|Year : 2021 | Volume
| Issue : 6 | Page : 611-615
|Evaluation of the diffusion of calcium hydroxide-based medicaments through the dentinal tubules and apical foramen: A mass spectrometry study
Matthew Wong1, James Jarrett2, Alan White2, Laurence J Walsh3, Roy George1
1 School of Medicine and Densitry, Griffith University, Gold Coast, QLD 4215, Australia
2 School of Environment and Science, Griffith University, Nathan, QLD 4111, Australia
3 The University of Queensland School of Dentistry, Herston, QLD 4006, Australia
Click here for correspondence address and email
|Date of Submission||16-Aug-2021|
|Date of Decision||02-Dec-2021|
|Date of Acceptance||19-Jan-2022|
|Date of Web Publication||01-Apr-2022|
| Abstract|| |
Aims: This mass spectrometry study investigated the diffusion of hydroxyl and calcium ions from a calcium hydroxide intracanal medicament (Pulpdent®) when used alone or mixed in equal parts with a steroid–antibiotic paste (Ledermix®).
Materials and Methods: The pH (using pH meter) and calcium ion concentration (using inductively coupled plasma-mass spectrometry) of the diffused medicaments were assessed using endodontically prepared human extracted teeth with either a sealed or a patent apex, at time intervals of 1, 3, 8, 24, and 168 h.
Statistical Analysis: A one-way ANOVA was used to explore differences between and within groups, with Tukey–Kramer/Games–Howell posttests.
Results: In both situations tested, Pulpdent® showed greater release of both calcium and hydroxyl ions than when mixed in equal parts with Ledermix®. Greater initial release of both ions occurred in roots with a patent apex, but by 1 week there was no significant difference between the two. If a 50:50 combination of Pulpdent® paste and Ledermix® paste is used, there is a lower release of calcium ions and hydroxyl ions than using Pulpdent® paste alone.
Conclusion: With both Pulpdent® paste alone and Pulpdent® paste mixed equally with Ledermix® paste, the major pathway for movement of hydroxyl ions and calcium ions is diffusion through the dentine of the root, with the apex playing only a minor role.
Keywords: Apical foramen; calcium hydroxide; dentine; diffusion; endodontics; ledermix
|How to cite this article:|
Wong M, Jarrett J, White A, Walsh LJ, George R. Evaluation of the diffusion of calcium hydroxide-based medicaments through the dentinal tubules and apical foramen: A mass spectrometry study. J Conserv Dent 2021;24:611-5
|How to cite this URL:|
Wong M, Jarrett J, White A, Walsh LJ, George R. Evaluation of the diffusion of calcium hydroxide-based medicaments through the dentinal tubules and apical foramen: A mass spectrometry study. J Conserv Dent [serial online] 2021 [cited 2023 Jun 1];24:611-5. Available from: https://www.jcd.org.in/text.asp?2021/24/6/611/342478
| Introduction|| |
Calcium hydroxide medicaments have been used in endodontics for inter-appointment disinfection of the prepared root canal space and to stimulate hard tissue repair in the cases of vital pulp therapy, resorption, or incomplete root formation., The therapeutic effects are achieved through the release of both calcium (Ca2+) ions and hydroxyl (OH-) ions, both of which diffuse into the target tissues. Hydroxyl ions kill bacteria by damaging the bacterial cytoplasmic membrane, and denaturing proteins and DNA. Calcium hydroxide can also inactivate lipopolysaccharides, which play a fundamental role in the initiation and maintenance of periapical lesions, and in the differentiation of osteoclasts. Furthermore, it can activate alkaline phosphatase activity, reduce osteoclastic acid hydrolase activity, and modulate the differentiation of osteoclasts. An elevated calcium ion concentration in the interstitial tissues also stimulates hard tissue formation, and upregulates fibronectin gene expression.
For calcium hydroxide to be effective, both the hydroxyl and calcium ions must be able to diffuse throughout the root canal system and periapical tissues. Residual bacteria and their by-products are often located in inaccessible areas such as dentinal tubules, isthmuses, ramifications, deltas, accessory canals, lateral canals, cemental canals, and in the apical lesion itself. Together, an alkaline pH and calcium ions in the periapical tissues promote hard tissue formation.
