Long-term bond strength was not influenced by TiF 4 in aqueous or primer solution and concentrations.
Bond strength values to superficial bond were higher than values to deep dentin.
Premature failures were more frequent with the use of 4% TiF 4 in aqueous solution.
Aqueous solution of 2.5% and 4% TiF 4 showed a trend towards agglomeration over time.
TiF 4 incorporated into the primer presented an excellent stability, especially at 2.5%.
To evaluate the bond strength to superficial (SD) and deep (DD) dentin after the use of 2.5% (T2.5%) or 4% (T4%) titanium tetrafluoride (TiF 4 ) in aqueous solution as a dentin pretreatment, or when incorporated into the primer (T2.5%P and T4%P) of an adhesive system (Clearfil SE Bond/CL). Degree of conversion (DC), particle size (PS), polydispersity index (PI) and zeta potential (ZP) of the solutions were evaluated.
Fifty molars were sectioned longitudinally to obtain two slices of each tooth, which were demarcated into SD and DD. Treatments were applied (n = 10): CL; T2.5%; T4%; T2.5%P; T4%P. After 24 h or 180 days storage, microshear bond strength tests were performed. The DC values of T2.5%P and T4%P were evaluated by FTIR. PS, PI and ZP were measured using dynamic light scattering.
Analysis of mixed models showed significant effect of concentration of TiF 4 * solution * storage time (p = 0.0075). There were higher bond strength values in SD than in DD (p = 0.0105) for all treatments in both times. The failure mode showed adhesive failures in the majority of groups, irrespective of depth and time (p = 0.3746). The bond strength values were not affected by treatments. Lower average particle size was observed for T2.5%P and T4%P at baseline. T2.5% and T4% showed a trend towards agglomeration.
Higher bond strength values were achieved at SD for all treatments and times. The failure modes observed were adhesive. TiF 4 incorporation did not affect DC. T2.5%P and T4%P presented excellent stability over time.
Dentin biomodifiers and dentin pretreatment agents have been proposed for the purpose of reducing hybrid layer degradation, inhibiting the action of metalloproteinases present in the dentin matrix, diminishing the incidence of secondary caries lesions, and acting on bond strength stability . Among the dentin pretreatment agents, the application of an aqueous solution of titanium tetrafluoride (TiF 4 ) promotes the formation of a massive structure composed of titanium oxides or organometallic complexes, and a stable, modified, acid resistant smear layer . It is suggested that the incorporation of TiF 4 in the hybrid layer formed by adhesives could enhance the underlying dentin by tissue-specific biomodification with greater durability . This TiF 4 solution has been used in studies that evaluated the anticariogenic and antierosive effect, with concentrations varying from 2.5% to 4%, which can suggest that these concentrations may also be used as dentin pretreatment.
Dündar et al. verified that pretreatment with an aqueous solution of 2.5% titanium tetrafluoride (TiF 4 ) interfered in the bond strength of adhesive cements to dentin. For etch-and-rinse adhesive systems, Devabhaktuni and Manjunath observed that the application of a 4% aqueous solution of TiF 4 before or after acid etching when using a 3-step adhesive system had no influence on the bond to dentin, whereas Tranquilin et al. verified that the use of pretreatment with 2.5% TiF 4 promoted higher bond strength values of a two-step adhesive system to dentin. In addition, a hybrid layer micromorphology with the formation of more numerous and larger diameter tags was obtained, showing the favorable effects of using this pretreatment.
For self-etching adhesive systems, dentin pretreatment with 2.5% TiF 4 may not interfere in the values of bond strength to dentin , possibly due to the fact that these adhesives have moderate to slight degrees of acidity, and may penetrate through the glaze-like layer formed after the application of TiF 4 , although the micromorphology of the hybrid layer can be affected with the presence of tags in smaller diameter and depth. However, the differences in TiF 4 concentrations used (2.5 or 4%) to be applied as dentin pretreatment has not yet been elucidated.
