Method for the Fabrication of Elastomeric Polyester Scaffolds for

Subscriber access provided by MT ROYAL COLLEGE

Methods/Protocols

A Method for the Fabrication of Elastomeric Polyester Scaffolds for Tissue Engineering and Minimally Invasive Delivery Miles Montgomery, Locke Davenport Huyer, Dawn Bannerman, Mohammad Hossein Mohammadi, Genevieve Conant, and Milica Radisic ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.7b01017 • Publication Date (Web): 21 Feb 2018 Downloaded from http://pubs.acs.org on February 25, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Biomaterials Science & Engineering is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 33 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

ACS Biomaterials Science & Engineering

A Method for the Fabrication of Elastomeric Polyester Scaffolds for Tissue Engineering and Minimally Invasive Delivery Miles Montgomery1,2, Locke Davenport Huyer1,2, Dawn Bannerman1,2, Mohammad Hossein Mohammadi1, Genevieve Conant1, Milica Radisic1,2,3* 1

Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario, Canada Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Ontario, Canada 3 Toronto General Research Institute, University Health Network, Toronto, Ontario, Canada

2

Abstract Using the methods described herein, we have demonstrated how scaffolds can be designed for a number of applications including tissue engineering, biomedical devices and injectable tissues. Details on the methods of polymerization, physical and chemical characterization of poly(octamethylene maleate (anhydride) citrate (POMaC) are described. Two POMaC polymer recipes with different monomer ratios of maleic anhydride and citric acid and used and compared. Mechanical testing was performed on scaffolds of two distinct anisotropic designs to show how scaffold design influences the apparent elasticity in the long and short axis. POMaC scaffolds of various patterns and geometries were fabricated to demonstrate: 1) scaffold function can be determined by scaffold design (e.g. inherent shape-memory or self-assembling tubular structures), and 2) the soft lithography approach to fabricating biodegradable elastomers described here can be used to suit a number of different potential applications.

Keywords: biomaterials, polymers, polyesters, microfabrication, soft lithography, scaffolds, shapememory, photocrosslinkable

*

Corresponding author: Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Ontario M5S 3G9, Canada. Tel.: +1-416-946-5295. Fax: +1-416-978-4317. E-mail address: [email protected].

ACS Paragon Plus Environment

ACS Biomaterials Science & Engineering 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

Table of Contents Abstract .................................................................................................................................... 1 Introduction ............................................................................................................................. 3 Materials .................................................................................................................................. 7 Reagents ............................................................................................................................................ 7 Polymer synthesis and characterization .................................................................................................... 7 Microfabrication ....................................................................................................................................... 7 Equipment ......................................................................................................................................... 7 General...................................................................................................................................................... 7 Polymer synthesis and characterization .................................................................................................... 7 Microfabrication ....................................................................................................................................... 8

Methods .................................................................................................................................... 9 Polymer synthesis .............................................................................................................................. 9 Polymer characterization.................................................................................................................. 12 NMR spectroscopy ................................................................................................................................. 12 FTIR measurement ................................................................................................................................. 12 Formulating the POMaC pre-polymer solution .................................................................................. 13 Microfabrication of scaffolds ............................................................................................................ 14 Fabrication of the SU-8 master mold ...................................................................................................... 14 Curing POMaC in PDMS moulds .......................................................................................................... 15 Mechanical characterization of POMaC Scaffolds .............................................................................. 18 Tensile testing using Myograph .............................................................................................................. 18 Using Matlab to automatically extract 7-segment display ..................................................................... 19

Anticipated Results ................................................................................................................ 23 Conclusion .............................................................................................................................. 26 Supplemental Information ..................................................................................................... 28 Movies ............................................................................................................................................. 28 Files ................................................................................................................................................. 28

References .............................................................................................................................. 29 For Table of Contents Graphic .............................................................................................. 33


Page 2 of 33

Page 3 of 33 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


Introduction Over the past decade, the fields of tissue engineering, stem cell biology, and biomaterials have converged, promising an exciting future for regenerative and precision medicine.1 Continued progress has been by made through simultaneous improvements in our understanding of fundamental cellular biology and tools enabling control over cellular microenvironments through physical cues and paracrine signalling. Scaffolds play an integral role in guiding and promoting functional tissue assembly by providing topographical, biological, and mechanical cues.2-6 Leveraging scaffold design, researchers have designed biomaterials for studying mechanobiology7; developing improved medical device,8-10 cell11-15 and tissue engineering based therapies16-19; and building organ-on-a-chip models including skin, lung, gastrointestinal tract, liver, kidney, heart, cornea, eye, blood vessels, spleen, pancreas, blood-brain-barrier, bone, bone marrow, placenta, female reproductive tract, and fat among several others.20-22 Several scaffold design criteria must be optimized to suit the intended biomedical application including: biocompatibility, mechanical properties, architecture (complex topographies/pattern/geometries), surface energy, biodegradation (rate, by-products, mechanism), porosity, and chemical composition. In order to satisfy these multi-factor design constraints, we have developed an adaptable method using microfabrication for precise control over structure and topography, and precisely tuned poly(octamethylene maleate (anhydride) citrate) (POMaC), a biodegradable elastomer, to meet desired functional properties. Here we will provide a detailed protocol for the microfabrication of such scaffolds, with the functional basis of a POMaC biomaterial. This material, originally developed by Tran et al., is a biodegradable elastomer that possesses both radical and condensation polymerization susceptible reactive moieties.23 The material is developed through one-pot bulk condensation polymerization, generating a liquid material that may be postpolymerized through multiple mechanisms. The vinyl groups in the polymer backbone allow for free radical polymerization with the addition of an ultra-violet (UV) sensitive photoinitiator and UV light. Pendant carboxylic acid and alcohol groups can be post-polymerized to form ester bonds, which with the addition of heat to accelerate the reaction, further crosslinking the polymer. Furthermore, the ester bonds are susceptible to hydrolytic degradation. As such, the rate of POMaC degradation can be tuned by modifying the monomer feed ratios to produce polymers with different amounts of ester bonds and vinyl groups.23 Furthermore, the pendant functional groups (hydroxyl and carboxylic acid) that are present in the POMaC allow for potential conjugation of bioactive peptides or proteins24. Scaffolds should ideally match the mechanical properties of the native tissue of interest. The high Young’s modulus of common materials from the polylactone family (~0.65-2.7 GPa25-26), often found in FDA approved devices, limits their applicability in developing solutions for engineered soft tissues.23, 27-28 For reference, the elasticity of soft tissues such as the healthy human liver ranges from 0.2-6 kPa,29-31 ~64-112 kPa for the bovine spinal chord,32 and ~20-500 kPa for the human myocardium over diastole and systole states.33-36 In contrast, stiff tissues can range between ~0.671.8 MPa for human articular hip cartilage37 and ~10.4-20.7 GPa for trabecular and cortical bone.38 Compliant biomaterials that intently match the local mechanical properties of the native tissue allow deformation and cellular arrangement to take place without significant irritation.39-40 Substrate



