Effects of fruit vinegars on root dentin microhardness and roughness.

AIMS: The aim of this in vitro study was to evaluate the effects of newly proposed irrigants; the pomegranate, apple cider, and grape vinegars in comparison with contemporary irrigants; sodium hypochlorite (NaOCl), chlorhexidine gluconate (CHX), and octenidine-hydrochloride (OCT) on microhardness and surface roughness of human root canal dentin. SUBJECTS AND METHODS: The crowns of the 105 mandibular incisor teeth were removed, and roots were separated longitudinally. Root halves were embedded in acrylic-resin and were ground flat. The specimens were randomly divided into following six test groups (n = 30); pomegranate vinegar, apple cider vinegar, grape vinegar, 2.5% NaOCl, 2% CHX, OCT and a control group based on the treatment time, samples were then divided into two subgroups (15 min or 30 min). Each specimen was first subjected to surface roughness and then Vickers microhardness testing. The data were statistically analyzed.
RESULTS: Pomegranate, apple cider, and grape vinegars exhibited similar roughness values (P > 0.05) and presented higher results than other groups (P < 0.05). There was no statistically significant difference between the microhardness values of the irrigant groups (P > 0.05). The microhardness decreased when the exposure time increased from 15 min to 30 min (P < 0.05).
CONCLUSIONS: The use of vinegar for endodontic irrigation may have a softening effect on root canal dentin with time and may increase dentin roughness.

INTRODUCTION

Bacteria and their products are the main etiological factors responsible for periradicular infections.[12] Endodontic procedures aim for the total elimination of all microorganisms, or, at least, for reducing the bacterial population to a level at which the host resistance can overcome it.[3] Root canal irrigation is one of the most important parts of endodontic therapy, and it contributes through root canal system disinfection and the mechanical flushing of debris from the canal. Solutions of sodium hypochlorite (NaOCl) and chlorhexidine gluconate (CHX) are the most common irrigants used during the irrigation step; however, NaOCl is widely preferred due to its high antibacterial activity and tissue dissolution capacity. CHX has been recommended as both a root canal dressing and final irrigant based on its antibacterial action and substantivity.[4] Octenidine hydrochloride (OCT) is an antiseptic agent that has broad-spectrum antimicrobial activity,[5] and until recent years, this molecule has been used in medicine. Due to its favorable properties, such as noncarcinogenicity, nonmutagenicity, and bactericidicity, OCT has been suggested as a root canal irrigant.[6]

Vinegar is a natural food preservative that has been used for thousands of years, and it is of interest to the field of medicine due to its beneficial health effects. Since vinegar has antibiotic and antiseptic properties, it has been used for the treatment of infected wounds.[7] Moreover, because it is bactericidal, vinegar could be an alternative root canal irrigant. Apple cider vinegar, which contains acetic and maleic acid, has been found to reduce approximately 30% of the Enterococcus faecalis cell population.[8] Apple cider vinegar has also been researched regarding its ability to remove the smear layer.[9]

It is crucial to evaluate the effects of irrigation solutions on the dentin surface because they stay in contact with the dentin during irrigation. Irrigants may alter the physical and chemical structure of dentin, and they may inhibit interactions with obturation materials and coronal restorations.[10] The microhardness and roughness, as physical indicators of dentin, are sensitive to the composition and surface changes of the tooth structures, which could be influenced directly by irrigants.[11]

To the best of the authors' knowledge, no previous studies have investigated the efficacies of different fruit vinegars and OCT on the root dentin microhardness and roughness. Therefore, the aim of this in vitro study was to evaluate the effects of some newly proposed irrigants, including pomegranate, apple cider, and grape vinegars (Kemal Kukrer, Eskisehir, Turkey), and compare them with the effects of more contemporary irrigants, including 2.5% NaOCl (Caglayan Kimya, Konya, Turkey), 2% CHX (Klorhex; Drogsan Ilaclari A. S., Ankara, Turkey) and OCT (Octenisept; Schülke and Mayr GmbH, Norderstedt, Germany), on the microhardness and surface roughness of human root canal dentin.

SUBJECTS AND METHODS

Specimen preparation

One hundred and five mandibular incisor teeth extracted for periodontal reasons, without caries, cracks, previous endodontic treatments, or coronal restorations, were collected for use in this study. The teeth were stored in distilled water for <3 months until further processing. The crowns of the teeth were removed using a diamond saw under water cooling, and the pulp tissue was extirpated. Then, the roots were separated longitudinally in a mesiodistal direction to produce a total of 210 specimens. The specimens were horizontally embedded in autopolymerizing acrylic resin, and they were ground flat with silicon carbide abrasive papers (to 2000 grit) under distilled water. The specimens were randomly divided into six test groups and a control group according to the irrigation solutions used (n = 30): (G1) pomegranate vinegar, (G2) apple cider vinegar, (G3) grape vinegar, (G4) OCT, (G5) 2.5% NaOCl, (G6) 2% CHX, and (G7) distilled water (control).

