|Year : 2015 | Volume
| Issue : 1 | Page : 1-7
Histopathological evaluation of human pulp response to two self-etching resins
Nevin Cobanoglu1, Fusun Ozer2, Mustafa Demirci3, Ozgür Ozdemir4, Satoshi Imazato5
1 Department of Restorative Dentistry, Faculty of Dentistry, University of Selcuk, Konya, Turkey
2 Department of Preventive and Restorative Sciences, University of Pennsylvania, Philadelphia, USA
3 Department of Restorative Dentistry, Faculty of Dentistry, University of Istanbul, Istanbul, Turkey
4 Department of Pathology, Faculty of Veterinary, University of Selcuk, Konya, Turkey
5 Department of Biomaterials Science, Faculty of Dentistry, University of Osaka, Osaka, Japan
|Date of Web Publication||27-Jan-2015|
Department of Restorative Dentistry, Faculty of Dentistry, University of Selcuk, Konya-42075
Source of Support: None, Conflict of Interest: None
Objective: The aim of this study was to evaluate the human pulp response following the application of two proprietary self-etch adhesive systems (Clearfil Protect Bond and Clearfil SE Bond). Materials and Methods: Deep Class V cavities were prepared on the teeth scheduled to be extracted. Cavities were covered with a composite resin (Clearfil APX) following the application of the bonding agents. The teeth were extracted after 7 or 90 days and prepared for histological investigation. The pulp response was categorized. Two cavities were restored with calcium hydroxide used as the control in the 7-day period. The data were submitted to two-way analysis of variance (ANOVA), Mann-Whitney U tests and Wilcoxon signed-rank test. Results: Inflammatory cell infiltration was not shown in most of the teeth. No significant statistical difference concerning all the histological evaluation criteria was observed among the groups of adhesive systems or between the control group and the groups of adhesive systems. (P > 0.05). Conclusion: Both bonding systems were found biologically compatible with pulp tissues in non-exposed cavities.
Keywords: Antibacterial adhesive, pulp response, self-etch adhesives
|How to cite this article:|
Cobanoglu N, Ozer F, Demirci M, Ozdemir O, Imazato S. Histopathological evaluation of human pulp response to two self-etching resins. J Res Dent 2015;3:1-7
|How to cite this URL:|
Cobanoglu N, Ozer F, Demirci M, Ozdemir O, Imazato S. Histopathological evaluation of human pulp response to two self-etching resins. J Res Dent [serial online] 2015 [cited 2020 Jun 4];3:1-7. Available from: http://www.jresdent.org/text.asp?2015/3/1/1/150013
| Introduction|| |
Clinical and in vivo studies have shown a low incidence of unfavorable effects from adhesive systems, but pathological changes of pulpal tissues, such as dilatation and congestion of blood vessels, production of irregular dentin, and inflammatory responses, can occur after placement of resin restorations. , Material cytotoxicity and bacteria are the two most important possible reasons for these pathological changes. 
Most non-polymerized organic matrix components of the adhesive systems have shown a cytotoxic effect when in direct contact with mammalian fibroblasts.  Several studies have shown that the degree of the monomer-polymer conversion of resin-based materials varied between 35% and 77%. , The dentin fluid also interferes with the adhesive's polymerization on deep wet dentin.  Additionally, leachable components are released from resin materials due to degradation or erosion over time. These unconverted and leachable components of resin materials in the cavity may diffuse to the pulp through dentinal fluid  and may cause adverse effects. ,
However, some studies have confirmed that the bacteria at the tooth-restoration interface are more important than material cytotoxicity for pulp inflammations. ,, Bacteria may produce acids and toxins that can be responsible for the pulp reaction. , A hermetic sealing of the cavity margins is desirable to prevent nutrient transport to these bacteria and to maintain metabolism and proliferation, but gaps in which bacteria can colonize occur in tooth-restoration interfaces due to polymerization shrinkage in composite resins.  These gaps were decreased using layer-by-layer photo polymerization technology. Improvements in resin-containing materials have reduced the shrinkage. New adhesive technologies led to the formation of a hybrid layer and diminished the interface to less than 1 μm.  However, this is still a large gap for some microorganisms, such as Streptococcus mutans, which are 0.5−0.75 μm in diameter.  In addition, unbound free monomers are excellent substrates for cariogenic bacteria.  It has been demonstrated that triethylene glycol dimethacrylate (TEDGMA) stimulates the growth of S. mutans and Streptococcus salivarius in a pH-dependent manner. 
