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 Table of Contents  
Year : 2023  |  Volume : 15  |  Issue : 1  |  Page : 71-77

Efficiency of orange oil solvent combined with disinfectants and bioactive glass (BAG) on the cleanness of root canal after endodontic retreatment: An in vitro study

1 Prosthetic Dentistry Department, Faculty of Dentistry, King Salman International University, El Tur, South Sinai, Egypt
2 Endodontics Department, Faculty of Dentistry, Ahram Canadian University, Giza, Egypt

Date of Submission26-May-2022
Date of Decision26-Oct-2022
Date of Acceptance06-Nov-2022
Date of Web Publication28-Feb-2023

Correspondence Address:
Dr. Ahmed Abdou
Prosthetic Dentistry Department, Faculty of Dentistry, King Salman International University, El Tur, South Sinai
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jioh.jioh_115_22

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Aim: Endodontic retreatment is associated with clinical complication with doubtful success rate due to limited removal and cleaning of remnants and bacteria. This study investigated the effect of orange oil solvent containing bioactive glass on the removal of root canal filling material and bacterial loading during root canal retreatment. Materials and Methods: Thirty single-rooted, freshly extracted human central teeth were collected. Access opening and instrumentation were done. Samples were injected with 10 µL bacterial suspension (Enterococcus faecalis [E. faecalis]) at 37°C for 48 h and then filled with root canal filling using lateral compaction technique. Upon removal of the filling materials by retreatment kits, samples were divided based on the type of irrigating solution into three groups (n = 10): G1, 5 mL orange oil solution only; G2, 5 mL of 5% sodium hypochlorite with an orange oil solvent solution; G3, 5 mL of orange oil solution with bioactive glass (10%). All groups were subjected to a turbidity analysis and tested for colony-forming units (CFU)/mL. Afterward, longitudinal sections of samples were obtained from the root, and scanning electron microscopy was used to scan root wall. Scanned images were analyzed by two evaluators using a scoring system. Data were then statistically analyzed using Kruskal–Wallis test. Results: Significant difference resulted between tested groups for bacterial count (log CFU) at P = 0.015. G2 showed the highest significant bacterial count compared with G3 (P = 0.018). G1 showed an insignificant difference with other groups (P > 0.05). For cleaning efficiency scores, the highest significant scores resulted for G2, which was significant with +ve ctrl group (P = 0.01). Conclusion: The addition of bioactive glass did not influence antibacterial activity of orange oil irrigants in the removal of residual root canal filling materials.

Keywords: Bioactive Glass, Orange Oil Solvent, Root Canal Irrigants, Scanning Electron Microscopy

How to cite this article:
Ashraf R, Badra H, Abdou A. Efficiency of orange oil solvent combined with disinfectants and bioactive glass (BAG) on the cleanness of root canal after endodontic retreatment: An in vitro study. J Int Oral Health 2023;15:71-7

How to cite this URL:
Ashraf R, Badra H, Abdou A. Efficiency of orange oil solvent combined with disinfectants and bioactive glass (BAG) on the cleanness of root canal after endodontic retreatment: An in vitro study. J Int Oral Health [serial online] 2023 [cited 2023 Apr 1];15:71-7. Available from:

  Introduction Top

Several factors can influence the success of endodontic retreatment; the ability to totally remove the previously used filling material and completely sterilize the canal can be out of the operator hands even using the usual retreatment protocols and appropriate retreatment kit. Thus, clinical or radiographic failure that requires root canal retreatment (RCR) represents a challenging condition with a doubtful success rate. RCR involves complicated procedure including: first, reassessing of the pulp cavity followed by removing the old endodontic filling material and then chemo-mechanical instrumentation and refilling of the root canal space.[1],[2]

Several methods have been proposed for removing of the old root canal filling material, including laser, manual, rotary ultrasonic activation, or reciprocating instruments, associated with chemical solvents to completely remove old gutta-percha and sealers.[3] However, the complete removal of gutta-percha with no remnants is still a challenge, and no single protocol was able to guarantee the success of RCR.[3],[4]

Clinical failure of endodontic treatment is associated with bacterial infections, which interfere with retreatment protocols. Throughout the procedures of RCR, necrotic materials, debris, and infected dentine must be eliminated, thus not only complete removal of gutta-percha and sealers is mandatory for successful retreatment of the canal, but also decrease in the bacterial loading and completely disinfecting the canals is a major target.[5]

Rotary instruments from retreatment systems are widely used for this purpose together with the irrigant, disinfectants, and gutta-percha solvents. However, it is still challenging to achieve this purpose with the current available techniques.[6]

Solvents for gutta-percha help in removing their remnants from the canal and an additional benefit would be achieved if these solvents would aid in canal disinfection; thus, natural solvents with additional benefits were introduced such as the orange oil.[7] Limonene is the main constituent of the orange oil, which is able to dissolve endodontic sealers. It was reported to be safe, biocompatible, and noncarcinogenic compared with other chloroform-based solvents.[8]

Recent microbiologic studies confirmed the presence of resistant microorganism such as E. faecalis in persistent canal infections and secondary infections in endodontic failures.

