Kinetics of apatite formation on a calcium-silicate cement for root-end filling during ageing in physiological-like phosphate solutions.

Abstract

The bioactivity of calcium silicate mineral trioxide aggregate (MTA) cements has been attributed to their ability to produce apatite in presence of phosphate-containing fluids. This study evaluated surface morphology and chemical transformations of an experimental accelerated calcium-silicate cement as a function of soaking time in different phosphate-containing solutions. Cement discs were immersed in Dulbecco's phosphate-buffered saline (DPBS) or Hank's balanced salt solution (HBSS) for different times (1-180 days) and analysed by scanning electron microscopy connected with an energy dispersive X-ray analysis (SEM-EDX) and micro-Raman spectroscopy. SEM-EDX revealed Ca and P peaks after 14 days in DPBS. A thin Ca- and P-rich crystalline coating layer was detected after 60 days. A thicker multilayered coating was observed after 180 days. Micro-Raman disclosed the 965-cm(-1) phosphate band at 7 days only on samples stored in DPBS and later the 590- and 435-cm(-1) phosphate bands. After 60-180 days, a layer approximately 200-900 μm thick formed displaying the bands of carbonated apatite (at 1,077, 965, 590, 435 cm(-1)) and calcite (at 1,088, 713, 280 cm(-1)). On HBSS-soaked, only calcite bands were observed until 90 days, and just after 180 days, a thin apatite-calcite layer appeared. Micro-Raman and SEM-EDX demonstrated the mineralization induction capacity of calcium-silicate cements (MTAs and Portland cements) with the formation of apatite after 7 days in DPBS. Longer time is necessary to observe bioactivity when cements are immersed in HBSS.

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ORIGINAL ARTICLE
Kinetics of apatite formation on a calcium-silicate cement
for root-end filling during ageing in physiological-like
phosphate solutions
Maria Giovanna Gandolfi & Paola Taddei & Anna Tinti &
Elettra De Stefano Dorigo & Piermaria Luigi Rossi &
Carlo Prati
Received: 27 January 2009 /Accepted: 10 November 2009 /Published online: 27 November 2009
# Springer-Verlag 2009
Abstract The bioactivity of calcium silicate mineral triox-
ide aggregate (MTA) cements has been attributed to their
ability to produce apatite in presence of phosphate-
containing fluids. This study evaluated surface morphology
and chemical transformations of an experimental accelerated
calcium-silicate cement as a function of soaking time in
different phosphate-containing solutions. Cement discs were
immersed in Dulbecco’s phosphate-buffered saline (DPBS)
or Hank’s balanced salt solution (HBSS) for different times
(1–180 days) and analysed by scanning electron microscopy
connected with an energy dispersive X-ray analysis (SEM-
EDX) and micro-Raman spectroscopy. SEM-EDX revealed
Ca and P peaks after 14 days in DPBS. A thin Ca- and P-rich
crystallinecoatinglayerwasdetected after60days.Athicker
multilayered coating was observed after 180 days. Micro-
Raman disclosed the 965-cm−1phosphate band at 7 days
only on samples stored in DPBS and later the 590- and
435-cm−1phosphate bands. After 60–180 days, a layer
∼200–900 μm thick formed displaying the bands of
carbonated apatite (at 1,077, 965, 590, 435 cm-1) and
calcite (at 1,088, 713, 280 cm−1). On HBSS-soaked, only
calcite bands were observed until 90 days, and just after
180 days, a thin apatite–calcite layer appeared. Micro-
Raman and SEM-EDX demonstrated the mineralization
induction capacity of calcium-silicate cements (MTAs and
Portland cements) with the formation of apatite after 7 days
in DPBS. Longer time is necessary to observe bioactivity
when cements are immersed in HBSS.
Keywords Calcium-silicatecements.MTAcements.
Portlandcement.Ageing.Phosphatesolutions.Apatite.
