Accepted Manuscript

Title: Biodegradable collagen sponge reinforced with

chitosan/calcium pyrophosphate nanoflowers for rapid
hemostasis

Authors: Tingsheng Yan, Feng Cheng, Xinjing Wei, Yudong

Huang, Jinmei He

PII: S0144-8617(17)30479-4
DOI: http://dx.doi.org/doi:10.1016/j.carbpol.2017.04.080
Reference: CARP 12268

To appear in:

Received date: 11-2-2017

Revised date: 25-4-2017
Accepted date: 25-4-2017

Please cite this article as: Yan, Tingsheng., Cheng, Feng., Wei, Xinjing.,
Huang, Yudong., & He, Jinmei., Biodegradable collagen sponge reinforced with
chitosan/calcium pyrophosphate nanoflowers for rapid hemostasis.Carbohydrate
Polymers http://dx.doi.org/10.1016/j.carbpol.2017.04.080

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Biodegradable collagen sponge reinforced with chitosan/calcium

pyrophosphate nanoflowers for rapid hemostasis

Tingsheng Yan 1, Feng Cheng 1, 2, Xinjing Wei1, Yudong Huang 1, Jinmei He 1, 2, *

1
MIITKey Laboratory of Critical Materials Technology for New Energy Conversion and Storage,

State Key Laboratory of Urban Water Resource and Environment, School of Chemistry and

Chemical Engineering, Harbin Institute of Technology, Harbin, Heilongjiang 150010, China

2
State Key Laboratory for Modification of Chemical Fibers and Polymer Materials, Donghua

University, Shanghai 201620, China

∗ Corresponding author. E-mail address: hejinmei@hit.edu.cn (J. He).

Interfacial
Chitosan/Calcium
Interaction
Pyrophosphate Blood
Nanoflowers

Freeze
Drying Pore:
Collagen rapid absorption
NH 2 -
NH 2 -

NH 2 -
NH 2 -

Nanoflowers: Hemostatic
facilitate clotting Evaluation

Graphical abstract. The preparation process of chitosan nanoparticle collagen

sponge and its hemostatic evaluation

Highlights
 A chitosan/calcium pyrophosphate nanoflowers coating collagen sponge was
designed.
 The sponge could promote the blood clotting and achieve hemorrhage control.
 The sponge can degrade in 3 weeks thus suitable for post-operative treatment.

ABSTRACT: Efficient and biodegradable hemostatic materials become increasingly

important in civilian and military clinical. However, traditional hemostatic materials

are difficult to achieve expected effects especially in parenchymal organs with rich

vascularity. In facing these challenges, we designed a biodegradable collagen sponge

reinforced with chitosan/calcium pyrophosphate nanoflowers (CPNFs-Col sponge) for

rapid hemostasis. With specific performances, such as rapid water absorption ability,

the positive surface rich in amino groups and high specific surface area (952.5 m2g-1),

the obtained CNPFs-Col sponge with optimized composition could activate the intrinsic

pathway of coagulation cascade, induce haemocytes and platelets adherence, promote

the blood clotting and achieve hemorrhage control in vitro and in vivo. In addition, the

CNPFs-Col sponge can be completely biodegraded in 3 weeks, which is suitable for

post-operative treatment and peritoneal adhesion prevention. It can be concluded that

the CPNFs-Col sponge would become a promising candidate for clinical hemostatic

applications.

KEYWORDS: Collagen sponge; Chitosan/calcium pyrophosphate nanoflower;

Biodegradable; Hemostasis
1. INTRODUCTION

Uncontrollable and extensive bleeding is very dangerous, as it might lead to death

without timely and effective solutions (Dai et al., 2009; Kozen, Kircher, Henao,

Godinez & Johnson, 2008). Many hemostatic methods are available in clinical, such as

straight seam, ligation (for phleborrhagia and arteriorrhagia) and electrocautery (for

capillary bleeding) (Quan, Li, Di, Yuan, Lei & Xing, 2015). However, the traumatic

hemorrhage in parenchymal organs with rich vascularity remains an intractable issue,

as the traditional hemostasis methods and materials clogs up and becomes inoperative.

New efficient hemostatic materials that can rapidly control bleeding are pressing

needed.

Scientists have long sought to design outstanding hemostatic materials with good

biocompatibility, rapid hemostatic ability and low manufacturing cost (Englehart et al.,

2008; Pusateri, Holcomb, Kheirabadi, Alam, Wade & Ryan, 2006). So far, various

natural and synthetic materials, such as chitosan, collagen oxidized regenerated

cellulose and polyglycolic acid (Ansorge et al., 2009; Ling et al., 2014; Sumita et al.,

2006) have been used to make hemostatic dressings or bandages. In these materials,

collagen sponge showed outstanding performances, owing to the porous structure for

the adsorption of haemocytes, the similar composition with the extracellular matrix, the

low immunogenicity and the efficiency to form different shapes (Cioca, 1983;

Juncosamelvin, Matlin, Holdcraft, Nirmalanandhan & Butler, 2007; Kim, Ji, Lee, Seo,

Hwang & Kim, 2014; Shen et al., 2014). All these features suggest that collagen

sponges have the potential to become a great hemostatic material.

