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Blood absorption improvement of a naturally derived hemostatic agent by atmospheric pressure plasma jet
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ScienceDirect Materials Today: Proceedings 17 (2019) 2088–2096
www.materialstoday.com/proceedings
MRS-Thailand 2017
Blood absorption improvement of a naturally derived hemostatic agent by atmospheric pressure plasma jet Jureeporn Jaifua,b,c, Kittiya Thunsiria,b,c, Suruk Udomsoma,b, Dheerawan Boonyawand, Wassanai Wattanutchariyab,c,* a
Biomedical engineering program, Faculty of Engineering, Chiang Mai University, Chaing Mai, Thailand b Biomedical engineering center(BMEC), Chiang Mai University, Chiang Mai, 50200, Thailand c Advanced manufacturing technology research center(AMTech), Department of Industrial Engineering, Chiang Mai University, Chiang Mai, Thailand d Plasma and beam physics research facility(PBP), Department of Physics and Materials, Faculty of Science, Chiang Mai University, Chiang Mai, Thailand
Abstract Hemostatic capability improvement of a chitosan hemostatic agent was achieved by surface modification using the atmospheric pressure plasma jet experiment. The chitosan hemostatic agent acquired high blood absorption rate, at 4.60 ml/m, when exposed to input power 10 W of the radiofrequency power supply, the argon flow rate of 4 L/m mixture with an oxygen gas of 10 ml/m, and treatment time of 30 s. A significant decreased was observed in the hemoglobin leak value, inducing early blood clotting ability of clotting in 30 s. The strong relation of gas plasma discharge with the OH radical and the atomic oxygen, exhibited an increase in the hydrophilic properties, which lead to the chitosan hemostatic agent being very effective.
© 2019 Elsevier Ltd. All rights reserved. Selection and/or Peer-review under responsibility of The First Materials Research Society of Thailand International Conference. Keywords: Hemostatic agent; Chitosan(CS); Blood absorption; Hemoglobin leak; Atmospheric pressure plasma jet(APPJ)
1. Introduction Uncontrolled hemorrhage is one of the important problems encountered during operations. Surgery interrupts the blood vessel status and function and increases the chances of occurrence of bleeding complications [1,2]. *Corresponding author. Tel.: +66 5394 4125, +66 5394 4183; fax: +66 5394 4185. E-mail address : [email protected] 2214-7853© 2019 Elsevier Ltd. All rights reserved. Selection and/or Peer-review under responsibility of The First Materials Research Society of Thailand International Conference.
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Management of rapid clotting is a primary concern in the procedures2 [3]. Traditionally, gauze product is generally used, but its blood absorption rate is dependent on the compressive force applied to the area that is operated on [4]. Prolonging this pressure, however, causes damage to nerves and cell tissues [1]. Thus, a hemostatic agent plays an important role as its use is the most effective method to stop bleeding by providing an agent that assists clot formation [3,5]. In the last few years, several hemostatic agents with high effectiveness have been developed to control bleeding at rapid rates [3,4]. Biomaterials such as gelatin, rice starch, and chitosan are widely used in medical application. Previous studies have presented effective hemostatic agent abilities of these biomaterials [3,5,6]. According to Chiang Mai University’s Science and Technology Research Institute’s mention of important factors as regards commercial hemostatic agents, the material should quickly stop bleeding, be absorbed effectively, be degraded in the body within a specific period, and have no side effects [7]. Gelatin (Gel) is the widely used commercial hemostatic agent because it is a fast biodegradable material [3,8]. A previous study presented the good performance of rice starch (RS) in absorption: Because starch granules have the property of gelatinization, the RS hemostatic agent provided high water absorption and good biodegradability [9]. Chitosan (CS) is a natural polymer that can be used as a hemostatic agent [6]. A previous study presented that CS has a hemostatic effect because of its electrostatic forces [10]. Moreover, CS has high water-binding capacities which could accelerate platelet adhesion, and red blood cell (RBC) aggregation leading to quick stopping of bleeding [11]. Therefore, mixing CS, Gel, and RS may yield effective blood absorption properties. Furthermore, the above-mentioned materials are local materials with low market value. Thus, fabrication of these materials into medical materials will add to the ways for value addition. Currently, plasma jet technology has been introduced and implemented as a material surface modification approach [12,13]. Atmospheric pressure plasma jet (APPJ) can enhance material surface ability without causing material structure interruption [13,14]. Furthermore, the mixture of Ar/O2 gas plasma generates the etching process, which decreases the surface tension and presents surface roughness. This effect increases the hydrophilicity and absorption properties of materials [15,16]. Also, the combination of CS, Gel, and, RS was fabricated in various ratios to investigate the most absorbent one. The most effective ratio was treated by APPJ and investigated for the difference with regard to before treatment and, after treatment. 2. Materials and Methods 2.1. Preparation and fabrication of naturally-derived hemostatic agents The selected materials in this study are CS, Gel, and RS. Each of the materials was prepared in a solution form and mixed in various ratios, as shown in Table 1. Table 1. Material Ratio of Naturally Derived Hemostatic Agents Chitosan solution
Gelatin solution
(2.0% w/v)
(2.5% w/v)
Rice starch solution (2.5% w/v)
1
0
0
0
1
0
0
0
1
1/2
1/2
0
0
1/2
1/2
1/2
0
1/2
