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Year : 2007  |  Volume : 10  |  Issue : 3  |  Page : 104-111
A confocal microscopic evaluation of the hybrid layer and resin tag formation of a total etch technique in comparison with self etching primers with three different pH


Department of Conservative Dentistry and Endodontics, Meenakshi Ammal Dental College, Maduravoyal Chennai 600 095, India

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   Abstract 

Aim: A Confocal Microscopic Evaluation of the hybrid layer and resin tag formation -of a total etch technique in comparison with self etching primers with three different ph.
Materials and Methods: 20 non carious human premolars stored in normal saline were used for the study. Occlusal surfaces of all the teeth were ground using slow speed diamond disc with copious water supply, so as to expose a flat surface of dentin with an approximate residual dentin thickness of 1.5-2mm. The teeth were then randomly divided into 4 groups of 5 specimens each. Group 1: (Blot dry)­ Prime and Bond NT (Dentsply), Group 2; Strong self etch adhesives usually have a ph of 1 or below { Adper Prompt L- Pop (3M ESPP)}, Group 3; Intermediary self etch adhesives have a ph of about 1.5 { AdheSE (Vvadent)}, Group 4; Mild self etch systems have a ph of about 2- {Cleatfil SE bond plus primer (knraray)}. Bonding agents mixed with Rodhamine B dye was applied according to the manufacture's instructions and cured using a light curing unit. All the bonded surfaces were restored with 2mm of resin composite Z 350 (3M) and cured for 40 seconds, to protect the bonding layer. All the specimens were stored in normal saline. After 24 hrs of storage the teeth were longitudinally sectioned using a slow speed diamond disc under copious water supply. The sectioned surface was polished' with a' 600 grit silicon carbide paper. The dentin/adhesive interfacial region was examined using a Confocal Laser Scanning Microscope (CLSM). Statistical analysis was done to compare the values among the groups using Kruskal-Wallis One way ANOVA appropriately by testing the means.
Results: Statistical analysis demonstrated that Mean length of resin tag formation and thickness of hybrid layer formation in Group I was maximum followed by Group IV; Group II and Group III respectively.
Conclusion: Within the parameters of this study the Prime and Bond NT (Group 1) performed better than all the three self etching primers in terms of resin tag formation and hybrid layer thickness.

How to cite this article:
Baweja PS, Hemamalathi S, Velmurugan N, Kandaswamy D. A confocal microscopic evaluation of the hybrid layer and resin tag formation of a total etch technique in comparison with self etching primers with three different pH. J Conserv Dent 2007;10:104-11

How to cite this URL:
Baweja PS, Hemamalathi S, Velmurugan N, Kandaswamy D. A confocal microscopic evaluation of the hybrid layer and resin tag formation of a total etch technique in comparison with self etching primers with three different pH. J Conserv Dent [serial online] 2007 [cited 2021 Dec 7];10:104-11. Available from: https://www.jcd.org.in/text.asp?2007/10/3/104/42270

   Introduction Top


The advent of resin composite restorative materials has brought about a revolution in the field of restorative dentistry. Resin composite material, with all its advantages, also poses a challenge to the clinician to find a perfect bonding system and technique.

Current dentin bonding theories suggest that there are two fundamental processes involved in bonding an adhesive to dentin. First, the mineral phase must be extracted from the dentin substrate without damaging the collagen matrix, and second, the voids left by mineral must be filled with an adhesive resin that penetrates the exposed collagen fibril network. If the exposed collagen collapses during the bonding procedure, the porosity of the dentin substrate is reduced, and many of the sites available for resin penetration are eliminated. [1]

The concept of hybridization of dentin is the benchmark of good bonding, according to Nakabayashi et al. The formation of hybridized dentin is greatly dependent upon the permeability of the dentin substrate to which a dentin bonding agent is applied, as well as the diffusion potential of the applied adhesive monomer. Hybridized dentin reduces the risk of microleakage, the incidence of secondary caries, and postoperative sensitivity that can be caused by such leakage. [2]

Based on the number of application steps, Van Meerbeek classified adhesives as: [3]

  • Etch and rinse adhesives
  • Self etch adhesives
  • Resin modified glass ionomer adhesives


