Assessment in vitro of brushing on dental surface roughness alteration by laser interferometry

Noncarious cervical lesions (NCCLs) are considered to be of multifactorial origin, normally associated with inadequate brushing. This study assessed the influence in vitro of simulated brushing on NCCL formation. Fifteen human premolars were submitted to brushing in the cementoenamel junction region, using hard-, mediumand soft-bristled brushes associated with a toothpaste of medium abrasiveness under a 200 g load, at a speed of 356 rpm for 100 minutes. The surface topography of the region was analyzed before and after brushing, by means of a laser interferometer, using “cut-off” values of 0.25 and considering roughness values in m. The initial roughness ( m) results for dentin (D / bristle consistency: 1 – soft, 2 – medium and 3 – hard) were as follows: (D1) 1.25 0.45; (D2) 1.12 0.44; (D3) 1.05 0.41. For enamel (E / bristle consistency: 1 – soft, 2 – medium and 3 – hard), the initial results were: (E1) 1.18 0.35; (E2) 1.32 0.25; (E3) 1.50 0.38. After brushing, the following were the values for dentin: (D1) 2.32 1.99; (D2) 3.30 0.96; (D3) Over 500. For enamel, the values after brushing were: (E1) 1.37 0.31; (E2) 2.15 0.90; (E3) 1.22 0.47. Based on the results of the ANOVA and Tukey statistical analyses ( = .05) it was concluded that soft, medium and hard brushes are not capable of abrading enamel, whereas dentin showed changes in surface roughness by the action of mediumand hard-bristled brushes. Descriptors: Tooth abrasion; Dentifrices; Toothbrushing. Alessandra Miranda de Azevedo(a) Heitor Panzeri(b) Célio Jesus do Prado(c) José Daniel Biasoli De-Mello(d) Carlos José Soares(e) Alfredo Julio Fernandes-Neto(f) (a)DDS, Graduate Student; (b)PhD, Department of Dental Materials and Prosthodontics – School of Dentistry of Ribeirão Preto, University of São Paulo. (c)PhD, Assistant Professor, Department of Removable Prosthodontics; (e)PhD, Department of Restorative Dentistry; (f)PhD, Professor and Chairman, Department of Occlusion, Fixed Prosthodontics, and Dental Materials – School of Dentistry, Federal University of Uberlândia. (d)PhD, School of Mechanical Engineering, Federal University of Uberlândia. Dental Materials Corresponding author: Alessandra Miranda de Azevedo Faculdade de Odontologia de Ribeirão Preto Universidade de São Paulo (USP) Departamento de Materiais Dentários e Prótese Av. do Café, s/n Ribeirão Preto SP Brazil CEP: 14040-904 E-mail: aleazevedopa@yahoo.com.br Received for publication on Aug 07, 2006 Accepted for publication on Nov 27, 2006 Assessment in vitro of brushing on dental surface roughness alteration by laser interferometry Braz Oral Res 2008;22(1):11-7 12 Introduction Noncarious cervical lesions (NCCLs), generically denominated tooth abrasion, present a variety of forms, and can affect vestibular, lingual and/or proximal surfaces, commonly being of multifactorial origin.1-4 Factors as acids, and occlusal and abrasive forces may interact or act separately, thus contributing to the appearance of cervical lesions.3,5-8 These lesions may be classified as: erosive, attritive, abfractive and abrasive.9 Dental erosion is tooth structure loss by nonbacterial chemical action;2,5,6,10 attrition is wear of one surface against another and abfraction is a wedge-shaped lesion, located at the cementoenamel junction, caused by stress generated by biomechanical force.1,7,11 Clinically, the term abrasion refers to pathological wear by objects repeatedly in contact with the teeth.2 Brushing with dentifrice is an example of a triple-body abrasion process, in which disaggregated particles slide between the tooth and brush bristles, the size of the abrasive particles and pressure being important factors in the speed at which the surface undergoes abrasion.12 However, definition of this process as an isolated etiologic factor for cervical lesions is still controversial. There are studies in which toothbrushing without dentifrice is apparently incapable of abrading enamel and dentin,7,9,13-17 as the toothpaste abrasiveness may be caused by a combination of its erosive effect and the mechanical effect of the toothbrush bristles,18 while other studies showed that toothbrushing without dentifrice may induce abrasion.19,20 Frequency and toothbrushing technique are also factors related to tooth abrasion.21 In view of this context, an hypothesis is set forth that brushing and the type of toothbrush result in topographic alteration of human enamel and dentin, characterizing this process as an etiologic factor of noncarious cervical lesions. In order to confirm this hypothesis, this study assessed the topography, in vitro, of human enamel and dentin before and after brushing with soft-, mediumand hard-bristled brushes associated with dentifrice. Material and Methods Tooth obtainment To conduct this study, 15 healthy human premolars were selected because they presented high incidence of abrasive lesions,3,22 but did not present any type of lesion on the vestibular enamel