Photo-Cross-Linked Poly(ethylene glycol) Diacrylate Hydrogels

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Applications of Polymer, Composite, and Coating Materials

Photo-cross-Linked Poly(ethylene glycol) Diacrylate Hydrogels: Spherical Microparticles to Bow Tie-shaped Microfibers Farrokh Sharifi, Bhavika B. Patel, Marilyn C McNamara, Peter J Meis, Marissa N Roghair, Mingchang Lu, Reza Montazami, Donald S. Sakaguchi, and Nicole N Hashemi ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 01 May 2019 Downloaded from http://pubs.acs.org on May 1, 2019

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ACS Applied Materials & Interfaces

Photo-cross-Linked Poly(ethylene glycol) Diacrylate Hydrogels: Spherical Microparticles to Bow Tie-shaped Microfibers

Farrokh Sharifi1,†, Bhavika B. Patel2, Marilyn C. McNamara1 , Peter J. Meis1, Marissa N. Roghair2, Mingchang Lu1, Reza Montazami1, Donald S. Sakaguchi2, Nicole N. Hashemi1,3*

1 Department 2 Department

of Mechanical Engineering, Iowa State University, Ames, Iowa 50011

of Genetics, Development and Cell Biology and Neuroscience Program, Iowa State University, Ames, Iowa 50011

3

Department of Biomedical Sciences, Iowa State University, Ames, Iowa 50011 University, Ames, Iowa 50011

ABSTRACT Bow tie-shaped fibers and spherical microparticles with controlled dimensions and shapes were fabricated with Poly(ethylene glycol) Diacrylate (PEGDA) hydrogel utilizing hydrodynamic shear principles and a photo-polymerization strategy. Decreasing the flow rate ratio between the core and sheath fluids from 25 (50:2) to 1.25 (100:80) resulted in increasing the particles size and reducing the production rate by 357% and 86%, respectively. The width of the fibers increased by a factor of 1.4 when the flow rate ratio was reduced from 2.5 to 1 due to the decrease of the shear force at the fluid/fluid interface. The stress at break and Young’s modulus of the fibers were enhanced by 32% and 63%, respectively, when the sheath-to-core flow rate ratio decreased from 100:40 to 100:80. The fiber fabrication was simulated using Finite Element Method (FEM) and

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the numerical and experimental results were in agreement. Adult hippocampal stem/progenitor cells (AHPCs) and bone marrow-derived multipotent mesenchymal stromal cells (MSCs) were seeded onto the fibrous scaffolds in vitro and cellular adhesion, proliferation and differentiation was investigated. Microgrooves on the fibers’ surface were shown to positively affect cell adhesion when compared to flat fibers and planar controls.

KEYWORDS: Microfluidics, bow tie-shaped fibers, photopolymerization, hydrodynamic focusing, finite element method, cell adhesion improvement

1. INTRODUCTION Hydrogels are insoluble three-dimensional cross-linked networks made from hydrophilic polymer chains that swell in aqueous solutions.1-2 They can be used in a wide range of biomedical applications such as cell encapsulation

3-6,

tissue engineering

7-9,

wound dressing

10-11,

and drug

delivery 12-14 due to their high biocompatibility and versatile mechanical properties. Additionally, the transport of oxygen, nutrients and other water-soluble metabolites is efficient through the hydrogel network, which supports cell health while diminishing the buildup of acid products in the hydrogels and makes them good candidates to be used as scaffolds in tissue engineering.15-16 The most common methods used to fabricate micro- and nano-fibers/particles over the past few decades are microfluidics and electrospinning.17-19 Electrospinning is governed by the electrohydrodynamic phenomena where a high voltage is applied to create an electric field between

the polymer and a collector, and the fibers and particles are created due to the solvent evaporation. Electrospinning is a powerful tool to mainly generate nano-fiber mats used in applications such as drug delivery and tissue engineering.18 Microfluidics, on the other hand, is an emerging technology 2 ACS Paragon Plus Environment

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since it uses small amounts of material and can play significant roles in a wide range of fields such as biomedical sciences and energy systems.20-29 Microfluidic particle/fiber fabrication is a costeffective, simple, and highly biocompatible platform.30-31 It can be used to fabricate polymeric materials with various architectures.32-39 Additionally, this approach is versatile, and it reliably produces different sizes, microstructures, and morphologies, while allowing for the tuning of mechanical properties within a desirable range.29, 40-44 There are various methods for synthesizing physical and chemical hydrogels.45-46 Photopolymerization is one of the main solidification strategies in microfluidic polymer fabrication due to the rapid polymerization process, which normally takes less than a second. Photopolymerization can be used in a biocompatible way, with low UV intensity and short irradiation of ultraviolet light (UV) and without harsh environmental conditions.47 In addition, there is no need for high or low temperatures, which can be important when the biological micromolecules such as cells, bacteria, and enzymes are incorporated into the prepolymer solution for encapsulation. In this process, liquid monomers or macromeres can be photopolymerized in the presence of photoinitiators (PI) via visible or ultraviolet (UV) light. The short polymerization time is useful for the conditions in which accurate nano- or micro-patterns are needed. In other solidification methods such as phase inversion or chemical cross-linking approaches, the crosssection of the fibers is affected by the surface tension in the two-phase systems due to their relatively slow solidification rates.35, 38, 48 In this study, we used the photopolymerization strategy in our microfluidic platform mainly due to its short process to fix the structure of the fibers, which helped minimize the fiber deformation time. Poly(ethylene glycol) diacrylate (PEGDA) was used in this study due to its various biomedical applications and its high biocompatibility. It is a PEG derivative that contains



double-bond acrylate groups at each end of its chain, which makes it possible to have free radical photopolymerization in the presence of a PI and form a chemical hydrogel via crosslinking reaction.38,