In order to tolerate the effects of bacteria in resin restorations, research has been conducted on the development of various antibacterial resins by incorporating monomer 12-methacryloyloxydodecyl pyridinium bromide (MDPB), which has antibacterial activities. , MDPB provides bacteriostatic properties after curing that act as an inhibitor to contact bacteria.  In addition, incorporation of this antibacterial monomer into a dentine adhesive system resulted in strong antibacterial effects against oral streptococci ex vivo due to its bactericidal activity at the unpolymerized stage. ,, Accordingly, it has been demonstrated that the adhesive system incorporating MDPB can show antibacterial effects before and after curing. ,,
The aim of this study was to compare the pulp response following application of two commercially available self-etch adhesive systems (antibacterial monomer MDPB-containing Clearfil Protect Bond, CPB; Kuraray Noritake Dental, and non-MDPB-containing Clearfil SE Bond, CSE; Kuraray Noritake Dental) in the cavities prepared in human teeth. The null hypothesis is that antibacterial monomer incorporation into the adhesive system increases pulp response.
| Materials and methods|| |
The study protocol was approved by the Local Ethics Committee, Selcuk University, Faculty of Dentistry, Konya, Turkey. Project no: (03/02).
Thirty-four caries-free human premolars scheduled to be extracted for orthodontic reasons from healthy patients, male or female, between 15 and 20 years of age were used in this study. The vitality of the teeth included in the study was evaluated using an electro-pulp tester device. Patients and their parents were informed with a written statement about the clinical procedures and the possible inconvenience that could arise. Those who agreed to participate in the research signed an acceptance form. The experimental procedures should have no effects on the therapeutic treatment of the patients and should not modify their treatment plans.
The teeth selected for the experiment were scaled and polished with a rubber cup on the day of the operative procedure. Local anesthesia (Ultracain D-S Forte, Aventis, Turkey) was provided prior to cavity preparation. With a diamond bur (801-014C, Diatech Dental Ac, Switzerland), which previously had its active tip limited to 2.5 mm with a resin stop to allow minimum differences among cavities depth, Class V cavities were prepared at high speed with copious water irrigation in the cervical region on the buccal surface. The final cavity dimensions were 3 mm in length, 2.5 mm in depth, and 1.5 mm in width. New burs were used for each procedure. Teeth were rinsed with sterile distilled water and isolated with a rubber dam, and saliva was controlled through high-speed evacuation. All procedures were performed by the same operator.
The adhesive materials used in this study and their application procedures are shown in [Table 1].
|Table 1: Application procedure, composition, pH and batch numbers of the used adhesive systems|
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Teeth were randomly divided into five experimental groups, as described in [Table 2], according to the materials used for treatment and the time period of evaluation. In Groups 1-4, after application of the bonding procedures according to the manufacturer's instructions, the cavities (with bonding) were restored with a hybrid restorative resin composite (Clearfil APX; Kuraray Noritake Dental, Tokyo, Japan). In Group 5 (control), the cavity floor was protected with hard-setting calcium hydroxide cement (Dycal-Dentsply, Milford, DE, USA) and restored with zinc oxide-eugenol cement (IRM-Dentsply, Caulk, Milford, DE, USA).
