UNIVERSIDADE ESTADUAL PAULISTA

“JÚLIO DE MESQUITA FILHO”

Instituto de Ciência e Tecnologia

Campus de São José dos Campos

ORIGINAL ARTICLE DOI: https://doi.org/10.4322/bds.2024.e4007

Braz Dent Sci 2024 Apr/June;27 (2): e4007

This is an Open Access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in

any medium, provided the original work is properly cited.

Cytotoxic eﬀects of bioceramic materials on stem cells from human

exfoliated deciduous teeth (SHED)

Efeitos citotóxicos de materiais biocerâmicos em células-tronco de dentes decíduos esfoliados humanos (SHED)

Bárbara Luísa Silva OLIVEIRA1 , Ana Beatriz Vieira da SILVEIRA1 , Mariel Tavares Oliveira Prado BERGAMO2 ,

Eloá Cristina Passucci AMBROSIO3 , Paula Karine JORGE3 , Mayara BRINGEL1 , Natalino LOURENÇO NETO1 ,

Maria Aparecida Andrade Moreira MACHADO1 , Thais Marchini OLIVEIRA1 

1 - Universidade de São Paulo, Faculdade de Odontologia de Bauru, Departamento de Odontopediatria, Ortodontia e Saúde Coletiva,

Bauru, São Paulo, Brazil.

2 - University of Michigan, School of Dentistry, Department of Pediatric Dentistry. Ann Arbor, USA.

3 - Universidade de São Paulo, Hospital de Reabilitação de Anomalias Craniofaciais, Bauru, São Paulo, Brazil.

How to cite: Oliveira BLS, Silveira ABV, Bergamo MTOP, Ambrosio ECP, Jorge PK, Bringel M et al. Cytotoxic effects of bioceramic

materials on stem cells from human exfoliated deciduous teeth (SHED). Braz Dent Sci. 2024;27(2):e4007. https://doi.org/10.4322/

bds.2024.e4007

ABSTRACT

Objective: This study aimed to evaluate stem cell from human deciduous teeth (SHED) viability after exposure

to different bioceramic materials. Material and Methods: Discs were constructed to obtain the material extracts

according to the following groups: G1 - Bio-C Repair, G2 - MTA Repair HP, G3 - TheraCal LC, and G4 – Biodentine.

Positive and negative control group were respectively maintained with αMEM + 10% FBS and αMEM + 1%

FBS. SHED obtained through primary culture were in contact with material extracts for 24, 48, and 72h. MTT

assay evaluated cell viability. Groups were plated in triplicate and the cell viability assay were repeated three

times. Data were analyzed by two-way ANOVA followed by Tukey test (p<0.05). Results: The treatment and

period comparisons showed statistically signicant differences (p<0.000). G2 (MTA Repair HP) had greater

cell viability values than the other experimental groups and negative control. MTA Repair HP and the control

groups exhibited a similar behavior with cell viability values decreasing from 24h to 48h and increasing from

48h to 72h. Bio-C Repair, Biodentine, and Theracal LC did not show statistically signicant differences among

periods. Conclusions: SHED increased viability values after contact with MTA Repair HP in comparison with

other bioceramic materials.

KEYWORDS

Cell viability; Cytotoxicity; Materials testing; SHED; Stem cells.

RESUMO

Objetivo: O objetivo desse estudo foi avaliar a viabilidade de células-tronco de dentes decíduos humanos (SHED)

após o contato com diferentes materiais biocerâmicos. Material e Métodos: Foram confeccionados discos para

obtenção dos extratos dos materiais de acordo com os seguintes grupos: G1 - Bio-C Repair, G2 - MTA Repair

HP, G3 - TheraCal LC e G4 - Biodentine. Grupo de controle positivo e negativo foram mantidos respectivamente

com αMEM + 10% FBS e αMEM + 1% FBS. SHED obtidas por cultura primária entraram em contato com os

extratos de materiais por 24, 48 e 72h. O ensaio MTT avaliou a viabilidade celular. Os grupos foram semeados

em triplicata e o ensaio de viabilidade celular foi repetido três vezes. Os dados foram analisados por ANOVA a

dois critérios seguido pelo teste de Tukey (p<0,05). Resultados: As comparações de tratamentos e períodos

mostraram diferenças estatisticamente signicativas (p<0,000). O G2 (MTA Repair HP) apresentou maiores

valores de viabilidade celular que os demais grupos experimentais e controle negativo. O MTA Repair HP e os

grupos controle exibiram um comportamento semelhante com os valores de viabilidade celular diminuindo de 24h

Braz Dent Sci 2024 Apr/June;27 (2): e4007

Oliveira BLS et al.

Cytotoxic effects of bioceramic materials on stem cells from human exfoliated deciduous teeth (SHED)

Oliveira BLS et al. Cytotoxic eﬀects of bioceramic materials on stem cells from

human exfoliated deciduous teeth (SHED)

INTRODUCTION

Stem cell are undifferentiated cells with high

proliferation and self-renewal potential capable of

differentiating in many cell types [1]. The possibility

of isolating mesenchymal stem cells from dental

pulp enables the application of bioengineering for

pulp regeneration, allowing promising treatment

options [2,3]. Human deciduous teeth are source of

stem cells that may be isolated and cultured in vitro.

