DR.

HANI NAZZAL (Orcid ID : 0000-0002-6220-8873)

Accepted Article
Article type : Original Scientific Article

A prospective clinical study of regenerative endodontic treatment of traumatised immature

teeth with necrotic pulps using bi-antibiotic paste

Nazzal H1, Kenny K1, Altimimi A1, Kang J2, Duggal MS1

1
Department of Paediatric Dentistry and 2Department of Oral Biology, School of Dentistry, University

of Leeds, Leeds, UK.

Running Title: Regenerative endodontic treatment of traumatised teeth.

Key Words: Regenerative endodontic treatment, Dental trauma, Non-vital teeth, Immature Teeth

Corresponding author:

Dr. Hani Nazzal

Lecturer and Consultant in Paediatric Dentistry, Department of Paediatric Dentistry, School of

Dentistry, University of Leeds, Worsley Building, Clarendon Way, Leeds, LS2 9LU, UK

Tel: +44 1133436228

Fax: +44 1133436264

Email: [email protected]

This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/iej.12808
This article is protected by copyright. All rights reserved.
 Abstract

Aim To evaluate the treatment outcomes of a revitalisation endodontic technique (RET) for the
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management of traumatised immature teeth with necrotic pulps in children.

Methodology Fifteen healthy children (age range=7-10 years) with traumatised immature maxillary

incisors with necrotic pulps treated with bi-antibiotic revitalisation endodontic techniques were

prospectively assessed over approximately two years (mean=22 months). One operator undertook all

treatments, clinical reviews and standardised radiographic exposures with radiographic analysis being

carried out by two calibrated experienced clinicians. Crown colour change was assessed using an

objective published methodology. Wilcoxon signed-rank test was used to compare root lengths, root

dentinal widths and apical foramen widths over time.

Results Inter-operator measurement reliability was consistently strong for all measurements. There

was no significant difference in root lengths or root dentinal wall widths following RET. A significant

difference in apical foramen widths was observed after 2 years (p=0.013) with resolution of clinical

signs of infection in all cases. Despite omitting Minocycline and using Portland cement (Non Bismuth

containing cement), a noticeable crown colour change (yellower, redder and lighter), as measured by

an objective colour measurement system with ΔE=7.39, was recorded. Most patients, however, were

satisfied with the aesthetic outcome.

Conclusions: Traumatised immature teeth with necrotic pulps treated with revitalisation endodontic

techniques did not demonstrate continuation of root development or dentine formation when assessed

by periapical radiographs. However, apical closure and periodontal healing was observed. A

measurable change in crown colour (yellower, redder and lighter), with mostly no aesthetic concern to

the patients/parents, was also observed.

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 Introduction

Despite the growing use of revitalisation endodontic technique (RET) in the management of immature
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teeth with necrotic pulps in the last decade, these techniques have unpredictable outcomes especially

with regards to continuation of root development and thickening of root dentinal walls (Moreno-

Hidalgo et al. 2013, Kontakiotis et al. 2014). Although a RET protocol has been published by the

European Society of Endodontology and the American Association of Endodontics (American

Association of Endodontics 2016, European Society of Endodontology 2016), several RET

techniques, medicaments and success criteria have been reported (Kontakiotis et al. 2014).

With the use of this approach, successful apical healing has been consistently reported in the literature

(79 -100%), while continuation of root development and thickening of root dentinal walls have only

been achieved in 27.3-47% and 20-57% respectively of cases reported in the literature (Nazzal &

Duggal 2017). Furthermore, early studies have reported outcomes of RET regardless of the reason for

pulpal necrosis, in other words combining outcomes for traumatised teeth, carious teeth and those

with dental anomalies. This could be one of the reasons that has contributed to these conflicting

results as damage to the Hertwig Epithelial Root Sheath (HERS) following trauma could result in

failure in continuation of root development and thickening of dentinal walls (Nazzal & Duggal 2017).

Recently published studies on the use of RET in managing traumatised immature teeth with necrotic

pulps have reported the least successful results mainly in terms of continuation of root development

(Nagata et al. 2014, Saoud et al. 2014). These results suggest that traumatised teeth might be less

likely to respond to RET than those teeth with pulps that have become necrotic due to developmental

anomalies such as dens- invaginatus/evaginatus.

Several RET protocols have been proposed (Kontakiotis et al. 2015) making it difficult to compare

the results of various studies. The use of a tri-antibiotic mixture, in particular those containing

Minocycline, is associated with tooth discolouration as is the use of Mineral Trioxide Aggregate

(MTA) which is often used in achieving a coronal seal in this technique (Reynolds et al. 2009, Kim et

al. 2010 ). Alternatives to Minocycline have been proposed, including omitting Minocycline

altogether from the antibiotic mixture (Thibodeau & Trope 2007). This is supported by some data that

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 describes similar antibacterial effects of the bi-antibiotic paste consisting of a mixture of 100 mg

Metronidazole and 100 mg Ciprofloxacin to that of tri-antibiotic paste (Twati et al. 2011).
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The aim of this prospective study was to assess the success of RET using bi-antibiotic paste for the

management of traumatised immature teeth with necrotic pulps.

