Influence of toothbrushing on enamel softening and abrasive wear of eroded bovine enamel : an in situ study

This study assessed the surface softening and abrasive wear of eroded bovine enamel with or without the influence of toothbrushing. Five volunteers took part in this in situ study of 5 days. They wore acrylic palatal appliances containing 6 bovine enamel blocks divided in two rows with 3 blocks, which corresponded to the studied groups: erosion without toothbrushing (GI) and erosion with toothbrushing (GII). The blocks were subjected to erosion by immersion of the appliances in a cola drink for 10 minutes, 4 times a day. After that, no treatment was performed in one row (GI), whereas the other row was brushed (GII). The appliance was then replaced into the mouth. Enamel alterations were determined using profilometry and microhardness tests. Data were tested using paired Students t test (p < 0.05). The mean wear values (microm) and percentage of superficial microhardness change (%SMHC) were respectively: GI--2.77 +/- 1.21/91.61 +/- 3.68 and GII--3.80 +/- 0.91/58.77 +/- 11.47. There was a significant difference in wear (p = 0.001) and %SMHC (p = 0.001) between the groups. It was concluded that the wear was more pronounced when associated to toothbrushing abrasion. However, toothbrushing promoted less %SMHC due to the removal of the altered superficial enamel layer.


INTRODUCTION
While the incidence of the major dental disease, caries, has declined in developed countries 27 , other dental lesions such as erosion are becoming increasingly important 15 .Dental erosion is defined as the loss of tooth substance by chemical processes not involving bacteria 20 caused by a variety of extrinsic and intrinsic factors 9,25 .
Intrinsic factors are the result of endogenous acids, generally gastric acids that become in contact with dental surface during nervous anorexia, nervous bulimia and gastroesophagic disturbances 28,29 .Extrinsic factors are related to frequent consumption of acidic foodstuffs or beverages and exposure to acidic contaminants in the working environment 22 .In modern societies the extrinsic factors are becoming more important, due to the increased consumption of acidic drinks as soft drinks, sport drinks, fruit juices and fruit teas 22 .
The acidic attack leads to irreversible loss of dental hard tissue accompanied by a demineralization and softening of the surface 2,9,17,25 .This softened zone, particularly of enamel, appears to be more susceptible to removal by mechanical forces, like attrition and abrasion, which have little or no effect on the intact tissues 1,4,5,8,18 .Toothbrushing is a very important procedure to prevent dental caries.On the other hand it represents a mechanical force that can led to abrasion.Thus, the analysis of alterations that can be promoted by toothbrushing of previously eroded enamel is important and necessary.
The methods used for measurement of the superficial alterations of dental enamel can be quantitative (superficial microhardness, profilometry) and qualitative (scanning electron microscopy) 14 .Microhardness testing permits the measurement of the degree of softening of the surface, but does not allow the quantification of the height of surface loss.The profilometric method uses a depth-sensing instrument, which gives the height of this surface loss.Since the dental enamel can be softened (altering the microhardness) in association with dental wear, a combined study, using microhardness and profilometry would be able to quantify the alterations resulted from erosion and abrasion 6,14 .
Taking into account the association between erosion and abrasion and the importance of combined methodologies, the purpose of this study was to assess in situ the surface softening and abrasive wear of eroded bovine enamel with or without the influence of toothbrushing, by using profilometric and microhardness analyses.

Experimental design
This in situ study was approved by the Research and Ethics Committee, School of Dentistry of Bauru, University of São Paulo.Five healthy adult volunteers with a mean age of 23 yr (range 19-28 yr) (Figure 1I), with normal salivary flow (1 ml/min, pH 7.0), and residing in a fluoridated area (0.70 mgF/L) took part in this study, during 5 days 11 .They wore acrylic palatal appliances, which contained two rows of 3 bovine dental enamel blocks each.The rows corresponded to one of these conditions: erosion without toothbrushing (GI) and erosion with toothbrushing (GII).The use of two conditions in the same intraoral palatal appliance was supported by the absence of a cross-effect in previous studies 5,15,21 .Enamel mineral loss was determined by profilometric and surface microhardness tests.Each group comprised 5 experimental units (volunteers), which were compared using paired Student's t test.

