Content uploaded by Paolo De Angelis
Author content
All content in this area was uploaded by Paolo De Angelis on Feb 12, 2020
Content may be subject to copyright.
930
Abstract. – OBJECTIVE: The objective of this
work is to compare cellular toxicity in vitro of
two resins for orthodontic use: an auto-polym-
erizable composite and a photo-polymerizable
composite.
MATERIALS AND M ETHODS: Samples were
obtained by joining a couple of steel ortho-
dontic brackets by using auto-polymerizing
or photo-polymerizing resin. We used a hal-
ogen lamp, a mini LED lamp and a fast LED
lamp used for orthodontics cure for 40 seconds.
The 3T3 Swiss cellular line of broblasts was
used. The samples obtained were used to deter-
mine the cellular toxicity in vitro using the Neu-
tral Red Up -take (NRU) and the 3-(4,5-dimeth-
ylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide
(MTT ) ass ay.
RE S ULT S : Toxicity of the extract appraised at
a low level at MTT and NRU assays. There were
statistically relevant differences between the
toxicity induced by the auto-polymerizing mate-
rial and the toxicity induced by the photo-polym-
erizing composite material, polymerized with the
blue-light lamp (p < 0.001) and with the mini LED
lamp (p < 0.05).
CONCLUSIONS: From the data collected in
this study, we can conclude that both resins
show a low level of cytotoxicity that, in the case
of photochemical polymerizing resin, depends
on the characteristics of the lamp.
Key Words:
Orthodontic, Composites, Monomers, Cytotoxici-
ty, Fibroblasts.
Introduction
In recent years brackets replaced bands in or-
thodontics, although they are only used from the
front teeth to the premolars and, consequently,
composites have become a very important topic
in orthodontics, being the most used adhesive
system.
Composites and cements have the following
characteristics:
– Optical properties: they contain lithium, bari-
um, strontium or other elements. They absorb
X-rays and they can appear radio-opaque (ra-
diodense) to X-rays;
– Mechanical properties: when weight is applied,
the structure deforms because of the compres-
sion of its connection that can be otherwise
pulled or cut;
– Biological properties: exposition time and po-
tentially toxic substance rate are two important
clinical factors that determine toxicity. When
using these materials on patients, the biological
properties that can cause toxicity and sensitiv-
ity reactions, both locally and systematically,
must be known.
Biocompatibility and properties of the com-
posite resins are linked to the release of mono-
mers and reagents (activators, initiators, stabi-
lizers, inhibitors, etc.) present in the materials.
Researchers1-4 have detected that monomers,
like bisphenol A-diglycidyl-dimethacrylate
(Bis-GMA), urethane-dimethacrylate (UD-
MA), comonomers like triethylene-glycol-di-
methacrylate (TEGDMA), 2-hydroxyeth-
yl-methacrylate (HEMA) and initiators, like
camphorquinone (CQ), are released by com-
posite resins, glass ionomer cement, and dentin-
al adhesive.
European Review for Medical and Pharmacological Sciences 2020; 24: 930-934
P.C. PASSARELLI1, S. SACCOMANNO2, P. DE ANGELIS1, A. ROMEO1,
G.B. PICCIRILLO1, V. DESANTIS1, C. GRIPPAUDO2, A. D’ADDONA1
1Department of Head and Neck, Division of Oral Surgery and Implantology, Institute of Clinical
Dentistry, Università Cattolica del Sacro Cuore, and 2Department of Head and Neck, Division
of Orthodontics, Institute of Clinical Dentistry; Università Cattolica del Sacro Cuore, Fondazione
Policlinico Universitario Gemelli IRCCS, Rome, Italy
Pier Carmine Passarelli and Sabina Saccomanno have contributed equally to this work
Corresponding Author: Pier Carmine Passarelli, DDS; e-mail: piercarminepassarelli@hotmail.it
Study of cellular toxicity in vitro of two resins
for orthodontic use
Study of cellular toxicity in vitro of two resins for orthodontic use
931
Dental monomers can be released in the oral
cavity and the tooth/material interface, due to an
incomplete polymerization and to the resinous
nature of the matrix.
Furthermore, once released, dental monomers
can be quickly absorbed by the body, forming
intermediate metabolites that can be more toxic
than the monomer itself, after its release5.
Some biological problems, connected to res-
in-based dental materials (RBDM), prove their
apparent biocompatibility.
Some studies have shown potential risks,
linked to the monomer release, such as local im-
munological effects6, apoptotic reactions7,8 , and
inammatory reactions9.
