Research Paper
Ions-modified nanoparticles affect functional remineralization and energy dissipation through the resin-dentin interface

https://doi.org/10.1016/j.jmbbm.2017.01.026Get rights and content

Highlights

  • Ion-doped NPs increased the crystallinity and maturity at the resin-dentin interface.

  • Ca-NPs provoked favorable dissipation of energy with minimal stress concentration.

  • Zn-NPs released the stress by breaking the remineralized resin-dentin interface.

  • Raman signals were not registered below the hybrid layer with ion-doped NPs, at 21 d.

Abstract

The aim of this study was to evaluate changes in the mechanical and chemical behavior, and bonding ability at dentin interfaces infiltrated with polymeric nanoparticlesstandard deviations and modes of failure are (NPs) prior to resin application. Dentin surfaces were treated with 37% phosphoric acid followed by application of an ethanol suspension of NPs, Zn-NPs or Ca-NPs followed by the application of an adhesive, Single Bond (SB). Bonded interfaces were stored for 24 h, submitted to microtensile bond strength test, and evaluated by scanning electron microscopy. After 24 h and 21 d of storage, the whole resin-dentin interface adhesive was evaluated using a Nano-DMA. Complex modulus, storage modulus and tan delta (δ) were assessed. AFM imaging and Raman analysis were performed. Bond strength was not affected by NPs infiltration. After 21 d of storage, tan δ generally decreased at Zn-NPs/resin-dentin interface, and augmented when Ca-NPs or non-doped NPs were used. When both Zn-NPs and Ca-NPs were employed, the storage modulus and complex modulus decreased, though both moduli increased at the adhesive and at peritubular dentin after Zn-NPs infiltration. The phosphate and the carbonate peaks, and carbonate substitution, augmented more at interfaces promoted with Ca-NPs than with Zn-NPs after 21 d of storage, but crystallinity did not differ at created interfaces with both ions-doped NPs. Crosslinking of collagen and the secondary structure of collagen improved with Zn-NPs resin-dentin infiltration. Ca-NPs-resin dentin infiltration produced a favorable dissipation of energy with minimal stress concentration trough the crystalline remineralized resin-dentin interface, causing minor damage at this structure.

Introduction

To promote adhesion to dentin, the mineral phase from the substrate has to be removed and the voids left by mineral should be filled with the adhesive resin that undergoes complete in situ polymerization to form the hybrid layer (HL) (Nakabayashi and Pashley, 1998). The ideal hybrid layer would be characterized as a three-dimensional collagen-resin biopolymer that provides both a continuous and stable link between the bulk adhesive and dentin substrate (Misra et al., 2004). A volume of demineralized/unprotected collagen remains at the bottom of the hybrid layer (BHL), susceptible of degradation, attributed to the action of host-derived matrix metalloproteinases (MMPs) (Hashimoto et al., 2003, Hebling et al., 2005, Pashley et al., 2004). This jeopardizes the longevity of bonded restorations, compromising the bonding efficacy over time (Breschi et al., 2010, Carrilho et al., 2009). Thus, remineralization of demineralized dentin has important consequences for the improvement of bonding stability.

The ultimate goal in the design and refinement of dental adhesives is to render a stronger and durable adhesion to dental tissues, despite the severe conditions in the oral environment (Profeta, 2014). Dentin adhesives should not only be long-lasting, but promoters of both protection and remineralization of resin-dentin interfaces, triggering the bioactive nature of dentin matrix, by releasing bound bioactive molecules. Minimally invasive dentistry, within the current conservative dental practice is permanently emphasizing on arresting and remineralizing demineralized dentin with either tissue engineering approaches or solution chemical therapy (Toledano and Osorio, 2015). To get ion exchange and mineral precipitation within the hybrid layer, multiple approaches or bioactive materials have been used for functionalization of adhesives and chemical remineralization of dentin, e.g., phosphoproteins, casein phosphopeptide-amorphous calcium phosphate, bioactive glass particles, colloidal nano-beta-tricalcium phosphate, carboxylic acid-containing polyelectrolytes. All of them claim relative success with respect to the observation of regrowth of minerals at the demineralized dentin. Ceramic bioactive nanospheres, such as hydroxyapatite (HAp) and other engineered nanoparticles (NPs) have also been proposed as a resin filler (Besinis et al., 2014), but they do not possess a controllable release and optimal degradation kinetics (Wu et al., 2011).

