Indian Journal of Dental Research

: 2019  |  Volume : 30  |  Issue : 3  |  Page : 414--419

Diffusion of hydroxyl ion on external root surface using different irrigating solutions: An In vitro study

Monique Costa Moreira França, Tânia Mara da Silva, Renata Camila Queiroz, Cláudia Villela Bin, Flávia Goulart Da Rosa Cardoso, Márcia Carneiro Valera 
 Department of Restorative Dentistry, Institute of Science and Technology, São Paulo State University, SP, Brazil

Correspondence Address:
Dr. Tânia Mara da Silva
Avenida Engenheiro Francisco José Longo, 777 - Jardim São Dimas - 12245-000 - São José Dos Campos, SP


Aims: The aim of this study was to evaluate the diffusion of hydroxyl ion to the external root surface using different irrigating solutions and intracanal medication with calcium hydroxide. Materials and Methods: Sixty bovine tooth roots were randomly divided into six experimental groups (n = 10), according to the irrigating substance used during biomechanical preparation: 12% glycolic propolis extract (PROP); 20% glycolic ginger extract (GENG); 2% sodium hypochlorite with surfactant (NaOClS); 2% chlorhexidine gel (CLX); 2.5% sodium hypochlorite (NaOCl); and physiological saline solution. After filling the root canals with calcium hydroxide paste, pH measurements were taken directly at the external cavities over time intervals of up to 30 days. Statistical Analysis Used: Data were submitted to two-way ANOVA and Tukey's test (P < 0.05). Results: The pH of the external root surface was increased when the surfactant associated with NaOCl was used. However, the pH values were very close for the different groups. Hydroxyl ion diffusion up to the external root surface did not exceed the pH value of 8.5, and in the hollow passage of the canal, the pH was higher than 12. Conclusions: Hydroxyl ion diffusion of calcium hydroxide paste through the dentinal tubules up to the external root surface allows minimal alkalinization of this surface, and it is greater when using NaOCl with surfactant during biomechanical preparation.

How to cite this article:
Moreira França MC, da Silva TM, Queiroz RC, Bin CV, Da Rosa Cardoso FG, Valera MC. Diffusion of hydroxyl ion on external root surface using different irrigating solutions: An In vitro study.Indian J Dent Res 2019;30:414-419

How to cite this URL:
Moreira França MC, da Silva TM, Queiroz RC, Bin CV, Da Rosa Cardoso FG, Valera MC. Diffusion of hydroxyl ion on external root surface using different irrigating solutions: An In vitro study. Indian J Dent Res [serial online] 2019 [cited 2022 Dec 8 ];30:414-419
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Intracanal medication is used to complement disinfection of the root canal system in cases of pulp necrosis, dental trauma and apexification of permanent teeth.[1] The mechanism of action of calcium hydroxide depends on the ionic release of this substance with consequent increase in pH.[2],[3]

However, the root canal walls form a complex medium containing organic and inorganic compounds that may exert a buffer effect against alkalis, thus having their action limited in clinical use. Moreover, the ionic diffusion of calcium hydroxide may be lower as a result of the number and diameter of dentinal tubules, especially in the apical third of the root.[4],[5]

The microorganisms present in infected root canals may be significantly reduced by biomechanical preparation associated with chemical substances with antimicrobial action.[6] Sodium hypochlorite at different concentrations is the most used and accepted irrigant substance, due to its tissue solvent capacity, powerful antimicrobial action,[6] low surface tension, detergent action,[7] high alkalinity, and germicidal properties. Association of a surfactant with the sodium hypochlorite solution increases the wettability of this solution due to the reduction in surface tension and may favor the penetration of the intracanal medication to be used. The previous study presented that NaOCl with surfactant was the most effective irrigant against endotoxins and microorganisms.[8] However, some authors did not observe any differences in penetration of NaOCl with and without surfactants into dentin tubules.[9]

Chlorhexidine at 2% is effective against Gram-positive and Gram-negative bacteria and yeasts, in addition to being biocompatible and presenting residual antimicrobial activity.[4],[5] It also has the capacity of adsorption by dentin and slow release of active substances, thus prolonging its residual activity (substantivity).

At present, natural extracts have been evaluated for possible use as irrigating substances.[10],[11],[12] Glycolic propolis extract used as an intracanal irrigant has shown antimicrobial activity and as intracanal medication has revealed effectiveness against microorganisms commonly found in root canals such as Enterococcus faecalis and Candida albicans.[11],[12]

Zingiber officinale (ginger) due to its healing, anti-inflammatory, and antimicrobial action has been used in association with other natural substances such as honey, propolis, or pomegranate in solutions or sprays used in the oral cavity, and these are indicated for tonsillitis, coughs, and halitosis.[8],[10],[12] In some previous in vitro studies, the effectiveness of glycolic ginger extract used as intracanal medication was shown against E. faecalis, C. albicans, and Escherichia coli.[12]

In spite of natural extracts having been shown to have effective antimicrobial action and against endotoxins, the physical-chemical properties of these products are still not known, especially with regard to the interference of these chemical substances in the diffusion of intracanal medication. However, there are no studies that evaluate the action of irrigating solution in the hydroxyl ion diffusion of calcium hydroxide paste directly onto the external root surface.

