An In vitro Evaluation of Antimicrobial Efficacy of new Nano-zinc Oxide Eugenol (NZOE)

Document Type: Original Article

Authors

1 dental Research Center, Mashhad University of Medical Sciences, Mashhad, Iran

2 Dental Materials Research Center, Mashhad University of Medical Sciences, Mashhad, Iran

3 Dental Research Center, Mashhad University of Medical Sciences, Mashhad, Iran

4 Assistant professor in Microbiology, Kashmar Center of Higher Health Education, Mashhad University of Medical Sciences, Mashhad, Iran

5 Assistant professor in Molecular Microbiology, Kashmar Center of Higher Health Education, Mashhad University of Medical Sciences, Mashhad, Iran

Abstract

Introduction: This interventional in-vitro study aimed to evaluate the antimicrobial activity of a new Nano Zinc OxideEugenol (NZOE) sealer in comparison with AH26 and Pulpdent common root canal sealers against endodontic pathogens. Methods: The antimicrobial efficacy of three sealers(NZOE, AH26 and Pulpdent)  against Enterococcus faecalis (ATCC 29212), Escherichia coli (ATCC 25922), Streptococcus mutans (ATCC700610), Candida albicans (ATCC 90028), and Staphylococcus aureus (ATCC 25923)  were evaluated by Direct Contact Test (DCT) at five  different time intervals 0, 2, 14, 48 hours and 7 days. The results were statistically analyzed using SPSS software and Kruskal–Wallis at 5% significance level. Results: All three tested sealers had antimicrobial activity against microorganisms involved in this study. Fresh NZOE sealer eliminated all microorganisms tested, except the strain of E. faecalis colony which was reached to zero after 2 hours. However, AH26 and Pulpdent failed to completely kill all of the E. faecalis colony during the entire observation period. Also, NZOE showed a significant antimicrobial action (P<0.05) in comparison with other two sealers by effectively eliminating the Candida albicans colonies at zero time and 7 days. Conclusion: Highest antimicrobial effect of NZOE sealer was shown followed by Pulpdent sealer and AH 26 against Candida albicans and Enterococcus faecalis.
 
 

Keywords

Main Subjects


Introduction

     Microorganisms and their byproducts are the main etiological factors of pulpits and apical periodontitis. The main goal in endodontic therapy is the elimination of microorganisms from the root canal system by instrumentation, irrigation, and intra-canal medication. Unfortunately, complete elimination of microorganisms and their by-products from the root canal system is impossible (1). So, antimicrobial activity of root canal filling materials is considered to be a beneficial property for reduction of the remaining microorganisms’ number (2).

There are the numbers of root canal sealers currently available that possess quite different formula. Zinc-oxide-Eugenol based sealers are the most commonly used sealers in root canal treatment. Studies have shown that, nanotechnology is extensively used in manufacturing dental materials (3). Some advantages such as penetration into dentinal tubules, antibacterial activity and low micro-leakage property of nano-particles have drawn attention in endodontics (4,5).

A new endodontic sealer with Nano-sized ZOE powder particles (NZOE) has been developed in the Dental Material Research Center, Mashhad University of Medical Sciences, Mashhad, Iran. This sealer is similar to various ZOE-based sealers, but with different sizes of ZOE nanoparticles (3). This sealer exhibit cyto-compatibility (6, 7), similar tooth discoloration compared to resin sealers (3), and proper sealing ability (8).

The aim of the current in vitro study was to evaluate the antimicrobial activity of NZOE, compared with two common endodontic sealers AH26 (resin-based) and Pulpdent (ZOE-based) against Enterococcus faecalis, Streptococcus mutans, Staphylococcus aureus, Escherichia coli and Candida albicans at five different time intervals.

 

Materials and Methods

Endodontic sealers used in this study were AH26 (Dentsply Sirona, Germany), Pulpdent (Watertown, USA) and NZOE (Dental Material Research Center, Mashhad Iran). All the sealers were prepared in accordance with the manufacturer’s recommendations. Enterococcus faecalis (ATCC 29212), Escherichia coli (ATCC 25922), Streptococcus mutans (ATCC700610), Candida albicans (ATCC 90028), and Staphylococcus aureus (ATCC 25923) strains were used. All the reference strains were obtained from the Department of Microbiology, Pasteur Institute of Iran, Tehran.