Other studies have also demonstrated that
RBDM can have a systemic estrogenic effect10
or they can cause allergic reactions11 or they can
even have a carcinogenic effect12. Therefore, when
biological materials are used, they need to be as
compatible as possible. The most used materials
are micro-lled acrylic resins, available in various
forms and distinguished by contents, lling, and
polymerization (chemical or photoinduced).
Auto-Polymerizable Composites
In orthodontics, auto-polymerizable composite
resins are available in two different components:
groundwood pulp-catalyst and resin bonding
agent-catalyst, that are blended together before
using. From that moment, there is a limited time
for handling it and for clinical use of the material
before the polymerization process begins.
Photo-Polymerizable Composites
The light source provides energy able to inter-
act with photosensitive activators present in the
material, causing free radicals formation. Free
radicals can open carbon chain double binding
in the monomers of the composite, making them
available for the polymerization process that ex-
plodes in a chain reaction.
Photopolymerization requires a certain quanti-
ty of energy and it is produced by the radiant ux
by the ow time of the radiant ux itself.
The photo-polymerizing light must have a
wavelength between 400 and 500 nm (blue light)
because the photo-initiator (camphorquinone/ter-
tiary ammine), present in most composites, is
sensitive to wavelengths near 470 nm. There are
also other photo-initiators (light-activated) that
are specically sensitive to other wavelengths,
always in the indicated span. Light sources used
for photoinitiation are halogen, LED or plasma.
Purpose of the Work
This work aims to compare and contrast cel-
lular toxicity in vitro of two resins used in ortho-
dontics: one auto-polymerizable (Orthocryl, Den-
taurum, Ispringen, Germany) and another one
photo-polymerizable composite (Transbond XT
Unitek, 3M, Maplewood, MN, USA). This was
accomplished by cytotoxicity assay 3-(4,5-di-
methylthiazol-2-Yl)-2,5-diphenyltetrazolium bro-
mide (MTT) on the murine cellular line 3T3
Swiss.
Materials and Methods
Cells and Treatments
The 3T3 Swiss cellular line of broblasts was
grown in incubator at an atmosphere with 5%
CO2 at 37°C in Dulbecco’s Modied Eagle’s
Medium (DMEM) with hepes (10 mM), glucose
(1 g/L), NaHCO3 (3,7 g/L), penicillin (100 U/
ml), streptomycin (100 μg/ml) and 10% Fetal calf
serum (FCS).
The samples were obtained by joining a couple
of steel orthodontic brackets (Sweden & Martina,
Due Carrare, PD, Italy) by using auto-polymer-
izing resin following producers’ instructions or
by using the photopolymerizing resin following
the producers’ instructions and using different
lamps.
We used a halogen lamp (Blue-light pro, Mec-
tron, Loreto, AN, Italy) for an illumination period
of 40 seconds, a mini LED lamp (Mini LED,
Acteon, Mérignac, France) and a fast LED lamp
(Ortholux, 3M, Maplewood, MN, USA) used for
orthodontics cure also for 40 seconds.
Samples obtained following these premises
were used to determine the cellular toxicity in
vitro using the Neutral Red Uptake (NRU) and
the MTT assay.
Toxicity of the Eluates After 24 Hours
Every sample was immersed in 1 ml of DMEM
and left in situ for 24 hours, at a temperature of
37°C. At the same time, 10.000 3T3 Swiss bro-
blasts were sowed in each well of a 96 well plate
and put into a culture for 24 hours, up to the for-
mation of a monomolecular layer.
After incubation, 200 μL of DMEM, contain-
ing what had been released by the composite resin
to the cellular monomolecular layer, were added.
After the other 24 hours, the cellular viability
was judged with the MTT assay and the NRU
(Figure 1).
P.C. Passarelli, S. Saccomanno, P. De Angelis, A. Romeo, G.B. Piccirillo, et al.
932
MTT Assay
The MTT assay was executed following the
procedure described by Wataha et al13: 20 μl of
MTT was dissolved in phosphate-buffered saline
(PBS), at a concentration of 5 mg/ml. The solu-
tion was added to the culture medium and, after
a 4 hourlong incubation at 37°C, the intracellu-
lar formazan crystals produced were solubilized
with a muriatic acid solution in isopropanol.
The absorbance of the solution in each well was
determined by using an automatic exposure meter
for microplates (Packard Spectracount, Packard
BioScience Company, Meriden, CT, USA) at the
wavelength of 570 nm. The NRU was executed
according to Borenfreund and Puerner14.
A water solution of neutral red (0,4%) was add-
ed until it reached a concentration of 50 μg/ml.
