Gelatin-Based Photocurable Hydrogels for Corneal ... - ACS Publications

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Biological and Medical Applications of Materials and Interfaces

Gelatin-based photo curable hydrogels for corneal wound repair Lingli Li, Conglie Lu, Lei Wang, Mei Chen, Jacinta F. White, Xiaojuan Hao, Keith M McLean, Hao Chen, and Timothy C Hughes ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 05 Apr 2018 Downloaded from http://pubs.acs.org on April 5, 2018

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ACS Applied Materials & Interfaces

Gelatin-Based Photo Curable Hydrogels for Corneal Wound Repair Lingli Li1, 3, Conglie Lu1, Lei Wang3, Mei Chen1, Jacinta White2, Xiaojuan Hao2, Keith M. McLean2, Hao Chen1, 3*, Timothy C. Hughes2* 1

School of Ophthalmology & Optometry, Eye Hospital, Wenzhou Medical University, Wenzhou,

Zhejiang Province, PRC, 325000 2

CSIRO Manufacturing, Clayton, Victoria, Australia, 3169

3

Wenzhou Institute of Biomaterials and Engineering, Wenzhou, Zhejiang Province, PRC, 325001

* Corresponding authors: Timothy C. Hughes CSIRO Manufacturing, Clayton, Australia Bayview Avenue, Clayton VIC 3168, Australia Phone +61 3 95452614 Fax: +61 3 95458106 Email: [email protected] Hao Chen

School of Ophthalmology & Optometry, Eye Hospital, Wenzhou Medical University, Zhejiang Province, PRC No.270 Xueyuan Road, Wenzhou, Zhejiang Province, 325000, China Phone +86 577 88833805 Fax: +86 57788067962 1

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Email: [email protected] Abstract

In this study, an injectable, photo-curable gelatin system, consisting of acrylated gelatin and thiolated gelatin, with tunable mechanical, biodegradation and biological properties was used as a potential cell supportive scaffold for the repair of focal corneal wounds. The mechanical property of hydrogels can be readily modified (post-cure shear modulus of between 0.3 and 22 kPa) by varying the ratio of acrylate to thiol groups, photo intensity and solids content, and the biodegradation times also varied with change of solids content. More importantly, the generated hydrogels exhibited excellent cell viability in both cell seeding and cell encapsulation experiments. Furthermore, the hydrogels were found to be biocompatible with rabbit cornea and aided the regeneration of new tissue under a focal corneal wound (exhibiting epithelial wound coverage in < 3d), while UV irradiation did not have an obvious harmful effect on the cornea and posterior eye segment tissues. Along with their injectability, and tunable mechanical properties, the photo curable thiol-acrylate hydrogels showed promise as corneal substitutes or substrates to construct new corneal tissue.

Keywords Thiol-acrylate chemistry; acrylated gelatin; thiolated gelatin; photocrosslink; corneal wound repair

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Introduction

The cornea is the transparent exterior tissue of the eye that covers the pupil, iris and anterior chamber. It protects the eye from the external environment and pathogens, and plays a pivotal role in light transmission and refraction. Many corneal trauma and diseases such as chemical/thermal burns, Stevens-Johnson’s syndrome, ocular cicatricial pemphigoid, corneal ulcers, pseudophakic bullous keratopathy, and Fuch’s endothelial dystrophy affect corneal transparency and may pose a serious risk of vision loss.1 So far, penetrating or lamellar keratoplasty are the most common treatment for restoring ocular integrity and avoiding further complications.2 In a classical penetrating keratoplasty, the whole cornea is replaced, including the epithelium, stromal and endothelial layers, while in a lamellar keratoplasty, only the diseased layer is replaced. However, in some corneal dysfunctions, the infections or injury occurs within a well-defined focal area, leaving the surrounding corneal tissue healthy and intact. In these cases, replacing only the damaged tissue would be attractive as the use of donated grafts requires extensive and multiple invasive surgeries3-5 and in many countries there is a limited supply of suitable donor tissue and accompanying infrastructure to collect, store and distribute the tissue.

