Acta Biomaterialia 10 (2014) 1167–1176

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Development of high refractive ZnS/PVP/PDMAA hydrogel nanocomposites for artificial cornea implants Quanyuan Zhang a,b,1, Kai Su a,1, Mary B. Chan-Park a, Hong Wu c,⇑, Dongan Wang a, Rong Xu a,⇑ a

School of Chemical & Biomedical Engineering, Nanyang Technological University, N1.2, 62 Nanyang Drive, Singapore 637459, Singapore Ministry of Education Key Laboratory for the Green Preparation and Application of Functional Materials, Hubei University, Wuhan, Hubei 430062, China c Department of Ophthalmology, Second Hospital of Jilin University, Changchun 130041, China b

a r t i c l e

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Article history: Received 2 May 2013 Received in revised form 8 December 2013 Accepted 10 December 2013 Available online 25 December 2013 Keywords: Polymers Hydrogel ZnS nanoparticles Refractive indices Cornea implants

a b s t r a c t A series of high refractive index (RI) ZnS/PVP/PDMAA hydrogel nanocomposites containing ZnS nanoparticles (NPs) were successfully synthesized via a simple ultraviolet-light-initiated free radical co-polymerization method. The average diameter of the ZnS NPs is 3 nm and the NPs are well dispersed and stabilized in the PVP/PDMAA hydrogel matrix up to a high content of 60 wt.% in the hydrogel nanocomposites. The equilibrium water content of ZnS/PVP/PDMAA hydrogel nanocomposites varied from 82.0 to 66.8 wt.%, while the content of mercaptoethanol-capped ZnS NPs correspondingly varied from 30 to 60 wt.%. The resulting nanocomposites are clear and transparent and their RIs were measured to be as high as 1.58–1.70 and 1.38–1.46 in the dry and hydrated states, respectively, which can be tuned by varying the ZnS NPs content. In vitro cytotoxicity assays suggested that the introduction of ZnS NPs added little cytotoxicity to the PVP/PDMAA hydrogel and all the hydrogel nanocomposites exhibited minimal cytotoxicity towards common cells. The hydrogel nanocomposites implanted in rabbit eyes can be well tolerated over 3 weeks. Hence, the high RI ZnS/PVP/PDMAA hydrogel nanocomposites with adjustable RIs developed in this work might potentially be a candidate material for artificial corneal implants. Ó 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction The human cornea, the main refractive element of the eye and a prerequisite for good vision, is a clear and transparent tissue which functions like a window that controls the entry of light into the eye. It consists of a highly organized group of cells and proteins which are arranged in three main cellular layers for different functions: an outermost multilayered protective epithelium, a central stroma with keratocytes consisting primarily of water (78%) and collagen (16%) [1], and an innermost single-layer endothelium that governs fluid and solute transport and maintains water balance for optical transparency. Myopia and other forms of cornea-related refractive errors represent the commonest cause of visual disability today. It has been reported that corneal disease and damage are the primary reasons for vision loss caused in 10 million people worldwide [2]. To date, the most common medical treatment for vision correction is laser-assisted in situ keratomileusis (LASIK) surgery, which is the irreversible reshaping of the cornea by laser energy. Furthermore, corneal function is related to its transparency and any corneal disease or damage to the stroma or endothelium may reduce the transparency, leading to the loss of vision or ⇑ Corresponding authors. 1

E-mail addresses: [email protected] (H. Wu), [email protected] (R. Xu). These authors contributed equally to this work.

blindness. The widely adopted treatment for most forms of corneal blindness is corneal transplantation with human donor tissue. However, due to the fact that the demand far exceeds the supply in many parts of the world, corneal transplantation with donor corneas is not always possible [3]. The critical shortage of human corneal donor tissue has resulted in various efforts to develop corneal substitutes as an urgent need [4]. An artificial cornea (keratoprosthesis), which was first developed over a century ago, has great potential application in treating cornea-related diseases and vision errors [5,6]. Moreover, the use of a refractive implant made of biocompatible materials is relatively obvious as the preferred method for creating a successful long-term refractive change of the cornea, either for myopia or hyperopia. The ability to specify an accurate refractive change would be enhanced, and most importantly the procedure is additive and reversible, not removing tissue [7,8]. In recent years, many research groups have attempted to develop various materials as corneal replacements [9–17]. Hydrogels were the first type of biomaterials developed for use in the human body and have wide applications in biomedical areas such as drug delivery, soft contact lenses, tissue engineering scaffolds, biosensors and soft tissue replacement [18–22]. Hydrogels with a crosslinked network structure can absorb large amounts of water while maintaining their network structures to form water-swollen polymeric materials. Hydrogel biomaterials resemble hydrodynamic properties of cells and tissues due to their soft tissue-like physical

