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Table of Contents
Year : 2021  |  Volume : 17  |  Issue : 2  |  Page : 34-44

An in-vitro comparative evaluation of quantitative release of transforming growth factor β-1 from dentin upon the action of endodontic irrigants, medicaments, ultrasonic activation, and low-level laser irradiation

1 Department of Conservative Dentistry and Endodontics, Amrita Institute of Medical Sciences and Research Centre, Amrita Vishwa Vidyapeetham, Kochi, Kerala, India
2 Department of Biostatistics, Amrita Institute of Medical Sciences and Research Centre, Amrita Vishwa Vidyapeetham, Kochi, Kerala, India
3 Centre for Nanosciences and Research, Amrita Institute of Medical Sciences and Research Centre, Amrita Vishwa Vidyapeetham, Kochi, Kerala, India
4 Al Hinaee Health Centre, Musandam, UAE

Date of Submission31-Mar-2021
Date of Acceptance27-May-2021
Date of Web Publication09-Aug-2021

Correspondence Address:
Dr. Anilkumar Akhila
Sreebhavan HSRA C-56, Kalady, Karamana, P.O. Thiruvananthapuram, Kerala.
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/AMJM.AMJM_11_21

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Aim: The aim of this article is to evaluate the amount of transforming growth factor beta-1 (TGF β-1) released from dentin upon the action of various endodontic irrigants, medicaments, ultrasonic activation, and low-level laser irradiation. Materials and Methods: To assess the effect of endodontic irrigants and medicaments on TGF β-1 release, 200 dentin disks of 1 µm thickness prepared from human mandibular premolars were divided into five groups of 40 each. The specimens in the test groups were treated with four reagents: Group A: (2% chlorhexidine gluconate); Group B: (2.5% sodium hypochlorite); Group C: [calcium hydroxide powder (Ca(OH)2)]; Group D: [triple antibiotic paste (TAP) (minocycline 100 mg + ciprofloxacin 200 mg + metronidazole 500 mg)]; and one control reagent group, i.e., Group E: (normal saline). Dentin disks were subsequently treated with 10% ethylenediaminetetraacetic acid (EDTA). To assess the effect of ultrasonic activation and low-level laser irradiation on TGF β-1 release, 90 dentin disks of 1 mm thickness obtained from mandibular premolar roots were divided into 6 groups of 15 disks each: Group 1: (10% EDTA +ultrasonic activation), Group 2: [10% citric acid (CA) + ultrasonic activation], Group 3: (10% EDTA + low-level laser), Group 4: (10% CA+ low-level laser), and two control groups, i.e., Group 5 (10% EDTA) and Group 6 (10% CA). Three subgroups were formed among main groups indicating the region from where the specimens were prepared, namely, coronal, middle, and apical thirds. The irrigation solutions from all the above groups were collected, frozen in liquid nitrogen, and stored at −80°C and later thawed and subjected to growth factor quantification by using an enzyme-linked immunosorbent assay test system for TGF β-1. Results: Root canal irrigant 2% chlorhexidine gluconate and intracanal medicament calcium hydroxide both showed an inducing effect on TGF β-1 release, giving a maximum value of 0.741 ng/mL. The least value of 0.0823 ng/mL was given by 2.5% sodium hypochlorite, showing its negative impact on growth factor release. TAP showed a neutral effect similar to that of the control group (normal saline), giving a value of 0.247 ng/mL. Ultrasonic activation and low-level laser irradiation of EDTA and CA have both improved TGF β-1 release from dentin. Conclusion: Chlorhexidine gluconate and calcium hydroxide exerted a positive influence on TGF β-1 release from dentin, whereas sodium hypochlorite retarded its release and TAP gave a neutral impact similar to normal saline. Ultrasonic activation and low-level laser irradiation can enhance TGF β-1 expression. There is no significant difference in the growth factor release among the different regions of root dentin.

