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Journal of Indian Society of Pedodontics and Preventive Dentistry Official publication of Indian Society of Pedodontics and Preventive Dentistry
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Year : 2020  |  Volume : 38  |  Issue : 4  |  Page : 374-380

Comparative evaluation of the efficacy of different herbal irrigants on the removal of smear layer of primary teeth: A scanning electron microscopy study

1 Department of Pedodontics and Preventive Dentistry, People's College of Dental Sciences and Research, Bhopal, Madhya Pradesh, India
2 Department of Pedodontics and Preventive Dentistry, Index Dental College, Indore, Madhya Pradesh, India
3 Department of Pedodontics and Preventive Dentistry, Bankey Bihari Dental College and Research Center, Ghaziabad, Uttar Pradesh, India
4 Department of Pedodontics and Preventive Dentistry, Lincoln University College, PJ, Malaysia
5 Department of Pedodontics and Preventive Dentistry, Mansarover Dental College, Bhopal, Madhya Pradesh, India

Date of Submission14-Jul-2020
Date of Decision17-Aug-2020
Date of Acceptance02-Sep-2020
Date of Web Publication5-Jan-2021

Correspondence Address:
Dr. Parimala Tyagi
Department of Pedodontics and Preventive Dentistry, People's College of Dental Sciences and Research, Bhopal - 462 010, Madhya Pradesh
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/JISPPD.JISPPD_315_20

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Aim and Objectives: The aim and objectives of this study are to evaluate and compare the effectiveness of Myristica fragrans – Nutmeg, Terminalia chebula – Myrobolan, Ocimum sanctum-tulsi, and 2.5% sodium hypochlorite (NaOCl) on the removal of the smear layer by the scanning electron microscope (SEM). Settings and Design: Endodontic treatment aims at disinfection and then obturation of root canal system in to prevent re-infection. Root canal irrigants play a pivotal role in the disinfection process. One of the important properties of an irrigant is the removal of complete smear layer and debris. Smear layer has the potential to protect bacteria within the dentinal tubules; therefore removal may be prudent. Smear layer removal increases the bond strength of resin sealers which results in better apical seal. Materials and Methods: Forty extracted single-rooted, primary teeth were allocated randomly into four groups of ten each: Group 1 – NaOCl, Group 2 – Nutmeg, Group 3 – Myrobolan, and Group 4 – Tulsi. Samples were stored in sterile saline (0.9% NaCl) and then decoronated at the level of the cementoenamel junction. Working length was determined followed by appropriate irrigation. The roots were split into two halves with a chisel and were stored in 2.5% glutaraldehyde solution for 24 h. After fixation, the samples were dehydrated in ethanol series (70, 90, and 95 and twice at 100%). Each specimen was mounted on Al stub and sputter coated with a 20 nm layer of gold. Samples were then examined using a SEM quantum 60 at magnification of ×2000. Results: Tulsi demonstrated the most statistically significant results followed by myrobolan and nutmeg extract. All herbal extracts were found to be significantly effective than 2.5% NaOCl. Conclusion: Tulsi, nutmeg and myrobolan can be effectively used as an irrigant in primary teeth.

Keywords: Myrobolan, nutmeg, primary teeth, pulpectomy, smear layer, sodium hypochlorite, tulsi

How to cite this article:
Mali S, Singla S, Tyagi P, Sharma A, Talreja N, Gautam A. Comparative evaluation of the efficacy of different herbal irrigants on the removal of smear layer of primary teeth: A scanning electron microscopy study. J Indian Soc Pedod Prev Dent 2020;38:374-80

How to cite this URL:
Mali S, Singla S, Tyagi P, Sharma A, Talreja N, Gautam A. Comparative evaluation of the efficacy of different herbal irrigants on the removal of smear layer of primary teeth: A scanning electron microscopy study. J Indian Soc Pedod Prev Dent [serial online] 2020 [cited 2022 Dec 5];38:374-80. Available from: http://www.jisppd.com/text.asp?2020/38/4/374/306218

