Construction of Tough, in Situ Forming Double-Network Hydrogels

Dec 28, 2016 - glycol (PEG)−agarose double-network (PEG−agarose DN) hydrogels with good biocompatibility. The hydrogels display excellent mechanic...
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Construction of Tough, in-situ Forming Double Network Hydrogels with Good Biocompatibility Yazhong Bu, Hong Shen, Fei Yang, Yanyu Yang, Xing Wang, and Decheng Wu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b15364 • Publication Date (Web): 28 Dec 2016 Downloaded from http://pubs.acs.org on January 3, 2017

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ABSTRACT: Hydrogels are required to have high mechanical properties, biocompatibility, and an easy fabrication process for biomedical applications. Double network hydrogels, although strong, are limited because of the complicated preparation steps and toxic materials involved. In this study, we report a simple method to

prepare

tough,

in-situ

forming

PEG/Agarose

double

network

(PEG/Agarose DN) hydrogels with good biocompatibility. The hydrogels display excellent mechanical strength. Because of the easily in-situ forming method, the resulting hydrogels can be molded into any form as need. In vitro and in vivo experiments illustrate that the hydrogels exhibit satisfactory biocompatibility, and cells can attach and spread on the hydrogels. Furthermore, the residual amino groups in the network can also be functionalized for various biomedical applications in tissue engineering and cell research.

INTRODUCTION

Hydrogels consisting of a large amount of water in their three-dimensional networks have attracted many attentions as drug release matrices, coating, tissue engineering scaffolds, and biosensors.1-5 However, most of hydrogels are generally mechanically soft or brittle, and they ought to be stronger and more functional to be applied in a complicated and practical way.6 A sea of efforts have been made to develop highly tough hydrogels including nanocomposite hydrogels, tetrapolyethylene glycol hydrogels (tetra-PEG hydrogels), sliding-ring hydrogels, hydrophobically associated hydrogels, ionically cross-linked hydrogels, hydrogen bonding or dipole−dipole 2 ACS Paragon Plus Environment

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enhanced hydrogels, and double-network hydrogels.7-15 Among these, double network (DN) hydrogels consisting of two cross-linked networks are the strongest synthetic soft materials with failure compressive nominal stress 20−60 MPa, and strain 90−95%.16 However, the construction of DN hydrogels usually involved multi-step free radical polymerization processes, including forming of the first network, swelling of the first network, diffusion of second monomers, and forming of the second network. The multi-step methods have some limitations: (i) these fabrication steps are tedious; (ii) the diffusion steps involved make molar ratio of the components randomly distributed and decrease the repeatability; (iii) it is difficult to fabricate hydrogels with different complex shapes because of the multi-steps; and (iv) most of the DN hydrogels use the toxic initiators and acrylamide starting materials, which prevents their use in the biological sciences.16-19 Some efforts have been made to prepare DN hydrogels in a one-pot method.12,17,20-22 Chen et al. developed a one-pot synthesis of highly recoverable DN hydrogels using thermoreversible sol-gel polysaccharide.17 Truong et al. used a dual-click approach to form tough in-situ hydrogels.16 Ionically and covalently crosslinking method has also been combined to achieve the one-pot goal.20 However, these methods still involve free radical polymerization17,20, require long preparation time and use toxic acrylamide materials17,20 or materials needing complicated synthesis16.

Different from DN hydrogels, tetra-PEG hydrogels have high mechanical strength because of its homogeneous structure.7,23-25 Because PEG is essentially nonimmunogenic, antifouling, and nontoxic, the resulting tetra-PEG hydrogels are 3 ACS Paragon Plus Environment

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biocompatible.26 What’s more, the tetra-PEG hydrogels could be formed easily just by mixing the two precursor solutions. These properties make tetra-PEG hydrogels potential biomaterials for biomedical applications such as tissue engineering, wound dressing and anti-fouling materials.26-29 What’s more, PEGs with different arms and functional groups are already commercially available.

Herein, to overcome the challenges for the synthesis of biocompatible DN hydrogels, we report a simple method to prepare tough, in-situ forming PEG/Agarose double network (PEG/Agarose DN) hydrogels (Scheme 1). By combining tetra-PEG and agarose, the resulting PEG/Agarose DN hydrogels display excellent mechanical strength. Because of the easily in-situ forming method, the hydrogels can be molded into any form as need. What’s more, the hydrogels exhibit satisfactory biocompatibility both in vitro and in vivo, on which cells can adhere and grow. The residual amino groups in the network may be functionalized for different applications.

