Research Article

pubs.acs.org/journal/ascecg

Preparation and Characterization of 2,2,6,6-Tetramethylpiperidine-1-

oxyl (TEMPO)-Oxidized Cellulose Nanocrystal/Alginate
Biodegradable Composite Dressing for Hemostasis Applications
Feng Cheng,† Changyu Liu,† Xinjing Wei,† Tingsheng Yan,† Hongbin Li,‡ Jinmei He,*,†
and Yudong Huang†
†
MIIT Key Laboratory of Critical Materials Technology for New Energy Conversion and Storage, School of Chemistry and Chemical
Engineering, Harbin Institute of Technology, Harbin 150001, People’s Republic of China
‡
School of Light Industry and Textile, Qiqihar University, Qiqihar 161000, People’s Republic of China
See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

*
S Supporting Information

ABSTRACT: Hemorrhage is common in surgery, and excessive bleeding is the main

Downloaded via NEW YORK UNIV on January 3, 2024 at 17:50:40 (UTC).

reason for trauma death. Effective control of bleeding is becoming more and more
important in military and civilian trauma. In this work, oxidized cellulose nanocrystal/
alginate composite films and sponges were successfully prepared and their usages as the
hemostatic materials were investigated. Carboxyl functionalization on the cellulose
nanocrystal surface not only played a fundamental role in the structural of composites,
but also contributed to absorb plasma and stimulate erythrocytes and platelets. Fourier
transform infrared (FTIR) and X-ray photoelectron spectroscopy (XPS) spectra
showed that the carboxyl groups were successfully introduced on the cellulose
nanocrystal surface by TEMPO-mediated oxidization. The oxidized cellulose nano-
crystals (TOCN)/alginate (SA) composites were in the presence of Ca2+ solution
cross-linking. Physical properties tests results indicated that the ultrahigh porosity
(sponge), surface homogeneity (film), water absorption ability, and chemical stability of
TOCN-30/SA composite sponge, as well as TOCN-30/SA composite film, were all
increased after ionic cross-linking, compared to the SA sponge and film, respectively. In vitro evaluation of the hemostatic effect,
hemostatic time, and the blood loss in two injury models exhibited that TOCN-30/SA composite sponge had the most excellent
hemostatic efficiency and could be biodegraded completely without inflammatory reaction after three weeks. In addition, the
potential hemostatic mechanism of TOCN/SA composites was discussed.
KEYWORDS: Cellulose nanocrystals, Oxidation, Alginate, Cross-linked sponge, Cross-linked film, Hemostatic, Biodegradability

■ INTRODUCTION
Excessive hemorrhage is the main cause of prehospital trauma
death during both military and civilian trauma, and effective
hemostatic materials can quickly prevent bleeding and thus
reduce the mortality.1,2 In recent years, numerous materials
have been widely developed to promote rapid bleeding
control,3 such as oxidized regenerated cellulose (ORC,
Surgicel), 4 HemCon chitosan-based dressing,5 and the
QuikClot zeolite powder.6 All of them have their own
advantages and limitations. For instance, the ORC materials,
which is implanted into the patient’s body, will damage the
nervous system when the carboxyl content is within the range
of 16%−24% and pH value is ∼3.1.7 Also, HemCon dressings
prepared as gauzes are difficult to conform to deep, narrow
wounds or irregularly shaped wounds.2 In addition, the study of
QuikClot agents indicated that the exothermic reaction and
poor biodegradability of QuikClot can even cause tissue injuries
and abnormal reaction to foreign bodies.1 Thus, the challenge
now is developing more alternative effective hemostatic
materials to control hemorrhage.
As a type of natural anionic polymer, alginate obtained from
brown seaweed has been widely studied and used in a variety of
biomedical applications,8 such as wound dressing9 and stable
gels,10 because of its good biocompatibility and low cytoxicity.
Especially, the calcium-induced gels with “egg-box” structure
have been proved to be formed under the cation interaction
between Ca2+ and guluronate blocks in alginate.11 Alginate
dressings with excellent hemostasis efficiency can absorb large
volumes of wound exudate and provide a physiologically moist
microenvironment for wound healing. Besides that, it can be
easily removed from the wound site.12,13 However, the poor
chemical stability, weak mechanical strength, and the uncontrol-
lable structure degradation of neat alginate film or sponge
limited their further application of neat alginate film or
sponge.14,15
Cellulose consisted of β-1−4-linked D-anhydroglucose units
as the most abundant renewable biopolymer composed and
Received: November 24, 2016
Revised: March 20, 2017
Published: March 24, 2017

