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Regeneration of periapical lesions post-endodontic treatment and periapical surgeries in

experimental animals utilizing thermo-responsive nano--tricalcium phosphate/chitosan

hydrogel: a proof of concept

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2017 Biomed. Mater. 12 045007

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 Biomed. Mater. 12 (2017) 045007 https://doi.org/10.1088/1748-605X/aa6f26

PAPER

Regeneration of periapical lesions post-endodontic treatment and

RECEIVED
6 December 2016
periapical surgeries in experimental animals utilizing thermo-
REVISED
12 April 2017
responsive nano-β-tricalcium phosphate/chitosan hydrogel: a proof
ACCEPTED FOR PUBLICATION
25 April 2017
of concept
PUBLISHED
5 July 2017
Wafa I Abdel-Fattah1 , Salma Hassan El Ashry2, Ghareib W Ali1, Mohamed Aiad Abdel Hamid3,
Amina Gamal El-Din4 and Bassma El-Ashry5
1
Emeritus, Refractories, Ceramics, Building Materials Dept.: Biomaterials Group, National Research Centre, Egypt
2
Endodontics, Faculty of Dentistry, Ain Shams University, Egypt
3
Surgery, Anesthesiology, and Radiology, Faculty of Veterinary Medicine, Cairo University, Egypt
4
Pathology Department, Medical Research Division, National Research Centre, Egypt
5
Restorative Dentistry & Dental Materials Department, Oral & Dental Research Division, National Research Centre, Egypt
E-mail: nrcfifi@yahoo.com

Keywords: β-tricalcium phosphate/chitosan, injectable, periapical surgery, periapical lesions, proof of concept

Abstract
Using phosphate nanoparticles/polymeric hydrogels presents an interesting approach, especially
concerning the reduced particle migration and enhanced biocompatibility. The current work aims to
achieve a proof of concept for the development of a thermo-sensitive nano β-tricalcium phosphate
(β-TCP)/chitosan (Cs)/glycerophosphate (Gl)/glyoxal (Gly) hydrogel to be applied in periapical
surgeries post endodontic treatment. Physicochemical characterization using x-ray powder diffrac-
tion, Fourier transform infrared, TEM and SEM was performed. Bone formation efficiency of the
achieved β-TCP/Cs/Gl/Gly hydrogel was followed. The composite gels were tested in vivo in dogs in
comparison with the commercially available and surgically applied Klipdent-PL® up to three months.
Radiographic examinations were performed. Histological evaluations were achieved through
histomorphological criteria being apical cementum surface, bone tissue resorption, apical PDL
thickness, the intensity of inflammatory reaction and osseous repair. The cytotoxicity results proved
the safety of the developed hydrogel. The thermo-sensitive hydrogel possessed comparable enhanced
biocompatibility with anti-inflammatory activity. New bone formation was clearly enhanced in the
infected teeth. Therefore, it can be directly applied in specific non-invasive dental surgeries.

1. Introduction prohibit fast degradation of the small granules [7–9].

Thermally responsive hydrogels are promising biome-
dical systems since their gelation and changes in
swelling are triggered by temperature changes [1].
These systems allow for the in situ hydrogel formation,
where they are delivered in solution via a minimally
invasive route and solidify inside the surgical site.
Their potential as in situ forming biomaterials has
rendered them very attractive. Recently, they are
applied in drug delivery and tissue engineering [2–4].
Beta-tricalcium phosphate (β-TCP) composites is
an adequate bone substitute reporting biocompatibility,
biodegradability and osteoconductivity [5, 6]. Its com-
posites are applied for periapical lesion treatment as they
The incorporation of bioactive phosphatic phases as
nano hydroxyapatite [10, 11] or β-TCP [12, 13] within
biopolymers as chitosan, improve their functionality,
physiochemical and biological properties for applica-
tions in bone regeneration.
Chitosan proved to function as a bioactive matrix
gel-like bone-substitute holding the β-TCP nano-
particles. Chitosan is reported for biomedical applica-
tions including bone tissue engineering [2], blood vessels
[14] and nerves [15]. The synergistic combination of
β-TCP nanoparticles within biocompatible chitosan
matrix provides an injectable, biodegradable material
with non-toxic degradation products, display self-healing
beside adhesiveness to mineral surfaces [12, 16–19].

