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Mechanotransduction through substrates engineering and microfluidic devices
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ScienceDirect Mechanotransduction through substrates engineering and microfluidic devices Stefano Giulitti1,2, Alessandro Zambon1,2, Federica Michielin1,2 and Nicola Elvassore1,2 Living cells are able to sense three-dimensional environmental properties, to integrate biophysical and biochemical stimulations and to adapt to dynamic changes of their physical surroundings for maintaining appropriate biological functions. Mechanotransduction — the mechanism by which a mechanical stimulation is transduced into a chemical signal — typically involves force-induced conformational changes in proteins or altered affinities to their binding partners but these interactions remain largely unexplored. New development in microengineering offers unique capabilities for in vitro controlling and perturbing both the biochemical and biomechanical cellular microenvironment. Here, we focus on studies and discoveries that — through innovative microtechnology — dissected and reconstructed biochemical and biophysical mechanisms underlying mechano-associated biological processes. These microengineered platforms provide endless possibilities for new insights into the role of mechanotransduction in physiology and pathophysiology of human tissues and organs, including tumorigenesis and metastasis. Addresses 1 Dept. of Industrial Engineering, University of Padova, via Marzolo 9, Padova 35131, Italy 2 Venetian Institute of Molecular Medicine, via Orus 2, Padova 35129, Italy Corresponding author: Elvassore, Nicola ([email protected])
Current Opinion in Chemical Engineering 2016, 11:67–76 This review comes from a themed issue on Biological engineering
Living cells are immersed in a three-dimensional structure based on biopolymers forming the extracellular matrix (ECM). A distinctive cell–ECM combination confers unique physiological and biological functions to human organs. Cells are able to sense three-dimensional environmental properties of the extracellular matrix and neighboring cells, to integrate biophysical and biochemical stimulations and, accordingly, to adapt to the dynamic changes of their physical surroundings for maintaining appropriate biological functions [1]. The cellular processes, which translate biophysical cues and biomechanical stimulations in biochemical signals are called mechanotransduction [2]. At the cellular level, mechanical signals influence cell motility, proliferation, apoptosis, differentiation, and maturation patterns. They also play a critical role during tissue and organ morphogenesis in the embryo state [3], and in the maintenance of functionalities in the adult organism. Impairments in these cellular processes contribute to the underlying causes of many diseases and pathological conditions [4], including tumorigenesis and metastasis. Since all cells can have mechano-sensitive elements able to perceive mechanical changes in different cell districts (Figure 2), mechanotransduction is present in all tissues and organs. For instance, mechanotransduction signaling has a crucial role in the maintenance of many mechanically stressed tissues, such as skeletal and cardiac muscle, bone, cartilage and blood vessels [5].
Edited by Sharon Gerecht and Kostas Konstantopoulos
http://dx.doi.org/10.1016/j.coche.2016.01.010 2211-3398/# 2016 Elsevier Ltd. All rights reserved.
Introduction Tissues and organs have a variety of functions, which depend on particular cell phenotypes and on their topological and hierarchical organizations within the human body. Besides biochemical signals, tissues and organs normally experience dynamic mechanical stresses dependent on their activities (Figure 1). The impossibility of some organs to exert or counteract specific forces could lead to the onset of a disease. www.sciencedirect.com
Conventionally, biological research investigates the cellular response to extrinsic soluble biochemical factors and biochemical properties of ECM. However, the rational understanding of the biochemical mechanisms by which the cells sense, transduce, and respond to mechanical stimulations and shape deformations is largely unexplored. Only recent investigations provide unambiguous knowledge about mechano-associated mechanisms, despite the importance of these in fundamental aspects of human physiology and pathophysiology and in several biomedical applications (such as cell-based therapy or regenerative medicine). The mechanisms by which the mechanical stimulation is transduced into a chemical signal was not confirmed until the presence of mechanosensing ion channels was reported, explaining the cellular depolarization after stretching of nerve cells [6]. Many additional mechanotransduction pathways have been identified; typically, the Current Opinion in Chemical Engineering 2016, 11:67–76
68 Biological engineering
Figure 1
(h) Lungs (chronic obstructive pulmunary disease and inflammation)
(a) Vascular endothelium (calcification, aterosclerosys)
(g) Skin (keloid)
(b) Heart (hypertophy, heart failure)
(f) Bladder, urethra (urodynamic disorder)
(c) Intestine (chronic intestinal pseudo-obstruction)
(e) Bone, cartilage, tendons (loss of bone mass)
(d) Skeletal muscle (muscular dystrophies)
Current Opinion in Chemical Engineering
Mechanical stimuli in human tissues and organs, and mechano-associated diseases. Cells of different tissues experience mechanical stimuli and deformations in the human body. Example of common forces, stresses, loads and deformations are represented clockwise reporting also possible mechano-associated diseases (arrows depict force/pressure directions; normal stress (s) or shear stress (t) are not reported in the text for organs subjected to extremely variable forces). (a) Vascular endothelium in blood vessels is continuously subjected to shear and hydrostatic stress exerted by blood flow (t = 0.07–13 Pa); (b) during heart beating, cardiomyocytes are subjected to active contraction during systole and relaxation during diastole; (c) peristalsis involves radial symmetrical contraction and relaxation of muscles during digestion (s = 2.6–6.6 kPa); (d) skeletal muscles generate uniaxial contraction; (e) compressive and tensile loads are experienced by cartilage, bones and tendons respectively; (f) in the urinary apparatus, bladder undergoes high dilatation providing compliance while maintaining constant hydrostatic pressure (s < 6 kPa) whereas urethra experiences shear stresses and osmotic forces; (g) skin is a multilayered organ which are exposed to mechanical pressure and osmotic stresses. Hair skin has also sensory units that respond to mechanical pressure or distortion; (h) epithelial/endothelial bilayer in lungs undergoes cyclic deformation (s = 0.3–9.8 kPa) preserving the body from inflammation and infections.
employed mechanisms involve force-induced conformational changes in a protein that result in opening of membrane channels [7,8] or altered affinities to binding partners, thereby activating signaling pathways [9,10]. The downstream targets of these signaling pathways could include ECM reorganization, changes in protein affinity and binding partners, cytoskeleton remodeling, chromatin reorganization and specific gene expression [1]. The experimental investigations of mechano-associated cellular processes are quite limited using conventional biological approaches. New tools and technologies are required to further investigate, understand, dissect and reconstruct the complex machinery that the cells use to Current Opinion in Chemical Engineering 2016, 11:67–76
respond and adapt to specific mechanical stimulations. New development in microengineering technology offers unique opportunities for in vitro controlling and perturbing the biochemical and biomechanical properties of three-dimensional environment surrounding cells. Microfabrication techniques are increasingly used for controlling the three-dimensional architecture of in vitro cellular microenvironment and for enabling accurate, quantitative measurements of cellular responses in high-throughput experiments [11]. These microengineered platforms provide possibilities for new insights into interactions between cells and their dynamically changing microenvironment, which underlies www.sciencedirect.com
Design of microtechnology to study mechanotransduction Giulitti et al. 69
the physiology and pathophysiology of human tissues and organs. Recent excellent reviews on mechanobiology and associated technology provided a general perspective of this emerging and rapidly evolving interdisciplinary field [12,13]. Our review focuses on the biological applications of micro-engineering tools specifically designed for studying, dissecting and reconstructing biochemical and biophysical mechanisms, which sustain mechano-associated biological processes. Here we review mechanotransduction studies, evidencing two main approaches in the field: mechanotransduction modulation through substrate engineering and active dynamic modulation of force-deformation using microengineering advances, including microfluidics. For over a decade, substrate engineering has resulted in soft hydrogels and micro-patterns that enabled considerable new insights in biological processes. In the first part we will overview recent biological discoveries in mechanotransduction field made possible through substrate engineering. In the second part, we will highlight emerging microengineering technologies that have been tailored on specific mechanotransduction problems and will found new bases on how to study mechanorelated biological activities.
