Potential of Trichloroacetic Acid in Smear Layer and Calcium Hydroxide Removal in Root Canals–Exploratory Pilot Studies In Vitro

Abstract

Introduction and aims

This study investigated whether trichloroacetic acid (TCA) can function as an adjuvant solution to facilitate pulp dissolution and removal of smear layer and calcium hydroxide (Ca(OH)₂) during endodontic procedures.

Methods

For pulp dissolution studies, fresh pulp was weighed before and after immersion in 90% TCA and/or 6% NaOCl. For smear layer or Ca(OH)₂ removal, each study utilized 25 extracted human teeth with prepared root canal systems. For Ca(OH)₂ removal, canals were filled with Ca(OH)₂ and stored at 37 °C for 14 days. Teeth were divided into five groups (n=5/group) and the canal irrigated with various solutions. Group I: saline; Group II: 17% EDTA; Group III-V: 90% TCA for 15, 30 or 60 seconds, respectively. Samples were subjected to scanning electron microscopy (SEM) or spectrum analysis.

Results

Under our experimental conditions, NaOCl alone consistently dissolved more pulp than TCA at all time points (1, 3, and 5 minutes) under static conditions at RT (P < .01 or < .001). Some TCA groups had significantly less smear layer than the saline or EDTA group in the coronal and middle thirds (P < .05 to < .001). In the apical third, there was significantly less smear layer in the TCA 15-second group than the control or EDTA group (P < .01 or P < .001). In Ca(OH)₂ removal studies, the coronal third showed that TCA 30-second group and middle third TCA 60-second group had significantly less Ca(OH)₂ than the EDTA group (P < .05).

Conclusion

These findings warrant further studies with a larger sample size to validate the potential use of TCA as an adjunctive agent to facilitate smear layer and Ca(OH)₂ removal from root canal walls,

Clinical relevance

This is the first study to evaluate whether TCA can be used as an adjunctive endodontic irrigant. Our findings suggest that a brief treatment with TCA is more effective than EDTA for canal smear layer removal and may facilitate Ca(OH) ₂ removal.

Introduction

Successful non-surgical endodontic therapy depends on optimal management of multiple procedural steps, including thorough cleaning and shaping of the root canal system to achieve effective infection control followed by a final hermetic seal. Various innovative approaches have been proposed and tested to enhance the cleaning and shaping procedures, such as the use of lasers and nanobubble water, as we have recently reported.¹ However, clinical challenges remain associated with smear layer removal and the elimination of intracanal medicament such as calcium hydroxide (Ca(OH)₂).

Although the clinical benefits of smear layer removal with regard to treatment outcomes remain uncertain,² it is widely regarded as a standard practice.^3,4 The use of ethylenediaminetetraacetic acid (EDTA) during canal preparation has been shown to enlarge dentinal tubule openings, as evidenced by scanning electron microscope (SEM) studies.^5,6 Additionally, EDTA increases root dentin permeability, thereby enhancing the efficacy of intracanal medicaments.⁷

Ca(OH)₂ is the most commonly used intracanal medicament in endodontics. Despite its widespread application, it presents certain limitations. One well-documented drawback is its incomplete removal, even after thorough irrigation with saline, NaOCl and EDTA.⁸ Although ultrasonic agitation of NaOCl has been shown to be more effective than irrigation alone, it still fails to completely eliminate residual Ca(OH)₂.⁹ Remaining Ca(OH)₂ can adversely affect the setting properties of zinc oxide-eugenol-based sealers by interacting with eugenol, resulting in a more brittle, granular structure and a compromised apical seal.¹⁰ Additionally, residual Ca(OH)₂ has been shown to have a negative effect on the bond strength of AH-26, AH Plus and EndoSequence BC Sealer.^11,12 Therefore, when Ca(OH)₂ is used during root canal therapy, its persistence within the canal may negatively impact the quality of obturation and potentially compromise treatment outcomes.

Other acids besides the gold standard EDTA have been studied and showed varying levels of efficacy, such as citric acid, maleic acid, and phosphonic acid.13, 14, 15 Trichloroacetic acid (TCA), which has been used in endodontics primarily for the management of external resorption, has been reported for other endodontic applications, including use as a canal irrigant to facilitate canal navigation, preparation, and Ca(OH)₂ removal.^16,17 However, experimental investigation of the potential of TCA for these clinical applications is lacking.

