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 Table of Contents  
ORIGINAL ARTICLE
Year : 2017  |  Volume : 9  |  Issue : 2  |  Page : 98-104

Antibacterial activity of four root canal sealers against Enterococcus Faecalis at 1st, 3rd, 5th, and 7th day duration: A In vitro study


1 Department of Conservative Dentistry and Endodontics, BJS Dental College and Hospital, Ludhiana, Punjab, India
2 Department of Conservative Dentistry and Endodontics, Maratha Mandal Dental College and Research Centre, Belgaum, Karnataka, India
3 Department of Microbiology, Jawaharlal Nehru Medical College, Belgaum, Karnataka, India
4 Department of Periodontology and Oral Implantology, MMCDSR, Mullana, Ambala, Haryana, India
5 Department of Oral and Maxillofacial Surgery, BJS Dental College and Hospital, Ludhiana, Punjab, India
6 Department of Prosthodontics, SKSS Dental College, Sarabha, Ludhiana, Punjab, India

Date of Web Publication26-May-2017

Correspondence Address:
Bhuvanesh Tandon
Department of Conservative Dentistry and Endodontics, BJS Dental College and Hospital, Ludhiana, Punjab
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/IJDS.IJDS_22_17

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  Abstract 

Background: Root filling plays an important role in control of reinfection by entombing the residual organisms through antimicrobial activity of endodontic sealers. Several endodontic sealers have also been found to have antibacterial properties, depending on their chemical contents. Aim: Aim of the present study was to assess the antibacterial activity of 4 endodontic sealers i.e AH Plus (Group 1), Sealapex (Group 2), Roekoseal (Group 3) and ZOE (Group 4) against E. faecalis at 1hr, 1st, 3rd, 5th and 7th day duration. Materials and Methods: Antimicrobial activities of four root canal sealers were evaluated against E. faecalis at various time intervals by direct contact test. Freshly mixed sealers were placed in 96-well microtitre plates. Plates were placed for incubation at 37°C and thereafter placed in microplate reader wherein optical density in each well was measured at 630 nm. Readings were taken at regular intervals. i.e after 1 hr (fresh specimen), then on 1st, 3rd, 5th and 7th day. Statistical Analysis: Data was collected by recording optical density, considering the fact that as, bacterial population increases, absorbance reading, i.e. Optical Density (O.D.) given by spectrophotometer increases. Data were recorded, then plotted and statistically analyzed using 2 way -ANOVA (Analysis of Variance) and Newman-Keuls Post hoc procedure. Result: Intragroup comparison in various groups showed greatest antibacterial effect at 1 hr, followed by a decrease in the effectiveness at 1st day, 3rd day, 5thday, 7th day. In intergroup comparison, GROUP 2 (Sealapex) showed least inhibition of bacterial growth at 1 hr interval followed by further decrease at different studied intervals. Conclusion: It was concluded that Antibacterial activity of tested endodontic sealers on E. faecalis in an ascending order was as follows: Sealapex, Roekoseal, AH Plus and Zinc Oxide Eugenol sealer.

Keywords: Antibacterial property, endodontic sealers, Enterococcus faecalis


How to cite this article:
Tandon B, Uppin VM, Hogade SA, Chaudhry S, Bansal S, Bansal S. Antibacterial activity of four root canal sealers against Enterococcus Faecalis at 1st, 3rd, 5th, and 7th day duration: A In vitro study. Indian J Dent Sci 2017;9:98-104

How to cite this URL:
Tandon B, Uppin VM, Hogade SA, Chaudhry S, Bansal S, Bansal S. Antibacterial activity of four root canal sealers against Enterococcus Faecalis at 1st, 3rd, 5th, and 7th day duration: A In vitro study. Indian J Dent Sci [serial online] 2017 [cited 2019 Oct 20];9:98-104. Available from: http://www.ijds.in/text.asp?2017/9/2/98/207105


