Journal of Applied Biomaterials & Biomechanics 2009; Vol. 7 no. 3, pp.

160-170 original article

© Società Italiana Biomateriali

Biomimetic calcium-silicate cements aged in simulated body

solutions. Osteoblast response and analyses of apatite coating
Maria Giovanna Gandolfi1,4, Gabriela Ciapetti2, Francesca Perut2, Paola Taddei3, Enrico Modena3,
Piermaria L. Rossi4, Carlo Prati1
1
Department of Odontostomatological Sciences, Endodontic Clinical Section, Laboratory of Endodontic Materials -
Alma Mater Studiorum, University of Bologna, Bologna - Italy
2
Laboratory for Physiopathology of Orthopedic Implants, Istituti Ortopedici Rizzoli, Bologna - Italy
3
Department of Biochemistry - Alma Mater Studiorum, University of Bologna, Bologna - Italy
4
Department of Earth Sciences - Alma Mater Studiorum, University of Bologna, Bologna - Italy

ABSTRACT: Purpose: Calcium-silicate cements have been recently proposed for application in dentistry as root-end filling and
root-perforation repair materials. The aim of this study was to investigate the effect of ageing of experimental calcium-silicate ce-
ments on the chemistry, morphology and in vitro bioactivity of the surface, as well as on osteoblast viability and proliferation.
Methods: Two experimental cements (wTC-Bi, containing bismuth oxide and wTC), mainly based on dicalcium-silicate and
tricalcium-silicate, were prepared and tested for their bioactivity after soaking in Dulbecco’s phosphate buffered saline
(DPBS), used as simulated body fluid. Human marrow stromal cells (HMSC) were seeded on the cements maintained in DPBS
for 5 hr (non-aged group), 14 and 28 days (aged group). Cell viability was assessed by the Alamar blueTM test and morphol-
ogy by scanning electron microscopy (SEM) at different time endpoints. The surface of the soaked cements was analyzed
by environmental scanning electron microscopy or SEM coupled with energy dispersive X-ray microanalysis (ESEM/EDX or
SEM/EDX respectively) and the micro-Raman technique.
Results: The ESEM/EDX results showed a uniform surface composed of CSH hydrogel (mainly derived from the hydration of
belite and alite) on both non-aged cements. Micro-Raman spectroscopy revealed the presence of calcium carbonate, anhy-
drite, ettringite, alite and belite. The SEM/EDX data showed an irregular calcium-phosphate multi-layered biocoating with
many sharp and protruding crystals on both the aged cements. Micro-Raman spectroscopy revealed crystalline apatite and
calcite. The osteoblast response results showed that both the experimental cements exerted no acute toxicity in the cell assay
systems. The non-aged samples promoted greater cell growth. SEM showed cells well spread and adherent to the non-aged
materials. A reduced number of attached cells was noticed on the aged cements. Bismuth oxide-containing cement allowed
a reduced cell viability suggesting some cytotoxic effects. However, the thick biocoating formed on the 28-day aged samples
lowered the deleterious effect of bismuth oxide on cell growth. Actually, micro-Raman spectroscopy revealed progressive
bismuth oxide depletion on the wTC-Bi surface, due to the increased thickness of the apatite deposit.
Conclusions: The study demonstrated that (1) these materials support osteogenic cells growth and may induce early bone
formation, (2) the ageing in DPBS reduced the growth of HMSC, but eliminated the deleterious effect of the bismuth oxide
on cell growth. In conclusion, the experimental cements have adequate biological properties to be used as root-end/root
repair filling materials or pulp capping materials. (Journal of Applied Biomaterials & Biomechanics 2009; 7: 160-70)

Key words: Calcium-silicate cement, Endodontic cements, Human marrow stromal cells, Biocompatibility, Biocoating

Received: 29/04/09; Revised: 04/09/09; Accepted: 03/11/09

INTRODUCTION
Different root-end filling materials have been used to
seal the apical root cavities during surgical procedures, in-
cluding amalgam, zinc oxide-eugenol cements, glass ion-
omer cements, epoxy resins and calcium-silicate Portland-
based cements. Many of them have demonstrated clinical
limitations reducing the success rate, and none of them
have fulfilled all the criteria for an ideal retrograde fill-
ing. A material for retrograde filling procedures should be
easy to use, biologically inert or bioactive, should induce
fast bone formation, and must be stable and inexpensive.
Earlier in vivo and clinical studies suggest that mineral tri-
oxide aggregate (MTA) and other calcium-silicate cements
allow better clinical results than the other materials (1-5).
New calcium-silicate materials are under development for
clinical use in endodontic practice to extend their use to
other clinical applications (6-10).
All calcium-silicate cements are hydraulic materials
able to set in the presence of moisture (11). This allows
their use in oral surgery (i.e. root-apex removal) and in
all conditions where blood and fluid contamination ham-
pers the correct use of other cements/materials. Calcium-
silicate cements have demonstrated suitable biological

