Effect of Tricalcium Aluminate on the Physicochemical Properties, Bioactivity, and Biocompatibility of Partially Stabilized Cements
Biocompatibility of Partially Stabilized Cements. PLoS ONE 9(9): e106754. doi:10.1371/journal.pone.0106754
Effect of Tricalcium Aluminate on the Physicochemical Properties, Bioactivity, and Biocompatibility of Partially Stabilized Cements
Chun-Pin Lin 0
Kai-Chun Chang 0
Chia-Chieh Chang 0
Ying-Chieh Huang 0
Min-Hua Chen 0
Feng-Huei Lin 0
Jie Zheng, University of Akron, United States of America
0 1 Graduate Institute of Clinical Dentistry, School of Dentistry and National Taiwan University Hospital, National Taiwan University , Taipei, Taiwan , 2 Institute of Biomedical Engineering, National Taiwan University , Taipei , Taiwan
Background/Purpose: Mineral Trioxide Aggregate (MTA) was widely used as a root-end filling material and for vital pulp therapy. A significant disadvantage to MTA is the prolonged setting time has limited the application in endodontic treatments. This study examined the physicochemical properties and biological performance of novel partially stabilized cements (PSCs) prepared to address some of the drawbacks of MTA, without causing any change in biological properties. PSC has a great potential as the vital pulp therapy material in dentistry. Methods: This study examined three experimental groups consisting of samples that were fabricated using sol-gel processes in C3S/C3A molar ratios of 9/1, 7/3, and 5/5 (denoted as PSC-91, PSC-73, and PSC-55, respectively). The comparison group consisted of MTA samples. The setting times, pH variation, compressive strength, morphology, and phase composition of hydration products and ex vivo bioactivity were evaluated. Moreover, biocompatibility was assessed by using lactate dehydrogenase to determine the cytotoxicity and a cell proliferation (WST-1) assay kit to determine cell viability. Mineralization was evaluated using Alizarin Red S staining. Results: Crystalline phases, which were determined using X-ray diffraction analysis, confirmed that the C3A contents of the material powder differed. The initial setting times of PSC-73 and PSC-55 ranged between 15 and 25 min; these values are significantly ( p,0.05, ANOVA and post-hoc test) lower than those obtained for MTA (165 min) and PSC-91 (80.5 min). All of the PSCs exhibited ex vivo bioactivity when immersed in simulated body fluid. The biocompatibility results for all of the tested cements were as favorable as those of the negative control, except for PSC-55, which exhibited mild cytotoxicity. Conclusion: PSC-91 is a favorable material for vital pulp therapy because it exhibits optimal compressive strength, a short setting time, and high biocompatibility and bioactivity.
Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its
Supporting Information files.
Funding: This study was supported by a grant from the Ministry of Science and Technology, Taiwan (99-2628-B-002-065-MY3). The funders had no role in study
design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
The pulpal tissue in teeth has various functions, including (1)
physiological deposition of dentin by odontoblasts; (2) nutritional
supply through microcirculation; (3) protection and sensation of
nerve endings in dentin, and (4) repair under stimulation by
forming tertiary dentin. When pulpal tissue is subjected to
trauma, caries, or iatrogenesis, the tooth can be treated with root
canal treatment or pulp capping to prevent further pulpal
inflammation or infection. Although the success rate of root canal
treatment is high, the absence of pulpal tissue prevents teeth
sensation or further repair. To maintain the functions of pulpal
tissue, vital pulp therapy (VPT) is a less invasive alternative
treatment to root canal therapy. VPT involves direct pulp capping
or partial pulpotomy, in which a biomaterial is used to maintain
the vitality of the pulp of a tooth and establish an environment in
which the dentin-pulp complex can form. An ideal material for
VPT must exhibit favorable sealing ability, nontoxicity to pulp
tissue, antibacterial properties, high biocompatibility, stability in
tissue fluids, a short setting time, adequate mechanical strength,
and favorable handling properties.
