doi:10.1111/iej.

12328

Biocompatibility of root filling pastes used in

primary teeth

nior2, M. S. Rizzo3, R. D. Moura4, M. S. Moura5, M. D. M.

C. C. B. Lima1, A. M. Conde Ju
5 5
Lima & L. F. A. D. Moura
1
Postgraduate Programme in Dentistry, Federal University of Piau
ı (UFPI), Teresina; 2Department of Morphology, Federal
University of Piau
ı (UFPI), Teresina; 3Department of Clinic and Veterinary Surgery, Federal University of Piau
ı (UFPI),
Teresina; 4Postgraduate Programme in Pathology, University of S~ ao Paulo; and 5Department of Pathology
ao Paulo (USP), S~
and Dentistry Clinic, Federal University of Piau
ı (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.
Aim To evaluate the biocompatibility of two pastes
designed to fill the root canals of primary teeth.
Methodology A study group of 54 mice received
subcutaneous tissue implants of polyethylene tubes
containing CTZ or calcium hydroxide paste or, as a
negative control, empty tubes. Biocompatibility was
evaluated on days 7, 21 and 63, yielding a total of nine
groups of six animals each. Following the experimental
intervals, the implant areas were removed and sub-
jected to histologic processing. After the tissues were
stained with HE and Masson trichrome, two patholo-
gists performed a histologic analysis of the samples in a
blinded manner. Collagen fibre formation, tissue
thickness and inflammatory cell infiltration were
analysed qualitatively. Quantitative morphometry was
performed for the thickness, perimeter length and tissue
area of the region in direct contact with the open tube.
ANOVA with the Tukey post-test and Kruskal–Wallis
analysis followed by Dunn’s post-test, with significance
established as P < 0.05, were used for data analysis.
Results At 7 days, all groups had severe acute
inflammatory infiltrates. Inflammation was reduced at
21 days in the CTZ paste group. Mild chronic inflam-
matory infiltrates were observed after 63 days in the
CTZ and Ca(OH)2 paste groups; these groups also
showed a significant decrease in collagen fibre density
(P < 0.05), which was not observed in the control
group. The average tissue thickness, perimeter length
and area in contact with the tube decreased during
the experimental periods in all groups.
Conclusion The CTZ and calcium hydroxide pastes
demonstrated biocompatibility with subcutaneous tis-
sue in this experimental model.
Keywords: biocompatibility, calcium hydroxide,
primary teeth, root canal filling materials, subcutane-
ous tissue.
Received 26 August 2013; accepted 29 May 2014

canal disinfection in primary teeth with pulp necrosis

Introduction
The complex morphology of root canal systems and
the root resorption of primary molars are uncontrollable
factors that may hinder proper chemical–mechanical
Correspondence: L
ucia de F

atima Almeida de Deus Moura,
Department of Pathology and Dentistry Clinic, Federal Uni-
versity of Piau
ı, Rua Ang
elica, 1650, Bairro de F
atima,
CEP 64049-532, Teresina, Piau
ı, Brazil (Tel.: +55 86 3233
3050; e-mail: mouraiso@uol.com.br).
(Harini Priya et al. 2010, de Sousa et al. 2011).
These conditions may also impede the development
of permanent tooth follicles as well as the establish-
ment of accurate working length (Molander &
Dahl
en 2003, Barja-Fidalgo et al. 2011, Gondim
et al. 2012).
Most of the recommended techniques for filling root
canals of primary teeth propose the use of instrumenta-
tion and the chemo-mechanical preparation of the root
canal system (American Academy of Pediatric

