11 Nanosynthesized calcium-silicate-based biomaterials in endodontic treatment of young permanent teeth










CHAPTER
11
Nanosynthesized calcium-
silicate-based biomaterials
in endodontic treatment
of young permanent teeth
Dejan Markovi
´
c
1
, Bojana
´
Cetenovi
´
c
1
, Ana Vukovi
´
c
1
, Vukoman Jokanovi
´
c
2
and Tatjana Markovi
´
c
3
1
Department of Pediatric and Preventive Dentistry, School of Dental Medicine, University of
Belgrade, Belgrade, Serbia
2
Department of Atomic Physics, Vinca Institute of Nuclear Sciences,
University of Belgrade, Belgrade, Serbia
3
Institute for Medicinal Plants Research “Dr Josif
Pan
ˇ
ci
´
c,” Belgrade, Serbia
11.1 INTRODUCTION
Dental pulp pathology is a severe and most common complication of traumatic
dental injuries or less frequently a complication of dental caries in children and
adolescents. The final stage of the disease is necrosis of the pulp which may have
a degenerative impact on root growth and development.
Necrosis of the pulp tissue is a consequence of a tissue collapse caused by
overloads of both bacterial and host defense cells, in infected tissue. Compared to
teeth with a mature root canal system, young permanent teeth are able to persist
much longer as their healing and regenerative ability is higher compared to the
mature teeth (
Huang, 2009). Although the microbiological profile of immature
teeth does not distinguish them from teeth with closed apices, the pathogenesis of
endodontic apical disease may differ. This may be explained with widely open
apices which allow better blood supply, collateral circulation and ability of young
pulp to overcome pulpal blood flow fluctuations, as well as higher prevalence of
progenitor cells and neuropeptides (
Dahlen, 2009; Huang, 2009).
Endodontic treatment in children and adolescents with young immature per-
manent teeth is quite difficult due to particularities of their immature root canal
system. Besides the specific treatment procedure, adequate choice and seque nce
of root canal irrigation, dressing, and sealing are essential for a favorable out-
come. Although calcium hydroxide (CH) has been considered for a long time as a
“golden standard” for the treatment of immature teeth, new technologies have
269
Nanobiomaterials in Dentistry. DOI: http://dx.doi.org/10.1016/B978-0-323-42867-5.00011-4
© 2016 Elsevier Inc. All rights reserved.

enabled the development of novel, and improvement of old, procedures and mate-
rials with more applicable clinical and mechanical properties. Also, intermediary
replacements of CH dressings in monthly or 3- to 6-month intervals, depending
on treatment protocol, may bring about reinfection and leakage if a temporary fill-
ing is not appropriate.
The first bioactive calcium-silicate-based material, deriving from basic Portland
cement (PC), was mineral trioxide aggregate (MTA). MTA was first presented as a
root-end filling material since it achieves good apical sealing, sets in the presence
of moisture and acquires high pH (10.212.5) in the first 3 h following mixing. It
shows several disadvantages related to manipulation difficulties caused by its dry
consistency, low flowability, and long setting time, compared to similar materials.
A new strategy of the synthesis of MTA-like materials utilizing nanotechnology
might be beneficial to eliminate the above-mentioned disadvantages.
Specific materials based on calcium silicate, such as Biodentin, also represent a
material that could be suitable for this type of treatment. However, research has to
be conducted and the obtained clinical results will estimate its therapeutic efficacy.
11.2 CHEMICAL PROPERTIES OF CALCIUM-SILICATE-BASED
MATERIALS
11.2.1 PORTLAND CEMENT
The first bioactive calcium-silicate-based material that appeared on the market
was MTA, which was introduced by
Abedi and Ingle (1995) and Torabinejad
et al. (1995a)
. It is derived from basic PC.
The main advantage of PC is its hydraulic nature which allows binding in a
humid environment. Although MTA has a similar chemical composition to PC
(
Table 11.1), there are certain limitations to the use of PC in everyday dental
practice. The restrictions for the clinical application of PC are a consequence of
its relatively high degree of solubility (
Islam et al., 2006), lack of X-ray contrast
(
Bortoluzzi et al., 2009; Vivan et al., 2009), heavy metal content (Duarte et al.,
2003; Bramante and Duarte, 2009), high degree of expansion during setting, and
distribution of different particle sizes of the powder (Damm aschke et al., 2005
).
The main components of PC are tricalcium silicate (CaO)
3
SiO
2
, dicalcium
silicate (CaO)
2
SiO
2
, tricalcium aluminate (CaO)
3
Ai
2
O
3
, and tetracalcium
aluminoferrite (CaO)
4
Al
2
O
3
Fe
2
O
3
.
Tricalcium silicate (C
3
S) is the main PC component responsible for its hydra-
tion, and consequently, for calcium silicate hydrate (CSH) formation. Tricalcium sil-
icate influences development of the early PC strength (
Greeberg and Chang, 1965).
The polymorphism of tricalcium silicate is defined by the presence of impurities,
known as alite (
Bye, 1983; De Noirfontaine et al., 2003). The symmetry of the
tricalcium silicate crystals increases with a rise of temperature during its synthesis,
and its structure is stabilized due to the presence of impurities in the raw material.
270 CHAPTER 11 Nanosynthesized calcium-silicate-based biomaterials

Dicalcium silicate (C
2
S) hydrates significantly slower than tricalcium silicate,
thus it is responsible for the development of the late PC strength. Similarly to
C
3
S, the most frequent form of dicalcium silicate with impurities present in nature
is known as belite (
Bye, 1983; Odler, 2000).
Tricalcium aluminate (C
3
A) is the most reactive component of PC, and even
though it quickly reacts while in contact with water, it has little influence on PC
strength. Tetracalcium aluminoferitte (C
4
AF) is the PC constituent that shows a mod-
erate reactivity with water, which increases with a rise in Al content (
Bye, 1983). At
the same time, C
4
AF is the only PC constituent that may affect teeth discoloration.
11.2.2 MINERAL TRIOXIDE AGGREGATE
MTA was developed as a root-end filling material at Loma Linda University, dur-
ing the last decades of the twentieth century. It is defined as a mixture of 75% PC,
20% bismuth oxide added as radiopaquer, and 5% gypsum, with trace amounts of
SiO
2
,CaO,MgO,K
2
SO
4
,andNa
2
SO
4
(Torabinejad et al., 1995a). Although PC
and MTA have similarities in their chemical compositions, there are still some dif-
ferences. One of the main differences is almost two times lower content of gypsum
and aluminum in MTA, which is a reason for its prolonged setting time, the major
MTA disadvantage (
Dammaschke et al., 2005). Even though MTA has a smaller
size of particles, which creates a finer powder, this, “sandy consistency causes dif-
ficulties during its clinical application (
Asgary et al., 2006). In addition, analysis
revealed the presence of bismuth oxide, lack of potassium, and smaller amounts of
heavy metals in MTA (
Song et al., 2006; Asgary et al., 2006).
The original formulation of a gray MTA (GMTA) was partially changed in
2002, and resulted in a synthesis of a white MTA (WMTA), as an aesthetically
acceptable formulation. In contrast to GMTA, which consists of dicalcium silicate,
tricalcium silicate, and bismuth oxide, WMTA primarily consists of tricalcium sili-
cate and bismuth oxide (
Camilleri et al., 2005)(Table 11.2). Since WMTA is
Table 11.1 Composition of PC (%)
Component Content (%) Phase Content (%)
SiO
2
19.7 C
3
S55
CaO 63.2 C
2
S15
CaO (free) 0.46 C
3
A 7.9
Al
2
O
3
4.7 C
4
AF 8.1
Fe
2
O
3
2.67 CaO 0.46
K
2
O 1.12 CaCO
3
4.4
MgO 1.85 CaSO
4
4.5
CO
2
1.93 K
2
SO
4
1.6
SO
3
3.35 NaSO
4
0.096
SrO 0.07
27111.2 Chemical Properties of Calcium-Silicate-Based Materials

synthesized in order to meet aesthetic criteria, smaller amounts of Fe, Al, and Mg
oxide are included in its composition (
Asgary et al., 2005, 2006, 2009; Camilleri
et al., 2005; Song et al., 2006). Iron oxide is considered to be the main cause of the
tooth crown discolorations in the case of GMTA, when present in sufficient quanti-
ties and due to the presence of easily excitatory electrons (Estrelaetal.,2000;
Funteas et al., 2003; Dammaschke et al., 2005; Camilleri et al., 2005; Roberts
et al., 2008). The WMTA powder particles are eight times smaller than those of
GMTA, which supports the larger surface hydration, stimulates development of the
early strength, and formation of a smoother surface. The presence of smaller parti-
cles leads to the conclusion that the WMTA setting time is probably shorter.
11.2.3 HYDRATION MECHANISM
Setting reaction of calcium-silicate-based materials is an extremely complicated
process that depends on the respective phases, purity of the phases, and tempera-
ture of the mixtures (
Table 11.3). A series of reaction products used to be formed,
not only due to kinetics of different processes but also due to influences of physi-
cal barriers of reaction products on the surface of the particles, which disturb the
process of water diffusion. In addition, dissolution, recrystallization, and interac-
tion of the products also take place (
Darvell and Wu, 2011).
Hydration of the powder particles produces porous CSH gel and CH. It takes
place according to the following reactions:
2ð3CaOUSiO
2
Þ 1 6H
2
O-3CaOU2SiO
2
U3H
2
O 1 3CaðOHÞ
2
2ð2CaOUSiO
2
Þ 1 4H
2
O-3CaOU2SiO
2
U3H
2
O 1 CaðOHÞ
2
CSH particles are precipitated on the surface of the cement as a result of
tricalcium silicate hydrolysis. CSH is poorly crystallized and porous, and is
described as a rigid colloidal gel. CH is a major soluble fraction of cement,
responsible for the high pH value.
Table 11.2 Composition of WMTA and GMTA (%)
Chemical GMTA (wt%) WMTA (wt%)
CaO 40.45 44.23
SiO
2
17.00 21.20
Bi
2
O
3
15.90 16.13
Al
2
O
3
4.26 1.92
MgO 3.10 1.35
SO
3
0.51 0.53
FeO 4.39 0.40
P
2
O
5
0.18 0.21
H
2
O 1 CO
2
13.72 14.49
272 CHAPTER 11 Nanosynthesized calcium-silicate-based biomaterials

At this stage, tricalcium aluminate also responds quickly, typically producing
series of hydrates affecting formation of diffusion barriers. If sulfate (S) is pres-
ent, the occurring competing reactions prevent further reactions with water until
sulfate is completely consumed:
C
3
A 1 S 1 H-C
6
AS
3
H
32
-C
4
ASH
12
These reactions are responsible for the early setting of material. The process
of water diffusion through a formed layer of nonpor ous matrix-gel and its swell-
ing eventually leads to formation of porous and poorly crystall ized solid gel.