49-51

Interfacial tension between two fluids was changed via using miscible and

immiscible fluids and micro-particles and micro-fibers were created, respectively. Additionally, shear force at the at the interface between two fluids was changed by using different sheath-tocore flow rate ratios to tune the size of micro-particles and fibers. In addition to interfacial tension and shear force, we changed the components of shear fluid velocity in parallel and vertical directions, with respect to the microchannel main direction, by embedding four chevrons in the microchannel design to change the lateral and vertical hydrodynamic focusing forces, from the sheath fluid to core fluid, and fabricate fibers with controlled size and structure. The tensile properties of single PLGA microfibers were measured for the first time, and it was shown that a wide range of mechanical properties can be obtained by changing the flow rate ratio between the two fluids. Apart from experimental work, we developed a numerical work, as a tool to model the microfluidic fiber fabrication using Finite Element Method (FEM) and predict the structure of the resulting fibers fabricated using different operating parameters both qualitatively and quantitatively. The grooved and flat fibers were used to create aligned PEGDA fibrous scaffolds and investigate the effect of the grooves on the cell adhesion and alignment with a potential application in neural tissue engineering. Studies have shown that aligned fibrous scaffolds significantly affect neurite outgrowth and facilitating nerve regeneration.52-53 To date, stem cells have been used in preclinical and clinical trials to investigate their potential in replacing damaged or lost cells, but often have low survival rates resulting in little functional benefit.54-56 In this study, we introduced a controlled microfluidic platform to design shape and size of the products by tuning the interactional tension between sheath and core fluids, shear force at the


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fluid/fluid interface, and velocity components along the microchannel. This platform was accompanied by a numerical work as a helpful tool for predicting the shape and size of the products. The effect of the fibers’ longitudinal grooves on the adhesion and alignment of marrowderived multipotent mesenchymal stromal cells (MSCs) and adult hippocampal-derived progenitor/stem cells (AHPCs) was studied to investigate the potential of micro-grooves in neural tissue engineering. It was found that the grooves play a significant role on AHPCs attachment compared to the flat fibers and planar controls, which has a significant effect for cell therapy procedures for use in neural tissue engineering. Using this approach, it is hopeful that more cells could be transplanted due to better efficacy for using biomaterials, leading to more functional benefit for those suffering from neurodegenerative diseases.

2. EXPERIMENTAL SECTION 2.1 Materials Poly(ethylene glycol) diacrylate (PEGDA) (Mn=575) and polyethylene glycol (PEG) (Mn = 20,000) were purchased from Sigma-Aldrich (St. Louis, MO). Irgacure 2959 was kindly provided by Chidley & Peto Company (Arlington Heights, IL). For cell culture, alpha minimum essential media (αMEM) and GlutaMAX were purchased from Gibco BRL Thermo Fisher Scientific (Waltham, MA), and fetal bovine serum (FBS) was purchased from Atlanta Biologicals (Atlanta, GA). Antibiotic–antimycotic and Earle’s Balanced Salt Solution (EBSS) were purchased from Invitrogen Thermo Fisher Scientific (Waltham, MA). Poly-L-ornithine (Sigma-Aldrich, St. Louis, MO) and purified mouse laminin (R&D Systems, Minneapolis, MN) were used to coat flasks. Delbucco’s Modified Eagle Medium (F-12, 1:1) was purchased from Omega Scientific (Tarzana,



CA), and basic fibroblast growth factor (human recombinant bFGF) was purchased from Promega Corporation (Madison, WI).

2.2 Microfluidic channel In order to create the microfluidic channel, Polydimethylsiloxane (PDMS) was patterned by a mold created using a SU-8 photoresist, deposited via soft photolithography onto a silicon wafer. Two silicon wafers were used to make the channels with its chevron grooves embedded at the top and bottom of the channel. The height and width of the channel were 130 μm × 390 μm. Four diagonal grooves with the height and width of 130 μm × 100 μm were 200 μm apart. The combination of Sylgard 184 elastomer base and cross-linker agents in a 10:1 ratio was used to make Polydimethylsiloxane (PDMS). Then, the mixture was cured on the mold by placing it within an oven held at a constant 85 °C for 25 minutes; two halves of the mold were then joined using plasma cleaning.

2.3 Microfluidic particle/fiber fabrication The core fluid was made by mixing different concentrations of PEGDA and Irgacure 2959 (PI) with deionized (DI) water and ethanol at room temperature. The UltraViolet (UV) light was provided by a Dymax, BlueWave 200, light-curring spot-lamp system. Miscible fluids were used for the microfiber fabrication. For the core fluid, we used 30 v% PEGDA, 70 v% DI water, 2 wt% Irgacure 2959, and ultraviolet light (UV) light intensity of 153 mW. 5 w% PEG solution (PEG in DI water) was used for the sheath fluid and DI water for the bath. Two different micro-channel


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designs, with and without chevron grooves, were used to create bow tie-shaped and flat microfibers, respectively. For the particle fabrication, the same solution was used for the core fluid, but mineral oil was used for the sheath fluid and the bath, which is immiscible to the core fluid. An UV intensity of 342 mW was used, which was higher than the one required for fiber fabrication due to the lower surface area of the spherical particles, compared to microfibers. The solutions were introduced into the microchannel via a double syringe pump (Cole-Parmer, Veron Hills, IL) with different flow rates. The double syringe pump helped us design a compact unit for fabrication of micro-particles/fibers via a microfluidic approach.

2.4 Simulation of Microfluidic Fiber Fabrication The process of microfluidic fiber fabrication was simulated to investigate the effect of shear force at the core fluid/sheath fluid interface on the shape and dimensions of the fibers. COMSOL Multiphysics was applied to create the 3D model of the microchannel and numerically simulate the microfluidic fiber fabrication using Finite Element Method (FEM). Due to symmetry, only one fourth of the microchannel was modelled to reduce the processing time. The software used the 3D Navier-Stokes equations and Fick’s equation to solve the momentum balance and diffusive transport in the microchannel, respectively. The flow regime in the microchannel was assumed to be laminar because all of the flow rates were at low Reynolds number. Therefore, the motion of the fluid was described by the linear reversible Stokes equation due to the negligible inertial forces. The problem was solved with the steady state assumption due to the continues fiber fabrication process.