Stem cells from human deciduous teeth (SHED)

are a high proliferative cell population with varied

differentiation capacity [4]. Pulp therapy repair

mechanism occurs through migration, proliferation,

and differentiation of stem cells from dental pulp

into odontoblasts that account for the synthesis and

secretion of tertiary dentin [2,5].

Pulp vital treatment requires biomaterials

to protect the exposed vital pulp [6]. For

this purpose, the ideal material should be

biocompatible; bactericidal; capable of promoting

pulp healing; and should not interfere in the

normal exfoliation of deciduous teeth [7,8].

To date, different materials for pulp therapy of

deciduous teeth are available, including calcium

silicate bioceramic cements. These cements are

biocompatible and bioinductive, there is, when

in contact with the injured pulp, they have the

appropriate bioactivity to induce the repair and

formation of mineralized tissue [9].

The introduction of bioceramic materials

represented a pivotal advancement in the

evolution of regenerative endodontic therapy.

Currently, the available literature evaluate the

differences between recently introduced silicate-

based materials such as Biodentine, MTA or

even more traditional materials such as calcium

hydroxide. However, few studies have evaluated

newer materials such as Bio-C Repair and

Theracal LC. Additionally, there is more extensive

literature available on stem cells originating from

the dental pulp of permanent teeth, revealing a

knowledge gap regarding pulp from deciduous

teeth. The introduction of new bioceramic

materials combined with additives requires up-to-

date research in the area. The evaluation of the

bioactivity of these materials concerning dental

pulp stem cells relies on in vitro tests or animal

studies. These methods, while imperfect, are

crucial for mimicking relevant clinical scenarios

to a certain extent [10].

In this context, previous studies evaluate the

cytotoxicity of vital pulp treatment materials on

stem cells from the pulp of permanent teeth, but

few used SHED [2,4,11]. Thus, this study aimed

to evaluate stem cell from human deciduous

teeth (SHED) viability after exposure to four

different bioceramic materials: Bio-C Repair,

MTA Repair HP, Biodentine, and Theracal. The

null hypothesis is that the materials would show

similar biocompatibility.

MATERIALS AND METHODS

Study design

This was a two-factor study: treatment (6

levels: Bio-C Repair, MTA Repair, Theracal LC,

Biodentine, negative control, and positive control)

and periods (3 levels: 24, 48, and 72 horas).

Ethical issues

This study was submitted and approved by

the Institutional Review Board (protocol CAAE:

29177820.9.0000.5417). All participants and

their legal guardians read and signed a free and

claried consent form to donate the exfoliated

deciduous teeth.

Cell culture and isolation

The cells were obtained from primary cell

culture and characterized following a previous

study [12]. SHED were plated on 25-cm2 culture

para 48h e aumentando de 48h para 72h. Bio-C Repair, Biodentine e Theracal LC não apresentaram diferenças

estatisticamente signicativas entre os períodos. Conclusões: SHED aumentou os valores de viabilidade após o

contato com o MTA Repair HP em comparação com outros materiais biocerâmicos.

PALAVRAS-CHAVE

Viabilidade celular; Citotoxicidade; Teste materiais; SHED; Células-tronco.

Braz Dent Sci 2024 Apr/June;27 (2): e4007

Oliveira BLS et al.

Cytotoxic effects of bioceramic materials on stem cells from human exfoliated deciduous teeth (SHED)

Oliveira BLS et al. Cytotoxic eﬀects of bioceramic materials on stem cells from

human exfoliated deciduous teeth (SHED)

ask (Corning, Union City, CA) containing Alpha-

MEM supplemented with 20% FBS, incubated

at 37°C and 5% CO2. The cells were cultivated

at 80% ask conuence (passage O). The cells

were washed with phosphate buffered saline

solution (PBS) (Gibco Invitrogen) and detached

with 0.25% trypsin-EDTA (Gibco Invitrogen) for

5 min at 37°C. Culture medium was added to

inactivate trypsin activity. Finally, the cells were

centrifugated at 1200 rpm, for 5 min and plated

on de 75-cm2 asks at density of 1 × 104 cm2,

for cell expansion. SHEDs at passages 4th and 8th

were used in the experiments.

Pulp capping materials

Four bioactive materials were analyzed:

Bio-C Repair (Angelus, Londrina, PR, Brazil),

MTA Repair HP (Angelus, Londrina, PR, Brazil),

Theracal LC (Bisco Inc., Schaumburg, IL, USA),

and Biodentine (Septodont, Saint-Maur-des-

Fosses, France) (Table I).

Sample preparation

The cements extracts were prepared

following previous studies and ISO standard

10993 [11,13-16]. The materials were prepared

according to the manufacturer’s instructions.

MTA Repair pack content (0.17g) was mixed

with two drops of the liquid for 40 seconds to

obtain a homogenous cement. Five drops of

liquid were added to Biodentine capsule and

agitated at 4000 rpm for 30 seconds. Bio-C Repair

and TheraCal LC are ready for use. All samples

were prepared in aseptic conditions, with the

aid of sterile rubber molds (5 mm diameter,

3 mm height) and incubated at 37º C for 6 h.

Elapsed that time, the samples were removed

from the molds and sterilized by ultraviolet light

for 1h inside biosafety cabinets. Each sample

was immersed into 1 mL of αMEM (Invitrogen,

Carlsbad, California) + 10% FSB (Thermo Fisher

Scientic, USA) + 1% antibiotics and antifungals

(Anti-Anti - Gibco, Grand Island, NY, USA) and

incubated at 5% CO2 for 3 days. Elapsed that

period, the material discs were discarded and

the supernatants were collected and ltered with

0.22-mm sterile lter (Sigma-Aldrich, St Louis,

MO). The collected supernatants were referred

as extracts (1:1).

