The Anti-biofilm Activity of Nanometric Zinc doped Bioactive Glass against Putative Periodontal

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Research Article

Nasrin Esfahanizadeh, Mohammad Reza Nourani, Abbas Bahador, Nasrin Akhondi, and Mostafa Montazeri*

The Anti-biofilm Activity of Nanometric Zinc doped Bioactive Glass against Putative Periodontal

Pathogens: An in vitro Study

https://doi.org/10.1515/bglass-2018-0009

Received Feb 11, 2018; revised Oct 10, 2018; accepted Oct 14, 2018

Abstract: Colonization of periodontal pathogens on the surgical sites is one of the primary reasons for the failure of regenerative periodontal therapies. Bioactive glasses (BGs) owing to their favorable structural and antimicrobial properties have been proposed as promising materials for the reconstruction of periodontal and peri-implant bone defects. This study aimed to investigate the anti- biofilm activity of zinc-doped BG (Zn/BG) compared with 45S5 Bioglass^r(BG^r) on putative periodontal pathogens.

In thisin vitro experimental study, the nano BG doped with 5-mol% zinc and BG^r were synthesized by sol-gel method. Mono-species biofilms ofAggregatibacter actino- mycetemcomitans (A. a),Porphyromonas gingivalis (P. g), andPrevotella intermedia (P. i)were prepared separately in a well-containing microplate. After 48 hours of exposure to generated materials at 37^∘C, the anti-biofilm potential of the samples was studied by measuring the optical density (OD) at 570nm wavelengths with a microplate reader.

Two-way ANOVA then analyzed the results.

Both Zn/BG and BG^rsignificantly reduced the biofilm formation ability of all examined strains after 48 hours of incubation (P=0.0001). Moreover, the anti-biofilm activity of Zn/BG was significantly stronger than BG^r(P=0.0001), which resulted in the formation of a weak biofilm (OD<1)

*Corresponding Author: Mostafa Montazeri:Department of Pe- riodontics, Islamic Azad University of Tehran, Faculty of Dentistry, Tehran, Iran; Email: [email protected]; Tel: +98-917-7153048 Nasrin Esfahanizadeh:Department of Periodontics, Islamic Azad University of Tehran, Faculty of Dentistry, Tehran, Iran; Dental Im- plant Research Center, Dental Research Institute, Tehran University of Medical Sciences, Tehran, Iran

Mohammad Reza Nourani:Division of Genomics, Systems Biology Institute, Baqiyatallah University of Medical Sciences, Tehran, Iran Abbas Bahador:Dental Implant Research Center, Dental Research Institute, Tehran University of Medical Sciences, Tehran, Iran Nasrin Akhondi:Department of Mathematics, South Tehran Branch, Islamic Azad University, Tehran, Iran

compared with a moderately adhered biofilm observed with BG^r(1<OD<2).

Zn/BG showed a significant inhibitory effect on the biofilm formation of all examined periodontal pathogens. Given the enhanced regenerative and anti-biofilm properties of this novel biomaterial, further investigations are required for its implementation in clinical situations.

Keywords: Periodontitis, Biomaterial(s), Microbiology, Antimicrobial(s), Periodontal regeneration

1 Introduction

Periodontitis and peri-implant diseases are strongly associated with the accumulation of oral biofilm which mainly consists of anaerobic gram-negative bacteria [1], in a way that the rate of these microorganisms significantly increases compared to physiologic conditions [2]. Hence, the colonization of such species at surgical sites is a primary concern following regenerative periodontal proce- dures [3]. Indeed, biofilms are responsible for the occur- rence of most oral infections [4, 5], and the organisms in a biofilm are 1000 to 1500 times more resistant to antibiotics compared to planktonic bacteria [6, 7]. Among the identified periodontal pathogens,Porphyromonas gingivalis (P.

g) [8], Prevotella intermedia (P. i) [9], andAggregatibac- ter Actinomycetemcomitans (A. a)[10] play the most cru- cial role in the initiation and development of periodontal and peri-implant associated bone destruction [11–13]. One of the essential prerequisites for bone regeneration relies on the prevention of microbial colonization of the surgical field [12, 14] and studies have shown that the high levels of periodontal pathogens negatively affect the outcomes of regenerative approaches [13, 15].

