d e n t a l m a t e r i a l s 3 3 ( 2 0 1 7 ) e221–e238

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Osteoinductive potential and bone-bonding ability

of ProRoot MTA, MTA Plus and Biodentine in rabbit
intramedullary model: Microchemical
characterization and histological analysis

M.G. Gandolfi a,b,∗ , G. Iezzi c , A. Piattelli c , C. Prati b , A. Scarano c

a Laboratory of Biomaterials and Oral Pathology, Dental School, Department of Biomedical and NeuroMotor Sciences,
University of Bologna, Bologna, Italy
b Endodontic Clinical Section, Dental School, Department of Biomedical and NeuroMotor Sciences, University of

Bologna, Bologna, Italy

c Department of Medical, Oral and Biotechnological Sciences, University of Chieti-Pescara, Chieti Scalo, Italy

a r t i c l e i n f o a b s t r a c t

Article history: Objective. To study the in vivo osteoinductive potential, bone-bonding ability (bioactivity) and
Received 8 March 2016 bone biomineralization of current hydraulic calcium silicate cements used as graft materials
Received in revised form and placed in contact with medullary bone.
10 January 2017 Methods. ProRoot MTA, MTA Plus and Biodentine were used to fill surgical bone defects (2-
Accepted 31 January 2017 mm diameter through the entire cortical thickness to reach the medullary bone) in the tibia
of mature male rabbits. Tibiae were retrieved after 30 days and submitted to histological
analysis and microchemical characterization using Optical Microscopy (OM) and Environ-
Keywords: mental Scanning Electron Microscopy with Energy Dispersive X-ray analysis (ESEM-EDX).
Hydraulic calcium silicate cements Bone neoformation and histomorphometric evaluations, degree of mineralization (by Ca/P,
ProRoot MTA Ca/N and P/N ratios) and the diffusion of material elements were studied.
MTA Plus Results. Bone neoformation was observed in response to all materials. No sign of necrosis
Biodentine were found on the walls of the pre-existing cortical bone. No osteoclasts and no formation
Osteoinduction of fibrous tissue were evident. Sign of angiogenesis were present.
Bone-bonding (bioactivity) EDX (element content, line profile and element mapping) showed the increase in Ca and
Mineralization degree P and decrease in C, S and N from the mature bone towards the mineralizing interface.
Ca/P ratio Ca/P, Ca/N and P/N ratios showed differences in the degree of mineralization/maturation
Ca/N ratio stage of bone.
P/N ratio MTA Plus and ProRoot MTA exhibited close contact with the pre-existing bone and good
bone-bonding with neoformed bone juxtaposed on the medullary side of the materials with-
out interposed connective tissue or resorption lacunae or gaps. The materials showed a
dense appearance with 100% of residual materials and no colonization by fluids and cells.
No migration of Bi or Al material elements to the newly formed bone was found.
Biodentine showed newly formed trabecular bone with marrow spaces and sparse traces
of residual material (≈9%).
Significance. The in vivo osteoinductive properties with dynamic biomineralization processes
around these calcium silicate materials extruded in medullary bone in appropriate animal

∗
Corresponding author.
E-mail address: mgiovanna.gandolfi@unibo.it (M.G. Gandolfi).
http://dx.doi.org/10.1016/j.dental.2017.01.017
0109-5641/© 2017 The Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.
e222 d e n t a l m a t e r i a l s 3 3 ( 2 0 1 7 ) e221–e238

model have been demonstrated by ESEM-EDX in association with OM. Good biocompatibility
was evident as only slight inflammatory infiltrate and no sign of necrosis at the interface
with the pre-existing bone were found.
MTA Plus and ProRoot MTA exhibited bioactive potential as they can bond to bone directly
without interposed connective tissue. Biodentine was replaced by newly formed bone.
Clinical significance. The results of the study demonstrate the capacity of calcium silicate
cements to allow osteoid matrix deposition by activated osteoblasts and favour its biomin-
eralization, and to achieve a direct bond between the (bioactive) materials surface and the
mineralized bone matrix.
© 2017 The Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

