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 Table of Contents  
ORIGINAL ARTICLE
Year : 2020  |  Volume : 32  |  Issue : 4  |  Page : 193-197

Elastic analysis of Gutta percha cones in 50 μg/ml and 80 μg/ml concentration of silver nanoparticles and 5.25% sodium hypochlorite by atomic force microscope: In vitro study


1 Priyesh Dental Clinic and Super Speciality Centre, Pratapgarh, India
2 Department of Conservative Dentistry and Endodontics, People's Dental Academy, Bhopal, Madhya Pradesh, India
3 Department of Orthodontics and Orthopedic Surgery, Babu Banarasi Das College of Dental Sciences, Lucknow, Uttar Pradesh, India

Date of Submission06-Jun-2020
Date of Decision20-Jul-2020
Date of Acceptance03-Nov-2020
Date of Web Publication18-Jan-2021

Correspondence Address:
Dr. Priyesh Mishra
Priyesh Dental Clinic and Super Speciality Centre, City Road, Meerabhawan, Pratapgarh - 230 001, Uttar Pradesh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/endo.endo_62_20

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  Abstract 


Introduction: Gutta percha (GP) should be sterilized and chemical sterilization is the most reliable method, but the surface topographical alteration is observed on GP; hence in this study, comparison and evaluation of the changes in modulus of elasticity of GP cones after disinfecting it with 5.25% sodium hypochlorite (NaOCl) and 50 μg/ml and 80 μg/ml concentration of silver nanoparticles (AgNPs) for three different durations were performed.
Materials and Methods: Ninety GP cones (Dentsply) were taken from the same batch and were randomly selected for the study. GP points were cut 3 mm from their tip and affix to a glass base with a rapid-setting cyanoacrylate glue. Then, the samples were divided into three treatment groups: Group 1, Group 2, and Group 3 comparison between 50 μg/ml and 80 μg/ml AgNPs and 5.25% NaOCl in 1, 5, and 10 min. Untreated GP points were used as control. The samples were positioned in the atomic force microscope (AFM). The AFM analyses were performed on 12 different regions located between 1 and 2 mm from the specimen's tip. One-way ANOVA was used for the statistical analysis.
Results: Group 1 – root mean square (RMS) value of 50 μg/ml in 1 min is 8.2, in 80 μg/ml is 8.0, and 5.25% is 6.4; Group II – RMS value of 50 μg/ml in 5 min is 7.94, 80 μg/ml is 7.11, and 5.25% is 5.34; Group III – RMS value of 50 μg/ml in 10 min is 6.38, 80 μg/ml is 6.24, and 5.25% is 4.82; and Group IV – untreated GP cones showed RMS value of 8.3. A statistically significant difference was found among groups.
Conclusion: NaOCl despite of being the ideal material for GP sterilization showed some flaws when it comes to surface topographical and physical changes. Changes are, A – NaOCl solution at 5.25% would increase the modulus of elasticity and left a numerous pitting on the surface of GP cones and B –where as AgNPs solution at 50 and 80 μg/ml did not significantly affect mechanical properties and surface texture of GP even after 10 min of disinfection exposure. Therefore, AgNPs can be seen as a safe disinfectant.

Keywords: Atomic force microscope, Gutta percha, modulus of elasticity, silver nanoparticles, sodium hypochlorite


How to cite this article:
Mishra P, Tyagi S, Tripathi D. Elastic analysis of Gutta percha cones in 50 μg/ml and 80 μg/ml concentration of silver nanoparticles and 5.25% sodium hypochlorite by atomic force microscope: In vitro study. Endodontology 2020;32:193-7

How to cite this URL:
Mishra P, Tyagi S, Tripathi D. Elastic analysis of Gutta percha cones in 50 μg/ml and 80 μg/ml concentration of silver nanoparticles and 5.25% sodium hypochlorite by atomic force microscope: In vitro study. Endodontology [serial online] 2020 [cited 2021 Mar 2];32:193-7. Available from: https://www.endodontologyonweb.org/text.asp?2020/32/4/193/307316