Scheme 1. Schematic of the PEG/Agarose DN hydrogel systems formed by mixing 4-arm-PEG-NH2 and 4-arm-PEG-NHS in 2% agarose solutions using a dual syringe.

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EXPERIMENTAL SECTION

Materials. 4-Arm poly(ethylene glycol) succinimidyl (4-arm-PEG-NHS, Mw=20 kDa, Mw/Mn=1.03) and 4-arm poly(ethylene glycol) amine (4-arm-PEG-NH2, Mw=20 kDa, Mw/Mn=1.03) were purchased from SINOPEG, China. Agarose was purchased from ENERGY CHEMICAL. All chemicals were used as received. All other chemicals were of analytical grade.

Preparation of Hydrogels. PEG/Agarose hydrogels were prepared in the phosphate buffer solution (PBS) (pH 7.4). Briefly, agarose (200 mg, melting point 120 oC) was added into a tube with 10 mL PBS and heated to 120 oC in an oil bath. After heating for several minutes, the transparent agarose solution was cooled to 50 oC. Then precursor solution 1 was prepared by dissolving 4-arm-PEG-NH2 with the agarose solution in a sample bottle, while precursor solution 2 was prepared by dissolving 4-arm-PEG-NHS in another sample bottle with the agarose solution. By using a dual syringe, the same volume of precursor solution 1 and 2 were simultaneously injected into the molds and then cooled down at room temperature to form hydrogels. The syringe and molds were heated to 50 oC by the oven before use. Similarly, pristine PEG hydrogels were prepared by mixing 4-arm-PEG-NH2 and 4-arm-PEG-NHS without addition of agarose.

Mechanical Testing. Hydrogels were prepared in cylindroid molds (15 mm in diameter and 7.5 mm in height). The compression testing was performed using a universal tensile machine (3365 Instron, USA). The dimensions of each sample were 5 ACS Paragon Plus Environment

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measured using a digital caliper before testing. The rate of compression testing was set at 1 mm/min until the sample fractured. The PEG hydrogels without agarose were used as the control. Every kind of hydrogels was tested for 5 times.

Scanning Electron Microscopy (SEM). Hydrogels were prepared and then freeze-dried at -50 oC for 24 hours. Then the dried samples were carefully stuck onto the conducting resin with double-sided adhesive and were sputter-coated with a thin layer of Pt for 120 s to make the sample conductive before testing. Field emission scanning electron microscopy (SEM) images were obtained at acceleration voltage of 5 kV on a JSM-6700F microscope (JEOL, Japan).

Fourier Transform Infrared (FTIR) Spectra. The PEG/Agarose DN hydrogels were prepared according to methods described above and then freeze-dried at -50 oC for 24 hours. The tetra-PEG hydrogels and agarose were used as the control samples. Fourier transform infrared (FTIR) spectra of the dried samples were obtained using TENSOR-27 spectrometer (Bruker, German) in the frequency range 4000-400 cm-1 at a resolution of 2 cm-1 with a total of 32 scans by the potassium bromide tableting technique.

Dynamic Light Scattering Measurements. The dynamic light scattering measurements were performed on the 3D LS Spectrometer from LS Instruments (Switzerland) consisting of a goniometer system with the incident light (Lumentum 114/P HeNe laser 21 mW) of 632.8 nm according to method reported by others.30 The measurements are conducted at a 90o angle with the temperature of 50 oC. The 6 ACS Paragon Plus Environment

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measured samples are prepared by injecting the 4-arm-PEG-NH2 and 4-arm-PEGNHS agarose solutions into the sample cells and kept at 50 oC until being measured.

Cell Viability. Cell viability was measured using quantitative MTT cytotoxicity assay and was assessed by contacting extracts of the hydrogels. 3T3 mouse fibroblasts were suspended in cell culture medium and seeded into 96-well microculture plates with a density of 1 × 104 cells/ 100 μL/ well and incubated for 24 h at 37 oC in a 5% CO2 humidified incubator to obtain a monolayer of cells. Cell medium was replaced with hydrogel extracts and further incubated for an additional time (48 h or 96 h). The sample solution was removed and the cells were incubated with 50 μL of 1 mg/mL of MTT in PBS solution for 2 h. Finally, the PBS solution was replaced with 100 μL of DMSO to dissolve formazan, and the absorbance of the DMSO solution was detected at 570 nm (reference 650 nm). The relative cell viability was calculated as the ratio between the mean absorbance value of the sample and that of cells cultured in the medium. Samples with relative cell viability less than 70% were deemed to be cytotoxic. For each sample, 6 independent cultures were prepared and cytotoxicity test was repeated 3 times for each culture.

