Histologic evaluation and immunohistochemical localization of STRO-1 and BMP-4 in rat immature teeth: A comparison between vital and induced pulp necrosis

Histologic evaluation and immunohistochemical localizationof STRO-1 and BMP-4 in rat immature teeth: A comparisonbetween vital and induced pulp necrosis

Daiana Elisabeth Bottcher a,d,*, Roberta Kochenborger Scarparo b,d, Eraldo Luiz Batista Jr.c,d,Anna Christina Medeiros Fossati c,d, Fabiana Soares Grecca b,d

aConservative Dentistry Department, Faculty of Dentistry, Federal University of Rio Grande do Sul (UFRGS), BrazilbClinical Department, Faculty of Dentistry, Pontifical Catholic University of Rio Grande do Sul (PUCRS), Av. Ipiranga 6681 – Bld. 06,

Porto Alegre, RS 90619-900, BrazilcDepartment of Diagnostics and Surgical Sciences, Faculty of Dentistry, University of Manitoba, D344B-790 Bannatyne Avenue, Winnipeg,

MB R3E 0W2, CanadadMorphological Sciences Department, Faculty of Dentistry, Federal University of Rio Grande do Sul (UFRGS), Brazil

a r c h i v e s o f o r a l b i o l o g y 5 8 ( 2 0 1 3 ) 1 1 7 4 – 1 1 7 9

a r t i c l e i n f o

Article history:

Accepted 2 April 2013

Keywords:

Endodontics

Dental pulp

Immature necrotic teeth

Mesenchymal stem cells

a b s t r a c t

Objective: To assess histological features and the expression of STRO-1 and BMP-4 in dental

pulp and periapical tissues in vital or necrotic rat immature teeth.

Design: The lower left first molars of male Wistar rats ageing four weeks (n = 24) had their

pulps exposed to the oral environment for 3, 6, 9 and 12 weeks (animals ageing 7, 10, 13 and

16 weeks-old, respectively; n = 24). The right lower first molars served as control untouched

teeth. After sample harvesting the jaws were dissected and processed for histology and

immunodetection of STRO-1 and BMP-4.

Results: Necrotic teeth had root development arrested, while control animals showed

development of dental tissues. Immunohistochemistry showed that detection of BMP-4

was restricted to vital pulps. For both groups, STRO-1 expression was evident around blood

vessels walls. Neither BMP-4 nor STRO-1 was observed in the apical papilla region.

Conclusion: STRO-1-positive precursor cells were not detected in the apical papilla. BMP-4

expression has not been detected during infection.

# 2013 Elsevier Ltd. All rights reserved.

Available online at www.sciencedirect.com

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1. Introduction

Dental caries or traumatic injuries to young permanent teeth

can lead to pulp necrosis and tooth development arrest.

Several reports1–6 demonstrate apical healing and root

completion after the treatment of necrotic immature teeth,

suggesting that repair may occur even in adverse conditions.

* Corresponding author at: Conservative Dentistry Department, FacultyRamiro Barcelos, 2492 Porto Alegre, CEP: 90035-003, Brazil. Tel.: +55 5

E-mail addresses: [email protected], [email protected]

0003–9969/$ – see front matter # 2013 Elsevier Ltd. All rights reservehttp://dx.doi.org/10.1016/j.archoralbio.2013.04.001

Some studies have identified the presence of stem cells (SC)

on dental tissues by the immunodetection of markers such as

STRO-1.7–9 The existence of SC in the dental pulp (DP) has been

established since the year 2000.7 Nevertheless, their presence in

the apical papilla has been verified only in the last years, being

suggested as a probably source of undifferentiated cells, which

could explain the clinical phenomenon of root formation in

non-vital teeth.8,9 In this regard Sonoyama et al.10 stated that it

of Dentistry, Federal University of Rio Grande do Sul (UFRGS), Av.1 84485602; fax: +55 51 33085002.m.br (D.E. Bottcher).

d.

a r c h i v e s o f o r a l b i o l o g y 5 8 ( 2 0 1 3 ) 1 1 7 4 – 1 1 7 9 1175

is likely that stem cells from the apical papilla (SCAP) survive

infection because of their close proximity to periapical tissues.

