Log in Register

Login to your account

Username *
Password *
Remember Me

Create an account

Fields marked with an asterisk (*) are required.
Name *
Username *
Password *
Verify password *
Email *
Verify email *
Captcha *

Captcha Image Reload image challenge



Authors : Dr. Niladri Maiti Dr. Utpal Kumar Das.

Abstract :

Microorganisms invading the root canal system of a tooth may interact with the host tissue and cause pulpo-periapical pathosis.The goals of root canal treatment are to disinfect the root canal system and to prevent subsequent re-infection.The disinfection is attempted with endodontic instruments,irrigants and medications.

Due to the complex canal anatomy ,microorganisms can persist even after thorough disinfection regimens are used.After disinfection,the canal is sealed with a root filling material along with sealers.To curtail residual microorganisms,root-filling materials should ideally be bactericidal ,as well as biocompatible.

In addition,the root-filling materials are expected to act as a physical barrier to prevent the leakage of substrate to any residual microorganisms and the ingress of additional microorganisms.The aim of the present study was to assess the antimicrobial activity of various Endodontic sealers on the Enterococcus faecalis.Enterococcus faecalis is a microorganism commonly detected in asymptomatic,persistent endodontic infections.In the changing face of dental care,continued research on E.faecalisand its elimination from the dental apparatus may well define the future of the endodontic speciality.

INTRODUCTION

Successful root canal treatment requires proper cleaning and shaping of the root canal system, as well as hermetic sealing of the canal space with an inert, dimensionally stable and biologically compatible material.1 Therefore the search has continued for an endodontic sealer that fulfills the requirements for the ideal physicochemical and biological properties.

Microbes and their products are the main etiologic factors of root canal diseases2. Therefore, the main objective of endodontic therapy is the elimination of microorganisms from the root canal system and the prevention of subsequent re-infection 3. However, mechanical root canal instrumentation, antibacterial irrigation, adequate filling of the root canal space, and use of inter-appointment antimicrobial dressing do not result in complete disinfection of root canal space 4. Grossman advocated that ideal root canal filling material should be bacteriostatic 5.Therefore, antimicrobial activity of endodontic filling materials such as sealers play an important role in reducing or avoiding growth of these remaining microorganisms and therefore improve the success rate of endodontic treatment.

Microorganisms infecting the root canal dentine might adhere superficially to the dentinal wall or penetrate deeper into the dentinal tubules6.Enterococcus faecalis is a resilient bacterium frequently recovered from obturated root canals with signs of apical periodontitis7.The presence of Enterococcus faecalis at the time of obturation can significantly reduce the success rate of root canal treatment. When established in the dentinal tubules,it is difficult to eliminate this species through root canal medication. Therefore,it might be advantageous if the sealer exerts some antimicrobial activity as the last element in the treatment regimen.

The purpose of this study was to evaluate the antimicrobial activity of four different sealers based on zinc oxide-eugenol , calcium hydroxide, silicon and MTA, on Enterococcusfaecalis most commonly isolated in failed root canal cases.

MATERIALS AND METHODS

I)Preparation of the medium for Enterococcus faecalis

  • The strains of microorganisms used for the study were standard strains of Enterococcus faecalis which were obtained from ATCC29212 and were subcultured in blood agar plate and were incubated at 370C for 24 hours.
  • A pure,single Enterococcus faecalis colony was isolated from the same cultured plate and Gram’s staining was done to confirm its growth, observed under microscope and then inoculated with a Brain Heart Infusion (BHI) broth.
  • The BHI broth was incubated at 370C for a 24-hour period and checked for bacterial growth by changes in turbidity.
  • A drop of BHI containingEnterococcus faecalis was placed into saline solution and checked for correct bacterial concentration with a spectrophotometer.
  • The density of the bacterial suspension is standardized by comparing the broth at a density equivalent to the barium sulphate standard of 0.5 McFarland units,which is equivalent to 1.5x108 colony forming units per millilitre(CFU/ml).

