The Use of Vacuum-Dried Maral Blood in the Treatment of Purulent Wounds

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INTRODUCTION

Purulent wounds of soft tissues remain one of the main problems of the modern surgery [1–3]. More than 30.0% of patients of surgical hospitals and more than 60.0–70.0% of requests for primary surgical care are cases of infectious complications of wounds. According to prognosis, these complications will cause more than 10 million annul deaths, among other things, due to the growth of antibiotic-resistant microorganisms [4]. Treatment of infectious complications is also associated with a high incidence of disability and mortality and with high cost [5, 6]. To solve these problems, various methods of treating purulent wounds, wound dressings, medical drugs and antibiotics [7–10], biologically active drugs, for example, platelet-enriched autoplasma [11–13], are being actively developed.

The use of various biogenic growth factors, which have a low cost, seems promising for potentiating reparative processes [14]. Thus, preparations of maral, in particular, vacuum-dried maral blood (VDMB), are noted by a number of authors as a medicinal raw material with proven high effectiveness [15–17]. The concentration of various growth factors in them, such as IGF-I, IGF-II, TGF-β, EGF, group B, PP vitamins, amino acids, testosterone, growth hormone, makes these medicinal substances a potential stimulator of reparative processes in soft tissue injuries [18, 19]. However, the complexity and multifactorial nature of their composition do not allow a confident prediction of the extent of their effectiveness in reparative processes in various tissues.

The aim of this study to effectiveness of use of vacuum-dried maral blood in the treatment of purulent wounds in experiment.

MATERIALS AND METHODS

The experimental study was conducted from December 2022 to October 2023 of the Research Institute of Experimental Biology and Medicine (Voronezh). The study was conducted in strict accordance with the requirements of the Federal Law of the Russian Federation of May 14, 1993 No. 4979-1 ‘On Veterinary Medicine’ (as amended on July 02, 2021), Directive 2010/63/EU of the European Parliament and of the Council of the European Union ‘On the Protection of Animals Used for Scientific Purposes’, GOST No. 33216-2014 ‘Guidelines for the Maintenance and Care of Laboratory Animals, Rules for the Maintenance and Care of Laboratory Rodents and Rabbits’, and is based on the approval of the Study Protocol by the Local Ethics Committee of the N.N. Burdenko Voronezh State Medical University (Protocol No. 7 of December 13, 2022).

The study was conducted on 90 Wistar rats standardized by gender, weight and age, and randomized into three groups:

- in control group 1 (n=30) — no treatment of wounds was carried out;

- in control group 2 (n=30) — daily dressings were carried out with 0.01% benzyldimethyl-myristoylamino-propylammonium (BMP) solution,

- in experimental group (n=30) — daily dressings were carried out with 0.01% BMP plus application of VDMB at a dose of 0.7 g/cm2 (particle size 100–150 µm).

The purulent wound was modeled according to the following scheme. In the first stage, under inhalation narcosis, (isoflurane, induction dose — 3.0–5.0%, maintenance dose — 1.5–3.0%), the rear surface of the neck was shaved at first with a trimmer, then with a disposable shaving stick, the prepared surgical field was twice treated with 0.01% BMP solution [20]. In the second stage, using a plastic round template 1.3 cm in diameter, soft tissues and superficial fascia were excised with a scalpel. For contamination, S. aureus culture (1 ml, 10⁹ microbial bodies) was applied on a cotton-gauze tampon and placed in the zone of wound defect. Then, 3–4 interrupted sutures were applied to the edges of the wound until the defect was completely closed. On day 2, pronounced hyperemia and edema in the wound area, and the appearance of tissue discharge were noted. On day 3, the sutures were removed, significant purulent exudation was detected, the wound surface was washed with 0.01% solution of BMP, non-viable tissues were removed and treatment was started. After modeling, all animals were kept in individual cages with the same nutrition and care conditions.

Animals were withdrawn from the experiment and biological material was collected for histological and histochemical examination on days 1, 3, 5, 7 and 14 after modeling.

Hyperemia, edema of soft tissues in the zone of defect, the kind and amount of discharge, appearance of epithelialization and granulation, surface cleansing (fibrinolysis, necrolysis) were visually assessed daily. Planimetric examinations with the calculation of the wound surface areas were performed using WoundVision personal computer software (IIMEDSCAN, Russia) and a RealSeance D415 camera (Intel, China). Morphological analysis of the material of the rat skin dermis was conducted with hematoxylin and eosin staining, Giemsa staining, impregnation with silver and toluidine blue. The dynamics of mast cells, collagen formation processes, and inflammatory infiltrate areas were also assessed.

