Microsc. Microanal. 21, 214–230, 2015 doi:10.1017/S1431927614013658

© MICROSCOPY SOCIETY OF AMERICA 2014

Bond Strength and Bioactivity of Zn-Doped Dental Adhesives Promoted by Load Cycling Manuel Toledano,* Fátima S. Aguilera, Estrella Osorio, Inmaculada Cabello, Manuel Toledano-Osorio, and Raquel Osorio Faculty of Dentistry, Dental Materials Section, University of Granada, Colegio Máximo de Cartuja s/n, 18071 Granada, Spain

Abstract: The purpose of this study was to evaluate if mechanical loading influences bioactivity and bond strength at the resin–dentin interface after bonding with Zn-doped etch-and-rinse adhesives. Dentin surfaces were subjected to demineralization by 37% phosphoric acid (PA) or 0.5 M ethylenediaminetetraacetic acid (EDTA). Single bond (SB) adhesive—3M ESPE—SB + ZnO particles 20 wt% and SB + ZnCl2 2 wt% were applied on treated dentin to create the groups PA + SB, SB + ZnO, SB + ZnCl2, EDTA + SB, EDTA + ZnO, and EDTA + ZnCl2. Bonded interfaces were stored in simulated body fluid for 24 h and tested or submitted to mechanical loading. Microtensile bond strength (MTBS) was assessed. Debonded dentin surfaces were studied by high-resolution scanning electron microscopy. Remineralization of the bonded interfaces was assessed by atomic force microscope imaging/nanoindentation, Raman spectroscopy/cluster analysis, and Masson’s trichrome staining. Load cycling (LC) produced reduction in MTBS in all PA + SB, and no change was encountered in EDTA + SB specimens, regardless of zinc doping. LC increased the mineralization and crystallographic maturity at the interface; a higher effect was noticed when using ZnO. Trichrome staining reflected a narrow demineralized dentin matrix after loading of dentin surfaces that were treated with SB-doped adhesives. This correlates with an increase in mineral platforms or plate-like multilayered crystals in PA or EDTA-treated dentin surfaces, respectively. Key words: dentin, remineralization, load cycling, doped adhesives, bond strength

I NTRODUCTION Dentin is a complex hydrated fiber-reinforced biological composite. Its structure is composed of about 50 vol% mineral in the form of sub-micrometer to nanometer-sized carbonate-rich, calcium-deficient apatite crystallites (~5 ×30 × 100 nm) that are dispersed between parallel, micrometer-sized, hypermineralized, collagen-poor hollow cylinders that form the dentinal tubules containing peritubular dentin (Marshall et al., 1997). The organic matter represents ~30 vol%, which is largely a felt-work of type I collagen (90%) and noncollagenous proteins (10%) such as dentin matrix protein and dentin phosphoproteins, the major portion of the noncollagenous proteins with a potent modulating effect on biomineralization (Xu & Wang, 2011). Demineralization of dentin, or dentin conditioning, is the process of removing mineral ions from the apatite latticework leaving the collagen fibers without support except for the water contained within the dentin. The aim is to improve monomer spreading onto the substrate, and to increase diffusion into the dental tissue in order to get subsequent entanglement of the adhesive resin into the dentinal components to achieve a hybrid layer (HL) (Nakabayashi et al., 1982). Etch-and-rinse adhesives require treating dentin with phosphoric acid (PA) to remove the smear layer and to demineralize the underlying dentin, to a depth of ~5 μm. Received August 4, 2014; accepted October 30, 2014 *Corresponding author. [email protected]

This exposes a dense filigree of organic-matrix fibrils and is followed by the application of a primer/bonding. Milder conditioners (i.e., ethylenediaminetetraacetic acid, EDTA) eliminate the smear layer and plugs, causing dentinal erosion, but remove less calcium from the dentin surface. This promotes shallow demineralization and induces favorable chemical modifications (Habelitz et al., 2002). With both PA or EDTA agents, a volume of demineralized and nonresin infiltrated collagen remains at the bottom of the hybrid layer (BHL). This unprotected collagen may become the site for collagen hydrolysis by host-derived matrix metalloproteinase (MMP) enzymes (Osorio et al., 2011). Zinc has been widely used in dentistry and has been experimentally incorporated into several resin adhesives to reduce MMP-mediated collagen degradation (Toledano et al., 2012b), to induce dentin remineralization at the bonded interface (Toledano et al., 2013), and to preserve the bonding efficacy over time (Toledano et al., 2012b). In vitro effects of mechanical stimuli have promoted remineralization at both HL and BHL in the resin–dentin interface (Toledano et al., 2014a). The outcomes might be understood as resin–dentin interfaces with new mineral crystals embedded within a preserved collagen network. Thereby, the experimental clinical exploitation of combining dentin conditioning, Zn-doped dentin adhesives, and in vitro mechanical stimulation might provide improved bonding efficacy and therapeutic approaches for management of dental diseases.

Zinc-Doped Adhesives Performance After Loading

The purpose of this study was to evaluate the resin– dentin bond strength and the ability of etch-and-rinse zinc-doped adhesives to induce remineralization at the bonded dentin interface created by using two different demineralization procedures of the dentin surface, and an in vitro mechanical loading application. This study tested the two null hypotheses that, (1) load cycling has no effect on the microtensile bond strength (MTBS) of the zinc-doped adhesives to dentin and (2) remineralization of the resin– dentin interface obtained with zinc-doped adhesives is not produced or affected by the different etching procedures tested, after load cycling.

M ATERIALS AND M ETHODS Specimen Preparation, Bonding Procedures, and Mechanical Loading In all, 48 human molars extracted for surgical reasons were obtained within informed consent from donors (20–40 years of age), under a protocol approved by the Institution Review Board. Molars were stored at 4°C in 0.5% chloramine T for up to 1 month before use. A flat mid-coronal dentin surface was exposed using a hard tissue microtome (Accutom-50; Struers, Copenhagem, Denmark) equipped with a slow-speed, watercooled diamond wafering saw (330-CA RS-70300; Struers). A 180-grit silicon carbide (SiC) abrasive paper mounted on a water-cooled polishing machine (LaboPol-4; Struers) was used to produce a clinically relevant smear layer (Koibuchi et al., 2001).

Table 1.

215

An etch-and-rinse adhesive system, Single Bond (SB) Plus (3M ESPE, St Paul, MN, USA), was tested. It was zinc doped by mixing the bonding resin with 10 wt% ZnO microparticles (Panreac Química, Barcelona, Spain) (SB-ZnO) or with 2 wt% ZnCl2 (Sigma Aldrich, St Louis, MO, USA) (SB-ZnCl2). To achieve complete dissolution of ZnCl2 and dispersion of ZnO nanoparticles, adhesive mixtures were vigorously shaken for 1 min in a tube agitator (Vortex Wizard, Ref. 51075; Velp Scientifica, Milan, Italy). The complete process was performed in the dark. The chemical and adhesive descriptions are provided in Table 1. The specimens were divided into the following groups (n = 4) based on the adhesive systems tested and dentin-etching procedure: (i) SB was applied on 37% PA-treated dentin, 15 s (PA + SB); (ii) SB was applied on EDTA-treated dentin, 0.5 M, 60 s (EDTA + SB); (iii) SB-ZnO was applied on 37% PA-treated dentin; (iv) SB-ZnO was applied on EDTA-treated dentin, 0.5 M, 60 s; (v) SB-ZnCl2 applied on 37% PA; (vi) SB-ZnCl2 applied on EDTA-treated dentin, 0.5 M, 60 s. The bonding procedures were performed in moist dentin following the manufacturer’s instructions. A flowable resin composite (X-FlowTM, Dentsply, Caulk, UK) was placed incrementally in five 1 mm layers and light cured with a Translux EC halogen unit (Kulzer GmbH, Bereich Dental, Wehrheim, Germany) for 40 s. Half of the teeth were stored for 24 h in simulated body fluid solution (SBF) (Osorio et al., 2014), and the other half were submitted to mechanical loading in SBF (100,000 cycles, 3 Hz, 49 N) (S-MMT-250NB; Shimadzu, Tokyo, Japan) (Toledano et al., 2014a). During the

Materials and Chemicals Used in This Study and Respective Manufacturers, Basic Formulation, and Mode of Application.

