Biomimetic substrate control of cellular mechanotransduction
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Extracellular mechanophysical signals from both static substrate cue and dynamic mechanical loading have strong potential to regulate cell functions. Most of the studies have adopted either static or dynamic cue and shown that each cue can regulate cell adhesion, spreading, migration, proliferation, lineage commitment, and differentiation. However, there is limited information on the integrative control of cell functions by the static and dynamic mechanophysical signals. For example, a majority of dynamic loading studies have tested mechanical stimulation of cells utilizing cultures on flat surfaces without any surface modification. While these approaches have provided significant information on cell mechanotransduction, obtained outcomes may not correctly recapitulate complex cellular mechanosensing milieus in vivo. Several pioneering studies documented cellular response to mechanical stimulations upon cultures with biomimetic substrate modifications. In this min-review, we will highlight key findings on the integrative role of substrate cue (topographic, geometric, etc.) and mechanical stimulation (stretch, fluid shear) in modulating cell function and fate. The integrative approaches, though not fully established yet, will help properly understand cell mechanotransduction under biomimetic mechanophysical environments. This may further lead to advanced functional tissue engineering and regenerative medicine protocols.
KeywordsBiomimetic substrate Mechanical stimulation Mechanotransduction Functional tissue engineering
intracellular calcium concentration
atrial natriuretic factor
mesenchymal stem cell
phosphorylated focal adhesion kinase
small interference RNA
transcriptional co-activator with PDZ-binding motif
Mechanical loading plays a vital role in tissue homeostasis [1, 2]. Also for the regeneration of a more biomechanically-competent tissue constructs, physiologically relevant, controlled mechanical loading is critically needed. A wide variety of cell functions such as orientation, migration, proliferation, lineage commitment, and differentiation has been shown to respond to different modes of mechanical loading, as in our group’s reports [3, 4, 5, 6]. Many other studies have also reported that mechanical loading, such as stretch, fluid shear, compression, and others, could contribute to successful regeneration of mechanically functional tissues such as cardiac, muscle, vasculature, ligament, tendon, bone, and so on [7, 8, 9, 10, 11, 12]. Different loading mode can be a purpose-specific regulator of cellular systems, e.g., mechanical strain contributed to mesenchymal stem cell (MSC) differentiation into smooth muscle cells and chondrocytes [13, 14] while fluid shear stress could induce their differentiation towards endothelial cells . To take advantage of mechanical loading for the functional tissue engineering, several types of bioreactors have been developed that provide different loading modes such as shear flow, tension, torsion, or combination of these .
In addition to dynamic mechanical loading, static mechanophysical signals given by the cell culture substrates also have a strong potential to affect cell function and fate. It has long been established that changes in substrate topographic and geometric features (e.g., isotropic and anisotropic topographic patterns, micro and nanoscale surface patterning, etc.) can direct cellular adhesion, spreading, orientation, alignment, and migration, and via this affect downstream cell behaviors including cell survival and apoptosis, cell-cell interaction, lineage specification, and terminal differentiation (see more details in our previous review ). Significant developments in substrate fabrication techniques have allowed the investigation of cell behaviors on substrates with a more biomimetic characteristic. These include photo- and electron beam lithography, soft lithography, nanoimprint lithography, electrospinning, polymer demixing, 3D printing, etc. [17, 18, 19, 20, 21, 22].
Although each mechanical stimulation and substrate induction are well recognized as described above, little is known in regard to their integrative control of cellular functions. It is true that conventional cell mechanotransduction studies have dealt with cells cultured on plain surfaces, for example, mechanical stretching of cells seeded on elastic, flat membranes or fluid flow of cells seeded on glass slides. While these approaches provide advantages in assessing cellular mechanotransduction pathways via allowing easiness in imaging and RNA and protein sample collection, tests on simple flat surfaces would not necessarily recapitulate complex cellular mechanosensing environments in vivo, thus potentially depreciating the usefulness of the identified molecular mechanisms. Several studies reported pioneering data on cellular responses to mechanical stimulations upon cultures with biomimetic substrate modifications. In this mini-review, rather than in-depth technical or mathematical description of various mechanical cell stimulation methods or substrate modification techniques, we will highlight key findings on cellular responses to mechanical stimuli on biomimetically modified substrates. Specifically, how cell sensing of and response to mechanical stretch and fluid shear can be modulated via biomimetic substrate cultures will be focused. Understanding the crosstalk between engineered substrate and mechanical loading in affecting cellular mechanotransduction under correctly combined conditions could be of benefit for both biomaterials science and mechanobiology. This approach will further advance the theories and applications of functional tissue engineering and regenerative medicine.
