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Different chemical groups modification on the surface of chitosan nonwoven dressing and the hemostatic properties
International Journal of Biological Macromolecules 107 (2018) 463–469
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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Different chemical groups modification on the surface of chitosan nonwoven dressing and the hemostatic properties Dong Yan a , Shihao Hu a , Zhongzheng Zhou a , Shah Zeenat a , Feng Cheng b , Yang Li a , Chao Feng a , Xiaojie Cheng a,∗ , Xiguang Chen a,∗ a b
College of Marine Life Science, Ocean University of China, 5 Yushan Road, 266003 Qingdao, PR China Center Blood Station of Qingdao, 9# Longde Road, 266071 Qingdao, PR China
a r t i c l e
i n f o
Article history: Received 25 April 2017 Received in revised form 6 July 2017 Accepted 5 September 2017 Available online 6 September 2017 Keywords: Chitosan nonwoven Surface modification Hemostatic property
a b s t r a c t The hemostatic properties of surface modified chitosan nonwoven had been investigated. The succinyl groups, carboxymethyl groups and quaternary ammonium groups were introduced into the surface of chitosan nonwoven (obtained NSCS, CMCS and TMCS nonwoven, respectively). For blood clotting, absorbance value (0.105 ± 0.03) of NSCS1 nonwoven was the smallest (CS 0.307 ± 0.002, NSCS2 0.148 ± 0.002, CMCS1 0.195 ± 0.02, CMCS2 0.233 ± 0.001, TMCS1 0.191 ± 0.002, TMCS2 0.345 ± 0.002), which indicated the stronger hemostatic potential. For platelet aggregation, adenosine diphosphate agonist was added to induce the nonwoven to adhered platelets. The aggregation of platelet with TMCS2 nonwoven was highest (10.97 ± 0.16%). Further research of blood coagulation mechanism was discussed, which indicated NSCS and CMCS nonwoven could activate the intrinsic pathway of coagulation to accelerate blood coagulation. NSCS1 nonwoven showed the shortest hemostatic time (147 ± 3.7 s) and the lowest blood loss (0.23 ± 0.05 g) in a rabbit ear artery injury model. These results demonstrated that these surface modified chitosan nonwoven dressings could use as a promising hemostatic intervention, especially NSCS nonwoven dressing. © 2017 Elsevier B.V. All rights reserved.
1. Introduction Hemorrhage in trauma has associated with the increased mortality rate, which is a leading cause of the majority of deaths on the battlefield and civilian settings as well [1,2]. In common condition, the blood coagulation cascade process of the body could occur following these procedures. Initially, platelets are activated and aggregated and adhere to the exposed subendothelial matrix. Plasma proteins and small molecules are released to form a bridge and generated an initial hemostatic plug to reduce the loss of blood. Subsequently, a series of coagulation cascade are triggered to form fibrin clot which reinforce the platelet plug to heal the wound [3,4]. However, the severe or uncontrollable hemorrhage cannot be stopped by the body’s natural clotting mechanism, which results in the requirement of a hemostatic intervention. Currently, Synthetic or natural hemostatic agents including bandage, powder, sponge and gel have been applied to control hemorrhages. Among them, chitosan, a natural polymeric mate-
∗ Corresponding authors. E-mail addresses: [email protected] (F. Cheng), [email protected] (X. Cheng), [email protected] (X. Chen). https://doi.org/10.1016/j.ijbiomac.2017.09.008 0141-8130/© 2017 Elsevier B.V. All rights reserved.
rial which derived of deacetylated chitin, that has the excellent properties such as biocompatibility, biodegradability, antimicrobial properties, non-toxicity, and so on [5–7]. The hemostatic property of chitosan is owing to the electrostatic interaction with negatively-charged cell membranes of erythrocytes and platelet, which distincts from the body’s natural clotting mechanism [8,9]. Therefore, chitosan has been widely used as hemostatic agent and wound dressing with multiple forms, such as film, hydrogel, sponge, fiber and nonwoven which is spun by fibers [6,10,11]. Among these forms, fiber or nonwoven is the main form because it allow gaseous exchange and be removed easily. Although numerous studies about the hemostatic capacity of chitosan have been reported, there still are limitations including insolubility and weak antibacterial activity which make it an inappropriate therapy [12,13]. Therefore, the surface chemically modified chitosan fibers by processes such as carboxymethylation [14], quaternization [15], succinylation [16], have been investigated to improve the water solubility and antibacterial activity to use as wound dressings. But few studies have been conducted to investigate the hemostatic property of these surface modified chitosan fibers. In the present work, the chitosan nonwoven was chosen to use as raw material because of the better mechanical property and
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Fig. 1. Different chemical groups modification on the surface of chitosan nonwoven dressing, CS, NSCS, CMCS and TMCS nonwoven.
then NSCS, CMCS and TMCS nonwoven were prepared. To study the hemostatic effect, the different degree of substitution (DS) of nonwoven samples varied by regulating reaction conditions, characterizations such as chemical structure, blood compatibility and cytotoxicity were investigated. In the following work, nonwoven samples were evaluated in a rabbit ear artery injury model. The results suggested that the NSCS, CMCS and TMCS nonwoven exert a better hemostatic property than CS nonwoven.
2. Materials and methods 2.1. Materials Chitosan nonwoven (CS nonwoven, viscosity = 1092 cps, degree of deacetylation = 91.5%, linear-density = 3.1 cN) were purchased from Weifang Yingke Group Co., Ltd. (Shandong, China). Succinic anhydride was purchased from Yuanhang Chemical Co. Ltd (Tianjin, China). Chloroacetic acid was got from Tianjin Kaixin Chemical Industry Co., Ltd. Methyl iodide (CH3 I), dimethyl sulfoxide (DMSO) and sodium hydroxide (NaOH) were obtained from Qingdao Yunshan Biotechnology Co., Ltd. Rabbits were purchased from Qingdao Institute for Drug Control. All other chemicals used in this work were of analytical grade.
