In Situ Forming, Cytocompatible, and Self-Recoverable Tough

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In Situ Forming, Cytocompatible, and Self-recoverable Tough Hydrogels Based on Dual Ionic and Click Crosslinked Alginate Mohammad Hossein Ghanian, Hamid Mirzadeh, and Hossein Baharvand Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b00140 • Publication Date (Web): 29 Mar 2018 Downloaded from http://pubs.acs.org on March 30, 2018

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Biologically-inspired design of tough hydrogels based on dual crosslinked (DC) alginate. 209x166mm (300 x 300 DPI)

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Synthesis and chemical characterization of furan functionalized alginate. 209x168mm (300 x 300 DPI)


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Calcium and click crosslinking ability of furan derivatives of alginate with varied furan substitutions. 166x297mm (300 x 300 DPI)



Optimization of the ionic/click crosslink balance for toughening the FAlg hydrogels. 209x279mm (300 x 300 DPI)


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Self-recovery and self-healing of the dual crosslinked (DC) hydrogels. 209x232mm (300 x 300 DPI)



“One-pot” gelation, moldability and injectability of the dual crosslinked (DC) hydrogels. 209x128mm (300 x 300 DPI)


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Bioconjugation ability of the DC hydrogels. 209x297mm (300 x 300 DPI)



Cytocompatibility of the dual crosslinking strategy. 209x85mm (300 x 300 DPI)


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In Situ Forming, Cytocompatible, and Selfrecoverable Tough Hydrogels Based on Dual Ionic and Click Crosslinked Alginate Mohammad Hossein Ghanian1, Hamid Mirzadeh1,*, Hossein Baharvand2,3,4,* 1

Department of Polymer Engineering and Color Technology, Amirkabir University of Technology, Tehran, Iran

2

Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute, ACECR, Tehran, Iran 3

Department of Cell engineering, Cell Science Research Center, Royan Institute, ACECR, Tehran, Iran

4

Department of Developmental Biology, University of Science and Culture, Tehran, Iran

*Corresponding authors: E-mail: [email protected] and [email protected]



Abstract

A dual crosslinking strategy was developed to answer the urgent need for fatigue-resistant, cytocompatible, and in situ forming tough hydrogels. Clickable, yet calcium-binding derivatives of alginate was synthesized by partial substitution of its carboxyl functionalities with furan, which could come into Diels-Alder click reaction with maleimide end groups of a four arm poly(ethylene glycol) crosslinker. Tuning the cooperative viscoelastic action of transient ionic and permanent click crosslinks within the single network of alginate provided a soft tough hydrogel with a set of interesting features: (i) immediate self-recovery under cyclic loading, (ii) highly efficient and autonomous self-healing upon fracture, (iii) in situ forming ability for molding and minimally invasive injection, (iv) capability for viable cell encapsulation, and (v) reactivity for on-demand biomolecule conjugation. The facile strategy is applicable to a wide range of natural and synthetic polymers by introducing the calcium binding and click reacting functional groups and can broaden the use of tough hydrogels in load-bearing, cell-laden applications such as soft tissue engineering and bioactuators.

Keywords: Tough hydrogel, Dual crosslinked alginate, Injectable, Self-recovery, Self-healing, Click reaction

1. Introduction Tough hydrogels are of tremendous interest as mechanically strong materials to engineer loadbearing soft tissues or develop mechanically active biodevices such as bioactuators. Prior to their application, a set of requirements should be simultaneously addressed: (i) cytocompatibility and bioorthogonality of the gel formation process, (ii) in situ forming ability for minimally invasive


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injection or shaping by molding or extrusion, (iii) biomimetic stiffness and strength, (iiii) fast and autonomous recovery under cyclic loading, and (iv) ability to self-heal upon fracture. Over the past decade, tough hydrogels have been developed by different approaches according to a common general principle: “dissipating mechanical energy to save the original crosslinks”.1 One of the most successful strategies to toughen hydrogels is the incorporation of sacrificial weak and permanent strong crosslinks into polymer networks. Under loading, the weak bonds break to dissipate energy and support the strong bonds which are responsible for strength and shape recovery of the hydrogel. This strategy has been initially introduced by the development of double network (DN) hydrogels that consisted of two intertwined brittle and ductile networks synthesized via a two-step sequential free-radical polymerization. The DN hydrogels exhibited a high degree of toughness by breakage of the covalent bonds in the brittle network.2 However, irreversible breakage of the covalent crosslinks softens the network and hampers the use of DN hydrogels for sustained loading situations such as cyclic stresses. Alternatively, weak physical interactions have been proposed as reversible sacrificial bonds to produce recoverable tough hydrogels.

The

physical

crosslinks

are

able

to

associate/dissociate

reversibly

by

loading/unloading and lead to hydrogels with high toughness as well as recoverable mechanical properties. In this regard, several attempts have been devoted to develop recoverable tough hydrogels by introduction of different reversible physical crosslinks such as ionic bonding,3-4 crystal

formation,5-6

hydrogen

bonding,7

host–guest

interactions,8-9

and

hydrophobic

interactions10 within single network or interpenetrating double networks of permanent covalent crosslinks. Three challenges hamper the use of convectional physicochemical tough hydrogels in cellladen, sustained load-bearing applications. First, the covalent crosslinking mostly occurs via



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multistep chemical reactions under harsh conditions.6 Particularly, very low or high temperature, toxic reagents, or UV radiation are ubiquitous conditions in most of the reports which precludes their use as in situ forming, cytocompatible hydrogels. Second, the convectional physicochemical tough hydrogels suffer from inefficient self-healing after complete fracture as they are mostly synthesized by irreversible covalent crosslinking.11 Third, the physicochemical hydrogels typically exhibit slow recovery of mechanical properties under cyclic loading. For example, Suo et al. have reported that after the first cycle of loading/unloading, the DN hydrogel of alginate/polyacrylamide (PAAm) was much weaker if the second loading was applied immediately (observed as negligible hysteresis), and recovered significantly (74% of work on reloading) if sufficient recovery duration (1 day) and thermal treatment (80°C) is allowed.12 A similar delayed recovery of more than one hour has been reported for other physicochemical hydrogels.3-4, 13-18 Therefore, there is an urgent need to develop a physicochemical crosslinking strategy that allow for cytocompatible, in situ formation of gels with capability for efficient selfhealing and rapid self-recovery under physiological conditions. The kinetics of recovery in physicochemical hydrogels is mainly governed by dynamics of physical association. A number of physical interactions are inherently slow under mild conditions. Examples include hydrogen bonding of agar or crystallization of polyvinyl alcohol which exhibit optimal kinetics at high or low temperatures, respectively, that lead to delayed recovery of toughness under mild conditions.17-18 However, most physical interactions, such as ionic boding or host-guest interactions are able to rapidly associate under mild conditions.9, 19 In these cases, the physical association may be slowed down by sterical hindrance of a secondary network as with the DN hydrogels,20 or sever reorganization of the physical netwroks.4,

21

Therefore, an extended period of time, elevated temperature, or swelling in solvents is required


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for the dissociated chains to rearrange throughout the mixed network and recouple the physical pairs4, 12-13 Inspired from a natural protein, titin, we hypothesized that incorporation of a low density of covalent crosslinks within a highly transient physical network would lead to dual crosslinked (DC) hydrogels with fast self-recovery aided by the elastic action of the permanent covalent crosslinks and rapid reassociation of the sacrificial physical pairs (Scheme 1A). Titin is a skeletal muscle protein that dominates the mechanical strength and toughness of muscles.22 This long chain protein is constructed from folded repeating units of E-rich peptide PEVK motifs which are surrounded by immunoglobulin (Ig)-like segments. In the activated state, transient association of carboxyl groups (on E; glutamate) with calcium ions (Ca2+) forms reversible stiff zones of the E-rich motifs and determine the resistant against the unfolding.22 Under loading, the folded motifs dissipate energy by unfolding and the Ig-like domains mostly bind to actin and myosin filaments to form strong cross-bridges which prevent the massive unfolding of Ig motifs and store the energy for rapid recovery upon unloading by an elastic action (elastic active zones are pink shaded in Scheme 1A).23 In an analogous configuration, the natural polysaccharide, alginate, is a linear copolymer consisting of guluronic acids (G) and mannuronic acids (M) monosaccharides which is able to Ca2+ association by zipping the G-rich domains and covalent crosslinking by chemical modification (Scheme 1). Under loading, the ionic crosslinks can work as sacrificial bonds which support the covalent bonds by effective dissipation of energy through unzipping,12 while the covalent crosslinks are supposed to resist high stresses and prevent massive rearrangement of the dissociated chains. Once the load is released, the alginate chains are expected to return rapidly to their original arrangement, guided by memory of the elastically active permanent crosslinks and fast re-zipping of the dissociated pairs, which may lead to