rigidity is sensed by many cell types and has strong influence over cellular behaviours affecting cytoskeletal arrangement,41 migration,41 stem cell differentiation,42 extracellular matrix deposition,43 and muscle contractility.5, 44 Importantly, softer materials may be more suitable for in vivo purposes as they can lead to a reduction in the activation of macrophages and foreign body response.45 Thus, soft, elastic, biodegradable materials, such as POMaC, that can be tuned to match the mechanical properties of native tissues are a great asset. We have found additional benefits to using POMaC as a base material as it yields properties of a soft elastomer, making it well-suited for supporting and allowing for the contractile behaviour of cardiac tissue. POMaC is also robust for handling and manipulation with tweezers, unlike some hydrogels that may be fragile or difficult to handle. Additionally, POMaC’s capacity for photocrosslinking enables a broad range of material geometries through micromolding in capillaries, a process used in conjunction with standard photo and soft lithographic techniques to produce materials with features that can be controlled in the micron-scale. We have found this fabrication process to be useful as the many degrees of freedom the researcher has to adapt the microfabrication process to their specific research project goals, primarily through changing the physical design of the scaffold or by tuning the elastomeric material. With a view of in vivo application, anisotropic POMaC scaffolds served as a guide to cardiac tissue assembly as cells compacted around scaffold struts.46 By introducing T-shaped hooks that protruded from the surface of scaffolds, tissues could be “clicked” together into desired horizontal or vertical patterns (Figure 1a).46 Montgomery et al. developed a flexible shape-memory scaffold capable of delivering functional tissues in vivo in a minimally invasive manner, without the need for a major surgery (Figure 1b).47 POMaC scaffolds of various designs were fabricated using the methods described herein to identify the scaffold design that produced optimal shape-memory and anisotropic mechanical (69.3 ± 17.4 kPa and 14.7 ± 1.56 kPa in long and short direction, respectively) properties that closely matched the adult rat myocardium (43 ± 9 kPa and 12 ± 5 kPa in long and short myofiber direction, respectively).47-48 Using a rat myocardial infarction (MI) model, the hearts that received cardiac patches cultured on the POMaC scaffold had significantly improved cardiac function six weeks post-MI (5 weeks of patch therapy). A similar biomaterial host response was seen when comparing POMaC and polyethylene glycol (PEG) scaffolds of identical shape implanted onto the healthy rat epicardium.47 The applicability of the microfabricated scaffolds can be extended further still by modifying the POMaC material to tune biodegradation, surface energy, porosity, and mechanical properties. By adjusting the levels of factors such as the monomer ratio of citric acid to maleic anhydride, porogen content, UV energy, and heat energy post-UV exposure, the Young’s Modulus of the bulk POMaC can be tuned from 50-1423 kPa.48 Moreover, the fabrication process can be achieved with biomaterials other than POMaC. For example, poly(octamethylene maleate (anhydride) 1,2,4-butanetricarboxylate) (124-polymer) was designed for cardiac tissue applications to provide a high degree of elasticity (Figure 1c).49 124polymer scaffolds made using the aforementioned microfabrication process supported the formation of contractile cardiac tissue, were non-cytotoxic, and elicited a similar host response subcutaneously compared to poly(l-lactic acid) (PLLA) controls. To improve the electrical conductivity and structural integrity of scaffolds, carbon nanotubes (CNTs) were added to 124-


Page 4 of 33

Page 5 of 33 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


polymer (Figure 1d).50 Cardiac tissues cultured on 124-polymer scaffolds with CNTs led to tissues with significantly reduced excitation thresholds compared to 124-polymer only controls, suggesting a higher degree of tissue maturation was achieved with improved conductance. Here we provide detailed methods on the microfabrication of elastomeric POMaC scaffolds that we have published in previous work. Exemplary results for two POMaC polymers synthesized from different monomer ratios and examples of various scaffold designs are presented. We expect the information presented in this manuscript will provide researchers the ability to design scaffolds to suit a diverse number of potential applications.



Figure 1. Microfabricated scaffolds for organ-on-a-chip engineering, tissue engineering and minimally invasive delivery. a, Zhang and Montgomery et al. developed a hook-and-loop system for interlocking scaffolds together to facilitate the on-demand assembly of tissue patches.46 Reproduced with permission from reference 46. Copyright 2015 Science Advances. b, Montgomery et al. generated a shape-memory scaffold capable of supporting the formation of functional engineered tissue patches that can collapse and be injected through an orifice as small as 1mm and


Page 6 of 33

Page 7 of 33 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


regain their initial shape, allowing for the minimally invasive delivery of functional tissues.47 Reproduced with permission from reference 47. Copyright 2017 Nature Materials. c, Davenport Huyer et al. synthesized a novel biodegradable, highly elastic polyester that exhibited favourable host response compared to PLLA. This biomaterial, known as 124 polymer, was photocrosslinkable and anisotropic scaffolds made of 124 polymer were capable of supporting functional cardiac tissue.49 Reproduced with permission from reference 49. Copyright 2015 American Chemical Society. d, Ahadian and Davenport Huyer et al. dispersed CNTs into 124 polymer to yield scaffolds with improved conductance, which supported functional cardiac tissue assembly.50 Reproduced with permission from reference 50. Copyright 2017 Elsevier.

Materials Reagents Polymer synthesis and characterization - 1,8-octanediol (OD) (Sigma-Aldrich, O3303) - Maleic anhydride (MA) (Sigma-Aldrich, 63200) - Citric acid (CA) (Caledon Laboratory Chemicals, 2980170) - Deionized distilled water (MilliQ, Millipore, USA) - Ethanol (Commercial Alcohols, P016EAAN) - 1,4-Dioxane (Sigma Aldrich, CAS# 123911) - Deuterated dimethyl sulfoxide (DMSO-d6) (Sigma-Aldrich, 156914) - Nitrogen gas - Silicone oil (Sigma-Aldrich, 146153) - Compressed air (optional) - Liquid nitrogen (optional) Microfabrication - Sylgard 184 Silicone Elastomer Kit (Dow Corning, USA, 3097358-1004) - 2-hydroxy-1-[4(hydroxyethoxy)phenyl]-2-methyl-1-propane (Irgacure 2959) (Sigma Aldrich, 410896) - Poly(ethylene glycol) dimethyl ether (PEGDM) (Mn ~ 500, Sigma-Aldrich, 445886) - 4” or 6” Silicon Wafers - SU-8, SU8-2000 Series (MicroChem, USA) - SU8 Developer (MicroChem, USA) Equipment General - Lab coat - Gloves - Safety goggles Polymer synthesis and characterization - Triple-neck flask (250-500mL recommended) - Magnetic stir bar (35mm)



-

-

Large Pyrex dish for oil bath Aluminum foil Temperature controlled oil bath with stir plate Mass Balance (0.0001g sensitivity) Rubber stoppers Needles (25G) Proton nuclear magnetic resonance (1H-NMR) spectrometer (e.g. Agilent DD2 spectrometer, Agilent, USA) NMR Analysis Software (e.g. MNova software v. 9.0.0 (Mestrelab Research, Spain)) NMR tubes Attenuated total reflectance-Fourier-transform infrared (ATR-FTIR) Spectrometer (e.g. Spectrum One FTIR spectrometer with a fast recovery deuterated triglycine sulfate detector, PerkinElmer Inc., USA) FTIR Analysis Software (e.g. OMNIC Series Software, Thermo Scientific, USA) Laboratory stand Laboratory clamp Separatory funnel 1000mL beaker Glass container for sample Lyophilizer (optional)

Microfabrication -

AutoCAD (2013 or newer recommended) (Autodesk, USA) Photomask printer or outsource (e.g. CAD/Art Services, Inc., USA) Spin coater UV safety goggles Clean suit Oven Vacuum Chamber Polystyrene dish Mass balance (0.01g and 0.0001g sensitivity) Nitrogen feed UV mask aligner with 365nm bulb (Model 30 UV Light Source, OAI, USA) UV 365nm filter UV intensity sensor Uniform hot plates (65oC and 95oC) Orbital shaker (developing) Glass cylindrical container (for developing SU8 master molds) Stereoscope Bright field microscope Razor blades Scalpel Glass slides (e.g. 75x50mm, Corning, 2947-75X50) Scotch tape Syringe (optional)


Page 8 of 33

Page 9 of 33 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


-

Tygon tubing (optional) Tensile tester (e.g. Myograph (Kent Scientific, USA))

Methods Polymer synthesis POMaC is synthesized via bulk melt polyesterification (step-growth polymerization). A random branched network polymer is formed through reactions between monomer units based on OD and MA being difunctional and CA possessing a functionality of 4. The mechanisms of step-growth polymerization dictate that a high degree of polymerization is required for high chain length. As the reaction proceeds the viscosity of the melt will slowly increase initially with time and then exponentially increase as the critical extent of polymerization (Pc) or gel point is reached for the step-growth polymerization of a branched polymer. Thus, care must be taken to monitor the progress of the polyesterification and to stop the reaction prior to reaching the Pc, as beyond this stage the polymer will become a solid, making it unusable for the injection molding we require. One method for predicting an approximate Pc is performed by rearranging the modified Carothers equation for branched polymers51 as follows: =