 Due to the antimicrobial, biocompatibility and intrinsic hemostatic properties,
chitosan has been regarded as a prominent candidate for wound care in clinical
(Dowling, Kumar, Keibler, Hess, Bochicchio & Raghavan, 2011; Rao & Sharma, 1997;
Yang, Tian, Wang, Wang, Zeng & Chen, 2008). A chitosan derivative hemostatic
bandage has been developed using lyophilization method by HemCon Medical
Technologies. This bandage could attract the negatively charged residues on haemocyte
membranes based on the protonated amine groups of chitosan molecules. The chitosan
molecular chain could also easily adsorb the fibrinogens and plasma proteins. However,
the HemCon® bandage could merely work efficiently on flat surfaces while not flexible
to the post-operative persistent hematuria or secondary hemorrhage, which limited the
broader utilization in severe situations (Whang, Kirsch & Zhu, 2005).
In facing with these challenges, we designed a chitosan/calcium pyrophosphate
nanoflowers (CPNFs) coated collagen sponge to improve the hemostatic properties.
The organic-inorganic hybrid nanoflowers have been applied in many fields, such as
drug delivery (Kumari & Singh, 2012), enzyme immobilization (Ansari & Husain, 2012;
Peng, Pan, Xie, Liu, Xiang & Liu, 2016) and chemical testing probe (Du & He; Huang
et al., 2016). In this work, the CPNFs were synthesized by an improved one-pot
preparation method (Wang et al., 2014) and then mixed with collagen to form CPNFs-
Col sponge via a lyophilization method (Scheme. 1). The hemostatic properties of the
CPNFs-Col sponge were evaluated both in vitro and in vivo. The biodegradable
behavior, biocompatibility and histological examination of the sponges were also
investigated.

2. EXPERIMENTAL SECTION

2.1 Materials

Chitosan (Mw = 100 kDa, deacetylation degree (DD) = 82.5%) was obtained from

Zhejiang Golden-Shell Biological Co., Ltd., China. Bovine tendon was purchased from

Heilongjiang Binxi Industry Co., Ltd., China. Pepsin from porcine gastric mucosa,
lysozyme, collagenase type I and dialysis bags (membrane molecular weight cut off 3,

8 and 12~16 kDa) were obtained from Sigma-Aldrich Co., LLC. Glacial acetic acid,

CCK-8 kit, citric acid, sodium citrate, D-glucose, calcium chloride, sodium carbonate,

1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC), N-hydroxysuccinimide

(NHS) and sodium tripolyphosphate (TPP) were purchased from Shanghai Aladdin

Bio-Chem Technology Co., Ltd. Standard gauze (W1912) were purchased from

Johnson & Johnson Medical (China) Ltd. SPF ICR mice (20~25 g) and SPF New

Zealand white rabbits were provided by the Animal Experiment Center of Harbin

Medical University (Harbin, Heilongjiang Province, China). The protocol was

approved by the ethics committee of Harbin Medical University (Permission number:

SYXK (HEI) 2016-007). All animals were handled according to the Chinese National

Institutes of Health Guidelines for the Care and Use of Laboratory Animals.

2.2 Preparation of hybrid nanoflower

The preparation method of CPNFs was optimized by an improved one-pot

preparation method (Wang et al., 2014). Some 0.25 g chitosan was dissolved in 500 mL

of 0.02 % (v/v) acetic acid. After all the chitosan has dissolved, 50 mL of 0.11 mg/mL

CaCl2 was added into the solution and kept stirring for 10 min. Then, 150 mL of 0.5

mg/mL TPP was added dropwise to the mixture within 10 min. The suspension was

stirred at 30°C for 4 h, then centrifuged at 8000 g for 10 min, washed with deionized

water (3 times) and freeze-dried. An amount of 0.23 g CPNFS was synthesized with a

yield 69.7%. The prepared powder was analyzed by scanning electron microscopy
(SEM; JSM-6360LV, JEOL, Japan ) and dynamic light scattering (DLS; Nano-Z,

Malvern Instrument).

2.3 Extraction and purification of type I collagen

Type I collagen was extracted from bovine tendon according to the approach of

reference (Maturana, Zanon, Pierucci, Vidal & Oliveira, 2011; Vogel, Paulsson &

Heinegård, 1984). 50 g of small fragments of defatted and cleaned bovine tendons were

immersed in an aqueous solution of 0.1% sodium carbonate at 4°C for 12 h. Then,

transferred to a three-necked, round-bottomed flask containing a mixture of 1000 mL

0.3% acetic acid and 0.5 % pepsin (activity 3000 U/mg) aqueous solution, stirring at

0~4°C for 72 h. The crude products were centrifuge at 9600 g for 20 min. The

supernatant was collected to a beaker. A certain amount of NaCl (with a final

concentration of 10%) was added to the supernatant to reconstitute collagen fibers. The

obtained collagen fibers were then swelled in 0.1M Tris-HCl solution for 12 h and

dialyzed (membrane molecular weight cut off 12~16 kDa) against distilled water at 4°C

for 6 days. The outside bath water was changed every 24 h. The purified collagen was

frozen at -20°C for 24 h and then frozen-dried at -50°C in vacuum for 48 h. An amount

of 12.4 g collagen was collected with a yield calculated about 51.6% (Equation S1).