Differently, a mild-to-moderate chronic inflammatory cell

1. Introduction infiltration consisting of lymphocytes, macrophages, fibrob-
lasts, and some giant cells was present in a thin fibrous
Root-end filling materials are placed in contact with the
capsule generated at 30 days as response to MTA Angelus
medullary alveolar bone when used to seal resected root
inside polyethylene tubes in rat alveolar sockets [20]. The
apices, to plug root perforations and to fill wide open apices.
fibrous capsule was present near the tube was thin, and the
Wide open apices in nonvital immature teeth and in over-
bone tissue with dystrophic calcification was close to the
instrumented root canals with a resected apex need the
material [20]. The intensity of the inflammation reduced with
orthograde placement of appropriate sealing materials as api-
time and was absent at 90 days.
cal barrier/plug [1–3]. A critical point is the biological response
Similarly, bone tissue reaction to ProRoot MTA filling
of the periapical medullary bone, as large amounts of mate-
implantation cavities studied in a rat femur model showed
rial can be extruded into the periapical tissue when used
a decrease of the number of inflammatory cells together with
to plug wide apices and root perforations, producing toxic
the increase of the new bone formation with the implantation
effects, (additional) tissue inflammation and foreign body
time [21].
reactions. Therefore, the use of biocompatible osteoconduc-
Otherwise, both ProRoot MTA and an experimental
tive (preferably osteoinductive) materials [4–6] is advocated to
dicalcium silicate implanted in distal mesial rabbit femur
avoid extrusion-related complications or the impairment of
incorporated well with the surrounding tissue and exhibited
the bone healing process.
no inflammatory response, rejection or necrosis in the adja-
Currently, hydraulic calcium silicate cements represent the
cent host tissue [22].
golden standard among the materials for root-end filling to
Bone healing and minimal inflammatory response adja-
seal/close the root in root apex resection, root perforation
cent to MTAs implanted in proximal rabbit femur were
repair and apexification in relationship to their specific and
observed at 3–12 weeks [23].
suitable chemical–physical (they are non resorbable materials
MTAs and Portland cement implanted in rabbit mandible
setting in fluid/blood contaminated environments [7,8] having
showed bone healing and regeneration [24].
slight degradability and low solubility [6,9,10]) and biointerac-
Experimental calcium silicates cements implanted in bone
tive properties correlatable with their positive biological effect
defects in rabbit tibiae showed bone repairing capacity and
[6]. Their ability to release biologically relevant ions [10,11] and
osteoconductive potential [25].
the property to nucleate calcium phosphates/apatite [11,12]
In the present study current hydraulic calcium silicate
suggest their pivotal role in mineral tissue regeneration by
cements (Biodentine, MTA Plus and ProRoot MTA) have been
activating the osteogenic potential and promoting the dif-
used as graft materials in bone defects and intentionally
ferentiation of mineralizing-cells as human bone marrow
placed in contact with the medullary cavity through the entire
stromal cells, human orofacial bone marrow mesenchymal
cortical thickness of rabbit tibia.
stem cells [13–15] and osteoblasts [16,17].
The histological analysis associated with the microchem-
Previous histological studies on the intraosseous place-
ical characterization by ESEM-EDX have been performed to
ment of hydraulic calcium silicate cements in different animal
study their osteoinductive potential and bone-bonding ability
models showed contradictory results.
(bioactivity) and the morphostructural features of the heal-
Bone healing and minimal inflammatory response adja-
ing bone tissue at the interface with cancellous/medullary
cent to the implants were observed as response to freshly
bone.
mixed ProRoot MTA or Portland cement inserted (inside cylin-
drical Teflon applicators, 2 mm diameter and 2 mm length)
into the bone cavities in guinea pigs mandible [18].
2. Materials and methods
A toxicity level diminishing with elapsing time and excel-
lent biological qualities with bone growth in close contact with
2.1. Surgical procedure
the material and no interposing connective tissue has been
reported for ProRoot MTA implanted in the lower jaw symph-
Skeletally mature (9 month old, 3.5 kg weigh) pathogen free
ysis of guinea-pigs [19].
(SPF) and virus antibody free (VAF) male New Zealand white
 d e n t a l m a t e r i a l s 3 3 ( 2 0 1 7 ) e221–e238 e223

