March 2025 Vol. 14 No. 1 Postgraduate Medical Journal of Ghana A COMPARATIVE SCANNING ELECTRON MICROSCOPIC STUDY OF THE EFFICACY OF THREE GUTTA-PERCHA SOLVENTS USED DURING ENDODONTIC RETREATMENT Goka RY1*; Nyako EA2; Ampofo PC2; Hewlett SA2; Acheampong AO34; Ndanu TA3; Konadu AB2; Adu-Ampomah NF2; Ankoh SE2 1Dental Department, Ho Teaching Hospital, Ho, Ghana; 2Department of Restorative Dentistry; 3Department of Community and Preventive Dentistry, University of Ghana Dental School, College of Health Sciences, University of Ghana, Accra, Ghana; 4Department of Oral and Maxillofacial Surgery, KNUST School of Medicine and Dentistry, Kumasi, Ghana Abstract Objective: This study sought to compare the number of unfilled dentinal tubules in the root canals using a Scanning Electron Microscope (SEM) after the use of K-files and the respective solvents (Chloroform, Eucalyptol, D-limonene). Methodology: The study was a prospective comparative study. Single-rooted teeth extracted because of various dental conditions were root-treated using the rotary ProTaper system and stored in normal saline. After three months, the teeth were randomly assigned to the three solvent groups (Chloroform, Eucalyptol, and D- limonene). Gutta- percha was removed using the solvent and K-files. The teeth were then divided longitudinally and the root canal surfaces (apical third, middle third, and coronal third) were examined using a scanning electron microscope to determine the number of unfilled dentinal tubule orifices per unit area. Results: D-limonene had the greatest number of unfilled dentinal tubules across the three segments of the divided teeth while Chloroform had the least number of unfilled dentinal tubules Conclusion: D-limonene had superior gutta-percha removal abilities as compared to Eucalyptol and Chloroform. Key words: Endodontic Retreatment, Chloroform, Eucalyptol, D-limonene, SEM Introduction Endodontic retreatment is a procedure performed on a previously root-treated tooth which requires additional new endodontic treatment to achieve a successful outcome.1,2 Its main objective is to remove all the obturation material from the root canal system and to regain access to the apical third of the root canal system.3 Remnants of the previous obturation material may harbour microorganisms, cause their growth and multiplication and subsequently lead to failure of the retreatment.4,5 The clinical success rate of endodontic retreatment has been estimated to vary between 50% and 90%.6 Endodontic retreatment is indicated in any of the following scenarios: when clinical symptoms are present, when radiographic signs of failure are shown, when elective root canal treatment is indicated, and when endodontic treatment is incomplete even if clinical or radiographic signs are not present.3 Gutta-percha (GP) has been the most used root canal obturation material for more than a century.7,8 In Ghana 98.4% of dentists studied, used gutta-percha as a root canal obturation material.9 Several techniques can be employed to remove gutta- percha from the root canals during endodontic retreatment. e.g. the use of heated instruments, mechanical e.g. rotary and hand files, ultrasonic tips and files, chemical e.g. solvents, lasers or a combination of any of these.3,8,10 Regardless of the GP removal technique used, studies have shown that the root canal dentine walls are never completely free of GP.11,12,13 In a study by Ampofo et al. in Ghana9, 61.3% of respondents reported using hand files in combination with solvents and Gates-Glidden burs to remove gutta- percha during re-endodontic treatment. Chloroform, Eucalyptol, and Orange oil (D-limonene) were found to be the three most commonly used solvents by Ghanaian dentists9. Dental gutta-percha is composed of 18.9% to 21.8% gutta-percha, 59.1% to 75.3% zinc oxide filler material, 1.5% to 17.3% metal sulphates which confer radio- opacity, and 1.0% to 4.1% wax and/or resin which act as plasticizers.14,15,16 Gutta-percha is a non-polar material. Effective gutta-percha solvents are weakly polar or non- polar. These solvent molecules can effectively penetrate the gutta-percha polymer strand and cause their separation and subsequent softening or dissolution.17 Chloroform is a polar solvent but has weakly non-polar properties. Both D-limonene and Eucalyptol are non- polar solvents.18 When solvents are applied to a polymer, the polymer absorbs the solvent and swells. The swelling softens the polymer and when more solvent is added, dissolution begins.19 Chloroform and Eucalyptol are the most widely used solvents by dentists worldwide but they have some undesirable properties. Chloroform has been categorized as a 2B carcinogen by the International Corresponding Author: Dr. Ruby Yayra Goka Dental Department, Ho Teaching Hospital Email Address: rubygoka@gmail.com