TCA is a soft tissue cauterizing agent widely used in medicine for treatment of various dermatological conditions.^18,19 It reduces corneocyte cohesion and keratinocyte plugging, contributing to its therapeutic effects.²⁰ In dentistry, 90% TCA is employed for the management of external cervical resorption, as it can effectively infiltrate small channels and recesses to eliminate invasive tissue from areas inaccessible to mechanical instrumentation.^21,22 This effect is primarily attributed to its strong protein-denaturing and tissue-dissolving properties as a potent organic acid.²³ Because of this property, TCA has also been used for gingival haemorrhage control and as an adjunct in pulp capping procedures.^24,25 Low concentrations (eg, 0.5%) of TCA have been reported to promote the regeneration of oral soft tissues.²⁶ To date, no adverse effects have been reported with the use of TCA in the treatment of oral tissues.

To explore additional potential applications of TCA in endodontic treatment, we conducted a pilot in vitro study to evaluate whether TCA can facilitate pulp digestion and serve as a final brief rinse for the removal of smear layer and residual Ca(OH)₂ from root canal walls. We hypothesized that the coagulative properties of TCA, together with its strong acidity, would enhance the pulp tissue digestion when used in combination with NaOCl and improve the removal of smear layer and Ca(OH)₂. To the best of our knowledge, this is the first study exploring these potential applications of TCA.

Materials and methods

Sample collection

Extracted teeth were collected according to an exempt protocol approved by the Institutional Review Board of UTHSC (#19−06855 NHSR). To investigate multiple aspects of potential TCA capacities, the sample size was determined based on an exploratory pilot design to ensure the project could be feasibly conducted.

Pulp tissue dissolution experiment

Freshly extracted human teeth were collected and sectioned open with a flexible serrated 365 cutting disk. The pulp was removed and then placed in saline in 1.5 mL microcentrifuge tubes until use. Pulp tissue was cut into fragments weighing approximately 10 mg each. TCA (90%, Delasco, Plano TX, USA) and 6% NaOCl were used for the experiment. A lower concentration of NaOCl was used because our pilot studies showed that 8.25% was too strong and dissolved pulp too rapidly for a meaningful time-based comparison.

A post hoc power analysis based on the current study data yielded power values ranging from 0.762 to 0.990, indicating that the sample size was sufficient for the present pilot study.

Experimental groups:

Incubation at room temperature (22 °C), static condition:

• Group 1. Dissolution of pulp by NaOCl (0.1 mL) alone for 1, 3, or 5 minutes.

• Group 2. Dissolution of pulp by TCA (0.1 mL) alone for 1, 3, or 5 minutes.

• Group 3. Dissolution of pulp by TCA (0.1 mL) for 1 min then NaOCl (0.1 mL) for 1, 3, or 5 minutes.

• Group 4. Dissolution of pulp by TCA (0.1 mL) + NaOCl (0.1 mL) for 1, 3, or 5 minutes. TCA and NaOCl were mixed homogeneously.

Incubation at 37 °C, agitated conditions:

• Group 5. Dissolution of pulp by NaOCl (0.1 mL) alone for 1 or 3 minutes.

• Group 6. Dissolution of pulp by TCA (0.1 mL) alone for 1 or 3 minutes.

• Group 7. Dissolution of pulp by TCA (0.1 mL) + NaOCl (0.1 mL) for 1 or 3 minutes. TCA and NaOCl were homogeneously mixed.

Weighing samples: The pulp sample was gently blotted with Kimwipe paper to remove excess surface liquid and then inserted into an empty 1.5 mL microcentrifuge tube. The tube containing the pulp sample was placed on the scale which was reset to 0. The scale had an accuracy of 0.01 mg (Mettler-Toledo XS205 Dual Range Analytical Balance, Columbus, OH, USA). After which, 0.1 mL of NaOCl and/or TCA were added into the tube and the tube lid was closed. Samples stayed at room temperature for the specified incubation period. At the end of each time point, the solution was aspirated out thoroughly, and the remaining pulp weighed again. For agitation groups, the tube was placed on a shaker/incubator (Eppendorf Thermomixer 5436 Incubator Shaker, Enfield, CT, USA) set at 37 °C with 700 rpm. This shaker was used to simulate, in a simplified manner, the agitation produced by the EndoActivator during intracanal irrigation. Although it does not fully replicate clinical conditions, all experimental groups were subjected to identical treatment protocols to ensure valid comparison and to detect any meaningful differences between groups.

