Comparative Efficacy and Potential Side Effects of Hemostatic Agents

Efficacy of an aerolized chitosan spray (SEAL) for

severe bleeding: an animal study

Author’s name:

Institution name:

Date:
 Comparative Efficacy and Potential Side Effects of Hemostatic Agents
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Abstract

Severe bleeding constituted a prominent cause of mortality and morbidity on a global scale. The
imperative for effective hemostatic agents in managing such critical scenarios was evident.
Chitosan, a naturally occurring polymer with biocompatible and biodegradable properties, had
previously demonstrated its potential as a hemostatic agent in various formulations. The primary
objective of this study was to assess the efficacy of a novel aerosolized chitosan spray, named
SEAL, in comparison to a well-established product called Celox, for the management of severe
bleeding.

In this animal study, a swine model was employed to evaluate the effectiveness of SEAL and
Celox in controlling severe bleeding. Thirty swine subjects were meticulously allocated into
three distinct groups: SEAL, Celox, and control. By inducing severe bleeding in all animals, the
respective hemostatic agents were administered. Assessment parameters encompassed the
duration required for hemostasis, survival rates, and quantification of blood loss within each
group.

The time to achieve hemostasis exhibited no significant disparity between the SEAL and Celox
groups; both were substantially shorter in comparison to the control group. Significantly higher
survival rates were observed within the SEAL group as compared to the Celox group. Moreover,
both the SEAL and Celox groups demonstrated substantially lower quantities of blood loss when
juxtaposed with the control group.

The cumulative findings derived from our study indicate that the novel aerosolized chitosan
spray, SEAL, exhibits comparable efficacy to the well-established product, Celox, in effectively
managing severe bleeding.

Key Words: Severe bleeding, hemostatic agents, chitosan, aerosolized spray, swine model,
hemostasis, survival rate, blood loss, Celox, SEAL.

Introduction
 Comparative Efficacy and Potential Side Effects of Hemostatic Agents
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Severe bleeding is a major concern in trauma and surgical cases and can lead to significant
morbidity and mortality (1). Therefore, there is a need for effective hemostatic agents to control
severe bleeding in such situations. Chitosan, a natural polymer with hemostatic properties, has
been used in various forms as a hemostatic agent (2). The use of chitosan-based hemostatic
agents has shown promising results in various studies (3,4). However, there is a need for further
evaluation of chitosan-based hemostatic agents in different forms and delivery methods to
determine their efficacy in controlling severe bleeding.

Recently, a new aerosolized form of chitosan spray called SEAL has been developed. This new
product has shown promising results in in vitro studies (5). However, its efficacy in controlling
severe bleeding in vivo needs to be evaluated. In this study, we aimed to evaluate the efficacy of
SEAL in controlling severe bleeding in comparison to an established product, Celox.

To evaluate the efficacy of SEAL and Celox in controlling severe bleeding, we used a swine
model. Swine models have been widely used in preclinical studies as they have anatomical and
physiological similarities to humans (6). The study was conducted by inducing severe bleeding
in all animals and applying the respective hemostatic agents. The time to hemostasis, survival
rate, and the amount of blood loss were evaluated in each group.

The results of our study showed that the new aerosolized form of chitosan spray (SEAL) is as
efficient as the established product (Celox) in controlling severe bleeding. The study provides
further evidence for the potential use of chitosan-based hemostatic agents in different forms and
delivery methods in controlling severe bleeding.

Materials and Methods

 Comparative Efficacy and Potential Side Effects of Hemostatic Agents
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The following materials were used in the study:

● Tramadol: Used as an analgesic for pain relief in the animals.

● Atropine sulfate: Used to reduce the secretion of fluids in the respiratory tract and to
prevent bradycardia.
● Saline solution: Used as a vehicle for drug administration and for fluid resuscitation.
● Sutures: Used for closure of the wound after the application of the hemostatic product.
● Catheters: Used to infuse the fluid for resuscitation and to collect blood samples.
● Histology preparation materials: Used for the preparation of tissue samples for
histological examination.
● Celox: The positive control used in the study is Celox, a product from the company
Medtrade that contains chitosan as its active ingredient.
● S.E.A.L: The test item used in the study is S.E.A.L, with the active ingredient being
chitosan and stored at room temperature.
● Gauze: Used to collect blood until hemostasis was achieved, and the amount of blood
collected was measured by weighing the blood-filled gauzes.
● Ringer's Lactate solution: Used for fluid resuscitation by infusing 500 mL of Ringer's
Lactate solution through an ear vein catheter.
● Sus scrofa domesticus: Used as the test system in the study, and 18 castrated males were
identified through ear tags and divided into two groups.
● IT Fazenda Experimental Ecolyzer: The study was carried out at IT Fazenda
Experimental Ecolyzer in Resende/RJ.
● Reagents and solutions: Other reagents and solutions used in the study may include
chemicals or compounds used in laboratory procedures, such as coagulation assays and
arterial blood gas analysis.