  Introduction Top


Root canal treatment (RCT) is accomplished by chemomechanical preparation, intracanal disinfection, and three-dimensional obturation, which is essential to avoid reinfection of root canal space.[1] Thus, root filling plays an important role in the control of reinfection by entombing the residual organisms through antimicrobial activity of endodontic sealers.[2]

Ideal requirements of sealers according to Grossman are biocompatibility, excellent seal, adequate adhesion, and antimicrobial property.[3] Several endodontic sealers have also been found to have antibacterial properties, depending on their chemical contents.[4],[5] Antibacterial properties may be advantageous in clinical situations of persistent or recurrent infection.[6],[7] Facultative anaerobic microorganisms may interact with strict anaerobes, causing changes in nutritional relationship, shifts in redox potential, and oxygen tension which determine the microbial–survival relationship.[8]

Enterococcus faecalis is a Gram-positive, Group D streptococci. It constitutes only a small proportion of the initial flora in untreated canals,[9] but this genus is most commonly recovered from root canals with failed root treatment.[10],[11] Ability of these organisms to cause periapical disease and chronic failure of an endodontically treated tooth is due to its ability to bind to collagen of dentinal tubule and remain viable within the tubule.[12]

Enterococci possess a number of virulence factors that permit adherence to host cells and extracellular matrix, facilitate tissue invasion, effect immunomodulation, and cause toxin-mediated damage.[13] These microorganisms have the ability to utilize opportunities created by removal of other microbes and also to grow in a low-nutrient environment, surviving in root canals as a monoinfection.[14]

E. faecalis is a recalcitrant candidate among causative agents of failed endodontic treatment. According to Sundqvist et al., 38% of failed root canal systems were contaminated with E. faecalis.[15] Therefore, it might be advantageous if the sealer exerts some antimicrobial activity as the last element in treatment regimen.[16]

With the introduction of direct contact test (DCT) by Weiss et al., antibacterial activity of endodontic sealers is been tested based on measuring the effect of close contact between test bacteria and tested material.[17],[18]

  • DCT has been used to evaluate in vitro antibacterial activities of numerous endodontic sealers
  • Zinc oxide eugenol (ZOE)-based sealers have shown to possess persistent antimicrobial activity [Figure 1][19]
  • Calcium hydroxide-based sealers have pH-dependent action which as per studies decreases with time [Figure 2][20]
  • Resin-based sealers (AH Plus) have been found to have antimicrobial properties [20] but of short duration only [Figure 3]
  • Silicone-based sealers (Roekoseal) have recently been introduced in dental community, and sufficient data are not available with regard to its antibacterial properties [Figure 4].[8]
Figure 1: Resin-based sealer (AH Plus).

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Figure 2: Calcium hydroxide-based sealer.

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Figure 3: Silicone-based sealer (Roekoseal).

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Figure 4: Zinc oxide eugenol sealer.

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Thus, the objective of this study was to analyze in vitro antibacterial properties of four root canal sealers against E. faecalis at 1 h, 1st, 3rd, 5th, and 7th day duration.


  Materials and Methods Top


This in vitro study was conducted at the Department of Conservative Dentistry and Endodontics, KLE V. K. Institute of Dental Sciences, Belgaum, and Department of Microbiology, Jawaharlal Nehru Medical College, Belgaum, Karnataka, India.

Source of data

E. faecalis was obtained from the Department of Microbiology, Jawaharlal Nehru Medical College, Belgaum, Karnataka, India.

Method of collection of data

Data were collected by recording optical density (OD) and measurement of turbidity, with the help of a spectrophotometer.

Study materials

Four commercially available root canal sealers were used in this study:

  1. ZOE-based sealer
  2. Ca(OH)2-based sealer - Sealapex
  3. Resin-based sealer - AH Plus
  4. Silicon-based sealer - Roekoseal.


Sealers were prepared in strict compliance with manufacturer's recommendation.

