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 Table of Contents  
REVIEW ARTICLE
Year : 2021  |  Volume : 12  |  Issue : 3  |  Page : 146-151

Disinfection in regenerative endodontics and effect of commonly used antimicrobials on stem cell fate: A comprehensive review


Department of Conservative Dentistry and Endodontics, Vokkaligara Sangha Dental College and Hospital, Bengaluru, Karnataka, India

Date of Submission12-Aug-2020
Date of Decision08-Jun-2021
Date of Acceptance18-Jun-2021
Date of Web Publication17-Sep-2021

Correspondence Address:
Dr. Anitha Kumari Rangappa
Dept. No. 6, 2nd Floor, Department of Conservative Dentistry and Endodontics, Vokkaligara Sangha Dental College, V. V. Puram, Bengaluru, Karnataka
India
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DOI: 10.4103/srmjrds.srmjrds_76_20

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  Abstract 

The endodontic management of necrotic immature permanent teeth is often challenging for the clinician. Regenerative endodontic treatment has emerged as an important alternative to the traditional apexification treatment in such cases. In regenerative endodontics, elimination of microbes from the root canal is important for the survival, proliferation, and differentiation of stem cells. Translational studies suggest that irrigants and medicaments used for root canal disinfection may adversely affect stem cells and ultimately, the regenerative outcome. Conventionally, irrigants and medicaments have been chosen for their maximum antimicrobial effect without considering their effects on stem cells and the dentinal microenvironment. In this review, advances in disinfection for regenerative endodontics are discussed.

Keywords: Disinfection, irrigants, medicaments, regenerative endodontics, stem cells


How to cite this article:
Rai S, Rangappa AK. Disinfection in regenerative endodontics and effect of commonly used antimicrobials on stem cell fate: A comprehensive review. SRM J Res Dent Sci 2021;12:146-51

How to cite this URL:
Rai S, Rangappa AK. Disinfection in regenerative endodontics and effect of commonly used antimicrobials on stem cell fate: A comprehensive review. SRM J Res Dent Sci [serial online] 2021 [cited 2021 Dec 4];12:146-51. Available from: https://www.srmjrds.in/text.asp?2021/12/3/146/326212




  Introduction Top


Trauma, dental caries, and developmental dental anomalies such as dens evaginatus may lead to pulpal necrosis in immature permanent teeth.[1] Conventionally, apexification with calcium hydroxide or mineral trioxide aggregate was the treatment of choice in such cases,[2] Although apexification procedures appear to resolve the signs and symptoms of the pathosis, they have no potential to promote root maturation. Furthermore, long-term use of calcium hydroxide for root apexification might increase the risk of root fracture.[3]

Regenerative endodontic procedures (REPs) have emerged as an alternative treatment modality to apexification. REPs aim to promote normal pulpal functions by regeneration of the pulp–dentin complex. These include continued root development, nociception, and immune defense. Numerous case reports on REPs have been published since the first reported case in 2001.[4] However, great variability in treatment protocol has been observed in these reports.[5],[6]

Optimal disinfection of the root canal system is crucial for achieving favorable outcomes in REPs. Previous study has shown that successful regeneration of the pulp–dentin complex can occur only when the canal space is free from bacteria.[7] However, it is evident from recent regenerative endodontic studies that commonly used root canal disinfectants may have direct or indirect effect on stem cells. In addition, these studies have significantly contributed to the currently recommended regenerative endodontic treatment protocol.[8]

Dentin conditioning with 17% ethylenediaminetetraacetic acid (EDTA) promoted differentiation of dental pulp stem cells and stem cells of apical papilla by increasing the intracanal bioavailability of dentin derived growth factors.[9] Sodium hypochlorite (NaOCl) at higher concentrations (5%–6%) adversely affected stemcells from the apical papilla (SCAP) survival.[10] Commonly used paste-like preparations (1000 mg/ml) of triple antibiotic paste (TAP) and double antibiotic paste (DAP) has been found to be toxic for SCAP survival.[11] This review aims to comprehensively discuss the commonly used disinfectants in regenerative endodontics and their role in determining stem cell fate.


