PVA Composite Hydrogels with Jellyfish Gel

Jul 31, 2014 - Construction of Chitin/PVA Composite Hydrogels with Jellyfish Gel-Like ... †Department of Chemistry and ‡College of Basic Medical S...
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Construction of Chitin/PVA Composite Hydrogels with Jellyfish GelLike Structure and Their Biocompatibility Meng He,† Zhenggang Wang,† Yan Cao,† Yanteng Zhao,‡ Bo Duan,† Yun Chen,‡ Min Xu,§ and Lina Zhang*,† †

Department of Chemistry and ‡College of Basic Medical Science, Wuhan University, Wuhan, 430072, China § Department of Physics, East China Normal University, Shanghai, 200062, China S Supporting Information *

ABSTRACT: High strength chitin/poly(vinyl alcohol) (PVA) composite hydrogels (RCP) were constructed by adding PVA into chitin dissolved in a NaOH/urea aqueous solution, and then by cross-linking with epichlorohydrin (ECH) and freezing−thawing process. The RCP hydrogels were characterized by field emission scanning electron microscopy, FTIR, differential scanning calorimetry, solid-state 13C NMR, wideangle X-ray diffraction, and compressive test. The results revealed that the repeated freezing/thawing cycles induced the bicrosslinked networks consisted of chitin and PVA crystals in the composite gels. Interestingly, a jellyfish gel-like structure occurred in the RCP75 gel with 25 wt % PVA content in which the amorphous and crystalline PVA were immobilized tightly in the chitin matrix through hydrogen bonding interaction. The freezing/thawing cycles played an important role in the formation of the layered porous PVA networks and the tight combining of PVA with the pore wall of chitin. The mechanical properties of RCP75 were much higher than the other RCP gels, and the compressive strength was 20× higher than that of pure chitin gels, as a result of broadly dispersing stress caused by the orderly multilayered networks. Furthermore, the cell culture tests indicated that the chitin/PVA composite hydrogels exhibited excellent biocompatibility and safety, showing potential applications in the field of tissue engineering.

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derived from the oligo squid pen; γ-Chitin exists in cocoon fibers of the Ptinus beetle and the stomach of Loligo.10 However, the most widely available α-chitin has been seldom utilized and studied in the material field because of its poor solubility. Only a few solvents such as LiCl/N,N-dimethylacetamide (DMAc) mixtures, CaCl2/CH3OH and ionic liquids can dissolve chitin for the preparation of a few materials.11−14 In our laboratory, chitin has been successfully dissolved in a NaOH/urea aqueous solution at low temperature to fabricate novel hydrogels, aerogels, films, and fibers.7,15−17 These chitinbased materials regenerated directly from its solution have been proven to promote adhesion and proliferation of cells, showing good biocompatibility and no cytotoxicity. It is not hard to imagine that chitin-based material is a good candidate in the tissue engineering field, because chitin is an important natural polymer with biological functions.18 However, the chitin gels at low concentrations were still fragile, so an improvement of their mechanical properties is essential for their successful applications. As is known to us, the mechanical properties of biopolymers can be improved by being cross-linked or blending with nanoparticles, nanowhiskers, or other polymers.6,19−21 Poly(vinyl alcohol) (PVA) is a nontoxic, water-soluble,

INTRODUCTION Hydrogels are composed of a three-dimensional hydrophilic polymer network, which can absorb a large amount of water without dissolution. Recently, hydrogels have been studied extensively for biomedical applications, because their structures are similar to the macromolecular-based human tissue.1 Due to the advantages such as good biocompatibility and biodegradability,2 biopolymer-based hydrogels, including collagen,3 cellulose,4 gelatin,5 chitin6,7 and chitosan8 are of considerable interest for applications in the biomedical field. However, the strength of most biopolymer-based hydrogels is relative weak, which largely restricts their applications as biomaterials. Thus, the construction of the biopolymer materials with high mechanical strength is essential for the successful applications in the tissue engineering field. It is noted that “The Chemistry of Natural Resources” was the theme of the 241st American Chemical Society National Meeting. Recently, the utilization of raw materials from bioresources such as chitin and cellulose have attracted increasing attentions because these raw materials are safe, sustainable, biocompatible, and biodegradable.7 As one of the most abundant biomasses, chitin is perhaps the least exploited source to date.9 Chitin exhibits three different crystalline polymorphic forms depending on the source: α-chitin, β-chitin, and γ-chitin.10 The α-chitin has been found widely in arthropods, fungi, and the cysts of Entamoea; β-chitin is © XXXX American Chemical Society