Numerous materials, such as zinc oxide eugenol, glass ionomer
or resin-modifed glass ionomer, and calcium hydroxide, have been
used in VPT; however, none of these materials have achieved
the requirements for an ideal material. Calcium silicate
cements, such as Portland cement or mineral trioxide aggregate
(MTA), currently exhibit substantial potential for use as
biomaterials in VPT.[9,10] MTA is a bioactive material that features
excellent apatite-forming ability; thus, it exhibits a substantial
clinical advantage over traditional cements. In addition,
numerous studies have shown that MTA exhibits excellent sealing
ability, a high pH, radiopacity, biocompatibility, and an ability to
stimulate dentin matrix protein expression. However, the
poor handling properties and long setting time of MTA limit its
clinical application.[4,16] Effort is required to overcome the
shortcomings of MTA as a biomaterial used in VPT.
In previous studies, we developed a partially stabilized cement
(PSC) that exhibits similar properties to MTA. PSC is an
innovative biomaterial developed to reform some of the weaknesses of
MTA. PSC is a refined calcium silicate cement of which the major
chemical constituents are tricalcium silicate (3CaONSiO2; C3S),
dicalcium silicate (2CaONSiO2; C2S), tricalcium aluminate
(3CaONAl2O3; C3A), and calcium aluminoferrite (4CaONAl2O3NFe2O3;
C4AF), with specific ratios of each component. Among these
components, C3S is associated with long-term mechanical strength.
However, C3S and C2S exhibit long setting times and low
mechanical strength at the early stages of hydrated calcium silicate
cement. Nevertheless, C3A exhibits the fastest hydration rate and
provides the initial mechanical strength of calcium silicate
cement. Therefore, the C3A/C3S ratio may play a crucial role
in the early hydration reaction of PSC, producing an accelerating
effect on setting time. The sol-gel process is a useful method for
preparing ceramics that enables easy control of the compositions of
mixtures. Therefore, PSCs with different molar ratios of C3A were
fabricated using a one-step sol-gel process featuring a low processing
temperature, high chemical homogeneity, uniform phase distribution
in a multicomponent system, and high reactivity of the
The purposes of this study were to investigate the effect of C3A
on PSCs and compare the PSCs with MTA by using X-ray
diffraction analysis (XRD) and scanning electron microscopy
(SEM) to observe the microstructures and hydration behavior of
PSCs in a physiological environment and by evaluating the pH
variation, setting time, mechanical properties, and
biocompatibility of the cements.
Materials and Methods
PSC powder was prepared using the sol-gel process as
previously described. Figure 1 shows schematic diagrams of
the preparation. Aluminum sec-butoxide (ASB, Al(OBus)3), an Al
precursor mixed with acetylacetone (acac), was used as the
complex ligand for modifying Al(OBus)3. The mixture was stirred
and reacted for 4 h in a complexing ratio (x) equal to 1. A
Al(OBus)3/(acac) complex was formed before conducting further
sol-gel process reaction. The complexing ratio (x) represented the
molar ratio of acac to Al(OBus)3 (Al(OBus)3/acac). After the
surface of Al(OBus)3 was modified, tetraethyl orthosilicate
(Si(OEt)4) was added as a Si precursor to the solution, which
was subjected to continual stirring. An aqueous solution of
Ca(NO3)2 as a Ca precursor and Fe(NO3)3 as a Fe precursor
was subsequently added to the solution. Ammonia water was
added as a catalyst to facilitate reaction between alkoxides. A gel
was formed and maintained for 24 h until gelation occurred. The
gel was dried at 110uC, and then heated at 1400uC for 2 h and
quenched in air. All of the reagents and chemicals used in this
study were purchased from Sigma-Aldrich Co (St. Louis, MO,
USA). White MTA was obtained from Dentsply, Tulsa Dental
Products (Tulsa, OK, USA).
