In vitro analysis of biphasic calcium phosphate and hydroxyapatite as bone implants

IN VITRO ANALYSIS OF BIPHASIC CALCIUM PHOSPHATE
AND HYDROXYAPATITE AS BONE IMPLANTS

RAHMI SOLIHAT

GRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2011

STATEMENT ON THESIS
I hereby declare that the thesis of “In vitro analysis of biphasic calcium phosphate
and hydroxyapatite as bone implants” 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source of information is derived or quoted from the published or unpublished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.

Bogor, 2011

Rahmi Solihat

G751090011

ABSTRACT
RAHMI SOLIHAT. In Vitro Analysis of Biphasic Calcium Phosphate and
Hydroxyapatite as Bone Implants. Supervised by KIAGUS DAHLAN and BOY
M. BACHTIAR.
Hydroxyapatite (HA) and Biphasic Calcium Phosphate (BCP) are widely
used as bone implant materials because of its biocompatibility. The minimum
requirement of biocompatible materials is nontoxic. The reduction of MTT (3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to a purple formazan
is used to measure toxicity of implant materials that exposed to the cell. The
biocompatibility also can be observed from the attachment between cell and
implant materials under electron microscope. This research reported that HA and
BCP derived from eggshell using precipitation and hydrothermal method were
nontoxic. MTT toxicity analysis was held during 1, 2 and 3 days immersion of
HA and BCP on MG63-osteoblast cells. The viability cells immersed with HA
and BCP were higher than 100% for 1, 2, and 3 days which were 476.12%,
380.60%, 182.59% and 307.21%, 128.36%, 155.47% respectively. This result
means that HA and BCP were categorized as nontoxic and induced the cells to
grow. The viability cells were decreased as longer immersion time because of
osteocalcifications that secret protein collagen were appeared. This MTT analysis

result was appeared in a good agreement with Scanning Electron Microscope
(SEM) characterization, that showed the attachments of either HA or BCP to the
osteoblast cells after 1 day immersion. The SEM photos after incubated 3 days
showed that cells start to calcify and secret protein. The calcification and secretion
of protein collagen performed much better after 14 days immersion. In conclusion,
HA and BCP derived from eggshell were nontoxic and performed good adhesion
interaction to the host cells in vitro.
Keywords: hydroxyapatite, biphasic calcium phosphate, in vitro, MTT analysis,
SEM

ABSTRAK
RAHMI SOLIHAT. Pengujian kalsium fosfat dua fasa dan hidroksiapatit secara in
vitro sebagai implan tulang. Dibimbing oleh KIAGUS DAHLAN dan BOY M.
BACHTIAR.
Hidroksiapatit (HA) dan Kalsium Fosfat Dua Fasa (KFDF) digunakan
secara luas sebagai bahan implan tulang karena sifat biokompatibilitasnya.
Persyaratan minimum bahan biokompatibel adalah tidak toksik. Reduksi 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) menjadi formazan
yang berwarna ungu digunakan untuk mengukur toksisitas bahan implan yang
diberikan terhadap sel. Biokompatibilitas juga dapat diamati melalui adanya
pelekatan antara sel dan bahan implan melalui mikroskop elektron. Penelitian ini

melaporkan bahwa HA dan KFDF yang diperoleh dari cangkang telur melalui
metode presipitasi dan hidrotermal bersifat tidak toksik. Pengujian toksisitas
dilakukan dengan perlakuan perendaman HA dan KFDF di dalam sel osteoblas
MG-63 selama 1, 2, dan 3 hari. Viabilitas sel yang direndam dengan HA dan
KFDF lebih dari 100% untuk 1, 2, dan 3 hari perendaman, yakni 476.12%,
380.60%, 182.59% dan 307.21%, 128.36%, 155.47%. Hasil ini menunjukkan
bahwa HA dan KFDF bersifat tidak toksik dan menginduksi sel-sel untuk tumbuh.
Viabilitas sel cenderung menurun karena terjadinya proses osteokalsifikasi yang
mensekresikan protein kolagen. Hasil pengujian MTT ini sesuai dengan hasil
karakterisasi Scanning Electron Microscope (SEM) yang menunjukkan terjadinya
pelekatan antara HA atau KFDF dengan sel osteoblas setelah 1 hari perendaman.
Foto SEM sampel setelah inkubasi selama 3 hari menunjukkan bahwa sel mulai
mengalami kalsifikasi dan mensekresikan protein. Kalsifikasi dan sekresi protein
kolagen semakin terlihat setelah perendaman selama 14 hari. Jadi, HA dan KFDF
yang diperoleh dari cangkang telur bersifat tidak toksik dan memiliki interaksi
adesi yang baik dengan sel secara in vitro.
Kata kunci : hidroksiapatit, kalsium fosfat dua fasa, in vitro, pengujian MTT,
SEM

SUMMARY

RAHMI SOLIHAT. In Vitro Analysis of Biphasic Calcium Phosphate and
Hydroxyapatite as Bone Implants. Supervised by KIAGUS DAHLAN and BOY
M. BACHTIAR.
The minimum requirement of synthetic biomaterial is must be
biocompatible. Biocompatibility means that material is nontoxic and able to
interact with the host cells. The analysis of 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) assay was used to detect toxicity. Interaction
between cells and bone implants were characterized by Scanning Electron
Microscopy (SEM). Previously, hydroxyapatite is well known as biocompatible
implantable materials. But, the dense hydroxyapatite is almost non-resorbable
when used as bone implant. In the other hand, the porous β-tricalcium phosphate
(TCP) displays affinity for high speed biological degradation. TCP was later
identified as Biphasic Calcium Phosphate (BCP) consisting of hydroxyapatite
(HA) and tricalcium phosphate (β-TCP).
Cytotoxicity analysis of bone implants were exposed to the osteoblast
cells. The bone implants are powder of HA and BCP. HA synthesis was using
precipitation method as Dewi’s work (2009). BCP synthesis was using
hydrothermal method as Fajriyah’s work (2010). Both HA and BCP were using
CaO and (NH4)2PO4 as the raw materials. CaO was derived from chicken
eggshells that rich of calcium carbonates (CaCO3). Chicken eggshells were
calcined at 1000°C for 5 hours based on Nurlaela’s work (2009). Calcinations of

