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graphene oxide hydrogel
Accepted Manuscript Title: Preparation and characterization of Oxidized Konjac Glucomannan/Carboxymethyl Chitosan/Graphene Oxide hydrogel Author: Lihong Fan Jiayan Yi Jun Tong Xiaoyu Zhou Hongyu Ge Shengqiong Zou Huigao Wen Min Nie PII: DOI: Reference:
S0141-8130(16)30453-6 http://dx.doi.org/doi:10.1016/j.ijbiomac.2016.05.042 BIOMAC 6103
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
8-3-2016 8-5-2016 11-5-2016
Please cite this article as: Lihong Fan, Jiayan Yi, Jun Tong, Xiaoyu Zhou, Hongyu Ge, Shengqiong Zou, Huigao Wen, Min Nie, Preparation and characterization of Oxidized Konjac Glucomannan/Carboxymethyl Chitosan/Graphene Oxide hydrogel, International Journal of Biological Macromolecules http://dx.doi.org/10.1016/j.ijbiomac.2016.05.042 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Preparation
and
characterization
of
Oxidized
Konjac
Glucomannan/Carboxymethyl Chitosan/Graphene Oxide hydrogel
Lihong Fana, Jiayan Yia, Jun Tonga, Xiaoyu Zhoua, Hongyu Gea, Shengqiong Zoua, Huigao Wena, Min Nieb, * a
School of Chemistry, Chemical Engineering and Life Sciences, Wuhan
University of Technology, Wuhan 430070, China b
The State Key Laboratory Breeding Base of Basic Science of Stomatology
(Hubei-MOST) & Key Laboratory of Oral Biomedicine Ministry of Education, School & Hospital of Stomatology, Wuhan University, Wuhan 430079, China *
Corresponding author. Tel.: +86 27 62773634; fax: +86 27 87859019
E-mail addresses: [email protected] (M. Nie) Abstract Polysaccharide hydrogels have been widely used as biomaterials in biomedical field. In this article,
composite hydrogels were prepared through the Schiff-base
reaction between the aldehyde of oxidized konjac glucomannan (OKGM) and the amino of carboxymethyl chitosan (CMCS). Meanwhile, different amount of graphene oxide (GO) was added as nano-additive. The hydrogels have been characterized by various methods including Fourier transform infrared spectroscopy (FT-IR) and Surface morphology (SEM). Through the observation of SEM, the hydrogels’ scaffolds present a homogeneous interconnected pore structure after lyophilizing. In
addition, the influence of different GO content on properties including gelation time, swelling ability, water evaporation rate and mechanical properties were investigated. The results indicate that the hydrogels have short gelation time, appropriate swelling ability and water evaporation rate. Especially, the compressive strength and modulus increase 144% and 296% respectively as the GO content increase from 0 to 5 mg/ml. Moreover, MTT assay was applied to evaluate the biocompatibility of hydrogels. The result indicate that hydrogels with GO show better biocompatibility. Therefore, due to the appropriate water absorption capacity, the similar compressive modulus with soft tissue and excellent biocompatibility, the composite hydrogels have potential application in wound dressings.
Keywords: oxidized konjac glucomannan; carboxymethyl chitosan; graphene oxide; hydrogel; mechanical properties; wound healing
1. Introduction Skin is an important natural barrier organ for protecting internal organs from the external environment and preventing body dehydration, and it would lose its protected mechanism upon damage
[1]
. In the same way, an intact barrier following the
occurrence of a wound is of critical importance
[2]
. In recent years, as an important
class of biomaterials, hydrogels have a wide range of applications in pharmaceutical and biomedical areas due to its unique properties including biocompatibility,
biodegradability, non-toxicity
[3]
. The capacity of hydrogels to maintain a moist
environment is important to facilitate the wound-healing process
[4]
. Their high water
content as well as swelling ability to absorb large amount of body fluid facilitates also creating a moist environment that encourages rapid granulation tissue formation and reepithelialization
[5]
. Furthermore, hydrogels are structurally similar to the native
extracellular matrices (ECM)
[6, 7]
, they can provide three dimensional structures for
cell adhesion, proliferation, transportation of cytokines, nutrients and metabolic waste [8, 9]
. Therefore, hydrogels are very suitable to be used as wound dressings. Natural polymers have similar components with native extracellular matrices
(ECM) and are widely used for biomedical applications. Chitosan and its derivatives are among the most frequently used biomaterials
[10]
. Chitosan (CS) is composed of
N-acetyl-Dglucosamine and D-glucosamine units linked by β-(1–4) bonds. It has always been used as a wound healing promoter [11-14]. However, it is suffering due to its poor water-solubility which largely restricts its application
[15, 16]
. As a modified
chitosan, carboxymethylated chitosan (CMCS) is an important water-soluble chitosan derivative, which exhibit low toxicity, biodegradability, biocompatibility, stability in blood and a good ability to form films and hydrogels
[17-19]
. So CMCS has been
extensively used in many biomedical materials such as moisture-retention agents, bactericides, wound dressings, artificial tissue, blood anticoagulants and drug-delivery matrices
[20-22]
. Furthermore, CMCS is capable of stimulating the extracellular
lysozyme activity of fibroblasts, promoting the proliferation of normal skin fibroblasts
and inhibiting the proliferation of keloid fibroblasts
[16]
. There are many reports on
cross-linking of CMCS to form hydrogels, where small molecular cross-linking agents are generally involved but they have cytotoxicity potential [23-25]. Konjac glucomannan (KGM) is a natural polysaccharide and it is composed of D-mannose and D-glucose units linked by β-(1, 4) bonds. Being a β-(1, 4) linked polysaccharide, KGM can be oxidized by reacting with sodium periodate to produce OKGM
[26, 27]
. Through the
oxidation reaction, carbon-carbon bonds of the cis-diol group in the KGM molecular chain are cleaved and generate reactive aldehyde functions, which can chemically cross-link with CMCS via Schiff-base reaction between the free amino groups of CMCS and the aldehyde groups of the OKGM. Conventional hydrogels consist of natural or synthetic polymers, usually exhibit relatively poor mechanical properties, which limits their practical applications as dressings
[28]
. To deal with this issue, researchers have paid special attention to
graphene oxide, a precursor of chemically converted graphene, which consists of a two-dimensional sheet and has a large number of oxygen-containing groups such as hydroxyl, epoxide and carboxyl groups on the basal planes and edges
[29, 30]
. These
oxygen-containing groups impart GO sheets with the function of strong interaction with polymers to form GO intercalated or exfoliated composites
[31]
. Meanwhile, this
property makes GO can be also easily combination with polymers for enhancing the mechanical properties of hydrogels
[32]
.
In addition, studies have shown that
graphene and graphene oxide own the ability to support cellular proliferation,
adhesion, and differentiation with little or non-cytotoxic effects has been reported that GO shows pro-angiogenic properties
[36]
[33-35]
. Moreover, it
, which can promote
wound healing. Therefore, we consider adding graphene oxide into CMCS/OKGM hydrogels to improve the mechanical properties and biocompatibility. In this article, we have prepared CMCS/OKGM/GO composite hydrogels, which were verified by FT-IR and SEM. And the effects of different GO content on the properties such as gelation time, swelling ability, water evaporation rate and mechanical properties were studied. In addition, the biocompatibility was evaluated by methyl thiazolyl tetrazolium (MTT) assay. On account of their biomimic composition and green fabrication procedure, the composite hydrogels are expected to have potential applications as wound dressings. 2. Experimental 2.1 Materials Konjac glucomannan (The content of glucomannan is above 85%) was purchased from Konson konjac Corp. (Wuhan, Hubei, China). Chitosan (degree of deacetylation=92%) was purchased from Zhejiang Yuhuan Ocean Biochemistry Co. Let. (China). Graphite power was purchased from Aladdin. Monochloro acetic acid, sodium hydroxide, sodium periodate, ethylene glycol, potassium permanganate, Diphosphorus pentaoxide, Sodium nitrate, 30%wt H2O2 and other reagent used in this article were of analytical grade and without further purification. They were purchased from Sinopharm Group Chemical Reagent Corp.
