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Table of Contents   
ORIGINAL ARTICLE  
Year : 2022  |  Volume : 33  |  Issue : 1  |  Page : 69-74
Cytokine expression and anti-microbial effectiveness of different calcium hydroxide dilutions: An In Vitro study


Department of Restorative Dental Sciences, Division of Endodontics, College of Dentistry, King Saud University, Riyadh, Kingdom of Saudi Arabia

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Date of Submission12-Jan-2020
Date of Decision03-Mar-2022
Date of Acceptance12-Apr-2022
Date of Web Publication09-Aug-2022
 

   Abstract 


Aims: To determine the cytokine expression by human gingival fibroblasts in response to different calcium hydroxide (Ca(OH)2) dilutions and test the effectiveness of these dilutions in root canal dentin infected with Enterococcus faecalis (E. faecalis). Methods: UltraCal XS Ca(OH)2 dilutions were prepared (60, 10, and 1 mg\mL) and co-cultured with gingival fibroblasts for 24 and 48 hours. Untreated cells were used as controls. Expressions of interleukin (IL-1β), tumour necrosis factor alpha (TNF-α), transforming growth factor beta (TGF-β), and IL-10 were analysed with real-time polymerase chain reaction (PCR). Root canals of extracted human teeth were inoculated with E. faecalis. After 21 days, canals were medicated with Ca(OH)2 dilutions for 7 days. Samples were taken to determine bacterial reduction using quantitative PCR. Analysis of variance, Tukey post-test, and Wilcoxon matched pair test were used for statistics. Results: IL-1β and TNF-α expressions of all Ca(OH)2 dilutions were higher at 24 and 48 hours compared to the control. Similarly, all Ca(OH)2 dilutions induced TGF-β expression at 24 hours compared to the control and continued to be higher in 60 mg/mL groups at 48 hours. In contrast, IL-10 was constitutively expressed by untreated cells in the control group and was down-regulated significantly by all Ca(OH)2 dilutions at 24 and 48 hours. All dilutions demonstrated a significant E. faecalis reduction (P < 0.001) with no significant difference between dilution groups (P > 0.05). Conclusions: All Ca(OH)2 dilutions had a differential inflammatory effect on fibroblasts and had a down-regulation effect to IL-10. All dilutions tested were effective against E. faecalis, with 60 mg/mL having the highest bacterial reduction.

Keywords: Anti-bacterial, calcium hydroxide, human fibroblasts, inflammatory response, intra-canal medication

How to cite this article:
Binanzan N, Alsalleeh F. Cytokine expression and anti-microbial effectiveness of different calcium hydroxide dilutions: An In Vitro study. Indian J Dent Res 2022;33:69-74

How to cite this URL:
Binanzan N, Alsalleeh F. Cytokine expression and anti-microbial effectiveness of different calcium hydroxide dilutions: An In Vitro study. Indian J Dent Res [serial online] 2022 [cited 2022 Oct 4];33:69-74. Available from: https://www.ijdr.in/text.asp?2022/33/1/69/353535



   Introduction Top


Microorganisms are the aetiology of dental pulp diseases that lead to peri-apical lesions.[1] Calcium hydroxide (Ca(OH)2) is known to eliminate bacteria which may survive chemo-mechanical instrumentation.[2] Recently, Ca(OH)2 was advocated for the treatment of regenerative cases as it significantly increases stem cell survival and proliferation.[3],[4] However, Ca(OH)2 is known to interfere with the healing phase and may cause excessive inflammatory response that will lead to cellular damage when it is accidentally injected outside the root canal system.[5]

Fibroblasts play an important role during endodontic-peri-apical infections through activation and release of several pro- and anti-inflammatory cytokines: tumour necrosis factor alpha (TNF-α), interleukins (ILs), and transforming growth factor beta (TGF-β).[6] The effect of IL-1β includes enhancement of bone resorption, inhibition of bone formation, stimulation of lymphocytes, potentiation of neutrophils, and activation of the production of prostaglandins and proteolytic enzymes.[7] TNF-α had a direct cytotoxic effect and similar local effects to IL-1β.[8] IL-10 has a role in the healing process and induces down-regulation of pro-inflammatory cytokines.[9] TGF-β is also involved in the repair process of peri-apical lesions.[6] Thus, these mediators play a vital role in the pathogenicity of apical periodontitis; therefore, it is important to evaluate the role of Ca(OH)2 on inflammatory cytokine expression to control tissue inflammation and preserve cell viability. However, the evidence lacks whether diluted Ca(OH)2 preparations induce a less inflammatory response and maintain the anti-bacterial effect. The purpose of the study was to evaluate the effect of different Ca(OH)2 dilutions on cytokine expression by human gingival fibroblasts and evaluate their anti-microbial effectiveness against E. faecalis.


