Cellular Durotaxis Revisited: Initial-Position-Dependent Determination

Sep 19, 2018 - Laboratory of Biomedical and Biophysical Chemistry, Institute for Materials Chemistry and Engineering, Kyushu University , 744 Moto-oka...
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Cellular Durotaxis Revisited: Initial-Position-Dependent Determination of the Threshold Stiffness Gradient to Induce Durotaxis Kousuke Moriyama and Satoru Kidoaki*

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Laboratory of Biomedical and Biophysical Chemistry, Institute for Materials Chemistry and Engineering, Kyushu University, 744 Moto-oka, Nishi ku, Fukuoka, Japan ABSTRACT: Directional cell movement from a softer to a stiffer region on a culture substrate with a stiffness gradient, socalled durotaxis, has attracted considerable interest in the field of mechanobiology. Although the strength of a stiffness gradient has been known to influence durotaxis, the precise manipulation of durotactic cells has not been established due to the limited knowledge available on how the threshold stiffness gradient (TG) for durotaxis is determined. In the present study, to clarify the principles for the manipulation of durotaxis, we focused on the absolute stiffness of the soft region and evaluated its effect on the determination of TG required to induce durotaxis. Microelastically patterned gels that differed with respect to both the absolute stiffness of the soft region and the strength of the stiffness gradient were photolithographically prepared using photo-cross-linkable gelatins, and the TG for mesenchymal stem cells (MSCs) was examined systematically for each stiffness value of the soft region. As a result, the TG values for soft regions with stiffnesses of 2.5, 5, and 10 kPa were 0.14, 1.0, and 1.4 kPa/μm, respectively, i.e., TG markedly increased with an increase in the absolute stiffness of the soft region. An analysis of the area and long-axis length for focal adhesions revealed that the adhesivity of MSCs was more stable on a stiffer soft region. These results suggested that the initial location of cells starting durotaxis plays an essential role in determining the TG values and furthermore that the relationship between the position-dependent TG and intrinsic stiffness gradient (IG) of the culture substrate should be carefully reconsidered for inducing durotaxis; IG must be higher than TG (IG ≥ TG). This principle provides a fundamental guide for designing biomaterials to manipulate cellular durotaxis.

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INTRODUCTION Directional cell movement, so-called cellular taxis, is an essential phenomenon in living organisms. Tactic movements are typically observed not only in normal physiological processes such as embryo development and wound healing1,2 but also in pathological conditions such as cancer metastasis.3 In general, the driving factors of taxis are asymmetric cues consisting of various kinds of tactic attractants or repellants in the extracellular environment such as gradients of chemicals, stiffness, electric current, light, gravity, and so forth. Among these inducers of taxis, a stiffness gradient in the cell culture matrix leads cells toward the stiffer region, which is known as durotaxis.4 Durotaxis is an important form of taxis for several developmental processes and the progression of pathological diseases in vivo.5−8 What conditions of stiffness gradients are required to induce durotaxis? In addition, what factors determine these required conditions? Since the first observation by Lo et al.,4 many studies have reported the conditions of stiffness gradients required to induce durotaxis as well as the biomaterials used, adhesion molecules on the substrate surface, absolute stiffness range, and cell type.9 Though the literature provides useful © XXXX American Chemical Society

information on the required stiffness gradient, there is some discrepancy among the reported values. For example, Wong et al. reported that the durotaxis of vascular smooth muscle cells (VSMCs) was induced on a stiffness gradient of 0.001 kPa/μm for a collagen-coated substrate.10 On the other hand, the biased migration of VSMCs was not observed on a larger gradient of 0.01 kPa/μm.11 In the case of fibroblast cells, durotaxis was strongly induced on a stiffness gradient of 0.8 kPa/μm on gelatinous gels, whereas biased migration was suppressed on a 2 kPa/μm gradient.12 These results suggest that some other factors contribute to durotaxis other than the strength of the stiffness gradient. Regarding this issue, an important consideration is the effect of the absolute basal stiffness in the softer region. For VSMCs, the absolute stiffness of the softer region was set at 2.5 kPa for Special Issue: Interfaces and Biology 1: Mechanobiology and Cryobiology Received: July 26, 2018 Revised: September 18, 2018 Published: September 19, 2018 A