© 2017 IOP Publishing Ltd

Biomed. Mater. 12 (2017) 045007
β-glycerol phosphate is a neutralizing agent for
chitosan salt solutions resulting, therefore, hydrogels
with sol–gel transition at human body temperature
[20, 21]. It reacts with phosphatic phases leading to
electrostatic interactions via hydrogen bonding, elec-
trostatic attractions, and hydrophobic effects. The
required amounts of β-GP to impart a change in
charge density is roughly proportional to the polymer
concentration [22].
Glyoxal (Gly) is a cross-linker applied to enhance
the mechanical properties for the chitosan composites
[23]. In gel formulation, glyoxal is essential as it reacts
with chitosan hydroxyl and amino groups.
Periapical lesions resulting from the necrotic den-
tal pulp are among the most frequently occurring
pathologies reported in alveolar bone. The exposure of
the dental pulp to bacteria and their by-products acts
as antigens, which elicit nonspecific inflammatory
responses and specific immunological reactions in the
periarticular tissues causing, therefore, the periapical
lesions [11]. The elimination of the infectious agent by
root canal treatment would allow the lesion healing.
However, incomplete eradication of the infection and
the remaining of periapical lesion is considered as a
treatment failure. In such cases; dental surgeon should
consider either retreatment of the canal, periapical
surgery or extraction of the affected tooth. Therefore,
an arising consideration for developing a biomaterial
to be applied in regenerating periapical lesions exists.
A previous work by Wafa et al reported an inject-
able thermosensitive CS hydrogels crosslinked with
both Gly and Gl, and reinforced with beta tricalcium
phosphate nanoparticles [24]. Their rheological analy-
sis showed a strong gelation behavior. Mechanical
testing proved their elasticity and ability to support
mechanical loads due to their 3D crosslinked network.
The hydrogels possessed biological features that
enhance cellular adhesion and proliferation [24].
The present work aims to satisfy a proof of concept
for β-TCP/Cs/Gl/Gly thermosensitive hydrogel for-
mulated to be applied in periapical surgeries post
endodontic treatments avoiding, therefore, particle
migration. Owing to the presence of Gl and Gly, the
resulting system undergoes sol–gel transition at body
temperature. In vivo comparison with the commercially
available Klipdent-PL and the presently formulated
hydrogel on the particularly regeneration of endodonti-
cally treated teeth in dogs was performed. The feasibility
of materials injection and the corresponding tissue
reactions were analyzed radiographically, histologically
and histo-morpho-metrically.
2. Materials and methods
β-TCP/Cs/Gl/Gly hydrogel was successfully achieved
with a concentration of 1% (w/w) of βTCP. βTCP was
previously prepared as reported by Wafa et al [25].
2
W I Abdel-Fattah et al
Briefly, calcium hydroxide (Ca(OH)2) (ANALAR, UK)
and diammonium hydrogen phosphate (NH4)2HPO4
(NORMAPUR, CE-EMB) were used as chemical
precursors for calcium and phosphorus, respectively.
Respective synthesis temperature maintained at 90 °C
during the precipitation process. The precipitate was
matured for 16 hours without stirring in the mother
liquor. Then, the solution and the precipitate were
subjected to a domestic microwave oven (2450 MHz,
350 W) for one hour to release ammonia gas. The
mother liquor was decanted and the powder was
filtered under vacuum. The dried synthesized powders
were calcined at 900 °C for three hours.
Chitosan (85% deacetylated, Oxford Chemical Co.)
was dissolved in acetic acid (1%). Initially, a suspension
of the synthesized nano β-TCP powder in a chitosan
solution 1.4% (w/v) was achieved by magnetic stirring
[25]. β-Gp solution (β-glycerol 2-phosphate disodium
salt hydrate, Mw = 306.25, Sigma-Aldrich) was prepared
by dissolving 0.6 M in 5 ml of bi-distilled water and chil-
led at 4 °C overnight. Further, in an ice bath (4 °C), β-Gp
solution was added to the nano β-TCP/chitosan suspen-
sion dropwise with stirring to achieve pH of 7–7.4.
Diluted glyoxal hydrate bifunctional aldehyde (0.8 ml)
(40%, Merck) was added dropwise with stirring for half
an hour. The mixed media were incubated in a thermo-
static bath at 37 °C to allow gelation followed through
inverted test tube observation [24, 26]. The achieved
gels were stored at 4 °C. The commercially available
Klipdent-PL (granulated resorbable beta-tricalcium
phosphate in the polylactide-glycolide matrix) was pur-
chased and applied for comparison.
3. Characterization
3.1. Physico-chemical characterization
X-ray powder diffraction (XRD) analysis was achieved
using Cu Kα radiation (l = 1.5418 Å) at a scanning
speed of 0.3 S (Philips X’pert Pro x-ray powder
diffractometer). The applied voltage and current were
40 kV and 40 mA respectively. Fourier transform
infrared (FTIR) spectra were recorded using JASCO
430 FTIR (Japan) spectrometer equipped with TGS
detector. The samples were prepared by compressing
powders with KBr (2/198 mg). The spectra were taken
at 4.0 cm−1 resolution. 64 scans were accumulated to
get a reasonable signal to noise ratio. High-resolution
transmission electron microscopy (HRTEM) (JEOL-
1230) was used at an accelerating voltage of 100 kV.
