12328
Postgraduate Programme in Dentistry, Federal University of Piau (UFPI), Teresina; 2Department of Morphology, Federal
University of Piau (UFPI), Teresina; 3Department of Clinic and Veterinary Surgery, Federal University of Piau (UFPI),
ao Paulo (USP), S~
ao Paulo; and 5Department of Pathology
Teresina; 4Postgraduate Programme in Pathology, University of S~
and Dentistry Clinic, Federal University of Piau (UFPI), Teresina, Brazil
Abstract
nior AM, Rizzo MS, Moura RD,
Lima CCB, Conde Ju
Moura MS, Lima MDM, Moura LFAD. Biocompatibility
of root filling pastes used in primary teeth. International
Endodontic Journal, 48, 405416, 2015.
Introduction
The complex morphology of root canal systems and
the root resorption of primary molars are uncontrollable
factors that may hinder proper chemicalmechanical
Correspondence: L
ucia de F
atima Almeida de Deus Moura,
Department of Pathology and Dentistry Clinic, Federal University of Piau, Rua Angelica, 1650, Bairro de Fatima,
CEP 64049-532, Teresina, Piau, Brazil (Tel.: +55 86 3233
3050; e-mail: mouraiso@uol.com.br).
2014 International Endodontic Journal. Published by John Wiley & Sons Ltd
405
406
Animal model
A total of 54 mice (Mus musculus) were used. The
animals were Swiss albino males from 6 to 8 weeks of
age that weighed between 15 and 30 g. Throughout
the experiment, the animals were housed in a vivarium with standard rations and access to water ad libitum. All animals were examined every 2 days and
monitored for local or systemic changes.
The animals were wormed with Basken suspension (14.5 mg pyrantel pamoate + 9.5 mg oxantel
pamoate kg 1), which was administered orally in a
single dose 7 days prior to the onset of the experimental procedural.
2014 International Endodontic Journal. Published by John Wiley & Sons Ltd
2014 International Endodontic Journal. Published by John Wiley & Sons Ltd
Histologic analysis
Following fixation, the polyethylene tubes were
removed from the fixative, dehydrated in increasing
concentrations of alcohol, cleared in xylene, embedded in paraffin and blocked. The blocks were cut
using a microtome (Leica RM 2245) to yield 5-lmthick longitudinal semi-serial sections. Tissues were
stained with haematoxylin and eosin (HE) and Masson trichrome (Putt 1972).
The sections were examined under a binocular light
microscope (Olympus, Tokyo, Japan) at 100, 200 and
4009 magnification and digitally photographed.
A histologic analysis was performed by two pathologists, who were blind to the experimental conditions,
at different times. Both examiners analysed the same
fields. The Masson trichrome and HE stains were used
to analyse the collagen fibre formation and thickness,
respectively, at 2009 magnification. The inflammatory
infiltrate was analysed at 4009 magnification with HE
stain. The intra-examiner agreement was based on two
histologic evaluations performed at different intervals
on 10% of the sections, which were arranged in order
from the smallest to the largest increase (j = 0.80 and
0.81). The interexaminer agreement (j = 0.79) indicated interexaminer reliability.
The tissues that contacted the open end of the
tubes were analysed and graded for the following histologic findings: 1. formation of collagen fibres, 2. tissue thickness and 3. inflammatory infiltrate. The
density of collagen fibre formation was graded as 0
(absent), 1 (mild collagen fibres arranged similarly
to normal connective tissue), 2 (moderate individual
collagen fibres were observed, but did not display linear features or a typical wavy microanatomy) and 3
(severe very dense, individual collagen fibres cannot
be distinguished) (Silva et al. 2009, Queiroz et al.
2011).
The tissue thickness was assessed by the extent of
fibroblast cell layers formed and the presence of
macrophages in the peripheral contact region of the
tube and classified as follows: 0 (normal no layer),
1 (slightly increased 1 to 3 layers), 2 (moderately
increased 4 to 10 layers) and 3 (intensely increased
more than 10 layers). The inflammatory infiltrate
was analysed qualitatively to assess the characteristics
and concentration of polymorphonuclear cells (PMN)
and mononuclear cells, which were classified as
407
References
American Academy of Pediatric Dentistry (2012) Guideline
on pulp therapy for primary and young permanent teeth.
Pediatric Dentistry 34, 2229.
Andolfatto C, da Silva GF, Cornelio AL et al. (2012) Biocompatibility of intracanal medications based on calcium
hydroxide. ISRN Dentistry 2012, 16.
Barja-Fidalgo F, Moutinho-Ribeiro M, Oliveira MAA, Oliveira
BH (2011) A systematic review of root canal filling materials for deciduous teeth: is there an alternative for zinc
oxide-eugenol? ISRN Dentistry 1, 17.