1/3
1/3
1/3
2.1.1. Preparation of chitosan solution In this study, the squid pen CS was purchased from TAMING ENTERPRISE CO., LTD. (The degree of deacetylation was 94.69%). The 2% (w/v) CS solution was prepared by soaking 2 g of CS in 100 ml deionized water
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with 1% acetic acid, and the solution was kept at room temperature for 3 days [6,17]. After 3 days, the CS was found to have completely dissolved, and homogeneously gelatinized, ready for mixing. 2.1.2. Preparation of gelatin solution Pork Gel was purchased from Nitta Gelatin Inc. The Gel solution was prepared by dissolving 2.5 g of Gel powder in 100 ml of deionized water. The mixing solution was continuously stirred at 80ºC for 15 min to form a 2.5% (w/v) Gel gelatinized solution [9]. 2.1.3. Preparation of rice starch solution Medical grade RS was purchased from Erawan Pharmaceutical Research, and Laboratory Co., Ltd., Thailand. The RS solution was prepared by dissolving 2.5 g of RS powder in 100 ml of deionized water. The mixing solution was continuously stirred at 80ºC for 2 h until it was gelatinous to form a 2.5% (w/v) RS solution [9]. 2.1.4. Fabrication of naturally derived hemostatic agents The prepared solutions of CS, Gel, and RS were mixed in the ratio shown in Table 1. Then, a cross–linking agent, 2.5% glutaraldehyde, was added for 1 ml/100 ml of the solution and mixed until the solution became homogeneous. Afterward, 1 ml of the cross–link solution was poured into 24–well plates. Then, the samples were prefrozen at –80ºC in the freezer for 24 h prior to the freeze-dry process which was carried out at –50ºC for 48 h [3,9]. 2.2. Characterization of naturally derived hemostatic agents 2.2.1. Biodegradation testing Biodegradation was performed to confirm the degradation ability in the conditions mimicking human body of the fabricated hemostatic agent in every ratio. The degradation was determined by weight loss after immersing the samples in 3 ml of PBS containing lysozyme (1.6 µl/ml) and incubated at 37°C for 7 days. Then, the degraded samples were repeatedly washed by deionized water for 3 times. The samples were pre-frozen at –80ºC for 24 h in the freezer again before the freeze–dry process for 48 h [11,17]. The rate of degradation was compared between the final weights of the sample after the freeze drying (Wo) and the initial weight of the sample (Wt). The percentage of biodegradation was calculated by the following equation:
%D
Wt Wo x 100 Wt
2.2.2. Blood collection The donated blood which was collected by the licensing nurse was kept in an anticoagulant agent tube (3.2% sodium citrate) with 0.9 ml in each tube for avoiding blood clotting. The donated blood was used within an hour of collection [11]. 2.2.3. Maximum volume of blood absorption testing In this study, blood absorption volume was a decision factor for the material ratio selection. The ratio that provided the highest blood absorption volume as used in the APPJ experiment and the difference was compared between before treatment and after treatment. The testing started with the preparation of the fabricated hemostatic
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agent (n=6, each ratio). The whole blood was dropped on the hemostatic agent and the blood volume was measured simultaneously until the absorption limit of the hemostatic agent was reached. 2.2.4. Blood absorption rate testing After the material ratio which provided the highest blood absorption volume was selected. The blood absorption rate was tested on the selected hemostatic agent and the rates between before the APPJ experiment, and after the APPJ experiment were compared. The APPJ conditions are presented in Table 2. The absorption rate of the hemostatic agent is defined as the time required for the absorption of 0.2 ml blood. The APPJ condition that provided the highest blood absorption rate was used in the hemoglobin leak test [6]. 2.2.5. Hemoglobin leak testing This test was performed after the best fabricated hemostatic agent ratio, and the APPJ condition were selected. The test was performed before and after the APPJ experiment. A volume of 0.9 ml of citrate collected blood was recalcified by mixing it with 0.1 ml of 16 mM CaCl2 [11]. The decalcified blood was pipetted for 1.0 ml and dropped into the hemostatic agent. The blood clotting was observed by the leakage of hemoglobin from the samples after the blood was dropped into the sample for 30, 60, 90, 120, 150, and 180 s. The sample with the blood on was soaked in 10 ml of deionized water and the leakage of hemoglobin from the samples was observed. The RBCs that were not entrapped in the clot were hemolyzed with the deionized water. The soaked water of each sample was measured for absorbance at 540 nm (UV–Vis spectrophotometer) [6]. 2.3. Atmospheric pressure plasma jet treatment The schematic diagram of the APPJ setup is shown in Figure 1. The plasma discharge was driven by a radiofrequency (RF) power supply and set up from a combination of argon and oxygen gases under the atmospheric pressure plasma jet (APPJ). The copper inner electrode was inserted into the middle of the quartz capillary and the outside of quartz capillaries was wrapped with aluminum outer electrode while it worked as the grounding electrode. The complete electrode system was enveloped by Teflon. The gap between the quartz capillaries and the sample was 5 mm [16,18]. However, the necessity of the experiment was that it should carry out the spectrum analysis by the OES (optical emission spectroscopy) software and lead to the appropriate plasma treatment condition [14,19]. The argon gas flow rate was fixed as 4 L/min. The oxygen gas flow rate (ml/min), the input power (W), and the treatment times were varied in the different conditions, as shown in Table 2.
Figure 1. The schematic of the atmospheric pressure plasma jet.
