Densitometric and Histomorphometric Analysis for Testing New Bone Substitutes (BLUE BONE®), a Comparative Study on Rabbits' Femoral Defect

Abstract

Aims: to evaluate the osteogenic regeneration ability of new nanometric biphasic hydroxyapatite and tricalcium phosphate (80/20) BLUE BONE. Materials and methods: twenty domestic rabbits were used in the present study with two defects were created in the rabbit's femur, one filled with BLUE BONE material and the other defect were left empty as negative control. Densitometric and histomorphometric analysis measured at 3 days, 7 days, 14 days, and 28 days. Results: showed positive osteogenic properties of the BLUE BONE due to the osteoconduction properties. Conclusions: this study supports that BCP may have better application prospects for bone repair.

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Karrar A. Zaker Department of Oral and Maxillofacial Surgery
BDS, (Master student) College of Dentistry, University of Mosul
Ziad H. Delemi Department of Oral and Maxillofacial Surgery
BDS., FIBMS.,MF.(Asst. Prof.) College of Dentistry, University of Mosul
Dr.Ammer A. Taqa Department of dental basic science
BSc, MSc, PhD, (Prof.) College of Dentistry, University of Mosul
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ABSTRACT
Aims: to evaluate the osteogenic regeneration ability of new nanometric biphasic hydroxyapatite and
tricalcium phosphate (80/20) BLUE BONE. Materials and methods: twenty domestic rabbits were used in
the present study with two defects were created in the rabbit's femur, one filled with BLUE BONE material
and the other defect were left empty as negative control. Densitometric and histomorphometric analysis
measured at 3 days, 7 days, 14 days, and 28 days. Results: showed positive osteogenic properties of the
BLUE BONE due to the osteoconduction properties. Conclusions: this study supports that BCP may have
better application prospects for bone repair.
Keywords: Bone Substitutes, osteogenic regeneration
Zaker KA., Delemi ZH., Taqa AA. Densitometric and Histomorphometric Analysis for Testing New Bone
Substitutes (BLUE BONE®), a Comparative Study on Rabbits' Femoral Defect. Al–Rafidain Dent J.
2020;20(2):221-232. ©2020, College of Dentistry, University of Mosul
Received: 22/5/ 2020 Sent to Referees: 30/5/ 2020 Accepted for Publication: 25/ 6/ 2020
This is an open access article under theCCBY4.0license (http://creativecommons.org/licenses/by/4.0/
INTRODUCTION
Regeneration of bones is a daily faced
problem in dentistry. Musculoskeletal diseases
rise from trauma, surgical interventions and
diseases regarded as second largest disabilities
throughout the world as recognized by World
Densitometric and Histomorphometric
Analysis for Testing New Bone
Substitutes (BLUE BONE®), a
Comparative Study on Rabbits' Femoral
Defect
ISSN: 1812–1217
E- ISSN: 1998-0345
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Health Organization (WHO) (1). About 1.2
million people lose their life due to the lack of
bone reconstruction facilities (2). Different
biomaterials have been introduced for bone
regeneration purpose. Among all these
biomaterials autogenous bone graft is the gold
stander because it has the ideal requirements
for bone regeneration as osteoinduction,
osteoconduction and the availability of
osteoprogenitors cells (3,4). All biomaterials
share the same property of osteoconduction (5).
There are many types of alloplastic
biomaterials that has been introduced among
these biomaterials the biphasic calcium
phosphate (BCP) show promising results (6,7).
Hydroxyapatite and tricalcium phosphate are
the most calcium ceramic applied as bone graft
(8,9). BCP composed of hydroxyapatite and
tricalcium phosphate of different ratios. The
sapience of this combination is to produce
material with optimum dissolution rate
obtained from highly resorption rate of TCP to
replace by new bone (10,11) and slowly
degradable HA for providing mechanical
support under load and to maintain volume (1).
BLUE BONE is alloplastic biomaterial
composed pf HA/TCP of ratio of 80/20
characterized by nano-metric particles ranged
from 195.5 nm-348.2nm.
The aim of this study is to evaluate the bone
regeneration capacity of the new biomaterial
BLUE BONE.
MATERIALS AND METHODS
The study was accomplished at Mosul
University College of dentistry. Twenty rabbits
weighted 1.3-1.5 Kg and aged 3-4 months were
chosen. The blue bone® graft is a synthetic
compound of 80% hydroxyapatite and 20%
tricalcium phosphate. Blue bone consists of
nanometric particles with homogeneous shapes
and sizes (thickness and height) as shown in
(Figures1 and 2).
Figure (1): Blue bone material package
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Figure (2): Scanning electron microscope of blue bone material.
