Polymer 123 (2017) 194e202

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Polymer
journal homepage: www.elsevier.com/locate/polymer

Biodegradable polycaprolactone nanofibres with b-chitosan and

calcium carbonate produce a hemostatic effect
Jun-Yong Park a, Kyu-Hong Kyung a, Kosuke Tsukada a, Sae-Hoon Kim b,
Seimei Shiratori a, *
a
School of Integrated Design Engineering, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama, Kanagawa 223-8522,
Japan
b
Department of Advanced Ceramic Materials Engineering, Gangneung-Wonju National University, 123 Jibyeon-dong, Gangneung, Gangwon-do, South
Korea

a r t i c l e i n f o a b s t r a c t

Article history: We newly developed high-performance blood coagulation nanofibres using Polycaprolactone (PCL)
Received 2 May 2017 including Calcium carbonate (CaCO3) and b-chitosan. The most important feature for a wound dressing is
Received in revised form that it is harmless to the human body. Here, we fabricated the nanofibre using all human-safe materials.
27 June 2017
We used a PCL nanofibre mat as a substrate and then synthesized artificial CaCO3 from sodium carbonate
Accepted 4 July 2017
and calcium chloride. The CaCO3 was then added to the fibre solution to create PCL/CaCO3 nanofibres. We
Available online 4 July 2017
coated PCL and PCL/CaCO3 nanofibres with b-chitosan as a hemostatic material via a spray method. For
the more uniform coating, we used ultrasonic spray coating method and then compared the blood
Keywords:
Electrospinning
coagulation abilities of the PCL and PCL/CaCO3 nanofibres. We found that the PCL/CaCO3 nanofibres
Polycaprolactone sprayed with b-chitosan had a greater effect on blood coagulation than the PCL nanofibres. In the result
Nanofibre of animal experiment, b-chitosan have a key role in changing of surface wettability from hydrophobic to
Calcium carbonate hydrophilic. Moreover, this is contributed to enhance blood coagulability. PCL/CaCO3 nanofibres sprayed
Hemostatic effect with b-chitosan therefore offer promise in medical applications.
b-chitosan © 2017 Elsevier Ltd. All rights reserved.

1. Introduction engineering [8,9], enzyme immobilization, and wound dressing

In the bio-application, porous structures and materials have
been attracted by their unique property such as high specific sur-
face area, highly porous structure, controllable morphology [1].
Although there are variety methods for fabrication porous struc-
ture, feasible methods are nanoscale/microscale porous structures
and material, nanofibre. In particular, nanoscale/microscale porous
structures and materials have highly advantage in the drug de-
livery, responsible to high efficiency and selectivity [2,3]. However,
these methods cannot satisfy from human-safety because of their
synthesis process and stability in vivo/vitro and limits their clinical
applications. Nanofibre provides a simple method for creating
porous structure from a rich variety of materials, including poly-
mers, composites, self-standing, which can be used for a variety of
bio-application such as the native extracellular matrix (ECM),
enhancing cell migration and proliferation [4e6], sensing [7], tissue
* Corresponding author.
E-mail address: shiratori@appi.keio.ac.jp (S. Shiratori).
[10,11].
Hemostasis is necessary for treating military traumas or con-
ducting surgical operations, else uncontrolled hemorrhages can
lead to death. Wound dressing therefore plays an important role in
hemostasis in several situations [12]. Substances allowing the for-
mation and stabilization of blood clots are essential in effective
wound dressing. Fibrin sealant, collagen and fibrin sponges, and
thrombin have all been studied for their effectiveness in wound
dressing [13,14]. Biomaterials can either be naturally derived (e.g., a
blood vessel or protein such as collagen) or synthetic (e.g., a poly-
mer, metal, or ceramic) materials that comprise all or part of a living
structure or biomedical device. Biopolymers, such as biodegradable
and biocompatible polymers, are non-toxic to the human body and
environmentally friendly. For these reasons, biopolymers are useful
for wound dressing and drug delivery [15]. Biopolymers such as
polycaprolactone (PCL), polylactic acid (PLA), polyglycolic acid
(PGA), poly(L-lactide-co-glycolide) (PLGA), and sodium alginate are
suitable for nanofibre wound dressing because they exhibit histo-
compatibility, biodegradability, and lack of antigenicity in humans.
In particular, many researchers have demonstrated the