1722-6899/160-11$25.00/0
Gandolfi et al
properties (8, 12-16), good marginal adaptation and seal-
ing ability (6, 17, 18), antimicrobial effect (19) and the
absence of genotoxicity (16, 20).
Recent studies have demonstrated that silicates and
calcium-silicate materials may produce a biocoating,
mainly composed of apatite, when immersed in simulated
body fluids (SBF) (21-25), but not in water. All calcium-
silicate Portland-based cements may leach calcium from
the dissolution of calcium-hydroxide (named portlandite)
formed during their hydration reaction (26), which may
react with phosphate ions in SBF creating the conditions
for an early and fast precipitation of apatite deposits (21-
25). Therefore, it is important to evaluate the biological re-
sponse of these cements before and after the formation of
the apatite coating, since the biological response to fresh
cement may be partially or completely different from that
of aged/mature cement.
In this study two experimental calcium-silicate ce-
ments usable as root-end and root-repair filling materials
were devised. The purpose of the study was to evaluate
the cell viability and growth on the experimental calcium-
silicate cements. Human marrow stromal cells (HMSC)
were seeded on the cements soaked in Dulbecco’s phos-
phate buffered saline (DPBS) for different time periods (5
hr, 14 and 28 days) to evaluate the influence of ageing on
the HMSC proliferation and viability. The in vitro bioactiv-
ity of the surfaces was assessed using environmental scan-
ning electron microscopy coupled with energy dispersive
X-ray microanalysis (ESEM/EDX), micro-Raman technique
and SEM/EDX.
MATERIALS AND METHODS
Materials
Two experimental calcium-silicate cements were pre-
pared and identified as wTC and wTC-Bi. The calcium-
silicate powder (CEM I white Aalborg, Aalborg, Denmark)
was subjected to thermal and mechanical treatments, and
added to calcium chloride (wTC) and with bismuth oxide
(wTC-Bi).
The cement powder was mixed for 15 sec with DPBS
(Lonza, Walkersville, Inc) using the powder/liquid ratio
2:1 by weight, and layered on a plastic coverslip (Therma-
nox, diameter 1.2 cm) to obtain standard disks. Mechani-
cal vibrations were used to make the disk surfaces flat and
regular, with a 1.13 ± 0.1 cm2 exposed surface area.
After preparation, the disks were immediately immer-
sed in DPBS at 37 °C for 5 hr to obtain a partial rigidity
and cohesiveness (non-aged samples = t0), or 14 days
(14-day aged samples = t14) or 28 days (28-day aged
samples = t28).
Before cell seeding, the materials were placed in
24-well culture plate and treated for 2 hr with 1% an-
tibiotic/antimycotic solution (10.000 U penicillin, 10 μg
streptomycin, 25 μg amphotericin B per mL), washed twi-
ce with phosphate buffered saline (PBS) and finally pre-
wetted with culture medium containing 10% fetal bovine
serum (FBS) for 24 hr at 37 °C to reproduce the in vivo
situation when proteins from blood rapidly coat the mate-
rial surface at implantation (27).
Cement surface morphology
The surface morphology of non-aged cements was
assessed in real time using an environmental scanning
electron microscope connected with an energy dispersi-
ve X-ray microanalysis system (ESEM/EDX Zeiss EVO 50,
Germany). The surface morphology of 14- and 28-day
aged samples was assessed using a conventional scanning
electron microscope (SEM/EDX Philips, Eindhoven, The
Netherlands). Additional 24-hr aged samples were also
observed using conventional SEM.
Micro-Raman spectroscopy
Micro-Raman spectra were obtained using a Ra-
man Spectrometer Jasco NRS-2000C (Jasco, Easton, MD,
USA) connected to a microscope with 20x magnification
(in these conditions the laser spot size was about 5 µm).
All the spectra were recorded in back-scattering condi-
tions with 5 cm-1 spectral resolutions using the 488 nm
line (Innova 70, Coherent Inc, CA, USA) with a power of
50 mW. A 160 K cooled CCD detector from Princeton In-
struments Inc (Trenton, NJ, USA) was used. The cements
were analyzed as wet samples, when maintained in their
storage media. To minimize variability deriving from
the possible sample inhomogeneity, five spectra at least
were recorded on the surface of each specimen. Raman
spectra were also recorded on unhydrated cements.
Cell culture
HMSC (Cambrex, Charles City, IA, USA) were cul-
tured in DMEM, supplemented with 10% v/v heat-inac-
tivated FBS, 1% penicillin-streptomycin, 50 μg/ml ascor-
bate 2-phosphate, 10-8 M dexamethasone and 2mM
L-glutamine, in 5% CO2 humidified atmosphere at 37
°C.
To test cell growth on materials, 1 x 104 cells/cm2
were seeded on disks placed in 24-well microplates and
cultured in 1 mL of complete medium for up to 28 days,
with medium change twice a week. The pH of the me-
dium was measured twice a week.
Cell viability
Alamar blueTM (BioSource International, USA) dye
was used for cell viability. After 24 hr, 6, 13, 22 and 28
161
Bioactivity of MTA cements
days of culture, the Alamar blueTM reagent was added to
the culture wells (1:10 v/v) and maintained for 4 hr. After
transferring the supernatants to 96-well plates, fluorescence
was read at 490 excitation-540 emission wavelengths using
a Cytofluor 2350 fluorimeter (Millipore Corporation, Bed-
ford, MA, USA). Cells plated on polystyrene culture wells
(TCPS) provided the controls. The results were expressed as
relative fluorescence units (RFU) and are reported as mean
± standard error of n = six replicates.
The differences were analyzed using the Wilcoxon
test with a significance level of p<0.05. Statistical anal-
ysis was performed with StatView™ 5.0.1 software for
Windows (SAS Institute Inc, Cary, NC, USA).
Cell morphology
The cells on differently aged cement surfaces were
visualized after 28 days of culture using a conventional
scanning electron microscope (SEM - Jeol 5200, Jeol,
Japan). The samples were washed in 0.2 M phosphate
buffer at pH 7.3 and fixed in Karnovsky’s fixative (1%
paraformaldehyde, 1.5% glutaraldehyde and 0.1 M so-
dium cacodylate buffer, pH 7.2-7.4).