  Introduction Top


In clinical practice, the dentist is occasionally faced with the problem of re-infection after endodontic treatment.[1] One possible explanation for the reinfection may be the introduction of contaminated gutta percha (GP) cones into the root canal.[1] Therefore, much care must be taken during this procedure to prevent contamination of filling materials.[1],[2] Therefore, the handling of GP cones must follow the basic principles of infection control. In addition, GP cones that have been in contact with patients should be discarded.[1] Owing to the thermoplastic characteristic of GP cones, they may not be sterilized by the conventional process in which moist or dry heat is used because this may cause alteration to the GP structure.[2],[3],[4],[5]

Sodium hypochlorite (NaOCl) is one of the most widely used endodontic solutions, either as an irrigant or for rubber dam and cone decontamination. Its concentration ranges from 0.5% (Dakin solution) to 5.25%.[6],[7] The recommended method consists of treating the cones using a 5.25% sodium hypochlorite for 1 min (Milton's solution) or 3% sodium hypochlorite for 5 min (Dakin's solution).[8] However, sodium hypochlorite yields crystal deposition within the canals and might cause the dilapidation of GP points, including elevated depth of surface irregularities and loss of elasticity which can impede the obturation.[6] Therefore, the ideal disinfectant should be the one that can be used routinely in dental clinics, delivering a accelerated disinfection without altering the structure of the cone.[7]

Nanoparticles have gained popularity because of their broad spectrum of activity and biocompatibility.[8] Silver nanoparticle (AgNP) shows antibacterial effect; it also exhibits novel physicochemical and biological activities.[9] When AgNPs are present in a solution, they secrete a small amount of silver ions, which will have an additional contribution to the bactericidal effect of AgNPs.[9],[10] Its disinfection capability is well–documented,[9] but its effect on GP physical property has never been seen.

Today, the atomic force microscope (AFM) is the most commonly used scanning probe technique for materials characterization.[11] The major advantages of AFM are that it has a combination of high resolution in three dimensions, the sample does not have to be conductive, and there is no requirement for operation within a vacuum.

Hence, the intent of this article is to compare and evaluate the changes in the physical property (modulus of elasticity) of GP cones after disinfecting it with 50 μg/ml and 80 μg/ml concentration of AgNPs and 5.25% sodium hypochlorite at three different time durations.


  Materials and Methods Top


  1. GP cones – 90 cones were taken of size 80 (Dentsply Maillefer, Ballaigues, Switzerland)
  2. Two different concentrations of AgNPs – 50 μg/ml and 80 μg/ml (Banaras Hindu University, Varanasi, Uttar Pradesh, India)
  3. 5.25% sodium hypochlorite (HiMedia)
  4. The elastic analysis was done by AFM (MANIT, Bhopal, Madhya Pradesh, India).


Ninety GP cones (Dentsply Maillefer, Ballaigues, Switzerland) were taken from the same batch and were randomly selected for the study. It was made sure that all samples used were before the expiration date. GP points were dichotomized 3 mm from their tip and affix to a glass base with a rapid-setting cyanoacrylate glue. Following these procedures, the samples were divided into three treatment groups:

  • Group 1. Comparison between 50 μg/ml and 80 μg/mlAgNPs and 5.25% NaOCl in 1 min
  • Group 2. Comparison between 50 μg/ml and 80 μg/ml AgNPs and 5.25% NaOCl in 5 min
  • Group 3. Comparison between 50 μg/ml and 80 μg/ml AgNPs and 5.25% NaOCl in 10 min.


Untreated GP points were used as control. After cumulative treatment times of 1, 5, and 10 min for each solution, the samples were positioned in the AFM. The AFM analyses were performed on 12 different regions located between 1 and 2 mm from the specimen's tip. For each period of immersion, fresh aliquots (10 mL) of 5.25% NaOCl or AgNPs were used. The samples were thoroughly rinsed with 5 mL of Nano Pure water after the immersion and then dried with filter paper.