The samples were efficiently dispersed in an ultrasonic
homogenizer for 6 min. One drop was placed on the
carbon coated copper grids and left to dry at ambient
temperature. Field emission scanning electron micro-
scopy (FESEM) of the achieved composites were
performed on a Quanta FEG 250-type microscope
equipped with an energy dispersive x-ray attachment
(EDAX/Genesis device).
Biomed. Mater. 12 (2017) 045007
Table 1. Different tested and control groups.
Subgroups Klipdent-PL Nano-β TCP/Cs/Gl/Gly
One month 7 7
Two month 7 7
Three month 7 7
Total samples number 21 21
3.2. In vitro studies
In vitro cytotoxicity studies were investigated against
Wish normal cells, hepatocellular carcinoma (HEPG2)
and breast cancer (MCF7) cell lines by MTT assay 3-[4,
5-dimethylthiazol-2-yl]-3, 5-diphenyltetrazolium bro-
mide dye. Cells were inserted in a 96-well plate at a density
of 1 × 104 cells per well. The media were supplemented
with 1% antibiotic–anti-mycotic mixture. It is composed
of 10 000 U ml−1 potassium penicillin, 10 000 μg ml−1
streptomycin sulfate, 25 μg ml−1 amphotericin B and 1%
L-glutamine (Bio west, USA). Incubation at 37 °C was
performed in humidified atmosphere with 5% CO2. After
the cells attachment, the media were replaced by the
hydrogel samples for 72 h. The cells were incubated with
the MTT solution (5.0 mg ml−1) and further, the plates
were incubated at 37 °C for four hours. The developed
purple formazan crystals were dissolved in 100 μl
dimethyl sulfoxide (DMSO). An ELISA reader was used.
The IC50 values (the concentration required to produce
50% inhibition of cell growth) were calculated using SPSS
statistical analysis software package/version 11.0, SPSS
Inc., (IL), Chicago, USA.
The cell viability % was calculated applying the fol-
lowing equation:
ODtest - OD blank
Viability% = ´ 100.
ODcontrol - OD blank
ODtest is the mean value of sample absorbance
measured at 570 nm.
ODblank is the mean value of blank absorbance
measured at 570 nm.
ODcontrol is the negative control.
3.3. In vivo studies
3.3.1. Pre-surgical phase
The animal investigation provides primary screening
examination of tissue reaction to the newly introduced
bone grafting materials. All animal experiments were
conducted following the approved guidelines of the
Animal Care Committee of National Research Centre
(13/77). Six adult mongrel dogs of both sexes were used.
The selected dogs were apparently healthy, with an
average weight of 20 kg and were one year of age. The
dogs were thoroughly examined upon selection and kept
under observation for two weeks as a pre-surgical phase
to exclude the diseased animals. They were quarantined
in separate cages at the department of veterinary surgery;
3
W I Abdel-Fattah et al
Groups
Control group
Negative intact Positive infected Total number of samples
7 7 28
14
14
7 7 56
faculty of veterinary medicine; Cairo University, Egypt.
The dogs were put under general anesthesia. The kennels
were sprayed with 6/1000 ml neocidal diazinone (Diazi-
none 60 EC; Ciba-Geigy, Switzerland) and the dogs were
bathed in 1/1000 neocidal diazinone. The dogs were
injected by ivermectine (Ivomec; Merck, Sharp and
Dome, USA). 0.1 mg kg−1 body weight subcutaneously
to guard against ectoparasitic and endoparasitic infec-
tions. The dogs were pre-medicated with atropine sulfate
(Atropine, Cairo, Egypt) that was injected subcuta-
neously (10–30 min) before operations in a dose of
0.05 mg kg−1 body weight. The dogs were put under
general anesthesia. The anesthetic agent used for induc-
tion of anesthesia was a mixture of Xylazine HCL
(Rompun, Bayer, Germany) 1 mg kg−1 body weight and
Ketamine HCL (Ketalar, Park Davis, USA) 5 mg kg−1
body weight. The mixture was injected via a 23 gauge IV
cannula through the cephalic vein. The anesthesia was
maintained through the operative time by the injection
of sodium thiopental (thiopentone sodium 2.5% Novar-
tis, Egypt) 25 mg kg−1 body weight via the cannulated
cephalic vein. The respiratory airway was kept patent by
applying the endotracheal tube.
The selected teeth were the lower premolars. The
specimens were assigned into two experimental
groups (n = 42) randomly according to the applied
material and two control groups. Group one (n = 21);
bony lesions were filled surgically with Klipdent-PL
while group two (n = 21) bony lesions were filled with
the new formulation of nano β-TCP/Cs/Gl/Gly gel.
The control groups (n = 14) were divided into two
subgroups: negative control; intact teeth (n = 7), teeth
with unprepared root canal receiving no treatment and
serving as the negative control and positive control;
infected, not treated teeth (n = 7), and teeth receiving
no obturation (table 1). The infected teeth were not
treated and the root canal openings were sealed with an
intermediate restorative material (IRM).
Preoperative radiographs of teeth under invest-
igation were performed, so radiographic images
emphasized that the teeth were intact with no apical
radiolucency and the apical bone was intact
(figure 1(A)). The access cavity was done using a round
bur size three, flaring was performed using a finely
tapered stone. Initially, the root canals were explored
with no. 20 K-files and the dental pulps were removed
Biomed. Mater. 12 (2017) 045007 W I Abdel-Fattah et al

