Apatite-forming ability (bioactivity) of ProRoot MTA

Apatite-forming ability (bioactivity) of ProRoot MTA

M. G. Gandolfi1, P. Taddei2, A. Tinti2 & C. Prati1

1Laboratory of Biomaterials and Oral Pathology, Endodontic Clinical Section, Department of Odontostomatological Science,

University of Bologna; and 2Department of Biochemistry, University of Bologna, Bologna, Italy

Abstract

Gandolfi MG, Taddei P, Tinti A, Prati C. Apatite-forming

ability (bioactivity) of ProRoot MTA. International Endodontic

Journal, 43, 917–929, 2010.

Aim Apatite-forming ability, considered as an index of

bioactivity (bond-to-bone ability), was tested on Pro-

Root MTA cement after immersion in phosphate-

containing solution (DPBS).

Methodology Disk samples were prepared and

immersed in DPBS for 10 min, 5 h, 1 and 7 days.

The cement surface was studied by attenuated total

reflectance Fourier transform infrared (ATR-FTIR)

spectroscopy, by micro-Raman spectroscopy and by

environmental scanning electron microscope with

energy dispersive X-ray (ESEM-EDX) analyses. The pH

of the storage solution was also investigated.

Results Spectroscopic analyses revealed calcium

phosphate bands after 5-h immersion in DPBS. After

1 day, an even coating composed of apatite spherulites

(0.1–0.8 micron diameter) was observed by ESEM/EDX.

After 7 days, its thickness had increased. Apatite

nucleation had already occurred after 5-h immersion.

At this time, the presence of portlandite (i.e. Ca(OH)2,

calcium hydroxide) on the cement surface was also

observed; at longer times, this component was released

into the medium, which underwent a remarkable pH

increase.

Conclusions The study confirms the ability of

ProRoot MTA to form a superficial layer of apatite

within hours. The excellent bioactivity of ProRoot MTA

might provide a significant clinical advantage over the

traditional cements used for root-end or root-perfora-

tion repair.

Keywords: apatite-forming ability, bioactivity,

calcium carbonate (calcite), calcium hydroxide (port-

landite), calcium-silicate cements, carbonate apatite,

dicalcium silicate (belite), Portland cement, ProRoot

MTA, tricalcium silicate (alite).

Received 14 December 2009; accepted 1 May 2010

Introduction

A material can be considered bioactive if it evokes a

positive response from the host (BSI 2007), and a

bioactive material must be able to elicit a biological

response at the interface and induce the formation of a

bond between tissue and the material (Hench & West

1996).

The concept of bioactivity is closely correlated with

biointeractivity, i.e. the ability to exchange information

within a biological system (BSI 2007). This means that

a bioactive material reacts chemically with body fluids

in a manner compatible with the repair processes of the

tissue.

An artificial material can bind to living bone by the

formation of a bone-like apatite layer on its surface in

the body environment (Kokubo & Takadama 2006) or

by biofunctionalization (i.e. chemical and/or physical

modifications of the surface of a non-biological

material to make it biointeractive) (Kumar 2005,

BSI 2007, Schiephake & Scharnweber 2008). Few

materials directly bond to living bone without the

formation of detectable apatite on their surfaces

Correspondence: Dr. Maria Giovanna Gandolfi, DBiol, DSc,

MSc, PhD, Biomaterials Designer, Manager of Research,

Laboratory of Biomaterials and Oral Pathology of Endodontic

Clinical Section, Department of Odontostomatological Sciences

- University of Bologna, Via San Vitale 59, Bologna, Italy

(Tel.:+39 051 2088 184; e-mail: mgiovanna.gandolfi@

unibo.it).

