Original Research

Comparative in vitro
Volume 23
2024
e243355

analysis of the antifungal

activity of different
calcium silicate-based
endodontic sealers
Luiz Felipe Nunes Moreira1 , Fernando Peña-Bengoa2* ,
Sven Eric Niklander3 , Carlos Eduardo da Silveira
Bueno4 , Alexandre Sigrist de Martin4 , Daniel
Guimarães Pedro Rocha4,5

1
Faculty of Dentistry, Vale do
Rio Doce University, Governador
Valadares, Minas Gerais, Brazil
Aim: This study aimed to perform an in vitro comparative
2
Departament of Endodontics,
Dentistry Faculty, Andres Bello analysis of the antifungal activity of different calcium silicate-based
University, Viña del Mar, Chile. endodontic sealers against three fungal species. Methods:
3
Unit of Oral Pathology and The antifungal properties of three calcium silicate-based
Medicine, Dentistry Faculty, Andres sealers were tested: Bio-C Sealer, Cambiar a Sealer Plus BC,
Bello University, Viña del Mar, Chile.
and MTA-Fillapex. Two commonly used sealers were used as
4
Department of Endodontics,
controls: AH Plus and Endomethasone. An agar diffusion test
Faculty of Dentistry, São Leopoldo
Mandic College of Campinas, was performed to analyze the antifungal activity of the sealers
Campinas, São Paulo, Brazil. against Candida albicans, Candida glabrata, Candida tropicalis,
5
Department of Endodontics, and a mixed microbial culture medium. The results were
Faculty of Dentistry, Pontifical
analyzed using ANOVA (p <0.05). Results: Endomethasone
Catholic University of Campinas,
Campinas, São Paulo, Brazil. exhibited the highest inhibition against all strains examined,
maintaining a consistent level of inhibition throughout 7 days.
Corresponding author: MTA-Fillapex demonstrated the best performance among
Fernando Peña-Bengoa the calcium silicate-based sealers for the three fungal species
Department of Endodontics,
Universidad Andres Bello.
(p < 0.05), maintaining stable values over the 7 days, surpassing
Quillota 980, torre E, 2531015, that of Endomethasone. Nevertheless, MTA-Fillapex only exhibited
Viña del Mar, Chile. antimicrobial effect against the mixed culture for the first
Phone number: 569 85481196
ORCID:0000-0001-9389-0468 24 hours, and no antimicrobial activity was observed at 48 hours,
E-mail: [email protected], being surpassed by all tested sealers (p < 0.05). Conclusion:
[email protected]
Of all silicate-based sealers tested, only MTA-Fillapex exhibited
Editor: Dr. Altair A. Del Bel Cury promising antifungal activity. Nevertheless, care must be taken
when extrapolating these results, as MTA-Fillapex exhibited poor
Received: May 4, 2023 antimicrobial activity when tested in mixed microbial cultures.
Accepted: November 30, 2023
Keywords: Endodontics. Bacteria. Candida albicans. Root canal
filling materials.

http://dx.doi.org/10.20396/bjos.v23i00.8673355 Braz J Oral Sci. 2024;23:e243355 1

 Moreira et al.

Introduction

Microbiological factors are the main reason for endodontic failure1,2. The complexity
of endodontic microbiota and their ability to colonize irregularities in the root canal
system (RCS) pose challenges for clinicians, particularly in areas where instrumen-
tation, irrigation, or intracanal medication may be difficult for anatomical reasons3.
Although endodontic treatments have a high success rate, most failures are associ-
ated with persistent or secondary infections3,4.
Bacteria are the main cause of endodontic infections; however, other microorgan-
isms, such as fungi may be involved3. The proportion of Candida spp. in infected
root canals ranges from 0.5% to 55%, with C. albicans being the most frequently
involved species5. Other species, such as C. tropicalis, which is the most common
non-albicans-candida species found in the oral cavity of healthy people6, may also
be involved in apical periodontitis7. Fungi in endodontic infections are commonly
associated with secondary or persistent infections; however, they may also be pres-
ent in primary infections1,8. Although fewer in number compared to bacteria, they are
aggressive enough to maintain periapical disease because of their ability to adapt to
the environment, adhere to surfaces, produce hydrological enzymes, undergo mor-
phological transitions, form biofilms, and resist calcium hydroxide, the most com-
monly used intracanal medication9.
Calcium silicate-based endodontic sealers have become popular in recent years
because of their excellent properties and simplicity of use10. Their biocompatibil-
ity, high pH, and low cytotoxicity are just some of their favorable properties11. In
addition, silicate-based endodontic sealers do not shrink after insertion and form
hydroxyapatite, establishing a chemical bond with dentin12-14. This made it possible
to simplify the filling stage by complying with all parameters of adequate root canal
obturation. However, the use of these sealers in teeth treated for secondary or per-
sistent endodontic infections, where fungi may be present, must be handled with
care given that the setting of these materials depends on the local conditions of
the RCS15. The persistence of fungi and their byproducts cause inflammation of the
periradicular tissues and a reduction in the local pH, which can alter the properties
of these sealers16.
Considering the complexity of fungal infections and their resistance to endodontic
procedures, it is essential to understand the antifungal properties of endodontic
sealers. Calcium silicate-based sealers are frequently studied for their antibacterial
properties; however, studies on their antifungal properties are limited. This study
aimed to perform a comparative in vitro analysis of the antifungal activities of differ-
ent calcium silicate-based sealers against three Candida spp. species.