© 2017 American Chemical Society 3819 DOI: 10.1021/acssuschemeng.6b02849

ACS Sustainable Chem. Eng. 2017, 5, 3819−3828
ACS Sustainable Chemistry & Engineering
almost inexhaustible raw material has been used in a wide
variety of applications,16,17 such as food, paper, and medicine.
Especially, cellulose nanocrystal (CN) with nanoscale features,
high specific surface area, unique morphology, low density,
mechanical strength, renewability, and biodegradability,18−21
has attracted a great deal of interest during the past decade.
Furthermore, abundant active hydroxyl groups on the CN
surface are suitable for chemical modification, such as oxidation
and polymer grafting.22 For instance, a recent study reported
that the presence of 2,2,6,6-tetramethylpiperidine-1-oxyl
(TEMPO)-oxidized cellulose nanofibers can regulate the
post-prandial blood metabolic variables and showed promising
hemocompatibility and unique biological activities.23 TEMPO-
mediated oxidized bacterial cellulose-sodium alginate compo-
sites have also been prepared as a type of biomedical material
for cell encapsulation, in which the TEMPO-mediated oxidized
bacterial cellulose improved the mechanical stability, could
exhibit good chemical stability of the composites, and would be
a potential candidate for many biomedical applications.24
For the above-mentioned reasons, a type of cellulose
nanocrystal/alginate composites was designed based on the
cross-linking by external gelation in CaCl2 ethanol/water
solution as a co-solvent. In addition, as the C6 primary
hydroxyls made from TEMPO-oxidized materials converted to
carboxyl groups on the cellulose surface, oxidized cellulose
nanocrystal (TOCN) provided the possibility of participating in
the construction of the cross-linking network from alginate-
based composites and plays an important role in the structural,
mechanical, and chemical stability of the composites.25 So far,
there was no specific report referred to the hemostatic
properties and biological degradable performance of TEMPO-
mediated oxidized cellulose nanocrystal (TOCN)/alginate
composites. In this study, the morphology, chemical and
physical properties, hemostatic efficiency of TOCN/SA
composites both in vitro and in vivo, and the degradation in
vivo were studied. It is expected that this study is useful for
understanding the possible hemostatic mechanism of TOCN/
SA composites and designing an effective hemostatic material
for the wound healing.
■ MATERIALS
Microcrystalline cellulose (99%) was purchased from Shanghai Luan
Biological Technology Co., Ltd. (China). Sodium alginate was
provided by Sinopharm Chemical Reagent Co., Ltd. (China). Sodium
hypochlorite (NaClO) solution was purchased from Shuang Shuang
Chemical Co., Ltd. (Yantai, China). TEMPO (C9H18ON, 98%),
sodium bromide (NaBr), and calcium chloride (CaCl2) were
purchased from Sinopharm Chemical Reagent Co., Ltd. (China). All
of the reagents were of analytical grade and used without further
purification. Healthy rabbits and human blood were supplied by
animal experiment center of the second affiliated hospital of Harbin
medical university (Harbin, Heilongjiang Province, China). The
protocol was approved by the ethics committee of the Harbin Medical
University. All animals were handled according to the Chinese
National Institutes of Health Guidelines for the Care and Use of
Laboratory Animals.
Preparation of Cellulose Nanocrystals (CNs). Procedure for the
preparation of CN was the same as those described previously.25−28
Briefly, acid hydrolysis was prepared at 35 °C with 64 wt % H2SO4
(225 mL) for 2 h under vigorous stirring and then MCC powder (10
g) was slowly added into the suspension. The hydrolysis was
terminated by adding a large amount of distilled water (more than
10 times the volume of the H2SO4 solution used). Subsequently, the
mixture was placed overnight at 4 °C and the supernatant was
discarded. After that, the system was centrifuged (10 000 rpm) for 10
3820
Research Article
min each step (washed repeated more than three times), and then
dialyzed with distilled water for ∼3−5 days until a neutral pH
environment, followed by ultrasonic treatment. Finally, the CN power
was obtained after freeze drying.
TEMPO-Mediated Oxidation of Cellulose Nanocrystals
(TOCN). TEMPO-mediated oxidation of CN was followed by using
the method described in the literature.25,29−31 Briefly, ∼0.5 g CN was
suspended in 50 mL of distilled water, followed by ultrasonic
dispersion treatment for 15 min. TEMPO (50 mg, 0.32 mmol) and
NaBr (500 mg, 4.86 mmol) were also dissolved in 50 mL of distilled
water and the solution were added dropwise slowly into the CN
dispersion. A certain quantity of 12 wt % NaClO (15 mL, 46.5 M)
solution was then added slowly into the mixed solution for the
oxidizing reaction; meanwhile, the pH value of the mixture remained
at 10.8 by adding 0.5 M NaOH. The oxidation reaction was terminated
by adding ethanol (1 mL). Meanwhile, the pH was adjusted to 7 with
0.5 M HCl. Finally, the aqueous dispersion of oxidized CN (TOCN)
was washed thoroughly with distilled water more than three times and
then freeze-dried to obtain TOCN powders.
Preparation of Cellulose Nanocrystal/Alginate Cross-Linked
Composite Sponges (Films). TOCN was introduced in a certain
amount of alginate solution for the preparation of cross-linked sponges
and films. The detailed procedure was illustrated in Figure 1. TOCN
Figure 1. Scheme of preparation and application for TOCN/SA
composite sponge (film).
solution (3 wt %) was prepared by adding the TOCN powder in
distilled water under vigorous stirring for 30 min under room
temperature. Similarly, sodium alginate (SA) was dissolved in distilled
water and stirred for 2 h to get a 3 wt % SA solution. Then, TOCN
solution was dropwise added into the prepared SA solution, followed
by vigorous stirring for another 3 h. The TOCN/SA composite
suspension was then cast in Petri dish plates, and a portion of them
was freeze-dried and another portion was vacuum-dried. Cross-linked
TOCN/SA composite sponges (films) were prepared by immersion in
a CaCl2/H2O/C2H5OH (1.5 g/80 mL/20 mL) solution for 1 h.32 In
addition, the TOCN/SA composites were washed with distilled water
to remove residual Ca2+ ions and freeze-dried again to obtain the
cross-linked sponges (dried cross-linked film at 25 °C). The obtained
composite sponges and films were stored in a desiccator for more than
48 h before their use. The weight ratios and contents of TOCN/SA
cross-linked samples are shown in Table S1 in the Supporting
Information.
Characterization. Fourier Transform Infrared (FT-IR) Analysis.
Fourier transform infrared (FT-IR) spectra were used to confirm the
characteristics of CN powders and TOCN/SA samples. FT-IR was
DOI: 10.1021/acssuschemeng.6b02849
ACS Sustainable Chem. Eng. 2017, 5, 3819−3828
ACS Sustainable Chemistry & Engineering
measured using the Nicolet−Nexus 670 spectrometer at room
temperature. All the powder samples were recorded at the range of
4000−500 cm−1, with a resolution of 2 cm−1.
X-ray Photoelectron Spectroscopy (XPS) Analysis. The elemental
composition of unmodified and oxidized CNs surface was obtained
from the X-ray photoelectron spectroscopy (XPS) under the ultrahigh
vacuum conditions. The device was equipped with a VG Scientific
ESCALAB system (220i-XLT, UK) and an X-ray source (Al Kα).
X-ray Diffraction Analysis. Crystallinity of unmodified and oxidized
cellulose nanocrystal was measured by X-ray diffraction (XRD)
method using an XRD-6000X diffractometer with Cu Kα X-radiation.
The XRD patterns of the samples were recorded in the 2θ range of
0°−40°.
Morphology Analysis. Scanning electron microscopy (SEM) and
energy-dispersive spectroscopy (EDS) analysis on the TOCN/SA
sponges were carried out using Quanta 200 FEG scanning electron
microscope (FEI, Hong Kong) equipped with an EDS detector. The