Figure 1. (A) A radiograph showing the investigated teeth preoperative. (B) A photograph showing the files inserted in the canals. (C)
Access openings sealed with IRM, a thick quick setting zinc oxide and eugenol based cement. (D) Post-operative radiograph to verify
the quality of the obturation, adaptation with the dentin walls, and homogeneity of the filling. (E) Photographs showing the surgical
procedures on dogs, identical width and length was marked using chisel and hammer. (F) Exposed roots and periapical lesions.
Photograph showing the placement of the bone grafting materials in the bony defect. (G) The placement of Klipdent PL. (H) The
placement of the formulated hydrogel which was injected in the surgical site showing gelation and setting at body temperature.

with no. 30/40 Hedströem files depending on their
diameters (figure 1(B)). The root canals were exposed
to the oral cavity for seven days to allow microbial con-
tamination. Access openings were then sealed with
IRM, a thick quick setting zinc oxide and eugenol
based cement (figure 1(C)). Radiographs of teeth
under investigation post 45 days, where radiographic
images show periapical radiolucency indicating that
there is a periapical reaction (periapical periodontitis)
(figure 1(D)) [14–27].
The infected teeth in the experimental groups
along with the positive control one were recessed with
the animals under general anesthesia. Upon the IRM
removal, teeth were instrumented following the stan-
dard instrumentation. Root canal enlargement was
done using K-files until clean dentinal shavings were
obtained. The canals were rinsed with 5.25% con-
centrated sodium hypochlorite (NaOCl) sterile saline
solution. Gates Glidden drills (size two and three) were
used to prepare the coronal 2/3 of the root canal. The
apical 1/3 was done with K-files. The master gutta-
percha cones were selected according to the master
apical files. A zinc oxide and a eugenol root canal sealer
were properly mixed. The root canals were efficiently
sealed following the classical technique and com-
plemented by active lateral condensation using size 25

4
Biomed. Mater. 12 (2017) 045007
finger spreaders with an adaptation of auxiliary cones
(size 25) until the canal was filled. The coronal open-
ings were sealed permanently with IRM. Teeth were
radiographed to verify the obturation quality, adapta-
tion with the dentin walls, and homogeneity of the fill-
ing. The animals were kept in separated cages and fed
with a soft diet.
3.3.2. Surgical phase
The surgical phase was carried out one-week post
obturation. A submandibular incision was performed;
the periosteum was incised horizontally at the level of
the inferior border of the mandible and elevated until
the cement/enamel junction of the four premolars
exposed the alveolar crest (figure 1(F)). The buccal
plate of the bone opposite to the four premolars was
removed using the chisel (figure 1(E)). Granulation
tissue was curetted from the periapical region of each
root, and Klipdent-PL was packed in on one side of the
surgical site of the dog’s mandible (figure 1(G)). On
the other side of the same dog, the nano β-TCP/Cs/
Gl/Gly was injected into the surgical sites
(figure 1(H)). Radiographs were taken immediately
post surgeries after repositioning the flaps.
3.3.3. Post-surgical phase
After surgical intervention, the dogs were kept within
confinement and watched carefully until recovery and
then the animals were returned carefully to the cages.
Each animal received an intramuscular injection of
vetrocine (penicillin 600.000 I.U and streptomycin
2 g) every 24 h for 6 or 7 successive days. The animals
were divided into groups and sacrificed 1, 2, and three
months post surgeries to evaluate the healing due to
material placement. Block sections were obtained
including each tooth with its surrounding bone (1, 2,
and 8). Jaw blocks containing the treated teeth were
resected and fixed in 10% buffered formalin. The
collected samples were regularly processed for decalci-
fication and staining with hematoxylin and eosin for
the light microscopic examination by two blind
evaluators. The formation of the new bone, cementum
and periodontal ligament along with healing in the
infected periapical tissues were followed histologically.
3.3.4. Histopathological examinations
The histomorphological criteria considered for the
evaluation of the achieved injected materials covered
the following criteria [28, 29]:
(i) Apical cementum surface whether regular and
healed or irregular and resorbed. (ii) Active bone tissue
resorption or active resorption areas. (iii) Apical PDL
thickness whether thin with no widening, moderate
thickness or thick and hyperplastic or the presence of
the apical PL destruction. (iv) The intensity of inflam-
matory reaction whether mild (inflammatory cells
only close to the foramen) or moderate (inflammatory
cells in part of the periodontal ligament thickness) or
5
W I Abdel-Fattah et al
severe (inflammatory cells in all the periodontal liga-
ment thickness) or absent. (v) Osseous repair whether
there was no new bone formation or newly formed
woven bone and osteoblastic rimming.
3.3.5. Statistical analysis
The data were presented as mean, standard deviation
(SD), frequency and percentage when appropriate using
the two materials at each period. Data were explored for
normality using Kolmogorov–Smirnov and Shapiro–
Wilk tests. The significance level was set at P „ 0.05.
Statistical analysis was performed with IBM® SPSS® (SPSS
Inc., IBM Corporation, NY, USA) Statistics Version 23
for Windows and MidCalc® Version 12.2.1 (MedCalc
Software bvba, Ostend, Belgium).
4. Results
4.1. Physicochemical assessment
4.1.1. XRD analysis
XRD diffractogram of the deride gel powder is shown
in figure 2(A). XRD analyses exhibited the two
characteristic peaks indicating the crystalline nature of
chitosan at 2θ = 11.31° and 20.20°. The dominance of
β-TCP phase (JCPDS #09-0169) was apparently
registered within chitosan matrix. The diffraction
peaks at 2θ values of 22°, 25.9°, 27.3°, 29.2°, 31.8°,
34.2°, 39.8°, 41.1°and 48° are corresponding to (0 2 4),
(1 0 1 0), (2 1 4), (3 0 0), (0 2 1 0), (2 2 0), (1 0 1 6), (3 0 1
2) and (4 0 1 0) h k l planes for βTCP respectively [12].
Additional peaks corresponding to the secondary
precipitated phases of calcium-deficient hydroxyapa-
tite are recorded, to being pyrophosphate (Ca2P2O7),
and some residual calcium carbonate (CaCO3) at
2θ = 30°, 33° and 37.5°. Moreover, the peaks at
2θ = 26° and 30° indicate traces of di-calcium
phosphate anhydrous phase.
4.1.2. FTIR
The synthesized calcium phosphate functional groups
as well as those of chitosan are depicted in the FTIR
spectrum (figure 2(B)). The absorption band recorded
at 1561 cm−1 is characterizing amide II bending of CS.
The peaks at 1663 and 1666 cm−1 are assigning the
glucosamine as N-acetyl-glucosamine spectra. The
CH3 symmetric bending of CS amide groups is
recorded at 1360 cm−1, whereas the peak located at
1442 cm−1 is assigned to the CH2 vibration in the ring,
triggered off by the rearrangement of hydrogen bonds
in the OH groups orientation. Additionally, the
symmetric and asymmetric CH2 stretching vibrations
are detected at 2870 cm−1, 1330 and 1250 cm−1. The
stretching vibration intense bands of both hydroxyl
groups and intermolecular H-bonds within the CS
network raised at 3400 cm−1. The vibration mode
observed at 1079 cm−1 can be assigned to –C–O–C– in
CS glycosidic linkage. Moreover, the crosslinking of
Biomed. Mater. 12 (2017) 045007 W I Abdel-Fattah et al