Nowadays, biomaterials have become an essential part of the treatment modalities aimed at reconstructing the periodontal and peri-implant bone defects. One of the promising materials proposed for the regeneration of bone

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lesions is bioactive glass (BG) that due to its structural, regenerative and antimicrobial properties has gained the at- tention of researchers [16].

Henchet al.first introduced the BG^r at the Univer- sity of Florida in 1969 [17]. He discovered that this type of glass made such a strong bond with the bone that it was not possible to detach them except by fracturing the bone [17]. Subsequentlyin vivostudies demonstrated that BGs exhibit osteoinductive and osteoconductive properties through the formation of a carbonated hydroxyl- apatite (CHA) with the bone [18]. Since then, despite much research have taken place in this field, the original Hench’s compound is still used extensively [16].

Presently BGs are manufactured by a method known as sol-gel, which utilizes a solvent at low tempera- tures [19]. This approach has several known benefits, in- cluding the porous structure and high bioavailability of the generated material and the possibility of producing a variety of glass-ceramics with different additives [19, 20].

Perio-Glas^r(NovaBone^r, Florida, USA 1992) was the first particle-sized (710-90 microns) BG used to reconstruct the jaw and periodontal defects [21]. Since then, BGs in various commercial brands, alone or in combination with different metal ions, have been used to reconstruct jaw-related bone defects [18].

One of the desired characteristics of BGs in regenerative dental medicine is their anti-biofilm activity [22–24].

Biofilms are composed of microbial cells encased within a matrix of extracellular polymeric substances, such as polysaccharides, proteins, and nucleic acids [5–7]. It is hypothesized that following the dissolution of glass and release of cations, pH increases, and the produced alka- line condition exert anti-biofilm effects [25]. The number of studies has shown that BG^rand different modifications of its original formula yield particular antimicrobial activity against various strains ofStreptococciandStaphylo- cocci[3]. However, the studies on periodontal pathogens are scarce [25, 26].

Moreover, the implementation of various chemical agents such as metal ions in BGs has been widely studied [27]. The incorporation of such agents has been shown not only to enhance the structural and mechanical properties of BG but also to improve its antimicrobial fea- tures [28–30]. In a recent study by Galarraga-Vinuezaet al.[31], bioactive glass doped with 5wt% CaBr2had a re- markable anti-biofilm effect against oral bacteria such as P. gandFusobacterium nucleatum. In the context of tissue engineering, BGs doped with metal ions such as silver, copper, zinc, and strontium have been proposed for the treatment of periodontal and peri-implant defects [30, 32, 33]. One of the most potent anti-biofilm and antifun-

gal ions is zinc, whose salts inhibit the pathogens in the microbial plaque [34]. Chemical addition of zinc to the structure of BGs enhances its mechanical and regenerative properties [30, 33, 35]. In a recent study on the Zn/BG, authors found that the release of Zn²⁺ions accelerated bone formation and the material demonstrated better mechanical properties compared with the control group [36]. Fur- thermore, zinc-containing glasses and ceramics have been shown to have antimicrobial properties againstStaphylo- coccus aureusandEscherichia coli[30, 37, 38], but there is no study on the antimicrobial activity of zinc-containing glasses against periodontal pathogens [27].

According to the studies above, the elimination of microorganisms from the operation field is necessary to increase the amount of the regenerated bone [13, 15].

This study aimed to investigate the anti-biofilm activity of Zn/BG compared with BG^r on putative periodontal pathogens.