  Challenges in Disinfection for Regenerative Endodontics Top


Optimal disinfection of large infected canals in REPs possesses multiple challenges. The roots of such teeth are incompletely developed and are characterized by thin, fragile dentinal walls with an open apex. The presence of open apex increases the chance of irrigant extrusion. On the other hand, thin, fragile dentinal walls contraindicate mechanical instrumentation. In long-standing cases, the microbial colonies are often present in the form of tightly adherent biofilms, the removal of which requires stringent disinfection protocol.

Translational research in disinfection for REPs suggests that some irrigants and intracanal medicaments, when used at higher concentrations, may adversely affect stem cell survival, attachment, and differentiation.[10],[12],[13] Furthermore, alteration of dentin matrix (stem cell substrate) by chemicals used during disinfection may affect stem cell attachment.[14] Long-term placement of calcium hydroxide intracanal medicament in such cases may further weaken the already weak dentinal walls.[15]


  Irrigants and Their Role in Stem Cell Fate Top


Sodium hypochlorite

NaOCl remains the most widely used irrigant in REPs.[4] In endodontics, NaOCl is commonly used in concentrations between 0.5% and 6%, all of which possess antibacterial property. Excellent antibacterial property and tissue-dissolution capacity make NaOCl an excellent irrigant for disinfecting necrotic immature teeth in REPs. However, the use of higher concentrations of NaOCl is not advocated, owing to its toxic effect on the survival and differentiation of stem cells.

In an in vitro study, it was observed that 6% NaOCl adversely affected SCAP survival.[10] Studies have also shown that NaOCl at concentrations of 5%–6% prevented dental pulp stem cells from differentiating into an odontoblast-like phenotype.[12],[16] Moreover, a study suggested that NaOCl has both direct and indirect toxic effects on stem cells.[13] Thus, the use of higher concentrations of NaOCl in REPs diminishes stem cell survival and differentiation.

Dentin-derived growth factors such as vascular endothelial growth factor and transforming growth factor-beta 1 promote stem cell differentiation and/or proliferation. NaOCl, at higher concentrations, is known to denature these growth factors.[17] In one study, it was concluded that 1.5% NaOCl had minimal to no adverse effects on SCAP survival and differentiation.[18] The findings of this study were in accordance with that of another study which suggested that the use of 1.5% NaOCl in REPs was both effective and safe.[19]

Stem cell survival, proliferation, and differentiation are also influenced by the substrate on which they grow. Alteration in the composition of dentin (stem cell substrate) due to irrigants and medicaments may adversely affect the attachment of stem cells to the dentine surface. NaOCl when used at a high concentration decreases the carbon and nitrogen content in the dentin matrix. In contrast, 1% NaOCl did not significantly alter the composition of dentin.[20] In a study, dentin surface treated with NaOCl showed lowest amount of stem cell attachment compared to the groups treated with Morinda citrifolia and aquatine endodontic cleanser.[14] Thus, evidence suggests that a high concentration of NaOCl may have deleterious effects on stem cell attachment, and hence, a low concentration (1%–1.5%) is recommended.

Ethylenediamine tetraacetic acid

EDTA is a strong chelating agent and has excellent smear layer removal capacity. EDTA also exerts its antibacterial property by extracting bacterial surface proteins by combining them with metal ions from the cell membrane which can eventually lead to bacterial death.[21] Seventeen percent EDTA also has the ability to detach the tightly adherent root canal biofilms. This antibiofilm property of EDTA helps in decreasing the intracanal microbial load.[22]

EDTA is also known to increase the bioavailability of growth factors residing in the dentin matrix.[9] Some of these growth factors, especially vascular endothelial growth factor and transforming growth factor-beta 1, are known to promote the proliferation and differentiation of mesenchymal stem cells into odontoblast-like phenotypes. In an independent study, it was found that dentin disks conditioned with 17% EDTA showed a significant increase in odontoblastic markers such as dentin matrix protein-1 and dentin sialophosphoprotein.[12] Thus, the use of 17% EDTA in REPs may allow clinicians to harness the inductive properties of dentin-derived growth factors.

In an in vitro study, it was found that 17% EDTA promoted the survival of stem cells of apical papilla.[10] An ex vivo study demonstrated that conditioning of dentin with 17% EDTA promoted the differentiation of dental pulp stem cells into pulp-like tissue.[16] The use of 17% EDTA in regenerative endodontics also promotes the attachment of stem cells to the dentin surface. In a study, groups, in which dentin surfaces were treated with EDTA, showed maximum stem cell attachment.[14] Thus, the use of 17% EDTA, as an irrigant, offers several advantages in REPs.