All of the groups were then divided into two subgroups of 15 specimens each. The specimens in the first subgroup were treated with each irrigation solution for 15 min, while those in the second subgroup were treated for 30 min. Each specimen was first subjected to a computerized roughness tester (Mitutoyo Surftest Analyzer; Matsuzawa Seiki Co., Ltd., Tokyo, Japan) to determine the surface roughness (Ra, μm). Three tracings were performed at different locations, and the Ra mean, and standard deviation were measured.

After determining the roughness, each specimen was subjected to the Vickers Hardness Tester (MHT2, High-Quality Microhardness Tester; Matsuzawa Seiki Co., Ltd.). Three separate indentations were made on the coronal, middle, and apical regions of the root dentin surface with 300 g of loading and a dwell time of 20 s. The values were averaged and converted into Vickers numbers.

Scanning electron microscopy

One sample from each vinegar group was observed under the scanning electron microscope (SEM) (EVO LS10; Carl Zeiss Meditec AG, Oberkochen, Germany) to visualize the dentin surface after the vinegar treatment. The SEM micrographs were obtained at ×1000 in representative areas of the samples [Figure 1].

Figure 1

Scanning electron microscope micrographs showing different irrigation regimen's effect on dentin surfaces after 30 min; (a) Pomegranate vinegar, (b) Apple cider vinegar, (c) Grape vinegar (×1000)

Statistical analysis

The data were analyzed using IBM SPSS Statistics for Windows version 19 (IBM Corp., Armonk, NY, USA). A two-way analysis of variance and post hoc Tukey test were used to compare the different groups, with a significance level of P < 0.05.

RESULTS

The means and standard deviations of the root dentin roughness values for the experimental groups and control group are listed in Table 1. Significant differences were observed in the roughness values among the groups tested. The pomegranate vinegar, apple cider vinegar, and grape vinegar were similar to each other (P > 0.05), and they presented higher results (P < 0.05). However, the time had no effect on the roughness of the root canal dentin (P > 0.05).

Table 1

The means and standard deviations of the root dentin roughness values for test and control groups

Groups	Mean±SD
	Roughness (15 min)	Roughness (30 min)
Pomegranate vinegar	0.61±0.16c	0.55±0.16
Apple cider vinegar	0.44±0.10b,c	0.55±0.12
Grape vinegar	0.52±0.17c	0.63±0.14
OCT	0.34±0.12a	0.36±0.15
NaOCl	0.43±0.18a,b	0.36±0.14
CHX	0.31±0.10a	0.33±0.15
Distilled water	0.37±0.15a,b	0.38±0.19

Different superscript letters represent statistically significant differences between the test groups (α=0.05). SD: Standard deviation, OCT: Octenidine-hydrochloride, NaOCl: Sodium hypochlorite, CHX: Chlorhexidine gluconate

The means and standard deviations of the root dentin microhardness values for the experimental groups and control group are shown in Figure 2. There was no statistically significant difference between the microhardness values of the irrigant groups (P > 0.05). However, the application time had an effect on the microhardness, which decreased when the exposure time increased from 15 min to 30 min (P < 0.05).

Figure 2

Microhardness values (means and standard deviations) of test and control groups. PV: Pomegranate vinegar, AV: Apple cider vinegar, GV: Grape vinegar, OCT: Octenidine hydrochloride, NaOCl: Sodium hypochlorite; CHX: Chlorhexidine gluconate, DW: Distilled water

DISCUSSION

This study was designed to evaluate and compare the effects of irrigation solutions with bactericidal actions on the dentin roughness and microhardness. The results of the present study indicated that the vinegars tested significantly increased the dentin roughness, and all of the irrigants affected the dentin microhardness over time.