Review: mechanical cell stimulation on biomimetic substrates
Mechanical stretching of cells on biomimetic substrates
Cells in vivo are often exposed to aligned extracellular matrix (ECM) architectures and respond to them by orienting and elongating themselves along the anisotropic matrix direction, i.e., contact guidance . Various synthetic ridge and groove topographies have been produced to mimic anisotropic in vivo architectures, and studies using these synthetic topographies demonstrated that contact-guided cell alignment could be replicated in vitro. On the mechanical loading side, studies have shown that in response to mechanical stretching the cells actually aligned perpendicular to the stretch direction [23, 24, 25]. A potential cellular mechanism of the perpendicular cell orientation to the stretch, e.g., to relieve cellular tension under stretch loading, is described in our review . Combining the two results, i.e., cell alignments along the groove direction and perpendicular to the stretch direction, it would be interesting to test how cells will be aligned under two superposed cues. The design will include the case in which the stretch is applied to the direction parallel or traverse to the anisotropic groove. For this, stretchable microgroove topographies were fabricated by using elastic substrates, e.g., custom-made silicone dishes [26, 27]. It was observed in these studies that cell alignment may be more affected by topographic guidance relative to stretch signal. When fibroblasts cultured on microgrooved substrates were subjected to cyclic uniaxial stretching, the cells did not alter their contact-guided alignment by the additional stretch cue regardless of the stretching direction. Another study also concluded that substrate control may play a primary role in cell shaping. In the study using two different stretchable topographies, a 10 μm wide square-groove and 40 μm wide V-groove, fibroblasts primarily adjusted their orientation according to the anisotropic substrates while stretching only played a secondary role .
In addition to microgrooved substrates, aligned electrospun nanofibers may also provide cell alignment signal . Utilizing this capability, cells seeded on nanofibers have also been tested for the stretch sensitivity [36, 37, 38]. The evolution of intracellular calcium concentration ([Ca2+]i), one of the markers of cellular mechano-responsiveness, was assessed for meniscus fibrochondrocytes (MFCs) cultured on aligned nanofibers and exposed to longitudinal stretch (along the aligned nanofibers) . The [Ca2+]i in response to stretch on aligned nanofibers was substantially different from that in the native meniscus tissue, e.g., significantly more frequent Ca2+ peaks on nanofibers than the native tissue. Further, taking advantage of nanofibers that can be used as tissue engineering scaffolds, co-control of MSC differentiation by substrate (nanofiber) and mechanical stretch was attempted . The differentiation of MSCs to ligament fibroblasts could be accomplished when MSCs were cultured on aligned nanofibers and co-stimulated with longitudinal stretching. However, MSCs seeded on random nanofibers failed to undergo such differentiation even in the presence of stretch.
Other than anisotropic substrate cues (grooves, lane micropatterns, aligned nanofibers, etc.) as described above, isotropically modified substrates have also been used for testing cellular sensitivity to the stretch signal. Isotropic substrate modifications, e.g., randomly or uniformly distributed topographic features (islands, pits, etc.) both at micro and nanoscale, have been widely utilized as another biomimetic platform for cell culture . However, only a few studies attempted their integration with mechanical stretch. For instance, a combined effect of uniformly distributed microisland surfaces and mechanical stretch on cellular neurogenesis was examined . Microisland textures were found to promote neurite outgrowth under low or static stretch condition, but interestingly the effect was decreased at high strains. In a study using randomly roughened stainless steel surfaces, cultured human MSCs could be exposed to mechanical forces via an electromagnet system that uses magnetic collagen-coated particles . MSCs cultured on rough surfaces showed a rapid upregulation in phosphorylated focal adhesion kinase (p-FAK at Tyr-397) by the mechanical stimuli, which was not observed on smooth surfaces. This suggests that FAK activation may be required for MSC mechanical sensing and functioning on metallic implants with rough surfaces.
Fluid shear stimulation of cells on biomimetic substrates
The potential of nanofibrous substrates to mimic ECM nanofilamentary architecture can be integrated with microfluidic platforms that can generate spatially and temporally defined flow microenvironments. The nanofiber-microfluidic integration may thus provide biomimetic cell growth environments required for regenerative medicine, as proposed and developed by Wallin et al. . Another study also developed a nanofiber-microfluidic device via which MSC responses seeded on aligned nanofibers could be examined at varying fluid flow directions (0°, 45°, 90°) to the aligned nanofibers . Their results suggested that MSC morphology and fate decision may depend on the fluid shear magnitude and direction to the aligned nanofibers. Specifically, when the fluid shear was perpendicular to the aligned nanofibers, it was conducive to MSC fibrochondrogenesis. On the other hand, the parallel flow allowed MSCs to show fibroblastic phenotype. In signaling pathway studies, RhoA kinase (ROCK) and yes-associated protein (YAP)/transcriptional co-activator with PDZ-binding motif (TAZ) were proposed to govern the nanofiber-fluid shear induction of MSC fibrochondrogenesis, since the differentiation was disrupted by Y-27632, a ROCK inhibitor, and the small interference RNA (siRNA) of YAP/TAZ.
