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UDC 616.314-002.4-084
Shnaider Stanislav Arkadievich
Doctor of medical Sciences, Professor, Odessa National Medical University, State Establishment «The Institute of Stomatology andMaxillo-Facial Surgery
National Academy of Medical Science of Ukraine» Salekh Anuar Yusefovych corresponding author
Makarenko Ol'ga Anatolievna
Doctor of biology Odessa National Medical University DOI: 10.24412/2520-2480-2020-3082-56-60 CARIES OF THE TEETH AND GERD - POSSIBILITIES OF PREVENTION
Шнайдер Станислав Аркадиевич
доктор медицинских наук, профессор Одесский национальный медицинский университет,
Государственное учреждение «Институт стоматологии и челюстно-лицевой хирургии Национальной академии медицинских наук Украины» Салех Ануар Юсефович corresponding author Макаренко Ольга Анатольевна доктор биологических наук Одесский национальный медицинский университет
КАРИЕС ЗУБОВ И ГЭРБ - ВОЗМОЖНОСТИ ПРОФИЛАКТИКИ
Abstract.
Today gastroesophageal reflux disease (GERD) is attributed to diseases of the 21st century and great attention is paid to the relationship of reflux esophagitis and the pathology of other organs and systems of the body. Pathological manifestations in the oral cavity include aphthous stomatitis, changes in the papillae of the tongue, burning of the tongue, enamel erosion, caries, halitosis, a feeling of dry mouth, cheilitis, voluminous plaque on the tongue, dry lips.
Objective: to substantiate experimentally and develop a complexfor the prevention of dental caries in patients with GERD.
Materials and methods: 30 Wistar rats (females). The caries model was reproduced using Stefan's cariogenic diet. A group 1 - intact control on a standard diet of the vivarium; a group 2 - model of caries and GERD; a group 3 - model of caries and GERD + developed preventive complex. Preventive measures in the 3rd group of rats were carried out daily. They included taking Biotrite-plus tablets and Bactoblis, then rinsing the mouth with 1 ml of Remodent solution; applying mucosal gel "Osteovit" and gel "Biotrit-Dent" to the tissues of the oral cavity
Results. The developed preventive complex made it possible to prevent a decrease in the activity of alkaline phosphatase of the pulp, the processes of demineralization of the hard dental tissues, inhibit the development of the carious process and carry out effective prevention of dental caries in case of GERD.
Аннотация.
Сегодня гастроэзофагеальную рефлюксную болезнь (ГЭРБ) относят к заболеваниям ХХ1 века и уделяют повышенное внимание связи рефлюкс-эзофагита и патологии других органов и систем организма. Патологические проявления в полости рта включают афтозный стоматит, изменения сосочков языка, жжение языка, эрозии эмали, кариес, галитоз, ощущение сухости во рту, хейлиты, обильный налета на языке, сухость губ.
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Цель исследования: экспериментально обосновать и разработать комплекс для профилактики кариеса зубов при ГЭРБ.
Материалы и методы: 30 крыс (самки) линии Вистар. Модель кариеса воспроизводили при помощи кариесогенного рациона Стефана. 1 группа - интактный контроль на стандартном рационе вивария; 2 группа - модель кариеса и ГЭРБ; 3 группа - модель кариеса и ГЭРБ + разработанный лечебно-профилактический комплекс. Лечебно-профилактические мероприятия в 3-ей группе крыс проводили ежедневно. Они включали приём таблеток Биотрит-плюс и Бактоблис, затем ополаскивание полости рта 1 мл раствором Ремодента; нанесение на ткани полости рта мукозального геля «Остеовит» и геля «Биотрит-Дента».
Результаты: Разработанный лечебно-профилактический комплекс позволил предотвратить снижение активности щелочной фосфатазы пульпы, процесс деминерализации твердых тканях зубов, затормозить развитие кариозного процесса и осуществить эффективную профилактику кариеса зубов при ГЭРБ.
