The study of anti-inflammatory action of various remedies in the treatment of chronic periodontitis

Yu. N. Battalova; Батталова Ю. Н.; M. A. Postnikov; Постников М. А.; S. E. Chigarina; Чигарина С. Е.; L. T. Volova; Волова Л. Т.; N. K. Osina; Осина Н. К.; E. I. Pugachev; Пугачев Е. И.; T. V. Starikova; Старикова Т. В.; N. A. Ryabov; Рябов Н. А.; S. A. Goncharenko; Гончаренко С. А.

doi:10.17816/pmj42238-46

The study of anti-inflammatory action of various remedies in the treatment of chronic periodontitis

Authors: Battalova Y.N.¹, Postnikov M.A.², Chigarina S.E.², Volova L.T.², Osina N.K.², Pugachev E.I.², Starikova T.V.², Ryabov N.A.², Goncharenko S.A.²
Affiliations:
1. National Medical Research Center for High Medical Technologies – A.A. Vishnevsky Central Military Clinical Hospital
2. Samara State Medical University
Issue: Vol 42, No 2 (2025)
Pages: 38-46
Section: Original studies
Submitted: 24.11.2024
Published: 27.05.2025
URL: https://permmedjournal.ru/PMJ/article/view/642193
DOI: https://doi.org/10.17816/pmj42238-46
ID: 642193

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Abstract

Objective. To study the anti-inflammatory effects of the “Alvostaz-sponge” medical product for the treatment of chronic periodontitis.

Materials and methods. The culture of juvenile human dermal fibroblasts was treated with TNF-α (1st series of experiments) and a mixture of TNF-α with “Alvostaz-sponge” extract (2nd series of experiments). The concentration of cytokines IL-6, MCP-1 in the culture medium of fibroblasts was assessed using enzyme immunoassay (ELISA). The examination and treatment of 74 patients (49 women and 25 men), with a diagnosis of chronic generalized periodontitis of moderate severity were carried out.

Results. In response to TNF-α stimulated inflammation, fibroblasts sharply increased the production of both chemokine MCP-1 and the inflammatory mediator IL-6 into the culture medium. A dose-dependent rise of “Alvostaz-sponge” extract content in the culture medium correlated with a decrease in the concentration of MCP-1, which indicates a pronounced inhibitory effect of “Alvostaz-sponge” extract on the production of MCP-1 cytokine. An increase in the concentration of IL-6 correlated with a dose-dependent increase in the extract of “Alvostaz sponge” in the culture medium of TNF-α-stimulated fibroblasts.

Conclusions. The results of the study demonstrated “Alvostaz-sponge” medication to be a powerful inhibitor of chemokine MCP-1 production and, it is due to this effect that it restricts the monocyte cells migration into the periodontal pocket zone. The obtained results of the treatment show accelerated healing of the periodontal complex when using the “Alvostaz-sponge” remedy.

Keywords

In vitro, cytokines, fibroblasts, periodontal pocket, chronic periodontitis of moderate severity

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Introduction

Periodontal tissues include a complex of tissues that surround the tooth and provide the dentogingival junction. The development of chronic periodontitis is associated with manifestations of specific subgingival bacterial flora from the deep sections of the gingival sulcus: exotoxins and endotoxins produced by periodontopathogenic microorganisms, which lead to long-term inflammation and destruction of periodontal tissues with subsequent formation of periodontal pockets [1; 2].

Inflammation is mainly mediated by cells of the so-called monocyte link - neutrophils, monocytes/macrophages and T- and B-lymphocytes, which move to the site of inflammation by chemotaxis, attracted by special molecules called chemokines. These cells produce inflammatory mediators that promote tissue degradation and bone resorption as a result of the attraction of monocytic cells to the site of inflammation [3–5]. One of the known triggers of inflammation is tumor necrosis factor (TNF-α), which initiates local release of cytokines, stimulates adhesion, migration and activation of leukocytes at the site of invasion. Interleukins represent the most numerous family of cytokines that participate in intercellular communication. It is known that, gingival fibroblasts are capable of producing cytokines, chemokines and matrix metalloproteinases that participate both in maintaining bone tissue homeostasis and in the pathogenesis of inflammation together with infiltrating inflammatory cells (monocytes/macrophages) [6–8]. Periodontal ligament fibroblasts, as key connective tissue cells, play an important role in paracrine inflammatory signaling, providing regulation of various cellular functions and processes, such as inflammation, tissue regeneration and remodeling [9; 10]. Paracrine signaling is a form of cellular communication in which fibroblasts secrete signaling molecules (cytokines, growth factors and other mediators) and have an effect on neighboring cells within a local area. All this contributes to: attracting immune cells (monocytes, macrophages) to the site of inflammation, increasing vascular permeability, which allows leukocytes and plasma to penetrate tissues and stimulate regenerative processes, promoting the restoration of damaged tissues. “Alvostaz-sponge” is a special medication used for the prevention and treatment of alveolitis in clinical practice.