The antimicrobial activity of the endodontic sealers was performed by means of the modified Direct-Contact Test (9) against above reference strains, freshly mixed at five different time intervals 0, 2, 14, 48 hours and 7 days. The tested sealers were prepared according to the manufacturer’s instructions. . Each sealer was placed on the side wall of the Micro-tubes (Eppendorf 2.0 mL) by using a cavity liner applicator. The sealers immediately after preparation was considered as T0; the others were allowed to set for 2 hours (T2), 14 hours (T14), 48 hours (T48) and 7 days (D7). Afterwards, the Micro-tubes were incubated at 37°C in humidity for five different time intervals 0, 2, 14, 48 hours up to 7 days.

The microorganisms inoculated in Brain Heart Infusion (Condo, Spain) and incubated for 24 h at 37 °C.  After incubation, the broth cultures were adjusted to 0.5 McFarland (1.5 × 108 CFU; colony-forming units) by using McFarland standard. Ten µL of bacterial suspension was placed on the surface of each fresh aged and sealers in Micro-tubes and exposed one hour in an incubator shaker at 37ºC (available humidity). All interval time Micro-tubes was done as well.

Consequently, 240 µL of BHI broth was added to each interval time Micro-tubes, mixing well for one minute and transfer 10 µL of each diluted samples are then spread onto BHI agar.

The BHI agar plate incubated at 37°C for 24 hours. The microbial culture was prepared for each interval time Micro-tubes on BHI agar. After incubation, the CFU/mL was calculated from bacteria dilution.

All experiments were performed in triplicate. Microbial suspension without cements used as positive control and sealer in addition to BHI without microbial suspension, considered as negative control. The normality was checked by shapiro-wilk test and the data distribution was not normal. Kruskal–Wallis comparison tests were used for the statistical analysis. The data were processed using SPSS software and the level of significance was set at 5% (P < 0.05).

 

Result

The results of the antimicrobial effects of three endodontic sealers are presented in Tables (1-5). Fresh sealers and sealers set for 2, 14, 48 hours, and 7 days showed differences in their antimicrobial effects. In fresh sealers, significant difference in antimicrobial activity had present only against E.coli and Candida albicans (Table3, 4). Fresh NZOE eradicated all bacteria except E. faecalis. But after two hours of contact, NZOE eliminated all E. faecalis strains.

NZOE, AH-26, and Pulpdent killed all S. mutans in 0, 2, and 14 hrs. Of contact, and in 48 hrs. no microbial colony was performed in Pulpdent and NZOE.

 

 

After 7 days, the significant difference were only present against S. aureus and candida. Against s. aureus, AH26 showed the most activity and Pulpdent was the worst. Against candida, NZOE presented the best antimicrobial activity and Pulpdent was the worse.

However, after 7 days most sealers had lost much of their antimicrobial effect.

 

 

 

  

 Table 1. Mean and standard deviation of colony count for sealers in different intervals in E.faecalis

Time

Materials

Min max

Mean ± SD

Min

Max

P-Value

T0

Pulpdent

10000

40000±51962

10000

100000

P=0.304

NanoZno

10000

40000±51962

10000

100000

AH26

10000

7000±5196

1000

10000

2h

Pulpdent

100000

70000±51962

10000

100000

P=0.304

NanoZno

0

0

0

0

AH26

0

17±29

0

50

14h

Pulpdent

100000

70000±51962

10000

100000

P=0.044

NanoZno

1000

1000

1000

1000

AH26

1000

4000±5196

1000

10000

48h

Pulpdent

400

367±58

300

400

P=0.025

NanoZno

1000

4000±5196

1000

10000

AH26

1000000

7000000±5196152

1000000

10000000

7d

Pulpdent

10000

40000±51962

10000

100000

P=0.050

NanoZno

10000

40000±51962

10000

100000

AH26

100000000

70000000±51961524

10000000

100000000

 

 

 

 

 

 

 

 

 

 

 

 

 

Table 2. Mean and standard deviation of colony count for sealers in different intervals in S.aureus 

Time

Materials

Min max

Mean ± SD

Min

Max

P-Value

T0

Pulpdent

10000

40000±51962

10000

100000

P=0.304

NanoZno

10000

40000±51962

10000

100000

AH26

10000

7000±5196

1000

10000

2h

Pulpdent

100000

70000±51962

10000

100000

P=0.304

NanoZno

0

0

0

0

AH26

0

17±29

0

50

14h

Pulpdent

100000

70000±51962

10000

100000

P=0.044

NanoZno

1000

1000

1000

1000

AH26

1000

4000±5196

1000

10000

48h

Pulpdent

400

367±58

300

400

P=0.025

NanoZno

1000

4000±5196

1000

10000

AH26

1000000

7000000±5196152

1000000

10000000

7d

Pulpdent

10000

40000±51962

10000

100000

P=0.050

NanoZno

10000

40000±51962

10000

100000

AH26

100000000

70000000±51961524

10000000

100000000

 

