oxidized konjac glucomannan hydrogel

International Journal of Biological Macromolecules 108 (2018) 376–382

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Preparation and characterization of chitosan – collagen peptide / oxidized konjac glucomannan hydrogel Liangling Liu a , Huigao Wen a , Ziqie Rao a , Chen Zhu a , Meng Liu a , Lian Min a , Lihong Fan a,∗ , Shengxiang Tao b,∗∗ a b

School of Chemistry, Chemical Engineering and Life Sciences, Wuhan University of Technology, Wuhan 430070, China Department of Orthopedics, Zhongnan Hospital of Wuhan University, Wuhan 430070, China

a r t i c l e

i n f o

Article history: Received 11 July 2017 Received in revised form 23 September 2017 Accepted 19 November 2017 Available online 21 November 2017 Keywords: Oxidized konjac glucomannan Chitosan-collagen peptide Hydrogel Blood clotting activity Wound healing

a b s t r a c t In this paper, the microbial transglutaminase (MTGase) was used as a catalyst to graft the collagen peptide (COP) molecules on the amino group of chitosan to obtain water-soluble chitosan-collagen peptide (CS-COP) derivatives. The preparation of composite hydrogel was via the Schiff-base reaction between the amino of CS-COP and the aldehyde of oxidized konjac glucomannan (OKGM). The hydrogels were characterized by various techniques including Fourier transform infrared spectroscopy (FT-IR) and scanning electron microscopy (SEM). The results of SEM showed that the hydrogel sample had a clear and stable three-dimensional network structure. Meanwhile, these effects of the addition of OKGM on gelation time, swelling behaviors, water evaporation rate and blood coagulation capacity were investigated. The shortest gelation time for hydrogels was 99.3 s. The hydrogels showed a good swelling ability and appropriate water retention capacity. The maximum swelling ratio of the hydrogel was 265%. Dynamic blood clotting test showed that the hydrogels materials had good blood coagulation capacity. Moreover, The biocompatibility of hydrogels was evaluated with NIH-3T3 cells by MTT method. The results indicated that the hydrogels exhibited better biocompatibility. Therefore, this hydrogel has a promising potential to be applied as wound dressing. © 2017 Published by Elsevier B.V.

1. Introduction Human skin is an important organ of the body against the external environment. One of its most vital functions is protection against external mechanical aggressions, which is ensured by using reversible deformation of its structure [1,2]. The loss that large portions of the skin would lead to a lesion can bring out major disability or even death [3]. Hydrogels with characteristic properties such as desired functionality, reversibility, sterilizability and biocompatibility have many incredible purposes in tissue engineering, biology and pharmaceutical sciences [4–7]. Hydrogels can maintain a moist environment at the wound interface which is important for the wound-healing process [8]. Hydrogels can also absorb body fluids and simultaneously prevents their excessive loss, being permeable to oxygen, nutrients, and other water-soluble metabolites [9,10]. Furthermore, there are many similarities between hydro-

∗ Corresponding authors. ∗∗ Corresponding author. E-mail addresses: [email protected] (L. Fan), [email protected] (S. Tao). https://doi.org/10.1016/j.ijbiomac.2017.11.128 0141-8130/© 2017 Published by Elsevier B.V.

gels and the native extracellular matrix (ECM) [11,12], and they are a three-dimensional network structure which is beneficial to cell adhesion, proliferation, transportation of cytokines, nutrients and metabolic waste [13].Thus, hydrogels are well suited for use as wound dressings. As a unique triple-helical structural protein, collagen is the primary component of extracellular matrices existing in all multicellular animals [14].Therefore collagen has been widely used in biomedical applications fields including hemostatic [15], drug delivery systems [16], wound dressings [17], and tissue engineering scaffolds [18]. COP is hydrolyzed from collagen, which has good antioxidant properties [19] and could be particularly beneficial for wound healing application [20]. Chitosan is a natural polymer derived from crustacean shells, insect exoskeletons, fungi, and algae, can effectively promote blood coagulation [21]. Chitosan possesses desirable properties, such as biodegradable, biocompatible, nontoxic and bioactive [22]. So, it has a beneficial effect as a wound healing promoter [23–25]. However, it is suffering due to its poor solubility in water which restricts biomedical application to some extent [26,27]. Accordingly, to exploit its application, increasing the solubility of chitosan will