In this regard, the “Alvostaz-sponge” medication is proposed for administration in the periodontal pocket for the treatment chronic generalized periodontitis of moderate severity.

The aim of the study is to investigate the anti-inflammatory effect of the “Alvostaz-sponge” medication for the treatment of chronic periodontitis.

Materials and Methods

The biological testing methodology was carried out in the laboratory of cells cultures of the scientific research institution Biotekh SamSMU, and the “Fibrotest-system” developed by the employees was selected as an in vitro cell model for studying the molecular mechanism of action of a special tool-hemostatic and antiseptic compress for the alveoli “Alvostaz-sponge”. “Fibrotest-System” is based on the use of the primary culture of human fibroblasts of the juvenile nature, which, in response to triggers of inflammation, produce MCP-1, IL-8 chemokines and the IL-6 cytokine of the pleiotropic action to the culture medium [11; 12]. This paper evaluates the mediated effect of the “Alvostaz-sponge” medication on cells by the level of secretion of IL-6 and MCP-1 cytokines, which took part in the development of periodontal inflammation and maintaining bone-tissue homeostasis.

The study was conducted in two stages:

selection of the optimal concentration of TNF-α (TNF) for the induction of IL-6 and MCP-1 production;
exposure to an extract with the optimal concentration of TNF-α to determine the effect of the medication.

The preparation of the extract from the “Alvostaz-sponge” medication was carried out in accordance with GOST R ISO 10993.12-99. The “Alvostaz-sponge” medication was placed in the M199 culture fluid (“Biolot”, Russia) and left for 24 hours while stirring by a magnetic mixer. The extract was filtered through 0.22 μm filter for sterilization, then serial breeding of the extract was done in concentrations of 2x, 4x, 8x, 16x, 32x and 64x.

The culture of human juvenile fibroblasts was obtained as described in [13].

The cells’ culture after defrosting was cultivated in the nutritional medium 199 (“Biolot”, Russia), containing 10 % fetal calf serum (FCS, Biolot, Russia) and 40 μg/ml of gentamicin (“Dalhimfarm”, Russia), until the subcontuational Monolayer was formed and cultured again in the 96-well cell plate (TPP, Switzerland) using versene-trypsin (“Biolot”, Russia) for conducting TNF-α (SciStore, Russia) stimulation.

Cell viability was assessed using the MTT assay, which measures the ability of viable cells to convert a soluble tetrazolium salt into an insoluble purple formazan sediment. Optical density was measured using a spectrophotometer at a wavelength of 540 mm.

Titration of the TNF-α inflammation trigger was performed by serial 10-fold dilution of TNF-α. 200 μl of TNF-α solution at a final concentration of 200; 20; 2; 0.2; 0.02; 0.002; 0.0002 ng/ml were added to cell plates containing human fibroblast cell culture.

To test the effect of “Alvostaz-sponge” medication, a mixture of 5 ng/ml TNF-α with serially diluted “Alvostaz-sponge” extract was added to cell plates containing culture of human fibroblast cells, followed by incubation of the cells in a CO2 incubator. After 20 ± 2 h, the nutrient media were collected from the 96-well cell plate into Eppendorf tubes. Culture media were stored at -20 °C for subsequent enzyme-linked immunosorbent assay (ELISA).

The ELISA analysis was performed according to the manufacturer's instructions for the IL-6 ELISA and MCP-1 ELISA kits (“Vector-Best”). The sensitivity of the ELISA was 0.5 pg/ml for IL-6 and 15 pg/ml for MCP-1.