autonomous rapid self-recovery (Scheme 1A). In this scenario, an optimal recoverable toughness might be achieved by tuning the balance of ionic/click crosslinking. Alginate was selected due to its biocompatibility, potential for chemical modification, and most importantly, the inherent capability for ionic crosslinking through cytocompatible, highly transient calcium-carboxyl complexation, which allows for reversible energy dissipation by fast dissociation/ reassociation under loading/ unloading at physiological conditions.19 To the best of our knowledge, this great capability has not been already harnessed for development of single network tough hydrogels. Clickable, yet Ca2+-binding derivatives of alginate (FAlg) can be produced by partial substitution of the carboxyl groups with furan. Click coupling of the Ca2+crosslinked FAlg chains by four-arm poly(ethylene glycol)-maleimide (4arm-PEG-Mal) as the elastic crosslinker may produce a DC network with self-recoverable toughness (Scheme 1B). The cycloaddition between furan and maleimide, known as the Diels-Alder (DA) reaction, is a bioorthogonal click reaction that occurs under physiological conditions (aqueous media, 37 °C, pH 7.4) without the use of any catalysts or UV-radiation and production of any byproducts. Due to these unique characteristics, DA has been recently considered as an situ forming and highly cytocompatible crosslinking strategy to develop biomolecule or cell-laden hydrogels.24 Additionally, due to dynamic nature of the DA covalent bonds under physiological conditions,25 the DC alginate hydrogels are expected to autonomously re-establish their original mechanical properties via self-healing of the fractured covalent bonds, even after complete failure. DA kinetics is reasonably slow to allow good polymer mixing and easy handling before application. Herein, upon mixing the aqueous solutions of the polymer precursors and Ca2+, ionic crosslinking is expected to start rapidly, providing an injectable or moldable pregel as a template for DA curing which may proceed autonomously on a slower timescale to increase the strength and


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elasticity of the hydrogel. This new combination of sequential ionic and click crosslinking allows for cytocompatible and “one-pot” formation of tough hydrogels, and does not require multistep exogenous stimulations for sequential network formation. We have stated that tandem incorporation of the orthogonal, recoverable, physiologically possible, and cytocompatible covalent and physical crosslinking into a single network would provide an innovative “one-pot” dual crosslinking strategy which has not been explored yet to answer the urgent need for in situ formation of cytocompatible, fatigue resistant, and self-healing tough hydrogels.

Scheme 1. (A) Biologically-inspired design of tough hydrogels based on dual crosslinked (DC) alginate. In an analogous to titin configuration in muscles, the long chains of furan-alginate consist of G-rich domains that form calcium-crosslinked stiff zones surrounded by PEG-



mediated covalent crosslinks. Under loading, the stiff zones are reversibly dissociated to dissipate energy, while the permanent PEG crosslinks prevent massive plastic deformation of chains and work as elastically active zones (pink shaded area) to store energy for rapid selfrecovery upon unloading. (B) Photographic and schematic illustration of the ionic/click DC alginate hydrogel: ionic crosslinking by calcium ions and Diels-Alder click crosslinking by a four-arm PEG crosslinker. 2. Experimental Section Materials Dichloromethane (DCM) and dimethyl formamide (DMF) were purchased from Merck and were dehydrated before use. All other chemicals were provided by Sigma-Aldrich and used as received, otherwise mentioned. Synthesis of furan substituted alginate (FAlg). Amidation reaction was utilized to couple furfurylamine (FA) to the carboxyl groups of alginate under the catalysis of EDC/NHS (Figure 1A). Briefly, alginic acid sodium salt (Sigma; 0.2 g, 1.01 mmol in terms of the repeating unit) was dissolved in 50 mL of 2-(N-Morpholino)ethanesulfonic acid (MES) buffer (100 mM, pH 6), after which predetermined amounts of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrocholoride (EDC) and N-hydroxysuccinimide (NHS) were added. The reaction mixture was stirred at room temperature for 45 min to fully activate the carboxylic groups of alginate followed by addition of furfurylamine (0.4 mL, 4.04 mmol). The solution was stirred for 24 h at room temperature, dialyzed against deionized water for 5 days (cutoff: 3500 Da), and lyophilized to obtain the product as pale yellow color foams. The furan substitution was verified by comparing the spectra of neat alginate with FAlgs through 1H NMR (Bruker Avance, DRX 300 MHz), UV spectrophotometry (Thermo Fisher Scientific, Multiskan GO Microplate


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spectrophotometer), and ATR-FTIR (Bruker-Equinox 55 FTIR spectrometer equipped with ATR accessories) as shown in Figures 1B-1D. The degree of substitution (DS) of the products, i.e., percentage of carboxylic acid groups in alginate coupled with furan was calculated based on elemental analysis (Carlo Erba, EA1100) data (table S1) using the Equation 1:26 =

⁄

× 100

(1)

Where MAlg, MN, MNaOH, and MFur correspond to molar weight of alginate, N element, NaOH, and furfurylamine, respectively, and WN is the mass fraction of N in FAlg that was obtained by the elemental analysis. DSs of the products and their synthesis conditions are reported in Table 1. Synthesis of four arm-poly(ethylene glycol) (4arm-PEG) crosslinker. 4-arm PEG (MW = 10000; JenKem, USA) was functionalized at the OH-termini with maleimide through multistep reactions (Figure S2A) as described following. The success of conversion on the OH-termini was confirmed by NMR spectroscopy as shown in Figure S2B. To quantify the conversion of end groups, the protons of a specific functional group were calculated by integration of the characteristic peak of new end group (color-shaded, Figure S2B), and were divided by the functional group protons calculated based on the area of PEG repeating unit (10H, gray-shaded, Figure S2B). 1. Synthesis of tosylated 4arm-PEG (4arm-PEG-OTs). Pre-dried 4arm-PEG-OH (MW: 10000 g/mol, 5 g, 0.5 mmol) was dissolved in 25 mL anhydrous dichloromethane and trimethylamine (12 mmol, 1.663 mL) and p-toluenesulfonyl chloride (TsCl) (4 mmol, 0.762 g) were added to the above solution under N2 atmosphere. The mixture was stirred for 24 h at room temperature. The reaction mixture was filtered and the filtrate was washed twice with saturated ammonium chloride solution. The organic phase was dried over anhydrous magnesium sulfate (MgSO4) and concentrated by rotary evaporation. The concentrated filtrate was dropped into



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excess cold diethyl ether to precipitate the product. The white precipitate was filtered and vacuum dried at 40 °C to obtain 4arm-PEG-OTs (conversion 100%). 1H NMR (CDCl3, 300 MHz, δ): 2.42 (s, 12H, -CCH3), 3.65 (s, 909H, PEG backbone, -OCH2-), 4.16 (t, 8H, -CH2OTs), and 7.35-7.80 (d, 16H, aromatic rings) (figure S2B). 2. Synthesis of amine functionalized 4arm-PEG (4arm-PEG-NH2). The 4arm-PEG-OTs (2.5 g, 0.25 mmol) was added to 200 mL ammonia (32%) and stirred for 5 days at room temperature. The aqueous phase was twice extracted using dichloromethane. The separated organic phase was dried over anhydrous MgSO4, concentrated and dropped into excess cold diethyl ether. The 4arm-PEG-NH2 was obtained as a white powder (conversion 94%). 1H NMR (CDCl3, 300 MHz, δ): 1.90 (broad peak, 8H, -NH2), 3.41 (t, 8H, end -OCH2- group), and 3.65 (s, 909H, PEG backbone, -OCH2-) (figure S2B). 3. Synthesis of 3-(Maleimido) propionic acid N-hydroxysuccinimide ester (NHS-activated maleimide). β-alanine (1.8 g, 20 mmol) and maleic anhydride (2 g, 20 mmol) was added into 30 mL anhydrous dimethylformamide (DMF) and stirred at room temperature for 3 h. After complete

dissolution,

dicyclohexylcarbodiimide

(DCC,

4.6

g,

24

mmol)

and

N-

hydroxysuccinimide (NHS, 2.3 g, 20 mmol) were added into the solution and the mixture was stirred for 24 h at room temperature. The reaction mixture was filtered and the precipitate was washed with water (100 mL) and dichloromethane (100 mL). The filtrate was collected and the organic layer was washed with 5% NaHCO3 (3×50 mL) and once with brine. The organic layer was dried over anhydrous MgSO4 and dichloromethane was completely removed by rotary evaporator to obtain a white solid as product (yield 55%). 4. Synthesis of maleimide functionalized 4arm-PEG (4arm-PEG-Mal). To a round-bottom flask, 0.312 g (1.5 equivalents, 1.2 mmol) of 3-(Maleimido) propionic acid N-


