Applied Materials Today 13 (2018) 228–241

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Shape-adaptive composite foams with high expansion and absorption used for
massive hemorrhage control and irregular wound treatment
Yansen Wang a,1 , Cai Wang a,1 , Longxue Qiao b , Jingxuan Feng a , Yudong Zheng a,∗ , Yong Chao b,∗ ,
Wei He a , Yajie Xie a , Wanjun Shuai b , Minglu Li a
a
School of Material Science and Engineering, University of Science and Technology Beijing, Beijing 100083, PR China
b
First Affiliated Hospital of Chinese PLA General Hospital, Beijing 100037, PR China

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

Article history: Effective bleeding control is becoming more and more important in the military field and civilian trauma
Received 23 July 2018 arena. However, traditional hemostats present limitations, they have been proven to be less useful
Received in revised form for irregular, deep or penetrating wounds with uncontrollable hemorrhage caused by minor-caliber
14 September 2018
weapons, shrapnel or explosive devices. Here, a series of porous polyvinyl alcohol (PVA)/alginate com-
Accepted 17 September 2018
posite foams (PACFs) were prepared through crosslinking reaction and lyophilization process. The effect
of different content of alginate on the physicochemical properties and hemostatic function of the PACF
Keywords:
were evaluated. The results demonstrated that PACF could not only rapidly absorb plasma, but also stim-
Polyvinyl alcohol
Alginate
ulate blood cells, thus further promoting blood coagulation. In addition, the PACF with higher alginate
Composite foams content showed increased capability to enhance thrombus generation, blood clotting, and blood cells
Hemostasis adhesion and aggregation. More importantly, the PACF exhibited over 2000% volume expansion, high
flexibility and shape-adaptive ability, and could therefore expand to entirely fill and conform to the
shape of wound cavity. The in vivo hemostatic tests further demonstrate the high hemostatic efficiency
of PACF. These results suggest that PACF has strong potential as a hemostatic material to be applied for
massive hemorrhage control and deep/irregular wounds treatment.
© 2018 Elsevier Ltd. All rights reserved.

1. Introduction and severe bleeding control [8–11]. Currently, the common used
hemostatic materials can be divided into organic and inorganic
Massive and uncontrollable hemorrhage caused by severe hemostats. Hemostats made from inorganic materials, including
®
trauma is a global health problem. Hemorrhage control remains zeolites (i.e., QuikClot , Z-Medica Corporation, Wallingford, CT),
a major concern of military and civilian across the world. Excessive can lead to thermal tissue injuries by the exothermic reaction and
bleeding usually increases the risk of suffering hemorrhagic shock immune response owing to their poor biocompatibility [12,13].
®
coagulopathy, infection, and multiple organ failures. As reported, Clay-based hemostatic products (i.e., WoundStat ), which belong
hemorrhage results in 30–40% of deaths, causing more than 5.8 to another group of inorganic hemostats, also present limitations in
million deaths worldwide annually [1,2], which is predicted to rise achieving rapid hemostasis, which would result in extensive blood
to over 8 million by 2020 [3,4]. Effective hemostatic materials that loss [14,15]. Organic hemostats such as collagen-based hemostatic
can rapidly stop severe bleeding are crucial to the wound healing products produce allergic reactions and can cause nerve compres-
or gain time for further rescue [5,6]. Thus, developing novel hemo- sion and damage due to excessive swelling [16]. They have also
static materials with practicality and ideal hemostatic performance proven to be ineffective in controlling arterial bleeding [14]. Other
is important for rapid and effective control of hemorrhage. organic hemostats which are primarily used in battlefield and
In recent years, plenty of materials with different proper- civilian trauma arena, are the chitosan-based hemostatic agents
®
ties have been widely developed to enhance wound healing [7] (i.e., HemCon , HemCon Medical Technologies, Inc., Portland, OR
and CELOXTM , SAM Medical, Tualatin, OR). They have demon-
strated better efficiency than other hemostats in stropping bleeding
∗ Corresponding author.
and inducing hemostasis [17]. However, high re-bleeding rates
®
E-mail addresses: zhengyudong@[Link] (Y. Zheng),
have been reported for the wounded treated with HemCon and
TM
CELOX [18,19]. Additionally, although each kind of hemostat has
yongchg@[Link] (Y. Chao).
1
The authors contributed equally to this work.

[Link]
2352-9407/© 2018 Elsevier Ltd. All rights reserved.
 Y. Wang et al. / Applied Materials Today 13 (2018) 228–241 229