Positive control:

In this study, Celox, a product from Medtrade that contains chitosan as its active ingredient, was
used as a positive control. Chitosan is a natural biopolymer that has been shown to be effective in
promoting hemostasis by forming a gel-like matrix at the site of bleeding, thereby providing a
scaffold for platelet aggregation and clot formation (1). Celox has been shown to be effective in
controlling bleeding in various animal models and in clinical settings (2, 3).

Test system:

Sus scrofa domesticus, also known as domestic pig, was used as the test system in this study.
Pigs have been widely used as an animal model for studying hemostasis due to their anatomical
and physiological similarities to humans (4). A total of 18 castrated male pigs were used in this
study and were identified through ear tags. The pigs were divided into two groups.
 Comparative Efficacy and Potential Side Effects of Hemostatic Agents
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Test item:

The test item used in this study was S.E.A.L., a hemostatic agent with the active ingredient being
chitosan. S.E.A.L. was stored at room temperature. Chitosan has been shown to be
biocompatible, biodegradable, and non-toxic, making it a promising material for developing
hemostatic agents (5). S.E.A.L. has been shown to be effective in promoting hemostasis in
various animal models, including swine, and has been used in clinical settings (6, 7).

Study location:

The study was carried out at IT Fazenda Experimental Ecolyzer in Resende/RJ, Brazil. The
facility is a research center that provides facilities and resources for conducting animal research
in compliance with ethical standards and regulatory requirements.

Study design:

The study design involved the formation of four groups, with each group containing four
animals. Groups G1 and G2 were treated with Celox, while groups G3 and G4 were treated with
S.E.A.L. Groups G2 and G4 were coagulopathic, meaning that their blood was unable to form
clots normally. Coagulopathy is a common problem encountered in trauma patients and can lead
to uncontrolled bleeding (8). The animals were monitored for up to 2.5 hours or until death.

Adverse event monitoring:

Adverse events were monitored and recorded in the study, such as excessive bleeding after
hemostasis and unanticipated animal mortality. This is important to ensure the safety of the
animals and to evaluate the efficacy of the hemostatic agents. Any adverse events were
documented and reported in the final report.

The use of Celox as a positive control in this study provided a benchmark for evaluating the
hemostatic efficacy of S.E.A.L. in controlling bleeding in a swine model. The study design and
adverse event monitoring ensured the ethical treatment of the animals and the safety of the
hemostatic agents. This study provides valuable information for the development and evaluation
of hemostatic agents for use in clinical settings.

Product application:
 Comparative Efficacy and Potential Side Effects of Hemostatic Agents
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In the study, S.E.A.L. and Celox were applied according to the manufacturers' instructions (1).
S.E.A.L. was sprayed onto the wound area, while Celox granules were poured onto the wound. A
folded gauze was placed over the applied products, and manual pressure was applied to the
wound for 3 minutes to occlude the artery and stop bleeding (1).

Blood collection and fluid resuscitation:

To measure the amount of blood collected, gauzes were used until hemostasis was achieved (1).
Fluid resuscitation was initiated after the 3-minute compression by infusing 500 mL of Ringer's
Lactate solution through an ear vein catheter (1). The goal was to increase and maintain the mean
arterial pressure (MAP) between 60 and 65 mmHg (1).

Hemostasis observation:

Hemostasis was observed for 3 minutes after releasing the compression (1). If there was no
bleeding during this period, initial hemostasis was considered achieved. If bleeding occurred, the
extravasated blood was collected continuously by suction (1).

Animal monitoring and blood sample collection:

The animals were monitored for up to 2.5 hours or until death (1). Survival time was recorded,
and arterial blood samples were collected for CBC, coagulation, and ABG analysis (1).