Some studies on nanofiber-fluid shear combination reported potential cell detachment from the nanofibers under high shears. When the neurite outgrowth behavior of PC-12 cells was assessed using nanofibrous culture and fluid flow, higher shear stresses preferably enhanced cell alignment and thus neurite outgrowth but increased shear stress would sometimes result in the detachment of neuronal cells from nanofibers . In an endothelial cell culture on electrospun nanofibers and under fluid shear, cells cultured on aligned nanofibrous scaffolds had greater resistance to detachment compared with those on random nanofibers . Combined with this result, increased F-actin bundle formation and VE-cadherin expression by fluid shear on aligned nanofibers suggested that aligned topographical guidance could be an effective mean to enhance endothelial cell adhesion for functional vascular tissue engineering.
One recent study reported that MSC lineage specification could be governed by cellular contractile forces that are determined by topography-fluid shear cues . They utilized both anisotropic (gratings) and isotropic (wells) topographies. Human MSCs seeded on 1 μm wells showed higher cell contractility, and displayed under fluid shear osteogenesis. On the other hand, MSCs seeded on 2 μm gratings had lower contractility and remained multipotent even under fluid shear stimulation. Related focal adhesion formation was also changed, e.g., MSCs seeded on wells had focal adhesions with increased area and number. With an inhibition of actomyosin, MSC differentiation was not detected regardless of topographic or fluid shear stimulation, suggesting the potential role of topography-flow-induced cellular contractility in MSC fate determination.
Conclusions and perspective
All data taken together, cells may sense and respond to both substrate cues and mechanical stimuli in a simultaneous manner. Depending on the substrate cues, such as grooves and aligned nanofibers (anisotropic) or randomly/uniformly distributed topographic features (isotropic), cells display differential morphological adaptations (alignment, spreading, migration) and then altered downstream behaviors (growth, lineage commitment, differentiation). The studies highlighted in this article suggest a strong possibility that such cellular reactions to substrate cues could be modulated by external mechanical stimulations, stretch and fluid shear. Depending on the varying regimens of the mechanical stimuli (strain, shear stress, oscillatory or steady, etc.) and correlation with the substrate cue (e.g., direction/angle of stretch or flow), the mechanical stretch or fluid shear either synergistically or competitively regulated cellular responses. In addition to observations that cell-substrate interaction could be actively modulated by added mechanical stimuli, the integrative approaches using substrate-stretch and substrate-fluid shear will help correctly recapitulate complex cellular mechanosensing environments in vivo. This may thus provide significantly improved understanding of cellular mechanotransduction behaviors accounting biomimetic mechanophysical conditions.
On the other hand, with some limited number of reports on the substrate-mechanical integrative control, there still exist considerations to be addressed. First, a more extensive and systematic studies with using various substrate parameters and loading regimens are required. Currently, it is quite difficult to compare each data from different reports due to the wide varieties of substrate properties and loading conditions. The need becomes even more significant when considering the reports that the sensitivity of substrate-mechanical integrative control of cells may be highly dependent on the scale of substrate topographies and the level of mechanical forces from stretch and shear, as described above. Also, a consideration of the other loading mode, such as compression or impulsive pressurization, and the combinatory loadings thereof may help fully describe in vivo mechanical environments.
Technically, lacking information includes the exact quantification of the mechanical loading under the substrate-combined situations. For example, fluid shear will definitely alter from unperturbed laminar flows to more turbulent flows if applied on substrates with varying micro and nanotopographies. Also, depending on the properties of the topographic features (shape and modulus), local stain values at varying substrate topographic positons might be different to each other and from the apparently imposed macroscopic stains. Mechanical stretch of the substrates within the cell culture media will also give rise to fluid flows originally not planned. These changes have not been calculated yet, and their potential effects on cell behaviors not addressed either.
From the standpoints of mechanobiology and functional tissue engineering, perhaps the more important consideration may be how to regulate cellular mechanosensitivity in response to external mechanical loading. The topic of this review article, substrate-mechanical integrative control, may answer to the question. As hypothesized in our previous study , the question to be answered can be “Does specific substrate culture (topography, patterning, nanofiber, etc.) will increase cellular responsiveness to mechanical stimulations (stretch, fluid flow)?” and if so, “What are the specific substrate topographic/geometric cues or dimensions to induce such upregulation in cellular mechanosensing?” Furthermore, taking into account that conventional mechanotransduction pathway studies have only dealt with plain surface cultures, an important question will be “What are the molecular mechanosensors that govern the substrate-mechanical integrative control of cells?” Answering these questions will lead to a proper description of cells in vivo that are exposed to complex ECM-mechanical integrative conditions. This may then significantly help design advanced functional tissue engineering and regenerative medicine protocols.
We thank the funding support from NSF (DMR-1310534, CMMI-1463636), NIH (1R01HL125736-01; MPI: Kamenskiy/MacTaggart), and ONR (N000141410663) (all to Dzenis); NSF CAREER Award (1351570), Osteology Foundation Grant (12–006), NE DHHS Stem Cell Research Project (Stem Cell 2015–06), Nebraska Tobacco Settlement Biomedical Research Seed Grant, and Nebraska Research Initiative (all to Lim). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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