Accepted Article

Received Date : 14-Sep-2013

Accepted Date : 02-Nov-2014
Article type

: Case Report

Regenerative endodontic treatment of an immature tooth with a necrotic pulp and apical
periodontitis using platlet-rich plasma (PRP) and mineral trioxide aggregate (MTA): a case report

G S Sachdeva, L T Sachdeva , M Goel, S Bala

Department of Conservative Dentistry and Endodontics, Himachal Dental College and Hospital,
Sundernagar, Himachal Pradesh, INDIA

RUNNING TITLE: Regenerative endodontics

KEYWORDS: Incisor; mineral trioxide aggregate (MTA); platelet-rich plasma (PRP); regeneration;
revascularisation

Address for Correspondence:

Dr. Gurmeet Singh Sachdeva,
Department of Conservative Dentistry and Endodontics, Himachal Dental College and Hospital,
Sundernagar, District Mandi, Himachal Pradesh 175002, INDIA
Ph: 01905 221355, 09418492355. Fax: 01907 266093
E mail: lizagurmeet2002@yahoo.co.in or lizagurmeet2002@gmail.com

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
an 'Accepted Article', doi: 10.1111/iej.12407
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Accepted Article

Abstract
Aim To report the successful clinical and radiographic outcome of a regenerative endodontic
treatment.
Summary A 16-year-old male patient presented with a discoloured, nonvital maxillary left
lateral incisor. Radiographic examination revealed an incompletely developed root with an
open apex. Under local anaesthesia and rubber dam isolation, an access cavity was prepared
and the necrotic pulpal remnants were removed. The canal was disinfected without
mechanical instrumentation with 5.25% NaOCl solution and dried with sterile paper points. A
triple antibiotic (metronidazole, ciprofloxacin, and minocycline) mixed with distilled water
was packed in the canal and left for 28 days. Ten millimetres of whole blood was drawn by
venipuncture from the patients antecubital vein for preparation of platelet-rich plasma (PRP) .
After removal of the antibiotic mixture, the PRP was injected into the canal space up to the
cementoenamel junction level. Three millimetres of white MTA was placed directly over the
PRP clot. Two days later, the tooth was restored with permanent filling materials. The patient
was recalled for 3, 6, 12, 24 and 36 months clinical/radiographic follow-up. A three year
follow-up radiograph revealed resolution of the periapical lesion, increased thickening of the
root walls, further root development and continued apical closure of the root apex. The tooth
was not responsive to cold tests; however sensitivity tests with an electric pulp tester (EPT)
elicited a delayed positive response.

Key learning points

Regeneration is a viable treatment modality that allows continued root development

of immature teeth with open apices and necrotic pulp.

Platelet-rich plasma appears to be potentially a suitable scaffold for regeneration of

vital tissues in teeth with a necrotic pulp and an associated periapical lesion.

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Accepted Article

periapical inflammatory lesion using platelet-rich plasma and hydroxyapatite. Journal of

Endodontics 34, 10204.
Hoshino E, Kurihara-Ando N, Sato I, et al (1996) In-vitro antibacterial susceptibility of
bacteria taken from infected root dentine to a mixture of ciprofloxacin, metronidazole and
minocycline. International Endodontic Journal 29, 12530.
Huang G (2009) Apexification: the beginning of its end. International Endodontic Journal
42, 85566.
Huang GT, 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, 64551.
Kawase T, Okuda K, Wolff LF, Yoshie H (2003) Platelet-Rich Plasma-Derived Fibrin Clot
Formation Stimulates Collagen Synthesis in Periodontal Ligament and Osteoblastic Cells In
Vitro. Journal of Periodontology 74, 858-64.
Kim J, KimY, 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,
108691.
Lazarski MP, Walker WA 3rd, Flores CM, Schindler WG, Hargreaves KM (2001)
Epidemiological evaluation of the outcomes of nonsurgical root canal treatment in a large
cohort of insured dental patients. Journal of Endodontics 27, 791 6.
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.
Nakashima M, Akamine A (2005) The application of tissue engineering to regeneration of
pulp and dentin in endodontics. Journal of Endodontics 31, 7118.

This article is protected by copyright. All rights reserved.

Accepted Article

biologically based regenerative approach unlike apexification and artificial apical barrier
techniques, allows continuation of root development (Huang 2009, Hargreaves & Law 2011)
resulting in an increase in both root wall thickness and the length of the root. Additionally,
the treatment time may be completed in fewer visits than traditional apexification procedures.