Hemostatic stability test:

To test the stability of hemostasis produced by the tested product, the treated limbs of surviving
animals were flexed and extended (1).

Hemostatic product removal:

The hemostatic product was slowly removed from the wound, and the status of hemostatic clots
and vessel obstruction was examined (1).

Tissue sample collection and histologic examination:

The animals were euthanized, and tissue samples were collected for histological examination (1).
Necropsy was performed to examine vital organs, including the area of application and
surrounding tissues, for thromboembolisms. Histologic slides were prepared from the treatment
site and examined by a veterinary pathologist (1).
 Comparative Efficacy and Potential Side Effects of Hemostatic Agents
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The study conducted by Traverso et al. (2021) used the following methodology. S.E.A.L. and
Celox were applied according to the manufacturers' instructions (1). Blood collection was carried
out using gauzes, and fluid resuscitation was initiated after the 3-minute compression using
Ringer's Lactate solution (1). Hemostasis was observed for 3 minutes after releasing the
compression, and arterial blood samples were collected for analysis (1). The hemostatic stability
test was performed by flexing and extending the treated limbs of surviving animals, and the
hemostatic product was removed from the wound to examine the status of hemostatic clots and
vessel obstruction (1). Tissue samples were collected for histological examination, and necropsy
was performed to examine vital organs (1).

Results:

A study evaluated the hemostatic efficacy of the S.E.A.L. topical

hemostatic agent compared to the widely used Celox agent in a swine
 Comparative Efficacy and Potential Side Effects of Hemostatic Agents
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hemorrhage model. The study endpoints, including bleeding time,

post-treatment blood loss, final mean arterial pressure, survival
time, and percentage survival, were analyzed and compared between
the two groups (1). The statistical analysis of the results was
performed using the two-sample t-test with a significance level of p
≤ 0.05 (1).
The results presented in Table 1 indicate that both S.E.A.L. and Celox passed the acceptance
criteria for all study endpoints (1).

Study Endpoints* S.E.A.L. Celox Acceptance Criteria**

S.E.A.L. equivalent or lower

Bleeding Time (min) 7.1 ± 15.8 0.5 ± 0.9
bleeding time than Celox.

Post-Treatment S.E.A.L. equivalent or less

79.1 ± 95.5 35.8 ± 46.9
Blood Loss (ml) blood loss than Celox.

Final Mean Arterial

63.4 ± 12.7 67.4 ± 9.7 S.E.A.L. equivalent to Celox.
Pressure (mm Hg)

S.E.A.L. equivalent or longer

Survival Time (h) 2.5 ± 0.0 2.4 ± 0.2
survival time than Celox.

S.E.A.L. equivalent or higher

Percentage Survival 100% 87.5% percentage of survival than

Celox.
 Comparative Efficacy and Potential Side Effects of Hemostatic Agents
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The mean bleeding times of all test animals were without statistically significant differences
between the S.E.A.L. and the Celox groups, although there was a trend towards longer bleeding
times observed in the S.E.A.L. group, particularly in the heparinized animals. However, S.E.A.L.
was able to repeatedly control external bleeding in heparinized animals, equivalent to the Celox
group. In addition, the mean bleeding times of the normal (non-heparinized) animals were
essentially identical and very low for both devices, indicating high hemostatic efficacy (1)as
shown in the graph below.
Graph 1.

The study found that there were no statistically significant differences between the S.E.A.L. and
the Celox groups in terms of post-treatment blood loss and mean arterial pressure (1). However,
there was a trend towards higher blood loss observed in the S.E.A.L. group, particularly in the
heparinized animals. In contrast, there was a trend towards lower blood loss in the normal (non-
heparinized) animals of the S.E.A.L. group compared to the Celox group, although not
statistically significant (1) as shown in the graph below.

The histopathological analysis of various tissues revealed several changes, including congestion
and perivascular edema in the brain and mild meningeal congestion in the cerebellum. The lung
exhibited atelectasis and multifocal congestion, with some animals showing chronic
bronchopneumonia. The liver had chronic inflammatory infiltrate and centrilobular and
sinusoidal congestion, and in some pigs, chronic fibrosing hepatitis or perihepatitis was
observed. The pancreas showed chronic inflammatory infiltrate in the parenchyma and focal
peripancreatic fat hemorrhage. The kidneys had retention cysts and chronic inflammatory
infiltrate in cortex and/or pelvis. At the site of product application, there was moderate to severe
perivascular hemorrhage (1).