Product Details

Basic Formulation

Mode of Application

Adper Single Bond Plus (SB) (3M ESPE, St Paul, MN, Bis-GMA, HEMA, dimethacrylates, ethanol, water, Dentin conditioning USA) a novel photoinitiator system, a methacrylate 37% H3PO4 (15 s) 0.5 M EDTA (60 s) functional copolymer of polyacrylic, polyitaconic acids Adhesive application Rinse with water Adhesive application (30 s) Light activation (15 s) Zinc oxide (Panreac Química SA, Barcelona, Spain) Zinc chloride 2-hydrate powder (Sigma Aldrich, St Louis, MO, USA) Phosphoric acid 37% (Braun Medical SA, Barcelona, Spain) EDTA (Sigma Aldrich, St Louis, MO, USA) X-FlowTM (Dentsply, Caulk, UK)

Strontium alumino sodium fluorophosphorsilicate glass, di- and multifunctional acrylate and methacrylate resins, DGDMA, highly dispersed silicon dioxide UV stabilizer, ethyl-4dimethylaminobenzoate camphorquinone, BHT, iron pigments, titanium dioxide

Bis-GMA, bisphenol A diglycidyl methacrylate; HEMA, 2-hydroxyethyl methacrylate; CQ, camphorquinone; DGDMA, diethyleneglycol dimethacrylate phosphate; BHT, butylated hydroxytoluene; H3PO4, phosphoric acid; EDTA, ethylenediaminetetraacetic acid.

216

Manuel Toledano et al.

24-h time period the loaded specimens were kept in SBF at 37°C.

(independent factors are mechanical loading and adhesive type) and Student–Newman–Keuls multiple comparisons (p < 0.05).

MTBS After the different procedures, 24 teeth (two from each group) were sectioned into serial slabs, and further into beams with cross-sectioned areas of 1 mm2. Specimens were attached to a modified Bencor Multi-T testing apparatus (Danville Engineering Co., Danville, CA, USA) with a cyanoacrylate adhesive (Zapit/Dental Venture of America Inc., Corona, CA, USA) and stressed to failure in tension (Instron 4411/Instron Inc., Canton, MA, USA) at a crosshead speed of 0.5 mm/min. The cross-sectional area at the site of failure of the fractured specimens was measured to the nearest 0.01 mm with a pair of digital calipers (Sylvac Ultra-Call III, Fowler Co. Inc., Newton, MA, USA). Bond strength values were calculated in MPa. Fractured specimens were examined with a stereomicroscope (Olympus SZ-CTV; Olympus, Tokyo, Japan) at 40× magnification to determine the mode of failure. Failure modes were classified as adhesive or mixed. Representative specimens of each group were maintained for 48 h in a desiccator (Sample Dry Keeper Simulate Corp., Tokyo, Japan), mounted on aluminum stubs with carbon cement, and sputter-coated with pure gold by means of a sputtercoating Unit E500 (Polaron Equipment Ltd., Watford, England). Prepared specimens were observed with a scanning electron microscopy (HRSEM Gemini; Carl Zeiss, Oberkochen, Germany) at an accelerating voltage of 20 kV in order to observe the morphology of the debonded interfaces. MTBS values were analyzed by two-way analysis of variance (ANOVA) (independent factors are mechanical loading and adhesive type) and Student–Newman–Keuls multiple comparisons tests. For all tests, statistical significance was set at a = 0.05.

Atomic Force Microscope (AFM) Imaging and Nanoindentation An AFM (Nanoscope V; Digital Instruments, Veeco Metrology Group, Santa Barbara, CA, USA) equipped with a Triboscope indentor system (Hysitron Inc., Minneapolis, MN, USA) and a Berkovich indenter (tip radius ~20 nm) was employed for the imaging and indentation processes in a fully hydrated status (Sauro et al., 2012). For each subgroup, three slabs were tested. On each slab, five indentation lines were executed in five different mesio-distal positions along the interface in a straight line starting from the adhesive layer down to the intertubular dentin. Indentations were performed with a load of 4,000 nN and a time function of 10 s. The distance between each indentation was kept constant by adjusting the distance intervals in 5 µm (±1) steps and the load function (Toledano et al., 2013). Hardness (Hi) and modulus of elasticity (Ei) data were registered. Mean nanohardness and Young’s modulus values were obtained in GPa. Data were analyzed by two-way ANOVA

Raman Spectroscopy and Cluster Analysis A dispersive Raman spectrometer/microscope (Horiba Scientific Xplora, Villeneuve d’Ascq, France) was also used to analyze bonded interfaces. A 785-nm diode laser (100 mW sample power) through a X100/0.90 NA air objective was employed. The Raman signal was acquired using a 600-lines/mm grating centered between 900 and 1,800 cm–1. Chemical mapping of the interfaces were performed. For each specimen a 45 × 45 μm area of the interfaces was mapped using 2 μm spacing at X-axis and 1 μm at Y-axis. A total of 1,100 points were performed per map. The resolution of the mapping was 6.25 cm–1. BHL depth was between 0.25 and 2.25 μm and HL ranged from 2.00 to 6.00 μm, approximately. Chemical mapping was submitted to K-means cluster (KMC) analysis using the multivariate analysis tool (ISys Horiba), which includes statistical patterns to derive the independent clusters. Hypotheses concerning the number of clusters formed in resin-bonded interfaces were previously obtained (Toledano et al., 2014b, 2014c). However, Ward’s method was employed to get some sense of the number of clusters and the way they merge as seen from the dendrogram. The aim of a factor analysis lies in the effective reduction of the data set dimension while maintaining a maximum of information. This method was used to model the data and to determine spectral variances associated with data differentiation. It resulted in the calculation of a new coordinate system whereby variations in the data set are described via new axes, principal components (PC). KMC is a cluster analysis based on a centroid model that partitions n observations into k clusters in which each observation belongs to the cluster with the nearest mean (Almahdy et al., 2012). The natural groups of components (or data) based on some similarity and the centroids of a group of data sets were found by the clustering algorithm once calculated by the software. To determine cluster membership, this algorithm evaluated the distance between a point and the cluster centroids. The output from a clustering algorithm was basically a statistical description of the cluster centroids with the number of components in each cluster. The biochemical content of each cluster was analyzed using the average cluster spectra. Four clusters were identified and values for each cluster such as adhesive, HL, BHL, and dentin, within the interface, were independently obtained. Principal component analysis decomposed the data set into a bilinear model of linear independent variables, the PCs. Two PCs were selected for the present study at the HL and BHL interfaces. Raman spectra were acquired from a minimum of two different sites on each sample. The observed spectra were described at 900–1,800 cm–1 with ten complete overlapping Gaussian lines, suggesting homogeneous data for further calculations (Nakabayashi, 1992; Ager et al., 2005). Gaussian–Lorentzian

