Biochemical assessment of nanostructures in human trabecular bone: Proposal of a Raman microspectroscopy based measurements protocol

Background: Improvements to the understating of the compositional contributions of bone mineral and organic components to the competence of trabecular bone are crucial. The purpose of this study was to propose a protocol to study biochemical composition of trabecular bone, based on two combined Raman analysis methodologies. Material and Methods: Both cluster and single point Raman mappings were obtained, in order to assess bone degeneration associated with aging, disease, or injury, and to help in the evaluation and development of successful therapies. In this study, human trabecular bone has been analysed throughout a) Raman cluster analysis: bone mineral content, carbonate-to-phosphate ratio (both from the mineral components), the crosslinking and nature/secondary structure of collagen (both from the organic components); and b) Single point Raman spectra, where Raman points related to the minerals and organic components were also obtained, both techniques were employed in spectra attained at 400 to 1700 cm- 1. Results: Multivariate analysis confirmed: 1) the different spectral composition, 2) the existence of centroids grouped by chemical affinity of the various components of the trabecular bone, and 3) the several traces of centroids and distribution of chemical compositional clusters. Conclusions: This study is important, because it delivers a study protocol that provides molecular variations information in both mineral and collagen structure of trabecular bone tissue. This will enable clinicians to benefit knowing the microstructural differences in the bone subjected to degeneration of their patients.Project MAT2017-85999-P supported by the Ministry of Economy and Competitiveness (MINECO) of Spanish Government and European Regional Development Fund (ERDF)

Effect of zinc-doping in physicochemical properties of dental adhesives

Purpose: To evaluate changes in the physicochemical properties, water sorption (WS), solubility (SO), modulus of elasticity (E), ultimate tensile strength (UTS), and microhardness (MH) tests were undertaken in zinc-doped dental adhesives.Methods: Two bonding resins, Adper Single Bond Plus (SB) and Clearfil SE Bond (SEB), were zincdoped by mixing them with 5, 10 or 20wt% of ZnO powder, or with 1 or 2wt% ZnCl2. Resin disks were made of each adhesive blend for the evaluation of WS, SO, and MH, and dumbbell-shaped specimens were prepared for E and UTS testing.Results: An increase in WS and SO was observed for adhesives doped with ZnCl2. A reduction in WS was observed for the adhesive blends containing 10% or 20wt% ZnO, while the SO was not altered in any of the ZnO-doped adhesives. An increase in E values was observed only for the SB adhesive doped with ZnCl2. For SEB-blends, the incorporation of zinc compounds did not alter the E values. UTS values decreased when SEB was doped with ZnO. SBblends doped with 20wt% ZnO significantly increased their MH, and the addition of zinc to the SEB-blends augmented the MH values in all cases.This investigation was supported by grants CICOM/FEDER MAT2014-52036P and AUIP-JA 201

Ex vivo detection and characterization of remineralized carious dentin, by nanoindentation and single point Raman spectroscopy, after amalgam restoration

The aim of this study was to assess the mechanical and chemical performance of sound and caries-affected dentin (CAD), after Zn-free vs containing amalgam restorations placement and thermocycling. Dentin surfaces were studied by Atomic Force Microscopy (AFM) analysis for surface morphological characterization (including fibril diameter assessment), nano-indentation (to measure nano-hardness-Hi and modulus of Young-Ei), and single point Raman spectroscopy for chemical analysis. Measurements were performed before amalgam placement, after amalgam removal, and after 3 months of thermocycling (100,000cy/5 ºC and 55 ºC). Restorations increased both Hi and Ei at intertubular dentin of CAD. The highest values of Hi were achieved at intertubular dentin after restoring with Zn-containing amalgams. Remineralization of dentin was attributed to the increase of both amorphous and crystalline new mineral, as lower degrees of crystal imperfections in junction with crystal disorders, and improvement in structural stability of collagen were found. Higher presence of minerals were also confirmed after the decrease of fluoridated apatite and the increase of the total water content. Proteoglycans, lipids, and proteins, augmented in both sound and CAD, providing support for the mineral growing. The increase of bands assigned to vibration of carbonate calcium phosphate contributed to a decrease of crystallinity.Project MAT2014-52036-P supported by the Ministry of Economy and Competitiveness (MINECO) and European Regional Development Fund (FEDER)

In vitro mechanical stimulation facilitates stress dissipation and sealing ability at the conventional glass ionomer cement-dentin interface.

