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Biological properties of dialdehyde carboxymethyl cellulose crosslinked gelatin–PEG composite hydrogel fibers for wound dressings
Carbohydrate Polymers 137 (2016) 508–514
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Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol
Biological properties of dialdehyde carboxymethyl cellulose crosslinked gelatin–PEG composite hydrogel fibers for wound dressings Defu Li a,∗ , Youxin Ye a , Derong Li b , Xinying Li c , Changdao Mu a,∗ a
School of Chemical Engineering, Sichuan University, Chengdu 610065, PR China People’s Hospital of Lanshan District, Linyi 276000, PR China c College of Chemistry and Environment Protection Engineering, Southwest University for Nationalities, Chengdu 610041, PR China b
a r t i c l e
i n f o
Article history: Received 30 July 2015 Received in revised form 4 September 2015 Accepted 6 November 2015 Available online 10 November 2015 Keywords: Hydrogel fiber Dialdehyde carboxymethyl cellulose Gelatin Gel-spinning Wound dressings
a b s t r a c t Gelatin-based composite hydrogel fibers were prepared by gel-spinning with PEG6000 as the modifier. Dialdehyde carboxymethyl cellulose (DCMC), as an ideal crosslinking reagent for protein, was used to fix the composite hydrogel fibers. Then the biological properties of the hydrogel fibers for wound dressings were evaluated. The results indicate that the hydrogen bond interactions and C N linkages between gelatin and DCMC can be formed. The addition of DCMC can efficiently improve the mechanical properties, enzymatic stability and blood compatibility of the hydrogel fibers. Crosslinking with DCMC can reduce the degree of swelling of the hydrogel fibers, which is beneficial for hydrogel fibers to avoid undesired reduction in mechanical properties. Moreover, the composite hydrogel fibers present three-dimensional structure, porous networks and low cytotoxicity. The study suggests that DCMC is an effective crosslinking reagent for biomaterials fixation. The developed composite hydrogel fibers can be well-suited for biomedical applications such as wound dressings. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction Gelatin is an animal protein obtained by a controlled hydrolysis of fibrous insoluble collagen, which is generated as waste during animal slaughtering and processing (Guo, Li, Mu, Zhang, Qin, & Li, 2013). Gelatin is an effective biomaterial as wound dressings since it can absorb wound exudates and provide moist environment to accelerate wound healing (Kavoosi, Dadfar, & Purfard, 2013). It also provides immediate haemostasis and helps to prevent wound contracture and contour deformities associated with conventional wound healing (Rullan, Vallbona, Rullan, Mansbridge, & Morhenn, 2011; Wong, MacRobert, Cheema, & Brown, 2013). Positive biological responses facilitating cell adhesion and proliferation are the properties of gelatin that should also be highlighted (Saik, Gould, Watkins, Dickinson, & West, 2011; Young, Wong, Tabata, & Mikos, 2005). However, the main disadvantages of gelatin are connected with its poor mechanical properties and low thermal stability, which limit its applications as wound dressings (Dash, Foston, & Ragauskas, 2013; Weng, Pan, & Chen, 2007). Therefore,
∗ Corresponding authors. Tel.: +86 28 8540 5221; fax: +86 28 8540 5221. E-mail addresses: [email protected] (D. Li), [email protected] (C. Mu). http://dx.doi.org/10.1016/j.carbpol.2015.11.024 0144-8617/© 2015 Elsevier Ltd. All rights reserved.
many researchers are working at the realm of reinforcement of gelatin. To date, many attempts have been made to modify the poor properties of gelatin including crosslinking, blending, compounding with natural fibers and nanosized clay (Martucci & Ruseckaite, 2010). Glutaraldehyde (Ulubayram, Cakar, Korkusuz, Ertan, & Hasirci, 2001), N-[3-(dimethylamino)propyl]-N ethylcarbodiimide (EDC) (Choi et al., 2001), transglutaminase (Ito et al., 2003), plant polyphenols (Rocasalbas et al., 2013), formaldehyde (De Carvalho & Grosso, 2004), oxidized chondroitin sulfate (Dawlee, Sugandhi, Balakrishnan, Labarre, & Jayakrishnan, 2005), oxidized alginate (Balakrishnan, Mohanty, Fernandez, Mohanan, & Jayakrishnan, 2006; Balakrishnan, Mohanty, Umashankar, & Jayakrishnan, 2005; Xu, Li, Yu, Gu, & Zhang, 2012), dialdehyde starch (Mu et al., 2010) and cellulose nanowhiskers (Dash et al., 2013) are the effective crosslinking reagents to reinforce gelatin in order to increase the resistance to thermal degradation and improve the mechanical properties. Note that oxidized polysaccharides have received an increasing attention as ideal crosslinking reagents of protein in recent years. The oxidation of polysaccharides by periodate is characterized by the specific cleavage of the C2 C3 bond of glucose residues. This cleavage results in the formation of two aldehyde groups per glucose unit, forming
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2,3-dialdehyde polysaccharides (Li, Wu, Mu, & Lin, 2011). The aldehyde groups in dialdehyde polysaccharides can crosslink with -amino groups of lysine or hydroxylysine side groups of gelatin by C N linkages (Schiff’s base) to improve the properties of gelatin (Ge et al., 2015; Guo, Ge, Li, Mu, & Li, 2014). Dawlee et al. (2005) had reported a new class of gelatin hydrogel using aldehyde chondroitin sulfate as a crosslinking reagent. This new class of hydrogel without employing any extraneous crosslinking reagents was expected to have potential as wound dressing materials. Dialdehyde carboxymethyl cellulose (DCMC), a new kind of dialdehyde polysaccharide, had been successfully prepared in our previous work, which showed some properties as short chain dialdehyde and good solubility in water (Li et al., 2011). It was observed that the DCMC crosslinked gelatin edible films showed better properties than gelatin films (Mu, Guo, Li, Lin, & Li, 2012). More importantly, the crosslinking effects of DCMC on decellularized porcine aortas and collagen cryogel had been evaluated. The results demonstrated that DCMC was an effective crosslinking reagent for biological tissue fixation with low cytotoxicity (Tan et al., 2015; Wang, Wang, Li, Gu, & Yu, 2015). So far, gelatin has been widely used for numerous biomedical applications as hard or soft capsules, hydrogels, microspheres and hydrogel fibers (Dash et al., 2013). Among those applications, gelatin-based composite hydrogel fiber has attracted more and more attention due to its wide application field (Hu et al., 2009, 2010). Hydrogel fiber has the similar properties as hydrogel. Moreover, it can be further processed into other forms of biomedical materials. In our previous studies, polyethylene glycol (PEG) was added into gelatin spinning solutions as a modifier. It was found that PEG could significantly improve the spinnability of gelatin (Mu, Li, Guo, Bi, & Li, 2013). In the present work, gelatin-based composite hydrogel fibers were prepared by gel-spinning using PEG6000 as the modifier. To improve the thermal and mechanical properties of gelatin–PEG composite hydrogel fibers, DCMC was incorporated into spinning solutions during gel-spinning process as the crosslinking reagent. Then the biological properties of the hydrogel fibers for wound dressings were evaluated.