   Introduction Top

The preservation of integrity of primary dentition is the most important aspect of preventive dentistry, and it is vital to retain the primary dentition until its normal exfoliation, as it is essential for the normal growth of facial-skeletal complex. One of the common reasons for the premature loss of primary teeth is dental caries and an alternative to avoid such loss would be an endodontic treatment, in which the main goal is to preserve the teeth in the same position. A clean root canal system along with a three-dimensional seal is the clinician's path to success for maintaining the primary tooth in the arch. Irrigation plays a main role in complete debridement and disinfection of the root canal preparation of primary teeth since there is a formation of dentinal shavings composing of organic materials and microorganisms forming the smear layer. It consists of organic and inorganic components which covers the root canal surfaces and blocks the dentinal tubules openings.[1] The inorganic material is composed of tooth structure and some nonspecific inorganic contaminants, whereas the organic components consist of heat coagulated proteins; necrotic or viable pulp tissue, and odontoblastic processes plus saliva, blood cells, and microorganisms. It harbors microorganism's that infect dentinal tubules, impedes penetration or diffusion of antibacterial irrigants and medicaments into the dentinal tubules and compromises the seal between the filling materials and the dentinal wall.[2] Because of its potential contamination and adverse effects on the outcome of root canal treatment, smear layer removal is recommended.

The most widely used irrigant for root canal treatment is sodium hypochlorite (NaOCl) at concentrations of 0.5%–5.25%. The tissue-dissolving capacity and microbicidal activity of NaOCl make it an excellent irrigating solution.[3] It has some undesirable characteristics such as tissue toxicity, allergic potential, and disagreeable smell and taste and also causes serious damage when allowed to enter periradicular tissues even in small amounts.[4] Hence, to reduce these effects, especially taste and smell, which is very important aspect for pediatric patients, plant products are assayed for their antibacterial properties and potential to smear layer owing to the fact that they have minimal side effects.

Herbal products and there derivatives as root canal irrigants are gaining popularity owing to their desirable properties. Natural extracts such as Myristica fragrans-Nutmeg, Terminalia chebula – Myrobolan, and Ocimum sanctum – Tulsi reported to exert antimicrobial, anti-inflammatory, and antioxidant properties.[5] Tulsi/Basil (O. sanctum), a sacred plant in India have several medicinal properties and its extracts showed almost 100% inhibition of growth of  Escherichia More Details coli.[6] The main constituents of Nutmeghave been found to be alkyl benzene derivatives (myristicin, elemicin, safrole, etc.,), terpenes, alpha-pinene, beta-pinene, myristic acid,[7] trimyristin, neolignan (myrislignan), and macelignan. It has strong anticariogenic activity, possessed antibacterial effect against oral microorganisms such as Streptococcus species, and Lactobacillus species, and exhibited weak activity for Actinomyces viscosus, Porphyromonas gingivalis, and Staphylococcus aureus.[8] It is widely distributed throughout India, Burma, and Sri lanka. They have antioxidant and free radical scavenging activities.[9] It is useful as an anticaries agent[10] and both aqueous and ethanolic extract of Nutmeg have strong antimicrobial activity against the uropathogen E. coli. The role of all the above-mentioned natural extracts for endodontic purpose in the smear layer removal is still questionable and to be evaluated. Therefore, validating the need for this study, where we aim to assess and compare the efficacy of NaOCl 2.5% (gold standard), and herbal extracts such as Nutmeg, Myrobolan, and Tulsi, in removal of the smear layer and debris from the root canal walls.

   Materials and Methods Top

The present study was conducted in the department of pediatric dentistry, and it was conducted in two parts. In vitro herbal irrigant preparation and effect of irrigants on the removal of smear layer of primary teeth by the scanning electron microscopy. The ethical clearance of the study was obtained from the institutional review board, and informed consent was obtained from the parents of the participants.

Preparation of extracts

Seeds of nutmeg, myrobolan, and Tulsi were subjected to shade drying and grounded into fine powder. 45 g of powder were than subjected to Soxhlet extraction with 90% ethanol as solvent. Soxhletion process was allowed to carry out till the complete exhaustion of sample material use for extraction with the maintenance of temperature 70°C the boiling points of the solvents used. After complete phytochemical exhaustion, the extract in the round bottom flask was transferred into clean and preweighed universal tubes. The tubes were then stored at 4–8°C in the refrigerator. Percentage yield was calculated as dividing the initial weight of raw material taken by the final weight of extracts.

Where n indicates the final weight of nutmeg, myrobolan, tulsi extract.

