Dental Materials 40 (2024) 1515–1523

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Dental Materials
journal homepage: www.elsevier.com/locate/dental

Cytotoxicity and microbiological behavior of universal resin

composite cements
Uros Josic a, Gabriella Teti a , Andrei Ionescu b, Tatjana Maravic a, Claudia Mazzitelli a ,
Stevan Cokic c, Bart Van Meerbeek c , Mirella Falconi a , Eugenio Brambilla b, Annalisa Mazzoni a ,
Lorenzo Breschi a,*
a
Department for Biomedical and Neuromotor Sciences, University of Bologna, Bologna, Italy
b
Oral Microbiology and Biomaterials Laboratory, Department of Biomedical, Surgical and Dental Sciences, University of Milan, Milan, Italy
c
KU Leuven (University of Leuven), Department of Oral Health Sciences, BIOMAT & University Hospitals Leuven (UZ Leuven), Dentistry, Leuven, Belgium

A R T I C L E I N F O A B S T R A C T

Keywords: Objectives: To investigate the cytotoxicity on human dental pulp cells (HDPCs) and Streptococcus mutans (S.
Cytotoxicity mutans) biofilm formation on universal resin composite cements (UCs).
Universal resin cements Methods: Three UCs (RelyX Universal, 3 M Oral Care - RXU; Panavia SA Cement Universal, Kuraray Noritake -
Cytokines
PSAU; SoloCem, Coltene - SCM) and one ‘gold-standard’ multi-step cement (Panavia V5, Kuraray Noritake - PV5)
Polymerization
Biofilm formation
were used following two polymerization protocols (light-cured - LC; self-cured - SC). Cytotoxicity (MTT) tests
Bioreactor were performed after 1, 3 and 7 days of direct contact. Carboxy-2′,7′-dichlorodihydrofluorescein diacetate was
used to detect the release of reactive oxygen species (ROS), and interleukin 6 (IL-6) expression was analyzed by
IL-6 proquantum high sensitivity immunoassay. S. mutans biofilms were grown on UCs samples in a bioreactor
for 24 h, then adherent viable biomass was assessed using MTT assay. For microbiological procedures, half of UCs
samples underwent accelerated aging. Data were statistically analyzed (α = 0.05).
Results: The highest cytotoxicity was observed for PSAU SC, RXU SC, and PV5 SC at day 1, then for SC RXU after 3
days, and SC PSAU, LC PV5 and SCM after 1-week (p < 0.05). There was no increase in IL-6 expression after 1
day, while it increased depending on the group at 3 and 7 days. The highest ROS expression after 12 h was
recorded for PSAU SC, PV5 SC and PV5 LC. Biofilm formation was as follows: RXU > > PSAU = PV5 > SCM,
while light-curing systematically decreased biofilm formation (≈− 33 %). Aging leveled out differences between
UCs and between polymerization protocols.
Significance: The choice of cement brand, rather than category, and polymerization protocol influence cell
viability and microbiological behavior. Light-curing is beneficial for reducing the harmful pulpal effect that UCs
may possess.

1. Introduction polymerization reaction [3]. Other features displayed by UCs are the
implementation of adhesion-promoting functional monomers and/or
Resin composite cements can be classified into 3 main categories: silane in their formulation, and dual-cure polymerization mechanisms
primer/adhesive-assisted or multi-step, self-adhesive/one-step, and (auto/photo) [1].
universal resin composite cements (UCs) [1]. The latter have most To date, only three UCs are available on the market [1], with more
recently been introduced following the tendency of dental industry to- expected to come shortly. Each of them has peculiar characteristics
wards simplification and versatility in everyday practice [2]. UCs can be regarding their chemical composition: the incorporation of a silane
essentially viewed as the latest generation of self-adhesive cements. coupling agent alongside one functional monomer such as 10-methacry-
However, unlike their predecessors, UCs can be successfully coupled loyloxydecyl dihydrogen thiophosphate (10-MDP) in the case of Panavia
with their own representative universal adhesive without issues related SA Cement Universal (‘PSAU’, Kuraray Noritake, Tokyo, Japan); the
to chemical incompatibility and consequent adverse effects on integration of two functional monomers (10-MDP and

* Correspondence to: Department for Biomedical and Neuromotor Sciences, DIBINEM, University of Bologna, Via San Vitale 59, Bologna 40125, Italy.
E-mail address: [email protected] (L. Breschi).

https://doi.org/10.1016/j.dental.2024.07.004
Received 13 December 2023; Received in revised form 8 July 2024; Accepted 22 July 2024
Available online 25 July 2024
0109-5641/© 2024 The Authors. Published by Elsevier Inc. on behalf of The Academy of Dental Materials. This is an open access article under the CC BY-NC-ND
license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
U. Josic et al. Dental Materials 40 (2024) 1515–1523