Cell Attachment Experiments. The gel samples were placed in 24-well plates and incubated with 500 μL of 1× 105 cells/well suspension of 3T3 mouse fibroblasts at 37 °C and 5% CO2. After 24 h substrates were rinsed with PBS and fresh medium to remove unattached cells. Cells were digested from the substrates and counted using a cell counter. Cells on the substrates were stained with 2% FDA solution and fixed 7 ACS Paragon Plus Environment

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with 2.5% glutaraldehyde solution. Fluorescence images were taken with a laser confocal microscopy.

In vivo Biocompatibility. Healthy, weight-matched Sprague−Dawley rats of two months old obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. were used in this experiment. Tetra-PEG and PEG/Agarose DN hydrogels were molded into discs with the diameter of 5 mm and the thickness of 1.5 mm and were bilaterally implanted subcutaneously in the backs of the rats along the dorsal midline. Before the implanting, hydrogels were disinfected by the ethanol-based sterilization. After 3, 7, 14 and 28 days of implanting, rats were sacrificed and hydrogels along with surrounding tissues were collected, after which the tissues were fixed in formalin for 3 days. Following fixation, the sample was embedded in paraffin, cut into 3-5 μm and stained with H&E and Masson’s trichrome staining. The histological imaging results were obtained by an Olympus microscope.

Functionalization of Hydrogels. Rhodamine B with carboxyl (COOH) was used to modify the hydrogels through amidation.31 The hydrogel was submerged in a CH3OH solution of rhodamine B (1 mg mL-1) for 12 h at 60 oC to allow complete reaction of the remaining amino functional groups within the hydrogel. Subsequently, the hydrogel was washed with excess water for 6 h with frequent water changes until no UV absorbance of rhodamine B in the water was observed by the ultraviolet absorption spectrum. Then, the hydrogels were freeze-dried at -50 oC for 24 hours and imaged by using a laser confocal microscope (excitation 554). 8 ACS Paragon Plus Environment

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RESULTS AND DISCUSSION

Synthesis and Characterization of Hydrogels. Our strategy to the easy preparation of DN hydrogels uses tetra-PEG hydrogels and the agarose (Scheme 1). Briefly, PEG/Agarose DN

hydrogels

can be

constructed

by simply mixing the

4-arm-PEG-NH2 agarose solution (50 oC) and the 4-arm-PEG-NHS agarose solution (50 oC), and then cooling down to room temperature. The tetra-PEG hydrogel forms a dense network and the agarose forms a loose network. By using a dual syringe, the DN hydrogels can be formed in any mold with desired shape and used as injectable materials of free-shapeable properties. The clover, snowflake, letters ‘ICCAS’ (in red) and heart-shaped hydrogels were manufactured easily (Figure 1A-E) by the injection method. The injectable properties make the hydrogels of potential interest for various biomedical applications such as drug delivery vehicles or tissue engineering matrices.32 Particularly, although the heart-shaped hydrogels endured large deformations, the hydrogel could quickly recover to its original shape and maintain the integrity (Figure 1D and 1E), demonstrating the excellent deformation-recovery ability of PEG/Agarose DN hydrogels.

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Figure 1. PEG/Agarose DN hydrogels can be shaped freely by using a dual syringe: (A) clover; (B) snowflake; (C) letters ‘ICCAS’. (D) Top view and side view (E) of a heart-shaped hydrogel, under pressing by a piece of glass and then removing the deformation force.

