Characterization of Biphasic Calcium Phosphate with Ratio of 70/30 Before and After Implanted into Sheep’s Bone

CHARACTERIZATION OF BIPHASIC CALCIUM
PHOSPHATE WITH RATIO OF 70/30 BEFORE AND AFTER
IMPLANTED INTO SHEEP’S BONE

DINI NOVIALISA

DEPARTMENT OF PHYSICS
FACULTY OF MATHEMATICS AND NATURAL SCIENCES
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2014

STATEMENT ON THESIS
I hereby declare that thesis entitled characterization of biphasic calcium
phosphate with ratio of 70/30 before and after implanted into sheep’s bone is my
work under direction of the supervising committee and has not been submitted in
any form to any college. The source of information is derived or quoted from the
published or unpublished work by other authors mentioned in the text and listed in
the reference at the end of this thesis.
I hereby assign the copyright of my papers to the Bogor Agriculture
University.

Bogor, June 2014
Dini Novialisa
G74100046

ABSTRACT
DINI NOVIALISA. Characterization of Biphasic Calcium Phosphate with Ratio
of 70/30 Before and After Implanted into Sheep’s Bone. Supervised by KIAGUS
DAHLAN.
Nowadays, biphasic calcium phosphate is used for biomaterial to cure bone
damage. This study focuses only on mixing ratio of 70% mass of hydroxyapatite
and 30% mass of tricalcium phosphate with chitosan and acetic acid as the
hardening. Pure HA had done by wet precipitation in sintering process at
temperature of 900 oC while β-TCP is at temperature of 1000 oC. Mechanical
method of BCP is proven to be a non toxic material and could absorb by the
mineral body. Implanted BCP was characterized with XRD, FTIR, SEM-EDS and
bone characterization with Vickers Hardness. In XRD analysis, the result is
adding chitosan makes BCP less accuracy of pure HA and β-TCP. FTIR shows
composition of BCP sample with HA, TCP, H2O, AKB, and N-H bending. SEMEDS releases ratio Ca/P of implant in post operation has irregularity caused by
interaction with bone that supported by Vickers Hardness number. HV for 1

month of post operation have larger number for both implanted and control bone.
Keywords: BCP, HA, β-TCP

CHARACTERIZATION OF BIPHASIC CALCIUM
PHOSPHATE WITH RATIO OF 70/30 BEFORE AND AFTER
IMPLANTED INTO SHEEP’S BONE

DINI NOVIALISA

a paper submitted
in partial fulfillment of the requirement
for bachelor degree
Faculty of Mathematic and Natural Sciences

DEPARTMENT OF PHYSICS
FACULTY OF MATHEMATICS AND NATURAL SCIENCES
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2014

Title
Name
NIM

: Characterization of Biphasic Calcium Phosphate with Ratio of
70/30 Before and After Implanted into Sheep’s Bone
: Dini Novialisa
: G74100046

Approved by

Dr Kiagus Dahlan
Supervisor

Known by

Dr Akhiruddin Maddu
Head of Physics Department

Graduated at:

PREFACE
Alhamdulillah, blessing to God and The prophet Muhammad the author
prayed that can be given to completed this research proposal entitled
“Characterization of Biphasic Calcium Phosphate With Ratio of 70/30 Before and
After Implanted into Sheep’s Bone”. The proposal is structured as a condition of
graduation degree program in Physics Departement of Bogor Agricultural
University.
The author would like to express sincere appreciation of my parents who
always support me, Dr. Kiagus Dahlan for his guidance, Setia Utami Dewi, M.Si
for suggestions to help completing this research and Nur Aisyah Nuzulia, M.Si for
her patience to teach theories of this research. I am also thankful to M. N. Indro
M.Sc and the other lecturers for the advices and encouragement. For all of my
friends at Physics students, IKPMR, Student Executive Board, I feel grateful to
have you in my life.
Nonetheless, the author also welcome for any critical feedback and advice
from readers in order to maintain it as a successful project. I hope this paper could
be useful and become reference of the other researches.

Bogor, June 2014

Dini Novialisa

CONTENT
TABLE LIST

viii

FIGURE LIST

viii

APPENDIX LIST

viii

INTRODUCTION

1

Background

1

Hypotheses

2

Objective

2

Benefit

2

MATERIALS AND METHOD

3

Place and Time

3

Materials and Equipments

3

Experimental Method

3

RESULTS AND DISCUSSION

5

XRD Analysis of BCP Implant

6

FTIR Analysis of BCP Implant

8

Analysis of Post Operation Material

8

Bone Characteristics of Post Operation
CONCLUSIONS AND SUGGESTION

12
14

Conclusions

14

Suggestions

14

REFERENCES

15

Curriculum Vitae

30

TABLE LIST
1 Lattice parameter of BCP sample
2 Macroscopic changes in implant and bone after implanted.*
3 Vickers Hardness Number of Implanted Bone in Three Month

7
12
13

FIGURE LIST
1

2
3
4
5
6
7
8

XRD patterns of HA and β-TCP sample
XRD patterns of BCP powder
XRD patterns of BCP pellet
FTIR spectra of BCP powder and BCP pellet
FTIR spectra of implanted samples in each month
SEM image showing physical structures of 1 month after implanted
SEM image showing physical structures of 2 months after implanted
SEM image showing physical structures of 3 months after implanted

5
7
7

8
9
10
11
11

APPENDIX LIST
1
2
3
4
5
6
7
8

Flowchart of Research
Pictures of Research Properties
Lattice parameter formulation match to HA
Lattice parameter formulation match to TCP
JCPDS references
FTIR Spectra of Samples
SEM Characterization
Vickers Hardness Number of Implanted Bone

