Archives of Oral Biology 71 (2016) 122–128

Histologic characterization of regenerated tissues after pulprevascularization of immature dog teeth with apical periodontitis usingtri-antibiotic paste and platelet-rich plasma

Carlos Stambolsky, DDS MSc PhDa, Soledad Rodríguez-Benítez, DDS MSc PhDa,José Luis Gutiérrez-Pérez, MD DDS PhDb, Daniel Torres-Lagares, DDS PhDa,Jenifer Martín-González, DDS PhDa, Juan José Segura-Egea, MD DDS PhDa,*aDepartment of Stomatology, School of Dentistry, University of Sevilla, C/Avicena s/n, 41009 Sevilla, SpainbDepartment of Stomatology, School of Dentistry, Virgen Macarena University Hospital, Andalusian Health Service, University of Sevilla, C/Avicena s/n, 41009Sevilla, Spain

A R T I C L E I N F O

Article history:Received 15 November 2015Received in revised form 12 May 2016Accepted 25 July 2016

Keywords:Apical negative pressure irrigationImmature toothMineral trioxide aggregatePlatelet-rich plasmaPulp regenerationPulp revascularizationScaffoldTri-antibiotic paste

A B S T R A C T

Introduction: This study evaluates histologically the efficacy of 4 revascularization protocols in necrotic-infected immature dog teeth with apical periodontitis (AP).Methods: Forty double-rooted immature premolar teeth from 4 female Beagle dogs aged 5 months wereused. Four teeth were left untouched as negative controls; the other 36 teeth were infected to developpulp necrosis and AP. Four teeth were left untreated and assigned to the positive control group. The last28 teeth were randomly assigned into four experimental groups of 8 teeth, each one treated with adifferent treatment protocol: A1, sodium hypochlorite (SH) + blood clot (BC); A2, SH + platelet-richplasma (PRP); B1, SH + modified tri-antibiotic paste (mTAP) + BC; B2, SH + mTAP + PRP. The animals weresacrificed, histologic sections were prepared and three parameters were assessed: (1) presence orabsence of new hard tissue on the internal root dentinal walls, (2) presence or absence of continued apicalclosure, and (3) presence or absence of vital tissue within the canal space.Results: Significant differences (p < 0.05) between the four experimental groups were evident in thepercentage of teeth showing histological apical closure (34.5%) and vital tissue within the canal space(68.8%). Group B2 showed the maximal improvement in the three variables assessed (p < 0.05). Group A1showed the minimum percentages in the three parameters assessed (p < 0.05).Conclusions: These results suggest that an intracanal dressing of mTAP, and the use of PRP as scaffold,improves the success rate of the revascularization procedure.

ã 2016 Elsevier Ltd. All rights reserved.

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1. Introduction

Apexification with a long-term calcium hydroxide applicationhas been the most accepted treatment option for immaturepermanent tooth with pulp necrosis (Rafter, 2005). However, thistreatment has several disadvantages: requires multiple visitsduring a long period, hindering patient’s follow-up (Kleier andBarr, 1991). Moreover, long-term calcium hydroxide therapy mayleave thin dentinal walls even more prone to fracture (Andreasen,Farik, & Munksgaard, 2002).

* Corresponding author at: Facultad de Odontología, Universidad de Sevilla, C/Avicena s/n, 41009 Sevilla, Spain.

E-mail address: segurajj@us.es (J.J. Segura-Egea).

http://dx.doi.org/10.1016/j.archoralbio.2016.07.0070003-9969/ã 2016 Elsevier Ltd. All rights reserved.

An alternative to conventional calcium hydroxide apexificationwas suggested by Torabinejad and Chivian (1999), who proposed toseal the open apex with mineral trioxide aggregate (MTA) in onevisit. Nevertheless, this technique could promote apical repair butdoes not reduce the risk of future fracture because the root widthwill not increase (Jeeruphan et al., 2012).

