APPLICATION OF THE RAMAN FLUORESCENCE METHOD TO STUDY THE EFFECTS OF CHEMICAL, PHYSICAL AND RADIATION FACTORS ON THE MINERALIZATION OF HARD DENTAL TISSUES

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Introduction

The factors and processes leading to demineralization of hard tooth tissues are diverse. One of these factors is radiation therapy. The use of radiation therapy methods plays an important role in the clinical practice of cancer treatment. It is worth noting that this type of therapy can cause severe complications that reduce the possibility of its use. The current direction of improving radiation therapy is considered to reduce the negative consequences: lesions, reactions. At the same time, ionizing radiation affects the tissues of the oral cavity, causing complications. Most often, dentists in their clinical practice meet with such complications as telangiectasia, xerostomia, chronic pain syndrome, atrophy and changes in the sensitivity of the mucous membrane, changes in teeth [1, 2, 3]. Taking into account the growing success of oncologists in the treatment of malignant neoplasms, the life expectancy of such patients is expected to increase [4], and their total number at dental appointments increases.

After exposure to the radiation factor, the mineralization and resistance of hard tooth tissues to caries decreases due to many factors. One of them is deterioration of the quality of individual oral hygiene. Due to this, more plaque remains on the surface of hard tissues, a favorable environment is created for the development of microorganisms, including caries-causing ones, and acidity increases (pH 4-5 under plaque), which ultimately triggers the process of demineralization of hard tooth tissues. First of all, there may be signs of hypersensitivity (reaction to temperature, tactile, food stimuli) and changes in the color of teeth (from light gray to dark gray and loss of shine). With further deterioration of the situation, the amount of dense sticky plaque increases. As a result, the teeth can become brittle. A feature of radiation-induced dental lesions is the rapid rate of progression and location in atypical locations. Such as-bumps of the chewing group of teeth, cutting edge of incisors, cervical region. [5, 6].

Both in Russia and abroad, the diagnosis, treatment and prevention of dental changes after exposure to radiation methods of cancer therapy are considered very popular areas in dental practice. At the same time, about 50% of patients face the problem of radiation damage to the teeth after radiation treatment.

Taking into account the high rates, the problem of changes in the hard tissues of teeth caused by radiation should be considered urgent. At the same time, objective, pathogenetically oriented studies and digital technologies have not yet been proposed that adequately and rapidly assess the main manifestations of the mechanisms of caries development in patients with CHLO ZNO on the background of radiation therapy. In particular, its manifestations such as the level of hygiene and qualitative composition of oral microorganisms, salivation, mineralization/demineralization processes in the complex and in their interrelation [7]. For the above-mentioned purposes, quantitative express medical technologies "on the spot" that confirm the effectiveness of a particular remineralizing drug in situ for each patient during radiation therapy of CHLO in oncological pathology have not been developed experimentally and clinically.

Thanks to the development of quantum electronics, prerequisites are being created for the introduction of various laser technologies into medicine for practical and scientific purposes. Laser methods with diagnostic, preventive, and therapeutic focus are being actively introduced in dentistry [8]. Dentists face the problem of early instant diagnosis of changes in hard tooth tissues with high sensitivity [9].

Some of the existing methods of spectrum estimation based on the infrared, fluorescence, and Raman fields are currently the main "on-site" express technologies for studying the structural features, clinical status, and organo-mineral components of CHLO, in particular, bone and dental tissues. These rapid methods are recommended for their development and implementation in the clinic by the Presidium of the Russian Academy of Medical Sciences .

Thus, it is of scientific interest to use a fundamentally new technical and methodological basis to study the main pathogenetic factors affecting the mineralization of hard dental tissues in patients with CHLO ZNO at the stages of radiation therapy and to develop, on this basis, an effective algorithm for its sanogenetic assessment. prevention measures.

At the same time, methodologically and methodically, in order to obtain objective results and exclude other interpretations, it is necessary to carry out measurements and register them simultaneously for all these interaction components. To solve this important problem for dentistry, we used modern domestic technologies – Raman fluorescence diagnostics.

Based on the presented concept, the purpose of the study was formulated.

The aim of the work isstudy the effect of physical, chemical and radiation factors on the degree of mineralization of the surface of hard tooth tissues using spectroscopy.

Materials and methods of research

In accordance with the purpose of the study, a multi-factor multi-stage study was conducted on the basis of the Department of Orthopedic Dentistry and Orthodontics of the South Ural State Medical University of the Ministry of Health of the Russian Federation (YUGMU).

