2019 SCIENCELINE

Journal of Life Science and Biomedicine

J Life Sci Biomed, 9 (1): 19-25, 2019

ISSN 2251-9939

New hemostatic preparation made of

the cellulose derivatives

Rustam Abrarovich Sadykov, Bahodir Abdimusaevich Ismailov^, and Olga Valerevna Kim

¹Republican Specialized Surgery Centre named after Academician V.Vakhidov, Tashkent city, Uzbekistan.

Corresponding author’s Email: bahodirismailov@gmail.com

ABSTRACT

Original Article

Aim. The aim of this study was to investigate the indexes of biocompatible pellicle

hemostatic coverage in vitro and in vivo conditions. Methods. Samples of pellicle

hemostatic coverage on the basis of the cellulose derivatives were used in researches.

Breaking strength, estimation of the implant’s structure and adhesion power were

evaluated according to the Ts 05957837-28:2014 instructions and documents of the National

certification system of the Republic of Uzbekistan and with the using the apparatus "Zwick"

(Germany) and atomic-power microscope of Agilent technologies (USA). Hemostatic activity

of the coverage on the basis of the cellulose derivatives was estimated by the

Lee and White test for the blood coagulation time. For in vivo research, 30 mature rats were

required. Operations were performed under inhalation anesthesia, and the wound of liver

was formed. Both macroscopic and microscopic studies had been undertaken.

Morphological changes were studied in terms of 3 and 12 hours and then on the 1^st, 3^d, 7^th,

14^thand 30^thday after an operation. Results. An adhesion power of the pellicle coverage on

the basis of the cellulose derivatives was 7.3±0.2 N/cm², breaking strength was 390±4.8

kGf/cm². In presence of polymer, a coagulation time on Lee and White test was shortened by

as many as 2.1 times in relation to control that made up 2.4±0.6 min. In in vivo conditions

hemostasis started during 3-5 sec. A weak inflammatory reaction of tissue was

histologically determined. Further observations over dogs showed that an hour after an

operation, an implant had been preserved on the surface of liver as a white pellicle and had

not been separated from the wound surface. Bleeding signs were not marked. An abdominal

cavity remained intact. Conclusion. Rapid enough biodegradation of polymer along with the

unexpressed inflammatory reaction allows preventing the infecting related to the presence

of foreign body. The rapid forming of fibrotic tissue in a zone of lesion makes it possible to

obtain a durable hemostasis. A poorly expressed reaction was also marked from the side of

peritoneum and surrounding organs. Recommendations. The oxidized regenerated cellulose

can result in an intensive inflammation of the surrounding tissues because of the low level

of pH that had not happened in the present research due to the selected correlation of

ingredients of hemostatic pellicle.

PII: S225199391900004-9

Rec.

Rev.

Pub.

29 Dec 2018

24 Jan

25 Jan

2019

Keywords

Hemostasis,

Morphology,

Carboxymethylcellulose,

Oxidized Cellulose,

Hemostatic Substance.

INTRODUCTION

Cellulose is a natural polymer of vegetable origin, not water-soluble. It possesses a good biocompatibility, does

not cause an allergic reaction, and in this connection is widely used in medicine and pharmaceutical industry.

Oxidized regenerated cellulose (ORC) – is the water-soluble cellulose’s derivative which is synthesized by the

influence of oxidizing agents. ORC in the organism of a human biodegrades without the forming the toxic

substances and does not cause the expressed inflammatory reaction of tissue [1 ]. Additionally ORC possesses an

antibacterial effect initiating the forming the unfavorable pH environment. Bio-absorption of ORC proceeds

during two weeks [2 ]. ORC is successfully used in plastic surgery because it does not cause an edema or

deformation of tissue [3 ]. Mechanisms of the favorable healing and fibrogenesis are explained by the ORC’s

absorption of free radicals, ions of metals, stabilizing of growing factors and others [4 ].

