International Scholarly Research Network
ISRN Dentistry
Volume 2012, Article ID 904963, 6 pages
doi:10.5402/2012/904963

Research Article

Biocompatibility of Intracanal Medications Based on
Calcium Hydroxide

Carolina Andolfatto,1 Guilherme Ferreira da Silva,1 Ana Livia Gomes Cornélio,1

Juliane Maria Guerreiro-Tanomaru,1 Mario Tanomaru-Filho,1 Gisele Faria,1

Idomeo Bonetti-Filho,1 and Paulo Sérgio Cerri2

1 Department of Restorative Dentistry, Araraquara Dental School, Universidade Estadual Paulista (UNESP),
14801-903 Araraquara, SP, Brazil

2 Laboratory of Histology and Embryology, Department of Morphology, Araraquara Dental School,
Universidade Estadual Paulista (UNESP), 14801-903 Araraquara, SP, Brazil

Correspondence should be addressed to Paulo Sérgio Cerri, pcerri@foar.unesp.br

Received 6 November 2012; Accepted 27 November 2012

Academic Editors: H. S. Cardash, J. H. Jeng, and G. Mount

Copyright © 2012 Carolina Andolfatto et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.

Objective. The aim of this study was to evaluate the rat subcutaneous tissue reaction to calcium hydroxide-based intracanal
medicaments, UltraCal XS (calcium hydroxide, barium sulphate, aqueous matrix), Hydropast (calcium hydroxide, barium
sulphate, and propyleneglycol), and Calen (Calcium hydroxide, zinc oxide, colophony, and polyethyleneglycol), used as a control.
Methods. Forty-eight rats (Rattus Norvegicus Holtzman) were distributed in three groups: Calen, UltraCal XS, and Hydropast.
Polyethylene tubes filled with one of the medicaments were implanted in the dorsal subcutaneous. After 7 and 30 days, the implants
were removed and the specimens were fixed and embedded in paraffin. Morphological and quantitative analyses were carried out
in the HE-stained sections. The numerical density of inflammatory cells in the capsule was evaluated and statistical analyses were
performed (P ≤ 0.05). Results. At 7 days, all materials induced an inflammatory reaction in the subcutaneous tissue adjacent to
the implants. In all groups, a significant reduction in the number of inflammatory cells and giant cells was verified in the period
of 30 days. Conclusion. These results indicate that the calcium hydroxide-based medicaments evaluated present biocompatibility
similar to Calen.

1. Introduction

The success of endodontic treatment of teeth with periapical
lesion depends on the reduction or elimination of the
intraradicular infection [1, 2]. The root canal mechanical
preparation is not enough to eliminate this infection because
many microorganisms are not only in the main root canal,
but also disseminated throughout the root canal system.
Therefore, the use of an intracanal dressing to eliminate the
microorganisms is indicated [3–6].

Antimicrobial activity and biocompatibility are charac-
teristics that an ideal intracanal dressing has to show [7].
Calcium hydroxide [Ca(OH)2] has been widely used for its
biological and antimicrobial activity [4, 8], ability to dissolve
organic tissue [9], and capacity to inactivate bacterial

endotoxin [10, 11]. Despite these properties, the Ca(OH)2

has no satisfactory physical properties such as radiopacity to
visualize on dental radiographs and flow capacity to facilitate
its insertion in the root canal [12, 13]. For this reason, it
needs the incorporation of a radiopacifying agent and a
vehicle to improve these characteristics [8, 14].