Revascularization procedure (RP) is a conservative and effectivemethod for inducing maturogenesis in necrotic immature teeth,increasing thickening of the canal walls by deposition of hardtissue and encouraging continued root development (Jadhav, Shah,& Logani, 2012). Revascularization has been proposed to treatimmature permanent teeth with necrotic pulp tissue and/or apicalperiodontitis/abscess (Iwaya, Ikawa, & Kubota, 2001). In RP ofimmature permanent teeth the disinfection protocol is a mainaspect because the tooth is not mechanically cleaned to its fulllength (Iwaya et al., 2001). A copiously irrigation of root canal and

C. Stambolsky et al. / Archives of Oral Biology 71 (2016) 122–128 123

dressing with antimicrobial agents are needed. Amongst thedisinfection protocols proposed in RP are the following: conven-tional irrigation with sodium hypochlorite in combination of tri-antibiotic paste (TAP) (a mixture of ciprofloxacin, metronidazole,and minocycline) (Thibodeau et al., 2007; Windley et al., 2005) theintra-canal dressing with calcium hydroxide (Chueh and Huang,2006), and most recently the irrigation with apical negativepressure (ANP) has shown similar bacterial reduction to conven-tional irrigation with sodium hypochlorite plus intra-canaldressing with TAP (Hockett, Dommisch, Johnson, & Cohenca,2008; Cohenca et al., 2010). A problem that often accompanies theintracoronal use of TAP containing minocycline is dentin discolor-ation (Rodríguez-Benitez, Stambolsky, Gutiérrez-Pérez, Torres-Lagares, & Segura-Egea, 2015), but it has been confirmed that theincorporation of cefaclor in the TAP, instead of minocycline, avoidsdiscoloration (Miller et al., 2012).

Initial studies on revascularization, after disinfection of the rootcanal, induced a hemorrhage to form a blood clot into the canal toact as a scaffold to aid the in-growth of new tissue into the emptycanal space (Thibodeau et al., 2007). It is hypothesized that theblood clot serves as a matrix for migration of progenitor cells intothe canal, possibly from the apical papilla (Thibodeau et al., 2007).Collagen solutions (Thibodeau et al., 2007; Yamauchi et al., 2011)and platelet–rich plasma (PRP) (Torabinejad & Faras, 2012;Torabinejad & Turman, 2011) have been mentioned as a potentiallyideal scaffold for RP.

If a thorough disinfection of the canal space is accomplishedbefore filling it with a scaffold, then successful revascularization ofimmature permanent human teeth with apical periodontitis hasbeen demonstrated, just as it is for the uninfected canals in case oftooth avulsion (Banchs and Trope, 2004; Cvek et al., 1990; Petrinoet al., 2010).

The aim of this study was to assess histologically the ability offour different protocols, combining two type of scaffolds (BloodClot and PRP) and two disinfection procedures (Sodium Hypochlo-rite with ANP using the Endovac1 system and Tri-Antibiotic Paste)to obtain revascularization of necrotic-infected immature dogteeth with apical periodontitis.

2. Materials and methods

2.1. Study groups

This study was made with the approval of the EthicalCommittee of the University. Forty double-rooted premolar teethfrom 4 female Beagle dogs aged 5 months were randomly dividedinto 4 experimental groups of 8 teeth each (16 roots), a positivecontrol group (4 teeth, 8 roots) and a negative control group (4teeth, 8 roots).

Before any interventions, the involved teeth were radiographi-cally throughout (Kodak RVG 61001 Digital Radiography System,Carestream Health, Inc. Rochester, NY, USA) using radiographparalleling devices (Dentsply Rinn, Elgin, IL, USA) to confirmincomplete root formation and open apices. These radiographicaids were used for all subsequent radiographs to improve thealignment and position of the films and x-ray beam for directcomparison of the radiographs with minimal distortion ormagnification.

All interventions were made under general anesthesia (induc-tion by Zolazepam hydrochloride [Zoletil 100, Virbac España, S.A.,Spain] 0.1 mL/kg intravenously and intubation and maintenancewith isoflurane [Isoflo, Abbott Laboratories Ltd., Berkshire, UK.])supplemented with local anesthesia (Lidocaine 5%, B. BraunMedical, S.A, Barcelona, Spain).

