Regenerative potential following revascularization of immature permanent teeth with necrotic pulps

Regenerative potential following revascularizationof immature permanent teeth with necrotic pulps

H. Tawfik1, A. M. Abu-Seida2, A. A. Hashem1 & M. M. Nagy1

1Endodontic Department, Faculty of Dentistry, Ainshams University, Cairo; and 2Depatment of Surgery, Anesthesiology &

Radiology, Faculty of Veterinary Medicine, Cairo University Giza, Egypt

Abstract

Tawfik H, Abu-Seida AM, Hashem AA, Nagy MM.

Regenerative potential following revascularization of immature

permanent teeth with necrotic pulps. International Endodontic

Journal, 46, 910–922, 2013.

Aim To assess the regenerative potential of immature

teeth with necrotic pulps following revascularization

procedure in dogs.

Methodology Necrotic pulps and periapical pathosis

were created by infecting 108 immature teeth, with

216 root canals in nine mongrel dogs. Teeth were

divided into three equal groups according to the evalu-

ation period. Each group was further subdivided into

six subgroups according to the treatment protocol

including MTA apical plug, revascularization protocol,

revascularization enhanced with injectable scaffold,

MTA over empty canal. All root canals were disinfected

with a triple antibiotic paste prior to revascularization

with the exception of control subgroups. After disinfec-

tion, the root length, thickness and apical diameter

were measured from radiographs. Histological evalua-

tion was used to assess the inflammatory reaction, soft

and hard tissue formation.

Results In the absence of revascularization, the

length and thickness of the root canals did not

change over time. The injectable scaffold and growth

factor was no more effective than a revascularization

procedure to promote tooth development following

root canal revascularization. The tissues formed in

the root canals resembled periodontal tissues.

Conclusion The revascularization procedure

allowed the continued development of roots in teeth

with necrotic pulps.

Keywords: immature teeth, necrotic pulp, regener-

ation, revascularization.

Received 21 July 2012; accepted 2 February 2013

Introduction

Treatment of immature permanent teeth with pulp

necrosis and apical pathosis constitutes a challenge

for endodontists. Teeth with a necrotic pulp are com-

monly encountered in cases of trauma to the anterior

teeth or untreated carious lesions. Such conditions

are challenging, not only in root canal debridement

and filling, but also for the thin dentinal walls

increasing the risk of subsequent fracture (Deutsch

et al. 1985, Cvek 1992).

Management of such cases was achieved histori-

cally by apexification procedures using calcium

hydroxide. Such treatment requires long-term place-

ment of calcium hydroxide inside the root canal to

induce formation of an apical hard tissue barrier

(Kerekes et al. 1980, Sheehy & Roberts 1997, Abbott

1998).

The placement of apical plugs has been advocated

but do not solve the problem of thin and weak den-

tinal root canal walls (Simon et al. 2007, Holden

et al. 2008, Witherspoon et al. 2008).

Periapical tissues around immature teeth are rich

in blood supply and contain stem cells that have rela-

tive potential to regenerate in response to tissue

injury. Bacterial eradication from the canal space is

essential for successful revascularization procedures.

Reports using antibiotics revealed that a combination

Correspondence: Prof. Dr. Ashraf Abu-Seida, Professor of

Surgery, Anesthesiology & Radiology, Faculty of Veterinary

Medicine, Cairo University, Giza, Egypt (Tel.: +2 01001997359;

e-mail: [email protected]).

© 2013 International Endodontic Journal. Published by John Wiley & Sons LtdInternational Endodontic Journal, 46, 910–922, 2013

doi:10.1111/iej.12079

910

of metronidazole, minocycline and ciprofloxacin can

be effective against common dental pathogens in vitro

and in vivo (Sato et al. 1996, Hoshino et al. 1997,

Windley et al. 2005). Advances in tissue engineering

research focused upon three key elements for tissue

regeneration (Tabata 2004, Murray et al. 2007).

First, the adult stem cells, which have the ability for

proliferation and differentiation. Second, the scaffolds;

which are three dimensional structures that support

cell organization and vascularization. Third, the

growth factors which are the extracellular secreted

signals governing morphogenesis.

Several case reports have been published concern-

ing revascularization procedures (Huang 2009, Petri-

no et al. 2010, Iwaya et al. 2011, Nosrat et al.

2011). However, few experimental studies have been

carried out evaluating the histological characteriza-

tion of the regenerated tissues (Camp & Fuks 2006,

Thibodeau et al. 2007, Wang et al. 2010). Further-

more, lack of randomized clinical trials prevents wide-

spread application of this treatment protocol.

The aim of the present investigation was to assess

radiographically and histopathologically the regenera-

tive potential of young permanent immature teeth

with necrotic pulps following several treatment proto-

cols namely (a) MTA apical plug, (b) revascularization

by blood clot, (c) revascularization by blood clot and

injectable scaffold coated with basic fibroblast growth

factor (bFGF), (d) MTA closure of an empty root

canal, (e) pulp exposed teeth (positive control) and (f)

untouched teeth (negative control).

Materials and methods

A total of nine mongrel dogs aged 6–9 months were

selected. Three premolars in each quadrant were

included to give a total of 108 teeth. Teeth

were divided into three equal groups according to the

post-treatment evaluation period including Group I

(1 week), Group II (3 weeks) and Group III

(3 months).

Each group included 36 teeth which were subdi-

vided into four experimental subgroups and two con-

trol groups according to the treatment protocol. The

subgroups included MTA apical plug (subgroup a),

revascularization by blood clot (subgroup b), revascu-

larization by blood clot and injectable scaffold coated

with bFGF (subgroup c), MTA closure of an empty

root canal (subgroup d), pulp exposed teeth (positive

control) (subgroup e) and untouched teeth (negative

control) (subgroup f). All subgroups were represented

in each dog and in a randomized manner. Each indi-

vidual root was taken as the unit of measure for sta-

tistical purposes.