Tissue engineering provides a new promising approach by which a scaffold can be designed for minimally invasive aesthetic and functional reconstruction of damaged tissues.6,7 Hydrogels in their hydrated state allow diffusion of oxygen, water and glucose through their networks and their physicochemical characteristics can simulate the cell’s natural microenvironment.8, 9 An ideal

3



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hydrogel for corneal defects would mimic the properties of the cornea having high transparency (78%),10 a water content of 78-82%11 and a shear modulus of 2-8 kPa.12 In addition, it is desirable that the hydrogel afford high cell viability, is biodegradable and implantable with minimally invasively surgery. Over the last few decades, photo polymerization has become an attractive approach utilized in the development of hydrogels for biomedical applications.13, 14 The use of visible or UV light to crosslink macromolecules into hydrogel networks and to pattern materials with enhanced functionality has significant advantages, including rapid cure rates under physiological conditions, minimal heat production and the ability to be performed using aqueous biocompatible precursors.15-18 Photo intensity, irradiation duration and location of exposure may be manipulated so that a single premixed formulation can be crosslinked into hydrogels with various desirable mechanical properties. In addition, the formulations are easy to prepare and handle as the precursors remain stable and unreacted after mixing and can be readily kept prior to light exposure.15 Moreover, in situ curable hydrogels can be readily moulded into diverse shapes and depths of tissue defects to create patient-specific scaffolds, which will improve the integration of as-prepared scaffolds with tissues.

Photointiated thiol-ene chemistry has been an important part of photo polymerization, attracting much attention from the field of regenerative medicine owning to its spatiotemporal controllability. 19 The thiol-ene chemistry was first identified more than one century ago, but its utility in biomedical areas was not fully realized until recently.20 Thiol-ene chemistry meets all the requirements of preparation of in-situ formed hydrogel, as it involves simple reactive moieties, 4


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mild conditions, and importantly does not produce any by-products.21 Meanwhile, it is highly efficient, and can be employed in reactions with versatile substrates, such as small molecule derivatization,22,23 bioconjugation chemistry,24 and polymerization.18 In particular, thiol-ene chemistry has attracted additional attention for tissue defect repair as they hold a promise to allow for complete control over their material properties and response to different environmental stimulus.7,15 Typical photo polymerizations, such as the acrylate/methacrylate systems, are vulnerable to oxygen inhibition19 and are driven by radical chain-growth polymerization that results in networks with heterogeneous crosslinks made up by poly(meth)acrylate chains of varying size25,26 with low inherent capacity to biodegrade. In contrast, thiol-ene chemistry is oxygen insensitive and proceeds via step-growth mechanisms that form crosslinks between reactive groups resulting in more homogeneous networks that might have superior mechanical properties compared to those of chain-growth networks.26,27 Thiol-acrylate chemistry is a sub-set of thiol-ene chemistry involving the specific reaction of a thiol with an acylate.28 As such, thiol-acrylate reactions can occur via both chain-growth and step-growth mechanisms (‘mixed mode’) depending on the relative ratios of starting monomers.29

A number of hydrogels successfully adopting the thiol-ene chemistry have been reported and relevant photochemical methods for biological applications have been demonstrated to be more cytocompatible than chain growth mechanisms.30, 31 Synthetic polymers, such as polyethylene glycol32, have been the main macromolecular precursors employed for thiol-ene crosslinkable hydrogels, however, synthetic polymer scaffolds often lack biofunctionality and may not actively 5



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interact with cells and often resist biodegradation. Naturally derived polymers such as collagen33, gelatin9, laminin34, hyaluronic acid13 and alginate35 offered an interesting choice for providing potential cell interaction sites and desirable mechanical properties to tissue engineering scaffolds.

Owing to its excellent transparency, gelatin hydrogels have been used in ocular tissue engineering. 36, 37

In order to minimize cell damage from exogenous chemicals,26, 38 we were interested to

explore the use of pure gelatin hydrogels in ocular applications using thiol-acrylate chemistry. Despite the popularity of thiol-ene chemistry there are relatively few reports of its use to form gelatin hydrogels, the majority of reports involve co-polymers with polyethylene glycol derivatives (either vinyl or thiol functionalized) 39-46 with only 1 report of a pure gelatin system46. Likewise, few reports have explored application of thiol-ene crosslinked hydrogels beyond in vitro cell culture, including 3D printing8, two-photon microfabrication15 and only one report of in vivo study examining rate wound model16.