In the first treatment session, the teeth of the negative controlgroup were left untouched for natural development for

comparison with the experimental and positive control teeth.The pulps of 32 experimental and 4 positive control teeth wereinfected according to the protocol described previously byLeonardo et al. (1993). The pulps were mechanically exposedusing a #12 diamond bur in a high-speed hand-piece with copioussaline solution. Then, each pulp was disrupted with a #20 sterilestainless steel endodontic hand file (Colorinox1, DentsplyMaillefer. Ballaigues. Switzerland). This procedure was repeatedindividually on each dog and the root canals were left exposed tothe oral cavity for 7 days to allow microbial contamination. Theanimals were given analgesics (Torbugesic 0.2 mg/kg; ButorphanolTartrate, Fort Dodge Animal Health, Fort Dodge, IA) postoperativelyfollowing this and all operative procedures and were monitored inthe postoperative period. After 1 week, the coronal access wassealed with Cavit1 (ESPE, Norristown, PA), without intracanaldressing. The teeth were monitored radiographically by usingparalleling devices until there was radiographic evidence of apicalperiodontitis (AP), which occurred within 15–25 days. Once theinjuries were radiographically visible, 32 teeth were randomlyassigned into 4 groups of 8 teeth, each following differenttreatment protocols, and 4 teeth were assigned to the positivecontrol group, in which no further treatment was carried out.

Under general and local anesthesia, all previously infected teethwere isolated with a rubber dam, and the operative field wasdisinfected with 30% hydrogen peroxide until no bubblingoccurred. All surfaces were then coated with tincture of iodineand allowed to dry. The temporary restoration was removed with asterilized round bur #12 in a high-speed handpiece. Using #40 K-file (Colorinox1, Dentsply Maillefer, Ballaigues. Switzerland), theworking length (WL) was established radiographically 1 mm shortof the radiographic apex. Then, four teeth were left with no furthertreatment as positive controls, and each experimental group wastreated according to 4 different protocols, as follows:

Group A1: disinfection with sodium hypochlorite (SH) andblood clot (BC) as scaffold; Group A2: disinfection with SH and PRPas scaffold; Group B1: disinfection with SH and a modified tri-antibiotic paste (mTAP) dressing during 15 days, and BC blood clotas scaffold; Group B2: disinfection with SH and mTAP dressing, andPRP as scaffold.

2.1.1. Group A1: sodium hypochlorite/blood clotThe canals were disinfected with 20 mL of 1.25% NaOCl (sodium

hypochlorite; Sigma-Aldrich Química SA, Madrid, Spain) using theapical negative pressure irrigation ANP-Endovac1 system (DiscusDental, Culver City, CA, USA). Taking into account that immatureteeth with open apices were being treated, the ANP-Endovac1

system was modified to avoid the extrusion of the sodiumhypochlorite solution to the apical tissues, as described by Cohencaet al. (2010). Canals were irrigated using the macro-cannula onlyafter being gauged to fit the apical size of the root.

The canals were left filled with sodium hypochlorite solution1.25% for 3 min and then irrigated with 10 mL sterile saline solutionto remove the rest of sodium hypochlorite. Then, a final irrigationwas accomplished using 1 mL of 17% EDTA (Ultradent Products Inc.South Jordan, Utah, USA) for 60 s (Yamauchi et al., 2011).

The root canals were dried with sterile paper points and asterile #30 K-file (Colorinox1, Dentsply-Maillefer, Ballaigues.Switzerland) was used to stimulate bleeding for clot formation.The bleeding was stopped at the level of the cementum-enameljunction by using a small cotton pellet soaked with sterile saline.After 10 min, the blood clot was formed. Over the clot, a collagensponge (Collacote1, Integra Lifesciences Corporation, Plainsboro,NJ, USA) was set. Next, a 4 mm plug of MTA (Proroot1, Denstply,Tulsa Dental, Johnson City, TN, USA) was inserted into the canalsusing an MTA carrier (Hartzell & Son, CA, USA) to seal the root canalat the cervical level. The MTA plug was verified radiographically.

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Then, a moist cotton pellet was placed over the MTA and the accesscavity was double sealed using IRM1 (Dentsply, Caulk, Milford, DE)and glass ionomer cement (Vitrebond1, 3 M ESPE, Seafeld,Germany).