morphologies of CN and TOCN were examined via transmission
electron microscopy (TEM) analysis (Model JEM-F 200, Japan) at an
accelerating voltage of 80 kV.
Swelling Degree of TOCN/SA Composite Sponges (Films). The
degree of swelling of the sponges and films was measured
gravimetrically. The sponge and film sample was cut into dimensions
of 3.0 cm × 3.0 cm and immersed in the phosphate buffered saline
(PBS) (pH 7.2−7.4, 37 °C) for 30 min. Then, the residual PBS was
removed from the wet sample surface with filter paper and the
obtained sample was immediately weighted (Wd). The swelling degree
(Sd) was calculated using the following equation:33
Ww − Wd
Sd (%) = × 100
Wd (1)
where Ww and Wd represents the wet and dry weight of the sample,
respectively. An average value of five replicates for each sponge sample
was determined.
Porosity of TOCN/SA Composite Sponge. The porosity of the
sponge sample was determined by the reported method.33 Briefly, the
sponge samples were immersed in a certain amount of ethanol for 30
min. Subsequently, the sponge samples were weighed before and after
immersion in alcohol. The porosity (P) was calculated with the
following equation:
W2 − W1
P (%) = × 100
ρV0 (2)
where W1 and W2 represent the weight of sponge before and after
immersion in alcohol, respectively. V0 is the sponge volume and ρ is
the density of ethanol (0.785 g/cm3). Five samples were measured for
each type of TOCN/SA composite.
Measurement of Mechanical Properties. The materials were cut
into pieces with dimensions of 25 mm × 10 mm. The tensile strength
was examined at a speed of 2 mm/min and recorded with an electronic
universal testing machine (CMT, No. 03000227, New Think Materials
Testing Co., Ltd., Shenzhen, China). At least 10 replicates were run for
each sample.
Hemolysis Assay In Vitro. The hemolytic study was performed on
composite sponges and films extract in vitro. Sterile normal saline (NS)
(20 mL) was added into the test tube which contained 25 mg sponges
and films samples with incubation at 37 °C for 72 h. Hemolytic test
was determined by rabbit erythrocyte suspension in vitro. In brief, 2.5
mL of sample extract solution, TOCN (1 mg/mL), TOCN (4 mg/
mL), SA (4 mg/mL) and SA (6 mg/mL) solutions were added into
2.5 mL of 2% (v/v) rabbit erythrocyte (diluted with NS) suspension
incubated at 37 °C for 3h, respectively. Meanwhile, distilled water and
NS was used as the positive and negative control, respectively.
Whereafter, the mixture erythrocyte suspensions were separated at
1500 rpm for 10 min and determined the hemolytic rate with the UV/
vis spectrophotometer at 540 nm. The hemolytic rate (H) was
calculated with the following equation:33
3821
Research Article
Dt − Dnc
H (%) = × 100
Dpc − Dnc (3)
where Dt is the absorbance of test sample, and Dnc and Dpc are the
absorbance of negative and positive control, respectively. An average
value of three replicates for each test sample was determined.
Cytocompatibility Assay. The sterilized materials were cut into
several 1.0 mm × 1.0 mm × 1.0 mm cubes, and immersed in PBS
buffer solution (5, 2.5, 0.5, and 0.1 mg mL−1) for 72 h in an air-tight
glass container at 37 °C. After draining the fluid, the supernatant was
sterilized for 30 min under 253.7 nm UV light. The group without any
material was set as a control. Single-cell suspensions of 5000 Hela cells
in 100 μL DMEM medium containing 10% (v/v) fetal bovine serum
(FBS) and 1% (v/v) penicillin−streptomycin (Invitrogen), were
added in a 96-well plate and incubated for 48 h at 37 °C under 5%
CO2. Then, 10 μL material extraction supernatant was added and
incubated for another 48 h and the cell-seeded wells were washed
twice with PBS to remove unattached cells. Then, 100 μL of DMEM
was added and the cells were cultured with a cell counting kit-8 (CCK-
8) for 2 h at 37 °C. Finally, the 96-well plates were placed in a
microplate reader to measure absorbance at 450 nm. The relative
growth rate (RGR) of cells cultured in each group was measured to
calculate the cell viability, according to the following formula:
Abs450 test
RGR (%) = × 100
Abs450 control (4)
All results were estimated from six individual experiments and are
expressed as the mean ± the standard deviation (SD).
Blood Cells and Platelet Adhesion. The blood cells and platelet
adhesion were conducted as described in the reported literature.34,35 In
brief, for whole blood cell and platelet adhesion determination, the
TOCN/SA composite sample was cut into 1 cm × 1 cm dimensions,
followed by immersion into PBS (pH = 7.2−7.4) for 1 h at 37 °C.
Subsequently, the whole blood was added dropwise into the sample
and then incubated for 5 min at 37 °C. Platelet-rich plasma (PRP) was
separated from the whole blood by centrifugation of blood at 800 rpm
for 10 min. The PRP was then added dropwise into the sample and
incubated for 1 h at 37 °C. All samples were then washed with PBS
solution three times to remove the physical adhered blood cells and
platelets, and then fixed by 2.5% glutaraldehyde for another 2 h. After
that, blood cells and platelets were dehydrated with 50%, 60%, 70%,
80%, 90%, and 100% ethanol solution, with the interval for 10 min.
Finally, the TOCN/SA samples were obtained after drying and SEM
images were taken.
Hemostatic Evaluation. Rabbit Liver Trauma Model. The
hemostatic behavior of the TOCN/SA composite sponges and films
was estimated by covering them on the abraded livers of male New
Zealand White rabbits (4 months old and ∼3.5 kg). The composite
samples were cut into pieces of required size (2.0 cm × 2.0 cm) and
sterilized by the ultraviolet radiation for evaluating the hemostatic
efficiency. Before undergoing an abdominal incision, the rabbits were
fixed on the surgical cork board and then the ear marginal veins were
injected with 3% pentobarbital sodium aqueous solution (30 mg/kg)
to anaesthetize them. The composite samples were applied to the liver
wound immediately when the liver was pricked with a needle (the
diameter is 2 mm, and the pricked depth is 3 mm), respectively. The
hemostatic evaluation was detected every 30 s, then the hemostatic
time and blood loss was recorded accordingly.
Rabbit Ear Artery Model. After the anesthesia of ear marginal veins
through the injection of a pentobarbital sodium solution, the auricular
artery of the rabbit in the middle of rabbit ear was prepared and
sterilized, and the blood vessels were torn by the scalpel blade. The
composite samples then were covered on the wound, and the
hemostatic time and blood loss were recorded accordingly.
In Vivo Degradation Behavior. The male New Zealand White
rabbits (4 months old and ∼3.5 kg) were selected for the degradation
test in vivo. Rabbits were randomly divided into a treatment group and
a control group. All composite samples were cut into pieces of
required size (1.0 cm × 1.0 cm) and sterilized by the ultraviolet
radiation for testing the degradation behavior. After the anesthesia of
DOI: 10.1021/acssuschemeng.6b02849
ACS Sustainable Chem. Eng. 2017, 5, 3819−3828
ACS Sustainable Chemistry & Engineering
ear marginal veins through the injection of sodium pentobarbital, part
of the operation area was sterilized, and then the sample was
implanted across the ham muscle. Three rabbits from each group were
examined at 7 d, 14 d, and 21 d, respectively. All the animals were
carefully nurtured until the terminals were implanted. Finally, the
implants and surrounding tissues were carefully removed, fixed in 10%
formaldehyde solution, embedded in paraffin, and then sectioned and
stained with hematoxylin and eosin (H&E).
Statistical Analysis. For graphs and texts, values were expressed as
mean ± standard deviation (SD). For the comparison of two groups, a
Student’s t-test was used to measure the statistical significance. The
probability values of p < 0.05 (using one-way analysis of variance