doi:10.1111/j.1365-2591.2010.01768.x

ª 2010 International Endodontic Journal International Endodontic Journal, 43, 917–929, 2010 917

https://www.researchgate.net/publication/7324687_How_Useful_is_SBF_in_Predicting_in_vivo_Bone_Bioactivity?el=1_x_8&enrichId=rgreq-767e4d3b-e276-47fd-969a-a5d4d8b7fc20&enrichSource=Y292ZXJQYWdlOzQ1Mjc3MTk5O0FTOjEwMzA5MTc2MDI3MTM2NUAxNDAxNTkwMzk5NTUw

whereas most do so through the absorption of

biological molecules such as proteins. The examina-

tion of apatite formation on a material in simulated

body fluid (SBF) is a commonly accepted method to

predict its in vivo bone bioactivity (Kokubo & Takad-

ama 2006), although the use of SBF for bioactivity

testing may lead to false-positive and false-negative

results (Boher & Lemaitre 2009), and many materials

will not form an apatite layer in the laboratory but

will be bioactive in vivo and vice versa.

ProRoot MTA (Dentsply Maillefer, Tulsa, OK, USA)

has been extensively studied and has provided inter-

esting results in both laboratory (Torabinejad et al.

1995a,b) and in vivo tests (Holden et al. 2008, Pace

et al. 2008, Saunders 2008, Mente et al. 2009).

ProRoot MTA has demonstrated a wide range of

clinical applications including root-end filling and root

perforation repair.

As stated by the patent (US Patent Number,

5,769,638;1995) (Torabinejad & White 1995), Pro-

Root MTA (Mineral Trioxide Aggregate) is a hydraulic

calcium-silicate cement 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 (Torabinejad & White

1995). The active ingredients (clinker particles) of

ProRoot MTA are similar to those reported for other

Portland-based cements (Camilleri et al. 2005, Santos

et al. 2005, Camilleri 2007, 2008, Gandolfi et al.

2007), modified to improve radiopacity (Camilleri &

Gandolfi 2010), handling and setting time.

The bioactivity of Portland cements in phosphate-

buffered solutions has been reported recently when

immersed in phosphate solutions (Sarkar et al. 2005,

Tay et al. 2007, Coleman et al. 2009, Gandolfi et al.

2009a).

In this study, the bioactivity of ProRoot MTA was

investigated as a function of soaking time in a

phosphate solution (Dulbecco’s Phosphate Buffered

Saline, DPBS). Environmental scanning electron

microscopy connected to energy dispersive X-ray

(ESEM-EDX), FTIR and micro-Raman analyses were

performed to analyse the formation of apatite as an

index of bioactivity of the cement.

The study was aimed at gaining insight into the

bioactivity of ProRoot MTA as a function of short

soaking times (up to 7 days) in phosphate solution. The

hypothesis was that ProRoot MTA possesses early-stage

bioactivity with the ability to form apatite within a

few hours after immersion in phosphate-containing

solution.

Materials and methods

Sample preparation

The cement (ProRoot MTA, Dentsply Maillefer – lot n.

08003395) was mixed with water and placed on a

plastic coverslip of 13 mm diameter (Thermanox Plas-

tic, Nalgene Nunc Thermo Scientific, Rochester, NY,

USA) to obtain standard disks. Mechanical vibration

was used to obtain a flat, regular surface. The exposed

surface area of the disks was 1.9 ± 0.1 cm2 with a

thickness of approx. 0.9 mm. Immediately after prep-

aration, the samples were placed in DPBS (Dulbecco’s

Phosphate Buffered Saline) at 37 �C.

DPBS (Dulbecco’s Phosphate Buffered Saline; Lonza,

Lonza Walkersville Inc, Walkersville, MD, USA, 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 mmol L)1 and HPO4

2)

8.06 mmol L)1).

In DPBS, the release of calcium ions from the

cements was the limiting parameter in the precipitation

reaction, and the large amount of phosphate represents

the continuous replenishment of phosphate ions from

tissue fluids. A commercial medium was selected

because of standardized and its availability. The end-

point times were 10 min (freshly prepared samples),

5 h, 1 and 7 days.