2.5 Cell culture



Bone marrow-derived multipotent mesenchymal stromal cells (MSCs) were isolated and cultured from Brown Norway rats (Rattus norvegicus) as described previously.57 Briefly, cells were propagated in maintenance media (MM) containing αMEM supplemented with 20% FBS, 2% 200 mM solution GlutaMAX and 1% antibiotic–antimycotic (Invitrogen Thermo Fisher Scientific). The adult rat hippocampal stem/progenitor cells (AHPCs) were a generous gift from Dr. F.H Gage (Salk Institute for Biological Sciences, La Jolla, CA) and isolated from Fischer 344 rats. The AHPCs were infected with a retrovirus in order to induce the expression of green fluorescent protein (GFP).58 Cells were cultured in T-75 flasks coated with poly-L-ornithine (10 µg/mL) and purified mouse laminin (5 µg/mL) diluted in Earle’s Balanced Salt Solution (EBSS). Maintenance media consisted of DMEM/F12 with 2.5 mM GlutaMAX, N2 supplement (Gibco BRL Thermo Fisher Scientific), and 20 ng/mL basic fibroblast growth factor. Both types of cells were maintained at 37 ˚C in 5% CO2 and 95% humidified air atmosphere. Cells were passaged every 2 to 3 days until 70% confluency.

2.6 PEGDA fibrous scaffold preparation Glass coverslips (12 mm, Fisher Scientific) were cleaned using RBS 35 detergent (Thermo Scientific) diluted (1:50) in deionized water and boiled for 15 min. After this, the coverslips were rinsed with DI water, and were allowed to air dry before being exposed to UV light for sterilization. Microfibers were attached using small droplets of medical adhesive placed at opposite sides of the coverslips, and a parallel array of microfibers placed across the coverslips and attached to the medical adhesive droplets. Small pieces of broken glass coverslips were then used to secure the microfibers to the cover glass at a fixed height from the surface. The microfibers were fixed at opposite ends and loose across the middle of the cover glass. PEGDA hydrogels were incubated


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in EBSS for 5 minutes, and samples were disinfected using 70% ethanol for 20 minutes, followed by 2 washes in EBSS for 5 minutes. Samples were then air-dried for 5 minutes. To aid in cell adhesion, all samples were coated with Entactin Collagen Laminin (ECL; 10 µg/mL) (Millipore/Chemicon, Temecula, CA) substrate overnight at 4 °C.

2.7 Plating of MSCs and AHPCs on PEGDA fibrous scaffolds Cells were collected using 0.05% trypsin-EDTA (Invitrogen, Carlsbad, CA) and centrifuged at 500 rpm (MSCs) or 800 rpm (AHPCs) for 5 minutes, then re-suspended with fresh media. Viable cell counts were performed using a hemocytometer and Trypan blue dye (T8154, Sigma Aldrich). Cells were plated at a density of 50,000 cells per sample and maintained at 37 ˚C in 5% CO2 and 95% humidified air atmosphere. MSCs were cultured for 3 days in vitro (DIV). After 1 DIV, AHPCs were switched to differentiation media (MM without bFGF) and cultured for 6 more days. For feeding, half of the media was exchanged every other day. As a control for cells growing on flat or grooved PEGDA fibers, cells were seeded on Poly-L-Ornithine (10 µg/mL; Sigma-Aldrich) and Laminin (10 µg/mL; R&D Systems) coated 12 mm glass coverslips.

2.8 Immunocytochemistry Samples were rinsed using 0.1 M phosphate buffer two times and fixed in cold 4% paraformaldehyde (Thermo Fisher Scientific, Hampton, NH) in 0.1 M PO4 buffer for 20 minutes. Samples were rinsed with phosphate buffered saline (PBS; Invitrogen Thermo Fisher Scientific) 3 times for 7 minutes each. Samples were incubated for 1 hour in blocking solution consisting of 5% normal donkey serum (NDS, Jackson ImmunoResearch, West Grove, PA), 0.4% bovine serum albumin (BSA; Sigma Aldrich), and 0.2% triton X-100 (Thermo Fisher Scientific) in PBS. Primary



antibodies were applied overnight at 4 °C (Table 1). The next day, cells were rinsed 4 times for 8 minutes each with PBS. Secondary antibodies (donkey anti-rabbit Cy3 or donkey-anti mouse Cy3 (1:500, Jackson ImmunoResearch, West Grove, PA) were diluted in blocking solution and were applied for 90 minutes at room temperature in the dark. All samples with MSCs were stained with Alexa Fluor 488 Phallodin (AF488; Invitrogen Thermo Fisher Scientific) to visualize the filamentous actin (F-actin) cytoskeleton. All samples were stained with DAPI (4',6-diamidino-2phenylindole) (8 µg/mL, Invitrogen Thermo Fisher Scientific), a nuclear stain. Samples were rinsed 4 times for 8 minutes in PBS and mounted on microscope slides using DAPI Fluoromount-G mounting media (Southern Biotech, Birmingham, AL) and stored at 4 °C until imaging.

