2.1

Preparation of the experimental resin and biomimetic primers

A control co-monomer blend (Res.CTR) was formulated using urethane dimethacrylate (UDMA: 30 wt%), ethoxylated bisphenol-A dimethacrylate (Bis-GMA: 5 wt%), triethyleneglycol dimethacrylate (TEGDMA: 25 wt%) (Esstech, Essington, PA), hydroxyethyl methacrylate (HEMA: 18 wt%), absolute ethanol (15 wt%), camphorquinone (CQ: 1.0 wt%) and ethyl-4-dimethylaminobenzoate (EDAB: 1.0 wt%) (Sigma-Aldrich, St. Louis, MO). A control primer (Pr.CTR) was also created by solvating Res.CTR (50 wt%) with absolute ethanol (50 wt%). A type I Portland cement (Italcementi Group, Italy) (80 wt%) was mixed with 20 wt% β-TCP (Sigma–Aldrich) and deionized water (ratio 2:1). After setting (24 h), the cement was incubated at 40 ̊C for 12 h, milled, sieved to <30 μm and incorporated as a filler into the unfilled resin control (filler/resin ratio: 40/60 wt%) to create the experimental ion-releasing resin (Res.IR) .

The first biomimetic primer solution was created by dissolving 150 μg/mL of poly- l -aspartic acid (PLA) with a molecular weight of 27 kDa (Sigma-Aldrich) as previously described . A second biomimetic primer solution (TMP + PLA) was created by dissolving 10 wt% sodium trimetaphosphate (TMP; Mw 367.8, Sigma-Aldrich), an analog of dentin matrix proteins, and 150 μg/mL of PLA in deionized water; the pH of both solutions was adjusted to 7.1 . This experiment was designed to have the presence of such chemical species released by the bioactive ion-releasing filler and from the artificial saliva solution (AS) used in this study. The composition of artificial saliva was 0.7 mM/L CaCl ₂ , 0.2 mM/L MgCl ₂ , 4.0 mM/L KH ₂ PO ₄ , 30.0 mM/L KCl, 20.0 mM/L HEPES .

2.2

ATR-FTIR analysis of dentin collagen

Six sound human third molars were obtained with informed consent from donors and used in accordance with the ethical guidelines of the Research Ethics Committee (REC) for medical investigations. The teeth were stored at 4 °C in 0.5% chloramine-T up to 30 days before use. Two middle-dentin specimens (2 × 2 × 1.5 mm) were obtained from each tooth using a hard tissue microtome (Accutom-50; Struers, Copenhagen, Denmark) equipped with a slow-speed water-cooled diamond wafering saw (330-CARS-70300, Struers, Copenhagen, Denmark). A 320-grit silicon carbide (SiC) abrasive paper mounted on a water-cooled polishing machine (LaboPol-4, Struers, Copenhagen, Denmark) was used to produce a clinically relevant smear layer on both surfaces of the specimens. The dentin specimens were immersed in a 37% H ₃ PO ₄ solution and etched for 15 s; they were subsequently rinsed with distilled water (5 s). These specimens (no. sp. =2/each group) were used to evaluate the chemical changes (phosphate uptake) induced by the two experimental biomimetic primers compared to the single control primer (no biomimetic agents) when applied onto demineralized dentin for 1 min and subsequently rinsed with distilled water (5 s); etched-dentin specimens pre-treated with deionized water were also analyzed. Three different positions of each specimen were analyzed using the ATR-FTIR Spectrometer (Spectrum One, Perkin-Elmer, UK) in the region of 650–4000 cm ⁻¹ with a resolution of 4 cm ⁻¹ and 64 scans per spectrum. A moderate pressure was applied (5 psi) to establish good contact between the specimen and the ATR surface .