Studies of the patterns of diffusion of calcium and hydroxyl ions from medicament pastes have produced varying results. With Pulpdent® paste (Puldent Corp., Waterton, MA), a pH change in the cervical outer dentin from 7.6 to 9.4 has been seen after 3 days. With Calasept® (Scania Dental, Knivsta, Sweden), the pH in the outer root dentin did not rise for 7 days, and a period of 3 weeks was required before the pH reached its peak pH of 9.3 cervically and 9.0 apically. Conversely, other studies have shown no pH changes on the root after 10 days or after 28 days. Similarly, results for diffusion of calcium ions through roots over periods of up to 1 week are also variable. Diffusion of ions from the root canal system influenced the size of the apical foramen. No past studies have examined the diffusion of hydroxyl and calcium ions for the situation where a calcium hydroxide medicament is mixed in equal parts with a corticosteroid–antibiotic paste (CAP), with and without apical foramen patency. The concept of such mixtures was first advocated by Schroeder in 1981, to overcome the limitations of the individual medicaments.,,
The objective of the current study was to assess the diffusion of calcium ions and hydroxyl ions in human tooth roots filled with either Pulpdent® or with a 50:50 (v/v) mixture of Pulpdent® paste with Ledermix® paste (Ozdent, Sydney, Australia), a CAP containing 1.0% (w/w) triamcinolone acetonide and 3% (w/w) demeclocycline hydrochloride. The experimental design also explored the effect of apical foramen patency.
| Materials and Methods|| |
Preparation of teeth
Extracted human premolar teeth with fully developed single root canals (n = 41) were collected with institutional human ethics approval. The teeth had no previous restorative or endodontic treatment and were free of visible defects or cracks. The root surface was debrided gently with an ultrasonic scaler to remove remnants of periodontal ligament, and care was taken not to remove cementum. The crown was removed, to give roots of a consistent length of 14 mm.
An ISO 10 K file (Dentsply Sirona, Tulsa, OK) was introduced passively into the canal until the tip was observed at the major constrictor. The working length was established as 1 mm short of this measurement. The canals were prepared using nickel-titanium rotary files (ProTaper® Universal, Dentsply Sirona, Tulsa, OK) in the following sequence: SX, S1, S2, F1, and F2. An ISO 15 K file was used to recapitulate to the working length between each file sequence. During preparation, canals were irrigated constantly with 1% NaOCl (Endosure Hypochlor, Dentalife, Ringwood, Australia). After using the final F2 file, canals were rinsed with 15% EDTA/C (EndoPrep, Dentsply Sirona, Tulsa, OK) for 1 min. During this step, the EDTA solution was activated with using an EndoActivator® (Dentsply Sirona, Tulsa, OK) with a medium tip and a 2–3 mm vertical plunging action, as per the manufacturer's instructions. This was followed by a further rinse with 1% NaOCl for 1 min at a rate of 2 mL/min, followed by distilled water for 3 min at a rate of 2 mL/min.
Following canal preparation, the roots were assigned using stratified random sampling based on weight, to reduce variations in dentine thickness and other potential confounding variables [Table 1]. They were then mounted on the lids of 6.5 mL scintillation vials, with 12 mm of the root extending into the vial when the lid was fully tightened. There were five roots in Group A (control roots with no medicaments and a patent apex). There were nine roots per group in each of the four treatment groups (Pulpdent with sealed apex (B) or patent apex (C) and the Pulpdent + Ledermix mixture with sealed apex (D) or patent apex (E).
Preparation of medicaments and teeth
To prepare the combination medicament, equal volumes of Pulpdent® and Ledermix® were mixed on a glass slab for 30 s to achieve homogeneity. A syringe with a 27G needle was used to place the materials within the prepared canals, until they were seen to extrude from the apical foramen. Excess material was thoroughly cleaned off. To simulate a blocked apical foramen for Groups B and D, the apex was sealed using sticky wax (Ainsworth Dental, Sydney, Australia) by dipping 1 mm of the root surrounding the apex into molten wax, which was then left to harden. Cavit G (3M, St Paul, MN) was then used to seal the coronal portion of roots in Groups B–E.
