Nanomaterials 14 00274 v2

nanomaterials
Article
Dentine Remineralisation Induced by “Bioactive” Materials
through Mineral Deposition: An In Vitro Study
Marta Kunert 1 , Ireneusz Piwonski 2 , Louis Hardan 3 , Rim Bourgi 3,4 , Salvatore Sauro 5,6 ,
Francesco Inchingolo 6 and Monika Lukomska-Szymanska 1, *
1 Department of General Dentistry, Medical University of Lodz, 251 Pomorska St., 92-213 Lodz, Poland;
[email protected]
2 Department of Materials Technology and Chemistry, Faculty of Chemistry, University of Lodz,
163 Pomorska St., 90-236 Lodz, Poland; [email protected]
3 Department of Restorative Dentistry, School of Dentistry, Saint-Joseph University, Beirut 1107 2180, Lebanon;
[email protected] (L.H.); [email protected] (R.B.)
4 Department of Biomaterials and Bioengineering, INSERM UMR_S 1121, University of Strasbourg,
67000 Strasbourg, France
5 Dental Biomaterials and Minimally Invasive Dentistry, Departamento de Odontología, Facultad de Ciencias
de la Salud, Universidad CEU-Cardenal Herrera C/Del Pozo ss/n, Alfara del Patriarca, 46115 Valencia, Spain
6 Department of Interdisciplinary Medicine, University of Bari “Aldo Moro”, 70124 Bari, Italy;
[email protected]
* Correspondence: [email protected]; Tel.: +48-42-675-7461
Abstract: This study aimed to assess the ability of modern resin-based “bioactive” materials (RBMs)
to induce dentine remineralisation via mineral deposition and compare the results to those obtained
with calcium silicate cements (CSMs). The following materials were employed for restoration of
dentine cavities: CSMs: ProRoot MTA (Dentsply Sirona), MTA Angelus (Angelus), Biodentine
(Septodont), and TheraCal LC (Bisco); RBMs: ACTIVA BioACTIVE Base/Liner (Pulpdent), ACTIVA
Presto (Pulpdent), and Predicta Bioactive Bulk (Parkell). The evaluation of the mineral deposition
was performed through scanning electron microscopy (SEM) and energy-dispersive X-ray spec-
troscopy (EDX) on the material and dentine surfaces, as well as at the dentine–material interface
Citation: Kunert, M.; Piwonski, I.;
after immersion in simulated body fluid. Additionally, the Ca/P ratios were also calculated in all the
Hardan, L.; Bourgi, R.; Sauro, S.;
Inchingolo, F.; Lukomska-Szymanska,
tested groups. The specimens were analysed after setting (baseline) and at 24 h, 7, 14, and 28 days.
M. Dentine Remineralisation Induced ProRoot MTA, MTA Angelus, Biodentine, and TheraCal LC showed significant surface precipitation,
by “Bioactive” Materials through which filled the gap between the material and the dentine. Conversely, the three RBMs showed
Mineral Deposition: An In Vitro Study. only a slight ability to induce mineral precipitation, although none of them was able to remineralise
Nanomaterials 2024, 14, 274. https:// the dentine–material interface. In conclusion, in terms of mineral precipitation, modern “bioactive”
doi.org/10.3390/nano14030274 RBMs are not as effective as CSMs in inducing dentine remineralisation; these latter represent the
Academic Editors: Takahiro Kanno only option to induce a possible reparative process at the dentin–material interface.
and Shintaro Sukegawa
Keywords: bioactive dental materials; calcium silicate materials; Ca/P ratio; EDS; pulp capping;
Received: 18 December 2023
MTA; SEM; vital pulp therapy
Revised: 24 January 2024
Accepted: 25 January 2024
Published: 27 January 2024
1. Introduction
In modern, minimally invasive restorative dentistry, the preservation of pulp vitality
Copyright: © 2024 by the authors. represents one of the main clinical aims. Moreover, according to the European Society
Licensee MDPI, Basel, Switzerland. of Endodontology (ESE), a more selective caries excavation should be prioritised to com-
This article is an open access article plete caries excavation in order to avoid unnecessary pulp exposure, which may lead to
distributed under the terms and the necessity for root canal treatment (RCT) [1–3]. There is a growing interest in dental
conditions of the Creative Commons research on innovative materials for the management of pulp using vital pulp therapies
Attribution (CC BY) license (https:// (VPTs)—namely pulp capping (PC) [4]. This is because in our modern society, where life
creativecommons.org/licenses/by/ span has increased significantly, minimally invasive clinical procedures fulfil the task of
4.0/).
Nanomaterials 2024, 14, 274. https://doi.org/10.3390/nano14030274 https://www.mdpi.com/journal/nanomaterials

Nanomaterials 2024, 14, 274 2 of 26
postponing non-vital treatments and tooth loss [5]. Indeed, there has been a rapid devel-
opment of more biocompatible materials with improved “bioactive” properties. Ideally,
such materials should be able to stimulate remineralisation of underlying dentine, thereby
protecting and preserving pulp vitality.
In general, the term “bioactivity” refers to the ability of a biomaterial to elicit
a specific biological response at the interface between living tissues and the material [6,7].
In restorative dentistry, a material is considered bioactive if it can evoke the precipitation
of apatite-like crystals when exposed to body fluids such as saliva [6,8]. The ability of
material to release a specific amount of calcium (Ca2+ ) and phosphate (PO3 4− ) ions plays
an important role in dentine remineralisation via apatite-like deposition [9,10].
Currently, most of the materials indicated as “bioactive” are mainly employed as
dental pulp-capping (PC) agents, which are either placed directly on the exposed pulp
(direct pulp capping; DPC) or as a cavity liner placed over hypomineralised carious dentine
(indirect pulp capping; IPC). Depending on their composition, the materials investigated in
this study can be assigned to four clinically significant groups: calcium silicate materials
(CSMs), namely ProRoot MTA (Dentsply Sirona, York, PA, USA), MTA Angelus (Angelus,
Londrina, Brazil), Biodentine (Septodont, Saint-Maur-des-Fossés, France), and a light-cured
calcium silicate-based material TheraCal LC (Bisco, Schaumburg, IL, USA). However,
there is a group of resin-based “bioactive” materials (RBMs) in this study constituted by
resin-modified glass-ionomer cement (RMGIC) ACTIVA BioACTIVE base liner (Pulpdent
Corporation, Watertown, MA, USA), a light-curing composite containing modified calcium
phosphate, ACTIVA Presto (Pulpdent Corporation, Watertown, MA, USA), and a dual-cure,
bulk-fill resin composite, Predicta Bioactive Bulk (Parkell, Inc., Edgewood, NY, USA) [11,12].
According to the safety data sheets, the only materials recommended for DPC are those be-
longing to the CSM group; the remaining four resin based materials (TheraCal LC, ACTIVA
BioACTIVE, ACTIVA Presto, Predicta Bioactive Bulk) may be suitable only for IPC proce-
dures. ProRoot MTA has been extensively demonstrated to be highly biocompatible and
bioactive in dentine remineralisation [13,14]. Due to its main drawbacks, such as the long
setting time and high costs, new types of MTA-based materials have been developed [15].
MTA Angelus was introduced with the advantage of a significantly reduced final setting
time (24–83 min), and placement that can be performed on a single visit [16,17]. The afore-
mentioned bioactivity and sealing ability of MTA are strongly associated with the calcium
ions released from such materials, which then reacts with pulp fluid and induce mineral
precipitation and dentine “reparation” [18]. Several studies showed that MTA may be able
to induce the formation of hydroxyapatite crystals when immersed in a phosphate buffered
saline (PBS), as well as the production of amorphous calcium phosphate (ACP) [19,20].
To address the difficulties of using MTA in VPT, novel materials have been designed
for more predictable clinical application. For instance, Biodentine (BD), was marketed as
a permanent, biocompatible dentine substitute, either facilitating application in one session,
or immediately followed by placement of final restoration [21]. Despite the manufacturer
claim of a reduced setting time of 9–12 min, it was proven that it only set completely
after 45 min; while an intrinsic maturation process continues for up to 14 days [22–24].
The interaction of BD with the dentine leads to a marginal sealing by evoking tertiary
dentine production and dentine bridge formation [25,26]. ProRoot MTA and MTA Angelus
showed some greater toxicity compared to Biodentine, Activa BioActive, and Predicta Bulk
Bioactive. Biodentine is the material with the least toxicity and simultaneously the best
properties for both indirect and direct pulp capping [27]. BD has become the most used
agent for VPT, although long-term clinical studies are still needed to confirm Biodentine as
the gold standard material for VPT [28].
TheraCal LC was introduced as a light-cured, resin-modified calcium silicate-based
agent that can be used in a single session; it can achieve immediate bonding to resin-based
restorative materials [29,30]. The current literature indicates that TheraCal LC is less ef-
ficient than MTA and BD as a pulp capping material; this seems to be mainly due to the
lower quality of calcific barrier formation, a higher inflammatory effect, less favourable
odontoblastic layer formation, and lower Ca-releasing ability [28,31,32]. Moreover, Thera-
Cal LC demonstrates cytotoxic and genotoxic effects on SC cells, making it inappropriate
for biological treatment of pulp diseases. A possible cause for its toxicity is the presence of
Bis-GMA monomers in its composition [27].
Bioactive materials which facilitate remineralisation of the tooth structure and seal
margins by depositing Ca, P, and fluoride (F) ions blur the lines between restorative and pre-
ventive dentistry; these are in high demand nowadays. ACTIVA BioACTIVE base liner is a
resin-modified glass ionomer cement (RMGIC) containing a modified calcium-phosphate
(MCP), which presents a favourable setting time that is attributed to its dual-setting mecha-
nism. It can also be used for one-visit treatment [33]. According to the manufacturer, the
bioactive properties of ACTIVA BioACTIVE are attributed to the presence of MCP, as well
as the mechanism by which the material responds to pH cycles and releases/recharges
significant amounts of Ca, P, and F ions. According to the available literature, ACTIVA
BioACTIVE shows lower fluoride-releasing potential than a traditional RMGIC, but higher
release of Ca ions as well as a relatively constant phosphate ion release [34,35].
One of the latest developments in bioactive restorative materials is ACTIVA™ PRONTO™,
which is available in the US and EU as ACTIVA™ Presto™. It is a resin-based composite
that can be used as a bulk-fill restorative material and it contains the patented molecule,
Crysta™, which is a modified calcium phosphate functionalised with methacrylate (MCP).
It is a stabilised calcium phosphate that is trapped in a transitional state that enables it
to deliver the necessary Ca and P ions to the adjacent tooth structure and induce rem-
ineralisation. The manufacturer also claims that the material continuously releases and
recharges such ions, and supplements those ions naturally present in the saliva and/or
provided by dietary sources. Crysta™ also acts as a precursor for nucleation sites that
attract and bind Ca and PO ions in the environment, stimulating a slow and natural process
of mineralisation.
Predicta Bioactive is a resin-based material recently introduced to the market. The
safety data sheet indicates that the resin matrix contains a poly(2-hydroxyethyl methacry-
late) (Poly-2-HEMA) monomer, and that it is free of Bisphenol A-glycidyl methacrylate
(BisGMA)-based compounds [36]. However, there is limited information available about
its properties, especially with regard to its interaction with the pulp and dentine. Although
available research states that Predicta does not exhibit cytotoxic or genotoxic potential,
further investigation is necessary to determine its real biocompatibility, bioactivity, and
dentine remineralisation capacity [27].
It is worth emphasising that there are no studies that evaluated the incidence of
mineral deposition as bioactivity markers for novel bioactive materials such as ACTIVA
BioACTIVE, ACTIVA Presto, and Predicta Bioactive Bulk in comparison to gold standard
CSMs. Thus, this study is aimed to assess the ability of modern resin-based “bioactive”
materials (RBMs) to remineralise dentine via mineral deposition and compare the results
to those obtained with calcium silicate cements (CSMs). This aim was accomplished by
evaluation of the mineral deposition at the dentine–material interface, as well as on the
dentine and material surfaces after immersion in simulated body fluid (DPBS) through
scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDX).
Additionally, the Ca/P ratios were also calculated in all the tested groups. The hypothesis
of this study was that RBMs would have similar dentine remineralising ability to that of
CSMs when immersed in DPBS.
2. Materials and Methods