Objective: The aim of this study was to evaluate the induced changes in the chemical and mechanical performance at the glass-ionomer cement-dentin interface after mechanical load application. Methods: A conventional glass-ionomer cement (GIC) (Ketac Bond), and a resin-modified glass-ionomer cement (RMGIC) (Vitrebond Plus) were used. Bonded interfaces were stored in simulated body fluid, and then tested or submitted to the mechanical loading challenge. Different loading waveforms were applied: No cycling, 24 h cycled in sine or loaded in sustained hold waveforms. The cement-dentin interface was evaluated using a nano-dynamic mechanical analysis, estimating the complex modulus and tan δ. Atomic Force Microscopy (AFM) imaging, Raman analysis and dye assisted confocal microscopy evaluation (CLSM) were also performed. Results: The complex modulus was lower and tan delta was higher at interfaces promoted with the GIC if compared to the RMGIC unloaded. The conventional GIC attained evident reduction of nanoleakage. Mechanical loading favored remineralization and promoted higher complex modulus and lower tan delta values at interfaces with RMGIC, where porosity, micropermeability and nanoleakage were more abundant. Conclusions: Mechanical stimuli diminished the resistance to deformation and increased the stored energy at the GIC-dentin interface. The conventional GIC induced less porosity and nanoleakage than RMGIC. The RMGIC increased nanoleakage at the porous interface, and dye sorption appeared within the cement. Both cements created amorphous and crystalline apatites at the interface depending on the type of mechanical loading.This work was supported by the Ministry of Economy and Competitiveness (MINECO) and European Regional Development Fund (FEDER) [Project MAT2017-85999-P]

Self-etching Zinc-doped adhesives improve the potential of caries-affected dentin to be functionally remineralized

The aim of this study was to evaluate if mechanical cycling influences bioactivity at the resin-carious dentin interface after bonding with Zn-doped self-etching adhesives. Caries-affected dentin (CAD) surfaces were bonded with: Clearfil SE Bond (SEB), and 10 wt% ZnO nanoparticles or 2 wt% ZnCl2 were added into the SEB primer or bonding components. Bonded interfaces were stored during 24 h, and then tested or submitted to mechanical loading. Microtensile bond strength (MTBS) was assessed. Debonded dentin surfaces were studied by field emission scanning electron microscopy (FESEM). Remineralization of the bonded interfaces was evaluated through nanohardness (Hi) and Young’s modulus (Ei), Raman spectroscopy/cluster analysis, and Masson's trichrome staining technique. Load cycling increased the percentage of adhesive failures. New precipitation of minerals composed of zinc-base salts and multiple Zn-rich phosphate deposits were observed in samples infiltrated with the Zn-doped adhesives. At the hybrid layer, specimens treated with ZnO incorporated in the primer (SEB·P-ZnO), after load cycling, attained the highest Ei and Hi. Load cycling increased Ei at the bottom of the hybrid layer when both, SEB un-doped and SEB with ZnCl2 included in the bonding (SEB·Bd-ZnCl2), were used. ZnO incorporated in the primer promoted an increase in height of the phosphate and carbonate peaks, crystallinity, relative mineral concentration, and lower collagen crosslinking. ZnCl2 included in the bonding attained similar results, but relative mineral concentration decreased, associated to higher crosslinking and restricted collagen maturation. Staining techniques permitted to observe no signs of exposed protein at the resin-dentin interface after using SEB·PZnO.This work was supported by grant MINECO/FEDERMAT2014-52036-P

Advanced zinc-doped adhesives for high performance at the resin-carious dentin interface