2.2 Preparation of Carboxymethyl Chitosan (CMCS) CMCS was prepared according to our previously research with slight modification [15]. Briefly, chitosan (6g) was added into 50%wt NaOH solution and the mixture was kept at -20℃ for 24h. Then the thawed chitosan was dispersed in isopropanol and monochloro acetic acid (9 g) was added. The mixture was stirred vigorously at room temperature. Then the mixture was heated to 60℃ for 5h. The reaction product was dialyzed against distilled water for 3d through the 8000–10,000 molecular weight cut-off dialysis tubing, and vacuum-dried at 50℃ to obtain the purified CMCS and the dried samples were stored in vacuum desiccators for further use. The reaction is shown in Scheme 1. 2.3 Synthesis of Oxidized Konjac Glucomannan (OKGM) OKGM was prepared according to the previous research with slight modification [26]
. Konjac glucomannan was oxidized using sodium periodate. In 600 ml of 1% (w/v)
aqueous dispersions of KGM, 1.58g of sodium periodate was added and the mixture was stirred vigorously at 30℃ in the dark for 12h. Then 10ml ethylene glycol was added to reaction mixture to reduce unreacted periodate and stirred for another 2h. The reaction product was dialyzed against distilled water for 3 d until the dialysate was free from iodate (checked with silver nitrate). Then the reaction product was centrifuged for 20min at 3000rpm. The supernatant was vacuum-dried at 50℃ to obtain the purified OKGM and the dried samples were stored in vacuum desiccators for further use. The procedures of synthesizing OKGM were as follows in Scheme 2.
2.4 Synthesis of Graphene Oxide (GO) sheets GO was prepared from graphite power by a modified Hummers’ method
[29, 37]
.
Graphite powder (2 g), K2S2O8 (1g), P2O5 (1g) were put into a flask, concentrated H2SO4 (20ml) were added into the flask to preoxidize graphite power. The mixture was stirred vigorously at 80℃ for 5h. Then the mixture was slowly cooled to room temperature over a period of 6h. The mixture was filtered and washed until the filtrate was neutral. Then the filter cake was dried overnight at 50℃. The preoxidize graphite power (2g), NaNO3 (2g) and H2SO4 (100ml) were put into a flask with stirring in an ice bath. Meanwhile KMnO4 (6g) was added slowly in portions to keep the reaction temperature below 10℃. After stirring for 30 min, the mixture diverted to an oil bath with further stirring for 5 h at 35 °C, then the mixture was diluted with distilled water (500ml) and followed by 15ml of H2O2 (30%wt) was gradually added to terminate the reaction. This product was washed by diluted hydrochloride acid (1:10 in volume). After centrifugation at 8000 rpm for 10min, the precipitation was washed until the supernatant was neutral. Then the product was dispersed in distilled water and sonicated for 3h at 100W and followed by the dispersion was dialyzed against distilled water for 3d. The dispersion was freeze-dried to obtain the purified GO. 2.5 Preparation of the OKGM/CMCS/GO hydrogels A certain amount of CMCS and OKGM was added to distilled water, magnetic stirred continuously at room temperature until dissolved to a final concentration of 5%wt, respectively. Then the solutions were stored at 4℃ for further use.
The different amount of dried GO was added to OKGM solutions (5%wt) to a final concentration of 0mg/ml, 1mg/ml, 3mg/ml, 5mg/ml, respectively. In this way, four kind of GO/OKGM solutions were obtained for preparing the composite hydrogels. Different OKGM/GO solutions (2ml) were mixed with CMCS solution (2ml), respectively. Then the mixture was stirred at 155 rpm using a Corning model PC-320 hot plate/stirrer for 15s to ensure graphene oxide uniformly distributed in the mixture and followed by keeping at 4℃ for 24h. According to the amount of GO, the hydrogel samples were coded as GO-0, GO-1, GO-3, GO-5, respectively. The procedures of synthesizing hydrogels were as follows in Scheme 3. 2.6 Gelation time test The gelation time test was performed according to the previously reported method [38]. A mixture of CMCS and OKGM/GO solution was put on a petri-dish (100 ×20 mm2, International VWR, Shanghai, China), and a magnetic stirring bar (Teflon fluorocarbon resin, 5×2 mm2, Fisher Scientific, Shanghai, China) was placed in the center of the solution droplet. The solution was stirred at 155 rpm using a Corning model PC-320 hot plate/stirrer. The gelation time was decided when the solution formed a solid globule that completely separated from the bottom of the dish. Data from each sample was calculated using triplicate measurement. 2.7 Swelling measurements The hydrogel samples (column, diameter 20mm and height 10mm) were put in a
vacuum at 50℃ and dried to a constant weight. Then the samples were immersed in PH=7.4 Phosphate Buffered Saline and kept at room temperature for 48h. The swollen gels were removed and the excess of liquid on the surface was absorbed by filter paper. Then the weight of the hydrogels was measured. The swelling ratio (SR) was calculated from the formula: SR =
(𝑊𝑠 − 𝑊𝑑 ) × 100% 𝑊𝑑
where 𝑊𝑠 and 𝑊𝑑 are the weights of the samples in swelling state and in the dry state, respectively. Data from each sample was calculated using triplicate measurement. 2.8 Water evaporation rate The obtained fresh hydrogel samples (column, diameter 20mm and height 10mm) were immersed in distilled water at room temperature until equilibrium of swelling had been reached. Then the samples were taken out and weighted. The samples were placed in an incubator at 50°C and 50% relative humidity for 24h. The weight of the samples was measured at regular intervals until it became constant. The water evaporation rate was calculated by the following formula: Water lost =
𝑊1 − 𝑊2 × 100% 𝑊1 − 𝑊3
where 𝑊1 , 𝑊2 , 𝑊3 are the initial weight, measured weight and the final weight of the samples, respectively. Data from each sample was calculated using triplicate measurement. 2.9 Mechanical properties of hydrogels
The mechanical properties of the hydrogels were determined by measuring their compression strength and their compression modulus using a universal testing machine. To prepare the samples for the assay, molds were used to shape hydrogels into disks of 20mm in diameter and 10mm thick. A uniaxial compression test was performed on the cylindrical samples with a 5KN load cell at a strain rate of 10mm/min and the load was applied until the samples were crushed completely. The compression modulus was calculated from the initial liner region (0.10-0.20 strain) of the stress-strain curve
[39]
. Data from each sample was calculated using triplicate
measurement. 2.10 Characterizations Transmission electron microscopy (TEM) analysis of GO was performed using a JEOL JEM-2100F (Japan) transmission electron microscope. The samples were prepared by putting drops of the diluted GO aqueous solution on a TEM grid. The copper grids were freeze-dried and observed at an accelerating voltage of 200 kV. Raman spectroscopy analysis of GO was performed using a RENISHAW INVIA Raman Spectrometer at room temperature with an excitation laser source of 532 nm. Spectra were recorded from 300 to 3300 cm-1. FT-IR spectra of CS, KGM, CMCS, OKGM, GO and hydrogel samples were recorded with a Nicolet 170SX Fourier transform infrared spectrophotometer (USA) in the wavenumber ranging from 400 to 4000 cm-1. All the test samples were prepared by the KBr disk method.