An examination and treatment of 74 patients on their request with a diagnosis of chronic generalized periodontitis of moderate severity (according to ICD-10 K05.31) was conducted, including 49 women and 25 men, the age of the patients was from 32 to 67 years. The patients were divided into two groups. The main group (n = 30): “Alvostaz-sponge” medication was injected into periodontal pockets, and in the comparison group (n = 44), the osteoplastic biocomposite material “KollapAn M” (composition: artificial hydroxyapatite, collagen, antimicrobial agent) was injected.

Results and Discussion

The optimal concentration of TNF-α was selected to stimulate inflammation in fibroblast cell culture to determine the optimal TNF-α dose to stimulate the synthesis of IL-6 and MCP-1 cytokines in the first series of experiments. The proinflammatory effect of TNF-α on human fibroblast culture was assessed by the level of secretion of IL-6 and MCP-1 cytokines, which are involved in the development of periodontal inflammation. Conditioned medium from cells not treated with TNF-α (0) contained trace quantities of these factors (Fig. 1). After treatment of cells with TNF-α at concentrations from 0.2 to 200 ng/ml, the cytokine content in the conditioned medium increased proportionally to the doses of TNF-α. Control fibroblasts (0 ng/ml of TNF) produced 2.08 ± 0.47 pg/ml of IL-6 and 18.27 ± 1.66 pg/ml of MCP-1 (see Fig. 1). Analysis of the results showed that TNF-α doses in the range of 2–10 ng/ml were optimal for stimulating the production of both cytokines.

Fig. 1. Production of MCP-1 (a) and IL-6 (b) in response to stimulation with the TNF-α inflammatory trigger. The concentration of cytokines in the culture medium of primary fibroblast cell culture increased proportionally to the increase in the concentration of TNF-α (0.2–200 ng/ml)

The presence of TNF-α in the culture medium at a concentration of 5 ng/ml leads to the accumulation of MCP-1 and IL-6 fibroblasts of up to 7500 and 1000 pg/ml, respectively. Thus, TNF-α-stimulated fibroblasts produce hundreds of times more MCP-1 and IL-6 into the cellular medium compared to control, non-stimulated cells.

The MTT test was performed 48 hours after treatment of TNF-α-stimulated fibroblasts with “Alvostaz-sponge” extract at a dilution of 1x, 2x, 4x, 8x, 16x, 32x, 64x to determine the cytotoxicity of “Alvostaz-sponge”. Control cells (100% viability) are cells that were grown in vitro in a medium that did not contain the “Alvostaz-sponge” medication. It is noteworthy that, starting from an 8x dilution of the extract, the cells demonstrate stable viability comparable to the control data (Fig. 2).

Fig. 2. Determination of the viability of human fibroblast culture in the presence of the extract of the "Alvostaz-sponge" medication using the MTT test

Thus, in the 2nd series of experiments, stimulation with the TNF-α (5 ng/ml) inflammation trigger was carried out in the presence of serial dilutions of the “Alvostaz-sponge” extract (8x–64x), at which the cells demonstrated stable viability within 80–90 %.

Unstimulated fibroblasts (without TNF) demonstrate the absence of IL-6 cytokine production. The concentration of IL-6 in the culture medium of TNF-α-stimulated fibroblasts varies depending on the degree of dilution of the extract (8x, 16x, 32x, 64x), as shown in Fig. 3.

Fig. 3. ELISA analysis of IL-6 in the culture medium of unstimulated fibroblasts (without TNF-α) and stimulated with TNF-α (+TNF-α) inflammatory trigger

IL-6 is a pleiotropic cytokine that is involved not only in inflammatory reactions, but also, in the regulation of metabolic and regenerative processes [10]. The pleiotropic biological effects of IL-6 are determined by a unique signaling system that includes IL-6 receptors (membrane analogue IL-6R or cytoplasmic analogue sIL-6R) and gp130 signaling molecules.

IL-6, acting through membrane-bound IL-6R, has regenerative and anti-inflammatory effects. Responses to IL-6 through sIL-6R are proinflammatory (Fig. 4). In classical signaling, IL-6 stimulates target cells through the membrane-bound interleukin-6 receptor (IL-6R), which is expressed only on certain cells (macrophages, neutrophils, etc.). A soluble (s) form of sIL-6R is present in the bloodstream and some tissues, which in combination with IL-6 activates trans-signaling by binding to the gp130 receptor, which is present on virtually all cells in the human body. It is believed that “trans-signaling” is more responsible for the “pathogenic” effects of IL-6 than “classical” signaling [11].

