1
Development and Assessment of Hemostasis Chitosan Dressings 1
Pei-Leun Kang1, Shwu Jen Chang2, Ioannis Manousakas2, Chen Wei Lee2, Chun-Hsu Yao3,*, 2
Feng-Huei Lin1,*, Shyh Ming Kuo2,* 3
4
1
Institute of Biomedical Engineering, National Taiwan University, Taipei, Taiwan 5
2
Department of Biomedical Engineering, I-SHOU University, Kaohsiung County, Taiwan 6
3
Department of Biomedical Imaging and Radiological Science, China Medical University, 7
Taichung, Taiwan 8
ABSTRACT 9
The aim of this study was to prepare and evaluate chitosan dressings treated with sodium 10
hydroxide (NaOH) and/or sodium tripolyphosphate (Na5P3O10) for haemostatic use. The pure 11
sodium hydroxide-gelated chitosan dressings (CS-B) presented water content (about 95%) 12
and porosity (about 85%) similar to those of commercially available chitosan-based products. 13
The CS-B dressing also exhibited homogeneously sized and penetrating pores throughout, 14
whereas the commercially available Clo-Sur PAD showed porous lamellar structures inside 15
and Instant Clot Pad exhibited heterogeneously distributed pores. Additionally, the CS-B 16
dressing was flexible and resilient, free of odour and able to recover completely after 17
compression in a hydrated state. Finally, the CS-B sponge absorbed blood quickly, 18
accelerating blood clotting, enhancing red blood cell adhesion and maintaining its original 19
shape after haemostatic testing. 20
Keywords: chitosan, dressing, haemostasis, gelation 21
22
*Corresponding author. Tel.: +886 7 6577711x6715; fax: +886 7 6577056. 23
E-mail: smkuo@isu.edu.tw (S.M. Kuo), double@ha.mc.ntu.edu.tw (F.H. Lin) 24
Equal co-corresponding author: Chun-Hsu Yao 25
26 27
2
1. Introduction 28
Chitosan is a biocompatible, antimicrobial material derived from the alkaline 29
N-deacetylation of chitin, a natural biopolymer originating from crustacean shells. This partial 30
deacetylation gives rise to chitosan, a linear polysaccharide with interspersed D-glucosamine 31
and acetyl-D-glucosamine units. For example, chitosan has been found to promote tissue 32
growth and to accelerate wound healing (Brown, Daya & Worley, 2009; Peter et al., 2010). 33
Moreover, its efficient gel-forming properties and ability to be shaped or incorporated into 34
hydrogels, microspheres and spongy dressings expand its potential applications in 35
biomedicine (Dai et al., 2009; Kranokpiraksa et al., 2009; Muzzarelli, 2009; Muzzarelli, 36
2010). 37
There are two commercially available haemostatic dressings in Taiwan: the Clo-Sur PAD 38
(Scion Cardio-Vascular, Inc., Florida, U.S.A.) and the Instant Clot Pad (Cosmo Medical Inc., 39
Taiwan). Both are composed of chitosan and are often used to stop trauma-related arterial 40
bleeding, as well as routinely applied post-angioplasty and after wound debridement. 41
Clinically, when the chitosan dressing makes contact with a wound, it adheres to and covers 42
the site and attracts red blood cells, forming a seal that prevents further haemorrhage. The 43
haemostatic mechanism of chitosan involves the agglutination of red blood cells, possibly due 44
to its intrinsic polycationic properties and nonspecific binding to cell membranes (Fischer, 45
Bode, Demcheva, & Vournakis, 2007; Okamoto et al., 2003; Rao & Sharma, 1997). Some 46
reports indicate that chitosan also accelerates coagulation in vivo by influencing the activation 47
of platelets (Baldrick, 2010; Chou, Fu, Wu, & Yeh, 2003; Muzzarelli et al., 2007). 48
These two chitosan products have several shortcomings that may limit their clinical 49
applications, such as having a trace acidulous odour due to use of acetic acid as a processing 50
solvent, which is potentially allergenic; being too fragile to retain proper shape under 51
compression; and needing a long period of compression to stop bleeding after angioplasty. 52
We previously reported on chitosan membrane and sponge-like devices that were prepared 53
3
by thermally induced phase separation, followed by non-toxic sodium hydroxide 54
(NaOH)-based gelation. Finally, sodium tripolyphosphate (Na5P3O10) was employed as a 55
crosslinking agent to increase mechanical strength. Here, a series of chitosan sponges was 56
fabricated by adjusting the molecular weight of chitosan and the crosslinking conditions. The 57
dressings’ basic properties and haemostatic efficacy were then evaluated against the two 58
commercially available products, which served as controls. 59