Three specimens (PSCs) were prepared in C3S (x)/C3A (y)
molar ratios of 9/1, 7/3, and 5/5 (PSC-91, PSC-73, and PSC-55)
by using sol-gel processes. De-ionized water (D.I. water) was then
added to obtain PSC homogeneous pastes. The liquid-to-powder
ratio (L/P) for all specimens was 0.5 mL/g. The mixtures were
mixed for 5 min and then placed into a Teflon mold. The
specimens were subsequently retrieved from the mold and stored
in a sealed container with 100% relative humidity at 37uC to
solidify further. The hydration products of all of the specimens
were mixed with D.I. water and hydrated with a simulated body
fluid (SBF) solution at 4 h, 12 h, 1 day, 3 days, 7 days, 10 days,
and 28 days. After incubating for a period of time, the specimens
were soaked immediately in an anhydrous ethanol to stop the
hydration reaction and enable them to be subjected to material
X-ray diffraction analysis
The crystalline phases of all specimens before and after
hydration were determined using a Rigaku X-ray powder
diffractometer (Rigaku Geigerflex, Japan) with CuKa radiation
(l = 1.54 A) and a Ni filter which was generated at 30 kV and
20 mA. The scanning rate of the specimens was 3u/min, and the
scanning range (2h) was 10u to 60u. The XRD patterns were
collected and analyzed according to a model automatched to the
standard JCPDS database by using Jade 6.0 software.
Scanning electron microscope observation
The microstructures of the hydration products on the specimen
surfaces were examined using a field-emission scanning electron
microscope (FE-SEM, Hitachi S-4700) operated at 15 kV. Three
specimens (PSCs) with different molar ratios of C3A were
prepared for morphological observation conducted using a
FESEM. After the specimens were air dried for 24 h at room
temperature, the surfaces of the hydrated cement specimens were
coated with a gold film by using sputtering physical vapor
deposition and examined using the FE-SEM.
Vicat setting time
The initial and final setting times of the PSCs and MTA were
measured using a Vicat needle apparatus. This test was based on
International Standard ISO 9597. The Vicat needle was
cylindrical and 2.0 mm in diameter. The needle was initially
fixed on a 100-g moveable rod and moved in a vertical alignment.
Cement was placed in a mold and the needle penetrated 35 mm
above the bottom of cement paste. The final setting time was
determined using the needle (1.13 mm in diameter) loaded on a
300-g moveable rod, which no longer penetrated or indented the
surface of the paste. Five cement specimens were measured, and
the values are expressed as mean 6 standard deviation (mean 6
The pH values of all PSCs and the MTA were measured using
the temperature-compensated electrode of a pH meter. Six
samples of each cement were subjected to measurement. Each
specimen was placed in a tube containing 10 mL of D.I. water and
sealed in a container. The pH of the D.I. water in the tube was
assessed using a pH meter at 2, 4, 12, 24, 72, 168, and 240 h. After
each test, the samples were removed from the container and
placed in a new container with the same volume of D.I. water.
Mechanical strength measurements
The mechanical strength of all tested cements was evaluated
according to the compressive strength. The specimen was placed
into a cylindrical Teflon mold (4 mm in diameter 6 6 mm in
height) and stored in an environment of 100% relative humidity
for 24 h at 37uC. Tensile strength data were collected by
measuring the diameter and height of the specimens by using a
micrometer. The specimens were fractured at a cross-head speed
of 1.0 mm/min by using a universal testing machine (Instron
5566, Canton, MA, USA). The maximal load required to fracture
each specimen was measured, and the compressive strength, s,
was calculated using the formula
where P is the maximum load applied to the specimen in Newtons,
and A is the area in millimeters squared. Statistical analysis was
conducted using a one-way ANOVA in which p values (*) ,0.05
Culture of human dental pulp cells
Primary human dental pulp cells were used in this study, and
approved by the National Taiwan University Hospital (NTUH)
Research Ethics Committee (REC) and all patients signed written
informed consent, which was obtained from all subjects, dental
pulp tissues were obtained from freshly extracted premolars and
third molars without caries or pulpal diseases. A tissue explant
technique was processed to cultivate dental pulp cells as described
previously.[22,23] Briefly, pulp tissues were minced into small
pieces (approximately 1 mm3) and then digested with 3 mg/mL
collagenase type I (Sigma, St Louis, MO, USA) and 4 mg/mL
dispase (Sigma, St Louis, MO, USA) for 1 h at 37uC. The human
dental pulp cells were cultured in Dulbeccos modified Eagles
medium (DMEM), which contained 4 mM L-glutamine,
4500 mg/L of glucose, 1 mM sodium pyruvate, and 1500 mg/L
of sodium bicarbonate. The culture medium was supplemented
with 10% fetal bovine serum, and the dental pulp cells were
incubated in a humidified atmosphere of 5% CO2 at 37uC. When
the dental pulp cells proliferated to 90% confluence in DMEM,
the confluent cultures were detached using 0.25% Trypsin-EDTA
and subcultured in a flask of DMEM to enable expansion.
