Dynamic Mechano-Regulation of Myoblast Cells on Supramolecular Hydrogels Cross-Linked by Reversible Host-Guest Interactions AbstractA new class of supramolecular hydrogels, cross-linked by host-guest interactions between 尾-cyclodextrin (尾CD) and adamantane, were designed for the dynamic regulation of cell-substrate interactions. The initial substrate elasticity can be optimized by selecting the molar fraction of host- and guest monomers for the target cells. Moreover, owing to the reversible nature of host-guest interactions, the magnitude of softening and stiffening of the substrate can be modulated by varying the concentrations of free, competing host molecules (尾CD) in solutions. By changing the substrate elasticity at a desired time point, it is possible to switch the micromechanical environments of cells. We demonstrated that the Young鈥?/b>s modulus of our 鈥?/b>host-guest gels鈥?/b>, 4鈥?1鈥塳Pa, lies in an optimal range not only for static (ex situ) but also for dynamic (in situ) regulation of cell morphology and cytoskeletal ordering of myoblasts. Compared to other stimulus-responsive materials that can either change the elasticity only in one direction or rely on less biocompatible stimuli such as UV light and temperature change, our supramolecular hydrogel enables to reversibly apply mechanical cues to various cell types in vitro without interfering cell viability. IntroductionAmple experimental evidence suggested that biological cells do not only sense and respond to biochemical cues from the surrounding environment but also actively react to the mechanical properties (elasticity) of the extracellular microenvironments1,2. There are two signaling pathways regulating such mechano-sensing machineries. First, the clusters of integrin receptors, called focal adhesions, trigger the downstream cascades of intracellular signaling pathways, called 鈥渙utside-in鈥?signaling. This leads to mechanical force generation via contraction of actin-myosin (actomyosin) complexes3. The resistance of the extracellular matrix (ECM) against the traction force determines the connection between integrin clusters and actomyosin complexes, mediated by talin and vinculin. Second, the mechanical stimulation of actomyosin complexes by external cues can also cause the conformational change in the cytoplasmic domains of integrin and strengthen the binding to the extracelluar matrix, called as 鈥渋nside-out鈥?signaling4.To date, various ECM models based on chemically cross-linked hydrogels have been designed5 to investigate the regulatory principles of mechano-sensing. Through fine adjustment of cross-linker concentrations and the reaction time6,7, one can control the bulk elasticity (Young鈥檚 modulus) of a given gel substrate 鈥?i>ex situ鈥? Such materials have been used to gain insight into the vital role of elasticity compliance between cells and ECM in optimizing the cell morphology8,9,10, regulating the migratory behavior11,12, and controlling the stem cell differentiation13,14. On the other hand, many in vivo studies and experiments using organotypic cultures demonstrated that dynamic changes in the ECM stiffness influence various key functions of cells. For example, transplanted mesenchymal stem cells exhibit a remarkable enhancement of bone regeneration upon the degradation of soft alginate matrix15. It was also found that mesenchymal stem cells transplanted near the liver tissue did not adhere or repair the damaged tissue, but settled down in the periportal space16. Another medically relevant example is a clear correlation between the ECM density and the migration pattern of cancer cells17. These findings inspired the design of a new ECM model, whose mechanical properties can be altered in a time-dependent manner18.Recent studies in the field of supramolecular chemistry have shown that reversible bonds formed through specific intermolecular interactions can impart dynamic and adoptive properties to polymer materials19,20. To date, a number of polymer materials that respond to external stimuli such as light, redox reaction, pH level, heat, and electric/magnetic fields, have been designed to regulate their mechanical properties21,22. However, the external stimuli used in many cases, such as UV light23 and temperature change24,25, are cytotoxic and interfere with the cell viability. Several recent studies demonstrated that the thiolated hyaluronic acid26 and gelatin27 form chemical gels via disulfide bonds, whose Young鈥檚 modulus decreased by adding dithiothreitol. However, such materials have a fundamental drawback: the change in the substrate stiffness by the cleavage of disulfide bonds goes only in one direction. Once a bond is cleaved, it can hardly be recovered. Polymers increasing the density of covalent cross-links by external stimuli share the same problem. Once a covalent bond is formed, it is very hard to cleave it.Previously, we proposed the use of physically cross-linked hydrogels composed of an inter-connected micellar network of triblock copolymer chains possessing pH-responsive blocks at two ends. The elasticity of pH-responsive gels can be modulated by pH titration. The morphological dynamics and adhesion