Bioengineered in vitro tissue model of Fibroblast activation for

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Tissue Engineering and Regenerative Medicine

Bioengineered in vitro tissue model of Fibroblast activation for modeling Pulmonary Fibrosis Aswin Sundarakrishnan, Heather Zukas, Jeannine M. Coburn, Brian Bertini, Zhiyi Liu, Irene Georgakoudi, Lauren Baugh, Queeny Dasgupta, Lauren D. Black, and David L Kaplan ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b01262 • Publication Date (Web): 10 Apr 2019 Downloaded from http://pubs.acs.org on April 11, 2019

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ACS Biomaterials Science & Engineering

Bioengineered in vitro tissue model of fibroblast activation for modeling Pulmonary Fibrosis Aswin Sundarakrishnan1, Heather Zukas1, Jeannine Coburn1,5, Brian T. Bertini3, Zhiyi Liu1,6, Irene Georgakoudi1, Lauren Baugh1, Queeny Dasgupta1, Lauren D. Black1,4 and David L. Kaplan1, * 1Department

of Biomedical Engineering, Tufts University, 4 Colby Street, Medford, MA 02155, of Chemical and Biological Engineering, Tufts University, 4 Colby Street, Medford, MA 02155, 4Department of Cell, Molecular & Developmental Biology, Sackler School of Graduate Biomedical Sciences, Tufts University, 136 Harrison ave., Boston, MA 02111, 5Department of Biomedical Engineering, Worcester Polytechnic Institute, 60 Prescott Street, Worcester, MA 01605, 6Wellman Center for Photomedicine, Massachusetts General Hospital, 40 Blossom St, Boston, MA 02114. 3Department

*Corresponding Author: David L. Kaplan, PhD Department of Biomedical Engineering Tufts University 4 Colby Street Medford, MA 02155 [email protected]

Keywords: pulmonary fibrosis; idiopathic pulmonary fibrosis; fibroblast activation; in vitro models; threedimensional culture; anti-fibrotic drug testing

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ABSTRACT Idiopathic Pulmonary Fibrosis (IPF) is a complex disease of unknown etiology with no current curative treatment. Modeling pulmonary fibrotic tissue (PF) has the potential to improve our understanding of IPF disease progression and treatment. Rodent animal models do not replicate Hum-FF pathology and current iterations of in vitro model systems (e.g., collagen hydrogels, polyacrylamide hydrogels and fibrosis-onchip systems) are unable to replicate the three-dimensional (3D) complexity and biochemical composition of human pulmonary fibrotic tissue. Herein, we have fabricated a 3D bioengineered pulmonary fibrotic (Eng-PF) tissue utilizing cell laden silk-collagen-type I dityrosine crosslinked hydrogels and Flexcell bioreactors. We show that silk-collagen-type I hydrogels have superior stability and mechanical tunability compared to other hydrogel systems. Using customized Flexcell bioreactors we have reproduced human fibroblastic foci (Hum-FF) like pathology with airway epithelial and microvascular endothelial cells. EngPF tissues can model myofibroblast differentiation and permit evaluation of anti-fibrotic drug treatments. Further, Eng-PF tissues could be used to model different facets of IPF disease including epithelial injury with the addition of bleomycin and cellular recruitment by perfusion of cells through the hydrogel microchannel.

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1. INTRODUCTION Idiopathic pulmonary fibrosis (IPF) is a fatal disease of unknown etiology affecting older adults (55-75 years), with an estimated median survival of 2-5 years.1 Fibroblasts are considered the key effector cells and fibroblastic foci (FF) are the histopathological hallmark of IPF disease, preceding end-stage fibrosis, characterized by a stiff collagenous extracellular matrix (ECM) layer containing myofibroblasts separating the airway epithelial cells from the endothelium.2 Impaired epithelial-fibroblast signaling has been implicated in IPF disease progression and injured epithelial cells with denuded basement membrane are found covering proliferating myofibroblasts at FF sites.3-5 While the lack of significant inflammatory cell infiltrate is characteristic IPF pathology, immune cells from both innate and adaptive immune processes have been shown to influence fibroblast phenotype, hence contributing to IPF disease.6 IPF is therefore a complex disease involving cellular signaling from multiple cell types including fibroblasts, epithelial, endothelial and immune cells. While IPF disease has no cure, there is also a paucity of Federal Drug Administration (FDA) approved drugs for ameliorating disease.7, 8 One of the reasons for this discrepancy is that pre-clinical animal models that have aided in the identification of key mediators of IPF disease (e.g., Transforming Growth Factor-β1 (TGF-β1))9 are not an accurate representation of human disease and drug candidates identified using such models have often failed human trials.10, 11 In vitro tissues can be used to model IPF disease using human cells, but current iterations are limited two-dimensional (2D) cell culture models and do not recapitulate the complex three-dimensional (3D) morphology of human FF (Hum-FF), with critical cell-cell and cell-ECM interactions.12 The maturation of fibrotic lung processes is analogous to wound healing and by parallelism fibroblast migration/accumulation precedes fibroblast proliferation and differentiation. 2, 3 Collagen-type I is one of the predominant ECM proteins found within mature FF3 and therefore, 3D cultures of fibroblasts within collagen-type I hydrogels is a common in vitro approach to study cellular signaling associated with IPF disease. Collagen hydrogels replicate important cell-ECM interactions and such systems have been