Chlorhexidine gluconate

Chlorhexidine gluconate (CHX) is a wide spectrum antimicrobial, active against Gram-positive and Gram-negative bacteria and yeasts.[23] In endodontics, CHX has been extensively studied both as an endodontic irrigant and intracanal medication. In vitro studies have demonstrated that 2% CHX solution, when used as an irrigant, is able to effectively eliminate bacteria from root canals.[24],[25]

In regenerative endodontics, 2% CHX solution has been successfully used for disinfection, either alone or in combination with other irrigants. Two published case report of REP demonstrated favorable outcomes when 2% CHX was used for disinfection of the root canal system.[26],[27] However, recent in vitro studies suggest that 2% CHX negatively impacts the survival[10] and attachment of stem cells. In a study by Ring et al., dentin surfaces treated with 2% CHX showed the lowest amount of stem cell attachment.[14] The deleterious effects of 2% CHX on stem cells may be attributed to its direct cytotoxic effects, leading to loss in cellular viability. Furthermore, the substantivity property of CHX may be the reason for diminished cell attachment.[10] Thus, evidence suggests that the use of 2% CHX as an irrigant in regenerative endodontics may lead to unfavorable outcomes.


  Intracanal Medicaments and Their Role in Stem Cell Fate Top


Antibiotic medicaments

Chemical debridement remains the mainstay of disinfection for regenerative endodontics. Previous study have reported that NaOCl irrigation alone is not sufficient in creating conditions favorable for regeneration.[7] An interappointment dressing with an intracanal medicament helps to effectively eliminate the microbes and create a favorable environment for subsequent regeneration.

Intracanal medicaments have been used in almost all published cases of regenerative endodontics.[4],[28] TAP a mixture of ciprofloxacin, metronidazole, and minocycline is a commonly used antibiotic intracanal medicament in endodontics. In regenerative endodontics, TAP was first used in 2004.[29] At present, it is the most commonly used medicament in regenerative endodontics. In an in vitro study, 0.1 mg/mL of each antibiotic of TAP (0.3 mg/mL of mixture) effectively eliminated bacteria from specimens. Furthermore, no bacteria were recovered from the infected dentin after 48 h, indicating significant drug dentin penetration.[30] In another study, it was found that a 2-week application of TAP in immature dog teeth rendered 70% of all canals free of cultivable microorganisms.[31]

In a retrospective study, it was observed that necrotic immature teeth, in which TAP was used as medicament, showed a greater increase in root thickness when compared to calcium hydroxide.[6] Recently, concerns regarding the toxicity of TAP on stem cells have been raised by few authors. In one study, it was observed that the paste-like consistency (1000 mg/ml) of TAP commonly used in endodontics was toxic for SCAP survival. However, at concentrations of 0.1 mg/ml and 0.01 mg/ml, the drugs showed minimal to no toxicity.[11] These findings correlated with the findings of another study, in which low concentrations of TAP such as 1, 0.1, and 0.01 mg/ml eradicated Enterococcus faecalis colonies, with no adverse effect on stem cell viability.[32]

The action of other antibiotic medicaments such as DAP (ciprofloxacin, metronidazole), a modified TAP (ciprofloxacin, metronidazole, cefaclor) and augmentin (amoxicillin and clavulanic acid) on stem cells, have also been tested. All these antibiotic formulations have been found to be toxic to SCAP in a concentration dependent manner. Upon increasing the concentration of antibiotic mixtures, the percentage of viable SCAP decreased, with only 10% live cells at 100 mg/ml antibiotic concentration.[11] This is clinically important since the medicament contacting the apical papilla may affect SCAP survival. In addition, medications remaining on the dentin walls after irrigation may impact stem cells after the evoked bleeding step.