Calcite
Introduction
Calcium-silicate cements, such as mineral trioxide aggre-
gate (MTA) and others [1], demonstrated a wide range of
clinical applications including root-end filling material, root
perforation repair, pulp capping and dentine hypersensitiv-
ity [2–6]. As stated by the patent, MTA is a derivative of a
type I ordinary Portland cement with 4:1 proportions of
bismuth oxide added for radiopacity. Portland cement is the
active ingredient in white MTA [7]. White Portland
cements are hydraulic materials mainly composed of di-
and tricalcium silicate (2CaO.SiO2belite and 3CaO.SiO2
alite), tricalcium aluminate 3CaO.Al2O3 and gypsum
CaSO4·2 H2O hydrophilic powders. When hydrated, the
silicate phases of Portland cements (and MTA) undergo a
series of physicochemical reactions resulting in the forma-
M. G. Gandolfi:C. Prati
Laboratory of Biomaterials and Oral Pathology,
Department of Odontostomatological Science,
Endodontic Clinical Section, University of Bologna,
Bologna, Italy
M. G. Gandolfi (*):P. L. Rossi
Department of Earth Sciences, University of Bologna,
Bologna, Italy
e-mail: mgiovanna.gandolfi@unibo.it
P. Taddei:A. Tinti
Department of Biochemistry, University of Bologna,
Bologna, Italy
E. De Stefano Dorigo
Unit of Dental Sciences and Biomaterials, Department of
Biomedicine, University of Trieste,
Trieste, Italy
Clin Oral Invest (2010) 14:659–668
DOI 10.1007/s00784-009-0356-3

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tion of a nanoporous matrix/gel of calcium silicate hydrates
(“C–S–H phases”) and of a soluble fraction of calcium
hydroxide Ca(OH)2or portlandite [1, 8].
The bioactivity of Portland cement in phosphate buffered
solutions has been recently reported [9–12]. Recent inves-
tigations have reported that Portland cements release
calcium and when immersed in phosphate solutions induce
apatite formation and produce high alkalinity in the
surrounding environment [9–12]. Moreover, Portland
cements may remineralize the partially demineralized
dentine and induce calcium-phosphate deposits formation
[13].
Besides these advantages, Portland cements and MTA
also have some drawbacks, when used as endodontic
material. The major problem lies in their extended setting
time [14–16] and difficult handling. Reduction in setting
time could be advantageous in endodontics, as allows an
earlier dental reconstruction. Therefore, the composition of
Portland cements should be adjusted and properly modified
to avoid these drawbacks. Calcium chloride accelerates the
reaction between tricalcium aluminate and gypsum, and it
has been demonstrated that the addition of calcium chloride
reduce the setting time of Portland cements [17–19].
In the present study, a new accelerated cement based on
white Portland cement has been prepared. Both scanning
electron microscopy connected with energy dispersive X-
ray analysis (SEM-EDX) and micro-Raman analyses were
used to investigate the surface morphology and composi-
tion of the cement after ageing for different times in two
physiological-like phosphate solutions.
To our knowledge, no previous research has assessed the
apatite-like formation directly on the cement surface and
analysed the bioactivity of modified Portland cements.
Furthermore, this is the first study regarding the kinetics of
formation–transformation of biocoating on Portland cement
over time (from few hours to 180 days). The originality of
this investigation is: (1) the long-term study on the
bioactive behaviour of Portland cement, (2) the use of both
high and low phosphate-containing soaking solutions, (3)
the chemical and morphological typification of the outer
surfaces over time and (4) the use of micro-Raman
technique.
Materials and methods
Cement samples
An experimental thermally and mechanically treated
calcium-silicate cement (identified as wTC) composed of
white Portland cement (CEM I, Aalborg, Denmark) and
calcium chloride 5% w/w was prepared. The white Portland
cement contains the main active ingredients (tricalcium
silicate and dicalcium silicate) of MTAs (white ProRoot
MTA and MTA-Angelus). Commercial MTAs were not
used in this study for the interferences of bismuth oxide
(added as radio-opacifier agent) with micro-Raman analysis
(in particular with the detection of the portlandite band).
The cement was mixed with water and layered on a
plastic cover-slip with 13 mm diameter (Thermanox Plastic,
Nalge Nunc International, NY, USA) to obtain standard
discs. Mechanical vibrations were used to obtain a flat
regular surface. The exposed surface area of the discs was
1.9±0.1 cm2with a thickness of approx. 0.9 mm. Imme-
diately after preparation, the samples were placed in
physiological-like storage solutions at 37°C, Dulbecco’s
phosphate-buffered saline (DPBS) and Hank’s balanced salt
solution (HBSS).
DPBS (Cambrex Bio Science Verviers s.p.r.l., Belgium,
cat. n.BE17-512) is a physiological-like buffered (pH 7.4)
Ca- and Mg-free solution with the following composition
(mM): K+(4.18), Na+(152.9), Cl−(139.5), PO43−(9.56)
(sum of H2PO4−1.5 mM and HPO42−8.06 mM).