In an effort to reduce the number of steps involved in bonding procedures, but at the same time retain the effectiveness of dentin adhesives, a self-etch approach was identified in the early 90's. In this approach, the steps etching, rinsing and bonding was reduced to etching and bonding, thereby eliminating one step. This 'No rinse technique' lessens clinical operating time. [4]

Additionally, self etching primers are less likely to result in a discrepancy between the depth of demineralization and depth of resin infiltration because self etching primers demineralize and infiltrate dentin simultaneously. Self etching primers do not remove the smear layer from dentin completely, which is the main reason that they result in less postoperative sensitivity than total etch adhesives. [5]

Development in the microscopic technology has improved the study of hybridization. The confocal microscope, with its advantage of obtaining laser penetrated thin optical sections below the surface of intact specimens, and eliminating the artifacts that arise with manual sectioning, offers superior images of the resin dentin interface. [6],[7]

The aim of this study was to evaluate the hybrid layer and resin tag formation of a total etch technique in comparison with self etching primers with three different ph using a con focal microscope.


   Materials and Methods Top


20 non carious human premolars (5 in each group) stored in normal saline were used for the study. Collection, storage, sterilization and handling of extracted teeth to be used in the study followed the Occupational Safety and Health Administration (OSHA) and the Centre for Disease Control and Prevention (CDC) recommendations and guidelines.

Occlusal surfaces of all the teeth were ground using slow speed diamond disc with copious water supply, so as to expose a flat surface of dentin with an approximate residual dentin thickness of 1.5-2mm. A flat bonding surface devoid of enamel remnants or pulp exposure was then prepared by polishing each dentin surface with 600 grit silicon carbide paper.

The teeth were then randomly divided into 4 groups of 5 specimens each.

Group 1:

(Blot dry) -Prime and Bond NT (Dentsply)

The dentin surfaces were etched using 36% phosphoric acid etchant and rinsed with distilled water for 20 seconds.

A small piece of absorbent paper was then used to blot out the excess water that remained after rinsing off the acid conditioner. Thus the dentin was maintained in a moist condition. Bonding agent was then applied and cured for 15 sec's.

In group 1, 2 and 3 the SEP was mixed according to the manufactures instructions and applied to the prepared dentin surfaces.

Group 2:

Strong self etch adhesives having a ph of 1 or below {Adper Prompt L- Pop (3M ESPE)}

It involves a single step application technique. The adhesive was applied on the prepared dentin surface, for a period of 15 sec followed by gentle air drying. The adhesive was then light cured for 10 sec's.

Group 3:

Intermediary self etch adhesives having a ph of 1.5 {AdheSE (Vivadent)}

Here the adhesive is supplied as primer and bond and involves a two step application procedure. First, the primer is applied on the prepared dentin surfaces for 15 sec followed by a strong stream of air to disperse the primer. Secondly, bond is applied and uniformly spread with a weak stream of air and immediately polymerized by light curing for 10 sec's.

Group 4:

Mild self etch systems having a ph of 2- {Clearfil SE bond plus primer (kuraray)}

Is supplied as primer and bond and involves a two step application procedure. First, the primer is applied on the prepared dentin surfaces and left in place for 20 sec followed by drying with mild airflow. Secondly, bond is applied and uniformly spread with a weak stream of air and immediately polymerized by light curing for 10 sec's.

Fluorescent labeling of bonding agent

The visualization of the distribution of bonding agents can be greatly enhanced by the incorporation of fluorescent labels. In this study Rhodamine B dye was used. The dye was mixed with bonding agent before its application.

Visible light cure unit

In this study Translux-CL (kulzer) visible light cure unit was used to cure all the specimens.

All the bonded surfaces were restored with 2mm of resin composite Z 350 (3M) and cured for 40 seconds, to protect the bonding layer. All the specimens were stored with normal saline. After 24 hrs of storage the teeth were longitudinally sectioned using a slow speed diamond disc under copious water supply. The sectioned surface was polished with a 600 grit silicon carbide paper. The dentin/adhesive interfacial region was examined using a Confocal Laser Scanning Microscope (CLSM)

Confocal Laser Scanning Microscope

Confocal Laser optical Microscope has recently emerged as a significant new technique which exhibits several advantages over conventional optical microscopy. The most important of these arise from the fact that out of focus blur is essentially absent from confocal images, giving the capability for direct non invasive serial optical sectioning of intact and even living specimens.