After gel bursting, simple hydration reactions continue to occur. Meanwhile, C
3
S
powder reacts with water to provide a basic, but slower , solidification mechanism
with the involvement of C
2
S:
C
3
S; C
2
S 1 H-CSH 1 CH
The source of CH production is quite cont radictory; while Camilleri et al.
(2005)
consider that CH is formed from dicalcium and tricalcium silicate after
mixing the powder wi th water,
Dammaschke et al. (2005) state that CH is a prod-
uct of tricalcium aluminate hydration. Bismuth oxide also influences CH precipi-
tation following MTA hydration (
Dammaschke et al., 2005). It is detected as a
constituent of CSH, as well as in the form of unreacted filler (11.6%), which may
represent a problem from the aspect of MTA biocompatibility.
CSH phase is not so well defined, with varying stoichiometric values in its
composition, and it is predominantly amorphous, while CH is a clear crystal
phase of a set material (the C:S ratio of CSH is less than 2:1, usually ranging
between 0.8 and 2.1). As the ratio of C:S increas es, the hydration product of a
high degree of crystallization, portlandite, is formed (
Lee et al., 2007). Generally,
the setting reaction of CSH involves formation of more or less crystalline
hydrated calcium aluminate and calcium sulfate in an amorphous matrix of
hydrated calcium silicate (
Lee et al., 2007).
Table 11.3 The Principal Phases of MTA (%)
Phases Chemical Formula CCN Proportion
Tricalcium silicate 3CaOSiO
2
C
3
S 51.9
Dicalcium silicate 2CaOSiO
2
C
2
S 23.2
Tricalcium aluminate 3CaOAl
2
O
3
C
3
A 3.8
Tetracalcium aluminoferrite 4CaOAl
2
O
3
Fe
2
O
3
C
4
AF Nil
Calcium sulfate dihydrate CaSO
4
2H
2
O 1.3
Calcite, bismuth oxide, alkali metal
oxides
CaCO
3
,Bi
2
O
3
, CaO,
MgO
19.8
27311.2 Chemical Properties of Calcium-Silicate-Based Materials

11.2.4 CHEMICAL PROPERTIES OF OTHER TYPES
OF CALCIUM-SILICATE-BASED MATERIALS
Bioaggregate is a biomater ial based on calcium silicates with phosphates and
without aluminum. In addition to calcium silicates and its oxides, Bioaggregate is
composed of some other constituents, such as: hydroxyapatite (phosphate compo-
nent), calcium phosphate silicate, calcilite, and tantalum oxide as radiopaquer
(
Park et al., 2010; Saghiri et al., 2013).
Biodentine represents a biomaterial which, in addition to the predominantly
present tricalcium silicate, is also composed of dicalcium silic ate, calcium carbon-
ate, calcium oxide, and zirconium oxide as radiopaquer. The liquid with which
powder is mixed contains calcium chloride and a hydrosoluble polymer based on
polycarboxylates, which is responsible for the low water content and the mixture
consistency (
Burgess et al., 2009).
11.3 NANOTECHNOLOGY IN THE PROCESS OF SYNTHESIS
OF NEW CALCIUM-SILICATE-BASED MATERIALS
11.3.1 SYNTHESIS OF HIGHLY ACTIVE CALCIUM SILICATES
In order to obtain active calcium silicate phases a stoichiometric amount of
CaCl
2
3 5H
2
O (35.59 g) and silica sol (15 g of 30% solution) are used. The
synthesized calcium tricalcium silicate phase (Ca
3
SiO
5
-C
3
S) and dicalcium sili-
cate phase (2β-CaSiO
4
-C
2
S) should be used in the ratio 2:1. A small amount of
Al(C
2
H
3
O
2
) (4.55g) used to be added in order to achieve preparation of an active
C
3
A phase. While NH
4
NO
3
(71.3 g) is used as the oxidizing agent,
C
6
H
8
O
7
3 H
2
O (53.51g) is used during the reactio n, as a fuel.
Furthermore, a gel phase is obtained by drying the samples at a temperature of
80
C. Excess water should be removed from the samples by exposure to a tem-
perature of 150
C, which leads to formation of viscous gel. Total dehydration of
the gel is achieved at a temper ature close to 180
C.
Transformation of the mixture into the final form is performed by the method
of strong self-propagation combustion reaction, which causes a release of large
amounts of gases and a rise of temperature. Afterwards, samples have to be
rapidly cooled using a copper plate to minimize crystallization of the obtained
calcium silicate phases and to ensure their high reactivity. The product of this
reaction is a black powder, calcined during a 4-hour period at the very high tem-
perature (650
C), in order to remove excessive carbon (Nikoli
´
c et al., 2009;
Colovic et al., 2013).
Results of XRD analysis of silicate phases are presented in
Figure 11.1. The
authors (
Colovic et al., 2013) used the Scherrer equation for calculation of crys-
tallite sizes of the phases, and the obtained values were about 19.9 nm [calculated
for highly pronounced planes at 32.37
;(2121) for β-C
2
S and (2715) for C
3
S].
274 CHAPTER 11 Nanosynthesized calcium-silicate-based biomaterials

SEM analysis of the calcium silicate phase (Figure 11.2) shows that it mostly
consists of spherical and ellipsoidal particles (117477 nm) which form agglom-
erates of several micrometers in diameter. Such hierarchical structures seem to be
promising for applications in biological systems. This may be explained by the
fact that they are not biologically destructive and their nano-elements (crystallites)
facilitate very pronounced activity, especially quick bonding, so they are
suitable for use in endodontic therapy.
EDS analysis shows that chemical composition of calcium silicate (22.21%
Ca, 8.22% Si, and 69.7% O) corresponds to the ratio Ca:Si B2.7 at.% (
Nikoli
´
c
et al., 2009; Colovic et al., 2013).
11.3.2 SYNTHESIS OF NANOSTRUCTURED BIOMATERIALS BASED
ON HIGHLY ACTIVE CALCIUM SILICATES AND
CALCIUM CARBONATES
Mineral polyoxide carbonate aggregat e (ALBO-MPCA), CaCO
3
, and Bi
2
O
3
with
calcium silicate phases (Ca
3
SiO
5
and 2β-CaSiO
4
), in the ratio 2:2:1 are used in
synthesis of novel nanostructured materials.
FIGURE 11.1
XRD spectra of calcium silicate phases.
Adapted from Jokanovi
´
c et al. (2014b) with permission.
27511.3 Process of Synthesis of New Calcium-Silicate-Based Materials

The synthesis of calcium carbonate includes several steps, as follows.
Foremost, calcium chloride tetrahydrate (CaCl
2
3 4H
2
O) is used as a precursor.
Using the ultrasound effect at temperature of 40
C, 5 mmol of CaCl
2
3 4H
2
Ois
dissolved in 50 mmol of ethylene glycol. Alongside, 10 mM of NaHCO
3
is
dispersed into 50 ml of ethylene glycol, with constant mechanical stirring.
Furthermore, this dispersion should be preheated for 30 min at 40
C and then
calcium carbonate is isolated by centrifugation (9000 rpm, 30 min). Moreover, the
calcium carbonate is washed several times with a mixture of water and ethanol
(1:4), and finally rinsed only with water. Sulfonyl dodecyl sulfate (0.5%) is used
as an agent against agglomeration, and then the resulting nanoparticles of the
powder are exposed to ultrasound for 30 min, with vigorous mechanical stirring
for 5 h. Then, the powder is dried at 120
C for 5 h and heated at 500
C for an
hour, to obtain calcium carbonate phase.
The monoclinic Bi
2
O
3
, used as the radiocontrast agent, is obtaine d by
calcinations of Bi(NO
3
)
3
at a temperature of 450
C for 20 h, to ensure the
stable tetragonal Bi
2
O
3
phase (Jokanovi
´
c et al., 2014b).
XRD investigations of ALBO-MPCA before hydration are presented in
Figure 11.3. The peaks at 32.37, 47.77, and 56.53
[with corresponding planes
FIGURE 11.2
Typical appearance of calcium silicate phases.
Adapted from Jokanovi
´
c et al. (2014b) with permission.
276 CHAPTER 11 Nanosynthesized calcium-silicate-based biomaterials

(2121), (222), and (2303), respectively] correspond to β-C
2
S phase. The planes
(422), (2606), and (2822) corresponding to C
3
S phase are identified at angles of
29.65, 32.41, and 34.57
, respectively. Calcite phase is identified at 29.49, 34.88,
47.63, and 48.69
[planes (104), (110), (018), and (116), respectively]. And finally,
planes (002), (120), and (200) corresponding to monoclinic Bi
2
O
3
are identified at
angles 29.49, 34.88, and 48.69
,respectively(Jokanovi
´
cetal.,2014b).
SEM investigation of ALBO-MPCA before and after hydration (
Figures 11.4
and 11.6) reveals polygonal particles elongated in one direction, between 300 and
500 nm. They form larger spherical and rod-like aggregates, about 3 μm in size
(Jokanovi
´
c et al., 2014b
).
XRD investigat ions of ALBO-MPCA samples hydrated for 1, 3, 7, and 28
days (
Figure 11.5) revealed that the quantity of hydrated tobermorite phase
increased in correlation with hydrating time, while the quantities of other phases,
calcite and Bi
2
O
3
, remained almost the same. These changes are clearly noticed
while observing characteristic tobe rmorite planes (112), (110), (019), and (020),
corresponding to angles of 29.75 , 30.01, 43.69, and 48.8
, respectively. The plane
(110) is chosen as the most appropriate to present the changes inside the mixture
FIGURE 11.3
XRD patterns of ALBO-MPCA (B-Bi
2
O
3
, C-calcite) before hydration.
Adapted from Jokanovi
´
c et al. (2014b) with permission.
27711.3 Process of Synthesis of New Calcium-Silicate-Based Materials

FIGURE 11.4
SEM analysis of ALBO-MPCA before hydration (calcium silicate phase predominates).
Adapted from Jokanovi
´
c et al. (2014b) with permission.
FIGURE 11.5
XRD patterns of ALBO-MPCA hydration for various times (B-Bi
2
O
3
, C-calcite, T-tobermorite,
P-portlandite).
Adapted from Jokanovi
´
c et al. (2014b) with permission.
278 CHAPTER 11 Nanosynthesized calcium-silicate-based biomaterials

during its hydration. As expected, portlandite phase is also present [the planes
(100), (011), and (012), at the angles 28.42, 34.71, and 47.44
, respectively]
(
Jokanovi
´
c et al., 2014b).