It should be considered that aqueous solutions of TiF 4 for pretreatment are unstable , and there may be the formation of agglomerates, as demonstrated by Basting et al. when using concentration of 2.5%. Therefore, the incorporation of TiF 4 into the primer of the two-step self-etching adhesive system may be an alternative for seeking the greater stability of the product and making it easy to use, reducing the clinical steps, and observing that it does not appear to have any influence on the bond strength to superficial dentin, or the degree of conversion , suggesting that future studies use TiF 4 incorporated into the primer.
However, the bond of adhesive systems to deep dentin has been reported to be lower than the values shown for superficial dentin due to the lower quantity of intertubular dentin and collagen fibrils, and larger quantity of water in deep dentin. Therefore, it would appear that intertubular dentin pays an important role during hybrid layer formation, sine the bond strength is directly proportional to the quantity of intertubular dentin available for bonding . Moreover, it must be considered that over time, the bond may be harmed due to the presence of matrix metalloproteinases (MMPs), especially the gelatinases of the MMP-2 and MMP-9 types that lead to hybrid layer degradation . Niu et al. observed that MMP-2 was more concentrated in deep dentin and at the amelo-dentin junction, while MMP-9 presented a decreasing distribution from deep dentin in the direction towards superficial dentin, capable of influencing the longevity of the bond in the different depths of the dentin substrate .
Therefore, the aim of this study was to evaluate the longevity of the bond to superficial and deep dentin; failure mode, hybrid layer micromorphology when using TiF 4 at the concentrations of 2.5% or 4% in an aqueous solution for dentin pretreatment, or incorporated into the primer of a two-step self-etching adhesive system; the degree of conversion of adhesive systems containing TiF 4 , as well as the particle size, polydispersity index and zeta potential of the TiF 4 solutions. The null hypotheses to be tested were: (1) short-term bond strength to dentin at different dentin depths, fracture mode and micromorphological features of the hybrid layer when using 2.5% or 4% TiF 4 incorporated into the primer or when using dentin pretreatment with 2.5% or 4% aqueous solutions of TiF 4 would be similar; (2) the degree of conversion, particle size, polydispersity index and zeta potential of the two-step adhesive system would not be affected by the incorporation of 2.5% or 4% TiF 4 into the primer/aqueous solution.
Materials and methods
Microshear bond strength test
Fifty sound third molars were obtained after patient consent and approval by the local ethics committee (CAAE 46933615.3.0000.5374). The teeth were cleaned and stored in a 0.1% thymol solution until the beginning of the experimental phase, and for a period not exceeding six months.
The specimens were prepared according to the methodology presented by Zhang et al. . Two sections, 2 mm thick ( Fig. 1 A) were first made along the longitudinal axis of the tooth to remove the lingual and buccal tooth tissues ( Fig. 1 A). The remaining middle portion of each crown was then sectioned in the middle part, along the same direction, to obtain two slabs (approximately 2.0 mm thick each) ( Fig. 1 B) from the same tooth.
The two slices obtained from each tooth were embedded in polyester resin (Massa Fix, Royal Polímeros Indústria e Comércio de Produtos Químicos Ltda, SP, Brazil). The dentin surfaces were planed with abrasive papers impregnated with 600 grit aluminum oxide (3 M do Brasil, Sumaré, SP, Brazil) fitted to a water cooled, pneumatic rotary electric polishing machine (Ecomet 250, Buehler Ltda, Lake Bluff, IL, USA). The blocks were ultrasonically cleaned in deionized water for 10 min. After this, one slice of each tooth, which corresponded to the time of 24 h, and another slice from the same tooth, corresponding to the time of 180 days were identified, totaling 50 slices for each time interval.