2.4 Preparation of composite sponges

A new type of collagen sponge reinforced by CPNFs was synthesized by the

following steps: A certain amount of CPNFs (0, 15%, 30% and 40%) and 0.5 g of
collagen was dissolved into 100 mL distilled water. EDC and NHS were added into

these mixtures at a weight percentage of 0.5%, following by stirring at 100 rpm for 4 h.

The products were dialyzed (membrane molecular weight cut off 8 kDa) against

distilled water at 4°C for 48 h. The mixed solution of dialysed collagen and CPNFs was

rapidly frozen at -80°C in a glass plate, followed by freeze drying at -50°C in vacuum

for 48 h to form composite sponges (with a yield more than 85%, Table S3).

The water absorption ability and the absorption rate of the sponges were tested.

The water absorption capacity test was conducted according to Lee and Chen’s method

(Lee & Chen, 2001) by the following equation:

𝑚1 − 𝑚0
water absorption = × 100%
𝑚0

where, m0 means the initial weight of the sponges and m1 means the weight after

adequately absorbing water.

The structures were identified by Fourier transform infrared spectrophotometric

(FTIR), scanning electron microscopy (SEM) and thermogravimetry (TG)

measurements. FTIR measurements were performed using a Perkine Elmer

System2000 spectrometer (Nicolet Magma 550 series II, Midac, USA) in the range of

4000~400 cm-1 with a resolution of 4 cm-1. The morphology of the sponges was

observed by JSM-6360LV, JEOL, Japan, at 10 kV after gold sputter coating. Thermal

weight loss of the sponges was measured using a SDTQ600TG analyzer from 20°C to

800°C at a heating rate of 10°C min-1 under N2 atmosphere. The specific surface area

of the sponges were measured according to the methylene blue (MB) method (Deng,

Yang, Tao & Dai, 2009; Zhao, Ren & Cheng, 2012).
 2.5 Blood collection and platelets isolation

The whole blood was collected from the rabbit marginal ear vessels with acid

citrate dextrose (20 mM citric acid, 110 mM sodium citrate, 5 mM D-glucose) at a ratio

of 9:1 (v/v), as per-protocol approved by the DSO Institutional Animal Care and Use

Committee (IACUC). The platelet rich plasma was separated by centrifuging at 80 g

for 20 min. Then, the platelet rich plasma was centrifuged at 1000 g for 20 min to collect

condensed platelets. At last, the platelets were resuspended in PBS buffer at a

concentration of 3×105 platelets/mL.

2.6 The safety evaluation in vitro and vivo

Hemolytic tests were performed using rabbit haemocyte suspension. 5 mL of

material extract (See Supplementary materials) was mixed with 5 mL of 2% rabbit

haemocyte suspension (diluted with 0.9% NaCl solution (NS)), followed by incubating

at 37°C for 3 h. Then the haemocyte suspensions were centrifuged at 240 g for 10 min

and detected on a UV/Vis spectrophotometer at 545 nm to determine the hemolytic ratio.

Double distilled water was employed as a positive control while NS was employed as

a negative control. The hemolytic ratio was calculated according to the following

equation:
Test sample − Negative control
Hemolytic Ratio = × 100%
Positive control − Negative control
Intracutaneous stimulation tests were performed in SPF New Zealand white

rabbits (weight about 2.5-3.0 kg). 0.1 mL of sample extraction was injected
intradermally on the back of the rabbits. The primary irritation index (PII) was

evaluated (Table S1, S2) after observing at the rated point (24 h). NS and 4%

formaldehyde solution were employed as the negative and positive control, respectively.

Acute toxicity tests were evaluated in SPF ICR mice. A certain amount of sample

extraction (50 g/kg body weight) was injected into the mice through cauda vein. The

general conditions (the activity, body weight, behavior pattern, mortality of the animals

and other clinical signs) were observed carefully at determined time point (12 h, 24 h

and 48 h). NS and 4% formaldehyde solution were employed as the negative and

positive control, respectively.

The cytotoxicity evaluation was carried out using human umbilical vein

endothelial cells (HUVEC) by CCK-8 assay. 10 μL of the sample extraction (10, 5, 2.5,

1.25 mg mL-1) was mixed with 100 μL DMEM medium containing 10% (v/v) fetal

bovine serum (FBS) and 1% (v/v) antibiotics. HUVEC were incubated with the mixture

in a 96-well plate for 48 h. The cell number was determinated by CCK-8 assay (See

Supplementary materials). The relative growth rate (RGR) of cells cultured in each

group was measured according to the following formula:

Abs 490 test
RGR(%) = × 100%
Abs 490 control
In vivo biocompatibility was carried out using SPF New Zealand white rabbits. A

slice of the sponge (1 × 1 × 0.6 cm3) was implanted across the thigh muscle. All the

animals were carefully nurtured until the implanting terminals were reached (0, 1, 2, 3

week).The implants and surrounding tissues were carefully removed, fixed in 10 %

formaldehyde solution, embedded in paraffin, sectioned and stained with hematoxylin

and eosin for histopathological examination.

2.7 Interfacial interaction between blood cells and sponges

To evaluate the interfacial interaction between the blood cells and sponges, 100

μL of whole blood with anticoagulant citrate dextrose (ACD)was added to a slice of the

sponge (1 × 1 × 0.6 cm3) and incubated for 1, 3, 5 min at 37 °C. The sample was washed

with PBS (pH 7.4) 3 times to remove the physically adhered cells. The blood cells were

immobilized with 2.5% glutaraldehyde for 3 h, dehydrated with ethanol, freeze dried

and observed with SEM.