ESEM-EDX analysis

Samples were examined with an Environmental Scan-

ning Electron Microscope (ESEM, Zeiss EVO 50; Carl

Zeiss, Oberkochen, Germany) connected to a secondary

electron detector for Energy Dispersive X-ray analysis

(EDX; Oxford INCA 350 EDS, Abingdon, UK) computer-

controlled software Inca Energy Version 18, using an

accelerating voltage of 20–25 kV. The elemental anal-

ysis (weight % and atomic %) of samples was performed

applying the ZAF correction method. At 25-kV accel-

eration, the X-ray electron beam penetration of ESEM-

EDX (inside a material with a density of about

3 g cm)3) proved to be 2.98 micrometers, and conse-

quently the volume excited and involved in the

emission of characteristic X-rays from the constituting

elements was considered to be 10 micrometers3.

Cement disks were placed directly onto the ESEM stub

and examined without preparation (the samples were

not coated for this analysis). All samples were initially

analysed at low-vacuum conditions (9.9 Torr, 500 Pa),

Bioactivity of ProRoot MTA Gandolfi et al.

International Endodontic Journal, 43, 917–929, 2010 ª 2010 International Endodontic Journal918

100% relative humidity (RH) and 4 �C. After the initial

examination, each sample was inspected at higher

vacuum (2.9 Torr, 74 Pa), 40% RH and 4 �C.

Micro-Raman and ATR/FTIR spectroscopy

Micro-Raman spectra were obtained using a Jasco NRS-

2000C instrument (Jasco Inc., Easton, MD, USA)

connected to a microscope with 20 · magnification.

In these conditions, the laser spot size (i.e. the excita-

tion source) was a few microns. All the spectra were

recorded in back-scattering conditions with 5 cm)1

spectral resolutions using the 488-nm line (Innova

Coherent 70; Coherent Inc., Santa Clara, CA, USA)

with a power of 50 mW. A 160-K-frozen charge-

coupled detector (CCD) (Princeton Instruments Inc.,

Trenton, NJ, USA) was used.

IR spectra were recorded on a Nicolet 5700 FTIR

(Thermo Electron Scientific Instruments Corp., Madi-

son, WI, USA), equipped with a smart orbit diamond

attenuated total reflectance (ATR) accessory and a

deuterated triglycine sulphte (DTGS) detector; the

spectral resolution was 4 cm)1, and the number of

scans was 64 for each spectrum. The ATR area had

2 mm diameter. The IR radiation penetration was

about 2 microns.

To minimize the variability deriving from possible

sample inhomogeneity, at least five spectra were

recorded on five different points on the upper surface

of each specimen. Raman and IR spectra were also

recorded on unhydrated cement powder. The Raman

spectra were recorded on wet cement samples (i.e.

when maintained in their storage media).

pH measurements

The pH of the soaking solution DPBS was measured

with a Denver Intrument Basic pH meter (New York,

NY, USA) equipped with a Hamilton liquid–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.

Results

SEM-EDX analyses

ESEM-EDX analysis showed different surface morpho-

logies depending on the soaking time. EDX and the

elemental analysis on unhydrated cement powder

(Fig. 1) revealed Ca (calcium), Si (silicon), S (sulphur),

Al (aluminium), O (oxygen) and Bi (bismuth) and the

absence of P (phosphorus).

Freshly prepared samples (Fig. 2) had an uneven

surface with irregular granules. EDX and the elemental

analysis displayed high Ca Si, S, Al and Bi peaks. The

map of elements (Fig. 3) showed the distribution of O,

Al, Si, Ca, Bi and S on the freshly prepared cement.

After 1 day of storage in DPBS, the outer surface

was amorphous and irregular, with many visible

deposits composed of aggregates of apatite nano-

spherulites (Fig. 4a,d). Elongated structures (needle-

like crystals of ettringite) were occasionally disclosed.

Many spherulites (0.1–0.8 microns) precipitated on

the cement surface were packed to form clusters of

spheroidal bodies randomly distributed on the cement

surface. EDX and the elemental analysis showed Ca,

(a) (b) (c)

Figure 1 Morphology of unhydrated powder of ProRoot MTA. (a) Particles of different sizes are well visible. (b) EDX on marked

area revealed Ca, Si, S, O, Bi and Al and the absence of P in the cement composition. (c) Elemental analysis of marked area.

