Musculoskeletal mechanobiology: Interpretation by external force and engineered substratum

Mechanobiology aims to discover how the mechanical environment affects the biological activity of cells and how cells’ ability to sense these mechanical cues is converted into elicited cellular responses. Musculoskeletal mechanobiology is of particular interest given the high mechanical loads that musculoskeletal tissues experience on a daily basis. How do cells within these mechanically active tissues interpret external loads imposed on their extracellular environment, and, how are cell–substrate interactions converted into biochemical signals? This review outlines many of the main mechanotransduction mechanisms known to date, and describes recent literature examining effects of both external forces and cell–substrate interactions on musculoskeletal cells. Whether via application of external forces and/or cell–substrate interactions, our understanding and regulation of musculoskeletal mechanobiology can benefit by expanding upon traditional models, and shedding new light through novel investigative approaches. Current and future work in this field is focused on identifying specific forces, stresses, and strains at the cellular and tissue level through both experimental and computational approaches, and analyzing the role of specific proteins through fluorescence-based investigations and knockdown models.     

Cellular mechanobiology and cancer metastasis

The primary cause of cancer treatment failure is invasion and metastasis, and invading tumor cells utilize many of the motility patterns that have been documented for normal morphogenesis. Recently, the role of mechanical forces in guiding various tissue and cell movements in embryonic development has been systematically analyzed with new experimental and computational methods. The tissue and cellular mechanobiology approach also holds promise for increasing the understanding of tumor invasion. In fact, the mechanical stiffness of tumors has correlated with invasiveness, and manipulation of extracellular matrix (ECM) stiffness in vitro has suppressed the cancer phenotype. Several important signaling molecules reside on the cytoskeleton, which is affected by external stress imparted by the ECM, and deformation of the nucleus can trigger the activation of certain genes. All these observations suggest that a synthesis of the biology of cancer cell invasion and cellular mechanobiology may offer new targets for the treatment of malignant disease. Accordingly, sensitive and relevant in vivo models and methods to study cancer mechanobiology are needed.     

The emergence of ECM mechanics and cytoskeletal tension as important regulators of cell function

The ability to harvest and maintain viable cells from mammalian tissues represented a critical advance in biomedical research, enabling individual cells to be cultured and studied in molecular detail. However, in these traditional cultures, cells are grown on rigid glass or polystyrene substrates, the mechanical properties of which often do not match those of the in vivo tissue from which the cells were originally derived. This mechanical mismatch likely contributes to abrupt changes in cellular phenotype. In fact, it has been proposed that mechanical changes in the cellular microenvironment may alone be responsible for driving specific cellular behaviors. Recent multidisciplinary efforts from basic scientists and engineers have begun to address this hypothesis more explicitly by probing the effects of ECM mechanics on cell and tissue function. Understanding the consequences of such mechanical changes is physiologically relevant in the context of a number of tissues in which altered mechanics may either correlate with or play an important role in the onset of pathology. Examples include changes in the compliance of blood vessels associated with atherosclerosis and intimal hyperplasia, as well as changes in the mechanical properties of developing tumors. Compelling evidence from 2-D in vitro model systems has shown that substrate mechanical properties induce changes in cell shape, migration, proliferation, and differentiation, but it remains to be seen whether or not these same effects translate to 3-D systems or in vivo. Furthermore, the molecular “mechanotransduction” mechanisms by which cells respond to changes in ECM mechanics remain unclear. Here, we provide some historical context for this emerging area of research, and discuss recent evidence that regulation of cytoskeletal tension by changes in ECM mechanics (either directly or indirectly) may provide a critical switch that controls cell function.     

Techniques for assessing 3-D cell–matrix mechanical interactions in vitro and in vivo

Cellular interactions with extracellular matrices (ECM) through the application of mechanical forces mediate numerous biological processes including developmental morphogenesis, wound healing and cancer metastasis. They also play a key role in the cellular repopulation and/or remodeling of engineered tissues and organs. While 2-D studies can provide important insights into many aspects of cellular mechanobiology, cells reside within 3-D ECMs in vivo, and matrix structure and dimensionality have been shown to impact cell morphology, protein organization and mechanical behavior. Global measurements of cell-induced compaction of 3-D collagen matrices can provide important insights into the regulation of overall cell contractility by various cytokines and signaling pathways. However, to understand how the mechanics of cell spreading, migration, contraction and matrix remodeling are regulated at the molecular level, these processes must also be studied in individual cells. Here we review the evolution and application of techniques for imaging and assessing local cell–matrix mechanical interactions in 3-D culture models, tissue explants and living animals.     

