Comparative Analysis of Calcium and Hydroxyl Ion Release from Pulp Dressing Materials in Pulpotomized Premolars

Introduction

Pulpotomy, also known as partial pulp amputation, is one of the most widely used minimally invasive vital pulp therapies (VPT) for primary and young permanent teeth. It is defined by the American Academy of Endodontics (AAE) as removal of the coronal portion of a vital pulp to preserve the vitality and function of the remaining radicular portion. It can be performed as an emergency procedure for either temporary relief of symptoms in cases diagnosed with normal pulp or reversible pulpitis or therapeutic treatment in case of trauma (Cvek pulpotomy) [1,2]. Root canal treatment is performed to remove the infected pulp tissue or prevent further infection of a root canal system that is irreversibly damaged and cannot be preserved [3].

However, with the introduction of bioactive materials, many studies revealed the effectiveness of coronal pulpotomy in managing permanent teeth with irreversible pulpitis, with good clinical success rates on long-term follow-up [4–7]. Accordingly, employing VPT in mature teeth could help prevent further root canal therapies, preserving the vitality of the dental pulp, which is crucial for the defensive mechanisms and structural integrity of the tooth [8].

Many biocompatible/bioactive materials, such as calcium hydroxide, calcium silicate (MTA, Biodentine), tricalcium phosphate, and calcium aluminate, have recently been used for different VPT procedures [9,10]. Calcium hydroxide was primarily used for VPT due to its ability to release Ca++ and OH−. However, the use of calcium silicate-based materials like MTA recently became popular in response to its ability to generate calcium hydroxide and release Ca++ and OH− ions during the hydration process [11].

Some studies have reported that MTA usually has a higher success rate than calcium hydroxide in treating mature permanent teeth undergoing pulpotomy [12–16]. Despite its biocompatibility and ability to promote dentin bridge formation, MTA exhibits certain drawbacks, including tooth discoloration, prolonged setting time, and difficult manipulation [17]. To overcome these challenges, MTA-Angelus was developed, offering a shorter setting time and improved handling while maintaining the superior performance of traditional MTA [18].

Calcium silicate-based bio-ceramics were introduced to replace MTA for root perforation repair, root end filling, and pulp capping, as well as pulpotomy cases [19]. This hydrophilic material can form hydroxyapatite after solidification, with low risk of color change [20]. ESRRM is an insoluble, radiopaque, and aluminum-free calcium silicate preparation with superior handling characteristics like MTA [21,22]. Clinically, ESRRM can preserve pulp vitality following the pulpotomy procedure in permanent teeth with irreversible pulpitis [23], and it shows biocompatibility and mineralization potential like those reported for MTA [24].

The literature indicates that the therapeutic effects of any repair material depend on the dissociation of calcium ions that stimulate the differentiation of the pulp stem cells into odontoblast-like cells, and on the availability of hydroxyl ions that sustain a bactericidal alkaline medium [25,26]. However, there has been no consensus on which is the best pulp dressing material. Therefore, the aim of this study was to assess and compare the capabilities of the most frequently used materials – Dycal, MTA-Angelus, and the most recently introduced material, ESRRM, which releases both Ca++ and OH− at different times following their use as pulp orifices capping materials in pulpotomy in permanent premolars. The null hypothesis was that there would not be a difference in the performance of these materials in terms of Ca++ and OH− release at the testing times.

Material and Methods

STUDY DESIGN, ETHICS APPROVAL, AND SAMPLE SIZE CALCULATION:

Fifty freshly extracted, caries-free mandibular first premolars were collected following IRB approval (IRB/KKUCOD/ETH/2020-21-031) from patients treated at the Orthodontic outpatient clinics, College of Dentistry, Khalid University. All teeth were extracted due to orthodontic reasons, subjected to thorough ultrasonic cleaning, and disinfected in 4% thymol solution for safe future handling. Sample size was calculated using G*Power software (version 3.1; University of Dusseldorf). The effect size (d) was 0.5, α was 0.05, and 1−β (power) was 0.80. The required sample size was 50, as calculated according to previous studies [1,14,21].