Cell viability assay

Cell viability was analyzed by 3-(4,5-dimethyl-

thiazol-2-yl)-2,5-diphenyltetrazolium bromide

(MTT). SHED at density of 1 x 10 4 were seeded

in 96-well plates (Corning #3595) with 1 mL of

culture medium and incubated for 24h, at 37ºC

and 5% CO2 for cell adhesion. Elapsed that period,

the culture medium was changed by the materials

extracts. SHED was exposed to material extracts

for 24, 48, and 72h. Positive and negative control

cells were maintained in αMEM + 10% FSB and

1% SFB, respectively. Elapsed the study times,

the culture medium was removed, and the cells

were washed with PBS 1x. Next, 110ul of MTT

(0.5mg/mL) was added to each well. The plates

were covered with aluminum foil and incubated

at 37º C, 5% CO2, for 4 h. After that, MTT solution

was discarded and 200 ul of Dimethyl sulfoxide

(DMSO, Fisher Scientific, Hampton, VA, EUA)

were added per well. After 30 minutes, absorbance

was measured by spectrophotometer (Synergy

Mx; BioTek Instruments, USA), at 570 nm wave-

length. Groups were plated in triplicate and the cell

viability assay were analyzed in three independent

experiments. All statistical analyses were obtained

Table I - Main components of the study materials

Material Manufacturer Composition

Bio-C Repair Angelus, Londrina, PR, Brazil Calcium silicates, calcium aluminate, calcium oxide, zirconium oxide, iron

oxide, silicon dioxide, and dispersing agent.

MTA Repair HP Angelus, Londrina, PR, Brazil

Powder: Tricalcium silicate; Dicalcium silicate; Tricalcium aluminate;

Calcium Oxide; Calcium Tungstate.

Liquid: Water and Plasticizer.

Theracal LC BiscoInc, Schaumburg, IL, USA

Tricalcium silicate particles; Urethane-Dimethacrylate (UDMA); Bisphenol

A-glycidyl Methacrylate (Bis-GMA) and Triethylene Glycol Dimethacrylate

(TEDGMA); Hydroxyethyl Methacrylate (HEMA) and Polyethylene Glycol

Dimethacrylate (PEDGMA).

Biodentine Septodont, Saint- Maur-des-

Fosses, France

Powder: Tricalcium Silicate; Zirconium Oxide; Calcium Oxide; Calcium

Carbonate; Yellow Pigment; Red Pigment; Brown Iron Oxide.

Liquid: Calcium Chloride Dihydrate; polycarboxylate. Puriﬁed Water.

Braz Dent Sci 2024 Apr/June;27 (2): e4007

Oliveira BLS et al.

Cytotoxic effects of bioceramic materials on stem cells from human exfoliated deciduous teeth (SHED)

Oliveira BLS et al. Cytotoxic eﬀects of bioceramic materials on stem cells from

human exfoliated deciduous teeth (SHED)

with Statistica 10.0 software for Windows. Data

were analyzed by two-way ANOVA followed by

Tukey test (p = 0.05).

RESULTS

Cell viability assay showed statistically

signicant differences between materials and

times (p<0.000). At 24h, MTA Repair HP showed

statistically signicant higher cell viability values

than all the other materials and negative control

(C-), followed by Biodentine, Bio-C Repair, and

Theracal LC (Table II). Positive control (C+)

had the greatest statistically signicant viability

means than that of the other groups (p<0,05),

except for MTA Repair HP. Negative control

group (C-) exhibited statistically significant

differences with all studied groups (Table II)

At 48h, SHED viability values signicantly

decreased with differences between MTA

Repair HP and the other materials and negative

control group (Table II). At 72h, the cell

viability increased, as follows: MTA Repair

HP>Biodentine>Bio-C Repair>Theracal LC

(Table II). We observed that SHEDs in contact

with MTA Repair HP, negative and positive

controls exhibited similar patterns of statistically

signicant viability decreasing from 24h to 48h,

followed by a statistically signicant increasing

at 72h (24h>48h; 72h>48h; 24h=72h). SHED

in contact with Bio-C Repair, Theracal LC, and

Biodentine did not show statistically signicant

differences between periods (Table II).

DISCUSSION

Biocompatibility is an important property

to be considered when selecting a material for

pulp therapy due to the direct contact with vital

tissues [11,17]. As science advances, current

bioinductive materials show promising outcomes

in pulp therapy for deciduous and permanent

teeth. Previous studies have demonstrated

the effects of Bio-C Repair, MTA Repair HP,

Theracal LC, and Biodentine on human pulp

tissue alone or together with other materials with

variable success rates [9,15,17,18]. However, the

literature lacks studies on the direct contact of

these materials with SHEDs.

Cell culture techniques are an excellent choice

for analyzing the biocompatibility of different

materials. Many quantitative and qualitative

in vitro methods evaluate the cytotoxicity of

biomaterials and the potential side effects on

cellular mechanisms [11]. In vitro tests offer a

comprehensive analysis of the biological properties

of the materials cultured with cells aiming at

predicting the clinical behavior [19]. Minimum

Essential Medium (MEM) is a widely recognized

medium utilized for culturing mammalian

cells. MEMα, as per the manufacturer, does not

contain any proteins, lipids, or growth factors.