The purpose of this study was to evaluate the remineralization ability of an etch-and-rinse Zn-doped resin applied on caries-affected dentin (CAD). CAD surfaces were subjected to: (i) 37% phosphoric acid (PA) or (ii) 0.5M ethylenediaminetetraacetic acid (EDTA). 10wt% ZnO nanoparticles or 2wt% ZnCl2 were added into the adhesive Single Bond (SB), to create the following groups: PA+SB, PA+SB-ZnO, PA+SB-ZnCl2, EDTA+SB, EDTA+SB-ZnO, EDTA+SB-ZnCl2. Bonded interfaces were submitted to mechanical loading or stored during 24h. Remineralization of the bonded interfaces was studied by AFM nano-indentation (hardness and Young׳s modulus), Raman spectroscopy [mapping with principal component analysis (PCA), and hierarchical cluster analysis (HCA)] and Masson׳s trichrome staining technique. Dentin samples treated with PA+SB-ZnO attained the highest values of nano-mechanical properties. Load cycling increased both mineralization and crystallographic maturity at the interface; this effect was specially noticed when using ZnCl2-doped resin in EDTA-treated carious dentin. Crosslinking attained higher frequencies indicating better conformation and organization of collagen in specimens treated with PA+SB-ZnO, after load cycling. Trichrome staining technique depicted a deeper demineralized dentin fringe that became reduced after loading, and it was not observable in EDTA+SB groups. Multivariate analysis confirmed de homogenizing effect of load cycling in the percentage of variances, traces of centroids and distribution of clusters, especially in specimens treated with EDTA+SB-ZnCl2.Project MAT2014-52036-P supported by the Ministry of Economy and Competitiveness (MINECO) and European Regional Development Fund (FEDER)

Functional and molecular structural analysis of dentine interfaces promoted by a Zn-doped self-etching adhesive and an in vitro load cycling model

The aim of this study was to evaluate if mechanical cycling influences bioactivity and bond strength of resin–dentine interface after bonding with Zn-doped self-etching adhesives. Sound dentine surfaces were bonded with Clearfil SE Bond (SEB), 10 wt% ZnO microparticles or 2 wt% ZnCl2 were added into the SEB primer (P) or bonding (Bd) for Zn-doping. Bonded interfaces were stored in simulated body fluid (24 h), and then tested or submitted to mechanical loading. Microtensile bond strength testing was performed. Debonded dentine surfaces were studied by scanning electron microscopy. Remineralisation of the bonded interfaces was assessed by nano-indentation, Raman spectroscopy, and Masson׳s trichrome staining. Load cycling (LC) increased the percentage of adhesive failures in all groups. LC increased the Young׳s modulus (Ei) at the hybrid layer (HL) when SEB, SEB·P-ZnO and SEB·P-ZnCl2 were applied, but decreased when both ZnO and ZnCl2 were incorporated into the bonding. Ei was higher when Zn compounds were incorporated into the primer (SEB·P). ZnO promoted an increase, and ZnCl2 a decrease, of both the relative presence of minerals and crystallinity, after LC. LC increased collagen crosslinking with both SEB·P-ZnO and SEB·P-ZnCl2. The ratios which reflect the nature of collagen increased, in general, at both HL and BHL after LC, confirming recovery, better organisation, improved structural differences and collagen quality. After loading, trichrome staining reflected a deeper demineralised dentine fringe when Zn-doped compounds were incorporated into SEB·Bd. Multiple Zn-rich phosphate deposits and salt formations were detected. Mineral precipitates nucleated in multilayered platforms or globular formations on peritubular and intertubular dentine.This work was supported by grant MINECO/FEDER MAT2011-24551 and MAT2014-52036-P

Silver improves collagen structure and stability at demineralized dentin: a dynamic-mechanical and Raman analysis.

Objective: This study aimed to evaluate the effect of silver loaded nanoparticles (NPs) application on dentin remineralization. Methods: Polymethylmetacrylate-based NPs and silver loaded NPs (Ag-NPs) were applied on demineralized dentin surfaces. Dentin was characterized morphologically by scanning electron microscopy, mechanically probed by a nanoindenter to test nanohardness and Young modulus, and chemically analyzed by Raman spectroscopy after 24 h and 7 d of storage. Untreated surfaces were used as control. Data were submitted to ANOVA and Student-Newman-Keuls multiple comparisons tests (P<0.05). Results: After Raman analysis, dentin treated with Ag-NPs obtained the lowest mineralization and intensity of stoichiometric hydroxyapatite when compared with specimens treated with undoped-NPs. The lowest relative mineral concentration, expressed as the ratio phosphate or carbonate/phenyl group, and crystallinity was attained by dentin treated with with Ag-NPs, after 7 d. Ag-NPs application generated the highest values of collagen crosslinking (intensity at 1032 cm-1 band). The molecular conformation of the collagen’s polypeptide chains, amide-I and CH2 also attained the highest peaks in dentin treated with Ag-NPs. Staggered and demineralized collagen fibrils were observed covering the dentin surfaces treated with Ag-NPs, at both 24 h and 7 d. Samples treated with Ag-NPs attained the lowest values of nanohardness and Young’s modulus at 7 d of storage. Conclusions: Peritubular and intertubular dentin were remineralized when using undoped-NPs. After 7 d, collagen treated with NPs was remineralized but dentin treated with Ag-NPs attained an improved collagen matrix structure and stability but the lowest mineralization and crystallinity.This work was supported by the Ministry of Economy and Competitiveness (MINECO) and European Regional Development Fund (FEDER). Project MAT2017-85999-P MINECO/AEI/FEDER/UE

