Synthesis of Chicken Eggshells-Based β-Tricalcium Phosphate Bioceramics and Their Biocompatibility Test as A Tooth Filler
SYNTHESIS OF CHICKEN EGGSHELLS-BASED
β-TRICALCIUM PHOSPHATE BIOCERAMICS AND
THEIR BIOCOMPATIBILITY TEST AS A TOOTH FILLER
NUR AISYAH NUZULIA
GRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2014
STATEMENT ON THESIS
I hereby declare that the thesis of “Synthesis of Chicken Eggshells-Based
β-Tricalcium Phosphate Bioceramics and Their Biocompatibility Test as A Tooth
Filler” is my work with the direction of the supervising committee and has not
been submitted in any form to any college. Source of information derived or
quoted from the work published or not published 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 thesis to Bogor Agricultural University.
Bogor, April 2014
Nur Aisyah Nuzulia
ID G751110071
RINGKASAN
NUR AISYAH NUZULIA. Sintesis Biokeramik β-Tricalcium Phosphate Berbasis
Cangkang Telur Ayam dan Uji Biokompatibilitasnya sebagai Tooth Filler.
Dibimbing oleh KIAGUS DAHLAN, GUNANTI, dan SRIHADI
AGUNGPRIYONO.
Preservasi tulang alveolar setelah ekstraksi gigi merupakan langkah penting
dalam praktek gigi sebelum pemasangan implan gigi. Penelitian ini melaporkan
tentang sintesis biokeramik β-Tricalcium Phosphate (β-TCP) dan uji
biokompatibilitasnya sebagai tooth filler untuk mempertahankan dimensi alveolar
ridge dan merangsang proses penyembuhan tulang di daerah hilangnya gigi.
Biokeramik β-TCP dihasilkan secara langsung melalui metode presipitasi
cangkang telur ayam yang telah dikalsinasi sebagai sumber kalsium dan asam
fosfat. β-TCP tersebut dicampur dengan kitosan 2 % untuk mendapatkan β-TCP
dalam bentuk pelet yang digunakan dalam pengujian biokompatibilitas. Pengujian
biokompatibilitas β-TCP dilakukan pada gigi seri bawah kelinci dan domba
sebagai hewan uji.
Dalam studi ini, hasil x-ray diffraction (XRD) menunjukkan bahwa pola
puncak XRD cangkang telur ayam yang dikalsinasi pada suhu 1000oC bersesuaian
dengan fase kalsium oksida (CaO). Adapun fase CaO bertransformasi menjadi
fase Ca(OH)2 selama masa penyimpanan lebih dari tiga bulan. Profil XRD juga
membuktikan bahwa biokeramik β-TCP yang dihasilkan memiliki pola yang sama
dengan referensi data base dan β-TCP komersil yang tersedia. Spektra fourier
transform infrared (FTIR) sampel konsisten dan memperkuat hasil karakterisasi
XRD. Selain itu, rasio Ca/P sebesar 1.48 yang diperoleh dari hasil atomic
absorption spectroscopy (AAS) menunjukkan kemurnian sampel yang sesuai
dengan standar biokeramik β-TCP. Adapun pola puncak XRD dan spektra FTIR
yang berulang jelas menunjukkan sifat reproducibility sampel.
Alveolar socket domba yang diisi dengan produk β-TCP menunjukkan hasil
karakteristik yang diharapkan. Hasil radiografi sinar-x pada rahang domba
menunjukkan tidak adanya perubahan mesio-distal di edentulous area. Selain itu,
hasil histologi juga konsisten terhadap hasil radiografi dan gambaran makroskopik
yang dibuktikan dengan adanya pembentukan tulang baru. Namun, hasil histologi
dari rahang kelinci menunjukkan hal yang berlawanan dengan hasil radiografi dan
gambaran makroskopik. Hasil histologi pada rahang kelinci menunjukkan tidak
adanya pertumbuhan gigi pada kelinci yang dibuktikan dengan daerah ekstraksi
yang terisi penuh oleh lipid dan kolagen. Pengujian biokompatibilitas biokeramik
β-TCP menunjukkan bahwa produk β-TCP mampu meminimalis hilangnya tulang
serta mampu mempertahankan dimensi alveolar ridge.
Kata kunci: β-tricalcium phosphate, cangkang telur ayam, domba, kelinci, tooth
filler.
SUMMARY
NUR AISYAH NUZULIA. Synthesis of Chicken Eggshells-Based β-Tricalcium
Phosphate Bioceramics and Their Biocompatibility Test as A Tooth Filler.
Supervised
by KIAGUS
DAHLAN,
GUNANTI,
and
SRIHADI
AGUNGPRIYONO.
Preservation of alveolar bone following tooth extraction is an important step
in dental practices before dental implant placement. This study reported synthesis
of β-Tricalcium Phosphate (β-TCP) bioceramics and its biocompatibility test as a
tooth filler to maintain the desired alveolar ridge dimension and stimulate the
bone healing processes in the area of tooth loss. β-TCP bioceramics was resulted
directly from precipitation of calcinated chicken eggshells as calcium source and
phosphoric acid. The corresponding β-TCP was mixed with chitosan 2% to get
β-TCP pellet for biocompatibility testing. Biocompatibility testing of β-TCP on
lower incisor was performed in rabbits and sheep as animal model.
In this study, the x-ray diffraction (XRD) profiles showed that 1000 oC
calcinated chicken eggshells correspond to calcium oxide (CaO). There was also
transformation phase of CaO into Ca(OH)2 observed after more than three months
stored. The XRD profiles proved that obtained β-TCP bioceramics by
precipitation has the same pattern with that of reference data base and commercial
β-TCP. Fourier transform infrared (FTIR) spectra were completely consistent with
the result of crystallization processes observed by XRD measurements. Moreover,
the purity of the product shown by the atomic absorption spectroscopy (AAS)
resulted in Ca/P ratio of 1.48 which was closed to that of standard β-TCP
ceramics. The repeatable XRD peaks patterns and FTIR spectra obviously showed
the sample reproducibility.
Sheep’s alveolar socket that is filled with the corresponding β-TCP
demonstrated the expected results. The x-ray radiograph of sheep’s jaw indicated
no change on mesio-distal of the edentulous area. Moreover, the histological
results were completely consistent with the x-ray radiograph and macroscopic
result which is proved by the formation of new bone. In the contrary with sheep’s
result, histological results showed no tooth growth on rabbit proved by the
presence of lipid and collagen on the extraction site. This result did not
correspond to the x-ray radiograph and macroscopic results that demonstrated the
tooth growth and erupted tooth. It is clear that the corresponding β-TCP could
minimize the bone loss and maintain the dimension of alveolar ridge.
Key words: β-tricalcium phosphate, chicken eggshells, rabbit, sheep, tooth filler.
© Copyright of IPB, the year 2014
Copyright reserved by the law
Forbidden to quote part or all of these writings without including or mentioning
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Prohibited announced and reproduce part or the whole paper in any form
without permission from Bogor Agricultural University.
SYNTHESIS OF CHICKEN EGGSHELLS-BASED
β-TRICALCIUM PHOSPHATE BIOCERAMICS AND
THEIR BIOCOMPATIBILITY TEST AS A TOOTH FILLER
NUR AISYAH NUZULIA
Thesis
submitted in partial fulfillment of the requirement for
Master Degree
in
Biophysics Program
GRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2014
External examiner :
drh Deni Noviana, PhD
Thesis title : Synthesis of Chicken Eggshells-Based β-Tricalcium Phosphate
Bioceramics and Their Biocompatibility Test as A Tooth Filler
Name
: Nur Aisyah Nuzulia
ID
: G751110071
Approved by
The Commission of Supervisors
Dr Kiagus Dahlan
Supervisor
Dr drh Gunanti, MS
Co-Supervisor
drh Srihadi Agungpriyono, PhD
Co-Supervisor
Certified by:
Head of Biophysics
Graduate Program
Dean of the IPB Graduate School
Dr Agus Kartono
Dr Ir Dahrul Syah, MSc Agr
Tanggal Ujian:
30 Januari 2014
Tanggal Lulus:
PREFACE
Alhamdulillah, all praises be to Allah because by His blessing I could finish
this thesis successfully, which is one of biophysics project of Physics Department
funded by Decentralization Research Fund Project 2013. This research
synthesized β-Tricalcium Phosphate as a tooth filler from chicken eggshells as the
calcium source that is expected to be applied in dental surgery. The result of this
research has been presented in International Seminar on Sciences 2013 in Bogor
on November 15-17, 2013.
The author would like to express sincere appreciation of Dr Kiagus Dahlan,
Dr drh Gunanti, MS, and drh Srihadi Agungpriyono, PhD for the guidances and
advices in directing the research, drh Deni Noviana as external examiner for the
valuable suggestion and criticism, Dr Akhiruddin Maddu as representative of
Biophysics Program for the suggestion, Setia Utami Dewi, M.Si for the help in the
laboratory sample preparation and useful discussion. I also thank to drh. Riki
Siswandi, drh. Heryudianto Vibowo for the help in biocompatibility test,
Biomaterials team and FVM team for the cooperation of this research. The author
is also grateful to the Ministry of Education and Culture for awarding her a
scholarship under the programme Beasiswa Unggulan and Hibah Desentralisasi
for funding this research. So many thankful to Yessie Widya Sari and Husin
Alatas for the useful discussion, 2011’s Biophysics, Hafif, Buan, Wisma Intan
family, colleagues and students at Nurul Fikri for the support and kindness. To all
of these people, I owe its whole-hearted gratitude that impossible to describe.
I am also thankful to Bapak, Ibu, Mas Aix, Mbak Ria, Adek Agus, Adek Aris,
Adek Abdu, Kimia and whole families, 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. I do hope this thesis could be useful.
Bogor, April 2014
Nur Aisyah Nuzulia
TABLE OF CONTENS
TABLE LIST
vi
FIGURE LIST
vi
APPENDICES LIST
viii
1 INTRODUCTION
Background
Hypotheses
Objective
Benefit
1
1
3
3
3
2 MATERIALS AND METHODS
Place and Time Schedule
Materials and Equipments
Experimental Method
Calcination of Eggshells
β-TCP Synthesis
Biocompatibility Testing
3
3
3
4
4
4
5
3 RESULTS AND DISCUSSION
Calcination of Chicken Eggshells
Chicken Egshells-Based β-Tricalcium Phosphate
Biocompatibility Test on Rabbits
Biocompatibility Test on Sheep
6
6
7
12
19
4 CONCLUSION AND SUGGESTION
Conclusion
Suggestion
29
29
29
REFERENCES
29
APPENDICES
33
TABLE LIST
1
2
3
4
5
β-Tricalcium Phosphate Bioceramics Codes
Precipitate Mass of β-Tricalcium Phosphate Bioceramics
Lattice parameter and accuracy of β-TCP products
AAS results of β-TCP products
Mesio-distal of the edentulous area
8
8
8
11
23
FIGURE LIST
1 Calcination process of chicken eggshells
2 β-TCP synthesis through precipitation method
3 X-ray diffraction (XRD) pattern of calcinated chicken eggshells at
1000oC for 5h (a) and peak matching of calcinated chicken eggshells
with database PCPDF 37-1497 (b)
4 XRD pattern of β-TCP samples with five repetition and commercial
TCP, RTR Septodont
5 FTIR spectra of β-TCP samples with five repetition
6 XRD pattern of calcinated chicken eggshells after three months stored
7 Temperature of rabbits both control (untreated) and treated with β-TCP
product during the healing process post extraction
8 Pulse rate of rabbits both control (untreated) and treated with β-TCP
product during the healing process post extraction
9 Respiration rate of rabbits both control (untreated) and treated with βTCP product during the healing process post extraction
10 The laterolateral (LL) view radiograph of rabbit’s jaw pre-operation.
Scale bar 12mm
11 The laterolateral (LL) view radiograph of rabbit’s jaw that unfilled with
β-TCP product (control) at day+0 post operation(a). There was tooth
growth that is shown by increasing opacity of extracted area at day+7
post operation (b) and the presence of margin and higher opacity of
alveolar socket at day+30 post operation indicates the formation of
tooth germ (c). Scale bar 12mm
12 The laterolateral (LL) view radiograph of rabbit’s jaw that filled with βTCP product. There was high opacity at day+0 post operation due to the
similar inorganic of sample with bone (a) and gradually decreased
because of the resorption of sample by body fluid at day+7 post
operation (b). The formation of complete tooth was showed by clear
margin on the extracted area at day+30 post operation (c). Scale bar
10mm
13 Macroscopic results of rabbit’s mandible at Day+30 harvesting. Sketch
of rabbit’s mandible was shown to describe the anatomy of lower jaw.