11.3.3 SYNTHESIS OF SUPERPLASTIC, QUICK-BONDING
ENDODONTIC MIXTURES
In order to obtain superplastic, quick-bonding material for application in endodon-
tics, two mixtures are synthesized, ALBO-MPSA I and II. The ALBO-MPSA I
mixture contains the following components: β-C
2
SandC
3
Sphases(24%ofthe
total quantity of mixture), hydroxyapatite (40%), gypsum dehydrate (24%), and
BaSO
4
(12%) (Jokanovi
´
c et al., 2006, 2008, 2009; Opa
ˇ
ci
´
c-Gali
´
cetal.,2013). The
composition of ALBO-MPSA mixture II is as follows: 40% hydroxyapatite, 20%
C
2
SandC
3
S mixture (in ratio 2:1), 20% gypsum dihydrate, and 20% BaSO
4
.In
addition, 0.65% of 2-phosphonobutane-1,2,4-tricarboxylic acid (PBTCA), as super-
plasticizer, is added to both mixtures. After common milling for better homogeniza-
tion, the mixtures are finally combined with water to produce cement pastes with a
water-to-mixture ratio of 0.5 (
Jokanovi
´
c, 2012; Jokanovi
´
cetal.,2014a).
FIGURE 11.6
SEM analysis of ALBO-MPCA after hydration.
Adapted from Jokanovi
´
c et al. (2014b) with permission.
27911.3 Process of Synthesis of New Calcium-Silicate-Based Materials

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CHAPTER11Nanosynthesized calcium-silicate-based biomaterialsin endodontic treatmentof young permanent teethDejan Markovi´c1, Bojana´Cetenovi´c1, Ana Vukovi´c1, Vukoman Jokanovi´c2and Tatjana Markovi´c31Department of Pediatric and Preventive Dentistry, School of Dental Medicine, University ofBelgrade, Belgrade, Serbia2Department of Atomic Physics, Vinca Institute of Nuclear Sciences,University of Belgrade, Belgrade, Serbia3Institute for Medicinal Plants Research “Dr JosifPanˇci´c,” Belgrade, Serbia11.1 INTRODUCTIONDental pulp pathology is a severe and most common complication of traumaticdental injuries or less frequently a complication of dental caries in children andadolescents. The final stage of the disease is necrosis of the pulp which may havea degenerative impact on root growth and development.Necrosis of the pulp tissue is a consequence of a tissue collapse caused byoverloads of both bacterial and host defense cells, in infected tissue. Compared toteeth with a mature root canal system, young permanent teeth are able to persistmuch longer as their healing and regenerative ability is higher compared to themature teeth (Huang, 2009). Although the microbiological profile of immatureteeth does not distinguish them from teeth with closed apices, the pathogenesis ofendodontic apical disease may differ. This may be explained with widely openapices which allow better blood supply, collateral circulation and ability of youngpulp to overcome pulpal blood flow fluctuations, as well as higher prevalence ofprogenitor cells and neuropeptides (Dahlen, 2009; Huang, 2009).Endodontic treatment in children and adolescents with young immature per-manent teeth is quite difficult due to particularities of their immature root canalsystem. Besides the specific treatment procedure, adequate choice and seque nceof root canal irrigation, dressing, and sealing are essential for a favorable out-come. Although calcium hydroxide (CH) has been considered for a long time as a“golden standard” for the treatment of immature teeth, new technologies have269Nanobiomaterials in Dentistry. DOI: http://dx.doi.org/10.1016/B978-0-323-42867-5.00011-4© 2016 Elsevier Inc. All rights reserved. enabled the development of novel, and improvement of old, procedures and mate-rials with more applicable clinical and mechanical properties. Also, intermediaryreplacements of CH dressings in monthly or 3- to 6-month intervals, dependingon treatment protocol, may bring about reinfection and leakage if a temporary fill-ing is not appropriate.The first bioactive calcium-silicate-based material, deriving from basic Portlandcement (PC), was mineral trioxide aggregate (MTA). MTA was first presented as aroot-end filling material since it achieves good apical sealing, sets in the presenceof moisture and acquires high pH (10.212.5) in the first 3 h following mixing. Itshows several disadvantages related to manipulation difficulties caused by its dryconsistency, low flowability, and long setting time, compared to similar materials.A new strategy of the synthesis of MTA-like materials utilizing nanotechnologymight be beneficial to eliminate the above-mentioned disadvantages.Specific materials based on calcium silicate, such as Biodentin, also represent amaterial that could be suitable for this type of treatment. However, research has tobe conducted and the obtained clinical results will estimate its therapeutic efficacy.11.2 CHEMICAL PROPERTIES OF CALCIUM-SILICATE-BASEDMATERIALS11.2.1 PORTLAND CEMENTThe first bioactive calcium-silicate-based material that appeared on the marketwas MTA, which was introduced byAbedi and Ingle (1995) and Torabinejadet al. (1995a). It is derived from basic PC.The main advantage of PC is its hydraulic nature which allows binding in ahumid environment. Although MTA has a similar chemical composition to PC(Table 11.1), there are certain limitations to the use of PC in everyday dentalpractice. The restrictions for the clinical application of PC are a consequence ofits relatively high degree of solubility (Islam et al., 2006), lack of X-ray contrast(Bortoluzzi et al., 2009; Vivan et al., 2009), heavy metal content (Duarte et al.,2003; Bramante and Duarte, 2009), high degree of expansion during setting, anddistribution of different particle sizes of the powder (Damm aschke et al., 2005).The main components of PC are tricalcium silicate (CaO)3SiO2, dicalciumsilicate (CaO)2SiO2, tricalcium aluminate (CaO)3Ai2O3, and tetracalciumaluminoferrite (CaO)4Al2O3Fe2O3.Tricalcium silicate (C3S) is the main PC component responsible for its hydra-tion, and consequently, for calcium silicate hydrate (CSH) formation. Tricalcium sil-icate influences development of the early PC strength (Greeberg and Chang, 1965).The polymorphism of tricalcium silicate is defined by the presence of impurities,known as alite (Bye, 1983; De Noirfontaine et al., 2003). The symmetry of thetricalcium silicate crystals increases with a rise of temperature during its synthesis,and its structure is stabilized due to the presence of impurities in the raw material.270 CHAPTER 11 Nanosynthesized calcium-silicate-based biomaterials Dicalcium silicate (C2S) hydrates significantly slower than tricalcium silicate,thus it is responsible for the development of the late PC strength. Similarly toC3S, the most frequent form of dicalcium silicate with impurities present in natureis known as belite (Bye, 1983; Odler, 2000).Tricalcium aluminate (C3A) is the most reactive component of PC, and eventhough it quickly reacts while in contact with water, it has little influence on PCstrength. Tetracalcium aluminoferitte (C4AF) is the PC constituent that shows a mod-erate reactivity with water, which increases with a rise in Al content (Bye, 1983). Atthe same time, C4AF is the only PC constituent that may affect teeth discoloration.11.2.2 MINERAL TRIOXIDE AGGREGATEMTA was developed as a root-end filling material at Loma Linda University, dur-ing the last decades of the twentieth century. It is defined as a mixture of 75% PC,20% bismuth oxide added as radiopaquer, and 5% gypsum, with trace amounts ofSiO2,CaO,MgO,K2SO4,andNa2SO4(Torabinejad et al., 1995a). Although PCand MTA have similarities in their chemical compositions, there are still some dif-ferences. One of the main differences is almost two times lower content of gypsumand aluminum in MTA, which is a reason for its prolonged setting time, the majorMTA disadvantage (Dammaschke et al., 2005). Even though MTA has a smallersize of particles, which creates a finer powder, this, “sandy” consistency causes dif-ficulties during its clinical application (Asgary et al., 2006). In addition, analysisrevealed the presence of bismuth oxide, lack of potassium, and smaller amounts ofheavy metals in MTA (Song et al., 2006; Asgary et al., 2006).The original formulation of a gray MTA (GMTA) was partially changed in2002, and resulted in a synthesis of a white MTA (WMTA), as an aestheticallyacceptable formulation. In contrast to GMTA, which consists of dicalcium silicate,tricalcium silicate, and bismuth oxide, WMTA primarily consists of tricalcium sili-cate and bismuth oxide (Camilleri et al., 2005)(Table 11.2). Since WMTA isTable 11.1 Composition of PC (%)Component Content (%) Phase Content (%)SiO219.7 C3S55CaO 63.2 C2S15CaO (free) 0.46 C3A 7.9Al2O34.7 C4AF 8.1Fe2O32.67 CaO 0.46K2O 1.12 CaCO34.4MgO 1.85 CaSO44.5CO21.93 K2SO41.6SO33.35 NaSO40.096SrO 0.0727111.2 Chemical Properties of Calcium-Silicate-Based Materials synthesized in order to meet aesthetic criteria, smaller amounts of Fe, Al, and Mgoxide are included in its composition (Asgary et al., 2005, 2006, 2009; Camilleriet al., 2005; Song et al., 2006). Iron oxide is considered to be the main cause of thetooth crown discolorations in the case of GMTA, when present in sufficient quanti-ties and due to the presence of easily excitatory electrons (Estrelaetal.,2000;Funteas et al., 2003; Dammaschke et al., 2005; Camilleri et al., 2005; Robertset al., 2008). The WMTA powder particles are eight times smaller than those ofGMTA, which supports the larger surface hydration, stimulates development of theearly strength, and formation of a smoother surface. The presence of smaller parti-cles leads to the conclusion that the WMTA setting time is probably shorter.11.2.3 HYDRATION MECHANISMSetting reaction of calcium-silicate-based materials is an extremely complicatedprocess that depends on the respective phases, purity of the phases, and tempera-ture of the mixtures (Table 11.3). A series of reaction products used to be formed,not only due to kinetics of different processes but also due to influences of physi-cal barriers of reaction products on the surface of the particles, which disturb theprocess of water diffusion. In addition, dissolution, recrystallization, and interac-tion of the products also take place (Darvell and Wu, 2011).Hydration of the powder particles produces porous CSH gel and CH. It takesplace according to the following reactions:2ð3CaOUSiO2Þ 1 6H2O-3CaOU2SiO2U3H2O 1 3CaðOHÞ22ð2CaOUSiO2Þ 1 4H2O-3CaOU2SiO2U3H2O 1 CaðOHÞ2CSH particles are precipitated on the surface of the cement as a result oftricalcium silicate hydrolysis. CSH is poorly crystallized and porous, and isdescribed as a rigid colloidal gel. CH is a major soluble fraction of cement,responsible for the high pH value.Table 11.2 Composition of WMTA and GMTA (%)Chemical GMTA (wt%) WMTA (wt%)CaO 40.45 44.23SiO217.00 21.20Bi2O315.90 16.13Al2O34.26 1.92MgO 3.10 1.35SO30.51 0.53FeO 4.39 0.40P2O50.18 0.21H2O 1 CO213.72 14.49272 CHAPTER 11 Nanosynthesized calcium-silicate-based biomaterials At this stage, tricalcium aluminate also responds quickly, typically producingseries of hydrates affecting formation of diffusion barriers. If sulfate (S) is pres-ent, the occurring competing reactions prevent further reactions with water untilsulfate is completely consumed:C3A 1 S 1 H-C6AS3H32-C4ASH12These reactions are responsible for the early setting of material. The processof water diffusion through a formed layer of nonpor ous matrix-gel and its swell-ing eventually leads to formation of porous and poorly crystall ized solid gel.After gel bursting, simple hydration reactions continue to occur. Meanwhile, C3Spowder reacts with water to provide a basic, but slower , solidification mechanismwith the involvement of C2S:C3S; C2S 1 H-CSH 1 CHThe source of CH production is quite cont radictory; while Camilleri et al.