Thus, thiol-ene chemistry provides a method to crosslink gelatin with minimal use of chemicals which can greatly reduce harm to its physiochemical property or biocompatibility. In this study, a facile method has been developed to modify gelatin with acrylate anhydride and cysteamine respectively by grafting free vinyl groups and thiol groups to the gelatin backbone to prepare acrylated gelatin and thiolated gelatin, respectively. The mixtures of the two precursors were then crosslinked via a photoinitiated thiol-acrylate reaction. These crosslinked gelatin-based hydrogels allow easy mechanical property modulation through varying precursor ratios, solid content, and

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irradiation intensity. 2D and 3D Cell morphology and proliferation studies were carried out to investigate the effect of preparation conditions on the in vitro biocompatibility of the gelatin hydrogels. Lastly, the use of a thiol-acrylate gelatin formulation was investigated in the repair of focal corneal defects in vivo.

Experimental

2.1 Materials

Type-A porcine skin gelatin (Bloom 100), cysteamine (98%), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide

hydrochloride

(EDC),

N-hydroxysuccinimide

(NHS),

initiator

2-Hydroxy-4’-(2-hydroxyethox)- 2-methylpropiophenone (Irgacure 2959), cell counting kit 8 (CCK8), and human recombinant insulin were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Acrylic anhydride (90%) was purchased from Chengdu Xi Ya Chemical Co. Ltd. (Chengdu, China). Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F-12), Fetal Bovine Serum (FBS), Penicillin-Streptomycin, LIVE/DEAD® Viability/Cytotoxicity Kit and

1,1'-Didodecyl-3,3,3',3'-Tetramethylindocarbocyanine

Perchlorate

(DiIC12(3))

were

purchased from Life Technology. Milli-Q grade water (Millipore, Bedford, MA, USA) was used in the preparation of solutions. All the reagents used in the study were used as purchased without further purification.

2.2 Synthesis of acrylated gelatin Acrylated gelatin was synthesized as described previously.35 Briefly, type A porcine skin gelatin 7



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was mixed at 10% (w/v) into Dulbecco’s phosphate buffered saline (DPBS) (Sigma) at 50 ºC and stirred until its components were fully dissolved. Subsequently, acrylic anhydride (AA) was added dropwise with stirring to the gelatin solution at a rate of 0.5 mL/min. Final concentration of 1% AA (v/v) was used and the reaction was heated at 50 ºC for 3 h to form acrylated gelatin (GE-AA) solutions. The solution was then dialyzed against distilled water by using 1000 kDa cutoff dialysis tubing at 40 ºC for one week. The purified product was frozen at -80 ºC and lyophilized. The grafting ratio was characterized by 1H NMR.

2.3 Synthesis of thiolated gelatin

Thiolated gelatin (GE-SH) was obtained as described below. 7.5g of type A porcine skin gelatin was dissolved into 750 mL DPBS at 50 ºC and stirred until it was fully dissolved, 5-fold molar excess of EDC/NHS (~3.5g/0.7g) and 2.5-fold molar excess of cysteamine (~23.2g) were added to above solution. The reaction was kept at room temperature under nitrogen for 8 hrs. The raw products were subjected to dialysis against milli-Q water (MWCO 1000) under nitrogen bubbling for 3 days to remove unreacted cysteamine and other side products. The purified products were frozen at -80 ºC and lyophilized. The grafting ratio was characteriazed by 1H NMR.

2.4 NMR Compositions of modified gelatin, grafting ratios of GE-AA and GE-SH were determined by 1H NMR spectroscopy (Bruker, 400 MHz, Germany). In all cases, the samples were prepared by dissolving in D2O at a concentration of about 10 mg polymer per mL. The extent of acrylation 8


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and thiolation was determined using NMR spectroscopy47 using the peaks at δ 6.45, 5.90, for acrylated materials and δ 2.9 for thiolyated materials.

2.5 Hydrogel fabrication

Thiol-acrylate crosslinked hydrogels were prepared by photo-crosslinking GE-AA with GE-SH at different ratios. Firstly, a stock solution of 5% (w/v) initiator (solution 1) was prepared by dissolving Irgacure 2959 in mixed solution of phosphate buffer saline (PBS) and ethanol with the ratio of 1:1(v/v). Secondly, GE-AA and GE-SH were dissolved in PBS at a predetermined ratios of C=C (ene)/HS (thiol) (solution 2). Then the required amount of gelatin mixture (solution 2) and initiator (solution 1) were mixed together and cast into custom-made molds (20 mm × 100 µm discs), which was then exposed to 365 nm UV light (at predetermined photo intensity) for 2 minutes to form the hydrogel. Light intensity was measured using a Molectron Power Meter (PowerMax 5200, USA) with a Molectron PM10 detector. All of the hydrogels were peeled off and kept in sterilized PBS at 4℃ for further use.