certain effect on preventing hemorrhage, they have proven less 2. Materials and methods
useful for irregular, deep or penetrating wounds with massive hem-
orrhage caused by minor-caliber weapons, shrapnel or devices [20]. 2.1. Materials and reagents
Thus, the challenge now is developing more effective hemostatic
materials to control hemorrhage and treat specific wounds. Sodium alginate was purchased from Tianjin Guangfu Fine
As a natural anionic polymer, alginate obtained from seaweed Chemical Research Institute. PVA powder (polymerization degree
has been deeply studied and widely used in a variety of biomedi- of 1750 and 99% hydrolyzed) was supplied by Beijing Xisi Chem-
cal applications [21], such as wound dressing and in vivo injectable icals Co. Ltd. Calcium chloride was purchased from Aladdin
gels [22–24], because of its excellent biocompatibility. Especially, Reagent Co., Ltd. (Shanghai, China). Fibrinogen was purchased from
metal cations, for example Ca2+ , Cu2+ and Fe2+ , could interact Sigma–Aldrich Co. LLC (MO, USA). LDH was provided by Beijing
with the guluronate blocks in alginate to form polymers with Institute of Nanoenergy and Nanosystem, Chinese Academy of Sci-
particular “egg-box” structure [25]. In addition, alginate compos- ences. APTT and PT assay reagents were purchased by Tianjin MD
ites possesses negative charges generated by its carboxyl groups. Pacific Technology Co., Ltd. All other reagents were analytical grade
Alginate products have proven having the ability to absorb large and used as received.
amount of wound exudate and provide a physiologically moist All animal experiment procedures were ethically approved by
microenvironment for wound healing [26]. Alginate based compos- the Institutional Animal Care and Use Committee of the First Affil-
ites with excellent biocompatibility, promotion of wound healing iated Hospital of Chinese PLA General Hospital and the animal
and hemostatic function, are widely used for hemostatic applica- ethical permission registration No. 2013-07. All procedures of ani-
tions [27,28]. Alginate composite has been used to treat different mal experiment were in full compliance with recommendations
types of wounds and is particularly suitable for treating open on animal studies of the Helsinki Declaration of World Medical
wounds and mechanical trauma owing to its high water absorp- Association.
tivity and gelation ability [29,30], which can help relieve pain and
prevent secondary bleeding from the wound site. Additionally, algi-
nate composites have been also used to control bleeding in tooth 2.2. Preparation of PVA foam and the PACFs
sockets. However, the poor chemical stability, weak mechanical
strength and lacking long-term efficacy restrict the further appli- PF: PVA particles were dissolved in distilled water to prepare
cations of alginate-based composites for hemostasis, particularly PVA aqueous solution with concentration of 12.5% (W/V). Then PVA
against massive hemorrhage. solution was chemically cross-linked by glutaraldehyde in the pres-
In recent years, composite foams have shown strong poten- ence of H2 SO4 as catalyst at room temperature to obtain PF. The
tial in hemostatic application owing to their unique properties, molar ratio of PVA and glutaraldehyde was 1:0.8.
such as high porosity, high surface area and pore volume, and fast PACFs: Alginate with mass fractions of 10%, 20%, 30%, 40% and
water absorption [31–35]. In our previous study, we also devel- 50% were added into 12.5% PVA solution to prepare a gel-like solu-
oped macroporous foams based on the cross-linked PVA [36]. The tion. CaSO4 (1 g) was dissolved in distilled water (100 ml), left
prepared foams possess favorable water absorption capacity and standing for 30 min and centrifuged for 5 min to obtain supernatant.
biocompatibility. Notably, our present investigations indicate that The Ca2+ concentration in the CaSO4 solution is 0.02 mol/L. Subse-
through using different crosslink agents and changing the synthetic quently, the CaSO4 solution together with glutaraldehyde (molar
method, composites could maintain the uniform porous structure ratio of PVA and glutaraldehyde was 1.25) were added into the
while obtaining higher water absorption capacity, better hemo- gel-like solution in the presence of H2 SO4 and several drops of
static and expansion properties than most of the common used Triton X-100 at room temperature until well mixed. Triton X-100
hemostatic agents. was used as a non-ionic surfactant. Finally, the mixture was put
For the above-mentioned reasons, in the following work, we into molds and directly treated through a lyophilization process.
attempt to develop a series of hemostatic and shape-adaptive com- The obtained composite foams with different alginate mass frac-
posite foams composed of glutaraldehyde cross-linked PVA and tions were named as PACF-1, PACF-2, PACF-3, PACF-4 and PACF-5,
CaSO4 cross-linked alginate. We aim to combine multiple hemo- respectively
static mechanism into one composite material. The PACF with
porous structure could quickly absorb plasma and concentrate the
blood cells; the introduction of alginate could stimulate the sur- 2.3. Physicochemical characterization of PF and PACFs
face interaction between the PACF and blood cells through negative
charges and Ca2+ , thus enhancing the hemostatic efficiency of the Chemical structures of PF and PACFs were spectroscopically
PACF. Therefore, introducing alginate into porous foam should be measured by Fourier Transform Infrared (FT-IR) Spectrometer
effective for a reinforced hemostasis, especially through combin- (TENSOR II, Bruker, Germany) with a range of frequency from
ing fast plasma absorption and surface effect as well as charge 4500 cm−1 to 900 cm−1 and a resolution of 4 cm−1 .
stimulation. Furthermore, the PACF also inherits the flexibility and The surface of PF and PACFs were observed through an optical
deformable ability of foam materials, and it can be conformed to any microscope with a VHX-5000 digital microscope (Life Technologies,
three-dimensional and curvilinear surface through fast absorbing Eugene, OR, USA). The morphology and microstructure of the sam-
liquid to obtain the shape adaption to wound cavity (Scheme 1). ples were characterized by Scanning Electron Microscopy (SEM)
In this regard, the PVA/alginate composite foams with differ- (AURIGA Cross Beam FIB/SEM Station, Carl Zeiss, Germany) at an
ent alginate contents (10, 20, 30, 40 and 50 wt%) were prepared accelerating voltage of 15 kV, after carbon sputtering treated.
through crosslinking reaction and lyophilization method. The effect Contact angle (CA) measurements were conducted at room tem-
of alginate content on the physicochemical properties of PACFs as perature using 2 ␮L drops of distilled water. The water droplets
well as the interactions between blood cells and PACFs were inves- were dropped onto the samples for 5 s, and then photographed the
tigated. The hemostatic efficacy of the PACF was also tested and contact angle in the fifth second. Contact angle of each sample was
compared to that of some commercial hemostatic products. Fur- recorded by an optical contact angle meter (OCA20, Dataphysics
ther, in vivo hemostatic performance and shape-adaptive ability of Inc.). All the measurements were conducted under the same exper-
the PACF were evaluated using a hemorrhaging liver mouse model imental conditions. Each sample was tested for five times to ensure
and a swine femoral artery gunshot injury model. the accuracy of the results.
230 Y. Wang et al. / Applied Materials Today 13 (2018) 228–241

Scheme 1. Schematic representation of the fabrication process of the PACF and its hemostatic performance. First, the PACF was prepared through crosslinking method and
formed a double network. Second, the PACF could be inserted into the wound and then expand to completely fill the irregular wound cavity. Finally, two animal experimental
models were used to evaluate the hemostatic performance of PACF.

The surface roughness of different samples were characterized After pre-loading, 37 ◦ C PBS solution was added to initiate sample
by confocal laser microscope (LSCM, KEYENCE, VK), and the surface expansion. The distance between the compression platen and the
roughness as well as surface height difference was then calculated. operate board remained constant throughout the test, while the
The electrokinetic potential of the PF and PACFs have a great expansion force exerted by the expanding composite was continu-
influence on the adsorption capacity and hemostatic perfor- ously measured for 1 h. The buoyancy force exerted by PBS solution
mance via electrostatic interaction. Zeta-potential (␨-potential) was also measured. The expansion forces were calculated through
was tracked by a Zeta Potential Analyser (DelsaTM Nano, Beckman subtracting the buoyancy force from the raw force.
Coulter, USA).

2.4. Liquid absorption capacity and expansive performance 2.5. Fibrinogen adsorption

The liquid absorption capacity of the samples was tested using Fibrinogen (FIB) solutions with a certain concentration were
phosphate buffer saline (PBS, 0.05 mol/L, pH = 7.4 ± 0.2) solution at prepared using PBS. 10 mL of pre-determined concentration FIB
37 ◦ C. The dry weight (Wdry ) of each sample was tested before the aqueous solution and 1 disk of the adsorbent (PF or PACFs) were
test. Then the dry samples were then immersed in PBS solution, mixed in a stoppered glass tube and shaken at 120 rpm in a ther-
followed by incubation for 1 h at 37 ◦ C. The foams were then taken mostatic oscillator at 37 ◦ C. Samples were taken out at same time
out and gently dabbed on filter papers to remove the excess liquid intervals and then the absorption of FIB was determined by UV–vis
on the surface, and the wet weight of the samples was immediately spectrophotometry (HP 8453, USA) at 280 nm. The morphologies
measured (Wwet ). Liquid absorption of the samples (%) was finally of the absorbed FIB on the surface of samples at 15 h and 30 h were
calculated by Eq. (1). also observed by SEM, respectively.
(Wwet − Wdry )
(1)
Wdry
2.6. In vitro platelet adhesion
The volume expansion (VE ) of PF and PACFs was analyzed using
E. (2): Citrated anticoagulated blood was collected from ear vein of
healthy rabbits, and platelet-rich plasma (PRP) was obtained using
Va
VE (%) = × 100% (2) common method [37]. The samples were immersed in the platelet
Vb suspension and incubated at 37 ◦ C for 30 min. The samples were
where Vb and Va refer to the volumes of the foams before and then rinsed in PBS for three times and incubated with 1% Triton X-
after expansion, respectively. All the samples were measured in 100 in PBS at 37 ◦ C for 1 h. The morphology of the adhered platelets
triplicates for each group. was observed by SEM after the platelets were fixed using 2.5% glu-
The expansion force of PACFs and PF was analyzed according to taraldehyde, dehydrating in a graded series of ethanol and going
the method based on ISO 25539-2 (n = 2) [34]. Briefly, samples were through freeze-drying. The number of platelets on the samples
placed between the compression platen and the operate board of was also quantified using a Lactate dehydrogenase (LDH) assay
the Texture Analyzer (TAXT plus, Stable Micro Systems, UK). Trig- kit (Sigma, Aldrich) by measuring the LDH released from the lysed
ger force of 0.1 N was applied to the foams before the test started. platelets [38].
 Y. Wang et al. / Applied Materials Today 13 (2018) 228–241 231