HBSS solution (pH 7.4, Cambrex Bio Science Verviers
s.p.r.l., Belgium, cat. n.10-527) reported the following
composition (mM): Ca2+(1.27), Cl−(144.7), K+(5.8) Na+
(141.6), Mg2+(0.81), HCO3−(4.17), SO42−(0.81), PO43−
(0.776) (sum of H2PO4−0.44 mM and HPO42−0.336 mM).
DPBS is a calcium-free and phosphate-rich (9.56 mM)
solution where the release of calcium ions from the cements
was the limiting parameter in the precipitation reaction [12],
and the high amount of phosphate represents the continuous
replenishment of phosphate ions from tissue fluids. Differ-
ently, HBSS contains calcium and lower phosphate.
Commercial media were selected because they are stand-
ardised and readily available.
The endpoint times were 1, 3, 7, 14, 21, 28, 60, 90 and
180 days. The soaking media were renewed weekly.
SEM-EDX
All samples were removed from the solution and dried at
37°C for 12 h. Specimens were then coated with carbon
before being examined by a scanning electron microscope
equipped with EDX (SEM-EDX 515, Phillips, Eindhoven,
The Netherlands) using an accelerating voltage of 5–20 keV.
The electron beam penetration at 20-keV acceleration was
2.98 μm, and the volume excited with a density of 3 g/cm3
must be considered 2.30 μm3. The x-axis of the EDX
spectra displays the energy of the X-ray in KeV; the y-axis
displays the intensity of the X-ray signal (counts).
Micro-Raman spectroscopy
Micro-Raman spectra were obtained using a Jasco NRS-
2000C instrument connected to a microscope with ×20
660Clin Oral Invest (2010) 14:659–668

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magnification (in these conditions the laser spot size
was about 5 μm). All the spectra were recorded in
back-scattering conditions with 5 cm−1spectral resolu-
tions using the 488 nm line (Innova Coherent 70) with a
power of 50 mW. A 160 K frozen CCD detector from
Princeton Instruments, Inc. was used. The laser penetra-
tion in cement was about 10 μm. To minimise the
variability deriving from possible sample inhomogeneity,
five spectra at least were recorded on five different points
of each specimen area (i.e. upper surface, inner fractured
side). In addition, the powder dispersed in the medium at
selected storage times was isolated and at least five spectra
were recorded. Raman spectra were also recorded on
unhydrated powder of cements. The cements were
analysed as wet samples when maintained in their storage
media.
pH measurements
The pH of the soaking solutions was measured with a
Denver Intrument Basic pH metre equipped with a
Hamilton liq-glass electrode and ±0.01 resolution. The
electrode was inserted into the soaking solutions at room
temperature (24°C). Each measurement was repeated three
times. The mean pH was then plotted against time.
Results
SEM
SEM-EDX analysis showed different surface morpholo-
gies depending on the soaking solution and ageing time.
The Ca/Si and Ca/P ratios calculated from the spectra have
not been judged reliable, since unfortunately, the EDX
spectra never reflected the composition of a unique phase.
Calcite can be considered present in all the spectra, and it
is hard to eliminate its contribution. At the same time, the
high X-ray penetration under thin deposits made detect-
able the Si peak assignable to the underneath cement
matrix.
Samples aged in DPBS for 1 day showed an amorphous
irregular outer surface free from visible deposits. EDX
detected high calcium (Ca) and somewhere silicon (Si)
peaks. Samples aged for 3–7 days presented many
prismatic/cubic crystals on the surface. EDX revealed Ca
peaks. Diffuse larger hexagonal and cubic crystals were
detected on the cement surface after 14 days of DPBS
immersion (Fig. 1a). Many thin bright cloud-like deposits
were detected on the cement surface. EDX of the area
revealed Ca and low P reflexes. Punctual microanalyses
detected high Ca and P peaks on cloud-like deposits and
only Ca peaks on prismatic crystals (Fig. 1a). A thick
continuous granular layer showing a porous finely granular
structure was detected after 28–60 days of DPBS immer-
sion. EDX revealed Ca and P peaks on the entire surface.
After 90–180 days, the surface deposit appeared thicker
(Fig. 2a) and adherent to the cement surface as a thick
coating layer. EDX revealed Ca and P peaks. Silicon
reflexes were no longer detectable after 21 days of
immersion.