2.1.2. Group A2: sodium hypochlorite/PRPThe disinfection protocol and the final irrigation with EDTA was

the same of group A1. Then, the root canals were dried with sterilepaper points and PRP was used as scaffold instead of blood clot. ThePRP was obtained following the protocol described previously byAnitua (1999). Briefly, 5 mL sample of whole venous blood wasdrawn from the dogs and deposited in 3.8% w/v sodium citrate1:9 v/v to prevent blood coagulation. A one-stage centrifuge wasused at a low speed (460 g) for 15 min at room temperature (PRGFSystem II, BTI, Vitoria, Spain). After the blood sample had beencentrifuged and the plasma had been separated, the 0.5 mL plasmafraction 3, located immediately over a layer of red blood cells, wascollected. This fraction 3 is designated as PRP because it contains aconsiderable amount of platelet. For activation, aggregation anddegranulation of platelets, releasing GFs, 50 microliters of 10% w/vcalcium chloride was added to each tube containing 1 mL of PRP.Then, the PRP was condensed into the canal until the cemento-enamel junction using a hand plugger (Dentsply Maillefer, USA).Over the PRP, the MTA placement and the coronal seal was set asdescribed in group A1.

2.1.3. Group B1: sodium hypochlorite/tri-antibiotic paste/blood clotIn this group, the teeth were disinfected in two sessions. In a

first treatment session, the root canals were irrigated with sodiumhypochlorite using the ANP irrigation system Endovac1 asdescribed in group A1. Then, the canals were irrigated with10 mL sterile saline to remove the sodium hypochlorite, and weredried with sterile paper points.

After that, a modified triple antibiotic paste dressing wasprepared immediately mixing ciprofloxacin, metronidazole andcefixime (mTAP) in sterile distilled water into creamy mixture atconcentration of 20 mg of each antibiotic (Hoshino et al.,1996). Themodified tri-antibiotic paste was delivered into the root canal withsterile lentulo spiral filler (Caulk, Milford, DE) following thetechnique described previously by Windley et al. (2005). All excessof the mTAP in the pulp chamber was removed and a sterile cottonpellet was placed. The access cavity was then doubling sealed withCavit 1 (3 M ESPE, St Paul, MN) and glass ionomer cement (3 MESPE, Seafeld, Germany). The intracanal dressing was left in thecanals during 15 days.

At the second treatment session, the coronal seal was removedin aseptic conditions with sterile high speed diamond round burunder copious water cooling followed by flushing of the pulpchamber with 20 mL saline solution to remove the tri-antibioticpaste intracanal dressing. Then, a final irrigation was accomplishedusing 1 mL of 17% EDTA (Ultradent Products Inc. South Jordan,Utah, USA) for 60 s.

The root canals were dried with sterile paper points. In thisgroup, a blood clot was used as scaffold following the sameprotocol used in group A1, and the coronal seal was accomplishedusing MTA, IRM1 and glass ionomer cement, as describedpreviously.

2.1.4. Group B2: sodium hypochlorite/tri-antibiotic paste/PRPThe disinfection protocol was accomplished in two sessions as

described in group B1. Then, after removal the tri-antibiotic paste,the canals were dried with sterile paper points, irrigated with 1 mLof EDTA for 60 s., and once again dried with sterile paper points. Inthis group, PRP was used as scaffold instead of blood clot followingthe same protocol used in group A2 and the coronal seal was

accomplished using MTA, IRM1 and glass ionomer cement asdescribed, previously

2.2. Histological evaluation

Teeth were monitored radiographically on a monthly basis for 6months before the animals were sacrificed. The preparation oftissue and sections for the histological evaluation was carried outfollowing the method described previously by Thibodeau et al.(2007). The jaws with the involved teeth were resected and placedin formaldehyde (Fisher Scientific, Fair Lawn, NJ). After removal ofall soft tissue and excess hard tissue from the specimens, they werenext placed in Formical (Decal Chemical Corporation, Congers, NY)for decalcification for 6 days, including one change of the solution.The specimens were subsequently decalcified in Immunocal (DecalChemical Corporation, Tallman, NY) for 2 months, undergoing fourchanges of the solution over that time. On removal from thedecalcification solution, the specimens were placed under arunning tap water wash for 20 min followed by immersion in70% ethyl alcohol. The specimens were then dehydrated throughascending gradations of ethanol and processed on a Leica TP 1020dip n’ dunk processor (Leica, Wetzlar, Germany) at 45 min perstation in the following manner: one cycle of 70% ethanol, twocycles of 80% ethanol, two cycles of 95% ethanol, two cycles of 100%ethanol, two cycles of xylene, and two cycles of Paraplast paraffin(Fisher Scientific, Fair Lawn, NJ) at 58 �C. The specimens were thenprocessed, embedded in paraffin, longitudinally sectioned alongthe long axis of the teeth every 5 mm through the apical foramen ofthe roots, and stained by the Masson’s trichrome protocol andharvested for histological examination.