Received: June 5, 2014 Revised: July 31, 2014

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ratios at 0 °C under mechanical agitation, and the bubbles were removed by ultrasonication at low temperature using an ice bath. The resultant chitin/PVA solutions were kept at 25 °C for 12 h to form gels, which were partially chemically cross-linked. Subsequently, the gels were frozen at −20 °C for 24 h and thawed at room temperature for five cycles to fully form PVA crystals and physically cross-link the PVA.23,26 The preparation process is shown in Scheme S1. The gels were taken out carefully and washed with distilled water to completely remove alkali, urea, and excess ECH. The total concentration of chitin and PVA was about 6 wt %, and the resultant chitin/PVA composite hydrogels (RCP) were coded as RCP100, RCP83, RCP79, RCP75, RCP70, RCP50, RCP25, and RCP0 according to the weight percentage of chitin (from 100 to 0 wt %), respectively. Characterization. The field emission scanning electron microscopy (FESEM, SIRION TMP, FEI) was used for morphology observations of the chitin powder and gels at an accelerating voltage of 20 kV. The gels were frozen in liquid nitrogen, immediately snapped and then freeze-dried. The fracture surface (cross-section) of the gels was sputtered with gold, observed, and then photographed. Wide angle X-ray diffraction (WAXD) measurements were carried out on a WAXD diffractometer (D8-Advance, Bruker, U.S.A.). The patterns with Cu Kα radiation (λ = 0.15406 nm) at 40 kV and 30 mA were recorded in the region of 2θ from 4 to 40°. The freeze-dried gels were cut into powder and dried in a vacuum oven at 60 °C for 48 h before testing. The crystallinity Xc (%) of the samples was estimated according to the Lorentz−Gaussian peak separation method, and was then calculated by using the following equation33

biocompatible, and biodegradable synthetic polymer, and the elastic PVA hydrogel can be fabricated by the freezing−thawing process.22−24 It is worth noting that the intermolecular interaction of PVA can yield small crystalline nuclei, and the freezing/thawing cycles can promote crystallization to crosslink PVA chains and form a dense three-dimensional structure.24−26 PVA has been blended with β-chitin,27,28 cellulose,29 gellan,30 and chitosan31 to construct gels or films with good mechanical properties and biocompatibility. In view of the above circumstances, a worthwhile endeavor would be to construct high strength hydrogels by using hydrophilic PVA and chitin, in which a multilayered porous structure can be formed through hydrogen bonds and chemical cross-linking, as well as microphase separation caused by the crystallization. In the present work, α-chitin powder was dissolved in NaOH/urea aqueous solution by a freezing− thawing process, and then composite hydrogels were prepared by adding PVA solution into 6 wt % α-chitin solution. The chitin and PVA in the blend solutions were partially crosslinked with ECH. The freezing−thawing process was used to promote the formation of PVA crystals and a combination of chitin with PVA, creating a layered porous structure of the composite hydrogels. The whole preparation process was environmentally friendly because there were no organic solvents and toxic chemicals used. The structure of the resultant chitin/PVA hydrogels was characterized, and their mechanical strength and biocompatibility were also studied to evaluate the potential applications as biomaterials.