II)Preparation of the disks

  • Fifteen blood agar plates (15x100mm) were prepared for inoculation.
  • The plates were incubated at 370C in ambient atmosphere for 24 hours to check any external contamination.
  • These plates were inoculated with prepared E. faecalis suspension by evenly swabbing the plates with a sterile cotton swab to obtain a lawn culture.
  • Amoxiclav disks (10 micrograms)were used as the control.
  • The filter paper disks (Whatman no.1) were standardized to 3mm in diameter.
  • The sealers were manipulated according to manufacturer’s recommendation to get homogeneous consistency using a glass slab and spatula or paper mixing pad and agate spatula.
  • Sterile filter paper disks soaked in sealer(Fig.no 1) with the help of sterile forceps and pressed gently to ensure even contact with the medium.

  • Fig.no.1 Sealer soaked filter paper

  • Around 100 microliter (0.1ml) of each sealer is placed with the help of micropipettes.
  • The plates containing the sealer impregnated disks along with control disks were kept for incubation at 370C in ambient atmosphere for 24 hours period(agar diffusion method).
  • The Petri dishes containing the sealer impregnated disks along with the microorganisms namely Enterococcus faecalis were incubated for 48 hours at 370C in an incubator.
  • Zones of inhibition were measured at the end of 24 hours and 48 hours period.(Table no.1,2)

  • TABLE

    Sealers

    Specimens

     

    1

    2

    3

    4

    5

    6

    7

    8

    9

    10

    11

    12

    13

    14

    15

    Endofill

    1

    2

    2

    2

    1

    1

    1

    2

    1

    2

    2

    2

    1

    2

    1

    MTAFilapex

    1

    0.5

    0.5

    0.5

    0.5

    1

    0.5

    0.5

    0.5

    0.5

    0.5

    0.5

    0.5

    0.2

    0.5

    Roekoseal

    0

    0

    0

    0

    0

    0

    0

    0

    0

    0

    0

    0

    0

    0

    0

    Apexit plus

    0.5

    0.5

    0.2

    0.2

    0.5

    0.5

    0.2

    0.2

    0.2

    0.2

    0.5

    0.5

    0.5

    0.2

    0.2

    Amoxiclav

    8

    7

    8

    8

    8

    7

    7

    8

    7

    8

    8

    8

    7

    8

    7

    Table 1 : Zone of inhibition measured after 24 hours(measured in mm)


    Sealers

    Specimens

    1

    2

    3

    4

    5

    6

    7

    8

    9

    10

    11

    12

    13

    14

    15

    Endofill

    1

    2

    2

    2

    1

    1

    1

    2

    1

    2

    2

    2

    1

    2

    1

    MTAFilapex

    1

    0.5

    0.5

    0.5

    0.5

    1

    0.5

    0.5

    0.5

    0.5

    0.5

    0.5

    0.5

    0.2

    0.5

    Roekoseal

    0

    0

    0

    0

    0

    0

    0

    0

    0

    0

    0

    0

    0

    0

    0

    Apexit plus

    0.5

    0.5

    0.2

    0.2

    0.5

    0.5

    0.2

    0.2

    0.2

    0.2

    0.5

    0.5

    0.5

    0.2

    0.2

    Amoxiclav

    8

    7

    8

    8

    8

    7

    7

    8

    7

    8

    8

    8

    7

    8

    7

    Table 2 : Zone of inhibition measured after 48 hours(measured in mm)

  • The diameter of the zone of inhibition of growth was measured in millimetres with the help of an inhibition zone measuring scale and the values recorded.(Table no.3)

  • Sealers

    Mean (±SD)

    Endofill

    1.53(±0.5164)

    MTAFilapex

    0.54(±0.1995)

    Roekoseal

    0.00

    Apexit plus

    0.34(±0.1549)