Therefore, MEMα requires supplementation,

usually with 10% Fetal Bovine Serum (FBS).

MEMα uses a sodium bicarbonate buffer system

(2.2 g/L), requiring a 5–10% CO2 environment

to maintain physiological pH (Thermo Fisher

Scientic, USA – Safety Data Sheet). Selecting

the appropriate culture medium and determining

the appropriate percentage of Fetal Bovine Serum

(FBS) are crucial steps in establishing an optimal

environment that does not interfere with the

differentiation of stem cells [20]. According to

the manfacturer and previous studies, FBS 10%

concentration can maintain the growth of the

cells with no interference on the proliferation,

which could be a bias in the study [12,21]. A

reduced percentage of FBS can create a distinct

environment for the cells, potentially mimicking

suboptimal conditions for cell growth and

proliferation, thereby serving as a negative

control [12,21].

Table II - Cell viability intragroup and intergroup comparisons

Materials

Periods Bio-C Repair

(mean ± SD)

MTA Repair HP

(mean ± SD)

Theracal LC

(mean ± SD)

Biodentine

(mean ± SD)

Negative

control

(mean ± SD)

Positive

control

(mean ± SD)

24 h 0.074 ± 0.004a0.366 ± 0.029e0.078 ± 0.004 a0.088 ± 0.004 a0.287 ± 0.016 d0.378 ± 0.041 e

48 h 0.098 ± 0.006a0.242 ± 0.007c0.091 ± 0.001 a0.110 ± 0.007 a0.197 ± 0.011 b0.264 ± 0.019 cd

72 h 0.102 ± 0.011a0.351 ± 0.008e0.095 ± 0.004 a0.108 ± 0.004 a0.244 ± 0.012 cd 0.355 ± 0.037 e

Diﬀerent superscript letters in the same column and row indicate statistically signiﬁcant diﬀerences in intragroup and intergroup comparisons.

(two-way ANOVA followed by Tukey test; p<0.05). Standard Deviation.

Braz Dent Sci 2024 Apr/June;27 (2): e4007

Oliveira BLS et al.

Cytotoxic effects of bioceramic materials on stem cells from human exfoliated deciduous teeth (SHED)

Oliveira BLS et al. Cytotoxic eﬀects of bioceramic materials on stem cells from

human exfoliated deciduous teeth (SHED)

New biomaterial formulations have been

constantly launched into the market for clinical

use. Currently, silicate and calcium phosphate-

based materials have been used due to their

capability of stimulating tissue repair through

the deposition of mineralized tissue. Thus, they

have been studied regarding their cytotoxicity

and bioactivity on cell cultures [15,22].

Ghilotti et al. [17] tested the cytotoxicity of

Biodentine, Bio-C Repair, and ProRoot MTA on

human dental pulp cells from permanent teeth

and reported through the production of formazan

that the materials were not cytotoxic to hDPCs.

Unexpectedly, undiluted Biodentine exhibited

signicantly higher levels of relative formazan

formation than the control group at all tested time

points. Bio-C Repair showed formazan production

similar to that of untreated cells. The more

diluted ProRoot MTA extracts showed higher

formazan formation than control group only at 72

hours. The studies of Youssef et al. [23] showed

that the cell viability of DPSCs was measured

using MTT assay, exhibiting variable cytotoxicity

against DPSCs compared to the control; MTA

was more cytocompatible than Biodentine,

which showed signicant cytotoxicity against

DPSCs compared to the control, corroborating

the results of the present study [24,25]. To

achieve tooth regeneration, a comprehensive

understanding of tooth stem cells is essential

for better application of tissue engineering. A

systematic review carried out by Sanz et al. [10],

evaluated the bioactivity of bioceramic materials

in relation to dental pulp stem cells (DPSCs), a

total of 37 articles were included in the review. A

systematic review carried out by Sanz et al. [10],

evaluated the bioactivity of bioceramic materials

in relation to dental stem cells (DSC), a total

of 37 articles were included in the review. The

authors concluded that the differences between

DSC justies the need for individual assessment of

the biological response of dental biomaterials to

different DSC variants. The study points out that

SHED generally exhibited adequate levels of cell

viability, proliferation, migration and an increase

in the formation of mineralized nodules after

incubation with various calcium silicate-based

compositions, acting as supporting evidence for

their use in endodontic procedures [10,17,23].

In this present study, the tested materials

showed statistically significant different cell

viability results. MTA Repair HP was biocompatible

while TheraCal LC signicantly decreased cell

viability values. Therefore, the null hypothesis of

this in vitro study was rejected. Although using

different methodologies, other studies showed

comparable results [17,22,26].

The biocompatibility of MTA HP Repair has

been shown by previous studies, suggesting its

benecial clinical use [16,27,28]. MTA HP Repair

main components (tricalcium silicate, calcium

oxide, tricalcium oxide, silicate, and aluminate)

are similar to dentinal tissue components and

account for its low cytotoxicity [29]. In this study,

SHED in contact with MTA HP Repair behaved

better at 24 and 72h, showing the highest cell

viability values. Previous studies demonstrate

that MTA cements are biocompatible with dental

pulp stem cells. Tomás-Catalá et al. [30] studied

the effects of MTA HP Repair on dental pulp stem

cells, through MTT assay, and found high cell

viability rates, corroborating our results.