The research proposal was approved by the review

board, Faculty of Dentistry Ainshams University. The

procedures were carried out at the Faculty of Veteri-

nary Medicine, Cairo according to regulations of Fac-

ulty of Veterinary Medicine, Cairo University. General

anaesthesia was administrated after pre-medication

using 0.05 mg kg�1 atropine sulphate injected subcu-

taneously and 1 mg kg�1 XylazineHCl (Xylaject;

ADWIA Co., Cairo, Egypt) injected intramuscularly.

Anaesthesia was induced by using Ketamine HCl (Kei-

ran; EIMC pharmaceuticals Co., Cairo, Egypt) injected

intravenously using a cannula in the cephalic vein at

a dose of 5 mg kg�1 body weight. The anaesthesia

was maintained by using Thiopental sodium at a dose

of 25 mg kg�1 body weight 2.5% injected intrave-

nously (dose to effect).

After general anaesthesia, all teeth were examined

radiographically to confirm incomplete root formation

and to establish base line working lengths for further

comparison. Endodontic access cavities were prepared

in all experimental and positive control teeth. Expos-

ing the pulp chamber was achieved using size no. 2

diamond burs in a conventional speed handpiece. A

size 40 sterile file was used to disrupt the pulp tissue

in the canals. A piece of cotton was inserted into the

entrance of each canal, and the access was left open

for 2 weeks. Carprofen tablets were given orally for

15 days at a dose of 4.4 mg kg�1 per once daily as

post-operative analgesic.

After the infection period, the triple antibiotic paste

was prepared using metronidazole 500 mg tablets

(Flagyl 500 mg, Aventis, Cairo, Egypt), ciprofloxacin

250 mg tablets (Ciprocin 250 mg, EPICO, Cairo,

Egypt) and doxycycline 100 mg capsules (Vibramy-

cin, Pfizer, Cairo, Egypt). Under general anaesthesia,

the previously infected experimental teeth were

re-entered under aseptic conditions and cotton roll

isolation. The soaked cotton was removed, and canals

were irrigated using 10 mL of 2.6% sodium hypochlo-

rite and then dried using paper points. Injection of

1–2 mL of the prepared antibiotic paste into each

canal was carried out until the canal was filled. The

access cavity was then sealed using a temporary res-

toration (Coltosol F, Coltene Whaledent, Altst€atten,

Switzerland) for 3 weeks.

The teeth were then re-entered under the same

anaesthesia and aseptic conditions. The antibiotic

paste was removed by copious irrigation using 10 mL

Tawfik et al. Regenerative potential following revascularization

© 2013 International Endodontic Journal. Published by John Wiley & Sons Ltd International Endodontic Journal, 46, 910–922, 2013 911

of 2.6% sodium hypochlorite. Canals were dried and

treated according to different treatment modalities as

follows:

Subgroup (a): MTA apical plug. MTA (MTA

Angelus, Londrina, Brazil) was mixed according to

the manufacturer instructions and inserted into

the canal using a suitable sized amalgam carrier.

The material was packed apically using a plugger

to fill the apical half of the canal with a

4–5 mm plug. Teeth were radiographed to check

the MTA apical plug. A sterile wet cotton pellet

was then placed in the access cavity. Glass iono-

mer was used to fill the access cavity.

Sub group (b): Revascularization by blood

clot. A size 60 hand file was inserted past the

canal terminus until bleeding was induced to fill

the canal space reaching the level of the cement–

enamel junction. An MTA plug was used to seal

the canal orifice. The access cavity was then filled

using glass ionomer.

Subgroup (c): Revascularization by blood clot

and injectable scaffold coated with bFGF. A

mixture of 150 lg of bFGF (Kaken Pharmaceutical

Co Tokyo, Japan) and 300 lL phosphate-buffered

saline was used to form a suspension

10 mg mL�1. The suspension was dropped onto

2 mg dried gelatin hydrogel (Nitta Gelatin Co,

Osaka, Japan). The mixture was left for one hour

at 37 °C. Induction of bleeding was carried out as

described in subgroup (b) then the prepared hydro-

gel was inserted into the canals using a suitable

sized plugger. Root canal orifices were covered

with MTA. Access cavities were then sealed using

glass ionomer.

Subgroup (d): MTA closure of an empty root

canal. An MTA plug (2–3 mm) was used to seal

the empty canal orifice and covered by a small wet

cotton pellet before the access cavity was filled

using glass ionomer.

Subgroup (e): Pulp exposed teeth (positive con-

trol). Cotton pellets were inserted into the access

cavity, and teeth were left open without a tempo-

rary filling

Subgroup (f): Untouched teeth (negative control).

Normal teeth were left for future comparison with-

out any intervention

Radiographic evaluation

Periapical radiographs were taken after induction of

the periapical lesion and compared with follow-up

radiographs taken according to each group, at

1 week, 3 weeks and 3 months.

Periapical radiographs were digitized using a trans-

parency scanner (HP Scanjet G3110, Hewlett-Packard

Development Company, Palo Alto, CA, USA). Digital

image files were converted to 32-bit TIFF files using

Image-J analysis software (Image-J v1.44, US National

Institutes of Health, Bethesda, MD, USA). TurboReg

plug-in (Biomedical Imaging Group, Swiss Federal

Institute of Technology, Lausanne, Switzerland) was

used to transform nonstandardized pre-operative and

post-operative radiographs into standardized images

(Bose et al. 2009, Nosrat et al. 2011). The following

criteria were assessed.