(ANOVA) on SPSS) were considered to be statistically significant.
■
RESULTS AND DISCUSSION
TEMPO-Mediated Oxidation of Cellulose. The FT-IR
spectra of the unmodified MCC, CN, TEMPO-treated TOCN-
COONa, and acidic form TOCN-COOH are presented in
Figure 2a. The peak at 3410 cm−1 was attributed to the
Figure 2. (a) FTIR spectra for unmodified microcrystalline cellulose
(MCC), cellulose nanocrystals (CNs), oxidized cellulose nanocrystals
(TOCN-COONa), and acidic form of oxidized cellulose nanocrystals
(TOCN−COOH). (b) XPS wide spectra of unmodified cellulose
nanocrystals (CN), and oxidized cellulose nanocrystals (TOCN).
hydrogen bond stretching vibration of −OH groups, and the
absorption at 1640 cm−1 was ascribed to the bending vibration
of absorbed H2O. In addition, the peak at 2900 cm−1 was
attributed to the stretching vibration of C−H. Compared to the
MCC, no new peaks appeared in the FT-IR spectrum of CN,
except a slight stretching vibration shoulder peak of C−H at
2900 cm−1. After TEMPO oxidization on the surface hydroxyl
groups of CN, some apparent changes on the spectrum of the
modified cellulose nanocrystal can be found. The new peak
attributed to the stretching band of the sodium carboxylate
groups (−COONa) can be observed at 1612 cm−1, which is the
most important change of the carboxyl groups (CO)
3822
Research Article
appeared. Moreover, as shown in the spectrum of TOCN-
COOH, the peak located at 1735 cm−1 was the characteristic
peak of free carboxyl groups (acidic form-COOH), indicating
that the carboxylate-COONa groups in TOCN-COONa were
successfully converted to free carboxyl groups (acid-COOH)
via HCl treatment on oxidized CN. Therefore, the acid
treatment was helpful for eliminating the interference with the
absorbed water band (1640 cm−1).
The XPS wide scan spectra are shown in Figure 2b; these
spectra further demonstrate that the compositions on the
surface of CN and TOCN also contribute to the O 1s spectrum
and C 1s spectrum to some extent. Table 1 shows the XPS
Table 1. XPS Analysis of CN and TOCN
Peak (eV) Concentration (atomic %)
sample C O C O
CN 284.8 531.9 75.69 24.31
TOCN 285.9 532.1 62.11 37.16
semiquantified elemental concentration for CN and TOCN. It
can be found the relative amount of carboxyl groups increased
after cellulose nanocrystal oxidation. With the TEMPO
modification on CN, the oxygen content was increased from
24.31% to 37.16%. Therefore, XPS data indicate that TEMPO
modification is a selectivity oxidation reaction that occurs on
C6 with the appearance of carboxyl groups.
The changes of morphology and dimension of the CN before
and after oxidization were observed by TEM and using a
Sepctrex laser particle counter. As shown in Figure 3a, the
unmodified CN possesses a typical rodlike shape, with a length
of 100−300 nm and a width of 3−15 nm, which is consistent
with previous reports by other researchers.36,37 After the
oxidization process, the average length is decreased from 275.5
nm (CN) to 233.8 nm (TOCN) (see Figure S1 in the
Supporting Information), while the samples still maintain the
rod-like shape shown in Figure 3b. These results demonstrate
that CN and TOCN can maintain original morphologies and
geometrical sizes of cellulose nanocrystal.
The XRD patterns of CN before and after oxidation are
shown in Figure 3c. It can be found that the main diffraction
characteristics of TOCN from cellulose nanocrystals were quite
similar (high peak at 2θ = 22.8°) and were typical for the
cellulose I (2θ = 14.9° and 2θ = 16.7°), although the intensities
were slightly decreased.38,39 Furthermore, the crystallinity index
(Ic) values of cellulose were 88.9% (CN), 86.5% (TOCN),
according to the Segal method.40 This result demonstrated that
the original crystalline structure of CN can be still preserved
after the chemical modification.
Structure and Properties of TOCN/SA Composite.
FTIR spectra of neat SA and Ca2+ cross-linking TOCN/SA
composite sponges are presented in Figure 4. For neat SA, the
peak at 1602 cm−1 is attributed to the stretching vibration of
the carbonyl bond (−CO). And, the peaks at 1421 and 1020
cm−1 in both SA and cellulose were assigned to stretching
vibration of the carboxyl (−COO−) and −C−O−C groups,
respectively. However, some obvious difference can be
observed in the spectrum of TOCN/SA composites (Figure
4). After the alginate was cross-linked with Ca2+, the
asymmetric −CO and symmetric −COO− adsorption
bands were shifted to higher wavenumbers, from 1602 cm−1
to 1607 cm−1 and from 1421 cm−1 to 1434 cm−1, and the
TOCN ratio in composite sponge was also increased. These
DOI: 10.1021/acssuschemeng.6b02849
ACS Sustainable Chem. Eng. 2017, 5, 3819−3828
ACS Sustainable Chemistry & Engineering
Figure 3. TEM images for (a) CN and (b) TOCN. (c) XRD spectra of CN and TOCN.
Figure 4. FTIR spectra for neat SA, and TOCN/SA composites with
different CN contents.
results could be attributed to the strong interaction between the
carboxylic groups on oxidized cellulose nanocrystal, alginate,
and the Ca2+ ions, according to the cross-linking.
SEM Analysis. The SEM micrographs of the cross-section
of sponges are shown in Figure 5. It was clear observed that the
Figure 5. SEM images of the cross-section morphology of cross-linked
sponges: (a) SA and (b) TOCN-30/SA.
neat alginate sponge (SA) possesses a stratiform pore structure
(Figure 5a), which is basically consistent with the previous
reports.41 When cellulose nanocrystals (TOCN) were intro-
Figure 6. (A) Degree of swelling of wet sponge and film, (B) porosity evaluation of sponge, and (C) tensile strength.
3823
Research Article
duced into SA, the rodlike nanocrystals appeared in the hole
wall of sponge (Figure 5b). In addition, there was no obvious
self-aggregation or microphase separation in the sponge. The
results of EDS mapping showed that the Ca 2+ was
homogeneously distributed on both the superficial and cross-
sectional surfaces of film and sponge. These results further
indicated that Ca2+ not only cross-linked on the TOCN/SA
composites surface (FT-IR spectra confirmed) but also that
Ca2+ easily diffused into the material structure via thermody-
namic driving forces, leading to the internal cross-linking
reaction within the TOCN/SA composites (Figure S2 in the
Supporting Information). The introduction of TOCN was
built-in using the regular internal three-dimensional (3D)
network of cross-linked TOCN/SA sponge. The 3D network
structures were useful for nutrients metabolism of metabolic
waste and were beneficial to the growth and transport of cells,
and the vessels and tissues could grow successfully with
materials implanted. Thus, the TOCN/SA composite sponge
might accelerate the healing of the tissues within a shorter time
biodegradation in vivo.
Swelling Degree and Porosity. The swelling degree of
the sponge and film was measured gravimetrically. The swelling
degree of sponges and films is shown in Figure 6A. The
TOCN-30/SA composite film and sponge had the most
outstanding water absorption in all testing materials (75.2%,
1399.1%), while the alginate film or sponge was the lowest
(43.35%, 825.9%). The swelling degree value of sponges was far
higher than that of films. Water absorption is an important
property for biomaterials and it is highly dependent on their
intrinsic structure and morphology. The water molecules could
smoothly pass through and penetrate the sponge, according to
the sponges composed of the regular internal 3D network and
high porosity. On the other hand, the introduced TOCN might
disrupt the hydrogen bonding between the alginate and be
conducive to increased water absorption ability with the TOCN
content. Compared with the neat SA sponge (82.3%), TOCN/
SA composite sponges possess a higher density of ordered
DOI: 10.1021/acssuschemeng.6b02849
ACS Sustainable Chem. Eng. 2017, 5, 3819−3828
ACS Sustainable Chemistry & Engineering Research Article