Materials and Methods

This project was approved by the local ethics committee, which authorized its devel-
opment under protocol number 2018/0997.

Braz J Oral Sci. 2024;23:e243355 2

 Moreira et al.

Tested Materials
Three calcium silicate-based endodontic sealers were tested: Bio-C Sealer (Ange-
lus, Londrina, PR, Brazil), Sealer Plus BC (MK Life, Porto Alegre, RS, Brazil), and
MTA-Fillapex (Angelus, Londrina, PR, Brazil). Two of the most commonly used end-
odontic sealers, AH Plus (Dentsply Sirona, Ballaigues, Switzerland) and Endometha-
sone (Septodont, Saint-Maur-Des-Fossés, France), were used as controls.

Microbiological analysis
The methodology used in this study was adapted from Damasceno et al.17 (2008),
Gomes et al.18 (2004), and Rahman et al.19 (2017). An agar diffusion test was used
to determine inhibitory effects against three fungal species: Candida albicans
(ATCC 10231), Candida glabrata (ATCC 90030), and Candida tropicalis (ATCC 750).
The initially lyophilized fungi were reactivated, cultured, and maintained in trypticase
soy broth (TSB) (Difco, Detroit, MI, USA). A 24-hour culture was used as the inocu-
lum. To standardize the inoculum, TSB medium was prepared according to the 0.5
McFarland turbidity standard (1.5 × 108 colony-forming units/mL20).
Petri dishes containing TSA medium were inoculated with 0.1 mL culture of each
fungal species using sterile swabs rubbed across the surface of the TSB medium.
A mixed microbial culture collected from human saliva was used as a compara-
tive parameter for antifungal tests. Saliva was diluted to a concentration of 1 mL
in 4 mL of TSB. After the inoculation of fungal species and the mixed microbial
culture on the TSB medium, three 6-mm-diameter and 5-mm-deep holes (dictated
by the thickness of the agar layer) were made with a sterile metal hole punch.
Endodontic sealers were manipulated simultaneously according to the manufac-
turer’s instructions. Premixed sealers (Bio-C Sealer and Sealer Plus BC) were used
directly from the syringes with dispensing tips, whereas MTA–Fillapex was pre-
pared using the automix tip designed for the dual syringe of the sealer. For the
preparation of AH Plus, equal volumes (1:1) of pastes A and B were dispensed on
a glass slab and mixed using a metal spatula until a homogeneous consistency
was obtained. Endomethasone was prepared by progressively pouring the powder
into the liquid at a ratio of two spoons of powder to four drops of liquid until a
homogeneous mixture was obtained. Sealers were introduced into the agar holes
to be tested in triplicate. The plates were kept for 2 hours at room temperature to
allow the diffusion of the sealers through the agar, and then they were transferred
to a 37 °C aerobic incubation environment for 24 hours to allow microbial growth
(Tanomaru-Filho et al.21 (2007), Gomes et al.18 (2004)). Growth inhibition zones
were measured daily for 7 days using a manual caliper (Damasceno et al.17 (2008),
Rahman et al.19 (2017)).