Statistical processing of the obtained data was performed using the Statistica 10.0 (Stat Soft Inc., USA) and Excel 2010 (Microsoft, USA) software packages. The normality of distribution was checked and the following descriptive statistics methods were used: calculation of the mean (M) of the obtained results, standard deviation (SD) within the study groups. To determine the reliability of differences, Student's t-test was used to evaluate indicators with a normal distribution, Mann–Whitney — indicators whose distribution did not correspond to the normal, Wilcoxon t-test — for dependent samples in case of non-compliance with the normal distribution. The significance level was taken as 5.0% (p <0.05).

RESULTS

The average time of relief of local inflammation signs in treatment of purulent wounds is given in Table 1, and the dynamics of reduction of the wound area is given in Table 2.

 

Table 1. Time (M±SD) of relief of local inflammation signs in the control and experimental study groups, days

Wound process symptoms	Study group			р
	1	2	3
Necrolysis	4.3±0.2	3.9±0.2	3.6±0.2	р1-2=0.075 р1-3=0.032 р2-3=0.041
Skin hyperemia	4.7±0.3	4.5±0.2	3.8±0.2	р1-2=0.064 р1-3=0.021 р2-3=0.038
Edema	4.5±0.2	4.4±0.2	3.8±0.2	р1-2=0.072 р1-3=0.043 р2-3=0.037
Fibrinolysis	5.5±0.2	5.2±0.2	4.2±0.3	р1-2=0.078 р1-3=0.029 р2-3=0.031
Appearance of granulations	4.2±0.2	3.9±0.2	3.1±0.2	р1-2=0.066 р1-3=0.019 р2-3=0.032
Start of epithelialization	5.1±0.3	4.8±0.2	4.2±0.2	р1-2=0.061 р1-3=0.024 р2-3=0.039
Reduction of discharge to scanty	6.8±0.2	5.7±0.3	4.5±0.3	р1-2=0.042 р1-3=0.014 р2-3=0.027

 

Table 2. Dynamics of reduction (M±SD) of the wound area in control and experimental study groups, mm²

Timepoint of study	Wound area after modeling			р
	1	2	3
Day 1	93.7±6.1	88.4±5.9	81.2±5.8	р1-2=0.057 р1-3=0.043 р2-3=0.052
Day 3	54.2±6.3	53.5±5.4	45.7±5.6	р1-2=0.073 р1-3=0.037 р2-3=0.046
Day 5	47.3±5.6	41.5±5.1	29.5±5.2	р1-2=0.053 р1-3=0.018 р2-3=0.024
Day 7	34.4±4.8	27.3±4.6	20.7±4.7	р1-2=0.043 р1-3=0.014 р2-3=0.044
Day 14	13.1±1.3	9.8±1.5	2.2±1.2	р1-2=0.035 р1-3=0.006 р2-3=0.012

 

After modeling a purulent wound and removing the sutures, the microbial contamination was on average 10¹⁰–10¹³ of microbial bodies per ml of exudate (Table 3). In the experimental group, a marked decrease in this parameter was noted on day 3 of the study to 10²–10³ microbial bodies per ml of exudate, which indicates suppression of bacterial growth in the wound canal when using a combination of VDMB and 0.01% BMP solution.

 

Table 3. Dynamics of bacterial contamination (M±SD) of wound surface in the control and main study groups, microbial bodies per milliliter of exudate

Timepoint of study	Study group			р
	1	2	3
Day 1	109–1010	109–1010	108–1010	р1-2=0.087 р1-3=0.047 р2-3=0.059
Day 3	105–108	105–106	102–103	р1-2=0.063 р1-3=0.025 р2-3=0.039
Day 5	105–107	104–105	102–103	р1-2=0.038 р1-3=0.021 р2-3=0.033
Day 7	104–105	103–104	101–102	р1-2=0.044 р1-3=0.013 р2-3=0.019
Day 14	104–106	101–102	101–102	р1-2=0.021 р1-3=0.010 р2-3=0.086

 

In morphological analysis on day 3 of the study, in control groups 1 and 2, a pronounced inflammatory reaction, accumulation of neutrophilic-lymphocytic cells, mainly segmented and band neutrophils, and formation of microthrombi in the parietal vessels were determined (Table 4). In the experimental group, small foci of formed granulations, single fibroblasts and macrophages were visualized, indicating the onset of regeneration processes.