Each individual root was taken as a unit of measurement/assessment for the histological evaluations. After a training sessionexplaining the gold standard of the different evaluation param-eters, two evaluators, blinded to the experimental groups,examined the histological slides of the roots and graded themfor the following three parameters: (1) presence or absence of newhard tissue on the internal root dentinal walls, (2) presence orabsence of continued apical closure, and (3) presence or absence ofvital tissue within the canal space. The evaluators examined thesame slide independently at the same time under identical lightingand magnification conditions by using a multi-headed microscope.When the results of evaluation of a parameter were notunanimous, a consensus was reached through discussion, asdescribed previously by Thibodeau et al. (2007).

2.3. Radiographic evaluation

Teeth were monitored radiographically on a monthly basis for 6months before the animals were sacrificed. Each individual rootwas taken as the unit of measure. Radiographic evaluation wascarried out according to the method described previously byThibodeau et al. (2007). Briefly, after a training session explainingthe gold standard of the three evaluation parameters, twoexaminers evaluated the radiographs independently, regardinghealing of radiolucencies, thickening of the dentinal walls andapical closure of roots. Digital radiographs were saved to acomputer in jpg format and the examiners, blinded to theexperimental groups, separately viewed the image exposedpreoperatively with an image exposed postmortem. Each examinergraded each root for the following three parameters: (1)diminished size or absence of periapical radiolucency, (2) presenceor absence of continued thickening of radicular walls, and (3)presence or absence of apical closure. When there was notagreement between both evaluators, a discussion was undertakenuntil a consensus was reached. The kappa statistic valuescomparing the two evaluators were as follows: 0.80 for evaluation

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of periapical radiolucencies, 0.72 for evaluation of thickness ofradicular walls, and 0.68 for evaluation of apical closure, allindicating good agreement between the two evaluators.

2.4. Statistical analysis

The data were analyzed using Fisher’s exact test, with the levelof significance set at p < 0.05.

3. Results

No animal showed any established signs of undue distress fromthe treatment procedures. Because of a gross mobility of the toothoccurred after the initial infection procedure, one animal (positive

Fig. 1. A – Tooth from negative control group (untouched) showing normal developmagnification 10�). B – Tooth from A2 group (SH + PRP) showing slightly thickened root wvital tissue (VT) within the root canal. Bb – Cementum matrix (CM) and bone islands (BI) (development, and healing of apical periodontitis (10�). Ca – Vital tissue (VT) and bone itissue (BT) are evident. D – Tooth from B2 group (SH + mTAP + PRP). Complete developmPercentage of roots in experimental groups with and without vital tissue within the canaand without hard-tissue deposition on radicular dentin walls assessed histologically. III –

histologically. (p values were calculated using Fisher’s exact test).

control group) lost two experimental teeth during the course of thestudy, and another animal (positive control group) also lost oneexperimental tooth. These roots were excluded in the final analysis.

None of the healed cases have shown partial survival of pulptissue. Histologic evidence of hard-tissue deposition on internalroot canal walls, apical closure, and new vital tissue within thecanal spaces were seen in roots from all experimental groups. Inthe control group, teeth were allowed to develop normally and theroots reached their maturity as shown in Fig. 1A. The root dentinacquired its functional thickness, which resulted in narrowing ofthe pulp space. In contrast, teeth in the experimental groupsshowed different grades of arrested dentin development (Fig. 1, B–D). There was a gain of thickness of the apical third of some rootsbecause of the ingrowth of hard-tissue (bone-like or cementum-

ment of the roots, with thickened root walls and apical development (originalalls, apical development, and healing of apical periodontitis (10�). Ba – New formed10�). C – Tooth from group B1 (SH + mTAP + BC) showing thickened root walls, apicalslands (BI) within the root canal. Cb – Tooth apex. Connective tissue (CT) and boneent of the roots, with thickened root walls and vital tissue are evident (10�). I –

l space assessed histologically. II – Percentage of roots in experimental groups withPercentage of roots in experimental groups with and without apical closure assessed

126 C. Stambolsky et al. / Archives of Oral Biology 71 (2016) 122–128

like tissues) from the root surface into the canal walls (Fig. 1, B andC). Bone islands (BI), bone tissue (BT), cementum matrix (CM) andcementum tissue (CT), as well as vital tissue (VT) were evidentwithin the root canal (Fig. 1, Ba, Bb, Ca and Cb). Bone-like tissuewith trabecular formation was scattered in the canal space in manycases of the experimental groups (Fig. 1, Ca, Cb). Almost normaldevelopment of the roots, with thickened root walls and vital, wereevident in some teeth of the B2 group (Fig. 1, D).