Nevertheless, inflammatory mediators up-regulated in

injured tissues seem to interfere with the signals needed for

root development.11 Growth factors, like BMP-4, are among the

triggered signalling networks during dental morphogenesis

that may be negatively affected by inflammation.12 In

assessing the role of BMP-4 in Hertwig’s epithelial root sheath

(HERS) formation, Hosoya et al.13 verified that this protein

plays an important role in regulation of root formation,

preventing elongation and maintaining cell proliferation.

Few studies focused on alterations of tissues affected by

pulp necrosis in immature teeth.14,15 Moreover, in necrotic

teeth, the immunolocalization of precursor cells and growth

factors that have an influence on root development have yet to

be explored.

Thus, the purpose of the present study was to assess

histological features and the expression of STRO-1 and BMP-4

in dental pulp and periapical tissues of rat immature teeth

with vital or induced pulp necrosis.

2. Methods

2.1. Experimental protocols

This study was approved by the Institutional Review Board and

by the Research Ethics Committee of the School of Dentistry of

the Federal University of Rio Grande do Sul (Protocol #19001) and

is in accordance with the European Convention for the

Protection of Vertebrate Animals used for Experimental and

Other Scientific Purposes. The sample consisted of 24 Wistar

male rats, and a split-mouth experimental design was adopted.

Prior to the experimental procedures, the animals were

anesthetized intraperitoneally with ketamine 80 mg/kg and

xylazine 20 mg/kg (Virbac do Brasil, Juruatuba, SP, Brazil). The

procedures adopted herein were described elsewhere.15 Briefly,

right mandibular first molars were used for the observation of

natural morphogenesis (control group – CG). Pulp necrosis was

induced on the left mandibular first molars (test group – TG)

during the initial stage of root development (animals ageing 4

weeks-old). Dental pulps were exposed by drilling cavities on

the central portion of the occlusal surface with a 1011 HL round

bur in high speed (KG Sorensen, Cotia, SP, Brazil) to a depth

nearly equal to the bur diameter (1 mm). Teeth were left open to

the oral environment throughout the course of the experiment.

Time required for detection of periapical lesions was confirmed

by radiographs taken as previously reported.15,16 Animals were

euthanized by inhalation of CO2 at 3, 6, 9 or 12 weeks post pulp

exposure (animals ageing 7, 10, 13 and 16 weeks-old; n = 24).

Jaws were dissected for histologic and immunohistochemical

evaluation.

Immediately after euthanasia, samples were fixed in

buffered phosphate 10% paraformaldehyde for 24 h, decalci-

fied in 17% EDTA for 5 weeks, dehydrated in ascending

concentrations of ethanol, and embedded in paraffin. Five-

micrometre sections were cut and stained with haematoxylin–

eosin or processed for immunohistochemistry. Three sections

were selected per sample, so the central portion of the roots,

including the apex, was visible.

2.2. Histological evaluation

Histological evaluation (n = 35) was based on a descriptive

analysis on pulp tissue. The presence of inflammatory cells, as

well as features related to vascularity, odontoblast organiza-

tion, degeneration/destruction processes were considered.

Additionally, other phenomena including root resorptions and

closure of apical foramen were considered.

Periapical inflammation was classified by calibrated exam-

iners (k = 0.79, P < 0.001) according to the following scores: (0)

absence – absence of inflammatory reaction, (1) mild –

inflammatory cells restricted to the root canal space, (2)

intense – inflammatory cells in form of infiltrate in periapical

region, and (3) severe – inflammatory cells in form of infiltrate

in periapical region with abscess formation.