®

Zinc-Doped Adhesives Performance After Loading

peaks summed to match small regions of the spectrum were obtained by a nonlinear peak-fitting routine that employs the Levenberg–Marquardt algorithm and a first-order polynomial (Awonusi et al., 2007; Milly et al., 2014). As the cluster centroids are essentially means of the cluster score for the elements of cluster, the mineral and organic components of dentin HLs were examined for each cluster. A comparison of the spectra that were collected from the two specimens which compose each subgroup indicated complete overlap, suggesting similarity between both measurements. The relative mineral component of dentin was assessed as follows (Toledano et al., 2014a): –1

217

2. Ratio pyridinium/phenyl (1,032/1,001 cm–1): the higher the ratio, the greater the extent of collagen crosslinking (Jastrzebska et al., 2003; Xu & Wang, 2012). 3. Ratio 1,003 (phenyl)/1,450 (CH2) arises preceding deposition of hydroxyapatite (HAP) crystals within the structure (Wang et al., 2009). 4. Advanced glycation end products (AGEs)-pentosidine at 1,550 cm–1 were interpreted as a marker of the aging process (Sell & Monnier, 1989).

Nature of Collagen

–1

1. Phosphate (960 cm ) and carbonate (1,070 cm ) peaks and areas of their bands. Peak heights were processed in absorbance units. 2. Relative mineral concentration (RMC) (i.e., mineral-tomatrix ratio). It was inferred from the visible ratio of the intensities of the peaks at 960 cm–1 (phosphate) (PO34 − ) and 1,003 cm–1 (phenyl group), the aromatic ring of phenylalanine residues in collagen. These indexes are concerned with the maximum relative degree of mineralization (Karan et al., 2009; Schwartz et al., 2012). In addition, peaks at 960 and 1,450 cm–1 (CH2) or 1,070 and 1,450 cm–1 can be used (Wang et al., 2009). Crystallinity was evaluated based on the full-width at half-maximum (FWHM) of the phosphate band at 960 cm–1 and carbonate band at 1,070 cm–1. These indeces expressed the crystallographic or relative atomic order, as narrower peaks suggest less structural variation in bond distances and angles (Schwartz et al., 2012). In general, the narrower the spectral peak width, the higher the degree of mineral crystallinity (Karan et al., 2009). The gradient in mineral content (GMC) or carbonate content of the mineral crystallites was assessed as the relationship between the ratio of heights at 1,070 cm–1 (carbonate, CO23 − ) to 960 cm–1 (phosphate, PO3− 4 ), indicating carbonate substitution for phosphate (Schwartz et al., 2012). Phosphate peaks ratios (PPR) were used to assess the ratio between the mineral peak at 960 cm–1 (PO3− 4 ), within the demineralized zone and the mineral peak (PO3− 4 ) within the healthy substratum (Milly et al., 2014). The organic component of dentin was analyzed by examining the following parameters. The phenyl group peak at 1,003 cm–1, which is assigned to C–C bond in the phenyl group, was used for normalization (Xu & Wang, 2011). Crosslinking 1. Pyridinium ring vibration: in the spectra, the peak at 1,030/1,032.7 cm–1 was assigned to the C–C in pyridinium ring vibration, which has a trivalent amino acid crosslinking residue (Daood et al., 2013). The relative intensity of this peak increased after the crosslinking formation (Jastrzebska et al., 2003).

1. Peaks at 1,246/1,270, 1,450, and 1,655/1,667 cm–1, assigned to amide III, CH2, and amide I, respectively, are sensitive to the molecular conformation of the polypeptide chains (Jastrzebska et al., 2003; Xu & Wang, 2011). The decrease in amide I peak indicates damage or removal of collagen fibrils (Xu & Wang, 2012). 2. The amide I/amide III ratio is concerned with the organization of collagen. 3. The amide III/CH2 ratio wagging mode indicates structural differences (Salehi et al., 2013). 4. The amide I/CH2 ratio indicates altered collagen quality (Salehi et al., 2013). 5. The amide III and I/AGEs-pentosidine ratios indicate the glycation reaction versus collagen scaffolding (Salehi et al., 2013). 6. The 1,340 cm–1 peak was assigned to protein α-helices where intensity is sensitive to molecular orientation (Wang et al., 2009).

Light Microscopy–Masson’s Trichrome Staining The resin–dentin-bonded slices of each group were used for histo-morphological evaluations. The medial aspects of each resin–dentin-bonded slice was fixed in a glass holder with a photo-curing adhesive (Technovit 7210 VLC; Heraeus Kulzer GmbH Co., Werheim, Germany) and ground with SiC papers of increasing fine grits (800, 1,000, 1,200, and 4,000) in a polisher (D-2000; Exakt Apparatebau, Norderstedt, Germany) until its thickness was ~10 mm. Slices were stained with Masson’s trichrome for differentiation of resin and nonresin encapsulation of the exposed collagen. This dye has a high affinity for cationic elements of normally mineralized type I collagen, resulting in staining collagen green, and when demineralized, resulting in different coloration, generally red. Collagen coated with adhesive stains orange and pure adhesive appears beige. Slices with adherent stained sections were dehydrated through ascending ethanol series and xylene. The sections were cover slipped and examined by light microscopy (BH-2; Olympus) at 100× magnifications. Three slices were prepared from each specimen, and images were digitized in a scanner (Agfa Twin 1200; Agfa-Gevaert NV, Mortsel, Belgium). In each specimen, the presence or absence of a red band (that would correspond to demineralized dentin) was observed. A qualitative assessment of the collagen

218

Manuel Toledano et al.

Table 2. Mean and Standard Deviation of Microtensile Bond Strength Values (MPa), and Percentage Distribution (%) of Failure Mode (A and M), Obtained for the Different Experimental Groups. Unloaded

PA + SB PA + SB-ZnO PA + SB-ZnCl2 EDTA + SB EDTA + SB-ZnO EDTA + SB-ZnCl2

Loaded

Mean (SD) (MPa)

A (%)

M (%)

36.95 (4.89) A1 35.15 (4.62) A1 34.95 (5.95) A1 28.1 (4.2) B1 19.69 (2.43) C1 22.77 (3.20) C1

27 22 31 55 62 59

73 78 69 45 38 41

Mean (SD) (MPa) 29.12 (4.4) a2 27.51 (7.92) ab2 23.33 (3.97) b2 27.5 (9.7) a1 18.79 (5.1) b1 19.59 (5.75) b1

A (%)

M (%)

63 71 65 57 60 62

37 29 35 43 40 38

Identical letters indicate no significant difference in columns and numbers in rows, after Student–Newman–Keuls or Student’s t tests (p < 0.05). A, adhesive; M, mixed; EDTA, ethylenediaminetetraacetic acid; PA, phosphoric acid; SB, single bond; ZnO, zinc oxide; ZnCl2, zinc chloride.

encapsulation was completed by observing color differences within the interfacial zones of resin–dentin interfaces (Toledano et al., 2012a).

