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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Abstract

Objective. To study the effect of physical, chemical and radiation factors on the degree of mineralization of the hard dental tissues surface using spectroscopy.

Materials and methods. The degree of mineralization of hard dental tissues was studied by the Raman spectroscopy using hardware-software complex InSpectrum M on the teeth removed according to clinical indications (in vitro): in varying degrees of moisture (dry and wet); before and after exposure to acid and remineralizing drug; before and after exposure to radiation

Results. The data obtained demonstrated the difference in the indicators in dry (1801 [1800; 1802]) relative units, and moistened (591.5 [591; 592]) teeth. The mineralization of teeth according to Raman Intensity before exposure to citric acid, was (602 [601; 602]) rel. units, and after the exposure it was (152 [152; 153]) rel. units, after application of the cm-2 plate it was (423 [422; 423]) rel. units. The identified differences are reliable (p < 0.001). Thus, it is confirmed that organic acids (citric acid) contribute to the enamel demineralization by more than two times, and the use of calcium-containing plates contributes to remineralization almost to the initial state. At the same time, there were no significant differences in the level of mineralization (according to the Raman method of fluorescence spectroscopy) in teeth before and after direct radiation exposure, regardless of dose (2 Gy, 70 Gy, 110 Gy) in any of the functional groups (incisors, canines, premolars, molars).

Conclusions. 1. Raman-fluorescence spectroscopy has a high sensitivity and is capable of detecting mineralization, de- and re-mineralization of hard dental tissues. 2. It is recommended to determine the hard tooth tissues mineralization by Raman-fluorescence spectroscopy in a moistened rather than a dry form. 3. Organic acids contribute to the demineralization of enamel, and the use of calcium-containing plates promotes its remineralization. 4. Direct radiation exposure does not have a direct effect on the mineralization of the surface of the hard dental tissues. regardless of the dose applied in all functional groups (incisors, canines, premolars, molars), in all areas of the teeth (equator, cutting edge, gum border).

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Introduction

The factors and processes leading to demineralization of hard dental tissues are varied. One of these factors is radiation therapy. The use of radiation therapy methods plays a major role in the clinical practice of treating oncological diseases. It is worth noting that this type of therapy can cause serious complications that reduce the possibilities of its use. A current direction for improving radiation therapy is considered to be the reduction of negative consequences: lesions and reactions. At the same time, ionizing radiation affects the tissues of the oral cavity, causing complications. Most often, dentists in their clinical practice encounter complications such as telangiectasia, xerostomia, chronic pain syndrome, atrophy and changes in the sensitivity of the mucous membrane, and changes in the teeth [1–3]. Considering 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 is increasing.

After exposure to radiation, mineralization and resistance of hard dental tissues to caries decrease due to many factors. One of them is the 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 cariogenic ones, and acidity increases (pH 4–5 under plaque), which ultimately triggers the process of demineralization of hard dental tissues. First of all, signs of increased sensitivity (reaction to temperature, tactile, food irritants) and changes in tooth color (from light gray to dark gray and loss of shine) may appear. As the situation worsens, the amount of thick, sticky plaque increases. As a result, teeth can become brittle. A characteristic feature of radiation lesions of teeth is the rapid rate of progression and location in atypical localizations, such as the tubercles of the chewing group of teeth, the cutting edge of the incisors, the gingival margin [5; 6].

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

Given the high rates, the problem of changes in hard dental tissues caused by radiation should be considered relevant. However, objective, pathogenetically oriented studies and digital technologies have not yet been proposed that adequately and expressly assess the main manifestations of the mechanisms of caries development in patients with malignant neoplasms of the maxillofacial region (MFR) against the background of radiation therapy. In particular, such manifestations as the level of hygiene and the qualitative composition of microorganisms in the oral cavity, the processes of salivation, mineralization / demineralization in a complex and in their interrelationship [7]. Quantitative express medical technologies “on site” have not been experimentally and clinically developed for the above-mentioned purposes, confirming the effectiveness of a particular remineralizing drug in situ for each patient during radiation therapy of the maxillofacial region for oncological pathology.

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

Some of the existing methods of spectrum evaluation, based on infrared, fluorescent and Raman fields, are currently the main express technologies "on site" in studying the features of the structure, clinical status, organ-mineral components of the maxillofacial region, in particular bone and dental tissues. These express methods are recommended for their development and implementation in the clinic by the Presidium of the Russian Academy of Medical Sciences. These express methods are recommended for development and implementation in the clinic by the Presidium of the Russian Academy of Medical Sciences.