The time frame of the study covers the period from 2020 to 2024.

At the experimental stage, a study was performed on teeth removed for clinical indications (in vitro):

- in different degrees of humidity (dry and wet);

- before, after exposure to acid and remineralizing agent;

-before and after exposure to the radiation factor in three different total doses (2 Gy, 70 Gy, 110 Gy).

The study included teeth without hard tissue defects removed in middle-aged patients (from 30 to 50 years) who did not have severe somatic pathology.

At the first stage of the study, it was necessary to select the parameters of the studied objects that were suitable for further research. For this purpose, the mineralization of teeth was studied by Raman spectroscopy at the InSpectr M agro-industrial complex in different degrees of moisture content (dry and wet). We studied 10 dry (air-dried teeth for at least 3 days) and 10 wet (teeth that were in saline for at least 600 seconds) teeth.

At the second stage, to understand the process of changing the level of mineralization of hard tooth tissues under the influence of chemical factors, it was decided to use a demineralization factor (citric acid) and a complex of remineralizing therapy. Teeth (incisors, 20 pcs) were studied in the equator region by Raman spectroscopy at the Inspection M industrial complex. Then, they were alternately exposed to citric acid (exposure for 1 day) and a remineralization complex (CM-2 plates), repeating the study at each stage.

At the third stage, in order to understand the effect of direct radiation exposure on the surface of hard tooth tissues, in particular on the level of mineralization, it was necessary to check it in the absence of possible secondary factors (such as salivation, hygiene level, etc.). To solve this problem, we performed studies on extracted teeth (in vitro) exposed to radiation in three different doses (2 Gy, 70 Gy, 110 Gy). The teeth immediately after removal were divided into three study groups: (280 teeth were subjected to Raman spectroscopy in three areas (equator, cervical region, cutting edge (incisors, canines) or occlusal surface (premolars, molars)) on the agroindustrial complex "Inspector M" and are divided into 3 sub-groups (20 pcs each, depending on the planned further radiation dose of 2 Gy, 70 Gy, 110 Gy). Later, on the basis of the State Medical Institution "Chelyabinsk Regional Clinical Center of Oncology and Nuclear Medicine", the examined teeth were exposed to radiation therapy by a radiotherapy specialist on the LNK-268 X-ray unit After the teeth were irradiated, according to the dose, in each group, the teeth were re-examined by Raman spectroscopy in the same areas.

In the course of the study, AIC "InSpectr M" (wavelength - 514 nm) was used according to the proposed scheme [2]. (Figure 1)

Fig. 1. Scheme of operation of the agricultural complex "InSpektrM" with a light-guide nozzle

Fig. 1. Hardware and software complex "InSpectrM" with a light guide attachment

To analyze the Raman radiation of the solid tissue surface, data were recorded at different power levels (minimum and maximum). The Raman intensity (Ms) was calculated as the difference of data with different power levels. (Fig. 2.)[4].

Figure 2. Finding Raman lines

Fig. 2. Finding Raman lines

As a result of the study, data was obtained, which are displayed in the form of graphs and tables. Calculations were made according to the rules of medical statistics using the IBM SPSS Statistics 22 software package and Microsoft Excel 2020. Quantitative and ordinal indicators were calculated using descriptive statistical methods and displayed as the mean value (M) and standard deviation (m). The results of the calculation are presented in the form M ± m. In cases where the distribution of indicators differs from normal (according to the Shapiro-Wilk criterion), medians and quartiles are presented (Me [Q1;Q3]). For comparison, the Mann-Whitney test was used, since two unrelated groups are compared on a quantitative basis. All the material presented in the study was obtained and analyzed personally by the author. The work was performed in accordance with modern principles of evidence-based medicine.

Results and discussion

The obtained data from the study of teeth with different degrees of moisture content (Table 1) allow us to see the difference in dry (1801[1800; 1802]rel.units), moistened (591.5[591; 592]). Based on the results obtained, it was chosen to conduct further studies in a moistened form, taking into account their greater compliance with clinical measurements of mineralization.

Thus, at the first stage, the .