There was no presence of erupted tooth on control (b) but the erupted
tooth on the treatment showed the complete tooth growth (c)
14 A lateral section photomicrograph of lower rabbit incisor (filled with βTCP product) at Day+30 post operation. The presence of fibrous
connective tissue in the ground portion of socket (a) and the presence of
5
5
7
9
10
11
12
13
13
14
14
15
15
15
16
17
18
19
20
21
22
23
24
blood clot in the middle portion of socket indicate blood vessel
rupture(b). Hematoxylin Eosin Stained, x40, Scale bar 100μm
A lateral section photomicrograph of lower rabbit incisor (control) at
Day+30 post operation. Tooth growth was indicated by the presence of
pulp chamber with odontoblastic layer (
) in the apical portion of
socket (a) and fully filled with fat cells in the middle portion (b).
Hematoxylin Eosin Stained, x40, Scale bar 100μm
Temperature of sheep both control (untreated) and treatments (filled
with β-TCP product and commercial β-TCP) during the healing
process post extraction
Pulse rate of sheep both control (untreated) and treatments (filled with
β-TCP product and commercial β-TCP) during the healing process post
extraction
Respiration rate of sheep both control (untreated) and treatments (filled
with β-TCP product and commercial β-TCP) during the healing
process post extraction
The radiograph of dorsoventral (DV) position of sheep ’s jaw preoperation. Scale bar 15mm
The radiograph of dorsoventral (DV) position of sheep’s jaw of control
(unfilled with sample). There was large mesio-distal and low opacity on
the extraction site ( ) at (a) day+0 post operation that become narrower
at day+7 post operation (b) and there was high alveolar bone resorption
shown by significant change on mesio-distal at day+30 post operation
(c). Scale bar (a) 10mm (b) 8mm (c) 11mm
The radiograph of dorsoventral (DV) position of sheep (filled with
β-TCP product). High opacity on the extracted area ( ) at day+0 post
operation showed the alveolar socket filled with sample (a). The same
width of mesio-distal indicated there was no alveolar bone resorption
day+7 post operation and there was resorption of sample showed by
decreasing opacity (b). Resubstituting process of sample and
preservation of alveolar ridge was clearly showed by the same width of
mesio-distal on the edentulous area at day+30 post operation (c). Scale
bar (a) 3mm (b) 10mm (c) 7mm
The radiograph of dorsoventral (DV) position of sheep (filled with
commercial β-TCP, RTR Septodont). High opacity on the extracted
area ( ) at day+0 post operation showed the the alveolar socket filled
with commercial product (a). Decreasing opacity on the extracted area
showed the resorption of materials at day+7 post operation (b) and there
was alveolar bone resoption on the horizontal direction at day+30 post
operation(c). Scale bar (a) 7mm (b) 7mm (c) 10mm
Macroscopic results of sheep’s mandible at day+30 harvesting.
Narrower mesio-distal occurred on control (a) but the width of mesiodistal remained same on the treatment filled with β-TCP product (b).
There was alveolar bone resorption in the horizontal direction on the
treatment filled with commercial β-TCP, RTR Septodont (c)
A lateral section photomicrograph of alveolar bone healing (control) at
Day+30 post operation. Alveolar bone resorption occurs that was
showed by connective tissue in the upper portion of extracted area (a)
17
18
19
20
20
21
21
22
22
23
and a cavity in the middle portion (b). Hematoxylin Eosin Stained, x40,
Scale bar 100μm
25 A lateral section photomicrograph of alveolar bone healing (filled with
β-TCP product) at Day+30 post operation. Biocompatibility of tooth
filler seen from infiltration of fibrous connective tissue within β-TCP
product in the middle portion of extracted area (a). Bone remodeling
occurs in the formation of woven bone (b) via intramembranous bone
development under layer of connective tissue (c) in the upper portion
near gingiva. Hematoxylin Eosin Stained x40 (a, b) x100 (c). Scale bar
100μm
26 A lateral section photomicrograph of alveolar bone healing (filled with
commercial β-TCP) at Day+30 post operation. Biocompatibility of
tooth filler seen from the presence of vessel in the upper portion of
extracted area but the presence of blood clot indicated vessel rupture(a).
Alveolar bone resorption occurs in horizontal direction shown by the
presence of collagen and fibrous connective tissue near gingiva (b).
Repairing process by connective tissue in the middle portion shown by
the presence of vessel (c). Hematoxylin Eosin Stained x40. Scale bar
100μm
24
26
28
APPENDIX LIST
1 Flow chart of the research
2 Joint committee on powder diffraction
Calcium Oxide
3 Joint committee on powder diffraction
β-Tricalcium Phosphate
4 Joint committee on powder diffraction
Calcium Phosphate (Ca2P2O7)
5 Joint committee on powder diffraction
Ca(OH)2
34
standards (JCPDS) database of
35
standards (JCPDS) database of
35
standards (JCPDS) database of
36
standards (JCPDS) database of
36
1 INTRODUCTION
Background
Oral health is essential to general health and quality of life. It means a
condition of being free from any diseases that may impact to our whole body.
Based on the report of World Health Organization (WHO) website on April 2012,
the most common oral diseases are dental cavities, periodontal (gum) disease, oral
cancer, oral infectious diseases, trauma from injuries, and hereditary lesions.
Worldwide, dental cavities happen to 60–90% of school children and nearly 100%
of adults and severe periodontal (gum) disease is found in 15–20% of middle-aged
(35-44 years) adults. Complete loss of natural teeth is widespread and particularly
affects older people (WHO 2013). In Indonesia, health survey conducted by
Ministry of Health Republic of Indonesia in 2001 showed that about 70% of
Indonesian aged over than 10 years old have tooth decay (Pratiwi et al. 2013).
Among these diseases, dental cavities and periodontal disease are the major
causes of tooth loss. Physiologically, there is the loss of alveolar bone as a result
of human tooth loss process. The rate of alveolar bone resorption is fairly high as
a reduction of about millimeters happens in the first 6 months of the healing
process and two-thirds of reduction occurs in the first 3 months (Brkovic et al.
2008, Kokovic and Todorovic 2011). It is observed that over 12 months the
reduction is about 50% in both horizontal and vertical directions (Brkovic et al.
2008). A very progressive bone resorption is able to reduce the dimensions of the
socket and lead to the bone athropy and cause difficulty on implant or prostheses
placement (Calixto et al. 2007). Therefore, preservations of bone volume in the
area of tooth loss are necessary to maintain the dimensions of the alveolar ridge.
In general, grafting procedures are also necessary for improving the bone
tissue ability to regenerate. For this purpose, several approaches have been
attempted for defect filling and subsequent regeneration, including autogeneous
and xenogeneous bone grafting and synthetic biomaterials (Coimbra et al. 2009).
Compared to autogeneous and xenogenous procedures, biomaterials eliminate
problems of donor scarcity, supply limitations, pathogen transfer, and immune
rejection. In the field of dental surgery, biomaterials have been used include
filling of pockets and maxillary ridge augmentation, deficiencies caused by loss of
dentition with advancing age or various diseases (Costa et al. 2008). The use of
filling biomaterials is one way to preserve the adequate bone volume that
commonly used by clinicians.
Nowadays, bioceramics have been widely studied for orthopedic and
dental applications due to their good biocompatible and osseoconductive
properties (Coimbra et al. 2009). Then, because the mineral phase of bones and
teeth is composed of calcium phosphate salts, the use of some Ca-P materials for
bone substitution, augmentation, and repair has gained clinical acceptance in
many aspects of orthopedics and dentistry (Dai et al. 2004). Among calcium
phosphates, hydroxyapatite (HA) and tricalcium phosphate (TCP) have been
widely used as biomaterials in dentistry and medicine (Abdurrahim and Sopyan
2008) due to their close chemical similarity to the inorganic component of bone
and tooth (Tazaki et al. 2009). β-TCP is the most frequently observed TCP
2
polymorphs in the field of bioceramics (Shi 2004) with Ca/P ratio of 1.5 that has
been shown to exhibit good biocompatibility and osteoconductivity (Ain et al.
2008 & Sader et al. 2009) in both animal and clinical studies (Oi et al. 2009)
while HA is considered as bioactive and nonbiodegradable bone replacement
materials. Moreover, β-TCP appears to be advantageous in comparison to HA for
the property of absorption (Tazaki et al. 2009). The faster dissolution/resorption
of β-TCP may allow a gradual biological degradation over a period time and a
progressive replacement by the natural host tissue (Coimbra et al. 2009).
β-TCP itself can be obtained from several methods in which the method of
wet precipitation is one of the widely used methods (Boilet et al. 2013, NasiriTabrizi and Fahami 2013, Farzadi et al. 2011, Koç et al. 2004). In this method,
synthetic materials such as CaCO3 and Ca(NO3)2 are commonly used as calcium
source for obtaining β-TCP ceramics (Park and Bronzino 2000, Wei and Akinc
2007). Previous study reported that β-TCP can not be synthesized directly in
aqueous solution (Belouafa et al. 2006). Wet precipitation in aqueous solution
would synthesize apatitic TCP, Ca9(HPO4)(PO4)5(OH). Following precipitation
from aqueous solution, sintering at high temperatures above 750 C was required
to transform apatitic TCP into β-TCP (Kannan et al. 2006, 2009). Different
synthesis approach applying natural resources material could obtain bioactive
β-TCP that might elicit a direct bond between the bioceramics and living host
tissue. The chicken eggshells are one of natural resources that commonly
considered as waste, contain much of calcium in the form of CaCO3 that
potentially could be used as calcium source in synthesizing β-TCP bioceramics.
The previous study showed that chicken eggshells as the corresponding calcium
source could obtain the other kind of calcium phosphate (hydroxyapatite,
hydroxyapatite/carbonate apatite) by precipitation method (Dahlan et al. 2012).
Therefore, it is clear that β-TCP might be produced from natural resources and
synthesized directly by precipitation method.
Prior to the clinical use, biocompatibility and mechanical stability of new
materials should be test under both initial in vitro and in vivo conditions (Pearce
2007). In vitro testing is used primarily as a first stage test for acute toxicity and
cytocompatibility while in vivo testing is used to demonstrate the tissue response
to materials (Arora et al. 2011). Previous study reported that calcium phosphates
derived from eggshells were nontoxic and performed good adhesion interaction to
the host cells in vitro (Solihat 2011). Therefore, advanced research under in vivo
condition is required to observe the living host tissue response to new materials.
Rabbits and sheep are animal models that could be applied to observe the
biocompatibility and mechanical stability of β-TCP bioceramics. Rabbits are one
of the most commonly used animals for medical research despite of easy handling
and sheep represent human bone relatively closely (Pearce 2007). Rabbits are
lagomorph with hypselodont teeth that could be used as animal model for cases of
premature tooth loss while sheep are for observing cases of permanent tooth loss
posextraction due to the similarity on alveolar ridge with human.