    Amoxiclav

    7.6(±0.5070)

    Table 3: Mean values

  • The point of abrupt diminution of growth,which corresponds to the point of complete inhibition of growth,is taken as the zone edge.
  • Sealers used in the study were Endofill , MTA-Filapex, Apexit Plus and Roekoseal. (Fig.no. 2)

  • Fig.no.2 Endodontic Sealers

RESULTS

The results were tabulated after 24 and 48 hours respectively and statistically analyzed by Kruskal-Wallis one-way ANOVA used to calculate the p-value and Mann-Whitney U-test was used to identify the significant groups at 5% level after correcting the p-values for comparison by Bonferroni correction method. (Table no.4)

Mean difference95% confidence interval

Sealers(I)

Sealers(J)

(I-J)

Std.error

Sig.

Lower bound

Upper  bound

 

Tukey HSD

Endofill

MTAFilapex

1.0824*

0.1182

0.000

0.7524

1.4123

 

Roekoseal

1.5882*

0.1182

0.000

1.2583

1.9182

Apexit plus

1.2294*

0.1182

0.000

0.8995

1.5594

Amoxyclav

-5.9412*

0.1182

0.000

-6.2711

-5.6112

MTAFilapex

Endofill

-1.0824*

0.1182

0.000

-1.4123

-0.7524

Roekoseal

0.5059*

0.1182

0.000

0.1759

0.8358

Apexit plus

0.1471

0.1182

0.726

-0.1829

0.4770

Amoxyclav

-7.0235*

0.1182

0.000

-7.3535

-6.6936

Roekoseal

Endofill

-1.5882*

0.1182

0.000

-1.9182

-1.2583

MTAFilapex

-0.5059*

0.1182

0.000

-0.8358

-0.1750

Apexit plus

-0.3588*

0.1182

0.026

-0.6888

-2.8874E-02

Amoxyclav

-7.5294*

0.1182

0.000

-7.8594

-7.1995

Apexit plus

Endofill

-1.2294*

0.1182

0.000

-1.5594

-0.8995

MTAFilapex

-0.1471

0.1182

0.726

-0.4770

0.1829

Roekoseal

0.3588*

0.1182

0.026

2.887E-02

0.6888

Amoxyclav

-7.1706*

0.1182

0.000

-7.5005

-6.8406

Amoxyclav

Endofill

5.9412*

0.1182

0.000

5.6112

6.2711

MTAFilapex

7.0235*

0.1182

0.000

6.6936

7.3535

Roekoseal

7.5294*

0.1182

0.000

7.1995

7.8594

Apexit plus

7.1706*

0.1182

0.000

6.8406

7.5005

Dunnet t(2-sided) a

Endofill

Amoxyclav

-5.9412*

0.1182

0.000

-6.2357

-5.6466

 

MTAFilapex

Amoxyclav

-7.0235*

0.1182

0.000

-7.3181

-6.7290

Roekoseal

Amoxyclav

-7.5294*

0.1182

0.000

-7.8240

-7.2349

Apexit plus

Amoxyclav

-7.1706*

0.1182

0.000

-7.4651

-6.8760

The mean difference is significant at the 0.05 level
A Dunnet t-tests treat one group as a control and compare all other groups against it
Table 4: Multiple comparisons Dependent variable: Inhibition zone


Graphs were plotted presenting mean values of sealers at 24 and 48 hours.(Graph no.1)

GRAPH

Graph no.1


The disk impregnated with the sealer,which exhibited the maximum zone of inhibition was considered as having the most efficient antimicrobial activity.

DISCUSSION

Chemo-mechanical preparation is of paramount importance in successful endodontic treatment.However,this does not negate the importance of the quality of the obturation,in which the sealer has a role to play.According to Grossman,a requirement and characteristic of a sealer should be bacteriostatic or at least not encourage bacterial growth.8

The most common reason for the failures in conservative root canal therapy are problems in instrumentation,however,occasionally bacteria resistant to conservative therapy of good quality may also be involved.9 Hence,a three-dimensional seal with the antimicrobial property of the sealer is critical for endodontic success.