© 2014 International Endodontic Journal. Published by John Wiley & Sons Ltd International Endodontic Journal 1
 Materials biocompatibility Lima et al.
Dentistry 2012). The use of calcium hydroxide (Ca
(OH)2) paste predominates over other techniques for the
pulp therapy of primary teeth. As a root filling material,
Ca(OH)2 paste demonstrates antibacterial action, bio-
compatibility and tissue repair capacity (Hauman &
Love 2003b, Mohammadi & Dummer 2011).
Alternative pulp therapies that have been developed
include those that recommend restricting treatment
to the pulp chamber and methods that may promote
the disinfection or sterilization of root canals with
necrotic pulps, as in the use of antibiotic-containing
pastes (Cappiello 1964, Takushige et al. 2004, Pinky
et al. 2011, Trairatvorakul & Detsomboonrat 2012).
Cappiello (1964) developed a paste for filling root
canals in primary teeth. The paste comprises chloram-
phenicol, tetracycline and zinc oxide with eugenol as a
vehicle and is currently known as CTZ paste (powder
base provided by a pharmacy in proportions of 1 : 1 : 2
chloramphenicol/tetracycline/zinc oxide, respectively).
This method obviates the need for canal instrumenta-
tion, thereby facilitating its use in young children and
users of public health services (Cappiello 1964).
Successful endodontic treatment of teeth with pulp
necrosis requires reducing or eliminating infection
within the root canal system. The medicaments used
in the pulp therapy of primary teeth should possess
antimicrobial activity and be biocompatible, particu-
larly given the proximity of contact in the furcation
and periapical regions (Silva et al. 2010, Barja-Fidalgo
et al. 2011).
Biocompatibility is the ability of a material that is
in contact with a tissue to promote an appropriate
biological response with minimal inflammatory reac-
tions. Methods that deploy substances in the subcuta-
neous tissues of laboratory animals are commonly
used to evaluate the biocompatibility of endodontic
materials (Hauman & Love 2003a, Viola et al. 2012).
The purpose of this study was to evaluate the bio-
compatibility of two pastes for the root canal treat-
ment of primary teeth (CTZ and calcium hydroxide
pastes). The null hypothesis was that there is no dif-
ference between the biocompatibility of CTZ and cal-
cium hydroxide pastes in mouse subcutaneous tissue.
Materials and methods
Ethical approval
The procedures used in this study were in accordance
with a protocol reviewed and approved by the Ethics
Committee on Animal Experimentation, Federal
2 International Endodontic Journal
University of Piau
ı (UFPI) (opinion No. 067/12) and
with the ethical guidelines for animal experimentation
advocated in the ‘European Communities Council
Directive’ of 24 November 1986 (86/609/EEC) regard-
ing recommendations for the care and use of labora-
tory animals.
This experimental trial began in November 2012
and was concluded in May 2013 upon euthanasia of
the last test animal.
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 vivar-
ium with standard rations and access to water ad libi-
tum. All animals were examined every 2 days and
monitored for local or systemic changes.
The animals were wormed with Baskenâ suspen-
sion (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 experi-
mental procedural.
Root canal pastes
The biocompatibility of the CTZ and calcium hydrox-
ide pastes was tested using an empty tube as a neg-
ative control. Histological analysis was performed on
the tissue samples for each material at three different
intervals: 7, 21 and 63 days. The tissue response
was analysed for nine groups with six animals per
group, which were distributed as follows: Group I –
six animals received a polyethylene tube containing
CTZ paste for 7 days; Group II – six animals received
a polyethylene tube containing CTZ paste for
21 days; Group III – six animals received a polyeth-
ylene tube containing CTZ paste for 63 days; Group
IV – six animals received a polyethylene tube contain-
ing calcium hydroxide paste for 7 days; Group V – six
animals received a polyethylene tube containing
calcium hydroxide paste for 21 days; Group VI – six
animals received a polyethylene tube containing cal-
cium hydroxide paste for 63 days; Group VII – six ani-
mals received an empty polyethylene tube for 7 days;
Group VIII – six animals received an empty polyethyl-
ene tube for 21 days; and Group IX – six animals
received an empty polyethylene tube for 63 days.
The micronized powder base for the CTZ paste was
provided in 250-mg capsules by a compounding
© 2014 International Endodontic Journal. Published by John Wiley & Sons Ltd

