polymers
Article
Effects of Postcuring Temperature on the Mechanical Properties
and Biocompatibility of Three-Dimensional Printed Dental
Resin Material
Enkhjargal Bayarsaikhan 1 , Jung-Hwa Lim 2 , Seung-Ho Shin 2 , Kyu-Hyung Park 2 , Young-Bum Park 1 ,
Jae-Hoon Lee 1 and Jong-Eun Kim 1, *






Citation: Bayarsaikhan, E.; Lim,
J.-H.; Shin, S.-H.; Park, K.-H.; Park,
Y.-B.; Lee, J.-H.; Kim, J.-E. Effects of
Postcuring Temperature on the
Mechanical Properties and
Biocompatibility of Three-Dimensional
Printed Dental Resin Material.
Polymers 2021, 13, 1180. https://
doi.org/10.3390/polym13081180
Academic Editor: Paul F. Egan
Received: 15 February 2021
Accepted: 4 April 2021
Published: 7 April 2021
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Copyright: © 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
Polymers 2021, 13, 1180. https://doi.org/10.3390/polym13081180
1 Department of Prosthodontics, Yonsei University College of Dentistry, Yonsei-ro 50-1, Seodaemun-gu,
Seoul 03722, Korea; ejbayar@gmail.com (E.B.); drybpark@yuhs.ac (Y.-B.P.); jaehoon115@yuhs.ac (J.-H.L.)
2 Oral Research Science Center, BK21 FOUR Project, Department of Prosthodontics, College of Dentistry,
Yonsei University, Yonsei-ro 50-1, Seodaemun-gu, Seoul 03722, Korea; erin850313@gmail.com (J.-H.L.);
shin506@prostholabs.com (S.-H.S.); khyungpark@yuhs.ac (K.-H.P.)
* Correspondence: gomyou@yuhs.ac; Tel.: +82-2-2228-3160
Abstract: Three-dimensional (3D) printing is an attractive technology in dentistry. Acrylic-based 3D
printed resin parts have to undergo postcuring processes to enhance their mechanical and biological
properties, such as UV-light and thermal polymerization. However, no previous studies have revealed
how the postcuring temperature influences the biocompatibility of the produced parts. Therefore,
we postprocessed 3D printed denture teeth resin under different postcuring temperatures (40, 60
and 80 ◦ C) for different periods (15, 30, 60, 90 and 120 min), and evaluated their flexural properties,
Vickers hardness, cell cytotoxicity, cell viability, and protein adsorption. In addition, confocal laser
scanning was used to assess the condition of human gingival fibroblasts. It was found that increasing
the postcuring temperature significantly improved the flexural strength and cell viability. The flexural
strength and cell viability were 147.48 ± 5.82 MPa (mean ± standard deviation) and 89.51 ± 7.09%,
respectively, in the group cured at 80 ◦ C for 120 min, which were higher than the values in the 40
and 60 ◦ C groups. The cell cytotoxicity increased in the 40 ◦ C groups and for longer cultivation
time. Confocal laser scanning revealed identifiable differences in the morphology of fibroblasts. This
study has confirmed that the postcuring temperature influences the final mechanical and biological
properties of 3D printed resin.
Keywords: additive manufacturing; three-dimensional printing; rapid prototyping; CAD/CAM;
postcuring; flexural strength; degree of conversion; cell viability; cytotoxicity; confocal laser scanning
1. Introduction
The advent of computer-aided design (CAD) and computer-aided manufacturing
(CAM) has brought a new modality to restorative dentistry, which has developed re-
markably over the past 2 decades [1]. CAD/CAM has mainly utilized in subtractive
manufacturing (SM), which is now being used routinely in various dental applications. By
trimming a prefabricated block or disk, SM can be used to fabricate prostheses that are
comparable with conventional prostheses with regard to biological properties, mechanical
performance, and accuracy [2,3]. Although SM has some benefits such as standardized
industrially fabricated materials that offer high quality finished products [4], SM technol-
ogy has several drawbacks, such as greater waste of the raw material, wear of milling
instruments, restrictions in the geometric details of the fabricated product in terms of
undercuts and intaglio geometry, and only one unit being produced at a time [5]. Addi-
tive manufacturing (AM) is thus of greater interest than SM for the production of dental
prostheses [6].
Noteworthy technological improvements in digital dentistry are now occurring in
a method of additive manufacturing called three-dimensional (3D) printing due to its
https://www.mdpi.com/journal/polymers
Polymers 2021, 13, 1180 2 of 17

potential for rapid prototyping and the production of desired complex structures in a
layer-by-layer manner from printable biomaterials [6,7]. Myriad photosensitive resins have
been approved and used widely in restorative dentistry due to their good biocompatibility.
The applications of 3D printing in dentistry include the production of dental casts, sur-
gical guides, orthodontic and bite splints, temporary fixed dental prostheses, dentures,
castable patterns and custom impression trays [8–11]. Moreover, numerous 3D printers,
which utilize many AM methods such as stereolithography apparatus (SLA)/digital light
projection (DLP), Multijet/Polyjet, selective laser sintering (SLS), and fused filament fabri-
cation (FFF)/fused deposition modeling (FDM), are now commercially available for dental
photosensitive resins [12]. In brief, SLA/DLP utilize UV lasers or UV LED light source to
polymerize liquid photopolymers as the vat moves up or down, and thus generating 3D
structures. Multijet/Polyjet printing, micrometer sized droplets of liquid photopolymer
are dispensed from multiple print heads using 3-axis stages to build. SLS, on the other
hand, the high temperature of the laser light is used to either sinter or weld specific regions
of polymer powders. Lastly, FFF/FDM methods utilizes one or more heated nozzles that
spatially distribute extruded polymer as a fine filament [12].
The type of 3D printer, materials, various printing and manufacturing parameters
are known to influence the accuracy of printed objects [13,14]. There are biocompatible
soft polymers such as silicone, polyurethane which can be fully cured during DLS/DLP
printing process. Thus, they do not require postprocessing treatment [15]. However,
postprocessing treatment such as thermal or UV-light curing is indicated for acrylic-based
photosensitive resin in order to crosslink unreacted monomers and thereby complete the
polymerization process after printing, which improves its final thermal and mechanical
properties [16–18] depending on 3D printer type. Printable photosensitive resins consist
essentially of monomers that are mostly based on (meth)acrylates [19], photo-initiators,
and additives [20]. Once exposed to the appropriate light source, the polymerization
involves free-radical reactions in which the material is transformed from a viscous to a
rigid state. The terminal aliphatic C=C bonds are broken and converted to primary C–C
covalent bonds between methacrylate monomers during the polymerization process [21].
The amount of polymerization is quantified as the degree of conversion (DC). Obtaining
a higher DC usually provides better mechanical properties and biocompatibility [22,23],
with residual monomer being decreased [24]. This is more critical for 3D printed dentures,
temporary prostheses, and splints that are in contact with soft and hard tissues for much
longer times than are implant surgical guides.
For these reasons, the postpolymerization process after printing process can play a
crucial role in the final outcome. Previous studies have found that postpolymerization
is highly dependent on the type of curing chamber, the frequency and intensity of the
UV light, the exposure time, and the composition and color of the photosensitive resin,
which can potentially affect the mechanical strength and biocompatibility of the 3D printed
resin [25], as well as its accuracy [26]. Salmoria et al. revealed that thermal curing of the
resin used in an SLA 3D printer can reduce the cure heterogeneity and anisotropy [27]. It
was also demonstrated that the postcuring time and temperature influenced the DC [26].
Kumar et al. performed postpolymerization at curing temperatures from 80 to 140 ◦ C
for different postcuring times, which revealed that postcuring at 140 ◦ C for 6 hours (h)
resulted in better thermal and mechanical properties [28]. Furthermore, 3D printed resins
when applied for provisional fixed dental prostheses and dentures must withstand occlusal
forces, thus ensuring sufficient mechanical properties for clinical application is important.
Reymus et al. investigated fracture load of 3D printed provisional three-unit bridge and
concluded that postcuring affects mechanical properties of 3D printed resin material [29].
However, no previous study has investigated the biocompatibility of the final 3D printed
dental resin products postpolymerized under various curing temperatures. Moreover,
mechanical properties of the final 3D printed dental resin are not extensively evaluated in
previous studies.
s 2021, 13, x FOR PEER REVIEW 3 of 18