162
Fig. 1 - ESEM analysis of non-
aged wTC-Bi cement inspected at
7.09 Torr (low vacuum) at 100%
RH and 5 °C. (a) The cements
resulted covered by a water film
and a semi-transparent hydrogel
visible on the top of the surface.
(b) Granules and round-shaped
deposits mixed with small needle-
like crystals (c) appeared on the
surface when it was observed at
higher vacuum and 20-40% RH.
Small porosities were observed.
(d) EDX showed Ca, Si, Al, Cl
and O peaks, the latter compat-
ible with the presence of water.
No P peak was detected.
RESULTS
Cement surface morphology
Non-aged samples of both cements were analyzed us-
ing ESEM. Each sample was initially inspected at 9.9-5.6
Torr (low vacuum procedure) at 100% relative humidity
(RH) and 4 °C. Under these conditions the cements result-
ed in being covered by a film of water (Fig. 1a) and a semi-
transparent hydrogel visible on the top of the surface. EDX
showed Ca and O peaks, the latter compatible with the
presence of water. Granules and round-shaped deposits
(Fig. 1b) mixed with small needle-like crystals (Fig. 1c) ap-
peared on the surface when it was observed at 3.1-2.1 Torr
at 4 °C and 20-40% RH. Small porosities were observed.
EDX revealed Ca, Si, Cl, Al and O peaks (Fig. 1d). No P
peak was detected on any cement.
SEM/EDX analysis of 24-hr aged cements revealed an
irregular surface with rounded formations of 2-10 microns
in diameter (Fig. 2a-d). EDX demonstrated the presence
of Ca, Si, and traces of S, Al, Cl and P. Peaks of Bi were
sporadically detected on wTC-Bi cement.
After 14 days in DPBS, a coating layer composed of
Gandolfi et al
Fig. 2 - SEM micrographs of the ce-
ment surface of wTC after 24 hours in
DPBS. (a,c) surface showing round-
shaped small formations of different siz-
es. White/black scale bar, 10 microns.
(b,d) EDX demonstrated the presence
of Ca, Si, traces of S, Al, Cl and P.
aggregate micro-globules or spherulites (0.2-0.6 microns
in diameter) mixed with polygon- and cube-shaped crys-
tals covered the surface of both cements. Ca and P peaks
and traces of Si were detected. The surface presented large
irregularities and a high number of pores. Many differently
shaped crystals, including diffuse small globular crystals
and aggregates of spherulites, larger polygon- and cube-
shaped crystals were detected on the surface of both ce-
ments (Fig. 3a-f).
After 28 days in DPBS a multi-layered irregular coating
mainly composed of aggregated spherulites was observed.
Agglomerates and precipitates increased the irregularity
and the unevenness, and changed the surface topography
of the cement. EDX revealed Ca and P peaks (Fig. 4a-d),
while Bi, S and Al were not detected.
Micro-Raman spectroscopy
Figure 5 shows the micro-Raman spectra recorded on
the two unhydrated powder cements as well as on the sur-
face of the two cements after storage for different times in
DPBS.
The spectra of both the unhydrated powders revealed
the presence of calcium carbonate, anhydrite, gypsum,
belite and alite, as shown by the respective marker bands
indicated in the spectra (28, 29). In addition, the spectrum
of unhydrated wTC-Bi showed strong bands assignable
to bismuth oxide. No phosphate band (about 962 cm-1)
was detected in the unhydrated powder of any of the ce-
ments.
The micro-Raman spectra recorded on the surface of
the freshly mixed cements (10’) revealed the presence of
ettringite (about 990 cm-1), anhydrite (about 1015 cm-1),
gypsum (about 1000 cm-1), alite and belite (855-840 cm-1
range), and calcium carbonate (about 1085 cm-1).
After 5 hr of soaking in DPBS, the ettringite content in-
creased, as revealed by the relative intensity of its marker
band. The anhydrite was found to hydrate more quickly
than gypsum so that its marker band at about 1015 cm-1
became nearly undetectable after 5 hr of soaking; at the
same time, alite appeared decreased with respect to be-
lite, confirming its higher hydration rate.
The spectra recorded on the surface of the cements
soaked for 24 hr in DPBS were revealed as major compo-
nents of ettringite (about 990 cm-1), alite and belite (855-
840 cm-1 range) and calcite and/or aragonite (about 1085
163
Bioactivity of MTA cements
cm-1). All the cements showed the band at about 962 cm-1
due to calcium phosphate, indicating the presence of an
apatite deposit. However, this deposit was still inhomoge-
neous, since the 962 cm-1 band was observed in only one
spectra of the five recorded on each cement. The band
due to hydrated silicates (670 cm-1), observed at longer
storage times was not detected since it was intrinsically
very weak. The marker band of portlandite at 360 cm-1
was never observed on the surface of the cements, due to
its release into the surrounding water medium.
At longer soaking times (14 and 28 days) the bands of
the cements became undetectable, while the 962 cm-1 ap-
atite band increased in intensity and the 1075 cm-1 com-
164
Fig. 3 - Cement surface of 14 days-aged wTC ce-
ment analyzed by SEM/EDX. White/black scale
bar, 10 microns (a,c) A layer composed of many
round-shaped precipitates mixed with cubic-
like crystals covered the surface. (b, d) Ca and
P peaks and traces of Si were detected. (e,f)
The surface showed irregularities and pores.
Many differently shaped crystals, including dif-
fuse small globular crystals (apatite spherulites)
0.2-0.6 microns diameter deposited on larger
polygonal- or cubic-shaped crystals (calcite)
5-20 microns diameter were detected.
ponent typical of B-type carbonated apatite appeared;
the presence of calcite was revealed by the sharp band at
about 1085 cm-1 occasionally accompanied by the weak
711 cm-1 component. Interestingly, for wTC-Bi, at increas-
ing storage times, the marker bands of the bismuth oxide
progressively decreased in intensity due to the increased
thickness of the deposit.
Cellular attachment/adhesion and morphology
The morphology and the number of cells on the non-
aged, 14 and 28-day aged cements were assessed.
An appreciable cell growth to semi-confluence was
Gandolfi et al