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J. Jaifu et al. / Materials Today: Proceedings 17 (2019) 2088–2096 Table 2. Atmospheric Pressure Plasma Jet Treatment Condition APPJ treatment (Fixed argon flow rate 4 L/m) Input power (W)
Oxygen flow rate (ml/m)
Treatment time (s)
10
10
30
10
10
60
10
10
90
10
30
30
10
30
60
10
30
90
15
10
30
15
10
60
15
10
90
15
30
30
15
30
60
15
30
90
3. Statistical All experiments were repeated 6 times, and the data presented are expressed as mean ± SD. The statistical analysis was employed using ANOVA, and the multiple comparison test was conducted using the MINITAB software program. The difference was considered statistically significant when the p–value was less than 0.05 4. Results and Discussion 4.1. Fabricated naturally derived hemostatic agent All the material ratios in the study can be fabricated to form a naturally derived hemostatic agent. The structure and the appearance of each ratio is presented in Figure 2. However, the appearances of the–pure Gel and the Gel–RS specimens were such that they looked easily deformed and fragile. A previous study reported that low concentrations of the Gel solution and glutaraldehyde (GA) were found to increase the property of fragility [5]. Thus, the concentration of 2.5% (w/v) Gel solution and 0.025% of GA were not appropriate to fabricate a hemostatic agent. At the meantime, RS was also observed to have poor mechanical properties and be not uniform [7,9].
Figure 2. The naturally derived hemostatic agent in each of the ratios between CS:Gel:RS.
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4.2. Biodegradation of naturally derived hemostatic agent After the naturally derived hemostatic agent was fabricated, the biodegradation test was performed. The degradation results after 7 days in the conditions mimicking human body are shown in Figure 3a. Most of the samples degraded by more than 50%, except the sample with the 1/3:1/3:1/3 ratio. The sample with the ratio of only Gel was found to have completely degraded before 7 days, which is a finding that is in accordance with the reports of the previous studies that presented the fast biodegradability of the Gel hemostatic agent [3,8]. Biodegradability is one of the important properties in tissue engineering. The trend of biodegradability is one of the properties exhibited by newly developed materials. A previous study reported that proteases and lysozymes can also break the glycosidic bonds of the polysaccharide of CS, and could degrade the particle of CS to oligosaccharides, following which they combine with the metabolic pathway and are excreted [1,3,20,21]. The results also confirm the degradation ability of the fabricated naturally derived hemostatic agent which is sure to degrade in the surgical area after use. 4.3. Maximum volume of blood absorption Fast absorption is one of the factors recommended factors for hemostatic agents [7]. In fact, the effectiveness of the hemostatic agent is defined by its ability to absorb large volumes of blood. For that reason, this study identified the best ratio of naturally derived hemostatic agent of the CS, Gel, and RS solution based on a test of the maximum volume of blood absorption. As mentioned previously, the ratio which provided the highest blood absorption volume was used in the APPJ experiment. The results are shown in Figure 3b. The largest volume of blood was absorbed by the CS hemostatic agent, which absorbed a volume of 1.2±0.1 ml. In contrast, the hemostatic agent which contained Gel could not completely absorb the blood within 10 min. Thus, ratios which could not absorb blood within 10 min were classified as non–absorbent ratios. The low concentration of the Gel solution and its property of being fragile were the causes of it having the lowest blood absorbability [3,6]. The CS hemostatic agent exhibited higher absorbance than all of the ratios because it has the specific advantage of having an excellent swelling machanism [22]. CS is composed of polar groups, which are an interaction effect between amine (NH2) and hydroxyl (OH). The hydrophilic property is mainly because of the amine group, the hydrogen, and the covalent bond interaction [10,22,23,24]. The swelling property of CS abled it to effectuate high absorption of the serum proteins and fluids in the RBCs [11]. All of the ratios indicated significant difference (P<0.05). Therefore, in this experiment, the CS hemostatic agent was selected as the hemostatic agent of choice to investigate the effective plasma treatment condition. As mentioned previously, the ratio that provided the highest blood absorption volume was used in the APPJ experiment. The results are shown in Figure 3b. The highest blood absorption volume was presented by the CS hemostatic agent which absorbed a volume of blood of 1.2±0.1 ml. Interestingly, the hemostatic agent which contained Gel could not completely absorb the blood within 10 min. Fast absorption is one of the recommendations with regard to factors essential for a good hemostatic agent [7]. Thus, ratios which could not absorb the blood within 10 min were classified as non–absorbent ratios. Based on the results of this experiment, the hemostatic agent selected was the CS hemostatic agent.
Figure 3. (a) Biodegradation of the naturally derived hemostatic agents, and (b) the maximum volume absorption of human whole blood of the naturally derived hemostatic agents.
