Influences of Polymeric Magnetic Encapsulated Nanoparticles on the Adhesive Layer for Composite Materials Used for Class I Dental Fillings

CRISTIAN ZAHARIA1, COSMIN SINESCU1, ALIN-GABRIEL GABOR1, VLAD SOCOLIUC2, SERBAN TALPOS1, TAREQ HAJAJ1, PAULA SFIRLOAGA3, ROXANA OANCEA1*, MARINELA MICLAU3, MEDA-LAVINIA NEGRUTIU1 1Victor Babes University of Medicine and Pharmacy, Faculty of Dentistry, 9 Revolutiei 1989 Blvd., 300070, Timisoara, Romania 2Romanian Academy -Timisoara Branch, Center for Fundamental and Advanced Technical Research, Laboratory of Magnetic Fluids, 24 Mihai Viteazu Av., 300223, Timisoara, Romania 3Institute for Research and Development in Electrochemistry and Condensed Matter, 1 P. Andronescu Str. 300224, Timisoara, Romania

The development of adhesive techniques in dental medicine has increased the requirements of aesthetic dentistry as well as increasing the number of minimally invasive restorations made on dental surfaces [1].
Contemporary dentistry has been revolutionized by the appearance of dental adhesives, through it, other research directions have emerged for their improvement: the development of methacrylate-based composites, the development of hydrophilic resins and the modification of acid-engraved dental surfaces. Surfaces of dental adhesives are susceptible to biodegradation. This biodegradation includes the interaction of bacterial enzymes, endogenous enzymes and dental biofilm. Acidity and hydrophilicity of resins increase the degree of degradation of the adhesive at its interface, and modified forms of dentin and enamel can affect the adhesion of the adhesive to enamel or dentin [2].
Composite resins have surpassed amalgam and have become the most used materials for dental restorations made by direct technique. Adhesive composite restorations are threatened by secondary caries, the degradation of the adhesive layer present at the interface between the tooth and by the various defective restorationmaterials, which will then be infested by fluids, bacteria and secondary bacterial products, leading ultimately to the failure of the composite filling. Therefore the durability over time of the connections between dentin and adhesives become an issue [3].
Thus, considering the fact that the main reason for the loss of a composite restoration by bacterial colonization and the appearance of secondary caries was generated by the microfissures at the edge of the restoration, the researchers developed the first dental dhesive material that has self-healing properties with antibacterial and remineralizing action. It has increased dental adhesion qualities, but its most important property is the self-healing of fractures at the adhesive interface layer [4]. Dental adhesives have been modified over time to alleviate the deficiencies they have acquired since the first generations, with methacrylamides being added to their component, the main purpose of which is to increase the resistance to hydrolytic and enzymatic degradations occurring at the interface of the adhesive [5].
In the formula of theadhesives components, lysine has been incorporated, which has resulted in good pH modulation outcomes, the effect of this modification can improve the durability of composite dental restorations [6].
Previous studies show that the thickness of the adhesive layer at the interface between the tooth and the restoration material is between 0.02 mm and 0.3 mm [7][8][9].
The presence of resin tags and the thickness of the hybrid layer do not greatly influence adhesion. [10,11]The strength of adhesion may vary depending on the action time of the acid on the dental surface. At the same time, the surface of the hybrid layer changes, and the long time action of the acid on the surface of the tooth can influence the adhesion in a negative way [12].
Stress at the dentine-adhesive interface may be influenced by the type of adhesive used, with increased stress levels for self-etch adhesives compared to etch-andrinses and self-etch-primer, both adhesive systems presenting partial demineralized dentin [13].