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2.3. Preparation of DCMC crosslinked gelatin–PEG composite hydrogel fibers The DCMC crosslinked gelatin–PEG hydrogel fibers were prepared using a method similar to the conventional procedure (Mu, Li, Guo, Bi, & Li, 2013). Certain amount of gelatin and PEG6000 (Wgelatin :WPEG = 10:1, total weight = 30 g) were mixed in 70 mL distilled water. After fully swelling, the mixture was continuously stirred at 60 ◦ C for 1 h. Then desired amount of DCMC solution (10 wt%) was added in the mixture. After another 1 h with stirring at 60 ◦ C, the blend spinning solution was degassed and extruded into saturated sodium sulfate bath through a single nozzle 0.3 mm in internal diameter under a constant force. Then the raw fibers were drawn with a drawing ratio of 3 and dried in air. Finally the fibers were washed in distilled water and dried in air again. The diameter of the fibers was about 0.23 ± 0.03 mm. The samples were named GeP, GeP-D1000, GeP-D500, GeP-D100 and GeP-D50 when the ratio of DCMC to gelatin was 0, 1/1000, 1/500, 1/100 and 1/50, respectively. 2.4. FT-IR study FT-IR spectra of the samples were obtained from discs containing ∼2.0 mg sample in ∼20 mg potassium bromide (KBr). The measurements were carried out on a Perkin–Elmer Spectrum One FT-IR spectrophotometer at the resolution of 4 cm−1 in the wave number region 400–4000 cm−1 . 2.5. Mechanical properties measurement After dried in air, the tensile strength and elongation at break of the composite hydrogel fibers were measured on a fiber electron tensile tester (YG061, Laizhou Electronic Machine Co., Ltd., China). The gauge length was 100 mm and crosshead speed was 100 mm/min. 2.6. Swelling study
2. Materials and methods 2.1. Materials Gelatin type B was purchased from Aladdin Reagent Database Inc with Bloom 250. (Shanghai, China). Carboxymethyl cellulose sodium (CMC), sodium periodate and PEG6000 were purchased from Kelong Chemical Reagent Company (Chengdu, China). The average Mw of CMC was ∼250,000. The degree of substitution (DS) of CMC was ∼0.90 according to Fourier transform infrared (FT-IR) analysis. Sodium periodate and PEG6000 were of analytical grade. 2.2. Preparation of DCMC DCMC was prepared using a method similar to the conventional procedure (Guo et al., 2013). About 1.0 g of CMC was dissolved in 20 mL distilled water in the flask which was immersed in a temperature controlled water bath with a magnetic stirrer. Then, 10 mL of periodate solution (0.11 g/mL) was added to the CMC solution under stirring. The pH value was adjusted to 3.0 with 1 M sulfuric acid solution. After the mixture was stirred in the dark at 35 ◦ C for 4 h, the oxidized product, referred to DCMC was precipitated by pouring the solution into a large amount of ethanol. It was then recovered and cross-washed with distilled water and ethanol until all iodic compounds were removed. The product was dried at 37 ◦ C to constant weight for the subsequent use.
The swelling process was performed at room temperature by immersing the samples in phosphate buffered saline (PBS, pH 7.4, 0.1 M). PBS was prepared by dissolving 17.97 g of di-sodium hydrogen phosphate, 5.73 g of monosodium hydrogen phosphate and 9 g of sodium chloride in 1L of distilled water. The swelling ratio (SR) was experimentally determined using the following equation (Balakrishnan et al., 2005; Dawlee et al., 2005): SR (%) =
[Ws − Wd ] × 100 Ws
(1)
where Ws and Wd represent the weight of swollen and dry sample, respectively. All experiments were done at least in triplicate. 2.7. Evaporation of water from swollen hydrogel fibers After fully swelling in PBS, the samples were gently blotted with a filter paper and kept at 37 ◦ C and 35% relative humidity. The weight was noted at regular intervals. Weight remaining was found out by the equation: Weight Remaining (%) =
Wt × 100 W0
(2)
where W0 and Wt are the initial weight and weight after time t respectively.
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2.8. Enzymatic degradation The enzymatic degradation behavior was obtained by immersing the samples into the trypsin enzyme solutions (1 g trypsin in 100 mL PBS solution) at 37 ◦ C. The absorbance values of enzyme solutions against reference solution (PBS solution) at 275 nm were measured at regular intervals, which were used to reflect the enzymatic degradation behavior of composite hydrogel fibers (Dalev, Vassileva, Mark, & Fakirov, 1998). 2.9. Blood clotting test The blood clotting test was carried out using a method similar to the conventional procedure (Dey & Ray, 2003; Zhou & Yi, 1999). Acid citrate dextrose (ACD) solution was prepared by mixing 0.544 g of anhydrous citric acid, 1.65 g of trisodium citrate dihydrate and 1.84 g of dextrose monohydrate to 75 mL of distilled water. Then 1 mL of ACD solution was added to 9 mL of fresh human blood to obtain ACD blood. The samples were hydrated to equilibrium in normal saline solution (0.9% sodium chloride solution in water) at 37 ◦ C. Then 0.25 mL of ACD blood was dropped on the surface of samples followed by the addition of 0.02 mL of CaCl2 solution (0.2 mol/L). The blood samples were incubated in a thermostat at 37 ◦ C for 5 min. The blood clotting test was carried out by spectrophotometric measurement of the relative absorbency of blood sample that had been diluted to 50 mL with distilled water at 542 nm after addition of CaCl2 . The absorbance of solution of 50 mL of distilled water and 0.25 mL of ACD blood at 542 nm was assumed to be 100, which was used as reference value. The blood clotting index (BCI) of samples can be quantified by following equation:
100 L of extraction liquid obtained from various fibers was added into each well. The cell culture was maintained in a humidified incubator at 37 ◦ C with 5% CO2 in air. Each sample was photographed using an inverted light microscope on the 4th day to observe the morphology of L929. 3. Results and discussion 3.1. Preparation of the gelatin–PEG composite hydrogel fibers Gelatin is a widely used material for numerous biomedical applications as hard or soft capsules, hydrogels, microspheres and fibers (Dash et al., 2013). Among those applications, gelatin-based composite fibers have attracted more and more attention due to their wide application fields. Moreover, the composite fibers can be further knitted into other forms for different applications, such as hydrogels and films. However, it is difficult to prepare pure gelatin fibers due to the poor spinnability of gelatin itself. In our previous studies, polyethylene glycol (PEG) was added into gelatin spinning solutions as a modifier. It was found that PEG could significantly improve the spinnability of gelatin (Mu et al., 2013a,b). In addition, it has been reported that gelatin fibers can be prepared by the gel-spinning method. The use of the method and the drawing in a gel state were effective in inducing segmental orientation in gelatin fibers to present good mechanical performances (Fukae, Maekawa, & Sangen, 2005). Hence, the gel-spinning method was used to prepare gelatin-based composite hydrogel fibers with PEG6000 as the modifier in the present work. To improve the thermal and mechanical properties of the fibers, DCMC was incorporated into spinning solutions as a crosslinking reagent since PEG played a poor role in improving the thermal and mechanical properties of gelatin fibers.
