Silica

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Biological and Medical Applications of Materials and Interfaces

High Performance Photopolymerized Poly (vinyl alcohol)/ Silica Nanocomposite Hydrogels with Enhanced Cell Adhesion Can Zhang, Kaili Liang, Ding Zhou, Hongjun Yang, Xin Liu, Xianze Yin, Weilin Xu, Yingshan Zhou, and Pu Xiao ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b09026 • Publication Date (Web): 26 Jul 2018 Downloaded from http://pubs.acs.org on July 27, 2018

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ACS Applied Materials & Interfaces

High Performance Photopolymerized Poly (vinyl alcohol)/Silica Nanocomposite Hydrogels with Enhanced Cell Adhesion Can Zhang 1, Kaili Liang 1, Ding Zhou 1, Hongjun Yang 1, Xin Liu 1, Xianze Yin 1, Weilin Xu 1, Yingshan Zhou 1, *, Pu Xiao 2, *

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Key Laboratory of Green Processing and Functional Textiles of New Textile Materials, Ministry of Education, Wuhan Textile University, Wuhan 430073, People’s Republic of China 2 Research School of Chemistry, Australian National University, Canberra, ACT 2601, Australia Corresponding Authors: [email protected]; [email protected]

Abstract: Poly (vinyl alcohol) (PVA) hydrogels have been considered as promising implants for various soft tissue engineering applications due to their tissue-like viscoelasticity and biocompatibility. However, two critical barriers including lack of sufficient mechanical properties and non-tissue-adhesive characterization limit their application as tissue substitutes. Herein, PVA is methacrylated with ultralow degrees of substitution of methacryloyl groups to produce PVA-GMA. Subsequently, the PVA-GMA/methacrylate-functionalized silica nanoparticles (MSi) based nanocomposite hydrogels are developed via the photopolymerization approach. Interestingly, both PVA-GMA based hydrogels and PVA-GMA/MSi based nanocomposite hydrogels exhibit outstanding compressive properties, which cannot be damaged through compressive stress-strain tests in the allowable scope of tensile tester. Moreover, PVA-GMA/MSi based nanocomposite hydrogels demonstrate excellent tensile properties compared to neat PVA-GMA based hydrogels, and 15-fold, 14-fold, and 24-fold increase in fracture stress, elastic modulus and toughness, respectively, is achieved for the PVA-GMA/MSi based hydrogels with 10 wt% of MSi. These remarkable enhancements can be ascribed to the amount of long and flexible polymer chains of PVA-GMA and the strong interactions between the MSi and PVA-GMA chains. More interestingly, exciting improvements in the cell adhesion can also be successfully achieved by the incorporation of MSi nanoparticles.

Keywords: hydrogel; poly (vinyl alcohol); photopolymerization; nano-silica; nanocomposite

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1. Introduction Poly (vinyl alcohol) (PVA) hydrogels have been widely investigated as promising implants for various soft tissue engineering applications such as artificial articular cartilages

1,2

and

cardiovascular tissue applications 3,4 due to their tissue-like viscoelasticity and biocompatibility 2,5. PVA hydrogels are often prepared by various chemical or physical crosslinking approaches. However, the reported neat PVA hydrogels obtained via these techniques commonly suffer from the poor mechanical properties. Even though the PVAs with various molecular weights and degrees of hydrolysis were used in these studies, their compressive strength of 5.5×10-3 ~ 9.9×10-1 MPa 2,6-8 was far less than expected. And the disadvantage always limits their applications in in vivo translation. To improve the mechanical performances, the introduction of nanomaterials into PVA hydrogels is a promising approach 9. Recently, nanomaterials such as cellulose nanofibrils 10, carbon nanodot 9, and graphene oxide nanosheets 11 have been reported to be incorporated into the PVA hydrogels to enhance the mechanical strength. Despite these efforts, these nanocomposite hydrogels still cannot be extensively used as implants in vivo because of the dose-dependent cytotoxicity of carbon-based materials

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or trace residual of toxic crosslinking agents e.g.

glutaraldehyde 12. In addition, this kind of hydrogels are usually not tissue-adhesive and lacking of the integration with surrounding living tissues, which are attributed to PVA hydrogels’ intrinsical cell non-adhesive characterization 13,14. Great efforts have been made to endow the hydrogels with tissue adherence by developing hydrogels with bioactive nanocomponents such as nano-hydroxyapatite

