Subscriber access provided by UNIV OF DURHAM
Article
Diels-Alder Click-Crosslinked Hydrogels with Increased Reactivity Enable 3D Cell Encapsulation Laura J. Smith, S. Maryamdokht Taimoory, Roger Y Tam, Alexander E.G. Baker, Niema Binth Mohammad, John F. Trant, and Molly S Shoichet Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b01715 • Publication Date (Web): 14 Feb 2018 Downloaded from http://pubs.acs.org on February 15, 2018
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Biomacromolecules is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Biomacromolecules
1
Diels-Alder Click-Crosslinked Hydrogels with
2
Increased Reactivity Enable 3D Cell Encapsulation
3
Laura J. Smith,1 S. Maryamdokht Taimoory, 2 Roger Y. Tam, 1 Alexander E. G. Baker, 1 Niema
4
Binth Mohammad, 1 John F. Trant,2* Molly S. Shoichet1*
5
1
Department of Chemical Engineering & Applied Chemistry, Donnelly Centre, University of
6
Toronto, 160 College Street, Toronto, ON M5S3E1 2
7
Department of Chemistry, University of Windsor
8 9
KEYWORDS: hydrogel, 3D cell culture, biomaterials, Diels-Alder, click chemistry
10 11
AUTHOR INFORMATION
12
Corresponding Author
13
*correspondence to:
14
[email protected] 15
and
16
[email protected] 17
ACS Paragon Plus Environment
1
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Biomacromolecules
Page 2 of 30
1
ABSTRACT
2
Engineered hydrogels have been extensively used to direct cell function in 3D cell culture
3
models, which are more representative of the native cellular microenvironment than conventional
4
2D cell culture. Previously, hyaluronan-furan and bis-maleimide polyethylene glycol hydrogels
5
were synthesized via Diels-Alder chemistry at acidic pH, which did not allow encapsulation of
6
viable cells. In order to enable gelation at physiological pH, the reaction kinetics were
7
accelerated by replacing the hyaluronan-furan with the more electron-rich hyaluronan-
8
methylfuran. These new click-crosslinked hydrogels gel faster and at physiological pH, enabling
9
encapsulation of viable cells, as demonstrated with 3D culture of 5 different cancer cell lines.
10
The methylfuran accelerates Diels-Alder cycloaddition yet also increases the retro Diels-Alder
11
reaction. Using computational analysis, we gain insight into the mechanism of the increased
12
Diels-Alder reactivity and uncover that transition state geometry and an unexpected hydrogen-
13
bonding interaction are important contributors to the observed rate enhancement. This
14
crosslinking strategy serves as a platform for bioconjugation and hydrogel synthesis for use in
15
3D cell culture and tissue engineering.
16
Introduction
17 18
It is broadly recognized that conventional 2-dimensional (2D) cell culture does not adequately
19
represent the native cellular microenvironment, necessitating the design of 3-dimensional (3D)
20
cell culture strategies.1,2
21
microenvironments and direct cell function. These materials can be engineered to mimic
22
different characteristics of the extracellular matrix (ECM) including: mechanical properties,
Hydrogels are used extensively to engineer 3D cellular
ACS Paragon Plus Environment
2
Page 3 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Biomacromolecules
1
biochemical composition, and architecture.3 These biomimetic strategies enable improved
2
understanding of the role of the ECM in disease progression and allow for additional functional
3
readouts in higher content drug screens.3,4 For the latter, 3D cell culture allows cell survival as
4
well as cell invasion into the hydrogel to be probed, which is critical to understanding highly
5
invasive diseases, yet impossible to ascertain in conventional, 2D cell culture.5,6
6
Hydrogels for 3D cell culture comprise bio-inert polymers, such as PEG, or bio-active