BCI (%) =
3.2. FT-IR studies
Absorbancy of blood which had been in contact with sample Absorbancy of solution of distilled water and ACD blood
× 100
(3)
2.10. Haemolysis ratio measurement After hydrated to equilibrium in normal saline solution at 37 ◦ C, the sample (∼0.5 g) was added in 10 mL of normal saline solution at 37 ◦ C for 60 min. Then 0.2 mL of diluted ACD blood was added (8 mL ACD whole blood in 10 mL normal saline solution). After another 60 min, the absorbance of the upper solution that was centrifuged at 100 g for 5 min was measured by a spectrophotometer at 542 nm. Distilled water and normal saline solution were used as positive and negative controls, respectively. The haemolysis ratio was obtained from the following equation (Zhou and Yi, 1999): Haemolysis Ratio (%) =
AS − AN × 100 AP − AN
Fig. 1 shows the FT-IR spectra of the DCMC crosslinked gelatin–PEG composite hydrogel fibers. The bands at ∼3400, 2930, 1650 and 1540 cm−1 are denoted as A, B, I and II amide bands, respectively. Generally, the amide A and B bands are mainly associated with the stretching vibrations of N H groups. The amide I band is originated from C O stretching vibrations coupled to N H bending vibration. The amide II band arises from the N H bending vibrations coupled to C N stretching vibrations (Mu et al., 2013a,b). Fig. 1 shows that the positions of B, I, and II amide bands in composite hydrogel fibers are nearly unchanged while the amide A band shifts to low-frequency shoulder in comparison with GeP. The
(4)
where AS is the absorbance of sample; AP and AN are the absorbance of positive and negative controls, respectively. 2.11. Evaluation of cytotoxicity The effect of composite hydrogel fibers on cell proliferation was evaluated in vitro using a mouse-derived established cell line of L929 fibroblasts (Xu et al., 2013). The extraction liquid was obtained by immersing the samples in saline at 37 ◦ C for 24 h in 5% CO2 . Highdensity polyethylene extraction was used as a negative control and phenol solution as a positive control. L929 fibroblasts were seeded in 96-well plates at the density of 2 × 103 cells/well in 100 L of Dulbecco’s modified eagle medium (DMEM) supplemented with 10% FBS, 100 U/mL penicillin and 100 mg/mL streptomycin. Meanwhile,
Fig. 1. FT-IR spectra of the DCMC crosslinked gelatin–PEG composite hydrogel fibers with various DCMC contents. (A) GeP, (B) GeP-D1000, (C) GeP-D500, (D) GeP-D100 and (E) GeP-D50.
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Table 1 Tensile strength and elongation at break of the DCMC crosslinked gelatin–PEG composite hydrogel fibers with various DCMC contents. Samples GeP GeP-D1000 GeP-D500 GeP-D100 GeP-D50
Tensile strength (cN/dtex) 0.66 + 0.10 1.04 + 0.13 1.30 + 0.20 1.36 + 0.18 2.15 + 0.21
Elongation at break (%) 13.2 + 1.3 12.3 + 1.0 11.6 + 1.5 10.7 + 1.7 10.2 + 0.8
result should be due to the hydrogen bond interactions between gelatin and DCMC. Note that the amide A of composite hydrogel fibers shifts to low frequency position with the increase of DCMC content. It suggests that the more DCMC addition in hydrogel fibers may promote more hydrogen bond interactions formation. CMC was oxidized in the presence of sodium periodate to yield the corresponding C2 C3 dialdehyde product. It was reported that DCMC had two characteristic IR bands at 1738 cm−1 and 886 cm−1 , which were assigned to the aldehydic carbonyl groups and the formation of hemiacetal bonds between the aldehyde groups and neighbor hydroxyl groups, respectively (Li et al., 2011). As shown in Fig. 1, the absorption bands at 1738 cm−1 for DCMC in composite hydrogel fibers are disappeared, indicating the formation of Schiff’s base between -amino groups of lysine or hydroxylysine side groups of gelatin and the aldehyde groups in DCMC. However, the peak at about 1660 cm−1 for C N (Schiff’s base) cannot be observed, which is masked by the strong amide I band (Mu et al., 2010). In addition, Fig. 1 shows the shift of the sensitive crystalline bands at 1050–1200 cm−1 in the composite hydrogel fibers, which demonstrates that the crosslinking by DCMC may disturb the crystallization of gelatin. 3.3. Mechanical properties Table 1 shows the tensile strength and elongation at break of the DCMC crosslinked gelatin–PEG composite hydrogel fibers with various DCMC contents. The data show that the addition of DCMC can greatly improve the mechanical properties of the composite hydrogel fibers, which indicates the occurrence of crosslinking between gelatin and DCMC. Note that the increase of DCMC content in hydrogel fibers causes tensile strength to increase while elongation at break to decrease. Increased tensile strength of materials due to crosslinking is often accompanied by reduced elongation at break (less extensible films) as the crosslinking results in a more rigid structure. For example, the greater tensile strength and lower elongation at break than control films had been reported for collagen films crosslinked by glutaraldehyde (Weadock, Olson, & Sliver, 1983). In our previous studies, the same phenomenon had been observed for DCMC crosslinked gelatin edible films (Mu et al., 2012). It is worth noting that although more DCMC addition can give higher tensile strength of the hydrogel fibers, it is disadvantageous for the spinnability of the spinning solutions. More DCMC addition will cause higher viscosity of the spinning solutions. It is difficult to spin for spinning solutions with too high viscosity. Therefore, the addition of DCMC in the present spinning solutions is at most 1/50 (DCMC to gelatin, w/w).
Fig. 2. Swelling behavior of the DCMC crosslinked gelatin–PEG composite hydrogel fibers in PBS.
for using as wound dressings since they can absorb wound exudates and provide moist environment for wound to accelerate wound healing. The water in hydrogel can be divided into bound water, half-bound water and free water. The quantity of bound water and half-bound water is connected with the number of hydrophilic groups in hydrogel while the content of free water is connected with the three-dimensional structure and pore volume of hydrogel (Mu, Zhang, Lin, & Li, 2013). The high water absorption capacity of the hydrogel fibers reveals the presence of the threedimensional structure and porous networks, which is beneficial to facilitate cell adhesion and proliferation (Cheng et al., 2014). Note that the swelling behavior of GeP cannot be obtained during the measurements. Gelatin is soluble in aqueous solution and a few minutes of storage in physiological solution is sufficient to induce considerable swelling (Bigi, Cojazzi, Panzavolta, Roveri, & Rubini, 2002). Crosslinking with DCMC markedly reduces the degree of swelling of gelatin. Moreover, swelling is often accompanied by a reduction in mechanical properties. The suppression of swelling is beneficial for hydrogel fibers to be used in wild biomaterial fields. To examine the extent of water loss from the hydrogel fibers, the extent of evaporative loss of water with time from hydrogel fibers was examined and shown in Fig. 3. The water transport in the hydrogel, in particular, is closely related to the free water content. In the hydrogel, the free water is different from bound water and half-bound water, which has a very high degree of freedom and is easy to be lost. Fig. 3 shows that the decrease in weight is nearly linear with time up to 4 h and thereafter does not change significantly. Evaporative water loss is about 85% within 10 h. The results
3.4. Swelling behavior The swelling behavior of the DCMC crosslinked gelatin–PEG composite hydrogel fibers is studied and presented in Fig. 2. It shows that the equilibrium swelling ratio of the hydrogel fibers is very high, which is found to be between 89% and 93%. The result indicates that the hydrogel fibers are effective biomaterials
Fig. 3. Evaporative water loss from the DCMC crosslinked gelatin–PEG composite hydrogel fibers.
