in animal models of the disease. Recently, it has also been demonstrated that the mechanical stimulation sensed by osteocytes changes significantly during osteoporosis. Specifically, it was shown osteoporotic bone cells experience higher maximum strains than healthy bone cells after short durations of oestrogen deficiency. However, in long-term oestrogen deficiency there was no significant difference between bone cells in healthy and normal bone. The mechanisms by which these changes arise are unknown. In this study, we test the hypothesis that complex changes in tissue composition and lacunar-canalicular architecture during osteoporosis alter the mechanical stimulation of the osteocyte. The objective of this research is to employ computational methods to investigate the relationship between changes in bone tissue composition and microstructure and the mechanical stimulation of osteocytes during osteoporosis. By simulating physiological loading, it was observed that an initial decrease in tissue stiffness (of 0.425 GPa) and mineral content (of 0.66 wt% Ca) relative to controls could explain the mechanical stimulation observed at the early stages of oestrogen deficiency (5 weeks post-OVX) during in situ bone cell loading in an oestrogen-deficient rat model of post-menopausal osteoporosis (Verbruggen et al. 2015). Moreover, it was found that a later increase in stiffness (of 1.175 GPa) and mineral content (of 1.64 wt% Ca) during long-term osteoporosis (34 weeks post-OVX), could explain the mechanical stimuli previously observed at a later time point due to the progression of osteoporosis. Furthermore, changes in canalicular tortuosity arising during osteoporosis were shown to result in increased osteogenic strain stimulation, though to a lesser extent than has been observed experimentally. The findings of this study indicate that changes in the extracellular environment during osteoporosis, arising from altered mineralisation and lacunar-canalicular architecture, lead to altered mechanical stimulation of osteocytes, and provide an enhanced understanding of changes in bone mechanobiology during osteoporosis.

Chronic organ injury leads to fibrosis and eventually organ failure. Fibrosis is characterized
by excessive synthesis, remodeling, and contraction of extracellular matrix produced by
myofibroblasts. Myofibroblasts are the key cells in the pathophysiology of fibrotic disorders
and their differentiation can be triggered by multiple stimuli. To develop anti-fibrotic
therapies, it is of paramount importance to understand the molecular basis of the signaling
pathways contributing to the activation and maintenance of myofibroblasts. Several
signal transduction pathways, such as transforming growth factor (TGF)-β, Wingless/Int
(WNT), and more recently yes-associated protein 1 (YAP)/transcriptional coactivator with
PDZ-binding motif (TAZ) signaling, have been linked to the pathophysiology of fibrosis.
Activation of the TGF-β1-induced SMAD complex results in the upregulation of genes
important for myofibroblast function. Similarly, WNT-stabilized β-catenin translocates
to the nucleus and initiates transcription of its target genes. YAP and TAZ are two
transcriptional co-activators from the Hippo signaling pathway that also rely on nuclear
translocation for their functioning. These three signal transduction pathways have little
molecular similarity but do share one principle: the cytosolic/nuclear regulation of its
transcriptional activators. Past research on these pathways often focused on the isolated
cascades without taking other signaling pathways into account. Recent developments
show that parts of these pathways converge into an intricate network that governs the
activation and maintenance of the myofibroblast phenotype. In this review, we discuss
the current understanding on the signal integration between the TGF-β, WNT, and YAP/
TAZ pathways in the development of organ fibrosis. Taking a network-wide view on
signal transduction will provide a better understanding on the complex and versatile
processes that underlie the pathophysiology of fibrotic disorders.

type and the degree of stretch. In NRK-52E cells, it was mediated through both JNK and p38 SAPK-2 pathways, and inhibition of either pathway reduced the degree of stretch-induced apoptosis. Stretched cells showed increased activity of caspase-3 and -9 but not -2 or -8. Stretch-induced apoptosis was modulated by inhibition of caspase-3 and to a lesser extent by caspase-9. Conclusion: These fi ndings suggest that stretch induces apoptosis in renal epithelial cells through the specifi c activation of JNK/SAPK and p38 SAPK-2 pathways and is dependent on the activation of caspase-3 and -9.

