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27MEDICAL SCIENCES
THE ORAL MUCOSA REACTION ON THE SUTURE MATERIALS AND THE COMBINED METHOD OF CONNECTING SURGICAL WOUNDS IN THE EXPERIMENT
Pavlenko O.
Shupyk National Healthcare University of Ukraine, Kyiv, Ukraine, Head of the department of dentistry,
MD, professor.
ORCID ID: https://orcid.org/0000-0001-6816-6830
Boiko M.
Shupyk National Healthcare University of Ukraine, Kyiv, Ukraine, postgraduate student of the department
of dentistry, corresponding author. ORCID ID: https://orcid.org/0000-0003-2179-4703
Savitskaya I.
Shalimov National Institute of Surgery and Transplantology, National Academy of Medical Sciences of Ukraine, Kyiv, senior research officer of department of experimental surgery, PhD.
ORCID ID: https://orcid.org/0000-0020-2749-7825
Geylenko O.
Shalimov National Institute of Surgery and Transplantology, National Academy of Medical Sciences of Ukraine, Kyiv, research officer of department of experimental surgery, PhD.
ORCID ID: https://orcid.org/0000-0001-5342-3881
Abstract
Introduction. Hermetic connection of wound edges and ensuring quality hemostasis is the final stage of any surgery. The connection of tissues and the influence of connecting elements on them, including in oral surgery, is an urgent problem of modern dentistry. Despite the invention of new methods of connection and modification of existing ones, there is no consensus of experts on their usage in each clinical situation. The suture technique is one of a choice. The stage of selection suture material is one of the main points in surgical treatment, on which depends the course of the postoperative period and the timing of wound healing. Due to the ambiguous views of specialists, as an alternative to suturing techniques, a medical adhesive composition was created, which facilitated the surgeons work. However, given the ambiguity of views on the usage of each of the methods, this issue remains debatable to this day.
Objectives. Based on the assessment of histological material and analysis of morphometric changes in the oral mucosa (OM) to investigate and study the course of regeneration processes after suturing 5/0 based on polyester, 5/0 polyester in combination with medical adhesive composition and 5/0 nylon.
Material and methods. The studies were performed on 76 adult outbred rabbits of different sexes with a body weight of 2, 6 to 4, 2 kg due to the similarity of the histological structure of the mucous membrane with the human. A series of incisions of the soft tissues of the vestibulum of the oral cavity of the maxilla were made. Rabbits were divided into 3 groups. In group 1, the joints of wound edges were made by applying knotted sutures made of 5/0 polyester. In group 2, a combined method of joining wound edges was performed by applying 5/0 polyester sutures and using the medical adhesive composition "Histoacryl". In group 3, oral mucosa connections were performed by applying knotted sutures made of nylon 5/0. On days 3, 7, 14 and 21, the animals were removed from the experiment. Tissue samples in the affected area were removed so that the wound canal with a deviation of 3 mm to healthy tissues fell within its boundaries, histological examination of the taken material was performed.
Results. In the course of the study, 45 calculations of the U-test of Mann-Whitney were performed. It was found that in 38 cases there are significant differences in the levels of morphometric parameters. Analysis of the dynamics of the average values studied during the experiment reveals that in rabbits of group 1 (polyester 5/0) they were all recorded at the highest level. In rabbits of group 2 (polyester 5/0 in combination with the adhesive composition) there was a significant difference in quantitative indicators for each of the days in comparison with groups 1 and 3, faster sutures erruption, moderate inflammatory-cellular reaction of tissues. The lowest values at all terms of observation were observed in group 3 (nylon 5/0), the lowest inflammatory-cellular reaction of tissues.
Conclusions. The high reliability of differences in the levels of morphometric parameters between groups of rabbits on each of the days was revealed, as evidenced by the calculated values of the U-Mann-Whitney test. In 37 cases, which is 97.4%, the significance level was p<0.01, in one case - p<0.05. Rabbits of group 3 had the lowest values in all indicators, a decrease in the levels of all studied cells from day 7 was recorded, which indicates a lower inflammatory-cellular response of oral mucosa and a reduction in the time of postoperative wound healing.