http://dx.doi.org/10.1016/j.polymer.2017.07.013
0032-3861/© 2017 Elsevier Ltd. All rights reserved.
 J.-Y. Park et al. / Polymer 123 (2017) 194e202
effectiveness of PCL in wound management [16], initiating a
gradual increase in the use of PCL as a biomaterial in tissue engi-
neering [17,18]. Compared to other biodegradable polymers such as
PLA and PGA, PCL exhibits some unique physical properties that
make it ideal for medical applications, such as low melting tem-
perature, high blend compatibility, low cost, and Food and Drug
Administration (FDA) approval [19,20]. Bioactive materials are
categorized based on the nature of their activity. Bioactive materials
include proteoglycans, collagen, non-collagenous proteins, algi-
nates, or chitosan. b-chitosan is produced by deacetylation of b-
chitin and exhibits similar properties to b-chitin [21]. Owing to
their high biocompatibility, b-chitin and b-chitosan have been
employed in wound healing management and drug delivery sys-
tems [22]. Since acceleration of wound healing is associated with
collagenase activity, and because chitosan exhibits higher collage-
nase activity than chitin [23], b-chitosan demonstrates high wound
healing abilities [24e31]. PCL does not usually dissolve in water-
based solutions. In previous research, an organic solvent was
used to dissolve PCL [32e34], but this solution exhibited poor hy-
drophilicity. As wound dressings are in direct contact with the
wound and blood, they require a high contact area and low surface
wettability. To our knowledge, b-chitosan demonstrates a higher
hydrophilicity than PCL. b-chitosan is generally dispersed in
aqueous solutions with low pH derived from acetic acid [35,36].
The calcium ion, a divalent metal ion, is present at a high con-
centration in human bone and tissue. Calcium ions participate in
hemostasis and accelerate the formation of blood clots by aiding in
the conversion of prothrombin to thrombin, as well as catalysing
many other coagulation-related reactions [37]. CaCO3 has been
used in many fields, particularly biology, as a source of calcium ions
[38,39]. Furthermore, CaCO3 can promote tissue regeneration in
acidic condition [40] and degrade in human body [41].
In this study, we focused on the fabrication of wound dressing
with all human-safety materials. We used three components: PCL,
CaCO3, b-chitosan. First, PCL is biodegradable material, however, it
had limited characteristics for wound dressing because of its hy-
drophobicity. Second, CaCO3 is natural safety material that plays an
important role to accelerate blood coagulation property. Third, b-
chitosan is used for surface property modification in order to
permeate blood easily into nanofibre and to increase the contact
area on the fibre. In addition, b-chitosan has wound healing
property. Moreover, by using these three components, electrospun
nanofibers were fabricated in order to increase the surface area to
contact blood or animal wounded skin. Hear, we used a PCL
nanofibre mat as a substrate and added CaCO3 to the fibre solution.
We then coated the fibre surface with b-chitosan via an ultrasonic
spray method. Blood coagulation characteristics of PCL and PCL/
CaCO3 nanofibres were compared. We found that PCL/CaCO3 fibres
with b-chitosan exhibited a higher blood coagulation rate than
those of PCL nanofibres without CaCO3 or b-chitosan. PCL/CaCO3
nanofibres with b-chitosan are therefore expected to be useful for
diverse medical applications.
2. Experimental section
2.1. Materials
Polycaprolactone (Sigma-Aldrich, St. Louis, MO, USA;
MW ¼ 80 K), b-chitosan (Arabio Co., Ltd., Korea; average
MW ¼ 180 K), and acetic acid (Kanto Chemical Co., Inc., Malaysia;
purity ¼ min. 99.7%) were used without further purification. Highly
purified water was obtained by deionization and filtration
(resistivity > 18.2 MU$cm). Calcium chloride (CaCl2, Junsei Chem-
