|DENTAL SCIENCE - ORIGINAL ARTICLE
|Year : 2015 | Volume
| Issue : 6 | Page : 616-622
Influence of erbium, chromium-doped: Yttrium scandium-gallium-garnet laser etching and traditional etching systems on depth of resin penetration in enamel: A confocal laser scanning electron microscope study
Vishal Vijayan1, K Rajasigamani2, K Karthik2, Sasidharan Maroli1, Jitesh Chakkarayan1, Mohamed Haris3
1 Department of Orthodontics and Dentofacial Orthopaedics, Kannur Dental College, Kannur, India
2 Department of Orthodontics and Dentofacial Orthopaedics, Rajah Muthiah Dental College and Hospital, Annamalai University, Chidambaram, Tamil Nadu, India
3 Department of Orthodontics and Dentofacial Orthopaedics, P. S. M College of Dental Science and Research, Thrissur, Kerala, India
|Date of Submission||28-Apr-2015|
|Date of Decision||28-Apr-2015|
|Date of Acceptance||22-May-2015|
|Date of Web Publication||1-Sep-2015|
Dr. Vishal Vijayan
Department of Orthodontics and Dentofacial Orthopaedics, Kannur Dental College, Kannur
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Objective: This study was performed to assess the resin tag length penetration in enamel surface after bonding of brackets to identify which system was most efficient. Methodology: Our study was based on a more robust confocal microscopy for visualizing the resin tags in enamel. Totally, 100 extracted human first and second premolars have been selected for this study and were randomly divided into ten groups of 10 teeth each. In Group 1, the buccal enamel surface was etched with 37% phosphoric acid (3M ESPE), Group 2 with 37% phosphoric (Ultradent). In Groups 5, 6, and 7, erbium, chromium-doped: Yttrium scandium-gallium-garnet (Er, Cr: YSGG) laser (Biolase) was used for etching the using following specifications: Group 5 (1.5 W/20 Hz, 15 s), Group 6 (2 W/10 Hz, 15 s), and Group 7 (2 W/20 Hz, 15 s). In Groups 8, 9, and 10, Er, Cr: YSGG laser (Biolase) using same specifications and additional to this step, conventional etching on the buccal enamel surface was etched with 37% (3M ESPE) after laser etching. In Groups 1, 5, 6, 7, 8, 9, and 10 3M Unitek Transbond XT primer was mixed with Rhodamine B dye (Sigma-Aldrich, Germany) to etched surface and then cured for 20 s. In Group 2, Ultradents bonding agent was mixed with Rhodamine B. In Group 3, 3M Unitek Transbond PLUS, Monrovia, USA, which was mixed with Rhodamine B dye (Sigma-Aldrich, Germany). Group 4, with self-etching primer (Ultradent-Peak SE, USA) was mixed with Rhodamine B dye (Sigma-Aldrich, Germany). Later (3M Unitek, Transbond XT, Monrovia USA) [Figure 1] was used to bond the modified Begg brackets (T. P. Orthodontics) in Groups 1, 3, 5, 6, 7, 8, 9, and 10. In Groups 2, 4 Ultradent-Peak LC Bond was used to bond the modified brackets. After curing brackets were debonded, and enamel depth penetration was assessed using confocal laser scanning microscope. Results: Group J had a mean maximum depth of penetration of 100.876 μm, and Group D was the least having a maximum value of 44.254 μm. Conclusions: Laser alone groups had comparable depths of penetration to that of self-etching groups but much lower than conventional acid etched groups.
Keywords: Acid etching, bonding, confocal laser scanning microscope, enamel etching, erbium, chromium-doped: Yttrium scandium-gallium-garnet laser, lasers, self-etching primers
|How to cite this article:|
Vijayan V, Rajasigamani K, Karthik K, Maroli S, Chakkarayan J, Haris M. Influence of erbium, chromium-doped: Yttrium scandium-gallium-garnet laser etching and traditional etching systems on depth of resin penetration in enamel: A confocal laser scanning electron microscope study. J Pharm Bioall Sci 2015;7, Suppl S2:616-22
|How to cite this URL:|
Vijayan V, Rajasigamani K, Karthik K, Maroli S, Chakkarayan J, Haris M. Influence of erbium, chromium-doped: Yttrium scandium-gallium-garnet laser etching and traditional etching systems on depth of resin penetration in enamel: A confocal laser scanning electron microscope study. J Pharm Bioall Sci [serial online] 2015 [cited 2019 Dec 10];7, Suppl S2:616-22. Available from: http://www.jpbsonline.org/text.asp?2015/7/6/616/163571
In 1955 Buonocore,  introduced the use of phosphoric acid for etching and in 1965 Newman  first tried to bond orthodontic brackets to teeth using the acid etch technique and an epoxy-derived resin.