Keywords: oral mucosa, oral surgery, polyester, adhesive composition, nylon, wound healing, morphometry, histology.
Introduction
The responsible stage of each surgical intervention is the hermetic connection of the wound edges and ensuring quality hemostasis [1-3]. The issue of connection of tissues and the influence of connecting elements on them, including in oral surgery, is an urgent problem of modern dentistry, as evidenced by the large number of printed sources [1-7]. Despite the invention of new suture materials and modification of existing ones, the search for the best in terms of characteristics continues, due to various data presented in the scientific literature [8-12]. The most common method of choice is the suture technique of joining the edges of the wound [1315]. The stage of selection of suture material is one of the most important in surgical treatment, on which depends the course of the postoperative period and the timing of wound healing [7, 16-18]. Due to the ambiguous views of specialists and the development of the chemical industry, as an alternative to the suture technique, a medical adhesive composition was created, which facilitated the work of surgeons [19-21]. However, it is not always possible to connect tissues in anatomical and topographical areas that are prone to dynamic movements, there are cases of allergic reactions, the limit of widespread using is the high price [22-27]. Because of the lack of consensus of experts on the usage of each methods, this issue remains debatable to this day and needs further study. In this regard, it was decided to compare the two suture materials, the combination of the suture material with the adhesive composition and the reaction of the wound after application of them.
Objectives. Based on the assessment of histologi-cal material and analysis of morphometric changes in the oral mucosa (OM), to investigate and study the regeneration processes after suturing 5/0 based on polyester, 5/0 polyester in combination with medical adhesive composition "Histoacryl" and nylon 5/0.
Statistical analysis
Statistical analysis was performed using the standard software package SPSS 17.0. For each group, the average values of morphometric parameters and their errors (M ± m) were calculated, and a statistical analysis of the dynamics of changes in the levels of morpho-metric parameters was performed. To assess the reliability and statistical significance of differences in the levels of morphometric parameters determined for each group on each of the days, the nonparametric Mana-Whitney method was used.
Material and methods
Experimental studies were performed on 76 adult, outbred rabbits of different sexes and ages with body weight from 2260 to 4650 g (average weight 3565 ± 100 g) due to the similarity of the histological structure of the mucous membrane with the human [28]. All animals were veterinary examined and had a group health passport with the necessary preventive measures. Ways of acquisition, maintenance conditions, methods of anesthesia complied with the Rules of work performance with experimental animals. All experimental work was performed in compliance with ethical and humane
treatment of animals in accordance with the provisions of the Council of Europe Convention on Biomedicine. During the experiment, they were guided by the standards of the Guide for the core and Use of Laboratory Animals (National Academy Press, Revised, 1996) and the American Heart Association's "Guidelines for the Use of Animal in Reseach" [29,30]. The surgery was performed in sterile conditions under general and local anesthesia.
After anesthesia, which was achieved by intraper-itoneal administration of 5% sodium thiopental solution at a rate of 40 mg / kg body weight and intravenous administration of 1% propofol solution at a rate of 5 mg / kg body weight, surgery was performed. During each of the series of operations, the animals were fixed to the operating table, the surface of the oral mucosa was treated with 0.05% chlorhexidine solution. Local anesthesia was achieved by infiltration of 0.3 ml of 1% li-docaine.
Made a cut longitudinal surgical wound 1.3-1.5 cm in length and 0.5-0.7 cm in depth with a scalpel blade № 15. The mucous membrane and submucosal base were separated to periosteum.