Enamel blocks and palatal appliance preparation
Sixty enamel blocks (4 x 4 x 3 mm) were prepared from bovine incisors sterilized by storage in a 2% formaldehyde solution (pH 7.0) for 30 days at room temperature 11 .Using one diamond disk (Isomet 1000; Buehler, Lake Bluff, IL, USA) the crowns were sectioned from the roots (Figure 1A).Next, using two parallel diamond disks separated by a 4-mm spacer, one slice was cut from the crown of each bovine incisor (Figure 1B).The enamel surface of the blocks (Figure 1C) was ground flat with water-cooled carborundum discs (320, 600 and 1,200 grades of Al 2 O 3 papers; Buehler, Lake Bluff, IL, USA), and polished with felt paper wet by diamond spray (1 µm; Buehler) (Figures 1D and 1E) for surface microhardness determination (five indentations in different regions of the blocks, 25 g, 5 s, HMV-2000; Shimadzu Corporation, Tokyo, Japan) (Figure 1F).
Thirty blocks, with a mean (SD) surface microhardness of 353 (± 23) KHN, were randomly divided into two groups.In order to maintain reference surfaces for lesion depth determination, two layers of nail varnish were applied on half of the surface of each block (Figure 1G).
On the left and right sides of the intraoral palatal appliances, 6 cavities of 5 x 5 x 3 mm were made, and into each of them, one block of enamel was randomly fixed with wax (Figure 1H).The position of two groups (right or left) on the appliance was randomly chosen.

Treatments
During the experimental phase, the volunteers brushed their teeth with fluoride-free dentifrice 6 .The palatal device was worn for five consecutive days 16,30 .On the first 12 hours, specimens were not subjected to erosive and abrasive processes, to allow the formation of a salivary pellicle 15 .On the following 5 days, erosive and abrasive challenges were made extraorally 4 times a day and at predetermined times (8:00, 12:00, 16:00 and 20:00 h) 5,16,30 .
In order to submit the enamel blocks to erosion, the volunteers were instructed to remove the appliance and immerse it in a cup containing 150 ml of a freshly opened bottle of Coke  (pH 2.8, Spal, Porto Real, RJ, Brazil) for 10 minutes (Figure 1J) 3,5 .After that, no treatment was performed in one row (GI), whereas the other row (GII) was immediately brushed by the volunteers (Figure 1K).The brushing procedure consisted of 30 extraoral brushing strokes with a soft end-rounded toothbrush (Bitufo; Sanifil, Jundiaí, SP, Brazil) with a small portion (approximately 0.3 g) of the dentifrice without fluoride (Crest  pH 6.8, silica as abrasive) 7 .Volunteers were trained and instructed to carefully perform this procedure and to avoid a carry-across effect of the treatments.The brushed blocks were washed under deionized water and before the appliance was replaced into the mouth, the volunteers were instructed to take one sip of the beverage 24 .
The volunteers received instructions to wear the appliances continuously, including at night, but to remove them during meals (3 times a day), when oral hygiene was performed 11,15 .Plaque control on the blocks was achieved by dipping the appliance in 0.2% chlorhexidine gluconate solution (pH 6.8) for 5 minutes at the end of each study day 16,30 .

Wear assessment
After 5 days, the volunteers stopped wearing the appliances.The enamel blocks were removed from the appliances and the nail varnish over the surfaces was carefully cleaned with acetone-soaked cotton wool 4 .The blocks were dried and the enamel wear was determined in relation to the reference surface, by profilometry using a rugosimeter (Hommel Tester T1000, VS, Schwenningen, Germany) (Figures 1M and 1N).Five readings were performed on each specimen through a scanning from the reference to the exposed surface.The average wear depth of each group by volunteer (experimental unit) was computed using the 15 readings per- formed in micrometers (µm): three blocks x five readings.

Percentage of superficial microhardness change assessment
After the wear assessment, enamel surface microhardness was measured as described earlier (Figure 1L) and an average per volunteer was obtained.Ten indentations on each specimen were made, five on the previously protected enamel surface (SMH) and five on the experimental area (SMH 1 ).Using these measurements, the percentage of superficial microhardness change (%SMHC) was calculated (%SMHC = [[SMH 1 -SMH]/SMH] × 100).

Scanning electron microscopy
Four specimens whose wear and %SMHC values were closest to the mean of each group were selected for scanning electron microscopic observations.The specimens were mounted and covered with palladium-gold by ion sputtering in a Hammer VI cathodic evaporator (Anatech LTD, Alexandria, USA) (Figures 1O and 1P).Then, they were studied and photographed in a JEOL JSM T220A scanning electron microscope (Tokyo, Japan) operating at 15 kV (Figure 1Q).

Statistical analysis
Each group comprised 5 experimental units (volunteers), which were statistically analised.First, the assumptions of equality of variances and normal distribution of errors were checked for the response variables tested.Since the assumptions were satisfied, the paired Student's t test was carried out for statistical comparisons.The significance level was set at 5%.