=

∙

(1)

− ∙

(2)

is the average degree of polymerization, = where

∑ ∑

is the average functionality, where

and represent the moles and functionality of the i monomer, respectively, and P is the extent approaches infinity and equation 2 reduces to: of reaction. As the theoretical gel point occurs, th

=

(3)

Therefore, 1:4 and 3:2 CA:MA (mol:mol) POMaC recipes will have a theoretical of approximately 90.9% and 70.9% (,: = 11/5, , : = 13/5), respectively. Alternative theoretical models could be used, and ultimately experimental models must be conducted to determine the precise ; however, we chose this simple model to indicate directionally the influence of the determined by the CA:MA ratio used. In practical terms this suggests that when carrying out the 3:2 polymerization a more viscous solution will result after less polymerization time due to the greater degree of branching. The polymerization has traditionally been carried out in our lab with equimolar proportions of alcohol and carboxylic acid functional groups. However, nonstoichiometric ratios of alcohol to carboxylic acid groups could be investigated to explore the potential to control the degree of polymerization. To prepare a new batch of POMaC, the initial moles of each monomer in the feed can be calculated using stoichiometry with alcohol (OH) and carboxylic acid groups (COOH) equimolar as follows: ∑ ,"# = ∑ ,$""# ACS Paragon Plus Environment

(4)


2"#' "& + 1"#' $) + 0"#' "&

$)

+)

+) = 0$""#'

+) =

"& =

"&

,-

"& + 3$""#' $) + 2$""#'

.,-'

$)

+)

+) (5) (6)

/-

,- ∙0

Page 10 of 33

1 3 2,'/-

(7)

where ni,x is the initial moles of either OH or COOH added to the reaction vessels for the ith monomer, nx represents the initial moles of either OD, CA, or MA added to the reaction vessel, and 4$)' is the molar ratio of CA to MA monomer in the feed. Therefore, to calculate the moles of +) each monomer, a total batch size (mass) can be selected (e.g. 30g) and the moles of each monomer can be calculated. Alternatively, the researcher may choose a basis mass of CA, and calculate the appropriate moles of each monomer. 1. The bulk melt polyesterification reaction takes place in a triple-neck flask in a temperature controlled oil bath at 140oC on a magnetic stirrer (Figure 2a,i). Pre-heat the oil bath to 160oC (to melt all the monomers initially), the reaction is continuously stirred at 200RPM. Troubleshooting Tip: Use a digitally controlled hotplate for precise temperature control to avoid the risk of evaporating OD; melting point of CA is ~156°C, which is close to the 172°C boiling point of OD. 2. Weigh each monomer (OD, CA, MA) carefully according to the desired ratio (example amounts for molar ratio of 1:4 or 3:2 CA:MA given in Table 1). Add each to the triple-neck flask, add a magnetic stir bar, and mix by hand to make a uniform distribution. Troubleshooting Tip: The total mass of the polymer batch can be increased or decreased to suit the amount of polymer needed. Set-up a spreadsheet with the mass of CA as an input, to quickly adjust the amount of CA to get the desired total mass of the batch. 3. Attach rubber stoppers to the triple-neck flask. Develop a nitrogen flow setup in the angled necks, with an inlet flow from a gas flow line and an outlet relief needle in opposing necks. (Figure 2a,i). 4. Once a clear liquid solution has formed (indicating the monomers have melted) set the oil bath to140oC for 3 hours for POMaC 1:4 or ~2 hours for POMaC 3:2 and protect from light with tin foil. Once polymerization is complete a viscous and optically clear polymer will have formed (Figure 2a,ii). Turn off oil bath and remove flask to allow contents to cool. Troubleshooting Tip: Viscosity does not increase appreciably after extended polymerization (>3 hours for POMaC 1:4 or >2 hours for POMaC 3:2): - Once a clear solution has formed, immediately reduce temperature to improve batch-tobatch polymerization consistency. - Ensure the temperature in the reaction vessel is at 140°C. If the temperature is too low reaction kinetics will be reduced, whereas if the temperature is too high OD monomers may evaporate. - Ensure the nitrogen purge is running continuously throughout the reaction to help remove water vapor to drive the reaction forward (Le Chatelier's principle).


Page 11 of 33 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


-

5.

6.

7.

8.

9.

Dry triple-neck flask in oven prior to polymerization to ensure no moisture present in the flask. - Take care when measuring out the mass and transferring each monomer to the tripleneck flask to avoid a stoichiometric imbalance. To reduce risk of spilling monomer, prepare a funnel to assist transferring of monomer from weigh boat to triple-neck flask. In order to remove any unreacted monomer or oligomer, dissolve the contents of the tripleneck flask in ~30-50mL of 1,4-dioxane (wait for the flask to cool for 10-15min at room temperature prior to adding solvent), allow the polymer to dissolve with the help of stirring, and transfer solution into a separation funnel (Figure 2b,i). Troubleshooting Tip: To clean glass vessels that came into contact with the POMaC, soak them in concentrated NaOH to quickly degrade POMaC Begin purifying via drop-wise precipitation by opening the separatory funnel slightly allowing drops to fall at ~1 drop per second into room temperature deionized distilled water in a 1 or 2L beaker (Figure 2b,ii, Movie 1). After drop-wise precipitation is complete, carefully decant the water from purified polymer that accumulates at the bottom of the beaker. Remove as much water as possible without losing the polymer (pour water into an appropriate chemical waste bottle) and transfer the remaining polymer into a glass jar. Troubleshooting tip: When decanting, it is important to move in one direction of flow. Once decanting has started, continue to pour slowing until you have removed as much water as possible. Stopping and starting will disturb the separation and reduce the yield significantly. To remove remaining solvent and water, either snap-freeze and lyophilize for 72 hours or dry under air flow (~24-72 hours depending on POMaC batch size and how much residual water/1,4-dioxane present). The purified polymer should appear optically clear and free from solvent odour. The obtained purified polymer is a relatively transparent and viscous liquid. (Figure 2c, Movie 2). Store at 4oC and protect from light. A shelf-life is 4-6 months at 4oC in the dark is recommended, however, the actual shelf-life may be longer. Troubleshooting tip: The purified polymer may optionally be stored with a desiccant to potentially increase shelf-life if longer storage times are desired.



Table 1. Summary of structure and physical properties of monomers used in polyesterification reaction of POMaC and example recipes with monomer proportions.

Polymer characterization NMR spectroscopy 1. 2. 3. 4.

Dissolve polymer samples in deuterated dimethyl sulfoxide (DMSO-d6) at ?@ A%6 B, where < is the absorbance (a.u.). 6. Plot spectrum and reference literature to identify and assign peak locations to functional groups.

Formulating the POMaC pre-polymer solution Purified POMaC can be photocrosslinked through a free radical polymerization by adding a photoinitiator (such as Irgacure 2959) and exposing the sample to UV light. A porogen, such as poly(ethylene glycol) dimethyl ether (PEGDM), is miscible with POMaC and water (allowing it to be leached out after POMaC has been crosslinked) and can be mixed with POMaC to: 1) improve the workability of the POMaC, which is relatively viscous, and 2) control the porosity of POMaC, which in turn will modify POMaC Young’s Modulus and diffusivity (higher porosity for increased mass transfer). 1. Place a known mass of POMaC into a glass container and add PEGDM at desired proportion (e.g. typically in the range of 0-80wt%). Mix thoroughly. (e.g. 2g POMaC, 3g PEGDM for a batch containing 60% porogen) 2. Dissolve Irgacure 2959 at 5wt% (of the total mass). Be sure all of the Irgacure is dissolved by thoroughly mixing until no small particulates are seen. If needed, the solution can be heated slightly on a hotplate to ~60-120oC while mixing. It may take 5-10minutes of mixing to fully dissolve the Irgacure. Minimize prolonged exposure at elevated temperature to avoid further cross-linking through polycondensation. Protect from light with tin foil/opaque tape. 3. Store at room temperature if using within 2 weeks, otherwise store in the dark at 4oC and use within 6 weeks to prevent undesired polymerization. Troubleshooting tip: If the POMaC pre-polymer sample has been stored in fridge at 4oC, allow sample temperature to reach room temperature and mix solution to avoid possibility of phase separation during storage.