Fig. 4 - Cement surface of 28

days-aged wTC cement ana-
lyzed by SEM/EDX. White/black
scale bar, 10 microns (a,b) A
thick multi-layered coating was
observed on the cement surface,
with polygonal and many sphe-
roidal precipitates (0.2-0.6 mi-
crons diameter) and aggregates
of apatite spherulites, which
changed the surface topography
by the increase of surface irregu-
larities and unevenness. (c) Frac-
tured surface showing the thick
coating on cement surface. (d)
EDX revealed Ca and P peaks.

observed on the non-aged wTC cement (Fig. 6a, b).
Cells were spread, flattened on the cement surface, with
many anchoring processes from the cells to the material
surface and to other cells, and some cytoplasmic exten-
sions entering the cement asperities. Cells penetrating
within aggregates could be observed on the wTC.
The wTC-Bi had a reduced number of cells with few
morphological differences. The cells appeared partially
flattened and spread on the cement surface.
The surface of 14-day aged cements resulted in be-
ing more irregular with many pores and surface asperi-
ties. In several samples the coating layer resulted in
being partially delaminated and completely free from
cells (Fig. 6c, d). Both cements showed a reduced num-
ber of cells.
The 28-day aged cements showed few spread cells
and a markedly uneven coating layer with a great num-
ber of precipitates. wTC-Bi was almost free from cells
(Fig. 6e, f). The control cells on the TCPS surface were
very close to confluence and showed a fully spread
morphology with many filopodia and thin cell exten-
sions among them.
Cells number and viability
The pH of the culture medium was measured every 3
days and ranged between 7.0 and 8.0.
The viability of the cells on solid surfaces after 24 hr, 6,
13, 22 and 28 days of culture was assessed using the Alamar
blueTM assay. Figure 7 shows the results of cell viability report-
ing the mean ± standard error of six replicates for each type of
material, expressed as Relative Fluorescence Units.
The cells on both experimental cements had a good
viability, but cell adhesion was not favored by 14 days of
aging in DPBS.
Statistical analysis of Alamar blueTM data after 24 hr
of culture gave significant differences between wTC t0
vs. wTC t14 (p=0.0262), and wTC-Bi t0 vs. wTC-Bi t14
(p=0.0414).
In contrast, after 6 and 13 days of culture no signifi-
cant differences were found among the tested samples.
After 22 days of culture, the 28-day aged samples had
the lowest values; statistically significant differences in
cell viability were found comparing wTC t14 vs. wTC t28
(p=0.0277), and wTC t14 vs. wTC-Bi t14 (p=0.0277).