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hydroxysuccinimide ester (NHS-activated maleimide) was added into 30 mL anhydrous dimethylformamide (DMF) and stirred at room temperature until the reagent was completely dissolved. To this solution, 20 mL solution of 4arm-PEG-NH2 (2 g, 0.2 mmol) in anhydrous DMF was added dropwise. To above solution, triethylamine (1.5 equivalents, 1.2 mmol, 0.17 mL) was then added and the resulting mixture was stirred under N2 at room temperature for 24 h. The solvent was removed from the crude product mixture by rotary evaporation, and the residue was dissolved in 10 mL dichloromethane. The resulting solution was filtrated, concentrated by rotary, and dropped into cold diethyl ether to precipitate the crude product. After separation by filtration and drying in vacuum, the crude product was dissolved again in 20 mL of water. The aqueous solution was filtered to remove debris, and the filtrate was lyophilized to obtain the product as a white powder (conversion 84%). 1H NMR (CDCl3, 300 MHz, δ): 2.50 (t, 8H, CH2NH), 3.40 (t, 8H, end -CH2N- group), 3.50 (t, 8H, end CH2O- group), 3.65 (s, 909H, PEG backbone, -OCH2-), and 6.70 (s. 8H, -CH=CH-) (figure S2B). Synthesis of thiolated fluorescein (FITC–SH). To prepare a fluorescently trackable, thiolbearing molecule as a model for illustration of bioconjugation ability of hydrogels, fluorescein (FITC) was thiolated through coupling reaction between amine and isocyanate. Briefly, Cystamine dihydrochloride (0.013 mmol, 3 mg) and triethylamine (TEA; 0.13 mmol, 18 µl) were mixed in 200 µl DMSO in a 2 mL vial, and vortexed until cysteamine hydrochloride was dissolved. Fluorescein isothiocyanate isomer I (FITC; 0.026 mmol, 10 mg) was dissolved in 200 µl DMSO. The cystamine solution was added into the FITC solution, and stirred for 4 hrs under dark conditions, followed by addition of dithiothreitol (DTT; 0.031 mmol, 9 mg) and sirring for 1 h. The reaction mixture was dropped into cold ethyl ether to precipitate FITC-SH. The precipitate was washed with water and again with ether. The ether was decanted and the residue



was diluted with DMSO to the concentration of 10 mg/mL. The thiolation was verified by observation of yellow color using Ellman’s assay. Hydrogel Formation. For chemically crosslinked hydrogels (DA), equal volumes of FAlg (4 wt%) and 4arm-PEG-Mal (10 wt%) solutions were mixed by vortex to ensure a homogenous solution and the DA cycloaddition was allowed to proceed overnight at 37 °C to ensure complete reaction and obtain hydrogels with 7 wt% overall concentration. During the gelation, the samples were monitored by time sweep rheometry and vial tilting method to determine gel time (Figure S2). For dual crosslinked hydrogels (DC), the DA hydrogels were incubated in CaCl2 (5 wt%) solution overnight. The DC hydrogels with one-pot strategy were prepared by mixing a 2x PEG solution with equal volume of freshly prepared slurry of CaCO3/D-(+)-Gluconic acid δ-lactone (GDL) to provide an initial rapid ionic gelation followed by sustained release of calcium during overnight incubation at 37 °C. Calcium crosslinked neat alginate hydrogels were prepared by mixing equal volume of the fresh slurry of CaCO3/GDL and solution of alginate (4 wt%) followed by overnight incubation at 37 °C. To enable visible tracking of PEG crosslikers for injection (movie S2), approximately 10% of the maleimide functionality was reacted with the FTIC-SH. In this way, the FTIC-SH was first introduced to the 4arm-PEG-Mal stock solution 1 hour prior to polymerization to allow conjugation via thiol-meleimide Michael addition reaction. Shear Rheology. Viscoelastic properties of hydrogels were determined using a stress controlled rheometer (Paar-Physica, MCR300 SN599139). Hydrogel disks (10 mm diameter, 1 mm thickness) were swollen in PBS and transferred on the stage plate, and a solvent trap was used to minimize solvent evaporation. The upper plate (20 mm diameter) was lowered until contacting the gel surface, and the examination was performed by oscillatory frequency sweeps (0.1-600 s-1, 0.5% strain). The gel time for the DA hydrogels was determined using the


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rheometer fitted with a 25 mm 2° acrylic cone using parallel plate geometry and a solvent trap to reduce evaporation. The hydrogel solutions were loaded onto the rheometer and the upper plate was lowered to a gap size of 50 µm. The margin of the samples was covered by silicon oil to inhibit evaporation. A time sweep was performed at 37 °C and 0.5% strain for 2 h to allow for gelation via DA reaction. The time at which G’ (shear storage modulus) dominated G” (shear loss modulus) was taken as the gel time. Compressive Mechanical Analysis. Hydrogel cylinders (10 mm diameter, 5 mm height) were swollen in PBS and analyzed using a universal testing machine (Santam, STM-20, 6 N load cell). Samples (n ≥ 6) were secured via a preload (0.02 N) and compression performed (1 mm.min-1) for determination of elastic moduli (slope from 5-15% strain), failure strain, and failure stress. Hysteresis testing was performed at 1 mm.min-1 with repeated loading and unloading cycles to 50% strain. Similar cyclic testing, with maximum strain progressively increased by 10% per cycle was also performed. Stress-strain profiles were integrated by geometric approximation (Graphpad Prism) to determine hydrogel toughness, work on loading, and work on unloading. For self-healing analysis, the ruptured samples were sealed to prevent dehydration and allowed to heal by overnight incubation at 37 °C. The healed samples were analyzed by compression test and healing efficiency was determined based on toughness as follows: Healing efficiency (%) = (Toughness healed / Toughness original) × 100

(2)

Scanning Electron Microscopy (SEM). The DA and DC alginate hydrogels were swollen in PBS overnight, frozen at -20 °C, and lyophilized for 48 hours. The resulting sponges were sputter coated with gold and were observed using a scanning electron microscope (AIS 2300C, Seron Technology, Korea).



Cell Encapsulation and Viability. Human cardiac progenitor cells (hCPCs) were generated by differentiation of human embryonic stem cells under a proprietary protocol.27 On the day 4 of differentiation, the differentiated cardiospheres were dissociated to single cells with accutase (GIBCO), followed by dispersing in the growth media (RPMI; Invitrogen, containing B-27, βmercaptoethanol, glutamine, non-essential amino acids and penicillin/streptomycin) at a density of 5×106 cells per mL. Prior to cell encapsulation, precursors were sterilized by UV exposure (254 nm, 2 cm distance, 15 min) for FAlgs or through syringe filters (0.22 µm) for PEG and CaCl2 solutions in the supplemented RPMI media. The DC hydrogels were prepared as described, with 10% of the total volume reserved to account for addition of a concentrated cell suspension (5×106 cells/mL) to the components immediately before onset of gelation. The encapsulated cells were maintained in culture for 24 hours (day 1) or an additional 3 and 7 days, and stained for Live/Dead following manufacturer’s instructions (Molecular Probes). At the desired days post-encapsulation, culture media was replaced with PBS containing calcein AM (2 µM) and ethidium homodimer-1 (4 µM) for Live/Dead staining. Hydrogels were incubated in Live/Dead stain for 45 min and visualized with an Olympus DP72 digital camera that was mounted on the microscope with a 10X objective. To assess metabolic activity, the cells (4000 cells/well; 96-well plate) were seeded onto the plate (as control) or encapsulated within the gels as described above and were examined for differences in metabolism by MTS assay at the desired days of culture. The cell-seeded plates or cell-laden gels (n = 4) were incubated for 4 hours with MTS reagent (Promega) and the supernatant was analyzed for absorbance at 490 nm. Biomolecule conjugation. Solutions (1.5 wt%) of neat alginate, FAlg4 and FAlg4/4arm-PEGMal were individually dropped through a 24G needle into CaCl2 (5 wt%) bath. The microspheres were incubated overnight in the bath to ensure complete gelation. The microgels were transferred


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to an aqueous solution of FITC-SH (as a model of thiol-bearing biomolecule) and incubated for 1 hr under shaking. After washing with PBS, the FITC-loaded microgels were incubated in PBS at 37 °C for 10 days and were imaged at desired time intervals by fluorescent microscopy. Statistical Analysis. All data are reported as mean ± standard deviation. Statistical significance was determined by two-way ANOVA with post hoc Tukey honestly significant difference (HSD) test to compare between groups unless otherwise stated. In all cases, significance was determined at P < 0.05. 3. Results and discussion 3.1. Synthesis of clickable yet calcium chelating furan-alginate (FAlg) Amidation reaction via EDC chemistry was successfully utilized to couple furfurylamine (FA) to the carboxyl groups of alginate as shown by furan peaks in 1H NMR, UV-spectrophotometry and ATR-FTIR spectra (Figure 1). FAlgs with different degrees of carboxyl substitutions with furan (DSfur) were synthesized by adjusting the stoichiometric ratio of the reactants (Table 1). The DSfur describes percentage of carboxylic acid groups in alginate coupled with furan and was calculated based on elemental analysis data (table S1) using the Equation 1. In terms of the maximum DSfur of ~60%, we could expect maintenance of the Ca2+-gelation capability for all FAls.9 However, further investigation was required to confirm this property. Alginate is a linear copolymer of G and M monosaccharides and only the carboxyls that reside on the latter are responsible for gelation with divalent cations such as Ca2+. The M/G ratio is thus the determining factor of Ca2+-gelation capacity.9 To investigate the conformation of neat and furanfunctionalized alginates (Alg and FAlg), FTIR spectra were recorded as shown in Figure 1D. The peak at 945 cm−1 and the peaks at 887 cm−1 and 810 cm−1 were attributed to the G and M residues, respectively.28-30 By normalizing the intensity of M and G-specific peaks to another



characteristic peak of alginate (1025 cm−1 for COC of saccharides), we could quantitatively investigate the proportion of each monosaccharide that dealt with the chemical modification. Table 1 shows the normalized transmittance of M and G of FAlgs, expressed as fold change, versus Alg. An increase in the ratio was realized by the modification of the monosaccharide. The data showed relatively equal values of elevated transmittance for both G and M in all FAlgs, which indicated a non-preferential contribution of either monosaccharides in furan functionalization. With regards to the initial value of ~40% G in the row material (stated by the manufacturer), it could be concluded from the FTIR spectra that a portion of G residues on the alginate remained intact after furan functionalization. Therefore, the Ca2+-gelation ability of FAlgs was reasonably expected to be well maintained.