It is generally believed that the significant cause of root canal treatment failure is the persistence of microorganisms in the apical third of root filled teeth.10 Approximately,apical one-third of the canals of root filled teeth with persistent periapical lesions have shown high proportion of Enterococcus faecalis.11.Significant increase in Enterococcus faecaliscontamination in retreatment cases has been shown as they gain entry into the canal during root canal treatment,due to poor temporary seals between appointments or inadequate aseptic techniques.9

And other probable reasons for the isolation of Enterococcus faecalis in failed root canal treated teeth may be due its survive even with scant amounts of substrate and without the support of other microorganisms,and grow to establish monoinfections.

Traditionally, agar diffusion method and agar dilution method are commonly employed for detecting antimicrobial susceptibility. In this study, Kirby-Bauer method(agar disk diffusion method)was chosen instead of agar dilution method.The disadvantage of the agar dilution method is that this technique can alter some of the properties of the sealers being tested.

Moreover, some sealers cannot be homogeneously dissolved and is a difficult and slow technique. Hence , the agar disk diffusion method was used in this method.The chemical properties of the sealers are not changed and the antimicrobial resistance can be detected by challenging bacterial isolates with antimicrobial disks.Moreover,this is an easy and less technique-sensitive method.12

Amoxyclav was chosen as the control against E. faecalisas it is a potent bactericidal causing lysis of the bacterial cell wall.

Among the test groups ,Endofill showed statistically significant antimicrobial efficacy against E.faecalis.

A sealer with an antimicrobial activity can be considered advantageous in order to eliminate the remaining microbes present in the root canal after chemo-mechanical preparation of the root canal system and to prevent reinfection.

Zinc-oxide eugenol-based sealers have been traditionally the most commonly employed sealants.They have served as the benchmark with which other sealers are compared , as it reasonably meets most of Grossman’s requirements for sealers.

Luebke and Ingle in 1976 forecast a new paradigm for endodontics involving a broader use of calcium hydroxide in medicating and sealing the root canal.

This led to the introduction of several calcium hydroxide-based sealers namely Metapex , Sealapex , Apexit and Apexitplus.The calcium hydroxide-based sealer, Apexit plus showed zones of inhibition against E. faecalis,which was statistically lesser than the zones of inhibition produced by both Endofill and MTA based sealers.

Esterela et al hypothesized that in calcium hydroxide,the antimicrobial mechanism is influenced by its speed of dissociation into calcium ions and hydroxyl ions.13

This dissociation into hydroxyl ions creates a high pH environment , which raise the pH to above 12.5which inhibits enzymatic activities that are essential for microbial metabolism , growth and cellular division. As the calcium hydroxide sealer sets, the pH declines to about 9.14, resulting in loss of the sealer’s effectiveness.14

The use of materials that provide high alkalinity favours hard tissue mineralization as well as offers good antimicrobial activity. Accordingly, MTA-based sealers present alkaline pH, high calcium ion releasing [15] and adequate biocompatibility 16.

A MTA endodontic sealer (MTA Fillapex, Angelus SoluçõesOdontológicas, Londrina, PR, Brazil) was recently created.

According to the manufacturer, its composition after mixture is basically MTA, salicylate resin, natural resin, bismuth and silica. To date, there is a lack of studies evaluating the material, concerning to its physico-chemical ,biological and antimicrobial properties.

Kuga et al.(2011) Showed At the 14th day of assessment, MTA Fillapex’s calcium releasing was similar to white MTA, but lower than gray MTA.MTA Fillapex showed a tendency towards maintaining the calcium releasing relatively constant until 14 days.17

Endofill, is a zinc oxide-eugenol-based sealer, and has shown good antibacterial activity when compared with calcium hydroxide-based sealers.18 According to the results of this study the Endofill had the greatest antimicrobial effect against E.faecalis.