17
18
19
21
22
23
25
28

1

INTRODUCTION
Background
The development of medical engineering to solve problems in bone
fracture or bone tissue repair has increased. The materials that mostly used in
medical engineering are apatite materials which have been known as the main
component of bone and tooth.1 Sources that can be used to synthesize apatite
materials as implant are stone, coral, eggshells and etc, have same characteristics
of apatite materials as bone graft such as bioactive, biocompatible, and
bioresorbable.2 It can be used to repair, restore, and replace damage bone tissue.3
One of the apatite materials that have same component with calcium
phosphate minerals in bone are hydroxyapatite (HA), Ca10(PO4)6(OH)2.4 HA as
bone filler can induce more new bone growth and accelerate bone healing
processes. Problems can arise in clinical situations due to the slow resorption rate
of pure HA.5 Nowadays, to overcome this problem some research have used
another calcium phosphate minerals to get better result for bone repair. Tricalcium
phosphate (β-TCP), Ca3(PO4)2, has biodegradability in faster replacement of the
material with bone tissue.6 Precipitation process by mixing a solution of
phosphate resources at higher sintering temperature successfully formed β-TCP
phase. It showed biodegradability or bioresorbability that is more readily than HA
ceramics.5,7
One common way to enhance the degradation properties of calcium
phosphate scaffolds is to combine a high soluble phase (β-TCP) with an insoluble
phase (HA) to create material called BCP ceramics.8 A mixture of HA and β-TCP
produces biphasic calcium phosphate (BCP) which possesses the reactivity of βTCP and the stability of HA, providing more bioactivity, involving more new
bone growth, and ensuring better resistance of the implants to strain.9 In
mechanical process, it will be expected that mixing of both calcium phosphate
phases can form materials that have osteoconductive and osteoinductive
properties which increase bone growth from fracture condition.2 The concept is
based on an optimum balance of the more stable phase of HA and more soluble βTCP. The material is soluble and gradually dissolves in the body, seeding new
bone formation as it releases calcium and phosphate ions into the biological
medium.10
Synthetic material is well crystallized, while bone mineral is a mixture of
amorphous and crystalline phase. With HA 70:30 β-TCP ratio, different structure and
component of bone will have same quality of crystalline because of biomaterial.
From Yessie (2007) research about in vivo study, bone apatite crystalline has a
nonlinear relation to the age, however younger rats is more crystalline than
older.11 Making a material that has osteoconductive and osteoinductive should be
adapted to what the bone needs. Young bone need more calcium to its growth,
with a high amount of HA will give more stability to bone’s healing. This
research suggests a new kind of biomaterial components that generate BCP with
highly successful for the present of 70 HA/30 β-TCP.
BCP materials have been largely used and made by factory-scale in some
developed country to meet the demand for bone damage. Commercial BCP (e.g.

2
Biomatlante ) has capability of resorbable and active properties that are well
suited to human bone. Based on economic point, the material is considered
expensive and still imported, so the material is not affordable by our Indonesian
society.12 In this thesis, it is used chicken eggshell as starting material to have
lower of the cost production and can be reachable by whole society. Utilization of
eggshell has proven by wise drop methods to form a phase of HA and β-TCP.7,13
Adding chitosan with acetic acid as solvent of BCP is for forming BCP so it can
be implanted into the bone. Picture of BCP pellet can be seen in Appendix 2.
Results of bone remodeling will show in X-Ray Diffraction (XRD),
Fourier Transform Infra Red (FTIR) and Scanning Electron Microscopy with
Energy Dispersive X-Ray Spectroscopy (SEM-EDS) analysis. Data of bone
analysis is obtained by Hardness Vickers Test. Hardness is usually redefined as
the resistance of material to indentation by another solid body under static or
dynamic loading. Hardness or indentation test measures hardness by driving an
indenter with a specific geometry into the polished surface of material with a
known load for a specific time.14
Hypotheses
Ratio 70/30 of HA/β-TCP will determine the ability of material for
accelerating the bone healing process in young bone sheep. It will drive more
bone tissue to appear. With 30% β-TCP of BCP material, the absorption of
material occurs after postoperative implantation. After the callus appeared that
shows bone healing process, the bone will absorb the material and speed up the
formation process of new bone tissue by the stability properties of 70% HA.
Objective
This research is conducted to analyze BCP material with ratio of 70/30
that was implanted into sheep’s bone. Ability to regenerate bone tissue by
material to be shown in the results will determine future research for better
improvement in bone healing. Characterization with X-Ray diffraction (XRD),
Fourier Transform Infra Red (FTIR), Scanning Electron Microscopy-Energy
Dispersive X-ray Spectroscopy (SEM-EDS), and Vickers Hardness Test will
show that material have great connection to bone for the healing process.
Benefit
It is expected from this research to obtain the best ratio of BCP for
biocompatibility, bioresorbability, and good osteoconductive and osteoinductive
of bone implant. Future medical material implant from biological waste can be
selected to have advantage in economical way. Besides it has shorter time to make
biomaterial than the synthetic one.