TEETH GROUPING AND PULPOTOMY CAVITIES PREPARATION:

Ten teeth were incubated intact in deionized water at 37±1°C for reference purposes (G1). The remaining 40 teeth were subjects of standardized pulpotomy procedures. Occlusal cavities were first deeply prepared to expose the pulp using flat-end diamond burs (847F.FG.012, JOTA, Switzerland) in a high-speed handpiece. Using #2 round burs (808R.FG.014, JOTA, Switzerland), pulp deroofing was then carried out in all teeth before removing the coronal pupal tissue using a sharp endodontic spoon excavator (# L31, ST Instrument USA, Inc, Brooklyn, NY, USA).

TEETH FILLINGS FOR DIFFERENT GROUPS:

The prepared cavities were then flooded with 4 mL of 18% EDTA for 3 min at room temperature to simulate the clinical condition as EDTA conditioning of pulp cavity dentinal walls after pulpotomy induced dentinogenic activity in the root pulp [27], thoroughly washed out with deionized water, and gently dried with cotton pellets before starting the restorative procedure in 4 different groups. In the Control group (G2, n=10) specimens, cavities of the pulpotomized teeth were lined with a 0.5-mm-thick layer of the resin-modified glass-ionomer liner (RMGI, Vitrabond, 3M ESPE, St. Paul, MN). The powder and liquid were mixed on a paper pad using a plastic spatula and the mixed material was applied directly against the floor of the pulp chamber using a calcium hydroxide hand applicator and light-cured for 10 s using an Elipar LED curing unit (Elipar S10, 3M ESPE, Neuss, Germany) [1,13].

At the time of restoring teeth in Groups 3, 4, and 5 (n=10 each), the root canal orifices were capped using a 2-mm-thick layer of Dycal (DENTSPLY-SIRONA, York, PA, USA) (Figure 1A), EndoSequence Root Repair Material (ESRRM) (BUSA Bioceramix, Inc, Burnaby, BC, Canada) (Figure 1B), and MTA-Angelus (Angelus, Londrina, PR, Brazil) pulp dressing materials (Figure 1C). In G2, equal amounts of the Dycal base and catalyst pastes were thoroughly mixed over a paper pad using plastic spatula to reach a homogenous consistency and a uniform color. Using a calcium hydroxide applicator, the mixed material was incrementally placed against the root canal orifice until the desired thickness (2–3 mm) was reached [21,22]. In G3, the fast-set ESRRM paste was continuously injected against the root canal orifice until reaching the desired thickness. In G4, the MTA-Angelus bioceramic powder was mixed with distilled water according to the manufacturer’s recommended liquid/powder ratio and mixed to a homogenous consistency. The mixed material was applied against the root canal orifice in two 1-mm-thick increments, each adapted in place using cotton pellets wetted with distilled water and squeezed to help its setting process according to the manufacturer’s instructions.

Following the pulp dressing procedure in each tooth, a layer of the RMGI liner was applied, following the same previously described protocol, to cover the dressing material before finalizing the coronal restorations [24,26]. To finalize the coronal restorations following the capping and lining procedures, enamel margins of all tooth access cavities were selectively etched with 35% phosphoric acid gel (Scotchbond Universal Etchant, 3M ESPE, St. Paul, MN, USA) for 15 s, rinsed using water-air spray for another 10 s, and gently air dried for 10 s before agitating all cavity walls with 2 layers of the universal resin adhesive (Scotchbond Universal Adhesive, 3M ESPE, St. Paul, MN, USA), then left in contact for 15 s before light-curing took place. All cavities were then completely restored using only 2 layers of the paste-like bulk-fill resin composite (Filtek One bulk-fill Restorative, 3M ESPE, St. Paul, MN), and each was light-cured for 20 s. The performed restorations were thoroughly finished and polished to avoid leaching out of any chemical at the time of assessment.