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Figure 1. Synthesis and chemical characterization of furan functionalized alginate (FAlg). (A) Schematic of EDC-mediated amidation reaction for coupling of alginate carboxyls with furfurylamine. Modification of FAlg was confirmed by (B) 1H NMR spectra revealing the ethyl multiplet of furan, (C) UV-spectrophotometry spectra revealing furan peak in the FAlgs, with different intensities depending on DSfur (inset: furfurylamine spectra), and (D) ATR-FTIR spectra revealing characteristic peaks of furan (red dotted lines) and alginate (black dotted lines).



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Table 1. Chemical characteristics of the furan derivatives of alginate (FAlg). Fold change vs. Algb)

Molar ratio vs. COOH Sample

EDC

NHS

FA

DS (%)a) G

M1

M2

FAlg1

2

1

4

61.5

1.49

1.44

1.40

FAlg2

1

0.5

4

43.2

1.41

1.37

1.35

FAlg3

0.5

0.25

4

31.9

1.21

1.18

1.15

FAlg4

0.2

0.1

4

23.9

1.06

1.04

1.03

a)

Calculated from elemental analysis data via Equation 1.

b)

Calculated from the characteristic peaks of G and M monosaccharides in ATR-FTIR.

We sought to examine the possible Ca2+-gelation of FAlgs through the intact G residues by utilizing an encapsulation process. Upon dropping of the FAlg solutions (1.5 wt. %) into a CaCl2 (100 mM) bath, we observed the immediate formation of spherical particles (Figure 2A) that confirmed the retention of Ca2+-chelating G-rich domains in all four FAlgs. However, the morphological differences suggested that the extent of Ca2+-gelation depended on DSfur. More spherical and smaller particles were formed at lower furan functionalization such that FAlg4 was the most similar group to the neat Alg particles. Ca2+-gelation of alginate is described by an “egg-box” model of zipping the G-rich domains. The egg-box formation might result in a packed organization of alginate chains;31 hence, a higher Ca2+-gelation might be realized from more packed and smaller particles. The available G residues for Ca2+-gelation might control gelation kinetics. More round particles might thus be formed by rapid gelation, while delayed gelation might cause deformation of the incoming droplets due to their miscibility with the aqueous bath. We concluded that the Ca2+-gelation capacity of the FAlgs was regulated by DSfur.


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In addition, DSfur can govern the elastic properties of FAlg hydrogels by determining the available furan groups for the DA reaction. The covalent crosslinking of FAlgs was performed by using a proprietary synthesized 4arm-PEG-Mal (Figure S1). Of note, alginate and 4arm-PEG were fully miscible such that dissolution time of the hardly soluble alginate was considerably shortened (from ~2 hours to less than 2 minutes) by presence of the 4arm-PEG (see Movie S1). It was speculated that the hydrogen boding between carboxyl and hydroxyl groups of sodium alginate and ether oxygen on PEG backbone32 might replace some of the pre-existing intermolecular and intramolecular hydrogen bonds of alginate chains, leading to fast dissociation of alginate chains by incorporation of the readily water soluble PEG chains. Upon volumetric mixing of the separate solutions of FAlg (4% w/v) and 4arm-PEG-Mal (10% w/v), the furan and maleimide groups underwent a cycloaddition reaction to form the chemical DA hydrogels over a period of several minutes to a few hours, depending on DSfur (Figure S2). The gel time was determined by time sweep rheometry and simple method of vial tilting. The storage shear modulus (G’) and loss shear modulus (G”) determine the viscous and elastic behavior of the polymer solution, respectively. Upon mixing the FAlg and 4armPEG-Mal solutions, a viscous polymer solution with low elasticity (closed G’ and G”) was obtained (Figure S2A). The initial weak elasticity might be related to hydrogen bonding between the polymer chains that acts as physical weak crosslinks. As DA reaction proceeded, the elasticity (G’) was governed by chemical crosslinks. The gel time can be defined as the time point that the elastic behavior (G’) dominates the viscous nature (G”) of solution in rhoemetry test.33 In this way, gel time for DA hydrogels were measured ranging from 15 to 55 minutes (Figure S2B). In addition, the required time for sol-gel transition was visually determined (ranging from 45 to 140 minutes) as no flow was observed in vial titling method (Figure S2C).



We investigated the possibility of Ca2+-gelation in the preformed network of DA crosslinks. DC hydrogels were fabricated by incubating the DA-crosslinked cylindrical samples in the CaCl2 solution (Figure S3), after which we measured the swelling ratio in PBS as the mass increase (Figure 2B). In contrast to the chemical crosslinked DA networks, the DC hydrogels showed considerably low swelling in PBS, which indicated the packing induced by egg-box formation. This phenomena is important for mechanical performance as the swelling severely weakens hydrogels and compromised their behavior in the wet environments such as in-vivo applications.34-35 The regulating effect of DSfur on each ionic/click crosslinking could be quantitatively assessed by rheology analysis of viscous and elastic characteristics of the gels. To explore the viscoelasticity upon the crosslinking, storage modulus G’ and loss modulus G” for chemical crosslinked (DA) and dual crosslinked (DC) alginate hydrogels were determined by frequency sweep rheometry (Figures 2C and 2D). Comparing DA and DC hydrogels indicated that Ca2+-crosslinking increased the modulus (G’ and G”), strongly dependent on the DSfur (Figure 2C). Importantly, due to the unzipping (viscous) behavior of the calcium-carboxyl crosslinks, the loss modulus was more affected compared to the storage modulus upon Ca2+crosslinking (Figure 2C). This finding confirmed that DSfur determines the available carboxyls for Ca2+-binding, in agreement with Figure 1A. The frequency-independent, linear behavior of G’ revealed the highly elastic nature of all the DA and DC hydrogels (Figure 2D). As expected, the elastic behavior was mainly governed by covalent crosslink density as G’ showed a clear correlation with DSfur in both the DA and DC hydrogels.


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Figure 2. Calcium and click crosslinking ability of furan derivatives of alginate (FAlg) with varied furan substitutions. (A) Morphological characterization of the microspheres prepared by dropping neat alginate (Alg) or FAlgs into a CaCl2 bath. (n=30; *, p < 0.05, **, p < 0.01, ***, p < 0.001, and ****, p < 0.0001; n.s., not significant). Scale bars: 500 µm. (B) Equilibrium swelling ratio of the chemical crosslinked (DA) and dual ionic/chemical crosslinked (DC) FAlg hydrogels (n=3) after overnight incubation in PBS. (C) A comparison of the change in storage



modulus (G’) and loss modulus (G”) of the DA FAlg hydrogels after calcium crosslinking. The data was calculated as an average of values at 0.1- 10 s-1, Ԑ= 2% (n= 16 points; ****, p < 0.0001) (D) Storage modulus as frequency changes for the DA and DC FAlg hydrogels. 3.2. Optimization of toughness by tuning Diels-Alder/calcium crosslink balance Unconfined compression studies were conducted to further investigate the viscoelastic behavior of the hydrogels (Figures 3A and 3B). Consistent with the rheometry results (Figure 2D), the mechanical properties of the chemically crosslinked DA hydrogels was governed by covalent crosslink density, as the compressive modulus and strength were gradually increased by DSfur (Figure 3C). However, all DA hydrogels exhibited weak strength (below 80 kPa) and a sudden break into small fragments upon reaching strain values of 50-60% (Figure 3A). This observation could be attributed to the fragile nature of the covalent crosslinks and unhindered propagation of the covalent rupture points in the absence of ionic crosslinks.11, 35-36 The addition of ionic crosslinking enabled the mechanical properties of the DC hydrogels to be superior to those of the chemically crosslinked hydrogels (Figure 3B). The strength and fracture strain of the DC hydrogels gradually increased by the proportion of the ionic crosslinks where maximum values of 351±124 kPa and 75.2±3.5%, respectively, were achieved for FAlg4 (Figure 3C). To quantify toughness, the area under the stress-strain curve (fracture energy) was measured. The results showed that toughness exceedingly increased by Ca2+-crosslinking and reached a maximum value of 137±27 J.m-2 for the DC hydrogel of FAlg4, which was 30 times higher than that of the chemically cross-linked FAlg4 hydrogel (Figure 3C), demonstrating a significant reinforcement in toughness.7 The high strength and toughness of the DC alginate hydrogels could be attributed to high dissipation of energy by the transient ionic crosslinks which retarded rupture of the covalent crosslinks. However, the DC hydrogels with predominantly higher covalent