In regenerative endodontics, ideal concentration of the antibiotic mix would be the one that provides maximum antibacterial effect without causing toxicity to the stem cells. The AAE Regenerative Committee recommends the use of low concentration (0.1–1 mg/ml) of Triple or DAP as an interappointment medicament to preserve the viability of stem cells.[33] However, such low concentrations are very difficult to measure clinically. Furthermore, the antibacterial efficacy of such low concentrations of antibiotic mixtures has been questioned by few authors. According to the European Society of Endodontology position statement, the use of antibiotics in REP should be avoided since there is a lack of strong evidence favoring its use.[34]

Calcium hydroxide

Calcium hydroxide has its own limitations as an intracanal medicament. Due to its low solubility and diffusibility, calcium hydroxide is not very effective in eliminating microbes from dentinal tubules and the anatomical complexities within the root canal system. Buffering ability of dentin decreases the pH of calcium hydroxide, thereby reducing its antimicrobial effectiveness.[35]

The use of calcium hydroxide as an interappointment medicament dressing in REPs has been advocated by many authors. One advantage of calcium hydroxide over TAP/DAP is that calcium hydroxide is nontoxic to stem cells. One study demonstrated that unlike TAP, commercial preparations of calcium hydroxide promoted survival and proliferation of SCAP.[11] Another study evaluated the effect of residual calcium hydroxide or TAP on the survival of SCAP. It was found that TAP (paste-like consistency) remaining in the canal following irrigation had a detrimental effect on stem cell survival. Calcium hydroxide, on the other hand, promoted survival and proliferation.[36]


  Choice of Irrigation Technique Top


Positive pressure irrigation, sonic irrigation, passive ultrasonic irrigation, and negative pressure irrigation are some of the commonly used irrigation techniques in endodontics. In positive pressure irrigation, irrigant is delivered with the help of a syringe or needle under positive pressure. For the irrigant to reach the apex, the needle should be positioned 1–2 mm from the working length. The positive pressure exerted near the apical foramen may cause irrigant to extrude beyond the apex. This may lead to serious complications such as NaOCl accident. Negative pressure irrigation on the other hand uses negative pressure to deliver the irrigants. The canal is continuously irrigated and the microcannulae aspirate the irrigants to remove the debris along with the irrigant.

In the literature, there is lack of robust evidence as to which irrigation technique in REPs is superior. However, few authors have advocated the use of apical negative-pressure irrigation as it minimizes the risk of irrigant extrusion and is superior in terms of cleaning ability.[37],[38] A recent study on dog teeth suggested that the regenerated tissue in the groups which received EndoVac irrigation was histologically more organized than those that received conventional syringe irrigation.[39]

The use of apical negative pressure irrigation in REPs (immature teeth with open apex) may be advantageous over other irrigation techniques as it minimizes irrigant extrusion,[40] thereby reducing patient discomfort as well as direct toxicity to stem cells of the apical papilla. However, further research in this field is required to fully standardize the irrigation protocol.


  Clinical Considerations in Disinfection for Regenerative Endodontics Top


Based on the current evidence available in the literature, the following things must be considered during disinfection for REPs.

A low concentration of NaOCl preferably 1.5% should be used for irrigating the canal.[18] After copious irrigation with 1.5% NaOCl, the canal should be copiously irrigated with 17% EDTA.[41] An interappointment dressing with calcium hydroxide or preferably TAP/DAP is beneficial in creating a conducive environment for regeneration. TAP/DAP should be used at concentrations ≤1 mg/ml, so as to avoid stem cell toxicity. In the second appointment, a final irrigation with 17% EDTA increases the bioavailability of dentin-derived growth factors, thereby promoting stem cell proliferation and differentiation. [Table 1] summarizes the commonly used irrigants and medicaments in REPs along with their important features and clinical considerations.
Table 1: Important properties/clinical considerations of irrigants and medicaments used in regenerative endodontic procedures

Click here to view



  Conclusions Top


The choice of antimicrobials in regenerative endodontics should be such that they can bring about maximum disinfection, without causing toxicity to stem cells. A thorough understanding of the direct and indirect effects of irrigants and medicaments on stem cells and dentinal microenvironment will help standardize the disinfection protocol, which might help in improving the regenerative outcome. In the past, numerous studies have been conducted to examine the effect of commonly used antimicrobials on stem cell fate. However, additional clinical studies are required to understand the interrelationship between various aspects of REPs.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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Abstract
Introduction
Challenges in Di...
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Intracanal Medic...
Choice of Irriga...
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