2.1. Sample Preparation and Incubation in DPBS
Sixty-four dentine slices (2 mm thick) were obtained from extracted caries-free human
teeth. Institutional Ethical Committee approval was obtained for this study (RNN/173/23/KE).
Prior to extraction, patients were informed about the use of the teeth for research purposes,
which was confirmed with their written consents. The dentine slices were prepared using
a round bur #16 (bur S6801.016 Komet Gebr. Brasseler GmbH & Co. KG z.H., Lemgo,
& Co. KG z.H., Lemgo, Germany) to create a cavity of 3 mm in diameter and 1.6 mm deep.
Sof-Lex™ (3M Oral Care, St. Paul, MN, USA) discs in coarse, medium, fine, and superfine
grits were used to create a standardised smear layer. In order to reduce debris, all speci-
mens were cleaned in an ultrasonic cleaner with deionised water.
Nanomaterials 2024, 14, 274 The specimens were then divided into 8 experimental groups (Scheme 1, 4Appendix of 26
A Table A1) and the prepared cavities were filled with the following materials: ProRoot
MTA (Dentsply Sirona, York, PA, USA), MTA Angelus (Angelus, Londrina, Brazil), Bio-
Germany) to create a cavity of 3 mm in diameter and 1.6 mm deep. Sof-Lex™ (3M Oral
dentine (Septodont, Saint-Maur-des-Fossés, France), TheraCal LC (Bisco, Schaumburg, IL,
Care, St. Paul, MN, USA) discs in coarse, medium, fine, and superfine grits were used to
USA), ACTIVA BioACTIVE base liner (Pulpdent Corporation, Watertown, MA, USA), AC-
create a standardised smear layer. In order to reduce debris, all specimens were cleaned in
TIVA Presto (Pulpdent
an ultrasonic cleaner withCorporation,
deionised water.Watertown, MA, USA), Predicta Bioactive Bulk
(Parkell,
TheInc., Edgewood,
specimens NY, divided
were then USA) (Scheme 1). The control
into 8 experimental groups dentine
(Scheme (control)
1, Appendixwas A etched
forTable
15 s A1)
withand37%theorthophosphoric acidfilled
prepared cavities were to eliminate the smearmaterials:
with the following layer and exposeMTA
ProRoot the den-
tinal tubules.
(Dentsply The specimens
Sirona, in the
York, PA, USA), groups
MTA Angelus ACTIVA
(Angelus,Presto and Predicta
Londrina, Bioactive Bulk
Brazil), Biodentine
(Septodont,
were Saint-Maur-des-Fossés,
etched, and then bonded as per France), TheraCal instructions,
manufacturer LC (Bisco, Schaumburg, IL, USA),bond-
using a universal
ACTIVA BioACTIVE base liner (Pulpdent Corporation, Watertown,
ing agent (3M Single Bond Universal, 3M ESPE, Seefeld, Germany) (Appendix MA, USA), ACTIVA A, Table
Presto (Pulpdent Corporation, Watertown, MA, USA), Predicta Bioactive Bulk (Parkell, Inc.,
A1). The resin-based materials (TheraCal LC, ACTIVA BioACTIVE, ACTIVA Presto, Pre-
Edgewood, NY, USA) (Scheme 1). The control dentine (control) was etched for 15 s with
dicta Bioactive Bulk) were light-cured using a hand light-curing unit (Elipar™ DeepCure-
37% orthophosphoric acid to eliminate the smear layer and expose the dentinal tubules.
L, The
3M ESPE, Seefeld,
specimens in theGermany),
groups ACTIVAwhile Presto
the CSMs and were
PredictaleftBioactive
undisturbed
Bulk until final setting
were etched,
(ProRoot
and then MTA,
bonded MTA Angelus,
as per Biodentine)
manufacturer according
instructions, using toathe manufacturer’s
universal bonding agentspecifications
(3M
(Appendix
Single Bond Universal, 3M ESPE, Seefeld, Germany) (Appendix A, Table A1). The resin- 2 mL
A, Table A1). The specimens were placed in Eppendorf tubes containing
calcium-free DPBS
based materials (Dulbecco’s
(TheraCal Phosphate
LC, ACTIVA Buffered
BioACTIVE, Saline;Presto,
ACTIVA Lonza, LonzaBioactive
Predicta Walkersville
Bulk)
Inc., were light-cured
Walkersville, using aimmediately
MD, USA) hand light-curing
after unit (Elipar™
reaching the DeepCure-L,
setting time3M ESPE,
stated by the
Seefeld, Germany), while the CSMs were left undisturbed until final
manufacturer. DPBS is a physiological-like buffered (pH 7.4) Ca- and Mg-free solution setting (ProRoot MTA,
MTA Angelus, Biodentine) according to the manufacturer’s specifications (Appendix A,
with the following composition (mM): K+ (4.18), Na+ (152.9), Cl− (139.5), PO43− (9.56, sum
Table A1). The specimens were placed in Eppendorf tubes containing 2 mL calcium-free
of H2PO4) 1.5 mM and HPO42− 8.06 mM). Specimens were stored at 37 °C. The evaluation
DPBS (Dulbecco’s Phosphate Buffered Saline; Lonza, Lonza Walkersville Inc., Walkersville,
wasMD,performed at various
USA) immediately incubation
after reaching thetimes: after
setting timesetting,
stated by 24the
h, manufacturer.
7, 14, and 28DPBS days. The
samples were examined without prior washing to avoid any potential
is a physiological-like buffered (pH 7.4) Ca- and Mg-free solution with the following removal of the de-
posited minerals.
composition + + − 3 −
(mM): K (4.18), Na (152.9), Cl (139.5), PO4 (9.56, sum of H2 PO4 ) 1.5 mM
and HPO4 2− 8.06 mM). Specimens were stored at 37 ◦ C. The evaluation was performed at
various incubation times: after setting, 24 h, 7, 14, and 28 days. The samples were examined
without prior washing to avoid any potential removal of the deposited minerals.
64 specimens
ACTIVA
Control group ProRoot MTA MTA Angelus Biodentine TheraCal LC Predicta Bioactive ACTIVA Presto
BioACTIVE
8 specimens 8 specimens 8 specimens 8 specimens 8 specimens 8 specimens 8 specimens
8 specimens
SEM SEM SEM SEM SEM SEM SEM SEM

4 specimens 4 specimens 4 specimens 4 specimens 4 specimens 4 specimens 4 specimens 4 specimens
EDX EDX EDX EDX EDX EDX EDX EDX

4 specimens 4 specimens 4 specimens 4 specimens 4 specimens 4 specimens 4 specimens 4 specimens
Scheme 1. Experimental groups.
2.2. SEM Imaging

SchemeAll1. Experimental groups.were sputter-coated with 20 nm of gold and then examined
of the 32 specimens
using a scanning electron microscope (SEM, FEI Nova NanoSEM 450, FEI, Hillsboro, OR,
2.2.USA),
SEMwith
Imaging
an accelerating voltage of 5 kV, WD = 4.8 ± 0.2 mm. Representative images
were captured on the surfaces of the tested materials and at the material–dentine interface
to evaluate the reparative/sealing ability of the tested materials. Moreover, the tested
materials were also analysed to assess whether they could release enough ions which could
reprecipitate on the adjacent dentine surface. The representatives SEM images for each
study group were captured at 200×, 500×, 1000×, 3500×, 5000×, and 5000× magnification
for the characterisation of the specific microstructure. The assessment was carried out after
24 h and after prolonged incubation in DPBS (7-, 14-, and 28 days).
to evaluate the reparative/sealing ability of the tested materials. Moreover, the tested ma-
terials were also analysed to assess whether they could release enough ions which could
reprecipitate on the adjacent dentine surface. The representatives SEM images for each
study group were captured at 200×, 500×, 1000×, 3500×, 5000×, and 5000× magnification for
Nanomaterials 2024, 14, 274 the characterisation of the specific microstructure. The assessment was carried out5 of after
26
24 h and after prolonged incubation in DPBS (7-, 14-, and 28 days).
2.3.EDX
2.3. EDXAnalysis
Analysis
For elemental analysis,
For elemental analysis, a further
a further 32 specimens
32 specimens werewere examined
examined with
with an an energy-dis-
energy-dispersive
persive spectrometer
spectrometer (EDX, EDAX/AMETEK,
(EDX, EDAX/AMETEK, Materials
Materials AnalysisAnalysis
Division,Division, Model Octane
Model Octane Super,
Super, Mahwah, NJ, USA), using an accelerating voltage of 20 kV. The
Mahwah, NJ, USA), using an accelerating voltage of 20 kV. The specimens were then placed specimens were
then placed directly onto the SEM stub and examined without any previous
directly onto the SEM stub and examined without any previous coating procedures. The coating pro-
cedures. The surface calcium-to-phosphorus (Ca/P) ratio was then calculated
surface calcium-to-phosphorus (Ca/P) ratio was then calculated at 24 h and after 28 days at 24 h and
after
of 28 days in
incubation of DPBS.
incubation
For ain DPBS. For a comprehensive
comprehensive evaluation,
evaluation, SEM–EDX SEM–EDX
analysis analysis
was also per-
was also
formed onperformed
the material on and
the material and on dentine
on the adjacent the adjacent dentine
(Figure (Figure
1) after 1) after
28 days 28 days
incubation.
incubation.
The The latter
latter analysis wasanalysis was also1 performed
also performed 1 mmthe
mm away from away from the dentine–material
dentine–material interface to
interface to analyse if the tested materials could release enough
analyse if the tested materials could release enough ions, which could ions,reprecipitate
which couldon repre-
the
cipitate on the occlusal dentine surface as mineral-like crystals. For each specimen,
occlusal dentine surface as mineral-like crystals. For each specimen, an average of three an av-
erage of three
independent independent in
measurements measurements in different
different locations locations were considered.
were considered.
A
B
Figure1.1.Cross-section
Figure Cross-sectionsample
sampleoverview
overviewwith
withlocation
locationofofSEM
SEMimages.
images.(A)
(A)dentin–material
dentin–materialinterface
interface
(B) material surface and (C) dentin surface 1 mm adjacent to the material.
(B) material surface and (C) dentin surface 1 mm adjacent to the material.
2.4.Statistical
2.4. StatisticalAnalysis
Analysis
TheShapiro–Wilk
The Shapiro–Wilk test
test was used
used totoconfirm
confirmthe
thenormality
normalityofof the data.
the TheThe
data. Scheffé test
Scheffé
(post
test hoc)hoc)
(post waswas
usedused
to compare the repeated
to compare measurement
the repeated measurementof ionofchanges overtime
ion changes in the
overtime
tested
in materials.
the tested All statistical
materials. analyses
All statistical were were
analyses evaluated with the
evaluated withstatistical software
the statistical pack-
software
age Statistica
package v. 13.1
Statistica (StatSoft,
v. 13.1 Inc.,Inc.,
(StatSoft, Tulsa, OK,OK,
Tulsa, USA), andand
USA), statistical significance
statistical was was
significance con-
sidered at pat<p0.05.
considered < 0.05.
3. Results
3.1. SEM Imaging
The control dentine (control) presented the clear presence of open dentinal tubules
during SEM analysis (Figure 2). The SEM analysis also showed that the control dentine
presented (Figure 2B,C) some mineral precipitation after 28 days of incubation in DPBS,
which suggests that some sort of remineralisation via mineral precipitation can occur
spontaneously in simulated body fluid (Figure 2D).
3. Results
3.1. SEM Imaging
3. Results
The control dentine (control) presented the clear presence of open dentinal tubules
3.1. SEMSEM
during Imaging
analysis (Figure 2). The SEM analysis also showed that the control dentine
presented (Figuredentine
The control (control)
2B,C) some presented
mineral the clear
precipitation presence
after of of
28 days open dentinalintubules
incubation DPBS,
during SEM analysis
which suggests (Figure
that some 2). of
sort The SEM analysis also
remineralisation via showed
mineral that the control
precipitation can dentine
occur
presented (Figure
spontaneously 2B,C) some
in simulated mineral
body fluidprecipitation
(Figure 2D). after 28 days of incubation in DPBS,
which suggests that some sort of remineralisation via mineral precipitation can occur
24 h spontaneously
7 days in simulated body 14 fluid (Figure 2D).
days 28 days
24A
h B
7 days 14C
days 28D
days
A B C D
Dentine
Dentine
20 μm 20 μm 20 μm 20 μm
Figure 2. SEM images of dentine morphology after (A) 24 h, (B) 7-, (C) 14-, and (D) 28-day incubation
20 μm
Figure 2. SEM
periods. images of5000×
Magnification dentine
20 μm
morphology after (A) 24 h, (B) 7-, (C) 14-, and (D) 28-day incubation
(control).
20 μm 20 μm
periods. Magnification 5000 × (control).