Test microorganism and growth conditions

E. faecalis was obtained from the Department of Microbiology, Jawaharlal Nehru Medical College, Belgaum, Karnataka, India. Bacteria were grown aerobically from frozen stock cultures in brain–heart infusion (BHI) broth at 37°C. Inoculum was prepared by resuspension of washed cells to predetermined optical densities which relate to known concentrations. Suspension was adjusted spectrophotometrically at 800 nm to match transmittance of 90T (equivalent to 0.5 McFarland scale = 1.5 × 108 CFU).

Direct contact test

DCT is based on turbidimetric determination of bacterial growth in 96-well microtiter plates [Figure 5]. Kinetics of outgrowth in each well was monitored at 630 nm at 37°C and recorded using a spectrophotometer (ELISA reader: Stat Fax 2100; Awareness Technology).
Figure 5: 96-well microtiter plate (Hi Media).

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  • Of the 96 wells of microtiter plate, two sets of four wells were utilized per se aler, namely “Subgroup 1” wells (with the sealer; four wells) and “Subgroup 2” wells (without the sealer; four wells)
  • Subgroup 1 wells were held vertically, i.e., plate's surface was maintained perpendicular to the floor plane and side wall was coated with freshly mixed tested material
  • Even and thin coating was achieved using a cavity liner applicator
  • Special care was taken to avoid material's flow to bottom of the well, which would interfere with the path of light through microplate well and result in false readings.


Approximately 20 min later that corresponded to the recommended setting time of sealers, 10 μL bacterial suspension (108 CFU) was placed on the test material.

The plate was held in the vertical position and wells were inspected for evaporation of suspension's liquid, which occurred within 1 h at 37°C. This ensured direct contact between bacteria and tested material. BHI broth (245 μL) [Figure 6] was added to each of these wells and gently mixed for 2 min.
Figure 6: Brain heart infusion agar (Hi Media).

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Nearly 15 μL of broth was then transferred from Subgroup 1 wells to an adjacent set of “Subgroup 2” wells that already contained fresh BHI medium (215 μL). This resulted in two sets of four wells for each tested material containing an equal volume of liquid medium so that bacterial outgrowth could be monitored both in the presence and in the absence of tested material. Plate was placed for incubation at 37°C and thereafter placed in microplate reader wherein OD in each well was measured at 630 nm. Readings were taken at regular intervals, i.e., after 1 h (fresh specimen), then on 1st, 3rd, 5th, and 7th day.

Data were collected by recording OD, considering the fact that as bacterial population increases, absorbance reading, i.e., OD given by spectrophotometer increases. Data were recorded, and then plotted and statistically analyzed using two-way - analysis of variance and Newman–Keuls post hoc procedure.

The whole experiment was carried out under aseptic conditions and was repeated three times to ensure reproducibility.


  Results Top


  • Group 1: AH Plus which was further divided into subgroup 1 and subgroup 2
  • Group 2: Sealapex which was further divided into subgroup 1 and subgroup 2
  • Group 3: Roekoseal which was further divided into subgroup 1 and subgroup 2
  • Group 4: ZOE which was further divided into subgroup 1 and subgroup 2 [Graph 1],[Graph 2],[Graph 3],[Graph 4],[Graph 5].

,

,

,

,



On intergroup comparison, Group 1 (AH Plus) showed greatest inhibition of bacterial growth among different sealers at 1 h interval followed by decrease in inhibition on the 5th and 7th day test period. Intragroup comparison showed greatest antibacterial effect at 1 h, followed by a decrease in the effectiveness at 1st, 3rd, 5th, and 7th days.

On intergroup comparison, Group 2 (Sealapex) showed least inhibition of bacteria at 1 h interval among the other groups and decreased further on the 1st day, followed by a constant decrease in efficacy at 3rd, 5th, and 7th days.

Intragroup comparison depicted that freshly mixed sample had more efficacy than samples at 1st, 3rd, 5th, and 7th days.