Two types of resin tags are found. Macro-tags that fills the space surrounding the enamel prisms and micro-tags from resin infiltration is formed within the tiny etch-pits of the enamel prisms. 
Self-etch primers are user-friendly and technique sensitive. It does not remove the smear layer from dentin completely, thus results in less postoperative sensitivity than total-etch adhesives. 
Three types of lasers are commonly used in dentistry is the erbium laser. Enamel and dentin surfaces etched with erbium, chromium-doped: Yttrium scandium-gallium-garnet (Er, Cr: YSGG) lasers show microirregularities and no smear layer. 
The confocal laser scanning microscope (CLSM) can penetrate laser in thin optical sections below the surface of intact specimens and offers superior images of the resin dentin interface. , CLSM is able to identify the tissue-emitting fluorescent signal, which exhibits natural autofluorescence. Coupled with photomultipliers that have high quantum efficiency in the near-ultraviolet, visible, and near-infrared spectral regions, these microscopes can examine fluorescence emission in the range of 400-750 nm. 
This study depth of penetration of resin tags by direct measurement on an intact resin-enamel surface were evaluated for different etching systems which include conventional etching, self-etching primer (SEP), laser etching, and laser with conventional etching group.
| Methodology|| |
Hundred extracted human first and second premolars have been selected for this study. Selection of teeth was based on lack of enamel defects, morphological defects, decalcification, and fluorosis, a tooth that was not previously bonded and had no cracks caused by extraction forceps and tooth extracted for the orthodontic purpose alone.
The samples were divided into ten groups of 10 teeth each based on the mode of enamel conditioning which is elaborated in [Table 1] and stored in 0.7% thymol solution. The buccal enamel surfaces of the teeth were pumiced, washed for 30 s, and dried for 10 s with a moisture-free air spray [Table 1].
In Group A, the buccal enamel surface was etched with 37% phosphoric acid for 15 s using 3M ESPE's Scotchbond. The samples were rinsed with water and gentle air spray for 15 s, and dried for another 15 s. The etched enamel showed a uniform dull, frosty appearance. 3M Unitek Transbond XT primer was then mixed with Rhodamine B dye (Sigma-Aldrich, Germany) at a concentration of 0.1 mmol/L as a thin film to the etched surface and then cured for 20 s. This dye is a fluorescent dye, which aids in visualization of the resin tags under the confocal microscope. Later adhesive paste (3M Unitek, Transbond XT, Monrovia USA) was used to bond the modified Begg brackets (T. P. Orthodontics, La Porte, Indiana, USA) with modified premolar band material of 0.125 × 0.003 inch welded to the bracket base [Figure 2]. It was then cured using 3M ESPE Elipar 2500 Halogen Curing Light, USA as per manufacturer's instructions.
In Group B, the buccal enamel surface was etched with 37% phosphoric acid for 15 s using Ultradent's Ultra Etch, USA. Ultradents bonding agent was mixed with Rhodamine B and applied to etched enamel. Later adhesive paste Ultradent-Peak LC Bond, USA was used to bond the modified Begg brackets (T. P. Orthodontics, La Porte, Indiana, USA) and then cured.
In Group C, the buccal enamel surface was etched with SEP 3M Unitek Transbond Plus, Monrovia, USA which was mixed with Rhodamine B dye (Sigma-Aldrich, Germany) at a concentration of 0.1 mmol/L as a thin film to the etched surface and then cured for 20 s. The self-etch primer was applied into the tooth surface as per the manufacturer's instructions. Later adhesive paste (3M Unitek, Transbond XT, Monrovia USA) was used to bond the modified Begg brackets (T. P. Orthodontics, La Porte, Indiana, USA) and then cured.
In Group D, the buccal enamel surface was etched with SEP using Ultradent-Peak SE, USA which was mixed with Rhodamine B dye (Sigma-Aldrich, Germany) at a concentration of 0.1 mmol/L as a thin film to the etched surface and then cured for 20 s. The self-etch primer was applied into the tooth surface as per the manufacturer's instructions. Later adhesive paste Ultradent-Peak LC Bond, USA was used to bond the modified Begg brackets (T. P. Orthodontics, La Porte, Indiana, USA) and then cured.