2.8 Hemoglobin adsorption

The hemoglobin adsorption test was conducted according to the previous methods

(Behrens, Sikorski, Li, Wu, Griffith & Kofinas, 2013; Ong, Wu, Moochhala, Tan & Lu,

2008). A 100 μL of whole blood solution (per 100 μL blood with 8 μL of 0.2 M CaCl2)

was added onto the CPNFs-Col sponge (1 × 1 × 0.6 cm3) in a glass dish and incubated

for a certain period of time (1, 2, 3, 4 and 5 min) at 37°C (100 μL of whole blood was

directly added onto the glass dish as a negative control).Then, 20 mL of distilled water

was added to dissolve the hemoglobin in free haemocytes. Collagen sponge, freeze

dried chitosan sponge from 2% w/w chitosan in 2% acetic acid aqueous solution,

pyrophosphate powder, and standard gauze were utilized as control samples. The
content of hemoglobin on the samples was measured using an UV-Vis

spectrophotometer at 542 nm by the following equation:

Hemoglobin adsorption rate = (𝐼𝑟 − 𝐼𝑠 )/𝐼𝑟 × 100%

Where, Is means the absorbance of hemoglobin in suspension and Ir means the

absorbance of the reference value. As a reference value, 100 μL of unreacted blood was

dropped directly in 20 mL of distilled water to measure its ultraviolet absorption value.

2.9 Platelets adhesion

A 100 μL of the platelets suspension was added to the CPNFs-Col sponges (1 × 1

× 0.6 cm3) and incubated at 37°C for 0.5 h. The samples were rinsed in 2 mL Tyrode’s

solution for 10 min to remove the unattached platelets. The concentration of platelets

in the suspension was determined with a Dark-Line Hemacytometer. Collagen sponge,

freeze dried chitosan sponge, pyrophosphate powder, and standard gauze were utilized

as control samples. The platelet adhesion rate was measured according to the following

equation:
𝐶0 − 𝐶1
Platelet Adhesion Rate = × 100%
𝐶0
where, C0 represents the initial concentration of the platelets and C1 represents the

concentration of the platelets in suspension after adhesion.

2.10 In vitro and in vivo degradation

The in vitro degradation tests were performed in simulated physiological

conditions. 50 mg of the samples was immersed in 10 mL of PBS (pH 7.4) and 10 mg

of lysozyme or collagenase I was added into it. The mixture was incubated at 37°C and
transfered to a dialysis bag (membrane molecular weight cut off 3 kDa) at determined

time points (1, 2, 4, 6, 8, 10, 12, 16 or 24 day). After dialyzed against distilled water for

48 h, the samples were lyophilized and weighed (Wt). The weight loss percentage

(∆W%) at each time interval was determined according to the following equation:
𝑊0 − 𝑊𝑡
∆𝑊(%) = × 100%
𝑊0
Where, W0 is the initial weight of each sample.

In vivo degradation was carried out using SPF New Zealand white rabbits. A slice

of the sponge (1 × 1 × 0.6 cm3) was implanted across the thigh muscle. All the animals

were carefully nurtured until the implanting terminals were reached (0, 1, 2, 3 weeks).

The degradation level of the implants was observed by anatomy and tissue paraffin

section.

2.11 Hemostatic evaluation in vivo

The hemostatic efficiency was tested in rabbit hepatic trauma model and in rabbit

ear artery model. In rabbit ear artery model, the middle auricular artery of a rabbit was

transversely cut by a scalpel blade after anesthesia with pentobarbital sodium. A piece

of conventional sterile gauze was used to absorb the blood flowing out in the beginning.

Then, one layer of the CPNFs-Col sponge was covered on the wound immediately. The

hemostatic time and the blood loss were recorded. In hepatic trauma model, the

abdomen of an anaesthetized rabbit was opened to expose its liver. The wound in the

anterior lobes of the liver was induced using a scalpel, measuring approximately 0.5

cm × 0.5 cm × 0.3 cm. The blood flowing out in the beginning was wiped off and one
layer of the CPNFs-Col sponge was covered on the wound immediately. Collagen

sponge, freeze dried chitosan sponge, pyrophosphate powder, and standard gauze were

utilized as control samples. The hemostatic time and the blood loss were recorded. The

blood loss was measured by weigh the weight of the samples before and after the

treatment.

2.12 Statistical analysis

All data were expressed as the mean ± SD. Differences between means were

analyzed for statistical significance using one-way ANOVA on SPSS 19.0. P values <

0.05 were considered significant and P values < 0.01 were considered extremely

significant (Li et al., 2014).

3 RESULTS

3.1 Preparation of CPNFS and composite sponges

The CPNFs were successfully synthesized via an improved one-pot preparation

method as described above. The reaction mechanism of this method was shown in

Figure 1a. As the adding of TPP, tiny Ca2P2O7 crystals were formed as seeds to provide

a well-defined template. Chitosan molecules were then adsorbed on the anisotropic

nano-crystals, resulting in the formation of hybrid nanoflowers. The mean diameter of

the CPNFs was calculated as 322 nm. As shown in Fig 1b (inset), the CPNFs presented

a blooming flower structure.