Gandolfi et al. Bioactivity of ProRoot MTA


Si and P peaks, suggesting the presence of calcium

phosphate deposits (Fig. 4b,c). Punctual microanaly-

ses on spherulite clusters detected high Ca and P

peaks (Fig. 4e,f). Si was absent from the surface,

suggesting its accumulation mainly in the subsurface

calcium silicates hydrate (CSH) phase and a certain

release into the solution. The high O peak is

ascribable to the presence of water, Cl and Na

(traces) to the soaking medium. The Ca/P ratio from

the microanalysis of the area (Ca/P 1.89) was higher

than that obtained from the punctual microanalysis

(Ca/P 1.32), because the ratio value was affected and

increased by the contribution of calcium from

calcium carbonate (detectable by Raman and FTIR

but not by EDX).

After 7 days in DPBS, apatite deposits formed a layer

evenly distributed on the entire surface (Fig. 5a). At

high magnification, the deposits appeared to be

composed of aggregates of spherulites (Fig. 5c,d).

Punctual microanalyses and the elemental analysis

on spherulites showed Ca and P (Fig. 5e,f).

The Ca/P ratio from the elemental analysis of

punctual EDX on apatite spherulites increased over

the soaking time (Ca/P 1.32 after 1 day and 1.98 after

7 days) indicating the maturation of a carbonated-

apatite phase, where carbonate ions replaces phosphate

ions in the apatite structure (carbonate ion can

substitute both for OH in the apatite channel (type A

CAp) and for the phosphate ion (type B CAp).

Micro-Raman analyses

Several chemical modifications were observed on the

cement surface during ageing (Fig. 6). Band assign-

ments (Table 1) were made in accordance with the

literature (Taddei et al. 2009a,b).

(a)

(d) (e) (f)

(b) (c)

Figure 2 ProRoot MTA surface observed under environmental scanning electron microscope with energy dispersive X-ray 10 min

after preparation. (a,d) The surface appeared covered by particles of different sizes. (b) EDX on area displayed the presence of Ca, Si,

S, Bi, O and traces of Na, K and Al. (c) Elemental analysis of area. (e) Punctual EDX on marked formation displayed the presence of

Ca, Si, S, Bi, O and traces of K, Na and Al. (f) Elemental analysis of area.



The spectrum of the MTA unhydrated powder

showed the presence of alite (tricalcium silicate,

Ca3SiO5 sometimes formulated as 3CaO Æ SiO2) and

belite (dicalcium silicate, Ca2SiO4, sometimes formu-

lated as 2CaO Æ SiO2), calcium sulphate prevalently as

anhydrite (CaSO4), calcium carbonate (CaCO3) as

calcite and aragonite and bismuth oxide. No phosphate

band was detected in the unhydrated powder.

Freshly prepared samples showed the hydration of

alite, belite and anhydrite with formation of ettringite

(3CaO Æ Al2O3 Æ 3CaSO4 Æ 31H2O) and gypsum

(CaSO4Æ2H2O). The CSH phase (i.e. the hydration

product of alite and belite) had weak Raman bands,

detectable only at long storage times (Taddei et al.

2009a,b). The bands of anhydrite, alite and belite were

still observable. Calcium carbonate in different crystal-

line phases (calcite, aragonite, vaterite) was revealed.

The cements stored for 5 h in DPBS had apatite

deposits, although not well distributed over the entire

cement surface; anhydrite had disappeared.

At increasing storage times, the deposit became

progressively thicker and more homogeneous. After

one day, the band typical of an HPO42) -containing

apatite appeared; this result is consistent with a Ca/P

ratio of 1.32.

After 7 days of storage in DPBS, a calcite/aragonite

and B-type hydroxycarbonate apatite (HCA) coating

was present on the entire surface, and the cement

bands were significantly reduced in intensity because of

the increased thickness of the deposit.

The portlandite (i.e. calcium hydroxide, Ca(OH)2)

band at 360 cm)1 was never detected because of the

overlapping of the strong bands of bismuth oxide.

FTIR analyses

The spectrum of the MTA unhydrated powder con-

firmed the Raman results. Band assignments (Table 2

and Fig. 7) were made in accordance with the litera-

ture (Taddei et al. 2009a).