On the other hand, Theracal LC was the most

cytotoxic cement because it signicantly reduced

SHED viability values in comparison with negative

control, at all studied times, in agreement with the

literature [16,31-33]. The literature has indicated

that TheraCal LC has shown worst results than

MTA and Biodentine, due to low quality of the

dentin barrier, great inammatory effect, less

favorable odontoblastic layer formation, and

small capacity of calcium release. The non-

polymerized resin monomer accounts for such

results, leading to inammation and toxicity to

pulp tissue [34-38]. Moreover, heating during

photopolymerization can potentially induce

unfavorable pulp reactions [39]. The study

of Camilleri et al. [40] on calcium hydroxide

releasing of pulp capping materials found a

relation between calcium hydroxide releasing and

pulp tissue regeneration. These authors reported

that Theracal LC calcium releasing directly

depends on its hydration, so enough calcium

hydroxide may be not produced and consequently

released. pH is another issue. The material pH is

an essential physical property that is related with

pulp response [41]. The releasing of hydroxyl ions

increases the surrounding environment pH leading

to pulp tissue inammation, but it accounts for

the bactericidal effect of the material, which may

explain the grater cytotoxicity values [32].

Biodentine showed smaller viability values

than MTA Repair HP and positive and negative

control groups, which is in agreement with

previous studies [9,17,24]. The literature reports

Braz Dent Sci 2024 Apr/June;27 (2): e4007

Oliveira BLS et al.

Cytotoxic effects of bioceramic materials on stem cells from human exfoliated deciduous teeth (SHED)

Oliveira BLS et al. Cytotoxic eﬀects of bioceramic materials on stem cells from

human exfoliated deciduous teeth (SHED)

that cell viability may signicantly depend on the

extracts’ concentration, that is, less extraction

dilution leads to small cell viability values [15].

Thus, we consider Biodentine concentrations

lower than that used in this present study (1:1)

would be ideal to increase SHED regenerative

potential [25]. Sequeira et al. [42] analyzed

apical papilla cell viability after exposure of 100%

Biodentine and found results similar to those

of this present study, with significantly small

viability values at 24h, 48h, and 72h. Possible

explanations for this result would be that 1) all

newly-prepared calcium silicate cements (resin

free) are initially cytotoxic mainly due to their

alkalinity [43]; 2) this material may increase

stem cell differentiation, which may change

outcomes dependent on cell proliferation, but

it may show the regenerative potential of the

material. Therefore, further studies are necessary

to nd the bioactive properties inuencing on

differentiation because they can indirectly impact

on proliferation and interfere in cell viability.

We emphasize that the material dilution is

justified to mimic its contact with the tissue

resulting in dilution by the extracellular uids

and progressively decreasing of its concentration.

According to Ghilotti et al. [17], Bio-C Repair

biocompatibility is similar to that of Biodentine,

which agrees with the results of this present study.

However, Bio-C Repair cell viability values were

signicantly smaller than that of MTA Repair

HP. We hypothesize that longer setting time

and high solubility favored cytotoxicity because

they indicate the greater releasing of toxic

components. Remnants of some biomaterials

in the extracts may negatively influence cell

cultures [44]. Youssef et al. [23] emphasizes

that the mechanisms involved in cytotoxicity

are not clearly understood, suggesting the initial

calcium ion releasing, ionic activity, presence of

toxic components, or pH changing may affect the

cell behavior. This would explain the different

cytotoxic effect of the tested materials.

The proper cement choice should consider

not only the biological behavior but also other

parameters, such as antimicrobial, physical,

and chemical properties. Although these results

cannot be directly applied to clinical situations

in humans, they are scientifically significant

because they represent an appropriate prototype

for evaluating various initial features of dental

materials. Further research using in vivo animal

models is necessary to conrm the results, and

enable direct outcome comparisons and clinical

application, aiming at the best cement choice.

CONCLUSION

In conclusion, SHED increased viability

values after contact with MTA Repair HP in

comparison with other bioceramic materials.

The results suggested that MTA Repair HP is

still considered the gold standard among the

materials studied and can be indicated for use

in clinical regenerative procedures of the dentin-

pulp complex.

Author’s Contributions

BLS: Data curation, Methodology, Investigation,

Writing – Original Draft Preparation. ABVS:

Supervision, Writing – Original Draft Preparation.

MTOP: Conceptualization, Writing – Review &

Editing. ECPA: Formal Analysis, Writing – Review

& Editing. PKJ: Formal Analysis, Validation, Writing

– Review & Editing. MB: Methodology, Writing –

Original Draft Preparation. NLN: Conceptualization,

Methodology, Writing – Review & Editing. MAAMM:

Conceptualization, Writing – Review & Editing.

TMO: Conceptualization, Fundation Acquisition,

Supervision, Project Administration, Writing –

Review & Editing.

Conict of Interest

The authors have no proprietary, nancial,

or other personal interest of any nature or kind

in any product, service, and/or company that is

presented in this article.

Funding

This study was totally supported by The

São Paulo Research Foundation - FAPESP.

[2021/10002-7, 2021/08730-4].

Regulatory Statement

This study was conducted in accordance

with all the provisions of the local human

subjects oversight committee guidelines and

policies of: Research Ethics Committee of the

Faculty of Dentistry of Bauru, University of

São Paulo. The approval code for this study is:

29177820.9.0000.5417.

Braz Dent Sci 2024 Apr/June;27 (2): e4007

Oliveira BLS et al.