used for studying potential FF triggers including growth factor proteins, ECM proteins or matrix stiffness on myofibroblast phenotype.13 However, collagen hydrogels are unable to replicate normal/fibrotic ECM stiffnesses (mechanical tunability) and hydrogel contraction/degradation limit the use of these systems to short-term studies (1-2 weeks).14 Two-point-five dimensional (2.5D) transwell cell culture models permit long-term ( ≥ 1 month) in vitro culture and have aided in the identification of hydrogen peroxide (H2O2) as a diffusible signal inducing apoptosis in airway epithelial cells grown in conjunction with IPF fibroblasts.15 Although, transwell systems lack mechanical tunability, 3D integrin adhesions and biomechanical strain/perfusion stimuli.12 Lung-on-chip models overcome most of these limitations and a fibrosis-on-chip model with an integrated collagen-type I hydrogel has been proposed for replicating the morphology of 3D Hum-FF with strain/perfusion capacity, albeit these systems are still under development and their utilization for in vitro anti-fibrotic drug testing has not been realized.16 Thus, there is a need for advanced engineered in vitro models with mechanical tunability, long-term culture capacity and the ability to apply biomechanical stimuli that could be used to model chronic events related to pulmonary fibrosis formation including persistent epithelial injury, exaggerated matrix production, matrix remodeling and feedback signaling, typical of human IPF disease.12 We hypothesized that a 3D engineered pulmonary fibrotic (Eng-PF) tissue model generated using silk-collagen-type I dityrosine crosslinked scaffolds seeded with human pulmonary cells in a bioreactor would provide suitable tissue systems to model human IPF disease. Our results show that silk-collagentype I hydrogels replicate Hum-FF biochemical composition with 3D culture capacity and possess superior mechanical tunability compared to collagen-type I and polyacrylamide hydrogel systems. A custom bioreactor fabrication enabled reproduction of Hum-FF cellular biodistribution and induced parallel alignment of fibroblasts within Eng-PF tissues, replicating Hum-FF-like pathology. We show that Eng-PF tissues can be used to model myofibroblast differentiation with the exogenous addition of TGF-β1 cytokine and furthermore be used to test anti-fibrotic drugs agents for their ability to attenuate myofibroblast phenotype. Finally, we show that Eng-PF tissues can be used to model epithelial injury under co-culture conditions with antineoplastic drug bleomycin and can also be used to study immune cell recruitment by 4 ACS Paragon Plus Environment

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supporting perfusion of THP-1 monocytes through the hydrogel microchannel. Together, we present a novel in vitro Eng-PF model with mechanical tunability, long-term culture capacity and the ability to apply biomechanical stimuli that can be used to model different aspects of IPF disease. 2. EXPERIMENTAL SECTION 2.2 Preparation of silk-collagen-type I dityrosine crosslinked hydrogels: Solubilized silk protein was