Integrins in development and cancer

The correct control of cell fate decisions is critical for metazoan development and tissue homeostasis. It is established that the integrin family of cell surface receptors regulate cell fate by mediating cell–cell and cell–extracellular matrix (ECM) interactions. However, our understanding of how the different family members control discrete aspects of cell biology, and how this varies between tissues and is temporally regulated, is still in its infancy. An emerging area of investigation aims to understand how integrins translate changes in tension in the surrounding microenvironment into biological responses. This is particularly pertinent due to changes in the mechanical properties of the ECM having been linked to diseases, such as cancer. In this review, we provide an overview of the roles integrins play in important developmental processes, such as proliferation, polarity, apoptosis, differentiation and maintenance of “stemness”. We also discuss recent advances in integrin mechanobiology and highlight the involvement of integrins and aberrant ECM in cancer.     

Concentration independent modulation of local micromechanics in a fibrin gel

Methods for tuning extracellular matrix (ECM) mechanics in 3D cell culture that rely on increasing the concentration of either protein or cross-linking molecules fail to control important parameters such as pore size, ligand density, and molecular diffusivity. Alternatively, ECM stiffness can be modulated independently from protein concentration by mechanically loading the ECM. We have developed a novel device for generating stiffness gradients in naturally derived ECMs, where stiffness is tuned by inducing strain, while local mechanical properties are directly determined by laser tweezers based active microrheology (AMR). Hydrogel substrates polymerized within 35 mm diameter Petri dishes are strained non-uniformly by the precise rotation of an embedded cylindrical post, and exhibit a position-dependent stiffness with little to no modulation of local mesh geometry. Here we present the device in the context of fibrin hydrogels. First AMR is used to directly measure local micromechanics in unstrained hydrogels of increasing fibrin concentration. Changes in stiffness are then mapped within our device, where fibrin concentration is held constant. Fluorescence confocal imaging and orbital particle tracking are used to quantify structural changes in fibrin on the micro and nano levels respectively. The micromechanical strain stiffening measured by microrheology is not accompanied by ECM microstructural changes under our applied loads, as measured by confocal microscopy. However, super-resolution orbital tracking reveals nanostructural straightening, lengthening, and reduced movement of fibrin fibers. Furthermore, we show that aortic smooth muscle cells cultured within our device are morphologically sensitive to the induced mechanical gradient. Our results demonstrate a powerful cell culture tool that can be used in the study of mechanical effects on cellular physiology in naturally derived 3D ECM tissues.     

Discoidin domain receptors: Microenvironment sensors that promote cellular migration and invasion

ABSTRACT

Extracellular matrix (ECM) provides cells scaffolding for cell migration and microenvironment for various cellular functions. Collagens are major ECM components in tissue and discoidin domain receptors (DDRs) are receptor tyrosine kinases (RTK) that recognise fibrillar collagens. Unlike other RTK, their ligands are solid ECM the that are abundantly present in the pericellular environment in various tissue, and thus its activation and regulations are unique amongst RTK family. It is emerging that DDRs may be the sensors that monitor and detects changes in ECM microenvironment and determines the cellular fates upon tissue injuries. In this mini-review, recent findings on the role of DDRs as microenvironment sensor and their roles in cell migration and invasion are discussed.     

Modulation of epithelial morphogenesis and cell fate by cell-to-cell signals and regulated cell adhesion

Cell-cell and cell-extracellular matrix (ECM) interactions control many developmental decisions of epithelial cell fate and morphogenesis. Protein tyrosine kinases are one class of regulatory molecules that have been implicated in the modulation of these processes. Several protein tyrosine kinases co-localize with cell-cell (cadherin) and cell-ECM (integrin) adhesion molecules at specific adhesion domains of epithelial cells. Protein tyrosine kinases may regulate epithelial development by modulating cell-cell and cell-ECM interactions and by relaying signals initiated by these interactions to other cellular components that determine cell structure and function.     