2.3

Dentin specimen preparation and bonding procedures

Thirty smear-layer covered middle-dentin specimens obtained from additional sound human third molars were prepared as previously described. After acid-etching (37% H ₃ PO ₄ ; 15 s), the specimens were pre-treated by immersion in deionized water (DW) (no. sp. = 10) or were immersed in one of the two biomimetic primer solutions tested in this study: (i) PLA ( n = 10); (ii) TMP + PLA ( n = 10). Subsequently, these specimens were washed with DW (5 s) air-dried (3 s) and immediately primed using two consecutive layers of the adhesive control primer Pr.CTR. Two consecutive layers of the filler-free Res.CTR (no. sp. = 5) or ion-releasing resin (Res.IR) (no. sp. = 5) were applied and light-cured for 30 s using a Translux EC halogen light-curing unit (KulzerGmbH, BereichDental, Wehrheim, Germany) at a distance of 1 mm. The bonding procedures were completed by applying the resin composite (TetricEvo Cerams, Ivoclar Vivadent, Schaan, Liechtenstein) with five 1 mm layer increments and light-cured for 40 s. Further details about the bonding procedures are depicted in Fig. 1 .

Schematic illustration of the specimen preparation, adhesive procedures, AFM nano-indentation and Raman microscopy evaluation. Human third molars were used to prepare standardized dentin surfaces. The two different biomimetic primers were water solutions containing either poly- l -aspartic acid (PLA) alone or a combination of sodium trimetaphosphate plus PLA (TMP + PLA); deionized water was used as control pre-treatment. An ethanol-based resin primer and a control filler-free resin or an experimental ion-releasing resin containing a 40 wt% Portland cement (calcium silicates) doped with β-TCP were applied onto the dentin surface and light-cured. After resin build-ups, the specimens were sectioned into slab and immersed in artificial saliva (AS) for different period of times in order to evaluate the dentin remineralization at the resin–dentin interface through AFM nano-indentation and Raman microscopy [image modification from Toledano et al. (26)].

2.4

AFM imaging and nano-indentation

The resin-bonded dentin specimens prepared for each group were longitudinally sectioned into multiple 1.5-mm-thick slabs (three each specimen) and polished through ascending SiC papers from #800 up to #4000-grit. Further final polishing procedure was performed using diamond pastes (Buheler-MetaDi, Buheler Ltd., Lake Bluff, IL, USA) through 1 μm down to 0.25 μm. The specimens were cleaned in ultrasonic bath (Model QS3, Ultrawave Ltd, Cardiff, UK) containing deionized water [pH 7.4] for 5 min at each polishing step.

An atomic force microscope (AFM-Nanoscope V, Digital Instruments, Veeco Metrology group, Santa Barbara, CA, USA) equipped with a Triboscope indentor system (Hysitron Inc., Minneapolis, MN) and a Berkovich indenter (tip radius ∼20 nm) was employed for the imaging and nano-indentation processes in a fully hydrated status as recently described by Sauro et al. . Briefly, five indentations with a load of 4000 nN and a time function of 10 s were performed after different AS storage periods (24 h, 30 days, 90 days) in a straight line starting from the top of hybrid layer down to the underlying intertubular dentin in order to evaluate changes in the modulus of elasticity (Ei) of hybrid layers. Three indentation lines 15 (±5) μm from each other were made in five different mesio-distal positions along the interface. The apical-occlusal distance between each indentation was kept constant (5 ± 1 μm) by adjusting the distance intervals steps ( Fig. 1 ) and the load function .

A Kolmogorov–Smirnov test was conducted to assess data distribution ( p > 0.05) and data were analyzed by ANOVA including analysis of interactions ( p < 0.05). Bonding procedure and storage period in AS were considered as independent variables. Student’s t -test ( p < 0.001) and Student–Newman–Keuls ( p < 0.05) were used for post hoc comparisons.

2.5

Dye-assisted confocal laser scanning microscopy evaluation (micropermeability analysis)

Three acid-etched dentin specimens for each group were bonded as previously described with the tested resin-based systems mixed with 0.2 wt% Rhodamine B (Sigma–Aldrich). After 24 h, all the specimens had their pulp chambers (PCs) exposed and immediately filled with 0.1 wt% sodium fluorescein solution (Sigma–Aldrich) for 3 h. The PCs of the other half part of specimens were immersed in AS for 90 days and only subsequently the PCs were filled with the fluorescein solution; the AS was replaced every 72 h .

