|Year : 2015 | Volume
| Issue : 1 | Page : 77-81
|Scanning electron microscopy analysis of microstructure of the adhesive interface between resin and dentin treated with papain gel
Anderson Jana Rosa1, Eduardo Moreira da Silva2, Monica Almeida Tostes3
1 Department of Radiology, Fluminense Federal University, Campus Valonguinho, Niteroi, Rio de Janeiro, Brazil
2 Analytical Laboratory of Restorative Biomaterials (LABiom-R), Fluminense Federal University, Campus Valonguinho, Niteroi, Rio de Janeiro, Brazil
3 Department of Pediatric Dentistry, School of Dentistry, Fluminense Federal University, Campus Valonguinho, Niteroi, Rio de Janeiro, Brazil
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|Date of Submission||29-Oct-2014|
|Date of Decision||06-Dec-2014|
|Date of Acceptance||19-Feb-2015|
|Date of Web Publication||11-May-2015|
| Abstract|| |
Aim: The aim of this in vitro study was to observe and compare the microstructure of the adhesive interface between resin and dentin treated with Papacarie® using scanning electron microscopy (SEM).
Materials and Methods: Totally, 10 human dentin slabs were randomly distributed into two groups. The control group (n = 5) was subjected to etching with 37% phosphoric acid for 20 s and washed for 30 s, dried with absorbent paper, and the bonding agent was applied along with low viscosity resin. In turn, the experimental group (n = 5) was subjected to the same procedure, but Papacarie® (Fσrmula and Aηγo, Sγo Paulo, Brazil) was added for 30 s prior to etching. Adper™ Single Bond 2 (3M ESPE, Sγo Paulo, Brazil) adhesive was applied to both groups following manufacturer instructions. The specimens were prepared for observation under SEM with ×1.000, ×2.000, ×2.200 and ×5.000 magnification. The micrographs were evaluated with respect to the formation of the hybrid layer, thickness, shape and length of the tags and microtags.
Results: In the experimental group there was the formation of more fine hybrid layer and tags with average of similar length to the control group; microtags in less number and without formation of lateral branches. The resin tags presented conical, smooth and uniform characteristics.
Conclusions: When Papacarie® was used prior to the application of a bonding agent it could interfere with the formation of the hybrid layer without changing the length of the tag. Moreover, the morphology in the experimental group was found to be more uniform and regular.
Keywords: Dentin, dentin-bonding agents, papain
|How to cite this article:|
Rosa AJ, da Silva EM, Tostes MA. Scanning electron microscopy analysis of microstructure of the adhesive interface between resin and dentin treated with papain gel. Indian J Dent Res 2015;26:77-81
Recent scientific and technological advances have substantially altered the treatment of carious lesions. Removal of the carious tissue, which was traditionally performed by means of mechanical instruments, is currently carried out by different methods with a view to preserving the healthy tooth structure. Among these new methods, the chemical removal of caries by substances that soften the carious dentin for subsequent removal is often recommended.
|How to cite this URL:|
Rosa AJ, da Silva EM, Tostes MA. Scanning electron microscopy analysis of microstructure of the adhesive interface between resin and dentin treated with papain gel. Indian J Dent Res [serial online] 2015 [cited 2019 Nov 13];26:77-81. Available from: http://www.ijdr.in/text.asp?2015/26/1/77/156816
Carisolv™, which consists of a gel based on sodium hypochlorite and amino acids associated with the use of special manual instruments, has been widely studied and effectiveness in the removal of decay and well accepted by patients. ,, Although, the steps necessary for the application of the material and final price of the product, may be obstacles to regular use in clinical routine.  Papacarie® (Formula and Aηγo, Sγo Paulo, SP, Brazil), consisting of a papain-based gel, toluidine blue, and chloramine, was launched in Brazil in 2003. The material is indicated for removal of carious dentin, thus eliminating infected and necrotic tissues. The gel removes caries effectively and is well accepted by patients, but the various aspects involving the chemical removal of caries and adherence warrant further evaluation. 