4-methacryloxyethyl trimellitic anhydride (4-META)) in SoloCem Table 1

(‘SCM’, Coltène, Alstätten, Switzerland), and the absence of bisphenol A The composition of the materials used in the present study.
(BPA) derivates as reported by the patent literature for RelyX Universal Resin cement, shade and batch Composition
(‘RXU’, 3 M Oral Care, Seefeld, Germany). Furthermore, RXU is char- number
acterized by the presence of glycerol phosphate dimethacrylate (GPDM). RelyX Universal, 3 M ESPE A3O γMPTES, reaction products with vitreous silica,
The physical properties and bonding performance of PSAU have been (LOT 7182977) DUDMA, TEGDMA, Mixture of GPDMA,
investigated in recent laboratory studies [4–7], along with one clinical bisGPDMA and trisGPDMA, silane treated silica,
case reporting its use during luting procedures to enamel [8]. Similarly, t-Amyl Hydroperoxide, 2,6-Di-tert-butyl-p-
cresol, HEMA, Methyl Methacrylate, Acetic acid,
data from in vitro studies about biomechanical, optical, and adhesive copper(2 +) salt, monohydrate, Ytterbium (III)
properties of SCM [9,10] and RXU can also be found in literature fluoride Silane-treated glass powder, L-Ascorbic
[11–17]. Current scientific literature generally encourages using UCs acid, 6-hexadecanoate, hydrate (1:2), silane
since they showed comparable or improved bonding properties treated silica, Titanium Dioxide Triphenyl
Phosphite
compared to their predecessors. Furthermore, they have proven to be a
Panavia SA Cement Universal, 10-MDP, Bis-GMA, TEGDMA, HEMA, silanted
reliable luting material for indirect restorations, as they can achieve Kuraray Noritake A2 (LOT barium glass filler, silanted colloidal silica,
good bond strength values to various restorative materials such as AT0139) Catalysts, Pigments, Paste B Hydrophobic
composites and ceramics [1]. aromatic dimethacrylate, Silane coupling agent
From a biological point of view, it is essential to mention that all (LCSi proprietary monomer), Silanated barium
glass filler, Aluminum oxide filler, Surface
resin composite cements can display cytotoxic properties towards treated sodium fluoride (Less than 1 %),
mesenchymal cells and fibroblasts, independently from being primer/ Camphorquinone Accelerators Pigments,
adhesive-assisted or self-adhesive [18]. Indeed, the most frequent bio- Peroxide, Camphorquinone
logical complications seen in indirect restorations are the need for SoloCem, Coltène/Whaledent 2,6-di-tert-butyl- 4-methylphenol, 10-MDP,
Dentin (LOT L48519) dibenzoyl peroxide (BPO initiator), 4-META,
endodontic therapy and secondary caries [19]. The cytotoxic effect is
TEGDMA, DUDMA, Bis-GMA, HEMA, zinc oxide,
especially noticeable when composite cements are not properly poly- ytterbium(III) fluoride
merized. The lower degree of conversion (DC) observed when dual-cure Panavia V5, Kuraray Noritake, Bis-GMA, TEGDMA, aromatic multifunctional
cements are not adequately light-cured facilitates leakage of potentially A2 (LOT 970213) monomer, aliphatic multifunctional monomer,
toxic monomers from the cement’s matrix [18,20]. Considering that UCs new chemical polymerization accelerator, dl-
camphor quinone, photopolymerization
were the last to be launched into the dental market, it is still not known if
accelerator, surface treated barium glass,
these materials may cause reduction in cell viability and lead to in- fluoroaluminosilicate glass, fine particulate
flammatory reaction when in close contact with dental pulp cells. filler
Furthermore, it is unknown to what extent biofilm masses of car- Abbreviations: 10-MDP, 10-Methacryloyloxydecyl dihydrogen phosphate; 4-
iopathogens (such as S. mutans) can be formed to UCs surface. For that META, 4-methacryloxyethyl trimellitic anhydride; APTES, (3-aminopropyl)
reason, this laboratory study aimed to investigate the potential cytotoxic triethoxysilane; Bis-GMA, Bisphenol A diglycidylmethacrylate; bisGPDMA, bis
effect of universal resin composite cements depending on their poly- (gliceryldimethacrylate) phosphate; DEGDMA, Diethylene glycol dimethacry-
merization protocol (light-cure [LC] or self-cure [SC]) and in compari- late; DUDMA, diurethane dimethacrylate; GPDMA, glycerol phosphate dime-
son to a ‘gold-standard’ [21] primer-assisted cement (Panavia V5, thacrylate; HEMA, 2-Hydroxymethacrylate; LCSi, long carbon-chain silane
Kuraray Noritake: ‘PV5’). The production of inflammatory cytokines coupling agent; TEGDMA, Triethyleneglycol dimethacrylate; trisGPDMA, tris
(Interleukin 6 [IL6]) and generation of reactive oxygen species (ROS) (glyceryldimethacrylate) phosphate; γMPTES, 2-Propenoic acid, 2-methyl-, 3-
from human dental pulp cells (HDPCs) in the presence of the investi- (trimethoxysilyl)propyl ester.
gated cements were additionally analyzed. The null hypotheses tested
were that: (1) UCs do not exhibit cytotoxic potential and ability to air-dried, and sterilized under UV light for 30 min (15 min from each
trigger an inflammatory response from HDPCs, and (2) the polymeri- side) [22]. For each time point, 4 disks per cement were prepared, and
zation protocol does not influence HDPCs viability and microbiological the experiments were repeated 3 times.
properties of UCs.
2.3. Resin composite cement specimen preparation for microbiological
2. Materials and methods analysis