   Results Top


Statistical analysis was done to compare the values among the groups using Kruskal-Wallis One way ANOVA appropriately by testing the means.

Mann- Whitney U test was employed to identify the significant groups at 5% level.

From the above results it can be concluded

  • Mean length of resin tag formation in Group I was maximum followed by Group IV, Group II and Group III respectively.
  • Mean thickness of hybrid layer formation in Group I was maximum followed by Group IV, Group II and Group III respectively.



   Discussion Top


The American Society for Testing and Materials (ASTM; specification D 907) defines adhesion as "the state in which two surfaces are held together by interfacial forces which may consist of valence forces or interlocking forces or both. " The word adhesion comes from the Latin word adhaerere ("to stick to") [3]

The key element for adhesion is the intimate bond that develops between the adhesive and the substrate. So, the interaction of tissues and biomaterials at biologic interfaces is extremely important, but it is very difficult to connect natural tissues (Enamel, Dentin) with artificial materials as reported by Nakabayashi et al [8],[9] . To solve this problem in teeth, a liquid must first be used to form an intimate contact between the biomaterial and the solid hard tissue which is then converted to a solid via polymerization. This is the approach that is selected in an attempt to provide retention of biocompatible monomer with tooth substrates. [2],[9]

Mineralized dentin usually does not permit much monomer diffusion into its substance. Therefore, dentin must be suitably conditioned to permit diffusion of monomers, which should have a good affinity for demineralized dentin, into the substrate. [9] Prepared dentinal surfaces are covered with a smear layer that adheres weakly to the underlying intact dentin. Dentin conditioning involves the removal or modification of the smear layer to permit monomer diffusion into the demineralized collagen matrix. [10],[11],[12]

Earlier used dentin bonding agents became multi step systems with more complicated, time consuming and technique sensitive application procedures. In the early 90s, the selective enamel etching technique was replaced by a total etch concept. Now more recent research and developments efforts have focused on simplifying the multi step bonding process and reducing its technique sensitivity. [13]

In an Etch and rinse technique the biggest challenge for the hybrid layer and resin tag formation comes in the form of smear layer. Unless the smear layer is removed, neither hybridization nor resin tag formation can occur. Hence, it becomes necessary to acid etch the dentin to remove the smear layer and smear plug. Acid etching removes the smear layer, opens the dentinal tubules, increases dentinal permeability and decalcifies the inter-tubular and peritubulardentin. [2],[14]

As acid etching is absolutely essential for good dentin bonding, [15] the question arises as to whether the acid etched dentin must be dehydrated or rehydrated or do we stop in-between.

Both conditions of extreme overwetting of dentin and total dehydration can be detrimental to dentin bonding [2],[14] . So, the concept of moist bonding came into existence. [16]

In order to overcome the phenomenon of over wetting and over drying Nakabayashi and his colleagues hypothesized that if the bonding step with acidic conditioner to remove smear layer was eliminated, then the bonding procedure would become simpler. This led to the development of self-etching/ self-­priming system, where the monomer could penetrate into underlying intact dentin to form both hybridized smear layer and hybridized dentin. These bonding systems do not employ separate acidic conditioning solutions as mentioned earlier. Instead they are applied directly to smear layer. [17],[18],[13] In addition to simplifying the bonding technique, the elimination of the rinsing and drying step reduces the possibility of over wetting or over drying which can have a negative influence in adhesion. [19],[20],[21] The actual rationale behind these systems is to superficially demineralize dentin and to simultaneously penetrate it to the depth of demineralization with monomers that can be polymerized in situ. [15],[22]

These reactive components of self-etching primers are basically esters from bivalent alcohols with methacrylic acid and phosphoric acid or their derivatives. [23] The phosphate residue is thought to etch the enamel, while the methacrylate component of the molecule is available for co-polymerization with the bonding agent and composite resin. With this process there is no need to rinse off reaction products or residual phosphoric acid ester, because both are subsequently polymerized into bonding layer. [15],[22]