(2005)consider that CH is formed from dicalcium and tricalcium silicate aftermixing the powder wi th water,Dammaschke et al. (2005) state that CH is a prod-uct of tricalcium aluminate hydration. Bismuth oxide also influences CH precipi-tation following MTA hydration (Dammaschke et al., 2005). It is detected as aconstituent of CSH, as well as in the form of unreacted filler (11.6%), which mayrepresent a problem from the aspect of MTA biocompatibility.CSH phase is not so well defined, with varying stoichiometric values in itscomposition, and it is predominantly amorphous, while CH is a clear crystalphase of a set material (the C:S ratio of CSH is less than 2:1, usually rangingbetween 0.8 and 2.1). As the ratio of C:S increas es, the hydration product of ahigh degree of crystallization, portlandite, is formed (Lee et al., 2007). Generally,the setting reaction of CSH involves formation of more or less crystallinehydrated calcium aluminate and calcium sulfate in an amorphous matrix ofhydrated calcium silicate (Lee et al., 2007).Table 11.3 The Principal Phases of MTA (%)Phases Chemical Formula CCN ProportionTricalcium silicate 3CaOSiO2C3S 51.9Dicalcium silicate 2CaOSiO2C2S 23.2Tricalcium aluminate 3CaOAl2O3C3A 3.8Tetracalcium aluminoferrite 4CaOAl2O3Fe2O3C4AF NilCalcium sulfate dihydrate CaSO42H2O 1.3Calcite, bismuth oxide, alkali metaloxidesCaCO3,Bi2O3, CaO,MgO19.827311.2 Chemical Properties of Calcium-Silicate-Based Materials 11.2.4 CHEMICAL PROPERTIES OF OTHER TYPESOF CALCIUM-SILICATE-BASED MATERIALSBioaggregate is a biomater ial based on calcium silicates with phosphates andwithout aluminum. In addition to calcium silicates and its oxides, Bioaggregate iscomposed of some other constituents, such as: hydroxyapatite (phosphate compo-nent), calcium phosphate silicate, calcilite, and tantalum oxide as radiopaquer(Park et al., 2010; Saghiri et al., 2013).Biodentine represents a biomaterial which, in addition to the predominantlypresent tricalcium silicate, is also composed of dicalcium silic ate, calcium carbon-ate, calcium oxide, and zirconium oxide as radiopaquer. The liquid with whichpowder is mixed contains calcium chloride and a hydrosoluble polymer based onpolycarboxylates, which is responsible for the low water content and the mixtureconsistency (Burgess et al., 2009).11.3 NANOTECHNOLOGY IN THE PROCESS OF SYNTHESISOF NEW CALCIUM-SILICATE-BASED MATERIALS11.3.1 SYNTHESIS OF HIGHLY ACTIVE CALCIUM SILICATESIn order to obtain active calcium silicate phases a stoichiometric amount ofCaCl23 5H2O (35.59 g) and silica sol (15 g of 30% solution) are used. Thesynthesized calcium tricalcium silicate phase (Ca3SiO5-C3S) and dicalcium sili-cate phase (2β-CaSiO4-C2S) should be used in the ratio 2:1. A small amount ofAl(C2H3O2) (4.55g) used to be added in order to achieve preparation of an activeC3A phase. While NH4NO3(71.3 g) is used as the oxidizing agent,C6H8O73 H2O (53.51g) is used during the reactio n, as a fuel.Furthermore, a gel phase is obtained by drying the samples at a temperature of80C. Excess water should be removed from the samples by exposure to a tem-perature of 150C, which leads to formation of viscous gel. Total dehydration ofthe gel is achieved at a temper ature close to 180C.Transformation of the mixture into the final form is performed by the methodof strong self-propagation combustion reaction, which causes a release of largeamounts of gases and a rise of temperature. Afterwards, samples have to berapidly cooled using a copper plate to minimize crystallization of the obtainedcalcium silicate phases and to ensure their high reactivity. The product of thisreaction is a black powder, calcined during a 4-hour period at the very high tem-perature (650C), in order to remove excessive carbon (Nikoli´c et al., 2009;Colovic et al., 2013).Results of XRD analysis of silicate phases are presented inFigure 11.1. Theauthors (Colovic et al., 2013) used the Scherrer equation for calculation of crys-tallite sizes of the phases, and the obtained values were about 19.9 nm [calculatedfor highly pronounced planes at 32.37;(2121) for β-C2S and (2715) for C3S].274 CHAPTER 11 Nanosynthesized calcium-silicate-based biomaterials SEM analysis of the calcium silicate phase (Figure 11.2) shows that it mostlyconsists of spherical and ellipsoidal particles (117477 nm) which form agglom-erates of several micrometers in diameter. Such hierarchical structures seem to bepromising for applications in biological systems. This may be explained by thefact that they are not biologically destructive and their nano-elements (crystallites)facilitate very pronounced activity, especially quick bonding, so they aresuitable for use in endodontic therapy.EDS analysis shows that chemical composition of calcium silicate (22.21%Ca, 8.22% Si, and 69.7% O) corresponds to the ratio Ca:Si B2.7 at.% (Nikoli´cet al., 2009; Colovic et al., 2013).11.3.2 SYNTHESIS OF NANOSTRUCTURED BIOMATERIALS BASEDON HIGHLY ACTIVE CALCIUM SILICATES ANDCALCIUM CARBONATESMineral polyoxide carbonate aggregat e (ALBO-MPCA), CaCO3, and Bi2O3withcalcium silicate phases (Ca3SiO5and 2β-CaSiO4), in the ratio 2:2:1 are used insynthesis of novel nanostructured materials.FIGURE 11.1XRD spectra of calcium silicate phases.Adapted from Jokanovi´c et al. (2014b) with permission.27511.3 Process of Synthesis of New Calcium-Silicate-Based Materials The synthesis of calcium carbonate includes several steps, as follows.Foremost, calcium chloride tetrahydrate (CaCl23 4H2O) is used as a precursor.Using the ultrasound effect at temperature of 40C, 5 mmol of CaCl23 4H2Oisdissolved in 50 mmol of ethylene glycol. Alongside, 10 mM of NaHCO3isdispersed into 50 ml of ethylene glycol, with constant mechanical stirring.Furthermore, this dispersion should be preheated for 30 min at 40C and thencalcium carbonate is isolated by centrifugation (9000 rpm, 30 min). Moreover, thecalcium carbonate is washed several times with a mixture of water and ethanol(1:4), and finally rinsed only with water. Sulfonyl dodecyl sulfate (0.5%) is usedas an agent against agglomeration, and then the resulting nanoparticles of thepowder are exposed to ultrasound for 30 min, with vigorous mechanical stirringfor 5 h. Then, the powder is dried at 120C for 5 h and heated at 500C for anhour, to obtain calcium carbonate phase.The monoclinic Bi2O3, used as the radiocontrast agent, is obtaine d bycalcinations of Bi(NO3)3at a temperature of 450C for 20 h, to ensure thestable tetragonal Bi2O3phase (Jokanovi´c et al., 2014b).XRD investigations of ALBO-MPCA before hydration are presented inFigure 11.3. The peaks at 32.37, 47.77, and 56.53[with corresponding planesFIGURE 11.2Typical appearance of calcium silicate phases.Adapted from Jokanovi´c et al. (2014b) with permission.276 CHAPTER 11 Nanosynthesized calcium-silicate-based biomaterials (2121), (222), and (2303), respectively] correspond to β-C2S phase. The planes(422), (2606), and (2822) corresponding to C3S phase are identified at angles of29.65, 32.41, and 34.57, respectively. Calcite phase is identified at 29.49, 34.88,47.63, and 48.69[planes (104), (110), (018), and (116), respectively]. And finally,planes (002), (120), and (200) corresponding to monoclinic Bi2O3are identified atangles 29.49, 34.88, and 48.69,respectively(Jokanovi´cetal.,2014b).SEM investigation of ALBO-MPCA before and after hydration (Figures 11.4and 11.6) reveals polygonal particles elongated in one direction, between 300 and500 nm. They form larger spherical and rod-like aggregates, about 3 μm in size(Jokanovi´c et al., 2014b).XRD investigat ions of ALBO-MPCA samples hydrated for 1, 3, 7, and 28days (Figure 11.5) revealed that the quantity of hydrated tobermorite phaseincreased in correlation with hydrating time, while the quantities of other phases,calcite and Bi2O3, remained almost the same. These changes are clearly noticedwhile observing characteristic tobe rmorite planes (112), (110), (019), and (020),corresponding to angles of 29.75 , 30.01, 43.69, and 48.8, respectively. The plane(110) is chosen as the most appropriate to present the changes inside the mixtureFIGURE 11.3XRD patterns of ALBO-MPCA (B-Bi2O3, C-calcite) before hydration.Adapted from Jokanovi´c et al. (2014b) with permission.27711.3 Process of Synthesis of New Calcium-Silicate-Based Materials FIGURE 11.4SEM analysis of ALBO-MPCA before hydration (calcium silicate phase predominates).Adapted from Jokanovi´c et al. (2014b) with permission.FIGURE 11.5XRD patterns of ALBO-MPCA hydration for various times (B-Bi2O3, C-calcite, T-tobermorite,P-portlandite).Adapted from Jokanovi´c et al. (2014b) with permission.278 CHAPTER 11 Nanosynthesized calcium-silicate-based biomaterials during its hydration. As expected, portlandite phase is also present [the planes(100), (011), and (012), at the angles 28.42, 34.71, and 47.44, respectively](Jokanovi´c et al., 2014b).11.3.3 SYNTHESIS OF SUPERPLASTIC, QUICK-BONDINGENDODONTIC MIXTURESIn order to obtain superplastic, quick-bonding material for application in endodon-tics, two mixtures are synthesized, ALBO-MPSA I and II. The ALBO-MPSA Imixture contains the following components: β-C2SandC3Sphases(24%ofthetotal quantity of mixture), hydroxyapatite (40%), gypsum dehydrate (24%), andBaSO4(12%) (Jokanovi´c et al., 2006, 2008, 2009; Opaˇci´c-Gali´cetal.,2013). Thecomposition of ALBO-MPSA mixture II is as follows: 40% hydroxyapatite, 20%C2SandC3S mixture (in ratio 2:1), 20% gypsum dihydrate, and 20% BaSO4.Inaddition, 0.65% of 2-phosphonobutane-1,2,4-tricarboxylic acid (PBTCA), as super-plasticizer, is added to both mixtures. After common milling for better homogeniza-tion, the mixtures are finally combined with water to produce cement pastes with awater-to-mixture ratio of 0.5 (Jokanovi´c, 2012; Jokanovi´cetal.,2014a).FIGURE 11.6SEM analysis of ALBO-MPCA after hydration.Adapted from Jokanovi´c et al. (2014b) with permission.27911.3 Process of Synthesis of New Calcium-Silicate-Based Materials The hydration process of the mixtures is analyzed by XRD method, over-induced phase changes during various hydration times (24 h, 3, 7, and 28 days).The XRD patterns (Figure 11.7) of the mixture I shows the presence of followingprevailing phases: gypsum dihydrate (planes (020), (2121), (031), (2141), and(051), values 12.03, 21.13, 23.15, 29.51, and 33.14, respectively), barite (planes(111), (112), (122), and (312), at the angles of 23.65, 31.94, 42.94, and 43.35,respectively), and tobermorite (planes (110) and (112), values of 29.3 and29.51). The phases of portlandite, Ca(OH)2, (planes (011), (012), and (110),values 34.86, 48.29, and 50.62, respective ly), and ettringite (planes (110), (304),and (226), values 15.17, 33.14, and 39.9, respectively) are pres ent in small quan-tities. The presence of hydroxyapatite, as a basic component in the mixture I,with its characteristic planes (200), (002), (121), (300) , and (123), values 21.05,26.23, 31.87, 33.16, and 49.36, respectively, is also confirmed. Therefore it isreasonable to assume that the prevailing mechanism of the mixture hydration pro-cess is the Ca(OH)2phase adsorption on the surface of hydrated calcium silicatephases and formation of a well-known tobermorite phase, as a dominant and anew one. XRD analysis