Conflict of Interest: None Declared This is an Open Access Article under the CC BY License 22 doi:10.60014/pmjg.v14i1.400 mailto:rubygoka@gmail.com March 2025 Goka RY et al. Efficacy of Three Gutta-Percha Solvents Agency for Research of Cancer.20 Eucalyptol has a pungent odour which is discomforting to some patients and needs to be heated before it can soften gutta-percha maximally.21 These undesirable properties have led many dentists to turn to alternative gutta-percha solvents from the essential oil family. D-limonene is refined orange oil.22 It is safe, biocompatible, has low cytotoxicity, and is non-carcinogenic.22 In dentistry, it has been used for the dissolution of zinc oxide cemented root fillings.7,22 This study sought to compare the number of unfilled dentinal tubules in the root canals using a Scanning Electron Microscope (SEM) after the use of K-files and the respective solvents (Chloroform, Eucalyptol, D- limonene). Materials and Methods This was a prospective comparative study which was performed on 42 extracted maxillary single-rooted whole human teeth which had been extracted because of poor periodontal support. The clinical component of the study was carried out in the Clinical Simulation Laboratory (Phantom Head Clinic) of the Restorative Department of the University of Ghana Dental School (UGDS), Accra. The Scanning Electron Microscope evaluation and analysis was carried out at the Environmental and Safety Engineering Department of the University of Mines and Technology (UMaT), Tarkwa. Adult maxillary central and lateral incisors with lengths between 19mm to 25mm were included in the study. Teeth with the following characteristics were excluded from the study: root or crown fractures, the presence of internal or external resorption, caries, root canal or pulp chamber calcifications, dilaceration of roots, open apices, previously root-treated teeth, and cervical tooth surface loss that involved the pulp. The teeth used in the study were obtained from the Tooth Bank of the Oral Diagnosis and Maxillofacial clinics of the University of Ghana Dental School, Korle- Bu. A total of 42 whole teeth comprising 24 central incisors and 18 lateral incisor teeth were selected. The teeth were transferred into a storage container containing 10% formalin. Soft tissue and calculus were manually removed from the tooth surfaces with a universal scaler. A periapical x-ray machine (Carestream CS2 100, Japan) was used to take digital periapical x-rays using the paralleling technique to confirm the patency of the canals and also to confirm the absence of internal resorption. The cleaned teeth were subsequently embedded in wax moulds that contained a mixture of dental plaster and sawdust to enable easy handling of the teeth during the root canal procedure. Endodontic treatment was carried out using the DTE Endo Radar Plus Endodontic Motor (Guilin Woodpecker Medical Instrument Company Limited, China) and ProTaper Universal Files (Dentsply Maillefer, Switzerland) and using the ProTaper crown down filing technique. Irrigation was done with 2.5% sodium hypochlorite (Milton, Procter and Gamble, United Kingdom) using a size 27-gauge side-vented needle (Eoskyo, Guangzhou, China). Finishing file F1 was used to complete the shaping and cleaning of the lateral incisor canals and Finishing files F1 and F2 were used for the central incisor canals. 17% ethylenediamine tetraacetic acid (EDTA), (Prevest DenPro Limited, India) was used to lubricate the canals during filing. A final irrigation of the canal was done with 2.5% sodium hypochlorite. The canals were dried with paper points (Technical &General Ltd, London, England) and master cones corresponding to the finishing files were lightly coated with Sealapex (Kerr, Italy) and positioned into the canal. Excess gutta-percha was removed with the use of a heated instrument. The access cavities were restored with Glass Ionomer Cement (Prevest DenPro Limited, India). The teeth were taken out of the moulds with the sawdust and plaster fillings and cleaned. Post-obturation x-rays were then taken to confirm the canal spaces were well-obturated and without voids. The root-treated teeth were kept in normal saline at room temperature for three months to simulate intraoral conditions. After three months, the teeth were separated into two groups; maxillary central incisors and maxillary lateral incisors. A simple randomisation technique was used to allocate the root-treated teeth into the three solvent groups i.e., Chloroform (VWR Chemicals BDH, France), Eucalyptol (Silver Bird Eucalyptus oil, Bells, Sons & Co. United Kingdom), and D-limonene (Carvene, Prevest DenPro Ltd. India). This process involved first writing the names of the solvents to be used i.e., Chloroform (VWR Chemicals BDH, France), Eucalyptol (Silver Bird Eucalyptus