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xc =

∫0

∞

s 2Ic(s)ds /

∫0

∞

s 2I(s)ds

(1)

where s is the magnitude of the reciprocal-lattice vector and is given by s = (2 sin θ)/λ, λ is the X-ray wavelength, I(s) is the intensity of the coherent X-ray scattering from a specimen (both crystalline and amorphous), Ic(s) is the intensity of the coherent X-ray scattering from the crystalline region. Solid-state 13C NMR spectra of freeze-dried hydrogels were recorded on a BRUKER AVANCE III spectrometer operated at a 13 C frequency of 75 MHz using the combined technique of magic angle spinning (MAS) and cross-polarization. The spinning speed was set at 5 kHz for all samples. The contact time was 3 ms, the acquisition time 50 ms, and the recycle delay 3s. A typical number of 1024 scans were acquired for each spectrum. FT-IR spectra were recorded on a FT-IR spectrometer (1600, Perkin−Elmer Co., MA) in the wavelength range from 4000 to 400 cm−1. The freeze-dried gels were cut into powder and then vacuum-dried for 24 h before measurement. The test specimens were prepared by the KBr disk method. Differential scanning calorimetry (DSC) experiments were carried out on a DSC 200PC (NETZSCH, Germany) with a heating rate of 10 °C/min. The temperature was controlled with liquid nitrogen, and the freeze-dried hydrogels were put in a tightly sealed aluminum cell. The hydrogel properties at equilibrium in water were affected by both the chemical composition and the gelation procedures. To reach the swelling equilibrium, the samples were incubated in distilled water for over 24 h at 37 °C. The equilibrium swelling ratio (ESR) was calculated as

EXPERIMENTAL SECTION

Materials. Chitin powder was purchased from Jinke Chitin Co. Ltd. (Zhejiang, China), and it was purified by treating the raw material in 5 wt % NaOH, 7 wt % HCl, 5 wt % NaOH for 12 h, and bleached in 4 wt % H2O2 at pH = 8 (pH meter, BEBCH/PHS-25) and 75 °C step by step. The resultant chitin powder was washed repeatedly to neutral with distilled water after each step. The purified chitin powder was dried at 60 °C and kept in a desiccator, coded as PCP. The degree of acetylation (DA) for PCP was calculated by FTIR spectra to be 87% according to the value of (A1740/A1030) × 100%, which is the ratio of the absorption bands at 1740 and 1030 cm−1, respectively.32 The weight-average molecular weight (Mw), measured by dynamic light scattering (DLS, ALV/CGS-8F, ALV, Germany) in 5% LiCl/DMAc (w/w), was 3.0 × 105. 3,3′-Dioctadecyloxacarbocyanineperchlorate (DiO) for cell membrane staining was purchased from Beyotime Institute of Biotechnology (Jiangsu, China). NaOH and urea (Shanghai Chemical Reagent Co. Ltd., China) were used as received. Epichlorohydrin (ECH, Chemical Agents, Ltd. Co. Shanghai, China; density, 1.18 g/mL) was of analytical grade and used without further purification. PVA was purchased from a commercial source, with an average degree of polymerization (DP) of 1750 ± 50. For the cell culture test and 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) assay, L929 cells were obtained from China Typical Culture Center (Wuhan University) and were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Sigma), supplemented with 4 mM L-glutamine, 10% fetal bovine serum (FBS), and antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin) at 37 °C in a humidified air atmosphere containing 5% CO2. Madin Darby canine kidney (MDKC) cells were maintained in DMEM-high glucose (HG-DMEM) supplemented with 5% FBS, 100 U/mL of penicillin, and 100 mg/mL of streptomycin at 37 °C in a humidified atmosphere with 5% CO2. Preparation of Chitin/PVA Hydrogels. The 6 wt % chitin solution was prepared by dissolving the purified chitin powder in 11 wt % NaOH/4 wt % urea solvent through the freezing/thawing method. The 6 wt % PVA aqueous solution and ECH (1 g for 20 mL solutions) were added dropwise into the chitin solution with different volume

ESR =

Ws‐Wd Wd

(2)

where Ws is the weight of the swollen gel at 37 °C and Wd is the weight of the gel at the dry state. The compression strength of the gels was measured on a universal tensile tester (CMT 6503, Shenzhen SANS Test machine Co. Ltd., Shenzhen, China) according to ISO527−3−1995 (E) at a speed of 1 mm/min. Because the strength data are related to the environmental temperature and humidity, these data were obtained under the same conditions. Cell Viability Assay. The RCP hydrogels were cut into powder and sterilized by autoclaving and then used to prepare the extract. According to ISO 10993−5, a cell line of L929 was resuspended in the culture medium and plated (200 μL/well) into 96-well micrometer plates at 1 × 104 cells/well, which were incubated at 37 °C in a 5% B

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Figure 1. Solid-state 13C NMR of the samples: PCP powder (a), RCP100 (b), RCP0 (c), RCP75 (d), RCP50 (e), and RCP25 (f).