of mixture II shows the presence of the same phases andcharacteristic planes as in mixture I. The presence of tobermorite, an importantnew phase formed during the hydration process, is confirmed in both mixtures.FIGURE 11.7The XRD patterns of the mixtures.Adapted from Jokanovi´c et al. (2014a) with permission.280 CHAPTER 11 Nanosynthesized calcium-silicate-based biomaterials The absence of starting cement phases β-C2S and C3S is also evident. These factsare very important for theoretical explanat ion of the process of hydration thattakes place in mixtures I and II (Jokanovi´c et al., 2014a).SEM morphology following hydration of cement mixtu re I reveals irregularparticle morphology (Figure 11.8). Mean particle diameter ranges between 62 and332 nm, and the most dominant are the particles with a mean diameter close to75 nm. The particles in cement mixture II (Figure 11.9) are packed very close toeach other; therefore, it is difficult to precisely determine their shape and size.However, they are irregular in shape, and their mean diameter approximatelyranges between 170 and 260 nm (Jokanovi´c et al., 2014a).11.3.4 MECHANICAL PROPERTIES OF NANOSTRUCTUREDMATERIALS BASED ON CALCIUM SILICATESThe measured compressive strength values for the ALBO-MPCA (cylinders20 3 10 mm in diameter) are as follows: 23.1 MPa (1 and 3 days), 35 MPa(7 days), and 42.5 MPa (28 days). The measured setting time of ALBO-MPCA isaround 70 min (Jokanovi´c et al., 2014b).The measured compressive strength values for ALBO-MPSA I are29.5 6 0.5 MPa (1 day), 31.4 6 0.5 MPa (3 days), 34.5 6 0.5 MPa (7 days), and35.7 6 0.5 MPa (28 days), and for ALBO-MPSA II are 32.6 6 0.5 MPa (1 day),35.2 6 0.5 MPa (3 days), 35.8 6 0.5 MPa (7 days), and 37.4 6 0.5 MPa (28 days).The calculated setting time of ALBO-MPSA I is 10 6 0.15 and of ALBO-MPSAII is 15 6 0.15 min (Jokanovi´c et al., 2014a).FIGURE 11.8Scanning electron microscopy of cement mixture I following hydration.Adapted from Jokanovi´c et al. (2014a) with permission.28111.3 Process of Synthesis of New Calcium-Silicate-Based Materials Recent research showed that superplastic properties of the endodontic mixturesare influenced by PBTCA (Jokanovi´c et al., 2014a). As presented in Figure 11.10,viscosity of the mixtures containing PBTCA quickly drops to a very low value,while viscosity of the mixtures without it is significantly higher, showing similarbehavior with increase in the shear rate.11.3.5 HYDRATION REACTIONSHydration of calcium silicate phase is the most important parameter that influ-ences binding and mechanical properties of the MTA-like mixtures. As the moist-ening of CS particles take place, the rapid release of Ca21and OH2ions fromthe surface of each grain occurs (Jokanovi´c, 2012). Transformation of C3S in theamorphous CSH, known as tobermorite gel, and calcium hydroxide (Ca(OH)2), isillustrated by the following reaction:2C3S 1 7H2O-CSH 1 3CaðOHÞ2FIGURE 11.9Scanning electron microscopy of cement mixture II following hydration.Adapted from Jokanovi´c et al. (2014a) with permission.282 CHAPTER 11 Nanosynthesized calcium-silicate-based biomaterials Similarly, the reaction between β-C2S and water may be expressed as follows:2C2S 1 5H2O-CSH 1 CaðOHÞ2CSH, obtained in both reactions, is the last member of the series, describedas silica calcium hydrate, and it can be of variable composition. The lack ofcharacterization of the exact reactions of hydration is associated with difficultiesin determining the exact tobermorite structure and its corresponding formula.The assumed ideal formula of tobermorite could be 4CaO 3 Ca(OH)23 4H2O 3 6SiO2. It is well known that the central part of each toberm oritelayer is similar to the octahedral layer in the clay mineral, which could bedescribed as a distorted CH without hydrogen atoms (CaO2). This layer is sur-rounded, on both sides, with the parallel rows of wollastonite chains pinched in aperpendicular plane (Jokanovi´c, 2012).When preparing a paste based on calcium silicates, C3S phase proba bly reactsvery quickly (in a few minutes), and water suddenly becomes saturated with Ca(OH)2. The same reaction happens with β-C2S, but more slowly.The hydration reaction leads to the formation of Ca(OH)2, and a member oftobermorite G subgroup. If silicates are however hydrated in a large excess ofwater so that the concentration of lime in the solution is below saturation, theformed CSH is not a member of tobermorite (G) and the usual reaction productFIGURE 11.10Dependence of viscosity and shear rate for ALBO-MPSA I and ALBO-MPSA II.Adapted from Jokanovi´c et al. (2014a) with permission.28311.3 Process of Synthesis of New Calcium-Silicate-Based Materials Ca(OH)2is missing. CaO/SiO2ratio in the CSH depends on the final concentra-tion of lime in the solution; its lower limit is close to 0.8, and the upper about 1.5(at or near saturation value for lime) (Jokanovi´c, 2012).11.4 PHYSICAL AND ANTIMICROBIAL PROPERTIESOF CALCIUM-SILICATE-BASED MATERIALS11.4.1 SETTING TIME, SETTING CONDITIONSDue to the hydraulic nature of calcium-silicate-based materials and the fact thatthe humidity of the surrounding tissues acts as an activator of chemical reactionsduring the setting, these materials are convenient for application in endodonticprocedures. However, because of the chemical composition and lasting hydration,initial setting of calcium-sili cate-based materials lasts several hours, which limitstheir clinical application.Initial setting time of MTA, when mixing the powder with sterile water inratio 3:1, rates for 165 6 5 min (Torabinejad et al., 1995a). Although GMTAmanifests a longer setting time then WMTA (Chng et al., 2005; Islam et al.,2006) both materials display longer setting time in comparison to PC, due to areduced content of sulfur and tricalcium aluminate (Dammaschke et al., 2005). Inorder to overcome the disadvantage of the longer setting time, the investigatorsattempted to replace water as a liquid agent with other solutions but the effortsdid not give the desired results. While mixing MTA with anesthetic solutionprolongs the setting time (Storm et al., 2008), the application of 5% calcium chlo-ride or NaOCl gel reduced it to 2025 min, though it also leads to an undesiredreduction of the compressive strength of MTA (Antunes et al., 2006).Accordingly, it may be expected that Biodentine possesses a lower setting ti mebecause of the addition of calcium chloride to the mixin g liquid; however, itsfinal setting time is about 45 min, which is, from a clinical point of view, stillquite a long period. Recent investigation shows that material with mor e phosphatecomponents, such as Bioaggregate, requires an even longer time (1260 min) forits final setting in comparison to MTA (Grech et al., 2013).11.4.2 SOLUBILITYThe long setting time of calcium-silicate-based materials may represent a certainrisk of the material’s dissolution at the site of its application, which can furtherjeopardize the entire endodontic treatment. The solubility of this material isdirectly associated to its porosity, related to the amount of water used during themixing procedure, residual air bubbles during preparation of the material, and pHof the medium. The solubility is measured by changes in the mass of the materialstored in a humid environment and it should not exceed 3% of its total weight.During setting, some water is consumed in the course of chemical reactions, while284 CHAPTER 11 Nanosynthesized calcium-silicate-based biomaterials some water remains captured in the set cement. The constant tendency of waterand Ca21ions release from the set cem ents exists because of the fact that water issaturated with Ca(OH)2, which is a basic soluble component (Lee et al., 2004). Onthe other hand, voids and microchannels inside the set cements bind water fromthe surrounding environment and gain its weight again. According to the literaturedata, 0.33 g of water with 1 g of MTA provides an optimum mix, and with anincrease in water to powder ratio, the degree of MTA solubility and porosity rises(Fridland and Rosado, 2005; Budig and Eleazer, 2008). The degree of MTA solu-bility is insignificant, as well as of both Bioaggregate and Biodentine (Torabinejadet al., 1995a; Islam et al., 2006; Grech et al., 2013; De Souza et al., 2013).11.4.3 SEALING ABILITYThe ideal root-end filling material should obtain hermetic obturation and preventmovement of periapical tissue fluids into the root canal, as well as the residualmicroorganism penetration and vice versa. Furthermore, the adequate apical sealshould provide long-lasting success of endodontic treatment.When compared to other materials using fluid filtration method, MTA exhibitsthe same sealing ability as reinforced zinc oxide cement such as Super EBA andbetter sealing ability than amalgam, the most commonly used material for root-endfillings (Bates et al., 1996). Enhanced sealing ability of MTA could be explainedby its expansion during hydration which contributes to the better adaptation to thedentinal walls. The studies on bacterial microleakage indicate that MTA displaysless or no microleakage in comparison to amalgam, Super EBA, and IRM(Torabinejad et al., 1995b; Fischer et al., 1998). This is very important because thepresence of residual microorganisms in dentinal tubules is considered a main reasonfor endodontic treatment failure.Calcium-silicate based materials used to be placed in the environment that isusually inflamed. With regard to this,Roy et al. (2001) examined the influe nce ofacidic pH on the MTA sealing property, and reported that it does not affect thesealing ability of MTA with calcium phosphate cement matrix. When using MTAin the treatment of teeth with necrotic pulps and immature roots, it is preferableto acquire at least a 4-mm- thick apical plug, because it demonstrates significantlyless microleakage compared to 1-, 2-, or 3-mm-thick apical plug (Valois andCosta, 2004). The sealing ability of MTA may also be increased by mixing mate-rial with calcium chloride (Bortoluzzi et al., 2006), though the microleakage isstill more pronounced in the case of Bioaggregate (El Sayed and Saeed, 2012).11.4.4 COMPRESSIVE STRENGTHCompressive strength is defined as the abili ty of material to resist the direct pres-sure of applied compression force. Voids and microchannels inside the hydrauliccement provide the adequate hydration of the material and subsequently increaseits compressive strength. MTA may be used in a wet environment (Chogle et al.,28511.4 Physical and Antimicrobial Properties 2007), which is an important property assuming that in a number of indications itis impossible to achieve a completely dry working field. Because of the hydraulicnature of calcium-silicate-based cements, immediately following a mixing proce-dure, the com pressive strengt h of MTA use to be significantly lower in compari-son to those of IRM, Super EBA, and amalgam; however, 3 weeks later, nosignificant differences are observed (Torabinejad et al., 1995a). These results maybe explained by the slower hydration process of dicalcium silicates than trical-cium silicates. Precisely, the conflicting results, considering the compressivestrengths of WMTA and GMTA, could be a consequence of different contents ofdicalcium and tricalcium silicates and their hydration mechanisms (Torabinejadet al., 1995a; Holt et al., 2007). Given that the MTA is not exposed to directeffects of pressure and that condensation