165
Bioactivity of MTA cements
After 22 and 28 days of culture the cells on the experi-
mental cement wTC were more viable compared to the
wTC-Bi-seeded cells.
DISCUSSION
Biomaterials placed in contact with bone should have
a favorable response in terms of cell adhesion, proliferation
and differentiation. Topography and chemistry of the ce-
ment surface play an important role in osteoblast adhesion
to biomaterials (30, 31). It is recognized that the cellular
response to a biomaterial begins with protein adsorption,
followed by the attachment phase which involves physical-
chemical linkage between cells and materials (32).
One of the characteristics of bioactive materials (eg cal-
cium phosphates, calcium silicates, bioactive glass cements
and glass-ceramic cements) is the ability to form an apatite-
like layer on their surfaces when in contact with physiological
fluids in vivo or with SBF in vitro (21, 22, 24, 25, 33-38).
To this purpose, advanced analytical techniques,
such as ESEM/EDX, Raman and Fourier Transform Infra-
red (FTIR) spectroscopy, are needed to assess the chemi-
cal and morphological characteristics of the newly formed
surface layers on materials soaked in SBF. Therefore, in
this study the morphology and composition of the cement
surface were inspected by ESEM equipped with EDX and
micro-Raman spectroscopy.
166
Fig. 5 - Micro-Raman spec-
tra of the wTC and wTC-
Bi cements: unhydrated
powders, freshly prepared
(10’), and after 5 hours, 1
day, 14 and 28 days of stor-
age in DPBS. The bands
due to belite (◊), alite (☐),
anhydrite (◆), gypsum (*),
calcium carbonate (●),
calcium phosphate (°) bis-
muth oxide (#) have been
indicated.
The purpose of this study was to evaluate the bioactivity
of two experimental calcium-silicate cements, and to cor-
relate the cell response with the chemistry/morphology of
the surface as it changes with the ageing time in a soaking
solution (non-aged, 14 and 28-day aged samples). An ad-
ditional purpose was to study the effect of the presence of
bismuth oxide (added as radiopacifier) (5, 10, 11) on cell
viability. The experimental cement wTC-Bi was composed
of the same active constituents as ProRoot MTA (10, 11), ie
dicalcium and tricalcium silicates, and anhydrite and Bi2O3,
while the wTC cement was formulated without Bi2O3.
As a first result, the study demonstrated that the morphol-
ogy of the cement surfaces was influenced by the soaking time
in DPBS. ESEM/EDX showed that the surfaces of non-aged
materials were formed by a hydrogel primarily composed of
water, silicon and calcium. SEM analysis disclosed different-
ly-shaped particles on the surfaces of the cements, including
diffuse small crystals, larger hexagonal crystals and globular
formations. The globular appearance of crystals on calcium-
silicate MTA was previously described by other authors (13).
The initially homogeneous surface of non-aged cement, with
several rounded formations and voids (calcium carbonate and
calcite), was replaced after 2 weeks by a surface completely
covered by precipitates frequently arranged as cloudy-like de-
posits. Using the micro-Raman technique, bands indicative
of apatite deposits were detected. After 28 days thick multi-
layered precipitates were observed on the cement surfaces.
EDX displayed Ca and P peaks only on 14- and 28-day aged
Gandolfi et al

Fig. 6 - (a) SEM observation of cells seeded

on non-aged fresh wTC cement after 28
days of culture. The surface is covered by a
layer of well-spread confluent cells. (b) SEM
observation of cells seeded on fresh wTC-
Bi cement and cultured for 28 days. Sparse
round-shaped cells are observed on the
surface of the cement. (c) Few spread cells
are observed on 14 days-aged wTC cement
after 28 days of culture. (d) Very few cells
are observed on 14 days-aged wTC-Bi ce-
ment after 28 days of culture. (e) Very lim-
ited amount of spread cells was observed
on 28 days-aged wTC cement after 28 days
of culture. (f) No cells were observed on
cement surface on 28 days-aged wTC-Bi ce-
ment after 28 days of culture.

samples, and the presence of B-type carbonated apatite and
calcite was confirmed by the micro-Raman technique. Evi-
dence of biological bone-like apatite formation on calcium-
silicate cements has been recently provided using other meth-
odologies and different calcium silicate cements (24, 25). It
has been reported that Si-OH groups on the hydrogel surface
act as nucleation centers of apatite precipitation (37, 38). The
elution of calcium originated from the dissolution of a hy-
dration product (i.e. portlandite) of calcium silicate particles
could significantly assist apatite growth by promoting local
Ca supersaturation, which accelerated the nucleation rate of
apatite. A synergistic effect between the CSH hydrogel as an
effective apatite nucleator and Ca as an apatite precipitation
accelerator caused the fast apatite precipitation.
The second finding of this study was that cell number
and viability was higher in non-aged groups, despite the
presence of apatite deposits on aged samples. The aged
samples showed a rough surface with many irregularities
and edges, as observed by SEM: this may be correlated with
the reduced number of attached cells. Possibly the non-
aged samples had a surface topography more suitable for
cell adhesion and proliferation than the aged samples, and

