Effect of continuous chelation irrigation on transforming growth factor-β1 release: An in vitro assay

Abstract

Context:

Root canal irrigants and medicaments influence the growth factors released from dentin and create a conducive environment for regeneration.

Aim:

This study assesses the transforming growth factor-beta 1 (TGF-β1) release after using clodronate and etidronate as continuous chelating agents with two different medicaments calcium hydroxide (CH) and triple antibiotic paste (TAP).

Settings and Design:

This study involves an in vitro study.

Materials and Methods:

Ninety standard-sized dentin cylinders with apical sizes of 1 mm and 12 mm length were made from freshly extracted single-rooted premolars. Samples were randomly divided (45 each) and subjected to two intracanal medicaments, CH (Groups 1, 2, and 3) and TAP (Groups 4, 5, and 6). Three different irrigation protocols were followed: Group 1 and 4: Sequential use of 1.5% sodium hypochlorite (NaOCl) followed by 17% ethylenediaminetetraacetic acid (EDTA); Group 2 and 5: Etidronate + 1.5% NaOCl mixture; Group 3 and 6: 7.6% clodronate + 1.5% NaOCl mixture. The samples were stored in phosphate-buffered saline and subjected to enzyme-linked immunosorbent assay for TGF-β1quantification.

Statistical Analysis Used:

One-way analysis of variance and Tukey’s post hoc test with significance value <0.05.

Results:

Growth factor release was significantly higher in the clodronate irrigation groups irrespective of the intracanal medicament used (P < 0.05). There was no significant difference in TGF-β1 release when comparing the two intracanal medicaments among all three irrigation groups.

Conclusions:

In the context of growth factor release, etidronate, and clodronate, which are regarded as substitutes for EDTA in regenerative endodontic procedures, performed equivalent to the gold standard irrigant, regardless of the medicament used.

INTRODUCTION

Regenerative endodontic procedures that reinstate tooth viability are a worthwhile therapeutic option for necrotic immature permanent teeth.[1] The pulp-dentin complex regeneration strategy based on cell homing has been scientifically proven to be more effective in clinical settings than cell transplantation.

Cell homing-based revascularization procedure recruits host endogenous stem cells to the injured site utilizing the biological signaling molecules. Growth factors, in particular transforming growth factor-beta1 (TGF-β1), play an integral role in the regenerative triad.[2] Research findings illustrate that TGF-β1 primarily regulates pluripotent stem cell migration, proliferation, and differentiation into odontoblasts.[3]

In the recently released clinical considerations for a regenerative procedure, the American Association of Endodontists (AAE) recommends using 17% ethylenediaminetetraacetic acid (EDTA) in the second appointment before inducing bleeding.[4] This irrigation regimen conditions the dentin and unveils the growth factors that were sequestered into the dentinal matrix during dentinogenesis.[5] Due to its hexadentate ligand attributes, EDTA dissolves calcium ions in dentin by forming a chelate with them. As a result, the bound growth factors are released, and the superficial intertubular dentin is decalcified by 1–5 µ, eroding the canal walls.[6,7]

Evidence strongly suggests the detrimental effects of strong chelators on the biomechanical properties of root dentin and the resulting alteration of stress distribution patterns in root-filled teeth. This possibly harms the longevity of teeth, particularly in immature teeth with thin dentinal walls.[8,9,10] An alternative irrigation strategy known as continuous chelation was put forth a few years ago to overcome all these disparities. This technique is claimed to be innocuous with dentin and effective in smear layer elimination. Continuous chelation irrigation requires the concurrent utilization of a weak chelator and a proteolytic agent mixture.[11]