1742-7061/$ - see front matter Ó 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.actbio.2013.12.017

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properties and can also improve the biocompatibility due to their surface hydrophilicity and high intrinsic mobility of the polymer chains [23,24]. However, due to the low refractive index (RI) of water at 1.33, hydrogel biomaterials with high water contents generally have low RIs. The incorporation of high RI inorganic building blocks such as TiO2, ZnO and ZnS into organic matrices is an effective way of increasing the RIs of the polymeric materials [25–27]. In a preceding paper, we developed a high RI organic–inorganic interpenetrating network (IPN) hydrogel nanocomposite incorporating ZnS nanoparticles (NPs) (ZnS/PHEMA/PAA). The nanocomposite exhibited a high RI of 1.49 in the hydrated state with an equilibrium water content of 60.2% [28]. In this work, we further developed a series of high RI organic–inorganic hybrid ZnS/PVP/ PDMAA hydrogel nanocomposites with tunable ZnS NPs and water contents. Nanometer-sized ZnS particles were incorporated into a PVP/PDMAA hydrogel matrix. Different from the previous PHEMA/PAA system, the RIs of the ZnS/PVP/PDMAA nanocomposites are easily adjustable by varying the contents of ZnS NPs. The physical and biological properties of the resultant hydrogel nanocomposites were extensively characterized and their suitability as potential artificial corneal implants was also investigated. 2. Materials and methods 2.1. Materials Zinc acetate dihydrate (Zn(Ac)22H2O, 98%, Alfa-Aesar), mercaptoethanol (ME, 98%, Alfa-Aesar), thiourea (99%, Alfa-Aesar), N-vinyl-2-pyrrolidone (NVP, 99%, Acros Organics), N,N-dimethylacrylamide (DMAA, 99.5%, Alfa-Aesar), 2-hydroxy-2-methylpropiophenone (Darocur 1173, 97%, Sigma–Aldrich), triethylene glycol dimethacrylate (TEGDMA, 97%, Sigma–Aldrich), bovine serum albumin (BSA, Sigma–Aldrich), bicinchoninic acid solution (Sigma–Aldrich), copper (II) sulfate solution (Sigma–Aldrich, 4% (w/v) prepared from copper (II) sulfate pentahydrate) and BSA (Quick Start™, standard set, BIO-RAD) were purchased and used without further purification. N,N-dimethylformamide (DMF, Fisher Scientific) was of high-performance liquid chromatography grade and purified by vacuum distillation prior to use. All other solvents were of analytical grade and were used as-received. Cell culture dishes and flasks, centrifuge tubes and serological pipettes were purchased from Becton Dickinson (Franklin Lakes, NJ). Dulbecco modified Eagle’s medium (DMEM), Ham’s F-12, HEPES, penicillin and streptomycin, L-glutamine, 0.05% trypsin0.02% ethylenediaminetetraacetic acid (EDTA) solution were acquired from Invitrogen-GIBCO BRL (Grand Island, NY). Fetal bovine serum (FBS) was purchased from Hyclone (Logan, UT). Mouse NIH 3T3 fibroblasts (ATCC CCL 92) were acquired from American Type Culture Collection (ATCC, Rockville, MD). Dispase II was purchased from Roche (Mannheim, Germany). Mitomycin-C, bovine insulin, human transferrin, hydrocortisone, human epidermal growth factor (EGF), cholera toxin and other reagents were from Sigma–Aldrich (St Louis, MO). CellTiter 96Ò AQueous One Solution Cell Proliferation Assay kit (MTS) was obtained from Promega (Madison, USA). 2.2. Synthesis of ME capped ZnS NPs The synthesis procedure of ME capped ZnS NPs was the same as that in our previous work [28], which was adopted from the literature [29]. A three-necked round-bottom flask 500 ml in volume, equipped with a magnetic stirrer, a condenser and nitrogen purging, was charged with Zn(Ac)22H2O (22.0 g, 0.1 mol), ME (11.6 g, 0.148 mol), thiourea (5.5 g, 0.072 mol) and DMF (300 ml). The solution was refluxed at 160 °C for 10 h under continuous stirring and