Figure 2. SEM images of dentine morphology after (A) 24 h, (B) 7-, (C) 14-, and (D) 28-day incubation
ProRoot
periods. MTA after
Magnification 5000× 7 days of DBPS immersion induced the precipitation of a layer of
(control).
ProRoot
globular MTA after
crystallites. From 7 days
day 14 ofofDBPS immersion
incubation, induced
irregular the precipitation
aggregates clustered into of a layer of
glob-
ular ProRoot
globular structures MTA
crystallites. after
From
of increased 7 days
day of
14DBPS
thickness immersion
of incubation,
and diameter induced
(Figure the
irregular precipitation
aggregates
3(A2–A4, ofwere
clustered
B2–B4)) a layer
into of
seen.globular
globular
After 24 h,
structures crystallites.
ofthe gap atFrom
increased the day 14 of
thickness incubation,
dentine–material
and irregular
interface
diameter aggregates
was evident,
(Figure 3(A2–A4, whileclustered
after 7,were
B2–B4)) intoand
14, glob-28 After
seen.
24ular
days structures
h, theof gap at of
storage, theincreased
the gaps were thickness
filled and
dentine–material and diameter
covered
interface by
was(Figure
minerals3(A2–A4,
evident, (Figure B2–B4))
after 7,were
while3(A2–A4)). seen.
After
14, and 2428 days
After
of h, 24 h,
the surface
storage, the
the ofgap at
ProRoot
gaps the
wereMTA dentine–material
filledexhibited
and covered interface
a regular, was evident,
but non-uniform
by minerals while
(Figuresurface after
3(A2–A4)). 7, 14,
with the and 28
pres-
After 24 h, the
days storage,
ence ofofno mineraltheprecipitation
gaps were filled and3(B1)).
(Figure covered byin
Also minerals
this case,(Figure
from 3(A2–A4)).
day 14with After 24
of incubation,
surface ProRoot MTA exhibited a regular, but non-uniform surface the presence of
h,
thethe surfacewere
surfaces of ProRoot
coatedMTA by the exhibited a regular,aggregates
aforementioned but non-uniform
(Figuresurface
3(B2–B4)).withMoreover,
the pres-
no mineral precipitation (Figure 3(B1)). Also in this case, from day 14 of incubation, the
ence of no mineral
the material surface precipitation
was not only (Figure
covered 3(B1)). Also inaggregates,
by mineral this case, from
butday
after1414ofdays
incubation,
smaller
surfaces
the werewere
surfaces coated by by the aforementioned aggregates (Figure3(B2–B4)).
3(B2–B4)). Moreover, the
crystals depositedcoated
uniformlythe onaforementioned
the entire surface aggregates (Figure
of the dentine, and not only Moreover,
adjacently
material
the
to the surface
material surfacewas
material–dentine wasnot notonly
onlycovered
interface bymineral
mineral
covered3(C1–C4)).
(Figure by aggregates,
aggregates,
The SEM images but revealed
but also
after after
14 days 14smaller
days smaller
specific
crystals
crystals deposited
flake-likedeposited uniformly
uniformly
shape deposits, on
onwith
along the entire
the entire
globular surface
surface of the dentine,
of the dentine,
structures, which were and
andonly
not onlynot only
seen adjacently adjacently
in the case
totothe
thematerial–dentine
material–dentine interface
interface
of this particular MTA (Figure 3(D3)). (Figure
(Figure 3(C1–C4)).
3(C1–C4)). The The
SEM SEM
images images
also also
revealed revealed
specific specific
flake-like
flake-likeshape
shape deposits, alongwith
deposits, along with globular
globular structures,
structures, which which
werewere onlyinseen
only seen in the case
the case
24 h ofofthis
thisparticular
particular7 MTA
MTA
days (Figure
(Figure3(D3)).
3(D3)). 14 days 28 days
1000×1000×
A1 24 h dentine A2 7 days
dentine A3 14 days dentine A4 28 days
dentine
A1 dentine A2 dentine A3 dentine A4 dentine

Interface
5000×5000×Interface
ProRoot ProRoot ProRoot ProRoot

100 μm 100 μm 100 μm 100 μm
B1 100 μm
ProRoot B2 100 μm
ProRoot B3 100 μm
ProRoot B4 100 μm
ProRoot
B1 B2 B3 B4
Material
1000×1000× Material
20 μm 20 μm 20 μm 20 μm
C1 20 μm
C2 20 μm
C3 20 μm
C4 20 μm
C1 C2 C3 C4
Dentin
Dentin
100 μm 100 μm
100 μm 100 μm
100 μm 100 μm
100 μm
D4 100 μm
Figure 3. Cont. D4
Nanomaterials 2024, 14, x FOR PEER REVIEW 7 of 27
D1 D2 D3
5000×5000×
D1 D2 D3
Dentin
Dentin
20 μm 20 μm
20 μm 20 μm
20 μm Figure 3. SEM images of ProRoot MTA morphology after 24 h, 7-, 14-, and 28-day incubation peri-
20 μm 20 μm 20 μm
Figure 3. SEM images of ProRoot MTA morphology after 24 h, 7-, 14-, and 28-day incubation periods.
ods. Scans of (A) dentine–material interface 1000×, (B) material morphology 5000× and dentine sur-
Figure
Scans of 3.
(A)SEM
face adjacent toimages of ProRoot
dentine–material
the material MTA morphology
interface
in magnification, ×,1000×,
1000(C) (B)after 24(D)
material
and h, 7-, 14-, and
morphology
5000×. 28-day
Arrow 5000 × and
incubation
indicates peri- surface
thedentine
specific
adjacent to the material in magnification, (C) 1000×, and (D) 5000×. Arrow indicates thesur-
ods.
for Scans
ProRoot of (A)
MTA dentine–material
flake-like interface
deposits. 1000×, (B) material morphology 5000× and dentine specific for
face adjacent to the material in magnification, (C) 1000×, and (D) 5000×. Arrow indicates the specific
ProRoot MTA flake-like deposits.
for ProRoot
As withMTA flake-like
ProRoot MTA,deposits.
MTA Angelus also exhibited the presence of irregular mineral
deposition
As with after 7 daysMTA,
ProRoot of incubation.
MTA However,
Angelus from day 14 of theincubation aof
uniform and
As with ProRoot MTA, MTA Angelus alsoalso exhibited
exhibited presence
the presence irregular
of irregular mineralmineral
dense
depositiondeposition of globular crystallites was detected. The globular structures—firstly
depositionafter
after 7 daysof
7 days ofincubation.
incubation. However,
However, fromfrom
day day
14 of14 of incubation
incubation a uniform
a uniform and and
seen after
dense 7 days (Figure 4(B2,C2))—were later detected.
covered with Thefiner particles. Mineral coat- seen
densedeposition
depositionofofglobular
globular crystallites
crystallites waswas detected. The globular
globular structures—firstly
structures—firstly
ing was seen on the surface of the material as well as on adjacent dentine (Figure 4(B3,C3)).
after
seen7 after
days7(Figure 4(B2,C2))—were
days (Figure 4(B2,C2))—were laterlater
covered
covered with
with finer particles.
finer particles.Mineral
Mineral coating
coat- was
The dentine–material interface was filled and covered with the precipitates (Figure 4(A2–
seen on the
ing was seensurface of the of
on the surface material as well
the material as on
as well as onadjacent
adjacentdentine (Figure4(B3,C3)).
dentine (Figure 4(B3,C3)). The
A4)). The material surface itself had larger particles than ProRoot MTA.
The dentine–material
dentine–material interface
interface waswas filled
filled andand coveredwith
covered with the precipitates
precipitates(Figure
(Figure4(A2–
4(A2–A4)).
24 h A4)).
The The material
material surface surface
7 days itself itself
had had
largerlarger particles
particles
14 daysthanthan ProRoot
ProRoot MTA.
MTA. 28 days
dentine dentine
A1 24 h dentine A2 7 days A3 14 days A4 28 days dentine
1000×1000×
dentine dentine
A1 dentine A2 A3 A4 dentine
Interface
5000×5000×Interface
MTA Angelus MTA Angelus MTA Angelus MTA Angelus

100 μm 100 μm 100 μm 100 μm
MTA Angelus MTA Angelus MTA Angelus MTA Angelus

B1 100 μm
B2 100 μm
B3 100 μm
B4 100 μm
B1 B2 B3 B4
Material
10,000×Material
20 μm 20 μm 20 μm 20 μm
C1 20 μm
C2 20 μm C3 20 μm
C4 20 μm
C1 C2 C3 C4
10,000×
Dentin
Dentin
10 μm 10 μm 10 μm 10 μm
10 μm Figure 4. SEM images of MTA Angelus morphology after 24 h, 7-, 14-, and 28-day incubation peri-
10 μm 10 μm 10 μm
ods. Scans of (A) dentine–material interface 1000×, (B) material morphology 5000×, and (C) dentine
Figure 4.
surface SEM images
adjacent to the of MTA Angelus
material morphology
in magnification after 24 h, 7-, 14-, and 28-day incubation peri-
10,000×.
Figure 4. SEM
ods. Scans images
of (A) of MTA Angelus
dentine–material morphology
interface after 24morphology
1000×, (B) material h, 7-, 14-, and 28-day
5000×, and incubation
(C) dentine periods.
Scans of
surface (A) dentine–material
adjacent
Although to
thethe interface
materialsilicate
calcium in 1000×
magnification
materials , (B) material
10,000×.
promoted morphology
the formation of a5000 ×, and
mineral (C) dentine
barrier
surface
at the adjacent to the material
dentine–material in magnification
interface, the SEM images for×Biodentine
10,000 . after 7 days incubation
Although the calcium silicate materials promoted the formation of a mineral barrier
showed the most evident sealing effect (Figure 5(A2)). The presence of globular crystalline
at the dentine–material
Although the to interface,
calcium thematerials
silicate SEM images for Biodentine after 7 days
of aincubation
structures, similar those observed in ProRoot promoted
MTA and MTA the Angelus,
formation were mineral
clearly de- barrier
showed
attected the most evidentinterface,
the dentine–material sealing effect
the(Figure
SEM 5(A2)). The
images for presence of globular
Biodentine after 7 crystalline
days incubation
on the surface of Biodentine (Figure 5(B2,B3)). Over time, such deposits became
structures,
showed the similar
most to thosesealing
evident observed in ProRoot
effect (Figure MTA and The
5(A2)). MTApresence
Angelus, of
were clearly crystalline
globular de-
progressively thicker and more homogeneous, and the adjacent dentine was also coated
tected on
structures, the surface
similar of Biodentine (Figure 5(B2,B3)). Over time, such deposits became
with a uniform layertoofthose observed
mineral in (Figure
crystallites ProRoot MTA and MTA Angelus, were clearly
5(C2–C4)).
detected on the surface of Biodentine (Figure 5(B2,B3)). Over time, such deposits became
with a uniform layer of mineral crystallites (Figure 5(C2–C4)).
with a uniform layer of mineral crystallites (Figure 5(C2–C4)).
Nanomaterials
Nanomaterials 14, 274
2024, 2024, 14, x FOR PEER REVIEW 8 of 27 8 of 26
24 h 7 days 14 days 28 days
A1 24 h dentine A2 7 days dentine A3 14 days dentine A4 28 days dentine