On modeling and nano-analysis of caries-affected dentin surfaces restored with Zn-containing amalgam and in vitro oral function

The aim of this research was to assess the influence of mechanical loading on the ability of Zn-free vs Zn-containing amalgams to promote remineralization at the dentin interface. Sound (SD) and caries-affected dentin surfaces (CAD) were restored using Zn-free or Zn-containing dental amalgams. Mid-coronal dentin surfaces were studied by 1) Atomic Force Microscopy (AFM) analysis (including plot and phase imaging, nanoindentation test (modulus of Young (Ei), nano-roughness measurements, and fibril diameter assessment), 2) Raman spectroscopy/cluster analysis, 3) X-ray diffraction (μXRD2), 4) field emission electron microscope (FESEM) and energy-dispersive analysis (EDX), for morphological, mechanical, and physico-chemical characterization. Analyses were performed before amalgam placement and after amalgam removal, at 24 hours and 3 weeks of load cycling. Zn-free and Zn-containing amalgams restorations promoted an increase in the modulus of Young of CAD surfaces, after three weeks of load cycling; at this time, Zn-containing amalgams attained higher Ei than Zn-free restorations. Zn-containing amalgams induced tubular occlusion after load cycling, in both sound and CAD. Zn free-amalgams promoted remineralization of both intertubular and peritubular dentin in CAD substrata. These minerals were identified as calciumphosphate deposits, and crystals as hydroxyl-apatite with augmented crystallographic maturity but with some components of lattice distortion. Crosslinking of collagen diminished and secondary structure of collagen increased in CAD substrate restored with Zn-containing amalgam after 3 w of load cycling, indicating an advanced preservation, molecular organization and orientation of collagen fibrils after load cycling. Plot and phase images permitted to observe the topographical changes which were promoted by the mineral deposits; in general, the indexes related to higher remineralization gave rise to a decrease of nano-roughness and an augmentation of the bandwidth of the collagen fibrils. Zn-containing amalgam restorations submitted to mechanical stimuli promote remineralization of the partially mineral-depleted subjacent substrate at the caries-affected dentin.This work was supported by Grant Nos. MINECO/FEDER MAT2014-52036-P and FIS2013-41821-R

The mineralizing effect of zinc oxide-modified hydroxyapatite-based sealer on radicular dentin

Objective To evaluate the remineralization ability of three endodontic sealer materials at different root dentin regions. Material and methods Cervical, medial and apical root dentin surfaces were treated with two experimental hydroxyapatite-based cements, containing sodium hydroxide (calcypatite) or zinc oxide (oxipatite); an epoxy resin-based canal sealer, AH Plus; and gutta-percha. Remineralization, at the inner and outer zones of dentin disk surfaces, was studied by nanohardness (Hi) and Raman analysis. Nano-roughness and collagen fibrils width measurements were performed. Numerical data, at 24 h or 12 m, were analyzed by ANOVA and Student-Newman-Keuls (P<0.05). Results At the outer and inner zones of cervical dentin treated with oxipatite, the highest Hi after 12 m of immersion was achieved. The same group showed the highest intensity of phosphate peak, markers for calcification and crystallinity. Nanoroughness was lower and fibrils diameter was higher at the inner zone of dentin treated with oxipatite. Dentin mineralization occurred in every region of root dentin treated with oxipatite and calcypatite, especially at inner zone of dentin after 12 m. Conclusions Oxipatite, reinforced the inner root zone at any third of radicular dentin, by increasing both nanohardness and remineralization. When using calcypatite, the highest nanohardness was found at the apical third of the inner root dentin, but the lowest mechanical performance was obtained at the cervical and the medial thirds of the roots. Therefore, application of oxipatite as sealing cement of root canals is recommended. Clinical relevance Oxipatite, when used as endodontic sealing material, strengthens radicular dentin.Project MAT2017-85999-P MINECO/AEI/FEDER/UE supported by the Ministry of Economy and Competitiveness (MINECO) and European Regional Development Fund (FEDER)