Experimental study of the antibacterial effect of anodic dissolution of a copper electrode used in endodontic dental treatment.

Full Text

The purpose of the study was to study the comparative antibacterial activity of anodic dissolution of copper and silver-copper electrodes used in endodontic dental treatment in an experiment.

The experimental study was carried out by implementing a technique for the automatic cultivation of microorganisms in liquid nutrient media. To conduct the study, we used clinical isolates of individual strains of bacteria and yeast fungi, namely: S. constellatus , P. intermedia , C. albicans , as well as mixed cultures: 1) S. constellatus + F. nucleatum ; 2) Streptococcus sanguis + Enterococcus faecium , obtained from the root canals of teeth in the treatment of chronic forms of pulpitis.

The results of the study showed that anodic dissolution of both silver-copper and copper electrodes has a pronounced and, in general, unidirectional antibacterial effect. It was found that, if in relation to the clinical isolate of P. intermedia the use of a silver-copper electrode is more effective, then in relation to strains of S. constellatus , C. Albicans , as well as mixed cultures of pathogenic microorganisms S. constellatus + F. nucleatum and Streptococcus sanguis + Enterococcus faecium, anodic dissolution of the copper electrode showed a more pronounced antibacterial effect.

The results of the experimental microbiological study indicate that in the endodontic treatment of teeth with partially obliterated root canals, along with the anodic dissolution of silver-copper electrodes, it is possible to use the anodic dissolution of copper electrodes as a means that can have a pronounced antibacterial effect.

Rationale: endodontic treatment of teeth with obliterated root canals represents a serious problem [1, 2, 3], where one of the possible ways to improve the quality of treatment of teeth with obliterated root canals is the use of transcanal direct current [4, 5, 6]. The antibacterial effect of such procedures when using metal electrodes connected to the positive side of the current source is associated with the anodic dissolution of the metal from which the electrode is made [7, 8, 9]. The high antibacterial efficiency of silver-copper electrodes used in apexphoresis has been proven [ 10]. However, along with silver-copper electrodes, when carrying out this procedure, electrodes made of other metals can be used, for example, copper electrodes [11]. In this regard, the study of the antibacterial effectiveness of anodic dissolution of copper electrodes is of great scientific and practical interest.

Target: study of the comparative antibacterial activity of anodic dissolution of copper and silver-copper electrodes used in endodontic dental treatment in an experiment.

Methods: An experimental study of the antibacterial activity of the anodic dissolution of copper electrodes used in endodontic dental treatment was carried out by implementing a technique for the automatic cultivation of microorganisms in liquid nutrient media. To conduct the study, we used clinical isolates of individual strains of bacteria and yeasts , namely: S. constellatus , P. intermedia , C. albicans , as well as mixed cultures: 1 ) S. constellatus + F. nucleatum ; 2) Streptococcus sanguis + Enterococcus faecium , obtained from the root canals of teeth in the treatment of chronic forms of pulpitis.

The cultivation of microorganisms was carried out in a bioreactor with an interactive option for controlling the growth of microorganisms - “Reverse Spinner RTS-1” ( BioSan , Latvia ). To interpret the results, the optical density ( OD ) was automatically measured at a wavelength of λ=850 nm. The result was interpreted in McFarland turbidity units .

To cultivate microorganisms in a bioreactor, we used a set of liquid nutrient media produced by HiMedia Laboratories Pvt . Limited (India).

For each experiment, a bacterial suspension in a total amount of 4 ml was prepared separately in sterile 5 ml tubes. The optical density of the resulting suspension was measured using a DEN-1B densitometer ( BioSan , Latvia), and for all samples it was 0.5±0.3 IU Mcf , which in terms of CFU is 1.5x108 ⁱⁿ 1 ml.

During each experiment, cultivation was carried out in several different parallels.

To cultivate microorganisms in a bioreactor, 50 ml centrifuge tubes were used, into which 20 ml of nutrient medium and 1 ml of microbial suspension were placed.

To study the antibacterial activity of the anodic dissolution of a silver-copper electrode, 2 silver-copper electrodes used in apex phoresis were placed in a test tube . One of the electrodes, which was stripped of insulation 2 mm from the end surface of the electrode, was placed at the bottom of the test tube. This electrode was connected to the “+” of the current source. The second electrode, stripped of insulation 1 cm from the end of the electrode, was placed in the upper part of the test tube so that the part of the electrode stripped of insulation was completely immersed in a nutrient medium with microorganisms. This electrode was connected to the “-” current source.

When studying the antibacterial activity of the anodic dissolution of a copper electrode, 2 copper electrodes were placed in test tubes, which were stripped of insulation, placed in test tubes and connected to a current source in the same way as when studying the effectiveness of the anodic dissolution of a silver-copper electrode.