2.1. Resin composite cements Microbrush fine tips (Microbrush International, Grafton, WI, USA)
were used to evenly distribute a fixed amount of each cement on the flat
Three commercially available UCs (Panavia SA Cement Universal, bottoms of 96-well, flat-bottom, tissue culture-treated black plates
Kuraray Noritake - ‘PSAU’; RelyX Universal, 3 M Oral Care - ‘RXU’; (Corning CLS3991 Microtiter plates, Thermo Scientific Italy, Rodano,
SoloCem, Coltène - ‘SCM’) and one ‘gold-standard’ primer-assisted resin MI, Italy). A total of 32 wells were obtained for each cement. Two holes
composite cement (Panavia V5, Kuraray Noritake - ‘PV5’), which served (diameter=3.0 mm) were produced on top of the lid of each 96-well
as a control, were investigated in this study. Table 1 details the plate. The holes provided openings for polyethylene tubing that was
composition of the cements investigated with their batch numbers. connected to cylinders containing nitrogen gas. The lids were then
hermetically sealed to the plate using impression material (Express™ 2
2.2. Resin composite cement specimen preparation for cytotoxicity testing Light Body Standard, 3 M ESPE, Seefeld, Germany). A constant flow of
nitrogen gas (1000 ml/min) was maintained over the surface of the
On the day of the experimental procedure, resin composite cement specimens for 10 min to obtain the desired oxygen-depleted atmosphere
disks (5 ×1 mm) were prepared by applying the cement into a custom- [23], then, half of the samples (n = 16) were LC using the same poly-
made polyvinyl mold positioned between two microscopic glass slides to merization protocol previously applied, fitting the tip of the light source
prevent the formation of an oxygen inhibition layer. For each cement, 2 over the top of the well, precisely matching its diameter. The other
polymerization protocols were applied: light-curing (LC: Elipar Deep unpolymerized wells (n = 16) were covered with black cardboard until
cure, 3 M Oral Care, operating at 1470 mW/cm2 for 40 s in close contact LC procedures were finished, upon which they were left in a dark
with the specimen surfaces) and self-curing (SC: 90 min in a dark chamber at 37 ◦ C for 90 min (SC). During storage in the dark, nitrogen
chamber at 37 ◦ C). Subsequently, the disks were removed from their flow was kept to a minimum positive air pressure to ensure maintaining
molds, disinfected with 80 % ethanol, rinsed with sterile distilled water, the oxygen-depleted atmosphere. Subsequently, the disks were removed

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from the wells that acted as molds and stored in a dark room under 2.6. Detection of ROS generation
running water for 24 h. Then, they were divided into two additional
subgroups (n = 8), depending on the aging procedures. Non-aged sam- The total reactive oxygen species generation was determined 12 h
ples were dried and stored in a dark room for 30 days, while plates after HDPCs’ contact with the cements by employing the fluorescent
containing the samples undergoing accelerated aging were immersed in probe 5-(and-6)-carboxy-2′,7′-dichlorodihydrofluorescein diacetate
excess absolute ethanol at 37 ◦ C for 24 h (500 ml), and then stored under (DCFDA). Positive controls were introduced by adding 50-μM hydrogen
running water for 30 days [24]. peroxide on HDPCs 30 min before the reading procedure by the GloMax
Discover Microplate Reader (Promega Italia, Milan, Italy) (excitation/
2.4. Cell cultures and exposure of cells to resin composite cements emission = 485/535 nm, respectively).