pharmacy (Teresina, PI, Brazil), and the paste com-
prised chloramphenicol 62.5 mg, tetracycline
62.5 mg and zinc oxide 125 mg with 0.1 mL of
eugenol vehicle (Biodin^ amica, Ibipor~a, PR, Brazil).
Calcium hydroxide (Ca(OH)2) powder (analytical
grade) (Biodin^amica) was provided in 250-mg cap-
sules and reconstituted in 0.2 mL of distilled water
(Isofarma, Eus
ebio, CE, Brazil).
To prepare the two pastes, the powders were dis-
pensed onto sterile glass plates and mixed with their
respective vehicles by a metal spatula at the time of
use.
Experimental design for subcutaneous implantation
Animals were pre-medicated with Tramadol at a dose
of 2 mg kg 1 (Tramadol hydrochloride 50 mg mL 1,
Hipolaborâ; Sabar
a, MG, Brazil) by deep intramuscu-
lar injection with a 1-mL syringe. After 10 minutes,
anaesthesia was induced using a combination of
xylazine (10 mg kg 1) (xylazine 2% Xilazin â; Syntec,
Cotia, SP, Brazil) and ketamine (80 mg kg 1) (keta-
mine hydrochloride 50 mg mL 1, Clortaminaâ; Bio-
Chimico, Itatiaia, RJ, Brazil) prepared in the same
syringe and administered by deep intramuscular injec-
tion. The back of the animal was shaved, and the sur-
gical region was disinfected with a 1% solution of
chlorhexidine gluconate.
In preparation for the dorsal implantation in each
animal, polyethylene tubes that were 1 mm in inter-
nal diameter (peripheral catheter type scalp No. 27
Solidorâ; Shijiazhuang, China) were sterilized in ethyl-
ene oxide and closed at one end with an Allis forceps.
The tubes were heated, sliced into 1-cm segments and
filled with the material to be tested. After a 0.5-cm lin-
ear incision was made at the caudal lumbar dorsum,
the soft tissue was dissected, and a tube was inserted
into the tissue. The skin incision was closed with 5.0
nylon sutures (Shalonâ Sutures; S~ ao Luis de Montes
Belo, GO, Brazil).
For the first 24 h following implantation, the ani-
mals received tramadol analgesia (2 mg kg 1), which
was administered by deep intramuscular injection
every 6 h.
At the end of 7, 21 or 63 days, animals from each
group were pre-medicated with tramadol (2 mg kg 1)
by deep intramuscular injection and euthanized with
an anaesthetic combination of xylazine (10 mg kg 1)
and ketamine (80 mg kg 1) prepared in the same
syringe and administered by deep intramuscular injec-
tion. The implant area, which comprised polyethylene
© 2014 International Endodontic Journal. Published by John Wiley & Sons Ltd
Lima et al. Materials biocompatibility
tubing, subcutaneous tissue and skin, was removed
and fixed in buffered 4% paraformaldehyde for 24 h.
Histologic analysis
Following fixation, the polyethylene tubes were
removed from the fixative, dehydrated in increasing
concentrations of alcohol, cleared in xylene, embed-
ded in paraffin and blocked. The blocks were cut
using a microtome (Leica RM 2245) to yield 5-lm-
thick longitudinal semi-serial sections. Tissues were
stained with haematoxylin and eosin (HE) and Mas-
son 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 patholo-
gists, 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) indi-
cated interexaminer reliability.
The tissues that contacted the open end of the
tubes were analysed and graded for the following his-
tologic findings: 1. formation of collagen fibres, 2. tis-
sue 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 lin-
ear 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
International Endodontic Journal 3
 Materials biocompatibility Lima et al.
follows: 0 (no cells), 1 (mild – 1 to 10 cells), 2
(moderate – 11 to 20 inflammatory cells) and 3
(intense – inflammatory cell concentration ≥21 cells)
(Silva et al. 2009, Queiroz et al. 2011).
Morphometric analysis
Morphometry, via quantitative analysis of the tissue
area that directly contacted the open part of the tube,
was performed using imaging software (Leica QWin –
Image Processing and Analysis Software Leica D-
1000, version 4.1; Cambridge, UK). Estimated values
for the tissue thickness (lm), perimeter (lm) and area
(lm2) were obtained by image analysis of the photo-
micrographs.
Statistical analysis
Data were tabulated and analysed using SPSS version
18.0 (SPSS Inc., Chicago, IL, USA). The Shapiro–Wilk
test was used to test the null hypothesis. ANOVA with
the Tukey post-test was used to verify differences
between the areas and perimeter thicknesses, and the
data grading scores were analysed by the Kruskal–
Wallis test followed by Dunn’s post-test, with signifi-
cance established as P < 0.05 (Fig. 1).
4 International Endodontic Journal
Results
Upon removal, all implants were covered with a visi-
ble, palpable and transparent fibrous capsule. There
were no signs of rejection in the specimens. The tubes
containing CTZ paste displayed progressive darkening
over time.
The results from the qualitative histologic analysis
of the collagen fibre formation, thickness and inflam-
matory infiltration of connective tissue adjacent to
the open ends of the polyethylene tubes are presented
in Table 1 and Figs 2, 3 and 4.
When the same treatment was compared for differ-
ent experimental periods (7, 21 and 63 days), signifi-
cant differences were observed in the reduction of
collagen fibre formation (P < 0.05) and inflammatory
cell infiltration (P < 0.05) for the specimens that were
treated with calcium hydroxide paste. In the group
implanted with an empty tube, increased formation of
collagen fibres (P < 0.05) was observed, and no sig-
nificant difference was observed with the CTZ paste at
different times (P > 0.05).
The mean values with standard deviations for the
thickness (lm), perimeter (lm) and area (lm2) of the
reactive tissue in direct contact with the open tubes
are presented in Table 2.
Figure 1 Study flowchart.
© 2014 International Endodontic Journal. Published by John Wiley & Sons Ltd
 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