Smear layer removal experiment

This experiment described in this section was performed by one investigator. Twenty-five human anterior and premolar teeth with single canals, relatively straight roots (9-14 mm long), and small apices less than #30 were collected to minimize anatomical variability. They were decoronated at the CEJ to expose the root canal system. The teeth were then accessed, patency was gained by protruding a #10 C-file 1.0 mm out of the apical foramen, and working length was measured to 0.5 mm within the apex. Each tooth was instrumented to a size 30.06 in the same sequence. The sequence started with 10 K-file to patency, followed by a 13.02 Path File (Dentsply, Long Island City, NY, USA), a 17.04 Brasseler EndoSequence CM file (Savannah, GA, USA), and 20.04, 20.06, 25.06 and 30.06 DC Taper H files (SS White, Lakewood, New Jersey, USA), while irrigating with a full 1.0 mL of 8.25% NaOCl between each file (the clinically available maximum NaOCl concentration was used for the purpose of canal cleaning and shaping). A Max-1-Probe 30G side vented needle (Dentsply) was used for NaOCl irrigation. The canals were kept flooded with NaOCl during all instrumentation.

Afterward, each group of teeth had a final irrigation protocol specific to the group. A Max-1-Probe 30G side-vented needle was also used for both the TCA and sterile saline administration during the final rinses. In the procedures below, 8.25% NaOCl, 17% EDTA and 90% TCA were used. We used 8.25% NaOCl to irrigate the root canal based on reported studies aiming at stronger disinfection capacity at this concentration.27, 28, 29

• •Group I, Control (n = 5): Final rinse with NaOCl for 15 seconds, then 2 mL of sterile saline.
• •Group II, EDTA (n = 5): Final rinse with NaOCl for 15 seconds, EDTA for 60 seconds, then 2 mL of sterile saline.
• •Group III, TCA 15 seconds (n = 5): Final rinse with NaOCl for 15 seconds, TCA for 15 seconds, then 2 mL of sterile saline.
• •Group IV, TCA 30 seconds (n = 5): Final rinse with NaOCl for 15 seconds, TCA for 30 seconds, then 2 mL of sterile saline.
• •Group V, TCA 60 seconds (n = 5): Final rinse with NaOCl for 15 seconds, TCA for 60 seconds, then 2 mL of sterile saline.

Ca(OH)₂ removal experiment

This experiment described in this section was performed by one investigator. Twenty-five single-canaled human teeth selected with the same criteria above were decoronated at the CEJ. Working length was established by extruding a #10C file out of the apex. All canals were instrumented using the same protocol with a combination of a Brasseler EndoSequence Scout file (17.04) and SS White DC Taper files (20.04, 20.06, 25.06, and 30.06) until the tips of the files were visualized beyond the apex. Canals were kept flooded with 8.25% NaOCl during instrumentation, and final irrigation was completed.

Teeth were then dried with paper points and canals were filled with Ca(OH)₂ (Ultracal XS, Ultradent Products Inc, South Jordan, UT, USA) by inserting a purple capillary tip and expressing until Ca(OH)₂ extruded out the apex to ensure the entire canal was filled with Ca(OH)₂, followed by back filling. Fuji Triage (GC Dental, Alsip, IL, USA) was used to cover both the orifice and apex. Teeth were stored in phosphate-buffered saline at 37 °C for 14 days. After which, teeth were randomly divided into 5 groups (n = 5/group) and re-accessed and irrigated as the following using 8.25% NaOCl, 17% EDTA and 90% TCA.

• •Group 1, control, irrigated with saline for 30 seconds (negative control).
• •Group 2, EDTA, irrigated with EDTA for 60 seconds, agitated with #10C hand file, and rinsed with saline for 30 seconds.
• •Group 3, TCA 15 seconds, irrigated with TCA for 15 seconds, agitated with #10C hand file, and rinsed with saline for 30 seconds.
• •Group 4, TCA 30 seconds, irrigated with TCA for 30 seconds, agitated with #10C hand file, and rinsed with saline for 30 seconds.
• •Group 5, TCA 60 seconds, irrigated with TCA for 60 seconds, agitated with #10C hand file, and rinsed with saline for 30 seconds.