Van Meerbeek classified self etching primers based on their pH into strong, intermediary and mild self­etch adhesives. [24]

  • Strong self etch adhesives usually have a ph of 1 or below e.gAdperPromtL-Pop (ph-0.4).
  • Intermediary self etch adhesives have a ph of about 1.5 e.g OptiBond Solo Plus SE primer (ph­1.5),AdheSe (ph-1.4).
  • Mild self etch systems have a ph of about 2 e.g Clearfil SE Bond plus primer (ph-2.0), Unifil Bond primer (ph-2.2).


Van Meerbeek classified self etching primers based on their pH into strong, intermediary and mild self­etch adhesives. [24]

Common methods used to visualize bonding structures such as hybrid layer and resin tags in dentin are transmission electron microscopy (TEM), scanning electron microscopy (SEM), Fourier Transform Infrared spectroscopic analysis (FTIR) and Micro-Raman Spectroscopy. TEM requires an embedding process. SEM can show only the surface details brought into relief by an etching process. [25] Drawbacks of FTIR technique are the spectral interference from water and the limited spatial resolution. [8] Fluorescence from the sample can obscure the Raman signal in some parts of the spectrum. [5]

The confocal microscope has an advantage of obtaining laser penetrated thin optical sections below the surface of intact specimens. This eliminates the artifacts that arise with manual sectioning and offers superior images of the resin dentin interface. [25],[26],[27] Another advantage of using confocal microscopy is that out of focus blur is absent from confocal images, giving the capability for direct non invasive serial optical sectioning of intact and even living specimens. [7]

Hence we chose this technique for analyzing the hybrid layer and resin tag formation of different bonding agents employed in our study.

In our study Prime and Bond NT (Group I) showed the maximum values when compared to self etching primers and the values were statistically significant.

Possible reasons could be, in case of Group I the specimens were etched with phosphoric acid and the reaction products were rinsed off. This results in complete removal of smear layer and smear plugs from the dentin, [28] so the dentin permeability increases and there is better infiltration of resin, and hence aids in the better penetration of the resin tags and also thick hybrid layer. [11]

But in case of self etching primers (Group II, III, and IV) as the primer is not rinsed after application but air dried only. The calcium and phosphate ions that were dissolved from hydroxyapatite crystals must have been suspended in the watery solution of the primer. When the water is evaporated during air drying the concentrations of solubilized calcium and phosphate ions within the primer may exceed the solubility constant for a number of calcium phosphate salts. [15],[29]

Presumably minerals will then precipitate within the primer. This high concentration of calcium phosphate will tend to limit further dissolution of apatite due to the common ion effects of calcium and phosphates and thereby limit the depth of surface demineralization. [15],[29]

The present findings are corroborated by the report of R.H. Sundfeld et al, in 2005 which revealed that as self-etching adhesive materials eliminate the need of rinsing the tooth structure, the byproducts of dentin yielded by the low pH of the adhesive system may lead to a limited demineralizing action, restricting penetration of the adhesive system to the most superficial dentin layers. Moreover, the mineral components from the smear layer may neutralize the acidity of these self-etching systems. [30]

Another reason can be as the Prime and Bond was applied using a wet bonding technique. In wet bonding technique water is retained in collagen fibrils and acetone was able to diffuse and dislocate water from the moist collagen network and thus promoting the infiltration of resin monomers through the nano-spaces of the collagen web and resulting in dense hybrid layer and also enhancing the bond strength. [2],[16]

Similar results were early reported by Mohan et al (2005) who stated that maximum width of hybrid layer formation and maximum length of resin tags was best achieved when the dentin was kept moist by blotting and when acetone based adhesive was used. [2]

Among the self etching primer Clearfil SE (Group IV) which has mild pH (2.0) performed better than Adper Promt (Group II) with strong pH (.4) and AdheSe (Group III) with intermediary pH (1.4) performed the least.

