Autonomously Self-Adhesive Hydrogels as Building Blocks for

Nov 23, 2017 - We report a simple method of preparing autonomous and rapid self-adhesive hydrogels and their use as building blocks for additive ...
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Article Cite This: Biomacromolecules 2018, 19, 62−70

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Autonomously Self-Adhesive Hydrogels as Building Blocks for Additive Manufacturing Xudong Deng,†,‡ Rana Attalla,§ Lukas P. Sadowski,‡ Mengsu Chen,‡ Michael J. Majcher,‡ Ivan Urosev,‡ Da-Chuan Yin,† P. Ravi Selvaganapathy,§ Carlos D. M. Filipe,*,‡ and Todd Hoare*,‡ †

Key Laboratory for Space Bioscience and Biotechnology, School of Life Sciences, Northwestern Polytechnical University, Xi’an, 710072, People’s Republic of China ‡ Department of Chemical Engineering and §Department of Mechanical Engineering, McMaster University, 1280 Main Street West, Hamilton, Ontario L8S 4L7, Canada S Supporting Information *

ABSTRACT: We report a simple method of preparing autonomous and rapid selfadhesive hydrogels and their use as building blocks for additive manufacturing of functional tissue scaffolds. Dynamic cross-linking between 2-aminophenylboronic acidfunctionalized hyaluronic acid and poly(vinyl alcohol) yields hydrogels that recover their mechanical integrity within 1 min after cutting or shear under both neutral and acidic pH conditions. Incorporation of this hydrogel in an interpenetrating calciumalginate network results in an interfacially stiffer but still rapidly self-adhesive hydrogel that can be assembled into hollow perfusion channels by simple contact additive manufacturing within minutes. Such channels withstand fluid perfusion while retaining their dimensions and support endothelial cell growth and proliferation, providing a simple and modular route to produce customized cell scaffolds.

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INTRODUCTION Hydrogels prepared using dynamic chemistries that facilitate continuous formation and degradation of cross-links offer unique opportunities to apply hydrogels in nontraditional ways.1,2 A variety of dynamic cross-linking chemistries has been developed in recent years by multiple groups3−5 (including ours6,7) to facilitate both injectable delivery (via in situ gelation) as well as potential self-healing or self-adhesion following damage or fracture.8 Such hydrogels offer significant advantages in drug delivery,9 antibiofouling materials,10 and mimicking the dynamic extracellular matrix.11 However, most self-healing or self-adhesive approaches based on covalent cross-linking require external stimuli or energy input, significantly limiting their applicability in vivo. As such, developing gel chemistries that can rapidly and autonomously self-adhere under physiological conditions offers potential to more effectively translate the promise of self-adhesive hydrogels into clinical applications.12−14 This is particularly true if selfadhesive approaches are to be used together with additive manufacturing to build complex biomedical devices such as 3D vascularized tissue scaffolds,15,16 an application in which maintaining high cell adhesion and viability during processing is essential. Methods to achieve functional vascularization within hydrogels include needle-based subtractive prevascularization17 or 3D printing of a dissolvable prevascular network of sacrificial materials.18,19 These methods need at least one additional step to remove the sacrificial material from the hydrogel. Furthermore, indirect 3D printing of a prevascular structure © 2017 American Chemical Society