Thus, it is of scientific interest to study, on a fundamentally new technical and methodological basis, expressly, “on site”, objectively (digital method), in interconnection, the main pathogenetic factors influencing the mineralization of hard tissues of teeth in patients with malignant neoplasms of the maxillofacial region at the stages of radiation therapy, and to develop on this basis an effective algorithm for sanogenetic prevention. At the same time, methodologically, in order to obtain objective results and exclude other interpretations, it is necessary to conduct measurements and record them simultaneously for all the specified components of interaction. 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 is to study the impact of physical, chemical and radiation factors on the degree of mineralization of the surface of hard dental tissues using spectroscopy.

Materials and methods

In accordance with the purpose of the work, a multifactorial multi-stage study was conducted at the Department of Orthopedic Dentistry and Orthodontics of the State Budgetary Educational Institution of Higher Education “South Ural State Medical University” of the Ministry of Health of Russia (SUSMU).

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

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

– in varying degrees of moisture (dry and wet);

– before, after exposure to acid and remineralizing agent;

– before and after exposure to radiation factors in three different total doses (2; 70; 110 Gy).

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

At the first stage of the study, it was necessary to select parameters of the objects under study suitable for further research. For this purpose, a study of tooth mineralization was conducted using Raman spectroscopy on the InSpectr M hardware and software system at different levels of moisture (dry and wet). Ten dry (air-dried teeth for at least 3 days) and ten wet (teeth that had been in a physiological solution for at least 600 s) teeth were studied.

At the second stage, in order to understand the process of changing the level of mineralization of hard dental tissues under the influence of chemical factors, it was decided to use a demineralization factor (citric acid) and a complex of remineralizing therapy. The teeth (incisors, 20 pcs.) were studied in the equatorial region using Raman spectroscopy on the InSpectr M hardware and software system. Then they were alternately exposed to citric acid (exposure – one day) and a remineralization complex (CM-2 plates), repeating the study at each stage.

At the third stage, in order to understand the influence of the direct impact of the radiation factor on the surface of hard dental tissues, in particular on the level of mineralization, it was necessary to test it in the absence of possible secondary influencing factors (such as salivation, level of hygiene, etc.). To solve the problem, a study was conducted on removed teeth (in vitro), exposed to radiation at three different doses (2; 70; 110 Gy). The teeth were immediately divided into three study groups after extraction: 280 teeth were subjected to Raman spectroscopy in three areas: equator, cervical region, cutting edge (incisors, canines) or occlusal surface (premolars, molars) on the InSpectr M hardware and software system, and divided into 3 subgroups (20 pieces each, depending on the planned further radiation dose of exposure in 2; 70; 110 Gy). Subsequently, at the State Autonomous Healthcare Institution “Chelyabinsk Regional Clinical Center of Oncology and Nuclear Medicine”, a specialist in radiation therapy exposed the examined teeth to radiation using an LNK-268 X-ray machine. After irradiation of the teeth according to the dose in each group, the teeth were subjected to repeated Raman spectroscopy examination in the same areas.

During the study, the InSpectr M hardware and software system (wavelength – 514 nm) was used according to the proposed scheme [2] (Fig. 1).

 

Fig. 1. Scheme of operation of the "InSpectr M" hardware and software system with a light guide head

 

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

 

Fig. 2. Finding Raman lines

 

The study resulted in the data being presented in the form of graphs and tables. The calculations were made according to the rules of medical statistics using the IBM SPSS Statistics 22 and Microsoft Excel 2020 software package. Quantitative and ordinal indicators were calculated using descriptive statistical methods and are presented as mean (M) and standard deviation (m). The calculation results 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 (Me [Q1; Q3]) are presented. The Mann – Whitney test was used for comparison, since two unrelated groups are compared based on a quantitative characteristic. All material presented in the study was obtained and analyzed personally by the authors. The research was done in accordance with modern principles of evidence-based medicine.

Results and discussion

The information we obtained in the study helped us to better understand the process of changing the mineralization of the surface of hard dental tissues. The obtained data from the study of teeth with different degrees of moisture (Table 1) allow us to see the difference in indicators in dry (1801 [1800; 1802] relative units) and moistened form (591.5 [591; 592]). Based on the obtained results, it was decided to conduct further studies in a moistened form, taking into account their greater correspondence with clinical measurements of mineralization.

 

Table 1

Spectral characteristics of hard dental tissues at different degrees of moisture (dry and wet)

Comparison Object

Upper Level

Lower Level

Raman Intensivity
(Relative Units), Мavg.

Dry sample

y = 4021.5 [4020.75; 4022];

x = 963 cm-1

y = 2220 [2220; 2221.25];

x = 963 cm-1

y = 1801 [1800; 1802],

Wet sample

y = 3503 [3501.75; 3503];

x = 963 cm-1

y = 2911.00 [2912; 2910];

x = 963 cm-1

y = 591.5 [591; 592],

р

p < 0.001

p < 0.001

p < 0.001

 

Thus, at the first stage, using the InSpectr M software package, a study of the Raman fluorescence spectrum was substantiated in a humidified rather than a dry form.

