MDP forms MDP-Ca 2+ salts with hydroxyapatite.
MDP inhibits the activation of MMPs.
Combining chlorhexidine with MDP interferes with the formation of MDP–Ca salts.
This study aimed to evaluate the potential interaction of chlorhexidine (CHX) and 10-methacryloyloxydecyl dihydrogen phosphate (MDP) and its effects on the durability of dentin bonding.
Two commercial adhesives were tested: a MDP-free adhesive (Single Bond 2, SB2) and a MDP-containing adhesive (Single Bond Universal, SBU). Teeth were randomly assigned to six groups and tested for micro-tensile bond strength (μTBS): Ctr, direct bonding with SB2; CHX, CHX conditioning and SB2; MDP, MDP conditioning and SB2; CHX + MDP, combined CHX and MDP conditioning and SB2; SBU, direct bonding with SBU; CHX + SBU, CHX conditioning and SBU. The potential interaction of CHX and MDP was assessed by measuring nanoleakage, in situ zymography, and chemoanalytic characterization via Fourier transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS) and nuclear magnetic resonance (NMR). Specimens for μTBS and nanoleakage tests were first subjected to water storage for 24 h or 6 months.
The initial μTBS values of the Ctr and CHX groups were significantly lower than those of the other four groups (P < 0.05). Water storage for 6 months significantly weakened all groups (P < 0.05), with the Ctr group showing the lowest μTBS. This group also showed more obvious nanoleakage than the other five groups. In situ zymography revealed that the Ctr group showed the strongest fluorescence and that the CHX + MDP group showed greater fluorescence than either CHX or MDP group. FTIR, XPS, and NMR indicated that MDP can interact with hydroxyapatite. NMR detected no Ca 2+ salt peak for MDP when it was combined with CHX.
The application of either CHX or MDP alone can improve dentin bond durability. However, CHX may interfere with the formation of MDP–Ca salts.
Modern dentin adhesives form very strong short-term bonds between resin and dentin (around 30–50 MPa), but the limited durability of the bonded interface represents a clinically significant challenge [ ]. Clinically, nearly 75% of resin filling fail, mainly owing to insufficient resin–dentin bond durability, resulting in secondary caries, pulp sensitivity and the loss of restorations [ , ]. The goal of dentin bonding is to form and preserve a stable resin–adhesive–dentin interface system, which will provide sufficient retentive strength, marginal sealing and clinical durability [ ].
Dentin bonding procedures are either etch-and-rinse or self-etching [ ]. In either case, dentin bonding depends on the formation of a hybrid layer (HL) characterized as an interlocking anchor composed of a mixture of demineralized collagen, resin, residual water, and hydroxyapatite (HA) crystallites that forms when the resin penetrates dentin tubules [ ]. The HL created in dentin is unstable in aqueous environments, as over time water disrupts the demineralized collagen matrix and hydrolyzes the resin [ ]; nonetheless, the HL has a leading role in dentin bonding [ ]. Matrix metalloproteinases (MMPs) are typical endogenous dentin proteases widely distributed in oral saliva and dentin matrices [ ]. They can be activated by etch-and-rinse and self-etching adhesive systems [ ], and can progressively degrade exposed collagen fibrils, thus degenerating the integrity of the HL [ ]. The main sites of MMPs’ activity are in the inner tubular walls and at the bottom of the HL, which are the weakest areas along the adhesive interface and the most susceptible to degradation over time [ , ]. Therefore, MMP inhibitors have been applied to inhibit and reduce collagen degradation in the early stage [ ]. Chlorhexidine (CHX) is a commonly used broad-spectrum antimicrobial agent and the most widely studied non-specific MMP inhibitor [ ]. In vitro studies have confirm that it inhibits the activity of various MMPs (such as MMP-2, -8, -9) and prevents or minimizes the reduction of resin–dentin bonding [ , ]. CHX is incorporated into commercial adhesives to preserve the bonding interfaces more permanently; however, its use may affect the efficacy of other components in the adhesive, reducing the bond strength; ultimately, CHX might potentially be chemically incompatible with adhesives [ ].