pressure does not have a significantimpact on the compressive strength, raises the question whether the compressivestrength is relevant to its clinical application. In the case of no direct exposure toa pressure, accelerators such as 5% calcium chloride or NaOCl gel, could beapplied, since they reduce MTA setting time (Bortoluzzi et al., 2006). On theother hand, due to a low water to powder ratio, Bioden tine expresses higher com-pressive strength (67.18 MPa) compared to Bioaggregate (16.34 MPa) and MTA(B40 MPa) (Torabinejad et al., 1995a; Grech et al., 2013). In acidic conditions,the mean compressive strengths of both, WMTA and Bioaggregate, decrease sig-nificantly (Saghiri et al., 2013), so the plac ement of composite resins after theaccomplishment of pulp capping or pulpotomy with these materials is not recom-mended for at least 96 h, which substantially prolongs the treatment time.11.4.5 FLEXURAL STRENGTHFlexural strength is the maximum tension that can be loaded on the material with-out causing its fracture. In WMTA it significantly increases when material isexposed to moisture on both sides (Walker et al., 2006). Therefore, placement ofwet cotton pallet over MTA is recommended when it is used for perforation repair,as a pulp capping agent, or an apical plug. The exposure to moisture should lastonly 24 h, while 72 h later the achieved effect is exactly the opposite (Walkeret al., 2006). Since the procedure delays accomplishment of the mentioned inter-ventions, it is obvious why it is not preferable in everyday clinical practice.11.4.6 PUSH-OUT STRENGTHPush-out strength is an import ant property of perforation repair materials, as toothfunction may easily dislodge the appl ied material. According toLoxley et al.(2003), MTA has a lower push-out strength compared to those of IRM and SuperEBA. Mixing MTA with anesthetic solution increases this property after 72 h(Sluyk et al., 1998), but since it also prolongs the setting time, it restrains its clin-ical use. Placement of the wet cotton pallet over MTA significantly increases itspush-out strength (Gancedo-Caravia and Garcia-Barbero, 2006), which confirms286 CHAPTER 11 Nanosynthesized calcium-silicate-based biomaterials the importance of moi sture for this MTA property. However, removal of a wetcotton pallet implies potential reinfection of a root canal, also inducing delay intreatment time. The 24 h push-out strength of MTA is lower compared toBiodentine (Aggarwal et al., 2013), and higher, but more influenced by acidicconditions, compared to Bioaggregate (Hashem and Wanees Amin, 2012).Therefore, Biodentine may be considered for application as a perforation repairmaterial due to its higher push-out strength and shorter setting time.11.4.7 DISPLACEMENT (EFFECT OF CONDENSATION ON MTA)The effect of applied condensation pressure, when MTA is indicated for perforationrepair as a pulp-capping agent, root-end-filling material, and especially an apicalplug in teeth with immature root development, is usually uncontrolled. It is clearthat the condensation pressure should be significantly reduced when MTA isapplied as an apical plug to avoid its extrusion in periapical tissue and interferencewith tissue regeneration. It is established that a 4-mm-thick MTA apical plug showssignificant resistance to displacement when compared to a 1-mm-thick material(Hachmeister et al., 2002). Because of the above stated, it is preferable to achieveadequate thickness of MTA during clinical application. It is not defined yet whetherthere is an influence of condensing pressure on compressive strength (Nekoofaret al., 2007), still a condensation pressure affects the surface microhardness, whichis explained by limitation of the space required for hydration of material, whichthus becomes more compact. Uncontaminated blood samples of WMTA showstatistically significant resistance to displacement (Vanderweele et al., 2006), whichusually do not fulfill the requirements for endodontic treatment materials.11.4.8 pH VALUE OF MTAImmediately following the mixing procedure, the expressed pH value of MTA is10.2, and it increases to 12.5 during the next 3 h (Torabinejad et al., 1995a).According to Fridland et al., (2005), due to a constant release of calcium ions,MTA maintains high pH value for a long period of time, thus enabling goo dbiological and antibacterial properties of this material. Immediately after mixing,both WMTA and GMTA manifest statistically higher pH in comparison to PC;however, 30 min later, the difference is no longer registered, whereas by the endof the next 60 min GMTA displays significantly lower pH value than those ofWMTA and PC (Islam et al., 2006). In addition, although it is obvious that differ-ent periods of time influence the pH of MTA, it should be also noted that theMTA complete setting was not finalized during the investigated time.11.4.9 RADIOPACITYRadiopacity is an essential requirement for the endodontic application of a mate-rial; according to ISO standar d 6876/2001 it should be greater than 3-mm-thick28711.4 Physical and Antimicrobial Properties aluminum. In the case of MTA, the most common chemical component used asan X-ray contrast is bismuth oxide.Torabinejad et al. (1995a) demonstrated thatMTA is less radiopaque than Super EBA, IRM, amalgam, and gutta-percha, whileShah et al. (1996) confirmed just the opposite. Some other authors proclaim thatWMTA is more radiopaque then GMTA (Chng et al., 2005; Islam et al., 2006),which is probably due to a slightly higher content of bismuth oxide in WMTA.However, the radiopacities of Biodentine and Bioagg regate (which possess asradiopaquers, zirconium oxide and tantalum oxide, respectively) are greater com-pared to 3-mm-thick aluminum and regard this as indicating that they aresuitable for use in endodontics (Grech et al., 2013).11.4.10 PARTICLE SIZEThe mixed MTA has granular consistency and it is quite complicated for clinicalmanipulation. On the other hand, the smaller particle size although allows easiermanipulation and also affects MTA consistency. To a certain extent, this explainsway investigators try so hard to synthesize nanostructure materials based oncalcium silicates. According toLee et al. (2004), the most common GMTA parti-cles range in size from 1 to 10 μm, whileCamilleri (2007) states that WMTApowder is composed of particles which are less than 1 μm. The particle size andtheir structure may affect the physical properties of the material. Since it is quitewell known that particles below 1.5 μm are smaller than a diameter of dentinaltubules (Komabayashi and Spangberg, 2008), it is not surprising that they play acrucial role in the good sealing properties of a material.11.4.11 MICROHARDNESSMicrohardness or indentation hardness is the hardness of the material exposed to lowapplied loads. Upon hydration MTA consists of needle-like, predominantly cubic,crystals; in acidic environment the crystals disintegrate causing loss of MTA micro-hardness (Lee et al., 2004). According to Matt et al. (2004), the microhardness of a5-mm-thick MTA sample is significantly higher compared to that of 2 mm; onceagain it is obvious that achieved thickness of a material provides its desired physicalproperties. However, it has been also presented that an increase of condensation pres-sure reduces surface hardness of MTA (Necroofar, 2007), which can be explained bylimitation of the space required for hydration of material. Considering this, insistingon a better compaction of MTA may cause a diverse effect.11.4.12 FRACTURE RESISTANCE (EFFECT OF MTAON THE STRENGTH OF ROOT DENTINE)It is well known that, following endodontic treatment, teeth lose their biologicalqualities and become more prone to fractures. Therefore, it is of cruc ial288 CHAPTER 11 Nanosynthesized calcium-silicate-based biomaterials importance that root-filling materials do not cause further root dentine weakening.Study on extracted sheep teeth confirmed that those filled with MTA after 1 yearhave significant resistance to fracture compared to those filled with CH(Hatibovic-Kofman et al., 2008). In the samples with MTA, the same researchersconfirm the presence of the matrix metalloproteinase inhibitor, TIMP-2, whichinhibits collagen fiber destruction.11.4.13 ANTIMICROBIAL ACTIVITYConsidering the ability of residual microorganisms to provoke periapical irritations,antibacterial activity of a sealing material seems to be a desirable property.Contradictory research results from the literature regarding antimicrobial effects ofcalcium-based materials are probably due to differences in methodology, chemicalcomposition, forms, concentrations of used materials, etc. Thus, while some studiesverified that MTA-based materials and PC possess antimicrobial activity againstMicrococcus luteus, Staphylococcus aureus, Escherichia coli, Pseudomonas aerugi-nosa, Candida albicans,andEnterococcus feacalis (Tanomaru-Filho et al., 2007;Asgary and Kamrani, 2008), other studies conclude that MTA exhibits non-antibacterial activity (Miyagak et al., 2006; Yasuda et al., 2008). The antibacterialactivity of Bioaggregate is similar to MTA (Zhang et al., 2009), and when freshlymixed, it also possesses activity against C. albicans (Dohaithem et al., 2011).11.5 MICROBIOLOGICAL PROFILE OF ROOT CANALSASSOCIATED WITH PERIAPICAL PATHOSIS11.5.1 ENDODONTIC APICAL DISEASE: INFECTION OF TISSUESNowadays, etiology of endodontic apical disease is well known; it is an infectiousdisease. Microorganisms of infected root canals are related to invasion and estab-lishment in root canal systems and dentinal tubules of the root canal walls, whichare normally a sterile area (Cohenca and Lez Amaro, 2014; Ballal andGopikrishna, 2009). Microorganisms, their endotoxins, and by-products maycause symptomatic or asymptomatic inflammation and destruction of apical peri-odontal tissues which usually appears as a radiolucent area in the radiography(Cohenca and Lez Amaro, 2014).Research and implementation of new concepts, such as biofilm formation inendodontic microbiology, could provide a better understanding of the root canalinfection and endodontic apical disease etiology, pathogenesis, and process. Theapplication of contemporary knowledge is necessary in order to enable effectiveprevention, modern treatment strategies, adequate infection management, and betterresponse to treatment. Therefore, understanding and identifying microorganismsassociated with endodontic infections illustrates a framework which could providedesign of preventive programs and straightforward therapeutic protocols.28911.5 Microbiological Profile of Root Canals Associated 11.5.2 MICROBIAL COMMUNITY: BIOFILMBiofilm formation represents a natural process in all planktonic microbes’ nativeenvironments. Many researches made an effort to explain factors which influencethe transition of microbes from planktonic to biofilm lifestyles and their adhesionto the surface (Davies and Geesey, 1995; Li and Burne, 2001; Svensater andBergenholtz, 2004). If environmental conditions become adverse, oral bacteriamay exhibit the capacity to alter their gene expression and express new pheno-types in order to enable different behaviors, which will enhance their survival(Davies and Geesey, 1995).It may be assumed that although organization and composition of comprisingmicroorganisms within biofilms in distinct natural environments may differ, theprocess of its formation will follow the same