strength of several different cell types could be achieved28,29,30. However, although we observed no major interference with cell viability, a new class of hydrogel substrates that can reversibly alter the elasticity at pH 7.4 is more desirable for broader applications. In this study, we selected cyclodextrin (CD) as a milder chemical stimulus, which is a cyclic oligosaccharide that can act as the 鈥渉ost鈥?for different hydrophobic 鈥済uest鈥?molecules. Harada et al. have utilized host-guest interactions of CD, and designed a rich variety of hydrogels that are cross-linked with the host-guest interactions31,32. Since the association and dissociation of host-guest pairs can readily be achieved by adding either host or guest molecules in solutions, the host-guest polymer materials allow a number of applications33,34, such as stimuli-responsive actuators35 and self-healing materials31,36.We functionalized the side chain of acrylamide monomers with 尾-cyclodextrin (尾CD), and used them as the host. As the guest moiety, we selected adamantane, which fits well into the hollow cavity inside 尾CD. By optimizing the mixing ratio between pure acrylamide monomers (matrix) and acrylamide monomers modified with host/guest moieties, we fabricated hydrogels that possess the elastic modulus suited for the culture of myoblasts. This substrate material is advantageous over other commonly used hydrogels: First, the poylacrylamide backbones have widely been utilized for the culture of various cell types. Second, both 尾CD and adamantane are known to be non-cytotoxic37,38. This enabled us to use free cyclodextrin molecules as a molecular stimulus (competitor) that can fine-adjust the chemical equilibrium of cross-links and thus can reversibly alter the substrate elasticity without damaging cells. Details of the experimental findings are summarized in the following sections.Materials and MethodsMaterialsAcrylamide (AAm), CDCl3, and D2O were purchased from Wako Pure Chemical Industries, Ltd. 尾CD was obtained from Junsei Chemical Co., Ltd. Triethylamine (Et3N), sodium hydroxide, hydrochloric acid, ammonium peroxodisulfate (APS), N,N,N鈥?N鈥测€搕etramethylethylenediamine (TEMED), and N,N鈥测€搈ethylenebis(acrylamide) (MBAAm) were purchased from Nacalai Tesque Inc. Acryloyl chloride and vinyltrimethoxysilane were purchased from Tokyo Chemical Industry Co., Ltd. DMSO鈥?i>d6 was obtained from Merck Co., Inc. RPMI-1640 medium, fetal bovine serum, penicillin and streptomycin were purchased from Sigma-Aldrich, Co. LifeAct-TagGFP2 and the Torpedo庐 lipofection reagent were purchased from ibidi GmbH. A highly porous synthetic resin (DIAION HP鈥?0) used for column chromatography was purchased from Mitsubishi Chemical Co., Ltd. Water used for the preparation of the aqueous solutions was purified with a Millipore Integral MT system. Other reagents were used without further purification.Sample preparationRound cover glass (鈭?/span>鈥?鈥?5鈥塵m) were cleaned using a modified RCA method39. The substrates were immersed in a 5% (v/v) solution of vinyltrimethoxysilane in toluene and shaken for 18鈥塰 at room temperature. After the sequential rinsing in acetone, ethanol, and deionized water, the glass substrates were baked at 140鈥壜癈 for 1鈥塰 in air. The vinyl-silanized glass substrates were protected from UV irradiation until final use.尾CD鈥擜d gel (x,x) was prepared according to previous literatures32,40. In brief: 6鈥揳crylamido鈥撐睠D (尾CD鈥擜Am, x mol%) and adamantane-acrylamide (Ad鈥擜Am, x mol%) were mixed in water at 90鈥壜癈 in a silicone oil bath for 3鈥?鈥塰. After cooling, AAm (100鈥?x mol%) was added. Here, x represents mol% of 尾CD鈥揂Am and Ad鈥揂Am monomers. Cm represents the total concentration of 尾CD鈥擜Am, Ad鈥擜Am, and AAm. After dissolving all monomers, APS and TEMED were added, and a 50鈥壩糒 portion of the mixed solution was injected in a gap between a vinyl-silanized glass substrate and a hydrophilic, plasma-treated cover glass and kept for 15鈥塵in. After removing the cover glass, the sample was successively soaked in excess of DMSO/water (1:1, v/v) for 24鈥塰 and in water for 48鈥塰 to remove the residual chemicals. The surface of hydrogel substrates was functionalized with fibronectin via bifunctional cross-linker Sulfo-SANPAH (Thermo Scientific), following previous publications (Supporting InformationS1)41,42.Mechanical properties of host-guest gelsThe bulk elasticity of host-guest gels was measured by a Rheoner RE鈥?3005 creep meter (Yamaden Ltd., Tokyo). The elasticity of host-guest gels near the surface was determined by nano-indentation with an atomic force microscope (NanoWizard, JPK Instruments, Berlin). As reported previously, the density of hydrated polymers near the surface is diffusive and thus cannot be treated like a clear boundary with a Gaussian roughness43,44,45. In order to avoid the artifacts caused by indenting diffusive interface that has a larger length scale than the curvature radius of cantilevers (typically 20鈥?0鈥塶m), we indented the sample with a Pyrex-nitride spherical colloidal probe (R鈥?鈥?