Figure 7. (a) Hemolysis assay of TOCN, SA, and TOCN/SA composite extracts. (b) Hemolysis ratio in each group (n = 3). (c) Cytocompatibility
of materials on Hela cells.

pores (>90%), while the porosity of TOCN-50/SA was slightly

decreased (Figure 6B). It was suggested that the high porosity
of TOCN/SA sponges were attributed to the internal network
microstructure of the interconnection between alginate and
TOCN chains (as shown in the SEM results). In addition, the
highly homogeneous dispersion of nanocrystal contents may
have an effect on material porosity. Furthermore, the highly
porous character of the sponges would be useful to facilitate the
adsorption of wound exudates.
A tensile strength test was used to measure the material
characteristics under an axial tensile load. Figure 6C
demonstrates that the Ca2+ cross-linked composites filled
with oxidized cellulose nanocrystals were generally stronger and
more robust than neat alginate. The highest mechanical
performance was obtained for TOCN-30/SA composites
(sponge and film, respectively). The TOCN-30/SA composite
film and sponge could withstand maximum stresses, which were
261.23 ± 21.33 MPa, and 35.16 ± 10.59 MPa, respectively. A
possible reason might be attributed to the TOCN participating
in cross-linking and have a good “egg-box” conjunction zone
between TOCN and alginate during the cross-linking process.
However, if superfluous CNs were added in alginate, the self-
aggregation of nanoparticles may cause the reinforcing effect to
be shaded-off.
Hemolysis Assay In Vitro. Hemolysis tests were conducted
using 2% rabbit erythrocyte suspension. As shown in Figure 7,
the TOCN/SA composite extracts, TOCN solution (1 and 4
mg/mL), and SA solution (4 and 6 mg/mL) did not cause any
hemolysis, while the apparent hemolysis can be observed in the
positive control (distilled water). In addition, although the
hemolysis ratio of the positive control (distilled water) was set
to 100%, the TOCN/SA composite extracts, TOCN solution
(1 and 4 mg/mL), and SA solution (4 and 6 mg/mL) were
observed to exhibit <2% hemolysis. It demonstrates that the
TOCN, SA, and TOCN/SA composites have excellent
hemocompatibility and may be suitable for wound dressing.
The cytocompatibility tests were performed using the CCK-8
method for 48 h (Figure 7c). The cell viability on the culture
plate (control) was set as 100%. As presented, the cell viabilities
were more than 99% after 48 h of culture, which meant that all
materials were noncytotoxic to Hela cells with concentrations
(0.1, 0.5, 2.5, and 5 mg/mL) and were suitable for the following
studies.
Blood Cells and Platelet Adhesion. In order to further
reveal the hemostatic mechanism, the surface adhesion and
morphologies of blood cells and platelets on the composite
samples were observed by SEM.42,43 A large amount of
erythrocytes gathered on the TOCN/SA composite surface and
kept their distinctive biconcave disks, while none of them
exhibited deformation or aggregation (see Figures 8a and 8b).
3824
Figure 8. SEM images of hemocyte and platelet adhesion: (a) TOCN/
SA composite sponge hemocyte adhesion, (b) TOCN/SA composite
film hemocyte adhesion, (c) TOCN/SA composite sponge platelet
adhesion, and (d) TOCN/SA composite film platelet adhesion.
Moreover, there were no spiny pseudopodia for erythrocytes
adsorbed on the material surface. The results indicated that the
TOCN/SA composite sponge and film would not affect the
physiological action of the blood cells. Incubation with platelet-
rich plasma (PRP) further demonstrated the platelets adhesion
on the TOCN/SA composites surface, which was attributed to
the carboxyl groups, which could attract and activate the
platelets. The amount of platelets adsorbed on the composites
material surface can be observed in Figures 8b and 8d, and
most of them were activated. Compared to TOCN/SA
composite film (Figure 8c), larger platelet aggregates and
more platelets altered their shape from irregular distinctive
disks to circular deformation on TOCN/SA composite sponge
(Figure 8d). These results also might indicate that the porous
structure and good degree of swelling of the sponge (Figure 6)
were helpful to improve the adsorption ability for platelets and
erythrocytes to achieve a rapid hemostatic effect.
Hemostatic Evaluation. Hemostatic properties of neat SA
and TOCN/SA composite materials was exhibited by the
amount of bleeding and hemostatic time both in the rabbit liver
injury model and the ear artery injury model, and the results are
shown in Figure 9 and Table 2. In the rabbit liver injury model,
the amount of bleeding on the gauze, which was 1.735 ± 0.055
g (p < 0.05), was significantly different from the other groups,
and the amount of bleeding of TOCN-30/SA composite film
(0.826 ± 0.075 g) and sponge (0.539 ± 0.069g) were
DOI: 10.1021/acssuschemeng.6b02849
ACS Sustainable Chem. Eng. 2017, 5, 3819−3828
ACS Sustainable Chemistry & Engineering Research Article

Figure 9. Hemostatic effect of neat SA, TOCN/SA composite films, and TOCN/SA composite sponges on the different trauma of the rabbit: (a) the
liver and (b) the ear artery. Also shown is the amount of bleeding of (c) neat SA and TOCN/SA composite films and (d) sponges. The mean value
was obtained from the testing, replicated six times for each material (*p < 0.05, compared to the control group).