The slices of each time interval were randomly divided into five groups, according to the adhesive treatments (n = 10) ( Table 1 ).
|Group/abbreviation lot number/||Number of slices — time 24 h||Number of slices — time 180 h||Composition/pH||Treatment||Protocol for use||Manufacturer (City, State, Country)|
|Clearfil SE Bond/CL 051553||10||10||Primer: 10-methacryloyloxydecyl dihydrogen phosphate (MDP), 2-hydroxyethyl methacrylate (HEMA), hydrophilic dimethacrylate, Camphorquinone, N , N -Diethanol p -toluidine, water||Application of 2-step self-etching adhesive system (without incorporation of TiF 4 )||– Apply primer actively for 20 s;||Kuraray Medical Inc. (1621 Sakazu, Kurashiki, Okayama, Japan)|
|pH 1.76||– Apply light jet of air;|
|Bond: 10-methacryloyloxydecyl dihydrogen phosphate (MDP); Bisphenol A-diglycidyl dimethacrylate (Bis-GMA); 2-hydroxyethyl methacrylate (HEMA), hydrophilic dimethacrylate, Camphorquinone, N , N -diethanol p -toluidine, Silanized Colloidal Silica||– Apply bond;|
|pH 1.81||– Light activation for 10 s|
|Pretreatment with TiF 4 2,5% + Clearfil SE Bond/T2.5%/MKBR9299V/051553||10||10||Pretreatment: 2.5% TiF 4 , distilled and deionized water||Dentin pretreatment with aqueous solution of 2.5% TiF 4 , followed by application of the two-step self-etching adhesive system (without incorporation of TiF 4 )||– Apply pretreatment actively for 60 s;||Sigma Aldrich (Saint Louis, MO, USA)/Kuraray Medical Inc. (1621 Sakazu, Kurashiki, Okayama, Japan)|
|pH 1.04 + Primer + Bond||– Apply primer actively for 20 s;|
|– Apply light jet of air;|
|– Apply bond;|
|– Light activation for 10 s.|
|Pretreatment with TiF 4 4% + Clearfil SE Bond/T4% MKBR9299V/ 051553||10||10||Pretreatment: 4% TiF 4 , distilled and deionized water||Dentin pretreatment with aqueous solution of 4% TiF 4 , followed by application of the two-step self-etching adhesive system (without incorporation of TiF 4 )||– Apply pretreatment actively for 60 s;||Sigma Aldrich (Saint Louis, MO, USA)/Kuraray Medical Inc. (1621 Sakazu, Kurashiki, Okayama, Japan)|
|pH 0.87 + Primer + Bond||– Apply Primer actively for 20 s;|
|– Apply light jet of air;|
|– Apply bond;|
|– Light activation for 10 s.|
|2.5% TiF 4 incorporated into primer of Clearfil SE Bond/T2.5%P MKBR9299V/ 051553||10||10||2.5% TiF 4 incorporated into Primer||Application of the primer with 2.5% TiF 4 incorporated into it, followed by the application of the bond of the two-step self-etching adhesive system (without incorporation of TiF 4 )||– Apply primer with TiF 4 incorporated into it actively for 20 s;||Sigma Aldrich (Saint Louis, MO, USA)/Kuraray Medical Inc. (1621 Sakazu, Kurashiki, Okayama, Japan)|
|pH 1.82 + Bond||– Apply light jet of air;|
|– Apply bond;|
|– Light activation for 10 s.|
|4% TiF 4 incorporated into primer of Clearfil SE Bond/T4%P MKBR9299V/ 051553||10||10||4% TiF 4 incorporated into primer||Application of the primer with 4% TiF 4 incorporated into it, followed by application of the bond of the two-step self-etching adhesive system (without incorporation of TiF 4 )||– Apply primer with TiF 4 incorporated into it actively for 20 s;||Sigma Aldrich (Saint Louis, MO, USA)/Kuraray Medical Inc. (1621 Sakazu, Kurashiki, Okayama, Japan)|
|pH 1.61 + Bond||– Apply light jet of air;|
|– Apply bond;|
|– Light activation for 10 s.|
In Group CL, the adhesive system was applied in accordance with the two-step self-etching adhesive system manufacturer (Clearfil SE Bond) ( Table 1 ). For the T2.5% and T4% Groups, dentin pretreatment was performed with 2.5% or 4% titanium tetrafluoride. The titanium tetrafluoride was acquired in the pro-analysi (P.A.) form, and manipulated at the concentration of 2.5% or 4% in deionized distilled water (weight:volume) and applied as presented in Table 1 . After pretreatment, the adhesive system was applied and light activated, in accordance with the manufacturer’s instructions. For Groups T2.5%P and T4%P, the titanium tetrafluoride was acquired in the pro-analysi (P.A.) form, and manipulated at the concentration of 2.5% or 4% diluted in the primer (weight:volume) of the two-step self-etching adhesive system and was actively applied (rubbing) with a disposable paint brush as presented in Table 1 .