Key words: gastroesophageal reflux disease, carious process, preventive complex.
Ключевые слова: гастроэзофагеальная рефлюксная болезнь, кариозный процесс, лечебно-профилактический комплекс.
Introduction. Gastroesophageal reflux disease (GERD) is a chronic recurrent disease characterized by a morphological change in the mucous membrane of the esophagus due to retrograde reflux of the gastric or gastrointestinal content, manifested by esophageal and extra-esophageal symptoms.
Symptoms of GERD are revealed in almost half of the adult population of the developed countries, and lately the incidence has been steadily increasing. This gives grounds for many experts to attribute GERD to diseases of the 21st century [1-4] and to pay greater attention to the relationship between reflux esophagitis and the pathology of other organs and systems of the body. Clinical examination of the patients with GERD revealed the presence of pathological manifestations in the oral cavity in the mucous membrane of the oral cavity and lips in the form of soft tissue lesions (aphthous stomatitis, changes in the papillae of the tongue, burning of the tongue, cheilitis, voluminous plaque on the tongue, dry lips), non-carious lesions of the hard dental tissues (enamel erosion), caries, halitosis, dry mouth. Extra-esophageal manifestations of GERD are observed in approximately 60 % of representatives of the older age group, this pathology is rejuvenating at an alarming rate, and now researchers note an increase in the incidence of GERD even in children, which ranges from 8.7 to 49 % [2].
The objective of the study is to develop and substantiate the possibility of using a new therapeutic and prophylactic anticariosis complex in experimental gas-troesophageal reflux disease.
Materials and methods. The experiment was carried out on Wistar female rats with a mean weight of 85 ± 9 g. 30 rats were used in the experiment, which were divided into three groups (n = 10).
The caries model was reproduced using Stefan's cariogenic diet, onto which 1-month-old animals were transferred. The diet contained 32 % of milk powder, 66% of sugar, 2% of dry beef liver [3]. Additionally, the reflux pathology was simulated in animals by acidification of drinking water with hydrochloric acid to pH 3.5 with the introduction of the microbial renin enzyme 0.5 g / L (Microbial meito rennet, Japan).
A group 1 - intact control on a standard diet of vivarium;
A group 2 - model of caries and reflux disease;
A group 3 - model of caries and reflux disease + developed medical and preventive complex.
Treatment and preventive measures in the 3rd group of rats were carried out daily, starting from the first day of the experiment:
• Biotrit-plus 300 mg / kg as oral tablets in the morning (Odessa Biotechnology, NVA and Bactoblis 1.3 g / kg (Medico domus d.o.o, Republic of Serbia) as tablets, then rinsing the mouth with 1 ml of Remodent solution (3 g / 100 ml of water) (TOV "Kyiv Pharmaceutical Partnership", Ukraine);
• 1 ml of the mucosal gel "Osteovit" (NVA "Odessa Biotechnology", Ukraine) (NVA "Odessa Biotechnology", Ukraine) on the tissue of the oral cavity for 2 weeks in the evening, the next 2 weeks the medications were replaced by Biotrit-Denta gel (NVA "Odessa) Biotechnology", Ukraine).
The experiment lasted 30 days, after that the rats were killed by total bloodletting from the heart under thiopental anesthesia (20 mg / kg), and blood serum was collected. Blocks of the jaws with the teeth, the pulp from the lower incisors, and gums were isolated.