Thus, the aim of this study was to evaluate the diffusion of hydroxyl ion attained onto the external root surface in the apical and middle thirds when the canals were prepared with different irrigating solutions and intracanal medication with calcium hydroxide. The hypotheses tested were (i) there is no significant difference in hydroxyl ion diffusion between root thirds and (ii) there is no significant difference in hydroxyl ion diffusion among irrigating solutions.

 Materials and Methods

Sample preparation

This study was approved by the Local Institutional Review Board (Protocols numbers 012/2010-PA/CEP and 013/2010-PA/CEP). Sixty bovine lateral incisors were selected, cleaned, immersed in physiological saline solution (SS), and frozen (−4°C) until the time of use.[13] Roots with incomplete apex or very wide foramen (over Kerr-type file No. 40) was excluded from the study. Teeth crowns were sectioned with a carborundum disc to standardize root length at 20 ± 0.5 mm. Remnants of pulp tissue were removed with endodontic file (70 LK; Maillefer Instruments SA-CH 1338, Ballaigues, Switzerland).

First, the teeth were submitted to digital radiographs to measure the dentinal wall thickness using software program (Image Tool ® for Windows 3.0, Texas, USA).[14] Next, root canal preparation was performed using Gates Glidden burs (n. 3-4 Maillefer Instruments SA-CH 1338, Ballaigues, Switzerland) up to 15 mm of the length of the canal. The apical third was prepared from the anatomic diameter of the canal up to 40 LK. Then, the teeth were radiographed again to verify standardization of root dentin thickness at 1.0 mm ± 0.2 in the apical third (5 mm from the apex) and 1.5 mm ± 0.2 in the middle third and to ensure that all roots had similar thickness between the canal preparation and the root canal wall.[15]

The sixty roots were randomly divided into six experimental groups (n = 10), according to the irrigating solutions used during biomechanical preparation [Table 1]. In the PROP, GENG, and CLX groups,[10],[12],[16] root canals were filled with the respective irrigating solutions during instrumentation and irrigated 10 mL of physiological saline solution (Equiplex, GO, Brazil) at each change of file. In the NaOClS, NaOCl, and SS groups,[8] root canals were irrigated with 10 mL of the respective irrigating solution, at every change of file.{Table 1}

Throughout the biomechanical preparation, canal was irrigated with the irrigating substance according to the group it belongs. The canal was filled with calcium hydroxide paste (Biodinâmica, Ibiporã, PR, Brazil) associated with physiological SS, in a ratio of 1:1 by volume.[15],[17] The cervical opening and apical foramen were sealed with light-cured composite resin (Z350, 3M ESPE, St Paul, MN, USA) and the roots were stored in an incubator at 37°C, 100% relative humidity in SS for 30 days.[18]

After canal biomechanical preparation, two “windows” were made in the mesial external root surface: 1) in the apical third at 5 mm from the root apex and 2) in the middle third at 12 mm from the root apex. To prepare these cavities, a wheel-type bur was used (KG Sorensen, São Paulo, Brazil) 2 mm in diameter and 0.5 mm depth. The “windows” were filled with EDTA (Asfer Indústria Química Ltda, São Caetano do Sul, SP, Brazil) for 3 min, to remove residues and any smear layer that could have possibly formed following which the windows were rinsed with physiological saline solution (Equiplex, GO, Brazil).

The external root surfaces were coated with two layers of nail varnish, except for the windows [Figure 1], to waterproof the entire surface and not interfere in the diffusion of the hydroxyl ions by root dentin tubules.{Figure 1}

pH measurement

To measure the pH, a microelectrode (Thermo-Electron Corporation, Orion Products, Beverly, USA) coupled to a potentiometer was used. The microelectrode was calibrated using known solutions with pH 4.0, pH 7.0, and pH 9.0. In each measurement, the microelectrode was washed and cleaned with distilled water. pH measurements were taken directly at the “windows” by the microelectrode at time intervals of 0 h (immediately), 12 h, 24 h, 48 h, 7 days, 15 days, and 30 days after filling the canals with hydroxide paste.[17],[18] During the entire period, the windows were filled with deionized water. The pH value obtained after 30 days was submitted to statistical analysis.

Statistical analysis

The response variable was the value of the area of the pH curve versus time (up to 30 days), obtained with the use of the software program MedCalc (version 11.2.0, Acacialaan, Ostend, Belgium).

