SUPPLEMENTARY INFORMATION

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1 Cell-mediated fibre recruitment drives extracellular matrix mechanosensing in engineered fibrillar microenvironments Brendon M. Baker 1,2,*+, Britta Trappmann 1,2,*, William Y. Wang 1, Mahmut S. Sakar 3, Iris L. Kim 4, Vivek B. Shenoy 5, Jason A. Burdick 4, Christopher S. Chen 1,2,+ 1 Tissue Microfabrication Lab Department of Biomedical Engineering Boston University Boston, MA Wyss Institute for Biologically Inspired Engineering Center for Life Science Boston Building, 5th Floor 3 Blackfan Circle Boston, MA Institute of Robotics and Intelligent Systems Eidgenössische Technische Hochschule Zürich CH-8092 Zürich, Switzerland 4 Department of Bioengineering University of Pennsylvania Philadelphia, PA Department of Material Science and Engineering University of Pennsylvania Philadelphia, PA * authors contributed equally to this work + Corresponding Authors: Christopher S. Chen, M.D., Ph.D. Department of Biomedical Engineering, Boston University SLB201, 36 Cummington Mall Boston, MA Phone: (617) Fax: (617) cschen@bu.edu Brendon M. Baker, Ph.D. Department of Biomedical Engineering, Boston University SLB304, 36 Cummington Mall Boston, MA Phone: (617) Fax: (617) bambren@bu.edu NATURE MATERIALS 1

2 Supplementary Figures and Legends Supplementary Figure 1: 1 H NMR spectrum (D 2 O) of methacrylated dextran (DexMA). DexMA was characterized by 1H-NMR and the degree of functionalization was calculated as the ratio of the averaged methacrylate proton integral (6.174 ppm and ppm in D2O) and the anomeric proton of the glycopyranosyl ring (5.166 ppm and ppm). Since the signal of the anomeric proton of α- 1,3 linkages (5.166 ppm) partially overlaps with other protons, a pre-determined ratio [ref. 11] of 4% α-1,3 linkages was assumed and the total anomeric proton integral was calculated solely based on the integral at ppm. A methacrylate:dextran repeat unit ratio of 0.7 was determined. 2 NATURE MATERIALS

3 SUPPLEMENTARY INFORMATION Supplementary Figure 2: Multiscale mechanical testing of fibrous materials. a, Schematic (top) and image (bottom) of single fiber three-point bending tests performed with AFM. Cross-sectional (b) and top-down (c) views of microfabricated PDMS trough substrates used to isolate individual electrospun fibers for testing. d) Lateral translation (in direction of arrow) of DexMA fiber demonstrating fixation at trough edges and validating the assumption of fixed boundary conditions required for the calculation of Young s modulus. e, Example deflection-indentation curves for DexMA fibers exposed to varying amounts of UV light. f, Schematic of network testing performed with a calibrated, microfabricated cantilever. Side (g) and top-down (h) views of tungsten rod with adhered cylindrical indentor fabricated by photolithography. i, Image of DexMA fiber network during indentation. Prescribed displacement of the indentor was applied to the center of networks via micromanipulator and resulting indentor deflection and network deformation were recorded. j, Confocal x-, y-, and z-projections of fiber network during testing showing indentation profile. k, Force response as a function of indentation depth of networks exposed to varying amounts of UV light. An additional set of soft (0 mj) networks were exposed to humidified conditions to weld network fibers. Scale bars: 50 μm (a-d), 500 μm (g-j). NATURE MATERIALS 3

4 Supplementary Figure 3: Relative quantification of RGD density coupled to DexMA fiber networks of different stiffnesses. a, Confocal maximum projections of DexMA fibers coupled with RGD (left) and FITC-RGD (middle) via a Michael type addition. Additional samples were incubated in identical conditions with FITC to detect the background fluorescence due to diffusion and passive adhesion (right). b, Soft and stiff (as described in the main text) fiber networks coupled with a range of FITC-RGD concentrations, demonstrating RGD coupling is independent of network stiffness. c, Quantification of fluorescence intensity from confocal stacks. No statistically significant differences were identified when comparing soft and stiff at a given RGD concentration (mean ± s.d., n 5 ROI, significance set at P < 0.05). 4 NATURE MATERIALS

5 SUPPLEMENTARY INFORMATION Supplementary Figure 4: DexMA fibers are protein adsorption resistant and require RGD functionalization for cell spreading. Cell spreading on stiff DexMA fiber networks (as described in main text) as a function of RGD density after one day of culture. Cells have been stained for F-actin (green) and nuclei (blue) with phalloidin-alexa 488 and Hoechst 33342, respectively; DexMA fibers have been labeled with rhodamine methacrylate (red). Cell spread area was quantified for n > 37 cells using a custom image analysis tool in Matlab. Scale bar: 50 μm. 5 5 NATURE MATERIALS

6 Supplementary Figure 5: Macroscale contraction of cell seeded DexMA fiber networks and collagen substrates. hmsc-seeded DexMA fiber and collagen constructs after 3 days of culture, stained for F-actin (green) and nuclei (blue) with phalloidin-alexa 488 and Hoechst 33342, respectively. DexMA fibers have been labeled with rhodamine methacrylate (red). Fiber constructs were formed as ~500 μm thick mats without boundary constraints to allow contractile cell forces to deform the material. Collagen gels were cast in silanized PDMS wells to prevent adhesion of the gel to the walls of the well. The original size of constructs at the time of seeding is shown with a dashed white outline. Scale bars: 500 μm. 6 NATURE MATERIALS

