In Situ Effect of Dentifrices Associated to CO 2 Laser in the Permeability of Eroded Root Dentin

Abstract

Objective: The aim of this study was to evaluate, through a crossover 2×2 in situ trial, the effect of a desensitizing dentifrice associated with CO₂ laser irradiation to control the permeability of eroded root dentin. Background data: Facing the increased prevalence of erosive lesion and the need for preventive means to control painful symptoms related to them. Methods: Eighty slabs of bovine root dentin were subjected to initial erosive challenge (citric acid 0.3%, 2 h), followed by a remineralizing period in artificial saliva (24 h). Specimens were then divided according to dentin treatment: desensitizing dentifrice, desensitizing dentifrice+CO₂ laser, fluoride anticavity dentifrice. and fluoride anticavity dentifrice+CO₂ laser. After a 2-day lead-in period, 10 volunteers wore an intraoral palatal appliance containing four root dentin slabs, in two phases of 5 days each. During the intraoral phase, one side of the appliance was immersed in 0.3% citric acid, and the opposite side was immersed in deionized water, four times a day. One hour after the immersions, all specimens were brushed with dentifrice slurry provided by the researcher. After a 7-day washout period, volunteers were crossed over on the different dentifrice group. Each phase having been completed, the specimens were evaluated for permeability through an optical microscope. Results: Data were analyzed using ANOVA and no significant difference (p=0.272) was found between the surface treatments performed on bovine root dentin. Conclusions: It can be concluded that fluoride anticavity or desensitizing dentifrice, regardless of the association with the CO₂ laser irradiation, was able to control the permeability of eroded root dentin.

Introduction

T he increase of noncarious cervical lesions, including dental erosion, has become common in clinical practice. Such lesions are developed by chemical agents, without bacterial involvement, by means of intrinsic factors and/or extrinsic factors and may lead to irreversible loss of tooth structure.¹

Because of the excessive consumption of acidic beverages, the occurrence of these lesions becomes more frequent as the contact of acidic substances removes the smear layer and tubular plugs, increasing dentin permeability,² which may cause hypersensitivity. Different stimuli cause the movement of fluids within the dentinal tubules, causing a painful sensation.³ The obliteration or sealing of tubules can decrease dentin permeability and therefore decrease fluid movement,⁴ thereby reducing hypersensitivity.

The use of desensitizing agents that occlude dentinal tubules aims at reducing the movement of dentinal fluid and creating resistance to acid dissolution by precipitating calcium and protein crystals in the entrance or inside tubules.⁵

In vivo studies have demonstrated considerable reduction of painful hypersensitivity when teeth were brushed with dentifrices that contained desensitizing agents.^6
–8 During brushing, it is likely that dentin surface undergoes changes, forming microprecipitates that make it insoluble, acid resistant, and remineralized.⁹ Another advantage is that the incorporation of desensitizing agents in dentifrices provides action that is more durable during daily use.¹⁰

Another treatment option for reducing hypersensitivity arises with CO₂ laser because of its ability to partially or totally occlude dentinal tubules by decreasing the hydraulic conductance¹¹ and consequently the painful symptoms.^11

–14 In vitro studies with CO₂ laser showed a reduction in the number of open tubules,¹⁵ and in tubule diameter after irradiation.¹⁶ Moritz et al.¹⁷ observed the almost complete occlusion of dentinal tubules in all irradiated teeth.

Considering the ability of desensitizing agents and CO₂ laser irradiation in occluding dentinal tubules, the association of these therapies could present favorable results for tubule sealing with a possible reduction of dentin erosion. Therefore, the aim of the present study was to evaluate in situ the effect of associating desensitizing dentifrice with CO₂ laser to control the permeability of eroded root dentin.

