Periodontal tissue changes under chronic tobacco intoxication in an experimental model of gingival recession: a laser Doppler flowmetry study in rats

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Background

Inflammatory diseases of the periodontal tissues remain a major concern in modern dentistry. According to the World Health Organization (WHO, 2024), approximately three-quarters of the global population are affected by periodontal diseases. The prevalence and severity of pathological changes in the periodontium increase with patient age. Since the microcirculatory network plays key compensatory and trophic roles, assessing its condition may serve as the first diagnostic step toward effective treatment of periodontitis. Laser Doppler flowmetry (LDF) provides a rapid, noninvasive method to detect disturbances in periodontal tissues, allowing for timely therapeutic intervention.

Aim

To evaluate periodontal changes in rats with gingival recession under conditions of chronic tobacco intoxication (CTI), using laser Doppler flowmetry (LDF).

Methods

Study Design

It was an experimental study on adult male Wistar rats.

Participants

The study included 75 adult male Wistar rats (Rattus norvegicus domestica) weighing 250–350 g. Animals were randomly assigned to the following groups using a random number generator:

• Group 1 (n = 15): intact animals (control).
• Group 2 (n = 15): experimental animals under CTI.
• Group 3 (n = 15): experimental animals with gingival recession induced under CTI.
• Group 4 (n = 15): animals with gingival recession under CTI that underwent surgical correction using a modified tunnel technique to restore keratinized soft tissues as a method aimed at improving microcirculation.
• Group 5 (n = 15): animals with gingival recession under CTI in which tobacco exposure was discontinued, followed by correction using the modified tunnel technique.

Intervention

Before the start of the study, the experimental animals were allowed to rest for 25 minutes under controlled environmental conditions at 25 °C. Parameters were recorded for 1 minute (30 seconds in each examined tissue area) using a laser wavelength of 632.8 nm. To ensure measurement accuracy, each recording was repeated three times at the same site, and mean arithmetic values were calculated.

In all experimental groups, except the control, the rats were subdivided into three subgroups according to the observation period: 14, 30, and 60 days.

Microcirculatory blood flow was assessed using LDF with a LAKK-02 laser analyzer, version 4 (Scientific Production Enterprise Lazma, Moscow, Russia). Microcirculation was measured in the marginal and attached gingiva.

The following parameters were evaluated:

• Mean blood flow rate over time (M), reflecting changes in tissue perfusion;
• Coefficient of variation (Kv), indicating vasomotor activity of the microvessels;
• Parameter representing oscillations in erythrocyte flow [1, 2].

The experimental model of CTI was based on the protocol described by Yen et al. (2008) and on the studies of Grebenyuk and Shilkova (2019). Each animal was placed in an exposure chamber where smoke from two cigarettes (each containing 1.3 mg of nicotine and 16 mg of tar) was introduced for 7 minutes every 30 minutes, 6 hours a day, daily for 6 weeks [3, 4].

Outcomes Registration

LDF is a qualitative, noninvasive diagnostic method used to assess the microcirculatory network, based on the application of laser irradiation that penetrates biological tissues to several millimeters. The main advantage of this technique is its ability to perform contact-free measurement of blood flow and, consequently, hemodynamics, without influencing the natural parameters of capillary circulation. Another important feature of LDF is its capability to record up to one thousand measurements per minute, with real-time data registration and analysis, enabling continuous monitoring of rhythmic microvascular activity within the capillary network [5, 6]. Since the blood supply of the periodontium by small vessels ensures its trophic function, microcirculatory assessment enables evaluation of the regenerative potential of periodontal tissues following dental interventions.

The development of LDF aimed to create an advanced in vivo diagnostic method capable of detecting tissue alterations by analyzing changes within the microcirculatory network. A series of studies were conducted to establish this diagnostic approach, which is based on the following physical principles:

Laser light penetrates tissues to a depth sufficient for signal detection (several millimeters);

The Doppler effect reflects the ability of moving erythrocytes to scatter laser radiation, allowing quantification of microcirculatory intensity within the examined tissue region;

Flowmetry represents the recording of microvascular blood flow variations related to the presence of cellular components, hemoglobin, bilirubin, and other chromophores in the bloodstream [7, 8].