Figure 2. (A) XRD pattern of the developed hydrogel, (B) FTIR spectrum, (C) HR-TEM image of the β-TCP/Cs/Gl/Gly hydrogel
with its SAED on the right side and (D) FESEM image of the β-TCP/Cs/Gl/Gly hydrogel with corresponding EDAX analyses.

CS and Gl resulting from the reaction between the CS
amine groups and the Gl aldehyde groups is high-
lighted by peaks at 1663 and 1665 cm−1. In this
domain, a Schiff base having an absorption band
representative of C=N imine stretching is achieved.
The protonation of the phosphate groups of β GP by
the positively charged (NH+ 3 ) groups of CS is repre-
sented by small peaks at 1170 and 1200 cm−1. Further,
the presence of calcium phosphate was depicted by the
vibration modes of phosphate ions (PO43 -). The
phosphate ions possess four bonding vibrations v1, v2,
v3, and v4 due to different stretching vibration modes.
The peaks at 975 and 480 cm−1 are characteristic of v1
and v2 modes, whereas the bands observed at 1090,
1035, 607, and 550 cm−1 are attributed to v3 and v4
vibration modes, respectively. The bands attributed to
carbonate ions (CO3-2) of the ceramic phase are
recorded at 448, 1410, and 1640 cm−1 [22].
4.1.3. HRTEM
TEM images and their corresponding SAED of the
β-TCP powders are shown in figure 2(C). It is proved
that β-TCP comprised nano-sized particles. Nano
β-TCP crystals are around 8–17 nm within the chitosan
composite. Its high crystallinity is confirmed via SAED

