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Effective hemorrhage control is vital for reducing mortality after major trauma both in civilian life and in
the military. In recent years microporous starch (MS) has been used as a hemostatic agent. However, MS
has an insufficient hemostatic capacity to stop severe bleeding. To improve its hemostatic performance,
in this study calcium-modified microporous starch (CaMS) was firstly developed via oxidization and self-
assembly with calcium ions (Ca2+) on MS, and the hemostasis efficiency and degradation behaviour
were evaluated. The results showed that the carboxyl groups and Ca2+ had been modified successfully
onto MS. MS and CaMS both initiated the hemostatic response by rapid absorption and swelling due to
their porous structure and high surface area. It is noteworthy that CaMS activated an intrinsic pathway of
coagulation cascade and induced platelet adhesion because of the modified Ca2+ and carboxyl groups.
The synergistic effects of the chemical activation mechanism and physical absorption mechanism
Received 4th February 2015, resulted in a dramatic improvement in the hemostatic capacity of CaMS, and thus achieved an effective
Accepted 8th April 2015 hemorrhage control in rabbit liver and femoral artery injuries. Additionally, the degradation of CaMS was
DOI: 10.1039/c5tb00250h improved greatly by the modification. In conclusion, CaMS effectively improved hemostatic performance
and degradability. CaMS is a promising candidate for designing hemostatic agents in more extensive
www.rsc.org/MaterialsB clinical applications.
which allows for internal hemorrhaging applications. Combined Additionally, the degradable behavior of the CaMS was also
with its inherent biosafety, simplicity of preparation and long investigated by immersion in a modified simulated body fluid
shelf life during storage, MS has become one of the most (mSBF).
attractive hemostatic agents.
Although effective in hemorrhage control for low bleeding
Experimental
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water three times and dried in a vacuum-freeze dryer. 6CaMS a humidified atmosphere with 5% CO2 at 37 1C for 7 days, with
and 9CaMS were then prepared and stored in a desiccator at the solution being changed daily. When the solution was
room temperature for further analysis. All preparations were changed, the sample was filtered, gently rinsed with deionized
made in triplicate. water, and dried at 80 1C to a constant weight. The final weight
of each sample was measured and recorded as Wt. The weight
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Characterization of CaMS loss rate of each sample was calculated according to eqn (2).
The morphology of CaMS was examined through scanning Each test was repeated three times and the average value was
electron microscopy (SEM, JSM-6360LV, JEOL, Japan) and coating calculated.45
with gold at an acceleration voltage of 15 kV. The phase composi-
W% = [(W0 Wt)/W0] 100% (2)
tions were analyzed with X-ray diffraction (XRD, Rigaku, Japan)
using Cu-Ka radiation. Fourier-transform infrared spectroscopy The absorption ratio of samples depended on the maximum
(FTIR, Nicolet 380, Thermo, USA) was used to study the structural amount of liquid per unit of materials absorbed in the mSBF.3,28
organization of CaMS. To remove the moisture inside, all samples were vacuum-dried at
(1) Determination of the carboxyl content. The carboxyl 80 1C, and then each sample was weighed and recorded as W0.
content of OMS was determined by alkaline titration. OMS (2 g) Afterwards, the samples were placed on filter paper in a funnel,
was stirred in 30 mL of 0.1 M aqueous HCl for 30 min. The and deionized water was continuously added drop-wise to the
slurry was then filtered and washed with distilled water until samples at a rate of 8 mL min 1 until the samples reached their
free of chlorine ions, which was verified using AgNO3 solution. limit, which was determined when the first drop of fluid fell
The slurry was carefully transferred into a beaker with 300 mL into the filter flask. The weight of sample at its maximal water
deionized water and heated in a boiling water bath with con- absorption was recorded as W1. The absorption ratio of each
tinuous stirring for 20 min to ensure complete gelatinization. sample can be calculated according to eqn (3):
The hot starch was titrated with 0.1 M NaOH using phenol-
phthalein as an indicator. A blank determination for MS was A% = [(W1 W0)/W0] 100%. (3)
carried out in the same manner. In the blank determination the
Cold-water solubility and swelling properties of the samples
sample had previously been stirred in 30 mL deionized water
were analyzed in triplicate following the method of Dhital
instead of 0.1 M HCl. The carboxyl content can be calculated
et al.29 In brief, starch samples (200 mg, recorded as W) were
according to eqn (1)
suspended in 10 mL distilled water in a tared centrifuge tube.