Figure 2. Polymer synthesis and purification. a, i) Bulk melt polyesterification set-up used for POMaC synthesis in a triple-neck flask and digital temperature controlled oil bath. ii) Final polymerization product is a clear viscous polymer solution, b, i) Drop-wise purification set-up for POMaC into deionized distilled water. ii) Purified droplets of POMaC fall and accumulate at the bottom of the flask. c, Collected purified POMaC before and after lyophilizing or air drying to remove excess solvent. Final solution is optically clear and no odor of the solvent should be noticeable. Microfabrication of scaffolds A visual overview of the process of the microfabrication steps for producing a scaffold is shown in Movie 3. Fabrication of the SU-8 master mold 1. Use AutoCAD to create photomask designs for a silicon wafer size of your preferred diameter (e.g. 4”). Prepare photomask designs such that when PDMS is cured over the SU-8 master mould, negative scaffold features appear in the PDMS. Include injection ports extending from base design (can be cut-off once scaffolds have been cured) (Figure 3a). Two example scaffold designs are shown in Figure 3 and the AutoCAD files are provided


Page 14 of 33

Page 15 of 33 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


(Supplemental Information File 1 and File 2). Note: If designing multilayered scaffolds (e.g. channels) include alignment markers on the mask. Troubleshooting Tip: To avoid issues when printing the photomasks, ensure there are no gaps in the AutoCAD design by performing the following commands: Select command Pedit Multiple Objects press Enter Convert Lines, Arcs and Splines to polylines Y (Yes) Join set a fuzz distance = 0 press Enter 2. Print photomasks using mask writer or through outsourcing (recommended dpi: 20,000). 3. In a cleanroom, perform microfabrication with SU-8 according to the manufacturer guidelines and follow conventional techniques52-53. We recommended beginning with a seed layer with SU-8 25. For improved control over feature thickness, use spin coating speeds (RPM) within the linear range from the SU-8 datasheets. Troubleshooting Tip: Only open silicon wafer box inside clean room and before spin coating SU-8 onto the silicon wafer always blow off any potential dust with nitrogen air. 4. Once the SU-8 master mould has been developed and dried, tape the master mould to the bottom of a polystyrene petri dish.

Curing POMaC in PDMS moulds This procedure outlines the steps for producing PDMS negative moulds. PDMS is first poured and degassed on the SU-8 master mould, allowed to cure, and carefully peeled-off/released from the mould. 1. Add a weight ratio of 10 parts PDMS base agent to 1 part PDMS curing agent to a mixing container and mix thoroughly. 2. The mixture is poured onto the SU-8 master mould, and place in the vacuum chamber to degas (~1 hour), ensuring all air bubbles have been dispersed. 3. The mould is removed from the vacuum apparatus and placed in the 80°C oven for 1 hour. Alternatively, the mixture can be left at room temperature to cure for 2 to 3 days. 4. Once cured, the PDMS may be removed from the master mould using a scalpel to score around the edge of the silicon wafer. Care should be taken to avoid damaging the PDMS or silicon wafer during its removal. 5. Cut/trim the PDMS to fit onto glass slides. Optional: Cut PDMS into individual rectangular squares so there is an individual scaffold design per PDMS rectangle as this can make it easier to remove PDMS moulds from the glass slide post-UV curing in subsequent steps. 6. Clean a glass slide and the feature side of the PDMS mould with tape to remove dust/particulates. 7. Cap the PDMS mould onto the glass slide feature side down, this will form a closed network for the scaffold (micromolding in capillaries). 8. Place a drop of POMaC pre-polymer over an inlet and allow POMaC to enter mould via capillary perfusion (usually left overnight to ensure complete perfusion). Troubleshooting tip: To ensure complete perfusion of POMaC throughout the PDMS mould, visually inspect the mould to observe POMaC has flowed through outlet and no air bubbles are visible. Alternatively, a syringe with a needle and Tygon tubing attached (over the needle) may be loaded with POMaC pre-polymer and the Tygon tubing end inserted into the PDMS inlet.



By applying pressure, the POMaC pre-polymer may be slowly injected into the PDMS mould. Troubleshooting Tip: POMaC will not flow through the PDMS mold: - POMaC viscosity is likely too high, try increasing porogen content or warming POMaC pre-polymer solution to reduce viscosity. - If relying on capillary force to perfuse PDMS with POMaC, glass slide can be placed on an angle to increase the rate of perfusion. - Try directly injecting POMaC pre-polymer via a syringe and tubing, if injecting manually by hand or syringe pump, inject very slowly otherwise pressure of the POMaC in the PDMS mould may cause the PDMS to rupture and detach from the glass slide. 9. For each batch of POMaC pre-polymer, expose scaffolds to a pre-determined amount of UV energy (typically around 3600mJ). After the dose of UV energy, the PDMS mould is removed and the photocrosslinked POMaC scaffold remains attached to the glass slide. Additional UV energy can be delivered following mould removal to tune final elasticity. Troubleshooting Tips: - When removing the PDMS from the glass slide post-UV exposure and POMaC is uncured, try increasing the UV exposure time. - When removing the PDMS mould from the glass slide and the POMaC scaffold is getting caught in the PDMS mould and/or scaffold is detaching from the glass slide: o Carefully peel PDMS at a slow and constant speed. o The scaffold may have been over exposed to UV, try reducing UV exposure time. o The PDMS may be too stiff, try decreasing the ratio of base:curing agent or baking for less time/lower temperature to make a softer PDMS. o Trial and error may be required to find the optimal UV cure time where the POMaC is sufficiently cured, but not over cured. If the POMaC is over exposed when the PDMS mold is removed, the POMaC may remain caught in the PDMS and detach from the glass slide, which will cause the scaffold to likely tear. Generally, for 1:4 POMaC with 60% porogen content, 3600mJ can be used for the first exposure and 3600mJ again after the PDMS has been removed. This exposure can be tuned depending on the batch. 10. Leave the scaffolds on the glass slide and optionally bake for the desired time (e.g. 80°C for 1 day) to further increase scaffold stiffness. 11. After baking, release the scaffold from the glass slide by soaking in PBS (~15min). A razor blade can be used to help release scaffold from the glass slide if needed. To leach porogen, place scaffolds into fresh PBS and soak for a minimum of 24 hours (e.g. in a 6-well plate), followed by three washes of fresh PBS.


Page 16 of 33

Page 17 of 33 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


Figure 3. AutoCAD images of photomask designs for two anisotropic scaffolds. a, Example AutoCAD photomask designs to fit onto 4” silicon wafers for a two-layered POMaC scaffold: a base scaffold layer (L1) and channel layer (L2). b-e, Higher magnification views of scaffold base layer (b, d) and channels (c, e), f-g, AutoCAD design of an alternative anisotropic scaffold design. Scale bars: 5cm (a), 5mm (b, c, f), 1mm (d, e, g).