Table 1

Spectral characteristics of hard tooth tissues in different degrees of moisture content (dry and wet)

Table 1. Spectral characteristics of hard tooth tissues in varying degrees of moisture content (dry and wet)

At the second stage of the study (examination of teeth before and after exposure to acid and a remineralizing drug), data on changes in mineralization were obtained (Table 2). Before exposure to citric acid, the mineralization according to Raman Intensity is (602 [601; 602]) rel. units, after exposure to citric acid (152 [152; 153]) 423 [422; 423])revealed differences are significant (p<0.001). Thus, it is confirmed that organic acids (citric acid) contribute to demineralization of enamel more than twice, and the use of calcium-containing plates contributes to remineralization almost to the initial state. In other words, it is shown that the InSpectr M agribusiness system really has a high sensitivity and is able to register mineralization, de - and re - mineralization of hard tooth tissues.

Table 2

Spectral characteristics of hard tooth tissues: before and after exposure to acid and remineralizing agent

Table 2. Spectral characteristics of hard tooth tissues: before, after exposure to acid and remineralizing drug

The data obtained at the third stage helped to study the process of changing the mineralization of the surface of hard tooth tissues under the direct influence of radiation exposure in various doses. According to the results of this study,there were no significant differences in the level of mineralization (according to the Raman fluorescence spectroscopy method) in teeth before and after direct radiation exposure,regardless of the dose (2 Gy, 70 gy, 110 Gy) in any of the functional groups (incisors, canines, premolars, molars). All available differences are within the statistical margin of error. The results obtained, divided according to the dose of radiation exposure, are presented in tables (Tables 3,4, 5). At the same time, the data on mineralization of hard tooth tissues in different parts of the tooth, in all functional groups, were very different. Thus, the study shows that there is a difference in the mineralization of the surface of the hard tissues of the teeth in different areas of the hard tissues of the teeth (pis. 3.).

However, there was no direct effect of radiation exposure
(regardless of the dose) on the mineralization of the surface of the hard tissues of the teeth. These data suggest that there is an indirect mechanism of influence of radiation therapy on the occurrence of radiation damage to the teeth, due to a decrease in the level of hygiene, salivation, etc.

At the same time, this study shows different levels of mineralization in different parts of the tooth, both before and after radiation exposure.

As can be seen from the results of the study, there are differences in different areas of the surface of hard tissues of the teeth (equator, neck, cutting edge) (Fig. 3.)lowest mineralization according to the data (Raman intensity) is present in the neck of the teeth (Incisors-y=145± 1.5, x=963 cm-1, Canines -y=141± 1.1, x=963 cm-1, Premolars y=142± 1.8, x=963 cm-1, Molars-y=143± 1.3, x=963 cm-1) average in the area of the cutting edge and occlusal surface, (Incisors-y=374± 1.7, x=963 cm-1, Canines-y=377± 1.3, x=963 cm-1, Premolars-y=375± 1.2, x=963 cm-1, Molars-y=375± 1.1, x=963 cm-1) and maximum in the equator region (Incisors-y=413± 1.1, x=963 cm-1, Canines-y=414± 1.9, x=963 cm-1, Premolars-y=415± 1.7, x=963 cm-1, Molars-y=419± 1.6, x=963 cm-1). At the same time, in each of the regions, the level of mineralization does not change after radiation exposure, regardless of the dose.

Table 3

Spectral characteristics of hard tooth tissues after radiation exposure (2 Gy)

Table 3. Spectral characteristics of hard tissues of the tooth (2 Gy)

Table 4

Spectral characteristics of hard tooth tissues after radiation exposure (70 Gy)

Table 4. Spectral characteristics of hard tissues of the tooth (70 Gy)

Table 5

Spectral characteristics of hard tooth tissues after radiation exposure (110 Gy)

Table 5. Spectral characteristics of hard tissues of the tooth (110 Gy)

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Fig. 3. Spectral characteristics of hard tooth tissues by sections

Raman Intensity (M wed)

Fig. 3. Spectral characteristics of hard tissues of the tooth area

        Conclusions

1. Raman fluorescence spectroscopy is highly sensitive and can detect mineralization, de-and re-mineralization of hard tooth tissues.

2. Determination of the mineralization of hard tooth tissues by Raman-fluorescence spectroscopy is recommended to be carried out in moist, rather than in dry form.

3. Direct radiation exposure does not significantly change the level of mineralization of hard tissues of the teeth, regardless of the dose used in all functional groups (incisors, canines, premolars, molars), in all areas of the teeth (equator, cutting edge, cervical region).

About the authors

German Belyakov

Author for correspondence.
Email: belyakov-95@mail.ru
ORCID iD: 0000-0002-1927-0751

Natalia NURIEVA

Email: natakipa@mail.ru

Dmitriy TEZIKOV

Email: tezia_tooth@mail.ru

References

Supplementary files