Housing and feeding for all rabbits were the
same and all rabbits were examined by
veterinary physician to check the animals
health condition. Each rabbit was given general
anesthesia. It was a mixture of ketamine of
0.6mg/kg and Xylazine of 0.3ml/kg injected
intramuscularly. Few minutes later, the animal
had lost its consciousness lost. Then the rabbit
positioned on his left side and the area over the
right femur shaved and cleaned with povidone
iodine. A small of 1.5 cm created over the
femur bone near its head by surgical blade
no.15 avoiding any trauma to muscle after that
the femur bone exposed by blunt dissection.
Two holes of 2 mm dimensions depth and
diameter created under copious irrigation with
distilled water in the femur using 2 mm carbide
bure connected to slow motion dental engine.
About 5 mg of BLUEBONE material were
mixed with drop of distilled water to create
pasty material for better application, one hole
was filled with material and the other left
empty. The rabbits were left to heal at different
time intervals. Animals were divided into 4
groups and sacrificed at different time intervals
at 3 days, 7 days, 14 days, and 28 days. After
the end of each time interval, the rabbits at each
group had been sacrificed and the femoral bone
was isolated and cut into two pieces, one
contained the control defect and the other
contained the treated defect each defect
radiographed at standard alignment and
distance from the X-ray source, the
radiographic digital system was Carestream®.
The setting of the machine was 60 kV, 10 mA
and 0.30 seconds. Measures were managed by
drawing line from the cortical bone crossing the
defect by Cs imagining software 7.0.3. Each
specimen kept in formalin 10%, labeled and
sent for histological preparation and
examination by specialist.
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Statistical analysis done by using SPSS
program version 26, Data were presented as
means ± SE (standard error) of mean and
analyzed by independent T-test at significant
level < 0.05.
RESULTS
1.Radiographic results
The radiographic results showed an increased
radioopacity of treated group as compared with
the periods of study. At 3 days after surgery,
both defects recognized a higher radioopacity
of treated group. As shown in (Figure 3).
Figure (3): Treated and control defects at 3 days after surgery.
At 7 days after surgery, the control defect
borders were clearly detected, whereas the
treated defect showed obstructed borders as
shown in (Figure 4). At 2 weeks after surgery,
the control group borders were still detected,
while the borders of treated group barely
detected with Material still detected as shown in
(Figure 5). At 28 weeks after surgery, the
borders of both groups were not detected, and
the treated group showed higher radioopacity as
shown in (Figure 6).
Figure (4): Treated and control defect at 7 days after surgery.
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Figure (5): Treated and control defects at 14 days after surgery.
Figure (6): Treated group at 28 days after surgery.
2.Histological results
At 3 days after surgery control defect showed
sever infiltration of large number of
inflammatory cells, no bone spicules found.
Granulation tissue were formed with new
vascularization. While the defect treated with
BLUE BONE showed defect filled with
granulation tissue and moderate infiltration of
inflammatory cells. Small bone spicules were
formed with good vascularization and the
biomaterial still founded as shown in (Figures 7
and 8).
Figure (7): Histological section of control group 3 days after surgery at 10 X magnification.
Arrows show inflammatory areas.
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Figure (8): Histological section of BLUE BONE material group at 10X magnification at 3 days
after surgery. The selected area shows areas of newly formed bone. The arrow shows the
biomaterial.
At 7 days after surgery, the control group
showed few osteoblasts started to form bone
spicules i.e. cellular activity with moderate
infiltration of inflammatory cells. The treated
defect was filled with granulation tissue with
the formation of new bone trabeculae with mild
infiltration of inflammatory cells. Few
biomaterials still found in the defect area as
shown in (Figures 9 and 10).
Figure (9): Histological section of control group at one week at 7 days of 40X magnification.
Selected area indicates newly formed bony spicules. Arrows show osteoblast.
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Figure (10): Histological section of 10X magnification of BLUE BONE material group at 7 days.
The selected areas show the newly formed bone trabeculae. The arrow shows the biomaterial.
At 14 days after surgery, well recognized bone
trabeculae were found in the defect area with
granulation tissue, no inflammatory infiltration
was seen. The treated defect showed a very
well-formed bone trabeculae the biomaterial
still found with no inflammatory cells
infiltration as shown in (Figures 11 and 12).
Figure (11): Histological section of 4x magnification of control group at 14 days. Arrows show the
newly formed bone.
Figure (12): Histological section of 4X of BLUE BONE material group at 14 days. The
arrows show the newly formed bone trabeculae. The circle shows the remaining biomaterial.
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At 28 days after surgery, formation of new
compact, bone that closed the defect with no
inflammation seen. The treated defect showed
formation of thick compact bone that close the
defect area with no inflammation founded as
shown in (Figure 13 and 14).
Figure (13): Histological section of 4X magnification of control group at 28 days. The selected
areas show formation of new compact bone closing defect.