In intergroup comparison GROUP 3 (Roekoseal) showed similar antibacterial effects to AH Plus and ZOE but greater than Sealapex followed by a constant decrease in antibacterial efficacy at 1st, 3rd, 5th and 7th day.

On intergroup comparison, Group 4 (ZOE) showed a potent antibacterial effect at 1 h duration similar to Roekoseal and AH Plus and a significant difference compared to Sealapex. At 1st day, it showed greatest antibacterial effect among the other groups which remained constant throughout the study period.


  Discussion Top


Elimination of microorganisms from root canal has always been an important part of endodontic therapy. Bystrom et al. and Sjögren et al. stated that, if these microorganisms persist in the root canal at the time of root filling or if they penetrate into canal after filling, there is a higher risk of treatment failure.[21],[22]E. faecalis, most frequently recovered microorganism from refractory periapical periodontitis,[23] has been used in numerous studies of antibacterial properties of disinfecting agents because of its resistance to some medicaments and its ability to survive conventional root canal therapy.[8],[16] DCT has many advantages over agar diffusion test and has been studied previously by Weiss et al. and Fuss et al.[17],[18] It is a quantitative assay which allows water-insoluble materials to be tested.

Results of this study were in line with previous studies with freshly mixed specimen showing greater antimicrobial efficacy followed by lesser but consistent efficacy over 1st, 3rd, 5th, and 7th day duration. Consistent antimicrobial efficacy over 1st, 3rd, 5th, and 7th day duration can be explained by the fact that even after the material sets, surface hydrolysis of the chelate (zinc eugenolate) results in release of eugenol, thus explaining effective antibacterial activity of this substance even after 72 h. This is in accordance with the previous studies which have shown that ZOE-based sealers possess a strong and persistent antimicrobial activity.[7],[8],[16]

Calcium hydroxide-based sealer (Sealapex) showed the least inhibition of bacteria at 1 h interval among the other groups that decreased further on 1st day, followed by a constant decrease in efficacy at 3rd, 5th, and 7th days. However, intragroup comparison depicted that freshly mixed samples showed antibacterial effect. Antibacterial action of Sealapex might be associated with its ionization, which releases hydroxyl ions that increases pH to above 12.5, thus rendering the environment unfavorable for growth of microorganisms.[23]

Initial rise in pH might explain the antibacterial efficacy of freshly mixed samples. However, samples at 1st, 3rd, 5th, and 7th days showed a decrease in antibacterial activity. These results are in contradiction with the other studies stating that antimicrobial effect of calcium hydroxide-based sealers increases with time.[1],[17],[18] Under the test conditions, lesser antibacterial components may have been released from the sealer to affect bacteria by the time.[19] To render antibacterial effect, E. faecalis should maintain a pH level >12.5.[24] As the calcium hydroxide sealer sets, the pH declines to about 9.14, resulting in loss of the sealer's effectiveness.[7],[24] Zhang et al.[1] found that the pH of Sealapex over the 1st, 3rd, and 7th day duration does not rise above 10.5, thus indicating reduced efficacy of the sealer. Furthermore, low solubility of calcium hydroxide and pH neutralizing the effect of culture media make it difficult for the sealer to reach the critical pH which might also be a factor for the decreased antimicrobial effect.[6]

Roekoseal showed similar antibacterial effects to AH Plus and ZOE in freshly mixed samples but greater effect than Sealapex followed by a constant decrease in the antibacterial efficacy at 1st, 3rd, 5th, and 7th days among different groups. Intragroup comparisons revealed a decrease in antibacterial efficacy at 1st, 3rd, 5th, and 7th days. These results are only in partial accordance with the findings of Cobankara et al.[8] Observations by Saleh et al.[7] stated a limited ability of Roekoseal automix to kill E. faecalis. Slutzky-Goldberg et al.[20] noted no bacterial growth inhibition by Roekoseal.

