Influence of chlorhexidine on longitudinal bond strength to dentin : in vitro study

Objetivo: Este estudo avaliou o efeito da solução de gluconato de clorexidina 0,2% (CHX) usado como primer terapêutico sobre a resistência de união longitudinal de adesivo convencional a dentina. Material e Métodos: dentes bovinos foram desgastados para obter uma área de dentina, e foram divididos em 2 grupos: Grupo C (Controle) ácido fosfórico à 37%) + 0,2% CHX por 30s + Single Bond. Blocos de resina composta form fabricados e armazenados por 24 h e 6 meses, seccionados e submetidos ao teste de resistência a microtração. Os resultados foram analisados por ANOVA dois-fatores seguido pelo teste de Tukey. Resultados: Valores de média (±Desvio-padrão) (MPa): Grupo CHX/24h 41,8(±2,62)A; Grupo C/24h 40,8(±3,35)AB; Grupo CHX/6 meses – 36,4(±3,52)B; Grupo CHX/6 meses 26,1(±1,54) C. Resultados: Valores de média (±Desvio-padrão) (MPa): CHX (24 h) – 41,76 (±2,62)a; C (24h) – 40,81 (±3,35)ab; GT (24h): 37,38(2,98)abc; CHX (6 meses) – 36,04 (±3,52) bcd; EGCG (24h) – 35,91 (±4,82)cd; EGCG (6 meses) – 35,75 (±4,44)cd; GT (6 meses) – 31,95 (±3,40)de; C (6 meses): 30,05 (±1,54)e. Conclusão: CHX aumentou a resistência de união imediata da interface dentina-resina e significantemente reduziu a perda de resistência de união após 6 meses de armazenagem em água quando comparado ao grupo controle. AbstRAct


Influence of chlorhexidine on longitudinal bond strength to dentin: in vitro study
Fonseca BM et al.

intRoDuction
A fter nearly 4 decades of research, advances in the adhesive systems and bond techniques allow less wear of sound tooth structure and contributed to the increase of bond strength between tooth and restorative material.[1] However, the longevity of hybrid layer has been still widely questioned as a consequence of hydrolysis of the adhesive interface over time.[2][3][4] Degradation of the hybrid layer may be triggered by various factors.The degradation of the collagen fibers present in the dentin organic matrix which were left unprotected after the process of demineralization by acid etching and not infiltrated by the adhesive monomers is the main factor related to decreases in bond strength.[5,6] Dentine has proteolytic enzymes, also called matrix metalloproteinases (MMPs) that can be activated by acid etching during adhesive procedure.[7,8] These MMPs are responsible for rapidly degrade denatured collagen fibers.Among the MMPs identified in the organic matrix dentin are gelatinases (MMP-2 and -9) collagenase (MMP-8) and enamelysin (MMP-20) [9].It is believed that these MMPs are related to degradation of the adhesive interface [10].
Thus, inhibition of MMPs could be effective in the preservation of the hybrid layer [10].Studies have shown that the use of MMP inhibitors improves the integrity of the hybrid layer [7].Thus, the use of chlorhexidine digluconate (CHX) disinfectant solution prior to the use of a dentin bonding agent have been investigated in order reduce the degradation of the hybrid layer [11,12] due to their MMP inhibitory properties [13].Previous studies have shown that the degradation of the organic matrix infiltrated with resin can be delayed through the use of CHX [7,12,14,15].Therefore, longitudinal evaluation of the bond strength using this disinfectant solution has become of primordial importance.The aim of this study was to evaluate in vitro, the influence of CHX on the microtensile bond strength to dentin of the two-step total-etch adhesive at time intervals of 24 h and after a 6 months period of storage in water at 37 ºC.
This study tested two null hypotheses: 1) CHX applied prior to the adhesive system does not affect bond strength to dentin; 3) the storage period does not affect the bonding effectiveness of two-step total-etch adhesive to dentin.