The Potok-1 apparatus (Russia) was used as a direct current source.

The amount of electricity during anodic dissolution of silver-copper and copper electrodes was 5 mA x min.

After anodic dissolution, the electrodes were removed from the test tubes, and the test tubes were placed in a bioreactor .

the McFarlane optical standard (ED Mcf), which was recalculated to the number of microbial cells in 1 ml, for example, 10 ⁸ CFU/ml (colony forming units). The results were processed by the method of variation statistics (the error in recording the optical indicator was ±0.3 Umcf .).

 

Results: When cultivating the studied culture of S. constellatus after anodic dissolution of a silver-copper electrode ( Ag + Cu ) in one sample and anodic dissolution of a copper electrode ( Cu ) in another sample, there was a significant delay in the onset of exponential cell development in both samples (up to 6 and 8 hours of cultivation, respectively).

The periods of intensity of generative activity did not have tendentious differences among themselves, however, the rate of increase in bacterial biomass was lower than in the control sample, which was reflected in the construction of the development curve. The achievement of the main key development points during cultivation had a number of differences: in relation to the sample where the anodic dissolution of the silver-copper electrode ( Ag + Cu ) was carried out, the presence of the α indicator (14 hours) was traced - 2.23±0.3 Umcf , and indicator β (16 hours) – 2.76±0.3 Umcf ; in the sample where anodic dissolution of the copper electrode ( Cu ) was carried out, only M-concentration was noted during cultivation - the β indicator (18 hours) - 2.32±0.3 Umcf . A significant decrease in the rate of logarithmic development in the P-2 segment and the formation of periodic fragmentation with subsequent registration of the α indicator were not observed. In both cases, there was a significant decrease in optical density in relation to the control sample (average OD in the stationary phase of cultivation): for the sample ( Ag + Cu ) - a decrease of 55.66%, for the sample ( Cu ) - a decrease of 63.64 %. Among themselves, the samples also differed in the difference in the values of key indicators of optical density, and in the prolongation of the adaptive phase, which was more pronounced when applying exposure using a copper electrode.

According to the results of culturing a clinical isolate of P. intermedia after anodic dissolution of a silver-copper electrode ( Ag + Cu ) in one sample and anodic dissolution of a copper electrode ( Cu ) in another sample, an antibacterial effect was noted in both test samples.

At the same time, there was a difference in the trend of the initial development of cell cultures: when exposed to ( Cu ), the initial change in optical density was noted already from the 6th hour of the experiment (earlier than the control sample by 2 hours), the initial stages of microbial development did not have clear boundaries relative to the exponential jump, which in turn, was not intense and not significant. Index α for sample ( Cu ) (14 hour) – 1.22±0.3 Umcf (reduction relative to control by 68.6%), index β (16 hour) – 1.32±0.3 Umcf (reduction relative to control by 69.44%).

Particular attention should be paid to the short duration of recording data from the main periods of increase in crop biomass. The ( Ag + Cu ) sample demonstrated a significant prolongation of the culture lag position, which was 2 times higher than the ( Cu ) sample and 1.5 times higher than the control sample. The characteristic period of accelerated development of bacterial cells (12-16 hours) was clearly visible against the background of a subsequent logarithmic increase in optical density, while the rate of change in optical number in the P-2 period was significantly lower relative to the control tube, which indicates a lower rate of development of cellular agents . The α index and the β index were recorded at one point (20 hours) due to the absence of a period of negative acceleration. The optical value at M-concentration for the sample ( Ag + Cu ) (20 hours) is 1.89±0.3 Umcf (56.25% reduction relative to the control). The stationary phase of population development was noted for its duration in comparison with all cultivation samples, with the presence of a slight fluctuation in optical density, which was not statistically significant. The average OD in the P-4 period is 1.99±0.3 Umcf (20-28 hours).

When cultivating the studied culture of C. albicans after anodic dissolution of a silver-copper electrode ( Ag + Cu ) in one sample and anodic dissolution of a copper electrode ( Cu ) in another sample, a pronounced antibacterial effect was observed in both samples compared to the control.

There were no differences in the prolongation of the adaptive period, either in comparison with the control sample or when comparing the test tubes with each other. A significantly reduced rate of generative activity contributed to a shortening of the exponential phase, namely in the P-2 period: up to 6 hours for the sample ( Ag + Cu ), and up to 8 hours for the sample ( Cu ). Indicator α (peak of true logarithmic increase, end of period P-2): for sample ( Ag + Cu ) – 6 hours (1.47±0.3 Umcf ), for sample ( Cu ) – 8 hours (0.99±0 ,3 Umcf ). In these samples, there was a significant increase in the duration of the period of negative acceleration (P-3), at the end of which the cells reached the M-concentration (β indicator): for the sample (Ag + Cu) ( 10 hours ) – 1.85±0.3 Umcf (lower, relative to the control sample by 54.09%), for the sample ( Cu ) (14 hours) – 1.25±0.3 Umcf (lower, relative to the control sample by 68.98%).