MEASUREMENTS OF THE RELEASED CALCIUM AND HYDROXYL IONS:

The entire surfaces of the completely restored teeth received 2 coats of nail polish, leaving only 1 mm surrounding the root apices. Using a wax gun, the occlusal surface of each specimen was thoroughly attached to the inner surface of a polystyrene tube lid. Each tube was filled with 20 mL of deionized water (pH=6.5±0.1), which was previously assessed for its pH and calcium ions content to ensure the accuracy of the measurements to be collected. The sealed tubes were then stored inverted at 37±1°C (Ehret KBK 4200, Burladingen, Germany) to create a simulated intra-pulpal pressure equivalent to 20 cmH2O [28]. The pH meter used in this study is presented in Figure 1D.

MEASUREMENTS OF RELEASED CALCIUM IONS:

The amount of the leached-out Ca++ into the immersing water was measured using flame photometer (JENWAY PFP7, T Equipment, Manasquan, USA) (Figure 1E), which measures the intensity of the emitted spectra from the exited calcium element. The analysis of the Ca++ concentration took place at 24 h, 1 week, 4 weeks, and 8 weeks following immersion of the samples [21,22,24,26]. Figure 2 summarizes the steps of the study and the different groups with the materials used. The value of the released OH− ions was qualitatively measured by determining the actual pH of the immersion solution using a pH meter (Beckman PHI 40, Beckman Coulter, Brea, USA) (Figure 1E) at the same storage time intervals.

STATISTICAL ANALYSIS:

The mean±standard deviation (SD) was determined for all samples in each group, and the normal distribution of each data set was confirmed by Kolmogorov-Smirnov tests. The value of the released OH⁻ ions was qualitatively measured at different timepoints by determining the actual pH of the immersion solution using a pH meter (Beckman PHI 40, Beckman Coulter, Brea, USA) at the same storage timepoints. All the collected data were statistically analyzed using ANOVA and Tukey’s comparisons at α=0.5 to determine the significance of the detected differences among groups.

Results

AMOUNT OF RELEASED CALCIUM IONS:

The mean and standard deviation of the calcium ion concentration through the proposed testing times are summarized in Table 1. Two-way ANOVA was used to detect significant differences between test groups (P<0.05) and between the testing times (P<0.05) in addition to a significant interaction between the 2 variables (P<0.05). Only Groups 3–5 (Dycal, ESRRM, and MTA-Angelus, respectively) showed significant changes in calcium ion concentration through different testing intervals.

At 24 h after immersion of specimens, no difference in Ca⁺⁺ ion concentration was detected among Groups 1, 2, and 3 (Reference, Control, and Dycal) (Tukey’s, P>0.05), although higher concentrations were detected in Groups 4 and 5 (ESRRM and MTA-Angelus) (Tukey’s, P<0.05). One week later, the highest Ca⁺⁺ ion concentration was in Groups 4 and 5, and G3 also showed higher concentration than the Reference and the Control groups (Tukey’s, P<0.05).

After 4 weeks of immersion, the highest Ca⁺⁺ ion concentration was in G4, followed by Groups 3 and 4 (Tukey’s, P<0.05), and the Control and Reference groups had lower concentrations in comparison to other test groups (Tukey’s, P<0.05). At 8 weeks, the highest Ca⁺⁺ ion concentration was in Groups 4 and 5, and G3 had a higher concentration than the Control and Reference groups (Tukey’s, P<0.05).