nitrogen purging. The resultant mixture was concentrated to 80 ml by rotary evaporation, after which the solid was precipitated using excess ethanol. The solid precipitate was collected and washed thoroughly with methanol before being dried in vacuum. 2.3. Synthesis of ZnS/PVP/PDMAA hydrogel nanocomposites The powder of ME capped ZnS NPs with desired weight ratio (30, 40, 50 and 60 wt.% in the final composites at dry state) was dispersed in the mixture consisting of DMF and DMAA. After stirring for 0.5 h at room temperature, the monomer NVP was added into the mixture. The weight ratio of DMAA:NVP:DMF was 2:2:1. The resultant mixture was stirred for another 0.5 h. After adding the photoinitiator (Darocur 1173, 1 vol.%, with respect to the monomer) and the cross-linking agent (TEGDMA, 1 vol.%, with respect to the monomer), the mixture was ultrasonicated for 30 s and then the transparent precursor solution was transferred into a Teflon spacer (250 mm in thickness and 20 mm in inner diameter) positioned on a glass plate (1.0 mm thick). After a second glass plate was put on top of the spacer, the solution was exposed to an ultraviolet (UV) light source (200–2500 nm) for 10 min, during which free-radical-induced gelation occurred and a transparent hydrogel was formed which is insoluble in DMF. The resultant ZnS/PVP/PDMAA hydrogel nanocomposites were extensively washed with water to exchange the solvent in the hydrogel and then immersed in deionized water for at least 3 days to eliminate any unreacted components, as well as to attain equilibrium water content in the swelling state. 2.4. Materials characterization Fourier transform infrared (FTIR) spectra were recorded on a Bio-Rad digilab FTS 3100 spectrometer. The FTIR pellets were made from 2 mg of the sample and 100 mg of KBr. Thermogravimetric analysis (TGA) was carried out in a Perkin Elmer Diamond TG/DTA instrument at a heating rate of 10 °C min1 from ambient temperature to 700 °C under a flow of nitrogen at 200 ml1 min. The RIs of the IPN hydrogel nanocomposites at a wavelength of 589 nm were measured on a NAR-4T and NAR-1T solid abbe refractometer at 20 °C, using methylene iodide containing sulfur solution (for dry state) and monobromonaphthalene (for hydrated state) as contact liquids. The sample was cut to 20–30 mm in length, 8 mm in width and 3–10 mm in height, followed by washing and surface polishing for RI measurement. The RIs of three duplicates cut from the same sample were measured for consistency checking. 2.5. Swelling studies The equilibrium water content of the ZnS/PVP/PDMAA hydrogels was estimated by comparing the dry and the swollen weights. The swollen gels soaked in deionized water were taken out and patted dry and the weight was measured regularly until the equilibrium was reached. The equilibrium water percentage was calculated using Eq. (1):

W% ¼ ðW s  W d Þ=W s  100%

ð1Þ

where Ws and Wd are the weights of swollen and dry nanocomposites, respectively. 2.6. Viability/cytotoxicity tests The in vitro viability/cytotoxicity of the ZnS/PVP/PDMAA hydrogel nanocomposites was studied using WST-1 {4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate} assay and LIVE/DEADÒ assay. The WST-1 test was carried out using a Transwell cell culture system, which is an indirect

