Evaluation of the Flexural Strength of Orthodontic Acrylic Resin incorporated with Propolis Nanoparticles: An In Vitro Study

Evaluation of the flexural strength of orthodontic acrylic resin incorporated with propolis nanoparticles: an in vitro study


INTRODUCTION
In fact, high prevalence of malocclusion amongst growing children entails interceptive orthodontic treatments [1][2][3][4] that are commonly performed using removable appliances.In addition, removable appliances have been used as retainers since the 1920s and still remain as practical means for retention. [5]These appliances are mostly fabricated from PMMA due to its various advantages including ease of processing, fitting accuracy, convenience, and reasonable cost. [6]Despite all perfect features, it is highly prone to colonization of microorganisms [7][8][9] which may lead to dental caries, periodontal diseases, and chronic atrophic candidiasis.Therefore, several efforts have been employed to introduce antibacterial efficacy to PMMA.In this sense, PMMA has been incorporated with different antimicrobial agents such as silver nanoparticles [10] , silicon dioxide and titanium dioxide [11] , carbon nanotubes [12] , chlorhexidine diacetate [13] , 2-methacryloyloxyethyl phosphorylcholine (MPC), and quaternary ammonium dimethylaminohexadecyl methacrylate (DMAHDM) [14] , polyethylene oxide (PEO) [15] , nanodiamonds [16] and so on .Despite the incorporation of PMMA with a range of antibacterial agents, there is still lack of consensus in terms of their clinical effectiveness. [17]In this regards, the contemporary trend towards natural products has drawn attentions to propolis (bee glue) as a promising ingredient for both medical and dental applications. [18,19]Propolis has a wide range of outstanding biological scopes including antioxidant and anti-ageing [20] , anticancer [21] , immunomodulatory [22] , antidiabetic [23] , anti-inflammatory and anti-allergic [24] properties along with wound healing promotion [25] .Moreover, several investigations have confirmed its activity against wide spectrum of bacteria [26] , viruses [27] and fungi [28] .Additionally, propolis has been utilized vastly in dentistry to alleviate dentin hypersensitivity and aphthous stomatitis, prevent dental caries, pulp capping, storing the avulsed teeth and to culture the PDL cells [29] and so on.Propolis mouthwash and paste inhibit Streptococci mutans and Lactobacilli.Also, propolis paste improves the healing of periodontal socket after extraction by 90% in human subjects. [30]The inhibitory effect of 300 µg/mL propolis nanoparticle against E. faecalis for the purpose of root canal disinfection is comparable with 6% NaOCL and 2% CHX. [31]Furthermore, PMMA containing 1 or 2% of propolis nanoparticles has presented antibacterial activity against common oral pathogens such as Streptococcus mutans, Streptococcus sanguinis, Lactobacillus acidophilus, and Candida albicans. [32]Therefore, propolis can be considered as a promising agent to incorporate with PMMA due to its numerous beneficial attributes.In spite of that, the effect of propolis on the mechanical properties of PMMA should be taken into account as a crucial requirement in order to endure the loads imposed in the oral cavity.To the best of our knowledge, there is lack of evidence in this respect.

AIM
Thus, this study aimed to investigate the effect of incorporating nanopropolis (NPS) on flexural strength of PMMA in order to induce antimicrobial features in PMMA without compromising its mechanical properties.

Nanopropolis preparation
Pure propolis was purchased from Gold Zagros (Lorestan, IRAN).Twenty grams of pure propolis was dissolved in 100 ml of ethanol for 7 days at room temperature and then filtered through filter papers (Wattman-40Ashless-Germany) to remove rough particles.Afterwards, pure propolis particles were isolated by adding the solution to distilled water at 1:10 ratio.The suspension was placed in an ultrasonic bath for 20-30 minutes to obtain propolis nanoparticles.The achieved colloidal NPS was centrifuged at 9000 rpm for 20 minutes using a centrifuge machine (HeroLab-22000 rpm, Germany) and then filtered by filter papers.Nanoparticles were verified under scanning electron microscope (SEM; Zeiss, Oberkochen, Germany) at ×65000 magnification (Fig. 1).The colloid was poured to plates and placed in a freezer at −80°C for 20 minutes followed by freeze drying (Freeze dryer, LYOTRAP, LTE scientific, UK) at −70°C for 24 hours to obtain powder form of NPS particles.