Cultured human dental pulp cells in passage number 310 were
used for the following studies.
Cytotoxicity assay and cell viability assay
The cytotoxicity of all tested cements were measured using the
Cyto Tox Non-Radioactive Cytotoxicity Assay detection kit
(Promega, Madison, WI, USA). The lactate dehydrogenase
(LDH) activity was determined using a spectrophotometric assay.
Methods for determining LDH involved combining tetrazolium
salts with diaphorase. The chemical reactions of the assay are
listed as follows:
NADHztetrazolium salt?NADzzformazan red color
The cell cytotoxicity percentage was calculated by quantifying
the amount of LDH in the medium from dead cells and dividing
the result by the total amount of LDH in the medium and target
cell lysate in the sample. Cell cytotoxicity results are expressed as
the percentage of LDH released. The cytotoxicity of the cement
was tested in accordance with ISO 10993-5. Briefly, 0.2 g of the
sample was soaked in 1 mL of DMEM and incubated in 37uC for
72 h. Dental pulp cells were seeded at a density of 56103 cells per
well in a 96-well culture plate for 24 h. After 24 h, the extract
solution of the sample containing the medium at various
concentrations was then added to the culture plate. The plates
were incubated for 24 h and 72 h. The LDH released from the
medium was measured using an ELISA reader (optical density at
Dental pulp cells were used in a cell proliferation and viability
assay. Cells at 36103 cells/well were cultured in a 96-well culture
plate containing 100 mL of DMEM per well for 1 day and 3 days.
The cell viability of all tested cements was assessed by using a
water-soluble tetrazolium salt-1 (WST-1) cell proliferation assay kit
(Roche Diagnostics, Mannheim, Germany). This assay depends on
cleavage of WST-1 by mitochondrial dehydrogenase in viable
cells. The formazan dye produced by viable cells can be
quantified. The number of viable cells was measured
colorimetrically by using an ELISA reader at an absorbance (optical density,
O.D.) of 440 nm. The results were expressed as the mean O.D. of
experimental groups (n = 6) vs. negative control (normal DMEM).
The mean O.D. of the control group was set to represent 100%
Alizarin red S staining
Mineralization of human dental pulp cells was assessed using
Alizarin Red S staining (Sigma-Aldrich, St. Louis, MO, USA).
Human dental pulp cells (56103 cells/well) were cultured in
DMEM containing 10% FBS and treated with the extracts of all
tested cements for 21 days for a mineralized nodule assay. The
pulp cells were cultured with a culture medium containing extracts
of the cements, and the culture medium was replaced every 3 days.
After 21 days of treatment, the cells were rinsed twice with
phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde
for 15 min at room temperature, and stained with a 2% (w/v)
Alizarin Red S staining solution at a pH level of 4.2 and a
temperature of 37uC. Images of Alizarin Red S staining were
viewed and photographed under a light microscope.
Data are expressed as the mean 6 standard deviation (SD).
Statistically significant differences from the control group were
determined using a one-way factorial ANOVA. Differences with p
values (*) ,0.05 were considered significant.