All the specimens immersed in fluorescein were treated in an ultrasonic bath for 2 min, sectioned into slabs (1.5 mm) and finally polished using a 1200-grit SiC paper for 1 min. A confocal laser scanning microscope (DM-IRE2 CLSM; Leica, Heidelberg, Germany) equipped with a 63×/1.4 NA oil immersion lens and a 488-nm argon/krypton (fluorescein excitation) or 568-nm helium/neon ions laser (rhodamine B excitation) was used to analyze the ultra-morphology and micropermeability along the bonded-dentin interfaces; the emission fluorescence was recorded at 512–538 and 585–650 nm, respectively. Reflection and fluorescence optical images were captured 5 μm below the outer surface up to 25 μm depth (1 μm z-step) and converted into topographic and/or single projections using the Leica SP2 CLSM image-processing software (Leica, Heidelberg, Germany) . The configuration of the system was standardized and used at the same level throughout the entire investigation.

2.6

Qualitative Raman spectroscopy and quantitative cluster analysis

The same specimens submitted to AFM imaging/nano-indentation were also analyzed using a dispersive Raman spectrometer/microscope (Horiba Scientific Xplora, Villeneuve d’Ascq, France) after 24 h and 90 days of AS storage ( Fig. 1 ). A 785-nm diode laser (100 mW sample power) equipped with a ×100/0.90 NA water-immersion objective was employed. Raman signals were acquired using a 600-lines/mm grating centered between 900 and 1700 cm ⁻¹ . Three chemical mappings were captured along the interfaces (two in proximity of the pulpal horns and one in the middle of the pulpal floor) and submitted to K-means cluster (KMC) analysis as described by Toledano et al. , using multivariate analysis (ISyss Horiba), which includes statistical patterning to derive independent clusters; the biochemical content of each cluster was analyzed using the average cluster spectra. Principal component analysis (PCA) decomposed data were set into a bilinear model of linear independent variables, the so-called principal components (PCS). Five to six clusters were identified but only three representative clusters, always present in all the specimens before and after AS storage, were considered and values for each cluster such as demineralized dentin, sound dentin and resin-collagen hybrid zone within the interface were independently obtained. At this point, the mineral matrix ratio (MMR: the ratio of integrated areas of the phosphate P–O symmetric stretch [ v ₁ —961 cm ⁻¹ ] and contour of the CH ₂ deformation of the collagen [1465 cm ⁻¹ ]) was measured for the same cluster in each group, in order to quantitatively calculate the extent of mineralization along the interface. Data were analyzed by ANOVA and Tukey’s post hoc test for pairwise comparisons ( p < 0.05).

2.7

Micro-tensile bond strength (μTBS) and fracture ultra-morphology analysis (FEG-SEM)

A further five resin-bonded dentin specimens were prepared for each group and sectioned in both X and Y directions to obtain multiple bonded-sticks of 0.9 mm ² . Their microtensile bond strengths were tested after 24 h or 90 days of AS storage using a customized microtensile jig on a linear actuator (SMAC Europe Ltd., Horsham, West Sussex, UK). Mean bond strength values were analyzed by ANOVA, which was performed including the bond strength (MPa) as the dependent variable. Bonding groups (CTR, PLA and TMP + PLA) and period of AS storage (24 h and 90 days) were considered as independent variables. Analysis of interactions was also conducted. Student–Newman–Keuls test was used for multiple comparisons. Statistical analysis was set at a significance level of α = 0.05. Modes of failure were classified as percentage of adhesive (A) or mixed (M) or cohesive (C) failures using a stereo microscope (magnification ×60). Three representative fractured specimens for each group were mounted on aluminum stubs using carbon tape, gold-sputter-coated and finally imaged using FEG-SEM (Gemini, Carl Zeiss, Oberkochen, Germany) at 3 kV and a working distance of 15 mm.