Minimal inhibitory concentration determination for extracts

One gram of ethanolic extract of Tulsi, nutmeg and myrobolan was dissolved in 10 ml of distilled water separately, in this way an extract concentration of 100 mg per ml was obtained afterward each stock extract as passed from 0.22μ syringe filter to get sterile solution. To prepare 5 dilutions of Tulsi extract, 0.5 ml of extract was taken in sterile micro-tube, which was then diluted with distilled water at a ratio of 1:1, 1:2, 1:3, 1:4, 1:5 similar dilution was done for nutmeg and myrobolan extract, this yielded serially diluted concentration of 100, 50, 25, 12.5 and 6.25 mg/ml for each extract. Broth cultures of the pure culture isolates of Enterococcus faecalis were prepared by transferring a loop of culture into sterile nutrient broth and incubated at 37°C for 48 h. From the prepared broth culture of pure bacterial stains, with the help of a sterile cotton swab inoculum was taken and seeded onto sterile Muller-Hinton agar plates and leaving it for 5 min before punching holes. After incubation this will develop into diffused heavy lawn culture in inoculated plates. Well diffusion method was used to determine the antibacterial activity of the extracts prepared from the seed extract preparation using standard procedure. The wells were then punched on agar plate with a diameter of 6 mm and stock extracts were taken into the micropipette and 20 μL of extract was discharged onto the well, incubated at 37°C for 48 h. After incubation, appearance of zone of inhibition that is an area of an agar plate where the growth of an organism is prevented by tulsi, nutmeg and myrobolan on the agar surface after the incubation period was measured using Vernier caliper. The minimum concentration at which the tulsi, nutmeg and myrobolan extracts achieved was at 25%, 50% and 50% respectively against E. faecalis.

Preparation of irrigants

Based upon the results obtained from the antimicrobial activity i.e., minimum inhibitory concentration (MIC) a weighted amount of extract was taken in sterile beaker and 100 ml of distilled water was mixed, thereafter the beaker was placed on hot plate magnetic stirrer at 60° Celsius to dissolve the extract in solvent till a homogenous solution is obtained, thus obtained solution was mixed with 900 ml distilled water to make a final volume of 1000 ml in a clean sterile measuring cylinder. The solution was then transferred to sterile plastic bottle and labeled; the same procedure was done for both the extracts. The final extract was stored in amber-colored bottle under refrigeration for further use.

Preparation of sample and irrigation protocol

Forty extracted single-rooted, primary human teeth were allocated at random into four experimental groups of 10 teeth each. Over-retained single rooted primary anterior teeth with at-least 2/3rd root length were included in the study and primary teeth with internal or external resorption, root with multiple canals, presence of cracks, presence of caries were excluded. The teeth were stored in distilled water before the initiation of the experiment and then decoronated at the level of the cement–enamel junction using a carborundum disk. The working length was determined by placing a No. 10 H-file into the root canal performed manually by a single operator (Dentsply Maillefer) using the step-back technique. The apical stop was prepared to size 40. During instrumentation, 2 ml of the selected irrigant was used for at least 10 s after each file, and 10-ml final flush with appropriate irrigant was carried out for 2 min. The irrigant was delivered using a 30G endod-tipped needle placed in the canal without binding.

Smear layer evaluation

Longitudinal grooves were made on the buccal and palatal surfaces of the root segments without penetrating into the canal. The roots were then split into two halves with a Metacut DCM machine. The split halves were stored in 2.5% glutaraldehyde solution for 24 h. After fixation, the samples were dehydrated in ethanol series (70, 90, 95, and twice at 100%) and then dried using dry ice. Each specimen was mounted on an aluminium stub and sputter coated with a 20-nm layer of gold, to render a conductive surface. The specimens were examined using a scanning electron microscope (SEM) quantum 60 at magnification of ×2000. The specimen was then blind coded. Representative photographs from each root third were taken for all the samples.[11]

  • Score 1: Dentinal tubules completely opened
  • Score 2: More than 50% of dentinal tubules opened
  • Score 3: Less than 50% of dentinal tubules opened
  • Score 4: Nearly all the dentinal tubules covered with smear layer.

Statistical analysis

The scoring of both the data were then analyzed by using the Statistical Package for the Social Sciences (SPSS) software version 21(IBM Corporation, Bhopal, Madhya Pradesh, India) for Windows. Descriptive statistics included computation of percentages which were analyzed using the Kruskal–Wallis test followed by the Mann–Whitney U-test for pairwise comparison. Statistical Significance was set at P < 0.05.