oil, Bells, Sons & Co. United Kingdom), and D-limonene (Carvene, Prevest DenPro Ltd. India) on slips of paper, scrambling them up, and putting them in a bowl. With the operator’s eyes closed, a slip of paper was picked from the bowl. The name of the solvent on the piece of paper (Chloroform) was assigned to group one. The second solvent (Eucalyptol) was assigned group two, and the third solvent (D-limonene) was the third group. At the end of the allocation, each solvent group comprised of eight maxillary central incisors and six maxillary lateral incisors. The crowns of the teeth were sectioned to obtain a uniform length of 18mm from the apex to standardize the root lengths. Removal of the residual coronal Glass Ionomer Cement was done with a high-speed round diamond bur. Removal of the coronal 2-3mm of gutta- percha within the canal to create a reservoir for the root canal solvents was done using sizes 1 and 2 Gates- Glidden burs (Henry Schein, Switzerland). This is an Open Access Article under the CC BY License 23 doi:10.60014/pmjg.v14i1.400 March 2025 Vol. 14 No. 1 Postgraduate Medical Journal of Ghana Two drops (10µL each) of the selected solvent were placed in the created reservoir. The same dropping pipette was used for all three solvents to ensure the same amount of solvent was delivered each time. The solvents were left in the created reservoirs for two minutes to allow the solvent to wet the surface of the gutta-percha adequately, soften the gutta-percha and percolate down within the canal. A crown-down instrumentation technique was used to remove the gutta- percha from the root canals, starting with a size 50 K- file. When the resistance to the progress of the file was encountered, the canal was irrigated and the next lower file was used to remove the GP. Debris from filing and softened GP were rinsed out using 2.5% sodium hypochlorite with a side-vented 27-gauge needle. During this process, the solvent was replenished as required. Filing to clean the walls with the K-files followed by irrigation and replenishing of the solvent continued until there were no more gutta-percha particles on the flutes of the file. A pair of magnification loupes (3.5X, Aries Outlet, China) was used by the operator in examining the file to ensure there were no traces of gutta-percha present on the flutes. The canal was then irrigated with 17% EDTA, (Prevest DenPro Limited, India) and a final irrigation of the canal was done with 2.5% sodium hypochlorite. After complete removal of the gutta- percha from the root canals of the teeth, longitudinal grooves of 1mm depth were prepared on the labial and palatal surfaces of the teeth with a diamond bur and separating discs (NTI-Kahla GmbH, Germany) in a laboratory handpiece (Foshan Tuo Kang Medical Instruments Co., Ltd. Guangdong, China) was used to divide the teeth longitudinally. The sectioned halves were then placed into labelled Eppendorf vials. The sectioned halves were attached to a conductive carbon adhesive tape to enable the samples to adhere to stud holders which were then mounted on a SEM specimen holder. Seven samples at a time were attached to individual studs and mounted on the SEM specimen holder. The specimen holder with the attached studs was then placed in a vacuum chamber in a sputter machine (Quorom Sputter Coater, UK). The chamber was filled with an inert gas (Argon) at 0.5 mbar for thirty minutes. The samples were sputtered with a thin layer of gold-palladium at a current of 10 mA for 3 minutes. Argon was used to prevent the gold-palladium alloy from reacting with any other element in the chamber. The gold-palladium coat enhanced the production of secondary electrons by the samples. The specimen holder with the gold-palladium coated samples was then transferred to the high vacuum sample chamber of the SEM machine (EVO MA15, ZEISS, Oberkochen, Germany). The samples were then positioned properly for the maximum effect of the primary beam of electrons. After a vacuum was generated, the samples were viewed under a magnification of X2500 that corresponded to an area of 9845.165µm2. For evaluation purposes, the samples were digitally divided into cervical, middle, and apical zones. The central region of each zone was located digitally and a SEM micrograph was taken (Figures 1, 2, and 3). The number of unfilled dentinal tubules per unit area was quantified using the ImageJ software (National Institutes of Health and the Laboratory for Optical and Computational Instrumentation (University of Wisconsin), United States of America). Figure 1: A scanning electron micrograph of the apical third of a root canal treated with Chloroform. Blue arrows show unfilled dentinal tubules. Red arrows show filled dentinal tubes. Figure 2: A scanning electron micrograph of the apical third of a root canal treated with Eucalyptol. Blue