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4.4. Blood absorption rate CS exhibited hydrogen bonding, and induced high water uptake, because of the internal effect between the amine (NH2) and the hydroxyl (OH) groups of CS, while they combined with the covalent bond of the external interaction [20,22]. At the same time, several studies have reported that since the amine group of CS is positively charged, it could have interacted with the negative charge of the RBC membrane due to by the electrostatic force, leading to rapid blood absorbance [5,6,10]. The amine (NH2) and the hydroxyl (OH) groups are the components of CS which that exhibit hydrogen bonding and induce high water uptake [22,25]. In this study, the CS hemostatic agent was used to investigate the effective plasma treatment condition, APPJ in various conditions, as presented in Table 2. The comparison of each APPJ condition is shown in Figure 4a: the highest blood absorption rate of 4.60 ml/m, was found in the APPJ condition of input power 10 W, Ar flow rate 4 L/m of the mixture with the O2 gas 10 ml/m, and treatment time in 30 s. At the meantime, all of the plasma treatment conditions with treatment times of 30 s and 60 s presented higher volumes of blood absorbed than that in the non–treated condition, whereas the high input power of 15 W and the prolonged treatment time of 90 s were found to have damaged the surface of the samples, which decreased the blood absorption to less than that of the non–treated condition, while the factor response of the O2 flow rate and the treatment time did not indicate significant difference (P>0.05) and it was not found to be able to affect the blood absorption rate properties. The APPJ experiment in this work excited the ions and discharged the argon/oxygen gas mixture, which reactivated the OH radical (308 nm) and the atomic oxygen (777 nm): this increased the surface energy and decreased the surface tension, and they exhibited the wettability of the CS surface. Thus, blood absorption abilities might be increased due to this mechanism. 4.5. Hemoglobin leak The blood clotting ability could be observed from the hemoglobin absorbance (540 nm) which was the results of leakage from the sample. A previous study could utilize the observation of hemoglobin leak by dropping 0.2 ml of blood on the sample before soaking it in water [6]. But in this study, the 0.2 ml of blood was completely entrapped by the CS hemostatic agent before 10 s (Figure 5.). Thus, the hemoglobin leak could not be observed. The blood volume was increased from 0.2 ml to 1.0 ml to increase the chances of hemoglobin leak. The CS hemostatic agent itself could absorb high amounts of blood and accelerated the blood clotting time. The comparison between with and without APPJ treatment and the negative control demonstrated that the observations were totally different in terms of the hemoglobin leak value. The absorbance values of the CS hemostatic agent with and without plasma treatment indicated significant difference (P<0.05) in that the values of leakage of hemoglobin were lower than the those without plasma treatment and the negative control, and it could induce early blood clot in 30 s. The CS hemostatic agent has the specific property of having an excellent swelling mechanism because it has the effect of internal interaction between the amine (NH2) and the hydroxyl (OH) groups, so the hydrophilic property was mainly effected by the amine group, the hydrogen, and the covalent bonding interaction. At the same time, the hydroxyl (OH) groups of CS could have reacted with the water molecules at the external site [1,22,23,24]. The swelling property of CS could enable the immersion and the high absorption of the serum proteins or fluids in the blood [11]. At the same time, the hemostatic agent of CS can relate to the positive charge of the amino backbone (NH2); therefore, it interacts with the negative charge of the RBC membrane by electrostatic mechanism [5,10]. The OH bonding increased the absorbability of the water molecule, the ionic complex, and the serum protein of the blood on the CS surface, while the RBC particle could slowly pass into the small pores of CS and accelerate blood clotting [10,11,20,26]. Additionally, the plasma treatment enhanced the high amount of blood absorption and accelerated the clotting time of the blood onto the CS surface. A previous study reported that Ar plasma treatment was effective with the amine groups of CS membranes as it increased the physical property through etching of surface roughness. At the same time, Ar gas plasma has been found to discharge free radicals on the sample surface, following which the free radicals would react with the O2 functional group from the atmosphere: these chemical properties of Ar plasma allow for ionic permeability and blood clotting properties [11,15,27,28]. Therefore, the APPJ treatment is effective enough to improve blood absorption abilities and accerelate the rapid blood clotting of the CS hemostatic agent.
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Figure 4. (a) Absorption rate of 0.2 ml of human whole blood of naturally derived hemostatic agents with plasma jet treatment condition, and (b) comparison of hemoglobin absorbance of 1.0 ml of human whole blood of naturally derived hemostatic agents between with and without plasma treatment.
Figure 5. Hemoglobin leak testing of the hemostatic agent from the control (no speciment) to the 3 min period (30 s interval).
5. Conclusion The CS hemostatic agent exhibited high swelling ability between the internal reaction of an amine (NH2) and hydroxyl (OH) groups with covalent bonding of the water molecule at the external site. Thus, the swelling property of CS enabled it to absorb the high volume of the serum proteins and fluids in the blood. At the same time, the hemostatic agent of CS can relate to the positive charge of the amino backbone, and it then interacts with the negative charge of the RBC membrane through electrostatic force. Furthermore, CS has good biodegradability, so the proteases and the lysozyme could also break the glycosidic bonds of the polysaccharide of CS, and could degrade the particles of CS to oligosaccharides; thereafter, they combine with the metabolic pathway, and get excreted, thus promoting non-toxicity. Therefore, CS is appropriated to fabricate the naturally derived hemostatic agent. This study achieved the improvement of blood absorption properties with APPJ. The plasma treatment is an effective technique to enhance the blood absorbance ability of the CS hemostatic agent. Non-thermal plasma of the APPJ experiment is an alternative to surface modification. This study achieved the hemostatic capacity performance with an input power of 10 W, Ar flow rate of 4 L/m mixture with O2 gas at 10 ml/m, and treatment time of 30 s. The Ar/O2 gas excited the ions and discharged the OH radicals (308 nm) and atomic oxygen (777 nm), which increased the hydrophilic property. This plasma condition increased the blood absorption rate to 4.60 ml/m. In addition, acceleration of early blood clotting to within 30 s was also successful. Thus, plasma treatment has its advantages, and it could contribute to improving the blood absorption ability of the CS hemostatic agent in surgical applications.