Data were submitted to statistical analysis using the computer software Statistica for Windows (version 5.5, Statsoft, Tulsa, OK, USA) and Minitab for Windows (version 16.1, College State, PA, USA). The inferential statistics consisted of two-way ANOVA (irrigating solutions and root third) followed by Tukey's test. The level of significance level was set at 5%.


Analyses of the pH measurement were performed using the area of the pH curve; the overall pH mean values are shown in [Figure 2]. [Figure 3] demonstrates the descriptive analysis of the mean and standard deviation of the area values (pH vs. time) according to the irrigating solution and taking into consideration the apical and middle thirds. It can be observed that roots treated with NaOClS and GENG obtained greater mean values of pH. Furthermore, there were no differences between the root thirds.{Figure 2}{Figure 3}

ANOVA showed statically significant difference for irrigating solutions (F = 7.32; P < 0.0001) on the area values of pH curve. In relation to root third, there were no significant differences between the apical and middle thirds [Table 2]. Regarding the irrigating solutions, as shown in [Table 3], NaOClS group resulted in higher pH values, differing significantly from the CLX and PROP groups. GENG presented an intermediate behavior. Considering only middle third, the SS, NaOCl, and GENG did not differ significantly from the NaOClS group.{Table 2}{Table 3}


Studies have shown that the penetration and therapeutic effect of chemical substances used are directly related to the number and diameter of dentinal tubules.[19],[20] Therefore, detailed knowledge of these structures is essential for us to understand the process of dentin tissue permeability, and consequently, the efficacy of intracanal medications, and filling cements used in endodontics.

A large variety of techniques have been used to study the anatomy and structural details of dentinal tubules including histochemistry, immunofluorescent microscopy, light microscopy, and scanning electron microscopy.[21] Moreover, studies have been conducted to measure the number and diameter of dentinal tubules. However, researches that evaluate tubular distribution according to root thirds are still scarce in the literature.[22] Furthermore, the ethical aspect of using human teeth in in vitro studies are difficult, since the use favors the extraction and sale of dental organs, thereby violating law 9434 of 1997/02/04, which prohibits this type of procedure.[23] Thus, the use of bovine teeth has become an alternative to resolve this problem as these are easily obtained and present a reduced caries index, making it possible to use them in innumerable investigations in recent times.[19],[20] In addition, they are easy to handle, due to the same genetic lineage and similar ages, grow in the same oral conditions with same diet, presenting greater homogeneity in the mineral composition, unlike human teeth, which come from different donors with age and intake food variations.[24] Furthermore, some studies have demonstrated that there is little or no difference between human and bovine teeth, both at macroscopic and microscopic levels.[19] Anido-Anido et al. concluded that there was a similarity in behavior between human and bovine teeth, according to depth of dentin.[25]

Camargo et al.[14] evaluated the number and diameter of dentinal tubules in root canals in the cervical, middle, and apical thirds of human and bovine teeth. The authors concluded that the cervical root thirds presented the highest mean values for both number and diameter of dentinal tubules, followed by the middle and apical thirds. The bovine specimens presented a significantly higher mean value, in comparison with human specimens regarding the number of dentinal tubules; however, no statistically significant difference was found with regard to the dentinal tubule diameters studied in the two samples. Therefore, the data obtained in the present study will be extremely useful for in vitro studies intended to simulate in vivo conditions.

In addition, the pH value attained on the external root surface depends directly on dentin permeability. However, this variable is absolutely related to the distribution and diameter of the dentinal tubules present in the different root thirds. An evaluation of dentin morphology in root canals as regards the density and orientation of dentinal tubules revealed a high density of these in the cervical third in comparison with the middle and apical thirds.[26] As the number of tubules is reduced in the crown in the direction toward the root apex, the permeability of calcium hydroxide, and consequently, the pH value attained on the external root surface may vary among the different root thirds of one and the same tooth.[14] Nevertheless, in the present study, the differences in pH values were not verified when the two regions evaluated were compared. Root third did not show statistically significant difference, as observed in [Table 2]. This finding suggests that the nature of dentin in the root canal wall or the tubular density might not significantly influence the permeability of the apical and middle thirds. This could also be due to the similarity between the number and diameter of the dentinal tubules present in the two analyzed thirds. Then, the first hypothesis of this study was accepted.