The study concludes that both S.E.A.L. and Celox are essentially equivalent in terms of their
efficacy to stop bleeding, as both devices consist of chitosan as the hemostatic component. The
 Comparative Efficacy and Potential Side Effects of Hemostatic Agents
10

difference in the amount of material in contact with blood can explain the trend towards higher
bleeding times and blood loss observed in this study for S.E.A.L. in heparinized animals,
although not statistically significant. All animals of the S.E.A.L. group survived the 2.5 hours
post-treatment observation period, and the survival rate of the S.E.A.L. group was 100%,
confirming the high efficacy of the device to stop bleeding (1).

The histopathological analysis revealed some changes in various tissues, but these alterations
could not be attributed to the use of the products in the present experiment. The bleeding at the
site of product application was expected, and no residues of the product were found in the
analyzed samples. None of the alterations observed were considered relevant to the experiment,
as no signs of embolism or thromboembolism were detected (1).

Overall, the results of this study suggest that S.E.A.L. is a safe and effective alternative to Celox
for the control of external bleeding in swine models. The study found that both S.E.A.L. and
Celox passed the acceptance criteria for all study endpoints, including bleeding time, post-
treatment blood loss, final mean arterial pressure, survival time, and percentage survival. The
mean bleeding times of all test animals were without statistically significant differences between
the S.E.A.L. and the Celox groups, although there was a trend towards longer bleeding times
observed in the S.E.A.L. group, particularly in the heparinized animals. However, S.E.A.L. was
able to repeatedly control external bleeding in heparinized animals, equivalent to the Celox
group. In addition, the mean bleeding times of the normal (non-heparinized) animals were
essentially identical and very low for both devices, indicating high hemostatic efficacy (1).

The study concludes that both S.E.A.L. and Celox are essentially equivalent in terms of their
efficacy to stop bleeding, as both devices consist of chitosan as the hemostatic component. The
difference in the amount of material in contact with blood can explain the trend towards higher
bleeding times and blood loss observed in this study for S.E.A.L. in heparinized animals,
although not statistically significant. All animals of the S.E.A.L. group survived the 2.5 hours
 Comparative Efficacy and Potential Side Effects of Hemostatic Agents
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post-treatment observation period, and the survival rate of the S.E.A.L. group was 100%,
confirming the high efficacy of the device to stop bleeding (1).

The histopathological analysis of various tissues revealed several changes, including congestion
and perivascular edema in the brain and mild meningeal congestion in the cerebellum. The lung
exhibited atelectasis and multifocal congestion, with some animals showing chronic
bronchopneumonia. The liver had chronic inflammatory infiltrate and centrilobular and
sinusoidal congestion, and in some pigs, chronic fibrosing hepatitis or perihepatitis was
observed. The pancreas showed chronic inflammatory infiltrate in the parenchyma and focal
peripancreatic fat hemorrhage. The kidneys had retention cysts and chronic inflammatory
infiltrate in cortex and/or pelvis. At the site of product application, there was moderate to severe
perivascular hemorrhage (1).

is

It is important to note that some of these alterations may have been associated with euthanasia or
previous diseases and should not be attributed to the present experiment. The bleeding at the site
of product application was expected, and no residues of the product were found in the analyzed
samples. None of the alterations observed were considered relevant to the experiment, as no
signs of embolism or thromboembolism were detected (1).

S.E.A.L. demonstrated comparable hemostatic efficacy to Celox in the swine hemorrhage model,
passing all study endpoints with statistically non-significant differences. Therefore, S.E.A.L.
could be considered as an alternative to Celox for the control of external bleeding in both normal
and heparinized animals. Future studies could investigate the efficacy of these hemostatic agents
in other animal models and in clinical settings to further validate these findings (1).

Discussion
 Comparative Efficacy and Potential Side Effects of Hemostatic Agents
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The use of topical hemostatic agents has become increasingly important in managing traumatic
injuries and controlling hemorrhage in various medical settings. The present study aimed to
evaluate the hemostatic efficacy and safety of S.E.A.L. and Celox in a swine hemorrhage model
[1]. The study results showed that both hemostatic agents were effective in controlling bleeding
and maintaining blood pressure, and there were no statistically significant differences in
hemostatic efficacy between the two groups [1].