After 7 or 90 days, the teeth were extracted under local anesthesia (Ultracain D-S Forte, Aventis, Turkey) and prepared for histological analysis. Immediately after extraction the roots were sectioned in half with a high-speed hand piece under intensive water spray to allow penetration of the fixative. The teeth were fixed in a 10% neutral-buffered formalin solution for seven days. The specimens were then demineralized in 9% EDTA for five months. Finally, the teeth were embedded in paraffin and serially sectioned through the pulp at 7 μm thickness. All sections coming through the cavity floor were either stained with hematoxylin-eosin to assess soft tissue organization and tertiary dentine formation, or were subjected to Taylor's modified Brown-Brenn's technique to detect the presence of Gram-positive and Gram-negative microorganisms. All sections were evaluated blindly for five histologic features: Inflammatory cellular response, soft tissue disorganization, reparative dentin formation, bacteria, and the remaining dentin thickness (RDT). The pulp response was evaluated by light microscope using the criteria defined in [Table 3].
The data were submitted to two-way analysis of variance (ANOVA), Mann-Whitney U tests and Wilcoxon signed-rank test.
| Results|| |
In [Table 4], the mean and standard deviations of the minimum RDT in each group of teeth are presented. The RDT was compared between the groups using a two-way ANOVA, and a statistically significant difference was not found (P > 0.05).
|Table 4: Mean value of the minimum remaining dentine thickness and standard deviation values|
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Histopathological findings are presented in detail in [Table 5] and illustrated in [Figure 1] and [Figure 2].
|Figure 1: Observation period: 7 days. (a) Group CSE; RDT 1400 μm. Formation of vacuoles in the odontoblast layer. Presence of hyperemic pulpal vessels. Absence of inflammatory cell infiltration (H and E, ×40). (b) Group CSE; RDT 890 μm. Severe reduction in the odontoblast number. Absence of inflammatory cell infiltration (H and E, ×200). (c) Group CPB; RDT 1249 μm. Presence of some displaced odontoblastic nuclei into the dentinal tubules (arrow) and reduction in the odontoblast number. (d) Group CPB; RDT 1340 μm. Presence of slight inflammatory cell infiltration (arrow) (H and E, ×40). (e) Group Control; RDT 1807 μm. Absence of inflammatory cell infiltration. Slight reduction in the odontoblast number in a region of the cavity. (H and E, ×400). (f) Group CPB; RDT 1276 μm. Absence of inflammatory cell infiltration. Presence of normal histological appearance of pulp tissue (H and E, ×100). D: Dentin; PD: Predentin; OL: Odontoblast Layer|
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|Figure 2: Observation period: 90 days. (a) Group CSE; RDT 1112 μm. Presence of hyperemic pulpal vessels, absence of inflammatory cell infiltration (H/E, ×40). (b) Higher magnification of reparative dentin layer in a. (H/E, ×100) (c) Group CPB; RDT 1500 μm. Presence of slight inflammatory cell infiltration (arrow) (H/E, ×100). (d) Group CSE; RDT 1468 μm. The pulp tissue is histologically normal in appearance and a layer of the predentin is observed; cavity located on the down side. (H/E, ×40). D: Dentin; PD: Predentin; RD: Reparative dentin|
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Inflammatory cell infiltration was not found in the teeth except for a slight chronic inflammatory cell infiltration that was found in only one tooth in both Groups CPB-7 and CPB-90.
No significant statistical difference concerning the inflammatory cell infiltration was observed among the groups of teeth restored using the adhesive systems, either between the postoperative periods or between the adhesive systems themselves (P > 0.05).
A small disorganization in the soft tissue that formation of vacuoles in the odontoblast layer, the presence of hyperemic pulpal vessels and edema were seen for all materials in some of the teeth at the seven-day period, but these changes in the soft tissue were not classified as severe tissue damage. No significant statistical difference concerning the changes in soft tissue was observed among the groups of teeth restored with adhesive systems, either between the postoperative periods or between the adhesive systems themselves (P > 0,05).