2.6 Photo-rheometry

Dynamic viscoelastic measurement was carried out on an ARES photo-rheometer (TA Instruments, USA) connected to an EXFO Acticure 4000 light source via a liquid light-guide. A Peltier temperature controller was also connected to the rheometer to maintain the cure temperature at 37 ℃. The sample was loaded in the center of two parallel plates of 20 mm in diameter. The gap between the two plates was set at 0.3 mm. The in situ cure kinetics were 9



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studied at a constant temperature of 37 ℃ for at least 10 min. For the first 180 s, the light source (365nm) was controlled in off-mode to get a baseline and automatically converted to on-mode with an predetermined intensity for 9 min. Parameters such as storage shear modulus (G′) and the loss shear modulus (G″) were measured as a function of time at a constant frequency of 100 rad/s and a strain of 1.0% at a data acquisition rate of 2 measurements per second.

2.7 Microscopic morphology of prepared hydrogels

Morphology of the hydrogels was examined by field emission scanning electron microscopy (FE-SEM, Philips Electronics N.V. Holland). The cross-sectioned samples were frozen in liquid nitrogen and quickly fractured to expose their inner structures, followed by freeze-drying and sputter-coating with platinum. 2.8 Transparency

Light transmission through formed hydrogels was measured with a UV-Vis spectrophotometer (Paradigm Absorbance Detection, Beckman Coulter). The measurements were repeated with triplicate samples and the background was determined with air alone.

2.9 Water content

The equilibrium water content of the formed hydrogels was defined as the weight ratio of water content to the swollen hydrogels. All of the hydrogels were soaked in PBS (pH 7.4) for 24 h at 37 °C. Then the hydrogels were removed from the buffer solution, placed between two pieces of dried filter paper to remove excess solution, and then weighed (WS). The swollen hydrogels were 10


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weighted and the swelling ratio (g/g) was calculated by the following equation35.

Swelling ratio / =

(1)

where Ws and Wi are the weights of the swollen and the initial (as prepared) hydrogels , respectively.

The equilibrium water content (EWC) was calculated from equilibrium hydrogel weight (We) and the dried hydrogels (Wd) according to equation (2):

EWC =

× 100

(2)

The measurements were repeated with triplicate samples.

2.10 In vitro biodegradation

The in vitro degradation behavior of hydrogels was investigated by incubating pre-formed hydrogels (20 mm × 100 µm discs) in PBS (pH 7.4) in the presence of collagenase (50IU/mL) at 37 °C. At pre-determined time intervals, the hydrogels were taken out and rinsed with deionized water three times. Then, the samples were freeze dried for two days and their weight were measured immediately. The degradation rate was calculated using equation (3):

Remaining weight % =

" #

× 100

(3)

where Wt is the dry weight of the hydrogel after degradation and W0 (the initial dry weight of hydrogel). The measurements were repeated with triplicate samples.

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2.11 In vitro Cytotoxicity

L929 mouse fibroblast cells were cultured to assess the in vitro biocompatibility of the hydrogels. Cells were cultured in 75 cm2 T–flask (Nunc) using DMEM (Gibco, Life Technology, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, Life Technology, USA). All cultures were maintained at 37 °C and 5% CO2. The in vitro biocompatibility of the thiol-acrylate hydrogels was investigated in two model systems. For cured hydrogels, all of the hydrogels precursors were sterilized and cured in 24-well tissue culture plates, the hydrogel surfaces were seeded with L929 at a density of 5000 cells/well. The Cell Counting Kit-8 (CCK8) was adopted to detect cytotoxicity by analyzing OD at 492 nm at 1st, 3rd, 5th and 7th days. For uncured formulations, L929 cells were co-cultured with 100 mg/mL uncured precursors for 24 hrs and stained with LIVE/DEAD® Assay. Three replicate experiments were performed with four parallel samples in each group.