Batista RF, Hidalgo MM, Hernandes L et al. (2007) Microscopic analysis of subcutaneous reactions to endodontic
sealer implants in rats. Journal of Biomedical Materials
Research Part A 81, 1717.
Bruno GB, Alves APN, Menezes VA, Maia MCG, Bruno JA,
Viana GSB (2007) Biocompatibility evaluation of an antibiotic paste after pulpotomy in dogs. Brazilian Journal of
Oral Sciences 6, 1397401.
Cappiello J (1964) Tratamentos pulpares em incisivos
prim
arios. Revista de la Asociacion Odontologica Argentina
52, 13945.
Fuks AB (2000) Pulp therapy for the primary and young
permanent dentitions. Dental Clinics of North America 3,
57196.
Gomes-Filho JE, Duarte PC, de Oliveira CB et al. (2012) Tissue reaction to a triantibiotic paste used for endodontic tissue self-regeneration of nonvital immature permanent
teeth. Journal of Endodontics 38, 914.
Gondim JO, Avaca-Crusca JS, Valentini SR, Zanelli CF, Spolidorio DM, Giro EM (2012) Effect of a calcium hydroxide/
chlorhexidine paste as intracanal dressing in human primary teeth with necrotic pulp against Porphyromonas
gingivalis and Enterococcus faecalis. International Journal of
Paediatric Dentistry 22, 11624.
Harini Priya M, Bhat SS, Sundeep Hegde K (2010) Comparative evaluation of bactericidal potential of four root canal
filling materials against microflora of infected non-vital
primary teeth. Journal of Clinical Pediatric Dentistry 35,
239.
Hauman CHJ, Love RM (2003a) Biocompatibility of dental
materials used in contemporary endodontic therapy: a
review. Part 1. Intracanal drugs and substances. International Endodontic Journal 36, 7585.
2014 International Endodontic Journal. Published by John Wiley & Sons Ltd
415
7a
2014 International Endodontic Journal. Published by John Wiley & Sons Ltd
63c
21b
P: KruskalWallis.
There was no statistically significant difference between groups (P > 0.05).
There was no statistically significant difference between groups (P > 0.05).
c
There was a statistically significant difference between the groups in the collagen fibre formation during this period (P < 0.05).
Material tested
Table 1 Qualitative histologic analysis of the collagen fibre formation, thickness and inflammatory cell infiltration of the connective tissue adjacent to the open ends of the
polyethylene tubing
409
Ca(OH)2 paste
Empty tube
CTZ paste
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)
(i)
7th Day
21st Day
63rd Day
(j)
Moderate
Mild
Severe
da
y
ay
3r
d
1s
tu
be
-6
-2
tu
y
pt
Em
Em
pt
y
pt
Em
td
da
y
be
tu
be
-7
3r
d
)2
H
O
a(
C
th
da
y
ay
-6
-2
)2
H
a(
O
a(
C
td
1s
th
-7
)2
H
TZ
C
da
y
da
y
3r
d
-6
-2
TZ
C
TZ
-7
1s
th
td
ay
da
y
100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%
Figure 2 (ai) Photomicrographs stained with Masson trichrome showing the formation of collagen fibres in the connective tissue adjacent to the open ends of the polyethylene tubes in contact with the empty tube (a, d, g), calcium hydroxide paste (Ca
(OH)2) (b, e, h) and CTZ paste (c, f, i), at 7, 21 and 63 days, respectively (2009 magnification). (j) Groups for the qualitative
analysis of the collagen fibre formation. (1) Space occupied by the tube and (2) subcutaneous tissue.
Discussion
A great challenge facing Paediatric Dentistry is to
develop biomaterials that possess antimicrobial properties, in addition to being biocompatible and resorb-
410
2014 International Endodontic Journal. Published by John Wiley & Sons Ltd
Empty tube
Ca(OH)2 paste
CTZ paste
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)
(i)
7th Day
21st Day
63rd Day
(j)
Slighty increased
Moderately increased
Intensely increased
3r
d
td
-6
tu
be
y
tu
be
-2
1s
th
-7
da
y
ay
da
y
Em
pt
Em
pt
H
O
a(
C
)2
tu
be
-6
-2
)2
H
O
a(
Em
pt
1s
3r
d
td
da
y
ay
da
y
-7
H
O
a(
C
TZ
)2
-6
-2
TZ
C
th
3r
d
td
1s
th
-7
TZ
C
da
y
ay
da
y
100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%
Figure 3 (ai) Photomicrographs showing the HE staining for the tissue thickness adjacent to the open end of polyethylene tube
in contact with the empty tube (a, d, g), calcium hydroxide paste (Ca(OH)2) (b, e, h) and CTZ paste (c, f, i), at 7, 21 and 63 days,
respectively (2009 magnification). (j) Groups for the qualitative analysis of tissue thickness. (1) Space occupied by the tubing, (2)
subcutaneous tissue, (*) neovascularization, (f) fibroblasts, (l) lymphocytes, (m) macrophages and (n) neutrophils.