into hydrogels has to date required deposition of the precursor ink within a sacrificial support scaffold;20 direct template-free 3D printing of hollow channel structures remains difficult to realize.21,22 Layer-by-layer additive strategies to form prevascular hollow structures have been demonstrated but are slow, requiring at least a 1 h chemical sealing step to adhere the prechannel structures.23,24 Rapid and autonomously selfadhesive gels thus offer an appealing alternative to these current methods by allowing sequential casting of building blocks with desired shapes and subsequent assembly of those building blocks via self-adhesion. In the context of biomedical applications, hyaluronic acid (HA) is frequently used as a backbone polymer for self-healing and self-adhesive polymer structures given its excellent gelforming properties and biological activity.25−27 While multiple chemistries have been explored to prepare self-healing HA, phenylboronic acid (PBA) interactions with cis-diol containing carbohydrates or polymers with multiple pendant −OH groups offer significant advantages in terms of facilitating rapid, highly reversible, and cytocompatible covalent cross-link formation.28 For example, 3-aminophenylboronic acid-conjugated HA can undergo gelation and subsequent self-healing with maltosefunctionalized HA at physiological pH.29 However, the mechanical strength of such hydrogels was low (G′ < 500 Pa); indeed, stiffer mechanics (i.e., higher cross-linking density) Received: August 29, 2017 Revised: November 23, 2017 Published: November 23, 2017 62

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solutions were loaded separately into two barrels of a double-barrel syringe and coextruded through a static mixer into a silicone or acrylic mold of dimensions specified for each subsequent test. The formed gels were then fully submerged into a 100 mM calcium chloride bath to gel the alginate phase. For preparing IPN hydrogels with 0.5% or 1% alginate, the concentration of alginate stock solution was changed into 5% or 10% w/v, respectively. Swelling and Degradation. Hydrogel disks for swelling and degradation experiments were prepared by mixing the precursor polymers inside cylindrical silicone rubber molds (diameter = 11.5 mm, height = 3.5 mm). The mixtures were incubated at room temperature for at least 4 h to ensure complete gelation prior to testing. The resulting gel disks were subsequently placed into cell culture inserts, after which the insets were placed in the wells of a 12well cell culture plate and the hydrogel samples were completely submerged with PBS (4 mL/well). At predetermined times, the cell culture inserts were removed, excess PBS inside the inset was drained off, the hydrogel was wicked with a Kimwipe to remove any excess PBS, and the weight of the hydrogel + insert was measured; decreases in weight corresponded to degradation or deswelling, while increases in weight corresponded to swelling responses. The cell culture inserts were subsequently resubmerged into a fresh 4 mL of PBS and tested repeatedly until no gel fraction was left in the cell culture insert (i.e., complete degradation). Error bars represent the standard deviation of the replicate measurements (n = 4). Mechanical Analysis. The rheological properties of the hydrogel were characterized using a Mach-1 Mechanical Tester (Biomomentum Inc., Laval, QC) operating under parallel plate geometry at room temperature. Hydrogel disks (diameter = 11.5 mm, height = 3.5 mm) were allowed to gel overnight and then transferred from the silicone mold to the mechanical tester. Shear testing was performed by precompressing the gels to 75% of the sample height and subsequently subjecting them to a strain sweep test using amplitudes ranging from 0.1 to 2.2° at 0.5 Hz to determine the linear viscoelastic region (LVE) of the gel. The gels were subsequently subjected to a dynamic frequency sweep (0.1 to 2.2 Hz) within the linear viscoelastic range (LVE) to determine their shear storage modulus (G′). All experiments were repeated in triplicate; reported results represent the average of these replicates, with error bars representing one standard deviation from the mean. The strain sweep healing tests were conducted by switching the applied strain from being relatively small (1.468° amplitude, 1.468 Hz) to large (to disrupt the gel structure: 29.36° amplitude, 1.468 Hz, 10 cycles), back to small (to track self-healing: 1.468° @ 1.468 Hz), measuring G′ as a function of time. Fabrication of Gel Microchannels. The master mold used to fabricate the hollow channel gel structures was 3D printed using a ProJet HD3000 printer and a UV-curable acrylic plastic (VisiJet EX200) as the ink. Precursor solutions of PVA + alginate and HA2APBA + alginate (prepared as described previously) were rapidly mixed, after which 1 mL of the resulting pregel solution was immediately cast onto the patterned mold. After 5 min (allowing for HA-2APBA/PVA gelation), the entire mold + casted gel was fully submerged into a 100 mM calcium chloride bath to gel the alginate phase. After the patterned gel was removed from the mold, it was placed on a flat gel film (prepared using the same hydrogel composition with dimensions 15 mm long × 15 mm wide × 1 mm high) without any additional applied pressure for 5 min to achieve adhesion, with the 5 min adhesion time chosen to be consistent with the self-healing experiments in the strain sweep healing tests; note that functional adhesion was observed at any healing time >1 min. Perfusion Tests in Gel Microchannels. Channels were perfused by pushing an aqueous solution containing blue food coloring as an indicator through the channels using a 3 mL syringe equipped with a 22 gauge needle. Images were taken 1 min after the start of perfusion using a camera, eliminating any competing effects associated with simple diffusion of the colorimetric probe into the hydrogel phase. The same methods and time points were used for perfusion and imaging of hollow channel constructs formed solely of alginate-calcium. Crosssection images of the hollow channels were taken using cryo-

