Antibacterial response of oral microcosm biofilm to nano-zinc oxide in adhesive resin


  • •High-challenge biofilm model was used against ZnO-loaded adhesives.

  • •ZnO-loaded adhesives were tested against mature saliva-derived microcosm biofilm.

  • •Group with 7.5 wt.% of ZnO decreased mature biofilm formation on adhesives.

  • •ZnO affects the degree of conversion and mechanical properties of the adhesives.

  • •Suitable physico-chemical properties were achieved with any concentration of ZnO.



Various nanoparticles are currently under investigation to impart biointeractivity for dental materials. This study aimed to: (1) formulate an experimental dental adhesive containing ZnO nanoparticles; (2) evaluate its chemical and mechanical properties; and (3) assess the antibacterial response against oral microcosm biofilm.


Nanosized ZnO was chemically and morphologically evaluated. ZnO was incorporated at 0 (G CTRL ), 2.5 (G 2.5% ), 5 (G 5% ) and 7.5 (G 5% ) wt.% in an experimental dental adhesive. The adhesives were evaluated for the degree of conversion (DC), flexural strength (FS), and elastic modulus (E). The antibacterial activity was evaluated using a 48 h-microcosm biofilm model after the formation of acquired pellicle on samples’ surfaces. Colony-forming units (CFU), metabolic activity, and live/dead staining were assessed.


Nanosized ZnO presented characteristic peaks of Zn-O bonds, and the particles were arranged in agglomerates. The DC ranged from 62.21 (±1.05) % for G Ctrl to 46.15 (±1.23) % for G 7.5% ( p < 0.05). G 7.5% showed lower FS compared to all groups ( p < 0.05). Despite achieving higher E ( p < 0.05), G 2.5% did not show differences for G Ctrl regarding the FS ( p > 0.05). G 7.5% had lower CFU/mL compared to G Ctrl for mutans streptococci ( p < 0.05) and total microorganisms ( p < 0.05), besides presenting lower metabolic activity ( p < 0.05) and higher dead bacteria via biofilm staining.


The dental adhesives’ physicochemical properties were similar to commercial adhesives and in compliance with ISO recommendations. G 7.5% restricted the growth of oral microcosm biofilm without impairing the physicochemical performance.


Increasing demand for improved long-term performance of dental adhesives has led to intense investigations on strategies for them to express biointeractivity . In this way, dental adhesives may have the potential to improve the longevity of the bonding interface by reducing dental plaque or promoting bioactivity . Besides antibacterial activity and stimulation of dentin remineralization, dental adhesives aimed to be modified to present higher hydrolytic stability and to inhibit collagen degradation by endogenous proteases .

Many types of nanoparticles have emerged as a new class of inorganic agents due to their pronounced effect in reducing bacterial proliferation . As bioactive agents, nanoparticles can facilitate mineral growth with a direct result in bonding interface degradation . Zinc oxide (ZnO) nanoparticles have attracted much consideration due to their multifaceted and promising applications . In dentistry, this oxide presented in microsized particles has well-recognized use in sealers and cements (zinc oxide-eugenol and zinc phosphate cements) . Some researchers have oriented studies toward bioactivity promoted by zinc oxides . Nevertheless, ZnO nanoparticles present not only bioactivity via the formation of calcium phosphates on its surface and excellent biocompatibility , but also can negatively affect metalloproteinases and oral bacteria .

The antimicrobial behavior of nanoparticles is mainly due to their smaller size and the high surface area to volume ratio, i.e., the the nanoparticle’s large surface area enhances their interaction with the bacteria to carry out broad-spectrum antibacterial efficacy . ZnO nanoparticles have shown bactericidal effects on Gram-positive and Gram-negative oral bacteria, as well as against fungal spores . Specifically, studies have demonstrated the superior antimicrobial activity of ZnO nanoparticles against Streptococcus mutans in comparison to ZnO in the particle size range of microns . Moreover, the literature has shown that ZnO nanoparticles have toxicity toward bacteria andhave minimal effect on human cells . These outcomes are exciting and can help investigators determine whether a candidate nanoparticle has scientific merit to justify further applicability.