significantly lower than other materials (see Figures 9c and 9d). TOCN-30/SA composite sponge (70 ± 5.93 s) was the
There was no significant difference between the bleeding shortest. The difference was statistically significant, as p < 0.05
amount on the TOCN-10/SA sponge (0.815 ± 0.079 g) and
(as demonstrated from Table 2). The hemostatic time of the
the TOCN-50/SA sponge (0.789 ± 0.056 g), and both of them
were lower than that on the TOCN-10/SA film (1.005 ± 0.096 TOCN-30/SA composite film was 75 ± 5.33 s, which was
g) and TOCN-50/SA film (0.998 ± 0.083 g), respectively. In significantly more rapid than that of the TOCN-10/SA
addition, the hemostatic time of the TOCN-30/SA composite composite film (108 ± 5.99 s) and TOCN-50/SA composite
sponge (76 ± 8.13 s) was much shorter than that of other
film (80 ± 7.43 s).
groups. The difference was statistically significant as P < 0.05
(as demonstrated from Table 2).
Table 2. Mean Hemostatic Time of SA and Different
In the rabbit ear injury model, as shown in Figures 9c and 9d,
TOCN/SA Composites in Two Rabbit Injury Modelsa
the blood loss of sponges was lower than that of films.
Compared to the gauze (1.012 ± 0.068 g), SA film (1.132 ± Average Hemostatic Time (s)
0.356 g), TOCN-10/SA composite film (0.728 ± 0.062 g), and liver injury ear artery injury
TOCN-50/SA composite film (0.653 ± 0.055 g), the blood gauze 179 ± 8.99 130 ± 5.89
loss of the TOCN-30/SA composite film (0.615 ± 0.053 g) was SA film 207 ± 10.26b 159 ± 6.44b
the lowest one (p < 0.05) (see Figure 9c). The amount of TOCN-10/SA film 132 ± 5.69b 108 ± 5.99b
bleeding of all sponge groups was significantly lower than that TOCN-30/SA film 99 ± 8.83b 75 ± 5.33b
of gauze (1.012 ± 0.068 g), and the average value of TOCN- TOCN-50/SA film 102 ± 9.16b 80 ± 7.43b
30/SA composite sponge was the lowest (0.404 ± 0.058 g) SA sponge 186 ± 12.05b 123 ± 13.38b
(Figure 9d). There was no significant difference between TOCN-10/SA sponge 92 ± 7.59b 85 ± 10.37b
TOCN-10/SA (0.503 ± 0.058 g) and the TOCN-50/SA TOCN-30/SA sponge 76 ± 8.13b 70 ± 5.93b
composite sponge (0.498 ± 0.065 g). Moreover, when it came TOCN-50/SA sponge 80 ± 11.38b 69 ± 10.08b
to the hemostatic speed of all materials, the sponge groups were
more rapid than films, and the average hemostatic speed of a
N = 6. bp < 0.05, compared to the control group.

