The article is published in Ukrainian in the journal.
The English text is given in the author's version.

UDC 616.314.17: 616.311-006: 615.454.1

STUDY OF THE INFLUENCE OF PHOTOSENSITIZER AND COMBINED EFFECTS OF LOW-INTENSITY LASER RADIATION ON THE COMPOSITION OF THE MICROFLORA OF THE DENTAL PLAQUE

N.I. Filimonova *,O.G. Geiderikh *, R.S. Nazarian * *, K.Y. Spiridonova **

*-the National University of pharmacy

**-the Kharkiv National Medical University

Keywords: ethacridine lactate, low-intensity laser radiation, the microflora of dental plaque, a photosensitizer, activity.

Introduction. The high prevalence and intensity of pathologies of the oral cavity in the form of gingivitis, periodontitis, dental caries in children and adults brings the problem of their treatment and prevention to the forefront of modern dentistry. Recently, special attention deserves such pathology as tooth decay that can lead to the development of allergic reactions, disorders of the circulatory system, reducing masticatory function, removal of teeth and, as a result, any disease of the digestive tract. At the present time, the leading role in the development of caries belongs to the microorganisms that inhabit the oral cavity. According to the literature, more than 70 % of the microorganisms have on streptococci, 15 % of veillonella and Neisseria, the remaining component of the microflora - diphtheroids, lactobacilli, staphylococci, leptotrichia, Fusobacterium, actinomyces, yeast and other fungi[2, 3, 9, 10].

According to the modern theory of caries cariogenic microorganisms of the oral cavity during the fermentation of low molecular weight carbohydrates produce organic acids, which leads to a pH decrease and, as a consequence, progressive demineralization of dental tissues. Existing, currently, a wide range of antimicrobials used including dentistry, has several side effects: their use leads to mass destruction of the flora of the entire habitat of the oral cavity and the selection of resistant strains of microorganisms[1, 7, 8, 11, 12].In the last few years introduced a new method of antimicrobial therapy - photo-disinfection (FAD), is based on the selective destruction of pathogenic organisms, sensitised special preparation and activated by laser light with a specific wavelength[4, 5, 6, 13].

The aim of our study was to investigate in vitro the effect of combined exposure of the photosensitizer and low-intensity laser radiation on the level of expression of antimicrobial action against pathogenic microflora of dental plaque.

Materials and methods study. The material for analysis were taken from 20 patients without concomitant somatic pathology, age 6-7 years with different levels of dental caries. Fence material produced using standard dental instruments.

The study of the action of the laser radiation. The total microflora of the root canals were made in sugar broth in a ratio of 1:10. For the quantitative evaluation results of the obtained suspension by microdonation made fence in a volume of 0.05 ml, made culture Petri dishes on a surface of a 5 %blood agar, one of which served as control, while others were exposed to laser radiation in different modes. The Cup was placed in a thermostat at 36-48 hours, then gave a qualitative assessment of laser radiation on the microflora.

After irradiation, the content drops carefully to break up with a spatula on the surface of the medium. Crops were incubated in a thermostat at a temperature of 370C, within 36-48 hours, and then counted the number of grown colonies.

The study of the action of the photosensitizer. The microflora of root canals was emulsiable in 0.9 ml sugar broth. Microdonation took 0.05 ml were sown in a Cup with 5 %blood agar. This seeding served as a control. After that the tube was made of 0.1 ml of 0.1%solution rivanol (ethacridine lactate).

After the exposure time (1-3 min) ethacridine lactate with microbial cells microdonation suspended in a volume of 0.05 ml was transferred into a test tube with 1 ml of sugar broth. The concentration of ethacridine lactate decreased 20 times that were excluded in further antimicrobial effect even 1%solution rivanol. From this tube of 0.05 ml of the suspension were sown on plates with 5 %blood agar. Cups were placed in a thermostat at 370C for 24-36 hours, then take into account the presence and intensity of growth.