2.3. Immunohistochemistry

The sections were deparaffinized and immersed in 3.0%

hydrogen peroxide in absolute methanol (two incubations of

15 min each) in order to obtain the endogenous peroxidase

blocking, followed by three washing cycles in PBS, pH 7.2.

Sections were washed in PBS and incubated either with a

monoclonal mouse anti-human STRO-1 antibody (R&D Sys-

tems Inc., Minneapolis, MN, USA) or mouse monoclonal anti-

rat BMP-4 (Novocastra, Newcastle, Northumberland, Reino

Unido) at 4 8C overnight. The primary antibody was diluted to

1:100 (according to a pilot study) with antibody diluent and

background reducing agents as described by the manufacturer

(Dako, Carpinteria, CA, EUA) for both markers.

The sections were washed in PBS pH 7.2 and incubated with

a secondary conjugated antibody as recommended by the

manufacturer (Picture Max, HRP Polymer Conjugate Broad

Spectrum, Invitrogen, Carlsbad, CA, USA) during 30 min. The

sections were rinsed with PBS for 2 min, 3 times, and

immunoreactivity was visualized after incubation with 3,30-

diaminobenzidine (DAB) solution (Dako Liquid DAB Substrate

Chromogen System, Dako, Carpinteria, CA, EUA), and then

counterstained with Harry’s haematoxylin.

Immunohistochemical control was obtained by incubating

with nonimmune mouse IgM (Rockland Immunochemicals,

Gilbertsville, PA, USA) as the primary antibody. None of the

control sections were positive for the target markers (not

shown). Positive controls were alveolar bone for BMP-4 and

umbilical cord cells for STRO-1.

3. Results

3.1. Histological analysis

Pulp exposure to the oral environment resulted in periapical

lesions and root development arrest, i.e., open apices and thin

dentinal walls, regardless the experimental period. Complete

pulp tissue destruction was observed in all samples (Fig. 1a

and c). On the other hand, histological evaluation of vital teeth

showed uneventful, progressive apical closure. In these cases,

root development was completed in the 13-week-old animals,

and continuous dentine and cementum deposition could be

observed, respectively, in the internal and external root

Fig. 1 – Pulp necrosis and tissue destruction: establishment of periapical inflammatory reaction – score 3 – and abscess

formation zones ("). Complete destruction of pulp tissue (*). (a) Seven-week test group animal (3 weeks after pulp exposure)

(40T). (c) Thirteen-week test group animal (9 weeks after pulp exposure) (40T). (b) Periapical inflammatory reaction:

increased magnification from periapical region of (a) (100T). (d) Increased magnification from periapical region of (c) (100T).

Scale bars = 100 mm.

Fig. 2 – Developing tooth: presence of periodontal ligament

(PL), cementum (C), dentine (D), pulp (P), odontoblasts (O),

pre-dentine (PD), cell-rich zone (CRZ), apical papilla (AP)

and Hertwig’s epithelial root sheath (HERS). Animal from

control group ageing 7 weeks-old (100T). Scale

bars = 100 mm.

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surfaces. Apart from features mentioned above, pulp tissue

presented similar characteristics, regardless of the evaluation

period. Dental pulps showed the presence of collagen fibres,

intense vascularity and aligned layers of polarized odonto-

blasts (Fig. 2). Both hyalin and calcific degenerations were

found in vital teeth. In TG, during the first experimental

periods, remnants of pulp tissue were detected in conjunction

with the presence of some calcific degeneration.

Periapical inflammatory reaction was observed only in the

TG and scored 2 or 3 in most of the samples (Fig. 1b and d). As

expected, resorption zones were also found. When pulp tissue

was not assessed (CG), dentine and cementum tissues

developed in such a way that enabled the complete develop-

ment of the dental root walls. Moreover, periodontal fibres

were functionally inserted on dental tissues. In samples with

incomplete root formation, Hertwig’s epithelial root sheaths

(HERS) and the apical papilla (AP) were detected in CG (Fig. 2).