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be a prerequisite. Compared to chemical modification, Enzymecatalyzed reactions are constantly investigated as an attractive alternative to toxic, environmentally unfriendly and non-specific chemical approaches [28]. MTGase, an extracellular enzyme of the class of transferases [29], is used in many applications to attach proteins and peptides to small molecules, polymers, surfaces, DNA, as well as to other proteins at present [30]. And it catalyses an acyl transfer reaction between ␥–carboxyamide groups of peptidebound glutamine residues and ␧-amino groups of lysine or primary amino groups of some polyamines [31]. In the previous work, we used MTGase as the catalyst to graft the COP on the amino group of chitosan to obtain the CS-COP derivative, and its solubility was greatly improved [32]. Konjac glucomannan (KGM) is a natural polysaccharide that is made up of ␤-1,4-linked D-mannose and D-glucose [33]. KGM can be prepared into various derivatives easily because of its good biocompatibility and biodegradable activity [34]. As a ␤-(1,4) linked polysaccharide, KGM can react with sodium periodate to obtain OKGM [35]. After the oxidation reaction, the carbon–carbon bonds of the cis-diol group in the molecular chain of KGM are cleaved and forms an aldehyde structure, which can react with CS-COP via Schiff-base reaction. In this paper, we have prepared composite hydrogels from OKGM and CS-COP without employing any extraneous crosslinking agents, which were verified by FT-IR and SEM. The physical properties of the resulting hydrogels, such as gelation time, swelling ability, water evaporation rate were studied by varying the amount of OKGM. The hemostatic properties of the hydrogels were also evaluated in vitro. In addition, Cytotoxicity of gels was evaluated by methyl thiazolyl tetrazolium (MTT) assay. 2. Experimental 2.1. Materials Konjac glucomannan (The content of glucomannan is above 85%) was purchased from Konson konjac Corp. (Wuhan, Hubei, China). Chitosan (Mw 520,000) with a 92% degree of deacetylation was purchased from Zhejiang Yuhuan Ocean Biochemistry Co. Let. (China). Collagen peptide (Mw 800) was purchased from Sichuan Mingrang Biological Technology Co. Ltd., Sichuan, China, without further purification. Microbial transglutaminase was purchased from Huashun Biological Technology Co. Ltd., Wuhan, China. Sodium hydroxide, sodium periodate, ethylene glycol, acetic acid, sodium dihydrogen phosphate, disodium hydrogen phosphate, calcium chloride 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 oxidized konjac glucomannan (OKGM) The preparation of OKGM is described in [36] with slight modification. Into 5 g KGM dissolved in 500 mL distilled water. Fifty milliliter 0.15 mol/L NaIO4 solution was added dropwise and the mixture was stirred vigorously at 30 ◦ C in the dark for 12 h. The degree of oxidation was found by determining the concentration of unreacted periodate by iodometry after 12 h [37]. Briefly, an aliquot (5 mL) of the reaction mixture was neutralized with 10 mL of 10 wt% sodium bicarbonate solution, and iodine was liberated by the addition of 20% potassium iodide solution (2 mL).After keeping in dark for 15 min, the liberated iodine was titrated with standardized sodium thiosulphate solution using starch as the indicator. And the oxidation degree of OKGM was determined to be 28%.Then 10 mL ethylene glycol was added to reaction mixture to remove unreacted periodate and stirred for another 2 h. After reaction,

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solutions were dialyzed against distilled water for 3 d with several changes of water till the dialyzate was periodate-free (checked with silver nitrate). The dialyzate was then centrifuged for 20 min at 3000 rpmThe supernatant was dried at 50 ◦ Cto constant weight for the subsequent measurements. The procedures of synthesizing OKGM were as follows in Supplementary Data S1.