3.1. Vital tissue within canal spaces

Vital tissue within the canal space was found in 68.8% of allteeth, with significant differences between the four groups(p = 0.020) (Fig. 1. I). Group B2 showed the maximal percentageof roots with vital tissue within the canals (87.5%) and groups A2and B1 the minimum (25%).

When the individual experimental groups were compared witheach other using Fisher’s exact tests in 2 � 2 tables with 1 � offreedom for the presence of vital tissue, there were significantdifferences between groups A1 and A2 (p = 0.031), groups A1 andB1 (p = 0.031) and groups A1 and B2 (p = 0.004). Comparing allteeth of groups B1 and B2 (disinfected in two session with tri-antibiotic paste) with all teeth of groups A1 and A2 (disinfected inone session only with sodium hypochlorite), there were significantdifferences, with higher percentage of presence of vital tissue inthe teeth of groups B1 and B2 (81.3%) compared to the teeth ofgroups A1 and A2 (56.3%) (p = 0.022). Comparing all the teeth fromgroups A1 and B1 (25%) with all teeth from groups A2 and B2 (50%)there also were significant difference (p = 0.022). There were noother significant differences between the experimental groupswith respect to presence of vital tissue within canal spaces.

3.2. Histological hard-tissue deposition on radicular dentin walls

The percentage of teeth of all experimental groups showingdeposition of hard-tissue on radicular dentin walls was 53.1%, withsignificant differences between the four groups (p = 0.011) (Fig. 1.II). Group B2 showed the maximal percentage of hard-tissue-deposition (87.5%) and groups A1 and A2 the minimum (37.5%).

When the individual experimental groups were compared witheach other for hard-tissue deposition on the dentinal walls, therewere significant differences between groups A1 and B2 (p = 0.004),groups A2 and B2 (p = 0.004) and groups B1 and B2 (p = 0.024).Comparing all teeth of groups B1 and B2 (disinfected in two sessionwith mTAP) with all teeth of groups A1 and A2 (disinfected in onesession only with SH), there were significant differences, withhigher percentage of hard-tissue deposition in the teeth of groupsB (68.8%) compared to the teeth of groups A (37.5%) (p = 0.009).Comparing all teeth from groups 1 (BC as scaffold) with all teethfrom groups 2 (PRP as scaffold) there were no significantdifferences (p = 0.066). There were no other significant differencesbetween the experimental groups with respect to hard-tissue-deposition.

3.3. Histological apical closure

Only 34.4% of teeth of all experimental groups showedhistological apical closure, with significant differences betweenthe four groups (p = 0.023) (Fig. 1. III). Group B2 showed themaximal percentage of histological apical closure (62.5%) andgroup A1 the minimum (12.5%).

When the individual experimental groups were compared witheach other using Fisher’s exact test in 2 � 2 tables with 1 � offreedom for histological apical closure, there were significantdifferences between groups A1 and B2 (p = 0.004), and groups A2and B2 (p = 0.031). Comparing all teeth of groups B1 and B2

(disinfected in two session with tri-antibiotic paste) with all teethof groups A1 and A2 (disinfected in one session only with sodiumhypochlorite), there were significant differences, with higherpercentage of histological apical closure in the teeth of groups B1and B2 (50.0%) compared to the teeth of groups A1 and A2 (18.8%)(p = 0.007). Comparing all the teeth from groups A1 and B1 (bloodclot) with all teeth from groups A2 and B2 (PRP as scaffold) therewere no significant difference (p = 0.062). There were no othersignificant differences between the experimental groups withrespect to histological apical closure.

4. Discussion

The results of the present study add new experimental evidenceshowing that revascularization of necrotic-infected immature dogroot canals with AP can be attained.

In this study, as in other previous studies, the dog was selectedas the animal model because of its similarities in radicularstructure to immature human teeth in their open apex character-istics (Thibodeau et al., 2007; Windley et al., 2005; Wang,Thibodeau, Trope, Lin, & Huang, 2010; Gomes-Filho et al., 2012;Khademi et al., 2014; Zhang et al., 2014). In addition, the dog has ahigh growth rate, so results are obtained in shorter experimentalperiods (Citome, Kaminski, & Heuer, 1979).