Figure 3 Map of the elements detected on the surface of freshly prepared ProRoot MTA. Calcium is present on the entire external

surface. Bismuth oxide particles are homogeneously distributed. The oxygen peak represents an index of water presence on fresh

samples.



After 5 h in DPBS, the cement surface showed the

formation of ettringite, portlandite, CSH phase and an

apatite deposit; the bands of calcium carbonate (as

calcite, aragonite, vaterite), belite and alite were still

observable.

After 1 day, the bands typical of the cement compo-

nents were no longer detected in the spectrum; the

bands observed were all attributable to the presence of

B-type HCA and calcium carbonate deposit thicker

than 2 lm (penetration depth of the IR radiation) and

homogeneously distributed on the surface (all five

spectra recorded were coincident).

After 7 days, an even coating of more mature B-type

HCA was present; calcium carbonate was revealed in

higher amounts and in different chemical phases

(mainly calcite and aragonite).

pH measurements

The pH of the soaking solution DPBS was 7.4. After

immersion of the ProRoot MTA sample, the pH of DPBS

increased and attained alkaline values: pH approx. 9.9

after 10 min and pH approx. 11.3 from 5 h until

7 days.

Discussion

This study clearly demonstrated that ProRoot MTA

cement possesses high bioactivity and showed that

surface morphology and chemical composition are

rapidly modified by immersion in phosphate solution.

The significant finding of this study is that apatite

formation (revealed by the fast formation of calcium

(a)

(d) (e) (f)

(b) (c)

Figure 4 ProRoot MTA surface observed after 1 day of storage in DPBS. (a) Cement surface observed at low vacuum. Many

deposits composed of apatite spherulites were visible on cement surface. Elongated needle-like ettringite crystals (hydrated calcium-

aluminium-sulphate) and round-shaped calcium-silicate particles (alite, belite) were present on the surface. (b) EDX on area

displayed O, Ca, P and traces of Al, Na, Cl. No Si were noticed. The high O peak is ascribable to the presence of water. (c) Elemental

analysis of the area marked in picture a. (d) Cement surface observed at low vacuum (300 Pa) showing the mineral deposits. (e)

Punctual EDX on white deposits displayed O, Ca and P peaks. Cl and Na (traces) are because of the soaking medium. No Si was

detected. (f) Elemental analysis of a white deposit.



phosphate deposits of apatite nano-spherulites) starts

after 5 h of immersion into a phosphate-containing

solution, and a uniform thickness of the apatite layer is

formed in 7 days.

Recent studies have demonstrated the capacity of

calcium-silicate cements (Sarkar et al. 2005, Zhao et al.

2005, Bozeman et al. 2006 Coleman et al. 2007 and,

2009, Huan & Chang 2007, Tay & Pashley 2008, Ding

et al. 2009, Gandolfi et al. 2009a) to induce the

formation of apatite deposits. It has been reported that

the formation of CSH and HCA layer is used to define

the laboratory behaviour of the material (Hench &

West 1996, Rehman et al. 2000, Saravapavan et al.

2003, Orefice et al. 2009).

At first, Sarkar et al. (2005) immersed a non-

specified MTA in a phosphate-buffered saline solution

for 3 days and 2 weeks and showed the formation of

apatite by XRD analyses in the precipitates recovered

from the soaking solution. Then, Bozeman et al. (2006)

reported the formation of apatite on white and grey

MTA immersed for 40 days in a calcium-free PBS. No

previous study investigated either the apatite-forming

ability of ProRoot MTA at the early stages of hydration

or the kinetics formation of bone-like apatite (HCA) on

ProRoot MTA.

It was subsequently proved that apatite deposits

form on modified calcium silicate/tetrasilicate cements

(Gandolfi et al. 2009a, Taddei et al. 2009a,b, Torrisi

et al. 2010), white Portland cement (Coleman et al.

2009), tricalcium-silicate cements (Liu & Chang

2009) and experimental dicalcium-silicate cements

(Ding et al. 2009, Chen et al. 2009). In particular, the

bioactivity of a white Portland cement previously

cured for 28 days and ground then immersed in

simulated body fluid has been demonstrated using

FTIR spectroscopy and SEM-EDX (Coleman et al.