3825 DOI: 10.1021/acssuschemeng.6b02849

ACS Sustainable Chem. Eng. 2017, 5, 3819−3828
ACS Sustainable Chemistry & Engineering
All of the above-mentioned results indicated that both the
TOCN-30/SA film and sponge have the potential to
significantly improve the hemostatic efficiency, especially the
TOCN-30/SA sponge. The electric charges on the surface of
TOCN were exposed, which could rapidly adhere to protein
and damaged erythrocytes, and activate the platelets. The
carboxyl groups on TOCN may have strong complexation
ability to the Fe atom at the ferrous state from the damaged
erythrocytes, which leads to nonspecific aggregation of
hemocyte or platelets and promote the generation of blood
clot.44 The hemostatic effect of TOCN-30/SA composite
sponge was better than TOCN-30/SA film due to its excellent
hemostatic properties, such as the blood loss and the
hemostatic time (see Figures 9c and 9d, as well as Table 2).
First, TOCN-30/SA composite sponge had a porous structure
(Figure 5), which was beneficial to quickly absorb the blood on
the material surface with large capacity and made it more
conducive to promoting the aggregation of platelets and
inducing erythrocytes to accelerate blood clotting.45 Second,
the TOCN-30/SA composite sponge with hydrophilic carboxyl
groups could combine Fe3+ in blood fluid to form the brown gel
and stimulate erythrocytes and platelets (GPIIb/IIIa) on the
blood absorption process and also provide a rapid hemostatic
effect. Furthermore, TOCN/SA composite material cross-
linking with Ca2+ would activate blood coagulation by
stimulating platelets and the clotting factors VII, IX, and X in
the blood clotting process.46,47 In this study, when TOCN/SA
composite materials was used on the surface of the wound, the
materials containing Ca2+ ions with excellent physical proper-
ties would absorb water from the blood and aggregate the
clotting factors to achieve hemostasis. It means that a rapid
coagulation rate would reduce the amount of bleeding and clots
formation. Thus, we chose the TOCN-30/SA composite
sponge and film to evaluate the biological degradation.
Evaluation of Biological Degradation. Histopathological
evaluations of the experimental tissue wounds of the
subcutaneous implantation in the rabbit inner thighs reveal
the different responses of the commercial Surgicel and TOCN-
30/SA composite film and sponge at different implante periods.
Figure 10 presents the typical histological images of each
sample after 7, 14, and 21 days of the subcutaneous
implantation. The implanted materials would change and
become irregular lumps or scattered pieces, and the color of the
samples should be stained to deep purple or deep red after
Figure 10. Histopathological examination of Surgicel, TOCN-30/SA
composite film and TOCN-30/SA composite sponge after 7, 14, and
21 days of implantation.
3826
Research Article
H&E staining, which was darker than normal tissue. However,
some large-size samples revealed that no coloring occurred
during the process of sectioning and staining, because of the
lack of materials or unstained area. In the first 7 days, there
were many fibroblasts, inflammatory, eosinophils, and tissue
cells around the Surgicel samples and within muscle tissue
(Figure 10a). The inflammatory cells were observed around the
unstained TOCN/SA composite film material. Moreover, they
replaced fibrous connective tissue with the partial muscle fiber
necrosis tissue (Figure 10b). The small pieces of particles on a
few areas and have been stained, and the inflammatory cells
around unstained TOCN/SA composite sponge material are
observed (Figure 10c). After implantation for 14 days, the
inflammatory cells decreased in all three groups, and the fibrous
tissue was observed in TOCN-30/SA composite sponge groups
(Figure 10f) However, the hyperplasia of the fibrous tissue
appeared in TOCN-30/SA composite film (Figure 10e).
Moreover, the residue Surgicel and TOCN-30/SA composite
film materials are found in Figures 10d and 10e, respectively.
Furthermore, the multinucleate giant cells could be observed in
the Surgicel group. After implantation for 21 days, there is
neither the residual material remaining in the muscle tissue nor
the obvious tissue reactions on the implanted sites in all groups
(see Figures 10f and 10g).
The results demonstrated that the materials would initiate
inflammatory response and local tissue necrosis in the first
week after implantation, and then the fibroblast proliferation
increased and the hyperplasia of fibrous tissue was caused. In
the second week, the inflammatory cells decreased, indicating
that, without a foreign body reaction remaining reaction would
help accelerate the wound healing. On the 21th day, the
inflammatory cells disappeared, showing that all testing
materials were absorbed completely. Compared to the Surgicel
group, more granulation tissue grew in the TOCN-30/SA
composites, especially in the TOCN-30/SA composite sponge,
where the greatest amount was observed. In short, the TOCN-
30/SA composite sponge was the best group in the process of
wound repair.
■ CONCLUSION
2,2,6,6-Tetramethylpiperidine-1-oxyl (TEMPO)-mediated oxi-
dized cellulose nanocrystals/alginate composite films and
sponges were successfully prepared and simultaneously
performed traditional Ca2+ cross-linked. In vivo degradation
tests indicate that all materials could be biodegraded completely
and without inflammatory reaction after three weeks. Especially,
the TOCN-30/SA composite sponge without cytotoxicity had
higher porosity and tensile strength, and was helpful to absorb a
large abundance of wound exudate and improved the
adsorption ability for platelets and erythrocytes to achieve a
rapid hemostatic effect. As a result, the hemostatic efficiency of
the TOCN-30/SA composite sponge is the highest one (∼70
s) with the least blood loss in the rabbit ear artery and liver
trauma models, respectively. The hemostatic mechanism of the
TOCN/SA composite is the combination of physical
adsorption and physiological hemostasis. Therefore, further
study is essential for investigating the material hemostatic
mechanism and improving the materials’ hemostatic efficiency
used in the field of wound healing.
DOI: 10.1021/acssuschemeng.6b02849
ACS Sustainable Chem. Eng. 2017, 5, 3819−3828
ACS Sustainable Chemistry & Engineering
■
*
ASSOCIATED CONTENT
S Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acssusche-
meng.6b02849.
Composition of various TOCN/SA films and sponges
and the size of CN and TOCN; SEM and EDS spectrum
of Ca2+ cross-linking for TOCN/SA composite film and
sponge (PDF)
■
AUTHOR INFORMATION
Corresponding Author
*Tel.: +86 0451 86414806. Fax: +86 0451 86414806. E-mail:
hejinmei@hit.edu.cn.
ORCID
Feng Cheng: 0000-0002-4555-554X
Yudong Huang: 0000-0002-9998-557X
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
This work was financially supported by the State Key
Laboratory for Modification of Chemical Fibers and Polymer
Materials, Donghua University (No. LK1510).
■ REFERENCES
(1) Kauvar, D. S.; Lefering, R.; Wade, C. E. Impact of hemorrhage on
trauma outcome: An overview of epidemiology, clinical presentations,
and therapeutic considerations. J. Trauma 2006, 60, S3−S9.
(2) Alam, H. B.; Burris, D.; DaCorta, J. A.; Rhee, P. Hemorrhage
Control in the Battlefield: Role of New Hemostatic Agents. Mil. Med.
2005, 170, 63−69.
(3) Cheng, F.; Gao, J.; Wang, L.; Hu, X. Y. Composite chitosan/
poly(ethylene oxide) electrospun nanofibrous mats as novel wound
dressing matrixes for the controlled release of drugs. J. Appl. Polym. Sci.
2015, 132, 42060−42067.
(4) Ashworth, D. R.; Whear, N. M. The haemostatic poultice: an aid
to microvascular anastomosis. Br. J. Oral. Maxil. Surg. 2003, 41, 353−
354.
(5) Wedmore, I.; McManus, J. G.; Pusateri, A. E.; Holcomb, J. B. A
special report on the chitosan-based hemostatic dressing: experience in
current combat operations. J. Trauma 2006, 60, 655−658.
(6) Rhee, P.; Brown, C.; Martin, M.; Salim, A.; Plurad, D.; Green, D.;
Chambers, L.; Demetriades, D.; Velmahos, G.; Alam, H. QuikClot use
in trauma for hemorrhage control: case series of 103 documented uses.
J. Trauma 2008, 64, 1093−1099.
(7) Watt, P. W.; Harvey, W.; Wiseman, D.; Light, N. C. O. J.; Ltd, J.
M.; Saferstein, L.; Cini, J. Wound dressing materials comprising collagen
and oxidized cellulose. Eur. Patent EP1325754, 2007.
(8) Sun, J.; Tan, H. Alginate-Based Biomaterials for Regenerative
Medicine Applications. Materials 2013, 6, 1285−1309.
(9) Hrynyk, M.; Martins-Green, M.; Barron, A. E.; Neufeld, R. J.
Alginate-PEG sponge architecture and role in the design of insulin
release dressings. Biomacromolecules 2012, 13, 1478−85.
(10) Gombotz, W. R.; Wee, S. F. Protein release from alginate
matrices. Adv. Drug Delivery Rev. 1998, 31, 267−285.
(11) Goh, C. H.; Heng, P. W. S.; Chan, L. W. Alginates as a useful
natural polymer for microencapsulation and therapeutic applications.
Carbohydr. Polym. 2012, 88, 1−12.
(12) Rowley, J. A.; Madlambayan, G.; Mooney, D. J. Alginate
hydrogels as synthetic extracellular matrix materials. Biomaterials 1999,
20, 45−53.
(13) Thomas, A.; Harding, K. G.; Moore, K. Alginates from wound
dressings activate human macrophages to secrete tumour necrosis
factor-α. Biomaterials 2000, 21, 1797−1802.
3827
Research Article
(14) Kalyani, S.; Smitha, B.; Sridhar, S.; Krishnaiah, A. Pervaporation
separation of ethanol−water mixtures through sodium alginate
membranes. Desalination 2008, 229, 68−81.
(15) Kaklamani, G.; Cheneler, D.; Grover, L. M.; Adams, M. J.;
Bowen, J. Mechanical properties of alginate hydrogels manufactured
using external gelation. J. Mech. Behav. Biomed. 2014, 36, 135−42.
(16) Kanjanamosit, N.; Muangnapoh, C.; Phisalaphong, M. Biosyn-
thesis and characterization of bacteria cellulose-alginate film. J. Appl.
Polym. Sci. 2010, 115, 1581−1588.
(17) Cheng, F.; He, J. M.; Yan, T. S.; Liu, C. Y.; Wei, X. J.; Li, J. W.;
Huang, Y. D. Antibacterial and hemostatic composite gauze of N,O-
carboxymethyl chitosan/oxidized regenerated cellulose. RSC Adv.
2016, 6, 94429−94436.
(18) Moon, R. J.; Martini, A.; Nairn, J.; Simonsen, J.; Youngblood, J.
Cellulose nanomaterials review: Structure, properties and nano-
composites. Chem. Soc. Rev. 2011, 40, 3941−3994.
(19) Azizi Samir, M. A. S.; Alloin, F.; Dufresne, A. Review of Recent
Research into Cellulosic Whiskers, Their Properties and Their
Application in Nanocomposite Field. Biomacromolecules 2005, 6,
612−626.
(20) Dufresne, A. Polysaccharide nano crystal reinforced nano-
composites. Can. J. Chem. 2008, 86, 484−494.
(21) Ben Azouz, K.; Ramires, E. C.; Van den Fonteyne, W.; El Kissi,
N.; Dufresne, A. Simple Method for the Melt Extrusion of a Cellulose
Nanocrystal Reinforced Hydrophobic Polymer. ACS Macro Lett. 2012,
1, 236−240.
(22) Lin, N.; Huang, J.; Chang, P. R.; Feng, J.; Yu, J. Surface
acetylation of cellulose nanocrystal and its reinforcing function in
poly(lactic acid). Carbohydr. Polym. 2011, 83, 1834−1842.
(23) Shimotoyodome, A.; Suzuki, J.; Kumamoto, Y.; Hase, T.; Isogai,
A. Regulation of postprandial blood metabolic variables by TEMPO-
oxidized cellulose nanofibers. Biomacromolecules 2011, 12, 3812−3818.
(24) Park, M.; Lee, D.; Hyun, J. Nanocellulose-alginate hydrogel for
cell encapsulation. Carbohydr. Polym. 2015, 116, 223−228.
(25) Lin, N.; Bruzzese, C.; Dufresne, A. TEMPO-Oxidized
Nanocellulose Participating as Crosslinking Aid for Alginate-Based
Sponges. ACS Appl. Mater. Interfaces 2012, 4 (9), 4948−4959.
(26) Lokanathan, A. R.; Uddin, K. M.; Rojas, O. J.; Laine, J. Cellulose
nanocrystal-mediated synthesis of silver nanoparticles: role of sulfate
groups in nucleation phenomena. Biomacromolecules 2014, 15, 373−
379.
(27) Song, J.; Fu, G.; Cheng, Q.; Jin, Y. Bimodal Mesoporous Silica
Nanotubes Fabricated by Dual Templates of CTAB and Bare
Nanocrystalline Cellulose. Ind. Eng. Chem. Res. 2014, 53, 708−714.
(28) Jin, E.; Guo, J.; Yang, F.; Zhu, Y.; Song, J.; Jin, Y.; Rojas, O. J.
On the polymorphic and morphological changes of cellulose
nanocrystals (CNC-I) upon mercerization and conversion to CNC-
II. Carbohydr. Polym. 2016, 143, 327−335.
(29) Jiang, F.; Hsieh, Y.-L. Self-assembling of TEMPO Oxidized
Cellulose Nanofibrils As Affected by Protonation of Surface Carboxyls
and Drying Methods. ACS Sustainable Chem. Eng. 2016, 4, 1041−
1049.
(30) Jiang, F.; Hsieh, Y.-L. Super water absorbing and shape memory
nanocellulose aerogels from TEMPO-oxidized cellulose nanofibrils via
cyclic freezing−thawing. J. Mater. Chem. A 2014, 2, 350−359.
(31) Koga, H.; Saito, T.; Kitaoka, T.; Nogi, M.; Suganuma, K.; Isogai,
A. Transparent, conductive, and printable composites consisting of
TEMPO-oxidized nanocellulose and carbon nanotube. Biomacromole-
cules 2013, 14, 1160−1165.
(32) Li, J. W.; He, J. M.; Huang, Y. D.; Li, D. L.; Chen, X. T.
Improving surface and mechanical properties of alginate films by using
ethanol as a co-solvent during external gelation. Carbohydr. Polym.
2015, 123, 208−216.
(33) Li, L.; Wang, N.; Jin, X.; Deng, R.; Nie, S.; Sun, L.; Wu, Q.; Wei,
Y.; Gong, C. Biodegradable and injectable in situ cross-linking
chitosan-hyaluronic acid based hydrogels for postoperative adhesion
prevention. Biomaterials 2014, 35, 3903−3917.
(34) Ong, S. Y.; Wu, J.; Moochhala, S. M.; Tan, M. H.; Lu, J.
Development of a chitosan-based wound dressing with improved
DOI: 10.1021/acssuschemeng.6b02849
ACS Sustainable Chem. Eng. 2017, 5, 3819−3828
ACS Sustainable Chemistry & Engineering Research Article

