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Microfabrication of Photo-crosslinked Hyaluronan Hydrogels by Single- and Two-photon Tyramine Oxidation Claudia Loebel1,2 †, Nicolas Broguiere2†, Mauro Alini1, Marcy Zenobi-Wong2*, David Eglin1*
1
AO Research Institute Davos, Clavadelerstrasse 8, Davos Platz, 7270, Switzerland
2
ETH Zurich, Cartilage Engineering + Regeneration, Department of Health Sciences and
Technology, Otto-Stern-Weg 7, Zürich, 8093, Switzerland
†
These authors contributed equally to this work
*
Corresponding authors
KEYWORDS Single-photon-lithography, Two-photon-polymerization, Hyaluronic acid, Hydrogels, Actuators
ABSTRACT Photo-crosslinking of tyramine-substituted hyaluronan (HA-Tyr) hydrogels is demonstrated for the first time. HA-Tyr hydrogels are fabricated via a rapid photosensitized process using visible light illumination. Non-toxic conditions offer photo-encapsulation of human mesenchymal 1 ACS Paragon Plus Environment
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stromal cells (hMSCs) with high viability. Macroscopic gels can be formed in less than 10 s, and one and two-photon photo-patterning enable 2D and 3D microfabrication. Different degrees of crosslinking induce different swelling/shrinking, allowing for light induced microactuation. These new tools are complementary to the previously reported horseradish peroxidase / hydrogen peroxide crosslinking and allow sequential crosslinking of HA-Tyr matrices.
INTRODUCTION Advances in light-mediated hydrogel patterning have achieved micrometer-scale control over the distribution of biochemical1-4 and biophysical5, 6 signals in defined synthetic hydrogel systems. The vast majority of the photo-patterned hydrogels have been based on vinyl derivatives and free-radical chemistry.7 Guvendiren and Burdick demonstrated temporal stiffening of methacrylated hyaluronic acid (HA) hydrogel, applying UV light to locally trigger additional crosslinking.8 However, highly modified HA backbone of photo-pattern hydrogels and their unreacted acrylates may impair biofunctionality9,
10
, while use of low substituted hyaluronan
cross-linked hydrogels have not yet been reported. A photo-crosslinkable HA-hydrogel is described which has a broad range of gel mechanics while maintaining cell compatibility and the ability to be cleaved enzymatically. We and others have reported the introduction of hydroxyphenyl groups on natural polymers, such as hyaluronic acid-tyramine (HA-Tyr).11-13 Here, photo-crosslinkable, biodegradable, HATyr hydrogels are introduced allowing temporal and micrometer-scale control of fabrication with single-photon lithography. Visible light has been described for crosslinking of modified polyethylene glycol (PEG)14-16 and hyaluronic acid17 with eosin Y (EO) as photo-initiators. However, the use of cytotoxic co-
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initiator e.g. triethanolamine for generating radicals in this case limits the applicability.15 Recently, Shih and colleagues described a rapid and orthogonal thiol-ene photoclick gelation between a dithiothreitol and PEG-norbornene macromers with visible light and EO as only photo-initiator.18 Recent studies have shown that visible light can be used for gelation of tyramine functionalized synthetic polymers and tyrosine rich proteins with ruthenium (Ru(II)) and sodium persulfate.19, 20 Here we use visible light to induce cytocompatible polymerization of rose bengal (RB) and EO sensitized HA-Tyr conjugates. Photo-crosslinked HA-Tyr hydrogels are prepared without the use of horseradish peroxide (HRP) and hydrogen peroxide (H2O2). In addition, the sequential enzymatic and light crosslinking of HA-Tyr conjugates is also reported for precise spatial control and accurate time-dependent stiffening of the hydrogels. As single-photon lithography is limited by the lack of depth control, two-photon polymerization (2PP) holds promise for precisely fabricating 3D hydrogels with user-defined architecture.21 Most 2PP studies use synthetic polymers with highly reactive acrylate groups such as poly(ethylene) glycol diacrylate (PEG-DA)21 or highly modified HA macromers requiring supportive thiol-ene chemistry (dithiothreitol)22 and addition of PEG-DA,23 respectively. Here we report on fabrication of high-resolution hyaluronan hydrogel microstructures using twophoton polymerization and their ability to actuate upon swelling.