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method and can show the effect of any harmful leachable substances from the hydrogel on cells. The hydrogel nanocomposites were cut into small circular pieces (diameter of 6 mm and thickness of 4 mm) followed by sterilization in an ethanol–water mixture (70:30, v/v) under UV light at room temperature for 1 day and then soaked with sterilized phosphate-buffered saline (PBS) under UV light for another day for completely displacement of ethanol with PBS in the hydrogel nanocomposites. After the cells were seeded onto a 96-well tissue culture polystyrene (TCPS) (5  103 cells per well) and incubated at 37 °C (at 33 °C for osteoblasts (OB)) in 5% CO2 atmosphere for 24 h, the small pieces of ZnS/ PVP/PDMAA hydrogel nanocomposites were then added to the plate wells. The TCPS well was used as a positive control and PVP/PDMAA hydrogel without ZnS NPs was used for comparison. The medium was changed every 2 days. Cells were stained using a LIVE/DEADÒ Viability/Cytotoxicity Assay Kit (Molecular Probe), containing calcein AM and ethidium homodimer (EthD-1) for the identification of live and dead cells, respectively. After a certain period of cell seeding time ranging from 1 to 7 days, the medium was discharged and the culture was washed with PBS. Then ‘‘LIVE/DEAD’’ solution containing 2 lM calcein AM and 4 mM EthD-1 was added and the mixture was incubated at 37 °C for 30 min. The populations of live and dead cells in the stained cultures were analyzed using fluorescent microscopy (Olympus IX71 microscope system). WST-1 assay was used to examine mitochondrial function and cell proliferation. WST working solution was prepared by mixing a tenth of WST regent with the cell media. After the cells were cultured for certain time periods ranging from 1 to 7 days, the culture medium was removed and then 220 ll of WST working solution was added to each well of the 96-well plate containing hydrogel nanocomposites. The culture media were incubated on a shaker at 37 °C (at 33 °C for OB) for 1.5 h for color development. After that, the WST work solution was transferred to a new 96-well assay plate (100 ll per well) and absorbance was measured using a Thermo Multiskan Spectrum at a wavelength of 490 nm. Four parallels were averaged for each sample. The effects of the hydrogel nanocomposite samples on human limbal epithelial stem cells (LESCs) viability was examined using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy-methoxyphenyl)-2-(4sulfophenyl)-2H-tetrazolium (MTS) assay. Cells were seeded in 24well plates (1.0  104 cm2 per well) in 1 ml of serum-free medium. Cells were allowed to attach overnight and then treated with the samples. For each sample, 200 ll of MTS solution was added at 1 and 5 days after treatment. After incubation for 3 h at 37 °C in an atmosphere with 5% CO2, the light absorbance was measured at 490 nm with a microplate reader. The cell viability was calculated by the ratio of the number of viable cells with hydrogel sample treatment to that without treatment. Experiments were repeated in triplicate for consistency. 2.7. Protein adsorption The protein adsorption test was carried out in a 48-well TCPS, the circular hydrogel samples were 0.5 cm in diameter and 0.2 cm thick, the total BSA solution was 0.2 ml, and 0.1 ml of the sample solution was used for protein adsorption tests. An appropriate amount of BSA was dissolved in PBS solution to prepare a BSA solution with a concentration of 3 mg ml1. The working solution was prepared by mixing the bicinchoninic acid (BCA) and copper (II) sulfate solution at a ratio of 50:1 v/v. The hydrogel nanocomposite samples were cut into small circular pieces and soaked in the PBS solution for 1 h. The PBS solution-welled samples were immersed in the BSA solution and incubated on a shaker at 37 °C for 4 h. The concentration of BSA in the solution before and after adsorption was measured using a BCA assay kit. Briefly,

