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Endodontic Topics 2015, 32, 4785

All rights reserved

2015 John Wiley & Sons A/S.

Published by John Wiley & Sons Ltd

What do different tests tell about

the mechanical and biological
properties of bioceramic materials?

A wide selection of commercial bioceramic (MTA-type) materials is now available for dentin replacement, pulp
capping, pulpotomy, creation of apical barriers in teeth with open apices, repair of root perforation and
resorptive defects, as well as orthograde or retrograde root canal fillings. A variety of experiments with
different models and methods employed by researchers in the endodontic community have been developed for
studying the properties of bioceramic materials in order to understand and predict their bioactive behavior and
how they fulfill the mechanical, chemical, and microbiological goals set to these materials. Objective criteria
for testing bioceramic materials should be developed by the International Organization for Standardization
(ISO) or the American Society for Testing and Materials (ASTM) to accurately describe the biological,
chemical, and mechanical characteristics of the bioceramic cements and sealers that are available for use in
various endodontic applications. This article is an overview of those methods and devices that have been and
will be used in the literature for testing bioceramic materials.
Received 13 April 2015; accepted 17 April 2015.

Bioceramic materials are described as ceramic products or components employed in medical and dental
applications, mainly as implants and replacements,
that have osteoinductive properties (1). The first bioceramic hydraulic calcium silicate-based cement
(HCSC) patented for endodontic applications was
mineral trioxide aggregate (MTA; Dentsply Tulsa
Dental Specialties, Johnson City, TN, USA) (2),
which is essentially a derivative of Portland cement.
It has attracted considerable attention (35) owing
to its excellent sealing ability, biocompatibility,
regenerative capabilities, and antibacterial properties
(3,4,68). The main hydraulic components in
HCSCs are tricalcium silicate (Ca3SiO5 or C3S) and
dicalcium silicate (Ca2SiO4 or C2S). HCSCs have
been widely used as both endodontic repair materials
and dentin substitutes (9). An increasing number of

publications report that these cements produce an

apatite-rich surface layer after they contact simulated
body fluids (5,6,10). Several HCSC-based root
repair materials have been developed following the
introduction of MTA and are available clinically for
dentists. These include ProRoot (Dentsply Tulsa
Dental Specialties) and MTA Plus (Prevest-Denpro,
Jammu City, India) (Table 1). Calcium phosphate
silicate cement (CPSC) is a new generation biological cement that consists of phosphate salts in addition to hydraulic calcium silicates. The reason for its
development was the expectation that the hydration
process would enhance the cements mechanical
properties and biocompatibility (11). As examples of
CPSCs (12), BioAggregate (Innovative Bioceramix,
Vancouver, Canada), EndoSequence Root Repair
Material Putty (ERRM Putty; Brasseler USA, Savannah, GA, USA), EndoSequence Root Repair Material Paste (ERRM Paste; Brasseler USA), and iRoot


Shen et al.
Table 1: List of bioceramic cements for endodontic use



White ProRoot Mineral

Trioxide Aggregate

Dentsply Tulsa Dental

Specialties, Johnson
City, USA

Powder: tricalcium silicate, dicalcium silicate, bismuth oxide, tricalcium

aluminate, calcium sulfate dihydrate or gypsum
Liquid: water

Gray ProRoot Mineral

Trioxide Aggregate

Dentsply Tulsa Dental

Specialties, Johnson
City, USA

Powder: tricalcium silicate, dicalcium silicate, bismuth oxide, tricalcium

aluminate, calcium sulfate dihydrate or gypsum, calcium aluminoferrite
Liquid: water

Nano White Mineral

Trioxide Aggregate

Kamal Asgar Research

Center, Tehran, Iran

Powder: tricalcium silicate, dicalcium silicate, bismuth oxide, tricalcium

aluminate, calcium sulfate dihydrate or gypsum, disodium hydrogen
phosphate, strontium carbonate, zeolite
Liquid: water

MTA Angelus

~ es
Angelus Soluco
Londrina PR, Brazil

Powder: tricalcium silicate, dicalcium silicate, bismuth oxide, tricalcium

aluminate, calcium oxide, aluminum oxide, silicon dioxide
Liquid: water

MTA Plus

Jammu City, India
Avalon Biomed Inc.,
Bradenton, FL, USA

Powder: similar to white ProRoot MTA

Liquid: water-based gel with water-soluble thickening agents and

Gray MTA Plus

Jammu City, India
Avalon Biomed Inc.,
Bradenton, FL, USA

Powder: similar to gray ProRoot MTA

Liquid: water-based gel with water-soluble thickening agents and

Mechanically Mixed
Mineral Trioxide
Aggregate (MM-MTA)

Besancon, France

Powder: a modified Portland cement with added calcium carbonate

Liquid: water

Calcium enriched mixture

cement (CEM)

Bionique Dent,
Tehran, Iran

Powder: different compounds of calcium, including oxide, sulfate,

phosphate, carbonate, silicate, hydroxide, and chloride compounds
Liquid: water-based solution

Endocem MTA

Maruchi, Wonju,
Republic of Korea

A silicate-based material containing fine-size particles of pozzolan

Endocem Zr

Maruchi, Wonju,
Republic of Korea

A zirconium oxide-containing white MTA-like material

Retro MTA

BioMTA, Seoul,
Republic of Korea

Powder: calcium carbonate, silicon oxide, aluminum oxide, hydraulic

calcium zirconia complex
Liquid: water

Ortho MTA

BioMTA, Seoul,
Republic of Korea

Powder: tricalcium silicate, dicalcium silicate, bismuth oxide, tricalcium

aluminate, free calcium oxide, calcium aluminoferrite
Liquid: water

Tech Biosealer capping

Isasan, Rovello
Porro, CO, Italy

Powder: white Portland cement, calcium chloride, montmorillonite,

calcium sulfate
Liquid: Dulbeccos Phosphate-Buffered Solution (DPBS)

Tech Biosealer root end

Isasan, Rovello
Porro, CO, Italy

Powder: white Portland cement, calcium chloride, montmorillonite,

calcium sulfate, sodium fluoride, bismuth oxide
Liquid: Dulbeccos Phosphate-Buffered Solution (DPBS)


Bisco Inc.,
Schaumburg, IL,

45% wt. mineral material (type III Portland cement), 10% wt. radiopaque
component, 5% wt. hydrophilic thickening agent (fumed silica), 45%
metacrylic resin


Mechanical and biological properties of bioceramic materials

Table 1: Continued




Septodont, SaintMaur-des-fosses
Cedex, France

Powder: tricalcium silicate, dicalcium silicate, calcium carbonate, zirconium

oxide, calcium oxide, iron oxide
Liquid: calcium chloride, a hydrosoluble polymer, water


Innovative Bioceramix,
Vancouver, Canada

Powder: tricalcium silicate, dicalcium silicate, tantalum pentoxide, calcium

phosphate monobasic, amorphous silicon oxide
Liquid: deionized water

EndoSequence Root
Repair Material Paste
(ERRM Paste)

Brasseler USA,
Savannah, GA, USA

Calcium silicates, zirconium oxide, tantalum oxide, calcium phosphate


EndoSequence Root Repair

Material Putty
(ERRM Putty)
iRoot BP Plus Root
Repair Material

Brasseler USA,
Savannah, GA, USA
Vancouver, Canada

Calcium silicates, zirconium oxide, tantalum oxide, calcium phosphate


iRoot FS Fast Set

Root Repair Material
(iRoot FS)

Innovative Bioceramix,
Vancouver, Canada

Calcium silicates, zirconium oxide, tantalum oxide, calcium phosphate


QuickSet (QS)

Avalon Biomed Inc.,

Bradenton, FL, USA

An experimental calcium aluminosilicate material mixed with

anti-washout gel


Binderware, S~ao
Carlos, SP, Brazil

Aluminum oxide, calcium oxide, silicon dioxide, magnesium oxide,

iron oxide

FS Fast Set Root Repair Material (iRoot FS; Innovative Bioceramix) have been developed as new bioceramic materials (Fig. 1).

Endodontic sealers are used in the obturation of

root canal systems to achieve a fluid-tight or hermetic seal throughout the canal including the apical

Fig. 1. Bioceramic cements. (A) Gray MTA. (B) White MTA. (C) Biodentine. (D) ERRM Putty. (E) iRoot FS.


Shen et al.

Fig. 2. Bioceramic sealers. (A) EndoSequence BC Sealer. (B) MTA Plus. (C) BioRoot RCS.

foramen and canal irregularities and minor discrepancies between the dentinal wall of the root canal
and the core filling material (13). Therefore, sealers
help prevent leakage, reduce the possibility of residual bacteria from the canal invading the periapical
tissues, and resolve the periapical lesion (13,14).
According to Grossman (15), an ideal root canal sealer should provide the following: an excellent seal
when set, dimensional stability, a slow setting time
to ensure sufficient working time, insolubility to tis-

sue fluids, adequate adhesion with canal walls, and

biocompatibility. Nowadays, various kinds of endodontic sealers are available, including sealers based
on glass ionomer, zinc oxideeugenol, resin, calcium
hydroxide, silicone, and bioceramic-based root canal
sealers (Fig. 2). In particular, bioceramic-based sealers (Table 2) that usually contain calcium silicate
and/or calcium phosphate have attracted considerable attention because of their physical and biological properties such as their alkaline pH, chemical

Table 2: List of bioceramic and hybrid sealers for endodontic use




EndoSequence BC Sealer
(iRoot SP root
canal sealer)

Brasseler USA, Savannah, GA,

USA (Innovative Bioceramix,
Vancouver, Canada)

Tricalcium silicate, dicalcium silicate, zirconium oxide, colloidal

silica, calcium phosphate monobasic, calcium hydroxide


Isasan, Rovello Porro, CO, Italy

Powder: white Portland cement, calcium chloride,

montmorillonite, sodium fluoride, bismuth oxide
Liquid: Dulbeccos Phosphate-Buffered Solution (DPBS)

BioRoot RCS

Septodont, Saint-Maur-des-fosses
Cedex, France

Pure mineral formulation and resin-free


EGEO SRL, MTM Argentina SA,

Buenos Aires, Argentina

Powder: tricalcium silicate, tricalcium oxide, tricalcium aluminate

and other oxides
Liquid: saline solution and calcium chloride

ProRoot Endo

Dentsply Tulsa Dental Specialties,

Dentsply/Maillefer, Ballaigues,

Powder: tricalcium silicate, dicalcium silicate, calcium sulfate,

bismuth oxide, tricalcium aluminate
Liquid: viscous aqueous solution of water soluble polymer

MTA Fillapex

~es Odontol
Angelus Soluco
Londrina PR, Brazil

After the mixture: MTA, salicylate resin, natural resin, diluting

resin, bismuth oxide, nanoparticulated silica, pigment

MTA Plus

Prevest-Denpro, Jammu City, India

Avalon Biomed Inc.,
Bradenton, FL, USA

Powder: similar to white ProRoot MTA

Liquid: a proprietary salt-free polymer gel and water

MTA Obtura

Angelus, Angelus Odontologica,

Londrina, PR, Brazil

Powder: Portland cement clinker and bismuth oxide

Liquid: a liquid resin

Pure bioceramic sealers

Hybrid bioceramic sealers


Mechanical and biological properties of bioceramic materials

stability within the biological environment, and lack
of shrinkage. Hybrid bioceramic sealers are defined
as a resin-based sealer with a bioceramic component.
They are also non-toxic and biocompatible (1618).
Despite the advertising claims of superiority of
those bioceramic materials, few have been supported
by objective well-designed studies in the literature. A
variety of models and methods have been used to
investigate the properties and behavior of bioceramic
cements. This article aims to present an overview of
these methods and devices that have been used in
the endodontic literature for studying bioceramic
cements. We attempt to explain how differences in
experimental methods may affect the mechanical and
biological properties, as well as to provide cuttingedge information on recent developments.