鈥壩糾) attached to a silicon-nitride cantilever with a nominal spring constant of 0.08鈥塏/m (CP-PNP-BSG; Olympus Optical). The Young鈥檚 modulus E of the gel was calculated from the nonlinear least-square fitting of the force-indentation curves46,47:$$F=4E{R}^{1/2}cdot {[3{(1-nu )}^{2}]}^{-1}cdot {delta }^{3/2}$$ where F is the force applied to the indenter, 谓鈥?鈥?.5 the Poisson鈥檚 ratio, and 未 the indentation depth48.Unless stated otherwise, all the data points are from more than three independent measurements, and the error bars in each figure correspond to the standard deviations.Cell cultureMouse myoblast cells (C2C12, 20 passages, RIKEN BRC Cell Bank) were cultured in RPMI-1640 medium supplemented with 10鈥墂t% of fetal bovine serum, 100鈥塙/mL penicillin and 100鈥壩糶/mL streptomycin. The cells were detached from the culture-flasks by enzymatic digestion using trypsin-EDTA (0.25%, Sigma), and 1鈥壝椻€?04 cells were seeded on the fibronectin-coated substrate 24鈥塰 before the observation. The actin filaments in live cells were visualized by transfecting C2C12 with LifeAct-GFP following the manufacturer鈥檚 protocol (ibidi GmbH). Prior to the experiments, we confirmed that free 尾CD molecules in solutions have no cytotoxicity by the WST assay (Supporting InformationS2).Morphology analysisCell dynamics on hydrogels were monitored by an inverted fluorescence microscope (Zeiss) and a Fast-Scan Confocal Fluorescence Microscope (Nikon, A1R). Morphological parameters, such as projected area A, aspect ratio AR (the ratio between major and minor axes), and circularity$$C=4pi A/{(perimeter)}^{2}$$ Of the adherent C2C12 cells were calculated using algorithms written in Matlab (Mathworks). The nematic order parameter of actin cytoskeletons S is calculated from the actin filaments as described before29,49,50. The original image was convoluted with the n eLoG kernels and the maximum response image was calculated for each pixel, as$${I}_{{rm{max }}}(x,y)=,max ,[eLoG(n)times I(x,y)]$$ Imax was processed by an intensity-threshold to obtain the image of the segmented stress fibers51, and the fibers with the same rotational direction with less than 10 pixels were removed. The order parameter$$langle Srangle =langle ,cos ,2theta rangle $$ was calculated from the histogram of fiber orientations that were scaled by the corresponding fluorescence intensities to account for the local concentration of actin filaments.Data availabilityAll data generated or analyzed during this study are included in this published article (and its Supplementary Information files).Results and DiscussionFigure1a shows preparation and chemical structure of the host-guest gel (尾CD鈥揂d gel (x,x)) used as cell culture substrates in this study. The gel was prepared by radical polymerization after dissolving the hydrophobic guest monomers (Ad鈥揂Am) by forming the inclusion complex with the host monomer (尾CD鈥揂Am). These monomers were terpolymerized with AAm in aqueous solution, forming a hydrogel. x represents mol% of 尾CD鈥揂Am and Ad鈥揂Am monomers in the monomer solution. Figure1b shows how the fraction of 尾CD鈥揂d cross-linkages can be modulated by the presence of free 尾CD molecules in aqueous solution. Free 尾CD molecules act as competitors and reduce the number of 尾CD side chains conjugated with Ad. As a consequence, one can fine-adjust the degree of cross-linking and thus the Young鈥檚 modulus E of gels by adjusting 尾CD concentration under equilibrium. It is notable that the gel can recover the original elasticity value once immersed into 尾CD-free solution.Figure 1Working principle of host-guest gel. (a) Preparation and chemical structure of 尾CD鈥揂d gel (x,x). x represents the mol% contents of 尾CD and Ad units. (b) Reversible switching of 尾CD鈥揂d bonds in response to free 尾CD molecules (purple) in solution. Under equilibrium, the fraction of 尾CD鈥揂d bonds connected to the polymer chains depends on 尾CD concentration, which allows for the dynamic modulation of Young鈥檚 modulus E.Full size imageFigure2a represents the influence of total concentration of monomers Cm on the optical transparency and the elastic modulus obtained by the compression test with a creep meter (Ebulk) at a constant fraction of host- and guest monomers (x鈥?鈥?鈥塵ol%). As presented in the figure, the sample seemed opaque at a low monomer concentration, Cm鈥?鈥?.0鈥塵ol鈥塳g鈭?. The increase in Cm to 2.0鈥塵ol鈥塳g鈭? led to a monotonic increase in the elastic modulus to Ebulk鈥?鈥?5鈥塳Pa, and the gel became transparent at Cm鈥夆墺鈥?.5鈥塵ol鈥塳g鈭?. Further increase in Cm did not result in any remarkable change in Ebulk. Previously, Horkay et al. reported that the saturation of Ebulk occurs at a volume fraction (蠁) of 蠁鈥夆増鈥?.2 in the case of poly(acrylamide)52. In our experimental system, Cm鈥?gt;鈥?.0鈥塵ol鈥塳g鈭? corresponds to 蠁鈥?gt;鈥?.2. Thus, we concluded Cm鈥?鈥?.0鈥塵ol鈥塳g鈭? is optimal for our purpose. Figure2b shows the influence of the molar fraction of host- and guest monomers x on Ebulk, while keeping Cm constant at 2.0鈥塵ol鈥塳g鈭?. A clear increase in Ebulk in accord with the increase in x from 1 to 3鈥塵ol%. However, we found that the gel becomes opaque and Ebulk took a much lower value at a high molar fraction, x鈥?gt;鈥?鈥塵ol%. Based on these two experimental findings, we kept Cm鈥?鈥?.0鈥塵ol鈥塳g鈭? and x鈥?鈥?