Scanning electron microscope (SEM) images were taken with a scanning electron microscope (JEOL JSM-5610, Japan). The hydrogels were freeze-dried to prepare the SEM samples. The test samples were coated with a gold layer and followed by the experiment was performed at an accelerating voltage of 25 KV. 2.11 Cell culture and sample preparation The NIH-3T3 mouse embryonic cells were cultured using Dulbecco’s Modified Eagle Medium (DMEM, High glucose, HyClone) supplemented with 10% Fetal Bovine Serum (FBS) and antibiotics containing 100U/ml penicillin, 100μg/ml streptomycin. Cells were incubated in a humidified atmosphere containing 5% CO2 at 37℃ and the medium was renewed every 2-3 days. Exponentially growing cells were detached with 0.25% trypsin and then replanted in fresh culture medium to create a new cell suspension for further inoculation [40]. To prepare the samples for the cytotoxicity assay, materials were all sterilized and all operations were carried out under sterile conditions. Molds were used to shape hydrogels into disks of 10mm in diameter and 2mm thick. The disks were exposed to ultraviolet light (70μw/cm2) for 2h on each surface in a clean bench to ensure sterility for the further use. 2.12 Cytotoxicity assay An indirect cytotoxicity assay was performed using an elution method as previously
[41]
. Briefly, NIH-3T3 cells were seeded (in quintuplicate) to 96-well
culture plates at a density of 5000 cells/well and incubated for 24h to achieve
attachment of the cells. At the same time, the sterilized hydrogel samples were transferred into a 24-well tissue culture plates containing cell culture medium and incubated in humidified atmosphere containing 5% CO2 at 37℃ for 24h. Then the medium in the cell plates was removed and cells were rinsed twice with PBS. The hydrogel sample’s extraction elution solution (200μl) was added into each well. After incubation for 1d, 3d, 5d respectively, 20μl MTT solution (5mg/ml in PBS) was added to each well and the plates were incubated another 4h at 37℃. Then the medium in the cell plates was removed and a certain amount of dimethylsulfoxide (DMSO) was added to each well, followed by the plates were shaken for 10min. The optical density (OD) at 490nm was measured using microplate reader. The cells were incubated without the hydrogel sample’s extraction elution solution as a positive control group and the wells only contain DMSO as a background group. The cell viability rate was calculated by the following formula: Viable cell =
(𝑂𝐷𝑠 − 𝑂𝐷𝑏 ) × 100% (𝑂𝐷𝑐 − 𝑂𝐷𝑏 )
where 𝑂𝐷𝑠 , 𝑂𝐷𝑐 , 𝑂𝐷𝑏 are optical density values from sample wells, positive control wells and background wells, respectively. 3. Results and discussion 3.1 Characterization of GO The FTIR spectrum of GO powders is shown in Fig. 1. The FTIR spectrum of GO shows characteristics bands at 3399cm-1, 1731cm-1, 1623cm-1, 1400cm-1 and 1052cm-1, which are attributed to the O-H stretching vibration, C=O stretching
vibration of the carboxylic, C=C stretching mode of the sp2 network, O-H deformations of the C-OH groups and C-O-C stretching vibration, respectively [33, 42]. The TEM image of the raw GO is shown in Fig. 2(a). It can be seen that GO contains several graphitic layers, some of which fold to induce wrinkles. The wrinkles are very important for preventing aggregation of graphene caused by van der Waals forces during drying process
[43]
. The digital photo of GO (1mg/ml)/OKGM (5%wt)
solution is shown in Fig. 2(b). The GO sheets are homogeneous dispersion in OKGM solution and the solution is stable for several weeks with no precipitation or color change occurring. Raman spectroscopy is utilized to investigate the carbon structure of graphite during the oxidation process. A typical Raman spectrum of GO is obtained in Fig. 3. The characteristic peaks of the G band and D band were 1590 cm-1 and 1350 cm-1, respectively. The intensity ratio of the D and G band (ID/IG) is 1.03, which are similar to the literature values for GO
[44, 45]
. These characterizations confirm that graphene
oxide has been successfully prepared. 3.2 Gelation time The gelation time test was performed to monitor the gelation of hydrogels. The results of gelation time of the hydrogels are shown in Fig. 4. It can be seen that all the hydrogels show extremely short time of gelation. During the formation of hydrogels, the OKGM acts as a macromolecular chemical cross-linker which makes spontaneous and fast chemical crosslinking reaction between the aldehyde of OKGM and the
amino of CMCS. So the formation of crosslinked network structure is very speedy. Furthermore, the gelation time of hydrogels decreased from 35.8s to 18.6s with the GO content increased from 0 to 5 mg/ml. This fact was explained as related to the hydrogen bonds between GO nanosheets and the polymer chains. GO nanosheets are hydrophilic and contain numerous –COOH and –OH groups, which can form hydrogen bonds with the CMCS and OKGM macromolecules. Meanwhile, the higher content of GO will produce a higher amount of hydrogen bonds which facilitate the formation of network structure and lead to a shorter gelation time. 3.3 Studies of swelling Swelling behavior of the hydrogels in PBS was investigated to evaluate their capacity to absorb wound exudation fluid. The swelling ratio（SR）of the hydrogels in PBS (PH=7.4) at room temperature are shown in Fig. 5. All the samples show good swelling ability. It can be explained that OKGM, CMCS and GO are hydrophilic polymers which contain a large amount of hydrophilic groups such as –OH, –NH2 and –COOH. So the free water could come into the network of the hydrogels. Furthermore, the hydrogels swelling properties were influenced by the amount of GO. The SR value decreased from 236% to 212% when the amount of GO increased from 0mg/ml to 5mg/ml. This is probably due to the hydrogen bonds between GO nanosheets and the polymer chains [46]. The –COOH and –OH groups on the GO nanosheets can form hydrogen bonds with the CMCS and OKGM macromolecules. In this case, the capacity of hydrogels to form hydrogen bonds with water molecules descend.
Therefore, the swelling ability of hydrogels decreased with the increase of GO content. 3.4 Water evaporation rate Generally, the maintenance of moist wound bed has been widely accepted as the most ideal environment for effective wound healing. Therefore, the wounding dressing that possess a smaller water evaporation rate can provide ideal environment for wound healing. Compared with saline dressings, hydrogel dressings exhibit more advantages such as quicker healing, less pain and cost savings that were confirmed in previous studies [47, 48]. The water loss percentage of different hydrogels are shown in Fig. 6. It can be seen that the water loss percentage increased sharply in initial 8h. And it reached a constant percentage in 24 hours. After 24 hours, all of the hydrogels still have retained 5% to 15% of water. It shows that the hydrogels have good water evaporation rate. Furthermore, the water loss percentage increased with the increase of GO content. 3.5 Mechanical properties As the wound dressings, hydrogels must have appropriate mechanical properties. The compressive strength and modulus of hydrogels with different GO loadings are shown in Fig. 7. It can be seen that the compressive strength and modulus significantly increased with the increase of GO content. The compressive strength and modulus increased from 85.1 kPa to 208.2 kPa (more than 144%) and increased from 0.28 MPa to 1.11 MPa (more than 296%) as the GO content increased from 0 to 5
mg/ml, respectively. The enhancement of mechanical properties can be mainly attributed to the homogeneous dispersion of GO sheets in the polymer matrix and the strong interfacial interactions, specifically, hydrogen bonds formed between GO nanosheets and polymer chains. This provides better load transfer between the polymer matrix and the GO nanosheets, and is beneficial for mechanical enhancement [46]
. Furthermore, it can be seen that the compressive strength and modulus increased
rapidly with the GO content increased from 0 to 3 mg/ml and increased slowly with the GO content increased from 3 to 5 mg/ml. This phenomenon can be explained that the GO sheets trend to form irreversible agglomerates due to the van der Waals force at high GO content, which lead to a reduced capacity of GO sheets to disperse in the polymer matrix [33]. 3.6 FT-IR analysis The FTIR spectra of CS, CMCS are shown in Fig. 8(a). In the FTIR spectrum of CS, the bands at 3419cm-1, 1651cm-1, 1605cm-1, 1382cm-1, 1093cm-1 are attributed to the O-H stretching vibration, C=O stretching mode of N-acetylguscosamine, N-H bending vibration, C-H bending vibration, C-O stretching vibration, respectively. From the FTIR spectrum of CMCS, it could be observed that characteristics bands at 1596cm-1, 1410cm-1, 1065cm-1, which are due to the asymmetric and symmetric stretching vibration of COO-, and the stretching vibration of C-O-C, respectively. This demonstrated the introduction of COO- group to CS chains [15]. The FTIR spectra of KGM, OKGM are shown in Fig. 8(b). In the FTIR spectrum
of KGM, the bands at 3419cm-1, 1734cm-1 are ascribed to the O-H stretching vibration, C=O stretching vibration of the acetyl group, respectively. Obviously, two characteristic bands around 1730cm-1 and 895cm-1 are observed in the spectrum of OKGM. The former is due to the aldehyde symmetric vibrational band and the latter is due to the hemiacetal structure [26, 27]. The FTIR spectra of GO-0, GO-1, GO-3, GO-5 hydrogels are shown in Fig. 8(c). Compared with GO-0 hydrogel CMCS and OKGM, the C=O stretching vibration of the acetyl group band at 1731cm-1 was disappeared and the characteristic band of hemiacetal structure at 895cm-1 was practically disappeared, along with the stretching vibrations of C=N band at 1640cm-1 was appeared. It indicates that the crosslinking reaction is followed between –CHO groups of OKGM and –NH2 groups of CMCS. For hydrogels, it can be seen that some peaks are enhanced but no obvious change of peak position occurs in the spectra, which indicates that the formation of hydrogen bonding between the oxygen-containing functional groups of GO and the hydroxyl groups on the CMCS and OKGM molecular chains [33, 49] 3.7 Structure and morphology analysis The digital photo of composite hydrogels with different GO loadings is shown in Fig. 9(A). It can be seen that the GO sheets are homogeneous dispersion in hydrogels. Meanwhile, with the GO content increased, the white hydrogels first turn to brown and then to black.
