The impact of tooth decay prevention measures on the pro-antioxidants system

Pages: 07-28

Assoc. Prof., Dr. Aurelia Spinei (1), Assoc. Prof., Dr. Iurie Spinei (2)

(1) „Nicolae Testemitanu” State University of Medicine and Pharmacy, Chișinău, Republic of Moldova, (2) „Nicolae Testemitanu” State University of Medicine and Pharmacy, Chișinău, Republic of Moldova,

Summary

The purpose of the work: to assess the impact of the tooth decay prevention measures on the pro-antioxidants system.

Material and method: The controlled clinical study was performed on a sample of 120 psychosomatic healthy children, aged between 7 and 12, having a high or extreme cavity risk, caused by the high cariogenic activity of the dental biofilm. The antibacterial photodynamic therapy (APDT) was performed in children from groups 1 and 2. The photosensitizing agent (PhA) in L1 was the methylene blue solution 1%, and in L2 the anthocyanin grape extract 5%. The tooth decay prediction and the complex assessment of the cavity risk was performed with the help of the Cariogram software. The determination of the antiradical activity (AA) of the oral fluid (OF) was performed by using the free radical 1,1 diphenyl-1-picrylhydrazyl, DPPH˙.

Results: after performing APDT by using the anthocyanin grape extract (L2, AA=80.09± 1.57%), AA of OF increased 12.83 times compared to using the methylene blue solution (L1).

Conclusions: The results obtained suggest that the anthocyanin grape extract contributes to the rapid collection of free radicals (possibly produced when performing the APDT) and demonstrates the advantages of using it as PhA when performing the APDT.

Key words: tooth decay, cavity risk, children, biofilm, anthocyans.

Introduction

The oxidative stress (OS) has an extremely negative impact over living organisms. In a classical manner, the OS is defined as an imbalance between oxidants and antioxidants, generated by the free radicals [1-3], which, in their turn, are formed during the metabolism and energy production within the body. Free radicals (FR) are involved in multiple processes, such as transduction and expression of genes, activating receptors and nuclear transcription factors, oxidizing deteriorations of cellular components [1, 2] and others. As a response reaction to the OS, the cell activates its antioxidant mechanisms, in order to counteract the effect of the oxidative stress, thus re-establishing the balance between the reactive oxygen species (ROS) and the action of the antioxidant barrier, for an adequate operation of the human and animal body, determining the synthesis of multiple antioxidant enzymes which protect the cells and tissues against OS by converting ROS into non-reactive species [1, 4].

It was determined that the OS has a significant role in developing oral diseases. In the specialty literature, the increase of antioxidant system indicators is mentioned for the subjects affected by tooth decay (TD), including for children [5, 6]. In our previous studies, in the case of children with TD, the oral fluid (OF) showed a significant increase of the glucosyltransferase activity and of the low glutathione concentration, which is considered to be the biomarker of the cariogenic activity of bacteria within the dental biofilm and of the high or extreme cavity risk [7, 8].

For the past couple of decades, the antimicrobial photodynamic therapy (APDT) has been used to destroy bacteria and viruses. The bactericidal and bacteriostatic action of APDT over the pathogen agents is achieved through the singlet oxygen generation and through the peroxide radicals by photosensitive exogenous and endogenous substances, with a chain of photochemical reactions [9]. APDT is currently and adjacent therapeutic method for treating dental problems, including tooth decay and its complications. The latest studies show a reduction in the number of pathogen bacteria by 92-100 %, without using antiseptics and antibiotics, which can have side effects [10, 11] and by regaining the physiological balance between the aerobic and anaerobic microflora in the oral cavity, after performing APDT, in a ratio of 75:25 % [12, 13].

Our previous studies tested the APDT action, by using different photosensitizing agents (PhA) on the cariogenic dental biofilm composition, under in vitro and experimental conditions [14-20]. At the same time, the research performed in vitro established a reduction of the bacterial culture increase, which was determined not only directly by the APDT effect, but also by the oxidative stress, adjacent to this operation.  The capacity of the bacterial cell to survive after the effect of the oxidative stress depends on the activity of the bacterial superoxide dismutase or on the amount and activity of heat shock proteins, which produce, under the oxidative stress, HSP-70 and HSP-90 [21, 22]. Therefore, it is essential to assess the antioxidant barrier – a complex enzyme system, elements and substances that are formed in the aerobic bodies in order to protect them from the high oxygen concentrations.