2.3. Preparation of chitosan – collagen peptide (CS-COP) The details of preparation of CS-COP can be found in our previous report [32] .Briefly, appropriate amount of MTGase was dissolved in 50 mL of PBS (0.2 mol/L, pH 6.0) buffer solution. After the solution was centrifuged, the supernatant was vacuum filtered. The filtrate was dialyzed through the 8000–10,000 molecular weight cut-off dialysis tubing for 72 h and lyophilized to obtain purified MTGase lyophilized powder. Two grams of chitosan dissolved in 1% acetic acid solution was mixed with COP (2 g) dissolved in PBS (0.2 mol/L, pH 6.0) and pH was adjusted to 6.0. Then 0.2 g of purified MTGase lyophilized powder was added and mechanically stirred at 40◦ C for 1.5 h. Then the solution was treated in boiling water for 10 min and later cooled to room temperature. The CS-COP solution was obtained after filtering. Subsequently, the solution was neutralized with 20% (w/w) aqueous NaOH. Finally, the solution was dialyzed with distilled water through the 8000–10,000 molecular weight cut-off dialysis tubing for 72 h, and freeze-dried to obtain the purified CS-COP. The degree of substitution of CS-COP was confirmed by ultraviolet-visible spectroscopy.CS-COP (0.05 g/l) was dissolved in deionized water, and its absorption was measured at 200 nm. A standard curve of collagen was established in the range of 0.001–0.05 g/l. The degree of substitution of CS-COP is 0.554.The reaction is shown in Supplementary Data S2.

2.4. Preparation of CS-COP/OKGM hydrogel A certain amount of CS-COP and OKGM was added to distilled water, magnetic stirred continuously at room temperature until dissolved to a final concentration of 6%wt, respectively. Volumes of 2, 4, 6, 8 and 10 mL of OKGM solution were added to the CS-COP solution. The mixture stood for some time to get the CS-COP/OKGM hydrogel. According to the amount of OKGM, the hydrogels were marked as OKCP-2, OKCP-4, OKCP-6, OKCP-8, and OKCP-10, respectively. The procedures of synthesizing hydrogels were as follows in Scheme 1.

2.5. Gelation time test In this study, gelation time was assessed according to the previously reported method [38]. A mixture of CS-COP and OKGM solution was added in a 15 mL flat bottom vial (diameter 26 mm) and stirred using a Teflon magnetic stir bar (diameter 5 mm, length 10 mm) at 155 rpm. Gelling time was noted as the time required for the stir bar to stop using a stop watch.The experiments were performed in triplicate

2.6. Swelling measurements The hydrogel samples (column, diameter 20 mm and height 10 mm) were lyophilized with a freeze-dryer. Then the samples were immersed in PBS buffer solution (PH = 7.4) at room temperature. After a period of time, the hydrogel can achieve swelling equilibrium, and the samples were removed from the tube and gently absorbed with filter paper to remove the excess of liquid

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Scheme 1. Schematic illustration of the synthesis route of the hydrogel.

on the surfaces, and weighed. The swelling rate of the hydrogel is calculated according to the following equation. mt − m0 × 100% SR = m0 Where mt and m0 are the weights of the samples at the equilibrium swelling state and lyophilized state, respectively. All experiments were done in triplicate.

2.7. Water evaporation rate The lyophilized gel sample (column, diameter 20 mm and height 10 mm) was immersed in distilled water. After the hydrogel reaches the swell equilibrium, the hydrogel surface moisture is completely removed and weighed, denoted as m1 . The sample was then placed in a constant temperature oven (oven ambient parameter set at: 50 ◦ C, 50% humidity),and weighed at regular intervals until it became constant. The water evaporation rate was calculated by the following formula: m1 − m2 Waterlost% = × 100% m1 − m3 Where m1 , m2 , m3 are the initial weight, measured weight and the final weight of the hydrogels, respectively. The data for each sample was calculated using triplicate measurement.

2.8. Physical characterization of the hydrogel FTIR spectra of CS, KGM, CS-COP, OKGM and OKCP-6 hydrogel samples were measured with a Nicolet 170SX Fourier transform infrared spectrophotometer (USA) in the wavenumber ranging from 400 to 4000 cm−1 . The test samples were prepared by the KBr-disk method.