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0.1 ml of the sample solution was added into 1 ml of the working solution and incubated at 30 °C for 15 min in a water bath. The color changed from green to purple. After the incubation, the solution was analyzed with UV spectroscopy (UV-2450, Shimadzu), and the absorption intensity at 560 nm was used to estimate the concentration of the protein. The measurement was carried out three times to obtain the average of the readings. 2.8. Cell adhesion The procedures for cell culture and the treatment of ZnS/PVP/ PDMAA hydrogel nanocomposites were the same as above. Hydrogel nanocomposites were placed into 96-well TCPS and washed three times with PBS and one time with the cell media. The wells were seeded with cells (pCCs) followed by incubation at 37 °C for 24 h. The seeding density was 1.0  105 cells per well. After incubation, the surfaces of the wells were washed three times with the cell media and cell adhesion was assessed in a phase contrast microscopy (Olympus IX71 microscope system). 2.9. Transplantation study New Zealand white rabbits (12 rabbits), of 2–3 kg, were used and the guidelines of the Association for Research in Vision and Ophthalmology (ARVO) statement for the use of animals in ophthalmic and vision research were followed. The experimental protocol was approved by the Institutional Animal Care and Use Committee of Biological Resource Centre, Jilin University. All rabbits were anesthetized by intramuscular injection with ketamine (New South Wales, Australia) at 40 mg kg1 and xylazine (New South Wales, Australia) at 8 mg kg1. The hydrogel samples were obtained using a 4 mm diameter punch from the sheet of hydrogel (1 mm thick) and the samples were implanted by lamellar keratoplasty (LKP); silk sutures were used. To evaluate scaffold biocompatibility, the hydrogel samples in the form of lamellae were implanted into the corneas of the right eyes of the rabbits. A lamellar stromal pocket (5  4 mm) was created in the center of the right eye cornea, and the sample lamellae were implanted into the pocket. Three kinds of hydrogel materials (0, 40 and 60 wt.% ZnS) were chosen and in each hydrogel we implanted four samples in the right eyes of the rabbits. After transplantation for 1 and 3 weeks, rabbits were euthanized before the corneal specimens were harvested; at each time, two corneal specimens were harvested and examined by hematoxylin and eosin staining. Slit-lamp examination was also used to assess the corneal optical clarity and neo-vascularization (compare the left non-implanted eyes after 3 weeks). 2.10. Statistical analysis Data analysis was performed using ANOVA and Student’s t-test. For each study, the sample size was 4 (n = 4) and a p < 0.05 was considered a statistically significant difference. Data were presented as mean ± standard deviation (SD). 3. Results and discussion 3.1. Properties of the ZnS/PVP/PDMAA hydrogel nanocomposites The ME capped ZnS NP powders were readily dispersible in DMF to obtain stable and transparent solutions at different concentrations for the preparation of transparent hydrogel nanocomposites with tunable ZnS contents. As reported in our earlier study, the as-formed cubic phased ZnS NPs have a good crystallinity despite their smaller average diameter of 3.0 nm [28]. Besides

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Fig. 3. Variation of RI (d dry state and w hydrated state) and water content (j) with the content of ME capped ZnS NPs. Fig. 1. TGA curves of ME capped ZnS NPs, PVP/PDMAA hydrogel and ZnS/PVP/ PDMAA hydrogel nanocomposites with different ZnS contents from 30 to 60 wt.%.

DMF, it has been reported that polar organic molecules, for example DMAA, can effectively stabilize and disperse ZnS NPs due to the coordinative interaction with the surface of ZnS NPs [30–33]. NVP, which has a similar molecular structure to DMF or DMAA, is expected to play a similar role due to its good complexation with the metal ions. Moreover, both NVP and DMAA also serve as monomer precursors to form the hydrogel network. The ZnS/PVP/ PDMAA hydrogel nanocomposites with different ZnS contents were successfully obtained via a simple UV-initiated free radical co-polymerization by UV irradiation of the mixed solution of ME capped ZnS NPs, DMF, NVP and DMAA monomers. The content of ME capped ZnS NPs in the resultant nanocomposites ranged from 30 to 60 wt.%. PVP and PDMAA can well stabilize and disperse ME capped ZnS NPs into the polymer matrix by interacting with the surface of ZnS NPs via (i) complexation with Zn2+ ions and (ii) intermolecular hydrogen bonding interaction with hydroxyl groups of ME molecules on the surface of ZnS NPs. The thermal stabilities of the ME capped ZnS NPs, PVP/PDMAA hydrogel and ZnS/PVP/PDMAA hydrogel nanocomposites were studied and their TGA curves are shown in Fig. 1. Due to the unstable surface ME molecules, ZnS NPs started to lose weight at 200 °C and the decomposition of these organic species completed at 300 °C. Based on weight loss data, the estimated molar ratio of ME:Zn2+ is as high as 1:1.45. It is therefore suggested that abundant ME molecules capped on the surface ZnS NPs can improve the compatibility of ZnS NPs with the polymer matrix to avoid phase separation so as to obtain homogeneous and transparent nanocomposites. The decomposition of the PVP/PDMAA polymer matrix occurred during the temperature range of 330–450 °C. When ZnS NPs are incorporated into the PVP/PDMAA polymer matrix, the nanocomposites exhibited two different stages of weight loss cor-