Although the Ca(OH)2 shows an excellent biocom-
patibility, the addition of other substances can affect its
biological properties [8, 12]. In the last years, it has been
marketed the calcium hydroxide-based medicaments such
as UltraCal XS (Ultradent Products, Inc.) and Hydropast
(Biodinâmica Quı́mica e Farmacêutica Ltda., Brazil). Ultra-
Cal XS is basically composed by 35% of calcium hydrox-
ide, a radiopacifier, and a vehicle which does have not
the proportions related by the manufacturer. Studies have



2 ISRN Dentistry

demonstrated that UltraCal XS has a high pH value [15]
and an effective antimicrobial activity against common
endodontic bacteria of teeth with pulp necrosis [16]. On the
other hand, Brazilian paste Hydropast, composed by 38% of
calcium hydroxide, barium oxide, as a radiopacifying agent,
and propilenoglycol as vehicle, is a recent material, and,
therefore, until now, there are no studies of its biological
properties.

Considering the recommendation of International Orga-
nization for Standardization [17], it is necessary in vitro
and/or in vivo studies for evaluation of the biocompatibility
of these new materials. Thus, the purpose of this study was to
evaluate the tissue reaction of these calcium hydroxide-based
medicaments in rat subcutaneous.

2. Materials and Methods

2.1. Animals and Experimental Proceedings. This study was
performed in accordance with the principles of animal care
on animal experiments. The research protocol was autho-
rized by the Ethical Committee for Animal Research of the
São Paulo State University, Brazil (Dental School, UNESP,
Araraquara).

Forty-eight male Holtzman rats (Rattus norvegicus albi-
nus), weighing 250 ± 10 g, were kept in individual stainless
steel cages under 12 : 12 light-dark cycle at controlled
temperature (23± 2◦C) and humidity (55 + 10%), with food
and water provided ad libitum. The animals were randomly
distributed into three groups (n = 16) on the basis of the
intracanal medicaments analysed, Calen group, Calen paste
(S. S. White Artigos Dentários Ltda., Rio de Janeiro, RJ,
Brazil), used as control group; UltraCal group, UltraCal XS
paste (Ultradent Products, Inc., South Jordan, UT, USA),
and Hydropast group, Hydropast (Biodinâmica Quı́mica e
Farmacêutica Ltda., Ibipora, PR, Brazil). The composition of
these materials is described at Table 1.

The polyethylene tubes (Embramed Ind. Com. Ltda.,
São Paulo, SP, Brazil) with 10.0 mm length and 1.5 mm
diameter previously sterilized with ethylene oxide were filled
with Calen, UltraCal XS paste, or Hydropast intracanal
medicaments.

The animals were anaesthetized with an intraperitoneal
injection containing 80 mg/Kg of body weight of ketamine
(União Quı́mica Farmacêutica Nacional S/A-Brazil) and
4 mg/Kg of body weight of xylazine (Virbac do Brasil
Indústria e Comércio Ltda., Brazil). After shaved and
disinfection with 5% iodine solution, a 20 mm-long incision
in a head-to-tail orientation was made using a scalpel (no.
15, Fibra Cirúrgica, Joinvile, SC, Brazil) in the dorsal skin.
Subsequently, the polyethylene tube containing Calen paste,
UltraCal XS, or Hydropast was immediately implanted into
the dorsal subcutaneous connective tissue. After implan-
tation, the skin was closed with 4.0 silk suture (Vicryl;
Johnson & Johnson: Ethicon Inc., New Brunswick, NJ, USA).
One polyethylene tube filled with an intracanal paste was
implanted in each animal and left for periods of 7 and 30
days.

Table 1: Tested materials and composition.

Composition Manufacturer

Calen

49.77% calcium hydroxide,
50.23% of zinc oxide,
colophony, and
polyethylene glycol 400

S. S. White Artigos
Dentários Ltda., Rio de
Janeiro, RJ, Brazil

UltraCal XS
35% calcium hydroxide
and a radiopacifier

Ultradent Products,
Inc., South Jordan, UT,
USA

Hydropast
38% of calcium hydroxide,
62% of barium oxide, and
propilenoglicol

Biodinamica Quimica e
Farmaceutica Ltda.,
Ibipora, PR, Brazil

After experimental periods, the animals were killed by
overdose of anesthetic solution, and the tubes were removed
with surrounding connective tissue and prepared for paraffin
embedding.