Tuning mechanotransduction through substrates engineering The ability of cells to sense the biochemical and biophysical properties of the ECM affects the regulation of cellular activities in development, tissue homeostasis, regeneration, as well as malignant transformation (Figure 2a). Pioneering works show that mesenchymal stem cells are committed to osteogenic fate when cultured on stiff substrates, whereas they express a neurogenic phenotype when adhere on softer substrate [14]. Several other works show that the biophysical properties of the substrate also regulate proliferation, cell motility and cell spreading. Only recently, some works reported experimental evidence that the cells are able to respond to specific substrate properties by translating mechanical cues into biochemical signals that activates specific signaling pathways, including transcription factors, and regulates gene expression [15–17]. However, the molecular mechanisms responsible for sensing microenvironment mechanical changes remain poorly understood and substrates micro-engineering offers an endless potential of probing and investigating molecular mechanisms underlying the mechanobiology. Figure 2b shows the main technological advances, such as cell-adhesive patterned surfaces, substrates with tunable stiffness or micro-pillared surfaces, that have been successfully used for controlling the architecture and adhesiveness of a cell and facilitating the study at three levels: firstly, cell–substrate interactions; secondly, cell–cell interactions; and finally, nuclear mechanotransduction. www.sciencedirect.com
Owing to the large number of works developed, here we report more recent intriguing aspects of cell mechanobiology with a particular focus on probing mechanotransduction mechanisms through the use of microengineered substrates and surface chemistry. Cell–substrate interactions
Micropatterning techniques have been used to spatially control cell–substrate interactions, to manipulate the biochemical adhesiveness of substrates and to patterning specific ECM proteins through microcontact printing [18] or microfluidic patterning [19]. On the other hand, relatively few approaches in substrates engineering have been developed to specifically investigate the effects of the mechanical forces of cell adhesion on the regulation of cell signaling and function (Figure 3). The main technique to obtain substrates with tunable stiffness consists in using one or more polymerizable chemical species with a variable molar ratio compared to a crosslinking agent. Specifically, the substrate elastic modulus can be varied by orders of magnitude, with a typical physiological range from 1 kPa to 100 kPa. One of the most used material, polyacrylamide (PA), forms an inert transparent and biocompatible polymer of acrylamide crosslinked with metilen-bis-acrylamide in a water-rich environment [14]. Both micropatterning and substrate stiffness regulation and their combinations were successfully used in studying cell proliferation, differentiation, apoptosis, migration and tumorigenesis [13]. In a very simplified model, sensing and adaptation to matrix rigidity is determined by the bond dynamics of different integrin types, which activate RHO and its downstream transducer ROCK (RHO kinase), connecting the adhesion plaque to the acto-myosin cytoskeleton, and promoting its contractility. Through this mechanism cells actively pull on the ECM and probe its resistance developing internal tensional forces that are proportional to the ECM elasticity. Thus, cell contractility enables cells to ‘measure’ the mechanical status of the ECM, and to behave accordingly [3,20]. Cell contractility also underlies the ability of cells to perceive their shape or geometry. Moreover, several cytoplasmic and cytoskeletal signaling pathways have emerged as important mediators of mechanical signaling. These pathways involve small GTPases, focal adhesion kinases and transforming growth factor beta, as well as transcription factors such as NFkB and beta-catenin, and are regulated by specific mechanical inputs (cell stretching or cell compression) or directly by actin, as demonstrated for the MAL/MRTF cofactors of SRF in response to serum stimulation. Recently, we reported the identification of the transcriptional coactivators YAP and TAZ as important downstream transducers of ECM elasticity and cell geometry Current Opinion in Chemical Engineering 2016, 11:67–76
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Figure 2
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Signal transduction network Plasma membrane
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Cell mechanosensing elements and technologies for mechano-associated perturbations. (a) Signal transduction network involves different cellular districts and sensing elements at cytoplasm and membrane level. Cells interact with extracellular elements, such as the extracellular matrix and neighboring cells through sensing molecular complexes. Within the cell, cytoskeletal elements interact with the nuclear envelop to provide mechanical support, transduction and control on gene expression. Different sensing elements are mutually interconnected each other providing information on intracellular and extracellular dynamics and triggering biochemical responses. Here we highlight three relevant cellular interfaces with their main sensors and mediators: cell–substrate interface, cell–cell interface and nuclear envelope. Each domain and complex comprises
Current Opinion in Chemical Engineering 2016, 11:67–76
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Design of microtechnology to study mechanotransduction Giulitti et al. 71
[16,21], affecting proliferation and differentiation in vitro. By drastically reducing cell spreading, mechanotransducing signals induce YAP/TAZ exclusion from the nucleus and degradation in the cytosol (Figure 3). Either topological control of adhesive areas or extremely soft hydrogels were capable to trigger mechano-suppression of YAP/TAZ activity and inducing adipogenesis in mesenchymal stem cells (MSC) rather than osteogenesis in spread/stiff conditions. Interestingly, mechanotransduction seems to operate also in a memory-dependent manner: when cells are passaged from stiff to soft substrates, expression levels of YAP/TAZ and RUNX2 genes decrease with an inverse correlation to the culture time on stiff hydrogels [22]. Moreover, when exposed for longer times to stiff substrates, cells lose responsiveness to soft materials. It is tempting to speculate that the duration of alterations in mechanotransduction pathways are a key component of in vivo pathogenesis onset. Intriguingly, other mechanotransduction pathways evidence that tumorigenesis and metastatic process are sustained by matrix stiffening through a TWIST-mediated mechanotransduction pathway [23]. Remarkably, the assignment of cell shape in undifferentiated cells can be a guidance to generate differentiated cells resembling the imposed shape. MSCs cultured on elongated nanogratings differentiate toward the neuronal and myogenic lineages without the use of any biochemical factor [24]. It is worth to notice that mechanotransduction becomes even a visual phenomenon when scientists have to measure cellular contractility, and cardiomyocytes are probably the best known example. In this context, deflection of flexible cantilever was used to probe cardiac cells forces [25]. Cell–cell interactions and cell topology
The micro-technological advances in growing multi-cellular structures and tissues on specialized surfaces that mimic the stiffness of ECM in vivo could be used to better understand tissue homeostasis in vitro. A major challenge is to understand how cells distribute their load between cell–ECM and cell–cell contacts. Microfabricated devices (with characteristic lengths down to mm-scale) are used to study cell and tissue structure and function under controlled conditions [26].