n = [(V1 V0)/2] c, (1) The starch suspension was incubated at 25 1C for 30 min with
stirring to extract the cold-water soluble materials. Afterwards
where n represents the carboxyl content (mmol); V1 and V0
the suspension was centrifuged at 3000 rpm for 20 min, the
represent the consumption volume of NaOH (mL) for the
supernatant was decanted carefully and dried at 105 1C to a
titration of OMS and MS, respectively; and c represents the
constant weight (A). The residue was also dried and the final
concentration of NaOH used (mol L 1).
weight was recorded as P. Swelling and solubility were determined
(2) Calcium content in CaMS. CaMS (1 g) was calcined at
using the following eqn (4) and (5):
600 1C for 4 h to remove organic compounds. CaCl2 solution
was formed by dissolving the sintered CaMS in 1 M HCl. The Solubility = A/W 100%, (4)
Ca2+ concentration in the CaCl2 solution was measured with
an inductively coupled plasma-atomic emission spectroscopy Swelling rate = P/W 100%. (5)
(ICP-AES, IRIS 1000, Thermo Elemental, USA).
Assessment of plasmatic coagulation in vitro
Degradation in vitro, absorption ratio, solubility and swelling Activated partial thromboplastin time (APTT) and prothrombin
In vitro degradation was performed by immersing the samples time (PT) were performed using a semi-automatic coagulation
in a modified simulated body fluid (mSBF). The degradation analyzer (Biomerieux, France). The study was approved by
rate of the sample was determined by its weight loss rate in the the Research Center for Drug Safety Evaluation of Shanghai
mSBF. Since endogenous levels of a-amylase are approximately University of Traditional Chinese Medicine. Fresh blood con-
40–220 U L 1 in the human body, mSBF contained 50 mg of taining 3.8% sodium citrate [sodium citrate : blood = 1 : 9 (v/v)]
2000 U g 1 a-amylase and 1 L of pure simulated body fluid was collected from Sprague-Dawley rats into a siliconized tube.
(SBF). SBF was prepared according to the procedure described The platelet poor plasma (PPP) was immediately obtained by
by Kokubo.27 The solution was buffered at pH 7.4 using centrifuging the blood at 3000 rpm for 15 min at 37 1C.
tris(hydroxymethyl) aminomethane ((CH2OH)3CNH2) and 1 M APTT:APTT:6MS or 6CaMS powder was incubated separately
HCl at 37 1C. with 0.1 mL APTT reagent and 0.1 mL PPP at 37 1C. After
Before immersion, the samples were dried at 60 1C for 24 h. incubation for 5 min, 0.1 mL CaCl2 was added in the test tube
The initial weight of each sample was accurately measured and and APTT was tested simultaneously. PT: 0.1 mL of PPP was
recorded as W0. The samples were immersed in the mSBF at a incubated separately with 6MS or 6CaMS powder at 37 1C in test
weight to volume ratio of 0.2 g : 1 mL in plastic bottles. The tubes. After incubation for 3 min, 0.2 mL of PT reagent was
bottles were continuously shaken in a water bath at 100 rpm in added and PT was measured simultaneously.
self-assembled with 6OMS to form 6CaMS. seconds until the bleeding stopped, and then the superficial
Platelet adhesions of 6MS and 6CaMS were determined with samples were wiped from the injury site. Samples after hemo-
a lactate dehydrogenase (LDH) assay. Before the assay, blood stasis were collected for SEM observation to explore the inter-
from the rabbits’ ear-rim vein was mixed with 0.109 M sodium action between the sample and blood. After completion of the
citrate solution at a ratio of 1 : 9 in test tubes. Platelet rich treatment, the abdomen was temporarily closed with a mono-
plasma (PRP) was obtained by centrifugation at 1500 rpm for filament suture, and the animal was monitored for 180 min
5 min. MS and CaMS granules were incubated at 37 1C for after injury or until death, whichever came first.