In Groups E, F, and G, Er, Cr: YSGG laser (Biolase Waterlase MD, USA) was used for etching the enamel surface with different power outputs and duration for Group E (1.5 W/20 Hz, 15 s), Group F (2 W/10 Hz, 15 s), and Group G (2 W/20 Hz, 15 s).
This device operates at a wavelength of 2780 nm; a pulse repetition rate of 20 pulses per second (20 Hz). Super short pulse was used. The average power output can be varied from 0.1 to 8 W. Two power settings (1.5 W and 2 W) were used. The air and water levels were 30% and 20%, respectively. The laser beam was perpendicular to the enamel at a distance of 3 mm [Figure 1] and [Figure 3].
After etching, 3M Unitek Transbond XT primer was then mixed with Rhodamine B dye (Sigma-Aldrich, Germany) at a concentration of 0.1 mmol/L as a thin film to the etched surface and then cured for 20 s. Later adhesive paste (3M Unitek, Transbond XT, Monrovia USA) was used to bond the modified Begg brackets (T. P. Orthodontics, La Porte, Indiana, USA) and then cured.
In Groups H, I, and J, Er, Cr: YSGG laser (Biolase Waterlase MD, USA) were used for etching the enamel surface with different power outputs and duration for Group H (1.5 W/20 Hz, 15 s), Group I (2 W/10 Hz, 15 s) and Group J (2 W/20 Hz, 15 s). In addition to this step, conventional etching on the buccal enamel surface was etched with 37% phosphoric acid for 15 s using 3M ESPE's Scotchbond after laser etching. The samples were rinsed with water and gentle air spray for 15 s. After etching, 3M Unitek Transbond XT primer was then mixed with Rhodamine B dye (Sigma Aldrich, Germany) at a concentration of 0.1 mmol/L as a thin film to the etched surface and then cured for 20 s. Later adhesive paste (3M Unitek, Transbond XT, Monrovia USA) was used to bond the modified Begg brackets (T. P. Orthodontics, La Porte, Indiana, USA) and then cured.
After that, the specimens were then stored in deionized water for 24 h before debonding. Debonding pliers were used to debond the brackets. Once debonding was done, the tooth was mounted on cylindrical acrylic jigs for the purpose of sectioning. Tooth sectioning was done using a hard tissue microtome (Leica Biosystems, SP 1600, Nussloch GmbH, Germany), by sectioning them in a labiolingual manner and parallel to the long axis of the tooth [Figure 4]. The instrument was set to section at a thickness of 300 μ. The basic purpose of this step was to reduce the amount of time required in optical sectioning of the tooth under the microscope. All the samples were then stored in amber colored bottles until further examination.
After the samples had been sectioned, they were examined under CLSM (Olympus, Fluoview FV1000, Japan) [Figure 5]. This laser scanning microscope receives its illumination from a Helium-Neon laser and was used for this study. The excitation wavelength of Rhodamine B was between 543 nm and 569 ± 5 nm and the emission wavelength that was collected by the microscope ranged between 543 μm and 640 μm. In this study, the hard tissue disks were sectioned close to the facial axis of the crown with an intact resin layer. The roots had been sectioned off as it was not a part of the study.
The samples were viewed by using a 10X objective and 60X oil immersion lens. The optical sectioning was done uniformly for the 200 μm depth and avoiding the superficial 50 μm. This was done as the layer could be scratched off from the sectioning procedure. The optical sectioning was done with a slice thickness of 1 μm. The resin tags were selected such that the tags have to be distinct, deepest, and continuously arising from the surface of the enamel extending into the tooth. In [Figure 6], E represents the enamel and RT represents the resin tags, and R represents root area. The quantification software that was used was FV10 ASW 3, and this software was also used to capture the images of the samples in OIB format.
Tools such as two-way analysis of variance (ANOVA) [Table 2] were used to analyze the data. Statistical analysis was performed, and the results were expressed as mean values ± standard deviation. One-way ANOVA used for multiple comparisons [Table 2] and [Table 3]. P < 0.05 was considered significant.