2 Material and methods

2.1 BG nano-powder synthesis

BG^r composed of SiO2, Na2O, CaO and P2O5and Zn/BG was prepared by the sol-gel method described in the previous studies [24, 39]. In a brief review, 0.064M (13.33 g) of tetraethyl orthosilicate (TEOS) was dissolved in 0.1M HNO3(30mL) as a catalyst and stirred for 30 minutes at room temperature to allow hydrolyzing to occur. After 45 minutes, triethyl phosphate (TEP), calcium nitrate tetrahy- drate, and zinc nitrate hexahydrate were added to the mix- ture and stirred for an hour at room temperature to complete the hydrolysis reaction. 5 mol% zinc nitrate hexahydrate was exclusively added for producing the zinc oxide particles in the structure of BG. The produced sol was stored in an isolated Teflon container for ten days to allow for the gelatinization and condensation of the products.

The gel was first dried at 70^∘C for three days and then at 120^∘C for two days. The dried gel passed through a 90-lm pore size filter (170 meshes). The product was then heated at 700^∘C for 24 hours to stabilize the glass structure. Fi- nally, the BG nanopowder was prepared by ball milling (SVD15IG5-1, LG Company, Germany) for 30 min. All chemical substances were purchased from Merck Company, Ger- many.

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2.2 BG nano-powder characterization

2.2.1 X-ray diffraction

As previously have been explained [24], the crystal structure of the nanopowder was assessed by X-ray diffraction (XRD) technique with Cu Kα= ¼ 1.54 Å wavelength (Philips, Germany) [24]. The resident time and size of each step were 1 second and 0.02^∘, respectively.

2.2.2 Fourier Transform Infrared Spectroscopy (FTIR)

FTIR spectroscopy (Bomem-MB Series Spectrometer) was used to determine the functional groups of the generated nanopowders. Briefly, one mg of the powder was carefully mixed with 300 mg of potassium bromide (KBr) (infrared grade) and pelletized under vacuum. Then, the pellet was analyzed in the range of 400- 4000 cm⁻¹at the scan speed of 22 scans/min with 4 cm⁻¹resolution [40].

2.2.3 Scanning Electron Microscopy (SEM)

The morphology of nanoparticle structures was studied by scanning electron microscopy (SEM- Philips XL30) at an accelerating voltage of 15kV. Before viewing, the conduits were coated with gold using a sputter coater (EMITECH K450X, England). Then the average diameter of the particles was measured from the SEM micrographs [30].

2.2.4 PH release test

To investigate the pH values of the nanopowders, 0.3 grams of each material (BG and Zn/BG) with the concentration of 20 mg/mL were dissolved in 200-cc distilled water. One test tube used as a control group containing only distilled water without BG. Using shaking incubator, test tubes were stirred at 37^∘C with a 75 rpm shaking frequency.

For each test tube, pH value was measured at room temperature (21^∘C) after 1 hour, 24 hours and 48 hours [33].

2.2.5 Cytotoxicity analysis

MTT assay was implemented to evaluate the biocompat- ibility and cytotoxicity of the synthesized powder by ex- posing the hamster ovarian cells to 20 mg/ml of BG powder [24, 39]. The MTT assay and trypan blue dyeing were carried out to measure the cell viability and prolifera-

tion [24]. As previously described, for the assessment of the cytotoxicity effects of BG nanopowders on mam- malian cells, Chinese hamster ovarian cells (Cho cell line) were used (purchased from the Pasteur Institute of Iran).

These cells were cultured in RPMI1640 medium (Invitro- gen) containing 10% fetal bovine serum and 1% peni- cillin/streptomycin at 37^∘C and 5% CO2. After the cells reached 90% confluency, the viable cells were counted with the trypan blue (0.4%, w/v; Sigma–Aldrich, Ger- many) stain; then 10⁵cells were transferred to each well of the 96-well plates. After 24 hours, the supernatant of each well was replaced with the medium containing BG nanopowder with the concentration of 20 mg/mL. After 24, 48, or 72 hours of incubation, the medium of each well was eliminated, and the wells were washed with phosphate- buffered saline three times. The cells were incubated with 20 µL/well of MTT (Sigma–Aldrich, Germany) reagent for four hours at 37^∘C to allow the mitochondrial enzyme of viable cells, succinate dehydrogenase, to convert the tetra- zolium ring to blue formazan. Then the supernatants were replaced with 150 µL dimethyl sulfoxide (Sigma–Aldrich, Germany). The plate was stirred to dissolve the formazan crystals. The optical density of each well was read by Elisa Reader (Tecan, Switzerland) at 540 nm. All the tests were done in triplicate. When staining the viable cells with trypan blue, the cells were first incubated with 20 mg/mL of BG nanopowder. Then they were collected using trypsin/EDTA and suspended in the new medium. The trypan blue dye was added to the cell suspension at a ratio of 1:1. The numbers of dead and viable cells were counted using a hemocytometer slide.