eggshell formed CaO and removed carbonate ion. Dewi (2009) was then
optimizing the molarities of CaO and (NH4)2PO4, which were 0.1, 0.2, 0.3, 0.4,
and 0.5 with Ca/P 1.67 as the characteristic of HA. X-Ray Diffraction (XRD)
characterizations showed that pure HA was produced from 0.3 M of CaO.
Fajriyah (2010) was done an optimation of sintering temperature in synthesizing
BCP, which were 2, 4, and 6 hours. XRD characterization showed that BCP
phase was formed by 6 hours of sintering. MG63-cell line was using as the
prototype of human osteoblast cells. The osteoblast cells are one type of human
bone cells.
Citotoxicity analysis was using MTT solution which is yellow and toxic.
MTT solution reacted to the cells indicated by color change from red to black. The
MTT solution added to the blank which contained only basic medium was not
resulted color change. Color change into black is because of the reduction of MTT
to the formazan product. Degree of blackness was measured by the absorbance
devices. Based on the calculation of absorbance, there is a percentage of cells
viability on cells, HA, and BCP samples treated by 1, 2, and 3 days of immersion.
One day immersion of cells either exposed by HA or BCP were higher than
samples which contained cells only. This result indicated that HA and BCP
implant acted as extracellular matrices and induced cells to grow and calcify.
Cells viability of cells exposed by either HA or BCP after 2 and 3 days of

immersion tend to decrease. This result means that HA and BCP could induce
osteocalcifications. Cells osteocalcifications were not appeared at cells samples

that proved by the increasing of cells viability after 2 and 3 days of immersion.
All samples that exposed by bone implants have higher than 100% of cells
viability which enable us to categorize either HA or BCP as nontoxic
biomaterials.
SEM photos show that either HA and BCP were small cubes crystallites.
In the other hand, SEM photos of cells only incubated for 1, 3, and 14 days show
that cells structure presented as small flatten balls-like morphology. Interaction
between cells and bone implants were showed by the adhesion that covered
almost all structure of HA or BCP after 1 day of immersion. Crystallite HA and
BCP structure were still be recognized for 1 day of immersion. SEM photos after
3 days of immersion show that cells start to calcify and secret sticky extracellular
matrices protein which covered all of HA and BCP structures. This
osteocalcifications were appeared in a good agreement as MTT result after 3 days
of immersion. SEM photos after 14 days of immersion show perfectly greater
interaction of secreted protein and bone implants.
In conclusion, HA and BCP derived from eggshell were nontoxic and
performed good adhesion interaction to the host cells in vitro.

Keywords: hydroxyapatite, biphasic calcium phosphate, in vitro, MTT analysis,
SEM

© Copyright of IPB, the year 2011
Copyright reserved by the law

Forbidden to quote part or all of these writings without including or mentioning
the source. Citing is only for educational purposes, research, paper writing,
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reproduce part or the whole paper in any form without permission from Bogor
Agricultural University.

IN VITRO ANALYSIS OF BIPHASIC CALCIUM PHOSPHATE
AND HYDROXYAPATITE AS BONE IMPLANTS

RAHMI SOLIHAT

A THESIS

in partial fulfillment of the requirements
for the degree of Master of Science of
Biophysics Program

GRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2011

APPROVAL SHEET
Theses Title :
Name
NRP

:
:

In Vitro Analysis of Biphasic Calcium Phosphate and
Hydroxyapatite as Bone Implants
Rahmi Solihat

G751090011

Approved by:
The Commission of Supervisors

Prof. drg. Boy M. Bachtiar, MS., Ph.D
Co-Supervisor

Dr. Kiagus Dahlan
Supervisor

Certified by:
Head of Biophysics
Postgraduate Program

Dean of the IPB Graduate School

Dr. Agus Kartono

Prof. Dr. Ir. Khairil Anwar Notodiputro, MS

Date of Examination: 8 Februari 2010

Date of Graduation:

External Examiner: Dr. Akhiruddin Maddu

ACKNOWLEDGEMENTS
The author would like to expresses sincere appreciation of Dr. Kiagus
Dahlan, Prof. drg. Boy M. Bachtiar, MS., Ph.D, and Prof. Djarwani Soejoko for
directing the research, Setia Utami Dewi, M.Si for the help in the laboratory
sample preparation, Maysyaroh, S.Si for in vitro analysis, Ir. Basril Abbas for
gamma radiation sterilization, and Wikanda for SEM characterization. The author
is also grateful to the National Education Department for awarding her a
scholarship under the programme Beasiswa Unggulan and Hibah Kompetensi for
funding this research. To all of these people, I owe its whole-hearted gratitude that
impossible to describe.
I am also thankful for Ibu, Bapa, Aa Diki & Teh Tina, De Rahmat, Aa
Rudy, Mama, Papa, Teh Mira & Ka Haris, De Putri and whole families, 2009’s
Biophysics for the pray and support in bringing about this thesis. I give all respect

that impossible to describe, thank you so much. Nonetheless, I also welcome any
critical feedback and advice from readers in order to maintain it as successful
project. Hope this thesis could be useful.

Rahmi Solihat

CURRICULUM VITAE
Rahmi Solihat was born on September 17th, 1986. She is the second
daughter from Mr. Aip Syarifuddin and Mrs. Titin Prihatini. She was a student in
SDN Teladan Bangka III Bogor in 1992-1998, SMP Insan Kamil Bogor in 19982001, SMA Negeri 5 Bogor in 2001-2004, Bogor Agricultural University, and
Physics major in 2004-2008. Trying to share her knowledge, she has been being
as a private teacher for both high school and first-year college student.