Mechanical characterization of POMaC Scaffolds Tensile testing using Myograph In the following section, we describe the methods of performing tensile testing on POMaC scaffolds using a Myograph (Figure 4a) to determine apparent elasticity. This particular method of mechanical testing is outlined in detail as it is tailored to the scaffolds fabricated in this paper, which do not conform to standard methods of mechanical testing as the scaffolds are porous. This method is also useful, as the user may be able to build a simple mechanical tester, in lieu of Myograph, using a basic perfusion chamber, hooks, a force transducer and a simple video recording device such as a smart phone without the need to purchase expensive mechanical testers. Since the scaffold design and shape are determinants of its mechanical properties, developing a reliable procedure was necessary to evaluate the mechanical properties of these specific designs. Thus, apparent elasticity is determined rather than the true material property which would be determined using continuous slabs of material. It should be noted that care must be taken when working with this instrument as the force transducer can be damaged when subjected to forces higher than the rated capacity. 1. Position video recording device for optimal view of transducer and displacement readings. Ensure there is no glare and the digits are in focus. Once the camera height has been set (e.g. using retort stand and a clamp to position a smart phone) do not adjust height until all videos have been taken. This makes downstream video analysis much easier. 2. Ensure the voltage meter is on 20g, then carefully place 10x10x3mm (length x width x height) 3D printed platforms (or 10x10x5mm stiff PDMS slabs) onto each metal rod. Print platforms with a rigid plastic such as VeroWhite (Stratasys, USA). The POMaC scaffolds will be placed and attached onto the platforms to hold the sample during mechanical testing. Adjust the platform heights using the Myograph adjustable arms such that they are level. 3. Move platforms together until they just touch. Zero the displacement. 4. Spread the platforms to approximately 1.00mm (this measurement represents the original sample length, Lo). 5. Remove scaffold from solution (e.g. culture media or PBS) using tweezers and place across PDMS slabs in the orientation of interest. Blot dry using a Kimwipe. 6. Affix the scaffolds to the PDMS slabs using cyanoacrylate glue to the ends of the scaffold, ensuring that no excess glue is applied. Troubleshooting tip: Place a small drop of cyanoacrylate glue over both ends of the scaffold and wait 5 minutes for the glue to dry. Ensure even coverage of the glue over the scaffold to avoid scaffold slippage/detachment from the PDMS slabs during tensile test. Excess glue can be removed/moved using a dry 2-20uL pipette tip. 7. If mechanical testing under liquid conditions is desired, raise glass well and fill with preferred liquid and using a pipette, create a bubble of liquid over the scaffold. 8. Measure width of scaffold with calipers if exact width is unknown. 9. Adjust transducer sensitivity (i.e 2g, 10g, 20g) as desired such that voltage over the entire tensile testing remains between 0-5V (linear range of the transducer). Typically, for a POMaC 1:4 scaffold 2g/5V settings are used. 10. On voltage box, adjust knob so that voltage reading is about 0.0. 11. Start recording device.


Page 18 of 33

Page 19 of 33 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


12. Slowly turn displacement knob until material failure at a strain rate of 1 mm/mm⋅min. Stop recording once test complete. Troubleshooting Tip: Scaffold is slipping from PDMS when strain is applied, check that: - A small amount of glue is used so it can dry sufficiently. - Scaffold is relatively dry before glue is applied. - Enough glue is covering entire scaffold area (on portions overlapping on PDMS only). 13. Carefully remove platforms and shut-down equipment. Using Matlab to automatically extract 7-segment display The stress-strain data can be extracted manually from the tensile test video by hand, however, this process can be time consuming and tedious. To facilitate this process, we developed a Matlab script to automatically extract the 7 segment displays from the video recordings into text, which can be exported to an Excel file. There are two Matlab scripts labelled “DataExtractMicromanipulator.m” and “DataExtractTransducer.m” (Supplemental Information File 3 and File 4), which are optimized for the micromanipulator and force transducer readouts, respectively. 1. Convert source video format to a TIFF stack (ignore and proceed to Step 2 if already a TIFF stack). If file type is .tif: Open file using ImageJ and convert by selecting: File > Save As > Tiff. Otherwise use MPEG Streamclip (file converting software, can be downloaded from http://www.squared5.com ). a. Drag and drop video into MPEG Streamclip. b. File > Export To Other Formats > Format: Image Sequence, Options… > Format: TIFF, Frames per second: 2, Sound: No. (Figure 4b). c. Click Ok and save image sequence to a separate folder. 2. Import Tiff stack into ImageJ. To import: File > Import > Image Sequence. Select the folder in which the image sequence is enclosed, ensure virtual stack option is selected. 3. Convert image type to 8-bit. To convert: Image > Type > 8-bit. Save this tiff stack. (File > Save As> Tiff) 4. Crop the TIFF stack twice so you get two separate videos; one for the force transducer and one for the digital micromanipulator display (Figure 4c). To crop: use the rectangle tool and select the rectangle shape you want (include an additional margin around the readout as this may be needed when rotating stack subsequent steps) > Image > crop. 5. Rotate the image so that the readout is straight (this is essential for the program to run properly). To rotate: Image > Transform > Rotate > input angles you need to rotate. This might take some trial and error, but aim to get digits as straight as possible. Select preview when choosing best angle. 6. Save each TIFF stack after rotating (i.e. SampleName_Force, SampleName_Disp). 7. Open Matlab and the appropriate script you need depending on which TIFF stack you are analyzing. 8. Click run. 9. DIALOG BOX 1: Select file. Select correct file for the script being used. 10. DIALOG BOX 2: Zoom in on region of interest (ROI) and then enter two points (click on the top left corner and bottom right corner of your readout) (Figure 4d i-ii).



11. DIALOG BOX 3: Select ROI for first digit. Select two points (top left and bottom right corners of the first digit). This first ROI will propagate rightwards across other digits and extract the number to text (Figure 4d iii). 12. Final results are saved as variable “Value”. Copy and paste values to Excel. Be sure to verify values from random sections of the recording are correct to ensure the program is working accurately. Troubleshooting Tip: If the Matlab scripts are not extracting data accurately, there may be several reasons: - Video is not straight, meaning that the region of interest is cutting off the digits. Rotate video in ImageJ. - Ensure there is sufficient lighting in the video recording and no glare on the display screens. - The ROI selected may be too small or large. - The space between the digits in the code is too big/small. To fix: Go into ImageJ, and use the line tool to measure the distance between the digits. Then change the variable “space” on line 46 of the Matlab code. Note: Because the readouts are different sizes, the space between digits will be different on both codes. (This is the most likely cause of issue). 13. In Excel, calculate the following strain and engineering stress data as follows: C =

∆E E − E; = A8B E; E;

O =

G G = A10B < P∙ℎ

G = H ∙ ? = 6IJIKKL ∙ ∆M ∙ ? = 6IJIKKL ∙ AM − M; B ∙ ? A9B

where the terms are described in Table 2. 14. Calculate the Young’s Modulus (for bulk material) or apparent elasticity (for patterned scaffold) from the slope of the linear portion of the stress-strain curve (typically from 0 to ~%20 strain) Table 2. Description of terms used in calculated strain and stress from tensile testing data Symbol Description Units strain at the nth data point unitless C original length of the sample m E; th length at the n data point m E force at the nth data point N G th equivalent mass at the n data point kg H gravitational acceleration 9.81m2/s ? 6IJIKKL Myograph transducer sensitivity used during kg/5V tensile testing per every 5V (e.g. 2e-3 kg/5V, 10e-3 kg/5V, or 20e-3 kg/5V) transducer voltage at the nth data point V M


Page 20 of 33

Page 21 of 33 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


M; O < P ℎ

starting transducer voltage engineering stress at the nth data point original cross-sectional area of the sample original sample width original sample height


V Pa m2 m m


Figure 4. Mechanical testing of POMaC scaffolds using a Myograph. a, i) Overview of the Myograph set-up, showing where sample is held, force transducer display, micromanipulator display, and position of camera to capture video of both displays simultaneously during the mechanical test, ii) Close-up view of how the scaffold is positioned in the Myograph for tensile testing on PDMS slabs. b, View of the graphical user interface from MPEG Streamclip video


Page 22 of 33

Page 23 of 33 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


converter with overlays to indicate the correct settings prior converting to a TIFF stack. c, Example of how micromanipulator and transducer displays should be cropped and saved as two separate TIFF stacks prior to processing in Matlab. d, In Matlab, i) the TIFF stack is first cropped for efficient processing (first selection point is red marker, second selection point is white marker). ii) Shows the cropped TIFF stack that will be used to extract digits to text. iii) The first ROI is selected around the leftmost digit (first selection point is red marker, second selection point is white marker).