2.3 Microorganisms and culture medium

Strains ofP. g(ATCC^r33277),A. a(ATCC^r33384) andP. i (ATCC^r49046) obtained from the Microbiology Research Center, Tehran University of Medical Sciences, Tehran, Iran. The organisms were grown in a fresh brain heart infusion (BHI) broth (Merck, Darmstadt, Germany) augmented with 0.6% (wt/vol) yeast extract (Merck, Darmstadt, Ger- many). Afterward, 1 mg/L menadione was added (Sigma- Aldrich, Steinheim, Germany) at 37^∘C in an anaerobic at- mosphere comprising 80% N2, 10% CO2, and 10% H2and set to a final concentration of 1.0×10⁸colony forming units (CFU)/mL per bacteria. This was confirmed by spectropho- tometry (optical density [OD]600: 0.08-0.13) [41, 42].

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Table 1:Comparisons of the Mean±SDs between the experimental groups. The level of significance set to 0.05.

Type of material/bacteria Mean±SD P-value (Type of

material)

P. gingivalis A.

actinomycetem- comitans

P. intermedia

Zn/BG 0.916±0.105 1.044±0.103 0.906±0.104 0.0001*

BG^r 1.55±0.186 1.968±0.087 1.782±0.100

Negative Control 2.872±0.041 2.994±0.094 2.474±0.131

P-value (Type of bacteria) 0.0001*

P-value (The interaction between the bacteria and the type of material)

0.0001*

Legend:Zn: Zinc, BG: Bioactive glass. * Denotes a statistically significant result.

2.4 Mono-species biofilm formation and evaluation of anti-biofilm activity of the BG nano-powders

Quantitative analysis and interpretation criteria of the biofilm formation ability ofP. g,A. a, andP. iperformed according to previous studies [41–43]. Briefly, 150µL aliquots of free-floating bacteria in planktonic suspension at a final concentration of 1.0×10⁸CFU/mL transferred to a flat- bottom, polystyrene 96 wells microplate (TPP, Trasadin- gen, Switzerland) and incubated at anaerobic conditions for 48 hours at 37^∘C to allow for the biofilm formation. Af- ter incubation, the microplate contents were emptied from each well and washed three times with phosphate buffered saline (PBS) (10 mM Na2HPO4, two mM NaH2PO4, 2.7 mM KCl, 137 mM NaCl, pH 7.4) to remove free-floating planktonic bacteria. Subsequently, 150 µL of BG powder and 150 µL of Zn/BG with a concentration of 20mg/mL dissolved in sterilized stilled water added to each bacteria-containing wells separately. Afterward, the cells in the biofilm were stained with 150 µL of 0.1% (wt/vol) crystal violet solution at room temperature for 15 minutes. After washing twice with PBS, 150 µL of 95% ethanol was added to each well, and the plates were incubated at 25^∘C for 10 minutes to fix the cells [43]. Afterward, the wells were rinsed three times with PBS and air-dried. For quantifying the biofilms, 150 µL of 33% (v/v) acetic acid was poured in each well, and the absorbance was identified at 570 nm using a microplate reader (Anthos 2020, Biochrom Ltd., UK). Each experiment carried out five times per bacteria per type of BG, and five wells were assigned as laboratory controls for each group.

2.5 Statistical analysis

The SPSS ver. 22 software used for statistical calculations and mean ± SD values for quantitative variables reported in Table 1. The one-sample Kolmogorov-Smirnov test then affirmed normality of the data. The significance of differ- ences among experimental groups then assessed by analysis of variance (ANOVA) followed by post hoc Bonferroni multiple comparisons. The significance level was set at 5%

as determined by ANOVA, but in multiple comparisons of the groups, the level of significance defined as 0.05/3 = 0.016.