Increase in length of root

A scale was set in the Image-J software by measur-

ing a known clinical dimension to its radiographic

dimension. The scale was calculated as number of

measured pixels per mm length. Root lengths were

measured as a straight line from the cement–enamel

junction to the radiographic apex of the tooth in

millimetre.

Increase in thickness of root

Using the preset measurement scale, the level of the

apical third was determined and fixed from the

cement–enamel junction. The root thickness and

the pulp space width were measured at this level in

millimetre. Dentine thickness was measured by

subtraction of the pulp space from the entire root

thickness.

Decrease in apical diameter of the canal

Using the preset measurement scale, the diameter of

the apical foramen was measured in millimetres.

The measurements were taken pre- and post-opera-

tively. The difference in length, thickness and apical

diameter of roots was calculated. Percentage of apical

closure and increase in root length and dentine thick-

ness were calculated.

Histopathological evaluation

The animals were sacrificed according to the post-

evaluation period using an anaesthetic overdose.

Teeth and surrounding periapical tissues were fixed

using 10% buffered formalin solution and decalcified

using 17% EDTA solution for 120 days. Decalcified

bone blocks were sectioned in bucco-lingual sections

of 6 lm thickness. Sections were stained using

Regenerative potential following revascularization Tawfik et al.


haematoxylin and eosin dye. The following histo-

pathological findings were evaluated:

1. Inflammatory cell count: (in the periapical tis-

sues)

For each slide, three representative fields were anal-

ysed at 9200 magnification. Fields were selected

conforming to the following criteria: (i) well-preserved

tissue with good architecture and no artefacts, (ii)

intense inflammatory cells infiltration. Total inflam-

matory cell number was counted using image analy-

sis software ‘Image-J software’. The colour-coding

threshold was adjusted to select the perimeter of the

whole range of inflammatory cells to exclude other

nondesired structures. Then, binary thresholds of the

selected colour coded inflammatory cells were com-

pleted prior to calculation. The total number of cells

was then counted as a factor of 103.

2. Presence or absence of vital tissues inside the

pulp space

Score 0: No tissue in-growth into the canal space

Score 1: Evidence of tissue in-growth into the apical

third of the canal

Score 2: Evidence of tissue in-growth extending to

the middle third of the canal

Score 3: Evidence of tissue in-growth extending to

the cervical third of the canal

3. Presence or absence of new hard tissue: (in the

canal space)

• Qualitative analysis:

Criteria for histological identification of hard struc-

ture (Huang 2009):

Dentine: presence of dentinal tubules

Cementum: adherence to dentine, absence of den-

tinal tubules and presence of cementocyte-like cells

Bone: presence of Haversian canals with uniformly

distributed osteocyte-like cells

Periodontal ligament: presence of Sharpey’s fibre

bridging cementum and bone

Inflamed tissues: presence of oedema and inflamma-

tory cells; lymphocytes

• Quantitative analysis:

Score 0: absence of new hard tissue formation

Score 1: partial formation of new hard tissues

Score 2: Complete formation of new hard tissues

Statistical analysis

Data were analysed using SPSS (Statistical Packages

for the Social Sciences 19.0, IBM, Armonk, NY, USA).

Numeric data were analysed by the Kruskal–

Wallis nonparametric analysis of variance, with Dunn

multiple comparison test to identify differences

between treatment groups. A P value <0.05 was con-

sidered significant. Nonnumerical data were analysed

with chi-square tests, with the level of significance set

at P � 0.05.

Results

Data are collected, tabulated and statistically analysed

(Tables 1–3). Radiographs of representative samples

of each group and subgroup are shown in (Fig. 1–6).

Table 1 Mean and percentage of root length increase (mm)

Group I Group II Group III

Subgroup (a) 0%Aa 0 � 0

(0%)Aa0 � 0 (0%)Aa

Subgroup (b) 0%Aa 1.1 � 0.14

(10.45%)Aa1.88 � 0.18 (17.8%)Bb

Subgroup (c) 0%Aa 1.06 � 0.11

(10%)Aa1.42 � 0.23 (14.1%)Bb

Subgroup (d) 0%Aa 0.5 � 0

(5%)Aa0.6 � 0 (6%)Aa

Subgroup (e) 0%Aa 0 � 0

(0%)Aa0 � 0 (0%)Aa

Subgroup (f) 0%Aa 1.26 � 0.16

(12.7%)Ab1.94 � 0.24 (19.4%)Bc

Significant at P � 0.05.

Different capital letters indicate significant difference between

groups within the same subgroup. Different small letters indi-

cate significant difference between subgroups within the same

group.

Table 2 Mean and percentage of root thickness increase

(mm)


Subgroup (a) 0%Aa 0 � 0

(0%)Aa0 � 0 (0%)Aa

Subgroup (b) 0%Aa 0.25 � 0.07

(8%)Aa0.36 � 0.11 (11.1%)Bb

Subgroup (c) 0%Aa 0.3 � 0.1

(9.2%)Aa0.35 � 0.11 (14.1%)Bb

Subgroup (d) 0%Aa 0.3 � 0

(10%)Aa0.3 � 0 (10%)Aa

Subgroup (e) 0%Aa 0 � 0

(0%)Aa0 � 0 (0%)Aa

Subgroup (f) 0%Aa 0.39 � 0.12

(12.2%)Ab0.55 � 0.15 (17.6%)Bc





group.



Increase in root length

Data were calculated and tabulated in Table 1. Statis-

tical analysis revealed no significant difference

between all subgroups in group I (after 1 week).