   Results Top

A significant proportion of specimens treated with Tulsi had dentinal tubules completely opened (score 1) especially at apical third [Figure 1]a. Images of myrobolan treated specimens revealed score 2, i.e., more than 50% of dentinal tubules opened [Figure 1]b. In nutmeg group, significantly higher percentage of score 3 was observed [Figure 1]c. Almost all dentinal tubules were covered with smear layer for NaOCl group [Figure 1]d. Significant difference between myrobolan, nutmeg, and tulsi and NaOCl irrigant at different root levels for smear layer scores were seen. However, there was no significant difference for smear layer scores in tulsi irrigant was seen at different root levels [Table 1]. Kruskal–Wallis test showed significant difference for smear layer scores (overall) between different irrigant groups (χ2 = 70.253, P = 0.000 [<0.001]). When the Mann–Whitney U-test was applied for pair wise comparison, it showed that significantly higher scores in NaOCl than nutmeg, myrobolan, and tulsi. In nutmeg scores were higher than myrobolan and tulsi. Furthermore, in myrobolan scores were higher than tulsi [Table 2]. It shows that almost all dentinal tubules were opened with the use of tulsi irrigant and almost all dentinal tubules were covered with smear layer with the use of NaOCl irrigant. Myrobolan irrigant shows no significant difference at cervical and apical root levels (P = 0.108 [>0.05]) [Figure 1]a, [Figure 1]b, yc, [Figure 1]d. However, significant difference was observed between middle and apical section scores (P = 0.030 [<0.05]). This indicates that Myrobolan was more effective at the cervical and middle level as compared to the apical section. Nutmeg irrigant shows significantly higher scores in the apical section than cervical (P = 0.000 [<0.001]). Significantly higher scores were observed in the apical section than the middle section (P = 0.000 [<0.001]). While cervical section scores were significantly higher than the middle section of the root (P = 0.000 [<0.001]). This indicates that Nutmeg was more effective at middle level as compared to apical and cervical sections [Table 3].
Figure 1: Scanning electron microscope images showing smear layer removal after using (a) Tulsi irrigant. (b) Myrobolan irrigant. (c) Nutmeg irrigant. (d) Sodium hypochlorite irrigant

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Table 1: Intra group comparison of smear layer removal at different root levels

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Table 2: Smear layer scores (overall) between different irrigant groups

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Table 3: Pair wise comparison between different irrigants at different root levels

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   Discussion Top

One of the fundamental steps in a pulpectomy treatment is the reduction of the pathogenic bacterial load to the minimum level within the root canals. Hence, the clinician must pay particular attention to the biochemical preparation of the complex pulp canal system characteristic of primary teeth to reduce the bacteria and their byproducts to a minimum, thus increasing the chances of a successful pulpectomy. Facultative anaerobic microorganisms such as Enterococcus faecalis are considered highly resistant species and therefore possible cause of failed root canal treatment.

In the present study, over-retained extracted teeth were stored in distilled water because various studies have found that distilled water is the best storage media for extracted teeth for the research purpose.[12] Reena et al. (2011)[13] found that immersion in distilled water might be the best option for storage and sterilization of bovine teeth that are to be used in dental bonding studies in vitro. In the present study, ethanolic extraction of tulsi, nutmeg and myrobolan seeds was done, as the polarity of solvent affects the quantity and composition of secondary metabolite of an extract, organic solvents give more consistent antimicrobial activity compared to aqueous extracts. Cowen reported that the ethanolic extracts show better antimicrobial properties. Alcohol has antibacterial property of its own, so to neutralize this effect the crude extracts were dissolved in dimethyl formamide, which is an inert solvent and is neutral toward the Streptococcus mutans and E. faecalis, following this the desired volume of irrigant was obtained by the method suggested by Agarwal et al.[14] Antibacterial activity was tested by agar well-diffusion method as it is more accessible and economical method. The MIC values were interpreted as the lowest concentration of the sample, which showed the maximum zone of inhibition.

Simple random selections of teeth were done to divide the teeth into different groups. Smear group were divided based on the irrigants used during shaping and cleaning. Irrigation was performed with 2 ml of the selected irrigant for at least 10 s and 10-ml final flush with appropriate irrigants were carried out for 2 min. Gupta[15] explained that the effect of the irrigation solution depends on waiting time in a root canal: One min waiting time removed only the surface smear layer, and more waiting time was needed to remove tubular plugs. Thus, in the present study, 2 min final flush was done for all the irrigants Teixeira et al.[16] demonstrated that canal irrigation with NaOCl for 1, 3, and 5 min were equally effective in removing the smear layer from the canal walls of straight roots. In the present study, the entire canal length was utilized to simulate the clinical situation and to test the efficacy of the solutions in all the segments of the root canal system of the primary teeth since entire obturating material has to be in direct contact with the root canal walls of primary teeth.