For instance, when different cells are cultured in confined bidimensional geometries, the adhesion site of each cell can also be responsible for its motility. In this condition, Ma et al. show that human embryonic stem cells cultured under differentiation stimuli are prone to higher motility in the center of the geometric spot, while cells at the edges are exposed to higher tension, upregulation of RhoA/ROCK associated mechano-pathways, proliferation and high cell density [27]. Using a similar setup, Warmflash et al. previously evidenced how these cells are able to generate self-organized zonation of three-germ layers occurring during embryo development. While their research focused on soluble signals gradients generated across culture islets, analog spatio-temporal patterns related to motility, cell density, tension and shape are present [28]. Perhaps cancer is the most intriguing of all mechanotransduction diseases related to 3D environment. Indeed, tumors contain a complex mixture of cell types and matrix components, which affect the physical environment of cancerous cells inside the tumor and the adjacent normal cells. For instance, Calvo et al. followed an in vitro and in vivo analysis of cancer-associated fibroblasts (CAF) that sustain cancer progression. Activation of transcription factors YAP is required in CAFs to promote matrix stiffening that trigger a feed-forward self-reinforcing loop that helps CAF in their proactive tumor sustainment [29]. Nuclear mechanotransduction
The nucleus is physically connected to the cell surface through the cytoskeleton and the linker of nucleoskeleton and cytoskeleton (LINC) complex, allowing rapid mechanical stress transmission from adhesions complexes to the nucleus [30]. This physical connection is essential for a broad range of cellular functions, including intracellular nuclear movement and positioning, cytoskeletal organization, cell polarization and cell migration, which are fundamental aspects during development, wound healing or cancer metastasis [31]. Even though mechanotransduction at the nucleus has not been widely studied, there is evidence of interactions between nuclear mechanics and a few pathways (e.g. SRF, WNT) [32,33]. For instance, Emerin — a transmembrane protein that links
(Figure 2 Legend Continued) cytoskeletal components, membrane proteins and soluble interactors that can be directly or indirectly correlated with the nucleus. Signal transduction eventually triggers the activation or repression genes (arrow in nuclear panel) related to one or more pathways. (b) Different external stimuli, either static or dynamic, can affect cell behavior by altering its adhesion and migratory capabilities on a substrate. Substrates can be designed to provide chemical and topological characteristics that affect cell adhesion and spreading. Water-enriched gels (hydrogels) at different stiffness can mimic the mechanics of different tissues in human body. Micropillars with tunable compliance represent an alternative method to probe forces exerted by cells. Dynamic stimuli can alter normal cell behavior over time with the support of microfluidics and Microtechnologies. Cell shapes and cell–cell contact can be easily perturbed by applying external forces, also with specific frequency. Shear stress can be generated by a flow above a cell layer. Migration and deformability can be evaluated using microchannels in presence of cell chemo-attractive molecules or differential pressure. www.sciencedirect.com
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Figure 3
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Current Opinion in Chemical Engineering
Applications of microengineering to mechanotransduction studies. Substrate engineering including cell patterning and substrate stiffness was successfully used to study mechanosensing and signaling pathways activation. Cell patterning, including multidimensional cell pattern, allows studies such as transcription factor, YAP, translocation according to cell spreading and skeletal muscle differentiation on cell pattern of soft matrix. Substrate stiffness has been shown to regulate transcription factor translation according to surface stiffness, which regulates cell shape, leading to osteogenic or adipogenic differentiation of mesenchymal stem cells on differ matrix stiffness. 3D organization determines developmental specification of multiple tissues according to cell spatial position, as well as 3D perturbation of nuclear shape induced by external mechanical constrains. Examples of biological study of cell stretching using deformable membrane include: YAP translocation and proliferation activation in confluent monolayer of epithelial cell, membrane integrity assay in human Duchenne Muscular dystrophy in vitro model. Shear flow-based approaches allow studying cell motility and contractility under chemical or pressure gradients, by analyzing cell migration within microfluidic devices. [16] Reprinted by permission from Macmillan Publishers Ltd: Nature [16], copyright 2011. [21] Reprinted from M. Aragona, et al., A mechanical checkpoint controls multicellular growth through YAP/TAZ regulation by actin-processing factors, Cell. 154 (2013) 1047–1059. [28] Reprinted by permission from Macmillan Publishers Ltd: Nature Methods [28], copyright (2014). [18] Reprinted with permission from S. Zatti, et al., Micropatterning Topology on Soft Substrates Affects Myoblast Proliferation and Differentiation, Langmuir. 28 (2012) 2718–2726. Copyright (2012) American Chemical Society. [35] Reprinted from Badique F et al. Directing nuclear deformation on micropillared surfaces by substrate geometry and cytoskeleton organization. Biomaterials 2013, 34:2991-3001, with permission from Elsevier Inc.[47] Reproduced from Ref. [47] with permission from The Royal Society of Chemistry. [39] Adapted from [39] with permission of The Royal Society of Chemistry. [53] Adapted from [53] with permission of The Royal Society of Chemistry. [54] Adapted from [54] with permission of The Royal Society of Chemistry.
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cytoskeleton and nuclear envelop to the nuclear lamina — is phosphorylated in response to nuclear tension and affects genes dependent on the serum response factor [34]. Microtechnologies could offer unique opportunities to study how nuclear architecture and the transfer of mechanical forces to the nucleus are defined in response to 3D environment and mechanical constrains. An elegant demonstration of the importance of surface topology and 3D organization is shown by Anselme and colleagues. While different cell types cultured on flat substrates do not show substantial differences in their nucleus distribution, cancer cells show exceptional plasticity when cultured on micropillars: their nucleus deforms subsiding down into micrometric grooves and forming different concavities and protrusions [35]. Notably, this capacity is independent of substrate chemistry and stiffness and correlates with the cytoskeletal organization of each cell type. Similarly, Nagayama et al. demonstrated that normal cells are not able to alter the shape of trapped nuclei to the same extent of cervical cancer HeLa cells and only transformed cells can replicate regardless of nuclear shape distortion [36]. Similarly, when cells are cultured on flat substrates with selective adhesion, actin filaments play an active role in nuclear shaping. Imposition of narrow cell shape induce nuclear stretching, lateral compression and significant loss of nuclear volume leading to DNA condensation [37]. It is evident that nuclear positioning has a huge impact on cell functionalities, sustaining also cellular maturation during differentiation [38]. Unbalancing the toolbox of proteins usually associated with nuclear stabilization and motility evidenced to trigger numerous diseases. Moreover, deformation of the nucleus during 3D migration is gathering increasing interest in the context of cancer metastasis, with the underlying hypothesis that a softer nucleus may aid to tumor spreading. The ability of cells to migrate through tissues and interstitial spaces could be easily probed by microfluidic devices composed of channels with precisely defined constrictions that mimic physiological environments. Intracellular mechanics and dynamics in physiologically relevant applications, ranging from cancer cell invasion to immune cell recruitment, can be studied integrating devices to study cell migration with high-resolution living imaging [39].
Dynamic biological stimulation through micro-devices Living tissues are continuously exposed to cyclic mechanical loads, which induce changes in their structure, composition, and function. For instance, endothelial cells forming the inner lining of the blood vessel, are www.sciencedirect.com
influenced by two distinct hemodynamic loads: cyclic strain due to vessel wall distension and shear stress due to the frictional forces generated by blood flow [40]. Muscle cells are also particularly responsive to mechanical forces; indeed, expression of skeletal muscle specific genes and skeletal muscle functional maturation, including sarcomeric organization and contractile structure development, can be modulated by applying an external mechanical stress. From a technological point of view cell-stretching devices have been developed to study cellular mechanoresponses in vitro. The most employed methodology is to use vacuum to actively deform a thin elastic membrane in which cells are cultured [41,42]. Beside the most common commercially available cell-stretching devices, a number of custom-made solutions have been proposed to perform mechanical stimulations on cell cultures in uniaxial and biaxial directions [43,44]. Many of them take advantage of microfluidic technology to achieve a better control of the strain induced to the cells while maintaining focal plane for in line detection. For instance, Shao et al. developed a cell stretching device combining dynamic directional cell stretch with in situ subcellular livecell imaging, demonstrating a rapid force-mediated focal adhesion assembly [45]. According to this design, several works reported on the role of cyclic stretch in myogenesis, tissue remodeling and sarcomere assembling in skeletal muscle [46]. Recently, we developed a microfluidic cell-stretching device to investigate the effect of cyclic mechanical stimulation on dystrophin-deficient human myotubes derived in vitro from healthy and diseased muscle biopsies [47] (Figure 3). Moreover, loading conditions can also play important roles in tissue and organ pathology, such as the processes of osteoporosis, atherosclerosis and fibrosis. In the cardiovascular field, increased mechanical stress is associated with cardiac dysfunction, such as myocardial hypertrophy, vessel wall thickening and fibrosis [12]. Cells including endothelial and smooth muscle cells experience cyclic circumferential stretch and compressive pressure generated by blood flow [11]. In this context, Zhou et al. observed activation of key components of SMAD1/SMAD2 and Wnt/b-catenin pathways in vascular cells precursors subjected to cyclic strains obtained through a microfluidic platform which simulates pulsatile blood flow [48]. Similarly, Suresh Babu et al. identified a stretch-induced signaling pathway in vascular cells involving the cytoskeletal transducer protein zyxin [49]. Moreover, microfluidics, characterized by laminar flow regime which allows local stimulation of cells with precise shear stress, is a perfect tool for studying vascular endothelium. Basically mechanical stimulus can be externally imposed by active force (shear stress, stretch, strain) controlling the flow rate or using pneumatic and electric systems [12]. Current Opinion in Chemical Engineering 2016, 11:67–76
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Mechanotransduction plays a critical role in cancer where cancerous cells are characterized by altered biophysical properties [50]. Microfluidic systems are particularly suited to investigate the role of interstitial flow (IF) due to elevated fluid pressure in tumor microenvironment, where cells exhibit altered biophysical properties and mobility. For instance, Polacheck et al. studied the effect of IF-induced fluid stresses imparted on cells embedded within collagen type I hydrogel [50]. Moreover, microfluidic tools can be successfully used to probe cell deformability and, consequently, for studying infiltration of immune cells and migration of metastatic cells into or from tumor, respectively [39]. Adamo et al. measured cell deformability and stiffness with a microfluidic device capable to resolve both cell travel time and relative cell diameter upon passage of cells through a micrometric constriction [51]. Byun et al. were able to identify the relative importance of deformability and surface friction between cell lines measuring the entry and transit velocity [52]. The use of microfluidic-based separation technique shows potential to conduct rapid and low cost cell analysis for disease diagnostics. An automatic micropipette that is capable to perform both deformation and relaxation measurements of individual cancer cells was recently proposed [53]. A further advancement in micropipette aspiration via microfluidic pipette array (mFPA) is able to conduct aspiration in a parallel manner [54]. The influence of cytoskeletal and nuclear structures on cell mechanical properties was demonstrated by the use of a micro-constriction array. While physical constrictions may not require specific substrate characteristics, the design of culture surface and its material is crucial in mechanotransduction studies. Independently from the chosen technology, substrate chemistry determines cell adhesion selectiveness depending on the cell type and strength at the cell– substrate interface. Non-fouling inert substrates (e.g. polyacrylamide and polyethyleneglycol-based hydrogels) offer a broad range in mechanical properties (e.g. stiffness) and have extensively been used as a mechano-assay with the aid of biochemical functionalization in order to attach ECM proteins and favor cell adhesion without compromising mechano-dependent cells responses. Biopolymer derivates (e.g. methacrylated gelatin and hyaluronic acid) are acquiring importance thanks to their biological activity and degradability for in vivo purposes [55]. Many of these materials are extremely versatile since they can be photo-activated and polymerized to topologically control cells within microdevices [56]. Beyond the modeling of bidimensional surfaces, 3-dimensional microengineering and mechanobiology are now Current Opinion in Chemical Engineering 2016, 11:67–76
gaining research focus thanks to new available technologies providing a more comprehensive definition of in vivo spatial organization and the opportunity to actively and dynamically perturb biological systems in real time [57].