The depth of carious lesions, the number of carious cavities in the teeth, by an average of 1 rat was considered. The degree of atrophy of the alveolar process in the jaws was calculated according to the Nikolaeva method [3]. The activity of acidic and alkaline phosphatases in the pulp homogenates (5 mg / ml) was determined by hydrolysis of paranitrophenyl phosphate [4]. The calcium content in the jaw homogenates (75 mg / ml) was determined by the reaction with ortho-cresolphthalein-complexone [5], and inorganic phosphorus by the reaction with molybdenum acid. The activity of catalase in the serum and gum homogenate (20 mg / ml) was studied by the residual amount of hydrogen peroxide [7]; the content of malondialdehyde MDA - by the reaction with 2-thiobarbituric acid [8], urease activity - by the formation of ammonia from urea, which was recorded using Nessler reagent [9], lyso-zyme activity - by clarification of a suspension of Mi-crococcus lysodeikticus culture [11], elastase activity -by hydrolysis of the substrate Nt-BOC-L-alanine-p-
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nitrophenyl ester (Sigma, USA) [11]. Statistical processing of the results was carried out in EXCEL programs (standard Microsoft Office package).
Results and discussion. The degree of development of the carious process in the experimental animals was evaluated by the number of carious cavities (average per rat) and the depth of the caries damage. Rat consumption of the cariogenic diet in combination with reflux modeling has led to an increase in the number of
Table 1
The carious process and the degree of atrophy of the alveolar process of the lower jaw of rats against the
carious lesions by 33.8%, and their depth increased by 28.4%. Daily treatment with preventive measures for rats of the 3rd group had a pronounced carioprophylac-tic effect, significantly reducing both the number of carious cavities and their depth (p1 <0.001 and p1 <0.002, respectively). In the rats of the 3rd group, both indices were significantly lower than the corresponding values in the intact animals (p <0.05 and p <0.01, Table 1).
Rat groups The number of caries cavities, average per 1 rat Depth of the caries affection, points Atrophy of the alveolar process, %
Intact 6.8 ± 0.5 8.8 ± 0.5 21.6 ± 1.3
Pathology 9.1 ± 0.4 p < 0.02 11.3 ± 0.7 p < 0.01 20.7 ± 0.6 p > 0.5
Pathology + prevention 5.6 ± 0.3 p < 0.05 p1 < 0.001 6.9 ± 0.2 p < 0.01 pi < 0.002 19.4 ± 0.9 p > 0.2 p1 > 0.25
Note. p - reliability of differences to the index in the "Intact" group; pi - reliability of differences to the index in the "Pathology " group.
Thus, rats' consumption of the cariogenic diet with excess sugar and acidified drinking water with rennin does not significantly affect the degree of atrophy of the alveolar process of the lower jaw of animals (p> 0.5). After the use of the treatment-and-prophylactic complex in the 3rd group of rats, this index also did not change both in relation to the values obtained in the group of the intact animals (p> 0.2) and in comparison with the level of atrophy in the rats with a combined pathology (p: > 0.25, table 1).
It is known that the intensity of mineralization of the hard dental tissues depends on the degree of activity
Table 2
The activity of acid and alkaline phosphatases in the pulp of rat incisors against the background of pathol-
of alkaline phosphatase (ALP) of the pulp, since this enzyme is involved in the formation of hydroxyapatite crystals [14]. The activity of acidic phosphatase (ACP), in turn, characterizes the degree of demineralization, since the latter destroys the hard dental tissues at low pH values. The ratio of alkaline activity to acidic phosphatase activity (ALP / ACP) characterizes the mineralizing function of the pulp. The results of the study of the activity of phosphatases and mineralizing index of the pulp of experimental animals are presented in table 2.
Rat groups Activity of acidic phosphatase, mkat/kg Activity of alkaline phosphatase, mkat/kg ALP/ACP
Intact 0.032 ± 0.002 2.10 ± 0.16 65.62
Pathology 0.055 ± 0.005 p < 0.001 1.41 ± 0.13 p < 0.01 25.63
Pathology+ prevention 0.047 ± 0.004 p < 0.01 p1 > 0.1 2.13 ± 0.17 p > 0.1 p1 < 0.01 45.32
Note. p - reliability of differences to the index in the "Intact" group; pi - reliability of differences to the index in the "Pathology" group.