AH Plus (epoxy resin-based sealer) showed that only fresh AH Plus samples possessed antibacterial activity, whereas 24-h and 7-day-old samples did not show antibacterial effect against E. faecalis. Similar results were reported by Kayaoglu et al.[16] and Slutzky-Goldberg et al.[20] Antibacterial effect of epoxy resin-based sealers might be related either to bisphenol-A diglycidyl ether, which was previously identified as a mutagenic component, or to the release of formaldehyde during the polymerization process.[20] The present study also showed that fresh AH Plus had significant antibacterial effect, whereas set samples did not show antibacterial activity.

Overall, ZOE-based sealers proved to be effective against the microorganisms at the time intervals studied. In the present study, eventually, all the sealers except ZOE lost their antibacterial effect in relation to their constituents.

The present study tested the antibacterial activity of four sealers against microorganisms considered to be resistant to endodontic treatment. Therefore, it means that, if a sealer is effective against microorganisms, it will probably be effective against the more susceptible ones.[25] None of the sealers tested totally inhibited the microbial growth, and antibacterial activity of each sealer decreased with time and depended on the microbial susceptibility to them. Thus, endodontic treatment must be carried out under aseptic conditions, using a powerful irrigant solution, an intracanal medicament when necessary, a sealer with antimicrobial activity, and an effective coronal seal to prevent coronal microleakage to increase the chances of successful RCT.[25]

The present in vitro study evaluated the antibacterial activity of four endodontic sealers on E. faecalis by DCT. In addition to antimicrobial activity, other properties such as biocompatibility, stability, and sealability must also be considered when selecting a root canal sealer.[16]


  Conclusions Top


The following conclusions were drawn from the present study:

Sealers had different inhibitory effects on E. faecalis during the incubation period as follows:

  • Group 1 (AH Plus) showed that only freshly mixed samples had significant antibacterial effect, whereas set samples did not show any antibacterial activity
  • Group 2 (Sealapex) showed least inhibition of bacteria at 1 h interval among the other groups followed by a decrease till the 7th day
  • Group 3 (Roekoseal) showed antibacterial activity only in freshly mixed samples with a decrease in activity over the 1st, 3rd, 5th, and 7th day duration
  • Group 4 (ZOE) possessed strong and consistent antibacterial activity over the time intervals studied
  • Antibacterial activity of tested endodontic sealers on E. faecalis in an ascending order is as follows: Sealapex, Roekoseal, AH Plus, and ZOE sealer
  • Based on the results of this in vitro study, it was concluded that technique, time, or ingredients of the tested materials affect the results of microbiological studies. Therefore, it is proposed that more than one assaying method should be used in the process of evaluating antibacterial properties of dental materials.[8] Further, more studies and research activities are needed to confirm the findings to assure the effectiveness of sealers in the outcome of endodontic therapy.


Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
  References Top

1.
Zhang H, Shen Y, Ruse ND, Haapasalo M. Antibacterial activity of endodontic sealers by modified direct contact test against Enterococcus faecalis. J Endod 2009;35:1051-5.  Back to cited text no. 1
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Peters LB, Wesselink PR, Moorer WR. The fate and the role of bacteria left in root dentinal tubules. Int Endod J 1995;28:95-9.  Back to cited text no. 2
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Grossman L. Endodontic Practice. 11th ed. Philadelphia: Lea & Febiger; 1988.  Back to cited text no. 3
    
4.
Rappaport HM, Lilly GE, Kapsimalis P. Toxicity of endodontic filling materials. Oral Surg Oral Med Oral Pathol 1964;18:785-802.  Back to cited text no. 4
    
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6.
Heling I, Chandler NP. The antimicrobial effect within dentinal tubules of four root canal sealers. J Endod 1996;22:257-9.  Back to cited text no. 6
    
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Saleh IM, Ruyter IE, Haapasalo M, Ørstavik D. Survival of Enterococcus faecalis in infected dentinal tubules after root canal filling with different root canal sealers in vitro. Int Endod J 2004;37:193-8.  Back to cited text no. 7
    
