UNIVERSITI PUTRA MALAYSIA DEVELOPMENT OF COCKLE …psasir.upm.edu.my/id/eprint/41860/1/FPV 2013...
Transcript of UNIVERSITI PUTRA MALAYSIA DEVELOPMENT OF COCKLE …psasir.upm.edu.my/id/eprint/41860/1/FPV 2013...
UNIVERSITI PUTRA MALAYSIA
KH. NURUL ISLAM
FPV 2013 4
DEVELOPMENT OF COCKLE SHELL-BASED NANOCOMPOSITE BIOMATERIAL BONE PASTE AND ITS EFFECTIVENESS FOR
BONE REPAIR IN RABBIT MODEL
© COPYRIG
HT UPM
DEVELOPMENT OF COCKLE SHELL-BASED
NANOCOMPOSITE BIOMATERIAL BONE
PASTE AND ITS EFFECTIVENESS FOR BONE
REPAIR IN RABBIT MODEL
KH. NURUL ISLAM
DOCTOR OF PHILOSOPHY
UNIVERSITI PUTRA MALAYSIA
2013
© COPYRIG
HT UPM
DEVELOPMENT OF COCKLE SHELL-BASED NANOCOMPOSITE
BIOMATERIAL BONE PASTE AND ITS EFFECTIVENESS FOR BONE
REPAIR IN RABBIT MODEL
By
KH. NURUL ISLAM
Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia
in Fulfilment of the Requirements for the Degree of Doctor of Philosophy
January 2013
© COPYRIG
HT UPM
COPYRIGHT
All material contained within the thesis, including without limitation text, logos, icons,
photographs and all artwork, is copyright material of Universiti Putra Malaysia unless
otherwise stated. Use may be made of any material contained within the thesis for non-
commercial purposes from the copyright holders. Commercial use of material may only
be made with the express, prior, written permission of Universiti Putra Malaysia.
Copyright@ Universiti Putra Malaysia
© COPYRIG
HT UPM
ii
DEDICATION
To my late parents who are in the garden of heaven for eternal peace
To my relatives
To my three sisters, two wives, four daughters and only one son Kh. Omar Faruque
© COPYRIG
HT UPM
iii
Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfilment
of the requirement for the degree of Doctor of Philosophy
DEVELOPMENT OF COCKLE SHELL-BASED NANOCOMPOSITE
BIOMATERIAL BONE PASTE AND ITS EFFECTIVENESS FOR BONE
REPAIR IN RABBIT MODEL
By
KH. NURUL ISLAM
January 2013
Chairman: Professor Md. Zuki Bin Abu Bakar@ Zakaria, PhD
Faculty: Veterinary Medicine
This study revealed the development of paste from cockle shell-based calcium
carbonate nanoparticles and in vivo evaluation using a rabbit model. Calcium
carbonate and its polymorphs from cockle shells (Anadara granosa) were
characterized using variable pressure scanning electron microscope (VPSEM), a
transmission electron microscope (TEM), an energy dispersive X- ray analyzer
(EDXA), X-ray diffraction (XRD) and Fourier transmission infrared spectroscopy
(FT-IR). Rod-like aragonite crystals of cockle shell powders were observed by both
SEM and TEM. The EDXA results showed that the cockle shells contained more
calcium and carbon than the commercial calcium carbonate. The FT-IR analyzes
revealed the presence of carbonate groups in cockle shell powders. The FT-IR
analyzes also showed the presence of aragonite in cockle shell powders. The FT-IR
and XRD analyzes showed that the cockle shell powders contained aragonite. The
© COPYRIG
HT UPM
iv
cockle shell powders were formed with good quality calcium carbonate and
contained calcium carbonate in the aragonite phase.
The calcium carbonate aragonite nanoparticles were synthesized from micron sized
cockle shell powders. The method involves a simple mechanical stirring of the
micron-sized cockle shells powders in the presence of a non-toxic and non-hazardous
biomineralization catalyst, dodecyl dimethyl betaine (BS-12). The method produced
rod-shaped aragonite nanoparticles with the diameter of 20-30 nm with good
reproducibility and without any additional impurities. This was confirmed by a
combined analysis of variable pressure scanning electron microscopy (VPSEM),
transmission electron microscopy (TEM), Fourier transmission infrared spectroscopy
(FTIR), Thermogravimetric analyzer (TGA), X-ray powder diffractometer (XRD)
and an energy dispersive X-ray analyzer (EDX). The method should find potential
applications in the industry for large scale synthesis of aragonite nanoparticles at low
cost from an abundant natural resource such as cockle shells. The calcium carbonate
nanoparticles in the aragonite phase were synthesized from the cheap and naturally
abundant cockle shells.