Herein we aimed to understand how nanoscale clustering of RGD ligands alters the mechano-regulation of their integrin receptors. We combined molecular tension fluorescence microscopy with block copolymer micelle nanolithography to fabricate substrates with arrays of precisely spaced probes that can generate a 10-fold fluorescence response to pN-forces. We found that the mechanism of sensing ligand spacing is force-mediated. This strategy is broadly applicable to investigating receptor clustering and its role in mechanotransduction pathways.

In working towards the goal of mimicking multiple features of the extracellular matrix (ECM), we developed a strategy to create adhesive protein patterns on polymer films with tunable viscous characteristics. The block copolymer films are generated by interfacial self-assembly with the presentation of dopant homopolymer, since the concentration of the latter is known to affect the lateral mobility of the film. The supported polymer films are subsequently surface-modified by microcontact printing using fibronectin (FN), yielding material surfaces which can potentially display independent control over mechanical and adhesive properties. This work demonstrates a new method for the design of materials with the potential to recapitulate the viscous component of the ECM in future in vitro studies.

Segmentation is a characteristic feature of the vertebrate body plan. The prevailing paradigm explaining its origin is the ‘clock and wave-front’ model, which assumes that the interaction of a molecular oscillator (clock) with a traveling gradient of morphogens (wave) pre-defines spatial periodicity. While many genes potentially responsible for these processes have been identified, the precise role of molecular oscillations and the mechanism leading to physical separation of the somites remain elusive. In this paper we argue that the periodicity along the embryonic body axis anticipating somitogenesis is controlled
by mechanical rather than bio-chemical signaling. Using a prototypical model we show that regular patterning can result from a mechanical instability induced by differential strains developing between the segmenting mesoderm and the surrounding tissues. The main ingredients of the model are the assumptions that cell–cell adhesions soften when overstretched, and that there is an internal length scale defining the cohesive properties of the mesoderm. The proposed mechanism generates a robust number of segments without dependence on genetic oscillations.

Chondrocytes are mechanosensitive cells that require mechanical stimulation for proper growth and function in in vitro culture systems. Ultrasound (US) has emerged as a technique to deliver mechanical stress; however, the intracellular signaling components of the mechanotransduction pathways that transmit the extracellular mechanical stimulus to gene regulatory mechanisms are not fully defined. We evaluated a possible integrin/ mitogen-activated protein kinase (MAPK) mechanotransduction pathway using Western blotting with antibodies targeting specific phosphorylation sites on intracellular signaling proteins. US stimulation of chondrocytes induced phosphorylation of focal adhesion kinase (FAK), Src, p130 Crk-associated substrate (p130Cas), CrkII and extracellular-regulated kinase (Erk). Furthermore, pre-incubation with inhibitors of integrin receptors, Src and MAPK/Erk kinase (MEK) reduced US-induced Erk phosphorylation levels, indicating integrins and Src are upstream of Erk in an US-mediated mechanotransduction pathway. These findings suggest US signals through integrin receptors to the MAPK/Erk pathway via a mechanotransduction pathway involving FAK, Src, p130Cas and CrkII. (E-mail: asubramanian2@unl.edu) Published by Elsevier Inc. on behalf of World Federation for Ultrasound in Medicine & Biology

Mechanobiology to date has focused on differentiated cells or progenitors, yet the effects of mechanical forces on early differentiation of pluripotent stem cells are still largely unknown. To study the effects of cellular deformation, we utilize a fluid flow bioreactor to apply steady laminar shear stress to mouse embryonic stem cells (ESCs) cultured on a two dimensional surface. Shear stress was found to affect pluripotency, as well as germ specification to the mesodermal, endodermal, and ectodermal lineages, as indicated by gene expression of OCT4, T-BRACHY, AFP, and NES, respectively. The ectodermal and mesodermal response to shear stress was dependent on stress magnitude (ranging from 1.5 to 15 dynes/cm2). Furthermore, increasing the duration from one to four days resulted in a sustained increase in T-BRACHY and a marked suppression of AFP. These changes in differentiation occurred concurrently with the activation of Wnt and Estrogen pathways, as determined by PCR arrays for si...