RESULTS AND D ISCUSSION The first null hypothesis was rejected, as load cycling decreased the MTBS and increased the percentage of adhesive failures in

all PA + SB formulations; in both groups (unloaded versus loaded), all subgroups performed similarly except PA + SB-ZnCl2 load cycled, which attained the lowest bond strength values (Table 2). A decrease in bonding efficacy in PA + SB has been previously reported after mechanical loading (Toledano et al., 2006), as well as after water degradation (De Munck et al., 2003). It has been pointed out that fatigue stress (Nikaido et al., 2002) produces a failure mostly at the top or beneath the

Figure 1. Mean and SD of nanohardness (Hi) and Young’s modulus (Ei) (GPa) measured at the experimental hybrid layers in sound dentin. Identical letters indicate no significant differences between unloaded restorations from the different experimental groups, identical numbers indicate no significant differences between load-cycled restorations from the different experimental groups, and * indicates significant differences between unloaded and load-cycled restorations from the same experimental group. PA, phosphoric acid; SB, single bond; EDTA, ethylenediaminetetraacetic acid; ZnO, zinc oxide; ZnCl2, zinc chloride.

Zinc-Doped Adhesives Performance After Loading

HL where demineralized collagen fibrils were exposed and the adhesive failed to envelop the collagen network properly (Prati et al., 1999; Toledano et al., 2006). PA + SB-doped groups with ZnO or ZnCl2 nanohardness (Hi) and Young’s modulus (Ei) at HL and BHL when compared with the control group (PA + SB) are shown in Figures 1 and 2. The improvement in both Hi and Ei at the resin–dentin interface may be associated with a remineralizing effect, as the mechanical properties of the dentin depend on the degree and on the quality of the mineralization (Balooch et al., 2008). This remineralization front after load cycling was also observed throughout the Masson’s trichrome images, which showed a reduction in red intensity (Figs. 3c, 3d), and a growing of some new canaliculi crossing the demineralized dentin (Fig. 3d), indicating remineralization of peritubular and intertubular dentin (Figs. 3d, 3f). High-resolution scanning electron microscopy corroborated the presence of new mineral formation at both intertubular and peritubular locations (Figs. 4a, 4b). Nevertheless, PA + SB-Zn-doped and then cycled specimens showed a general decrease in both nanomechanical properties (Hi and Ei)

219

(Fig. 1) and in the degree of mineralization related to the phosphate group, i.e., the mineral-to-matrix ratio (RMC) at both HL and BHL (Table 3a; Fig. 5Ag). RMC was inferred from the visible ratio of the intensities of the peaks at 960 cm–1 (PO34 − ) and 1,003 cm–1 (phenyl group) (Table 3b), the aromatic ring of phenylalanine residues in collagen (Schwartz et al., 2012). In the PA + SB-Zn-doped and load-cycled specimens, peaks at 960 cm–1 increased ~1.26 fold with respect to the control samples (PA + SB) (Table 3a; Figs. 5Ad, 5Af); peaks at 1,003 cm–1 increased proportionally (Table 3b), an average of ~1.31 fold in the same groups (PA + SB-Zn-doped and cyclic loaded). An increase in the protein-dependent spectral signal at phenylalanine preceded the appearance of HAP crystals (Wang et al., 2009; Fig. 5Ag). The relative composition of this new mineral or GMC varied from an increase (∼1.30 fold at HL and ∼1.5 fold at BHL) in PA + SB-ZnCl2, to a low in PA + SB-ZnO, both load cycled (Table 3a). The promoted change after PA + SB-ZnCl2 application means a higher carbonate/phosphate content at the resin–dentin interface (HL and BHL). Carbonated

Figure 2. Mean and SD of nanohardness (Hi) and Young’s modulus (Ei) (GPa) measured at the experimental bottom of the hybrid layer in sound dentin. Identical letters indicate no significant differences between unloaded restorations from the different experimental groups, identical numbers indicate no significant differences between load-cycled restorations from the different experimental groups, and * indicates significant differences between unloaded and loadcycled restorations from the same experimental group. PA, phosphoric acid; SB, single bond; EDTA, ethylenediaminetetraacetic acid; ZnO, zinc oxide; ZnCl2, zinc chloride.

220

Manuel Toledano et al.

Figure 3. Representative light micrographs of PA + SB specimens interface stained with Masson’s trichrome. Mineralized dentin is stained green, the adhesive is stained beige, and exposed protein is stained red. Original magnification: 150 × . a: PA + SB control (unloaded). b: PA + SB loaded. c: PA + SB-ZnO. d: PA + SB-ZnO load cycled. e: PA + SB-ZnCl2. f: PA + SB-ZnCl2 load cycled. Intense and wide (a, c), faint (b, e), or thin (d, f) evidence of partial demineralization and/or exposed protein (red stain) is detectable at the resin–dentin interface. New tubular dentin walls originate and are observed as a mineral canaliculi formation crossing the demineralized and noninfiltrated collagen. These new formations reproduce the sinusoidal primary curvatures (pointer) (d). Load cycling of both PA + SB-Zn-doped groups showed a reduction in red intensity and width, revealing slight and limited resin uncovered decalcified dentin (arrows) at the interface (d, f), permitting observation of the remineralization of the partially demineralized dentin layer (asterisk). PA, phosphoric acid; SB, single bond; ZnO, zinc oxide; ZnCl2, zinc chloride.

apatite is a precursor of HAP (Gandolfi et al., 2011). Similarly, the ratio of phosphate peak/healthy substratum (PPR), which detects differences between intact and demineralized substratum regions (Milly et al., 2014), also increased an average of ∼1.17 and ∼1.37 fold in both PA + SB-ZnO/Cl2 after loading, respectively. Therefore, bigger ratios of GMC and PPR after PA + SB-ZnCl2 application are associated, in the present work, to the lowest values of crystallinity (19.30 at both HL and BHL). Thus formed mineral gave rise to Raman bands characteristic of a poorly carbonated apatite (Wang et al., 2009), with a

higher decrement in FWHM and calcium/phosphate ratio than in PA + SB-ZnO. On the contrary, PA + SB-ZnO exhibited the lowest FWHM (19.26 and 16.05, at HL and BHL, respectively), with a greater crystallographic maturity, i.e., lower degree of imperfections after load cycling. Narrowing of the phosphate v1 peak (at ca. 960 cm–1), i.e., lower FWHM or higher relative crystallinity (Table 3a; Karan et al., 2009) was observed at both HL (~1.07 fold in control groups and ~1.00 fold in the load-cycled specimens) and BHL (~1.09 fold in both control and load-cycled specimens). PA + SB-ZnCl2 might have better oriented the crystalline HAP formation inside the