2.2. Preparation of nonwoven dressings NSCS, CMCS and TMCS nonwoven were synthesized through the modification on the surface of chitosan nonwoven [15–17]. Briefly, 1.0 g CS nonwoven was added into a mixture solution (0.75 g/1.5 g succinic anhydride and 50 mL DMSO) and stirred for 2 h at 40 ◦ C. 1.0 g CS nonwoven, 1.35 g sodium hydroxide, 40 mL of isopropanol and water (4:1 v/v) were added to a flask to swell and alkalize. After 1 h, 0.5 g/1 g chloroacetic acid dissolved in isopropanol was slowly added to the solution and reacted at 50 ◦ C for 20 min 1.0 g CS nonwoven was placed in a round-bottom flask with 40 mL of DMSO and 6 mL of 15% (w/v) NaOH solution for 10 min, subsequently, 1.5 mL/2 mL of methyl iodide was added and oscillated for 15 min
at 45 ◦ C. Thereafter, all nonwoven dressings were washed with 75% (v/v) ethanol and then dried. 2.2.1. The degree of substitution of nonwoven The DS of the NSCS, CMCS and TMCS nonwoven were measured by conductivity titration [18]. 2.2.2. The fourier transform infrared spectroscopy (FTIR) The chemical structure of nonwoven was analyzed using a Fourier transform infrared spectrophotometer. (NEXUE470, Nicolet, Madison, USA). 2.2.3. 1 H NMR spectroscopy 1 H NMR spectrum of CS, NSCS, CMCS and TMCS nonwoven were recorded on a Bruker AV300 instrument and dissolved in the mixed solvent DCl/D2 O, respectively. 2.3. Whole blood clotting test The blood clotting test was described by Ong et.al [19]. and Leslie W. Chan et.al [2]. Nonwoven samples (1 cm × 1 cm) were placed in glass dishes and kept the temperature to 37 ◦ C. 100 L of citrated whole blood was pipetted onto each nonwoven dressings followed by the addition of 10 L of 0.2 M CaCl2 solution and then incubation at 37 ◦ C for 5 min. Each dressing was then placed in a 50 mL of centrifuge tube containing 12.5 mL of distilled water. The tubes were inverted three times to rinse the unclotted blood cells, and the absorbance of the hemoglobin was measured at 540 nm by UV−vis spectrophotometer (UV-1200 MAPADA, China). Then the nonwoven samples were washed with phosphate buffer solution (pH 7.4) several times and immobilized with 2.5% glutaraldehyde for 2 h, dehydrated with a series of ethanol and dried with critical point drier, and then observed by SEM (JSM-6010LA, JEOL Ltd., Japan). 2.4. Platelet aggregation Blood was collected from the heart of rabbits under anesthesia by a syringe and added into a sodium citrated tube with anti-
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Fig. 2. FTIR spectra of CS, NSCS, CMCS and TMCS nonwoven.
pathway of coagulation (aPTT) was performed by mixing 100 L of PPP with 100 L of partial thromboplastin reagent and incubating at 37 ◦ C. Thereafter, nonwoven samples and 100 L of CaCl2 were added after 3 min of incubation and then measured. For PT determination, the extrinsic pathway, was performed by adding 100 L of PT reagent in the mixture, 100 L of PPP and nonwoven samples, which preincubated at 37 ◦ C for 3 min, and the clotting time then measured [22,23]. 2.6. Cytotoxicity assays
Fig. 3.
1
H NMR spectra of CS, NSCS, CMCS and TMCS nonwoven.
coagulant (9:1 v/v). The citrated whole blood was centrifuged at 3000 × g for 15 min at room temperature to obtain platelet poor plasma (PPP). Platelet rich plasma (PRP) in the upper layer was isolated from centrifugation of the citrated whole blood at 150 × g for 15 min. Platelet aggregation was determined by measuring changes in the optical density of stirred PRP after the addition of nonwoven dressings. 100 L of each nonwoven sample (10 mg/100 L normal saline) was added to a cuvette with 900 L of PRP accordingly, then the light transmittance was measured at 595 nm immediately and after a 20 min interval at 37 ◦ C by UV−vis spectrophotometer. Normal saline and adenosine diphosphate (ADP) were used as a negative and positive control respectively. 100 L of PRP and PPP were pipetted into wells for calibration [20,21]. The percent of aggregation was calculated as follows: Aggregation% =
L595,PRP − L595.test × 100% L595,PRP − L595,PPP
Where L595,PRP , L595,PPP and L595,test were the absorbance of the PRP, PPP and tested samples. 2.5. Blood coagulation activity Activated partial thromboplastin time (aPTT) and prothrombin time (PT) with different nonwoven samples were measured using a semiautomatic coagulation analyzer (TS6000, MD PACIFIC Ltd., China). The activity of each nonwoven dressing on the intrinsic
In vitro cytotoxicity of the nonwoven dressings were investigated by the MTT assay using L929 cell line [24–26]. The sterilized nonwoven samples were incubated in DMEM at 37 ◦ C for 24 h to obtain extracts of 0.1 g/mL. L929 cells were seeded in 96-well culture plate at a density of 1 × 104 cells per well and incubated under a 5% CO2 atmosphere at 37 ◦ C for 24 h. The culture medium was removed and replaced with the concentration of 25%, 50% and 100% extracts dilutions and incubated for another 24 h and 48 h. Then 10 L of MTT solution was added into each well. After 4 h incubation, the formazan salts were dissolved followed by addition of 150 L of DMSO for about 10 min on the shaker at 37 ◦ C. The absorbance at 490 nm was determined with an ELISA reader (SUN˘ RISE, BASIC TECAN, 5082 GrOdig, Austria). The relative cell viability (RCV) was expressed by the following equation: RCV% =
Abssample Abscontrol
× 100%
Where Abs sample and Abs control were the absorbance of the tested sample and the negative control. 2.7. Hemostatic assay on ear artery Rabbits were anesthetized by intravenous injection of 3% pentobarbital sodium (20 mg/kg) and shaved off the hair on the back of the ear to expose the ear artery. A 1 cm × 1 cm wound including the ear artery was created, and the pre-weighted nonwoven dressing (CS, NSCS1, CMCS1, TMCS1 and Gauze) was applied over the blooding area after free hemorrhage for 5 s and constant pressure (200 g) was exerted. The nonwoven dressing was removed and weighted until blood was absolutely coagulated and the hemostatic time was recorded. Total blood loss was also evaluated by the difference value between the weights of nonwoven dressing before and after the hemostatic assay [26,27]. All above mentioned experimental rabbits were treated with the National Research Council’s Guide for the care and use of laboratory animals.