HDPCs were purchased from Lonza (Euroclone, Milan, Italy). They 2.7. Microbiological procedures
were cultivated in high glucose Dulbecco’s Modified Eagle Medium
(DMEM) (Sigma-Aldrich, Munich, Germany), supplemented with 10 % The culture media and reagents were obtained from Becton-
Fetal Bovine Serum (FBS) (Gibco, Thermo Fisher Scientific, Monza, Dickinson (BD Diagnostics-Difco, Franklin Lakes, NJ, USA). An artifi-
Italy). The cells were then incubated at 37 ◦ C in a humidified atmo- cial saliva medium (ASM) simulating the average electrolyte composi-
sphere of 5 % CO2 in air and the medium was changed every 3 days until tion of human whole saliva was prepared from 0.1 L of 150 mM KHCO3,
the cells reached a sub-confluent state. HDPCs between 3rd and 9th 0.1 L of 100 mM NaCl, 0.1 L of 25 mM K2HPO4, 0.1 L of 24 mM
passages were used during all experiments. Na2HPO4, 0.1 L of 15 mM CaCl2, 0.1 L of 1.5 mM MgCl2 and 0.006 L of
In order to perform a 3-(4,5-dimethylthiazol-2-yl)− 2,5-diphenylte- 25 mM citric acid. The volume was made up to 1 L, and the pH was
trazolium bromide MTT (Sigma-Aldrich) assay, sub-confluent cells were adjusted to 7.0 by pipetting 4 M NaOH or 4 M HCl solutions under
detached with 0.25 % trypsin and 0.05 % EDTA for 1 min and seeded vigorous stirring [29]. A pure suspension of S. mutans strain ATCC 35668
into 24-plates at a concentration of 3 × 104 per well, into which 300 μl of in brain-heart infusion broth (BHI) was obtained at 37 ◦ C in a 5 %
fresh DMEM with 10 % (FCS) was added. The cells were then incubated supplemented CO2 environment after overnight incubation. Cells were
at 37 ◦ C for the next 24 h, after which the resin disks were gently placed harvested by centrifugation (1.500g, 19 ◦ C, 5 min), washed twice with
into wells under aseptic conditions [25]. Wells with HDPCs and no resin sterile ASM, and resuspended in the same medium. The cell suspensions
material served as control, while blank controls [26] consisted of wells were subsequently subjected to sonication (Sonifier model B-150;
with DMEM and FCS in which composite disks were placed. The plates Branson, Danbury, CT, USA; 7 W energy output, 30 s) to disperse the
were further incubated, and cytotoxicity tests were performed after 1, 3, bacterial chains; then each suspension was adjusted to 0.5 McFarland.
and 7 days of HDPCs contact with the tested cements. Briefly, 30 μl of Biofilm formation was obtained using a modified drip-flow biore-
MTT dye at a concentration of 5 mg/ml was added to the wells; after actor [30]. The bioreactor allowed the placement of PTFE sample car-
180-min incubation, the insoluble formazan produced by vital HDPCs riers on the bottom of the flow cells to completely submerge the surface
was dissolved with 300 μl di-methyl sulfoxide. After complete solubili- of the disks (n = 8/group and subgroup) under the flowing medium. The
zation of formazan crystals, optical density (OD=590 nm) was read samples were randomly allocated across the carriers and flow cells, and
using the GloMax Discover Microplate Reader (Promega Italia, Milan, the bioreactor was sterilized using a chemical peroxide-ion plasma
Italy). The OD values of the experimental groups were divided by the sterilizer (STERRAD, ASP, Irvine, CA, USA) at a maximum temperature
control and expressed as a percentage of the control. Cement’s cyto- of 45 ◦ C for one hour before starting the experiments. The bioreactor
toxicity was rated based on cell viability (%) relative to the respective was assembled inside a sterile hood. Then, each flow cell was inoculated
controls at each timepoint, according to the following equation [26–28]: with 10 ml of the previously obtained S. mutans suspension, and the
bioreactor transferred into an incubator to operate at 37 ◦ C. Time was

OD average of tested group − OD average of blank control

Cell viability = x 100%
OD average of negative control − OD average of blank control

allowed under static conditions to promote microbial adherence

For the purpose of visualizing the shape and viability of HDPCs, the (t = 4 h); then a nutrient medium flow through the bioreactor cells was
cells were cultured on glass slides within wells of a 12-well plate, as initiated using a multichannel, computer-controlled peristaltic pump
described above. Cell-viability staining (LIVE/DEAD Viability/Cyto- (RP-1, Rainin, Emeryville, CA, USA). The sterile culture medium
toxicity Assay kit, Invitrogen, Thermo Fisher Scientific) was performed included 2.5 g/L mucin (type II, porcine gastric), 2.0 g/L bacteriological
after 1 day of cell contact with the cements, upon which the specimens peptone, 2.0 g/L tryptone, 1.0 g/L yeast extract, 0.35 g/L NaCl, 0.2 g/L
were observed using a Nikon Eclipse E800 (Nikon, Melville, NY) KCl, 0.2 g/L CaCl2, 0.1 g/L cysteine hydrochloride,0.001 g/L hemin,
microscope. and 0.0002 g/L vitamin K1, and was supplemented with 1 wt% sucrose.
The flow rate was set to 20 ml/h. After 24 h, the culture medium flow
2.5. Analysis of IL-6 release was stopped, and all samples were gently extracted from the flow cells.
Assessment of the viable biomass adherent to the surface of the samples
HDPCs seeded in 24-well plates were exposed to resin composite was then performed.
cements as described above; the supernatant was collected on days 1, 3,
and 7 and stored until further measurements were performed. The level
(pg/ml) of IL-6 within the supernatant was analyzed using human IL-6 2.8. Viable microbial biomass assessment
pro quantum high sensitivity immunoassay (Invitrogen, Thermo Fisher
Scientific, Monza, Italy) following the manufacturers’ instructions. The viable biomass adherent to the surface of each sample was
assessed using a modification of the MTT test [30]. Briefly, two starter
stock solutions were prepared by dissolving, respectively, 5 mg/ml of