pattern (Svensater and Bergenholtz,2004)(Table 11.4).Biofilms provide support and numerous advantages to the colonizing microor-ganisms by enabling an appropriate environment for survival of its residents(Svensater and Bergenholtz, 2004; Tronstad, 2009). It provides protection fromexternal agents such as host defense, dehydration, different antimicrobial agents,and competing microorganisms. Bacteria within biofilms possess inherentlyincreased resistance to adver se agents compared to free-floating microorganismsin liquid environments which are more susceptible to antimicrobial agents such asantiseptics and antibiotics (Svensater and Bergenholtz, 2004). A very importantbenefit of the lifestyle within a multispecies biofilm is that its residents havemore pathogenic potential than in planktonic state due to a collective pathogeniceffect to the host. Recent scientific data suggest that microorganisms may discloseinteraction and genetic exchange between genetically different members, whichallows the forming of novel phenotypes due to a new gene expression (Siqueiraand Rocas, 2009b; Sedgley et al., 2008). Further benefits of community life fororal microorganisms may be their metabolic interaction which permits cross-feeding, easier uptake, and digestion of the nutrients (Tronstad, 2009). Withinbiofilms, different microorganisms could assemble a food web that allows a largeramount of nutrients. This could clarify survival of the root canal populationfollowing prolonged starvation periods (Svensater and Bergenholtz, 2004).Table 11.4 General Phases of Biofilm Formation1. Adsorption ofmacromoleculesFormation of the conditioning film which will influence selectionof attached microorganisms2. Adhesion andcoadhesionAdhesion of the specific early colonizers and after them othermicroorganisms can be recruited3. Multiplication andmetabolismStructural organization of microbial community4. Detachment Continuous process which enables spreading and colonization290 CHAPTER 11 Nanosynthesized calcium-silicate-based biomaterials Caries, gingivitis, and periodontal disease are typical infections caused by oralbacterial plaque or biofilms (Marsh, 2004; Netuschil et al., 1998). Also, traumaticdental injuries are considered one of the most frequent causes of pulp pathologyin young permanent teeth. Microbial invasion and infection of necrotic pulp tissueenables development of periapical disease and may compromise apexogenesis.One of the first papers identifying root canal microbial aggregates as a main causeof endodontic apical disease describes analysis of the root canals by transmissionelectron microscopy (Nair, 1987). Recent scientific data definitely confirm bacte-rial biofilms in root canals of tooth-associated apical periodont itis (Ricucci andSiqueira, 2010). Bacterial survival is possible due to mutual cooperation andinteraction within the metabolically integrated microbial community present inthe root canals. These communities are considered as a successful survival strat-egy that enables microorganisms to adapt to environmental changes and overcomestresses (Siqueira et al., 2012).11.5.3 GENERAL CHARACTERISTICS OF BIOFILMBiofilm represents a multicellular microbial aggregate comprised of microbialcells surrounded by heterogeneous extracellular matrix produced by the residentsthemselves and separated with water channels (Donlan and Costerton, 2002). Inmost biofilms, extracellular polymeric substance (EPS) matrix may contain morethan 90% of a dry mass, while bacterial cells comprise only up to 10% (Sedgleyand Jacinto, 2014).The matrix is constituted of various types of biopolymers—extracellular poly-meric substances (EPSs) . EPS matrix forms specific physical dividers which sepa-rate so-called biofilm microdomains. They may be altered themselves or activatedin altered environmental conditions (Lawrence et al., 2007). Polysaccharides, pro-teins, and DNA, which are major matrix components, are unequally arrangedbetween bacterial cells forming regions with different biochemical characteristicsand enzyme functions (Flemming and Wingender, 2010; Mayer et al., 1999).Biofilm morphology type (smooth, rough, or filamentous) and degree ofporosity are determined by the EPS matrix characteristics (Flemming andWingender, 2010). Maintenance of biofilm structure depends on the productionand quantity of the matrix. EPS matrix has an important role in determining thethree-dimensional organization of bacterial biofilm scaffold and providing tran-sient immobilization of bacterial cells, mechanical stability, integrity, and cohe-sion of all residents. This enables long-term existence of the multispeciescommunity.It was demonstrated that many factors controlled by EPS matrix, such ashydrodynamic conditions, concentration of nutrients, mobility of bacterial cells,cell-to-cell communication, polysaccharides, proteins, and interactions betweenanionic EPS, may influence biofilm architecture (Flemming and Wingender,2010). Therefore, it may be assumed that EPS matrix plays a crucial role in for-mation of biofilm and enables community lifestyle.29111.5 Microbiological Profile of Root Canals Associated 11.5.4 PARTICULARITIES OF BIOFILMS IN ENDODONTICMICROENVIRONMENTThe root canal is considered as a specific microenvironment. In order to enablebiofilm formation according to presumed scheme, as in other natural environ-ments, microbes should be in planktonic state before adhesion to the surface andcolonization. Nevertheless, this is possible only in some cases when pulp tissuebecomes necrotic and liquefied before bacterial invasion, because the root canalenvironment is not necessarily filled with fluid (Dahlen, 2009).Although caries is the most common way for microbial access to the pulptissue, other possible routes are shown inFigure 11.11.The etiology and pathogenesis of caries is well known; it is caused by micro-organisms in plankton ic state in saliva which organize communities on dental sur-faces. As the caries process develops, the biofilm from the caries lesion alsoexpands toward the pulp tissue through the dentinal walls. Finally, when the pulpbecomes exposed, histological studies observed that biofilm covers canal wallsand the surface of the inflamed pulp tissue and further progress toward the apicalsections (Ricucci and Siqueira, 2010). Pulp tissue under the biofilm is inflamed,necrotic, and the host’s immune defense is activated; polymorphonuclear cellshave the role to form a barrier between biofilm and other tissue. Inflammatoryexudates, produced in the inflamed area, represent ideal source of nutrients forbacterial cells within community, since it is rich in protei ns and glycoproteins(Siqueira et al., 2012). After pulp exposure, penetration of oral bacteria is up to2 mm into the vital pulp tissue after 2 weeks. Vital pulp and host defenseFIGURE 11.11Possible routes of oral microbes to pulp tissue: (a) dentinal tubules (caries process ordeep preparations); (b) pulp exposure (hard dental tissue traumatic injuries); (c) apicalforamen (periodontal disease or periodontal tissue traumatic injuries).292 CHAPTER 11 Nanosynthesized calcium-silicate-based biomaterials mechanisms prevent bacterial invasion, while empty dentinal tubules, in the casewhen pulp is already necrotic, allow rapid colonization of the pulp (Baumgartneret al., 2008). The pulpal tissue collapses and becomes necrotic due to scarce vas-cular supply through the apical foramen and overload with inflammatory and bac-terial cells within the root canal.The microbial community is located in avascular necrotic space, so they areprotected from host immu ne mechanisms and antimicrobial agents. Therefore, theimmune system will not be able to act until the infection reaches the root apexand periapical tissue s. However, in the meantime, the microbial community maybecome well organized and stable. The homeostasis of biofilm will remain con-stant until the eventual remarkable changes in environmental conditions occur(Siqueira et al., 2012).11.5.5 BACTERIAL DIVERSITY IN ENDODONTIC BIOFILMSOral microorganisms include almost 1000 different bacterial species (Paster et al.,2006; Paster and Dewhirst, 2009). However, more than half of them have not beencultivated, characterized, and named so far (Munson et al., 2002; Tronstad, 2009;Sakamoto et al., 2006). Also, analysis revealed that only around 200 of them maybe present at the same time in the oral cavity of one person (Paster et al., 2006).Recent evidence demonstrated that more than 400 different microbial specieshave been identified from teeth with endodontic apical disease (Siqueira and Rocas,2009a). Primary intraradicular infection is distinguished by the presence of 1030species of anaerobic bacteria in each root canal (Siqueira and Rocas, 2005). Also,the number of bacterial cells in each canal ranges from 103to 108(Sakamoto et al.,2007). These results demonstrate that high variability of oral microbes presented indifferent individuals cause heterogeneous etiology of periodontal apical disease.Molecular studies revealed that the most common microorganisms in endodonticinfections belong to following divisions (or phyla): Firmicutes, Bacteroidetes,Actinobacteria, Fusobacteria, Proteobacteria, Spirochaetes, Synergistetes,SR1,TM7,Chloroflexi, Deinococcus, Acidobacteria,andCyanobacteria (Siqueira and Rocas,2009a; Munson et al., 2002; Siqueira et al., 2005; Sakamoto et al., 2006, 2008).Recently, bacteria from Aquificae, Nitrospira, Planctomycetes,andThermomicrobiadivisions have been discovered in the oral environment by DNA microarray method,although they have not been identified yet (Huyghe et al., 2008; Dahlen, 2009).Almost 90% of root canal microbes are obligate anaerobes. Aerobic bacteriaused to be found in the initial phases of biofilm formation although they might beintroduced during endodontic treatment (Cohenca and Lez Amaro, 2014). Studieswhich use cultivation methods for isolation and identification of endodonticmicrobes demonstrate that Gram-negative anaerobic bacteria are most commonlyobserved in root canals of infected teeth (Siqueira and Rocas, 2009b).Microorganisms from phylum Spyrochaete proved to be often present in clinicalisolates from endodontic infections, and they are divided into following twogroups: saccharolitic (Treponema pectinovorum, T. socranskii, T. amylovorum,T. lecithinolyticum, T. maltophylum,andT. parvum) and asaccharolitic (T. denticola,29311.5 Microbiological Profile of Root Canals Associated T. medium, T. putidum,andT. vincentii)(Baumgartner et al., 2003). Fusobacteriumnucleatum, Dialister pneumosintes,andD. invisus are also commonly observed inspecimens from primary asymptomatic root canal infections and in endodonticabscesses (Siqueira and Rocas, 2009a). It is stated that F. nucleatum is a strategicmicroorganism that enables interactions and coaggregation with many speciessuch as Prevotella oralis, Streptococcus anginosus, Enterococcus faecalis,andPeptostreptococcus anaerobius (Khemaleelakul et al., 2006). Advanced molecularmethods revealed Prevotella and Porphyromonas species in higher frequencies(Prevotella endodontalis, P. gongivalis, P. intermedia, P. nigrescens, P. tannerae,P. multissacharivorax,andP. baroniae)(Sakamoto et al., 2006). These microbesmay be present in subgingival plaque and are strongly associated with periodontaldisease and may also cause endodontic infections (Neskovic, 2008; Goncalves et al.,1999). Tannarella