Cytotoxic effects of bioceramic materials on stem cells from human exfoliated deciduous teeth (SHED)

Oliveira BLS et al. Cytotoxic eﬀects of bioceramic materials on stem cells from

human exfoliated deciduous teeth (SHED)

REFERENCES

1. Lemischka IR. Stem cell biology: a view toward the future. Ann

N Y Acad Sci. 2005;1044(1):132-8. http://doi.org/10.1196/

annals.1349.017. PMid:15958706.

2. Araújo LB, Cosme-Silva L, Fernandes AP, Oliveira TM, Cavalcanti

BN, Gomes JE Fo,etal. Effects of mineral trioxide aggregate,

BiodentineTM and calcium hydroxide on viability, proliferation,

migration and differentiation of stem cells from human exfoliated

deciduous teeth. J Appl Oral Sci. 2018;26:e20160629. http://

doi.org/10.1590/1678-7757-2016-0629.

3. Gronthos S, Mankani M, Brahim J, Robey PG, Shi S. Postnatal

human dental pulp stem cells (DPSCs) in vitro and in vivo.

Proc Natl Acad Sci USA. 2000;97(25):13625-30. http://doi.

org/10.1073/pnas.240309797. PMid:11087820.

4. Athanasiadou E, Paschalidou M, Theocharidou A, Kontoudakis

N, Arapostathis K, Bakopoulou A. Biological interactions

of a calcium silicate based cement (BiodentineTM) with

Stem Cells from Human Exfoliated Deciduous teeth. Dent

Mater. 2018;34(12):1797-813. http://doi.org/10.1016/j.

dental.2018.09.014. PMid:30316525.

5. Ahmed GM, Abouauf EA, Abubakr N,Dörfer CE,El-Sayed KF.

Tissue engineering approaches for enamel, dentin, and pulp

regeneration: an update. Stem Cells Int. 2020;2020:1-15. http://

doi.org/10.1155/2020/5734539.

6. Prati C, Gandolfi MG. Calcium silicate bioactive cements:

biological perspectives and clinical applications. Dent Mater.

2015;31(4):351-70. http://doi.org/10.1016/j.dental.2015.01.004.

PMid:25662204.

7. Cuadros-Fernández C, Lorente Rodríguez AI, Sáez-Martínez S,

García-Binimelis J, About I, Mercadé M. Short-term treatment

outcome of pulpotomies in primary molars using mineral trioxide

aggregate and Biodentine: a randomized clinical trial. Clin Oral

Investig. 2016;20(7):1639-45. http://doi.org/10.1007/s00784-

015-1656-4. PMid:26578117.

8. Bossù M, Iaculli F, Di Giorgio G, Salucci A, Polimeni A, Di Carlo

S. Different pulp dressing materials for the pulpotomy of

primary teeth: a systematic review of the literature. J Clin

Med. 2020;9(3):838. http://doi.org/10.3390/jcm9030838.

PMid:32204501.

9. Dahake PT, Panpaliya NP, Kale YJ, Dadpe MV, Kendre SB, Bogar C.

Response of stem cells from human exfoliated deciduous teeth

(SHED) to three bioinductive materials - an in vitro experimental

study. Saudi Dent J. 2020;32(1):43-51. http://doi.org/10.1016/j.

sdentj.2019.05.005. PMid:31920278.

10. Sanz JL, Rodríguez-Lozano FJ, Llena C, Sauro S, Forner

L. Bioactivity of bioceramic materials used in the dentin-

pulp complex therapy: a systematic review. Materials

(Basel). 2019;12(7):1015. http://doi.org/10.3390/ma12071015.

PMid:30934746.

11. Collado-González M, García-Bernal D, Oñate-Sánchez RE,

Ortolani-Seltenerich PS, Álvarez-Muro T, Lozano A, etal.

Cytotoxicity and bioactivity of various pulpotomy materials

on stem cells from human exfoliated primary teeth. Int Endod

J. 2017;50(Suppl 2):e19-30. http://doi.org/10.1111/iej.12751.

PMid:28169432.

12. Oliveira Prado Bergamo MT, Vitor LLR, Lopes NM, Lourenço Neto

N, Dionísio TJ, Oliveira RC,etal. Angiogenic protein synthesis

after photobiomodulation therapy on SHED: a preliminary study.

Lasers Med Sci. 2020;35(9):1909-18. http://doi.org/10.1007/

s10103-020-02975-7. PMid:32056077.

13. International Standards Organization. ISO 10993-5 Avaliação

Biológica de Dispositivos Médicos – Parte 5: Testes para

citotoxicidade in vitro. Genebra: ISO; 2005.

14. Cintra LTA, Benetti F, Queiroz ÍOA, Lopes JMA, Oliveira SHP, Araújo

GS,etal. Cytotoxicity, biocompatibility, and biomineralization of

the new high-plasticity MTA material. J Endod. 2017;43(5):774-8.

http://doi.org/10.1016/j.joen.2016.12.018.

15. Benetti F, Queiroz ÍOA, Cosme-Silva L, Conti LC, Oliveira SHP,

Cintra LTA. Cytotoxicity, biocompatibility and biomineralization

of a new ready-for-use bioceramic repair material. Braz

Dent J. 2019;30(4):325-32. http://doi.org/10.1590/0103-

6440201902457. PMid:31340221.

16. Rodríguez-Lozano FJ, López-García S, García-Bernal D, Sanz

JL, Lozano A, Pecci-Lloret MP, etal. Cytocompatibility and

bioactive properties of the new dual-curing resin-modified

calcium silicate-based material for vital pulp therapy. Clin Oral

Investig. 2021;25(8):5009-24. http://doi.org/10.1007/s00784-

021-03811-0. PMid:33638052.