Conclusions The knowledge of the molecular players and interactions between cell signals and different mechanotransduction pathways are still far to be completely understood. Manipulating the extracellular environment through microengineered features affects the spatio-temporal distribution and interactions of biochemical structures at the cell–substrate and cell–cell interface. It is tempting to speculate that control of cell shape and induction of particular phenotypes are the consequence of spatial (alignment, juxtaposition, zonation or exclusion) or conformational reorganization of mechanosensors that can interact with different players inside the cell. In this view, emerging microengineering-based opportunities and challenges in providing means for generating external mechanical stimuli affecting a living cell with accurate control of three-dimensional spatial and temporal patterns are of paramount importance. Moreover, performing live-cell imaging by integrating these technological platforms with new tools capable of applying and measuring forces and displacements with picoNewton and nanometer resolutions (e.g. atomic force microscopy, optical and magnetic tweezers, biomembrane and substrate force probes) will be fundamental for further advances in the field. Despite their importance, several limitations are involved in the choice of microtechnological strategies that could dissuade the final user. Microtechnological approaches are still often adopted only on a lab scale and may require specialized instrumentation and expertise in bioengineering design. Moreover, the biological processing with microtechnology is not conventional, since implicates mismatching with standard methods, loss of historical context, requires ad hoc standards and controls, and is more challenging to compare data with published results. Microfluidic devices work with nano or microliter volume and require specific knowledge and protocols that often are incompatible with the rapid processing of large sample volumes with pipettes. Miniaturization has also the effect to increase the surface to volume ratio that enhances the surface adsorption losses of molecules that could cause misunderstanding and wrong data interpretations. Despite these issues, new tools in microtechnology will represent a key aspect of future research in mechanotransduction. Engineering of culture substrates has push forward biological research for more than 10 years. New advancements in microengineering and their biological application will ensure even more emphasis and detail in mechano-biology. www.sciencedirect.com
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24. Teo BKK, Wong ST, Lim CK, Kung TYS, Yap CH, Ramagopal Y et al.: Nanotopography modulates mechanotransduction of stem cells and induces differentiation through focal adhesion kinase. ACS Nano 2013, 7:4785-4798 http://dx.doi.org/10.1021/ nn304966z. 25. You J, Moon H, Lee BY, Jin JY, Chang ZE, Kim SY et al.: Cardiomyocyte sensor responsive to changes in physical and chemical environments. J Biomech 2014, 47:400-409 http:// dx.doi.org/10.1016/j.jbiomech.2013.11.013. 26. Pruitt BL, Dunn AR, Weis WI, Nelson WJ: Mechano-transduction: from molecules to tissues. PLOS Biol 2014, 12:e1001996 http:// dx.doi.org/10.1371/journal.pbio.1001996. 27. Ma Z, Wang J, Loskill P, Huebsch N, Koo S, Svedlund FL et al.: Self-organizing human cardiac microchambers mediated by geometric confinement. Nat Commun 2015, 6:7413 http:// dx.doi.org/10.1038/ncomms8413. 28. Warmflash A, Sorre B, Etoc F, Siggia ED, Brivanlou AH: A method to recapitulate early embryonic spatial patterning in human embryonic stem cells. Nat Methods 2014, 11:847-854 http:// dx.doi.org/10.1038/nmeth.3016. The authors introduce a patterning methodology to explain differentiation and self-organization of pluripotent cells into stratified germ layers, evidencing the importance of diffusive signals dependent of cell population size. 29. Calvo F, Ege N, Grande-Garcia A, Hooper S, Jenkins RP, Chaudhry SI et al.: Mechanotransduction and YAP-dependent matrix remodelling is required for the generation and maintenance of cancer-associated fibroblasts. Nat Cell Biol 2013, 15:637-646 http://dx.doi.org/10.1038/ncb2756. 30. Wang N, Tytell JD, Ingber DE: Mechanotransduction at a distance: mechanically coupling the extracellular matrix with the nucleus. Nat Rev Mol Cell Biol 2009, 10:75-82 http:// dx.doi.org/10.1038/nrm2594.
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mechanotransduction pathway in the nucleus. Nat Cell Biol 2014, 16:376-381 http://dx.doi.org/10.1038/ncb2927. 35. Badique F, Stamov DR, Davidson PM, Veuillet M, Reiter G, Freund J-N et al.: Directing nuclear deformation on micropillared surfaces by substrate geometry and cytoskeleton organization. Biomaterials 2013, 34:2991-3001 http://dx.doi.org/10.1016/j.biomaterials.2013.01.018. 36. Nagayama K, Hamaji Y, Sato Y, Matsumoto T: Mechanical trapping of the nucleus on micropillared surfaces inhibits the proliferation of vascular smooth muscle cells but not cervical cancer HeLa cells. J Biomech 2015, 48:1796-1803 http:// dx.doi.org/10.1016/j.jbiomech.2015.05.004. 37. Versaevel M, Grevesse T, Gabriele S: Spatial coordination between cell and nuclear shape within micropatterned endothelial cells. Nat Commun 2012, 3:671 http://dx.doi.org/ 10.1038/ncomms1668. 38. Gundersen GG, Worman HJ: Nuclear positioning. Cell 2013, 152:1376-1389 http://dx.doi.org/10.1016/j.cell.2013.02.031. 39. Davidson PM, Sliz J, Isermann P, Denais C, Lammerding J: Design of a microfluidic device to quantify dynamic intra-nuclear deformation during cell migration through confining environments. Integr Biol 2015, 7:1534-1546 http://dx.doi.org/ 10.1039/C5IB00200A. Here the authors developed an innovative microfluidic device for quantitative measurements of nuclear and cell deformability showing the implication of lamin A/C in nuclear deformability and providing a new approach to study cancer cell invasion and immune cell recruitment. 40. Wang JH-C, Thampatty BP: An introductory review of cell mechanobiology. Biomech Model Mechanobiol 2006, 5:1-16 http://dx.doi.org/10.1007/s10237-005-0012-z. 41. Tremblay D, Chagnon-Lessard S, Mirzaei M, Pelling AE, Godin M: A microscale anisotropic biaxial cell stretching device for applications in mechanobiology. Biotechnol Lett 2014, 36:657665 http://dx.doi.org/10.1007/s10529-013-1381-5. 42. Huh D, Leslie DC, Matthews BD, Fraser JP, Jurek S, Hamilton GA et al.: A human disease model of drug toxicity-induced pulmonary edema in a lung-on-a-chip microdevice. Sci Transl Med 2012, 4:159ra147 http://dx.doi.org/10.1126/ scitranslmed.3004249. 43. Hsieh H-Y, Camci-Unal G, Huang T-W, Liao R, Chen T-J, Paul A et al.: Gradient static-strain stimulation in a microfluidic chip for 3D cellular alignment. Lab Chip 2014, 14:482-493 http:// dx.doi.org/10.1039/c3lc50884f.