Keywords: Low-level laser, regenerative endodontic procedures, TGF β-1, ultrasonic activation

How to cite this article:
Akhila A, Prabath Singh V P, Varma KR, Vasudevan SV, Sukhithasri V, Sasikumar S. An in-vitro comparative evaluation of quantitative release of transforming growth factor β-1 from dentin upon the action of endodontic irrigants, medicaments, ultrasonic activation, and low-level laser irradiation. Amrita J Med 2021;17:34-44

How to cite this URL:
Akhila A, Prabath Singh V P, Varma KR, Vasudevan SV, Sukhithasri V, Sasikumar S. An in-vitro comparative evaluation of quantitative release of transforming growth factor β-1 from dentin upon the action of endodontic irrigants, medicaments, ultrasonic activation, and low-level laser irradiation. Amrita J Med [serial online] 2021 [cited 2021 Dec 9];17:34-44. Available from: https://www.ajmonline.org.in/text.asp?2021/17/2/34/323541

  Introduction Top

Dental caries and traumatic injuries of immature permanent teeth can result in necrosis of pulp and ensuing retardation of root development.[1],[2] The traditional line of management of these conditions includes apexification in which calcium hydroxide paste or mineral trioxide aggregate (MTA) is used for the induction of an apical barrier to attain closure of the root apex.[3] Though there occurs the resolution of signs and symptoms of the disease through these approaches, the restoration of normal pulpal defenses and nociception cannot be achieved.[4]

The goals of regenerative endodontic procedures (REPs) are to recreate dentin-pulp complex; resuscitate damaged coronal dentin; and revive damaged root, cervical, or apical dentin.[5],[6]

The success of REPs depends on the accomplishment of four key elements of tissue engineering, namely, the acquisition of stem cells, acceptable scaffolds, suitable signaling molecules, and a biomimetic environment for the stem cells to regenerate.[7]

The most promising group of stem cells employed in endodontic regeneration includes the autologous postnatal dental stem cells because of their prominent odontogenic ability and decreased immune rejection.[8],[9],[10],[11] Dental stem cells are obtained from various parts of the oral cavity, namely, the apical papilla, periodontal ligament, dental follicle, etc.[12] The dental pulp stem cells (DPSCs) have multi-potentiality and can differentiate into odontoblasts, osteoblasts, adipocytes, chondrocytes, or neural cells.[13]

Scaffolds can be synthetic as well as natural.[14] Natural scaffolds are more compatible biologically but have the disadvantages of procuring in large amounts and decreased mechanical stability.[15] A variety of scaffolds are used in regenerative endodontics, including moderately short-lasting polymers such as polyglycolic acid (PGA), polylactic acid (PLA), polyglycolic acid-poly-l-lactic acid (PGA-PLLA), as well as polylactic polyglycolic acid (PLGA).[16]

Growth factors are bioactive molecules, chemically proteins or polypeptides that modify cellular response through intercellular communication.[17] Action of various agents that cause dentin demineralization can release these molecules from their quiescent state within the extracellular matrix components.[18],[19] These molecules are able to evoke cellular response and to regulate immunodefense, angiogenesis, differentiation, and mineralization even at very low concentrations comparable to picograms.[20]

A diverse group of growth factors are present in the dentin matrix, namely, the transforming growth factor β-1 (TGF-β-1), fibroblast growth factor 2, bone morphogenetic protein (BMP) 2, platelet-derived growth factor, placenta growth factor, and vascular endothelial growth factor (VEGF), to name a few.[21] The TGF β family comprises a group of diverse growth factors including TGF β proteins, BMPs, growth and differentiation factors, anti-Mullerian hormone, and activin and nodal.[22] TGF β-1 is instrumental in signaling events concerned to proliferation, differentiation, and recruitment of stem/progenitor cells to the site of tooth injury, for the initiation of regeneration, thus facilitating repair of dental tissues.[23] The isoforms of TGF β (TGF β-1, TGF β-2, TGFβ-3) all have similar homology but varied genetic representation and are secreted as latent precursor molecules consisting of a latency-associated peptide region.[24],[25] TGF β-1 and β-3 have been shown to induce secretory activity in odontoblasts in vitro, upon direct application to the odontoblastic region of tooth disks in rat incisors and on transdentinal application in human teeth during culture.[26]

Organic acids and chelating agents like ethylenediaminetetraacetic acid (EDTA) can evoke the release of growth factors from the dentin matrix.[18] Sadaghiani et al.[27] in an in-vitro study demonstrated that the action of EDTA, citric acid (CA), and phosphoric acid on pulverized dentin can extract and immobilize the matrix-bound growth factors.