Tests for mechanical (physical)

It has been reported that investigations of simple
mechanical tests allow a correlation of mechanical
properties with clinical performance (19) and can
advise clinicians as to which cements need special
care or entail particular risks during treatment.

Bioceramic cements
Setting time
The setting time is defined as the length of time for
a material to transition from a fluid state into a hardened state (17). The setting time is one of the most
relevant factors clinically. A long setting time could
cause clinical problems because of the cements
inability to maintain shape and tolerate stresses during this time period. Most of the hydration of bioceramic cements occurs during the first several weeks,
although complete hydration may even take 1 or 2
years (5,10). An important factor in non-surgical as
well as surgical restorative repair in endodontics is to
achieve a fluid-tight seal between the tooth and the
repair material. In most cases a bioceramic material
is the restorative material of choice. In many cases,
dentists must allow time for MTA to set, which has
been reported to be anywhere between 75 minutes
(20) and 4 hours (21), and even up to 72 hours
(22), before proceeding to the next step in the procedure. The slow setting also results in challenging

handling characteristics (20,23), which may compromise the application as well, especially in supracrestal
areas. The possibility of the material being washed
out at the cervical/furcal area during a long setting
time needs to be considered. Irrigation of an area
containing newly placed MTA can lead to washout
of the material. In addition, early occlusal pressure
directed to the material, even in a deeper location,
may compromise the integrity of the seal.

Test methods
Several methods have been used to determine the
setting time of bioceramic cements; the terms initial
and final setting are also used. The ISO 6876 (24)
and ISO 9917-1 standards (25) use flat cylindrical
indenters of two sizes and two weights. The lighter
weight and larger diameter needle is specified in
ISO 6876 (24) for root canal sealers, and the heavier weight and smaller diameter needle is specified
for water-based cements in ISO 9917-1 (25). The
setting of bioceramic materials requires water; drying of the specimen must be avoided. The physical
properties of hydraulic cements depend on the water
to cement ratio. The materials are mixed and compacted into stainless steel molds (d = 10 mm and h
= 2 mm). Usually, specimens are wrapped in pieces
of gauze soaked in sterile distilled water. The whole
assembly is then transferred to an incubator (37C,
> 95% relative humidity). The ISO 6876 (24) setting procedure also requires adding excess water to
the sealers that require water for setting. Therefore,
for the pre-mixed (calcium phosphate silicate)
cements e.g. ERRM Putty and ERRM Paste (11),
which require continuous exposure to moisture during setting, a plaster of Paris mold with a cavity is
required. The mold is first stored at 37C in a water
bath for 24 hours, and then the calcium phosphate
silicate cement is poured into the mold. The whole
assembly is then stored in a water bath at 37C.
The setting time assessment tests are based on the
visual inspection of a Gillmore needles impression
into a cement surface. ASTM C266 (26) for Portland cements specifies the initial and final setting
time to be determined with a lighter, larger diameter
needle and then with a heavier, smaller diameter
needle. The Gillmore needle for testing the initial
setting time has a weight of 113.4 g and an active
tip of 2.12 mm in diameter (initial needle). The


Shen et al.
needle for the final setting time has a weight of
453.6 g and an active tip of 1.06 mm in diameter
(second needle) (27). The initial needle is applied
carefully, avoiding pressure on the surface of each
sample. For each sample, the time that elapsed
between the end of mixing and the unsuccessful
indentation is recorded in minutes and defined as
the initial setting time. The final setting time is
determined following the same procedures using the
second needle, with the 400 g load. The quantity of
material tested affects the results of setting time testing, which has also made comparison of data difficult
since most investigators adapt molds for testing
rather than using the precise mold specified by the
particular standard (2831). The ASTM C266-08
standard is used to determine the setting time; however, the use of molds according to the ISO
6876:2012 standard is established due to using a
reasonable amount of cement (32).

Many manufacturers have attempted to shorten the
setting time of hydrated MTA by introducing a
strong calcium-based electrolyte into the reaction
that completely dissociates in solution in order to
produce calcium ions to accelerate the dissolution
step. Calcium chloride (CaCl2) is often used to
improve the properties of Portland cement. It is
the main component of the additives of civil construction known as accelerators. The addition of
CaCl2 aims to reduce the setting time of Portland
cement and improve its physicochemical properties
(33). The setting of MTA is significantly accelerated by highly soluble calcium compounds (e.g.
CaCl2, calcium nitrate, or calcium formate)
(34,35). Wiltbank et al. (35) found that CaCl2 and
calcium formate with white-colored MTA (WMTA)
shortened the initial setting time to 35 minutes.
Kogan et al. (34) added various additives such as
chlorhexidine (CHX), K-Y Jelly (Johnson & Johnson, New Brunswick, NJ, USA), NaOCl gel, and
CaCl2 to the gray-colored ProRoot MTA powder
(Dentsply Tulsa Dental, Tulsa, OK, USA). After
mixing with water, the reagents used decreased the
setting time, which dropped from the original 50
minutes down to 20 minutes. However, there was
also a 3140% reduction in the compressive


In a study by Stowe et al. (36), chlorhexidine gluconate (0.12%) enhanced the antimicrobial effect of
white MTA as compared to MTA mixed with sterile
water. Kogan et al. (34) found that MTA mixed
with CHX gel did not set until the end of the observation period (4 h). In the clinical setting, saline and
local anesthetics are often mixed with MTA when
sterile water is not available. The same group (34)
also found that saline extended the setting time to
90 min. The setting time was more than double for
MTA mixed with 2% lidocaine compared to that
mixed with water.
Another less-than-ideal property of MTA is its handling characteristics. The manufacturer recommends
mixing MTA with sterile water. This produces a
grainy, sandy mixture that is sometimes challenging
to deliver to the required site and to compact adequately. The desire to change some of the properties
of MTA, especially handling and set time, has been
discussed (20,23,37). However, it is not known
whether altering its makeup would change the physical properties that make MTA so successful clinically.
One characteristic whose change would be of concern
is dimensional stability because it is likely related to
its ability to seal well. MTA has been shown to
expand slightly after setting fully (20). A change of
this property, possibly leading to contraction, would
likely have a negative impact on MTAs ability to seal
a root end or perforation site. However, if an altered
MTA were to expand to a greater degree, then delicate root ends might be at risk for fracture. Neither
scenario would be clinically acceptable.
Radiopacity was achieved for ProRoot MTA by
the addition of bismuth oxide (Bi2O3) to MTA powder (Portland cement) (2). MTA Angelus (Angelus
Dental Industry Products S/A, Londrina, PR, Brazil) is produced by adding Bi2O3 to white Portland
cement clinker to improve its radiopacity. Lower levels of radiopacifier (10.5%) are added to MTA Angelus powder (38). Portland cement clinker is made by
heating a homogeneous mixture of raw materials in
a rotary kiln at high temperature. Also, the material
does not contain calcium sulfate (gypsum) in its
composition in order to reduce the setting time.
Grazziotin-Soares et al. (27) studied the setting time
of white MTA Angelus and white MTA Angelus
without Bi2O3. The results showed that the mean
initial setting time for white MTA Angelus (18.3 
7.5 min) was longer than for white MTA Angelus

Mechanical and biological properties of bioceramic materials

without Bi2O3 (10 min). Bi2O3 in white MTA
Angelus (165  31.6 min) was associated with a significantly longer final setting time compared to white
MTA Angelus without Bi2O3 (38.3  9.8 min).
Biodentine is a newly developed tricalcium silicate
cement. The powder is composed of a tricalcium silicate cement as the main component, which makes it
similar to MTA. Small proportions of dicalcium silicate, calcium carbonate, and zirconium oxide as a
radiopacifier have also been added. The liquid for
mixing with the cement powder consists of calcium
chloride (as an accelerator to reduce setting time)
and a hydrosoluble polymer (39). Grech et al. (40)
reported an initial setting time of < 20 minutes and
a final setting time of 45 minutes. The same study
reported a high washout by water during the setting
for Biodentine in comparison to some other
cements. The relatively short initial setting time
makes it possible to use Biodentine as a dentin substitute in underfilling during restorative treatment.
Recently, iRoot FS Fast has been introduced for
use as a root canal repair material, as a fast-setting
white hydraulic pre-mixed bioceramic paste (http://
www.ibioceramix.com/products.html). iRoot FS is
an insoluble, radiopaque and aluminum-free material
based on calcium silicate, which requires the presence of water to set and harden. A quickly setting
cement could allow for a reduction in chair-side time
and the number of visits needed per treatment.
However, the fundamental properties of this
improved performance material are still unknown.
One study (41) evaluated the setting time of iRoot
FS, ERRM Putty, GMTA, WMTA, and intermediate
restorative material (IRM). The initial setting time of
iRoot FS, as defined by ASTM International Standard C266-03, was 18 minutes, which is less than
half of WMTA. iRoot FS was the only material of
the four bioceramic cements tested with a final setting time just below 1 hour.
Although excellent materials, bioceramic cements
could be even more useful if their initial and final
setting times would be shorter. When appropriate, a
dentist could complete a case in one appointment
and finalize the restoration.

Microhardness test
Indentation hardness as a principal parameter for the
mechanical characterization of materials has been

commonly used as a technique to measure mechanical properties of materials. Surface microhardness

provides some indication of the surface strength of
the material (42). The microhardness of a material is
not a measure of a single property. It is influenced
substantially by other fundamental properties of the
material, such as yield strength, tensile strength,
modulus of elasticity (43), and crystal structure stability (44). Thus, it can be used as an indicator of
the setting process and the overall strength or resistance to deformation when compared with baseline
information. It can also indicate the effect of various
setting conditions on the overall strength of a material (45).

Microhardness has been tested for bioceramic products, but is not required for endodontic materials by
ISO or ADA standards. Microhardness testing is performed by applying a load to a material surface using
a diamond indenter. There are two universal types of
microhardness test, Vickers and Knoop. The main
difference is attributed to the shape of the diamond
indenter. The shape of the Vickers diamond indenter
is a square pyramid whereas the shape of the Knoop
diamond indenter is an elongated pyramid shape.
Gong et al. (46) measured silicon nitride ceramic
samples and showed that Knoop hardness values
were generally lower than the corresponding values
for Vickers hardness. However, there is a strong correlation between these two methods that may be
related to elastic recovery occurring during the
indentation. The Vickers test is the more commonly
used of the two. The load and dwell time used are
selected according to the materials to be tested by a
pilot test showing that the load is able to create a
clear and reliable indent in all materials. Different
loads have been employed for different conditions
(4749). Bioceramic cements are composed of multiple phases of varied hardness that adds variation to
hardness measurements depending on where the
small diamond indenter is placed on the sample.
Two methods of Vickers microindentation hardness measurement are in practical use. In one
method, a hard indenter (i.e. a Vickers diamond pyramid) penetrates into the solid with a constant load
(Fig. 3). After unloading, the dimensions of the
indentation in the solid are measured and the


Shen et al.

Fig. 3. Device for Vickers and Knoop microhardness test. (A) General view. (B) Magnified view of the working
stage under the microscope.

hardness, defined as the ratio of the load to the facet

contact area of the indentation, is determined. This
method is known as conventional indentation; it is
commonly used for microhardness testing of bioceramic cements (48,49). One general disadvantage of
this method pertains to the facet contact area measured after the load is removed. It has been argued
that the area may be susceptible to elastic recovery.
Secondly, at loads of a few grams, the indentation
dimensions are about a few micrometers and the
penetration depths into the solid are about an order
of magnitude smaller. It is difficult to measure such
dimensions optically with a high degree of accuracy.
Therefore, conventional indentation hardness
obtained from microscopic observation of the
indented cavity cannot give reliable mechanical properties of materials. The second method for determining the mechanical properties of solids is the depthsensing indentation or dynamic indentation method
(Fig. 4). This method offers great advantages over

the conventional indentation test in two aspects.