2009). The apatite-forming ability of a novel dical-

cium-silicate cement prepared by sol-gel method using

(a)

(d) (e) (f)

(b) (c)

Figure 5 ProRoot MTA after 7 days of storage in DPBS observed at 300 Pa. (a) Cement surface was covered by a layer of

aggregated apatite spherulites. The deposits are evenly distributed on the entire surface. (b) EDX showed Ca, P, O and Cl peaks and

traces of Na. (c,d) At higher magnification, clusters of apatite spherulites were well visible, and the morphology of apatite

spherulites (diameter 0.5–2 microns) is well visible. (e) Punctual EDX on the marked spherulite displayed the presence of Ca and P

peaks. (f) Elemental analysis of the marked globular formation.



orthosilicate and calcium nitrate after 1–90 days in

Hanks’ solution (Ding et al. 2009) or after 1–30 days

of immersion in Kokubo solution (Chen et al. 2009)

was demonstrated using XRD, FTIR and SEM-EDX

techniques.

In bioactivity studies, micro-Raman and IR spectros-

copy are complementary techniques able to detect

silicate, sulphate, carbonate, hydroxyl and phosphate

vibrational modes, allowing the hydration process of

the cement as well as apatite deposition to be followed.

IR spectroscopy is more sensitive than Raman spec-

troscopy in identifying the CSH phase and portlandite

in the presence of bismuth oxide; on the other hand,

the latter technique detects the alite/belite bands at

longer times than the former. Micro-Raman configu-

ration allows the detection of changes in chemical

composition on a microscale, as the laser spot (i.e. the

excitation source) size is a few microns. ESEM-EDX

(Environmental Scanning Electron Microscopy con-

nected to Energy Dispersive X-ray analysis) analysis

has been used to study calcium-silicate and calcium-

phosphate bioactive materials (Meredith et al. 1995)

and bone-like calcium phosphate layers.

So, in this study, both ESEM-EDX and micro-Raman

spectroscopy were used to investigate in situ and in

real-time the hydration and the surface morphology of

wet ProRoot MTA in its humid ‘‘natural’’ state with no

sample manipulation and minimal interference from

environmental water (Martinez-Ramirez et al. 2006,

Taddei et al. 2009a,b).

Figure 6 Micro-Raman spectra recorded

on the surface of ProRoot MTA at t = 0

(unhydrated cement), freshly prepared

(10 min) and after 5 h, 1 and 7 days

of storage in DPBS. The bands because

of belite (B), alite (A), anhydrite (An),

gypsum (G), calcite (C), aragonite (Ar),

vaterite (V), ettringite (E), apatite (Ap)

and bismuth oxide (Bi) have been

indicated.



These techniques allowed the investigation of the

morphology, the microstructure and the elemental

composition of ProRoot MTA and monitoring of the

chemical transformations of its dynamic surface imme-

diately after preparation and after 5 and 24 h and

7 days of soaking in simulated body fluid.

This study clearly demonstrated that ProRoot MTA

cement possesses high bioactivity, as revealed by the

rapid formation of calcium phosphate deposits of

apatite nano-spherulites at ESEM-EDX and the detec-

tion of apatite bands in FTIR and micro-Raman

analyses. ProRoot MTA develops a calcium-phos-

phate-rich layer that contains carbonate ions within

a few hours of immersion in a simulated physiological

solution. After 1 day in DPBS, the deposit appeared

evenly distributed over the entire surface. This study

demonstrated that ProRoot MTA surface morphology

and chemical composition are rapidly modified by

the surrounding environment, i.e. the phosphate

solution.