Simulation of the combined pathology of caries and GERD leads to a decrease in alkaline phosphatase activity by 32.8% (p <0.01) against the background of a sharp (71.9%) increase in ACP activity (p <0.001). As a result, the mineralizing index of ALP/ ACP decreased 2.6 times. The data obtained indicate a disorder of the processes of mineralization of the hard dental tissues of the experimental animals and an increase in the processes of demineralization. An increase in the activity of destructive ACP and a simultaneous decrease in the activity of ALP in pulp in the rats of the 2nd group can explain the increase in the number and depth of carious lesions.
Daily prevention by the developed complex prevented a decrease in alkaline phosphatase activity in the pulp (p> 0.1 and p1 <0.01) of the third group of the experimental animals. However, ACP activity of the incisor pulp decreased unreliably (p1> 0.1) and did not reach the normal level (p <0.0.1, table 2).
As a result of treatment and preventive measures and changes in the activity of alkaline and acidic phos-phatase the mineralizing index in the pulp of the rat teeth increased, but not to the level of the intact animals (Table 2).
Table 3 shows the results of the determination of calcium and phosphorus, as the main mineral components in the alveolar bone of the experimental animals.
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The calcium content in the bone tissue of the jaw of the rats of the group 2, in which caries and reflux was modelled, increased by 11.5 % (p <0.01), which is associated with prolonged consumption of the cariogenic diet. After daily prevention, the level of calcium in the alveolar bone of the 3rd group of rats remained at a high level (p <0.05 and p1> 0.1). The phosphorus content in
the alveolar bone of rats of the 2nd and 3rd groups also increased (p <0.02 and p1> 0.1, table 3). The high values of calcium and phosphorus in the bone tissue of the jaws of rats of the group 3 may explain a slight decrease in the resorption of the alveolar process in animals that were simulated a combined pathology (Table 1).
Table 3
The content of calcium and phosphorus in the alveolar bone of rats against the background of pathol-
Rat groups Calcium content, mmol/g Phosphorus content, mmol/g
Intact 2.216 ± 0.063 1.520 ± 0.014
Pathology 2.472 ± 0.054 p < 0.01 1.585 ± 0.019 p < 0.02
Pathology+ prevention 2.466 ± 0.081 p < 0.05 p: > 0.1 1.587 ± 0.015 p < 0.01 p1 > 0.1
Note. p - reliability of differences to the index in the "Intact" group; p1 - reliability of differences to the index in the "Pathology " group.
Biochemical indices that characterized the inten- elastase and ACP; the presence of opportunistic micro-sity of lipid peroxidation (LPO) processes were studied biota - by urease activity; the state of non-specific an-in the gums of the rats - by the level of malondialde- timicrobial protection - according to the activity of ly-hyde (MDA) and the activity of the antioxidant system sozyme. The results of this study are presented in the (AOS) by the activity of catalase; the degree of inflam- table 4. mation was investigated by the activity of neutrophilic
Table 4
Biochemical indices in the gums of the rats against the background of pathology and after application of
Indices Rat groups
Intact Pathology Pathology + prevention
MDA content, mmol/kg 18.8 ± 1.2 29.2 ± 1.4 p < 0.001 24.7 ± 1.0 p < 0.002 pi < 0.02
Catalase activity, mcat/kg 8.55 ± 0.22 7.16 ± 0.47 p < 0.02 7.98 ± 0.35 p > 0.1 pi > 0.1
Etalase activity, mc-mcat/kg 57.80 ± 4.85 67.27 ± 1.74 p < 0.05 57.04 ± 1.52 p > 0.1 pi < 0.001
Acidic phosphotase activity, mc-mcat/kg 22.58 ± 1.25 31.79 ± 2.19 p < 0.002 26.78 ± 1.00 p < 0.02 pi < 0.05
Urease activity, mc-mcat/kg 0.55±0.06 0.81 ± 0.06 p < 0.01 0.60 ± 0.06 p > 0.1 Pi < 0.05
Lisozyme activity, U/kg 162 ± 17 116 ± 14 p < 0.05 136 ± 10 p > 0.1 pi > 0.1
Note. p - reliability of differences to the index in the "Intact" group; pi - reliability of differences to the index in the "Pathology " group.