167
Bioactivity of MTA cements
Fig. 7 - Results of cell viability (mean ± standard error) of six replicates for
each type of material, expressed as Relative Fluorescence Units (RFU).
The differences were analyzed using Wilcoxon test with a significance
level of p<0.05.
Statistical analysis of Alamar blueTM data after 24 hours of culture gave
significant differences between wTC t0 vs wTC t14 (p=0.0262), and wTC-
Bi t0 vs wTC-Bi t14 (p=0.0414). In contrast, after 6 and 13 days of culture
no significant differences were found among the tested samples.
the presence of crystals and a multi-layer coating on the
surface of the aged cements may be responsible for ham-
pering cell adhesion and/or reduction in cell viability.
It is well known that micro- and nano-topography (surface
roughness with different features including grooves, ridges,
wells, pits, pillars) adsorbed proteins and chemical patterns of
material directly affect cell behavior and activity, mainly cel-
lular adhesion and viability (30, 31, 39). The nanostructured
calcium phosphate surfaces have been shown to increase
osteoblast adhesion, proliferation and differentiation, as bone
itself is nanostructured (40), and the size and geometry of crys-
tals could modify the response of surrounding tissues to the
implants as well (41). Sharp edged apatite particles could have
led to cell membrane damage following cytoskeletal contrac-
tion during cell adhesion. This may explain the higher cell ad-
hesion observed in plastic substrates (41).
The third finding of this study was the correlation between
the chemistry (silicon and calcium) of non-aged groups and
cell number and viability. EDX showed the presence of Si
(calcium silicate hydrated CSH gel) only on non-aged sam-
ples; analogously, micro-Raman spectroscopy revealed the
presence of the cement bands (gypsum, ettringite, alite and
belite) only on the surface of these samples. Moreover, SEM
observations and viability data support the concept that cells
preferentially adhere to the fresh cement surface. It is known
that osteoprogenitor cells respond to critical concentrations
of calcium and silicon released by the fresh cement surface
(24, 37, 42), and that calcium and silicon leached by fresh
cement represent signals to stimulate osteoblast maturation
and cell growth (37, 42). Silicon is an important element in
the early stages of bone formation and may stimulate col-
lagen synthesis and osteoblastic differentiation in human os-
teoblast-like cells. Silicon plays an active role in the surface
bioactivity and also has a catalytic role in the mineralization
of new bone (37), and calcium ions showed stimulatory ef-
168
fect on cell proliferation (42, 43).
The fourth finding of this study was that the lower
crystallinity of the coating proved to be more favorable
for the adhesion/proliferation of osteogenic cells, and
the crystallinity of apatite deposits increase during age-
ing in SBF. As previously reported (33-36), the crystallin-
ity degree of the biocoating formed on the wTC cement
increased at increasing storage times, being composed of
amorphous calcium phosphate at earlier storage times.
It has been reported that amorphous calcium phos-
phate showed improved bioactivity compared to crystal-
line hydroxyapatite, and more adhesion and proliferation
of osteogenic cells are observed on the amorphous cal-
cium phosphate substrates. Moreover, surface coatings
with lower crystallinity may be favorable to cell adhesion,
while apatite coatings with higher crystallinity yielded
higher rates of cell proliferation (44).
The final finding of this study was that the biocoating for-
mation reduced the deleterious effect of bismuth oxide on
cells. In this study, the inclusion of bismuth oxide on cement
composition was responsible for a reduction in cell growth.
The negative effect of bismuth oxide was present only on non-
aged and on 14-day aged cements, suggesting that the forma-
tion of a thick biocoating may prevent its deleterious effect.
The effect of bismuth oxide on human cells is controversial:
it has been demonstrated that calcium-silicate cements (Portland
cements and MTA) containing bismuth oxide induced cytotox-
icity in dental pulp cells, and affected cell viability (45). In con-
trast, other researchers have found that the addition of bismuth
oxide to Portland cement does not affect the biocompatibility of
the material (46). Therefore, caution should be exercised before
drawing firm conclusions about Bi2O3 toxicity.
A number of studies have shown that most endodon-
tic materials exert some toxic effect especially when they
are fresh/unset (47-51). Instead, our findings show that
calcium silicate cements (like MTAs and Portland-derived
cements) show some osteoconductivity, i.e. cell adhesion
and growth, immediately after mixing.
CONCLUSIONS
The study demonstrated:
• Both the experimental cements showed bioactivity
when soaked in simulated body fluid solution (DPBS),
with the formation of an irregular calcium-phosphate
biocoating with many sharp and protruding crystals.
The non-aged cements showed a more uniform sur-
face composed of CSH hydrogel, alite, belite, ettring-
ite, anhydrite and calcium carbonate.
• Proliferation of HMSC was higher on non-aged (fresh)
samples than on aged samples.
• Cell viability was decreased on the bismuth-contain-
ing cement, confirming the moderate cytotoxicity of
bismuth oxide. However, the adverse effect of bismuth