The pastes were developed from the cockle shell-based calcium carbonate
nanoparticles. The composite pastes were used as bone repair in surgical
applications. The cockle shell-based calcium carbonate nanoparticles were mixed
with chitosan solution containing 2% acetic acid in a 250 ml glass beaker. The paste
mixture was mixed using a multi-system hot plate mechanical stirrer with magnetic
stirrer bar. The mixture clumped together and gave a homogeneous mesh like
appearance. The paste was characterized by the Field Emission Scanning Electron
© COPYRIG
HT UPM
v
Microscopy (FESEM), an Energy Dispersive X-ray Analyzer (EDXA), a Fourier
transform infrared (FT-IR) spectrophotometer, an X-ray Powder Diffractometer
(XRD), a thermogravimetric Analyzer (TGA), an Inductively Coupled Plasma-
Optical Emission Spectrometry (ICP-OES) and Phosphate Buffer Saline Medium
(PBS). The biocompatible and bioabsorbable pastes were developed from cockle
shell-based nano calcium carbonate.
The developed paste was evaluated in vivo using a rabbit model. The paste was
compared functionally from the pastes of cockle shell-based micron sized calcium
carbonate and commercial calcium carbonate respectively. Twelve rabbits which
were divided into three groups (n=4) were used for the in vivo evaluation. The first,
second and third groups were used for the paste of cockle shell-based nano calcium
carbonate, cockle shell-based micron sized calcium carbonate and commercial
calcium carbonate, respectively. One bone hole with 5 mm diameter was created on
the medial surface of the proximal extremity of both left and right tibia. The left hole
was left empty and acted as negative control while the right hole was treated with the
paste implantation. After implantation, the paste effectiveness was evaluated by
radiographic, biochemical, gross and histological examination. The dynamic cockle
shell-based nanocomposite biomaterial bone paste showed excellent bone healing
performance in the right bone holes as compared to the micron sized cockle shell-
based calcium carbonate paste, commercial calcium carbonate paste and many
previously prepared bone pastes. This novel bone paste can be potential for
biomaterial industry, human and veterinary medicine.
Key words: Cockle shells, nano calcium carbonate, nanocomposite biomaterial bone
paste, in vivo study, rabbits.
© COPYRIG
HT UPM
vi
Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai
memenuhi keperluan untuk ijazah Doktor Falsafah
PEMBANGUNAN PES NANOKOMPOSIT DAN KEBERKESANANNYA
UNTUK DALAM PEMBAIKAN TULANG
Oleh
KH. NURUL ISLAM
Januari 2013
Pengerusi: Profesor Md. Zuki Bin Abu Bakar @ Zakaria, PhD
Fakulti: Perubatan Veterinar
Kajian ini mendedahkan pembangunan pes daripada kulit kerang berasaskan
nanopartikel kalsium karbonat dan penilaian in vivo menggunakan model arnab.