mAteRiAl AnD methoDs
Forty freshly extracted bovine incisor teeth bovine were used in this study.The roots were sectioned with a steel diamond disc (KG Sorensen, Rio de Janeiro, Brazil) at the cementenamel junction.The buccal surfaces were worn using abrasive papers (granulation 600) coupled to a circular polishing machine (PA-10, Panambra, São Paulo, Brazil) under water cooling, to obtain a 5mm2 area of flat dentin.
The dental surface was worn to standardize the remaining dentin thickness at 2 mm, using a caliper thickness gauge (Golgran, São Caetano do Sul, Brazil) to measure it.The smear layer was standardized using 600 grit abrasive papers coupled to a circular polishing machine.

Bonding Procedures
The teeth were divided into 2 groups (n = 20), according to the dentin treatment performed: Group C (Control technique): The surfaces were etched for 15 s with 37% phosphoric acid gel, rinsed and the excess moisture was removed with absorbent paper.Two layers of Single Bond total-etch adhesive (3M ESPE, St. Paul, MN, USA) were applied on the surface actively for 15 s and gently air dried for 10 s.The adhesive was light activated for 10 s with a LED light unit (Emitter A, Schuster, Santa Maria, RS, Brazil) with power density of 600 mW/cm 2

Restoration placement
Composite resin blocks (Amelogen Plus, Ultradent, Indaiatuba, SP, Brasil), approximately 4 mm high were built on the treated surfaces.Each 2 mm portion was light activated for 40 s.Materials used in this study, chemical composition and manufacturers are presented in Table 1.
The bonded teeth were stored in distilled water at 37 ºC, for time intervals of 24 h or 6 months.The water was changed every week during the course of 6 months.[16]

Microtensile Bond Strength Test
The test specimens were cut into parallel sections measuring approximately 1 mm, made from the mesial to the distal, and from the cervical to the occlusal surface, using a diamond disc attached to a Labcut 1010 (Extec Technologies Inc., USA) cutting machine to obtain sticks, producing a minimum of 7 sticks per tooth.The sections were made at low speed under water cooling to prevent stress induction at the bond interface.
The sticks were attached to a microtensile device in a universal testing machine (DL-1000, EMIC, São José dos Pinhais, PR, Brazil), with a 10 kg load cell, at a cross-head speed of 0.5 mm/min, in accordance with the ISO 11405 Standard.The bond strength data were expressed in Megapascals (MPa).

Statistical Analysis
Pre-test failures (PTFs) were not included in statistical analyses.To the cohesive failures (dentin or composite), the specimens were discarded.The mean value for the sticks originating from each tooth was calculated and used for the statistical analysis.[17] Data, expressed in megapascal (MPa), were analyzed by two-way ANOVA (technique X storage time) followed by Tukey test (α 5%).

Scanning electron microscopy (SEM) examination
Two teeth from each group were prepared for SEM analysis.The specimens were sectioned perpendicularly to the bonding interface.The sections were polished with 2000 and 4000 mesh sheets.Phosphoric acid etchant was applied for 15 s, rinsed off with water for 10 s.Specimens were dehydrated, sputter-coated with goldpalladium and examined by SEM (high vacuum, 15KV, Everhart-Thornley Detector/ETD, 5000X magnification).

Results
Table 2 shows the results of the Tukey test for the interaction between the independent variables of "technique" and "storage time" (p=0.00) and type of fracture detected under microscopy for each group.
The Group CHX assessed after 24 h showed the highest tensile bond strength mean values.The Group C with storage interval of 6 months presented the worst tensile bond strength values.The Group CHX with storage interval of 6 months presented significantly lower mean bond strength values when compared with Group CHX with storage interval of 24 h, and higher mean bond strength values when compared with Group C with storage interval of 6 months.The adhesive and mixed fractures were the predominately detected types of fracture.

Materials
Figures 1A and 1B show SEM images obtained of the interfaces created by Group C (Control), respectively storage for 24 h and 6 month of storage.Figures 2A and 2B show SEM images obtained of the interfaces created by  Group CHX, respectively storage for 24 h and 6 month of storage.