2.9 Propidium Iodide Assay Propidium Iodide (PI, Thermo Fisher Scientific) staining was used to detect dead cells after 7 days in vitro. The stock solution of PI was diluted to 1.5 μM in AHPC culture medium. Culture media from samples was replaced with the PI solution and incubated for 20 minutes at 37 ̊C in 5% CO2/95% humidified air atmosphere. For a positive PI reagent control, one grooved and non-grooved microfiber sample were incubated in 70% ethanol for 5 minutes to cause cell death before adding the PI solution. Following incubation, samples were rinsed with 0.1 M phosphate buffer and then fixed with 4% PFA in 0.1 M phosphate buffer for 20 minutes at room temperature. Samples were rinsed with PBS and incubated in DAPI (1:50) diluted in PBS to label cell nuclei. Samples were rinsed with PBS and mounted on microscope slides with DAPI Fluoromount-G mounting media for imaging.

2.10 Characterization and imaging


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Imaging: Fluorescent images were captured on a Zeiss LSM700 Confocal microscope equipped with an AxioCam MRc5. Images were captured using a 20x objective and stacks of optical planes were taken for each PEGDA sample. To create a single image, ImageJ was used to condense the z-stacks into maximum intensity projections. Photoshop CC was used to make figures. Scanning electron microscopy (SEM; JCM-6000 NeoScope Benchtop scanning electron microscope) was applied to study the size and morphology of the particles and fibers. MSCs (after 3 DIV) and AHPCs (after 7 DIV) culturing on PEGDA fibers were dehydrated for SEM imaging. Samples were rinsed in a 50:50 solution of water and PBS for 10 minutes. Then, samples were dehydrated in a graded ethanol series from 0% to 100% ethanol (in 10% increasing increments). This was followed by 3 flushes with pure ethanol, then another series of 10-minute washes with a solution of ethanol and hexamethyldisilazane (HDMS; Electron Microscopy Sciences, Hatfield PA): 3:1 to 1:1 and 1:3 each for 10 minutes. The final step was to wash samples with 100% HDMS 3 times. Samples were air-dried overnight before imaging. The substrates were made conductive using iridium sputter-coating. The coating thickness of the samples was 2 nm. The cross sections of the bow tie-shaped fibers were measured using the SEM images and ImageJ.

Mechanical Properties of the fibers: Single fiber tensile testing was utilized to analyze the tensile properties of fibers. Standard extension tensile test procedures were used, with a load cell of 10 N and an extension rate of 20 mm/min, with an Instron Universal Testing Machine (Model 5569, Instron Engineering Corp., Canton, MA). Fibers were mounted onto rectangular paper frames with epoxy glue, which allowed for the machine to grip the samples properly. The sides of the frame were cut once the sample was installed in the machine so that only the tensile properties of the fiber were studied. The beginning length of the sample was 15 mm, and Bluehill software



was used to analyze results. A total of 10 samples were analyzed for each fiber type, and their averages were reported.

2.11 Quantitative Analysis After immunocytochemistry, samples were imaged using a Zeiss LSM700 Confocal microscope equipped with an AxioCam MRc5 for high-resolution images with a 10x objective. For the analysis, 5 microscopic fields were imaged per PEGDA sample. Stacks of optical planes were taken for each PEGDA sample and z-stacks were condensed into maximum intensity projections to obtain a single image. The maximum intensity projection was used to count the number of DAPI positive cells per PEGDA sample.

3. RESULTS AND DISCUSSIONS A schematic of the microfluidic particle and fiber fabrication is shown in Scheme 1 (a) and (b), respectively. Both the core and sheath fluids were introduced into a three-inlet microchannel. The flow regime was laminar and diffusion occurred at the fluid/fluid interface. The microchannel consisted of two main regions: the nozzle (upstream) and four chevrons (downstream). The photopolymerization took place at the outlet of the channel. Scheme 1 (c) illustrates the photopolymerization process of PEGDA hydrogel. The PI absorbed UV light and created free radicals, which reacted with PEGDA macromeres and broke the carbon-carbon double bond. Then, the reactive PEGDA macromeres connected with each other and form a large molecule.15, 59 The core and sheath fluids were introduced to the microchannel via a syringe pump and a UV light spot cure unit was used to polymerize the pre-polymer solution through a free radical photopolymerization. Scheme 1 (d) shows an enlarged view of the microchannel, UV light guide, and the collection 12 ACS Paragon Plus Environment

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bath. The microchannel was covered by Aluminum foil to prevent early polymerization of the core solution and clogging the channel. For the particle fabrication, sheath fluid wrapped around the core fluid due to the high interfacial tension between oil and water.60-61 In the fiber fabrication process, however, miscible fluids were used where the sheath fluid focused the core fluid laterally and vertically. The focusing force originated from the shear force that arises from velocity gradient at the interface between the core and sheath fluids. At the end of the nozzle region part of the microchannel, the core fluid changed to a vertical thin strip due to the lateral force from the sheath fluid. Then, the sheath fluid filled the chevron areas and exerted vertical force on the core fluid. In the chevron region, the hydrodynamic resistance perpendicular to the flow direction decreased. The hydrodynamic resistance is inversely dependent on the flow rate.62 Because the sheath fluid had a higher flow rate in this study, it experienced less resistance compared to the core fluid. Consequently, the sheath fluid filled the top and bottom of the channel, wrapped around the core fluid such that the core fluid was placed at the center of the channel and it was rapidly polymerized and the bow tieshaped structure was fixed at the outlet of the microchannel using UV light. In addition, to investigate the effect of grooves on cell adhesion, flat fibers were created using another channel design with no chevron grooves through which the vertical thin strip of core fluid made using the lateral hydrodynamic force did not turn to the bow tie shape cross-section due to the absence of the vertical force.