Rabbits were divided into 3 groups. In group 1, the tissues were joined by applying three knotted sutures with 5/0 suture material based on polyester (POLYESTER®). In group 2, tissues were joined by applying three knotted sutures with polyester-based 5/0 suture material in combination with «Histoacryl®» medical adhesive composition. In group 3, tissue connections were performed using nylon-based suture material 5/0 (PIRUS®). On days 3, 7, 14 and 21, the animals were removed from the experiment by intraperi-toneal injection of an excess of 5% sodium thiopental solution and tissue samples were excised for histologi-cal examination. The removed area where the intervention was performed, together with the wound canal and a gap of up to 3 mm, which included intact tissue, was fixed in 10% neutral formalin solution for 24 hours, dehydrated in ethyl alcohol increasing concentrations (from 50° to 96°), clarified in xylene for 30 minutes, kept for two hours at a temperature of 37°C in a mixture of xylene and paraffin (1: 1) and twice in paraffin for 30 min at 56°C, compacted in paraffin according to conventional methods, made histological sections 5 ^m thick, which were stained with hematoxylin and eosin, picrofuxin by van Gizon.
Histological examinations were performed by using a Leica DM 500 research optical microscope with a computer prefix and the possibility of morphometric studies. Photographing the preparations was performed using a Leica ICC50 HD camera. Morphometric processing was performed using a video analyzer and computer program "Paradise", developed by the research and production company "Eva". The density of neutro-philic granulocytes, lymphocytes, macrophages, giant foreign body cells in 8 fields of view at a magnification of 400 times, then recalculated the content of these cells in 1 mm2 and statistically processed the obtained values between groups (Table 1,2).
Table №1
The average values of cell density (1 mm2 / GFBC in the eyeshot)
Indicator day Group 1 Group 2 Group 3
A 3 562,33±60,11 344,06±32,51 317,14±59,13
A 7 318,64±30,72 396,93±30,16 186,71±29,53
A 14 293,45±29,01 128,31±9,48 94,15±15,91
A 21 98,25±79,04 84,02±7,49 78,51±9,64
B 3 75,33±15,91 88,17±10,03 51,55±7,69
B 7 153,55±14,63 122,69±11,73 82,31±12,08
B 14 101,29±9,47 95,91±10,04 68,36±17,04
B 21 77,15±7,58 63,44±6,61 59,37±10,11
C 3 9,34±0,90 10,08±1,16 8,31±0,95
C 7 26,16±1,99 16,84±1,71 14,48±2,63
C 14 33,92±3,57 9,65±1,16 10,46±6,25
C 21 29,21±2,44 5,61±0,55 4,89±0,51
D 3 0 1 0
D 7 3 3 2
D 14 3 2 1
D 21 2 1 1
Symbols: A - neutrophilic granulocytes. B - lymphocytes. C - macrophages. D - Giant foreign body cells (GFBC). 0 - the indicator of the phenomenon is equal to zero.
Results
Qualitative results were described according to the type of connection on days 3, 7, 14 and 21 after the intervention
Polyester 5/0
On day 3 rd, under the wound surface, the tissue is infiltrated mainly with neutrophilic granulocytes. The threads are visible both on the surface and in the depth of its own plate of the mucous membrane. On day 7th, a small amount of granulation tissue formed in some areas. In its own plate in the area of the threads there are defects in the area of the seams and the connective tissue capsule around the sutures. On the 14th day, the epithelial plate is partially restored, visible filaments located in the thickness of the newly formed connective tissue. The tissue is moderately vascularized by dilated blood vessels, closer to the surface the number of blood vessels increases significantly, they are sharply dilated,
full-blooded. In some areas, small granulomas containing macrophages and giant foreign body cells (GFBC) are observed in the thickness of the newly formed tissue. Infiltration is insignificant, focal, lymphocytic (Pic. 1). On day 21st, the epithelial plate in the area of the filaments had a conical depression, thickened relative to the intact areas. The epithelial plate in the suture area is restored, forming outgrowths in its own plate, as in the surrounding areas. The epithelial plate is restored, with deep ingrown into the own plate of the mucous membrane. Under the epithelium in its own plate around the thread a connective tissue capsule was formed infiltrated by neutrophilic granulocytes, lymphocytes, single macrophages. The surrounding connective tissue of its own plate is also infiltrated by neu-trophilic granulocytes.
Pic. 1. Polyester-based suturing area after 14 days. Hematoxylin and eosin staining. Increase 100.