arrows show unfilled dentinal tubules. Red arrows show filled dentinal tubes. This is an Open Access Article under the CC BY License 24 doi:10.60014/pmjg.v14i1.400 https://en.wikipedia.org/wiki/National_Institutes_of_Health https://en.wikipedia.org/wiki/National_Institutes_of_Health March 2025 Goka RY et al. Efficacy of Three Gutta-Percha Solvents Figure 3: A scanning electron micrograph of the apical third of a root canal treated with D-limonene. Blue arrows show unfilled dentinal tubules. Red arrows show filled dentinal tubes. Data Analysis The Statistical Package for Social Sciences (SPSS) Version 22 was used to analyse the results statistically. The medians of the unfilled dentinal tubule orifices of Results Comparison of the Unfilled Dentinal Tubules Between Solvent Type for Each Tooth Segment Using the Kruskal-Wallis test with the level of significance set at 0.05 , there was a significant difference in the median numbers of dentinal tubules free of gutta-percha in the coronal third (p= 0.007), middle third (p= 0.004), and apical third (0.001) of the teeth treated with the different solvents. Discussion SEM Comparison of the Different Tooth Segments SEM analyses in each of the segments of the divided teeth (coronal, middle, and apical third) showed there were significant differences in the number of unfilled dentinal tubules among the tested solvents (Table 1.0). Across the three tooth segments, D-limonene was the solvent with the most unfilled dentinal tubules. Chloroform was the solvent with the least unfilled dentinal tubules across all three tooth segments. In this study, D-limonene and Eucalyptol were found to be better solvents for the softening and removal of gutta- percha across the length of the tooth as compared to Chloroform. In a similar study by Scelza et al.23, although D-limonene had the most number of unfilled dentinal tubules per mm2 in the middle and apical thirds (6332.35/ 3552.05) compared to Chloroform (6160.46/ Table 1.0: Comparisons of the median values of the numbers of unfilled dentinal tubules in the different segments of the divided teeth across solvent groups Tooth Segment Solvent Median numbers of unfilled dentinal tubules (50/ numbers) Interquartile Range (25-75/ numbers) p-value Coronal Chloroform 79.00 35.75-102.00 0.007* Eucalyptol 128.00 91.00-160.25 D-limonene 134.50 94.75-236.25 Middle Chloroform 62.50 25.50-100.50 0.004* Eucalyptol 107.00 51.75-147.75 D-limonene 149.50 96.25-190.75 Apical Chloroform 37.00 22.25-52.75 0.001* Eucalyptol 62.00 50.00-166.25 D-limonene 99.50 65.50-119.50 *and bold indicate statistically significant difference using the Independent Kruskal-Wallis test (p<0.05) the three solvents (Chloroform, Eucalyptol, and D- limonene) in the three distinct root canal parts (coronal, middle, and apical) were compared using the Kruskal- Wallis test. The level of significance was set at p < 0.05. Ethical Considerations Ethical approval was obtained from the Ethics and Protocol Review Committee of the Korle Bu Teaching Hospital (KBTH-STC 00093/2021). Written permission was obtained from the Clinical Director of the University of Ghana Dental School to use teeth from the school’s Tooth Bank. 3575.53) and Eucalyptol (6286.96/ 3501.29), there was no statistical significant differences (p>0.05) in the cleaning abilities of the three solvents. These findings differ greatly from the current study where significant differences were obtained in the coronal, middle, and apical third of the teeth with D-limonene having the greatest number of unfilled dentinal tubules and Chloroform having the least number of unfilled dentinal tubules. The difference might be attributed to the fact that in the Scelza study23, a predetermined time of 5 minutes (300 seconds) was set for each solvent so it is possible the gutta-percha was not completely removed This is an Open Access Article under the CC BY License 25 doi:10.60014/pmjg.v14i1.400 March 2025 Vol. 14 No. 1 Postgraduate Medical Journal of Ghana from the root canals, while in our study, removal of the gutta-percha continued until no gutta-percha remnants/ debris was visibly present on the file. The time for the complete removal of gutta-percha varies for studies. In a study by Imura et al.2, a predetermined maximum time of 20 minutes (1200 seconds) was set for the removal of gutta-percha. However, in a study by Kasam et al.10, no time limits were set. The mean retreatment times in that study were between 261 seconds to 523 seconds for the various solvents used.10 No time limits were set in this study to simulate a clinical setting, where all gutta- percha must be removed from the root canal before it can be reshaped and refilled. In the current study, the obturated teeth were left in normal saline at room temperature to simulate intraoral conditions for a period of 