The porous microstructure of the scaffolds had significant influence on the ability of the cells intrusion, proliferation and function in tissue engineering
[50]
. The
SEM images are obtained to characterize the microstructure of the hydrogels. The cross-section images of the hydrogels at different magnifications are shown in Fig. 9. It can be observed that all the hydrogel samples have a continuous and stable three-dimensional network structure, which are similar to some earlier reported polysaccharide and GO hydrogels. Furthermore, all the hydrogel samples exhibit many micro-pores, which distribute uniformly in the internal of the hydrogels. The interconnection between pores could be attributed to the network formed by the cross-linking between CMCS and OKGM, and the hydrogen bonds between GO nanosheets and the polymer chains. Compared with GO-0(fig 9B1), GO-3(fig 9C1) and GO-5(fig 9D1), it also can be observed that the average pore diameter of the gels have no obvious change as the concentration of GO increased from 0 to 5mg/ml. However, it can be seen that the wall of GO-0 sample seems to be torn during the freeze drying process, which indicated that the hydrogels with GO have sufficient capability to avoid the structural collapse during dehydration. This result is consistent with the result of mechanical properties. It is believed that the strong hydrogen bonding interaction between GO and the polymer chains are the main reason for this result. The hydrogels with GO exhibit more stable network structure than the hydrogels without GO. 3.8 Cytotoxicity assay
A wound dressing designed to be placed in wound and direct contact with wound tissue should possess the highest possible biocompatibility. In vitro cytotoxicity assays have the advantages of being simple, reproducible, cost-effective, and suitable for the evaluation of basic biological aspects relative to biocompatibility. The cell viabilities of GO-0, GO-1, GO-3, GO-5 are shown in Fig.10. The cell viabilities of all the samples were higher than 90% during the whole incubation period, which could be classified to a scale 0 (non-toxicity). The cell viabilities were related with the GO content and incubation time. It can be seen that the cell viabilities of all the samples significantly increase with the increase of incubation time and the cell viabilities of GO-1, GO-3, GO-5 hydrogels were higher than GO-0 in the incubation period. In addition, cells incubated with the extract from GO-1 hydrogel showed the highest viabilities during the test period. The higher cell viabilities of GO-1, GO-3, GO-5 hydrogels than GO-0 hydrogel suggests that the addition of GO to hydrogels promotes cell growth without producing additional cytotoxicity. This result is consistent with previous studies, which have confirmed that GO have positive effects on adhesion, grown, proliferation and differentiation of cells
[51, 52]
. Furthermore, compared with GO-1, GO-3, GO-5, the
cell viabilities significantly decrease with the increase of GO content. This phenomenon may be due to the GO content in extracts increase with the increase of the GO content in hydrogels and GO display a significant cytotoxic effect at high concentration [53].
4. Conclusions In this article, graphene oxide (GO) was added as a nano-additive in CMCS/OKGM/GO composite hydrogels, which were prepared through the crosslinking reaction between oxidized konjac glucomannan (OKGM) and carboxymethyl chitosan (CMCS). Through a series of properties characterization, the hydrogels showed rapid gelation process, good swelling ability, appropriate water retention capacity and a stable three-dimensional network structure, which all satisfy with the application as a wound dressings. Especially, the compressive strength and modulus increased significantly with loading of GO, which can be attributed to the hydrogen bonding between GO sheets and the polymer chains. Moreover, the in vitro cytotoxicity assay demonstrated that all the hydrogels have good biocompatibility with a cytotoxicity of grade 0, and the hydrogels with GO have better biocompatibility than hydrogels without GO. Therefore, OKGM/CMCS/GO hydrogels have great clinical potential to be applied as a wound dressing. Acknowledgements The work was supported by the National Natural Science Foundation of China (Foundation No. 51173143, No. 51273156), The Special Funds Project of Major New Products of Hubei Province (Foundation No. 20132h0040), University-industry Cooperation Projects of The Ministry of Education of Guangdong province (Foundation No. 2012B091100437), The innovation fund project of the Ministry of Science and Technology of Small and Medium-sized Enterprises (Foundation No.
11C26214202642, No. 11C26214212743), Zhuhai Science and Technology Plan Projects (Foundation No. 2011B050102003), Wuhan Science and Technology Development (Foundation No. 201060623262), The Fundamental Research Funds for the Central Universities(Foundation No. 2014-zy-220). Reference [1] Z.J. Fan, B, Liu, J.Q. Wang, S.Y. Zhang, Q.Q. Lin, P.W. Gong, L.M. Ma, S.R. Yang, A Novel Wound Dressing Based on Ag/Graphene Polymer Hydrogel: Effectively Kill Bacteria and Accelerate Wound Healing, Adv. Funct. Mater. 24(25) (2014) 3933-3943. [2] J.E. Park, A. Barbul, Understanding the role of immune regulation in wound healing. Am. J. Surg. 187(187) (2004) 511–516. [3] J.Y. Drury, D.J. Mooney, Hydrogels for tissue engineering: scaffold design variables and applications, Biomaterials. 24 (2003) 4337-4351. [4] Y.J. Fan, H.N. Li, J. Yang, X.N. Hu, J. Liang, X.D. Zhang, Superabsorbent polysaccharide hydrogels based on pullulan derivate as antibacterial release wound dressing, J. Biomed. Mater. Res. A. 98(1) (2011) 31-39. [5] X. Huang, Y.Q. Zhang, X.M. Zhang, L. Xu, X. Chen, S.C. Wei, Influence of radiation crosslinked carboxymethyl-chitosan/gelatin hydrogel on cutaneous wound healing, Mat. Sci. Eng. C-Mater. 33(8) (2013) 4816-4824. [6] Z.Y. Li, B.M. Yuan, X.M. Dong, L.J. Duan, H.Y. Tian, C.L. He, X.S. Chen, Injectable polysaccharide hybrid hydrogels as scaffolds for burn wound healing,
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Transmittance (a.u.)
Figure Captions
GO
1731
1400
1623
1052
3999
4000
3500
3000
2500
2000
1500
Wavenumber (cm-1)
Fig. 1. FTIR spectra of the GO powders.
1000
500
Fig. 2. (a) TEM photo of GO aqueous solution, (b) digital photo of GO(1mg/ml)/OKGM(5%wt) solution.
G
Intensity (a.u.)
D
0
500
1000
1500
2000
2500
-1
Raman shift (cm )
Fig. 3. Raman spectrum of GO powders.
3000
3500
38 36 34 32
凝胶时间
30 28 26 24 22 20 18 GO-0
GO-1
GO-3
GO-5
Fig. 4. Gelation time of GO-0, GO-1, GO-3, GO-5 hydrogels.
240 GO-1
235
GO-3
230
GO-5
225
GO-0
Swelling ratio (%)
220 215 210 205 200 195 190 185 180 175 0
10
20
30
40
50
Time (h)
Fig. 5. Swelling percentage of composite hydrogels with different GO loadings.
110
GO-1 GO-3 GO-5 GO-0
100
Water lost (%)
90 80 70 60 50 40 30 0
5
10
15
20
25
Time (h)
Fig. 6. Water loss of GO-0, GO-1, GO-3, GO-5 hydrogels.
1.2
200
compressive strength compressive modulus
0.8
150
0.6
100 0.4
50
compressive modulus (MPa)
compressive strength (KPa)
1.0
0.2
0
0.0
GO-0
GO-1
GO-3
GO-5
Fig. 7. Compressive strength and modulus of composite hydrogels with different GO loadings. (a)
Transmittance
CS 1382 1651 1605 1155 2879
1093
3419
CMCS
1325 1596
4000
3500
3000
2500
2000
1410
1500
1065
1000
500
-1
Wavenumber(cm )
(b)
KGM
1734 1638
2925
Transmittance
3419
OKGM 1730 895 2925
1644
3420
4000
3500
3000
2500
2000
1500 -1
Wavenumber(cm )
(c)
1000
500
GO-5
Transmittance
2928 1640 1410
GO-3 3418
1063
2927 1640
GO-1
3423
1410 1064
2925
1410 1640 1064
3421
GO-0 2923
1640
1410 1065
3424
4000
3500
3000
2500
2000
1500
1000
500
-1
Wavenumber (cm )
Fig. 8. (a) FTIR spectra of the CS powders, CMCS. (b) FTIR spectra of KGM powders, OKGM. (c) FTIR spectra of GO-0, GO-1, GO-3, GO-5 composite hydrogel.
(A)
(B)
(B1) (50×)
(B2) (200×)
(B3) (1000×)
(C2) (200×)
(C3) (1000×)
(C)
(C1) (50×) (D)
(D1) (50×)
(D2) (200×)
(D3) (1000×)
Fig. 9. (A) Digital photo of composite hydrogels with different GO loadings, SEM images of the cross-section of the GO-0 hydrogel (B), GO-3 hydrogel (C) and GO-5 hydrogel (D) at different magnifications.
120 110 100
GO-0 GO-1 GO-3 GO-5
Cell viability (%)
90 80 70 60 50 40 30 20 10 0 Day 1
Day 3
Day 5
Fig. 10. The cell viabilities of NIH-3T3 cells after incubation for 1, 3, 5 days with extracts form GO-0, GO-1, GO-3, GO-5 hydrogels.