Polyester 5/0 + medical adhesive composition "Histoacryl"
On day 3 rd, a funnel-shaped depression was formed in the area of the glue and sutures in its own plate of the mucous membrane where filaments of threads and fragments of glue were visible, detritus masses were located between them, the epithelium was absent. The surrounding tissue is infiltrated mainly by neutrophilic granulocytes. Blood vessels are full-blooded, capillaries and venules around the sutures with signs of stasis. On day 7th, the filaments of the filaments were visible in the mucosal plate itself and were located on the surface of the epithelial plate (above the epithelium), where small defects in the suture area were noted. Between the filaments on the side of the connective tissue capsule formed around the filament, thin strands of fibroblasts and thin bundles of collagen fibers began to grow. Detritus masses and neutrophilic granulocytes were observed in the central part of the
multifilament filament. Complete blood vessels of the microvasculature with signs of atony. On day 14th, several sutures were missing from the wound. The epithelial plate in the area of the suture is partially restored, thinned. Venules and capillaries are full-blooded, without signs of stasis. The threads are located on the surface, fragments of adhesive masses are not noted (Pic. 2). On the 21st day, the epithelial plate is restored, thickened with branched deep ingrown into its own plate. In addition, in the area of the thread location there was an ingrown long wide epithelial cord into the connective tissue, in some areas the thread is visible. It was surrounded by epithelium, on the formation of a cyst around the thread was observed. The intrinsic mucosal plate between the cyst and the epithelial plate was compacted with a more compact arrangement of collagen fibers. The connective tissue around the thread was infiltrated with neutrophils and lymphocytes.
Pic. 2. Rabbit's oral mucosa 14 days after the usage of sutures based on polyester in combination with an adhesive composition. Hematoxylin and eosin staining. Increase 100.
Nylon 5/0
On the 3rd day, a thin layer of granulation tissue is formed under the surface of the wound, it and the surrounding tissues are abundantly infiltrated mainly by neutrophilic granulocytes and lymphocytes, macrophages are solitary. Signs of epithelial plate recovery are weak.
Full blood vessels with signs of stasis. On day 7th, a significant volume of granulation tissue is formed in the area of the suture, leukocyte-lymphocytic infiltration increases closer to the location of the sutures, and macrophages are present. The epithelial plate is partially restored. On the 14th day, the channels of the fila-
ments located in the thickness of the newly formed connective tissue are visible, along which the connective tissue capsule is observed. The epithelial plate is not completely restored, epithelial cells crawl to the edges of the wound, but complete epithelialization is not observed. Moderate leukocyte-lymphocytic infiltration of the scar zone on the surrounding tissues (Pic. 3). On the 21st day, the epithelial plate was restored, in different areas of uneven thickness, mostly thickened relative to intact areas, the basal layer formed outgrowths in the newly formed connective tissue of the suture area. The newly formed connective tissue is compacted. Small clusters of lymphocytes and macrophages were observed in the connective tissue around the filaments.
Pic. 3.
Suture area, 14 days after application of nylon sutures. Staining with hematoxylin and eosin. Increase 100.
Table 2
The results of pairwise assessments of the significance of differences between groups by the Mann-Whitney test
on morphometric parameters
Indicator Group/№ № 2 №3
Day Day
3 7 14 21 3 7 14 21
A №1 1 1 1 1 1 1 1 1
B 1 1 1 1 1 1 1 1
C 1 1 1 1 1 1 1 1
D - 0 0 0 - 0 1 2
A №2 X X X X 1 1 1 1
B X X X X 1 1 1 1
C X X X X 1 1 1 1
D X X X X - 0 0 0
Note: Calculations were performed using the Man-Whitney U-test; for significance levels p<0.05 and p<0.01 (according to Gubler E.I., Genkin A.A., 1973) critical values were determined. . x - pairwise assessments between the same groups does not make any sense.
