A STUDY OF SPLEEN TISSUE REACTION USING NEW SAMPLES OF POLYMERIC HEMOSTATIC MATERIALS

A STUDY OF SPLEEN TISSUE REACTION USING NEW SAMPLES OF POLYMERIC HEMOSTATIC MATERIALS

Lipatov V.A., Lazarenko S.V., Severinov D.A.

Kursk State Medical University, Kursk, Russia

 One of the main problems of abdominal surgery is search for appropriate and non-traumatic way for bleeding arrest in injuries and surgery for parenchymatous abdominal organs, efficiency of which determines the patients’ life and favorable postsurgical course [1]. The available techniques for bleeding arrest (hemostatic sutures, electro-/spray coagulation etc.) are considered as invasive (penetrating the organ to some degree, with traumatic influence on organ tissue) [2]. In its turn, the application hemostatic implants present the opposite (sutureless) approach to treatment of such bleedings, without organ injuries. It determines the important advantage of local polymer hemostatics: absence of additional organ injuries, a decrease in amount of postsurgical complications [3].
In comparison with other traditional techniques for intrasurgical arrest of bleeding, the use of application hemostatic implants shows much higher efficiency for parenchymal bleedings, with decreasing mortality after injuries to abdominal organ [4, 5]. Also hemostatic implants can be used as the carrier-matrix for antimicrobial and hemostatic substances (enhancing and prolonging the main hemostatic effect of the implant) [6].
Currently, the scientific literature describes a lot of various studies of different local hemostatic measures for surgical interventions for parenchymal organ injuries [7]. Collagen is the most studied basis for local hemostatic agents. One of the most important physicochemical properties of collagen is the ability to absorb water which is necessary for the local hemostatic agent. The action of collagen sponge is based on formation of matrix for capture of formed elements and for formation of the blood clot [8].
The examples of collagen-based local hemostatic agents, which have been implemented into daily practice of a surgical hospital, are collagen Tachocomb® (Takeda Pharmaceuticals, Linz, Austria), hemostatic collagen sponge (Belkozin, Luga, Russia), hemostatic collagen sponge (Zelyonaya Dubrava, Dmitrov, Russia). However, the hemostatic capabilities of collagen sponges are quite limited because of weak fixation to the wound surface that determines the long period of blood clot formation, and, therefore, increases the time of bleeding arrest and blood loss volume [9, 10].
Conversely, one of the main advantages of cellulose-based (sodium carboxymethylcellulose [CMC]) hemostatic implants are safety of use, low immune response, and fat resorption in the body [11]. The important feature of CMC-based materials is high degree of adhesion to adjacent tissues, which prevent displacement of the sponge as result of bleeding from an injured organ. Another important feature of CMC is pseudoplasticity (weakening of apparent viscosity with increasing degree of velocity gradient of parallel layers of the fluid in isothermic and reversible conditions), which provides the appropriate obstruction of bleeding vessels of parenchyma after approximation of edges of the organ with the hemostatic sponge between the edges [12]. After transition of implant’s hard substance to colloid mass by means of contact with fluid component of the blood, and pressure of edges of the organ, CMC penetrates the capillaries and arrests the bleeding [13].
Therefore, the important tasks of modern abdominal surgery are development of low traumatic techniques for bleeding arrest, experimental approbation and clinical implementation of new efficient biologically inert hemostatic agents for bleeding arrest in parenchymal organ injuries in abdominal cavity at the site of implantation of hemostatic agents.
Objective – to assess the splenic tissue responses when using the new samples of polymeric hemostatic materials in experiment in vivo.