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3-(4,5)-dimethylthiazol-2-yl-2,5- diphenyltetrazolium bromide (MTT), standard fibroblast like morphology. Cells exposed to LC composite ce-
or 0.3 mg/ml of N-methylphenazinium methyl sulfate (PMS) in sterile ments save their spindle-like morphology altough the numer seems
phosphate-buffered saline (PBS). The solutions were stored at 2 ◦ C in lightly reduced compared to control samples, in agreement with the
light-proof vials until the day of the experiment, when a measurement MTT data previously described. Cells exposed to SC materials showed a
solution (MS) was made by mixing 1 ml of MTT stock solution, 1 ml of reduced cellular density compared to control samples, suggesting
PMS stock solution, and 8 ml of sterile PBS. A lysing solution (LS) was cellular toxicity presumably connected with the SC polymerization
prepared by dissolving 10 % v/v of sodium dodecyl sulfate and 50 % v/v protocol.
of dimethylformamide in distilled water. Live and dead assay confirm the morphologidal data previously
Samples extracted from the bioreactor were washed 3 times with described and allow us to distinguish live vs. dead HDPCs after 1 day
sterile PBS at 37 ◦ C to remove loosely attached cells, then were placed exposition to resin cements. Fig. 3 demonstrates a high density of cells in
into 48-well plates containing 300 μl of MS each. They were left to react control samples, characterized by the normal fibroblast-like
at 37 ◦ C in a dark room for 1 h, and then the supernatant was gently morphology. On the contrary, the cellular density is noticeable
discarded before adding 300 μl of LS. After 30 min at 37 ◦ C in a dark reduced in cells exposed to all the SC materials where several cells
room followed by 30 min in an orbital shaker, 100 μl the solution was appear with a round shape morphology, likely connected to early cell
transferred to 96-well plates and read using a dual-wavelength spec- toxicitological events. In particular, in RXU SC and PSAU SC samples it is
trometer (550 nm and 630 nm, Genesys 10-S, Thermo Spectronic, possible to distinguish red round cells corresponding to dead cells.
Rochester, NY, USA), subtracting the second reading from the first one. After 3 days (Fig. 1), RXU SC continued to impair viability compared
In this way, turbidimetry increases by elution of leachates from the ce- to the control group (p < 0.05). Further, PSAU SC, SCM LC and PV5 LC
ments were prevented from influencing readings. led to a significant reduction in cell vitality (p < 0.05) after 1 week of
incubation compared to the control.
2.9. Statistical analysis
3.2. IL-6 release
After checking the normality (Kolmogorov–Smirnov) and the ho-
moscedasticity (modified Levene’s test) of the data, parametric or non- No increased level of IL-6 was detected after 24 h of cell exposure to
parametric tests were performed to analyze the data. In the experiments resin cements (p > 0.05) (Fig. 4). However, relative to the control,
where the data were normalized relative to the controls in each apar- HDPCs produced a significantly higher level of IL-6 when exposed to
teining time point (cell viability %, IL-6 release, ROS generation), in case RXU SC, PSAU SC and RXU LC after 3 days of incubation, with RXU SC
of normal distribution of the data (cell viability and ROS release), one- demonstrating a higher IL-6 release compared to the other groups
way analysis of variance (ANOVA) was performed for each time point (p < 0.05). After 7 days, the production of IL-6 decreased significantly
(if applicable), followed by Tukey’s multiple comparison tests to assess for RXU SC to a level comparable with that of the control group, while it
the differences between groups [31]. When the data were not normally notably increased for PSAU SC (p < 0.05). Overall, IL-6 results demon-
distributed (IL-6 release, failed Kolmogorov–Smirnov test; p < 0.05), strate a light influence of some resin cements in inducing the release of
the non-parametric Kruskal-Wallis and Dunn’s post-hoc tests were run. IL-6 up to 3 days, while the effect is almost absent after long exposition
The microbiological biomass data were normally distributed, and hence time, confirming the lack of any time dependet toxicity relation between
three-way ANOVA was performed considering the cement type, the HDPCs and resin cements.
polymerization protocol, and the aging as fixed factors. All the analyses
were performed by a statistician blinded to the groups using SigmaPlot 3.3. Generation of ROS after exposure to resin cements
14.0 (Systat Software, Chicago, IL, USA). The significance level was set
at α = 0.05. Cell exposure to PV5 SC, PV5 LC and PSAU SC generated higher ROS
(3.3-, 1.7- and 1.6-fold, respectively) compared to the control (p < 0.05)
3. Results (Fig. 5).