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Langmuir Table 1. Elastic Conditions of Microelastically Patterned Gels soft region samples S2.5/G0.04 S2.5/G0.14 S2.5/G0.3 S2.5/G0.4 S5/G0.3 S5/G0.6 S5/G1.0 S5/G1.6 S10/G0.4 S10/G0.6 S10/G0.9 S10/G1.4

first irradiation time (s) 250

320

480

stiff region

Young’s modulus (kPa)

second irradiation time (s)

± ± ± ± ± ± ± ± ± ± ± ±

20 40 50 70 65 80 95 100 120 150 180 210

2.8 2.4 2.5 2.2 5.0 4.7 5.7 4.6 10 12 13 13

0.1 0.2 0.2 0.1 0.3 0.6 1.2 1.0 1.1 0.6 1.0 2.2

stiffness gradient Young’s modulus (kPa)

(kPa/μm)

(kPa/100 μm)a

(kPa/mm)a

± ± ± ± ± ± ± ± ± ± ± ±

0.04 0.14 0.3 0.4 0.3 0.6 1.0 1.6 0.4 0.6 0.9 1.4

4 14 30 40 30 60 100 160 40 60 90 140

40 140 300 400 300 600 1,000 1,600 400 600 900 1,400

4.8 9.5 17 22 20 35 59 83 31 43 58 83

0.4 0.7 1.5 2.8 1.2 3.7 7.8 11 2.4 3.8 4.4 8.0

a

Values were converted to other units for comparison to those in the literature.

which clarifies a design principle for the mechanical field of a biomaterial surface to manipulate cell movement.

0.001 kPa/μm (durotaxis occurs)10 and 30 kPa for 0.01 kPa/ μm (durotaxis does not occur),11 indicating that stiffer soft regions suppress durotaxis. A similar tendency was also observed for fibroblast cells, i.e., durotaxis occurs for a stiffness gradient of 0.8 kPa/μm with a 10 kPa soft region but does not occur for a stiffness gradient of 2 kPa/μm with a 200 kPa soft region.12 Although these facts suggest that the absolute stiffness of the softer region essentially affects the stiffness gradient required to induce durotaxis, there has been no systematic investigation of the effect of the basal stiffness in the softer region on the modulation of cellular durotaxis. In the present study, to systematically characterize the effect of the absolute stiffness of the soft region on the induction of durotaxis and to identify the conditions that determine the threshold stiffness gradient (TG) required to induce durotaxis, cell movements were investigated on hydrogels with various conditions of both the stiffness gradient (0.04−1.6 kPa/μm) and the absolute stiffness in the soft region (2.5−10 kPa) prepared by using custom-made mask-free photolithography for photo-cross-linkable gelatin.13−16 As the cells used in the investigation, mesenchymal stem cells (MSCs) were adopted from the view of functional variety and growing interest in the mechanoresponsivity of cells, such as in matrix-stiffnessdependent lineage control.17−20 The TG required to induce the durotaxis of MSCs was evaluated under different conditions of stiffness for the soft region. As a result, the TG was found to be 0.14 kPa/μm for a 2.5 kPa soft region, 1 kPa/ μm for a 5 kPa soft region, and 1.4 kPa/μm for a 10 kPa soft region; i.e., TG increased with an increase in the stiffness of the soft region. To identify possible molecular causes of the TG, the degree of maturation of focal adhesions (FAs) was evaluated on plain soft gels of 2.5−10 kPa, which reflects the adhesion strength of the cells to the matrix. The area and longaxis length of FAs on stiffer gels were significantly increased, indicating that these FAs were more stabilized. More mature adhesions with stiffer soft regions were considered to keep cells in the soft region more stably, which provides a rationale for the increase in TG. These findings provide a more general explanation for the TG value required to induce durotaxis depending on the absolute stiffness of the initial location of cells in durotaxis. On the basis of these observations, a fundamental relationship between the initial location-dependent TG values and the intrinsic stiffness gradient (IG) of the culture substrate for the induction of durotaxis is discussed,