3
Objectives
1. Synthesizing β-TCP bioceramics obtained from chicken eggshells by
precipitation method
2. Observing the tooth growth and the bone healing using β-TCP bioceramics
and tissue response to materials by in vivo testing in rabbits and sheep
Hypotheses
1. Chicken eggshells can be used as calcium donor in synthesizing β-TCP
bioceramics that biocompatible for dentistry
2. Chicken eggshells-based β-TCP bioceramics as tooth filler can accelerate
the tooth growth on rabbits and stimulate the bone healing on sheep after
tooth extraction
Benefits
This research is expected to obtain natural resource-based β-TCP
bioceramics such chicken eggshells that generally known as waste in use as
calcium donor in synthesizing of β-TCP bioceramics. It may produce
biocompatible biomaterials with economical price for dentistry application in
Indonesia. This will subsequently assume to decrease the dependence on
importing biomaterial for bone and tooth substitution.
2 MATERIALS AND METHOD
Place and Time Schedule
This research was conducted from January 2013 through September 2013
which took place in Biophysics Laboratory-IPB in sample preparation, while
biocompatibility testing was done in Faculty of Veterinary Medicine-IPB. Sample
characterization was done in Forestry Development and Research Center-Bogor
for x-ray diffraction (XRD) characterization, in Laboratory of Material AnalysisIPB for fourier transform infrared (FTIR) analysis, and in Indonesian Soil
Research Institute-Bogor for atomic absorption spectroscopy (AAS). Histological
test and x-ray radiography analysis was done in Faculty of Veterinary MedicineIPB.
Materials and Equipments
The materials used in this research were calcium oxide obtained from
chicken eggshells, pro-analyze phosphoric acid (H3PO4), chitosan, aquabidest, and
aquadest. The equipments used were analytical balance, beaker glass, burette,
4
aluminium foil, pipette, magnetic stirrer, hotplate, furnace, digital thermometer,
filter paper, mortar, crucible, and vacuum.
Materials and equipments used for biocompatibility testing were rabbits
and sheep as animal model, minor surgery set, dental surgery tools, anesthetic
material, and surgery room for aseptical insertion. Then, materials used for
histopathological examination were formalin 10%, nitric acid 5%, aquadest,
aquabidest, alcohol, xylol, Hematoxilin-Eosin (HE), and Permount ®.
Experimental Methods
Calcination of chicken eggshells
The raw eggshells where their surface was initially cleaned were
calcinated in an air atmosphere at 1000oC for 5 hours as seen in Figure 1. This
calcination aimed to convert calcium carbonate (CaCO3) into calcium oxide
(CaO). To synthesize calcium phosphate powder, the corresponding eggshells
were crushed and milled in crucible then characterized by using x-ray diffraction
(XRD) Shimadzu diffractometer, Cu-Kα radiation, 40 kV, 30 mA and 0,02o s-1
scanning step. The chemical reaction of eggshell calcination is given below:
β-TCP synthesis
β-TCP bioceramics was prepared by precipitation of H3PO4 as the
phosphor (P) source. Then it was dropped into CaO as the calcium (Ca) source
which was synthesized further at 50oC with molar ratio of Ca:P as 1.2M : 0.8M
and the chemical reaction is given below:
The results were precipitated, filtered and heated at sintering temperature
1000 C for 7 hours (Figure 2). The final results were obtained in the form of white
powder and analyzed by using XRD and FTIR spectroscopy (ABB MB 3000)
characterization to identify the formed phase and the functional group of the
sample, respectively, as well as AAS spectroscopy to verify the purity of the
products. To assure the reproducibility of the sample the process was performed
five times. Samples used for biocompatibility testing were β-TCP pellet. To get
the pellet form, samples were mixed with chitosan 2%, molded in form of pellet
with 2 mm and 6 mm in diameter respectively for rabbits and sheep then heated in
incubator at 50oC for 6 hours.
o
5
Calcination at 1000 oC
The cleaned raw eggshells
Powder of CaO
Figure 1 Calcination process of chicken’s eggshells
Ca and P solution
Precipitation
Filtering
Sintering
Figure 2 β-TCP synthesis through precipitation method
Biocompatibility testing
Biocompatibility test of β-TCP bioceramics as a tooth filler was performed
by using rabbits and sheep as animal models. There are two 1 year old rabbits
used with 2.5 kg body weight and three 1 year old sheep. All experiments were
conducted along the institutional guidelines for the care and use of laboratory
animals. Prior to surgery, the animal models were observed closely for a week in
order to check their health status. They were maintained under identical
environment, management and standard diet with ad libitum supply of drinking
water. Then, surgery was done on the lower incisor. Before surgery, rabbits and
sheep were anaesthetized by injection of ketamine 10% and xylazin 10%. Under
aseptical conditions, the lower incisor was extracted and immediately after tooth
extraction, the alveolar socket was filled with synthesized β-TCP bioceramics that
were sterilized initially by exposure to ultraviolet light. As control requirement,
other rabbit’s and sheep’s lower incisor were extracted in the same manner and
unfilled with any tooth filler. Each surgery was performed under same veterinary
surgeon. Then, the rabbits and sheep were housed under a climate-controlled
environment in stall of animal used of FVM Bogor Agricultural University.
The alveolar sockets that filled with β-TCP tooth filler and unfilled with
any tooth filler were monitored using a set of x-ray radiographic apparatus in
day+0 pre operation, D+0 post operation (PO), D+7 PO and D+30 PO for
observing the tooth growth on rabbits and alveolar bone healing on sheep. Then,
the rabbits and sheep were euthanized on D+30 PO for macroscopic evaluation.
For histological evaluation, the mandibular was thoroughly washed with water
and fixed in 10% neutral formalin. After fixation, the mandibular was dissected
6
out, decalcified, and processed for paraffin embedding. The decalcification
process was carried out using 10% EDTA solution and the specimens was
dehydrated in ascending grades of ethyl alcohol, cleared in xylene and embedded
in paraffin wax. The histopathological examination was done for observing the
degree of tooth growth on rabbits and new bone formation on sheep. The
histological analysis was carried out with some parameters comprised the
presence and type of inflammatory infiltrate, reaction to foreign body, fibrous
connective tissue, osteoblastic activity, formation of osteoid and new bone.
Detailed flow chart of the research was shown in Appendix 1.
3 RESULTS AND DISCUSSION
Calcination of Chicken Eggshells
It was found that the calcinated chicken eggshells contain pure calcium
oxide with mass decomposition of 48.40%. The characteristics of the
corresponding calcium oxide atomic structure were examined by using XRD
where its pattern is shown in Figure 3a. It was observed that the highest peaks
were on 2θ = 32.31, 37.47, 53.98, 64.27, 67.51, and 79.76. Based on data from
joint committee on powder diffraction standards (JCPDS) database number
37-1497 (Appendix 2), these results clearly prove the presence of calcium oxide.
Figure 3b shows that the sample pattern was match to the database, since they
share similar peaks pattern. The absence of other phases in the sample pattern
indicates the high purity of CaO powder. The result in this study had higher
accuracy of CaO pattern compared to that of other studies (Saeed et al. 2011,
Balazsi et al. 2007). The XRD pattern of 950 oC calcined chicken eggshell
corresponds to CaO with Ca(OH)2 as the majority phase according to Miller
indices of (1 0 0), (1 0 1), and (1 0 2) (Saeed et al. 2011). In addition to Ca(OH)2,
MgO, which may influence the purity of β-TCP products, was also observed
which calcination was performed at 900 oC (Balazsi et al. 2007). It seems that the
calcinating temperatures of 900 oC and 950 oC are not enough to get pure CaO due
to the strong hydroxyl binding at eggshells.
7
intensity (counts)
1000
800
600
400
200
0
10
20
30
40
50
2θ (degree)
60
70
80
Intensity (counts)
(a)
2θ (degree)
(b)
Figure 3 X-ray diffraction (XRD) pattern of calcinated chicken eggshells at
1000oC for 5h (a) and peak matching of calcinated chicken eggshells
with database PCPDF 37-1497 (b)
Chicken Eggshells-Based β-Tricalcium Phosphate
Synthesis of chicken eggshells-based β-Tricalcium phosphate (β-TCP) by
precipitation method was performed five times with the sample codes are shown
in Table 1. The sintering temperature used to obtain β-TCP bioceramics was
1000oC. Referring to the previous study it was reported that the required
temperature to start phase transformation of calcium phosphate into β-TCP is
760oC and get stable at 1125oC (Salahi and Heinrich 2003, Shi 2004). The result
of this process was a white powder with mass precipitates are shown in Table 2.
8
In contrary with Belouafa et al. (2006), β-TCP could be synthesized
directly in aqueous solution as shown from its XRD profile (Figure 4). The three
important highest peaks pattern of the first three samples (TCP1, TCP2 and
TCP3) were completely similar to the peaks of β-TCP database number 09-0169
(Appendix 3). It was proved by high intensity on 2θ around 27.84, 31.06, and
34.41. The XRD peaks pattern of the corresponding β-TCP samples was the same
with that found by heating the apatitic TCP at temperature above 750oC (Kannan
et al. 2006, 2009). Moreover, it was similar with that one of commercial TCP
(RTR Septodont France) and sintered commercial TCP powder (Sader et al. 2009).
The determined lattice parameters of the corresponding β-TCP samples are shown
in Table 3 by fitting the peaks of identified reflections from the XRD pattern
using Cohen Method. It is clearly showed the formation of β-TCP bioceramics
proved by rhombohedral structure with lattice parameters correspond to the
reference a=b=10.42Å and c=37.38Å (Shi 2004).
Table 1 β-Tricalcium Phosphate Bioceramics Codes
Sample
Code
TCP (first repetition)
TCP1
TCP (second repetition)
TCP2
TCP (third repetition)
TCP3
TCP (fourth repetition)
TCP4
TCP (fifth repetition)
TCP5
Table 2 Precipitate Mass of β-Tricalcium Phosphate Bioceramics
Sample
Precipitate Mass (g)
TCP1
7.75
TCP2
7.62
TCP3
7.34
TCP4
6.94
TCP5
6.77
Table 3 Lattice parameter and accuracy of β-TCP samples
Sample
TCP1
TCP2
TCP3
Lattice parameter (Å)
a
c
10.44
37.44
10.39
37.22
10.42
37.37
Accuracy (%)
a
c
99.78
99.84
99.71
99.57
99.99
99.97
Intensity (counts)
9
Figure 4 XRD pattern of β-TCP samples with five repetition and commercial
TCP, RTR Septodont
In the meantime, the FTIR results as shown in Figure 5 were completely
consistent with the result of crystallization processes observed by XRD
measurements given by Figure 4. The broad IR band of FTIR spectra around 540580 cm-1, 690 cm-1 and 1010 cm-1 indicates the formation of a typical TCP
structure containing PO43- band. The absence of hydroxyl band in corresponding
samples around 3500 cm-1 shows better characteristics compared to Salahi and
Heinrich (2003) that used synthetic materials. The FTIR result of β-TCP obtained
10
% Transmittance
from synthetic materials was not pure that clearly showed by the existence of
hydroxyl band in sample sintered in 1000 oC (Salahi and Heinrich 2003). Other
study done by Ain et al. (2008) showed the presence of another phase
(hydroxyapatite) on the XRD results. It means that β-TCP bioceramics has been
formed directly in aqueous solution by precipitation method which also been
proved with a better characteristics compared to Salahi and Heinrich (2003) and
Ain et al. (2008) that used synthetic materials. Since the chicken eggshells are
considered as a raw material, the corresponding β-TCP samples should be more
economical. It is obvious that the corresponding β-TCP samples is reproducible
due to the repeatable XRD peaks patterns and FTIR spectra.
Wavenumber (cm-1)
Figure 5 FTIR spectra of β-TCP samples with five repetition
11
On the other hand, it should be noted that the last two samples (TCP4 and
TCP5) correspond to the other phase of calcium phosphate (Ca2P2O7) with
database number 09-0346 as seen in Appendix 4 which is occured probably due to
the different calcium source used in synthesizing β-TCP. The corresponding
calcium source was calcinated chicken eggshells powder that had been stored for
more than three months. This result is obviously depicted by its XRD profile as
shown in Figure 6 that refers to the phase of Ca(OH)2 with database number
44-1481 (Appendix 5). It is exhibited that there is phase transformation caused by
interaction of CaO with the environment during the storage at non-vacuum
condition. The existence of OH- functional group on Ca(OH)2 tends to release and
to form another phase of calcium phosphate as also been observed in the previous
study that synthesized biphasic calcium phosphate (BCP) with CaO and Ca(OH)2
as raw materials (Dahlan et al. 2011). Their research also showed that TCP only
appeared on samples synthesized with CaO that probably caused by the difference
binding type of each precursor.