3.1. Cell viability (MTT) test 3.4. Viable microbial biomass

Fig. 1 shows that, compared to the control, the viability was signif- Multi-way ANOVA showed a highly significant influence of cement
icantly reduced (p < 0.05) after 1 day when cells were exposed to RXU type, polymerization protocol, and aging on microbiological behavior
SC, PSAU LC, PSAU SC and PV5 SC. Microscopic analysis of HDPCs after (p < 0.0001), and a highly significant interaction between the type of
1 day of exposure to resin cements can be seen in Figs. 2 and 3. Light cement and aging, indicating that each cement had a different aging
microscopy images (Fig. 2) show control cells characterized by the behavior. No significant interaction was highlighted between the

Fig. 1. Mean percentage of viable HDPCs after 1, 3 and 7 days of exposure to resin cements activated with different polymerization protocols (light-cured [LC] and
self-cured [SC]). Data are expressed in percentages + standard deviation (y-axis), and are relative to the non-exposed HDPCs (control =100 % viability). * indicates
statistically significant difference compared to the contro (p < 0.05).

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Fig. 2. Light microscopy images and morphological changes of HDPCs after 1 day of exposure to the investigated resin cements.

Fig. 3. Live/dead (green/red) staining with subsequent microscopic analysis of viability and morphology of HDPCs after 1 day of exposure to the tested resin ce-
ments (magnification 200X in all the images).

cement type and the polymerization protocol (p = 0.23), indicating that ultimately leveling out the differences in the microbiological behavior of
all tested cements showed a similar tendency when comparing SC with the tested cements (Fig. 6). PV5 was significantly less colonized than all
LC. other tested cements (ca. 15 % less, p < 0.05). No influence of poly-
Considering the non-aged samples, the observed colonization was as merization protocols on microbiological behavior was recorded
follows: RXU > > PSAU = PV5 > SCM, with RXU showing about two (p = 0.12).
times higher viable biomass values than PSAU and PV5, while SCM
exhibited ca. 40 % lower colonization than PSAU and PV5 (Fig. 6). 4. Discussion
Light-curing significantly decreased the viable biomass by ca. 33 % for
PSAU, PV5 and SCM (p < 0.05). Various resin cements are routinely used for luting indirect restora-
Accelerated aging caused a significant decrease in the viable biomass tions. The need for post-restorative endodontic therapy is listed among
values of RXU and increased the values of all the other tested cements, the most frequent biological complications in fixed prosthodontics [19]

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Fig. 4. Analysis of IL-6 expression levels (y-axis) within supernatants from the experimental groups after 1, 3 and 7 days of HDPCs’ exposure to resin cements. The
levels of detected IL-6 were normalized to non-exposed cells (=1.0). * indicates statistically significant difference compared to the control (p < 0.05).

compared to that observed for the controls. The percentage of viable

cells in these groups was on average below 70 % (Fig. 1), which ac-
cording to ISO 10993–5:1999(E) [35] can be considered as a charac-
teristic of a cytotoxic material. Cement cytotoxicity is a direct
consequence of its chemical composition and polymerization efficacy,
since monomers can leach out from resin composite materials, damaging
the integrity of HDPCs [36]. In particular, it was reported that the
highest cytotoxic potential of resin-based dental materials is related to
the release of Bis-GMA, followed by UDMA, TEGDMA, and HEMA [18].
As seen in Table 1, all investigated resin cements contain at least one of
the mentioned molecules that could be responsible for causing cells’
death.
One might have expected to see superior biocompatibility of RXU
cement, since data retrieved from patent literature and safety data sheet
indicated that RXU is a Bis-GMA-free material (Table 1) and that it also
contains a novel amphiphilic polymerization-initiator system that en-
Fig. 5. Analysis of ROS generation from the experimental groups after 12 h of
HDPCs’ contact with resin cements. Mean fluorescence intensities (y-axis) were hances the polymerization process, thus decreasing the possibility that
normalized to non-exposed cells (=1.0). * indicates statistically significant the cement is inadequately cured. Indeed, a laboratory study confirmed
difference compared to the control (p < 0.05). that RXU could achieve adequate DC in both self- and light-cure mode,
[37] and that no bis-GMA traces were found in the eluates of this cement
and might be due to an harmful effect of the resin cement on pulpal [16]. However, the same study reported that the total amount of eluted
tissue [32]. Besides, secondary/recurrent caries mainly limit the monomers, which mainly consisted of UDMA, was significantly higher
longevity of prosthetic treatments, leading to the most frequent cause of for RXU SC compared to RXU LC, [16] therefore explaining greater cell
crown replacement [33,34]. Considering modifications in the chemical death caused by RXU SC in our experiment. Similarly, the lower per-
composition of the latest generation of UCs, we aimed to assess potential centage of viable HDPCs observed for PV5 SC can be justified by
harmful effects of these materials on HDPCs, as well as their microbio- hampered polymerization of this cement in the absence of light-curing,
logical properties. The results of the present study indicate that the since it has recently been demonstrated that this cement requires
newest group of resin cements can harm HDPCs and have different light-curing to achieve optimal DC [38]. Additionally, it was reported
microbiological responses, by which both null hypotheses were rejected. that PV5 SC released more bis-GMA than PV5 applied in LC mode [16];
Most significant changes in cell vitality were observed after 1 day: 3 this undoubtedly contributed to the reduction of viable cells observed in
resin cements in the SC group (RXU, PAN, and PV5) and 1 cement in the our study.
LC group (PSAU) led to a significant reduction in HDPC viability, as An interesting feature of PSAU is the integration of a long carbon-
chain silane coupling agent (LCSi), which should increase the