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and nano-silica

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. Nano-silica with excellent chemical stability,

thermodynamic properties, and water resistance has been widely studied and used as an inorganic

2


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nanomaterial 17,18. In particular, nano-silica has shown promising potential in the medical field due to the good bioactivity e.g. cell affinity 19. Recently, PVA/silica hybrid hydrogels have been reported to be fabricated by freeze-thaw cycles 20 or chemical corsslinking with boric acid as a crosslinking agent

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. However, cyclic freezing-thawing process is a multi-step and

time-consuming approach, which is not suitable to form tissue-adhesive hydrogels in situ, while the leachable toxic crosslinking agents used in the chemical corsslinking method can be harmful to the surrounding tissues and organs 12. To overcome the aforementioned issues, in this study, methacrylated PVA with ultralow degrees of substitution of methacryloyl groups is synthesized, which is expected to keep amounts of long, extendible, and flexible polymer chains that can move availably to dissipate of the crack energy under loading after crosslinking of methacrylated PVA. It is hypothesized that this kind of methacrylated PVA hydrogels with low crosslinking density illustrate a good compressive strength. Until now, few literatures have been reported on high compressive strength of the neat PVA hydrogels. Moreover, nano-silica is incorporated into methacrylated PVA hydrogels to enhance further its tensile strength and cell adhesion properties. On the other hand, to achieve the rapid formation of hydrogels at a defect site in vivo, photopolymerization technique is conducted in this study, attributed to its rapid and highly tunable gelation kinetics under mild aqueous conditions at physiological temperatures with spatial-temporal control

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. In this study, photocrosslinkable PVA-GMA/MSi nanocomposite

hydrogels are developed, and their related properties including rheological property, swelling kinetics, morphology, crystallinity and mechanical properties are investigated. The in vitro cytotoxic evaluation and cell culture of the hydrogels in combination with mouse fibroblasts are 3



also studied to demonstrate their potential as implants.

2. Experimental 2.1 Materials Poly (vinyl alcohol) (PVA, 1.7×105 Da, 88 % hydrolyzed) was obtained from Kuraray Co., Ltd., Japan. Glycidyl Methacrylate (GMA) was supplied by Adamas Reagent Co., Ltd. 4-Dimethylaminopyridine (DMAP) was purchased by Sinopharm Chemical Reagent Co, Ltd. Methacrylate-functionalized silica nanoparticles (Aerosil® R 7200) with a carbon content of 4.5～ 6.5 wt% was purchased from Evonik Specialty Chemicals (Shanghai) Co., Ltd. The photoinitiator Darocur 2959 (D-2959, 2-hydroxy-1-[4-(hydroxyethoxy) phenyl]-2-methyl-1-propanone) was donated from IGM Resins B.V. (Netherlands). Mouse fibroblasts (L929 cells) were obtained from Procell Life Science Co., Ltd., China. Other reagents were all A.R. grade. All commercially available solvents and reagents were used without further purification.

2.2 Synthesis of methacrylated poly (vinyl alcohol) (PVA-GMA) PVA-GMA was prepared according to the method reported by us earlier 23. Briefly, 5.0 g PVA was dissolved in 100 mL dimethyl sulfoxide (DMSO). DMAP and GMA were added into it at 1.0 mol % and 0.025 mol % (relative to the hydroxyl group of PVA), respectively. And the reaction mixture was stirred for 6 h at 60 ℃ (Table 1). After that, the mixture was precipitated by acetone, dried under vacuum for 2 days and stored at -5 ℃ in the dark. 1H NMR spectrum was recorded on a Bruker AV 400 NMR instrument.

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2.3 Preparation of PVA-GMA/methacrylate silica (MSi) nanocomposite hydrogels A 15 % (w/v) PVA-GMA solution was prepared by dissolving 15.0 g PVA-GMA in 100 mL deionized water. Methacrylate silica naoparticles (0%, 1%, 2.5%, 5%, and 10%) were added into the PVA-GMA solution and dispersed uniformly by stirring at a high speed to prepare the PVA-GMA/MSi solution systems PM0, PM1, PM2, PM3, and PM4 respectively. D-2959 was added into the mixture solution at the concentration of 0.05 wt % (relative to amount of the solution). After that, the blend solution was injected into a mold consisting of two glass microslides separated by a spacer, then irradiated with an Omnicure Series 1000 UV light source (maximum emission wavelength 365 nm, 60 mW/cm2, Exfo, Canada) for 15 min at ambient temperature. The photoinitiator D-2959 used in this study has been demonstrated to be cytocompatibility to various cells 24.