Furthermore, to verify the purity of the products, the β-TCP samples were
characterized by using atomic absorption spectroscopy (AAS). Based on the AAS
results, it is shown that Ca/P ratio of TCP3 corresponded to β-TCP bioceramics as
shown in Table 4. This result confirmed the XRD results and in accordance with
the theory of Ca/P ratio of β-TCP bioceramics (Shi 2004). Nevertheless, the AAS
measurements of TCP1 and TCP2 are not consistent with the XRD results. This
inconsistency probably occured due to the limited number of samples powder that
used for the AAS analysis.
Intensity (counts)
300
250
200
150
100
50
0
10
20
30
40
50
2θ (degree)
60
70
Figure 6 XRD pattern of calcinated chicken eggshells after three months stored
Table 4 AAS results of β-TCP products
Sample
TCP 1
TCP 2
TCP 3
TCP 4
TCP 5
Percentage
Ca
P
51.9
38.54
59.7
35.22
53.06
35.8
46.31
42.98
47.23
44.3
Ca/P ratio
1.34
1.69
1.48
1.08
1.07
12
Biocompatibility Test on Rabbits
Biocompatibility is the ability of a material to perform with an appropriate
host response in a specific application (Shi 2004). Biocompatibility test of β-TCP
as tooth filler on rabbits was performed to observe the tooth growth post
extraction. Biocompatible properties of new materials could be analyzed from
several examinations such as physical examination, x-ray radiographic analysis,
and histological evaluation.
The physical examination on rabbits that is observed for 30 days showed
that β-TCP product was biocompatible and non-toxic. There were no significant
influence on rabbits filled with β-TCP product and unfilled (control) in
temperature and pulse rate post operation. The results of physical examination on
rabbits were in normal range of each parameter (Figure 7 and Figure 8). The
normal temperature of rabbit is 38.9-40.5oC and its normal pulse rate is 120-250
waves/minute (Kelly 1984). There was temperature decreasing on rabbit filled
with β-TCP product post operation that might be influenced by the injection of
anaesthetic material. Thus, the temperature was in normal range on D+1 PO and
days after. It is worth to note that filling β-TCP product do not interfere the
physiological body temperature of rabbit. Nevertheless, the respiration of rabbits
as seen in Figure 9 both control and treatment were not in normal range that is
30-45 (Kelly 1984). It might be caused by condition of rabbits that are stress
during data collection process and made them excited. An increased demand for
oxygen after exercised might increase respiratory frequency (Kelly 1984).
Moreover, the longer period of expiration in healthy animals depends upon
whether the animal is relaxed and resting, or has recently been excited or
excercised. In short, postextraction preservation by β-TCP as a tooth filler do not
interfere the physiological condition of rabbit.
Temperature (oC)
42
41
40
39
Normal
38
Control (untreated)
37
Treated with B-TCP
Product
36
Observing time
Figure 7 Temperature of rabbits both control (untreated) and treated with β-TCP
product during the healing process post extraction
13
Pulse rate (waves/minute)
300
250
200
Control (untreated)
150
Treated with B-TCP
product
100
Observing time
Figure 8 Pulse rate of of rabbits both control (untreated) and treated with β-TCP
product during the healing process post extraction
250
Respiration
200
150
100
Control (untreated)
50
Treated with B-TCP
product
0
Observing time
Figure 9 Respiration rate of of rabbits both control (untreated) and treated with
β-TCP product during the healing process post extraction
The rabbits tooth growth and status of β-TCP product was monitored using
a set of x-ray radiographic apparatus. The radiograph of rabbit’s jaw pre operation
is shown in Figure 10 and the radiograph of rabbit’s jaw as control and treatment
is shown in Figure 11 and Figure 12. Based on the radiograph in Figure 11 and
Figure 12, these results show a tooth growth on rabbits at D+30 PO that was
demonstrated by the presence of margin and high opacity on an extracted tooth
area. It was clear that alveolar socket of lower incisors rabbit on D+0 PO (Figure
11a) had low opacity and then increase on D+7 PO as shown in Figure 11b.
Increasing of alveolar socket opacity indicates the formation of tooth germ
precursor tissue. It was also observed that in Figure 11b there was a tooth growth
14
on upper incisors rabbit. It might because rabbits are a lagomorpha with all teeth
continually growing. Rabbit is mammalian group with hypselodont teeth, evergrowing or open-rooted teeth that grow continuously during the lifetime of the
animal. They also called open rooted teeth that refers solely to the large apical
opening present in all continuously growing teeth and does not imply that the
tooth actually needs to have a root in a classical form (Ali and Mubarak 2012).
Moreover, the radiograph of alveolar socket’s rabbit on D+30 PO (Figure 11c)
demonstrated the formation of new teeth which was shown by increasing opacity
with apparent margin. Nevertheless, the empty space around alveolar socket of
lower incisors obviously indicated the incomplete tooth formation. In the
meantime, the macroscopic result of rabbit’s mandibular at D+30 harvesting as
shown in Figure 13b was in accordance with the radiograph result of alveolar
socket at D+30 PO. It is clear that there was unerupted tooth on D+30 harvesting
due to no appearance of tooth on gums surface.
Figure 10 The laterolateral (LL) view radiograph of rabbit’s jaw pre-operation.
Scale bar 12mm
(a)
(b)
(c)
Figure 11 The laterolateral (LL) view radiograph of rabbit’s jaw that unfilled with
β-TCP product (control) at day+0 post operation (a). There was tooth
growth that is shown by increasing opacity of extracted area at day+7
post operation (b) and the presence of margin and higher opacity of
alveolar socket at day+30 post operation indicates the formation of
tooth germ (c). Scale bar 12mm.
15
(a)
(b)
(c)
Figure 12 The laterolateral (LL) view radiograph of rabbit’s jaw that filled with
β-TCP product. There was high opacity at day+0 post operation due
to the similar inorganic of sample with bone (a) and gradually
decreased because of the resorption of sample by body fluid at day+7
post operation (b). The formation of complete tooth was showed by
clear margin on the extracted area at day+30 post operation (c). Scale
bar 10mm.
(a)
(b)
(c)
Figure 13 Macroscopic results of rabbit’s mandible at Day+30 harvesting. Sketch
of rabbit’s mandible was shown to describe the anatomy of lower jaw.
There was no presence of erupted tooth on control (b) but the erupted
tooth on the treatment showed the complete tooth growth (c).
On the contrary, the radiograph of alveolar socket filled with β-TCP as
shown in Figure 12 demonstrated the tooth growth on rabbit. As seen in Figure
12a, the alveolar socket was filled with β-TCP and there was higher opacity than
one in control at D+0 PO, which might be caused by the similar inorganic
component of β-TCP with bone. At the same time, there was jaw bone fracture
during tooth extraction shown by unclear margin that is different with the origin.
Figure 12b clearly shows resorption of β-TCP by the body fluids that is indicated
by volume decreasing of β-TCP pellet in alveolar socket. In addition, there was a
tooth growth of upper incisors rabbit at D+7 as well as at the control. However,
there were differences on rabbits alveolar socket filled with β-TCP at D+30 PO. It
was clear that β-TCP had been resorbed and could accelerate the formation of
complete tooth shown from its radiograph with clear margin at Figure 12c. In the
meantime, jaw bone healing process was observed indicated by bone margin
which was similar with that of pre-operation bone margin. Furthermore, this result
was supported by macroscopic result at D+30 PO as seen in Figure 13c that
16
indicates the complete tooth formation of rabbits lower incisors due to the erupted
tooth of 15 mm. it demonstrated a rapid growth of rabbits lower incisors and
relevant to the other studies that reported lower incisors growth is approximately
2-3 mm per week (Capello 2005, Meredith 2007). Furthermore, the upper incisors
growth of rabbit was shorter than that of control. It might be caused by the
presence of erupted tooth and makes slower growth on the rabbit upper incisors. It
is obvious that β-TCP could accelerate the formation of complete tooth and
stimulate the tooth growth. In short, it could be applied clinically in case of
premature tooth loss that commonly happen in children.
On the other hand, the histological results of treated extraction site that
filled with β-TCP product inconsistent with the x-ray radiograph and macroscopic
result. It is clearly proved by Figure 14 that showed the presence of fibrous
connective tissue at the ground portion lining the sample as seen in Figure 14a. As
seen in Figure 14a, blood clot filled the socket post extraction and was replaced
with fibrous connective tissue. Then, in the middle portion of the socket was fully
filled with mass of blood clod. Figure 14b showed that β-TCP product was partly
resorbed and degraded. It was clearly showed by distributed β-TCP product that
interacted with blood then will be replaced with fibrous connective tissue. The
formation of fibrous connective tissue is a reaction to chronic insult and result
from the activity of fibroblast cells. As explained by Banks (1986) connective
tissues serve numerous functions and one of them is insulate organs from
mechanical damage. It might be called as important line of defense. Moreover,
some connective tissues also play an important role in repair and regeneration.
The existence of blood clot might indicate the incomplete healing processes and
means no tooth growth on rabbit mandible. The erupted tooth in the macroscopic
result might be a remnant of unextracted tooth that forced by the blood clot and βTCP product.
The opposite microscopic result was showed by control (unfilled) rabbit in
Figure 15 that demonstrate the formation of tooth germ. It was showed by the
presence of pulp chamber as shown in Figure 15a. It was obvious from wide pulp
chamber that lined by odontoblastic layer in the apical region of rabbit incisor.
Odontoblastic layer was not covered by dentine and enamel on its external side.
These results showed the incomplete tooth formed proved by no cervical loop at
the labial side in rabbit incisor that has function to generate ameloblasts.
Ameloblast is cells that produce enamel on the labial tooth surface (Ali and
Mubarak 2012). Moreover, the extracted socket was also filled with fat cells as
seen in Figure 15b. These cells occur as single cells, clusters of cells, or as
massive sheets of cells associated with connective tissues, especially loose
connective tissue (Banks 1986). In addition, these cells appear as large empty
spaces surrounded by the peripheral portion of the cell and a flattened nucleus
(Copenhaver et al. 1978). These results support the radiograph result of untreated
rabbit (control) at day+30 post operation that indicated the formation of
incomplete tooth. In short, filling socket by β-TCP product after tooth extraction
does not stimulate the regrowth of rabbit incisor. It might be caused by rabbits as
animal used in this research have the same weight but not with the age so it might
cause different tooth growth process.
17
a
fibrous
bone
Β-TCP product
fibrous
b
Blood clot
β-TCP product
Figure 14 A lateral section photomicrograph of lower rabbit incisor (filled with
β-TCP product) at Day+30 post operation. The presence of fibrous
connective tissue in the ground portion of socket (a) and the presence
of blood clot in the middle portion of socket indicate blood vessel
rupture(b). Hematoxylin Eosin Stained, x40, Scale bar 100μm.
18
a
Odontoblastic
layer
Fat
cells
Pulp
chamber
b
Fat cells
Figure 15 A lateral section photomicrograph of lower rabbit incisor (control) at
Day+30 post operation. Tooth growth was indicated by the presence of
pulp chamber with odontoblastic layer (
) in the apical portion of
socket (a) and fully filled with fat cells in the middle portion (b).
Hematoxylin Eosin Stained, x40, Scale bar 100μm.