Figure 2. FTIR (a) and WAXD spectra (b) of the PCP powder, as well as RCP100, RCP75, RCP50, RCP25, and RCP0 gels. CO2 atmosphere for 24 h. The medium was then replaced by 50 μL/ well sterilized extract using the culture medium itself as a control. After incubating for 24, 48, and 72 h, the cells were treated with 20 μL/well of MTT (5 mg/mL in PBS filtered for sterilization) to reach a final concentration of 0.5 mg MTT per mL and incubated for a further 4 h at 37 °C in a 5% CO2 atmosphere. At this stage, the MTT was removed and 150 μL/well of dimethyl sulfoxide (DMSO) was added to dissolve the formazan crystals. The plates were placed in an incubator at 37 °C to shake for 10 min. The absorbance values were read in triplicate against a reagent blank at a test wavelength of 570 nm (Tecan GENios, Tecan Austria GmbH, Salzburg, Austria). Cell viability was calculated using the following equation:

cell viability(%) = A test /Acontrol × 100%

PBS. The RCP75 gels with cells treated by DiO were observed and photographed using a fluorescence microscope (Nikon ECLIPSETE 2000-U) at the corresponding excitation wavelength (Ex/Em = 484/ 501 nm). For MDKC cell culture, the RCP75 slices were put into 24well plates after sterilization. Subsequently, the MDKC cells were seeded into 24-well plates (2 × 105 cells/well) with or without the materials and incubated for 48 h before taking photographs. The 24well plates without the RCP75 gels for cell culture were denoted as the control.

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RESULTS AND DISCUSSION Cross-Linking Interactions in RCP Gels. To remove the impurities and proteins in the raw chitin powder from the industrial products, the chitin purification process was performed to give PCP. The photographs of the raw chitin powder and PCP (Figure S1) showed that the raw chitin powder was a little yellow as a result of the existence of pigments and impurities such as protein. After purification, the resultant PCP was white on the whole. Moreover, the PCP powder displayed the homogeneous surface morphology, compared with that of the raw chitin powder (see Figure S2). This suggested that the protein and other impurities in the native chitin34 were removed successfully. Thus, the purified chitin powder was suitable for the preparation of biomaterials. The cross-polarization/magic-angle spinning (CP/MAS) 13C NMR spectra of the PCP powder and the RCP gels are shown in Figure 1, and their chemical shifts are listed in Table S1 in

(3)

where Atest and Acontrol were the absorbance values of the test and control groups, respectively. L929 and MDKC Cell Culture. The RCP75 gels were cut into cylindrical slices with a thickness of about 0.1 cm, sterilized by autoclaving, soaked in the 0.1 mg/mL polylysine for 0.5 h, and then transferred to the bottom of 24-well plastic culture plates. A fresh fibroblast cells (L929 cell) suspension (1 mL) was added to each triplicate sample, and the RCP75 gels were submerged totally with the suspension. After 3−4 h, the samples were supplemented with 1 mL of DMEM containing 10% fetal bovine serum (FBS). The L929 cells, with a cell density of 2 × 105 cells cm−3, were cocultured with RCP75 gel in an incubator for 2 days, respectively. The cell membranes were stained by using 3,3′-dioctadecyloxacarbocy anineperchlorate (DiO) dye as follows. The RCP75 gels with cultured L929 cells were immersed in a 10 μM DiO solution at 37 °C and then washed 3× with C