The obtained PEG/Agarose DN hydrogels showed excellent mechanical properties. The PEG/Agarose DN hydrogel with 10 wt% PEG had a maximum compressive stress of 28.8 MPa about 10 times higher than that of the corresponding tetra-PEG hydrogels (2.7 MPa) (Figure 2A). For the PEG contents to be 4, 6, 8 and 10 wt%, the fracture energies (Figure 2B) are 278.6 kJ/m3, 370.1 kJ/m3, 754.3 kJ/m3, and 822.6 kJ/m3 for PEG/Agarose DN hydrogels and 65.3 kJ/m3, 95.7 kJ/m3, 134.1 kJ/m3, and 115.9 kJ/m3 for tetra-PEG hydrogels respectively, clearly illustrating the enhanced mechanical properties. Figure 2C demonstrated that the fracture strain had been improved approximately from 91% to 98% after the formulation of the DN hydrogels which was further vividly confirmed by the pictures of the compression experiments in Figure 2D. The tetra-PEG hydrogel was broken at the the compress strain of 95%, while the PEG/Ag arose DN hydrogel was still unspoiled manifesting the elevated 10 ACS Paragon Plus Environment

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mechanical properties. These results clearly declare that the excellent mechanical properties of PEG/Agarose DN hydrogels which are superior to the corresponding tetra-PEG hydrogels.

Figure 2. Mechanical and physical properties of hydrogels. (A) Fracture stress of PEG/Agarose DN and tetra-PEG hydrogels. (B) Fracture energy of PEG/Agarose DN and tetra-PEG hydrogels. (C) Fracture strain of PEG/Agarose DN and tetra-PEG hydrogels. (D) The pictures of the compression tests showing that PEG/Agarose DN can withstand larger deformation than tetra-PEG hydrogels (The compression strain is 95%).

For tetra-PEG hydrogels in our experiments, the compression stress increased at first then decreased with the increasing of PEG concentration and the maximum compression stress was achieved when the concentration of PEG was 8 wt%, which accorded with the homogeneity changes reported by Sakai et al.7 However, unlike traditional tetra-PEG hydrogels, the PEG/Agarose DN hydrogels had an increased strength when the PEG content was increased from 4 wt% to 10 wt%. So we hypothesized that in the range of these PEG concentrations, the DN hydrogels had

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similar homogeneity which has been proved by the DLS tests. The data in Figure 3A demonstrates that, although the PEG concentrations are different, the PEG/Agarose DN hydrogels have almost equal scattering intensity indicating the same distributed polymer structure. So the defects in the hydrogels are nearly the same and with the increasing of the solid content, more chemical bonds formed in the hydrogel network, leading to higher mechanical properties. However, when the concentration of PEG became 12 wt%, the scattering intensity increased almost three times larger and macroscopic defects appeared (Figure 3B-F), leading to the failure of testing the mechanical properties. We deduce that this happened because too high PEG concentration led to too fast gelation speed, making the gelation happen before polymer could be distributed evenly. So in our next experiments, the PEG concentration was fixed at 10 wt% unless specially mentioned.

Figure 3. (A) The average scattering intensity of PEG/Agarose DN hydrogels under different PEG concentrations. The pictures of the PEG/Agarose DN hydrogels when the PEG concentrations are (B) 4 wt%, (C) 6 wt%, (D) 8 wt%, (E) 10 wt%, and (F) 12 wt%.

To reveal the energy dissipation capacity of the PEG/Agarose DN and tetra-PEG hydrogels, the stress-strain curves of the two hydrogels were compared by using the cyclic loading experiments. Figure 4 showed that the PEG/Agarose DN hydrogels had 12 ACS Paragon Plus Environment

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a larger hysteresis loop than the tetra-PEG hydrogels in the first cycle, confirming more efficient energy dissipation of PEG/Agarose DN hydrogels. Then the second loading–unloading curves were immediately conducted and the hysteresis loop of PEG/Agarose DN hydrogels became smaller, indicating the softening occurrence. In contrast, tetra-PEG hydrogels showed overlapped hysteresis loops for the two cycles, demonstrating a typical rubber elastic behavior. The cyclic loading experiments results robustly illustrated that incorporating agarose into tetra-PEG hydrogels indeed improved the mechanical properties of the PEG/Agarose DN hydrogels. We deduce that when the PEG/Agarose DN hydrogels are compressed, the agarose loose network ruptures into small clusters and dissipates energy, contributing to the larger hysteresis loop, which has already been reported by the literature.17

Figure 4. The cyclic loading experiments of (A) PEG/Agarose DN hydrogels and (B) tetra-PEG hydrogels to 80% strain. (The concentration of PEG is 10 wt%)