2.7. Evaluation of thrombus formation and clot strength Computer angiography was used to observe the blood flow and
blood vessels around the wound site. Computer tomography was
Thrombelastography (TEG) was performed to observe the initial used to observe the morphology of samples in the wound cavity of
time of thrombus formation and evaluate the clot strength in vitro. swine. Finally, PACFs were removed from the wound site manually
The blood was collected from a healthy donor and put into antico- or with surgical forceps. After that, 1.5 L of physiological saline was
agulant tube contained citrate. The TEG Analyzer (TEG Hemostasis used to carefully clean the wound site. All swine survived during
Analyzer 5000, USA) was calibrated by quality control standards and after the test.
obtained from hemoscope at 37 ◦ C. This test was conducted accord-
ing to the reported literature [39]. Briefly, citrated whole blood 2.11. Statistical analysis
(1 mL each) was mixed with different samples (30 mg each) of in
plastic vials. The citrated blood mixed with CaCl2 was used as a con- All results were expressed as mean ± standard deviation (SD)
trol group. Samples were tested and tracing continued for 30 min and statistical analysis was performed by one-way analysis of vari-
after the formation of initial fibrin. The following parameters were ance (ANOVA). P < 0.05 was considered to be statistically significant.
measured in each sample: reaction time (R, min, initial fibrin forma-
tion time); clotting time (K, min, the time from the reaction time to 3. Results
the formation of clots); angle (degree, the kinetics of clot develop-
ment); and maximum amplitude (MA, mm, the maximum strength 3.1. Characterization of PVA foam and PVA/alginate composite
of the formed clot). foams

2.8. APTT and PT measurement The porous structures of materials used as hemostatic agents
play a vital role in inducing hemostasis since they can help to effec-
The in vitro coagulant ability of the samples was further eval- tively absorb wounds exudate, thus increasing the concentration
uated by APTT and PT assays. The blood was collected from the of the blood cells [44]. Fig. 1a confirmed the formation of highly
ear-rim auricular vein of rabbits. The tests were conducted accord- interconnected porous structures of the foams. The typical sur-
ing to the method our previous work described [23]. face morphology and microstructure of PF, and PACF are shown in
Fig. 1b. PF sample presented a smooth and uniform surface, and
2.9. In vitro whole blood clotting evaluation no particles was found on its surface or networks. On the con-
trary, large amount of calcium alginate particles were observed
A blood clotting test was used to evaluate the clotting proper- distributed on the surface and networks of PACF.
ties of the samples. 2 mL rabbit whole blood was added into vacuum Static water contact angles of the samples were shown in Fig. 1c.
blood collection tubes (contain anticoagulant sodium citrate inside) It can be seen that the contact angles of all the tested samples were
with PF (80 mg) and PACFs (80 mg) respectively. Whole blood with- less than 90◦ , which indicated that all the samples were hydrophilic.
out sample was used as a control group. Each sample interacted However, the PACFs possessed higher contact angles than PF, and
with blood for certain seconds and the blood coagulation time was the water contact angles raised from 25.3◦ to 71.0◦ with the increas-
recorded by a digital camera. ing content of alginate. Therefore, we can conclude that introducing
Then the samples were taken out of the tubes, and 15 mL of alginate in the PF matrix, at some extent, may lower the hydrophilic
distilled water was added into the tubes to dissolve the hemoglobin ability of the PACFs.
in red blood cells. A microplate reader (BMG LABTECH, Offenburg, Surface roughness has great influences on the blood cell adhe-
Germany) was subsequently used to determine the absorbance of sion and coagulant properties of materials and rough material
hemoglobin in the solution at 542 nm. As a reference value, 15 mL of surface would always be easier for more surface areas to be exposed
distilled water was directly added into the unreacted whole blood to blood. Besides, the rough surface also contributes to the platelet
to measure the absorption value. as well as plasma protein adhesion. As shown in Fig. 1d, with
the increasing alginate content, surface roughness value gradually
2.10. In vivo hemostatic performance increased, the surface became more uneven and the average surface
height difference became larger.
A hemorrhaging liver mouse model was employed Fig. 1e shows the surface potential of PF and PACF with differ-
(Sprague–Dawley rat, 200–240 g, male) to primarily evaluate ent alginate content in PBS (7.2). Since alginate possesses negative
the in vivo hemostatic performance of different samples. This test surface charges generated by its carboxyl groups, after combining
was conducted as previous literature described [40,41]. PACF-4 with PVA to form PACF, the negative charge density of the compos-
samples were used in this test and all the measurements were ite foams intensively strengthened as compared with the original
triplicate. PF (barely below 0 mV). With the increasing alginate content, the
Further, a swine femoral artery gunshot injury model was zeta potential of PACFs increased from −13 mV ± 0.7 mV (PACF-1)
employed to evaluate the safety and efficacy of samples (PACF-4) to −32.0 mV ± 1.5 mV (PACF-4).
[42,43]. Briefly, five male castrated swine weighing 47.0–52.0 kg
were provided by the First Affiliated Hospital of Chinese PLA Gen- 3.2. Liquid absorption capacity and expansion properties
eral Hospital (Beijing, China) and were treated in accordance with
the National Research Council’s Guide for the care and use of lab- High-speed digital imaging showed that 15 mL liquid could
oratory animals. All swine were anesthetized with 10% chloral be completely absorbed into the PACFs (10 mg) within 20 s, as
hydrate (intraperitoneal injection with 0.5 mL per 100 g) before shown in Fig. 2a and Movie S1, illustrating an ultrafast liq-
surgery. A firearm wound was created through gunshot in the groin uid absorption rate. In addition, the PACFs also exhibited rapid
area directly over the left femoral artery. The start time of injury expansion ability when contacting with liquid. Further, the PACFs
was recorded and the vessels was allowed to bleed freely for 30 s became flexible and deformable after absorbing liquid, and finally
before the samples were applied. PACFs particles were applied as completely adhered to the beaker boundary, presenting obvious
quickly as possible and as many as necessary until the bleeding shape-adaptive ability.
was stopped. The injury condition was observed every 10 s. When A comparison of liquid absorption capacities of PF and
the wound stopped bleeding, the hemostatic time were recorded. PACFs is shown in Fig. 2c. The results demonstrated that, after
232 Y. Wang et al. / Applied Materials Today 13 (2018) 228–241

Fig. 1. Characterization of PF and PACFs. Microscope images (a), SEM micrographs (b), contact angles (c), surface roughness (d) and zeta potential (e) of PF and PACFs with
different alginate content. Scale bar: 10 ␮m (d).

5 min of immersion in deionized water, the water absorp- The PF and PACFs were then analyzed for volume expansion
tion of PF, PACF-1, PACF-2, PACF-3, PACF-4 and PACF-5, was in 37 ◦ C PBS. The original and expanded shapes of the compos-
1792.3 ± 127.2%, 1636.1 ± 135.5%, 1587.2 ± 87.1%, 1567.9 ± 107.2%, ite foam are shown in Fig. 2b. The images showed that the shape
1381.6 ± 105.5% and 1141.7 ± 99.4%, respectively. Apparently, the of the composite foam could change from the original state to a
PACFs absorbed significantly more water compared with alginate highly expanded geometry upon contact with liquid. Within this
hydrogel (500–600%) and our previous developed composite foams time frame the foam volume could increase more than a 2000%
[36]. in average (Fig. S1, Movies S2 and S3). As shown in Fig. 2c, the
 Y. Wang et al. / Applied Materials Today 13 (2018) 228–241 233

Fig. 2. Liquid absorption capacity and expansion properties. (a) PACFs rapidly absorbed liquid and expanded until completely filled the beaker. (b) Representative image of
the appearance of PACF before (down) and after (up) absorbing liquid. Scale bar is in centimeters. (c) Water absorption rate and volume expansion rate. (d) Expansion force
exerted by PACFs during expansion in water.

