Pathomorphological analysis of the gum tissues in the application of the combination of Soderm ® - Forte gel with Cytoflavin ® in experimental periodontitis in rats

Introduction: Periodontitis is one of the most urgent problems of modern dentistry. The development of new paradigms and regimens of combination therapy of patients with periodontitis is a strategic task for pharmacologists and dentists. In view of this, pathomorphological examination is of high importance, since it allow us to conclude about the therapeutic effectiveness of the administered drugs with high objectivity. Aim of the study : to evaluate the effect of the composition of Soderm ® -Forte and Cytoflavin ® on the pathomorphological pattern of gum tissues of rats with experimental periodontitis (EP). Materials and Methods: EP was simulated in rats by ligature method. Study design: animals with intact periodontium; animals with untreated EP; animals with EP treated with traditional drug therapy (TDT); animals with EP treated with the combination of TDT and Soderm ® -Forte gel; and animals with EP treated with the combination of TDT, Soderm ® -Forte and Cytoflavin ® . For pathomorphological examination, biopsy specimen was taken from the gingival margin of the lower incisors. Slides were stained with hematoxylin and eosin, as well as by Masson. Computer morphometry was performed using the ImageJ software. Results and Discusion: In EP, TDT has a moderate positive effect on pathomorphological changes in the gum. The combination of TDT and Soderm ® -Forte and, to a greater degree, the combination of TDT, Soderm ® -Forte and Cytoflavin ® have high therapeutic efficacy, characterized by rapid regeneration of the gum tissues. Conclusion: The combination of TDT, Soderm ® -Forte and Cytoflavin ® in EP has a more pronounced therapeutic effect, manifested by early regression of pathological changes and acceleration of tissue regeneration in the gum.