Everything was placed in an incubator at 37°C for
4 hours, then, the supernatant was removed. The
neutral red captured by the viable cells was sol-
ubilized with 200 µL of a solution, made of eth-
anol at 50% and acetic acid at 1%. An automatic
photometer for microplates with a wavelength of
540 nm was used to calculate the optical density
(OD) of each well.
For each experiment, realized in quadruple
copies and repeated for three times, the cellular
toxicity was calculated through the equation de-
scribed by Hashieh et al15.
Statistical Analysis
All the values were expressed as mean and
standard error of mean (SEM). The means groups
were compared through a variance analysis
(ANOVA), followed by a multiple means compar-
ison through the Student-Newman-Keuls meth-
od. Following the t-Student method of means
comparison, p < 0.05 was considered statistically
signicant.
Results
Toxicity of the extracts appraised through the
MTT assay (Figure 2A and 2B): both materials
showed slight toxicity (inferior to 20%) without
signicative rate differences.
Extracts toxicity appraised through the NRU
assay (Figure 3A, 3B, and 3C): both materials
showed toxicity between 30% and 50% (depend-
ing on the lamp used for the polymerization)
(Figure 3A and 3B). There were statistically
relevant differences between the toxicity induced
by the auto-polymerizing material and the toxic-
ity induced by the photopolymerizing composite
material polymerized with the blue-light lamp (p
< 0.001) and with the mini LED lamp (p < 0.05)
(Fig ure 3C).
Discussion
This study evaluated the cytotoxicity of two
resins for orthodontic use: a chemically polym-
erized resin and a photochemically polymer-
ized one.
The cytotoxicity assays were characterized by
three factors16:
Figur e 1. MTT and NRU assays performing procedure.
Study of cellular toxicity in vitro of two resins for orthodontic use
933
1. Cellular culture
2. Cell/material contact
3. Final parameter to judge (it variates depending
on the nature of the examined material).
In this investigation, 3T3 Swiss broblasts
were used because they are recommended by
the International Standard Organization (ISO)17
among the cellular lines for in vitro studies of
dental materials.
The cell-material contact used was mediat-
ed with eluate because it requires only a set of
samples for each multiple measure, but above all,
since the evaluated materials were for orthodon-
tic use, the contact between cells and oral cavity
was mediated by saliva.
The different cytotoxicity assays value differ-
ent parameters, all inuenced in different ways,
depending on the chemical nature of the compo-
nents of the material.
We decided to use two assays because this
study concerns the composite resins, made of ma-
terials of various chemical nature, in particular
NRU, more sensitive to lipophilic substances, and
MTT, more sensitive to hydrophilic substances,
instead.
The NRU assay is used to evaluate the toxicity
of lipophilic substances because it evaluates the
cell wall integrity: the live cells, incubated in
presence of Neutral Red, capture and withhold
dyestuff; on the contrary, the cells with a dam-
aged membrane cannot retain the dye after the
washing and xation procedures18.
On the other hand, hydrophilic substances
do not damage cell walls, but can interact with
intracellular enzymes. For this reason, their ef-
fects can be evaluated with functional assays like
MTT19.
This study was based on the capacity of the
succinate dehydrogenase enzymes of live cells to
Figure 2. Toxicity induced by auto-polymerizable resin and by orthodontic brackets (A). Toxicity induced by photo-
polymerizable resin in different photo-polymerization conditions, and toxicity induced by brackets (B).
Figure 3. Toxicity induced by auto-polymerizable resin and by orthodontic brackets (A). Toxicity induced by photo-
polymerizable resin in different photo-polymerization conditions, and toxicity induced by brackets (B). Toxicity induced by
both resins (C) (*p < 0.05 vs. auto-polymer izable, **p < 0.01 vs. auto-polymerizable, ***p < 0.001 vs. auto-polymerizable).
P.C. Passarelli, S. Saccomanno, P. De Angelis, A. Romeo, G.B. Piccirillo, et al.
934
transform the soluble salt bromide of 3-(4,5-di-
metiltiazol-2-il)-2,5-difeniltetrazole into insolu-
ble formazan that precipitates inside the cells.
As expected, due to the preponderancy of li-
po-soluble substances in both the resins, the NRU
assay turned out to be more sensitive than the
MTT assay and, for this reason, the evaluation
between the two samples was made only for the
NRU assay.
A comparison between the results, obtained
through different conditions of polymerization
applied to the same material, was made for both
the assays. Besides, the higher sensibility of the
assay RNU was evident in this kind of analysis.