volume expansion rate of PACF-1, PACF-2, PACF-3, PACF-4 and the characteristic absorption peak of protein (amide I and amide
PACF-5, was 1650.5 ± 175.0%, 2210.2 ± 190.6%, 2730.5 ± 240.3%, II), were observed in the FT-IR spectrum of PACF-3, demonstrat-
2350.3 ± 225.6%, 1950.5 ± 190.4%, respectively. Owing to the high ing that fibrinogen had been absorbed into the composite foams.
volume expansion rate of PACFs, the PACFs in small size could be Fig. 3c shows the surface morphology images of foams after absorb-
easily delivered into the wounds and subsequently undergo over ing protein at different points in time. Protein fibers could be clearly
2000% expansion to entirely fill the wound cavity in short time. observed on the surface of PACF. The FIB amount after absorbing for
One important feature of ideal hemostatic materials used to 30 h was far greater than that of 15 h-absorption. Meanwhile, with
treat large wounds or penetrating wounds is the ability to apply time prolonging, dispersed protein particles gradually aggregated
sufficient radial force to offer durable and assistant pressure to together from irregular polygon shapes into thick fiber-like shape
blood vessels and prevent dislocation of the materials from the (the red rectangle in Fig. 3c).
wound bed under physiologic blood flow and pressure. The expan-
sion force exerted by the composites during expansion in 37 ◦ C
3.4. Platelets and red blood cells adhesion
water are summarized in Fig. 2d. The results indicated that, with the
increasing alginate content in the composites, the expansion force
As an important kind of blood cells, platelets mainly contribute
of the composites increased significantly. The expansion force of the
to inducing blood coagulation through the formation of platelet
PACFs ranged from 1.5 N (PACF-1 and PACF-2) to 8 N (PACF-5). The
plugs. After PACFs interacted with PRP suspensions for 30 min,
results indicated that the composite foams could rapidly expand to
we investigated the adhesion and aggregation of platelets on the
fill the wounds and provide a similar pressure exerted by compres-
PACFs. No adherent platelets were found on the PF surface. How-
sion to give assistant pressure on tissue and blood vessels to help
ever, large number of platelets were found on the surface of PACFs,
stop bleeding and prevent re-bleeding.
and especially, more platelets adhered onto the PACFs with a higher
amount of the alginate (PACF-3 and PACF-4), indicating their out-
3.3. Fibrinogen absorption standing ability to induce platelet adhesion and activation (Fig. 4b).
These results were further confirmed by SEM images that, plenty of
The PF and PACFs (with different alginate content) were placed platelets with lots of “pseudopods” stretching out were observed
in FIB solution and then the FIB absorption was measured by on the surface of PACF with higher alginate content (PACF-3 and
ultraviolet spectrophotometer. As shown in Fig. 3a, the FIB con- PACF-4), while only few activated platelets could be observed on
tent gradually increased with the prolongation of time. While the the other PACF samples (Fig. 4a).
absorption rate of each sample reduced to zero after nearly 30 h, To assess the effect of composite foams on red blood cells (RBCs)
which meant the amount of protein absorbed by composite foams adhesion, the test samples firstly interacted with fresh blood and
tended to be saturated. The protein adsorption values of PF close then were put into 0.9% saline solution. As seen in Fig. 4c, the leak-
to zero, indicating PF did not have the adsorption ability on FIB. age of RBCs was observed in the PF group. Similar phenomena were
It can be seen that the FIB absorption ability of PF could be sig- also observed for the PACF-2, although less RBCs leached out of the
nificantly improved through introducing alginate. Fig. 3b further foam. In contrast, RBCs were well entrapped inside PACF-3 and
proved the change of composites before and after absorbing pro- PACF-4, less RBCs were leached out in the two groups as com-
tein. Apparently, peaks at 1645 cm−1 and 1542 cm−1 , which were pared to the other groups, demonstrating better RBCs adhesion.
234 Y. Wang et al. / Applied Materials Today 13 (2018) 228–241

Fig. 3. (a) Absorption quantity of fibrinogen on different composites over time. FTIR spectrum (b) and surface morphology images (c) of PACF-3 after absorbing protein for
15 h and 30 h, respectively. Red rectangle indicates the corresponding area at high magnification. Scale bar: 10 ␮m.

Further, the absorbance of the hemoglobin-containing solution was thrombus formation time and the highest clot strength, which indi-
measured to evaluate the ability of samples to stimulate RBCs cated that PACF-4 had the best clotting capacities.
adhesion. In general, samples with a lower value of hemoglobin The APTT and PT assays were also conducted to evaluate the
absorbance have demonstrated a higher capability to trapping RBCs effect of materials on the intrinsic and extrinsic pathway of in vitro
and forming clots. The results showed that PACFs had a significant coagulation progress, respectively. Unreacted plasma was used as
lower absorbance value than PF. Notably, PACF-4 had the lowest a control group. The results of APTT and PT tests were depicted
absorbance value among all the tested samples, indicating it had in Fig. 5b and c. Generally, the normal range of APTT and PT were
the best ability to induce RBCs adhesion (Fig. S2). around 30 s and 15 s, respectively. The control group showed the
longest but normal APTT time (34 s) and PT time (14 s). While in
PACFs groups, through adding PACFs into the plasma, it is obvi-
3.5. Thrombelastogram analysis and APTT and PT measurement ous that the clotting time conspicuously decreased both in APTT
and PT test. Among all the samples, PACF-4 with the shortest APTT
Thrombelastogram (TEG) technology is an effective method to time (9 s) and PT time (6 s) exhibiting the most superior promoting
analyze the viscoelastic changes occurred during the coagulation of action in blood coagulation process.
the whole blood, and provide a visual way to evaluate the dynamic
process of thrombus formation (involving initiation, intermediate 3.6. In vitro whole blood clotting measurement
and formation) and the clot strength of blood. Thrombelastogram
has already been used to evaluate the in vitro hemostatic perfor- As a preliminary and basic hemostatic performance evalua-
mance of hemostatic materials [45]. The maximal amplitude (MA) tion, whole blood clotting test was performed to compare the
is the measurement of the highest point on the TEG curve, repre- coagulation efficiency between the PF and the PACFs. The citrated
senting the maximum clot strength [46]. As shown in Fig. 5a, no blood was used as control. As shown in Fig. 6b, no coagulation for
curve could be observed in the PF group after testing for 10 min, nearly 3 min was observed in both the control group and the PF
indicating no thrombus was formed. However, typical TEG curves group, indicating PF had no ability to promote blood coagulation.
were found in the control group and each PACF group. The initial In contrast, the whole blood clotting time of the PACFs was 78 s
position of the curve clearly shifted to left in each PACF group, (PACF-1), 66 s (PACF-2), 53 s (PACF-3), 48 s (PACF-4) and 60 s (PACF-
as compared with the control group, indicating the decrease of 5), respectively (Fig. 6b and c). Whole blood on PACFs demonstrated
reaction time (R). Besides, the clot formation rate (Angle) and progressively greater clotting with the increase of alginate content.
clot strength (MA) increased significantly with the increasing algi- Among all the samples, PACF-4 presented the shortest coagulation
nate content, especially, PACF-4 exhibited the largest clot strength time, which was consistent with the results of the APTT and PT
and clot formation rate. The results demonstrated that the clot assays and could be attributed to the favorable and stable physico-
strength of thrombus could be increased and the initial thrombus chemical properties of PACF-4. Notably, although PF had the highest
formation time could be greatly reduced by adding PACF into the average liquid absorption value, it had extremely poor blood coag-
blood. Among all the PACF samples, PACF-4 possessed the shortest ulation efficiency.
 Y. Wang et al. / Applied Materials Today 13 (2018) 228–241 235

Fig. 4. Platelets and RBCs adhesion. (a) Low and high magnification SEM images of platelets adhered to the surfaces of PF and PACFs with different alginate content. Red
rectangles indicate the black dotted rectangle areas at high magnification. Scale bar: 2 ␮m. (b) Quantitative analysis of platelet adhesion on the surface of materials. * represent
a significant difference in comparison to PF at P < 0.05. (c) Photographs depicting less RBCs leaked from PACF-3 and PACF-4 than that from PF and PACF-2. (For interpretation
of the references to color in this figure legend, the reader is referred to the web version of the article.)