60
2. Materials and methods 61
2.1. Materials 62
Chitosan of two different molecular weights, 300 kDa and 70 kDa, were purchased from 63
TCI (Tokyo, Japan). The degree of deacetylation of the chitosan was approximately 83%. 64
Sodium tripolyphosphate (Na5P3O10, 5%) and sodium hydroxide (NaOH, 1 N) were 65
purchased from SHOWA (Tokyo, Japan) and acetic acid was purchased from 66
Merck-Schuchardt (Hohenbrunn, Germany). All chemicals used in this study were of reagent 67
grade. 68
69
2.2. Fabrication of haemostatic chitosan dressing 70
Chitosan was dissolved in 0.1 N aqueous acetic acid to form a 2% (w/v) chitosan solution. 71
Part of this solution was neutralised to pH 6.0 by adding aqueous 1 N NaOH. Ten millilitres 72
of the acidic and neutralised chitosan solutions were put into 6 cm dish and frozen at -20C 73
for 8 h, followed by lyophilisation for at least 24 h. The resultant porous chitosan dressings 74
were further treated with a mixture solution of 1 N aqueous NaOH and 5% Na5P3O10 at a 75
volume ratio of 3:17 for 3 h to induce gelation and crosslinking (Table 1) (Lin et al., 2006, 76
Chang, Niu, Kuo, & Chen, 2007). The treated dressings were then washed with distilled water 77
three times, frozen at -20C for 4 h and lyophilised again. 78
4
2.3. SEM observation of morphology and blood coagulation 80
Scanning electron microscopy (SEM) was employed to examine the morphology of the 81
chitosan dressings, with emphasis on the porous characteristics resulting from different 82
NaOH/Na5P3O10 ratios. Prior to SEM, the samples were lyophilised and sputter-coated with 83
gold, followed by observation using a Hitachi S-2700 (Tokyo, Japan) instrument. 84
A sample of 0.5 mL of human whole blood or platelet-insufficient blood obtained from the 85
TBSF was added to each chitosan dressing. After incubation at 37C for a predetermined 86
amount of time, the dressings were fixed, dried and sputter-coated with gold for SEM studies. 87
Blood coagulation on a cover glass was used as a control. 88
89
2.4. Water content and equilibrium swelling testing 90
The water content of the chitosan dressings was determined by swelling the dressing in 91
phosphate buffered saline (PBS) at pH 7.4 at room temperature for 2 h. The wet weight (Wwet) 92
of the swollen dressing was measured immediately after gently blotting with filter paper to 93
remove surface liquid, followed by lyophilisation and reweighing (Wdry). The water content of 94
the dressing was calculated using the formula 95
WC (Wwet Wdry) / Wwet × 100% . 96
The equilibrium swelling ratio (ESR) of the dressing was also calculated, based on the 97
following equation: 98
ESR (Vw Vd) / VW × 100% . 99
Here, Vd is the exterior volume of the chitosan dressing (1 cm 1 cm), measured using a 100
vernier caliper, and Vw is the exterior volume of the dressing after swelling in distilled water 101 for 1 min. 102 103 2.5. Determination of porosity 104
The porosity of the prepared chitosan dressings was determined using Archimedes’ 105
5
principle. The exterior volume (Vd) of each chitosan dressing (1 cm 1 cm) was measured 106
using a vernier caliper. The sample was then immersed in a pycnometer containing 99% 107
ethanol solution. The actual volume of (Va) of the sample was calculated using the formula 108
Va [(Ww － Wo) － (Wt － Wp)] / (0.789 g/cm3 ) , 109
where Ww is the weight of ethanol and the pycnometer; Wo is the dry weight of the 110
pycnometer; Wt is the combined weight of the ethanol, the pycnometer and the dressing 111
sample; Wp is the combined weight of the dry pycnometer and dry dressing sample; and 0.789 112
g/cm3 is the density of 99% ethanol solution. The porosity of the chitosan dressing was then 113
determined using the following formula: 114
Porosity (%) (Vd － Va) / Vd × 100%. 115
Porosity values were expressed as means standard deviations (n 6). 116
117
2.6. Absorption and blood clotting testing 118
The absorption rate of the chitosan dressings was determined using distilled water and 119
human whole blood. The latter was obtained from the Taiwan Blood Services Foundation 120
(TBSF; Taipei, Taiwan). Dressings were cut into 1 cm 1 cm squares and placed into glass 121
bottles. Then, 0.4 mL of distilled water or human whole blood was dispensed onto the 122
dressing. The absorption rate was defined as the time required for the dispensed fluid to be 123
completely absorbed by the dressing. 124