   Material and Methods Top


Preparation of Ca(OH)2 dilutions

UltraCal XS (Ultradent Products Inc, South Jordan, UT), which contains 35% Ca(OH)2, was used. A saturated solution of the paste was made as follows: 2.5 g of UltraCal XS was mixed with 5 mL of distilled water, giving a concentration of 175 mg\mL, and the mixture was stirred for 4 hours at room temperature. For the cytokine expression experiment, the mixture was centrifuged at 3000 rpm for 15 minutes, and the aqueous supernatant layers were filter-sterilized using a sterile 25-mm syringe filter when used on cells (Fisher Scientific, Newark, DE). Dilutions were made using cell media for the cytokine expression experiment or in distilled water for the anti-bacterial experiment at specific concentrations (60, 10, and 1 mg\mL).[10] The pH of each dilution was measured using a Jenway 3540 pH and conductivity meter (Jenway, Staffordshire, UK). The outline of the experimental design is shown in [Figure 1].
Figure 1: The outline of the experimental design

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Inflammatory cytokine analysis

Cell culture

The study was approved by the Institutional Ethics Committee (PR0061) and was performed following the minimum information and quality standards for conducting, reporting, and organising in vitro research.[11] Primary human gingival fibroblasts were grown in Dulbecco's modified Eagle's medium (DMEM) (ATCC, Manassas, VA, USA), supplemented with 10% fatal bovine serum, streptomycin (50 g/mL), and 1% antibiotic/antimycotic (300 U/mL, 300 mg/mL streptomycin, 5 mg/mL amphotericin 100 g/mL) under standard cell culture conditions (37°C, 100% humidity, 95% air and 5% CO2). Cells between the fourth and sixth passages were used.

A total of 2 × 106 cells were plated with the culture medium onto six-well plates and grown overnight in 5% CO2 at 37°C. On the next day, the non-adherent cells were removed from the plates, and a fresh culture medium was added. Culture plates containing fibroblasts were then divided into

  • Cells kept in the fresh medium to serve as the negative control.
  • Cells co-cultured with various dilutions of Ca(OH)2 (60, 10, and 1 mg\mL).


Fibroblasts were incubated for 24 and 48 hours, and RNA was extracted using the standard protocol and using a BioFACT™ Total RNA Prep Kit (Biofact Biofactory, Daejeon, Korea) following the manufacturer's instructions. cDNAs were prepared from 1 μg of RNA using a FIREScript RT cDNA synthesis KIT (Solis Biodyne, Tartu, Estonia). The RNA concentration was measured using a spectrophotometer. The mRNA expressions of IL-1β, TNF-α, TGF-β, and IL-10 by fibroblasts were quantified using quantitative real-time polymerase chain reaction (qRT-PCR) with 5× HOT FIREPol® EvaGreen® qPCR Supermix (Solis Biodyne, Tartu, Estonia) on an ABI 7500 Real-time PCR instrument (Applied Biosystems) in a total reaction volume of 20 μL. Quantitative PCR (qPCR) was run with primers incubated at 95°C for 12 minutes, followed by 40 cycles of 95°C for 15 seconds, 65°C for 30 seconds, and 72°C for 30 seconds. Cycle thresholds (Cts) were normalised to Ct for housekeeping gene GAPDH (glyceraldehyde-3-phosphate dehydrogenase) for each cDNA and expressed by fold increase using the formula 2-ΔΔCt.[12] The primers were used according to previous publications [Table 1].[13],[14] Each experiment was independently performed three times in triplicate on separate days.
Table 1: Primers sequences

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Anti-bacterial effect

Teeth selection and preparation

A total of 94 human-extracted single-rooted teeth were collected and stored in saline. Teeth were sectioned above the cementoenamel junction to standardise the length. A K-type hand file (Dentsply Maillefer, Ballaigues, Switzerland) of size 15 was used to determine the working length to the clinically visible apical foramen. Each canal was instrumented to the full length using rotary 0.04 taper K3XF nickel titanium files up to a size 35 file (SybronEndo, Orange, Calif.). Irrigation with 2 mL of 5.25% sodium hypochlorite (NaOCl) was delivered with a 30-gauge needle (NaviTip; Ultradent, South Jordan, UT). A final rinse occurred with 2 mL of 17% ethylenediaminetetraacetic acid for 1 minute, followed by 2 mL of 5.25% NaOCl to remove the smear layer. The teeth were then autoclaved at 121°C for 20 minutes.[15] All teeth were covered with layers of nail varnish, and the apical foramen was sealed with a composite resin.