Methods

Experimental design

This crossover double-blind in situ study, was conducted after a 2-day lead-in period, in two phases of 5 days each, with a 7-day washout period between them. The factors under study were dentin treatment at four levels (desensitizing dentifrice, desensitizing dentifrice+CO₂ laser, fluoride anticavity dentifrice, and fluoride anticavity dentifrice+CO₂ laser) and condition of the substrate at two levels (eroded and uneroded [control]). The experimental sample was composed of 80 fragments of bovine root dentin, randomly assigned to 10 volunteers, and each volunteer was considered as a statistical block (n=10). The response variable was obtained through the analysis of permeability, in percentage.

Ethical aspects and selection criteria

This study was approved by the Ethics Committee of the School of Dentistry of Ribeirão Preto (Process n° 2010.1.217.58.3). After the information was received, the volunteers signed an informed consent, agreeing to participate and cooperate with the experiment. For the study, we selected 10 volunteers of both sexes (4 males and 6 females) between 22 and 47 years of age with normal salivary flow, no evidence of active cavity and noncarious lesions, and with salivary buffer pH between 6.5 and 7.0. Volunteers who had dentures or braces, who presented with systemic diseases and digestive disorders, who were smokers or pregnant, or who were undergoing chemotherapy or radiation therapy were all excluded.

Root dentin sample preparation

Bovine incisors were scraped with a curette to remove the periodontal tissue and stored in 0.1% thymol solution at 4°C.¹⁸ These were cleaned with the aid of pumice and water, with Robinson Brushes mounted on a low speed turbine and examined by stereomicroscope (Leica S6 D Stereozoom, Mycrosystems Leica AG, Switzerland) with an increase of 40×, discarding those with cracks or structural anomalies.

Dentin fragments were obtained from the cervical third of the root, with dimensions of 3×3×2 mm. Specimens were coated with an acid-resistant nail varnish (Colorama, SP, Brazil) in two layers, except on the buccal surface (9 mm²), which were subjected to the erosive challenges.

Slabs were sterilized with ethylene oxide¹⁹ and carefully inspected for surface defects using the stereomicroscope, with 80 pieces selected.

Initial erosive challenge

Each fragment was individually immersed in 50 mL of 0.3% citric acid (pH=3.2) and placed in an orbital shaker (CT155, Cientec, Piracicaba, SP, Brazil), with a stirring velocity of 50 rpm for 2 h²⁰ to form erosive lesions. Afterwards, specimens were rinsed for 10 sec with deionized water and stored in 10 mL of artificial saliva at 37°C during 24 h.

The artificial saliva was similar to that described by McKnight-Hanes and Whitford²¹ and modified by Amaechi et al.²²

Dentin treatment

After the initial erosive challenges, specimens were then divided according to dentin treatment (desensitizing dentifrice, desensitizing dentifrice+CO₂ laser, fluoride anticavity dentifrice, and fluoride anticavity dentifrice+CO₂ laser) and subdivided according to the condition of the substrate (eroded and uneroded [control]). The dentifrices used in treatment of dentin and their composition are described in Table 1.

Table 1.

Characterization of the Dentifrices

Dentifrices	Composition	Manufacturer
Colgate® Sensitive Pro-Alívio (desensitizing dentifrice)	Arginine 8%, sodium monofluorophosphate 1.10% (1450 ppm fluor), calcium carbonate, water, sorbitol, arginine bicarbonate, sodium lauryl sulfate, sodium monofluorophosphate, aroma, cellulose gum, sodium bicarbonate, potassium acesulfame, sodium silicate, xanthan gum, sucralose, and titanium dioxide.	Colgate-Palmolive, Osasco, SP, Brazil
Colgate^® Máxima Proteção Anticáries (fluoride anticavity dentifrice)	Sodium monofluorophosphate (1500ppm fluor), calcium carbonate, sodium lauryl sulfate, sodium saccharin, tetrasodium pyrophosphate, sodium silicate, polyethylene glycol, sorbitol, carboxymethyl cellulose, methylparaben, propylparaben, aromatic composition, and water.	Colgate-Palmolive, Osasco, SP, Brazil