Results
When analyzing the performance of the sealers in terms of their ability to inhibit
fungal growth over time, Endomethasone exhibited the highest inhibition against all
strains examined, maintaining a consistent level of inhibition throughout the entire
7-day period. MTA-Fillapex demonstrated the best performance among the calcium

Braz J Oral Sci. 2024;23:e243355 3

 Moreira et al.

silicate-based sealers for the three fungal species, maintaining stable values over 7
days, and was only surpassed by Endomethasone. Bio-C Sealer, Sealer Plus BC, and
AH Plus exhibited similar levels of inhibition as MTA-Fillapex against C. albicans and
C. tropicalis only after 24 hours, and at 48 hours, the antifungal activities of Sealer
Plus BC and BIO-C Sealer decreased. At all the time points evaluated, AH Plus failed to
exhibit any inhibitory effect against C. glabrata (Fig. 1).

A B
40 40
AH Plus
Inhibition halo (mm)

Inhibition halo (mm)

30 30 Endomethasone
Bio-C Sealer
20 20 Sealer Plus BC
MTA-Fillapex
10 10

0 0
ys

ys
6 s
ys

6 s
7 s
48 s

48 s
72 s
96 s
5 s

72 s
96 s
5 s
y

y
y
hr

hr
hr
hr
hr

hr
hr
hr
da

da
da
da

da
da
24

24
7

C. albicans ATCC 1031 C. tropicalls ATCC 750

C D
40 15
Inhibition halo (mm)

Inhibition halo (mm)

30
10
20
5
10

0 0
ys

ys
6 s
ys

6 s
7 s
48 s

48 s
72 s
96 s
5 s

72 s
96 s
5 s
y

y
y
hr

hr
hr
hr
hr

hr
hr
hr
da

da
da
da

da
da
24

24
7

C. glabrata ATCC 90030 Mix culture

Figure 1. Variation over time of the inhibition halos produced by the sealers for the different species studied.

When analyzing the performance of calcium silicate-based sealers at 24 hours,

MTA-Fillapex displayed the highest inhibition halo against C. albicans, exhibiting sig-
nificant differences compared to Bio-C Sealer and Sealer Plus BC (p<0.008). However,
for C. tropicalis, all three calcium silicate-based sealers demonstrated similar values.
Concerning C. glabrata, the highest inhibition halo was achieved by Sealer Plus BC,
showing significant differences compared to MTA-Fillapex (p<0.008) but not when
compared to Bio-C Sealer (Fig. 2).

Braz J Oral Sci. 2024;23:e243355 4

 Moreira et al.

**
****
***
**** **
**** *** ****
**** **** ** ****
****
****
* ****
**** **
**** ****
40 40 40 ** 15
**** ****
**** **** ** **** ****
Inhibition halo (mm)

Inhibition halo (mm)

Inhibition halo (mm)

Inhibition halo (mm)

30 30 30
10
20 20 20
5
10 10 10

0 0 0 0
Bi has s
al Se e
M Plu r
-F BC

Bi has s
al Se e
M Plu r

x
-F BC

Bi has s
al Se e
M Plu r

x
-F BC

Bi has s
al Se e
M Plu r

x
-F BC
e

e
pe

pe

pe

pe
et Plu

et Plu

et Plu

et Plu
o- on

o- on

o- on

o- on
er al

er al

er al

er al
TA s

TA s

TA s

TA s
illa

illa

illa

illa
AH

AH

AH

AH
Se C

Se C

Se C

Se C
m

m
do

do

do

do
En

En

En

En
24 hrs. C. albicans ATCC 10231 24 hrs. C. tropicalls ATCC 750 24 hrs. C. glabrata ATCC 90030 24 hrs. Mix culture

****<0.0001; ***<0.0008; **<0.008; *<0.05

Figure 2. Inhibition halos at 24 hours for the different sealers

At 48 hours, MTA-Fillapex displayed larger inhibition halos against C. albicans than

Bio-C Sealer (p<0.0008), whereas Sealer Plus BC failed to exhibit any inhibition. Only
MTA-Fillapex produced inhibition halos for C. tropicalis and C. glabrata, an effect that
was sustained only for fungal species, as no inhibition halos were observed for mixed
bacterial cultures (Fig. 3).