Explore FAD. The total microflora of dental plaque were made in 0.9 ml sugar broth. In a test tube made of 0.1 ml of rivanol so as to obtain a final concentration of 0.1%. Before making ethacridine lactate and after the exposure time (time sensitivity) of the test-tube with microdonation was collected, suspended in a volume of 0.05 ml and brought to the surface of the Cup with 5 %blood agar.

Experienced Petri dishes were subjected to irradiation with low-intensity laser radiation (NEELY). The exposure duration was 30, 60 and 120 seconds. After exposure, the material was carefully divided with a spatula over the surface of the agar. Cups were placed in a thermostat at 36-48 hours at 370C, and then counted the number of grown colonies. Comparing data control (original number before adding the photosensitizer) and experience (after irradiation photosensibilisation cells) to judge the action of laser radiation on sensitized by rivanol microflora of dental plaque.

The total microflora of dental plaque was treated with the composition of a photosensitizer and NEELY. After that, they were sustained over time, is required for efficient binding of the composition to the cells of the microorganisms (60 sec). Then on the specified area within 60 and 120 sec., wrought optical radiation with a wavelength of 445 nm, which corresponded to the maximum absorption of the photosensitizer and a power density of 100 mW/cm2 required for the activation of the composition.

Analysis carried out by counting the number of colony forming units (CFU) after 24 to 72 hours incubation at 37°C. Control are suspended bacteria, not treated with sensitizer and not subjected to irradiation.
For statistical processing of the results obtained was used the software package STATISTICA 6.0 company StatSoft Inc. for personal computer in Windows.

Research results and their discussion. The composition of the microflora of dental plaque in clinically healthy patients and patients with inflammation periodontal tissues has significant differences that bind primarily to the fact that the species specificity of microorganisms is variable and depends on both endogenous and exogenous factors. Of particular interest is the ratio of pathogenic and nonpathogenic microorganisms. When the choice of tactics of treatment of caries with the use of antimicrobial drugs should be given their inhibitory effect I only pathogenic but also on the "helpful" microorganisms. In the present research studied the effect of LLLT on the background of a photosensitizer (ethacridine lactate) on the selective elimination of pathogenic and conditionally pathogenic microorganisms.

Given that ethacridine lactate inherent antiseptic properties ought to set the maximum exposure time of ethacridine lactate as a photosensitizer. It was found that after exposure of rivanol (ethacridine lactate) for 1.5-3 minutes was observed bactericidal effect on microorganisms. The impact of antiseptics in the interval from 30 to 60 s was not accompanied by a marked quantitative changes in microbial populations.

The second phase of the study was to determine the sensitivity of microorganisms to different concentrations of photosensitizer. Used an aqueous solution of ethacridine lactate concentrations 0,1; 0,05; 0,01%. The obtained results allow to conclude that the solution of ethacridine lactate in a concentration of 0.1% increases the sensitivity of microorganisms to the action of low-intensity laser radiation.

In the course of the experiment, it was determined the antimicrobial activity ofthe combined impact of a 0.1%solution of ethacridine lactate and laser blue spectrum, which is manifested by a decrease in the number of CFU/ml of the total microflora of dental plaque. The average number of CFU decreased from a value of 14.3±0,12×103/ml to a value of 2.4±0,3× 102 ml after irradiation (table 1). Comparing monitoring data (the initial number of grown colonies) and experience (the number of colonies that grew after conducting photo disinfection) we established that the antimicrobial action photo disinfection is directly dependent on the duration of exposure. So the effectiveness of the combined use of a photosensitizer with NEELY 1.2 times the activity of ethacridine lactate (exposure time of 60 seconds) and 2.0 times the antimicrobial effect of laser radiation in the blue spectrum (exposure time of 120 seconds).

The conclusions. Thus, obtained in the framework of the conducted experiment, the results indicate a pronounced antimicrobial action of laser radiation in the blue spectrum on the microflora of dental plaque, sensitized with a solution of ethacridine lactate, which allows to prove the possibility of elimination of pathogenic microflora of the oral cavity by such method and requires further study.