2014 International Endodontic Journal. Published by John Wiley & Sons Ltd
411
Empty tube
Ca(OH)2 paste
CTZ paste
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)
(i)
7th Day
21st Day
63rd Day
Mild
(j)
Moderate
Intense
da
y
1s
3r
d
td
ay
da
y
th
tu
be
-6
-2
be
Em
pt
y
tu
y
pt
Em
Em
O
a(
C
pt
y
tu
)2
be
-6
-2
)2
H
O
a(
-7
3r
d
td
1s
th
-7
H
)2
O
da
y
ay
da
y
da
y
3r
d
C
a(
TZ
-6
-2
TZ
C
TZ
-7
1s
th
td
ay
da
y
100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%
Figure 4 (ai) Photomicrographs of HE-stained tissue showing inflammatory infiltrate in the connective tissue adjacent to the
open end of the polyethylene tube in contact with the empty tube (a, d, g), calcium hydroxide paste (Ca(OH)2) (b, e, h) and
CTZ paste (c, f, i), at 7, 21 and 63 days, respectively (4009 magnification). (j) Groups for the qualitative analysis of the inflammatory reaction. (1) Space occupied by the tubing, (2) subcutaneous tissue, (*) neovascularization, (f) fibroblasts, (l) lymphocytes, (m) macrophages and (n) neutrophils.
412
2014 International Endodontic Journal. Published by John Wiley & Sons Ltd
Table 2 Mean values with standard deviations for thickness (lm), perimeter (lm) and area (lm2) of tissue reaction
Thickness (lm)
CTZ
Ca(OH)2
Control
P
Perimeter (lm)
CTZ
Ca(OH)2
Control
P
Area (lm2)
CTZ
Ca(OH)2
Control
P
Day 7
M (SD)
Day 21
M (SD)
Day 63
M (SD)
135.9 (59.0)
84.7 (17.2)
108.3 (41.6)
0.091
121.4 (69.6)
92.0 (28.5)
87.1 (22.6)
0.389
88.8 (41.0)
76.4 (26.1)
70.4 (31.8)
0.634
0.376
0.552
0.121
1046.1 (252.3)
884.4 (253.8)
1079.6 (73.1)
0.261
950.0 (211.8)
894.3 (143.0)
880.4 (115.2)
0.738
941.2 (179.0)
916.7 (173.4)
763.8 (190.9)
0.218
0.656
0.958
0.004
53554.9 (16442.2)
32132.6 (11382.7)
49362.2 (9999.3)
0.027
47344.8 (27953.0)
32404.8 (9832.4)
32561.9 (12713.6)
0.308
33816.3 (13998.1)
24996.0 (1335.6)
23483.9 (13930.3)
0.396
0.261
0.473
0.008
P*
2014 International Endodontic Journal. Published by John Wiley & Sons Ltd
413
414
2007, Silva et al. 2010, Marques et al. 2011, GomesFilho et al. 2012).
Other materials are used as pastes for the root
canal treatment of primary teeth, including pastes
containing zinc oxide and eugenol (ZOE) with iodoform or a paste that combines iodoform and calcium
hydroxide (Ranly & Garcia-Godoy 2000, American
Academy of Pediatric Dentistry 2012). Studies with
ZOE as a root canal filling material reported a chronic
inflammatory reaction and slow resorption in relation
to the tooth (Fuks 2000, Hauman & Love 2003b).
The iodoform pastes, when combined with calcium
hydroxide, exhibit good biological response and antimicrobial properties and are more easily reabsorbed
at the periapical region (Ranly & Garcia-Godoy
2000). However, the techniques proposed for using
these materials recommend the use of instrumentation and chemicalmechanical systems for the root
canals of primary teeth (American Academy of Pediatric Dentistry 2012).
The limitations of this study are inherent to preclinical experimental studies. Although the results
presented here do not reflect a complete analysis of
the reactions that occur in the pulp, periapical or interradicular regions, the results are important for the
preliminary assessment of the irritative potential of
the tested materials and subsequent biological
responses.
Conclusion
The initial inflammatory reactions in mice induced by
CTZ and calcium hydroxide pastes regressed during the
experimental periods; in addition, these pastes resulted
in less-dense fibrous connective tissue, compared with
the results using the empty control. These reactions
support the biocompatibility of the two pastes.