final 3D printed dental resin products postpolymerized under various curing tempera-
tures. Moreover, mechanical properties of the final 3D printed dental resin are not exten-
Polymers 2021, 13, 1180 3 of 17
sively evaluated in previous studies.
Hence, in order to gain a better understanding of the effects of various postpolymer-
ization conditions on the mechanical strength and biocompatibility of 3D printed resin
Hence, inhow
specimens, we investigated order to gain a better
postcuring understanding
parameters influenceofthe
theflexural
effects of various postpolymer-
strength,
DC, and biocompatibility of 3D SLA-printed denture teeth resin. The null hypothesisof
ization conditions on the mechanical strength and biocompatibility was3D printed resin
specimens, we investigated how postcuring parameters influence
that postcuring parameters would significantly affect the mechanical properties, DC, and the flexural strength,
DC, and biocompatibility of
biological properties of a 3D printed object. 3D SLA-printed denture teeth resin. The null hypothesis was
that postcuring parameters would significantly affect the mechanical properties, DC, and
biological
2. Materials and Methods properties of a 3D printed object.
2.1. 3D CAD Design and 3D Printing
2. Materials and Methods
Specimens2.1.
were
3D designed
CAD Design using
andCAD software (Meshmixer, Autodesk, San Rafael,
3D Printing
CA, USA) prior to printing. For the mechanical-property
Specimens were designed using CAD software tests, a bar was designed
(Meshmixer, with aSan Rafael, CA,
Autodesk,
length of 25 mm,
USA)a width
prior toofprinting.
2 mm, and Forathe
thickness of 2 mm according
mechanical-property tests, ato the
bar ISO
was 10477 with a length
designed
standard. The surface
of 25 mm, a width of 2 mm, and a thickness of 2 mm according to the ISO a10477 standard.
hardness, DC, and biocompatibility test were investigated using
disk with a diameter of 9 mm
The surface and a thickness
hardness, DC, and of 2 mm. The specimen
biocompatibility test weredesigns were saved
investigated using a disk with
as STL (Standard Tessellation Language) files and exported into
a diameter of 9 mm and a thickness of 2 mm. The specimen designsa 3D printing software
were saved as STL
program (PreForm Software,
(Standard Formlabs,
Tessellation Somerville,
Language) files MA, USA). The
and exported bara 3D
into andprinting
disk speci-
software program
mens were prepared
(PreFormin 120° and 0° Formlabs,
Software, orientations, respectively,
Somerville, MA,which
USA).were Theprinted
bar andindisk
three
specimens were
dimensions using
prepared in 120◦ and
commercially 0◦ orientations,
available PMMA resin (Denture teeth
respectively, whichresin
wereA2, Formlabs)
printed in three dimensions
with a layer thickness of 50 μm using
using commercially a desktop
available PMMA SLA 3D printer
resin (Denture (Form
teeth3, Formlabs).
resin The with a layer
A2, Formlabs)
3D printer usedthickness
in this study was powered by Low Force Stereolithography (LFS)™ using
of 50 µm using a desktop SLA 3D printer (Form 3, Formlabs). The 3D printer
405 nm UV light with
used in athis
laser power
study wasofpowered
250 mW.by Low Force Stereolithography (LFS)™ using 405 nm UV
light with a laser power of 250 mW.
2.2. UV-Light Postcuring Protocol for 3D Printed Specimens
2.2. UV-Light
After printing, the support Postcuring Protocol
structures werefor 3D Printed
removed andSpecimens
the residual resin monomer
on the 3D printed After printing,
specimens wasthe support
cleaned structures
with were removed
90% isopropanol usinganda the
3D residual
printingresin monomer
on the 3DMEDIFIVE,
washer (Twin Tornado, printed specimens
Incheon,was cleaned
Korea) forwith 90%The
10 min. isopropanol
specimens using
werea then
3D printing washer
postcured in a (Twin Tornado,
UV-light MEDIFIVE,chamber
polymerization Incheon,(FormCure,
Korea) for 10Formlabs)
min. The specimens
using a 405 were
nmthen postcured
light source (13inmultidirectional
a UV-light polymerization
LEDs) andchamber
capable of(FormCure,
heating up Formlabs)
to 80 °C.using a 405 nm light source
The printed
(13postcured
multidirectional LEDs) andofcapable of heating ◦
specimens were at temperatures 40, 60 and 80 °C up
for to
15,8030,C.60,
The90printed
and 120specimens were
postcured at temperatures of 40, 60 and 80 ◦ C for 15, 30, 60, 90 and 120 min. The specimens
min. The specimens in the green state (GS) (without postcuring) were used as a control
in
group (Figure 1). the green state (GS) (without postcuring) were used as a control group (Figure 1).

Workflow
Figure 1.Figure of the overall
1. Workflow of theexperimental process, showing
overall experimental process, the postcuring
showing conditions
the postcuring and typesand
conditions of experiment.
types of experiment.
2.3. Flexural Strength and Flexural Modulus Test
Fifteen bar specimens per group (n = 15) were stored in at 37 ◦ C for 24 h according
to the ISO 10477 standard before testing. They were loaded using a universal testing
machine (Model 3366, Instron Corporation, Norwood, MA, USA) with a crosshead speed
of 1 mm/min. Each specimen was placed on two rounded supports with a span length of
20 mm parallel to each other. It was then uniaxially loaded in the middle until the specimen
failed, and the maximum force before fracturing was recorded in newtons.
 mm/min. Each specimen was placed on two rounded supports with
mm parallel to each other. It was then uniaxially loaded in the middl
failed, and the maximum force before fracturing was recorded in new
Polymers 2021, 13, 1180
The flexural strength (σ) in megapascals and the flexural modulu
4 of 17

were calculated as follows [13]:

3𝐹𝐿
The flexural strength (σ) in megapascals and the flexural𝜎= modulus (E) in gigapascals
were calculated as follows [13]: 2wℎ
σ =
3FL 𝐹𝐿 (1)
2wh2 𝐸=
FL3
4𝑤ℎ 𝑑
where F is the load at a selected E = point
4wh3 d
of the elastic region of the (2) stress
spanFlength
where is the loadbetween the
at a selected supports
point in millimeters,
of the elastic w is theplot,
region of the stress–strain width
L is of th
meters,
the h isbetween
span length the height of theinspecimen
the supports millimeters, winis millimeters,
the width of the and d isinthe def
specimen
millimeters, h is the height of the specimen in millimeters, and d is the deflection of the
men in millimeters (Figure 2).
specimen in millimeters (Figure 2).

Figure
Figure2. Schematics of the testof
2. Schematics setup
thefortest
ISOsetup
10447 flexural
for ISOstrength.
10447 flexural strength.
2.4. Vickers Hardness Test
2.4.Disks
Vickers
(n = Hardness Test with increasing fineness using SiC paper (2000 and
5) were polished
4000 grit, sequentially) and stored in a 37 ◦ C incubator for 24 h. A Micro Vickers hardness
Disks (n =Shimadzu
tester (HMV-G31ST, 5) wereCo. polished
Ltd., Kyoto,with
Japan)increasing finenessspecimen
was used to determine using SiC p
hardness by applying a force of 200 g (1.96 N) for 15 s [30]; an average
grit, sequentially) and stored in a 37 °C incubator for 24 h. A Micro Vic value was calculated
from five different locations on each specimen.
(HMV-G31ST, Shimadzu Co. Ltd., Kyoto, Japan) was used to determ
2.5.
nessDegree
byofapplying
Conversion a force of 200 g (1.96 N) for 15 s [30]; an average v
Printed disks were polished with SiC paper (4000 grit) and stored in ambient condi-
from five different locations on each specimen.
tions for 24 h. The spectrum of the postcured specimen and that of the unpolymerized
resin were obtained using a Fourier transform infrared spectrometer (Nicolet IS10, Thermo
Scientific,
2.5. Degree ThermoofElectron Scientific Instruments, Madison, WI, USA). The absorbance was
Conversion
measured under the following conditions: 16 scans, 4 cm−1 resolution, and wavelengths
from 750 to 4000−1disks
Printed cm. Forwere polished
measuring with SiCstate
the unpolymerized paperof the(4000 grit)
material, and
a drop of store
monomer resin was applied on a microscope slide and measured. Three measurements
tions for 24 h. The spectrum of the postcured specimen and that of
were performed on the top surface of each disk, and the average of these scans was ana-
resinThewere
lyzed. obtained
peak areas using
for aliphatic a Fourier
and aromatic C=C double transform
bonds at 1638infrared
and 1608 cmspectrom
−1 ,

respectively, were analyzedThermo

Thermo Scientific, [25,31]. TheElectron
DC was calculated as follows:
Scientific Instruments, Madison
sorbance was measured 1638under − 1 thecmfollowing
− 1 conditions: 16 scans, 4 c
" #
cm /1608 Peak height (cured )
DC (%) = 1 − − 1 − 1
× 100 (3)
wavelengths from 750 tocm4000
1638 −1 cm.
/1608 cm For measuring
Peak height (monomer ) the unpolymerize
rial,Mean
a drop of monomer
± standard resin
deviation values werewas applied
calculated onmeasurements
from the a microscope slide an
made at
measurements
the three positions. were performed on the top surface of each disk, and
scans
2.6. was analyzed.
Biocompatibility Test The peak areas for aliphatic and aromatic C=C d
andIn1608
2.6.1. Vitro cm
Cell −1, respectively,
Culture and Cell Linewere analyzed [25,31]. The DC was calcu
Primary human gingival fibroblasts (HGFs;1638 cm /1608
PCS-201-018, ATCC, cm
Manassas, VA, USA)
were cultured in a cell𝐷𝐶 (%) = 1 −
culture dish with Dulbecco’s modified Eagle’s medium (DMEM, Wel-( )
1638 cm /1608 cm (
Mean ± standard deviation values were calculated from the mea
the three positions.
Polymers 2021, 13, 1180 5 of 17