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Acknowledgments The authors would like to thank Assoc. Prof. Dr. Phisit Seesuriyachan from the Faculty of Agro–Industry, Chiang Mai University, Thailand, for laboratory support during the preliminary experiment. References [1] O.E. Ogle, J. Swantek, A. Kamoh, Hemostatic Agents, J. Dent. Clin N Am. 55 (2011) 433–439. [2] J.B. Glick, R.R. Kaur, D. Siegel, Achieving hemostasis in dermatology-Part II: Topical hemostatic agents, J. Indian Dermatol. 4(3)(2013) 172–176. [3] M. Kabiri, S.H. Emami, M. Rafinia, M. Tahriri, Preparation and characterization of absorbable hemostat crosslinked gelatin sponges for surgical application, J. Current Applied Physics. 11 (2011) 457–461. [4] J. Barnard, R. Millner, A review of Topical Hemostatic Agents for Use in Cardiac Surgery, J. Ann Thorac Surg. 88 (2009) 1378–1383 [5] M. Emilia, S. Luca, B. Francesca, et al, Topical hemostatic agents in surgical practice, J. Transfusion and Apheresis Science. 45 (2011) 305311. [6] P.L. Kang, S.J. Chang, I. 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ScienceDirect Materials Today: Proceedings 17 (2019) 2088–2096
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MRS-Thailand 2017
Blood absorption improvement of a naturally derived hemostatic agent by atmospheric pressure plasma jet Jureeporn Jaifua,b,c, Kittiya Thunsiria,b,c, Suruk Udomsoma,b, Dheerawan Boonyawand, Wassanai Wattanutchariyab,c,* a
Biomedical engineering program, Faculty of Engineering, Chiang Mai University, Chaing Mai, Thailand b Biomedical engineering center(BMEC), Chiang Mai University, Chiang Mai, 50200, Thailand c Advanced manufacturing technology research center(AMTech), Department of Industrial Engineering, Chiang Mai University, Chiang Mai, Thailand d Plasma and beam physics research facility(PBP), Department of Physics and Materials, Faculty of Science, Chiang Mai University, Chiang Mai, Thailand
Abstract Hemostatic capability improvement of a chitosan hemostatic agent was achieved by surface modification using the atmospheric pressure plasma jet experiment. The chitosan hemostatic agent acquired high blood absorption rate, at 4.60 ml/m, when exposed to input power 10 W of the radiofrequency power supply, the argon flow rate of 4 L/m mixture with an oxygen gas of 10 ml/m, and treatment time of 30 s. A significant decreased was observed in the hemoglobin leak value, inducing early blood clotting ability of clotting in 30 s. The strong relation of gas plasma discharge with the OH radical and the atomic oxygen, exhibited an increase in the hydrophilic properties, which lead to the chitosan hemostatic agent being very effective.
© 2019 Elsevier Ltd. All rights reserved. Selection and/or Peer-review under responsibility of The First Materials Research Society of Thailand International Conference. Keywords: Hemostatic agent; Chitosan(CS); Blood absorption; Hemoglobin leak; Atmospheric pressure plasma jet(APPJ)
1. Introduction Uncontrolled hemorrhage is one of the important problems encountered during operations. Surgery interrupts the blood vessel status and function and increases the chances of occurrence of bleeding complications [1,2]. *Corresponding author. Tel.: +66 5394 4125, +66 5394 4183; fax: +66 5394 4185. E-mail address : [email protected] 2214-7853© 2019 Elsevier Ltd. All rights reserved. Selection and/or Peer-review under responsibility of The First Materials Research Society of Thailand International Conference.
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Management of rapid clotting is a primary concern in the procedures2 [3]. Traditionally, gauze product is generally used, but its blood absorption rate is dependent on the compressive force applied to the area that is operated on [4]. Prolonging this pressure, however, causes damage to nerves and cell tissues [1]. Thus, a hemostatic agent plays an important role as its use is the most effective method to stop bleeding by providing an agent that assists clot formation [3,5]. In the last few years, several hemostatic agents with high effectiveness have been developed to control bleeding at rapid rates [3,4]. Biomaterials such as gelatin, rice starch, and chitosan are widely used in medical application. Previous studies have presented effective hemostatic agent abilities of these biomaterials [3,5,6]. According to Chiang Mai University’s Science and Technology Research Institute’s mention of important factors as regards commercial hemostatic agents, the material should quickly stop bleeding, be absorbed effectively, be degraded in the body within a specific period, and have no side effects [7]. Gelatin (Gel) is the widely used commercial hemostatic agent because it is a fast biodegradable material [3,8]. A previous study presented the good performance of rice starch (RS) in absorption: Because starch granules have the property of gelatinization, the RS hemostatic agent provided high water absorption and good biodegradability [9]. Chitosan (CS) is a natural polymer that can be used as a hemostatic agent [6]. A previous study presented that CS has a hemostatic effect because of its electrostatic forces [10]. Moreover, CS has high water-binding capacities which could accelerate platelet adhesion, and red blood cell (RBC) aggregation leading to quick stopping of bleeding [11]. Therefore, mixing CS, Gel, and RS may yield effective blood absorption properties. Furthermore, the above-mentioned materials are local materials with low market value. Thus, fabrication of these materials into medical materials will add to the ways for value addition. Currently, plasma jet technology has been introduced and implemented as a material surface modification approach [12,13]. Atmospheric pressure plasma jet (APPJ) can enhance material surface ability without causing material structure interruption [13,14]. Furthermore, the mixture of Ar/O2 gas plasma generates the etching process, which decreases the surface tension and presents surface roughness. This effect increases the hydrophilicity and absorption properties of materials [15,16]. Also, the combination of CS, Gel, and, RS was fabricated in various ratios to investigate the most absorbent one. The most effective ratio was treated by APPJ and investigated for the difference with regard to before treatment and, after treatment. 2. Materials and Methods 2.1. Preparation and fabrication of naturally-derived hemostatic agents The selected materials in this study are CS, Gel, and RS. Each of the materials was prepared in a solution form and mixed in various ratios, as shown in Table 1. Table 1. Material Ratio of Naturally Derived Hemostatic Agents Chitosan solution