prepared using previously established procedures.17, 18 All reagents were purchased from ThermoFisher Scientific (Waltham, MA) or Sigma-Aldrich (St. Louis, MO) unless specified. Briefly, aqueous silk protein was concentrated to 12-14% (w/v) by air drying in dialysis cassettes at room temperature. Insoluble particulates were removed by centrifugation and filtered sterilized using a 0.2 µm polyethersulfone (PES) bottle top filter before storage in 4°C. All silk solutions were used within 30 days or discarded. Silk-collagen-type I hydrogels were fabricated using a custom formula (Table S1) after modifications to previous protocols.18 To prepare 1000 µL of silk-collagen-type I hydrogel solution, 500 µL of Dulbecco's modified Eagle's medium (DMEM) media was mixed with 2% (w/v) silk solution, 1 mg/mL high concentration rat tail collagen-type I (Corning, Tewksbury, MA), 1M NaOH and ice-cold water. Tyrosine crosslinking was initiated by adding 10 µL of 1,000 U/mL stock solution of horseradish peroxidase (HRP) and 10 µL of 165 mM hydrogen peroxide. Cells (50 µL of cell suspension with a seeding density of 1x106 cells/mL) were added towards the end to ensure homogeneity and avoid ionic or pH shock. 2.3 Dityrosine crosslinking kinetics in collagen-type I and silk-collagen-type I hydrogels: To confirm

dityrosine formation, fluorescence time and wavelength sweeps were conducted in 96-well plates using a SpectraMax M2 plate reader (Molecular Devices, Sunnyvale, CA). Collagen-type I hydrogels and silk-collagen-type I hydrogels were fabricated using the custom protocol (Table S1). Cell free hydrogels were prepared by substituting water instead of cell suspension. Acellular 1, 2 or 5 mg/mL collagen-type I hydrogels were prepared by mixing collagen-type I with DMEM, water and 1M NaOH. Acellular silk-collagen-type I hydrogels were prepared with or without collagen (1mg/mL) and



different silk (0.5, 0.75, 1 and 2%) concentrations. For tyrosine crosslinking HRP and H2O2 were added. One hundred and fifty microliters (150 µL) of hydrogel mixture was added to each well (n = 4/group) in a 96-well glass bottom sensoplate (Greiner Bio-one, Monrow, NC) and fluorescence (excitation: 290 nm; emission: 415 nm) was measured every minute for 60 or 300 min at 37 °C. Collagen-type I mixtures with HRP enzyme, but without H2O2 served as negative controls. Using the same 96-well plate, fluorescence emission was measured from 350-600 nm at 1 nm increments (excitation: 290 nm). 2.4 Human pulmonary cell isolation and culture: Pulmonary human fibroblasts were isolated from

donor human lungs obtained from National Disease Research Interchange (NDRI) using established protocols19, 20, with institutional review board (IRB) approval (Tufts IRB approval # 1302015). Briefly, pieces of distal lung tissue (~0.3 cm3) were placed on tissue culture treated plastic dishes in DMEM/F12 conditioned medium containing 10% fetal bovine serum (FBS) and 100 U/mL penicillin/streptomycin (Penn/Strep). Media were changed 2 times/week for 2 weeks during which non-mesenchymal cells perished, but immune cells and fibroblasts remained (Figure S1). Differential adherence of fibroblasts was utilized for further purification and pure populations of fibroblasts (cytokeratin-, cluster of differentiation-68-, cluster of differentiation-31- and vimentin+) were either frozen or sub-cultured for further experiments. Pulmonary fibroblasts within passage 2-5 was used for all experiments. Airway epithelial cell line A549 obtained from human adenocarcinoma was purchased from ATCC Cell Bank. Cells were cultured using DMEM/F12 conditioned medium containing 10% FBS and 1xPenn/Strep. Human dermal microvascular endothelial cell line HMEC-1 (ATCC, Manassas, VA) was cultured in MCDB131 growth medium containing 10 ng/mL Epidermal Growth Factor (EGF), 1µg/mL hydrocortisone and 10% FBS. For detachment of cultured cells from tissue culture flasks, 0.05% Trypsin-ethylenediaminetetraacetic acid (EDTA) and trypsin neutralizing solution (Lonza, Walkersville, MD) were used. 2.5 AlamarBlue metabolic activity and viability: AlamarBlue metabolic activity was measured every

3rd day for 40 days. Briefly, pulmonary fibroblasts were trypsinized as described previously. 6 ACS Paragon Plus Environment