CH2OH
CH 2OCH2COONa O
O
O
ClCH2COOH
OH
O
OH
NaOH n
n
NH2
NH2
CS
CMCS
Scheme 1. The synthesis of Carboxymethyl Chitosan
OH
OH O
O
O
OH
OH O
NaIO4
OH n
OH
OH
OH O
O
O
O
CH O
KGM Scheme 2. The synthesis of Oxidized Konjac Glucomannan
OH
OH
n
OH
OKGM
OKGM
OH
OH
OH O
O
O
O
CH
OH
OH
HO
n
O
O
OH
HO
O COOH
+
COOH OH
HOOC CH2 OCH2 COONa
OH
O COOH COOH OH COOH
HO
OH OH
O
OH O
OH
O
O
n NH2
O
CH
OH COOH
HO
+ OH
OH HO
COOH
n OH
O OH
O
HO
OH
OH
OH
N
CMCS O COOH COOH OH COOH
O
n
CH2 OCH 2COONa
OH O O
OKGM-CMCS-GO Hydrogel
COOH COOH
OH
HO
COOH O
HO
O
COOH
HO
OH COOH
HO OH
GO 2D sheet
Scheme 3. Schematic illustration of the synthesis route of the hydrogel
S0141-8130(16)30453-6 http://dx.doi.org/doi:10.1016/j.ijbiomac.2016.05.042 BIOMAC 6103
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
8-3-2016 8-5-2016 11-5-2016
Please cite this article as: Lihong Fan, Jiayan Yi, Jun Tong, Xiaoyu Zhou, Hongyu Ge, Shengqiong Zou, Huigao Wen, Min Nie, Preparation and characterization of Oxidized Konjac Glucomannan/Carboxymethyl Chitosan/Graphene Oxide hydrogel, International Journal of Biological Macromolecules http://dx.doi.org/10.1016/j.ijbiomac.2016.05.042 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Preparation
and
characterization
of
Oxidized
Konjac
Glucomannan/Carboxymethyl Chitosan/Graphene Oxide hydrogel
Lihong Fana, Jiayan Yia, Jun Tonga, Xiaoyu Zhoua, Hongyu Gea, Shengqiong Zoua, Huigao Wena, Min Nieb, * a
School of Chemistry, Chemical Engineering and Life Sciences, Wuhan
University of Technology, Wuhan 430070, China b
The State Key Laboratory Breeding Base of Basic Science of Stomatology
(Hubei-MOST) & Key Laboratory of Oral Biomedicine Ministry of Education, School & Hospital of Stomatology, Wuhan University, Wuhan 430079, China *
Corresponding author. Tel.: +86 27 62773634; fax: +86 27 87859019
E-mail addresses: [email protected] (M. Nie) Abstract Polysaccharide hydrogels have been widely used as biomaterials in biomedical field. In this article,
composite hydrogels were prepared through the Schiff-base
reaction between the aldehyde of oxidized konjac glucomannan (OKGM) and the amino of carboxymethyl chitosan (CMCS). Meanwhile, different amount of graphene oxide (GO) was added as nano-additive. The hydrogels have been characterized by various methods including Fourier transform infrared spectroscopy (FT-IR) and Surface morphology (SEM). Through the observation of SEM, the hydrogels’ scaffolds present a homogeneous interconnected pore structure after lyophilizing. In
addition, the influence of different GO content on properties including gelation time, swelling ability, water evaporation rate and mechanical properties were investigated. The results indicate that the hydrogels have short gelation time, appropriate swelling ability and water evaporation rate. Especially, the compressive strength and modulus increase 144% and 296% respectively as the GO content increase from 0 to 5 mg/ml. Moreover, MTT assay was applied to evaluate the biocompatibility of hydrogels. The result indicate that hydrogels with GO show better biocompatibility. Therefore, due to the appropriate water absorption capacity, the similar compressive modulus with soft tissue and excellent biocompatibility, the composite hydrogels have potential application in wound dressings.
Keywords: oxidized konjac glucomannan; carboxymethyl chitosan; graphene oxide; hydrogel; mechanical properties; wound healing
1. Introduction Skin is an important natural barrier organ for protecting internal organs from the external environment and preventing body dehydration, and it would lose its protected mechanism upon damage
[1]
. In the same way, an intact barrier following the
occurrence of a wound is of critical importance
[2]
. In recent years, as an important
class of biomaterials, hydrogels have a wide range of applications in pharmaceutical and biomedical areas due to its unique properties including biocompatibility,
biodegradability, non-toxicity
[3]
. The capacity of hydrogels to maintain a moist
environment is important to facilitate the wound-healing process
[4]
. Their high water
content as well as swelling ability to absorb large amount of body fluid facilitates also creating a moist environment that encourages rapid granulation tissue formation and reepithelialization
[5]
. Furthermore, hydrogels are structurally similar to the native
extracellular matrices (ECM)
[6, 7]
, they can provide three dimensional structures for
cell adhesion, proliferation, transportation of cytokines, nutrients and metabolic waste [8, 9]
. Therefore, hydrogels are very suitable to be used as wound dressings. Natural polymers have similar components with native extracellular matrices
(ECM) and are widely used for biomedical applications. Chitosan and its derivatives are among the most frequently used biomaterials
[10]
. Chitosan (CS) is composed of
N-acetyl-Dglucosamine and D-glucosamine units linked by β-(1–4) bonds. It has always been used as a wound healing promoter [11-14]. However, it is suffering due to its poor water-solubility which largely restricts its application
[15, 16]
. As a modified
chitosan, carboxymethylated chitosan (CMCS) is an important water-soluble chitosan derivative, which exhibit low toxicity, biodegradability, biocompatibility, stability in blood and a good ability to form films and hydrogels
[17-19]
. So CMCS has been
extensively used in many biomedical materials such as moisture-retention agents, bactericides, wound dressings, artificial tissue, blood anticoagulants and drug-delivery matrices
[20-22]
. Furthermore, CMCS is capable of stimulating the extracellular
lysozyme activity of fibroblasts, promoting the proliferation of normal skin fibroblasts
and inhibiting the proliferation of keloid fibroblasts
[16]
. There are many reports on
cross-linking of CMCS to form hydrogels, where small molecular cross-linking agents are generally involved but they have cytotoxicity potential [23-25]. Konjac glucomannan (KGM) is a natural polysaccharide and it is composed of D-mannose and D-glucose units linked by β-(1, 4) bonds. Being a β-(1, 4) linked polysaccharide, KGM can be oxidized by reacting with sodium periodate to produce OKGM
[26, 27]
. Through the
oxidation reaction, carbon-carbon bonds of the cis-diol group in the KGM molecular chain are cleaved and generate reactive aldehyde functions, which can chemically cross-link with CMCS via Schiff-base reaction between the free amino groups of CMCS and the aldehyde groups of the OKGM. Conventional hydrogels consist of natural or synthetic polymers, usually exhibit relatively poor mechanical properties, which limits their practical applications as dressings
[28]
. To deal with this issue, researchers have paid special attention to
graphene oxide, a precursor of chemically converted graphene, which consists of a two-dimensional sheet and has a large number of oxygen-containing groups such as hydroxyl, epoxide and carboxyl groups on the basal planes and edges
[29, 30]
. These
oxygen-containing groups impart GO sheets with the function of strong interaction with polymers to form GO intercalated or exfoliated composites
[31]
. Meanwhile, this
property makes GO can be also easily combination with polymers for enhancing the mechanical properties of hydrogels
[32]
.
In addition, studies have shown that
graphene and graphene oxide own the ability to support cellular proliferation,
adhesion, and differentiation with little or non-cytotoxic effects has been reported that GO shows pro-angiogenic properties
[36]
[33-35]
. Moreover, it
, which can promote
wound healing. Therefore, we consider adding graphene oxide into CMCS/OKGM hydrogels to improve the mechanical properties and biocompatibility. In this article, we have prepared CMCS/OKGM/GO composite hydrogels, which were verified by FT-IR and SEM. And the effects of different GO content on the properties such as gelation time, swelling ability, water evaporation rate and mechanical properties were studied. In addition, the biocompatibility was evaluated by methyl thiazolyl tetrazolium (MTT) assay. On account of their biomimic composition and green fabrication procedure, the composite hydrogels are expected to have potential applications as wound dressings. 2. Experimental 2.1 Materials Konjac glucomannan (The content of glucomannan is above 85%) was purchased from Konson konjac Corp. (Wuhan, Hubei, China). Chitosan (degree of deacetylation=92%) was purchased from Zhejiang Yuhuan Ocean Biochemistry Co. Let. (China). Graphite power was purchased from Aladdin. Monochloro acetic acid, sodium hydroxide, sodium periodate, ethylene glycol, potassium permanganate, Diphosphorus pentaoxide, Sodium nitrate, 30%wt H2O2 and other reagent used in this article were of analytical grade and without further purification. They were purchased from Sinopharm Group Chemical Reagent Corp.