X-ray diffraction analysis
Figure 2 shows the XRD powder patterns of unhydrated and
hydrated cements stored in D.I. water. Unhydrated PSC powder
was obtained using sol-gel processes after calcination at 1400uC for
2 h and was characterized using XRD (Figure 2 A). Crystalline
phases were characterized using standard data from the JCPDS
database. Most of the diffraction peaks were identified as
structures of C3S, C2S, C3A, and C4AF, which are the major
components of PSC. The peaks at the position 2h = 33.2 and 47.6u
corresponded to a C3A structure, and the peak intensity increased
as the C3A molar ratio increased. The peaks at 2h = 23.2u, 32.1u
32.7u corresponded to C2S and C3S structures. A similar pattern
was observed among the unhydrated MTA samples. In addition,
the peak at 2h = 37.3u and 53.9u corresponded to a CaO structure,
and that at 2h = 27.4u corresponded to a Bi2O3 structure.
For the hydrated PSCs, three phases of C3S, C2S, and C3A
were identified at the same peaks at 2h. However, the relative
intensities of the peaks of these three phases decreased over time
after the hydration reaction, as shown in Figure 2 (B)(D).
Portlandite hydrated products and calcium silicate hydrate
(CSH) were observed in each experimental group after the
samples were stored in D.I. water. Portlandite is a major hydration
product of calcium silicate cement. The peaks at the position
2h = 18u and 34.1u corresponded to Ca(OH)2, and the relative
intensity of the peak increased as the degree of hydration
increased. CSH (2h = 28.6u, 29.1u, and 31.6u) was identified in
all PSCs because of the hydration of C3S. Figure 2 (C) and
Figure 2 (D) show the Ca3Al2(OH)12 (C3AH6) hydration product
(2h = 17.3u, 39.2u, and 44.4u). The relative intensity of the peak of
PSC-55 was higher than that of PSC-73 because the amount of
Characterization of hydration products soaking in
simulated body fluid by using scanning electron
The PSC specimens, of which the microstructures are illustrated
in Figure 3, were stored in an SBF environment for 1 day and 7
days. Morphological observations indicated that the hydrated
PSC-91 soaked in the SBF for 1 day exhibited a cubic
microstructure containing mesh-like crystals, as shown in Figure 3
(A). Hydroxyapatite-like crystals, which covered the specimen
surface, were the hydration products of PSC-91; no cubic structure
was observed. By contrast, the hydrated PSC-73 and PSC-55 that
soaked in SBF for 1 day exhibited both a slight amorphous silk-like
structure and a crystalline hexagonal structure, as shown in
Figures 3 (B) and 3 (C). A silk-like structure is the initial hydration
product of calcium silicate with a low Ca/Si ratio and crystallinity,
whereas a crystalline hexagonal structure is a product of
portlandite, which has a crystallinity that is higher than that of
CSH. Figure 3 (E) shows the surface morphology of PSC-91
hydrated in SBF for 7 days; a large portion of the surface was
covered with mesh-like HAp crystals. A hydroxyapatite-like
structure began to form after only 1 day. After 7 days, aging
completely covered the PSC-91 and MTA surface, as shown in
Figure 3 (D) and (H). Figure 3 (G) shows that mesh-like crystals
embedded the ball-like structure in the matrix of the PSC-55
specimen that was soaked in SBF for 7 days. The hydration
products of PSC-55 included more hydroxyapatite-like crystals
compared with the specimen that was soaked in SBF for 1 day. A
gradual change in the microstructure of PSC-73 was observed
using FE-SEM, as shown in Figure 3 (F); additional ball-like
crystals and few hydroxyapatite-like crystals covered the matrix.
Physicochemical properties of the cements
The physicochemical properties of all tested cements are shown
in Figure 4. The change in pH as a function of time for all
materials is shown in Figure 4 (A). The initial pH value of all tested
cements after mixing was approximately 7.5 and rose to 12.4
12.7. The initial pH value of PSC-91, PSC-73, and PSC-55 at 4 h
was higher than that of the MTA, and the alkalinity of these PSCs
tended to increase over time. All cements exhibited a high alkaline
pH. The highest value was that of PSC-91, and the mean was 12.7
at 240 h. The pH values of PSC-91, PSC-73, and PSC-55 did not
differ significantly from those of MTA after 72 h.