Methods

Ethical approval was obtained from the National Research Ethics Service (NRES) Committee,

Yorkshire & the Humber - Leeds East, UK. The legal guardians of fifteen healthy children (age

range=7-10 years, mean=8.3 years) with traumatised immature maxillary incisors with necrotic pulps

consented to take part. Patients were considered for the study if they met the following inclusion

criteria:

 Fit and healthy (ASA I and II);

 Cooperative enough to allow treatment under local analgesia;

 Had a minimum of one immature traumatised permanent incisor with necrotic pulp;.

Revitalisation endodontic treatment using a mixture of two antibiotics (ciprofloxacin and

metronidazole), was performed by one operator (HN) as follows:

First treatment visit

1. Local analgesia was given when indicated using lidocaine 2%+epinephrine 1:80,000.

2. The tooth was isolated using dry dam, accessed and the necrotic pulp extirpated using

barbed broaches.

3. The canal was then negotiated with minimal or no filing to prevent further weakening of

dentinal walls.

4. The root canal system was then irrigated slowly with 0.5% sodium hypochlorite (NaOCl)

with the needle introduced into the root canal at a point 2 mm short of the apical foramen.

5. The canal was then dried using paper points.

6. Metronidazole (100 mg) and Ciprofloxacin (100 mg) (TriBiodent, Royal Victoria

Infirmary, Newcastle, UK), were mixed with distilled water and introduced into the canal

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 using the plastic part of a green cannula (VasofixR Safety, B Braun, Melsungen,

Germany).
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7. A cotton pellet was then placed to cover the root canal orifice and the access filled with a

glass ionomer cement to prevent coronal leakage or contamination of the root canal with

oral microorganisms.

Second treatment visit: (after 2 weeks)

1. Where needed, plain local analgesia (no vasoconstrictor) was administered and the tooth

isolated and re-accessed as described above.

2. The antibiotic mixture was then flushed out of the root canal by irrigation with copious

amounts of normal saline.

3. Following this the root canal was dried thoroughly with paper points.

4. This was then followed by insertion of a sterile sharp instrument (finger spreader) with a

length of 2 mm beyond the working length, past the confines of the root canal, into the

periapical tissues to intentionally induce bleeding into the root canal. The bleeding was

then allowed to fill the root canal.

5. Once the root canal was filled with blood, a cotton pledget was placed in the pulp

chamber and a clot was allowed to form in the root canal.

6. Once the clot had formed, the pulp chamber in the coronal part was thoroughly cleaned to

remove remnants of blood, which could cause discolouration in the future.

7. The access cavity was then hermetically sealed with three layers of material to prevent

coronal leakage and contamination: Portland cement (Medcem, Weinfelden,

Switzerland), followed by glass ionomer (Fugi IX, GC Corporation, Tokyo, Japan), and

composite resin (SpectrumTPH Compules®, Dentsply, Weybridge, UK).

Patients were reviewed clinically and radiographically post treatment by the same assessor (HN).

Clinical assessment involved evaluation of the presence of signs of infection such as pain; abscess

formation; presence of a sinus tract; tenderness to palpation and percussion; and sensibility testing

using ethyl chloride cold test (Axongesic ®, BTC Invest, Praha, Czech Republic) and electric pulp

testing (Electric Pulp tester, SybronEndo, Sybron Dental Specialties, Glendora, California, USA).

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 Radiographs were taken, by the same operator, and standardised using a paralleling device (Dentsply

Rinn, Elgin, IL), the same x-ray machine and technique (Jadhav et al. 2012, McTigue et al. 2013,
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Nagy et al. 2014, Narang et al. 2015). Radiographic images were viewed and measurements were

performed using Infinitt digital radiograph software, Seoul, Korea. Radiographic assessment was

performed by two calibrated specialists in paediatric dentistry using two techniques; digital

measurement and visual assessment.

Using visual assessment methods (McTigue et al. 2013, Narang et al. 2015), the assessors reported on

continuation of root development, thickening of dentinal root walls and closure of apical foramen.

Any discrepancy between the two assessors was resolved by discussion and agreement. Assessment of

inter examiner reliability was performed using Kappa scores.

Using the digital measurement method, a modification of the line measurement technique (Jeeruphan

et al. 2012, Nagy et al. 2014) was used with the assessors recording the following (Figure 1):

1) Root length: The distance between the mesial cemento-enamel junction and the mesial and distal

ends of the radiographic root apex. The average of the mesial and distal root length measurements was

recorded, to the nearest 0.01, as root length (Figure 1a)

2) Apical foramen width: The distance between the mesial and distal apical root ends (Figure 1b).

3) Dentinal wall width: The difference between the outer root thickness (Figure 1c) and the inner pulp

canal width (Figure 1d), at two thirds root length measured from the cemento-enamel junction.

An average of the measurements performed by both assessors was used as the final measurement and

inter examiner reliability was measured using intra-class correlation.

Comparison between root lengths, root dentinal wall widths and apical foramen widths was performed

in order to assess root development between baseline radiographs, taken immediately after RET, and

those taken at the 2 year follow-up (Nagy et al. 2014)(mean 22 months).

Crown colour changes were measured based on the method described by Day et al. (2011) using an

objective digital camera system (IKAM). IKAM photographs were taken at baseline (immediately

after treatment) and 2 years (18-27 months, mean= 24 months). Using MATLAB software, colour

changes over time using CIELAB scores (L*, a*, b* and ΔE) were measured using a validated

objective method (Day et al. 2011) by one assessor (AA). At 2 years, patients were asked to report

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 their perception of traumatised tooth colour change following treatment and whether it affected them.