2.2 Preparation of Carboxymethyl Chitosan (CMCS) CMCS was prepared according to our previously research with slight modification [15]. Briefly, chitosan (6g) was added into 50%wt NaOH solution and the mixture was kept at -20℃ for 24h. Then the thawed chitosan was dispersed in isopropanol and monochloro acetic acid (9 g) was added. The mixture was stirred vigorously at room temperature. Then the mixture was heated to 60℃ for 5h. The reaction product was dialyzed against distilled water for 3d through the 8000–10,000 molecular weight cut-off dialysis tubing, and vacuum-dried at 50℃ to obtain the purified CMCS and the dried samples were stored in vacuum desiccators for further use. The reaction is shown in Scheme 1. 2.3 Synthesis of Oxidized Konjac Glucomannan (OKGM) OKGM was prepared according to the previous research with slight modification [26]
. Konjac glucomannan was oxidized using sodium periodate. In 600 ml of 1% (w/v)
aqueous dispersions of KGM, 1.58g of sodium periodate was added and the mixture was stirred vigorously at 30℃ in the dark for 12h. Then 10ml ethylene glycol was added to reaction mixture to reduce unreacted periodate and stirred for another 2h. The reaction product was dialyzed against distilled water for 3 d until the dialysate was free from iodate (checked with silver nitrate). Then the reaction product was centrifuged for 20min at 3000rpm. The supernatant was vacuum-dried at 50℃ to obtain the purified OKGM and the dried samples were stored in vacuum desiccators for further use. The procedures of synthesizing OKGM were as follows in Scheme 2.
2.4 Synthesis of Graphene Oxide (GO) sheets GO was prepared from graphite power by a modified Hummers’ method
[29, 37]
.
Graphite powder (2 g), K2S2O8 (1g), P2O5 (1g) were put into a flask, concentrated H2SO4 (20ml) were added into the flask to preoxidize graphite power. The mixture was stirred vigorously at 80℃ for 5h. Then the mixture was slowly cooled to room temperature over a period of 6h. The mixture was filtered and washed until the filtrate was neutral. Then the filter cake was dried overnight at 50℃. The preoxidize graphite power (2g), NaNO3 (2g) and H2SO4 (100ml) were put into a flask with stirring in an ice bath. Meanwhile KMnO4 (6g) was added slowly in portions to keep the reaction temperature below 10℃. After stirring for 30 min, the mixture diverted to an oil bath with further stirring for 5 h at 35 °C, then the mixture was diluted with distilled water (500ml) and followed by 15ml of H2O2 (30%wt) was gradually added to terminate the reaction. This product was washed by diluted hydrochloride acid (1:10 in volume). After centrifugation at 8000 rpm for 10min, the precipitation was washed until the supernatant was neutral. Then the product was dispersed in distilled water and sonicated for 3h at 100W and followed by the dispersion was dialyzed against distilled water for 3d. The dispersion was freeze-dried to obtain the purified GO. 2.5 Preparation of the OKGM/CMCS/GO hydrogels A certain amount of CMCS and OKGM was added to distilled water, magnetic stirred continuously at room temperature until dissolved to a final concentration of 5%wt, respectively. Then the solutions were stored at 4℃ for further use.
The different amount of dried GO was added to OKGM solutions (5%wt) to a final concentration of 0mg/ml, 1mg/ml, 3mg/ml, 5mg/ml, respectively. In this way, four kind of GO/OKGM solutions were obtained for preparing the composite hydrogels. Different OKGM/GO solutions (2ml) were mixed with CMCS solution (2ml), respectively. Then the mixture was stirred at 155 rpm using a Corning model PC-320 hot plate/stirrer for 15s to ensure graphene oxide uniformly distributed in the mixture and followed by keeping at 4℃ for 24h. According to the amount of GO, the hydrogel samples were coded as GO-0, GO-1, GO-3, GO-5, respectively. The procedures of synthesizing hydrogels were as follows in Scheme 3. 2.6 Gelation time test The gelation time test was performed according to the previously reported method [38]. A mixture of CMCS and OKGM/GO solution was put on a petri-dish (100 ×20 mm2, International VWR, Shanghai, China), and a magnetic stirring bar (Teflon fluorocarbon resin, 5×2 mm2, Fisher Scientific, Shanghai, China) was placed in the center of the solution droplet. The solution was stirred at 155 rpm using a Corning model PC-320 hot plate/stirrer. The gelation time was decided when the solution formed a solid globule that completely separated from the bottom of the dish. Data from each sample was calculated using triplicate measurement. 2.7 Swelling measurements The hydrogel samples (column, diameter 20mm and height 10mm) were put in a
vacuum at 50℃ and dried to a constant weight. Then the samples were immersed in PH=7.4 Phosphate Buffered Saline and kept at room temperature for 48h. The swollen gels were removed and the excess of liquid on the surface was absorbed by filter paper. Then the weight of the hydrogels was measured. The swelling ratio (SR) was calculated from the formula: SR =
(𝑊𝑠 − 𝑊𝑑 ) × 100% 𝑊𝑑
where 𝑊𝑠 and 𝑊𝑑 are the weights of the samples in swelling state and in the dry state, respectively. Data from each sample was calculated using triplicate measurement. 2.8 Water evaporation rate The obtained fresh hydrogel samples (column, diameter 20mm and height 10mm) were immersed in distilled water at room temperature until equilibrium of swelling had been reached. Then the samples were taken out and weighted. The samples were placed in an incubator at 50°C and 50% relative humidity for 24h. The weight of the samples was measured at regular intervals until it became constant. The water evaporation rate was calculated by the following formula: Water lost =
𝑊1 − 𝑊2 × 100% 𝑊1 − 𝑊3
where 𝑊1 , 𝑊2 , 𝑊3 are the initial weight, measured weight and the final weight of the samples, respectively. Data from each sample was calculated using triplicate measurement. 2.9 Mechanical properties of hydrogels
The mechanical properties of the hydrogels were determined by measuring their compression strength and their compression modulus using a universal testing machine. To prepare the samples for the assay, molds were used to shape hydrogels into disks of 20mm in diameter and 10mm thick. A uniaxial compression test was performed on the cylindrical samples with a 5KN load cell at a strain rate of 10mm/min and the load was applied until the samples were crushed completely. The compression modulus was calculated from the initial liner region (0.10-0.20 strain) of the stress-strain curve
[39]
. Data from each sample was calculated using triplicate
measurement. 2.10 Characterizations Transmission electron microscopy (TEM) analysis of GO was performed using a JEOL JEM-2100F (Japan) transmission electron microscope. The samples were prepared by putting drops of the diluted GO aqueous solution on a TEM grid. The copper grids were freeze-dried and observed at an accelerating voltage of 200 kV. Raman spectroscopy analysis of GO was performed using a RENISHAW INVIA Raman Spectrometer at room temperature with an excitation laser source of 532 nm. Spectra were recorded from 300 to 3300 cm-1. FT-IR spectra of CS, KGM, CMCS, OKGM, GO and hydrogel samples were recorded with a Nicolet 170SX Fourier transform infrared spectrophotometer (USA) in the wavenumber ranging from 400 to 4000 cm-1. All the test samples were prepared by the KBr disk method.