Regarding group II (after 3 weeks), significant differ-

ences (P = 0.002) between subgroups (f) untouched

teeth (negative control) versus all subgroups were

found.

Group III (after 3 months) showed no significant

difference between subgroup (b) revascularization by

blood clot and subgroup (c) revascularization by blood

clot and injectable scaffold coated with bFGF. Also no

significant difference was noticed between subgroup

(a) MTA apical plug, (d) MTA closure of an empty

root canal and (e) pulp exposed teeth (positive

control). Significant differences were found between

subgroups (b) and (c) versus subgroups (a), (d) and

(e). Subgroup (f) untouched teeth (negative control)

was significantly higher than all subgroups.

No significant difference was found between groups

I, II and III (all periods) in subgroups (a) MTA apical

plug, (d) MTA closure of an empty root canal and

(e) pulp exposed teeth (positive) control. Significant

difference was found between group II (after 3 weeks)

and III (after 3 months) in subgroups (b) revasculari-

zation by blood clot, (c) revascularization by blood

clot and injectable scaffold coated with bFGF and (f)

untouched teeth (negative control).

Increase in root thickness

Data were calculated and tabulated in Table 2. Statis-

tical analysis showed that there was no significant dif-

ference between subgroups in group I (after 1 week).

There was no significant difference between subgroups

(b) revascularization by blood clot, (c) revasculariza-

tion by blood clot and injectable scaffold coated with

bFGF, (d) MTA closure of an empty root canal and (f)

untouched teeth (negative control) in group II (after

3 weeks). However, significant difference was noticed

between the former subgroups and subgroup (e) pulp

exposed teeth (positive control).

As regards group III (after 3 months), no significant

difference was found between subgroups (b) revascular-

ization by blood clot, (c) revascularization by blood clot

and injectable scaffold coated with bFGF and (d) MTA

closure of an empty root canal. Significant difference

was noticed between the former subgroups and

subgroup (f) untouched teeth (negative control) and

significant difference was recorded towards subgroup

Table 3 Mean and percentage of decrease in apical foramen

diameter (mm)


Subgroup

(a)

0 � 0 (0%)Aa 0 � 0

(0%)Aa0 � 0 (0%)Aa

Subgroup

(b)

0 � 0 (0%)Aa 0.39 � 0.11

(6.5%)Aa0.85 � 0.6 (29.9%)Bb

Subgroup

(c)

0 � 0 (0%)Aa 0.34 � 0.09

(5.7%)Aa0.98 � 0.6 (32%)Bb

Subgroup

(d)

0 � 0 (0%)Aa 0.27 � 0.04

(4.5%)Aa0.78 � 0.33 (10%)Aa

Subgroup

(e)

0 � 0 (0%)Aa 0 � 0

(0%)Aa0 � 0 (0%)Aa

Subgroup

(f)

0 � 0 (0%)Aa 0.79 � 0.12

(13.2%)Ab1.39 � 0.6 (46.5%)Bc





group.

(a) (b) (c) (d)

Figure 1 Representative radiographs of subgroup (a) (MTA apical plug); pre-operative (a) and 1 week (b), 3 weeks (c) and

3 months (d) following MTA application.



(e) pulp exposed teeth (positive control). Significant dif-

ference was found between subgroup (f) untouched

teeth (negative control) and (e) pulp exposed teeth

(positive control). No significant difference was found

between groups I, II and III (all periods) in subgroups

(a), (d), and (e). Significant difference was found

between group II (after 3 weeks) and III (after

3 months) in subgroups (b), (c) and (f) (P = 0.019).

(a) (b) (c) (d)

Figure 2 Representative radiographs of subgroup (b) (revascularization by blood clot); 0 days (a), 7 days

(b), 21 days (c) and 90 days (d).

(a) (b) (c) (d)

Figure 3 Representative radiographs of subgroup (c) (revascularization by blood clot and injectable scaffold coated with basic

fibroblast growth factor 0 days (a), 7 days (b) 21 days (c) and 90 days (d).

(a) (b) (c) (d)

Figure 4 Representative radiographs of subgroup (d) (MTA closure of an empty root canal); 0 days, (a), 7 days (b), 21 days

(c) and 90 days (d).



Decrease in apical diameter

Radiographs were evaluated for decrease in apical

diameter and percentage of apical closure. Data were

calculated and tabulated in Table 3

There was no significant difference among all sub-

groups in group I (after 1 week).

Regarding group II (after 3 weeks), there was no

significant difference between subgroups (b) revascu-

larization by blood clot, (c) revascularization by blood

clot and injectable scaffold coated with bFGF, (d) MTA

closure of an empty root canal and (f) untouched

teeth (negative control). However, a significant differ-

ence was noticed between the former subgroups and

subgroup (e) pulp exposed teeth (positive control)

(P = 0.02).

In group III (after 3 months), no significant differ-

ence was found between subgroups (b), (c) and (d).

Significant difference (P = 0.006) was noticed

between the former subgroups and subgroup (f), and

high significant difference (P = 0.0019) was recorded

towards subgroup (e) pulp exposed teeth (positive

control). Significant difference (P = 0.02) was also

found between subgroup (f) untouched teeth

(negative control) and (e) pulp exposed teeth (positive

control).

No significant difference was found between all sub-

groups in group I (after 1 week) and group II (after

3 weeks). In group III (after 3 months), a significant

difference (P = 0.016) was found between subgroups

[(a) MTA apical plug and (e) pulp exposed teeth (posi-

tive control)] versus subgroups [(b) revascularization

by blood clot and (c) revascularization by blood clot

and injectable scaffold coated with bFGF] and sub-

group (f) untouched teeth (negative control). No

significant difference was found between subgroups

(a) and (e). Also no significant difference was found

between subgroups (b) and (c). No significant differ-

ence was found between groups I, II and III (all peri-

ods) in subgroups (a), (d), and (e). Significant

difference (P = 0.003) was found between group II

(a) (b) (c) (d)

Figure 5 Representative radiographs of subgroup (e) pulp exposed teeth (positive control); 0 days (a), 7 days (b), 21 days (c)

and 90 days (d).