Each irrigation took 1-2 mL per canal of the respective irrigant, which filled the canal during the specified period. The irrigation time for EDTA was restricted to 60 seconds in order to compare with the TCA which is only suited for very short irrigation time.

Specimen processing for SEM

The teeth for smear layer or Ca(OH)₂ removal were then processed for SEM. Teeth were fixed in 2.5% glutaraldehyde (in 0.1 M sodium cacodylate buffer at pH 7.4) and stored at room temperature until the next step. The following steps were all performed at room temperature. Teeth were split longitudinally into two halves using a diamond disc, chisel, and surgical mallet. Split half fragments from the same tooth were kept together and stored in the same 2.5% glutaraldehyde until processing. Halved fragments were placed in 0.2 M sodium cacodylate buffer at pH 7.4 for 1 hour with changes to fresh solution every 20 minutes (3 times), then rinsed in water for 1 minute, followed by dehydration in ascending grades of ethanol (25%, 50%, 75%, and 95%) for 15 minutes each, and a final soak in 100% ethanol for 30 minutes. Lastly, the fragments were immersed in hexamethyldisilazane (HMDS) for 10 minutes and placed in a vacuum overnight.

A few tooth samples were damaged during splitting procedures and were not analysed with SEM. Three teeth per group were randomly chosen for SEM analysis with the EVO HD 15 SEM (Carl Zeiss, Oberkochen, Germany). One half-segment of each tooth was chosen based on the quality of the split. Chosen segments were mounted on aluminium stubs using double-sided copper tape, gold coated and placed in the SEM for image acquisition. To streamline the workload for SEM analysis, three samples per group were selected for SEM imaging.

SEM analysis

Smear layer removal groups

Analysis was accomplished by dividing each specimen into thirds (coronal [C], middle [M], and apical [A]), and two locations per third were chosen at random for imaging at 3000x, 1000x, 500x, 100x, and 50x magnifications. Each location was labelled as C1, C2, M1, M2, A1, or A2. Each segment was examined for the presence of the smear layer using a semi-quantitative grading method. The level of smear layer was graded on a scale of 0-5. Three calibrated investigators independently scored the images.

• •0 = no smear layer
• •1 = sparsely scattered (≤ 10%) layer(s) of crystalline, amorphous materials
• •2 = scattered (10%-30%) layer(s) of crystalline, amorphous materials
• •3 = scattered (30%-50%) layer(s) of crystalline, amorphous materials
• •4 = scattered (50%-80%) layer(s) of crystalline, amorphous materials
• •5 = heavy coverage (>80%) by thick smear layer(s), no dentinal tubule openings seen

Inter-examiner reliability was evaluated to ensure examiner calibration using the intraclass correlation coefficient (ICC) test.

Ca(OH)₂ removal groups

Analysis was accomplished by dividing the specimen into thirds as above, and two locations per third, spaced as evenly as possible, were chosen for imaging at 1000x magnification. The specific imaging site within the location was randomly selected and the acquisition of the images was blinded to experimental groups. To determine whether the debris was Ca(OH)₂ or dentin particles, at least 6 sites per location at 1000x magnification images were subjected to spectrum analysis which was accomplished by using Energy Dispersive Spectroscopy (EDS by Oxford, High Wycombe, UK). This provided elemental and chemical analysis of the sample inside the SEM with the Aztec software on the EDS system.

Barium is used as an opacifier in Ca(OH)₂ paste. Therefore, detection of barium was to confirm the presence of Ca(OH)₂ compared to dentin debris, which does not contain barium. The spectrum analysis revealed the percentage of barium in Ultracal XS present in each analysed site ranged from 15% to 41% showing heterogeneous distribution of barium. Spectrum analysis measures the percentage of barium in each site analysed. Samples were recorded as either having barium-positive or barium-negative. For samples positive for barium, the percentage of barium present was categorized as less than 10%, between 10% and 25%, or greater than 25%. The percentage of barium that was present ranged from 0% to over 60%.

Data analysis

All SEM images were independently scored by three investigators, and their consistency was evaluated and confirmed using the Intraclass Correlation Coefficient (ICC). The ICC test was used to assess the inter-rater reliability of smear layer evaluation. We obtained the ICC value of 0.977, indicating a high level of agreement among the three scorers. The average value of the 3 scorers was used for the statistical analysis.