 The prepared CPNFs were further used for the modification of collagen sponge to

improve the hemostatic performance. The collagen sponge and CPNFs modified

collagen sponge (CPNFs-Col sponge) were prepared with a lyophilization method. As

shown in Figure 2 (a, d), the CPNFs-Col sponge presented a pale yellow surface after

coating with CPNFs. Figure 2 (b, e) exhibited the SEM images of the unordered porous

cavity interior structure of the sponges. The inner surface of CPNFs-Col sponge was

very rough with a plurality of protrusions (Figure 2f), which was different from the

smooth inner surface of collagen sponges (Figure 2c). With the increase of CPNFs

content, the specific surface area of the CPNFs-Col sponge was increased and then

decreased with a peak appeared at 30%. As shown in Table 1, the water absorbability

of sponges showed a similar trend as the specific surface area, while the pore size kept

growing with the increase of CPNFs content.

Figure 3a showed the FTIR spectra of CPNFs, collagen sponge and CPNFs-Col

sponge-30. In the spectrum of CPNFs, besides the correlative characteristic peaks of

chitosan, a new peak at 1127 cm-1 was observed. This was assigned to the asymmetrical

stretching vibration of P-O in TTP. The spectrum of collagen sponge and CPNFs-Col

sponge-30 showed three characteristic absorption peaks at 1659 (C=O) cm-1, 1552 (N-

H) cm-1 and 1393 (C-N) cm-1, corresponding to the amide band I, II and III, respectively.

The presence of a new absorption peak at 1127 cm-1 in the spectrum of CPNFs-Col

sponge-30 was assigned to the P-O in CPNFs, reconfirming the successful graft of

CPNFs.
 The successful coating of CPNFs on the surface of collagen sponge was also

confirmed by TG-DTG analysis in Figure 3b. The relevant TG curve of CPNFs showed

a three-stage weight loss in the range of 0 to 800°C. The first stage ranged below 122°C

with about 13.2% weight loss, which are corresponding to the loss of adsorbed and

bound water in CPNFs. The second stage ranging from about 170°C to 500°C showed

41.2% weight loss. This can be attributed to the degradation of the main chain of the

chitosan. The last stage was ranged from 520°C to 700°C with about 26.5% weight loss,

owing to the oxidative degradation of all organic molecules. Data from the collagen

sponge was presented for comparison. Very little weight loss (about 3%) was exhibited

during the low temperature stage. A relatively continuous weight loss (about 80%)

during the medium and high temperature stage was presented, due to the degradation

of collagen helix and amino acids from 180°C to 700°C. With the modification of

CPNFs, the relevant TG curve of CPNFs-Col sponge-30 showed a continuum between

the CPNFs and the uncoated collagen sponge.

The water absorption test showed (Figure 3c) that a droplet of water could be

absorbed into CPNFs-Col sponge-30 within 120 ms. The CPNFs-Col sponge-30 could

absorb water 50 times more than its own weight. The time required for the uncoated

collagen sponge to absorb equal water was more than 180 ms. The rapid water

absorption capacity of the CPNFs-Col sponge provided a theoretical foundation for

rapid hemostasis.

3.2 Biocompatibility of the sponges in vitro and in vivo

 The biocompatibility was studied in terms of intracutaneous stimulation test,

hemolysis assay, acute systemic toxicity assay and cytotoxicity assay. The results of

intracutaneous stimulation test showed (Figure 4a) that none of the CPNFs, collagen or

CPNFs-Col sponge samples had pathological reaction. The PIIs were all calculated to

zero (Figure 4a). Hemolysis tests were conducted using 2% rabbit haemocyte

suspension. As presented in Figure 4b, obvious hemolysis appeared in distilled water

while no evident hemolysis was detected in the collagen sponge, CPNFs-Col sponge-

30, CPNFs extract and NS. In order to describe the hemolysis more directly, the positive

control (distilled water) was set as 100% of hemolysis. The collagen and CPNFs-Col

sponge-30 showed excellent hemocompatibility with less than 0.6% hemolytic ratio.

In the acute toxicity test, none of the mice showed adverse reactions such as slow

movement or reducing food take during the following 3 consecutive days. All mice

were sensitive to sound, light and other stimulations without running nose, eye secretion

or edema symptom. The body weights (Figure 4c) of the collagen, CPNFs or CPNFs-

Col sponge-30 treated group showed no significant difference with the control group.

According to the results of cytotoxicity test (Figure 4d), all the collagen and CPNFs-

Col sponge-30 extract were non-cytotoxic to HUVEC cells with all concentrations

(0.125, 0.25, 0.5 and 1 mg/mL) in 2 days.

3.3 Hemoglobin adsorption and platelets adhesion

In order to find out the influence of CPNFs on blood-clotting, collagen sponges

with different content of CPNFs were tested in vitro. The hemoglobin adsorption rate
on the sponges was shown in Figure S1, in which the concentration of the hemoglobin

in 100 μL blood was set as 100%. A higher hemoglobin adsorption indicated the blood

was clotted more completely. The hemoglobin adsorption rate in the control group was

less than 8%, which means that the blood clots were difficult to form on culture dish in

5 min. A positive linear relationship between hemoglobin adsorption rates and CPNFs

content was found in the initial 2 min. This numerically proved that the modification of

CPNFs would enhance the blood coagulation effects in the very beginning. However,

high levels of CPNFs content were not conducive to the total hemoglobin adsorption in

5 min. The collagen sponge decorated with 30% CPNFs showed a highly

comprehensive hemoglobin adsorption ability.