2.12 Encapsulation, proliferation and viability of cells within hydrogel GE-AA and GE-SH were prepared in sterilized PBS containing 0.5% Irgacure 2959. L929 cells were trypsinized in 0.25% Trypsin-EDTA (Gibco, USA), centrifuged (×1000rpm) and re-suspended with PBS containing 0.5% Irgacure 2959. Cell suspension was then added to hydrogel precursor solutions with gentle agitation to yield a final concentration of 1×106 cell/mL. 100 μL of cell encapsulated precursor solution was transferred to 96-well TCPS and subject to 12


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photopolymerization with 365 nm UV light (intensity 35 mW/cm2) for 2 min. The viability of L929 cells encapsulated in the hydrogel samples was determined after 1, 5, 8 days of incubation via LIVE/DEAD® stain. Viable and nonviable cells were counted in quadrants and central part assigned to each image using Image J software, with the average number fraction of live cells reported. Due to the plane of focus, the results are represented as a ratio of live to total cells and these results are not normalized to materials area or volume. All cell encapsulation experiments were performed in duplicates with five parallel samples in each group. Cell tracking was carried out by pre-staining L929 cells with a lipophilic dye, DiLC12(3). 10 μ g/mL DiLC12(3) stock solution was prepared and added to a cell monolayer at 80% confluence. Cells were incubated at 37 °C in CO2 incubator for 60 min and DiLC12(3) media was rinsed from cells thoroughly with DPBS. The DiLC12(3) pre-stained cells were trypsinised from flask and re-suspended in fresh media. The pre-stained cells encapsulated within the hydrogel were cultured for 7 days, during which the culture media was changed every other day. After 1, 4, 7 days in hydrogel discs, DiLC12(3) stained cells were visualized with a confocal microscope imaging system (Carl Zeiss, LSM710, Germany).

2.13 Animal implantation and clinical evaluation

2.13.1 In-vivo focal corneal injury rabbit model

In all experimental procedures, animals were housed and treated in accordance with the ARVO Statement for the use of Animals in Ophthalmic and Vision Research and with ethics approval 13



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from Wenzhou Medical University. To produce a focal corneal injury, New Zealand White Rabbits weighing 2.4-2.8 kg were anaesthetized via peritoneal administration of xylazine and pentobarbital sodium. Subsequently, a 3.0 mm corneal trephine was applied to remove certain part of corneal epithelium-stroma (d = 3 mm, thickness = 200 μm). Sterilized formulations of thiol-acrylate hydrogel (150 mg/mL) was applied onto the wound site and allow to gel (6 eyes) under 365 nm UV irradiation (intensity 35 mW/cm2) (UVLED, Shanghai Machine Optoelectronic Technology Co., Ltd, China) for 2 minutes. A soft contact lens was used to cover and provide structural support to the hydrogel. In the control eyes (6 eyes), only focal corneal wound was established and no hydrogel was applied.

2.13.2 Clinical evaluation

Follow-up examinations were performed on a daily basis for the first week following surgery, then weekly. Slit-lamp examinations were performed to check the abnormality in anterior segment of eyes. At each stage, the area of the corneal epithelial defect was monitored using fluorescent staining paper and photographed. Optical Coherence Tomography (RTVue OCT, Inc., Fremont, CA) was utilized to observe the detailed information of tissues of anterior and posterior segments. Specular microscopy was used to detect changes in the corneal endothelial cells. At 4 weeks, the animals were humanely sacrificed and their eyes taken for histological analysis. Following fixation in 4% paraformaldehyde solution and embedding in paraffin and sectioning, the sections were strained with hematoxylin and eosin (H&E) staining and examined using

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optical microscope (LEICA, German) for the structure and inflammation in different groups.

2.14 Expression of results and statistics

All quantitative data were expressed as the mean±standard deviation in triplicate, unless otherwise stated. One-way Anova was used to compare mean values from two groups. p < 0.05 was considered as statistically significant and marked with an asterisk. All analyses were performed with Origin 8.0 software.

3 Results and discussion

3.1 Functionalization of gelatin

Gelatin has a long history of application as an ocular tissue engineered material due to its excellent biocompatibility.44,

45

Being derived from collagen, gelatin contains amino and

carboxylic groups which can be modified with different functional groups. In this study, the functionalized gelatin was synthesized via two methods: (1) through acrylation of gelatin to generate acrylated gelatin (GE-AA) by a similar protocol as reported;46 (2) thiolation of gelatin to produce a thiol-gelatin (GE-SH) by EDC/NHS bioconjugation with cysteamine (Fig.1). The introduction of acrylate groups and cysteamine to the polymer backbone is an important step enabling gelatin to be crosslinked on-demand upon exposure to light. The extent of modification of gelatin after functionalization was determined by 1H NMR spectroscopy (Fig. S1). 47 In the 1H

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NMR spectrum of GE-AA, signals appeared at δ 6.45 and 5.90 ppm corresponds to the vinylic protons while the peaks at δ 2.9 ppm attributed to methylene adjacent to amino groups in lysine decreased. In the 1H NMR spectrum of GE-SH, a signal appeared at 2.9 ppm is typical for methylene protons next to thiol functional groups. Based upon an average lysine content of 2.9% 48

, 100% lysine was converted to acrylates calculated from 1H NMR spectrum, therefore about 4%

acrylate functionalization happens in GE-AA, however, 50% lysine were thiolated, so about 2% thiol functionalization occurred in GE-SH.