Thus, LDF employs a fiber-optic probe with three optical fibers to detect and record both steady-state and fluctuating components of the signal, which are displayed on the monitor as an integrated microcirculatory index.

Main Study Outcome

After the laboratory rats were divided into groups and the experiment was completed, it was found that CTI had a negative effect on the gingival healing period, both in the absence (group 2) and presence (group 3) of experimentally induced gingival recession. However, according to LDF data, only a slight decrease in connective tissue cell density was observed in group 4, followed by gradual recovery of these parameters in group 5.

Statistical Analysis

Statistical data analysis was performed using the Statistica 8.0 software package (StatSoft, Russia) for Windows. The Shapiro–Wilk test was used to assess the normality of distribution of quantitative variables within groups. Descriptive statistics included both parametric (mean and standard deviation) and nonparametric (median and quartile) analyses. Differences between groups were evaluated using the Student t-test (for normally distributed data) or the Mann–Whitney U test (for non-normally distributed data). A p-value < 0.05 was considered statistically significant.

Results and discussion

Comparative evaluation of the number of connective tissue cells and the epithelial layer thickness in groups 1–3 demonstrated a marked decrease in both parameters among rats exposed to CTI and gingival recession: from 75.8 to 67.1 connective tissue cells per field of view and from 25.6% to 11.2%, respectively (see Table 1). In group 4, in which gingival recession was corrected under CTI conditions, only a slight decrease in connective tissue cell count was observed (from 63.1 to 60.0 per field of view), along with partial normalization of the epithelial layer to 15.1%. By day 60, this value did not reach 25.6%, corresponding to the level observed in the control group (see Table 2). In group 5, morphological improvement was noted, characterized by recovery of connective tissue cell density and improvement in the epithelial layer thickness, reaching 68.3 cells per field of view and 18.1%, respectively (see Table 3).

 

Table 1. Comparative characteristics of laser doppler flowmetry parameters in groups 1–3

Parameters	Group ١	Group ٢	Group ٣
ADMA, µmol/L, mean (SD)	0.35 (0.08)	0.46 (0.09)	0.43 (0.05)
Number of connective tissue cells per field of view, median [Q1; Q3]	75.8 [74.2; 77.6]	67.1 [65.7; 67.3]	69.4 [66.9; 70.5]
Relative area of connective tissue, mean (SD), %	82.3 (2.4)	84.8 (1.6)	88.6 (2.8)
Cross-sectional area of blood vessels, median [Q1; Q3], %	12.4 [12.1; 12.7]	6.9 [5.8; 8.1]	5.8 [5.7; 7.1]
Relative area of muscle tissue, mean (SD), %	5.8 (1.4)	4.9 (0.8)	3.7 (1.2)
Epithelial layer thickness, median [Q1; Q3], %	25.6 [24.8; 26.4]	17.9 [17.7; 18.2]	11.2 [10.7; 11.8]

Note: ADMA, asymmetric dimethylarginine.

 

Table 2. Comparative characteristics of laser doppler flowmetry parameters in group 4 at different time points

Parameters	Time point, days
	14	30	60
Leukocytes per field of view	40.2 (1.1)	15.6 (2.0)	3.0 (3.0; 3.1)
Number of connective tissue cells per field of view, mean (SD), median [Q1; Q3]	63.1 (2.0)	60.1 [59.7; 62.7]	60.0 (1.2)
Relative area of connective tissue, mean (SD), %	61.1 (1.7)	70.7 (0.8)	71.1 (0.3)
Cross-sectional area of blood vessels, median [Q1; Q3], %	2.0 [2.0; 2.2]	4.1 [4.0; 5.0]	6.0 [4.2; 6.3]
Relative area of muscle tissue, mean (SD), median [Q1; Q3], %	3.4 (0.4)	4.0 [4.0; 4.2]	5.0 [4.9; 5.4]
Epithelial layer thickness, median [Q1; Q3], %	12.1 [11.9; 12.7]	14.4 (1.7)	15.1 [14.0; 15.4]