 

Table 4. Dynamics of number (M±SD) of cells of inflammatory infiltrate and connective tissue fibers in the zone of wound defect in the control and experimental study groups

Timepoint of study	Клеточные элементы	Группа исследования			р
		1	2	3
Day 3	Inflammatory infiltrate cells, U/mm2	194.0±7.7	158.0±4.3	131.0±5.11	р1-2=0.036 р1-3=0.022 р2-3=0.027
	Connective tissue fibers > 1 µm, %	8.3±0.2	9.8±0.4	14.2±0.2	р1-2=0.024 р1-3=0.018 р2-3=0.015

Continuation of the table

Day 5	Inflammatory infiltrate cells, U/mm2	147.0±9.5	102.0±7.3	84.0±11.4	р1-2=0.023 р1-3=0.016 р2-3=0.028
	Connective tissue fibers > 1 µm, %	10.1±0.2	19.7±0.4	27.4±0.5	р1-2=0.022 р1-3=0.012 р2-3=0.021
Day 7	Inflammatory infiltrate cells, U/mm2	93.0±6.9	85.0±9.2	51.0±3.7	p1-2=0.041 p1-3=0.014 p2-3=0.019
	Connective tissue fibers > 1 µm, %	15.5±0.2	31.2±0.3	39.7±0.3	р1-2=0.020 р1-3=0.009 р2-3=0.037
Day 14	Inflammatory infiltrate cells, U/mm2	41.0±3.4	33.0±1.2	15.0±5.6	р1-2=0.032 р1-3=0.017 р2-3=0.013
	Connective tissue fibers > 1 µm, %	44.7±0.2	61.3±0.2	74.1±0.5	p1-2=0.016 p1-3=0.013 p2-3=0.025

 

On day 5 of the study, the formation of epithelium at the wound edges was recorded in the experimental group. In control group 1 without treatment, inflammatory infiltrates were visualized not only in the tissue, but also in the walls of newly formed vessels, which may indicate the secondary bacterial dissemination due to untimely rejection of the scab. Also, among the key participants in fibrillogenesis and tissue remodeling after injury are mast cells, the growth of which was noted in the experimental group of the study (Table 5).

 

Table 5. Dynamics of the number (M±SD) of mast cells and fibroblastic differon cells in the wound defect zone and surrounding tissues in the control and main groups, units/mm²

Timepoint of study	Клеточные элементы	Study group			р
		1	2	3
Day 3	Mast cells	112.0±5.4	107.0±4.2	99.1±5.7	р1-2=0.067 р1-3=0.033 р2-3=0.056
	Fibroblastic differon cells	11.0±2.3	15.0±5.4	21.0±5.3	р1-2=0.074 р1-3=0.042 р2-3=0.059
Day 5	Mast cells	96.0±9.5	87.0±3.7	81.2±3.9	р1-2=0.081 р1-3=0.046 р2-3=0.055
	Fibroblastic differon cells	17.0±4.3	25.0±3.3	36.0±1.8	р1-2=0.037 р1-3=0.023 р2-3=0.029
Day 7	Mast cells	91.0±7.3	82.0±4.4	77.3±1.8	р1-2=0.063 р1-3=0.031 р2-3=0.057
	Fibroblastic differon cells	27.0±3.2	34.0±5.7	29.0±4.1	р1-2=0.044 р1-3=0.082 р2-3=0.077
Day 14	Mast cells	75.0±6.4	69.0±3.1	61.3±5.4	р1-2=0.085 р1-3=0.032 р2-3=0.039
	Fibroblastic differon cells	26.0±4.2	23.0±3.2	17.0±3.6	р1-2=0.089 р1-3=0.026 р2-3=0.052

 

On day 7, with the use of a combined staining method toluidine blue and silver impregnation allowing for assessment of the remodeling process in the dermis structure in the experimental group using VDMB, formed collagen fibers were visualized coinciding with the morphological architectonics of the surrounding dermis outside the wound. In the control groups, the different thickness of the fibers and their chaotic arrangement was noticeable (Tables 4, 5). In the micropreparations of control group 1, there were single inflammatory infiltrates, mainly consisting of lymphocytes and macrophages (Table 4).

On day 14, in the experimental group, a complete epithelial layer was formed after the rejection of the scab, and in the dermis, there was a large number of hair follicles in the mature anagen phase, and newly formed sebaceous glands.

In control group 1, the wound bed is in places devoid of the epithelial coating, however, in the dermis, single germs of hair follicles are visualized, the surrounding tissue is moderately infiltrated with lymphocytes. In control group 2, healing occurred with a predominance of fibroblastic differon cells over others, single hair follicles were formed (Table 4).

DISCUSSION

VDMB, containing high concentrations of various growth factors, hormones, vitamins and microelements, is of considerable interest for use in the treatment of soft tissue wounds.

The conducted study showed that the introduction of 1 ml of S. аureus culture at a concentration of 109 microbial bodies according to the proven method, led to the formation of a purulent wound with a considerable purulent discharge by day 3. In the model without treatment (control group 1), epithelialization of wounds started on average on day 5.1±0.3, the wound area after modeling reduced to (34.4±4.8) mm²; microbial contamination of the exudate on day 7 reduced to 10⁴–10⁵ microbial bodies per ml.