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MATERIALS AND METHODS

Preparation of a Photocurable Gel Precursor Solution. Photocurable styrenated gelatin (StG) was synthesized as shown in a previous report.13−15 StG (30 wt %) and sulfonyl camphorquinone (SCQ, Toronto Research Chemicals, ON, Canada; 1.5 wt % gelatin) were dissolved in phosphate-buffered saline (PBS). The mixture was centrifuged (MX-301; TOMY, Tokyo, Japan) at 17 800g for 1 h to remove the insoluble colloidal coagulate, and the supernatant was stored at −80 °C. Before the preparation of a microelastically patterned gel, the sol solution was warmed to 45 °C. StG sol was aspirated for 20 min for degassing and conditioned using a deforming agitator (AR-100, THINKY, Tokyo, Japan). Finally, the StG sol was equilibrated in a nitrogen atmosphere for 30 min to exclude dissolved oxygen prior to photoirradiation. Photolithographic Microelasticity Patterning of a Gelatinous Gel. A vinylmethoxyxilane-modified glass (vinylated glass, 18 mm in diameter) was used for the substrate to fix the StG gel sample.13−15 The StG solution was spread between vinylated glass and normal glass (22 × 22 mm2) coated with poly(N-isopropylacylamide) (pNIPAAm, Sigma-Aldrich, St. Louis, MO, USA) as a sacrificial layer to promote the detachment of StG gel from the normal glass and then plated on a warm plate at 45 °C. First, to set the stiffness of the basal gel, the sample was irradiated by uniform visible light with an intensity of ca. 60 mW/cm2 at 488 nm (first irradiation, MME-250 light source; Moritex Saitama, Japan). The first irradiation time was varied from 250 to 480 s. Second, stiff regions were prepared by irradiation with patterned visible light (second irradiation, ca. 200 mW/cm2 at 488 nm) with square geometry (400 × 400 μm2) using a custom-built, mask-free photolithography system.13,21 The stiffness of stiff regions was also adjusted by varying the second irradiation time. The first and second irradiation times are summarized in Table 1. After photoirradiation, the gel samples were detached from normal glass and immersed in PBS with gentle shaking at room temperature overnight to remove adsorbed pNIPAAm. Measurement of the Surface Elasticity of an Elastically Patterned Gelatinous Gel. The distribution of the elastic modulus near the elastic boundary was examined by the nanoindentation method. Force−indentation curves were measured using an atomic force microscope (AFM, Nano Wizard 4, JPK Instruments, Germany). A tetrahedral cantilever (BioLever mini, Olympus, Japan) with a normal spring constant of 0.1 N/m was used. Young’s moduli were calculated from force−indentation curves by nonlinear least-squares fitting to the Hertz model in the case of a conical indenter (semivertical angle (α), 18°; Poisson ratio (μ), 0.5):22−24 B

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Langmuir F=

2E tan α 2 δ π(1 − μ2 )

stiffness gradient are ca. 2.5 kPa and ca. 0.04 kPa/μm, respectively. Figure 1 shows representative phase-contrast images of the fabricated gels with a microelastically patterned square stiff