3 Results

In this,in vitroexperimental study, the anti-biofilm effect of Bioglass 45S5 and Zn/BG was evaluated against certain strains of periodontal pathogens, includingP.g,A.a, andP.i. Five wells assigned to each bacteria per type of nanopowder. With the total of 45 wells utilized, the biofilm formation ability of each cluster was evaluated.

According to the results of the Kolmogorov-Smirnov test, the biofilm formation ability of all experimental groups followed a normal distribution (Appendix table 1).

Since homogeneity of the variances held and the biofilm formation ability of all experimental groups followed a normal distribution, two-way analysis of variance (ANOVA) used. The results ANOVA showed that both BG and Zn/BG significantly reduced the biofilm formation ability of all examined strains (P=0.0001) (Table 1).

Moreover, in a pair-wise comparison between the materials, Zn/BG showed a statistically significant more re-

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Table 2:Multiple comparisons of experiment groups regarding the type of the material used. The level of significance set to 0.016.

Type of Material Mean difference

Std. error Significance 95% confidence interval Lower bound Upper bound

BG^r Zinc-doped BG 0.8113 0.04571 0.0001* 0.6965 0.9261

BG^r Control −1.0133 0.04571 0.0001* −1.1281 −0.8985

Zn/BG Control −1.8247 0.04571 0.0001* −1.9395 −1.7099

Legend: Zn: Zinc, BG: Bioactive glass. * Denotes a statistically significant result.

duction in the biofilm formation ability of all strains compared with BG (P=0.0001) (Table 2).

Furthermore, A. a showed the highest mean difference of OD withP. iandP. g, respectively (P=0.0001) (Ap- pendix Table A2), but there is no significant difference observed between optical densities ofP. gandP. i(P=0.62).

These data indicate that BG nano-powders show the most inhibitory effect on the biofilm formation ability ofA. a, which significantly showed more reduction in the biofilm formation than the other two strains (P=0.0001).

Figure 1 shows the anti-biofilm activity of Zn/BG and BG on the examined strains includingP. i,P. g,andA. a.

0/916 1/044 0/906

1/55

1/968

1/782

2/872 2/994

2/474

0 0/5 1 1/5 2 2/5 3 3/5

P. gingivalis A. actinomycetemcomitans P.intermedia

Optical Density (OD) at 570 nm wavelength

Periodontal Pathogens

Zinc-doped Bioactive glass Bioglass 45S5 Control

**

Figure 1:Graph showing the anti-biofilm activity of bioactive glass nano-powders on the strains of P. gingivalis, A. actinomycetemcomitans, and P. intermedia. ** Denotes an extremely significant result compared to negative control of each bacteria.

3.1 XRD results

Figure 2 shows the analysis of samples of BG (15266-1) and Zn/BG (15266-2). The Zn/BG sample has a much lower crystalline phase compared to the BG sample and yields an entirely amorphous structure. The bulk of BG consists of Ca2Na2O9Si3, which is consistently stable. This sample also revealed CaNaO4P structures. On the other hand, in the Zn/BG sample, the most structure is composed of

Figure 2:XRD analysis of samples of BG (15266-1) and Zn/BG (15266-2). BG: Bioactive glass, Zn/BG: Zinc-doped bioactive glass.

Figure 3:FTIR spectral analysis of the BG nanopowders in the range of 400- 4000 cm⁻¹at the scan speed of 22 scans/min with 4 cm⁻¹ resolution.

Na2Ca4(PO4)2SiO4, which indicates the presence of phosphorus atoms within complex structures.

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Figure 4:Scanning electron micrographs: (A) BG^rpowder, (scale bar 2, 1µm). (B) Zn/BG powder (Scale bar 200nm).