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crosslinks (FAlgs 1-3) ruptured into numerous small pieces (brittle fracture; Figure S4A), likely due to the intrinsic fragile properties of the dominant covalent bonds. Surprisingly, by providing an optimum balance of the covalent and physical crosslinking (FAlg4), the DC hydrogel demonstrated exceptional shape recovery after fracture (shown on inset of Figure 3A) with only minor and localized defects observed (shown by arrows in Figure S4A), indicative of a tough fracture. The trend could be explained by different viscoelastic behavior of each crosslink. It is well-known that chemical crosslinks contribute to the elasticity of the network while weak physical crosslinks tend to induce viscous properties.1 In the networks where both physical and chemical crosslinks co-exist, the elasticity becomes dominated and eventually energy dissipation deteriorates as the proportion of chemical crosslinks exceed a critical threshold.9,

12

Hence,

balancing the viscoelasticity is critical to obtain a hydrogel with optimal toughness.7 Herein, DSfur was found as the key parameter that regulated the balance of DA covalent crosslinking and calcium ionic crosslinking (Figure 2). To study the viscoelastic behavior of the DA and DC gels by DSfur, the G”/G’ (loss tangent) was determined as the frequency changes (Figures 3D and 3E). The plateau curves of the highly crosslinked chemical hydrogels (FAlgs 1-3; Figure 3D) suggested that the alginate chains were tightly coupled together by covalent crosslinks which might severely hinder chain mobility required for energy dissipation by unzipping the subsequent Ca2+-crosslinked zones. In contrast, the low crosslink density (FAlg4) might allow the chains to move against each other more efficiently because the G”/G’ demonstrated a peak (Figure 3D) which reflected the typical relaxation behavior of low density crosslinked networks.37 Upon additional crosslinking with the transient ionic crosslinks, the viscous behavior of the DC gels gradually increased by increasing the Ca2+-gelation capacity of the FAlgs (Figure 3E). However, this increase was surprisingly more pronounced when the optimal balance of ionic/click



crosslinks was provided (i.e. FAlg4). It is noteworthy that the G’ values was still at least 10 times higher than G” demonstrating highly elastic nature of the FAlg4 which is necessary for shape recovery. The dynamic nature of the Ca2+-crosslinks at this balance was determined by the sudden drop of the loss tangent at higher frequencies (~ 10 s-1) of shear stress (Figure 3E). At low frequencies, the dissociated chains could reassociate before complete stress relaxation. However, at high strain rates, the experiment time might exceed the lifetime of the Ca2+-carboxyl association and did not allow reformation of the dissociated bonds.19, 21 The elastic function of the permanent chemical crosslinks as well as the energy dissipation provided by the transient ionic crosslinks could be better understood when the DC hydrogels were compared with the physically crosslinked and chemically cross-linked alginate hydrogels under loading-unloading cycles with varying maximum compressions (Figures 3F-3I). The physically crosslinked FAlg hydrogels were too weak to examine their compression behavior. Alternatively, the calcium alginate hydrogel exhibited pronounced hysteresis and significant permanent deformation after each loading-unloading cycle due to high density of the ionic bonds and lack of any elastic crosslink (Figure 3F). In contrast, the chemically cross-linked (DA) hydrogel exhibited a small hysteresis-like cycle and a substantially smaller plastic deformation after unloading; however, the stress and strain at fracture were low (Figure 3G). In comparison, the DC alginate hydrogels showed both pronounced hysteresis and very small permanent deformation after unloading (Figure 3H). Hysteresis, defined as energy dissipated during a cycle (Uhist), was determined for each cycle to compare the DA and DC hydrogels (Figure 3I). The hysteresis of the DC hydrogels increased exponentially with the applied successive strain up to 60%, suggesting that its energy dissipation was more effective than those of the chemically cross-linked DA hydrogels during the loading/unloading cycles.38 Measurements of the


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hysteresis for all the DA and DC hydrogels was performed by loading/unloading compression at 50% strain (Figure S6). For chemically crosslinked (DA) hydrogels (Figure S6A), the mechanical behavior was governed entirely by an elastic response with little hysteresis, which was likely due to energy dissipation caused by physical events such as reversible hydrogen bonding between the ethereal oxygen on the PEG backbone and hydroxyl groups of alginate,39 or pressurization of the interstitial water in the swollen network.40 In comparison, dual crosslinked (DC) gels (Figure S6B) demonstrate considerable hysteresis, indicative of significant dissipation of the applied stresses. A comparison of the results (Figure S6C) suggested that the higher energy dissipation capacity of FAlg4 DC hydrogel might be the main reason underlying its high toughness. Herein, by tuning the DSfur at ~20%, a mechanically strong and tough DC alginate hydrogel was obtained. Of note, the more increase in Ca2+-crosslinking capability was found ineffective in fabrication of tough and recoverable alginate hydrogels, as the DC hydrogel with lower density of chemical crosslinks (FAlg5, DSfur=15%) ruptured at lower stresses and was irreversibly compressed upon loading (Figure S4B). Possibly, at a high density of Ca2+-bonds, the alginate chains were tightly crosslinked in Ca2+-zipped zones which are only stressed enough to break and unzip at high energy transfer. As a result, the elastic DA bonds might break before unzipping these zones, leading to irreversible deformation of the hydrogel. On the other hand, compression of FAlg5 was accompanied by extreme water expelling and this phenomena was more severe in the control hydrogel of Ca2+-crosslinked neat Alg, which was deformed as a compact sheet upon compression (as shown in inset of Figure 3F). The hybrid PEG/Alg hydrogel networks were expected to hold water more efficiently due to high hydrophilicity of the hydrogen bond acceptor



PEG chains and water entrapment within the mesh constructed by the elastic active PEG chains.40 The toughening mechanism can be more understood by studying microstructure of the hydrogels. The swollen DA and DC hydrogels were subjected to freeze-drying procedure to prepare samples for SEM characterization. The SEM images of different samples were shown in Figure S5. All the DA gels were macroporous as large pores were observed on the surface. The macroporosity can be attributed to the spacing caused by discrete chemical crosslinks. Upon ionic crosslinking, the large pores were disappeared at high density of Ca2+-zipped zones (FAlgs 2-4). The nearly smooth surface is the typical microstructure of Ca2+-crosslinked alginate hydrogels41 which might be caused by packing induced by egg-box formation via tight Ca2+crosslinks and shrinkage of small pores during freeze-drying.42 The more homogenous distribution of small pores may result in efficient stress distribution throughout the network as an underlying reason of toughening in the DC hydrogels. Altogether, the incorporation of physically and chemically crosslinked domains within alginate hydrogels not only increased the stress at fracture, but also allowed for higher deformation. The toughening mechanism is understood as follows. In both the DA-crosslinked and DC-crosslinked alginate hydrogels, the covalent crosslinks construct the elastic scaffold of the network which governs the shape recovery and resists against high stresses under loading. The DA-crosslinked alginate hydrogels are brittle because there is no temporary physical crosslink to dissipate energy and lower the applied load on the covalent bonds. Hence, at a specific stress, a fraction of the elastic crosslinks breaks down into tiny cracks which run quickly throughout the network and result in a brittle fracture observed as numerous small pieces after failure.3, 6 By contrast, in the DC hydrogel, the ionic crosslinks work as sacrificial bonds which can dissipate the stored energy


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by unzipping before the DA bonds break and avoid the catastrophic crack propagation. In this scenario, the covalent crosslinks work as energy transfer bridges in the network, resulting in a dramatic increase in the number of chains that participate in energy dissipation.6 Under loading, the applied stress is distributed throughout the network of chains connected together by elastic covalent bonds. Thus a larger fraction of alginate chains would be subjected to stress and dissipate energy by unzipping ionically crosslinked zones. This phenomenon leads to a tough fracture by preventing stress concentration in the localized crack zones as shown by the shape recovery after failure.3 Hence, in the DC strategy, a minimum number of covalent crosslinks is required for elastic deformation, shape recovery, water entrapment, and energy transfer throughout the network and a minimum density of breakable physical zones of coupled chains is necessary to sufficiently dissipate energy.



Figure 3. Optimization of the ionic/click crosslink balance for toughening the FAlg hydrogels.