Reactionary dentin deposition was observed in two teeth in Group CSE-90 and in one tooth in Group CPB-90. The differences between the reactionary dentin depositions in Groups CPB and CSE were not statistically significant (P > 0.05).
Small amounts of bacterial staining along the cavity walls were observed in two teeth in Groups CSE-7 and CSE-90, and one tooth in Groups CPB-7 and CPB-90. No significant statistical difference concerning the bacterial staining was observed among the adhesive system groups (P > 0.05).
In the present study, two teeth restored with CH + ZnOE were used as the control in the seven-day period. No statistically significant difference concerning all the histological evaluation criteria used in this study was observed between the seven-day period control group and the groups of adhesive systems in the same postoperative period (P > 0.05).
| Discussion|| |
In the present study, human pulp reactions were examined following the restoration of the cavities with two self-etching adhesive systems. The teeth restored with CH + ZnOE were used as the control. This is due to CH having long been employed as a liner or base because of its antibacterial activity, thermal isolation, and biocompatibility to the pulp tissue. In addition, ZnOE has excellent biological sealing characteristics and antimicrobial activity due to the effect of eugenol. 
The null hypothesis of our study was rejected. The results of this study showed that there were no statistically significant differences among groups of tested material regarding pulp inflammation and bacterial infiltration. Inflammation was not observed in most of the teeth; however, slight soft tissue disorganization characterized by the formation of vacuoles in the odontoblast layer, decrease of the odontoblast number and hyperemic pulpal vessels and edema in the region localized to the affected dentinal tubules were observed in the seven-day groups. These pulp reactions were not observed at 90 days. These slight pulp reactions seen for all test materials, including CH , in the seven-day groups may be due to cavity preparation trauma. Similar changes in pulp tissue have also been observed in some studies after cavity preparations with adequate water cooling. , Damage to the pulp tissue during cavity preparation may be caused by factors such as the mechanical irritation of the odontoblastic processes and heat generation, which is created by continuous cutting or the inadequate water-cooling or air-drying of exposed dentin. ,,
The presence of bacteria within the dentinal tubules or along the cavity walls is considered to be the most significant factor related to the pulp reaction under a resin-based restorative material in non-exposed cavities. ,,, In this study, very small amounts of bacteria could be found in the cavity walls of some of the specimens. The inflammatory cell infiltrations were not seen in all teeth with bacteria staining. A few inflammatory cell infiltrations were seen in only one tooth with bacterial staining in both adhesive systems after 90 days.
The antibacterial properties of the adhesives used in this study have been shown previously in vitro.  CSE and CPB have a short-term mild antibacterial effect due to the acidic nature of the primer (pH = 2). Additionally, an antibacterial monomer (MDPB) is included in CPB in order to achieve long-term residual bacteria inhibition and protection against bacterial infiltration. However, no difference was found between the bacterial staining of the adhesive systems used in this study. These results may be due to the non-extreme cavity depth and good marginal sealing provided by both adhesive bonding systems.
Another reason for pulpal inflammation is chemical irritation of non-polymerized monomers, which can penetrate pulp tissue via dentinal tubules. Tay et al., demonstrated the diffusion of resin globules across dentinal tubules in human teeth using a scanning electron microscope.  In their studies, resin globules of various sizes were observed clustering around the unmineralized collagen fibers within the predentin and adjacent to the odontoblasts and their processes. It was predicted that the bonding agents and their components inhibited mitochondrial respiration, interfering with the cell metabolism. ,, Additionally, lack of supply of oxygen to odontoblast cells interfered with their dentinogenic activity.  Costa et al. also speculated that the bonding agent present in the pulpal space triggered a remarkable inflammatory response associated with tissue disorganization.  In the present study, inflammation was not observed in most of the teeth for both adhesive systems. This may be due to the RDT between the cavity floor and the pulp tissue. RDT influences the amount of bonding agent that penetrates through dentin into the pulpal space. The residual dentin layer absorbs non-polymerized monomers and, therefore, contributes to the decrease in cytotoxicity of the material (3). A dentin thickness of 0.5 mm can reduce material toxicity to 75%, and 1 mm dentin can reduce toxicity to 90% of the control value when dentin is not present.  In this study, RDT was greater than 1 mm in the majority of the samples.