Cell Culture. Human MSCs (Lonza, Inc., Switzerland) were expanded in MSC growth medium (MSCGM, Lonza). Cells were maintained on tissue culture polystyrene dishes at 37 °C in a humidified atmosphere containing 5% CO2. MSCs below passage 6 were used for all experiments. Time-Lapse Observations of Cell Migration. Cell migration on an elastically patterned gel was observed using a BZ-X700 all-in-one fluorescence microscope (Keyence, Osaka, Japan) at 37 °C in a humidified chamber containing 5% CO2 (Tokai Hit Co., Shizuoka, Japan). Prior to imaging, cells were seeded on gel at low density (1500 cells/cm2) to exclude the contribution of cell−cell interactions and cultured overnight in low-glucose Dulbecco’s Modified Eagle’s Medium (DMEM, Nacalai Tesque, Kyoto, Japan) supplemented with 10% fetal bovine serum (FBS, Gibco BRL, Grand Island, NY, USA), penicillin, and streptomycin in an atmosphere containing 5% CO2. Images were captured every 15 min for 24−48 h. Three or four gel samples with 12 square patterns per gel were used for each condition (N = 36−48). A total of 30 cells were randomly chosen and analyzed from 5 to 10 patterns per gel (n = 30). The coordinates and the moving trajectories of the cells were tracked manually using ImageJ software (NIH, USA). Immunofluorescence Staining of Vinculin. MSCs were cultured on gels of 2.5−10 kPa for 2 days and then fixed with 4% paraformaldehyde in PBS for 20 min. Fixed cells were permeabilized with 0.1% Triton-X100 (Sigma-Aldrich) containing 1% bovine serum albumin (BSA, Wako Pure Chemicals, Osaka, Japan) and 10% donkey serum (Millipore, MA, USA) for 1 h at room temperature. After being washed with 1% BSA, cells were incubated with 8 μg/mL rabbit antivinculin antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4 °C and then incubated with secondary antibody (Alexa 488-antirabbit IgG, Invitrogen, CA, USA) for 1 h at 37 °C. The samples were observed using a confocal laser scanning microscope (CLSM, LSM 510META MAITAI, Carl Zeiss, Germany) with a 40× oil-immersion objective lens. Focal Adhesion Analysis. CLSM images of FAs were quantitatively characterized by image analysis using MetaMorph version 7.6 software (Molecular Devices, Inc., San Jose, CA, USA). Raw 16-bit images were treated by flattening the background to reduce significant noise, and then threshold processing was performed at certain lower criteria to offset the constant background level. Finally, the area and long-axis length of FAs were measured by integrated morphometric analysis. FA areas smaller than 0.4 μm2 were excluded as small focal complexes and noise. These treatments were applied to CLSM images obtained under standardized constant parameters for confocal microscopy to enable the relative quantitative comparison of different images. Projected Cell Area. Mean projected cell areas were measured from phase-contrast images using ImageJ software (NIH, USA). At least 100 isolated cells were analyzed for each condition from 2 separate samples. Statistical Analysis. The statistical significance was evaluated by one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test using Kaleidagraph software (Synergy Software, Reading, PA, USA). p < 0.05 was considered to be statistically significant.

Figure 1. (Left) Phase-contrast images of representative microelastically patterned gels with elasticity conditions of S2.5/G0.4 (top), S5/G1.6 (middle), and S10/G1.4 (bottom). Scale bars: 200 μm. (Right) Distribution of Young’s moduli around the elastic boundaries measured along the white broken lines in the phase-contrast images. Light blue, gray, and red regions in graphs show soft, boundary, and stiff regions, respectively.

domain (Figure 1, left) and the distribution of the elastic modulus around the elasticity boundaries for all of the gel samples listed above (Figure 1, right). The width of the elasticity boundaries in the gels was adjusted to between 40 and 50 μm. The visibility of the elasticity boundary in phasecontrast images is attributable to the height differences between the stiff and soft regions depending on their different swelling states. The surface topography of the elasticity boundary may hinder natural durotaxis13 and thus should be evaluated carefully. To check this point, cross-sectional images of patterned gels stained with fluorescein-labeled albumin were obtained by confocal laser scanning microscopy (Figure 2).

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RESULTS Fabrication of Microelastically Patterned Gels with Soft Regions with Different Elastic Moduli. To evaluate the influence of the stiffness of the soft region on the durotaxis of MSCs, we prepared microelastically patterned gels with soft regions with different elastic moduli (2.5−10 kPa) and stiffness gradients (0.04−1.6 kPa/μm), as summarized in Table 1. In the following paragraphs, the nomenclature “S2.5/G0.04 gel” denotes a gel for which the elasticity of the soft region and the

Figure 2. Confocal laser scanning microscopic images around the elastic boundary. Scale bars are 25 μm. C