and root dentin faces, and had been indicated for extraction due to periodontal problems or orthodontic purposes. Teeth that presented any damage resulting from forceps during extraction were excluded.20 This study was approved by the Research Ethics Committee, Federal University of Uberlandia (Protocol No. 224/04). Sample obtainment The selected teeth were embedded in polystyrene resin (Aerojet, São Paulo, SP, Brazil), in the proportion of 12% monomer to 2% catalyzer. The teeth were placed horizontally with the vestibular face penetrating approximately 1 mm into a utility wax slide, and afterwards, enveloped by a rectangular 25 x 10 x 10 cm aluminum matrix. Polystyrene resin was poured in till it was full, and when the resin was completely polymerized, the set was detached from the wax and the matrix, removed, with the result that the tooth, except for the vestibular face, was embedded in resin. The samples received a finishing procedure to remove excess resin and to clean them of wax. The samples were identified and stored in distilled water at a temperature of 37 C in an oven and then randomly divided into three groups: 1 – use of soft-bristled brushes; 2 – use of medium-bristled brushes, and 3 – use of hard-bristled brushes. To define the type of substrate, D was designated to identify Dentin and E, to identify enamel. Initial surface topography determination Initially, the samples were metal-coated (Emitech K550, Emitech Technologies Ltd., Kent, England), by deposit of a thin layer of gold, equivalent to 10-6 mm, in order to increase surface reflectivity. Next, the samples were examined by laser interferometry (Microfocus Expert IV, UBM Corporation, Sunnyvale, CA, USA), and measurements were taken by optic reading of pre-determined 4.0 mm2 areas in enamel and 3.0 mm2 areas in dentin. Readings were taken in the central area of the two substrates, 0.5 mm from the cementoenamel junction. The gross data obtained were Azevedo AM, Panzeri H, Prado CJ, De-Mello JDB, Soares CJ, Fernandes-Neto AJ Braz Oral Res 2008;22(1):11-7 13 analyzed by specific software (Mountains Map 3, Besançon, France), enabling this surface to be characterized with regard to shape and undulation and to calculate surface roughness parameters, using “cut-off” values of 0.25.23 The roughness parameter assessed for numerical characterization of the surface was as follows: Sq, standard deviation of the distribution of surface peak and valley heights,24,25 associated with assessment of the functional parameters: Ssk, symmetry coefficient, the parameter used to measure the symmetry of a profile in relation to the mean plane, and Sk, flattening coefficient, which describes the form of topography height distribution. The values found for each parameter were statistically analyzed, the parameter Sq being submitted to the parametric ANOVA and Tukey tests ( =.05) and Sk and Ssk expressed in frequency. Simulated brushing After determining initial topography, the samples were washed under running water to remove the gold layer. Next, they were placed inside an ultrasonic vibration device (Thornton, Vinhedo, SP, Brazil) containing distilled water and stayed there for 10 minutes. Then they were washed with soap and water, alcohol and distilled water and then fixed horizontally in the receptacles by means of modeling compound. To perform the abrasion tests, a brushing machine was used comprised of six stainless steel compartments to put the samples in. The test specimen is placed on the internal base of the receptacle, fixed to a metal plate by means of modeling compound (DFL, Rio de Janeiro, RJ, Brazil). The appliance has a support to which the toothbrush is fixed, aligned parallel to the plate, regulated by screws positioned on the sides and top. The machine was set to run a horizontal course of 3.8 cm, applying a 200 g load at a speed of 356 rpm for 100 minutes, corresponding to 2 years of normal standard tooth brushing. Similar toothbrushs (Tek, Johnson & Johnson, São José dos Campos, SP, Brazil), with small, oval-headed and round-tipped synthetic bristles of soft, medium and hard consistency were fixed to the supports and adjusted so that a largest number of bristles would come into contact with the sample. Fifteen milliliters of a suspension prepared with 70 ml of distilled water and 70 g of dentifrice of medium abrasiveness (Contente, Uberlândia, MG, Brazil) were poured into each tray containing the sample, in order to perform brushing for 100 minutes. Final surface topography determination When the brushing ended, the samples were washed under running water and then submitted to ultrasonic vibration (Thornton, Vinhedo, SP, Brazil) for 10 minutes to remove the abrasive particles. The samples were metal-coated again and the surface topography parameters were obtained again, in accordance with the same measuring methodology used initially. Results Mean and standard deviation values of the parameter Sq for the human enamel and dentin are presented in Table 1. The data were submitted to the an