2.2. Histological Procedures and Analysis. The specimens
containing the implanted polyethylene tubes were fixed
in 4% formaldehyde (prepared from paraformaldehyde)
buffered at pH 7.2 with 0.1 M sodium phosphate at room
temperature for 48 hours. Subsequently, the specimens were
dehydrated and embedded in paraffin. Serial 6 µm-thick
sections were made parallel to the tube long axis and stained
with hematoxylin and eosin (H&E) for morphological and
morphometric analyses. The morphological analysis of the
capsule in contact with the material on the opening of the
tube was performed considering the following parameters:
presence of inflammatory process, main cells (inflammatory
cells or fibroblasts) present in the capsule, presence of
multinucleated giant cells, and presence of collagen fibers.

The numerical density of inflammatory mononucleated
cells and multinucleated giant cells was undertaken using
a light microscope (BX51, Olympus, Tokyo, Japan) and an
image analysis system (Image Pro-Express 6.0, Olympus).
Three H&E-stained sections per animal were selected at
intervals of at least 100 µm; in each section, a standardized
field of 0.09 mm2 of the connective tissue adjacent to the
opening of the tube implanted was analyzed, totaling
0.27 mm2 per animal. In each area, the total number of
inflammatory cells was counted using the image analysis sys-
tem at ×40 magnification; in each animal, the total number
of inflammatory cells was divided by total area, and, then,
the number of inflammatory cells/mm2 was obtained. The
differences between the groups were statistically analyzed
using SigmaStat 2.0 software (Jandel Scientific, Sausalito, CA,
USA); the data were submitted to ANOVA and Tukey test.
The significance level accepted was P ≤ 0.05.

3. Results

3.1. Morphological and Quantitative Analyses. After 7 days
of implantation, the capsule adjacent to the opening of the
tubes filled with Calen, UltraCal XS, and Hydropast paste
showed numerous inflammatory cells, mainly lymphocytes
and macrophages. Usually, the inflammatory infiltration was



ISRN Dentistry 3

I

BV

BV

CG

CG

IC

IC

I

Fb CF

Fb

CF

BV
BV

(a)

(b)

(c)

Figure 1: Light micrographs of sections showing portions of capsules adjacent to the opening of the tubes (I) filled with Calen paste after 7
(a) and 30 days (b and c) of implantation in the subcutaneous. In (a), numerous inflammatory cells (ICs) are observed in the inner portion
of the capsule adjacent to the tube opening (I). The inset of the outlined area shows multinucleated giant cells (GCs) in close juxtaposition
to the material implanted. BV, blood vessels. ×110; inset: ×250 (b) shows the capsule exhibits several fibroblasts (Fb) dispersed among
the collagen fiber bundles (CF) ×130. In (c), outlined area in (b), shows some inflammatory cells (arrows), mainly lymphocytes, situated
adjacent to the blood vessels (BV) ×250.

I

BV

BV

I

IC

IC

CG

BV

IC

(a)

I

BV

BV

FC

Fb

Fb

Fb

(b)

Figure 2: Light micrographs of sections showing portions of capsules adjacent to the opening of the tubes (I) filled with UltraCal XS paste
after 7 (a) and 30 days (b) of implantation in the subcutaneous. In (a), The capsule exhibits several inflammatory cells (IC) and blood vessels
(BV). Note that dense masses of inflammatory cells (ICs) are observed in the inner portion of the capsule, adjacent to the tube opening (I).
The inset, outlined area, shows multinucleated giant cells (GCs). ×120; inset: ×230. (b) The capsule contains several fibroblasts (Fbs) and
numerous blood vessels (BV). Inflammatory cells (arrows), lymphocytes and macrophages are situated mainly in the inner portion of the
capsule and next to the blood vessels ×150.

evident in the innermost portion of the capsule, that is, in
close juxtaposition to the materials (Figures 1(a), 2(a) and
3(a)). According to Table 2, no significant difference was
verified in the numerical density of inflammatory cells
among the groups. Multinucleated giant cells were also
observed in the capsules formed in all groups (Figures
1(a), 2(a), and 3(a)). However, in the Hydropast group the
number of multinucleated giant cells was significantly higher

in comparison to other groups; on the other hand, the
capsule of the Calen group exhibited the lower number of
multinucleated giant cells (Table 2).

