Mechanobiology
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Most cited papers in Mechanobiology
Intervertebral disc degeneration is a major cause of low back pain. Despite its long history and large socio-economical impact in western societies, the initiation and progress of disc degeneration is not well understood and a generic... more
Intervertebral disc degeneration is a major cause of low back pain. Despite its long history and large
socio-economical impact in western societies, the initiation and progress of disc degeneration is not well
understood and a generic disease model is lacking. In literature, mechanics and biology have both been
implicated as the predominant inductive cause; here we argue that they are interconnected and amplify
each other. This view is supported by the growing awareness that cellular physiology is strongly affected
by mechanical loading. We propose a vicious circle of mechanical overloading, catabolic cell response,
and degeneration of the water-binding extracellular matrix. Rather than simplifying the disease, the
model illustrates the complexity of disc degeneration, because all factors are interrelated. It may however
solve some of the controversy in the field, because the vicious circle can be entered at any point,
eventually leading to the same pathology. The proposed disease model explains the comparable efficacy
of very different animal models of disc degeneration, but also helps to consider the consequences of
therapeutic interventions, either at the cellular, material or mechanical level.
socio-economical impact in western societies, the initiation and progress of disc degeneration is not well
understood and a generic disease model is lacking. In literature, mechanics and biology have both been
implicated as the predominant inductive cause; here we argue that they are interconnected and amplify
each other. This view is supported by the growing awareness that cellular physiology is strongly affected
by mechanical loading. We propose a vicious circle of mechanical overloading, catabolic cell response,
and degeneration of the water-binding extracellular matrix. Rather than simplifying the disease, the
model illustrates the complexity of disc degeneration, because all factors are interrelated. It may however
solve some of the controversy in the field, because the vicious circle can be entered at any point,
eventually leading to the same pathology. The proposed disease model explains the comparable efficacy
of very different animal models of disc degeneration, but also helps to consider the consequences of
therapeutic interventions, either at the cellular, material or mechanical level.
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... more
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.
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... more
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.
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.
Solid stress and tissue stiffness affect tumour growth, invasion, metastasis and treatment. Unlike stiffness, which can be precisely mapped in tumours, the measurement of solid stresses is challenging. Here, we show that 2D spatial maps... more
Solid stress and tissue stiffness affect tumour growth, invasion, metastasis and treatment. Unlike stiffness, which can be precisely mapped in tumours, the measurement of solid stresses is challenging. Here, we show that 2D spatial maps of the solid stress and the resulting elastic energy in excised or in situ tumours with arbitrary shapes and a wide range of sizes can be obtained via three distinct and quantitative techniques that rely on the measurement of tissue displacement after disruption of the confining structures. Application of these methods in models of primary tumours and metastasis revealed that (i) solid stress depends on both cancer cells and their microenvironments, (ii) solid stress increases with tumour size and (iii) mechanical confinement by the surrounding tissue substantially contributes to intratumoral solid stress. Further study of the genesis and consequences of solid stress, facilitated by the engineering principles presented here, may lead to new discoveries and therapies. I ncreased tissue stiffness is a widely accepted and actively studied biomechanical property of fibrotic tumours and has been linked to several hallmarks of cancer, including growth, metabolism, invasion and metastasis 1–7. However, the abnormal mechanics of tumours are not limited to tissue stiffening. We recently demonstrated that solid stress represents a new mechanopathology that is consistently elevated in mouse and human tumours 8,9. Solid stress is distinct from interstitial fluid pressure, as the former is contained in—and transmitted by—solid and elastic elements of the extracel-lular matrix and cells rather than fluids 10. Therefore, tumours are not only more rigid than many normal tissues, but cancer cells also produce and are exposed to these physical forces. Composed of a combination of tension and compression, these forces are significant in tumours, but negligible in most normal tissues. Early evidence for the existence of solid stress in tumours came from the discovery that blood and lymphatic vessels are mechanically compressed 11–13. This can contribute to hypoxia 9,14 , promote tumour progression and decrease the efficacy of chemo-, radio-and immunotherapies 15. In addition to these physiological consequences, forces can directly affect tumour biology: the exog-enous application of solid stress in vivo can mechanically stimulate tumorigenic pathways, increasing β-catenin signalling in colon epithelia 16 , and the controlled application of compressive forces in vitro affects the growth 8 of cancer cells and promotes their collective migration 17. Strategies designed to reduce solid stress and decompress blood vessels by reducing levels of collagen and hyal-uronic acid 14,18,19 have led to therapeutic approaches for enhancing the efficiency of conventional anticancer treatments and are currently being tested in clinical trials 20,21. Despite the important implications of solid stress and the immense potential for finding new mechanically activated pathways and targets, there are currently no high-resolution methods for quantifying solid stress in experimental or human tumours. Unlike stiffness, which can be measured using widely available mul-tiscale techniques, measuring solid stress in biological tissues has proved challenging. Previous studies in our laboratory 8,9 , based on previous observations 22,23 that arterial wall tissue relaxes if the contained forces are surgically released, demonstrated the presence of residual tissue stresses in tumours. However, this approach is based on a partial cut through a spherical model of the tumour, which makes the precise release of solid stress and the measurement of the ensuing deformation challenging. This method is also limited to bulk estimation of solid stress and is not applicable in situ. The optical depth limitations of alternative imaging-based methods, such as fluorescent oil microdroplet injection 24 and single-molecule fluorescent force sensors 25 , restrict their use to cellular-and subcellular-scale force detection. We have developed experimental and mathematical frameworks to provide 2D mapping of solid stress in tumours (planar-cut method), sensitive estimations of the solid stress in small tumours with small magnitudes of solid stress, as is the case for metastatic lesions (slicing method), and in situ quantification of solid stress in tumours, which retains the effects of the normal surrounding tissues (needle-biopsy method). All three methods are based on the concept of releasing the solid stress in a controlled way with a defined geometry and then quantifying the stress-induced deformation via high-resolution ultrasonography or optical microscopy. Given the specific topography of the stress relaxation and the geometric
The most common cause of death in the developed world is cardiovascular disease. For decades, this has provided a powerful motivation to study the effects of mechanical forces on vascular cells in a controlled setting, since these cells... more
The most common cause of death in the developed world is cardiovascular disease. For decades, this has provided a powerful motivation to study the effects of mechanical forces on vascular cells in a controlled setting, since these cells have been implicated in the development of disease. Early efforts in the 1970 s included the first use of a parallel-plate flow system to apply shear stress to endothelial cells (ECs) and the development of uniaxial substrate stretching techniques (Krueger et al., 1971, “An in Vitro Study of Flow Response by Cells,” J. Biomech., 4(1), pp. 31–36 and Meikle et al., 1979, “Rabbit Cranial Sutures in Vitro: A New Experimental Model for Studying the Response of Fibrous Joints to Mechanical Stress,” Calcif. Tissue Int., 28(2), pp. 13–144). Since then, a multitude
of in vitro devices have been designed and developed for mechanical stimulation of vascular cells and tissues in an effort to better understand their response to in vivo physiologic
mechanical conditions. This article reviews the functional attributes of mechanical bioreactors developed in the 21st century, including their major advantages and disadvantages.