MATERIALS AND METHODS

Experimental samples of CMC-based local hemostatic agents developed by the authors in cooperation with Lintex (Saint Petersburg, Russia), and hemostatic collagenic sponges (HCS) (Zelyonaya Dubrava, Dmitrov, Russia) were used as the study materials.
The experimental samples of local hemostatic materials were produced with the technique, which is described in “A Way for Production of Porous Film Materials Based on Carboxymethyl Cellulose” (the Patent of RF No. 2509784, March 20, 2014; the authors: Zhukovsky V.A., Nemilov V.E., Akhmetshina O.Z., Zhukovskaya I.I., Edomina N.A., Krasiy Yu.A., Sosina I.M., Lipatov V.A.), with modifications, i.e. introduction of 3 % solution (from mass of polymer) of aminoacetic acid during production.
The test subjects were mature males of Soviet chinchilla rabbits (weight of 2.3-2.5 kg, 50 subjects). The animals were under quarantine, and then were in conditions of the experimental and biological clinic of Kursk State Medical University. The laboratory animals were distributed into five experimental groups (10 subjects in each group:
Group 1 – control group (intact liver of laboratory animals);
Group 2 – injury model (injury modeling, bleeding arrest, omentum packing);
Group 3 – Na-CMC (injury modeling; bleeding was arrested with application of the sponge based on Na-CMC on the injured part of the organ);
Group 4 – Na-CMC+AAA (injury modeling; bleeding was arrested with application of the sponge based on Na-CMC with 3 % (from mass of polymer) solution of aminoacetic acid on the injured organ);
Group 5 – HCS (injury modeling; bleeding was arrested with application of the hemostatic collagen sponge (produced by Zelyonaya Dubrava) including collagen, boric acid, aminocaproic acid, argovit).

Premedication included Chloropyramine (0.4 mg/kg intramuscularly), Platyphyllin (0.07 mg/kg subcutaneously), Ketorol (0.1 ml intramuscularly), Xyla (0.2 ml/kg intramuscularly). All surgical interventions were conducted under general inhalation anesthesia (narcosis drug R340 Isoflurane, China; Isoflurane level (Baxter, USA) in inhaled level – 3 %, air flow – 0.8 l/min) with adherence to the international and local standards of humane treatment with laboratory animals: the order 2010/63/EU of European Parliament and Council of the European Union from September 22, 2010, for protection of animals used for scientific purposes, the order of Healthcare Ministry of Russia No. 199n from April 1, 2016, “About confirmation of rules for appropriate laboratory practice”, the order by Healthcare Ministry of USSR No. 755 from August 12, 1977 “About measures for further improvement in organizational forms of work with use of experimental animals” and others. All studies were conducted under supervision of the regional ethical committee of Kursk State Medical University.
The laboratory animals were exposed to midline laparotomy and to a superficial injury to the spleen in sterile conditions in the surgery block of the research institute of experimental medicine of Kursk State Medical University. The injury was modeled with use of the special plate with a hole of 7×12 mm [14]. At the moment of force exertion to the plate, the organ tissues extending from the hole were resected with the scalpel, which was moved in parallel to its plane. As result, superficial parenchymal bleeding appeared, which was arrested with application of the tested samples of local hemostatic materials (2×2 cm). After achievement of hemostasis, the wound was sutured with interrupted stitch in layer-by-layer manner.
The experiment was completed with narcosis overdose on 14th day after surgery. The injured part of the liver with implanter hemostatic agent was autopsied. The biological samples were fixed in 10 % of neutral formalin. After fixation, the smaller pieces of tissues with fragments of implanted materials were resected. After washing, dehydration and standard saturation with paraffin, and microtoming, the slices (thickness of 10-12 µm) were made in manner to permit the visualization of the region of contact between the implant and subjacent tissues and were stained with hematoxylin-eosine with standard protocols. 10 microsamples were received from each animal.
Microscopic examination and photographing (40-fold magnification) of microsamples were performed with the medical microscope MICMED-6 (LOMO, Saint Petersburg, Russia). The photos of spleen tissues and tested samples were used for measurement (px) of thickness of the capsule, square of lymphoid follicles, square of reactive center and size of T-zone [15].
The statistical analysis was conducted with methods of descriptive and variance statistics. The mean (M), error of the mean (т) (M ± m), and n – 10 were calculated. The trial version of Statistica 10.0 (Dell Software Company, USA) was used for statistical analysis. Owing to the small size of the sample (n < 30) in the experimental groups, and abnormal distribution of the sample (according to Kolmogorov-Smirnov’s test), the non-parametrical test of Mann-Whitney was used for estimation of confidence of differences. The critical level of significance (p) was 0.05 (acceptable value for medicobiological studies).
The study was conducted under supervision of the regional ethical committee of Kursk State Medical University with compliance with the present local and international ethical standards.