3.2. STRO-1 immunolocalization

Throughout natural root development, expression of STRO-1

was observed in pulp cells (Fig. 3a) and in the perivascular area

a r c h i v e s o f o r a l b i o l o g y 5 8 ( 2 0 1 3 ) 1 1 7 4 – 1 1 7 9 1177

(Fig. 3b). Moreover, periapical blood vessels walls showed

immunoreactivity to STRO-1 in both vital and necrotic

samples. In CG, STRO-1 expression was observed regardless

of the experimental period. On the other hand, in test groups,

expression occurred only at 3 weeks of exposure to oral

environment. At this period, some test samples presented

immunoexpression of STRO-1 in perivascular cells of the

periapical region (Fig. 3c). Noteworthy, some samples in both

groups did not show any expression at all.

Fig. 3 – Immunohistochemical localization of STRO-1: (a)

STRO-1 expression in pulp cells ("). Animal from control

group ageing 7 weeks-old (200T). (b) Peripheral blood

vessels cells from pulp tissue ("). Animal from control

group ageing 7 weeks-old (200T). (c) Expression in the

peripheral blood vessels cells from periapical region (").Animal from test group ageing 7 weeks-old (3 weeks after

pulp exposure) (100T). Scale bars: (a) and (b) = 50 mm;

(c) = 100 mm.

3.3. BMP-4 immunolocalization

Immunoreactivity to BMP-4 was observed in CG (Fig. 4a) at all

time points and along the entire crown and root. BMP-4

expression was detected in pulp blood vessels walls, odonto-

blasts and the sub-odontoblast layer, near to predentin

(Fig. 4b). No immunoreactivity to BMP-4 was observed in

samples from TG.

4. Discussion

The present study showed, for the first time, that stem cells

located in the perivascular area of the apical periodontium

could maintain their viability during root canal infection.

Earlier studies7–9,17–19 showed SC presence in dental tissues of

vital teeth, but its possible survival during bacterial triggered

inflammation had yet to be established. In this regard, short

periods of infection were more likely to present STRO-1

positive cells. These findings bring insights into the impor-

tance of the continuous effect of inflammatory modulation

over the tissues ability to respond to injuries and repair. In this

regard, the present data might be clinically relevant when

comparing the prognosis of immature teeth presenting recent

or long-term infections. On the other hand, the current results

fails to demonstrate a correlation between the intensity of

inflammatory reaction and the presence of STRO-1 positive

cells. Thus, the duration of noxious stimuli appears to present

a greater influence on this feature compared to the inflam-

mation intensity.

Mesenchymal stem cells have been often characterized with

several surface markers, since a single labelling might not be

sufficient for their precise definition.20,21 Kaneko et al.19 used

CD146, CD105 and CD166 for the assessment of SC in dental

pulp. Besides, STRO-1, a cell surface antigen expressed by bone

marrow stromal cells and erythroid precursors,22 has been

frequently used in dental tissues.8,23 The same marker was used

herein since it is specific for stem cells/pericytes from DP,24

bringing additional information to the current knowledge on

the differences between vital and necrotic teeth.

At the periapical region, STRO-1 labelling was verified

mainly surrounding blood vessels, even in non-infected teeth.

As well as in other investigations,9,10,17,18,23,25 this protein was

detected in both DP cells and vessels. The present study showed

that apart from dental pulp, the apical periodontium also

presented STRO-1 positive perivascular niches. Hereupon, this

immunoreaction was not evidenced in later stages of infection.