Scanning electron microscope (SEM) images were taken with a scanning electron microscope (JEOL JSM-5610, Japan). The hydrogels were freeze-dried to prepare the SEM samples. The test samples were coated with a gold layer and followed by the experiment was performed at an accelerating voltage of 25 KV. 2.11 Cell culture and sample preparation The NIH-3T3 mouse embryonic cells were cultured using Dulbecco’s Modified Eagle Medium (DMEM, High glucose, HyClone) supplemented with 10% Fetal Bovine Serum (FBS) and antibiotics containing 100U/ml penicillin, 100μg/ml streptomycin. Cells were incubated in a humidified atmosphere containing 5% CO2 at 37℃ and the medium was renewed every 2-3 days. Exponentially growing cells were detached with 0.25% trypsin and then replanted in fresh culture medium to create a new cell suspension for further inoculation [40]. To prepare the samples for the cytotoxicity assay, materials were all sterilized and all operations were carried out under sterile conditions. Molds were used to shape hydrogels into disks of 10mm in diameter and 2mm thick. The disks were exposed to ultraviolet light (70μw/cm2) for 2h on each surface in a clean bench to ensure sterility for the further use. 2.12 Cytotoxicity assay An indirect cytotoxicity assay was performed using an elution method as previously
[41]
. Briefly, NIH-3T3 cells were seeded (in quintuplicate) to 96-well
culture plates at a density of 5000 cells/well and incubated for 24h to achieve
attachment of the cells. At the same time, the sterilized hydrogel samples were transferred into a 24-well tissue culture plates containing cell culture medium and incubated in humidified atmosphere containing 5% CO2 at 37℃ for 24h. Then the medium in the cell plates was removed and cells were rinsed twice with PBS. The hydrogel sample’s extraction elution solution (200μl) was added into each well. After incubation for 1d, 3d, 5d respectively, 20μl MTT solution (5mg/ml in PBS) was added to each well and the plates were incubated another 4h at 37℃. Then the medium in the cell plates was removed and a certain amount of dimethylsulfoxide (DMSO) was added to each well, followed by the plates were shaken for 10min. The optical density (OD) at 490nm was measured using microplate reader. The cells were incubated without the hydrogel sample’s extraction elution solution as a positive control group and the wells only contain DMSO as a background group. The cell viability rate was calculated by the following formula: Viable cell =
(𝑂𝐷𝑠 − 𝑂𝐷𝑏 ) × 100% (𝑂𝐷𝑐 − 𝑂𝐷𝑏 )
where 𝑂𝐷𝑠 , 𝑂𝐷𝑐 , 𝑂𝐷𝑏 are optical density values from sample wells, positive control wells and background wells, respectively. 3. Results and discussion 3.1 Characterization of GO The FTIR spectrum of GO powders is shown in Fig. 1. The FTIR spectrum of GO shows characteristics bands at 3399cm-1, 1731cm-1, 1623cm-1, 1400cm-1 and 1052cm-1, which are attributed to the O-H stretching vibration, C=O stretching
vibration of the carboxylic, C=C stretching mode of the sp2 network, O-H deformations of the C-OH groups and C-O-C stretching vibration, respectively [33, 42]. The TEM image of the raw GO is shown in Fig. 2(a). It can be seen that GO contains several graphitic layers, some of which fold to induce wrinkles. The wrinkles are very important for preventing aggregation of graphene caused by van der Waals forces during drying process
[43]
. The digital photo of GO (1mg/ml)/OKGM (5%wt)
solution is shown in Fig. 2(b). The GO sheets are homogeneous dispersion in OKGM solution and the solution is stable for several weeks with no precipitation or color change occurring. Raman spectroscopy is utilized to investigate the carbon structure of graphite during the oxidation process. A typical Raman spectrum of GO is obtained in Fig. 3. The characteristic peaks of the G band and D band were 1590 cm-1 and 1350 cm-1, respectively. The intensity ratio of the D and G band (ID/IG) is 1.03, which are similar to the literature values for GO
[44, 45]
. These characterizations confirm that graphene
oxide has been successfully prepared. 3.2 Gelation time The gelation time test was performed to monitor the gelation of hydrogels. The results of gelation time of the hydrogels are shown in Fig. 4. It can be seen that all the hydrogels show extremely short time of gelation. During the formation of hydrogels, the OKGM acts as a macromolecular chemical cross-linker which makes spontaneous and fast chemical crosslinking reaction between the aldehyde of OKGM and the
amino of CMCS. So the formation of crosslinked network structure is very speedy. Furthermore, the gelation time of hydrogels decreased from 35.8s to 18.6s with the GO content increased from 0 to 5 mg/ml. This fact was explained as related to the hydrogen bonds between GO nanosheets and the polymer chains. GO nanosheets are hydrophilic and contain numerous –COOH and –OH groups, which can form hydrogen bonds with the CMCS and OKGM macromolecules. Meanwhile, the higher content of GO will produce a higher amount of hydrogen bonds which facilitate the formation of network structure and lead to a shorter gelation time. 3.3 Studies of swelling Swelling behavior of the hydrogels in PBS was investigated to evaluate their capacity to absorb wound exudation fluid. The swelling ratio（SR）of the hydrogels in PBS (PH=7.4) at room temperature are shown in Fig. 5. All the samples show good swelling ability. It can be explained that OKGM, CMCS and GO are hydrophilic polymers which contain a large amount of hydrophilic groups such as –OH, –NH2 and –COOH. So the free water could come into the network of the hydrogels. Furthermore, the hydrogels swelling properties were influenced by the amount of GO. The SR value decreased from 236% to 212% when the amount of GO increased from 0mg/ml to 5mg/ml. This is probably due to the hydrogen bonds between GO nanosheets and the polymer chains [46]. The –COOH and –OH groups on the GO nanosheets can form hydrogen bonds with the CMCS and OKGM macromolecules. In this case, the capacity of hydrogels to form hydrogen bonds with water molecules descend.
Therefore, the swelling ability of hydrogels decreased with the increase of GO content. 3.4 Water evaporation rate Generally, the maintenance of moist wound bed has been widely accepted as the most ideal environment for effective wound healing. Therefore, the wounding dressing that possess a smaller water evaporation rate can provide ideal environment for wound healing. Compared with saline dressings, hydrogel dressings exhibit more advantages such as quicker healing, less pain and cost savings that were confirmed in previous studies [47, 48]. The water loss percentage of different hydrogels are shown in Fig. 6. It can be seen that the water loss percentage increased sharply in initial 8h. And it reached a constant percentage in 24 hours. After 24 hours, all of the hydrogels still have retained 5% to 15% of water. It shows that the hydrogels have good water evaporation rate. Furthermore, the water loss percentage increased with the increase of GO content. 3.5 Mechanical properties As the wound dressings, hydrogels must have appropriate mechanical properties. The compressive strength and modulus of hydrogels with different GO loadings are shown in Fig. 7. It can be seen that the compressive strength and modulus significantly increased with the increase of GO content. The compressive strength and modulus increased from 85.1 kPa to 208.2 kPa (more than 144%) and increased from 0.28 MPa to 1.11 MPa (more than 296%) as the GO content increased from 0 to 5
mg/ml, respectively. The enhancement of mechanical properties can be mainly attributed to the homogeneous dispersion of GO sheets in the polymer matrix and the strong interfacial interactions, specifically, hydrogen bonds formed between GO nanosheets and polymer chains. This provides better load transfer between the polymer matrix and the GO nanosheets, and is beneficial for mechanical enhancement [46]
. Furthermore, it can be seen that the compressive strength and modulus increased
rapidly with the GO content increased from 0 to 3 mg/ml and increased slowly with the GO content increased from 3 to 5 mg/ml. This phenomenon can be explained that the GO sheets trend to form irreversible agglomerates due to the van der Waals force at high GO content, which lead to a reduced capacity of GO sheets to disperse in the polymer matrix [33]. 3.6 FT-IR analysis The FTIR spectra of CS, CMCS are shown in Fig. 8(a). In the FTIR spectrum of CS, the bands at 3419cm-1, 1651cm-1, 1605cm-1, 1382cm-1, 1093cm-1 are attributed to the O-H stretching vibration, C=O stretching mode of N-acetylguscosamine, N-H bending vibration, C-H bending vibration, C-O stretching vibration, respectively. From the FTIR spectrum of CMCS, it could be observed that characteristics bands at 1596cm-1, 1410cm-1, 1065cm-1, which are due to the asymmetric and symmetric stretching vibration of COO-, and the stretching vibration of C-O-C, respectively. This demonstrated the introduction of COO- group to CS chains [15]. The FTIR spectra of KGM, OKGM are shown in Fig. 8(b). In the FTIR spectrum
of KGM, the bands at 3419cm-1, 1734cm-1 are ascribed to the O-H stretching vibration, C=O stretching vibration of the acetyl group, respectively. Obviously, two characteristic bands around 1730cm-1 and 895cm-1 are observed in the spectrum of OKGM. The former is due to the aldehyde symmetric vibrational band and the latter is due to the hemiacetal structure [26, 27]. The FTIR spectra of GO-0, GO-1, GO-3, GO-5 hydrogels are shown in Fig. 8(c). Compared with GO-0 hydrogel CMCS and OKGM, the C=O stretching vibration of the acetyl group band at 1731cm-1 was disappeared and the characteristic band of hemiacetal structure at 895cm-1 was practically disappeared, along with the stretching vibrations of C=N band at 1640cm-1 was appeared. It indicates that the crosslinking reaction is followed between –CHO groups of OKGM and –NH2 groups of CMCS. For hydrogels, it can be seen that some peaks are enhanced but no obvious change of peak position occurs in the spectra, which indicates that the formation of hydrogen bonding between the oxygen-containing functional groups of GO and the hydroxyl groups on the CMCS and OKGM molecular chains [33, 49] 3.7 Structure and morphology analysis The digital photo of composite hydrogels with different GO loadings is shown in Fig. 9(A). It can be seen that the GO sheets are homogeneous dispersion in hydrogels. Meanwhile, with the GO content increased, the white hydrogels first turn to brown and then to black.