The action of Papacarie® gel on the dentin has not been well established yet. According to Bussadori et al.,  it acts selectively on carious tissues only there are tissue that contain partially degraded collagen fibers, whereas healthy tissues contain an antiprotease named alpha-1-anti-trypsin, which prevents the action of proteolytic enzymes. Other authors found that chloramine - a substance present in papain gel - can partially degrade collagen and affect the bonding mechanism.  According to Piva et al.,  bond strength is compromised when carious dentin is treated with papain gel. A similar result was found by Cecchin et al.,  regardless of the chemical agent used. Gianini et al.,  found no difference in the microtensile of demineralized dentin, whereas Botelho et al.,  found no decrease in bond strength, but a hybrid layer was formed differently in response to the different adhesives tested, although this finding did not interfere with the application of papain gel. Chemical methods (papain gel) form an irregular, amorphous layer that is similar to the smear layer with low exposure of tubular dentin compared to the conventional method, which produced a smooth, regular exposure of the dentinal tubules. 
Therefore, the purpose of this study is to examine the adhesive-dentin interface with the aid of a scanning electron microscope (SEM), and study the morphology and thickness of the hybrid layer in healthy dentin of premolars, with and without the use of Papacarie® combined with Adper™ Single Bond 2 dental bonding agent.
| Materials and methods|| |
Totally, 10 healthy human premolars were selected for this study. Tissue remnants and debris were removed from the teeth, and the teeth were refrigerated for up to 1-month in a 0.05% chloramines disinfectant solution. This research protocol received previous consent from the Research Ethics Committee, School of Dentistry, Federal Fluminense University (protocol number 175/06).
The occlusal surface of each tooth was ground flat with a water-cooled. Half of the crown height was removed to expose mid-coronal dentin. Dentin was polished with wet 240, 400 and 600-grit. In order to obtain dentin slices, teeth were ground across the crown and after exposing the area of the dentin, a cut with 2 mm thick was made parallel to the surface, which had been ground with a diamond disc (KG Sorensen, Cotia, Sγo Paulo, Brazil). The discs were divided into two groups, that is, the control group (n = 5) and experimental group (n = 5).
The dentin was etched for 20 s with 37% phosphoric gel (Magic acid, Vigodent, Bonsucesso, Rio de Janeiro, Brazil), rinsed with water and gently air dried for 30 s. The dentin was dried with absorbent paper. Adper™ Single Bond 2 adhesive (3M ESPE, Sumarι, Sγo Paulo, Brazil) was applied to the etched dentin surfaces for 10 s and gently air dried, and light-cured for 20 s. A low-viscosity resin (Natural Flow, DFL, Rio de Janeiro, Brazil) was applied on the adhesive with a thickness of 1 mm and light-cured for 20 s (Degulux® Soft start, Degussa Hulls) and radiometer (Optilux 400; Demetron Research Corporation, Danbury, Connecticut, USA).
Papacarie® gel was applied for 30 s and removed with cotton balls soaked in water. Thereafter, etching acid, bonding agent and low viscosity resin (Natural Flow, DFL, Rio de Janeiro, Brazil) were applied as described for the control group.
To evaluate the ultrastructure of the dentin and the characteristics of the hybrid layers which were formed after surface treatment, the disks were cut transversely to the adhesive surfaces with double-sided diamond discs under refrigeration, giving rise to dentin sticks. Each of the cross sections was polished along the cut surface with a series silicon carbides disks (#400, 800, 1000, 1200) and rinsed under water. The sections were treated with 37% orthophosphoric acid for 5 s, rinsed with water and treated with 5% NaOCl for 10 min. The samples were then left at room temperature for desiccation and gold-sputter coated for evaluation under SEM (JEOL model JSM-5800) with magnification ranging from ×1000 to ×5000. The micrographs were evaluated with respect to the formation of the hybrid layer, thickness, shape and length of the tags and microtags, and then measured with Image Tools software (UTHSCSA Image Tool version 3.0, Dental Diagnostic Science, San Antonio, USA).
| Results|| |
Sample results of the experimental and control groups are shown in the micrographs.