The Eugenol is a potent antimicrobial agent, and therefore, the activity of ZOE- based sealers may be attributable to the free eugenol released from the set materials. 19

Eugenol ,a phenolic compound acts on microorganisms by protein denaturation whereby the protein becomes non-functional.The antimicrobial effect of zinc oxide eugenol sealers can be gauged by the results of the following studies.

Andre Mickel et al found that zinc oxide eugenol-based sealant exhibited larger zone of inhibition against E. faecalis when compared with calcium hydroxide-based sealer Metapex and epoxy resin-based sealer AH Plus and AH26.20

The result of the present study are consistent with the Gomes et al. (2004) study, in that the ZOE-based sealer such as Endofill demonstrated the highest antimicrobial activity than epoxy resin-based sealers.21

RoekoSeal is a silicon-based root canal sealer. In the present study RoekoSeal did not exhibit any bacterial growth inhibition. These results are only in partial accordance with the findings of Cobankara et al , who tested the antibacterial properties of RoekoSeal by using both ADT and DCT; they showed that in the ADT, RoekoSeal had no antibacterial activity, whereas other sealers, including AH plus, did inhibit bacterial growth. 22

CONCLUSION

  1. Endofill ,zinc oxide –based sealer showed significantly greater antimicrobial effect against E.faecalis.
  2. MTAFilapex , MTA -based sealer showed significantly greater( but less than Endofill ) antimicrobial effect against E.faecalis
  3. There was no significant difference between the antimicrobial activity of RoekoSeal and Apexit Plus sealer on E. faecalis.
  4. Apexitplus,a calcium hydroxide-based sealer is less effective against E. faecalis.
  5. RoekoSeal , a silicon-based sealer showed no antimicrobial activity on E. faecalis.
  6. There was no difference in the zones of inhibition between 24 hours and 48 hours time periods.

REFERENCES

  1. ScarparoRK, GreccaFS ,Fachin EV. Analysis of tissue reactions to methacrylate resin-based, epoxy resin-based, and zinc oxide-eugenol endodontic sealers. J. Endod2009., 35: 229-232.
  2. BergenholtzG. Micro-organisms from necrotic pulp of traumatized teeth. Odontol. Revy1974., 25: 347-358.
  3. Pizzo G, Giammanco GM, CumboE, NicolosiG ,Gallina G . In vitro antibacterial activity of endodontic sealers. J Dent. 2006, 34: 35-40.
  4. Abdulkader A, Duguid R, Saunders EM . The antimicrobial activity of endodontic sealers to anaerobic bacteria. Int. Endod. J1996., 29: 280-283.
  5. Grossman L . Antimicrobial effect of root canal cements. J. Endod1980., 6: 594-597.
  6. 4.Peters LB,WesselinkPR,BuijsJF,vanWinkelhoff AJ. Viable bacteria in root dentinal tubules of teeth with apical periodontitis.Journal of Endodontics 2001;27:76-81.
  7. SundqvistG,FigdorD,PerssonS,SjogrenU.Microbiologic analysis of teeth with failed endodontic treatment and the outcome of conservative retreatment.OralSurgery,OralMedicine,Oral Pathology Oral Radiology and Endodontics 1998;85:86-93.
  8. Grossman LI.Endodonticpractice(10thed).1982,297.
  9. Siren EK,HaapasaloMPP,RantaK,SalmiP,KerosuoENJ.Microbiological findings and clinical treatment procedures in endodontic cases selected for microbiological investigation.International Endodontic Journal 1997;30:91-95.
  10. Evans M,DaviesJK,SundqvistG,FigdorD.Mechanisms involved in the resistance of Enterococcus faecalis to calcium hydroxide.International Endodontic Journal 2002;35:221-28.
More references are availabe on request.

Add comment


Security code
Refresh