and rapid self-healing/self-adhesion are difficult to simultaneously achieve. While approaches such as double networks,30,31 slide-ring gels,32 and ideally homogeneous networks33 have been applied to enhance the strength of conventional hydrogels, these approaches have not yet been widely investigated for self-healing/self-adhesive hydrogels. Herein, we report the preparation and self-adhesive properties of a hydrogel prepared by mixing 2-aminophenylboronic acid (2APBA)-conjugated HA and poly(vinyl alcohol) (PVA) as well as an interpenetrating network (IPN) hydrogel combining this self-adhesion approach together with alginatecalcium ionotropic gelation. The formation of the IPN facilitates significantly enhances gel mechanics without compromising rapid self-adhesion upon contact, enabling the use of these gels as self-sealing materials for additive manufacturing.

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EXPERIMENTAL SECTION

Materials. Sodium hyaluronate (Lot #010600, Mn = 337 kDa, Đ = 1.386 by GPC, see Supporting Information in ref 34) was obtained from Fidia Farmaceutici S.p.A. (Abano Terme, Italy). N-Ethyl-N′-(3(dimethylamino)propyl) carbodiimide hydrochloride (EDC, 98%), paraformaldehyde, 2-amino phenylboronic acid hydrochloride (2APBA, 95%), poly(vinyl alcohol) (PVA; Mw = 85000−124000, 99+% hydrolyzed), and 2-(N-morpholino)ethanesulfonic acid (MES) hydrate (99.5%) were all purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.) and were used without further purification. Fluorescent probes FITC-phalloidin and Hoechst 33342 were purchased from Sigma-Aldrich and Thermo Scientific Pierce (Rockford, IL, U.S.A.), respectively. Dulbecco’s modified Eagle’s medium (DMEM) basal medium (high glucose) and fetal bovine serum were obtained from Gibco (Gaithersburg, U.S.A.). The water used in all experiments was purified by a Milli-Q purification system, with a resistivity of >18.2 MΩ cm. Acetate buffer (0.2 M, pH 4.0) was prepared by mixing 18 mL of 0.2 M sodium acetate solution with 82 mL of 0.2 M acetic acid solution. Acetate buffer (0.2 M, pH 5.5) was prepared by mixing 89 mL of 0.2 M sodium acetate solution with 11 mL of 0.2 M acetic acid solution. Tris buffer (0.1 M, pH 7.0) was prepared by dissolving 1.21 g of Tris into 100 mL of Milli-Q water and adjusting the pH to 7.0 by adding 0.1 M HCl or 0.1 M NaOH. Synthesis and Characterization of HA-2APBA. HA (80 mg) was dissolved in 100 mL of 10 mM MES buffer. An aqueous solution of EDC (95.8 mg) was added into the solution, and the pH was adjusted to 4.75 by the addition of 1 M NaOH. After waiting for 10 min, 2APBA (259 mg) was then added to the mixture, and the pH of the reaction mixture was maintained at 4.75 via addition of 0.1 M NaOH or HCl over the 4 h reaction time (all at room temperature). The reaction was stopped by the addition of 0.1 M NaOH to adjust the pH to 7.0, after which the modified HA was purified by dialysis against a large excess of water using a prewashed dialysis membrane (MWCO = 14 kDa). The dialysis was stopped when the conductivity of the dialysate was less than 5 μS·cm−1, after which the HA-2APBA product was recovered by freeze-drying. Characterization of the 2APBA graft density was performed by 1H NMR using a Bruker AVANCE 700 MHz spectrometer and D2O (99.96% D, Cambridge Isotope Laboratories) as the solvent. Integration of the NMR signals arising from the anomeric protons of HA (δ = 4.2−4.7 ppm), sugar ring protons of HA (δ = 3.0−4.2 ppm) and the aromatic protons of the PBA group (δ = 7.0−7.7 ppm) indicated a degree of substitution of 0.13 ± 0.01 PBA groups per disaccharide repeat unit. Preparation of HA-2APBA/PVA + Alginate/Calcium IPN Hydrogels. For preparing HA-2APBA/PVA + alginate/calcium IPN hydrogels with 0.2 wt % alginate, precursor solutions of PVA + alginate (consisting of 90% v/v of a 1.5% w/v PVA stock solution well mixed with 10% v/v of a 2% w/v alginate stock solution) and HA-2APBA + alginate (containing 90% v/v of a 1.5% w/v HA-2APBA stock solution well mixed with 10% v/v of a 2% w/v alginate stock solution) were prepared in the same 0.1 M Tris pH 7.0 buffer. The two precursor 63