Gene, Daegu, Korea) supplemented with penicillin/streptomycin (Penicillin-Streptomycin,

100X, WelGene), MEM nonessential amino acid solution (MEM Non-Essential Amino Acid
Solution, 100X, WelGene), and 10% fetal bovine serum (FBS, Thermo Scientific, Waltham,
MA, USA) at 37 ◦ C in 5% CO2 , 95% air atmosphere, and 100% relative humidity. The cell
culture medium was changed every 2–3 days. When the cells reached 85–90% confluency,
they were treated with trypsin-ethylenediaminetetraacetic acid solution (Trypsin-EDTA,
1X, WelGene) and seeded into 48-well plates at a density of 5 × 104 cells/well for each
specimen. Cells were allowed to attach for 24, 48 and 72 h at 37 ◦ C in 5% CO2 before cell
viability and cytotoxicity assays.

2.6.2. Cell Viability and Cytotoxicity Assays

Cell viability was evaluated using a CELLOMAX™ viability kit based on the tetra-
zolium salt (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-
tetrazolium and monosodium salt [WST-8]; Precaregene, Hanam, Kyungido, Korea). After
24, 48 and 72 h of incubation, 50 µL of CELLOMAX™ solution (CELLOMAX™ viability
assay kit, Precaregene) was added to the 48-well plate with the specimen and incubated for
90 min at 37 ◦ C according to manufacturer’s instructions. Subsequently, 100 µL aliquots of
the media from each well were transferred to the 96-well plate and their absorbance was
recorded using a microplate reader (VERSA max, Molecular Devices, Sunnyvale, CA, USA)
at a wavelength of 450 nm. The percentage of cell viability was calculated as follows:

ODtest sample − ODblank

 
Cell viability (%) = × 100 (4)
ODcontrol − ODblank

The cytotoxicity of the 3D printed resin on HGFs was measured using a lactate
dehydrogenase (LDH) release assay (Quanti-LDH™ Cytotoxicity Assay Kit, BIOMAX,
Seoul, Korea) according to the manufacturer’s protocol. After 24, 48 and 72 h, 20 µL of
the culture medium were collected and centrifugated at 7000 rpm for 3 min. Then, 10 µL
of the obtained supernatant was transferred to a new 96-well plate, to which 100 µL of
the LDH substrate mixture was added. After 30 min of incubation at room temperature,
the absorbance of the resultant solution was measured at 450 nm. For the cell cytotoxicity
assay, the media were not changed until 72 h. The percentage of cytotoxicity was calculated
as follows:
  
ODcells with 3D printed resin − ODbackground control − ODlow control
Cytotoxicity (%) =   × 100 (5)
ODhigh control − ODlow control

2.6.3. Cytoskeleton Staining and Confocal Laser Scanning Microscopy

After a cultivation time of 24 h, specimens were washed three times with PBS and
the cells were fixed with 4% paraformaldehyde in PBS for 15 min at room temperature.
The specimens were then washed again three times, permeabilized with 0.1% Triton™
X-100 in PBS for 15 min, washed once again three times with PBS. For the cytoskeleton
quantification, phalloidin (Alexa Flour 488® Phalloidin, Invitrogen, Thermo Scientific)
was used to fluorescently stain the cytoskeleton via its binding to β-actin, in accordance
with manufacturer’s instructions. The specimens were subsequently mounted in antifade
mounting media (VECTASHIELD, Vector Laboratories, Burlingame, CA, USA) and scanned
using confocal laser scanning microscopy (CLSM; LSM 700, Carl Zeiss Microscopy, Jena,
Germany). Co-expression was confirmed in Z-stacked serially captured images.

2.7. Protein Adsorption Assay

Three specimens per group were placed into 48-well plate for the protein adsorption.
A 1-mg/mL protein stock solution was prepared from bovine serum albumin (BSA; Sigma-
Aldrich, St. Louis, MO, USA). Each 3D printed disk was immersed into 500 µL of the
 A 1-mg/mL protein stock solution was prepared from bovine serum albumin (BSA; Sigma
Aldrich, St. Louis, MO, USA). Each 3D printed disk was immersed into 500 μL of the protein
stock solution. After 24 h of incubation under sterile humidity conditions at 37 °C in 5% CO2
any unadhered protein was removed by washing three times with PBS. Diluted BSA stand
Polymers 2021, 13, 1180 6 of 17
ards and bicinchoninic acid (BCA) working reagent were prepared according to the manu
facturer’s protocol (Pierce™ BCA Protein Assay Kit, Thermo Fisher Scientific, Rockford, IL
USA). A total of 1 mL micro BCA working reagent was used for the colorimetric detection
protein stock solution.
and quantitation After
of total 24 h offollowed
protein, incubationby under sterileathumidity
incubation 37 °C inconditions
5% CO2 for at 30 min
37 ◦ C in 5% CO , any unadhered protein was removed by washing three times with PBS.
Thereafter, 100 μL aliquots were transferred to 96-well plate and the absorbance was rec
2
Diluted BSA standards and bicinchoninic acid (BCA) working reagent were prepared
orded using a microplate reader (VERSA max, Molecular Devices) at 562 nm. Standard
according to the manufacturer’s protocol (Pierce™ BCA Protein Assay Kit, Thermo Fisher
curves were prepared using the BSA standard. The protein concentration was used to cal
Scientific, Rockford, IL, USA). A total of 1 mL micro BCA working reagent was used for the
culate the amount
colorimetric ofand
detection protein adsorbed
quantitation on the
of total surface
protein, of 3D printed
followed disks.at 37 ◦ C in
by incubation
5% CO2 for 30 min. Thereafter, 100 µL aliquots were transferred to 96-well plate and the
2.8. Statistical
absorbance wasAnalysis
recorded using a microplate reader (VERSA max, Molecular Devices) at
562 nm.
Data for thecurves
Standard were
flexural preparedflexural
strength, using themodulus,
BSA standard. The hardness
Vickers protein concentration
test, DC, and pro
was used to calculate the amount of protein adsorbed on the
tein adsorption were analyzed using one-way ANOVA and two-waysurface of 3D printed
ANOVAdisks. followed
by the
2.8. Bonferroni
Statistical pairwise comparison and Tukey multiple-comparisons test. The data
Analysis
fromData
the for
cellthe
viability and cytotoxicity assays and for protein adsorption were analyzed
flexural strength, flexural modulus, Vickers hardness test, DC, and protein
using three-way
adsorption ANOVAusing
were analyzed and one-way
the t-testANOVA
using standard statistical
and two-way ANOVA software (version
followed by 25.0
SPSS Statistics, IBM, Armonk, NY, USA) (α < 0.05).
the Bonferroni pairwise comparison and Tukey multiple-comparisons test. The data from
the cell viability and cytotoxicity assays and for protein adsorption were analyzed using
3. ResultsANOVA and the t-test using standard statistical software (version 25.0, SPSS
three-way
Statistics, IBM,
3.1. Flexural Armonk, NY, USA) (α < 0.05).
Strength
Two-way ANOVA was performed to verify the effects of postcuring temperature and
3. Results
timeFlexural
3.1. on theStrength
flexural strength. It was found that both the postcuring temperature (F =
113.416, p < 0.001)
Two-way ANOVA andwas
the performed
postcuringtotime (F the
verify = 1297.688,
effects ofppostcuring
< 0.001) significantly
temperature affected
the flexural
and time on strength (Figure
the flexural 3). There
strength. It waswas alsothat
found a significant interaction
both the postcuring effect between the
temperature
(F = 113.416, p < 0.001) and the postcuring time (F = 1297.688, p < 0.001)
postcuring temperature and time (F = 5.876, p < 0.001). Overall, the flexural significantly affected
strength did
the
notflexural strength
significantly (Figure more
improve 3). There
than was
60also
minaof significant interaction
postcuring effect between
time (129.12–137.3 the (Figure
MPa)
postcuring temperature and time (F = 5.876, p < 0.001). Overall, the flexural strength did not
3B). Figure 4 shows the flexural strength in groups with different postcuring times fo
significantly improve more than 60 min of postcuring time (129.12–137.3 MPa) (Figure 3B).
each postcuring temperature. The flexural strength varied significantly with the curing
Figure 4 shows the flexural strength in groups with different postcuring times for each
time and temperature,
postcuring temperature. The being highest
flexural for 120
strength variedmin at 80 °C (147.48
significantly with the±curing
5.82 MPa) and 90 min
time and
at 80 °C (145.13
temperature, being± highest
8.73 MPa) (Figure
for 120 min at4C).
80 For
◦ the groups
C (147.48 ± 5.82 with
MPa) 15andmin of postcuring
90 min at 80 ◦ C time
the flexural
(145.13 ± 8.73strength was4C).
MPa) (Figure significantly lower
For the groups withfor15postcuring temperatures
min of postcuring time, the of 40 °C (100.70
flexural
± 6.65 MPa),
strength 60 °C (115.00
was significantly lower± for
10.55 MPa), temperatures
postcuring and 80 °C (121.35of 40 ◦ C±(100.70 ± 6.65than
8.70 MPa) MPa),the othe
60 ◦ ◦
± 10.55 MPa), and 80 C (121.35 ± 8.70 MPa) than the other postcuring time.
C (115.00time.
postcuring