Surface roughness is an important factor for adhesion, and ideal roughness is necessary to create optimal wetting.[12] An increase in roughness would result in a decrease in contact angle which means better wettability.[1314] In addition, an increase in the surface roughness could have clinical benefits because it contributes to the micromechanical bonding of the root canal sealers that require the presence of root canal wall irregularities.[1516] However, rough surfaces could promote bacterial adherence, which might lead to plaque formation.[17] Furthermore, comprehensive understanding of the surface characteristics, including chemical composition, roughness, hydration state, etc., is essential to obtain the adhesion of root canal sealers that have different physicochemical characteristics.[16] Such chemicals like irrigation solutions or root canal medicaments could affect the interaction at the interface between root canal sealers and dentin via changing the surface composition and roughness. Our findings showed that the pomegranate, apple cider, and grape vinegars presented higher roughness values, which were similar to each other. Acetic acid is the main component of fruit vinegar, and apple cider vinegar also contains maleic acid. Maleic acid has been studied previously with regard to its efficacy on the dentin microhardness and roughness, and consistent with the present findings, it was found to cause maximum roughness of the root canal dentin surface.[18] To the best of the authors' knowledge, no study has evaluated pomegranate and grape vinegars as irrigation solutions, other than here in the present study. Probably due to the similar contents of the different fruit vinegars, they exhibited similar roughness values. Another explanation for the high roughness values in the vinegar groups could be that vinegar has a low pH, which is associated with dental erosion.[9] In addition, longer contact with the vinegars did not alter the dentin roughness.

Our results indicated that the NaOCl, CHX, and OCT had no effects on the root dentin roughness, and similar roughness values were demonstrated between these and the control group values. Consistent with our findings, one previous study showed that the 0.2% CHX did not alter the dentin roughness.[19] However, contrary to our results, the 5.25% and 2.5% NaOCl did increase the dentin roughness.[1920] As for the OCT, this was the first study to evaluate its effects on the dentin roughness and microhardness.

Based on the results of the present study, no significant reduction in the dentin microhardness occurred after 15 min of treatment with any of the irrigants tested. However, it was verified that the microhardness was lower after 30 min of irrigation than after 15 min of irrigation. In addition, all of the irrigants displayed similar microhardness values. In contrast to the present study, the NaOCl was proven to reduce the dentin microhardness in several studies.[1921] Moreover, there are conflicting results regarding the effects of CHX on the dentin microhardness. Oliveira et al.[22] reported that 2% CHX reduced the microhardness, whereas another study revealed that the CHX had no effect on the root canal dentin microhardness.[19] As we stated above, the effects of the OCT on the dentin microhardness have not been researched previously.

A recent study revealed that maleic acid reduced the dentin microhardness significantly,[18] and this effect was attributed to its strong acidic pH and demineralization capacity.[18] The grape and pomegranate vinegars were not evaluated by direct contact with the dentin; however, their main component, acetic acid, has been studied. The apple cider vinegar, acetic acid, and malic acid demonstrated similar microhardness values, and they presented intermediate results.[23] Methodological differences could be responsible for the conflicting data. In our methodological setup, we created smooth dentin surfaces on horizontally located root halves. To achieve this, the root canal space was removed by grinding. Moreover, the measurements were made on more superficial dentin. An inverse correlation has been found previously between the dentin microhardness and the tubular density.[24] As the tubular density decreases from the inner to the outer dentin, the dentin becomes less susceptible to biomechanical stress.

The SEM micrographs of the vinegars showed open dentin tubules and the absence of a smear layer on the dentin surface. Similar to our observations, a previous study demonstrated that contact with vinegar using different application methods removed the smear layer and exposed the dentin tubules.[9] In addition, another study showed that apple cider vinegar has the capacity to remove the smear layer.[25]

The degree of mineral content and the amount of hydroxyapatite in the intertubular substance are considerable factors in determining the intrinsic hardness characteristics of dentin structure.[11] As we stated above, in vinegar groups, SEM micrographs indicated removed smear layer and open dentin tubules. The increased number of widely opened dentine tubules free of peritubular dentin near the pulp showed little resistance to the testing indenter.[26] Therefore, the demineralizing ability of irrigants decreased the microhardness of dentin with time. In addition, as a result of the mineral loss and dissolution at the intertubular area after applying these solutions to the endodontic surfaces, dentine tubules become patent and the surface roughness increases. Confirming the relation between roughness and mineral content, in a recent research, a potential cavity disinfectant-acidic electrolyzed water was shown to increase dentin roughness and decrease the Ca/P ratio, suggesting that decalcification occurred. Another solution-neutral electrolyzed water was shown to cause neither marked dissolution of calcium nor an increase in surface roughness.[27] Accordingly, we can reveal that there is a correlation between surface roughness and Ca/P ratio of dentin. This histological pattern probably contributes to both microhardness reduction and an increase in surface roughness through demineralizing capacity. Moreover, prolonged immersion in the irrigation solutions led to greater softening effect in dentin depending on the acidity of vinegars.

CONCLUSIONS

Within the limitations of this in vitro study, it can be concluded that pomegranate, grape, and apple cider vinegars may have softening effects on the root canal dentin over time, and they may increase the dentin roughness. With these favorable effects, vinegars could be further evaluated in comparison with chelating agents for other endodontic applications.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.