Introduction
Pathomorphological studies using modern light microscopy and computer morphometry, as a rule, play a pivotal role in the study of both the toxic and therapeutic effects of potential drugs in preclinical studies. This approach to the development of new drugs and regimens of their administration in periodontology, in particular in the treatment of patients with inflammatory lesions of the periodontal complex (PC), is also often preferred (Bedrosova et al. 2018;Leontiev et al. 2020).
It is known that the etiological factors of periodontitis can be both violations of hydrodynamics in the PC (Kopitov and Leontiev 2022) and the microecology of the oral cavity; the presence of periodontopathogenic microorganisms of the first and second importance, coinfecting agents and opportunistic species (King et al. 2019;Ushakov and Tsarev 2019;Tsarev et al. 2020;Balmasova et al. 2021), which initiate and support the inflammatory process. Inflammatory process negatively affects not only the physiological state of the PC, but also the immunological reactivity of the body (Guerra et al. 2018;Tsepov et al. 2018).
To date, it is generally accepted in medical practice that the use of many antimicrobial drugs, in particular antibiotics, and antiseptic drugs leads to the development of resistance to them of many pathogenic microorganisms (Orekhova et. al. 2020;Zhuravleva et al. 2021). This encourages pharmacologists in collaboration with synthetic chemists and clinicians (including dentists) to search for and develop new highly effective drugs and their combinations capable to inhibit a wide range of pathogenic microorganisms, without causing resistance to them, as well as to possess positive pleiotropic properties (anti-inflammatory, antihypoxic, antioxidant, etc.), which would have a positive effect on the complex treatment of some diseases caused by pathogenic microorganisms, including periodontitis (Bakhit et al. 2018;Tsepov et al. 2018;Kuzmina et al. 2019;Ushakov and Tsarev 2019).
In the light of the above, application of Soderm ® -Forte and Cytoflavin ® in the complex treatment of periodontitis seemed promising.
Soderm ® -Forte is a mixed-type micellar gel containing nanocluster zero valent metallic silver in the form of Agn K+ monomers and monomer micelles, the structure of which consists of a metal core and a surface double electric layer (emulsion micelles formed by a mixture of nonionic surfactants (polyethylene glycols)), oil and an aqueous phase including superoxide dismutase (SOD). anosilver and drugs containing it, including Soderm ® -Forte, have a wide spectrum of antimicrobial activity (Ahmad et al. 2020;Yunusov et al. 2020;Farheen et al. 2021;Bharkhavy et al. 2022;, including antibiotic-resistant gram-positive and gram-negative strains (Talapko et al. 2020;Enas et al. 2021), antifungal activity (Novikova and Russkih 2018;Kowalczyk et al. 2021;) and antiviral activity Dung et al.2020;Kowalczyk et al. 2021), as well as antiinflammatory effect (Singh et al. 2018;Shin et al. 2018;Fehaid et al. 2020;Kubyshkin et al. 2020;Farheen et al. 2021). It has been shown that nanosilver can be successfully used in the treatment of periodontitis (Kadam et al. 2020;Leontiev et al. 2020;Fernandez et al. 2021). SOD is an enzyme of the first line of the body antioxidant defense, which has a regulating effect on the antioxidant-prooxidant system, which go out of balance due to increased production of free radicals, activation of oxidative stress and damage to cellular structures, during inflammatory and destructive processes in various tissues, including PC. It has been shown that Soderm ® -Forte as a multi-target agent has significant therapeutic efficacy in complex pharmacotherapy of EP in rats .
Cytoflavin ® is a combination drug (it contains metabolites -succinic acid and riboxin, and coenzymes -riboflavin and nicotinamide). Pharmacotherapeutic properties of Cytoflavin ® are based on synergistic action of its components. Succinic acid in significantly larger concentration compared to other succinatescontaining drugs phosphorylates a large number of proteins, increases the tissue oxygenation and significantly improves the cell respiration (Shapovalov et al. 2020;Bhattacharjee and Banerjee 2020). Riboxin suppresses the production of pro-inflammatory cytokines, interferon-ɣ, IL-1 and IL-12, tumor necrosis factor-alpha (TNF-α) in macrophages (activated) and spleen cells (Kondratiev et al. 2020). Riboflavin inhibits the nuclear factor-KAPPA B (NF-kB), having an anti-inflammatory effect, also reduces TNF-α and p r o t e a s o m a l e l i m i n a t i o n o f t h e u b i q u i t o u s phosphorylated inhibitor-kappa (P-Iκ), and increases the production of nitric oxide (Badawy 2020). Nicotinamide plays the role of a highly active modulator of pro-inflammatory cytokines IL-1, IL-6 and IL-8, TNF-α (Ungerstedt et al. 2003). A variety of pharmacological effects of Cytoflavin ® components result in its antihypoxant, antioxidant, antiinflammatory, anti-dystrophic and regenerative activities, activating effect on the redox enzymes of the mitochondrial respiratory chain and resynthesis of highenergy compounds, as well as a positive effect on energy formation in tissues and a decrease in the production of free radicals and restore the activity of antioxidant defence enzymes (Marashly and Bohlega 2017;Olenskaya 2021;Orlov et al. 2021;Maksimova et al. 2022).
According to Leontiev et al. (2012) additional application of Cytoflavin ® to TDT significantly increases the treatment success in patients with chronic generalized periodontitis and has a beneficial effect on PC tissues.
To date, it has been established that the combination of the TDT with Soderm ® -Forte and Cytoflavin ® in the EP in rats can significantly increase the pharmacotherapy effectiveness .
Based on the above, it seemed important to conduct comparative studies of the effect of TDT, combinations of TDT with Soderm ® -Forte and TDT with Soderm ® -Forte and Cytoflavin ® on the pathomorphological pattern of gum tissues in EP in rats.
Aim of the study was to evaluate the effect of the combination of Soderm ® -Forte and Cytoflavin ® on the pathomorphological pattrern of the gum tissues in EP in rats.

Experimental animals
The studies were performed from September to December. The experiments were performed in 50 white male Wistar rats weighing 230-250 g. The animals were kept in the vivarium in the standard lighting and food ration in accordance with the requirements of the Law of the Russian Federation "On the Animal Protection from Animal Cruelty'" dated 06/24/1998, Good Laboratory Practice in Preclinical Studies in the Russian Federation Prior to the study, the rats were quarantined (for at least 15 days), according to the requirements of the European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes. After the quarantine was completed, healthy animals with clear and shiny hair, moderate locomotor activity, no signs of lesions of the conjunctiva, mucous membranes of the nose and the mouth (inclusions) were selected. Unhealthy rats with signs of lesions from the above mentioned tissues were rejected (exclusions).