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Fig. 4. (A) Blood absorption behavior of nonwoven samples. (B) The effects of different nonwoven samples on blood clotting. *P < 0.05 compared to the CS group. SEM images of interaction between RBCs and CS (C), NSCS1 (D), NSCS2 (E), CMCS1 (F), CMCS2 (G), TMCS1 (H), TMCS2 (I).
Table 1 Reaction conditions and DS (%) for the nonwoven dressing. Samples
Reagents
Groups
DS (%)
NSCS1 NSCS2 CMCS1 CMCS2 TMCS1 TMCS2
succinic anhydride succinic anhydride chloroacetic acid chloroacetic acid methyl iodide methyl iodide
-CO(CH2 )2 COOH -CO(CH2 )2 COOH -CH2 COONa -CH2 COONa -(CH3 )3 -(CH3 )3
17.5 39 19 36.5 17.3 38.5
2.8. Statistical analysis All the data were expressed as the mean ± standard deviation (SD), and assessed by a one-way analysis of ANOVA to demonstrate differences between groups. P values <0.05 were considered to be statistically significant. 3. Results and discussions
amino groups of chitosan [29]. CMCS nonwoven was confirmed by the new absorption bands in 1598 cm−1 and 1454 cm−1 [30,31]. For the TMCS nonwoven, the peak at 1597 cm−1 reduced (N H stretching) and 1454 cm−1 (C H stretching) observably increased [15], which indicated the introduction of quaternary ammonium groups onto chitosan chain. The 1 H NMR spectra (Fig. 3) showed the successful synthesis of NSCS, CMCS and TMCS nonwoven. The 1 H NMR assignments of chitosan nonwoven was as follows [30,32]: 1 H NMR (D2O/DCl) ı=4.75 (H1), ı=3.0 (H2), ı=3.43-3.81 (H3, H4, H5, H6). The 1 H NMR assignments of NSCS nonwoven was as follows: 1 H NMR (D2O/DCl) ␦ = 2.90 (H2), ␦ = 3. 50-3.82 (H1, H3, H4, H5, H6), ␦ = 2.3 (H*) [33]. The characteristic proton signals of CMCS nonwoven appeared in the range of ı=4.0-4.1, which indicated that the amino groups and hydroxyl groups were partly carboxymethylated [28], and the characteristic peak of TMCS appeared in ı=3.1-3.3 [13,34]. According to the ratio of the integral peak of characteristic peak of simple nonwoven and H2 in chitosan nonwoven structure, it could be known that the degree of substitution [35].
3.1. Synthesis and characterization of NSCS, CMCS and TMCS nonwoven dressing
3.2. Whole blood clotting test
NSCS, CMCS and TMCS nonwoven were synthesized by introduction of succinyl groups, carboxymethyl groups and quaternary ammonium groups into the −NH2 or OH of the glucosamine units on the surface of CS nonwoven, respectively (Fig. 1). The degree of substitution (DS) could be varied by regulating reaction conditions (Table. 1). FTIR analysis demonstrated the chemical structure on the CS, NSCS, CMCS and TMCS nonwoven (Fig. 2). The CS nonwoven spectrum showed that the principal absorption bands appeared at 3440 cm−1 (O H stretch), 1652 cm−1 (C O stretch), 1593 cm−1 (N H bend), 1153 cm−1 (bridge O stretch), 1077 cm−1 (O H stretch) [28]. Compared with FTIR spectrum of CS, the reduced intensity of the peak at 1598 cm−1 (-NH2 bending) and the new absorption band of 1413 cm−1 ( COO symmetric stretch) and 1559 cm−1 (Amide II) indicated that succinyl introduced onto the
Blood absorption behavior was observed after application of 100 L of citrated whole blood through pipetting onto each nonwoven samples. Chitosan nonwoven is relatively hydrophobic when applied blood immediately and 5 min later (Fig. 4A). Blood was not absorbed in the TMCS2 nonwoven sample as well, which due to the presence of more positive charge leading to the occurrence of hemolysis [36]. All other nonwoven samples exhibited a better blood absorption because of the surface hydrophilic. The recalcified blood was applied onto nonwoven samples, and the unclotted blood rinsed by distilled water was determined at 540 nm (Fig. 4B). The smaller absorbance value indicated the stronger hemostatic potential of the samples. The absorbance value of TMCS2 nonwoven (0.345 ± 0.003) was the greatest among the samples, which resulted from more introduced quaternary ammonium cationic group that would lead to the hemolysis. The irregular
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Fig. 5. Platelet aggregation by nonwoven samples. *P < 0.05 compared to the negative and positive control group.
shape erythrocytes were found on the surface of TMCS2 nonwoven samples in the SEM image (Fig. 4I). Meanwhile, CS nonwoven had a slightly higher absorbance value (0.307 ± 0.002), due to the solid state form has a disability to induce the aggregation [8]. And its poor absorption was observed in blood absorption behavior test. Similarly, the SEM images showed only a few RBCs were absorbed on the CS nonwoven (Fig. 4C). Compared with all the nonwoven samples, NSCS1 had the best hemostatic activity (0.105 ± 0.003), as a result of the negative charges on the surface promoting the intrinsic pathway of coagulation [9].Moreover, the CMCS nonwoven could also promote blood coagulation, but exhibited a slightly lower absorbance value than that of NSCS nonwoven. Notably, for the same sample, the higher degree of substitution had a higher absorbance value, which indicated lower blood absorption on the surface of samples, and this might be because the repulsive force between the negative charges on the nonwoven surface and erythrocyte membrane hindered the blood absorption behavior of nonwoven samples. 3.3. Platelet aggregation In aggregation test, the adherence and aggregation properties of platelet with different nonwoven samples were evaluated. As shown in Fig. 5, the ability to induce platelet aggregation of CS nonwoven sample (7.746 ± 0.05) was stronger than NSCS and CMCS nonwoven samples beacause of the increasing Ca2+ mobilization and enhancing expression of GPIIb/IIIa complex on platelet membrane surfaces [37]. The platelet aggregation of TMCS nonwoven samples (TMCS1 8.746 ± 0.054, TMCS2 10.979 ± 0.163) was the strongest among all the samples. What’s more, the platelet aggregation increased with the increasing of DS. TMCS nonwoven samples had the highest value of platelet aggregation likely due to the positive charges on the surface which could enhance the ability of samples adhesion [20]. Furthermore, the ability to induce platelet aggregation of NSCS nonwoven samples was not significantly different from CMCS nonwoven samples. 3.4. Blood coagulation activity The blood coagulation cascade involves intrinsic, extrinsic, and common pathways. PT and aPPT were investigated to evaluate the effects of the nonwoven samples on the extrinsic and intrinsic coagulation pathways, respectively. Compared to the control group, the results showed that no statistically significant difference of all the nonwoven samples which were observed in the PT assay (Fig. 6A). It indicated that the extrinsic pathway of coagulation was not affected by these nonwoven samples. CS nonwoven sample did not change
Fig. 6. PT (A) and aPPT (B) measurement of CS, NSCS, CMCS and TMCS nonwoven samples. *P < 0.05 compared to the control group.