hemostatic and antimicrobial properties. Biomaterials 2008, 29, 4323−

4332.
(35) Quan, K.; Li, G.; Tao, L.; Xie, Q.; Yuan, Q.; Wang, X.
Diaminopropionic Acid Reinforced Graphene Sponge and Its Use for
Hemostasis. ACS Appl. Mater. Interfaces 2016, 8, 7666−7673.
(36) Habibi, Y.; Lucia, L. A.; Rojas, O. J. Cellulose Nanocrystals
Chemistry, Self-Assembly, and Applications. Chem. Rev. 2010, 110,
3479−3500.
(37) Isogai, A.; Saito, T.; Fukuzumi, H. TEMPO-oxidized cellulose
nanofibers. Nanoscale 2011, 3, 71−85.
(38) Saito, T.; Hirota, M.; Tamura, N.; Kimura, S.; Fukuzumi, H.;
Heux, L.; Isogai, A. Individualization of Nano-Sized Plant Cellulose
Fibrils by Direct Surface Carboxylation Using TEMPO Catalyst under
Neutral Conditions. Biomacromolecules 2009, 10, 1992−1996.
(39) Saito, T.; Kimura, S.; Nishiyama, Y.; Isogai, A. Cellulose
Nanofibers Prepared by TEMPO-Mediated Oxidation of Native
Cellulose. Biomacromolecules 2007, 8, 2485−2491.
(40) Segal, L.; Creely, J. J.; Martin, A. E., Jr.; Conrad, C. M. An
Empirical Method for Estimating the Degree of Crystallinity of Native
Cellulose Using the X-ray Diffractometer. Text. Res. J. 1959, 29 (10),
786−794.
(41) Yuan, N. Y.; Lin, Y. A.; Ho, M. H.; Wang, D. M.; Lai, J. Y.;
Hsieh, H. J. Effects of the cooling mode on the structure and strength
of porous scaffolds made of chitosan, alginate, and carboxymethyl
cellulose by the freeze-gelation method. Carbohydr. Polym. 2009, 78,
349−356.
(42) Quan, K.; Li, G.; Luan, D.; Yuan, Q.; Tao, L.; Wang, X. Black
hemostatic sponge based on facile prepared cross-linked graphene.
Colloids Surf., B 2015, 132, 27−33.
(43) Dai, C.; Yuan, Y.; Liu, C.; Wei, J.; Hong, H.; Li, X.; Pan, X.
Degradable, antibacterial silver exchanged mesoporous silica spheres
for hemorrhage control. Biomaterials 2009, 30 (29), 5364−5375.
(44) Martina, B.; Kateřina, K. O.; Miloslava, R. k.; Jan, G.; Ruta, M.
Oxycellulose: Significant characteristics in relation to its pharmaceut-
ical and medical applications. Adv. Polym. Technol. 2009, 28 (3), 199−
208.
(45) Lan, G.; Lu, B.; Wang, T.; Wang, L.; Chen, J.; Yu, K.; Liu, J.;
Dai, F.; Wu, D. Chitosan/gelatin composite sponge is an absorbable
surgical hemostatic agent. Colloids Surf., B 2015, 136, 1026−1034.
(46) Xia, Q.; Liu, Z.; Wang, C.; Zhang, Z.; Xu, S.; Han, C. C. A
Biodegradable Trilayered Barrier Membrane Composed of Sponge
and Electrospun Layers: Hemostasis and Antiadhesion. Biomacromo-
lecules 2015, 16, 3083−3092.
(47) Spronk, H. M.; Govers-Riemslag, J. W.; Ten Cate, H. The blood
coagulation system as a molecular machine. BioEssays 2003, 25, 1220−
1228.

3828 DOI: 10.1021/acssuschemeng.6b02849

ACS Sustainable Chem. Eng. 2017, 5, 3819−3828