First, apart from hardness (or strength), the method
can provide well-defined mechanical parameters such
as the elastic modulus (Fig. 5) of the interfacial
zone. Second, the load and depth of an indentation
are continuously monitored and optical observation
and measurement of the diagonal lengths of the
indent/impression, which can be difficult and subjected to inaccuracy, are no longer required (50).
Difficulties in perforation repair are associated with
the physiological and chemical environment at the
perforation site. As the root repair material is usually
placed in root canals where an acidic environment
may have been generated by bacteria or inflammation, a low pH becomes a factor that may reduce the
hardness and weaken the ultrastructure of the perforation repair materials (47,51). In order to simulate
infectious and normal in vivo conditions, butyric
acid and phosphate-buffered saline (PBS) have been
used. Butyric acid is one of the by-products of the

Fig. 4. Device for dynamic microhardness test. (A) General view. (B) Magnified view of the working stage.


Mechanical and biological properties of bioceramic materials

Fig. 5. Dynamic microhardness test under loadunload mode of elastic modulus for Biodentine hydrated in PBS
for 4 days.

metabolism of anaerobic bacteria, the dominant bacteria in endodontic infections (52). Therefore, to
simulate infectious situations in laboratory studies,
the use of butyric acid has been suggested (47,53).
PBS is a simulated tissue fluid containing phosphate
that can be used for the purpose of mimicking normal in vivo conditions in laboratory studies (42,54).
The test surface is polished prior to the microhardness measurement in order to obtain a smooth,
scratch-free area that allows for a more reliable measurement of the indentations (55). However, the
polishing procedure inevitably removes the most
superficial layer that was directly exposed to the acid
environment, resulting in higher microhardness values on the polished surface. Conversely, the pressure
from polishing may lead to the formation of subsurface microdefects that may negatively interfere with
the surface hardness and therefore compensate for
the hardness increase caused by polishing (56). Polishing may also result in sample thickness reduction.
Vickers hardness (HV) is a calculated using the following equation in accordance with the ASTM E384
standard for Vickers microhardness:
HV 1:854  F =d 2
where F is the load (kg1) and d is the mean of the
two diagonals produced by the indenter in millime-

ters. There is no relationship between the initial thickness of the cement and the microhardness values.

Various mixing techniques

MTA is a type of hydraulic cement that can set in
the presence of water. In simple terms, hydraulic
cements are finely ground materials (powders) that,
when mixed with water, gradually or instantly set
and harden in air or in water; the reaction results in
the formation of hydrated compounds whose
strength increases with time (57,58). The hydration
process is a complex phenomenon that, if modified,
might influence the biological, chemical, and physical properties of the resulting product (57).
ProRoot MTA and MTA Angelus are available as
powder and water systems. When one component
in the system is a liquid, the achievement of a
homogenous mixture becomes less predictable (59).
In addition, depending on the setting and hardening mechanisms of the material, manual mixing may
result in inconsistencies that could be prevented by
mechanical mixing of the constituents (60).
Mechanical mixing (in a mixer) has the potential to
reduce air spaces between adjacent particles, resulting in a more thorough wetting of the powder particles, and lead to an improvement in the
unification of the resultant paste (61). Trituration is


Shen et al.
one method of mechanical mixing that is used
extensively for various dental materials. Initially, the
individual components of the material are manually
placed into a mixing device. Then, in an attempt to
enhance consistency, various dental materials including amalgam, glass ionomer, self-cured composite
resin, zinc phosphate, and calcium hydroxide
cements became available in capsules containing
pre-set proportions of their components that were
then triturated prior to use. The capsule can also
contain a small rod-like pestle, which improves the
mechanical mixing. This system has the potential to
produce a consistently uniform, void- or pore-free
mixture (59). Encapsulation of pre-set proportions
of MTA powder and water appears advantageous as
it enables the powder to liquid ratio and the mixing
technique to be regularized by the manufacturer.
Nekoofar et al. (62) evaluated the influence of
various mixing procedures including ultrasonic
vibration, trituration of customized encapsulated
MTA, and condensation on the Vickers surface microhardness of white and gray MTA and MTA
Angelus. As for ultrasonic vibration, the tip of the
ultrasonic device was placed in the center of the
material and not in contact with the walls or floor
of the mold. The results demonstrated that the
application of ultrasonic energy to MTA produced a
significantly higher surface microhardness value
compared to trituration and condensation techniques at both 4 and 28 days after mixing. This
could be explained by the dispersion effect of the
ultrasonic energy on the material particles that
might provide sufficient space for water molecules
and better water diffusion, producing a greater
degree of hydration and subsequently a higher surface microhardness value. The total reactive surface
area and particle interactions are increased by ultrasonic energy and this may decrease setting time
(63,64). The findings for the condensed MTA
group may be explained by the concept that compacting the material limits the formation of microchannels, compromising the ingress of water to
hydrate the material adequately. Irrespective of mixing technique, WMTA and gray MTA Angelus had
the highest surface microhardness values using the
Vickers microhardness test. The main drawback of
MTA Angelus concerns the clinker that is produced
using a raw mix with a poor burnability with an
unsuitable thermal profile. This leads to a low


amount of tricalcium silicate and the presence of less

reactive dicalcium silicate as some calcium oxide and
silicon oxide remain unreacted. MTA Angelus with
a low surface microhardness value may be related to
the higher variation in particle size in Angelus compared to MTA (65).

Effect of the environment

Microhardness testing is based on the evaluation of
the resistance of materials to deformation. A variety
of factors such as the surrounding pH (42,4749),
material thickness (66), mixing techniques (62), condensing pressure (67), powder particle size (68),
etching (55), blood and serum contamination (69),
and temperature (70) can affect bioceramic cement
Butyric acid significantly reduced the microhardness of cements even at a neutral pH when compared
to specimens in distilled water. The possible mechanism for the lower microhardness is that butyric acid
interferes with hydroxyapatite crystal formation (53)
and the hydration reaction (71) during the setting of
the bioceramic materials. In a clinical situation, the
acidotic metabolism of butyric acid may suppress the
activity of alkaline phosphatases and the development
of bone mineralization (72). Scanning electron
microscopy has shown that a lower environmental
pH can be detrimental to bioceramic root repair
materials. The nucleation rates of the components of
MTA (tricalcium silicate, tricalcium aluminate, tricalcium oxide, silicate oxide, mineral oxide, and bismuth
oxide) may differ in terms of the crystallization of
hydrates in an interlocking mass, shown as the cubic
and needle-like crystals (73). This crystallization consists of the formation of calcium silicate hydrates (CS-H) and Portlandite (crystalline calcium hydroxide)
(42). Giuliani et al. (71) indicated that four phases
might exist in the hardened MTA cement, including
unhydrated grains, calcium hydroxide, hydration production, and pores. Specimens at low pH exhibited a
significantly lower microhardness value, and the
microstructure revealed that the crystal clusters were
dissolved, indicating structural weakening and adverse
effects on the hydration process of MTA (Fig. 6).
The needle-like crystals are believed to have an
important role in interlocking the entire mass of
material, and their disappearance can cause the material hardness to decrease (42).

Mechanical and biological properties of bioceramic materials


Fig. 6. SEM images of cross-sections of MTA and ERRM Putty exposed to butyric acid at pH 5.4 and 7.4 after 7
days of setting. (A) MTA exposed to pH 5.4. (B) MTA exposed to pH 7.4. (C) ERRM Putty exposed to pH 5.4.
(D) ERRM Putty exposed to pH 7.4. Reproduced from Wang et al., 2015 (49).

When MTA is exposed to an acidic environment,

it appears to be more sensitive than Biodentine,
which showed higher surface hardness, compressive
strength, and bond strength than MTA (74). The
influence of an acidic environment on MTA has been
attributed to the changes in its ultrastructure
(75,76). Wang et al. (49) investigated the microhardness and microstructural features of three tricalcium silicate materials (MTA, ERRM Putty, and
ERRM Paste) after exposure to a range of acidic
environments. The results showed that at pH 5.4,
MTA displayed the highest microhardness value of
23.3, followed by ERRM Putty (15.8), ERRM Paste
(11.7), and the control material IRM (5.0). When
compared to MTA, the reason for the lower microhardness values of ERRM Putty and Paste may be due
to the low pH environment that inhibited crystallization in the hydration reaction of these two materials
(53). In comparison to MTA, ERRM materials contain calcium phosphate and tantalum oxide but lack
aluminum. The absence of the aluminate phase may
result in fewer formed ettringite crystals, which

interlock with cubic crystals. Another possible explanation could be that the setting time accelerator in
ERRMs interferes with the hydration reaction of the
cements, especially at low pH values where the crystalline structures of the hydrated cement appear less
cohesive (71).
An ideal reparative material should be dimensionally stable, adhere to the cavity walls, resist dislodging forces, and remain unaffected in the presence of
tissue fluids. Bioceramic cements used as a root-end
filling come into contact with tissue fluid before
complete hydration is achieved. When MTA is in
contact with tissue fluid, the setting time is extended
and, in certain cases, the material may not set at all
(77,78). Bolhari et al. (48) compared the surface microhardness of BioAggregate, ProRoot MTA, and
CEM Cement when exposed to an acidic environment or PBS. The results showed that the surface
microhardness of BioAggregate, ProRoot MTA, and
CEM Cement was greater when exposed to PBS
compared to butyric acid (pH: 5.4) and distilled
water. The increase in microhardness values after


Shen et al.
exposure to PBS may be due to the formation of
carbonated apatite crystals (79).
Bioceramic materials may often come into contact with blood when used for root perforations.
Blood may in fact be mixed with the cement,
depending on the clinical situation and method
used in placing the material. Nekoofar et al. (69)
studied the setting reaction of MTA in the presence of blood and found that blood contamination
had a detrimental effect on the surface microhardness of MTA and caused a change in the surface
microstructure. Therefore, there is a basis for the
recommendation that clinicians should attempt to
control bleeding when placing MTA in any clinical
situation. Interestingly, white MTA had greater
hardness than gray MTA irrespective of the level of
blood contamination. However, quite unexpectedly,
white MTA mixed with blood had a greater hardness than white MTA only exposed to blood, and
was harder than all other experimental groups. The
reason for this difference is unclear, and further
investigation is warranted. Therefore, if contamination with blood is unavoidable, white MTA may be
a preferred choice over gray MTA. The same study
also found that the surface microhardness values of
MTA after 6 months were similar to those after 4
days (69).

Compressive strength
Compressive strength is the capacity of a material
to withstand axially directed pressure generating
compressive stress as a result of compression force
(80). The compressive strength of bioceramic
cements is a measure of multiple material properties including the hydration reaction that is critical
to the setting of hydraulic silicate cements
(3,55,81). It is one of the indicators of the setting
and strength of a material (3,55,81). Although
dimensional stability and adequate sealing are of
great importance to the clinician when choosing a
root-end filling material, compressive strength is
also an important property that may affect the clinical performance of the material (82). It has been
reported that a high compressive strength in a root
repair material could enable it to withstand loads
causing deformation and shrinkage (21). Torabinejad et al. (21) reported that the compressive
strength of gray MTA was 40.0 MPa. Holt et al.


(83) and Watts et al. (84) showed that white MTA

had a higher compressive strength than gray MTA.
The compressive strength of MTA is affected by
the type of MTA (28), mixing liquid (69,83), condensation pressure (67), acid etching procedures
(55), and mixing technique (62).