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(A-B) Representative compressive stress–strain curves of the FAlg hydrogels crosslinked with: A) Diels-Alder (DA) chemical crosslinks and B) Calcium/DA dual crosslinks (DC). The insets present a representative DA gel (A) or the FAlg4 DC gel (B) after fracture. (C) Mechanical properties of the DA and DC FAlg hydrogels obtained by compression testing (n=6; a, significant difference (P < 0.005) between DA and DC gel in each group, determined by t-test; A, p < 0.001 in comparison with the previous DC groups; B, p < 0.0001 in comparison with other DC groups). (D-E) Viscoelastic behavior of the DA (D) and DC (E) FAlg hydrogels by varying frequencies of the shear strain. (F-H) A comparison of the: F) physically crosslinked Alg hydrogel, G) chemically crosslinked FAlg4 hydrogel, and H) DC FAlg4 hydrogel under loadingunloading cycles with varying maximum compressions. The insets present: F) the Alg sample after compression at the last cycle and G-H) the results over an initial narrow strain range. (I) Quantified hysteresis energy of the successive cycles for the DA and DC hydrogels of FAlg4. 3.3. Self-recovery of dual crosslinked tough hydrogels Except for toughness, the self-recovery property of a hydrogel at room temperature is also important for fatigue resistance during sustained uses. Under successive strain, the loading– unloading curve (Figure 3H) of the DC hydrogels showed that reloading stress in the subsequent cycle exceeds the unloading stress in the former cycle so that the loading curves lie between the curves of the former cycles. In contrast, for a sample with no fatigue resistance, the reloading curve always follows the previous unloading curve, a phenomenon commonly observed in irreversible DN hydrogels.35 This different behavior suggested that reformation of the broken ionic bonds occurs during the unloading process, as noted previously.12,

17

To investigate the

recoverability, hydrogels were subjected to two successive compression cycles with a set strain of 50% (Figures S6A and S6B). The chemically crosslinked hydrogels exhibited a highly elastic



response with a recoverable little hysteresis (Figures S6C and S6D). These samples could be reloaded with almost no change in the loading/unloading behavior. In comparison, dual crosslinked gels demonstrated a considerable hysteresis, along with an elastic recoverability (Figure S6D). To more investigate the recoverability of the tough gel, DC hydrogel of FAlg4 was subjected to several successive compression cycles with a set strain of 50% (Figure 4A and Figure S7). The loading–unloading cycles showed that neither serious plastic deformation nor strength attenuation occurred in the DC hydrogels after cyclic compression, which indicated excellent shape recovery and fatigue resistance properties of the DC alginate hydrogels. The mechanical recoverability was then quantified as the ratio (Rn) of the nth cycle’s Uhist to that of the (n-1)th compressive cycle (Figure 4B). Importantly, we obtained recovery rate as high as R2 ~ 70% which was in the range of previously reported recoverable polysaccharide-based DN hydrogels with R2 values of 82% (gellan gum/gelatin), 74% (alginate/PAAm), 65% (agar/PAAm), and 53% (gellan gum/PAAm)13-14, 17, 20 despite the fact that we did not apply any delayed conditioning. Mechanical recoverability was likely caused by cooperative action of the ionic and covalent crosslinks, the former by fast re-formation of the fractured ionic bonds during unloading and the latter by elastic action of the covalent crosslinks to recover the deformed area.12 However, reduction in the recovered hysteresis could be partially attributed to the observed water expelling during the initial loading cycle. This could be indicative of a deswelling effect, contributing to the observed stiffening of the gels (67% increase in the modulus). The other possibility is that the first loading might trigger some irreversible network reconstruction such as disentanglement of entrapped polymer strands or scission of a number of active elastic covalent bonds,14, 20, 43 which would dissipate a large amount of energy and would not occur in subsequent loading cycles. Interestingly, we observed that although the DC


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hydrogels might not fully recover from the first compressive cycle, they could almost fully recover from subsequent cycles as the curves almost overlapped (Figure 4A). The recovery rate between cycles 2 and 3 was obtained as R3=83% and similar results were obtained for the other subsequent compressive cycles (Figure 4B). This finding agreed with those reported for the Alg/PAAm DN hydrogels,14 despite their use of delayed rest periods between the cycles. Hydrogels that consist of ionic and covalent crosslinked polymers are typically regarded as highly tough and recoverable due to the potential for transient ionic crosslinks to rapid association/dissociation. However, the recovery rate strongly depends on the inherent kinetics of physical association and, more importantly, the availability of physical binding pairs after dissociation.15 For example, the calcium alginate based DN hydrogels have shown a slow recovery (∼74% work from the first loading after 24 h storage at 80 °C), likely due to the limited dynamics of rearranged dissociated alginate chains within the chemical network of PAAm after unloading.12 Delayed recovery has been also reported for similar Ca2+-polysaccharide based DN hydrogels (storage times of 80 minutes for gellan gum/PAAm and 300 minutes for xanthan gum/PAAm and gellan gum/gelatin).13-15 In contrast, we have observed a significant recovery of hysteresis immediately after unloading without any exogenous treatment, which is highly important for fatigue resistance in cyclic conditions. This surprising result could be ascribed to the differences in action by covalent crosslinks in the alginate-based DC and DN hydrogels. In DN hydrogels, covalent and physical crosslinks act independently in each network with no covalent crosslinks between the chains of the physical network. As a result, by unzipping under loading, these chains may freely slide against each other and rearrange throughout the chemical network.20 In order to reform Ca2+-crosslinks, the long alginate chains require sufficient dynamics to find and recouple G residues, and reconstruct a new network.44 Although this



mechanism is partially restricted in such mixed systems, the long-term, high temperature conditions favor the very slow chain dynamics.15, 44 In addition, the alginate network undergoes plastic deformation when the calcium crosslinks dissociate and reform elsewhere.45 As a result, the recovered network would not necessarily reproduce its previous properties.12, 20, 38 In contrast to DN hydrogels that are constructed from two individual networks, in the present DC hydrogel, the covalent binding sites have been incorporated into a physically crosslinked network to form a homogeneous macromolecular structure on which the both crosslinkings were imposed in a single network. This may lead to homogenous distribution of the elastic covalent crosslinks along the main polymer chains at the molecular level.4 Therefore, the Ca2+-crosslinked zones are surrounded by highly restricted polymer areas which may work as topological constraints at the molecular level along the chain backbone to avoid the plastic flow and severe rearrangement of the dissociated chains during loading. (Scheme 1A). As a result, the movement of the polymer chains during unloading is not restricted by a secondary network of covalent crosslinks; rather, it might be more facilitated by elastic memory of the covalent crosslinks. Moreover, the permanent covalent crosslinks may change the chain dynamics by avoiding the translational movements (reptation and diffusion) of polymer chains and limiting the macromolecular dynamics to more rapid, local (segmental) movements inside a restricted space (e.g., the tube defined by the Rouse model).21, 44 As a result, the molecular rearrangement becomes strongly limited in space so that once the load is released, the dissociated physical pairs are expected to return rapidly to their original positions (Scheme 1A). The proposed mechanism agreed well with a previous report on DC hydrogels of Ca2+-carboxyl crosslinked triblock copolymers which showed nearly full recovery immediately after unloading.19 In addition, Azevedo et al. recently reported faster recovery of DC networks compared to DN hydrogels of physicochemical chitosan hydrogels that


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were physically crosslinked by catechol-Fe3+ coordination and chemically by genipin.11 However, toughening by breakage of the relatively strong metal-ligand interaction was hindered by the weak strength of the low-density chemical crosslinks, which led to limited energy dissipation before failure in the DC hydrogels. It is worthy to note that carboxyl association with cationic metals is also possible with trivalent ions (e.g., Fe3+) or divalent transition metals (e.g., Zn2+), leading to a stronger interaction and subsequently stiffer hydrogels.16 However, the strong metal-ligand interaction and weak strength of the low-density chemical crosslinks may result in dominant elastic behavior and limited energy dissipation before fracture of the chemical crosslinks in DC hydrogels.11 Whereas, the weaker complexation with group II metals, especially Ca2+, may lead to much faster and less stable transient association, which is favorable for fast, efficient recovery of energy dissipation.19 On the other hand, the carboxylic– metal interaction with trivalent cations such as Fe3+ is more complex, existing in different forms (e.g., mono-, bi-, or tridentates for Fe3+). Only the tridentate coordinates have been shown to lead to secondary ionic crosslinking and subsequent toughening of a chemically crosslinked network.4 Accordingly, DC hydrogels of PAAm-co-AAc that were covalently crosslinked by free radical polymerization and physically by carboxyl-Fe3+ coordination exhibited immediate recovery of only 20% after unloading which was attributed to rearrangement of the coordinates and replacement of a part of the tridentates by the elastically inactive coordinates. Hence, a storage time of 4 hours was required for reorganization of the coordinates to reform trivalents and gain recovery of hysteresis (up to 87.6%).4 Therefore, the simple and highly transient Ca2+-carboxyl interaction is a key factor to achieve soft and tough hydrogels with fast self-recovery. In addition, the PEG crosslinkers in our design might work as small spacers, providing a steric spacing of Ca2+-crosslinking zones in the alginate, which may