1000×

1000×
Interface
5000× Interface
Biodentine Biodentine Biodentine Biodentine

100 μm 100 μm 100 μm 100 μm
Biodentine Biodentine Biodentine Biodentine

B1 100 μm
B2 100 μm
B3 100 μm
B4 100 μm
B1 B2 B3 B4
5000×
Material
5000× Material
20 μm 20 μm 20 μm 20 μm
C1 20 μm
C2 20 μm
C3 20 μm
C4 20 μm
C1 C2 C3 C4
5000×
Dentin
Dentin
20 μm 20 μm 20 μm 20 μm
20 μm Figure 5. SEM images of Biodentine morphology after 24 h, 7-, 14-, and 28-day incubation periods.
20 μm 20 μm 20 μm
Scans5.ofSEM
Figure images of Biodentine
(A) dentine–material interfacemorphology after 24
1000×, (B) material h, 7-, 14-,5000×,
morphology and 28-day
and (C)incubation
dentine sur-periods.
Figure 5. SEM images of Biodentine morphology after 24 h, 7-, 14-, and 28-day incubation periods.
Scans
face of (A) dentine–material
adjacent interface 1000
to the material in magnification ×, (B) material morphology 5000×, and (C) dentine
5000×.
Scans of (A) dentine–material interface 1000×, (B) material morphology 5000×, and (C) dentine sur-
surface adjacent
face adjacent to to
thethe material
material 5000×.5000×.
in magnification
in magnification
TheraCal LC also exhibited at 24 h an interface characterised by the presence of a gap
which after 28LC
TheraCal
TheraCal days
LC was
also
also covered at
exhibited
exhibited and sealed
at2424
hh anan by minerals
interface
interface (Figure 6(A1–A4)).
characterised
characterised by the Characteristic
bypresence
the presence of a gap
of a gap
plate-like
which
which after precipitants
after28
28days
days waswere observed
coveredand
was covered on
andsealedthe
sealedsurface
byby of the
minerals
minerals material
(Figure
(Figure after 7 days
6(A1–A4)).
6(A1–A4)). of Characteristic
incuba-
Characteristic
tion (Figure
plate-like
plate-like 6(B2)). Maturation
precipitants
precipitants were of suchon
wereobserved
observed crystallites
on thesurface
the resulted
surface ofofthe inmaterial
a thicker
thematerial layer
after
after 7ofdays
7 days denser and
of incubation
of incuba-
finer
(Figure particles (Figure
6(B2)).6(B2)).
tion (Figure Maturation6(B1–B4)).
of such
Maturation After 28 days,
crystallites
of such the surface
resulted
crystallites of the
resultedininaathicker adjacent
thicker layer dentine
ofdenser
layer of was
denserandand finer
evenly
finer covered(Figure
particles with the sporadic After
6(B1–B4)). presence of globular
28 days, structures
the surface (Figure
of the 6(C4)).
adjacent dentine was
particles (Figure 6(B1–B4)). After 28 days, the surface of the adjacent dentine was evenly
evenly with
covered covered
the7with the sporadic
sporadic presence
presence of globular structures (Figure 6(C4)).
of globular
24 h days 14 daysstructures (Figure 6(C4)).
28 days
dentine
A4
1000×
A1 24 h A2 7 days dentine A3 14 days dentine 28 days dentine

dentine
A4
1000×
A1 A2 dentine A3 dentine dentine

Interface
Interface
TheraCal TheraCal TheraCal TheraCal

100 μm 100 μm 100 μm 100 μm
TheraCal TheraCal TheraCal TheraCal

B3
10,000×
B1 100 μm
B2 100 μm 100 μm
B4 100 μm
B3
10,000×
B1 B2 B4
Material
5000× Material
10 μm 10 μm 10 μm 10 μm
C1 10 μm C2 10 μm
C3 10 μm C4 10 μm
C1 C2 C3 C4
5000×
Dentin
Dentin
20 μm 20 μm 20 μm 20 μm
20 μm 20 μm 20 μm 20 μm
Figure 6. SEM images of TheraCal LC morphology after 24 h, 7-, 14-, and 28-day incubation periods.
Scans of (A) dentine–material interface 1000×, (B) material morphology 10,000×, and (C) dentine
surface adjacent to the material in magnification 5000×.
Nanomaterials 2024, 14, 274 Figure 6. SEM images of TheraCal LC morphology after 24 h, 7-, 14-, and 28-day incubation periods.
9 of 26
Figureof6. (A)
Scans SEMdentine–material
images of TheraCal LC morphology
interface 1000×, (B) after 24 h,morphology
material 7-, 14-, and 28-day incubation
10,000×, and (C) periods.
dentine
Scans ofadjacent
surface (A) dentine–material
to the material interface 1000×, (B)
in magnification material morphology 10,000×, and (C) dentine
5000×.
Higher
Highermagnification
magnification images
images of of all
allcalcium
calcium silicate-based materials tested
silicate-based materials testedinin the
the study
arestudyHigher
presented magnification
are presented
in images
Figurein7.Figure
All of of
of7.them
All all calcium
them
generated silicate-based
generated
spherical materials
spherical
surface surfacetested in the with
precipitates
precipitates coated
study are
coated
a uniform with presented
a uniform
layer inlayer
of mineral Figure 7. All ofafter
of mineral
crystallites themthegenerated
crystallites
28after spherical
daysthe 28 dayssurface
incubation precipitates
incubation
period. period.
SEM revealed
coated
SEM with a specific
revealed uniformflake-like
layer of mineral
shapes incrystallites
the ProRootafter
MTAthe (Figure
28 days7(A1,B1))
incubationandperiod.
char-
specific flake-like shapes in the ProRoot MTA (Figure 7(A1,B1)) and characteristic plate-like
SEM revealed
acteristic specific
plate-like flake-like
structures shapes in the ProRoot
groupMTA (Figure 7(A1,B1)) and char-
structures in the TheraCal LCingroup
the TheraCal
(FigureLC 7(A4,B4)). (Figure 7(A4,B4)).
acteristic plate-like structures in the TheraCal LC group (Figure 7(A4,B4)).
ProRoot MTA MTA Angelus Biodentine TheraCal LC
ProRoot MTA MTA Angelus Biodentine TheraCal LC
A1 A2 A3 A4
A1 A2 A3 A4
15,000×
15,000×
5 μm 5 μm 5 μm 5 μm
B1 B2 B3 B4
B1 B2 B3 B4
25,000×
25,000×
Figure 7. Ultrastructure of ProRoot MTA, MTA Angelus, Biodentine, and TheraCal LC surface pre-
Figure
Figure
cipitates Ultrastructure
7.7.after
Ultrastructure ofof ProRoot
ProRoot
28 days incubation MTA,
MTA,
period (A) MTA
inMTA Angelus,
Angelus,
15,000× and Biodentine,
Biodentine,
(B) 25,000×and and TheraCal
TheraCal LC surface LC
magnification. pre-surface
precipitates after
cipitates after 2828
daysdays incubation
incubation period
period in (A)in15,000×
(A) 15,000 × and
and (B) 25,000× magnification.
(B) magnification.
25,000×
In contrast to the CSMs, Activa BioActive exhibited less potential for mineral precip-
InIn
itation contrast
contrast
in totothe
the presentedthe CSMs,
CSMs, ActivaBioActive
study Activa
(Figure BioActive
8). As in in exhibited
exhibited
the of less
casesless potential
potential
the CSMs, for for mineral
mineral
a gap theprecipi-
precip-
between
tation in
itation the
in presented
the presented study
study (Figure
(Figure 8).
8). As
As inin
in in
the the cases
cases of of
the
tooth and material was observed, which was only reduced over time compared to the the
CSMs,CSMs,
a gap a gap between
between the the
tooth
tooth andand material
material waswas observed,
observed, which
which was
was only
only reduced
reduced over
over
CSMs tested in this study (Figure 8(A1–A4)). The SEM images showed that ACTIVA in- time
time compared
compared to
to the
the CSMs
CSMs
tested
duced intested
no in thismineral
thisevident
study study (Figure
(Figure 8(A1–A4)).
8(A1–A4)).
precipitation The The
SEM
neither onSEM
images images
showed
the material showedthethat
nor that ACTIVA
ACTIVA
dentine in-
induced
surface no
duced mineral
during
evident no
theevident mineral
entireprecipitation
period precipitation
of incubation
neitherup neither
ontothe on the
28 days material
(Figure
material nor8).thenor the dentine
dentine surface
surface during the
during
entire the entire
period period of incubation
of incubation up to 28 daysup to(Figure
28 days8). (Figure 8).
1000×

1000×
Interface
5000× Interface
Activa BioActive Activa BioActive Activa BioActive Activa BioActive

100 μm 100 μm 100 μm 100 μm
Activa BioActive Activa BioActive Activa BioActive Activa BioActive
100 μm 100 μm 100 μm 100 μm
B1 B2 B3 B4
B1 B2 B3 B4
Material
Material 5000×

20 μm 20 μm 20 μm 20 μm
20 μm 20 μm 20 μm 20 μm
C1 C2 C3 C4
Dentin 5000×
20 μm 20 μm 20 μm 20 μm
Figure 8. SEM images of Activa BioActive Base Liner morphology after 24 h, 7-, 14-, and 28-day
incubation periods. Scans of (A) dentine–material interface 1000×, (B) material morphology 5000×,
incubation periods.
and (C) dentine Scans
surface of (A) to
adjacent dentine–material interface 1000
the material in magnification ×, (B) material morphology 5000×,
5000×.
and (C) dentine surface adjacent to the material in magnification 5000×.
The SEM images of the dentine at the Activa Presto interface revealed a stable adhe-
siveThe SEM
layer images
with no gapofafter
the dentine at the
24 h (Figure Activa
9(A1)). Presto
After interface
28 days, revealedcracks
spontaneous a stable adhesive
at the
layer with no gap
resin–dentine afterwere
interface 24 hidentified
(Figure (Figure
9(A1)).9(A4)).
AfterIn28 days,
terms spontaneous
of mineral cracks
deposition, no at the
remineralisation was observed at the material’s surface during the 28-day incubation pe-
riod (Figure 9(B1–B4)). After 28 days, only a few precipitates were observed at the dentine
adjacent to the material (Figure 9(C4)).

Dent
20 μm 20 μm 20 μm 20 μm
incubation periods. Scans of (A) dentine–material interface 1000×, (B) material morphology 5000×,
Nanomaterials 2024, 14, 274 and (C) dentine surface adjacent to the material in magnification 5000×. 10 of 26
The SEM images of the dentine at the Activa Presto interface revealed a stable adhe-
sive layer with no gap after 24 h (Figure 9(A1)). After 28 days, spontaneous cracks at the
resin–dentine
resin–dentineinterface wereidentified
interface were identified (Figure
(Figure 9(A4)).
9(A4)). In termsIn
of terms
mineralof mineral no
deposition, deposition,
no remineralisation
remineralisation was was observed
observed at theatmaterial’s
the material’s surface
surface during during
the 28-day the 28-daype-
incubation incubation
period
riod(Figure 9(B1–B4)).
(Figure 9(B1–B4)). AfterAfter 28only
28 days, days, only
a few a few precipitates
precipitates were observedwere
at the observed
dentine at the
adjacent
dentine to the material
adjacent (Figure 9(C4)).
to the material (Figure 9(C4)).