A modified version of the ANSI/ADA No. 96 (85)
or ISO 9917-1 has been used to test the compressive strength of bioceramic cements by many
researchers and manufacturers. The ADA 57 or ISO
6876 standards for root canal sealers do not have a
requirement for compressive strength. The ANSI/
ADA No. 96 (85) standard specifies requirements
and test methods for powder/liquid acidbase dental
cements intended for permanent cementation, lining,
and restoration. This standard is an adoption of ISO
9917-1:2007. The compressive strength testing
method is performed on the 28th day for ANSI/
ADA No. 96 (57). The ISO 9917 standard requires
testing performed after 24 h, which is much too
short for the original bioceramic cements; therefore
longer times are used. A modified test of compressive strength for endodontic bioceramic cements
with the ISO 9917 standard is done after mixing
and setting at 37C in 95% humidity for 7 days
(27,86). Samples for compressive strength measurement are made in cylinders and tested at an appropriate loading rate using a testing jig attached to a
console. Usually a two-piece split mold is used to
make cylindrical samples. The mold has previously
been isolated with vaseline and protected at the bottom surface using a strip. The materials are slowly
inserted through the matrix and then another strip is
placed on the upper surface followed by a 2-mmthick glass slab manually pressed to obtain a regular
material surface. After unmolding, each specimen is
abraded with fine-grain sandpaper to create flatended samples for testing. Defect-free samples are
loaded by compressive pressure to failure, with an
Instron universal testing machine along the longitudinal axis of each cylinder, at a crosshead speed of 1
mm/min (Figs. 7 and 8). The load-at-fracture
and the precise sample dimensions are used to calculate the compressive strength. The compressive
strength rc [MPa] is calculated using the following

Mechanical and biological properties of bioceramic materials


Fig. 7. Device for compressive strength test. (A) General view. (B) Magnified view of the working stage.

rc 4P=pD 2
where P is the maximum load and D is the mean
diameter of the specimen in millimeters.
The size and shape of the tested specimens are
two of the most important parameters that influence
the results of compressive strength. There are basically three shapes of test specimen: cube, cylinder,
and prism. With cylindrical specimens, it is of utmost
importance to secure the standardized shape of the
specimens in order to prevent any variation in the
contact area between the specimen and the bearing
plate. Specimen dimensions are 6 mm x 4 mm
according to ANSI/ADA Specification No. 96 for
dental water-based cements. These molds have been
used in various studies investigating the compressive
strength of MTA (21,27,67,87,88). Other researchers have used different-sized molds (12 mm in

length and 6 mm in diameter) (28). Cylindrical

specimens with a height to diameter ratio of 2 are
commonly used in Portland cement research
(57,89). Specimens that are longer may bend or
even break under testing, and a correction factor
may be required for specimens which are shorter
than the recommended height to diameter ratio of 2
(Fig. 9).

Condensation pressure
In the manufacturers instruction for MTA, there is
no information about the minimum or maximum
condensation pressure that should be applied.
Because of this lack of standardization and the use
of uncontrolled hand placement methods, the results
obtained in some studies may be inconsistent
(21,23,9094). When placing MTA with varying

Fig. 8. Compressive test with crosshead speed 1 mm/min for iRoot Fast Setting (FS) and IRM stored in PBS after
28 days (cylindrical specimens with 6 mm diameter and 12 mm high). Significantly higher maximum loading force
is shown for iRoot FS than for IRM, indicating much higher compressive strength for iRoot FS.


Shen et al.

Fig. 9. General relationship between height/diameter ratio and strength ratio. Graph shows that the strength ratio
is more sensitive to h/d < 1.5 than for h/d > 1.5.

condensation pressures, this becomes an uncontrolled variable and can affect material properties and
performance (42,51,75,93,9598). For instance,
when used as a root-end filling, pressure will be
applied during placement, both as it is packed into
various carrying devices (MTA gun) and also when
aliquots of MTA are manipulated in situ in the rootend cavity. A corresponding situation would presumably exist for surgical repair of perforations. When
MTA is used for the non-surgical repair of perforations, as an apical barrier in the treatment of immature teeth with open apices, or as a pulp-capping/
pulpotomy material, only gentle condensation pressure is employed to prevent the material from being
forced into the periodontal ligament or pulp tissue.
Based on various clinical applications, moisture may
be available from adjacent periodontal or pulpal tissues and from a damp cotton or sponge pellet that is
placed on the material. It is possible that the degree
of condensation pressure may change the molecular
distance between water molecules and MTA particles
and change the space required for the hydration
reaction; this in turn may change the optimum water
to powder ratio (99).
Nekoofar et al. (67) found, perhaps unexpectedly,
that when greater pressures were applied to MTA
during placement, its surface hardness was significantly reduced. Conversely, its maximum compressive strength occurred with the minimum pressure.


It was anticipated that a greater condensation pressure would result in a harder material, although the
result showed that when the condensation pressure
was more than 3.22 MPa, surface hardness was
reduced. This may occur because of insufficient intermolecular space for the ingress of water to hydrate
the material adequately. SEM images also demonstrated that higher condensation pressures were associated with fewer voids that could result in a less
than optimal volume of intermolecular space with a
negative effect on the hydration process. Thus,
applying a greater pressure in an attempt to achieve
a harder material appears futile. The role of water
molecules during the setting reaction is crucial. It
not only mixes with powder, but also chemically
binds with various phases of the cement and has a
direct effect on the setting process (23,93,100). In
other words, MTA hardens and gains strength as it
hydrates; this process occurs rapidly at first and then
slows down with time. When MTA powder is mixed
with water, a special structure of microchannels is
created. The continuity of microchannels is damaged
during the setting process (98). The hardened
cement presents pores and broken microchannels.
Therefore, when only light condensation pressure is
used in certain clinical situations, such as repair of
perforations, placing apical barriers within immature
apices, and during direct pulp capping, the resultant
material will be stronger.

Mechanical and biological properties of bioceramic materials

Mixing and placement techniques

Correct proportioning and mixing are essential to
ensure that cements attain their optimum physical
properties (101). Few studies have examined variations in mixing and placement techniques and their
effects on the properties of bioceramic materials.
Hachmeister et al. (96) reported that the placement
technique was more important than the material
itself. When comparing manual and mechanical mixing, Nekoofar et al. (62) revealed that the application of ultrasonic energy to MTA produced a
significantly higher surface microhardness value. Basturk et al. (87) evaluated the effect of various mixing
techniques including mechanical and manual mixing
as well as the effect of indirect ultrasonic agitation
during placement on the compressive strength of
MTA. For the indirect ultrasonic agitation group, an
endodontic plugger was applied by placing it in the
center of the material and avoiding contact with the
walls or floor of the mold. The results showed that
mechanical mixing enhanced the compressive
strength of MTA compared with manual mixing and
placement. This could be explained by the concept
that mechanical mixing created a less grainy mixture
with fewer unhydrated particles, resulting in better
water diffusion. Conversely, manual mixing and
placement were associated with inadequate hydration
by limiting microchannel formation inside the material and compromising the ingress of water to
hydrate the material (62). However, these results
may be in contradiction to earlier results where
higher condensation pressure during material placement resulted in reduced microhardness (67). The
compressive strength values reported for ProRoot
MTA have been significantly greater than those of
MTA Angelus. MTA Angelus particles have a wide
size distribution compared with ProRoot MTA particles (65), and as a result, ProRoot MTA is more
homogeneous. Incongruities in the cements microstructure could result in larger local water-to-powder
distances, which are inversely related to the strength
of the material (43). Smaller particles are better able
to absorb moisture (102). Therefore, the difference
in the compressive strength of ProRoot MTA and
MTA Angelus could be attributed to the differences
in particle shape and size, which may affect both
flexural (103) and compressive strength (87).

According to the manufacturers instructions for

ProRoot MTA, 1 g of powder supplied in one
packet should be mixed with a 0.34-g aliquot of distilled water. This mixture results in a waste of the
cement because only a small amount of MTA slurry
is needed for most cases. In an attempt to overcome
this waste, the manufacturer of MTA Angelus developed smaller packets containing 0.14 g of MTA
powder. Yet the amount of liquid mixed with the
MTA powder is still dependent on the clinicians
choice. Thus, clinicians tend to prepare a mixture
according to their best estimations rather than the
manufacturers recommended guidelines. This variation in water-to-powder (WP) ratio will have an
effect on the compressive strength of MTA in the
clinical setting. Ultrasonication has been reported to
enhance the compressive strength (87), surface microhardness (62), and sealing ability (67) of MTA.
However, in a study evaluating the adaptation of
MTA using hand compaction or ultrasonication,
Aminoshariae et al. (91) concluded that the manufacturers recommended WP ratio of 1:3 for MTA
may not be the most favorable for ultrasonic placement and that it may cause voids inside the material.
Recently, Basturk et al. (88) investigated the influence of WP ratio variations on the compressive
strength of MTA and MTA Angelus placed by hand
or using ultrasonic instruments. The results revealed
that MTA in the 0.34 WP ratio had higher compressive strength than when the 0.40 WP ratio was used,
and the compressive strength values of ProRoot
MTA were significantly greater than those of MTA
Angelus. In this study, ultrasonic placement had no
significant effect on the compressive strength of
either formulation of MTA.
The compressive strength of bioceramic cements
can be an important factor, especially in applications
such as pulp capping or repair of furcation perforations in which the material can be subjected to considerable occlusal loading. Clinicians should be
encouraged to use the recommended WP ratio when
preparing MTA cement in clinical practice.

Porosity is a common characteristic of cements and
manifests as microscopic spaces between the unhydrated cement grains. As the hydration reaction


Shen et al.
progresses, the hydration products fill these gaps and
reduce the porosity; however, if too high a water to
cement ratio is used during mixing, excess water
eventually dries off and leaves voids that are not
filled with hydration products. Thus, porosity
increases with an increase in the WP ratio (104) and
decreases as the cement ages (105). Porosity of an
endodontic material is an important property to consider because in theory it could be related to the solubility, degradation, and longevity of the cement.
Because of the complexity of the pore structure of
the bioceramic cements, measuring total porosity
and size distribution is difficult. Pores are classified
in different classes depending on their size: micropores (pore diameter smaller than 2 nm), mesopores
(pore diameter 250 nm), and macropores (pore
diameter larger than 50 nm). Pores may be further
classified according to how accessible they are to
external fluids as closed (not accessible externally),
blind (open at one end), and through (open at
both ends). The open porosity class includes blind
and through pores. Closed pores influence macroscopic properties of the material such as bulk density, elasticity, mechanical strength, and thermal
conductivity) (106). The importance of open porosity is in its direct impact on the possibility of penetration of bacteria and bacterial toxins into
unprotected dentin (107), as well as enhancement of
bacterial attachment by providing a medium. However, so far there is no evidence suggesting that
pores in the commonly used bioceramic materials
allow bacterial penetration deep into the material or
even through it.
Various techniques and devices have been used for
porosity evaluation, including scanning electron
microscopy (SEM) (108,109,112), mercury intrusion porosimetry (MIP) (105,110), nitrogen perfusion (111), capillary flow porometry (113), and the
Archimedes principle method (75,114). However,
all of these techniques have drawbacks. The SEM
method, although reliable for evaluating surface
porosity, cannot relate to the bulk of these materials.
The methods commonly used assume that the
geometry of the pores is regular, that the pores are
interconnected, and that the size distribution is not
affected by the loss of water in the pores upon drying. A single technique is not able to characterize
the entire range of pore sizes found in the bioceramic cement materials. MIP can measure larger capillary


pores (0.0510 lm), whereas the adsorption techniques can describe the C-S-H gel porosity (< 0.03