Forty pieces of root dentin were irradiated with a CO₂ laser. The parameter settings used were: λ=10.6 μm, 0.5 W input power, 0.44 W output power, 0.04 J/cm² energy density. The beam spot size was 0.8 mm². The average output power was measured by means of a measuring instrument of electrical power (Fied-Top Max II laser power/energy meter, Coherent Radiation, Palo Alto, CA). The model used for irradiation was the PC015-A (Shanghai Jue Hua Laser Tech. Development Co., Ltd.), at a distance of 4 mm²³ from the dentin surface, in continuous mode (defocused). The entire surface was scanned by the laser with the aid of a device that sets the pen during the laser irradiation, which moves the second commands previously established through a computer connected to the scanning machine (MPC ElQuip, San Carlos, California, Brazil), allowing the radiation to reach the entire area evenly. In regard to beam diameter and irradiated area, the total irradiation time was ∼10 sec per sample.

Preparation and installation of removable intraoral appliance and intraoral phase

For each volunteer, a removable intraoral appliance was constructed in acrylic resin, with two sites (15×5×3 mm, one in each side of the midline).

The fragments were fixed with wax. The positioning was performed randomly to either side of intraoral appliance.

A 2-day lead-in period was allowed prior to the beginning of the intraoral phase. Volunteers were instructed to brush their teeth exclusively with the toothbrush and dentifrice supplied. All volunteers used a fluoridated dentifrice (Colgate^® Máxima Proteção Anticáries, Colgate-Palmolive, Osasco, São Paulo, Brazil) and the toothbrush (Oral-B Indicator 35, Gillette do Brasil Ltda., Manaus, Amazonas, Brazil), provided by the researchers.

Erosive challenges started 24 h after installation of the devices.

After this time, one side of the device, determined by lot, was immersed in 50 mL of 0.3% citric acid pH 3.2, four times a day (8, 12, 16, 20 h) for 90 sec, for 5 days. We used a screen made of EVA (Cortiarte, Diadema, SP, Brazil), which was adapted in the middle region of the palatal appliance, in order to prevent the other side from coming in contact with citric acid. The fragments of the contralateral side of the device were immersed in 50 mL of deionized water, using the same immersion protocol.

At the end of the first phase, specimens were removed from the devices and a 7-day washout period was implemented, during which volunteers did not wear the intraoral device but brushed their teeth with the same toothbrush and dentifrice supplied.

After the washout period, dentin slabs from the groups not tested in the first phase were placed into the palatal appliance and volunteers started the second phase, where the volunteers were crossed on the dentifrice groups.

In both experimental phases, the use of the devices was continuous, including during the night,²⁴ except during meals (four times daily, 1 h per meal), beverage consumption, and oral hygiene procedures.^25
–27 In order to avoid dehydration of specimens during these periods, the appliance was covered with humid cotton gauze.

The control of the biofilm on the surface of the specimens was performed at the beginning and end of each experimental day, dripping chlorhexidine 0.2% on the fragments, for 1 min, and rinsing them with tap water, according to the protocol described by West et al.²⁸

Application of dentifrice

Each specimen was brushed for 15 sec extraorally²⁶ with the aid of an electric toothbrush (Oral B Cross Action Power, Gillette do Brazil Ltda. Manaus, Amazonas, Brazil) and slurry (3 g dentifrice/10 mL water).²⁹ This procedure was performed four times a day, 1 h after the erosive challenges. Volunteers were trained and instructed to perform this procedure. The specimens were rinsed after brushing for 20 sec under running water, and the palatal appliance was reinserted in the mouth.