7
polymers, such as polypeptides or glycosaminoglycans. Crosslinking strategies for these
8
materials often rely on thiol-based chemistries including Michael-type addition and radical-
9
mediated thiol-ene chemistry; however, thiols are sensitive to oxidation, which can complicate
10
these reactions.7 Strain-promoted azide-alkyne cycloaddition (SPAAC) is a more reliable
11
alternative, but is synthetically complex.8 Diels-Alder chemistry, such as the inverse electron
12
demand tetrazine-norbornene system, has been effectively used to crosslink PEG hydrogels9, and
13
provides a simpler alternative to SPAAC with the same advantages of long-term stability,
14
efficient conversion due to favourable thermodynamics, and a clean reaction profile.10
15
Hyaluronan hydrogels have been formed by the Diels-Alder reaction between furan-modified
16
hyaluronan with either bismaleimide-poly(ethylene glycol) or bismaleimide-peptides.3,5,11-14 This
17
reaction, while useful in the synthesis of pre-formed gels on which cells are cultured on the
18
surface, is unsuitable for cell encapsulation because it is inefficient at physiological pH 7.4,
19
requiring a low pH of 5.5 for a timely reaction; however, these conditions are too acidic and too
20
slow for live cell encapsulation.11 With a view toward utilizing these hyaluronan hydrogels for
21
3D cell culture, a more efficient crosslinking reaction is required at physiological pH. Although
22
reaction kinetics can be accelerated by increasing reaction temperature, polymer concentration,
23
and the degree of bioorthogonal functional group substitution, changing these parameters alters
ACS Paragon Plus Environment
3
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Biomacromolecules
Page 4 of 30
1
the porous architecture of the system, which may lead to changes in cell mechanosensing, cell-
2
confinement, and downstream pathways.15 An alternative approach to changing the reaction
3
kinetics is to use more reactive coupling agents.
4
We developed HA-based hydrogels that are crosslinked at physiological pH 7.4, thereby
5
enabling encapsulation and 3D culture of viable cells (Fig. 1). The Diels-Alder (DA) reaction
6
was accelerated at pH 7.4 by replacing furan with the more electron-rich diene, methylfuran, that
7
couples with maleimide crosslinkers.10,16,17 Using both experimental and computational studies,
8
we gain a thorough understanding of the chemistry of the crosslinked system and demonstrate its
9
utility for 3D cell culture.
10
11 12
Figure 1: (A) Covalently crosslinked hydrogels can be fabricated using furan-maleimide Diels-
13
Alder (DA) chemistry. (B) Replacing furan with methylfuran accelerates the DA reaction at pH
14
7.4.
15 16
Experimental
17
ACS Paragon Plus Environment
4
Page 5 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Biomacromolecules
1
Materials: Lyophilized sodium hyaluronate (HA) (289kDa) was purchased from Lifecore
2
Biomedical (Claska, MN, USA). Bis-maleimido-poly(ethylene glycol) ((Mal)2PEG) was
3
purchased from RAPP Polymere GmbH (Tülbingen, Germany). 5-methylfurfurylamine, and 4-
4
(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) was purchased
5
from TCI (Boston, MA, USA). Sodium chloride and 2-(N-morpholino)-ethanesulfonic acid
6
(MES) were purchased from BioShop Canada Inc. (Burlington, ON, Canada). Hyaluronidase,
7
deuterium oxide (D2O) and Dulbecco’s phosphate buffered saline (PBS) were purchased from
8
Sigma-Aldrich (St. Louis, MO, USA). Dialysis membranes were purchased from Spectrum
9
Laboratories (Rancho Dominguez, CA, USA).
10 11
Synthesis and Characterization of Furan-modified Hyaluronan (HA-FA) and Methylfuran10
12
Modified Hyaluronan (HA-mF): HA-FA was prepared as previously reported.
