The microbiota of carious dentin during the treatment of teeth with burs of various grain sizes

Introduction

The prevention and treatment of dental diseases is of crucial importance because of their high prevalence, complex diagnostics and detrimental effect on human health [1—3]. A number of factors should be taken into account when choosing a method for deep caries treatment, viz. the presence of microbial associations in the tooth decay cavity, dentinal tubules and pulp, as well as the structural changes in the dental pulp tissue [4—8]. Clinical studies have established that dental manipulations, medications and the exposure of a tooth to various types of radiation or critical temperatures cause a reaction of the tooth pulp [9—11].

Tooth preparation is an integral stage of dental therapy, the efficacy of which is the key to the successful treatment of caries, which necessitates the constant improvement of the odontotrypy techniques and instruments. That said, mechanical pre-treatment remains the most widely used and efficient method in caries therapy [12—14].

The key factor of the successful deep caries therapy is preventing any infectious or traumatic inflammatory processes in the dental pulp. Dental ivory traumas during the preparation process, or open dentinal canals serve as the paths through which microorganisms can easily penetrate under pressure during cavity preparation. It is a known fact that though not all microorganisms are mobile, they can move along the dentinal canals by means of recurring cytokinesis and under pressure during meals [15—17]. To date, various methods of hard dental tissue preparation are used in dental practice [18—20].

In case of a deep or acute process, it is a good practice to remove dentin in carious cavities with steel burs, SmartPrep burs or an excavator [21, 22]. A lot of research has been done on the nature of the change in the dentin structure during the preparation, which is reflected in domestic and foreign publications. The results of the application of particular methods and instrument types are studied by microscopic examinations [23—26].

Thus, according to the findings of a scanning electronic microscopic study, the use of burs with supercoarse and coarse grains of diamond coating for the preparation of carious cavities leads to dentin destruction with the formation of many dentin fragments and a smear layer. Dentin preparation with fine-gain diamond coating burs results in the formation of a small amount of a dense smear layer. The use of burs with the regular grain size of diamond coating for the dentin preparation causes the formation of an inapparent smear layer [27, 28]. Despite the use of state-of-the-art techniques and materials in caries treatment, the hard tooth tissues may remain infected after the carious cavity preparation [29—31]. Diseases of hard tooth tissues are most probably caused by multispecies microbiocenoses [32], therefore, the improvement of the deep caries treatment methods and the methods for detecting and identifying the causative agents of dental disorders is very important today [33—36]. It was of interest to study the dentin microflora in various areas of a carious cavity in the process of preparing a tooth for restoration with burs of various grain sizes, using a new technology, the method of gas chromatography mass spectrometry (GCMS).

Objectives: to study the components and the count of the dentin microflora of a carious cavity and its changes in the process of preparation with burs of various grain sizes.

Materials and methods

Seven apparently healthy patients aged 20 to 30 were selected for the clinical study. Basic and secondary methods of study were used during the examination: survey, examination, probing, thermometry, percussion, radiography and electroodontometry. The oral health status was assessed using the Greene—Vermillion OH1-S index as satisfactory. Then, on the basis of biased and unbiased examination methods, the first molars of the upper jaw with diagnosed chronic deep caries (I.G. Lukomsky's zonal classification) of class 1 according to Black (ICD-K02.1) were selected. The dental treatment and material sampling took place on the basis of the Department of Dental Therapy and Periodontology of the Pavlov First Saint Petersburg State Medical University with informed consent of patients. The beginning of the therapy involved professional oral hygiene treatment of the patient, anesthesia and rubber dam imposition to exclude the ingress of oral microflora into the sample. The carious cavity was drilled out using an air-turbine handpiece with mandatory water cooling. This included three stages: opening the cavity, necrotomy, and forming the walls and the floor of the carious cavity. A visual and tactile assessment of the removed material quality was used. After the microbiological test sampling, all dental cavities were treated with a 0.2% chlorhexidine antiseptic solution and restored with a filling composite.

Sampling for the assessment of the dentin microflora components and count was carried out using a sterile dental plier and sterile foam swabs (PeleTim No. 1, Voco). A swab was placed in a sterile test tube and delivered to the laboratory on the same day. Twenty-one swabbed samples were taken from the dentin surface. The material under study was sampled at three stages of the preparation: first, from the surface of the untreated softened dentin after the carious cavity had been opened with a sterile diamond bur of the regular grain size (ISO 524). Then the material was sampled after necrotomy performed with a sterile fine-grain diamond bur (ISO 524), and then after the formation of the walls and the floor of the cavity, for which purpose a sterile ultra-fine grain diamond bur (ISO 504) was used. The characteristics of the burs used for the preparation of carious cavities are given in Table 1.

Swabbed samples from the dentin surface were examined by the GCMS method using the 7820N—5975 Agilent Technologies (USA) system at the Microbial Chromatography Laboratory, Medbazis LLC (St. Petersburg, Russia). The χ² test was used to compare the signs detection rate, and the Mann—Whitney test to compare the absolute values.