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Pulmonary fibroblast (1x106 cells/mL) laden hydrogels were cast into 24-well cell culture plates and incubated in cell culture medium with 10% AlamarBlue reagent for 2h at 37°C. Following incubation, aliquots of 200 µL were placed into black 96-well plates (n = 4/group) and fluorescence quantified using a spectrofluorometer (excitation: 570 nm; emission: 585 nm). Acellular hydrogels were used to adjust for background fluorescence. Cellular viability and distribution at the end of AlamarBlue metabolic assessment was performed by incubating cell laden hydrogels in 10 µM of CellTracker Green CMFDA in serum free media (DMEM/F12+0.5% bovine serum albumin) for 20 min at 37°C. Hydrogels were fixed using 4% paraformaldehyde (PFA) in 1xPBS for 30 min and washed 3x in 1x PBS. All hydrogels were subjected to a clearing protocol for fluorescence imaging permitting better visualization of cells within otherwise opaque hydrogels. Briefly, hydrogels were dehydrated in graded alcohols (25%, 50%, 75% and 100% EtOH) and finally cleared in BABB (benzyl alcohol: benzyl benzoate; 1:2 ratio) before imaging using confocal microscopy. 2.6 Hydrogel contraction assay: Human pulmonary fibroblasts (1x106 cells/mL) were encapsulated using

either collagen-type I or silk-collagen-type I hydrogels and seeded within Flexcell Tissue Train plates as per manufacturer’s protocol. Encapsulated hydrogels were floated by cutting nylon tethers and cultured floating in DMEM/F12 conditioned medium. Media was replaced every 3rd day and all hydrogels were weighed at day 0, 1, 4, 7, 10, 13 and 16 to assess contraction due to water loss (n = 3/group). 2.7 Mechanical testing of Normal, IPF lung and engineered tissue: Mechanical testing was conducted

on hydrogels using an Instron Model 3366 with a 10 N load cell and pneumatic grips with 100 grit sandpaper. Acellular or fibroblast laden (1x106 cells/mL) silk-collagen-type I hydrogels and collagentype I hydrogels were cut from nylon tethers of Flexcell Tissue Train plates and tested on days 1, 4, 7, 10 and 13 (n = 3/group). Hydrogels were kept hydrated in 1xPBS at room temperature until testing. Samples were subjected to tensile test until failure at 1% strain/s after applying a pre-load of 0.005N. Stress-Strain curves were generated and tensile elastic modulus at 10% strain was calculated using MATLAB. Surface moduli measurements using Atomic Force Microscopy (AFM) were used for 7 ACS Paragon Plus Environment


calibrating silk-collagen-type I hydrogel moduli to normal/IPF lung tissue moduli. Normal and fibrotic lung samples with pathological confirmation of human IPF disease were procured through a Lung Tissue Research Consortium (LTRC) (Tufts IRB approval # 1302015). Surface moduli of pulmonary fibroblast laden silk-collagen-type I hydrogels on days 1 (n = 3) and day 13 (n = 7) were measured using a Veeco Dimension 3100 atomic force microscope (AFM). Additionally, three normal human lung samples and three fibrotic lung samples were measured on the same AFM. Each sample was measured over a 9 μm x 9 μm area using a 16 x 16 grid with a total of 256 force curves taken per measurement. Stiffness measurements were taken using a 10 μm borosilicate glass beaded probe tip (Novascan, Ames, IA; spring constant of 0.06 N/m). Before each measurement session, the tip was calibrated for sensitivity (nm/V) and the spring constant was determined using thermal calibrations. Each force curve was used to calculate a Young’s Modulus (E) using the Hertz model. For the Hertz model, the Poisson’s ratio was taken to be ν = 0.5 by assuming that the scaffolds were incompressible.21 The modulus values for the force curves of the 16 x 16 measurement grid were averaged to give a single Young’s Modulus value for each area taken. 2.8 Multi-photon microscopy and Second Harmonic Generation (SHG): Fibroblast (1x106 cells/mL)

laden silk-collagen-type I hydrogels were cultured attached to nylon tethers inside the Flexcell bioreactor. Hydrogel constructs were harvested at days 1, 5, 10, 20 (n = 3/group) and live stained using 10 µM CellTracker Green CMFDA in serum free media and subsequently fixed, cleared before imaging using multiphoton microscopy. Second Harmonic Generation (SHG) and Two Photon Excitation Fluorescence (TPEF) images were obtained using a Leica TCS SP2 confocal microscope equipped with a tunable (710–920 nm) titanium-sapphire laser (Mai Tai; Spectra Physics; Mountain View, CA). TPEF images of fibroblasts were acquired with an excitation wavelength of 760 nm and recorded by the 525 ± 25 nm photomultiplier tube (PMT) detector, and SHG images were excited using 800 nm and recorded by the 400 ± 10 nm PMT detector. A water-immersion 25x objective (NA 0.95; 2.4 mm working distance) was used to obtain z-stacks of ~250 µm thickness with a 2 µm step size.