 

 

 

 

 

 

 

 

 

 

 

 

Table 3. Mean and standard deviation of colony count for sealers in different intervals in E.coli

Time

Materials

Min max

Mean ± SD

Min

Max

P-Value

T0

Pulp dent

0

42±72

0

125

P=0.560

NanoZno

0

7±12

0

20

AH26

100

75±66

0

125

2h

Pulp dent

300

267±153

100

400

P=0.044

NanoZno

0

0

0

0

AH26

400

467±208

300

700

14h

Pulp dent

10000

7000±5196

1000

10000

P=0.0610

NanoZno

60

60±20

40

80

AH26

60

70±26

50

100

48h

Pulp dent

100000

67000±57158

1000

100000

P=0.044

NanoZno

20

17±6

10

20

AH26

0

7±12

0

20

7d

Pulp dent

100000000

70000000±51961524

10000000

100000000

P=0.027

NanoZno

3000

11667±15885

2000

30000

AH26

500

523±166

370

700

 

 

 

 

 

 

 

 

 

 

 

 

Table 4. Mean and standard deviation of colony count for sealers in different intervals in Candida albicans 

Time

Materials

Min max

Mean ± SD

Min

Max

P-Value

T0

Pulpdent

0

13±23

0

40

P=0.046

NanoZno

0

0

0

0

AH26

80

73±31

40

100

2h

Pulpdent

100

400±520

100

1000

P=0.123

NanoZno

700

667±153

500

800

AH26

1000

1000

1000

1000

14h

Pulpdent

0

7±12

0

20

P=0.035

NanoZno

0

0

0

0

AH26

150

167±126

50

300

48h

Pulpdent

200

300±173

200

500

P=0.139

NanoZno

300

300±100

200

400

AH26

1000

800±346

400

1000

7d

Pulpdent

100

400±520

100

1000

P=0.289

NanoZno

800

833±153

700

1000

AH26

600

600±100

500

700

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Table 5. Mean and standard deviation of colony count for sealers in different intervals in S.mutans 

Time

 

Materials

Min max

Mean ± SD

Min

Max

P-Value

T0

 

Pulpdent

500

467±351

100

800

P=0.022

 

NanoZno

0

0

0

0

 

AH26

0

0

0

0

2h

 

Pulpdent

100

83±76

0

150

P=0.105

 

NanoZno

0

0

0

0

 

AH26

0

0

0

0

14h

 

Pulpdent

1000

700±520

100

1000

P=0.051

 

NanoZno

1000

700±520

100

1000

 

AH26

100000

70000±51962

10000

100000

48h

 

Pulpdent

100000

70000±51962

10000

100000

P=0.670

 

NanoZno

10000

40000±51962

10000

100000

 

AH26

100000

70000±51962

10000

100000

7d

 

Pulpdent

1000000

700000±519615

100000

1000000

P=0.040

 

NanoZno

1000

4000±5196

1000

10000

 

AH26

10000

40000±51962

10000

100000

 

 

 

 

 

 

 

 

 

 

 

 

 

Discussion

Residual microorganisms are the most important cause of failure in endodontic treatment. Thus, in addition to biocompatibility, sealing ability, and dimensional stability, antimicrobial effect of sealers is one of the critical properties (2).

In this study, we investigated the antimicrobial properties of AH26, pulpdent, and NZOE against facultative bacteria and yeast, which are predominant in persistent or refractory periapical lesions. E. faecalis is robust microorganism that may infect root canal (9) and are more likely to be found in cases of failed endodontic therapy than in cases of primary infection (10). E.coli is sometimes recovered from root canals and represent a standard organisms used in antimicrobial testing (11). C. albicans has the ability to form biofilms on different surface, and may be involved in cases of persistent and secondary infection (12). S. mutans may have a major influence on both the initial pulpal lesion and subsequent pulpal pathology (13).