rabbits (Crl:KBL(NZW)) obtained from Charles River Labo-

ratories (Lieu-dit Oncins, France) were used. The protocol
conformed to the guiding principles of ISO 10993-2 (Animal
welfare requirements) and ISO 10993-1 (Part 6: tests for local
effects after implantation) [26] and was approved by the Ethi-
cal Committee of University of Chieti-Pescara, Italy.
The rabbits were anesthetized with intramuscular injec-
tions of fluanizone (0.7 mg/kg body weight) and diazepam
(1.5 mg/kg body weight), and local anaesthesia was given using
1 mL of 2% lidocain/adrenalin solution. The medial surface of
the tibiae was exposed via a skin excision with a periosteal
flap [27–29]. Care was taken to split the muscular layer by
blunt dissection and to keep the periosteum intact apart from
the longitudinal excision. Cylindrical noncritical-sized bone
defects (2 mm in diameter) were performed in the medial dia-
physeal face of rabbit tibia using a 2 mm diameter drill working
at 300 rpm under constant copious saline irrigation. The bone
excision was performed through the entire cortical thickness
in order to reach the medullary space. Defects were filled
with Biodentine (Septodont, Saint-Maur-des-Fossés, France;
batch number B01767), MTA Plus with gel (Prevest Detpro Lim-
ited, Jammu, India; lot n. 41001) or ProRoot MTA (Dentsply
Tulsa, Johnson City, TN, USA; batch number 09003850) as graft
materials (n = 6 for each material in 6 different animals, fol-
lowing ISO 10993-1) [26]. All cements were prepared following
the manufacturer directions and inserted manually into the
bone defects using a stainless steel spatula, and extruded into
the medullary area. In the control group the noncritical-sized
defects were left empty and were allowed to heal sponta-
neously.
Fig. 1 – Surgical procedure. Cylindrical noncritical-sized
The overlying soft tissues, periostium and fascia, were
bone defects (2 mm in diameter) performed in the medial
sutured with catgut and the skin with silk suture. Eight bone
diaphyseal face of rabbit tibia and filled with Biodentine,
defects were created in each animal, 4 in the right tibia, and 4
MTA Plus or ProRoot MTA.
in the left tibia (Fig. 1).
Oxytetracycline dihydrate (Terramicina long Acting by
Pfizer Italia srl) 100 mg/kg single dose and analgesics with
tramadol hydrochloride (Altadol Abiogen Pharma S.p.A Italia) diamond disc at about 150 ␮m and ground down to about
were given for 1 week. Sutures were removed 2 weeks after 30 ␮m. Then, the slides were stained with acid fuchsin and
surgery. Postsurgical visits were scheduled daily to check the toluidine blue.
course of healing. No complications or deaths occurred in the
postoperative period. 2.3. Histological analysis
The animals were pharmacologically euthanized 30 days
after surgery (following ISO 10993-1 for prolonged-permanent 2.3.1. EDX microchemical analysis and ESEM examination
long-term contact of implant devices for bone tissue) with an The histological sections were examined using an envi-
overdose of intravenous Tanax (Intervet Italia srl, Peschiera ronmental scanning electron microscope (ESEM, Zeiss EVO
Borromeo, Mi, Italy) under general anaesthesia with intra- 50; Carl Zeiss, Oberkochen, Germany) connected to a sec-
muscular injections of fluanizone (0.7 mg/kg body weight) and ondary electron detector for energy dispersive X-ray analysis
diazepam (1.5 mg/kg body weight). Then, the bone samples (EDX; Oxford INCA 350 EDS, Abingdon, UK) using computer-
were retrieved and processed for histological analysis. controlled software (Inca Energy Version 18). The sections
were examined uncoated at low vacuum (100 Pascal), 20 kV
2.2. Histological specimen processing accelerating voltage, 8.5 mm working distance, 0.5 wt% detec-
tion level, 133 eV resolution, 100 microseconds amplification
The retrieved specimens were immediately stored in 10% time, measuring time: 600 s for element mapping and 60 s for
buffered formalin and processed to obtain thin ground sec- spectra. The resulting electron beam penetration inside was
tions with the Precise 1 Automated System (Assing, Rome, approx. 2 ␮m.
Italy) [30,31]. They were dehydrated in an ascending series of ESEM histological observations were performed at 100×,
alcohol rinses and embedded in a glycolmethacrylate resin 1500× and 3000× magnification.
(Technovit 7200, VLC, Kulzer, Wehrheim, Germany). After EDX microchemical analysis (elemental X-ray microanaly-
polymerization, the specimens were sectioned longitudinally sis) was carried out at random in areas of approx. 50 × 50 ␮m to
along the major axis of the rabbit tibiae with a high-precision evaluate the relative element content. Microanalysis (weight
e224 d e n t a l m a t e r i a l s 3 3 ( 2 0 1 7 ) e221–e238

Fig. 2 – ESEM showed the surgical defect completely filled by compact ProRoot MTA and the neoformed bone juxtaposed to
the material on its medullary side.
EDX on the material displayed its constitutive elements i.e., Ca, Si, Bi and traces of Al, pre-existing trabecular bone showed
Ca and P, neoformed bone revealed Ca, P, absence of Bi, and traces of Si deriving from the material. The Ca/N, P/N and Ca/P
ratios are reported. Ca/N and P/N showed higher values in the pre-existing bone than in the newly formed bone; Ca/P in
neoformed trabeculae approached the value of the mature bone.

% and atomic %) with ZAF correction method was performed
in full frame and spot mode to analyze entire areas or specific
points respectively. The Ca/P, Ca/N and P/N ratios were calcu-
lated from the data to evaluate the degree of mineralization of
the newly formed bone.
EDX element mapping was performed at the bone-material
interface to detect the element distribution within the mature
and neoformed bone and the presence in the surrounding tis-
sues of elements from the implanted material.
Element mapping was performed using 512 × 384 pixel
matrix, 30–40 frames, 100 ␮s dwelling time, 600–700 total read-
ing time.
Line profile (line scans) through the bone-interface-
material were performed to detect the variation content of Ca,
P, S, C and N from the mature pre-existing bone towards the
implanted material.
Scan line was carried out using 300 s reading time.
2.3.2. OM examination and histomorphometry
Histological analysis and histomorphometry of the percent-
age of newly formed bone, marrow spaces and residual
biomaterial was carried out using a light microscope (Leitz
Laborlux, Wetzlar, Germany) connected to a high resolu-
tion video camera (3CCD, JVC KY-F55B, JVC, Yokohama,
Japan) and interfaced to a monitor and PC (Intel Pentium
III 1200 MMX, Intel, Santa Clara, CA, USA). This optical sys-
tem was associated with a digitizing pad (Matrix Vision
GmbH, Oppenweiler, Germany) and a histometry software
package with image capturing capabilities (Image-Pro Plus,
Media Cybernetics Inc., Immagini e Computer Snc, Milano,
Italy).
 d e n t a l m a t e r i a l s 3 3 ( 2 0 1 7 ) e221–e238 e225

Fig. 3 – Interface between ProRoot MTA and newly formed bone at high magnification. The perfect contact with absence of
gaps of the neoformed trabeculae with the material and the absence of Bi or Al in the newly formed bone are evident. Note
in the BS image the bright small points of Bi.