Mercury intrusion porosimetry

MIP is a widely used technique for characterizing
the distribution of pore sizes in cement-based materials (105,115,116). It is a simple and quick indirect
technique. MIP is based on the premise that a nonwetting liquid (one having a contact angle greater
than 90) will only intrude capillaries under pressure.
The samples are dried for 4 days in an incubator at
60C. The porosity is measured in a two-stage process using a mercury intrusion porosimeter. Calibrated mercury displacement pycnometry is followed
by high pressure mercury intrusion porosimetry. The
volume of mercury intruded in the pores of the specimen is measured. Porosimetry data is processed
using the software supplied with the machine, which
gives the pore volume and size distribution from the
pressure versus intrusion data using a method based
on the Washburn equation. The relationship
between the pressure and the capillary diameter is
described by Washburn as:
D 4c cos h=P
where D is the pore diameter, c is the surface tension of mercury (485 dynes/cm), h is the contact
angle (130) between the mercury and the sample,
and P is the applied pressure.
This technique estimates porosity based on the
behavior of a non-wetting liquid (mercury), which
does not enter pores spontaneously but is forced
into pores by an external pressure. Because the pressure required for mercury to intrude into a pore
depends on the diameter of the pore, plotting intrusion volume against intrusion pressure will give
information about the pore size distribution and the
total open porosity of the material. However,
because mercury must pass through the narrowest
pores connecting the pore network, the method can
therefore not provide a true pore size distribution
(104). It must be taken into account that MIP measures the diameter of the pore entrance rather than
the pore diameter itself (117), and hence large cavities connected by smaller entrances are registered
as pores having the diameter of the entrances. The

Mechanical and biological properties of bioceramic materials

values of total porosity given by MIP can also differ
from those obtained by other techniques. The relationship between MIP results and the actual pore
distribution and connectivity can be better understood with the use of image analysis.
Formosa et al. (115) investigated the porosity and
interfacial characteristics of MTA Plus mixed with
water or anti-washout gel. The results demonstrated
that anti-washout-type MTA Plus had a lower initial
porosity than MTA Plus mixed with water, although
this trend was reversed after 28 days of immersion in
physiological fluid. Camilleri et al. (105) reported
that BioAggregate exhibited greater porosity (36%)
than Biodentine and IRM. Biodentine, which had
less porosity (13%) than BioAggregate, exhibited
more shrinkage and developed cracks within the bulk
of the material when it dried in the experimental

Archimedes principle method

Porosity can be determined using the test method
described in the ASTM Standard C830-00 (118).
Kerosene has been chosen as the saturation liquid
instead of water to avoid any reaction with the specimen (118). The air-dried specimens are dried in an
oven at 105C to a constant weight and the dry
weight, D, is determined (for all weight measurements, the gram is the unit used with an accuracy of
0.001 g). The test specimens are then placed in a
beaker containing kerosene and located in a vacuum
chamber with an absolute pressure of not more than
6.4 kPa for 60 min. The suspended weight, S, is
determined for each test specimen suspended in kerosene. The saturated weight, W, is determined by
removing all drops of liquid from the surface using a
wet smooth linen. The exterior volume (V1) of the
specimen in cubic centimeters is calculated by:
V1 W  S=c
and the volume of the open pores (V2) is calculated
V2 W  D=c
where c is the density of kerosene, 0.80 g/cm3. The
apparent porosity (P) of the specimen in percent is
calculated by:

P V2 =V1  100%
Gandolfi et al. (114) compared the porosity of
WMTA, MTA Plus, and Dycal after immersion in
simulated body fluid using the Archimedes principle
method. The results showed that both MTA materials were more porous than Dycal. WMTA had a
lower porosity (29.36%) than MTA Plus (40.34%).
The difference in particle size may play an important
role in the amount of porosity. Cutajar et al. (119)
found that the addition of zirconium oxide powder,
which was used to replace bismuth oxide, reduced
the overall porosity in MTA. Both microscopy and
the evaluation of porosity indicated a degree of
porosity consisting mainly of capillary pores and
entrapped air voids.

Micro-computed tomography
Micro-computed tomography (lCT) is a nondestructive, three-dimensional imaging technique
that can be used as an alternative means of determining both porosity and pore size distribution
(120). Even individual closed pores inside the material can be visualized, and the true internal morphology of granules can be revealed (120). It can be
used to evaluate the porosity percentage of the specimens. In a study comparing the efficacy of amalgam, Fuji-Plus, Geristore, and ProRoot MTA as
intraorifice barriers, lCT was used by Zakizadeh
et al. (121). They reported that MTA was significantly less porous compared with Fuji-Plus and Geristore. Later, Basturk et al. (103) used highresolution lCT to measure the percentage by volume of the total porosity in MTA specimens. Even
though the difference was not statistically significant,
ultrasonicated groups had higher porosity percentages (1.57%) than non-ultrasonicated groups
(1.37%). Aminoshariae et al. (91) reported that ultrasonication caused more voids than hand condensation and concluded that the manufacturers
recommended powder-to-liquid ratio for MTA may
not be ideal for ultrasonic placement and might be
the reason for voids that resulted with this technique. The same trend was also observed for
mechanically mixed groups showing higher porosity
values (1.49%) than those mixed manually (1.42%).
Using zinc phosphate cement, Fleming et al. (101)


Shen et al.
reported that mechanical mixing of encapsulated
cement resulted in air entrapment in the cement
mix, which manifested itself as porosity. Even
though different cement types might show different
porosity levels, air entrapment caused by the
mechanical mixing motion might be the reason for
the higher porosity values versus manual mixing.
Interestingly, a medium negative correlation was
found between flexural strength values and
total porosity percentages on MTA and MTA Angelus. Therefore, the authors (103) concluded that
although mechanical mixing of encapsulated cements
was quicker and provided more consistent mixes,
this technique along with ultrasonic agitation was
not associated with a significant advantage in terms
of flexural strength and total porosity over manual

The solubility of the cements is tested as a percentage of the mass of specimen material removed from
the distilled water compared to the original mass of
the specimens. According to ISO 6876 (24), 1.5mm-thick stainless steel rings with an inner diameter
of 20 mm are used for sample preparation. The rings
are filled with the cement and supported by a glass
plate covered with a cellophane sheet, and the filled
mold is placed in an incubator (37C, >95% relative
humidity) for a period of time 50% longer than the
final setting time. The samples are removed from the
mold and weighed three times each with an accuracy
of 0.0001 g. Two samples are put in a Petri dish,
which is weighed before use and which contains 50
mL distilled water. After 24 hours in the incubator
(37C, >95% relative humidity), the samples are
rinsed with 23 mL distilled water and the washings
are allowed to drain back into the Petri dish. The
samples are then discarded, and the Petri dishes are
dried in an oven at 110C, cooled in the desiccator
to room temperature, and reweighed. The amount
of sample removed from each specimen is calculated
as the difference between the initial mass and the
final mass of the Petri dish. The ISO requirement
for solubility is less than 3%. Grech et al. (40)
reported that BioAggregate and Biodentine demonstrated negative solubility values. The solubility of
ProRoot MTA was similar to Portland cement


Both ISO 4049; 2009 (122) and ISO 6876; 2012

(24) include procedures on testing solubility; ISO
4049 includes procedures for testing both sorption
and solubility. Fluid uptake tests are not included in
either standard; however, this test can easily be performed together with testing sorption and solubility.
ISO 4049 (122) could easily be modified to conduct
sorption, solubility, and water uptake simultaneously
and more accurate results could be achieved. The
main differences between the two standards include
the specimen size and the volume of storage solution
used. ISO 6876 (24) suggests the use of discs 20
mm in diameter immersed in 50 mL solution. All of
the readings are calculated from this solution, not
taking into consideration the rate of evaporation of
relative humidity, which can play a significant role in
the results. ISO 4049 suggests the use of discs 15
mm in diameter immersed in 10 mL solution. The
calculations take into consideration the specimen
mass as cast, after immersion in fluids and drying to
constant mass (123).

In accordance with ISO 6876 (ISO 6876 clause 7.8
for dental root canal sealing materials) (24), completely set samples (10  0.1 mm diameter; 1.0 
0.1 mm height) are radiographed using a radiographic unit with reference aluminum step wedges at
3 mm increments. A standard x-ray machine is used
to irradiate x-rays onto the specimens. A digital
image of the radiograph is obtained. The gray pixel
value on the radiograph of each step in the step
wedge is determined using an imaging program, and
a graph of the thickness of aluminum versus the gray
pixel value on the radiograph is then plotted with the
best-fit logarithmic trend line. The equation of the
trend line gives the gray pixel value of an object on
the image as a function of the objects thickness in
millimeters of aluminum. This equation is inverted to
express the objects thickness as a function of its gray
pixel value on the radiograph. The gray pixel values
of the specimens are then determined, and the equivalent radiopacity is expressed in millimeters of aluminum. A radiopacity 3 mm Al is suggested by ISO
6976. Zirconium oxide is used as a radiopacifier in
Biodentine. Camilleri et al. (124) found that Biodentine had a lower radiopacity (4.8) than MTA Angelus
(6.9) after immersion in water for 1 day.

Mechanical and biological properties of bioceramic materials

Differential scanning calorimetry

Differential scanning calorimetry (DSC) is a thermal
analysis technique well suited to the study of chemical reactions and phase transformations in a wide
range of materials. DSC can be used to study the
setting of cements by measuring the temperature
(i.e. the exothermic heat) during the early stages of
setting, as well as monitoring the reaction products
that form via their decomposition upon heating
(125,126). Isothermal DSC analysis can provide a
more complete understanding of the setting property
of the cements.
An isothermal calorimeter can be used to evaluate
the kinetics of the setting reactions of the samples at
a constant temperature of 37C (124). The samples
are mixed and manipulated in accordance with the
manufacturers instructions. The mixtures are transferred to pre-weighed 40-mL aluminum crucibles
and weighed in an analytical balance so that the
amount of mixture in each can be calculated. The
sample preparation process is completed in 1 minute. The heat flux is automatically recorded every 2
seconds. Each crucible is fitted with a lid to prevent
water evaporation and placed in the DSC to analyze
any exothermic peaks associated with the setting
reactions. As a reference, an empty 40-mL aluminum crucible is used. All resulting DSC thermograms are evaluated with the DSC manufacturers
Camilleri et al. (127) studied the kinetics of
hydration of BioAggregate and MTA Angelus using
DSC. The heat flux generated for BioAggregate
during hydration in water at 37C presented an initial endothermic peak that is due to the wetting of
the surface. This endothermic effect was not
observed for MTA Angelus because it was overcome
by the strong endothermic effect induced by the
dissolution of tricalcium silicate, tricalcium aluminate, and free lime. The main exothermic phase for
BioAggregate started at 8 h and ended at 19 h. Its
maximum at 14 h had a very low intensity, indicating a low reaction rate. Both the presence of an initial endothermic peak and a small and delayed
exothermic peak indicate the very low initial reactivity of BioAggregate when compared to MTA Angelus. The same group (124) also found that the heat
flux generated during tricalcium silicate hydration in

Fig. 10. Graphical representation of heat flux generated over time for the different materials.

water at 37C presented an exothermic peak starting at 90 min and passing through a maximum at
210 min. The first 90 min corresponded to the
induction period followed by the initial setting and
then the hardening of tricalcium silicate paste. Biodentine paste displayed one strong exothermic peak,
which was narrower and more intense than that of
tricalcium silicate paste. This indicated that the
kinetics of hydration for Biodentine was faster than
that of pure tricalcium silicate. In WMTA, two exothermic peaks were found (Fig. 10). The first peak
possibly correlates with the initial water absorption
on the calcium silicate particle surfaces, followed by
their dissolution and the start of hydration of the
calcium silicates in the cements. The second peak
can be related to the start of calcium hydroxide precipitation, mostly on the surface, which is a byproduct of calcium silicate hydration. Interestingly,
while GMTA had one intense exothermic peak,
WMTA had two peaks. The hydration mechanism
of GMTA is expected to be the same as WMTA,
but the chemical components and particle size distribution could be different, thus affecting the
hydration kinetics.