Interface 500×
Activa Presto Activa Presto Activa Presto Activa Presto

200 μm 200 μm 200 μm 200 μm
B3 B4
Material 10,000×
B1 B2
10 μm 10 μm 10 μm 10 μm
C1 C2 C3 C4
Dentin 5000×
20 μm 20 μm 20 μm 20 μm
Figure 9. SEM images of Activa Presto morphology after 24 h, 7-, 14-, and 28-day incubation periods.
Figure 9. SEM
Scans of (A) images of Activa
dentine material Presto500×,
interface morphology after
(B) material 24 h, 7-, 10,000×
morphology 14-, and 28-day
and incubation
(C) dentine sur- periods.
Scans of (A) dentine material interface 500×, (B) material morphology 10,000× and (C) dentine
face adjacent to the material in magnification 5000×.
Predicta Bulk created a uniform and well-sealed interface with dentine after 24 h,
which then Bulk
Predicta deteriorated
createdafter
a 7, 14 and 28and
uniform dayswell-sealed
(Figure 10(A1–A4)). The with
interface inhomogeneous,
dentine after 24 h,
but regular material surface, exhibited no signs of mineralisation during incubation. The
which then deteriorated after 7, 14 and 28 days (Figure 10(A1–A4)). The inhomogeneous,
dentine surface presented only the sporadic presence of irregular mineral precipitants
but (Figure
regular10(C1–C4)).
material surface, exhibited no signs of mineralisation during incubation. The
dentine surface presented only the sporadic presence of irregular mineral precipitants
(Figure 10(C1–C4)).
A1 A2 A3 A4
Interface 1000×
Predicta Bulk Predicta Bulk Predicta Bulk Predicta Bulk

100 μm 100 μm 100 μm 100 μm
B3 B4
Material 10,000×
B1 B2
10 μm 10 μm 10 μm 10 μm
C1 C2 C3 C4
Dentin 5000×
20 μm 20 μm 20 μm 20 μm
Figure 10. SEM images of Predicta Bulk morphology after 24 h, 7-, 14- and 28-day incubation peri-
Figure
ods.10. SEM
Scans of images of material
(A) dentin Predictainterface
Bulk morphology after 24
1000×, (B) material h, 7-, 14- 10,000×,
morphology and 28-day incubation
and (C) dentin periods.
Scans of (A)
surface dentin
adjacent material
to the materialinterface 1000×5000×.
in magnification , (B) material morphology 10,000×, and (C) dentin
3.2. EDX Analysis
ESEM–EDX elemental analysis of ProRoot MTA showed different surface composi-
tion depending on the incubation time. The elemental analysis of the specimens after 24 h
immersion revealed prevalently C (Carbon), O (Oxygen), Ca (Calcium), Si (Silicon), and
Bi (Bismuth) and slight traces of F (Fluoride), Na (Sodium), Mg (Magnesium), and Al (Al-
uminium). Elemental analysis performed after 28 days showed higher Ca, Si, O, and P
Dent
20 μm 20 μm 20 μm 20 μm
Figure 10. SEM images of Predicta Bulk morphology after 24 h, 7-, 14- and 28-day incubation peri-
Nanomaterials 2024, 14, 274 ods. Scans of (A) dentin material interface 1000×, (B) material morphology 10,000×, and (C) dentin
11 of 26
3.2.
3.2.EDX
EDXAnalysis
Analysis
ESEM–EDX
ESEM–EDX elemental analysis of
elemental analysis ofProRoot
ProRootMTA MTAshowed
showeddifferent
different surface
surface composi-
composition
tion depending
depending on the
on the incubation
incubation time.
time. TheTheelemental
elementalanalysis
analysisofofthe
the specimens
specimens after
after 24
24 hh
immersion revealed prevalently C (Carbon), O (Oxygen), Ca (Calcium),
immersion revealed prevalently C (Carbon), O (Oxygen), Ca (Calcium), Si (Silicon), and Si (Silicon), and
Bi
Bi(Bismuth)
(Bismuth)and andslight
slighttraces
tracesof of
F (Fluoride),
F (Fluoride),Na Na(Sodium),
(Sodium),Mg Mg
(Magnesium),
(Magnesium),and Aland(Al-
Al
uminium). Elemental analysis performed after 28 days showed higher
(Aluminium). Elemental analysis performed after 28 days showed higher Ca, Si, O, and Ca, Si, O, and P
content.
P content.
AACa/P
Ca/P ratio of 5.33
ratio waswas
of 5.33 obtained
obtainedduring the elemental
during analysis
the elemental of the mineral
analysis spher-
of the mineral
ulites after 28 days (Figure 11, Table 1) which is very far from the Ca/P ratio
spherulites after 28 days (Figure 11, Table 1) which is very far from the Ca/P ratio of apatite. of apatite.
Because
Becauseof ofthe
theinsignificant
insignificantamounts
amountsof ofPPin
inthe
thefreshly
freshlyprepared
preparedmaterial,
material,assessment
assessmentwas was
only
only made after a 28-day maturation period. Elements that significantly decreasedduring
made after a 28-day maturation period. Elements that significantly decreased during
incubation
incubationwerewereas asfollows:
follows: C, C,O,
O,Na,
Na,and
andBi,
Bi,whereas
whereasaasignificant
significantincrease
increasewas
wasobserved
observed
for
forSi,
Si,P,
P, and
and Ca (p << 0.05)
0.05) (Table
(Table 1).
1).
Figure
Figure 11.
11. Scanning
Scanning electron
electron microscopic
microscopic (SEM)
(SEM) images
images of
of the
the material
material surface
surface with
with the
the EDX
EDX results
results
of ProRoot MTA after (A) 24 h incubation period and (B) 28 days.
MTA Angelus after 24 h incubation showed high Ca and O content and traces of Mg,
Table 1. Comparison of elemental analysis (% weight) of all tested materials after 24 h and 28 days
Si, Cl, and F. Subsequent to 28 days in DPBS, the surface was characterised by a higher
incubation period. Significant p < 0.05 increase (↑) and decrease (↓) in elemental analysis during the
presence of Ca (33.51% wt) and P (14.81% wt) with a Ca/P ratio of 2.26. Bismuth was de-
incubation period were marked.
tected after 28 days only (0.21% wt) (Figure 12, Table 1). The most significant alterations
in composition were observed in the following: O, F, Al, Si, Ca (decrease) and in C, Na,
Activa BioActive
ProRoot MTA MTA Angelus Biodentine TheraCal Base Liner Activa Presto Predicta Bulk
Mg, Zr, P (increase) (p < 0.05) (Table 1).
24 h 28 d 24 h 28 d 24 h 28 d 24 h 28 d 24 h 28 d 24 h 28 d 24 h 28 d
C 11.30 3.42 ↓ 4.82 6.42 0.98 4.99 ↑ 6.06 1.69 ↓ 47.77 49.28 33.44 33.04 36.74 38.43
O 50.49 39.86 ↓ 41.96 A
37.61 ↓ 38.72 42.43 38.58 37.22 37.28 32.90 ↓ 35.60 33.24 46.44 48.95
F 2.97 3.48 3.85 1.93 ↓ 3.07 3.27 3.93 3.51 2.92 3.25 1.01 0.67 3.42 2.78
Na 2.18 0.30 ↓ 0.40 1.83 ↑ 0.56 0.86 1.68 1.78 1.37 1.67 1.27 1.58 1.58 1.22

Table 1. Cont.
ProRoot MTA Activa BioActive Activa Presto

MTA Angelus Biodentine TheraCal Base Liner Predicta Bulk
24 h 28 d 24 h
B28 d 24 h 28 d 24 h 28 d 24 h 28 d 24 h 28 d 24 h 28 d
Mg 1.09 1.80 0.13 1.06 ↑ 0.23 3.09 ↑ 0.47 0.92 0.30 0.46 0.43 0.34 0.69 0.88
Al 1.18 0.86 2.03 0.05 ↓ 0.16 0.04 0.27 0.06 1.32 2.12 ↑ 3.66 3.81 1.99 1.64
Si 5.57 7.98 ↑ 11.81 1.23 ↓ 7.21 7.08 1.57 0.22 3.87 4.28 14.52 15.63 4.81 3.87
Zr 0.26 0.28 0.00 1.05 ↑ 1.88 2.20 0.00 1.91 ↑ 0.33 0.35 0.19 0.19 0.25 0.47
P 0.14 5.96 ↑ 0.14 14.81 ↑ 0.19 0.49 16.39 17.00 1.61 0.70 0.40 0.42 1.32 0.16
Bi 4.86 3.50 ↓ 0.01 0.21 0.07 0.02 0.00 0.00 0.00 0.31 ↑ 0.23 0.22 0.15 0.33
Cl 0.14 0.66 0.00 0.16 2.14 0.36 ↓ 0.09 0.09 0.01 0.10 0.22 0.47 0.05 0.10
35.09 35.48
Ca 19.70 31.78 ↑ 34.64 33.51 ↓ 44.73 ↓ 30.78 ↑ 2.14 1.72 0.55 0.58 1.58 0.62
Ba 0.12 0.11 0.20 0.13 0.07 0.07 0.19 0.12 1.36 2.86 ↑ 8.49 9.83 ↑ 0.98 0.54
Ca/P 247.43Figure 71.61
ratio
140.71 5.33 ↓ 2.26 11.
↓ Scanning
235.42 electron
↓
microscopic
1.87 (SEM) images
2.08 3.50 of 2.45
the material
1.36surface with the
1.38 EDX results
1.20 3.38
MTA
MTAAngelus
Angelusafter
after24
24hhincubation
incubationshowed
showedhigh
highCa CaandandOOcontent
contentand
andtraces
tracesofofMg,
Mg,
Si,
Si,Cl,
Cl,and
andF.F. Subsequent
Subsequentto to28
28 days
days inin DPBS,
DPBS, the
the surface
surfacewaswascharacterised
characterisedby byaahigher
higher
presence
presenceofofCa Ca(33.51%
(33.51%wt)wt)and
andP P(14.81%
(14.81%wt) with
wt) witha Ca/P
a Ca/P ratio of 2.26.
ratio Bismuth
of 2.26. was was
Bismuth de-
tected after 28 days only (0.21% wt) (Figure 12, Table 1). The most significant
detected after 28 days only (0.21% wt) (Figure 12, Table 1). The most significant alterationsalterations
in
incomposition
composition were observed
observed in inthe
thefollowing:
following:O,
O,F,F,Al,
Al,Si,Si,
CaCa (decrease)
(decrease) andand in Na,
in C, C, Na,
Mg,
Mg,
Zr, PZr, P (increase)
(increase) (p < (p < 0.05)
0.05) (Table
(Table 1). 1).
Figure
Figure12.
12.Scanning
Scanningelectron
electronmicroscopic
microscopic(SEM)
(SEM)images
imagesofofthe
thematerial
materialsurface
surfaceand
andEDX
EDXresults
resultsof
of
MTA Angelus after (A) 24 h incubation period and (B) 28 days.
MTA Angelus after (A) 24 h incubation period and (B) 28 days.
Biodentine
Biodentineafter
after24
24hhimmersion
immersionrevealed
revealedhigh
highCa,
Ca,Si,
Si,and
andOOcontent
contentandandthe
theabsence
absence
of
ofP.P.Zr
Zrwas
wasalso
alsodetected
detectedininamounts
amounts constant over
constant time.
over After
time. 28 days,
After this this
28 days, material ex-
material
hibited
exhibitedsimilar proportions
similar proportionsof of
thethe
aforementioned
aforementionedelements
elementswith
withstatistically
statisticallysignificant
significant
increased C and Mg content, but with a lower presence of Ca and Cl (p < 0.05) (Figure 13,
Table 1).

increased C and Mg content, but with a lower presence of Ca and Cl (p < 0.05) (Figure 13,
Table 1).
Figure
Figure13.
13.Scanning
Scanningelectron
electronmicroscopic
microscopic(SEM)
(SEM)images
imagesofofthe
thematerial
materialsurface
surfaceand
andEDX
EDXresults
resultsof
of
Biodentine after (A) 24 h incubation period and (B) 28 days.
Biodentine after (A) 24 h incubation period and (B) 28 days.
TheraCal
TheraCalLC LCafter 24 24
after h incubation
h incubationin DPBS revealed
in DPBS high Ca,
revealed highP and
Ca, OP content
and O (Figure
content
14, Table14,
(Figure 2).Table
SEM–EDX analysis performed
2). SEM–EDX after 28 days
analysis performed aftershowed
28 dayssimilar
showedresults toresults
similar those
observed
to those at 24 h, butatwith
observed statistically
24 h, significant increases
but with statistically in Ca
significant and Zr in
increases butCaa decrease in
and Zr but
Ca (p < 0.05) (Table
decrease in C (p1). The Ca/P
< 0.05) (Tableratio obtained
1). The Ca/Pduring the elemental
ratio obtained duringanalysis of precipitates
the elemental analysis
showed a valueshowed
of precipitates of 1.87 after 24 hofand
a value 1.872.08 after
after 24 h28and
days.
2.08 after 28 days.
Figure 14. Cont.