Carboxymethylcellulose (CMC) is a nontoxic and biocompatible polymer owing to what it finds a wide use

in pharmaceutical, cosmetic, and food industry [5-7 ]. Ca-CMC unlike Na-CMC is not dissolved to the end in

water, swells and forms a gel substance [8 ]. The mechanism of hemostatic action of Ca-CMC is in a point that at

To cite this paper: Sadykov RA, Ismailov BA, and Valerevna KO. 2019. New hemostatic preparation made of the cellulose derivatives. J. Life Sci. Biomed. 9(1): 19-

25; www.jlsb.science-line.com

a contact with blood the ions of calcium accelerate the coagulation process however the surplus amount of

calcium can provoke a burn and necrosis of tissue [9 ].

The aim of this study was to investigate the indexes the indexes of biocompatible pellicle hemostatic coverage in vitro

and in vivo conditions.

MATERIAL AND METHODS

The authors together with the scientific research center of chemistry and physics of polymers under the

Academy of sciences of the Republic of Uzbekistan, worked out a new composite polymer material (CPM)

possessing hemostatic property for the surgery of liver (30 white mature outbred male rats weighing 198±2.7 gr

were used). (Patent No.IAP 20160273 "Bio-absorbable surgical hemostatic substance", registration from

13.06.2016, Nazirov F.G., Rashidova S.Sh., Sadykov R.A., Sarymsakov A.A., Alimov M.M., Ismailov B.A., Li Yu.B.). It

presents itself as a semi-transparent flexible pellicle moderately water-soluble coverage (Picture 1). The

temperature of melting is 220°C. It is stable at pH 5-7. Rapidly hydrolyzing in an alkaline environment and more

steady in an acidic environment.

At the light microscopy of implant fibers of the oxidized cellulose were located evenly on the entire surface

of pellicle. Methodologies of the evaluating the breaking strength, estimation of the implant’s structure and the

determining the adhesion power were conducted according to the Ts 05957837-28:2014 instructions and

documents of the national certification system of the Republic of Uzbekistan and using the apparatus "Zwick"

(Germany) and atomic-power microscope of Agilent Technologies (USA).

At the studying the blood coagulation time (BCT) in presence of hemostatic coverage on the basis of the

cellulose derivatives on Lee-White test a polymer in the kind of a pellicle with the size one cm ²was placed in a

test tube with one ml of venous blood. The test tube was placed in water bath at t - 37° and incubated within

two minutes, and then after every 30 sec the test tube was inclined under the angle of 45°. The time of the

coagulum appearance was fixed. Control samples were investigated by analogy without adding the polymer to

the whole blood.

Experimental studies of the new composition hemostatic coverage were undertaken in accordance with the

Russian national standard ISO 10993-6-2011. Guideline: 30 white mature outbred male rats weighing 198±2.7 gr

were used. Operations were performed under inhalation anesthesia (Isofluranum) with the modeling the wound

of liver with an active parenchymatous bleeding. A hemostatic effect was achieved by the application of pellicle

coverage (Picture 2). Morphological changes were studied in terms of 3 and 12 hours and then on the 1^st, 3^d, 7^th,

14^thand 30^thday after an operation. Macroscopic and microscopic studies were undertaken.

Picture 1. Sample of the Composite polymer material

Picture 2. Stopping the bleeding with pellicle coverage

(Composite polymer material)

Methodology of operation

Under inhalation anesthesia a supramedian laparotomy was performed with 3-4 cm lengthwise. A left

hepatic lobe was withdrawn in a wound. On the surface of liver a flat wound with a diameter up to one cm and a

depth up to 0.3 cm had been modeled. From the hepatic wound an active parenchymatous bleeding was marked.

The pellicle coverage was applied on the bleeding surface. The control over the possible renewal of bleeding was

kept by the supervision during ten minutes. A postoperative wound was sutured by interrupted sutures.

Animals were withdrawn from anesthesia, and subsequently a dynamic supervision over the post-operational

state was conducted.

To cite this paper: Sadykov RA, Ismailov BA, and Valerevna KO. 2019. New hemostatic preparation made of the cellulose derivatives. J. Life Sci. Biomed. 9(1): 19-

25; www.jlsb.science-line.com

In the fixed terms animals were withdrawn from the experiment for the estimating the macroscopic

changes, and also for the intake of material for histological researches. Euthanasia was carried out according to

the Provisions of ISO 10993-2 under the general anesthesia. During the experiment a macroscopic estimation of

changes in an abdominal cavity was analyzed at the dissection of animals after euthanasia. For the getting

ready the morphological preparations the tissue of liver was excised and fixed in 10% solution of neutral

formalin. After expiration of the fixing terms bioptate was inundated by paraffin. Paraffin blocks had been

manufactured. Series of sections with a thickness of three-four µm were made. Histological preparations were

painted by hematoxylin-eosin.