For the observation of prepared samples for smear layer removal, teeth sectioning was performed in the present study using Metacut Diamond cutting machine (DCM) machine, which had negligible alteration of the root content. This technique is less destructive than the cross-section, which provides knowledge about the adaptation of irrigation solution. Furthermore, no attempts were made to split the teeth with a chisel, due to the small diameter of primary teeth cross-section, which provides knowledge about the adaptation of irrigation solution but ends up with a destroyed sample.[17]

In this study, the SEM has been used to determine the effectiveness of various irrigants to remove the smear layer.[18] Under SEM, the smear layer is viewed as a uniform, dense layer of an amorphous structure that completely obliterates the entrance to the dentinal tubules and drastically reduces the permeability of the dentin.[19]

NaOCl is till date, the most commonly employed root canal irrigant. The antimicrobial activity of NaOCl is by the release of hypochlorus acid and its oxidative action on sulfhydryl groups of bacterial enzymes, thereby disrupting the metabolism of the microorganisms. NaOCl at all concentration was effective in eliminating resistant endodontically relevant microbes including E. faecalis.[4] From a cleansing perspective, lower concentration of NaOCl still retained substantial tissue dissolution capacity and are effective in cleaning root canals. Although it is an effective antibacterial agent, NaOCl is toxic when extruded to the perirarticular tissues and has unpleasant odor and taste. Therefore, research for new irrigant continues.[4] The results of the present study demonstrated that conventional irrigation with 2.5% NaOCl alone was comparatively ineffective in achieving thorough smear layer removal. NaOCl had least efficacy in comparison with tulsi, nutmeg, and myrobolan, and the difference in the smear layer removal was found to be highly significant. This corroborates with earlier studies done by McComb and Smith,[20] Sadr Lahijani et al.,[21] Venkataram et al.[22] also demonstrated that conventional irrigation with 2.5% NaOCl alone was comparatively ineffective in achieving thorough removal of the smear layer. In this study, at cervical, middle, and apical root level, almost all dentinal tubules were covered with smear layer, and score were significantly higher than nutmeg, myrobolan, and tulsi.

In the present study, tulsi irrigant can effectively remove smear layer from the canal especially at apical level than nutmeg, myrobolan and NaOCl irrigant. Also at cervical and middle root level tulsi were effective than other irrigants. The antimicrobial activity of tulsi can be attributed to essential oils like eugenol, chavicol linalol, carvacrol. Eugenol (1-hydroxyl-2 methoxy– 4 allybenzene), is the active constituent present is largely responsible for therapeutic potential of tulsi.[23] An important characteristic of essential oils and their components is their hydrophobicity, which enables them to partition in the lipids of bacterial cell membrane and mitochondria, disturbing the structures and rendering them more permeable as a result of which leakage of ions and other cell contents can then occur. Despite abundance of literature on antibacterial activity of tulsi, none have attempted to evaluate the effect of tulsi irrigant on smear layer removal.

There was no significant difference seen for smear layer scores at different root level for nutmeg group. Nutmeg irrigant were effective in smear layer removal especially at apical root level followed by Cervical and middle level of root. Antimicrobial activity of Nutmeg oils can be attributed to essential oils which contain 85%–93% monoterpene hydrocarbons, 6.6%–12% oxygenated monoterpenes and sesquiterpenes and 3.5% aromatic ethers, while the corresponding values for the mace oils were 75%–94%, 4.7%–17.6% and 0%–5.9%, respectively.[24]

Myrobolan showed significant difference at different root levels but dentinal tubules were open in middle one third followed by cervical root level. Less than fifty percent tubules were open in apical group.

The limitation of present study is that it did not evaluate in vivo effects. It was necessary to conduct the study for confirmation of clinical utility of these extracts as irrigant or intracanal medicament. All the herbal irrigants were biocompatible and also well accepted by all the subjects. To advocate the use of these irrigants, it is necessary to carry out a long-term in vivo study on a large sample to evaluate its efficacy on removal of smear layer.

   Conclusion Top

Tulsi as well as nutmeg and myrobolan irrigants can be effectively used as an irrigant in primary teeth as they can remove smear layer. This would be attributed to the activity of active ingredients present in the extracts i.e., anti-inflammatory, wound healing, antimicrobial properties. They all are having good anti-bacterial property. Hence, microbiologically, it is clear that these irrigants are effective against E. faecalis Tulsi was more effective when compared to nutmeg and myrobolan irrigant. Almost all the dentinal tubules were open in apical section.

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Conflicts of interest

There are no conflicts of interest.

   References Top

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Mahajan VA, Kamra AI, Dahiwale SS. The effect of 17% EDTA and MTAD on smear layer removal and on erosion of root canal dentin when used as final rinse: An in vitro SEM study. J Intern Clin Dent Res Organ 2010;2:113-8.  Back to cited text no. 2
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  [Figure 1]

  [Table 1], [Table 2], [Table 3]


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