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the Supporting Information. The 13C NMR spectrum of the purified chitin displayed eight main peaks: sharp peaks at 174.0 and 23.5 ppm were assigned to the carbonyl and methyl carbons, respectively; the peaks at 104.5, 83.9, 76.0, 73.6, 61.3, and 58.1 ppm were ascribable to the resonances of C1, C4, C5, C3, C6, and C2 on the N-acetyl-D-glucosamine unit of chitin, respectively. The chemical shifts of the carbon atoms confirmed that PCP was α-chitin (Figure 1a).35 In Figure 1b, the C6 peak (62.7 ppm) was broadened, and the peak at 75.5 ppm was the combination of C5 (76.0 ppm) and C3 (73.3 ppm) for chitin in the RCP100 hydrogels. These changes could be attributed to the cross-linking reaction between chitin and ECH.7 In the 13C CP/MAS NMR spectrum of PVA in the solid state (c), the 13C NMR signal for the CH carbon with three splitting peaks (77.2, 71.0, 64.7 ppm) was observed. The most downfield peak (peak I), the central peak (peak II), and the most upfield peak (peak III) come from the CH carbons, which could form two kinds of hydrogen bonds with or without a hydroxyl group for PVA, respectively.36,37 Interestingly, the observable chemical shifts of the chitin peaks (C1−C5) of the RCP 75, RCP 50, and RCP 25 gels shifted to a higher magnetic field than that of RCP 100 without PVA, demonstrating that the formation of the strong hydrogen bonds between chitin and PVA, leading to the increase of electron density around these carbons. However, the C6 peak of the RCP100, RCP 75, RCP 50, and RCP 25 gels shifted to a lower magnetic field than that of PCP, indicating the occurrence of the chemical cross-linking, which led to a decrease of the electron density of the carbon. Figure 2 shows the FTIR and WAXD spectra of the PCP powder and RCP gels. The O−H, N−H (asymmetric), and N− H (symmetric) stretching vibrations for the chitin powder were located at 3461, 3267, and 3106 cm−1, respectively, coinciding with the reported data of chitin.38 Absorption bands of OCNH around 1660, 1550, and 973 cm−1 were characteristic of chitin, which were assigned to the amide I, II, and III bonds, respectively.39 The amide I for the PCP powder was split into 1662 and 1625 cm−1 because of the intrasheet and intersheet hydrogen bonding for PCP (Figures 2a and S3). This result further confirmed that chitin in the PCP powder existed as the α-chitin crystalline form.10 The amide I for the RCP gels was around 1660 cm−1 and singlet, indicating that the cross-linking reaction broke the hydrogen bonds of chitin. The sharp band at 1378 cm−1 was assigned to the −CH3 symmetrical deformation mode, this band and the N−H stretching bands became weaker and disappeared gradually in the RCP gels with an increase in the PVA content.20 As shown in Figure 2b, there were six peaks at 2θ = 9.3, 12.7, 19.3, 20.6, 23.4, and 26.3° for PCP, which were assigned to (020), (021), (110), (120), (130), and (013) of Bragg reflections, respectively. This further confirmed the crystalline structure of α-chitin in the PCP powder.40 The crystallinity of α-chitin for PCP was about 75.8% according to eq 1. It was noted that these initial characteristic peaks of αchitin disappeared in the RCP gels, indicating a transition from the crystalline structure to amorphous states during the dissolution and the cross-linking process. The RCP0 gel had a clear peak at 19.8°, whereas it became weaker with a decrease of the PVA content in the gels from RCP25 to RCP100. Interestingly, the appearance of PVA crystal peak still existed in the RCP hydrogels, suggesting the existence of physical crosslinking caused by PVA in the RCP gels. In view of these results, there were two kinds of the interactions including chemical and physical cross-linkings in the chitin/PVA composite hydrogels, referred to as bicrosslinking.

Morphologies and Structure of RCP Gels. Figure 3 shows the photographs of the RCP100, RCP50, and RCP0 gels.

Figure 3. Photographs of the RCP100 (a), RCP50 (b), and RCP0 (c) gels.