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Fig. 4. Time dependence of the absorbance of enzymatic degradation solution of hydrogel fibers in PBS at 275 nm.
show that the DCMC crosslinked gelatin–PEG composite hydrogel fibers have strong ability to absorb free water due to the threedimensional structure and porous networks. Note that GeP-D1000 has the lowest weight remaining while GeP-D50 has the highest value. The result indicates that GeP-D1000 has more porous network structures to hold free water while GeP-D50 is much more compact. 3.5. Enzymatic degradation behavior The biodegradation behavior usually plays an important role in the engineering process of the biomedical materials (Sionkowska & Kozłowska, 2013). Fig. 4 shows the in vitro biodegradation profiles of gelatin–PEG composite hydrogel fibers with different DCMC content. It can be observed that the hydrogel fibers degrade very soon in the first four hours and slow down subsequently. Meanwhile, the hydrogel fibers with higher DCMC content degrade slower than those with lower DCMC content. Similar to the enzymatic resistance mechanism of genipin crosslinked collagen/chitosan scaffolds, the intramolecular and intermolecular crosslinking networks in the hydrogel fibers may hinder the enzyme to access the reaction sites in gelatin (Yan et al., 2010). The result suggests that the addition of DCMC can efficiently improve the enzymatic stability of gelatin–PEG composite hydrogel fibers. 3.6. Blood clotting test The platelet aggregation and blood clotting play an important role during the hemostasis process (Cha et al., 2014). The blood clotting test is an efficient way to reflect the variation of antithrombotic activity of biomaterials. Generally, the larger blood clotting index (BCI) demonstrates the slower clotting rate and the better blood compatibility of the materials (Zhou and Yi, 1999). Fig. 5 shows the effect of DCMC content on the blood clotting index of hydrogel fibers when the samples are in contact with the ACD blood for 5 min. As showed in Fig. 5, the BCI is 56.09%, 65.32%, 66.71% and 67.74% for GeP-D1000, GeP-D500, GeP-D100 and GeP-D50, respectively. The BCI value of the hydrogel fibers increases accordingly with the increase of DCMC content. The results suggest that the addition of DCMC can properly increase the blood compatibility of the hydrogel fibers.
Fig. 5. Effect of DCMC content on the blood clotting index of hydrogel fibers. (Contacting time of blood sample: 5 min).
sample contacting with blood (Tan et al., 2015; Zhou and Yi, 1999). Hence, lower haemolysis ratio means better blood compatibility. According to ASTM standard (Wang et al., 2003), the permissible haemolysis ratio of the materials should be less than 5% for medical applications. Fig. 6 shows the haemolysis ratio of the DCMC crosslinked gelatin–PEG composite hydrogel fibers. The extent of haemolysis of all the hydrogel fibers is lower than the permissible level of 5%, indicating non-hemolytic property of the materials. Moreover, it can be seen that the haemolysis ratio decreases with the increase of DCMC content, which may be ascribed to the reduction of active groups in gelatin ( NH2 ) induced by crosslinking. The same phenomenon had been reported for DCMC crosslinked collagen cryogel (Tan et al., 2015). The result indicates that the suitable addition of DCMC can improve the blood compatibility of the hydrogel fibers. The hydrogel fibers can be well-suited for biomedical applications such as wound dressings. 3.8. Cytotoxicity of the gelatin–PEG composite hydrogel fibers The biocompatibility is crucial for the applications of biomaterials. The cytotoxicity of the composite hydrogel fibers was evaluated in vitro using a mouse-derived established cell line of L929 fibroblasts. The observation of cell morphology cultured in the extraction liquid of hydrogel fibers is showed in Fig. 7. The negative control culture of high-density polyethylene extraction displays regular, dense and well spread L929 cells. The cells present elongated, spindle-shaped and long slender appearances. In contrast, the cells exposed to phenol in the positive control culture are
3.7. Haemolysis ratio The blood compatibility of the hydrogel fibers was evaluated in vitro by measuring the haemolysis ratio when they contacted with ACD blood. The haemolysis ratio represents the extent of fracture and dissolution of the red blood cell caused by the
Fig. 6. Haemolysis ratio of the DCMC crosslinked gelatin–PEG composite hydrogel fibers.
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Fig. 7. Observation of L929 cell morphology cultured in the extraction liquid of hydrogel fibers and blank control.
in the process of dying. Nearly all the cells are significant retracted and rounded, with distinct enlargement of intercellular space. The color of the cells is transparent in negative control while relative dark in positive control. The cytotoxicity test results indicate that the extraction liquid of composite hydrogel fibers is less cytotoxic than the positive control. Nevertheless, the presence of DCMC still plays some disadvantageous influence on the cells as showed in GeP-D50 and GeP-D100. Some cells in GeP-D50 and GeP-D100 are in the process of dying with retracted and rounded shapes, leading to a certain enlargement of the intercellular space. But then the most of cells do not show any differences compared to negative control and the growth of the cells is better than that of the positive control. The situation becomes different when the content of DCMC decreases to a certain level. As showed in GeP-D500, no obvious dead cells can be seen and the result shows near normal fibroblast morphology. Furthermore, GeP-D1000 exhibits no noticeable difference no matter in quantity or morphology of the cells. The results indicate that DCMC might be an effective crosslinking reagent for biomaterials fixation with
low cytotoxicity. The same results had been reported by previous studies (Tan et al., 2015; Xu et al., 2013).
4. Conclusions Gelatin–PEG composite hydrogel fibers were prepared by gelspinning. Dialdehyde carboxymethyl cellulose (DCMC) was used as the crosslinking reagent to fix the composite hydrogel fibers. The results indicate that the mechanical properties, enzymatic stability and blood compatibility of the composite hydrogel fibers are efficiently improved due to the addition of DCMC. The hydrogel fibers are effective biomaterials for using as wound dressings since they can absorb wound exudates and provide moist environment. Moreover, the composite hydrogel fibers present low cytotoxicity. The study indicates that DCMC is an effective crosslinking reagent for biomaterials fixation. The DCMC crosslinked gelatin–PEG composite hydrogel fibers can be well-suited for biomedical applications such as wound dressings.