Histochemical staining method

After completion of the intraoral phase, dentin specimens were individually immersed in 1 mL of a 10% aqueous solution of copper sulfate for 30 min. Then, each specimen was dried on absorbent paper and immersed in 1 mL of a 1% alcoholic solution of rubeanic acid for 30 min. Copper ions were revealed by the rubeanic acid, resulting in a specific coloration that ranged from dark blue to black, depending upon the amount of copper ion penetration. After being stained, specimens were rinsed with distilled water for 15 sec, dried, and kept individually in a sealed container with a piece of cotton that had been moistened in ammonia for 7 days. A similar histochemical staining protocol was described by Carrasco et al.³⁰ and validated for erosion lesions by Turssi et al.³¹

Permeability assessment

The specimens were sectioned transversely using a water-cooled diamond saw (Isomet 1000; Buehler, Lake Bluff, IL). We obtained three sections from each sample with an average thickness of 400 μm. These were ground on 600- and 1200-grit Al₂O₃ papers up to the thickness of 200 μm. The images for permeability analysis were obtained with a digital camera (AxioCam MRC, Carl Zeiss, Jena, Germany) coupled to the optical microscope (AxiostarPlus, Carl Zeiss, Jena, Germany). The evaluation of the permeability was performed using software (Axiovision 4.8, Carl Zeiss, Germany) for capture and image analysis. In each of three sections obtained, we performed five evaluations of penetration of the tracer solution. The average of these five values represented the permeability for each section. For each specimen, we obtained 15 values of relative permeability, calculated based on the extent of copper ion penetration and the thickness of dentin. Permeability=[(extent of copper ion penetration)/(thickness of dentin) ×100].

Statistical analysis

The mean values of permeability of each specimen were analyzed, and presented normal distribution and homogeneity of variance. Therefore, analysis of variance (ANOVA) was performed using SPSS 12.0 for Windows (SPSS Inc., Chicago, IL) with a significance level of 5%.

Results

The results of permeability showed that the surface treatments performed in bovine root dentin did not differ statistically significantly among groups (p=0.272). Table 2 shows permeability of eroded root dentin after different treatments.

Table 2.

Mean (Standard Deviation) of Percentage of the Permeability in Dentin After Erosive Challenges for the Different Experimental Groups

	Laser
	Irradiated		Nonirradiated
	Anticavity dentifrice with fluoride	Desensitizing dentifrice	Anticavity dentifrice with fluoride	Desensitizing dentifrice
Eroded	2.87 (0.35)	2.82 (0.40)	2.76 (0.45)	2.89 (0.32)
Uneroded	2.66 (0.43)	2.74 (0.27)	2.42 (0.39)	2.56 (0.65)

Discussion

The need for methods that control the dentin hypersensitivity stimulated research that evaluated the effect of dentifrices^6,8,10,32,33, as well as the use of CO₂ laser^11

–14 in reducing painful symptoms. However, the association of these treatments with the control of permeability in eroded root dentin has not been observed, and according to Pashley,³⁴ an effective treatment for hypersensitivity is the occlusion of tubules reducing fluid movement and dentin permeability.

In this study, regardless of CO₂ laser association, both dentifrices, fluoride anticavity and desensitizing, presented similar mean values of percentage of dye penetration, which indicates that the desensitizing dentifrice does not show any additional effect on reducing the eroded dentin permeability.

Desensitizing dentifrices that contain in their composition arginine and calcium carbonate together, accelerate mechanisms of occlusion and help form a layer rich in calcium and phosphate on the surface and inside the dentinal tubules, thereby sealing them.³⁵ The dentin surface has a negative charge that attracts arginine, facilitating its adhesion to the calcium carbonate from the surface and also in the tubules. In an in situ environment, this association stimulates endogenous calcium and phosphate ions to deposit and occlude the dentinal tubules even more.³⁶

Regarding the fluoride anticavity dentifrices, it is likely that the presence of fluoride in their composition has created a barrier by precipitation of CaF₂ on the surface of eroded dentin,³⁷ reducing the dentin permeability, which is in agreement with Ponduri et al.³⁸ and Magalhães et al.²⁶ who found that the presence of fluoride on dentifrices reduced dentin wear, providing protection, even if small, against erosion.