ical Co., Ltd., Japan; purity ¼ min. 99.5%), sodium carbonate
(Na2CO3, Junsei Chemical Co., Ltd., Japan; purity ¼ min. 99.5%),
195
were used without further purification.
2.2. Synthesis of calcium carbonate
We synthesized CaCO3 using the typical experimental process
[42]. Briefly, 100 mL of 0.2 M CaCl2 aqueous solution was poured
into an aqueous solution of 0.2 M Na2CO3, followed by vigorous
stirring for 5 min. The solution was then allowed to stand for 24 h at
room temperature. When the reaction was finished, the precipitate
was washed three times with distilled water and dried at 60  C
before collection of CaCO3.
2.3. Fabrication of electrospun nanofibres
For fabrication of PCL nanofibres, a PCL pellet was dissolved in a
mixture of 75 wt% chloroform and 25 wt% methanol [43]. We
prepared two types of PCL solutions: I) pure PCL solution, and II)
PCL/CaCO3 solution. For the PCL/CaCO3 solution, CaCO3 was added
to the PCL solution at a with aluminum foil. Electrospinning con-
ditions included a voltage of 20 kV, a tip-to-collector distance of
130 mm, and a hydrostatic pressure in the syringe of 1 mL/h. All
fibres were fabricated at a weight ratio of 1:3 PCL: CaCO3. Electro-
spinning solutions were degassed before being loaded into a 30 mL
syringe with an 18 G (1.2 mm) stainless needle (Terumo Co., Ltd.,
Japan). An electrode was clamped onto the needle and connected to
a power supply. The grounded counter electrode was connected to
a Cu collector covered room temperature and 33% humidity. Elec-
trospinning proceeded for 3 h; nanofibre mats were detached from
the aluminum foil when capable of self-standing. After electro-
spinning, the aluminum foil with the nanofibre mat was removed
from the collector. PCL and PCL/CaCO3 nanofibre morphologies
were measured by field emission scanning electron microscope
(FE-SEM, Hitachi, S-4700) and confirmed the EDAX (Ametek., Inc.
Octane pro). We measured the CaCO3 content in the nanofibre by
thermogravimetric analysis (TGA) and differential thermal analysis
(DTA) analyzer (Exstar 6000). We used 300 mL nitrogen (N2) gas
and an aluminum sample holder with an initial temperature of
25  C, a final temperature of 430  C, and a temperature gradient of
40  C/min. The specific surface area and pore size were measured
using the Brunauer-Emmett-Teller (BET, Micrometritcs ASAP 2010)
method after degassing under N2 gas at 40  C. For confirmation of
stability of fibre, we soaked fibre mat in phosphate-buffered saline
(PBS) aqueous solution (9.57 mM, pH 7.35e7.65) and mouse blood
from 0 to 24 h. After that, the fibre mat was taken from PBS solution
and mouse blood at every 6 h. And then, fibre mat was rinsed with
distilled water and dried at room temperature.
2.4. Fibre spraying with b-chitosan
When spray-coating nanofibres, a spray nozzle is used with an
air pump for spraying substances onto the substrate. However, a
typical spray nozzle emits a large amount of solution and large-
diameter droplet. An ultrasonic spray machine consists of a sy-
ringe pump, air pump, ultrasonic spray nozzle, and ultrasonic
generator. When the solution droplet is being emitted from the
nozzle tip, the ultrasonic spray nozzle causes the droplet of solution
to vibrate. The frequency of the ultrasonic spray nozzle can be
controlled by controlling the frequency of the ultrasonic generator.
The diameter of the solution droplet can thus be reduced to coat the
substrate uniformly. Moreover, the ultrasonic spray machine uses a
syringe pump, allowing for a small amount of solution to be
sprayed onto the substrate without completely wetting the sample.
For coating of nanofibres, we used an ultrasonic spray machine
manufactured by Ceratorq (Korea; schematic shown in Fig. 1). For
ultrasonic spraying, we prepared a b-chitosan aqueous solution and
196 J.-Y. Park et al. / Polymer 123 (2017) 194e202