46. Kook S-H, Lee H-J, Chung W-T, Hwang I-H, Lee S-A, Kim B-S et al.: Cyclic mechanical stretch stimulates the proliferation of C2C12 myoblasts and inhibits their differentiation via prolonged activation of p38 MAPK. Mol Cells 2008, 25:479-486. 47. Michielin F, Serena E, Pavan P, Elvassore N: Microfluidicassisted cyclic mechanical stimulation affects cellular membrane integrity in a human muscular dystrophy in vitro model. RSC Adv 2015, 5:98429-98439 http://dx.doi.org/10.1039/ C5RA16957G. 48. Zhou J, Niklason LE: Microfluidic artificial ‘‘vessels’’ for dynamic mechanical stimulation of mesenchymal stem cells. Integr Biol Quant Biosci Nano Macro 2012, 4 http://dx.doi.org/ 10.1039/c2ib00171c. 49. Suresh Babu S, Wojtowicz A, Freichel M, Birnbaumer L, Hecker M, Cattaruzza M: Mechanism of stretch-induced activation of the mechanotransducer zyxin in vascular cells. Sci Signal 2012, 5:ra91 http://dx.doi.org/10.1126/scisignal.2003173. 50. Polacheck WJ, German AE, Mammoto A, Ingber DE, Kamm RD: Mechanotransduction of fluid stresses governs 3D cell migration. Proc Natl Acad Sci U S A 2014, 111:2447-2452 http:// dx.doi.org/10.1073/pnas.1316848111. 51. Adamo A, Sharei A, Adamo L, Lee B, Mao S, Jensen KF: Microfluidics-based assessment of cell deformability. Anal Chem 2012, 84:6438-6443 http://dx.doi.org/10.1021/ac300264v. 52. Byun S, Son S, Amodei D, Cermak N, Shaw J, Kang JH et al.: Characterizing deformability and surface friction of cancer cells. Proc Natl Acad Sci U S A 2013, 110:7580-7585 http:// dx.doi.org/10.1073/pnas.1218806110. 53. Mak M, Erickson D: A serial micropipette microfluidic device with applications to cancer cell repeated deformation studies. Integr Biol Quant Biosci Nano Macro 2013, 5:1374-1384 http:// dx.doi.org/10.1039/c3ib40128f. 54. Lee LM, Liu AP: A microfluidic pipette array for mechanophenotyping of cancer cells and mechanical gating of mechanosensitive channels. Lab Chip 2015, 15:264-273 http://dx.doi.org/10.1039/c4lc01218f. 55. Malda J, Visser J, Melchels FP, Ju¨ngst T, Hennink WE, Dhert WJA et al.: 25th anniversary article: engineering hydrogels for biofabrication. Adv Mater 2013, 25:5011-5028 http://dx.doi.org/ 10.1002/adma.201302042.
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Current Opinion in Chemical Engineering 2016, 11:67–76
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ScienceDirect Mechanotransduction through substrates engineering and microfluidic devices Stefano Giulitti1,2, Alessandro Zambon1,2, Federica Michielin1,2 and Nicola Elvassore1,2 Living cells are able to sense three-dimensional environmental properties, to integrate biophysical and biochemical stimulations and to adapt to dynamic changes of their physical surroundings for maintaining appropriate biological functions. Mechanotransduction — the mechanism by which a mechanical stimulation is transduced into a chemical signal — typically involves force-induced conformational changes in proteins or altered affinities to their binding partners but these interactions remain largely unexplored. New development in microengineering offers unique capabilities for in vitro controlling and perturbing both the biochemical and biomechanical cellular microenvironment. Here, we focus on studies and discoveries that — through innovative microtechnology — dissected and reconstructed biochemical and biophysical mechanisms underlying mechano-associated biological processes. These microengineered platforms provide endless possibilities for new insights into the role of mechanotransduction in physiology and pathophysiology of human tissues and organs, including tumorigenesis and metastasis. Addresses 1 Dept. of Industrial Engineering, University of Padova, via Marzolo 9, Padova 35131, Italy 2 Venetian Institute of Molecular Medicine, via Orus 2, Padova 35129, Italy Corresponding author: Elvassore, Nicola ([email protected])
Current Opinion in Chemical Engineering 2016, 11:67–76 This review comes from a themed issue on Biological engineering
Living cells are immersed in a three-dimensional structure based on biopolymers forming the extracellular matrix (ECM). A distinctive cell–ECM combination confers unique physiological and biological functions to human organs. Cells are able to sense three-dimensional environmental properties of the extracellular matrix and neighboring cells, to integrate biophysical and biochemical stimulations and, accordingly, to adapt to the dynamic changes of their physical surroundings for maintaining appropriate biological functions [1]. The cellular processes, which translate biophysical cues and biomechanical stimulations in biochemical signals are called mechanotransduction [2]. At the cellular level, mechanical signals influence cell motility, proliferation, apoptosis, differentiation, and maturation patterns. They also play a critical role during tissue and organ morphogenesis in the embryo state [3], and in the maintenance of functionalities in the adult organism. Impairments in these cellular processes contribute to the underlying causes of many diseases and pathological conditions [4], including tumorigenesis and metastasis. Since all cells can have mechano-sensitive elements able to perceive mechanical changes in different cell districts (Figure 2), mechanotransduction is present in all tissues and organs. For instance, mechanotransduction signaling has a crucial role in the maintenance of many mechanically stressed tissues, such as skeletal and cardiac muscle, bone, cartilage and blood vessels [5].
Edited by Sharon Gerecht and Kostas Konstantopoulos
http://dx.doi.org/10.1016/j.coche.2016.01.010 2211-3398/# 2016 Elsevier Ltd. All rights reserved.