DOI: 10.1021/acs.langmuir.8b02529 Langmuir XXXX, XXX, XXX−XXX

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Langmuir Though the soft regions (Figure 2, left) were swollen compared to the stiff regions (Figure 2, right) for all the gels, in all cases the height differences between them were less than 25 μm and the soft and stiff regions were connected smoothly. We previously confirmed that smoothly connected elasticity boundaries without any valley/hill topography do not affect natural cellular migration even if the height differences are on the order of several tens of micrometers.15 Therefore, we could confirm that all of the elastically patterned gels fabricated in this study could be used to systematically evaluate the effect of the elasticities in Table 1 on durotaxis without the influence of the surface topography. In addition, concerning the influence of surface chemical conditions such as the difference in the amount of adsorbed adhesive proteins, which is one of the most common criticisms of cell characterization on an elastically patterned substrate, we confirmed that the amount of adsorbed fibronectin is almost the same between the stiff and soft regions in our patterned gelatinous gels.13 Soft-Region-Dependent Determination of the Threshold Stiffness Gradient for Inducing Durotaxis. MSCs were seeded on the above-fabricated microelastically patterned gels with different conditions of the elastic modulus in the soft regions (S2.5, S5, and S10). Cell migration was observed for 24−48 h, and the trajectories of cells close to the elasticity boundaries were monitored for 12 h. To clearly analyze the movement of each cell around the elasticity boundaries, the starting position of each trajectory was set at the origin of the graphs in Figures 3−5, in which the Y axis corresponds to the elasticity boundary with a stiff region of X > 0 (light red) and a soft region of X < 0 (light blue). Soft-region-dependent modulation of the induction of durotaxis was clearly reflected in the X−Y trajectories of MSCs, as shown in the left panels of Figures 3−5. On S2.5 gels, the G0.04 condition did not induce any biased migration (Figure 3a, left), while durotaxis was induced under conditions G0.14, G0.3, and G0.4 (Figure 3b−d, left). This indicates that TG should exist around G0.14 for an S2.5 base. The TG was markedly modulated depending on the condition for the soft region, which is around G1.0 for an S5 base and G1.4 for an S10 base (Figures 4 and 5, left). An increase in stiffness in the soft region tended to increase the TG for inducing durotaxis. To precisely evaluate the TG, the strength for inducing durotaxis was characterized in a time-course analysis of the X position of each cell (Figures 3−5, top right) and of the ensemble average and standard deviations (Figures 3−5, bottom right). On S2.5 gels, a significant bias in the average displacement of the X position was confirmed above G0.14 (Figure 3b−d, right). On S5 gels, a significant bias in the X average was clearly recognized above G1.0 (Figure 4c,d, right). Finally, on S10 gels, this bias was observed above G1.4 (Figure 5d, right). These results indicated that TG strongly depends on the absolute stiffness of the soft region. Interestingly, the TG value increased with an increase in the elastic modulus of the soft region. Characterization of the Adhesivity of MSCs on Plain Soft Gels. One of the major driving forces of durotaxis is the generation of cell polarity based on stiffness-gradient-induced asymmetric adhesivity between the anterior and posterior parts in single cells.4,13,25 When the anterior part entering a stiff region across an elasticity boundary is stabilized due to stronger adhesivity than in the posterior part of the softer region, the cell moves toward the stiff region. This means that the anterior/posterior relative adhesivity determines whether

Figure 3. Migration of MSCs on a microelastically patterned gel with a soft region of 2.5 kPa. (Left) Trajectories of each cell around the elasticity boundaries. (Right) Time-course analysis for (top) trajectories in the X direction of each cell and (bottom) ensembleaveraged X-direction trajectories with the standard deviation. The starting position was mapped at the origin of each graph. X > 0 (light red) and X < 0 (light blue) show stiff and soft regions, respectively. Analyzed numbers of cells and patterns: n = 30 and N = 36−48.

durotaxis occurs. On the other hand, the absolute adhesivity can be modulated depending on the matrix stiffness level; i.e., the greater the stiffness, the stronger the adhesivity.26,27 Thus, if the stiffness of the soft region increases, then the absolute adhesivity of cells on the soft region will also increase. How do the changes in absolute adhesivity in the soft region affect the change in the TG required to induce the durotaxis observed above? To evaluate the relationship between the adhesivity in the soft region and TG, the maturity of FAs and the projected cell area on S2.5−10 gels were characterized by immunofluorescence staining of vinculins and cell shape observation, and the speed of cell movement was measured from time-lapse observations. D

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Figure 4. Migration of MSCs on a microelastically patterned gel with a soft region of 5 kPa. (Left) Trajectories of each cell around the elasticity boundaries. (Right) Time-course analysis for (top) trajectories in the X direction of each cell and (bottom) ensembleaveraged X-direction trajectories with the standard deviation. The starting position was mapped at the origin of each graph. X > 0 (light red) and X < 0 (light blue) show stiff and soft regions, respectively. Analyzed numbers of cells and patterns: n = 30 and N = 36−48.