Regeneration can be achieved through activity of cells from the pulp, periodontium
vascular or immune systems, or stem cells. It is now thought that stem cells from the apical
papilla (SCAP) are responsible for the continuation of root development in immature teeth in
the absence of intracanal infection (Huang et al. 2008). Most of the current therapies involve
the use of the hosts own pulp or vascular cells. There are obvious advantages to a
regenerative approach as it is technically simple and can be completed using currently
available equipment and medicaments as compared to a tissue engineering that involves a
controlled delivery of stem/progenitor cells as well as a scaffold and growth factors
(Nakashima & Akamine 2005). Also, there is a decreased possibility of immune rejection and
pathogen transmission than if replacing the pulp with a tissue engineered construct. There are
numerous case reports and case series in the literature reporting to use these procedures to
treat permanent immature teeth with necrotic pulps. These reports show radiographic
evidence of increased root thickening and length of the root walls with apical closure (Banchs
& Trope 2004, Hargreaves et al. 2008, Ding et al. 2009)

The protocol of these treatments vary considerably but most of them follow the same
principle as suggested by Murray et al (2007) i.e. minimal instrumentation, irrigation with
NaOCl, intracanal medication, bleeding or introduction of blood products into the canal
space, MTA placement and closure.

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Accepted Article

A regenerative endodontic case is reported where a necrotic immature tooth

with apical periodontitis was successfully treated using platelet-rich plasma (PRP) and
mineral trioxide aggregate (MTA). PRP was used as a physical scaffold since it is known to
support cell growth and differentiation of the vital tissues in the canal after disinfection and it
increases the concentrated delivery of growth factors (Hiremath et al. 2008). This minimally
invasive technique of revascularization may be an effective treatment modality for managing
immature permanent teeth with a compromised structural integrity.

Case report
A 16-year-old male patient presented for treatment with a chief complaint of a discoloured
maxillary left lateral incisor (tooth 22). Medical history was noncontributory. The patient had
experienced intermittent spontaneous pain in that area for several months and gave a history
of trauma approximately eight years earlier, involving a direct impact on the maxillary
anterior teeth.

Clinical examination revealed Tooth 22 to be discoloured and sensitive to percussion

and palpation. A draining sinus tract was also observed on its buccal aspect. The patients
oral hygiene was poor but no periodontal pocketing was detected. Thermal and electrical pulp
testing using heated gutta-percha and electric pulp tester (Parkell, Edgewood, NY, USA)
respectively elicited a negative response.

Radiographic examination revealed that tooth 22 had an open apex associated with a
large radiolucency (Fig. 1a). The sinus tract was traced by using a gutta-percha cone (size 20)
into the lesion. On the basis of the clinical and radiographic findings, the diagnosis of an
immature tooth with a necrotic pulp with symptomatic apical periodontitis was made.

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Accepted Article

Considering various treatment options for tooth 22, it was felt that initially an attempt
to achieve regeneration of the pulp should be made with the aid of PRP. The patient was
informed that this treatment was an attempt to initiate further root development and that the
proposed treatment might not be successful. Written informed consent was obtained from the
patient.

Local anaesthesia was obtained by using 2% lidocaine (Septodont, St Maur Des

Fosses Cedex, France) with 1:80,000 epinephrine and the tooth was isolated with a rubber
dam. An access cavity was prepared using a diamond-coated fissure bur (Diatech, Heerbrugg,
Switzerland) and a high-speed handpiece with copious water spray. No haemorrhage was
observed from the root canal. The necrotic pulpal remnants were removed with the help of Hfiles (Mani, Inc, Tochigi, Japan) under copious irrigation with sterile saline. Coronal flaring
was carried out with numbers 2 & 3 Gates Glidden drills (Mani, Inc, Tochigi, Japan) (Fig. 1b)
and working length was determined with the help of an apex locator (Root ZX, Morita,
Tokyo, Japan), which was later confirmed with a radiograph (Fig. 1c). The root canal was
irrigated copiously with 5.25% sodium hypochlorite (Sigma-Aldrich Chemicals, Bangalore,
India) for 20 minutes followed by sterile saline without any instrumentation. The canal was
then dried with sterile paper points (Dentsply Maillefer, Baillaigues, Switzerland). Equal
proportions of ciprofloxacin, metronidazole and minocycline were ground and mixed with
distilled water to a thick paste and placed in the canal using a lentulo spiral to the working
length (Mani, Inc). The walls of the access cavity were cleaned with a sterile cotton pellet and
the access cavity was filled with Cavit (3M ESPE AG, Seefeld, Germany).

The patient returned after 28 days and was asymptomatic with tooth 22 being non
responsive to both percussion and palpation tests. Clinical examination revealed further

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Accepted Article

discoloration of the clinical crown. After administration of local anaesthesia and rubber dam
isolation, the temporary restoration was removed and the antibiotic mixture was washed out
using sterile saline (approximately 20ml). The canal was dried with sterile paper points
(Dentsply Maillefer, Baillaigues, Switzerland).