The blood clotting test was modified from Ong et al. (2004). Dressings were cut into 1 cm 125
1 cm squares and placed into glass bottles. Next, 0.25 mL of human whole blood 126
(containing the anticoagulant citrate dextrose at a 1:6 ratio) was slowly dispensed onto the 127
surface of the dressings. The bottles containing the samples were then incubated at 37C. 128
After a predetermined amount of time (30, 60, 90, 120 or 180 sec), 20 mL of distilled water 129
were carefully added by dripping water down the inside wall of the bottles, preventing 130
disruption of the clotted blood. Red blood cells that were not entrapped in the clot were 131
6
haemolysed with distilled water and the absorbance of the resultant haemoglobin solution was 132
measured at 540 nm (UV-VIS spectrophotometer Agilent 8453, Santa Clara, California, USA). 133
The absorbance of 0.25 mL of whole blood in 20 mL of distilled water was used as a 134 reference value. 135 136 2.7. Statistical analysis 137
All quantitative data were expressed as means standard deviations. Differences between 138
means were analysed for statistical significance using the Student’s t test. P-values less than 139
0.05 were considered statistically significant. 140
141
3. Results and discussion 142
Rapid blood absorption and effective coagulation by the prepared chitosan dressings were 143
our main concerns. We first tested the dressings’ absorption of distilled water prior to further 144
examination (Fig. 1). Among the chitosan sponges that we prepared (Table 1), the ones treated 145
with pure 4% NaOH solution absorbed 0.4 mL of distilled water in less than one second 146
(CS-A, CS-B, CS-G and CS-H dressings). In contrast, dressings treated with pure 5% 147
Na5P3O10 solution or with a mixture of NaOH and Na5P3O10 at a volume ratio of 3/17 148
exhibited a slower absorption rate. Specifically, the sponges treated with pure Na5P3O10 149
needed over 10 minutes to absorb 0.4 mL of distilled water (CS-C, CS-D, CH-I and CS-J 150
dressings). 151
It was observed that chitosan dressing treated with 4% NaOH was flexible and resilient, as 152
assessed macroscopically. Measurement of resistance to compression (hardness) indicated that 153
the chitosan dressings treated with 5% Na5P3O10 were slightly tougher. In general, these 154
dressings had a high degree of recovery to original shape upon immersion in distilled water or 155
whole blood than other sponges. 156
7
All chitosan dressings prepared in this study exhibited a high water content of about 89% to 157
95%, whereas the commercially available chitosan-based products had a slightly higher water 158
content of 96% (Table 1). The porosity of the prepared dressings was also determined, falling 159
in the range of 44.4% to 85.6%. Variation among the porosities of the chitosan dressings was 160
observed, related to whether the chitosan solutions were titrated to pH 7.0 (Table 1). The two 161
titrated chitosan samples, with molecular weights of 300 kDa and 70 kDa, respectively, 162
exhibited lower porosity than the non-treated ones due to the pre-gelation reaction of the 163
NaOH solution with the soluble chitosan molecules. SEM observation also demonstrated 164
fewer pores and more lamellar structures in the pH-titrated CS-A and CS-G samples (data not 165
shown). The CS-A as well as CS-B dressings exhibited lower swelling ratios than the 166
commercially available chitosan-based products, Clo-Sur dressings and Inst-Clot dressings. 167
Based on our preliminary results regarding the water content, absorption rate, porosity, 168
macroscopic characteristics and ease of preparation of various chitosan dressings, we chose 169
those treated with pure NaOH for further examination and used the Clo-Sur and Inst-Clot 170
dressings for comparison. 171
Photographs of the CS-B dressing and commercially available chitosan-based products are 172
shown in Fig. 2. The CS-B sponge had a white appearance, while the Clo-Sur and Inst-Clot 173
dressings were more yellow and brown in colour and had an acidulous odour. Scanning 174
electron micrographs of these dressings are presented in Fig. 3, indicating a porous structure. 175
The CS-B dressing had a more homogenous pore-size distribution and displayed penetrating 176
pores both on the surface and in the cross-sectional view. On the other hand, the Clo-Sur 177
dressing exhibited lamellar sheet structures on the inside, while a heterogeneous pore 178