Figure 5. Migration of MSCs on a microelastically patterned gel with a soft region of 10 kPa. (Left) Trajectories of each cell around the elasticity boundaries. (Right) Time-course analysis for (top) trajectories in the X direction of each cell and (bottom) ensembleaveraged X-direction trajectories with the standard deviation. The starting position was mapped at the origin of each graph. X > 0 (light red) and X < 0 (light blue) show stiff and soft regions, respectively. Analyzed numbers of cells and patterns: n = 30 and N = 36−48.

Mature FAs were observed at the edges of cells on each gel, and larger FAs were formed on stiffer gels (Figure 6, left). To quantitatively analyze the maturity of FAs, the area and longaxis length of FAs were measured by image analysis and were confirmed to increase significantly as the stiffness of the plain soft gels increased from 2.5 to 10 kPa (Figure 6, right top and middle), indicating that MSCs adhere more stably to stiffer gels. The projected cell area increased with increasing stiffness of the plain soft gel (Figure 6, right bottom). The movement speed of MSCs decreased with increasing stiffness of the plain soft gel (Figure 7), suggesting that the adhesivity was strengthened. The decrease in the speed of cell movement generally means that the cell adheres to the substrate surface

with stronger adhesive force and needs a longer time to translocate their positions. Concerning the relationship between the speed of cell movement and cell adhesivity on an elastic substrate, two strong pieces of evidence have been reported: (1) When lamellipodial fluctuation is decreased on the stiffer gels under enhanced tyrosine phosphorylation of the focal adhesion proteins, the movement speed is suppressed because of the slower dynamics of the stabilized lammellipodia.28 (2) The adhesive force of cells was measured on StG gels with different stiffness values than in our previous study, which increased on the stiffer gels according to an intrinsic power law of adhesive force ∼ (elastic modulus)0.53.29 These results E

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Figure 8 shows that there is a roughly proportional relationship between the area of FAs for MSCs on the plain

Figure 8. Correlation between the mean area of FAs on plain soft gels and the TG required to induce the durotaxis of MSCs on gelatinous gels.

soft gel and the TG required to induce durotaxis. From the approximate straight line, the increment of the TG per unit area of FAs was estimated to be ca. 0.6 kPa/μm3 on gelatinous gels. This means that the TG can be predicted from the mean area of FAs on the soft region, while appropriately taking into account the composition of the culture substrate, which is critically affected by the absolute area of FAs.

Figure 6. Characterization of cell adhesivity on each soft gel. (Left) Confocal microscopic images of MSCs stained with vinculin on StG gels with S2.5−10 kPa. Scale bars are 10 μm. (Right) Quantification of the area (top) and long-axis length (middle) of FAs. FAs (200− 400) were measured under each condition. (Right bottom) Quantification of the projected cell area. More than 100 cells were measured under each condition. Error bars denote standard deviations. (*p < 0.05 and **p < 0.005.)

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DISCUSSION Although the strength of the stiffness gradient of a culture substrate has been recognized to be an important factor for inducing cellular durotaxis,11,13,30 it is still quite difficult to precisely manipulate the direction of movement and the local repositioning of durotactic cells by regulating only the stiffness gradient. The required condition for inducing durotaxis, i.e., the TG for inducing durotaxis, has been somewhat confusing and has remained unclear in previous studies, even for the same types of cells, as mentioned in the Introduction. Thus, the design criteria of the culture substrate for manipulating durotaxis have not yet been established. In the present study, to reveal the criteria for manipulating durotaxis, we focused on the effect of the absolute stiffness of the soft region on the TG for MSCs. The degree of such modulation of the TG was systematically clarified; the TG was substantially up-regulated (from 0.14 to 1.4 kPa/μm) as the stiffness of the soft region increased (from 2.5 to 10 kPa), which suggested that cell adhesivity in the soft region strongly contributed to the determination of the TG. On the basis of the obtained results, we should discuss the following three issues: (1) a mechanistic understanding of the effect of the soft region on the TG, (2) the principle that underlies the determination of TG in a stiffness gradient, and (3) the impact of the present finding for the manipulation of MSCs. Regarding the first issue, why does the TG used to induce durotaxis increase with an increase in the absolute stiffness of the soft region? As shown in the analysis of the maturity of FAs, the TG value exhibited an almost linear correlation with