3

MATERIALS AND METHOD
Place and Time
This research was conducted from June to November 2013 which took
place in IPB-Biophysics Materials Laboratory while sample analysis (XRD and
FTIR) were performed in IPB-Analysis of Materials Laboratory. Characterization
of SEM-EDS and Vickers Hardness Test was done in BATAN. Bone implant
operation was done in IPB-Surgical and Radiology Laboratory, Faculty of
Veterinary.
Materials and Equipments
The Equipments are erlenmeyer, beaker glass, crucibles, mortar, aluminum
foil, whatman paper, Mohr pipette, magnetic stirrer, hotplate, analytical scales,
furnace, digital thermometer, Furnace Nebhertherm, Furnace Vulcan, burette,
infusion device, X-ray Diffraction (XRD), Fourier Transform Infrared
Spectroscopy (FTIR), Scanning Electron Microscopy and Vickers Hardness Tool.
The materials are eggshells, pro-analysis ammonium hydrogen phosphate
((NH4)2HPO4) and phosphoric acid (H3PO4), chitosan (C6H11NO4), acetic acid
(CH3COOH), aqua bides and aquades.
Experimental Method
Preparations
Preparation is started by cleaning up the eggshells from any kind of dirt
then drying it in room temperature for a while. Next process is calcinations, a
thermal treatment process in presence of air applied to solid materials to bring
about a thermal decomposition of calcium carbonate (CaCO3) into calcium oxide
(CaO) at 1000 oC. Calcination is be heated for about 3 hours of temperature
increment and 5 hours of holding time using Furnace Nebertherm.
Synthesis of Biphasic Calcium Phosphate by Mechanical Method
Hydroxyapatite (HA) synthesis was precipitated in wise drop method for
90 minutes. A solution of 0.3 M (NH4)2HPO4 in 100 ml of distilled water was
slowly added into 0.5 M CaO in 100 ml of distilled water while it was stirred
about 60 minutes and then aged for overnight. Samples then filtered by using
Whatman paper and dried in furnace with temperature 110 oC with holding time
for 5 hours. It followed by sintering process at temperature of 900 oC with holding
time for 5 hours. Samples were crushed and get characterization by using XRD
and FTIR.
Synthesis of β-Tricalcium Phosphate (β-TCP) started with precipitation by
using burette for 2 hours. A solution of 1.2 M CaO in 100 ml of distilled water
and solution of 0.8 M H3PO4 in 100 ml of distilled water was stirred in hot plate
about 50 oC of temperature. The sample then filtered by using whatman paper and
dried in the furnace at temperature of 110 oC and held for 5 hours. It followed by

4
sintering process at a temperature of 1000 oC with holding time for 5 hours. After
that, sample was be mashed and then characterized by using XRD and FTIR.
HA and β-TCP samples were mixed by using magnetic stirrer. For each
implant of 10 gram BCP, it has 7 gram of HA and 3 gram of β-TCP. These
samples were mixed with 0.2 gram of chitosan and acetic acid to form a gel.
Ensure to get the sample is in tube shaped with a diameter and length of 6-6.5 mm
(see pictures in Appendix 2) and get it into incubator with 50 oC of temperature
for sterilization. Flowchart of research is shown in Appendix 1.
Implantation of BCP into Tibia Sheep’s bone
BCP is implanted in 9 sheep aged 1-2 years old. All male sex of sheep
divided into 3 groups of 3 animals each for 30 days, 60 days and 90 days
observation the healing process in bone. Before the operation, BCP materials were
sterilized by exposure to ultraviolet light. The sheep were anaesthetized by
intravenous injection of xylazin 2%. Right os tibia of sheep is used for material
implant and the left os tibia for control requirement. Materials were implanted
with about 6 mm length or should not be entered into the bone marrow. Each
material implantation was performed under the same veterinary surgeon. The
edges of the bone membrane sutured with a traumatic suture and the incision was
closed with silk suture. Antibiotics were injected into the sheep post surgery. All
operations and postoperative procedures already had followed according to the
animal welfare act and the NIH guide for care and use of laboratory animals.
Animals were euthanized at 3 months, postoperatively.
XRD and FTIR Characterization
BCP before and after formed into pellet is characterized by both XRD and
FTIR to know the component phase of the sample. XRD analysis is used for
determining phase and lattice parameter of both samples. FTIR characterization is
used to get functional group of sample and support the results of XRD data.
After 3 months BCP had implanted, material implant is characterized to
know the macroscopic and microscopic changes of BCP. By using XD-610
Shimadzu XRD characterization, it had Cu as target with wavelength 1.54060 x
10-10 m and then discharge the sample of 200 mg on aluminum plate with
diameter of 2 cm. XRD pattern was being recorded in the range of 2θ from 10° to
80°. Results were compared with data analysis Joint Committee on Powder
Diffraction Standards (JCPDS). Lattice parameter is measured by angle references
emergence of HA and β-TCP. FTIR characterization used ABB MB 3000 model
FTIR Spectroscopy. Two milligram of sample compacted into a pellet with a
hundred of KBr to be irradiated by infra red with wave number in a range of
4000-400 cm-1.
SEM-EDS Characterization and Vickers Hardness Test
Postoperative characterization was FTIR, SEM-EDS and Vickers
Hardness. After getting FTIR analysis, implant was characterized by using
scanning electron microscope with energy dispersive X-ray spectroscopy, which

5
was using Phenom Machine. Some spot were used to get percent concentration of
each composition in the sample.
Vickers Hardness used HV-1000 with magnification lens by 40 and using
50 gram of indenter with 10 second of suppression time. Sample measurement by
Vickers Test is formed in (0.5 x 0.25 x 0.02) cm3 size and giving buffer with resin
and hardness after had being polishing to refine the surfaces. Hardness Vickers
Number (HV) is measured with 5 different spot area, 3 spots at the surfaces of
bone and the other 2 spots at the inner bone. Each spot have different HV that
were representing the hardness of bone.