Therefore, approaches other than MMP inhibitors have been proposed to improve the bonding’s strength and durability. Examples include the use of cross-linking agents [ ], removal of unbound/residual water within the HL [ ], and remineralization at both intertubular and peritubular dentin [ ]. Phosphate ester monomers can chemically interact with hydroxyapatite, and can improve dentin bonds (but cannot facilitate interlocking between collagen fibrils and permeated resin tags [ ]). They can potentially enhance adhesives’ resistance to collagen degradation [ ], and have become an important and irreplaceable functional component of dental adhesives [ ]. 10-Methacryloyloxydecyl dihydrogen phosphate (MDP), the most widely used example, has a pH value of 2.64, and can etch tooth substrates (partially dissolving the smear layer) and enhance monomer penetration [ , ]. In addition to improving bonding stability, MDP can also self-assemble into a “nano-layering” structure through the deposition of MDP–Ca salts, which has been considered responsible for preventing HL degradation [ ]; however, this remains controversial, and nano-layering is rarely observed at the resin–dentin interface formed by commercial MDP-containing adhesives [ ]. As phosphate ester monomers like MDP are common in dental adhesives, they often clinically interact with CHX, but few studies have focused on the effects of the combination of MDP and CHX on dentin bonding. MDP forms insoluble calcium salts during dentin demineralization, which could limit the amount of ionized calcium in the HL created by the adhesive: it needs to be investigated whether MDP will antagonize or compete with CHX.
This study evaluates the potential interaction of CHX and MDP through micro-tensile bond strength testing (μTBS), analyses of nanoleakage and in situ zymography and chemoanalytic characterization. The null hypothesis tested is that the combination of MDP and CHX does not alter their individual effects on dentin bonding.
Materials and methods
Sixty freshly extracted non-carious human third molars were used for in vitro research based on a protocol approved by the Ethical Committee of the Nanjing Medical University, China. The teeth were cleaned by removing the remaining soft tissues and debris, and then immediately stored in Hanks’ solution at 4 °C to reduce demineralization.
A flat dentin surface (approximately 3 mm thick) was prepared perpendicular to the long axis of each tooth with a low-speed saw (Isomet 1000, Buehler Ltd., Lake Bluff, IL, USA) under water cooling. The enamel and superficial dentin were removed to expose the mid-coronal dentin, which as then wet-polished with 400-grit silicon carbide paper to create a standardized smear layer [ ]. All the dentin surfaces were carefully examined using a dental microscope (OMS2350 Dental Microscope, Zumax, China) to ensure that there was no residual enamel or pulp exposure. Then, the exposed dentin was stored in saline solution (0.09%) for the preparation of dentin bonding.
MDP-containing experimental primer was prepared in ethanol with the following formulation: 10 wt% MDP (DM Healthcare Products, USA); 88.8 wt% ethanol; 0.3 wt% camphorquinone (CQ, Aladdin, China); and 0.9 wt% 4-dimethylamino-benzoic acid ethyl ester (4EDMAB, Aladdin, China) [ ].
All the dentin surfaces were etched with 35% phosphoric acid etchant (Gluma Etch 35 Gel) for 15 s, and then rinsed with water thoroughly for 30 s to ensure that the etchant was completely removed following etch-and-rinse processing. The dentin surface was gently dried with absorbent paper. The 60 pretreated teeth were then randomly distributed into six equal groups based on whether CHX was applied and which adhesive was used (detailed in Table 1 ).
|Group||Dentin bonding strategy|
|Ctr||Acid etch + Single Bond 2 (MDP-free)|
|CHX||Acid etch + CHX conditioning + Single Bond 2 (MDP-free)|
|MDP||Acid etch + MDP-containing experimental primer conditioning prior to Single Bond 2 (MDP-free)|
|CHX + MDP||Acid etch + CHX conditioning + MDP-containing experimental primer conditioning prior to Single Bond 2 (MDP-free)|
|SBU||Acid etch + Single Bond Universal (MDP-containing)|
|CHX + SBU||Acid etch + CHX conditioning + Single Bond Universal (MDP-containing)|
CHX conditioning was performed through pre-treating the acid-etched dentin surface with 2% CHX aqueous solution for 60 s and blot-drying.
The adhesive was spread thinly with moisture-free air and light-cured for 20 s with a light-curing unit (Elipar™ S10, 3M ESPE, USA). Two commercial adhesive were used in this study: a phosphate ester monomer free adhesive (Single Bond 2) and a MDP-containing universal adhesive (Single Bond Universal). Table 2 lists their compositions.
|Single bond 2||Bis-GMA, HEMA, dimethacrylates, ethanol, water||3M ESPE, USA|
|Single bond universal||MDP, phosphate monomer, HEMA, dimethacrylate resins, Vitrebond copolymer, filler, ethanol, water, initiators, silane||3M ESPE, USA|
Then composite resin (Filtek Z250, 3M ESPE, USA) was built-up on the adhesive-applied dentin surface in two 2-mm-thick increments, and each increment was compressed firmly and light cured for 40 s. These resin–dentin bonded specimens for each group were stored in distilled water at 37 °C for either 24 h or 6 months.
Microtensile bond strength (μTBS)
Each bonded specimen was vertically sectioned into 0.9 mm-thick slabs with a low-speed diamond saw under water irrigation after storage for 24 h or 6 months in water. The two central slabs of each specimen were sectioned into 0.9 mm × 0.9 mm sticks containing the resin-dentin adhesive joint in the center of each stick. The three longest sticks from each slab were selected for μTBS testing.