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Li, H., Wu, B., Mu, C., & Lin, W. (2011). Concomitant degradation in periodate oxidation of carboxymethyl cellulose. Carbohydrate Polymers, 84(3), 881–886. Martucci, J. F., & Ruseckaite, R. A. (2010). Biodegradable three-layer film derived from bovine gelatin. Journal of Food Engineering, 99(3), 377–383. Mu, C., Guo, J., Li, X., Lin, W., & Li, D. (2012). Preparation and properties of dialdehyde carboxymethyl cellulose crosslinked gelatin edible films. Food Hydrocolloids, 27(1), 22–29. Mu, C., Li, X., Guo, J., Bi, C., & Li, D. (2013). Effects of montmorillonite on the structure and properties of gelatin–polyethlene glycol composite fibers. Journal of Applied Polymer Science, 129, 773–778. Mu, C., Liu, F., Cheng, Q., Li, H., Wu, B., Zhang, G., et al. (2010). Collagen cryogel cross-linked by dialdehyde starch. Macromolecular Materials and Engineering, 295, 100–107. Mu, C., Zhang, K., Lin, W., & Li, D. (2013). Ring-opening polymerization of genipin and its long-range crosslinking effect on collagen hydrogel. Journal of Biomedical Materials Research, 101(2), 385–393. Rocasalbas, G., Francesko, A., Tourino, S., Fernandez-Francos, X., Guebitz, G. M., & Tzanov, T. (2013). Laccase-assisted formation of bioactive chitosan/gelatin hydrogel stabilized with plant polyphenols. Carbohydrate Polymers, 92(2), 989–996. Rullan, P. P., Vallbona, C., Rullan, J. M., Mansbridge, J. N., & Morhenn, V. B. (2011). Use of gelatin sponges in mohs micrographic surgery defects and staged melanoma excisions: A novel approach to secondary wound healing. Journal of Drugs in Dermatology, 10, 68–73. Saik, J. E., Gould, D. J., Watkins, E. M., Dickinson, M. E., & West, J. L. (2011). Covalently immobilized platelet-derived growth factor-BB promotes angiogenesis in biomimetic poly(ethylene glycol) hydrogels. Acta Biomaterialia, 7(1), 133–143. Sionkowska, A., & Kozłowska, J. (2013). Properties and modification of porous 3-D collagen/hydroxyapatite composites. International Journal of Biological Macromolecules, 52, 250–259. Tan, H., Wu, B., Li, C., Mu, C., Li, H., & Lin, W. (2015). Collagen cryogel cross-linked by naturally derived dialdehyde carboxymethyl cellulose. Carbohydrate Polymers, 129, 17–24. Ulubayram, K., Cakar, A., Korkusuz, P., Ertan, C., & Hasirci, N. (2001). EGF containing gelatin-based wound dressings. Biomaterials, 22, 1345–1356. Wang, X., Wang, Y., Li, L., Gu, Z., & Yu, X. (2015). Feasibility study of the naturally occurring dialdehyde carboxymethyl cellulose for biological tissue fixation. Carbohydrate Polymers, 115, 54–61. Wang, X. H., Li, D. P., Wang, W. J., Feng, Q. L., Cui, F. Z., Xu, Y. X., et al. (2003). Crosslinked collagen/chitosan matrix for artificial livers. Biomaterials, 24(19), 3213–3220. Weadock, K., Olson, R., & Sliver, F. (1983). Evaluation of collagen crosslinking techniques. Biomaterials Medical Devices and Artificial Organs, 11, 293–318. Weng, L., Pan, H., & Chen, W. (2007). Self-crosslinkable hydrogels composed of partially oxidized hyaluronan and gelatine: In vitro and in vivo responses. Journal of Biomedical Materials Research, 85A, 352–365. Wong, J. P., MacRobert, A. J., Cheema, U., & Brown, R. A. (2013). Mechanical anisotropy in compressed collagen produced by localised photodynamic cross-linking. Journal of The Mechanical Behavior of Biomedical Materials, 18, 132–139. Xu, Y., Li, L., Wang, H., Yu, X., Gu, Z., Huang, C., et al. (2013). In vitro cytocompatibility evaluation of alginate dialdehyde for biological tissue fixation. Carbohydrate Polymers, 92(1), 448–454. Xu, Y., Li, L., Yu, X., Gu, Z., & Zhang, X. (2012). Feasibility study of a novel crosslinking reagent (alginate dialdehyde) for biological tissue fixation. Carbohydrate Polymers, 87(2), 1589–1595. Yan, L., Wang, Y., Ren, L., Wu, G., Caridade, S. G., Fan, J., et al. (2010). Genipin-cross-linked collagen/chitosan biomimetic scaffolds for articular cartilage tissue engineering applications. Journal of Biomedical Materials Research, 95A(2), 465–475. Young, S., Wong, M., Tabata, Y., & Mikos, A. G. (2005). Gelatin as a delivery vehicle for the controlled release of bioactive molecules. Journal of Controlled Release, 109(1–3), 256–274. Zhou, C., & Yi, Z. (1999). Blood-compatibility of polyurethane/liquid crystal composite membranes. Biomaterials, 12, 2093–2099.
Contents lists available at ScienceDirect
Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol
Biological properties of dialdehyde carboxymethyl cellulose crosslinked gelatin–PEG composite hydrogel fibers for wound dressings Defu Li a,∗ , Youxin Ye a , Derong Li b , Xinying Li c , Changdao Mu a,∗ a
School of Chemical Engineering, Sichuan University, Chengdu 610065, PR China People’s Hospital of Lanshan District, Linyi 276000, PR China c College of Chemistry and Environment Protection Engineering, Southwest University for Nationalities, Chengdu 610041, PR China b
a r t i c l e
i n f o
Article history: Received 30 July 2015 Received in revised form 4 September 2015 Accepted 6 November 2015 Available online 10 November 2015 Keywords: Hydrogel fiber Dialdehyde carboxymethyl cellulose Gelatin Gel-spinning Wound dressings
a b s t r a c t Gelatin-based composite hydrogel fibers were prepared by gel-spinning with PEG6000 as the modifier. Dialdehyde carboxymethyl cellulose (DCMC), as an ideal crosslinking reagent for protein, was used to fix the composite hydrogel fibers. Then the biological properties of the hydrogel fibers for wound dressings were evaluated. The results indicate that the hydrogen bond interactions and C N linkages between gelatin and DCMC can be formed. The addition of DCMC can efficiently improve the mechanical properties, enzymatic stability and blood compatibility of the hydrogel fibers. Crosslinking with DCMC can reduce the degree of swelling of the hydrogel fibers, which is beneficial for hydrogel fibers to avoid undesired reduction in mechanical properties. Moreover, the composite hydrogel fibers present three-dimensional structure, porous networks and low cytotoxicity. The study suggests that DCMC is an effective crosslinking reagent for biomaterials fixation. The developed composite hydrogel fibers can be well-suited for biomedical applications such as wound dressings. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction Gelatin is an animal protein obtained by a controlled hydrolysis of fibrous insoluble collagen, which is generated as waste during animal slaughtering and processing (Guo, Li, Mu, Zhang, Qin, & Li, 2013). Gelatin is an effective biomaterial as wound dressings since it can absorb wound exudates and provide moist environment to accelerate wound healing (Kavoosi, Dadfar, & Purfard, 2013). It also provides immediate haemostasis and helps to prevent wound contracture and contour deformities associated with conventional wound healing (Rullan, Vallbona, Rullan, Mansbridge, & Morhenn, 2011; Wong, MacRobert, Cheema, & Brown, 2013). Positive biological responses facilitating cell adhesion and proliferation are the properties of gelatin that should also be highlighted (Saik, Gould, Watkins, Dickinson, & West, 2011; Young, Wong, Tabata, & Mikos, 2005). However, the main disadvantages of gelatin are connected with its poor mechanical properties and low thermal stability, which limit its applications as wound dressings (Dash, Foston, & Ragauskas, 2013; Weng, Pan, & Chen, 2007). Therefore,
∗ Corresponding authors. Tel.: +86 28 8540 5221; fax: +86 28 8540 5221. E-mail addresses: [email protected] (D. Li), [email protected] (C. Mu). http://dx.doi.org/10.1016/j.carbpol.2015.11.024 0144-8617/© 2015 Elsevier Ltd. All rights reserved.