Data that did not meet the assumptions of normality and homoscedasticity were analysed using the Kruskal–Wallis test (rank sum test), followed by Dunn’s post hoc comparison tests. For data that met normality assumptions, one-way ANOVA on Ranks with Holm-Sidak method was performed. Chi square and rank sum tests were also applied as indicated. Statistical analyses were conducted using SigmaPlot 14.5 or GraphPad Prism 10.0, with a significance level set at P < .05.

For box plot graph presentation, the box plot line within the middle of each box denotes the median. Boxes represent the interquartile range with the first quartile (25th percentile) of the data to the third quartile (75th percentile). Bars in the plot represent the 'whiskers,' which are data outside of the interquartile range (below the 25th and above the 75th percentiles).

Results

Trichloroacetic acid did not enhance the pulp dissolving capacity of NaOCl

Pilot tests were conducted to evaluate pulp dissolution using TCA and NaOCl at room temperature and at 37 °C. Complete tissue dissolution occurred in roughly 10 minutes, so we limited the incubation period to a maximum of 5 minutes. Using 8.25% NaOCl would have been too aggressive, digesting the pulp too rapidly for the experimental setting of this study.

The results are shown in Figure 1. Under static conditions at room temperature, the NaOCl group exhibited significantly greater pulp dissolution than the other groups at 1, 3, and 5 minutes (P < .01 to P < .001), with 1.1- to 1.7-fold differences from other groups (Figure 1A-C).

Fig. 1.

Pulp dissolution after TCA and/or NaOCl treatment. Box plot graphical results for difference in percentage weight reduction after 1 minute (A), 3 minutes (B), and 5 minutes (C). Statistical tests were one-way ANOVA on Ranks with Holm-Sidak method (1, 3, and 5 minutes), N = 4-6, *P < .05, **P < .01, ***P < .001. The box plot line within the middle of each box denotes the median values for each group. The X within each box symbolizes the mean value of each group. The circle standing alone represents data points for each group.

Under agitated conditions at 37 °C, tissue dissolution with TCA + NaOCl (62.2% and 60.2% at 1 and 3 minutes, respectively) was comparable to that with TCA alone and significantly less than NaOCl alone at 3 minutes (P < .001) with 1.1- to 1.5-fold differences from other groups (Figure 1A, and B).

When comparing groups between static (room temperature) and agitated (37 °C) conditions, NaOCl under agitation dissolved pulp significantly more than TCA or TCA + NaOCl under static conditions at 1 minute (P < .01, 1.3- to 1.4-fold differences), and more than TCA, TCA followed by NaOCl, or TCA + NaOCl at 3 minutes (P < .001, 1.4- to 1.7-fold differences) (Figure 1A, and B).

TCA was more effective in smear layer removal than EDTA

Representative SEM images are presented in Figure 2A, showing the smear layer on the canal surface at coronal, middle or apical locations. The semi-quantitative analysis is presented in Figure 2B.

Fig. 2.

Smear layer levels after EDTA or TCA irrigation. (A) Scanning electron microscope analysis of root canal surface. Representative images all at 3k x from each group (GI-V) are shown. Ctrl, control. (B) Semi-quantitative analysis of smear layer. Each SEM image location was scanned at 50x, 100x, 1000x, and 3000x. Scoring data analysed by Kruskal–Wallis test; N = 6, three teeth each had two locations/observations measured. *P < .05, **P < .01, ***P < .001.

There were no significant differences at the coronal, middle, or apical thirds between the saline (control) and EDTA groups, whereas the TCA groups (15 and 60 seconds) had significantly less smear layer than the saline or EDTA group in the coronal third (P < .01 or P < .001, 1.70- to 2.0-fold differences). In the middle third, the TCA groups (15 and 30 seconds) had significantly less smear layer than the saline or EDTA group (P < .05 or P < .01, ∼2-fold differences). There was significantly less smear layer in the TCA 15-second group than the saline/EDTA groups in apical third (P < .01 or P < .001, ∼1.5-fold differences). These findings indicate a brief 15-second TCA rinse is more efficient than EDTA irrigation. In the TCA 60-second group, some areas of the dentin surface exhibited exposed collagen fibres. This indicates the benefit of a 15 second treatment than a longer irrigation time.