The cross-sectional area of each stick was measured to the nearest 0.01 mm with a digital caliper (MNT-150, Meinaite, China). Each stick was bonded to a testing jig with cyanoacrylate glue, and stressed at a cross-head speed of 1 mm/min until failure using a microtensile testing device (Micro Tensile Tester, BISCO, USA). The μTBS was expressed in MPa, as derived from dividing the imposed force (N) at the time of fracture by the bond area of the individual specimen (mm 2 ).
The μTBS of each group for 24 h and 6 m water-storage was recorded, and mean and standard deviation values were calculated. Statistical analysis of μTBS was performed using SPSS 21.0 statistical software (IBM SPSS Inc., Chicago, IL, USA). The data sets were non-linearly transformed to satisfy the normality and equal variance assumptions, prior to using parametric statistical methods. Two-way analysis of variance (two-way ANOVA) then evaluated the effects of water storage time (i.e. 24 h or 6 months) and bonding strategy (i.e. the six groups), and also the interaction of the two factors on dentin bonding performance. The statistical significance level was set at P = 0.05.
Three bonded slabs for each storage condition from each group were observed by scanning electron microscopy (SEM). The slabs were wet-polished using 600-grit silicon carbide paper and coated with two layers of nail varnish applied to within 1 mm of the bonded interfaces. The slabs were immersed for 24 h in ammoniacal silver nitrate solution—which was prepared as described by Tay et al. [ ]—rinsed thoroughly in distilled water, and subsequently immersed in photodeveloping solution for 8 h under a fluorescent light to reduce silver ions to metallic silver grains. These slabs were coated with Au and examined by SEM (TESCAN MAIA3, Kohoutovice, Czech Republic) in backscattered electron imaging mode to evaluate nanoleakage.
The potential interaction between CHX and MDP was chemically characterized via Fourier transform infrared (FTIR) spectroscopy, X-ray photoelectron spectroscopy (XPS) and 31 P solid-state nuclear magnetic resonance (NMR) spectroscopy.
For FTIR spectroscopy, three centrifuge tubes containing 0.2 g hydroxyapatite (HA) powder were selected and treated with CHX, MDP or CHX + MDP; a fourth was used as control. The three treated HA powders (CHX-HA, MDP-HA and CHX + MDP-HA) were washed three times with acetone, centrifuged and then dried in air at ambient temperature for 48 h. The spectrometer (Nicolet 6700, Thermo Fisher Scientific, USA) recorded infrared signals in the spectral range of 2000–500 cm −1 .
Samples were grouped and treated for XPS and NMR spectroscopy in the same way as for FTIR. XPS (Escalab 250xi, Thermo Fisher Scientific, UK) used monochromatic A1 Kα radiation, with untreated HA as the control. The C1s spectra were recorded using XPS Peak 4.1 software with the Lorentz–Gauss ratio fixed at 80%.
31 P NMR spectra for the four sample groups were measured (AVANCE III HD 400M, Bruker, Germany), and the chemical shifts were recorded in ppm, with crystalline H 3 PO 4 powder serving as an external reference (0.0 ppm).
In situ zymography
Dentin specimens from each group were prepared as listed in Table 1 . A 1-mm-thick flowable composite (Filtek Flow, 3M ESPE, USA) was applied to the bonded dentin, light-cured for 20 s, and then cut vertically with a low-speed saw into 1-mm-thick slabs to expose the adhesive/dentin interfaces.
The slabs were ground to 500-μm-thick specimens and glued to microscope slides [ ]. In situ zymography was performed with quenched fluorescein-conjugated gelatin as the MMP substrate (EnzChek™ Gelatinase/Collagenase Assay Kit, Thermo Fisher Scientific, USA) according to a reported procedure [ ]. The fluorescent gelatin mixture was placed on the top of each slab and protected with a cover slip. The microscope slides were light-protected and incubated at 37 °C for 24 h. The activities of endogenous gelatinolytic enzymes such as MMPs were assessed by examination with a confocal laser scanning microscope (CLSM; Zeiss LSM880 with NLO & Airyscan, Germany).
Table 3 summarizes the means and standard deviations of μTBS results obtained from the six groups stored in water for either 24 h or 6 months.
|Group||24 h (MPa)||6 months (MPa)|
|Ctr||33.00(3.95) a||28.36(4.01) d|
|CHX||37.43(5.29) b||33.31(3.28) e|
|MDP||43.38(4.32) c||37.58(3.87) f|
|CHX + MDP||43.01(3.78) c||38.75(4.58) f|
|SBU||42.60(2.68) c||35.94(2.98) e,f|
|CHX + SBU||44.40(3.13) c||37.33(3.24) f|