Comparison of PACF and traditional hemostatic products to less than 35 s, demonstrating its significant hemostatic efficiency
® ®
(HemCon , QuikClot and CELOXTM ) on clotting time was further (Fig. 6f and g).
performed. As shown in Fig. 6d and e, clotting time of the sam-
®
ples (80 mg) was 48 s (PACF-4), 79 s (CELOXTM ), 99 s (HemCon )
®
and 366 s (QuikClot ), respectively. Obviously, PACF exhibited the 3.7. In vivo hemostatic performance
shortest clotting time, indicating its best clotting capability among
all the hemostats. In addition, we explored the effect of mass of sam- PACF-4 was chosen for in vivo hemostatic evaluation because
®
ples on coagulation performance. PACF-4 and HemCon , which had it simultaneously possessed good surface properties, rapid liquid
been demonstrated above having better coagulation ability, were absorption capacity, high volume expansion rate and excellent in
used in the test. With the increasing mass of samples (100 mg), vitro clotting capability. The in vivo evaluations of hemostatic per-
clotting time was significantly shortened both in the two groups. formance include laceration (liver or skin) model, vascular (artery
Especially, whole blood clotting time in the PACF-4 group decreased or vein) injury model, and so on [41,47,48]. Generally, mouse is
236 Y. Wang et al. / Applied Materials Today 13 (2018) 228–241

Fig. 5. (a) The thrombelastograms of a healthy donator’s blood after being mixed with PF and PACFs, respectively. The blood sample mixed with CaCl2 solution was used as
control. The APTT (b) and PT (c) assays of PACFs with different alginate content.

usually used as a primary animal model, followed by larger animals treatment. The PACF-4 samples were cut into particles and filled in
as a superior model. the wound to make sure them fully contacts with the bleeding site.
Therefore, we firstly chose the hemorrhaging liver mouse model The blood was absorbed rapidly by the PACFs and after 65.0 ± 9.0 s,
to evaluate the hemostatic efficiency of PACF-4. The amount of the wound stopped bleeding. Clots were observed formed on the
blood loss after applying PACF-4 or without treatment on the interface of the wound bed, suggesting that the whole hemostatic
hemorrhaging site was measured. The PACF-4 group presented progress completed. Notably, 1 week after surgery, the legs of the
excellent hemostatic performance showing almost no obvious swine which suffered from gunshot recovered normal motor func-
bloodstain on the filter paper (Fig. 7a3), while the control group tion, and no swine were died during and after the experiment.
exhibited a large area of bloodstain on the filter paper (Fig. 7a2).
The quantitative results of the amount of blood loss were in consis- 3.8. Computer tomography images and angiography analysis
tent with the macroscopical results (Fig. 7a1). PACF-4 group had a
total blood loss of 52.7 ± 5.1 mg, while the blood loss of the control As a kind of advanced detecting technology, computer tomog-
group was 399.3 ± 27.9 mg, which was much higher than PACF-4 raphy (CT) angiography was conducted to detect the blood flow
group (P < 0.05). and blood vessels of the experimental swine. Fig. 8a illustrated
Hemostatic performance of the PACF was further evaluated the morphology of the swine’s blood vessels after treating with
using a swine femoral artery gunshot injury model (Fig. 7b). As PACFs. The blood vessels were clear when contrast agents flowed
reported, this kind of injury could cause continuously bleeding through them. The red circle in the right indicated the wound site
and lead to nearly 100% mortality in the battlefield if not receive created by gunshot, while the yellow circle in the left indicated the
 Y. Wang et al. / Applied Materials Today 13 (2018) 228–241 237

Fig. 6. Whole blood clotting time measurement. (a) Blood used in this test was collected from ear vein of rabbit. (b and c) Whole blood clotting time of different samples
(from left to right: control group, PF, PACF-1, PACF-2, PACF-3, PACF-4 and PACF-5). Red rectangle indicates the clotting time of PACF-4. (d and e) Comparison of PACF and
® ®
traditional hemostatic products (HemCon , QuikClot and CELOXTM ) on clotting time. (f and g) Effect of different mass of samples on clotting time (PACF-4 versus CELOXTM ).

same site in the normal tissue. Normal blood flow and blood vessels prolonged, the small sized PACF gradually expanded through
were clearly observed in the normal tissue. However, no blood flow absorbing blood to fill the whole wound cavity and achieved shape-
was observed around the wound site, which means the PACF had adaptive with the surrounding wound tissue (Fig. 8b). Once the
already expanded to completely block the vessels at the wound site sample completely adhered to the irregular-shaped wound bound-
and gave assistant pressure on the vessels to help stop bleeding. ary, the hemostasis and wound sealing process ended.
Further, Micro-CT images were used to observe the location of Finally, when the hemostatic progress ended, the wound com-
the PACF in the wound cavity and the changes of shape of the PACF pletely stopped bleeding. The PACFs were then carefully taken out
when contacting with blood during the whole hemostasis progress. from the wound on an operating table (Fig. S3). The white dotted
The PACF samples were inserted into the wound cavity in small circles indicated plenty of blood clots formed on the surface of the
size (red dotted circle in Fig. 8b). With the hemostasis process PACF samples at the wound site (Fig. 8c).
238 Y. Wang et al. / Applied Materials Today 13 (2018) 228–241

Fig. 7. In vivo hemostatic performance of PACFs. Hemostatic experiment in (a) a hemorrhaging liver mouse model and (b) a swine artery injury model. (a1) The amount
of blood loss without treatment (a2) or after applying PACF-4 (a3) on the hemorrhaging site. (b1–b4) The whole progress of applying PACFs for the treatment of massive
bleeding caused by gunshot.

Fig. 8. Hemostasis was achieved in swine artery injury model. (a) Computer tomography (CT) angiography was used to observe the blood vessels of swine. Red circle shows
the injury site, while the yellow one shows the corresponding site in normal tissue. (b) CT images showed the tissue conformability and shape-adaptive ability of PACF. (c)
The PACF samples were taken out from the wound. Blood clots were observed on the samples. (For interpretation of the references to color in this figure legend, the reader
is referred to the web version of the article.)
 Y. Wang et al. / Applied Materials Today 13 (2018) 228–241 239

Fig. 9. (a) Schematic representation of the potential synergy effect for hemostasis. (I) Rapid and high absorption. (II) Charge stimulation. (III) Surface effect. (V) Expansion
force. (b and c) The PACFs possess high flexibility and deformable ability and the unique shape-adaptive property.