In this study, the mild pulp response with both adhesive systems may also be attributed to the bonding characteristics of the adhesive systems in addition to their antibacterial properties. Pashley reported that a sterile chemical insult to odontoblast should recover within 48 h after the clinical procedure, as the dentinal tubules should be sealed by adhesive resin films.  CPB and CSE, both with an acidity of pH 2, are classified as 'mild' two-step self-etching adhesives. These adhesive systems demineralize dentin only in a very shallow manner, and, as a result, a shallow hybrid layer (1 μm thick) is formed with the formation of short resin tags. , Moreover, superficial demineralization with these mild self-etch adhesives occurs only partially, keeping residual hydroxyapatite still attached to the collagen.  CPB and CSE contain 10-MDP as a functional monomer, and the chemical bonding potential of 10-MDP with hydroxyapatite is significantly high and hydrolytically stable because of the production of Ca salt, which is barely soluble.  This superior and stable bonding effectiveness may also have contributed to the prevention of the bacterial microleakage. , Moreover, keeping hydroxyapatite around the collagen may have offered better protection for the collagen against hydrolysis, thus, early degradation of the bond. 
Additionally, a mild self-etch adhesive does not completely remove smear plugs from the dentin tubule.  The smear plugs fill the orifices of dentin tubules, decreasing dentin permeability by up to 86%.  The permeability of dentin plays an important role in the toxicity of adhesive materials. High permeability should increase the toxicity of adhesives by allowing increased diffusion of the released components through dentin and the outward fluid movement from the pulp onto the exposed dentin surface.  This fluid is expected to inhibit the monomer-polymer conversion.
In the present study, reactionary dentin was seen in small regions of the cavities in some teeth. Reactionary dentin is laid down by primary odontoblast cells, in response to specific stimuli, but it is not enough to kill those primary pulp cells.  This suggests that the RDT of cavity preparations is a more powerful stimulus for the initiation and progression of a tertiary dentinogenic response as formed by reactionary dentin than any of the other cavity-cutting and restoration variables. Maximal tertiary dentin deposition was found to take place when the RDT was between 0.5 and 0.25 mm, while cavities cut with an RDT of less than 0.25 mm or more than 0.5 mm appeared to result in more minimal tertiary dentin deposition.  In this study, cavity RDT was more than 0.5 mm, and the results related to reactionary dentin deposition are consistent with this explanation.
In a previous study, Tziafas et al., examined the human pulp reactions following treatment with CSE and an antibacterial adhesive system with 5% MDPB in infected, non-exposed deep cavities.  Similar to the results of this study, they did not find inflammation or complete tissue disorganization. But more tertiary dentin formation was seen in teeth treated with the antibacterial adhesive than in those treated with CSE. They speculated that the two adhesive systems tested differ only in their incorporation of the antibacterial monomer MDPB, therefore the benefit of the adhesive system containing MDPB in stimulating tertiary dentine formation might be attribute to its antibacterial properties. The current results with reactionary dentin deposition do not agree with the results of Tziafas et al. The amount of tertiary dentin deposition was the same for both bonding systems. The reason for this difference in results may be due to the different conditions of the cavities, such as infection and depth.
| Conclusion|| |
Within the limitations of this in vivo study, it was demonstrated that both bonding systems were biologically compatible with pulp tissue under the non-exposed cavities. However, further research is needed to evaluate the long-term antibacterial effect and influence of MDPB on pulp tissue in a clinical environment.
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[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]