Gelatin solution
(2.0% w/v)
(2.5% w/v)
Rice starch solution (2.5% w/v)
1
0
0
0
1
0
0
0
1
1/2
1/2
0
0
1/2
1/2
1/2
0
1/2
1/3
1/3
1/3
2.1.1. Preparation of chitosan solution In this study, the squid pen CS was purchased from TAMING ENTERPRISE CO., LTD. (The degree of deacetylation was 94.69%). The 2% (w/v) CS solution was prepared by soaking 2 g of CS in 100 ml deionized water
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with 1% acetic acid, and the solution was kept at room temperature for 3 days [6,17]. After 3 days, the CS was found to have completely dissolved, and homogeneously gelatinized, ready for mixing. 2.1.2. Preparation of gelatin solution Pork Gel was purchased from Nitta Gelatin Inc. The Gel solution was prepared by dissolving 2.5 g of Gel powder in 100 ml of deionized water. The mixing solution was continuously stirred at 80ºC for 15 min to form a 2.5% (w/v) Gel gelatinized solution [9]. 2.1.3. Preparation of rice starch solution Medical grade RS was purchased from Erawan Pharmaceutical Research, and Laboratory Co., Ltd., Thailand. The RS solution was prepared by dissolving 2.5 g of RS powder in 100 ml of deionized water. The mixing solution was continuously stirred at 80ºC for 2 h until it was gelatinous to form a 2.5% (w/v) RS solution [9]. 2.1.4. Fabrication of naturally derived hemostatic agents The prepared solutions of CS, Gel, and RS were mixed in the ratio shown in Table 1. Then, a cross–linking agent, 2.5% glutaraldehyde, was added for 1 ml/100 ml of the solution and mixed until the solution became homogeneous. Afterward, 1 ml of the cross–link solution was poured into 24–well plates. Then, the samples were prefrozen at –80ºC in the freezer for 24 h prior to the freeze-dry process which was carried out at –50ºC for 48 h [3,9]. 2.2. Characterization of naturally derived hemostatic agents 2.2.1. Biodegradation testing Biodegradation was performed to confirm the degradation ability in the conditions mimicking human body of the fabricated hemostatic agent in every ratio. The degradation was determined by weight loss after immersing the samples in 3 ml of PBS containing lysozyme (1.6 µl/ml) and incubated at 37°C for 7 days. Then, the degraded samples were repeatedly washed by deionized water for 3 times. The samples were pre-frozen at –80ºC for 24 h in the freezer again before the freeze–dry process for 48 h [11,17]. The rate of degradation was compared between the final weights of the sample after the freeze drying (Wo) and the initial weight of the sample (Wt). The percentage of biodegradation was calculated by the following equation:
%D
Wt Wo x 100 Wt
2.2.2. Blood collection The donated blood which was collected by the licensing nurse was kept in an anticoagulant agent tube (3.2% sodium citrate) with 0.9 ml in each tube for avoiding blood clotting. The donated blood was used within an hour of collection [11]. 2.2.3. Maximum volume of blood absorption testing In this study, blood absorption volume was a decision factor for the material ratio selection. The ratio that provided the highest blood absorption volume as used in the APPJ experiment and the difference was compared between before treatment and after treatment. The testing started with the preparation of the fabricated hemostatic
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agent (n=6, each ratio). The whole blood was dropped on the hemostatic agent and the blood volume was measured simultaneously until the absorption limit of the hemostatic agent was reached. 2.2.4. Blood absorption rate testing After the material ratio which provided the highest blood absorption volume was selected. The blood absorption rate was tested on the selected hemostatic agent and the rates between before the APPJ experiment, and after the APPJ experiment were compared. The APPJ conditions are presented in Table 2. The absorption rate of the hemostatic agent is defined as the time required for the absorption of 0.2 ml blood. The APPJ condition that provided the highest blood absorption rate was used in the hemoglobin leak test [6]. 2.2.5. Hemoglobin leak testing This test was performed after the best fabricated hemostatic agent ratio, and the APPJ condition were selected. The test was performed before and after the APPJ experiment. A volume of 0.9 ml of citrate collected blood was recalcified by mixing it with 0.1 ml of 16 mM CaCl2 [11]. The decalcified blood was pipetted for 1.0 ml and dropped into the hemostatic agent. The blood clotting was observed by the leakage of hemoglobin from the samples after the blood was dropped into the sample for 30, 60, 90, 120, 150, and 180 s. The sample with the blood on was soaked in 10 ml of deionized water and the leakage of hemoglobin from the samples was observed. The RBCs that were not entrapped in the clot were hemolyzed with the deionized water. The soaked water of each sample was measured for absorbance at 540 nm (UV–Vis spectrophotometer) [6]. 2.3. Atmospheric pressure plasma jet treatment The schematic diagram of the APPJ setup is shown in Figure 1. The plasma discharge was driven by a radiofrequency (RF) power supply and set up from a combination of argon and oxygen gases under the atmospheric pressure plasma jet (APPJ). The copper inner electrode was inserted into the middle of the quartz capillary and the outside of quartz capillaries was wrapped with aluminum outer electrode while it worked as the grounding electrode. The complete electrode system was enveloped by Teflon. The gap between the quartz capillaries and the sample was 5 mm [16,18]. However, the necessity of the experiment was that it should carry out the spectrum analysis by the OES (optical emission spectroscopy) software and lead to the appropriate plasma treatment condition [14,19]. The argon gas flow rate was fixed as 4 L/min. The oxygen gas flow rate (ml/min), the input power (W), and the treatment times were varied in the different conditions, as shown in Table 2.