Where a change of colour was reported, the parent’s opinion regarding need for intervention was
Accepted Article
sought. The assessor also reported on tooth colour in comparison to the contralateral non-traumatised

tooth.

Statistical analysis

Wilcoxon signed ranks test was used to assess the differences in root lengths, root dentinal wall

widths and apical foramen widths between baselines and follow up radiographs. McNemar’s Exact

test was used to assess the difference in tooth pulpal response to electric pulp testing between baseline

and follow up results. Descriptive statistics were used in reporting clinical findings at follow-up

appointment. Statistical analysis was performed using SPSS software version 20 (SPSS, IBM Corp.

Armonk, New York, USA).

Results

A summary of the participant demographics, type of trauma resulting in loss of pulp vitality, pre-

operative clinical and radiographic signs of infection, amount of pulpal bleeding during RET

procedures, pre-operative root development stage, and reason for exclusion are presented in Table 1.

Twelve patients (mean age=8.3 years, range 7-10) remained in the study and were followed up for

approximately two years (mean=22 months, range=18-27 months). Three patients were excluded: a

patient was lost to follow up despite several attempts at contacting them; a second patient developed a

calcific barrier probably due to long term use of calcium hydroxide while the tooth was

orthodontically repositioned following a severe intrusion injury; and the third patient sustained a

second trauma after 7 months of treatment which lead to recurrence of abscess and failure of

treatment.

Most teeth in the study had required endodontic intervention following enamel/dentine crown

fractures, except for two teeth that had been replanted following avulsion. Six patients had initially

presented with clinical and/or radiographic signs of infection in the form of apical abscess associated

with sinus formation and pus discharge in most cases. Eleven patients presented with 2/3-root

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 formation with over half showing parallel apical dentinal walls. During RET, adequate bleeding was

achieved in most cases with only 3 cases resulting in minimal bleeding, evident on paper points.
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Table 2 shows the clinical and radiographic outcomes of RET treatments. Healing, with no clinical or

radiographic evidence of infection, was observed in all cases before proceeding with second stage

RET and this status was maintained throughout the study period. Although none of the teeth were

responsive to cold testing after 2 years, one third of the teeth were responsive to electric pulp testing.

McNemar’s Exact test using binomial distribution, however, showed no significant effect of RET on

eliciting an EPT pulpal response after 2 years (p value = 0.508), due to small sample size (n=12).

Table 3 shows the results of inter-examiner reliability test between both assessors, when using the

visual and digital measurement techniques before and after treatment. These results indicate that

continuous digital measurements were a better indicator of agreement between assessors with high

intra-class correlation (ICC) scores and significant p-values indicating strong consistency between

measurements. The kappa scores were low in comparison to ICC for agreement, with a negative value

for dentinal wall width indicating complete randomness of visual assessment. Therefore, the results

for the digital measurement of continuation of root development, thickening of root dentinal walls and

apical foramen widths rather than the visual assessment results were reported in this study (Table 3).

The Wilcoxon signed rank test revealed no significant difference in root lengths nor root dentinal wall

widths (Table 4). There was, however, a significant difference in apical foramen widths after 2 years

(Table 4).

Over 2 years, IKAM recorded a global colour change of ΔE= 7.39 (SD = +/-3.25) with a mean change

in L*= 0.79 (lighter), a*= 1.76 (redder) and b*=5.19 (yellower) (Table 5). Operator and software

measurement error were calculated at ΔE=0.62. Despite the noticeable change reported by global

colour change of ΔE=7.39, Tables 6 and 7 report patient and assessor perceptions of the crown colour

change and patients’ satisfaction. Most patients reported their teeth to be darker in colour after

treatment which was similar to that reported by the assessor. However, despite the noticeable change,

only one patient and their parent were not happy with the darker colour of their tooth and requested an

intervention to lighten the tooth with most patients and parents being happy with the final outcome

(Table 7).

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 Discussion

The use of RET has gained popularity with several published case reports/series (Kontakiotis et al.
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2015), a growing base of randomised controlled trials (Nagata et al. 2014, Nagy et al. 2014, Bezgin et

al. 2015), several systematic reviews (Moreno-Hidalgo et al. 2013, Kontakiotis et al. 2014) and a

meta-analysis (Nicoloso et al. 2016).

Despite agreeing that the success of this technique in achieving regeneration rather than

revascularisation/repair/mutagenisis is dependent upon achieving four essential tissue engineering

principles mainly; 1) availability of suitable stem cells, 2) a scaffold for the stem cells to populate and

orient in 3 dimensional manner, 3) appropriate signalling molecules promoting stem cell

differentiation into the desired odontoblasts and 4) disinfection of the root canal system without

damage to stem cells, (Wigler et al. 2013, Galler 2015); several RET have been used and reported in

the literature (Kontakiotis et al. 2015).

Consequently, both the American Association of Endodontics (2016) and the European Society of

Endodontology (2016) have published guidelines/position statement to provide guidance on an

evidence based protocol for use in RET.

The RET protocol used in this study was based on the available evidence when this study was

conducted, the results of local studies conducted at the Leeds Dental Institute (Twati et al. 2009,

2011), and successful cases treated in the department using the same protocol used in this study

(Figure 2 ). The case presented in Figure 2 and Figure 3 e-f highlight the ability of the protocol used

in this study to promote continuation of root development and thickening of dentinal walls. However;

it should be highlighted that the current AAE and ESE recommendations are based on the available

evidence to date and should be consulted in conjunction with the available literature in any future

studies.