RESULTS AND DISCUSSION
Wise drop method to synthesis HA with 3 hours stirring from eggshell
proven showing pure HA by Putri.13 Figure 1 shows XRD pattern of HA sample.
XRD peak showed matching with JCPDS 09-0432 as HA reference major peaks
of 2θ at 31.78 , 32.196 and 32.92 . Impurities that changed phase of calcium
phosphate because of unequal increasing temperature shows one peak at 25.93 as
OCP (Octa Calcium Phosphate). Although this peak has 40% of highest relative
intensity, it not concluded as impurity. The presence of OCP will not affect BCP
material that will be explained later.
From Hardiyanti, synthesis of β-TCP has optimum temperature by 1000
C. This temperature indicates 2 phases of HA and β-TCP, which is more
dominated by β-TCP.7 Figure 1 shows XRD pattern of β-TCP sample. XRD peak
showed matching with JCPDS 09-0169 as β-TCP reference peaks of 2θ at 27.8o,
31.04o, and 34.38o. Impurities are also shown in β-TCP material that will affect in
BCP implant that will be explained later.

Intensity (Counts)

HA
β-TCP
OCP
HA

β-TCP
10

20

30

40

50

60

Degrees 2-Theta
Figure 1 XRD patterns of HA and β-TCP sample

70

80

6
XRD Analysis of BCP Implant
From the Bragg Law, the peak positions provide both of the crystal structure
and the lattice parameter for each phase that is contained in the powder sample.
The diffraction beam intensity provides a measure of the distribution and position
of atoms within the crystal.15 Reference major peaks 2θ of HA is at 31.8 (2 1 1),
32.2 (1 1 2), and 32.9 (3 0 0) meanwhile major peak of β-TCP is at 27.77 (2 1
4), 31.03 (2 0 10) and 34.37 (2 0 0) is attached in Appendix 5 for JCPDS
references.
In this research, it used mechanical method to mix samples of HA and βTCP. This method is successfully making BCP without any significantly changes
in material phases. Material implant shows BCP peak before formed into pellets
(BCP powder) and BCP peak after formed into pellets (BCP pellet) which consist
of HA, β-TCP and OCP. XRD pattern of BCP powder and BCP pellet is shown in
Figure 2 and Figure 3.
From Figure 2, it is found 36 experimental peaks matching with 87 HA
reference peaks meanwhile from 523 β-TCP reference peaks, 61 experimental
peak matching is found. Relative Intensity Ratio (RIR) at 31.0778 (2 1 1) is 61%
of HA, 32.968 (2 0 11) is 38.98% of β-TCP and the others is OCP. Whereas in
Figure 3 showed 36 experimental peak matching with HA and 37 experimental
peak matching with β-TCP. RIR of HA at 31.778 (2 0 11) and β-TCP at 32.981
(3 0 6) is 63% and 36.97% respectively. Chitosan also affected to shift of the
diffraction peaks that decreased peak matching with the references. Matching
peaks of samples by XRD characterization can be seen in Appendix 3 and
Appendix 4.
By using ratios and measuring peak areas, the RIR method can be used to
determine the concentrations. It can be concluded that higher the percentage
shows higher concentration of samples. The mismatch of BCP ratio was caused
by the presence of β-TCP that is also known to enhance decomposition of HA. It
is postulated that β-TCP accelerates HA decomposition due to the thermal
expansion coefficient mismatch between the intimately mixed phase.16 Existence
of OCP was also presented at HA and β-TCP materials. However, the existence of
OCP did not matter for ceramics used for implantation because synthetic OCP
showed its osteoconductive characteristics that is faster in biodegrade rate than βTCP.17
Lattice parameter of BCP sample is shown in Table 1 where it changed
because of chitosan presence. Accuracy of each sample is reduced caused by the
bonding of composite BCP-chitosan. One of the most important characteristics of
chitosan, for tissue engineering applications, is its ability to be shaped into various
structures, such as pellet to improve their process capability and mechanical
properties. Chitosan presents a wide range of properties that make it appropriate
for tissue engineering applications, namely, its biodegradability, biocompatibility,
antibacterial activity, wound healing properties, and bioadhesive character.18 The
bonding extension arise due to appear of chitosan ties. Adding more mass of
chitosan will decrease accuracy of lattice parameter and peak number of HA and
β-TCP due to ion bonding in the sample.

7
300

HA
β-TCP
OCP

275

Intensity (Counts)

250
225
200
175
150
125
100
75
50
25
0
10

20

30

40

50

Degrees 2-Theta

60

70

80

Figure 2 XRD patterns of BCP powder
HA
β-TCP
OCP

350

Intensity (Counts)

300
250
200
150
100
50
0
10

20

30

40

50

60

70

80

Degrees 2-Theta
Figure 3 XRD patterns of BCP pellet
Table 1 Lattice parameter of BCP sample
β-TCP

HAP
Sample name
BCP powder
BCP pellet

a (Å)
9.46
9.47

Accuracy c (Å)

Accuracy a (Å)

Accuracy c (Å)

6.92
6.92

10.55
10.43

37.87
37.32

(%)
99.51
99.45

(%)
99.52
99.49

(%)
98.74
99.88

Accuracy
(%)
98.69
99.84

8
FTIR Analysis of BCP Implant

91

71
61
51
41

BCP powder

PO43-

BCP pellet

31

Transmittance (%)