3.2 FTIR spectral analysis

Stretching vibrations of the Zn-O bond will be visible in the range of 400-800 cm⁻¹. Characteristic Zn-O spectrum (fingerprint-like) is merely visible. The explanation for this finding is that the amount of Zn-O (5 mol%) is so small so that it is covered by the spectrum of silicate bonds (Fig- ure 3).

3.3 Particle Size

Figure 4 demonstrates the microstructure of synthesized nano BG powder. The particle size of BG was shown to be approximately 20-50nm.

3.4 PH test results

According to the obtained data, the initial pH (7.0 for distilled water) was increased gradually from 1 hour to 48 hours after the immersion for all samples (P<0.05). How- ever, it was observed that pH values did not significantly differ in any of the time intervals between the groups (P=0.2) (Table 3).

Table 3:Results of the pH release test.

Time interval/Group

BG/Zn BG^r

Control value 7.0 7.0

1 hour 9.93 10.2

24 hours 9.66 9.59

48 hours 9.40 9.47

Legends: BG/Zn: Zinc-doped bioactive glass, BG: 45S5 bioactive glass.

3.5 Cytotoxicity analysis

The chart (Figure 5) shows the results of the MTT assay.

According to the chart, there was no significant difference between the results of BG and Zn/BG after 24, 48 and 72 hours (P = 0.05). This is, none of the materials showed cy- totoxic effects on hamster ovarian cells.

4 Discussion

Numerous studies have confirmed that one of the necessary conditions for bone regeneration is the prevention of microbial colonization of the surgical field [12], which can lead to post-surgical complications [13]. One of the major causes of dental implant failure is a microbial invasion of

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1

Legend: The orange bars indicate BG^® and the blue bars show Zn/BG.

0 0/5 1 1/5 2 2/5

24 48 72

Test Neg Co

Time (hours)

Absorbance (540 nm)

Figure 5:Results of the MTT assay after 24 hours, 48 hours and 72 hours. The orange bars indicate BG^rand the blue bars show Zn/BG.

the surgical site [12, 14]. Biomaterials exhibiting both regenerative and antimicrobial properties can successfully integrate with the surrounding bone, thereby ensuring the complete osseointegration of dental implants [14].

BGs have the highest bioactivity index compared to all other biomaterials [44]. One of the significant properties of BGs is their antimicrobial activity [22]. Studies demonstrate that following the dissolution of the glass, due to the release of cations, pH increases and therefore provides al- kaline conditions that can destroy the bacteria [25].

In the present study, Zn/BG showed a broad inhibitory effect on the biofilm formation ability ofP. g,A. aandP. i, which was significantly stronger than the anti-biofilm activity of BG. Also, it was shown that zinc doped bioactive glass demonstrates no cytotoxicity which is a desired feature in regenerative periodontal treatment. Several mechanisms may explain this anti-biofilm activity, such as the physical properties of the material, the release of Zn²⁺

ions, pH alterations, and BG surface reactions, which will be discussed below.

Stooret al.[25] reported thatActinomyces naeslundii lost its vitality within 10 minutes of exposure to S53P4 BG paste andP.g andA.adisappeared after 60 minutes.

The authors attributed this observation to the fact that the particle size in the BG paste was <45µm which produces a much higher surface-to-volume ratio than the conven- tional granular types (297-500 µm). This sizeable surface- to-volume ratio resulted in the faster release of the ions from the substance and thus more rapid pH rise during the first hour. BG^r has a lower surface-to-volume ratio than S53P4 type and shows decreased antimicrobial activ-

ity than S53P4 form, but the formation of the hydroxyap- atite layer and biological properties of the BG^ris more favorable than those introduced later [16, 26]. In the present study, nano-sized particles of approximately 20-50 nm were used to achieve a positive effect of the surface-to- volume ratio on the anti-biofilm properties while preserv- ing the mechanical properties of the material.