6
Biomed. Mater. 12 (2017) 045007
pattern with characteristic rhombohedral crystallo-
graphic structure [12].
4.1.4. FESEM
Figure 2(D) presents the SEM micrograph of the β-
TCP/Cs/Gl/Gly hydrogel. The particles are arranged
in a parallel manner on the surface. The powder purity
was confirmed by its EDAX. The elemental analysis
proves O, P and Ca of the calcium phosphates.
4.2. In vitro cytotoxicity
The general toxicity (in vitro cytotoxicity test) is one of
the four requirements needed for biomaterials assess-
ment to satisfy a proof of concept. It provides a measure
of cell death resulting from biomaterials or their extracts
and recognizes their limitations [30]. The viability
percentage of the developed β-TCP/Cs/Gl/Gly hydro-
gels was evaluated against normal Wish cells, HEPG2
and MCF7 cancer cells. Their viability % were 99, 98
and 99 at 100 ppm respectively indicating, therefore,
the safety feature for its intended application.
4.3. In vivo assessment
4.3.1. Histopathological findings
Histopathological examinations were performed to
assess the apical cementum surface, bone tissue
resorption, apical periodontal thickness, the intensity
of inflammatory reactions and osseous repair up to
three months.
• Post one month
The Klipdent-PL group microscopically revealed the
presence of moderate to severe inflammatory cell infil-
trates in the periapical region. The cementum surface and
the cementum dentin surfaces showed extensive resorp-
tion with giant cells mostly. Lacunae of the remaining
apical cementum were either intact or filled with necrotic
tissues. Mostly, complete destruction of the apical period-
ontal ligaments is recorded. The pre-existing periapical
bone showed active resorption and was lined with cells of
enormous size having osteoclastic histologic features.
Finally, no new bone formation was observed in any sam-
ple except for one (figures 3(A) and (B)).
Microscopic examination of the developed β-TCP/
Cs/Gl/Gly gel showed the absence of inflammatory cell
infiltrates in the periapical region in four samples while
the three others revealed average to lacunae of the apical
cementum were intact and vital. Also, most of the sam-
ples revealed widened and moderate thickness apical
periodontal ligament space. No resorption in the pre-
existing periapical bone was recorded in all samples.
Finally, new bone formation was clearly proved as bone
surfaces were lined with enormous sized cells. Osteo-
clastic histologic features from one side and osteoblastic
rimming on the other one, reflect, therefore, remodel-
ing activity (figures 3(C) and (D)).
7
W I Abdel-Fattah et al
• Post two months
The Klipdent-PL group revealed the presence of
severe inflammatory cell infiltration in the periapical
region in all samples. Mostly, the cementum surfaces
and the cementum-dentin surfaces suffered extensive
resorption with giant cells. Lacunae of the remaining
apical cementum were either intact or filled with
necrotic tissues. Moreover, samples suffered complete
destruction of the apical periodontal ligaments.
Resorption in the pre-existing periapical bone was not
recorded at all with apparent new bone formation but
in only three samples (figures 4(A)–(C)).
The developed β-TCP/Cs/Gl/Gly gel group
revealed the presence of mild to moderate to severe
inflammatory cell infiltrates in the periapical region
microscopically. Only one sample showed the absence
of inflammatory cell infiltrates. Mostly, the cementum
surface as well as the cementum-dentin surfaces
showed surface regularity reflecting, therefore, tissue
healing activity. Lacunae of the apical cementum were
intact and vital. Moreover, much of samples still reveal
thin to moderate thickness apical periodontal ligament
space. Resorption in the pre-existing periapical bone
could not be observed in all samples and new bone for-
mation was mostly recorded (figures 4(D)–(F)).
• Post three months
A severe inflammatory cell infiltrates in the peria-
pical region was revealed microscopically for the Klip-
dent-PL group. Extensive resorption with giant cells
was shown in cementum surfaces and the cementum-
dentin. Lacunae of the remaining apical cementum
were either intact or filled with necrotic tissues. A
complete destruction of the apical periodontal liga-
ments is registered in most of the cells. No resorption
was recorded in the pre-existing periapical bone at all
and the new bone formation was mostly proved
(figures 5(A)–(D)).
The β-TCP/Cs/Gl/Gly group proved the absence
of inflammatory cell infiltrates in the periapical region
in three samples while four samples showed moderate
inflammation. The cementum surface and the cemen-
tum-dentin surfaces showed surface regularity reflect-
ing, therefore, tissue healing activities in more than
half of the samples. Lacunae of the apical cementum
were intact and vital. Additionally, they revealed
widened and an average thickness of apical period-
ontal ligament space while the rest of the samples
showed complete periodontal ligaments destruction.
Resorption was absent in the pre-existing periapical
bone in majority of the samples proving no new bone
formation (figures 5(E)–(G)).
The positive (infected) group revealed severe
inflammatory cell infiltrates in the periapical region
microscopically. The cementum and the cementum-
dentin surfaces possessed extensive resorption with
giant cells mostly. Lacunae of the remaining apical
Biomed. Mater. 12 (2017) 045007
Figure 3. (A) A photomicrograph of Klipdent PL group (one month) showing the periapical area (×40). (B) Higher magnification
(×400) reveals severe inflammatory cell infiltrate. (C) A photomicrograph the formulated hydrogel (one month) showing the
periapical area (×40). (D) Higher magnification (×200) reveals absence of inflammatory cell infiltrate and beginning of new bone
formation (woven bone).
cementum were either intact or filled with necrotic tis-
sues. Mostly, the samples maintained complete
destruction of the apical periodontal ligaments. A
resorption in the pre-existing periapical bone in two
samples was recorded. New bone formation was
absent in all the samples (figures 6(A)–(C)).
Microscopically, the negative control group
revealed the absence of inflammatory cell infiltrates in
the periapical region in all the samples. Moreover, the
cementum surfaces and the cementum-dentin sur-
faces were regular and healed. Lacunae of the apical
cementum were intact and vital. All the samples
showed no hyperplasia in the apical periodontal liga-
ments. They remained thin without widening. Finally,
no resorption was shown in the pre-existing periapical
bone without new bone formation in all the samples
(figures 6(D) and (E)).
4.3.2. Histo-morphometric findings
One month
Klipdent-PL group presented only one sample
(14.3%) with a regular and healed apical cementum
surface while the other six samples (85.7%) showed an
irregular and desorbed apical cementum surface.
Therefore, a nonsignificant difference with the posi-
tive infected group, having two samples (28.6%) with a
regular and healed apical cementum surface while the
rest five samples (71.4%) possessed irregular and
resorbed one.
8
W I Abdel-Fattah et al
Klipdent-PL group possessed a significant differ-
ence in bone resorption criteria with the other tested
groups. Two samples (28.6%) recorded an absent
resorption while active resorption areas were pre-
sented in five samples (71.4%) at p = 0.006.
β-TCP/Cs/Gl/Gly gel samples did not record a
statistically significant difference as five samples
(71.4%) were regularly healed compared to the other
two (28.6%) showing an irregular and resorbed apical
cementum surface along with the negative control
group. On the other hand, the current gels possessed a
significant difference with the other tested groups as
five samples showed secondary bone score while the
other two are without a bone score at P = 0.003.
Considering the apical cementum criteria, a statis-
tically significant difference existed at P = 0.006.
A statistically significant difference exists between
the different groups in the apical PDL thickness at
P = 0.001. On the other hand, no statistically sig-
nificant difference existed between the experimented
groups and the positive control one.
The intensity of the inflammatory reaction showed
a statistically significant difference between the differ-
ent groups where P „ 0.001. Klipdent-PL group
showed the highest score for moderate intensity of
inflammatory response for four samples (57.1%)
while only two samples (28.6%) possessed an acute
inflammation score. The positive (infected) group
recorded the highest inflammation rate as six samples
(85.7%) clarified severe inflammation.
Biomed. Mater. 12 (2017) 045007 W I Abdel-Fattah et al

Figure 4. (A) Photomicrograph of Klipdent PL group (two months) showing the periapical area (×40). (B) Higher magnification
(×200) revealing new bone formation surrounded with severe inflammatory cell infiltrate. (C) Photomicrographs at (×20, ×200)
showing the remnants of Klipdent PL bone graft material not yet resorbed and surrounded by inflammatory cell infiltrates. (D) A
photomicrograph of the formulated hydrogel (two months) showing the periapical area (×20). (E) Higher magnification (×100)
revealing material remnant not yet resorbed and surrounded by inflammatory cell infiltrates. (F) Higher magnification (×100, ×200)
proving beginning of the new bone formation.