Figure3.3.Results
Figure Results from
fromtwo-way
two-way ANOVA.
ANOVA.Flexural strength
Flexural according
strength to postcuring
according (A) temperature
to postcuring (A) tempera-
and (B) time. Data are mean and standard-deviation values of 3D printed specimens.
ture and (B) time. Data are mean and standard-deviation values of 3D printed specimens. Different Differ-
lowercase lettersletters
ent lowercase indicate a significant
indicate difference.
a significant difference.
FOR PEER REVIEW
Polymers
Polymers 2021, 13,
2021, 13, 1180
x FOR PEER REVIEW 7 of 18
77 of 18
of 17

Figure 4. Results
Figure 4. from one-way
Results from one-way ANOVA.
ANOVA. Flexural
Flexural strength
strength according
according to
to postcuring
postcuring time for each
time for each postcuring
postcuring temperature.
temperature.
(A) ◦ ◦ ◦
(A) 40 °CC (B)
(B) 60
60°CCand
and(C)
(C)8080°C.C. Data
Data areare mean
mean and and standard-deviation
standard-deviation values
values of printed
of 3D 3D printed specimens.
specimens. Different
Different low-
ercase letters
lowercase indicate
letters a significant
indicate difference
a significant in the
difference same
in the group.
same group.
sults from one-way ANOVA. Flexural strength according to postcuring time for each postcuring temperature.
) 60 °C and (C) 80 °C. Data are mean and
3.2.
3.2. standard-deviation
Flexural
Flexural Modulus
Modulus values of 3D printed specimens. Different low-
s indicate a significant difference in the same group. ANOVA (Figure 5) revealed that the postcuring temperature (F =
The two-way
two-way
The ANOVA (Figure 5) revealed that the postcuring temperature (F = 50.191,
p50.191, p <and
< 0.001) 0.001)
theand the postcuring
postcuring time
time (F = (F = 309.135,
309.135, p <significantly
p < 0.001) 0.001) significantly
affected affected the
the flexural
3.2. Flexural Modulus
flexural modulus.
modulus. A significant
A significant interaction
interaction effect (F =effect
2.226,(F
p <= 0.05)
2.226,was
p <found
0.05) was found
between between
postcuring
postcuring temperature and time. Thestrength
flexural strength was significantly
higher higher for a post-
The two-waytemperature
ANOVA and time. 5)
(Figure
◦
The flexural
revealed that the was significantly
postcuring temperature
◦
for a postcuring
(F =
curing temperature
temperature of 80 Cof(1.02 80 °C±(1.02 ± 0.45pGPa,
0.45 GPa, p < 0.0001)
< 0.0001) than forthan forof
those those
40 of 40 °C±(0.80
C (0.80 0.36±GPa)
0.36
50.191, p < 0.001) and
GPa)the
and ◦C
60and
postcuring
60 °C±(0.86
(0.86 0.37±GPa
time (F
0.37(Figure
GPa= 309.135,
(Figure p <Specimens
5A).
5A). Specimens
0.001) significantly
in the
in the GS groupGS did affected
groupnotdid not
break
thebreak
during
flexural modulus. three-point
A significant
during three-point
bendinginteraction
bending
tests, effect
withtests, (F =the2.226,
with
the maximum maximum
exertedp load
< 0.05) was
exerted
being found
load beingbetween
significantly significantly
lower among
postcuring temperature
lower
all and(0.10
among
groups time. TheGPa)
all±groups
0.01 flexural
(0.10(Figure strength
± 0.01 GPa)
5B). was 5B).
(Figure
The flexuralsignificantly
The flexural
modulus higher
for each modulus forfor
postcuringa post-
each post-
tempera-
curing temperature of 80 °C (1.02 ± 0.45 GPa, p < 0.0001) than for those of 40 °C (0.80 ± 0.36resin
curing
ture is temperature
presented in is
Figurepresented
6. The in Figure
flexural 6. The
modulus flexural
of the 3Dmodulus
printed of the
resin 3D
used printed
in this study
increased
used in this with the postcuring
study increased time with and
the temperature,
postcuring time and and
reached a plateau after
temperature, 90 min ofa
and reached
GPa) and 60 °C (0.86 ± 0.37 GPa (Figure 5A). Specimens in the GS group
◦ did not break
postcuring.
plateau afterThe 90 flexural modulus was
min of postcuring. Thehighest
flexuralformodulus
90 min atwas 80 C (1.28 ±for
highest 0.13
90 GPa)
min at and
80 for
°C
during three-point120 bending
(1.28min at 80
± 0.13 tests,
GPa) andwith
◦ C (1.24
for 120the
± 0.16 min maximum
GPa),atand
80 °C (1.24exerted
lowest for 15 min
± 0.16 load
GPa), 40being
atand ◦ C (0.84significantly
lowest for ± 0.12
15 minGPa).
at 40 °C
lower among all groups (0.10
(0.84 ± 0.12 ± 0.01 GPa) (Figure 5B). The flexural modulus for each post-
GPa).
curing temperature3.3.is Vickers
presented Hardness Test
in Figure 6. The flexural modulus of the 3D printed resin
Figure 7 shows
used in this study increased with the postcuring result of two-ANOVA. time and The postcuring
temperature, temperature
and reached(F = 563.338,
a
p < 0.001) and the postcuring time (F = 1653.244, p < 0.001) significantly affected the Vickers
plateau after 90 min of postcuring. The flexural modulus was highest for 90 min at 80 °C
hardness. The Vickers hardness was 3.47 in the green state, and it increased to 14.89–17.51
(1.28 ± 0.13 GPa) and for40120
in the min at(Figure
◦ C groups 80 °C 8A), (1.24and ± 0.16 GPa), and
to 17.95–21.16 lowest
in the 60 ◦ C for
groups15 min at 8B),
(Figure 40 °C and to
◦
(0.84 ± 0.12 GPa). 19.4–25.7 in the 80 C groups (Figure 8C).

Figure 5. Results from two-way ANOVA. Flexural modulus data according to (A) postcuring tem-
perature and (B) time. Data are mean and standard-deviation values. Different lowercase letters
indicate a significant difference.