Analyzing the composition of both desensitizing and fluoride anticavity dentifrices, it can be observed that both present calcium carbonate as abrasive, and that these abrasives can often adhere to dentin surface and/or bind to tubules, producing a protective layer.³⁸ The abrasiveness of dentifrices could create, by brushing, a thin coating of smear layer able to occlude the tubules.^9,39

Some studies have used CO₂ laser to treat dentin hypersensitivity,^11

–14 in order to promote occlusion or narrowing of dentinal tubules.^15
–17,40 Irradiating dentin specimens with CO₂ laser, Gholami et al.¹⁶ observed a reduction of 42.3% in the average tubular diameter through the fusion of peritubular dentin because of the high absorption of laser by hydroxyapatite, melting and causing dentin recrystallization. Cakar et al.¹⁵ also observed a reduction in the number of opened dentinal tubules after CO₂ laser irradiation, and this result appears to be the result of the formation of a new structure caused by the melting of a dentinal substance that obstructed tubules.

In this study, CO₂ laser was irradiated with 0.5 W power in continuous mode, as Moritz et al.,¹⁴ using the same parameters, showed that CO₂ laser irradiation combined with fluoride gel reduced hypersensitivity in 96.5% of cases. These parameters can be safely applied, because even with the heat generated in the surrounding regions, this increase in temperature proved to be <5°C.^17,40 He et al.⁴¹ observed through systematic literature review, that in the treatment of dentin hypersensitivity, laser application does not lead to adverse effects if parameters are controlled.

Erosive challenges were performed with acidic substances able to remove the smear layer and open dentinal tubules,⁴² simulating situations in which dentinal hypersensitivity occurs. These challenges were performed ex vivo to avoid damage to the volunteers´ teeth, using citric acid because it is one of the agents used for exposure of dentinal tubules^42,43 and also because it is present in fruits, vegetables, and ready-to-drink juices.⁴⁴ The concentration of citric acid used in this study (0.3%, pH 3.2) was seen cause erosive dissolution of dentin.⁴⁵ Specimens were subjected to four challenges per day, to imitate a clinical condition of consumption of erosive drinks.⁴⁶ Applying repeated erosive challenges, they also increased the chances of obtaining reliable results, as changes can be detected after multiple exposures.⁴⁷

It is known that in situ models allow the formation of acquired pellicle through saliva, which is considered as a protective factor against dental erosion,⁴⁸ making erosive losses smaller in relation to in vitro studies.²⁹ Its protection effect against surface demineralization was demonstrated by Amaechi et al.,⁴⁹ who observed an inverse relationship between the level of erosion and saliva film thickness. Therefore, it can be considered that saliva was able to dilute erosive agents, neutralizing acid challenges, reducing demineralization, enhancing remineralization, and minimizing surface wear by brushing.⁵⁰

Considering the low values found for permeability, CO₂ laser irradiation associated with desensitizing dentifrices may be an alternative for controlling dentin erosion. However, the possibility of synergism between the different treatments was not observed. There is an absence of studies in the literature that evaluated the use CO₂ laser associated with desensitizing dentifrices in controlling the permeability of eroded dentin. Although treatment with CO₂ laser irradiation combined with NaF appears to induce a more compact surface layer when observed by scanning electron microscopy,¹⁵ clinically evaluating the association of these two therapies did not increase the effectiveness of treatment.¹¹

Conclusions

Based on the results of this study, it can be concluded that fluoride anticavity or desensitizing dentifrice, regardless of association with the CO₂ laser irradiation, was able to control the permeability of eroded root dentin.

Footnotes

Acknowledgments

The authors thank the State of São Paulo Research Foundation (FAPESP) for the scholarship awarded (Process n^o 2010/03180-1). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The authors also thank Abilio Borghi for the English language review.

Author Disclosure Statement

No conflicting financial interests exist.

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