Thus, knowing in detail the formation of ROS and the action mechanisms opens new perspectives in the treatment and prevention of different dental problems 5], including TD.

The purpose of the work

To assess the impact of tooth decay prevention measures over the pro-antioxidant system.

Material and method

The study was performed within the „Ion Lupan” Department of Pediatric Oro-Maxillo-Facial Surgeryand Pedodontics within the Nicolae Testemitanu” SUMF. In order to achieve the study objective, a sample was established, containing 120 psychosomatic healthy children, with ages between 7 and 12 years old and with a high or extreme cavity risk, caused by increased cariogenic activity of the dental biofilm. The children were allocated randomly in 4 structurally identical groups, the ratio between the groups being 1:1:1:1.

The study subject inclusion criteria: age 7- 12 years, conventionally healthy children, with high or extreme cavity risk, caused by the intense activity of the cariogenic bacteria branches from the dental biofilm (Cariogram score 0-40%, Streptococcus mutans number >105 UFC/ml, high and very high speed of bacterial plaque formation, the PFRI index >30%) and the informed agreement of the parents for their children participation in the study.

The study subject exclusion criteria: lack of informed agreement from parents for their children’s participation in the study, dental fluorosis, individual sensitivity to phototherapy or to the used photosensitizing agent, administration of antimicrobial treatment, participation in other TD prevention programs.

At the beginning of the study the antiradical capacity or the antioxidant activity (AA) of substances (compounds) used for performing the APDT was determined.

Table 1. Methodology of determining the antioxidant activity (AA,%) of tested substances

Depending on the applied preventive methods (Table 2), the children with high or extreme cavity risk caused by the increased cariogenic activity of the dental biofilm, which were included in the study, were randomly allocated in 4 identical groups, by sex, age and living environment. All participants in the study were trained to clean the oral cavity correctly. The children cleaned they oral cavity every day, using hygiene objects and remedies adequate to their age.

Table. 2. Study methodology

The antibacterial photodynamic therapy was performed after the complete removal of dental deposits. On dental surfaces and in the interdental areas, a PhA substance was applied for 1-2 min, then the treated surfaces were radiated for 30 sec with LED light, λ= 625-635 nm and the impulse power 2,0-3,0 W issued by the FotoSan 630 LAD pen device (CMS Dental,Denmark) (Fig.1). As PhA in children from group L1 0.5-1.0 ml methylene blue solution 1% was used, and for those in group L2 0,5-1,0 ml anthocyanin grape extract 5%, pH 8.0-9.0. In order to protect the visual apparatus of healthcare staff and of patients, special protection goggles were used (FotoSan 625-635 nm). APDT was then followed by lightly dabbing 2-6 drops of probiotic product, which contain  fidobacterium BB-12®, Lactobacillus rhamnosus GG®– 250 x108 UFC. For 30 days, probiotic products were administered per os, which contain Lactobacillus rhamnosus-LGG, Bifidobacterium BB-12 – 4,9 x109, 1-2 caps/day for 30 days [19, 20].

Figure 1.Performing antibacterial photodynamic therapy

The study was performed by applying data collection tools according to the sheet drawn up for registering the results of the cavity experience assessment, the cariogenic risk and the results of the laboratory studies. The prediction of the cariogenic risk was performed with Software Cariogram, which makes a correlation between the TD determining factors, the graphic expression of the cariogenic risk, the elaboration of specific prevention schemes based on allocating patients into risk groups (D.Bratthall, G.Hänsel Petersson, JR. Stjernswärd. Cariogram, Internet Version 2.01. April 2, 2004) [23]).

The quantification of the dental biofilm was performed by estimating the pH and the bacteriological study in which the total number of live germs was estimated in 1 g of substratum (NTG/g) and cariogenic bacteria were identified from the group Streptococcus viridans: Streptococcus mutans, Streptococcus mitis, Streptococcus sobrinus, Streptococcus gordonii and others, by using the GPI card (for gram-positive cocci) of the Vitek2 automated system [24]. The bacteriological investigations of the dental biofilm were performed during the initial phase, right after applying APDT (within 1-2 hours), at 7, 14-15 days and 1 month.