鈥塵ol% in the following.Figure 2Macroscopic and microscopic gel assessment. (a) Young鈥檚 modulus Ebulk of 尾CD鈥揂d gel (2,2) vs. total monomer concentration Cm鈥?鈥?.0, 1.5, 2.0, 2.5, and 3.0鈥塵ol鈥塳g鈭?, measured by a creep meter. (b) Ebulk plotted as a function of the mol% content of 尾CD鈥揂Am and Ad鈥揂Am monomers in the monomer solution (x), while keeping Cm鈥?鈥?.0鈥塵ol路kg鈭? constant. (c) EAFM of 尾CD鈥揂d gel (2, 2) vs. 尾CD concentration, measured at RT. The change in elasticity shows a clear dependence on 尾CD concentration; ({rm{Delta }}{E}_{AFM}propto ,mathrm{log}[beta CD]). (d) Correlation between Ebulk measured by a creep meter and EAFM measured by AFM indentation. 尾CD鈥揂d gel (2,2) with Cm鈥?鈥?.0鈥塵ol鈥塳g鈭? was immersed into RPMI 1640 medium containing 0, 2.5, 5.0, 10, and 15鈥塵M of 尾CD. (e) Temperature dependence of EAFM in the presence and absence of 5鈥塵M 尾CD. (f) In situ modulation of EAFM at T鈥?鈥?7鈥壜癈. Reversible switching of the substrate elasticity was confirmed by alternatively changing 尾CD concentration in the solution between 0 and 2.5鈥塵M (鈼?/span>) as well as 0 and 10鈥塵M (鈻?.Full size imageTo examine if one can modulate the elasticity of host-guest gels by adding host molecules, we measured the Young鈥檚 modulus of hydrogel samples by two methods: Ebulk was obtained from the linear regime of macroscopic stress-strain curves, and EAFM was determined by AFM nano-indentation. As presented in Fig.2c, EAFM exhibited a remarkable decrease by increasing 尾CD concentration, following the change in chemical potential of 尾CD,$${E}_{AFM}propto ,mathrm{log}[beta CD]$$ Figure2d represents the plot of Ebulk vs. EAFM measured at different 尾CD concentrations. It should be noted that the Ebulk value remained stable over 24鈥塰 when a hydrogel substrate was immersed in the medium with a defined 尾CD concentration, confirming that Ebulk was measured under equilibrium. Excellent agreement between Ebulk and EAFM implies that free 尾CD molecules in solution can diffuse inside the hydrogel samples, resulting in the modulation of the Young鈥檚 modulus of the whole hydrogel substrates with the thickness up to 200鈥壜祄. In the next step, EAFM was measured in the absence and presence of 5鈥塵M 尾CD under different temperature conditions (Fig.2e). The elastic modulus exhibited a clear decrease both in the presence and absence of free 尾CD in solution at elevated temperature. This finding suggests that the gels consist of three-dimensional networks of high molecular weight polymers like rubber and thus the elasticity is dominated by entropy53. Figure2f represents the dynamic modulation of EAFM in response to the repetitive exchange of media with and without free 尾CD in solutions at T鈥?鈥?7鈥壜癈. Initially, the elasticity of hydrogel substrates in the absence of free 尾CD in solutions ([尾CD]鈥?鈥?鈥塵M) was EAFM鈥夆増鈥?1鈥塳Pa, and the exposure to the medium containing 2.5鈥塵M free 尾CD in the solution (鈼?/span>) led to an abrupt decrease in the elasticity, EAFM鈥夆増鈥?鈥塳Pa. After confirming the equilibration, the medium was exchanged to 尾CD-free medium, which resulted in a recovery of the elasticity to the initial level, EAFM鈥夆増鈥?1鈥塳Pa. Several cycles were repeated to guarantee that the modulation of the substrate elasticity is fully reversible. The experiments with 10鈥塵M 尾CD (鈻? also exhibited a fully reversible modulation of EAFM. As expected from the results presented in Fig.2d, we obtained a lower EAFM鈥夆増鈥?鈥塳Pa with 10鈥塵M 尾CD, demonstrating that the magnitude of switching can be adjusted by 尾CD concentration.Figure3a represents the maximum swelling ratio [螖d/d0]max plotted as a function of 尾CD concentration. The acquired data can be represented empirically by a single negative exponential fit (Fig.3a, solid line) with the characteristic concentration [尾CD]c鈥?鈥?0.3鈥塵M. The addition of 10鈥塵M 尾CD led to an increase in the film thickness from 72鈥壜祄 to 133鈥壜祄, resulting in the swelling ratio of about 0.9. The insets show two confocal images of the gel (side views), whose surfaces were functionalized with the fluorescently labeled fibronectin. Figure3b shows the dynamic change in the swelling ratio (鈭?i>d/d0) as a function of time under repetitive exchanges of the 尾CD-free medium and the media with [尾CD]鈥?鈥?鈥塵M (鈼?/span>), 10鈥塵M (鈻?, and 20鈥塵M (鈻?. The swelling ratio reaches to its saturation level in about 1鈥塰 after the medium exchange in both directions. The apparent difference between the change in EAFM and the change in swelling ratio can be attributed to the difference in the length scales between the AFM indentation ( 1鈥壩糾) and 鈭?i>d ( 50鈥壩糾) as reported previously54.Figure 3Reversible switching of thickness by medium exchange. (a) Maximum swelling ratio [螖d/d0]max plotted as a function of 尾CD concentration. The insets show two representative cross-sectional images of gels at [尾CD]鈥?鈥? and 10鈥塵M, reconstructed from the confocal fluorescence microscopy images. Here, fibronectin labeled with HiLyte Fluor鈩?488 was used to confirm the uniform functionalization of gel surfaces. The black line is the fit of the equation (f([{rm{beta }}mathrm{CD}])propto 1-exp (-[{rm{beta }}mathrm{CD}]/{[{rm{beta }}mathrm{CD}]}_{c})) (6) with the characteristic concentration [尾CD]c鈥?鈥?0.3鈥塵M. (b) Reversible switching of 螖d/d0 by repetitive exchanges of media with different 尾CD concentration; 6鈥塵M (鈼?