Gandolfi et al
oxide on cell growth was reduced by the formation of
a biocoating on 28-day aged samples.
Therefore, it is likely that:
• The silicon exposed on non-aged (fresh) material, as
well as the calcium leached from portlandite dissolu-
tion, promote cell growth.
• The low crystallinity of the non-aged surfaces is favor-
able for adhesion/proliferation of osteogenic cells, and
the high crystallinity of apatite deposits on aged sam-
ples is jointly responsible for the reduced cell growth.
• The surface topography, such as pillars, sharp crystals,
ridges, bulges and irregularities protruding from the
surface disturb and interfere cellular behavior. Cells
spreading and proliferation and mainly adhesion are
strongly affected on the surface of aged samples.
The findings of this study support the use of the experi-
mental calcium-silicate cements as root-end filling and
REFERENCES
1. Torabinejad M, Hong UJ, McDonald F, Pitt Ford TR. Physical
and chemical properties of a new root-end filling material. J
Endod 1995; 21: 349-53.
2. Saunders WP. A prospective clinical study of periradicular
surgery using mineral trioxide aggregate as a root-end
filling. J Endod 2008; 34: 660-5.
3. Pace R, Giuliani V, Pagavino G. Mineral trioxide aggregate
as repair material for furcal perforation: case series. J Endod
2008; 34: 1130-3.
4. Holden DT, Schwartz SA, Kirkpatrick TC, Schindler WG.
Clinical outcomes of artificial root-end barriers with mineral
trioxide aggregate in teeth with immature apices. J Endod
2008; 34: 812-7.
5. Pelliccioni GA, Vellani CP, Gatto MRA, Gandolfi MG,
Marchetti C, Prati C. Proroot mineral trioxide aggregate
cement used as a retrograde filling without addition of
water: an in vitro evaluation of its microleakage. J Endod
2007; 33: 1082-5.
6. Gandolfi MG, Sauro S, Mannocci F, et al. New tetrasilicate
cement as retrograde filling material: an in vitro study on
fluid penetration. J Endod 2007; 33: 742-5.
7. Gandolfi MG, Farascioni S, Pashley DH, Gasparotto G, Prati
C. Calcium silicate coating derived from Portland cement as
treatment for hypersensitive dentine. J Dent 2008; 36: 565-78.
8. Gandolfi MG, Perut F, Ciapetti G, Mongiorgi R, Prati C.
New Portland cement-based materials for endodontics
mixed with articaine solution: a study of cellular response.
J Endod 2008; 34: 39-44.
9. Santos AD, Moraes JCS, Araujo EB, Yukimitu K, Valerio
Filho WV. Physico-chemical properties of MTA and a novel
experimental cement. Int Endod J 2005; 38: 443-7.
10. Gandolfi MG, Iacono F, Agee K, et al. Setting time and
root-perforation repair materials. Further formulations may
be designed to obtain materials for root sealing and other
clinical applications. We recommend the exclusion of bis-
muth oxide from the composition when the cement must
be used in pulp capping or apicogenesis techniques.
Conflict of interest statement: None to declare.
Address for correspondence:
Maria Giovanna Gandolfi
Alma Mater Studiorum - University of Bologna
Department of Odontostomatological Science
Endodontic Clinical Section
Laboratory of Endodontic Biomaterials and Oral Pathology
Via San Vitale 59
40125 Bologna, Italy
mgiovanna.gandolfi@unibo.it
expansion in different soaking media of experimental
accelerated calcium-silicate cement and ProRoot MTA. Oral
Surg Oral Med Oral Pathol Oral Radiol Endod 2009, in press.
11. Camilleri J. Hydration mechanisms of mineral trioxide
aggregate. Int Endod J 2007; 40: 462-70.
12. Gandolfi MG, Pagani S, Perut F, et al. Innovative silicate-
based cements for endodontics: a study of osteoblast-like
cell response. J Biomed Mater Res 2008; 87: 477-86.
13. Mitchell PJC, Pitt Ford TR, Torabinejad M, McDonald F.
Osteoblast biocompatibility of mineral trioxide aggregate.
Biomaterials 1999; 20: 167-73.
14. Koh ET, Torabinejad M, Pitt Ford TR, Brady K, McDonald F.
Mineral trioxide aggregate stimulates a biological response in
human osteoblasts. J Biomed Mater Res 1997; 37: 432-39.
15. Koh ET, McDonalg F, Pitt Ford TR, Torabinejad M. Cellular
response to mineral trioxide aggregate. J Endod 1998; 24:
543-7.
16. Ribeiro DA, Durante MAH, Matsumoto MA, Marques MEA,
Salvadori TMF. Biocompatibility in vitro tests of mineral
trioxide aggregate and regular and white Portland cements.
J Endod 2005; 31: 605-7.
17. Lee SJ, Monsef M, Torabinejad M. Sealing ability of a mineral
trioxide aggregate for repair of lateral root perforations. J
Endod 1993; 19: 541-4.
18. Torabinejad M, Smith PW, Kettering JD, Pitt Ford TR.
Comparative investigation of marginal adaptation of
mineral trioxide aggregate and other commonly used root-
end filling materials. J Endod 1995; 21: 295-9.
19. Al-Hezaimi K, Al-Shalan TA, Naghshvbandi J, Oglesby S,
Simon JH, Rotstein I. Antibacterial effect of two mineral trioxide
aggregate (MTA) preparations against Enterococcus faecalis and
Streptococcus sanguis in vitro. J Endod 2006; 32: 1053-6.
20. Sumer M, Muglali M, Bodrumlu E, Guvenc T. Reactions of
connective tissue to amalgam, intermediate restorative material,
169
Bioactivity of MTA cements
mineral trioxide aggregate, and mineral trioxide aggregate
mixed with chlorhexidine. J Endod 2006; 32:1094-6.
21. Zhao W, Wang J, Zhai W, Wang Z, Chang J. The self-setting
properties and in vitro bioactivity of tricalcium silicate.
Biomaterials 2005; 26: 6113-21.
22. Sarkar NK, Caicedo R, Ritwik P, Moiseyeva R, Kawashima I.
Physicochemical basis of the biologic properties of mineral
trioxide aggregate. J Endod 2005; 31: 97-100.