Currently, a glance at the current literature reveals that antibacterial investigations on ZnO in adhesives have been performed by agar diffusion tests , direct contact tests with 8 or 24 h to evaluate the biofilm formation or the planktonic cells’ viability . Even though many studies previously evaluated ZnO’s harmful activity against oral microorganisms, the designs are usually simple compared to the intra-oral environment Bacterial biofilms inside the mouth present a more complex physical structure and occur with high resistance against antibacterial agents than their free-living counterparts . Besides the concern about the complexity of the biofilm model used, other conditions related to these interactions are not reproduced in vitro, such as the formation of salivary acquired pellicle on the surface of restorative materials . In vitro assays, using more complex biofilm models could help understand materials’ properties and help in the translation of knowledge from bench to clinical setting .

A growing awareness of the limitations of using single oral bacterial species, immature biofilms, and its inability to make reliable predictions led us to investigate nano-ZnO properties using a more complex biofilm model . In the process of tuning nano-ZnO in dental resins, one potential strategy to overcome the challenge faced by treating biofilms is to increase the concentration of the chosen agent. Conflicting with this approach, high concentrations of inorganic fillers may reduce the adhesive’s curing potential and jeopardize the mechanical properties. This study aimed to: (1) formulate an experimental dental adhesive containing ZnO nanoparticles; (2) evaluate its chemical and mechanical properties; and (3) assess the antibacterial response against human saliva-based oral microcosm biofilm.

Materials and methods

Nanoparticles of ZnO powder were initially appraised for chemical and morphological characterization. Further independent studies for their chemical, physical, and antibacterial properties were conducted using experimental dental adhesives containing ZnO nanoparticles. Fig. 1 depicts a schematic diagram of the experimental design and the steps of assessments pipeline employed in this study.

Fig. 1
Flowchart of the experimental design purposed in this study. There was a phase for ZnO characterization, followed by dental adhesives formulation. Then, the resins were chemically and physically evaluated. Finally, the antibacterial activity was analyzed using microcosms biofilm model from saliva-derived inoculum, formation of acquired pellicle from saliva and maturation of biofilm for 48 h. The ability to inhibit the biofilm formation on dental adhesives surfaces was evaluated via colony formaing units couting (CFU/mL), metabolic activity via MTT analysis and live/dead staining of biofilms.

Chemical and morphological characterization of ZnO

A commercially available nanopowder of ZnO was used in this study (Aldrich Chemical Company, St. Louis, Missouri, USA). In order to chemically characterize the powder of ZnO, Fourier Transform Infrared Spectroscopy (FTIR) and micro-Raman spectroscopy were used. For FITR analysis (Vertex 70, Bruker Optics, Ettlingen, Germany), the powder of ZnO was directly placed on the attenuated total reflectance device (ATR). Opus 6.5 software (Bruker Optics, Ettlingen, Germany) was used for processing the spectra in the range from 400 to 3000 cm −1 using 16 scans and 4 cm −1 of resolution. For micro-Raman spectroscopy (SENTERRA, Bruker OptikGmbH, Ettlingen, Baden-Württemberg, Germany), a 100 mW diode laser with wavelength at 785 nm and a spectral resolution of ∼3.5 cm −1 were used. The spectra were obtained in the range from 70 to 1500 cm −1 with three co-additions per 2 s.

Transmission electron microscopy (TEM) was used after mixing ZnO powder with water to evaluate the nano-ZnO morphological features. The nanoparticle solution was dropped (15 μL) onto a 300-mesh carbon-coated copper grid (Ted Pella, Redding, CA, USA). The TEM (FEI Tecnai T20, Hillsboro, OR) equipped with the Software Imaging System CCD camera (Gatan UltraScan 1000) was performed at 80 kV with a magnification of 30,000× and 110,000×, and 200 particles were counted by measuring each one in two axes (400 counts).