Legend:
A - neutrophilic granulocytes;
B - lymphocytes;
C - macrophages;
D - GFBC;
u
0 - the hypothesis is accepted - J J :■■ differences between groups cannot be considered significant;
1 - hypothesis is accepted -1 -differences between groups can be considered significant (p<0.01);
2 - the hypothesis is accepted fii - the differences between the groups can be considered significant (p<0.05);
Statistical analysis
As a result of statistical data processing and pair-wise comparisons of rabbits groups on morphometric parameters in order to verify the significance of differences in the levels of morphometric parameters obtained during the study, 45 calculations of the U-test of Mann-Whitney were performed (Table 2). It was found that in 38 cases there are significant differences in the
group 1
levels of morphometric parameters between individual groups for each day.
The reliability of differences in the levels of mor-phometric parameters between groups of rabbits on each of the days is quite high, as evidenced by the calculated values of the U-test. In 37 cases, which is 97.4%, there are significant differences in the levels of morphometric parameters between individual groups on each of the days where the level of significance was p<0.01, in one case - p<0.05 (table 2). In 7 cases, the differences between the groups in terms of GFBC can not be considered as significant.
The dynamic analysis of the average levels of neu-trophilic granulocytes shows that the most important of this morphometric indicator were rabbits of group 1 on day 3 and 2 on day 7. On days 7 and 14 in group 1 it decreased almost twice and abruptly fell on day 21, but remained the highest in comparison with rabbits of group 2 and 3. In groups 2 and 3 the average level of neutrophilic granulocytes remained almost at the same level, except day 7, when it increased slightly in group 2. (Pic. 4).
group 2 group 3
600,0
400,0
200,0
0,0
day 3
day 7
day 14
day 21
Pic. 4. Dynamics of average values of neutrophilic granulocytes (per 1 mm2)
Indicators of the average level of lymphocytes in all groups increased on the 7th day, the highest value of this indicator was in the group 1 - more than two times compared with the 3rd day. Gradually it decreased in all
groups, meanwhile remained higher in group 1. The lowest average level of lymphocytes on each of the days was recorded in group 3 (Pic. 5).
group 1 group 2 group 3
213,3
160,0
day 3 day 7 day 14 day 21
Pic. 5. Dynamics of average values of lymphocytes (per 1 mm2)
The dynamics of the average level of macrophages is characterized by a sharp increasing in the group 1 on day 7, day 14 and a slight decreasing on day 21. However, it remains quite high throughout the experiment in comparison with groups 2 and 3, more than 3 times,
40,0
especially at 14th days and 21st days. The average level of macrophages in group 2 and 3 differs slightly, on the 7th day it increases almost 1.5 times compared with 3rd and 14th days, then gradually decreases (Pic. 6).
Group 1 Group 2 Group 3
day 3 day 7 day 14 day 21
Pic. 6. Dynamics of average values of macrophages (per 1 mm2)
As for the average level of GFBC, it is fixed from indicator is initially as high as in group 1, but then it day 7 and remains the highest in group 1 throughout the gradually decreases and on the 21st day reaches the study period. It is the lowest in group 3. In group 2 this same level as in group 3 (Pic. 7).
40,0
Group 1 Group 2 Group 3
day 3 day 7 day 14 day 21
Fig. 7. Dynamics of average values of GFBC in the eyeshot
Discussion
The results of our study are confirmed by the data obtained by J. S. Kim et al. [31], who compared and evaluated the severity of inflammatory reactions caused by three types of sutures (silk, polyglycolide, nylon), which are widely used in oral surgery on keratinized gums and mucous membranes of the dog's cheeks. It was found that the monofilament suture material based on nylon has the lowest inflammatory reaction compared to other suture materials.
T. Okamoto et al., A. Rached et al. [18,33], in an experiment on animals studied the effect of four types of suture material on the healing of the post-extraction wound in rats. It was found that nylon gave the best biological results in the healing of the mucous membrane and the cavity of the removed tooth. Healing was slightly prolonged with a polyester-based material, with a pronounced inflammatory-cellular reaction at the initial stage.