Twin Kleen (MAARC Dental, Maharashtra, India) is a commercially available capsule containing 9% etidronic acid.[12] Clodronate is another weak nonnitrogenous chelator; both contain phosphorous molecules instead of nitrogen and will react less with the chlorine ions of sodium hypochlorite (NaOCl), making it a highly stable mixture.[13,14] According to the literature, clodronate mixtures maintain the free available chlorine levels at root canal temperatures better than the etidronate mixtures for a clinically applicable period.[15,16] Furthermore, studies have confirmed that the mixtures of etidronate and clodronate are persuasive for maintaining the physical properties of teeth.[17] The utilization of continuous chelation in revascularization and its ability to release growth factors from the dentin matrix has not yet been investigated. The present study evaluates the role of etidronate and clodronate in a NaOCl mixture on TGF-β1 release. Intracanal medications inevitably play a role in regenerative endodontic treatment for disinfection.[4] This study has additionally assessed how AAE-recommended medications, such as calcium hydroxide (CH) and triple antibiotic paste (TAP), impact dentin’s ability to release growth factors in the future.

The research hypothesis investigates whether continuous chelation irrigation with a mixture of etidronate and clodronate has an altered release of growth factors and is influenced by the use of the two aforementioned intracanal medicaments.

MATERIALS AND METHODS

Approval for the research protocol was obtained from the institutional ethical committee. The sample size was estimated with an effect size (f) of 0.467, α error of 5% (0.05), and actual power of 0.95, using G*Power software latest version 3.1.9.7 (Heinrich Heine Universität Düsseldorf, Düsseldorf, Germany); Heinrich-Heine-Universität Düsseldorf, Düsseldorf, Germany). Freshly extracted single-rooted premolars without any anatomic malformations were included, and teeth with caries, cracks, dilacerated roots, and resorptive defects were excluded from this study.

Sample preparation

A total of 90 dentin cylinders of length 12 mm were prepared by making horizontal sections using a slow-speed diamond saw under constant water cooling. To simulate an open apex with a 1 mm apical diameter, the samples were instrumented using hand files up to 100 K-file (Mani Corp, Japan) with 0.9% normal saline (Puniska Injectables Pvt. Ltd., India) as an intermittent irrigant. A nail varnish was used to cover the external surface, thus exposing only the inner surface to the irrigants. The samples were mounted into a cylindrical mold using putty impression material (Zhermack Zetaplus C Silicone L Intro kit, Zhermack, Italy) to simulate periodontium.

Placement of intracanal medicaments

The samples were randomly allocated into two intracanal medicament groups (n = 45). In groups 1, 2, and 3 – aqueous-based CH paste (RC Cal, Prime Dental Products Pvt. Ltd., India) was placed and in groups 4, 5, and 6 TAP prepared in the concentration of 1 mg/ml mixed using normal saline was placed. The medicaments were stored at 37°C for 7 days. Subsequently, the medicament was removed using saline irrigation and passive ultrasonic agitation.

Preparation of irrigants

Etidronate solution was freshly prepared by mixing two capsules of Twin Kleen (MAARC Dental, Shiva Products, India) with 10 ml of 1.5% NaOCl. Clodronate solution was prepared using pure disodium clodronate tetrahydrate salt (D4160, Tokyo Chemical Industry Pvt. Ltd., India) and was adjusted to pH 10.7 using sodium hydroxide (Chemico Glass and Scientific Company, India). This was mixed with an equal volume of 3% NaOCl (Prime Dental Products Pvt. Ltd., India) to create a final irrigant containing 0.26M (7.6%) clodronate and 1.5% NaOCl.

Irrigation protocol

The samples from each medicament group were further subdivided into three groups based on the irrigation regimen (n = 15). Irrigation was done using a 30-gauge closed-end side-vented needle (Neoendo side vent needle, Orikam Healthcare Pvt. Ltd., India).

• Group 1 and 4 – Sequential chelation done using 20 ml of 1.5% NaOCl and 17% EDTA for 5 min followed by a final flush with 5 ml of normal saline

• Groups 2 and 5 – Continuous chelation with 20 ml of the mixture of 9% etidronate and 1.5% NaOCl for 5 min followed by a final flush with 5 ml of normal saline

• Groups 3 and 6 – Continuous chelation with 20 ml of the mixture of 7.6% clodronate and 1.5% NaOCl for 5 min followed by a final flush with 5 ml of normal saline.