Zinc-Doped Adhesives Performance After Loading

Figure 4. High-resolution scanning electron microscopy images of failures after bonding and MTBS testing. a: PA + SB-ZnO load. b: PA + SB-ZnCl2 load. c: EDTA + SB-ZnO unload. d: EDTA + SB-ZnCl2 unload. e, f: EDTA + SB-ZnO load. g, h: EDTA + SB-ZnCl2 load. Mixed failures and fracture at the bottom of the hybrid layer (a) or at the hybrid layer (b) may be observed in PA + SB-Zn-loaded samples. Collagen fibers are clearly observed, appearing mineralized (asterisk), and mineral platforms grow toward the entrance of tubules (pointer) (a). Some tubules are empty and open (arrow) or mineral filled, protruding from the entrance of tubules (pointer) (b). EDTA + SB-Zn-doped samples showed the collagen network covered by mineral, but allowing visual observation of fibrils (asterisk) (c), or precipitation of mineral on the surface of the dentin matrix, entirely covering the intertubular region and some part of tubules (pointer) (d). EDTA + SB-ZnO samples load cycled revealed an extended clump of mineral precipitates throughout the dense network of plate-like multilayered crystals on intertubular dentin and at the entrances of tubules (arrow) (e) or an extensive labyrinth of anastomoses, cavities, and hollows, perceptible at a nanometric scale (arrow). The typical staggered pattern of collagen fibrils (pointer) owing to the characteristic 67 nm periodicity was visible at the fibers that covered the tubular wall (f). EDTA + SB-ZnCl2 load-cycled samples produced a dentin surface totally covered by mineral deposits in strata (asterisks), as it became visible completely mineralized at both peritubular and intertubular locations, though differentiated. In the gradient zone the mineral formed a partial collar around each detected tubule lumen (arrow). Unaffected peritubular dentin was also observed (double arrows) (g, h). The prototypical D-periodicity banding of collagen fibrils was observed in detail (pointers) (h). MTBS, microtensile bond strength; PA, phosphoric acid; SB, single bond; ZnO, zinc oxide; ZnCl2, zinc chloride; EDTA, ethylenediaminetetraacetic acid.

221

222

Manuel Toledano et al.

Table 3a.

Mineral Gradients in Phosphoric Acid (PA)-Treated Dentin Surfaces Plus Single Bond (SB) Adhesive Application (PA + SB). Relative Presence of Mineral Phosphate (961)

PA + SB Control HL BHL Load cyclic HL BHL PA + SB-ZnO Control HL BHL Load cyclic HL BHL PA + SB-ZnCl2 Control HL BHL Load cyclic HL BHL

Carbonate (1,070)

FWHM

Peak

Area

RMC

Peak

Phosphate

GMC ratio (C/P)

PPR/healthy substratum

16.93 62.66

484.9 1,545.3

7.43 18.38

5.24 8.28

22.48 19.27

0.31 0.13

0.18 0.65

40.02 62.72

983.7 1,543.5

8.02 24.99

8.03 9.9

19.30 19.30

0.20 0.16

0.41 0.65

27.82 60.39

681.5 1,245.4

5.36 16.78

5.6 9.63

19.33 16.11

0.20 0.16

0.29 0.63

33.96 68.66

838.04 1,418.6

6.34 14.04

6.7 10.41

19.26 16.05

0.20 0.15

0.35 0.71

20.74 44.72

594.02 1,163.02

5.24 14.57

4.78 5.3

22.49 19.27

0.23 0.12

0.21 0.46

27.49 64.34

676.43 1,528.38

4.10 10.78

8.15 11.85

19.30 19.30

0.30 0.18

0.28 0.67

Peak intensities are expressed in cm–1. RMC, relative mineral concentration between mineral/phenyl (1,003); FWHM, full-width at half-maximun; GMC, gradient in mineral content; PPR, phosphate peaks ratio; ZnO, zinc oxide; ZnCl2, zinc chloride; HL, hybrid layer; BHL, bottom of the hybrid layer.

fibrils (Nudelman et al., 2010) as the increment in crystallinity was greater than that obtained by PA + SB-ZnO (Table 3a), probably because the dissolution rate of ZnCl2 is faster in comparison with that of ZnO (Osorio et al., 2014). The lower increment in crystallinity attained by PA + SB-ZnO may be consistent with a slower Zn+ + liberation rate from ZnO-resin doped, which will permit Ca and P deposits for delayed remineralization (Osorio et al., 2014). As a result, improvement of nanomechanical properties might be supported on this displayed long-range order among its component atoms and amorphous material (Wang et al., 2009), more than in the RMC performance. In general, ratios concerning the nature of collagen reflected a movement toward higher frequencies, denoting a rise in crosslinking of collagen that results after nucleation (Table 3b). Load cycling increased crosslinking in PA + SB-ZnCl2, as ratios 1,032 (pyridinium), 1,032/ 1,001, 1,004/1,450 (phenyl/CH2) were also augmented. However, 1,550 cm–1 (AGEs-pentosidine) decreased, as in PA + SB after loading. PA induced some changes in dentin collagen conformation mostly associated with denaturation processes and phase transition into gelatinous matrix (Pashley et al., 2000) and PA + SB-ZnO application promoted opposite results. The orientation-sensitive signals of α-helix decreased in PA + SB-ZnO, a common event when

mineralization is allowed to proceed (Wang et al., 2009; Table 3b). Considering unloaded groups, EDTA-treated surfaces attained a lower MTBS than PA + SB formulations, with the greatest percentage of adhesive failures (Table 2). Load cycling did not affect the bond strength results in EDTA groups. Among loaded subgroups, EDTA + SB (control) showed the highest bonding efficacy, similar to PA + SB and PA + SB-ZnO. SB-Zn-doped subgroups performed similarly, regardless of the type of conditioning (Table 2). EDTA caused shallow and minor dentinal demineralization, especially inside the tubules (Fig. 4h), without shifting dentin proteins. This avoided major alterations in the collagen fibrillar structure and conferred stability to such organic matrices, preserving the spongy character of the etched collagen matrix and consequently improving resin infiltration (Prati et al., 2000; Erhardt et al., 2008). Nanomechanical properties significantly increased in EDTA + SB-Zn-doped specimens after load cycling, but in lower proportion than the control undoped loaded group (Figs. 1, 2). Therefore, the second null hypothesis was, similarly, rejected as load cycling promoted remineralization of the resin–dentin interface obtained with both etching procedures. The higher amount of residual apatite crystallites left within the collagen matrix in EDTA-treated

Table 3b.

Organics Gradients in Phosphoric Acid (PA)-Treated Dentin Surfaces Plus Single Bond (SB) Adhesive Application (PA + SB). Crosslinking

Normalization Ratio Pyrid (1,032/ Phenyl (1,003) (1,032) 1,001

Ratio of phenyl/CH2 (1,003/CH)

AGEspentosidine (1,550)

A III (1,246– 1,270)

CH2 (1,450)

AI (1,655– 1,667)

Ratio of A Ratio of A I/AGEsRatio of Ratio of A Ratio of A III/AGEsA I/A III III/CH2 pentosidine pentosidine I/CH2

α-helices (1,340)

2.28 3.41

3.19 4.29

1.40 1.26

0.12 0.18

5.43 5.31

14.09 17.43

18.66 18.46

3.14 5.74

0.22 0.33

0.76 0.94

0.17 0.31

2.59 3.28

0.58 1.08

6.28 8.29

4.99 2.51

5.25 3.82

1.05 1.52

0.17 0.20

4.92 4.64

20.36 15.52

28.91 12.42

9.13 7.94

0.45 0.51

0.70 1.25

0.32 0.64

4.14 3.34

1.86 1.71

18.61 16.35

5.19 3.60

4.25 5.38

0.82 1.49

0.61 0.53

0.84 1.25

7.58 8.18

8.54 6.76

4.83 9.12

0.64 1.11

0.89 1.21

0.57 1.35

9.02 6.54

5.75 7.30

4.38 4.60

5.36 4.89

4.11 3.77

0.77 0.77

0.54 0.83

1.05 1.54

9.53 7.74

9.99 5.89

6.31 5.27

0.66 0.68

0.95 1.31

0.63 0.89

9.08 5.03

6.01 3.42

4.02 3.64

3.96 3.07

3.06 2.88

0.77 0.94

0.29 0.38

3.08 2.45

11.59 9.89

13.61 8.16

2.87 3.12

0.25 0.32

0.85 1.21

0.21 0.38

3.76 4.04

0.93 1.27

5.28 3.25

6.70 5.97

7.1 6.82

1.06 1.14

0.36 0.44

2.32 2.07

16.97 15.69

18.43 13.50

6.70 6.37

0.39 0.41

0.92 1.16

0.36 0.47

7.31 7.58

2.89 3.08

6.86 6.68

Peak intensities are expressed in cm–1. A, amide; Pyrid, pyridinium; AGEs, advanced glycation end products; HL, hybrid layer; BHL, bottom of the hybrid layer; ZnO, zinc oxide; ZnCl2, zinc chloride.