Clearly, all of the composite hydrogels exhibited good processability and appearance. The RCP100 was the most transparent, showing a homogeneous structure as a result of the cross-linking network associated with ECH and chitin, similar to that reported by our work previously.7 The freezing− thawing process can induce crystallization of the abundant PVA crystallites, which act as knots of the gel network and ensure a high dimensional stability of the gel to create elastic properties.41 Thus, RCP0 composed of PVA without chitin was totally white and opaque, because most of PVA chains were cross-linked physically. It was worth noting that the RCP50 gel consisted of chitin and PVA (1:1) was semitransparent. This could be explained that the existence of partially physically cross-linked PVA in the chemically cross-linked chitin networks led to the microphase separation, resulting in optical loss from light reflections and refractions.31 The morphologies and the three-dimensional network structure of the gels can be well investigated by FESEM.42 The FESEM images of the cross-section for the freeze-dried RCP gels are shown in Figure 4. All of the gels exhibited a homogeneous porous structure with different pore sizes, showing certain miscibility between chitin and PVA. Interestingly, large pores existed in RCP100 (without PVA), and their

Figure 4. FESEM images of cross-section for the RCP100 (a), RCP83 (b), RCP75 (c), and RCP25 (d) gels. The blue arrows indicate the layer. D

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average pore size was about 30 μm. In this case, the microphase separation between chitin-rich phase and the chitin-lean phase during the cross-linking reaction could induce the macropore formation. However, there were two different types of pores in RCP83, the larger pores mainly consisted of the chitin and the smaller ones probably consisted of PVA, which was created through hydrogen bonding between PVA chains during the freezing−thawing process. In our findings, a little amount of PVA could form physically cross-linked mesh containing PVA crystallites, which were created during the freezing−thawing process and embedded in the large chitin pores. It has been reported the structure of freezing−thawing PVA hydrogels in terms of a porous polymer network where the crystals act as knots and the polymer segments ensure the connectivity all over the macroscopic gel sample filling with free water in the pores.43 Thus, layered porous structure of the chitin/PVA hydrogels appeared in the FESEM images in Figure 4b−d, and the PVA networks were immobilized in the chitin pores. Interestingly, a jellyfish gel-like structure appeared in RCP75, which was similar to the freeze-dried jellyfish mesogloea.44 Namely, when the PVA content was 1/4 (25 wt %), the RCP75 gel with orderly layered porous structure and small pore size of about 4 μm (Figure 4c) could be constructed as a result of the just occupation of the PVA crystals along the chitin pore wall to form the second kind of pores. Such regular jellyfish-like gel is very flexible and robust with high water content, which has a much higher mechanical strength than normal synthetic hydrogels due to its layered porous structure with pore walls.44 As indicated by Gong et al., a two-phase composite structure can be formed here with improved mechanical strength and toughness.45 The morphologies for RCP70 and RCP79 are shown in Figure S4, obviously, the layered structures appeared but they were not good compared with RCP75. This could be explained that a small amount of PVA could not form complete PVA networks, as shown in Figures 4b and Figure S4a, whereas excessive PVA content led to the smaller pore size and the formation of continuous accumulation of the PVA crystals and chains due to space hindrance (Figure 4d). Thus, both RCP 83 and RCP 25 displayed imperfect bicrosslinked structure. In our findings, the intransigent chitin acted as the main backbone, which combined tightly with the hydrophilic PVA to form layered micropores during the freezing−thawing process. The freezing/thawing cycles can not only improve the formation of PVA crystals,41 but can also induce the tight immobilization of PVA in the chitin pore wall through hydrogen bonds because the PVA chains could form dense and aligned network cross-linked by resultant PVA crystals during the freezing process,24−26 leading to the higher stability of bicrosslinked networks structure. It was not hard to imagine that the intermolecular hydrogen bonds could form easily and were stable at low temperature (especially freezing). The freezing/thawing cycles played an important role in the formation of the layered PVA networks and the tight combination of the PVA chains and crystals with the pore wall of chitin. The equilibrium swelling ratio (ESR) of RCP gels can also reflect the change in the pore size. The ESR values of the RCP gels are shown in Figure 5. The ESR value for RCP100 was 52.5, and it decreased gradually with an increase in the PVA content, indicating that the structure of RCP gels with PVA was denser than that of the pure chitin gel (RCP100). According to the analysis of these results, both the introduction of PVA and the freezing−thawing process led to the formation of a dense

Figure 5. Equilibrium swelling ratio of the gels at 37 °C in distilled water.