Analyses with atomic force microscope

Force modulation method

They are using the AFM for nanoindentation method to determine elastic properties like elastic modulus of GP cones samples. The Hertz model approximates the sample as an isotropic and linear solid occupying an indefinitely extending half-space. Furthermore, it is assumed that the indenter is nondeformable and there are no additional interactions between indenter and samples. The data obtained by indentation measurement (force spectroscopy method) are usually plots of force against piezo displacement. The Hertz model is only valid for small indentation, which may be 200–500 nm. The JPK IP software offers automatic fitting for all the indenter. The Hertz model assumes absolute elastic behavior as well as homogeneity of the sample. The energy delivers by the indenter is not completely given back by a substrate but dissipates owing to plastic behavior.

The cantilever was selected should be used depends on the stiffness of the sample. A spherical probe was used since the force was applied to a wider sample area, whereas pyramidal shape probe or conical probe measured only smaller area. In this way, the penetration of the GP was prevented. Spherical indenters were purchased from Novascan (0.6–25 um).

The microsphere probe was mounted and aligned as usual on the AFM head. The cantilever was then calibrated, i.e., the spring constant determined to be able to exactly specify the force to be applied to the sample. Using the NanoWizard, the calibration manager of the JPK SPM software leads through the calibration process; once the calibration is done, the desired set point force can be entered in Newtons (nano-Newtons). Now, the experiment was started.

For the purpose of comparison, the root mean square (RMS) was used to investigate the elastic analyses of the GP points. All statistical analyses were performed with Stat View for Windows 10 Software (SAS Institute, Cary, NC, USA). The mean and standard error of the mean values of the RMS parameters achieved from FMM was calculated.

Data analysis

Statistical analyses were performed on all data using a one-way ANOVA test.

The following experimental conditions were compared for significant differences:

  • Group 1. Comparison between 50 μg/ml and 80 μg/ml AgNPs and 5.25% NaOCl in 1 min,
  • Group 2. Comparison between 50 μg/ml and 80 μg/ml AgNPs and 5.25% NaOCl in 5 min and
  • Group 3. Comparison between 50 μg/ml and 80 μg/ml AgNPs and 5.25% NaOCl in 10 min.



  Results of Elastic Analyses Assessment Through Atomic Force Microscope Top


  • Group 1: [Table 1] shows the comparison between 50 μg/ml of AgNPs, 80 μg/ml of AgNPs, and 5.25% NaOCl in 1 min.
  • Table 1: The comparison between 50 ug/ml of silver nanoparticles, 80 ìg/ml of silver nanoparticles, and 5.25% sodium hypochlorite in 1 min

    Click here to view


    F-stat value was 110.2 with P = 0.

  • Group II: [Table 2] shows the comparison between 50 μg/ml of AgNPs, 80 μg/ml of AgNPs, and 5.25% NaOCl in 5 min.
  • Table 2: The comparison between 50 ug/ml of silver nanoparticles, 80 ìg/ml of silver nanoparticles, and 5.25% sodium hypochlorite in 5 min

    Click here to view


    F-stat value was 383.5 with P = 0,

  • Group III: [Table 3] shows the comparison between 50 μg/ml of AgNPs, 80 μg/ml of AgNPs, and 5.25% NaOCl in 10 min.
  • Table 3: The comparison between 50 ìg/ml of silver nanoparticles, 80 ìg/ml of silver nanoparticles, and 5.25% sodium hypochlorite in 1 min

    Click here to view


    F-stat value was 189.4 with P = 0,

  • Group IV – Untreated GP cones showed an RMS value of 8.3.


The composite image of disinfectants showing the RMS value in different time duration and since p value is very low, it also shows a statistically significant difference between 50 μg/ml and 80 μg/ml of AgNPs and 5.25% NaOCl.

Greater the value of RMS lesser the modulus of elasticity, indirectly decrease in percent of elongation and increase in tensile strength. In this study, as the duration increases the stiffness also increases; thereby, the probability of GP cone breakage is far more significant. Hence, in this study, it is clearly shown that 50 μg/ml AgNPs causes least amount of physical discrepancy of GP cones, followed by 80 μg/ml AgNPs as compared to 5.25% NaOCl in 1, 5, and 10 min.