About 17% EDTA is conventionally used in the conditioning of the canal walls prior to initiating the incorporation of extraneous growth factors and scaffold or before initiating bleeding as in the case of revascularization procedures.[28] However, certain studies have evaluated the use of 10% aqueous EDTA and 10% CA in REPs with promising results.[18],[27]

There have been various studies which have investigated the use of agents that can induce and enhance the release of bioactive molecules from dentin.[18],[22],[27],[29] Duncan et al.[30] in an in-vitro study demonstrated that histone deacetylase inhibitors (HDACis) like valproic acid released TGF β-1, although the concentration was less compared with the amount released by EDTA. Similarly, prolyl hydroxylase inhibitors like L-mimosine can stimulate the production of growth factors such as VEGF, which plays a crucial role in the angiogenesis and regulation of inflammatory reactions.[31]

Latent TGF-β can be activated by various physiological mechanisms including the use of denaturing agents like heat, extremes of pH, chaotropic agents, and detergents and multifunctional proteins like thrombospondin-1.[32] A classic study by Arany et al.[33] had shown that low-level laser therapy at varying fluences from 10 s (0.1 J/cm2) to 600 s (6 J/cm2) can be used to activate the latent TGF-β complexes in vitro. Widbiller et al.[34] in 2017 substantiated that ultrasound waves caused increased ingression of the irrigants into the dentinal tubules accounting for increased growth factor exposure on the dentin surface.

In-situ growth factor expression in the root canals can bring about guaranteed results in REPs. The present study aims to investigate the effect of various root canal disinfectants and intracanal medicaments on the release of a particular growth factor TGF β-1 from dentin samples and also whether their expression on dentin surface can be enhanced by ultrasonic activation and low-power laser application.

  Materials and Methods Top

The present in-vitro study was conducted following approval and ethical clearance from the Institutional Ethics Review Board (IERB) at Amrita Institute of Medical Sciences and Research Centre. The specimen preparation was done in the Department of Conservative Dentistry and Endodontics, Amrita School of Dentistry. The enzyme-linked immunosorbent assay (ELISA) test and subsequent TGF β-1 quantification were performed at the Amrita Centre for Nanosciences and Molecular Medicine, Amrita Institute of Medical Sciences and Research Centre, Cochin, Kerala.

Collection of the samples

Freshly extracted human mandibular premolars without any morphological abnormalities, developmental defects, or carious lesions were chosen for the study. The teeth were subjected to ultrasonic scaling and cleaning with hydrogen peroxide.

Sample preparation

For the initial part of the study, a sample of 200 dentin disks of 1 µm thickness was prepared from the root portion of the teeth by sectioning transversely. The sections were prepared using a hard tissue microtome (Leica) [Figure 1]A.
Figure 1: (A) Hard tissue microtome producing 1 µm-thick dentin disks. (B) IsoMet diamond saw used to form 1 mm-thick dentin disks. (C) The Human TGF β-1 Quantikine Elisa kit. (D) Dentin samples undergoing ultrasonic activation. (E) Dentin samples undergoing laser irradiation. (F) ELISA well plates being loaded with reagents

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For the second part of the study, 90 dentin disks were prepared from different regions of the root. Dentin disks of 1 mm thickness were prepared from the coronal, middle, and apical portions of the root by transverse sectioning using the IsoMet diamond wheel saw under constant water flow at 600 rpm [Figure 1B]. The thickness was confirmed using digital calipers.

Storage of dentin specimens and interventions

The dentin disks for both parts of the study were collected using a camel brush and stored in 0.5% chloramine solution, which was exchanged with double distilled water 24 h before experimentation. For the initial part of the study, the reagents used included five groups: four test groups and one control group. The test groups consisted of two root canal disinfectants: (Group A: 2% chlorhexidine gluconate solution, Group B: 2.5% sodium hypochlorite solution) and two intracanal medicaments [Group C: calcium hydroxide powder and Group D: triple antibiotic paste (TAP) (minocycline 100 mg + ciprofloxacin 200 mg + metronidazole 500 mg)] and Group E: 0.9% normal saline which served as the control.

The dentin conditioning agent used was 10% wt/vol EDTA; 40 dentin disks were added into each group.

For the second part of the study, the reagents used included dentin conditioning agents: 10% wt/vol EDTA and 10% wt/vol CA. A total of six groups were included: four test groups: Group 1: (10% EDTA +ultrasonic activation), Group 2: (10% CA + ultrasonic activation), Group 3: (10% EDTA + low-level laser), Group 4: (10% CA+ low-level laser); and two control groups: Group 5: (10% EDTA) and Group 6: (10% CA). Each of the six groups were further subdivided into three groups according to the region from where the dentin disk was prepared, namely, coronal, middle, and apical thirds. Five dentin disks were incorporated into the individual subgroups; thus a total of 90 dentin disks were used. The ELISA kit for the quantification of the TGF β-1 not only detects the presence of TGF β-1 but also quantifies the amount of the growth factor released from the samples (Ray Biotech) [Figure 1C].