3 months. The storage time of the teeth varied in studies from periods of between 1 to 2 weeks to 2 to 3 months.2,23, 24,25 A longer period of 3 months was used in this study because anecdotal evidence by the authors of this paper based on their years of clinical practice, has shown that for gutta-percha that is laterally condensed, it is possible in some cases, to remove the gutta-percha whole/ as an intact piece after about 1 to 2 weeks. In this study, the apical third had the least dentinal tubules free of gutta-percha compared to the middle and coronal thirds. This is similar to results obtained by Scelza et al23 and Horvath et al25 in studies done in Brazil and Germany respectively. The reason for this could be that anatomically, the density of dentinal tubules decreases from the coronal to the apical segments of the tooth26,27 so fewer dentinal tubules (filled or unfilled) are expected at the apical third of the root compared to the coronal and middle thirds. Another reason is that anatomically, the root canal narrows as it approaches the apex. This narrowing promotes the accumulation and compaction of debris in the apical third of the root canal if recapitulation isn’t done during treatment resulting in more filled dentinal tubules. A third reason is that there is better adherence of the gutta-percha to sealer and the dentinal walls in the apical third of the tooth because the master cone fits snuggly in the apical third. Adequate quantities of the solvent might not reach these apical regions of the tooth to soften the gutta-percha. The lower number of unfilled dentinal tubules in the apical region is consistent with studies that have demonstrated that despite which gutta-percha removal technique is used, the apical third of the root canal is the least instrumented part of the root canal system.28,29,30 Hovarth et al25 who also obtained their teeth from a tooth bank, postulated that the dentinal tubules in the apical third of the roots might have been sclerosed since the ages of the samples were not known. In a study done in Switzerland by Paque et al31, they concluded that tubular sclerosis in the apical part of the root canal could account for the lesser numbers of unfilled dentinal tubules in their study. Since the ages of the extracted teeth were not known in the present study, dentinal tubular sclerosis might also account for the lesser numbers of unfilled dentinal tubules in the apical region. Another reason for the higher efficacy of D-limonene across all the tooth segments might be because D- limonene is a non-polar solvent,18 its molecules might be able to penetrate the non-polar gutta-percha molecule more effectively than Chloroform and Eucalyptol. It also has the additional benefit of dissolving zinc oxide filler materials which account for 59.1% to 75.3% of dental gutta-percha,14,15,16 so it is able to soften and dissolve most of the zinc oxide filler material which makes it easier for gutta-percha removal. The main limitation of this study was the high cost of SEM scanning the eighty-four divided tooth samples. Further studies will be needed to be conducted on a larger sample size. Of the three solvents, Eucalyptol is the most readily obtained on the Ghanaian market. Though D-limonene has more desirable properties, only a few dental supply companies stock it. Compared to Eucalyptol, the initial purchasing cost is more expensive but since fewer drops will be required per procedure, it might last longer than Eucalyptol and in the long term and might prove be more cost-beneficial. Conclusion D-limonene had the greatest number of unfilled dentinal tubules across all three tooth segments as compared to Chloroform and Eucalyptol. and is therefore recommended for use in endodontic retreatment. Further research on a larger sample size will however need to be done. Acknowledgements Heartfelt appreciation to my supervisors and staff of the University of Ghana Dental School and to the staff of the University of Mines and Technology. Data Availability Data is available upon written request to the principal investigator. References 1. Cordeiro KF, Silva DF, Filho ID, Castro FPL. Current protocols for endodontic retreatment : A Review. J Odontol 2018; 2: 111–116. 2. Imura N, Kato AS, Hata GI, Uemura M, et al. 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Intraradicular bacteria and fungi in root-filled, asymptomatic human teeth with therapy-resistant periapical lesions: A long-term light and electron microscopic follow-up study. J Endod 1990; 16: 580–588. 31. Paqué F, Luder HU, Sener B, Zehnder M. Tubular sclerosis rather than the smear layer impedes dye penetration into the dentine of endodontically instrumented root canals. Int Endod J 2006; 39: 18–25. This is an Open Access Article under the CC BY License 27 doi:10.60014/pmjg.v14i1.400