Neoformed bone juxtaposed and strictly in contact with the

3. Results material on its medullary side demonstrated the osteoinduc-
tive properties of ProRoot MTA.
3.1. ProRoot MTA EDX microchemical characterization (Fig. 2) on the material
displayed its constitutive elements i.e., Ca, Si, Bi and traces of
The histological section observed by ESEM (Fig. 2) showed
Al.
the surgical defect completely filled by compact material.
e226 d e n t a l m a t e r i a l s 3 3 ( 2 0 1 7 ) e221–e238

Fig. 4 – EDX element mapping showed ProRoot MTA elements, mainly Ca and Si that clearly marks the filled area. Note the
presence of P only in bone, whilst Si, amounts of Al and two granules of Bi only in the material.

Fig. 5 – EDX profile across the interface, from the inner of the material to the newly formed bone showed the decrease of Si
and Bi and the increase of Ca and P. No diffusion of Bi from ProRoot MTA into the new trabeculae was detected. The
presence of Si into the first 100 microns of the newly formed bone was found.
The interface can be identified by the fall of the P and by the rise of Si and Al, all at the same point (i.e., approx. at 270 ␮m in
the abscissa scale).
 d e n t a l m a t e r i a l s 3 3 ( 2 0 1 7 ) e221–e238 e227

Fig. 6 – Surgical bone defect filled with ProRoot MTA. The
pre-existing bone (PB) showed osteocytes (Oc) inside their
lacunae. A neat demarcation could be observed at the
pre-existing bone-material interface without interposed
connective tissue or resorption lacunae on its surface.
Toluidine blue and acid fuchsin. Magnification 40×.
Fig. 7 – In the marrow space (MS), a moderate
inflammatory infiltrate (II) was observed, although spicules
of newly formed bone (NB) could be seen. A tight contact
between ProRoot MTA and bone is present in the medullary
diaphyseal portion.
Toluidine blue and acid fuchsin. Magnification 200×.

Pre-existing trabecular bone showed Ca and P. Neoformed

bone revealed Ca, P, absence of Bi, and traces of Si deriving
from the material.
The Ca/N and P/N ratios were markedly higher in the
pre-existing mature bone that in the newly formed bone,
in particular in the new trabeculae adjacent to the material
(likely the newest bone).
The Ca/P ratio in neoformed trabeculae was quite high,
approaching the value of the mature bone, likely due to a more
mature bone and/or to a diffusion of Ca (similarly to Si) to the
surrounding/adjacent tissues. In addition, the high Ca/P ratio
calculated in some interface analysis (see points 12 and 15)
can be affected and raised by the constitutive Ca of the filling
material.
The analysis of the interface at high magnification (Fig. 3)
displayed the perfect contact and continuity of the neoformed
trabeculae with the material and well displayed the absence
of gaps. No Bi or Al was detected in neoformed bone but only
at the interface. Fig. 8 – ProRoot MTA 30 days. A fragment of material (RM)
EDX (Fig. 4) mapping showed the material elements, mainly surrounded by new bone (NB) could be observed in the
Ca and Si, that clearly mark the filled area. Amounts of Al and medullary side (MS). Osteoblasts (Ob) rimming the newly
sparse Bi charged particles were visible into the cement. formed bone as signs of bone neoformation could be seen
EDX profile across the interface (Fig. 5), from the inner part (arrow). Inflammatory cells (IC) could be detected in the
of the material to the newly formed bone showed the decrease marrow space (MS).
of Si and Bi and the increase of Ca and P. In particular, no Toluidine blue and acid fuchsin. Magnification 100×.
diffusion of Bi from the material into the new trabeculae was
found, whilst a migration of Si into the first 100 microns of the
newly formed bone was detected.
OM histological analysis showed the bone defects com-
pletely filled with the material, which showed a dense bone (Fig. 7), inflammatory cells were detected in the marrow
appearance and tight contact with pre-existing bone with- space together with material fragments partially surrounded
out interposed connective tissue or resorption lacunae on by bone and trabeculae of newly formed bone (Fig. 8). No
its surface (Fig. 6). The material on its medullary side was multinucleated cells were evident. Signs of angiogenesis with
in contact for the 20% of its surface with newly formed formation of capillaries close to the material were observed.
e228 d e n t a l m a t e r i a l s 3 3 ( 2 0 1 7 ) e221–e238

Fig. 9 – Surgical bone defect filled with MTA Plus after 30 days. EDX detected low Ca (1.74–1.9 wt%) in the material (points 7,
8, 11). No S was found in the material and its content was higher in the newly formed bone (0.16–0.33 wt%) than in the
pre-existing bone (0.0–0.13 wt%).
The Ca/N and P/N ratios were higher in the pre-existing bone (0.54–0.71 and 0.33–0.39 wt% respectively) than in the newly
formed bone (0.33–0.38 and 0.2–0.22 wt% respectively). Similar behavior was obtained for Ca/P ratio.