Methylfuran-
13
modified HA (HA-mF) was synthesized following the same strategy. Briefly, HA was dissolved
14
in MES buffer (100 mM, pH 5.5) at a concentration of 1.00% w/v. DMTMM was then added
15
followed by the dropwise addition of 5-methylfurfurylamine. The molar equivalencies of
16
DMTMM and furfurylamine were varied to achieve different degrees of furan substitution. After
17
24 hours, the reaction mixture was sequentially dialyzed against 0.1M NaCl for 1 day then
18
distilled water for 2 days (Mw cutoff 12-14kDa). The contents of the dialysis bag were
19
lyophilized to isolate the HA-mF as a white fibrous material. The degree of furan substitution
20
was determined from 1H NMR spectra by comparing the ratio of the integrations of the
21
methylfuran proton signals (5.98, 6.04 and 6.28 ppm), to that of the N-acetyl glucosamine peak
22
at 2.01 ppm. 1H NMR spectra were measured in D2O on an Agilent DD2-500 MHz NMR
23
spectrometer (Santa Clara, CA, USA). Hydrogels were then prepared by combining HA-mF or
ACS Paragon Plus Environment
5
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Biomacromolecules
Page 6 of 30
1
HA-FA with bis-maleimide PEG in either PBS buffer (0.1 M Phosphate) at pH 7.4 or MES
2
buffer (0.1 M) at pH 5.5. All hydrogels were synthesized using HA-mF or HA-F with DS
3
between 54-60% at a final concentration of HA of 1.50% w/v with a 1:0.6 molar ratio of furan to
4
maleimide.
5 6
Rheology: The viscoelastic properties of the HA-mF or HA-FA/ PEG hydrogels were
7
determined using an AR-1000 rheometer fitted with a 20 mm 1° acrylic cone using parallel plate
8
geometry and a solvent trap to reduce evaporation (TA Instruments, New Castle, DE, USA). The
9
hydrogel solutions were loaded onto the rheometer and the upper plate was lowered to a gap size
10
of 20 µm. A time sweep was performed at 37 °C and 0.2% strain for 8 h for HA-FA and 3 h for
11
HA-mF to allow for complete gelation. The time at which G’ (shear storage modulus) crossed G”
12
(shear loss modulus) was taken as the gel time, and the average G’ from the last 10 minutes of
13
the time sweep was taken to be the final G’ (Fig S1). Following the time sweep, a strain sweep
14
was conducted to ensure the strain used was within the linear viscoelastic region for the
15
hydrogels.
16 17
Swelling: To examine the equilibrium swelling properties of the hydrogels, 100 µL of HA-FA
18
or HA-mF hydrogels were prepared in either pH 5.5 or pH 7.4 buffer in pre-weighed vials, and
19
allowed to react overnight. Samples (n=3) were then weighed to give M0 and then incubated in
20
200 µL PBS at 37 °C. At select time points, the swelling ratio was determined by measuring the
21
mass (Mt) following removal of the buffer and gentle drying with a Kim wipe. Hydrogels were
22
then replenished with fresh buffer. Swelling ratios were calculated as Mt/M0.
23
ACS Paragon Plus Environment
6
Page 7 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
1
Biomacromolecules
1
H NMR determination of coupling efficiency: To examine the Diels-Alder coupling efficiency
2
in situ, 1H NMR was used to quantify the amount of remaining unreacted furan and methylfuran
3
on the HA backbone following gelation with (Mal)2PEG. 100 µL hydrogels were prepared as
4
above. The Diels Alder reaction was stopped at the selected time points by the addition of pH 9.0
5
phosphate buffer (100 mM), which quenched the maleimide partner via ring-opening
6
hydrolysis16,17 (Fig S2). The quenched hydrogels were then digested by hyaluronidase (200
7
U/100 µL gel) over 2 days, lyophilized, and resuspended in D2O for 1H NMR analysis of the
8
unreacted furan peaks (at 6.28 ppm for HA-mF and 7.54 ppm for HA-FA). In order to calculate
9
the percentage of furan (or methylfuran) groups that had reacted, the number of furan groups
10
available before ((DSinitial) and after (DSfinal) gelation were compared (equation [1]) and
11
normalized to the equivalent maleimides present:
12
[1]
(%DSfinal – %DSinitial)/60%mal/furan x 100%.