reduce the net avidity between the polymer chains.46 As a result, the applied stress would be relaxed faster and more efficiently,46 leading to a more sensitive response of the physical crosslinks to stress and more effective energy dissipation before the breakage of covalent crosslinks, all of which are important for toughening of DC hydrogels. 3.4. Self-healing of dual crosslinked tough hydrogels In addition to the molecular self-repair, a macroscopic self-healing after fracture was also expected by the dynamic covalent crosslinks. Importantly, convectional physiochemical tough hydrogels are mostly not recoverable after complete failure due to irreversible and permanent breakage of the covalent crosslinks.15 In contrast, DA crosslinks are known as dynamic covalent bonds with the capability to reform autonomously after fracture, even under physiological conditions.25 This would lead to self-healing capability of the damaged DC alginate hydrogels (Figure 4C). In order to assess healing efficiency, DA and DC alginate hydrogels were allowed to heal after compression failure by keeping the fractured pieces in contact together in a sealed polyethylene bag, overnight incubation at 37 °C, and subsequently analyzed. Surprisingly, the healed hydrogels reproduced mechanical behavior of their parent virgin hydrogels (Figure 4D). The physicochemical tough hydrogels in literature are mostly not able to self-heal efficiently after damage. Indeed, the convectional physicochemical hydrogels are self-recoverable before complete fracture thanks to reversible physical crosslinks, however, the efficient self-healing upon macroscopic fracture can be hindered by irreversible break of chemical crosslinks.9, 47-48 Many physicochemical DN hydrogels have been reported as self-recoverable tough hydrogels, however, the healing has not been possible or efficient, likely due to irreversible fracture of the covalent bonds.4-5,

9, 49-51

Alternatively, fully physical crosslinked DN hydrogels have been

proposed to achieve fully healable tough hydrogels. For example, several physical DN hydrogels


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have been developed by introducing hydrophobically associated polyacrylamide into different physically crosslinked networks such as agar, polyvinyl alcohol, and xanthan gum.15,

52-53

In

these systems, the efficient self-healing has been achieved after thermal treatment for enough time to favor molecular dynamics. For example, the physical DN hydrogels based on Ca2+xanthan gum/PAAm showed a healing efficiency of ~60% after a 10-hour incubation period at 70°C.15 Similarly, several attempts of developing self-healable tough hydrogels included the introduction of dynamic and reversible metal-ligand coordination interaction in the nanocomposite hydrogels. Highly efficient healing has been reported previously for nanocomposite tough hydrogels using nanoparticles as multifunctional physical crosslinkers.47, 54-55

For example, harnessing thiolate-gold coordination interaction as multifunctional dynamic

crosslinks, Qin et al. developed tough hydrogels with a fast, efficient healing (96% under nearinfrared irradiation in 1 min).55 Herein, we have incorporated both dynamic physical and chemical crosslinks within a single network to produce an efficient self-healable tough hydrogel. The dynamic covalent bonds are quite rare and DA is one which can be reformed autonomously after fracture under physiological conditions. Importantly, our DC alginate hydrogel was efficiently healed (79% recovery of toughness, calculated by Equation 2), under mild conditions (37 °C) without any external stimulus to trigger the healing process. The pronounced effect of the reversible DA covalent bonds in the healing process was confirmed by the significant healing of DA hydrogel which showed a healing efficacy as high as 85%. To the best of our knowledge, this is the first time that a physicochemical tough hydrogel is produced by both self-healable physical and chemical crosslinks. To demonstrate the self-healing behavior in liquid state, the compression fractured pieces of DA and DC hydrogels were kept in contact together in a bath of PBS or water. After 24 hours,



the healed samples were analyzed under compression and compared with their original state (Figure S8). Interestingly, the DA hydrogels were healed more efficiently in liquid state (healing efficiency of 109%). It has been shown that addition of water may improve healing efficiency of hydrogels likely due to elevated dynamics of polymer chains in the fractured zone.48 Additionally, DC hydrogels were healed efficiently in liquid state (healing efficiency of 83% and 80% in PBS and water, respectively). The results suggested that the DA and DC alginate hydrogels can be used as efficient self-healing structures in aqueous situations such as in vivo applications.

Figure 4. Self-recovery and self-healing of the dual crosslinked (DC) hydrogels. (A) Five successive loading–unloading cycles of the FAlg4 DC hydrogel. The inset represents the sample before and after each cycle. (B) Recovery efficiency of the hysteresis energy and work of loading after each cycle. (C) Schematic representation and photographs of the healing process under


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physiological conditions for two colored cuts of the DC hydrogels. (D) Compression stress– strain curves of the original and healed samples of the DA and DC hydrogels. 3.5. In situ forming capability of dual crosslinked tough hydrogels DA kinetics is reasonably slow (e.g., 160 minutes for FAlg4) to allow for adequate polymer mixing and convenient handling before application. In contrast, chemical hydrogels that rely on fast reactions (e.g., Michael-type addition of thiol-maleimide) can be exclusively injected by double barrel syringes which could lead to inefficient mixing, heterogeneous gelation, and inconsistent mechanical properties.56 However, when DA hydrogels are intended to be used as in situ forming scaffolds, loss of components before complete gelation at the site of injection might be a concern. This can be addressed by designing an initial fast gelation mechanism prior to DA curing. Due to its orthogonality and regioselectivity, the DA reaction has been found to proceed efficiently in the presence of preformed gels.57-59 Herein, we speculated that an initial Ca2+gelation might provide a network for retention of components intended to follow additional DA crosslinking for gel reinforcement. However, we were concerned about the efficiency of the DA reaction in the preformed network of a Ca2+-crosslinked alginate. To address this concern, the efficiency and kinetics of the DA reaction were compared in the solution state and in the calcium gel of FAlg4 by measurements of UV absorbance of maleimide60 over a period of time (Figure 5A). The results indicated that the DA reaction might be slightly slower in the presence of calcium crosslinks, likely due to steric hindrances and elevated viscosity caused by Ca2+gelation.61 However, no significant difference in efficiency was observed at the end of reaction as reflected by almost complete consumption of maleimide (Figure S9). This result was confirmed by mechanical analysis which showed closed compression behavior of the DC gels prepared by starting from either DA or Ca2+-gelation (Figure 5B). Moreover, by selective



dissociation of calcium crosslinks in the DC hydrogel, the remaining DA network reproduced the compression behavior of the original DA hydrogels under compression, which confirmed a lack of disturbance in DA reaction by the calcium crosslinks (Figures 5C and 5D). These observations confirmed the hypothesis that hydrogel curing can be caused by covalent DA crosslinking while already in the gel state.59, 62 The developed autonomous, one-pot gelation potentiates our system for molding into specific shapes or injection in wet environments. Ca2+-crosslinking of the 4arm-PEG-Mal/FAlg mixtures produces pregels that are expected to be moldable or injectable materials. The moldability was assessed by applying the pregel onto a mold of conical shaped microwells, followed by centrifuging to push it down to the microwells. The filled mold was left overnight to ensure curing of the pregel via an autonomous DA reaction. The molded DC hydrogel was successfully removed as an array of microposts with precisely recapitulated features (Figure 5E). The advantage of “one-pot” dual crosslinking for space filling is that neither swelling nor deformation occurs during molding. To illustrate the injectability of this system, the mixed precursor solutions of FAlg and FITC-labeled 4arm-PEG-Mal was partially crosslinked with Ca2+ and injected through a syringe needle into a water bath. The pregel was successfully injected without any leakage of the unreacted PEG component (trackable by the fluorescent label) into water (Figure 5F, see Movie S2). This finding showed that the initial Ca2+-crosslinked network could serve for retention of unreacted components at the injection site. The ability of rapid gelation and retention of the PEG crosslinker by Ca2+-crosslinking may provide us with another interesting feature, fabrication of Ca2+-gelation shaped structures, in which the physical network could work as a template that allows chemical reactions to cure the shaped sample by covalent crosslinks.63 The Ca2+-crosslinks might be of interest to obtain tough


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DC hydrogels or could be leached out by a cytocompatible process of Ca2+-chelating64 to leave chemical crosslinked hydrogels with the shaped structures. To prove this concept, DC microspheres were fabricated by dropping the mixed solution of the polymers into a CaCl2 bath which allowed for rapid formation of microspheres, followed by autonomous DA-curing within the microspheres over a 5-hour period of time (Figure S10). After the removal of Ca2+-crosslinks by chelating, the microspheres remained intact, indicating the efficient function of the Ca2+crosslinked network as a temporary template for retention of PEG component and DA-curing. Similarly, the cytocompatible and “one-pot” synthetic approach may allow for fabrication of cell-laden, tough 3D structures using extrusion bioprinting, as previously reported for the use of alginate/PEG-diacrylate as tough bioinks.65 In these systems, Ca2+-gelation provides suitable viscosity to print a specific shape after which the additional covalent crosslinking is typically induced by UV irradiation to reinforce the structure.66-68 However, the ability of self-reinforcing post printing can be an advantage of our system over the previous photo-curable tough bioinks.