Introduction Tissues and organs have a variety of functions, which depend on particular cell phenotypes and on their topological and hierarchical organizations within the human body. Besides biochemical signals, tissues and organs normally experience dynamic mechanical stresses dependent on their activities (Figure 1). The impossibility of some organs to exert or counteract specific forces could lead to the onset of a disease. www.sciencedirect.com
Conventionally, biological research investigates the cellular response to extrinsic soluble biochemical factors and biochemical properties of ECM. However, the rational understanding of the biochemical mechanisms by which the cells sense, transduce, and respond to mechanical stimulations and shape deformations is largely unexplored. Only recent investigations provide unambiguous knowledge about mechano-associated mechanisms, despite the importance of these in fundamental aspects of human physiology and pathophysiology and in several biomedical applications (such as cell-based therapy or regenerative medicine). The mechanisms by which the mechanical stimulation is transduced into a chemical signal was not confirmed until the presence of mechanosensing ion channels was reported, explaining the cellular depolarization after stretching of nerve cells [6]. Many additional mechanotransduction pathways have been identified; typically, the Current Opinion in Chemical Engineering 2016, 11:67–76
68 Biological engineering
Figure 1
(h) Lungs (chronic obstructive pulmunary disease and inflammation)
(a) Vascular endothelium (calcification, aterosclerosys)
(g) Skin (keloid)
(b) Heart (hypertophy, heart failure)
(f) Bladder, urethra (urodynamic disorder)
(c) Intestine (chronic intestinal pseudo-obstruction)
(e) Bone, cartilage, tendons (loss of bone mass)
(d) Skeletal muscle (muscular dystrophies)
Current Opinion in Chemical Engineering
Mechanical stimuli in human tissues and organs, and mechano-associated diseases. Cells of different tissues experience mechanical stimuli and deformations in the human body. Example of common forces, stresses, loads and deformations are represented clockwise reporting also possible mechano-associated diseases (arrows depict force/pressure directions; normal stress (s) or shear stress (t) are not reported in the text for organs subjected to extremely variable forces). (a) Vascular endothelium in blood vessels is continuously subjected to shear and hydrostatic stress exerted by blood flow (t = 0.07–13 Pa); (b) during heart beating, cardiomyocytes are subjected to active contraction during systole and relaxation during diastole; (c) peristalsis involves radial symmetrical contraction and relaxation of muscles during digestion (s = 2.6–6.6 kPa); (d) skeletal muscles generate uniaxial contraction; (e) compressive and tensile loads are experienced by cartilage, bones and tendons respectively; (f) in the urinary apparatus, bladder undergoes high dilatation providing compliance while maintaining constant hydrostatic pressure (s < 6 kPa) whereas urethra experiences shear stresses and osmotic forces; (g) skin is a multilayered organ which are exposed to mechanical pressure and osmotic stresses. Hair skin has also sensory units that respond to mechanical pressure or distortion; (h) epithelial/endothelial bilayer in lungs undergoes cyclic deformation (s = 0.3–9.8 kPa) preserving the body from inflammation and infections.
employed mechanisms involve force-induced conformational changes in a protein that result in opening of membrane channels [7,8] or altered affinities to binding partners, thereby activating signaling pathways [9,10]. The downstream targets of these signaling pathways could include ECM reorganization, changes in protein affinity and binding partners, cytoskeleton remodeling, chromatin reorganization and specific gene expression [1]. The experimental investigations of mechano-associated cellular processes are quite limited using conventional biological approaches. New tools and technologies are required to further investigate, understand, dissect and reconstruct the complex machinery that the cells use to Current Opinion in Chemical Engineering 2016, 11:67–76
respond and adapt to specific mechanical stimulations. New development in microengineering technology offers unique opportunities for in vitro controlling and perturbing the biochemical and biomechanical properties of three-dimensional environment surrounding cells. Microfabrication techniques are increasingly used for controlling the three-dimensional architecture of in vitro cellular microenvironment and for enabling accurate, quantitative measurements of cellular responses in high-throughput experiments [11]. These microengineered platforms provide possibilities for new insights into interactions between cells and their dynamically changing microenvironment, which underlies www.sciencedirect.com
Design of microtechnology to study mechanotransduction Giulitti et al. 69
the physiology and pathophysiology of human tissues and organs. Recent excellent reviews on mechanobiology and associated technology provided a general perspective of this emerging and rapidly evolving interdisciplinary field [12,13]. Our review focuses on the biological applications of micro-engineering tools specifically designed for studying, dissecting and reconstructing biochemical and biophysical mechanisms, which sustain mechano-associated biological processes. Here we review mechanotransduction studies, evidencing two main approaches in the field: mechanotransduction modulation through substrate engineering and active dynamic modulation of force-deformation using microengineering advances, including microfluidics. For over a decade, substrate engineering has resulted in soft hydrogels and micro-patterns that enabled considerable new insights in biological processes. In the first part we will overview recent biological discoveries in mechanotransduction field made possible through substrate engineering. In the second part, we will highlight emerging microengineering technologies that have been tailored on specific mechanotransduction problems and will found new bases on how to study mechanorelated biological activities.
Tuning mechanotransduction through substrates engineering The ability of cells to sense the biochemical and biophysical properties of the ECM affects the regulation of cellular activities in development, tissue homeostasis, regeneration, as well as malignant transformation (Figure 2a). Pioneering works show that mesenchymal stem cells are committed to osteogenic fate when cultured on stiff substrates, whereas they express a neurogenic phenotype when adhere on softer substrate [14]. Several other works show that the biophysical properties of the substrate also regulate proliferation, cell motility and cell spreading. Only recently, some works reported experimental evidence that the cells are able to respond to specific substrate properties by translating mechanical cues into biochemical signals that activates specific signaling pathways, including transcription factors, and regulates gene expression [15–17]. However, the molecular mechanisms responsible for sensing microenvironment mechanical changes remain poorly understood and substrates micro-engineering offers an endless potential of probing and investigating molecular mechanisms underlying the mechanobiology. Figure 2b shows the main technological advances, such as cell-adhesive patterned surfaces, substrates with tunable stiffness or micro-pillared surfaces, that have been successfully used for controlling the architecture and adhesiveness of a cell and facilitating the study at three levels: firstly, cell–substrate interactions; secondly, cell–cell interactions; and finally, nuclear mechanotransduction. www.sciencedirect.com
Owing to the large number of works developed, here we report more recent intriguing aspects of cell mechanobiology with a particular focus on probing mechanotransduction mechanisms through the use of microengineered substrates and surface chemistry. Cell–substrate interactions
Micropatterning techniques have been used to spatially control cell–substrate interactions, to manipulate the biochemical adhesiveness of substrates and to patterning specific ECM proteins through microcontact printing [18] or microfluidic patterning [19]. On the other hand, relatively few approaches in substrates engineering have been developed to specifically investigate the effects of the mechanical forces of cell adhesion on the regulation of cell signaling and function (Figure 3). The main technique to obtain substrates with tunable stiffness consists in using one or more polymerizable chemical species with a variable molar ratio compared to a crosslinking agent. Specifically, the substrate elastic modulus can be varied by orders of magnitude, with a typical physiological range from 1 kPa to 100 kPa. One of the most used material, polyacrylamide (PA), forms an inert transparent and biocompatible polymer of acrylamide crosslinked with metilen-bis-acrylamide in a water-rich environment [14]. Both micropatterning and substrate stiffness regulation and their combinations were successfully used in studying cell proliferation, differentiation, apoptosis, migration and tumorigenesis [13]. In a very simplified model, sensing and adaptation to matrix rigidity is determined by the bond dynamics of different integrin types, which activate RHO and its downstream transducer ROCK (RHO kinase), connecting the adhesion plaque to the acto-myosin cytoskeleton, and promoting its contractility. Through this mechanism cells actively pull on the ECM and probe its resistance developing internal tensional forces that are proportional to the ECM elasticity. Thus, cell contractility enables cells to ‘measure’ the mechanical status of the ECM, and to behave accordingly [3,20]. Cell contractility also underlies the ability of cells to perceive their shape or geometry. Moreover, several cytoplasmic and cytoskeletal signaling pathways have emerged as important mediators of mechanical signaling. These pathways involve small GTPases, focal adhesion kinases and transforming growth factor beta, as well as transcription factors such as NFkB and beta-catenin, and are regulated by specific mechanical inputs (cell stretching or cell compression) or directly by actin, as demonstrated for the MAL/MRTF cofactors of SRF in response to serum stimulation. Recently, we reported the identification of the transcriptional coactivators YAP and TAZ as important downstream transducers of ECM elasticity and cell geometry Current Opinion in Chemical Engineering 2016, 11:67–76
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Figure 2
(a)
Signal transduction network Plasma membrane
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signal transduction Gene expression Actin
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Cell mechanosensing elements and technologies for mechano-associated perturbations. (a) Signal transduction network involves different cellular districts and sensing elements at cytoplasm and membrane level. Cells interact with extracellular elements, such as the extracellular matrix and neighboring cells through sensing molecular complexes. Within the cell, cytoskeletal elements interact with the nuclear envelop to provide mechanical support, transduction and control on gene expression. Different sensing elements are mutually interconnected each other providing information on intracellular and extracellular dynamics and triggering biochemical responses. Here we highlight three relevant cellular interfaces with their main sensors and mediators: cell–substrate interface, cell–cell interface and nuclear envelope. Each domain and complex comprises
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Design of microtechnology to study mechanotransduction Giulitti et al. 71
[16,21], affecting proliferation and differentiation in vitro. By drastically reducing cell spreading, mechanotransducing signals induce YAP/TAZ exclusion from the nucleus and degradation in the cytosol (Figure 3). Either topological control of adhesive areas or extremely soft hydrogels were capable to trigger mechano-suppression of YAP/TAZ activity and inducing adipogenesis in mesenchymal stem cells (MSC) rather than osteogenesis in spread/stiff conditions. Interestingly, mechanotransduction seems to operate also in a memory-dependent manner: when cells are passaged from stiff to soft substrates, expression levels of YAP/TAZ and RUNX2 genes decrease with an inverse correlation to the culture time on stiff hydrogels [22]. Moreover, when exposed for longer times to stiff substrates, cells lose responsiveness to soft materials. It is tempting to speculate that the duration of alterations in mechanotransduction pathways are a key component of in vivo pathogenesis onset. Intriguingly, other mechanotransduction pathways evidence that tumorigenesis and metastatic process are sustained by matrix stiffening through a TWIST-mediated mechanotransduction pathway [23]. Remarkably, the assignment of cell shape in undifferentiated cells can be a guidance to generate differentiated cells resembling the imposed shape. MSCs cultured on elongated nanogratings differentiate toward the neuronal and myogenic lineages without the use of any biochemical factor [24]. It is worth to notice that mechanotransduction becomes even a visual phenomenon when scientists have to measure cellular contractility, and cardiomyocytes are probably the best known example. In this context, deflection of flexible cantilever was used to probe cardiac cells forces [25]. Cell–cell interactions and cell topology
The micro-technological advances in growing multi-cellular structures and tissues on specialized surfaces that mimic the stiffness of ECM in vivo could be used to better understand tissue homeostasis in vitro. A major challenge is to understand how cells distribute their load between cell–ECM and cell–cell contacts. Microfabricated devices (with characteristic lengths down to mm-scale) are used to study cell and tissue structure and function under controlled conditions [26].
