Vascular smooth muscle cell durotaxis depends on extracellular matrix composition Christopher D. Hartmana, Brett C. Isenberga, Samantha G. Chuaa, and Joyce Y. Wonga,1 aDepartment of Biomedical Engineering, Boston University, Boston, MA 02215 Edited by Robert Langer, Massachusetts Institute of Technology, Cambridge, MA, and approved August 9, 2016 (received for review July 14, 2016) Mechanical compliance has been demonstrated to be a key de- terminant of cell behavior, directing processes such as spreading, migration, and differentiation. Durotaxis, directional migration from softer to more stiff regions of a substrate, has been observed for a variety of cell types. Recent stiffness mapping experiments have shown that local changes in tissue stiffness in disease are often accompanied by an altered ECM composition in vivo. However, the importance of ECM composition in durotaxis has not yet been explored. To address this question, we have developed and charac- terized a polyacrylamide hydrogel culture platform featuring highly tunable gradients in mechanical stiffness. This feature, together with evidence that both stiffness and ECM composition can modulate VSMCs between a quiescent, contractile phenotype typical of healthy tissue and a synthetic, proliferative, and migratory phenotype observed in vascular disease (32–34). Fibronectin and laminin have opposing effects on VSMC phenotype, with fibronectin driving cells away from the contractile phenotype in vitro, whereas laminin has been shown to conserve it (35–38). A loss of laminin and an increase in fibronectin surrounding smooth muscle cells has been observed during the progression of neointima formation in vascular disease, suggesting these proteins may play an important role in regulation of cell phenotype in disease (39). Additionally, increasing stiffness has been shown to drive smooth muscle cells toward the synthetic, mi- gratory phenotype observed in atherosclerosis (40, 41), This evidence that VSMC phenotype is modulated by both matrix stiffness and composition, coupled with our previous observations of matrix type- dependent responses to stiffness by VSMCs, led us to hypothesize that the durotactic migratory behavior of VSMC s on substrates with mechanical gradients may also be differentially modulated by fi- bronectin and laminin. To test this hypothesis, we have developed a method of gener- ating polyacrylamide hydrogels with tunable gradients in substrate stiffness and independent control of matrix composition. Previously described methods to fabricate polyacrylamide mechanical gradient gels have been limited due to the difficulty of independently con- Fig. 1. Schematic of gradient generator device preparation and use. (A)Glass trolling the absolute stiffness range and gradient steepness (8, 14–16, slide coated in S1818 photoresist (orange). (B) Maskless lithography with visible 19, 42–44). However, by carefully controlling hydrogel geometry, light exposes device pattern onto photoresist-coated slide. (C) Photoresist re- cross-linker diffusion, and UV photopolymerization (Fig. 1), we are moved by development in TMAOH. (D) Exposed glass is coated with OTS to form a able to easily and independently adjust both absolute stiffness and hydrophobic barrier. (E) Remaining photoresist is removed with acetone. (F)Ex- gradient steepness in our system. Maskless lithography, which unlike posed glass is backfilled with hydrophilic and adhesive silane mixture (teal). μ traditional lithographic techniques does not require a custom-made, (G) The gradient generator device is assembled with 250- m Teflon spacers be- physical photomask and thus allows one to rapidly modify pattern tween the silane-coated generator slide and a top sacrificial slide coated in DCDM. Solutions of acrylamide solutions with high (blue) and low (white) concentrations dimensions with ease, is used to micropattern glass slides with hy- of bis-acrylamide cross-linker are injected into the reservoir regions through drophobic and hydrophilic silanes (45); this difference in hydro- 32-gauge needles. (H) The two acrylamide solutions meet in the middle of the phobicity gives the geometric constraint necessary for fine control device with their boundary constrained by silane patterning. The needles are re- over the location and degree of pregel solution mixing before po- moved and diffusion of cross-linker proceeds until (I) the acrylamide solutions are lymerization. Accordingly, a dumbbell- shape pattern was developed polymerized by exposure to UV light. (J) The sacrificial slide and spacers are re- in which the “weights” of the dumbbell consisted of large reservoir moved leaving a gradient hydrogel with a well-defined linear gradient in elasticity. regions filled with a high-concentration cross-linker