RESULTS

After implantation of the hemostatic sponge based on Na-CMC (the group 3) one could observe an evident increase in the square of lymphoid follicles of the spleen as compared to the control group (by 36 %) and to the injury model (by 280 %) (Fig. 1, the table 1). An evident decrease in the square of lymphoid follicles was found after use of the Na-CMC based implant with aminoacetic acid (the group 4). The decrease was determined by high amount of new follicles as compared to the control group. This is manifestation of the normal response of splenic tissue to the injury since no evident differences from the injury model group were found.

Figure 1. Change in square of lymphatic follicles (px) of the spleen of laboratory animals (rabbits) in the studied groups




Table 1. Values of histologic changes in spleen tissues in the studied groups, M ± m

Group

Value

Group name

n

Square of lymphatic follicles, px2

Reactive center square, px2

T-zone size,
px

Capsule thickness, px

1

Control group

10

92512.6 ± 2836.2

6404.6 ± 211.9

80.8 ± 0.2

21.1 ± 0.1

2

Injury model

10

33050.7 ± 2036.6

13482.78 ± 1667.8

185 ± 1.8

64.3 ± 4.3

3

Na-CMC

10

125360.8 ± 19540.3

13579.19 ± 1403.7

130.8 ± 0.4

46.1 ± 0.1

4

Na-CMC + AAA

10

33341,3 ± 3415,5

11108 ± 896.1

187 ± 3.2

55.9 ± 3.5

5

Hemostatic collagen sponges

10

59936,5 ± 4632

12058.2 ± 1219.6

202.2 ± 3.2

62.2 ± 5.5

Note: the level of statistical significance in the studied groups was estimated with Mann-Whitney’s test; the results are presented in the table 2-5.

After use of the collagenic sponge, we noted an evident decrease in the square of lymphoid follicles as compared to the control group (by 35 %), and an increase as compared to the injury model (by 81 %) (the table 2). These signs may testify the late stage of the organ response to the injury, which is characterized by gradual attenuation of white pulp, and decrease in sizes of follicles to previous sizes or smaller.
The examination of the square of reactive centers (Fig. 2, the table 3) did not find any reliable differences between the experimental groups and the injury model. Therefore, the increase in the square of reactive centers is a physiological response to the organ injury after implantation of hemostatic materials in comparison with the control group.

Table 2. Achieved level of statistical significance in differences of square of lymphatic follicles in the studied groups

Group name

Group

2

3

4

5

Injury model

Na-CMC

Na-CMC + AAA

HCS

1

Control group

0.54

0.0003*

0.00001*

0.000004*

2

Injury model

0.36

0.0011*

0.099

0.00073*

3

Na-CMC

0.90

0.99

0.51

0.0004*

4

Na-CMC + AAA

0.72

1.0

0.79

0.00001*

Note: * sign notes the statistically significant differences in p value in Mann-Whitney’s test.

Table 3. Achieved level of statistical significance in differences of reactive center square in the studied groups

Group name

Group

2

3

4

5

Injury model

Na-CMC

Na-CMC + AAA

HCS

1

Control group

1.0

0.33

0.08

0.81

2

Injury model

0.97

1.2

0.19

0.92

3

Na-CMC

0.07

0.93

0.68

1.4

4

Na-CMC + AAA

0.23

0.99

0.24

0.39

Note: * sign notes the statistically significant differences in p value in Mann-Whitney’s test.

Figure 2. Change in sizes of reactive center (px) of the spleen of laboratory animals in the studied groups


 

The response to the injury is characterized by an evident increase in T-zone (by 2.3 times) (Fig. 3, the table 4). After implantation of hemostatic materials based on Na-CMC, one can observe a slight decrease in T-zone as compared to the injury model. The group 4 with the injury model did not show any reliable differences in sizes of T-zone after addition of aminoacetic acid. The group of collagen use (group 5) showed an evident increase in T-zone in comparison with the injury model. It can testify a response of spleen tissue to collagen.