8.
Cobankara FK, Altinöz HC, Ergani O, Kav K, Belli S.In vitro antibacterial activities of root-canal sealers by using two different methods. J Endod 2004;30:57-60.  Back to cited text no. 8
    
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Siqueira JF Jr., Rôças IN, Souto R, de Uzeda M, Colombo AP. Actinomyces species, streptococci, and Enterococcus faecalis in primary root canal infections. J Endod 2002;28:168-72.  Back to cited text no. 9
    
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Rôças IN, Siqueira JF Jr., Santos KR. Association of Enterococcus faecalis with different forms of periradicular diseases. J Endod 2004;30:315-20.  Back to cited text no. 11
    
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Kayaoglu G, Erten H, Østavik D. Growth at high pH increases Enterococcus faecalis adhesion to collagen. Int Endod J 2005;38:389-96.  Back to cited text no. 12
    
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Portenier I, Waltimo T, Haapasalo M. Enterococcus faecalis the root canal survivor and 'star' in post treatment disease. Endod Topics 2003;6:135-59.  Back to cited text no. 13
    
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Trope M, Delano EO, Orstavik D. Endodontic treatment of teeth with apical periodontitis: Single vs. multivisit treatment. J Endod 1999;25:345-50.  Back to cited text no. 14
    
15.
Sundqvist G, Figdor D, Persson S, Sjögren U. Microbiologic analysis of teeth with failed endodontic treatment and the outcome of conservative re-treatment. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1998;85:86-93.  Back to cited text no. 15
    
16.
Kayaoglu G, Erten H, Alaçam T, Ørstavik D. Short-term antibacterial activity of root canal sealers towards Enterococcus faecalis. Int Endod J 2005;38:483-8.  Back to cited text no. 16
    
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Weiss EI, Shalhav M, Fuss Z. Assessment of antibacterial activity of endodontic sealers by a direct contact test. Endod Dent Traumatol 1996;12:179-84.  Back to cited text no. 17
    
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Fuss Z, Weiss EI, Shalhav M. Antibacterial activity of calcium hydroxide-containing endodontic sealers on Enterococcus faecalis in vitro. Int Endod J 1997;30:397-402.  Back to cited text no. 18
    
19.
Neelakantan P, Subbarao CV. An analysis of the antimicrobial activity of ten root canal sealers – A duration based in vitro evaluation. J Clin Pediatr Dent 2008;33:117-22.  Back to cited text no. 19
    
20.
Slutzky-Goldberg I, Slutzky H, Solomonov M, Moshonov J, Weiss EI, Matalon S. Antibacterial properties of four endodontic sealers. J Endod 2008;34:735-8.  Back to cited text no. 20
    
21.
Sjögren U, Figdor D, Persson S, Sundqvist G. Influence of infection at the time of root filling on the outcome of endodontic treatment of teeth with apical periodontitis. Int Endod J 1997;30:297-306.  Back to cited text no. 21
    
22.
Bystrom A, Happonen RP, Sjogren U, Sundqvist G. Healing of periapical lesions of pulpless teeth after endodontic treatment with controlled asepsis. Endod Dent Traumatol 1987;3:58-63.  Back to cited text no. 22
    
23.
da Silva LA, Leonardo MR, da Silva RS, Assed S, Guimarães LF. Calcium hydroxide root canal sealers: Evaluation of pH, calcium ion concentration and conductivity. Int Endod J 1997;30:205-9.  Back to cited text no. 23
    
24.
Mickel AK, Nguyen TH, Chogle S. Antimicrobial activity of endodontic sealers on Enterococcus faecalis. J Endod 2003;29:257-8.  Back to cited text no. 24
    
25.
Gomes BP, Pedroso JA, Jacinto RC, Vianna ME, Ferraz CC, Zaia AA, et al. In vitro evaluation of the antimicrobial activity of five root canal sealers. Braz Dent J 2004;15:30-5.  Back to cited text no. 25
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]



 

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