Good performance of Clearfil SE (Group IV) on dentin can be explained by its specific and adapted composition and the use of the functional monomer 10-MDP (10-methacryloyloxy methacrylate), which has been shown to exhibit high chemical interaction capacity to hydroxyapatite as stated by Yoshida et al. [12],[23]

Although current approach for bonding appear to depend on its effective penetration of partially demineralized dentin (hybrid layer formation) and intra-tubular dentin (resin tag formation), chemical interaction between the bonding agent and the substrate have also been reported as one of the contributing factor by Van Meerbeek et at. [24]

In 2006 Salz et al reported that aggressiveness of the self etching adhesives not only depends on pH value but also on several factors, such as water content, the amount and type of acidic monomer, polarity, and composition of the co-monomers and solvents [21] , This could be one of the reason for better results of Clearfil SE (Mild) than Adper Promt (Strong) and AdheSe (Mild), in our study.

Although the values of Adper Promt (Group II) were greater than AdheSe (Group III) in hybrid layer formation but these values were not statistically significant.

Within the limitations of our study total etch technique performed better than self etch but more studies are needed to evaluate the clinical performance of self etching primers.


   Conclusion Top


In our study amongst the groups tested for resin tag formation and hybrid layer thickness, the Prime and Bond NT (Group I) performed better than all the three self etching primers.

Among the self etching primers Clearfil SE (Group IV) with mild pH (2) performed better than Adper Prompt (Group II) with pH (.4) and AdheSe (Group III) with pH (1.4).