The pH values of the aqueous solutions and primers manipulated with TiF 4 in the different concentrations, and the primer and bond of the adhesive system used, were measured in triplicate with a microelectrode (Model 2A14, Analyser Instrumentação Analítica, São Paulo, SP, Brazil) and pH-meter (Model MPA 210, MS Tecnopon Instrumentação, Piracicaba, SP, Brazil).
On each slice, before the adhesive system was light activated, Tygon type microcylinders were positioned (internal diameter 0.8 mm and height 2 mm) ( Fig. 1 D) in different localizations of the surfaces treated, denominated superficial dentin (DS) and deep dentin (DP). For the purpose of delimiting DS and DD dentin, the surface of the slab was marked by a line to delineate two zones of equal width to represent different dentin depths . There was no way of placing the microcylinders tangentially to the roof of the pulp chamber or to the dentin-enamel junction, and leaving at least 1 mm distance from the roof of the pulp chamber or from the dentin-enamel junction. After light activating the adhesive with a LED light appliance (Valo, Ultradent, South Jordan, UT, USA; wavelength range of 395–480 nm at an intensity of 1000 mW/cm 2 ), the hybrid resin composite (Filtek MR Z250 XT, 3 M ESPE, Irvine, CA, USA, shade A2, lot number: 228214) was carefully inserted into the tubes ( Fig. 1 E) and light activated at once to avoid influence of multiple light activations. Light activation was performed for 20 s — the time recommended by the resin composite manufacturer — by applying the light parallel to the long axis of the cylinder. In general, 3 cylinders were positioned in superficial dentin, and 2–3 cylinders in deep dentin ( Fig. 1 F).
After storage in artificial saliva at 37 °C for 24 h, the tubes belonging to the group that would be evaluated in the 24-h time interval were carefully removed with a No.11 scalpel blade, leaving the resin composite cylinder apparent. For the group evaluated in the 180-day time interval, the test specimens were stored in individual receptacles in an artificial saliva solution (pH 7; 1.5 mM calcium; 0.9 mM phosphorous; 0.15 M potassium chlorate; 0.02 M tris buffer) that was changed on a weekly basis, to be tested after the storage period.
For the microshear bond strength tests, the blocks containing the tooth slices were fixed to the universal test machine device (Ez-LX 5 kN, Kyoto, Japan). The resin composite cylinders remained aligned to the load cell of 20 kgf. A stainless steel metal wire 0.2 mm in diameter was simultaneously looped around the prolongation of the machine load cell and one of the resin composite cylinders. The wire was kept in contact with the bottom semicircle of the cylinders as closely as possible to the area of the bond to the composite resin surface. A speed of 0.5 mm/min was used, until fracture of the cylinder occurred. The values at the time of fracture were recorded in N and converted into MPa.
Failure mode analysis
The fracture type was evaluated under a stereoscopic lens (Model EK3ST, Eikonal Equip Ópticos e Analíticos, São Paulo, São Paulo, Brazil) at 20× magnification. The fracture types were classified into adhesive, cohesive in dentin or in resin, or mixed. The fracture types were classified as cohesive when there was predominance of over 50% of the fracture in the body of dentin, or body of resin; adhesive when over 50% of the failure occurred at the junction between the dentin and resin; and mixed when it was adhesive and cohesive at the same time. Representative images of the fractures were acquired by scanning electron microscopy (Phenom Pro X, Phenom-World BV, Eindhoven, Denmark) at 330× magnification, used in standard mode under vacuum, and operating at a voltage of 10 kV.