The histomorphometric analysis showed 100% residual
biomaterial, 0% of newly formed bone and 0% of marrow
spaces inside the defect.
3.1.1. MTA Plus gel
The histological section observed by ESEM showed that the
surgical defect was completely filled with MTA Plus and tra-
beculae of newly formed bone were grown on its medullary
side (Fig. 9).
EDX on the material displayed its constitutive elements i.e.,
Ca, Si, S, Bi and traces of Al (Fig. 9). Pre-existing trabecular bone
showed higher Ca and P content, traces of S and absence of Bi.
Newly formed bone revealed Ca, P, S, and minimal traces of Si
and Bi deriving from the material. No diffusion of the new tra-
beculae with Bi or Al was detected, likely in relationship with
the compactness and anti-washout property of the material.
The Ca/N and P/N ratios were higher in pre-existing mature
bone (0.54–0.71 and 0.33–0.39 respectively) than in newly
formed bone (0.33–0.38 and 0.2–0.22 respectively). Similar
behaviour for Ca/P ratio.
The analysis of the interface at high magnification (Fig. 10)
showed the continuity between the material and the newly
formed bone tissue, without gaps and confirmed the absence
of Bi and Al in the newly formed bone at the interface
with the biomaterial. EXD analyses on MTA Plus (points 10
and 11) showed the silicatic component of the material (Si
4.89–5.51 wt%) and the presence of high Bi (3.37–5.06 wt%) and
traces of Al.
 d e n t a l m a t e r i a l s 3 3 ( 2 0 1 7 ) e221–e238 e229

Fig. 10 – Interface at high magnification. No diffusion of Al and Bi was found at the interface and in the new bone whilst
traces of Si were detected. EXD analyses on MTA Plus (points 10 and 11) showed the silicatic component of the material and
the presence of Bi and high Si and traces of Al. Note in the BS image the bright small points of Bi.

EDX element mapping (Fig. 11) showed the MTA Plus ele-
ments, mainly Si, Bi, Al that clearly marked the filled area. Ca
and P were displayed by the pre-existing bone.
EDX profile across the interface (Fig. 12), from the inner
part of the material to the newly formed bone showed the
decrease of Si and Bi and the increase of Ca and P. No migration
of material elements from MTA Plus to the newly formed bone
was found.
EDX on the outer (periosteum) side (Fig. 13) displayed a Ca/P
ratio of immature bone (approx. 1.16) and the presence of S.
No Bi was found and only traces of Si were detected. Inter-
estingly, the material showed the formation of bone on the
outer (periosteum) side of the graft (Fig. 13), demonstrating
osteoinductive properties.
Microanalyses displayed low Ca (1.77 wt%) together with
high Bi (7.13 wt%) and Si (7.89 wt%), and traces of Al. No dif-
fusion of Bi or Al was revealed in the newly formed bone at
the outer (periosteum) side (points 1–3) nor at the interface
(points 4, 6). Pre-existing bone showed high Ca and P (points
7–9) and higher Ca/P, Ca/N and P/N ratios compared to the
new bone (points 1–3). S content was higher in the new bone
(0.28–0.46 wt%) than in the pre-existing bone (0.16–0.18 wt%).
At OM histological analysis the bone defects appeared com-
pletely filled by residual biomaterial (Fig. 14), which was dense
and not colonized by fluids and cells.
A neat demarcation could be seen at the preexisting bone-
biomaterial interface with no signs of bone neoformation, but
e230 d e n t a l m a t e r i a l s 3 3 ( 2 0 1 7 ) e221–e238

Fig. 11 – EDX element mapping showing MTA Plus elements, mainly Si, Bi, Al marking the filled bone defect, and Ca and P
displayed by the pre-existing bone. Note the presence of Si only in the material, with traces of Bi and Al.

Fig. 12 – EDX profile across the interface from the inner of the material to the newly formed bone showing the decrease of Si
and Bi and the increase of Ca and P. No migration of MTA Plus elements from MTA Plus to the newly formed bone was found.
The sudden rise of the P and by the fall of Si, Al and Bi identify the interface (at approx. at 240 ␮m in the abscissa scale).

the bone was vital as osteocytes could be seen inside the lacu-
nae (Fig. 15).
MTA Plus on its medullary side was surrounded by newly
formed bone for 60.5% of its surface, although the biomaterial
margins were not defined. Multinucleated cells were absent
and only few inflammatory cells were present; in some areas
osteoblasts were observed (Fig. 16).
Close to the newly formed bone and to the biomaterial,
small vessels were present.
The histomorphometric analysis showed 100% residual
biomaterial, 0% of newly formed bone and 0% of marrow
spaces inside the defect.
 d e n t a l m a t e r i a l s 3 3 ( 2 0 1 7 ) e221–e238 e231

Fig. 13 – EDX on the outer (periosteum) side displayed a Ca/P ratio approx. 1.16 (immature bone), the presence of S, the
absence of Bi was found and only traces of Si. The formation of bone on the periosteal side demonstrated the
osteoinductive properties of MTA Plus.