They are frequently isolated from pathological root canals and considered the main cause of failure after endodontic treatment.[9] Mechanical debridement of root canal with antibacterial irrigant is essential protocol for the treatment of endodontic infections. The irrigation during RCR should have the potential to remove the debris, increase dentinal tubules permeability, and have a bactericidal or bacteriostatic effect. A lack of irrigation or insufficient irrigation leads to accumulation of debris, which decreases the efficiency of mechanical instrumentation preventing the optimal adaptation of final filling materials to canal walls.[10]

Sodium hypochlorite (NaOCl: 0.5%–5.25%) is a potent antimicrobial and widely used irrigating solution due to its cellular metabolism alteration and phospholipid destruction. Additionally, the chloramines formation interferes with cellular metabolism, and oxidative action causes an inactivation of bacteria. Together with the low surface tension, lubricating capacity, and organic dissolution properties favors its choice as an irrigating solution for root canal treatment/RCR protocols.[11],[12],[13]

Studies have recently identified bioactive glass (BAG) as a possible alternative to calcium hydroxide (which was previously used as a bacteriostatic intracanal dressing but was proven to be effective against E. faecalis). BAG as an intracanal dressing can enhance dentin mineralization without altering the mechanical properties of teeth. In aqueous suspension, BAG particles employ an antimicrobial effect, which results in an increased pH and osmotic pressure of the surroundings, interfering with microbial viability. Furthermore, the release of silica, Ca++, and P ions appears to exert bactericidal effects.[14],[15]

Recently, a new concept has evolved, which employs the use of a combination of antibacterial drugs and antimicrobial agents in conjunction with the irrigating solutions to clean the root canal in an attempt to get an empty, clean, and sterile space that is ready for filling in one step.[16]

The aim of this study was to investigate the influence of the combination of orange oil and BAG as a different protocol for the removal of filling materials and disinfection of the canal during RCR. The null hypothesis is that there is no difference between orange oil, orange oil plus sodium hypochlorite, and orange oil plus BAG in canal disinfection and cleanliness during RCR procedure.

  Materials and Methods Top

Teeth preparation

Thirty freshly extracted upper central human permanent teeth with mature apices were used for this study (ethical approval: Cairo University, 37-3-2022). All teeth were collected from living human subjects, and the extraction was done for periodontally affected teeth that requires removal or for arch clearance to prepare a full arch removable denture. A written ethical informed consent was signed by all the participants for the use of extracted teeth for anonymous research purposes. All teeth were cleaned with an ultrasonic scaler to remove any remaining bone, calculus, or soft tissue. Initial radiographs were taken to confirm root canal patency. Teeth crowns were cut using low-speed hand piece with diamond disc to remove the coronal interference and to standardize the length to 14 mm.

Root canals then were prepared using crown-down technique with nickel-titanium rotary instruments (ProTaper Universal, Dentsply-Maillefer, Ballaigues, Switzerland), following the manufacturer’s instructions up to size F3 instrument. The ProTaper system was connected to an endodontic micromotor (X-SMART, Dentsply-Maillefer, Ballaigues, Switzerland).

Irrigation of each canal with 2 mL of 5.25% sodium hypochlorite (NaOCl) with a 27-gauge needle at each cleaning step. Upon completion of root canal preparation, each canal was irrigated with 5 mL of 17% ethylenediaminetetraacetic acid (EDTA) for 60 s. The samples were then dried using paper point and were sterilized and autoclaved and then stored in a sterile pouch until use to ensure the sterility of the specimen before bacterial inoculation.