responding to decomposition of ME molecules and polymer matrix, respectively. As can be seen from the trend of the curves, the first segment of weight loss started at 220 °C while the second started at 300 °C for all the nanocomposite samples. With an increasing content of ZnS NPs, the residue weight percentage in the TGA curves increased correspondingly from 19.6 to 39.1%. As the weight percentage of ZnS in ME capped ZnS NPs is 64.8%, these data are in good accordance with the theoretical composition of ME capped ZnS (30–60 wt.%) in the nanocomposites. All the as-prepared ZnS/PVP/PDMAA hydrogel nanocomposites are optically clear and transparent at both dry and hydrated states, even at the highest content of 60 wt.% ZnS NPs as shown in Fig. 2. The change in sample size due to swelling is quite obvious. The equilibrium water content of ZnS/PVP/PDMAA hydrogel nanocomposites varied from 82.0 to 66.8% while the contents of ZnS NPs varied from 30 to 60 wt.%, respectively. High water contents render the hydrogel nanocomposites with high permeability to water-soluble metabolites including glucose, oxygen, and other nutrients [34,35]. The RIs of the resulting nanocomposites were measured to be as high as 1.58–1.70 and 1.38–1.46 in the dry and hydrated states, respectively, which increased from 1.48 and 1.35 for PVP/ PDMAA hydrogel. Fig. 3 shows the variation of RI and equilibrium water content with percentage of the ME capped ZnS for the asprepared hydrogel nanocomposites. As the content of ZnS NPs in the polymer matrix increases, the refractive index of the nanocomposites in both dry and hydrated state almost increases linearly, suggesting that the current approach is effective in enhancing and adjusting the RI of the hydrogel nanocomposites. We also investigated the RI of the hydrogel samples after immersing in deionized water for 1 month and the RI values didn’t change, indicating that the hydrogel nanocomposites are stable and no ZnS NPs leached. Furthermore, the RIs of all the hydrogel nanocomposites are higher than that of human corneas (1.373–1.380 [36]). In par-

Fig. 2. Photo images of ZnS/PVP/PDMAA hydrogel nanocomposites (with 60 wt.% ZnS NPs) at dry (left) and hydrated (right) states.

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ticular, the RI of the hydrogel nanocomposite with 40 wt.% ZnS NPs in the hydrated state is 1.41, which is superior to that of human corneal stroma at 1.373–1.380, while its equilibrium water content of 75.2% is just slightly lower than that of human cornea. The high RIs of the hydrogel nanocomposites obtained in this work can facilitate the use of a thinner lens optic portion for cornea implants and make it possible to insert thereof through a smaller incision in the cornea of an eye in a surgical procedure.

3.2. Viability/cytotoxicity tests of the ZnS/PVP/PDMAA hydrogel nanocomposites The in vitro viability/cytotoxicity of the ZnS/PVP/PDMAA hydrogel nanocomposites was determined by the WST-1 test and ‘‘LIVE/ DEAD’’ assay. The cytotoxicity study of ZnS/PVP/PDMAA hydrogel nanocomposites was first conducted with pCCs to assess the influence of the content of ZnS. Cells were cultured and seeded onto the 96-well culture plate as described earlier. Small pieces of ZnS/PVP/PDMAA hydrogel nanocomposites with different ZnS

1 days

3 days

Fig. 4. WST activities of porcine chondrocytes cultured for up to 7 days, incubated with ZnS/PVP/PDMAA hydrogel nanocomposites with different contents of ZnS NPs.

contents (30, 40, 50 and 60 wt.%) were added to the wells. Cells cultured in the cell medium without hydrogel nanocomposites

5 days

7 days

60%

50%

40%

30%

0

control

Fig. 5. Fluorescence micrographs of calcein AM and EthD-1 stained porcine chondrocytes at different periods of cell culture. Live cells are stained green, and dead cells in red. Scale bar represents 100 lm. Cell seeding numbers: 5000 cells per well.