Figure 7. Movement speed of MSCs on StG gels with S2.5−10 kPa, calculated from the total distance along each trajectory for 6 h for >60 cells (*p < 0.01).

demonstrated that MSCs interacted more strongly with the stiffer soft region. F

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Langmuir the area of FAs as an indicator of both their maturity and adhesivity in the cell−substrate interface; as adhesivity in the soft region increases, TG increases. This tendency is reasonable from the perspective of the work required for a single cell to detach from the soft region. In principle, for a cell to move from the soft region to the stiff region, i.e., durotaxis, the cell must detach from the soft region and then attach to the stiff region between the initial and final conditions, though of course the actual process proceeds gradually and sequentially. Here, the stronger adhesivity of the cell to the soft region should increase the work required for the cell to detach. The work of detachment can be determined from the area of cell adhesion that is approximately the summation of the area of all of the FAs in the adhesion interface, which is a positive value. On the other hand, the work required for attachment to the stiff region is also calculated from the area of the new adhesion interface, which is a negative value because of more stable adhesion on the stiffer substrate.26 Thus, the strength of inducing durotaxis is increased as the sum of these factors becomes increasingly negative. The TG used to induce durotaxis should reflect this strength, which can be called durotactic work. Actually, the cell adhesion area exhibits a significant dependency on the stiffness of the elastic substrate. The cell adhesion area becomes larger on the stiffer substrate,29 as also confirmed in the present study (Figure 6, bottom right). Therefore, the work of cell attachment in the stiffer region increases while the work of cell detachment in the softer region is smaller, which makes the durotactic work certainly more negative. Our present results indicate that durotactic work is sensitively affected by the adhesivity on the soft region in terms of the interfacial free energy. Concerning the second issue regarding the principle that underlies the determination of the TG, the soft region was clarified to strongly influence the durotactic work, which suggests, more generally, that the absolute stiffness at the initial location of an adhered cell starting durotaxis is critical. In our system, the initial location of the cells has a uniform stiffness (Figure 9a, left). On the other hand, many previous studies have used a culture substrate with a stiffness gradient (e.g., 10 kPa/millimeter) in which the cells are often located on a nonuniform field of stiffness at the initial location for starting durotaxis (Figure 9a, right).7,10,11,30,31 In the latter situation, the absolute stiffness felt by the cells starting durotactic movement is position-dependent and thus should vary depending on where the cell is located. This means that durotactic work and the TG required to induce durotaxis are also critically dependent on the position of the cell on a diffuse stiffness gradient. Durotactic work should differ for a single cell at different time points and positions as it moves along the stiffness gradient and also for different cells located in different regions along the gradient. For example, if we consider an MSC located in a region with a stiffness of between 2.5 and 10 kPa (Figure 9b, center), then a durotactic cell will require a TG of between 0.14 and 1.4 kPa/μm based on the obtained results (Figures 3 and 5). If the cell is positioned in a region with a stiffness of 10 kPa (Figure 9b, right), the TG is up-regulated to above 1.4 kPa/μm, suggesting that durotaxis should be suppressed as cells move up the slope against the stiffness gradient. On the basis of the above example, the essential conditions for durotaxis are now clear. For cellular durotaxis, the relation

Figure 9. Schematic representations of different gradient systems for durotaxis. (a) Starting positions of durotaxis are in the region of plain stiffness. (b) TG depending on the starting position in the stiffness gradient in the case of MSCs. (c) Different conditions of the relative strength of IG (blue arrow) and TG in inducing durotaxis (red arrows).