Zinc-Doped Adhesives Performance After Loading

PA + SB Control HL BHL Load cyclic HL BHL PA + SB-ZnO Control HL BHL Load cyclic HL BHL PA + SB-ZnCl2 Control HL BHL Load cyclic HL BHL

Nature of Collagen

223

224

Manuel Toledano et al.

Figure 5. A: Three-dimensional (3D) micro-Raman map of the phosphate peak (961 cm–1) intensities at the dentin-bonded interface of phosphoric acid (PA)-treated dentin surfaces plus single bond (SB) adhesive application (PA + SB): unloaded (left) or load cycled (right). a, b: Not doped; (c, d) SB-ZnO doped; (e, f) SB-ZnCl2 doped. g: Raman spectra of principal components (PCs): BHL, bottom of the hybrid layer for each PA + SB group. B: 3D micro-Raman map of the phosphate peak (961 cm–1) intensities at the dentin-bonded interface of EDTA-treated dentin surfaces plus SB adhesive application (EDTA + SB): unloaded (left) or load cycled (right). a, b: Not doped; (c, d) SB-ZnO doped; (e, f) SB-ZnCl2 doped. g: Raman spectra of PCs: BHL, bottom of the hybrid layer for each EDTA + SB group. HL, hybrid layer; EDTA, ethylenediaminetetraacetic acid; ZnO, zinc oxide; ZnCl2, zinc chloride.

Zinc-Doped Adhesives Performance After Loading

225

Figure 5. (Continued)

dentin and the functionalized monomers of the adhesive contributed to improvement in the dentin remineralization and prevented the denaturation of collagen (Kremer et al.,

1998; Toledano et al., 2014b). In general, at the HL, EDTA + SB-ZnCl2-doped specimens produced greater Hi and Ei (Fig. 1) than ZnO; this resin–dentin interface

226

Manuel Toledano et al.

Figure 6. Representative light micrographs of EDTA + SB specimens interface stained with Masson’s trichrome. Mineralized dentin is stained green, adhesive is stained beige, and exposed protein is stained red. Original magnification: 150 × . a: EDTA + SB control (unloaded). b: EDTA + SB loaded. c: EDTA + SB-ZnO. d: EDTA + SB-ZnO load cycled. e: EDTA + SB-ZnCl2. f: EDTA + SB-ZnCl2 load cycled. Absence of an unprotected collagen layer was detected in the majority of specimens (e, b, f). Exposed proteins were observed at both the resin–dentin interface and tubular area (c) (pointers). Any signs (f) of demineralization and/or exposed protein (red stain) and the thinnest (arrows) (d, f) uncovered decalcified dentin are detectable at the resin–dentin interface (asterisk). EDTA, ethylenediaminetetraacetic acid; SB, single bond; ZnO, zinc oxide; ZnCl2, zinc chloride.

corresponded with much thinner unprotected collagen layers in load cycling EDTA + SB-ZnCl2 (Fig. 6f) than seen for EDTA + SB-ZnO samples (Fig. 6d), and showed a complete absence of unprotected collagen layers representative of the advanced remineralization front in plate-like multilayered crystals (Fig. 4f) within the partially demineralized layer at BHL. The reduction in red intensity (less exposed protein) was accompanied with a maximum phosphate peak at the BHL in EDTA + SB-ZnCl2 load cycled (Table 4a), and with a typical mineralization pattern in strata that affects both intertubular and peritubular dentin (Figs. 4g, 4h).

As in the PA group, FWHM was lower in EDTA + SB-ZnO than in EDTA + SB-ZnCl2, both load cycled (Table 4a). This is attributed (Krajewski et al., 2005; Awonusi et al., 2007) to an increase in crystallographic perfection in the apatite unit cell as carbonate substituted for phosphate (Schwartz et al., 2012). The carbonate peak drops from 9.17 (EDTA + SB-ZnCl2) to 6.72 (EDTA + SB-ZnO), i.e., an increase in the GMC from 0.16 to 0.27, respectively (Table 4a). It is noteworthy that this GMC growth does not affect these nanomechanical properties, probably because the scarce new mineral deposited, though more crystalline, is not sufficient to increase both Hi and Ei. RMC was shown to

Zinc-Doped Adhesives Performance After Loading Table 4a.

227

Mineral Gradients in EDTA-Treated Dentin Surfaces Plus Single Bond (SB) Adhesive Application (EDTA + SB). Relative Presence of Mineral Phosphate (961)

EDTA + SB Control HL BHL Load cyclic HL BHL EDTA + SB-ZnO Control HL BHL Load cyclic HL BHL EDTA + SB-ZnCl2 Control HL BHL Load cyclic HL BHL

Carbonate (1,070)

FWHM

Peak

Area

RMC

Peak

Phosphate

GMC ratio (C/P)

PPR/healthy substratum

42.93 84.72

1,137.47 2,003.81

12.37 18.22

7.05 12.93

19.30 19.30

0.16 0.15

0.44 0.88

46.33 84.16

1,328.17 2,047.3

3.09 18.14

13.04 13.83

19.30 19.30

0.28 0.16

0.48 0.87

47.46 65.39

1,168.3 1,609.7

11.81 13.77

9.35 11.82

19.33 16.11

0.20 0.18

0.49 0.68

24.9 57.62

713.15 1,406.37

6.60 22.69

6.72 9.18

19.26 16.05

0.27 0.16

0.26 0.60

23.68 41.65

676.41 1,025.5

9.25 15.15

5.24 6.77

22.49 19.27

0.22 0.16

0.25 0.43

57.59 77.72

1,417.2 1,912.6

18.22 19.38

9.17 11.78

19.30 19.30

0.16 0.15

0.60 0.81

Peak intensities are expressed in cm–1. RMC, relative mineral concentration between mineral/phenyl (1,003); FWHM, full-width at half-maximum; GMC, gradient in mineral content; PPR, phosphate peaks ratio; HL, hybrid layer; BHL, bottom of the hybrid layer; ZnO, zinc oxide; ZnCl2, zinc chloride; EDTA, ethylenediaminetetraacetic acid.

be ~1.17 fold superior in EDTA + SB-ZnO than in EDTA + SB-ZnCl2, though the phosphate peak was ~0.74 fold inferior, respectively (Figs. 5Bd, 5Bf). This resulted after the proportional increase (~1.58 fold) of the phenyl peak (1,003 cm–1) in EDTA + SB-ZnCl2 with respect to EDTA + SB-ZnO (Table 4b; Fig. 5Bg). Nevertheless, the ratio 1,003 (phenyl)/1,450 (CH2) increases preceding deposition of HAP crystals within the structure (Wang et al., 2009) and in EDTA + SB-ZnO is ~1.21 fold higher than in EDTA + SBZnCl2. In general, ratios concerning both crosslinking and the nature of collagen reflected a movement toward higher frequencies in EDTA + SD-ZnCl2 adhesive after load cycling (Table 4b). Bands around 1,550 cm–1 (AGEs-pentosidine) performed inversely proportional, i.e., increased with ZnO and decreased in ZnCl2 after load cycling. Furthermore, the signal at 1,340 cm–1, assigned to protein α-helices, increased the intensity, indicating that they are more sensitive to molecular orientation in order to enhance further crystallization (Wang et al., 2009; Table 4b; Fig. 5Bg).