TABLE OF CONTENTS

Page
TABLES LIST.......................................................................................... xxiii
FIGURES LIST.........................................................................................xxv
APPENDICES LIST................................................................................ xxvii
INTRODUCTION................................................................................... 1
Background....................................................................................... 1
Hypotheses........................................................................................ 2
Objective.............................................................................................2
Benefit................................................................................................ 2
LITERATURE REVIEW........................................................................ 3
Hydroxyapatite (HA)…..………………………………………...... 3
HA Properties…………………..………………………….. 3
HA Synthesis……..………………………………………... 4
Biphasic Calcium Phosphate (BCP)……………………………….. 5
BCP Properties…………………..…………………………. 5
BCP Synthesis……..…………………………………………6
In vitro Study.................................................................................... 7
MATERIALS AND METHODS............................................................. 9
Place and Time Schedule.................................................................. 9
Materials and Equipments................................................................. 9
Experimental Method........................................................................ 11
HA Synthesis.......................................................................... 11
BCP Synthesis........................................................................ 12
Preparation for In vitro analysis............................................. 13
Cells Culture..................................................................13
Cells Concentration Counting........................................13
Sample Preparation for SEM Characterization………….......15
Sample Characterization................................................................... 15
MTT analysis...........................................................................15
Scanning Electron Microscopy (SEM) characterization...... 15
RESULT AND DISCUSSION................................................................ 17
HA characterization............................................................................17
BCP characterization...........................................................................18
MTT analysis..................................................................................... 18
SEM characterization........................................................................ 21
CONCLUSION AND SUGGESTIONS.................................................. 25
REFERENCES..........................................................................................27
APPENDICES...........................................................................................29

TABLES LIST
Page
1.
2.
3.
4.

Family of calcium phosphate compounds...........................................
Concentration and volume in culturing cells.......................................
Absorbance of cells, HA, and BCP after 1, 2, and 3 days of
immersion……………………………………………………………
Cells viability of cells, HA, and BCP samples………………………

4
19
20
20

FIGURES LIST
Page
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.

The crystal structure of hydroxyapatite…….......................................
XRD pattern of HA from wet precipitation method………………...
BCP X-Ray Diffraction Pattern from 9000C sintering........................
SEM picture showing the morphology of osteoblast cells on BCP
implant materials after 2 days immersion in vitro..............................
Precipitation process in HA synthesis……………………………….
Hydrothermal process in BCP synthesis…………………………….
Scheme of grid on hemocytometer glass board……………………..
Scheme of 96-well plates for MTT analysis………………………...
Visible spectrophotometer for absorbance analysis………………....
(a) JEOL JCM-35C scanning electron microscope and (b) Ion
Sputter JFC-1100 machine…………………………………………..
XRD Pattern of HA from precipitation method……………………..
XRD Pattern of BCP from hydrothermal method…………………...
Color change effect of MTT solution dropped to the samples………
Percentage of cells viability of cells, HA, and BCP samples 1, 2,
and 3 days of immersion.....................................................................
SEM photos of bone implants (a) HA (b) BCP powder with 5000
times-magnification............................................................................
SEM photos of cells only after (a) 1, (b) 3, and (c) 7 days of
immersion with 5000 times-magnification........................................
SEM photos of cells exposed by BCP after (a) 1, (b) 3, and (c) 14
days of immersion with 5000 times-magnification.............................
SEM photos of cells exposed by HA after (a) 1, (b) 3, and (c) 14
days of immersion with 5000 times-magnification.............................

3
5
6
7
12
12
14
14
14
15
17
17
19
21
21
22
23
23

APPENDICES LIST
Page
1.
2.

Flow Chart of BCP Synthesis…………………………….…………
Flow Chart of Hydroxyapatite Synthesis…………………..……..…
Flow Chart of (a) cell culture (b) cytotoxicity analysis by MTT
3.
analysis ……………………………………………………………...
4. Experimental Equipments of HA and BCP synthesis ……………....
5. Experimental Equipments of cell Culture and cytotoxity analysis.....
6. Experimental Equipments of sterilization...........................................
7. JCPDS Reference of HA and TCP…………………………………..
8. Cost Estimation of HA and BCP product……………………………
9. BCP and HA SEM photos of various magnification…………...……
10. Cells SEM photos of various magnification……………..………….
11. Cells exposed by HA-SEM photos of various magnifications………
12. Cells exposed by BCP-SEM photos of various magnifications……..

31
32
33
34
35
36
37
38
39
40
41
42

INTRODUCTION
Background
Recent developments on bone reconstruction concern mainly in
implantable synthetic materials, although there are different kind materials in bone
reconstruction as like as autograft, allograft and xenograft. Autograft are the
materials from others body part of patient itself for bone reconstruction. However,
the clinical use involves some difficulties such as septic complication, viral
transmission and the unavailability of native bone (Daculsi 2004). On the other
hand, allografts are more readily available than autograft since it is from the
donor. In spite of that, allograft bone has revealed a risk of disease transmission
(such as HIV and hepatitis), post-surgery pain, increased blood loss, secondary
surgical wounds, risk of thrombosis and it is also difficult to shape (Daculsi 2004
& Ooi et al 2007). A significant additional limitation of allograft is the delayed
remodeling by the host. In the case of very large defects, the allograft may remain
in the implant site throughout the patient’s life, creating area more prone to
fracture or infection (Ooi et al 2007). Likewise, xenograft also bears limitation
since it is from others species as like as animals, which has different characteristic
in mineral bone composition (Dewi 2009). In short, synthetic biomaterials were
preferable in answering others method limitation in bone reconstruction.
The minimum requirements of synthetic biomaterial include the following:
(1) the material must be biocompatible, such as nontoxic, blood- or tissuecompatible, noncarciogenic; (2) the material must not leach or release harmful
components into the living system; (3) the mechanical and physical properties of
the material, such as strength, elasticity, durability, and stability, must be
appropriate for the intended application; and (4) the desired mechanical properties
must last for the expected life of the implant; (5) the materials must be sterilizable
(Shi et al 2004).
Biocompatibility is the ability of a material to perform with an appropriate
host response in a specific application (Shi et al 2004). Previously, hydroxyapatite
is well known as biocompatible implantable materials. But, the dense
hydroxyapatite is almost non-resorbable when used as bone implant (Victoria &

Gnanam 2002). While, the porous β-tricalcium phosphate (TCP) displays affinity
for high speed biological degradation, they are bioactive and bioresorbable
materials (Victoria & Gnanam 2002). TCP were later identified as Biphasic
Calcium Phosphate (BCP) consisting of hydroxyapatite (HA) and tricalcium
phosphate (β-TCP) (Li et al 2003). Recently, there is a growing interest in
developing Biphasic Calcium Phosphate (BCP) ceramics as implant materials
because they are more effective in bone repair or regeneration which proved in
vitro and in vivo (Ramay & Zhang 2004).