Figure 1. The schematic of the atmospheric pressure plasma jet.
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J. Jaifu et al. / Materials Today: Proceedings 17 (2019) 2088–2096 Table 2. Atmospheric Pressure Plasma Jet Treatment Condition APPJ treatment (Fixed argon flow rate 4 L/m) Input power (W)
Oxygen flow rate (ml/m)
Treatment time (s)
10
10
30
10
10
60
10
10
90
10
30
30
10
30
60
10
30
90
15
10
30
15
10
60
15
10
90
15
30
30
15
30
60
15
30
90
3. Statistical All experiments were repeated 6 times, and the data presented are expressed as mean ± SD. The statistical analysis was employed using ANOVA, and the multiple comparison test was conducted using the MINITAB software program. The difference was considered statistically significant when the p–value was less than 0.05 4. Results and Discussion 4.1. Fabricated naturally derived hemostatic agent All the material ratios in the study can be fabricated to form a naturally derived hemostatic agent. The structure and the appearance of each ratio is presented in Figure 2. However, the appearances of the–pure Gel and the Gel–RS specimens were such that they looked easily deformed and fragile. A previous study reported that low concentrations of the Gel solution and glutaraldehyde (GA) were found to increase the property of fragility [5]. Thus, the concentration of 2.5% (w/v) Gel solution and 0.025% of GA were not appropriate to fabricate a hemostatic agent. At the meantime, RS was also observed to have poor mechanical properties and be not uniform [7,9].
Figure 2. The naturally derived hemostatic agent in each of the ratios between CS:Gel:RS.
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4.2. Biodegradation of naturally derived hemostatic agent After the naturally derived hemostatic agent was fabricated, the biodegradation test was performed. The degradation results after 7 days in the conditions mimicking human body are shown in Figure 3a. Most of the samples degraded by more than 50%, except the sample with the 1/3:1/3:1/3 ratio. The sample with the ratio of only Gel was found to have completely degraded before 7 days, which is a finding that is in accordance with the reports of the previous studies that presented the fast biodegradability of the Gel hemostatic agent [3,8]. Biodegradability is one of the important properties in tissue engineering. The trend of biodegradability is one of the properties exhibited by newly developed materials. A previous study reported that proteases and lysozymes can also break the glycosidic bonds of the polysaccharide of CS, and could degrade the particle of CS to oligosaccharides, following which they combine with the metabolic pathway and are excreted [1,3,20,21]. The results also confirm the degradation ability of the fabricated naturally derived hemostatic agent which is sure to degrade in the surgical area after use. 4.3. Maximum volume of blood absorption Fast absorption is one of the factors recommended factors for hemostatic agents [7]. In fact, the effectiveness of the hemostatic agent is defined by its ability to absorb large volumes of blood. For that reason, this study identified the best ratio of naturally derived hemostatic agent of the CS, Gel, and RS solution based on a test of the maximum volume of blood absorption. As mentioned previously, the ratio which provided the highest blood absorption volume was used in the APPJ experiment. The results are shown in Figure 3b. The largest volume of blood was absorbed by the CS hemostatic agent, which absorbed a volume of 1.2±0.1 ml. In contrast, the hemostatic agent which contained Gel could not completely absorb the blood within 10 min. Thus, ratios which could not absorb blood within 10 min were classified as non–absorbent ratios. The low concentration of the Gel solution and its property of being fragile were the causes of it having the lowest blood absorbability [3,6]. The CS hemostatic agent exhibited higher absorbance than all of the ratios because it has the specific advantage of having an excellent swelling machanism [22]. CS is composed of polar groups, which are an interaction effect between amine (NH2) and hydroxyl (OH). The hydrophilic property is mainly because of the amine group, the hydrogen, and the covalent bond interaction [10,22,23,24]. The swelling property of CS abled it to effectuate high absorption of the serum proteins and fluids in the RBCs [11]. All of the ratios indicated significant difference (P<0.05). Therefore, in this experiment, the CS hemostatic agent was selected as the hemostatic agent of choice to investigate the effective plasma treatment condition. As mentioned previously, the ratio that provided the highest blood absorption volume was used in the APPJ experiment. The results are shown in Figure 3b. The highest blood absorption volume was presented by the CS hemostatic agent which absorbed a volume of blood of 1.2±0.1 ml. Interestingly, the hemostatic agent which contained Gel could not completely absorb the blood within 10 min. Fast absorption is one of the recommendations with regard to factors essential for a good hemostatic agent [7]. Thus, ratios which could not absorb the blood within 10 min were classified as non–absorbent ratios. Based on the results of this experiment, the hemostatic agent selected was the CS hemostatic agent.
Figure 3. (a) Biodegradation of the naturally derived hemostatic agents, and (b) the maximum volume absorption of human whole blood of the naturally derived hemostatic agents.