Fig. 4. Pro- and anti-inflammatory reactions of IL-6

Taking into account the results of clinical studies, the presence of the “Alvostaz-sponge” medication in the periodontal space leads to the activation of IL-6 classical cascade of signaling reactions that mediate the anti-inflammatory and regenerative effect of the drug.

Fibroblasts not stimulated by an inflammatory trigger demonstrate low constitutive levels of MCP-1 chemokine production (Fig. 5). However, in the presence of TNF-α, MCP-1 production increases sharply: the concentration of MCP-1 in the fibroblast culture medium changes depending on the degree of dilution of the extract (8х, 16х, 32х, 64х) (see Fig. 5). High doses (8x) of “Alvostaz-sponge” extract completely inhibit MCP-1 production to the level of production by control fibroblasts. Reducing the concentration of the “Alvostaz-sponge” extract (8x, 16x, 32x, 64x) in the culture medium leads to a dose-dependent increase in the synthesis of MCP-1.

Fig. 5. ELISA analysis of МСР-1 in the culture medium of unstimulated fibroblasts (without TNF-α) and stimulated with TNF-α (+TNF-α) inflammatory trigger

Thus, the extract of the "Alvostaz-sponge" medication possesses MCP-1 inhibitory characteristics. To relieve the inflammatory process, an active search is underway for antagonists of MCP-1, the action of which is aimed at limiting the migration of leukocytes. The anti-inflammatory properties of MCP-1 inhibitory monoclonal antibodies, mutant recombinant forms of MCP-1, viral inhibitors, etc. are being studied.

The results of clinical observation showed that the proposed “Alvostaz-sponge” medication relieves inflammation and pain, has a therapeutic effect for several hours, after which it begins to dissolve gradually. (http://omegadent.ru/catalog/gemostatiki/483/). It should be noted that at a single administration of the "Alvostaz-sponge" medication patients did not complain after 48 hours, the gingiva was pale pink, tightly adjoined to the teeth, there was no exudate in the periodontal pockets, the depth of the periodontal pockets was less than 3 mm, and before treatment it was determined up to 5 mm.

In the comparison group, patients after treatment of periodontal pockets using the "KollapAn M" agent experienced discomfort in the oral cavity, minor hyperemia in the area of the gingival margin and bleeding persisted, there was no exudate, tooth mobility persisted for five days, a gradual decrease in the depth of periodontal pockets was noted from 5 mm (before treatment) to 3.5 mm (after treatment).

Thus, the molecular mechanism of action of the "Alvostaz-sponge" medication in the treatment of periodontal pockets allows to stop inflammation in periodontal tissues and achieve longer periods of remission for a short period of time, and the prolonged antimicrobial effect promotes the reverse development of the inflammatory-destructive process of periodontal tissues.

Conclusions

The studies which we conducted demonstrated that the “Alvostaz-sponge” medication is a potent inhibitor of the production of the MCP-1 chemokine, which limits the migration of monocytic cells into the periodontal space and leads to the prevention of the inflammatory cascade mediated by monocytes/ macrophages. The “Alvostaz-sponge” medication stimulates moderate production of IL-6 in parallel with the MCP-1 inhibitory effect. It can be assumed on the bases of the results of clinical studies which demonstrate accelerated restoration of the periodontal complex that moderate stimulation of IL-6 production mediates the classic IL-6 cascade of activation of signaling molecules, which is associated with anti-inflammatory and regenerative action.

Analysis of the results of treatment of patients with chronic generalized periodontitis of moderate severity using the "Alvostaz-sponge" medication allows to increase the effectiveness of treatment and reduce the rehabilitation period of patients.

About the authors

Yu. N. Battalova

National Medical Research Center for High Medical Technologies – A.A. Vishnevsky Central Military Clinical Hospital

Author for correspondence.
Email: battalovastoma@yandex.ru
ORCID iD: 0009-0000-2378-0378

Dentist-Therapist

Russian Federation, Moscow