Each of these systems has been categorized in terms of their primary loading modality: fluid shear stress (FSS), substrate distention, combined distention and fluid shear, or other applied forces. The goal of this article is to provide researchers with a survey of useful methodologies that can be adapted to studies in this area, and to clarify future possibilities for improved research methods.
of in vitro devices have been designed and developed for mechanical stimulation of vascular cells and tissues in an effort to better understand their response to in vivo physiologic
mechanical conditions. This article reviews the functional attributes of mechanical bioreactors developed in the 21st century, including their major advantages and disadvantages.
Each of these systems has been categorized in terms of their primary loading modality: fluid shear stress (FSS), substrate distention, combined distention and fluid shear, or other applied forces. The goal of this article is to provide researchers with a survey of useful methodologies that can be adapted to studies in this area, and to clarify future possibilities for improved research methods.
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... more
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
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... more
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 .
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... more
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.
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.
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... more
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.
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... more
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.
ABSTRACT Stem cell responsiveness to extracellular matrix (ECM) composition and mechanical cues has been the subject of a number of investigations so far, yet the molecular mechanisms underlying stem cell mechano-biology still need full... more
ABSTRACT Stem cell responsiveness to extracellular matrix (ECM)
composition and mechanical cues has been the subject of a number of
investigations so far, yet the molecular mechanisms underlying stem
cell mechano-biology still need full clarification. Here we demonstrate
that the paralog proteins YAP and TAZ exert a crucial role in adult
cardiac progenitor cell mechano-sensing and fate decision. Cardiac
progenitors respond to dynamic modifications in substrate rigidity and
nanopattern by promptly changing YAP/TAZ intracellular localization.
We identify a novel activity of YAP and TAZ in the regulation of
tubulogenesis in 3D environments and highlight a role for YAP/TAZ in
cardiac progenitor proliferation and differentiation. Furthermore, we
show that YAP/TAZ expression is triggered in the heart cells located at the infarct border zone. Our results suggest a fundamental role for the YAP/TAZ axis in the
response of resident progenitor cells to the modifications in microenvironment nanostructure and mechanics, thereby contributing to the maintenance of
myocardial homeostasis in the adult heart. These proteins are indicated as potential targets to control cardiac progenitor cell fate by materials design.
• Mosqueira D, Pagliari S, Uto K, Ebara M, Romanazzo S, Escobedo-Lucea C, Nakanishi J, Taniguchi A, Franzese O, Di Nardo P, Goumans M J, Traversa E, Pinto-Do-Ó P, Aoyagi T & Forte, G. Hippo Pathway Effectors Control Cardiac Progenitor Cell Fate by Acting as Dynamic Sensors of Substrate Mechanics and Nanostructure. ACS Nano (2014), 8(3):2033-47. DOI:10.1021/nn4058984.
KEYWORDS: adult cardiac progenitor cell . substrate nanotopography . mechano-transduction . cardiac differentiation . YAP/TAZ
composition and mechanical cues has been the subject of a number of
investigations so far, yet the molecular mechanisms underlying stem
cell mechano-biology still need full clarification. Here we demonstrate
that the paralog proteins YAP and TAZ exert a crucial role in adult
cardiac progenitor cell mechano-sensing and fate decision. Cardiac
progenitors respond to dynamic modifications in substrate rigidity and
nanopattern by promptly changing YAP/TAZ intracellular localization.
We identify a novel activity of YAP and TAZ in the regulation of
tubulogenesis in 3D environments and highlight a role for YAP/TAZ in
cardiac progenitor proliferation and differentiation. Furthermore, we
show that YAP/TAZ expression is triggered in the heart cells located at the infarct border zone. Our results suggest a fundamental role for the YAP/TAZ axis in the
response of resident progenitor cells to the modifications in microenvironment nanostructure and mechanics, thereby contributing to the maintenance of
myocardial homeostasis in the adult heart. These proteins are indicated as potential targets to control cardiac progenitor cell fate by materials design.
• Mosqueira D, Pagliari S, Uto K, Ebara M, Romanazzo S, Escobedo-Lucea C, Nakanishi J, Taniguchi A, Franzese O, Di Nardo P, Goumans M J, Traversa E, Pinto-Do-Ó P, Aoyagi T & Forte, G. Hippo Pathway Effectors Control Cardiac Progenitor Cell Fate by Acting as Dynamic Sensors of Substrate Mechanics and Nanostructure. ACS Nano (2014), 8(3):2033-47. DOI:10.1021/nn4058984.
KEYWORDS: adult cardiac progenitor cell . substrate nanotopography . mechano-transduction . cardiac differentiation . YAP/TAZ
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... more
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.
The strain-generated potential (SGP) is a well-established mechanism in cartilaginous tissues whereby mechanical forces generate electrical potentials. In articular cartilage (AC) and the intervertebral disc (IVD), studies on the SGP have... more
The strain-generated potential (SGP) is a well-established mechanism in cartilaginous tissues whereby mechanical forces generate electrical potentials. In articular cartilage (AC) and the intervertebral disc (IVD), studies on the SGP have focused on fluid-and ionic-driven effects, namely Donnan, diffusion and streaming potentials. However, recent evidence has indicated a direct coupling between strain and electrical potential. Piezoelectricity is one such mechanism whereby deformation of most biological structures, like collagen, can directly generate an electrical potential. In this review, the SGP in AC and the IVD will be revisited in light of piezoelectricity and mechanotransduction. While the evidence base for physiologically significant piezoelectric responses in tissue is lacking, difficulties in quantifying the physiological response and imperfect measurement techniques may have underestimated the property. Hindering our understanding of the SGP further, numerical models to-date have negated ferroelectric effects in the SGP and have utilised classic Donnan theory that, as evidence argues, may be oversimplified. Moreover, changes in the SGP with degeneration due to an altered extracellular matrix (ECM) indicate that the significance of ionic-driven mechanisms may diminish relative to the piezoelectric response. The SGP, and these mechanisms behind it, are finally discussed in relation to the cell response.