Fig. 1. Schematic of the ultrasonic spray machine and one cycle of the spraying process. The ultrasonic spray machine was composed of an air pump, ultrasonic generator, and
syringe pump. The frequency of the ultrasonic pump is determined by the ultrasonic generator (120 kHz). The flow rate of the solution can be adjusted with the syringe pump. The
substrate is moved vertically and horizontally. All steps are computer controlled. (Syringe pump excepted).

adjusted it to pH 3.0 with acetic acid. The nanofibre mat was cut to
the size of a glass slide and attached to the glass slide substrate
(76  26 mm2). The glass slide substrate was then attached to the
target area, and the b-chitosan solution was sprayed onto the PCL
nanofibre mat using the following ultrasonic spray conditions:
frequency, 120 kHz; nozzle-to-substrate distance, 5 cm; syringe
feeding rate, 0.5 mL/min. The substrate was repeatedly moved
vertically and horizontally, ensuring uniform coverage. A single
cycle of the spraying process is shown in Fig. 1. We prepared PCL
nanofibres sprayed with 1.25 mL/2.5 mL of b-chitosan and PCL/
CaCO3 nanofibres sprayed with 1.25 mL/2.5 mL of b-chitosan,
resulting in a total of four samples. The entire nanofibre fabrication
method including ultrasonic spraying is shown in Fig. 2. For com-
parison, we also hand-sprayed another nanofibre sample with the
same amounts of b-chitosan used in 1.25 mL/2.5 mL of ultrasonic
spraying. Particle analysis was used by ImageJ (Version 1.50i;
National Institutes of Health, Bethesda, MD, USA) with SEM image.
Fourier transform infrared spectroscopy (FT-IR) analysis were ob-
tained in RockSolid™ interferometer (ALPHA-T, Bruker) with KBr
(potassium bromide) in range 500e4000 cm1 with resolution
2 cm1. 16 scans were performed for each measurement. Sample
and KBr were mixed and loaded into sample holder. A background
spectrum was measured for KBr.
2.5. Whole-blood coagulation assessment
Coagulation assessments of whole pig blood were conducted
according to published methods [44,45]. We first investigated the
effect of CaCO3 on blood clotting by dropping 3 mL blood, or 3 mL
blood with 0.01 g CaCO3, on glass slides. All blood coagulation
processes were then assessed with a digital microscope. For
assessment of blood coagulation by nanofibres, nanofibre mat

Fig. 2. Nanofibre fabrication and spraying method. First, we fabricated the nanofibre via electrospinning. A solution of b-chitosan was then sprayed on to the PCL nanofibre and PCL/
CaCO3 nanofibre, and nanofibre mats were removed from the aluminum foil when they were capable of self-standing.
 J.-Y. Park et al. / Polymer 123 (2017) 194e202 197

2.6. Assessment of coagulation in mouse blood

All animal experimental protocols were approved by the Animal

Care Committee of Keio University School of Medicine (Protocol No.
14089). Male (BALB/c) mice were anaesthetized with urethane
(800 mg/kg) and chloralose (80 mg/kg). Whole blood was with-
drawn from the inferior vena cava with a 1 mL syringe, and 20 mL
was used immediately to prevent blood coagulation.

3. Results and discussion

3.1. Effect of calcium carbonate on blood clotting

We investigated the effect of CaCO3 on blood clotting and

captured the process on video using a digital microscope (Fig. 4).
Blood with CaCO3 began coagulating after 5 min, while blood
without CaCO3 started coagulating after 7 min. At 7 min, the sample
with CaCO3 contained red blood cells, while the blood-only sample
did not. Instead, the sample without CaCO3 contained small red
dots. This indicates that CaCO3 does indeed have an effect on blood
Fig. 3. Coagulation assessment of whole blood. Nanofibre mat samples were attached
coagulation.
to glass slides for same condition of rinsing in water bath.

3.2. Nanofibre morphology and CaCO3 content

samples were cut to 1.5 cm2 and attached to glass slides (Fig. 3). For
We successfully fabricated PCL and PCL/CaCO3 nanofibres via
the same condition of rinsing. We dropped 0.1 mL blood onto each
electrospinning using synthesized CaCO3. PCL and PCL/CaCO3
sample before incubating at 37  C for 3 min. Samples were then
nanofibre morphologies were measured by FE-SEM (Fig. 5). The PCL
dried at room temperature for 10 min. To remove non-clotted
and PCL/CaCO3 nanofibre diameters were approximately 500
blood, glass slides containing nanofibre mats were placed in a
nme1 mm. The PCL nanofibre is an ultrafine fibre without beads. In
50 mL distilled water bath with magnetic stirring at 250 rpm for
contrast, the PCL/CaCO3 nanofibre includes CaCO3 beads. A higher
15 s. A sample of 1.5 mL of solution was taken from the 2 mL bottle
magnification image is shown in Fig. S1. The PCL/CaCO3 nanofibre
and was centrifuged at 1000 rpm for 1 min. The optical density of
had a predicted weight ratio of 1:3 PCL: CaCO3. We experimented
the centrifuged solution was measured by ultraviolet visible (UV-
various weight ratio of PCL: CaCO3 (From 1:1 to 1:3). Over the
vis) spectrophotometer (Shimadzu UV mini-1240) at 540 nm.
weight ratio of PCL: CaCO3 (1:3) was too hard to fabricate the fibre.