The histologic analysis has shown that 53% of the roots in thefour experimental groups (previously infected canals) had newhard tissue deposited on the internal root walls. This percentage issimilar to the results of other studies (Anitua, 1999; Rodríguez-Benitez et al., 2015; Wang et al., 2010), but slightly higher than thatfound by Thibodeau et al. (2007) and Khademi et al. (2014), whofound 44% and 40%, respectively, of infected roots with new hardtissue on inner dentin walls. However, this percentage is lowerthan that found by Gomes-Filho et al. (2012), who found 72% ofhard tissue deposition. The teeth which were disinfected in twosessions with mTAP (groups B1 and B2) showed a significantlyhigher percentage of hard-tissue deposition (69%) compared toteeth disinfected in only one session with SH (groups A1 and A2)(37.5%) (p = 0.009). Moreover, the group B2 (SH + TAP + PRP)showed the maximal percentage of radicular wall thickening(87.5%), significantly higher than the others groups.

Recent studies advocate the use of 17% EDTA in order todecalcify the surface of the root canal dentin to expose the collagenfibers and release growth factors from the dentine (Galler et al.,2011; Yamauchi et al., 2011). Collagen is necessary because itimproves the adhesion of new cells (Murray, García- Godoy, &Hargreaves, 2007). Trevino showed that irrigation with 17% EDTAand 6% NaOCl was compatible with stem cell survival (Trevinoet al., 2011). Taking into account that, in the present study, EDTAhave been used in the protocol, it can be conclude that theexposure of dentin matrix together with mTAP disinfection andPRP as scaffold might provide an efficient approach to generate avital support structure for the treatment of infected immatureteeth.

The present results suggest that the use of PRP as scaffold couldincrease hard tissue formation in revascularization procedures, asRodríguez-Benitez et al. (2015) have proposed after find signifi-cantly more mineralized tissue formation when a scaffold wasused. Moreover, Zhang et al. (2014) has proposed that PRPapplication could be an option for clinical cases in which littleor no bleeding were found when irritating the apical tissue duringrevascularization procedures. As previously suggested Thibodeauet al. (2007), the new hard tissue deposited on the internal rootcanal walls, could represent the histologic measure that corre-sponds to the radiographic measure of thickening of root walls.This might indicates that the radiographic measure of thickenedcanal walls (normally the only measurable indication of successful

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revascularization available to the clinician) is representative of theactual histological outcome.

Based on the histologic examination in the present study,mainly two types of tissue were generated in the canal space:cementum-like tissue along the dentinal walls causing thethickening of the root, and bone-like tissue. In all groups, rootcanal wall thickening and apical closure were accomplished by thedeposition of bone-like, cementum-like and/or connective-liketissues after the experimental protocols. Similar findings havebeen reported by Wang et al. (2010) who found in the canal spacecementum or cementum-like tissues, similar to cellular cemen-tum, and bone or bone-like tissue. These findings are in agreementwith the results recently reported by Zhu, Wang, Liu, Huang, &Zhang (2014) who, in the dog mature root canals after regenerativeendodontic procedures, found no pulp tissues but mainly bone,cementum and periodontal tissues.

Histological apical closure has been found in 34.4% of the rootsin the experimental groups. Other studies have found higherpercentages of apical closure (55–70%) (9,26,27). There werestatistical differences between the four experimental groups, andGroup B2 (SH + TAP + PRP) showed the higher percentage (63%) ofroots with evidence of histological apical closure. This result are inagreement with the finding of Thibodeau et al. (2007), who found59% of roots with histological evidence of apical closure when ablood clot was used, and with the findings of Zhang et al. (2014),who reported a higher percentage of histological apical closurewhen PRP was used (75%) in comparison with BC (65%).

In a previous study with the same experimental sample(Rodríguez-Benitez et al., 2015), 43.8% of radiographic apicalclosure and 34.4% of histological apical closure were found. Thedisagreement between radiological and histological results may beexplained by the higher accuracy of the histological studycompared to radiographic study in assessing apical closure. Similarresults have been reported by Wang et al. (2010) who found 78% ofradiographic apical closure, but only 69% of histological apicalclosure.