Nanomaterials 2024,
Nanomaterials 14, x274
2024, 14, FOR PEER REVIEW 1414ofof27
26
Figure
Figure14.
14. Scanning
Scanningelectron
electronmicroscopic
microscopic(SEM)
(SEM)images
imagesof ofthe
thematerial
materialsurface
surfaceand
andEDX
EDXresults
results of
of
TheraCal LC after (A) 24 h incubation period and (B) 28 days.
TheraCal LC after (A) 24 h incubation period and (B) 28 days.
TableActiva Bioactive
2. Elemental Base
analysis (% Liner
weight)showed stable
of the dentin surface
adjacent composition
to all of the tested and morphologies
materials after 28-day
during the Measurements
incubation. different incubation
taken 1 periods.
mm from It revealed
the the prevalent
dentin material presence of O, Al, Si, P,
interface.
and C. A statistically significant decrease was observed in O and increases in Al, Bi, and
Ba (p < 0.05) (Table ProRoot
1). The Ca/P ratio from the elemental Activaperformed
MTA TheraCal analysis
BioActive Activaon the mate-
Predicta
Control MTA Angelus Biodentine LC Presto Bulk
rial surface decreased over time in DPBS storage (Ca/P—3.50 after
Base Liner1 day and 2.45 after 28
days).
C However,
8.69 the5.01
Ca and P2.34
levels were5.87significantly
5.49 lower8.32
than in all 1.82
the other 4.03
CSMs
O
(Figure 15, 45.32
Table 1). 39.07 37.81 39.64 38.45 51.00 45.61 39.93
F 3.18 2.27 1.60 2.52 3.76 5.02 4.21 2.79
Na 3.59 1.00 3.14 1.10 2.07 4.40 2.74 1.88
A
Mg 1.76 0.42 1.40 2.76 1.34 1.89 1.71 1.42
Al 0.56 0.08 0.01 0.00 0.27 0.82 0.51 0.33
Si 1.33 0.37 0.96 1.23 0.17 0.63 0.26 0.24
Zr 0.16 1.57 0.57 0.21 2.32 6.28 7.67 4.51
P 12.73 12.43 14.31 10.25 14.90 6.86 9.64 12.85
Bi 0.28 0.80 0.00 0.08 0.18 0.36 0.32 0.37
Cl 0.18 0.74 1.04 0.38 0.16 0.23 0.25 0.28
Ca 20.49 36.13 36.69 35.85 30.73 14.03 25.13 31.01
Ba 0.15 0.11 0.14 0.12 0.16 0.14 0.12 0.36
Ca/P 1.61 2.91 2.56 3.50 2.06 2.04 2.60 2.41
ratio
B Activa Bioactive Base Liner showed stable surface composition and morphologies
during the different incubation periods. It revealed the prevalent presence of O, Al, Si,
P, and C. A statistically significant decrease was observed in O and increases in Al, Bi,
and Ba (p < 0.05) (Table 1). The Ca/P ratio from the elemental analysis performed on the
material surface decreased over time in DPBS storage (Ca/P—3.50 after 1 day and 2.45
after 28 days). However, the Ca and P levels were significantly lower than in all the other
CSMs (Figure 15, Table 1).
ACTIVA Presto showed an unchanged surface morphology after incubation in DPBS.
The EDX and the elemental analysis of the specimen after 24 h immersion revealed C,
O, and Si as the main components and the presence of Ba (Barium). The study unveiled
no particular dynamic in elemental configuration during the 28-day incubation period
except statistically significant increase in the Ba content (p < 0.05). The Ca/P ratio remained
Figure 15. (Figure
constant Scanning16,
electron
Table microscopic
1). (SEM) images of the material surface and EDX results of
Activa Bioactive Base Liner after (A) 24 h incubation period and (B) 28 days.

The EDX and the elemental analysis of the specimen after 24 h immersion revealed C, O,
and Si as the main components and the presence of Ba (Barium). The study unveiled no
particular dynamic in elemental configuration during the 28-day incubation period except
during the different incubation periods. It revealed the prevalent presence of O, Al, Si, P,
and C. A statistically significant decrease was observed in O and increases in Al, Bi, and
Ba (p < 0.05) (Table 1). The Ca/P ratio from the elemental analysis performed on the mate-
rial surface decreased over time in DPBS storage (Ca/P—3.50 after 1 day and 2.45 after 28
days). However, the Ca and P levels were significantly lower than in all the other CSMs
(Figure 15, Table 1).
Figure
Figure 15.
15. Scanning
statistically
Scanning electron
significant
increase in the (SEM)
microscopic Ba images
content
(SEM) (pof
images < the
of the material
0.05). surface
The Ca/P
material and
ratio
surface and EDX
EDX results
remained con-
results of
of
Activa
stant Bioactive
Activa(Figure Base
16,Base
Bioactive Liner
Table after (A) 24 h incubation period and (B) 28 days.
1). after (A) 24 h incubation period and (B) 28 days.
Liner

TheAEDX and the elemental analysis of the specimen after 24 h immersion revealed C, O,
and Si as the main components and the presence of Ba (Barium). The study unveiled no
particular dynamic in elemental configuration during the 28-day incubation period except
Figure
Figure 16.
16. Scanning
Scanningelectron
microscopic (SEM)
(SEM) images
images of
of the
the material
material surface
surface and
and EDX
EDX results
results of
of
Activa Presto after (A) 24 h incubation period and (B) 28 days.
Predicta Bulk revealed no statistically significant changes in composition during the