For instance, when different cells are cultured in confined bidimensional geometries, the adhesion site of each cell can also be responsible for its motility. In this condition, Ma et al. show that human embryonic stem cells cultured under differentiation stimuli are prone to higher motility in the center of the geometric spot, while cells at the edges are exposed to higher tension, upregulation of RhoA/ROCK associated mechano-pathways, proliferation and high cell density [27]. Using a similar setup, Warmflash et al. previously evidenced how these cells are able to generate self-organized zonation of three-germ layers occurring during embryo development. While their research focused on soluble signals gradients generated across culture islets, analog spatio-temporal patterns related to motility, cell density, tension and shape are present [28]. Perhaps cancer is the most intriguing of all mechanotransduction diseases related to 3D environment. Indeed, tumors contain a complex mixture of cell types and matrix components, which affect the physical environment of cancerous cells inside the tumor and the adjacent normal cells. For instance, Calvo et al. followed an in vitro and in vivo analysis of cancer-associated fibroblasts (CAF) that sustain cancer progression. Activation of transcription factors YAP is required in CAFs to promote matrix stiffening that trigger a feed-forward self-reinforcing loop that helps CAF in their proactive tumor sustainment [29]. Nuclear mechanotransduction
The nucleus is physically connected to the cell surface through the cytoskeleton and the linker of nucleoskeleton and cytoskeleton (LINC) complex, allowing rapid mechanical stress transmission from adhesions complexes to the nucleus [30]. This physical connection is essential for a broad range of cellular functions, including intracellular nuclear movement and positioning, cytoskeletal organization, cell polarization and cell migration, which are fundamental aspects during development, wound healing or cancer metastasis [31]. Even though mechanotransduction at the nucleus has not been widely studied, there is evidence of interactions between nuclear mechanics and a few pathways (e.g. SRF, WNT) [32,33]. For instance, Emerin — a transmembrane protein that links
(Figure 2 Legend Continued) cytoskeletal components, membrane proteins and soluble interactors that can be directly or indirectly correlated with the nucleus. Signal transduction eventually triggers the activation or repression genes (arrow in nuclear panel) related to one or more pathways. (b) Different external stimuli, either static or dynamic, can affect cell behavior by altering its adhesion and migratory capabilities on a substrate. Substrates can be designed to provide chemical and topological characteristics that affect cell adhesion and spreading. Water-enriched gels (hydrogels) at different stiffness can mimic the mechanics of different tissues in human body. Micropillars with tunable compliance represent an alternative method to probe forces exerted by cells. Dynamic stimuli can alter normal cell behavior over time with the support of microfluidics and Microtechnologies. Cell shapes and cell–cell contact can be easily perturbed by applying external forces, also with specific frequency. Shear stress can be generated by a flow above a cell layer. Migration and deformability can be evaluated using microchannels in presence of cell chemo-attractive molecules or differential pressure. www.sciencedirect.com
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Figure 3
Mechanical stimuli
Applications [28]
[16]
[18]
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unpatt.
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osteocytes
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[28]
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control
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YAP/TAZ Proliferation (%) Localization (%)
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Current Opinion in Chemical Engineering
Applications of microengineering to mechanotransduction studies. Substrate engineering including cell patterning and substrate stiffness was successfully used to study mechanosensing and signaling pathways activation. Cell patterning, including multidimensional cell pattern, allows studies such as transcription factor, YAP, translocation according to cell spreading and skeletal muscle differentiation on cell pattern of soft matrix. Substrate stiffness has been shown to regulate transcription factor translation according to surface stiffness, which regulates cell shape, leading to osteogenic or adipogenic differentiation of mesenchymal stem cells on differ matrix stiffness. 3D organization determines developmental specification of multiple tissues according to cell spatial position, as well as 3D perturbation of nuclear shape induced by external mechanical constrains. Examples of biological study of cell stretching using deformable membrane include: YAP translocation and proliferation activation in confluent monolayer of epithelial cell, membrane integrity assay in human Duchenne Muscular dystrophy in vitro model. Shear flow-based approaches allow studying cell motility and contractility under chemical or pressure gradients, by analyzing cell migration within microfluidic devices. [16] Reprinted by permission from Macmillan Publishers Ltd: Nature [16], copyright 2011. [21] Reprinted from M. Aragona, et al., A mechanical checkpoint controls multicellular growth through YAP/TAZ regulation by actin-processing factors, Cell. 154 (2013) 1047–1059. [28] Reprinted by permission from Macmillan Publishers Ltd: Nature Methods [28], copyright (2014). [18] Reprinted with permission from S. Zatti, et al., Micropatterning Topology on Soft Substrates Affects Myoblast Proliferation and Differentiation, Langmuir. 28 (2012) 2718–2726. Copyright (2012) American Chemical Society. [35] Reprinted from Badique F et al. Directing nuclear deformation on micropillared surfaces by substrate geometry and cytoskeleton organization. Biomaterials 2013, 34:2991-3001, with permission from Elsevier Inc.[47] Reproduced from Ref. [47] with permission from The Royal Society of Chemistry. [39] Adapted from [39] with permission of The Royal Society of Chemistry. [53] Adapted from [53] with permission of The Royal Society of Chemistry. [54] Adapted from [54] with permission of The Royal Society of Chemistry.
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Design of microtechnology to study mechanotransduction Giulitti et al. 73
cytoskeleton and nuclear envelop to the nuclear lamina — is phosphorylated in response to nuclear tension and affects genes dependent on the serum response factor [34]. Microtechnologies could offer unique opportunities to study how nuclear architecture and the transfer of mechanical forces to the nucleus are defined in response to 3D environment and mechanical constrains. An elegant demonstration of the importance of surface topology and 3D organization is shown by Anselme and colleagues. While different cell types cultured on flat substrates do not show substantial differences in their nucleus distribution, cancer cells show exceptional plasticity when cultured on micropillars: their nucleus deforms subsiding down into micrometric grooves and forming different concavities and protrusions [35]. Notably, this capacity is independent of substrate chemistry and stiffness and correlates with the cytoskeletal organization of each cell type. Similarly, Nagayama et al. demonstrated that normal cells are not able to alter the shape of trapped nuclei to the same extent of cervical cancer HeLa cells and only transformed cells can replicate regardless of nuclear shape distortion [36]. Similarly, when cells are cultured on flat substrates with selective adhesion, actin filaments play an active role in nuclear shaping. Imposition of narrow cell shape induce nuclear stretching, lateral compression and significant loss of nuclear volume leading to DNA condensation [37]. It is evident that nuclear positioning has a huge impact on cell functionalities, sustaining also cellular maturation during differentiation [38]. Unbalancing the toolbox of proteins usually associated with nuclear stabilization and motility evidenced to trigger numerous diseases. Moreover, deformation of the nucleus during 3D migration is gathering increasing interest in the context of cancer metastasis, with the underlying hypothesis that a softer nucleus may aid to tumor spreading. The ability of cells to migrate through tissues and interstitial spaces could be easily probed by microfluidic devices composed of channels with precisely defined constrictions that mimic physiological environments. Intracellular mechanics and dynamics in physiologically relevant applications, ranging from cancer cell invasion to immune cell recruitment, can be studied integrating devices to study cell migration with high-resolution living imaging [39].