19
Biocompatibility test on sheep
Temperature (oC)
Biocompatibility test was also done in sheep to observe the alveolar bone
healing process by resubstituting process of the corresponding β-TCP. It is clear
that the corresponding β-TCP was biocompatible and non-toxic. Based on the
clinical examination, there was no inflammation on the extracted area of each
sheep. The result of physiological examination in sheep was the same wi
β-TRICALCIUM PHOSPHATE BIOCERAMICS AND
THEIR BIOCOMPATIBILITY TEST AS A TOOTH FILLER
NUR AISYAH NUZULIA
GRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2014
STATEMENT ON THESIS
I hereby declare that the thesis of “Synthesis of Chicken Eggshells-Based
β-Tricalcium Phosphate Bioceramics and Their Biocompatibility Test as A Tooth
Filler” is my work with the direction of the supervising committee and has not
been submitted in any form to any college. Source of information derived or
quoted from the work published or not published 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 thesis to Bogor Agricultural University.
Bogor, April 2014
Nur Aisyah Nuzulia
ID G751110071
RINGKASAN
NUR AISYAH NUZULIA. Sintesis Biokeramik β-Tricalcium Phosphate Berbasis
Cangkang Telur Ayam dan Uji Biokompatibilitasnya sebagai Tooth Filler.
Dibimbing oleh KIAGUS DAHLAN, GUNANTI, dan SRIHADI
AGUNGPRIYONO.
Preservasi tulang alveolar setelah ekstraksi gigi merupakan langkah penting
dalam praktek gigi sebelum pemasangan implan gigi. Penelitian ini melaporkan
tentang sintesis biokeramik β-Tricalcium Phosphate (β-TCP) dan uji
biokompatibilitasnya sebagai tooth filler untuk mempertahankan dimensi alveolar
ridge dan merangsang proses penyembuhan tulang di daerah hilangnya gigi.
Biokeramik β-TCP dihasilkan secara langsung melalui metode presipitasi
cangkang telur ayam yang telah dikalsinasi sebagai sumber kalsium dan asam
fosfat. β-TCP tersebut dicampur dengan kitosan 2 % untuk mendapatkan β-TCP
dalam bentuk pelet yang digunakan dalam pengujian biokompatibilitas. Pengujian
biokompatibilitas β-TCP dilakukan pada gigi seri bawah kelinci dan domba
sebagai hewan uji.
Dalam studi ini, hasil x-ray diffraction (XRD) menunjukkan bahwa pola
puncak XRD cangkang telur ayam yang dikalsinasi pada suhu 1000oC bersesuaian
dengan fase kalsium oksida (CaO). Adapun fase CaO bertransformasi menjadi
fase Ca(OH)2 selama masa penyimpanan lebih dari tiga bulan. Profil XRD juga
membuktikan bahwa biokeramik β-TCP yang dihasilkan memiliki pola yang sama
dengan referensi data base dan β-TCP komersil yang tersedia. Spektra fourier
transform infrared (FTIR) sampel konsisten dan memperkuat hasil karakterisasi
XRD. Selain itu, rasio Ca/P sebesar 1.48 yang diperoleh dari hasil atomic
absorption spectroscopy (AAS) menunjukkan kemurnian sampel yang sesuai
dengan standar biokeramik β-TCP. Adapun pola puncak XRD dan spektra FTIR
yang berulang jelas menunjukkan sifat reproducibility sampel.
Alveolar socket domba yang diisi dengan produk β-TCP menunjukkan hasil
karakteristik yang diharapkan. Hasil radiografi sinar-x pada rahang domba
menunjukkan tidak adanya perubahan mesio-distal di edentulous area. Selain itu,
hasil histologi juga konsisten terhadap hasil radiografi dan gambaran makroskopik
yang dibuktikan dengan adanya pembentukan tulang baru. Namun, hasil histologi
dari rahang kelinci menunjukkan hal yang berlawanan dengan hasil radiografi dan
gambaran makroskopik. Hasil histologi pada rahang kelinci menunjukkan tidak
adanya pertumbuhan gigi pada kelinci yang dibuktikan dengan daerah ekstraksi
yang terisi penuh oleh lipid dan kolagen. Pengujian biokompatibilitas biokeramik
β-TCP menunjukkan bahwa produk β-TCP mampu meminimalis hilangnya tulang
serta mampu mempertahankan dimensi alveolar ridge.
Kata kunci: β-tricalcium phosphate, cangkang telur ayam, domba, kelinci, tooth
filler.
SUMMARY
NUR AISYAH NUZULIA. Synthesis of Chicken Eggshells-Based β-Tricalcium
Phosphate Bioceramics and Their Biocompatibility Test as A Tooth Filler.
Supervised
by KIAGUS
DAHLAN,
GUNANTI,
and
SRIHADI
AGUNGPRIYONO.
Preservation of alveolar bone following tooth extraction is an important step
in dental practices before dental implant placement. This study reported synthesis
of β-Tricalcium Phosphate (β-TCP) bioceramics and its biocompatibility test as a
tooth filler to maintain the desired alveolar ridge dimension and stimulate the
bone healing processes in the area of tooth loss. β-TCP bioceramics was resulted
directly from precipitation of calcinated chicken eggshells as calcium source and
phosphoric acid. The corresponding β-TCP was mixed with chitosan 2% to get
β-TCP pellet for biocompatibility testing. Biocompatibility testing of β-TCP on
lower incisor was performed in rabbits and sheep as animal model.
In this study, the x-ray diffraction (XRD) profiles showed that 1000 oC
calcinated chicken eggshells correspond to calcium oxide (CaO). There was also
transformation phase of CaO into Ca(OH)2 observed after more than three months
stored. The XRD profiles proved that obtained β-TCP bioceramics by
precipitation has the same pattern with that of reference data base and commercial
β-TCP. Fourier transform infrared (FTIR) spectra were completely consistent with
the result of crystallization processes observed by XRD measurements. Moreover,
the purity of the product shown by the atomic absorption spectroscopy (AAS)
resulted in Ca/P ratio of 1.48 which was closed to that of standard β-TCP
ceramics. The repeatable XRD peaks patterns and FTIR spectra obviously showed
the sample reproducibility.
Sheep’s alveolar socket that is filled with the corresponding β-TCP
demonstrated the expected results. The x-ray radiograph of sheep’s jaw indicated
no change on mesio-distal of the edentulous area. Moreover, the histological
results were completely consistent with the x-ray radiograph and macroscopic
result which is proved by the formation of new bone. In the contrary with sheep’s
result, histological results showed no tooth growth on rabbit proved by the
presence of lipid and collagen on the extraction site. This result did not
correspond to the x-ray radiograph and macroscopic results that demonstrated the
tooth growth and erupted tooth. It is clear that the corresponding β-TCP could
minimize the bone loss and maintain the dimension of alveolar ridge.
Key words: β-tricalcium phosphate, chicken eggshells, rabbit, sheep, tooth filler.
© Copyright of IPB, the year 2014
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, writing papers,
drafting reports, writing criticism, or review an issue, and citing it does not harm
the interests of fair Bogor Agricultural University.
Prohibited announced and reproduce part or the whole paper in any form
without permission from Bogor Agricultural University.
SYNTHESIS OF CHICKEN EGGSHELLS-BASED
β-TRICALCIUM PHOSPHATE BIOCERAMICS AND
THEIR BIOCOMPATIBILITY TEST AS A TOOTH FILLER
NUR AISYAH NUZULIA
Thesis
submitted in partial fulfillment of the requirement for
Master Degree
in
Biophysics Program
GRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2014
External examiner :
drh Deni Noviana, PhD
Thesis title : Synthesis of Chicken Eggshells-Based β-Tricalcium Phosphate
Bioceramics and Their Biocompatibility Test as A Tooth Filler
Name
: Nur Aisyah Nuzulia
ID
: G751110071
Approved by
The Commission of Supervisors
Dr Kiagus Dahlan
Supervisor
Dr drh Gunanti, MS
Co-Supervisor
drh Srihadi Agungpriyono, PhD
Co-Supervisor
Certified by:
Head of Biophysics
Graduate Program
Dean of the IPB Graduate School
Dr Agus Kartono
Dr Ir Dahrul Syah, MSc Agr
Tanggal Ujian:
30 Januari 2014
Tanggal Lulus:
PREFACE
Alhamdulillah, all praises be to Allah because by His blessing I could finish
this thesis successfully, which is one of biophysics project of Physics Department
funded by Decentralization Research Fund Project 2013. This research
synthesized β-Tricalcium Phosphate as a tooth filler from chicken eggshells as the
calcium source that is expected to be applied in dental surgery. The result of this
research has been presented in International Seminar on Sciences 2013 in Bogor
on November 15-17, 2013.
The author would like to express sincere appreciation of Dr Kiagus Dahlan,
Dr drh Gunanti, MS, and drh Srihadi Agungpriyono, PhD for the guidances and
advices in directing the research, drh Deni Noviana as external examiner for the
valuable suggestion and criticism, Dr Akhiruddin Maddu as representative of
Biophysics Program for the suggestion, Setia Utami Dewi, M.Si for the help in the
laboratory sample preparation and useful discussion. I also thank to drh. Riki
Siswandi, drh. Heryudianto Vibowo for the help in biocompatibility test,
Biomaterials team and FVM team for the cooperation of this research. The author
is also grateful to the Ministry of Education and Culture for awarding her a
scholarship under the programme Beasiswa Unggulan and Hibah Desentralisasi
for funding this research. So many thankful to Yessie Widya Sari and Husin
Alatas for the useful discussion, 2011’s Biophysics, Hafif, Buan, Wisma Intan
family, colleagues and students at Nurul Fikri for the support and kindness. To all
of these people, I owe its whole-hearted gratitude that impossible to describe.
I am also thankful to Bapak, Ibu, Mas Aix, Mbak Ria, Adek Agus, Adek Aris,
Adek Abdu, Kimia and whole families, 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. I do hope this thesis could be useful.
Bogor, April 2014
Nur Aisyah Nuzulia
TABLE OF CONTENS
TABLE LIST
vi
FIGURE LIST
vi
APPENDICES LIST
viii
1 INTRODUCTION
Background
Hypotheses
Objective
Benefit
1
1
3
3
3
2 MATERIALS AND METHODS
Place and Time Schedule
Materials and Equipments
Experimental Method
Calcination of Eggshells
β-TCP Synthesis
Biocompatibility Testing
3
3
3
4
4
4
5
3 RESULTS AND DISCUSSION
Calcination of Chicken Eggshells
Chicken Egshells-Based β-Tricalcium Phosphate
Biocompatibility Test on Rabbits
Biocompatibility Test on Sheep
6
6
7
12
19
4 CONCLUSION AND SUGGESTION
Conclusion
Suggestion
29
29
29
REFERENCES
29
APPENDICES
33
TABLE LIST
1
2
3
4
5
β-Tricalcium Phosphate Bioceramics Codes
Precipitate Mass of β-Tricalcium Phosphate Bioceramics
Lattice parameter and accuracy of β-TCP products
AAS results of β-TCP products
Mesio-distal of the edentulous area
8
8
8
11
23
FIGURE LIST
1 Calcination process of chicken eggshells
2 β-TCP synthesis through precipitation method
3 X-ray diffraction (XRD) pattern of calcinated chicken eggshells at
1000oC for 5h (a) and peak matching of calcinated chicken eggshells
with database PCPDF 37-1497 (b)
4 XRD pattern of β-TCP samples with five repetition and commercial
TCP, RTR Septodont
5 FTIR spectra of β-TCP samples with five repetition
6 XRD pattern of calcinated chicken eggshells after three months stored
7 Temperature of rabbits both control (untreated) and treated with β-TCP
product during the healing process post extraction
8 Pulse rate of rabbits both control (untreated) and treated with β-TCP
product during the healing process post extraction
9 Respiration rate of rabbits both control (untreated) and treated with βTCP product during the healing process post extraction
10 The laterolateral (LL) view radiograph of rabbit’s jaw pre-operation.
Scale bar 12mm
11 The laterolateral (LL) view radiograph of rabbit’s jaw that unfilled with
β-TCP product (control) at day+0 post operation(a). There was tooth
growth that is shown by increasing opacity of extracted area at day+7
post operation (b) and the presence of margin and higher opacity of
alveolar socket at day+30 post operation indicates the formation of
tooth germ (c). Scale bar 12mm
12 The laterolateral (LL) view radiograph of rabbit’s jaw that filled with βTCP product. There was high opacity at day+0 post operation due to the
similar inorganic of sample with bone (a) and gradually decreased
because of the resorption of sample by body fluid at day+7 post
operation (b). The formation of complete tooth was showed by clear
margin on the extracted area at day+30 post operation (c). Scale bar
10mm
13 Macroscopic results of rabbit’s mandible at Day+30 harvesting. Sketch
of rabbit’s mandible was shown to describe the anatomy of lower jaw.