AMOUNT OF RELEASED HYDROXYL IONS:

The means and standard deviations of the pH levels at testing times are summarized in Table 2. Two-way ANOVA indicated significant differences among test groups (P<0.05) and between the testing times (P<0.05), in addition to a significant interaction between the 2 variables (P<0.05). Groups 3–5 (Dycal, ESRRM, and MTA, respectively) showed stable pH at all testing times; however, the reference group showed lower pH levels at 4 and 8 weeks. One day after immersion, no difference was noticed among the pH levels in all groups (Tukey’s, P>0.05). At 1 week, only Groups 4 and 5 showed higher pH than the other groups (Tukey’s, P<0.05). At weeks 4 and 8, all test groups showed higher pH than the reference group (Tukey’s, P<0.05).

Discussion

Pulpotomy is usually performed in case of deep caries or traumatic exposures in primary and immature permanent teeth either to preserve the vitality of the primary tooth until the permanent successor erupts or to continue root development of the immature permanent tooth [1]. Pulpotomy is also considered as a successful alternative to root canal treatment in mature permanent teeth with closed apices [29]. This approach is usually based on preserving the vitality of irreversibly inflamed pulp using ion-leaching biomaterials such as calcium hydroxide, calcium silicate-based biomaterials, and bioceramic pastes [4–7]. In the presence of fluids, these materials can release free Ca++and OH− ions, which have beneficial biological effects on pulpal stem cells, surrounding tissues, and infecting bacteria [11,26].

Although the optimal pulp dressing material is unknown, the current study aimed to assess the ability of Dycal, ESRRM, and MTA-Angelus to leach out more Ca++ and OH− ions at 4 different test timepoints representing the usual inspection times of pulp capping outcomes. The selected materials usually have favorable outcomes when used in vital pulp therapy procedures44–7]. On the other hand, the flame photometry technique has been selected to precisely assess the quantity of the released Ca++ ion in respect to the other known techniques such as atomic absorption spectrometry, Ca++ selective electrode, and fluorometry [30–33]. This technique is commonly used to measure the mineral concentration in fluids based on absorption at high temperature and excitation of the alkaline-earth metal ions, which on cooling release the absorbed energy in the form of ion-specific radiation emission proportional to the concentration of ions present in the immersion liquid [34]. Higher pH causes release of the hydroxyl ions, which has antimicrobial and antifungal effects, and the assessment of the OH− ions release was directed by measuring the immersion solution’s pH at the selected test timepoints [11,35].

We rejected the null hypothesis of no difference being detected among the tested materials at different timepoints seems a must, as the recorded results revealed the ability of both ESRRM and MTA-Angelus to release variably higher amounts of Ca++ ions, in comparison to Dycal, at the various testing timepoints (Table 1). These findings are supported by the findings of previous studies, which suggested similar levels of bioactivity of these materials [31,32]. Despite the differences detected in pH values among the tested orifice capping materials (Dycal, MTA-Angelus, and ESRRM) after 1 and 7 days of immersion, no differences were evident after 4 and 8 weeks of immersion, contrary to the results of some previous studies [33,35] that found significant differences in the pH values among these materials at all testing timepoints. This could be attributed to the chemical composition between the used materials in different studies as well as the form of the materials used.

In the current study, the immersion water of both Reference and the Control groups (G1 and G2) showed equal traces of Ca++ ions (Tukey’s comparisons, P>0.05), in agreement with the reports of Gandolfi et al (2012) [11], Misra et al (2017) [30], and Sáez et al (2017) [33], who explained this finding by the possible minimal dissociation of the hydroxyl apatite crystals of the tooth enamel and dentin in the immersion solution. However, all the tested pulp dressing materials (G3–G5) showed higher release of Ca++ ion in comparison to both the Reference and the Control groups (G1 and G2), exactly as recorded by Gandolfi et al (2012) [11]. This could be related to the time intervals used for Ca++ ion release.

Even though ESRRM released the highest amount of Ca++ ions 4 weeks after immersion, the same material, in addition to the MTA-Angelus, showed higher Ca++ ion release than did the Dycal (G3) at most of the other test intervals. These findings came in agreement with the results of Gandolfi et al (2012)4[11], Talabani et al (2020) [32], and Natale et al (2015) [36], who reported that both MTA-Angelus and ESRRM are hydraulic water-based calcium silicate materials, which can form a sticky calcium silicate-hydrate gel and produce calcium hydroxide during their hydration and setting processes, but ESRRM usually shows more calcium ion release.