Figure 5. Results from two-way ANOVA. Flexural modulus data according to (A) postcuring temperature and (B) time.
Figure 5. Results from two-way ANOVA. Flexural modulus data according to (A) postcuring tem-
Data are mean and standard-deviation values. Different lowercase letters indicate a significant difference.
perature and (B) time. Data are mean and standard-deviation values. Different lowercase letters
indicate a significant difference.
 Polymers 2021, 13, 1180 8 of 17
Polymers 2021, 13, x FOR PEER REVIEW 8 of 18

Figure 6. Results from one-way ANOVA. Flexural modulus according to postcuring time for each postcuring temperature.
(A)from
Figure 6. Results 40 °C (B) 60 ANOVA.
one-way °C and (C) 80 °C. modulus
Flexural Data are according
mean and to
standard-deviation
postcuring time forvalues. Different lowercase
each postcuring letters indicate a
temperature.
(A) 40 °C (B) 60significant difference
°C and (C) in theare
80 °C. Data same group.
mean and standard-deviation values. Different lowercase letters indicate a
significant difference in the same group.
3.3. Vickers Hardness Test
3.3. Vickers Hardness Test 7 shows result of two-ANOVA. The postcuring temperature (F = 563.338, p <
Figure
Figure 70.001)
showsand result
theofpostcuring
two-ANOVA. time The
(F = postcuring
1653.244, p temperature (F = 563.338,
< 0.001) significantly p < the Vickers
affected
0.001) and thehardness.
postcuring time (F = 1653.244, p < 0.001) significantly affected the Vickers
The Vickers hardness was 3.47 in the green state, and it increased to 14.89–17.51
Figure
Figure 6. hardness.
6. Results TheinVickers
from one-way the
ANOVA. hardness
40 °C Flexuralwas
groups 3.478A),
(Figure
modulus inaccording
the
andgreen state, and ittime
toto17.95–21.16
postcuring inincreased
the
for60 °Cto
each
each 14.89–17.51
groups (Figure
postcuring
postcuring 8B), and to
temperature.
temperature.
(A) 40 ◦°C
(A) 40 (B)in
C (B) 60 ◦ C 40
60the
°C and
and°C(C)
(C) 80 ◦°C.
groups
80 C. (Figure
19.4–25.7 Data
Data are8A),
in are
the mean
meanand
80 °C andtostandard-deviation
and 17.95–21.16
groups in thevalues.
(Figure 8C).
standard-deviation 60 °C groups
values. Different (Figure 8B),letters
lowercase
lowercase and toindicate
letters indicate aa
significant 19.4–25.7
significant difference
difference inin
in thethe
the same
same80group.
°C groups (Figure 8C).
group.

3.3. Vickers Hardness Test

Figure 7 shows result of two-ANOVA. The postcuring temperature (F = 563.338, p <
0.001) and the postcuring time (F = 1653.244, p < 0.001) significantly affected the Vickers
hardness. The Vickers hardness was 3.47 in the green state, and it increased to 14.89–17.51
in the 40 °C groups (Figure 8A), and to 17.95–21.16 in the 60 °C groups (Figure 8B), and to
19.4–25.7 in the 80 °C groups (Figure 8C).

Figure 7. Results from two-way ANOVA. Vickers hardness data according to postcuring (A) tem-
Figure 7. Results from
Figure two-way
7. Results ANOVA.
perature
from and (B)Vickers
two-way time.
ANOVA.hardness
Data data
are mean
Vickers according
and
hardness datatoaccording
postcuring
standard-deviation (A) temperature
tovalues. Different
postcuring and (B) time.
lowercase
(A) tem- letters
Data are mean and standard-deviation
indicate
perature and (B) values. Different
a significant
time. Data are mean lowercase
difference. letters indicate a significant difference.
and standard-deviation values. Different lowercase letters
indicate a significant difference.

Figure 7. Results from two-way ANOVA. Vickers hardness data according to postcuring (A) tem-
perature and (B) time. Data are mean and standard-deviation values. Different lowercase letters
indicate a significant difference.

Figure 8. Results from one-way ANOVA. Vickers hardness according to postcuring time for each postcuring temperature.temperature.
(A) 40 ◦°C
40
(A)from
Figure 8. Results C (B)
one-way60 ◦°C
(B) 60 C and
and (C)
ANOVA. 80 ◦°C.
80 C. hardness
(C)Vickers Data
Data are
are mean
mean and standard-deviation
andto
according standard-deviation values.
values.
postcuring time for Different lowercase
each postcuring lowercase letters
letters indicate
temperature. indicate aa
(A) 40 °C (B) 60significant
°C and (C)
significant difference in the
80 °C. Data
difference in theare
same
mean
same group.
and standard-deviation values. Different lowercase letters indicate a
group.
significant difference in the same group.
3.4. Degree of Conversion
The results for the DC are shown in the Figure 9. The two-way ANOVA revealed that
the postcuring temperature (F = 97.507, p < 0.001) and the postcuring time (F = 1856.26,
p < 0.001) significantly affected the DC. There was also a significant interaction effect
Figure 8. Results from one-way ANOVA.
between Vickers
curing hardness according
temperature and time to (F
postcuring p < for
= 7.475,time each postcuring
0.001). The DC was temperature.
significantly
(A) 40 °C (B) 60 °C and (C)higher
80 °C. in
Data 60 ◦mean
the are C (58.80 7.22%) and 80 ◦ C values.
and±standard-deviation Different
(58.76 ± lowercase
7.17%) groups letters
than 40 ◦ C group
indicate
in the a
significant difference in the (56.70
same group.
± 6.29%). Relative to GS specimens, the polymerization rate increased dramatically
in the postcuring temperature, and was highest for 120 min at 60 ◦ C (63.37 ± 0.42%) and
lowest for 15 min at 40 ◦ C (58.07 ± 1.28%) among the postcuring groups. However, after
60 min of postcuring, the polymerization rate did not vary significantly (p = 0.837) with the
postcuring temperature.
 tween curing temperature and time (F = 7.475, p < 0.001). The DC was significantly higher
in the 60 °C (58.80 ± 7.22%) and 80 °C (58.76 ± 7.17%) groups than in the 40 °C group (56.70
± 6.29%). Relative to GS specimens, the polymerization rate increased dramatically in the
postcuring temperature, and was highest for 120 min at 60 °C (63.37 ± 0.42%) and lowest
for 15 min at 40 °C (58.07 ± 1.28%) among the postcuring groups. However, after 60 min
Polymers 2021, 13, 1180 9 of 17
of postcuring, the polymerization rate did not vary significantly (p = 0.837) with the post-
curing temperature.

Figure 9. Degree of conversion (DC) of the 3D printed resin specimens that were postcured under dif-
Figure 9. Degree of conversion (DC) of the 3D printed resin specimens that were postcured under
ferent postcuring conditions. DC gradually increased after 15 min of postcuring for all temperatures.
different postcuring conditions. DC gradually increased after 15 min of postcuring for all tempera-
Data are mean and standard-deviation values.
tures. Data are mean and standard-deviation values.
3.5. Biocompatibility Test
3.5.3.5.1.
Biocompatibility
Cell ViabilityTestAssay
3.5.1. Cell Viability Assay
The HGFs were cultured on 3D printed specimens, and after 24, 48 and 72 h, cell
The HGFs
viability assays were cultured ondifferent
demonstrated 3D printed specimens,
results according and after 24, 48 temperature
to postcuring and 72 h, cell and
time (Figure
viability assays 10). The three-way
demonstrated ANOVA
different revealed
results that cell
according viability differed
to postcuring significantly
temperature and
with
time the cultivation
(Figure time (F ANOVA
10). The three-way p < 0.001),
= 36.204,revealed thatpostcuring temperature
cell viability (F = 79.775,
differed significantly
p <the
with 0.001), and postcuring
cultivation time (F =time (F =p57.386,
36.204, p <postcuring
< 0.001), 0.001). There was a significant
temperature interaction
(F = 79.775, p<
effect
0.001), andbetween cultivation
postcuring time (Ftime, postcuring
= 57.386, temperature,
p < 0.001). There was aand postcuring
significant time (F effect
interaction = 1.580,
p < 0.05).
between Cell viability
cultivation time,was lowest intemperature,
postcuring the GS group, andand increased time
postcuring continuously
(F = 1.580,with
p <the
postcuring
0.05). time (Figure
Cell viability was lowest10C).inAfter
the GS 24group,
h, the cell
andviability
increased was highest for 120
continuously withmin
the at 80 ◦ C
post-
(89.51 ± 7.09%) ◦ ± 14.58%)
curing time (Figureand lowest
10C). Afterfor2415h,min
the at 40viability
cell C (48.53was highest (Figure
for 120 11A).
min atAt8048°C
and
72 h, the cell viability was higher in the 80 ◦ C group (69.73 ± 33.96% and 55.58 ± 25.36%,
(89.51 ± 7.09%) and lowest for 15 min at 40 °C (48.53 ± 14.58%) (Figure 11A). At 48 and 72
◦ C groups (Figure 11B,C). According to t-tests, at 48 h of
h, respectively) thanwas
the cell viability in the 40 andin60the
higher 80 °C group (69.73 ± 33.96% and 55.58 ± 25.36%,
cell culturing, the ◦ C group and the
respectively) than incell
theviability
40 and 60 did°Cnot differ(Figure
groups significantly between
11B,C). the 80
According to t-tests, at 48 h
Polymers 2021, 13, x FOR PEER REVIEW
positive control (PC) 10 of 18
of cell culturing, the cell(p > 0.05, Figure
viability did not11B),
differwhich refers tobetween
significantly natural cell viability
the 80 without
°C group and a
3D printed specimen during the experiment.
the positive control (PC) (p > 0.05, Figure 11B), which refers to natural cell viability without
a 3D printed specimen during the experiment.