Discussion
The first null hypothesis was rejected, because the 0.2% CHX technique presented higher bond strength values compared with the Control technique.The second null hypothesis was rejected, because the storage time of 24 h presented higher bond strength values in comparison with the storage time of 6 months.
Adhesive systems have being modified over the years mainly as a result of technological advances, however, the possibility of failures in hybrid layer formation can be observed.Degradation of the hybrid layer may be triggered by various factors.When excessive acid etching is performed, it enables a very deep etched dentin surface to be formed; the adhesive is unable to infiltrate into it completely, giving rise to a region where the exposed collagen fibers are weakened and more susceptible to hydrolysis.[18] The MMPs present in dentin matrix are zinc-and calcium-dependent enzymes, which are responsible for physiology regulation and pathogenic metabolism of collagen-based tissues [19,20].Previous studies have detected the presence of MMP-2 and -9 in the dentin matrix [9,21], and when released and activated, play an important role in the degradation of collagen fibers exposed within the hybrid layer.Collagenolytic and gelatinolytic activity of these enzymes are likely responsible for rapidly degrade denatured collagen which were left unprotected after the process of demineralization by acid etching and not infiltrated by the adhesive monomers [21], resulting in gaps in the adhesive interface over time.[10] Several in vitro and in vivo studies have been accomplished with the aim of improving the adhesive interface using inhibitors of the MMPs in the adhesive protocol [12,15,22].CHX is one of the main substances used as potential MMP inhibitor, which has been introduced as a complementary step in the adhesive protocol, with good results in in vitro studies [7,14,15,18,22,23].
CHX is an antiseptic and antimicrobial solution [24], which inhibits bacterial adhesion to dental surfaces by competing with calcium or retention sites.Therefore, this solution can