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Table 1

Comparative characterization of the influence of the combined effect of photosensitizer and NEELY on the sensitivity of microorganisms, n=6

№ / CFU /ml
Control / The impact factor/ Exposure time, seconds
Ethacridine lactate (60s) / NEELY (120s) / Ethacridine lactate + NEELY (120s)
1 / 6,2±0,14× 103 / 2,1±0,07× 103 / 6,2±0,09× 103 / 3,6±0,2× 102
2 / 14,3±0,12× 103 / 5,7±0,13× 103 / 14,5±0,2× 103 / 2,4±0,3× 102
3 / 7,9±0,1× 103 / 3,1±0,04× 103 / 7,8±0,13× 103 / 2,6±0,2× 102
4 / 5,2±0,08× 103 / 4,0±0,07× 103 / 5,1±0,07× 103 / 5,2±0,14× 102
5 / 11,8±0, 2× 103 / 8,2±0,03× 103 / 11,9±0,1× 103 / 4,2±0,14× 102
6 / 6,5±0,25× 103 / 5,2±0,07× 103 / 6,7±0,17× 103 / 3,8±0,26× 102
7 / 8,3±0,17× 103 / 5,3±0,06× 103 / 8,0±0,307× 103 / 5,1±0,24× 102
8 / 6,0±0,19× 103 / 2,3±0,04× 103 / 4,5±0,1× 103 / 3,2±0,28× 102
9 / 5,9±0,09× 103 / 3,1±0,11× 103 / 6,0±0,1× 103 / 3,1±0,15× 102
10 / 7,9±0,04× 103 / 5,6±0,05× 103 / 8,2±0,04× 103 / 4,2±0,14× 102
11 / 6,8±0,13× 103 / 2,4±0,3× 103 / 5,4±0,06× 103 / 2,8±0,1× 102
12 / 7,2±0,11× 103 / 3,8±0,02× 103 / 6,9±0,17× 103 / 3,0±0,09× 102
13 / 12,3±0,14× 103 / 4,6±0,07× 103 / 5,2±0,08× 103 / 2,7±0,13× 102
14 / 5,6±0,09× 103 / 3,5±0,12× 103 / 6,1±0,09× 103 / 4,1±0,2× 102
15 / 6,7±0,2× 103 / 4,2±0,11× 103 / 5,3±0,05× 103 / 3,2±0,13× 102
16 / 7,6±0,12× 103 / 5,7±0,03× 103 / 7,9±0,12× 103 / 4,6±0,1× 102
17 / 8,6±0,15× 103 / 6,8±0,1× 103 / 9,2±0,1× 103 / 3,3±0,26× 102
18 / 9,3±0,16× 103 / 4,1±0,06× 103 / 5,3±0,05× 103 / 4,4±0,16× 102
19 / 11,4±0,13× 103 / 7,2±0,12× 103 / 10,9±0,13× 103 / 5,2±0,12× 102
20 / 5,8±0,1× 103 / 3,4±0,2× 103 / 5,7±0,08× 103 / 4,4±0,21× 102

INFORMATION ABOUT THE AUTHORS

Filimonova Natalia Igorevna - MD, Professor, Head. Department of Microbiology, Virology and Immunology, National University of Pharmacy, Kharkov
706-30-67
Geiderikh Olga G., MD, assistant professor of microbiology, virology and immunology of the National University of Pharmacy, Kharkov
706-30-67

Nazaryan Rozana Stepanovna - Doctor of Medicine, professor, head of department of pediatric dentistry, children's oral and maxillofacial surgery and implantology, KharkivNationalMedicalUniversity

764-05-96

Spiridonova Kseniya Y. - graduate student of pediatric dentistry, children's oral and maxillofacial surgery and implantology, KharkivNationalMedicalUniversity

764-05-96, e-mail: .