Experimental part
In this study, 15 teeth were used, that had Class I Black cavities. The materials used in this study were: phosphoric acid for tooth demineralisation, Evetric Bond (Ivoclar) dental adhesive, Brilliant Flow (Coltene) photopolymerizable composite flow and multicore-shell Fe 3 O 4 -SiO 2 magnetic nanoparticles ( fig.1).
After preparing the cavities on the surface of the teeth, they were restored using the adhesive technique. Demineralisation of dental surfaces was done with phosphoric acid for 15 s for dentin and 30 s for the enamel.
The acid was washed with water for 20 s and the surface wasdried with air. After demineralization, the dental adhesive doped with nanoparticles was applied by brushing onto the prepared surface of the cavity ( fig.2).
After applying the adhesive, it was photopolimerized with the blue light lamp for 40 s, resulting in anaesthetic filling with slightly colored edges due to the nanomagnetic particles ( fig.4). For 5 teeth the adhesive was applied by brush, and for the other 10teeth after brushing the adhesive, a magnetic field was applied to the entire teeth circumference as follows: for 5 teeth the magnetic field was applied for 2 min, and for the other 5 the magnetic field was applied for 5 min (fig.3). Further, all 15 samples were sectioned and analysed with the help of FEI Inspect S scanning electron microscope (SEM), optical microscope andEnergy Dispersive X-ray analysis(EDX). The scanning electrone microscope has the following characteristics: tungsten filament mounted in the tetrode cannery assembly with a resolution of 3.0 nm on standard specimen with gold particles separated on a carbon substrate. Focus domain is between 3 and 99 mm with a magnification from 6x to >1.000.000x.
SEM analyzes generated images at 200x magnification in which the dental adhesive layer was observed between the two interfaces of the composite resin and the surface of the tooth structure.
Samples showing the adhesive loaded with magnetic nanoparticles that was applied to the tooth surface by conventional technique were analyzed using SEM, resulting in images at a 200x magnification. EDX quantitative analysis is showing internal components ( fig.5).
The interfaces in which the dental adhesive loaded with magnetic nanoparticles was applied in the magnetic field for 2 and 5 min were also analyzed, generating high resolution images at the same magnification. The internal components diagram was also created (figs. 6, 7). All the samples were analyzed with A377 optical microscope. The microscope has a magnification range between 20X and 800X and an CMOS aquisition sensor of 2MPX. The focus is between 0 mm and 40 mm and the connection with the computer is made with USB 2.0 port. Luminosity on sample probes is adjusted manually with 10 LED lights (figs. 8-10). Fig.7. SEM and EDX analisys for the probes with adhesive reinforced with nanoparticles applied on teeth with magnetic field for 5 min Fig.8. Optical microscope analisys for the probes with adhesive reinforced with nanoparticles applied on teeth without magnetic field Fig.9. Optical microscopy measurements for the samples that presented dental adhesive with magnetic nanoparticles applied in magnetic field for 2 min Fig.10. Optical microscopy measurements for the samples that presented dental adhesive with magnetic nanoparticles applied in magnetic field for 5 min

Results and discussions
After recording images using SEM and optical microscopy, they were imported and analyzed using ImageJ software (Wayne Rasband, National Institutes of Health, USA). As a result of the measurements made on the samples where the dental adhesive was loaded with nanoparticles and applied to the surface of the teeth without magnetic field, have resulted thicknesses of the adhesive layer ranging from 10 to 25 microns (table 1).
For adhesives loaded with magnetic nanoparticles and applied in magnetic field for 2 min, the measurements generated adhesive layer sizes between 14 -36 microns (table 2).
For adhesives loaded with magnetic nanoparticles and applied in the magnetic field for 5 min, the measurements generated adhesive layer sizes between 2-12 microns.
Based on the analysis performed with optical microscopy, the thickness of adhesive layer for samples loaded with magnetic nanoparticles and applied to nonmagnetic dental surfaces were between 16-29 microns (table 4).    For samples using a magnetic field for 2 min, the analyzes generated thicknesses of adhesive layer between 13-33 microns (table 5).
For samples using a magnetic field for 5 min, the analyzes generated adhesive layer thicknesses between 3 -10 microns (table 6).
EDX semi quantitative analysis for samples with adhesive reinforced with nanoparticles applied on teeth without magnetic field has highlighted the presence of C Measurements made using scanning electron microscopeand optical microscopy generated results that fall within the same intervals.

Conclusions
The use of magnetic nanoparticles after incorporation into dental adhesives, can reduce the thickness of the adhesive layer by 30% by applying a magnetic field on the tooth surface for 2 min and by 86.5 % for applying the same magnetic field for 5 min compared to the application of dental adhesives by conventional techniques.
Further studies are needed for adhesion strength evaluations.