The mean value in μm of each etching group is taken. The graphical representation is plotted as different groups in x-axis and μm in y-axis are given in [Graph 1]. The overall analysis of groups with each other is given in [Table 3] for which Groups A, B and Groups C, D and Groups E, F, G and Groups H, I, J are clubbed into Group 1, Group 2, Group 3, and Group 4, respectively.
| Results|| |
This study depth of penetration of resin tags by direct measurement on an intact resin-enamel surface was evaluated for different etching systems which include conventional etching, SEP and lasers alone, and laser with conventional etching groups. The results were expressed as micrometers (μm) and shown in [Table 2], [Table 3] and [Table 4].
|Table 4: Mean, SD, and maximum and minimum values of depth of penetration of all 10 groups|
Click here to view
Group J had a maximum mean depth of penetration of 100.876 μm followed by Group 1 at 74.282 μm, Group A at 63.05 μm, Group H at 60.537, Group G at 59.387 μm, Group E at 55.673 μm, Group B at 55.654 μm, Group F at 55.273 μm, Group C at 46.247 μm, and Group D was the least having a maximum value of 44.254 μm.
| Discussion|| |
Phosphoric acid treatment has been implicated in decalcification and loss of enamel.  Etching tooth enamel with phosphoric acid creates surface microporosities and irregularities in to which low-viscosity resins can readily flow. A disadvantage of enamel acid etching is the demineralization of the outermost layer. ,
The urgent need to find alternative methods of etching resulted in SEP. Rinsing of enamel is not required thus reduces the clinical steps. Various investigators have suggested that SEP's produce a characteristic shallow and conservative etch pattern with reduced loss of enamel. , However, this phenomenon does not seem to affect the bond strength. ,
A single type of bracket and base design was used for all samples tested, as this would allow us to maintain an intact resin-enamel interface thereby preventing any form of fracture of resin layer from the undercuts of the bracket base. This was the reason why shear bond strengths of the samples were not conducted.
Buccal surfaces of the samples were considered for bonding in this study and the middle of the clinical crown was selected for bonding the brackets, in order to eliminate any difference in surface and prism morphology, which could lead to variations in etch patterns and tag lengths. 
The fluorescent dye (Rhodamine B) can be mixed individually with each of the components of the bonding system, thus, the behavior of each component can be studied individually.  While mixing the fluorescent dye with the primer component care was taken so that the concentration of the dye was kept as low as possible so that the dye would not overpower the solution. This could lead to a reduction in the fluorescence resulting in misleading results. 
The reasons why CLSM has been selected for this study was due to its ability to produce optical tomograms of thin (>0.35 μm) and thick (>1 μm) sections thus has been widely used in the field of dentistry to study the extent of caries lesions, and resin primer penetration.  It does not necessarily require fine sectioning and eliminates the cumbersome procedures of scanning electron microscopy. Moreover the samples can be viewed dry, and the stain would last for a very long period thereby prolonging the examination under a microscope is possible. , Extremely high-quality images and three dimensional reconstructions can be obtained from specimens prepared for conventional optical microscopy. 
In Orthodontics, lasers are mainly used for etching the enamel surface, curing and debonding the brackets. For the ER, Cr: YSGG laser working at 2 W a pattern similar to the type III acid etching pattern was described.  The mean shear bond strength and enamel surface etching obtained with Er, Cr: YSGG laser (operated at 1 W or 2 W for 15 s) is comparable to that obtained with acid etching.  It has the ability of laser irradiation to remove the smear layer has been reported.  Since 1965 studies have showed that exposure of enamel to laser irradiation imparts some degree of protection against demineralization under acid attack.  It also has the advantage of working on hard and soft tissues without anesthesia,  possible to ablate dental hard tissues like enamel and dentin without thermal side effects,  prevents caries formation  and is also safe for the pulp. 
In Er, Cr: YSGG laser enamel underwent physical changes including melting and recrystallization, thus forming numerous pores and small bubble-like inclusions.  These lasers produce super rough and micro cavitated surfaces and etches due to the vaporization of the water trapped within the hydroxyapatite matrix.  It causes surface roughening and irregularity similar to that of acid etching to a depth of 10 to 20 um, depending on the type of laser and the energy applied to the surface. 