The development of predictive mathematical models can contribute to a deeper understanding of the specific stages of bone mechanobiology and the process by which bone adapts to mechanical forces. The objective of this work was to predict, with spatial accuracy, cortical bone adaptation to mechanical load, in order to better understand the mechanical cues that might be driving adaptation. The axial tibial loading model was used to trigger cortical bone adaptation in C57BL/6 mice and provide relevant biological and biomechanical information. A method for mapping cortical thickness in the mouse tibia diaphysis was developed, allowing for a thorough spatial description of where bone adaptation occurs. Poroelastic finite-element (FE) models were used to determine the structural response of the tibia upon axial loading and interstitial fluid velocity as the me

One of the major unsolved mysteries of biological science concerns the question of where and in what form information is stored in the brain. I propose that memory is stored in the brain in a mechanically encoded binary format written into the conformations of proteins found in the cell-extracellular matrix (ECM) adhesions that organise each and every synapse. The MeshCODE framework outlined here represents a unifying theory of data storage in animals, providing read-write storage of both dynamic and persistent information in a binary format. Mechanosensitive proteins that contain force-dependent switches can store information persistently, which can be written or updated using small changes in mechanical force. These mechanosensitive proteins, such as talin, scaffold each synapse, creating a meshwork of switches that together form a code, the so-called MeshCODE. Large signalling complexes assemble on these scaffolds as a function of the switch patterns and these complexes would both stabilise the patterns and coordinate synaptic regulators to dynamically tune synaptic activity. Synaptic transmission and action potential spike trains would operate the cytoskeletal machinery to write and update the synaptic MeshCODEs, thereby propagating this coding throughout the organism. Based on established biophysical principles, such a mechanical basis for memory would provide a physical location for data storage in the brain, with the binary patterns, encoded in the information-storing mechanosensitive molecules in the synaptic scaffolds, and the complexes that form on them, representing the physical location of engrams. Furthermore, the conversion and storage of sensory and temporal inputs into a binary format would constitute an addressable read-write memory system, supporting the view of the mind as an organic supercomputer.

Fetal kicking and movements generate biomechanical stimulation in the fetal skeleton, which is important for prenatal musculoskeletal development, particularly joint shape. Developmental dysplasia of the hip (DDH) is the most common joint shape abnormality at birth, with many risk factors for the condition being associated with restricted fetal movement. In this study, we investigate the biomechanics of fetal movements in such situations, namely fetal breech position, oligohydramnios and primiparity (firstborn pregnancy). We also investigate twin pregnancies, which are not at greater risk of DDH incidence, despite the more restricted intra-uterine environment. We track fetal movements for each of these situations using cine-MRI technology, quantify the kick and muscle forces, and characterise the resulting stress and strain in the hip joint, testing the hypothesis that altered biomechanical stimuli may explain the link between certain intra-uterine conditions and risk of DDH. Kick force, stress and strain were found to be significantly lower in cases of breech position and oligohydramnios. Similarly, firstborn fetuses were found to generate significantly lower kick forces than non-firstborns. Interestingly, no significant difference was observed in twins compared to singletons. This research represents the first evidence of a link between the biomechanics of fetal movements and the risk of DDH, potentially informing the development of future preventative measures and enhanced diagnosis. Our results emphasise the importance of ultrasound screening for breech position and oligohydramnios, particularly later in pregnancy, and suggest that earlier intervention to correct breech position through external cephalic version could reduce the risk of hip dysplasia.

Graphical Abstract Highlights d Force-generating actin networks adapt to changing mechanical resistance d Resistance increases network density and power output without altering composition d Force-feedback strengthens load-bearing networks and gives them mechanical memory d Both external and internal material properties control network motor activity

In all biological systems, a balance between cell proliferation/growth and death is required for normal development as well as for adaptation to a changing environment. To affect their fate, it is essential for cells to integrate signals from the environment. Recently, it has been recognized that physical forces such as stretch, strain, and tension play a critical role in regulating this process. Despite intensive investigation, the pathways by which mechanical signals are converted to biochemical responses is yet to be completely understood. In this review, we will examine our current understanding of how mechanical forces induce apoptosis in a variety of biological systems. Rather than being a degenerative event, physical forces act through specific receptor-like molecules such as integrins, focal adhesion proteins, and the cytoskeleton. These molecules in turn activate a limited number of protein kinase pathways (p38 MAPK and JNK/SAPK), which amplify the signal and activate enzymes (caspases) that promote apoptosis. Physical forces concurrently activate other signaling pathways such as PIK-3 and Erk 1/2 MAPK, which modulate the apoptotic response. The cell phenotype and the character of the physical stimuli determine which pathways are activated and, consequently, allow for variability in response to a specific stimulus in different cell types.