In general, minerals elute from the resins, producing a rapid decrease in their chemical-mechanical properties and bond strength (Sauro et al., 2013). At these conditions, ion liberation used to be rapid and not maintained over time. A controllable release and optimal degradation kinetics are crucial but difficult to achieve (Hoppe et al., 2011). Dentin infiltration with polymeric nanoparticles as calcium and phosphate sequestering materials (i.e., carboxylate functionalized polymer particles that bind calcium) previous to the bonding procedure has been proposed (Osorio et al., 2014). These polymers may also act as carriers of other biological factors for the management of tissue mineralization, permitting a controlled ion release rate (Wu et al., 2011). These polymers should bind to collagen and facilitate amorphous calcium and phosphate precursors precipitation, at the hybrid layer. The use of engineered NPs has also become the focus of much research in this field (Besinis et al., 2016).

Zinc has been demonstrated to reduce MMPs-mediated collagen degradation (Osorio et al., 2011), to inhibit dentin demineralization (Takatsuka et al., 2005) and to induce dentin remineralization at the bonded interface (Toledano et al., 2013a). Zinc influences signaling pathways and stimulates a metabolic effect in hard tissue mineralization (Hoppe et al., 2011) and remineralization processes (Barcellos et al., 2016, Lynch et al., 2011). Zinc elicits a specific biological response at the interface of the material which results in the formation of a bond between tissue and material (Kokubo et al., 1990). The formula for stoichiometric HAp is Ca10(PO4)6(OH)2. However, biological apatite is calcium deficient and contains substantial amounts of carbonate. Carbonated apatite is a precursor of HAp, but when it is precipitated in the presence of zinc an exchange between Zn2+ and Ca2+ occurs in vitro forming a substituted apatite compound (Mayer et al., 1994). The binding constant of Zn is 8.7 and that of the calcium is 6.8. An isomorphous substitution can be obtained when Ca2+ is replaced by Zn2+ into dentin HAp (Vasant and Joshi, 2011). The radii of doped ions of Zn corresponds with 0.074 nm, smaller than Ca, i.e., 0.099 nm; thereby, it is easy for zinc to fill in the vacancy of crystal lattice presenting as lose of certain electrical neutrality (Song et al., 2003). Incorporating zinc into the chemical formulation of resin adhesives increases the potential for intrafibrillar remineralization at partially demineralized collagen matrices (Toledano et al., 2016a).

The degree and the quality of the mineralization will affect the mechanical properties of dentin. Indeed, the extrafibrilar minerals act as a granular material that can withstand load, but in the absence of intrafibrilar mineralization. Intrafibrilar mineralization is the key factor for ensuring that collagen fibrils have the same high modulus of elasticity as occurs in natural biomineralized dentin (Balooch et al., 2008). Therefore, the increase of the elastic modulus of the partially demineralized collagen is directly related to the precipitation of minerals at the resin-dentin interface has (Li et al., 2012), and more specifically at the intrafibrillar compartment (Balooch et al., 2008, Bertassoni et al., 2009). Atomic force microscopy (AFM) nano-indentation is the most commonly applied means of testing the mechanical properties of materials or substrates (Poon et al., 2008), and it was deemed to be a suitable method for the determination of the visco-elasticity of hard tissues (Balooch et al., 2008, Bar-On and Wagner, 2012), at nanoscale (Hu et al., 2015). Viscoelastic materials, as dentin (Toledano et al., 2015) deform according to a combination of these properties and, as such, exhibit time-dependent strain (Hayot et al., 2012). The complex modulus, as a measure of the resistance of a material to dynamic deformation (Ryou et al., 2013), can be decomposed into storage (elastic) and loss (damping) modulus components (Wilkinson et al., 2015). The storage modulus E′ (also called dynamic stiffness) characterizes the ability to store energy by the sample during a cycle of loading (Hayot et al., 2012), which is then available for elastic recoil. The storage modulus is the measure of the sample's elastic behavior. Any resulting phase lag between the force applied and the displacement is related to a loss of energy known as the loss modulus or damping E″ (Hayot et al., 2012). The ratio of the loss to the storage is the tan delta (δ) and is often called damping. Even more, the nano-DMA analysis shows that the dampening (or viscous) behavior of the tissue is much more sensitive to the structural changes that occur with the oral function and than the quasi-static behavior (Ryou et al., 2015). Thereby, it requires the capacity to absorb mechanical shock waves and alleviate stresses at these locations in order to prevent crack propagation across the boundary between the two phases of dentin and thus, may serve as useful biomimetic models for joining mechanically dissimilar biomaterials to restore form and function (Marshall et al., 2001).

Scanning probe microscopes and, in particular AFM have facilitated the imaging and analysis of biological surfaces with little or no sample preparations (Habelitz et al., 2002). AFM operates in a near field with a sharp probe by scanning, enabling characterization of three-dimensional surface morphology with minimal sample preparation and high resolution. AFM has been widely used to visualize the dentin matrix and to determine the spatial relationship between mineral and collagen and their morphology/topology as well (Hu et al., 2015). By integrating AFM and nano-DMA, both morpho and nanomechanical properties can be obtained. In particular, biological sample systems resemble complex biochemical and biophysic architectures (Rettler et al., 2013).