Conclusions
In line with the data collected, both resins
show a low level of cytotoxicity that, in the case
of photochemical polymerizing resin, depend on
the lamp’s characteristics.
Conflict of Interest
The Authors declare that they have no conict of interests.
References
1) Geurtsen W, Lehma nn F, spahL W, Le yhausen G. Cy-
totoxicity of 35 dental resin composite monomers/
additives in permanent 3T3 and three human pri-
mary broblast cultures. J Biomed Mater Res
19 98; 41: 474 -480 .
2) san terre Jp, shaJii L, LeunG BW. Relation of dental
composite formulations to their degradation and
the release of hydrolyzed polymeric-resin-derived
products. Crit Rev Oral Biol Med 2001; 12: 136-
151.
3) micheL sen VB, Kopperud hB, LyGre GB, BJörK man L,
Jensen e, KLeV en is, sVahn J, LyGre h. Detection and
quantication of monomers in unstimulated whole
saliva after treatment with resin-based composite
llings in vivo. Eur J Oral Sci 2012; 120: 89-95.
4) ma riGo L, spaGnuoLo G, maL ar a F, ma rtora na Ge,
cordaro m, Lupi a, nocca G. Relation between
conversion degree and cytotoxicity of a ow-
able bulk-ll and three conventional owable res-
in-composites. Eur Rev Med Pharmacol Sci 2015;
19: 4469-4480.
5) reichL FX, seiss m, Buters J, Behrendt h, hicKeL r,
durner J. Expression of CYP450-2E1 and for-
mation of 2,3-epoxymethacrylic acid (2,3-EMA)
in human oral cells exposed to dental materials.
Dent Mater 2010; 26: 1151-1156.
6) Jont eLL m, hanKs ct, BrateL J, BerGenho Ltz G. Ef-
fects of unpolymerized resin components on the
func-tion of accessory cells derived from the rat
incisor pulp. J Dent Res 1995; 74: 1162-1167.
7) JanKe V, Von neuhoFF n, schLeGeLBerGer B, Leyh au-
sen G, Geurtsen W. TEGDMA causes apoptosis in
primary human gingival broblasts. J Dent Res
2003; 82: 814-818.
8) spaGnuoLo G, d’antò V, renGo s. Citotossicità dei
monomeri dentari. Giornale Italiano di Conserva-
tiva 2006; 1: 18-32.
9) heBLi nG J, Giro em, costa ca. Biocompatibility of
an adhesive system applied to exposed human
dental pulp. J Endod 1999; 25: 676-682.
10) schaFer te, La pp ca , ha nes cm, LeWis JB, Wataha
Jc, schuster Gs. Estrogenicity of bisphenol A and
bisphenol A dimethacrylate in vitro. J Biomed Ma-
ter Res 1999; 45: 192-197.
11) Katsuno K, man aBe a, itoh K, na Kam ura y, WaKu-
moto s, hisamitsu h, yoshida t. Contact dermatitis
caused by 2-HEMA and GM dentin primer solu-
tions applied to guinea pigs and humans. Dent
Mater J 1996; 15: 22-30.
12) schWeiKL h, schm aLz G, spruss t. The induction of
micro nuclei in vitr o by unpolymer ize d resin mono-
mers. J Dent Res 2001; 80: 1615-1620.
13) Wataha Jc, cra iG rG, han Ks ct. Precision of and
new methods for testing in vitro alloy cytotoxicity.
Dent Mater 1992; 8: 65-70.
14) BorenFreund e, puerner Ja. A simple quantitative
procedure using monolayer cultures for Cytotox-
icity assay (HTD/NR-90). J Tiss Cult Meth 1984;
9: 7-9.
15) hashieh ia, cosse t a, Franquin Jc, ca mps J. In vitro
cytotoxicity of one-step dentin bonding systems.
J Endod 1999; 25: 89-92.
16) schmaLz G. Use of cell cultures for toxicity testing
of dental materials--advantages and limitations. J
Dent 1994; 22 Suppl 2: S6-S11.
17) iso 7405. Dentistry – preclinical evaluation of bio-
compatibility of medical devices used in dentistry
- test methods for dental materials. International
Standards Organization, 1996.
18) BaBich h, BorenFreun d e. Applications of the neu-
tral red cytotoxicity assay to in vitro toxicology.
ATLA 1990; 18: 129-144.
19) edmondson Jm, armstronG Ls, martine z ao. A rap-
id and simple MTT-based spectrophotometric as-
say for determining drug sensitivity in monolalay-
er cultures. J Tiss Cult Meth 1988; 11: 15-17.