Epithelial cell-cell junctions remodel in response to mechanical stimuli to maintain barrier function. Previously, we found that local leaks in tight junctions (TJs) are rapidly repaired by local, transient RhoA activation, termed "Rho... more
Epithelial cell-cell junctions remodel in response to mechanical stimuli to maintain barrier function. Previously, we found that local leaks in tight junctions (TJs) are rapidly repaired by local, transient RhoA activation, termed "Rho flares," but how Rho flares are regulated is unknown. Here, we discovered that intracellular calcium flashes and junction elongation are early events in the Rho flare pathway. Both laser-induced and naturally occurring TJ breaks lead to local calcium flashes at the site of leaks. Additionally, junction elongation induced by optogenetics increases Rho flare frequency, suggesting that Rho flares are mechanically triggered. Depletion of intracellular calcium or inhibition of mechanosensitive calcium channels (MSCs) reduces the amplitude of calcium flashes and diminishes the sustained activation of Rho flares. MSC-dependent calcium influx is necessary to maintain global barrier function by regulating reinforcement of local TJ proteins via junction contraction. In all, we uncovered a novel role for MSC-dependent calcium flashes in TJ remodeling, allowing epithelial cells to repair local leaks induced by mechanical stimuli.
Studying chemomechanical coupling at interfaces is important for fields ranging from lubrication and tribology to microfluidics and cell biology. Several polymeric macro-and microscopic systems and cantilevers have been developed to image... more
Studying chemomechanical coupling at interfaces is important for fields ranging from lubrication and tribology to microfluidics and cell biology. Several polymeric macro-and microscopic systems and cantilevers have been developed to image forces at interfaces, but few materials are amenable for molecular tension sensing. To address this issue, we have developed a gold nanoparticle sensor for molecular tension-based fluorescence microscopy. As a proof of concept, we imaged the tension exerted by integrin receptors at the interface between living cells and a substrate with high spatial (<1 μm) resolution, at 100 ms acquisition times and with molecular specificity. We report integrin tension values ranging from 1 to 15 pN and a mean of ∼1 pN within focal adhesions. Through the use of a conventional fluorescence microscope, this method demonstrates a force sensitivity that is 3 orders of magnitude greater than is achievable by traction force microscopy or polydimethylsiloxane micropost arrays, 1 which are the standard in cellular biomechanics.
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... more
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.
The role of substrate mechanics in guiding mesenchymal stem cell (MSC) fate has been the focus of much research over the last decade. More recently, the complex interplay between substrate mechanics and other material properties such as... more
The role of substrate mechanics in guiding mesenchymal stem cell (MSC) fate has been the focus of much research over the last decade. More recently, the complex interplay between substrate mechanics and other material properties such as ligand chemistry and substrate degradability to regulate MSC differentiation has begun to be elucidated. Additionally, there are several changes in the presentation of these material properties as the dimensionality is altered from two- to three-dimensional substrates, which may fundamentally alter our understanding of substrate-induced mechanotransduction processes. In this review, an overview of recent findings that highlight the material properties that are important in guiding MSC fate decisions is presented, with a focus on underlining gaps in our existing knowledge and proposing potential directions for future research.
How glycoprotein (GP)Ib-IX complex on the platelet surface senses the blood flow through its binding to the plasma protein von Willebrand factor (VWF) and transmits a signal into the platelet remains unclear. Here we show that optical... more
How glycoprotein (GP)Ib-IX complex on the platelet surface senses the blood flow through its binding to the plasma protein von Willebrand factor (VWF) and transmits a signal into the platelet remains unclear. Here we show that optical tweezer-controlled pulling of the A1 domain of VWF (VWF-A1) on GPIb-IX captured by its cytoplasmic domain induced unfolding of a hitherto unidentified structural domain before the dissociation of VWF-A1 from GPIb-IX. Additional studies using recombinant proteins and mutant complexes confirmed its existence in GPIb-IX and enabled localization of this quasi-stable mechano- sensitive domain of ∼60 residues between the macroglycopeptide region and the transmembrane helix of the GPIba subunit. These results suggest that VWF-mediated pulling under fluid shear induces unfolding of the mechanosensitive domain in GPIb-IX, which may possibly contribute to platelet mechanosensing and/or shear resistance of VWF-platelet interaction. The identification of the mechanosensitive domain in GPIb-IX has significant implications for the pathogenesis and treatment of related blood diseases.
Pluripotent embryonic stem cells (ESCs) are a potential source for cell-based tissue engineering and regenerative medicine applications, but their translation into clinical use will require efficient and robust methods for promoting... more
Pluripotent embryonic stem cells (ESCs) are a potential source for cell-based tissue engineering and regenerative medicine applications, but their translation into clinical use will require efficient and robust methods for promoting differentiation. Fluid shear stress, which can be readily incorporated into scalable bioreactors, may be one solution for promoting endothelial and hematopoietic phenotypes from ESCs. Here we applied laminar shear stress to differentiating ESCs using a 2D adherent parallel plate configuration to systematically investigate the effects of several mechanical parameters. Treatment similarly promoted endothelial and hematopoietic differentiation for shear stress magnitudes ranging from 1.5 to 15 dyne/cm2 and for cells seeded on collagen-, fibronectin-, or laminin-coated surfaces. Extension of the treatment duration consistently induced an endothelial response, but application at later stages of differentiation was less effective at promoting hematopoietic phenotypes. Furthermore, inhibition of the FLK1 protein (a VEGF receptor) neutralized the effects of shear stress, implicating the membrane protein as a critical mediator of both endothelial and hematopoietic differentiation by applied shear. Using a systematic approach, studies such as these help elucidate the mechanisms involved in force-mediated stem cell differentiation and inform scalable bioprocesses for cellular therapies.