The experimental group (papain gel) showed uniform formation of resin tags with penetration and cylindrical shape, with the length ranging from 8.58 to 45.79 μm (average of 23.03 μm) [Figure 1]a]. There was the formation of a thinner hybrid layer compared to the control group, with a large formation of lateral microtags as shown in the sample from the control group [Figure 1]b]. In the control group [Figure 1]b] tag length ranged between 12.14 and 42.17 μm, with a mean of 23.69 μm.
|Figure 1: Scanning electron microscopy micrograph of the sample. Dentin treated with papain gel (a) low viscosity resin (LVR), dentin adhesive (Ad), cylindrical tag (T) formation is clearly observed. Dentin without papain gel (b) hybrid layer (HL)|
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[Figure 2] depicts the smooth and regular surfaces of tags in the experimental group (a) compared to the control group (b). [Figure 2]b shows clearly the formation of tags with a mean diameter of 3.18 μm at the entrance of the dentinal tubules and microtags with 2.50 μm average length. There was hybrid layer formation, and tags had more regular and smooth surfaces, tending toward more uniform cylindrical shapes without bulb formation, while microtags were well defined, suggesting that the bonding agent penetrated directly into the tubules.Tag diameter at the entrance of the dentinal tubules ranged from 2.14 to 4.20 μm with a mean of 3.15 μm [Figure 2]b].
|Figure 2: Scanning electron microscopy micrograph (×5000) of the sample. Dentin treated with papain gel (a) and without papain gel (b)|
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In assessing the thickness of the hybrid layer, the mean value recorded was 3.04 μm, as it ranged from 2.50 to 3.75 μm. Tag formation occurred conically with plenty of lateral microtags and abundant lateral branches [detailed in [Figure 3]b].
|Figure 3: Scanning electron microscopy micrograph (×2200) of the sample. Dentin treated with papain gel (a) lateral microtags (MT); and without papain gel (b) lateral branches (LB)|
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[Figure 4]a and b are shown here with the purpose of highlighting the differences and similarities between features observed in the experimental and control groups.
|Figure 4: Scanning electron microscopy micrograph (×2000) of the sample. Dentin region treated with papain gel (a) and was not treated with papain gel (b)|
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| Discussion|| |
The chemomechanical caries removal, when compared with removal using conventional rotary instrument, seems to preserve healthy tooth with less trauma to the patient.  The usage of minimally invasive procedures and attention to patient comfort are of great importance, especially for dental treatment in small children. 
The hybrid layer is formed by the interpenetration of monomers into the dentin and subsequent polymerization. It is considered that the extent of demineralization and the texture of the dentin surface treated with Papacarie® are an important factor influencing monomer penetration. Studies have showed that the smear layer formed after treatment with papain gel may appear irregular and with open tubules or with minimal formation of smear layer on both primary and permanent teeth, respectively. , Corrκa et al.,  obtained an irregular, rough surface in primary teeth and emphasized that the action of Papacarie® on this substrate is different due to the characteristics of dentin in primary teeth. In this study, the surfaces treated with Papacarie® exhibited two different patterns of remaining dentin: A regular surface with little smear layer and exposed dentinal tubules ,, and a very irregular and rough surface covered by an amorphic layer indicating the presence of smear layer obliterating the dentinal tubules. , It should be emphasized that primary teeth have a less mineralized dentin than permanent teeth, which makes them more sensitive to acid, thereby yielding a thicker hybrid layer. ,
The smear layer on the dentin substrate of the present study was produced using 600-grit silicon carbide paper and it was previously been demonstrated that smear layer produced using this method are thicker than layers obtained using a carbide bur.  SEM analysis showed a continuous hybrid layer with resin tags and lateral branches in the group without Papacarie® while, in the experimental group and a thin hybrid layer, no was formation of lateral branches.