The initial and final setting times of all of the materials are
shown in Figure 4 (B). The initial setting times for PSC-91,
PSC73, and PSC-55 were between 15.5 and 80.5 min, and the final
setting times ranged between 68.5 and 160 min. C3A exhibited an
accelerated setting effect during hydration of the PSCs compared
with MTA. When the molar ratios of C3A in the PSC were
increased from 10% to 50%, the initial and final setting times of
the PSCs decreased dramatically compared with those of MTA.
Figure 4 (C) shows the compressive strength of all of the tested
cements. PSC-91 and PSC-73 exhibited similar compressive
strength in the early stage in 4 h. The compressive strength of
PSC-55 was slightly lower than that of the other experimental
samples at 4 h. After being set in D.I. water for 24 h, PSC-91
exhibited higher compressive strength (27.1 MPa) than PSC-73
and PSC-55 did. PSC-91 exhibited the highest compressive
strength among the PSCs, 51.5 MPa, after it was set for 168 h.
Reducing the amount of C3A increased the mechanical strength of
the PSCs. The compressive strength of PSC-73 and PSC-55 did
Figure 2. XRD powder patterns of unhydrated cements and hydrated PSCs stored in D.I. water for 1, 3 and 7 day. (A) the unhydrated of
PSC-73; (D) the hydrated patterns of PSC-55. [w C3A; C3S; Bi2O3; & Ca(OH)2; m CSH; N C3AH6].
PSC-91, PSC-73, and PSC-55 after calcined at 1400uC for 2 h and unhydrated MTA; (B) the hydrated patterns of PSC-91; (C) the hydrated patterns of
Figure 3. SEM micrograph of the surface of specimens stored in simulated body fluid (SBF) various durations. Soaked in SBF after 1
day: (A) PSC-91 (B) PSC-73 (C) PSC-55 (D) MTA; Soaked in SBF for 7 days: (E) PSC-91 (F) PSC-73 (G) PSC-55 (H) MTA.
not differ significantly after the specimens were set for 168 h. The
results revealed that the content of C3A in the PSCs exerted an
obvious effect on the compressive strength of the cement.
The cytotoxicity of all tested cements was determined by
conducting an LDH assay. The cytotoxicity of the control group
and all of the tested biomaterials increased over time, as shown in
Figure 5 (A). PSC-91 and PSC-73 exhibited no statistically
significant difference from the MTA at 1 day and 3 days.
However, the cytotoxicity of PSC-55 was significantly higher than
that of the other experimental samples at 1 day and 3 days. The
results of the LDH assay indicated that the PSCs exhibited low
levels of cell cytotoxicity.
Cell viability was evaluated according to the mitochondrial
function (WST-1 assay) of the cells. The O.D. value was directly
proportional to cell number. As shown in Figure 5 (B), the O.D.
value of MTA group was significant higher than that of the
negative control group at day 1 and 3. The O.D. value of PSC-91
was significantly increased compared with negative control group
at day 3. The cell numbers of PSC-55 at 1 day and 3 days
exhibited a statistically significant difference (p,0.05) from those
of MTA. Increasing the molar ratios of C3A in the PSCs decrease
the activity of mitochondria (cell viability).
Mineralized nodule formation
The effect of all of the tested cements on the formation of
calcification nodules in human dental pulp cells that were
incubated for 21 days and examined using Alizarin Red S staining
is shown in Figure 6. PSC-91 and MTA exhibited a significant
increase in the area of calcified nodules compared with PSC-73
and PSC-55, whereas no clear mineralization was observed in the
In this study, three PSCs containing C3A in different ratios were
fabricated using sol-gel processes. The sol-gel process is a useful
method for preparing ceramics and glass  and features high
chemical homogeneity, uniform phase distribution in
multicomponent systems, high reactivity of the product, and low synthesis
temperatures. According to the XRD results (Figure 2 (A)), the
major components of the synthesized unhydrated PSCs were C3S,
C2S, and C3A; this composition is similar to those of MTA and
portlandite. The XRD peak intensity of C3A increased as the
molar ratio of C3A in PSC increased, indicating that a proportion
of raw materials reacted homogenously during the sol-gel process.