Anticipated Results Two batches of POMaC were polymerized according to the recipes for POMaC 1:4 and 3:2 in Table 1. Polymerization at 140oC for POMaC 1:4 (Yield: 74%) and 3:2 (Yield: 71%) was carried out for 3 hours and 1 hour and 50 minutes, respectively. The 1H-NMR of each batch confirms expected resonance of hydrogen atoms in the polymer backbone. Both spectra possess similar peaks with a greater area under the curve for vinyl peaks (1) (between 6-7 ppm)23 for the 1:4 POMaC as expected due to the increased MA content, and larger peaks (3) (2.79 ppm)23 associated with –CH2– of CA in the 3:2 spectrum as anticipated due to the greater moles of CA (Figure 5a). In future studies, it would be useful to determine the polymerization kinetics as a function of temperature. Comparable FTIR spectra are seen between the POMaC 1:4 and 3:2 (Figure 5b). We assigned the broad peaks at 2932 cm-1 and 720 cm-1 to methylene groups arising from CA and OD and pendant carbonyl groups, respectively.23 A greater presence of pendant hydroxyl groups (3570 cm-1) were seen in the POMaC 3:2 spectrum compared to POMaC 1:4 as expected. Furthermore, a reduced presence of vinyl groups (1647 cm-1)23 in the polymer backbone is observed in the POMaC 3:2 spectrum compared to POMaC 1:4 as expected due to the reduced molar ratio of MA in the 3:2 POMaC. Representative stress-strain curves of two anisotropic POMaC 1:4 scaffold designs (Brick and Diamond pattern) in the long and short axis are shown in Figure 5c. Both Brick and Diamond scaffold designs had similar apparent elasticities in the short axis (6.0 vs. 5.4 kPa), but the Diamond design had a higher apparent elasticity in the long axis (44.1 vs. 60.1kPa) (Figure 5d). The anisotropic ratio (long/short) of the Brick and Diamond scaffolds tested here were 7.4 and 11.1, respectively. The ability to control the anisotropic properties to match a suited application in vivo (e.g. placing an anisotropic scaffold onto the heart), may be desirable.



Figure 5. POMaC characterization. a, Representative 1H-NMR spectra for two POMaC recipes (1:4 and 3:2). b, Representative FTIR spectra of purified POMaC 1:4 and 3:2. c, Typical stressstrain curves for two anisotropic scaffold designs made of 60% Porogen and 40% POMaC 1:4 in


Page 24 of 33

Page 25 of 33 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


the x (long axis) and y (short axis) directions. d, Measured apparent elasticities of Brick and Diamond patterned scaffolds in the x (long axis) and y (short axis) directions. n=1 tensile test per direction. To illustrate the flexibility of this method, scaffolds of various patterns and sizes were made (Figure 6a, b). Interestingly, design iv, a 13x23mm POMaC 1:4 sheet (using same base design from Zhang and Montgomery et al.46), spontaneously self-assembled into a tubular structure. The shape-memory effect of the diamond-shaped POMaC scaffold design ii is demonstrated in Figure 6c. The ability to influence scaffold function based on the scaffold design could potentially lead to a number of interesting applications, including minimally invasive delivery of materials and tissues into the body that would spontaneously open up from a small orifice.

Figure 6. Microfabrication of POMaC scaffolds. a, Various patterns and scaffolds sizes (area/height) (designs i-iv) can be made (Scale bars: 1cm). b, Higher magnifications images of designs i-iv (Scale bar: 1mm). c, Series of images (i-iv) showing a POMaC scaffold eject through a glass pipette tip. Note: scaffolds were soaked in Trypan Blue to enhance visibility.



Conclusion Up to date, majority of tissue engineering products require a surgical approach to deliver tissues into the body, limiting their utility in areas such as cardiac tissue engineering since open heart surgery is available to only a small number of patients due to various risks and possible complications. Thus, marrying tissue engineering and minimally invasive delivery would greatly increase the prospects of wide adoption of tissue engineered products. Using microfabrication and photocrosslinkable polymers described herein, we developed new shape-memory scaffolds that enable injection of fully functional tissues on the heart, liver and aorta in large animals (pigs) without major surgery, as well as under the skin in mice and rats.47 Tissues were grown on the scaffolds, constructed of POMaC, with photocrosslinkable, biodegradable, nontoxic, and minimally inflammatory citric acid-based polymer properties that make it optimal for this application.23 In addition to our own experience in working with POMaC, Tran et al. has demonstrated POMaC possesses elastomeric properties.23 Cardiac tissues cultured on anisotropic, elastic POMaC scaffolds (10 x 10 mm) were able to deform through a 1 mm orifice and regain their original scaffold shape without damaging the tissue. In a rat MI model, cardiac patch placement led to significantly improved cardiac function relative to MI-only controls. The methods described herein can be adapted through the use of photolithography to fabricate moulds, for the production of numerous complex geometries/topographies. For example, PoMAC scaffolds were fabricated with built-in engineered vasculature capable of supporting thick engineered cardiac tissue.48 The lumens of this scaffold, known as Angiochip, were coated with endothelial cells and directly anastomosed to the rat hind limb, allowing for the immediate perfusion of host blood into the implant. The concept of engineered vasculature was also applied to a multi-well platform known as InVADE (Integrated Vasculature for Assessing Dynamic Events) where 3D tissues were connected and cultured around a PoMAC tube structure with microscopic through holes.54 By placing tumor and liver tissues in series, inter-organ crosstalk led to an increase in the tumor toxicity for the chemotherapeutic agent, Tegafur, highlighting the role of liver metabolism and how it can modulate the effect of drugs. Furthermore, the platform may allow for detailed studies on the mechanisms of cancer metastasis as it was shown that cancer cells from the solid tumor were able to escape and travel into the downstream liver tissue. Thus, the methods presented here yield the ability to control the precise physical design and material properties of scaffolds for a number of biomedical applications.


Page 26 of 33

Page 27 of 33 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


Acknowledgments This work was funded by the Canadian Institutes of Health Research (CIHR) Operating Grant MOP-137107, the National Sciences and Engineering Research Council of Canada (NSERC) Steacie Fellowship to M.R., University of Toronto McLean Award to M.R., NSERC Vanier Scholarship to M.M. and L.D.H., NSERC Ontario Graduate Scholarship to M.M., Training Program in Organ-on-a-Chip Engineering & Entrepreneurship (TOeP) NSERC CREATE Scholarship to M.M., NSERC Alexander Graham Bell Canada Graduate Scholarships-Doctoral Program (CGS D) to D.B., NSERC Postgraduate Scholarships-Doctoral Program (PGS D) M.H.M., CIHR Operating Grant (MOP-126027), the Heart and Stroke Foundation Grant G-16-00012711, NSERC Discovery Grant (RGPIN-2015-05952), Canada Foundation for Innovation Grant (226225) and Ontario Institute for Regenerative Medicine New Ideas Grant (500235). Author Contributions M.M. designed and performed the experiments and wrote the manuscript. L.D.H. and D.B. performed chemical characterization and edited the manuscript. M.H.M. produced the microfabrication overview movie. G.C. developed the mechanical testing Matlab script. M.R. supervised the project and edited the manuscript.



Supplemental Information This section includes movies on the synthesis and purification of POMaC, and an overview of the fabrication required steps in the soft lithography procedure to make elastomeric scaffolds. AutoCAD photomasks of example scaffold designs and Matlab scripts used for tensile tested are also provided. Movies Movie 1. Video demonstrating dropwise purification of freshly polymerized POMaC into ddH2O. Movie 2. Final purified POMaC is an optically clear, viscous liquid. Movie 3. Animation highlighting major steps involved in fabricating POMaC scaffolds.