There are several sources of stem cells in the oral cavity (Hargreaves et al. 2013) with some

researchers implicating that stem cells of the apical papilla (SCAP) as having a major role in RET

(Huang et al. 2008). The present study involved promoting stem cell population of the root canal

system through induction of bleeding from the periapical area. Lovelace et al. (2011) reported a 400-

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 600 fold increase in mesenchymal stem cells markers in blood collected from the root canal, similar to

the technique used in the present study, in comparison to levels found in systemic blood samples.
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In tissue engineering, a scaffold is an endogenous or transplanted material that provides a 3

dimensional microenvironment promoting cell growth, differentiation, adhesion, and migration

(Nakashima & Akamine 2005). Several synthetic and natural scaffold for use in RET have been

reported in the literature (Murray et al. 2007, Gathani & Raghavendra 2016).

Promoting a blood clot in RET has the advantage of transporting stem cells from the apical area into

the root canal system while allowing these cells to differentiate in a 3 dimensional manner. However,

referring to a blood clot as a scaffold is controversial. Although popular in the literature (Huang 2009,

Kontakiotis et al. 2015) and seems to fulfil the criteria of a scaffold, a blood clot is considered an

uncontrolled environment that promotes healing rather than regeneration. Promoting bleeding and clot

formation in the root canal system was performed in this study in order to promote stem cell migration

into the root canal system (Lovelace et al. 2011).

In order to disinfect the root canal system, minimal instrumentation of the canals was performed to

prevent damage to the thin enamel walls followed by irrigation with 0.5% sodium hypochlorite and

application of a bi-antibiotic paste containing 100 mg Ciprofloxacin and 100 mg Metronidazole to the

root canal system.

Sodium hypochlorite has been shown to be an effective root canal irrigant due to its bactericidal,

bacteriostatic effect in addition to its tissue dissolution properties (Martin 1975). Sodium hypochlorite

has been used as the only irrigant or in combination with other irrigants in 97% of RET studies

published till May 2014 with concentrations ranging from 1-6% (Kontakiotis et al. 2015). Some

laboratory studies have investigated the effect of sodium hypochlorite concentration on stem cells

(Trevino et al. 2011, Martin et al. 2014). Martin et al. (2014) assessed the effect of several sodium

hypochlorite concentrations (0.5%, 1.5%, 3%, and 6%) followed by either 17% EDTA or normal

saline and reported the negative effects of high concentration of sodium hypochlorite on the survival

and differentiation of stem cells of the apical papilla (SCAP) and recommended the use of 1.5%

sodium hypochlorite followed by 17% EDTA.

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 Therefore, the AAE (2016) and ESE (2016) recommend using sodium hypochlorite concentrations of

1.5% and 1.5–3%, respectively, followed by the use of 20 mL gentle irrigation with 17% EDTA in
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RET.

The RET protocol used in this study involved the use of 0.5% sodium hypochlorite with no use of

17% EDTA irrigation as the evidence of the effect of sodium hypochlorite concentration and EDTA

effect was only evident following the start of this study (Trevino et al. 2011). Initial work by Trevino

et al. (2011) suggested the possible positive effect of 17% EDTA irrigation on the viability of stem

cells following irrigation with high concentrations of sodium hypochlorite. However, such an effect

was only established after the work of Martin et al (2014) whereby the use of 17% EDTA irrigation

following different sodium hypochlorite concentrations (0.5%, 1.5%, 3%, or 6%) had shown a

protective effect on stem cell viability.

Never the less, the use of 0.5% sodium hypochlorite irrigation without EDTA, in that study, reported

a reduction on SCAP cell viability of 37% rather than completely diminishing SCAP viability (Martin

et al. 2014).

The availability of signalling molecules capable of promoting stem cell differentiation is crucial for

successful regeneration of pulp tissues. EDTA is a chelating agents able to demineralise the

superficial dentine layer, therefore releasing dentinal growth factors such as transforming growth

factor-β, and bone morphogenetic protein 2, consequently promoting stem cell differentiation into the

desired odontoblasts (Galler et al. 2011).

The RET protocol used in this study did not involve the use of EDTA following sodium hypochlorite

irrigation. This protocol was based on the available evidence at the start of this study where EDTA

was not used before 2012 (Kontakiotis et al. 2015). Successful cases treated at the department using

this protocol (Figure 2) coupled with continuation of root formation in some of the cases treated as

part of this study (Figure 3 e-f) indicate stem cell differentiation.

The use of sodium hypochlorite (1.5-3%) followed by 17% EDTA is currently recommended in order

to reduce effect of sodium hypochlorite on stem cells (Martin et al. 2014) and promote release of

dentinal signalling molecule (Galler et al. 2011), therefore those irrigants should be used in future

RET studies.

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 The use of antibiotic paste has been reported in 88% of studies (Jeeruphan et al. 2012, Nagy et al.

2014, Bezgin et al. 2015, Kontakiotis et al. 2015) with few researchers using the same antibiotic
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mixture used in the present study (Iwaya et al. 2001, Hargreaves et al. 2013) while others replaced

Minocycline with other antibiotics such as Cephaclor (Bezgin et al. 2015), Amoxicillin (Kahler et al.