(a) (b) (c) (d)

Figure 6 Representative radiographs of subgroup (f) (untouched teeth (negative control)); 0 days (a), 7 days (b), 21 days (c)

and 90 days (d).



(after 3 weeks) and III (after 3 months) in subgroups

(b), (c) and (f).

Inflammatory cell count: (in periapical tissues)

Statistical analysis showed that there was no signifi-

cant difference between experimental subgroups after

1 week (group I) (Fig. 7).

For group II (after 3 weeks) (after 3 weeks), no sig-

nificant difference was found between experimental

groups except for subgroup (d) MTA closure of an

empty root canal and (e) pulp exposed teeth (positive

control) which were significantly higher (P = 0.004)

than the rest of experimental groups.

Regarding group III (after 3 months), no significant

difference was found between subgroups (a) MTA api-

cal plug, (b) revascularization by blood clot and (c)

revascularization by blood clot and injectable scaffold

coated with bFGF. Subgroups (d) MTA closure of an

empty root canal and (e) pulp exposed teeth (positive)

control were significantly higher (P = 0.005) than

other subgroups. Negative control samples recorded

significantly (P = 0.002) lower cell count than all

other subgroups (Table 4).

Nature and extent of tissue in-growth in the

pulp space

The histopathological analysis revealed connective

tissue in-growth in some samples (Fig. 8). The nat-

ure of this tissue resembles periodontal connective

tissue with variable amounts of inflammatory cell

infiltration and noticeable angiogenic activity in

some samples. The extent of in-growth was tabulated

(Table 5).

Statistical analysis showed that there was no signif-

icant difference between all subgroups in group I

(after 1 week).

Regarding group II (after 3 weeks), statistical anal-

ysis showed that there was no difference detected

between subgroups (b) revascularization by blood

clot, (c) revascularization by blood clot and injectable

scaffold coated with bFGF and (e) untouched teeth

(negative control). Also no significant difference was

found between subgroups (a) MTA apical plug and

(d) MTA closure of an empty root canal MTA clo-

sure of an empty root canal. Significant difference

was found between subgroups (a) and (d) versus

subgroups (b), (c) and (e) (P = 0.004).

(a) (b)

Figure 7 (a) Photomicrograph of subgroup I (b) showing dense inflammatory cells infiltration, numerous dilated blood vessels

and areas of oedema in the periapical tissues (H&E 9 400). (b) Photomicrograph of subgroup II (c) showing noticeable increase

in blood vessels in the periapical tissues 3 weeks after application of growth factor (H&E 9 100).

Table 4 Mean inflammatory cell count for all experimental

groups


Subgroup (a) 35.4 � 7.3Aa 27.6 � 3.5Aa 10.2 � 5.2Ba

Subgroup (b) 37.7 � 5.7Aa 28.1 � 6.6Aab 12.1 � 4.7Ba

Subgroup (c) 36.2 � 3.6Aa 28.7 � 7.8Aab 14.3 � 3.9Ba

Subgroup (d) 35.8 � 4.4Aa 36.7 � 9.9Aab 30.7 � 7.5Ab

Subgroup (e) 39.5 � 9.2Aa 42.5 � 7.2Ab 43.9 � 10.2Ac

Subgroup (f) 2.1 � 0.5Ab 2.3 � 1.3Ac 2.1 � .8Ad





group.



Regarding group III, statistical analysis showed that

there was no difference detected between subgroups

(b), (c) and (e). Also no significant difference was found

between subgroups (a) and (d). Significant difference

was found between subgroups (a) and (d) versus

subgroups (b), (c) and (e) (P = 0.002).

Formation of mineralized hard tissue: (in the canal

space)

Data were tabulated in Table 6. Several samples had

hard tissue formation in the canal space of different

thicknesses and shapes resembling cementoid tissue

(Fig. 9) in which there are incremental calcifications

with occasional cellular inclusions. In case of sub-

group (a) MTA apical plug, hard tissue formed at

the outermost aspect of MTA. Statistical analysis

showed that there was no significant difference

between subgroups (b), (c), (d) and (e) in group II

(3-week period). Subgroup (a) was significantly

higher than all subgroups (P = 0.02).

Regarding group III, statistical analysis showed that

no significant difference was found between sub-

groups (a), (b) and (c). Subgroups (d) and (e) were

significantly lower (P = 0.0031) regarding hard tissue

deposition scores.

Discussion

Management of immature teeth with necrotic pulps

has been considered a challenge. Interrupted root

development leads to weak and thin dentinal walls

liable to fracture, beside the difficulty to achieve an

adequate apical seal using conventional root canal

therapy (Andreasen et al. 2002).

Historically, treatment of such cases was achieved

using calcium hydroxide apexification (Citrome et al.

1979). However, long-term use of calcium hydroxide

(a) (b)

Figure 8 (a) Photomicrograph of subgroup III (c) showing tissue in-growth up to apical third of the canal (arrow) (H&E 9 40). (b)

Photomicrograph of subgroup III (e) showing tissue in-growth with apical root resorption (arrows) (H&E 9 100).