Figure 5. “One-pot” gelation, moldability and injectability of the dual crosslinked (DC) hydrogels. (A-D) A comparison of the Diels-Alder (DA) efficiency in calcium-jellified or solution state of FAlg/4arm-PEG-Mal mixture by: (A) UV-spectrophotometry analysis of maleimide absorbance (290 nm) over time in the two states. (B) Compression analysis of the DC hydrogels prepared by starting from DA or Ca2+-gelation. (C) Morphological and (D) compression analysis of the DA hydrogels prepared originally or by starting from Ca2+-gelation, followed by removal of calcium by chelators (EDTA/sodium citrate). (E) Photograph and microscopic image of the micromolded DC hydrogels. (F) A video frame captured from Movie S2, where the pregel of Ca2+-crosslinked FAlg4/FITC-labeled 4arm-PEG-Mal (schematically shown) was injected through a syringe needle into a water bath. 3.6. Biological potentials of dual crosslinked alginate hydrogels To further explore the biological interest of the developed hydrogels, the cytocompatibility and potential for biological modification were examined. The ability of these hydrogels to encapsulate viable cells was assessed by mixing cardiac progenitor cells (CPCs) with the polymeric precursor solutions and dual crosslinking for gel formation in the presence of viable cells. After gel formation, Live/Dead staining showed that the cells had a uniform distribution within the DC hydrogels and a high viability rate (>90%) as noted by the limited numbers of red cells during incubation at physiological conditions up to 7 days (Figure 6A). The encapsulated CPCs had comparable metabolic activity to those cultured on the control tissue culture plate (TCP) as analyzed by the MTS assay (Figure 6B). The results proved the cytocompatible character of the dual crosslinking strategy and emphasized its potential to create cell-laden artificial substitutes of native soft-tissues.


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Figure 6. Cytocompatibility of the dual crosslinking strategy. Cardiac progenitor cells were mixed with the polymer precursors followed by dual crosslinking and culture for 7 days. Cytocompatibility was assessed through (A) Live/Dead staining (viable cells appeared in green whereas dead cells were stained in red) and (B) by comparing metabolic activity of the cells, encapsulated within DC hydrogels or cultured onto tissue culture plate (TCP) through MTS assay (n=6; ***, P < 0.0001). In addition to biocompatibility, another interesting feature of the hydrogels for biomedical applications is their capability for covalent binding of biomolecules such as peptides, growth factors or drugs to achieve a specific biological function. Herein, the unreacted maleimide groups are expected to work as binding sites24 for on demand, post-gelation immobilization of thiol-bearing biomolecules (e.g., cysteine residues of proteins) via a fast, selective, and physiologically possible reaction known as the thiol-maleimide Michael addition (Figure 7A).69 The conjugation capability was investigated by incubating the DC hydrogel spheres in an aqueous solution of a thiol-bearing model (FITC-SH) for 30 minutes and subsequently assessing stability of the immobilized agent over a 10-day period of time (Figure 7B). The results showed successful conjugation and prolonged stability of FITC-SH in the DC alginate microspheres.



Importantly, the star shaped PEG crosslinkers can work as spacers within the network, constructing large enough mesh size (15.2 nm for FAlg4, calculated based on G’ values70) which would allow for diffusion of large macromolecular agents into the bulk structure.

Figure 7. Bioconjugation ability of the DC hydrogels. (A) Schematic illustration of the possibility of post-gelation conjugation of thiol-bearing molecules (FITC-SH as model) through Michael addition reaction with unreacted PEG-Mal branches. (B) Microspheres of calcium crosslinked Alg and FAlg4, and dual crosslinked FAlg4 were incubated within the FITC-SH solution, then transferred to a PBS bath (start point) and monitored by fluorescent microscopy to explore the conjugation stability over 10 days. Scale bars correspond to 500 µm.


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Of note, the present DC alginate tough hydrogel is reasonably soft with a modulus of ~30 kPa that can mimic the stiffness of load-bearing soft tissues such as cardiac or skeletal muscles. The importance of soft and tough hydrogels comes from the possibility to sustain cyclic loads and simultaneously ensure mechanical harmonization with the surrounding soft tissue.71 Tough hydrogels that achieve high toughness primarily through a great degree of stiffness and/or extensibility are not suitable for mechanical replacements of some load-bearing soft tissues, such as cardiac muscle, which are not so stiff (e.g., 10-50 kPa for healthy heart muscle) and do not operate under such extreme strains (e.g., 15-22% in a beating heart).71 In these situations, it is important to have rapid and autonomous recovery of toughness for stable function under cyclic stress rather than extreme toughness at high loading or stretching.72 Our soft and tough alginate hydrogels are well suited to work in a mechanically dynamic soft environment, such as the heart, and have the capability to sustain and recover from deformation due to the elasticity that arises from covalent crosslinks, and recapitulate the nonlinear elasticity of heart muscle71 by recoverable energy dissipation. 4. Conclusion A dual crosslinking strategy was developed to answer the urgent need for fatigue-resistant, cytocompatible, and in situ forming tough hydrogels. Clickable, yet calcium-binding derivatives of alginate was synthesized by partial substitution of its carboxyl functionalities with furan, which could come into Diels-Alder (DA) click reaction with maleimide end groups of a four arm poly(ethylene glycol) (4arm-PEG-Mal) crosslinker. The degree of substitution (DS) tuned the toughness of the dual crosslinked (DC) hydrogels by balancing the ionic and covalent crosslinking where each had a specific viscoelastic role: Under loading, the calcium-crosslinked zones dissociated to dissipate energy, while the covalent crosslinks avoided sever plastic



deformation and provided strength and shape fidelity. Once the load was released, the deformed gel returned to its initial position guided by elastic covalent crosslinks and immediately recovered its dissipative capacity by fast reformation of the calcium crosslinks. The optimal balance was achieved at DS ~20% (toughness of 136.7 J.m-2). Repeated compression loadings of the DC hydrogels revealed their ability to immediate self-recovery of shape and energy dissipation capacity. Additionally, thanks to dynamic nature of both the ionic and DA covalent crosslinks, the hydrogels exhibited significant recovery of mechanical properties (~80% of toughness) even after complete fracture. Calcium crosslinking of the precursors provided a pregel template for autonomous additional curing by DA reaction. This “one-pot” sequential ionic and click crosslinking allowed for sphere formation, molding into microfeatures, and administration by minimally invasive injections. The potential for biological applications was shown by encapsulation of viable cardiac cells and on-demand bioconjugation of thiol-bearing molecules. In summary, the DC alginate hydrogel had a set of interesting features including selfrecoverable toughness under cyclic loading, autonomous self-healing upon rupture, moldability and injectability, viable cell encapsulation, and bioconjugation ability under physiological conditions which distinguished it from the convectional physicochemical tough hydrogels. The soft and tough hydrogel can be used to replace or repair load-bearing soft tissues and support sustained mechanical functions in bioactuators. ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website.


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Elemental analysis results for furan derivatives of alginate; Synthetic procedure and 1H NMR spectra of 4arm-PEG-OTs, 4arm-PEG-NH2, and 4arm-PEG-Mal, Gel time of Diels-Alder (DA) FAlg hydrogels; Photographs of cylindrical samples of the as-prepared FAlg hydrogels and the PBS-swollen samples of the DA chemical crosslinked and dual crosslinked hydrogels; Photographic illustration of compression behavior of the dual crosslinked FAlg hydrogels and calcium crosslinked neat alginate hydrogel; SEM images of freeze-dried DA and DC hydrogels; Evaluation of energy dissipation and recoverability of the DA chemical crosslinked and dual crosslinked hydrogels by cyclic compressive loading; Fattigue resistance of FAlg4 DC hydrogel under 100 cycles of susccessive loading; Self-healing of DA and DC hydrogels in aqous media; Comparison of DA reaction efficiency in solution or calcium gel state of the FAlg4 by UV spectrophotometry; An illustration of templating role of the calcium crosslinking in sphericalshaped structures (PDF) Dissolution of sodium alginate in water without PEG or in presence of linear or four arm PEG10000 (Movie S1.avi) Injectability of the pregel precursor of dual crosslinked, tough alginate hydrogel (Movie S2.avi) AUTHOR INFORMATION Corresponding Authors *Hamid Mirzadeh Department of Polymer Engineering and Color Technology, Amir Kabir University of Technology (Tehran Polytechnic), Hafez Ave., Tehran 15875-4413, Iran



Tel: 009821-64542420 E-mail: [email protected] *Hossein Baharvand Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Banihashem Square, Banihashem Street, Resalat Highway, Tehran 1665659911, Iran Tel: 009821-23562504 Fax: 009821-23562507 E-mail: [email protected] Conflict of interest The authors declare no conflict of interest. Acknowledgements This work was supported by a grant from Royan Institute, the Iran National Science Foundation (INSF) and Iran Science Elites Federation to H.B. The authors express their appreciation to Dr. Sara Rajabi and Mr. Hassan Ansari for their technical support for the cell culture, and Dr. Hamid Sadeghi Abendansari for his support with material synthesis and productive discussions.


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In Situ Forming, Cytocompatible, and Self-Recoverable Tough

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