It should be noted that in this study, the samples were evaluated at 0, 2, 14, 48 hrs. and 7days by the Direct-Contact Test (DCT) method. The aim for selecting these intervals was that the antibacterial effects of sealers were evaluated in fresh and set manner. The setting time for pulpdent, AH26, and Nano-Zno is 2, 14, and 48 hours, respectively. And finally, we choose 7days for evaluating aging effect on antibacterial effect of sealers. The DCT is a quantitative and reproducible method that simulates the contact of the test microorganism with endodontic sealers inside the root canal (14). The effect of sealers at various stages of the setting reaction on microbial viability can be evaluated (15). The method also allows for better control of possible confounding factors than ADT. In DCT, the turbidimetric method allows detecting the prevention of growth (bacteriostatic effect). Also, in cases in which carryover effect is controlled, turbidimetric measurements in DCT can show whether all (100%) bacteria have been killed (16).

In our study, the colony count of Candida was zero by Nano-Zno and AH26 sealers in 0 and 2 hours that showed these two sealers had similar and also strong antifungal properties. But fresh pulpdent sealer could not eradicate all microorganisms. Zhang et al. (15) showed that nano sealers were effective against E.coli, Candida, and S. aureus.

Fresh Nano-Zno sealer killed all microorganisms except for E. faecalis.  In addition, Nano-Zno sealer eradicated all E. faecalis within 2 hours of contact that was better than AH26. AH26 and pulpdent could not eliminate all E. faecalis in any of intervals. Beyth et al. (17) in their microscopic evaluation reported that direct contact of nano particle with E. faecalis caused cellular lysis and immediate bacterial killing, while in contact to the other sealers, ability of  biofilm formation still would present.

Also, significantly more colony reduction of S. aureus was observed by Nano-Zno in comparison to AH26 during the first 2 hours. Nano-Zno in fresh manner and also within 14 hours of contact killed all E.coli while average colonies were significantly higher for AH26.

Antimicrobial effect of ZOE refer to free eugenol that is Phenolic Substrata that can effect on myocyte in vegetative form (6). Nano-sized ZO powder particles (NZO) are biocompatible material that was broadly used in medicine industry and also have profound antibacterial effect that because of these favorable properties, utilization of nanoparticles in production of endodontic sealers has become the center of interest (7).

Cobankara et al. (18) showed that ZOE-based sealer in combination with Nano-Zno particle could penetrate deeply in dentinal tubules in addition to releasing antibacterial particles that sealer without nano particle did not have this properties.

Nair et al. (19) reported that particle size reduction and increase of sealer concentration could increase the antimicrobial effect of sealers against E.coli and Staphylococous ureous. In Rad Ms et al. study (20), similar to the present study, Nano sealer had better antimicrobial properties against Enterococous in comparison to pulp-dent sealer.

Cobankara et al. (18) showed that Nano sealers had antimicrobial effect after 90 days that associated to nano –particle concentration. Shrestha et al. (21) reported that Nano-Zno particles still had antimicrobial properties against E. faecalis. Samadi et al. (16) demonstrated that resin sealer had antimicrobial effect against E. faecalis immediately after mixing, but after 48 hours the bacterial colony was increased. The present study showed that antimicrobial effect of sealers were strong within 2 hours of contact and then until 7 days reduced gradually that was in accordance with the findings of Ehsani et al. (22) and Heling et al. (23). Altogether, antimicrobial effect of sealers against E. faecalis was lower than the other organisms.  Nano-sealer in immediate contact to microorganism showed profound antimicrobial properties that this characteristic was reduced by passing the time.  NPs offer improved properties to classical organic antibacterial agents. One reason lies in their high surface area to volume ratio, resulting in appearance of new mechanical, chemical, electrical, optical, magnetic, electro-optical, and magneto-optical properties of the NPs that are different from their bulk properties (24).

The electrostatic properties of both NPs and biofilms influence how they interact (25). Moreover, Versiani et al. (4) have demonstrated that glass surfaces coated with ZnO nanoparticles are able to produce reactive oxygen species (ROS) that interfere with E. coli and S. aureus biofilm formation. Nanoparticles are able to attach to the membrane of bacteria by electrostatic interaction and disrupt the integrity of the bacterial membrane (26). Nanotoxicity is generally triggered by the induction of oxidative stress by free radical formation, that is, the ROS, following the administration of NPs (2).

 

Conclusion

Based on the results of the present study, it can be concluded that Nano-ZNO sealer had higher antimicrobial properties compared to AH26 and Pulpdent sealers. Thus, in addition to another satisfactory characterize of   Nano-ZNO, can be recommended to substitute the conventional sealer.

 

Acknowledgment

This study was supported by a grant from the Vice Chancellor of Research Council of Mashhad University of Medical Sciences, Iran. The results presented in this study have been taken from a student thesis (no. 582) in Mashhad University of Medical Sciences (MUMS). The authors deny any conflicts of interest related to this study.

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