Introduction
][7][8] These lesions may be classified as: erosive, attritive, abfractive and abrasive. 9Dental erosion is tooth structure loss by nonbacterial chemical action; 2,5,6,10 attrition is wear of one surface against another and abfraction is a wedge-shaped lesion, located at the cementoenamel junction, caused by stress generated by biomechanical force. 1,7,11linically, the term abrasion refers to pathological wear by objects repeatedly in contact with the teeth. 2 Brushing with dentifrice is an example of a triple-body abrasion process, in which disaggregated particles slide between the tooth and brush bristles, the size of the abrasive particles and pressure being important factors in the speed at which the surface undergoes abrasion. 12However, definition of this process as an isolated etiologic factor for cervical lesions is still controversial.There are studies in which toothbrushing without dentifrice is apparently incapable of abrading enamel and dentin, 7,9,[13][14][15][16][17] as the toothpaste abrasiveness may be caused by a combination of its erosive effect and the mechanical effect of the toothbrush bristles, 18 while other studies showed that toothbrushing without dentifrice may induce abrasion. 19,20Frequency and toothbrushing technique are also factors related to tooth abrasion. 21n view of this context, an hypothesis is set forth that brushing and the type of toothbrush result in topographic alteration of human enamel and dentin, characterizing this process as an etiologic factor of noncarious cervical lesions.In order to confirm this hypothesis, this study assessed the topography, in vitro, of human enamel and dentin before and after brushing with soft-, medium-and hard-bristled brushes associated with dentifrice.

Tooth obtainment
To conduct this study, 15 healthy human pre-molars were selected because they presented high incidence of abrasive lesions, 3,22 but did not present any type of lesion on the vestibular enamel and root dentin faces, and had been indicated for extraction due to periodontal problems or orthodontic purposes.Teeth that presented any damage resulting from forceps during extraction were excluded. 20This study was approved by the Research Ethics Committee, Federal University of Uberlandia (Protocol No. 224/04).

Sample obtainment
The selected teeth were embedded in polystyrene resin (Aerojet, São Paulo, SP, Brazil), in the proportion of 12% monomer to 2% catalyzer.The teeth were placed horizontally with the vestibular face penetrating approximately 1 mm into a utility wax slide, and afterwards, enveloped by a rectangular 25 x 10 x 10 cm aluminum matrix.Polystyrene resin was poured in till it was full, and when the resin was completely polymerized, the set was detached from the wax and the matrix, removed, with the result that the tooth, except for the vestibular face, was embedded in resin.The samples received a finishing procedure to remove excess resin and to clean them of wax.The samples were identified and stored in distilled water at a temperature of 37 C in an oven and then randomly divided into three groups: 1use of soft-bristled brushes; 2 -use of medium-bristled brushes, and 3 -use of hard-bristled brushes.To define the type of substrate, D was designated to identify Dentin and E, to identify enamel.