the PT and aPPT owing to the properties of chitosan to agglutinate RBCs rather than interfere with the coagulation factors [9,38]. The values of aPTT were shorter treated with NSCS and CMCS nonwoven samples compared with the control group. The shortening of aPTT could be attributed to the negative charges on the surface which could activate the intrinsic pathway of coagulation along with cofactors HWK-kininogen and prekallikrein [39]. Recent studies had reported that positive charges could increase aPTT [20], which may be the reason for the changes of aPPT treated with TMCS nonwoven samples. 3.5. Cytotoxicity assays To investigate the cytotoxicity of NSCS, CMCS and TMCS nonwoven, the MTT method of L929 cells was adapted for in vitro cytotoxicity assay (Fig. 7). It could be seen that, at 0.1 g/mL of NSCS2, the viability of the cell was lower than those of another concentration. That was probably owing to the ionization of the COOH on the surface of NSCS nonwoven in the DMEM, caused the extracts solution to be acidic and inhibited the growth of cells. But there was another reason, the sticky extracts solution formed by the dissolution of the higher DS of NSCS2 nonwoven in the DMEM also affected the growth of cells. However, the viability of the cell was high ( > 80%) after treated with the diluted concentration of NSCS2 nonwoven extracts, which indicated that NSCS2 nonwoven sample was relatively low toxic to L929 cells. Moreover, no statistically significant differences were observed in the cell activity of L929 cell within 48 h in all other nonwoven samples, which demonstrated that they were no toxic to L929 cells. All the results suggest that NSCS, CMCS and TMCS nonwoven had good biocompatibility to be used as hemostatic materials. 3.6. Hemostatic assay on ear artery The hemostasis of the lower DS of nonwoven samples (NSCS1, CMCS1 and TMCS1) was analyzed by measuring the hemostatic
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Fig. 7. Cell viability measured by MTT assay for nonwoven samples extract of different concentrations after 24 h (A) and 48 h (B) of incubation. Data are presented as the mean ± SD (n = 5).
Fig. 8. The rabbit ear artery injury model among the Gauze, CS, NSCS1, CMCS1 and TMCS1 (A).The hemostatic time and the total blood loss (B) of the treatments of Gauze, CS, NSCS1, CMCS1 and TMCS1 of rabbit ear artery injury. * P < 0.05 compared to the gauze group. Data are presented as the mean ± SD (n = 5).
time and the amount of blood loss of rabbit ear artery (Fig. 8). Above results mentioned, the lower DS of nonwoven exhibited better hemostasis property and could maintain the original fiber morphology. Therefore, the NSCS1, CMCS1 and TMCS1 nonwoven samples were chosen for the hemostatic assay on ear artery. Compared with the Gauze, CS nonwoven sample could reduce the hemostatic time
and the blood loss, owing to the property of promoting platelet aggregation. Moreover, all modified chitosan nonwoven exhibited better hemostatic property including the hemostatic time and the amount of blood loss. In particularly, the time for hemostatic of NSCS1 (147 ± 3.7 s) was the shortest in those of other groups, and the total blood loss of the treatment with the NSCS1 (0.23 ± 0.05 g)
D. Yan et al. / International Journal of Biological Macromolecules 107 (2018) 463–469
was less than other hemostatic dressings as well. The results demonstrated that NSCS1 nonwoven had better hemostatic property, which may be caused by the influence of intrinsic pathway of coagulation. 4. Conclusions The NSCS, CMCS and TMCS nonwoven were successfully synthesized in the present work. Moreover, the NSCS, CMCS and TMCS nonwoven with different hemostatic mechanism exert a better hemostatic property than CS nonwoven. In the blood clotting test and blood coagulation activity, the negative charges on the surface of NSCS and CMCS could activate the intrinsic pathway of coagulation, along with the hemostatic mechanism of chitosan, and thus accelerated blood coagulation. But the repulsive force generated by the excessive negative charges between nonwoven surface and erythrocyte membrane hindered the blood absorption behavior of high DS of nonwoven samples. Moreover, the lower blood absorption of the TMCS nonwoven might be owing to the introduced quaternary ammonium cationic group of TMCS nonwoven that would lead to the hemolysis. The ability of inducing platelet aggregation for TMCS nonwoven samples was strongest among all the samples, which would contribute to initiating hemostasis. However, the ability to induce platelet aggregation of NSCS nonwoven samples was weak and not significantly different from CMCS nonwoven samples. The results of the rabbit ear artery injury model demonstrated that NSCS, CMCS and TMCS nonwoven exhibited a better hemostatic property than gauze and CS nonwoven, and especially, the NSCS nonwoven had an excellent hemostatic effect, compared to other nonwoven samples. Consequently, It is expected that this study is useful for understanding the hemostatic mechanism of these surface modified chitosan nonwoven dressings, which could use as promising hemostatic intervention. Acknowledgments This work was supported by the National Natural Science Foundation of China (81671828), China Postdoctoral Science Foundation (2016M592246), Applied Basic Research Plan of Qingdao (16-5-170-jch), and the Taishan Scholar Program, China. References [1] D.S. Kauvar, R. Lefering, C.E. Wade, J. Trauma 60 (2006) S3–11. [2] L.W. Chan, C.H. Kim, X. Wang, S.H. Pun, N.J. White, T.H. Kim, Acta Biomater. 31 (2016) 178–185. [3] K.F. Earl, W. Davie, Walter. Kisiel, Biochemistry 30 (1991) 10363–10370. [4] A.M. Behrens, M.J. Sikorski, P. Kofinas, J. Biomed. Mater. Res. Part A 102 (2014) 4182–4194. [5] M. Rinaudo, Prog. Polym. Sci. 31 (2006) 603–632.