TCA was capable of removing Ca(OH)₂

SEM spectrum analysis was used to detect barium in the Ca(OH)₂ paste. Representative SEM images are shown in Figure 3 with coronal, middle and apical thirds for each group. Circled areas are randomly selected spots subjected to spectrum analysis, which provided the percentage of barium in each spot.

Fig. 3.

Detection of barium content on root canal surface by electron microscopy combined with spectral analysis. Representative images all at 1k x from each group (GI-V) are shown. Sites of spectrum analysis are circled with different colours based on the percentage of barium. Absence of barium was indicated by outlining the site of analysis in green, less than 10 % was outlined in yellow, between 10% and 25% was outlined in orange, and greater than 25% was outlined in red.

Based on the readout of the percentage of barium in each spot from the spectrum analysis, we categorized the barium-positive sites as 0% (green), <10% (yellow), 10% to 25% (orange), or >25% (red) and counted the number of spots in each range (see color-coded circles in Figure 3 and legend, and Supplemental Tables). To compare the amount of Ca(OH)2 remaining in the canal in each group, two quantitative approaches were used.

The first approach was by counting the number of barium- positive spots out of all the spots analysed per tooth sample and per group, thereby deriving a percentage of barium-positive sites. The results are presented in Figure 4, showing the total number of analysed sites and those in which barium was detected in the coronal, middle and apical thirds. Using this approach, EDTA and TCA samples had significantly lower barium counts in the coronal and middle thirds than the saline group, indicating the removal of Ca(OH)₂ in these regions (P < .05 to P < .001, 1.4- to 7.5-fold differences). In the coronal third, the TCA 30-second group and in the middle third, the TCA 60-second group showed significantly lower barium-positive counts compared with the EDTA group (P < .05, 2- to 3-fold differences). In the apical third, neither TCA nor EDTA had lower barium counts than the saline controls.

Fig. 4.

Analysis of the number of sites with barium present. Number of sites analysed (blue), Number of Sites with barium present (orange), Percentage of barium present per group (green line). Chi square test analysis was performed. *P < .05, **P < .01, ***P < .001. Thirty-six to forty-five data points analysed per group (3 teeth with 2 locations/observations and ≥ 6 sites per location or ≥ 36 sites per group. See Supplemental Materials). Multiple analysis sites were selected at the coronal third, middle third and apical third of each tooth. The blue bars represent the total number of analysed sites in each group.

The second approach was based on the readout of the percentage of barium in each spot from the spectrum analysis. We calculated the mean percentage of barium at each analysed site based on the spectrum analysis readouts and compared these values between groups, as shown in Figure 5. In the coronal third, all TCA groups showed significantly lower barium percentages at the analysed sites compared to the saline group (P < .001, ∼10-fold differences), whereas the EDTA group had a significantly higher barium percentage than the saline group. In the middle third, all TCA groups had significantly lower barium than the saline group (P < .01 or P < .001, ∼6- to 11-fold differences). Although the EDTA group also showed significantly lower barium levels than the saline group, it did not have lower levels than the TCA groups and, in fact, had significantly higher barium than the TCA 60-second group. In the apical third, none of the EDTA or TCA groups had lower barium than the saline group. These findings indicate that a brief treatment with TCA may be as effective or more than using EDTA except the apical third, which may need validation with a larger sample size.

Fig. 5.

Analysis of the percentage of barium in each analysed site. Rank sum test analysis was performed, *P < .05, **P < .01, ***P < .001. Thirty-six to forty-five data points analysed per group (3 teeth with 2 locations/observations, and ≥ 6 sites per location or ≥36 sites per group. See Supplemental Materials).

Discussion

To our best knowledge, this is the first study to evaluate TCA as a potential adjunctive endodontic irrigant. Our findings suggest that a brief application of TCA is more effective than EDTA in removing the smear layer and may facilitate the removal of Ca(OH)₂. The results partially support our hypothesis regarding the potential of TCA to enhance smear layer and Ca(OH)₂ removal. However, TCA did not improve pulp tissue dissolution when used in combination with NaOCl. This finding is consistent with the report by Naenni et al., which demonstrated that NaOCl dissolves significantly larger amounts of tissue compared to the other acids tested.³⁰