The poor prognosis of glioblastoma (GBM) is associated with a highly invasive stem-like subpopulation of tumor-initiating cells (TICs), which drive recurrence and contribute to intra-tumoral heterogeneity through differentiation. These... more
The poor prognosis of glioblastoma (GBM) is associated with a highly invasive stem-like subpopulation of tumor-initiating cells (TICs), which drive recurrence and contribute to intra-tumoral heterogeneity through differentiation. These TICs are better able to escape extracellular matrix-imposed mechanical restrictions on invasion than their more differentiated progeny, and sensitization of TICs to extracellular matrix mechanics extends survival in preclinical models of GBM. However, little is known about the molecular basis of the relationship between TIC differentiation and mechanotransduction. Here we explore this relationship through a combination of transcriptomic analysis and studies with defined-stiffness matrices. We show that TIC differentiation induced by bone morphogenetic protein 4 (BMP4) suppresses expression of proteins relevant to extracellular matrix signaling and sensitizes TIC spreading to matrix stiffness. Moreover, our findings point towards a previously unappreciated connection between BMP4-induced differentiation, mechanotransduction, and metabolism. Notably, stiffness and differentiation modulate oxygen consumption, and inhibition of oxidative phosphorylation influences cell spreading in a stiffness- and differentiation-dependent manner. Our work integrates bioinformatic analysis with targeted molecular measurements and perturbations to yield new insight into how morphogen-induced differentiation influences how GBM TICs process mechanical inputs.
- by Jasmine Hughes and +2
- •
- Stem Cells, Metabolism, Cancer, Cancer stem cells
Both hydrostatic pressure (HP) and cell-matrix interactions have independently been shown to regulate the chondrogenic differentiation of mesenchymal stem cells (MSCs). The objective of this study was to test the hypothesis that the... more
Both hydrostatic pressure (HP) and cell-matrix interactions have independently been shown to regulate the chondrogenic differentiation of mesenchymal stem cells (MSCs). The objective of this study was to test the hypothesis that the response of MSCs to hydrostatic pressure will depend on the biomaterial within which the cells are encapsulated. Bone-marrow-derived MSCs were seeded into either agarose or fibrin hydrogels and exposed to 10 MPa of cyclic HP (1 Hz, 4 h per day, 5 days per week for 3 weeks) in the presence of either 1 or 10 ng ml(-1) of TGF-β3. Agarose hydrogels were found to support a spherical cellular morphology, while MSCs seeded into fibrin hydrogels attached and spread, with clear stress fiber formation. Hydrogel contraction was also observed in MSC-fibrin constructs. While agarose hydrogels better supported chondrogenesis of MSCs, HP only enhanced sulfated glycosaminoglycan (sGAG) accumulation in fibrin hydrogels, which correlated with a reduction in fibrin contraction. HP also reduced alkaline phosphatase activity in the media for both agarose and fibrin constructs, suggesting that this stimulus plays a role in the maintenance of the chondrogenic phenotype. This study demonstrates that a complex relationship exists between cell-matrix interactions and hydrostatic pressure, which plays a key role in regulating the chondrogenic differentiation of MSCs.
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... more
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.
Cellular responses to chemical cues are at the core of a myriad of fundamental biological processes ranging from embryonic development to cancer metastasis. Most of these biological processes are also influenced by mechanical cues such as... more
Cellular responses to chemical cues are at the core of a myriad of fundamental biological processes ranging from embryonic development to cancer metastasis. Most of these biological processes are also influenced by mechanical cues such as the stiffness of the extracellular matrix. How a biological function is influenced by a synergy between chemical concentration and extracellular matrix stiffness is largely unknown, however, because no current strategy enables the integration of both types of cues in a single experiment. Here we present a robust microfluidic device that generates a stable, linear and diffusive chemical gradient over a biocompatible hydrogel with a well-defined stiffness gradient. Device fabrication relies on patterned PSA (Pressure Sensitive Adhesive) stacks that can be implemented with minimal cost and lab equipment. This technique is suitable for long-term observation of cell migration and application of traction force microscopy. We validate our device by testing MDCK cell scattering in response to perpen- dicular gradients of hepatocyte growth factor (HGF) and substrate stiffness.
- by Jerome Noailly and +1
- •
- Microfluidics, Cell Migration, Mechanobiology, Chemotaxis
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... more
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.
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... more
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.
Mechanisms by which blood cells sense shear stress are poorly characterized. In platelets, glycoprotein (GP)Ib–IX receptor complex has been long suggested to be a shear sensor and receptor. Recently, a relatively unstable and... more
Mechanisms by which blood cells sense shear stress are poorly characterized. In platelets, glycoprotein (GP)Ib–IX receptor complex has been long suggested to be a shear sensor and receptor. Recently, a relatively unstable and mechanosensitive domain in the GPIba subunit of GPIb–IX was identified. Here we show that binding of its ligand, von Willebrand factor, under physiological shear stress induces unfolding of this mechanosensory domain (MSD) on the platelet surface. The unfolded MSD, particularly the juxtamembrane 'Trigger' sequence therein, leads to intracellular signalling and rapid platelet clearance. These results illustrate the initial molecular event underlying platelet shear sensing and provide a mechanism linking GPIb–IX to platelet clearance. Our results have implications on the mechanism of platelet activation, and on the pathophysiology of von Willebrand disease and related thrombocy-topenic disorders. The mechanosensation via receptor unfolding may be applicable for many other cell adhesion receptors.
- by Renhao Li and +1
- •
- Mechanobiology, Platelets, Thrombocytopenia
Focal adhesion kinase (FAK) is a key molecule in focal adhesions and regulates fundamental processes in cells such as growth, survival, and migration. FAK is one of the first molecules recruited to focal adhesions in response to external... more
Focal adhesion kinase (FAK) is a key molecule in focal adhesions and regulates fundamental processes in cells such as growth, survival, and migration. FAK is one of the first molecules recruited to focal adhesions in response to external mechanical stimuli and therefore is a pivotal mediator of cell mechanosignaling, and relays these stimuli to other mechanotransducers within the cytoplasm. Yes-associated protein (YAP) has been identified recently as one of these core mechanotransducers. YAP translocates to the nucleus following changes in cell mechanics to promote the expression of genes implicated in motility, apoptosis, proliferation, and organ growth. Here, we show that FAK controls the nuclear translocation and activation of YAP in response to mechanical activation and submit that the YAP-dependent process of durotaxis requires a cell with an asymmetric distribution of active and inactive FAK molecules.
