After the research project was approved by the Research Ethics Committee of the Necmettin Erbakan University), 42 third molars were selected and cleaned with 4C distilled water. Reasons for the extractions were not related to the purpose of this study. Crowns were sectioned at the enamel–cement junction, using double-faced diamond discs (Isomet Low Speed Saw, Buehler Ltd., Lake Bluff, IL, USA), with water cooling at low speed. The buccal and lingual cervical enamel was removed with a diamond disc under tap water for cooling exposing the underlying dentin. Two cervical windows with 3 mm thick dentin discs were made in each sample in opposite positions. Eighty four dentine specimens were divided into seven groups. Group 1 (Control), Group 2 (Gluma Desensitizer), Group 3 (Teethmate Desensitizer), Group 4 (Nd:YAG laser), Group 5 (Er:YAG), Group 6 (Gluma + Er:YAG laser), Group 7 (Teethmate + Er:YAG laser) respectively, evaluated under SEM.
The smear layer were removed by a one-minute application of 18% ethylene diamine tetra-acetic acid (EDTA) (Ultradent Products Inc., USA). Teeth were rinsed with distilled water and dried with a five-second air blast.
The first group was only treated with EDTA. Gluma desensitizer(Group 2), (Gluma Desensitizer; Heraeus, Germany) and teethmate desensitizer (Group 3), (TMD; Kuraray Noritake Dental Inc., Tokyo, Japan) were applied onto the dentin discs using an applicator tip as per the manufacturer’s instructions and left for 30-40 second. The surface was then dried for a minute, after rinse gently with distiled water. The dentin specimens in the group 4 (Nd:YAG groups) were emitted for 60 s/cm2 with 15 Hz and 1-W laser without coolant and in noncontact mode (the distance between the optical fiber and the irradiated surface was 1 to 2 mm), two times. The dentin discs in the groups 5,6 and 7 (Er:YAG groups) were emitted for 60 s/cm2 with 30 Hz and 100 mJ with coolant and in noncontact mode (the distance between the optical fiber and the irradiated surface was 1 to 2 mm), two times, in VLP mode and by RO7 handpiece (Fidelis Plus Fotona, Slovenia).24,25 For all laser groups; the specimens were placed on a flat surface, the optical fiber was moved by the same dentist tangentially at approximately 1 mm/sec speed.
The dentine specimens were fixed in 2.5% glutaraldehyde in 0.1 M phosphatebuffered saline (pH 7.2) for 24 h at room temperature, washed with distilled water, and air dried. The samples were placed in an ionsputtering device. Under a high vacuum, ions are discharged from the gold target to the cathode. The specimens were coated with a thin layer of gold in a vacuum using a fine coat ion sputter (JFC-1100, JEOL, Tokyo, Japan).
Samples will examine using a scanning electron microcope at x2000, x1000 and x500 magnification. Total of twelve photographs from samples will take for each group. The number of open, partially occluded and total visible (open+partially occluded) dentin tubules will counte on each photograph. The tubules were counted in photographs. The photographs comprised a square frame with a border length representing 100 µm on the dentine specimen, allowing calculation of the number of tubules per 100 µm2. Atomic Force Microscopy (AFM) has been used to further substantiate the blocking mechanism. AFM was used to view the surface of dentin samples with open tubules, and those that had been occluded by different treatment groups. Prior to AFM analysis, the dentin disks were washed with deionized water and dried under nitrogen gas. Samples were imaged dry using a Nanoscope V Dimension 3100/5000 AFM (Veeco, Santa Barbara, CA, USA). Images were captured in the tapping mode with Veeco FESP/RFESP cantilevers. All images were amplitude images with pixel resolution between 1024 1024 and 2048 and were scanned at 0.4 Hz for 5 micron images and 0.2 Hz for 10 micron images.
All data were analyzed using the SPSS 13.0 statistical package for Windows (SPSS Inc., Chicago, IL). The measurements were analyzed using the Kolmogorov–Smirnov test at a p 0.05 level of significance to assess the distribution of the data. The Kruskal–Wallis and Mann–Whitney U tests were used with a level of significance of p 0.05.
The total number of tubules was counted from the various images captured by the SEM. Out of the total tubules, those that were completely sealed, partially sealed, and open tubules were counted. The data showed that all seven groups showed a reduction in radius of dentinal tubules to various technic compared to control group.
The mean values and standard deviations of the diameters of open dentinal tubules and the numbers of open tubules per 100 µm2 are presented in Table 1. Significant differences between groups were observed for both parameters ( p < 0.05). All test groups presented significantly superior tubule occlusion compared to control group ( p < 0.05). Group 7 demonstrated significantly more tubule occlusion when compared to all other groups. Compared to the partial or total closure of tubules, Groups 3, 4, 5 and 6 were better than group 2 ( p < 0.05). Figure 1 shows representative illustrations of dentine irradiated with all the different applications.
Scanning Electron Microscop
The dentin specimens of the control groups (Groups 1) which were only treated with EDTA showed a dentinal surface without the smear layer and wide open tubules (Figure 1A).
In Group 2, the specimens were treated with Gluma. Partially open tubules were observed, the dentinal tubules were partially sealed.
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