2.1

Preparation of the experimental resin and biomimetic primers

A control co-monomer blend (Res.CTR) was formulated using urethane dimethacrylate (UDMA: 30 wt%), ethoxylated bisphenol-A dimethacrylate (Bis-GMA: 5 wt%), triethyleneglycol dimethacrylate (TEGDMA: 25 wt%) (Esstech, Essington, PA), hydroxyethyl methacrylate (HEMA: 18 wt%), absolute ethanol (15 wt%), camphorquinone (CQ: 1.0 wt%) and ethyl-4-dimethylaminobenzoate (EDAB: 1.0 wt%) (Sigma-Aldrich, St. Louis, MO). A control primer (Pr.CTR) was also created by solvating Res.CTR (50 wt%) with absolute ethanol (50 wt%). A type I Portland cement (Italcementi Group, Italy) (80 wt%) was mixed with 20 wt% β-TCP (Sigma–Aldrich) and deionized water (ratio 2:1). After setting (24 h), the cement was incubated at 40 ̊C for 12 h, milled, sieved to <30 μm and incorporated as a filler into the unfilled resin control (filler/resin ratio: 40/60 wt%) to create the experimental ion-releasing resin (Res.IR) .

The first biomimetic primer solution was created by dissolving 150 μg/mL of poly- l -aspartic acid (PLA) with a molecular weight of 27 kDa (Sigma-Aldrich) as previously described . A second biomimetic primer solution (TMP + PLA) was created by dissolving 10 wt% sodium trimetaphosphate (TMP; Mw 367.8, Sigma-Aldrich), an analog of dentin matrix proteins, and 150 μg/mL of PLA in deionized water; the pH of both solutions was adjusted to 7.1 . This experiment was designed to have the presence of such chemical species released by the bioactive ion-releasing filler and from the artificial saliva solution (AS) used in this study. The composition of artificial saliva was 0.7 mM/L CaCl ₂ , 0.2 mM/L MgCl ₂ , 4.0 mM/L KH ₂ PO ₄ , 30.0 mM/L KCl, 20.0 mM/L HEPES .

2.2

ATR-FTIR analysis of dentin collagen

Six sound human third molars were obtained with informed consent from donors and used in accordance with the ethical guidelines of the Research Ethics Committee (REC) for medical investigations. The teeth were stored at 4 °C in 0.5% chloramine-T up to 30 days before use. Two middle-dentin specimens (2 × 2 × 1.5 mm) were obtained from each tooth using a hard tissue microtome (Accutom-50; Struers, Copenhagen, Denmark) equipped with a slow-speed water-cooled diamond wafering saw (330-CARS-70300, Struers, Copenhagen, Denmark). A 320-grit silicon carbide (SiC) abrasive paper mounted on a water-cooled polishing machine (LaboPol-4, Struers, Copenhagen, Denmark) was used to produce a clinically relevant smear layer on both surfaces of the specimens. The dentin specimens were immersed in a 37% H ₃ PO ₄ solution and etched for 15 s; they were subsequently rinsed with distilled water (5 s). These specimens (no. sp. =2/each group) were used to evaluate the chemical changes (phosphate uptake) induced by the two experimental biomimetic primers compared to the single control primer (no biomimetic agents) when applied onto demineralized dentin for 1 min and subsequently rinsed with distilled water (5 s); etched-dentin specimens pre-treated with deionized water were also analyzed. Three different positions of each specimen were analyzed using the ATR-FTIR Spectrometer (Spectrum One, Perkin-Elmer, UK) in the region of 650–4000 cm ⁻¹ with a resolution of 4 cm ⁻¹ and 64 scans per spectrum. A moderate pressure was applied (5 psi) to establish good contact between the specimen and the ATR surface .

2.3

Dentin specimen preparation and bonding procedures

Thirty smear-layer covered middle-dentin specimens obtained from additional sound human third molars were prepared as previously described. After acid-etching (37% H ₃ PO ₄ ; 15 s), the specimens were pre-treated by immersion in deionized water (DW) (no. sp. = 10) or were immersed in one of the two biomimetic primer solutions tested in this study: (i) PLA ( n = 10); (ii) TMP + PLA ( n = 10). Subsequently, these specimens were washed with DW (5 s) air-dried (3 s) and immediately primed using two consecutive layers of the adhesive control primer Pr.CTR. Two consecutive layers of the filler-free Res.CTR (no. sp. = 5) or ion-releasing resin (Res.IR) (no. sp. = 5) were applied and light-cured for 30 s using a Translux EC halogen light-curing unit (KulzerGmbH, BereichDental, Wehrheim, Germany) at a distance of 1 mm. The bonding procedures were completed by applying the resin composite (TetricEvo Cerams, Ivoclar Vivadent, Schaan, Liechtenstein) with five 1 mm layer increments and light-cured for 40 s. Further details about the bonding procedures are depicted in Fig. 1 .