3.1 Microfluidic PEGDA microparticle fabrication The experimental set-up for the fabrication of spherical particles is shown in Figure 1 (a). Similar to Scheme 1 (d), The microchannel was vertically positioned at the right side of the figure,



and the UV light was positioned on the left side such that it was irradiating the core fluid once it was leaving the microchannel at the outlet with the intensity of 340 mW. The rest of the microchannel was covered by aluminum foil to prevent premature polymerization and clogging of the microchannel. Figure 1 (b) shows the SEM image of one of the particles. The effect of flow rate ratio on the diameter (± Standard Error) and production rate of the particles are demonstrated in Figure 1 (c) and (d), respectively. This figure shows that particles with a wide range of diameter and formation rate could be created by changing the flow rate ratio between the two fluids. When the shear-to-core flow rate (µL/min) ratio decreases from 25 (50:2) to 1.25 (100:80), the diameter of the particles increased by a factor of 4.6 and the frequency of formation of the particles decreased by a factor of 7.4. The balance between shear force and interfacial tension between the core (dispersed) and sheath (continuous) fluids results in formation of the particles with different diameters and production rate.63 In this study, the material properties, such as viscosity and interfacial tension, were kept constant for both core and sheath fluids. In this condition, when the flow rate ratio between two fluids increased, the shear force at the fluid/fluid interface became stronger and broke the core fluid into smaller particles with higher production rate.64 In addition, the low standard errors demonstrate the uniformity of the particles, which is one of the advantages of using the microfluidic as a controllable and versatile technique to create monodispersed particles.

3.2 Microfluidic PEGDA fiber fabrication Miscible fluids were applied to create a continuous core flow stream, which can be polymerized in the form of microfibers at the end of the microchannel. Figure 2 (a) shows the experimental set-up used for PEGDA microfiber fabrication. The arrangement of the UV light


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guide, microchannel, fluids inlets, and the collection bath were similar to Scheme 1 (d) and Figure 1 (a). The only difference between Figure 1 (a) and Figure 2 (a) was changing the sheath fluid and the collection bath to 5 w% PEG solution, which is miscible to the core fluid with lower interfacial tension between them compared to the mineral oil and core fluid. Although the core and sheath fluids used for making fibers were miscible, they were not mixed in the microchannel. Instead, the interaction between them were the shear force from the sheath fluid to the core fluid occurred at the fluid/fluid interface due to the laminar flow regime in the microchannel. After being hydrodynamically focused from two directions by the sheath fluid, the core fluid was polymerized right at the outlet of the microchannel. The red stream in the collection bath in Figure 2

(a)

demonstrates

the

polymerized

microfluidically-spun

fibers

made

with

the

photopolymerization strategy. One of the advantages of using UV is the rapid polymerization that help design and fix microfibers with various noncircular cross-sections. The SEM image of the PEGDA fibers with longitudinal view is shown in Figure 2 (b). This figure demonstrates that there is no shape gradient across the length of the fiber due to the rapid polymerization rate. The crosssectional SEM images of microfibers fabricated with different flow rate ratios are depicted in Figure 2 (c-f). In these figures, the results of hydrodynamic focusing force from the sheath fluid to the core fluid in both lateral and vertical directions on the cross-section of the fibers can be clearly observed. The lateral force was originated from the flow rate ratio between two fluids, which changed the shear force at the fluid/fluid interface. The width of the fiber increased from 148 µm to 216 µm as the sheath-to-core flow rate ratio decreased from 100:40 to 100:100. Similar behavior was reported for all three microfluidic fiber fabrication strategies of solvent extraction 29, chemical cross-linking

65,

and photo-polymerization.66 That is because when the flow rate ratio

reduced, the velocity gradient at the interface decreased, which resulted in reduction of the lateral



shear force exerted on the core fluid from the sheath fluid. Consequently, the core fluid expanded laterally in the channel, which resulted in the formation of fibers with larger width. The microfluidic fiber fabrication process was also simulated using COMSOL Multiphysics. Figure 2 (c3-f3) shows the concentration distribution of the core and sheath fluids at the outlet of the channel. The red and blue colors represent the core and sheath fluids, respectively. The numerical results also showed that the cross-section of the resulting fibers fabricated using the same microchannel and flow rate ratio, between the core and sheath fluids, forms a bow-tie shaped fiber. In addition to a qualitative comparison, the numerical results were validated with the experimental results in terms of the dimensions of the resulting fibers (Figure 2 (g)) demonstrating that the numerical and experimental results follow the same behavior by changing the flow rate ratio. This demonstrates the capability of the numerical tool to predict the shape and dimensions of the fibers. The width of the fiber from experimental results were 19% and 17% larger than the width obtained from numerical results for the flow rate ratio of 100:40 and 100:100, respectively. Additionally, both of the experimental and numerical results consistently showed that the height of the fibers did not change significantly due to the fact that the vertical hydrodynamic force is mostly dependent on the number and height of the chevrons 62, which were kept constant in this study. Figure 3 shows the tensile properties of the PEGDA fibers made at different flow rate ratios between the core and sheath fluids. Stress at break (MPa), strain at break (%), and Young’s modulus (MPa) of different types of PEGDA fibers were averaged and listed in Table 2. Similar to the literature, the stress-strain curves of the PEGDA fibers, fabricated in different conditions, were found to not have a plastic region.67-68 This behavior is due to the cross-linking of PEGDA during the fiber fabrication process, in which covalent bonds link polymer chains together. This


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prevents polymer molecules slide over each other, which results in improving the toughness and reducing the flexibility of the polymers. When the core flow rate increased from 40 μL/min to 80 μL/min and the sheath flow rate was kept constant at 100 μL/min, the strain at break (%) decreased by a factor of 1.3, whereas the stress at break (MPa) and Young’s modulus (MPa) enhanced by the factors of 1.3 and 1.6, respectively. That was expected because when higher core flow rate was used, the cross-linking density increased, which resulted in more bonds and angles in the network. In this condition, more energy was needed to reach the same strain. This confirmed the fact that Young’s Modulus was enhanced when networks with higher cross-link densities were formed in the network. Additionally, higher strain concentration was created in the network with higher cross-link density under the same tensile strain. Thus, fibers made with the core flow rate of 80 μL/min had lower strain at break due to the high cross-link density and strain concentration in the network.68-71 The tensile properties of PEGDA single fibers have not been reported in the literature. Comparing the mechanical properties of the PEGDA fibers with PEGDA samples in different geometries, it was found that the stress at break, strain at break, and Young’s modulous of the fibers made by this technique was higher.67-68,