17. Ghilotti J, Sanz JL, López-García S, Guerrero-Gironés J, Pecci-

Lloret MP, Lozano A,etal. Comparative surface morphology,

chemical composition, and cytocompatibility of Bio-C Repair,

biodentine, and ProRoot MTA on hDPCs. Materials (Basel).

2020;13(9):2189. http://doi.org/10.3390/ma13092189.

PMid:32397585.

18. Nowicka A, Lipski M, Parafiniuk M, Sporniak-Tutak K, Lichota D,

Kosierkiewicz A,etal. Response of human dental pulp capped

with biodentine and mineral trioxide aggregate. J Endod.

2013;39(6):743-7. http://doi.org/10.1016/j.joen.2013.01.005.

PMid:23683272.

19. Pedano MS, Li X, Yoshihara K, Landuyt KV, Van Meerbeek B.

Cytotoxicity and bioactivity of dental pulp-capping agents

towards human tooth-pulp cells: a systematic review of in-vitro

studies and meta-analysis of randomized and controlled

clinical trials. Materials (Basel). 2020;13(12):2670. http://doi.

org/10.3390/ma13122670. PMid:32545425.

20. Zakrzewski W, Dobrzyński M, Szymonowicz M, Rybak Z. Stem

cells: past, present, and future. Stem Cell Res Ther. 2019;10(1):68.

http://doi.org/10.1186/s13287-019-1165-5. PMid:30808416.

21. Vitor LLR, Prado MTO, Lourenço N No, Oliveira RC, Sakai VT,

Santos CF,etal. Does photobiomodulation change the synthesis

and secretion of angiogenic proteins by different pulp cell

lineages? J Photochem Photobiol B. 2020;203:111738. http://

doi.org/10.1016/j.jphotobiol.2019.111738. PMid:31954290.

22. Sanz JL, Soler-Doria A, López-García S, García-Bernal D,

Rodríguez-Lozano FJ, Lozano A,etal. Comparative biological

properties and mineralization potential of 3 endodontic materials

for vital pulp therapy: theracal PT, theracal LC, and biodentine

on human dental pulp stem cells. J Endod. 2021;47(12):1896-906.

http://doi.org/10.1016/j.joen.2021.08.001. PMid:34425148.

23. Youssef AR, Emara R, Taher MM, Al-Allaf FA, Almalki M,

Almasri MA,et al. Effects of mineral trioxide aggregate,

calcium hydroxide, biodentine and Emdogain on osteogenesis,

Odontogenesis, angiogenesis and cell viability of dental

pulp stem cells. BMC Oral Health. 2019;19(1):133. http://doi.

org/10.1186/s12903-019-0827-0. PMid:31266498.

24. Zaen El-Din AM, Hamama HH, Abo El-Elaa MA, Grawish ME,

Mahmoud SH, Neelakantan P. The effect of four materials

on direct pulp capping: an animal study. Aust Endod

J. 2020;46(2):249-56. http://doi.org/10.1111/aej.12400.

PMid:32129919.

25. Hasweh N, Awidi A, Rajab L, Hiyasat A, Jafar H, Islam N,etal.

Characterization of the biological effect of BiodentineTM on

primary dental pulp stem cells. Indian J Dent Res. 2018;29(6):787-

93. http://doi.org/10.4103/ijdr.IJDR_28_18. PMid:30589009.

26. Manaspon C, Jongwannasiri C, Chumprasert S, Sa-Ard-Iam N,

Mahanonda R, Pavasant P,etal. Human dental pulp stem cell

responses to different dental pulp capping materials. BMC Oral

Health. 2021;21(1):209. http://doi.org/10.1186/s12903-021-

01544-w. PMid:33902558.

27. Cintra LTA, Benetti F, de Azevedo Queiroz ÍO, de Araújo Lopes

JM, Penha de Oliveira SH, Sivieri Araújo G,etal. Cytotoxicity,

Braz Dent Sci 2024 Apr/June;27 (2): e4007

Cytotoxic eﬀects of bioceramic materials on stem cells from

human exfoliated deciduous teeth (SHED)

Oliveira BLS et al.

Cytotoxic effects of bioceramic materials on stem cells from human exfoliated deciduous teeth (SHED)

Oliveira BLS et al. Cytotoxic eﬀects of bioceramic materials on stem cells from

human exfoliated deciduous teeth (SHED)

Date submitted: 2023 Aug 21

Accept submission: 2024 June 13

Thais Marchini Oliveira

(Corresponding address)

Universidade de São Paulo, Faculdade de Odontologia de Bauru, Departamento de

Odontopediatria, Ortodontia e Saúde Coletiva, Bauru, São Paulo, Brazil.

Email: marchini@usp.br

biocompatibility, and biomineralization of the new high-

plasticity MTA Material. J Endod. 2017;43(5):774-8. http://doi.

org/10.1016/j.joen.2016.12.018. PMid:28320539.

28. Nam OH, Kim JH, Choi SC, Kim Y. Time-dependent response of

human deciduous tooth-derived dental pulp cells treated with

TheraCal LC: functional analysis of gene interactions compared

to MTA. J Clin Med. 2020;9(2):531. http://doi.org/10.3390/

jcm9020531. PMid:32075286.