incubation period. EDX showed a high presence of O and C, and low Si and F content,
along with traces of Al, Na, P, Ca, and Mg. The Ca/P ratio from the elemental analysis
increased over the incubation period from 1.20 after 1 day to 3.38 after 28 days due to a
notable decrease in P and Ca (Figure 17, Table 1).
Nanomaterials 2024, 14, 274 Figure 16. Scanning electron microscopic (SEM) images of the material surface and EDX results
16 of of
26
Predicta
Predicta Bulk
Bulk revealed
revealed nono statistically
statistically significant
significant changes
changes in
in composition
composition during
during the
the
incubation
incubation period. EDX showed a high presence of O and C, and low Si and FF content,
period. EDX showed a high presence of O and C, and low Si and content,
along with traces of Al, Na, P, P, Ca,
Ca, and
and Mg.
Mg. The
The Ca/P
Ca/P ratio
ratio from the elemental analysis
increased over
over the
the incubation
incubationperiod
periodfrom
from1.20
1.20after
after11day
daytoto3.38
3.38after
after2828days
daysdue toto
due a
notable decrease
a notable decreaseininP Pand
andCaCa(Figure
(Figure17,
17,Table
Table1).1).
Figure
Figure 17.
17. Scanning
Scanning electron
microscopic (SEM)
(SEM) images
images of
of the
the material
material surface
surface and
and EDX
EDX results
results of
of
Predicta Bulk after (A) 24 h incubation period and (B) 28 days.
Predicta Bulk after (A) 24 h incubation period and (B) 28 days.
For
For a comprehensive
comprehensive comparison
comparison of of the
the analysed
analysed materials,
materials, allall of
of the collected
collected data
data
was summarised in Table 1
For a more detailed evaluation of the impact that the tested materials had on the
Table 1. Comparison
adjacent of elementalanalysis
dentine, SEM–EDX analysis (%
wasweight) of all tested
performed aftermaterials after 24
24 h (1 mm awayh and 28 days
from the
incubation period. Significant p < 0.05 increase (↑) and decrease (↓) in elemental
interface); it was possible to see the presence of Ca content in all the specimens treated analysis during the
incubation period were marked.
with CSMs, but with lower P content (Table 2). It was interesting to note that there was an
increase in the Ca/P ratio compared to the Activa non-CSM agents.
BioAc-
ProRoot MTA MTA Angelus Biodentine TheraCal Activa Presto Predicta Bulk
tive Base Liner
4. Discussion
24 h 28 d 24 h 28 d 24 h 28 d 24 h 28 d 24 h 28 d 24 h 28 d 24 h 28 d
In the present study, ProRoot MTA, MTA Angelus, Biodentine, and TheraCal LC
C 11.30 3.42 ↓ 4.82 6.42 0.98 4.99 ↑ 6.06 1.69 ↓ 47.77 49.28 33.44 33.04 36.74 38.43
induced important mineral deposition, while ACTIVA BioACTIVE, ACTIVA Presto, and
O 50.49 39.86 ↓ 41.96Predicta ↓ 38.72 did42.43
37.61 Bioactive not show38.58
any strong 37.28 32.90 ↓ activity.
37.22 remineralisation 35.60 The 33.24obtained
46.44EDX 48.95
data
F 2.97 3.48 3.85 does not↓substantiate
1.93 3.07 3.27 3.93that any
the notion 3.51of the2.92
examined3.25 materials
1.01 possessed
0.67 3.42 2.78
the capability
Na 2.18 0.30 ↓ 0.40 to initiate
1.83 ↑ apatite-like
0.56 0.86 1.68 when
deposition 1.78 immersed
1.37 in 1.67
DPBS.1.27 1.58
Nevertheless, 1.58 1.22
the observed
Mg 1.09 1.80 1.06 ↑in the0.23
0.13 increase amount of P serves
3.09↑ 0.47 as0.92
evidence 0.30for the0.46
formation0.43 of a0.34
phosphate-based
0.69 pre-
0.88
Al 1.18 0.86 2.03 cipitant.
0.05 ↓All the
0.16tests 0.04
performed0.27in this study1.32
0.06 confirmed
2.12 ↑the low
3.66 remineralisation
3.81 1.99 potential
1.64
Si 5.57 7.98 ↑ 11.81of non-CSM
1.23 ↓ materials.
7.21 Therefore,
7.08 1.57 the0.22use of 3.87
such materials
4.28 14.52 should 15.63
be considered
4.81 inferior
3.87
Zr 0.26 0.28 0.00 to both
1.05 ↑MTA1.88products and Biodentine,
2.20 0.00 1.91 ↑and0.33 their use should
0.35 be limited
0.19 0.19 to0.25indirect0.47
pulp
treatment or to restorative procedures. Hence, the hypothesis tested in this in vitro study
P 0.14 5.96 ↑ 0.14 14.81 ↑ 0.19 0.49 16.39 17.00 1.61 0.70 0.40 0.42 1.32 0.16
was rejected. Previous in vivo studies conducted using hDPCS showed that multilayered
Bi 4.86 3.50 ↓ 0.01 cultures
0.21 of cells
0.07had satisfactory
0.02 0.00attachment
0.00 0.00
to both0.31 ↑ 0.23
ProRoot MTA and 0.22Biodentine
0.15 [37–40].
0.33
Cl 0.14 0.66 0.00 0.16 2.14 0.36 ↓ 0.09 0.09 0.01 0.10 0.22 0.47 0.05 0.10
Ca 19.70 31.78 ↑ 34.64 33.51 ↓ 44.73 35.09↓ 30.78 35.48 ↑ 2.14 1.72 0.55 0.58 1.58 0.62
Ba 0.12 0.11 0.20 0.13 0.07 0.07 0.19 0.12 1.36 2.86 ↑ 8.49 9.83 ↑ 0.98 0.54
Ca/P
140.71 5.33 ↓ 247.43 2.26 ↓ 235.42 71.61 ↓ 1.87 2.08 3.50 2.45 1.36 1.38 1.20 3.38
ratio
Indeed, the results of the present study revealed that Biodentine is superior to ProRoot
MTA and also MTA Angelus in terms of Ca content within the material composition, which
may result in more efficient biological and bioactive effects when in contact with biological
fluids. On the other hand, MTA Angelus presents higher P content than ProRoot MTA and
Biodentine, as demonstrated in previous studies [39,41]. These results are in accordance
with the current data from dentine adjacent to the material (Table 2). Interestingly, the
Ca/P ratio of the dentine in contact with the cements after 28 days was higher in the
Biodentine group (3.50) than both ProRoot MTA (2.91) and MTA Angelus (2.56). This may
indicate an increase in mineral uptake from the storage media. Nonetheless, due to a lack of
long-term observational studies, it is challenging to definitively determine which material,
MTA or Biodentine, is superior in terms of dentine remineralisation, as well as longevity
of treatments. However, if one considers factors such as handling and cost-effectiveness,
Biodentine seems to be more favourable than MTA cements [42].
It has been advocated that when calcium silicate-based cements (MTA products and
Biodentine) are in contact with physiological fluids that contain phosphates, they may
produce apatite-like crystals or its precursors [19,41]. The resulting “apatite coating” is
believed to be the foundation of the positive biocompatibility of these materials, as well
as playing a crucial role in cell attachment, differentiation, and tissue repair associated
with mineralised tissue formation [43]. The hydroxyapatite generated by CSMs plays
an important role in creating a tight seal between the dentinal walls and the material,
which is crucial for the success of most of the endo-resto treatments [44]. Furthermore, the
presence of hydroxyapatite encourages the attachment of hard tissue-forming cells to the
material surface through the selective adsorption of fibronectin. This, in turn, facilitates
the development of biological hard tissue barriers that serve to deter the infiltration of oral
cavity bacteria into the dental pulp [45].
The present study revealed that all the tested CSMs generated the precipitation of
minerals on the surface of both the material and the dentine, with a spherical appearance
and acicular microprojections, containing Ca and P, as their principal elements. These
globular crystalline structures were regular but non-homogenous in terms of dimensions
and morphology. During the hydration process, amorphous calcium silicate hydrates
(CSH) were also formed. In fact, EDX analysis revealed the presence of Ca, Si, and O,
which are indicative of CSH [39]. Elemental analysis performed after 28 days also showed
higher P content, suggesting the presence of calcium phosphate deposits (Figure 11).
Interestingly, TheraCal LC, a light-curable CSM, also produced surface precipitates, but
with characteristic plate-like structures (Figure 7). These results are in accordance with
a recent paper by Maciel et al. [3]. According to the analysis performed on the surfaces
of non-CSM materials (ACTIVA BioACTIVE, ACTIVA Presto, and Predicta Bioactive),
they remained relatively unaffected during the 28-day incubation period due to their low
ability to induce minerals precipitation. These findings are also in accordance with previous
studies [42,46]. It is important to consider that the interface between the tested materials and
the dentine may have been affected by artifacts due to specimen preparation. For instance,
100% humidity incubation in DPBS and the vacuum needed for gold-sputtering and SEM
examination may have altered the structure of such an interface. However, the evident
formation of an interfacial layer in all calcium silicate materials has been widely proven and
it confirms the results observed in previous studies [47]. The SEM images of the material–
dentine interface obtained in the current study clearly demonstrated that Biodentine was
the most effective in gap “repairing” and may promote the best results in terms of dentine
sealing. In the case of the tested materials applied in combination with adhesive systems
(Activa Presto and Predicta Bulk) the results were quite different compared to those of
the CSMs. Indeed, the Activa Presto interface was characterised by a stable adhesive
layer with no gap after 24 h (Figure 9(A1)). Only after 28 days were spontaneous cracks
within the interface observed, but with no presence of mineral precipitation (Figure 9(A4)).
Predicta Bulk had a sound bonding interface after 24 h, which then deteriorated after
7, 14 and 28 days (Figure 10(A1–A4)). This indicates that such an interface is quite prone
to degradation over time, when immersed in simulated biological fluids [48]. For all the
RBMs tested in this study, including ACTIVA BioACTIVE, ACTIVA Presto, and Predicta
Bioactive, the SEM analysis of the material–dentine interface showed no evident mineral
precipitation. This result should be taken into consideration in further clinical studies when
evaluating the risk of microleakage of modern bioactive materials.
ProRoot MTA and MTA Angelus are known for their high levels of Ca, making them
ideal for use in VPT procedures such as pulp capping. Biodentine and TheraCal LC also
have good release of calcium ions and suitable mechanical and handling properties [49].
The current study demonstrated that CSMs (ProRoot MTA, MTA Angelus, Biodentine,
TheraCal LC) induce precipitation of Ca/P crystals on adjacent dentine. It is well known
that the presence of calcium ions can promote stem cells differentiation into odontoblast-
like cells and migration; these represent crucial issues involved in pulp healing [50,51].
In the EDX analysis, the concentration of Ca was higher in the CSM groups than in the
ACTIVA BioACTIVE, ACTIVA Presto, and Predicta Bioactive groups (Table 2). Those
findings are in agreement with a previous study that reported the great release of Ca and
P ions from Biodentine, which remained constant during a 14-day incubation period [37].
Moreover, according to the available literature, Biodentine and TheraCal LC have a higher
release of free Ca ions and diffusion levels compared to both ProRoot MTA and MTA
Angelus [52,53]. In the current study, calcium exhibited statistically significant changes
between the initial and final measurements for the following materials: ProRoot MTA
(increase), MTA Angelus (decrease), Biodentine (decrease), and TheraCal (increase). CSM
materials also provide significantly more alkaline pH (9.5–13) than RBM materials; this may
indicate an antibacterial capability and enhanced mineralisation [41,54,55]. The highest
levels of phosphorous were detected in TheraCal LC (16.39 after 24 h and 17.00 after
28 days) followed by MTA Angelus (0.14 after 24 h and 14.81 after 28 days), ProRoot MTA
(0.14 after 24 h and 5.96 after 28 days), Activa BioActive Base/Liner (1.61 after 24 h and 0.70
after 28 days), Predicta Bulk, Biodentine, and Activa Presto. According to presented study,
phosphorus showed statistically significant changes between the incubation times for the
following materials: ProRoot MTA (increase) and MTA Angelus (increase). It is worth
emphasising that CSMs have no phosphate in their composition; therefore, its presence
comes from the DPBS. Such an element may be an indicator of phosphates content in the
material; these ions are crucial for the neutralisation of the acidic pH created by bacteria
and to bind to Ca ions and form calcium–phosphates complexes [56].
It is worth emphasising that the Ca/P ratio may impact both the physical and me-
chanical properties of the dental materials, including their strength, hardness, and biocom-
patibility [57]. In general, a Ca/P ratio of approximately 1.5–2.0 is considered to be ideal
for maintaining the mineralisation and strength of teeth and bones. This is based on the
fact that the hydroxyapatite mineral, which is the main mineral in teeth and bones, has
a Ca/P ratio of approximately 1.67 [57]. Therefore, maintaining a minimum ratio within
this range can ensure the optimal mineralisation and strength of dental hard tissues. In the
present study, the Ca/P ratio from the EDX elemental analysis revealed the highest score
for ProRoot MTA (5.33 after 28 days). Generally, the CSM materials obtained higher Ca/P
ratios than the non-CSM agents in the following (descending) order: ACTIVA BioACTIVE
(2.45 after 28 days), MTA Angelus (2.26 after 28 days), TheraCal LC (2.08 after 28 days),
Predicta Bioactive (1.72 after 28 days), and ACTIVA Presto (1.38 after 28 days; which may be
the Ca/P ratio of the MCP inside ACTIVA) (Table 1). For Ca/P ratio, statistically significant
changes were observed between the initial and final measurements for the following materi-
als: ProRoot MTA, MTA Angelus, and Biodentine (all cases showed a decrease). The results
obtained in the current study showed no apatite deposition in any of the tested materials.
The first comparable assessment in ProRoot MTA was made after a 28-day maturation
period. The absence or trace quantity of P in CSMs is supported by other available studies
that suggested how the P content in following incubation periods are sourced from storage
media (e.g., DPBS) [39,41,58]. Given the initial absence of P and following measurements
after 28 days correlated with P intake from the DPBS solution, the Ca/P ratio assessment
might be more reliable in the dentin adjacent to the bioactive restoration as described below.
The current study did not explicitly address the calcium-to-phosphorus (Ca/P) ratio of the
mineral precipitated exclusively at the interface. However, throughout the course of the
study, comparative analysis of the elemental results revealed that deposits covering the
entire surface exhibited remarkably similar characteristics.
Although ACTIVA BioACTIVE has recently been shown to present some remineralisa-
tion ability compared to compomer and glass-ionomer cements, its bioactivity needs to be
considered inferior to CSM in terms of mineral deposition [59,60]. For a full evaluation of
the impact that the tested materials may have on the adjacent dentine, SEM–EDX analysis
was also performed 1 mm from the dentine–material interface. Higher Ca content in dentine
was observed for all those specimens in contact with CSMs and lower P, compared to the
non-CSM agents. The Ca/P ratio of the group Biodentine was 3.50, followed by ProRoot
MTA (2.91), Activa Presto (2.60), MTA Angelus (2.56), Predicta Bulk (2.41), Theracal LC
(2.06), and Activa BioActive Base Liner (2.04). Nevertheless, all the tested materials showed
a Ca/P ratio above the aforementioned range of 1.67 characteristic for hydroxyapatite.
The microstructural EDX analysis is in line with other studies, except for those el-
ements with a low proportion (which may be considered “trace elements”) within the
materials’ composition [27,40,61]. However, slight variations in the reported chemical
composition determined through EDX/EDS analyses may arise due to differences in the
equipment utilised and the measurement protocol. Data reported in the present study
were based upon the mean result from the three measurements that were carried out for
each specimen. In the CSM materials, there were a few compounds that might serve as
a radiopacifiers, namely bismuth oxide (ProRoot MTA, MTA Angelus), zirconium oxide
(Biodentine), and in newer generations calcium tungstate, which does not cause tooth
structure discoloration. Interestingly, in the MTA Angelus group the expected presence
of bismuth from bismuth oxide was not detectable after 24 h, however it did register after
28 days (0.21% wt). Those findings are in accordance with previous studies [62]. Bismuth
demonstrated statistically significant alterations in measurements from the 24 h to 28-day
incubation period for the following materials: ProRoot MTA (showing a decrease) and
Activa BioActive Base Liner (showing an increase). As Biodentine contains zirconium
oxide as a radiopacifier, Zr were detected in amounts constant over time. According to the
conducted study, zirconium exhibited a statistically significant increase between the initial
and final measurements for the following materials: MTA Angelus and TheraCal.
ESEM–EDX analysis of Activa Bioactive Base Liner showed stable surface composition
and morphology over a period of 28-day storage in DPBS. The specimen revealed the
presence of O, Al, Si, P, and Ca, which can be attributed to the oxides of aluminosilicate
glass and other glass fillers [63,64]. The Ca and P content detected in Activa Bioactive
Base Liner was significantly smaller than in the calcium silicate materials, but the highest
in the non-CSM group. The low amount of phosphorus provides evidence to support
the manufacturer’s assertion that the product contains a water-friendly ionic resin with
phosphate acid functionality and antibacterial properties, which can also facilitate interac-
tions between resin-glass fillers and the tooth structure. This mechanism can lead to the
formation of a strong resin-apatite complex, by replacing hydrogen ions with Ca through
the phosphate group [65]. Elemental analysis of Activa Presto revealed the highest Al, Si,
and Ba peaks among the tested non-CSM group, but the lowest Ca, P and F content. This is
probably the reason why it showed less ability to induce mineral precipitation compared to
the other tested CSMs. Barium is not typically known for its biological activity but its use
as a radiopacifier is a well-established practice in dentistry. In the conducted study, Activa
Presto revealed relatively high Ba content (8.49% after 24 h and a statistically significant
increase to 9.83% after 28 days) in comparison to the other study groups. During the study,
barium also exhibited a statistically significant increase in the Activa BioActive Base Liner
group. The concentration of barium in dental materials requires further investigation to
ensure that it meets biocompatibility standards.
The limitations of the present study are related to the in vitro characteristics of the
experiments performed to accomplish the aims of this study. The established objectives
were based on available research, which suggest that apatite formation may occur in vivo
in the presence of specific phosphate-containing fluids such as the constant circulation
of biological fluids and blood at the surgical site [38]. In the presented study, bioactive
materials provided a source of Ca and P for the remineralisation process as well as the
DPBS solution used as a soaking medium. In a clinical environment, there are several
variables that could affect leaching of the tested elements, such as saliva composition,
dietary source, and also intake from prophylactic products. In this study, the bioactivity of
the materials was investigated as a function of the soaking time in DPBS. The DPBS used in
this study may not have completely resembled saliva (i.e., its pH and ionic composition),
although such DPBS can simulate certain characteristics of the oral cavity. However, testing
the bioactivity of dental materials in phosphate buffer solutions has been reported to be
a suitable method to induce the precipitation of apatite deposition [19,38,66]. An alternative
to a PBS solution is a simulated body fluid (SBF), although its use for bioactivity testing
seems unreliable [67].
To analyse the formation of apatite as an index of bioactivity of the cement, other ad-
vanced characterisation techniques that also allow in situ observations of physio-chemical
properties and the transition kinetics of the researched materials might be used, namely
X-ray diffraction (XRD), rheometric analysis, Fourier transform infra-red (FTIR), and Ra-
man spectroscopies [41,68,69]. The visco-elastic behaviour of dental agents can be moni-
tored with rheometric analysis, while phase transformation kinetics, especially essential
in the CSM group, may be observed during X-ray diffraction (XRD). To assess the apatite-
forming abilities of the cements, other complementary research may revolve around Ca2+ -
releasing capability or the pH of the storage solution controlling its antibacterial activity
and leaching abilities [54,60].
A further limitation of the in vitro study is its inability to replicate the long-term
clinical performance of materials after they have been present in the cavity for many years.
The in vitro model used here does not account for factors such as long-term effects, the
absence of a dentine barrier, the immune response in human tissues, and other factors like
the age of the patient, all of which have been demonstrated to be significant in determining
the success rate of VPT [70].
The bioactive materials tested in this study require further investigation, since their
exact compositions are not yet clear, making it difficult to understand their real bioactive
properties. Nonetheless, the in vitro characterisation of the tested materials presented in
this study can serve as an initial evaluation of their potential biological behaviour. Further
studies that compare the in vitro tests with the clinical performance of the materials is
highly recommended to expand the possibilities of “smart” dental materials, accelerating
the shift from passive materials to active fillings as the widely recognised standard of
dental care.
5. Conclusions
Within the limitations of the present study, it can be concluded that the investigated
bioactive dental materials exhibited different remineralisation potential, as follows:
1. ProRoot MTA, MTA Angelus, Biodentine, and TheraCal LC showed significant surface
precipitation. Consequently, these materials also formed an interfacial layer between
the material and the dentin, filling the gap with precipitates, and demonstrated
a higher concentration of Ca within the material. However, Biodentine exhibited the
most evident sealing effect at the interfacial site.
2. Thanks to an evident bioactivity, ProRoot MTA, MTA Angelus, Biodentine, and
TheraCal LC may be suitable for remineralisation of caries dentine and pulp capping
in vital pulp therapies.
3. ACTIVA BioACTIVE, ACTIVA Presto, and Predicta Bulk exhibited inferior mineral
precipitation compared to the CSMs so they should be used only for indirect pulp
capping and/or restorative procedures.
Author Contributions: Conceptualisation, M.L.-S., S.S., M.K. and I.P.; methodology, I.P. and M.K.; formal
analysis, M.L.-S., F.I. and I.P.; investigation, M.K. and I.P.; resources, M.L.-S. and I.P.; writing—original
draft preparation, M.K. and M.L.-S.; writing—review and editing, M.L.-S., R.B, L.H., S.S. and I.P.;
visualisation, R.B., L.H. and M.K.; supervision, M.L.-S. and I.P.; project administration, M.L.-S. and
I.P.; funding acquisition, M.L.-S. All authors have read and agreed to the published version of
the manuscript.
Funding: Financed under the project № POWR.03.02.00-00-I029/16 from European Union funds.
Data Availability Statement: The data presented in this study are available on request from the
corresponding author.
Conflicts of Interest: The authors declare no conflicts of interest. The funders had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript or
in the decision to publish the results.
Appendix A
Table A1. Study groups and composition.
Group Name Composition Application and