Bioceramic sealers
Most of the methods for the evaluation of mechanical properties of bioceramic sealers are similar to
those used for bioceramic cements, because the
cements and sealers share some standards, e.g.
ANSI/ADA No. 57 and ISO 6876 standards.


Shen et al.

Setting time evaluation with pre-mixed

The introduction of a pre-mixed calcium phosphate
silicate-based sealer eliminates the potential of heterogeneous consistency during on-site mixing. Because
the sealer is pre-mixed with non-aqueous but watermiscible carriers, the water-free paste will not set
during storage in the syringe and only hardens on
exposure to an aqueous environment (128). According to its distributor, EndoSequence BC Sealer uses
the moisture that remains within the dentinal
tubules after canal irrigation to initiate and complete
the setting reaction. In 2011, Loushine et al. (17)
investigated the setting time of BC Sealer in the
presence of different moisture contents (09 wt%).
In this study, metal washers with an internal diameter of 13 mm and a thickness of 2 mm were secured
to microscope glass slides using Zapit and used as
molds for sealer placement. Approximately 0.5 mL
of sealer was weighed to determine the amount of
water to be added to the respective group. The
results demonstrated that BC Sealer required at least
168 hours to reach the final setting using the Gillmore needle method. BC Sealer is a material that
needs moisture during the setting process. Therefore, in one study (129), a plaster of Paris mold for
determining the setting time of BC Sealer was used
and stored at 37C and >95% relative humidity for
24 hours before use. The height of the cavity used
(height = 1 mm) was also different from other materials that do not require moisture for setting (height
= 2 mm) according to ISO 6876/2012. The setting
time of BC Sealer determined by using this method
in this study was shorter (2.7 h) than that in other
study (17) because of the different testing methods.
Recently, Xuereb et al. (130) evaluated the setting
time of BC Sealer under different conditions:
immersed in Hanks balanced salt solution (HBSS),
and using the dentin pressure model for 14 days. In
the HBSS model, the sealers were dispensed into
molds measuring 10 mm in diameter and 2 mm in
height. The molds were immediately placed in an
incubator at 37C immersed in HBSS or allowed to
set in a dry environment. The results demonstrated
that BC Sealer failed to set when stored dry, which
is in accordance with the manufacturers instructions.
Both BC Sealer (22.3 h) and MTA Fillapex (19.3 h)
set when immersed in HBSS. There was no statistical


difference in the setting time of these sealers. Using

the dentinal fluid pressure system resulted in an adequate flow of dentinal fluid that allowed BC Sealer
to set inside the root canal. The possible reasons for
the variations amongst the studies may be due to
different methodologies in material setting and the
incubation environment.

Different obturation techniques

To date little evidence exists to support one method
of obturation as being superior to another and the
influence of treatment technique on success/failure
has yet to be determined. Previous studies
(131,132) have suggested that warm vertical compaction may be superior to lateral compaction; however, definitive evidence is lacking. Camilleri et al.
(127) investigated the suitability of selected sealers
with warm gutta-percha obturation techniques. The
effect of temperature during the warm vertical compaction technique was evaluated by testing the sealer
properties after 1 minute to 100C or 37C. The
results showed that the experimental tricalcium silicate-based sealer (Septodont; Saint Maur-des-Fosses,
France) and Apexit Plus contained calcium hydroxide
peaks after setting, which were absent in MTA Fillapex. The properties of AH Plus and the experimental sealer were modified by heat; the setting time was
reduced, and the film thickness increased. AH Plus
had diminished nitrogen to hydrogen bond groups
when heated to 100C for 1 minute. MTA Fillapex,
Septodont sealer, and Apexit Plus were unaffected
by the application of heat. The authors concluded
that the choice of sealer should be considered when
selecting the obturation technique. The tricalcium
silicate-based sealer is recommended for obturation
using cold laterally condensed gutta-percha whereas
MTA Fillapex and Apexit Plus were suitable for
warm gutta-percha obturation techniques.
The push-out test is commonly used to evaluate
bond strength between the sealer and canal walls.
Important factors to consider when designing a
push-out study include material stiffness, pin diameter, and sample orientation (133). Because of the
good flowability and dimensional stability, both
manufacturers recommend using a single-cone (SC)
obturation technique with MTA Plus and BC Sealer.
Despite the manufacturers recommendations, many

Mechanical and biological properties of bioceramic materials

practitioners may feel uncomfortable using the SC
technique and still prefer to use a thermoplasticized
technique with these new calcium silicate-based sealers. DeLong et al. (134) evaluated the push-out
bond strengths of MTA Plus Sealer and EndoSequence BC Sealer when they were used with a thermoplastic technique, e.g. continuous wave (CW)
technique. The roots were sectioned into 1.0-mmthick slices and bond strengths were measured using
a standardized push-out test. Three pin sizes were
available for the push-out test. The pin that most
closely approximated 90% of the canal diameter was
selected for each slice. The results showed that the
MTA PlusCW had statistically significant lower
bond strengths than all other groups. The BCSC
group had statistically higher bond strengths than
the MTA PlusSC and AH PlusCW groups. No
significant differences were seen amongst the other
groups. Modes of failure were predominately cohesive or mixed except for the MTA PlusCW group,
where nearly half of the specimens had no visible
sealer. The investigators drew the conclusions that
BC Sealer and MTA Plus showed favorable bond
strengths when used with an SC technique. The CW
obturation technique decreased the bond strengths
of these sealers.

Antimicrobial effects
The use of root fillings with sealers with antibacterial
activity is considered beneficial in the effort to further reduce the number of remaining microorgan-

isms or even eradicate the infection completely. In

earlier studies, the agar diffusion test was commonly
used to assess the antimicrobial activity of endodontic sealers (135,136), but it is no longer recommended for this purpose because of its lack of
reliability (137). The agar diffusion test has since
been replaced by the direct contact test (DCT),
which better reflects the true antimicrobial potential
of the various sealers in standardized settings (18).
However, the DCT also has limitations in predicting
clinical performance because several important factors such as the microanatomy and chemistry of the
tooth and biofilm formation are not part of the
experimental design (138,139).
Recently, a new dentin infection model was introduced in order to establish standardized, deep penetration of bacteria into dentinal tubules by
centrifugation (140). This model is used to measure
the effectiveness of disinfecting solutions against
Enterococcus faecalis biofilms in dentin and has produced reproducible results under standardized conditions by using viability staining and confocal laser
scanning microscopy (CLSM) (141,142). In 2014,
Wang et al. (143) evaluated the antimicrobial effects
of root canal sealers [AH Plus, BC Sealer, and pulp
canal sealer EWT (EWT)] on E. faecalis biofilms in
dentinal tubules using the dentin infection model
(Fig. 11). They showed that BC Sealer and AH Plus
resulted in significantly more dead cells in dentin
than EWT did. There was no statistically significant
difference between BC Sealer and AH Plus at any
time point. Thirty days of exposure to BC Sealer and

Fig. 11. Confocal laser scanning microscope images of 3-week-old E. faecalis-infected dentin after exposure to BC
Sealer; viability staining. (A) 18% of the bacteria were killed at 1 day. (B) After 7-day exposure, 37% of the bacteria
were killed. (C) After 30-day exposure, 48% of the bacteria were killed.


Shen et al.
AH Plus resulted in significantly more dead bacteria
in dentin than 7-day and 1-day exposures in the biofilms, whereas no statistically significant increase in
the proportion of dead bacteria was detected
between 7-day and 30-day samples with EWT. The
long-lasting antibacterial ability of BC Sealer may be
due to the biomineralization process induced by calcium silicates/phosphates from the sealer together
with the participation of dentin mineral. Moisture
from dentin promotes the hydration reaction to produce calcium silicate hydrogel and calcium hydroxide
to elevate the pH (18). Silica dissolved in a high pH
environment may also directly inhibit bacterial viability (144). Calcium hydroxide subsequently reacts
with the phosphate to form hydroxyapatite and
water. This water is supposed to participate in the
reaction cycle again to produce more calcium silicate
hydrogel and calcium hydroxide (145). The continuous diffusion of calcium hydroxide expanded into
the dentinal tubules may thus explain the continued
killing of bacteria throughout the 30-day period.

Analysis of chemical composition

Rietveld XRD analysis
Rietveld XRD analysis is able to identify the crystalline phases of cements (54,58), but not amorphous
structures. The principle of Rietveld analysis is to
compare the experimental pattern with a pattern simulated based on the presumed amounts, crystal
parameters and equipment parameters of a mixture of
known phases. Rietveld refinement enables the
amounts of different phases in anhydrous cementitious materials to be determined to a high degree of
precision. The Rietveld XRD is used to semi-quantitatively identify and quantify the main phases related
to the MTA hydration process: tricalcium silicate,
dicalcium silicate, calcium hydroxide, ettringite, bismuth oxide, and tricalcium aluminate (Fig. 12). Tricalcium silicate and dicalcium silicate, that is, the
main crystalline phases involved in the hydration of
MTA (23,58,146,147), were detected in the Rietveld
XRD analysis. Diffraction patterns provide information on the chemical characterization of cements,
which is relevant to the understanding of the materials performance (58). One disadvantage of XRD is
that it may not be accurate in some compounds with
ingredients in quantities of less than 5% (146).


The hydrated material is crushed to a fine powder

before analysis (148). Phase compositions of MTA
specimens from each group are determined using an
x-ray diffractometer and CuKa radiation. Scans are
undertaken in the 1080 2 range. To identify
crystalline compounds, all patterns are matched
using the database of the International Centre for
Diffraction Data. The Rietveld refinement tool is
used for the quantitative analysis of phases.
It has already been reported that tricalcium silicate
is one of the main phases present in unhydrated
cements, accounting for a large portion of the MTA
(58,146,147) and Portland cement powder
(23,58,149). The percentage of calcium silicate will
depend on the type of cement and on the manufacturing process (150). Although a large proportion of
calcium silicate in relation to other phases was
observed in the powders (58,147), the real percentage of the compound was not calculated due to the
presence of several uncharacterized peaks resulting
from an amorphous phase or a combination of
phases with low crystallinity. The use of internal reference patterns in the XRD analysis would allow the
identification and quantification of amorphous
phases (58).