4. Discussion properties of both materials to enhance the hemostatic perfor-

mance of the composite foams.
Recently, hemostatic materials with various hemostatic mech- The PACFs could rapidly absorbed liquid (Fig. 2a). The aver-
anisms have been widely developed. Common used hemostatic age liquid absorption rate of the composite foams was more than
agents such as zeolite and medical sponge achieve hemostasis 1200%, which was favorable for rapidly absorbing plasma. Expan-
mainly by absorbing plasma to increase the concentration of blood sion experiment results indicated that the PACFs could be delivered
cells. Besides, chitosan or collagen based hemostatic products pro- into the wound cavity in the shape of small particles and undergo
mote blood clotting through charge stimulation on coagulation more than 20× volume expansion to fill large cavities, which is
factors. The three globally most widely used hemostatic products more than ten times larger than common used hemostatic materi-
®
were HemCon (HemCon Medical Technologies, Inc., Portland, OR), als on the market (Fig. 2b and c) [43]. Measurement of the expansion
®
QuikClot (Z-Medica Corporation, Wallingford, CT) and CELOXTM force exerted when the composite foams expanded demonstrated
(SAM Medical, Tualatin, OR). Although each product has proven an average expansion force of 6 N, which is sufficient to provide
to be effective in stop bleeding, they are less useful for uncontrol- durable pressure to the vessels to help stop re-bleeding and prevent
lable hemorrhage, irregular, deep or penetrating wounds caused by the dispersion of the materials under the blood flow (Fig. 2d).
minor-caliber weapons, shrapnel and explosive devices. Currently, Increasing the concentration of alginate imparted a greater
there are many available hemostatic agents, including polypeptide protein adhesion response; specifically, we observed a significant
composites, montmorillonite composite sponge, graphene sponge increase in adsorbed protein following the increasing addition of
and functional hydrogels. However, the efficacy of these hemostats alginate to PACFs (Fig. 3). We conclude that the enhanced ability
may vary significantly and has not been assessed by vigorous clini- of protein adhesion of the composite foams could contribute to
cal trials, some of them are reported to complicate tissue healing by the blood cells adhesion. Further, in the platelet and RBCs adhe-
forming a nidus for infection and abscess formation in severe hem- sion analysis, the PACFs enhanced the platelet and RBCs adhesion
orrhage. Most importantly, most of them are effective only for mild compared with PF. SEM images also demonstrated large number
to moderate bleeding. In general, an ideal hemostatic material is of activated platelets with protruding pseudopods were observed
capable of providing effective hemostasis to large vessels, requires on the surfaces of PACFs (Fig. 4). The results of the thrombelastog-
just simple preparation, is easy to apply, is stable in extreme envi- raphy (Fig. 5) further confirmed that the PACFs exhibited shorter
ronments, causes no harm to wound bed, and is low cost. However, thrombus formation time and higher clot strength with the increase
none of the materials currently used for hemostasis meet all of of alginate content. The formation of thrombus may be related to
these requirements. Therefore, deeper and more detailed research the negative charged surface and the improved platelet and RBCs
is required to improve the efficacy of the material and guarantee adhesion ability of the PACFs.
that the materials could not only stop bleeding but also provide All the experimental results affirmed that the high and fast
durable pressure on the vessels and simultaneously seal the wound. absorption capacity as well as the interfacial stimulation of the
In this study, a series of hemostatic composite foams composed PACF could work together to enhance the hemostatic efficiency.
of glutaraldehyde cross-linked PVA and CaSO4 cross-linked algi- On one hand, during the actual hemostasis process, the surface
nate were developed. We believe that incorporating alginate as a structure of the PACF surface could continuously stimulate ery-
natural component into PVA foam could combine the outstanding throcytes and platelets that have been separated with the plasma
240 Y. Wang et al. / Applied Materials Today 13 (2018) 228–241

based on the adsorption-concentration process. On the other hand, massive bleeding according to the shape and size of the wound.
the Ca-alginate microspheres which contain calcium ions and the Therefore, PACFs showed many advantages over the SMP foams and
negative charges of the PACF could also activate clotting factors XStatTM , and would be a promising hemostatic agent for controlling
thereby promoting the coagulation cascade. massive hemorrhage and treating large or irregular wound.
Therefore, we conclude that the PACF achieves rapid hemostasis
through fast and high absorption (I), charge stimulation (II), sur- 5. Conclusions
face effect (III) and expansion force (V) (Fig. 9a). (I) Fast absorption.
Rapid and high water absorption capacity of PACFs contribute to PVA/alginate composite foams with different alginate con-
absorbing plasma in the wounded area, concentrating blood cells, tent were prepared through a double crosslinking method and a
and enhancing the blood coagulation. The PACF fast absorbs plasma lyophilization technique. Our results indicated that although incor-
and enriches blood cells on its surface and then the absorbed plasma poration of alginate led to slightly decreased water absorption
could also enter the interior of the PACF and contact with internal capacity and porosity of the PACFs, it was in favor of enhancing
network of the PACF. The porous PACF can be considered as an infi- the hemostatic performance. The PACF promoted blood coagulation
nite space. More and more plasma can be absorbed efficiently into not only by quickly absorbing plasma to increase the concentra-
the PACF, thus increasing the interactions between Factor XII and tion of red blood cells and platelets on the wound surface, but also
negative charges or Ca2+ of the PACFs. (II) Charge stimulation. Factor by triggering the intrinsic pathway of blood coagulation through
XII, which is a serine protease zymogen in the plasma, could interact its surface effect and surface charges. In addition, the PACF could
with the negative charged networks of PACF. Then Factor XII was undergo up to over 20-fold volume expansion upon contact with
activated to change conformation and formed Factor XIIa. At last, fluid, and exhibited unique shape-adaptive ability that could be
the formed Factor XIIa would diffuse back into the plasma and trig- used to fill large, irregular and deep wound. The in vivo hemostatic
ger the intrinsic pathway of blood coagulation [49,50]. The result evaluation demonstrated the PACFs could rapidly stop bleeding in
of whole blood clotting test further suggested that PACF-4 which a hemorrhaging liver mouse model and a gunshot swine artery
contained more negative charges exhibited better clotting capac- injury model. This study demonstrates the potential application of
ity. The PACFs also had the ability to releasing Ca2+ ions from the PACFs in accelerating hemostasis and filling irregular wound. Fur-
network into the blood. Ca2+ ions not only contribute to the activa- thermore, this composite foam can act as a platform to introduce
tion of the intrinsic pathway but also help to produce thrombin and different hemostatic mechanisms, providing broad space for future
generate fibrin clot. Therefore, the calcium alginate microspheres development of hemostatic materials. The PACF studied here could
on the network of PACFs which contain Ca2+ ions could also con- also lead to a vastly improved technology for treating hemorrhage
tribute to the formation of thrombus and blood clotting [51]. (III) in the battlefield and civilian trauma arena.
Surface effect. The ability of the PACFs to enhance blood coagulation
can also be attributed to the surface effect. The porous surface of
Conflict of interest
PACF with high roughness could contribute to fibrinogen adsorp-
tion and blood cells adhesion, thereby promoting the formation of
The authors declare no competing financial interest.
thrombus in seconds. (V) Expansion force. The expansion force with
average 6 N enabled PACFs to rapidly expand to fill the wounds and
provide a durable pressure to the large vessels to help stop bleeding. Acknowledgements
Finally and more importantly, the PACFs possessed the unique
shape-adaptive property. The stretching, bending and twisting This work was financially supported by National Natural Science
images are performed to show the high flexibility and deformable Foundation of China (Grant No. 51773018 and 51473019) and Key
ability of PACFs. The PACFs were rather flexible after absorbing Research and Development Projects of People’s Liberation Army
liquid and it can be conformed to any three-dimensional or curvi- (BWS17J036).
linear surface (Fig. 9b). Therefore, incorporating alginate into PVA
substantially improves the stretchability, refines the deformable Appendix A. Supplementary data
ability, and extends the scalability of PACFs thereby endowing
PACFs the shape-adaptive ability to accommodate arbitrary sur- Supplementary data associated with this article can be found, in
faces. In addition, the ability of PACFs to deform larger than 20× the online version, at doi:10.1016/[Link].2018.09.009.
volume during expansion would enable insertion of a small sam-
ple into the irregular and deep wound that could rapidly expand References
through contacting blood until the sample entirely adheres to the
irregular-shaped wound boundary (Fig. 9c). [1] L.W. Chan, X. Wang, H. Wei, L.D. Pozzo, N.J. White, S.H. Pun, A synthetic fibrin
cross-linking polymer for modulating clot properties and inducing
Through investigation we found that two composite materials
hemostasis, Sci. Transl. Med. 7 (277) (2015) 277ra29.
has the similar expansion properties and hemostatic mechanism [2] R. Rossaint, B. Bouillon, V. Cerny, T.J. Coats, J. Duranteau, E.
with PACFs. One is the recently reported composite foams com- Fernández-Mondéjar, D. Filipescu, B.J. Hunt, R. Komadina, G. Nardi, The
European guideline on management of major bleeding and coagulopathy
posed of shape memory polymer (SMP) [34], and the other is
following trauma, Crit. Care 20 (1) (2016) 100.
the previously developed and marketed XStatTM (RevMedx, Inc., [3] I. Abubakar, T. Tillmann, A. Banerjee, Global, regional, and national age-sex
Wilsonville, OR) [43]. They both possess high volume expansion specific all-cause and cause-specific mortality for 240 causes of death,
rate and good water absorption capability and can rapidly expand 1990–2013: a systematic analysis for the Global Burden of Disease Study
2013, Lancet 385 (9963) (2015) 117–171.
to fill deep and non-compressible wounds [52]. Compared with [4] C.J. Murray, A.D. Lopez, Alternative projections of mortality and disability by
SMP foams and XStatTM , PACFs with higher volume expansion cause 1990–2020: global burden of disease study, Lancet 349 (9064) (1997)
rate could generate higher expansion force and better deforma- 1498–1504.
[5] H.B. Alam, E. Koustova, P. Rhee, Combat casualty care research: from bench to
tion performance, thereby helping rapidly stop large-vessel arterial the battlefield, World J. Surg. 29 (1) (2005) S7–S11.
bleeding. Besides, alginate and calcium ions in PACF could help to [6] H.B. Alam, D. Burris, J.A. DaCorta, P. Rhee, Hemorrhage control in the
promote blood clotting, which can accelerate wound hemostasis. battlefield: role of new hemostatic agents, Milit. Med. 170 (1) (2005) 63–69.
[7] X. Wang, J. Chang, C. Wu, Bioactive inorganic/organic nanocomposites for
Additionally, PACFs is convenient to use for both the wounded indi- wound healing, Appl. Mater. Today 11 (2018) 308–319.
vidual and the one giving aid. More importantly, PACF has different [8] B.D. Ippel, P.Y. Dankers, Introduction of nature’s complexity in engineered
shapes (Fig. S1). A suitable shape of PACF could be selected to treat blood-compatible biomaterials, Adv. Healthc. Mater. 7 (1) (2018) 1700505.
 Y. Wang et al. / Applied Materials Today 13 (2018) 228–241 241