Setting Mechanism
Control Dentinal Discs
Powder: tricalcium silicate,
ProRoot MTA dicalcium silicate, calcium
(Dentsply Sirona) dialuminate, calcium sulfate
dehydrated calcium phosphate, Left for setting for 4 h.
(PR)
calcium oxide, silica, bismuth
oxide. Liquid: distilled water.
Powder: potassium oxide,
aluminum oxide, sodium oxide,
MTA Angelus iron oxide, sulfur trioxide, Left for setting for
calcium oxide, bismuth oxide, 15 min.
(Angelus) (MA) magnesium oxide, potassium
sulfate, sodium sulfate, silica.
CSM Liquid: distilled water.
Powder: tricalcium silicate,
dicalcium silicate, calcium
Biodentine oxide, calcium carbonate, Left for setting for
(Septodont) (BD) zirconium oxide, iron oxide. 12 min.
Liquid: calcium chloride,
water-soluble polymer, water.
Liner single paste containing
calcium oxide, calcium silicate
particles (type III Portland
LCSM TheraCal LC (Bisco) cement), strontium glass,
(THC) Light-cured for 20 s.
fumed silica, barium sulphate,
barium zirconate and resin
containing Bis-GMA and
PEGDMA.
Table A1. Cont.
Group Name Composition Application and

Setting Mechanism
Control Dentinal Discs
Diurethane dimethacrylate, bis
(2-(methacryloyloxy) ethyl)
phosphate, barium glass,
RMGIC ACTIVA BioACTIVE ionomer glass, polyacrylic Self-cured for 20 s.
Liner (Pulpdent) (AB) acid/maleic acid copolymer, Light-cured for 20 s.
dual-cure chemistry, sodium
fluoride, colorants
Tooth-shade.
Etching
Nanomaterials 2024, 14, x FOR PEER REVIEW 23 of 27 Bonding agent—3M
Blend of diurethane and other Single Bond
ACTIVA Presto methacrylate resins,
(Pulpdent) (AP) Universal and
amorphous silica.
Etching light-cured for 10 s.
Bonding agent—3M Single BC light cured for
Blend of diurethane and 20 s/increment.
ACTIVA Presto Bond Universal and light-
other methacrylate resins,
(Pulpdent) (AP) cured for 10 s.
Poly(oxy-1,2-
amorphous silica.
BC light cured for 20 s/in-
ethanediyl),.alpha.′ -[(1-
crement.
methylethylidene)di-4,1-
Poly(oxy-1,2-
phenylene]bis[.omega.-[(2-
ethanediyl),.alpha.′-[(1- Etching
methyl-1-oxo-2-propenyl)oxy],
methylethylidene)di-4,1- Bonding agent—3M
BC
2-Propenoic acid, 2-methyl-,
phenylene]bis[.omega.-[(2- Single Bond
methyl-1-oxo-2-pro- 1,6-hexanediyl ester, Universal and
BC Predicta Bioactive Etching
penyl)oxy],
Bulk (Parkell) 2-Propenoic acid,
(PB) 2-Propenoic 2-methyl-, (1- light-cured for 10 s.
Bonding agent—3M Single
methylethylidene)bis[4,1-
acid, 2-methyl-, 1,6-hex- Self-cured for 30 s.
Bond Universal and light- Light-cured for 20 s.
Predicta Bioactive anediyl ester, 2-Propenoic phenyleneoxy(2-
cured for 10 s.
hydroxy-3,1-propanediyl)]
Bulk (Parkell) (PB) acid, 2-methyl-, (1- meth- PB self-cured for
Self-cured for 30 s. 5 min.
ester, 2,6-Di-tert-butyl-4-
ylethylidene)bis[4,1-phe-
Light-cured for 20 s.
methylphenol,
nyleneoxy(2- hydroxy-3,1- Dibenzoyl
PB self-cured for 5 min.
propanediyl)] ester,peroxide,
2,6- 2-Propenoic acid,
Di-tert-butyl-4-methylphe-
2-methyl-, 1,6-hexanediyl ester
nol, Dibenzoyl peroxide,
Bisphenol A-glycidyl methacrylate (Bis-GMA), Polyethylene glycol dimethacrylate (PEGDMA), Calcium silicate-
2-Propenoic acid, 2-me-
based materials (CSM), Light-cured calcium silicate-based material (LCSM), Resin-modified glass-ionomer
thyl-, 1,6-hexanediyl ester
(RMGIC), Bioactive composite (BC).
Bisphenol A-glycidyl methacrylate (Bis-GMA), Polyethylene glycol dimethacrylate (PEGDMA), Cal-
cium silicate-based materials (CSM), Light-cured calcium silicate-based material (LCSM), Resin-
modified glass-ionomer (RMGIC), Bioactive composite (BC).
Appendix B
Appendix B
TheraCal LC 24 h
Figure A1. Cont.

Nanomaterials
Nanomaterials 2024,
2024, 14,
14, xx FOR
FOR PEER
PEER REVIEW
REVIEW 24
24 of
of 27
27
LC28
TheraCalLC
TheraCal days
28days
Figure A1. Three independent EDX results of TheraCal LC after 24 h incubation period and 28
Figure A1. Three independent EDX results of TheraCal LC after 24 h incubation period and 28
Figure
days.
days.
A1. Three independent EDX results of TheraCal LC after 24 h incubation period and 28 days.
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Nanomaterials 2024, 14, 274. https://doi.org/10.3390/nano14030274 https://www.mdpi.com/journal/nanomaterials

2. Materials and Methods

SEM SEM SEM SEM SEM SEM SEM SEM

EDX EDX EDX EDX EDX EDX EDX EDX

Scheme 1. Experimental groups.

2.2. SEM Imaging

periods. Magnification 5000 × (control).

A1 dentine A2 dentine A3 dentine A4 dentine

ProRoot ProRoot ProRoot ProRoot

Nanomaterials 2024, 14, x FOR PEER REVIEW 7 of 27

MTA Angelus MTA Angelus MTA Angelus MTA Angelus

MTA Angelus MTA Angelus MTA Angelus MTA Angelus

Nanomaterials 2024, 14, x FOR PEER REVIEW 8 of 27

24 h 7 days 14 days 28 days

A1 24 h dentine A2 7 days dentine A3 14 days dentine A4 28 days dentine

A1 dentine A2 dentine A3 dentine A4 dentine

Biodentine Biodentine Biodentine Biodentine

Biodentine Biodentine Biodentine Biodentine

A1 24 h A2 7 days dentine A3 14 days dentine 28 days dentine

A1 A2 dentine A3 dentine dentine

TheraCal TheraCal TheraCal TheraCal

TheraCal TheraCal TheraCal TheraCal

A1 dentine A2 dentine A3 dentine A4 dentine

Activa BioActive Activa BioActive Activa BioActive Activa BioActive

Nanomaterials 2024, 14, x FOR PEER REVIEW 10 of 27

24 h 7 days 14 days 28 days

A1 dentine A2 dentine A3 dentine A4 dentine

Activa Presto Activa Presto Activa Presto Activa Presto

Predicta Bulk Predicta Bulk Predicta Bulk Predicta Bulk

Nanomaterials 2024, 14, x FOR PEER REVIEW 12 of 27

Nanomaterials 2024, 14, x FOR PEER REVIEW 12 of 27

ProRoot MTA Activa BioActive Activa Presto

Nanomaterials 2024, 14, x FOR PEER REVIEW 13 of 27

Figure 14. Cont.

ACTIVA Presto showed an unchanged surface morphology after incubation in DPBS.

Nanomaterials 2024, 14, x FOR PEER REVIEW 15 of 27

ACTIVA Presto showed an unchanged surface morphology after incubation in DPBS.

Predicta Bulk revealed no statistically significant changes in composition during the

Nanomaterials 2024, 14, x FOR PEER REVIEW 16 of 27

Table A1. Study groups and composition.

Group Name Composition Application and

Table A1. Cont.

Group Name Composition Application and

Figure A1. Cont.

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