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Scheme 1. (a) Synthesis of HA-2APBA and Hypothesized Mechanism of Hydrogel Formation between HA-2APBA with PVA at Neutral pH; (b) Preparation of HA-2APBA/PVA Hydrogels (Excluding the Calcium Chloride Bath Step) and HA-2APBA/PVA + Alginate/Calcium IPN Hydrogels (Including the Calcium Chloride Bath Step)

sectioning 30 min after structure fabrication. Cryo-sectioning samples were submerged in 5 mL of liquid nitrogen for 10 s, immediately removed, and sectioned into ∼1 mm slices using a razor prior to imaging with a light microscope. Casted gel layers were first adhered to one another and then immediately perfused to confirm that self-healing between layers was effective. The hollow channels were subsequently flushed with at least five times in order to remove any residual blue dye. The samples were then stored for 3 days in either air (at 100% relative humidity inside a sealed container) or submerged inside a water bath, after which the perfusion experiment was completed to confirm the stability of the adhesive bond formed. Endothelial Cell Culture on Hydrogels. Human umbilical vein endothelial cells (HUVEC, ATCC) were cultured at an initial density of 25000 cells/well in DMEM high glucose medium containing 10% (v/v) fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin in a six-well plate maintained at 37 °C in 5% CO2. After reaching 80% confluence, the cells were detached using 0.25% tripsin− 1 mM EDTA and resuspended in fresh culture medium. For preparing a thin layer of the HA-2APBA/PVA + alginate/calcium IPN gel at the bottom of each well of a 24-well plate, a 50 μL aliquot of a precursor solution containing 1.35 wt % PVA and 0.2 wt % alginate was first added to each well followed by another 50 μL aliquot of a precursor solution containing 1.35 wt % HA-2APBA and 0.2 wt % alginate. Next, 200 μL of calcium chloride solution (100 mM) was added to cover the gel for 2 h, after which it was aspirated off the now-IPN hydrogel thin film and discarded. A total of 1 mL of suspended cells was subsequently seeded on the thin layer gels at a density of 5000 cells/well (2500 cells/cm2); cells seeded on a bare polystyrene well were used as controls. After 6, 24, and 48 h of culture, cell growth in the wells was monitored using light microscopy. In addition, the nucleus and F-actin in the cells were stained with fluorescent probes to allow direct visualization of the degree of F-actin expression per cell on both the gel and control surfaces. For staining the nucleus, the culture