The porous microstructure of the scaffolds had significant influence on the ability of the cells intrusion, proliferation and function in tissue engineering
[50]
. The
SEM images are obtained to characterize the microstructure of the hydrogels. The cross-section images of the hydrogels at different magnifications are shown in Fig. 9. It can be observed that all the hydrogel samples have a continuous and stable three-dimensional network structure, which are similar to some earlier reported polysaccharide and GO hydrogels. Furthermore, all the hydrogel samples exhibit many micro-pores, which distribute uniformly in the internal of the hydrogels. The interconnection between pores could be attributed to the network formed by the cross-linking between CMCS and OKGM, and the hydrogen bonds between GO nanosheets and the polymer chains. Compared with GO-0(fig 9B1), GO-3(fig 9C1) and GO-5(fig 9D1), it also can be observed that the average pore diameter of the gels have no obvious change as the concentration of GO increased from 0 to 5mg/ml. However, it can be seen that the wall of GO-0 sample seems to be torn during the freeze drying process, which indicated that the hydrogels with GO have sufficient capability to avoid the structural collapse during dehydration. This result is consistent with the result of mechanical properties. It is believed that the strong hydrogen bonding interaction between GO and the polymer chains are the main reason for this result. The hydrogels with GO exhibit more stable network structure than the hydrogels without GO. 3.8 Cytotoxicity assay
A wound dressing designed to be placed in wound and direct contact with wound tissue should possess the highest possible biocompatibility. In vitro cytotoxicity assays have the advantages of being simple, reproducible, cost-effective, and suitable for the evaluation of basic biological aspects relative to biocompatibility. The cell viabilities of GO-0, GO-1, GO-3, GO-5 are shown in Fig.10. The cell viabilities of all the samples were higher than 90% during the whole incubation period, which could be classified to a scale 0 (non-toxicity). The cell viabilities were related with the GO content and incubation time. It can be seen that the cell viabilities of all the samples significantly increase with the increase of incubation time and the cell viabilities of GO-1, GO-3, GO-5 hydrogels were higher than GO-0 in the incubation period. In addition, cells incubated with the extract from GO-1 hydrogel showed the highest viabilities during the test period. The higher cell viabilities of GO-1, GO-3, GO-5 hydrogels than GO-0 hydrogel suggests that the addition of GO to hydrogels promotes cell growth without producing additional cytotoxicity. This result is consistent with previous studies, which have confirmed that GO have positive effects on adhesion, grown, proliferation and differentiation of cells
[51, 52]
. Furthermore, compared with GO-1, GO-3, GO-5, the
cell viabilities significantly decrease with the increase of GO content. This phenomenon may be due to the GO content in extracts increase with the increase of the GO content in hydrogels and GO display a significant cytotoxic effect at high concentration [53].
4. Conclusions In this article, graphene oxide (GO) was added as a nano-additive in CMCS/OKGM/GO composite hydrogels, which were prepared through the crosslinking reaction between oxidized konjac glucomannan (OKGM) and carboxymethyl chitosan (CMCS). Through a series of properties characterization, the hydrogels showed rapid gelation process, good swelling ability, appropriate water retention capacity and a stable three-dimensional network structure, which all satisfy with the application as a wound dressings. Especially, the compressive strength and modulus increased significantly with loading of GO, which can be attributed to the hydrogen bonding between GO sheets and the polymer chains. Moreover, the in vitro cytotoxicity assay demonstrated that all the hydrogels have good biocompatibility with a cytotoxicity of grade 0, and the hydrogels with GO have better biocompatibility than hydrogels without GO. Therefore, OKGM/CMCS/GO hydrogels have great clinical potential to be applied as a wound dressing. Acknowledgements The work was supported by the National Natural Science Foundation of China (Foundation No. 51173143, No. 51273156), The Special Funds Project of Major New Products of Hubei Province (Foundation No. 20132h0040), University-industry Cooperation Projects of The Ministry of Education of Guangdong province (Foundation No. 2012B091100437), The innovation fund project of the Ministry of Science and Technology of Small and Medium-sized Enterprises (Foundation No.
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Transmittance (a.u.)
Figure Captions
GO
1731
1400
1623
1052
3999
4000
3500
3000
2500
2000
1500
Wavenumber (cm-1)
Fig. 1. FTIR spectra of the GO powders.
1000
500
Fig. 2. (a) TEM photo of GO aqueous solution, (b) digital photo of GO(1mg/ml)/OKGM(5%wt) solution.
G
Intensity (a.u.)
D
0
500
1000
1500
2000
2500
-1
Raman shift (cm )
Fig. 3. Raman spectrum of GO powders.
3000
3500
38 36 34 32
凝胶时间
30 28 26 24 22 20 18 GO-0
GO-1
GO-3
GO-5
Fig. 4. Gelation time of GO-0, GO-1, GO-3, GO-5 hydrogels.
240 GO-1
235
GO-3
230
GO-5
225
GO-0
Swelling ratio (%)
220 215 210 205 200 195 190 185 180 175 0
10
20
30
40
50
Time (h)
Fig. 5. Swelling percentage of composite hydrogels with different GO loadings.
110
GO-1 GO-3 GO-5 GO-0
100
Water lost (%)
90 80 70 60 50 40 30 0
5
10
15
20
25
Time (h)
Fig. 6. Water loss of GO-0, GO-1, GO-3, GO-5 hydrogels.
1.2
200
compressive strength compressive modulus
0.8
150
0.6
100 0.4
50
compressive modulus (MPa)
compressive strength (KPa)
1.0
0.2
0
0.0
GO-0
GO-1
GO-3
GO-5
Fig. 7. Compressive strength and modulus of composite hydrogels with different GO loadings. (a)
Transmittance
CS 1382 1651 1605 1155 2879
1093
3419
CMCS
1325 1596
4000
3500
3000
2500
2000
1410
1500
1065
1000
500
-1
Wavenumber(cm )
(b)
KGM
1734 1638
2925
Transmittance
3419
OKGM 1730 895 2925
1644
3420
4000
3500
3000
2500
2000
1500 -1
Wavenumber(cm )
(c)
1000
500
GO-5
Transmittance
2928 1640 1410
GO-3 3418
1063
2927 1640
GO-1
3423
1410 1064
2925
1410 1640 1064
3421
GO-0 2923
1640
1410 1065
3424
4000
3500
3000
2500
2000
1500
1000
500
-1
Wavenumber (cm )
Fig. 8. (a) FTIR spectra of the CS powders, CMCS. (b) FTIR spectra of KGM powders, OKGM. (c) FTIR spectra of GO-0, GO-1, GO-3, GO-5 composite hydrogel.
(A)
(B)
(B1) (50×)
(B2) (200×)
(B3) (1000×)
(C2) (200×)
(C3) (1000×)
(C)
(C1) (50×) (D)
(D1) (50×)
(D2) (200×)
(D3) (1000×)
Fig. 9. (A) Digital photo of composite hydrogels with different GO loadings, SEM images of the cross-section of the GO-0 hydrogel (B), GO-3 hydrogel (C) and GO-5 hydrogel (D) at different magnifications.
120 110 100
GO-0 GO-1 GO-3 GO-5
Cell viability (%)
90 80 70 60 50 40 30 20 10 0 Day 1
Day 3
Day 5
Fig. 10. The cell viabilities of NIH-3T3 cells after incubation for 1, 3, 5 days with extracts form GO-0, GO-1, GO-3, GO-5 hydrogels.
CH2OH
CH 2OCH2COONa O
O
O
ClCH2COOH
OH
O
OH
NaOH n
n
NH2
NH2
CS
CMCS
Scheme 1. The synthesis of Carboxymethyl Chitosan
OH
OH O
O
O
OH
OH O
NaIO4
OH n
OH
OH
OH O
O
O
O
CH O
KGM Scheme 2. The synthesis of Oxidized Konjac Glucomannan
OH
OH
n
OH
OKGM
OKGM
OH
OH
OH O
O
O
O
CH
OH
OH
HO
n
O
O
OH
HO
O COOH
+
COOH OH
HOOC CH2 OCH2 COONa
OH
O COOH COOH OH COOH
HO
OH OH
O
OH O
OH
O
O
n NH2
O
CH
OH COOH
HO
+ OH
OH HO
COOH
n OH
O OH
O
HO
OH
OH
OH
N
CMCS O COOH COOH OH COOH
O
n
CH2 OCH 2COONa
OH O O
OKGM-CMCS-GO Hydrogel
COOH COOH
OH
HO
COOH O
HO
O
COOH
HO
OH COOH
HO OH
GO 2D sheet
Scheme 3. Schematic illustration of the synthesis route of the hydrogel