Fig. 4. Video image capture of blood coagulation process without (a) and (b) with CaCO3 on a glass slide. Red boxes indicate the beginning of the blood coagulation process.
198 J.-Y. Park et al. / Polymer 123 (2017) 194e202
Fig. 5. Field emission scanning electron microscope images of (a) the PCL nanofibre and (b) the PCL/CaCO3 nanofibre.
Because an amount of CaCO3 hindered to flow out polymer solution
on the syringe needle. As shown in Fig. S2, a clear distinction be-
tween contained CaCO3 in the nanofibre. At the increasing weight
ratio of CaCO3, existence of CaCO3 was increased. However, mor-
phologies of fibres were almost same in the weight ratio. From this
reason, the weight ratio of PCL: CaCO3 (1:3) is the appropriate
condition to fabricate nanofiber. Furthermore, the content of CaCO3
was higher than that of the other weight ratio. We verified the
CaCO3 content in the fibre using TG-DTA, and the results are shown
in Fig. 6. First, we analyzed the CaCO3, which had a melting point of
over 1000  C. The decrease in the TGA curve is attributed to the
decomposition of CaCO3 to calcium oxide (CaO) and removal of
water. The small endothermic peak at 430  C in the DTA curve
stems from the phase transformation of CaCO3 from veterite to
calcite. Before measurement, the CaCO3 sample weight was 8.4 mg,
and after analysis, it was 8.1 mg. Next, we analyzed the PCL nano-
fibre. PCL has a low melting point of approximately 60  C. The
endothermic peak at 60  C in the DTA curve reflects the phase
transition of PCL from solid to liquid. The large decrease in the TGA
curve could be attributed to the thermal decomposition of PCL.
Before measurement, the PCL sample weight was 6.5 mg, and after,
it was 0 mg. Lastly, we analyzed the PCL/CaCO3 nanofibre. The large
decrease in the TGA curve may be attributed to the thermal
decomposition of PCL and the decomposition of CaCO3 to CaO.
There were three endothermic peaks in this curve. The peak at
60  C arises from the phase transition of PCL from solid to liquid.
The peak at 350  C reflects the thermal decomposition of PCL, and
the peak at 420  C derives from the phase transformation of CaCO3
from veterite to calcite. Before analysis, the PCL/CaCO3 nanofibre
sample weight was 9.7 mg, and it was 7.2 mg after analysis. This
Fig. 6. The result of thermogravimetric (TG) and differential thermal analysis (DTA).
Table 1
Results of specific surface area, pore size, fibre diameter.
Specific surface area Pore size Fibre diameter
(m2/g) (mm) (nm)
PCL 2.5855 ± 1.1135 2.26 ± 0.77 900 ± 480
PCL/CaCO3 10.9252 ± 0.1437 1.58 ± 0.57 600 ± 240
indicates that the sample contained approximately 7.2 mg CaCO3
and 2.5 mg PCL, closely matching the predicted weight ratio. Spe-
cific surface area analysis, pore size, fibre diameter had shown in
Table 1. PCL/CaCO3 nanofibre have lager specific surface area than
that of PCL nanofibre. This is because CaCO3 which contained PCL
nanofibre had effect on increasing specific surface area. On the
other hand, pore size and fibre diameter slightly decreased. Pore
size indicates void which means space between fiber and fiber. PCL/
CaCO3 nanofibre has smaller fibre diameter than PCL nanofibre.
Because of fibre fabrication process. As shown in Fig. 7, When PCL/
CaCO3 fibre fabricated, the aggregation occurred on the tip of
needle. Outlet diameter of needle slightly was decreased by ag-
gregation. The fibre with smaller diameter has larger density than
that with bigger diameter. As the result, PCL/CaCO3 nanofibre has
small pore size. Nevertheless, PCL/CaCO3 has large specific surface
area. We confirmed the fibre morphology for stability of fibre.
Soaked fibre mat morphology was measured by FE-SEM. As shown
in Fig. S3, the morphology of fibre had maintained their fibre for-
mation without breaking and without dissolving in PBS solution
and mouse blood even after 24 h. Furthermore, fibre mat which
soaked in mouse blood (18 h, 24 h) had shown the absorption of
ingredient of blood such as protein, white blood cell and so on. This
absorption was formed membrane-like film on the surface of fibre.
From these experimental facts, it was demonstrated that the
nanofibre has stability in aqueous media and biological condition.
3.3. Ultrasonic spray coating vs. hand spraying
The nanofibres were coated with b-chitosan using an ultrasonic
spray machine, which can be used to control the specific coating
area and the flow rate of solution from the syringe pump. Moreover,
the diameter of the solution droplet can be reduced for uniform
coating. For comparison, we also hand-sprayed the nanofibres us-
ing the same amount of b-chitosan. The sample that had been
sprayed using the ultrasonic spray machine was not overly wet,