Energy-dispersive x-ray
SEM, accompanied by x-ray analysis, is considered a
relatively rapid, inexpensive, and basically nondestructive approach to surface analysis (Fig. 13). It
is often used to survey surface analytical problems
before proceeding to techniques that are more surface-sensitive and specialized. Energy-dispersive x-ray
(EDX) is an analytical technique that qualitatively
and quantitatively identifies the elemental composition of materials analyzed in an SEM, which generally analyzes the top two microns of the sample with
a spatial resolution of one micron. EDX analysis is
used to identify essential compounds of the bioceramic cements, such as calcium, aluminum, bismuth,
silicon, and sulfur. Zhu et al. (151) found that the
particle size of the original iRoot BP Plus paste was
1050 nm, which was more homogeneous and finer
than the MTA powder (Fig. 12). The elemental
analysis revealed carbon (C), oxygen (O), sodium
(Na), Si, P, sulfur (S), chlorine (Cl), and Ca in both
iRoot BP Plus and MTA. The major difference was
that iRoot BP Plus contained a significant amount of

Mechanical and biological properties of bioceramic materials


Fig. 12. Nano-sized surface observation and apatite-forming ability of iRoot BP Plus. (A) SEM observation for
iRoot BP Plus and MTA in unhydrated forms. Surface-formed apatite SEM observation (B), EDX analysis (C),
Ca/P ratio based on EDX spectrum (D), XRD, ATR-FTIR, and micro-Raman (E) analysis of iRoot BP Plus soaked
in SBF for 7 days. Reproduced from Zhu et al., 2014 (151).

tantalum (Ta) and zirconium (Zr), but did not

include aluminum (Al) or Bi.
An SEM is a powerful tool for microstructural
evaluation of dental materials. In the SEM technique, an electron beam scans the surface of the
sample to produce a variety of signals, the characteristics of which depend on many factors including the
energy of an electron beam and the nature of the
sample. Three types of signals provide the greatest
amount of information in SEM: the secondary electrons (SE), back-scattered electrons (BSE), and
x-rays (152). SE are emitted from the atoms on the
surface, which produce a readily interpreted image,
the contrast of which is determined by the sample
morphology. The small diameter of the primary electron beam helps in obtaining a high resolution
image. Back-scattered electrons are primary beam

electrons that are reflected from atoms. The atomic

number of the sample elements determines the
image contrast of a back-scattered micrograph (153).
Therefore, the image obtained with the back-scattered mode will show the distribution of different
chemical phases in a sample. However, the resolution in such an image is not as good as that obtained
through the secondary electrons due to the emission
of electrons from different depths in the sample
The hydration dynamics and mechanisms of bioceramic materials can be analyzed by several different
methods including microscopy, elemental analysis,
and phase analysis (57,58,124,149,155). The x-ray
diffractogram identifies crystalline and particular
phases in the materials. Scanning electron microscopy (SEM) is well suited for the study of material


Shen et al.

Fig. 13. A scanning electron microscope (SEM) with energy dispersive x-ray (EDX) analysis function. (A) General
view. (B) Magnified view.

surface microstructures. Energy dispersive x-ray

(EDX) analysis allows qualitative analysis of the various elements on the surface layers of the materials.
Laser-Raman spectroscopy is a method close to
infrared spectroscopy, but the interpretation of the
spectra is simpler (156); e.g. characteristic spectra
can be detected for different components in Portland
cement by Raman spectroscopy. In the in vivo situation, bioceramic cements are often in contact with
moisture from tooth structures or with body fluids
(e.g. perforation repair, retrograde fillings). FT-IR
and Raman spectroscopy both are methods to examine the interaction of calcium silicate cements with
various liquids (79,157159) (Figs. 14 and 15).

According to Camilleri et al. (124) These techniques are an adjunct to phase analysis by XRD and
aid to verify the phases identified where peak overlap
exists which is the main disadvantage with the use of
XRD to analyze cement based materials. To overcome this problem, a method developed by Rietveld
et al. (160) can be employed. In this method, powder diffraction analysis is standardized using calculated reference diffraction patterns that are based on
models of crystal structures (161).
Several physicochemical characterization methods,
such as SEM, XRD, ATR-FTIR, and micro-Raman,
have been used to investigate the fine structure of
iRoot BP Plus and MTA after immersion in simulated

Fig. 14. Fourier-transform infrared spectrometer (FT-IR). (A) General view. (B) Magnified view of working stage.


Mechanical and biological properties of bioceramic materials

Fig. 15. Absorption Fourier-transform infrared spectroscopy of hydrated WMTA stored in PBS for 4 days. The
graph displays a tricalcium silicate peak at 875 cm-1. Weak bands at 821-842 cm-1 and 900-920 cm-1 can be
assigned to Si-O stretching vibrations. A set of bands at 1400-1600 cm-1 are attributed to calcium hydroxide. Calcium carbonate peak at 1400 cm-1 is also shown.

body fluid (SBF) (Fig. 12) (151). iRoot BP Plus and

MTA have an apatite-forming ability in SBF. After
being soaked in SBF for 1 day, the surfaces of iRoot
BP Plus and MTA became rough and some nanosized particles (granules and round crystals) were
deposited (151). With increased soaking time, wormlike apatite clusters were formed on the surfaces of
iRoot BP Plus and MTA. After 7 days in SBF, a
multi-layered irregular coating composed mainly of
aggregated spherulites was observed. Agglomerates
and precipitates changed the surface topography of
the cements by increasing the irregularities and
unevenness. The apatite crystals formed on the iRoot
BP Plus surfaces appeared to be larger than those on
the MTA surfaces. XRD analysis confirmed that the
nanoparticles or microparticles formed were composed mainly of the apatite phase, according to the
reference pattern of natural hydroxyapatite (JCPDS:
09-0432) (Fig. 12E). ATR-FTIR and micro-Raman
spectra showed that after the materials were soaked in
SBF, new apatite peaks were formed in the spectrum
patterns (151). The peak intensity of the apatite layer
on iRoot BP Plus was much stronger than that on

Biocompatibility and cytotoxicity

Biocompatibility traditionally describes the ability of
a material or a substance to perform with an appropriate host response when applied as intended. Cytotoxicity is defined as the capacity of a material to

impact on cellular viability. Therefore, cytotoxicity

tests are primary biocompatibility tests that determine the lysis of cells, the inhibition of cell growth,
and other effects on cells caused by test substances.
This means that cytotoxicity only describes a single
aspect of biocompatibility (162).

A proper assessment of cytotoxicity requires precise
in vitro laboratory tests. The traditional MTT [3(4,5-dimethyl-thyazol-2-yl)-2,5-diphenyltetrazolium
bromide] assay can be used for preliminary screening
of materials for cytotoxic effects since it is relatively
inexpensive and easy to perform. MTT is transformed by mitochondrial dehydrogenases in living
cells to a non-water-soluble formazan precipitate. In
practice, a small part of the formazan may possibly
be lost when removing the supernatant and potentially affect the accuracy of the test. Loushine et al.
(17) found that all set sealers (AH Plus, BC Sealer,
and Pulp Canal Sealer EWT) exhibited severe cytotoxicity at 24 hours using the MTT test on MC3T3E1 mouse osteoblasts. The cytotoxicity of AH Plus
gradually decreased over the 6-week period and
became non-cytotoxic as early as the third week. In
contrast, BC Sealer remained moderately cytotoxic
up to the fifth week and became mildly cytotoxic
only at the sixth week. EWT remained severely cytotoxic over the entire examination period. The cytotoxicity results of the study by Loushine et al. (17)


Shen et al.
were opposite to those reported by Zhang et al.
(163). In that study, BC Sealer was less toxic than
AH Plus sealer to L929 mouse fibroblasts. The difference may be related to the type of cell line used
for the study as well as the method used to expose
the fibroblasts to the extracts. Prior to final setting,
sealers can release by-products that are cytotoxic and
cause tissue irritation, thereby producing a negative
impact on healing.
The CCK-8 assay is a new type of assay used to
evaluate cell viability with highly water-soluble tetrazolium salt (164). WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2Htetrazolium, monosodium salt] produces a water-soluble formazan dye upon reduction in the presence
of an electron carrier. The detection sensitivity of
CCK-8 may be higher than other tetrazolium salts
such as MTT, XTT, MTS, or WST-1 (165).
MTT assays may underestimate cellular damage
and detect cell death only at the stage of apoptosis
when cellular metabolism is substantially reduced
(166); the CCK-8 and MTT assays have poor linearity with the cell number and low sensitivity to environmental conditions (167,168). Among the
methods and strategies available for cytotoxicity assay
of materials, flow cytometry provides a fast, costeffective, safe, and sensitive assessment to the cytotoxic events (169) (Fig. 16). The flow cytometer
analyzes the stained cells with different fluorescent
characteristics at a single-cell level and assesses the
relative population viability at the end of the assay
(170). The determination of cell viability depends
on the physical and biochemical properties of the
cells. Calcein AM is a detection probe for live cells
that readily enters cells; the intracellular esterase
activity of live cells converts it to calcein and produces an intense green fluorescence. EthD-1 is a
detection probe for dead cells that enters cells with
damaged membranes and binds to nucleic acids to
produce bright red fluorescence. Therefore, cell viability can be determined by flow cytometry analysis
using calcein AM and EthD-1 fluorescent stains
(171). Cell viability depends on the type of material,
culture medium, and incubation time to which the
cells are exposed. MTA Fillapex is a combination of
calcium silicate with a resin component and a bioceramic sealer. Zhou et al. (171) found that fresh
MTA Fillapex was clearly more cytotoxic than BC
Sealer, and the set Fillapex was more cytotoxic than


both BC Sealer and AH Plus when tested using the

flow cytometry method. This indicates the different
influence of constituent components other than calcium silicate on the cytotoxicity of the sealers. MTA
Fillapex was cytotoxic throughout the 4-week test
period. It can be speculated that the initial cytotoxicity is caused by the resin component and the longlasting cytotoxic effect is caused by other substances
released from the sealer, such as lead (172).
Paracelsus early recognition that all substances
have the capacity to be poisonous depending upon
dosage has laid the foundation for modern toxicology (173). Nowadays, cytotoxicity testing of any
new drug or material that may come into contact
with human tissues is important in order to determine the level of safety (and risks) for these compounds. Animal models have been widely used to
test cytotoxicity and tissue toxicity of materials; however, for cytotoxicity, various in vitro methods have
largely replaced animal models. Despite its shortcomings, in vitro testing has several advantages: it
can be better standardized, interpretation of results
is usually straight-forward, and testing is cheaper.
According to Horvath (174), the material tested is
considered cytotoxic if it prevents cellular attachment, causes dramatic morphological changes,
adversely affects replication rates, or leads to a reduction in overall viability. Other, new tests for cytotoxicity measure biomarkers of cellular stresses or
specific signaling events that are common in the initial phases of cytotoxic events. Among the weaknesses of in vitro cytotoxicity tests is the fact that
cell cultures are typically represented by only one cell
type; therefore interactions with other cell types and
tissue components are missing. Another important
matter to consider is that culture conditions are not
homeostatic, and toxic substances are not eliminated
as they are in the in vivo situation. The lack of biotransformation capabilities is probably the bestknown limitation in cell culture systems (175),
although the lack of defense mechanisms probably
has a stronger impact on the precision of toxicity
estimations (176). In contrast to the in vitro condition, lymphatic system and periapical defenses such
as polymorphonuclear leukocytes, plasma cells, and
macrophages help eliminate toxic substances in vivo
(177). These mechanisms are not present in a culture plate and should be taken into account when
interpreting the cytotoxicity results of BC Sealer

Mechanical and biological properties of bioceramic materials


Fig. 16. iRoot BP Plus extracts promoted cell migration of human dental pulp stem cells (DPSCs) without cytotoxicity. (A) Horizontal cell migration of the DPSCs exposed to extracts from iRoot BP Plus and MTA evaluated by
an in vitro scratch wound healing model and acridine orange staining. (B) Vertical cell migration of the DPSCs
exposed to the extracts from iRoot BP Plus and MTA evaluated using a Transwell migration assay and crystal violet
staining. The horizontal (C) and vertical (D) migrated cells were counted and presented as percentages relative to
the control group. (E) Cell apoptosis of the DPSCs exposed to the extracts from iRoot BP Plus and MTA measured
by flow cytometry after Annexin V-PI staining. Reproduced from Zhang et al., 2015 (187).


Shen et al.


Mechanical and biological properties of bioceramic materials

in vitro. For example, EWT has been shown to be
quite cytotoxic in cell culture studies. However, it
has been reported to have milder tissue reactions
than AH Plus after subcutaneous implantation in rat
connective tissues (178). In conclusion, in vitro
cytotoxicity studies should always be interpreted
with caution.
Simulation of the in vivo conditions using an in vitro study for the evaluation of cytotoxicity is challenging. According to ISO standard 10993-5 (179),
the surface/medium ratio must be 1.25 to 6.0 cm2/
mL. This standard allows for more surface contact
with the sealer than that occurring in vivo where the
apical foramen is usually is about 0.20.3 mm in
diameter. The ISO standard may not have evolved
enough to accurately assess biocompatibility in the
reality of the root canal, which may result in overestimation of sealer cytotoxicity.