Cements soaked in HBSS for 1 day showed an
amorphous outer surface. EDX revealed Ca peaks and rare
traces of Si. At 3–7 days of immersion, the surface became
more homogeneous with small crystals. EDX revealed high
Ca peak. Rounded-globular and prismatic-cubic Ca-rich
crystals varying in size were observed on samples stored for
14–28 days (Fig. 1b). After 60–90 days cloud-like deposits
were occasionally detected on a prismatic-cubic crystal
layer (Fig. 2b). The deposits, not uniformly distributed,
were composed of light P-containing precipitates and cube-
like and/or polygonal Ca-rich crystals. EDX of the area
revealed Ca peaks. Punctual EDX analysis of cloud-like
deposits revealed Ca and P peaks (Fig. 2b). After 180 days,
the surface was covered by a porous inhomogeneous Ca-
and P-rich coating layer formed by a light porous matrix
with interspersed polygonal crystals partially inserted into
the coating.
A dense homogeneous structure with rare porosities was
observed in the inner area in all the fractured samples aged
for 1–180 days in both solutions. Needle-like crystals were
occasionally disclosed. EDX of the internal area revealed
Ca, Si and traces of aluminium (Al) peaks (Fig. 3).
Micro-Raman spectroscopy
Many chemical modifications were observed on the cement
surface in relation to the soaking solution and the ageing
time (Tables 1 and 2 and Figs. 4 and 5). Band assignments
reported in the Tables and Fig. 4 have been made according
to the literature [20–24].
No phosphate band (965 cm−1, ν1PO43−stretching mode
[20]) was detected in the unhydrated powder (t=0, Fig. 4a,
spectrum a).
The micro-Raman spectra recorded on the surface of the
samples stored for 1–3 days in DPBS (Fig. 3a, spectrum b)
disclosed the bands of ettringite (991 cm−1ν1SO42−stretching
mode [21]), gypsum (1,004 cm−1ν1SO42−stretching mode
[22]), alite and belite (860–848 cm−1ν1SiO44−[23]) and
hydrated silicates (670 cm−1, Si–O–Si bending mode [23],
very weak and only occasionally detected), carbonate ions in
different chemical phases (ν1 CO32−stretching bands at
1,088 cm−1for calcite and/or aragonite and at 1,075 cm−1
for vaterite [25]) and the disappearance of the anhydrite bands
at 1,019 and 677 cm−1(ν1SO42−stretching and ν4SO42−
bending modes, respectively [22]). No bands of portlandite
Clin Oral Invest (2010) 14:659–668 661

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(360 cm−1, due to Ca-O polyhedra vibrations) [25] or
phosphate (965 cm-1) [20] were detected. The 965-cm−1
phosphate band was detected in the powder isolated from the
storage medium. After 7 days of storage in DPBS, this band
occasionally appeared also on the surface of the cement
(Fig. 4a, spectrum c), while after 14 days it was detected in all
the recorded spectra indicating that the apatite deposit was
present on the entire surface. After 7–14 days in DPBS,
cement bands at 991 and 860 cm−1were still visible and
crystalline calcite (bands at 1,088, 713, 280 cm−1, assignable
to ν1, ν4CO32−and lattice vibrations, respectively [25]) was
present. No portlandite band (360 cm−1) was detected.
The samples stored in DPBS for longer times (after
28 days, Fig. 4a, spectrum e) also displayed the phosphate
bands at 590 and 435 cm−1(ν2PO43−and ν4PO43−bending
modes, respectively [20]). The band at 1,077 cm−1(ν1CO32−
stretching) typical of carbonated apatite [20] was detected
after 60 days (Fig. 4a, spectra f and g).
At 180 days, the cement bands were no longer visible
due to the thicker surface deposit: only carbonated apatite
and calcite were detected (Fig. 4a, spectrum g). Raman
analysis showed that at increasing storage times in DPBS,
the apatite deposit became progressively thicker, as
revealed by the progressive increase in intensity of the
965 cm−1band with respect to the cement bands (Fig. 4a).
The I965/I990phosphate/cement intensity ratio was calculat-
ed to evaluate the thickness of the deposit (Fig. 5): the rise
in this ratio indicated the increase in the deposit thickness
on the cement surface. The thickness of the apatite deposit
was estimated by recording micro-Raman spectra every
100 μm in the inner fractured side beginning at the surface.