Results and discussion

The GCMS method [37] makes it possible to detect the quality and quantity of the microbial markers content of more than 50 species and genera of bacteria, micromycetes and viruses in the sample under study, and the results of measuring the concentrations of microbial markers in blood with the subsequent reconstruction of the microbial community make it possible to determine the microecological status, as well as the composition of the mixed infection in the lesion focus. In accordance with the developed statistical test for counting the findings of the GCMS method described in the literature, it was assumed that an abnormality becomes clinically significant when the microbial count doubles or halves [38]. As there were no data on the normal values of the content of microbial markers in the studied carious cavities, the content of microbial markers in the nasopharynx biotope, provided by Medbazis LLC, was taken as the normal value. The results of measuring microbial markers by the GCMS analysis of 21 swabbed samples from the dentin surface of carious cavities have made it possible to determine changes in the microecological status: when comparing the findings obtained with the normal values for the nasopharynx biotope, above-limit values were found for some of the parameters (Table 2).

It can be noted that as a result of the study of microbial markers and the reconstruction of the microbial community in the studied samples of the carious cavity in the highest concentration, typical causative agents of caries, Streptococci, were detected. The Staphylococcus, Nocardia and Alcaligenes concentrations were also 4 to 6 times higher than the normal value. Besides, the above-limit values of the Epstein—Barr and Herpes simplex viruses counts call attention to themselves. Markers of anaerobic microorganisms — Clostridia bacilli, a part of the normal intestinal microbiota of the human body, — were identified. The leading representative of the genus was C. perfringens, which is capable of forming more than ten varieties of toxins, whose targets are biological membranes in tissues.

It was of interest to compare the presence of microorganisms at various levels of preparing the carious cavity for restoration. It was established that the detection rate of the dominating causative agents of caries did not change when the cavity was prepared with burs of different grain sizes. Thus, Streptococcus mutans and Streptococci in general were detected in all samples under study. The microbial marker isolation rate of fungi, Staphylococcus epidermidis, Clostridium perfringens, Ruminococcus spp. and other microorganisms decreased insignificantly or fluctuated within various limits, which did not allow revealing any significant differences, with the exception of Alcaligenes spp., Clostridium coccoides and Propionibacterium freudenreichii, which are not considered clinically significant in dentistry. Possibly, this is accounted for by the fact that not so many samples were taken and requires further research (Table 3).

The comparison of quantitative values did not allow to draw a conclusion as to any advantage of using burs of various grain sizes at different levels of cavity preparation, either. A marked decrease in the value was detected only for certain microorganisms, whose role in caries development was not established. Thus, the Nocardia, Prevotella and Propionibacterium genera were found only in the samples obtained after the opening of the carious cavity. There was a decrease in the Propionibacterium freudenreichii count from 3214±3232×10⁵ CFM/g of the biomaterial after the opening of the carious cavity to 591±1011×10⁵ CFM/g of the biomaterial after necrotomy and to ×10⁵ CFM/g of the biomaterial after the formation of the walls and the floor of the cavity (Table 4).

At this stage, a high concentration presence of at least 16 out of 58 studied species and genera of microorganisms was detected. The typical causative agents of caries (Streptococcus spp., Streptococcus mutans) prevailed among them, with an occasional occurrence of micromycetes, viruses and anaerobic bacteria. At the same time, no significant differences have been found in the isolation rate or the count of the typical pathogens of the infectious inflammatory process in the samples obtained from a carious tooth cavity at various boring levels using burs of different grain sizes.

Conclusions

1. During the study of samples swabbed from the dentin surface of a carious cavity by the GCMS method, a high concentration presence of at least 16 out of 58 studied species and genera of microorganisms was detected. The typical causative agents of caries (Streptococcus spp., Streptococcus mutans) prevailed among them, with an occasional occurrence of micromycetes, viruses and anaerobic bacteria. When comparing these findings with the nasopharynx biotope data taken as the reference values, a significant (3 to 10-fold) predominance of the leading pathogens in the studied samples was revealed.
2. According to the findings of the GCMS analysis, the microbiocenosis of the swabbed samples from the dentin surface of a carious cavity included a wide range of pathogens, including bacteria, viruses, and fungi. Such typical causative agents of caries as Streptococcus mutans, Streptococcus spp. and Staphylococcus epidermidis were found in almost all samples. Besides, micromycetes, Clostridium perfringens, Ruminococcus spp. and Lactobacillus spp. were well represented, as well as other microorganisms uncharacteristic of the range of caries causative agents.
3. In the process of preparing the carious cavity with burs of different grain sizes, a marked decrease was found only for some of the microorganisms, whose role in the development of caries has not been established. Thus, the Nocardia, Prevotella and Propionibacterium genera were found only in the samples obtained after the opening of a carious cavity. The count of Propionibacterium freudenreichii decreased from 3214±3232 cells/g × 10⁵ after the opening of the carious cavity to 591±1011 cells/g × 10⁵ after necrotomy and to 308±816 cells/g × 10⁵ after the formation of the cavity walls and floor.
4. Further research is required to determine the role of the GCMS method in studying the microbiocenosis of the carious cavity.

Thus, according to the findings of the GCMS study, it can be assumed that the range of possible causative agents of caries in the samples examined by us included well-studied pathogens, on the one hand, and anaerobic bacteria, viruses and fungi, on the other. In general, the GCMS study of the content of the carious cavity revealed in the material both typical causative agents of the infectious and inflammatory process in the tooth and other microorganisms whose role in the development of the disease has not been established. At the same time, it cannot be ruled out that the lipid components — microbial markers — found in the samples studied could belong to dead microorganisms of the intestinal and skin microflora, including viruses and micromycetes, and only specific markers, the concentration of which was proportional to the microbial count in the human body, penetrated into the lesion focus [39].