2.4 Rheological measurements The viscosities of the blend solutions were performed as shear rate swept from 0.01 to 1000 s-1 on an AR 2000ex rheometer (TA Instrument, USA) using a 40 mm Steel parallel plate geometry and 1 mm gap at 25 ℃. For photocrosslinking kinetics study, in-situ dynamic photorheology 25 was applied to measure the elastic and viscous moduli during the process of photopolymerization. A Haake Mars Rheometers (Thermo Fisher Scientific Inc.) was equipped with a UV curing attachment and 20 mm parallel plate geometry. The upper plate was made of an optically transparent quartz acting as filter for UV light with a cut-off of 320-480 nm. UV light intensity was set as 60 mW/cm2 and the gap setting was fixed as 1.0 mm. Time-sweep oscillatory tests were performed at 25 ℃ at strain 5



amplitude of 1.0 % and a 6.28 rad/s, which was within the linear viscoelastic region 26.

2.5 Swelling and Sol tests The dry photocrosslinked nanocomposite hydrogels (weighted as W0) were soaked in phosphate buffered saline (PBS) at 37 ℃. At regular intervals, the hydrogels were taken out of the PBS solution, dried superficially with filter paper, and weighted as Ws. The swelling ratio (Q) was determined as: (WS-W0)/W0. The dry photocrosslinked nanocomposite hydrogels (dry mass recorded as m0) were swollen for three days in purified water at 37 ℃, with the water replaced every day. The swollen gels were lyophilized and the final mass recorded as m1. The sol content was calculated as: Sol = (m0-m1)/m0.

2.6 Mechanical tests Compression tests. Unconfined compression testing of the nanocomposite hydrogels (cylindrical shape, 8 mm in diameter and 5 mm in height) was carried out on an Instron 5848 microtester (Instron, Norwood, MA, USA) with 10 kN load cell at a compression rate of 0.5 mm/min. Tensile tests. Dumbbell-shaped samples (4 mm wide at narrow space, 50 mm long and 3 mm thickness) was performed by using Instron Universal Testing Machine (5967, USA) at a 10 mm/min crosshead speed at room temperature. The fracture stress and strain were determined from the stress-strain curve, and the Young's modulus was calculated by determining the slope of the initial linear region of the strain (0～10%). Toughness was calculated from the integral of 6


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stress-strain curves till fracture point.

2.7 The morphology The cross-section morphology of the nanocomposite hydrogels were determined by scanning electron microscope (JSM-6510, JEOL Ltd., Japan) at accelerating voltage of 20 kV. The freeze-dried samples were loaded on the surface of an aluminium SEM specimen holder and sputter coated with gold before observation.

2.8 X-Ray Diffraction (XRD) The XRD patterns of the nanocomposite hydrogels were performed via X-ray diffractometer (XRD2000, Shimadzu, Japan) with Cu Kα characteristic radiation (wavelength λ=0.154 nm at 40 kV, 50 mA, and scan speed of 1°/min in the 2θ range of 5-70°)

2.9 Cytotoxicity assays To evaluate the cytotoxicity of the hydrogels, the sterilized nanocomposite hydrogels were incubated in culture medium at extraction ratio of 1.25 cm2/mL for 24 h at 37 ℃, after which the hydrogels were removed and the extraction medium was obtained. 200 µL of L929 cells suspension was seeded in 96–well plate at a concentration of 105 cells/well. After incubation at 37 ℃ for 1 day, the culture medium was instead of the extraction medium. After 1 day, the extract was removed and 20 µL of MTT solution was added to each well. L929 cells were allowed to incubate at 37 ℃ (5 % CO2) for 4 h, and then the formazan reaction products were dissolved in dimethyl sulfoxide (150 µL) and the plates were shaken for 10 min. The optical density of the 7



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formazan solution was read on an ELISA reader at 568 nm. The Dulbecco's modified eagle medium containing 10 % fetal bovine serum (FBS) was used as negative control for toxicity test. Results are depicted as mean ± standard deviation. Significance between the mean values was calculated using ANOVA one-way analysis (Origin 7.0 SRO, Northampton, MA, USA). A value of p

Silica

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