I. Evaluation of the effect of disinfectants and intracanal medicaments on TGF β-1 release from the samples

The 200 1 µm thickness dentin disks which were stored in double distilled water were equally divided into five groups: four test groups and one control group [Figure 2].
Figure 2: Flowchart summarizing the steps in the initial part of the study assessing the effect of irrigants and intracanal medicaments on TGF β-1 release

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Test groups

Treatment with disinfectants

The dentin disks were immersed in 500 µL each of 2% chlorhexidine digluconate and 2.5% sodium hypochlorite for a period of 10 min. After this step, the dentin disks were subsequently immersed in 500 µL of 10% EDTA solution at pH 7 for a period of 20 min. All these procedures were carried out in Eppendorf tubes.

Treatment with intracanal medicaments

Dentin disks were coated with a layer of intracanal medicament-calcium hydroxide and TAP and incubated at 37°C in a humidified atmosphere for 48 h. The disks were subjected to rinsing with phosphate-buffered saline three times and later immersed in 500 µL of 10% EDTA at pH 7 for a period of 20 min.

Control group

Dentin disks were immersed in 500 µL of normal saline for 10 min. After this step the disks were immersed in 10% EDTA for 20 min.

After dentin treatment, the irrigation solutions from all groups were removed separately, immediately frozen in liquid nitrogen, and stored at −80°C. The solutions were subsequently thawed, centrifuged, and subjected to growth factor quantification by using an ELISA test.

Quantification of growth factors

For running the ELISA test, initially, the standard solutions provided in the ELISA kit were prepared against which the test and control solutions were compared.

II. Evaluation of the effect of ultrasonic activation and low-level laser application on the growth factor release from dentin

Ninety dentin disks of 1 mm thickness which were prepared from different regions of extracted mandibular premolar roots were divided into six defined groups consisting of four test groups and two control groups [Figure 3].
Figure 3: Flowchart summarizing the steps in the second part of the study assessing the effect of ultrasonic activation and laser irradiation of irrigants

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Test groups

Groups undergoing ultrasonic activation

Initially, the dentin disks were immersed in 300 μL of 10% EDTA or 10% CA for a period of 20 min, after which activation per disk was performed with an ultrasonic file and the appendant unit in irrigation mode. During activation, the experimenter touched the dentin surface with the file continuously and, without pressure in a 30° angulation, performed circular motions over the samples. Each individual dentin disk underwent the respective protocol three times for a duration of 3 min [Figure 1D].

Groups undergoing laser activation

A diode laser was used for laser activation, which has a single probe laser handpiece at 940 nm. The dentin disks were initially immersed in 300 μL of 10% EDTA or 10% CA for a period of 20 min and subsequently irradiated with the laser device in a non-contact mode. The energy density applied to each dentin disk was adjusted to approximately 5 J/cm2 by applying 0.5 W output power with a beam angle of 90° for a period of 30 s per disk [Figure 1E].

Control groups

The dentin disks were immersed in 300 µL of both 10% EDTA and 10% CA for a period of 20 min, respectively.

After treatment, irrigation solutions were removed individually from the samples, immediately frozen in liquid nitrogen, and stored at −80°C and later thawed, centrifuged, and taken for growth factor quantification [Figure 1F].

Statistical analysis

The collected data were compiled by using Micro Excel 2010 and were analyzed by using SPSS 20.0 version statistical software. Quantitative variables were expressed as mean ± SD (standard deviation), and categorical variables were expressed as frequency and proportions. Comparison of means among the different groups was done by using non-parametric Mann–Whitney U-test for both parts of the study. Comparison was done between the values obtained from the standard solutions provided in the ELISA kit against those given by the solutions used in the study.

  Results Top

Effect of irrigants and intracanal medicaments on TGF β-1 release

The maximum amount of TGF β-1 released was from Group A (chlorhexidine digluconate) and Group C (calcium hydroxide) giving similar values of 0.741 ng/mL. The minimum value of 0.0823 ng/mL was given by Group B (2.5% sodium hypochlorite), and Group D (TAP) gave similar value of 0.027 ng/mL as that of control group [Figure 4]. Analysis of statistical data showed that P-value is greater than 0.05, which suggests that the difference among the groups is not statistically significant; however, it yields clinically relevant observations [Table 1] and [Table 2].
Figure 4: TGF β-1 released from the dentin following the action of irrigants and intracanal medicaments. EDTA = ethylenediaminetetraacetic acid, CHX = chlorhexidine, NaOCl = sodium hypochlorite, Ca(OH)2 = calcium hydroxide, TAP = triple antibiotic paste, ng/mL = nanogram per milliliter