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were used as a positive control and PVP/PDMAA hydrogel without ZnS NPs was also used for comparison. The cell viability was checked after 1–7 days of culture time. As shown in Fig. 4, although slightly lower than the positive control, the cells that were incubated with the hydrogel nanocomposites were allowed to proliferate and increase in number as a function of culture time, indicating a good biocompatibility of all the hydrogel nanocomposites. When ZnS NPs were incorporated into the PVP/PDMAA hydrogel at 30– 60 wt.%, the viability of the cells incubated with all these hydrogel nanocomposites was only slightly lower than that incubated with PVP/PDMAA hydrogel without ZnS NPs. Further, with the increasing ZnS content, it makes no significant difference in the cell viability profiles of these four hydrogel nanocomposites, suggesting that the ZnS NPs exert little cytotoxicity to the hydrogel nanocomposites towards pCCs even at the highest content of ZnS NPs of 60 wt.%. Fig. 5 depicts the morphology of the treated pCCs by ZnS/PVP/ PDMAA hydrogel nanocomposites and PVP/PDMAA hydrogel after calcein AM and EthD-1 staining. The stained living and dead cells

1 days

3 days

Fig. 6. WST activities of hFOB, hHepG2 and pSMSCs cultured for up to 7 days.

are displayed in green and red color under a fluorescence microscope, respectively. The majority of the pCCs cultured with the four ZnS/PVP/PDMAA hydrogel nanocomposites were alive, and dead

5 days

7 days

pSMSCs (40%)

pSMSCs (control)

hHepG2 (40%)

hHepG2 (control)

hFOB (40%)

hFOB (control)

Fig. 7. Fluorescence micrographs of calcein AM and EthD-1 stained hFOB, hHepG2 and pSMSCs for ZnS/PVP/PDMAA hydrogel nanocomposites with 40 wt.% ZnS NPs. Scale bar represents 100 lm. Cell seeding numbers: 5000 cells per well.

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Fig. 8. MTS activities of human limbal epithelial cells cultured for 1 day and 5 days.

cells were seldom found after 7 days, which confirmed high cell viability. Moreover, the cells spread quite well with no observable morphological alteration as compared to the positive control. The pCCs maintained a normal fibroblast cell shape for all the ZnS/ PVP/PDMAA hydrogel nanocomposites and the PVP/PDMAA hydrogel. Compared with the cells cultured with PVP/PDMAA

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hydrogel, the cells with all the ZnS/PVP/PDMAA hydrogel nanocomposites were similar in calcein AM stained (for live cells). Therefore, these results consistently show that the introduction of ZnS NPs into PVP/PDMAA hydrogel added little cytotoxicity towards pCCs. The effects of ZnS/PVP/PDMAA hydrogel nanocomposites on cell viability were also investigated for other cells including hFOB, hHepG2 and pSMSCs using ZnS/PVP/PDMAA hydrogel nanocomposites with 40 wt.% ZnS NPs. Cells cultured in the cell media without hydrogel nanocomposites were used as a positive control. Similar to chondrocytes, the viability of all the three type of cells that were incubated with hydrogel nanocomposites increased as a function of culture time, as shown in Fig. 6. Cells stained with ‘‘LIVE/DEAD’’ assay are shown in Fig. 7. Most of all the three cells were alive (with green fluorescence) after 7 days, indicating that the hydrogel nanocomposites presented good biocompatibility. Moreover, all the three cells remained in normal spreading cell shapes as compared to the positive control. The effects of hydrogel nanocomposites on LESCs viability was also examined using MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy-methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay. As shown in Fig. 8, the LESCs that were incubated with all the hydrogel nanocomposites were allowed to proliferate. Cells stained

Contr ol

0

30%

40%

50%

60%

Fig. 9. Fluorescence micrographs of calcein AM and EthD-1 stained human limbal epithelial cells after 5 days of cell culture. Scale bar represents 100 lm. Cell seeding numbers: 1.0  104 cells per well.