SEM was further used to characterize the enhancement mechanism of the PEG/Agarose DN hydrogels. Figure 5A and Figure 5B revealed that the tetra-PEG hydrogels had homogeneous surface structure with thick texture while the agarose had heterogeneous and porous surface structure. The PEG/Agarose DN hydrogel contained a morphology that most closely resembled that of tetra-PEG hydrogels, but 13 ACS Paragon Plus Environment

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had slim and fine texture (Figure 5C). PEG tends to gather together to crystallize, resulting the thick texture of tetra-PEG hydrogels.6 After agarose hydrogels are introduced, they interact with tetra-PEG hydrogels through H-bond between hydroxyl groups of agarose and ether groups of PEG,33 thus the crystallization of PEG weakened. The interaction between the two networks was further proved by the FTIR spectra. As is shown in Figure 5D, the O-H stretching in agarose was increased from 3357 cm-1 to 3466 cm-1, indicating the H-bonding interactions. The interactions between the two networks might significantly increase the toughness, as reported in other systems.10,34

Figure 5. SEM images of (A) tetra-PEG, (B) agarose and (C) PEG/Agarose DN hydrogels. (D) FTIR spectra of PEG/Agarose DN, tetra-PEG and agarose hydrogels. The characteristic peaks at 3466 and 3357 cm-1 referred to O-H stretching of PEG/Agarose hydrogel and agarose.

Cell experiments. Unlike traditional DN hydrogels containing toxic initiator and acrylamide, the DN hydrogels in our work are constructed by FDA-approved PEG and agarose. To clearly illustrate the cytotoxicity, the 3T3 mouse fibroblasts were 14 ACS Paragon Plus Environment

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cultured with exacts from the DN hydrogels for 2 and 4 days. As shown in Figure 6A, the cell viability remained over 90% when the concentrations of the hydrogels were up to 20 mg/mL, indicating the good biocompatibility of the DN hydrogels.

Figure 6. (A) Cytotoxicity of PEG/Agarose DN hydrogels in 3T3 fibroblasts after incubation for 1 and 3 days. (B) The amount of 3T3 fibroblasts 1, 2 and 3 days after seeding on tetra-PEG and PEG/Agarose DN hydrogels. (Values were presented as mean ±standard deviation, n = 5)

Cell culture on the surface of hydrogels has been widely used for basic research and preclinical studies, such as chondrocyte differentiation study, metastatic cell behaviors and so on.35-37 However, cells are difficult to attach on tetra-PEG hydrogels because of anti-fouling properties of PEG. Introduction of agarose which is a favorable substrate for cell growth will improve the cell attachment ability.38 Figure 15 ACS Paragon Plus Environment

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6B affirmed that the cells attaching on the surface of PEG/Agarose DN hydrogels were far more than that on tetra-PEG hydrogels. As the cell culture time increased, 3T3 mouse fibroblasts cells gradually proliferated on the PEG/Agarose DN hydrogels. The laser confocal microscopy and SEM were further used to detect the configuration of cells attaching on the hydrogels. As shown in Figure 7A and Figure 7B, after 3 days of cell culture, few cells could attach on tetra-PEG hydrogels and they were all in an unstretched state, demonstrating that tetra-PEG is not a proper material for the growth of the cells. In contrast, well-stretched and vigorous cells were observed on PEG/Agarose DN hydrogels, illustrating the enhanced ability to support cell growth (Figure 7A and 7C). The cell attachment experiments indicate that the PEG/Agarose DN hydrogels may be promising biomaterials for applications including cell therapy, stem cell research, in vitro cell diagnostics, tissue engineering and so on.39-41

Figure 7. (A) The laser confocal microscopy images of 3T3 mouse fibroblasts cells cultured on

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tetra-PEG or PEG/Agarose DN hydrogels for 3 days (bar scale = 40 μm). SEM images of freeze-dried (B) tetra-PEG and (C) PEG/Agarose DN hydrogels after being incubated with 3T3 fibroblasts cells for 3 days (cells are surrounded by the red dotted line).

In vivo biocompatibility. To further demonstrate the biocompatibility, PEG/Agarose DN hydrogels and tetra-PEG hydrogels were implanted subcutaneously into the rat for various exposure times. The H&E staining results were shown in Figure 8. Macrophages and monocytes were observed on day 3 for both PEG/Agarose DN and tetra-PEG hydrogels, indicating that there existed an inflammatory response. On day 7, the reduced inflammatory response was observed with PEG/Agarose DN and PEG hydrogels, indicating that both of the hydrogels would not lead to persistent inflammation. After 28 days, the number of inflammatory cells for both groups was negligible and the inflammatory response had resolved with the two hydrogels. The decreased inflammatory response of PEG/Agarose DN hydrogels is in agreement with the good biocompatibility of PEG and agarose.