2014) or Clindamycin (McTigue et al. 2013). The decision to eliminate Minocycline from the mixture

in the present study was due to it’s potential to discolour teeth (Kim et al. 2010 ) which was further

supported by recent work showing similar antimicrobial effect of the tri-antibiotic and bi-antibiotic

pastes (Twati et al. 2011). This antibiotic mixture is currently recommended by the AAE (2016) as

one of the potential intracanal medicaments used in RET. On the other hand, the ESE currently

recommends the use of calcium hydroxide instead of an antibiotic mixture ESE (2016). Although

there are currently few studies comparing the effect of calcium hydroxide versus antibiotic mixture

(Bose et al. 2009, Nagata et al. 2014), this recommendation is mainly based on recent studies showing

cytotoxicity to stem cells, and inability to remove antibiotic mixtures from root canal systems.

Ruparel et al. (2012) reported a reduction in the survival of stem cells of the apical papilla (SCAP)

when subjected to several antibiotic mixtures including tri-antibiotic and bi-antibiotic mixtures in the

high concentrations used for RET. This effect was not shown with lower concentrations of the

antibiotic mixtures (0.1-1mg/ml) and calcium hydroxide use (Althumairy et al. 2014).

Furthermore, Berkhoff et al. (2014) reported the inability of the current irrigation systems, including

the system used in this study, in removing antibiotic mixtures in comparison to calcium hydroxide.

Eighty-eight percent of the antibiotic mixture was retained in the root canal system regardless of the

irrigation system used while 98% of calcium hydroxide was removed.

The problems of antibiotic resistance, risk of sensitisation or even development of an allergic reaction

to the antibiotics used were other drivers to such recommendations.

Achieving a coronal seal is also crucial in maintaining a suitable root canal environment. The use of

MTA in achieving a coronal seal, hence preventing future contamination, has been associated with

crown discolouration. Most commercially available MTA contains agents, such as bismuth oxide,

which is used to enhance its radio-opacity. Bismuth Oxide is an agent known to cause discolouration

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 of teeth, therefore Portland cement, which has been shown to cause less tooth discolouration than

MTA (Lenherr et al. 2012), was used in the present study.

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The present study reports complete periapical healing of all teeth evident by the lack of clinical and

radiographic signs of infection, which is consistent with the results published in the literature (Nazzal

& Duggal 2017). However the effect on continuation of root development, thickening of dentinal

walls and apical foramen widths was inconsistent and warrants further discussion. Figure 3 shows

radiographic results of three of the patients treated in this study highlighting different outcomes

achieved in terms of continuation of root development, thickening of dentinal walls and apical

foramen widths.

The lack of continuation of root development, shown in the present study, is consistent with those

reported recently (Nagata et al. 2014, Saoud et al. 2014). With regards to thickening of dentinal walls,

the literature shows inconsistent results with thickening generally reported between 20-57% with a

minimum of 41% of traumatised teeth (Nagata et al. 2014, Saoud et al. 2014).

The lack of continued root development in the present study sample could be attributed to damage to

Hertwig’s epithelial root sheath (HERS) during trauma. It is a known biological process of

embryology that without the organising influence of the epithelium, undifferentiated mesenchymal

cells cannot differentiate into specialised cells, such as odontoblasts and cementoblasts.

While it is appropriate to hypothesise the viability of the HERS in discussing the results of this study

and the huge variability of the outcome of this technique in managing immature traumatised teeth

reported in the literature, further research is needed to test this hypothesis. In order to reduce post

treatment crown colour change, Minocycline was omitted from the protocol used in the present study

and Portland cement (a non-bismuth oxide containing cement) was used instead of the more widely

used MTA. Although changes in colour were still observed using CIELAB scores, patients and

parents were mostly satisfied with their tooth colour suggestive of minimal non-significant changes.

Conclusions

The use of a bi-antibiotic revitalisation endodontic technique in managing immature traumatised teeth

with necrotic pulps was not successful in promoting continuation of root development or thickening

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 of root dentinal walls over a period of 2 years. Survival of the teeth with no signs of infection, on the

other hand, was achieved successfully over the same period of time. Omitting Minocycline and using
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Portland cement resulted in a measurable crown colour change using an objective colour measurement

system although most patients were satisfied with the aesthetic outcome.

Acknowledgment

This study has been registered with clinicaltrials.gov under title Regenerative Endodontic

Treatment of Traumatised Teeth with identifier: NCT03045185. The authors would like to

acknowledge the Leeds Teaching Hospital Trust Charitable Foundation for their financial

support (Grant ref. 9R11/1207). We would like to thank Mr. Richard Steffen of Medcem,

Switzerland for supplying us with Portland cement for the trial and the editor of the European

Archives of Paediatric Dentistry for allowing us to reuse figures published in Nazzal and

Duggal 2017 in this manuscript.

Conflict of Interest statement

Dr. Nazzal, Dr. Kenny, Dr Altimimi and Dr Duggal report grants from Leeds Teaching Hospitals

Charitable Foundation and other from Medcem, Switzerland, during the conduct of the study. The

other author stated explicitly that there are no conflicts of interest in connection with this article

This article is protected by copyright. All rights reserved.

 References

Althumairy RI, Teixeira FB,Diogenes A (2014) Effect of dentin conditioning with intracanal
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medicaments on survival of stem cells of apical papilla. Journal of Endodontics. 40, 521-5.

American Association of Endodontics (2016) AAE Clinical Considerations for a Regenerative

Procedure [Online]. American Association of Endodontics. Available:

http://www.aae.org/uploadedfiles/publications_and_research/research/currentregenerativeendodonticc

onsiderations.pdf.