3.1.2. Biodentine
The histological section observed by ESEM (Fig. 17) showed the
trabeculae of newly formed bone filling the surgical defect.
The new bone was in close contact with the resected walls of
the pre-existing cortical bone. Wide lacunae were visible and
sparse traces of residual material were detected.
EDX (Fig. 17) showed the presence of the constitutive ele-
ments of Biodentine and bone. Traces of Biodentine (Zr and
Si) were noticed only in rare points. The S content was higher
in the newly formed bone whilst the Ca/P ratio (≈1.61) was
lower than in the pre-existing bone (1.72–2.13 wt%). Interest-
ingly, the newly formed bone showed a high mineralization
degree (high Ca/N and P/N ratios).
EDX element mapping (Fig. 18) showed residual constitu-
tive elements such as Si, Ca, Cl, and mainly Zr that clearly
marks the entire neoformed area.
EDX profile across the interface from the new trabeculae to
approx. 600 ␮m inside the cortical bone (Fig. 19), showed the
increase of Ca and P as expected going from the new immature
bone (bottom left) to the new more mature/calcified bone (top
e232 d e n t a l m a t e r i a l s 3 3 ( 2 0 1 7 ) e221–e238
Fig. 14 – Surgical bone defect filled with MTA Plus gel. The
bone defect was completely filled with the material (RM),
which showed a dense appearance and was partially
surrounded by newly formed bone (NB) on both the internal
(medullary) and outer (periosteal) sides.
Toluidine blue and acid fuchsin. Magnification 40×.
Fig. 15 – At the pre-existing bone-MTA Plus material
(PB-RM) interface, neither gaps, interposed connective
tissue nor signs of bone neoformation were evident.
Osteocytes (Oc) could be observed inside the lacunae of
cortical bone.
Toluidine blue and acid fuchsin. Magnification 100×.
right). N was almost stable whilst S, Si and Zr increased. Si and
Zr appeared incorporated into the newly formed bone during
the resorption of the material.
OM histological analysis showed newly formed trabecular
bone with marrow spaces and remnants of material inside
Fig. 16 – Newly formed bone (NB) surrounds the portion of
MTA Plus material (RM) facing the medullary side (MS) of
the tibia. The inflammatory infiltrate (II) was scarce. Small
vessels (V) were present close to the material.
Toluidine blue and acid fuchsin. Magnification 200×.
the bone defects (Fig. 20). Wide osteocyte lacunae, typical of
recently mineralized bone, were detected in the bone trabecu-
lae. In some areas, osteoid matrix undergoing mineralization,
but no osteoblasts were present. Osteons were only observed
in close proximity of the newly formed bone adjacent to pre-
existing bone (defect margins) (Fig. 21). Multinucleated giant
cells next to bone chips were evident, indicating bone remod-
eling. No remnants of the intentionally extruded material into
the medullary cavity were found.
In the marrow spaces inside the defects, several small rem-
nants of Biodentine were present and surrounded by bone
and osteoid matrix (Fig. 22). Multinucleated giant cells were
not observed. Mild inflammatory infiltrate was evident. Small
newly formed vessels were observed. The histomorphomet-
ric analysis showed 9 ± 4% of residual biomaterial, 45 ± 7% of
newly formed bone and 44 ± 7% of marrow spaces.
3.1.3. Control
The control bone defects appeared filled by newly formed
trabecular bone with small marrow spaces (Fig. 23), and no
inflammatory infiltrate. Osteoblastic activity could be detected
as well as osteoid matrix undergoing mineralization. EDX
analyses showed lower mineralization degree (Ca/N and Ca/P
ratios).
4. Discussion
In this study, current hydraulic calcium silicate cements
for oral surgery have been evaluated in vivo by OM and
ESEM-EDX analyses for their in vivo bone regeneration/repair
potential, bone-bonding ability and mineralizing properties in
noncritical-sized intramedullary bone defects in rabbit model
simulating the clinical application. Biodentine, MTA Plus and
ProRoot MTA used to fill bone defects through the entire cor-
 d e n t a l m a t e r i a l s 3 3 ( 2 0 1 7 ) e221–e238 e233

Fig. 17 – Surgical bone defect filled with Biodentine. Only traces of residual material (visible for the small white dots of Zr)
are present. The bone defect appeared completely filled by bone trabeculae in formation. The material showed very good
osteogenic bioactivity.
Microanalysis on points 7–9 showed the presence of residual material and displayed Zr (0.36–2.21 wt%), Ca (3.15–3.78 wt%)
and Si (1.76 wt%) only in point 7. Ca/P of the pre-existing bone (1.72–2.13 wt%) was higher than in the new one (≈1.61 wt%).
Interestingly, the P/N ratios of the points 10–13 of the new bone showed highly mineralized new bone i.e., higher P/N ratios
than in the pre-existing mature bone (≈0.14). Ca/N ratios as well were higher in the new bone (0.26–0.33 wt%) than in the
pre-existing bone (≈0.25). S content was higher in the new trabeculae (0.21–0.34 wt%) than in the pre-existing bone
(0.0–0.18 wt%).

tical thickness of rabbit tibia and intentionally extruded into
the intramedullary demonstrated osteoconductive and osteo-
productive behaviour (bone growth on material surface due to
enhanced osteoblasts activity).
This study proved the osteogenic effect of calcium sili-
cate cements as a marked bone neoformation with active
biomineralization processes around the materials intention-
ally extruded in the medullary region of the bone. Moreover,
the absence of necrotic processes of the pre-existing corti-
cal bone when in contact with the cements has been clearly
shown.
In this study EDX spectroscopy has been innovatively
used as a non-destructive, sensitive analytical method for
the assessment of the mineral content variations in micro-
scopic regions of bone [32]. Moreover, the present investigation
demonstrates that qualitative and semiquantitative analy-
sis of inorganic components of biological samples during the
bone repair process may be performed by Energy-dispersive
X-ray (EDX) spectroscopy. Specimens showed the relation
e234 d e n t a l m a t e r i a l s 3 3 ( 2 0 1 7 ) e221–e238

Fig. 18 – EDX element mapping showed residual constitutive elements of Biodentine as Si, Ca, Cl, and mainly Zr that clearly
marks the entire neoformed area. Note the bright small points of Zr.