/span>), 10鈥塵M (鈻?, and 20鈥塵M (鈻?. (c) The lateral displacement of fluorescent beads embedded in the gel caused by the incubation with 10鈥塵M 尾CD for 40鈥塵in. The magnitude of displacement is indicated by the color code, while the direction is indicated by the arrows. The histogram in the inset represents the distribution of displacements, implying that the lateral displacement is 鈮?鈥壜祄 for more than 90% of the beads.Full size imageBiological cells do not only deform the substrate by generating active traction forces55,56,57 but also react to the lateral strain of substrates58,59. Therefore, towards the dynamic regulation of cells, it is highly important to check if the swelling of hydrogels is isotropic. For this purpose, we embedded fluorescently labeled latex particles in the host-guest gel, and monitored the lateral displacement caused by swelling using confocal microscopy (Supporting InformationS4). Figure3c represents the lateral deformation of the gel analyzed by measuring the displacement of embedded fluorescent beads and using particle image velocimetry (PIV) after the incubation in the medium containing 10鈥塵M 尾CD. The magnitude of displacement is indicated by the color code, while arrows indicate the direction. Although the swelling of the film is 螖dmax鈥夆増鈥?0鈥壜祄, as shown in Fig.3a, the histogram of displacement (inset) implies that the lateral displacement is 鈮?鈥壜祄 for more than 90% of the beads. This finding confirms that exposure to 尾CD induces swelling of host-guest gels in the direction normal to the substrate but does not exert shear force on the cells caused by lateral deformation. As reported previously60, such an anisotropic swelling of hydrogels could be attributed to the physical constrain of the host-guest gel, whose bottom side is covalently anchored to the solid substrates.As the first step towards the mechanical regulation of cells using host-guest gel substrates, we monitored how C2C12 cells sense the substrate elasticity ex situ. Here, we seeded C2C12 myoblasts on four different substrates; glass (E鈥夆増鈥?0鈥塆Pa, control) and host-guest gel substrates equilibrated with [尾CD]鈥?鈥? (E鈥夆増鈥?1鈥塳Pa), 2.5 (E鈥夆増鈥?鈥塳Pa), and 10鈥塵M (E鈥夆増鈥?鈥塳Pa). Figure4a shows the representative confocal fluorescence images of C2C12 cells on four substrates, showing the overlay of vinculin (green), actin (red), and nuclei (blue). First, according to the increase in the substrate elasticity E, we found more concentrated focal adhesions and more pronounced actin stress fibers. Moreover, we found that the projected area of cells A monotonically increased according to the increase in E. The statistical reliability of the observed tendency was verified by taking the histogram from more than 23 cells for each substrate (Fig.4b), which can be well fitted with a log-normal function. As presented in Fig.4c, the dependence of projected area A on substrate elasticity E can be fitted by the empirical Hill equation (broken line):$$A(E)=frac{{A}_{max}}{({(frac{{E}_{frac{1}{2}}}{E})}^{m}+1)}+{A}_{min}.$$ Figure 4Ex situ mechano-sensing of C2C12. (a) Confocal fluorescence images of C2C12 cells on glass substrates (E鈥夆増鈥?0鈥塆Pa, control), and host-guest gel substrates with E鈥?鈥?1, 7, and 4鈥塳Pa. All the substrates were functionalized with fibronectin. Vinculin (green), actin (red), and nuclei (blue). (b) Histograms of the projected areas A of C2C12 cells at [尾CD]鈥?鈥? (green), 2.5 (red), and 10鈥塵M (blue), taken from a total number of n鈥?gt;鈥?50 cells. The distribution for each condition could be well fitted with a log-normal function. (c) Projected area as a function of the elastic modulus of the gel, fitted with the empirical Hill equation (broken line).Full size imageThe median area on the glass was taken as Amax鈥?鈥?217鈥壩糾2, while the median area on the softest substrate (E鈥夆増鈥?鈥塳Pa) was set as the minimum level Amin鈥?鈥?/sub>150鈥壩糾2. The half level, E1/2鈥夆増鈥?3鈥塳Pa, represents the characteristic E value for the cell spreading, while m鈥?鈥?/sub>2.2 is the cooperativity coefficient61. The obtained E1/2 value for C2C12 on host-guest gels seems to agree well with those of C2C12 on other materials reported previously, such as E1/2鈥夆増鈥?3鈥塳Pa on pH sensitive gels28 and E1/2鈥夆増鈥?.6鈥塳Pa on photo-crosslinked gelatin gels50. Actually, E1/2鈥夆増鈥?3鈥塳Pa agrees very well with the optimal elastic modulus of E鈥夆増鈥?2鈥塳Pa for actomyosin striations9 as well as for the stabilization of cardiac conduction62,63. Therefore, our host-guest gels are ideally suited to modulate the Young鈥檚 modulus of the substrates between the optimal level and the softer regime for myoblasts.In the next step, we focused on the dynamic, in situ response of C2C12 to the softening of the substrate elasticity from E鈥夆増鈥?1鈥塳Pa to 4鈥塳Pa by the transient transfection with LifeAct-GFP to visualize actin cytoskeletons. In Fig.5a, several representative snapshot images selected from the live cell imaging were presented. Initially, long actin cytoskeletons form bundles (stress fibers) near the cell periphery to maintain the tension on the substrate with E鈥夆増鈥?1鈥塳Pa (鈥渃ontractile鈥?state). Upon the exchange from 尾CD-free medium to medium containing 10鈥塵M of 尾CD at t鈥?