while the sample that had been hand-sprayed was completely wet.
According to the SEM images, hand spraying did not result in a
uniform amount of b-chitosan on the surface of the nanofibre
(Fig. 8). From the results of EDX (Fig. S4), PCL nanofibre was not
existed b-chitosan (N element). However, PCL nanofibre sprayed
 J.-Y. Park et al. / Polymer 123 (2017) 194e202
Fig. 7. Schematic of fabrication process of nanofibre. (a) PCL nanofibre, (b) PCL/CaCO3
nanofibre. The aggregation occurred on the tip of needle in fabrication process of PCL/
CaCO3 nanofibre.
with b-chitosan confirmed the existing of N element within b-
chitosan. From the results of particle analyzing, b-chitosan was
coated on the PCL nanofibre surface about 16.911% (Ultrasonic
spraying) and 3.411% (Hand spraying), respectively. A higher
magnification image is shown in Fig. S5. The functional properties
of fibre were analyzed by FT-IR (Fig. 9). In an analysis of fibre, the
spectrum for the b-chitosan showed absorption peaks at about
3436 cm1 and 1610 cm1. The strong band at 3436 cm1 corre-
sponded to OH and NH stretching of chitosan. The peak at
1610 cm1 was attributed to amide II bands of chitosan [46]. The
additional peak at 875 cm1 was attributed to CaCO3 [47]. These
major peaks were not detected in pure PCL fibre.
3.4. Assessment of whole-blood coagulation
We assessed the clotting ability of PCL and PCL/CaCO3 nano-
fibres on glass slides and measured the absorbance of the non-
Fig. 8. Field emission scanning electron microscope image of b-chitosan on PCL nanofibres after (a) hand spraying and (b) use of an ultrasonic spray machine. The using amount of
b-chitosan used is the same in the two images. however, b-chitosan is more homogenous in (b). Black box indicates the particle analysis.
199
clotted blood at 540 nm (Fig. 10). A lower absorbance reflects
less hemoglobin in the sample and therefore indicates a higher
coagulation rate. For comparison, the absorbance of pure distilled
water is 0.0559, and all sample absorbances were higher than
absorbance of pure distilled water. We assessed the absorbances of
PCL and PCL/CaCO3 nanofibres alone and after spraying with
1.25 mL, 2.5 mL of b-chitosan using an ultrasonic sprayer
(Fig. 10(a)) or hand spraying (Fig. 10(b)). The absorbance of bare
gauze was measured as a control and was almost the same as the
absorbance of the PCL nanofibre alone. However, PCL fibres with
sprayed with b-chitosan and all PCL/CaCO3 nanofibre samples
(whether sprayed with b-chitosan or not) exhibited lower absor-
bances than that of bare gauze. The fact that the PCL/CaCO3
nanofibre exhibited a higher clotting rate than that of the PCL
nanofibre confirms that CaCO3 has an effect on the blood coagu-
lation process. Similarly, b-chitosan improved coagulation, as the
PCL and PCL/CaCO3 nanofibres that underwent 2.5 mL of ultrasonic
spraying exhibited the highest clotting rates among all PCL and
PCL/CaCO3 nanofibre samples, respectively. Indeed, the PCL/CaCO3
nanofibre sprayed with 2.5 mL of b-chitosan exhibited the highest
clotting rate among all samples in the experiment. This reflects the
fact that b-chitosan increases red blood cell aggregation through an
electrostatic interaction in the blood. In comparing the hand- and
ultrasonically sprayed samples, we found that the former exhibited
consistently higher absorbances (Fig. 10(b)) than their ultrasoni-
cally sprayed counterparts. This is because hand spraying led to a
highly-wet surface, hindering the attachment of b-chitosan to the
surface of the fibres.
3.5. Assessment of coagulation in mouse blood
Because the pig blood in the above experiment contained an
anti-coagulant additive, we also assessed blood coagulation using
pure mouse blood taken by syringe. Again, absorbances of non-
clotted blood were measured at 540 nm (Fig. 11). For this exper-
iment, we used fibre samples that had been ultrasonically sprayed
with 2.5 mL of b-chitosan (PCL, PCL/CaCO3), and compared them to
non-sprayed fibres and bare gauze. Due to differences in the
amount of blood used in the experiment, the absorbance values
differed somewhat from those shown in Fig. 8. However, the re-
sults were similar, demonstrating that the PCL/CaCO3 nanofibre
sprayed with b-chitosan exhibits the highest blood clotting rate.
We had animal experiment for attaching directly at wound parts.
PCL/CaCO3 nanofibre mat sprayed with b-chitosan permeated
faster than PCL nanofibre mat (Fig. 12(a)). Mouse wound with PCL/
CaCO3 nanofibre mat sprayed with b-chitosan have also faster
blood clotting than that of PCL nanofibre (Fig. 12(c)). Rapid clotting
of the PCL/CaCO3 nanofibre mat sprayed with 2.5 mL of b-chitosan
200 J.-Y. Park et al. / Polymer 123 (2017) 194e202