In a similar study, Allanet al.[26] investigated the antimicrobial effect of particulate Bioglass^r against supra- and subgingival bacteria. All examined strains showed a reduction in counts after 1-hour exposure to BG and this antibacterial activity increased after 3 hours. Authors concluded that S53P4 and 45S5 BGs had an apparent bactericidal effect on oral microorganisms. In the present study, BG demonstrated an inhibitory effect on periodontal pathogens, which is in line with the mentioned studies. These early studies only evaluated the antibacterial properties of BGs within 3 hours, but current research showed that BGs maintain their anti-biofilm activity up to 48 hours after being exposed to bacteria. Furthermore, microorganisms in the oral cavity usually live in ecologic niches (biofilms) which earlier studies neglected this critical feature.

Currently, various ions are used to augment the properties of BGs. Enhanced glass-based scaffolds and grafts demonstrate improved antimicrobial effects against aer- obic microorganisms such as S. aureus, E. coli, S. mu- tansandLactobacillus casei^(30,45,46). However, there is no available study on the antimicrobial effect of Zn/BG on periodontal pathogens, which are primary anaerobic [27]. To the best understanding of the authors, this is the first study which investigates the anti-biofilm effects of Zn/BG on the critical periodontal pathogens.

The percentage of Zn²⁺added to glass and ceramics varies between 0.075 mol% to 10 mol% [33, 35]. It has recently been shown that doping 5-mol% Zn²⁺ on nano- glass scaffolds, increases the compressive strength, material density and stability and leads to a formation of apatite spherical particles with a calcium-to-phosphorus ratio of about 1.67. However, 10 mol% of zinc damaged the apatite layer structure and resulted in the creation of flake- like crystals [29, 33]. In the current study, doping 5 mol%

of Zn²⁺ in the structure of nano-sized BG, showed that this amount of Zn²⁺while improving material’s mechanical properties enhances its antimicrobial activity against periodontal pathogens.

One of the possible mechanisms explaining the increase in antimicrobial activity of Zn/BG is the release of Zn²⁺ions from the material. Zn²⁺is a metal ion that has significant effects on bone repair and formation, regula- tion of osseous remodeling, and shows inhibitory effects

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on osteoclasts [47]. Numerous studies have mentioned the antimicrobial mechanisms of the Zn²⁺ion. Zn²⁺can pre- vent active transport and metabolism of carbohydrates, and by dislodging magnesium ions (Mg²⁺) that are essential for plaque enzymatic activity, it interferes with the dental biofilm enzymatic system [48].

In a recent study by Echezarreta-López et al. [30], nanofiber glasses doped with Zn-Sr showed enhanced antibacterial effects onS. aureuscompared with BG^r, which is consistent with the results of the current study. The authors explained this improved activity by the alterations in pH induced by the release of Zn²⁺and Sr²⁺ions. BGs raise pH by exchanging Na⁺and Ca²⁺ions from the surface of the glass with H⁺ions in the environment [17]. Allanet al.

also attributed the enhanced antimicrobial properties of the S54P3 BG compared with BG to faster pH rise [26]. In a study by Bejaranoet al.[33] on zinc and copper-doped BGs, researchers used two types of glass base: 58S glass and NaBG glass (Standard). The researchers observed that 58S glass base raises pH more than NaBG glass, which is due to its less crystallinity and more cation release. Adding 1 mol% zinc to both types of glass bases increased the pH more than non-doped bases, but the addition of 5 mol%

and 10 mol% of Zn²⁺resulted in lower pH than that of non- reinforced bases. The authors explained that the addition of Zn²⁺to the glass reduces Ca²⁺and Na⁺release and pre- vents excessive pH rise. This finding means that Zn²⁺doping increases the stability of the glass structure [33].

In contrast, studies by Wanget al.[45] and Bellantone et al.[28] on the biological and bactericidal properties of silver-doped BG showed that the antibacterial action of the material only relates to the release of Ag²⁺ions. They re- jected the influence of pH or other ions released from BG in killing the bacteria. Therefore, authors concluded that the enhanced antibacterial properties of silver-doped BG are rooted in its porous structure, which allows the con- trolled release ions from the material. However, in a study by Fooladiet al.[24], authors suggested that the possible cause of the antibacterial activity of BG is the pH changes of the environment, as well as the release of other ions and free radicals from the glass.