RESULTS
All five volunteers completed the study.Table 1 shows that Group I (erosion) had a significantly higher %SMHC than Group II (erosion plus abrasion) (p = 0.001).Nevertheless the wear was less pronounced for Group I when compared to Group II (p = 0.001).
The scanning electron microscopy images permitted the visualization of a distinct line of demarcation at the test-control margin on both groups (Figures 2 and 3).Specimen surfaces that had been covered with nail varnish did not show alterations.At higher magnification of the test area of group I (erosion), prism core dissolution was observed (Figure 4).However, the test area of group II (erosion plus toothbrushing) showed a less altered   surface, more similar to that of the control area (Figure 5).

DISCUSSION
In order to simulate the everyday situation as closely as possible, an in situ model was chosen in the present study to test erosion plus abrasion on enamel.Bovine enamel was used in agreement with other studies 10,11 .It was observed that microhardness of this tissue is similar to that of humans, but bovine enamel is more affected by acid than is human enamel 3,10 .In addition, removal of the outermost layer of enamel during specimen preparation was thought to have influenced the severity of the erosion process 23 .Therefore the results presented may be overestimated when compared to those of human teeth in vivo, but since the study is capable of replicating the biological variations of the oral environment, the results must be an indicator of what would occur in vivo.
The formation of a salivary pellicle during the first 12 h aimed to protect the underlying enamel against erosive destruction.The erosive challenge was performed extraorally because the authors did not want to take the risk of demineralizing the natural teeth of the volunteers by frequent exposure to large amounts of acidic solution.
However, it was previously demonstrated that after rinsing with acidic solutions the pH at tooth surfaces drops below the critical pH for enamel dissolution for a period of about 2 min 24 .Moreover, rinsing with acidic beverages generates a sharp rise in parotid salivary flow rate, which returns to resting levels only within 6 min 24 .Considering these interactions of acidic substances with saliva, the participants of the present study were instructed to take one sip of the beverage before reinsertion of the intraoral appliance after the extraoral immersion of the specimens.
The volunteers were instructed not to touch the enamel blocks with the tongue, in order to avoid the abrasive effect of the tongue 13 .To standardize the parameter abrasion as much as possible, the specimens were brushed extraorally with a soft end-tufted toothbrush and the volunteers were trained and instructed to carefully perform this procedure with 30 brushing strokes without applying force 15,18 .It may be assumed that this brushing of a single tooth occurs only in the case of performing meticulous tooth cleaning.This fact should be considered when interpreting the total amount of enamel abraded recorded for the specimens in the present study.
The association of profilometric and microhardness analyses allowed the understanding of the erosive phenomenon and its association with abrasion 4,6 .The enamel blocks subjected to erosion alone presented higher percentage of microhardness change than the blocks subjected to erosion plus toothbrushing.On the other hand, toothbrushing of the enamel previously eroded promoted higher wear than erosion alone.
In order to explain these findings, the following hypothesis was formulated.Erosion may have resulted from some direct loss of the superficial  enamel layer, and in addition, may have softened the underlying layer, thus resulting in lower microhardness.This softened layer was vulnerable to mechanical forces, and was removed by the brushing procedure, resulting in exposure of a harder enamel surface.The scanning electron microscopy images also corroborate this hypothesis 26 .Due to this fact, the wear increased when eroded enamel was subjected to abrasion, but the %SMHC was lower when compared to that of erosion alone.This hypothesis is supported by previous investigations, which showed that softened enamel is extraordinarily susceptible to toothbrushing performed immediately after an erosive challenge 4,5,8,18 .
The results of the present study show that toothbrushing can potentialize the effects of an erosive challenge.Thus, a therapy to minimize this synergistic effect is required.Studies have shown that beverage modification, salivary stimulation by a chewing-gum and consumption of cheese or milk after the acid attack could decrease erosion 12,16,19 .Other studies have demonstrated that using fluoride mouth rinses before brushing procedures, or waiting at least one hour to brush the teeth after an erosive challenge, could decrease the enamel wear 4,5,7,15,18,21 .However, further studies should be conducted to establish preventive measures to decrease alterations by eroded enamel subjected to abrasion.

CONCLUSIONS
The enamel superficial wear was more pronounced when erosion was subjected to toothbrushing abrasion.However, the surface softening was smaller, when toothbrushing was performed, due to the removal of the altered superficial layer by erosion.

FIGURE 1 -
FIGURE 1 -Schematic illustration of the experimental sequence.

FIGURE 4 -
FIGURE 4 -Experimental area of specimen from group I (erosion).Severe erosion with prism core dissolution.The bar represents 10 µm (1,500 X).

FIGURE 5 -
FIGURE 5 -Experimental area of specimen from group II (erosion + abrasion).Surface roughening when compared with control area, which is smoother.The bar represents 10 µm (1,500 X).

TABLE 1 -
Means and SD of %SMHC and wear (µm) for the experimental groups (n = 5).