many researchers are working at the realm of reinforcement of gelatin. To date, many attempts have been made to modify the poor properties of gelatin including crosslinking, blending, compounding with natural fibers and nanosized clay (Martucci & Ruseckaite, 2010). Glutaraldehyde (Ulubayram, Cakar, Korkusuz, Ertan, & Hasirci, 2001), N-[3-(dimethylamino)propyl]-N ethylcarbodiimide (EDC) (Choi et al., 2001), transglutaminase (Ito et al., 2003), plant polyphenols (Rocasalbas et al., 2013), formaldehyde (De Carvalho & Grosso, 2004), oxidized chondroitin sulfate (Dawlee, Sugandhi, Balakrishnan, Labarre, & Jayakrishnan, 2005), oxidized alginate (Balakrishnan, Mohanty, Fernandez, Mohanan, & Jayakrishnan, 2006; Balakrishnan, Mohanty, Umashankar, & Jayakrishnan, 2005; Xu, Li, Yu, Gu, & Zhang, 2012), dialdehyde starch (Mu et al., 2010) and cellulose nanowhiskers (Dash et al., 2013) are the effective crosslinking reagents to reinforce gelatin in order to increase the resistance to thermal degradation and improve the mechanical properties. Note that oxidized polysaccharides have received an increasing attention as ideal crosslinking reagents of protein in recent years. The oxidation of polysaccharides by periodate is characterized by the specific cleavage of the C2 C3 bond of glucose residues. This cleavage results in the formation of two aldehyde groups per glucose unit, forming
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2,3-dialdehyde polysaccharides (Li, Wu, Mu, & Lin, 2011). The aldehyde groups in dialdehyde polysaccharides can crosslink with -amino groups of lysine or hydroxylysine side groups of gelatin by C N linkages (Schiff’s base) to improve the properties of gelatin (Ge et al., 2015; Guo, Ge, Li, Mu, & Li, 2014). Dawlee et al. (2005) had reported a new class of gelatin hydrogel using aldehyde chondroitin sulfate as a crosslinking reagent. This new class of hydrogel without employing any extraneous crosslinking reagents was expected to have potential as wound dressing materials. Dialdehyde carboxymethyl cellulose (DCMC), a new kind of dialdehyde polysaccharide, had been successfully prepared in our previous work, which showed some properties as short chain dialdehyde and good solubility in water (Li et al., 2011). It was observed that the DCMC crosslinked gelatin edible films showed better properties than gelatin films (Mu, Guo, Li, Lin, & Li, 2012). More importantly, the crosslinking effects of DCMC on decellularized porcine aortas and collagen cryogel had been evaluated. The results demonstrated that DCMC was an effective crosslinking reagent for biological tissue fixation with low cytotoxicity (Tan et al., 2015; Wang, Wang, Li, Gu, & Yu, 2015). So far, gelatin has been widely used for numerous biomedical applications as hard or soft capsules, hydrogels, microspheres and hydrogel fibers (Dash et al., 2013). Among those applications, gelatin-based composite hydrogel fiber has attracted more and more attention due to its wide application field (Hu et al., 2009, 2010). Hydrogel fiber has the similar properties as hydrogel. Moreover, it can be further processed into other forms of biomedical materials. In our previous studies, polyethylene glycol (PEG) was added into gelatin spinning solutions as a modifier. It was found that PEG could significantly improve the spinnability of gelatin (Mu, Li, Guo, Bi, & Li, 2013). In the present work, gelatin-based composite hydrogel fibers were prepared by gel-spinning using PEG6000 as the modifier. To improve the thermal and mechanical properties of gelatin–PEG composite hydrogel fibers, DCMC was incorporated into spinning solutions during gel-spinning process as the crosslinking reagent. Then the biological properties of the hydrogel fibers for wound dressings were evaluated.
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2.3. Preparation of DCMC crosslinked gelatin–PEG composite hydrogel fibers The DCMC crosslinked gelatin–PEG hydrogel fibers were prepared using a method similar to the conventional procedure (Mu, Li, Guo, Bi, & Li, 2013). Certain amount of gelatin and PEG6000 (Wgelatin :WPEG = 10:1, total weight = 30 g) were mixed in 70 mL distilled water. After fully swelling, the mixture was continuously stirred at 60 ◦ C for 1 h. Then desired amount of DCMC solution (10 wt%) was added in the mixture. After another 1 h with stirring at 60 ◦ C, the blend spinning solution was degassed and extruded into saturated sodium sulfate bath through a single nozzle 0.3 mm in internal diameter under a constant force. Then the raw fibers were drawn with a drawing ratio of 3 and dried in air. Finally the fibers were washed in distilled water and dried in air again. The diameter of the fibers was about 0.23 ± 0.03 mm. The samples were named GeP, GeP-D1000, GeP-D500, GeP-D100 and GeP-D50 when the ratio of DCMC to gelatin was 0, 1/1000, 1/500, 1/100 and 1/50, respectively. 2.4. FT-IR study FT-IR spectra of the samples were obtained from discs containing ∼2.0 mg sample in ∼20 mg potassium bromide (KBr). The measurements were carried out on a Perkin–Elmer Spectrum One FT-IR spectrophotometer at the resolution of 4 cm−1 in the wave number region 400–4000 cm−1 . 2.5. Mechanical properties measurement After dried in air, the tensile strength and elongation at break of the composite hydrogel fibers were measured on a fiber electron tensile tester (YG061, Laizhou Electronic Machine Co., Ltd., China). The gauge length was 100 mm and crosshead speed was 100 mm/min. 2.6. Swelling study
2. Materials and methods 2.1. Materials Gelatin type B was purchased from Aladdin Reagent Database Inc with Bloom 250. (Shanghai, China). Carboxymethyl cellulose sodium (CMC), sodium periodate and PEG6000 were purchased from Kelong Chemical Reagent Company (Chengdu, China). The average Mw of CMC was ∼250,000. The degree of substitution (DS) of CMC was ∼0.90 according to Fourier transform infrared (FT-IR) analysis. Sodium periodate and PEG6000 were of analytical grade. 2.2. Preparation of DCMC DCMC was prepared using a method similar to the conventional procedure (Guo et al., 2013). About 1.0 g of CMC was dissolved in 20 mL distilled water in the flask which was immersed in a temperature controlled water bath with a magnetic stirrer. Then, 10 mL of periodate solution (0.11 g/mL) was added to the CMC solution under stirring. The pH value was adjusted to 3.0 with 1 M sulfuric acid solution. After the mixture was stirred in the dark at 35 ◦ C for 4 h, the oxidized product, referred to DCMC was precipitated by pouring the solution into a large amount of ethanol. It was then recovered and cross-washed with distilled water and ethanol until all iodic compounds were removed. The product was dried at 37 ◦ C to constant weight for the subsequent use.
The swelling process was performed at room temperature by immersing the samples in phosphate buffered saline (PBS, pH 7.4, 0.1 M). PBS was prepared by dissolving 17.97 g of di-sodium hydrogen phosphate, 5.73 g of monosodium hydrogen phosphate and 9 g of sodium chloride in 1L of distilled water. The swelling ratio (SR) was experimentally determined using the following equation (Balakrishnan et al., 2005; Dawlee et al., 2005): SR (%) =
[Ws − Wd ] × 100 Ws
(1)
where Ws and Wd represent the weight of swollen and dry sample, respectively. All experiments were done at least in triplicate. 2.7. Evaporation of water from swollen hydrogel fibers After fully swelling in PBS, the samples were gently blotted with a filter paper and kept at 37 ◦ C and 35% relative humidity. The weight was noted at regular intervals. Weight remaining was found out by the equation: Weight Remaining (%) =
Wt × 100 W0
(2)
where W0 and Wt are the initial weight and weight after time t respectively.
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2.8. Enzymatic degradation The enzymatic degradation behavior was obtained by immersing the samples into the trypsin enzyme solutions (1 g trypsin in 100 mL PBS solution) at 37 ◦ C. The absorbance values of enzyme solutions against reference solution (PBS solution) at 275 nm were measured at regular intervals, which were used to reflect the enzymatic degradation behavior of composite hydrogel fibers (Dalev, Vassileva, Mark, & Fakirov, 1998). 2.9. Blood clotting test The blood clotting test was carried out using a method similar to the conventional procedure (Dey & Ray, 2003; Zhou & Yi, 1999). Acid citrate dextrose (ACD) solution was prepared by mixing 0.544 g of anhydrous citric acid, 1.65 g of trisodium citrate dihydrate and 1.84 g of dextrose monohydrate to 75 mL of distilled water. Then 1 mL of ACD solution was added to 9 mL of fresh human blood to obtain ACD blood. The samples were hydrated to equilibrium in normal saline solution (0.9% sodium chloride solution in water) at 37 ◦ C. Then 0.25 mL of ACD blood was dropped on the surface of samples followed by the addition of 0.02 mL of CaCl2 solution (0.2 mol/L). The blood samples were incubated in a thermostat at 37 ◦ C for 5 min. The blood clotting test was carried out by spectrophotometric measurement of the relative absorbency of blood sample that had been diluted to 50 mL with distilled water at 542 nm after addition of CaCl2 . The absorbance of solution of 50 mL of distilled water and 0.25 mL of ACD blood at 542 nm was assumed to be 100, which was used as reference value. The blood clotting index (BCI) of samples can be quantified by following equation:
100 L of extraction liquid obtained from various fibers was added into each well. The cell culture was maintained in a humidified incubator at 37 ◦ C with 5% CO2 in air. Each sample was photographed using an inverted light microscope on the 4th day to observe the morphology of L929. 3. Results and discussion 3.1. Preparation of the gelatin–PEG composite hydrogel fibers Gelatin is a widely used material for numerous biomedical applications as hard or soft capsules, hydrogels, microspheres and fibers (Dash et al., 2013). Among those applications, gelatin-based composite fibers have attracted more and more attention due to their wide application fields. Moreover, the composite fibers can be further knitted into other forms for different applications, such as hydrogels and films. However, it is difficult to prepare pure gelatin fibers due to the poor spinnability of gelatin itself. In our previous studies, polyethylene glycol (PEG) was added into gelatin spinning solutions as a modifier. It was found that PEG could significantly improve the spinnability of gelatin (Mu et al., 2013a,b). In addition, it has been reported that gelatin fibers can be prepared by the gel-spinning method. The use of the method and the drawing in a gel state were effective in inducing segmental orientation in gelatin fibers to present good mechanical performances (Fukae, Maekawa, & Sangen, 2005). Hence, the gel-spinning method was used to prepare gelatin-based composite hydrogel fibers with PEG6000 as the modifier in the present work. To improve the thermal and mechanical properties of the fibers, DCMC was incorporated into spinning solutions as a crosslinking reagent since PEG played a poor role in improving the thermal and mechanical properties of gelatin fibers.