Experimental period (days)

a
Material tested 7 21b 63c

Formation of collagen fibres (Fig. 2a–j)

Empty tube Mild to moderate (Fig. 2a) Individual collagen fibres and alternating extracellular Collagen fibres admixed with extracellular matrix
matrix without linear features or wavy fibres (Fig. 2b) without any typical linear or wavy forms; individual
collagen fibres could not be distinguished (Fig. 2c)
Calcium hydroxide paste Mild collagen fibre density (Fig. 2d) Mild collagen fibre density (Fig. 2e) Moderate (Fig. 2f)
CTZ paste Mild collagen fibre density (Fig. 2g) Individual collagen fibres observed as in normal Mild to moderate (Fig. 2i)
connective tissue (Fig. 2h)

© 2014 International Endodontic Journal. Published by John Wiley & Sons Ltd
Tissue thickness (Fig. 3a–j)
Empty tube Moderately increased (Fig. 3a) Severely increased (Fig. 3b) Moderately increased (Fig. 3c)
Calcium hydroxide paste Mild to moderately increased (Fig. 3d) Slightly increased (Fig. 3e) Mild to moderately increased (Fig. 3f)
CTZ paste Severely increased (Fig. 3g) Moderately increased, marked presence of fibroblasts Slightly increased (Fig. 3i)
(Fig. 3h)
Inflammatory infiltrate (Fig. 4a–j)
Empty tube Severe; presence of lymphocytes, Severe; presence of macrophages, lymphocytes, Severe; mononuclear and occasional neutrophils
macrophages, neutrophils, and newly neutrophils and newly formed vessels (Fig. 4b) present (Fig. 4c)
formed vessels (Fig. 4a)
Calcium hydroxide paste Moderate; mixed macrophages and Moderate; predominantly mononuclear, with the Mild; predominantly mononuclear with occasional
neutrophils present (Fig. 4d) possible presence of neutrophils (Fig. 4e) neutrophils (Fig. 4f)
CTZ paste Severe; predominantly mononuclear, Moderate; with macrophages, neutrophils and Mild; predominantly mononuclear with occasional
with neutrophils present (Fig. 4g) neovascularization (Fig. 4h) neutrophils (Fig. 4i)

P: Kruskal–Wallis.
a
There was no statistically significant difference between groups (P > 0.05).
b
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).
Lima et al. Materials biocompatibility

5 International Endodontic Journal

 Materials biocompatibility Lima et al.

Empty tube Ca(OH)2 paste CTZ paste

(a) (b) (c)

7th Day

(d) (e) (f)

21st Day

(g) (h) (i)

63rd Day

(j) Mild Moderate Severe

100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%
y

ay

ay

ay

y
da

da

da

da

da

da
td

td

td
th

th

th

d
1s

1s

1s
3r

3r

3r
-7

-7

-7
-2

-2

-2
-6

-6

-6
TZ

)2

be
TZ

)2

be
TZ

)2

be
H

tu
C

H
O

tu
C

tu
C

O
a(

y
a(

y
pt
a(

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pt

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Em

Em

Figure 2 (a–i) Photomicrographs stained with Masson trichrome showing the formation of collagen fibres in the connective tis-
sue 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 able, for primary teeth with necrotic pulps (Silva et al.
2010, Barja-Fidalgo et al. 2011, Queiroz et al. 2011).
A great challenge facing Paediatric Dentistry is to
CTZ paste has great potential as a treatment option
develop biomaterials that possess antimicrobial prop-
for endodontically compromised primary teeth in
erties, in addition to being biocompatible and resorb-

6 International Endodontic Journal © 2014 International Endodontic Journal. Published by John Wiley & Sons Ltd
 Lima et al. Materials biocompatibility

Empty tube Ca(OH)2 paste CTZ paste

(a) (b) (c)

7th Day

(d) (e) (f)

21st Day

(g) (h) (i)

63rd Day

(j) Slighty increased Moderately increased Intensely increased

100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%
y

ay

ay

ay

y
da

da

da

da

da

da
td

td

td
th

th

th

d
1s

1s

1s
3r

3r

3r
-7

-7

-7
-2

-2

-2
-6

-6

-6
TZ

)2

be
TZ

)2

be
TZ

)2

be
H

tu
C

H
O

tu
C

tu
C

O
a(

y
a(

y
pt
a(

y
C

pt

pt
C

Em

Em

Em

Figure 3 (a–i) 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 International Endodontic Journal 7
 Materials biocompatibility Lima et al.