 

Table 3. Comparative characteristics of laser doppler flowmetry parameters in group 5 at different time points

Parameters	Time point, days
	14	30	60
Leukocytes per field of view, mean (SD)	39.9 (1.1)	11.3 (2.4)	3.1 (0.1)
Number of connective tissue cells per field of view, mean (SD)	65.1 (1.1)	66.8 (0.9)	68.3 (1.2)
Relative area of connective tissue, mean (SD), %	63.4 (2.6)	72.8 (1.2)	80.1 (0.7)
Cross-sectional area of blood vessels, mean (SD), %	3.5 (0.9)	5.5 (1.2)	8.2 (0.3)
Relative area of muscle tissue, mean (SD), %	4.1 (0.6)	5.2 (1.4)	5.9 (0.4)
Epithelial layer thickness, mean (SD), %	13.3 (0.5)	14.5 (2.7)	18.1 (2.2)

 

Conclusion

The conducted experiment demonstrates the effectiveness of LDF in diagnosing the adverse effects of tobacco exposure on periodontal health, particularly in the presence of gingival recession and other periodontal diseases. The cessation of tobacco smoke exposure accelerates periodontal repair and healing, promoting the restoration of the trophic, supportive, protective, and barrier functions of the periodontal complex. In clinical practice, this approach may improve the quality of life of patients with periodontitis.

LDF enables early diagnosis of periodontal diseases and facilitates the timely detection of ongoing pathological processes within periodontal tissues.

Additional information

Author contributions: D.A. Trunin, E.D. Kostrigina: investigation, writing—original draft; O.A. Rubanenko: conceptualization, methodology; Yu.M. Zamyatin: writing—original draft, writing—review & editing; M.M. Suntseva: conceptualization; A.D. Eremeeva: investigation. All the authors approved the version of the manuscript to be published and agreed to be accountable for all aspects of the work, ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Acknowledgments: The authors express their sincere gratitude to Larisa A. Zyulkina, Dean of the Faculty of Dentistry, Penza State University. The authors also thank Andrey V. Eremin, Rector of Samara State Medical University.

Ethics approval: The experimental study involving laboratory animals was approved by the Local Ethics Committee of Penza State University (Protocol No. 5, dated March 1, 2019).

Funding sources: No funding.

Disclosure of interests: The authors have no relationships, activities, or interests for the last three years related to for-profit or not-for-profit third parties whose interests may be affected by the content of the article.

Statement of originality: No previously published material (text, images, or data) was used in this work.

Generative AI: No generative artificial intelligence technologies were used to prepare this article.

Provenance and peer-review: This paper was submitted unsolicited and reviewed following the standard procedure. The peer review process involved two external reviewers, a member of the editorial board, and the in-house scientific editor.

About the authors

Dmitry A. Trunin

Samara State Medical University

Author for correspondence.
Email: trunin-027933@yandex.ru
ORCID iD: 0000-0002-7221-7976
SPIN-code: 5951-4659

MD, Dr. Sci. (Medicine), Professor

Russian Federation, Samara

Ekaterina D. Kostrigina

Penza State University

Email: KostriginaED1987@yandex.ru
ORCID iD: 0000-0002-8232-5153
SPIN-code: 2173-4313
Russian Federation, Penza

Olesya A. Rubanenko

Samara State Medical University

Email: dmib@samsmu.ru
ORCID iD: 0000-0001-9351-6177
SPIN-code: 1546-2237

MD, Dr. Sci. (Medicine), Associate Professor

Russian Federation, Samara

Yuri M. Zamyatin

Penza State University

Email: yuran-best@yandex.ru
ORCID iD: 0009-0008-0683-2656
SPIN-code: 2575-4492
Russian Federation, Penza

Maria M. Suntseva

Penza State University

Email: mashakurbatova2346@gmail.com
ORCID iD: 0009-0002-0025-5276
Russian Federation, Penza

Anastasia D. Eremeeva

Penza State University

Email: anasteysha.eremeeva@bk.ru
ORCID iD: 0009-0003-1983-5868
Russian Federation, Penza

References

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