Abstract
1. Introduction
Designer substrates and devices have long been the heart of biomaterials, bioengineering, and a powerful tool for developmental biology, cell biology, biomedical studies. Especially, the high-cost of animal models and their concomitant long experimental cycle, ethical issues and poor reproducibility have cried out for in vitro substrates that mimic various in vivo conditions and tissue functions. The past decade has witnessed a surge in the interdisciplinary efforts in soft lithography, bio-inspired microfabrication, biochemistry and cell biology, allowing the design and production of sophisticated platforms that are capable to recapitulate natural contexts and elicit physiologically relevant events outside the body (Fig. 1). This emerging field that studies cell and tissue mechanics with biological and engineering approaches is coined as mechanobiology. Progress in this field has shed light on important biological processes of live systems at the interface of biology and artificial substrates, which are directly related to the design and advance of next generation biomaterials, biomedical devices, and large scale tissue engineering[
Figure 1.(Color online) Microengineered synthetic substrates for cell/tissue mechanics studies. The properties of a substratum can be modified to adjust the cell/material interactions, such as surface topographies, stiffness, and adhesiveness. In addition, mechanical probes can be integrated into the substrate to detect the force in tissue. These include microbeads in the traction force microscopy and elastomeric micro-pillars.
Live systems are made of cells that can support tissue development and homeostasis through processes such as self-replication, renewal, selective destruction and sensing of the microenvironment. These processes require the cells to actively react to the environmental inputs, while having sufficient mechanical stability to sustain shape and function, and at the same time adequate fluidity for remodeling. Supported by increasing evidences from reconstituted molecular systems and single cell studies, it is now known that these active properties can be attributed at the molecular level through ATP consumption and in particular, by the activity of cell cytoskeleton and molecular motors[
Such cell/tissue mechanosensing behaviors are influenced by a plethora of biochemical and biophysical cues[
In this review, we discuss how specific properties and functions of designer substrates/devices are achievable by various microfabrication methods and explain how underlying biological mechanisms due to cell-microenvironment interactions can be disclosed by these capabilities. In brief, we emphasize on semiconductor-based techniques that advance the study of cell/tissue mechanical responses to substrate adhesiveness, stiffness, topography, and shear flow. Moreover, we comment on the new concepts of measurement and paradigms for investigations of biological mechanotransduction that are yet to emerge due to on-going interdisciplinary efforts in the fields of mechanobiology and microengineering.
2. Engineering substrate adhesion
One important aspect of biologists’ concerns is the cell/substrate interface and their interaction. Attachment of cells to a surface via adhesion complexes (ACs, Fig. 2) provides important feedbacks that trigger a variety of signaling cascades and cellular behaviors. In vivo, cells take advantage of the heterogeneous distribution of extracellular matrix (ECM) cues to define many vital processes, such as wound healing[
Figure 2.(Color online) Molecular dynamics at adhesion complexes. The actin network as a mechanosensitive machine connecting the cell to its substrate and neighbors. The building of a stable focal adhesion (FA) complex for cell–substrate adhesion. Actomyosin forces apply on the FA at a fixed speed and the rate of force increase in the complex increases proportionally with the ECM stiffness. To avoid the destabilization and detachment of the FA, the binding-unbinding dynamics of the transmembrane protein, integrin, that connects the cells to the substrate needs to be equal to the force loading rate in the complex. Another force buffer and mechanosensor in the complex is Talin. Its unfolding at ~ 10 pN at the normal rate of force loading in cells lead to vinculin binding to recruit more actin fibers, thus reinforcing the FA.
Figure 3.(Color online) Methods for patterning adhesive surfaces. Semiconductor-based technologies has allowed the development of micro-contact printing and micro-stenciling for patterning biomolecules with define shapes. Later, researchers developed other techniques for this purpose, including Dip-pen lithography and UV-based patterning.
Patterning proteins is now made possible with multiple methods (Fig. 3) and has become a routine technique in many biological studies. As early as late 1990s, the Ingber group used μCP to constraint cells within arbitrary adhesive shapes containing fibronectin, a binding partner of integrin, and found that different areas of printing as well as its protein constituent results in dramatically modulation of cell growth and death[
3. Engineering substrate stiffness
The fact that FA growth is coupled to the cell–substrate interaction leads to the postulation that cells tune their contractility according to the substrate rigidity. In this context, a series of methods have been developed to adjust substrate stiffness. Conventional approaches involvethe modification of the cross-linking degree of the gels (Fig. 4), including polyacrylamide (PAA) and polydimethylsiloxane (PDMS). Typically, their Yong’s modulus can be changed over at least two orders of magnitudes, i.e., from 1 to 100 kPa, which is overlapping with the rigidity range of in vivo tissues[
Figure 4.(Color online) Methods for engineering substrate elasticity and viscosity. Conventionally, by controlling the cross-linking degree in elastomers, one could adjust the viscoelasticity of a gel. Another approach to change substrate rigidity involving photolithography is to pattern pillars of different shapes.