The thickness of the apatite deposit on cement was approx.
200 μm after 60 days and approx. 900 μm after 180 days.
Portlandite band (360 cm−1) was not found on the
cement upper surface (Fig. 4) and was detected only in the
inner fractured surface of the samples.
As regards ageing in HBSS, the bands of calcite deposit
were observed from 1 to 180 days (Fig. 4b). The cement
bands were no longer detectable on the sample surfaces
after 60 days (Fig. 4b, spectra c and d). No phosphate band
(965 cm−1) was detected on the upper surface until
180 days. However, the 965-cm−1phosphate band was
detected in the powder isolated from the storage medium
after 14 days. At 180 days, the phosphate band was also
detected on the upper surface (Fig. 4b, spectrum d),
indicating a sufficiently thick apatite deposit (less than
50 μm thick). In addition, many crystalline calcite deposits
were also noticed.
a
Outer surface of wTC stored in DPBS for 14 days
(1550x)
EDX of area
EDX of rounded crystals
b
Outer surface of wTC stored in HBSS for 14 days
(1550x)
EDX of area
EDX of white deposits
Fig. 1 SEM-EDX analysis of
wTC surface after a soaking
period of 14 days (bar 10 μm),
showing apatite and calcite
deposits in DPBS (a) and calcite
coating layer in HBSS (b). The
x-axis displays the energy of the
X-ray in KeV, and the y-axis
displays the intensity of the
X-ray signal (counts)
662Clin Oral Invest (2010) 14:659–668

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pH measurements
Figure 6 shows the pH values of the two ageing media. At
24 h, the pH was very high (approx. 11.9) in both solutions.
Thereafter, the pH was more than 12 until 7 days and
slowly decreased to about 9 at 28 days. At 180 days, the pH
was 9.0 in DPBS and 8.2 in HBSS.
Discussion
This study demonstrated that calcium-silicate cements aged in
physiological-like phosphate solutions produce a superficial
biocoating formed by carbonated apatite and calcite deposits.
A marked bioactivity of experimental cement was found
in DPBS. DPBS solution has been previously used in
Portland cement-DPBS systems [12, 26]. The high amount
of phosphate represents the continuous replenishment of
phosphate ions from tissue fluids [12, 26], and the absence
of calcium makes the release of calcium ions from the
cements the limiting parameter in the precipitation reaction
[12]. Long immersion time (i.e. 180 days) in DPBS created
the conditions for the formation of a thick carbonated
apatite layer (approx. 900 μm thick). Micro-Raman
Fig. 2 SEM-EDX analysis of
wTC surface after a soaking
period of 90 days (bar 10 μm),
showing a thick apatite coating
in DPBS (a), calcite and apatite/
calcite coating in HBSS (Si
reflex due to the underlying
cement is visible because of the
deep X-ray penetration under
the deposit) (b). X-ray energy
(KeV) on x-axis and X-ray
signal (counts) on y-axis
Fractured surface of wTC stored in DPBS for 28 days
biocoating on fractured surface
(3100x)
EDX on biocoating
Inner fractured area (1550x)
EDX of inner area
Fig. 3 Fractured disc showing the thick biocoating on cement surface
(bar 10 μm)
Clin Oral Invest (2010) 14:659–668 663

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spectroscopy identified the crystalline and amorphous
phases of both the outer surface and inner bulk of the
cement after hydration reaction whereas SEM-EDX
revealed their morphology and elemental composition.
The potential of Raman spectroscopy to characterise
cements has been used to investigate cement hydration in
situ and in real-time with no sample manipulation and
minimal interference from the water environment [25, 27].
Micro-Raman configuration monitors changes in chemical
composition and morphology on a micro-scale since the
laser spot (i.e. the excitation source) size is of the order of a
few microns. SEM-EDX may be used to detect cement
surface morphology [12] and to evaluate the chemical
modifications induced by immerging cements in body fluid
solutions for different times.
Although Raman spectroscopy has been widely
applied to the study of the hydration mechanism of
cements [24, 25, 27], this is the first study which uses
Raman technique to investigate the ageing of cements in
phosphate-containing solutions. The findings obtained in
the present study are in general agreement with the
results reported in the literature as concerns the formed
products [24, 25, 27]. As summarised in Table 1, after
1 day of ageing, the component at 848 cm−1(alite and belite)
was found to decrease in intensity with respect to the
860 cm−1band (belite); this trend confirmed that alite
hydrated more rapidly than belite, according to the literature
[24, 25, 27].