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Reconstruction of 3D images was performed by Image J (NIH, Bethesda, MD), and image analysis was performed using a 3D directional variance algorithm in MATLAB.22 2.9 Customized Flexcell bioreactor fabrication: Flexcell Tissue Train plates routinely used for strain

application were modified to support perfusion culture. Tissue Train plates (Flexcell International Corp., Burlington, NC) were purchased and custom holes were machined into the bioreactor plates (Figure S2 and S3). Briefly, machined plates were fitted with silicone tubing and a tubular sacrificial gelatin construct was created using established protocols.23 Fibroblast laden (1x106 cells/mL) silkcollagen-type I hydrogels were seeded around the gelatin construct. Incubation at 37 ºC resulted in simultaneous silk-collagen-type I hydrogel polymerization and gelatin melting, creating a hydrogel construct with a tubular channel. Subsequently, epithelial cell line A549 sourced from lung adenocarcinoma (2x106 cells/mL) and microvascular endothelial cell line HMEC-1 (2x106 cells/mL) were seeded on top and within the channel respectively. Perfusion through hydrogel channel and nutrient diffusion through hydrogel bulk were characterized using 3D models generated with finiteelement modeling (FEM) software COMSOL Multiphysics. To confirm laminar flow profile, Fluorescein isothiocyanate (FITC)-Dextran (4 kDa) was perfused through hydrogel channel at 5 µL/min and imaged using time-lapse epifluorescence microscopy. Mass-transfer through hydrogel were studied empirically and using FEM models. FITC-dextran (4 kDa and 120 kDa) was flowed through hydrogel channel at 5µL/min and images were captured using time-lapse epifluorescence microscopy every 2 min for 6 h or until steady-state (Figure S4). Image J software was used to plot pixel intensities and diffusivity values were calculated using Fick’s law.24 Further, empirically derived diffusivity values were used to generate FEM diffusion models for comparison (Figure S5). 2.10 Fibroblast phenotypic analysis and anti-fibrotic drug testing: 2D studies: Myofibroblast

differentiation can occur due to matrix stiffness and/or TGF-β1.25 Since silk-collagen-type-I hydrogels undergo dynamic stiffening and to establish parameters for induction of IPF phenotype in the current model, freshly isolated fibroblast cells were seeded on tissue culture treated plastic (TCP), collagen or silk-collagen-type I hydrogels at 0.2x106 cells/well. After 2 weeks of culture (post silk-collagen-type9 ACS Paragon Plus Environment


I hydrogel stiffening) media in all dishes were switched to serum free medium (DMEM/F12 + 0.5% BSA + 1x Insulin-Transferrin-Selenium). Twenty-four hours after serum starvation TGF-β1 (10 ng/mL; Cell signaling technology# 8915LC) was added to treated groups. Saline treated cells served as negative controls. Seventy-two hours after TGF-β1 treatment, hydrogels were fixed using 4% paraformaldehyde (PFA) and immunostained for fibroblast/myofibroblast phenotype. Experimental replicates (n = 3/group) were used for western blotting analyses. For 2D anti-fibrotic drug testing studies TCP plated fibroblasts were cultured for 2 weeks, serum starved and treated with TGF-β1 with or without anti-fibrotic drugs Pirfenidone (1 mM; Selleckchem, Houston, TX) and Nintedanib (5 µM; Selleckchem, Houston, TX. It is important to note that Pirfenidone (1mM) is always dosed much higher than Nintedanib (0.5-2 µM), and these dosages were obtained from previous studies which have successfully reduced myofibroblast markers.26 Negative controls received dimethyl sulfoxide (DMSO) of identical concentration. Seventy-two hours after treatments western blotting was performed to assess protein levels of fibroblast/myofibroblast markers. Fibroblast phenotype was assessed using antivimentin (Abcam#92547) and proto-myofibroblast, myofibroblast phenotype was assessed using antiα-SMA (Abcam#7817), anti-periostin (Abcam#14041), anti-collagen I (Abcam#138492) and anti-EDA fibronectin (Abcam#6328) primary antibodies. 3D studies: Fibroblast (1x106 cells/mL) encapsulated silk-collagen-type I hydrogels were cultured for 3 weeks within customized Flexcell bioreactor followed by serum starvation and treatment with TGF-β1 (10 ng/mL) with or without anti-fibrotic drugs pirfenidone (1mM) and nintedanib (5 µM). Negative controls received dimethyl sulfoxide (DMSO) of identical concentration. Seventy-two hours after treatments engineered constructs were flash frozen in liquid nitrogen and stored in −80°C until ready for Western blotting and immunostaining analyses. 2.11 Epithelial injury: Eng-PF tissues containing fibroblast, epithelial and endothelial cells were cultured