Initial surface topography determination
Initially, the samples were metal-coated (Emitech K550, Emitech Technologies Ltd., Kent, England), by deposit of a thin layer of gold, equivalent to 10 -6 mm, in order to increase surface reflectivity.Next, the samples were examined by laser interferometry (Microfocus Expert IV, UBM Corporation, Sunnyvale, CA, USA), and measurements were taken by optic reading of pre-determined 4.0 mm2 areas in enamel and 3.0 mm2 areas in dentin.Readings were taken in the central area of the two substrates, 0.5 mm from the cementoenamel junction.The gross data obtained were analyzed by specific software (Mountains Map 3, Besançon, France), enabling this surface to be characterized with regard to shape and undulation and to calculate surface roughness parameters, using "cut-off" values of 0.25. 23The roughness parameter assessed for numerical characterization of the surface was as follows: Sq, standard deviation of the distribution of surface peak and valley heights, 24,25 associated with assessment of the functional parameters: Ssk, symmetry coefficient, the parameter used to measure the symmetry of a profile in relation to the mean plane, and Sk, flattening coefficient, which describes the form of topography height distribution.The values found for each parameter were statistically analyzed, the parameter Sq being submitted to the parametric ANOVA and Tukey tests ( =.05) and Sk and Ssk expressed in frequency.

Simulated brushing
After determining initial topography, the samples were washed under running water to remove the gold layer.Next, they were placed inside an ultrasonic vibration device (Thornton, Vinhedo, SP, Brazil) containing distilled water and stayed there for 10 minutes.Then they were washed with soap and water, alcohol and distilled water and then fixed horizontally in the receptacles by means of modeling compound.To perform the abrasion tests, a brushing machine was used comprised of six stainless steel compartments to put the samples in.The test specimen is placed on the internal base of the receptacle, fixed to a metal plate by means of modeling compound (DFL, Rio de Janeiro, RJ, Brazil).The appliance has a support to which the toothbrush is fixed, aligned parallel to the plate, regulated by screws positioned on the sides and top.The machine was set to run a horizontal course of 3.8 cm, applying a 200 g load at a speed of 356 rpm for 100 minutes, corresponding to 2 years of normal standard tooth brushing.Similar toothbrushs (Tek, Johnson & Johnson, São José dos Campos, SP, Brazil), with small, oval-headed and round-tipped synthetic bristles of soft, medium and hard consistency were fixed to the supports and adjusted so that a largest number of bristles would come into contact with the sample.Fifteen milliliters of a suspension prepared with 70 ml of distilled water and 70 g of dentifrice of medium abrasiveness (Contente, Uberlândia, MG, Brazil) were poured into each tray containing the sample, in order to perform brushing for 100 minutes.

Final surface topography determination
When the brushing ended, the samples were washed under running water and then submitted to ultrasonic vibration (Thornton, Vinhedo, SP, Brazil) for 10 minutes to remove the abrasive particles.The samples were metal-coated again and the surface topography parameters were obtained again, in accordance with the same measuring methodology used initially.

Results
Mean and standard deviation values of the parameter Sq for the human enamel and dentin are presented in Table 1.The data were submitted to the analysis of normality and homogeneity and were shown to present normal and homogenous distribution for the parameter Sq.Therefore, statistical analysis was carried out by means of a two-way ANOVA.Post hoc comparisons among groups were done using the Tukey HSD test.Statistical significance was set at .05.The values Sk and Ssk were presented in the form of frequency, as they presented variation limits in positive and negative values.There was no statistically significant difference for the substrate enamel before and after simulated toothbrushing, irrespective of the toothbrush hardness, for all parameters analyzed, as well as for dentin with a soft toothbrush.However, the results of parameter Sq found for the medium brush showed a significant increase in dentin surface roughness after brushing.Dentin abrasion with the hard brush could not be analyzed because it was defined as being over 500 m, in excess of the laser interferometer reading capacity.For the parameter Ssk, enamel presented predominantly negative values, indicating a larger number of valleys before and after brushing; on the other hand, dentin presented predominantly positive values, indicating a larger number of peaks (Tables 2 and  3).The graphic representation of surface roughness along the analyzed area is represented in Figures 1  (A and B) and 2 (A and B), axonometric images that allow relief to be seen.