81

OH-

21
11

PO43-

1
4000

3500

3000

2500

2000

1500

1000

500

Wavenumber (cm-1)
Figure 4 FTIR spectra of BCP powder and BCP pellet
Figure 4 shows FTIR spectra of BCP powder and BCP pellet. The first
indication for HA formation that assigned to the stretching mode of hydroxyl
groups (OH-) is at 3572 cm-1 and 3448 cm-1. While it was at about 1041 cm-1
which arise due to factor group splitting of the stretching asymmetry vibration (ν3)
fundamental vibration mode of the PO43- band. The bands at 455 cm-1 and at 563671 cm-1 correspond to bending vibration (ν2) and bending asymmetry vibration
(ν4) of the PO43- ion, respectively. The asymmetry bands of HA in ν3 and ν4
indicate HA is not entirely amorphous as shown in XRD pattern. (See Appendix 6
for FTIR peak spectra)
OCP is presented by H2O band at 1643 cm-1 that followed by XRD peaks in
BCP pellets form. This band had growing amounts after combined with chitosan
because of temperature given while it was drying. The present of chitosan and
acetic acid in BCP pellet does not affect the emergence of a new bond. Besides of
too little amount, this bond is disappeared by temperature incubator of chitosan
melting point that affected on the lattice parameter accuracy only.
Analysis of Post Operation Material
In FTIR spectra of implant, many peaks showed interaction of implant
with bone. As seen in Figure 5 that shows FTIR spectra of implanted sample in
each month. The spectrum of bone exhibits all the most intense bands observed in
the spectrum of hydroxyapatite (at 500-700 cm-1 and 900-1200 cm-1) and that of
collagen (in the 1200-1700 cm-1 and 2800-3700 cm-1 regions), being nearly
coincident with the sum of the respective profiles. Nevertheless, there are some

9
160
implant after 1 month
140
implant after 2 months

Transmittance (%)

120

implant after 3 months

100
80
60

N-H

40

OH-

CO32-

PO434000

3500

3000

2500

2000

1500

PO43-

1000

20
0

500

Wavenumber (cm-1)
Figure 5 FTIR spectra of implanted samples in each month
new bands (namely at around 870 cm-1 and 1400-1450 cm-1) originated from
carbonate substitutions in the crystal lattice of hydroxyapatite.19 FTIR spectra of
sample is appropriate with Figueredo et al research about bone graft materials.19
Bands of BCP pellets still existed at 455-571 cm-1 and 602 cm-1 of bending
vibration (ν2) and bending asymmetry vibration (ν4) of the PO43- ion, respectively.
While band at 1041 cm-1 is ν3 PO43- stretching asymmetry as well as stretching
mode of hydroxyl groups (OH-) at 3572 cm-1 and about 3448 cm-1. On the other
hand, the collagen moiety of bone originates the typical Amide I and Amide II
bands at 1659 cm-1 and 1551 cm-1, respectively. Furthermore, the bands at 1412
and 1458 cm-1 show a different profile and higher intensity in the spectrum of
bone relative to its organic model compound. These bands correspond to
absorptions from CH2 wagging and bending vibrations superimposed with those
from asymmetric stretching (ν3, usually as a double band) vibrations of CO32groups, present as ionic substitutes in the apatite crystal.19 This band is
characteristic of a type A carbonated apatite. The bands at 2854 cm-1 and 29242932 cm-1 also showed CH2 asymmetry stretching.
Surface remodeling refers to the resorption or deposition of bone material
on the external surface of the bone.20 A special characteristic of bioactive
materials is their ability to form a direct bond between the tissue and the
implanted material resulting in a uniquely strong interface. Bioactivity has been
associated with the formation of bone apatite on the surfaces of biomaterial.21 The
addition of new bonds that appeared on the material after implanted, showed bone
remodeling process and the bioactivity of BCP.
By SEM-EDS characterization, ratio of calcium/phosphate a raw for each
month are 1.74, 1.02 and 1.60, respectively (see Appendix 7). The structures of

10
sample after having connection to bone could possibly cause irregularity in ratio
number. Besides, there is a natural tendency for a living organism to respond a
foreign object when the material was exposed to a living organism. It caused by
protein adsorption, cell adhesion and calcium-phosphate homeostasis.22 In bone
remodeling process, ions were the activator. Phosphate was the most needed ion
to accelerate bone healing process. High absorption of phosphate is showed in 1
month of material implant. In 2 months of material implant, calcium absorbed as
much as phosphate and it has same condition in 3 months of material implant.
Besides the ion activator, uneven mixing cause irregularity in ratio number
of Ca/P, as seen in Figure 6, 7, 8 by SEM of physical structure images for each
month after implantation. The grains size is not same in each location. To obtain
the same grains size, the mechanical method can be given a stirred in hot plate
rather than just mixing without any treatment.

Figure 6 SEM image showing physical structures of 1 month after implanted

11

Figure 7 SEM image showing physical structures of 2 months after implanted

Figure 8 SEM image showing physical structures of 3 months after implanted

12
The result of BCP macroscopic analysis also concluded, in 3 months postoperative of BCP 70:30 with 10% chitosan mixing with the implant still not
absorb completely in bone. Table 3 shows macroscopically changes of post
operation, in condition, had chosen best sample out of the three. Operations still
have visible threads after 60 days and implant protrusions at bone surfaces which
have white color. Growth tissue at internal implant that cover implant and
degradation sign of implant is happen in 60 days post operation. Some material
implant also found in bone marrow that means error operation or not suitable
material size to its implantation place.
Physical state of implant that was too tough caused the problems. Bone
cell is difficult to penetrate the implant so then bone healing became hampered.
Porosity in implants is also needed for bone engineering applications since it
facilitates transport of nutrients and oxygen and enables tissue infiltration into the
pores. The challenge however is to reach an optimum density which can provide
the desirable mechanical properties while still maintaining a porous structure.16
Table 2 Macroscopic changes in implant and bone after implanted.*
Evaluation Time (Day Post-Operation)
30
60
90
Implant Condition
Ud
Ud
Ud
Implant Color
White
White
Bone Color
Growth Tissue at Periosteum
+
++
*Best condition of three sample implanted bone; undegradated (Ud) degradated
(Deg) (-) none (+) little (++) enough (+++) more.
Characteristics