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Different chemical groups modification on the surface of chitosan nonwoven dressing and the hemostatic properties Dong Yan a , Shihao Hu a , Zhongzheng Zhou a , Shah Zeenat a , Feng Cheng b , Yang Li a , Chao Feng a , Xiaojie Cheng a,∗ , Xiguang Chen a,∗ a b
College of Marine Life Science, Ocean University of China, 5 Yushan Road, 266003 Qingdao, PR China Center Blood Station of Qingdao, 9# Longde Road, 266071 Qingdao, PR China
a r t i c l e
i n f o
Article history: Received 25 April 2017 Received in revised form 6 July 2017 Accepted 5 September 2017 Available online 6 September 2017 Keywords: Chitosan nonwoven Surface modification Hemostatic property
a b s t r a c t The hemostatic properties of surface modified chitosan nonwoven had been investigated. The succinyl groups, carboxymethyl groups and quaternary ammonium groups were introduced into the surface of chitosan nonwoven (obtained NSCS, CMCS and TMCS nonwoven, respectively). For blood clotting, absorbance value (0.105 ± 0.03) of NSCS1 nonwoven was the smallest (CS 0.307 ± 0.002, NSCS2 0.148 ± 0.002, CMCS1 0.195 ± 0.02, CMCS2 0.233 ± 0.001, TMCS1 0.191 ± 0.002, TMCS2 0.345 ± 0.002), which indicated the stronger hemostatic potential. For platelet aggregation, adenosine diphosphate agonist was added to induce the nonwoven to adhered platelets. The aggregation of platelet with TMCS2 nonwoven was highest (10.97 ± 0.16%). Further research of blood coagulation mechanism was discussed, which indicated NSCS and CMCS nonwoven could activate the intrinsic pathway of coagulation to accelerate blood coagulation. NSCS1 nonwoven showed the shortest hemostatic time (147 ± 3.7 s) and the lowest blood loss (0.23 ± 0.05 g) in a rabbit ear artery injury model. These results demonstrated that these surface modified chitosan nonwoven dressings could use as a promising hemostatic intervention, especially NSCS nonwoven dressing. © 2017 Elsevier B.V. All rights reserved.
1. Introduction Hemorrhage in trauma has associated with the increased mortality rate, which is a leading cause of the majority of deaths on the battlefield and civilian settings as well [1,2]. In common condition, the blood coagulation cascade process of the body could occur following these procedures. Initially, platelets are activated and aggregated and adhere to the exposed subendothelial matrix. Plasma proteins and small molecules are released to form a bridge and generated an initial hemostatic plug to reduce the loss of blood. Subsequently, a series of coagulation cascade are triggered to form fibrin clot which reinforce the platelet plug to heal the wound [3,4]. However, the severe or uncontrollable hemorrhage cannot be stopped by the body’s natural clotting mechanism, which results in the requirement of a hemostatic intervention. Currently, Synthetic or natural hemostatic agents including bandage, powder, sponge and gel have been applied to control hemorrhages. Among them, chitosan, a natural polymeric mate-
∗ Corresponding authors. E-mail addresses: [email protected] (F. Cheng), [email protected] (X. Cheng), [email protected] (X. Chen). https://doi.org/10.1016/j.ijbiomac.2017.09.008 0141-8130/© 2017 Elsevier B.V. All rights reserved.
rial which derived of deacetylated chitin, that has the excellent properties such as biocompatibility, biodegradability, antimicrobial properties, non-toxicity, and so on [5–7]. The hemostatic property of chitosan is owing to the electrostatic interaction with negatively-charged cell membranes of erythrocytes and platelet, which distincts from the body’s natural clotting mechanism [8,9]. Therefore, chitosan has been widely used as hemostatic agent and wound dressing with multiple forms, such as film, hydrogel, sponge, fiber and nonwoven which is spun by fibers [6,10,11]. Among these forms, fiber or nonwoven is the main form because it allow gaseous exchange and be removed easily. Although numerous studies about the hemostatic capacity of chitosan have been reported, there still are limitations including insolubility and weak antibacterial activity which make it an inappropriate therapy [12,13]. Therefore, the surface chemically modified chitosan fibers by processes such as carboxymethylation [14], quaternization [15], succinylation [16], have been investigated to improve the water solubility and antibacterial activity to use as wound dressings. But few studies have been conducted to investigate the hemostatic property of these surface modified chitosan fibers. In the present work, the chitosan nonwoven was chosen to use as raw material because of the better mechanical property and
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Fig. 1. Different chemical groups modification on the surface of chitosan nonwoven dressing, CS, NSCS, CMCS and TMCS nonwoven.
then NSCS, CMCS and TMCS nonwoven were prepared. To study the hemostatic effect, the different degree of substitution (DS) of nonwoven samples varied by regulating reaction conditions, characterizations such as chemical structure, blood compatibility and cytotoxicity were investigated. In the following work, nonwoven samples were evaluated in a rabbit ear artery injury model. The results suggested that the NSCS, CMCS and TMCS nonwoven exert a better hemostatic property than CS nonwoven.
2. Materials and methods 2.1. Materials Chitosan nonwoven (CS nonwoven, viscosity = 1092 cps, degree of deacetylation = 91.5%, linear-density = 3.1 cN) were purchased from Weifang Yingke Group Co., Ltd. (Shandong, China). Succinic anhydride was purchased from Yuanhang Chemical Co. Ltd (Tianjin, China). Chloroacetic acid was got from Tianjin Kaixin Chemical Industry Co., Ltd. Methyl iodide (CH3 I), dimethyl sulfoxide (DMSO) and sodium hydroxide (NaOH) were obtained from Qingdao Yunshan Biotechnology Co., Ltd. Rabbits were purchased from Qingdao Institute for Drug Control. All other chemicals used in this work were of analytical grade.