Schematic illustration of the specimen preparation, adhesive procedures, AFM nano-indentation and Raman microscopy evaluation. Human third molars were used to prepare standardized dentin surfaces. The two different biomimetic primers were water solutions containing either poly- l -aspartic acid (PLA) alone or a combination of sodium trimetaphosphate plus PLA (TMP + PLA); deionized water was used as control pre-treatment. An ethanol-based resin primer and a control filler-free resin or an experimental ion-releasing resin containing a 40 wt% Portland cement (calcium silicates) doped with β-TCP were applied onto the dentin surface and light-cured. After resin build-ups, the specimens were sectioned into slab and immersed in artificial saliva (AS) for different period of times in order to evaluate the dentin remineralization at the resin–dentin interface through AFM nano-indentation and Raman microscopy [image modification from Toledano et al. (26)].

2.4

AFM imaging and nano-indentation

The resin-bonded dentin specimens prepared for each group were longitudinally sectioned into multiple 1.5-mm-thick slabs (three each specimen) and polished through ascending SiC papers from #800 up to #4000-grit. Further final polishing procedure was performed using diamond pastes (Buheler-MetaDi, Buheler Ltd., Lake Bluff, IL, USA) through 1 μm down to 0.25 μm. The specimens were cleaned in ultrasonic bath (Model QS3, Ultrawave Ltd, Cardiff, UK) containing deionized water [pH 7.4] for 5 min at each polishing step.

An atomic force microscope (AFM-Nanoscope V, Digital Instruments, Veeco Metrology group, Santa Barbara, CA, USA) equipped with a Triboscope indentor system (Hysitron Inc., Minneapolis, MN) and a Berkovich indenter (tip radius ∼20 nm) was employed for the imaging and nano-indentation processes in a fully hydrated status as recently described by Sauro et al. . Briefly, five indentations with a load of 4000 nN and a time function of 10 s were performed after different AS storage periods (24 h, 30 days, 90 days) in a straight line starting from the top of hybrid layer down to the underlying intertubular dentin in order to evaluate changes in the modulus of elasticity (Ei) of hybrid layers. Three indentation lines 15 (±5) μm from each other were made in five different mesio-distal positions along the interface. The apical-occlusal distance between each indentation was kept constant (5 ± 1 μm) by adjusting the distance intervals steps ( Fig. 1 ) and the load function .

A Kolmogorov–Smirnov test was conducted to assess data distribution ( p > 0.05) and data were analyzed by ANOVA including analysis of interactions ( p < 0.05). Bonding procedure and storage period in AS were considered as independent variables. Student’s t -test ( p < 0.001) and Student–Newman–Keuls ( p < 0.05) were used for post hoc comparisons.

2.5

Dye-assisted confocal laser scanning microscopy evaluation (micropermeability analysis)

Three acid-etched dentin specimens for each group were bonded as previously described with the tested resin-based systems mixed with 0.2 wt% Rhodamine B (Sigma–Aldrich). After 24 h, all the specimens had their pulp chambers (PCs) exposed and immediately filled with 0.1 wt% sodium fluorescein solution (Sigma–Aldrich) for 3 h. The PCs of the other half part of specimens were immersed in AS for 90 days and only subsequently the PCs were filled with the fluorescein solution; the AS was replaced every 72 h .

All the specimens immersed in fluorescein were treated in an ultrasonic bath for 2 min, sectioned into slabs (1.5 mm) and finally polished using a 1200-grit SiC paper for 1 min. A confocal laser scanning microscope (DM-IRE2 CLSM; Leica, Heidelberg, Germany) equipped with a 63×/1.4 NA oil immersion lens and a 488-nm argon/krypton (fluorescein excitation) or 568-nm helium/neon ions laser (rhodamine B excitation) was used to analyze the ultra-morphology and micropermeability along the bonded-dentin interfaces; the emission fluorescence was recorded at 512–538 and 585–650 nm, respectively. Reflection and fluorescence optical images were captured 5 μm below the outer surface up to 25 μm depth (1 μm z-step) and converted into topographic and/or single projections using the Leica SP2 CLSM image-processing software (Leica, Heidelberg, Germany) . The configuration of the system was standardized and used at the same level throughout the entire investigation.