Hemoglobin adsorption of different materials was shown in Figure 5a. The

CPNFs-Col sponge-30 showed the best clotting ability compared to the other tested

groups. The chitosan sponge had a faster coagulation rates than the collagen sponge

with no CPNFs in 2 min, but lagged behind since 3 min. The standard gauze and

pyrophosphate powder possessed limited blood clotting ability. The platelets adhesion

test was also evaluated (Figure 5b). The platelets adhesion rate on the CPNFs-Col

sponge-30 was about 0.5 times more than that on the collagen sponge. The adhesion

rate of platelets on collagen sponge and chitosan sponge were nearly equal, while that

for the standard gauze was a little lower.

3.4 In vitro interfacial interaction

 The in vitro interfacial interaction was carried out to explore the hemostatic

mechanism. As shown in Figure 6, the blood coagulation process on CPNFs-Col

sponge-30 could be broadly divided into three phases. Phase I involved the interfacial

interaction between blood cells and the sponge surface after contact within 1 min. The

CPNFs-Col sponge-30 could affect haemocytes to change their physiological state.

Numerous haemocytes stretched out pseudopodia on the surface of CPNFs-Col sponge-

30, which enhanced their aggregating activity. Meanwhile, no pseudopodium was

observed in the collagen sponge group. Phase II involved the interfacial interaction

among haemocytes from 2 to 3 min. In this phase, the blood cells continued aggregating

on the materials, forming a haemocytes layer. It was found that haemocytes on the

CPNFs-Col sponge-30 were closely connected with each other through pseudopodia.

As contrast, no intercellular junction was found in the collagen sponge group. Phase III

involved the formation of fibrin from 4 to 5 min. In CPNFs-Col sponge-30 group, the

plasmatic fibrinogen was firstly activated and transformed to fibrin monomer. The

soluble fibrin monomers further cross-linked and formed fibrin clots. No fibrin clots

were observed in the collagen sponge group within 5 min.

3.5 In vivo hemostatic evaluation

The in vivo hemostatic efficiency was tested in rabbit hepatic trauma model and

ear artery model (Table 2 and Figure S2). The CPNFs-Col sponge-30 showed the

strongest hemostatic potential with the shortest hemostasis time and the least amount

of bleeding. Collagen sponge, chitosan sponge and standard gauze could achieve
successful hemostasis at the cost of longer time and more bleeding. The pyrophosphate

powder could not stop the bleeding in both models.

3.6 Biodegradability evaluation

Based on the available results, the shelf-life of the CPNFs-Col sponge is longer

than 8 months in shade and dry condition. Collagen and chitosan could be degraded by

collagenase and lysozyme, respectively. The in vitro degradation of collagen sponge

and CPNFs-Col sponge-30 were conducted in simulated physiological environment at

37°C, as shown in Figure 7 (a, b). In collagenase І solution, the CPNFs-Col sponge-30

was degraded rapidly with about 50% weight loss in 48 h. Meanwhile, collagen sponges

degraded less than 40% within the same time. Both collagen sponge and CPNFs-Col

sponge-30 were degraded more than 95% after 16 days. In lysozyme solution groups,

the degradation of CPNFs-Col sponge-30 was relatively fast in the beginning (in 48 h),

and then slowed down with approximately 50% weight loss in 16 days. At the same

time, the collagen sponges were only degraded less than 30% in lysozyme solution. In

PBS solution, all sponges showed weak degradation properties with about 20% weight

loss in 16 days. These results indicated that the collagen sponge and CPNFs-Col

sponge-30 were more susceptible to the collagenase І solution than the PBS and

lysozyme solution.

In vivo degradation was evaluated in the thigh muscle of New Zealand white

rabbits. According to the anatomic research and histological section (Figure 7c), the

collagen sponge and CPNFs-Col sponge-30 were first decreased into little pieces in 14
days. The implanted sponges can be completely degraded and absorbed after 21 days

without redness or swelling occurred around the implantation site. This might be

attributed to the combined effects of protease, collagenase and lysozyme.

Biocompatibility evaluation in the rabbits showed that collagen sponge and CPNFs-Col

sponge-30 have good biological compatibility with no evidence of inflammatory

change, tissue necrosis or fibrinoid degeneration.

4. DISCUSSION

It has been reported that chitosan and collagen sponge both exhibited hemostatic

effects (Rao & Sharma, 1997; Singla & Chawla, 2001; Yang, La, Cho, Shin, Yeo &

Kim, 2012). Chitosan primarily achieve hemostasis by an electrostatic driving

mucoadhesive mechanism that may involve tissue adherence, haemocyte binding and

platelet activation. Collagen can activate human plasma thrombocytes to induce clot

formation for hemostasis and also provide scaffold-like support for platelet adhesion.