At the second stage of the study (study of teeth before and after exposure to acid and remineralizing agent), data on changes in mineralization were obtained (Table 2). Before exposure to citric acid, mineralization according to Raman intensity is 602 [601; 602] relative units, after exposure to citric acid – 152 [152; 153] relative units, after application of the CM-2 plate – 423 [422; 423] relative units. The differences revealed are reliable (p < 0.001). Thus, it is confirmed that organic acids (citric acid) contribute to enamel demineralization more than twice, and the use of calcium-containing plates contributes to remineralization almost to the original state. That is, it has been shown that the InSpectr M hardware and software system really has high sensitivity and is capable of recording mineralization, de- and remineralization of hard dental tissues.

 

Table 2

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

Research Object, n = 20

Upper Level

Lower Level

Raman Rate
/ Relative Units

Enamel – before exposure

y = 8276 [8276; 8276];

х = 963 cm-1

y = 7675 [7674; 7675];

х = 963 cm-1

y = 602 [601; 602]

Enamel – exposure in acid (one day)

y = 3229.5 [3228.25; 3230];

х = 963 cm-1

y = 3077 [3076; 3078];

х = 963 cm-1

y = 152 [152; 153]

Enamel – plate application CM-2

y = 6967.5 [6966.25; 6968];

х = 963 cm-1

y = 6545 [6544; 6546];

х = 963 cm-1

y = 423 [422; 423]

p

p < 0.001

p < 0.001

p < 0.001

 

The data obtained at the third stage helped to better understand the process of changing the mineralization of the surface of hard dental tissues under the direct influence of radiation exposure in different doses.
According to the results of this study, no reliable differences in the level of mineralization (by the method of Raman fluorescence spectroscopy) were found in teeth before and after direct radiation exposure, regardless of the dose (2; 70; 110 Gy) in any of the functional groups (incisors, canines, premolars, molars). All existing differences are within the limits of statistical error. The obtained results, divided depending on the dose of radiation exposure, are presented in tables (Tables 3–5). At the same time, the data on the mineralization of hard dental tissues in different areas of the tooth in all functional groups differed greatly. Thus, the study showed that there is a difference in the mineralization of the surface of hard dental tissues in different areas (Fig. 3).

 

Table 3

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

Localization

Measurement

Radiation Dose

 Upper Level

Lower Level

Raman Rate
(Relative Units (Мavg.))

Maximum Fluorescence Rate
(Relative Units)

Occlusal surface

О Gy

y = 8271.6 ± 22.9; x = 963 cm-1

y = 7898.4 ± 29; х = 963 cm-1

y = 373 ± 12.8; х = 963 cm-1

y = 8455 ± 21.3

2 Gy

y = 8267.85 ± 24.6; х = 963 cm-1

y = 7892.45 ± 23.9; х = 963 cm-1

y = 375.4 ± 13.4; х = 963 cm-1

y = 8458.35 ± 22.2

Equator

О Gy

y = 6271.2 ± 29; х = 963 cm-1

y = 5856.8 ± 45.9; х = 963 cm-1

y = 414.4 ± 29.7; х = 963 cm-1

y = 6780.85 ± 65.5

2 Gy

y = 6259.1 ± 32.7; х = 963 cm-1

y = 5852.7 ± 45.3; х = 963 cm-1

y = 406.4 ± 23.6; х = 963 cm-1

y = 6772.35 ± 70.2

Cervical

region

vestibular surface

О Gy

y = 5109.85 ± 19.2; х = 963 cm-1

y = 4964.75 ± 19.3; х = 963 cm-1

y = 145 ± 8.8; х = 963 cm-1

y = 6043 ± 25.12

2 Gy

y = 5104.25 ± 15.4; х = 963 cm-1

y = 4958.25 ± 16.4; х = 963 cm-1

y = 146 ± 8.8; х = 963 cm-1

y = 6031.4 ± 24.2

p

 

p < 0.001

p < 0.001

p < 0.001

p < 0.001

 

Table 4

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

Localization

Measurement

Radiation Dose

 Upper Level

Lower Level

Raman Rate
(Relative Units (Мavg.))