medium was discarded and the cells were immersed in the Hoechst 33342 solution (10 μg/mL). After a 10 min incubation at 37 °C, cells were washed carefully for three times with PBS. For staining F-actin, cells were first fixed by 4% paraformaldehyde and then stained using FITC-phalloidin following conventional methods.35 The fluorescence from both stains was subsequently imaged using an inverted fluorescence microscope (Nikon TE2000) equipped with a Peltiercooled CCD camera. Nucleus staining was monitored using excitations of 345 nm and an emission range of 465−495 nm, while F-actin staining was monitored at excitations of 465−495 nm and an emission range of 515−555 nm.

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RESULTS AND DISCUSSION Precursor Polymer and Hydrogel Synthesis. PVA contains one pendant alcohol group on each monomer residue and can thus bind strongly with cis-diol binding PBA groups;36 it also has a long record of biomedical use.37,38 2APBAmodified HA (HA-2APBA) was obtained via a carbodiimidemediated conjugation reaction between HA and 2APBA (Scheme 1a). While para- or meta-functionalized phenylboronic acids have conventionally been used for creating selfhealing/self-adhesive materials,29,39 ortho-functionalized phenylboronic acids linked via an amide to the polymer backbone enable intramolecular coordination between the carbonyl oxygen of the amide and the boron of the boronic acid group (see Supporting Information (SI), Scheme S1),40,41 maintaining the tetrahedral form required for strong boronatecis diol interactions at neutral and even acidic pH values.40 In contrast, other PBA derivatives are either nonbonding at physiological pH or require coincorporation of coordinating functional groups (e.g., amines42 or carbonyls43) and/or 64

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Figure 1. Vial inversion assay result of (A) PVA solution (1.35 wt %), (B) HA-2APBA solution (1.35 wt %), and (C) gelation after mixing of HA2APBA and PVA solutions.

Figure 2. (a, b) Self-adhesion of (a) HA-2APBA with PVA (3 wt %, pH 7.0) and (b) HA-2APBA/PVA + alginate/calcium IPN hydrogel (1.35 wt % HA-2APBA/PVA, 0.2 wt % alginate, pH 7.0): (I) intact gel; (II) cut gel; (III) gel halves placed in contact for one minute after cutting; (IV) selfadhered gel suspended under its own weight immediately after the one minute healing time; (c, d) Rheological properties of self-adhesive hydrogels in response to a strain sweep at a fixed frequency (1.468 Hz) initiated at low shear (1.468° amplitude) followed by high shear (29.36° amplitude, 10 cycles - Damage), and returned to low shear (1.468° amplitude - Healing) for (c) HA-2APBA/PVA hydrogels (both at 3 wt %, pH 7.0) and (d) HA2APBA/PVA + alginate/calcium IPN hydrogels (1.35 wt % of HA-2APBA/PVA, 0.2 wt % of alginate, pH 7.0); n = 4 for all samples.

∼87% of −COO− groups on HA free to complex with grafted PBA groups to further assist in promoting tetrahedral boron geometry.

aromatic substitution of the PBA group44,45 to exhibit physiological activity. 1H NMR analysis indicated the degree of substitution of 2APBA was ∼13 mol % (Figure S1), leaving 65

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Figure 3. (a, b) Self-adhesion and recovery of HA-2APBA/PVA + alginate/calcium IPN hydrogels (1.35 wt % of both HA-2APBA and PVA, pH 7.0) at different alginate concentrations: (a) 0.5 wt % alginate and (b) 1.0 wt % alginate. (I) intact gel; (II) cut gel; (III) gel halves placed in contact for 1 min after cutting; (IV) healed gel suspended under its own weight immediately after the one minute healing time; (c, d) Rheological properties of HA-2APBA/PVA + alginate/calcium IPN self-healing hydrogels (1.35 wt % of both HA-2APBA and PVA) in response to a strain sweep initiated at low shear (1.468° amplitude, 1.468 Hz) followed by the application of high shear (29.36° amplitude, 1.468 Hz, 10 cycles, indicated as Damage), and then returned to low shear (1.468° amplitude, 1.468 Hz, indicated as Healing) at different alginate concentrations: (C) 0.5 wt % alginate; (D) 1.0 wt % alginate. n = 4 for all samples.