An SH3 domain-containing GTPase-activating protein for Rho and Cdc42 associates with focal adhesion kinase.

The integrin family of cell surface receptors mediates cell adhesion to components of the extracellular matrix (ECM). Integrin engagement with the ECM initiates signaling cascades that regulate the organization of the actin-cytoskeleton and changes in gene expression. The Rho subfamily of Ras-related low-molecular-weight GTP-binding proteins and several protein tyrosine kinases have been implicated in mediating various aspects of integrin-dependent alterations in cell homeostasis. Focal adhesion kinase (FAK or pp125FAK) is one of the tyrosine kinases predicted to be a critical component of integrin signaling. To elucidate the mechanisms by which FAK participates in integrin-mediated signaling, we have used expression cloning to identify cDNAs that encode potential FAK-binding proteins. We report here the identification of a cDNA that encodes a new member of the GTPase-activating protein (GAP) family of GTPase regulators. This GAP, termed Graf (for GTPase regulator associated with FAK), binds to the C-terminal domain of FAK in an SH3 domain-dependent manner and preferentially stimulates the GTPase activity of the GTP-binding proteins RhoA and Cdc42. Subcellular localization studies using Graf-transfected chicken embryo cells indicates that Graf colocalizes with actin stress fibers, cortical actin structures, and focal adhesions. Graf mRNA is expressed in a variety of avian tissues and is particularly abundant in embryonic brain and liver. Graf represents the first example of a regulator of the Rho family of small GTP-binding proteins that exhibits binding to a protein tyrosine kinase. We suggest that Graf may function to mediate cross talk between the tyrosine kinases such as FAK and the Rho family GTPase that control steps in integrin-initiated signaling events.     

A bit of give and take: the relationship between the extracellular matrix and the developing chondrocyte

The extracellular matrix (ECM), once thought to be a static structural component of tissues, is now known to play a complex and dynamic role in a variety of cellular functions in a number of diverse tissues. A significant body of literature attests to the ability of the ECM to communicate both spatial and temporal information to adherent cells, thereby directing cell behavior via interactions between the ECM and cell-surface receptors. Moreover, volumes of experimental data show that a great deal of communication travels in the opposite direction, from the cell to the ECM, allowing for regulation of the cues transmitted by the ECM. As such, the ECM, with respect to its components and their organization, is not a fixed reflection of the state the local microenvironment in which a cell finds itself at a particular time, but rather is able to respond to and effect changes in its local microenvironment. As an example of the developmental consequences of ECM interactions, this review gives an overview of the 'give and take' relationship between the ECM and the cells of the developing skeletal elements, in particular, the chondrocyte.     

Characterizing viscoelastic properties of breast cancer tissue in a mouse model using indentation

Breast cancer is one of the leading cancer forms affecting females worldwide. Characterizing the mechanical properties of breast cancer tissue is important for diagnosis and uncovering the mechanobiology mechanism. Although most of the studies were based on human cancer tissue, an animal model is still describable for preclinical analysis. Using a custom-build indentation device, we measured the viscoelastic properties of breast cancer tissue from 4T1 and SKBR3 cell lines. A total of 7 samples were tested for each cancer tissue using a mouse model. We observed that a viscoelastic model with 2-term Prony series could best describe the ramp and stress relaxation of the tissue. For long-term responses, the SKBR3 tissues were stiffer in the strain levels of 4–10%, while no significant differences were found for the instantaneous elastic modulus. We also found tissues from both cell lines appeared to be strain-independent for the instantaneous elastic modulus and for the long-term elastic modulus in the strain level of 4–10%. In addition, by inspecting the cellular morphological structure of the two tissues, we found that SKBR3 tissues had a larger volume ratio of nuclei and a smaller volume ratio of extracellular matrix (ECM). Compared with prior cellular mechanics studies, our results indicated that ECM could contribute to the stiffening the tissue-level behavior. The viscoelastic characterization of the breast cancer tissue contributed to the scarce animal model data and provided support for the linear viscoelastic model used for in vivo elastography studies. Results also supplied helpful information for modeling of the breast cancer tissue in the tissue and cellular levels.     