The scintillation vials were then loaded with 6 mL of fresh ultrapure Milli-Q water using a micropipette. The lid containing the root was screwed down into place, bringing the root into full contact with the water along its length. The assembly was kept in an incubator at 37°C with 100% humidity to simulate the oral environment. At the designated sampling times (1, 3, 8, 24, and 168 h), the lid was gently removed, and the water that had been exposed to the root was set aside for later analysis. The lid with the root was then placed onto a new scintillation vial filled with fresh Milli-Q water, to continue the experiment.
For pH measurements, a thin glass rod was used to gently stir the samples before measurements were taken. For the analysis of calcium ions, 1.0 mL of the sample water was analyzed by inductively coupled plasma-mass spectrometry (OPTIMA 8300 ICP-OES, PerkinElmer, Waltham, MA), with the aid of WinLab32 software (PerkinElmer, Inc, MA, USA) version 5.4.0.0687. A digital pH meter (TPS, Brendale, Australia) that had been calibrated with pH 7.00 and pH 9.00 buffer solutions was used to measure pH.
A one-way ANOVA was used to explore differences between and within groups, with Tukey–Kramer/Games–Howell posttests. For pH changes over time, the one-way repeated measures ANOVA with Bonferroni post hoc tests was undertaken, while for calcium ions, because the data sets did not follow a normal distribution, the Kruskal–Wallis H-test (with the Bonferroni-corrected Mann–Whitney U-test) and the Friedman test (with the Bonferroni-corrected Wilcoxon signed-rank test) were used. The threshold was set at P = 0.05. IBM SPSS version 25 (Version 220.127.116.11(142) IBM Corp(c) USA) statistical software was employed.
| Results|| |
pH changes occurred in the water surrounding the roots [Table 1], signifying the release of hydroxyl ions from the roots into the water. pH elevations were greater for Pulpdent® alone than for Pulpdent® + Ledermix® at 24 h, regardless of whether the root had a patent apex or not. The greatest changes occurred after 1 week.
Across all the four medicament groups, pH continued to rise between 24 h and 1 week. There was a tendency for samples with a patent apex (Groups C and E) to show greater pH elevation than those with a sealed apex (Groups B and D), but this difference did not reach the threshold for statistical significance for the majority of sampling times. When Pulpdent® was combined with Ledermix®, the pH change was significantly lower than that of Pulpdent® alone, regardless of whether the tooth had a patent apical foramen or not.
Calcium ions were released into the water surrounding the roots [Table 2], particularly in the period between 24 h and 1 week. Greater calcium ion release occurred from Pulpdent® alone than for Pulpdent® + Ledermix®. The maximum rate of release within the first hour was seen for the Pulpdent® + Ledermix® mixture with a patent apex (Group E), however by 7 days, release from these samples had been surpassed by other medicament treatment groups.
| Discussion|| |
To our knowledge, this is the first study to compare release of hydroxyl ions from roots when a calcium hydroxide paste is used alone or is combined with another medicament – in this case, a CAP. The majority of hydroxyl ions diffuse through the dentine of the root, where they interact with hydroxyapatite and dentin proteins which can buffer them. However, once the buffering effect of dentine is overcome, diffusion through the root then occurs. This explains why there is an initial delay of several days before pH elevations occur on the outer root surface, as has been seen in several studies.,, The consequence of this pattern of change is that longer periods of treatment with calcium hydroxide pastes may give greater changes in pH in the dentine, and thus greater inhibition of microbial growth. Given the documented powerful buffering actions of the dentine, both in its normal form,, and when used as a powder with a high surface area,, in some clinical situations, calcium hydroxide may need to be left for longer than 1 week to achieve the desired therapeutic effect.
In any study assessing pH changes in a water sample, the dissolution of atmospheric carbon dioxide gas, forming carbonic acid, will occur to some extent. As a result, the change in pH values recorded for the water is of greater relevance than the actual pH measurements themselves. While the pH was slightly higher and when the apex was patent, this was not significantly different to when it was closed. This indicates that the primary pathway for hydroxide ions is by diffusion through dentine, with only a minor contribution from diffusion though the apex. As a consequence, for maximum clinical performance of calcium hydroxide medicaments, it is essential to maximize dentinal tubule patency through removal of smear layer.