29. Bin CV, Valera MC, Camargo SEA, Rabelo SB, Silva GO, Balducci

I,etal. Cytotoxicity and genotoxicity of root canal sealers based

on mineral trioxide aggregate. J Endod. 2012;38(4):495-500.

http://doi.org/10.1016/j.joen.2011.11.003. PMid:22414836.

30. Tomás-Catalá CJ, Collado-González M, García-Bernal D, Oñate-

Sánchez RE, Forner L, Llena C,etal. Biocompatibility of New Pulp-

capping Materials NeoMTA Plus, MTA Repair HP, and Biodentine

on Human Dental Pulp Stem Cells. J Endod. 2018;44(1):126-32.

http://doi.org/10.1016/j.joen.2017.07.017. PMid:29079052.

31. Bakhtiar H, Nekoofar MH, Aminishakib P, Abedi F, Naghi Moosavi

F, Esnaashari E,etal. Human pulp responses to partial pulpotomy

treatment with TheraCal as compared with biodentine and

ProRoot MTA: a clinical trial. J Endod. 2017;43(11):1786-91. http://

doi.org/10.1016/j.joen.2017.06.025. PMid:28822566.

32. Arandi NZ, Rabi T. TheraCal LC: from biochemical and

bioactive properties to clinical applications. Int J Dent.

2018;2018(1):3484653. http://doi.org/10.1155/2018/3484653.

33. Adıgüzel M, Ahmetoğlu F, Ünverdi Eldeniz A, Tekin MG,

Göğebakan B. Comparison of cytotoxic effects of calcium

silicate-based materials on human pulp fibroblasts Mehmet. J

Dent Res Dent Clin Dent Prospect. 2019;13(4):241-6. http://doi.

org/10.15171/joddd.2019.037. PMid:32190206.

34. Manojlovic D, Dramićanin MD, Miletic V, Mitić-Ćulafić D,

Jovanović B, Nikolić B. Cytotoxicity and genotoxicity of

a low-shrinkage monomer and monoacylphosphine oxide

photoinitiator: comparative analyses of individual toxicity and

combination effects in mixtures. Dent Mater. 2017;33(4):454-66.

http://doi.org/10.1016/j.dental.2017.02.002. PMid:28258769.

35. Hebling J, Lessa FCR, Nogueira I, Carvalho RM, Costa CAS.

Cytotoxicity of resin-based light-cured liners. Am J Dent.

2009;22(3):137-42. PMid:19650592.

36. Lee H, Shin Y, Kim SO, Lee HS, Choi HJ, Song JS. Comparative

study of pulpal responses to pulpotomy with ProRoot MTA,

RetroMTA, and TheraCal in dogs’ teeth. J Endod. 2015;41(8):1317-

24. http://doi.org/10.1016/j.joen.2015.04.007. PMid:26015158.

37. Poggio C, Beltrami R, Colombo M, Ceci M, Dagna A, Chiesa M.

In vitro antibacterial activity of different pulp capping materials.

J Clin Exp Dent. 2015;7(5):e584-8. http://doi.org/10.4317/

jced.52401. PMid:26644833.

38. Jeanneau C, Laurent P, Rombouts C, Giraud T, About I. Light-

cured tricalcium silicate toxicity to the dental pulp. J Endod.

2017;43(12):2074-80. http://doi.org/10.1016/j.joen.2017.07.010.

PMid:29032813.

39. Kunert M, Rozpedek-Kaminska W, Galita G, Sauro S, Bourgi

R, Hardan L, etal. The cytotoxicity and genotoxicity of

bioactive dental materials. Cells. 2022;11(20):3238. http://doi.

org/10.3390/cells11203238. PMid:36291107.

40. Camilleri J, Laurent P, About I. Hydration of biodentine, Theracal

LC, and a prototype tricalcium silicate-based dentin replacement

material after pulp capping in entire tooth cultures. J Endod.

2014;40(11):1846-54. http://doi.org/10.1016/j.joen.2014.06.018.

PMid:25154317.

41. Paula A, Laranjo M, Marto CM, Abrantes AM, Casalta-Lopes J,

Gonçalves AC,etal. BiodentineTM Boosts, WhiteProRoot®MTA

Increases and Life® suppresses odontoblast activity. Materials

(Basel). 2019;12(7):1184. http://doi.org/10.3390/ma12071184.

PMid:30978943.

42. Sequeira DB, Seabra CM, Palma PJ, Cardoso AL, Peça J, Santos

JM. Effects of a new bioceramic material on human apical papilla

cells. J Funct Biomater. 2018;9(4):74. http://doi.org/10.3390/

jfb9040074. PMid:30558359.

43. Bortoluzzi EA, Niu LN, Palani CD, El-Awady AR, Hammond BD,

Pei DD,etal. Cytotoxicity and osteogenic potential of silicate

calcium cements as potential protective materials for pulpal

revascularization. Dent Mater. 2015;31(12):1510-22. http://doi.

org/10.1016/j.dental.2015.09.020. PMid:26494267.

44. Paula AB, Laranjo M, Coelho AS, Abrantes AM, Gonçalves AC,

Sarmento-Ribeiro AB,etal. Accessing the cytotoxicity and cell

response to biomaterials. J Vis Exp. 2021;2021(173):1. http://

doi.org/10.3791/61512. PMid:34309590.