Bone Characteristics of Post Operation
Bone analysis was done by data comparison of implanted and control bone
in Hardness Vickers Number (HVN or HV). Two spots are tested on the inner
surface table and three spots on the outer surface table were made on each
specimen. Inner and outer surfaces should have different measure in normal bone
(without case of remodeling bone). The average of HV is shown in Table 3.
Bone hardness depends on the levels of inorganic materials in the bone
matrix.23 Inorganic materials are calcium phosphate minerals. As mention above,
incompatibility value of Ca/P by SEM characterization is same with HV number
indicated in 1 month of post operation which has biggest number of HV.
Remodeling bone was activated by damage bone stimulated and blood released
the ions. The bone remodeling unit is composed of a tightly coupled group of
osteoclasts and osteoblasts that sequentially carry out resorption of old bone and
formation of new bone.24 Consequently bone damage should have minimum HV
because cells in healing condition that needed the nutrients. As implant given as
nutrients, implant material provided the components and the result is highest HV
for 1 month. In 3 months, bone cells have used all component availability to faster
healing process and HV number became higher than the previous month. After
remodeling bone is done that is a well known principle of orthopedics that
prolonged straining of a bone tends to make the bone stronger, that is to say,

13
stiffer and more dense.20 HV number of bone control showed decreasing caused
by unavailability of needed nutrients in bone and blood.
Table 3 Vickers Hardness Number of Implanted Bone in Three Month

1 Month Post-Operative
2 Month Post-Operative
3 Month Post-Operative

HV Bone Implant
Inner
Outer
surfaces
surfaces
17.68
23.29
14.60
18.06
14.73
20.38

HV Bone Control
Inner
Outer
surfaces
surfaces
14.02
13.22
16.37
18.31
11.01
15.94

14

CONCLUSIONS AND SUGGESTION
Conclusions
Characterization with FTIR, SEM-EDS and Vickers Hardness Test
showed a connection of material to bone for the bone healing process. Although
the ability of material to regenerate bone tissue is less fast than the control, BCP
material with ratio of 70/30 is proven to be a good material for implant in minor
damage bone because of its biocompatibility, bioresorbability, osteoconductive
and osteoinductive characteristic from biological waste synthesized. BCP material
also proved to providing more bioactivity, involving more new bone growth and
gradually dissolves in the body.
By using mechanical method to form BCP material, there is no phase
transformation happen because it changes only caused by thermal properties. BCP
analysis data showed that HA and β-TCP also brought out OCP, indicated by
XRD and FTIR characterization. Impurity in BCP ceramics was caused by result
of material before mixing process. In pellet formed, BCP have many peaks major
that showed HA, β-TCP and OCP. The presence of OCP did not matter in material
implantation because it biodegraded in faster than β-TCP. Adding more mass of
chitosan and acetic acid will decrease accuracy of lattice parameter and peak
number of HA and β-TCP due to ion bonding.
Implant characterization after implanted into sheep bone proved to have
protein adsorption, cell adhesion and calcium-phosphate homeostasis. These types
of responds are showed by the result of HV. Irregularity of HV was impacted by
unavailability of needed nutrients in bone and blood.
In SEM-EDS images showed implant for 1 month has the biggest Ca/P
because phosphate was firstly absorbed by bone. Damage bones need phosphate
to activated bone tissue for remodeling bone. It concluded that BCP with chitosan
and acetic acid showed biocompatibility properties because of showing any
inflammation but too slow for healing process and bioactive. Low absorption
capability of implant is showed with intact implant samples even after 3 months
of implantation. It caused by too hard of implant so that was difficult for bone
tissues and blood cells to penetrate. Which is means slow healing process due to
implant sample.
Suggestions
Using chitosan as hardener and to forming material into pellets is less
suitable in implant. Beside melting point of chitosan must be the incubator
temperature so that adding chitosan to formed material pellet would not changing
material characteristic such as XRD peaks to get the lattice parameter and any
other bending components beside HA and β-TCP. Mechanical method only used
in combining BCP implant with variation ratios of HA and β-TCP. It needs
another research to enhance better result in mechanical method, such as equitable
distribution of grains results.