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... more
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
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... more
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.
During metastasis, cancer cells are exposed to potentially destructive hemodynamic forces including fluid shear stress (FSS) while en route to distant sites. However, prior work indicates that cancer cells are more resistant to brief... more
During metastasis, cancer cells are exposed to potentially destructive hemodynamic forces including fluid shear stress (FSS) while en route to distant sites. However, prior work indicates that cancer cells are more resistant to brief pulses of high-level FSS in vitro relative to non-transformed epithelial cells. Herein, we identify a mechano-adaptive mechanism of FSS resistance in cancer cells. Our findings demonstrate that cancer cells activate RhoA in response to FSS, which protects them from FSS-induced plasma membrane damage. We show that cancer cells freshly isolated from mouse and human tumors are resistant to FSS, that formin and myosin II activity protects circulating tumor cells (CTCs) from destruction, and that short-term inhibition of myosin II delays metastasis in mouse models. Collectively, our data indicate that viable CTCs actively resist destruction by hemodynamic forces and are likely to be more mechanically robust than is commonly thought.
In lung fibrosis tissue architecture and function is severely hampered by myofibroblasts due to excessive deposition of extracellular matrix and tissue contraction. Myofibroblasts differentiate from fibroblasts under the influence of... more
In lung fibrosis tissue architecture and function is severely hampered by myofibroblasts due to excessive deposition of extracellular matrix and tissue contraction. Myofibroblasts differentiate from fibroblasts under the influence of transforming growth factor (TGF) b 1 but this process is also controlled mechanically by cytoskeletal tension. In healthy lungs, the cytoskeleton of fibroblasts is mechanically strained during breathing. In stiffer fibrotic lung tissue, this mechanical stimulus is reduced, which may influence fibroblast-to-myofibroblast differentiation. Therefore, we investigated the effect of cyclic mechanical stretch on fibroblast-to-myofibroblast differentiation.
- by Reinout Stoop and +2
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- Cystic Fibrosis, Cell Cycle, Health, Mechanobiology
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... more
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.
Immune cells can sense and respond to biophysical cues — from dynamic forces to spatial features — during their development, activation, differentiation and expansion. These biophysical signals regulate a variety of immune cell functions... more
Immune cells can sense and respond to biophysical cues — from dynamic forces to spatial features — during their development, activation, differentiation and expansion. These biophysical signals regulate a variety of immune cell functions such as leukocyte extravasation, macrophage polarization, T cell selection and T cell activation. Recent studies have advanced our understanding on immune responses to biophysical cues and the underlying mechanisms of mechanotransduction, which provides rational basis for the design and development of immune-modulatory therapeutics. This review discusses the recent progress in mechanosensing and mechanotransduction of immune cells, particularly monocytes/macrophages and T lymphocytes, and features new biomaterial designs and biomedical devices that translate these findings into biomedical applications.
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... more
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.
RATIONALE: PhosPhatidic-Acid-Phosphatase-type-2B (PPAP2B), an integral membrane protein that inactivates lysophosphatidic acid, was implicated in coronary artery disease (CAD) by genome-wide-association-studies (GWAS). However, it is... more
RATIONALE: PhosPhatidic-Acid-Phosphatase-type-2B (PPAP2B), an integral membrane protein that inactivates lysophosphatidic acid, was implicated in coronary artery disease (CAD) by genome-wide-association-studies (GWAS). However, it is unclear whether GWAS-identified CAD genes including PPAP2B participate in mechanotransduction mechanisms by which vascular endothelia respond to local athero-relevant hemodynamics that contribute to the regional nature of atherosclerosis.
OBJECTIVE: To establish the critical role of PPAP2B in endothelial responses to hemodynamics.
METHODS AND RESULTS: Reduced PPAP2B was detected in vivo in mouse and swine aortic arch endothelia exposed to chronic disturbed flow, and in mouse carotid artery endothelia subjected to surgically-induced acute disturbed flow. In humans, PPAP2B was reduced in the downstream part of carotid plaques where low shear stress prevails. In culture, reduced PPAP2B was measured in human aortic endothelial cells (HAEC) under athero-susceptible waveform mimicking flow in human carotid sinus. Flow-sensitive microRNA-92a and transcription factor KLF2 were identified as upstream inhibitor and activator of endothelial PPAP2B, respectively. PPAP2B suppression abrogated athero-protection of unidirectional flow; Inhibition of lysophosphatidic acid receptor 1 (LPAR1) restored the flow-dependent, anti-inflammatory phenotype in PPAP2B-deficient cells. PPAP2B inhibition resulted in myosin-light-chain phosphorylation and intercellular gaps, which were abolished by LPAR1/2 inhibition. Expression-quantitative-trait-locus-mapping demonstrated PPAP2B CAD risk allele is not linked to PPAP2B expression in various human tissues but significantly associated with reduced PPAP2B in HAEC.
CONCLUSIONS: Athero-relevant flows dynamically modulate endothelial PPAP2B expression through miR-92a and KLF2. Mechano-sensitive PPAP2B plays a critical role in promoting anti-inflammatory phenotype and maintaining vascular integrity of endothelial monolayer under athero-protective flow.
OBJECTIVE: To establish the critical role of PPAP2B in endothelial responses to hemodynamics.
METHODS AND RESULTS: Reduced PPAP2B was detected in vivo in mouse and swine aortic arch endothelia exposed to chronic disturbed flow, and in mouse carotid artery endothelia subjected to surgically-induced acute disturbed flow. In humans, PPAP2B was reduced in the downstream part of carotid plaques where low shear stress prevails. In culture, reduced PPAP2B was measured in human aortic endothelial cells (HAEC) under athero-susceptible waveform mimicking flow in human carotid sinus. Flow-sensitive microRNA-92a and transcription factor KLF2 were identified as upstream inhibitor and activator of endothelial PPAP2B, respectively. PPAP2B suppression abrogated athero-protection of unidirectional flow; Inhibition of lysophosphatidic acid receptor 1 (LPAR1) restored the flow-dependent, anti-inflammatory phenotype in PPAP2B-deficient cells. PPAP2B inhibition resulted in myosin-light-chain phosphorylation and intercellular gaps, which were abolished by LPAR1/2 inhibition. Expression-quantitative-trait-locus-mapping demonstrated PPAP2B CAD risk allele is not linked to PPAP2B expression in various human tissues but significantly associated with reduced PPAP2B in HAEC.