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Fig. 1 Schematic synthesis of GE-AA, GE-SH and Schematic description of the mechanism of network formation between GE-AA and GE-SH. Gelatin macromers containing primary amino groups were reacted with acrylic anhydride (AA) to attach acrylate pendant groups. Gelatin macromers containing carboxylic groups were reacted with cysteamine to produce thiolated gelatin.

Fig. 2 Photo-rheology profiles of cure process for GE-AA and GE-SH at different ratios (A), different photo intensity (B) and different solid content (C).

3.2 Photo-rheological properties of photo-curable hydrogel

Photo-rheology under UV irradiation has been established to examine the gelation kinetics of

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photocurable hydrogels.48,

50

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Such techniques are advantageous in measuring the real-time

changes in rheological behavior (G′, G″) upon exposure to light. In this study, we presented a rheological study of UV-activated crosslinked gelatin, investigating the ratios of thiol/ene groups, the effects of photo intensity, solid content on the gelation kinetics and final gel properties.

To analyze the effect of the ratios of thiol to ene groups, precursors of GE-AA and GE-SH were mixed at 1:2, 1:1, 2:1 and 3:1 molar ratio of acrylate group to thiol groups while maintaining a solids content at 200 mg/mL (w/v), the resulting photo-rheological data are presented in Fig 2(A) and Table 1. The oscillatory rheology time sweeps presented in Fig. 2(A) illustrates the rapid gelation of the precursor solutions when crosslinked with 365 nm light at 53 mW/cm2 irradiation intensity. In order to measure the pre-cure mechanical properties of the formulations, measurements were taken for 3 minutes before exposing the samples to UV light. The storage modulus increased immediately after the light was turned on. All of the samples could be fully cured and the storage moduli reached a plateau in 0.5-1 minute after UV light exposure whilst the ratio of 2:1 (GE-AA: GE-SH) attained the highest storage modulus (18.0±2.0 kPa). The different post-cure moduli may reflect the mixed modes of reaction mechanisms (step growth/chain growth) as a result of the different monomer feed ratios.29 Fig. 2(B) and Table 2 illustrate that the storage moduli could be varied by altering the photo intensity. For UV intensities investigated (18-71 mW/cm2), time sweeps show that G′increased from 18.5±1.5 kPa to 22.0±2.0 kPa while photo intensity decreased from 71 mW/cm2 to 18 mW/cm2, respectively. The increase in post-cure storage modulus with decreasing photo intensity may be a result of forming higher 18


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average molecular weight polymer chains under these conditions due to the lower initiator decomposition rate. To attain the highest post-modulus under moderate UV intensity with shortest irradiation, samples of GE-A/GE-SH = 2:1 was prepared and diluted to generate solid contents ranging from 200 mg/ml to 100 mg/ml and cured under 35 mW/cm2. The three hydrogels of different solid contents exhibited very different storage moduli according to Fig. 2(C) and Table 3; hydrogel containing 200 mg/mL solid content exhibited storage modulus of approximately 22.0±2.0 kPa; while the hydrogel containing 150 mg/ml solid content exhibited storage moduli of approximately 8.8±0.3 kPa, which gave hydrogels with similar shear modulus to that of natural cornea (Natural cornea 2-8 kPa)12. The storage modulus of the 100 mg/ml hydrogel was only 1.2±0.1 kPa.

Table 1 Photo rheology: effect of ene/thiol ratio (light intensity = 53 mW/cm2, 200 mg/ml solids content) Samples

G′(kPa)

Cure time (min)

GE-AA/GE-SH=1:2

0.3±0.3

0.5

GE-AA/GE-SH=1:1

6.0±1.3

1

GE-AA/GE-SH=2:1

18.0±2.0

0.5

GE-AA/GE-SH=3:1

11.5±0.5

1

ene/thiol ratio

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Table 2 Photo rheology: effect of light intensity (ene/thiol ratio = 2:1, 200 mg/ml solids content)

Light Intensity

Storage Modulus

(mW/cm2)

G′ (kPa)

71

18.5±1.5

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