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4.4. Blood absorption rate CS exhibited hydrogen bonding, and induced high water uptake, because of the internal effect between the amine (NH2) and the hydroxyl (OH) groups of CS, while they combined with the covalent bond of the external interaction [20,22]. At the same time, several studies have reported that since the amine group of CS is positively charged, it could have interacted with the negative charge of the RBC membrane due to by the electrostatic force, leading to rapid blood absorbance [5,6,10]. The amine (NH2) and the hydroxyl (OH) groups are the components of CS which that exhibit hydrogen bonding and induce high water uptake [22,25]. In this study, the CS hemostatic agent was used to investigate the effective plasma treatment condition, APPJ in various conditions, as presented in Table 2. The comparison of each APPJ condition is shown in Figure 4a: the highest blood absorption rate of 4.60 ml/m, was found in the APPJ condition of input power 10 W, Ar flow rate 4 L/m of the mixture with the O2 gas 10 ml/m, and treatment time in 30 s. At the meantime, all of the plasma treatment conditions with treatment times of 30 s and 60 s presented higher volumes of blood absorbed than that in the non–treated condition, whereas the high input power of 15 W and the prolonged treatment time of 90 s were found to have damaged the surface of the samples, which decreased the blood absorption to less than that of the non–treated condition, while the factor response of the O2 flow rate and the treatment time did not indicate significant difference (P>0.05) and it was not found to be able to affect the blood absorption rate properties. The APPJ experiment in this work excited the ions and discharged the argon/oxygen gas mixture, which reactivated the OH radical (308 nm) and the atomic oxygen (777 nm): this increased the surface energy and decreased the surface tension, and they exhibited the wettability of the CS surface. Thus, blood absorption abilities might be increased due to this mechanism. 4.5. Hemoglobin leak The blood clotting ability could be observed from the hemoglobin absorbance (540 nm) which was the results of leakage from the sample. A previous study could utilize the observation of hemoglobin leak by dropping 0.2 ml of blood on the sample before soaking it in water [6]. But in this study, the 0.2 ml of blood was completely entrapped by the CS hemostatic agent before 10 s (Figure 5.). Thus, the hemoglobin leak could not be observed. The blood volume was increased from 0.2 ml to 1.0 ml to increase the chances of hemoglobin leak. The CS hemostatic agent itself could absorb high amounts of blood and accelerated the blood clotting time. The comparison between with and without APPJ treatment and the negative control demonstrated that the observations were totally different in terms of the hemoglobin leak value. The absorbance values of the CS hemostatic agent with and without plasma treatment indicated significant difference (P<0.05) in that the values of leakage of hemoglobin were lower than the those without plasma treatment and the negative control, and it could induce early blood clot in 30 s. The CS hemostatic agent has the specific property of having an excellent swelling mechanism because it has the effect of internal interaction between the amine (NH2) and the hydroxyl (OH) groups, so the hydrophilic property was mainly effected by the amine group, the hydrogen, and the covalent bonding interaction. At the same time, the hydroxyl (OH) groups of CS could have reacted with the water molecules at the external site [1,22,23,24]. The swelling property of CS could enable the immersion and the high absorption of the serum proteins or fluids in the blood [11]. At the same time, the hemostatic agent of CS can relate to the positive charge of the amino backbone (NH2); therefore, it interacts with the negative charge of the RBC membrane by electrostatic mechanism [5,10]. The OH bonding increased the absorbability of the water molecule, the ionic complex, and the serum protein of the blood on the CS surface, while the RBC particle could slowly pass into the small pores of CS and accelerate blood clotting [10,11,20,26]. Additionally, the plasma treatment enhanced the high amount of blood absorption and accelerated the clotting time of the blood onto the CS surface. A previous study reported that Ar plasma treatment was effective with the amine groups of CS membranes as it increased the physical property through etching of surface roughness. At the same time, Ar gas plasma has been found to discharge free radicals on the sample surface, following which the free radicals would react with the O2 functional group from the atmosphere: these chemical properties of Ar plasma allow for ionic permeability and blood clotting properties [11,15,27,28]. Therefore, the APPJ treatment is effective enough to improve blood absorption abilities and accerelate the rapid blood clotting of the CS hemostatic agent.
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Figure 4. (a) Absorption rate of 0.2 ml of human whole blood of naturally derived hemostatic agents with plasma jet treatment condition, and (b) comparison of hemoglobin absorbance of 1.0 ml of human whole blood of naturally derived hemostatic agents between with and without plasma treatment.
Figure 5. Hemoglobin leak testing of the hemostatic agent from the control (no speciment) to the 3 min period (30 s interval).
5. Conclusion The CS hemostatic agent exhibited high swelling ability between the internal reaction of an amine (NH2) and hydroxyl (OH) groups with covalent bonding of the water molecule at the external site. Thus, the swelling property of CS enabled it to absorb the high volume of the serum proteins and fluids in the blood. At the same time, the hemostatic agent of CS can relate to the positive charge of the amino backbone, and it then interacts with the negative charge of the RBC membrane through electrostatic force. Furthermore, CS has good biodegradability, so the proteases and the lysozyme could also break the glycosidic bonds of the polysaccharide of CS, and could degrade the particles of CS to oligosaccharides; thereafter, they combine with the metabolic pathway, and get excreted, thus promoting non-toxicity. Therefore, CS is appropriated to fabricate the naturally derived hemostatic agent. This study achieved the improvement of blood absorption properties with APPJ. The plasma treatment is an effective technique to enhance the blood absorbance ability of the CS hemostatic agent. Non-thermal plasma of the APPJ experiment is an alternative to surface modification. This study achieved the hemostatic capacity performance with an input power of 10 W, Ar flow rate of 4 L/m mixture with O2 gas at 10 ml/m, and treatment time of 30 s. The Ar/O2 gas excited the ions and discharged the OH radicals (308 nm) and atomic oxygen (777 nm), which increased the hydrophilic property. This plasma condition increased the blood absorption rate to 4.60 ml/m. In addition, acceleration of early blood clotting to within 30 s was also successful. Thus, plasma treatment has its advantages, and it could contribute to improving the blood absorption ability of the CS hemostatic agent in surgical applications.
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