Integrin–ECM interactions and membrane-associated Catalase cooperate to promote resilience of the Drosophila intestinal epithelium

Balancing cellular demise and survival constitutes a key feature of resilience mechanisms that underlie the control of epithelial tissue damage. These resilience mechanisms often limit the burden of adaptive cellular stress responses to internal or external threats. We recently identified Diedel, a secreted protein/cytokine, as a potent antagonist of apoptosis-induced regulated cell death in the Drosophila intestinal midgut epithelium during aging. Here, we show that Diedel is a ligand for RGD-binding Integrins and is thus required for maintaining midgut epithelial cell attachment to the extracellular matrix (ECM)-derived basement membrane. Exploiting this function of Diedel, we uncovered a resilience mechanism of epithelial tissues, mediated by Integrin–ECM interactions, which shapes cell death spreading through the regulation of cell detachment and thus cell survival. Moreover, we found that resilient epithelial cells, enriched for Diedel–Integrin–ECM interactions, are characterized by membrane association of Catalase, thus preserving extracellular reactive oxygen species (ROS) balance to maintain epithelial integrity. Intracellular Catalase can relocalize to the extracellular membrane to limit cell death spreading and repair Integrin–ECM interactions induced by the amplification of extracellular ROS, which is a critical adaptive stress response. Membrane-associated Catalase, synergized with Integrin–ECM interactions, likely constitutes a resilience mechanism that helps balance cellular demise and survival within epithelial tissues.

A key feature of the resilience mechanisms that underlie the control of epithelial tissue damage is the balance between cell death and survival. This study shows that the anti-oxidant enzyme catalase can relocate to membranes in order to promote the resilience of the Drosophila midgut epithelium, synergizing with integrin-ECM interactions to prevent the spread of cell death.     

Receptor tyrosine kinases.

A major process through which environmental information is transmitted into cells is via activation of protein tyrosine kinases. Receptor tyrosine kinases contain extracellular ligand recognition, single membrane spanning, and cytoplasmic protein tyrosine kinase domains. The cytoplasmic kinase core is flanked by regulatory segments, which in some family members are also inserted into the core kinase domain. Ligand binding initiates receptor signaling from the cell surface. Activated receptors autophosphorylate to remove alternate substrate/inhibitory constraints and to provide loci for assembly of proteins that contain SRC homology regions. Information is transmitted and diffused by tyrosine phosphorylation of the assembled proteins and of cellular substrates that include protein kinases with specificity for serine/threonine residues. Signaling, which is strictly ligand-dependent, is attenuated by down-regulation of receptors and by feed-back inhibitory loops that involve receptor phosphorylation by cellular kinases. The tyrosine kinase receptors are essential for normal growth, development, and reparative processes. Mutations that remove normal regulatory constraints on the approximately 290 amino acid kinase core of these large proteins result in constitutive function and cell transformation.     

Cell mechanics studied by a reconstituted model tissue

Tissue models reconstituted from cells and extracellular matrix (ECM) simulate natural tissues. Cytoskeletal and matrix proteins govern the force exerted by a tissue and its stiffness. Cells regulate cytoskeletal structure and remodel ECM to produce mechanical changes during tissue development and wound healing. Characterization and control of mechanical properties of reconstituted tissues are essential for tissue engineering applications. We have quantitatively characterized mechanical properties of connective tissue models, fibroblast-populated matrices (FPMs), via uniaxial stretch measurements. FPMs resemble natural tissues in their exponential dependence of stress on strain and linear dependence of stiffness on force at a given strain. Activating cellular contractile forces by calf serum and disrupting F-actin by cytochalasin D yield "active" and "passive" components, which respectively emphasize cellular and matrix mechanical contributions. The strain-dependent stress and elastic modulus of the active component were independent of cell density above a threshold density. The same quantities for the passive component increased with cell number due to compression and reorganization of the matrix by the cells.     

细胞外基质与基质金属蛋白酶

细胞外基质（ECM）是存在于细胞之间的动态网状结构,由胶原、蛋白聚糖及糖蛋白等大分子物质组成.这些大分子物质可与细胞表面上的特异性受体结合,通过受体与细胞骨架结构直接发生联系或触发细胞内的一系列信号传导而引起不同的基因表达,从而导致细胞的生长和分化.作为降解ECM成分最重要的酶-基质金属蛋白酶（MMPs）及其组织抑制因子(TIMPs)在这一过程中起着非常重要的作用.MMPs是一类依赖金属离子锌并以ECM成分为水解底物的蛋白水解酶.其在转录水平的表达受到生长因子、细胞因子及激素等因素的严格调控,在蛋白质水平其活性也受到其激活剂和抑制剂的调节. MMPs通过对ECM成分的水解来影响其降解与重组的动态平衡而参与多种细胞的生理和病理过程.     