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0

40%

60%

Fig. 10. Cell adhesion on hydrogel nanocomposites at 10 magnification after 24 h incubation with porcine chondrocytes for PVP/PDMAA hydrogel and ZnS/PVP/PDMAA hydrogel nanocomposites with 40 and 60 wt.% ZnS NPs.

0

40%

60 %

Fig. 11. In vivo implantation in rabbit eyes after 3 weeks for ZnS/PVP/PDMAA hydrogel nanocomposites without ZnS NPs and with 40 and 60 wt.% ZnS NPs.

0

60%

Fig. 12. Histological sections of rabbit cornea at 3 weeks after transplantation of the hydrogel nanocomposites without ZnS NPs and with 60 wt.% ZnS NPs.

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with ‘‘LIVE/DEAD’’ assay are shown in Fig. 9. The cells spread well for the PVP/PDMAA hydrogel and all the ZnS/PVP/PDMAA hydrogel nanocomposites. Moreover, most of the cells cultured with all the ZnS/PVP/PDMAA hydrogel nanocomposites were alive (with green fluorescence) after 5 days, indicating that the ZnS/PVP/PDMAA hydrogel nanocomposites exert minimal cytotoxicity towards human limbal epithelial cells. Based on the above assay results, it can be summarized that the introduction of ZnS NPs in PVP/PDMAA hydrogel showed little cytotoxicity towards common cells in vitro.

3.3. Protein adsorption and cell adhesion The protein adsorption study showed that no protein adsorption was detected on all the four ZnS/PVP/PDMAA hydrogel nanocomposites with different contents of ZnS NPs and PVP/PDMAA hydrogel in the absence of ZnS NPs. The excellent resistance to protein adsorption of all the hydrogel materials with and without ZnS NPs suggested that the introduction of ZnS NPs into PVP/PDMAA hydrogel matrix does not change the properties of protein adsorption of the hydrogel matrix [37]. The cell adhesion of the hydrogel nanocomposites was also investigated and the results are shown in Fig. 10. Only a few cells adhered on the surfaces of ZnS/PVP/ PDMAA hydrogel nanocomposites and there were no cells spread, indicating that the hydrogel nanocomposites exhibited good resistance to cell adhesion.

3.4. Transplantation study In vivo implantation of different hydrogel nanocomposites in rabbit eyes is shown in Fig. 11. It was found that the hydrogel nanocomposite implants with 40 and 60 wt.% ZnS NPs were well tolerated over 3 weeks of the study, with no evidence of wound leakage, infection, inflammation or neovascularization. Histological sectioning results (Fig. 12) of both hydrogel corneal implants (without ZnS NPs and with 60% ZnS NPs) show that the transplanted corneal discs could remain in the rabbit cornea stroma after 3 weeks of transplantation without evidence of inflammation, edema or infection.

4. Conclusion In summary, a series of high RI ZnS/PVP/PDMAA hydrogel nanocomposites containing ZnS nanoparticles (NPs) have been successfully fabricated via a simple UV-initiated free radical copolymerization method. The equilibrium water contents of ZnS/ PVP/PDMAA hydrogel nanocomposites varied from 82.0 to 66.8% while the contents of ZnS NPs was increased from 30 to 60 wt.%. The RIs of the hydrogel nanocomposites were measured to be as high as 1.58–1.70 and 1.38–1.46 in the dry and hydrated state, respectively, which can be tuned easily by varying the ZnS NPs content. In vitro viability/cytotoxicity assays, protein adsorption, cell adhesion tests and in vivo implantation studies suggested that the ZnS/PVP/PDMAA hydrogel nanocomposites are biocompatible. Therefore, the inorganic/organic hybrid hydrogel nanocomposites with adjustable RIs developed in this work have a great potential as central optic materials in artificial cornea implants.

Acknowledgements This work was supported by National Medical Research Council (Grant No. NMRC/NIG/0022/2008) and SingHealth Foundation (Grant No. SHF/09/GMC(1)/012(R)), Singapore.

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Appendix A. Figures with essential colour discrimination Certain figures in this article, particularly Figs. 1–7 and 9, are difficult to interpret in black and white. The full color images can be found in the on-line version, at http://dx.doi.org/10.1016/ j.actbio.2013.12.017

Appendix B. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.actbio.2013. 12.017.

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