Files File 1. AutoCAD_diamond_design_photomask.dwg AutoCAD photomask design for multilayered Diamond patterned scaffold that fit onto a 4” silicon wafer. File 2. AutoCAD_brick_design_photomask.dwg AutoCAD design for Brick patterned scaffold. File 3. DataExtractMicromanipulator.m Matlab script for extracting 7-segment micromanipulator display from a TIFF stack of a tensile test video recording. File 4. DataExtractTransducer.m Matlab script for extracting 7-segment force transducer display from a TIFF stack of a tensile test video recording.


Page 28 of 33

Page 29 of 33 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


References 1. Takebe, T.; Zhang, B.; Radisic, M., Synergistic Engineering: Organoids Meet Organs-on-aChip. Cell stem cell 2017, 21 (3), 297-300. 2. Pahnke, A.; Montgomery, M.; Radisic, M., Spatial and Electrical Factors Regulating Cardiac Regeneration and Assembly. In Biomaterials for Cardiac Regeneration, Suuronen, E. J.; Ruel, M., Eds. Springer International Publishing: Cham, 2015; pp 71-92. 3. Discher, D. E.; Mooney, D. J.; Zandstra, P. W., Growth factors, matrices, and forces combine and control stem cells. Science 2009, 324 (5935), 1673-7. 4. Breuls, R. G.; Jiya, T. U.; Smit, T. H., Scaffold stiffness influences cell behavior: opportunities for skeletal tissue engineering. Open Orthop J 2008, 2, 103-9. 5. Bhana, B.; Iyer, R. K.; Chen, W. L.; Zhao, R.; Sider, K. L.; Likhitpanichkul, M.; Simmons, C. A.; Radisic, M., Influence of substrate stiffness on the phenotype of heart cells. Biotechnol Bioeng 2010, 105 (6), 1148-60. 6. Darnell, M.; Mooney, D. J., Leveraging advances in biology to design biomaterials. Nat Mater 2017, 16 (12), 1178-1185. 7. Li, L.; Eyckmans, J.; Chen, C. S., Designer biomaterials for mechanobiology. Nat Mater 2017, 16 (12), 1164-1168. 8. Sharma, U.; Concagh, D.; Core, L.; Kuang, Y.; You, C.; Pham, Q.; Zugates, G.; Busold, R.; Webber, S.; Merlo, J.; Langer, R.; Whitesides, G. M.; Palasis, M., The development of bioresorbable composite polymeric implants with high mechanical strength. Nat Mater 2017. 9. Singh, A.; Corvelli, M.; Unterman, S. A.; Wepasnick, K. A.; McDonnell, P.; Elisseeff, J. H., Enhanced lubrication on tissue and biomaterial surfaces through peptide-mediated binding of hyaluronic acid. Nat Mater 2014, 13 (10), 988-995. 10. Hook, A. L.; Chang, C. Y.; Yang, J.; Luckett, J.; Cockayne, A.; Atkinson, S.; Mei, Y.; Bayston, R.; Irvine, D. J.; Langer, R.; Anderson, D. G.; Williams, P.; Davies, M. C.; Alexander, M. R., Combinatorial discovery of polymers resistant to bacterial attachment. Nature Biotechnology 2012, 30 (9), 868-U99. 11. Eberli, D.; Aboushwareb, T.; Soker, S.; Yoo, J. J.; Atala, A., Muscle precursor cells for the restoration of irreversibly damaged sphincter function. Cell transplantation 2012, 21 (9), 2089-98. 12. O'Shea, T. M.; Wollenberg, A. L.; Bernstein, A. M.; Sarte, D. B.; Deming, T. J.; Sofroniew, M. V., CHAPTER 19 Smart Materials for Central Nervous System Cell Delivery and Tissue Engineering. In Smart Materials for Tissue Engineering: Applications, The Royal Society of Chemistry: 2017; pp 529-557. 13. Han, W. M.; Heo, S. J.; Driscoll, T. P.; Delucca, J. F.; McLeod, C. M.; Smith, L. J.; Duncan, R. L.; Mauck, R. L.; Elliott, D. M., Microstructural heterogeneity directs micromechanics and mechanobiology in native and engineered fibrocartilage. Nat Mater 2016, 15 (4), 477-+. 14. Vegas, A. J.; Veiseh, O.; Gurtler, M.; Millman, J. R.; Pagliuca, F. W.; Bader, A. R.; Doloff, J. C.; Li, J.; Chen, M.; Olejnik, K.; Tam, H. H.; Jhunjhunwala, S.; Langan, E.; Aresta-Dasilva, S.; Gandham, S.; McGarrigle, J. J.; Bochenek, M. A.; Hollister-Lock, J.; Oberholzer, J.; Greiner, D. L.; Weir, G. C.; Melton, D. A.; Langer, R.; Anderson, D. G., Long-term glycemic control using polymer-encapsulated human stem cell-derived beta cells in immune-competent mice (vol 22, pg 306, 2016). Nature Medicine 2016, 22 (4), 446-446.



15. Harkin, D. G.; Dunphy, S. E.; Shadforth, A. M. A.; Dawson, R. A.; Walshe, J.; Zakaria, N., Mounting of biomaterials for use in ophthalmic cell therapies. Cell transplantation 2017. 16. Marx, V., Tissue engineering: Organs from the lab. Nature 2015, 522 (7556), 373-7. 17. Lee, S. J.; Yoo, J. J.; Atala, A., Biomaterials and Tissue Engineering. In Clinical Regenerative Medicine in Urology, Kim, B. W., Ed. Springer Singapore: Singapore, 2018; pp 17-51. 18. Sadtler, K.; Estrellas, K.; Allen, B. W.; Wolf, M. T.; Fan, H.; Tam, A. J.; Patel, C. H.; Luber, B. S.; Wang, H.; Wagner, K. R.; Powell, J. D.; Housseau, F.; Pardoll, D. M.; Elisseeff, J. H., Developing a pro-regenerative biomaterial scaffold microenvironment requires T helper 2 cells. Science 2016, 352 (6283), 366-70. 19. Webber, M. J.; Khan, O. F.; Sydlik, S. A.; Tang, B. C.; Langer, R., A perspective on the clinical translation of scaffolds for tissue engineering. Ann Biomed Eng 2015, 43 (3), 641-56. 20. Bhatia, S. N.; Ingber, D. E., Microfluidic organs-on-chips. Nat Biotechnol 2014, 32 (8), 76072. 21. Esch, E. W.; Bahinski, A.; Huh, D., Organs-on-chips at the frontiers of drug discovery. Nature reviews. Drug discovery 2015, 14 (4), 248-260. 22. Zhang, B.; Radisic, M., Organ-on-a-chip devices advance to market. Lab Chip 2017, 17 (14), 2395-2420. 23. Tran, R. T.; Thevenot, P.; Gyawali, D.; Chiao, J. C.; Tang, L. P.; Yang, J., Synthesis and characterization of a biodegradable elastomer featuring a dual crosslinking mechanism. Soft Matter 2010, 6 (11), 2449-2461. 24. Gyawali, D.; Tran, R. T.; Guleserian, K. J.; Tang, L.; Yang, J., Citric-acid-derived photo-crosslinked biodegradable elastomers. J Biomater Sci Polym Ed 2010, 21 (13), 1761-82. 25. Weir, N. A.; Buchanan, F. J.; Orr, J. F.; Farrar, D. F.; Boyd, A., Processing, annealing and sterilisation of poly-L-lactide. Biomaterials 2004, 25 (18), 3939-49. 26. Gentile, P.; Chiono, V.; Carmagnola, I.; Hatton, P. V., An overview of poly(lactic-co-glycolic) acid (PLGA)-based biomaterials for bone tissue engineering. Int J Mol Sci 2014, 15 (3), 3640-59. 27. Vert, M., Lactic acid-based bioresorbable polymers: properties and traps. Med J Malaysia 2004, 59 Suppl B, 73-4. 28. Mikos, A. G.; Bao, Y.; Cima, L. G.; Ingber, D. E.; Vacanti, J. P.; Langer, R., Preparation of poly(glycolic acid) bonded fiber structures for cell attachment and transplantation. J Biomed Mater Res 1993, 27 (2), 183-9. 29. Yeh, W. C.; Li, P. C.; Jeng, Y. M.; Hsu, H. C.; Kuo, P. L.; Li, M. L.; Yang, P. M.; Lee, P. H., Elastic modulus measurements of human liver and correlation with pathology. Ultrasound Med Biol 2002, 28 (4), 467-74. 30. Rouviere, O.; Yin, M.; Dresner, M. A.; Rossman, P. J.; Burgart, L. J.; Fidler, J. L.; Ehman, R. L., MR elastography of the liver: preliminary results. Radiology 2006, 240 (2), 440-8. 31. Mueller, S.; Sandrin, L., Liver stiffness: a novel parameter for the diagnosis of liver disease. Hepat Med 2010, 2, 49-67. 32. Ichihara, K.; Taguchi, T.; Sakuramoto, I.; Kawano, S.; Kawai, S., Mechanism of the spinal cord injury and the cervical spondylotic myelopathy: new approach based on the mechanical features of the spinal cord white and gray matter. J Neurosurg 2003, 99 (3), 278-285. 33. Nagueh, S. F.; Shah, G.; Wu, Y.; Torre-Amione, G.; King, N. M.; Lahmers, S.; Witt, C. C.; Becker, K.; Labeit, S.; Granzier, H. L., Altered titin expression, myocardial stiffness, and left ventricular function in patients with dilated cardiomyopathy. Circulation 2004, 110 (2), 155-62.