2.9. Scanning electron microscopy (SEM) The morphology of the composite hydrogels were characterized by scanning electron microscopy (SEM). The hydrogels were freezedried using a freeze drier (Christ, Germany, Alpha 1–2) at −52 ◦ Cfor 6 h and then coated with a gold layer. The cross sections morphologies were performed with a JSM-5610 SEM (JEOL, Japan) at 20 kV accelerating voltage.

2.10. Dynamic blood clotting measurement The coagulation activity of the gel was investigated by BCI experiment. Samples of equal weight were placed into beakers and pre-warmed to 37 ◦ C. Then 0.1 mL anticoagulated blood was slowly dropped on the surface until the gel was completely covered following by adding 12.5 ␮L CaCl2 solution of 0.2 mol/L .The bottles were incubated in a thermostate incubator at 37 ◦ C. After 3 min, 5 min, 10 min, 15 min, 20 min, 10 mL DI water were carefully added along the sides of the bottles and centrifuged at 1000 rpm for 5 min. The absorbance of the supernatant was measured by UV–vis spectrophotometer at a wavelength of 545 nm. Each group of gel samples was repeated three times in parallel. Blood without hydrogel was used as a reference value. The BCI of the hydrogel is calculated based on the equation. BCI = Asample+blood /Ablood × 100 2.11. Cell culture and cytotoxicity assay The CS-COP and the OKGM were irradiated by UV for 2 h, then dissolved in sterile distilled water to prepare the solution. The hydrogel samples were prepared in sterile environment and were cut into cylindrical specimens with a diameter of 10 mm and a thickness of 2 mm. The hydrogel samples were placed in 6-well cell culture plates containing cell culture medium and incubated in humidified atmosphere containing 5% CO2 at 37 ◦ C for 24 hThen, the eluent in contact with the samples was then extracted by 0.22 ␮m membrane filtration and stored at −4 ◦ C before further use. Mouse embryonic fibroblasts cells were fed by Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS), 100U/ml penicillin, and 100 ␮g/ml streptomycin in a humidified atmosphere at 37 ◦ C and 5% CO2 ·For all cell lines the medium was renewed every 2 or 3 days. Exponentially growing cell were detached with 0.25% trypsin and then resuspended in fresh culture medium for further inoculation. The fourth generation of cells were seeded in 96-well plates at the density of 5000 cells/well and incubated for 24 h to allow cells to adhere. Then the medium was removed and cells were washed twice with PBS. 200 ␮L of hydrogel sample’s elution solution previously obtained was added to each well and was refreshed every day. After incubation for 1d, 3d, 5d respectively, 20 ␮L MTT solution (5 mg/ml) was injected into each well and the cells were incubated at 37 ◦ C for an additional 4 h. Then the medium in the cell plates were replaced with 150 ␮L dimethylsulfoxide (DMSO) and the plates were shaken for 15 min. The absorbance was measured

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0 Fig. 1. Gelation time of Composite hydrogels. Fig. 2. Swelling percentage of composite hydrogels with different amounts of OKGM.

at 490 nm using a microplate reader. The wells were not added the hydrogel sample’s extraction elution solution as a positive control group and only contain DMSO as a background group. Percent cell viability was calculated by the following equation: Viablecell =

ODs − ODb ×100% ODc − ODb

Where ODs is the absorbance of the test wells, ODb is the blank group, and ODc is the positive control group. 3. Results and discussion 3.1. Gelation time Gelation times of different amounts of OKGM are shown in Fig. 1. As shown in Fig. 1, with the increase in the amount of OKGM, the gelation time decreased first and then increased. The shortest gel time was 99.3 s at 6 mL. That was because when OKGM amounts lower than 6 mL, the number of the aldehyde groups increases with the increase of the amounts of OKGM, which reacts with the aminos of CS-COP to form the crosslinked network structure and correspondingly shorter reaction time. However, when the amount of OKGM was more than 6 mL, the aldehyde content in the system had reached saturation. Excessive OKGM solution reduces the overall concentration of the mixed solution. This resulted in a decrease in the rate of crosslinking reaction and a longer gel time [39].