After revascularization protocol, 68.8% of the previouslynecrotic infected roots of all experimental groups had new healthytissue within the canal spaces. Zhang et al. (2014) have foundhigher percentages of pulplike tissue formation in the canal space(89.6%). Group B2 (SH + TAP + PRP) again showed the maximalpercentage of roots with vital tissue within the canals (87.5%). Inaccordance with Thibodeau et al. (2007), this is a very significantoutcome. It highlights that the protocols rendered in previouslyinfected canals (Windley et al., 2005), allowed the in-growth ofnew vital tissue into the canal spaces. The regenerated soft vitaltissue in the canal space after revascularization protocols wasusually described as pulp-like tissue mainly because of the absenceof an odontoblast layer. In the present study, none case in the PRPand blood clot groups exhibited a normal odontoblast layer, and notypical pulp tissue or new dentin was found regenerated in all theexperimental samples. Other animal studies examining regenera-tion procedures have shown that the types of tissues found withinthe root are not pulp but consist of bone, cementum, andconnective tissue (Yamauchi et al., 2011; Zhu et al., 2014). Asclaimed by Khademi et al. (2014), some aspects of the long-termeffects of this regenerative procedure still remain unknown,including the possibility of continued deposition of hard tissue onthe dentinal walls until there is complete closure of the root canalspace. Further studies with longer follow-up intervals are neededto fully determine the long-term effects of this method.

Subsequent to disinfection, an appropriate scaffold is needed topromote cell growth and differentiation (Hargreaves, Giesler,Henry, & Wang, 2008; Aggarwal, Miglani, & Singla, 2012). Ostby(1961) showed that new vascularized tissue could be induced inthe apical third of the root canal through formation of a blood clot

as scaffold to support growth of new tissue into the unfilled portionof the root canal. The blood clot as a matrix is considered areservoir of growth factors that may play an important role in theregeneration process (Hargreaves et al., 2008; Marx, 2004). Thisrich supply of growth factors appears to stimulate the differentia-tion, growth and maturation of fibroblast, odontoblasts, andcementoblast from their undifferentiated precursors (Nagy,Tawfik, Hashem, & Abu-Seida, 2014). The use of collagen as ascaffold for the ingrowth of tissue into root canals has beenattempted (Nevins and Crespi, 1998). However, it has been foundthat the collagen acts as a passive scaffold and does not provide anyadvantage in the stimulation of the revascularization process as itdoes not contain the factors that promote cell growth anddifferentiation (Thibodeau et al., 2007).

Between group 1 (A1 + B1, BC as scaffold) and group 2 (A2 + B2,PRP as scaffold) there were no significant differences, except in thepercentage of roots with vital tissue within canal spaces (25% ingroup 1 and 50% in group 2, p = 0.022). Similar results have beenreported by Torabinejad et al. (2014) comparing the use of bloodclots and PRP as scaffolds in regenerative endodontics in a canineferret model, finding that the use of either PRP or blood clots leadsto the formation of intracanal bone-like tissue without continualroot maturation. Moreover, the results of the present study are ingood accordance with the recently published findings of Torabi-nejad, Milan, Shabahang, Wright, & Faras (2015) who have reportedthat both PRP or a BC, used as a scaffold, significantly increaseapical narrowing and hard tissue deposition in comparison to notusing a scaffold. The authors, although without statisticallysignificant differences, found a more prominent ingrowth of hardtissue in quality and quantity associated with the use of PRP.

Revascularization has not been found superior to otherapexification techniques in either clinical or radiographic out-comes (Alobaid et al., 2014) and the outcome of revascularizationprocedures remains somewhat unpredictable (Wigler et al., 2013).However, recent studies carried out in humans demonstrated thatpulp revascularization can attain root maturogenesis (Kahler et al.,2014) and is associated with significantly greater increases in rootlength and thickness in comparison with calcium hydroxideapexification and MTA apexification as well as excellent overallsurvival rates.

5. Conclusions

The histological findings reported in this study further supportthe concept that revascularization of necrotic-infected immatureteeth with AP can be attained. Tri-antibiotic paste and PRP seem tobe useful as disinfectant and scaffold, respectively, in pulprevascularization procedures. Further studies characterizing thedeveloped tissue are also necessary in order to better understandthe mechanisms of pulp revascularization.

Conflict of interest

None.

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