Bacterial inoculation of the specimen

A 24-h pure culture suspension of E. faecalis (ATCC 29212) was collected by centrifugation (1000g for 10 min); the pellet was then suspended in a sterile brain heart infusion (BHI) agar medium broth at 37°C. Afterward, inoculation of teeth with 50 μL suspension of E. faecalis (determined by comparing its turbidity to a 0.5 McFarland standard spectrophotometrically) and incubated at 37°C and 95% humidity for 48 h to grow in a planktonic form. Preirrigated sample was obtained by application of a sterile #25 paper point in root canal for 1 min and placed into glass tubes filled with 2 mL of sterile BHI. Subsequently, root canals were drying using paper points before obturation and gutta-percha with Adsealer endodontic seal (Meta Biomed, Cheongju, Korea) were used for obturation. Lateral compaction was done up to the level of the root canal orifice. Teeth were then stored at 37°C for 7 days to allow the sealer to set.

Root canal filling materials removal

For endodontic retreatment, root canal filling materials were removed with the retreatment system (ProTaper Universal Retreatment, Dentsply-Sirona, York, PA, USA): D1 (size 20, 0.09 taper) for the coronal third followed by D2 (25/.08) for the middle third and lastly D3 (20/.07) and F4 (40/.06) for the apical third using the X-smart motor at 600 rpm for ProTaper Universal Retreatment System instruments, and 300 rpm, with a torque setting 2 N/cm. Samples were randomly allocated into three groups according to the irrigation protocol into G1 (+ve ctrl): orange oil only; G2 (NaOCl): orange oil with 5% NaOCl; and G3 (BAG): orange oil with 5% BAG. The irrigation protocol was summarized in [Table 1].
Table 1: Irrigation protocols investigated

Click here to view

Irrigation of samples with the corresponding irrigant between each file exchange was then done for each group; the correspondent irrigant was inserted in the coronal third of samples and remained active for 1 min. Next, the coronal third was prepared with D1 instrument, followed by an irrigant for 1 min. D2 and D3 instruments were used sequentially with the corresponding irrigant as described in [Table 1]. All samples were prepared by the same operator. The canal was assumed to be fully removed when the gutta-percha was no more seen on the final instrument.

Assessment of antibacterial activity

Samples of bacteria were obtained using sterile paper points (S2). Three paper points were inserted individually inside each root canal for 1 min. Bacterial suspension (25 µL) was plated on BHI agar plate and incubated at 37°C in anaerobic jar for 24 h. Colonies were counted by a colony counter (Reichert-Jung). The process was carried out two times for each sample of each group, and the average was reported. After purity of the positive cultures was confirmed using gram-staining, the number of colony-forming units (CFUs) of each specimen was then determined.

Scanning electron microscopy evaluation

Root samples were vertically split with a diamond saw into two halves through preparation of two longitudinal grooves in the root. Afterward, both root halves were stored at 37°C for 7 days to be dehydrated, and then sputter-coating was done with gold (Desk IV Denton Vacuum, Moorestown, NJ, USA).

Scanning electron microscopy (SEM) was used to scan root canal walls at 100 Pa pressure conditions with voltages of 3 KV and 1000× magnification. Analysis of the images was done by two examiners to observe the efficiency of cleaning and removing the filling materials through measuring remnants in root canal walls and dentinal tubules. Using a scoring system[17] from 1 to 4, according to the percentage of root filling materials remnants per image area, analysis was evaluated as follows:

  • Score 1: remnants of filling materials ranging between 0% and 25%;

  • Score 2: remnants of filling materials ranging between 25% and 50%;

  • Score 3: remnants of filling materials ranging between 50% and 75%;

  • Score 4: remnants of filling materials ranging between 75% and 100%.

  • Statistical analysis

    Data were checked for normality using Shapiro–Wilk test; antibacterial test showed nonnormal distribution, so Kruskal–Wallis nonparametric test was used to compare between tested groups, followed by multiple comparisons with Dunn-Bonferroni correction. Statistical level was set at 0.05 (α = 0.05). Furthermore, the power of the study was calculated after the results were obtained (R Core Team (2020), R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria,

      Results Top

    Results of bacterial count are presented in [Table 2] and [Figure 1]. Significant difference resulted between tested groups for bacterial count (log CFU) at P = 0.015. Orange oil + NaOCl showed the highest significant bacterial count compared with orange oil + BAG (P = 0.018). Orange oil showed an insignificant difference with other groups (P > 0.05). The results showed high effect size (f = 0.6) based on the highest and lowest mean values, and the power of the study was 75% with current sample size. For cleaning efficiency scores [Figure 2], the highest significant scores resulted for orange oil+ NaOCl, which was significant with +ve ctrl group (P = 0.01).
    Table 2: Results of bacterial count (log CFU)

    Click here to view
    Figure 1: Box plot showing the log CFU for different groups

    Click here to view
    Figure 2: Pie chart showing the % of cleaning score for different groups

    Click here to view

      Discussion Top

    The current study investigated the effect of orange oil gutta-percha solvent in combination with NaOCl or BAG and their effect on the cleanness of root canal after RCR as well as their antibacterial effect. Based on the current results, the null hypothesis was rejected as the addition of NaOCl and BAG resulted in significant effect on the bacterial count and the cleanness of root canal after RCR.