3.3. Water evaporation rate The maintenance of a moist wound bed is of ideal environment for effective wound healing [40]. Therefore, water evaporation rate is one of the most important properties of hydrogel-based biomaterials, which represent the ability of the hydrogel to maintain the wound moist environment. The hydrogel dressing possess a smaller water evaporation rate, which leads to quicker healing, less pain and great cost savings[41]. Fig. 3 shows the evaporation rate of water for different hydrogels. It can be seen that the water loss rate of all hydrogels rapidly increased in initial 12 h. It reached a constant percentage in 24 hours and remained 5% to 15% of water. It shows that the hydrogels have good water evaporation rate. Furthermore, with the increase of the content of OKGM, the water loss rate of hydrogel gradually decreased. 3.4. FT-IR analysis FTIR spectra of CS, CS-COP, OKCP-6 are presented in Supplementary Data S3(a). As for the CS, the wide absorption band at 3426 cm−1 was assigned to the stretching vibration of O H groups, and the peaks at 1650 and 1598 cm−1 were attributed to amide I bands and N H bending vibration, respectively. In the FTIR spectrum of CS-cop, there is almost no band around 1598 cm−1 and 100 OKCP-2 OKCP-4 OKCP-6 OKCP-8 OKCP-10

3.2. Studies of swelling 90

Generally, a good hydrogel-based wound dressing needs to have the ability to absorb body fluid and transfer cell nutrients and metabolites, which can be characterized by swelling ratio of hydrogel. The swelling test results of different OKGM hydrogels in PBS (pH = 7.4) at room temperature are shown in Fig. 2.All the hydrogels show good swelling properties. It can be explained that the CS-COP, the OKGM contains a large amount of hydrophilic groups and the hydrogel has a highly porous network structure. So the free water could enter the porous network structure of the hydrogels. Generally, high concentrations of OKGM resulted in decrease of swelling ratio on composite hydrogels. These results were expected, because increasing OKGM concentration allows for higher crosslinking density. The relatively high density of hydrogel possess smaller pore size and the degree of swelling ratio decreases. It can be seen from Fig. 2 that when the amount of OKGM is 4 mL, the maximum swelling rate of the hydrogel is 265%.

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Fig. 3. Water loss of OKCP-2, OKCP-4, OKCP-6, OKCP-8,OKCP-10 hydrogels.

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Fig. 4. SEM images of the cross-section of the OKCP-2 hydrogel (A), OKCP-6 hydrogel (B) and OKCP-10 hydrogel (C) at different magnifications.

secondary amides (1552 cm−1 ) are formed. This demonstrated that the COP introduction reaction had taken place between amino group in CS and NH CO groups had been formed. Therefore, FT-IR spectra proved that CS-COP was synthesized [32]. The FTIR spectra of KGM, OKGM and OKCP-6 are shown in Supplementary Data S3(b). In the FTIR spectrum of KGM, the bands at 3418 cm−1 , 1736 cm−1 are ascribed to the O H stretching vibration, C O stretching vibration of the acetyl group, respectively. Comparing to the absorption of KGM, two new absorption peaks around

1730 cm−1 and 893 cm−1 were detected in the spectrum of OKGM. The former is consistent with the aldehyde symmetric vibrational band and the latter is due to the hemiacetal formation of free aldehyde groups [35,36]. However, these two strong absorption peaks weakened or even disappeared in the spectrum of OKCP-6 hydrogel, and the stretching vibrations of C N band at 1640 cm−1 was appeared. It suggests that the Schiff base reaction occured between CHO groups of OKGM and NH2 groups of CS-COP.

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Fig. 5. Effect of different amounts of OKGM on coagulation performance.

Fig. 6. The cell viabilities of NIH-3T3 cells after incubation for 1, 3, 5 days with extracts form OKCP-2, OKCP-4, OKCP-6, OKCP-8, OKCP-10 hydrogels.