The determination of the antioxidant activity (AA) of OF was done by using the free radical 1,1 diphenyl-2-picrylhydrazyl, DPPH˙(purity 90%) in accordance with the method proposed by Brand Williams W., Cuvelier M.E., Berset C. 1995, and quoted by Sturza A., 2012 [25]. The ethanol solution of 60 pM DPPH was prepared. For 4 ml DPPH solution 0.1 ml non-stimulated, collected OF was added. The control sample contained the same solvent volume and was used to measure the maximum absorption of DPPH. After 30 min of incubation in darkness, at room temperature, the absorbance was measured at 515 nm in order to determine the DPPH concentration. The DPPH radical collection activity was expressed as a percentage reduction of DPPH [25, 26].

The study was approved by the Ethics Research Committee of „Nicolae Testemitanu” SUMF and was performed in accordance with the ethical requirements, by obtaining the written approval of the children’s parents. For the statistical data analysis, the Epi Info software was applied.

Results

In the initial examination, all children included in the research had a high or extreme cavity risk, the chances of avoiding new cavities, assessed with the Software Cariogram varying between 27.2± 4.08% and 32.53± 3.57%. The results of determining NTG/g of dental biofilm in the initial phase and after performing the preventive measures were presented in Table 3. Thus, in the beginning of the study, between research and witness groups, there were no significantly statistic differences. The analysis of dental biofilm pH values (5.83±0.26 – 5.86±0.33) did not underline significant differences between the groups of children.

After performing the APDT supplemented with probiotics, in children from groups L1 and L2, there was a complete annihilation of all bacteria from the dental biofilm, followed by a further increase of the NTG/g, so that for 14-30 days NTG/g reaches norm levels and is maintained for 6 months: 6.133±0.079 log10 UFC/g, t=7,883, p<0.001 in group L1 and 6.34±0.088 log10 UFC/g, t=12.794, p<0.001 in group L2. Cleaning the oral cavity and gargling with antiplaque mouthwash contributed to the reduction of NTG/g down to 7.867±0.063 and respectively, 7.03±0.169 log10 UFC/g.

Table 3. Total number of germs in children’s dental biofilm (log10 UFC/g) depending on the applied preventive measures

             After the clinical study, it was established that right after performing APDT, combined with the local application and the oral administration of probiotics, the children presented the total annihilation of dental biofilm bacteria, followed by an increase of NTG/g, so that for 14-30 days NTG/g reached norm levels and was maintained for 5-6 months: 6.133±0.079 UFC/g, (p<0.001) in group L1 and 6.2±0.088 log10 UFC/g, (p<0.001) in group L2, a reduction of the cariogenic capacity of the dental biofilm, confirmed by a significant increase, of 1.1 times (p<0.001) in children from group L1 and of 1.16 times (p<0.001) in L2, of the bacterial plaque pH, thus insuring the considerable reduction of the cavity risk and significantly increasing the cavity-protecting effect, the chances of avoiding cavities increasing 2.194 times (p<0.001) in group L1 and 2.14 times (p<0.001) in L2.

By identifying the bacteria with the Vitek2 automatic system after performing the preventive treatment in children from L1 determined the change of the microbial scenery of the dental biofilm characterized by the significant statistical decrease, by 4.90 times of the Streptococcus mutans number (t=9.68, p<0.001) and by 2.80 times of the Streptococcus sobrinus number from the dental biofilm (t=4.87, p<0.001) at the same time with a significantly statistical increase, by 2.49 times of the Streptococcus salivarius  number (t=8.38, p<0.001) and by 5.25 times of  Streptococului sanguinis (t=-3.18, p<0.01) in relation to the initial level.

In the result of the preventive treatment performed in children from group L2 (performance of APDT by applying anthocyanin grape extract as PhA, followed by the administration of probiotics) the dental biofilm pH increased significantly, 1.14 times (t=-19.36, p<0.001), reducing the cariogenic capacity of the dental biofilm and the cavity risk from high to moderate and thus ensuring the increase of chances to avoid new cavities by 1.79 times, up to 59.71±1.90%, p<0.001.

Most children from group L3 presented an increase of 1.07 times of the Streptococcus mutans number (t=-1.58, p>0.05) and of 1.13 times of the Streptococcus salivarius number (t=-1.77, p>0.05), as well as the aggressive associations of streptococci, which decreased the dental biofilm pH 1.02 times (t=12.00, p<0.001), maintaining the high cavity risk, the chances to avoid new cavities formation being of only 38.48±3.86%, p<0.001. Thus, using antiplaque mouthwash was not sufficiently enough in reducing the intense cariogenic activity of the dental biofilm bacteria.