C3S and C2S are two major components of PSC. Their
hydration reaction can be expressed using Formulas (1)(3). The
hydration products are CSH and calcium hydroxide (CH). The
C3A is substantially influenced by the early hydration behavior of
PSC. The hydration of C3A is rapid and its hydration reactions
can be expressed using Formula (4). The hydration process in
this reaction involves several hydrates; (Ca3Al2(OH)12; C3AH6) is
one of the hydrates and is the most stable at high temperatures.
The hydration reaction of each component of PSC is expressed
C3AzH?gel?irregular flakes?hexagonol flakes?
C3AH6 single crystals?C3AH6 aggregates
where [A = Al2O3; C = CaO; CH = Ca(OH)2; H = H2O; S =
SiO2; C-S-H is amorphous hydrogen having variable composition
in terms of Ca/Si ratio and H2O/SiO2 ratios].
The hydrolysis of calcium silicates produces calcium hydroxide
and creates a less basic calcium silicate hydrate. Calcium
hydroxide precipitated during the hydration of PSC. The presence
of calcium hydroxide causes the hydrated PSC to be highly
alkaline (pH 12.5). All of the cements evaluated in this study
increased the pH values (7.5 at 12.7) through the release of
hydroxyl ions. The more C3A in PSC, the slower pH value rising
in the immersed solution. The curve of pH versus immersion time;
where slope of the initial stage was different depends on the C3A
molar ratio in PSCs. Figure 4 (A) shows the rising curve: PSC-91.
PSC-73.PSC-55.MTA. MTA contains gypsum, which hindered
the hydration reaction, causing the pH value to rise at slowest rate.
The highly alkaline environment has an antibacterial effect and
promotes cell remineralization.
One of the most clinically relevant factors is the setting time of
biomaterials, which is affected by numerous factors, such as the L/
P ratio, particle size of the cement, chemical content of the
cement, and cement additives. A long setting time may prevent the
material from being held at the operation site, causing a lack of
mechanical strength required for initial support. The initial
setting time of MTA is approximately 165 min. Because of this
long initial setting time, a two-step procedure must be used when
applying MTA in VPT. C3A is the most reactive component of
Portland cement, and increasing the molar ratio C3A in PSCs may
reduce the setting time and enhance the initial compressive
strength. PSC-55 has short initial (15.5 min) and final (68.5 min)
setting times (Figure 4 (B)) because the ratio of C3A to C3S is high;
however, the compressive strength of PSC-55 and PSC-73 was
lower than that of the other experimental groups during the 7-day
hydration reaction, as shown in Figure 4 (C). By contrast, C3S and
C2S play a crucial role in cement mechanical strength, and C2S
hydration occurs more slowly than C3S hydration does,
contributing to the long-term strength of the cement.
Calcium silicate materials exhibit dissolution and precipitation
behaviors during the hydration reaction. The calcium and
hydroxyl ions released from materials reacts with phosphate to
form a hydroxyapatite structure. Hydroxyapatite plays a
crucial role in tissue regeneration and maintaining function
because of its bioactive surface.
According to the SEM observation, the hydroxyapatite layer
precipitated on the surface of all of the PSCs after they were
exposed to the SBF environment for 7 days, as shown in Figure 3.
The mesh-like apatite layer was observed in PSC-91 and MTA at
1 day and 7 days. However, this layer was not observed in PSC-73
and PSC-55 until the samples were immersed in SBF for 7 days. In
addition, the hydration products were examined using XRD, and
the patterns indicated that Ca-P hydrated products and calcium
carbonate (CaCO3) were present in each experimental group after
the samples were soaked in SBF. In addition, the characteristic
peaks of hydroxyapatite crystals were present in the PSC-91 and
MTA groups, suggesting that hydroxyapatite precipitated on the
Figure 5. (A) Cytotoxicity assessment of PSC-91, PSC-73, PSC-55 and mineral trioxide aggregate (MTA) by LDH assay according to ISO-10993 protocol
standard. All tested cements on dental pulp cells were evaluated by LDH assay on 1day and 3 days. Each bar illustrated average absorbance
(A490 nm) 6 SD. No significant differences between PSC-91, PSC-73 and MTA (P.0.05); (B) Cell viability evaluation by WST-1 assay. Each bar
illustrated average absorbance (A440 nm) 6 SD.