The depth of resin penetration was first studied by Sheykholeslam and Buonocore.  There have been various studies ever since which was based on optical microscopes, but very few studies have been based on confocal microscopy. ,,, It has been concluded depth of penetration in acid etched teeth to be in the range of 5-50 μm. However, these studies were based on indirect decalcification procedures examined under optical microscopes. ,,, [Table 4] in our study, showed the mean depth of penetration for Group A and Group B to be 55.0739 μm and 50.34090 μm, respectively. These values obtained were higher than values obtained by Buonocore,  Wickwire and Rentz (25 μm),  Voss and Charbeneau (5-10 μm),  and Pahlavan (7 μm) et al.  but similar to values obtained by Retief,  Arakawa et al. (50 μm),  Asmussen,  Sundfeld et al. (53 μm),  and Ramesh Kumar et al. (53.9754 μm).  The reason for this increased tag length by phosphoric acid etched groups could be due to severe etching by unattenuated phosphoric acid.  However, intergroup comparison showed statistically less significant value (0.065).
In Group C and Group D the SEP samples showed mean tag lengths of 41.4921 μm and 37.2623 μm, respectively and were thereby comparatively shorter than phosphoric acid groups. The depth of penetration of the tags in this study co-relates with the studies conducted by Bishara et al.,  Dos Santos  and Ramesh Kumar et al. (53.9754 μm).  Intergroup comparison between the SEP's showed significant difference (0.002) such that PEAK SE had shorter tag lengths than Transbond PLUS SE thereby indicating that PEAK SE even though showed conservative etch pattern and depth of penetration, it might not be suitable for orthodontic bonding of brackets pending additional tests like bond strength test. In dentinal areas, greater bond strengths have been reported to occur in areas where dentin tubule density is high and long resin tag formation observed.  However, since longer tag lengths are not formed in these groups there has been a consistency in the tag lengths in Group C than in Group D, which thereby increased the surface area of mechanical undercuts thus making the system efficient.  Group D was included in the study as this could be shown as a parameter for comparison with already existing SEP for orthodontic use.
The power settings for laser groups had been selected on the basis of various studies on adequate shear bond strengths  and dentinal studies. , The mean depth of penetration for Group E, Group F, and Group G were 48.7958 μm, 46.6552 μm, and 51.3578 μm. However, studies pertaining to dentinal penetration, where the cavity was prepared and laser directly applied to dentin region showed, tag lengths greater than 100 μm were observed.  Thus, Group G using 2 W and 20 Hz power produced the maximum tag length in the laser alone group, which produced comparable results to that of SEP group (Group C). Studies by Türköz and Ulusoy have proved that laser etching followed by conventional etching provided greater SBS values.  Thus, this group had been included in the study. Groups H, I, and J gave mean penetration values of 54.8632 μm, 66.1526 μm, and 90.25830 μm. These groups provided greater tag lengths than compared to the laser alone etched groups. It, however, results in further roughening of enamel with the addition of acid etching.  Therefore, this group produced the least conservative etch depth patterns.
From [Table 3] mean comparison was done between all the groups and it was found that Acid Etch group (Group 1) showed 52.7074 μm, SEP groups (Group 2) showed 39.3772 μm, Laser alone group (Group 3) showed 48.9363 μm, and laser with acid etching group (Group 4) showed 70.4247 μm. Thus, it could be inferred that, though laser with etching group showed greater depths of penetration it was least conservative in terms of enamel demineralization and the operator time is increased with an additional step of conventional etching. Laser alone groups had comparable mean values to that of SEP groups and much lower than conventional acid etched groups. Nevertheless, additional studies have to be conducted in this area thereby leading to more innovative approach for bonding and much needed improvements in material science and laser technology.
| Conclusions|| |
Considering the results of the present in vitro study it was concluded that:
- Though laser with etching group showed greater depths of penetration it was least conservative in terms of enamel demineralization and the operator time is increased with an additional step of conventional etching
- Laser alone groups had comparable mean values to that of SEP groups and much lower than conventional acid etched groups. Thereby lasers do provide an alternative to regular etching procedures with its wide array of advantages
- Among the laser group, a 2 W and 20 Hz (Group G) provided the best results with consistency in the tag lengths and from various studies it has been proven from other studies to provide adequate bond strength
- CLSM is a useful tool in determining penetration depth of resin tags.
| Acknowledgments|| |
I would like to thank Center for Cellular and Molecular Platforms, Bangalore, India for providing access to the confocal laser scanning microscope. I would also like to thank Dr. Vidyaa Hari Iyer, Smile Dental Clinic, T. Nagar, Chennai, India for providing the Biolase Laser Machine for this study. Special thanks to Mr. Vishal Baxi for sponsoring Ultradent Products and Jamuna International, Chennai for providing 3M Unitek Products for this study.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
[Table 1], [Table 2], [Table 3], [Table 4]