Mold preparation
Stainless still molds were machined in 65×12×4 mm considering the shrinkage of polymer.Impressions were taken of molds using the putty-wash technique (Silicone impression material, Hydro, Detax, Germany).

Sample groups
Two commercial acrylic resin groups including Acropars (Marlic Medical Industries Co, Tehran, Iran) and Triplex (Ivoclar Vivadent AG, Schaan, Liechtenstein) were selected.Three subgroups, each including 15 samples, were prepared for the examination: one control group comprising acrylics without NPS and two experimental groups consisting of acrylic resins incorporated with 0.5% and 1% NPS.Acrylic preparation was preformed according to manufacturer's instruction and the doughy acrylic resins were inserted into the impression mold and pressed using glass slide until the completion of self curing at 26°C.Cured polymeric sam-ples were removed from molds after 1 hour and grinded to 64×10.0×3.3 mm using 60 and 80 grit sandpapers to meet the ISO 20795-2 (2013) standard of the polymeric base of orthodontic appliance. [33]Each sample was meausured three times using a digital caliper (Insize, USA) with a precision of 0.1 mm.Each sample was allocated a number from 1 to 90 (Fig. 2).

Flexural strength test
Samples were immersed in water and incubated at 37°C for 50±2 hours (Incubator, PECO-Iran).Then flexural strength test was carried out using a universal testing machine (Zwick Z250, Germany).Specimens were undertaken an increasing load at a crosshead speed of 5±1 mm/min to the failure point.Flexural strength was calculated according to the following equation: where F delegates the force in Newton at failure point, l stands for the distance between supports in millimeters with an accuracy of ±0.01 mm, b and h are the width and height in millimeters, respectively, at the center of the sample.

Statistical analysis
Statistical analysis was performed by IBM SPSS version 25 using one-way ANOVA, two-way ANOVA, Tukey HSD, and T-test.P less than 0.05 was considered statistically significant.