Hypotheses
1.

Both BCP and HA in vitro analysis show as nontoxic materials.

2.

There appear morphological properties change of BCP and HA after in vitro
indicating adhesion interaction between HA or BCP and cells.

Objective
In vitro analysis that has been held from 0 up to 14 days could explain the
biocompatibility. In this study, the biocompatibility of BCP compared to HA was
being performed through in vitro analysis by cytotoxicity screening by using MTT
analysis. Furthermore, the change of morphological properties after in vitro was
being characterized using Scanning Electron Microscopy (SEM). BCP was being
synthesized through hydrothermal method while HA through precipitation
method.

Benefit
It is expected from this research that BCP is able to serve as biocompatible
bone implant in vitro. The short-term responses of cells to an implant material in
vitro may provide valuable indicators of the long-term biocompatibility in vivo.

LITERATURE REVIEW
Hydroxyapatite (HA)

HA Properties
There are many apatite compounds, including fluorapatite, chlorapatite,
carbonate-apatite, and hydroxyapatite (Oliveira et al 2006). Hydroxyapatite is
chemically similar to the mineral component of bones and hard tissues in
mammals; its chemical formula is Ca10(PO4)6(OH)2 (Fernandes & Laranjeira
1999). The chemical nature of hydroxyapatite lends itcellsf to substitution,
meaning that it is common for non-stoichiometric hydroxyapatites to exist. The
most common substitutions involve carbonate, fluoride and chloride substitutions
for hydroxyl groups, while defects can also exist resulting in deficient
hydroxyapatites.
Hydroxyapatite is bioactive material; the ability to integrate in bone
structures and support bone ingrowths, without breaking down or dissolving.
Hydroxyapatite is a thermally unstable compound, decomposing at temperature
from about 800-1200°C depending on its stoichiometry. Hydroxyapatite is a
calcium phosphate including hydroxide, and its Ca/P ratio is represented as 1.67.
The structure of hydroxyapatite is hexagonal, which has unit cell size, a = 9.418 Å
and c = 6.883 Å (Shi et al 2004).

This structure can be assumed as ideal

hexagonal crystal structure (closed-packed) from PO4-3 ion, which is inserted by
Ca+2 ion and OH- ion among the empty space of PO4-3 ions (Figure 1) (Shi et al
2004).

Figure 1 The crystal structure of hydroxyapatite (Shi et al 2004).

31

Table 1 Family of calcium phosphate compounds (Shi et al 2004).
Mineral
name

Chemical Name

Chemical Formula

Ca/P

Monetite

Dicalcium phosphate (DCP)

CaHPO4

1.00

Brushite

Dicalcium phosphate dehydrate (DCPD)

CaHPO4.2H2O

1.00

Ca8(HPO4)2(PO4)4.5 H2O

1.33

Ca10(HPO4)(PO4)6

1.43

Tricalcium phosphate (TCP)

Ca3(PO4)2

1.50

Hydoxyapatite

Hydroxyapatite (HA)

Ca10(PO4)6(OH)2

1.67

Hillinstockite

Tetracalcium phosphate (TTCP)

Ca4P2O9

2.00

Whitlockite

Octacalcium phosphate (OCP)

There are different phases of calcium phosphate ceramics that can be used
in medicine, depending on whether a bioactive or a resorbable material is desired
(Table 1) (Shi et al 2004). Generally, dense hydroxyapatite does not have the
mechanical strength to enable it to succeed in long term load bearing applications.
But, hydroxyapatite may be employed as bone fillers in forms such as powders,
porous blocks or beads to fill bone defects or voids. These may arise when large
sections of bone have had to be removed (such as bone cancers) or when bone
augmentations are required (such as dental applications). The bone filler would
provide a scaffold and encourage the rapid filling of the void by naturally forming
bone and provides an alternative to bone grafts. It would also become part of the
bone structure and would reduce healing times compared to previous bone
prostheses.

HA Synthesis
Hydroxyapatite in particulate form can be produced by using a variety of
methods, such as wet method, dry method and hydrothermal method (Shi et al
2004). In this study, hydroxyapatite synthesis was being performed through wet
method that is precipitation. Santos et al (2004) have been succeeding in
synthesizing hydroxyapatite through wet precipitation method based on the
chemical reaction below:
10Ca(OH)2 + 6H3PO4 → Ca10(PO4)6(OH)2 + 18 H2O

32

Figure 2 XRD pattern of HA from wet precipitation method ( HA) (Santos et al
2004).
The 0.5 M Ca(OH)2 suspension was prepared using Ca(OH)2 powder. The
suspension was degassed, vigorously stirred and heated for one hour at 40°C
temperatures. The 0.3 M H3PO4 solution was dropped into the Ca(OH)2
suspension at same temperature for approximately one hour at the rate 6 mL/min.
The pH was adjusted become pH = 7 by addition of 1 M NH4OH solution at the
end of the precipitation process. The XRD result showed below match to the
hydroxyapatite pattern (Santos et al 2004).