Although H&E staining showed that there was no obvious distinction between the two hydrogels in inflammatory response, Masson staining revealed different results.

After two weeks of implantation, the formation of a cellular infiltration layer

composed of macrophages and fibroblasts between the hydrogels and the tissue was observed for both hydrogels (Figure 9). However, for tetra-PEG hydrogels, the infiltration layer was more compacted and thicker, indicating a more serious foreign-body reaction. After 28 days of implanting, a reduced infiltration layer was 17 ACS Paragon Plus Environment

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observed for tetra-PEG hydrogels, but that of PEG/Agarose DN hydrogels was nearly invisible indicating a reduced inflammatory response arising from the agarose. Meanwhile, the fibrous capsule around PEG/Agarose DN hydrogels was more densely compacted than that around tetra-PEG hydrogels. This may result from the enhanced activation and proliferation of fibroblasts because of the incorporation of agarose, which increased collagen production in the tissues surrounding the hydrogel.42 The Masson staining results indicated that there was a reduced foreign-body reaction to the PEG/Agarose DN hydrogels and incorporating of agarose accelerated the fibroblast proliferation.

Figure 8. H&E histological results of (A, C, E) tetra-PEG hydrogels and (B, D, F) PEG/Agarose DN hydrogels 3 (A, B), 7 (C, D) and 28 (E, F) days after implantation, respectively. The red

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triangles indicate neutrophils. The arrows indicate fibroblast. The asterisks indicate the hydrogels. (bar scale = 400 μm)

Figure 9. Masson histological results of (A, C) tetra-PEG hydrogels and (B, D) PEG/Agarose DN hydrogels 14 (A, B) and 28 (C, D) days after implantation, respectively. (bar scale = 400 μm). “Ir”, infiltration layer; “Fc”, fibrous capsule.

Function of the hydrogel. It is well-documented that amino is a popular functional group that can be easily modified for various biomedical applications in drug delivery, tissue engineering, fluorescence probe and cancer research.28,43-46. PEG tends to gather together in solutions, so unreacted amino groups resulting from aggregation exist in the hydrogel, which can be used for postfunctionalization of the PEG/Agarose DN hydrogels. To clearly illustrate that, fluorescent rhodamine-COOH was grafted onto the hydrogel through COOH-NH2 amidation in ethanol. After being freeze-dried, the PEG/Agarose DN hydrogels were tested by laser confocal microscopy (Figure 10). The strong red color of the rhodamine observed along the texture of the hydrogels

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demonstrated the successful functionalization approaches. The amino can also react with many other functional groups such as succinimide active carboxyl, aldehyde group, derivatives of methacrylate and so on. By using these reactions, the PEG/Agarose DN hydrogels can be easily modified with functional molecules or drugs.

Figure 10. Laser confocal microscopy images of PEG/Agarose DN hydrogels after postfunctionalization by rhodamine-COOH. (bar scale = 20 μm)

CONCLUSION

In this study, we reported a simple in-situ method to construct tough PEG/Agarose DN hydrogels with designable shapes and injectable properties. The in vitro cell experiments and in vivo subcutaneous implantation experiments indicate that PEG/Agarose DN hydrogels have good biocompatibility, on which cells can attach and spread. What’s more, because of the residual NH2 in the hydrogel network, the 20 ACS Paragon Plus Environment

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hydrogels can be postfunctionalized for biomedical applications in cell research and tissue engineering.

AUTHOR INFORMATION

Corresponding Author

*Email: [email protected].

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Notes

The authors declare no competing financial interests.

ACKNOWLEDGMENT

We are grateful to NSFC (21674120, 81630056, 21504096, 51573195, and 21474115), MOST (2014CB932200 and 2014BAI11B04), and the “Young Thousand Talents Program” for financial support.

REFERENCES (1) Ha, W.; Yu, J.; Song, X. Y.; Chen, J.; Shi, Y. P. Tunable Temperature-Responsive Supramolecular Hydrogels Formed by Prodrugs as a Codelivery System. ACS Appl. Mater. Interfaces 2014, 6, 10623-10630.

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