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Table 1: Mean and standard deviation values of TGF β-1 released from dentin samples treated with root canal irrigants and intracanal medicaments

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Table 2: Test statistics as per the non-parametric tests for the initial part of the study

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Effect of ultrasonic and laser activation on TGF β-1 release

Analyzing the TGF β-1 release from the various groups, it can be observed that the growth factor expression is improved by both ultrasonic activation and low-level laser irradiation of demineralizing agents like 10% EDTA and 10% CA than being used alone. A comparison of the different groups reveals that there is no significant correlation between TGF β-1 release and anatomical region of the tooth root [Figure 5][Figure 6][Figure 7]. Statistical estimation depicted a P-value less than 0.001, which showed that the difference between the groups is statistically highly significant [Table 3] and [Table 4].
Figure 5: TGF β-1 released from dentin following ultrasonic activation. EDTA = ethylenediaminetetraacetic acid, CA = citric acid, C = coronal third, M = middle third, A = apical third

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Figure 6: TGF β-1 released from dentin following low-level laser irradiation

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Figure 7: TGF β-1 released from dentin following treatment with EDTA and CA (control groups)

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Table 3: Mean and standard deviation values of TGF β-1 released from dentin samples subjected to ultrasonic activation and low-level laser irradiation of irrigants

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Table 4: Test statistics as per the non-parametric tests for the second part of the study

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  Discussion Top

The immature root with a necrotic pulp and apical periodontitis presents multiple challenges to successful treatment. These include the difficulty in proper disinfection of the canal system, lack of an apical barrier to facilitate obturation, and the thin dentinal walls which are susceptible to fracture.[35],[36] Before 1966, the clinical management of the “blunderbuss” canal with infected pulp usually required a surgical approach for the placement of an apical seal into the often fragile and flaring apex, which however further reduces the root length resulting in a very unfavorable crown–root ratio.[37]

There was a paradigm shift in the management of necrotic immature permanent teeth, with the experimental histologic study by Ostby[38],[39] in 1961 on the role of the blood clot in endodontic therapy, coupled with an understanding that revascularization is essential for continuation of root development after traumatic injuries. However, disinfection of the root canal system is of utmost importance in REPs which necessitate the use of root canal disinfectants and intracanal medicaments.[40]

The selection of suitable concentration of disinfectants and medicaments is crucial as this may have an impact on the potency of the growth factors and the viability of stem cells, both of which are essential for pulp-dentin regeneration.[18] The role of growth factors in REPs cannot be overemphasized. These bioactive molecules are important in regulating dental stem cell recruitment, migration, proliferation, and differentiation.[29] These factors can be acquired from various sources such as platelet-rich fibrin, platelet-rich plasma, blood clot, and most significantly the dentin matrix.[29],[41] Recruitment of these activation signals in situ is more desirable than their extraneous introduction into the root canals.[42]

Chlorhexidine has been widely used in REPs due to its antimicrobial and low cytotoxic properties along with its efficacy against most endodontic pathogens. Chlorhexidine also inhibits matrix metalloproteinases which are implicated in the development of periapical pathosis.[43] About 2% CHX gel is an effective agent in root canal disinfection in the revascularization of necrotic immature roots.[44] Chlorhexidine exerts both time- and concentration-dependent effects on the survival of DPSCs.[43] Thus these factors have to be carefully controlled when using CHX in the selected pulp regeneration technique. The influence of CHX on TGF β-1 release from dentin shows a favorable effect in the present study, which is in accordance with the results of the analysis by Galler et al.[18] Sodium hypochlorite (NaOCl) due to its antibacterial properties and ability to dissolve organic tissues remains the gold standard in endodontic disinfection.[45] NaOCl exerts a concentration-dependent effect on stem cell survival with reduced cell viability at higher concentrations.[46] In the present study, a concentration of 2.5% was chosen as it is the most widely used strength of NaOCl for root canal disinfection.[47] However, NaOCl has shown an inhibiting influence on TGF β-1 expression from dentin as per the results of the present study. According to a study done by Diogenes et al.,[40] 1.5% of NaOCl was found to have minimal effects on SCAP (stem cells of apical papilla) survival and differentiation. Thus it can be concluded that application of NaOCl should always precede EDTA treatment of dentinal walls which liberates the latent growth factors.