Cultivation of E. faecalis

E.faecalis (ATCC 29212) was grown in a brain heart infusion (BHI) broth for 24 hours. The optical density of the bacterial broth was measured using a spectrophotometer. Root canals were infected by inoculating the canals with 6.3 × 108 colony-forming units (CFUs) per mL of E. faecalis diluted in 4 mL of BHI. The fresh BHI broth was injected into canals weekly to ensure the viability of bacteria. All specimens were incubated aerobically at 37°C. After 21 days, bacterial growth was confirmed by culture. Eight teeth which were not infected served as negative controls.

Intra-canal medicament protocol

The sample size calculation revealed that at a 0.05 level of significance with a standard deviation of 1.5 × 104, a power of 0.9, and a maximum difference of 2%, the sample size should be a minimum of 18 teeth for each group. Infected teeth were randomly distributed to the experimental groups of different Ca(OH)2 dilutions (n = 20 per group). The pre-medication samples (S1) were taken as follows: a sterile solution (1 mL) of 0.85% saline was used to rinse the root canal, and a size 20 hand file was used to agitate the fluid. Samples were taken by the sequential use of three to five paper points placed to the WL. Each paper point remained in the canal for 1 minute and was transferred to tubes containing 1 mL of sterile saline. Medicament was introduced into each canal using NaviTip needles (30 g; 21 mm long) placed as close as possible to the WL until the paste was seen within the canal orifice, and a lentulo spiral (size 25) was used. The coronal access cavity was sealed with a sterile cotton pellet and closed with cavit (Cavit™ Temporary Filling Material, 3M United States). Specimens were stored in an incubator at 37°C and 100% humidity. After 1 week, the cavit was removed and canals were irrigated with 1 mL of 20% citric acid (Ultradent Products Inc, South Jordan, UT) to terminate the effect of Ca(OH)2. The post-medication samples (S2) were then taken as described above after a sterile H-file (size 25) was used to stroke it against the wall of the canals and agitate the fluid. Samples were agitated in a vortex for 1 minute.[16]

Real-time PCR was used for bacterial quantification. First, samples were subjected to DNA extraction using a HiGene™ Genomic DNA Prep Kit (Biofact Biofactory, Daejeon, Korea) according to the manufacturer's instructions. DNA extracts were frozen at –20°C until qPCR analysis. Second, 16S ribosomal RNA gene-targeted qPCR was performed as mentioned earlier. The published sequences of the forward and reverse primers of E. faecalis were 5′ CGCTTCTTTCCTCCCGAGT-3′ and 5′-GCCATGCGGCATAAACTG-3′, respectively.[17] Primers in a concentration of 0.2 μmol\L each and a DNA-extract volume of 2 μL were added to the PCR master mix in MicroAmp. In all experiments, appropriate negative controls were used, which consisted of the reaction mix and nuclease-free water instead of the sample. All measurements were taken in duplicate for samples and triplicate for standards. Bacterial cell counts were inferred for each sample based on constructed standard curves for direct bacterial quantification.[16]

Statistical analysis

The statistical significance of differences in cytokine expressions between the groups was determined by one-way analysis of variance (ANOVA), followed by Tukey post hoc test. A statistically significant difference of bacterial reduction between pre-medication samples (S1) and post-medication samples (S2) was determined by the Wilcoxon matched pair test. Inter-group quantitative analysis was performed using the Kruskal–Wallis test. All data were analysed using the Statistical Package for Social Studies (SPSS 22; IBM Corp., New York, NY, USA). P value ≤0.05 was considered statistically significant.


   Results Top


Alkalinity of different Ca(OH)2 dilutions

The pH of different Ca(OH)2 dilutions (60, 10, and 1 mg\mL) were measured at room temperature (25°C) and given the following values, respectively: 11.47, 9.33, and 8.93. All dilutions maintain alkalinity; however, as the concentration decreases, the alkalinity drops to a lower level.