Fig. 9. Results of FT-IR spectrum. (a) Wide range of FT-IR spectrum (500-4000 cm1). (b) Narrow range of FT-IR spectrum (1500-2500 cm1). Blue dash line indicates absorbance
peak at 1610 cm1. (c) Narrow range of FT-IR spectrum (500-1500 cm1). Red dash line indicates absorbance peak at 875 cm1.

3.6. Surface wettability vs blood coagulation

Surface wettability is important for blood coagulation.

Increasing the contact areas with nanofibre and wound or blood is
effect on fast blood clotting from the nanofibre including CaCO3 and
b-chitosan. We measured the blood contact angle and blood
coagulation with bare gauze and nanofibre samples.
The result was shown in Fig. 13. Surface wettability is almost
corresponded with the blood coagulation. Bare gauze has low blood
contact angle. However, blood coagulability is not higher than PCL/
CaCO3 nanofibre mat sprayed with b-chitosan. The reason is bare
gauze have not contained the increasing blood coagulation mate-
rials as CaCO3. As shown in Fig. 13, PCL have hydrophobic property
with blood. Contained the b-chitosan nanofibre mats is decreased
the blood contact angle changing to the hydrophilic surface prop-
erty. Compared with Figs. 11 and 13, nanofibre mat sprayed with
2.5 mL of b-chitosan is fast blood clotting rate. Furthermore, we
confirmed the optimum content of b-chitosan with increasing the
amount of b-chitosan up to 7.5 mL. As shown in Fig. S7, although the
amount of b-chitosan increased, blood contact angle is almost same

Fig. 10. Results of whole-blood coagulation experiment using pig blood on glass slides.
Nanofibre mats were fabricated via (a) ultrasonic spraying or (b) hand spraying. All
other experimental conditions are the same.