Hydrogels were subsequently formed by simple mixing of HA-2APBA and PVA solutions in buffers at neutral or acidic pH via coextrusion through a double-barrel syringe (Scheme 1b). HA-2APBA/PVA gelation kinetics were assessed using a vial inversion test (Figures 1 and S2). Solutions of either PVA or HA-2APBA alone did not gel, while gelation occurred quickly after mixing of HA-2APBA and PVA solutions (Figure 1), confirming that boronate-PVA cross-linking is occurring. In addition, at each of pH 4.0, 5.5, and 7.0, gelation occurred within 10 s after simple mixing at 1 wt % polymer concentration (Figure S2). Self-Adhesive Hydrogel Properties. The self-adhesive characteristics of HA-2APBA/PVA hydrogels were subsequently investigated using gels formed with 3 wt % of both polymers, with the higher concentration chosen to provide a more rigid matrix that can be handled easily with tweezers. The HA-2APBA/PVA gel was first assessed alone (i.e., without calcium-alginate gelation) using a qualitative healing test in which a hydrogel disk was cut in half and the two halves were moved back into contact without applying additional pressure.

The hydrogel showed rapid self-adhesion within one minute at room temperature at both pH 7.0 (Figure 2a) and 4.0 (Figure S3), as evidenced by the rapid disappearance of the scar at the damage site. The gels maintained their macroscopic dimensions during the healing process; furthermore, the healed gels were strong enough to support their own weight when suspended by tweezers only one minute after being moved back into physical contact (Figure 2a). Mechanical testing demonstrates the reversibility and kinetics of self-healing and self-adhesive HA-2APBA/PVA hydrogels (Figures 2c and S4). The introduction of large shear (t = 0, corresponding to a higher angular amplitude applied at a fixed 1.468 Hz frequency of rotation) coincides with a decrease in the measured G′ value from ∼800 to ∼350 Pa, indicating the disruption of dynamic gel cross-links. Following a return to low shear, the gel showed near- quantitative mechanical recovery within 1 min, consistent with the macroscopic observations in Figure 2a. Following, IPN hydrogels were fabricated by including alginate in both barrels and subsequently immersing the 66

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Biomacromolecules preformed HA-2APBA/PVA+alginate semi-IPN in a calcium chloride gelling bath (Scheme 1b). The gels became translucent following calcium cross-linking (Figure S5), especially at alginate concentrations > 0.5 wt %. Alginate concentrations of 0.2 and 0.5 wt % resulted in a slight increase in G′ (∼25− 30%) relative to the HA-2APBA/PVA gel alone, while the addition of 1 wt % alginate resulted in a ∼5-fold increase in G′ to the 3−5 kPa range (Figure S6). The self-adhesive properties of the hydrogel were not hindered by the interpenetrating alginate-calcium system, with visual self-adhesion still observed within one minute following physical contact (Figures 2b and 3a,b) and at >60% of the modulus recovered within 1 min >80% of the modulus recovered within 8 min following shear at all alginate concentrations tested (Figures 2d and 3c,d). As such, manipulation of the alginate concentration within the IPN hydrogel can alter the mechanics of the hydrogel over at least 1 order of magnitude without compromising the capacity of the IPN hydrogel for self-adhesion. Note that G′/G′′ is consistently >1 and, for most cases, >10, confirming the gel-like properties of the products formed (Figures S4 and S6); in addition, IPN gels showing even minor increases in bulk modulus demonstrated much higher interfacial stiffness, consistent with the higher calcium cross-link density present at the interface following immersion of alginate in a calcium bath.46 Additive Manufacturing of Hydrogel Microchannels. IPN hydrogels exhibit both the self-adhesive properties and bulk and interfacial mechanical stabilities required for the direct additive manufacturing of prevascular networks. Figure 4 illustrates the cast-heal strategy used to fabricate hollow channels within the self-adhesive IPN hydrogel.