3.7. Cytotoxicity assay 3.5. Structure and morphology analysis The role of porosity and interconnectivity in hydrogels is important for cells intrusion, proliferation and function in tissue engineering [42]. Those SEM images are obtained to characterize the microstructure of the hydrogels. Fig. 4 shows the SEM images of freeze-dried hydrogel samples at different magnifications. It can be observed that the hydrogels possess a continuous and stable three-dimensional network structure. Furthermore, all the hydrogel samples exhibit large amounts of micropores, which distribute evenly in the internal of the hydrogels. Compared with OKCS-2, OKCS-6 and OKCS-10, it’s obvious that the average pore diameter of hydrogel tends to decrease with the increase of the amount of OKGM. This may be due to crosslink density increases with the increase of the amount of OKGM, resulting in a denser hydrogel.

Wound dressings should have good biosecurity and be non-toxic to cells, for they are in direct contact with human tissue. Cytotoxicity assays is an important indicator of the impact of biomaterials in vitro cell viability and proliferation. Fig. 6 shows the cytotoxicity of OKCP-2, OKCP-4, OKCP-6, OKCP-8, OKCP-10 hydrogels on mouse embryonic fibroblasts. It can be seen from the figure that the cell viability of different samples during the 1, 3, 5 days culture period were higher than 90%, indicating that all hydrogel samples were not bio-toxic to NIH-3T3 cells. There was no significant difference in cell viability between the groups. When the amount of OKGM increased, the cell survival rate decreased slightly. It is possible that the uncrossed OKGM disrupts the nutrient balance of the culture medium and affects the cell proliferation. In addition, the cell viabilities of all the samples significantly increase with the increase of incubation time. Consequently it provides that CS-COP/OKGM hydrogel has good cytocompatibility and can promote the proliferation of NIH-3T3 cells.

3.6. Blood clotting measurement 4. Conclusion The blood clotting ability was conducted to assess the hemostatic potential of the material, which holds an important property of an ideal wound dressing material. In general, the lower the BCI index indicates the higher blood clotting ability of the material [43]. Fig. 5 shows the BCI of the OKCP-2, OKCP-4, OKCP-6, OKCP-8, OKCP-10 hydrogels. It is clear that all hydrogels have shown a good blood clotting ability. In particular, the OKCP-6 hydrogel has the best hemostatic effect with 0.034 blood clotting index. From the blood sample contacting time, the BCI value is rapidly declining. In addition, OKCP-6 hydrogel reached the lowest BCI value at 5 min, other hydrogel samples had similar coagulation effects and reached the lowest BCI value at 10 min. The hemostatic mechanism of the complex hydrogel is as follows. On the one hand, both chitosan and collagen peptides have a good hemostatic effect. Chitosan contains positively charged ammonium group (–NH3 +) which absorbs negatively charged blood cells and results in blood clotting [44]. Collagen can support a clotting response of platelet deposition, platelet-localized thrombin generation, and fibrin polymerization [45]. On the other hand, hydrogel has a stable porous structure and liquid absorption properties which can quickly absorb a large amount of blood from the wound. And it can be attached to a wound surface to block the ruptured of blood vessels.

In this paper, MTGase was effective in catalyzing collagen chains onto the chitosan backone. Hydrogels were obtained through the crosslinking reaction between the active aldehyde of OKGM and the amino of the CS-COP. This new class of hydrogels showed good equilibrium swelling properties, water evaporation rate, blood compatibility and no cytotoxicity. The results showed that these hydrogel dressings have the capacity to absorb and retain the wound fluid, which is necessary for wound debridement and maintenance of a moist wound environment for rapid wound healing. The hydrogel has a good swelling property and a maximum swelling rate of 265%.Water evaporation experiments show that hydrogels have the appropriate water retention capacity. Dynamic coagulation test demonstrated that all hydrogel materials had good coagulation performance and could be used as a hemostatic material. In addition, the results of cytotoxicity test showed that the hydrogel had excellent cell compatibility and promoted the proliferation of NIH-3T3 cells. Acknowledgements The work was supported by the National Natural Science Foundation of China (Foundation No. 51773161), The Special Funds

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