network structure of the RCP gels. Therefore, the RCP gels with different pore sizes from 300 nm to 30 μm and ESR values from 11.8 to 52.5 could be constructed by adjusting the ratio of chitin/PVA for various applications. In view of the above results, a schematic depiction of the layered porous structure with the bicrosslinked networks in the RCP75 gels is proposed in Scheme 1. The incomplete Scheme 1. Schematic Depiction of the Chemical CrossLinked RCP75 Gel (a) and Bicrosslinked RCP75 Gel through a Freezing−Thawing Process (b)

chemically cross-linked gel was constructed first, in which the chitin and PVA were partially chemical-cross-linked by ECH as well as physical-cross-linking, as shown in (a), supported by the results in Figures 1−3. The freezing−thawing process could promote PVA crystallization and combination of PVA with the chitin matrix through hydrogen bonds, because the intermolecular hydrogen bonds are more stable at low temperature than at room temperature.46,47 Thus, the tight connection of the micro- and nanopores from chitin and PVA appeared in the RCP75 to form an oriented architecture, leading to the formation of jellyfish gel-like structure during the freezing process (b), supported by the results in Figures 1−5. Therefore, an ordered bicrosslinking layered porous hydrogel (RCP75) was constructed successfully by using 75 wt % chitin and 25 wt % PVA, showing a jellyfish gel-like structure. In this case, a dense packing wall consisted of chitin and PVA as well as microphase separation caused by the crystalline ice and the PVA crystals occurred in RCP75 gels. Thermal Stability and Mechanical Properties of RCP Gels. Figure 6 displays the DSC curves of the gels from 100 to 260 °C. The RCP100 displayed no endothermic peaks as a E

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to broadly disperse stress near a crack tip, as indicated by Wang et al., ensuring that very large external loads are required to bring the force on crack tip up to that required for chain scission.44 Moreover, RCP70 and RCP79 also displayed much higher strength than RCP25, RCP50 and RCP83. This result further proved that the PVA (with 25% PVA content) could fully crystallize to form strong physical cross-linking, resulting in the jellyfish gel-like structure to significantly improve the mechanical strength. Therefore, high strength RCP gels were successfully constructed, and the layered porous structure with the bicrosslinked networks in the RCP gels played an important role in the improvement of the mechanical properties for chitin gels. The chitin-based composite hydrogels having good mechanical properties are of considerable interest for utilizations in the tissue engineering field, because their biocompatibility and biodegradability could appropriately meet the design criteria of tissue engineering. Biocompatibilities of RCP Gels. With excellent biocompatibility, antibacterial properties, and nontoxicity, chitin becomes one of the most promising candidates in biomaterials.17 However, the biocompatibility of the chitin/PVA composite hydrogels still needed to be evaluated after the ECH cross-linking and the freezing−thawing process. The cell cytotoxicity studies were performed to test their potential biomedical applications. Usually, the surface of the material can absorb extracellular protein when it is immersed in the cellculture medium, and then cells could adhere on its surface.49 The RCP75 gel with porous structure and excellent absorbing ability could absorb extracellular protein efficiently, providing the basis for cell adhesion. Figure 8 shows the experimental results of the cytotoxicity of the gels by MTT assay and the fluorescent image of L929 cells cultured on the RCP75 gel. The cell viability values on all gels were greater than 135%, indicating no cytotoxicity to the L929 cells. Moreover, fibroblast cells (L929 cell line) could adhere and proliferate on the RCP gels, showing good biocompatibility. The layered porous structure of the RCP gels was very important for biomaterials, because it can provide space for cell adhesion and spreading as well as nutriment permeability. MDKC is one kind of epithelial cells which grows and adheres to the surface. Thus, MDKC was used here to study the cell growth on the RCP75 gels and further evaluate the biocompatibility. Figure 9 shows the photographs of MDKC cells cultured on the RCP75 gel and the control after 48 h. Obviously, the MDKC cells could

Figure 6. DSC heating curves of the RCP100, RCP75, RCP25, and RCP0 gels; Scanning rate: 10 °C/min.