distribution was observed on the surface and inside of the Inst-Clot dressing. 179
180
3.1. Blood absorption 181
The NaOH-treated chitosan dressings presented faster absorption rates, absorbing 0.4 mL 182
8
of human whole blood in less than five seconds. Meanwhile, the Clo-Sur dressing required 183
more time (about 45 seconds) to completely absorb the same amount of blood (Fig. 4). The 184
distinctly rapid absorption rate of the CS-B dressing may be attributed to its homogeneous 185
and penetrating porous structure, while the Clo-Sur dressing contained lamellar sheets that 186
may have inhibited percolation of the blood. 187
To assess blood coagulation, the dressings were placed in 0.9 % saline solution. As seen in 188
Fig. 5, the Clo-Sur dressing swelled significantly in saline and blood leakage from the 189
dressing was observed. Similar phenomena were also observed for the Inst-Clot dressing, 190
although less blood leached out of the sponge. In contrast, blood was well entrapped inside 191
the CS-B dressing, despite slight swelling. 192
193
3.2. In vitro whole blood clotting test 194
To evaluate whether the CS-B dressing could increase the rate of blood clotting, human 195
whole blood containing a normal (about 250000 platelets/ml) or decreased number (60000 196
platelets/ml) of platelets was dripped on dressings for 30 to 180 seconds before haemolysis of 197
the RBCs that were not entrapped in the resultant clot. The absorbance of the resultant 198
haemoglobin-containing solution was measured, with a high absorbance value indicating a 199
slower clotting rate. As shown in Fig. 6, the CS-B dressing and Inst-Clot yielded significantly 200
lower absorbance values than the Clo-Sur dressing after 30 seconds of incubation with human 201
whole blood (p<0.05). The absorbance value for the Clo-Sur dressing was similar to that of 202
the other two dressings after 120 seconds. The platelet-insufficient blood could also form clots 203
on the CS-B dressing after 30 seconds. The Inst-Clot dressing exhibited a slightly higher 204
absorbance value, and thus slower clotting than the CS-B dressing, while the Clo-Sur sponge 205
could not form clots completely even after 180 seconds. 206
SEM evaluation of blood clot formation on the three chitosan dressings revealed that the 207
red blood cells formed larger aggregates (Fig. 7a and 7b). More specifically, the red blood 208
9
cells seemed to have coalesced into an erythrocyte clot or plug on the CS-B and Inst-Clot 209
dressings. Conversely, fewer aggregates were observed on the Clo-Sur dressing while more 210
extensive fibrin was noted on the surface, perhaps caused by attached platelets (Fig. 7c). 211
We have checked the pH changes of the solution resulting from immersion of the CS-B, 212
Clo-Sur and Inst-Clot dressings in distilled water for 120 minutes, using latex and 213
polypropylene film as control groups. The pH values decreased significantly from pH 6.8 to 214
pH 4.7 and 5.0, respectively, for the Clo-Sur and Inst-Clot dressings after 120-minute 215
immersion. On the other hand, the CS-B dressing, latex and polypropylene film did not cause 216
any obvious pH changes in the surrounding solution during testing. The decreased pH noted 217
for some of the dressings indicated their inherent acidity, which was achieved or retained 218 during preparation. 219 220 4. Conclusion 221
In the present study, we established a modified procedure for preparing chitosan dressings. 222
After freezing and lyophilisation to produce a porous chitosan matrix, the matrix was treated 223
with NaOH or a mixture of NaOH and Na5P3O10. The matrices were then washed and 224
lyophilised again to produce dressings. By modifying the gelation technique, the dressings’ 225
flexibility, texture, appearance, odour and blood absorption and coagulation could be 226
improved, especially in comparison with those of commercially available chitosan-based 227
products. Taken together, the results of the physical examination of the dressings and the 228
haemostatic assays demonstrated that the NaOH-treated chitosan dressing was optimal for 229
enhancing haemostasis. This preparation led to faster and more clotting and retained its 230
original shape and flexibility after contact with human blood. 231
232
References 233
10
Baldrick, P. (2010). The safety of chitosan as a pharmaceutical excipient. Regulatory 234
Toxicology and Pharmacology, 56(3), 290-299.