The different behaviour of the sulphate bands due to
gypsum and anhydrite (Table 1) indicated that the latter
reacted more rapidly than the former and after 1 day of
storage was completely consumed. An opposite result was
found by Gastaldi et al. [24] for calcium sulphoaluminate
cements aged in water. Our results indicated that ettringite
formed at expenses of anhydrite and possibly, more slowly,
of gypsum.
Table 1 Summary of the most significant information obtained by micro-Raman spectra recorded on the upper surface of the wTC cement during
ageing in DPBS and HBSS (0–7 days)
Ageing timeDPBS HBSS
t=0
unhydrated
cement
Alite: bands at 848 cm−1(ν1SiO44−), 539–523 cm−1(ν4SiO44−)
Belite: bands at 979 cm−1(ν3SiO44−), 856–848 cm−1(ν1SiO44−), 553–539–523 cm-1(ν4SiO44−)
Gypsum: band at 1,004 cm−1(ν1SO42−)
Anhydrite: bands at 1,019 cm−1(ν1SO42−), 677 cm−1(ν4SO42−)
Calcium carbonate: band at 1,089 cm−1(ν1CO32−)
Disappearance of the bands of anhydrite (1,019, 677 cm−1)
Appearance of the band at 991 cm−1(ettringite, ν1SiO44−): lower content than in the inner fractured side
Decrease in intensity of the component at 848 cm−1(alite and belite) with respect to the 860 cm−1band (belite): alite hydrates
more rapidly than belite
Carbonate ions in different chemical environments: calcite and/or aragonite (1,088 cm−1) and vaterite (1,075 cm−1)
No bands of portlandite (360 cm−1, Ca–O polyhedra vibrations)
No phosphate band detected (965 cm−1, ν1PO43−)
Phosphate band at 965 cm−1detected in the powder isolated from the
storage medium
Cement: ettringite (991 cm−1), gypsum (1,004 cm−1), alite and belite (860–848 cm−1), hydrated silicates (670 cm−1, Si–O–Si
bending)
Carbonate ions in different chemical environments: calcite and/or aragonite (1,088 cm−1) and vaterite (1,075 cm−1)
No bands of portlandite (360 cm−1)
No phosphate band detected (965 cm−1)
Phosphate band at 965 cm−1detected in the powder isolated from the
storage medium
Disappearance of the 1,004 cm−1band (gypsum consumed)
Bands of the cement still observable
Increase in crystallinity of the calcium carbonate component: calcite (1,088, 713, 280 cm−1)
No bands of portlandite (360 cm−1)
Appearance of the 965 cm−1phosphate band (one of the five recorded
spectra on the upper surface, poorly homogeneous apatite deposit)
1 day
Phosphate band at 965 cm−1not detected in
the powder isolated from the storage medium
3 days
Phosphate band at 965 cm−1not detected in
the powder isolated from the storage medium
7 days
No phosphate band detected (965 cm−1) either
on the upper surface or in the powder isolated
from the storage medium
Band assignments have been given according to the literature [20–24]
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The hydration reaction of Portland cement produces
calcium silicate (C–S–H) gel and a large amount of calcium
hydroxide [1, 8, 28]. The latter component can be easily
detected by Raman spectroscopy (if the cement is devoid of
bismuth oxide), while the former is characterised by a weak
Raman spectrum, due to the amorphous nature of C–S–H.
During the hydration reaction, the soluble calcium hydrox-
ide formed rapidly dissolves from the cement surface by
continuous washout in solution [11, 12], as demonstrated
by the absence of the portlandite Raman band (360 cm−1)
on the upper surface of the cement. Also, the pH increase of
the storage medium confirmed that calcium hydroxide was
washed away by the storage medium. At 24 h, the pH was
very high in both solutions (about 11.9) in agreement with
Tay et al. for PBS [12]. At 180 days, the pH was 9.0 in
DPBS and 8.2 in HBSS indicating that the amount of
portlandite released by the cement slowly decreased with
time and that Ca(OH)2was released faster into the HBSS
solution. Moreover, the OH−ions released are probably
partially buffered by the solution.