S UMMARY At the resin–dentin interface, PA + SB-Zn-doped groups remineralized the resin–dentin interface and the tubular

dentin walls, showing a reduction in uncovered or exposed protein substrata, and an increment of mineral platform growing on the intertubular dentin toward the entrance of tubules. The thinner unprotected collagen layers were produced after combining EDTA, SB-ZnCl2, and load cycling. These changes were accompanied with increases in both crystallinity and PPRs with gradients in poorly carbonated apatite content. PA or EDTA + SB-ZnO achieved the highest crystallographic maturity and the lowest α-helix signals after load cycling when the PA was used for dentin conditioning. The nature of collagen denoted a rise in crosslinking in EDTA or PA + SB-Zn-doped adhesives after loading. Though remineralized with dentin conditioning (PA versus EDTA), the resin–dentin interfaces promoted with PA application degraded after load cycling, as the bonding efficacy significantly diminished. The fatigue stress produced remineralization at both HL and BHL, but a failure at the top of the HL, i.e., at the adhesive layer, as reflected by the increment in adhesive failures. This is the first time that a reduction in dentin bond strength and an increase in dentin remineralization have been assessed at bonded interfaces after mechanical loading, regardless of the use of Zn-doped adhesives. The major beneficial effect attained after loading when dentin was EDTA treated deserves future research.

228 Manuel Toledano et al.

Table 4b.

Organics Gradients in EDTA-Treated Dentin Surfaces Plus Single Bond (SB) Adhesive Application (EDTA + SB). Crosslinking

Normalization Ratio Pyrid (1,031/ Phenyl (1,003) (1,032) 1,001) EDTA + SB Control HL BHL Load cyclic HL BHL EDTA + SB-ZnO Control HL BHL Load cyclic HL BHL EDTA + SB-ZnCl2 Control HL BHL Load cyclic HL BHL

Nature of Collagen

Ratio of phenyl/CH2 (1,003/CH)

AGEspentosidine (1,550)

A III (1,246– 1,270)

CH2 (1,450)

AI (1,655– 1,667)

Ratio of A Ratio of A I/AGEsRatio of Ratio of A Ratio of A III/AGEspentosidine pentosidine I/CH2 A I/A III III/CH2

α-helices (1,340)

3.47 4.65

4.53 6.32

1.31 1.36

0.22 0.56

2.7 3.14

12.82 12.03

16.11 8.25

4.34 4.61

0.34 0.38

0.80 1.46

0.27 0.56

4.75 3.83

1.61 1.47

3.91 5.22

14.97 4.64

11.56 6.71

0.77 1.45

0.43 0.29

12 5.03

18.09 17.06

35.17 15.90

10.84 9.82

0.60 0.58

0.51 1.07

0.31 0.62

1.51 3.39

0.90 1.95

19.06 14.08

4.02 4.75

4.72 6.76

1.17 1.42

0.69 0.73

3.23 2.81

8.98 9.38

5.85 6.48

3.28 3.92

0.37 0.42

1.54 1.45

0.56 0.60

2.78 3.34

1.02 1.40

5.43 4.78

3.77 2.54

4.38 3.49

1.16 1.37

0.52 0.32

4.56 2.17

8.11 10.26

7.20 8.03

2.36 3.29

0.29 0.32

1.13 1.28

0.33 0.41

1.78 4.73

0.52 1.52

5.62 5.89

2.56 2.75

2.86 3.36

1.12 1.22

0.24 0.44

3.24 3.53

8.40 8.23

10.45 6.23

1.85 2.80

0.22 0.34

0.80 1.32

0.18 0.45

2.59 2.33

0.57 0.79

5.90 6.02

3.16 4.01

4.37 5.8

1.38 1.45

0.43 0.57

3.18 3.08

11.75 11.87

7.31 7.02

3.70 4.25

0.31 0.36

1.61 1.69

0.51 0.61

3.69 3.85

1.16 1.38

4.95 4.69

Peak intensities are expressed in cm–1. A, amide; Pyrid, pyridinium; AGEs, advanced glycation end products; HL, hybrid layer; BHL, bottom of the hybrid layer; ZnO, zinc oxide; ZnCl2, zinc chloride; EDTA, ethylenediaminetetraacetic acid.

Zinc-Doped Adhesives Performance After Loading

ACKNOWLEDGMENTS This work was supported by grants MINECO/FEDER MAT2011-24551 and CEI-Biotic UGR. The authors have no financial affiliation or involvement with any commercial organization with direct financial interest in the materials discussed in this manuscript. Any other potential conflict of interest is disclosed.

REFERENCES AGER, J.W., NALLA, R.K., BREEDEN, K.L. & RITCHIE, R.O. (2005). Deep-ultraviolet Raman spectroscopy study of the effect of aging on human cortical bone. J Biomed Opt 10, 034012. ALMAHDY, A., DOWNEY, F.C., SAURO, S., COOK, R.J., SHERRIFF, M., RICHARDS, D., WATSON, T.F., BANERJEE, A. & FESTY, F. (2012). Microbiochemical analysis of carious dentin using Raman and fluorescence spectroscopy. Caries Res 46, 432–440. AWONUSI, A., MORRIS, M.D. & TECKLENBURG, M.M. (2007). Carbonate assignment and calibration in the Raman spectrum of apatite. Calcif Tissue Int 81, 46–52. BALOOCH, M., HABELITZ, S., KINNEY, J.H., MARSHALL, S.J. & MARSHALL, G. W. (2008). Mechanical properties of mineralized collagen fibrils as influenced by demineralization. J Struct Biol 162, 404–410. DAOOD, U., IQBAL, K., NITISUSANTA, L.I. & FAWZY, A.S. (2013). Effect of chitosan/riboflavin modification on resin/dentin interface: Spectroscopic and microscopic investigations. J Biomed Mater Res A 101, 1846–1856. DE MUNCK, J., VAN MEERBEEK, B., YOSHIDA, Y., INOUE, S., VARGAS, M., SUZUKI, K., LAMBRECHTS, P. & VANHERLE, G. (2003). Four-year water degradation of total-etch adhesives bonded to dentin. J Dent Res 82, 136–140. ERHARDT, M.C., OSORIO, R. & TOLEDANO, M. (2008). Dentin treatment with MMPs inhibitors does not alter bond strengths to caries-affected dentin. J Dent 36, 1068–1073. GANDOLFI, M.G., TADDEI, P., SIBONI, F., MODENA, E., DE STEFANO, E.D., PRATI, C. 2011). Biomimetic remineralization of human dentin using promising innovative calcium-silicate hybrid “smart” materials. Dent Mater 27, 1055–1069. HABELITZ, S., BALOOCH, M., MARSHALL, S.J., BALOOCH, G. & MARSHALL, G.W. Jr. (2002). In situ atomic force microscopy of partially demineralized human dentin collagen fibrils. J Struct Biol 138, 227–236. JASTRZEBSKA, M., WRZALIK, R., KOCOT, A., ZALEWSKA-REJDAK, J. & CWALINA, B. (2003). Raman spectroscopic study of glutaraldehydestabilized collagen and pericardium tissue. J Biomater Sci Polym Ed 14, 185–197. KARAN, K., YAO, X., XU, C. & WANG, Y. (2009). Chemical profile of the dentin substrate in non-carious cervical lesions. Dent Mater 25, 1205–1212. KOIBUCHI, H., YASUDA, N. & NAKABAYASHI, N. (2001). Bonding to dentin with a self-etching primer: The effect of smear layers. Dent Mater 17, 122–126. KRAJEWSKI, A., RAVAGLIOLI, A., TINTI, A., TADDEI, P., MAZZOCCHI, M., MARTINETTI, R., FAGNANO, C. & FINI, M. (2005). Comparison between the in vitro surface transformations of AP40 and RKKP bioactive glasses. J Mater Sci Mater Med 16, 119–128. KREMER, E.A., CHEN, Y., SUZUKI, K., NAGASE, H. & GORSKI, J.P. (1998). Hydroxyapatite induces autolytic degradation and inactivation of matrix metalloproteinase-1 and -3. J Bone Miner Res 13, 1890–1902.