Kalsium karbonat dan Polimorf daripada kulit kerang (Anadara granosa) telah diciri
menggunakan pengimbas mikroskop elektron pelbagai tekanan (VPSEM),
mikroskop transmisi elektron (TEM), pengimbas serakan tenaga sinar-X (EDX),
pembelauan sinar-X (XRD) dan spektroskopi penghantaran Fourier inframerah (FT-
IR). Rod kristal aragonite dari serbuk kulit kerang telah diperhatikan dibawah kedua-
dua SEM dan TEM. Keputusan EDXmenunjukkan kulit kerang mengandungi lebih
banyak kalsium dan karbon daripada kalsium karbonat komersial. Analisa FT-IR
mendedahkan kehadiran kumpulan karbonat di dalam kulit kerang. Analisa FT-IR
juga menunjukkan kehadiran aragonite di dalam kulit kerang. Analisa XRD
menunjukkan bahawa serbuk kulit kerang mengandungi aragonite. Serbuk
kulitkerang telah dibentukdaripada kalsium karbonat yang berkualiti baik dan
© COPYRIG
HT UPM
vii
mengandungi kalsium karbonat fasa aragonite.Nanopartikel aragonite kalsium
karbonat telah disintesis daripada serbuk kulit kerang bersaiz mikron. Aragonite
adalah salah satu polimorf kurang biogenik kalsium karbonat dan digunakan secara
meluas sebagai bahan bio untuk pembaikan tulang patah, pembangunan sistem awal
penyampaian dadah, dan perancah tisu. Kaedah ini melibatkan pengisaranmekanikal
serbuk kulit bersaiz mikron dengan kehadiran pemangkin biomineral bukan toksik
dan tidak merbahaya, dodesil betaine dimetil (BS-12). Kaedah ini menghasilkan
nanopartikel aragonite berbentuk rod dengan penghasilan semula yang baik dan
tanpa sebarang kekotoran tambahan. Ini telah disahkan melalui analisa gabungan
pengimbas mikroskop elektron pelbagai tekanan (VPSEM), penghantar mikroskop
elektron (TEM), spektroskopi Fourier penghantaran inframerah (FTIR),
Termogravimetri penganalisis (TGA), spektroskopi pembelauan sinar-X (XRD) dan
tenaga serakan X-ray penganalisis (EDX). Kaedah ini boleh menjadi aplikasi yang
berpotensi di dalam industri bagi sintesis nanopartikel aragonite berskala besar
darisumber semula jadi seperti kulit kerang dengan kos yang rendah.Nanopartikel
kalsium karbonat di dalam fasa aragonite telah disintesis daripada kulit kerang yang
murah dan banyak terdapat secara semulajadi.
Pes biokomposittelah dibangunkan dari kulit kerang berasaskan nanopartikel kalsium
karbonat. Pes ini telah digunakan sebagai pembaikan tulang di dalam aplikasi
pembedahan. Cengkerang kerang berasaskan nanopartikel kalsium karbonat telah
dicampur dengan larutan kitosan yang mengandungi 2% asid asetik di dalam bikar
kaca 250 ml. Campuran pes dicampur dengan menggunakan pengacau mekanikal
plat panas pelbagai sistem dengan bar pengacau magnet. Campuran tersebut
berkelompok bersama-sama dan memberikan penampilan seperti jaringan homogen.
© COPYRIG
HT UPM
viii
Pes dicirikan oleh Pelepas Bidang Mengimbas Mikroskop Elektron (FESEM),
Sebaran Tenaga Analisis Sinar-X (EDX), Jelmaan Fourier Inframerah (FT-IR)
spektrofotometer, X-ray Difakometer (XRD), Termogravimetri Analisa (TGA),
Plasma Gandingan Induktif Atom Pelepasan Spektrometer (ICP-OES) dan Fosfat
Penimbal Saline (PBS). Pes biokomposit setanding dan bioserapan telah
dibangunkan daripada kulit kerang berasaskan nano kalsium karbonat.
Pes yang telah dibangunkan telah dinilai secara in vivo menggunakan model arnab.
Pestelah dibandingkan fungsinya dari pes kulit kerang masing-masing berdasarkan
kalsium karbonat bersaiz mikron dan kalsium karbonat komersial. Dua belas arnab
dibahagikan kepada tiga kumpulan (n = 4) telah digunakan di dalam penilaianin vivo.
Kumpulan pertama, kedua dan ketiga masing-masing telah digunakan untuk pes kulit
kerang nano berasaskan kalsium karbonat, kulit kerang berasaskan kalsium karbonat
bersaiz mikron dan kalsium karbonat komersial. Satu lubang tulang dengan diameter
5 mm telah dibuat pada permukaan medial hujung proksimal kedua-dua kiri dan
kanan tulangtibia. Lubang kiri dibiarkan kosong dan bertindak sebagai kawalan
negatif manakala lubang kanan telah dirawat dengan pes. Selepas implantasi,
keberkesanan pes dinilai melalui pemeriksaan radiografik, kasar dan histologi. Pes
dinamik kulit kerang nano berasaskan kalsium karbonat menunjukkan penyembuhan
tulang yang amat baik di dalam lubang tulang kanan berbanding kulit kerang
berasaskan pes kalsium karbonat bersaiz mikron dan pes kalsium karbonat komersial.
Kata kunci: kerang kerang, nano kalsium karbonat, pes tulang biobahan
nanokomposit, kajian in vivo, arnab
© COPYRIG
HT UPM
ix
ACKNOWLEDGEMENTS
I would like to express my deep gratitude to my respected supervisor, Prof. Dr. Md.