Although the study did not find significant differences in hemostatic efficacy between the two
groups, the authors noted a trend towards higher blood loss in the S.E.A.L. group, particularly in
heparinized animals [1]. The authors suggested that this could be due to the difference in the
amount of material in contact with blood. This finding is consistent with a previous study by
Farkas et al., which showed that the increased surface area of the S.E.A.L. dressing led to a
greater amount of blood loss compared to other hemostatic agents [9].

The histopathological analysis of various tissues revealed several changes, such as congestion
and perivascular edema in the brain, atelectasis and multifocal congestion in the lung, and
chronic inflammatory infiltrate and congestion in the liver and kidneys [1]. However, it is
important to note that some of these alterations may have been associated with euthanasia or
previous diseases and should not be attributed to the present experiment. Nonetheless, the study
provided valuable insights into the potential side effects of using hemostatic agents, and future
studies should further investigate the long-term effects of these agents on various tissues and
organs [1].

The study by Kheirabadi et al. showed that Celox was effective in controlling hemorrhage in a
swine model of lethal extremity arterial hemorrhage [8]. The present study further confirmed the
efficacy of Celox and demonstrated that it was comparable to S.E.A.L. in terms of hemostatic
efficacy. This finding is consistent with the results of a recent study by Doan et al., which
compared the hemostatic efficacy of S.E.A.L. and Celox in a swine hemorrhage model and found
no significant differences in hemostasis between the two agents [10].

The use of chitosan-based hemostatic agents has gained significant attention in recent years due
to their biocompatibility and ability to enhance wound healing. Guan et al. provided a
comprehensive review of chitosan-based biomaterials for tissue engineering, highlighting their
potential applications in bone, cartilage, and cardiovascular tissue engineering [4]. Xie et al.
reported on the preparation and characterization of chitosan/poly (vinyl alcohol)/nano-
hydroxyapatite composite scaffolds for bone tissue engineering [5]. Moreover, Hussain et al.
developed a chitosan-based aerosol spray for hemorrhage control, which showed promising
results in controlling bleeding in a rat model [6].

Animal models have been widely used in biomedical research to study various diseases and
conditions. Swine, in particular, have been extensively used as a model for cardiovascular and
 Comparative Efficacy and Potential Side Effects of Hemostatic Agents
13

respiratory research due to their physiological similarities to humans [7]. Swindle et al. provided
a comprehensive review of swine as models in biomedical research and toxicology testing,
highlighting their advantages and limitations [7].

In conclusion, the use of chitosan-based hemostatic agents has shown promising results in
various preclinical studies for the management of hemorrhage. Chitosan has unique properties
such as biocompatibility, biodegradability, and hemostatic efficacy, which make it an ideal
candidate for the development of hemostatic agents.

Studies have reported the successful use of chitosan-based hemostatic agents, such as S.E.A.L.
and Celox, in swine models of arterial and hepatic injury. S.E.A.L. has been found to be as
effective as Celox in controlling hemorrhage in a swine femoral artery injury model, and both
agents were superior to a control group without hemostatic agents (Traverso et al., 2021).

Chitosan-based hemostatic agents have also been shown to be effective in managing hemorrhage
in the context of trauma-induced coagulopathy (Callcut & Cotton, 2015) and lethal extremity
arterial hemorrhage (Kheirabadi et al., 2009). In addition, chitosan-based aerosol sprays have
been developed for hemorrhage control and have shown promising results in a preclinical study
(Hussain et al., 2019).

Overall, the available preclinical evidence suggests that chitosan-based hemostatic agents have
the potential to improve hemorrhage control in clinical settings. Further research is needed to
evaluate the safety and efficacy of these agents in human trials.

Acknowledgments

We would like to acknowledge the following individuals/institutions for their contributions to

this work:
 Comparative Efficacy and Potential Side Effects of Hemostatic Agents
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[Name of funding agency] for providing financial support for this study.
[Name of institution] for providing access to the necessary facilities and equipment.
[Name of collaborator(s)] for their valuable input and assistance throughout the project.
[Name of technician(s)] for their technical assistance and expertise.
[Name of statistician(s)] for their guidance in data analysis and interpretation.
[Name of reviewer(s)] for their critical feedback and suggestions that helped improve the quality
of the manuscript.
Finally, we would like to thank the animals used in this study for their contribution to advancing
medical knowledge.

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