Figure (14): Histological section of 4X of BLUE BONE material group at 28 days. The arrows
show compact bone closing the defect.
3.Statistical analysis: all statistical results
represented mean ± standard error of mean, the
small letter refers the compression within
groups where change in the small letters means
that there was a significant statistical difference
while capital letters represent comparison
between groups where change in the capital
litters mean statistical significant difference.
1. Radiographic results: Statistical analysis
of radiographic results showed that there was
statistically significant difference between
defect filled with BLUE BONE material and
control defect as shown in (Table 1).
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Table (1): Statistical analysis of radiographical results, represented as mean ± standard error of
mean at significant level 0.05.
Small letters refer to comparison within group, their changes reflect statistically significant difference.
Capital letters refer to comparison between groups their change reflect statistically significant difference.
2. Newly formed bone below
Throughout the periods of study, the BLUE
BONE material showed greater bone formation
and statistically there was significant difference
as shown in (Table 2).
Table (2): Statistical analysis of new formed bone area represented as mean ± standard error of
mean at significant level 0.05.
Small letters refer to comparison within group, their changes reflect statistically significant difference.
Capital letters refer to comparison between groups their change reflect statistically significant difference.
3. Osteoblast numbers found:
The osteoblast numbers at treated defect were
greater than the numbers in control defect until
28 days. The number of osteoblasts at treated
group declined dramatically indicating that
most osteoblast changed to osteocyte. There
was statistically significant difference between
groups as shown in (Table 3).
Table (3): Statistical analysis of numbers of osteoblast represented as mean ± standard error of
mean at significant level 0.05.
3 Day
7 Day
14 Day
28 Day
Control
0.00 a A
11±0.7 b A
23.4±1.2 c
A
16.8±0.9 d
A
BLUE
BONE
12.6±0.7 a
B
26.6±1.08 b
B
41.5±1.06 c
B
10.8±0.8 a
B
Small letters refer to comparison within group, their changes reflect statistically significant difference.
Capital letters refer to comparison between groups their change reflect statistically significant difference.
3D
Mean ± SE
1W
Mean ± SE
2W
Mean ± SE
4W
Mean ± SE
CONTROL
90.8±3.2 a A
137±6.5 b A
179.6±12.9 c A
181.6±9.8 c A
Blue Bone
112.4±7.3 a B
159.2±5.1 b B
201.2± 9.6 c B
209.2±10.7 c A
3 Day
7 Day
14 Day
28 Day
Control
0.00
a A
28559.38±1679.3 b
A
111486.82±1818.6 c A
231346.76±4356.6 d A
BLUE
BONE
33187.66±2834.0
a C
141797.766 ± 887.96
bB
400577.04±14045.7 c B
606911.72±6197.6 dB
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4. Osteoclast number found
The osteoclasts numbers at treated defect were
greater than the numbers in control defect until
28 days the number of osteoclast at treated
group declined dramatically indicating that
most osteoclast finished their function. There
was statistically significant difference between
groups as shown in (Table 4).
Table (4): Statistical analysis of osteoclast number represented as mean ±standard error of mean at
significant level 0.05.
Small letters refer to comparison within group, their changes reflect statistically significant difference.
Capital letters refer to comparison between groups their change reflect statistically significant
difference.
DISCUSSION
Radiographic results revealed higher
radioopacity at treated group due to greater
bone formation and higher mineralization so
these results agreed with Chen et al 2017. The
current study displayed enhancement of bone
regeneration by using BLUE BONE material
by osteoconductive properties of material as the
biomaterial resorb free calcium and phosphate
ions released and the change in the ions
concentration stimulate formation and
differentiation of osteoblast and eventually
bone formation (12). Our results agreed with
Puttini et al. (2019) (13) who found greater bone
formation by using BCP. The treated group
showed greater cellular activity by presenting
higher numbers of osteoblast and osteoclast and
this high numbers of cells at treated group may
be due to porosities of material that permits
diffusion of cells, angiogenesis and nutrients
transportation. These porosities act as channels
for migration of cells and formation of new
blood vessels and this events founded by
Ebrahimi et al (2014) (14) who found that BCP
stimulate cellular accumulation. The
inflammatory response at the defect treated
with blue bone was lesser than that seen in
control group which showed sever
inflammatory response and this may be due to
anti-inflammatory action of both
hydroxyapatite and tricalcium phosphate. These
results agreed with Sadowska et al. 2019 (15)
3 Day
7 Day
14 Day
28 Day
COTROL
0.00 a A
2.2±0.3
b A
3.4±0.4
c A
1.8±0.1
b A
Blue bone
1.8±0.2
a B
5.5±0.7
b C
5.3±0.8
b B
0.7±0.1
a B
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who found lesser inflammatory response to
BCP. In conclusion, BLUE BONE material
accelerates bone formation and show high
potential capacity of bone regeneration.
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