forsythia (Bacteroides forsythus) is microorganism associatedwith periodontal disease which has never been detected in root canal specimens bycultivation methods. However, molecular methods confirm its frequent presence ininfected root canals (Fouad et al., 2002). Gram-positive species are also discoveredin high or moderate frequencies in primary endodontic infections (Pseudoramibacteralactolyticus, Filifactoralocis, Actinomyces species, Propionibacterium propionicum,Olsenellaulli, Parvimonas micra, S. oralis, S. mitis, S. sanguinis, Campylobacter rec-tus, C. gracilis, Catonellamorbi, Veillonellaparvula, Eikenella corrodens, Neisseriamucosa, Centipeda periodontii, Granulicatella adiacens, Gemella morbillorum,Capnocytophaga species, and anaerobic Lactobacilli)(Siqueira and Rocas,2003ad, 2004).Secondary root canal infections are triggered by microbes introduced duringendodontic treatment and such infections are mostly responsible for the treatmentfailure. Following Gram-positive microbes are most frequently connected withsecondary and persistent infections due to their resistance to antimicrobial factors:Enterococcus, Streptococcus, Lactobacillus, Actinomyces, Peptostreptococcus,Candida, Eubacterium alactolyticus, Propionibacterium propionicum, Dialisterpneumosintes, and Filifactoralocis (Sedgley and Jacinto, 2014).Although most endodontic infections are caused by bacteria, nowadays,researchers and clinicians are aware of possible fungal and viral involvement inthe etiology of endodontic apical disease (Baumgartner et al., 2000; Slots et al.,2003, 2004; Sabeti et al., 2003).11.6 MANAGEMENT OF TEETH WITH NECROTIC PULPSAND IMMATURE ROOT DEVELOPMENTEndodontic treatment and disinfection of a necrotic and infected imma ture rootcanal represent a real challenge since they face numerous difficulties. Root canalsof immature teeth are wide and usually in the shape of a funnel, which meansthey are often wider apically than coronally; because of this it is very difficult inimmature teeth to properly remove all necrotic tissue from dentine canals walls294 CHAPTER 11 Nanosynthesized calcium-silicate-based biomaterials and to accomplish adequate obturation (Andreasen et al., 2007). Wide apical fora-men and absence of the apical stop may cause trauma of periapical tissues withroot-filling material, jeopardizing favorable outcome of the treatment due to aleakage (Trope, 2008). A special issue, regarding endodontic treatment of imma-ture teeth, is represented by their thin dentinal walls which are prone to fractureduring treatment or following obturation (Garcia-Godoy and Murray, 2012 ).Because of thin and weak dentinal walls of immature root canal systems, lightbiomechanical instrumentation is recommended (Andreasen et al., 2007). Overthe past decades, the most commonly used material for intrac anal med ication wasCH; this shows strong antibacterial efficacy due to its high pH 5 12.5, bioacti-vity—hard dental tissue formation, and capacity to dissolve necrotic pulp tissue(Georgopoulou et al., 1993; Turkun and Cengiz, 1997). However, recentevidences state its disadvantage; it influences mec hanical properties of dentine,weakening its resistance to a fracture, formation of a necrotic layer in contactwith vital pulp tissue, reduction of pulp volume, and changes in fibroblasts thathave been in contact with this material (Andreasen et al., 2002; Yoldas et al.,2004; Petrovi´c et al., 2008). On the other hand newly synthesized bioceramicmaterials, such as hydroxyapatite, have a similar ratio of inorganic ions to that ofbone tissue or dentin, therefore they are widely accepted in endodontic treatmentof the immature teeth (Markovi´c et al., 2007; Petrovi´c et al., 2009). Also, MTA isgradually replacing CH as an apical plug or root canal obturation mater ial, andthe following advantages made it a good alternative: biocompatibility, nonmuta-genicity, good adaptation to dentin walls, low microleakage and bioactivity, withno adverse effect on dentinal fracture resistance (Moore et al., 2011; Torabinejadet al., 1993, 1995a; Bates et al., 1996; Murray et al., 2003;´Cetenovi´c et al.,2013). Utilization of modern technologies and development of new materials sup-port the apexogenesis during root canal treatment in immature teeth. One of therecently applied protocols is the use of triple antibiotic paste in the treatment ofteeth with immature root development, necrotic pulpal tissue, and apical disease.Although this procedure delivers favorable therapeutic outcomes, due to a poten-tial bacterial resistance and sensibilization it is associated with controversialexperimental and clinical results.The recent discovery of dental pulp stem cells and dental s tem cells fromapical papilla enable a better understanding of immature teeth further matura-tion following root canal dental treatment, allowing rapid p rogress in the fieldof endodontic regenerative procedures (Gronthos et al., 2000; Sonoyama et al.,2008). Recent evidence shows that pulp, periodontal tissue, vascular andimmune cells participate and en able endodontic regeneration (Lovelace et al.,2011). Although reg ene r ati on of necr o tic and infected pulp was consideredimpossible, current literat ure data all ow this possibility, provided an adequatetechnique is applied, including a proper root canal disinfection and coronal seal(Banchs and Trope, 2004; Chueh and Huang, 2006; Iwaya et al., 2001, 2011).The revascularization procedure may be improved by using a scaffold, althoughthis technique still aw aits FDA a pproval and clinical trials ( Garcia -Godoy andMurray, 2012).29511.6 Management of Teeth With Necrotic Pulps 11.7 USING CALCIUM-SILICATE-BASED MATERIAL ASAN APICAL PLUG IN THE TREATMENT OFTEETH WITH NECROTIC PULPS AND IMMATUREROOT DEVELOPMENT11.7.1 ENDODONTIC PROCEDUREBefore starting a procedure, immature teeth with nonvital pulps should be identi-fied using clinical and radiographic criteria. Also, it is considered necessary toobtain comprehensive medical and dental history prior to examination. Duringclinical and radiographic examination the following issues have to be determined:periodontal tissue status, oral status, width of root canal and its walls.Comprehensive clinical assessment of a young patient is essential because pro-longed and unpleasant dental treatment may cause anxiety and lack of coopera-tion, which may compromise the entire treatment.Current recommendations regarding the endodontic procedure in immatureteeth include gentle debridement of the root canals due to thin dentinal walls andpossible posttreatment fracture (Andreasen et al., 2007; Garcia-Godoy andMurray, 2012). Considering the fact that excessive removal of healthy tissue mayresult in increased tendency to fractures, it is proposed to use K-files only to elim-inate the necrotic tissue and softened layer of dentin. Recent case reports whichpresent management of immature teeth with abscesses and periapical lesions dem-onstrate root maturation after conservative treatment even when extensive puru-lence is noted (Iwaya et al., 2001; Banchs and Trope, 2004; Chueh and Huang,2006). Banchs and Trope (2004)advise delicate curettage of the periapical areausing barbed broaches in order to partially destruct the periapical lesion and pro-voke bleeding. Disinfection protocol of immature teeth with infected root canalsinvolves usage of 2.5% sodium hypochlorite, 0.12% chlorhexidine gluconate, andFIGURE 11.12The use of MTA in the treatment of immature teeth and extensive periapical lesions:(a) extensive periapical lesion in tooth with incomplete root development; (b) placement ofMTA apical plug.296 CHAPTER 11 Nanosynthesized calcium-silicate-based biomaterials 40% citric acid ( Huang, 2009). Recommended root canal dressings are triple anti-biotic paste (for a week) and CH (for 4 weeks). Following that time period, thepatient should be re-evaluated; if asymptomatic, the root canal should be irrigatedwith sterile saline and then dried carefully with sterile paper points.For final obturation of the apical parts of root canals MTA may be used, alwaysfollowing the manufacturer’s instructions during its mixing and application in orderto create an apical plug of 35mmofthickness(Figure 11.12). The remaining partof the canal should be filled with adequate sealer and gutta-percha points. Coronalparts of the root canal system should be sealed with a glass-ionomer cement (mini-mum thickness 1.52 mm), and should then be restored with adequate material.11.7.2 CLINICAL EVALUATIONPeriodical follow-up is essential since it will enable evaluation of the treatmentand provide observation of the tooth maturation. The following data should beobtained during examinations: presence and description of spontaneo us or pro-voked pain, tenderness to percussion, tenderness to palpation of the apical area,presence of a sinus tract or abscess, and discoloration of tooth. Though duringrecent decades, extensive periapical lesions in immature tooth have been consid-ered as an indication for apexification procedure, it might be possible that dentalpulp stem cells and dental stem cells from apical papilla survive enabling continu-ous root canal development. However, if clinical symptoms persist, than the apex-ification procedure seems to be a likely solution (Figures 11.1311.15).FIGURE 11.13A periapical radiograph of lower second premolar in a 12-year-old patient: (a) thebeginning of the therapy, presenting large periapical lesion and incomplete rootdevelopment; (b) radiographic examination after follow-up period of 36 months showingcomplete resolution of periapical pathosis and formation of partially calcified apical bridgewithout any clinical symptoms.29711.7 Using Calcium-Silicate-Based Material FIGURE 11.14Radiograph showing the case of a 10-year-old patient: (a) large periapical lesion in lowerincisor with incomplete root development; (b) 12 months after endodontic treatmentpresenting resolution of the periapical lesion without any clinical symptoms.FIGURE 11.15Clinical case presenting a 10-year-old patient: (a) periapical radiograph revealing diffuseperiapical lesion associated with upper first incisor with incomplete root development atthe beginning of the therapy; (b) after 12 months no clinical symptoms were observed andslightly extruded MTA did not obstruct regeneration of periapical tissues. 11.8 CONCLUSIONSMaintaining pulp vitality during tooth development is of the utmost importance.In the case of root canal infection and apical disease development, the longertime taken for infection to develop, the less stem cells and surviving pulp tissueremain. In such a case, it seems more desirable, that eventual apexogenesis is aless possible treatment option. In clinical practice, treatme nt and rehabilitationprocedures in immature root canal infection or early tooth loss are commonlyassociated with many particularities due to the specific morpholog y of the imma-ture root canal system of children and adolescents. Unfortunately, clinical studiesevaluating the success rate of endodontic treatments in immature teeth with peria-pical lesions are scarce. Development of appropriate prevention program designsand well-defined treatment protocols will certainly improve the final outcome.After such a challenging treatment is completed, it is necessary to performadequate teeth obturation with suitable materials. 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