    The effect of current irrigation protocol was investigated against E. faecalis bacteria as it is the most common bacterial species associated with endodontically treated teeth. Additionally, it is considered the most resistant bacteria to disinfecting medicaments in endodontics, together with the calcified biofilm developed in root dentine makes the RCR a success doubtful process.[18],[19]

    Using a potent antibacterial such as sodium hypochlorite may help reduce bacterial count and improves the cleanness of root canal.[20] Despite its popularity, NaOCl lacks the potential to remove or dissolve gutta-percha. Orange oil solvent was used during RCR to solubilize gutta-percha with no harm on dentine. The use of orange oil has been recommended as this solvent demonstrated to be effective in removing gutta-percha or different types of sealers in addition to being biocompatible.[21]

    The use of solvents together with irrigant was proven to be effective against various types of bacteria; the orange oil was found to be more potent with E. faecalis as neat extract than the methanolic extract.[22] This is in line with the report of Parekh et al. which states that some traditional medicines are more effective when used alone.[23]

    In the current study, there was no significant difference between using orange oil alone or with additives; however the addition of BAG had a significant effect in reduction of bacterial count in comparison to orange oil + NaOCl. However, all groups resulted in decreasing bacterial count compared with incubated bacterial dishes after the use of the orange oil solvent, which can be said to have potential antimicrobial activities.[24]

    NaOCl is potent antibacterial irrigant with several advantages in removing smear layer and thus cleans root canal effectively.[25] The action of NaOCl depends on its proteolytic activity, which helps dissolve inorganic materials. Furthermore, the free radicals elaborated form NaOCl oxidizing capabilities may has a strong antibacterial activity.[26] However, those effects were diminished when combining orange oil and NaOCl as it showed the least capability in the removal of remaining gutta-percha and organic remnants. Furthermore, the alkaline pH of NaOCl was neutralized with the weak acidic nature of orange oil.[27] NaOCl effect may also be counteracted after the use of EDTA in between files, which result in the formation of parachloroaniline (PCA). That may explain the reduction of antibacterial activity recognized upon the use of this mixture.[28],[29] Clinically, this precipitate may occlude the dentinal tubules impairing cleaning and sealing of the root canal system. The fact that the combination of both NaOCl and EDTA precipitates may have made it more difficult to readily flush out the PCA from the canal and occluded dentinal tubules.[30]

    The presence of orange oil solvent alone or with BAG during endodontic retreatment softens the gutta-percha and forms slurry that may penetrate the dentine tubules, thus obscuring proper cleaning of the root canal walls. Although that combination facilitates the penetration of the retreatment instruments and the removal of filling materials without the need to use the irrigating solution, it may be difficult to completely remove the filling remnants.[17] This could explain the presence of remnants of gutta-percha and precipitates of BAG found in SEM images [Figure 3].
    Figure 3: Representative sectional scanning electron microscopy images showing remnants of gutta-percha after root canal retreatment in (A) coronal and (b) middle root section

    Click here to view

      Conclusions Top

    A combination of orange oil and NaOCl might counteract the effect of both solutions. The addition of BAG did not affect the antibacterial efficiency of orange oil during RCR.


    Not applicable.

    Financial support and sponsorship


    Conflicts of interest

    There are no conflicts of interest.

    Authors’ contributions

    RA: conceptualization, methodology, investigation, writing original draft; HB: conceptualization, methodology, investigation, writing original draft; AA: methodology, investigation, visualization, formal analysis, writing review and editing. All authors reviewed the manuscript and gave final approval.

    Ethical policy and institutional review board statement

    The current study protocol ethical approval number is 37-3-2022 granted from the ethics committee of Cairo University. A written ethical informed consent was signed by all the participants for the use of extracted teeth in anonymous research purposes. All the guidelines and regulations were followed during all the steps of the current study.

    Patient declaration of consent

    Not applicable.

    Data availability statement

    The raw data required to reproduce these findings are available upon reasonable request for the corresponding author. The processed data required to reproduce these findings are available upon reasonable request from the corresponding author.

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      [Figure 1], [Figure 2], [Figure 3]

      [Table 1], [Table 2]


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