72-73

That might be due to the fact that the

microfluidic technique helps align the monomers in the flow direction. The alignment can increase the cross-linking density due to the higher possibility of functional end groups on PEGDA chains to form cross-link points in the solution, which results in formation of network with higher mechanical properties.67-68, 74 Comparing the geometry of the spherical particles and fibers, the fibers have a higher surface to volume ratio, which make them a good candidate in scaffolding to create fully aligned fibrous scaffolds for applications in tissue engineering and regenerative medicine. Additionally, in



this work, the surface of the microfibers was enhanced by designing a uniform microgroove in the longitudinal side of the fiber. The fibrous scaffolds that included the bow tie-shaped micro design on their surface were studied to understand their potential applications for cellular and neural tissue engineering. In addition to surface enhancement, it was hypothesized that the structure of the grooves can potentially collect more cells on its surface and navigate them in a desirable direction. The other advantage of fibers over the particles is that around 55% lower UV intensity is needed to polymerize the microfibers compared to the spherical particles, due to the low ratio of surface area to the volume, which is a crucial factor when biological entities, such as cells or bacteria, are incorporated into the process.

3.3 PEGDA microfibrous scaffolds support cellular adhesion and proliferation. Proliferation of the MSCs and AHPCs on PEGDA hydrogels was investigated by immunolabeling using a Ki-67 antibody. The Ki-67 antigen is expressed in the cell nucleus during the cell cycle and not detected during the resting phases. Ki-67-positive MSCs and AHPCs are shown in (Figure 4 (a1-a2)) and (Figure 5 (a1-a2)), respectively. The figures indicate that both of these stem cell types were able to proliferate on PEGDA hydrogels. To visualize the morphology of the MSCs, AF488 Phallodin was used to stain the F-actin cytoskeleton. MSCs grew along the edges of the bow tie-shaped fibers as well as within the grooves and showed a flattened morphology, consistent with their fibroblastic-like appearance in culture (Figure 4 (a2-c2)). The MSCs growing on the PEDGA hydrogels were further characterized by immunostaining for extracellular matrix (ECM) molecules that are highly expressed by MSCs. It was observed that cultured MSCs were able to produce an ECM that includes at least collagen type I and fibronectin (Figure 4 (b1-c1)). The F-actin cytoskeleton was visualized using AF488 Phallodin and the cells


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counter-stained with DAPI to detect cell nuclei. Collagen immunolabeling was found within the cell (Figure 4 (b1-b2)) and fibronectin immunolabeling is on the surface of the hydrogel, indicating the deposition of this ECM molecule (Figure 4 (c1-c2)). Figure 5 (a1-a2) shows that even under differentiation conditions, a subpopulation of AHPCs were immunolabeled with the Ki-67 antibody. AHPCs were characterized using an antibody directed against nestin, a class of intermediate filament proteins found in neural progenitor cells. AHPCs were observed growing on the PEGDA fibers and counter-stained with DAPI to visualize the cell nuclei. Many AHPCs were immunolabeled with the anti-nestin antibody and it was observed that a majority of cells had long processes (Figure 5 (b1-b2)). Early neurons were characterized using an antibody directed against class III β-tubulin (TuJ1) (Figure 5 (c1-c2)), primarily found in the neurites of the AHPCs. These results indicated that the AHPCs on the PEGDA hydrogels were able to retain their progenitor-like status, as well as begin differentiation towards a neuronal lineage. Together, these results demonstrate that PEGDA hydrogels support MSCs and AHPCs proliferation in vitro. Figure 6 shows the SEM images of the MSCs and AHPCs cultured on the bow tie-shaped fibers. It was observed that the AHPCs showed greater adherence to the PEGDA fibrous scaffolds than MSCs. This result may in part be due to the ECL substrate coating on the PEGDA microfibers. It is possible that other substrates, such as fibronectin, may provide greater cell adhesion for the MSCs. Figure 6 (b1-b2) demonstrates that the grooves embedded on the longitudinal direction of the fibers enhanced the number of the cells, attached on the surface of the fibers. To further assess the effect of microgrooves in the PEGDA fibers on cell adhesion, three different substrates were made: i. planar substrate, ii. fibrous scaffold with grooved fibers (Figure 7 (a)), and iii. fibrous scaffold with flat fibers (Figure 7 (b)). The cross-section of the flat fibers is



larger due to the absence of the vertical hydrodynamic force from the sheath fluid to the core fluid in the microchannel without the chevron grooves. Because the AHPCs had a better adherence on the PEGDA fibers compared to MSCs, AHPCs were cultured on PEGDA fibrous scaffolds precoated with poly-L-ornithine and laminin IV for 7 DIV. Results show grooved PEGDA fibers had significantly more attached AHPCs compared to the smooth fibers. Interestingly, there was no significant difference for cell adhesion when comparing planar controls and smooth PEGDA fibers (Figure 7 (c)). These results indicate grooved PEGDA fibers may provide greater support for cell adhesion of AHPCs due to the larger surface area of these fibers. In addition, the groove may also serve to reduce culture media movement across the inner surfaces of the grooved area of the microfibers and thus protect cells that have attached within the grooved region. Finally, a Propidium iodide (PI) live/dead cell assay was performed to assess the extent of possible cell death on PEGDA fibers (Figure 8). In comparison to the ethanol controls shown in Figure 8 (a1-a3 and c1-c3), very few PI-stained AHPCs were observed on smooth and grooved PEGDA fibers (Figure 8 (b1-b3 and d1-d3)), indicating extensive cell survival for AHPCs cultured on both smooth and grooved PEGDA fibers.