BCI (%) =
3.2. FT-IR studies
Absorbancy of blood which had been in contact with sample Absorbancy of solution of distilled water and ACD blood
× 100
(3)
2.10. Haemolysis ratio measurement After hydrated to equilibrium in normal saline solution at 37 ◦ C, the sample (∼0.5 g) was added in 10 mL of normal saline solution at 37 ◦ C for 60 min. Then 0.2 mL of diluted ACD blood was added (8 mL ACD whole blood in 10 mL normal saline solution). After another 60 min, the absorbance of the upper solution that was centrifuged at 100 g for 5 min was measured by a spectrophotometer at 542 nm. Distilled water and normal saline solution were used as positive and negative controls, respectively. The haemolysis ratio was obtained from the following equation (Zhou and Yi, 1999): Haemolysis Ratio (%) =
AS − AN × 100 AP − AN
Fig. 1 shows the FT-IR spectra of the DCMC crosslinked gelatin–PEG composite hydrogel fibers. The bands at ∼3400, 2930, 1650 and 1540 cm−1 are denoted as A, B, I and II amide bands, respectively. Generally, the amide A and B bands are mainly associated with the stretching vibrations of N H groups. The amide I band is originated from C O stretching vibrations coupled to N H bending vibration. The amide II band arises from the N H bending vibrations coupled to C N stretching vibrations (Mu et al., 2013a,b). Fig. 1 shows that the positions of B, I, and II amide bands in composite hydrogel fibers are nearly unchanged while the amide A band shifts to low-frequency shoulder in comparison with GeP. The
(4)
where AS is the absorbance of sample; AP and AN are the absorbance of positive and negative controls, respectively. 2.11. Evaluation of cytotoxicity The effect of composite hydrogel fibers on cell proliferation was evaluated in vitro using a mouse-derived established cell line of L929 fibroblasts (Xu et al., 2013). The extraction liquid was obtained by immersing the samples in saline at 37 ◦ C for 24 h in 5% CO2 . Highdensity polyethylene extraction was used as a negative control and phenol solution as a positive control. L929 fibroblasts were seeded in 96-well plates at the density of 2 × 103 cells/well in 100 L of Dulbecco’s modified eagle medium (DMEM) supplemented with 10% FBS, 100 U/mL penicillin and 100 mg/mL streptomycin. Meanwhile,
Fig. 1. FT-IR spectra of the DCMC crosslinked gelatin–PEG composite hydrogel fibers with various DCMC contents. (A) GeP, (B) GeP-D1000, (C) GeP-D500, (D) GeP-D100 and (E) GeP-D50.
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Table 1 Tensile strength and elongation at break of the DCMC crosslinked gelatin–PEG composite hydrogel fibers with various DCMC contents. Samples GeP GeP-D1000 GeP-D500 GeP-D100 GeP-D50
Tensile strength (cN/dtex) 0.66 + 0.10 1.04 + 0.13 1.30 + 0.20 1.36 + 0.18 2.15 + 0.21
Elongation at break (%) 13.2 + 1.3 12.3 + 1.0 11.6 + 1.5 10.7 + 1.7 10.2 + 0.8
result should be due to the hydrogen bond interactions between gelatin and DCMC. Note that the amide A of composite hydrogel fibers shifts to low frequency position with the increase of DCMC content. It suggests that the more DCMC addition in hydrogel fibers may promote more hydrogen bond interactions formation. CMC was oxidized in the presence of sodium periodate to yield the corresponding C2 C3 dialdehyde product. It was reported that DCMC had two characteristic IR bands at 1738 cm−1 and 886 cm−1 , which were assigned to the aldehydic carbonyl groups and the formation of hemiacetal bonds between the aldehyde groups and neighbor hydroxyl groups, respectively (Li et al., 2011). As shown in Fig. 1, the absorption bands at 1738 cm−1 for DCMC in composite hydrogel fibers are disappeared, indicating the formation of Schiff’s base between -amino groups of lysine or hydroxylysine side groups of gelatin and the aldehyde groups in DCMC. However, the peak at about 1660 cm−1 for C N (Schiff’s base) cannot be observed, which is masked by the strong amide I band (Mu et al., 2010). In addition, Fig. 1 shows the shift of the sensitive crystalline bands at 1050–1200 cm−1 in the composite hydrogel fibers, which demonstrates that the crosslinking by DCMC may disturb the crystallization of gelatin. 3.3. Mechanical properties Table 1 shows the tensile strength and elongation at break of the DCMC crosslinked gelatin–PEG composite hydrogel fibers with various DCMC contents. The data show that the addition of DCMC can greatly improve the mechanical properties of the composite hydrogel fibers, which indicates the occurrence of crosslinking between gelatin and DCMC. Note that the increase of DCMC content in hydrogel fibers causes tensile strength to increase while elongation at break to decrease. Increased tensile strength of materials due to crosslinking is often accompanied by reduced elongation at break (less extensible films) as the crosslinking results in a more rigid structure. For example, the greater tensile strength and lower elongation at break than control films had been reported for collagen films crosslinked by glutaraldehyde (Weadock, Olson, & Sliver, 1983). In our previous studies, the same phenomenon had been observed for DCMC crosslinked gelatin edible films (Mu et al., 2012). It is worth noting that although more DCMC addition can give higher tensile strength of the hydrogel fibers, it is disadvantageous for the spinnability of the spinning solutions. More DCMC addition will cause higher viscosity of the spinning solutions. It is difficult to spin for spinning solutions with too high viscosity. Therefore, the addition of DCMC in the present spinning solutions is at most 1/50 (DCMC to gelatin, w/w).