Fig. 6. S. mutans biofilm formation on the tested resin cements. The means ( ± 1 standard error) are shown on y-axis; different letters account for significant dif-
ferences between groups (Tukey’s test, p < 0.05). Universal cements were compared to the “golden standard” multistep PV5. Groups were divided by accelerated
aging treatment. The cement type markedly influenced biofilm formation, and light-curing systematically decreased adherent bacterial biomass in non-aged samples
(≈ − 33 %). Aging leveled differences between groups, leaving only PV5 slightly less colonized (≈ − 15 %) compared to the universal cements. No effect of poly-
merization protocol on biofilm formation was shown after aging procedures.

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universality of this material regarding the substrates it can bond to, be to predict the cytotoxic potential of resin cements based solely on
while reducing the number of clinical steps during cementation. ROS production.
Although the manufacturer does not reveal the exact chemical structure IL-6 is a pro-inflammatory cytokine that is upregulated when HDPCs
of LCSi, a previous paper hypothesized that LCSi is in fact trimethox- are exposed to stress [56]. This cytokine can be regarded as one of the
ysilyl long-chain alkyl methacrylate [4]. This suggestion was supported markers of the acute phase of inflammation, as elevated levels of IL-6
by a recent study [7] that detected methoxy groups attached to siloxane have also been reported in symptomatic pulpitis [57]. In deep cavities
oligomers and polymers, which may indicate incomplete silane hydro- with only a thin layer of dentin, small hydrophilic molecules such as
lysis and the presence of silanols on the cement surface. To the best of HEMA and TEGDMA (present in all tested materials, Table 1) can
our knowledge, no data can be found regarding the cytotoxic or anti- infiltrate dentin and diffuse towards the pulp, causing subsequent
microbial properties of commonly used silane coupling agents in inflammation [58]. According to the results of our study, there was no
dentistry. On the other hand, the negative effects induced by silanols significant increase in the production of IL-6 during the first 24 h of
have gained great interest in toxicology, and many efforts have been HDPC contact with resin cements. On the contrary, even though the
made to clarify the mechanism responsible for silica pathogenicity [39]. threshold for statistically significant difference was not reached, the
Today, it is known that some subfamilies, so-called “nearly free sila- concentration of IL-6 within supernatant was in some groups even lower
nols”, can lead to membrane lysis and induce the release of as compared to the control (Fig. 4). Although it may seem unexpected,
pro-inflammatory cytokines [40]. Consequently, we cautiously postu- our finding aligns with Cokic et al. [59], who observed lower levels of
late that the reduction in cell viability observed for PSAU is most likely IL-6 when bronchial cells were exposed to the dust of commonly used
due to silanols on the cement surface [7]; future in-vitro studies need to composite materials and which have potential to relese monomers in the
verify this hypothesis. environement [60]. As suggested by the author, we also support the
At day 3, only RXU SC showed a significant reduction in cell viability hypothesis that the reduction of cell viability in groups exposed to resin
compared to the controls (Fig. 1). Our study results align with previous cements could have led to lower production of IL-6 compared to the
research that found high cytotoxicity immediately after cell exposure to controls [59]. The actual irritative effect of resin cements on HDPCs
self-adhesive cements and composite materials, which gradually became evident at day 3, since the levels of the pro-inflammatory
decreased in most groups with time [41–43]. In order to better under- cytokine were significantly increased for RXU SC, PSAU SC, and RXU
stand the observed phenomenon, it is necessary to mention that the LC, as well as after one week for PSAU SC. The clinical relevance of this
acute release of monomers within the first 24 h [44,45], alongside the finding may be the occurrence of post-operative sensitivity within days
high acidity of UCs in the first hours of the setting phase [1], is most of the cementation procedure, indicating the possible development of
likely responsible for the maximum reduction in cell viability at this irreversible pulpitis [61,62] and the subsequent need for endodontic
early time point. Between days 1 and 3, the release of monomers from treatment.
resin cements is expected to decrease due to medium saturation [46], Eluates of EGDMA and TEGDMA were demonstrated to promote
while the cements’ initially low pH increases [47]. This allowed cells to proliferation of cariogenic bacteria, whereas BisGMA had the opposite
proliferate and regain viability rates above on average 70 % in most of effect [63]. Furthermore, composite resin degradation products from
the tested groups (Fig. 1). BisGMA upregulated the expression of genes associated with biofilm
It is well accepted that regular medium change keeps the cell cultures formation and other virulence factors in S. mutans [64]. In the present
healthy by providing fresh nutrients and eliminating waste products study, resin cements showed a significant reaction to light-curing that
generated by the cells [48,49]. After 7 days, the statistically significant consistently decreased biofilm formation in all tested formulations, as
drop in cell vitality in some LC groups (Fig. 1) compared to the control, compared to self-curing. The finding must most likely be attributed to
that was considered to have 100 % cell viability for each time-point, can the fact that light-curing produced better conversion, thus minimizing
be explained by the cells prolonged exposure to the medium, which had leakage of monomers that could promote biofilm formation. Besides,
not been refreshed. In this study, no medium change was done, as this each resin cement had its own formulation, including different monomer
would have removed the monomers released during the first days of cell blends, fillers, and antimicrobial agents, making it hard to separate the
exposure to the cements. The limited amount of nutrients between days contribution of each component to stimulation/inhibition of the biofilm
3 and 7, the accumulation of released monomers [36,50], as well as the formation observed.
accumulation of metabolic products and pro-inflammatory cytokines Data showed that microbial growth of aged specimens was similar in
could have negatively influenced the biological processes and impaired all groups, independently from the polymerization protocol applied.
the function of cellular enzymes [51], eventually having led to lower Aging procedures minimized monomer release, reducing the effect that
viability observed at day 7. such phenomenon may have had on biofilm formation. A deterioration
The generation of ROS induced by resin-based materials has been of the chemical-physical surface properties following surface aging
previously observed [52,53]. ROS synthesis is related to various procedures, as previously observed [24], may have produced surfaces
monomers that leach out from the materials and can disrupt the redox with similar characteristics, that ultimately leveled out biofilm forma-
equilibrium of cells [54]. Furthermore, the accumulation of ROS can tion between groups. It is unclear which composition difference could
lead to cell apoptosis and even DNA damage [55]. In our study, the have provided the slightly lower colonization of the considered
amount of total intracellular ROS was increased 3.3-, 1.7- and 1.6-fold gold-standard resin cement (PV5), when compared to all universal ce-
for PV5 SC, PSAU LC and PV5 LC, respectively; this was statistically ments. One may speculate on a slightly better degree of conversion,
higher compared to the non-exposed cells. The increased ROS levels possibly due to the absence of functional monomers (10-MDP, 4-META,
induced by PV5 SC and PSAU LC may be considered as one of the GPDM) or other agents (fluoride, butylated hydroxytoluene) that may
mechanisms responsible for reducing cell viability in the respective have decreased the overall polymerization reaction. Another arguably
groups after 24 h. As far as we are aware, only one study [52] investi- better explanation could be provided by PV5’s F-Al-Si glass filler con-
gated the potential of SCM and PV5 to induce ROS production. Contrary tent, which is widely known for releasing significant amounts of fluoride
to our results, the authors reported increased ROS production, followed and is the main mechanism of glassionomers’ most effective antimi-
by reduced viability (69 % of reduction compared to control) when el- crobial behavior. PSAU also contains fluoride in the form of
uates of SC SCM were added to the cell culture of human gingival fi- surface-treated sodium-fluoride particles. The latter characteristic could
broblasts. Additionally, they found no significant increase of ROS for be the reason of a similar microbiological behavior displayed by PV5
PV5, although the cement did produce higher cell death at day 1 and 3 as and PSAU [65]. On the other hand, fluoride contained in YbF3 fillers
compared to the control. Taking into consideration our and the previ- (SCM, RXU) is considered relatively stable, not providing much release
ously mentioned study [52], it becomes evident how complicated it can overall, especially after 14 days [66]. Actually, the main reason of its