can also be seen in photos of the blood clotting experiment Fig. 11. Results of whole-blood coagulation experiment using fresh mouse blood. b-
(Fig. S6). chitosan samples represent fibres sprayed ultrasonically for 2.5 mL.
 J.-Y. Park et al. / Polymer 123 (2017) 194e202
Fig. 12. Results of nanofibre mat attachment experiment. (a) Photo of attachment experiment. Left-hand is PCL nanofibre, right-hand is PCL/CaCO3 nanofibre sprayed with b-
chitosan. (b) Photo of wound after attachment PCL nanofibre. (c) Photo of wound after attachment PCL/CaCO3 nanofibre sprayed with b-chitosan. (d) Photo of nanofibre mat after
attachment experiment.
Fig. 13. Results of blood contact angle (mouse blood). We dropped the 1 mL of pig
blood droplet. After that, measured the blood contact angle.
value in both of PCL and PCL/CaCO3 over 2.5 mL of b-chitosan.
Though b-chitosan has wound healing property and effected sur-
face wettability, the key role for fastest blood clotting is existence of
CaCO3. Blood coagulation test (Corresponding to weight ratio of
PCL: CaCO3, optimum content of b-chitosan) is shown in Fig. S8. The
weight ratio of PCL: CaCO3 (1:3) has fastest blood clotting rate than
that of the other sample (Fig. S8(a)). This is because the high
amount of CaCO3 contained in the nanofibre. Over 2.5 mL of b-
chitosan, the absorbance showed almost the same values. This
shows blood clotting rate was saturated over the critical amount of
b-chitosan. b-chitosan has reached saturation in surface of fibre at
2.5 mL of solution.
Here, we discussed about the mechanism of blood coagulation.
When an area of the body (e.g., skin or blood vessel) is damaged,
resting platelets in the blood must become activated. Activated
platelets catalyse clot formation by converting the prothrombin on
their surfaces into thrombin. Thrombin acts as a serine protease
that converts soluble fibrinogen into insoluble strands of fibrin,
forming a blood clot. Blood clots contain platelets as well as other
blood cells, cover the damaged tissue, and halt bleeding [48]. In
generally, the mechanism of blood coagulation has quite a few
201
factors such as surface wettability, platelet activity and so on.
However, as mentioned above, the calcium ion is the most impor-
tant factor of hemostasis. Though CaCO3 is not ionic form, calcium
ion that contained in CaCO3 has effect on the blood coagulation. In
addition, the b-chitosan has a role to permeate blood into nanofibre
as well. The fiber surface fabricating by spraying b-chitosan has
faster blood clotting rate than that of non-sprayed surface. This
indicates that blood have a large contact area with nanofibre which
contained CaCO3. From this reason, the fast clotting rate shows high
amount of b-chitosan and CaCO3 in the nanofibre.
4. Conclusion
We fabricated a novel nanofibre using PCL and CaCO3 as
biocompatible materials and b-chitosan as a natural polymer. We
added CaCO3 to the PCL fibre fabricated via electrospinning to
fabricate an ultrafine PCL/CaCO3 fibre. Ultrasonic spraying was used
to coat the resulting nanofibres with b-chitosan while maintaining
their structures. From the results of SEM observation and whole-
blood coagulation assessments, we demonstrated that the combi-
nation of a PCL/CaCO3 nanofibre sprayed with b-chitosan exhibits
the highest blood clotting ability among tested samples. This is
because of the fact that the b-chitosan improves the surface
wettability of the fibre and CaCO3 speeds up the blood coagulation.
This can be expected to use not only urgent internal bleeding but
also external bleeding. Since CaCO3 and b-chitosan are human-safe,
natural materials for human, and PCL is a biodegradable material,
PCL/CaCO3 nanofibre sprayed with b-chitosan is used for wound
dressing, we may not have to worry about the operation for the
elimination of the threads that using the safety materials after
surgery. Moreover, the fabricated PCL/CaCO3 nanofibre sprayed
with b-chitosan is convenience and cost-effective. Furthermore, it
is easy to control its thickness and size to fit for the wounded area.
In addition, there is no limitation of substrate because of its self-
standing. In summary, we consider that the fabricated PCL/CaCO3
nanofibre sprayed with b-chitosan offer the opportunity for diverse
medical applications where rapid blood coagulation is required.
Acknowledgment
JYP thanks to the financial support of Ministry of Education,
202 J.-Y. Park et al. / Polymer 123 (2017) 194e202
Culture, Sports, Science and Technology, Japan.
Appendix A. Supplementary data
Supplementary data related to this chapter can be found at
http://dx.doi.org/10.1016/j.polymer.2017.07.013.
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