Empty tube Ca(OH)2 paste CTZ paste

(a) (b) (c)

7th Day

(d) (e) (f)

21st Day

(g) (h) (i)

63rd Day

(j) Mild Moderate Intense

100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%
y

ay

ay

ay

y
da

da

da

da

da

da
td

td

td
th

th

th

d
1s

1s

1s
3r

3r

3r
-7

-7

-7
-2

-2

-2
-6

-6

-6
TZ

)2

be
TZ

)2

be
TZ

)2

be
H

tu
C

H
O

tu
C

tu
C

O
a(

y
a(

y
pt
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y
C

pt

pt
C

Em

Em

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Figure 4 (a–i) 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 inflam-
matory reaction. (1) Space occupied by the tubing, (2) subcutaneous tissue, (*) neovascularization, (f) fibroblasts, (l) lympho-
cytes, (m) macrophages and (n) neutrophils.

8 International Endodontic Journal © 2014 International Endodontic Journal. Published by John Wiley & Sons Ltd
 Lima et al. Materials biocompatibility

Table 2 Mean values with standard deviations for thickness (lm), perimeter (lm) and area (lm2) of tissue reaction

Day 7 Day 21 Day 63

M (SD) M (SD) M (SD) P*

Thickness (lm)
CTZ 135.9 (59.0) 121.4 (69.6) 88.8 (41.0) 0.376
Ca(OH)2 84.7 (17.2) 92.0 (28.5) 76.4 (26.1) 0.552
Control 108.3 (41.6) 87.1 (22.6) 70.4 (31.8) 0.121
P 0.091 0.389 0.634
Perimeter (lm)
CTZ 1046.1 (252.3) 950.0 (211.8) 941.2 (179.0) 0.656
Ca(OH)2 884.4 (253.8) 894.3 (143.0) 916.7 (173.4) 0.958
Control 1079.6 (73.1) 880.4 (115.2) 763.8 (190.9) 0.004
P 0.261 0.738 0.218
Area (lm2)
CTZ 53554.9 (16442.2) 47344.8 (27953.0) 33816.3 (13998.1) 0.261
Ca(OH)2 32132.6 (11382.7) 32404.8 (9832.4) 24996.0 (1335.6) 0.473
Control 49362.2 (9999.3) 32561.9 (12713.6) 23483.9 (13930.3) 0.008
P 0.027 0.308 0.396

M = mean; SD = standard deviation.

P: ANOVA test, applied between the materials in same experimental period.
*
P: ANOVA test, applied for each material between the different experimental periods.

young children with behavioural problems. Another
application for CTZ paste is for users of public
health services because of the large patient loads in
such facilities and the ease with which general
practitioners can perform these procedures due to the
technical simplicity, excellent clinical outcomes and
cost-effectiveness (Cappiello 1964, Bruno et al. 2007).
There is a lack of scientific literature on the use of
CTZ paste in clinical settings. This study is part of a
macro-development project that encompasses research
on the performance and antimicrobial action of CTZ
paste and its components.
Biocompatibility is the ability of an applied material
or substance to trigger the intended response in the
host. That is, although a biocompatible material may
not be inert, the reaction caused by a test material
must not pose an unacceptable physiological hazard
when compared with other scientifically approved
materials (Wataha 2001, Peters 2013).
Three tests are recommended for evaluating the bio-
compatibility of endodontic materials: initial, second-
ary and application. The method used in this study
was a sub-type that is used to evaluate biological tis-
sue responses around the test material and to compare
such results to biocompatible controls with established
scientific acceptability (Olsson et al. 1981, ISO 7405
1996, ISO 10993-6 2007a, ISO 10993-12 2007b,
Hauman & Love 2003a, Silva et al. 2009, Pereira
et al. 2012). Subcutaneous implants, which were
introduced by Torneck (1966), are used most com-
monly in dentistry.
Compatibility with living tissue in the periapical or
interradicular region is one of the most important
properties for materials used in pulp therapy of pri-
mary teeth due to the occurrence of root resorption
and the presence of anatomical variations that may
jeopardize periodontal and periapical tissues, which
may in turn endanger the development of permanent
teeth (Lacativa et al. 2012).
Polyethylene tube implants are used experimentally
to simulate the relationship between the tooth apex
and periapical tissues because the material within the
tubes diffuses in a restricted area (Olsson et al. 1981,
International Organization for Standardization 2007a,
Silva et al. 2009, Khashaba et al. 2011, Marques
et al. 2011, Mutoh & Tani-Ishii 2011).
In this study, the groups in which empty polyethyl-
ene tubes were implanted had higher scores in the
qualitative histologic analysis for fibroplasia and had
significant differences in the perimeter and area of the
tissue regions in direct contact with the open tube.
This result was expected because no materials were
introduced that could induce an acute inflammatory
response or reaction. However, other studies have
reported responses to empty tubes, albeit less intense
than those observed for the experimental parameters
analysed at the time (Silva et al. 2009, Queiroz et al.
2011).
Significant reductions were observed in the density
of collagen fibres and the inflammatory reaction when
testing the calcium hydroxide paste for different
experimental periods. Calcium hydroxide is widely