Two months A statistically significant difference in the apical

The highest score of severe intensity of inflamma-
tion reaction in seven samples (100%) was recorded
for the Klipdent-PL group without a statistically sig-
nificant difference between the positive (infected) one.
On the other hand, the negative group did not show an
inflammation. Statistically significant difference with
all other tested groups was recorded.
cementum surface between the negative group on one
side and all other tested groups on the other side could
be traced at P = 0.018.
No statistically significant difference in bone tissue
resorption exists among all the tested groups where at
P = 0.101. Apical PDL thickness measurements recor-
ded a statistically significant difference between the

9
Biomed. Mater. 12 (2017) 045007 W I Abdel-Fattah et al

Figure 5. (A) A photomicrograph of Klipdent PL group (three months) showing the periapical area (×40). (B) Higher magnification
(×400) revealing severe inflammatory cell infiltrates. (C) Photomicrographs (×40, ×200) revealing new bone formation (osteoid).
(D) Photomicrographs (×40, ×200) revealing apical cementum surface was irregular and resorbed lined with enormous sized cells
having the histologic features of dentino-clasts. (E) A photomicrograph of the developed hydrogel (surgical) showing the periapical
area (×40). (F) Moderate inflammatory cell infiltrates (×400). (G) New bone formation (bone surfaces were lined with enormous
sized cells having the histological features of osteoblasts) (×200, ×400).

different groups where P „ 0.001. The negative con-
trol showed the highest rate of thin apical periodontal
ligaments without widening which has a statistically
significant difference with Klipdent-PL and the posi-
tive (infected) groups.
No statistically significant difference exists between
negative control group and β-TCP/Cs/Gl/Gly gel
group.
Conversely, a statistically significant difference in
osseous repair between all the different groups where
P = 0.007. β-TCP/Cs/Gl/Gly gel group possessed five
samples (71.4%) with a secondary bone without a sta-
tistically significant difference with Klipdent-PL group
which presented only three samples with secondary
bone (42.9%).
Three months
The highest score for severe inflammation reac-
tion intensity was recorded for Klipdent-PL and the
positive (infected) groups within six samples (85.7%)
without statistically significant difference in between.
The negative group presented an absence of inflam-
mation without statistically significant difference with
β-TCP/Cs/Gl/Gly gel group.
A statistically significant difference exists between
the different groups in apical cementum surface at
P = 0.009. The negative control group has no statisti-
cally significant difference with β-TCP/Cs/Gl/Gly gel
group while it possessed a statistically significant dif-
ference with the positive (infected) one. A higher rate
of irregular and resorbed apical cementum was pre-
sented by six samples (85.7%) by Klipdent-PL group
without statistically significance difference with any
other tested groups except for negative one.
There was no statistically significant difference
between all the tested groups in bone tissue resorption
where P = 0.212.
A statistically significant difference exists between
negative the control and all the other tested groups in
apical PDL thickness where P = 0.00, showing the
highest rate of thin apical periodontal ligament

10
Biomed. Mater. 12 (2017) 045007 W I Abdel-Fattah et al

Figure 6. (A) A photomicrograph of positive control group (infected) showing the periapical area proving complete destruction of the
apical periodontal ligaments and the apical cementum surface was irregular and resorbed (×40). (B) Higher magnification (×400)
revealing severe inflammatory cell infiltrate. (C) Photomicrographs (×40, 200) revealing bone resorption (bone surfaces were lined
with enormous sized cells having the histologic features of osteoclasts). (D) A photomicrograph of negative control group (intact
teeth) showing the periapical area having no widening nor hyperplasia of the apical periodontal ligaments (×40). (E) Higher
magnification (×400) clarifying absence of inflammatory cell infiltrate.

without widening. No statistically significant differ-
ence could be recorded between the tested groups with
each other. Klipdent-PL group showed the highest rate
of apical periodontal ligaments destruction of six sam-
ples (85.7%) followed by the actively infected group
then the β-TCP/Cs/Gl/Gly gel. A statistically sig-
nificant difference among all the different groups in
osseous repair where P = 0.002 could be achieved.
Klipdent-PL group have five samples (71.4%) with a
secondary bone formation. No statistically significant
difference with β-TCP/Cs/Gl/Gly gel exist (figure 7).
4.3.3. Comparing different periods within each main
group (level of significance 0.05)
β-TCP/Cs/Gl/Gly gel, recorded no statistically signifi-
cant difference in the apical cementum surface, bone
tissue resorption, apical periodontal ligament thick-
nesses, intensity of inflammatory reaction and the
osseous repair parameters post one, two and three
months (P-value was more than 0.05 for each parameter;
0.815, 0.122, 0.119, 0.143 and 1.00 respectively). No
statistically significant difference in the apical cementum
surface, apical periodontal ligament thicknesses and the
osseous repair parameters after one, two and three
months. P-value was more than 0.05 for each parameter;
recording 0.745, 0.535 and 0.108 respectively in Klip-
dent-PL group. There was a statistically significant
difference in bone tissue resorption parameter.
Only in the one month scores showed a significant
difference for the other time periods with five samples
(71.4%) showing active bone resorption at P = 0.002.
A statistically significant difference exists in inflamma-
tory reaction intensity between one month and the
other two periods (P = 0.009). The highest rate of
severe inflammation was recorded at two months fol-
lowed by the three months’ period (figure 8).