鈥?0鈥塵in, the cell started changing its shape after some lag time (鈭?i>t鈥夆増鈥?0鈥塵in). As a result, the area A reached to the steady state at t鈥夆増鈥?0鈥塵in, where the cell took a round shape (鈥渞esting鈥?state). As shown in Fig.5b, the actin cytoskeletons became shorter and more isotropic during the transition from the 鈥渃ontractile鈥?to 鈥渞esting鈥?states. To gain further insights into the dynamic response, changes in the standard morphometric parameters were plotted as a function of time; projected area A (Fig.5c), aspect ratio AR (Fig.5d), and circularity C (Fig.5e). The projected area A starts monotonical decreasing after t鈥夆増鈥?0鈥塵in, which coincides with the first detachment of focal adhesions. The fact that A did not show a remarkable increase seems reasonable from the lack of lateral expansion by the addition of competitive 尾CD (Fig.3c). This time lag 鈭?i>t is slightly different between cells because of the stochasticity of the bond detachment under the softening of the substrates. On the other hand, the aspect ratio AR first exhibited an increase until t鈥夆増鈥?0鈥塵in, followed by an abrupt decrease. The apparent increase in AR can be attributed to the strong pinning of the focal adhesions (indicated by white circles in Fig.5a), which remained even after the detachment of some other focal contacts (indicated by white arrows in Fig.5a). Nevertheless, AR rapidly decreased once these contacts were detached, reaching to AR鈥夆増鈥? (round, resting cell). The circularity C remained almost constant at a low level (C鈥夆増鈥?.2) until AR started dropping rapidly. This tendency seems consistent with the fact that C2C12 did not exhibit remarkable protrusions (filipodia) even at the contractile state. The identification of actin filaments from the live cell images (Fig.5b) further allows for the calculation of nematic order parameter of actin filaments, (langle Srangle =langle ,cos ,2theta rangle ) (4), where 胃 is defined as an azimuth angle between the actin filament and the major axis of the cell. As presented in Fig.5f, the nematic order parameter S showed a high level ( S 鈥夆増 0.6) at E鈥夆増鈥?1鈥塳Pa, which agrees well with the previous account that reported the optimal striation of actomyosin complexes at E鈥夆増鈥?2鈥塳Pa9. As shown in Fig.5f, S started decreasing at a much later time point than the shape adaptation as previously reported64. In fact, the actin filaments first got shorter before changing their orientation from nematic to isotropic, corresponding the distinct delay before S started decaying. This finding seems to agree well with the previous accounts, reporting the more pronounced alignment of actin fibers along the major axis according to the increase in substrate elasticity, because the alignment of actin cytoskeletons and the formation of stress fibers are regulated by the force transmission via actomyosin complexes30,49,65.Figure 5In situ mechano-response of C2C12. Dynamic response of C2C12 myoblasts upon the softening of 尾CD鈥揂d gel (2,2) from E鈥夆増鈥?1 to 4鈥塳Pa, caused by the exchange to the medium containing 10鈥塵M of 尾CD. (a) Snapshot images of a C2C12 cell from the live cell imaging (LifeAct-GFP). (b) Actin cytoskeletons extracted from the images. Changes in (c) projected cell area A, (d) aspect ratio AR, (e) circularity C, and (f) nematic order parameter of actin cytoskeleton S are plotted over time. The medium was exchanged at t鈥?鈥?0鈥塵in, and medium containing 尾CD was continuously supplied in the shaded time zone.Full size imageHowever, in contrast to our previous study using pH responsive gels functionalized with physisorbed fibronectin29, we observed no discontinuous change in the order parameter (referred to the break of symmetry) during the mechano-response of C2C12. The different observations might be explained potentially by the following several reasons. First, the characteristic time for the elasticity change caused by the competitive host-guest interaction in this system is much longer than that of hydrogels that change the elasticity by pH change, following a clear difference in the diffusibility of 尾CD vs. H+. In case of 鈥渟oftening鈥? the change in Young鈥檚 modulus completed after 10鈥?0鈥塵in (Fig.2f). This partially overlaps with the time window of cellular response (the lag time 鈭?i>t鈥夆増鈥?0鈥塵in, the time to reach the equilibrium鈥夆増鈥?0鈥塵in). This might cause a more progressive change in the cytoskeletal ordering. Another scenario is the magnitude of the change in substrate elasticity. In our previous system, the substrate elasticity was modulated from 40鈥塳Pa to 2鈥塳Pa, which covers a critical substrate elasticity proposed recently by Yip et al.; 20鈥塳Pa66. In this account, it has been suggested that mouse embryonic fibroblast remodels the actin cytoskeletons and retains the substrate strain at a constant level when the Young鈥檚 modulus of the substrate was below 20鈥塳Pa. On the other hand, they observed the conservation of stress when the Young鈥檚 modulus was above 20鈥塳Pa, which agrees well with the theoretical prediction49,67. Although these studies dealt with different cells (fibroblast and mesencymal stem cells), it is plausible that the critical substrate elasticity at which C2C12 myoblast switches the mechano-response may exist around this level. Actually, the conduction of cardiomyocyte tissues was significantly promoted near 13鈥塳Pa62. Further studies with substrates with higher elastic modulus (e.g. 