Performing only the oral cavity hygienic measures (L4) in children with high or extreme cavity risk, caused by the increase activity of the cariogenic bacterial branches did not ensure the envisioned cavity-preventive effect, so that the cavity risk remained high, a NTG/g increase being registered in the dental biofilm, especially in the number of the acidogenic bacteria, followed by a significant decrease, of 1.06 times (t=10.86, p<0.001) of the dental biofilm pH, the chances to avoid new cavities formation being lower (27.50±1.88%).

The results of determining the antioxidant activity (AA) of the tested substances compared to OF (control group) are represented in Table 4. The non-stimulated OF collected from conventionally healthy children with high or extreme cavity risk (L0) has moderate antioxidant/antiradical activity. The highest antioxidant/antiradical activity was established for the anthocyanin grape extract (AA=96.18±0.16 %), and the minimum one is for the methylene blue solution (AA=0.04±0.02%), both substances being used as PhA when performing APDT.

Table 4. Results of determining antioxidant activity (AA,%) of the tested substances

Taking into account the fact that OF offers a natural protection against ROS due to the content of antioxidants, during the clinical study, in the pre-testing phase, the AA of OF was assessed at 30 minutes after performing the TD prevention treatment in children who are conventionally healthy with high or extreme cavity risk caused by the increased cariogenic activity of the dental biofilm.

Table 5. Results of determining the antioxidant activity (AA, %) of the oral fluid after performing the tooth decay prevention measures

The results of assessing AA of OF right after cleaning the oral cavity (L4, AA=6.08±1.17%) and performing APDT by using the methylene blue solution of 1% as PhA (L1, AA=6.24±1.25%) indicated a low antioxidant/antiradical activity (Table 5). After performing APDT by using grape extract as PhA (L2, AA=80.09±1.57%), AA of OF increased 12.83 times in relation to using methylene blue. Administering probiotics after performing APDT did not significantly influence the antioxidant properties of OF.

Discussions

During this study, the impact of multiple tooth decay prevention measures on the pro-antioxidant system was assessed. Research was preceded by multiple studies performed by us in vitro and on laboratory animals. Thus, the efficiency of APDT action was assessed (by using different types of photosensitization agents) on streptococci isolated from dental biofilm of children with high cavity risk caused by the increased cariogenic capacity of the bacterial plaque. The higher efficiency was established for using the anthocyanin grape extract, compared to the norm, as PhA in APDT, resulting in annihilating the cariogenic microorganism Streptococcus mutans, Streptococcus mitis, Streptococcus gordonii, Streptococcus sobrinus and others. At the same time, the results obtained in the studies performed in vitro are difficult to transpose in vivo, because the oral cavity environment may interfere with the biological properties of PhA molecules. Therefore, additional research is necessary under experimental and clinical conditions for justifying the use of anthocyanin grape extract as photosensitizing substances in APDT, performing the APDT offering new opportunities in elaborating efficient TD prevention methods.

It is known that the bactericidal effect over the microorganisms exercised by APDT (in case of predomination of type I photochemical reactions) is ensured by ROS formation right nearby the outer membrane, which diffuses into the cell and destroy different cellular structures [27-31]. Therefore, in order to assess a possible unbalance between the oxidants and antioxidants, caused after performing APDT it is necessary to determine FR in the oral cavity. Determining FR is difficult because they are found in low ratios and have a short lifespan, many times indirect dosing methods being used, which intend to determine the antiradical capacity [32].

The results of the study have shown that non-stimulated OF collected from conventionally healthy children with high or extreme cavity risk have a moderate antioxidant/antiradical activity. The highest activity was established for the grape extract (AA=96.18±0.16 %), and the minimum one – for the methylene blue solution (AA=0.04± 0.02%), both substances being used as PhA for performing APDT.

In this research, we established that the use of the anthocyanin grape extract as PhA increased 12.83 times the ROS caption property in OF in the first 2 hours after performing APDT, unlike the APDT performance with using the reference substance (methylene blue solution). The data obtained confirm the results of multiple studies, which indicated the high hydrogen donation capacity of anthocyans, capable in a very short time to neutralize FR with hydrogen or through electron donation mechanisms and are in accordance with the results of researchers that studied the antioxidant activity of the polyphenolic compounds, in particular anthocyanins extracted from grapes or vineyard products from the Republic of Moldavia and Romania [34-38].