Cellular mechanical stimulators with multi-plexing and high-throughput capability often use multiple external pumps, which compromise simplicity and minia-turization. In this study, we report a bilayered microfluidic device driven by one single pump to deliver in-plane surface strains toward four membranes with proportional center strain magnitudes. The maximal strain magnitudes exhibited a constant proportionality (i.e., 1:2:3:4) under both static and dynamic loading conditions. The influences of the loading frequency and the total membrane number were examined. Proof-of-concept cell loading showed that the device can be used to investigate magnitude-dependent cell responses to mechanical stimuli, thus promising broad applications in cell mechanobiological studies where delivery of mechanical signals with controlled and varying magnitudes toward multiple loading sites is needed.

Facial wrinkles are clear markers of the aging process, being chronological, photo-induced or reflecting repetitive facial expressions. Recent in silico results demonstrated the role of stratum corneum (SC) biomechanics on the skin folding. Cumulative and localized mechanical stresses, induced by facial expressions, could generate biomechanical fatigue in deep skin layers and contribute to create wrinkles. To such an issue, computational models theoretically alleviate that a long-lasting softening of the SC should decrease the severity of facial wrinkles. The objective of our study was to assess clinically this hypothesis. We have used the combination of clinical, instrumental in vivo data, digital methodology and proteomic data to give new insights to the biophysical and biological mechanisms involved. The long-term efficacy of a 20% glycerol based moisturizer (GBM) versus its vehicle was evaluated on crow's feet skin hydration, crow's feet wrinkles visibility and skin elasticity. A significant increase of hydration was measured at D56 and D112 for both formulae, but 2 times higher with GBM vs its vehicle. A significant increase of the skin elasticity was observed on the crow's feet site at D56 or D112 always in favor of GBM, versus its vehicle. There was a significant decrease in the severity of crow's feet wrinkles scoring, depth and volume at D112 for both formulae, albeit higher with the GBM. Proteomic data revealed an association between the effect of GBM and upregulation of four proteins among which three enzymes associated with the desquamation process, the cell-glycan extracellular interactions and the glycation/oxidation of proteins. Three functions closely related to tissue mechanical/adhesion properties. Our results provide an in vivo demonstration that a cumulative skin surface softening treatment might have an anti-ageing impact, both on crow's feet wrinkle and skin elasticity. The use of high concentration of glycerol should bring additional performance to other conventional cosmetic anti-ageing formulations.

Talins are cytoskeletal linker proteins that consist of an N-terminal head domain, a flexible neck region and a C-terminal rod domain made of 13 helical bundles. The head domain binds integrin β-subunit cytoplasmic tails, which triggers integrin conformational activation to increase affinity for extracellular matrix proteins. The rod domain links to actin filaments inside the cell to transmit mechanical loads and serves as a mechanosensitive signalling hub for the recruitment of many other proteins. The α-helical bundles function as force-dependent switches – proteins that interact with folded bundles are displaced when force induces unfolding, exposing previously cryptic binding sites for other ligands. This leads to the notion of a talin code. In this Cell Science at a Glance article and the accompanying poster, we propose that the multiple switches within the talin rod function to process and store time- and force-dependent mechanical and chemical information.