Berkhoff JA, Chen PB, Teixeira FB,Diogenes A (2014) Evaluation of triple antibiotic paste removal

by different irrigation procedures. Journal of Endodontics. 40, 1172-7.

Bezgin T, Yilmaz AD, Celik BN, Kolsuz ME,Sonmez H (2015) Efficacy of platelet-rich plasma as a

scaffold in regenerative endodontic treatment. Journal of Endodontics. 41, 36-44.

Bose R, Nummikoski P,Hargreaves K (2009) A retrospective evaluation of radiographic outcomes in

immature teeth with necrotic root canal systems treated with regenerative endodontic procedures.

Journal of Endodontics. 35, 1343-9.

Day PF, Duggal MS, High AS, et al. (2011) Discoloration of teeth after avulsion and replantation:

results from a multicenter randomized controlled trial. Journal of Endodontics. 37, 1052-7.

European Society of Endodontology (2016) European Society of Endodontology Position Statement:

Revitalisation Procedures. International Endodontic Journal. 49, 717-23.

Galler KM (2015) Clinical procedures for revitalization: current knowledge and considerations.

International Endodontic Journal. 49, 926-36.

Galler KM, D'Souza RN, Federlin M, et al. (2011) Dentin conditioning codetermines cell fate in

regenerative endodontics. Journal of Endodontics. 37, 1536-41.

Gathani KM,Raghavendra SS (2016) Scaffolds in regenerative endodontics: A review. Dental

Research Journal. 13, 379-86.

Hargreaves KM, Diogenes A,Teixeira FB (2013) Treatment options: biological basis of regenerative

endodontic procedures. Journal of Endodontics. 39, S30-43.

Huang G (2009) Pulp and dentin tissue engineering and regeneration: current progress. Regenerative

Medicine. 4, 697–707.

This article is protected by copyright. All rights reserved.

 Huang G, Sonoyama W, Liu Y, Liu H, Wang S,Shi S (2008) The hidden treasure in apical papilla: the

potential role in pulp/dentin regeneration and bioroot engineering. Journal of Endodontics. 34, 645-
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51.

Iwaya SI, Ikawa M,Kubota M (2001) Revascularization of an immature permanent tooth with apical

periodontitis and sinus tract. Dental Traumatology. 17, 185-7.

Jadhav G, Shah N,Logani A (2012) Revascularization with and without platelet-rich plasma in

nonvital, immature, anterior teeth: a pilot clinical study. Journal of Endodontics. 38, 1581-7.

Jeeruphan T, Jantarat J, Yanpiset K, Suwannapan L, Khewsawai P,Hargreaves KM (2012) Mahidol

study 1: comparison of radiographic and survival outcomes of immature teeth treated with either

regenerative endodontic or apexification methods: a retrospective study. Journal of Endodontics. 38,

1330-6.

Kahler B, Mistry S, Moule A, et al. (2014) Revascularization outcomes: a prospective analysis of 16

consecutive cases. Journal of Endodontics. 40, 333-8.

Kim J, Kim Y, Shin S, Park J,Jung I (2010 ) Tooth discoloration of immature permanent incisor

associated with triple antibiotic therapy: a case report. Journal of Endodontics. 36, 86-91.

Kontakiotis EG, Filippatos CG,Agrafioti A (2014) Levels of Evidence for the Outcome of

Regenerative Endodontic Therapy. Journal of Endodontics. 40, 1045–53.

Kontakiotis EG, Filippatos CG, Tzanetakis GN,Agrafioti A (2015) Regenerative endodontic therapy:

a data analysis of clinical protocols. Journal of Endodontics. 41, 146-54.

Lenherr P, Allgayer N, Weiger R, Filippi A, Attin T,Krastl G (2012) Tooth discoloration induced by

endodontic materials: a laboratory study. International Endodontic Journal. 45, 942-9.

Lovelace TW, Henry MA, Hargreaves KM,Diogenes A (2011) Evaluation of the delivery of

mesenchymal stem cells into the root canal space of necrotic immature teeth after clinical regenerative

endodontic procedure. Journal of Endodontics. 37, 133-8.

Martin DE, De Almeida JF, Henry MA, et al. (2014) Concentration-dependent effect of sodium

hypochlorite on stem cells of apical papilla survival and differentiation. Journal of Endodontics. 40,

51-5.

This article is protected by copyright. All rights reserved.

 Martin H (1975) Quantitative bactericidal effectiveness of an old and a new endodontic irrigant.

Journal of Endodontics. 1, 164-7.

Accepted Article
McTigue DJ, Subramanian K,Kumar A (2013) Case series: management of immature permanent teeth

with pulpal necrosis: a case series. Pediatric Dentistry. 35, 55-60.

Moreno-Hidalgo MC, Caleza-Jimenez C, Mendoza-Mendoza A,Iglesias-Linares A (2013)

Revascularization of immature permanent teeth with apical periodontitis. International Endodontic

Journal. 47, 321-31.

Murray PE, Garcia-Godoy F,Hargreaves KM (2007) Regenerative endodontics: a review of current

status and a call for action. Journal of Endodontics. 33, 377-90.

Nagata JY, Gomes BP, Rocha Lima TF, et al. (2014) Traumatized immature teeth treated with 2

protocols of pulp revascularization. Journal of Endodontics. 40, 606-12.