11
Biomed. Mater. 12 (2017) 045007
Figure 7. Histograms showing the count of different histomorphometric parameters for all tested groups at each period.
5. Discussion
The present project is proposed to satisfy the proof-of-
concept to assess the feasibility of employing the
synthesized β-TCP/Cs/Gl/Gly based gel and ensure
its competence with the commercially available com-
modity (Klipdent-PL). It can pack defect and facilitate
wound suturing upon contact with blood or oral
fluids.
The general screening assays for biomaterials test-
ing are divided into four phases: 1—general toxicity
(in vitro cytotoxicity tests), 2—local tissue irritation
(animal implantation), 3—pre-clinical (proof of con-
cept in an animal model) and 4—clinical evaluation
(human trials) [3]. The research passed the early stages
of the investigation and the preclinical studies in dogs.
The project team is working towards the next phase of
clinical trials to apply in humans. Clinically, appro-
priate surgical procedure applying β-TCP/Cs/Gl/Gly
gel was adopted to successfully regenerate the periapi-
cal dental tissues with rich vasculature and a complex
histological structure imitating that of the native peri-
apical tissues.
The physicochemical characterizations proved
successful development of the present β-TCP/Cs/Gl/
Gly hydrogel. The XRD diffractogram of β-TCP
revealed narrow peaks indicating, therefore, its high
12
W I Abdel-Fattah et al
crystallinity. All the diffraction peak positions match
well with the standard XRD pattern of β-TCP (JCPDS
# 09-0169). Additionally, a minor amount of pyr-
ophosphate Ca2P2O7 (JCPDF# 9-346) was recorded
[31–33]. The chitosan narrow peaks indicate its crys-
talline nature.
The possible interaction of glycerophosphate with
the chitosan amino groups occurs via several types of
interactions such as electrostatic attraction, hydrogen
and hydrophobic bonding that triggered the gel for-
mation [22].
The in vivo periradicular regeneration on experi-
mentally induced periapical lesions of endodontically
treated teeth in dogs was assessed regarding (A) tissue
reaction and intraosseous biocompatibility and (B)
healing of the periapical lesions and osseous repair.
The treatment consists of the elimination of the
infectious agents by root canal treatment, allowing
lesion healing [33]. However, in non-eliminated infec-
tion, periapical lesions remain pointing to a treatment
failure. A consideration of either canal retreatment,
periapical surgery or extraction of the affected tooth
should be considered when faced with a periapical
lesion persisting post root canal treatment, even if
asymptomatic [34]. The periapical periodontitis per-
sists even if the canal is correctly cleaned and filled
observing a radio transparent image which may be
Biomed. Mater. 12 (2017) 045007
Figure 8. Histograms showing the count of different histomorphometric parameters for three months’ follow-up periods for Klipdent
PL and the developed hydrogel groups.
asymptomatic. The main reasons are the complex root
canal system, with accessory canals, ramifications, and
anastomoses, which cannot be accessed, cleaned or
even filled conventionally. Furthermore, six possible
biological factors were described as causing asympto-
matic apical periodontitis following root-canal treat-
ment being persistent intraarticular and extraarticular
infections (principally actinomycosis) and foreign body
reaction related to the root filling material. Addition-
ally, the accumulation of endogenous cholesterol crys-
tals irritates the periapical tissue, actual cystic lesions,
and scar tissue [35]. In this context, the β-TCP/Cs/Gl/
Gly thermo-sensitive gel nanocomposite was used for
the treatment of the alveolar bone defects combining
the advantages of several material classes. It remains
stable post application through a syringe mimicking,
therefore, the minimally invasive surgeries.
These results are very promising and supporting
the hypothesis that injectable bone substitute materi-
als having β-TCP/Cs/Gl/Gly hydrogel can be applied
as osteoconductive filling materials for successful bone
tissue augmentation. Accordingly, the application of
13
W I Abdel-Fattah et al
the present material to bone tissue reveal to what
extent it can influence bone regeneration.
6. Conclusion
A proof of concept for a resorbable injectable β-TCP/
Cs/Gl/Gly hydrogel was directed towards defining the
role of guided tissue regeneration to improve the
healing of osseous defects related to the induced
periapical lesions in dogs. It can be concluded that the
developed hydrogel possessed improved results com-
pared to the commercially available Klipdent-PL.
These injectable hydrogels function as bioactive fillers.
Moreover, injectable calcium phosphates gel is freely
moldable and adaptable. Its flowable nature can fill
complex bone cavities and undergo gelification in
physiological environment and thus adhering to tissue
cavities post gelation. The combination of high bio-
compatibility, easy-to-handle characteristics beside the
capacity to self-set under ambient conditions presents
it as an asset in repairing dental defects.
Biomed. Mater. 12 (2017) 045007
Acknowledgments
The financial support from the National Research Centre,
Giza, Egypt is appreciated for the performed research
project entitled ‘Nobel metal/Chitosan/Phosphatic Com-
posite hydrogels with antibacterial/anticancer functions
as bone repair for minimum invasive surgeries code
10070404 (2014–2016). Thanks are due to the Bioassay-
Cell Culture Laboratory (in vitro bioassays on human
tumor cell lines for drug discovery), National Research
Centre, Giza, Egypt.
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