Eukaryotic initiation factor 6 regulates mechanical responses in endothelial cells

The repertoire of extratranslational functions of components of the protein synthesis apparatus is expanding to include control of key cell signaling networks. However, very little is known about noncanonical functions of members of the protein synthesis machinery in regulating cellular mechanics. We demonstrate that the eukaryotic initiation factor 6 (eIF6) modulates cellular mechanobiology. eIF6-depleted endothelial cells, under basal conditions, exhibit unchanged nascent protein synthesis, polysome profiles, and cytoskeleton protein expression, with minimal effects on ribosomal biogenesis. In contrast, using traction force and atomic force microscopy, we show that loss of eIF6 leads to reduced stiffness and force generation accompanied by cytoskeletal and focal adhesion defects. Mechanistically, we show that eIF6 is required for the correct spatial mechanoactivation of ERK1/2 via stabilization of an eIF6–RACK1–ERK1/2–FAK mechanocomplex, which is necessary for force-induced remodeling. These results reveal an extratranslational function for eIF6 and a novel paradigm for how mechanotransduction, the cellular cytoskeleton, and protein translation constituents are linked.     

Mechanoregulation of gene expression in fibroblasts

Mechanical loads placed on connective tissues alter gene expression in fibroblasts through mechanotransduction mechanisms by which cells convert mechanical signals into cellular biological events, such as gene expression of extracellular matrix components (e.g., collagen). This mechanical regulation of ECM gene expression affords maintenance of connective tissue homeostasis. However, mechanical loads can also interfere with homeostatic cellular gene expression and consequently cause the pathogenesis of connective tissue diseases such as tendinopathy and osteoarthritis. Therefore, the regulation of gene expression by mechanical loads is closely related to connective tissue physiology and pathology. This article reviews the effects of various mechanical loading conditions on gene regulation in fibroblasts and discusses several mechanotransduction mechanisms. Future research directions in mechanoregulation of gene expression are also suggested.     

Extracellular matrix controls tubulin monomer levels in hepatocytes by regulating protein turnover.

Cells have evolved an autoregulatory mechanism to dampen variations in the concentration of tubulin monomer that is available to polymerize into microtubules (MTs), a process that is known as tubulin autoregulation. However, thermodynamic analysis of MT polymerization predicts that the concentration of free tubulin monomer must vary if MTs are to remain stable under different mechanical loads that result from changes in cell adhesion to the extracellular matrix (ECM). To determine how these seemingly contradictory regulatory mechanisms coexist in cells, we measured changes in the masses of tubulin monomer and polymer that resulted from altering cell-ECM contacts. Primary rat hepatocytes were cultured in chemically defined medium on bacteriological petri dishes that were precoated with different densities of laminin (LM). Increasing the LM density from low to high (1-1000 ng/cm2), promoted cell spreading (average projected cell area increased from 1200 to 6000 microns2) and resulted in formation of a greatly extended MT network. Nevertheless, the steady-state mass of tubulin polymer was similar at 48 h, regardless of cell shape or ECM density. In contrast, round hepatocytes on low LM contained a threefold higher mass of tubulin monomer when compared with spread cells on high LM. Furthermore, similar results were obtained whether LM, fibronectin, or type I collagen were used for cell attachment. Tubulin autoregulation appeared to function normally in these cells because tubulin mRNA levels and protein synthetic rates were greatly depressed in round cells that contained the highest level of free tubulin monomer. However, the rate of tubulin protein degradation slowed, causing the tubulin half-life to increase from approximately 24 to 55 h as the LM density was lowered from high to low and cell rounding was promoted. These results indicate that the set-point for the tubulin monomer mass in hepatocytes can be regulated by altering the density of ECM contacts and changing cell shape. This finding is consistent with a mechanism of MT regulation in which the ECM stabilizes MTs by both accepting transfer of mechanical loads and altering tubulin degradation in cells that continue to autoregulate tubulin synthesis.