Discussion
The hypothesis tested in this study was accepted only for dentin.The type of brush only influenced the dentin substrate topography and did not harm the enamel surface.Human tooth enamel behavior was similar for the three types of toothbrushes, and presented no significant variation for the parameter Sq among the groups, before and after simulated brushing.Dentin presented statistically similar results to those of enamel for the soft brush, but for the medium brush, there was increased surface roughness after brushing.Tooth structure abrasion with the hard-bristled brush was higher than 500 m, thus it was not possible to assess the roughness parameters of this structure by the methodology applied.
After the brushing procedure, the enamel surface was not abraded.Because of its highly mineralized content, enamel is extremely hard. 26However, when fracture occurs, it is reported as a result of enamel prism disorganization due to stress concentration in the cervical region of the tooth.The action of dentifrices and brush could result in fracture expansion only. 1,10,11With regard to surface form characterization, in this study, dentin presented a symmetry coefficient with a predominance of peaks, and it was more susceptible to abrasion in comparison with the enamel surface, in which valleys were predominant.
Abrasion on the dentin surface was observed in an abrasion test with soft-bristled brushes, 20 in contrast with the result obtained in the present study,

A B
in which this type of bristle did not result in abrasion of this structure.However, these authors used a load of 300 g while, in the present study, the load applied was 200 g.In an abrasion test by means of human dentin brushing, 14,16,27 there was no significant difference in the abrasion of this structure with regard to toothbrush bristle hardness.On the other hand, a reduction in abrasion was reported when hard bristles were used. 19This differs from the results of the present study, in which it was noted that hard-bristled brushes presented greater abrasion in dentin than the other types of bristles.Abrasion test studies did not observe enamel structure abrasion, 13 a result in agreement with that obtained in the present study, when soft-, mediumand hard-bristled brushes were used on this same substrate.Other studies related enamel abrasion and abrasion by brushing. 20However, in those researches, the abrasion tests were related to exposure to acid and lateral forces, respectively.
6][17] Nonetheless, abrasion may be caused by the corrosive effect of the dentifrice combined with the mechanics of the toothbrush bristles. 2,8,13As there was no variation in the type of dentifrice used in the present study, it was not possible to relate abrasion and abrasive concentration.However, the results showed that there was no enamel abrasion during the brushing procedure.On the other hand, in dentin, abrasion was observed with the use of medium and hard-bristled brushes using dentifrice of medium abrasiveness for both groups, which does show the influence of the type of brush.
Regarding the topography analysis, the roughness parameters can be calculated using two-dimensional (2D) or three-dimensional (3D) study. 242D parameters are used for profile analysis.However, digital techniques of surface analysis in 3D make possible the study of a three-dimensional area of the surface without contacting it.The accomplishment of digital analyses associated to a reading without contact by means of optical instruments in this study made possible the attainment of data without distortions or damages to the surface of enamel and dentine structures, 25 but the optical reading was sensitive, preventing the attainment of focus in the dentine surface after brushing with hard-bristled brushes due to a resultant wear superior to 500 m.

Conclusion
In accordance with the methodology used and based on the analysis of the data obtained in this study, it was possible to conclude that: Brushing with the use of soft-, medium-and hard-bristled brushes and dentifrice of medium abrasiveness is not capable of abrading human enamel.In dentin, medium-and hard-bristled brushes caused increased surface roughness.

Figure 1 ,
Figure 1, A and B -Surface Topography -x, y and z (length, width and height) of analyzed area.A -Dentin roughness before brushing, and B -Increase of dentin roughness after brushing with medium-bristled tooth brush.

Figure 2 ,
Figure 2, A and B -Surface Topography -x, y and z (length, width and height) of analyzed area.A -Enamel roughness before brushing, and B -Enamel roughness presented no significant variation before and after simulated brushing.

Table 1 -
Mean and standard deviation values of the parameter Sq and statistical categories -Tukey Test (P < 0.05).

Table 2 -
Values in frequency of Ssk/Sk for dentin according to type of brush used.

Table 3 -
Values in frequency of Ssk/Sk for enamel according to type of brush used.