A significant decrease in the number of inflammatory
cells and multinucleated giant cells was verified from 7 to
30 days, in all groups (Figures 1(b), 2(b), and 3(b); Table 2).
In the period of 30 days, the capsule adjacent to Calen paste
was formed by a dense connective tissue exhibiting typical



4 ISRN Dentistry

I

N

IC

IC

IC

BV

CG
CG

(a)

I

CF

BV

CF

BV

FC
BV

(b)

Figure 3: Light micrographs of sections showing portions of capsules adjacent to the opening of the tubes (I) filled with Hydropast after 7
(a) and 30 days (b) of implantation in the subcutaneous. In (a), numerous inflammatory cells (ICs) and multinucleated giant cells (GCs) are
present by thorough capsule. BVs, blood vessels ×120. (b) The capsule contains predominantly inflammatory cells (arrows); scarce collagen
fibers (FC) distributed irregularly are observed in the capsule. BVs, blood vessels ×120.

Table 2: Number of inflammatory cells and giant cells/mm2 of the capsule adjacent to the implants in the subcutaneous.

Inflammatory cells Giant cells

7 days 30 days 7 days 30 days

Calen 2,795.10 ± 939.90a,1 960.64 ± 433.22a,2 4.14 ± 0.90a,1 0.92 ± 0.18a,2

UltraCal XS 2,397.66 ± 613.14a,1 975.99 ± 227.71a,2 6.00 ± 1.03b,1 2.77 ± 1.05b,2

Hydropast 2,248.10 ± 315.41a,1 1,407.82 ± 870.89a,2 8.77 ± 1.46c,1 1.37 ± 0.87c,2

Values are expressed as mean ± standard deviation.
Equal letters indicate no statistically significant difference (P > 0.05) between the materials in the same experimental period.
Different numbers indicate difference statistically significant (P ≤ 0.05) of each material in the different experimental periods.

bundles of collagen fibers between fibroblasts; lymphocytes
and plasma cells were mainly present next to blood vessels
(Figure 1(b)). In the UltraCal group, the capsule contained
several fibroblasts among the inflammatory cells; usually,
bundles of collagen fibers were only present in the outermost
portion of the capsule (Figure 2(b)). The connective tissue
of the capsule of the Hydropast exhibited several cells and
scarce collagen fibers (Figure 3(b)). Although no significant
difference was found in the number of inflammatory cells
between the groups, the mean of the numerical density of
inflammatory cells in the Hydropast group was around
1,400 cells/mm2, whereas in the other groups was around
965 cells/mm2. Moreover, in the Calen group the number
of multinucleated giant cells was significantly lower in
comparison to UltraCal and Hydropast groups (Table 2).

4. Discussion

Implantation in the subcutaneous connective tissues of
experimental animals has been extensively used to evaluate
the biocompatibility of endodontic materials [18, 19]. Our
findings indicate that UltraCal XS and Hydropast exhibit
biological behavior similar to Calen (control group). At 7
days, an intense inflammatory reaction and foci of coag-
ulative necrosis were seen in the adjacent capsule to the
implanted materials. After 30 days, significant reduction

in the inflammatory process was verified in all analyzed
groups; usually, the capsule formed juxtaposed to the Calen
paste exhibited inflammatory cells among typical bundles of
collagen fibers and fibroblasts. The inflammatory reaction
observed in the period of the 7 days may be attributed to the
superficial necrosis promoted by calcium hydroxide-based
materials [20]. Calcium hydroxide has an alkaline pH [21]
and, when in contact with the connective tissue, induces the
formation of a coagulative necrosis zone [22]. Coagulative
necrosis refers to a spectrum of morphological changes in
living tissue resulting from the action of enzymes on lethally
injured cells. The mass of necrotic cells is characterized by
preservation of the basic outline of the coagulated cells for
a span of at least some days. As necrotic cells are unable to
maintain membrane integrity, their contents leak out and
elicit an inflammatory response that removes the cellular
debris by phagocytosis, followed by healing [20].