PRP was prepared using the procedure previously described (Okuda et al. 2003,
Kawase et al. 2003). Ten millilitres of whole blood was drawn by venipuncture from the
antecubital vein of the patients right arm. Blood was collected in a 15-ml sterile glass tube
coated with an anticoagulant (acid-citratedextrose). Whole blood was initially centrifuged
(2,400 rpm for 10 minutes) to separate PRP and platelet-poor plasma (PPP) portions from the
red blood cell fraction. PRP and PPP portions were again centrifuged (3,600 rpm for 15
minutes) to separate the PRP from the PPP. It was then injected into the canal space up to the
level of the cementoenamel junction (CEJ). White MTA (Pro-Root MTA; Dentsply Tulsa
Dental Specialties, Tulsa, OK, USA) was mixed according to the manufacturers instructions.
Three millimetres of MTA was placed directly over the PRP clot with the help of an MTA
Endo Carrier (Dentsply Maillefer) and an endodontic plugger. A moist cotton pellet was
placed over the MTA and the tooth was provisionally restored with Cavit.

The patient was seen two days later and after rubber dam application, the provisional
restoration and cotton pellet were removed and setting of the MTA was confirmed with a
blunt probe. The tooth was then filled with 2 mm of glass ionomer cement (GC Corporation,
Tokyo, Japan) and universal composite resin restorative material (3M ESPE, St Paul, MN,
USA) (Fig. 1d). The patient was recalled at 3, 6, 12, 24 and 36 month for
clinical/radiographic follow up.

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Accepted Article

Clinical evaluation after 12 months (Fig. 2a) revealed that the tooth 22 was
asymptomatic and was not sensitive to percussion or to palpation. No signs of oedema,
erythema or sinus tract were noted. The tooth was not responsive to cold testing with EndoFrost cold spray (Roeko; Coltene Whaledent, Langenau, Germany). Sensitivity test with an
electric pulp tester (EPT) (Parkell, Edgewood) now elicited a delayed positive response
compared to the contralateral tooth. Radiographic examination 36 months after initial
treatment revealed resolution of the periapical lesion, increased thickening of the root walls,
further root development and continued apical closure of the root apex (Fig. 2b). At this time
the tooth was again not responsive to cold testing with Endo-Frost cold spray (Roeko;
Coltene Whaledent). Sensitivity testing with an electric pulp tester still elicited a delayed
positive response. The tooth was functional with no signs of oedema, erythema or sinus tract
formation.

Discussion
Regeneration of pulp tissue in a tooth with a necrotic pulp and apical periodontitis has been
thought impossible (Ding et al. 2009). However, if the canals are effectively disinfected and a
scaffold conducive to tissue ingrowth is created, regeneration of odontogenic tissue might be
possible (Ding et al. 2009). The key factor for the success of this process is disinfection of
the root canal system. Owing to the extremely thin and weak root walls, disinfection relies
solely on irrigants and intracanal medicaments. In the case presented here, following copious
irrigation with 5.25% sodium hypochlorite, a triantibiotic paste was used (Hoshino et al.
1996). This triple antibiotic paste mixture consisting of metronidazole, ciprofloxacin, and
minocycline has been shown to be effective against the pathogens commonly found inside the
root canal system (Hoshino et al. 1996, Sato et al. 1996, Windley et al. 2005).

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Accepted Article

An ideal scaffold selectively binds and localizes cells, contains growth factors and
undergoes biodegradation over a period of time (Hargreaves et al. 2008). A blood clot as a
scaffold and as a source of stem cells has been used in several cases (Ding et al. 2009).
Revascularization of a necrotic root canal system by disinfection with 5.25% NaOCl and
Peridex (Chlorhexidine Gluconate 0.12%, Zila Pharmaceuticals, Phoenix, AZ, USA)
followed by establishing bleeding into the canal system via overinstrumentation has been
previously reported (Banchs & Trope 2004). A critical limitation to blod clot
revascularization approach is that the concentration and composition of cells trapped in the
fibrin clot is unpredictable. These variations in the cell concentraton and composition may
alter the treatment outcome (Murray et al. 2007).

PRP has been used in the field of dentistry for regenerative procedures (Hargreaves et
al. 2008, Ding et al. 2009). It contains growth factors, stimulates collagen production,
recruits other cells to the site of injury, produces anti-inflammatory agents, initiates vascular
ingrowth, induces cell differentiation, and improves soft and hard tissue wound healing
potential (Hiremath et al. 2008). In this case PRP was used as a 3-dimensional physical
scaffold to support cell growth and differentiation of vital tissues in the canal after
disinfection. It has also been suggested that PRP increases the concentrated delivery of
various growth factors (Hiremath et al. 2008).

Another physical scaffold suggested in regenerative endodontic procedures is platlet

rich fibrin (PRF). This high-density fibrin clot is associated with a slow and continuous
increase in cytokine levels (Tsay et al 2005). Leucocytes in PRF act as an immune response
regulator (Thibodeau & Trope 2007) and a source of vasculoendothelial growth factor
promoting angiogenesis.