Dressings with 0.01% BMP (control group 2) led to acceleration of epithelialization to (3.9±0.2) days, reduction of the defect area to (27.3±4.6) mm², reduction of microbial contamination of the exudate on day 7 to 10³–10⁴ microbial bodies per 1 ml.

Conducting standard treatment in combination with VDMB and 0.01% BMP solution (experimental group) led to a reduction in all the studied parameters when compared with both control groups 1 and 2. In particular, the onset of epithelialization decreased to (4.2±0.2) days, the area of the defect after modeling to (20.7±4.7) mm², microbial contamination of the exudate on day 7 reduced to 10¹–10² microbial bodies per ml of exudate. The obtained dynamics indicates an enhancement in regeneration processes with the use of VDMB, which can be explained by enhanced manifestations of local immunity confirmed by histological studies, and is one of the properties of VDMB.

CONCLUSION

The use of vacuum dried maral blood in complex treatment of purulent wounds of soft tissue made it possible to reduce the time of stopping the local inflammatory reactions, accelerate reduction of the wound area, reduce the activity of growth of bacterial microflora in the wound. Positive dynamics of cell elements and connective tissue fibers was also demonstrated, which indicates a more complete restoration of the dermis when using the proposed treatment method.

ADDITIONAL INFORMATION

Author contributions. N.O. Mikhaylov — conducting an experimental study, analysis and interpretation of data, writing the text; A.A. Andreev, A.A. Glukhov — concept and design of study, editing; V.V. Shishkina — analysis of morphological material, writing the text; O.V. Sudakov, D.V. Sudakov — analysis and interpretation of data, statistical processing. All authors approved the manuscript (the publication version), and also agreed to be responsible for all aspects of the work, ensuring proper consideration and resolution of issues related to the accuracy and integrity of any part of it.

Ethics approval. The study was approved from the Local Ethics Committee of the N.N. Burdenko Voronezh State Medical University (Protocol No. 7 of December 13, 2022).

Consent for publication. All participants of study and their representatives voluntarily signed an informed consent form before being included in the study.

Funding sources. No funding.

Disclosure of interests. The authors have no relationships, activities or interests for the last three years related with for-profit or not-for-profit third parties whose interests may be affected by the content of the article.

Statement of originality. The authors did not use previously published information (text, illustrations, data) when creating this work.

Data availability statement. The editorial policy regarding data sharing does not applicable to this work, and no new data were collected or created.

Generative AI. Generative AI technologies were not used for this article creation.

Provenance and peer-review. This work was submitted to the journal on its own initiative and reviewed according to the usual procedure. Two external reviewers, a member of the editorial board and the scientific editor of the publication participated in the review.

About the authors

Nikolay O. Mikhaylov

N.N. Burdenko Voronezh State Medical University

Author for correspondence.
Email: n.o.mikhailov@yandex.ru
ORCID iD: 0000-0002-1710-205X
SPIN-code: 6113-7105
Russian Federation, Voronezh

Aleksandr A. Andreev

N.N. Burdenko Voronezh State Medical University

Email: sugery@mail.ru
ORCID iD: 0000-0001-8215-7519
SPIN-code: 1394-5147

MD, Dr. Sci. (Medicine), Professor

Russian Federation, Voronezh

Aleksandr A. Glukhov

N.N. Burdenko Voronezh State Medical University

Email: glukhov-vrn@yandex.ru
ORCID iD: 0000-0001-9675-7611
SPIN-code: 3821-2175

MD, Dr. Sci. (Medicine), Professor

Russian Federation, Voronezh

Viktoriya V. Shishkina

N.N. Burdenko Voronezh State Medical University

Email: v.v.4128069@yandex.ru
ORCID iD: 0000-0001-9185-4578
SPIN-code: 9339-7794

MD, Cand. Sci. (Medicine), Assistant Professor

Russian Federation, Voronezh

Oleg V. Sudakov

N.N. Burdenko Voronezh State Medical University

Email: sudakov_ol@mail.ru
ORCID iD: 0000-0003-2677-2300
SPIN-code: 2640-7362

MD, Dr. Sci. (Medicine), Assistant Professor

Russian Federation, Voronezh

Dmitriy V. Sudakov

N.N. Burdenko Voronezh State Medical University

Email: sdvvrn@yandex.ru
ORCID iD: 0000-0003-4911-1265
SPIN-code: 1759-9075

MD, Cand. Sci. (Medicine)

Russian Federation, Voronezh

References

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