30 min in PRP with the number of platelets adjusted to 2 (2) Rabbit femoral artery injury experiment. 15 rabbits
108 platelets per mL. Then non-adherent platelets were thoroughly separated into three groups (6MS, 6CaMS and standard gauze)
washed by PBS (137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO412H2O, were anesthetized and their hair was shaved off to expose the
1.5 mM KH2PO4) at pH 7.4. Thereafter the samples were placed groin with their hind limbs extended. After transecting the
into a 24-well plate, and the adherent platelets were then lysed femoral skin and overlying muscles, the femoral artery was
with 0.25 mL 1% Triton X-100 in PBS at 37 1C for 1 h. The LDH exposed and a massive hemorrhage was created by clipping the
assay was performed on the lysates using the LDH/LD Kit (Sigma- femoral artery. Immediately, approximately 8 g of 6MS or
Aldrich, USA) and the number of adherent platelets was deter- 6CaMS were applied to the injury site. Manual compression
mined from the calibration curve. Previously, a calibration on the wound site was needed and given by a pad of gauze until
curve was obtained in a series of known concentrations of the bleeding ceased. The gauze was slightly uplifted to observe
platelets by measuring the OD between 490 nm and 650 nm the injury condition every five seconds. At the end of the study
spectrophotometrically (Multiskan MK3, Thermo Electron period, each groin was opened and both the liquid and clotted
Corporation, USA). Standard gauze was used as a control. For inguinal blood and materials were suctioned and measured.
each experimental condition, one set of samples (n = 5) was used To further evaluate the effect of hemostasis, the mortality rate
for the platelet adhesion measurement. for the femoral artery injury was calculated by division of the
The samples were fixed with 0.1 M phosphate-buffered number of dead rabbits by the total amount of rabbits within
solution containing 2.5% glutaraldehyde and 2.0% paraformal- 2 h after hemostasis. After being supervised for 2 h, the surviving
dehyde (pH 7.4), and then post-fixed with 1% osmium tetroxide rabbits were euthanized using an overdose of pentobarbital.
in the same buffer. After being dehydrated using a graded
series of ethanol solutions, the samples were examined via Statistical analysis
SEM, using the aforementioned experimental parameters. Results were expressed as means standard deviations. Except
special requests in the animal experiment, all data were generated
using more than three independent experiments. Statistical analysis
Hemostasis in an animal model
was conducted using one-way analysis of variance (ANOVA). A value
All animal experiments were approved by the ethical committee of p o 0.05 was considered to be statistically significant.