© 2014 International Endodontic Journal. Published by John Wiley & Sons Ltd International Endodontic Journal
Materials biocompatibility Lima et al.
used in dentistry due to its biocompatible properties
and antimicrobial, anti-inflammatory and reparative
activities (Hauman & Love 2003a,b, Mohammadi &
Dummer 2011, de Sousa et al. 2011, Gondim et al.
2012). The biocompatibility results obtained in this
study for calcium hydroxide paste are consistent with
those reported by several authors (Holland et al.
2001, Semenoff et al. 2008, Silva et al. 2010, Andolf-
atto et al. 2012).
In the present study, the tubes containing CTZ
paste displayed progressive darkening over time,
which may have been caused by the tetracycline
component. The CTZ paste was associated with an
initial intense inflammatory response during the first
days of the experiment. At the end of the experiment,
a mild chronic inflammatory process was observed,
with a qualitative and quantitative decrease in the
density of collagen fibres and tissue thickness. This
finding suggests that CTZ was biocompatible and
showed therapeutic properties. In a previous study,
histopathologic analysis of pulpotomy performed in
dogs’ teeth treated with CTZ paste demonstrated the
occurrence of an initial intense inflammatory process
in the coronal pulp with reduced inflammation at the
end of the experiment (Bruno et al. 2007). However,
the literature lacks studies on the biocompatibility of
CTZ paste; thus, it was not possible to compare the
results obtained in this study with those of other pub-
lished studies.
The normal reaction to any tissue injury tends to
manifest as an acute neutrophilic inflammatory infil-
trate (Serhan 2010). Given that acute histopathologic
tissue behaviour is similar across all groups, studies
that aim to test the biocompatibility of a drug that
may cause a tissue reaction should be focused on
chronic inflammatory reactions. In this study, the
pastes and the control group showed variable chronic
inflammatory processes at different experimental
times.
A histologic hallmark of successful pulp therapy is
the development of reparative tissue (Torneck 1966).
In the present study, a significant difference in the den-
sity of collagen fibres was observed between the two
pastes at the end of the experiment. The difference was
characterized by a decrease in the collagen fibres in
specimens containing either the CTZ or the calcium
hydroxide paste compared with the negative control.
This finding is important because the two pastes
behaved in manner that favours tissue repair. The
results are consistent with other studies that reported
similar methodologies (Sousa et al. 2004, Batista et al.
International Endodontic Journal
2007, Silva et al. 2010, Marques et al. 2011, Gomes-
Filho 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 iodo-
form 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 anti-
microbial 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 instrumenta-
tion and chemical–mechanical systems for the root
canals of primary teeth (American Academy of Pedi-
atric Dentistry 2012).
The limitations of this study are inherent to pre-
clinical experimental studies. Although the results
presented here do not reflect a complete analysis of
the reactions that occur in the pulp, periapical or in-
terradicular 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 pro-
ject 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

participated in the analysis and interpretation of
data and wrote the article. RDM participated in the
analysis of data. MSM revised the article. MDML
participated in the study preparation and revised
the article. LFADM conceived the study, participated
in the study preparation and coordination, and
wrote the article. All of the authors read and
approved the final manuscript.
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