For the estimating the histological changes in liver, a system of points was employed in accordance with

Russian national standard ISO 10993-6-2011 where the parameters of semi-quantitative estimation of the

number and distribution of cells characterizing the inflammatory process such as polymorphonuclear

neutrophils, lymphocytes, plasmatic cells, macrophages, eosinophiles and multinuclear cells were taken into

account. At the microscopy, the dynamics of the inflammatory reaction’s development, features of the liver’s

parenchyma regeneration as well as the degree of the investigated implant’s destruction, were estimated.

Ethical approval

The review board and ethics committee of RSCS named after acad. V.Vakhidov approved the study

protocol and informed consents were taken from all the participants. Experimental studies had been

undertaken with the observance of the rules accepted by the European convention for the protection of

vertebrate animals used for experimental and other scientific purposes (ETS N 123), Strasbourg, 18.03.1986.

Statistical analysis

The obtained results were subjected to the statistical processing with the using the standard package of

Microsoft Excel 2010software by the method of variation statistics with the estimation of indexes’ values (M±m)

RESULTS AND DISCUSSION

It was set by researches that adhesion required for medical aims had to provide the dense adherence of pellicle

to the wound surface that conditioned an instantaneous hemostatic effect in the final outcome. An adhesion

power of pellicle coverage made up 7.3±0.2 N/cm². Such a high adhesion of implant was related to its flexibility

and moderate hydrophilism that strengthens this effect in the aggregate.

At the estimating the breaking strength an attention was paid to the ability of implant to hold the edges of

the wound surface that provides the effect of the strengthening the tissue. It was set from the data of

comparative researches that the worked out implant had a limit of the breaking strength within the limits of

390±4.8 kGf/cm² that fully satisfied the requirements to the implants of a similar origin.

In the polymer’s presence BCT on Lee-White test was shortened by as many as 2.1 times in regards of the

control that made up 2.4±0.6 min. The obtained results testified to the strengthening the process of the donor

blood’s coagulation in vitro in the presence of implant due to the activating the factors of an external and

internal way of the coagulation’s hemostasis.

The results of in vivo researches, showed that hemostasis had started after the applying the polymeric

coverage within a short time. A complete hemostasis was marked during 3-5 sec. After the supervision during

10 minutes the resumption of bleeding was not marked.

Subsequently, an activation of factors of the strengthening the hemopexis led to the forming the fibrinous

pellicle on the surface of the hepatic wound. Further observations on animals showed that in an hour after an

operation an implant had been preserved on the surface of liver as a white pellicle and had not been isolated

from a wound surface. Bleeding signs were not marked. An abdominal cavity remained intact. In subsequent

terms a substitution of pellicle coverage for fibrin without a considerable inflammatory reaction had been

marked. It was paid attention to the absence of the expressed accretions in the place of the hemostatic coverage

application.

The result of dissection in 3 hours after an implantation showed that there was not free liquid in the

abdominal cavity, a renewal of bleeding had not been marked, fibrin pellicle was in the area of the wound. At the

microscopic research it was marked an inflammatory process which was characterized in visual field by a

presence of polymorphonuclear neutrophils – 18.1±0.6, lymphocytes – 14.3±0.8, plasmatic cells – 4.4±0.4,

macrophages – 2.5±0.2. The pellicle fitted closely to the hepatic tissue covering the zones with signs of the

To cite this paper: Sadykov RA, Ismailov BA, and Valerevna KO. 2019. New hemostatic preparation made of the cellulose derivatives. J. Life Sci. Biomed. 9(1): 19-

25; www.jlsb.science-line.com

stopped bleeding. The light microscopy of the wound surface of liver showed the presence of a considerable

number of hemolyzed erythrocytes testifying to their appearance in the moment of trauma, and also a lot of

micro-thrombus in vessels pointing to the hemostatic effect of implant.