Smear layer acts as a diffusion barrier, reducing dentin permeability by 25% to 49%.31, 32, 33 Its removal has been shown to increase dentin permeability^34,35 and reduce microleakage at the interface between the final root canal filling and the canal wall dentin.36, 37, 38 EDTA is commonly used for smear layer removal with an optimal irrigation time of 1 to 3 min.^39,40 It has been reported that 1 min of EDTA irrigation is sufficient for smear layer removal, whereas 10 min may cause dentin erosion; therefore, irrigation times exceeding 1 min are not recommended.⁴¹ Accordingly, the EDTA application time in our study was limited to 1 minute. Although the sample size in our study was limited, a significant difference was observed between the saline group and all three TCA-treated groups. Among the TCA groups, the 15-second application showed the greatest overall efficacy in smear layer removal. This short application time may represent a clinical advantage by reducing irrigation time while maintaining effectiveness.

Although our study provides evidence that TCA can facilitate the removal of Ca(OH)₂, residual Ca(OH)₂ was still observed in all teeth examined. The location within the canal appears to influence removal efficacy, as both EDTA and TCA were more effective in the coronal and middle thirds than in the apical third. Notably, TCA was more effective than EDTA in removing Ca(OH)₂ and exposing dentinal tubules in the coronal and middle thirds.

These findings warrant further investigation with a larger sample size. Although early signs of dentin structural changes were noted, which is aligned with previous research showing that 90% TCA reduces dentin microhardness,⁴² the clinical significance of this etching effect remains unclear. For example, TCA has been reported not to adversely affect adhesive bond strength to dental tissues.⁴³ The slight decalcification and opening of dentinal tubules may, in fact, benefit canal disinfection or sealer penetration.

In summary, in addition to the aforementioned limitations of a relatively small sample size, which necessitates further validation of the present findings with a larger sample size, future studies should investigate several key aspects of TCA use. These include determining optimal concentrations, application times, and safety considerations, as well as employing more rigorous in vitro experimental designs to facilitate its effective integration into endodontic treatment protocols. Further exploration of its potential novel properties is also warranted. These may include facilitating access to narrow or constricted canal orifices, reducing the risk of canal blockage during cleaning and shaping, serving as a final irrigant – potentially in conjunction with adjunctive methods such as ultrasonic activation, and providing haemostatic effects that could improve drying of the apical preparation prior to obturation.

Conclusions

This preliminary in vitro pilot demonstrates the potential of TCA as an adjunctive endodontic irrigant for the removal of the smear layer and Ca(OH)₂. Further validation with larger sample sizes, along with systematic investigation of optimal concentrations, application times and safety is necessary before its clinical application can be considered.

Funding

This work was supported in part by grants from the American Association of Endodontists Foundation (E.M., A.B., J.-B.C.R.) and funds from the University of Tennessee Health Science Center (G. T.-J.H.). B.X. was supported by Qingdao Key Medical and Health Discipline Project (2022-2024, 2025-2027), Qingdao Clinical Research Center for Oral Diseases (22-3-7-lczx-7-nsh) and Shandong Provincial Key Medical and Health Discipline of Oral Medicine (Qingdao University Affiliated Qingdao Stomatological Hospital, 2024-2027).

Ethics approval statement and document

Extracted teeth collection conformed to an exempt protocol approved by the Institutional Review Board of UTHSC (#19−06855 NHSR). No consent form was needed.

Availability of data and materials

All data generated or analysed during this study are included in this published article [and its supplementary information files].

Authorship contribution

E. Munoz, A. Benbow, J.-B. C. Russell: Experimental performance, data acquisition and interpretation, drafted the manuscript. B. R. Morrow: Experimental performance, data acquisition and interpretation. T. Pannkuk: Conceptualization, interpretation, drafted the manuscript, critically revised/edited the manuscript. B. Xin: Data and statistical analysis, interpretation, drafted the manuscript. G. T.-J. Huang: Conceptualization, design, supervision, data interpretation, drafted and critically revised/edited and finalized the manuscript.

Conflict of interest

All authors have no financial affiliation, or involvement with any commercial organization with direct financial interest in the subject or materials discussed in this manuscript, nor have any such arrangements existed in the past 3 years, except the co-author T. Pannkuk has a patent on the TCA use as irrigant (https://patents.google.com/patent/US11147747B2/en). The authors, except T. Pannkuk, deny any conflicts of interest related to this study.