Inflammatory cytokine analysis

Fibroblasts exposed to all Ca(OH)2 dilutions had higher expression of IL-1β compared to the control group at 24 hours; 10 mg\mL Ca(OH)2 dilutions induce significantly higher levels than the control and 60 mg\mL Ca(OH)2 groups (P = 0.0001). At 48 hours, IL-1β continued to be induced by all Ca(OH)2 dilutions, but none were significant (P = 0.072) [Figure 2]. Similarly, the expression of TNF-α among all Ca(OH)2 dilutions was significantly higher compared to the control at 24 hours (P = 0.000). At 48 hours, the expression of TNF-α in response to all Ca(OH)2 dilutions maintained higher levels than the control group, but the difference was not significant (P = 0.08).
Figure 2: mRNA expression of pro- and anti-inflammatory cytokines at 24 and 48 hours. Each column represents the mean ± standard error of the mean. Data were analysed with ANOVA and Tukey post hoc tests

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All dilutions induced expressions of TGF-β at 24 hours. The highest expression was observed in the 60 mg\mL Ca(OH)2 group, which was statistically significant (P = 0.0001). At 48 hours, the expression continued to be higher, but not significant, in 60 mg/mL Ca(OH)2 (P = 0.08) [Figure 2]. Interestingly, IL-10 was constitutively expressed by unstimulated fibroblasts and was down-regulated by all Ca(OH)2 dilutions used. The down-regulation effect was significant at 24 (P = 0.022) and 48 hours (P = 0.000) [Figure 2].

Overall, the anti-inflammatory TGF-β expression to different Ca(OH)2 dilutions followed the same pattern as the pro-inflammatory cytokines with the greater effect measured for the strongest dilution. However, the results were significant only at 24 hours. IL-10, on the other hand, was expressed in the control, and in each experimental group, the effect of all Ca(OH)2 dilutions was significant at 24 and 48 hours.

Anti-bacterial effect

Random sampling taken from root canals infected with E. faecalis and subjected to Gram staining confirmed the presence of microorganisms with a Gram-positive cocci morphology (data not shown). Furthermore, all samples (S1) rendered positive to E. faecalis, as analysed by qPCR.

The bacterial count in S1 samples showed no significant difference (P > 0.05). The results indicated that the method of bacterial coloniaation provided a homogeneous baseline of bacterial load. The bacterial reduction from S1 to S2 with different Ca(OH)2 dilutions demonstrated a highly significant bacterial reduction (P < 0.001) [Table 2]. Among the dilution groups, the mean reduction in bacterial count from S1 to S2 ranged between 86 and 96.5%. A lower percent reduction was found with the most diluted concentration. However, S2 samples revealed no significant difference between different dilution groups (P > 0.05) [Figure 3]. Thus, all Ca(OH)2 dilutions were effective against E. faecalis.
Table 2:  Enterococcus faecalis Scientific Name Search s before (S1) and after medicating the canals with different dilutions of calcium hydroxide (S2)

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Figure 3: Efficiency of bacterial elimination using different calcium hydroxide dilutions. Box plots indicate the bacterial reduction percentage by groups (n = 20 per group except in 1 mg\mL group; n = 19), where 1.00 is equal to 100%. The middle line in the box is the median; o individual observations that may be potential outliers. * indicates an extreme value

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   Discussion Top


It is believed that the cytotoxicity and tissue reaction of Ca(OH)2 are mediated by inflammatory cytokines produced by the host. Gingival fibroblasts are connective tissue cells that are located in the gingiva apical to the gingival epithelium. Inflammatory cytokines can regulate the functions of fibroblasts. Therefore, one of the aims was to determine the inflammatory cytokine expression by human gingival fibroblasts in response to different Ca(OH)2 dilutions.

The IL-1β and TNF-α expressions vary strongly between different Ca(OH)2 dilutions. At 48 hours, the TNF-α expression was decreased in all dilution groups, except in the 60 mg/mL group. This indicated that the inflammatory response of the material was diminished with time. Pereira et al.[3] supported this explanation as they reported no difference in expression of both genes between the Ca(OH)2 and empty tube control groups. Furthermore, a study which evaluated the cytotoxic effect of Ca(OH)2 on human fibroblasts showed no significant differences for cytokine gene expression levels, including IL-1β and TNF-α.[14]

Ca(OH)2 at 60 mg/mL had a more favourable effect on cells in terms of secreting and maintaining preferable TGF-β release with time than lower dilutions. It had been reported that Ca(OH)2 at a 1 mg/mL concentration increased stem cell proliferation and survival.[4] This is possibly because of TGF-β release by cells after being stimulated with Ca(OH)2. For IL-10, it was highly expressed by untreated fibroblasts and was down-regulated significantly by all Ca(OH)2 dilutions. This agrees with previous findings, which indicated that the expression of IL-10 is significantly down-regulated by endodontic infection and biofilms.[13] The decline in IL-10 expressions in response to Ca(OH)2 may contribute to the rise in pro-inflammatory cytokines, IL-1β and TNF-α.