Fig. 19 – EDX profile across the interface from the new immature bone (bottom left) to approx. 600 ␮m inside the more
mature/calcified bone (top right) showing the marked increase of Ca and P; N was almost stable whilst S, Si and Zr slightly
increased. Si and Zr appeared incorporated into the newly formed bone during the resorption of Biodentine material.
 d e n t a l m a t e r i a l s 3 3 ( 2 0 1 7 ) e221–e238
Fig. 20 – Surgical bone defect filled with Biodentine. Newly
formed trabecular bone (NB) with small marrow spaces
(MS) and remnants of material (RM) could be observed into
the bone defect.
Toluidine blue and acid fuchsin, magnification 40×.
Fig. 21 – Biodentine 30 days after implantation. Osteons (O)
were evident in the newly formed bone (NB) adjacent to
pre-existing bone (PB), and osteoid matrix (OM) undergoing
mineralization was present; residual material (RM) was
detected into the bone defect.
Toluidine blue and acid fuchsin, magnification 100×.
between calcium and phosphorus (Ca/P) closest to stoichio-
metric hydroxyapatite.
The X-ray microprobe and EDX mapping allowed to iden-
tify different mineralizing zones and to map the demarcation
between the mineralizing and the mineralized bone areas;
thus, differences in the mineral component reflect the dif-
ferences between cancellous and cortical bone and between
young and pre-existing mature bone. The markedly lower
Ca/P, Ca/N and P/N ratios of trabecular sections reflects the
e235
Fig. 22 – Small remnants of Biodentine material (RM)
surrounded by osteoid matrix were present in the marrow
spaces (MS). Mild inflammatory infiltrate was evident.
Small newly formed vessels were observed.
Toluidine blue and acid fuchsin, magnification 200×.
Fig. 23 – Control 30 days after osteotomy. Newly formed
trabeculae (NB) and osteoid matrix (OM) undergoing
mineralization together with interposed marrow spaces
(MS) could be observed.
Toluidine blue and acid fuchsin. Magnification 40×.
remodeling-induced formation of the “younger” trabecular
bone.
An increasing content of Ca and P and the decrease of C, S
and N from the mature bone toward the mineralizing interface
was showed by the element content, line profile and element
mapping.
Silicon was detected in the interface of ProRoot MTA and
MTA Plus. Increasing the distance with the interface, Si was
noticed in traces and disappeared at a distance of approx.
200 ␮m from the material surface. Interestingly, both MTA Plus
e236 d e n t a l m a t e r i a l s 3 3 ( 2 0 1 7 ) e221–e238
and ProRoot MTA after 30-day implantation showed a dense
appearance (100% of residual material in the surgical defect)
with no colonization by fluids or cells, and no diffusion of Bi
or Al. No Bi was detected in the neoformed bone.
Ca/P ratio obtained by EDX microanalysis for the new
formed bone/bone at the interface was in the range of the
values expected for apatites.
Interestingly, the MTA Plus showed the formation of bone
on the outer (periosteum) side of the graft, index of osteogenic
bioactivity and osteoinductivity.
The histological analyses showed that Biodentine, MTA
Plus and ProRoot MTA implanted intramedullary induced bone
neoformation, osteoblasts differentiation and angiogenesis.
All the materials demonstrated good biocompatibility as
only mild or moderate inflammatory infiltrate was evident
very close to the interface.
ProRoot MTA and MTA Plus can bond directly to bone with-
out encasement by a fibrous connective tissue: the in vivo
bioactive potential of these cements following their surgical
implantation in appropriate animal model has been proven.
A marked osteoinductivity with bone repair and dynamic
biomineralization processes was found for ProRoot MTA and
MTA Plus extruded in the medullary region. Clearly, the mate-
rials provided a suitable scaffold for osteoprogenitor cells
homing, osteoblasts differentiation, and bone growth process.
Differently, Biodentine was replaced by newly formed bone.
The osteoinductive effect and mechanism involved in
enhancing bone repair is likely correlated with the release of
biologically active ions from calcium silicate cements [10,11]
and the formation of calcium phosphates and apatite [6,11,12].
Bone-bonding is accomplished via the production of
osteoid matrix laid down on the surface of the scaffold by mes-
enchymal stem-osteoprogenitor cells migrated on the surface
and differentiated into active osteoblasts.
The study demonstrates the capacity of hydraulic calcium
silicate cements to allow osteoid matrix deposition by acti-
vated osteoblasts and favour its biomineralization, and to
achieve a direct bond between the (bioactive) materials surface
and the mineralized bone matrix.
The creation of a bone forming osteoblastic microenviron-
ment by calcium silicate cements (ion-exchanging interface
and solution-mediated effect on cellular activity) could
explain the good biological (osteogenic) response. It is known
that bioactivity is related to the chemical reactivity of the
material (biointeractivity) responsible for interface dissolu-
tion, precipitation and ion-exchange reactions [33] and that
bone formation by induction initiates by the invocation of
osteogenic insoluble signals or substrata that combined with
soluble molecular signals (as calcium) [34–37], trigger the cas-
cade of cell differentiation into osteoblastic cell lines secreting
bone matrix at site of surgical implantation [38].
Hydraulic calcium silicate cements form a hydrated silica-