Ischaemic mitral regurgitation (IMR), a frequent complication following myocardial infarction (MI), leads to higher mortality and poor clinical prognosis if untreated. Accumulating evidence suggests that mitral valve (MV) leaflets actively remodel post MI, and this remodelling increases both the severity of IMR and the occurrence of MV repair failures. However, the mechanisms of extracellular matrix maintenance and modulation by MV interstitial cells (MVICs) and their impact on MV leaflet tissue integrity and repair failure remain largely unknown. Herein, we sought to elucidate the multiscale behaviour of IMR-inducedMV remodelling using an established ovinemodel. Leaflet tissue at eight weeks post MI exhibited significant permanent plastic radial deformation, eliminatingmechanical anisotropy, accompanied by altered leaflet composition. Interestingly, no changes in effective collagen fibre modulus were observed, with MVICs slightly rounder, at eight weeks post MI. RNA sequencing indicated that YAP-induced genes were elevated at four weeks post MI, indicating elevated mechanotransduction. Genes related to extracellular matrix organization were downregulated at four weeks post MI when IMR occurred. Transcriptomic changes returned to baseline by eight weeks post MI. This multiscale study suggests that IMR induces plastic deformation of the MV with no functional damage to the collagen fibres, providing crucial information for computational simulations of the MV in IMR.

Talin is a mechanosensitive component of adhesion complexes that directly couples integrins to the actin cytoskeleton. In response to force, talin undergoes switch-like behaviour of its multiple rod domains that modulate interactions with its binding partners. Cyclin-dependent kinase-1 (CDK1) is a key regulator of the cell cycle, exerting its effects through synchronised phosphorylation of a large number of protein targets. CDK1 activity also maintains adhesion during interphase, and its inhibition is a prerequisite for the tightly choreographed changes in cell shape and adhesiveness that are required for successful completion of mitosis. Using a combination of biochemical, structural and cell biological approaches, we demonstrate a direct interaction between talin and CDK1 that occurs at sites of integrin- mediated adhesion. Mutagenesis demonstrated that CDK1 contains a functional talin-binding LD motif, and the binding site within talin was pinpointed to helical bundle R8 through the use of recombinant fragments. Talin also contains a consensus CDK1 phosphorylation motif centred on S1589; a site that was phosphorylated by CDK1 in vitro. A phosphomimetic mutant of this site within talin lowered the binding affinity of KANK and weakened the mechanical response of the region, potentially altering downstream mechanotransduction pathways. The direct binding of the master cell cycle regulator, CDK1, to the primary integrin effector, talin, therefore provides a primordial solution for coupling the cell proliferation and cell adhesion machineries, and thereby enables microenvironmental control of cell division in multicellular organisms.

Peri-acetabular bone ingrowth plays a crucial role in long-term stability of press-fit acetabular
cups. A poor bone ingrowth often results in increased cup migration, leading to aseptic loosening of the
implant. The rate of peri-prosthetic bone formation is also affected by the polar gap that may be introduced
during implantation. Applying a mechano-regulatory tissue differentiation algorithm on a two-dimensional
plane strain microscale model, representing implant-bone interface, the objectives of the study are to gain an
insight into the process of peri-prosthetic tissue differentiation and to investigate its relationship with
implant-bone relative displacement and size of the polar gap. Implant-bone relative displacement was found
to have a considerable influence on bone healing and peri-acetabular bone ingrowth. An increase in implantbone
relative displacement from 20 µm to 100 µm resulted in an increase in fibrous tissue formation from
22% to 60% and reduction in bone formation from 70% to 38% within the polar gap. The increase in fibrous
tissue formation and subsequent decrease in bone formation leads to weakening of the implant-bone
interface strength. In comparison, the effect of polar gap on bone healing and peri-acetabular bone ingrowth
was less pronounced. Polar gap up to 5 mm was found to be progressively filled with bone under favourable
implant-bone relative displacements of 20 µm along tangential and 20 µm along normal directions.
However, the average Young’s modulus of the newly formed tissue layer reduced from 2200 MPa to 1200
MPa with an increase in polar gap from 0.5 mm to 5 mm, suggesting the formation of a low strength tissue
for increased polar gap. Based on this study, it may be concluded that a polar gap less than 0.5 mm seems
favourable for an increase in strength of the implant-bone interface.