15

REFERENCES
1. Warastuti Y and Abbas B. 2011. Sintesis dan Karakterisasi Pasta Injectable
Bone Substitute Iradiasi Berbasis Hidroksiapatit. Jurnal Ilmiah Aplikasi
Isotop dan Radiasi, 7 (2011) No. 2.
2. Laurencin CT and Yusuf K. Bone Graft Substitute Materials. Orthopedic
Surgery. Expert Rev Med Devices (2006) 3(1) pp 49-57.
3. Darwis D. 2008. Biomaterial untuk Keperluan Klinis. http://nhc.batan.go.id/.
[September 13th 2013].
4. Wahl DA and Czrenuszka JI. Collagen-Hydroxyapatite for Hard Tissue
Repair. Europe Cells and Material, 11 (2006) pp 43-56.
5. Piatelli A, Scarano A and Manganot C. Clinical And Histologic Aspects Of
Biphasic Calcium Phosphate Ceramic (BCP) Used In Connection With
Implant Placement. Biomaterials 17 (1996) pp 1767-1770.
6. Velayati, CE. 2013. Sintesis Komposit Biomaterial (β-Ca3(PO4)2)–(ZrO)
Berbasis Cangkang Telur Ayam Ras Dengan Variasi Komposisi Dan
Pengaruhnya Terhadap Porositas, Kekerasan, Mikrostruktur, Dan
Konduktivitas Listriknya. [Skripsi]. Malang: Universitas Negeri Malang.
7. Hardiyanti. 2013. Sintesis Dan Karakterisasi Β-Tricalcium Phosphate Dari
Cangkang Telur Ayam Dengan Variasi Suhu Sintering. [Skripsi]. Bogor:
Institut Pertanian Bogor.
8. Legeros RZ et al. The Effect of Fluoride on the Bone Mineralization of
Young Wistar Rats. Advancement of Life Science. Amvo Publishing
Company, Taiwan (2003) pp 29-33.
9. Daculsi G et al. Macroporous Calcium Phosphate Ceramics For Long Bone
Surgery in Human Dogs. Clinical and Histological Study. Journal Biomedical
Material 11 (1990) pp 379-396.
10. Daculsi G. Biphasic Calcium Phosphate Concept Applied To Artificial Bone,
Implant Coating And Injectable Bone Substitute. Biomaterials 19 (1998) pp
1473-1478.
11. Sari YW et al. Nanostructure in Bone Apatite. Biomed 06, IFMBE
Proceedings 15 (2009) pp 118-121.
12. Yolanda, Henkky. 2013. Influence of Distribution of Hidroksiapatit (HA) On
The
Strength
Of
the
Composite
Matrix
Albumen.
th
http://library.gunadarma.ac.id. [March 14 2013].
13. Putri, AAM. 2011. Metode Single Drop Pada Pembuatan Hidroksiapatit
Berbasis Cangkang Telur. [Skripsi]. Bogor: Institut Pertanian Bogor.
14. Langton CM and Njeh CF. 2004. Series of Medical Physics and Biomedical
Engineering: Physical Measurement of Bone. London: Institute of Physics
Publishing.
15. Flewitt PEJ and Wild RK. 2003. Physical Method for Materials
Characterization. London: Institute of Physics Publishing.
16. Chetty, Avashnee et al. 2012. Hydroxyapatite: Synthesis, Properties, And
Applications. New York: Nova Science Publishers.
17. Nuzulia, NA. 2009. Study of Biphasic Calcium Phosphate Ceramics and HaChitosan Composite Implanted into Sheep’s Bone. [Skripsi]. Bogor: Institut
Pertanian Bogor.

16
18. Rita, Ana et al. Scaffolds Based Bone Tissue Engineering: The Role of
Chitosan. Tissue Engineering (2011), Part B 17 (5).
19. Figueiredo MM, Gamelas JAF and Martins AG. Characterization of Bone and
Bone-Based
Graft
Materials
Using
FTIR
Spectroscopy.
http:www.intechopen.com[September 13th 2013].
20. Cowin SC and Hegedus DH. Bone Remodeling I: Theory of Adaptive
Elasticity. Journal of Elasticity 6 (1976) No. 3.
21. Legeroz et al. Biphasic Calcium Phosphate Bioceramics: Preparations,
Properties and Application. Journal of Material Science: Materials in
Medicine 14 (2003) pp 201-209.
22. Shi, Donglu. 2004. Biomaterials and Tissue Engineering. Germany: SpringerVerlag Berlin Heidelberg
23. Elhaney et al. Mechanical Properties of Cranial Bone. Journal Biomehnzics 3
(1970) pp 495-511.
24. Mark
KK.
2010.
The
Skeletal
System:
Bone
Tissue.
http://www2.sunysuffolk.edu/kennym/. [March 27th, 2014].

17

Appendix 1 Flowchart of Research
Synthesis of
Hydroxyapatite

Synthesis of βTricalcium Phosphate

XRD and FTIR
Characterization

XRD and FTIR
Characterization
BCP Making by
Mechanical Method

XRD and FTIR
Characterization

BCP + Chitosan and
Acetic Acid (Pellet form)

XRD and FTIR
Characterization

Materials Implanted into
Tibia Sheep’s Bone

FTIR, SEM, and
Vickers Hardness Test

Data Analysis and
Thesis Writing

18

Appendix 2 Pictures of Research Properties

Presipitation

Pellets form of BCP

Position of Sample in Bone

Polisher tool

Hardness Test Tool

Bone sample to characterized (A) without
resin; (B) with resin as hardener

19

Appendix 3 Lattice parameter formulation match to HA

Where,

∑

∑

∑

∑

∑

∑

∑

∑

∑

∑

∑

∑

Intensity (Counts)

BCP Before Implanted

2
XRD patterns of BCP sample that matched with HA peak references

Intensity (Counts)

20

2
XRD patterns of BCP sample that matched with HA peak references

21

Appendix 4 Lattice parameter formulation match to β-TCP

Intensity (Counts)

BCP Before Implanted

2
XRD patterns of BCP sample that matched with β-TCP peak references

Intensity (Counts)

BCP After Implanted

2
XRD patterns of BCP sample that matched with β-TCP peak reference

22

Appendix 5 JCPDS references
a. Hydroxyapatite

b. β-Tri Calcium Phosphate

23

Appendix 6 FTIR Spectra of Samples
BCP Sample before Pellet Form

BCP Sample after Pellet Form

24

1 Month of Implant Sample

2 Months of Implant Sample

3 Months of Implant Sample

25

Appendix 7 SEM Image and Distribution of Element Concentrations
SEM image of 1 month implant sample

Distribution of element concentrations from 1 month implant sample
Element
Name
Calcium
Oxygen
Phosphorus
Carbon