Figure 10.Results
Figure10. Resultsfrom
fromthree-way
three-way ANOVA.
ANOVA.Cell viability
Cell datadata
viability are presented according
are presented to (A)tocultivation
according time, (B)
(A) cultivation postcuring
time, (B) post-
temperature, and (C) postcuring time. Mean and standard-deviation values are shown. Different lowercase
curing temperature, and (C) postcuring time. Mean and standard-deviation values are shown. Different lowercase letters indicate
letters
aindicate
significant difference.
a significant difference.
 Figure
Polymers 2021, 10. Results from three-way ANOVA. Cell viability data are presented according to (A) cultivation time, (B) post-
13, 1180 10 of 17
curing temperature, and (C) postcuring time. Mean and standard-deviation values are shown. Different lowercase letters
indicate a significant difference.

Figure 11. Results

Figure 11. Results from
from cell
cell viability
viabilityassays.
assays.Cell
Cellviability
viabilityincreased
increasedwith
withthe
thepostcuring
postcuringtemperature after
temperature (A)
after 2424
(A) h, h,
(B)(B)
4848
h
and (C)
h and 7272
(C) h of experimentation.
h of experimentation.TheThecell
cellviability
viabilitygradually
graduallyincreased
increasedwith
withthe
thepostcuring
postcuringtemperature
temperatureandandtime.
time. Mean
Mean
and standard-deviation
standard-deviation values
values are
are shown.
shown. Asterisk
Asterisk indicates
indicates aa significant
significant difference
differencefrom
fromthe
thePC
PCin thet-test.
inthe t-test.

3.5.2. Cell Cytotoxicity Assay

Figure 12 shows the cell cytotoxicity of the 3D printed printed resin
resin samples
samples according
according to
cultivation time, postcuring temperature, and postcuring time. The three-way ANOVA
revealed that cell cytotoxicity differed significantly
significantly with
with the cultivation
cultivation time
time (F = 2920.851,
2920.851,
p << 0.001),
0.001), postcuring
postcuring temperature
temperature (F (F = 327.132, p << 0.001),
0.001), and
and postcuring
postcuring time (F == 376.927,
376.927,
p < 0.001). There
There was
wasalso
alsoaasignificant
significantinteraction
interactioneffect
effect between
between cultivation
cultivation time,
time, post-
postcur-
ing temperature,
curing temperature,andandpostcuring
postcuringtime (F =(F13.687,
time p < p0.001).
= 13.687, Overall,
< 0.001). thethe
Overall, cellcell
cytotoxicity
cytotoxi-
was significantly
city was lower
significantly for afor
lower postcuring timetime
a postcuring of 90ofmin (14.41
90 min ± 11.17%)
(14.41 ± 11.17%)than forfor
than those of
those
15 min
of 15 (16.02
min ± 11.46%,
(16.02 ± 11.46%,p=p =0.01)
0.01)and
and3030min (15.96±± 11.99%,
min(15.96 11.99%,pp == 0.016), but did not differ
significantly from those in the 60 60 min
min group (15.42 ±
group (15.42 ± 11.31%, p = 0.268) or the 120 min min
group (14.44 ± 11.55%, p = 1.000) (Figure 12C). Figure 13 shows the results from the LDH
assays of HGFs cultured in the 3D printed specimens on different cultivation times. The
t-tests showed that the cytotoxicity at 24 h was lower in the 80 ◦ C (2.93 ± 4.89%) and 60 ◦ C
(3.41 ± 4.70%) groups than in the 40 ◦ C group (7.55 ± 3.11%), and that it did not differ
significantly from that in the PC (p > 0.05) except for 15 min at 60 ◦ C (p = 0.026) (Figure 13A).
However, from 48 h the cytotoxicity increased in all of the groups and differed significantly
from that in the PC (p < 0.05) (Figure 13B). The cytotoxicity was higher at 72 h than 48 h, but
the trend was similar to that at 48 h (Figure 13C). The cytotoxicity was lower for 120 min
at 80 ◦ C (7.04 ± 5.97%) throughout all cultivation times. We found that the cytotoxicity
decreased as the postcuring temperature increases.
 (3.41 ±± 4.70%)
(3.41 4.70%) groups
groups than
than in in the
the 40
40 °C
°C group
group (7.55
(7.55 ±± 3.11%),
3.11%), and
and that
that itit did
did not
not differ
differ
significantly from that in the PC (p > 0.05) except for 15 min at 60 °C (p
significantly from that in the PC (p > 0.05) except for 15 min at 60 °C (p = 0.026) (Figure = 0.026) (Figure
13A).However,
13A). However,from from48 48hhthethecytotoxicity
cytotoxicityincreased
increasedin inall
allof
ofthe
thegroups
groupsandanddiffered
differedsig-
sig-
nificantly from that in the PC (p < 0.05) (Figure 13B). The cytotoxicity was higher at 72 hh
nificantly from that in the PC (p < 0.05) (Figure 13B). The cytotoxicity was higher at 72
Polymers 2021, 13, 1180 than48
than 48h,h,but
butthe
thetrend
trendwaswassimilar
similartotothat
thatat
at48
48hh(Figure
(Figure13C).
13C).The
Thecytotoxicity
cytotoxicitywas was11 lower
of 17
lower
for 120 min at 80 °C (7.04 ± 5.97%) throughout all cultivation times.
for 120 min at 80 °C (7.04 ± 5.97%) throughout all cultivation times. We found that the We found that the
cytotoxicity decreased as the postcuring temperature
cytotoxicity decreased as the postcuring temperature increases. increases.

Figure 12. Results

Figure12. Results from
from three-way
three-way ANOVA.
ANOVA. Cell
Cell cytotoxicity according to
cytotoxicity according to (A)
(A) cultivation
cultivation time,
time, (B)
(B) postcuring
postcuringtempera-
temper-
Figure 12. Results from three-way ANOVA. Cell cytotoxicity according to (A) cultivation time, (B) postcuring tempera-
ature, and(C)
ture, and (C)postcuring
postcuringtime.
time.Mean
Meanand
andstandard-deviation
standard-deviation values
values areare shown.
shown. Different
Different lowercase
lowercase letters
letters indicate
indicate a
a sig-
ture, and (C) postcuring time. Mean and standard-deviation values are shown. Different lowercase letters indicate a sig-
nificant difference.
significant difference.
nificant difference.

Figure 13. Results

Resultsfrom
fromcell
cellcytotoxicity
cytotoxicityassays
assays after(A)
(A) 24h,
h,(B)
(B)48
48hhand
and(C)
(C) 72
72 h.
h. The
The cell cytotoxicity
cytotoxicity gradually
graduallyincreased
increased
Figure
Figure 13.
13. Results from cell cytotoxicity assays after
after (A) 24
24 h, (B) 48 h and (C) 72 h. The cell cytotoxicity gradually increased
withthe
with thecultivation
cultivationtime
timebut
butdecreased
decreasedwith
withthe
thepostcuring
postcuring temperature.
temperature. Mean
Mean andand standard-deviation
standard-deviation values
values are
are shown.
shown.
with the cultivation time but decreased with the postcuring temperature. Mean and standard-deviation values are shown.
Asteriskindicates
Asterisk indicatesaasignificant
significantdifference
differencefrom
fromthe
thepositive
positivecontrol
control(PC)
(PC)ininthe
thet-test.
t-test.
a significant difference from the positive control (PC) in the t-test.