鈮?0鈥塳Pa, Fig.2b) would enable us to determine the critical elasticity jump that causes the symmetry break of the nematic order parameter of actin filaments.Figure6a represents a series of phase contrast images of C2C12 that underwent the reversible switching of its morphology under a medium exchange cycle. Initially, cells were seeded in 尾CD-free medium. At t鈥?鈥?鈥塰 the medium was exchanged to the one containing 5鈥塵M of 尾CD, and the cell was kept under this condition for 3鈥塰. After confirming the equilibration of the system, the medium was exchanged back to the original 尾CD-free medium. The projected area A of C2C12 was plotted as a function of time in Fig.6b. The exchange from 尾CD-free to 尾CD-containing medium (corresponding to E from 11鈥塳Pa to 6鈥塳Pa) led to a monotonic decrease in A after a short lag time (鈭?i>t鈥?鈥?0鈥?0鈥塵in), which is comparable to that presented in Fig.5. The saturation level of projected area (A鈥夆増鈥?00鈥壜祄2) seems to agree well with the one obtained from the ex situ experiments, A ex situ 鈥?鈥?40鈥壜扁€?0鈥壜祄2 (Fig.4c), confirming that the system reached to equilibrium. At t鈥?鈥?鈥塰, the medium was exchanged back to 尾CD-free medium. Compared to the 鈥渟oftening鈥?of the host-guest gel substrate, the cellular response to the substrate 鈥渟tiffening鈥?was found to be much slower. As presented in the figure, the lag time before the visible response was almost 3鈥塰. Such an extremely long lag time here could be explained by the combination of two factors. First, as presented in Figs2f and 3b, the characteristic time required for the stiffening of host-guest gels is about 1鈥?.5鈥塰, which is in contrast to the softening that is completed after 10鈥?0鈥塵in. Moreover, it is plausible that the spreading of cells requires more time than the contraction. The spreading of cells requires both the establishment of focal contacts and the propagation of spreading fronts. In contrast, the contraction is dominated by cortical tension and the kinetics of actin depolymerization once the focal contacts are cancelled. In fact, the treatment of C2C12 on a stiff substrate (40鈥塳Pa) resulted in a fast contraction, while leaving the focal contacts behind64. Nevertheless, the projected area A increased to 600鈥?00鈥壜祄2, which is comparable to the initial level (A鈥夆増鈥?00鈥壜祄2 at t鈥?鈥?鈥塰) and the ex situ, equilibrium level, A ex situ 鈥?鈥?20鈥壜扁€?0鈥壜祄2 (Fig.4c). Previously, 尾CD has been used to deplete the cholesterol and influence the integrin clustering in heat-shocked, Drosophila cells68. In this study, we performed control experiments on fibronectin-coated glass substrates (Fig.S2) and polyacrylamide containing no host-guest side chains (Fig.S4), verifying that the presence of 尾CD does not cause major change in cell shape, adhesion area and cytoskeletal order parameter. Recent studies evidenced that not only the substrate elasticity but also the viscosity influences the dynamic cellular response, such as cell spreading and proliferation69,70. The combination with rheological study would enable the optimization of the time windows to investigate the interplay of elasticity and viscosity of substrates.Figure 6Reversible switching of cell morphology by mechanical commands. (a) Some representative phase contrast images are presented in subpanels A 鈥?G. C2C12 cells were exposed to medium containing 5鈥塵M of 尾CD from t鈥?鈥? to 5鈥塰 (shaded time zone). (b) Projected area A of C2C12 plotted over time. The response of C2C12 to the substrate 鈥渟oftening鈥?from E鈥夆増鈥?1 to 6鈥塳Pa occurred much faster than the reverse reaction. The lag time before the cell started responding was also found to be much shorter for the softening. (See Supporting Information Fig.S2 for other examples.)Full size imageConclusionsIn this study, we utilized a new class of hydrogel materials, cross-linked by host-guest interactions, and demonstrated the potential for the dynamic regulation of cell-substrate interactions. Taking the advantage of reversible, supramolecular cross-linkers based on 尾CD and adamantane, the substrate elasticity can be modulated by exchanging the culture medium with and without free host molecules (尾CD). As the modulation of magnitude and direction of the substrate elasticity can be achieved by varying the concentrations of free 尾CD in solutions, such 鈥渉ost-guest gels鈥?enable one to apply dynamic mechanical stresses to cells by altering the substrate elasticity at a desired time point. In this study, we fabricated a host-guest gel, whose elastic modulus can be adjusted between 4 and 11鈥塳Pa. In the first step, we systematically investigated the influence of monomer ratios between host, guest, and matrix monomers on the substrate elasticity. Second, we demonstrated that the elasticity of host-guest gels depends on free 尾CD concentrations. The projected area of C2C12 on substrates exhibited a clear correlation with the substrate elasticity, and the fitting of the experimental results with