According to Graham et al.,[48],[49] growth factors and other bioactive molecules, sequestered within the dentin matrix, may be released by the action of Ca(OH)2 and signal gene expression in pulp cells, which mediates the changes in cell behavior observed during regeneration. The conclusions of a study by Bose et al.[50] indicate that Ca(OH)2 and TAP, when applied as an intracanal medicament in immature necrotic teeth, can help in the functional development of the pulp-dentin organ as observed by changes in root morphology. The use of Ca(OH)2 in regenerative endodontics in collation to other frequently used antimicrobial combinations is recommended, as Ca(OH)2 showed an enhancing impact on SCAP survival.[51] A case series by Cehreli et al.[52] has shown that Ca(OH)2 can be successfully used as an intracanal medicament, enhancing the healing of immature necrotic teeth and restoring their vitality apart from MTA.

TAP has been extensively used to obtain a relatively sterile domain in the radicular space promoting tissue healing and repair.[53],[59] TAP on a scaffold system is a novel approach employed in REPs, which is gaining attention due to its potential to eliminate microorganisms and their biofilms (e.g., Actinomyces naeslundii).[54] TAP elicited appropriate response by providing the aseptic conditions within root canals that facilitate thickening of radicular walls, closure of apical foramen, and recovery of a relatively positive response to electric pulp testing.[53],[55] Discoloration of root canal walls and negative impact on SCAP are the two known disadvantages of TAP. The effect of antibiotics on the survival of stem cells was investigated by Ruparel et al.,[56],[57],[58] and they concluded that high concentrations of antibiotics have a detrimental effect on SCAP survival, whereas lower concentrations as well as Ca(OH)2 at all tested concentrations are conducive to SCAP survival and proliferation. The present study showed a rather neutral effect on growth factor release which could be attributed to the concentration used.

Passive ultrasonic irrigation (PUI) has emerged as an effective modality of eliminating the smear layer from the root canal walls.[59],[60] PUI relies on the transmission of acoustic energy from an oscillating file or smooth wire to an irrigant in the root canal, in which the energy thus produced by ultrasonic waves can cause acoustic streaming and cavitation of the irrigant.[61] Irrigant activation using ultrasonics produces a well-debrided root canal wall, which caters to the enhanced attachment of the stem cells and inherent growth factor expression.[62],[63] The present study demonstrates that ultrasonic activation of demineralizing acids such as EDTA and CA can bring about increased growth factor expression on the dentin surface of root canals, which is in accordance with the conclusions of a study by Widbiller et al. in 2017.

TGF-βs have a central role in tooth development, specifically in the pulp-dentin pathophysiology. Latent TGF β-1 (LTGF β-1) can be activated using different modalities including extreme pH, heat, ultrasound, integrin binding, ionizing radiation, and proteases.[34] Activation of LTGF β-1 is important in rendering its physiological and biochemical functions.[33] Low-power laser irradiation is capable of activating the LTGF β-1 complex in vitro and further enhances its expression as in wound healing.[64] Chen demonstrated that low-power laser-induced reactive oxygen species stimulated dentin production by activating TGF β, a signaling protein that can promote dental stem cell differentiation. Low-power laser apart from activating latent TGF β-1 also enhances the growth of SHEDs (stem cells from the human exfoliated deciduous teeth) during situations of nutritional deficiency.[65] This same principle is applied in the same study to analyze whether low-power laser can enhance dentin growth factor expression. A favorable response in the form of TGF β-1 release has been observed after exposure of dentin by low-power laser.

The results of the present study thus demonstrate that various factors which are involved in REPs can bring about either an enhancing or inhibitory effect on the release of growth factors from dentin, following the action of demineralizing solutions. However, appropriate conclusions can only be drawn following clinical studies. The use of appropriate concentrations of these determinants can bring about promising results in REPs.

  Conclusion Top

Within the limitations of the study, it can be concluded that various irrigants and intracanal medicaments used in endodontic disinfection may exert diverse effects on the release of entrapped growth factors within the dentin matrix. A favorable impact in growth factor expression has been shown by chlorhexidine gluconate and calcium hydroxide. While an inhibitory response was elicited by sodium hypochlorite, TAP showed a rather neutral effect. Ultrasonic activation and low-level laser application of demineralizing agents such as EDTA and CA can enhance the release of growth factors from dentin matrix. Thus the judicious use of the irrigants and intracanal medicaments and their influence on growth factor release and viability of stem cells both play a pivotal role in determining the success of REPs.

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Conflicts of interest

There are no conflicts of interest.

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  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]

  [Table 1], [Table 2], [Table 3], [Table 4]


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