Experimental formulations of dental adhesives

The formulations of experimental dental adhesives contain 66.66 wt.% of bisphenol A glycerolate dimethacrylate (BisGMA) manually mixed with 33.33 wt.% of 2-hydroxyethyl methacrylate (HEMA). Camphorquinone and ethyl 4-dimethylaminobenzoate at 1 mol% were added as a photoinitiator system. Butylated hydroxytoluene was added at 0.01 wt.% as a polymerization inhibitor . ZnO was incorporated at 2.5, 5, and 7.5 wt.% in the experimental formulation to obtain three test groups: G 2.5% , G 5% , and G 7.5% . One group remained without the ZnO addition to being used as control (G Ctrl ). The formulations were mixed using a mechanical mixer (DAC 150 Speed mixer, Flacktek, Landrum, SC, USA) at 2800 rpm for 1 min. All reagents used for the formulations were purchased from Aldrich Chemical Company (St. Louis, Missouri, USA) and used without further purification.

Degree of conversion

The degree of conversion (DC) of each dental adhesive ( n = 5) was measured via FTIR-ATR (Nicolet 6700, Thermo Fisher Scientific, Waltham, Massachusetts, USA). The dental adhesives were dispensed on the ATR crystal using a mold of polyvinylsiloxane to standardize samples’ thickness in 1 mm. Each sample was covered with a polyester strip and photoactivated during 20 s using a light-curing unit with 1000 mW/cm 2 (VALO Cordless, Ultradent Products, South Jordan, Utah, USA) and the tip as close as possible to the top of the sample. Each sample was analyzed before and after the photoactivation. The data were evaluated using OMNIC Series Software (Thermo Fisher Scientific), from 400 to 4000 cm −1 , with 32 scans and 4 cm −1 of resolution. We considered the intensity of the aliphatic carbon-carbon double bond (1638 cm −1 ) and the aromatic carbon-carbon double bond (1608 cm −1 ) from the dental adhesives to calculate the DC. Values before and after the photoactivation following the equation:

<SPAN role=presentation tabIndex=0 id=MathJax-Element-1-Frame class=MathJax style="POSITION: relative" data-mathml='DC%=100×peakheightofcuredaliphaticC=C/peakheightofcuredaromaticC=CpeakheightofuncuredaliphaticC=C/peakheightofuncuredaromaticC=C’>DC(%)=100×(peakheightofcuredaliphaticC=C/peakheightofcuredaromaticC=CpeakheightofuncuredaliphaticC=C/peakheightofuncuredaromaticC=C)DC%=100×peakheightofcuredaliphaticC=C/peakheightofcuredaromaticC=CpeakheightofuncuredaliphaticC=C/peakheightofuncuredaromaticC=C

Flexural strength and flexural modulus

Samples of each group ( n = 10) were prepared (2 × 2 × 25 mm), and the flexural strength and the flexural modulus were examined . Polyester strips were applied at the top and the bottom of each sample, and then, the samples were photoactivated (Valo grand, Ultradent Products Inc, South Jordan, UT, USA; standard mode; 1029 mW/cm 2 ) for 20 s on each side. The adhesive samples were stored at 37 °C for 24 h with distilled water. Three-point flexure with a 10 mm span and crosshead-speed of 1 mm/min (MTS 5500R, Cary, NC, USA) was used to examine the flexural strength and the flexural modulus of each sample. The following equation (2) was used to calculate the flexural strength ( F ):

<SPAN role=presentation tabIndex=0 id=MathJax-Element-2-Frame class=MathJax style="POSITION: relative" data-mathml='F(MPa)=3LS2WH2′>𝐹(MPa)=(3𝐿𝑆2𝑊𝐻2)F(MPa)=3LS2WH2
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Mar 21, 2021 | Posted by in Dental Materials | Comments Off on Antibacterial response of oral microcosm biofilm to nano-zinc oxide in adhesive resin
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