 
   References Top

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2.Bhuvaneswaran Mohan, Deivanayagam Kandaswamy. A Confocal Microscopic evaluation of resin-dentin interface using adhesive systems with three different solvents bonded to dry and moist dentin- An in vitro study. Quintessence Int 2005; 36; 511-521.  Back to cited text no. 2    
3.B Van Meerbek, M Vargas, S Inoue, Y. Yoshida, M Peumans et al. Adhesives and Cements to promote Preservation Dentistry. Operative dentistry 2000; 6; 119-144.  Back to cited text no. 3    
4.Sturdevant's. Art and Science of Operative Dentistry; 4 th edt  Back to cited text no. 4    
5.Cecilia Goracci, Fernanda Tranchesi Sadek, Francesca Monticelli, Paulo Eduardo Capel Cardoso, Marco Ferrari. Microtensile Bond Strength of Self-etching Adhesives to Enamel and Dentin. J adhes Dent 2004; 6; 313-318.  Back to cited text no. 5    
6.T. Pioch, S. Stotz, H.J. Staehle, H. Duschner. Applications of Confocal Laser Scanning Microscopy to Dental Bonding. Adv Dent Res 1997; 11(4);453-461.  Back to cited text no. 6    
7.Timothy F. Waston. Applications of Confocal Scanning Optical Microscopy to Dentistry. BDJ 1991;9;287-291.  Back to cited text no. 7    
8.F. R. Tay, A. J. Gwinnet, K. M. Pang et al. Structural Evidence of a sealed tissue interface with a Total Etch Wet bonding Technique in vivo. J Dent Res 1994; 73 (3); 629-636.  Back to cited text no. 8    
9.Nakabayashi N, Pashley DH. Hybridization of dental hard tissues. Chicago; Quintessence; 1998.  Back to cited text no. 9    
10.K.T. Maciel, R. M. Carvalho, R. D. Ringle et al. The Effects of Acetone, Ethanol, HEMA, and Air on the Stiffness of Human Decalcified Dentin Matrix. J Dent Res 1996;75(11); 1851-1858.  Back to cited text no. 10    
11.Sofia S.A. Oliveira, Megan K Pugach, Joan F. Hilton, Larry G. Watanabe, Sally J. Marshall et al. the influence of the dentin smear layer on adhesion; a self-etching primer vs a total-etch system. Dental Materials 2003; 19; 758-767.  Back to cited text no. 11    
12.Genevieve Gregoire, Yasin Ahmed. Evaluation of the enamel etching capacity of six contemporary self-etching adhesives. Journal of Dentistry 2007; 35; 388-397.  Back to cited text no. 12    
13.B Van Meerbek, M Vargas, S lnoue, Y. Yoshida, M Peumans et al. Adhesives and Cements to promote Preservation Dentistry. Operative dentistry 2000; 6; 119-144.  Back to cited text no. 13    
14.Takao Fusayama, Masato Nakamura, Norimasa Kurosaki, Masaaki Iwaku. Non-Pressure Adhesion of a NewAdhesive Restorative Resin. J Dent Res 1979; 58 (4); 1364- 1370.  Back to cited text no. 14    
15.M Hannig. K. J Reinhardt, B Bott. Self-Etching Primer vs Phosphoric Acid; An Alternative Concept for Composite to Enamel Bonding. Operative dentistry 1999,24; 172-180.  Back to cited text no. 15    
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18.Gordon J Christensen. Self- etching primers are here. JAm DentAssoc 200 1; 132(7); 1041-1043.  Back to cited text no. 18    
19.Satoshi Inoue, Marcos A Vargas, Yasuhiko Abe, Yasuhiro Yoshida et al. Microtensile Bond Strength of Eleven Contemporary Adhesives to Dentin. JAdhesive Dent 2001; 3; 237-245.  Back to cited text no. 19    
20.Sofia S.A. Oliveira, Megan K Pugach, Joan F. Hilton, Larry G. Watanabe, Sally J. Marshall et al. the influence of the dentin smear layer on adhesion; a self-etching primer vs a total-etch system. Dental Materials 2003; 19; 758-767.  Back to cited text no. 20    
21.Ulrich Salz, Angela Mucke, Jorg Zimmermann, Franklin R Tay, David H. Pashley. pKa value and Buffering Capacity of Acidic Monomers Commonly used in Self-etching Primers. J Adhes Dent 2006; 8; 143-150.  Back to cited text no. 21    
22.Fraklin R Tay, David H Pashely. Aggressiveness of contemporary self-etching systems. I; Depth of penetration beyond dentin smear layers. Dental Materials 2001; 17;296-308.  Back to cited text no. 22    
23.Y. Yoshida, K. Nagakane, R. Fukuda, Y. Nakayama et al. Comparative Study on Adhesive Performance of Functional Monomers. J Dent Res 2004; 83 (6); 454-458.  Back to cited text no. 23    
24.B Van Meerbeek, J De Munck, YYoshida, Sinoue, M vargas, P vijay, K Van Landuyt, P Lambrechts, G Vanherle. Adhesion to Enamel and dentin: Current Status and Future Challenges. Operative Dentistry 2003; 28 (3); 215-235.  Back to cited text no. 24    
25.T. Pioch, S. Stotz, H.J. Staehle, H. Duschner. Applications of Confocal Laser Scanning Microscopy to Dental Bonding. Adv Dent Res 1997; 11(4);453-461.  Back to cited text no. 25    
26.T. F. Waston. A Confocal Optical Microscope Study of the Morphology of the Tooth/Restoration Interface using Scotchbond 2 Dentin Adhesive. J Dent Res 1989, 68 (6)' 1124-1131.  Back to cited text no. 26    
27.T. F. Waston, D. M. de J. Wilmot. A Confocal microscopic evaluation of the interface between Syntac Adhesive and tooth tissue. J Dent 1992; 20;302-310.  Back to cited text no. 27    
28.F. R. Tay, A. J. Gwinnet, K. M. Pang et al. Structural Evidence of a sealed tissue interface with a Total Etch Wet bonding Technique in vivo. J Dent Res 1994; 73 (3); 629-636.  Back to cited text no. 28    
29.Jean Camps, David H Pashely. Buffering Action of Human Dentin. J Adhesive Dent 2000; 2; 39-50.  Back to cited text no. 29    
30.Renato Herman Sundfeld, Thiago Assuncao Valentino, Rodrigo Sversut de Alexandre et al. Hybrid layer and resin tag length of a self-etching adhesive bonded to sound dentin. Journal of Dentistry 2005; 33; 675-681  Back to cited text no. 30    

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Correspondence Address:
Parvinder Singh Baweja
Department of Conservative Dentistry and Endodontics, Meenakshi Ammal Dental College, Maduravoyal Chennai 600 095
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0972-0707.42270

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