Figure 5. Structure and functionality of additively manufactured selfadhered channels using HA-2APBA/PVA + alginate/calcium IPN hydrogels: (a) Gels can be patterned using a 3D printed mold; (b) Hollow channel structures can be formed by adhering the patterned gel to a flat gel support; (c) A cross-section of a 2 mm × 2 mm hollow channel shows no interface between the top and bottom gel layers, indicating self-adhesion; (d) Perfusion of water (dyed blue for visualization) through channels fabricated from alginate-calcium alone leak due to the lack of interfacial adhesion; (e) Perfusion of water through channels fabricated from an IPN hydrogel confirms functional adhesion at the interface via autonomous self-healing; (f) Hollow channel IPN gel samples stored in air at 100% relative humidity for 3 days can be successfully perfused without visible leaking; (g) Hollow channel IPN gel samples stored in water for 3 days can be successfully perfused without leaking. All scale bars = 5 mm, except (c) = 2 mm.

alone were too weak to support the channel structures and thus could not be perfused. In contrast, water could be perfused through the channels of the IPN gel without any leakage immediately after channel fabrication (Figure 5e), 72 h after fabrication followed by storage at 100% relative humidity in air (Figure 5f), and 72 h after fabrication followed by soaking of the IPN in water (Figure 5g). Note that the narrower channel dimension observed in Figure 5g is due to the slight (∼20%) deswelling of the IPN gel as a function of time upon aqueous storage over the 3-day incubation period (Figure 6), confirming that bulk gel swelling can occur without disrupting the selfadhered interface. Furthermore, functional gel degradation can be achieved in vitro on the time scale of 8−9 days (Figure 6), enabling clearance of the biomaterial at a rate relevant to

Figure 4. Schematic of the method used for fabricating hydrogels with integrated hollow channels: (a) the precursor polymers were mixed and spread evenly across the mold; (b) the cast hydrogel (still attached to the mold) was immersed in a calcium chloride bath to cross-link the free alginate to form an IPN gel; (c) the patterned IPN gel film was peeled from the mold while retaining the templated structure; (d) the patterned IPN gel was placed on a flat gel sheet of the same material and allowed to self-adhere to bond the channels.

A 3D-printed acrylic master mold was used to cast and pattern channel structures (Figure 5a). IPN hydrogels prepared with 1.35 wt % HA-2APBA/PVA and 0.2 wt % alginate were allowed to cure and then gelled in calcium chloride; these concentrations were found to offer high structural stability, transparency, and rapid self-adhesion. The IPN gel was then peeled from the mold and placed in contact with a flat gel sheet of the same chemistry. Functional adhesion between the two gel layers created a series of 15 mm length × 2 mm width × 2 mm height channels (Figure 5b,c). The boundaries between the flat and patterned gel films were not visible after five minutes of contact (Figure 5c), indicating effective self-adhesion between layers. To confirm the integrity of the seal produced, water dyed with blue food coloring was perfused through the channels. Channels created using alginate-calcium gels only showed substantial leakage during perfusion due to the lack of adhesion at the gel interface (Figure 5d), while HA-2APBA/PVA gels

Figure 6. Mass change as a function of time for HA-2APBA/PVA + alginate/calcium IPN hydrogel in phosphate buffered saline (1.35 wt % of both HA-2APBA and PVA, 0.2 wt % of alginate). n = 4 for all samples. 67