CONCLUSIONS The microfluidic approach coupled with the photopolymerization strategy was successfully used to fabricate PEGDA spherical particles as well as bow tie shaped fibers using immiscible and miscible fluids, respectively. The shear force at the core/sheath fluid interface was changed by using different core to sheath flow rate ratios of 50:2 to 100:100 to accurately tune the diameter of


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the particles and width of the fibers. The microfluidic fiber fabrication process was simulated, and the results were validated with the experimental data. Changing the core flow rate affected the mechanical properties of the fibers; as it increased, the stress at break and Young’s modulus enhanced due to the increase of the cross-link density. Fibrous scaffolds were created from the bow tie-shaped fibers in order to create a three-dimensional condition that can mimic the actual physiological environment in which the scaffold can be used for tissue engineering and regenerative medicine. It was found that the PEGDA fibers support the proliferation of MSCs and AHPCs. The grooves designed on the microfibers was shown to help localize cells to these regions of the microfibers. It was found that the grooves on the structure of the fibers can enhance AHPCs adhesion on the fibers compared to flat fibers and planar control. This unique design may serve not only to protect cells from disruption during in vitro culturing, but also during cell transplantation procedures, thus enhancing cell survival.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publication website at DOI: XXXX Microfluidic particle and fiber fabrication and generation of particles with different size by changing the flow rate ratio between the core and sheath fluids (Supporting Information 1).

AUTHOR INFORMATION Corresponding author: * E-mail address: [email protected], ORCID: Nicole Hashemi: 0000-0001-8921-7588



Present Address: † Department of Biomedical Engineering, Purdue University, West Lafayette, IN 47607

ACKNOWLEDGEMENT This work was partially supported by the Office of Naval Research Grant N000141612246, and Office of Naval Research Grant N000141712620. This research used the resources of W. M. Keck Metabolomics Laboratory at Iowa State University. The Authors would also like to thank Madeline Mathis for her help in creating the platform for the microfluidic fiber fabrication process using photo-polymerization. Lasitha Kurukula Arachchi and Ashley Christopherson are also acknowledged for illustrating the cover art and schematic of our particle and fiber fabrication technique, respectively.

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(63) Cristini, V.; Tan, Y.-C. Theory and Numerical Simulation of Droplet Dynamics in Complex Flows-a Review. Lab on a Chip 2004, 4 (4), 257-264, DOI: 10.1039/B403226H. (64) Teh, S.-Y.; Lin, R.; Hung, L.-H.; Lee, A. P. Droplet Microfluidics. Lab on a Chip 2008, 8 (2), 198-220, DOI: 10.1039/B715524G. (65) Shin, S.; Park, J. Y.; Lee, J. Y.; Park, H.; Park, Y. D.; Lee, K. B.; Whang, C. M.; Lee, S. H. "On the Fly" Continuous Generation of Alginate Fibers Using a Microfluidic Device. Langmuir 2007, 23 (17), 9104-9108, DOI: 10.1021/la700818q. (66) Jeong, W.; Kim, J.; Kim, S.; Lee, S.; Mensing, G.; Beebe, D. J. Hydrodynamic Microfabrication Via "on the Fly'' Photopolymerization of Microscale Fibers and Tubes. Lab on a Chip 2004, 4 (6), 576-580, DOI: 10.1039/b411249k. (67) Wu, C. J.; Wilker, J. J.; Schmidt, G. Robust and Adhesive Hydrogels from Cross‐Linked Poly(Ethylene Glycol) and Silicate for Biomedical Use. Macromolecular Bioscience 2013, 13 (1), 59-66, DOI: doi:10.1002/mabi.201200362. (68) Nakayama, Y.; Matsuda, T. Photocurable Surgical Tissue Adhesive Glues Composed of Photoreactive Gelatin and Poly(Ethylene Glycol) Diacrylate. Journal of Biomedical Materials Research 1999, 48 (4), 511-521, DOI: doi:10.1002/(SICI)1097-4636(1999)48:43.0.CO;2-V. (69) Zhao, J.; Yu, P.; Dong, S. The Influence of Crosslink Density on the Failure Behavior in Amorphous Polymers by Molecular Dynamics Simulations. Materials 2015, 9 (4), 234. (70) He, J. Y.; Zhang, Z. L.; Kristiansen, H.; Redford, K.; Fonnum, G.; Modahl, G. I. Crosslinking Effect on the Deformation and Fracture of Monodisperse Polystyrene-Co Divinylbenzene Particles. eXPRESS Polymer Letters 2013, 7 (4), 365-374. (71) Wu, J.; He, J.; Odegard, G. M.; Zhang, Z. Effect of Chain Architecture on the Compression Behavior of Nanoscale Polyethylene Particles. Nanoscale Research Letters 2013, 8 (1), 322, DOI: 10.1186/1556-276X-8-322. (72) Martínez‐Díaz, G. J.; Nelson, D.; Crone, W. C.; Kao, W. J. Mechanical and Chemical Analysis of Gelatin‐Based Hydrogel Degradation. Macromolecular Chemistry and Physics 2003, 204 (15), 1898-1908, DOI: doi:10.1002/macp.200350042. (73) Haryanto; Kim, S.; Kim, J. H.; Kim, J. O.; Ku, S.; Cho, H.; Han, D. H.; Huh, P. Fabrication of Poly(Ethylene Oxide) Hydrogels for Wound Dressing Application Using E-Beam. Macromolecular Research 2014, 22 (2), 131-138, DOI: 10.1007/s13233-014-2023-z. (74) López-Barrón, C. R.; Beltramo, P. J.; Liu, Y.; Choi, S.-M.; Lee, M.-J. Mechanical, Dielectric and Structural Characterization of Cross-Linked Peg-Diacrylate/Ethylammonium Nitrate Ionogels. Polymer 2016, 87, 300-307, DOI: https://doi.org/10.1016/j.polymer.2016.02.008.