Another possible mechanism of BG antibacterial activity is the release of Ca²⁺ions from the glass surface, which leads to the agglutination ofP.g [25]. However, as mentioned earlier, doping the Zn²⁺in glass structure reduces the space for Na+ and Ca²⁺ions, thus reducing the release of these ions. Altogether, it is likely that the enhanced antimicrobial activity of Zn/BG observed in the current study, is due to the direct antimicrobial activity of Zn²⁺and the role of the increase in pH or release of Ca²⁺ is question- able [30, 47].

One of the impressive verdicts of this study was the further reduction of the biofilm formation ability ofA.acompared to other bacteria. Numerous studies declared that gram-positive bacteria are more susceptible to Zn²⁺than gram-negative bacteria [27, 49]. However, in the present study, even BG^r showed anti-biofilm activity against gram-negative bacteria. Since there is no study on the direct effect of zinc on A.a, one can assume that the enhanced anti-biofilm impact of Zn/BG onA.amay be due to the interaction of zinc with the membrane of the bacteria or the more resistance ofP.gandP.ito Zn²⁺. An exact explanation of this finding requires further research on the in- teractions between Zn²⁺ions and periodontal pathogens.

One of the restrictions of this study was the use of mono-species biofilms instead of multispecies ones. Thus, it is recommended to use multi-species biofilm other than mono-species biofilm to mimic the oral ecologic condition.

5 Conclusions

Within the limitations of this study, the findings show that BG doped with 5 mol% zinc can significantly reduce the biofilm formation ability of gram-negative anaerobic periodontal pathogens includingP. g,P. i, andA. acompared to BG.

Acknowledgement: The authors thank Dr. Maryam Poorhajibagher for assistance in carrying out the microbi- ologic tests.

Conflict of Interests: The authors explicitly confirm that there is no other conflict of interests in connection with this article. The study is self-funded by the authors.

Ethical approval: The conducted research is not related to either human or animals use.

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Supplementary materials

Figure A1:Strains ofP. intermedia(ATCC 49046) (Left plate),P. gingivalis(ATCC 33277) (Central plate) andA. actinomycetemcomitans (33384) (Right plate) grown anaerobically on fresh modified brain heart infusion agar (Merck, Darmstadt, Germany).

Figure A2:Transferring the bacteria to flat-bottomed sterile polystyrene micro plates to allow for biofilm formation.

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Figure A3:Staining of the specimens with 0.1% (wt/vol) crystal violet solution.

Figure A4:Determination the anti-biofilm activity of nanopowders by a microplate reader (Anthos 2020, Biochrom Ltd., UK). The optical density of the samples is calculated at 570 nm wavelength.

OD: Optical density measured at 570 nm wavelength.

Figure A5:Flowchart of the study

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Table A1:Results of the one-sample Kolmogorov-Smirnov test. The values greater than 0.05 indicate the normality and homoscedasticity of the data.

Type of material Type of bacteria Biofilm

formation ability

Bioglass 45S5 A.actinomycetemcomitans N 5

Kolmogorov-Smirnov Z 0.448

P-value 0.988

P.gingivalis N 5

P-value 0.977

P.intermedia N 5

P-value 0.981

Zinc-doped BG A.actinomycetemcomitans N 5

P-value 0.974

P.gingivalis N 5

P-value 0.842

P.intermedia N 5

P-value 0.975

Control A.actinomycetemcomitans N 5

P-value 0.948

P.gingivalis N 5

P-value 0.990

P.intermedia N 5

P-value 0.928

Table A2:Multiple comparisons of experiment groups regarding the type of the bacteria used. The level of significance set to 0.016.

Type of bacteria Mean

difference

Std. error Significance 95% confidence interval Lower bound Upper bound A. actinomycetem-

comitans

P. gingivalis 0.2227 0.04571 0.0001* 0.1079 0.3375

A. actinomycetemcomitans

P. intermedia 0.2813 0.04571 0.0001* 0.1665 0.3961

P. gingivalis P. intermedia 0.0587 0.04571 0.623 −0.0561 0.1735

Legend: * Denotes a statistically significant result.