Micromorphological analysis of the hybrid layer
Ten sound third molars were used to evaluate the hybrid layer interface in the region of superficial dentin region. For this purpose, flat occlusal surfaces were obtained in dentin by removing the portion of occlusal enamel perpendicular to the long axis of the tooth, with a diamond disc mounted in a precision electric cutting machine(Isomet 1000 Precision Diamond Saw, Buehler Ltd, Lake Bluff, Illinois, USA). The fragments were taken to a water-cooled, rotary polishing machine (Politriz Aropol 2 V, Arotec, Cotia, SP, Brazil),with the use of 600 grit aluminum oxide abrasive papers (Imperial Wetordry, 3 M, Sumaré, SP, Brazil).
After this, the teeth were divided among the groups, according to the five adhesive treatments (n = 2) ( Table 1 ). After application of the adhesive treatments one resin composite block (Filtek Z350, 3 M ESPE, Saint Paul, MN, USA) measuring 4.0 mm high and 4.0 mm wide was fabricated on the tooth by the incremental technique. The first layer, approximately 2.0 mm thick was placed and light polymerized for 40 s. The second layer was placed and was also light activated for 40 s. After this, the resin was light activated for 20 s on each of its two sides.
The dentin/resin composite block sets were fixed onto acrylic plates for position in a metallographic cutting machine (Isomet 1000 Precision Diamond Saw, Buehler Ltd, Lake Bluff, Illinois, USA) to cut sections approximately 1 mm thick in the vestibular-lingual direction. One section of each tooth was prepared for analysis by scanning electron microscopy , with planing being performed with decreasing (400, 600, 1200) grits of aluminum oxide abrasive papers (Imperial Wetordry, 3 M, Sumaré, SP, Brazil) and polishing with diamond paste (Arotec SA Ind. E Com., Cotia, São Paulo, Brazil). After abundant rinsing, the specimens were demineralized, rinsed again, deproteinized, dehydrated in ethanol, chemically dried with HMDS (hexamethyldisilazane) and mounted on aluminum stubs. The specimens were examined by scanning electron microscopy (Phenom Pro X, Phenom-World BV, Eindhoven, Denmark), used in standard mode under vacuum, and operating at a voltage of 10 kV. The entire extent of the bond interface of each specimen was carefully scanned at 500× magnification. After this, the most representative area of each specimen was photographed at 1500× magnification. The interface morphology was analyzed in relation to hybrid layer formation, by analyzing its integrity and thickness, as well as presence, uniformity of size and disposition of resin tags.
Degree of conversion analysis
The degree of conversion of the primer solutions either containing TiF 4 , or not were dynamically analyzed by means of Fourier Transform Infrared Spectroscopy (FTIR, PerkinElmer Spectrum — One Fourier transform infrared spectrophotometer, Waltham, MA, USA) with resolution of 4 cm −1 . The technique consisted of collecting the radiation reflected from the interface between the solution and crystal (ATR), showing the transformation of the double carbon bonds (C C) in the range of intensity of 1638 cm −1 , in simple bonds (C C) in the range of 1608 cm −1 .
For each group, the quantity of 5 μL of primer of the self-etching adhesive system Clearfil SE Bond (with or without TiF 4 at the concentrations of 2.5 or 4%) was deposited on the ATR crystal of the equipment, followed by the addition of 5 μL of bond; the mixture between the components was performed with the use of an extra-fine disposable applicator brush (Cavibrush, FGM, Joinville, SC, Brazil) for the time of 10 s. Radiation was analyzed by collecting the first 10 spectra, with the addition of the LED light activating appliance (VALO Curing Light, Ultradent Products Inc., South Jordan, Utah, USA) 10 s, at a standardized distance of 2 mm. The degree of conversion was monitored for 2 min. The absorption-spectra were automatically collected by the equipment by means of the TimeBase (PerkinElmer) software, and saved in a format compatible with the Excel program for calculating the conversion data. The process of recording the spectra was repeated 5 times for each formulation, from which the mean degree of conversion was obtained for each experimental condition. The degree of conversion measurements were based on the baseline technique , in which:
D C ( % ) = ( 100 − ( 1638 c m − 1 1608 c m − 1 p o l y m e r i z e d 1638 c m − 1 1608 c m − 1 u n p o l y m e r i z e d ) ) × 100