2.2. Preparation of nonwoven dressings NSCS, CMCS and TMCS nonwoven were synthesized through the modification on the surface of chitosan nonwoven [15–17]. Briefly, 1.0 g CS nonwoven was added into a mixture solution (0.75 g/1.5 g succinic anhydride and 50 mL DMSO) and stirred for 2 h at 40 ◦ C. 1.0 g CS nonwoven, 1.35 g sodium hydroxide, 40 mL of isopropanol and water (4:1 v/v) were added to a flask to swell and alkalize. After 1 h, 0.5 g/1 g chloroacetic acid dissolved in isopropanol was slowly added to the solution and reacted at 50 ◦ C for 20 min 1.0 g CS nonwoven was placed in a round-bottom flask with 40 mL of DMSO and 6 mL of 15% (w/v) NaOH solution for 10 min, subsequently, 1.5 mL/2 mL of methyl iodide was added and oscillated for 15 min
at 45 ◦ C. Thereafter, all nonwoven dressings were washed with 75% (v/v) ethanol and then dried. 2.2.1. The degree of substitution of nonwoven The DS of the NSCS, CMCS and TMCS nonwoven were measured by conductivity titration [18]. 2.2.2. The fourier transform infrared spectroscopy (FTIR) The chemical structure of nonwoven was analyzed using a Fourier transform infrared spectrophotometer. (NEXUE470, Nicolet, Madison, USA). 2.2.3. 1 H NMR spectroscopy 1 H NMR spectrum of CS, NSCS, CMCS and TMCS nonwoven were recorded on a Bruker AV300 instrument and dissolved in the mixed solvent DCl/D2 O, respectively. 2.3. Whole blood clotting test The blood clotting test was described by Ong et.al [19]. and Leslie W. Chan et.al [2]. Nonwoven samples (1 cm × 1 cm) were placed in glass dishes and kept the temperature to 37 ◦ C. 100 L of citrated whole blood was pipetted onto each nonwoven dressings followed by the addition of 10 L of 0.2 M CaCl2 solution and then incubation at 37 ◦ C for 5 min. Each dressing was then placed in a 50 mL of centrifuge tube containing 12.5 mL of distilled water. The tubes were inverted three times to rinse the unclotted blood cells, and the absorbance of the hemoglobin was measured at 540 nm by UV−vis spectrophotometer (UV-1200 MAPADA, China). Then the nonwoven samples were washed with phosphate buffer solution (pH 7.4) several times and immobilized with 2.5% glutaraldehyde for 2 h, dehydrated with a series of ethanol and dried with critical point drier, and then observed by SEM (JSM-6010LA, JEOL Ltd., Japan). 2.4. Platelet aggregation Blood was collected from the heart of rabbits under anesthesia by a syringe and added into a sodium citrated tube with anti-
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Fig. 2. FTIR spectra of CS, NSCS, CMCS and TMCS nonwoven.
pathway of coagulation (aPTT) was performed by mixing 100 L of PPP with 100 L of partial thromboplastin reagent and incubating at 37 ◦ C. Thereafter, nonwoven samples and 100 L of CaCl2 were added after 3 min of incubation and then measured. For PT determination, the extrinsic pathway, was performed by adding 100 L of PT reagent in the mixture, 100 L of PPP and nonwoven samples, which preincubated at 37 ◦ C for 3 min, and the clotting time then measured [22,23]. 2.6. Cytotoxicity assays
Fig. 3.
1
H NMR spectra of CS, NSCS, CMCS and TMCS nonwoven.
coagulant (9:1 v/v). The citrated whole blood was centrifuged at 3000 × g for 15 min at room temperature to obtain platelet poor plasma (PPP). Platelet rich plasma (PRP) in the upper layer was isolated from centrifugation of the citrated whole blood at 150 × g for 15 min. Platelet aggregation was determined by measuring changes in the optical density of stirred PRP after the addition of nonwoven dressings. 100 L of each nonwoven sample (10 mg/100 L normal saline) was added to a cuvette with 900 L of PRP accordingly, then the light transmittance was measured at 595 nm immediately and after a 20 min interval at 37 ◦ C by UV−vis spectrophotometer. Normal saline and adenosine diphosphate (ADP) were used as a negative and positive control respectively. 100 L of PRP and PPP were pipetted into wells for calibration [20,21]. The percent of aggregation was calculated as follows: Aggregation% =
L595,PRP − L595.test × 100% L595,PRP − L595,PPP
Where L595,PRP , L595,PPP and L595,test were the absorbance of the PRP, PPP and tested samples. 2.5. Blood coagulation activity Activated partial thromboplastin time (aPTT) and prothrombin time (PT) with different nonwoven samples were measured using a semiautomatic coagulation analyzer (TS6000, MD PACIFIC Ltd., China). The activity of each nonwoven dressing on the intrinsic
In vitro cytotoxicity of the nonwoven dressings were investigated by the MTT assay using L929 cell line [24–26]. The sterilized nonwoven samples were incubated in DMEM at 37 ◦ C for 24 h to obtain extracts of 0.1 g/mL. L929 cells were seeded in 96-well culture plate at a density of 1 × 104 cells per well and incubated under a 5% CO2 atmosphere at 37 ◦ C for 24 h. The culture medium was removed and replaced with the concentration of 25%, 50% and 100% extracts dilutions and incubated for another 24 h and 48 h. Then 10 L of MTT solution was added into each well. After 4 h incubation, the formazan salts were dissolved followed by addition of 150 L of DMSO for about 10 min on the shaker at 37 ◦ C. The absorbance at 490 nm was determined with an ELISA reader (SUN˘ RISE, BASIC TECAN, 5082 GrOdig, Austria). The relative cell viability (RCV) was expressed by the following equation: RCV% =
Abssample Abscontrol
× 100%
Where Abs sample and Abs control were the absorbance of the tested sample and the negative control. 2.7. Hemostatic assay on ear artery Rabbits were anesthetized by intravenous injection of 3% pentobarbital sodium (20 mg/kg) and shaved off the hair on the back of the ear to expose the ear artery. A 1 cm × 1 cm wound including the ear artery was created, and the pre-weighted nonwoven dressing (CS, NSCS1, CMCS1, TMCS1 and Gauze) was applied over the blooding area after free hemorrhage for 5 s and constant pressure (200 g) was exerted. The nonwoven dressing was removed and weighted until blood was absolutely coagulated and the hemostatic time was recorded. Total blood loss was also evaluated by the difference value between the weights of nonwoven dressing before and after the hemostatic assay [26,27]. All above mentioned experimental rabbits were treated with the National Research Council’s Guide for the care and use of laboratory animals.