(Erdogan & van Gulik, 2008; Farndale, Sixma, Barnes & De Groot, 2004) In the present

study, chitosan and collagen was chosen to prepare CPNFs coated collagen sponges for

rapid hemorrhage control. The CPNFs were grafted to the collagen net by the amide

bonds between carboxyl groups of collagen and amino groups of chitosan chains

(Rashidova et al., 2004; Xi & Wu, 2006). Nanoflower structure was observed in the

CPNFs-Col sample (Figure 2). It has been reported that the deacetylation degree (DD)

and physical state of chitosan would influenced the hemostatic ability. Solid state

chitosan with approximately 80% DD exhibited good hemostatic activity (Fukasawa et

al., 1992; Yang, Tian, Wang, Wang, Zeng & Chen, 2008). Therefore, 82.5% DD

chitosan was employed in this study. Our study showed that the synthetic CPNFs-Col

sponge was efficient in producing rapid hemorrhage control by multiple modes of

action.

Topical hemostasis mainly involves the constriction of blood vessels, the adhesion

and activation of platelets, the generation of thrombin and finally the conversion of

fibrinogen to fibrin. Every effort to promote the above processes would be definitely

helpful for rapid hemostatic. The specific surface area, active groups and surface

charges play a critical role in the interaction between hemostatic materials and the blood

coagulation system (Fischer, Bode, Demcheva & Vournakis, 2007).

The exact hemostatic mechanism of the new CPNFs-Col sponge-30 remains

indistinct while several interpretations could be suggested. Firstly, the hydrophilic

CPNFs-Col sponge-30 can quickly absorb water from blood flow in the wound site.

This rapid water absorption can effectively improve the concentration of clotting factors

in blood flow. High levels of blood clotting factors would jump-start the coagulation

cascade and accelerate the subsequent formation of blood clot. This physical absorption

effect of CPNFs-Col sponge-30 can be attributed to the high specific surface area of the

water absorbing cavities (Figure 2).

Secondly, the protonated amine groups in the deacetylated repeat units (Yang, Tian,

Wang, Wang, Zeng & Chen, 2008) and the existence of calcium ions would be a

meaningful parameter for hemostatic activity (Brandenberg, Leibrock, Shuman,

Malette & Quigley, 1984). The haemocyte was coated with negatively-charged
neuraminic acid residues in the blood flow. The CPNFs-Col sponge can immobilize

large amounts of haemocytes by electrostatic interaction and cause irreversible

deformation thus to accelerate the clotting process.

Thirdly, the surface topography in the micron range could modulate the activation

of haemocytes and platelets, in terms of adhesion and platelet-selectin expression. The

surface topography changes caused by CPNFs may influence the activation of platelets

and plasmatic fibrinogen. (Ferraz, Carlsson, Hong & Ott, 2008; Flemming, Murphy,

Abrams, Goodman & Nealey, 1999).

In addition, the CPNFs-Col sponge possesses high biocompatibility and good

biodegradability, which is suitable for post-operative treatment and even peritoneal

adhesion prevention. Nevertheless, further optimization for biological applications,

wound healing, packaging and administrating approach remains to be studied,

particularly for the evaluation in more clinically relevant models.

5. CONCLUSION

In summary, a collagen sponge reinforced with chitosan/calcium pyrophosphate

nanoflowers has been designed for rapid hemorrhage control. The obtained CPNFs-Col

sponge with optimized composition possessed rapid water absorption ability, positive

surface rich in amino groups and high specific surface area. The CNPFs-Col sponge

could activate the intrinsic pathway of coagulation cascade, induce haemocytes and
platelets adherence, promote the blood clotting and achieve rapid hemorrhage control.

In addition, the good biocompatibility and biodegradability properties made it well-

suited for post-operative treatment and even peritoneal adhesion prevention. It can be

concluded that the CPNFs-Col sponge has the potential to be a promising candidate

clinical hemostatic applications.

ACKNOWLEDGEMENTS

The authors are indebted to the financial support from State Key Laboratory for

Modification of Chemical Fibers and Polymer Materials, Donghua University.

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a* O
O OH
+H N
3
OH
b
12
Chitosan/Calcium
Pyrophosphate Nanoflowers
O * 10

+H N OH O n
3 OH 8

Intensity (%)
O
Ca2+ O- O-
P O 6
-O
P O O
P Ca2+ 200 nm
O 4
-O O-
OH +H N
O 3 2 Mean diameter 322 nm
OH
* O
OH *
NH3+ HO O O n 0

100 1000 10000

1 Partical size (nm)

Figure 1. (a) The reaction mechanism of one-pot preparation method to synthesize CPNFs; (b)

The particle distribution and SEM image (inset) of the CPNFs.