13 14 15
Computational Methodology: Density functional theory (DFT) calculations employing hybrid
16
functional B3LYP/6-31G(d,p)11 and the IEF-PCM solvation model18 were carried out using the
17
Gaussian 09 package on Sharcnet (Shared Hierarchical Academic Research Computing
18
Network). The optimized geometries were then confirmed by frequency computations as minima
19
(zero imaginary frequencies) or transition states (one imaginary frequency). Single-point
20
calculations were performed at the M06-2X/6-311+G(d,p)19-21 levels using the B3LYP/6-31G(d)
21
optimized geometries. The HOMO and LUMO molecular orbitals and their associated energies
22
were calculated at this M06-2X/6-311+G(d,p) level of theory. Natural bond orbital analysis
23
(NBO) Version 3.1, as implemented in Gaussian 09, was applied. Structural visualizations were
ACS Paragon Plus Environment
7
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Biomacromolecules
Page 8 of 30
1
performed using GaussView v5.0.8.4. A topological analysis of the electron density was carried
2
out with Bader’s quantum theory of atoms in molecules (QTAIM) using the AIM2000
3
software.22 The absolute value of the reaction rate constant at 311 K in water was calculated from
4
the computed Gibbs free energy of activation.
5 6
Maintenance of Cancer Cell lines: Breast cancer cell lines (BT474, MCF7 and T47D) were
7
purchased from ATCC (Manassas, VA, USA). Cell lines were maintained in tissue culture flasks
8
in an incubator (37 °C, 5% CO2, 95% humidity) using their corresponding growth media: BT474
9
and MCF7 in Dulbecco’s modified Eagle’s medium Nutrient Mixture F-12 Ham, and T47D in
10
RPMI-1640. All cell culture media were supplemented with 10% FBS, penicillin (10 µmL-1) and
11
streptomycin (10 µg mL-1). T-47D and MCF-7 growth media were also supplemented with
12
insulin (10 µg mL-1).
13
Patient-derived glioma neural stem cell lines (G523, G411) were contributed by the
14
laboratory of Dr. Peter Dirks in accordance with Research Ethics Board approvals at the Hospital
15
for Sick Children, Toronto, and the University of Toronto. Cells were maintained in Corning
16
Primiaria flasks coated with poly(L-ornithine) and laminin (Sigma-Aldrich, St. Louis, MO,
17
USA) using serum-free media (NS-A media StemCell Technologies, Vancouver, BC, Canada)
18
supplemented with N-2, B27 (Life Technologies, Carlsbad CA,USA), EGF, FGF-2 and heparin
19
(Sigma Aldrich, St. Louis, MO, USA) as described previously for neural stem cells.23,24,25
20 21
3D Cell encapsulation: Hydrogels were prepared using HA-mF and (Mal)2PEG dissolved in
22
the appropriate cell culture medium at pH7.4.26 Cells were mixed at a concentration of 8,000
23
cells per 15 μL gel in wells of a 384-well plate. Gel solutions were mixed gently with a pipette,
ACS Paragon Plus Environment
8
Page 9 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Biomacromolecules
1
plated and then allowed to gel for 2 hours, after which 50 µL of media was added on top of the
2
gels. Media was changed every 2 days. For cell distribution analysis, cell-laden hydrogels were
3
washed with PBS twice then fixed with 4% paraformaldehyde (PFA) 24 hours following
4
preparation. Gels were washed twice more with PBS to remove excess PFA, then treated with a
5
1/500 solution of Hoechst nuclear counterstain in PBS (Cell Signalling Technologies, Danvers,
6
MA, USA). The gels were imaged using an Olympus Fluoview FV1000 confocal microscope
7
with xy scans every 20 µm in the z-direction. The number of cells in each imaged volume of
8
hydrogel was quantified using Imaris Bitplane software. For live/dead viability staining, a
9
mixture of MitoTracker Red ™ (1:3000) and Sytox Green (1:106) (Thermo Fisher Scientific,
10
Waltham, MA, USA) was added to the media and allowed to equilibrate for 2 hours, following
11
which, the gels were imaged. The number of live and dead cells in each imaged volume of
12
hydrogel was quantified using Imaris Bitplane software.