Bioactivity test
Bioactivity is the ability of a biomaterial to induce a
specific biological response. One of the characteristics of a bioactive material is its ability to form a
hydroxyapatite (6,180) or apatite-like layer (79) on
its surface when it comes in contact with phosphatecontaining fluids, a phenomenon called biomineralization. The ISO 23317 (Implants for surgeryIn
vitro evaluation for apatite-forming ability of implant
materials) method is used to evaluate layers precipitated on the materials. Molds are filled with prepared
cements. SEM with EDX provides qualitative and
semi-quantitative measurements of atomic calcium
and phosphorous to calculate the superficial calcium
to phosphorus (Ca/P) atomic ratios (114,181)
(Fig. 12). The formation of calcium carbonate
(CaCO3) is presumed when the Ca/P ratio exceeds
1.67. Gandolfi et al. (114) found that the Ca/P values after 28 days in Hanks Balanced Salt Solution
were 2.1, 1.84, and 1.64 for MTA Plus, ProRoot
MTA, and Dycal, respectively. They showed that the

Dycal material appeared to be less bioactive. Both

MTA products produced thicker precipitates with a
higher Ca/P ratio exceeding 1.67, indicating a coprecipitation of calcium carbonate formed from the
Ca(OH)2. The mechanism of apatite formation on
calcium silicate MTA cements in phosphate-containing solutions was summarized by Gandolfi et al.
(182) in 11 steps. The growth of a layer of apatite is
an ideal environment for stem cell and osteoblast differentiation and colonization to support new bone
formation. Apatite together with the epigenetic signals correlated to ion release may well explain the
excellent clinical outcomes of MTA cements (82).
Moreover, the ability to form apatite may provide
clinical advantages by improving their sealing by the
deposition of calcium phosphates at the interface
and inside the dentinal tubules of the root canal
when these cements are used as root canal filling
materials in association with gutta-percha (181).
The clinical relevance of data from cytotoxicity
tests depends on the aim of the study and the methods used. For a simple screening test, clinical relevance is not achieved; rather, basic biological
information is aimed at in a relative sense to compare a set of materials. The clinical relevance of more
specific tests depends on the degree of simulation
(e.g. the cellmaterial contact, the choice of site-specific cells). The mechanisms of interaction (e.g. inhibition
biomineralization, influences on the immune system)
can be evaluated in very special tests using specific
cells (e.g. odontoblast-like cells or macrophages).
The interactions between pulp-capping materials
and injured pulp tissues are complex but mainly
include the recruitment of dental stem/progenitor
cells (altered adhesion and migration), their attachment onto the biomaterial, and tertiary dentin formation at the damaged site above the pulp. Dental
pulp stem cells (DPSCs) are the main mesenchymal
stem/progenitor cell type derived from dental pulp,
and are especially promising for pulp repair and

Fig. 17. ERK 1/2, JNK, and Akt signaling pathways were required for the enhanced DPSC migration by iRoot BP
Plus extracts. DPSCs were pretreated with signaling inhibitors, including SB203580 (SB), PD98059 (PD),
SP600125 (SP), or LY294002 (LY), for 1 h and then treated with iRoot BP Plus extracts for 24 h. Horizontal (A)
and vertical (B) cell migrations were evaluated by the in vitro scratch wound healing model and Transwell migration assay. The horizontal (C) and vertical (D) migrated cells were counted and presented as percentages relative to
the control group. Reproduced from Zhang et al., 2015 (187).


Shen et al.
regeneration (183). Recently, Zhu et al. (151) characterized and determined the apatite-forming ability
of MTA and iRoot BP Plus, and investigated their
effects on the in vitro recruitment of DPSCs, as well
as their capacity to induce dentin bridge formation
in an in vivo model of pulp repair. In vitro primary
cultures of DPSCs were subjected to a cell apoptosis
assay, in vitro scratch wound healing model, Transwell assay, Western blot assay, and cell double-labeling immunofluorescence assay, after exposure to
extracts from the materials and signaling pathwayspecific inhibitors. This animal study for the in vivo
pulp repair model was designed to have pulp perforation that was repaired with bioceramic cements.
The maxillary segments were collected and subjected
to micro-computed tomography scanning, Masson
trichrome staining, immunohistochemistry, and tissue double-labeling immunofluorescence assay. The
possible effects of these extracts on the migration of
DPSCs by in vitro scratch wound healing and Transwell assays were investigated (Fig. 17). Cellular
adhesion and migration are functions controlled by
complex changes in cytoskeleton reorganization and
cellmatrix interaction (184). The dynamic structures of focal adhesions (FAs) consist of a complex
of proteins including vinculin, focal adhesion kinase
(FAK), and paxillin (185). Visualization of F-actin
with phalloidin revealed that dental pulp stem cells
(DPSCs) treated with iRoot BP Plus or MTA presented a highly organized and stretched stress fiber
assembly (Fig. 18). SEM showed the cellular morphology and distribution of DPSCs on the surfaces
of the test materials (Fig. 19) (151). DPSCs cultured on iRoot BP Plus and MTA specimens for 24
h appeared to be elongated and exhibited welldefined cytoplasmic extensions projecting from the
cells to adjacent cells or entering the cement asperities. On day 3, the cultured DPSCs in contact with
each other proliferated and became subconfluent.
The DPSCs on iRoot BP Plus seemed to be more
flattened and showed better spreading than the cells
on MTA. Micro-CT scans showed a reparative dentin bridge that was produced on the exposed pulp in
the iRoot BP Plus group after 1 month (Fig. 20A).
Distinct reparative structures were generated in both
MTA and iRoot BP Plus groups as shown by histological analysis (Fig. 20B). The reparative dentin
bridges were in continuity with primary dentin in
the iRoot BP Plus and MTA groups, with well dis-


Fig. 18. iRoot BP Plus extracts stimulated the cytoskeleton reorganization of DPSCs and upregulated focal
adhesion molecule expression via the ERK 1/2, JNK,
and Akt signaling pathways. (A) DPSCs were pretreated
with signaling inhibitors, including SB203580 (SB),
PD98059 (PD), SP600125 (SP), or LY294002 (LY),
for 1 h and then treated with iRoot BP Plus extracts for
3 h. The cytoskeleton was visualized with phalloidin
(red). (B) The relative expression ratio of p-FAK/FAK,
p-paxillin/paxillin, and vinculin/b-actin was calculated
based on the band intensity of Western blot. Reproduced from Zhang et al., 2015 (187).

tinguishable dentin tubules. By Masson trichrome,

Safranin O/Fast Green, and Sirius Red staining,
newly formed collagen fibers could be observed and

Mechanical and biological properties of bioceramic materials

Fig. 19. Optimized cell attachment of DPSCs on surface of two bioceramic cements. The cell attachment of DPSCs
on iRoot BP Plus or MTA specimens was observed by SEM after culturing for 1 or 3 days. Reproduced from Zhu
et al., 2014 (151).

appeared to be packed in bundles. Focal adhesion

molecule expression including vinculin and p-paxillin
was also detected (Fig. 20C). Moreover, the expression levels of odontogenic and focal adhesion molecules in the iRoot BP Plus group seemed to be
higher than those in the MTA group. The results
indicated that iRoot BP Plus possessed excellent apatite-forming ability, promoted in vitro recruitment
of DPSCs, and facilitated dentin bridge formation in
a pulp repair model in vivo (151). Moreover, iRoot
BP Plus extracts optimized the focal adhesion formation and stress fiber assembly of DPSCs.
The results from the above studies demonstrate
how cells with a high level of differentiation perform
their physiological secretory functions in the presence of the biomaterial studied; they do not provide
information on how that biomaterial affects the differentiation of those cells from their immature counterparts. Of particular interest to clinicians and
researchers is the ability of bioceramic materials to
induce osteogenic responses when they are applied
to bone defects (i.e. osteoactivity). Bone marrow is a

promising source of mesenchymal stem cells (MSCs)

for a broad range of cellular therapies in regenerative
medicine. Eid et al. (186) analyzed the effects of different tricalcium silicate cement formulations on the
temporal osteoactivity profile of human bone marrow-derived mesenchymal stem cells (hMW-MSCs).
These cells were exposed to GMTA, WMTA, gray
MTA Plus, and white MTA Plus formulations in
osteogenic differentiation medium. After 1, 3, 7,
and 10 days, quantitative real-time polymerase chain
reaction and Western blotting were performed to
detect the expression levels of the target osteogenic
markers ALP, RUNX2, OSX, OPN, MSX2, and
OCN. After 3, 7, 14, and 21 days, alkaline phosphatase assay was performed to detect changes in intracellular enzyme levels. An Alizarin Red S assay was
performed after 28 days to detect extracellular matrix
mineralization. In the presence of tricalcium silicate
cements, target osteogenic markers were downregulated at the mRNA and protein levels at all time
points. Intracellular alkaline phosphatase enzyme levels and extracellular mineralization of the experimen-


Shen et al.

Fig. 20. In vivo evaluation of iRoot BP Plus in a pulp repair model by lCT and histological analysis. (A) 3D visualization of the maxillary first molar with pulp capping by iRoot BP Plus or MTA. Representative 2D images in the
sagittal and frontal directions are presented. (B) HE and Masson trichrome staining were presented to observe
the formed reparative dentin bridge. #, biomaterial; db, dentin bridge. (C) Double immunofluorescent staining
of p-FAK co-localized with F-actin (phalloidin) in the dental pulp capped with iRoot BP Plus and MTA. d, dentin;
p, pulp. Reproduced from Zhu et al., 2014 (151).

tal groups were not significantly different from the

untreated controls. Quantitative polymerase chain
reaction results showed increases in the downregulation of RUNX2, OSX, MSX2, and OCN with
increasing time of exposure to the tricalcium silicate
cements, while ALP showed peak downregulation at
day 7. For Western blotting, OSX, OPN, MSX2,
and OCN showed increased downregulation with
increasing exposure time to the tested cements.
Alkaline phosphatase enzyme levels generally
declined after day 7. The results suggest that the
previously reported osteoactivity of tricalcium silicate
cements may be dependent upon the cellular differentiation stage, with a possible connection to the


activation of Wnt canonical and TGF-b signaling

pathways in MSCs (186). This also suggests that
fully or partially differentiated cell types with established secretory functions, such as osteoblasts and
pre-osteoblasts, may play a more substantial role in
the reported in vivo osteogenicity of tricalcium silicate cements than the osteogenic differentiation of
pluripotent stem cells.

The development of MTA and other hydraulic
calcium silicate cements has greatly improved the dentists possibilities to successfully treat cases such as

Mechanical and biological properties of bioceramic materials

pulp capping, pulpotomy, treatment of teeth with
open apices, apicoectomy (retrograde filling), and
repair of defects caused by accidental perforations and
resorptions. A wide selection of experimental models
and methods have been developed to study the physical, chemical, and bioactive properties and characteristics of bioceramic materials and their performance
in vitro and in vivo. A common challenge in the study
of bioceramic materials is that many established methods are not optimally suited to the study of this group
of materials, and that new methods and models may
favor one material over the other depending on what
aspect of the material is being studied and what kind
of methodology is employed. One can hope that in
the future more test methods specific to bioceramic
cements are developed and accepted as ISO or ASTM
standards, as this would further help us to understand
and improve the performance of materials in this
group. However, bioceramic materials have already
had a great impact on endodontic treatment for the
benefit of both dentists and patients.

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