Acknowledgements
This study was supported financial by a scholarship
grant from the Coordination of Improvement of
Higher Education Personnel (CAPES).
Author contributions
CCBL participated in the project design and data
collection and analysis and interpretation of data
and wrote the article. AMCJ participated in the project design and data collection and analysis and
interpretation of data and wrote the article. MSR
2014 International Endodontic Journal. Published by John Wiley & Sons Ltd
References
American Academy of Pediatric Dentistry (2012) Guideline
on pulp therapy for primary and young permanent teeth.
Pediatric Dentistry 34, 2229.
Andolfatto C, da Silva GF, Cornelio AL et al. (2012) Biocompatibility of intracanal medications based on calcium
hydroxide. ISRN Dentistry 2012, 16.
Barja-Fidalgo F, Moutinho-Ribeiro M, Oliveira MAA, Oliveira
BH (2011) A systematic review of root canal filling materials for deciduous teeth: is there an alternative for zinc
oxide-eugenol? ISRN Dentistry 1, 17.
Batista RF, Hidalgo MM, Hernandes L et al. (2007) Microscopic analysis of subcutaneous reactions to endodontic
sealer implants in rats. Journal of Biomedical Materials
Research Part A 81, 1717.
Bruno GB, Alves APN, Menezes VA, Maia MCG, Bruno JA,
Viana GSB (2007) Biocompatibility evaluation of an antibiotic paste after pulpotomy in dogs. Brazilian Journal of
Oral Sciences 6, 1397401.
Cappiello J (1964) Tratamentos pulpares em incisivos
prim
arios. Revista de la Asociacion Odontologica Argentina
52, 13945.
Fuks AB (2000) Pulp therapy for the primary and young
permanent dentitions. Dental Clinics of North America 3,
57196.
Gomes-Filho JE, Duarte PC, de Oliveira CB et al. (2012) Tissue reaction to a triantibiotic paste used for endodontic tissue self-regeneration of nonvital immature permanent
teeth. Journal of Endodontics 38, 914.
Gondim JO, Avaca-Crusca JS, Valentini SR, Zanelli CF, Spolidorio DM, Giro EM (2012) Effect of a calcium hydroxide/
chlorhexidine paste as intracanal dressing in human primary teeth with necrotic pulp against Porphyromonas
gingivalis and Enterococcus faecalis. International Journal of
Paediatric Dentistry 22, 11624.
Harini Priya M, Bhat SS, Sundeep Hegde K (2010) Comparative evaluation of bactericidal potential of four root canal
filling materials against microflora of infected non-vital
primary teeth. Journal of Clinical Pediatric Dentistry 35,
239.
Hauman CHJ, Love RM (2003a) Biocompatibility of dental
materials used in contemporary endodontic therapy: a
review. Part 1. Intracanal drugs and substances. International Endodontic Journal 36, 7585.
2014 International Endodontic Journal. Published by John Wiley & Sons Ltd
415
Pinky C, Shashibhushan KK, Subbareddy VV (2011) Endodontic treatment of necrosed primary teeth using two different combinations of antibacterial drugs: an in vivo
study. Journal of the Indian Society of Pedodontics and Preventive Dentistry 29, 1217.
Putt FA (1972) Manual of Histopathological Staining Methods.
New York: John Wiley and Sons.
Queiroz AM, Assed S, Consolaro A et al. (2011) Subcutaneous connective tissue response to primary root canal filling
materials. Brazilian Dental Journal 22, 20311.
Ranly DM, Garcia-Godoy F (2000) Current and potential
pulp therapies for primary and young permanent teeth.
Journal of Dentistry 28, 15361.
Semenoff TA, Semenoff Segundo A, de Figueiredo JA (2008)
Biocompatibility of different intracanal medications in rat bucal submucosa tissue. Journal of Applied Oral Science 16, 127.
Serhan CN (2010) Novel lipid mediators and resolution
mechanisms in acute inflammation: to resolve or not?
American Journal of Pathology 177, 157691.
Silva RA, Assed S, Nelson-Filho P, Silva LA, Consolaro A
(2009) Subcutaneous tissue response of isogenic mice to
calcium hydroxide-based pastes with chlorhexidine. Brazilian Dental Journal 20, 99106.
Silva LAB, Leonardo MR, Oliveira DSB et al. (2010) Histopathological evaluation of root canal filling materials for
primary teeth. Brazilian Dental Journal 21, 3845.
Sousa CJA, Loyola AM, Versiani MA, Biffi JCG, Oliveira RP,
Pascon EA (2004) A comparative histological evaluation
416
2014 International Endodontic Journal. Published by John Wiley & Sons Ltd