Biphasic Calcium Phosphate (BCP)

BCP Properties
Development of biphasic calcium phosphate (BCP), especially with
hydroxyapatite (HA: Ca10(PO4)6(OH)2) and tricalcium phosphate (TCP:
Ca3(PO4)2) has drawn considerable attention. HA and TCP, although have similar
chemical composition, they differ in their biological resorbing capacity. The dense
HA ceramics when used as bone implant as almost resorbable and bio-inert. While
the porous β-TCP containing ceramics displays affinity for high speed biological
degradation, they are bioactive and bioresorbable materials. The main attractive
feature of bioactive bone graft materials such as BCP ceramics is their ability to
form a strong direct bond with the host bone resulting in a strong interface
compared to bioinert or biotolerant materials which form a fibrous interface. The

33

bioactivity relies on physical and chemical properties of biphasic calcium
phosphate ceramics (Victoria & Gnanam 2002).
It is also noted that the presence of small amount of β-TCP 1100°C and
1200°C may be associated with the partial decomposition of HA phase. If HA is
annealed in air at 1200°C, it decomposed into the β-TCP phase according to
chemical reaction below (Ooi et al 2007).
Ca10(PO4)6(OH)2 → 3β-Ca3(PO4)2 + CaO + H2Ogas

BCP Synthesis
Kumar et al (2005) was succeed in synthesizing BCP from sintering
process. Firstly, the BCP granules were synthesized by the microwave. Calcium
hydroxide and diammonium hydrogen ortho phosphate (DAP) were used as raw
materials. The amounts of reactants used for the reaction were calculated based on
the Ca/P molar ratio of 1.58. Weighed amounts of the starting granules were
dissolved in water and the DAP solution was added to the calcium hydroxide
solution. The solution is then exposed to 900°C microwave irradiation in a
microwave oven during 20 minutes. The product was then dried in an oven. The
result of XRD has a major peak indicating TCP that is (0 2 0) peak as shown
below.

Figure 3 BCP X-Ray Diffraction Pattern from 9000C sintering (Kumar et al 2005).

34

In vitro study
Biocompatibility testing in vitro often involves the detection of cell
damage and death as like as cytotoxicity. Coelho et al (2000) was used the
reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to a
purple formazan product in the MTT assay to estimate cell viability. The
screening test is useful to detect over toxic effects of a test material in showing
incompatibility. For instance, the rate of growth, proliferation and differentiation
of cells on a material may be dependent on successful initial attachment and
spreading of the cells on the surface of the implant materials. In this respect, the
initial and short-term responses of cells to an implant material in vitro may
provide valuable indicators of the long-term biocompatibility in vivo (Lin et al
1997).
Riberio et al (2009) have been succeed in showing cell attachment to the
BCP implant materials within 2 days of immersion in osteoblast cells as SEM
pictures show below.

Figure 4 SEM picture showing the morphology of osteoblast cells on BCP implant
materials after 2 days immersion in vitro (Ribeiro et al 2009).

35

MATERIALS AND METHODS
Place and Time Schedule
This research was being conducted from February through September
2010 which took place in IPB-Biophysics Laboratory in sample preparation, while
in vitro sample analysis was done in Oral Biology Laboratory of Dental Faculty,
University of Indonesia. Sample sterilization was done in National Nuclear
Energy Agency (BATAN) Pasar Jumat. Sample characterization was done in
National Nuclear Energy Agency (BATAN) Serpong for SEM characterization.
Materials and Equipments
Materials
1. BCP synthesis materials :
a. Calcium oxide from chicken eggshell
b. Pro-analyze ammonium hydro phosphate; (NH4)2HPO4
c. Aquabides
2. MG-63 cell line as osteoblast cell
3. Cell culture medium materials :
a. Dulbecco’s Modified Eagle’s Medium (DMEM)
b. Fetal Bovine Serum (FBS)
c. Penicillin Streptomycin
d. Fungizone
4. Trypsin EDTA
5. Trypan blue
6. Washing medium materials : Phosphate Buffered Saline (PBS)
7. Cytotoxicity test materials :
a. MTT (3-(4,5-dimethylthiazol-2-yl)-2-5-diphenyltetrazolium
bromide)
b. Acidified Isopropanol
c. NaCl solution

37

8. SEM preparation materials :
a. Phosphate Buffered Saline (PBS)
b. 8% glutaraldehyde
c. Ethanol

Equipments
1.

BCP and HA synthesis equipments (Appendix 4) :
a. Analytical balance
b. Furnace
c. Heating plate
d. Hydrothermal reactor
e. Burette
f. Vacuum
g. Digital pHmeter
h. Beaker glass
i. Crucible
j. Mortar
k. Filter paper
l. Magnetic stirrer
m. Aluminum foil
n. Petri plate

2.

Gamma radiation sterilization with cobalt 60-radiation source
(Appendix 6)

3.

In vitro analysis equipments (Appendix 5) :
a. 0.2 m-sterile syiringe filter (Corning, Germany)
b. 50 mL-syringe (Terumo, Japan)
c. 15 mL- and 50 mL-tube (Falcon, USA)
d. Scrapper
e. Micropipette (Eppendorf, Germany)
f. Tips micropipette
g. Tube eppendorf (Axygen, USA)
h. Hemocytometer

38

i. Incubator (Memert)
j. Cell culture dish (35 mm×10 mm)
k. 96-well plates (NUNC, Denmark )
l. Microscope (Nikon Elipse 80i)
m. Biohazard safety cabinet (ESCO Micro PTE Ltd.)
n. Water bath
o. Centrifuge (Sorvall)
p. Vortexer (Bio-rad BR 2000)
q. Shaker (Certomat)
4.

Characterization equipments (Appendix 6) :
a. Scanning Electron Microscope (SEM) (JEOL JCM-35C)
b. Ion Sputter JFC-1100 machine
c. Bio-Rad Microplate Reader Benchmark Visible Spectrophotometer

Experimental Method
Experimental method is shown on flow chart on Appendices 1, 2, and 3.
Hydroxypatite synthesis
The same raw materials of CaO as synthesizing BCP that gained from
chicken eggshells were used to prepare 100 mL-CaO suspension which was seen
as white thick fluid. Chicken eggshells that mostly contain CaCO3 were calcined
at 1000°C for 5 hours as chemical reaction below:
CaCO3

CaO + CO2

0.3 M CaO suspension of eggshell product was dropped by 0.18 M of clear
(NH4)2HPO4-solution in 100 mL aquabides in 37°C while stirring at 300 rpm at
the rate of 7 mL/min. The final suspension was then be filtered under vacuum.
The filtered cake was then being dried in the furnace at 110°C during 5 hours. The
dried powder was then be sintered at 900°C during 5 hours by using furnace. The
chemical reaction of CaO and (NH4)2HPO4 was as below:
10CaO+6(NH4)2HPO4+14H2 O