EXPERIMENTAL SECTION Materials Hyaluronic acid sodium salt from Streptococcus equi (HANa) with weight-average molecular weight Mw = 290 kDa and poly-dispersion index Mw/Mn = 1.86, where Mn indicates the number-
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average molecular weight was purchased from Contipro Biotechs.r.o. (Czech Republic). 4-(4,6Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium
chloride
(DMTMM)
from
TDI
(Zwijndrecht, Belgium). Corgel®Kit (DSmol 5%, Mw > 1000 kDa) was purchased from LifeCore Biomedical (Chaska, MN, U.S.A.). Other chemicals were of analytical grade, purchased from Sigma Aldrich (Buchs, Switzerland) and used as received. HA-Tyr synthesis HA-Tyr was synthesized following a previously described procedure.13 Briefly, sodium hyaluronate (290 kDa) was dissolved in deionized H2O (1% w/v). HA-Tyr conjugates were prepared in a one-step reaction by adding 1.25 mmol DMTMM coupling agent and subsequently 1.25 mmol tyramine drop wise to the solution. The reaction was carried out at RT (DSmol 2.8%) or 37 °C (DSmol 7.8%) and under continuous stirring for 24 h. The product was purified via precipitation with 96% ethanol after adding 10 vol% saturated sodium chloride. Several wash steps were performed and the product kept under vacuum for 48 h. 1H NMR and UV-vis analysis were performed to confirm substitution of tyramine on HA (DSmol 7.8% and 2.8%). Corgel® was used as a commercial available source of high MW HA-Tyr conjugates with a DSmol of 5% as per manufacturer specifications. Equilibrium swelling/degradation assay To examine swelling properties, HA-Tyr was dissolved in PBS (3.5% (w/v)) and mixed with 0.05% (w/v) RB and 0.02% (w/v) EO, respectively. HA-Tyr solutions (50 µl) were photopolymerized in preformed polydimethylsiloxane (PDMS) molds (2x5 mm) for 5 min and 10 min, respectively. After several washing steps with PBS, samples were immersed in PBS (10 mM, pH 7.4) at 37 °C for 48 h until the swelling equilibrium had been reached. The swollen hydrogels were removed, carefully blotted to remove excess surface liquid, and the total swelled weight
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was measured. The samples were lyophilized overnight and the dry weight was measured. The swelling ratio was calculated by: swelling ratio = (weightwet- weightdry)/ weightwet. To determine stability, HA-Tyr precursor solutions (50 µl) containing 0.05% (w/v) RB and 0.02% (w/v) EO, respectively were photo-polymerized for 5 min in PDMS molds. After preswelling for 48 h at 37 °C, mass of the samples was measured and degradation performed using 10 Units/ml hyaluronidase in PBS at 37 °C.13 The percentage of hydrogel mass remaining was calculated in relation to the swollen mass after 48 h (weighttotal/weightswollen)*100). Characterization of HA-Tyr crosslinking kinetics and substrate mechanics Dynamic oscillatory time sweeps were performed using an Anton-Paar Rheometer equipped with a Peltier controller, a 120-watt metal halide light source (HXP120 350-700 nm, 134 mW/cm2; Zeiss) and plate-plate geometry, diameter 20 mm. The synthesized conjugates at 3.5% (w/v) were hydrated overnight at 4 °C in PBS. HA-Tyr precursor solution (100 µl) containing RB and EO respectively at various concentrations was placed in the gap and the upper plate was lowered to a spacing of 0.1 mm. A humid chamber was achieved by placing wet tissue paper around the platform and a chamber cover on top. A time-sweep oscillatory test was conducted for 10 min with a 1% sinusoidal strain in order to monitor the in situ liquid-to-solid transition (gelation point) of the solution during the photo-polymerization reaction. The mechanical spectra were carried out at 20 °C and 1 rad/ sec angular frequency to monitor the shear elastic modulus (G') and the loss modulus (G'') of the hydrogels. All in situ rheometry tests were performed with a 60 sec pre-conditioning cycle followed by visible light curing. Viability assay of human mesenchymal stromal cells (hMSCs) encapsulated in photo-crosslinked HA-Tyr hydrogels