Perivascular stem cells have been previously suggested as

having a role in repairing alveolar bone defects.26 Their

presence in necrotic teeth corroborates the assumption that

precursor cells may also be recruited from other sources than

dental pulp and apical papilla, triggering the accumulation of

undifferentiated cells into the canal space. In agreement, the

blood clot has been claimed as a possible source of these

cells,27,28 which seems plausible through the results of this

investigation. Nevertheless, the detection of root development

even when the blood clot is not induced,14,15 emphasizes the

importance of further investigations aiming at understanding

other alternative sources of undifferentiated cells. Unlike

previous investigations,8,10 stem cells from the apical papilla

Fig. 4 – Immunohistochemical localization of BMP-4: (a) BMP-4 expression in pulp cells ("). Animal from control group ageing

10 weeks-old (400T). (b) Peripheral blood vessels cells ("). Expression in odontoblasts and pre-dentine region (*). Animal

from control group ageing 7 weeks-old (400T). Scale bars = 10 mm.

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were neither found in vital pulp groups nor in the necrotic

ones. Similarly to the present study, Kaneko et al.23 localized

the STRO-1 human antigen in rat pulps and periodontal

tissues, although, STRO-1-positive cells were not identified

close to Hertwig’s epithelial root sheath. In this regard,

possible repositories of stem cells, such as the periodontal

ligament, as well as the pathways for mineralized-tissue

forming cells differentiation, should also be assessed.

As stated by Huang,28 apart from stem cells viability, the

root canal disinfection has a paramount importance in root

formation, something that has been already demonstrated by

others.15,29 The outcomes presented herein also emphasize

the role of root canal infection on the modifications of signs

related to root morphogenesis. The bone morphogenetic

protein 4 (BMP-4) is a growth factor related to tooth

development30 and, for this reason, it was chosen for

evaluation in the present study. As stated by Hosoya et al.13

and Gluhak-Heinrich et al.,31 BMP-4 was observed in regions of

developing mineralized tissues. In the CG, BMP-4 was

expressed by odontoblasts and near the pre-dentine region,

suggesting a role for this protein in dentine matrix formation.

Moreover, it was also expressed by alveolar bone tissue when

non-infected samples were observed.

The current outcomes suggest that BMP-4 expression is

sensible to the alterations induced by infection and inflam-

mation. When pulp necrosis was induced, there was no BMP-4

expression, indicating that inflammation of the apical period-

ontium arrests events associated with dentine formation. As a

matter of fact, this growth factor plays important roles during

odontoblast differentiation, and its occurrence has not been

identified as a current finding in protocols aiming root

development in non-vital teeth.15,27 Additionally, these strat-

egies have been shown to result in newly formed cementum

and bone,14,15,32 for which the expression of other growth

factors are certainly required and have yet to be characterized

on a qualitative and temporal basis.30,33

Taken together, the present outcomes highlight some

aspects that are important for understanding root formation

induction in necrotic teeth. In this regard, the duration of

infectious process appears to play an essential role on the fate

of mesenchymal stem cells. Moreover, original pathways of

root development are probably modified when root formation

is established in non-vital teeth. The characterization of this

signalling warrants further investigation. More sensitive

molecular techniques, to complement immunohistochemis-

try, should be performed in further investigations to provide

other insights into apical periodontium healing during

apexogenesis of necrotic teeth.

Funding

Supported by grants of the Coordination for the Improvement

of Higher Level Education Personnel (Capes) Brasilia, Distrito

Federal, Brazil.

Competing interests

The authors deny any conflicts of interest related to this study.

Ethical approval

This study was approved by the Institutional Review Board

and by the Research Ethics Committee of the School of

Dentistry of the Federal University of Rio Grande do Sul

(Protocol #19001) and is in accordance with the European

Convention for the Protection of Vertebrate Animals used for

Experimental and Other Scientific Purposes.

Acknowledgement

The authors gratefully acknowledge the contributions of Dr.

Renata Dornelles Morgental during the manuscript writing.

Appendix A. Supplementary data

Supplementary data associated with this article can be

found, in the online version, at http://dx.doi.org/10.1016/

j.archoralbio.2013.04.001.

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