Dynamic biological stimulation through micro-devices Living tissues are continuously exposed to cyclic mechanical loads, which induce changes in their structure, composition, and function. For instance, endothelial cells forming the inner lining of the blood vessel, are www.sciencedirect.com
influenced by two distinct hemodynamic loads: cyclic strain due to vessel wall distension and shear stress due to the frictional forces generated by blood flow [40]. Muscle cells are also particularly responsive to mechanical forces; indeed, expression of skeletal muscle specific genes and skeletal muscle functional maturation, including sarcomeric organization and contractile structure development, can be modulated by applying an external mechanical stress. From a technological point of view cell-stretching devices have been developed to study cellular mechanoresponses in vitro. The most employed methodology is to use vacuum to actively deform a thin elastic membrane in which cells are cultured [41,42]. Beside the most common commercially available cell-stretching devices, a number of custom-made solutions have been proposed to perform mechanical stimulations on cell cultures in uniaxial and biaxial directions [43,44]. Many of them take advantage of microfluidic technology to achieve a better control of the strain induced to the cells while maintaining focal plane for in line detection. For instance, Shao et al. developed a cell stretching device combining dynamic directional cell stretch with in situ subcellular livecell imaging, demonstrating a rapid force-mediated focal adhesion assembly [45]. According to this design, several works reported on the role of cyclic stretch in myogenesis, tissue remodeling and sarcomere assembling in skeletal muscle [46]. Recently, we developed a microfluidic cell-stretching device to investigate the effect of cyclic mechanical stimulation on dystrophin-deficient human myotubes derived in vitro from healthy and diseased muscle biopsies [47] (Figure 3). Moreover, loading conditions can also play important roles in tissue and organ pathology, such as the processes of osteoporosis, atherosclerosis and fibrosis. In the cardiovascular field, increased mechanical stress is associated with cardiac dysfunction, such as myocardial hypertrophy, vessel wall thickening and fibrosis [12]. Cells including endothelial and smooth muscle cells experience cyclic circumferential stretch and compressive pressure generated by blood flow [11]. In this context, Zhou et al. observed activation of key components of SMAD1/SMAD2 and Wnt/b-catenin pathways in vascular cells precursors subjected to cyclic strains obtained through a microfluidic platform which simulates pulsatile blood flow [48]. Similarly, Suresh Babu et al. identified a stretch-induced signaling pathway in vascular cells involving the cytoskeletal transducer protein zyxin [49]. Moreover, microfluidics, characterized by laminar flow regime which allows local stimulation of cells with precise shear stress, is a perfect tool for studying vascular endothelium. Basically mechanical stimulus can be externally imposed by active force (shear stress, stretch, strain) controlling the flow rate or using pneumatic and electric systems [12]. Current Opinion in Chemical Engineering 2016, 11:67–76
74 Biological engineering
Mechanotransduction plays a critical role in cancer where cancerous cells are characterized by altered biophysical properties [50]. Microfluidic systems are particularly suited to investigate the role of interstitial flow (IF) due to elevated fluid pressure in tumor microenvironment, where cells exhibit altered biophysical properties and mobility. For instance, Polacheck et al. studied the effect of IF-induced fluid stresses imparted on cells embedded within collagen type I hydrogel [50]. Moreover, microfluidic tools can be successfully used to probe cell deformability and, consequently, for studying infiltration of immune cells and migration of metastatic cells into or from tumor, respectively [39]. Adamo et al. measured cell deformability and stiffness with a microfluidic device capable to resolve both cell travel time and relative cell diameter upon passage of cells through a micrometric constriction [51]. Byun et al. were able to identify the relative importance of deformability and surface friction between cell lines measuring the entry and transit velocity [52]. The use of microfluidic-based separation technique shows potential to conduct rapid and low cost cell analysis for disease diagnostics. An automatic micropipette that is capable to perform both deformation and relaxation measurements of individual cancer cells was recently proposed [53]. A further advancement in micropipette aspiration via microfluidic pipette array (mFPA) is able to conduct aspiration in a parallel manner [54]. The influence of cytoskeletal and nuclear structures on cell mechanical properties was demonstrated by the use of a micro-constriction array. While physical constrictions may not require specific substrate characteristics, the design of culture surface and its material is crucial in mechanotransduction studies. Independently from the chosen technology, substrate chemistry determines cell adhesion selectiveness depending on the cell type and strength at the cell– substrate interface. Non-fouling inert substrates (e.g. polyacrylamide and polyethyleneglycol-based hydrogels) offer a broad range in mechanical properties (e.g. stiffness) and have extensively been used as a mechano-assay with the aid of biochemical functionalization in order to attach ECM proteins and favor cell adhesion without compromising mechano-dependent cells responses. Biopolymer derivates (e.g. methacrylated gelatin and hyaluronic acid) are acquiring importance thanks to their biological activity and degradability for in vivo purposes [55]. Many of these materials are extremely versatile since they can be photo-activated and polymerized to topologically control cells within microdevices [56]. Beyond the modeling of bidimensional surfaces, 3-dimensional microengineering and mechanobiology are now Current Opinion in Chemical Engineering 2016, 11:67–76
gaining research focus thanks to new available technologies providing a more comprehensive definition of in vivo spatial organization and the opportunity to actively and dynamically perturb biological systems in real time [57].
Conclusions The knowledge of the molecular players and interactions between cell signals and different mechanotransduction pathways are still far to be completely understood. Manipulating the extracellular environment through microengineered features affects the spatio-temporal distribution and interactions of biochemical structures at the cell–substrate and cell–cell interface. It is tempting to speculate that control of cell shape and induction of particular phenotypes are the consequence of spatial (alignment, juxtaposition, zonation or exclusion) or conformational reorganization of mechanosensors that can interact with different players inside the cell. In this view, emerging microengineering-based opportunities and challenges in providing means for generating external mechanical stimuli affecting a living cell with accurate control of three-dimensional spatial and temporal patterns are of paramount importance. Moreover, performing live-cell imaging by integrating these technological platforms with new tools capable of applying and measuring forces and displacements with picoNewton and nanometer resolutions (e.g. atomic force microscopy, optical and magnetic tweezers, biomembrane and substrate force probes) will be fundamental for further advances in the field. Despite their importance, several limitations are involved in the choice of microtechnological strategies that could dissuade the final user. Microtechnological approaches are still often adopted only on a lab scale and may require specialized instrumentation and expertise in bioengineering design. Moreover, the biological processing with microtechnology is not conventional, since implicates mismatching with standard methods, loss of historical context, requires ad hoc standards and controls, and is more challenging to compare data with published results. Microfluidic devices work with nano or microliter volume and require specific knowledge and protocols that often are incompatible with the rapid processing of large sample volumes with pipettes. Miniaturization has also the effect to increase the surface to volume ratio that enhances the surface adsorption losses of molecules that could cause misunderstanding and wrong data interpretations. Despite these issues, new tools in microtechnology will represent a key aspect of future research in mechanotransduction. Engineering of culture substrates has push forward biological research for more than 10 years. New advancements in microengineering and their biological application will ensure even more emphasis and detail in mechano-biology. www.sciencedirect.com
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