There was no presence of erupted tooth on control (b) but the erupted
tooth on the treatment showed the complete tooth growth (c)
14 A lateral section photomicrograph of lower rabbit incisor (filled with βTCP product) at Day+30 post operation. The presence of fibrous
connective tissue in the ground portion of socket (a) and the presence of
5
5
7
9
10
11
12
13
13
14
14
15
15
15
16
17
18
19
20
21
22
23
24
blood clot in the middle portion of socket indicate blood vessel
rupture(b). Hematoxylin Eosin Stained, x40, Scale bar 100μm
A lateral section photomicrograph of lower rabbit incisor (control) at
Day+30 post operation. Tooth growth was indicated by the presence of
pulp chamber with odontoblastic layer (
) in the apical portion of
socket (a) and fully filled with fat cells in the middle portion (b).
Hematoxylin Eosin Stained, x40, Scale bar 100μm
Temperature of sheep both control (untreated) and treatments (filled
with β-TCP product and commercial β-TCP) during the healing
process post extraction
Pulse rate of sheep both control (untreated) and treatments (filled with
β-TCP product and commercial β-TCP) during the healing process post
extraction
Respiration rate of sheep both control (untreated) and treatments (filled
with β-TCP product and commercial β-TCP) during the healing
process post extraction
The radiograph of dorsoventral (DV) position of sheep ’s jaw preoperation. Scale bar 15mm
The radiograph of dorsoventral (DV) position of sheep’s jaw of control
(unfilled with sample). There was large mesio-distal and low opacity on
the extraction site ( ) at (a) day+0 post operation that become narrower
at day+7 post operation (b) and there was high alveolar bone resorption
shown by significant change on mesio-distal at day+30 post operation
(c). Scale bar (a) 10mm (b) 8mm (c) 11mm
The radiograph of dorsoventral (DV) position of sheep (filled with
β-TCP product). High opacity on the extracted area ( ) at day+0 post
operation showed the alveolar socket filled with sample (a). The same
width of mesio-distal indicated there was no alveolar bone resorption
day+7 post operation and there was resorption of sample showed by
decreasing opacity (b). Resubstituting process of sample and
preservation of alveolar ridge was clearly showed by the same width of
mesio-distal on the edentulous area at day+30 post operation (c). Scale
bar (a) 3mm (b) 10mm (c) 7mm
The radiograph of dorsoventral (DV) position of sheep (filled with
commercial β-TCP, RTR Septodont). High opacity on the extracted
area ( ) at day+0 post operation showed the the alveolar socket filled
with commercial product (a). Decreasing opacity on the extracted area
showed the resorption of materials at day+7 post operation (b) and there
was alveolar bone resoption on the horizontal direction at day+30 post
operation(c). Scale bar (a) 7mm (b) 7mm (c) 10mm
Macroscopic results of sheep’s mandible at day+30 harvesting.
Narrower mesio-distal occurred on control (a) but the width of mesiodistal remained same on the treatment filled with β-TCP product (b).
There was alveolar bone resorption in the horizontal direction on the
treatment filled with commercial β-TCP, RTR Septodont (c)
A lateral section photomicrograph of alveolar bone healing (control) at
Day+30 post operation. Alveolar bone resorption occurs that was
showed by connective tissue in the upper portion of extracted area (a)
17
18
19
20
20
21
21
22
22
23
and a cavity in the middle portion (b). Hematoxylin Eosin Stained, x40,
Scale bar 100μm
25 A lateral section photomicrograph of alveolar bone healing (filled with
β-TCP product) at Day+30 post operation. Biocompatibility of tooth
filler seen from infiltration of fibrous connective tissue within β-TCP
product in the middle portion of extracted area (a). Bone remodeling
occurs in the formation of woven bone (b) via intramembranous bone
development under layer of connective tissue (c) in the upper portion
near gingiva. Hematoxylin Eosin Stained x40 (a, b) x100 (c). Scale bar
100μm
26 A lateral section photomicrograph of alveolar bone healing (filled with
commercial β-TCP) at Day+30 post operation. Biocompatibility of
tooth filler seen from the presence of vessel in the upper portion of
extracted area but the presence of blood clot indicated vessel rupture(a).
Alveolar bone resorption occurs in horizontal direction shown by the
presence of collagen and fibrous connective tissue near gingiva (b).
Repairing process by connective tissue in the middle portion shown by
the presence of vessel (c). Hematoxylin Eosin Stained x40. Scale bar
100μm
24
26
28
APPENDIX LIST
1 Flow chart of the research
2 Joint committee on powder diffraction
Calcium Oxide
3 Joint committee on powder diffraction
β-Tricalcium Phosphate
4 Joint committee on powder diffraction
Calcium Phosphate (Ca2P2O7)
5 Joint committee on powder diffraction
Ca(OH)2
34
standards (JCPDS) database of
35
standards (JCPDS) database of
35
standards (JCPDS) database of
36
standards (JCPDS) database of
36
1 INTRODUCTION
Background
Oral health is essential to general health and quality of life. It means a
condition of being free from any diseases that may impact to our whole body.
Based on the report of World Health Organization (WHO) website on April 2012,
the most common oral diseases are dental cavities, periodontal (gum) disease, oral
cancer, oral infectious diseases, trauma from injuries, and hereditary lesions.
Worldwide, dental cavities happen to 60–90% of school children and nearly 100%
of adults and severe periodontal (gum) disease is found in 15–20% of middle-aged
(35-44 years) adults. Complete loss of natural teeth is widespread and particularly
affects older people (WHO 2013). In Indonesia, health survey conducted by
Ministry of Health Republic of Indonesia in 2001 showed that about 70% of
Indonesian aged over than 10 years old have tooth decay (Pratiwi et al. 2013).
Among these diseases, dental cavities and periodontal disease are the major
causes of tooth loss. Physiologically, there is the loss of alveolar bone as a result
of human tooth loss process. The rate of alveolar bone resorption is fairly high as
a reduction of about millimeters happens in the first 6 months of the healing
process and two-thirds of reduction occurs in the first 3 months (Brkovic et al.
2008, Kokovic and Todorovic 2011). It is observed that over 12 months the
reduction is about 50% in both horizontal and vertical directions (Brkovic et al.
2008). A very progressive bone resorption is able to reduce the dimensions of the
socket and lead to the bone athropy and cause difficulty on implant or prostheses
placement (Calixto et al. 2007). Therefore, preservations of bone volume in the
area of tooth loss are necessary to maintain the dimensions of the alveolar ridge.
In general, grafting procedures are also necessary for improving the bone
tissue ability to regenerate. For this purpose, several approaches have been
attempted for defect filling and subsequent regeneration, including autogeneous
and xenogeneous bone grafting and synthetic biomaterials (Coimbra et al. 2009).
Compared to autogeneous and xenogenous procedures, biomaterials eliminate
problems of donor scarcity, supply limitations, pathogen transfer, and immune
rejection. In the field of dental surgery, biomaterials have been used include
filling of pockets and maxillary ridge augmentation, deficiencies caused by loss of
dentition with advancing age or various diseases (Costa et al. 2008). The use of
filling biomaterials is one way to preserve the adequate bone volume that
commonly used by clinicians.
Nowadays, bioceramics have been widely studied for orthopedic and
dental applications due to their good biocompatible and osseoconductive
properties (Coimbra et al. 2009). Then, because the mineral phase of bones and
teeth is composed of calcium phosphate salts, the use of some Ca-P materials for
bone substitution, augmentation, and repair has gained clinical acceptance in
many aspects of orthopedics and dentistry (Dai et al. 2004). Among calcium
phosphates, hydroxyapatite (HA) and tricalcium phosphate (TCP) have been
widely used as biomaterials in dentistry and medicine (Abdurrahim and Sopyan
2008) due to their close chemical similarity to the inorganic component of bone
and tooth (Tazaki et al. 2009). β-TCP is the most frequently observed TCP
2
polymorphs in the field of bioceramics (Shi 2004) with Ca/P ratio of 1.5 that has
been shown to exhibit good biocompatibility and osteoconductivity (Ain et al.
2008 & Sader et al. 2009) in both animal and clinical studies (Oi et al. 2009)
while HA is considered as bioactive and nonbiodegradable bone replacement
materials. Moreover, β-TCP appears to be advantageous in comparison to HA for
the property of absorption (Tazaki et al. 2009). The faster dissolution/resorption
of β-TCP may allow a gradual biological degradation over a period time and a
progressive replacement by the natural host tissue (Coimbra et al. 2009).
β-TCP itself can be obtained from several methods in which the method of
wet precipitation is one of the widely used methods (Boilet et al. 2013, NasiriTabrizi and Fahami 2013, Farzadi et al. 2011, Koç et al. 2004). In this method,
synthetic materials such as CaCO3 and Ca(NO3)2 are commonly used as calcium
source for obtaining β-TCP ceramics (Park and Bronzino 2000, Wei and Akinc
2007). Previous study reported that β-TCP can not be synthesized directly in
aqueous solution (Belouafa et al. 2006). Wet precipitation in aqueous solution
would synthesize apatitic TCP, Ca9(HPO4)(PO4)5(OH). Following precipitation
from aqueous solution, sintering at high temperatures above 750 C was required
to transform apatitic TCP into β-TCP (Kannan et al. 2006, 2009). Different
synthesis approach applying natural resources material could obtain bioactive
β-TCP that might elicit a direct bond between the bioceramics and living host
tissue. The chicken eggshells are one of natural resources that commonly
considered as waste, contain much of calcium in the form of CaCO3 that
potentially could be used as calcium source in synthesizing β-TCP bioceramics.
The previous study showed that chicken eggshells as the corresponding calcium
source could obtain the other kind of calcium phosphate (hydroxyapatite,
hydroxyapatite/carbonate apatite) by precipitation method (Dahlan et al. 2012).
Therefore, it is clear that β-TCP might be produced from natural resources and
synthesized directly by precipitation method.
Prior to the clinical use, biocompatibility and mechanical stability of new
materials should be test under both initial in vitro and in vivo conditions (Pearce
2007). In vitro testing is used primarily as a first stage test for acute toxicity and
cytocompatibility while in vivo testing is used to demonstrate the tissue response
to materials (Arora et al. 2011). Previous study reported that calcium phosphates
derived from eggshells were nontoxic and performed good adhesion interaction to
the host cells in vitro (Solihat 2011). Therefore, advanced research under in vivo
condition is required to observe the living host tissue response to new materials.
Rabbits and sheep are animal models that could be applied to observe the
biocompatibility and mechanical stability of β-TCP bioceramics. Rabbits are one
of the most commonly used animals for medical research despite of easy handling
and sheep represent human bone relatively closely (Pearce 2007). Rabbits are
lagomorph with hypselodont teeth that could be used as animal model for cases of
premature tooth loss while sheep are for observing cases of permanent tooth loss
posextraction due to the similarity on alveolar ridge with human.