3.5.3.CLSM
3.5.3. CLSM Analysis
CLSM Analysis
Analysis
CLSM observations of cell morphology on the surface of specimens are shown in
Figure 14. The GS group showed relatively a small number of adhered cells and poor
morphology. Differences of morphology and size became more obvious as the postcuring
temperature increased, which large numbers of multinucleate cells (stained blue), and
well-stretched cytoplasm and well-grown filopodias (stained green). All 40 ◦ C groups
exhibited fibroblasts with small and round morphology, and some cells in the specimens
processed at 40 ◦ C for 90 min and 40 ◦ C for 120 min remaining round with few filopodias
being produced at the cell edges. For 60 ◦ C groups, most cells showed an elongated shape
or dendritic morphology, although a very small number of the cells remained round. There
were noticeable differences between the 40 and 60 ◦ C groups. For all 80 ◦ C groups, many
 temperature increased, which large numbers of multinucleate cells (stained blue), and
well-stretched cytoplasm and well-grown filopodias (stained green). All 40 °C groups ex-
hibited fibroblasts with small and round morphology, and some cells in the specimens
processed at 40 °C for 90 min and 40 °C for 120 min remaining round with few filopodias
Polymers 2021, 13, 1180 being produced at the cell edges. For 60 °C groups, most cells showed an elongated 12 of 17shape
or dendritic morphology, although a very small number of the cells remained round.
There were noticeable differences between the 40 and 60 °C groups. For all 80 °C groups,
many more cell–cell contacts were detected on the surface of the specimens compared
more cell–cell contacts were detected on the surface of the specimens compared with the
with
40 and40
the 60◦and 60°C groups.
C groups. Increasing
Increasing the postcuring
the postcuring temperature temperature andintime
and time resulted moreresulted
cells in
more cells appearing
appearing stretched stretched andwith
and in contact in contact with cells.
neighboring neighboring cells.

FigureFigure 14. Confocal

14. Confocal laserlaser scanning
scanning microscopy(CLSM)
microscopy (CLSM) images
imagesofofhuman
human gingival fibroblasts
gingival cultures
fibroblasts cultures (54 ×cells/mL)
(5 × 10 on on
104 cells/mL)
3D printed specimens postcured at various temperatureand and times
times after ◦
3D printed specimens postcured at various temperature after2424hhofofincubation: (A)(A)
incubation: GS,GS,
(B) 40
(B) C 40for
°C15for
min,
15 min,
(C) 40(C)
°C40for◦ C30for 30 min,
min, (D) 40(D)°C ◦ C for 60 min, (E) 40 ◦ C for 90 min, (F) 40 ◦ C for 120 min, (G) 60 ◦ C for 15 min, (H) 60 ◦ C for
40 for 60 min, (E) 40 °C for 90 min, (F) 40 °C for 120 min, (G) 60 °C for 15 min, (H) 60 °C for 30
30 min, ◦ C for 60 min, (J) 60 ◦ C for 90 min, (K) 60 ◦ C for 120 min, (L) 80 ◦ C for 15 min, (M) 80 ◦ C for 30 min, (N) 80 ◦ C
min, (I) 60 °C(I)for
60 60 min, (J) 60 °C for 90 min, (K) 60 °C for 120 min, (L) 80 °C for 15 min, (M) 80 °C for 30 min, (N) 80 °C
◦ C for 90 min, and (P) 80 ◦ C for 120 min.
for 60for 60 min,
min, (O) 80 (O)°C
80for 90 min, and (P) 80 °C for 120 min.

3.6.3.6. Protein
Protein AdsorptionAssay
Adsorption Assay
Protein
Protein adsorptiononto
adsorption ontothe
the3D
3Dprinted
printedresin
resinsurfaces
surfaces is
is shown
shown inin Figure
Figure 15. The two-
15. The
two-way ANOVA test revealed a significant interaction effect between postcuring temper-
way ANOVA test revealed a significant interaction effect between postcuring temperature
ature and time (F = 5528.854, p < 0.001) (Figure 12A,B). The protein adsorption differed
and time (F = 5528.854, p < 0.001) (Figure 12A,B). The protein adsorption differed signifi-
significantly for 40 ◦ C (77.67–262.67 µg), 60 ◦ C (72.67–112.13 µg), and 80 ◦ C (22.83–36.33 µg)
cantly
Polymers 2021, 13, x FOR PEER REVIEW for According
groups. 40 °C (77.67–262.67 μg), 60
to t-tests, protein °C (72.67–112.13
adsorption μg), and
was significantly 80for
lower °C30,
(22.83–36.33
13 ofμg)
60, 90 and 18
groups. According
◦ to t-tests, protein adsorption was significantly lower for 30, 60,
120 min at 80 C, and did not differ significantly from that for the PC (p > 0.05) (Figure 12C). 90 and
120 min at 80 °C, and did not differ significantly from that for the PC (p > 0.05) (Figure 12C).

15. Results
Figure 15.
Figure Results from
fromprotein
proteinadsorption
adsorption assays.
assays.Mean
Mean and standard-deviation
and standard-deviation values are shown.
values (A) Two-way
are shown. ANOVA
(A) Two-way ANOVA
showed that the protein adsorption decreased with postcuring temperature and (B) did not differ
showed that the protein adsorption decreased with postcuring temperature and (B) did not differ significantlysignificantly after 30 min
after 30
of postcuring
min time. time.
of postcuring Different lowercase
Different letters letters
lowercase indicate a significant
indicate difference.
a significant (C) The protein
difference. (C) Theadsorption for 40, 60 and
protein adsorption for 40, 60
80 ◦ C80groups.
and Asterisk
°C groups. indicates
Asterisk a significant
indicates difference
a significant from the
difference PC in
from thethe
PCt-tests.
in the t-tests.

4. Discussion
In this study, the effects of different combinations of postcuring temperatures and
times were evaluated. From the above results testing the two key parameters of postcur-
ing temperature and time, we observed significant influence of change in temperature on
Polymers 2021, 13, 1180 13 of 17

4. Discussion
In this study, the effects of different combinations of postcuring temperatures and
times were evaluated. From the above results testing the two key parameters of postcuring
temperature and time, we observed significant influence of change in temperature on the
3D printed resin properties whereas for time differences were not discernible. Therefore,
the null hypothesis was partially accepted.
The present study found that the flexural strength of 3D printed denture teeth resin
increased significantly with the postcuring temperature. Postcuring temperature improves
the anisotropy that provides more-homogeneous curing for 3D printed specimens, which
results in better mechanical properties. In addition, a higher temperature would accelerate
polymerization by enhancing the diffusion of free radicals and thereby increasing the
potential for them to react throughout the specimen [32,33]. In the present study, the flexural
strength of the 3D printed specimens increased gradually until 120 min of postcuring
time, although there was no significant difference for postcuring times of longer than
60 min (Figure 3B). Another study also found that the flexural strength increased with
the postcuring time [34]. In our study the flexural strength increased abruptly compared
with the GS group after 15 min of postcuring. However, another previous study found
no significant differences in flexural strength between their GS group and the group with
15 min of postcuring [25]. Moreover, none of the specimens in the present GS group
fractured during the three-point bending test, which demonstrates the presence of flow
plasticity. The printing parameters of the 3D printer also appear to affect the mechanical
properties of printed specimens in their GS. In terms of the flexural modulus, the tendency
was similar to that for the flexural strength for both postcuring temperature and time.
The flexural modulus of 3D printed specimens increased significantly with the postcuring
temperature. This could be explained by the energy received by the system increasing
with the temperature so as to activate the monomers to further crosslink [28]. It was
observed that a higher postcuring temperature resulted in a smaller displacement being
required for fracturing the sample. Jindal et al. investigated the mechanical properties
of 3D printed clear aligners under different postcuring conditions and found that good
flexural properties could be achieved by using a higher temperature (80 ◦ C for 10 or 20 min)
with a shorter postcuring time compared with lower temperatures (60 ◦ C for 20 min or
40 ◦ C for 20 min) [10]. These findings are consistent with those of our study.
The Vickers hardness test demonstrated significant difference according to postcuring
temperature. Higher postcuring temperature improves surface hardness significantly.
Overall, Vickers hardness of the 3D printed specimens increased gradually until 120 min
of postcuring time. However, there was no significant difference for postcuring times of
longer than 60 min. This trend was similar with the result of flexural properties.
The DC is strongly influenced by several factors, including the exposure time, power
density, and wavelength of the light source, and the distribution, material composition,
and final characteristics of the resin [35]. Previous studies have also found that mechanical
and chemical properties are dependent on the DC, and thus may play crucial roles in
determining the ultimate success of the restoration [36,37]. In our study, no significant
difference was found in the DC between groups postcured at 60 and 80 ◦ C. Katheng et al.
evaluated the DC of 3D printed resin postcured at 40, 60 and 80 ◦ C, and found that it was
highest for 30 min at 60 ◦ C (90.92 ± 3.18%) [26]. In that study, the DC of resin that was
postcured for 15 and 30 min at 60 ◦ C differed significantly from that postcured for 15 min
at 40 ◦ C (79.42 ± 10.02%), 30 min at 40 ◦ C (72.19 ± 7.97%), 15 min at 80 ◦ C (73.94 ± 9.51%),
and 30 min at 80 ◦ C (75.72 ± 6.92%) [26]. The pattern of these results is similar to that
found in our study.
A particularly interesting finding of the present study was of no specific improvement
in the DC for the 80 ◦ C groups relative to the 60 ◦ C groups (Table S1). This might indicate
that an oxygen inhibition layer forms on the surface of the resin due to oxygen molecules
attaching to free radicals in the polymerization process, with this layer inhibiting the cure
process on the surface [38,39]. Such an inhibition process would be limited to the most-
Polymers 2021, 13, 1180 14 of 17