Study design
The rats were randomized into 5 groups of 10 individuals each: group 1 -the animals with intact periodontium (control-1); group 2 -the animals with EP (control-2); group 3 -the animals with EP treated with TDT, including irrigation of the oral cavity with a solution of chlorhexidine bigluconate (0.05%), Septo-Pack dentogingival dressing (Septodont, France); group 4 -the animals with EP treated with the combination of TDT with Soderm ® -Forte gel, which was injected into parodontal recess of the lower incisors (as well as in the 5 th group of animals) using a syringe and disposable syringe tips cannulae (Bisco Inc., USA); group 5 -the animals with EP treated with the combination of TDT with Soderm ® -Forte gel and Cytoflavin ® , which was administered intraperitoneally at a dose of 100 mg/kg (in terms of succinic acid). The animals with EP (groups 3-5) were treated for 12 days. The period of observation for groups 1-5 of rats was 42 days (Fig. 1).

Research methods
EP in rats was simulated by the ligature method described by Leontiev et al. (2020). A ligature with EVROLON 4/0 material (LLC "MZKRS Suture Materials", Russia) was applied to the necks of the lower incisors, followed by their immersion to the dento-gingival groove for 30 days. For secure fixation of the ligature, a light-activated flowable composite resin -Versa Flo (Cetntrics Inc., USA) was used. Biopsy material for pathomorphological examination was taken from the marginal part of the gum of the cervical region of the lower incisors on the 42 nd day of observation, i.e. 12 days after the start of pharmacotherapy.
Samples of the animals' gum were fixed in 10% neutral buffered formalin for 24 hours. After that, histological processing of the obtained samples was carried out using isopropyl alcohol and xylene in a Logos microwave tissue processor (Milestone, Italy). Then the samples of the gum were embedded in paraffin according to the generally accepted method; the sections were made 3-5 microns thick, using a rotary microtome Leica RM 2255 (Leica, Germany). Histological sections were stained with hematoxylin-eosin to obtain general pattern; Masson's trichrome was used for collagen fibers.
The obtained slides were studied using a Leica DM1000 microscope (Leica, Germany), and microphotography was performed with a Leica ICC50 E digital camera with Leica LAS Core software (Leica, Germany).
Morphometry was performed by computer image analysis using a Zeiss AxioLab.A1 microscope (Carl Zeiss Micro Imaging GmbH, Germany) with a personal computer and open source software "ImageJ" (US National Institutes of Health, USA). The epithelial thickness, acanthosis depth and diameter of blood vessels were measured using the "Straight Selection" software at the magnification of ×40. The number of capillaries was calculated using a fine tuned "Analyze particles" tool. At the same time, the slice area required for calculation was determined manually using the "Free hand selection" or " Polygon selection" tools. To quantify the fibrosis severity, the staining area coefficient (SAC) was calculated, which is the percentage ratio of the total area of collagen fibers detected by Masson to the total area of the biopsy section.

Statistical data processing
Statistical analysis of the morphometric study was carried out using "MedCalc Statistical Software" (Belgium). All samples were tested for the type of distribution using the Shapiro-Wilk W-test. Due to the absence of a normal distribution in most ordered samples, the results were described as a median (Me), lower and upper quartiles [Q1 and Q3, respectively]. Pearson's chi-squared test was used to determine the significance of the differences. The null hypothesis was rejected at the level of statistical significance p<0.05.