Calcium hydroxide has been used as intracanal medication in endodontics due to its high pH (12.5–12.8); antimicrobial action, tissue solvent and inducer of mineralization.[17] However, to enable calcium hydroxide to act throughout the root canal system in cases of infection and at the external root surface, this medication must be dissociated into calcium and hydroxyl ions, which in turn need to diffuse into the inside of dentinal tubules until they attain the external root surface.[2],[27]

It is known that in order for mineralized tissue resorption to occur, it is necessary for the local pH to be acidic. At an acidic pH, the acid hydrolases are active and demineralization of the mineral component of the tooth surface occurs. When calcium hydroxide is used inside the root canals, it diffuses up to the external root surface, and the acid products such as the lactic acid of the osteoclasts may be neutralized, thereby preventing the dissolution of the mineral component.[2]

In a similar manner, the optimum activity of the acid hydrolysis produced by the osteoclast occurs at a pH that ranges from 5.0 to 5.5. When calcium hydroxide is used, its activity is inhibited.[2] Therefore, calcium hydroxide interrupts this process of resorption and creates a favorable environment for hard-tissue formation.[2],[18] The hydroxyl ions derived from calcium hydroxide have the capacity to destroy the cell membranes of bacteria and their protein structure, thereby destroying the bacteria present inside the root canals.

However, the bacteria may penetrate into the interior of dentinal tubules to a depth of arpimd 3.75 mm.[28] Therefore, when the canal is filled with calcium hydroxide, it is possible that the interior of the root canal may present a highly alkaline pH, but in the interior of the tubules up to the external root surface, this pH may not be so alkaline. In the present study, it was verified that the pH on the external surface did not reach high levels of alkalinity.

Previous studies [2] verified that when calcium hydroxide is placed in the canal, this does not diffuse into the interior of the dentinal tubules and does not reach the external root surface. However, when another study [29] verified the diffusion of calcium hydroxide in the interior of the canal up to the external root surface, they considered that the dentin presents a buffer capacity, making it difficult for hydroxyl ions to diffuse. When there is external resorption, or when there is infection, even with the placement of calcium hydroxide medication, the pH at the external root surface may not reach significant levels of alkalinity to inhibit resorption, and the bacteria present in the dentinal tubules may not be eliminated, thereby contributing to the failure of endodontic therapy.

In the present study, it was verified that the external pH underwent little variation, when compared with the pH at the immediate time [Figure 2], confirming the difficulties in diffusing through dentin, presented by the hydroxyl ions.

It should be emphasized that to enable the ionic diffusion of calcium hydroxide to occur, the dentinal walls and the dentinal tubules must be open to allow contact with the tubules. In the present study, the pH values were higher in the NaOClS group [Table 3]. This may be due to the presence of the surfactant, which promotes greater wettability of the root walls, thereby reducing the surface tension of these walls and thus favoring the diffusion of hydroxyl ions. The surfactant is a detergent that possesses emulsifying properties. These properties facilitate the removal of debris from the dentin surface, which increases dentin wettability and facilitates instrumentation.[8] The wettability of the irrigant plays a major role in obtaining a suitable contact time between irrigant and root canal dentinal walls. In the current study, NaOCl with surfactant resulted in greater pH values, differing significantly from the CLX and PROP groups, as similar to a previous study [8] that obtained lower bacterial growth and greater endotoxin reduction using NaOCl with surfactant in comparison with CLX.

GENG presented an intermediate behavior, with no statistical differences among the groups. The glycolic ginger extract (GENG) showed similar pH values to NaOClS although they have different composition. This result suggests that GENG acts in dentinal tubules, but the real mechanism of the action inside the tubules is still unknown in the literature. It also verified that in the CLX group, the pH was slightly acidic at time intervals of 24 h and 48 h and after 30 days [Figure 2]. This may have occurred due to the gel present in this substance, which may make ionic diffusion difficult. Then, NaOClS and GENG substances differed from the conventional irrigating solutions (NaOCl, CLX, or SS). Hence, the second hypothesis was rejected.

However, the pH values were very close for the different groups, and these values were very low considering that in the hollow passage of the canal, the pH remained above 12, and at the external root surface, it did not exceed the value of 8.5; therefore, the difficulty of the diffusion of this paste through the dentinal tubules is evident. The vehicle for the calcium hydroxide paste used is physiological saline solution, which is a hydrosoluble vehicle, therefore allows rapid ionic release and rapid diffusion through the dentinal tubules. These ions would reach up to the external root surface and alkalinize it.[3],[15]

Within the limitations of this study, the irrigating solution may favor the ionic diffusion of calcium hydroxide; however, its action at the external root surface is limited. Thus, more investigations of mechanism of each irrigating solution through the dentinal tubules would be necessary to elucidate the pH changes during endodontic treatment.


Based on the methodology used, it can be concluded that (i) hydroxyl ion diffusion through the dentinal tubules up to the external root surface allows minimal alkalization of surface by pH measurement with no difference for root third and (ii) NaOCl with surfactant allowed higher pH values at the external root surface among irrigating solutions, regardless the root third.


The authors would like to thank Prof. Dr. Livia Tenuta for her assistance during pH analysis.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.


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