CONCLUSIONS: Athero-relevant flows dynamically modulate endothelial PPAP2B expression through miR-92a and KLF2. Mechano-sensitive PPAP2B plays a critical role in promoting anti-inflammatory phenotype and maintaining vascular integrity of endothelial monolayer under athero-protective flow.
- by Guohao Dai and +1
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- Mechanobiology, Endothelial dysfunction
The assembly and mechanics of actomyosin stress fibers (SFs) depend on regulatory light chain (RLC) phosphorylation, which is driven by Myosin Light Chain Kinase (MLCK) and Rho-Associated Kinase (ROCK). While previous work suggests that... more
The assembly and mechanics of actomyosin stress fibers (SFs) depend on regulatory light chain (RLC) phosphorylation, which is driven by Myosin Light Chain Kinase (MLCK) and Rho-Associated Kinase (ROCK). While previous work suggests that MLCK and ROCK regulate distinct pools of cellular SFs, it remains unclear how these kinases differ in their regulation of RLC phosphorylation or how phosphorylation influences individual SF mechanics. Here, we combine genetic approaches with biophysical tools to explore relationships between kinase activity, RLC phosphorylation, SF localization, and SF mechanics. We show that graded MLCK overexpression increases RLC mono-phosphorylation (p-RLC) in a graded manner and that this p-RLC localizes to peripheral SFs. Conversely, graded ROCK overexpression preferentially increases RLC di-phosphorylation (pp-RLC), with pp-RLC localizing to central SFs. Interrogation of single SFs with subcellular laser ablation reveals that MLCK and ROCK quantitatively regulate the viscoelastic properties of peripheral and central SFs, respectively. The effects of MLCK and ROCK on single-SF mechanics may be correspondingly phenocopied by overexpression of mono- and di-phosphomimetic RLC mutants. Our results point to a model in which MLCK and ROCK regulate peripheral and central SF viscoelastic properties through mono- and di-phosphorylation of RLC, offering new quantitative connections between kinase activity, RLC phosphorylation and SF viscoelasticity.
The cytoskeleton is a highly dynamical protein network that plays a central role in numerous cellular physiological processes, and is traditionally divided into three components according to its chemical composition, i.e. actin, tubulin... more
The cytoskeleton is a highly dynamical protein network that plays a central role in numerous cellular physiological processes, and is traditionally divided into three components according to its chemical composition, i.e. actin, tubulin and intermediate filament cytoskeletons. Understanding the cytoskeleton dynamics is of prime importance to unveil mechanisms involved in cell adaptation to any stress type. Fluorescence imaging of cytoskeleton structures allows analyzing the impact of mechanical stimulation in the cytoskeleton, but it also imposes additional challenges in the image processing stage, such as the presence of imaging-related artifacts and heavy blurring introduced by (high-throughput) automated scans. However, although there exists a considerable number of image-based analytical tools to address the image processing and analysis, most of them are unfit to cope with the aforementioned challenges. Filamentous structures in images can be considered as a piecewise composition of quasi-straight segments (at least in some finer or coarser scale). Based on this observation, we propose a three-steps actin filaments extraction methodology: (i) first the input image is decomposed into a 'cartoon' part corresponding to the filament structures in the image, and a noise/texture part, (ii) on the 'cartoon' image, we apply a multi-scale line detector coupled with a (iii) quasi-straight filaments merging algorithm for fiber extraction. The proposed robust actin filaments image analysis framework allows extracting individual filaments in the presence of noise, artifacts and heavy blurring. Moreover , it provides numerous parameters such as filaments orientation, position and length, useful for further analysis. Cell image decomposition is relatively under-exploited in biological images processing, and our study shows the benefits it provides when addressing such tasks. Experimental validation was conducted using publicly available datasets, and in oste-oblasts grown in two different conditions: static (control) and fluid shear stress. The proposed methodology exhibited higher sensitivity values and similar accuracy compared to state-of-the-art methods.
- by Ronnie Willaert and +1
- •
- Image Analysis, Mechanobiology, Human bone cells
Craniosynostosis is a bone developmental disease where premature ossifcation of the cranial sutures occurs leading to fused sutures. While biomechanical forces have been implicated in craniosynostosis, evidence of the efect of... more
Craniosynostosis is a bone developmental disease where premature ossifcation of the cranial sutures occurs leading to fused sutures. While biomechanical forces have been implicated in craniosynostosis, evidence of the efect of microenvironmental stifness changes in the osteogenic commitment of cells from the sutures is lacking. Our aim was to identify the diferential genetic expression and osteogenic capability between cells from patent and fused sutures of children with craniosynostosis and whether these diferences are driven by changes in the stifness of the microenvironment. Cells from both sutures demonstrated enhanced mineralisation with increasing substrate stifness showing that stifness is a stimulus capable of triggering the accelerated osteogenic commitment of the cells from patent to fused stages. The diferences in the mechanoresponse of these cells were further investigated with a PCR array showing stifness-dependent upregulation of genes mediating growth and bone development (TSHZ2, IGF1), involved in the breakdown of extracellular matrix (MMP9), mediating the activation of infammation (IL1β) and controlling osteogenic diferentiation (WIF1, BMP6, NOX1) in cells from fused sutures. In summary, this study indicates that stifer substrates lead to greater osteogenic commitment and accelerated bone formation, suggesting that stifening of the extracellular environment may trigger the premature ossifcation of the sutures.
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... more
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.
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.