Figure 2. Photograph of the collagen sponge (a) and CPNFs-Col sponge (d) (1.5 cm diameter,

0.5 cm thickness); The SEM image of the interior structure of the collagen sponge (b) and CPNFs-

Col sponge (e); The magnified SEM images of cavity inner surface of the collagen sponge (c) and

CPNFs-Col sponge (f).

 a 59 52
b 100 -2.0 c 80 220
27

93
16 15

Water Absorption Ability(times)

11 Water Absorption Ability

13
O -H O CPNFs
P-

-N
C= N Water Absorption Rate

Water Absorption Rate (ms)

Collagen sponge 200
80 70
CPNFs-Col sponge-30
-1.5

Transmittance

DTG(%/°C)
CPNFs-Col sponge-30 180

Weight(%)
60
60 637.0°C *
239.9°C -1.0
50
** 160

40
259.0°C
270.4°C
* 140
Collagen sponge 40
20 -0.5 120

513.8°C 639.6°C 30
CPNFs 0 ** 100
0.0
20 80
3500 3000 2500 2000 1500 1000 0 200 400 600 800
-1
Wavenumber(cm ) Temperature(°C) collagen sponge CPNFs-Col sponge-30

Figure 3. (a) The FTIR spectra of CPNFs, collagen sponge and CPNFs-Col sponge-30; (b) The

TG-DTG analysis of CPNFs, collagen sponge and CPNFs-Col sponge-30; (c) The water absorption

test of collagen sponge and CPNFs-Col sponge-30 (All data were expressed as the mean ± SD (n =

3). ** indicates extremely significant difference from the control group, p < 0.01, and * indicates

significant difference from the control group, p <0.05).

 a 5.0 b
110

105
4.35 0 0 0 0 100%

Skin irritation index

100
4.5

Hemolysls(%)
95

0
+ - - - - 1.4

-1 0.55% 0.60%
0.50%
0.7
0%
-2
0.0
l e e Distilled Normal CPNFs Collange CPNFs-Col
Methana Normal Saline CPNFs ollagen Spong NFs-Col Spong
C CP water Saline Sponge Sponge-30
30
c PBS
d
120
28 Collagen sponge
CPNFs-Col sponge

Cell viability(%)
26 CPNFs
Weight (g)

80 PBS
24
Collagen sponge
CPNFs-Col sponge-30
22 CPNFs
40
20

18
0 20 40 60 200 400 600 800 1000
Time (h) Concentration(μg/mL)

Figure 4. (a) The results of intracutaneous stimulation tests; (b) The results of hemolysis tests; (c)

The results of acute toxicity tests; (d) The results of cytotoxicity tests. All values are expressed as

the mean ± SD for each group.

a 100 b 100
Culture dish
**
Platelet adhesion rate (%)

Collagen sponge
Hemoglobin adsorption (%)

CPNFs-Col sponge-30 80
Pyrophosphate powder
Standard gauze
Chitosan sponge 60
* *
50

40

20

0 0
1 2 3 4 5 CPNFS-Col Collagen Standard Chitosan
Time (min) sponge-30 sponge gauze sponge

Figure 5. (a) The hemoglobin adsorption of different materials; (b) Platelet selective adhesion

test of different materials (All data were expressed as the mean ± SD (n = 5). ** indicates extremely

significant difference from the control group, p < 0.01, and * indicates significant difference from

the control group, p <0.05).

Figure 6. SEM images of interfacial interaction.
 a120 PBS solution Collagen sponge b120 PBS solution CPNFs-Col sponge-30
1 mg/mL lysozymle solution 1 mg/mL lysozymle solution
1 mg/mL collagenase I solution 1 mg/mL collagenase I solution

Weight loss (%)

Weight loss (%)

80 80

40 40

0 0
0 5 10 15 20 0 5 10 15 20
Time (day) Time (day)

Figure 7. In vitro degradation studies of the collagen sponge (a) and CPNFs-Col sponge-30 (b);

(c) In vivo degradation and biocompatibility of the collagen sponge and CPNFs-Col sponge-30 (the

sponges were indicated by arrows). All values are expressed as the mean ± SD for each group (n =

3).
 Chitosan/Calcium Hemostatic Sponge
Pyrophosphate Nanoflowers
Freeze
Drying
NH 2 -
Collagen NH 2 -

NH 2-
NH 2-

Blood
Polypeptide
Degradation
in vivo

Pore:
Oligochitosan rapid absorption

NH 2 -
NH 2 -

NH 2 -
NH 2 -

Nanoflowers:
facilitate clotting
Scheme. 1 Schematic illustration of the preparation process of hemostatic sponge and

mechanism for its degradation

 Table 1. The specific surface area, pore size and water absorbability of sponges

pore size
(μm)

Sample Chitosan content The specific surface Water absorption ability

(w/w %) area (m2 g−1) (times)

123.1±12.6

Collagen sponge 0 853.2±30.1 42.23±5.21

129.5±15.2

CPNFs-Col sponge-15 15 884.5±18.4 46.31±4.67

130.3±13.2

CPNFs-Col sponge-30 30 952.5±27.3 50.61±6.32

CPNFs-Col sponge-40 40 928.6±11.6 135.7±10.5 49.35±2.46

All values are expressed as the mean ± SD for each group (n = 3).
 Table 2. In vivo hemostatic evaluation in hepatic trauma model and ear artery model

Hepatic trauma model Ear artery model

Sample
Hemostasis Hemostasis time Amount of bleeding
Amount of bleeding (g)
time (s) (s) (g)

--
Pyrophosphate powder -- -- --

164 ± 10.3
Chitosan sponge 184 ± 10.3 1.48 ± 0.38 1.63 ± 0.25

173 ± 15.3
Standard gauze 233 ± 15.6 1.52 ± 0.40 1.84 ± 0.27

153 ± 11.0
Collagen sponge 138 ± 5.3 1.27 ± 0.10 1.32 ± 0.12

106 ± 7.3 0.95 ± 0.08 135 ± 8.0 1.04 ± 0.11

CPNFs-Col sponge-30