Nagy MM, Tawfik HE, Hashem AA,Abu-Seida AM (2014) Regenerative potential of immature

permanent teeth with necrotic pulps after different regenerative protocols. Journal of Endodontics. 40,

192-8.

Nakashima M,Akamine A (2005) The application of tissue engineering to regeneration of pulp and

dentin in endodontics. Journal of Endodontics. 31, 711-8.

Narang I, Mittal N,Mishra N (2015) A comparative evaluation of the blood clot, platelet-rich plasma,

and platelet-rich fibrin in regeneration of necrotic immature permanent teeth: A clinical study.

Contemporary Clinical Dentistry. 6, 63-8.

Nazzal H,Duggal MS (2017) Regenerative endodontics: a true paradigm shift or a bandwagon about

to be derailed? European Archives of Paediatric Dentistry. 18, 3-15.

Nicoloso GF, Potter IG, Rocha RO, Montagner F,Casagrande L (2016) A comparative evaluation of

endodontic treatments for immature necrotic permanent teeth based on clinical and radiographic

outcomes: a systematic review and meta-analysis. International Journal of Paediatric Dentistry. 27,

217-27.

Reynolds K, Johnson J,Cohenca N (2009) Pulp revascularization of necrotic bilateral bicuspids using

a modified novel technique to eliminate potential coronal discolouration: a case report. International

Endodontic Journal. 42, 84–92.

This article is protected by copyright. All rights reserved.

 Ruparel NB, Teixeira FB, Ferraz CCR,Diogenes A (2012) Direct Effect of Intracanal Medicaments on

Survival of Stem Cells of the Apical Papilla. Journal of Endodontics. 38, 1372–5.
Accepted Article
Saoud TM, Zaazou A, Nabil A, Moussa S, Lin LM,Gibbs JL (2014) Clinical and radiographic

outcomes of traumatized immature permanent necrotic teeth after revascularization/revitalization

therapy. Journal of Endodontics. 40, 1946-52.

Thibodeau B,Trope M (2007) Pulp revascularization of a necrotic infected immature permanent tooth:

case report and review of the literature. Pediatric Dentistry. 29, 47-50.

Trevino EG, Patwardhan AN, Henry MA, et al. (2011) Effect of irrigants on the survival of human

stem cells of the apical papilla in a platelet-rich plasma scaffold in human root tips. Journal of

Endodontics. 37, 1109-15.

Twati W, Percival R,Duggal M (2011) The antimicrobial effect of various root canal medicaments

used in management of immature non vital anterior teeth. International Journal of Paediatric

Dentistry. 21:S1 (Abstract O12-101), 35.

Twati W, Wood D, Liskiewicz T, Willmott N,Duggal M (2009) An evaluation of the effect of non-

setting calcium hydroxide on human dentine: a pilot study. European Archives of Paediatric

Dentistry. 10, 104-9.

Wigler R, Kaufman AY, Lin S, Steinbock N, Hazan-Molina H,Torneck CD (2013) Revascularization:

a treatment for permanent teeth with necrotic pulp and incomplete root development. Journal of

Endodontics. 39, 319-26.

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 Figure Legends

Figure 1 Radiographs showing root measurements (using Infinitt digital radiograph software, Seoul,
Accepted Article
Korea), a) Root length: the distance between the cemento-enamel junction and the radiographic apex

measured on mesial and distal sides of the root. The average of the mesial and distal root length

measurements was recorded as the root length. b) Apical foramen width: The distance between the

mesial and distal apical ends. c, d) Dentinal width: the outer root thickness and the inner pulp canal

width at two thirds root length were measured. The difference between the outer root thickness (c) and

the inner pulp canal width (d) was recorded as the root dentinal wall width.

Figure 2 Long cone radiographs of non vital immature UR2 with loss of vitality secondary to dense

invaginatus treated with RET using the same protocol used in this study. Radiographs showing

complete root formation and thickening of dentinal root walls over 32 months. (Published in (Nazzal

& Duggal 2017), approved for republication by European Archives of Paediatric Dentistry).

Figure 3 Long cone periapical radiographs of three patients treated in this study showing periapical

healing and different outcomes in terms of continuation of root development, thickening of root

dentinal walls and apical foramen widths. (a, b) show healing of PA area with no continuation of root

development, minimal thickening of dentinal walls and minimal closure of apical foramen in

immature UL2 with necrotic pulp following enamel/dentine crown fracture (Case # 6) (Published in

(Nazzal & Duggal 2017), approved for republication by European Archives of Paediatric Dentistry).

(c, d) show healing of PA area with minimal continuation of root development, minimal thickening of

dentinal walls, but an Apical barrier with apical foramen closure is evident in immature UR1 with

necrotic pulp following enamel/dentine crown fracture (Case # 13) (e, f) show healing of PA area with

continuation of root development, thickening of dentinal walls and closure of apical foramen in

immature UL1 with necrotic pulp following replantation (case #15) (Published in (Nazzal & Duggal

2017), approved for republication by European Archives of Paediatric Dentistry).

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ccepted Articl
Table 1 Showing participant demographics, type of trauma resulting in loss of vitality, pre-operative clinical and radiographic signs of infection, amount of pulpal bleeding during RET

procedure, pre-operative root development stage, and reason for exclusion.