Results and Discussion
Results of pathomorphological examination using light microscopy found that in the samples of the mucous membrane of the marginal part of the gum in group 1 of the rats with intact periodontium (control-1), the epithelial layer and submucous tissue did not have any pathological changes. Stratified squamous epithelium had a typical layering and cellular composition, as well as a pattern of uniform keratinization. The cells of the basal layer formed numerous outgrowths (rete ridges) in the underlying connective tissue. The proper mucous plate is represented by a superficial papillary layer formed by loose connective tissue and the deeper reticular layer, consisting of dense connective tissue.
The gum sections in the 2 nd group of the animals with EP (control-2) revealed changes in the epithelial layer and the proper mucous plate specific to proliferative inflammation.
Stratified squamous epithelium had keratinization disorder and hydropic degeneration. The thickening of the epithelial layer, hyperplasia of the basal layer, elongation of the rete ridges with the formation of acanthosis were observed. Focal or diffuse lymphocyte and macrophage infiltration with an admixture of single neutrophils and plasma cells was present in the proper mucous plate. Infiltrating cells reached the alveolar processes, destruction of the collagen fibers, areas of neoangiogenesis were observed (Figs 2B and 2C).  In two cases (20%), the inflammation had signs of a severe exudative component with areas of epithelial destruction and the formation of erosions, the presence of a large number of intraepithelial leukocytes.
In the 3 rd group of the rats with EP treated with TDT epithelial changes of the gingival mucosa in the form of hyperkeratosis and acanthosis, hyperplasia of the spinous layer, basal cell hyperplasia were less frequently observed compared with the 2 nd group (control-2). Inflammatory cellular infiltration was perivascular predominantly (Figs 2D and 2E).
In one casa (10%), necrotic changes of the mucous membrane persisted in the form of acute erosions. Fibrosis of the proper mucous plate was noted in one case (10%) -the formation of fibro-papillary overgrowths on the gum surface.
In the gingival samples of the 4 th group of the animals with EP treated with the combination of TDT with Soderm ® -Forte gel, epithelial acanthosis was low-grade, hyperplasia of the cells of the spinous layer, basal cell hyperplasia were observed. Inflammatory cell infiltration was reduced in comparison with the control group to focal mild and moderate, with a predominant presence in the rete ridges. In some cases, intraepithelial leukocytes and moderate stroma fibrosis were present (Fig. 2F).
In the samples of the 5 th group of the rats with EP treated with the combination of TDT with Soderm ® -Forte gel and Cytoflavin ® , in most cases there were no changes in the epithelial layer, fibrosis of the proper mucous plate was not pronounced; in rare cases, mild inflammatory cell infiltration persisted (Fig. 2G).
Computer morphometry revealed that in the 1 st group of the animals with intact periodontium, the median thickness of the epithelium was 124. 80 [110.19; 141.86     In the 2 nd group of the rats with EP, the thickness of the gingival epithelium decreased 5.4 times (p<0.05). The values of the other measured parameters increased as follows: the acanthosis depth -3.5 times (p<0.05), the number of capillaries per 1 mm 2 of the slice -2.0 times (p<0.05), the capillary diameter -1.3 times (p<0.05), and the fibrosis severity -2.8 times (p<0.05) (Table, Figs  3-7).
In the 3 rd group of the animals with EP treated with TDT, the thickness of the gingival epithelium increased 2.8 times (p<0.05) when compared with the 2 nd group (control-2). On the contrary, the acanthosis depth decreased 1.5 times (p<0.05); the number of capillaries per 1 mm 2 reduced 1.7 times (p<0.05); the capillary diameter decreased 1.3 times (p<0.05), and the fibrosis severity decreased 1.8 times (p<0.05) ( Table, Figs 3-7).
In the 4 th group of the rats with EP treated with the combination of TDT with Soderm ® -Forte, the gingival epithelium was 4.3 times thicker (p<0.05); the acanthosis depth decreased twofold (p<0.05); the number of capillaries per 1 mm 2 reduced 1.9 times (p<0.05); the capillary diameter decreased 1.4 times (p<0.05), and the fibrosis severity decreased 1.7 times (p<0.05) compared with the 2 nd group (control-2) (Table , Figs 3-7).
Thus, the performed pathomorphological studies show that the TDT has a moderate positive effect on pathomorphological changes in the gum in EP in rats. The combination of the TDT with Soderm ® -Forte and, more significantly, the combination of the TDT with Soderm ® -Forte and Cytoflavin ® have high therapeutic efficacy, characterized by rapid regeneration of the gum tissues.

Conclusion
The administration of the combination of the TDT with Soderm ® -Forte and Cytoflavin ® for 12 days in EP in rats has a pronounced regulating effect on pathomorphological changes in the gum tissues, characterized by resolution of inflammation due to the activation of regenerative processes.
According to the significance of the regenerative effect, the studied combinations and TDT can be arranged in the following decreasing range: TDT + Soderm ® -Forte + Cytoflavin ® > TDT + Soderm ® -Forte > TDT.
The data obtained in this study, as well as the results of the previous studies  indicate the promising future outlook of clinical studies of the combination of the TDT with Soderm ® -Forte and Cytoflavin ® to improve the treatment effectiveness in patients with periodontitis.

Conflict of interest
The authors declare no conflict of interests. Note: SAC -the staining area coefficient; *-significant difference from the group 1, p<0.05; **-significant difference from the group 2 (untreated EP), p<0.05.