Bite-force performance is an ecologically important measure of whole-organism performance that shapes dietary breadth and feeding strategies and, in some taxa, determines reproductive success. It also is a metric that is crucial to testing and evaluating biomechanical models. We reviewed nearly 100 published studies of a range of taxa that incorporate direct in vivo measurements of bite force. Problematically, methods of data collection and processing vary considerably among studies. In particular, there is little consensus on the appropriate substrate to use on the biting surface of force transducers. In addition, the bite out-lever, defined as the distance from the fulcrum (i.e. jaw joint) to the position along the jawline at which the jaws engage the transducer, is rarely taken into account. We examined the effect of bite substrate and bite out-lever on bite-force estimates in a diverse sample of lizards. Results indicate that both variables have a significant impact on the accuracy of measurements. Maximum bite force is significantly greater using leather as the biting substrate compared with a metal substrate. Less-forceful bites on metal are likely due to inhibitory feedback from mechanoreceptors that prevent damage to the feeding apparatus. Standardization of bite out-lever affected which trial produced maximum performance for a given individual. Indeed, maximum bite force is usually underestimated without standardization because it is expected to be greatest at the minimum out-lever (i.e. back of the jaws), which in studies is rarely targeted with success. We assert that future studies should use a pliable substrate, such as leather, and use appropriate standardization for bite out-lever.

In order to understand the sensitivity of alveolar macrophages (AMs) to substrate properties, we have developed a new model of macrophages cultured on substrates of increasing Young's modulus: (i) a monolayer of alveolar epithelial cells representing the supple (˜0.1 kPa) physiological substrate, (ii) polyacrylamide gels with two concentrations of bis-acrylamide representing low and high intermediate stiffness (respectively 40 kPa and 160 kPa) and, (iii) a highly rigid surface of plastic or glass (respectively 3 MPa and 70 MPa), the two latter being or not functionalized with type I-collagen. The macrophage response was studied through their shape (characterized by 3D-reconstructions of F-actin structure) and their cytoskeletal stiffness (estimated by transient twisting of magnetic RGD-coated beads and corrected for actual bead immersion). Macrophage shape dramatically changed from rounded to flattened as substrate stiffness increased from soft ((i) and (ii)) to rigid (iii) substrates, indicating a net sensitivity of alveolar macrophages to substrate stiffness but without generating F-actin stress fibers. Macrophage stiffness was also increased by large substrate stiffness increase but this increase was not due to an increase in internal tension assessed by the negligible effect of a F-actin depolymerizing drug (cytochalasine D) on bead twisting. The mechanical sensitivity of AMs could be partly explained by an idealized numerical model describing how low cell height enhances the substrate-stiffness-dependence of the apparent (measured) AM stiffness. Altogether, these results suggest that macrophages are able to probe their physical environment but the mechanosensitive mechanism behind appears quite different from tissue cells, since it occurs at no significant cell-scale prestress, shape changes through minimal actin remodeling and finally an AMs stiffness not affected by the loss in F-actin integrity. Cell Motil. Cytoskeleton 2006. © 2006 Wiley-Liss, Inc.

The objective of this study was to examine the interplay between matrix stiffness and hydrostatic pressure (HP) in regulating chondrogenesis of mesenchymal stem cells (MSCs) and to further elucidate the mechanotransductive roles of integrins and the cytoskeleton. MSCs were seeded into 1 %, 2 % or 4 % agarose hydrogels and exposed to cyclic hydrostatic pressure. In a permissive media, the stiffer hydrogels supported an osteogenic phenotype, with little evidence of chondrogenesis observed regardless of the matrix stiffness. In a chondrogenic media, the stiffer gels suppressed cartilage matrix production and gene expression, with the addition of RGDS (an integrin blocker) found to return matrix synthesis to similar levels as in the softer gels. Vinculin, actin and vimentin organisation all adapted within stiffer hydrogels, with the addition of RGDS again preventing these changes. While the stiffer gels inhibited chondrogenesis, they enhanced mechanotransduction of HP. RGDS suppressed the mechanotransduction of HP, suggesting a role for integrin binding as a regulator of both matrix stiffness and HP. Intermediate filaments also appear to play a role in the mechanotransduction of HP, as only vimentin organisation adapted in response to this mechanical stimulus. To conclude, the results of this study demonstrate that matrix density and/or stiffness modulates the development of the pericellular matrix and consequently integrin binding and cytoskeletal structure. The study further suggests that physiological cues such as HP enhance chondrogenesis of MSCs as the pericellular environment matures and the cytoskeleton adapts, and points to a novel role for vimentin in the transduction of HP.