[9] B.C. Streifel, J.G. Lundin, A.M. Sanders, K.A. Gold, T.S. Wilems, S.J. Williams, E. [32] K. Quan, G. Li, L. Tao, Q. Xie, Q. Yuan, X. Wang, Diaminopropionic acid
Cosgriff-Hernandez, J.H. Wynne, Hemostatic and absorbent polyHIPE–kaolin reinforced graphene sponge and its use for hemostasis, ACS Appl. Mater.
composites for 3D printable wound dressing materials, Macromol. Biosci. 18 Interfaces 8 (12) (2016) 7666–7673.
(5) (2018) 1700414. [33] S. Pourshahrestani, E. Zeimaran, N.A. Kadri, N. Gargiulo, H.M. Jindal, S.V.
[10] S. Laurence, R. Bareille, C. Baquey, J. Fricain, Development of a resorbable Naveen, S.D. Sekaran, T. Kamarul, M.R. Towler, Potency and cytotoxicity of a
macroporous cellulosic material used as hemostatic in an osseous novel gallium-containing mesoporous bioactive glass/chitosan composite
environment, J. Biomed. Mater. Res. A 73 (4) (2005) 422–429. scaffold as hemostatic agents, ACS Appl. Mater. Interfaces 9 (37) (2017)
[11] J. Wen, M. Weinhart, B. Lai, J. Kizhakkedathu, D.E. Brooks, Reversible 31381–31392.
hemostatic properties of sulfabetaine/quaternary ammonium modified [34] T. Landsman, T. Touchet, S. Hasan, C. Smith, B. Russell, J. Rivera, D. Maitland, E.
hyperbranched polyglycerol, Biomaterials 86 (2016) 42–55. Cosgriff-Hernandez, A shape memory foam composite with enhanced fluid
[12] D. Johnson, S. Agee, A. Reed, B. Gegel, J. Burgert, J. Gasko, M. Loughren, The uptake and bactericidal properties as a hemostatic agent, Acta Biomater. 47
effects of QuikClot combat gauze on hemorrhage control in the presence of (2017) 91–99.
hemodilution, U.S. Army Med. Dept. J. 3 (2) (2014) 21–25. [35] C.A. Murphy, J.B. Costa, J. Silva-Correia, J.M. Oliveira, R.L. Reis, M.N. Collins,
[13] A. Sairaku, Y. Nakano, N. Oda, Y. Makita, K. Kajihara, T. Tokuyama, Y. Kihara, Biopolymers and polymers in the search of alternative treatments for
Rapid hemostasis at the femoral venous access site using a novel hemostatic meniscal regeneration: state of the art and future trends, Appl. Mater. Today
pad containing kaolin after atrial fibrillation ablation, J. Interven. Cardiac 12 (2018) 51–71.
Electrophysiol. 31 (2) (2011) 157–164. [36] Y. Wang, Y. Zheng, W. He, C. Wang, Y. Sun, K. Qiao, X. Wang, L. Gao, Reprint of:
[14] S. Pourshahrestani, E. Zeimaran, I. Djordjevic, N.A. Kadri, M.R. Towler, preparation of a novel sodium alginate/polyvinyl formal composite with a
Inorganic hemostats: the state-of-the-art and recent advances, Mater. Sci. double crosslinking interpenetrating network for multifunctional biomedical
Eng. C 58 (2016) 1255–1268. application, Compos. B: Eng. 121 (2017) 9–22.
[15] B. Kheirabadi, Evaluation of topical hemostatic agents for combat wound [37] S. Pourshahrestani, E. Zeimaran, N.A. Kadri, N. Gargiulo, S. Samuel, S.V.
treatment, U.S. Army Med. Dept. J. (2011) 25–37. Naveen, T. Kamarul, M.R. Towler, Gallium-containing mesoporous bioactive
[16] H.E. Achneck, B. Sileshi, R.M. Jamiolkowski, D.M. Albala, M.L. Shapiro, J.H. glass with potent hemostatic activity and antibacterial efficacy, J. Mater.
Lawson, A comprehensive review of topical hemostatic agents: efficacy and Chem. B 4 (1) (2016) 71–86.
recommendations for use, Ann. Surg. 251 (2) (2010) 217–228. [38] G. Lan, B. Lu, T. Wang, L. Wang, J. Chen, K. Yu, J. Liu, F. Dai, D. Wu,
[17] B.L. Bennett, L.F. Littlejohn, B.S. Kheirabadi, F.K. Butler, R.S. Kotwal, M.A. Chitosan/gelatin composite sponge is an absorbable surgical hemostatic
Dubick, J.A. Bailey, Management of External Hemorrhage in Tactical Combat agent, Colloids Surf. B: Biointerfaces 136 (2015) 1026–1034.
Casualty Care: Chitosan-Based Hemostatic Gauze Dressings. TCCC Guidelines [39] J.-j. Rong, M. Liang, F.-q. Xuan, J.-y. Sun, L.-j. Zhao, H.-z. Zhen, X.-x. Tian, D. Liu,
Change 13-05, Army Inst of Surgical Research, Fort Sam Houston, TX, 2014, Q.-y. Zhang, C.-f. Peng, Alginate-calcium microsphere loaded with thrombin:
pp. 40–57. a new composite biomaterial for hemostatic embolization, Int. J. Biol.
[18] B.G. Kozen, S.J. Kircher, J. Henao, F.S. Godinez, A.S. Johnson, An alternative Macromol. 75 (2015) 479–488.
hemostatic dressing: comparison of CELOX, HemCon, and QuikClot, Acad. [40] E. Lih, J.S. Lee, K.M. Park, K.D. Park, Rapidly curable chitosan–PEG hydrogels as
Emerg. Med. 15 (1) (2008) 74–81. tissue adhesives for hemostasis and wound healing, Acta biomater. 8 (9)
[19] L.F. Littlejohn, J.J. Devlin, S.S. Kircher, R. Lueken, M.R. Melia, A.S. Johnson, (2012) 3261–3269.
Comparison of CeloxTM , ChitoFlex, WoundStat, and combat gauze hemostatic [41] X. Zhao, H. Wu, B. Guo, R. Dong, Y. Qiu, P.X. Ma, Antibacterial anti-oxidant
agents versus standard gauze dressing in control of hemorrhage in a swine electroactive injectable hydrogel as self-healing wound dressing with
model of penetrating trauma, Acad. Emerg. Med. 18 (4) (2011) 340–350. hemostasis and adhesiveness for cutaneous wound healing, Biomaterials 122
[20] J.J. Devlin, S. Kircher, B.G. Kozen, L.F. Littlejohn, A.S. Johnson, Comparison of (2017) 34–47.