Page 30 of 33

Page 31 of 33 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


34. Weis, S. M.; Emery, J. L.; Becker, K. D.; McBride, D. J., Jr.; Omens, J. H.; McCulloch, A. D., Myocardial mechanics and collagen structure in the osteogenesis imperfecta murine (oim). Circ Res 2000, 87 (8), 663-9. 35. Coirault, C.; Samuel, J. L.; Chemla, D.; Pourny, J. C.; Lambert, F.; Marotte, F.; Lecarpentier, Y., Increased compliance in diaphragm muscle of the cardiomyopathic Syrian hamster. J Appl Physiol (1985) 1998, 85 (5), 1762-9. 36. Omens, J. H., Stress and strain as regulators of myocardial growth. Progress in biophysics and molecular biology 1998, 69 (2), 559-572. 37. Athanasiou, K. A.; Agarwal, A.; Dzida, F. J., Comparative-Study of the Intrinsic MechanicalProperties of the Human Acetabular and Femoral-Head Cartilage. J Orthopaed Res 1994, 12 (3), 340-349. 38. Rho, J. Y.; Ashman, R. B.; Turner, C. H., Young's modulus of trabecular and cortical bone material: ultrasonic and microtensile measurements. J Biomech 1993, 26 (2), 111-9. 39. Wang, Y.; Ameer, G. A.; Sheppard, B. J.; Langer, R., A tough biodegradable elastomer. Nat Biotechnol 2002, 20 (6), 602-6. 40. Langer, R.; Vacanti, J. P., Tissue engineering. Science 1993, 260 (5110), 920-6. 41. Ghosh, K.; Pan, Z.; Guan, E.; Ge, S.; Liu, Y.; Nakamura, T.; Ren, X. D.; Rafailovich, M.; Clark, R. A., Cell adaptation to a physiologically relevant ECM mimic with different viscoelastic properties. Biomaterials 2007, 28 (4), 671-9. 42. Engler, A. J.; Sen, S.; Sweeney, H. L.; Discher, D. E., Matrix elasticity directs stem cell lineage specification. Cell 2006, 126 (4), 677-89. 43. Wells, R. G., Tissue mechanics and fibrosis. Biochim Biophys Acta 2013, 1832 (7), 884-90. 44. Engler, A. J.; Carag-Krieger, C.; Johnson, C. P.; Raab, M.; Tang, H. Y.; Speicher, D. W.; Sanger, J. W.; Sanger, J. M.; Discher, D. E., Embryonic cardiomyocytes beat best on a matrix with heart-like elasticity: scar-like rigidity inhibits beating. J Cell Sci 2008, 121 (Pt 22), 3794-802. 45. Blakney, A. K.; Swartzlander, M. D.; Bryant, S. J., The effects of substrate stiffness on the in vitro activation of macrophages and in vivo host response to poly(ethylene glycol)-based hydrogels. J Biomed Mater Res A 2012, 100 (6), 1375-86. 46. Zhang, B.; Montgomery, M.; Davenport-Huyer, L.; Korolj, A.; Radisic, M., Platform technology for scalable assembly of instantaneously functional mosaic tissues. Science advances 2015, 1 (7), e1500423. 47. Montgomery, M.; Ahadian, S.; Davenport Huyer, L.; Lo Rito, M.; Civitarese, R. A.; Vanderlaan, R. D.; Wu, J.; Reis, L. A.; Momen, A.; Akbari, S.; Pahnke, A.; Li, R. K.; Caldarone, C. A.; Radisic, M., Flexible shape-memory scaffold for minimally invasive delivery of functional tissues. Nat Mater 2017, 16 (10), 1038-1046. 48. Zhang, B.; Montgomery, M.; Chamberlain, M. D.; Ogawa, S.; Korolj, A.; Pahnke, A.; Wells, L. A.; Masse, S.; Kim, J.; Reis, L.; Momen, A.; Nunes, S. S.; Wheeler, A. R.; Nanthakumar, K.; Keller, G.; Sefton, M. V.; Radisic, M., Biodegradable scaffold with built-in vasculature for organ-on-a-chip engineering and direct surgical anastomosis. Nat Mater 2016, 15 (6), 669-78. 49. Huyer, L. D.; Zhang, B. Y.; Korolj, A.; Montgomery, M.; Drecun, S.; Conant, G.; Zhao, Y. M.; Reis, L.; Radisic, M., Highly Elastic and Moldable Polyester Biomaterial for Cardiac Tissue Engineering Applications. Acs Biomaterials Science & Engineering 2016, 2 (5), 780-788.



50. Ahadian, S.; Davenport Huyer, L.; Estili, M.; Yee, B.; Smith, N.; Xu, Z.; Sun, Y.; Radisic, M., Moldable elastomeric polyester-carbon nanotube scaffolds for cardiac tissue engineering. Acta Biomater 2017, 52, 81-91. 51. Carothers, W. H., Polymers and polyfunctionality. T Faraday Soc 1936, 32 (1), 0039-0053. 52. Huh, D.; Kim, H. J.; Fraser, J. P.; Shea, D. E.; Khan, M.; Bahinski, A.; Hamilton, G. A.; Ingber, D. E., Microfabrication of human organs-on-chips. Nat Protoc 2013, 8 (11), 2135-57. 53. Levario, T. J.; Zhan, M.; Lim, B.; Shvartsman, S. Y.; Lu, H., Microfluidic trap array for massively parallel imaging of Drosophila embryos. Nat Protoc 2013, 8 (4), 721-36. 54. Lai, B. F. L.; Huyer, L. D.; Lu, R. X. Z.; Drecun, S.; Radisic, M.; Zhang, B., InVADE: Integrated Vasculature for Assessing Dynamic Events. Advanced Functional Materials, 1703524-n/a.


Page 32 of 33

Page 33 of 33 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


For Table of Contents Use Only A Method for the Fabrication of Elastomeric Polyester Scaffolds for Tissue Engineering and Minimally Invasive Delivery Miles Montgomery, Locke Davenport Huyer, Dawn Bannerman, Mohammad Hossein Mohammadi, Genevieve Conant, Milica Radisic


Method for the Fabrication of Elastomeric Polyester Scaffolds for

Recommend Documents