In recent years, it has become increasingly evident that the cellular responses to microenvironmental signals go far beyond the chemical recognition through ligand-receptor interactions, and it encompasses a wide range of physical cues present at the adhesive interface between the cells and the surrounding ECM.37, 38 Cells respond to physical cues at the interfaces, such as the adhesive ligand density and pattern, ECM rigidity, and ECM dimensionality and anisotropy.39–43 In this Review, we introduce the biological effect of the topography and stiffness of the ECM.
In vivo, the basement membrane, composed of ECM components, is a complex network of pores, fibers, ridges, and other features of nanometer sized dimensions.44, 45 Topographical cues generated by the ECM, independent of biochemistry, have direct effects on cell behavior such as adhesion, migration, cytoskeletal arrangements, and differentiation.46–50 Cells are inherently sensitive to local microscale, mesoscale, and nanoscale topographic and molecular patterns in the ECM environment, a phenomenon called “contact guidance”.51–53 The development of microfluidics and micro-/nanofabrication methods to analyze the cellular response to substrate topography has provided new insights into the interactions of cells with their microenvironments.54–57
Grooves and pillars are the most common feature types employed in the study of the effects of surface structures on cells. The influence of groove patterns on the behavior of cell has been extensively investigated by using various cell types such as fibroblast,58 osteoblast,59 epithelial,60 myoblast,61 etc. A large number of studies revealed that cells tend to align to the long axis of the grooves.58, 60, 62 Kaiser et al. defined the role of groove/ridge dimensions on fibroblast cell migration.63 They found that surface structures significantly influenced cell orientation, migration direction, as well as migration speed in the directions parallel and perpendicular to the grooves/ridge in a surface structure dependent way (Figure 3A). Uttayarat et al. investigated the combination of flow shear stress and groove guidance on endothelial cell migration. When flow direction was oriented parallel to microgrooves, the cells migrated along the microgrooves. When microgrooves were oriented perpendicular to the flow, most cells migrated orthogonal to the grooves and downstream with the flow.64 Lee et al. reported that the nanoscale ridge/groove pattern arrays alone can effectively and rapidly induce the differentiation of human embryonic stem cells into a neuronal lineage without the use any differentiation-inducing agents, indicating the significant role of topography in determining cell fates.65 Apart from physical and chemical cues, the cell-cell interactions also influence cell behavior. We employed Madin–Darby canine kidney (MDCK), a cell line with relatively strong intercellular interactions, and NIH 3T3 fibroblast cells, a cell line with relatively weak intercellular interactions, to study the interplay between contact guidance and intercellular connections. The two types of cells were patterned onto polydimethylsiloxane (PDMS) substrates with microgrooves. Although MDCK cells migrate much more slowly than 3T3 cells on flat substrate, the velocity of migration of MDCK cells parallel to the grooves is higher than that of 3T3 cells perpendicular to the grooves and contact and form cell sheet (Figure 3B). Because MDCK cells have distinct group behavior, they contact each other very tightly, and the cell sheet acts as a barrier to prevent further migration of 3T3 cells. The 3T3 cell group could migrate only invade the space between them. After 72 h, MDCK cells had not contacted, but 3T3 cells had migrated around (Figure 3B). This experiment shows, for the first time, that both cell–cell and cell–substrate interactions simultaneously influent cell group behaviors.66
Figure 3. A) Migration speed of fibroblast cells in the directions parallel and perpendicular to the grooves/ridge in a surface structure dependent way (left: plane surface and right: structured suface). Reproduced with permission.63 Copyright 2006, Elsevier. B) Left panel: the MDCK cell matrix contacts and forms tight epithelial cell sheets, which trap 3T3 cells in the long narrow region. The 3T3 cell group only could migrate along the grooves. Right panel: the MDCK cell matrix did not contact before 3T3 cells drilled through. MDCK cells did not contact perpendicular to the grooves after 72 h. Reproduced with permission.66 C) Scatter graph of BLI values of recipient mice 1 month after transplantation with different numbers of Fluc MuSCs cultured for 7 days on either hydrogel (black) or plastic (red). Representative bioluminescence images of mice transplanted with each culture condition are shown. Reproduced with permission.41 Copyright 2010, American Association for the Advancement of Science. D) Scanning electron micrographs of hMSCs plated on PDMS micropost arrays of the indicated heights. Images at the bottom are magnifications of the boxed regions in the top images. Reproduced with permission.69 Copyright 2010, Nature Publishing Group.
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The influence of pillar patterns on the behavior of cells has also been extensively studied.67, 68 Mesenchymal stem cells (MSCs) preferentially differentiated and osteosarcoma cancer cells increased their malignant transformation due to the micropillar geometry.69, 70 In particular, increase of pillar heights from 1 to 10 μm affected the in vitro adhesion and guide morphology of fibroblasts by laminin expression enhancement.71 Furthermore, the spacing between 5 and 10 μm of pillars was shown to rearrange the actin cytoskeleton and govern fibroblast migration in vitro.72 Nanotopography alone can induce the differentiation of MSCs into neuronal lineage and induced a more significant upregulation of neuronal markers compared to microtopography, highlighting the importance of feature size in topography induced differentiation.73
Other micro-/nanosized features, such as nodes, pits, pores, and so forth have been reported to influence the behavior of cells.74, 75 The topography of the cell substratum plays an important role in regulating cellular behavior, and micro-/nanofabrication techniques provide useful tools for manipulating cells in both fundamental cell biology research and tissue engineering.
In tissues, adherent cells plus the ECM co-contribute to establish a relatively elastic microenvironment.76 Cells ranging from neurons to osteoblasts adhere, contract, and crawl within tissues where the stiffness of the ECM ranges from about 1 Kpa in brain to 100 Kpa in collageous bone.77 The stiffness of the ECM is known to impact on various cell activities from gene transcription, cytoskeleton remodeling, to cell-cell interactions.78–84 Most of the cells not only sense but also respond to the mechanical properties of the ECM by adjusting their focal adhesion structure, cytoskeleton organization, and overall state.83, 85–87 A study on how matrix stiffness couples with ligand density to modulate cellular responses suggested that substrate compliance and ligand density are orthogonal determinants.88 Stem cells that naturally reside in adult tissues exhibit robust regenerative capacity in vivo that is rapidly lost during in vitro culture. Engler and co-workers studied the effect of stiffness on MSC differentiation and indicated that soft matrices mimicking brain are neurogenic, stiffer matrices that mimic muscle are myogenic, and comparatively rigid matrices that mimic collagenous bone prove osteogenic.77 Gilbert et al. reported that muscle stem cells cultured on soft hydrogel substrates that mimic the elasticity of muscle (12 kPa) self-renew in vitro and contribute extensively to muscle regeneration when subsequently transplanted into mice (Figure 3C).41
Many studies used synthetic ECM analogs such as inert polyacrylamide gels in which the concentration of bis-acrylamide crosslinking sets the elasticity over several orders of magnitude, from extremely soft to stiff.89 The methods based on gels, however, have drawbacks, for example, altered cross-linker amount of synthetic gels, impacts not only bulk mechanics but also molecular-scale material properties including porosity, surface chemistry, backbone flexibility and binding properties of immobilized adhesive ligands.69 Fu et al. established a library of micromolded elastomeric micropost arrays to modulate substrate rigidity independently of effects on adhesive and other material surface properties. They demonstrated that micropost rigidity impacts cell morphology, focal adhesions, cytoskeletal contractility and stem cell differentiation (Figure 3D).69 Such studies provide evidence that by recapitulating physiological tissue rigidity, propagation of adult stem cells is possible, enabling future cell-based tissue repair and tissue engineering.