The rats' consumption of the unnatural cariogenic diet in combination with acidified water and rennin contributed to an increase in LPO intensity, as evidenced by a significant increase in the level of MDA in the gums of rats of the group 2 (by 55.3%, p <0.001). The increase in lipid peroxidation in the gums of the rats when modeling a combined pathology is most likely the result of depletion of antioxidant protection. This is evidenced by a decrease in catalase activity by 16.3% (p <0.02, table. 4).
A significant increase in inflammation markers was also observed in the gums of the rats of the 2nd group: elastase activity by 16.4% (p <0.05) and ACP by 40.8% (p <0.002). Urease activity in the gums of the animals that were simulated combined pathology increased almost 1.5 times (p <0.01), which is a consequence of a 1.4-fold decrease in lysozyme activity (p <0.05), which means non-specific antimicrobial protection (table 4).
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Comprehensive prevention, which was carried out daily to the rats of the 3rd group, had a pronounced positive effect on the studied indices. First of all, the proposed complex of drugs contributed to the complete normalization of indices characterizing non-specific resistance, namely, the activity of catalase (p> 0.1 and p1> 0.1) and lysozyme (p> 0.1 and p1> 0.1). The level of MDA in the gums of animals after the use of the complex decreased reliably (p1 <0.02), but did not reach the norm (p <0.002). After carrying out therapeutic and preventive measures in the gums of the rats of the 3rd group, markers of inflammation significantly decreased. For example, elastase activity dropped to normal (p> 0.1 and p: <0.001). The activity of ACP, despite a reliable decrease (p1 <0.05), slightly exceeded the values in the intact rats (p <0.02). Normalization of urease activity was also noted, and hence a decrease in microbial contamination of the rat oral tissues with the combined pathology (p> 0.1 and p1 <0.05).
In general, according to the results of the biochemical study of the rat gums, the cariogenic diet in combination with the GERD model leads to increased growth and reproduction of opportunistic microbiota, activation of lipid peroxidation and inflammation in the tissues of the oral cavity of the experimental animals. Most likely, these disorders were the result of depletion of antioxidant and antimicrobial protection of the oral cavity, which was induced by the cariogenic diet in combination with acidified water containing rennin. The preventive use of the developed complex effectively increases the nonspecific resistance of the oral cavity, reduces inflammation and the degree of lipid peroxidation, as well as inhibits the reproduction and growth of opportunistic microbiota in the gum of the rats.
Conclusions: 1. Reflux disease against the background of consumption of the high-carbohydrate diet of soft consistency causes the development of the carious process in the experimental animals, leads to deminer-alization of the hard dental tissues and disturbance of the pulp phosphatase activity, promotes the propagation of opportunistic microbiota, the intensification of inflammation and lipid peroxidation in the gum tissues and in the body as a whole, suppresses antimicrobial and antioxidant protection.
2. The developed treatment-and-prophylactic complex has a pronounced ability to prevent a pathological decrease in the activity of alkaline phosphatase of the pulp, the processes of demineralization of the hard dental tissues, and as a result, inhibits the development of the carious process induced by reflux disease and irrational nutrition with a high content of carbohydrates.
Prospects for further research. The use of the
proposed treatment and preventive complex will allow
to make the effective prevention of dental caries in case of reflux disease. The systemic administration of the complex of adaptogens and probiotics that make up this complex normalizes the mineralization of the hard dental tissues, inhibits the propagation of conditionally pathogenic microbiota, has an anti-inflammatory effect and prevents a decrease in nonspecific resistance both in the oral cavity and in the body as a whole.
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