**1. Introduction**

Chronic periodontitis is a serious and widespread periodontal pathology that causes significant impairment to dentoalveolar system functions, with damage to supporting tooth structures and the loss of teeth [1]. Periodontitis is mostly spread (60–65%) among people over age 30 [2]. However, the percentage of young patients with a severe form of chronic periodontitis has increased to 11.2%, and among people over age 65, it is 30% [3]. Current data indicate that periodontitis is a polyethiological disease [4]. Periodontitis is an insidious disease because the initial signs of inflammatory processes often remain unnoticed, and the chronic condition causes serious consequences not only for the dentoalveolar system, but for the patient as a whole.

Prompt diagnosis and prevention through the treatment of patients with periodontal diseases is essential. To improve this process and allow for a noninvasive diagnosis of periodontitis, it is necessary to identify the structural changes that occur in the tissues of teeth with this disease. Most studies that have investigated changes caused by this disease have not focused on changes in hard dental tissues, but have investigated the surrounding soft tissues [5,6], oral fluid [6–8], or osseous tissue regeneration during periodontitis treatment [9]. Few studies have focused on the tissues of teeth with periodontitis. The authors of [10] showed that the main structural change in the tissues of teeth with generalized periodontitis is dentin mineralization, and the process of root canal treatment of such teeth is recommended as an additional means of avoiding progression to dentin demineralization. The authors of [11] noted an increase in the micro-hardness of enamel in cases involving progression to periodontitis. Meanwhile, the authors of [10] showed that there are no apparent structural changes in the enamel in generalized periodontitis.

An analysis of the literature data showed that, for chronic periodontitis, the changes in hard dental tissues, especially in enamel, are unconfirmed, and having information about the changes in

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the composition of tooth enamel could allow the development of a noninvasive method to diagnose this disease and provide a correct treatment plan. Therefore, research of the tissues of teeth with periodontitis is urgently needed.

Biochemical analysis, scanning electron microscopy [10], fluorescence [7], and spectroscopy [10] are well-known current methods used for tooth tissue research. Biochemical analysis and scanning electron microscopy (SEM) provide quality images of the tooth tissue microstructure and are the most widely used for assessing tooth structure, but they require destructive preparation of the sample [9,10,12,13]. Fluorescence diagnosis in dentistry is based on the analysis of the spectra of fluorescence of hard dental tissues. While the main studied substance is hydroxyapatite, of which teeth are composed, detailed analysis of the composition of teeth is not possible [7]. The limitations of these hard dental tissue research methods could be overcome with the use of Raman spectroscopy. This is a simple, noninvasive, and rapid way of assessing dental tissue [9,12,13].

In [12], with the use of traditional routine histological methods and Raman spectroscopy, comparative research on mineralized tissues of the human jaw was carried out, and it was shown that the joint use of these methods allows significantly more data about pathological processes in the mineralized tissues (in the case of caries) to be collected, as well as allowing the features of mineralization under the conditions of directed bone regeneration to be defined. The authors of [9] studied the processes of bone healing and regeneration in periodontitis treatment using Raman spectroscopy. In our previous work [13], we used Raman spectroscopy to analyze the structure of teeth compared with synthetic apatites. Spectral lines related to the hard and soft tissues of teeth that provide important data for understanding the chemical structural properties of dentin and enamel were discussed. In [6,14–16], attention was paid to the study of the periodontal ligament after the application of orthodontic force and gingival slit fluid in periodontal disease. The authors showed the possibility of using Raman spectroscopy to monitor the periodontal condition at the biochemical level in subjects undergoing orthodontic treatment.

The aim of this work is to study the changes in the tissues of teeth with periodontitis using the Raman spectroscopy method for early, rapid diagnosis and the correction of treatment.

#### **2. Materials and Methods**

A randomized study design was used. Forty-two teeth (molars, premolars, and canines) from European patients aged 35–70 of both genders, that were removed due to chronic periodontitis (26 teeth) or for orthodontic reasons (control group, 16 teeth), were used as the materials of the study. Diagnosis of periodontitis was done clinically and after cone beam computed tomography (CT) analysis (the code of the disease according to ICD-10 (1997)—K05.3). The teeth removed due to severe chronic periodontitis with periodontal pockets of at least 6 mm deep and pathologic tooth mobility of grades III–IV were selected for the main group of study. Computed tomography showed a decrease in the bone tissue around the roots of removed teeth of more than half of the root length.

The study was carried out in accordance with the Declaration of Helsinki. The protocol was approved by the Ethics Committee (extract 20.05.2020 No. 207 of minutes of the meeting of the Committee on Bioethics of Samara State Medical University). The samples were collected within a period of 2 months. Measurements were taken immediately after the sampling.

The surfaces of teeth in 5 different areas were studied: enamel (a), dentin (b), in longitudinal slices), cementum (c), and dental calculus localized in the outer part of the teeth. The degree of intensity of the surface formation of the studied teeth corresponded to distinct under-gum (e) and above-gum (d) calculus [17].

Three spectra were investigated (with subsequent averaging) in every studied area at 3–5 different points of the surface of every tissue of each tooth. Samples were divided into 2 main groups: the control group (Figure 1I) and the group with periodontitis (Figure 1II).

**Figure 1.** The fragments of teeth following computed tomography that were (**I**) healthy and (**II**) diagnosed with periodontitis. (**III**) Photo of a tooth with the researched areas indicated: a—enamel, b—dentin, c—cementum, d—above-gum dental calculus, and e—under-gum dental calculus.

An in vivo study of the enamel of 22 teeth (molars, premolars, and canines) of one female volunteer patient was also carried out. One of the patient's teeth was diagnosed with localized periodontitis (disease code according to ICD-10 (1997)—K05.3).

The study was carried out using Raman spectroscopy, implemented using the process described in detail in [18].

The experimental process included the use of a semiconductor laser (LML-785.0RB-04, California, USA), an optical module for Raman spectroscopy (RPB-785, Changchun, China), a spectrograph (Sharmrock SR-303i, www.andor.oxinst.com) with an integrated digital camera (ANDOR DV-420A-OE, www.andor.oxinst.com) that was cooled to −60 ◦C, and a computer.

The use of this spectrograph provided a wavelength resolution of 0.15 nm with a low level of inherent noise. The method of subtracting the fluorescence component of polynomial approximation with additional filtration of random noise effects was used to exclude autofluorescence from the Raman spectrum. Analysis of the Raman spectra was carried out in the range of 350–2200 cm<sup>−</sup><sup>1</sup> in this work. The power of the laser radiation, 400 mW, within the used exposure time (30 s) did not cause any changes to the samples. The optical probe, positioned over the subject at a distance of 7 mm, was used for Raman spectrum registration [19].