DISCUSSION
Several attempts have been made to incorporate antimicrobial agents into PMMA to address the microbial colonization. [17]In this regard, NPS might serve as a promising alternative to metal antimicrobial agents due to its remarkable antibacterial, antiviral, and antifungal effectiveness along with biological safety and natural source. [34]Indisputably, PMMA incorporated with NPS should also meet the recommended flexural strength for clinical practice as well.[37] Various nanoparticles have been introduced to PMMA in several previous investigations.Nanodiamonds at a concentration of 0.5% improved flexural strength of PMMA due to the crystalline structure of diamond, intense chemical bonds, and suitable diffusion of nanoparticles in the resin matrix.However, higher concentration of nanoparticle resulted in lower flexural strength due to the inadequate ratio of polymer. [38]Addition of zinc oxide nanoparticles up to 1.4% increased the flexural strength of PMMA. [39]Incorporation of 2.5% and 5% zirconium oxide nanoparticles boosted flexural strength of PMMA regardless of resin thickness. [40]ilicon dioxide nanoparticles revealed a dose-dependent reducing effect on flexural strength, higher concentration leading to lower value due to the presence of voids and agglomeration of nanoparticles. [41]ince the antibacterial activity of NPS modified-PMMA against S. mutans, S. sanguinis, L. acidophilus, and C. albicans has been proven by the authors lately [32] , this study was designed to evaluate the effect of NPS incorporation on flexural strength of two acrylic resins namely Triplex and Acropars.In control groups, the mean flexural strength of Triplex was slightly higher than that of Acropars although the difference was insignificant and both were more than 50 MPa which is the minimum flexural strength base on ISO 20795-2:2013. [33]Current results indicated that the mean flexural strengths of Triplex+0.5% and 1% NPS were lower than that of control Triplex although the differences were insignificant (p=0.821 and p=0.258, respectively) and remained above the recommended value. [33]On the other hand, incorporation of both 0.5% and 1% NPS resulted in significant reduction of the mean flexural strength of Acropars (p<0.0001) to less than the accepted value.This may be explained by the interference of nanoparticles as impurities with resin polymerization.Moreover, nanoparticles may act as plasticizers and result in higher amount of unreacted monomers.
The differences between various acrylic resins have been considered in previous studies. [42]Triplex and Acropars self-curing acrylic resins opted in this study are different in terms of composition and concentration of oligomers, plasticizers, crosslinkers, initiators, and accelerators which may have to diverse effect of NPS on flexural strength of each.The present results for Acropars groups containing NPS are consistent with the previous reports with some other particles.Incorporation of higher percentage of Al 2 O 3 , TiO 2 , and SiO 2 (3-5%) [43] , 10% ratio of TiO 2 , ZrO 2 , Sic-nano, Si 3 N 4 and HA-nano [44] , 0.5 and 1% of either TiO 2 or SiO 2 [45] decreased flexural strength of PMMA significantly.Kul et al. revealed no significant differences in flexural strength of PMMA (Heraus Kulzer) following the addition of SiC, Al 2 O 3 or Ag by 10 wt% [44] which is in line with the current values achieved in 0.5 and 1% NPS enriched-Triplex.Nevertheless, there are controversies on how the incorporation of various antibacterial agents would affect the flexural strength of PMMA.For instance, in the study conducted by Yadav et al., incorporation of 10% mass of silver zinc zeolite, chlorhexidine (CHX), and fluconazole decreased the flexural strength of PMMA (Trevalon) significantly which was in accordance with our results in the Acropars groups.Their observations may be due to an increase in residual monomer in the zeolite porosities or disruption of the physical structure of the polymer by either CHX or large particles of fluconazole.It might also be attributed to the high percentage of the nanoparticles they used. [46]Contrariwise, in the research by Ratanajanchai et al., addition of 1.0% w/w of potassium sorbate, 0.5% w/w of sodium metabisulfite and 0.25% w/w of zinc oxide particles as antimicrobial agents increased the flexural strength and decreased the flexural modulus of PMMA but within the acceptable range. [47]Similarly, in a study by Lee et  al., incorporation of 0.5%, 1%, and 2% graphene-oxide nanosheets into PMMA introduced antimicrobial activity without any adverse effect on flexural strength.Besides, addition of 0.5% graphene-oxide improved flexural strength which may be due to the ability of graphene-oxide to deflect cracks. [48]Incorporation of 0.4%, 0.8%, and 1.6% of Galla Chinese extract into Acropars conferred antibacterial property along with improvement in the flexural strength of PMMA which the latter attributed to the covalent bonds creation between Galla extract and Acropars. [49]To our knowledge, despite promising addition of propolis to other substrates such as glass ionomer or resin composite, there is lack of evidence on mechanical properties of PMMA incorporated with NPS.Regarding the addition of propolis to other substrates, incorporation of 25% and 50% ethanolic extracts of propolis into glass ionomer inhibited S. mutans with no adverse effect on shear bond strength. [50]Similarly, 2% and 5% NPS incorporated into composite resin exhibited antibacterial effect against S. mutans and S. sanguinis beside acceptable shear bond strength. [51]Several factors including but not limited to nanoparticle's type, formulation, structure, and concentration along with the rate of dispersion in resin matrix and probability of interfering with the polymerization might affect the results.In addition, the properties of the matrix, which the nanoparticles introduced to such as formulation or mode of polymerization are other key factors in achieved data.Thus, further studies considering the aforementioned variables along with subsequent evaluation of antimicrobial efficiency, cytotoxic effect of nanoparticles, roughness of fractured surface and other major mechanical properties such as modulus of elasticity are recommended.In vitro experiments considering the effect of time, aging and so on to simulate clinical conditions are required.Ultimately, clinical investigations are essential to generalize the results to the practice.

CONCLUSIONS
The effect of NPS on flexural strength of acrylic resin is dependent on the commercial type and concentration of the nanoparticle.Addition of 0.5% and 1% propolis nanoparticles to Triplex does not have any adverse effect on its flexural strength.However, it drops that of Acropars below the acceptable value.

Figure 2 .
Figure 2. Control samples of Triplex acrylic resin on the left, and Triplex acrylic resin incorporated with 0.5% NPS on the right.

Table 2 .
Comparison between the mean flexural strength of Acropars and Triplex incorporated with 0%, 0.5%, and 1% of NPS.