After 12 hours, the presence of free liquid in the abdominal cavity was not revealed, a friable adhesion

process with the involving the anterior abdominal wall and a dense fibrin pellicle on the wound surface were

marked. The necrotic tissue with thickness 80 µm was marked microscopically. Around the hemostatic

coverage as a reaction on a foreign body an inflammatory process was marked with an increase in amount of

polymorphonuclear neutrophils up to 27.3±0.9, lymphocytes – 18.8±0.9, plasmatic cells – 5.8±0.3, macrophages -

10±0.3 in visual field.

At the macroscopic research on the 1^stday in an abdominal cavity a friable adhesion process with the

involving the epiploon and an expressed coverage of wound surface with fibrin were noted. Microscopically

subcapsularly a vacuolar dystrophy of hepatocytes with the expansion of sinusoid spaces was marked. The

amount of elements of inflammation in visual field made up: polymorphonuclear neutrophils – 29.8±0.7,

lymphocytes – 13.2±0.7, plasmatic cells – 2.9±0.4, macrophages –7.7±0.3. A vacuolar-granular dystrophy of

hepatocytes and a necrotic layer of tissue with thickness up to 120 µm were marked.

On the 3^dday in an abdominal cavity a moderate adhesion process with the epiploon’s areas was

registered. The loops of small intestine were not engaged in a process. An insignificant decrease in fibrin

pellicle was marked (Picture 3A). At the microscopy an abatement of inflammatory process was marked. The

vacuolar dystrophy of hepatocytes was visualized in a subcapsular zone (Picture 4A). The thickness of the

necrotic layer made up 93±4.6 µm.

On the 7^thday of the experiment in an abdominal cavity an adhesion process was preserved with the

surface of liver, epiploon and xiphoid process. A decrease in fibrin pellicle was marked (Picture 3B).

Microscopically the fibrosis of the hepatic capsule was marked with an abatement of inflammatory infiltrate

which was expressed as a reduction in the amount of polymorphonuclear neutrophils up to 4.7±0.6,

lymphocytes – 7.7±0.4, plasmatic cells – 1.5±0.3. The thickness of the necrotic layer made up 68±5.3 µm (Picture

4A).

Picture 3. Macroscopy. The 3^dday after application of composite polymer material, formation of fibrin pellicle

(A). The 7^thday after application of composite polymer material decrease in fibrin pellicle with the forming the

moderate adhesion process (B). The 14^thday after application of composite polymer material, calming down of

the adhesion process with the forming the pellicle coverage (C). The 30^thday after application of composite

polymer material, regeneration of the hepatic wound surface (D).

To cite this paper: Sadykov RA, Ismailov BA, and Valerevna KO. 2019. New hemostatic preparation made of the cellulose derivatives. J. Life Sci. Biomed. 9(1): 19-

25; www.jlsb.science-line.com

On the 14^thday the expressed adhesion process in the abdominal cavity was not marked. A thin

transparent fibrin pellicle was marked on the surface of liver (Picture 3C). Microscopically a moderate

lymphoid infiltration of the hepatic capsule was marked with decrease in visual field in amount of

polymorphonuclear neutrophils – 1.3±0.4, lymphocytes – 5.9±0.4, plasmatic cells – 1.2±0.2. The necrotic layer

was 19.8±2.3 µm. The fragments of polymer were determined that testified to its resolution (Picture 4C). On the

30^thday a moderate adhesion process was marked in the abdominal cavity. The wound surface of liver was of

soft consistency, smooth and without a cicatricial change and signs of inflammation (Picture 3D).

Microscopically a reduction in thickness of fibrotic pellicle up to 9.5±1.0 µm was marked. A decrease in the

amount of elements of inflammatory character (a rare quantity of lymphocytes) was noted. Complications and

lethal outcomes during the operation and after it had not been observed (Picture 4D).

Picture 4. Morphology. Light microscopy. Magnification х200. Coloration Hematoxylin-Eosin. The 3^dday after

application of composite polymer material, vacuolar dystrophy of hepatocytes (A). The 7^thday after application

of composite polymer material, reduction in inflammatory process (B). The 14^thday after application of

composite polymer material, calming down of inflammatory process, fragments of implant (C). The 30^thday

after application of composite polymer material, single elements of inflammation, regenerative process (D).

