Enhancement of Antibacterial and Mechanical Features of Glass Ionomer Restoration by Adding ZnO Nanoparticles Prepared by PLAL: In Vitro Study

The objective of this manuscript was to study the antibacterial and mechanical effect on composition ZnONPs-Glass Ionomer restoration. Zinc oxide nanoparticles prepared by laser ablation in liquid at different concentrations. After that, the glass ionomer was adding in calculated amount to get homogenous composition. The physical properties of resulted NPs were examined by FTIR, XRD, TEM, and AFM. The Antibacterial activity against streptococcus mutant and compressive strength were also measured. The result of the FTIR spectrum conrms the formation of the Zn-O bond. XRD pattern shows peaks at 28..55°, 31.21° and 42.70°, correspond to the (100) (101) and (102) crystal planes of cubic Zinc Oxide structure. TEM image exhibits spherical and homogenous ZnO nanoparticles with particle size <50nm. Also, the result shows a signicant increase in the antibacterial property of ZnO-GI with good modifying the mechanical properties. Thus adding ZnO nanoparticles to glass ionomer restoration can be considered as a better alternative to conventional GI.


Introduction
Secondary caries is a problem that occurs in the mouth within restoration that leads to fall of the restorative materials. One of the main causes of secondary caries was Oral Streptococcus mutans (SM) which cause dental plaque bio lm formation. So that, increase the tooth's reluctance to secondary decay an appropriate restorative material is used for prevention of dental caries by stop bacterial growth [1][2][3].
Glass ionomer popular restorative dental material based on acid base reaction [4,5]. It has wide dental applications as restoration of primary teeth, GI regarded a good chemical adhesion with tooth, low thermal expansion near to the tooth coe cient and has ability to uoride release Therefore, and it is considered an anti-bacterial with high e ciency [6][7][8][9]. In spite of the good features, GI high porosity with large internal cracks leading to weak mechanical properties that leading to form a secondary caries [10].
The adding nanoparticles which, in turn, are characterized by small particle size and large surface area [11] these properties allow a harmonious interaction with the micro cracks of GI-surface [12]. Zinc oxide nanoparticles good choice with GI because, was used as an antibacterial agent and incorporated in many dental products [13]. Zinc oxide Nanoparticles (ZnO-NPs) is a biocompatible material, non-toxic to human cells but more toxic to the bacteria. ZnO-NPs can inhibit the growth of oral SM and plaque formation [14][15]. Also, the antibacterial effect of ZnO-NPs increase by increase of the concentration and decrease in nanoparticles size [16]. Many techniques is used to fabricated nanoparticles for example: chemical and physical vapor deposition, sol-gel, and ame synthesis .These methods were complex setup and need special parameters such as temperature and pressure to get the desired composition and size distribution. Now days, laser ablation of materials was nearly new technique rst invented at 1993 by Fojtik et al has proven merits as the common e cient procedure for get nanoparticles [17][18][19]. This is the chosen method is characterized by its ease, e ciency and does not require complicated situations or speci c conditions to obtain a nanoscale solution [20]. At this promising method can control on size and concentration of fabricated ZnO nanomaterial via change the laser parameters. The study revolves around comparing and evaluates the mechanical and antibacterial activity of enhanced conventional glass ionomer restoration with ZnONPs.

Preparation of ZnONPs -GI Composition
ZnO nanoparticles synthesis by pulse laser ablation in liquid, rst the zinc bulk immersed in 1ml of double distilled water at room temperature. The Nd:Yag (Q-switched pulsed) with (1064 nm,1Hz ) laser energy 600 mJ to prepare ZnONPs. Laser beam focused with focal length =20cm of lens at distance =5cm about the target, gure (1). The glass container was rotated for prevent etching and get homogenous nanoparticles [21]. The samples divided into three groups ZnONPs (100,150 and 200) pulses and xed the laser energy. After that the glass ionomer( Riva, Australia) was mixed with P/L ratio 1:0.5:0.5 (one scoop powder and 0.5 drop of liquid+0.5 drop of ZnONPs). In order to obtain three groups with different concentration of zinc oxide nanoparticles changing laser pulses to (100,150 and 200) and xed laser energy then adding to constant amount of glass ionomer.

Antibacterial enhancement
The 1.9 gram of mueller hinton agar was dissolving in 50 mL of DDW then, shaking, heating and sterilization through autoclaving for 15 minute at temperature of 121° C and pressure of 15 lbs. After that cooled and divided on the petri-disk until be sold layer. Streptococcus mutans was sweep all the surface of petri-dish from all direction. The well making by using the tip of micropipette with 6mm diameter, ZnONPs samples and enhanced samples colloidal with 50μL lled the well. Then leaving the agars for 48h in incubation nally, inhibition zone diameter of the all samples antibacterial effect was calculated.

Tooth Preparation
Three fresh, free caries, for 20-35 years old patients and third premolars teeth extracted from dental clinic (Dr. Wissam, in Al-Harithiya). Teeth washed with de-ionized water, and then each tooth was wiped with acetone to remove any debris then stored with 50 ml of normal saline. Buckle surfaces of each tooth were coated with red nail varnish leaving circular hole with dimensions (4 * 4) mm. After that, the composition of enhance GI-restoration was lled the tooth, see gure2

Mechanical enhancement
The compressive strength was measured by using the Universal testing machine (England). The tooth specimens placed between the plates of the universal testing machine xed on enhanced restoration sides. A compressive load with maximum force applied on tooth samples until the specimen fractured.
The compressive strength of the samples was measured after 48 h in normal saline.