result of the chemically cross-linked structure. The RCP0 exhibited a critical PVA endothermic peak at 235 °C.48 Interestingly, there was a weak crystal melting endothermic peak at 228 °C for RCP75, indicating the existence of certain amount of PVA crystallites. This result confirmed that when PVA content was 1/4, it could form crystals at low temperature, which further supported the structure model in Scheme 1. Figure 7 shows the compressive stress−strain curves of the gels and the photographs of RCP75 gel. The RCP100 exhibited the lowest compressive strength (0.1 MPa) and strain at break (44%). With the addition of PVA, the mechanical properties of the RCP gels were improved, indicating the reinforcement role of PVA on chitin gels. The area under the compressive stress curves usually reflects the toughness. Clearly, the toughness of the RCP composite hydrogels increased with the addition of PVA, compared with RCP 100 without PVA. It was worth noting that the RCP75 gel exhibited the best mechanical properties (compressive strength of 2.1 MPa and strain at break of 80%), which were much higher than the other RCP gels. Obviously, the compressive strength of RCP75 was 20 times higher than that of the pure chitin gels, as a result of the reinforcement caused by the layered porous networks. This could be explained that RCP75 had a jellyfish gel-like structure

Figure 7. Compressive stress curves of the RCP100, RCP83, RCP79, RCP75, RCP70, RCP50, RCP25, and RCP0 gels (a), and the photographs for the normal (b) and twisted (c) RCP75. F

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Figure 8. Results of the cytotoxicity tests of the RCP100, RCP83, RCP75, RCP50, RCP25, and RCP0 gels (a) and the fluorescent image of L929 cells cultured on RCP75 gel for 48 h (b).

Figure 9. Optical photographs of MDKC cells cultured on the RCP75 gel (a) and control (b) for 2 days; magnification 40×.

by the ordered layered porous structure. Furthermore, the chitin/PVA hydrogels had excellent biocompatibility and safety. The chitin/PVA hydrogels would be of considerable interest for utilizations in the biomedical field, because its biocompatibility and biodegradability could appropriately meet the design criteria of tissue engineering.

adhere, spread and proliferate on the RCP75 gel well. Compared with the control, the MDKC cells cultured on RCP75 gel exhibited a normal state after a 48 h culture, which were more stereoscopic and clear due to the rough and uneven surface. This result was consistent with that from L929 cell culture, further confirming that the RCP75 gel could provide a matrix for cell adhesion and proliferation. As mentioned above, the combination of the chitin from arthropods in nature and biocompatible PVA endowed the RCP composite gels with good biocompatibility. Therefore, it can be expected that high strength and biocompatible RCP gels exhibit potential application in tissue engineering field.

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ASSOCIATED CONTENT

S Supporting Information *

The preparation process of RCP gels, SEM micrograph of the freeze-dried jellyfish, putative cross-linking reaction, photographs, and FESEM images of raw chitin powder and PCP powder, expanded FTIR spectrum of PCP powder, and FESEM images of cross-section for the RCP70 and RCP79 gels. This material is available free of charge via the Internet at http:// pubs.acs.org.

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CONCLUSIONS A novel chitin/PVA hydrogel with high strength and excellent biocompatibility was fabricated successfully from their blended solutions by cross-linking with ECH and treating with freezing−thawing process. When PVA content was 25 wt % for the RCP75 gel, a regular jellyfish gel-like structure occurred, as a result of immobilizing amorphous and crystalline PVA tightly in the chitin matrix through hydrogen-bonding interaction. The freezing/thawing cycles not only improved the formation of the PVA crystals, but also induced the tight combination of PVA with the chitin pore wall through hydrogen bonds, leading to the higher stability of the bicrosslinked networks. The RCP75 had higher compressive strength at breaking (2.1 MPa), and its mechanical properties were much higher than the other RCP gels and pure chitin gels as a result of the broadly dispersed stress near a crack tip caused

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AUTHOR INFORMATION

Corresponding Author

*Phone: +86-27-87219274. Fax: +86-27-68762005. E-mail: [email protected]; [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by National Basic Research Program of China (973 Program, 2010CB732203), the Major Program of National Natural Science Foundation of China (21334005), and the National Natural Science Foundation of China G

dx.doi.org/10.1021/bm500827q | Biomacromolecules XXXX, XXX, XXX−XXX

Biomacromolecules

Article

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(20874079, 81171480). We acknowledge Prof. Zhiling Zhang (Department of Chemistry, Wuhan University, China) for kindly providing the MDKC cells.

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