superficial layer of the surface [33]. During the postcuring process, UV light polymerizes
not only the surface of the resin but also penetrates a certain depth [40], and the uniformity
of the DC throughout the specimen has a decisive impact on both its biocompatibility and
mechanical properties. Furthermore, the maximum penetration of the spectrometer was
10 µm, and so only the absorption of the superficial layer of the resin could be measured [41].
For this reason, the present study was limited to measuring the DC on the specimen’s
outer surface.
Figure 7B indicates that the postcuring temperature had a significant impact on
the cell viability. Cell viability tended to increase gradually with the postcuring time,
but there were no significant changes after 60 min. A similar result was obtained in a
previous study [25]. At 48 h, the 3D printed specimens cured at 60 and 80 ◦ C did not differ
significantly from the PC (Figure 8B), whereas cell viability was significantly decreased at
72 h compared with 24 and 48 h. One possible explanation is that unpolymerized monomer
in the deeper layer of the resin leached out to induce a cytotoxic effect [41]. The cytotoxicity
of a material is determined mainly by the degree of polymerization on its surface, which
is the summation of the degree of polymerization induced during 3D printing plus the
further polymerization induced by postpolymerization [42]. As mentioned above, the
DC did not differ significantly between the 60 and 80 ◦ C groups despite the cytotoxicity
being lower in the latter group, and significant differences in cytotoxicity were found
between all postcuring temperatures. Park et al. investigated the influence of different
dental resin materials on fibroblasts, and found that 3D printed resin specimens showed
better biocompatibility than self-cured resin specimens [43]. The production of 3D printed
resin does not involve a chemical reaction, and so there is likely to be less cytotoxicity,
whereas self-curing technology does involve chemical reactions [43].
Fibroblasts are the most abundant cell type in connective tissues and they have
multiple functions and create complex cell signaling networks through their contacts with
other cells [44]. Additionally, gingival fibroblasts play essential roles in wound healing,
exhibit diversely responses to growth factors, and produce specific extracellular matrix
proteins [45]. The present CLSM images revealed clearly differences on the morphology,
size, and number of cells with the postcuring temperature. Well-developed morphology
and increased cell adhesion were observed in groups that were cured at high postcuring
temperatures such as 60 and 80 ◦ C, whereas cells on the specimens postcured at 40 ◦ C
demonstrated poor morphology. Substances leached from resin-based dental restorative
materials potentially exert adverse effects on basic cellular functions such as proliferation
and metabolism, as well as on cell morphology and membrane integrity [46]. Hence,
setting the optimal postcuring process is a challenging but crucial step to fulfilling the
biological requirement of 3D printed resin since it influences cytocompatibility. Our results
suggest that a higher postcuring temperature yields improved biocompatibility of the 3D
printed resin.
3D printed resin is increasingly being used in clinical practice due to its satisfactory
biocompatibility and mechanical strength, and simple processing. More plaque tends to
accumulate on resin materials in the intraoral cavity than on other restorative materials [47].
It is desirable that 3D printed resin prostheses accumulate less biofilm when they are used
in long-term applications such as dentures, temporary crowns, and bridges. The present
study found that the postcuring temperature significantly affects protein adsorption, with
less protein being adsorbed on the surface of the 3D printed resin specimen when us-
ing a higher postcuring temperature. However, no significant difference was found in
the specimens postcured for more than 30 min. We additionally observed the adhesive
characteristics on the surface of the specimens that were postcured at low temperature
or that had not undergone a postcuring process (i.e., GS). Previous studies found incom-
plete polymerization of the resin surface to be associated with atmospheric oxygen in the
curing process causing a sticky, uncured oxygen-inhibited layer [48,49]. Moreover, in the
presence of monomers, unpolymerized compounds can leach out, which may result in the
accumulation of biofilm [50,51]. The present findings indicate that postcuring at 80 ◦ C may
Polymers 2021, 13, 1180 15 of 17

be the most favorable postcuring condition and thus result in less biofilm formation. It is
plausible that there is an optimal postcuring temperature for enhancing the mechanical
properties and biocompatibility of the resin. A favorable postcuring temperature results
in resin being polymerized more efficiently by improving the molecular mobility of free
radicals and other reactive species [33].
The limitations of the current study included the use of only one type of 3D printed
resin material and one UV-light curing chamber. Using different curing chambers and 3D
printed materials may produce different outcomes. Future evaluations of the DC on the
cross-sectioned surface of printed resin and the two-dimensional mapping of the DC may
also provide further insight. In addition, this study only focused on mechanical and biolog-
ical properties, Vickers hardness and it is possible that the accuracy of the finally produced
3D printed parts would differ with the postcuring parameters. In particular, the accuracy of
small and thin 3D printed parts might be reduced at higher postcuring temperature, which
should be carefully investigated in future studies. Investigating monomer elution might be
useful for thoroughly interpreting the cytotoxic effects of 3D printed resin. Furthermore,
the effects of using various postcuring chambers on the properties of 3D printed resin
material should be determined in order to better understand the postcuring process.

5. Conclusions
This study investigated the effects of the postcuring temperature and time on the
mechanical properties, Vickers hardness, biocompatibility, and DC of 3D printed resin.
Within the limitations of this study, the following conclusions can be drawn: (1) increasing
the postcuring temperature results in significant enhancements of the flexural properties,
Vickers hardness, biocompatibility, and protein adsorption; (2) increasing the postcuring
time improves the flexural properties, Vickers hardness, and biocompatibility; and (3) a
longer postcuring time at a low temperature produces results similar to using a shorter
postcuring time at a higher postcuring temperature.

Supplementary Materials: The followings are available online at https://www.mdpi.com/article/

10.3390/polym13081180/s1, Table S1. Degree of conversion of three-dimensional printed resin under
various curing conditions. Data are mean ± standard deviation values. Different uppercase letters
indicate a significant difference in postcuring time for the respective postcuring-temperature group in
the same row. Different lowercase letters indicate a significant difference in postcuring temperature
according to postcuring time. Table S2. HGFs were seeded into 48-well plates at a density of
5 × 104 cells/specimen. The number of cells attached on each specimen (n = 3) was counted after
24, 48 and 72 h of incubation. Mean values are presented. Figure S1: Disk-shaped specimen, with a
diameter of 9 mm and a thickness of 2 mm, were designed using a 3D modeling software for Vickers
hardness, biocompatibility test and degree of conversion. Figures S2–S4: CLSM images of HGFs
cultured (1 × 105 cells/mL) on 3D printed specimens postcured at various temperatures and times
after 24, 48 and 72 h of incubation, respectively.
Author Contributions: Conceptualization, J.-E.K.; methodology, E.B., K.-H.P. and J.-E.K.; software,
E.B., S.-H.S. and K.-H.P.; validation, E.B. and J.-E.K.; formal analysis, E.B., K.-H.P. and J.-E.K.;
investigation, E.B. and J.-E.K.; data curation, E.B. and J.-H.L. (Jung-Hwa Lim); writing—original draft
preparation, E.B. and J.-E.K.; writing—review and editing, E.B., K.-H.P., Y.-B.P., J.-H.L. (Jae-Hoon
Lee) and J.-E.K.; visualization, E.B., Y.-B.P., J.-H.L. (Jae-Hoon Lee) and J.-E.K.; supervision, J.-E.K.;
project administration, J.-E.K.; funding acquisition, J.-E.K. All authors have read and agreed to the
published version of the manuscript.
Funding: This work was supported by the Korea Medical Device Development Fund grant funded by
the Korea government (the Ministry of Science and ICT, the Ministry of Trade, Industry and Energy,
the Ministry of Health & Welfare, the Ministry of Food and Drug Safety) (NTIS Number: 9991006910).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are available on request from the
corresponding author.
Polymers 2021, 13, 1180 16 of 17

Conflicts of Interest: The authors declare no conflict of interest.

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