between the IG of the culture substrate and the TG that is specifically required at each initial location should be considered; i.e., IG must be higher than TG. To explain this principle, various conditions of their relative strengths are illustrated in Figure 9c, where red arrows indicate the TG at each initial location. IG is constant and fixed, while TG, indicated by red arrows, gradually increases along the stiffness gradient. Durotaxis occurs in cases 1−3 (IG ≥ TG) but not in cases 4 and 5 (IG < TG). Here, let us consider three different situations of cell location in terms of the relative strength of IG/TG (regions I−III in Figure 9c): In region I, IG > TG at all locations; region II includes locations where IG > TG, IG = TG, and TG < TG; and in region III, IG < TG at all locations. The durotactic behaviors observed in previous studies are categorized into these three kinds of situations. For example, in the case of VSMCs, durotaxis was induced for IG = 0.001 kPa/ μm and an absolute stiffness of from 2.5 to 11 kPa,10 which indicates that TG is below 0.001 kPa/μm; i.e., this situation is categorized as region I (Figure 9c, left). On the other hand, durotaxis was not observed for IG = 0.01 kPa/μm and an absolute stiffness of from 30 to 50 kPa,11 categorized as region III (Figure 9c, right). Finally, for MSCs, different induction efficiencies of durotaxis depending on the location on the substrate were observed for IG = 1 kPa/mm and an absolute stiffness in the range of 1 to 13 kPa (Figure 2 in ref 7), where the induction efficiency of durotaxis decreased with an increase in the absolute stiffness of the initial location, which is categorized as region II (Figure 9c, center). By carefully considering the TG for the absolute stiffness at the initial location of the cells, we see that durotaxis can be precisely controlled at will, which makes it possible to design highly functional biomaterials for manipulating the direction of movement and position of cells. Finally, with regard to the third issue, in the present study we clarified the design criteria for manipulating the durotaxis of MSCs. MSCs have attracted considerable attention in the field of mechanobiology because of their outstanding mechanoresponsive stiffness-dependent lineage specifications17−19 and the G

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Langmuir

StG, photocurable styrenated gelatin; S, soft region; G, stiffness gradient

memory of the mechanical conditions of the culture environment.20 The ability to manipulate the durotaxis of MSCs in response to the stiffness of the culture matrix may be useful for the design of biomaterials. When mechanoresponsive MSCs migrate along a certain stiffness gradient under durotaxis, the MSCs will sense the position- and time-dependent flow of mechanosignal input from the stiffness gradient. This means that durotactic MSCs can receive a kind of spatiotemporally programmed mechanosignal input from a well-designed field of stiffness; i.e., MSCs can read a time-course program of mechanosignal input carved on the surface of an elastic substrate. Not only a simple stiffness gradient but also microelastically patterned fields should be effective for controlling durotactic MSCs and repositioning them in regions with different elasticity, based on the precise criteria for the TG required to induce durotaxis revealed in this study.

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CONCLUSIONS In the present study, the dominant role of the absolute stiffness of the soft region for the determination of the TG required to induce cellular durotaxis was quantitatively clarified. An increase in the absolute stiffness of the soft region from 2.5 to 10 kPa markedly increased the TG from 0.14 to 1.4 kPa/μm for MSCs. On the basis of an analysis of the maturity of FAs on each soft region, this increase in the TG level was attributed to more stabilized adhesions in the stiffer soft region. The observed strong influences of the soft region led us to conclude that the initial-location-dependent determination of the TG governs the strength of durotactic work for different cells at different initial locations along an elasticity gradient. Therefore, for the design of stiffness-gradient biomaterials used to manipulate durotactic cells, the relationship between the IG of the materials and the position-dependent TG required to induce durotaxis has to be precisely evaluated; the former must exceed the latter (IG ≥ TG), which is the most precise criterion for inducing cellular durotaxis. This principle should provide an essential guide for a wide range of applications for manipulating cell movement and repositioning on the microelastically patterned field of a cell culture matrix.

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +81-92-8022507. Fax: +81-92-802-2509. ORCID

Satoru Kidoaki: 0000-0001-9138-0018 Author Contributions

S.K. designed the research, K.M. performed all of the experiments under the supervision of S.K, and S.K. and K.M. discussed the results and wrote the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by a grant for Core Research for Evolutionary Medical Science and Technology from the Japan Agency of Medical Research and Development (AMEDCREST, JP18gm0810002).

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ABBREVIATIONS MSCs, mesenchymal stem cells; TG, threshold of the stiffness gradient; IG, intrinsic stiffness gradient; FAs, focal adhesions; H

DOI: 10.1021/acs.langmuir.8b02529 Langmuir XXXX, XXX, XXX−XXX

Article

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DOI: 10.1021/acs.langmuir.8b02529 Langmuir XXXX, XXX, XXX−XXX