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Figure 7. (a, b) Morphology of HUVECs cultured on (a) a polystyrene well plate and (b) HA-2APBA/PVA + alginate/calcium IPN gel; (c−f) Fluorescent staining of F-actin (green) and nucleus (blue) in HUVECs cultured for 48 h on (c, e) a polystyrene well plate and (d, f) HA-2APBA/ PVA + alginate/calcium IPN gel. All scale bars in a-f = 50 μm. (g) Relative green fluorescence intensity of image (e) and (f) measured by ImageJ 1.51k software (eight independent areas were measured in each image). *p < 0.01.

effective tissue regeneration, particularly in terms of mimicking the approximate timeline over which the presence of hyaluronic acid in a wound is significantly up-regulated.47 Pattern dimensions could easily be adjusted to compensate for such deswelling and ensure consistent channel size under different storage/preincubation conditions. Overall, stable adhesion was observed between the gel layers and the fabricated hollow channels could maintain their structural integrity under a range of storage conditions and times. Epithelialization of Additively Self-Adhered Microchannels. To assess the potential for functional blood vessel growth within the manufactured channels, human umbilical

vein endothelial cells (HUVEC) were cultured in the channel to assess the capacity of the IPN gel to support cell growth and proliferation. Functional cell confluence was achieved following plating of 2500 cells/cm2 after 48 h, with no significant differences in cell number between the IPN hydrogel and a flat polystyrene well plate (Figure 7a,b, both exhibiting ∼2.4 × 105/ cm2 cell density). Furthermore, the expression of actin microfilaments (indicated by green staining in Figure 7c−f) significantly increased when HUVEC cells were grown on the self-healing IPN hydrogel relative to a polystyrene well plate (Figure 7g), indicating strong focal adhesions to the IPN hydrogel. These results indicate that the self-healing hydrogels 68

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Biomacromolecules

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are both noncytotoxic and effective at supporting growth of endothelial cell monolayers. This high cytocompatibility, coupled with the fast, sacrificial material-free, and self-healing properties of our additive manufacturing approach, suggests that simultaneous channel formation and cell seeding on the inner surface of the channel/lumen may be possible. The modular nature of the assembly technique also introduces significant flexibility to adhere multiple functional subunits of any desired geometry and/or desired cell type(s), facilitating mimicking of tissue substructures of more complex organs (e.g., the heart).

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CONCLUSIONS A new strategy for synthesizing autonomous, rapid, and mechanically robust self-adhering hydrogels through dynamic cross-linking has been developed based on an interpenetrating network hydrogel of 2APBA-modified hyaluronic acid/poly(vinyl alcohol) and alginate/calcium. The hydrogel can selfadhere at both neutral and acidic pH to recover at least 60% of the original mechanical strength after fracture within 1 min while providing sufficient mechanical strength to stabilize hollow microchannels and support endothelial cell growth. This simple strategy opens the path for additive manufacturing of vascularized tissue scaffolds or organs with more complex spatial or biological subfeatures.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biomac.7b01243. 1 H NMR spectrum of HA-2APBA, vial inversion assays of hydrogel formation, visual appearance and rheological properties of hydrogels (PDF).

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: fi[email protected]. ORCID

Carlos D. M. Filipe: 0000-0002-7410-3323 Todd Hoare: 0000-0002-5698-8463 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank the Natural Sciences and Engineering Research Council of Canada (CREATE Biointerfaces Training Program-10532261, Discovery Grants RGPIN-356609-12 and RGPIN-261686-13), National Natural Science Foundation of China (NFSC U1632126), the Fundamental Research Funds for the Central Universities (3102017OQD039), the Canada Foundation for Innovation (Leaders Opportunity Fund #29131), and the Ontario Innovation Trust for financial support. T. H. holds the Canada Research Chair in Engineered Smart Materials.

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DOI: 10.1021/acs.biomac.7b01243 Biomacromolecules 2018, 19, 62−70

Article

Biomacromolecules

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DOI: 10.1021/acs.biomac.7b01243 Biomacromolecules 2018, 19, 62−70