Ca10(PO4)6(OH)2 + 12NH4OH + 10H2O

39

Figure 5 Precipitation process in HA synthesis.
BCP Synthesis
The solution of BCP in this research was prepared by a precipitation
method. A hundred milliliters of 0.67 M (NH4)2HPO4 solution was then be added
dropwise to the 100 mL of 1 M CaO solution at the rate of 7 ml/min. Previously,
CaO materials was prepared from chicken eggshell that calcined at 1000°C for 5
hours. The reaction was carried out at 300 rpm stirring. A white precipitate was
obtained at the end of the reaction. The precipitate was heated hydrothermally at
300°C for 8 hours while stirring at 300 rpm. Then, the precipitate was be aged for
12 hours without stirring until it cooled down to room temperature. The solution
was then be filtered under vacuum. The filtered cake was then being dried in the
furnace at 110°C during 5 hours. The dried powder was then be sintered at
1000°C during 6 hours.

Figure 6 Hydrothermal processes in BCP synthesis.

40

Preparation for In Vitro Analysis
BCP and HA need to be sterilized beforehand. Two milligram of BCP and
HA powder was being put in each glass bottle that was being sterilized by gamma
radiation with 25 kGy dozes.
Cells Culture
Culture medium was prepared in basic medium: DMEM supplemented
with 10% FBS, penicillin streptomycin and fungizones. All basic medium was
then melted beforehand inside 37°C-water bath within 15 minute. Osteoblast cell
was be taken from liquid nitrogen storage (-198°C). The cells was then melted
inside 37°C-water bath before being incubated for 24 hours at 37°C. The
osteoblast cell wells washed with PBS before added by 1 mL-trypsin EDTA in
order to release the attachment of cells from the bottom of the well. It was then be
incubated again for 10 min (37°C) before replaced to the 15 mL-tube and added
by basic medium. The 15 mL-tube was then centrifuge at 2000 rpm for 10 min
(24°C) in order to concentrate the cell become a small pellet. Its supernatant needs
to be removed and added by 5 mL of basic medium before homogenizing the
pellet cell by several times pipetting in order to get cell solution.
Cells Concentration Counting
80 L-cells solution, 10 L-FBS and 10 L-trypan blue was then mixed in
the 1.5 mL-eppendorf tube. Ten micro liters of solution in eppendorf tube was
then dropped to the hemocytometer glass board. The cells counting were done by
counting the cells on hemocytometer glass under optical microscope with 40
times-magnification. It has the separation grid to counting the cells as shown
Figure 7. A, B, C, D, and E is the result of cells counting manually under optical
microscope. The cells concentration was being calculated using Equation 1.
Cell suspension was prepared with a concentration of 2×105 cells ml-1 and
seeded into 96 well-plates. HA and BCP powder was being poured to well then be
incubated at 37°C in an atmosphere containing 5% of CO2 for 1, 2, and 3 days on
each sample in triplicate as scheme on Figure 8.
(1)

41

A

B

C

D

E

Figure 7 Scheme of grid on hemocytometer glass board
A is HA sample poured to the cells, B is BCP sample poured to the cells,
C is the cells only and D is a blank that only contains basic medium (Figure 8).
MTT test was performed to determine the cytotoxicity of BCP and HA. It was be
calculated using the Equation 2.

(2)

If the percentage of cell viability above 100 %, the materials exposed to the cell
would be categorized as nontoxic.

1

2

3

4

A

A

A

A

B

B

B

B

C

C

C

C

D

D

D

D

5

6

7

8

9

E
F
G
H

Figure 8 Scheme of 96-well plates for MTT analysis.

10

11

12

42

Sample preparation for SEM characterization
The surfaces and biocompatibility were examined by SEM. For this
purpose, after each culturing period, samples was being removed from culture,
washed in PBS, fixed in 2.5% glutaraldehyde, rinsed two times with PBS and
dehydrated in series of ethanol concentrations. The samples were then are dried at
room temperature and sputter coated with gold before observation under the SEM.
Sample characterization
MTT analysis
The absorbance of cells was being analysis by visible spectrophotometer at
wavelength 655 nm as shown on Figure 9. The output of absorbance measurement
is performed in optical density (OD).
Scanning Electron Microscopy (SEM) characterization
Sample surfaces were being examined using a Scanning Electron
Microscope after immersion in vitro (Figure 10a). Sample need to be coated by
gold-palladium (80% of Au and 20% of Pd) beforehand. Coating process is using
Ion Sputter JFC-1100 machine (Figure 10b). The magnification was being
performed in 5000, 10000, 20000, and 40000 times- magnification.

Figure 9 Visible spectrophotometer for absorbance analysis.

(a)

(b)

Figure 10 (a) Scanning electron microscope and (b) Ion Sputter machine.

43

RESULT AND DISCUSSION
HA characterization
XRD characterization was used to determine the presence of HA phase in
HA synthesis product. The determination of HA was based on JCPDS (Joint
Committee on Powder Diffraction Standards) database with number 09-0432
(Appendix 7). Figure 11 shows XRD pattern of HA from precipitation method.
This result proved that on 2θ 25.92, 31.8, and 32.96 have high intensity which
indicating the presence of HA. This XRD pattern of HA product was the same
with Santos et al (2004) that used Ca(OH)2 as its raw materials. This research has
more good economical value compare to Santos et al research, since the using of
eggshell as raw materials. Based on the estimation of cost calculation, every one
gram of HA synthesis consumed Rp104,636 (Appendix 8).

Figure 11 XRD Pattern of HA from precipitation method ( HA).

Figure 12 XRD Pattern of BCP from hydrothermal method ( TCP).

45

BCP characterization
The determination of BCP was based on JCPDS (Joint Committee on
Powder Diffraction Standards) of TCP database with number 09-0169 (Appendix
7). Figure 12 shows XRD pattern of BCP from precipitation method. This result
proved that on 2θ at 27.9, 31.14, and 34.56 have high intensity which indicating
the presence of TCP. This XRD pattern of HA product was the same Kumar et al
(2005) that used Ca(OH)2 as its raw materials. This research has more good
economical value compare to Kumar et al research, since the using of eggshell as
raw materials. Based on the estimation of cost calculation, every one gram of BCP
synthesis consumed Rp53,586 (Appendix 8).