(a)

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(c)

(b)

(e)

(d)

Scheme 1 Schematic of the microfluidic particle/fiber fabrication using a photopolymerization strategy: (a) Microparticle fabrication using immiscible fluids. (b) Bow-tie shaped-microfiber fabrication using miscible fluids. (c) The photopolymerization process of PEGDA hydrogels. (d) Enlarged view showing the arrangement of the microchannel, UV light guide, and collection bath.


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(a)

(b)

1 cm

2000

Diameter (m)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(c)

1500

1000

500

0

50:2

50:5

100:20

100:40

100:80

Flow Rate Ratio (Sheath:Core)

Figure 1 Microfluidic spherical PEGDA particle fabrication. (a) Experimental set-up: the microchannel is vertically positioned and the UV light is irradiating at the outlet of the channel. (b) SEM image of a particle made by the sheath-to-core flow rate of 100:40. (c) The diameter and (d) frequency of formation of the particles made by different flow rate ratios.



2 cm


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Page 31 of 40

400 350 300

Size (m)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


250

Knot Height Bow Height Width Width (Numerical) Bow Height (Numerical) Knot Height (Numerical)

(g)

200 150 100 50 0

100:40

100:60 100:80 100:100 Flow Rate Ratio (Sheath:Core)

Figure 2 Microfluidic PEGDA fiber fabrication. (a) The experimental set-up, similar to the set-up used for particle fabrication. (b) The longitudinal SEM image of the PEGDA fibers made by the sheath-to-core flow rate of 100:100 μL/min. (c1, 2-f1, 2) The cross-sectional SEM images of the fibers made by different flow rate (μL/min) ratios: (c) 100:40, (d) 100:60, (e) 100:80, and (f) 100:100. (c3-f3) The numerical results made by simulating the microfluidic fiber fabrication. Scale bar = 50 µm (g) Dimensions of the cross-section of PEGDA fibers made by different sheath-tocore flow rate ratios obtained from experimental and numerical results.



Figure 3 Tensile properties of PEGDA fibers fabricated with different flow rate ratios between the core and sheath fluids. The concentrations of PEGDA and PI for all the fibers were kept constant at 30 % and 2%, respectively.


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Figure 4 MSCs on PEGDA hydrogels. (a1-c1) Fluorescent images of MSCs illustrating immunoreactivity for (a1) Ki-67, (b1) Collagen and (c1) Fibronectin. (a2-c2) represent corresponding images merged with DIC. Scale bar = 50 µm. Abbreviations: DAPI, 4',6diamidino-2-phenylindole; DIC, differential interference contrast.



Figure 5 AHPCs on PEGDA hydrogels. (a1-c1) Fluorescent images of AHPCs illustrating immunoreactivity for (a1) Ki-67, (b1) Nestin, and (c1) TuJ1. (a2-c2) represent corresponding images merged with DIC. Scale bar = 50 µm. Abbreviations: TuJ1, βIII-tubulin; DAPI, 4',6-diamidino-2phenylindole; DIC, differential interference contrast.


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Figure 6 SEM images of the (a1-a2) MSCs and (b1-b2) AHPCs cultured on the bow tie-shaped PEGDA fibers. (a1-b1) Scale bar = 100 µm and (a2-b2) Scale bar = 50 µm.



(c)

Figure 7 SEM images of (a) bow tie-shaped and (b) flat fibers fabricated using a microfluidic channel with and without chevrons, respectively. (c) The cell density of AHPCs growing on PEGDA fibers. Fibrous scaffolds with grooved PEGDA samples had a higher cell density versus the flat PEGDA fibers and planar control samples. Error bars represent standard deviation. N = 1 independent experiment with 1 sample per condition. For analysis, 5 imaging fields were analyzed/sample. *Significantly different at p ≤0.05.


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Figure 8 Propidium iodide (PI) cell viability assay for AHPCs cultured on smooth and grooved PEGDA fibers. As a PI reagent control, AHPCs growing on PEGDA fibers were subjected to an ethanol incubation to cause cell death (a1-a3 and c1-c3). (Column a1-d1) Dead cells were stained red for PI. (Column a2-d2) Cell nuclei stained with DAPI (blue). (Column a3-d3) Merged images of PI-staining (red), DAPI nuclei counterstaining (blue), and DIC. Scale bar = 50 µm. 37 ACS Paragon Plus Environment


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Table 1 Antibodies used for immunolabeling MSCs and AHPCs on PEGDA hydrogels. Cell Type

Antibody

Dilution

Vendor

Marker

MSC and

Rab α Ki67

1:200

Abcam

Proliferation

Rab α CD29

1:1000

R&D

Cell Surface Adhesion

Systems

Molecules (Integrin β1)

R&D

Extracellular matrix

AHPC MSC

Rab α Collagen type I

1:200

Systems Rab α Fibronectin

1:1000

R&D

Extracellular matrix

Systems AHPC

Mo α Nestin

1:200

DSHB

Neural progenitor cells

Mo α TuJ1

1:200

R&D

Immature neurons

Systems

(Class III β-tubulin)


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Table 2 Tensile properties of the PEGDA fibers made with different flow rate ratios Flow Rate Ratio (Sheath:Core) 100:40 100:60 100:80

Stress at Break (MPa) 5.6 ± 1.1 5.9 ± 0.6 7.4 ± 2.1

Strain at Break (%) 43.6 ± 5.0 39.7 ± 2.7 34.2 ± 7.1


Young’s Modulus (MPa) 10.3 ± 1.1 14.1 ± 1.4 16.8 ± 1.2


For Table of Contents Only


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Photo-Cross-Linked Poly(ethylene glycol) Diacrylate Hydrogels

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