23. Bozeman TB, Lemon RR, Eleazer PD. Elemental analysis
of crystal precipitates from gray and white MTA. J Endod
2006; 32: 425-8.
24. Coleman NJ, Nicholson JW, Awosanya K. A preliminary
investigation of the in vitro bioactivity of white Portland
cement. Cem Concr Res 2007; 37: 1518-23.
25. Tay FR, Pashley DH, Rueggeberg FA, Loushine RJ, Weller
RN. Calcium-phosphate phase transformation produced by
the interaction of the Portland cement component of white
mineral trioxide aggregate with a phosphate-containing
fluid. J Endod 2007; 33: 1347-51.
26. Kamali S, Moranville M, Leclercq S. Material and environmental
parameter effects on the leaching of cement pastes: experiments
and modelling. Cem Concr Res 2008; 38: 575-85.
27. Sawyer AA, Hennessy KM, Bellis SL. The effect of adsorbed
serum proteins, RGD and proteoglycan-binding peptides on
the adhesion of mesenchymal stem cells to hydroxyapatite.
Biomaterials 2007; 28: 383-92.
28. Potgieter-Vermaak SS, Potgieter JH,Van Grieken R.The application
of Raman spectrometry to investigate and characterize cement,
Part I: a review. Cem Concr Res 2006; 36: 656-62.
29. Martinez-Ramirez S, Frias M, Domingo C. Micro-Raman
spectroscopy in white Portland cement hydration: long-
term study at room temperature. J Raman Spectrosc 2006;
37: 555-61.
30. Anselme K. Osteoblast adhesion on biomaterials. Bio-
materials 2000; 21: 667-81.
31. Martinez E, Engel E, Planell JA, Samitier J. Effects of artificial
micro- and nano-structured surfaces on cell behaviour. Ann
Anat 2009; 191: 126-35.
32. Ko HC, Han JS, Bächle M, Jang JH, Shin SW, Kim DJ. Initial
osteoblast-like cell response to pure titanium and zirconia/
alumina ceramics. Dent Mater 2007; 23: 1349-55.
33. Gandolfi MG, Taddei P, Tinti A, Siboni F, Prati C. Bio-coating
formation on bio-active endodontic materials derived from
portland cement. J Dent Res 2008; 87C (Spec. Iss): PEF-
Division Abstr. 762.
34. Taddei P, Tinti A, Gandolfi MG, Rossi PL, Prati C. Vibrational
study on the bioactivity of portland cement-based materials
for endodontic use. J Mol Structure 2009; 924-926: 548-54.
35. Taddei P, Tinti A, Gandolfi MG, Rossi PL, Prati C. Ageing of
calcium silicate cements for endodontic use in simulated body
fluids: a micro-Raman study. J Raman Spectrosc 2009; in press
(www.interscience.wiley.com) DOI 10.1002/jrs.2333.
36. Meseguer-Olmo L, Bernabeu-Esclapes A, Ros-Martinez E,
et al. In vitro behaviour of adult mesenchymal stem cells
seeded on a bioactive glass ceramic in the SiO2-CaO-P2O5
170
system. Acta Biomaterialia 2008; 4: 1104-13.
37. Ding SJ, Shie MY, Wang CY. Novel fast-setting calcium
silicate bone cements with high bioactivity and enhanced
osteogenesis in vitro. J Mater Chem 2009; 19: 1183-90.
38. Kokubo T, Takadama H. How useful is SBF in predicting in
vivo bone bioactivity? Biomaterials 2006; 27: 2907-15.
39. Hamilton DW, Brunette DM.The effect of substratum topography
on osteoblast adhesion mediated signal transduction and
phosphorylation. Biomaterials 2007; 28: 1806-19.
40. Hu Q, Tan Z, Liu Y, et al. Effect of crystallinity of calcium
phosphate nanoparticles on adhesion, proliferation, and
differentiation of bone marrow mesenchymal stem cells. J
Mater Chem 2007; 17: 4690-8.
41. Liu Y, Cooper PR, Barralet JE, Shelton RM. Influence of
calcium phosphate crystal assemblies on the proliferation
and osteogenic expression of rat bone marrow stromal cells.
Biomaterials 2007; 28: 1393-403.
42. Porter AE, Patel N, Skepper JN, Best SM, Bonfield W.
Comparison of in vivo dissolution processes in hydroxy-
apatite and silicon-substituted hydroxyapatite bioceramics.
Biomaterials 2003; 24: 4609-20.
43. Maeno S, Niki Y, Matsumoto H, et al. the effect of calcium
ion concentration on osteoblast viability, proliferation and
differentiation in monolayer and 3D culture. Biomaterials
2005; 26: 4847-55.
44. Chou L, Marek B, Wagner WR. Effects of hydroxylapatite
coating crystallinity on biosolubility, cell attachment efficiency
and proliferation in vitro. Biomaterials 1999; 20: 977-85.
45. Min KS, Chang HS, Bae JM, Park SH, Hong CU, Kim EC.
The induction of heme oxygenase-1 modulates bismuth-
oxide-indiced cytotoxicity in human dental pulp cells. J
Endod 2007; 33: 1342-6.
46. Kim EC, Lee BC, Chang HS, Lee W, Hong CU, Min KS.
Evaluation of the radiopacity and cytotoxicity of Portland
cements containing bismuth oxide. Oral Surg Oral Med
Oral Pathol Oral Radiol Endod 2008; 105: 54-57.
47. De Deus G, Ximenes R, Gurgel-Filho ED, Plotkowski MC,
Coutinho-Filho T. Cytotoxicity of MTA and Portland cement on
human ECV 304 endothelial cells. Int Endod J 2005; 38: 604-9.
48. Huang TH, Ding SJ, Hsu TC, Kao CT. Effects of mineral
trioxide aggregate (MTA) extracts on mitogen-activated
protein kinase activity in human osteosarcoma cell line
(U2OS). Biomaterials 2003; 24: 3909-13.
49. Huang TH, Yang CC, Ding SJ, Yan M, Chou MY, Kao CT.
Biocompatibility of human osteosarcoma cells to root end
filling materials. J Biomed Mater Res Part B Appl Biomater
2005; 72B: 140-5.
50. Huang TH, Yang CC, Ding SJ, Yeng M, Kao CT, Chou MY.
Inflammatory cytokines reaction elicited by root-end filling
materials. J Biomed Mater Res Part B Appl Biomater 2005;
73B: 123-8.
51. Lin CP, Chen YJ, Lee YL, et al. Effects of root-end filling materials
and eugenol on mitochondrial dehydrogenase activity and
cytotoxicity to human periodontal ligament fibroblasts. J
Biomed Mater Res Part B Appl Biomater 2004; 71B: 429-40.