****
**** ****
**** **** ****
**
*** **** ****
****
**** **** **** **** ****
*** **** **** **** **** **** ** ** ****
40 40 40 15
**** **** **** **** **** **** **** **** ****
Inhibition halo (mm)

Inhibition halo (mm)

Inhibition halo (mm)

Inhibition halo (mm)

30 30 30
10
20 20 20
5
10 10 10

0 0 0 0
Bi has s
Se -C one

M Plu r
-F BC

Bi has s
al Se e
M Plu r

x
-F BC

Bi has s
al Se e
M Plu r

x
-F BC

Bi has s
Se -C one

M Plu r

x
-F BC
e

e
pe

pe

pe

pe
et Plu

et Plu

et Plu

et Plu
o- on

o- on
er al

er al

er al

er al
TA s

TA s

TA s

TA s
illa

illa

illa

illa
al Se

al Se
AH

AH

AH

AH
Se C

Se C
o

o
m

m
do

do

do

do
En

En

En

En

48 hrs. C. albicans ATCC 10231 48 hrs. C. tropicalls ATCC 750 48 hrs. C. glabrata ATCC 90030 48 hrs. Mix culture

****<0.0001; ***<0.0008; **<0.008

Figure 3. Inhibition halos at 48 hours for the different sealers

After 7 days, only MTA-Fillapex maintained its inhibitory effect against the three
fungal species studied. In the mixed bacterial culture, Bio-C Sealer outperformed
Sealer Plus BC (p<0.008), and the values remained stable from 72 to 96 hours,
respectively (Fig. 4).

Braz J Oral Sci. 2024;23:e243355 5

 Moreira et al.

****
**** ****
**** **** **** *
**** **** **** ****
**** **** **** **** **
**** **** **** **** **** **** ** ****
30 40 40 15
**** **** **** **** **** ****
Inhibition halo (mm)

Inhibition halo (mm)

Inhibition halo (mm)

Inhibition halo (mm)

30 30
20 10
20 20
10 5
10 10

0 0 0 0
Bi has s
al Se e
M Plu r
-F BC

Bi has s
al Se e
M Plu r

x
-F BC

Bi has s
al Se e
M Plu r

x
-F BC

Bi has s
al Se e
M Plu r
-F BC

x
e

e
pe

pe

pe

pe
et Plu

et Plu

et Plu

et Plu
o- on

o- on

o- on

o- on
er al

er al

er al

er al
TA s

TA s

TA s

TA s
illa

illa

illa

illa
AH

do AH

do AH

AH
Se C

Se C

Se C

Se C
m

m
do

do
En

En

En

En
7 days. C. albicans ATCC 10231 7 days. C. tropicalls ATCC 750 7 days. C. glabrata ATCC 90030 7 days. Mix culture

****<0.0001; **<0.008; *<0.05

Figure 4. Inhibition halos at 7 days for the different sealers

Discussion
Understanding the antifungal properties of sealers is crucial, given their direct contact
with canal walls and the potential exposure to hard-to-reach areas that may be colo-
nized by fungi or other species. These areas, such as isthmuses, lateral canals, and
apical deltas, can serve as reservoirs for various microorganisms and contribute to
the development of persistent apical periodontitis3.
The simultaneous testing of the sealers and their measurement every 24 hours
allowed the analysis of the antimicrobial behavior of each sealer over time. Periodic
assessment of inhibition halos makes it possible to determine the time at which the
sealers exhibit fluctuations, time–pointing when further attention is needed. In this
study, we determined that the time points with the most significant fluctuations in
antimicrobial activity were 24, 48, and 7 days.
Endomethasone exhibited the highest antimicrobial activity, as it was the only sealer
that demonstrated inhibitory action against all fungal species and mixed micro-
bial cultures over 7 days. This sustained and potent antimicrobial activity can be
attributed to the combined effect of corticosteroids, formaldehyde, and zinc oxide
eugenol, all of which are present in the sealer’s composition18. AH Plus exhibited
greater inhibition than Sealer Plus BC and Bio-C Sealer against C. albicans and C.
tropicalis, with the highest inhibition observed at 48 hours. These findings can be
explained by the release of formaldehyde during the setting reaction of AH Plus.
Although AH Plus did not contain formaldehyde, it released small amounts of this
compound during the setting reaction, reaching its maximum level after 48 hours22.
Formaldehyde exhibits antimicrobial properties that alter the viability and growth
of bacteria and fungi22. No significant differences were observed between the anti-
fungal activity of AH Plus and MTA-Fillapex for C. albicans and C. tropicalis. These
results can be attributed to the high salicylate resin content in Fillapex, which
imparts antimicrobial properties to the sealer23.

Braz J Oral Sci. 2024;23:e243355 6

 Moreira et al.