Table 5 Mean tissue-in-growth scores among all subgroups


Subgroup (a) 0.0 � 0Aa 0.0 � 0Aa 0.0 � 0Aa

Subgroup (b) 0.0 � 0Aa 1.0 � 0.6Bb 1.1 � 0.66Bb

Subgroup (c) 0.0 � 0Aa 0.8 � 0.45Bb 1.2 � 0.57Bb

Subgroup (d) 0.0 � 0Aa 0.2 � 0.38Aa 0.3 � 0.45Aa

Subgroup (e) 0.0 � 0Aa 0.8 � 0.38Bb 0.9 � 0.28Bb





group.

Table 6 Mean mineralization scores among all subgroups


Subgroup (a) 0.0 � 0Aa 1.3 � 0.45Ba 1.2 � 0.38Ba

Subgroup (b) 0.0 � 0Aa 0.2 � 0.38Ab 0.8 � 0.62Ba

Subgroup (c) 0.0 � 0Aa 0.3 � 0.45Ab 0.9 � 0.66Ba

Subgroup (d) 0.0 � 0Aa 0.1 � 0.28Ab 0.2 � 0.38Ab

Subgroup (e) 0.0 � 0Aa 0.0 � 0Ab 0.0 � 0Ab





group.



has several drawbacks including multiple patient vis-

its, probability of canal contamination between visits

and increased dentine brittleness hence increased risk

of fracture (Andreasen et al. 2002).

Recently, the concept of apical plugs has been

advocated as a single visit procedure. This technique

involves placing an artificial apical barrier in the

apical portion of the canal using mineral trioxide

aggregate (MTA). This protocol has the advantages of

reduced number of visits, high patient compliance

and high success rate (Simon et al. 2007, Holden

et al. 2008, Witherspoon et al. 2008). However, the

problem of thin brittle roots remains.

Recent regenerative endodontics has gained atten-

tion as a biologically based alternative. Regenerative

approaches gain the advantage over apexification in

that it can allow for further root maturation in length

and thickness by the regenerated vital tissue. Revas-

cularization is considered a simple protocol by which

pulp regeneration can be enhanced (Hargreaves &

Law 2011).

Injectable scaffold is one of the treatment alterna-

tives for regenerative endodontics (Murray et al.

2007). The concept of an injectable scaffold includes

the use of a hydrogel incorporating bFGF aimed to

enhance revascularization processes via the drug

delivery system. The gelatin hydrogel acts as a resorb-

able scaffold and is a preferable candidate for a pro-

tein carrier for its biosafety and inertness towards

protein drugs (Kimura & Tabata 2007). The growth

factor was prepared in a basic form to be electrostati-

cally attached to the acidic gelatin. Thus, delivery of

the growth factor was controlled via degradation of

the hydrogel carrier not by simple diffusion.

The aim of the study was to evaluate the treatment

outcomes of immature teeth with necrotic pulps using

MTA apical barrier or revascularization protocols that

were either simple or enhanced using injectable scaf-

fold.

The choice of dogs as an animal model for biologi-

cal experiments in endodontics is based on the fact

that they have similar apical repair compared with

humans over short durations (average one-sixth of

human) due to the high growth rate (Mohammadi &

Abbott 2009).

Two rooted premolars were selected increasing the

whole number of samples for a reliable statistical

analysis. Premolars are considered accessible for end-

odontic procedures and have average-sized canals for

endodontic manipulation.

The age ranged between 6 and 9 months which

was suitable for the study of immature teeth, as pre-

molar teeth are immature at this age range and the

animal can withstand general anaesthesia.

The triple antibiotic paste has been demonstrated to

eradicate bacteria from infected root canals within

3 weeks (Banchs & Trope 2004). Similar findings

were recorded by (Pierce & Lindskog 1987, Sato et al.

1996, Hoshino et al. 1997).

Radiographic evaluation was standardized using

Image-J software including a TurboReg plug-in. This

computer software is used to standardize pre-operative

and post-operative radiographs. This is done by math-

ematical alignment of source and target images using

(a) (b)

Figure 9 (a) Photomicrograph of subgroup III (a) showing hard tissue deposition at the apical foramen (arrow) (H&E 9 40).

(b) Photomicrograph of subgroup III (c) showing newly deposited hard tissue on the canal walls (arrows) (H&E 9 200).



multiple identical points on the two images (Bose

et al. 2009).

Evidence of definite hard tissue deposition was

noticed at the apical third increasing the root length

and root thickness without significant difference

between subgroup (b) revascularization by blood clot

and (c) revascularization by blood clot and injectable

scaffold coated with bFGF. Subgroups (d) MTA closure

of an empty root canal and (e) pulp exposed teeth

(positive control) showed no hard tissue deposition.

This may be due to absence of regenerated tissue

responsible for hard tissue deposition. These findings

were in agreement with that reported by Thibodeau

et al. (2007) and Wang et al. (2010).

There was no significant difference between sub-

group (b) revascularization by blood clot and (c)

revascularization by blood clot and injectable scaffold

coated with bFGF. This is in agreement with the

results of Thibodeau et al. (2007) who reported the

importance of the scaffold in tissue growth and hard

tissue deposition.

Radiographic findings were in agreement with

Thibodeau et al. (2007) who stated that root elonga-

tion and thickening was evident radiographically.

However, Wang et al. (2010) concluded that radio-

graphic findings were not accurate regarding actual

increase in length or thickness of root. That was not

the case in the present study due to radiographic

standardization using the Image-J with TurboReg

plug-in.

After 3 months, no significant difference was found

between subgroup (b) and (c) regarding increase in

length and thickness. Approximately 67% of sub-

group (b) samples and 75% of subgroup (c) samples

had increased in length and thickness. This can be

attributed to hard tissue deposition and consequently

apical closure.

Group I specimens had mild to moderate inflamma-

tion. This finding can be attributed to the immediate

inflammatory reaction of the periradicular tissues to

the treatment protocols.