Influence of chlorhexidine on longitudinal bond strength to dentin: in vitro study
Fonseca BM et al.
prevent the formation of calcium-link between bacteria-dental surfaces or between bacteriabacteria [25].This mechanism may be effective in the inhibition of MMPs, since these are zinc and calcium-dependent [10].Recent studies have shown that the use of CHX prior to adhesive system, without rinsing, significantly inhibited the MMPs activity in the hybrid layer [7,15,23] even at low concentrations [13], which is in agreement with the results of our study.
When CHX is ionized, it is characterized with a strong base, with cationic properties.And this cation portion is able to bind to negatively charged surfaces / target sites of the substrate.Thus, CHX is able to remain on the substrate and re-mineralized dentin [26] independent of incubation time and it concentration.[27] This prevents that proteases bind collagen fibers not infiltrated by monomers, preserving the hybrid layer after long periods of exposure to water.[28] According to Gendron et al. [13], two mechanisms of action are involved in the inhibition of MMP; chelation inhibiting MMP-2 and -9 and the interaction with cysteines and/ or sulfhydryl groups present in the active site of MMP-8.
It can observed in this study that the use of CHX, as an adjuvant in dentin bonding, did not chemically interfere the immediate adhesion of resin materials (Figure 2A), because there is no difference in bond strength between 0.2% CHX technique and control technique after 24 h (Figure 1A).Also, 0.2% CHX technique after 6 months of storage presented lower bond strength when compared with the same technique at 24 h, but higher bond strength when compared with control technique with storage interval of 6 months.Therefore, one noted that the storage time did not interfere so strongly in the bonding of groups that used 0.2% CHX, when compared with those that did not use 0.2% CHX (Figure 1B), which can demonstrate a relative maintenance of the bond strength of the 0.2% CHX technique (Figure 2B) when compared to control technique.
According Carrilho et al. [29] and Brackett et al. [30], long-term action of CHX can be explained by their confinement in the adhesive interface, since their removal through the dentinal fluid flow is reduced by the formation of resin tags that obliterate the dentinal tubules.Thus, CHX may remain trapped between the collagen fibers during the impregnation of interfibrillar spaces for fluid resin during application of the adhesive.[31] CHX is a large molecule (molecular mass = 897.8g mol-1) compared to some monomers adhesive systems, such as Bis-GMA (molecular mass = 512.0g mol-1) and HEMA (molecular mass = 130.0 g mol-1) [32], however, in this study, CHX did not influence the resin monomers infiltration in dentin after acid etching, as observed in other studies.[29,[33][34][35] After 6 months of storage, both techniques showed decrease in bond strength values, however, the control technique showed the highest decrease, due to degradation of the hybrid layer.However, the 0.2% CHX technique was not also able to maintain the stability of the resin-dentin bond strength values over time.This decrease in bond strength may have occurred not only by the presence of MMPs but also by the incomplete polymerization of infiltrated monomers, or presence of free water in the dentine due to incomplete evaporation of residual solvents in the adhesive, as well as the presence of water in the saliva and in the intrinsic humidity of dentin resulting in a high osmolarity of the mixture of hydrophilic adhesive, creating water-filled channels within the adhesive, as observed by Stanislawczuk et al. [28] However, the better bond strength result of 0.2% CHX technique within 6 months of storage compared to control technique maybe due ability of CHX to inactive MMPs exposed in the hybrid layer, and their ability to be retained in the dentin matrix and their antimicrobial substantivity [27], allowed the formation of a hybrid layer which remained stable over time.The term substantivity is present in the literature much more with the residual antibacterial activity of CHX in human dentin [36,37] than for the inhibitory effect of CHX on MMP activity.
The antimicrobial substantivity of CHX is associated with the release of positively charged molecules from the substances treated with CHX and it ability to adsorb substances from the oral cavity.According to Loguercio et al. [23], it can also occur in the exposed collagen fibers within the hybrid layer, which can reduce degradation of the adhesive interface after exposure to water in the long term.
The use of 0.2% CHX and the storage time did not influence the type of fracture, and fractures were predominantly adhesive or mixed fractures.
Our study suggests that a low concentration of CHX (0.2%) applied for 30 s, is sufficient to preserve the adhesive interface for at least 6 months under in vitro conditions.Furthermore, in vivo studies are needed to clarify whether 0.2% CHX for 30 s is sufficiently able to preserve the adhesive interface after long time.
Therefore, within the limitations of this study, 0.2% CHX has a considerable potential in maintaining the stability of bond interface to dentin after 6 months of water storage.These results are in agreement with those of various studies [7,22,29,31], that observed a real potential MMP inhibitor of CHX, which can increase the time durability of adhesive restorations.However, new researches are necessary to finding an effective way of maintaining the adhesive interface in the long term.

conclusion
Within the limitations of this study it can be concluded that: • CHX used as a therapeutic primer improve the immediate bond strength of resindentin and significantly lowered the loss of bond strength after 6 months water storage as seen in the control bonds.

Figure 1 -
Figure 1 -A and B -Hybrid layer formed by Group C (Control), respectively storage for 24 h and 6 month of storage.A typical hybrid layer was created (Figure 1A).Adhesive resin with bubbles(arrows), which can indicate degradation due hydrolysis (Figure 1B).(Legends: CR -Composite resin; HL -Hybrid Layer; D -Dentin).
Influence of chlorhexidine on longitudinal bond strength to dentin: in vitro studyFonseca BM et al.

Figure 2 -
Figure 2 -A and B -Hybrid layer formed by Group CHX, respectively storage for 24 h and 6 month of storage.CHX used as a therapeutic primer did not interfere the formation of hybrid layer.No areas of degradation of hybrid layer, over time, were observed (Figure 2B).(Legends: CR -Composite resin; HL -Hybrid Layer; D -Dentin.)

Table 3
presents the fracture type results.

Table 2 -
Mean (±standard-deviation) bond strengths of Group C versus Group CHX tested 24 h or after 6 months of aging

Table 3 -
Results of fracture types