Fig. 2. Swelling behavior of the DCMC crosslinked gelatin–PEG composite hydrogel fibers in PBS.
for using as wound dressings since they can absorb wound exudates and provide moist environment for wound to accelerate wound healing. The water in hydrogel can be divided into bound water, half-bound water and free water. The quantity of bound water and half-bound water is connected with the number of hydrophilic groups in hydrogel while the content of free water is connected with the three-dimensional structure and pore volume of hydrogel (Mu, Zhang, Lin, & Li, 2013). The high water absorption capacity of the hydrogel fibers reveals the presence of the threedimensional structure and porous networks, which is beneficial to facilitate cell adhesion and proliferation (Cheng et al., 2014). Note that the swelling behavior of GeP cannot be obtained during the measurements. Gelatin is soluble in aqueous solution and a few minutes of storage in physiological solution is sufficient to induce considerable swelling (Bigi, Cojazzi, Panzavolta, Roveri, & Rubini, 2002). Crosslinking with DCMC markedly reduces the degree of swelling of gelatin. Moreover, swelling is often accompanied by a reduction in mechanical properties. The suppression of swelling is beneficial for hydrogel fibers to be used in wild biomaterial fields. To examine the extent of water loss from the hydrogel fibers, the extent of evaporative loss of water with time from hydrogel fibers was examined and shown in Fig. 3. The water transport in the hydrogel, in particular, is closely related to the free water content. In the hydrogel, the free water is different from bound water and half-bound water, which has a very high degree of freedom and is easy to be lost. Fig. 3 shows that the decrease in weight is nearly linear with time up to 4 h and thereafter does not change significantly. Evaporative water loss is about 85% within 10 h. The results
3.4. Swelling behavior The swelling behavior of the DCMC crosslinked gelatin–PEG composite hydrogel fibers is studied and presented in Fig. 2. It shows that the equilibrium swelling ratio of the hydrogel fibers is very high, which is found to be between 89% and 93%. The result indicates that the hydrogel fibers are effective biomaterials
Fig. 3. Evaporative water loss from the DCMC crosslinked gelatin–PEG composite hydrogel fibers.
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Fig. 4. Time dependence of the absorbance of enzymatic degradation solution of hydrogel fibers in PBS at 275 nm.
show that the DCMC crosslinked gelatin–PEG composite hydrogel fibers have strong ability to absorb free water due to the threedimensional structure and porous networks. Note that GeP-D1000 has the lowest weight remaining while GeP-D50 has the highest value. The result indicates that GeP-D1000 has more porous network structures to hold free water while GeP-D50 is much more compact. 3.5. Enzymatic degradation behavior The biodegradation behavior usually plays an important role in the engineering process of the biomedical materials (Sionkowska & Kozłowska, 2013). Fig. 4 shows the in vitro biodegradation profiles of gelatin–PEG composite hydrogel fibers with different DCMC content. It can be observed that the hydrogel fibers degrade very soon in the first four hours and slow down subsequently. Meanwhile, the hydrogel fibers with higher DCMC content degrade slower than those with lower DCMC content. Similar to the enzymatic resistance mechanism of genipin crosslinked collagen/chitosan scaffolds, the intramolecular and intermolecular crosslinking networks in the hydrogel fibers may hinder the enzyme to access the reaction sites in gelatin (Yan et al., 2010). The result suggests that the addition of DCMC can efficiently improve the enzymatic stability of gelatin–PEG composite hydrogel fibers. 3.6. Blood clotting test The platelet aggregation and blood clotting play an important role during the hemostasis process (Cha et al., 2014). The blood clotting test is an efficient way to reflect the variation of antithrombotic activity of biomaterials. Generally, the larger blood clotting index (BCI) demonstrates the slower clotting rate and the better blood compatibility of the materials (Zhou and Yi, 1999). Fig. 5 shows the effect of DCMC content on the blood clotting index of hydrogel fibers when the samples are in contact with the ACD blood for 5 min. As showed in Fig. 5, the BCI is 56.09%, 65.32%, 66.71% and 67.74% for GeP-D1000, GeP-D500, GeP-D100 and GeP-D50, respectively. The BCI value of the hydrogel fibers increases accordingly with the increase of DCMC content. The results suggest that the addition of DCMC can properly increase the blood compatibility of the hydrogel fibers.
Fig. 5. Effect of DCMC content on the blood clotting index of hydrogel fibers. (Contacting time of blood sample: 5 min).
sample contacting with blood (Tan et al., 2015; Zhou and Yi, 1999). Hence, lower haemolysis ratio means better blood compatibility. According to ASTM standard (Wang et al., 2003), the permissible haemolysis ratio of the materials should be less than 5% for medical applications. Fig. 6 shows the haemolysis ratio of the DCMC crosslinked gelatin–PEG composite hydrogel fibers. The extent of haemolysis of all the hydrogel fibers is lower than the permissible level of 5%, indicating non-hemolytic property of the materials. Moreover, it can be seen that the haemolysis ratio decreases with the increase of DCMC content, which may be ascribed to the reduction of active groups in gelatin ( NH2 ) induced by crosslinking. The same phenomenon had been reported for DCMC crosslinked collagen cryogel (Tan et al., 2015). The result indicates that the suitable addition of DCMC can improve the blood compatibility of the hydrogel fibers. The hydrogel fibers can be well-suited for biomedical applications such as wound dressings. 3.8. Cytotoxicity of the gelatin–PEG composite hydrogel fibers The biocompatibility is crucial for the applications of biomaterials. The cytotoxicity of the composite hydrogel fibers was evaluated in vitro using a mouse-derived established cell line of L929 fibroblasts. The observation of cell morphology cultured in the extraction liquid of hydrogel fibers is showed in Fig. 7. The negative control culture of high-density polyethylene extraction displays regular, dense and well spread L929 cells. The cells present elongated, spindle-shaped and long slender appearances. In contrast, the cells exposed to phenol in the positive control culture are
3.7. Haemolysis ratio The blood compatibility of the hydrogel fibers was evaluated in vitro by measuring the haemolysis ratio when they contacted with ACD blood. The haemolysis ratio represents the extent of fracture and dissolution of the red blood cell caused by the
Fig. 6. Haemolysis ratio of the DCMC crosslinked gelatin–PEG composite hydrogel fibers.
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Fig. 7. Observation of L929 cell morphology cultured in the extraction liquid of hydrogel fibers and blank control.
in the process of dying. Nearly all the cells are significant retracted and rounded, with distinct enlargement of intercellular space. The color of the cells is transparent in negative control while relative dark in positive control. The cytotoxicity test results indicate that the extraction liquid of composite hydrogel fibers is less cytotoxic than the positive control. Nevertheless, the presence of DCMC still plays some disadvantageous influence on the cells as showed in GeP-D50 and GeP-D100. Some cells in GeP-D50 and GeP-D100 are in the process of dying with retracted and rounded shapes, leading to a certain enlargement of the intercellular space. But then the most of cells do not show any differences compared to negative control and the growth of the cells is better than that of the positive control. The situation becomes different when the content of DCMC decreases to a certain level. As showed in GeP-D500, no obvious dead cells can be seen and the result shows near normal fibroblast morphology. Furthermore, GeP-D1000 exhibits no noticeable difference no matter in quantity or morphology of the cells. The results indicate that DCMC might be an effective crosslinking reagent for biomaterials fixation with
low cytotoxicity. The same results had been reported by previous studies (Tan et al., 2015; Xu et al., 2013).
4. Conclusions Gelatin–PEG composite hydrogel fibers were prepared by gelspinning. Dialdehyde carboxymethyl cellulose (DCMC) was used as the crosslinking reagent to fix the composite hydrogel fibers. The results indicate that the mechanical properties, enzymatic stability and blood compatibility of the composite hydrogel fibers are efficiently improved due to the addition of DCMC. The hydrogel fibers are effective biomaterials for using as wound dressings since they can absorb wound exudates and provide moist environment. Moreover, the composite hydrogel fibers present low cytotoxicity. The study indicates that DCMC is an effective crosslinking reagent for biomaterials fixation. The DCMC crosslinked gelatin–PEG composite hydrogel fibers can be well-suited for biomedical applications such as wound dressings.
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