However, the increase in pH and the high concentration
of Ca2+ions [11] enhance the supersaturation of the
solution with respect to apatite and promote the precipita-
tion of the carbonated apatite coating layer on the cement
surface. Calcium release may be biologically important in
several clinical applications such as pulp capping proce-
dures and apicogenesis [6, 29, 30]. Calcium hydroxide is a
well-known antibacterial agent of value in many clinical
conditions such as in infected root canals when used as root
canal dressing [31].
SEM-EDX of the samples immersed in DPBS for 3–
14 days disclosed Ca peaks in prismatic-cubic crystals,
likely calcite, as indicated by micro-Raman analysis.
Interestingly, after 14 days, cloud-like granular Ca- and P-
rich deposits appeared at SEM-EDX. The Raman band at
965 cm−1confirmed the formation of calcium phosphate
Table 2 Summary of the most significant information obtained by micro-Raman spectra recorded on the upper surface of the wTC cement during
ageing in DPBS and HBSS (14–180 days)
Ageing
time
DPBS HBSS
14 daysBands of the cement still observable
Calcite (1,088, 713, 280 cm−1, assignable to ν1, ν4CO32−and lattice vibrations, respectively)
No bands of portlandite (360 cm−1)
965 cm−1phosphate band observable in all the
recorded spectra (homogeneous apatite deposit)
Bands of the cement still observable
Calcite (1,088, 713, 280 cm−1)
No bands of portlandite (360 cm−1)
Apatite (bands at 965, 590 cm−1(ν2PO43−),
435 cm−1(ν4PO43−)) + calcite deposit
No phosphate band detected (965 cm−1) on the upper surface (phosphate
band detected in the powder isolated from the storage medium)
21-
28 days
Calcite deposit
No phosphate band detected (965 cm−1) on the upper surface (phosphate
band detected in the powder isolated from the storage medium)
60 daysCalcite (1,088, 713, 280 cm−1)
No bands of portlandite (360 cm−1)
Carbonated (1,077 cm−1, ν1CO32−, well visible)
apatite (∼200 μm thick) + calcite deposit
Bands of the cement still observable
Calcite deposit
No phosphate band detected (965 cm−1) on the upper surface (phosphate
band detected in the powder isolated from the storage medium)
Bands of the cement no longer detectable
90 daysCalcite (1,088, 713, 280 cm−1)
No bands of portlandite (360 cm−1)
Carbonated apatite (∼300 μm thick) + calcite
deposit
Bands of the cement still observable
Calcite deposit
No phosphate band detected (965 cm−1) on the upper surface (phosphate
band detected in the powder isolated from the storage medium)
Bands of the cement no longer detectable
180 daysBands of the cement no longer observable (thicker deposit)
No bands of portlandite (360 cm−1)
Carbonated apatite (∼900 μm thick) + calcite
deposit
Apatite (965 cm−1phosphate band detected also on the upper surface, less
than 50 μm thick) + calcite deposit
Band assignments have been given according to the literature [20–24]
Clin Oral Invest (2010) 14:659–668665

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(apatite) deposits on the cement surface. The data indicated
that apatite deposits are already present on 14-day DPBS-
aged samples. After 28 days, SEM-EDX on the samples
stored in DPBS showed a homogeneous Ca- and P-rich
coating layer primarily constituted by calcium-phosphate
(apatite) and calcium-carbonate (calcite) deposits and by
carbonated apatite and calcite after 90–180 days, as
demonstrated by micro-Raman. The thickness of the
apatite–calcite coating layer increased over soaking time
attaining approximately 900 μm after 180 days of ageing in
DPBS.
The bioactivity of Portland cement in highly concentrat-
ed phosphate-buffered solutions has been recently reported
both in medium-term [32] and short-term studies [12, 26,
32]. This is the first investigation on the bioactivity of
calcium-silicate cement after long soaking times in both
high- and low-phosphate-containing solutions, and the first
which analyses the biological activity of this cement using
Raman spectroscopy.
Coleman et al. [11, 32] investigated the in vitro apatite
layer formation in a ground Portland cement-SBF system,
whereas Tay et al. [12] showed the formation of calcium-
deficient apatite precipitates in a Portland cement-PBS
system. Gallego et al. [26] observed apatite-like and calcite
crystals on set Portland cement exposed to phosphate
solution and CO2 and apatite-like phase formation on
carbonated substrates. Calcium ions supplied by the
hydration-derived basic calcium hydroxide (portlandite)
can react with the environmental carbonate ions forming
calcium carbonate (calcite). The crystalline form of calcium
carbonate as calcite formed on cement surface by carbon-
ation process showed good biological activity [33, 34] and
positively affected the cytocompatibility [26]. Soluble
portlandite crystals allow the nucleation of calcium carbon-
ate polymorphs and/or metastable calcium salt crystals that
readily transform into the stable calcite phase in water [33].