When cultivating the studied mixed culture of S. constellatus + F. nucleatum after anodic dissolution of a silver-copper electrode ( Ag + Cu ) in one sample and anodic dissolution of a copper electrode ( Cu ) in another sample, there was a significant delay in the onset of exponential cell development only in relation to the sample Cu (up to 10 hours of cultivation). Compared to the control tube, the formed curves of the development kinetics of the bacterial population of the studied samples clearly reflected the difference in the change in optical density in the P-1 period, and the rate of generative activity of cells in the Cu sample was significantly lower relative to the Ag + Cu sample . Taking into account the prolongation of the adaptive phase and the primary periods of the exponential phase of the experiment, the logarithmic rise of the studied samples was much later than the control cultivation; however, the rate of bacterial growth was not statistically different. The maximum optical density at the end of the P-2 period was recorded at 16 hours ( Ag + Cu test sample) and 22 hours ( Cu test sample ), with an average decrease in optical number of 45.04% relative to the control tube.

At the same time, the graphs clearly showed the difference between the studied samples in the process of a decrease in the rate of cell development: the Ag + Cu sample was characterized by a period of negative acceleration of the culture (16-18 hours), at the end of which the M-concentration of the population was achieved, with a value of OD – 2.32±0.3 Umcf ; for the studied Cu sample this period was not observed, and the M-concentration was comparable to the α index - 2.43±0.3 Umcf (22 hours). The stationary development phase was short for these samples, with an average optical density: Ag + Cu – 2.34±0.3 Umcf (50% decrease relative to the control), Cu – 2.41±0.3 Umcf (decrease relative to the control by 48.5%). There was a significant delay in the onset of exponential cell development in both samples (up to 6 and 8 hours of cultivation, respectively).

When cultivating the studied mixed culture of Streptococcus sanguis + Enterococcus faecium , after anodic dissolution of a silver-copper electrode ( Ag + Cu ) in one sample and anodic dissolution of a copper electrode ( Cu ) in another sample, a difference in the time delay of the logarithmic phase was noted: when exposed to Ag + Cu , initial signs of cell development were already noted from the 4th hour of the experiment (control sample - 6th hour), while for the Cu sample the growth and initial division of populations was characteristic only from the 8th hour of cultivation.

Comparing the kinetics of development by changes in the optical density index for these samples, there was a significant decrease in OD indicators relative to the control tube, while the tendency for the curve to be plotted on the graph was relatively the same between the Ag + Cu and Cu samples , and differed in the difference in optical value.

For both samples, a difficult-to-distinguish picture of the transition of the culture from the period of accelerated development to true logarithmic growth was noted, while the beginning of the exponential jump (period P-2 of the F-2 phase) was at the same time: sample Ag + Cu - 10 hours ( 0.74 ± 0.3 Umcf ), Cu sample – 10 hours (0.14±0.3 Umcf ). The short duration of logarithmic development contributed to the rapid achievement of the maximum optical number by the samples in a given period of cultivation (index α) and the subsequent lengthening of the period of negative acceleration (period P-3). For the Cu sample , key optical density indicators were achieved simultaneously. Indicator α (peak of true logarithmic increase, end of period P-2): for the Ag + Cu sample – 14 hours (2.14±0.3 Umcf ), for the Cu sample – 18 hours (1.54±0.3 Umcf ) . Indicator β (M-concentration, end of period P-3): for the Ag + Cu sample (18 hours) – 2.63±0.3 Umcf (lower than the control sample by 53.04%), for the Cu sample (18 hour) – 1.54±0.3 Umcf (lower than the control sample by 72.50%). There was also a significant difference between the studied samples, most clearly visible in the optical density indicator in stationary equilibrium: for the Ag + Cu sample (18-22 hours) - 2.66 ± 0.3 Umcf , and for the Cu sample (18-28 hours) – 1.57±0.3 Umcf , which was 40.08% lower relative to the Ag + Cu sample and 72.46% lower relative to the optical density at the same point in the control tube.

Conclusion: The development and improvement of active methods of disinfection of dental root canals, especially those that allow the treatment of the obliterated part of the root canal, remains relevant at the present time. According to Sakko M , Tj ä derhane L at all , 2016, during repeated endodontic treatment of teeth, despite the use of modern irrigants , streptococci and enterococci are found in the root canals in association with other types of microorganisms, including fungi of the genus Candida , resistant to the antibacterial agents used [12].