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Fig. 4. (A) Blood absorption behavior of nonwoven samples. (B) The effects of different nonwoven samples on blood clotting. *P < 0.05 compared to the CS group. SEM images of interaction between RBCs and CS (C), NSCS1 (D), NSCS2 (E), CMCS1 (F), CMCS2 (G), TMCS1 (H), TMCS2 (I).
Table 1 Reaction conditions and DS (%) for the nonwoven dressing. Samples
Reagents
Groups
DS (%)
NSCS1 NSCS2 CMCS1 CMCS2 TMCS1 TMCS2
succinic anhydride succinic anhydride chloroacetic acid chloroacetic acid methyl iodide methyl iodide
-CO(CH2 )2 COOH -CO(CH2 )2 COOH -CH2 COONa -CH2 COONa -(CH3 )3 -(CH3 )3
17.5 39 19 36.5 17.3 38.5
2.8. Statistical analysis All the data were expressed as the mean ± standard deviation (SD), and assessed by a one-way analysis of ANOVA to demonstrate differences between groups. P values <0.05 were considered to be statistically significant. 3. Results and discussions
amino groups of chitosan [29]. CMCS nonwoven was confirmed by the new absorption bands in 1598 cm−1 and 1454 cm−1 [30,31]. For the TMCS nonwoven, the peak at 1597 cm−1 reduced (N H stretching) and 1454 cm−1 (C H stretching) observably increased [15], which indicated the introduction of quaternary ammonium groups onto chitosan chain. The 1 H NMR spectra (Fig. 3) showed the successful synthesis of NSCS, CMCS and TMCS nonwoven. The 1 H NMR assignments of chitosan nonwoven was as follows [30,32]: 1 H NMR (D2O/DCl) ı=4.75 (H1), ı=3.0 (H2), ı=3.43-3.81 (H3, H4, H5, H6). The 1 H NMR assignments of NSCS nonwoven was as follows: 1 H NMR (D2O/DCl) ␦ = 2.90 (H2), ␦ = 3. 50-3.82 (H1, H3, H4, H5, H6), ␦ = 2.3 (H*) [33]. The characteristic proton signals of CMCS nonwoven appeared in the range of ı=4.0-4.1, which indicated that the amino groups and hydroxyl groups were partly carboxymethylated [28], and the characteristic peak of TMCS appeared in ı=3.1-3.3 [13,34]. According to the ratio of the integral peak of characteristic peak of simple nonwoven and H2 in chitosan nonwoven structure, it could be known that the degree of substitution [35].
3.1. Synthesis and characterization of NSCS, CMCS and TMCS nonwoven dressing
3.2. Whole blood clotting test
NSCS, CMCS and TMCS nonwoven were synthesized by introduction of succinyl groups, carboxymethyl groups and quaternary ammonium groups into the −NH2 or OH of the glucosamine units on the surface of CS nonwoven, respectively (Fig. 1). The degree of substitution (DS) could be varied by regulating reaction conditions (Table. 1). FTIR analysis demonstrated the chemical structure on the CS, NSCS, CMCS and TMCS nonwoven (Fig. 2). The CS nonwoven spectrum showed that the principal absorption bands appeared at 3440 cm−1 (O H stretch), 1652 cm−1 (C O stretch), 1593 cm−1 (N H bend), 1153 cm−1 (bridge O stretch), 1077 cm−1 (O H stretch) [28]. Compared with FTIR spectrum of CS, the reduced intensity of the peak at 1598 cm−1 (-NH2 bending) and the new absorption band of 1413 cm−1 ( COO symmetric stretch) and 1559 cm−1 (Amide II) indicated that succinyl introduced onto the
Blood absorption behavior was observed after application of 100 L of citrated whole blood through pipetting onto each nonwoven samples. Chitosan nonwoven is relatively hydrophobic when applied blood immediately and 5 min later (Fig. 4A). Blood was not absorbed in the TMCS2 nonwoven sample as well, which due to the presence of more positive charge leading to the occurrence of hemolysis [36]. All other nonwoven samples exhibited a better blood absorption because of the surface hydrophilic. The recalcified blood was applied onto nonwoven samples, and the unclotted blood rinsed by distilled water was determined at 540 nm (Fig. 4B). The smaller absorbance value indicated the stronger hemostatic potential of the samples. The absorbance value of TMCS2 nonwoven (0.345 ± 0.003) was the greatest among the samples, which resulted from more introduced quaternary ammonium cationic group that would lead to the hemolysis. The irregular
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Fig. 5. Platelet aggregation by nonwoven samples. *P < 0.05 compared to the negative and positive control group.
shape erythrocytes were found on the surface of TMCS2 nonwoven samples in the SEM image (Fig. 4I). Meanwhile, CS nonwoven had a slightly higher absorbance value (0.307 ± 0.002), due to the solid state form has a disability to induce the aggregation [8]. And its poor absorption was observed in blood absorption behavior test. Similarly, the SEM images showed only a few RBCs were absorbed on the CS nonwoven (Fig. 4C). Compared with all the nonwoven samples, NSCS1 had the best hemostatic activity (0.105 ± 0.003), as a result of the negative charges on the surface promoting the intrinsic pathway of coagulation [9].Moreover, the CMCS nonwoven could also promote blood coagulation, but exhibited a slightly lower absorbance value than that of NSCS nonwoven. Notably, for the same sample, the higher degree of substitution had a higher absorbance value, which indicated lower blood absorption on the surface of samples, and this might be because the repulsive force between the negative charges on the nonwoven surface and erythrocyte membrane hindered the blood absorption behavior of nonwoven samples. 3.3. Platelet aggregation In aggregation test, the adherence and aggregation properties of platelet with different nonwoven samples were evaluated. As shown in Fig. 5, the ability to induce platelet aggregation of CS nonwoven sample (7.746 ± 0.05) was stronger than NSCS and CMCS nonwoven samples beacause of the increasing Ca2+ mobilization and enhancing expression of GPIIb/IIIa complex on platelet membrane surfaces [37]. The platelet aggregation of TMCS nonwoven samples (TMCS1 8.746 ± 0.054, TMCS2 10.979 ± 0.163) was the strongest among all the samples. What’s more, the platelet aggregation increased with the increasing of DS. TMCS nonwoven samples had the highest value of platelet aggregation likely due to the positive charges on the surface which could enhance the ability of samples adhesion [20]. Furthermore, the ability to induce platelet aggregation of NSCS nonwoven samples was not significantly different from CMCS nonwoven samples. 3.4. Blood coagulation activity The blood coagulation cascade involves intrinsic, extrinsic, and common pathways. PT and aPPT were investigated to evaluate the effects of the nonwoven samples on the extrinsic and intrinsic coagulation pathways, respectively. Compared to the control group, the results showed that no statistically significant difference of all the nonwoven samples which were observed in the PT assay (Fig. 6A). It indicated that the extrinsic pathway of coagulation was not affected by these nonwoven samples. CS nonwoven sample did not change
Fig. 6. PT (A) and aPPT (B) measurement of CS, NSCS, CMCS and TMCS nonwoven samples. *P < 0.05 compared to the control group.