Microscopically the expressed patho-histologic changes were not discovered in the hepatic tissue. A

capsule of liver was not incrassate and it contained longitudinally oriented fascicles of collagen fibers.

Interlobular connecting tissue was poorly developed, the signs of inflammatory infiltration and fibrosis of liver

were not found. Hepatocytes were of polygonal form with a centrally located nucleus, frequently a nucleolus

was determined. Quite often binuclear hepatocytes occurred. Sinusoid capillaries were of ordinary sizes.

Singular erythrocytes and leucocytes were determined in a lumen. In the wall of sinusoid hemocapillaries and

Disse spaces singular Kupffer cells having an intact structure were revealed at a large magnification. A

moderate dilation and blood filling of sinusoid hemocapillaries, central and underlobular veins were noted in

some cases. An endothelial lining was without destructive changes, in some places swelled endotheliocytes with

hyperchromic nucleus were marked. The structure of cholangiol and interlobular biliary ducts was without

pathological changes. And all this pointed to the fact that the studied preparation did not influence the

microscopic structures of liver negatively.

From the presented data it is possible to conclude that hemostatic coverage caused a morphological

reaction of liver as an inflammation and excrescence of the connecting tissue on the 1^stday. But these processes

calmed down quickly. A complete resolution of implant was being observed on the 7-14^thdays. To the 30^thday

after the implantation regenerator processes in the hepatic parenchyma, especially, in the zone of lesion were

To cite this paper: Sadykov RA, Ismailov BA, and Valerevna KO. 2019. New hemostatic preparation made of the cellulose derivatives. J. Life Sci. Biomed. 9(1): 19-

25; www.jlsb.science-line.com

observed that testified to the restoration of the tissue of liver after the applying the hemostatic coverage.

Nowadays the stopping the parenchymal bleeding from the abdominal cavity’s organs remained a problem

which had not entirely been solved especially when the question was about vast surfaces [10 ]. For the stopping

such bleeding various local hemostatic substances were used in an abdominal surgery. The most widely used of

them were as followed: collagen, gelatin, oxidized regenerated cellulose [9,10 ]. A substantial shortage of collagen

and gelatin was a potential risk of pathogenic transmission because of their animal origin. At the same time the

oxidized regenerated cellulose can result in an intensive inflammation of the surrounding tissues because of the

low level of pH that had not been happened in the present research thanks to the selected neat correlation of

ingredients of hemostatic pellicle. The following requirements were produced to the modern hemostatic

substances: a high hemostatic activity at a contact with a bleeding surface, a rapid absorption of blood, a

simplicity and comfort in use [1 ].

CONCLUSION

As a result of joint scientific researches an optimal composition and correlation of basic ingredients of a

hemostatic coverage had been worked out. A combination of three derivatives of cellulose allowed the creating

the implant with hemostatic properties that meets all criteria of the solution’s novelty and was the most

optimal for the stopping the bleeding from the wounds of liver. Rapid enough biodegradation of the polymer

along with an unexpressed inflammatory reaction made it possible to prevent the infecting related to the

presence of a foreign body. Rapid formation of the fibrotic tissue in a zone of lesion allows the obtaining the

stable hemostasis. The poorly expressed reaction from the side of peritoneum and surrounding organs was also

marked. According to the researches’ data the hemostatic coverage worked out on the basis of derivatives of

cellulose possessed high mechanical parameters with a hemostatic activity and a good biocompatibility.

DECLARATIONS

Acknowledgements

This work was supported by “Republican Specialized Scientific and Practical Medical Center of Surgery

named after Academician V.Vakhidov”, Uzbekistan.

Authors’ Contributions

All authors contributed equally to this work.

Competing interests

The authors declare that they have no competing interests.

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To cite this paper: Sadykov RA, Ismailov BA, and Valerevna KO. 2019. New hemostatic preparation made of the cellulose derivatives. J. Life Sci. Biomed. 9(1): 19-

25; www.jlsb.science-line.com