® ®
ChitoFlex , CELOXTM , and QuikClot in control of hemorrhage, J. Emerg. Med. [42] B.S. Kheirabadi, J.E. Mace, I.B. Terrazas, C.G. Fedyk, J.S. Estep, M.A. Dubick, L.H.
41 (3) (2011) 237–245. Blackbourne, Safety evaluation of new hemostatic agents, smectite granules,
[21] Y. Vueva, L.S. Connell, S. Chayanun, D. Wang, D.S. McPhail, F. Romer, J.V. and kaolin-coated gauze in a vascular injury wound model in swine, J. Trauma
Hanna, J.R. Jones, Silica/alginate hybrid biomaterials and assessment of their Acute Care Surg. 68 (2) (2010) 269–278.
covalent coupling, Appl. Mater. Today 11 (2018) 1–12. [43] G.R. Mueller, T.J. Pineda, H.X. Xie, J.S. Teach, A.D. Barofsky, J.R. Schmid, K.W.
[22] M. Hrynyk, M. Martins-Green, A.E. Barron, R.J. Neufeld, Alginate-PEG sponge Gregory, A novel sponge-based wound stasis dressing to treat lethal
architecture and role in the design of insulin release dressings, noncompressible hemorrhage, J. Trauma Acute Care Surg. 73 (2) (2012)
Biomacromolecules 13 (5) (2012) 1478–1485. S134–S139.
[23] Q. Yu, Y. Zheng, N. Yan, Y. Xie, K. Qiao, R. Jin, The gelation process and protein [44] A.M. Behrens, M.J. Sikorski, T. Li, Z.J. Wu, B.P. Griffith, P. Kofinas,
absorption property of injectable SA-CMBC hydrogel used for procoagulant Blood-aggregating hydrogel particles for use as a hemostatic agent, Acta
material, RSC Adv. 5 (129) (2015) 106953–106958. Biomater. 10 (2) (2014) 701–708.
[24] F. Cheng, C. Liu, X. Wei, T. Yan, H. Li, J. He, Y. Huang, Preparation and [45] B.S. Kheirabadi, J.W. Edens, I.B. Terrazas, J.S. Estep, H.G. Klemcke, M.A. Dubick,
characterization of 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO)-oxidized J.B. Holcomb, Comparison of new hemostatic granules/powders with
cellulose nanocrystal/alginate biodegradable composite dressing for currently deployed hemostatic products in a lethal model of extremity arterial
hemostasis applications, ACS Sustain. Chem. Eng. 5 (5) (2017) 3819–3828. hemorrhage in swine, J. Trauma Acute Care Surg. 66 (2) (2009) 316–328.
[25] C.H. Goh, P.W.S. Heng, L.W. Chan, Alginates as a useful natural polymer for [46] N. Sambu, A. Radhakrishnan, H. Dent, A.L. Calver, S. Corbett, H. Gray, I.A.
microencapsulation and therapeutic applications, Carbohydr. Polym. 88 (1) Simpson, N. Curzen, Personalised antiplatelet therapy in stent thrombosis:
(2012) 1–12. observations from the Clopidogrel Resistance in Stent Thrombosis (CREST)
[26] A. Thomas, K. Harding, K. Moore, Alginates from wound dressings activate registry, Heart 98 (9) (2012) 706–711.
human macrophages to secrete tumour necrosis factor-␣, Biomaterials 21 [47] Y. Bu, L. Zhang, J. Liu, L. Zhang, T. Li, H. Shen, X. Wang, F. Yang, P. Tang, D. Wu,
(17) (2000) 1797–1802. Synthesis and properties of hemostatic and bacteria-responsive in situ
[27] J. Jin, Z. Ji, M. Xu, C. Liu, X. Ye, W. Zhang, S. Li, D. Wang, W. Zhang, J. Chen, hydrogels for emergency treatment in critical situations, ACS Appl. Mater.
Microspheres of carboxymethyl chitosan, sodium alginate and collagen as a Interfaces 8 (20) (2016) 12674–12683.
hemostatic agent in vivo, ACS Biomater. Sci. Eng. 4 (7) (2018) 2541–2551. [48] Z. Karahaliloğlu, M. Demirbilek, İ. Ulusoy, B. Gümüşkaya, E. Baki Denkbaş,
[28] G. Xu, L. Cheng, Q. Zhang, Y. Sun, C. Chen, H. Xu, Y. Chai, M. Lang, In situ Hemostatic activities of nano/microporous bilayer dressings in a femoral
thiolated alginate hydrogel: instant formation and its application in artery bleeding rat model, J. Appl. Polym. Sci. 133 (28) (2016) 43657.
hemostasis, J. Biomater. Appl. 31 (5) (2016) 721–729. [49] A.L. Barrientos-Velázquez, S. Arteaga, J.B. Dixon, Y. Deng, The effects of pH,
[29] C. Wang, W. Luo, P. Li, S. Li, Z. Yang, Z. Hu, Y. Liu, N. Ao, Preparation and pepsin, exchange cation, and vitamins on aflatoxin adsorption on smectite in
evaluation of chitosan/alginate porous microspheres/Bletilla striata simulated gastric fluids, Appl. Clay Sci. 120 (2016) 17–23.
polysaccharide composite hemostatic sponges, Carbohydr. Polym. 174 (2017) [50] A.M. Behrens, M.J. Sikorski, P. Kofinas, Hemostatic strategies for traumatic and
432–442. surgical bleeding, J. Biomed. Mater. Res. A 102 (11) (2014) 4182–4194.
[30] M.B. Dowling, A. Chaturvedi, I.C. MacIntire, V. Javvaji, J. Gustin, S.R. Raghavan, [51] C. Dai, Y. Yuan, C. Liu, J. Wei, H. Hong, X. Li, X. Pan, Degradable, antibacterial
T.M. Scalea, M. Narayan, Determination of efficacy of a novel alginate dressing silver exchanged mesoporous silica spheres for hemorrhage control,
in a lethal arterial injury model in swine, Injury 47 (10) (2016) 2105–2109. Biomaterials 30 (29) (2009) 5364–5375.
[31] G. Li, K. Quan, Y. Liang, T. Li, Q. Yuan, L. Tao, Q. Xie, X. Wang, [52] J.F. Kragh, J.K. Aden, J. Steinbaugh, M. Bullard, M.A. Dubick, Gauze vs XSTAT in
Graphene–montmorillonite composite sponge for safe and effective wound packing for hemorrhage control, Am. J. Emerg. Med. 33 (7) (2015)
hemostasis, ACS Appl. Mater. Interfaces 8 (51) (2016) 35071–35080. 974–976.