the PT and aPPT owing to the properties of chitosan to agglutinate RBCs rather than interfere with the coagulation factors [9,38]. The values of aPTT were shorter treated with NSCS and CMCS nonwoven samples compared with the control group. The shortening of aPTT could be attributed to the negative charges on the surface which could activate the intrinsic pathway of coagulation along with cofactors HWK-kininogen and prekallikrein [39]. Recent studies had reported that positive charges could increase aPTT [20], which may be the reason for the changes of aPPT treated with TMCS nonwoven samples. 3.5. Cytotoxicity assays To investigate the cytotoxicity of NSCS, CMCS and TMCS nonwoven, the MTT method of L929 cells was adapted for in vitro cytotoxicity assay (Fig. 7). It could be seen that, at 0.1 g/mL of NSCS2, the viability of the cell was lower than those of another concentration. That was probably owing to the ionization of the COOH on the surface of NSCS nonwoven in the DMEM, caused the extracts solution to be acidic and inhibited the growth of cells. But there was another reason, the sticky extracts solution formed by the dissolution of the higher DS of NSCS2 nonwoven in the DMEM also affected the growth of cells. However, the viability of the cell was high ( > 80%) after treated with the diluted concentration of NSCS2 nonwoven extracts, which indicated that NSCS2 nonwoven sample was relatively low toxic to L929 cells. Moreover, no statistically significant differences were observed in the cell activity of L929 cell within 48 h in all other nonwoven samples, which demonstrated that they were no toxic to L929 cells. All the results suggest that NSCS, CMCS and TMCS nonwoven had good biocompatibility to be used as hemostatic materials. 3.6. Hemostatic assay on ear artery The hemostasis of the lower DS of nonwoven samples (NSCS1, CMCS1 and TMCS1) was analyzed by measuring the hemostatic
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Fig. 7. Cell viability measured by MTT assay for nonwoven samples extract of different concentrations after 24 h (A) and 48 h (B) of incubation. Data are presented as the mean ± SD (n = 5).
Fig. 8. The rabbit ear artery injury model among the Gauze, CS, NSCS1, CMCS1 and TMCS1 (A).The hemostatic time and the total blood loss (B) of the treatments of Gauze, CS, NSCS1, CMCS1 and TMCS1 of rabbit ear artery injury. * P < 0.05 compared to the gauze group. Data are presented as the mean ± SD (n = 5).
time and the amount of blood loss of rabbit ear artery (Fig. 8). Above results mentioned, the lower DS of nonwoven exhibited better hemostasis property and could maintain the original fiber morphology. Therefore, the NSCS1, CMCS1 and TMCS1 nonwoven samples were chosen for the hemostatic assay on ear artery. Compared with the Gauze, CS nonwoven sample could reduce the hemostatic time
and the blood loss, owing to the property of promoting platelet aggregation. Moreover, all modified chitosan nonwoven exhibited better hemostatic property including the hemostatic time and the amount of blood loss. In particularly, the time for hemostatic of NSCS1 (147 ± 3.7 s) was the shortest in those of other groups, and the total blood loss of the treatment with the NSCS1 (0.23 ± 0.05 g)
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was less than other hemostatic dressings as well. The results demonstrated that NSCS1 nonwoven had better hemostatic property, which may be caused by the influence of intrinsic pathway of coagulation. 4. Conclusions The NSCS, CMCS and TMCS nonwoven were successfully synthesized in the present work. Moreover, the NSCS, CMCS and TMCS nonwoven with different hemostatic mechanism exert a better hemostatic property than CS nonwoven. In the blood clotting test and blood coagulation activity, the negative charges on the surface of NSCS and CMCS could activate the intrinsic pathway of coagulation, along with the hemostatic mechanism of chitosan, and thus accelerated blood coagulation. But the repulsive force generated by the excessive negative charges between nonwoven surface and erythrocyte membrane hindered the blood absorption behavior of high DS of nonwoven samples. Moreover, the lower blood absorption of the TMCS nonwoven might be owing to the introduced quaternary ammonium cationic group of TMCS nonwoven that would lead to the hemolysis. The ability of inducing platelet aggregation for TMCS nonwoven samples was strongest among all the samples, which would contribute to initiating hemostasis. However, the ability to induce platelet aggregation of NSCS nonwoven samples was weak and not significantly different from CMCS nonwoven samples. The results of the rabbit ear artery injury model demonstrated that NSCS, CMCS and TMCS nonwoven exhibited a better hemostatic property than gauze and CS nonwoven, and especially, the NSCS nonwoven had an excellent hemostatic effect, compared to other nonwoven samples. Consequently, It is expected that this study is useful for understanding the hemostatic mechanism of these surface modified chitosan nonwoven dressings, which could use as promising hemostatic intervention. Acknowledgments This work was supported by the National Natural Science Foundation of China (81671828), China Postdoctoral Science Foundation (2016M592246), Applied Basic Research Plan of Qingdao (16-5-170-jch), and the Taishan Scholar Program, China. References [1] D.S. Kauvar, R. Lefering, C.E. Wade, J. Trauma 60 (2006) S3–11. [2] L.W. Chan, C.H. Kim, X. Wang, S.H. Pun, N.J. White, T.H. Kim, Acta Biomater. 31 (2016) 178–185. [3] K.F. Earl, W. Davie, Walter. Kisiel, Biochemistry 30 (1991) 10363–10370. [4] A.M. Behrens, M.J. Sikorski, P. Kofinas, J. Biomed. Mater. Res. Part A 102 (2014) 4182–4194. [5] M. Rinaudo, Prog. Polym. Sci. 31 (2006) 603–632.
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