The analysis of the results of calcium silicate-based sealers during the first 24 hours
showed that MTA-Fillapex, Bio-C Sealer, and Sealer Plus BC exhibited similar inhib-
itory effects on the three fungal strains studied. However, a sustained decrease in
inhibition was observed between 48 hours and 96 hours for the latter two. These
results demonstrate the resistance of fungi to pH changes, which could explain their
recognized resistance to the action of calcium hydroxide when used as an intraca-
nal medication8. Although calcium silicate-based sealers do not contain calcium
hydroxide in their composition, they contain calcium oxide. The interaction of the lat-
ter with tissue fluids produces calcium hydroxide, which can act as a source of the
calcium ions necessary for the growth and morphogenesis of Candida. It is import-
ant to consider that these results may have been influenced by the buffering ability
of the culture broth, which can neutralize the pH of these materials. This effect was
previously reported by Al Hezaimi et al.24 (2006) and Tanomaru Filho et al.21 (2007),
who observed a similar behavior for MTA (mineral trioxide aggregate). In relation to
the behavior of MTA-Fillapex, it presented a stable inhibitory action during the 7-day
period for all the fungal species analyzed. This activity can be mainly attributed to
the high percentage of salicylate resin present in its composition19,25.
Regarding the performance of premixed calcium silicate-based sealers in mixed
microbial cultures, stable inhibition halos were observed from 72 to 96 hours for
Sealer Plus BC and Bio-C Sealer. This might be explained by the alkalization of the
medium produced by these types of materials, which, from their hydration reaction,
produces calcium silicate and calcium hydroxide, releasing hydroxyl ions26. Based
on the obtained results, Bio C-Sealer produced greater inhibition halos than AH Plus
during the 7-day testing period, showing an incremental inhibition that stabilized
at 72 hours, at which time it showed inhibition halos similar to Endomethasone.
According to Sfeir et al.26 (2021), calcium silicate-based sealers have similar or
even greater antibacterial properties than conventional sealers; however, the lack
of standardized testing methologies makes it difficult to compare the results27. In
contrast, MTA-Fillapex presented inhibitory halos only in the first 24 hours. The anti-
microbial action of this sealer can be attributed to the resin and MTA present in its
composition; however, this effect is lost with setting time28. The limited alkalization
shown by MTA-Fillapex has been previously reported in other studies25, which can
be mainly attributed to the fact that it contains 13% of MTA in its composition23.
This low percentage of calcium silicate differentiates it from other calcium sili-
cate-based sealers that are characterized by high pH values over longer periods26.
This study aimed to compare the antifungal properties of three calcium
silicate-based sealers using an agar diffusion test. This test is commonly used
to compare and analyze the antimicrobial activity of materials, facilitating direct
comparisons between them28. However, this methodology is not exempt from lim-
itations because it is incapable of distinguishing between bacteriostatic and bac-
tericidal effects and can be influenced by the physical properties of the material.
Materials with high diffusibility may exhibit larger inhibition halos independent of
their antimicrobial ability29.

Braz J Oral Sci. 2024;23:e243355 7

 Moreira et al.

The scarce literature on the antifungal properties of calcium silicate-based sealers

makes it difficult to compare our results with those of other studies. Further studies
are required to understand and support the clinical relevance of these findings.
In conclusion, of all the silicate-based sealers tested, only MTA-Fillapex exhibited
promising antifungal activity. Nevertheless, care must be taken when extrapolating
these results, as MTA-Fillapex exhibited poor antimicrobial activity when tested in
mixed microbial cultures.

Acknowledgments
None.

Source of Funding
None.

Conflict of interest
The authors declare that there is no conflict of interest.

Data availability
Datasets related to this article will be available upon request to the corresponding
author.

Author Contribution
Luiz Felipe Nunes Moreira: Conceptualization, Investigation, Methodology, Project
administration
Fernando Peña-Bengoa: Investigation, Formal analysis, Methodology, Project admin-
istration, Writing - review & editing.
Sven Eric Niklander: Software, Formal analysis, Writing - review & editing.
Carlos Eduardo da Silveira Bueno: Conceptualization, Formal analysis, Methodology,
Alexandre Sigrist de Martin: Conceptualization, Formal analysis, Methodology.
Daniel Guimarães Pedro Rocha: Conceptualization, Formal analysis, Methodology
All authors approved the final version of the manuscript.

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