The second aim of this study was to evaluate the Ca(OH)2 dilution abilities to eliminate E. faecalis. The findings revealed that the intra-canal counts of E. faecalis were substantially reduced with all Ca(OH)2 dilutions, showing 86–96.5% bacterial reduction. However, the analysis showed no significant difference among dilutions tested. This finding was supported by previous studies that showed 1.6 mg/mL Ca(OH)2 as the minimum bio-film inhibitory concentration for E. faecalis.[10] A similar study reported that 0.1 mg/mL was the minimum inhibitory concentration, whereas 1 mg/mL was the minimum bactericidal concentration against planktonic E. faecalis.[18] A study reported that Ca(OH)2 preparations (0.1 g\mL) demonstrated a significant reduction of E. faecalis compared to (1g\mL) at different depths of dentinal tubules. This was explained by the higher ionic dissociation in low-viscosity Ca(OH)2 preparations.[19] The data reflect the efficiency of different Ca(OH)2 dilutions in killing E. faecalis that colonise root canals. Real-time PCR was used in the present study for bacterial quantification. Unlike the culture technique, qPCR has a higher sensitivity and specificity and is able to detect and quantify the bacterial count even if it is in the viable but non-cultivable state.[20]


   Conclusion Top


Fibroblasts expressed differential inflammatory cytokines in response to different Ca(OH)2 dilutions. The effect was significant regardless of concentration and subsided in all groups during the period occurring 24 to 48 hours from treatment. Nonetheless, the overall cytokine expression profile may favour Ca(OH)2 at a 60 mg/mL concentration owing to its proliferative effect on TGF-β. In contrast, the anti-bacterial effects against E.faecalis could be ranked from the strongest to weakest as follows: 60, 10, and 1 mg\mL. Within limitations of this study, Ca(OH)2 at 60 mg/mL could be the optimum concentration to be used in further studies.

Acknowledgements

The authors wish to thank Molecular and Cell Biology (MCB) Laboratory of Prince Naif bin Abdul Aziz Health Research Center (PNHRC).

Financial support and sponsorship

King Abdulaziz city for science and technology (KACST) (1-17-03-001-0031).

Conflicts of interest

There are no conflicts of interest.



 
   References Top

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Emmerich CH, Harris CM. Minimum information and quality standards for conducting, reporting, and organizing in vitro research. Handb Exp Pharmacol 2020;257:177-96.  Back to cited text no. 11
    
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Alsalleeh F, Williams S, Jaber H. Interaction of Candida albicans with periodontal ligament fibroblasts limits biofilm formation over elastomer silicone disks. Arch Oral Biol 2016;63:47-52.  Back to cited text no. 12
    
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Alsalleeh F, Young AC. Albicans biofilm formation is restricted by periodontal ligament cells and induces differential cytokines response compared to planktonic C. albicans. J Dent Appl Open 2014;1:139-44.  Back to cited text no. 13
    
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Yadlapati M, Souza LC, Dorn S, Garlet GP, Letra A, Silva RM. Deleterious effect of triple antibiotic paste on human periodontal ligament fibroblasts. Int Endod J 2014;47:769-75.  Back to cited text no. 14
    
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Western JS, Dicksit DD. A systematic review of randomized controlled trials on sterilization methods of extracted human teeth. J Conserv Dent 2016;19:343-6.  Back to cited text no. 15
[PUBMED]  [Full text]  
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Alves FR, Rocas IN, Almeida BM, Neves MA, Zoffoli J, Siqueira JF Jr. Quantitative molecular and culture analyses of bacterial elimination in oval-shaped root canals by a single-file instrumentation technique. Int Endod J 2012;45:871-7.  Back to cited text no. 16
    
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Santo Domingo JW, Siefring SC, Haugland RA. Real-time PCR method to detect enterococcus faecalis in water. Biotechnol Lett 2003;25:261-5.  Back to cited text no. 17
    
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Abbaszadegan A, Dadolahi S, Gholami A, Moein MR, Hamedani S, Ghasemi Y, et al. Antimicrobial and cytotoxic activity of cinnamomum zeylanicum, calcium hydroxide, and triple antibiotic paste as root canal dressing materials. J Contemp Dent Pract 2016;17:105-13.  Back to cited text no. 18
    
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Siqueira JF Jr, Rocas IN. Diversity of endodontic microbiota revisited. J Dent Res 2009;88:969-81.  Back to cited text no. 20
    

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Correspondence Address:
Dr. Fahd Alsalleeh
Department of Restorative Dental Sciences, Division of Endodontics, College of Dentistry, King Saud University, Riyadh
Kingdom of Saudi Arabia
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijdr.IJDR_41_20

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