2.6

Qualitative Raman spectroscopy and quantitative cluster analysis

The same specimens submitted to AFM imaging/nano-indentation were also analyzed using a dispersive Raman spectrometer/microscope (Horiba Scientific Xplora, Villeneuve d’Ascq, France) after 24 h and 90 days of AS storage ( Fig. 1 ). A 785-nm diode laser (100 mW sample power) equipped with a ×100/0.90 NA water-immersion objective was employed. Raman signals were acquired using a 600-lines/mm grating centered between 900 and 1700 cm ⁻¹ . Three chemical mappings were captured along the interfaces (two in proximity of the pulpal horns and one in the middle of the pulpal floor) and submitted to K-means cluster (KMC) analysis as described by Toledano et al. , using multivariate analysis (ISyss Horiba), which includes statistical patterning to derive independent clusters; the biochemical content of each cluster was analyzed using the average cluster spectra. Principal component analysis (PCA) decomposed data were set into a bilinear model of linear independent variables, the so-called principal components (PCS). Five to six clusters were identified but only three representative clusters, always present in all the specimens before and after AS storage, were considered and values for each cluster such as demineralized dentin, sound dentin and resin-collagen hybrid zone within the interface were independently obtained. At this point, the mineral matrix ratio (MMR: the ratio of integrated areas of the phosphate P–O symmetric stretch [ v ₁ —961 cm ⁻¹ ] and contour of the CH ₂ deformation of the collagen [1465 cm ⁻¹ ]) was measured for the same cluster in each group, in order to quantitatively calculate the extent of mineralization along the interface. Data were analyzed by ANOVA and Tukey’s post hoc test for pairwise comparisons ( p < 0.05).

2.7

Micro-tensile bond strength (μTBS) and fracture ultra-morphology analysis (FEG-SEM)

A further five resin-bonded dentin specimens were prepared for each group and sectioned in both X and Y directions to obtain multiple bonded-sticks of 0.9 mm ² . Their microtensile bond strengths were tested after 24 h or 90 days of AS storage using a customized microtensile jig on a linear actuator (SMAC Europe Ltd., Horsham, West Sussex, UK). Mean bond strength values were analyzed by ANOVA, which was performed including the bond strength (MPa) as the dependent variable. Bonding groups (CTR, PLA and TMP + PLA) and period of AS storage (24 h and 90 days) were considered as independent variables. Analysis of interactions was also conducted. Student–Newman–Keuls test was used for multiple comparisons. Statistical analysis was set at a significance level of α = 0.05. Modes of failure were classified as percentage of adhesive (A) or mixed (M) or cohesive (C) failures using a stereo microscope (magnification ×60). Three representative fractured specimens for each group were mounted on aluminum stubs using carbon tape, gold-sputter-coated and finally imaged using FEG-SEM (Gemini, Carl Zeiss, Oberkochen, Germany) at 3 kV and a working distance of 15 mm.

3.1

ATR-FTIR analysis of dentin collagen

Dentin before acid-etching presented phosphate bands at 885–1180 cm ⁻¹ (phosphate stretching mode) representative of mineral components, amide bands from organic components (1200–1725 cm ⁻¹ ) and O–H stretching of water (H ₂ O) at 3200–3400 cm ⁻¹ . When dentin was acid-etched, a clear presence of collagen at 1650 and 1540 cm ⁻¹ (C O—amide I and CNH—amide II, respectively) as well as very low-intensity peaks at 1083 and 1034 cm ⁻¹ identified the loss of the P–OR esters ( Fig. 2 ). The application of the biomimetic primers PLA or TMP + PLA biomimetic solutions increased the phosphate peaks at 1083 and 1034 cm ⁻¹ and induced the formation a further PO stretching peak at 967 cm ⁻¹ ( v ₃ —asymmetric stretching mode of hydroxyapatite) . The vibrational stretching of the CO C alcohol group (1048 cm ⁻¹ ) was also evident after biomimetic primer treatments, identifying the presence of HEMA (resin primer) which also showed the C O C peak at 1090 cm ⁻¹ .