Also Mirkovic S. et al. [32], in experimental conditions compared the intensity of the local inflammatory-tissue reaction after suturing based on nylon and silk. The advantage of monofilament seams made of nylon was revealed due to the wick properties of the multifilament suture material.
Fawad Javed et al. [8], investigated tissue reactions to different suture materials and composition used in oral surgery, found that different suture materials have different degrees of tissue reactions depending on several factors, including the surface properties of the material and the properties of bacterial adhesion.
Performing any surgical procedure, the surgeon should take into consideration such aspects as the type of suture material, the number and technique of suturing. Biological and clinical aspects must be considered.
Therefore, different suture materials can be used depending on the experience, judgments and training of the surgeon [8,34].
Conflict of interest
The authors declare no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Acknowledgement
This study did not receive any official financial support. The authors testify to the absence of a conflict of interest.
Conclusion
Determined the presence of significant differences in the levels of morphometric parameters between all groups for each day. The reliability of the discrepancies is quite high, as evidenced by the calculated values of the U-Mann-Whitney test. In 37 cases, which is 97.4% of the total, the significance level was p<0.01, in one case - p<0.05 - 2.6%. The lowest value of the average level of neutrophilic granulocytes, lymphocytes and macrophages was observed in rabbits of group 3 on each day during the whole experiment compared with rabbits of group 1 and 2. This also applies to the level of GFBC in the dynamics. Histological and morpho-metric examination indicated a better tissue reaction after suturing with nylon 5/0, compared with the use of polyester 5/0 and its combination with the adhesive composition. After application of the combined method of connection, premature eruption of sutures, moderate inflammatory reaction was observed. The experimental study indicates the presence of minor morphological and morphometric changes in the oral mucosa after using the nylon 5/0, which shows faster healing of the postoperative wound.
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ИССЛЕДОВАНИЕ ХИМИЧЕСКОГО СОСТАВА ЛИСТЬЕВ ЛОПУХА БОЛЬШОГО
(ARCTIUM LAPPA L.)
Дмитриева С.О.
Студент, ФГАОУ ВО Первый Московский государственный медицинский университет им. И.М. Сеченова Министерства здравоохранения Российской Федерации (Сеченовский Университет)
Бирюкова Н.В.
ФГАОУ ВО Первый Московский государственный медицинский университет им. И.М. Сеченова Министерства здравоохранения Российской Федерации (Сеченовский Университет)
STUDY OF THE CHEMICAL COMPOSITION OF THE LEAVES OF BURDOCK
(ARCTIUM LAPPA L.)
Dmitrieva S.
Student, I.M. Sechenov First Moscow State Medical University of the Ministry of Health of the Russian Federation (Sechenov University) Biryukova N.
I.M. Sechenov First Moscow State Medical University of the Ministry of Health of the Russian Federation
(Sechenov University)
Аннотация
Основываясь на литературных данных об ограниченности лечебных и профилактических средств, для оказания быстрой помощи пострадавшим спортсменам в сельской местности, которые получили травмы, связанные с поражением коленных суставов, поиск новых источников для производства продуктов данного назначения является актуальной проблемой современной формации. Анализ научной литературы показал, что при травматизации суставов в народной медицине использовались листья лопуха большого и различные средства на его основе. В официальной медицине данное растительное сырье не применяется из-за отсутствия нормативной документации. В связи с этим нами было проведено исследование по изучению химического состава листьев при помощи качественных реакций, проведенных химически-чистыми реактивами, объект исследования, листья лопуха большого, были предварительно собраны в Подмосковье летом 2020 года, высушены и измельчены, а также в соответствие с ОФС.1.4.1.0018.15 настои и отвары и ОФС.1.4.1.0019.15 Настойки, были получены водные и водно-спиртовые извлечения. В результате исследования перспективного лекарственного сырья были обнаружены биологически активные вещества, представленные группами: флавоноидов, (уменьшающие воспалительные процесс); дубильных веществ (обеспечивающие вяжущие свойства, кровоостанавливающие), аминокислот. Кроме того, мы сравнили результаты, полученные в ходе реакций с водно-спиртовым извлечением и свежими листьями, было