The ability of Dycal to release higher Ca++ ions than MTA-Angelus was evident 4 weeks after immersion, which may be due to the expected dissolution of the Dycal material. Sáez et al (2017) [33] also found higher Ca++ release from calcium hydroxide despite the crucial issue of using vehicles in their experimental groups, which may have influenced the results by promoting rapid Ca++ ion dissociation [37]. In contrast to the previous finding, Natale et al (2015) [36] found a reduction in Ca++ ion release at 4 weeks after insertion, which suggested a decline in the reactive property of the materials after application. To build upon these findings and explore the persistence of this behavior, the present study extended the measurement time to 8 weeks. This longer duration offers a more comprehensive understanding of the materials’ performance over a longer time, which is crucial for predicting their longevity and therapeutic value, although other studies focused on shorter testing intervals [11,31,32].

In all test groups, the Ca++ ion release started within the first 24 h of immersion. However, the maximum Ca++ ion release was in the ESRRM group (G3) 4 weeks after immersion, despite the maximum Ca++ ion release in the MTA-Angelus group (G4) observed earlier. Although these findings could be supported by those of Talabani et al (2020) [32], the subsequent reduction in the released Ca++ values were contrary to the findings of Herrera-Trinidad et al (2023) [31]. The differences between results of the current study and those of other studies might be related to the inherent roles of materials’ application, testing conditions, and the test duration, which can significantly influence the assessment outcomes [38].

Normally, the diffusion of the OH− ions into the pulpal and periapical areas is reflected by the increasing pH values of the surrounding tissues, which helps destroy bacteria, inactivate bacterial enzymes, reduce osteoclastic activity, and activate alkaline phosphatase, which is involved in mineralization [37]. These features should be found in an ideal pulp dressing material, but results of the current study indicated equal ability of the tested pulp dressing materials and the RMGI lining material in the Control group to maintain stable pH levels throughout all test timepoints (Tukey’s comparisons, P>0.05, Table 2). Although these results disagree with the results of Yazdanpanahi et al (2021) [35], which showed higher pH values in the calcium hydroxides group than those observed in the MTA group, results of the current study could be explained by the compositional nature and the specific elemental contents of the pulp dressing and lining materials (Table 3), which do not allow release of significant amounts of OH− ions. In addition, the recorded stability of the pH levels at various timepoints are supported by results of many previous studies [30,32,35].

Based on the outcomes of the current study, and due to the ability of ESRRM and MTA-Angelus to release considerable amounts of Ca++ ions and to maintain high and stable pH levels for a longer time, using either of them seems a valuable option to dress the root canal orifices of the pulpotomized permanent teeth. However, further laboratory studies and clinical trials are needed to confirm the findings of this study and to evaluate the long-term clinical outcomes of pulpotomy in permanent teeth when Dycal, ESRRM, or MTA-Angelus are used to directly cap the root canal orifices.

Additionally, some recently introduced compounds have a significant influence on the oral environment. The use of natural compounds [39] and ozone-based gels [40] or water [41] have been demonstrated to have significant effects on oral microbiota and they could be tested in the future in combination with pulp dressing materials to assess their possible mutual effects.

There are various limitations to this study that should be taken into account. Those included tooth type single or multirooted, longer follow-up periods after, 6 months and more, and the tissue response to different materials, as well as thermocycling period to simulate the clinical situation.

Conclusions

The current study concluded that the tested pulp dressing materials have equivalent and stable pH levels, but ESRRM and MTA-Angelus had the greatest Ca++ ion release at all tested timepoints. ESRRM and MTA-Angelus are potentially more active in pulpotomy cases that require higher Ca++ ion release and stable alkalinity for longer times.