Occlusal surface

О Gy

y = 8253.45 ± 17.4; x = 963 cm-1

y = 7876.6 ± 24.5; х = 963 cm-1

y = 376.85 ± 14.1; х = 963 cm-1

70 Gy

y = 8270.8 ± 24;
х = 963 cm-1

y = 7895.95 ± 25; х = 963 cm-1

y = 374.85 ± 14.9; х = 963 cm-1

Equator

О Gy

y = 6266.9 ± 30.2; х = 963 cm-1

y = 5855 ± 44.7;
х = 963 cm-1

y = 411 ± 25.5;
х = 963 cm-1

70 Gy

y = 6277.1 ± 27.2; х = 963 cm-1

y = 5867.25 ± 47.3; х = 963 cm-1

y = 409.85 ± 29.3; х = 963 cm-1

Cervical

region

vestibular surface

О Gy

y = 5109.4 ± 21.12; х = 963 cm-1

y = 4965 ± 22.7;
х = 963 cm-1

y = 144.4 ± 9.7;
х = 963 cm-1

70 Gy

y = 5105.85 ± 14.4; х = 963 cm-1

y = 4958.8 ± 16.8; х = 963 cm-1

y = 147 ± 8.6;
х = 963 cm-1

p

 

p < 0.001

p < 0.001

p < 0.001

 

Table 5

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

Localization

Measurement

Radiation Dose

Upper Level

Lower Level

Raman Rate (Relative Units (Мavg.))

Occlusal surface

О Gy

y = 8266.95 ± 25.4; x = 963 cm-1

y = 7894 ± 27.8;
х = 963 cm-1

y = 372.95 ± 14.1; х = 963 cm-1

110 Gy

y = 8264.35 ± 16.8; х = 963 cm-1

y = 7889.55 ± 20.7; х = 963 cm-1

y = 374.8 ± 5.7;
х = 963 cm-1

Equator

О Gy

y = 6279.6 ± 19.4; х = 963 cm-1

y = 5865.7 ± 34.8; х = 963 cm-1

y = 413.9 ± 27.6;
х = 963 cm-1

110 Gy

y = 6271.75 ± 32; х = 963 cm-1

y = 5869.8 ± 38.3; х = 963 cm-1

y = 402 ± 25.6; х = 963 cm-1

Cervical

region

vestibular surface

О Gy

y = 5113.8 ± 20.5; х = 963 cm-1

y = 4969.7 ± 21.2; х = 963 cm-1

y = 144.1 ± 9.8;
 х = 963 cm-1

110 Gy

y = 5110.55 ± 17.3; х = 963 cm-1

y = 4967.8 ± 21.8; х = 963 cm-1

y = 142.75 ± 8.8;
х = 963 cm-1

p

 

p < 0.001

p < 0.001

p < 0.001

 

Fig. 3. Spectral сharacteristics of dental hard tissues by raman rate areas (Мavg.)

 

However, the absence of a direct effect of irradiation (regardless of the dose) on the mineralization of the surface of hard dental tissues was revealed. These data allow us to assume that there is an indirect mechanism of the effect of radiation therapy on the occurrence of radiation damage to teeth due to a decrease in the level of hygiene, salivation, etc.

This study shows different levels of mineralization in different areas 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 dental tissues (equator, cervical zone of tooth, cutting edge) (see Fig. 3). The lowest mineralization according to the data (Raman intensity) is in the cervical zone of teeth (Incisors – y = 145 ± 1,5, х = 963 cm–1; canines – y = 141 ± 1,1, x = 963 cm–1; premolars y = 142 ± 1,8, х = 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 area (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 areas the level of mineralization does not change after radiation exposure, regardless of the dose.

Conclusions

  1. Raman fluorescence spectroscopy has high sensitivity and is capable of recording mineralization, de- and remineralization of hard dental tissues.
  2. It is recommended that the determination of mineralization of hard dental tissues by Raman fluorescence spectroscopy be carried out in a moistened rather than dry form.
  3. Organic acids promote enamel demineralization, and the use of calcium-containing plates promotes remineralization.
  4. Direct radiation exposure does not significantly change the level of mineralization of hard dental tissues, 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

G. I. Belyakov

South Ural State Medical University

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

Postgraduate Student of the Department of Orthopedic Dentistry and Orthodontics

Russian Federation, Chelyabinsk

N. S. Nurieva

South Ural State Medical University

Email: belyakov-95@mail.ru
ORCID iD: 0000-0002-5656-2286

DSc (Medicine), Professor of the Department of Orthopedic Dentistry and Orthodontics

Russian Federation, Chelyabinsk

D. A. Tezikov

South Ural State Medical University

Email: belyakov-95@mail.ru
ORCID iD: 0009-0007-2056-155X

PhD (Medicine), Assistant of the Department of Orthopedic Dentistry and Orthodontics

Russian Federation, Chelyabinsk

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1. JATS XML
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2. Fig. 1. Scheme of operation of the "InSpectr M" hardware and software system with a light guide head

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3. Fig. 2. Finding Raman lines

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4. Fig. 3. Spectral сharacteristics of dental hard tissues by raman rate areas (Мavg.)

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