the empirical Hill equation indicated that the elasticity of our host-guest gel covers the optimal range for regulating myoblasts. Such ex situ experiments under static conditions were further extended to monitor the dynamic, in situ response of cells to the change in substrate elasticity. In addition to the commonly used morphological parameters such as aspect ratio and circularity, we tracked the dynamic remodeling of cytoskeletons by calculating the nematic order parameter S of actin cytoskeletons from live cell images. The softening of substrates led to the detachment of focal adhesion contacts, followed by the morphological change from a stretched, contractile shape to a round shape, reflecting the depolymerization and disordering of actin cytoskeletons. Furthermore, we confirmed such a dynamic mechano-response of C2C12 is fully reversible under softening and stiffening of the gel substrate. Supramolecular hydrogels based on host-guest cross-linkers provide with a major advantage over recently reported materials that can either change the substrate elasticity only in a unidirectional manner or utilize more stressful cues for cells, such as temperature and UV light. 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Mater. 15, 326鈥?34 (2015).ADS聽 Article聽 PubMed聽 PubMed Central聽Google Scholar聽 Download referencesAcknowledgementsThis work was supported by MEXT (No. 26103521 to M.T.), JSPS (No. 26247070 to M.T., 26800223 and 16K05515 to A.Y., 16809998 and 16K14065 to M.N.), EU FP7 (ActiveSoft to M.T.), German Science Foundation (SFB873 to M.T.). We are thankful to the German-Japanese HeKKSaGOn Alliance for support. M.T. thanks T. Ichikawa, N. Kioka and M. Kengaku for insightful comments on the mechano-sensing of biological cells. P.L. thanks the HeKKSaGOn Program for the fellowship. M.T. is an investigator of German Cluster of Excellence 鈥淐ell Network鈥? WPI iCeMS supported by World Premier International Research Center Initiative (WPI), MEXT (Japan).Author informationAuthor notesMarcel H枚rningPresent address: Institute of Biomaterials and Biomolecular Systems (IBBS), University of Stuttgart, 70569, Stuttgart, GermanyMasaki NakahataPresent address: Division of Chemical Engineering, Department of Materials Engineering Science, Graduate School of Engineering Science, Osaka University, 1-3 Machikaneyama-cho, Toyonaka, Osaka, 560-8531, JapanMarcel H枚rning and Masaki Nakahata contributed equally to this work.AffiliationsInstitute for Integrated Cell-Material Science (WPI iCeMS), Kyoto University, Kyoto, 606-8501, JapanMarcel H枚rning,聽Akihisa Yamamoto聽 聽Motomu TanakaProject Research Center for Fundamental Sciences, Graduate School of Science, Osaka University, 1-1 Machikaneyama-cho, Toyonaka, Osaka, 560-0043, JapanMasaki Nakahata聽 聽Akira HaradaPhysical Chemistry of Biosystems, University of Heidelberg, D69120, Heidelberg, GermanyPhilipp Linke,聽Mariam Veschgini,聽Stefan Kaufmann聽 聽Motomu TanakaDepartment of Macromolecular Science, Graduate School of Science, Osaka University, 1-1 Machikaneyama-cho, Toyonaka, Osaka, 560-0043, JapanYoshinori TakashimaAuthorsMarcel H枚rningView author publicationsYou can also search for this author in PubMed聽Google ScholarMasaki NakahataView author publicationsYou can also search for this author in PubMed聽Google ScholarPhilipp LinkeView author publicationsYou can also search for this author in PubMed聽Google ScholarAkihisa YamamotoView author publicationsYou can also search for this author in PubMed聽Google ScholarMariam VeschginiView author publicationsYou can also search for this author in PubMed聽Google ScholarStefan KaufmannView author publicationsYou can also search for this author in PubMed聽Google ScholarYoshinori TakashimaView author publicationsYou can also search for this author in PubMed聽Google ScholarAkira HaradaView author publicationsYou can also search for this author in PubMed聽Google ScholarMotomu TanakaView author publicationsYou can also search for this author in PubMed聽Google ScholarContributionsM.T. and A.H. designed and directed the research. M.N. performed material synthesis and characterization, and M.H., P.L., and A.Y. performed biophysical experiments. M.V., S.K., and Y.T. contributed to the interpretation of results and reviewed the manuscript. M.H., M.N., P.L., and M.T. wrote the paper.Corresponding authorsCorrespondence to Akira Harada or Motomu Tanaka.Ethics declarations Competing Interests The authors declare that they have no competing interests. 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If material is not included in the article鈥檚 Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Reprints and PermissionsAbout this articleCite this articleH枚rning, M., Nakahata, M., Linke, P. et al. Dynamic Mechano-Regulation of Myoblast Cells on Supramolecular Hydrogels Cross-Linked by Reversible Host-Guest Interactions. Sci Rep 7, 7660 (2017). https://doi.org/10.1038/s41598-017-07934-xDownload citationReceived: 18 April 2017Accepted: 05 July 2017Published: 09 August 2017DOI: https://doi.org/10.1038/s41598-017-07934-x CommentsBy submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate. 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