As discussed above, most types of cells response to the resistance via their attachment to a surface. Light resistance on a soft, flexible substrate will not stretch the force-bearing proteins, e.g., talin and vinculin, and leads to diffuse and dynamic ACs[
4. Engineering substrate topography
Many biological studies are still carried out on planar and featureless substrates, while perfectly flat surfaces do not usually exist in vivo. Instead, cells in physiological environment often experience complex 3D architectures and out-of-plane curvatures. To mimic in vivo situations and to study cell interactions with these features, cells are seeded on synthetic substrate harboring precise surface topographies. In general, photolithography is combined with etching techniques to create prescribed 3D topographic structures and later, functional substrates are produced using replica molding from masters[
Previous studies revealed that cellular FAs have a broad range of size from about 10 nm to 10 μm, suggesting cell may employ dynamic FA mechanism to sense the topographical cues. For instance, cell migratory dynamics changes on an array of 10 μm size micropillars, where the cells move slower with higher persistence in comparison with that on a flat surface[
Figure 5.(Color online) Topography cues influences cell adhesion and migration. (a) Scanning electron micrographs (SEMs) showing cells aligned to the nano-lines (Reproduced from Ref. [
5. Microfluidic chip
One major goal of bioengineering is to establish in vitro models that emulate the architecture, function, microenvironment, and physiological processes of living tissues. The aforementioned techniques show various advantages in understanding various mechano-properties of cells/tissues, but they also face limitations. Recently, with advanced microfabrication methods there is a surge in the integration of multiple moduli that provide controls to various biochemical/physical cues into a single microfluidic chip[
The fluid shear stress is due to the flow imposed on the tissues such as epithelial sheets that line ducts and endothelia that line the vessels. Epithelial cells sense fluid flow via their primary cilia – as the primary cilia are bended, the Ca2+-signaling pathway is elicited[
Figure 6.(Color online) Shear stress influences cell adherens junction (AJ) and filopodia protrusion. (a) At AJs, a higher force transmitted from F-actin caused by other factors (such as shear) leads to
Such active behaviors have been attributed to active cellular force distribution changes under static or flow conditions measured using traction force microscopy (TFM), micropillars and/or FRET bio-sensor techniques[
Moreover, cancer cell migration and invasion can also be stimulated by hydrodynamic currents in the form of lymphatic flows experienced by cancers cells that have spread into the lymphatic system, and interstitial flows between the ECM of tissues. It was found that wall shear stress few orders of magnitude lower than that experienced by ECs (~ 1 Pa for ECs) induced by flow on a dense but non-confluent layer of PC3 prostate cancer cells on the inner collagen-coated surfaces of a cylindrical PDMS tube, could stimulate Yes-associated protein (YAP) activation and increase filopodia protrusion and migration[
6. Outlook
The recent advancement of mechanobiology has been largely based on the rapid development of material microfabrication and engineering methods to recapitulate aspects of in vivo cell/tissue milieus. Microenvironmental factors, including the surface affinity to cells, substrate rigidity and topography, as well as shear flow have been found to contribute significantly for cell adhesion, migration, polarization, and differentiation. Our ability to independently control a range of parameters has offered unprecedented insight into cell/tissue mechanical properties. This is further combined with conventional cell biology approaches, which results in many discoveries in novel molecular pathways that are related to various mechanosensing mechanisms. The latest efforts in this field begin to focus on even more complex microenvironmental controls[
Acknowledgements
The authors thank T. B. Saw and C. T. Lim from the Mechanobiology Institute, Singapore and the members of CAML group at Institut Jacques Monod, Paris for fruitful discussions. Financial supports from the European Research Council (ERC) under the European Union’s Seventh Framework Programme (FP7/2007-2013) / ERC grant agreement numbers 617233 (B.L.), Marie Skłodowska-Curie Actions (W.X., Individual Fellowship, Project: 846449), the Groupama Foundation – Research Prize for Rare Diseases 2017 (to D.D), the Fondation pour la Recherche Médicale (FRM) (to D.D.), the LabEx “Who Am I?” #ANR-11-LABX-0071 and the Université de Paris IdEx #ANR-18-IDEX-0001 funded by the French Government through its “Investments for the Future” program (to D.D), and the Human Frontier Science Program (RGP0038/2018) (to D.D) are gratefully acknowledged.
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