Calcium carbonate may precipitate at the surface and in the
cement paste porosity forming a protective layer of calcium
carbonate on the outer zone of cement discs [35]. The
protective calcium carbonate layer constrains or reduces the
ionic diffusion from cement bulk and probably decreases its
degradation and may improve the marginal sealing of root-
end filling restorations [9, 36].
The cement used in the present study revealed the
formation of a superficial Ca-rich layer (calcium carbonate
or calcite layer) on the surface after soaking in both
physiological-like phosphate solutions. Micro-Raman spec-
Fig. 4 Raman spectra showing the chemical transformations of the
upper surface of the wTC cement during ageing in DPBS (a) and
HBSS (b)
Fig. 5 Trend of the Raman I965/I991intensity ratio (average of five
measurements ± standard deviation) calculated from the spectra
recorded on the outer surface of wTC during ageing in DPBS
Fig. 6 pH (average of three measurements ± standard deviation) of
the two soaking solutions at different times
666 Clin Oral Invest (2010) 14:659–668

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troscopy confirmed the presence on the cement surface of
carbonate ions in different chemical phases, mainly as
calcite. Calcium ions supplied by the rapid dissolution of
portlandite and by the cement matrix can react with
environmental carbonate ions to form a calcium carbonate
superficial layer [35]. This Ca-rich layer may absorb
phosphate from the soaking solution allowing the crystal-
lisation of apatite crystals, as confirmed by micro-Raman
analysis. The apatite content in the coating layer increased
over soaking time. The high pH of both soaking media
detected at all the investigated ageing times reflects the
progressive hydration of silicate species and the rapid and
continuous dissolution of calcium hydroxide from the
cement matrix. Moreover, the Raman analysis of the
fractured samples aged in DPBS for 180 days detected
carbonated apatite until 900 μm from the cement disc
surface demonstrating the high thickness of the carbonated
apatite coating layer.
The cement bioactivity in HBSS was less noticeable than
in DPBS, according to the lower phosphate concentration
of HBSS. HBSS was selected to have a commercially
available standardised soaking medium mimicking the
composition of inorganic ions of human blood plasma,
without representing a supersaturated solution with respect
to apatite. HBSS contains lower calcium and same
phosphate concentration than human plasma and was
already used in bioactivity studies and in tissue engineering
or culture cell culture studies [37, 38]. The higher amounts
of calcite detected on the surface of cement soaked in
HBSS is related to the presence of carbonate ions in this
medium. The surface showed small Ca-rich crystals after 1–
14 days. Sparse cloud-like Ca- and P-containing deposits
appeared after 14 days in agreement with the 965-cm−1
phosphate band detected in the Raman spectrum of the
powder isolated from the storage medium. After 180 days
of ageing in HBSS, the deposit thickened (so that the 965-
cm−1band also became detectable on the surface) and the
entire surface appeared covered by a light porous matrix of
carbonated apatite with interspersed polygonal crystals of
calcite forming a porous and inhomogeneous apatite–calcite
coating layer less than 50 μm thick.
This study demonstrated the in vitro bioactivity of the
experimental calcium-silicate cement and supports the
hypothesis that the apatite biocoating formed on Portland
cements and MTAs may play a clinical positive role by
promoting the adsorption of proteins, the osteoblast
adhesion and proliferation [39–46] and bone bonding
and healing when used in vivo as root-end filling
material [47, 48]. The clinical implementation of
calcium-silicate portland cements may exploit the bioac-
tivity properties of bone-like apatite-forming materials
[41] for bone production [47, 48] and dentin bridge
formation [49, 50].
Conclusions
Calcium hydroxide (portlandite) was rapidly depleted from
the calcium-silicate cement matrix subsequently forming a
thick layer of calcium-carbonate (calcite) and calcium-
phosphate (apatite). SEM-EDX and micro-Raman analyses
showed the ability of these cements to produce a bone-like
apatite coating. The contact and the interaction of the
calcium-silicate hydraulic cement with different physiolog-
ical environments affect its surface morphology and surface
chemical composition.
Conflict of Interest
of interest.
The authors declare that they have no conflict
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