The ability of polyphenolic compounds to collect free radicals is partially determined by an electron’s reduction potential, which is a measure of antioxidants’ reactivity as hydrogen or electron donors. This is proven by the amount or oxidised molecules neutralized by a single antioxidant molecule. These properties are determined by the antioxidant structure and the reaction mechanism. The number of available OHgroups available, as well as the reaction products (dimers or quinines), can enter into a reaction with the free radicals and, thus, directly influence the antioxidant activity of polyphenols, significantly increasing it. The polyphenol molecule is made of two or more benzoic rings attached to the atoms by the hydroxyl groups, which determine the antioxidant effect of polyphenol [39-42].

 The studies performed in vitro defined the antioxidant potential of polyphenols, explained by many authors by their free radical collection capacity, such as super oxide radicals (O2˙), singlet oxygen (O˙), hydroxyl radical (OH˙), peroxide radical (HO2˙), nitrogen monoxide (NO˙) and peroxynitrate (ONOO), which, in their turn, are at the basis of different pathologies. The chemical structures, which contribute to the antioxidant activity of polyphenols (dihydroxy- or trihydroxy-), have the property of chelating metal ions by forming complexes and prevent free radical generation. This structure also allows the de-focalization of electrons, offering a high free radical destruction reactivity [42]. As a result, these compounds can inhibit the oxidising processes, protecting the biomolecules (lipids, proteins, DNA and others) from oxidising [38].

Anthocyanins belong to the class of vegetal secondary metabolites, known as flavonoids, from the greater class of polyphenols and have a complex chemical structure, they have a phenil-2-benzopirilium type structure, in which there is a chromium nucleus and a benzene nucleus [39]. The anthocyanins identified from grapes were mono-glycosides and mono-glycoside derivatives of five anthocyanidins: cyanidin, petunidin, delphinidin, peonidin and malvidin, and the derivatives were 6-O-acetyl and 6-O-cumaril. These compounds are antioxidant agents with an antioxidant capacity 50 times higher than the one of vitamin E and 20 times higher than that of vitamin C. Thus, the increased antioxidant activity of anthocyans can be explained by their chemical structure – high number of groupings – OH [40] and donating a free electron or a H atom to react to RL.

Thus, the clinical studies we performed including children with high and extreme cavity risk demonstrated that applying APDT with using anthocyanin grape extract as PhA does not have a negative impact on the glutathione/ glutathione transferase system (with antioxidant effects) and on the lacto peroxidase/thiocyanate system (with antitoxic effects) from OF [7, 8]. The grape extract increases the antioxidant capacity of OF and exercises an antiradical effect compared to RL, produced in excess, under OS conditions. The bacteriostatic action of APDT is a well-documented fact, also confirmed by the results of reducing the protein content in OF. Being a non-invasive and efficient control method for the cariogenic dental biofilm, which does not have side effects that can appear after using antiseptics, it is normal to use it for children who are not capable to clean their oral cavity (children with accentuated or severe psychosomatic disabilities) and with increased cariogenic activity of dental biofilm bacteria.

The vineyard products represent a cheap source of polyphenol compounds. Grapes contain significant amounts of polyphenols, including resveratrol (stilbene), catechins, flavonoids, flavonols and anthocyans [32, 34]. In recent years, researchers in our country have shown a special interest in the grapes skin and seeds, of which multiple natural bioactive, non-toxic, non-pollutant products can be obtained, with polyvalent benefits, which could represent a new source of antimicrobial substances efficient in controlling the dental biofilm [32, 37]. Thus, the vegetal polyphenols might be used at a reasonable price for producing oral cavity cleaning remedies [43].

Conclusions

The results of determining the antioxidant activity underlined the higher antioxidant/antiradical activity of anthocyanin grape extract (AA=96.18± 0.16%), in relation to methylene blue (AA=0.04±0.02%), both substances being used as PhA in performing APDT. The results obtained suggest that the anthocyanin grape extract contributes to rapid collection of free radicals (possibly produced when performing APDT) and proves the advantages of using it as PhA in performing the APDT. The APDT method with applying local photosensitising agents, supplemented with probiotics administration, represents a new therapeutic approach in the oral biofilm management and offers new opportunities in elaborating efficient methods for preventing TD in children with disabilities and high cavity risk.

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