Table 1 Correlation of the Raman

spectral changes observed for the

ProRoot MTA surface with the stages of

formation of a B-type hydroxycarbonate

apatite (HCA) layer

Wavenumber (cm)1) Vibrational mode Crystal phase

1083 CO32) stretching Calcite/aragonite

1077 CO32) stretching B-type HCA

1070 CO32) stretching Vaterite

1014 SO42) stretching Anhydrite

1005 SO42) stretching Gypsum

1002 HPO42) stretching HPO4

2)-containing apatite

990 SO42) stretching Ettringite

973 SiO44) stretching Belite

960 PO43) symmetric stretching Apatite

854 SiO44) stretching Belite

839 SiO44) stretching Alite/belite

827 SiO44) stretching Alite

711 CO32) bending Calcite

670 SiO44) bending CSH phase

605 and 580 PO43) bending Apatite

CSH, calcium silicate hydrate.

Table 2 Correlation of the IR spectral

changes observed for the ProRoot MTA

surface with the stages of formation of a

B-type hydroxycarbonate apatite (HCA)

layer

Wavenumber (cm)1) Vibrational mode Crystal phase

3640 OH stretching Portlandite

1470-1465 CO32) stretching Aragonite and/or vaterite

and B-type HCA

1415-1410 CO32) stretching Calcite and B-type HCA

1150 and 1130 SO42) stretching Anhydrite/gypsum

1110 SO42) stretching Ettringite

1081 CO32) stretching Aragonite

1025 PO43) asymmetric stretching Apatite

960 PO43) symmetric stretching Apatite

940 SiO44) stretching CSH

910 SiO44) stretching Alite

870 CO32) bending Calcite and/or vaterite

and B-type HCA

870 and 850 SiO44) stretching Belite

850 CO32) bending Aragonite

711 CO32) bending Calcite/aragonite

700 CO32) bending Aragonite

675 and 620 SO42) bending Anhydrite/gypsum

600 and 560 PO43) bending Apatite

595 SO42) bending Anhydrite

510-490 SiO44) bending CxS

445 SiO44) bending CSH

CSH, calcium silicates hydrate; CxS, calcium silicates.



Recent studies (Weller et al. 2008, Gandolfi & Prati

2010) demonstrated the sealing ability of calcium-

silicate cements used as root sealer in association with

gutta-percha. The formation of apatite may improve

the sealing ability and contribute to filling the marginal

porosities around restorations (Weller et al. 2008).

Moreover, apatite formation on the external surface

may contribute to cement expansion, as demonstrated

by another study with LVDT measurements (Gandolfi

et al. 2009a). Finally, this apatite may contribute to the

bio-remineralization of dentine just around the resto-

rations, an innovative biomechanism recently proposed

(Tay & Pashley 2008).

When immersed in fluids, ProRoot (like other

calcium-silicate cements) rapidly releases calcium ions

and creates an alkaline pH on the external surface

leading to the nucleation and crystallization of HCA on

the cement surface. The Ca/P ratio derived from the

apatite layer increased over soaking time (Ca/P 1.32

after 1 day and 1.98 after 7 days) indicating the

maturation of a carbonated-apatite phase (bone-like

apatite), where carbonate replaces phosphate ions (the

Ca/P ratio increased with increasing carbonate con-

tent).

When clinker grains of ProRoot MTA are mixed with

water (mixing solution), several reactions occur.

According to the literature (Camilleri 2007), the

silicate phase hydrates with formation of CSH and

portlandite. Raman spectra (Fig. 6) showed that alite

hydrates more quickly than belite: in the spectrum of

the freshly prepared cement, the bands attributable to

belite at 839 and 827 cm)1 significantly decreased in

intensity with respect to the belite band at 854 cm)1.

The formation of CSH and portlandite was not revealed

by Raman spectroscopy. In fact, the former is charac-

terized by weak bands, only observable at long storage

times, and the latter cannot be detected because of the

interference of bismuth oxide, which shows strong

bands superimposed on the 360 cm)1 portlandite

component. However, the increase in the pH of the

storage medium (9.9 after 10 min) indicates the release

of portlandite and the occurrence of the hydration

reaction.

Based on these findings, it can be hypothesized that:

1 A solid–liquid interface forms on the mineral

particles, and ion dissolution occurs almost immedi-

ately. Ca2+ions rapidly migrate into the mixing solution

and portlandite (i.e. Ca(OH)2) forms.