3
Objectives
1. Synthesizing β-TCP bioceramics obtained from chicken eggshells by
precipitation method
2. Observing the tooth growth and the bone healing using β-TCP bioceramics
and tissue response to materials by in vivo testing in rabbits and sheep
Hypotheses
1. Chicken eggshells can be used as calcium donor in synthesizing β-TCP
bioceramics that biocompatible for dentistry
2. Chicken eggshells-based β-TCP bioceramics as tooth filler can accelerate
the tooth growth on rabbits and stimulate the bone healing on sheep after
tooth extraction
Benefits
This research is expected to obtain natural resource-based β-TCP
bioceramics such chicken eggshells that generally known as waste in use as
calcium donor in synthesizing of β-TCP bioceramics. It may produce
biocompatible biomaterials with economical price for dentistry application in
Indonesia. This will subsequently assume to decrease the dependence on
importing biomaterial for bone and tooth substitution.
2 MATERIALS AND METHOD
Place and Time Schedule
This research was conducted from January 2013 through September 2013
which took place in Biophysics Laboratory-IPB in sample preparation, while
biocompatibility testing was done in Faculty of Veterinary Medicine-IPB. Sample
characterization was done in Forestry Development and Research Center-Bogor
for x-ray diffraction (XRD) characterization, in Laboratory of Material AnalysisIPB for fourier transform infrared (FTIR) analysis, and in Indonesian Soil
Research Institute-Bogor for atomic absorption spectroscopy (AAS). Histological
test and x-ray radiography analysis was done in Faculty of Veterinary MedicineIPB.
Materials and Equipments
The materials used in this research were calcium oxide obtained from
chicken eggshells, pro-analyze phosphoric acid (H3PO4), chitosan, aquabidest, and
aquadest. The equipments used were analytical balance, beaker glass, burette,
4
aluminium foil, pipette, magnetic stirrer, hotplate, furnace, digital thermometer,
filter paper, mortar, crucible, and vacuum.
Materials and equipments used for biocompatibility testing were rabbits
and sheep as animal model, minor surgery set, dental surgery tools, anesthetic
material, and surgery room for aseptical insertion. Then, materials used for
histopathological examination were formalin 10%, nitric acid 5%, aquadest,
aquabidest, alcohol, xylol, Hematoxilin-Eosin (HE), and Permount ®.
Experimental Methods
Calcination of chicken eggshells
The raw eggshells where their surface was initially cleaned were
calcinated in an air atmosphere at 1000oC for 5 hours as seen in Figure 1. This
calcination aimed to convert calcium carbonate (CaCO3) into calcium oxide
(CaO). To synthesize calcium phosphate powder, the corresponding eggshells
were crushed and milled in crucible then characterized by using x-ray diffraction
(XRD) Shimadzu diffractometer, Cu-Kα radiation, 40 kV, 30 mA and 0,02o s-1
scanning step. The chemical reaction of eggshell calcination is given below:
β-TCP synthesis
β-TCP bioceramics was prepared by precipitation of H3PO4 as the
phosphor (P) source. Then it was dropped into CaO as the calcium (Ca) source
which was synthesized further at 50oC with molar ratio of Ca:P as 1.2M : 0.8M
and the chemical reaction is given below:
The results were precipitated, filtered and heated at sintering temperature
1000 C for 7 hours (Figure 2). The final results were obtained in the form of white
powder and analyzed by using XRD and FTIR spectroscopy (ABB MB 3000)
characterization to identify the formed phase and the functional group of the
sample, respectively, as well as AAS spectroscopy to verify the purity of the
products. To assure the reproducibility of the sample the process was performed
five times. Samples used for biocompatibility testing were β-TCP pellet. To get
the pellet form, samples were mixed with chitosan 2%, molded in form of pellet
with 2 mm and 6 mm in diameter respectively for rabbits and sheep then heated in
incubator at 50oC for 6 hours.
o
5
Calcination at 1000 oC
The cleaned raw eggshells
Powder of CaO
Figure 1 Calcination process of chicken’s eggshells
Ca and P solution
Precipitation
Filtering
Sintering
Figure 2 β-TCP synthesis through precipitation method
Biocompatibility testing
Biocompatibility test of β-TCP bioceramics as a tooth filler was performed
by using rabbits and sheep as animal models. There are two 1 year old rabbits
used with 2.5 kg body weight and three 1 year old sheep. All experiments were
conducted along the institutional guidelines for the care and use of laboratory
animals. Prior to surgery, the animal models were observed closely for a week in
order to check their health status. They were maintained under identical
environment, management and standard diet with ad libitum supply of drinking
water. Then, surgery was done on the lower incisor. Before surgery, rabbits and
sheep were anaesthetized by injection of ketamine 10% and xylazin 10%. Under
aseptical conditions, the lower incisor was extracted and immediately after tooth
extraction, the alveolar socket was filled with synthesized β-TCP bioceramics that
were sterilized initially by exposure to ultraviolet light. As control requirement,
other rabbit’s and sheep’s lower incisor were extracted in the same manner and
unfilled with any tooth filler. Each surgery was performed under same veterinary
surgeon. Then, the rabbits and sheep were housed under a climate-controlled
environment in stall of animal used of FVM Bogor Agricultural University.
The alveolar sockets that filled with β-TCP tooth filler and unfilled with
any tooth filler were monitored using a set of x-ray radiographic apparatus in
day+0 pre operation, D+0 post operation (PO), D+7 PO and D+30 PO for
observing the tooth growth on rabbits and alveolar bone healing on sheep. Then,
the rabbits and sheep were euthanized on D+30 PO for macroscopic evaluation.
For histological evaluation, the mandibular was thoroughly washed with water
and fixed in 10% neutral formalin. After fixation, the mandibular was dissected
6
out, decalcified, and processed for paraffin embedding. The decalcification
process was carried out using 10% EDTA solution and the specimens was
dehydrated in ascending grades of ethyl alcohol, cleared in xylene and embedded
in paraffin wax. The histopathological examination was done for observing the
degree of tooth growth on rabbits and new bone formation on sheep. The
histological analysis was carried out with some parameters comprised the
presence and type of inflammatory infiltrate, reaction to foreign body, fibrous
connective tissue, osteoblastic activity, formation of osteoid and new bone.
Detailed flow chart of the research was shown in Appendix 1.
3 RESULTS AND DISCUSSION
Calcination of Chicken Eggshells
It was found that the calcinated chicken eggshells contain pure calcium
oxide with mass decomposition of 48.40%. The characteristics of the
corresponding calcium oxide atomic structure were examined by using XRD
where its pattern is shown in Figure 3a. It was observed that the highest peaks
were on 2θ = 32.31, 37.47, 53.98, 64.27, 67.51, and 79.76. Based on data from
joint committee on powder diffraction standards (JCPDS) database number
37-1497 (Appendix 2), these results clearly prove the presence of calcium oxide.
Figure 3b shows that the sample pattern was match to the database, since they
share similar peaks pattern. The absence of other phases in the sample pattern
indicates the high purity of CaO powder. The result in this study had higher
accuracy of CaO pattern compared to that of other studies (Saeed et al. 2011,
Balazsi et al. 2007). The XRD pattern of 950 oC calcined chicken eggshell
corresponds to CaO with Ca(OH)2 as the majority phase according to Miller
indices of (1 0 0), (1 0 1), and (1 0 2) (Saeed et al. 2011). In addition to Ca(OH)2,
MgO, which may influence the purity of β-TCP products, was also observed
which calcination was performed at 900 oC (Balazsi et al. 2007). It seems that the
calcinating temperatures of 900 oC and 950 oC are not enough to get pure CaO due
to the strong hydroxyl binding at eggshells.
7
intensity (counts)
1000
800
600
400
200
0
10
20
30
40
50
2θ (degree)
60
70
80
Intensity (counts)
(a)
2θ (degree)
(b)
Figure 3 X-ray diffraction (XRD) pattern of calcinated chicken eggshells at
1000oC for 5h (a) and peak matching of calcinated chicken eggshells
with database PCPDF 37-1497 (b)
Chicken Eggshells-Based β-Tricalcium Phosphate
Synthesis of chicken eggshells-based β-Tricalcium phosphate (β-TCP) by
precipitation method was performed five times with the sample codes are shown
in Table 1. The sintering temperature used to obtain β-TCP bioceramics was
1000oC. Referring to the previous study it was reported that the required
temperature to start phase transformation of calcium phosphate into β-TCP is
760oC and get stable at 1125oC (Salahi and Heinrich 2003, Shi 2004). The result
of this process was a white powder with mass precipitates are shown in Table 2.
8
In contrary with Belouafa et al. (2006), β-TCP could be synthesized
directly in aqueous solution as shown from its XRD profile (Figure 4). The three
important highest peaks pattern of the first three samples (TCP1, TCP2 and
TCP3) were completely similar to the peaks of β-TCP database number 09-0169
(Appendix 3). It was proved by high intensity on 2θ around 27.84, 31.06, and
34.41. The XRD peaks pattern of the corresponding β-TCP samples was the same
with that found by heating the apatitic TCP at temperature above 750oC (Kannan
et al. 2006, 2009). Moreover, it was similar with that one of commercial TCP
(RTR Septodont France) and sintered commercial TCP powder (Sader et al. 2009).
The determined lattice parameters of the corresponding β-TCP samples are shown
in Table 3 by fitting the peaks of identified reflections from the XRD pattern
using Cohen Method. It is clearly showed the formation of β-TCP bioceramics
proved by rhombohedral structure with lattice parameters correspond to the
reference a=b=10.42Å and c=37.38Å (Shi 2004).
Table 1 β-Tricalcium Phosphate Bioceramics Codes
Sample
Code
TCP (first repetition)
TCP1
TCP (second repetition)
TCP2
TCP (third repetition)
TCP3
TCP (fourth repetition)
TCP4
TCP (fifth repetition)
TCP5
Table 2 Precipitate Mass of β-Tricalcium Phosphate Bioceramics
Sample
Precipitate Mass (g)
TCP1
7.75
TCP2
7.62
TCP3
7.34
TCP4
6.94
TCP5
6.77
Table 3 Lattice parameter and accuracy of β-TCP samples
Sample
TCP1
TCP2
TCP3
Lattice parameter (Å)
a
c
10.44
37.44
10.39
37.22
10.42
37.37
Accuracy (%)
a
c
99.78
99.84
99.71
99.57
99.99
99.97
Intensity (counts)
9
Figure 4 XRD pattern of β-TCP samples with five repetition and commercial
TCP, RTR Septodont
In the meantime, the FTIR results as shown in Figure 5 were completely
consistent with the result of crystallization processes observed by XRD
measurements given by Figure 4. The broad IR band of FTIR spectra around 540580 cm-1, 690 cm-1 and 1010 cm-1 indicates the formation of a typical TCP
structure containing PO43- band. The absence of hydroxyl band in corresponding
samples around 3500 cm-1 shows better characteristics compared to Salahi and
Heinrich (2003) that used synthetic materials. The FTIR result of β-TCP obtained
10
% Transmittance
from synthetic materials was not pure that clearly showed by the existence of
hydroxyl band in sample sintered in 1000 oC (Salahi and Heinrich 2003). Other
study done by Ain et al. (2008) showed the presence of another phase
(hydroxyapatite) on the XRD results. It means that β-TCP bioceramics has been
formed directly in aqueous solution by precipitation method which also been
proved with a better characteristics compared to Salahi and Heinrich (2003) and
Ain et al. (2008) that used synthetic materials. Since the chicken eggshells are
considered as a raw material, the corresponding β-TCP samples should be more
economical. It is obvious that the corresponding β-TCP samples is reproducible
due to the repeatable XRD peaks patterns and FTIR spectra.
Wavenumber (cm-1)
Figure 5 FTIR spectra of β-TCP samples with five repetition
11
On the other hand, it should be noted that the last two samples (TCP4 and
TCP5) correspond to the other phase of calcium phosphate (Ca2P2O7) with
database number 09-0346 as seen in Appendix 4 which is occured probably due to
the different calcium source used in synthesizing β-TCP. The corresponding
calcium source was calcinated chicken eggshells powder that had been stored for
more than three months. This result is obviously depicted by its XRD profile as
shown in Figure 6 that refers to the phase of Ca(OH)2 with database number
44-1481 (Appendix 5). It is exhibited that there is phase transformation caused by
interaction of CaO with the environment during the storage at non-vacuum
condition. The existence of OH- functional group on Ca(OH)2 tends to release and
to form another phase of calcium phosphate as also been observed in the previous
study that synthesized biphasic calcium phosphate (BCP) with CaO and Ca(OH)2
as raw materials (Dahlan et al. 2011). Their research also showed that TCP only
appeared on samples synthesized with CaO that probably caused by the difference
binding type of each precursor.