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Human bone marrow was harvested from the iliac crest (ethical approval Freiburg, EK-326/08) and hMSCs isolated using previously described protocols.23 Passage 3 hMSCs (5 x106/ml) were suspended in PBS and encapsulated into HA-Tyr hydrogels (4x2 mm; DSmol 7.8%) using visible light induced crosslinking (Exfo X-Cite Series 120 lamp with a 395 nm long-pass filter/560 nm beam-splitter, power density at sample 134 mW/cm2, exposure for 5 min) in the presence of 0.02% (w/v) EO. Cell-seeded hydrogels (n=3) were directly added to DMEM low glucose with 10% FBS, 1% penicillin/streptomycin and cultured at 37 °C and 5% CO2. First media changes were performed after 3 h and subsequently every second day. Cell viability was assessed using live-dead staining (20 µM calcein-AM and 3 µM ethidium homodimer) and imaging on an inverted point-scanning confocal microscope (Zeiss, 510 LSM) at 10x magnification. Viability was quantified by counting live (green) and dead (red) cells in 6-9 microscopic fields from representative hydrogels. Fabrication of thin HA-Tyr hydrogel films HA-Tyr hydrogels (3.5% (w/v)) were prepared by adding H2O2 (0.11 mM) and 1 Unit/ml HRP to a precursor solution with 0.05% (w/v) RB and 0.02% (w/v) EO, respectively. Hydrogels were cast between coverslip glass slides using 150 µm spacers. After 15 min incubation in a humid chamber at RT, the obtained hydrogel films were utilized for hydrogel patterning. Detection of fluorescent di-tyramine bridges HA-Tyr hydrogels are autofluorescent upon exposure to UV light.11 Di-tyramine displays an excitation maximum of 285 nm and emission maximum of 415 nm. The hydrogels’ intrinsic fluorescence was used to qualitatively assess crosslinking density. Fluorescent microscopy images were acquired after overnight washing at 4 °C to ensure complete diffusion of the photosensitizer.
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Laser-assisted hydrogel patterning An inverted point-scanning confocal microscope (Zeiss, 510 LSM) equipped with a 488 nm enterprise laser (30 mW), a 365/415 nm argon laser and with a Plan-Neofluar 10x/0.30NA objective was employed for local illumination of the gels. The bleaching mode and user-defined ROI scanning option of the microscope software (Zen2009) allowed precisely controlled arbitrary patterns in xy direction. Precise control of specific laser intensities assigned to each ROI allowed generation of crosslinking gradients. Step-wise gradients were obtained by aligning a various number of contacting ROIs, each of which had assigned a different laser intensity (10010% of the maximal laser power) or exposure time. Solid 2D objects were printed out of a liquid precursor containing HA-Tyr 3.5% (w/v) and 0.05% (w/v) RB or 0.02% (w/v) EO buffered in PBS. Objects were gently washed with PBS to remove the un-crosslinked precursor solution and mounted on a glass cover slip. Images were acquired before and after free swelling in PBS. For 3D imaging, thirty sequential stacks of sections (10 µm depth) from the bottom of the construct were acquired. Two-photon excitation and fluorescence spectra of EO and HA-Tyr gels Spectra were acquired on a Leica SP8 multiphoton microscope. Excitation spectra were taken with a Mai Tai XF DeepSee (Spectra Physics) from 710 to 950 nm and an Insight DeepSee (Spectra Physics) from 950 to 1300 nm (Pulse width are