Figure 3. Change in sizes of T-zone (px) of the spleen of laboratory animals in the studied groups



Table 4. Achieved level of statistical significance in differences of  -zone sizes in the studied groups

Group name

Group

2

3

4

5

Injury model

Na-CMC

Na-CMC + AAA

HCS

1

Control group

0.82

0.0004*

0.0032*

0.98

2

Injury model

0.99

0.12

0.26

0.000001*

3

Na-CMC

1.2

0.45

1.0

0.000032*

4

Na-CMC + AAA

0.94

0.07

0.86

0.000014*

Note: * sign notes the statistically significant differences in p value in Mann-Whitney’s test.

The experimental groups (groups 3, 4, 5) with use of hemostatic implants showed an evident increase in thickness of the splenic capsule as compared to its mean thickness without damage (by 200 % for injury model, by 118 % for Na-CMC sponge, by 165 % for Na-CMC + AAA, by 195 % for the collagen sponge) (Fig. 4, the table 5).

Figure 4. Change in sizes (px) of splenic capsule of laboratory animals in the studied groups




Table 5. Comparison of values of capsule thickness in the studied groups

Group name

Group

2

3

4

5

Injury model

Na-CMC

Na-CMC + AAA

HCS

1

Control group

1.0

0.0002*

0.00001*

0.0031*

2

Injury model

0.09

0.07

0.43

0.06

3

Na-CMC

0.23

1.1

0.79

0.0004*

4

Na-CMC + AAA

0.11

0.85

0.31

0.002*

Note: * sign notes the statistically significant differences in p value in Mann-Whitney’s test.

There were not any differences in relation to the injury model in the groups. It supposes that an increase in the spleen size is the normal response of the organ’s tissue to trauma. The mean thickness of the splenic capsule reliably differs after implantation of Na-CMC sponges as compared to the collagen-based tested samples.

DISCUSSION

The response to the injury causes the evident decrease (by 64.2 %) in the square of lymphoid follicles as compared to the control group, as well as the increase in reactive centers by 115 %, T-zone – by 127 %, splenic capsule – by 204 % (p < 0.05). The use of Na-CMC implant with aminoacetic acid did not cause any evident differences from the injury model group (p > 0.05). According to our opinion, the changes in the square of reactive center and the splenic capsule after use of Na-CMC sponges without drugs and with addition of aminoacetic acid is determined by tissue response to trauma since no significant differences were found in the groups (p > 0.05).
The implantation of the hemostatic collagen sponge shows the evident increase in the square of lymphoid follicles by 81 % and in T-zone by 9 % as compared to the group with the injury model. There was a decrease in the square of lymphoid follicles as compared to the control group. These signs are common for the late stage of response of splenic tissues to the injury [7, 8, 16].
The addition of aminoacetic acid causes less intense response of splenic tissues with decreasing square of lymphoid follicles, reactive zones, and the increase in T-zone as compared to Na-CMC experimental samples. There were not any evident differences from the injury model group. Such response of splenic tissues can be determined by interaction between carboxyl groups of Na-CMC and the blood (with formation of the complexes preventing the continuation of bleeding which “obstruct’ small injured vessels), and by biological action of aminoacetic acid on injured tissues which manifests itself as pH “stabilization”. Preventing the increase in level of H+ ions in the region of contact between the implant and the organ, aminoacetic acid preserves the optimal pH for subsequent development of hemocoagulation processes.

CONCLUSION

The above-mentioned findings show that the use of the local hemostatic Na-CMC-based agent promotes the activation of elements of immune system and formation of adequate local immune response in conditions of modeling of spleen trauma. The manifestations include more intense morphological changes (increasing thickness of the capsule and T-zone, a decrease in the square of lymphoid follicles) in experimental use of samples of the collagen-based local hemostatic materials in comparison with estimated values in the control group.

Information on financing and conflict of interests

The study was conducted in compliance with the schedule of researches of Kursk State Medical University. The authors did not receive any financial support from producers of the medical agents and items.
The authors declare the absence of any clear and potential conflicts of interests relating to publication of this article.

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