After conditioning, the dentin samples were immediately placed in 1 ml of phosphate-buffered saline (PBS) and stored at −80°C in a deep freezer until the day of growth factor quantification.

Quantification of transforming growth factor – beta 1 array

The PBS solution from each sample was subjected to enzyme-linked immunosorbent assay (ELISA) analysis using an ELISA test system (TGFβ1 ELISA kit, Everon Life Sciences, India) for quantification of the released growth factor.

Statistical analysis

The data were statistically analyzed using SPSS software, Version 25.0 (Released 2017; IBM Corp., Armonk, New York, United States). A one-way analysis of variance (ANOVA) test followed by Tukey’s post hoc test was done to compare the mean TGF-β1 levels between the test groups with a level of significance (P value) <0.05.

RESULTS

The release assay of TGF-β1 was higher among clodronate (Groups 3 and 6) than in etidronate (Groups 2 and 5) when continuous chelation irrigation was followed. All the irrigation regimen groups in both the medicament categories had highly significant differences (P < 0.001) in one-way ANOVA [Table 1]. Multiple comparisons using the post hoc test [Figure 1] reveal statistical significance between Groups 2 and 6 (P = 0.003), Groups 3 and 4 (P = 0.004), and Groups 3 and 5 (P < 0.001). Multiple group comparisons [Figure 2] among the two medicament categories reveal statistical significance between Groups 1 and 3 (P = 0.003), Groups 2 and 3 (P < 0.001), and Groups 5 and 6 (P < 0.001).

Table 1.

Intergroup comparison of mean transforming growth factor - β1 values (pg/mL) by one-way analysis of variance

Groups	Mean	SD	95% CI for mean		P
			Minimum	Maximum
Group 1	1.0649	0.1890	0.748	1.412	<0.001*
Group 2	0.9432	0.0979	0.780	1.108
Group 3	1.3459	0.2279	0.952	1.779
Group 4	1.0769	0.1599	0.717	1.354
Group 5	0.8941	0.2048	0.490	1.218
Group 6	1.2193	0.2619	0.759	1.731

*High statistical significance (P<0.001). Group 1: Calcium hydroxide medicament and sequential chelation with EDTA, Group 2: Calcium hydroxide medicament and continuous chelation with etidronate, Group 3: Calcium hydroxide medicament and continuous chelation with clodronate, Group 4: Triple antibiotic paste medicament and sequential chelation with EDTA, Group 5: Triple antibiotic paste medicament and continuous chelation with etidronate, Group 6: Triple antibiotic paste medicament and continuous chelation with clodronate. SD: Standard deviation, CI: Confidence interval, EDTA: Ethylene diamine tetra acetic acid

Figure 1.

Tukey’s post hoc comparison of the mean Transforming growth factor-β1 values (in pg/ml) categorized according to three irrigation regimens under two different medicaments. TGF-β1: Transforming growth factor-β1

Figure 2.

Tukey’s post hoc comparison of the mean Transforming growth factor-β1 values (in pg/ml) categorized according to two medicaments. TGF-β1: Transforming growth factor-β1

DISCUSSION

Dentin is speculated to be a potent reservoir of growth factors, particularly TGF-β1. The growth factors emanate from their precursor during dentinogenesis and can bind covalently to a specific binding protein, which traps them in the dentin’s extracellular matrix in their inactive form. When the dentin experiences an insult that might lead to demineralization, these signaling molecules are expressed in its structure. Multiple strategies can be found in the literature that separate the inactive TGF-β1 from the latency-associated propeptide to biologically activate it. It is proven that exposure to pH extremes such as acidity and alkalinity will produce activity in the latent progenitors.[5,18] While conditioning the dentin in root canal space using a potent chelator like EDTA at neutral or slightly alkaline pH growth factors are released due to calcium chelation and demineralisation.[18]