The results of studying the antibacterial activity of the anodic dissolution of silver-copper and copper electrodes used during transcanal exposure to direct current, both in relation to clinical isolates of individual strains (bacteria and yeasts), and mix cultures of pathogenic microorganisms obtained from the root canals of teeth in the treatment of chronic forms pulpitis, showed that anodic dissolution of both silver-copper and copper electrodes has a pronounced and, in general, unidirectional antibacterial effect. It was found that, if in relation to the clinical isolate of P. intermedia the use of a silver-copper electrode is more effective, then in relation to strains of S. constellatus , C. Albicans , as well as mixed cultures of pathogenic microorganisms S. constellatus + F. nucleatum and Streptococcus sanguis + Enterococcus faecium, anodic dissolution of the copper electrode showed a more pronounced antibacterial effect.

The data obtained partly contradict the results of studies by Efanov O.I., Tsarev V.N. et al. (2006, 2008 ) , where they studied the antibacterial effect of anodic dissolution of electrodes made of various metals in relation to pathogenic facultative anaerobic microbiota of dental root canals [13, 14, 15]. The authors found that anodic dissolution of silver-copper electrodes has the greatest antibacterial activity. However, these experimental studies were carried out on solid nutrient media in Petri dishes. In our study, we studied the antibacterial activity of the anodic dissolution of metal electrodes in liquid nutrient media, which is more consistent with the vital activity of microorganisms in the root canals of teeth. In addition, these authors did not study the effect of anodic dissolution of electrodes on mixed cultures of microorganisms.

At the same time, our results are fully consistent with the research data of A.G. Volkov, V.F. Prikuls et al. (2019). These authors note a high antibacterial effect transchannel influences with direct current carried out from the anode [16]. In our study, we confirmed this fact using mixed cultures of microorganisms.

The greater antibacterial activity of anodic dissolution of copper electrodes compared to silver-copper electrodes in liquid nutrient media is obviously due to the fact that when using a silver-copper electrode, which is a copper core coated with a layer of silver, mainly silver is subjected to anodic dissolution, which is characterized by lower electrochemical solubility and electrophoretic mobility compared to copper [17].

Thus, the results of the experimental microbiological study indicate that in the endodontic treatment of teeth with partially obliterated root canals, along with the anodic dissolution of silver-copper electrodes, it is possible to use the anodic dissolution of copper electrodes as a means that can have a pronounced antibacterial effect.

About the authors

Andrei Tsarev

Федеральное государственное автономное образовательное
учреждение высшего образования «Российский университет дружбы
народов», г. Москва, Россия (Peoples’ Friendship University of Russia)

Author for correspondence.
Email: digreezvipru@gmail.com
ORCID iD: 0000-0002-1900-0962

Аспирант 4 года

Russian Federation

Natalya Zh. Dikopova

I.M. Sechenov First Moscow State Medical University (Sechenov University)

Email: zubnoy-doctor@yandex.ru
ORCID iD: 0000-0002-4031-2004
SPIN-code: 3635-2998

MD, Cand. Sci. (Med.), Associate Professor

Russian Federation, Moscow

Evgeniy V. Ippolitov

Moscow State Medical and Dental University named after A.I. Evdokimov

Email: ippo@bk.ru
ORCID iD: 0000-0003-1737-0887

MD, Dr, Sci. (Med.), professor

Russian Federation, Moscow

Alexander G. Volkov

The First Sechenov Moscow State Medical University (Sechenov University)

Email: parodont@inbox.ru
ORCID iD: 0000-0003-2674-1942
SPIN-code: 3391-0877

MD, Dr. Sci. (Med.), Professor

Russian Federation, 8, building 2, st. Trubetskaya, Moscow, 119991

Svetlana N. Razumova

RUDN University

Email: razumova_sv@mail.ru
ORCID iD: 0000-0003-3211-1357

MD, Dr. Sci. (Med.), Professor

Russian Federation, 6, Miklukho-Maklaya str., Moscow, 117198

Mikhail S. Podporin

Moscow State University of Medicine and Dentistry named after A.I. Evdokimov

Email: podporin.mikhail@yandex.ru
ORCID iD: 0000-0001-6785-0016
SPIN-code: 1937-4996

MD, Cand. Sci. (Med.), Research Associate

Russian Federation, Moscow

Tatyana V. Budina

Sechenov First Moscow State Medical University (Sechenov University)

Email: budina_tatiana@mail.ru
ORCID iD: 0000-0002-6957-5510
SPIN-code: 8217-9886

MD, Cand. Sci. (Med.)

Russian Federation, Moscow

References

Supplementary files