Soluble macromolecules present in the tumour microenvironment (TME) alter the physical characteristics of the extracellular fluid and can affect cancer cell behaviour. A fundamental step in cancer progression is the formation of a new... more
Soluble macromolecules present in the tumour microenvironment (TME) alter the physical characteristics of the extracellular fluid and can affect cancer cell behaviour. A fundamental step in cancer progression is the formation of a new vascular network which may originate from both pre-existing normal endothelium and cancer-derived cells. To study the role of extracellular macromolecules in the TME affecting endothelial cells we exposed normal and cancer-derived endothelial cells to inert polymer solutions with different physicochemical characteristics. The cancer cell line SK-HEP-1, but not normal human umbilical vein endothelial cells, responded to high-macromolecular-content solutions by elongating and aligning with other cells, an effect that was molecular weight-dependent. Moreover, we found that neither bulk viscosity, osmotic pressure, nor the fractional volume occupancy of polymers alone account for the induction of these effects. Furthermore, these morphological changes were accompanied by an increased extracellular matrix deposition. Conversely, cell-substrate adhesion was enhanced by polymers increasing the bulk viscosity of the culture medium independently of polymer molecular weight. These results show that the complex macromolecular composition of the extracellular fluid strongly influences cancer-derived endothelial cell behaviour, which may be crucial to understanding the role of the TME in cancer progression. Cancer cells require a constant supply of nutrients, oxygen, and the removal of waste products. Thus, the formation of a new vascular network, a process termed angiogenesis, is a pivotal step in tumour growth and progression 1,2. Moreover, angiogenesis also facilitates the penetration and circulation of cancer cells into the vascular network, which allows the spreading of cancer cells to adjacent and distant organs, a process known as metastasis. The formation of new vessels can originate from endothelial cells of pre-existing vessels, from newly recruited cells from the bone marrow, and can also derive from the angiogenic differentiation of tumour stem cells 3,4. To promote angiogenesis within tumours, cancer cells adapt the tumour microenvironment (TME) so as to acquire pro-angiogenic features. Cancer cells, and stromal cells influenced by the presence of cancer cells, are known to modify biophysical characteristics of the TME such as the stiffness and architecture of the extracellular matrix (ECM) by secreting ECM proteins and ECM-remodelling enzymes. These changes in the ECM have been reported to affect endothelial cell morphology and vascular permeability which can facilitate cancer cell intra-and extra-vasation 5,6. Endothelial cells also respond to haemodynamic forces including cyclic strain and shear stress. These biophysical signals have major effects on the structural organisation of the cytoskeleton, cell-cell junctions, and the adhesion of cells to their supporting ECM through mechano-sensitive structures such as focal adhesions, ion channels, and the junctional mechanosensory complex 7. The downstream signals of these structures converge in the regulation of the actin cytoskeleton through proteins involved in actin remodelling such as Rho GTPases 8-10. For instance, fluid shear stress induces cell alignment in the direction of the flow through a process mediated by the GTPases RhoA and Rac1 10 .
Mechanobiology seeks to understand and control mechanical and related biophysical communication between cells and their surroundings. While experimental efforts in this field have traditionally emphasized manipulation of the extracellular... more
Mechanobiology seeks to understand and control mechanical and related biophysical communication between cells and their surroundings. While experimental efforts in this field have traditionally emphasized manipulation of the extracellular force environment, a new suite of approaches has recently emerged in which cell phenotype and signaling are controlled by directly engineering the cell itself. One route is to control cell behavior by modulating gene expression using conditional promoters. Alternatively, protein activity can be actuated directly using synthetic protein ligands, chemically induced protein dimerization, optogenetic strategies, or functionalized magnetic nanoparticles. Proof-of-principle studies are already demonstrating the translational potential of these approaches, and future technological development will permit increasingly precise control over cell mechanobiology and improve our understanding of the underlying signaling events.
Epidermal growth factor receptor (EGFR) interacts with integrins during cell spreading and motility, but little is known about the role of EGFR in these mechanosensing processes. Here we show, using two diierent cell lines, that in... more
Epidermal growth factor receptor (EGFR) interacts with integrins during cell spreading and motility, but little is known about the role of EGFR in these mechanosensing processes. Here we show, using two diierent cell lines, that in serum-and EGF-free conditions, EGFR or HER2 activity increase spreading and rigidity-sensing contractions on rigid, but not soft, substrates. Contractions peak after 15–20 min, but diminish by tenfold after 4 h. Addition of EGF at that point increases spreading and contractions, but this can be blocked by myosin-II inhibition. We further show that EGFR and HER2 are activated through phosphorylation by Src family kinases (SFK). On soft surfaces, neither EGFR inhibition nor EGF stimulation have any eeect on cell motility. Thus, EGFR or HER2 can catalyse rigidity sensing after associating with nascent adhesions under rigidity-dependent tension downstream of SFK activity. This has broad implications for the roles of EGFR and HER2 in the absence of EGF both for normal and cancerous growth.
Background Leucine rich Aspartate motifs (LD motifs) are molecular recognition motifs on Paxillin that recognize LD-motif binding domains (LDBD) of a number of focal adhesion proteins in order to carry out downstream signaling and actin... more
Background
Leucine rich Aspartate motifs (LD motifs) are molecular recognition motifs on Paxillin that recognize LD-motif binding domains (LDBD) of a number of focal adhesion proteins in order to carry out downstream signaling and actin cytoskeleton remodeling. In this study, we identified structural features within LDBDs that influence their binding affinity with Paxillin LD motifs.
Methods
Various point mutants of focal adhesion targeting (FAT) domain of Focal Adhesion Kinase (FAK) were created by moving a key Lysine residue two and three helical turns in order to match the unique conformations as observed in LDBDs of two other focal adhesion proteins, Vinculin and CCM3.
Results
This led to identify a mutant of FAT domain of FAK, named as FAT(NV) (Asn992 of FAT domain was replaced by Val), with remarkable high affinity for LD1 (Kd = 1.5 μM vs no-binding with wild type) and LD2 peptides (Kd = 7.2 μM vs 63 μM with wild type). Consistently, the focal adhesions of MCF7 cells expressing FAK(NV) were highly stable (turnover rate = 1.25 × 10−5 μm2/s) as compared to wild type FAK transfected cells (turnover rate = 1.5 × 10−3 μm2/s).
Conclusions
We observed that the relative disposition of key LD binding amino-acids at LDBD surface, hydrophobic burial of long Leucine side chains of LD-motifs and complementarity of charged surfaces are the key factors determining the binding affinities of LD motifs with LDBDs.