Etidronate/HEBP (1-Hydroxyethylidene-1, 1-Bisphosphonate) and clodronate are well-known first-generation, nonnitrogenous bisphosphonates that are widely employed in the treatment of osteoporosis owing to their osteoclastic and anti-inflammatory properties. Optimal disinfection has been achieved with a mixture of these soft chelators and NaOCl, resulting in minimal accumulated hard tissue debris and homogeneous organic and inorganic content of the exposed dentin surface.[11,17]

The irrigating solutions used in the present study, possess a weak chelating property with an alkaline pH. Despite their weak chelating action, the possible explanation for the better results could be the alkaline pH of 11.8 and 10.7 for etidronate and clodronate, respectively, which promotes TGF-β1 dissociation from the LAP molecule.[13,18]

Etidronate and clodronate mixtures, in place of EDTA, efficiently eliminate the smear layer without inciting peritubular dentin erosion. As a nonnitrogenous bisphosphonate, clodronate further shows enhanced stability when combined with NaOCl without impairing its freely available chlorine content.[13,14,19] Previous studies have employed NaOCl in the concentration of 2.5%–5% when combined with a weak chelator.[14] However, the use of NaOCl at lower concentrations was recommended in the AAE clinical guidelines, mainly to prevent any detrimental effect on SCAP’s survival.[4] It has been established that the harmful impact of NaOCl is concentration-dependent and that implementing 1.5% NaOCl in the final rinse step of the procedure fosters the release of growth factors.[5,20] For that reason, a lower concentration of 1.5% NaOCl was utilized in this study.

Due to its anticoagulant properties, EDTA residues on the dentinal walls could adversely affect the blood clot quality during the revascularization process, which subsequently impacts the attachment and survival of stem cells.[21] Several authors have suggested using saline or PBS as the final rinse to remove the EDTA residues. Even though this step could decrease the number of growth factors released, it is more important to consider the stem cell’s viability and attachment since they ultimately determine the fate of regenerative endodontics.[22] All experimental groups in this study received a final flush of saline to neutralize the residues, presupposing this would have a negligible effect on the quantity of growth factor released. Furthermore, studies point out that the release of growth factors is delayed up to 5 min after dentin conditioning.[23]

The two intracanal medications used in this study showed insignificant differences. Conversely, Galler et al. proposed that compared to TAP; aqueous-based CH released more growth factors. The polypropylene glycol vehicle utilized, which culminated in 80% retention of TAP on dentinal walls, may be the underlying cause of the value disparity.[24] The deliberate inclusion of saline in TAP preparation in this study makes its removal easier and reflects the finding that intracanal medications by themselves did not affect growth factor release.

Previously, dentin disk and powdered dentin samples have been employed for assessing the growth factor release; however, it became apparent that the quantification of biological molecules had been overestimated in these samples. However, the use of dentin cylinders seems more appropriate as it mimics the clinical scenario.[4] Since the critical diameter for a favorable outcome in regenerative endodontics is 0.5–1.0 mm based on research, dentin cylinders made from freshly extracted teeth prepared with a size 100 k file were utilized in this study to establish an apical diameter of 1.0 mm.[25]

To the best of our knowledge, no research has been conducted regarding the potential benefits of a continuous chelation method of irrigation for revascularization of root canals. Since dentin samples were utilized in this study for growth factor release, evaluating its in vivo clinical effectiveness could be essential. Future research should focus on how these continuous chelating agents influence blood clot characteristics, fibrin density, stem cell viability, and attachment.

CONCLUSIONS

Within the limitations of the present study, it could be concluded that the continuous chelation regimen with etidronate and clodronate is capable of releasing TGF-β1 from the dentin matrix at levels equal to or higher than the sequential chelation regimen with EDTA. It could also be concluded that the intracanal medicaments did not influence the release of TGF-β1 from the dentin matrix.

Conflicts of interest

There are no conflicts of interest.

Funding Statement

Nil.