The vast majority of the research work has been focused on the histological, microscopic and mechanical aspects of dentin, but rarely on the underlying molecular structure which is integral to a full understanding of the adhesive-based therapy, especially its effect on the mineral content and collagen matrix (Liu et al., 2014). Even more, there exist contradictory results that arise from poor characterization of the NPs-loaded biomaterials and their interaction with the target site (Zhao et al., 2016). Active research in this area is helping to build a more solid base in understanding of NPs-tissue interaction. In this respect, Raman is a powerful tool in generating direct information about the molecules of a sample. Thereby, this study was complemented with Raman spectroscopy and cluster analysis, that offers nondestructive measures and provide an insight on biochemical nature and molecular structure, and emission spectroscopies of the tissue. It is used as a quantitative chemical assessment methodology for biological samples in conjunction with the fact that the Raman peak intensity is proportional to the number of molecules within the volume of the scanned area (Milly et al., 2014). Raman mapping, in combination with multivariate data analysis, is a label free imaging method for the analysis of dentin sections. This combined approach yields images depicting a semi-quantitative distribution of the biochemical species in the tissue with high resolution (Bonifacio et al., 2010). Micro-Raman mapping technique appeared to offer a powerful method to directly analyze the resin-dentin interface's constituents and their distribution after placing the restoration. Compared to the conventional histological and microscopic methods Raman spectroscopy and cluster analysis result advantageous because they are fast, non-intrusive, stain-free, quantitative and less prone to human subjectivity. The combination of various chemometric methods is essential in providing different images conveying complementary information about the tissue, for studying biochemical and morphological changes during resin-dentin interface degradation and remineralization.

The aim of the present study was to infiltrate calcium or zinc-loaded polymeric nanoparticles into phosphoric acid etched dentin, prior to the adhesive application, in order to assess the maintenance in bond strengths and a potential improvement of both chemical and mechanical properties at the short term (21 d), after mineral precipitation at the resin-dentin interface. The null hypotheses to be tested are that calcium and zinc loaded nanoparticles infiltration into etched dentin, (1) does not affect dentin bond strengths, (2) does not influence the dynamic mechanical behavior at the hybrid layer, after 21 days of SBFS storage and, (3) does not facilitate remineralization at the demineralized bonded interface.

Section snippets

Nanoparticles production

PolymP-n Active nanoparticles (NPs) were acquired from NanoMyP (Granada, Spain). Particles are fabricated trough polymerization precipitation. Precipitation polymerization is used to prepare polymeric nanospheres of uniform size and shape free of any added surfactant or stabilizer. This technique starts as a homogeneous mixture of monomer, initiator, and optional solvents, and during the polymerization, the growing polymeric chains are separated from the continuous medium by changes in the

Results

Mean MTBS values, standard deviations and modes of failure are reported in Table 1. Attained viscoelastic moduli are displayed in Table 2. Mineral, organic and adhesive components obtained through Raman analysis are found in Tables 3a, 3b, 4 and 5. FESEM images from debonded dentin surfaces are shown in Fig. 2. Fig. 3 contains scanning DMA analysis of SB resin/24 h and Ca-NPs+resin/24 h groups, and 3-D contour maps of NPs+resin/24 h and Zn-NPs+resin/24 h groups. Fig. 4 shows 3-D contour maps of SB

Discussion

Our results confirm that dentin infiltration with Ca-NPs before the adhesive application provoked a favorable dissipation of energy with minimal stress concentration trough the crystalline remineralized resin-dentin interface.

Infiltration of dentin with NPs did not exert changes in bond strength and the highest percentage of mixed failures among the tested groups (Table 1). When NPs were not applied, the failure existed at the bottom of the hybrid layer, where resin-uncovered collagen was

Conclusions

This work proved that ion doping in nanoparticles has a strong impact on resin-dentin interface behavior. Considering bonding efficacy, to incorporate NPs into the adhesive resin do not compromise the bond strength of the adhesive system to dentin. From a viscoelastic point of view, Ca-doping NPs caused an improved and favorable dissipation of energy when compared with Zn-doping NPs. On the other hand, dentin infiltrated with Zn-NPs released the stress by breaking the resin-dentin interface and

Acknowledgments

This work was supported by the Ministry of Economy and Competitiveness (MINECO) [Project MAT2014-52036-P]. The authors affirm that no actual or potential conflict of interest including any financial, personal or other relationships with other people or organizations within three years of beginning the submitted work that could inappropriately influence, or be perceived to influence, their work. Any other potential conflict of interest is disclosed.

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