At the resin-dentin interface, no treatment with Papacarie® yielded a thick hybridized complex with very long and thick tags extending almost through the entire-dentin inter-diffusion zone and lateral branches. In group, the formation of a great entanglement of tags occurred, and microtags with tapered bases could be seen as well as a few collagen fibrils infiltrated in the region where the tubules narrow down. In the Papacarie® group similar morphology was found but with different characteristic. SEM analysis didn't show lateral branches. Phosphoric acid decalcifies the superficial dentin, removes calcium and phosphate ions, and exposes the collagen. These micropores are formed by the action of phosphoric acid, which removes the smear layer and smear, demineralizing the peritubular dentin at the entrance of the dentinal tubules,  leaving them in the form of a funnel, ,, while exposing collagen, noncollagen fibers and proteoglycans reinforced by apatyte crystals  to form the hybrid layer. ,, In the Papacarie® group seems that the degree of the acid infiltration was lower. According to Piva et al.,  the remnants of the gel could stagnate on dentine surfaces, and hybrid layers may not be completely formed with potentially interferes with the bond mechanism. In addition, the smear layer removal and the collagen removal theoretically promotes more intimate contact between the adhesive and dentin. In the present study, tag formation and penetration into tubules were present in both without and with Papacarie® groups [Figure 3]a and b] but in samples from Papacarie® chemomechanical group, a similar tag formation occurred but not occurred complete demineralization in dentin intertubular and therefore did not provide lateral branches [Figure 3]a]. Arora et al.,  observed a thin smear layer in permanent teeth as well as the widespread formation of tags that were longer than in the present study. However, in other study, SEM analysis showed no interference of Papacarie® in the formation of the hybrid layer.  Corrκa et al.,  revealed innumerable elongated tags and a small amount of microtags.
Another important factor regarding the thickness of the hybrid layer is the type of bonding agent used.  Studies revealed that conventional single bond adhesive is associated with the thickest hybrid layers, while self-etching adhesives produced thinner layers. , In this study, a two-step-and-rinse (Adper Single Bond 2) was used and showed an intermediate hybrid layer thickness in the control group. The same results were found by Botelho et al.,  when identical adhesive was used. However, one-step self-etching showed no evidence of tag formation when Papacarie® was used. Research has been made to assess the formation of tags a according the type of adhesive system and removal of caries used. ,,
It is noteworthy that papain gel is indicated for the removal of carious dentin, and in this substrate, the results could potentially differ from those found in the present study. In the operative treatment of carious lesions in dentin, the morphology and nature of the prepared dentin surface influences bonding of adhesive restorative materials. Thereby, the bond strength to carious dentin of the self-etching system was negatively affected by chemo-mechanical excavation using the papain-based gel.  Nevertheless, caries removal with chemomechanical papain-based method did not interfere in the adhesion of the tested adhesive systems (etch-and-rinse, two-step self-etch and a one-step-self-etch) the demineralized dentin. 
According to Nakabayashi and Saimi  a quality hybrid layer plays an important role as a barrier to prevent demineralization by cariogenic agents. The absence of a hybrid layer and denaturation of collagen fibers can change dentin strength given that ideally the integrity of the collagen matrix should be preserved because it is a key factor in the durability of the dentinal connection.  Bertassoni and Marshall  concluded that intact nonmineralized type I collagen fibrils were partially degraded by papain gel and showed through mechanical data that the properties of normal dentin tend to decrease gradually as the papain gel applications were made.
Further studies are necessary to shed light on the effects that may result from the changes found in this study, notably their impact on durability of the bonding agent/dentin interface, microleakage and microhardness when using Papacarie® .
| Conclusion|| |
According to the methods employed in this study, the findings suggest that whenever Papacarie® is used prior to the application of a bonding agent it can interfere with the formation of the hybrid layer without changing the length of the tag. Moreover, the morphology in the experimental group was found to be more uniform and regular than in the control group, with a reduction in the number and size of microtags.
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Anderson Jana Rosa
Department of Radiology, Fluminense Federal University, Campus Valonguinho, Niteroi, Rio de Janeiro
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2], [Figure 3], [Figure 4]
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