MTT Analysis
Cytotoxicity analysis of bone implants exposed to the osteoblast cells. The
bone implants are powder of HA and BCP. HA synthesis was using precipitation
method as Dewi’s work (Dewi 2009). BCP synthesis was using hydrothermal
method as Fajriyah’s work (Fajriyah 2010). MG63-cell line was using as the
prototype of human osteoblast cells. The osteoblast cells are one type of human
bone cells (Kim et al 2004).
Both HA and BCP were using CaO and (NH4)2PO4 as the raw materials.
CaO was derived from chicken eggshells that rich calcium carbonates (CaCO3).
Chicken eggshells were calcined at 1000°C for 5 hours based on Nurlaela (2009).
Calcinations of eggshell formed CaO and removed carbonate ion. Dewi (2009)
optimizing the molarities of CaO and (NH4)2PO4, which were 0.1, 0.2, 0.3, 0.4,
and 0.5 with Ca/P 1.67 as the characteristic of HA. XRD characterization showed
that pure HA was produced from 0.3 M of CaO. Fajriyah (2010) was done an
optimation of sintering temperature in synthesizing BCP, which were 2, 4, and 6
hours. XRD characterization showed that BCP phase was formed by 6 hours of
sintering.

46

Table 2 Concentration and volume in culturing cells
C2 (cells/mL)

C1 (cells /mL)

V2 (mL)

V1 (mL)

medium (mL)

2×105

1.52×107

10

0.13

9.87

blank
Cells poured with HA
Cells poured with BCP
Cells only
Figure 13 Color change effect of MTT solution dropped to the samples.
Cells concentration derived from culturing cells after 1 day is 1.52×107
cells/mL. The concentration is enough over 3 days of immersion for MTT analysis
because this concentration is higher than 2×105 cells/mL (Table 2). The
concentration for immersion is based on Oliveira et al. (Oliveira et al 2006). Cells
volume was taken 0.13 mL from cells solution then 9.87 mL of basic medium was
added based on Equation 2. The function of basic medium was life medium and
nutrition for cells (Lin et al 1997).
Citotoxicity analysis was using MTT solution which is toxic and yellow.
MTT solution reacted to the cells indicated by color exchange from red to the
black. The MTT solution added to the blank which contained only basic medium
was not resulted color exchange (Figure 13). Color exchange become black is
because of the reduction of MTT to the formazan product (Ribeiro et al 2009).
Degree of black color was measured by the absorbance devices. Light source was
using red light so that it was absorbed when pointed to the black sample (contains
cells) and transmitted when pointed to the blank sample. Table 3 showed
absorbance measurement from spectrophotometer.

47

Table 3 Absorbance of cells, HA, and BCP after 1, 2, and 3 days of immersion
Immersion time (days)
1
2
3

Absorbance (OD)
Cells

HA

BCP

0.134
0.109
0.576

0.638
0.510
0.245

0.412
0.172
0.208

Table 4 Cells viability of cells, HA, and BCP samples
Immersion time (days)
1

2

3

Sample

Cells viability (%)

Cells (Control)

100.00

HA

476.12

BCP

307.21

Cells

81.09

HA

380.60

BCP

128.36

Cells

429.85

HA

182.59

BCP

155.47

Based on the calculation of absorbance into percentage of cells viability by
using Equation 3 (Table 4), so that the chart shows on Figure 14 representing the
change of viability cells on cells, HA, and BCP samples treated by 1, 2, and 3
days of immersion. One day immersion of cells either exposed by HA or BCP
sample were higher than samples which contained cells only. This result indicated
that HA and BCP implant acted as extra cellular matrices which inducing the cells
to grow (Lin et al 1997).
Cells viability of cells exposed by either HA or BCP after 2 and 3 days of
immersion tend to decrease (Figure 14). This result means that HA and BCP could
induce osteocalcification (Ribeiro et al 2009). Osteocalcification was not
appeared at cells only samples that proved by the increasing of cells viability after
2 and 3 days of immersion (Figure 14). All of samples exposed by bone implants
have higher than 100% of cells viability which enable us to categorize either HA
or BCP as nontoxic biomaterials.

48

Figure 14 Percentage of cells viability of cells, HA, and BCP samples 1, 2, and 3
days of immersion.

SEM Characterization
SEM photos show that either HA and BCP were small cubes crystallites
(Figure 15). In the other hand, SEM photos of cells only incubated for 1, 3, and 14
days show that cells structure presented as small flatten balls-like morphology
(Figure 16a, b, and c).

(a)

(b)

Figure 15 SEM photos of bone implants (a) HA (b) BCP powder with 5000 timesmagnification.

49

(a)

(b)

(c)

Figure 16 SEM photos of cells only after (a) 1, (b) 3, and (c) 7 days of immersion
with 5000 times-magnification.
Interaction between cells and bone implants were showed by the adhesion
that covered almost all structure of HA or BCP after 1 day of immersion as shown
on Figure 17a and 18a. Crystallite HA and BCP structure was still be recognized
for 1 day of immersion as shown on Figure 17a and 18a. SEM photos on Figure
17b and 18b after 3 days of immersion show that cells start to calcify and secret
sticky extra cellular matrices protein which covered all of HA and BCP structures
as Kim et al research (2004).
This osteocalcification was appeared in a good agreement as MTT result
after 3 days of immersion. SEM photos after 14 days of immersion show perfectly
greater interaction of secreted protein and bone implants as shown on Figure 17c
and 18c. Based on Figure 17 (c), BCP was appeared more degraded than HA as
shown on Figure 18 (c). This attachments between protein or cells and implants
are same as Riberio et al research. Riberio et al showed SEM photos BCP implant
materials within 2 days of immersion in osteoblast cells as shown on Figure 4.
BCP implants that immersed on osteoblast cells as Riberio et al research was in
densed shape of BCP scaffold while in this research in powder shape of HA and
BCP.