General significance
Our study will help in protein engineering of FAT domain of FAK by modulating FAK-LD motif interactions which have implications in cellular focal adhesions and cell migration.
Leucine rich Aspartate motifs (LD motifs) are molecular recognition motifs on Paxillin that recognize LD-motif binding domains (LDBD) of a number of focal adhesion proteins in order to carry out downstream signaling and actin cytoskeleton remodeling. In this study, we identified structural features within LDBDs that influence their binding affinity with Paxillin LD motifs.
Methods
Various point mutants of focal adhesion targeting (FAT) domain of Focal Adhesion Kinase (FAK) were created by moving a key Lysine residue two and three helical turns in order to match the unique conformations as observed in LDBDs of two other focal adhesion proteins, Vinculin and CCM3.
Results
This led to identify a mutant of FAT domain of FAK, named as FAT(NV) (Asn992 of FAT domain was replaced by Val), with remarkable high affinity for LD1 (Kd = 1.5 μM vs no-binding with wild type) and LD2 peptides (Kd = 7.2 μM vs 63 μM with wild type). Consistently, the focal adhesions of MCF7 cells expressing FAK(NV) were highly stable (turnover rate = 1.25 × 10−5 μm2/s) as compared to wild type FAK transfected cells (turnover rate = 1.5 × 10−3 μm2/s).
Conclusions
We observed that the relative disposition of key LD binding amino-acids at LDBD surface, hydrophobic burial of long Leucine side chains of LD-motifs and complementarity of charged surfaces are the key factors determining the binding affinities of LD motifs with LDBDs.
General significance
Our study will help in protein engineering of FAT domain of FAK by modulating FAK-LD motif interactions which have implications in cellular focal adhesions and cell migration.
- by Somsubhro Mukherjee and +1
- •
- Bioengineering, Biotechnology, Cancer, Mechanobiology
Human embryonic stem cells subjected to a one-time uniaxial stretch for as short as 30-min on a flexible substrate coated with Matrigel experienced rapid and irreversible nuclear-to-cytoplasmic translocation of NANOG and OCT4, but not... more
Human embryonic stem cells subjected to a one-time uniaxial stretch for as short as 30-min on a flexible substrate coated with Matrigel experienced rapid and irreversible nuclear-to-cytoplasmic translocation of NANOG and OCT4, but not Sox2. Translocations were directed by intracellular transmission of biophysical signals from cell surface integrins to nuclear CRM1 and were independent of exogenous soluble factors. On E-CADHERIN-coated substrates, presumably with minimal integrin engagement, mechanical strain-induced rapid nuclear-to-cytoplasmic translocation of the three transcription factors. These findings might provide fundamental insights into early developmental processes and may facilitate mechanotransduction-mediated bioengineering approaches to influencing stem cell fate determination.
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... more
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.
In order to identify the mechanisms by which skeletal maturity alters the mechanosensitivity of mes-enchymal stromal cells (MSCs) and, the implications for osteogenesis and angiogenesis during bone formation , we compared the response of... more
In order to identify the mechanisms by which skeletal maturity alters the mechanosensitivity of mes-enchymal stromal cells (MSCs) and, the implications for osteogenesis and angiogenesis during bone formation , we compared the response of MSCs derived from children and skeletally-mature healthy adults cultured on soft and stiff collagen-coated polyacrylamide substrates. MSCs from children were more mechanosensitive, showing enhanced angiogenesis and osteogenesis on stiff substrates as indicated by increased endothelial tubule formation, PGF production, nuclear-translocation of YAP, ALP activity and mineralisation. To examine these mechanisms in more detail, a customised PCR array identified an age-dependent, stiffness-induced upregulation of NOX1, VEGFR1, VEGFR2, WIF1 and, of particular interest , JNK3 in cells from children compared to adults. When JNK3 activity was inhibited, a reduction in stiffness-induced driven osteogenesis was observed – suggesting that JNK3 might serve as a novel target for recapitulating the enhanced regenerative potential of children in adults suffering from bone degener-ation. Statement of Significance We investigated the age-associated changes in the capacity of MSCs for bone regeneration involving the mechanosensitive signalling pathways, which reduce the ability of adult cells to respond to biophysical cues in comparison to cells from children, who are still undergoing bone development. Our results offer new insights into the mechanobiology of MSCs and sheds new light on age-altered mechanosensitivity and, on why children have such an immense capacity to regenerate their skeletal system. We have identified the mechanisms by which skeletal maturity alters the mechanosensitivity of mesenchymal stromal cells and an age-dependent, stiffness-induced upregulation of a number of prominent genes including, most notably, JNK3 in children cells, thus suggesting its potential to promote enhanced bone repair.
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... more
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.
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.
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... more
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.
Fibrin has been proposed as cell scaffold for numerous tissue engineering applications. While most of the studies have focused on fibrinogen and thrombin, other components of fibrin can also affect its properties. The present study aimed... more
Fibrin has been proposed as cell scaffold for numerous tissue engineering applications. While most of the studies have focused on fibrinogen and thrombin, other components of fibrin can also affect its properties. The present study aimed to evaluate the effects of buffer solution composition on fibrin biophysical properties. Fibrin scaffolds were synthesized with different calcium, chloride, and factor XIII (FXIII) final concentrations. Light transmission was determined as a relative, semi-quantitative estimator of fiber structure differences, and two compositions, resulting in translucent and opaque gels, were tested for mechanical and biological properties. Gels were seeded with mouse mesenchymal cells, C3H10T1/2, or bovine bone marrow-derived mesenchymal stromal cells and cultured up to 10 or 24 days, before cell number, morphology and distribution were evaluated. Calcium increased gel opacity (i.e., fiber thickness), while chloride and FXIII decreased it. Opaque gels displayed a fluid-like viscous behavior while translucent gels showed improved elastic properties. Both compositions supported survival of both cell types with opaque gels leading to better proliferation, but significant scaffold shrinkage after 17 days of culture. These results demonstrated that calcium, chloride, and FXIII modulate the biophysical properties of fibrin, and can be used to adjust mechanical and biological properties for tissue engineering applications.
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... more
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 signalling molecules. Together these studies show that the mechanical microenvironment may be an important regulator during early differentiation events, including gastrulation. This insight furthers understanding of normal and pathological events during development, as well as facilitates strategies for scale up production of stem cells for clinical therapies.
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