of the National Tissue Engineer R&D Center (Shanghai, China),
and were conducted in accordance with the ‘‘Regulations for
the administration of affairs concerning experimental animals’’ Results and discussion
issued by the Chinese Ministry of Science and Technology of
the 31 October 1988. Chemical modification is an effective method to generate
5-month old male New Zealand white rabbits (wt 3–4 kg) different surface properties. Protein adsorption is essential in
were purchased from Silaike Co. Ltd (Shanghai, China). the blood-borne hemostatic response of foreign materials. In
They were made to fast 24 h with water permitted only. All an initial response, during blood-material contact, the protein
samples were dried at 80 1C in a vacuum oven overnight and adsorption mechanism is greatly affected by the surface proper-
then sterilized using ultraviolet radiation (Heming Co. Ltd, ties of the material. It was reported that platelets adhered to
Shanghai, China) for 2 h before the experiment. Standard gauze positively charged surfaces,29,30 and that the negatively charged
was used as a control. All procedures were performed under surface induced coagulation was initiated via the auto-activation
sterile conditions. of coagulation factor XII.31 Therefore, chemically modified MS,
(1) Rabbit liver injury experiment. 15 rabbits separated an active hemostatic agent, could enhance the hemostatic
into three groups (6MS, 6CaMS and standard gauze) were capacity not only by its physical adsorption but also by activating
selected for hemostasis in rabbit liver injury. After the rabbits platelets. However, these modified MSs showed poor degrad-
were anesthetized via intravenous injection of pentobarbital ability or toxicity after modifications.23
and their abdominal hair was shaved off, the abdomens were Calcium ions (the fourth clotting factor) are important to
opened to expose the liver. A ‘‘+’’ shaped wound approximately activate the coagulation system to form thrombin, a key enzyme
time, and platelet adhesion were carried out. PTs and APTTs
Table 1 The physical properties of corn starch, 6MS and 6CaMS of samples were determined and the results are presented in
Corn starch 6MS 6CaMS Fig. 5. Compared with the control (standard gauze), the APTT of
6MS was higher than that of 6CaMS. When the amount of
Solubility (%) 0.21 8.01 11.71
Swelling (%) 153.74 398.32 420.42 6CaMS increased from 2 mg to 4 mg, the APTT of 6CaMS
Absorption ratio (%) 56.3 161.5 244.2 dropped to approximately half of its initial value (Fig. 5a).
In addition, Fig. 5b shows that the PTs of samples follows a
similar trend to that seen in APTT, with 6CaMS shortening PT
The degradation rate of all samples increased with time after more than 6MS. However, no significant dosage-dependent
immersion. The weight loss of the samples was greatest during relationship was observed in PT since it did not change
the first two days of immersion. Shortly thereafter the weight obviously with the use of 4 mg of sample compared to the
loss plateaued. Interestingly, although there was no significant 2 mg samples. The results indicated that the prepared CaMS
difference between 9MS and 9CaMS in weight loss during the could not only shorten the time of initial fibrin formation
first five days, 9MS (76%) had a higher weight loss than that of significantly, but it could also accelerate the activation of the
9CaMS (68%) on the sixth immersion day. After immersion for intrinsic coagulation system. With an increase in CaMS con-
7 days, 6CaMS possessed the greatest weight loss of 83.8%, tent, its coagulation system activation effects became obvious.
while 6MS showed the lowest weight loss of 63.4%. This may be
due to the porous structure of MS, the oxidization treatment, Platelet adhesion
and the self-assembly of calcium ions on the starch. Platelet adhesion to the samples was evaluated by agitation of
Fig. 4b shows the absorption ratio of MS and CaMS in the the samples in PRP followed by image capture of the samples
mSBF. The absorption ratio of CaMS was much higher than with adherent platelets by SEM (Fig. 6). It can be seen that the
that of MS at the same hydrolysis time. 6CaMS had the highest number of platelets adhered to the 6CaMS increased greatly.
absorption of 244.2%, nearly 50% higher than that of 6MS
(161.5%). The absorptions of 9CaMS and 9MS were 175.2% and
219.1%, respectively. 6CaMS exhibited a higher absorption
than 9CaMS. This may be due to the reduced surface area
caused by the structure collapse due to excessive oxidation.
Table 1 plots the physical properties of corn starch, 6MS and
6CaMS. It was obvious that 6CaMS had the highest solubility,
swelling and absorption ratio, which was conducive to improving
the efficacy in controlling bleeding. This result indicated that
chemical modification is an effective method to increase the
hemostatic activity of starch particles.
Due to the higher degradation and absorption in vitro of
6CaMS than 9CaMS, only 6CaMS, 6MS and the control group
were used in the following experiments.
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