235
Brown, M.A., Daya, M.R., & Worley, J.A. (2009). Experience with chitosan dressings in a 236
civilian EMS system, Journal of Emergency Medicine, 37(1), 1-7. 237
Chang, S. J., Niu, G. C. C., Kuo, S. M., & Chen, S. F. (2007). Preparation and preliminary 238
characterization of concentric of multi-walled chitosan microspheres. Journal of 239
Biomedical Materials Research Part A, 81A(3), 554-566.
240
Chou, T. C., Fu, E., Wu, C. J., & Yeh, J. H. (2003). Chitosan enhances platelet adhesion and 241
aggregation. Biochemical and Biophysical Research Communications, 302(3), 480-483. 242
Dai, T.H., Tegos, G.P., Burkatovskaya, M., Castano, A.P., Hamblin, M.R. (2009). Chitosan 243
acetate bandage as a topical antimicrobial dressing for infected burns. Antimicrobial 244
Agents and Chemotherapy, 53(2), 393-400.
245
Fischer, T. H., Bode, A. P., Demcheva, M., & Vournakis, J. N. (2007). Hemostatic properties 246
of glucosamine-based materials. Journal of Biomedical Materials Research Part A, 80(1), 247
167-174. 248
Kranokpiraksa, P., Pavcnik, D., Kakizawa, H., Uchida, B.T., Jeromel, M., Keller, F.S., & 249
Rosch, J. (2010). Hemostatic efficacy of chitosan-based bandage for closure of 250
percutaneous arterial access sites: An experimental study in heparinized sheep model. 251
Radiology and Oncology, 44(2), 86-91.
11
Lin, L. C., Chang, S. J., Chen, S. F., Chou, Y. J., & Kuo, S. M. (2006). Effects of pH of 253
Na5P3O10/NaOH reaction solution on the properties of chitosan microspheres. Biomedical 254
Engineering: Applications, Basic & Communications, 18(4),167-177.
255
Muzzarelli, R. A. A., Morganti, P., Morganti, G., Palombo, P., Palombo, M., Biagini, G., 256
Belmonte, M. M., Giantomassi, F., Orlandi, F., & Muzzarelli, C. (2007). Chitin 257
nanofibrils/chitosan glycolate composites as wound medicaments. Carbohydrate 258
Polymers, 70(3), 274-284.
259
Muzzarelli, R. A. A. (2009). Genipin-crosslinked chitosan hydrogels as biomedical and 260
pharmaceutical aids. Carbohydrate Polymers, 77(1), 1-9. 261
Muzzarelli, R. A. A. (2010). Chitins and chitosans as immunoadjuvants and non-allergenic 262
drug carriers. Marine Drugs, 8, 292-312. 263
Okamoto, Y., Yano, R., Miyatake, K., Tomohiro, I., Shigemasa, Y., & Minami, S. (2003). 264
Effects of chitin and chitosan on blood coagulation, Carbohydrate Polymers, 53(3), 265
337-342. 266
Ong, S. Y., Wu, J., Moochhala, S. M., Tan, M. H., & Lu, J. (2008). Development of a 267
chitosan-based wound dressing with improved hemostatic and antimicrobial properties. 268
Biomaterials, 29(32), 4323-4332.
269
Peter, M., Ganesh, N., Selvamurugan, N., Nair, S. V., Furuike, T., Tamura, H., & Jayakumar, 270
R. (2010). Preparation and characterization of chitosan–gelatin/ nanohydroxyapatite 271
12
composite scaffolds for tissue engineering applications. Carbohydrate Polymers, 80(3), 272
687-694. 273
Rao, S. B. & Sharma, C. P. (1997). Use of chitosan as a biomaterial: Studies on its safety and 274
hemostatic potential. Journal of Biomedical Materials Research, 34(1), 21-28. 275