With regard to calcium ions, the current results indicate that during the first 24 h, calcium ions diffuse through the apical foramen, because roots with a patent apex showed higher initial calcium concentrations. After that, the major pathway is diffusion through dentine, and this process continues over the following 6 days. Although the threshold concentration of calcium ions that is required to initiate and stimulate mineral formation is not known, the initial burst of calcium ions released from the medicament may accelerate this process, in the same way that higher concentrations of calcium ions have been shown to trigger odontoblast differentiation and dentine deposition. Furthermore, calcium ions can stimulate fibronectin gene expression, and can form calcium carbonate, which lowers the levels of carbon dioxide, and thus changes the conditions for bacterial growth.
Ledermix® paste uses a vehicle of polyethylene glycol 400 with several excipients, such as calcium chloride and sodium calcium edetate, that could contribute to calcium ion release. No significant calcium ion release would be expected to occur from the Cavit® temporary dressing, because of its location that is away from the water and its low content of calcium sulfate. Nevertheless, some minor release of calcium ions from the radicular dentine into the water did occur, as seen in the controls. It can be concluded that the majority of the released calcium ions that were detected came from the Pulpdent® paste, from where they have either passed through the apex or diffused through the root, to reach the water.
The addition in equal volume of Ledermix® paste to Pulpdent® paste lowered the release of hydroxyl ions, as seen with smaller pH elevations. There are several reasons for this. The first is that mass dilution reduced the total amount of calcium hydroxide that was loaded into the canal in the first instance. The second is that the mixture of two pastes will reduce the contact of the active ingredients with the walls of the root canal, and thus with the dentinal tubules. The net effect of a lower dose and a lower concentration gradient is reduced diffusion of hydroxide ions.
Demeclocycline is one of the strongest calcium-binding tetracyclines known. Mixing Ledermix and Pulpdent may however have less than 2% effect on the depletion of available calcium ions for diffusion. Assume that 1 g of Ledermix paste has been mixed with 1 g of Pulpdent paste. One gram of Pulpdent™ paste contains 40% calcium hydroxide, which is 400 mg, and thus 5399 micromoles. One gram of Ledermix™ paste contains 30 mg of demeclocycline HCl which is 59.84 micromoles. Each demeclocycline HCl molecule can bind 3 calcium atoms. On a molar ratio, there is a 90.22 fold of calcium to demeclocyline. Very little calcium is available because of the poor solubility of calcium hydroxide in the water vehicle of Puldent paste (1.48 g/l). Thus, in 1 g of Pulpdent, there would be 1.48 mg of dissolved calcium hydroxide, which is only 19.98 micromoles. There is a large excess of calcium hydroxide (27 times more in Pulpdent paste than can actually dissolve in its water vehicle), so some of the dissolved calcium ions from the calcium hydroxide will immediately bind to the demeclocycline HCl in the Ledermix paste and will not be available. However, once the calcium has bound to the demeclocycline HCl, the reservoir of excess calcium hydroxide is expected to dissolve and diffuse through the tubules, provided there is contact of the active ingredients with the walls of the root canal.
In the present study, an increased rate of calcium ion diffusion was observed within the first 24 h for the combination medicament with a patent apex. Although both medicaments are water soluble, a viscous methylcellulose vehicle is used in Pulpdent® paste, while a more fluid-like polyethylene glycol cream base is used for Ledermix®., The resulting two paste mixture may be less viscous than Pulpdent® paste, and thus may more readily dissolve in water, when the water meets the material via a patent apical foramen, causing the initial burst of calcium ion release.
| Conclusion|| |
With both Pulpdent® paste alone and Pulpdent® paste mixed equally with Ledermix® paste, the major pathway for movement of hydroxyl ions and calcium ions is diffusion through the dentine of the root. If a 50:50 combination of Pulpdent® paste and Ledermix® paste is used, there is lower release of calcium ions and hydroxyl ions than using Pulpdent® paste alone because of mass dilution of the calcium hydroxide.
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Conflicts of interest
There are no conflicts of interest.
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Prof. Roy George
School of Medicine and Dentistry, Griffith University, Parklands Drive, Southport, Gold Coast 4215
Source of Support: None, Conflict of Interest: None
[Table 1], [Table 2]
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