The vehicles mixed with calcium hydroxide powder play
an important role in the ionic dissociation process and so
in the disinfection of the root canal and biocompatibility
[8, 23]. There are three main types of vehicle: water-soluble
substances, viscous, and oil-based [8]. In the present study,
the vehicles of the different medicaments did not interfere
in their tissue reaction since the results were similar. In the
periods of 7 and 30 days, the number of giant cells was
significantly reduced in the Calen group in comparison to
UltraCal XS and Hydropast. At 30 days, the capsule adjacent



ISRN Dentistry 5

to Hydropast showed a significantly lower number of giant
cells than the UltraCal XS. The giant cells are derived from
the fusion of 20 or more monocytes/macrophages and are
formed for removing exogenous agents [24]. In the period
of 30 days, the high number of giant cells verified in the
UltraCal XS group suggests that this material may release
more irritant substances than the Hydropast and Calen.

In Calen paste, the calcium hydroxide is mixed to a
viscous vehicle, polyethylene glycol 400, one of the most
commonly used vehicles in root canal medicaments with low
toxicity, high solubility in aqueous solutions, low immuno-
genicity and antigenicity [25], and antibacterial activity [5].
This vehicle releases calcium and hydroxyl ions more slowly
and for longer periods than water-soluble and oil-based
materials [26]. Because the releasing of H+, the polyethylene
glycol 400 neutralizes the OH− released by calcium hydroxide
and, thereby, reduces the superficial necrosis area [27].

The propylene glycol is used as vehicle in the Hydropast;
the vehicle used in the Hydropast is classified as a viscous
vehicle with high molecular weight and, as well as polyethy-
lene glycol 400, prolong the action of calcium hydroxide in
the root canal system [26]. Moreover, propylene glycol shows
low toxicity and antimicrobial properties [28, 29]. It was
demonstrated that the addition of propylene glycol may not
interfere in the biocompatibility of MTA in rat subcutaneous
tissue [30].

Our results also suggest that the different radiopacifying
agents of the pastes did not interfere in the tissue reaction.
The endodontic materials should present sufficient radiopac-
ity to be distinguished from adjacent anatomical structures,
such as bone and teeth [31]. Zinc oxide, barium sulfate,
bismuth oxide, and other components with iodine and
bromine are some examples of radiopacifiers [12, 14, 27, 32].

The zinc oxide of Calen paste does not affect the
biological properties of calcium hydroxide [27, 33]. Barium
sulphate, radiopacifying agent of Hydropast, is also bio-
compatible because its cause no detrimental effect in rat
subcutaneous tissue [34] or in periapical tissue in association
with calcium hydroxide [14]; barium oxide in association
with the Norian, an skeletal repair system (SRS), in tibiae
defects of rats, maintains the properties of biocompatibility
and osteoconductive materials of the SRS [35]. Although the
manufacturer does not inform the radiopacifying agent of
the UltraCal XS, our results demonstrated that this material
has a good biological behavior.

Considering the methodology used in the present study,
our findings indicate that UltraCal XS and Hydropast are
biocompatible in subcutaneous tissue of rats.

Conflict of Interests

The authors declared that they have no any conflict of inte-
rests related to this study.

Acknowledgments

The authors thank Mr. Pedro Sérgio Simões for technical
support. This paper was supported by Coordenação de

Aperfeiçoamento de Pessoal de Nı́vel Superior (CAPES)
and Conselho Nacional de Desenvolvimento Cientı́fico e
Tecnológico (CNPq), Brazil.

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