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Accepted Article

MTA was placed over the PRP to isolate the root canal from the external surface of
the tooth and create a hard tissue barrier above the PRP. MTA has been shown to have
excellent sealing properties (Bates et al. 1996, Fischer et al. 1998) and may also provide
signaling molecules for the growth of stem cells (Torabinejad & Parirokh 2010). The access
was double sealed coronally with glass ionomer and a universal composite resin restorative
material to prevent salivary reinfection into the pulp space.

Revascularization treatment has several drawbacks. Drawing blood in young patients,

the skill of the operator, patient compliance, need of special equipment and medications to
prepare PRP, and the additional cost of treatment are some of the disadvantages of using
PRP. Coronal discoloration as a result of presence of minocycline in the triple antibiotic paste
(Kim et al 2010) and cervically placed MTA has been previously reported (Reynolds et al
2009, Parirokh & Torabinejad 2010, Petrino et al 2010, Felman & Parashos 2013). Also there
is a possibility of development of resistant bacterial strains (Slots 2002). Shin et al (2009)
suggested use of 6% NaOCl, followed by 2% chlorhexidine gluconate instead of triple
antibiotic paste to avoid both discoloration and development of resistant bacterial strains.

In addition, another technique for disinfection in immature teeth with necrotic pulp
using 2.5% NaOCl irrigation and apical negative pressure without using triple antibiotic paste
has been reported (da Silva et al 2010). The authors concluded that with this technique the
use of triple antibiotic paste might not be necessary. The replacement of minocycline with
cefaclor in triple antibiotic paste to prevent discoloration has also been suggested (Thibodeau
& Trope 2007). Another technique to prevent coronal discoloration is to seal the dentinal
walls of the access cavities with flowable composite resin before placement of the triple
antibiotic dressing (Reynolds et al. 2009).

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Accepted Article

The nature of the tissue formed in the canal space and its cellular composition is yet to
be identified (Hargreaves et al. 2008). Although radiographic evidence of hard tissue
deposition has been noticed, it has been theorized that this hard tissue could be due to the
ingrowth of dentine, cementum or bone (Chueh & Huang 2006, Thibodeau et al. 2007). Two
recent animal studies demonstrated that the vital tissue formed in the canal space was a
connective tissue similar to periodontal ligament (da Silva et al. 2010, Wang et al. 2010) and
the dentinal walls were thickened by the opposition of newly formed cementum-like tissue
(Wang et al. 2010). Recent study of a human immature permanent tooth reported that the
tissue formed in the canal of revascularized/revitalized human teeth was fibrous connective
tissue similar to that found in the periodontal ligament and cementum-like or bone-like tissue
(Becerra et al. 2014). Although definitive human histologic studies are required to verify the
exact nature of the hard tissue, the clinical outcome of these endodontic regeneration studies
appears to be promising.

Several studies have reported unfavourable outcomes of regenerative endodontic

treatments including absence of root development (Petrino et al. 2010, Nosrat et al. 2011),
root wall thickness (Petrino et al. 2010, Chen et al. 2012), or lack of formation of an apex
(Chen et al. 2012). Formation of a hard-tissue barrier inside the canal between the coronal
MTA plug and the root apex has also been reported (Chen et al. 2012).

In the presented case, thickening of the root wall and healing of the related lesion
occurred within 12 months, which suggests that PRP may have a potential role in
regenerative endodontics. The three year radiographic follow-up showed resolution of the
periapical lesion, increased thickening of the root walls, further root development and
continued apical closure. On the basis of the results of this case, PRP appears to be a

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Accepted Article

promising physical scaffold delivering signalling molecules for the management of immature
teeth with open apices and necrotic pulp tissue.

Conclusion
Regenerative endodontic procedures have the potential for regenerating both pulp and
the dental hard tissues and therefore may offer a viable treatment option for immature teeth
with a necrotic pulp. PRP is potentially a suitable scaffold for this procedure.

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Accepted Article

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Accepted Article

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Endodontics 34, 10204.
Hoshino E, Kurihara-Ando N, Sato I, et al (1996) In-vitro antibacterial susceptibility of
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This article is protected by copyright. All rights reserved.

Accepted Article

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Accepted Article

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Accepted Article

Figure legends
Figure 1 a Preoperative radiograph showing tooth 22 with an open apex and associated
periapical radiolucency. b Access opening view after coronal flaring. c Working length
determination. d Postoperative final radiograph after placement of PRP and MTA (arrow).
Tooth was then double-sealed with glass ionomer cement and universal composite resin
restorative material.

Figure 2 a 12 months recall radiograph. b 36 months recall radiograph showing resolution of

the periapical lesion, increased thickening of the root walls, further root development and
continued apical closure of the root apex.

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Accepted Article
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Accepted Article
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