surfaces of all of the specimens after they were soaked in SBF for 7
days. Both the SEM and XRD results indicated that the C3A and
C3S in PSCs facilitated the precipitation of a hydroxyapatite-like
active layer, suggesting that the PSCs feature favorable in vitro
bioactivity. The time required for C3A and C3S to induce a
hydroxyapatite-like structure increases when the C3A molar ratio
is over 30% in PSC. Small round particles were observed on the
surface of the PSCs after they were immersed in SBF for various
durations. XRD analysis revealed that the hydration products
were calcium-deficient carbonated apatite or had a calcium
The biocompatibility of dental materials can be evaluated using
numerous mammalian cell culture methods. Extracts of all of the
tested cements were used in this study. According to the ISO
10993-5 standard, all of the tested cements were evaluated using in
vitro tests of cytotoxicity, and the response of cells to the extracts
were assessed. Numerous studies have reported that MTA features
high biocompatibility. In this study, MTA was used as a
comparative biomaterial because PSC is chemically similar to
Portland cement, which exhibits a biological response similar to
that of MTA. Figure 6 (A) indicates that PSC-55 contained a
substantial amount of C3A, exhibiting a statistically significant
difference in the LDH assay compared with the other groups at 1
day and 3 days of incubation. Liu et al. determined that adding
0%15% C3A into C3A and C3S mixtures yields no significant
cytotoxicity within the extract concentration range between
3.125 mg/mL and 100 mg/mL. The extract concentration
in this study was 100 mg/mL and, according to the results of the
cell viability assay, all of the PSCs exhibited no cytotoxicity and
did not influence cell function for a long period of time. Finally,
LDH and WST-1 assay kits were used in this study, and the results
indicated that all of the PSCs and MTA are biocompatible.
Mineralized nodules were characterized by using Alizarin Red S
staining. After 21 days of induction, we observed that mineralized
nodules of human dental pulp cells formed. All of the tested
cements induced mineralization of the pulp cells. In addition, the
stain of mineralized nodules of PSC-91 and MTA was more
intense under light microscopy. These results indicated that
PSC91 can facilitate the formation of mineralized nodules of human
dental pulp cells.
In summary, increasing the C3A content from 30% to 50%
significantly improved the setting time of the PSCs; however, the
mechanical strength and biological properties of the PSCs
deteriorated. According to the results of this study, PSC-91
exhibits an appropriate setting time and high mechanical strength
as well as a cellular response similar to that of MTA, a commercial
product applied in VPT. Based on its physicochemical properties,
in vitro biocompatibility, and bioactivity, PSC-91 can be applied
In this study, three PSCs containing C3A in different molar
ratios were fabricated using sol-gel processes. Their
physicochemical properties, bioactivity, and biocompatibility were
characterized using XRD, SEM, pH-metry, an Instron machine, a Vicat
needle apparatus, Alizarin Red S staining, and a cytotoxicity assay
kit. The PSC containing 10% C3A (PSC-91) exhibited optimal
compressive strength and a setting time shorter than that of MTA.
The hydration properties of C3A in PSCs played a critical role at
the early stages, and the results indicated that PSC-91 facilitated
apatite formation when the cement was soaked in SBF. The results
of the Alizarin Red S staining and biocompatibility assay indicated
that PSC-91 exerts no effect on cell viability or cytotoxicity and
facilitates the formation of mineralized nodules of human dental
pulp cells. The physicochemical properties and biocompatibility of
PSC-91 indicated that this material has considerable potential for
use as a biomaterial in VPT. However, additional studies are
required to explore the physical properties, antimicrobial
properties, and in vivo effectiveness of this material.
Conceived and designed the experiments: KCC FHL CPL. Performed the
experiments: KCC CCC YCH MHC. Analyzed the data: CCC YCH.
Contributed reagents/materials/analysis tools: YCH MHC. Wrote the
paper: KCC CCC CPL.
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