Furthermore, to verify the purity of the products, the β-TCP samples were
characterized by using atomic absorption spectroscopy (AAS). Based on the AAS
results, it is shown that Ca/P ratio of TCP3 corresponded to β-TCP bioceramics as
shown in Table 4. This result confirmed the XRD results and in accordance with
the theory of Ca/P ratio of β-TCP bioceramics (Shi 2004). Nevertheless, the AAS
measurements of TCP1 and TCP2 are not consistent with the XRD results. This
inconsistency probably occured due to the limited number of samples powder that
used for the AAS analysis.
Intensity (counts)
300
250
200
150
100
50
0
10
20
30
40
50
2θ (degree)
60
70
Figure 6 XRD pattern of calcinated chicken eggshells after three months stored
Table 4 AAS results of β-TCP products
Sample
TCP 1
TCP 2
TCP 3
TCP 4
TCP 5
Percentage
Ca
P
51.9
38.54
59.7
35.22
53.06
35.8
46.31
42.98
47.23
44.3
Ca/P ratio
1.34
1.69
1.48
1.08
1.07
12
Biocompatibility Test on Rabbits
Biocompatibility is the ability of a material to perform with an appropriate
host response in a specific application (Shi 2004). Biocompatibility test of β-TCP
as tooth filler on rabbits was performed to observe the tooth growth post
extraction. Biocompatible properties of new materials could be analyzed from
several examinations such as physical examination, x-ray radiographic analysis,
and histological evaluation.
The physical examination on rabbits that is observed for 30 days showed
that β-TCP product was biocompatible and non-toxic. There were no significant
influence on rabbits filled with β-TCP product and unfilled (control) in
temperature and pulse rate post operation. The results of physical examination on
rabbits were in normal range of each parameter (Figure 7 and Figure 8). The
normal temperature of rabbit is 38.9-40.5oC and its normal pulse rate is 120-250
waves/minute (Kelly 1984). There was temperature decreasing on rabbit filled
with β-TCP product post operation that might be influenced by the injection of
anaesthetic material. Thus, the temperature was in normal range on D+1 PO and
days after. It is worth to note that filling β-TCP product do not interfere the
physiological body temperature of rabbit. Nevertheless, the respiration of rabbits
as seen in Figure 9 both control and treatment were not in normal range that is
30-45 (Kelly 1984). It might be caused by condition of rabbits that are stress
during data collection process and made them excited. An increased demand for
oxygen after exercised might increase respiratory frequency (Kelly 1984).
Moreover, the longer period of expiration in healthy animals depends upon
whether the animal is relaxed and resting, or has recently been excited or
excercised. In short, postextraction preservation by β-TCP as a tooth filler do not
interfere the physiological condition of rabbit.
Temperature (oC)
42
41
40
39
Normal
38
Control (untreated)
37
Treated with B-TCP
Product
36
Observing time
Figure 7 Temperature of rabbits both control (untreated) and treated with β-TCP
product during the healing process post extraction
13
Pulse rate (waves/minute)
300
250
200
Control (untreated)
150
Treated with B-TCP
product
100
Observing time
Figure 8 Pulse rate of of rabbits both control (untreated) and treated with β-TCP
product during the healing process post extraction
250
Respiration
200
150
100
Control (untreated)
50
Treated with B-TCP
product
0
Observing time
Figure 9 Respiration rate of of rabbits both control (untreated) and treated with
β-TCP product during the healing process post extraction
The rabbits tooth growth and status of β-TCP product was monitored using
a set of x-ray radiographic apparatus. The radiograph of rabbit’s jaw pre operation
is shown in Figure 10 and the radiograph of rabbit’s jaw as control and treatment
is shown in Figure 11 and Figure 12. Based on the radiograph in Figure 11 and
Figure 12, these results show a tooth growth on rabbits at D+30 PO that was
demonstrated by the presence of margin and high opacity on an extracted tooth
area. It was clear that alveolar socket of lower incisors rabbit on D+0 PO (Figure
11a) had low opacity and then increase on D+7 PO as shown in Figure 11b.
Increasing of alveolar socket opacity indicates the formation of tooth germ
precursor tissue. It was also observed that in Figure 11b there was a tooth growth
14
on upper incisors rabbit. It might because rabbits are a lagomorpha with all teeth
continually growing. Rabbit is mammalian group with hypselodont teeth, evergrowing or open-rooted teeth that grow continuously during the lifetime of the
animal. They also called open rooted teeth that refers solely to the large apical
opening present in all continuously growing teeth and does not imply that the
tooth actually needs to have a root in a classical form (Ali and Mubarak 2012).
Moreover, the radiograph of alveolar socket’s rabbit on D+30 PO (Figure 11c)
demonstrated the formation of new teeth which was shown by increasing opacity
with apparent margin. Nevertheless, the empty space around alveolar socket of
lower incisors obviously indicated the incomplete tooth formation. In the
meantime, the macroscopic result of rabbit’s mandibular at D+30 harvesting as
shown in Figure 13b was in accordance with the radiograph result of alveolar
socket at D+30 PO. It is clear that there was unerupted tooth on D+30 harvesting
due to no appearance of tooth on gums surface.
Figure 10 The laterolateral (LL) view radiograph of rabbit’s jaw pre-operation.
Scale bar 12mm
(a)
(b)
(c)
Figure 11 The laterolateral (LL) view radiograph of rabbit’s jaw that unfilled with
β-TCP product (control) at day+0 post operation (a). There was tooth
growth that is shown by increasing opacity of extracted area at day+7
post operation (b) and the presence of margin and higher opacity of
alveolar socket at day+30 post operation indicates the formation of
tooth germ (c). Scale bar 12mm.
15
(a)
(b)
(c)
Figure 12 The laterolateral (LL) view radiograph of rabbit’s jaw that filled with
β-TCP product. There was high opacity at day+0 post operation due
to the similar inorganic of sample with bone (a) and gradually
decreased because of the resorption of sample by body fluid at day+7
post operation (b). The formation of complete tooth was showed by
clear margin on the extracted area at day+30 post operation (c). Scale
bar 10mm.
(a)
(b)
(c)
Figure 13 Macroscopic results of rabbit’s mandible at Day+30 harvesting. Sketch
of rabbit’s mandible was shown to describe the anatomy of lower jaw.
There was no presence of erupted tooth on control (b) but the erupted
tooth on the treatment showed the complete tooth growth (c).
On the contrary, the radiograph of alveolar socket filled with β-TCP as
shown in Figure 12 demonstrated the tooth growth on rabbit. As seen in Figure
12a, the alveolar socket was filled with β-TCP and there was higher opacity than
one in control at D+0 PO, which might be caused by the similar inorganic
component of β-TCP with bone. At the same time, there was jaw bone fracture
during tooth extraction shown by unclear margin that is different with the origin.
Figure 12b clearly shows resorption of β-TCP by the body fluids that is indicated
by volume decreasing of β-TCP pellet in alveolar socket. In addition, there was a
tooth growth of upper incisors rabbit at D+7 as well as at the control. However,
there were differences on rabbits alveolar socket filled with β-TCP at D+30 PO. It
was clear that β-TCP had been resorbed and could accelerate the formation of
complete tooth shown from its radiograph with clear margin at Figure 12c. In the
meantime, jaw bone healing process was observed indicated by bone margin
which was similar with that of pre-operation bone margin. Furthermore, this result
was supported by macroscopic result at D+30 PO as seen in Figure 13c that
16
indicates the complete tooth formation of rabbits lower incisors due to the erupted
tooth of 15 mm. it demonstrated a rapid growth of rabbits lower incisors and
relevant to the other studies that reported lower incisors growth is approximately
2-3 mm per week (Capello 2005, Meredith 2007). Furthermore, the upper incisors
growth of rabbit was shorter than that of control. It might be caused by the
presence of erupted tooth and makes slower growth on the rabbit upper incisors. It
is obvious that β-TCP could accelerate the formation of complete tooth and
stimulate the tooth growth. In short, it could be applied clinically in case of
premature tooth loss that commonly happen in children.
On the other hand, the histological results of treated extraction site that
filled with β-TCP product inconsistent with the x-ray radiograph and macroscopic
result. It is clearly proved by Figure 14 that showed the presence of fibrous
connective tissue at the ground portion lining the sample as seen in Figure 14a. As
seen in Figure 14a, blood clot filled the socket post extraction and was replaced
with fibrous connective tissue. Then, in the middle portion of the socket was fully
filled with mass of blood clod. Figure 14b showed that β-TCP product was partly
resorbed and degraded. It was clearly showed by distributed β-TCP product that
interacted with blood then will be replaced with fibrous connective tissue. The
formation of fibrous connective tissue is a reaction to chronic insult and result
from the activity of fibroblast cells. As explained by Banks (1986) connective
tissues serve numerous functions and one of them is insulate organs from
mechanical damage. It might be called as important line of defense. Moreover,
some connective tissues also play an important role in repair and regeneration.
The existence of blood clot might indicate the incomplete healing processes and
means no tooth growth on rabbit mandible. The erupted tooth in the macroscopic
result might be a remnant of unextracted tooth that forced by the blood clot and βTCP product.
The opposite microscopic result was showed by control (unfilled) rabbit in
Figure 15 that demonstrate the formation of tooth germ. It was showed by the
presence of pulp chamber as shown in Figure 15a. It was obvious from wide pulp
chamber that lined by odontoblastic layer in the apical region of rabbit incisor.
Odontoblastic layer was not covered by dentine and enamel on its external side.
These results showed the incomplete tooth formed proved by no cervical loop at
the labial side in rabbit incisor that has function to generate ameloblasts.
Ameloblast is cells that produce enamel on the labial tooth surface (Ali and
Mubarak 2012). Moreover, the extracted socket was also filled with fat cells as
seen in Figure 15b. These cells occur as single cells, clusters of cells, or as
massive sheets of cells associated with connective tissues, especially loose
connective tissue (Banks 1986). In addition, these cells appear as large empty
spaces surrounded by the peripheral portion of the cell and a flattened nucleus
(Copenhaver et al. 1978). These results support the radiograph result of untreated
rabbit (control) at day+30 post operation that indicated the formation of
incomplete tooth. In short, filling socket by β-TCP product after tooth extraction
does not stimulate the regrowth of rabbit incisor. It might be caused by rabbits as
animal used in this research have the same weight but not with the age so it might
cause different tooth growth process.
17
a
fibrous
bone
Β-TCP product
fibrous
b
Blood clot
β-TCP product
Figure 14 A lateral section photomicrograph of lower rabbit incisor (filled with
β-TCP product) at Day+30 post operation. The presence of fibrous
connective tissue in the ground portion of socket (a) and the presence
of blood clot in the middle portion of socket indicate blood vessel
rupture(b). Hematoxylin Eosin Stained, x40, Scale bar 100μm.
18
a
Odontoblastic
layer
Fat
cells
Pulp
chamber
b
Fat cells
Figure 15 A lateral section photomicrograph of lower rabbit incisor (control) at
Day+30 post operation. Tooth growth was indicated by the presence of
pulp chamber with odontoblastic layer (
) in the apical portion of
socket (a) and fully filled with fat cells in the middle portion (b).
Hematoxylin Eosin Stained, x40, Scale bar 100μm.
19
Biocompatibility test on sheep
Temperature (oC)
Biocompatibility test was also done in sheep to observe the alveolar bone
healing process by resubstituting process of the corresponding β-TCP. It is clear
that the corresponding β-TCP was biocompatible and non-toxic. Based on the
clinical examination, there was no inflammation on the extracted area of each
sheep. The result of physiological examination in sheep was the same wi