7 SUPPLEMENTARY INFORMATION Supplementary Figure 6: Cell-mediated material deformation in soft hydrogels and fiber networks. a, Example bead displacements over a 3 hour period beginning with initial attachment of hmscs on soft (290 Pa) flat DexMA hydrogels (left) and soft (fiber: 140 MPa, network: 2.8 kpa) DexMA fiber networks (right). A subset of beads in each confocal image stack was manually tracked using an ImageJ plugin. b, Total displacement as a function of the initial distance between the bead and the center of the cell for soft hydrogels (left) and fiber networks (right). Quantification was performed on >50 beads for hydrogels and >16 beads for fiber networks, for 3 cells in each condition (red, green, blue). c, Displacements of five representative beads as a function of time. Projected distances were calculated along the vector defined by the center of the cell and the initial position of each bead. Motion towards the center of the cell would possess a negative value. Scale bar: 25 µm. NATURE MATERIALS 7

8 Supplementary Figure 7: Fiber diameter as a function of DexMA solution concentration. Histograms showing the distribution of diameter across a population of fibers formed from solutions at 0.6, 0.5, 0.4, and 0.3 mg/ml. For each concentration, diameters of n 36 fibers were quantified; fitted curves (blue) assume a Gaussian distribution. 8 NATURE MATERIALS

9 SUPPLEMENTARY INFORMATION Supplementary Figure 8: Influence of fiber stiffness on cell viability. a, Cell death two days following hmsc seeding as a function of fiber stiffness (soft and stiff conditions as defined in main text), determined by LIVE/DEAD staining (number of ethidium homodimer-1 positive cells normalized to calcein AM positive cells, n 8 ROI with totals of >350 cells analyzed). No statistical difference in cell death was found between soft and stiff fibers as determined by t-test (P = 0.87). b, LIVE/DEAD stained networks as quantified in a (green: calcein AM, red: ethidium homodimer-1). Scale bar: 500 µm. Supplementary Figure 9: Fiber diameter as a function of crosslinking. Histograms showing the distribution of diameter across a population of fibers formed from a 0.5 mg/ml solution UV crosslinked for varying durations to generate soft (blue) and stiff (red) fibers as described in the main text. For each fiber stiffness, diameters of n 172 fibers were quantified; fitted curves assume a Gaussian distribution. No statistical difference in mean diameter was found between soft and stiff fibers as determined by t-test (P = 0.63). NATURE MATERIALS 9

10 Supplementary Figure 10: hmsc proliferation as a function of initial network fiber density. a, Proliferation of hmscs over two days as determined by EdU incorporation; mean ± s.d., n 13 ROI with totals of cells analyzed, * P < b, Confocal maximum projections of networks at corresponding low, medium, and high densities. Note that networks used in studies presented in the main text were fabricated at low density. Scale bar: 50 µm. 10 NATURE MATERIALS

11 SUPPLEMENTARY INFORMATION Supplementary Figure 11: Custom electrospinning setup for fabricating fibrous networks. a, Image of electrospinning setup consisting of syringe pump, high voltage power supply, and fiber collection platform enclosed within a humidity and temperature controlled glove box. b, Schematic of collection platform for forming random fiber networks corresponding to inset in a. Image (c) and schematic (d) of rotating platen for fabricating networks of aligned fibers. NATURE MATERIALS 11

12 Supplementary Video Legends Supplementary Video 1 & 2: Tuning stiffness in DexMA fiber network can range between completely rigid to easily deformable under cell forces. 3T3 fibroblasts migrating on soft (Supplementary Video 1) and stiff (Supplementary Video 2) networks over a 6 hour time course. Supplementary Video 3: Cells actively recruit fibers during spreading on soft fiber networks. Fiber network underlying an hmsc spreading over a 3 hour duration following initial attachment. Supplementary Video 4 & 5: Long-range material deformations occur in soft fiber networks but not on soft flat hydrogels. Fluorescent microspheres were embedded within fibers or encapsulated throughout flat hydrogels. Microsphere displacements resulting from representative hmscs on fiber networks (Supplementary Video 4) and flat hydrogels (Supplementary Video 5). Timelapse images were acquired during hmsc spreading over a 3 hour duration after initial attachment. Images are sequentially color-coded to correspond with Figure 4b. Supplementary Video 6-8: Modulating fiber-fiber welding by altering network fabrication conditions. Fiber intersections in networks fabricated under normal conditions were a mixture of bound and unbound states when a micromanipulator-actuated probe laterally translated individual fibers, sliding of fibers across each other occurred (Supplementary Video 6). To maximize fiber-fiber welding, networks were placed in a controlled humidity environment and provided sufficient moisture to fuse all juxtaposed fibers. Laterally probing individual fibers caused translation of the entire network, revealing intersections to be solidly fused (Supplementary Video 7). UV crosslinking to stiffen individual fibers (to fabricate stiff networks as defined in main text) did not introduce fiber-fiber welds, demonstrating orthogonal control over inter-fiber and intra-fiber crosslinking (Supplementary Video 8). 12 NATURE MATERIALS