FTIR-ATR spectrum of acid-etched dentin treated with the experimental biomimetic solutions. TMP + PLA: the biomimetic primer containing poly- l -aspartic acid plus sodium trimetaphosphate. PLA: the biomimetic primer contains only poly- l -aspartic acid only. Res.Primer: control resin co-monomer primer formulated using UDMA, BisGMA, TEGDMA HEMA and 50 vol% absolute ethanol. Note the phosphate uptake by the demineralized collagen induced by the biomimetic primer TMP + PLA or PLA (1083 and 1034 cm ⁻¹ ) and the increase of vibration of P–O at 967 cm ⁻¹ indicating more presence of phosphate.

3.2

AFM Nano-indentation and dye-assisted CLSM imaging

The Young’s modulus of elasticity (Ei) of the hybrid layer in the second (HL) and the third (BHL) indentations were influenced by the main effects: ( p < 0.001) bonding procedure, ( p < 0.001) storage period. Interactions between factors were also significant ( p < 0.001) ( Table 1 ).

The first indentation performed along each resin–dentin interface was localized on the top part of the hybrid layer ( Fig. 3C and D ). There was no significant difference ( p > 0.05) between the Ei values of each bonding procedure at the same indentation position (1st hybrid layer). Moreover, the aging periods (30 and 90 days) induced no significant change ( p > 0.05) in any of the tested bonding strategies.

CLSM and AFM images of resin–dentin interface after 24 h storage. (A) CLSM 3D projection image captured in reflection/fluorescence showing the resin–dentin interface created with the experimental Res.IR. It is possible to see a strong reflection signal from the submicron filler particles within the adhesive layer (ad + FL) and inside the dentinal tubules (t). A clear micropermeability signal can be seen at the hybrid layer (HL). The brighter the fluorescein fluorescence, the higher the micropermeability of water throughout the hybrid layer. (B) CLSM 3D projection image (reflection/fluorescence) of the resin–dentin interface created with the control resin containing no filler. In this case, it is possible to note strong micropermeability within the hybrid layer (HL). No reflection signal is shown as there is no filler particles within the adhesive layer (ad) or inside the dentinal tubules (t). (C) AFM topographic image showing the indentations performed in straight lines along the hydrated resin–dentin interface created with the experimental Res.IR/PLA starting from the top of the hybrid layer (HL). The second indentation made in the middle of the hybrid layer (HL) and the third in the bottom of the hybrid layer (bHL). The remnant indentations (pointer) were performed within the underlying mineralized dentin (d). It is also possible to see the presence of 5-to-10 μm diameter filler particles (FL) within the adhesive layer (ad). (D) AFM topographic image showing the indentations in the resin–dentin interface created with the experimental Res/PLA starting from the top of the hybrid layer (HL). Also in this case the second indentation was performed in the middle of the hybrid layer (HL) and the third on the bottom of the hybrid layer (bHL). It is also possible to see the resin composite (c) layer above of the hybrid layer (HL) and underlying mineralized dentin (d); this latter characterized by the presence of resin tags (rt).

The resin–dentin interface created using the control resin (Res.CTR) showed a significant decrease in the nano-elasticity ( p < 0.05) of the hybrid layer (2nd indentation) both after 30 and 90 days of storage in AS ( Table 1 ); the bottom of the hybrid layer showed no change in the Ei values ( p > 0.05). The same resin applied after dentin pre-treatment using the primers containing PLA or PLA + TMP showed no significant change ( p > 0.05) in the Ei values of the 2nd indentation at the hybrid layer only after 30 days of AS storage; these values dropped significantly ( p > 0.05) after 90 days of AS immersion. However, the specimens created with Res.CTR/PLA and Res.CTR/TMP + PLA showed no significant Ei change ( p > 0.05) at the bottom of the hybrid layer both after 30 and 90 days of AS storage.

Table 1

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