rich layer, able to release calcium ions and increase local pH
[11], and therefore create at their interface a local microenvi-
ronment favourable for the apatite formation [6,12,14] and the
supplying of biologically active ions through their surface. In
addition, the presence of silicon on these materials could pro-
vide additional positive stimulus to bone neoformation as Si
ions could induce angiogenesis during bone regeneration by
increasing gene expression [39] and considering the intimate
relationship between invading capillaries and osteoblastic cell
differentiation, synthesis and matrix deposition [40,41].
Actually, in the present study the tested calcium silicate
cements allowed angiogenic activity with neovascularization
and formation of capillaries close to the materials.
In addition, MTA Plus and ProRoot MTA were in contact
with bone without interposed connective tissue or resorption
lacunae at both the interfaces with the pre-existing bone and
medullary side. Only few monocytes and lymphocytes were
observed in the marrow space and no osteoclasts or multin-
ucleate giant cells (osteoclast precursors) were found. We can
believe that the alkalinizing activity may have a strong effect
on osteoclasts recruitment and activation. Indeed, it has been
demonstrated that basic pH inactivates osteoclasts [34,42–44]
and previous studies showed a strong alkalinizing activity of
ProRoot MTA and MTA Plus, and less for Biodentine [6].
In the present study the rabbits were 9 months old and the
experimental time of 30 days was selected to obtain bone tis-
sue at the mineralizing/remodelling healing stage. Noncritical
size bone defects have been selected as the repair of critical-
size bone defects (6–8 mm diameter) in rabbit tibia occurs by
highly vascularised fibrous and granulation connective tissues
prevailing over healing/immature bony tissue.
Rabbit is a convenient model for skeletal research studies
[18,32,42] and has been extensively used to test the osteo-
conductive/osteoinductive reaction to implant biomaterials
[26,27,45–47]. In addition rabbit model provides an excellent
cost-effective animal model, their maintenance and housing
is simple and they recover very well postoperatively. Similar-
ity of bone composition between rabbit and human bone exist
[48].
Rabbit reaches skeletal maturity shortly at around 6
months of age and has fast skeletal change and bone turnover
(both intracortical and Haversian remodelling) with a peak of
bone neoformation at approximately 30 days from the surgery,
providing a rapid clinical response and so rabbits are widely
used to study implant materials for bone [48]. The remodeling
period consists of approx. 6 weeks and includes the resorp-
tion phase (1 week), the osteoclastic reversal i.e., shifting of
resorption processes into formative processes (0.5 week) and
the formation period (4.5 weeks). Consequently, it is expected
to have the maximum/peak of bone neoformation at approx. 4
weeks/30 days from the surgery and a mature bone at 60 days.
In a short-term test period (4–8 weeks) the healing of rabbit
bone is partial and involves immature bone tissue and fibrous
connective tissue. In these conditions, it is possible to eval-
uate how a biomaterial could affect the healing process and
whether the healing would be satisfactory in term of tissue
quantity and quality, as well as of the osteoconductive and/or
osteoinductive characteristics of testing materials.
5. Conclusions
The study innovatively proposed the microchemical and his-
tological characterization by ESEM-EDX associated with OM to
investigate the osteoinductive potential and neoformed bone
at the interface with hydraulic calcium silicate cements used
as graft materials in a rabbit intramedullary model.
 d e n t a l m a t e r i a l s 3 3 ( 2 0 1 7 ) e221–e238
Thein vivo osteogenic bioactivity of the tested hydraulic
calcium silicate cements following their surgical implantation
in appropriate animal model has been shown as these mate-
rials are able to bind directly to bone without encasement by
a fibrous connective tissue and to enhance/induce a marked
bone neoformation with dynamic biomineralization processes
around the materials extruded in the medullary region.
In addition, no evidence of destructive inflammatory pro-
cesses and the absence of necrotic processes at the interface
with the pre-existing bone was found; conversely angiogenic
activity with neovascularization and formation of capillaries
was demonstrated.
ProRoot MTA and MTA Plus provide a suitable scaffold
for bone marrow stem cells homing (osteoprogenitor cells in
bone marrow), osteoblasts differentiation, angiogenesis and
mineralized tissue formation and bone repair, demonstrat-
ing excellent fit properties as template for in situ bone tissue
regeneration. Biodentine demonstrates marked osteoinduc-
tivity and mineralizing properties.
The clinical significance of the results of this study demon-
strate the capacity of hydraulic calcium silicate cements to
allow osteoid matrix deposition by activated osteoblasts and
favor its biomineralization, and to achieve a direct bond
between the (bioactive) materials surface and the mineralized
bone matrix. The study supports the use of calcium silicate
cement to fill bone defects when large bone apical exposures
are present and also supports the possibility to expand the
clinical uses of these bioactive/osteogenic materials as bone
repairing materials in bony deficiencies.
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