Confi
dence
(%)
100
100
100
100
Sum

Spot 1

Spot 2

Spot 3

Spot 4

Spot 5

C

E

C

E

C

E

C

E

C

E

19
67.2
13.7
99.9

0.8
1.2
0.9
-

35.8
47.8
10.3
6
99.9

1
3.3
1.9
2.7

17.2
69.6
13.2
100

0.8
1.1
0.9
-

26.7
57
16.3
100

0.8
1.7
1
-

12.9
76.4
10.6
99.9

0.8
0.9
0.9
-

Ca/P =

Mean
22.32
63.6
12.82
6

26

SEM image of 2 months implant sample

Distribution of element concentrations from 2 months implant sample
Confid
Element ence
(%)
Ca
100
O
100
P
100
N
100
Sum

Spot 1

Spot 2

Spot 3

Spot 4

Spot 5

Spot 6

C

C

C

C

C

C

E

E

E

E

E

20 0.8 27.3 0.8 12.1 0.9 30.8 0.8 12.6 0.9
65.9 1.2 54.6 1.7 75.9 0.9 19.9 0.9 67.1 1.1
14.1 0.9 18.1 0.9 11.9 0.9 49.3 2.1 12.8 0.8
7.5 3.4
100
100
- 99.9 100
100
-

Ca/P =

18.4
68.5
13
99.9

E

Mean

0.8 20.2
1.1 58.65
0.9 19.86
7.5
-

27

SEM image of 3 months implant sample

Distribution of element concentrations from 3 months implant sample
Element
Name
Calcium
Oxygen
Phosphorus
Carbon

Confi
dence
(%)
100
100
100
100
Sum

Spot 1

Spot 3

Spot 4

C

E

C

E

C

E

C

E

36
41.9
22.1
100

0.8
2.6
0.9
-

18.8
67
14.2
100

0.8
1.3
0.9
-

32.6
44.6
16.3
6.5
100

0.9
2.6
1.2
2.2

15.5
73
11.5
100

0.8
1
1
-

Ca/P =
Note : C = Concentration (% Wt)
E = Error (%)

Spot 2

Mean
25.725
56.625
16.025
6.5

28

Appendix 8 Vickers Hardnes Number of Implanted Bone

d average =

HV =

Hardness Vicker Number of Bone in a Month after Implanted
Control Bone D-13
d1
d2
d average (d average)2
HV
HV average
3.38 3.38 0.08450
0.007140 12.98554
3.24 3.24 0.08100
0.006561 14.13199 14.022868
3.16 3.14 0.07875
0.006202 14.95107
3.32 3.3
0.08275
0.006848 13.54058
13.224813
3.39 3.39 0.08475
0.007183 12.90904

d1
2.77
2.82
3.14
2.48
2.57

d2
2.77
2.82
3.14
2.48
2.57

Implant Bone D-17
d average (d average)2
HV
HV average
0.06925
0.004796 19.33454
0.0705
0.004970 18.65500 17.678663
0.0785
0.006162 15.04645
0.062
0.003844 24.12071
23.290804
0.06425
0.004128 22.46090

Hardness Vicker Number of Bone in 2 Months after Implanted
d1
3.01
3.01
3.01
2.77
2.93

d1
3.13
3.16
3.28
2.78
2.96

d2
3.01
3.01
3.01
2.77
2.93

Control Bone D-8
d average (d average)2
HV
HV average
0.07525
0.005663 16.37421
0.07525
0.005663 16.37421 16.374212
0.07525
0.005663 16.37421
0.06925
0.004796 19.33454
18.307558
0.07325
0.005366 17.28057

d2
3.13
3.16
3.28
2.78
2.96

Implant Bone D-10
d average (d average)2
HV
HV average
0.07825
0.006123 15.14275
0.07900
0.006241 14.85659 14.596251
0.08200
0.006724 13.78941
0.06950
0.004830 19.19569
18.063881
0.07400
0.005476 16.93207

29

Hardness Vicker Number of Bone in 3 Months after Implanted

d1
d2
3.6 3.6
3.49 3.5
3.98 3.95
2.86 2.86
3.32 3.25

Control Bone D-2
d average (d average)2
HV
HV average
0.09000
0.008100 11.44691
0.08738
0.007634 12.14504 11.009457
0.09913
0.009826 9.436415
0.07150
0.005112 18.13683
15.942147
0.08213
0.006745 13.74747

d1
3.09
3.07
3.4
2.64
2.78

Implant Bone D-5
d average (d average)2
HV
HV average
0.07725
0.005968 15.53733
0.07675
0.005891 15.74043 14.728935
0.08475
0.007183 12.90904
0.06600
0.004356 21.28558
20.380242
0.06900
0.004761 19.47490

d2
3.09
3.07
3.38
2.64
2.74

30

Curriculum Vitae
The writer was born at Pekanbaru November 10th 1992.
She is the first of three children from Mr. Syafe’i and Mrs.
Lismar Yuhaimi. She studied at TK AISIYAH II in 1998, SDN
030 Pekanbaru and graduated in 2004, SMP N 1 Pekanbaru
and graduated in 2007, SMA N 1 Pekanbaru and graduated in
2010 and accepted as physics student at Bogor Agricultural
University by USMI (Undangan Seleksi Masuk IPB).
As a student, writer was active for being assistant of
physics laboratory at TPB IPB. In student organization, writer
was membered of Executive Student Board TPB IPB (2011),
Cybertron Asrama TPB IPB (2011), Executive Student Board
FMIPA IPB (2012) and Organisasi Mahasiswa Daerah IKPMR
Bogor (2013). During studying, writer had received Peningkatan Prestasi
Akademik (PPA) scholarship in 2011, Charoen Phokhand scholarship in 2012,
Bantuan Belajar Mahasiswa (BBM) scholarship in 2013-2014 and Yayasan
Amanah scholarship in 2013-2014.