229

MARSHALL, G.W. Jr., MARSHALL, S.J., KINNEY, J.H. & BALOOCH, M. (1997). The dentin substrate: Structure and properties related to bonding. J Dent 25, 441–458. MILLY, H., FESTY, F., WATSON, T.F., THOMPSON, I. & BANERJEE, A. (2014). Enamel white spot lesions can remineralise using bio-active glass and polyacrylic acid-modified bio-active glass powders. J Dent 42, 158–166. NAKABAYASHI, N. (1992). The hybrid layer: A resin-dentin composite. Proc Finn Dent Soc 88(Suppl 1), 321–329. NAKABAYASHI, N., KOJIMA, K. & MASUHARA, E. (1982). The promotion of adhesion by the infiltration of monomers into tooth substrates. J Biomed Mater Res 16, 265–273. NIKAIDO, T., KUNZELMANN, K.H., CHEN, H., OGATA, M., HARADA, N., YAMAGUCHI, S., COX, C.F., HICKEL, R. & TAGAMI, J. (2002). Evaluation of thermal cycling and mechanical loading on bond strength of a self-etching primer system to dentin. Dent Mater 18, 269–275. NUDELMAN, F., PIETERSE, K., GEORGE, A., BOMANS, P.H., FRIEDRICH, H., BRYLKA, L.J., HILBERS, P.A., DE WITH, G. & SOMMERDIJK, N.A. (2010). The role of collagen in bone apatite formation in the presence of hydroxyapatite nucleation inhibitors. Nat Mater 9, 1004–1009. OSORIO, R., CABELLO, I. & TOLEDANO, M. (2014). Bioactivity of zincdoped dental adhesives. J Dent 42, 403–412. OSORIO, R., YAMAUTI, M., OSORIO, E., RUIZ-REQUENA, M.E., PASHLEY, D., TAY, F. & TOLEDANO, M. (2011). Effect of dentin etching and chlorhexidine application on metalloproteinase-mediated collagen degradation. Eur J Oral Sci 119, 79–85. PASHLEY, D.H., ZHANG, Y., CARVALHO, R.M., RUEGGEBERG, F.A. & RUSSELL, C.M. (2000). H + -induced tension development in demineralized dentin matrix. J Dent Res 79, 1579–1583. PRATI, C., CHERSONI, S. & PASHLEY, D.H. (1999). Effect of removal of surface collagen fibrils on resin-dentin bonding. Dent Mater 15, 323–331. PRATI, C., PASHLEY, D.H., CHERSONI, S. & MONGIORGI, R. (2000). Marginal hybrid layer in class V restorations. Oper Dent 25, 228–233. SALEHI, H., TERRER, E., PANAYOTOV, I., LEVALLOIS, B., JACQUOT, B., TASSERY, H. & CUISINIER, F. (2013). Functional mapping of human sound and carious enamel and dentin with Raman spectroscopy. J Biophotonics 6, 765–774. SAURO, S., OSORIO, R., WATSON, T.F. & TOLEDANO, M. (2012). Therapeutic effects of novel resin bonding systems containing bioactive glasses on mineral-depleted areas within the bondeddentin interface. J Mater Sci Mater Med 23, 1521–1532. SCHWARTZ, A.G., PASTERIS, J.D., GENIN, G.M., DAULTON, T.L. & THOMOPOULOS, S. (2012). Mineral distributions at the developing tendon enthesis. PLoS One 7, e48630. SELL, D.R. & MONNIER, V.M. (1989). Structure elucidation of a senescence cross-link from human extracellular matrix. Implication of pentoses in the aging process. J Biol Chem 264, 21597–21602. TOLEDANO, M., AGUILERA, F.S., CABELLO, I. & OSORIO, R. (2014a). Remineralization of mechanical loaded resin-dentin interface: A transitional and synchronized multistep process. Biomech Model Mechanobiol 13, 1289–1302. TOLEDANO, M., AGUILERA, F.S., SAURO, S., CABELLO, I., OSORIO, E. & OSORIO, R. (2014b). Load cycling enhances bioactivity at the resin-dentin interface. Dent Mater 30, e169–e188. TOLEDANO, M., CABELLO, I., YAMAUTI, M., GIANNINI, M., AGUILERA, F.S., OSORIO, E. & OSORIO, R. (2012a). Resistance to degradation of resin–dentin bonds produced by one-step self-etch adhesives. Microsc Microanal 18, 1480–1493. TOLEDANO, M., OSORIO, E., AGUILERA, F.S., SAURO, S., CABELLO, I. & OSORIO, R. (2014c). In vitro mechanical stimulation promoted

230

Manuel Toledano et al.

remineralization at the resin/dentin interface. J Mech Behav Biomed Mater 30, 61–74. TOLEDANO, M., OSORIO, R., ALBALADEJO, A., AGUILERA, F.S., TAY, F.R. & FERRARI, M. (2006). Effect of cyclic loading on the microtensile bond strengths of total-etch and self-etch adhesives. Oper Dent 31, 25–32. TOLEDANO, M., SAURO, S., CABELLO, I., WATSON, T. & OSORIO, R. (2013). A Zn-doped etch-and-rinse adhesive may improve the mechanical properties and the integrity at the bonded-dentin interface. Dent Mater 29, e142–e152. TOLEDANO, M., YAMAUTI, M., RUIZ-REQUENA, M.E. & OSORIO, R. (2012b). A ZnO-doped adhesive reduced collagen degradation favouring dentine remineralization. J Dent 40, 756–765.

WANG, C., WANG, Y., HUFFMAN, N.T., CUI, C., YAO, X., MIDURA, S., MIDURA, R.J. & GORSKI, J.P. (2009). Confocal laser Raman microspectroscopy of biomineralization foci in UMR 106 osteoblastic cultures reveals temporally synchronized protein changes preceding and accompanying mineral crystal deposition. J Biol Chem 284, 7100–7113. XU, C. & WANG, Y. (2011). Cross-linked demineralized dentin maintains its mechanical stability when challenged by bacterial collagenase. J Biomed Mater Res B Appl Biomater 96, 242–248. XU, C. & WANG, Y. (2012). Collagen cross linking increases its biodegradation resistance in wet dentin bonding. J Adhes Dent 14, 11–18.