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presence in resin-based materials is to render dental products the desired application of the resin cement rather than being its permanent char-
radiopacity. acteristic. Adequate light-curing and the choice of the resin cement
The recent role that butylated hydroxytoluene (SCM, RXU) was brand (rather than cement class) are fundamental to avoid possible pulp
found to play as an antibiofilm agent in quorum sensing and regulation injuries and reduce biofilm formation in the first period after material
of biofilms is of note [67]. However, the results of the current study do application.
not allow us to hypothesize any significant influence of such molecule on
S. mutans biofilm formation, as other compounds can be responsible for
the decreased colonization shown by SCM, while the relatively highest Declaration of Competing Interest
colonization was observed on RXU specimens.
Zinc oxide seems to be the most responsible component for the The authors declare no conflict of interest. Uros Josic acknowledges
reduced biofilm formation observed on non-aged SCM surfaces, espe- Academy of Dental Materials Early Investigator Award.
cially the LC ones. The broad-spectrum antimicrobial effect of zinc
oxide, especially zinc-oxide nanoparticles, is indeed well-known in Acknowledgements
literature when incorporated into resin formulations [68,69]. The
complete loss of such activity by aged SCM specimens can be explained Uros Josic would like to acknowgledge Academy of Dental Materials
by its elution from the resin cement’s surface, given its relatively high 2022 Bowen Early-Stage Career Investigator Award.
solubility [70], especially at low pH.
Lastly, final considerations should be given to the methodology
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