  Discussion Top


The chemical composition of GP points varies, especially considering the proportions of GP and zinc oxide. This may lead to variations in brittleness, stiffness, tensile strength, and radiopacity,[12],[13],[14] and also in flow, plasticity, elongation, inherent tension force, and thermal behavior. Rigidity of GP cones upsurges with high percentage of inorganic components and low percentage of GP so it is very important to have accurate proportion of inorganic part and gutta percha in a cone to provide sufficient amount of elasticity and stiffness for obturation procedure.[15] GP cones should present at least 17% of GP and no more than 3% of wax/resin (Dentsply FM and Dentsply TP, 10.4 ± 0.11 and 4.0 ± 0.36, respectively), enabling a good thermal behavior during warm root canal filling. Hence, it is the reason why Dentsply GP cones were chosen for the present study.

The reason behind alteration in elasticity is unclear, but it is observed that GP contains trans 1,4-Polyisoprene (PI) component of GP cones which is 14.5–21.8%[16] and NaOCl in larger concentration and that too for longer period distort saturated bond of trans PI. NaOCl in higher concentration causes reduction of polymer component which leads to decrease in the tensile strength and since the polymer chain component gets reduced zinc oxide, it becomes the main component. NaOCl in higher concentration causes reduction of polymer chain component which leads to decrease in the tensile strength and since the polymer chain component gets reduced ZnO becomes the main component and because of the increase of ZnO proportion rigidity increases as ZnO is responsible for the increase in modulus of elasticity (increase stiffness) of the cone. Hence, this might increase the modulus of elasticity (increase stiffness) of the cone. Furthermore, increase on the rigidity will lead to a decrease on the elongation rate percentage of the cone. Hence, NaOCl at a high concentration of 5.25% would decrease tensile strength and increase elasticity.

Deterioration of GP points includes increased depth of surface irregularities and elasticity. Deep irregularities can create large interfacial gaps between the GP cone and the root canal wall, increasing the risk of leakage,[17] whereas increased modulus of elasticity could be associated with an increased tendency of GP points to fracture because of an increase in brittleness.

It has been observed that treating the cones using a 5.25% sodium hypochlorite for 1 min (Milton's solution) or 3% sodium hypochlorite for 5 min (Dakin's solution)[8] provides excellent removal of microbes from GP cones, but sodium hypochlorite also yields crystal deposition on cones along with surface irregularities and loss of physical properties which can hamper the proper bonding of resin sealer with GP.[6] Hence, it can be deduced that though NaOCl is an excellent material for GP cone disinfection because of its surface altering behavior, it can not be considered ideal material.

In the present study, the effects of two concentrations of AgNPs were compared and analyzed, i.e., 50 μg/ml and 80 μg/ml of AgNPs and 5.25% NaOCl on physical properties of the GP cone was investigated and compared by AFM. One of the primary attractions to the AFM is its ability to image insulating surfaces at high resolution in fluid. AFM also provides images three dimensionally.

The FMM parameter for AgNPs showed a similar average of elasticity for all evaluated immersion times and to untreated specimens. The elasticity results obtained for GP points suggest that 50 μg/ml and 80 μg/ml of AgNPs is a safe procedure during decontamination up to 10 min of exposure.

The extreme changes in AFM topographic features after 10 min of exposure to 5.25% NaOCl solution indicated aggressive deterioration of GP cone when compared with AgNPs, suggesting that AgNP is less prejudicial to GP structure. However, the clinical application of 5.25% NaOCl is usually for a much shorter time (1 min), and in the present study, it is clearly observed even 1-min exposure of sodium hypochlorite caused extreme damage to GP cones. Nevertheless, the rapid changes in the elastic properties of GP brought about by 5.25% NaOCl treatment imply that the use of AgNPs may be a safer alternative during root canal treatment.


  Conclusion Top


  1. AFM was done to investigate the change in modulus of elasticity in GP cones after disinfection with NaOCl and AgNPs, and it was found that there were significant changes which were observed in NaOCl. A statistically significant difference was observed in NaOCl when compared to untreated GP cones, although there was no significant change seen in AgNPs in 1 min compared to untreated GP cone.
  2. A. NaOCl solution at 5.25% would decrease tensile strength, increase the modulus of elasticity, decrease the percentage of elongation, and left numerous pitting on the surface of GP cones. Hence, it must not to be used for GP disinfection. B.- Solution at 50 and 80 μg/ml AgNP solution could not effect mechanical properties and surface texture of GP even after 10 min of disinfection. Therefore, they were considered as the safe disinfectant solutions for GP cones.


Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Dumani A, Yoldas O, Isci AS, Köksal F, Kayar B, Polat E. Disinfection of artificially contaminated resilon cones with chlorhexidine and sodium hypochlorite at different time exposures. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2007;103:82-5.  Back to cited text no. 1
    
2.
Ozalp N, Okte Z, Ozcelik B. The rapid sterilization of gutta-percha cones with sodium hypochlorite and glutaraldehyde. J Endod 2006;32:1202-4.  Back to cited text no. 2
    
3.
Redmerski R, Bulla JR, Moreno T, Garcia LB, Cardoso CL. Disinfection of gutta-percha cones with chlorhexidine. Braz J Microbiol 2007;38:649-55.  Back to cited text no. 3
    
4.
de Souza RE, de Souza EA, Sousa-Neto MD, Pietro RC. In vitro evaluation of different chemical agents for the decontamination of gutta-percha cones. Pesqui Odontol Bras 2003;17:75-7.  Back to cited text no. 4
    
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7.
da Motta PG, de Figueiredo CB, Maltos SM, Nicoli JR, Ribeiro Sobrinho AP, Maltos KL, et al. Efficacy of chemical sterilization and storage conditions of gutta-percha cones. Int Endod J 2001;34:435-9.  Back to cited text no. 7
    
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Neal AL. What can be inferred from bacterium-nanoparticle interactions about the potential consequences of environmental exposure to nanoparticles? Ecotoxicology 2008;17:362-71.  Back to cited text no. 8
    
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Kishen A, Shi Z, Shrestha A, Neoh KG. An investigation on the antibacterial and antibiofilm efficacy of cationic nanoparticulates for root canal disinfection. J Endod 2008;34:1515-20.  Back to cited text no. 9
    
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Liu HB, Ascencio JA, Alvarez PM, Yacaman MJ. Melting behavior of nanometer sized gold isomers. J Surf Sci 2001;491:88-98.  Back to cited text no. 10
    
11.
Yacamán MJ, Ascencio JA, Liu HB, Torresdey JG. Structure shape and stability of nanometric sized particles. J Vac Sci Technol 2001;19:109.  Back to cited text no. 11
    
12.
Friedman CE, Sandik JL, Heuer MA, Rapp GW. Composition and physical propertiesof gutta-percha endodontic filling materials. J Endod 1977;3:304.  Back to cited text no. 12
    
13.
Goldberg F, Massone EJ, Pruskin E, Zmener O. SEM study of surface architecture of gutta-percha cones. Endod Dent Traumatol 1991;7:15-8.  Back to cited text no. 13
    
14.
Ferreira MC, Valverde GB, Silva JB Jr, de Paula RC, Feitosa JP, de Souza-Filho FJ. Clinical relevance of trans 1,4-polyisoprene aging degradation on the longevity of root canal treatment. Braz Dent J 2007;18:97-101.  Back to cited text no. 14
    
15.
Gurgel-Filho ED, Andrade Feitosa JP, Teixeira FB, Monteiro de Paula RC, Araújo Silva JB, Souza-Filho FJ. Chemical and X-ray analyses of five brands of dental gutta-percha cone. Int Endod J 2003;36:302-7.  Back to cited text no. 15
    
16.
Chandra SS, Miglani R, Srinivasan MR, Indira R. Antifungal efficacy of 5.25% sodium hypochlorite, 2% chlorhexidine gluconate, and 17% EDTA with and without an antifungal agent. J Endod 2010;36:675-8.  Back to cited text no. 16
    
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Tagger M, Gold A. Flow of various brands of Gutta-percha cones under in vitro thermomechanical compaction. J Endod 1988;14:115-20.  Back to cited text no. 17
    



 
 
    Tables

  [Table 1], [Table 2], [Table 3]



 

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