solution on one side and low-concentration cross-linker solutions on the other (Fig. 1), and the “bar” of the dumbbell acted as a mixing region for cross- concentration of acrylamide monomer in solution and the extent linker diffusing between the two weights. After polymerization, the of cross-linking, which can be controlled by the concentration of resultant polyacrylamide gels were then functionalized with either bis-acrylamide cross-linker. In preliminary studies, we produced – SCIENCES fibronectin or laminin through an NHS–ester linker. The advantage uniform stiffness gels with 8 15% (wt/vol) acrylamide and bis- – of this design is that the cell movements observed in the constant acrylamide cross-linker concentrations ranging from 0.1 1%. We stiffness regions at the ends can serve as controls for cell migration found that after polymerization by photoinitiation, the elastic APPLIED BIOLOGICAL observed in the stiffness gradient region between them. Atomic modulus of the resulting gels was linearly proportional to the bis- force microscope nanoindentation of the end-product cell substrates acrylamide concentration over these ranges. We took advantage produced in this manner demonstrated the presence of linear and of this linear proportionality to generate hydrogels with linear highly reproducible mechanical gradients, and fluorescence imaging gradients in elastic modulus (Fig. 2). Briefly, the time allowed for demonstrated that homogeneous surface coatings of Alexa Fluor diffusion of cross-linker between reservoirs of high and low bis- 488-labeled fibronectin or laminin could be achieved. acrylamide concentration solutions was adjusted to yield linear SCIENCES We then used this system to directly compare the durotactic mi- gradients in bis-acrylamide across the region connecting the two APPLIED PHYSICAL gration of VSMCs on mechanical gradients coated with either fi- reservoirs. Nanoindentation was performed using an AFM in bronectin or laminin. The extent to which cells exhibited biased force contact mode to measure the stiffness of the gels as a migration in the direction of increasing substrate stiffness was com- function of position. It was determined that for gradient gels pared across all conditions by calculating a durotactic index, defined with 2- and 1-mm-long gradient regions, 40 and 10 min of diffu- as the length of migration in the direction of increasing substrate sion time, respectively, was sufficient to generate a linear gradient stiffness divided by the total migration path distance, for each cell in cross-linker concentration between the two reservoir regions. track. We found that cells seeded on fibronectin-coated gradient Results from the gels tested demonstrate that the gradient substrates exhibited durotactic behavior, whereas cells seeded on steepness can be changed both by altering the composition of the laminin-coated gradient substrates were capable of random move- pregel solutions while maintaining the same gradient geometry ment but did not exhibit durotaxis. This finding indicates that ECM and adjusting the gradient length. The stiffness gradients between composition is capable of modulating a cell’s motile response to the high- and low-stiffness regions (0.5–10 and 7–170 kPa, re- mechanical gradients. spectively, for the range of gel solutions tested) ranged from 2.9 to 142.6 kPa/mm (Fig. 2). Notably, an effective doubling of the Results gradient steepness was achieved by halving the distance between Generation and Characterization of Hydrogels with Defined Stiffness the reservoir regions, demonstrating that the stiffness gradient of Gradients. Polyacrylamide hydrogel stiffness is determined by the the gels can be tuned by adjusting the gel dimensions. Hartman et al. PNAS | October 4, 2016 | vol. 113 | no. 40 | 11191 Downloaded by guest on September 26, 2021 200 trajectory, where x is defined as parallel to the direction of in- creasing gradient stiffness (or a random direction in the case of 1mm Gradient 150 uniform controls), was calculated from migration tracks as a 142.6 kPa/mm function of time (Fig. 4). Cells on uniform stiffness control gels experienced an average x-directional displacement of zero as time 100 increased, with a wider range of displacements reflected in an increase in the SD of displacement as stiffness is increased. Cells 50 on gradient gels coated with fibronectin displayed a steady in- 18.6 kPa/mm crease in x-directional displacement as a function of time, reaching an average value of 37 μm after 18-h migration, whereas cells on 0 5.8 kPa/mm laminin-coated gradients migrated to an average displacement of 200 −2.63 μm after 18 h. Statistical testing of the displacements of cells at all time points by ANOVA and Tukey’s honest significant dif- 2mm Gradient