Regarding subgroups b and c, the mean inflamma-

tory cell count was higher than the other subgroups.

This might be attributed to the traumatic insult of

periapical tissues via overinstrumentation to induce

bleeding.

After 3 weeks, most of samples had none to mild

inflammation. No significant difference was found

between subgroups (a, b, c and d) regarding inflam-

matory cell counts. This is an indication of progressed

healing of the periapical lesion. This is in agreement

with previous studies (Gomes-Filho et al. 2011). For

subgroups (a, b, c and d), the decreased inflammatory

reaction might be due to the prolonged efficiency of

triple antibiotic paste in eradication of infection. Sub-

group (e) had a significantly more severe inflamma-

tory reaction due to progression of the infection

without any mean of eradication.

Tissue in-growth mechanisms are still unknown.

Many mechanisms have been postulated to explain

the mechanism of tissue regeneration inside the

canal, including the presence of few vital pulp cells at

the apical end (Banchs & Trope 2004), abundance of

dental pulp stem cells in immature teeth (Gronthos

et al. 2000), periodontal ligament stem cells (Seo et al.

2004), stem cells of apical papilla (SCAP) (Sonoyama

et al. 2008) and the blood clot itself, which acts as a

reservoir of growth factors (Wang et al. 2007).

In this study, histological evaluation of the newly

formed tissue revealed that it resembled periodontal

tissue in structure (Lieberman & Trowbridge 1983).

The newly deposited hard tissue resembled cementum,

characterized by inclusion of cementocyte-like cells,

direct attachment to dentine and fibrous attachment

to the neighbouring connective tissue. The role of

blood clot is considered beneficial as it supplies the

newly growing tissue with growth factors.

No significant difference was found between sub-

group b and c. This shows that the hydrogel scaffold

incorporating growth factor did not play a significant

role in the regeneration process. The present findings

were not supported by Kimura & Tabata (2007) who

found that the use of hydrogel incorporating bFGF

enhanced angiogenesis and regeneration in ischaemic

tissues. This conflict may be due to the difference in

substrate; their medical use of that drug delivery sys-

tem was concentrated on ischaemic tissues, which are

still vital and already supported by collateral circula-

tion and cellular supply is guaranteed. However, in

the present study, the pulp space was empty, enclosed

in hard tissue deprived from collateral circulation

with minimum cellular infiltration.

Subgroup (d) MTA closure of an empty root canal

recorded significantly low scores for tissue in-growth.

This may be due to the absence of a blood clot and

high level of inflammation rendering the conditions

unfavourable for tissue growth.

Some samples showed signs of hard tissue deposition

at the MTA – tissue interface. Few samples had com-

plete calcific barrier. This tissue response is believed to

be a result of good sealing ability, biomineralizing abil-

ity and alkaline pH (Torabinejad et al. 1995).



Regarding subgroup (d) of group III, the inflamma-

tory cell count was significantly higher than the other

subgroups. This might be attributed to the empty

canal space in which bacterial invasion via anachore-

sis increased with time.

Persistent inflammatory reaction was seen in sub-

group (e) due to continued bacterial irritation; Bezerra

da Silva et al. (2010) recorded the same finding.

Continued tissue growth was seen in subgroups (b)

and (c) without a significant difference between them.

These findings may be explained as the normal pro-

gression of the regenerated tissue. Similar findings

were mentioned by Thibodeau et al. (2007).

Although hard tissue deposition was noticed in the

apical third increasing the length and dentine thickness

of the root without significant difference between sub-

groups (b) and (c), subgroups (d) and (e) showed no

hard tissue deposition. This may be due to absence of

regenerated tissue responsible for hard tissue deposition.

Although both treatment protocols, MTA apical

plug and revascularization, were successful treatment

option with regard to closure of open apices, the MTA

apical plug treatment protocol did not enhance an

increase in root length or dentine thickness.

Conclusions

The revascularization procedure can continue the

development of the roots of teeth with a necrotic pulps.

References

Abbott PV (1998) Apexification with calcium hydroxide:

when should the dressing be changed? the case for regu-

lar dressing changes. Australian Endodontic Journal 24,

27–32.

Andreasen J, Farik B, Munksgaard E (2002) Long term cal-

cium hydroxide as a root canal dressing may increase risk

of root fracture. Dental Traumatology 18, 134–7.

Banchs F, Trope M (2004) Revascularization of immature

permanent teeth with apical periodontitis: new treatment

protocol. Journal of Endodontics 30, 196–200.

Bezerra da Silva L, Nelson-Filho P, Bezerra da Silva R (2010)

Revascularization and Periapical repair after endodontic

treatment using apical negative pressure irrigation versus

conventional irrigation plus triantibiotic intracanal dress-

ing in dogs, teeth with apical periodontitis. Oral Surgery,

Oral Medicine, Oral Pathology, Oral Radiology and Endodon-

tology 109, 779–87.

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.

Camp J, Fuks A (2006) Pediatric endodontics: endodontic

treatment for the primary and young permanent dentition.

In: Cohen S, Hargreaves K, Keiser K, eds. Pathways of the

pulp, 9th edn. St.Louis: Mosby Elsevier, pp. 822–82.

Citrome G, Kaminski E, Heuer M (1979) A comparative

study of tooth apexification in the dog. Journal of Endodon-

tics 5, 290–7.

Cvek M (1992) Prognosis of luxated non-vital maxillary

incisors treated with calcium hydroxide and filled with

gutta-percha. A retrospective clinical study. Endodontic and

Dental Traumatology 8, 45–55.

Deutsch A, Musikant B, Cavallari J (1985) Root fracture

during insertion of prefabricated posts related to root size.