50

(a)

(b)

(c)

Figure 17 SEM photos of cells exposed by BCP after (a) 1, (b) 3, and (c) 14 days
of immersion with 5000 times-magnification.

(a)

(b)

(c)

Figure 18 SEM photos of cells exposed by HA after (a) 1, (b) 3, and (c) 14 days
of immersion with 5000 times-magnification.

51

CONCLUSION AND SUGGESTIONS
Conclusion
Based on in vitro analysis, it proves that HA and BCP powder which was
synthesized in IPB Biophysics Laboratory was nontoxic through MTT analysis.
So, it is possible to examine for further analysis through in vivo. Cells viability
exposed by bone implants was higher than those which were not exposed by any
bone implants. This result means that bone implants could be act as extra cellular
matrices which induced the cells growth. The interaction between cells and
implants were further proved by SEM photos. That interaction was the
attachments cells on the implants which cover almost all implant structure.

Suggestions
Osteocalcification that secret protein collagen need to be proved by
analysis of alkaline phosphatase (ALP) activity which can measure protein
content in cells culture (Kim et al 2005). In vitro procedures need particular skill
and carefulness in order to avoid contamination to the cells.

53

54

REFERENCES
Coelho MJ, Cabral AT, Fernandes MH. 2000. Human bone cell cultures in
biocompatibility testing. Part I: osteoblastic differentiation of serially
passaged human bone marrow cells cultured in a-MEM and in DMEM.
Biomaterials 21: 1087-1094.
Daculsi G. 2004. Micro macroporous calcium phosphate bioceramics. Business
Briefing Global Surgery.
Dewi SU. 2009. Pembuatan komposit kalsium fosfat-kitosan dengan metode
sonikasi [tesis]. Bogor: Program Pascasarjana, Insitut Pertanian Bogor.
Fajriyah HI. 2010. Hydrothermal synthesis of biphasic calcium phosphate [tesis].
Bogor: Program Pascasarjana, Insitut Pertanian Bogor.
Fernandes GF, Laranjeira MCM. Calcium Phosphate Biomaterials from Marine
Algae, Hydrothermal Synthesis and Characterization. Journal of
Univeridade Federal de Sta Catarina, Florianopolis. 1999; 23 [4]
Kim HW, Knowles JC, Kim HE. 2005. Hydroxyapatite and gelatin composite
foams processed via novel freeze-drying and crosslinking for use as
temporary hard tissue scaffolds. J Biomed Mater Res 72A: 136–145.
Kumar KR, Kumar TSS, Sunder M, Babu NR, Victor SP. 2005. Biphasic calcium
phosphates for antibiotic release. Trends Biomater. Artif. Organ 18 [2] :
213-218.
Li S, Wijn JD, Li J, Layrolle P, Groot KD. 2003. Macroporous biphasic calcium
phosphate

scaffold

with

high

permeability/porosity ratio.

Tissue

Engineering 9 [3] : 535-548.
Lin FH, Liao CJ, Liu HC, Chen KS, Sun JS. 1997. Behavior of fetal rat
osteoblasts cultured in vitro on the DP-bioactive glass substratum.
Materials Chemist and Physics 49: 270-276.
Nurlaela A, Dewi SU, Dahlan K, Soejoko DS. 2009. The use of eggshells as
calcium sources for synthesis of bone mineral. Proceeding of the 1st
International Seminar on Science and Technology; 24-25 Jan 2009.
Oliveira JM, Rodrigues MT, Silva SS, Malafaya PB, Manuela E. Gomes ME,
Viegas CA, Isabel R. Dias IR, Jorge T. Azevedo JT, Mano JF, Reis RL.

55

2006. Novel hydroxyapatite/chitosan bilayered scaffold for osteochondral
tissue-engineering applications: Scaffold design and its performance when
seeded with goat bone marrow stromal cells. Biomaterials 27: 6123–6137.
Ooi CY, Hamdi M, Ramesh S. Properties of hydroxyapatite produced by
annealing of bovine bone. Ceramics International Elsevier. 2007 : 33 :
1171-1177
Ramay HRR, Zhang M. 2004. Biphasic calcium phosphate nanocomposite porous
scaffolds for load-bearing bone tissue engineering. Biomaterials 25 :
5171–5180.
Ribeiro C, Rojas-Cabrera WI, Marques M, Bressiani JC, Bressiani AHA. 2009. In
vitro characterization of porous ceramic based calcium phosphate
processing with albumin. Key Engineering Materials Vols 396-398 : 2730.
Santos MH, Oliveira M, Souza LPF, Mansur HS, Vasconcelos WL. 2004.
Synthesis control and characterization of hydroxyapatite prepared by wet
precipitation process. Materials Research 7 [4] : 625-630.
Shi D. 2004. Biocompatibility of materials. Di dalam: Wang M, Zhang M, Clare
A, Kasuga T, Lin Q. Biomaterials and Tissue Engineering. SpringerVerlag Berlin Heidelberg. Page 83-143.
Victoria EC, Gnanam FD. 2002. Synthesis and characterization of biphasic
calcium phosphate. Trends Biomater. Artif. Organs 16 [1] : 12-14.
Wang M. Bioactive Materials and Processing. Springer-Verlag Berlin Heidelberg;
2004 : 1-82

56

APPENDICES

57

58

Appendix 1 Flow Chart of BCP Synthesis

Materials and equipment preparation

Ready?

CaO dissolving

Precipitation of (NH4)2HPO4
and Ca(OH)2 while stirring

Hydrothermal synthesis
while stirring

Solution aging and filtering

Sample drying

Hydroxyapatite compound

Sample sintering

BCP compound

In vitro Analysis

SEM characterization

Data analysis
Report Arrangement

59

Appendix 2 Flow Chart of Hydroxyapatite Synthesis

Materials and equipment preparation

Ready?

CaO dissolving

Precipitation of (NH4)2HPO4
and Ca(OH)2 while stirring

Solution aging and filtering

Sample drying
Sample sintering

HA compound

In vitro Analysis

SEM characterization

Data