13 14
Statistical Analysis: Statistical analyses were performed using GraphPad Prism version 6 and 7
15
(GraphPad Software, San Diego, CA, USA). Statistical differences among two or more groups
16
were assessed using one-way ANOVA followed by a Tukey post-hoc test with 𝛼=0.05 as the
17
criterion for statistical significance. Graphs are annotated with p values represented as *p≤0.05,
18
**p≤0.01 and ***p≤0.001. All data are presented as mean ± standard deviation.
19 20
Results
21 22
HA-mF/Mal2PEG Hydrogel Synthesis and Characterization
ACS Paragon Plus Environment
9
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Biomacromolecules
Page 10 of 30
1
HA-mF was prepared by activating the carboxylate of HA with DMTMM, followed by
2
coupling with the primary amine of 5-methylfurfurlylamine (Fig 2A).27 The degree of methyl
3
furan substitution on HA was determined by 1H NMR, where the ratios of the integrals of the N-
4
acetyl glucosamine peak on the HA-backbone (Figure 2B, peak a) to the aromatic furan peaks
5
give the degree of substitution (Fig 2B, peaks b,c). The degree of substitution can be controlled
6
by varying reagent stoichiometric ratios and reaction time (Fig 2C).
7
ACS Paragon Plus Environment
10
Page 11 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Biomacromolecules
Figure 2: Synthesis and characterization of HA-mF: (A) Synthetic scheme of DMTMM mediated 1
coupling between HA-COOH and amine-functionalized furan; (B) H NMR spectrum of HA-mF. Degree of substitution (DS) is determined by the ratio of integration of HA-N-acetyl glucosamine peak (a) with aromatic methylfuran peaks (b and c); (C) DS can be controlled by varying stoichiometric equivalents of DMTMM and 5-methyl furfurylamine and reaction time.
1 2
We compared the bulk physical properties of the HA-FA and HA-mF crosslinked hydrogels in
3
terms of their rate of gelation, elastic storage modulus and equilibrium swelling. Rheological
4
measurements at pH 7.4 demonstrated that methylfuran-functionalized hydrogels increased the
5
gelation rate (12 ± 2 min) compared to furan-functionalized hydrogels (32 ± 9 min) (Fig 3A).
ACS Paragon Plus Environment
11
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Biomacromolecules
Page 12 of 30
1
This was consistent with our hypothesis that the increased electron density of methylfuran vs.
2
furan would increase the rate of DA reaction and therefore gelation rate. The differences in
3
gelation rate did not affect the bulk properties of the resulting network: both HA-FA and HA-mF
4
crosslinked hydrogels exhibited similar final storage moduli (G’) of 1000-1300 Pa (Fig 3B), as
5
determined from the rheological time traces (Fig S1). For the equilibrium swelling studies, HA-
6
FA and HA-mF hydrogels were crosslinked with (Mal)2PEG at either acidic pH 5.5 (0.1 M MES)
7
or physiological pH 7.4 (0.1 M PBS) and then monitored for change in mass over 16 days with
8
incubation in PBS (pH 7.4) at 37 °C. Gels crosslinked at pH 7.4 swelled more than those
9
crosslinked at pH 5.5 (Fig 3C,D) as there were likely fewer crosslinks formed at pH 7.4 due to
10
the hydrolysis of maleimides under slightly basic conditions (Fig S2). Interestingly, no
11
differences were observed between HA-FA and HA-mF swelling at each time point whether
12
crosslinked at pH 5.5 or pH 7.4 over the first 10 days. This is consistent with the rheological data
13
that show that the HA-mF and HA-FA hydrogels have similar initial bulk mechanical properties.
14
Notably, after 10 days, the HA-mF hydrogels seemed to swell more than HA-FA hydrogels (Fig
15
3C,D).
16
We investigated the impact of gelation time on swelling and stability and found differences in
17
neither swelling nor stability between HA-mF hydrogels that were allowed to crosslink at pH 7.4
18
for 45 min, 2 h, or 24 h before the addition of swelling buffer. This demonstrates that the
19
crosslinked HA-mF gels are stable, even when crosslinked for short periods of time (Fig 3E).
ACS Paragon Plus Environment
12
Page 13 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Biomacromolecules
Figure 3: Comparison of bulk properties of HA-FA and HA-mF hydrogels crosslinked with (Mal)2PEG (1.5% w/v HA 1:0.6 furan: maleimide molar equivalents). (A) Rheological determination of time to gelation of HA-mF and HA-FA (n=3, mean + standard deviation *p