FTIR Analysis
Fourier transformer infrared technique was used to identify the chemical bonding and component of substance. The spectrum peak of the ZnONPs was prepared by laser ablation in liquid listed in gure 3(A). The fundamental vibration mode was lie at (3000Cm -1 ) this is due O-H stretching bond stretching and distortion respectively assigned to the water absorption on the metal surface. The peak at rang 1000-1800 cm -1 refer to C=O asymmetric stretching bond while an absorption at 400-800cm -1 refer to indicate coordination of ZnONPs stretching vibrations [22][23]. While gure (3B) exhibits FTIR results of ZnONPs-GI restoration peaks attributable to polyacrylic acid (PAA) at 1600 cm -1 (C=O stretch), Peak representing water component at 3000cm -1 (O-H stretch) was also obtained. These peaks were looked broader and shifted to 300 cm -1 after GI modi cation with ZnONPs. It may be attributed to the incorporation of ZnONPs to the active C=O group in GI. This suggestion is con rmed by the decrease in the transmission observed after adding GI, which could be explained by the formation of polyacrylate salts [24].

XRD Measurement
X-ray diffraction of ZnONPs was estimated at gure 4, were the diffraction peaks calculated at position 2θ (10-80°) with angles of 28..55°, 31.21° and 42.70°,correspond to the re ection from the (100) (101) and (102) crystal planes with cubic zinc oxide structure [19]. The particles size calculated by using Scherer equation: D=0.89λ/βCosθ, Where D of the particles size, λ is the wavelength of Cu Kα,, β is the full width at the half-maximum (FWHM) by using of the ZnONPs line and θ is the diffraction angle by using xpert high score plus program. [20]. Table 2 calculated the mean grain size and lattice strain of the ZnONPs. The Williamson-Hall (W-H) measurement used to calculated lattice strain (ε) of the ZnONPs between β cosθ against 4sinθ was straight line gives information about the strain in the form of the slope. The crystal perfect when all atoms are at rest on their correct lattice positions in the crystal structure [25]. The variation of the lattice strains in samples due the variation in (hk.l) with linear behavior is observed because of the small difference in the average particle size distribution [26]. Figure 5 shows the TEM images for ZnONPs colloidal prepared at energies 600 mJ, a size of particles varies with domain a range between few tens of nanometers. These photos detect that the nanoparticles produced due to the interaction of laser pulse with zinc target are roughly uniform the images of the different shapes of the synthesized ZnO NPs [27]. The morphology of the NPs showed hexagonal, spherical and rod-shaped structures with some agglomerated large and small particles. The sizedistribution that indicates strong appearance of nanoparticle and aggregation at higher laser pulses according to the electrostatic attractive force between them, which fabricated because of electric double layer on the surface of nanoparticles [28].

Atomic Force Microscope Measurement
AFM images with three dimensions for zinc oxide colloidal and zinc oxide NPs-Glass ionomer restoration showed in gure 6. Larger number of laser pulses lead to a smaller particle size because of the longer ablation time leads to a narrowing of the nanoparticle size distribution due to the interaction of the ablating laser beam with the produced Nanoparticles [29,30] and The decrease in the particle size appear because of laser fragmentation effect. Since laser pulse causes larger particles to participate to smaller and smaller one. According to the AFM images ZnONPs-GI composition were smooth surface despite some small particles and grain clusters. Figure 7 shows the antibacterial activity of ZnONPs and ZnONPs-GI, the antibacterial activity of zinc oxide nanoparticles signi cantly increased with high concentration of nanoparticles. The ZnONPs synthesize by PLAL at xed laser energy (600mJ) and different laser pulse number show higher antibacterial activity against streptococcus mutans bacterial strains [31]. The antibacterial action of ZnONPs work via releasing reactive oxygen species (ROS), then interacting with the bacteria wall by electrostatic forces leading loss of extracellular content and bacterial cell death [32]. Also, the result show in gure 6 a signi cant antibacterial activity of GI after incorporated with different concentration of ZnONPs samples in comparison to the empty wells and GI. This result was approve with (Hojati et al.) [33]. That by increasing the nanoparticles concentration the antibacterial activity increase.

Compressive Strength
The mean compressive strength of the control and experimental groups after 48-hours after preparation in vitro study, see gure 8. The storage of tooth samples in normal saline is shown in Table 2 statistically enhanced signi cant differences after adding the ZnONPs. The enhanced restoration strength increased by increase ZnONPs concentration that's attributed to the increase in nanoparticles help to ll the micro cracking in surface layer of glass ionomer.

Conclusions
1-The incorporation ZnO-NPs into glass ionomer enhances its antibacterial activity against oral Streptococcus mutans and increase the compressive strength with increasing concentration of colloidal-ZnO.
2-The FTIR con rmed that, the interaction between the zinc oxide nanoparticles and glass ionomer was occurred through the C=O functional group of poly acrylic acid of glass ionomer.
3-Zinc oxide nanoparticles were formation with cubic structure detected by the X-ray diffraction and calculated the lattice strain takes linear behavior. The TEM image shows the homogenous distribution with almost spherical and other different shapes.