2 Silicates are attacked by OH) ions (hydrolysis of

SiO44) groups in alkaline environment), and a CSH

phase forms on mineral particles. CSH is a porous, fine-

grained and highly disorganized hydrated silicate gel

layer containing Si-OH silanol groups (Skibsted & Hall

2008) and negative surface charges that may act as

nucleation sites for apatite formation (Oliveira et al.

2003, Li et al. 2007).

3 The CSH contains an excess of calcium hydroxide

(formed by OH) ions from dissociated water molecules

with Ca2+ions from cement particles), as revealed by

the presence of portlandite inside the CSH phase of the

cement surface after 5 h of storage in DPBS. A

continuous flux of excess of portlandite formed in the

CSH structure occurs; portlandite diffuses outward and

passes into the storage solution, and a large increase in

pH occurs (pH approx. 9.9 after 10 min and pH approx.

11.3 since 5 h). Calcium ions in excess diffuse through

the initial CSH layer, causing a supersaturation with

Figure 7 Fourier transform infrared spectra recorded on the

surface of ProRoot MTA at t = 0 (unhydrated cement) and

after 5 h, 1 and 7 days of storage in DPBS. The bands due to

belite (B), alite (A), calcium silicates (CxS), hydrated calcium

silicates (CSH), portlandite (P), anhydrite (An), gypsum (G),

calcite (C), aragonite (Ar), vaterite (V), apatite (Ap) have been

indicated.



respect to Ca(OH)2; portlandite crystals nucleate

throughout the paste (Taddei et al. 2009a,b) and were

also detectable on the surface. The portlandite release

continues during storage time, as revealed by the pH of

the storage solution which remained constant at 11.3.

4 When exposed to a phosphate-containing solution

such as DPBS, a series of reactions take place on the

surface of ProRoot MTA between calcium from the

cement and phosphate from the solution, namely the

absorption of Ca and P ions on the silica-rich CSH

surface (as a result of local supersaturation) and the

precipitation of a HPO42)-containing apatite, which

matures into a B-type HCA phase at increasing storage

times. The apatite layer fills the superficial porosities

and may expand the mass of the cement.

During surgical procedures, blood and serum may

rapidly contaminate the external surface of cement

immediately after its placement in the root apical

cavity. So, it can be presumed that apatite formation

may occur in the presence of such fluids in vivo

(Holland et al. 2007, Reyes-Carmona et al. 2009). The

continuous flow of blood and biological fluids in the

surgical site constantly provides new phosphate and

may increase the amount of apatite formed on the

cement surface, as recently described by Gandolfi et al.

(2009a).

The cement proved to be a sort of remineralizing

agent and reservoir able to promote apatite deposition;

it can contribute to maintaining a stable seal when

placed in root-end cavities and promotes osteoblast

growth (Camilleri et al. 2005, Camilleri & Pitt Ford

2006, Gandolfi et al. 2008, Gandolfi et al. 2009c). The

previous in vitro studies on cell activity were probably

performed on a bioactive substrate because of the use of

phosphate-rich solutions. On the contrary, the evalu-

ation of mechanical properties, i.e. setting reaction,

flexural strength and sealing tests, should be re-

evaluated considering the effect of phosphate solutions

on material properties (Gandolfi et al. 2009a,b).

The excellent bioactivity (i.e. apatite formation abil-

ity) of ProRoot MTA might give a significant clinical

advantage over the traditional cements used for root-

end or root-perforation repairs and may be correlated

with its optimal biocompatibility, osteconductivity and

osteoinductivity demonstrated in many clinical and

laboratory studies.

Acknowledgement

The authors thank Dr Fabiola D’Amato for the precious

secretariat support.

Declaration

The authors deny any financial affiliations (e.g.

employment, direct payment, stock holdings, retainers,

consultantships, patent licensing arrangements or

honoraria), or involvement with any commercial

organization with direct financial interest in the subject

or materials discussed in this manuscript, nor have any

such arrangements existed in the past 3 years. Any

other potential conflict of interest is disclosed.

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Apatite-forming ability (bioactivity) of ProRoot MTA

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