Journal of Prosthetic Dentistry 53, 786–9.

Gomes-Filho JE, Costa MMT, Cintra LTA et al. (2011) Evalu-

ation of rat alveolar bone response to angelus MTA or

experimental light cured mineral trioxide aggregate using

fluorochromes. Journal of Endodontics 37, 250–4.

Gronthos S, Mankani M, Brahim J, Robey PG, Shi S (2000)

Postnatal human dental pulp stem cells (DPSCs) in vitro

and in vivo. Proceedings of the National Academy of Sciences

USA 97, 625–30.

Hargreaves K, Law A (2011) Regenerative endodontics. In:

Hargreaves K, Cohen S, eds. Pathways of the pulp, 10th

edn. St.Louis: Mosby Elsevier, pp. 602–19.

Holden D, Schwartz S, Kirkpatrick T (2008) Clinical out-

comes of artificial rootend barriers with mineral trioxide

aggregate in teeth with immature apices. Journal of End-

odontics 34, 812–7.

Hoshino E, Kurihara-Ando N, Sato I (1997) In-vitro anti-

bacterial susceptibility of bacteria taken from infected

root dentine to a mixture of ciprofloxacin, metronidazole

and minocycline. International Endodontic Journal 29, 125

–30.

Huang G (2009) Apexification: the beginning of its end.

International Endodontic Journal 42, 855–66.

Iwaya S, Ikawa M, Kubota M (2011) Revascularization of an

immature permanent tooth with periradicular abscess after

luxation. Dental Traumatology 37, 55–8.

Kerekes K, Heide S, Jacobsen I (1980) Follow-up examina-

tion of endodontic treatment in traumatized juvenile inci-

sors. Journal of Endodontics 6, 744–8.

Kimura Y, Tabata Y (2007) Experimental tissue regeneration

by DDS technology of bio-signaling molecules. Journal of

Dermatological Sciences 47, 189–99.

Lieberman J, Trowbridge H (1983) Apical closure of non

vital permanent incisor teeth where no treatment was

performed: case report. Journal of Endodontics 9, 257–60.

Mohammadi Z, Abbott P (2009) On the local applications of

antibiotics and antibiotic based agents in endodontic and

dental traumatology. International Endodontic Journal 42,

555–67.

Murray P, Garcia-Godoy F, Hargreaves K (2007) Regenera-

tive endodontics: a review of current status and a call for

action. Journal of Endodontics 33, 377–90.



Nosrat A, Seifi A, Asgary S (2011) Regenerative endodontic

treatment (Revascularization) for necrotic immature per-

manent molars: a review and report of two cases with

new biomaterial. Journal of Endodontics 37, 562–7.

Petrino J, Boda K, Shambarger S, Bowles W, McClanahan B

(2010) Challenges in regenerative endodontics: a case ser-

ies. Journal of Endodontics 36, 536–41.

Pierce A, Lindskog S (1987) The effect of an antibiotic/corti-

costeroid paste on inflammatory root resorption in vivo.

Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology

and Endodontology 64, 216–20.

Sato I, Kurihara-Ando N, Kota K, Iwaku M, Hoshino E

(1996) Sterilization of infected rootcanal dentine by topical

application of a mixture of ciprofloxacin, metronidazole

and minocycline in situ. International Endodontic Journal

29, 118–24.

Seo BM, Miura M, Gronthos S et al. (2004) Investigation of

multipotent postnatal stem cells from human periodontal

ligament. The Lancet 364, 149–55.

Sheehy E, Roberts G (1997) Use of calcium hydroxide for

apical barrier formation and healing in non-vital imma-

ture permanent teeth: a review. British Dental Journal 183,

241–6.

Simon S, Rilliard F, Berdal A (2007) The use of mineral tri-

oxide aggregate in one-visit apexification treatment: a pro-

spective study. International Endodontic Journal 40,

186–97.

Sonoyama W, Liu Y, Yamaza T et al. (2008) Characteriza-

tion of the apical papilla and its residing stem cells from

human immature permanent teeth: a pilot study. Journal

of Endodontics 34, 166–71.

Tabata Y (2004) Tissue regeneration based on tissue engi-

neering technology. Congenital Anomalies 44, 111–24.

Thibodeau B, Teixeira F, Yamauchi M, Caplan D, Trope M

(2007) Pulp revascularization of immature dog teeth with

apical periodontitis. Journal of Endodontics 33, 680–9.

Torabinejad M, Hong C, McDonald F, Pittford T (1995)

Physical and chemical properties of a new root-end filling

material. Journal of Endodontics 21, 349–53.

Wang Q, Lin XJ, Lin ZY, Liu GX, Shan XL (2007) Expression

of vascular endothelial growth factor in dental pulp of

immature and mature permanent teeth in human. Shang-

hai Journal of Stomatology 16, 285–9.

Wang X, Thibodeau B, Trope M, Lin L, Huang G (2010) His-

tologic characterization of regenerated tissues in canal

space after the revitalization/revascularization procedure

of immature dog teeth with apical periodontitis. Journal of

Endodontics 36, 56–63.

Windley W, Teixeira F, Levin L, Sigurdsson A, Trope M

(2005) Disinfection of immature teeth with a triple antibi-

otic paste. Journal of Endodontics 31, 439–43.

Witherspoon D, Small J, Regan J (2008) Retrospective analy-

sis of open apex teeth obturated with mineral trioxide

aggregate. Journal of Endodontics 34, 1171–6.



Regenerative potential following revascularization of immature permanent teeth with necrotic pulps

Documents

Transcript of Regenerative potential following revascularization of immature permanent teeth with necrotic pulps