Root development stage

Age* Infection Periapica
Sex Trauma type Bleeding** Inclusion
Years Pre-op l lesion Root Apex
length Morphology

1 M 8 E/D 11 No Yes (++) 3 Parallel Included

2 M 7 E/D C 11 Sinus+ pus No (+++) 3 Unclear Included

3 M 8 Avulsion 11 Sinus+ pus Unclear (+) 3 Parallel Included

4 M 10 E/D/R 11 Sinus+ pus Yes (+) 4 Convergent Included

5 M 9 E/D 22 No Yes Missing 3 Convergent Excluded: Second trauma

6 M 9 E/D 22 No Yes (++) 3 Divergent Included

7 M 9 E/D 11 Sinus tract Yes (+) 3 Divergent Excluded: Failed to return

8 M 7 E/D 21 No Yes (++) 3 Parallel Included

9 M 8 E/D 21 Sinus+ pus Yes (+) 3 Convergent Included

10 F 8 E/D 11 Sinus+ pus No Missing 3 Parallel Included

11 F 7 Intrusion No Yes (-) 3 Parallel Excluded: Apical stop

12 M 9 E/D11 No No (++) 4 Parallel Included

13 M 10 E/D C 11 No Yes (++) 4 Parallel Included

14 M 9 E/D 11 No No (++) 3 Parallel Included

15 F 7 Avulsion 21 (2h in milk) No No Missing 2 Parallel Included

*at start of treatment, **Bleeding (+: minimal-evident on paper point, ++: minimal bleeding-evident clinically, +++: perfuse bleeding), E=Enamel, D=Dentine, root length: 1= ¼ root length, 2=

½ root length, 3=2/3 root length, 4=complete root length with open apex, 5=complete root length with closed apex.

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ccepted Articl
Table 2 showing pre and post-operative clinical (signs of infection, response to cold and EPT tests) and radiographic (root lengths, root dentinal wall widths,

and apical foramen widths) outcomes of RET.

Root Apical
Infection Cold Root length
Infection Pre-op Cold 2 Y EPT BL EPT 2 Y Width foramen
2 years BL 22m
22m 22m

1 No No (-) (-) (+) (-) N N ↓

2 Sinus+Pus No (-) (-) (-) (-) ↑ ↑ ↓
3 Sinus+Pus No (-) (-) (-) (+) Res. Res. ↑
4 Sinus+Pus No (-) (-) (-) (-) N N ↑
5
6 No No (-) (-) (-) (+) N ↑ ↓
7
8 No No (-) (-) (-) (-) N N ↓
9 Sinus+Pus No (-) (-) (-) (-) ↑ N ↑
10 Sinus+Pus No (+) (-) (+) (-) ↑ ↑ ↓
11
12 No No (-) (-) (-) (+) ↑ ↑ ↓
13 No No (-) (-) (-) (+) ↑ ↑ ↓
14 No No (-) (-) (+) (-) ↑ N ↓
15 No No (+) (-) (-) (+) ↑ ↑ ↓
(-): Negative response, (+): Positive response, ↑: increased, ↓: decreased, N: No change

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ccepted Articl
Table 3 showing results of inter examiner reliability test of visual assessment and digital measurement methods using kappa and Intraclass correlation

respectively.

Root length Dentinal wall width Apical foramen width

Kappa score 0.333 -0.032 0.243

(Visual assessment method) p=0.116 p=0.849 p=0.04

ICC score 0.87 0.53 0.60

(Digital measurement method) 95% CI = 0.75-0.93 95% CI = 0.26-0.73 95% CI = 0.34-0.77

p <0.001 p <0.001 p <0.001

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ccepted Articl
Table 4 Results of radiographic measurements showing median values at baseline (immediately after RET) and follow up (average 22 months) showing no

statistical significant differences using Wilcoxon signed rank test (p> 0.05).

Median Wilcoxon signed rank test

Radiographic Measurement Stage
(mm) (p value)

Baseline 14.93
Root Length 0.79
Follow up 14.20

Baseline 2.51
Root Width 0.93
Follow up 2.67

Baseline 1.86
Apical foramen width 0.013
Follow up 1.20

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ccepted Articl
Table 5 Shows results of tooth crown colour change between baseline and 2 year photographs measured by CIELAB scores. Mean and standard deviation of

CIELAB scores (L*, a*, b* and ΔE) are reported over time indicating colour change based on CIELAB colour space.

Mean SD Colour

(n=9)

ΔL* 0.79 3.98 Lighter

Δa* 1.76 2.8 Redder

Δb* 5.19 3.34 Yellower

ΔE 7.39 3.25

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ccepted Articl
Table 6 shows the results of the subjective patient and dentist colour perception after 2 years. The results were reported for all patients (n=12) and for those

who had CIELAB scores (n=9).

Compared to CIELAB scores (n=9) All sample (n=12)

Patient colour perception Dentist opinion Patient perception Dentist opinion

Yellower 1 1 1 1

Whiter 1 0 1 0

Yellow grey 1 1 1 1

Darker 4 5 4 7

No change 2 2 3 2

Unable to comment 0 0 1 0

Missing Data 0 0 1 1

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ccepted Articl
Table 7 shows patients’ satisfaction of crown colour change post operatively after 22 months.

Patient colour perception Compared to CIELAB scores (n=9) All sample (n=12)

Happy with colour - 6 7

Patient deemed tooth yellow 1 1

Not affected Patient deemed tooth darker 1 1

Patient was unable to comment 0 1

Wants it lighter Patient deemed tooth darker 1 1

Missing - 0 1

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