for 3 weeks, as mentioned earlier. Preliminary experiments on 2D cultures revealed that bleomycin sulfate (Sigma-Aldrich) induced epithelial injury at 10 µg/mL without affecting fibroblast viability (Figure S6). Eng-PF tissues were cultured for 3 weeks and serum starved for 24 h and treated with 10 ACS Paragon Plus Environment

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serum-free media containing TGF-β1 + bleomycin 10 µg/mL or 20 µg/mL. Seventy-two hours after treatments, Eng-PF tissues were split into two and flash frozen separately for western blotting and immunostaining analyses of epithelial markers. Epithelial phenotype was assessed using anti-Ecadherin (BD#610405), anti-epithelial cell adhesion molecule (EpCAM) (Santacruz#25308), antioccludin (Fisher#71-1500) and anti-collagen-IV (Sigma#C1926). 2.12 Western blotting: Western blotting assessment of protein expression was carried out as previously

described27, 28 and all reagents were obtained from ThermoFisher Scientific unless specified otherwise. Engineered tissues were homogenized using a Fisherbrand 150 Handheld homogenizer. Tissue homogenates were placed inside tubes containing NP-40 cell lysis buffer and protease inhibitors. Following protein extraction using manufacturer’s protocol, protein quantification was performed using bicinchoninic acid (BCA) assay. Cell lysates were reduced, and equal amounts of protein were loaded into pre-cast 4-12% NuPAGE Bis-Tris protein gels followed by transfer onto PVDF membranes using an iBlot dry blotting system. Membranes were blocked by incubating in 5% bovine serum albumin (BSA) followed by overnight incubations in primary antibody solutions prepared in PBST (PBS + 0.1% tween-20) containing 0.5% BSA. Anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Abcam#181602) primary antibody was used as the loading control. Following primary antibody incubation, samples were washed in PBST+0.5% BSA and incubated in species specific HRP conjugated secondary antibodies. Images were acquired on the G:Box Chemi XR5 (Syngene, Cambridge, United Kingdom). Expression intensities were analyzed using Image J. When multiple blots were required to account for large sample size, expression intensities for each protein were normalized to the mean expression intensity per blot for that respective protein to allow for comparison between blots.27 2.13 Histology, immunostaining and microscopy: IPF lung paraffinized tissue sections from LTRC were

hematoxylin and eosin (H&E) stained. Brightfield images of Hum-FF were captured using a Keyence BZ-X700 microscope (Keyence, Itasca., IL). Engineered tissues were either processed for whole mount immunostaining or section immunostaining following embedding in OCT compound. For 11 ACS Paragon Plus Environment


immunostaining, fixed samples were washed 3 times with PBST and incubated in 0.1% TritionX-100 in PBST for 1 hour. Samples were blocked for 3 h in PBST containing 10% NGS and 0.5% BSA at room temperature. Primary antibodies were incubated in PBST containing 1%NGS, 0.5% BSA overnight in 4 ºC. Fibroblast and epithelial phenotype were assessed using respective markers. Endothelial phenotype was assessed using anti-Von Willebrand Factor (vWF) (Abcam#6994). Following primary antibody incubation, samples were washed in PBST+0.5% BSA five times for 1 hour each. Species specific secondary antibodies conjugated with Alexa Fluor were incubated (1% NGS, 0.5%BSA and PBST) overnight. Since silk-collagen-type I hydrogels are opaque, all samples were cleared prior to confocal or fluorescence imaging. Immunostained wholemounts and sections were imaged using a Leica TCS SP2 confocal microscope and Keyence BZ-X700 fluorescence microscope respectively. 2.14 Statistics: Data are expressed as mean and error bars denote standard deviation. One or two-way

analysis of variance (ANOVA) and Tukey post-hoc analysis was used to determine statistically significant differences. Statistical significance was accepted at the p < 0.05 level and indicated in figures as * p

Bioengineered in vitro tissue model of Fibroblast activation for

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