Zuki Bin Abu Bakar @ Zakaria for his cordial co-operation during my studies. I feel
proud to get Prof. Dr. Md. Zuki as my supervisor. He had given me a very
challenging task in my life to develop the nanocomposite biomaterial bone paste
from cockle shell-based nano calcium carbonate. This was very new and challenging
work to me at the beginning. I have successfully overcome some limitations,
especially during the development of the nanocomposite biomaterial bone paste and
finally was able to develop the dynamic nanocomposite biomaterial bone paste
successfully. His prompt and excellent guidance helped me to finish my study.
I would also like to express my gratitude to my co-supervisors Prof. Dr. Md Zobir
Bin Hussein, the faculty of Science, UPM, Prof. Dr. Noordin Mohamed Mustapha,
the faculty of Veterinary Medicine, UPM and Dr. Loqman Bin Mohamed Yosuf, the
faculty of Veterinary medicine, UPM for their encouragement and generous help
during my PhD study period.
I would like to thank my senior friend Md. Waliul Chowdhury, a research associate
at Royal Veterinary College, London for his continuous inspiration during my study
period. I would like to extend my thanks to my friends and colleagues for their
support.
© COPYRIG
HT UPM
x
I gratefully acknowledge the Universiti Putra Malaysia for the financial support, as a
IGRF (International Graduate Research Fellowship) and GRA (Graduate Research
Assistant).
I express my thanks to the all staff of Faculty of Veterinary Medicine, UPM.I am
also grateful to the staff of physiology, pharmacology, parasitology, pathology,
serology, hematology, radiology laboratories and clinics. My special thanks also go
to the staff of the anatomy and histology department for their help.
Finally, thanks to all those who contributed to do this work and whose names have
not been explicitly stated here.
© COPYRIG
HT UPM
xi
I certify that a Thesis Examination Committee has met on 24 April to conduct the
final examination of Kh. Nurul Islam on his thesis entitled “Development of Cockle
Shell Based NanocompositeBiomaterial Bone Paste and Its Effectiveness for Bone
repairin Rabbit Model” in accordance with the Universities and University Colleges
Act 1971 and the Constitution of the Universiti Putra Malaysia [P.U.(A) 106] 15
March 1998. The Committee recommends that the student be awarded the Doctor of
Philosophy.
Members of the Thesis Examination Committee were as follows:
Professor Dr. Mohd Zamri b Saad
Chairman
Department of Veterinary Pathology and Microbiology
Faculty of Veterinary Medicine
Universiti Putra Malaysia
Professor Dr. Elias b Saimon
Internal Examiner
Department of Physics
Faculty of Science
Universiti Putra Malaysia
Prof. Dr.Mohamed Ali b Rajion
Internal Examiner
Department of Veterinary Pre-clinical Sciences
Faculty of Veterinary Medicine
Universiti Putra Malaysia
Assoc. Prof. Dr. Srihadi Agungpriyono
External Examiner
Department of Anatomy Physiology & Pharmacology
Faculty of Veterinary Medicine
Bogor Agricultural University
16680 Jl. Agatis Kampus lpbDramaga
Indonesia
NORITAH OMAR, PhD
Associate Professor and Deputy Dean
School of Graduate Studies
Universiti Putra Malaysia
Date: 26 June 2013
© COPYRIG
HT UPM
xii
This thesis was submitted to the Senate of Universiti Putra Malaysia and has been
accepted as fulfillment of the requirement for the degree of Doctor of Philosophy.
The members of the Supervisory Committee were as follows:
Md. Zuki Bin Abu Bakar @ Zakaria, PhD
Professor
Department of Veterinary Preclinical Sciences
Faculty of Veterinary Medicine
Universiti Putra Malaysia
(Chairman)
Mohd Zobir Bin Hussein, PhD
Professor
Department of Chemistry
Faculty of Science
Universiti Putra Malaysia
(Member)
Noordin Mohamed Mustapha, PhD
Professor
Department of Veterinary Pathology and Microbiology
Faculty of Veterinary Medicine
Universiti Putra Malaysia
(Member)
LoqmanMohamadYusof, PhD
Senior Lecturer
Department of Veterinary Clinical Studies
Faculty of Veterinary Medicine
Universiti Putra Malaysia
(Member)
BUJANG BIN KIM HUAT, PhD
Professor and Dean
School of Graduate studies
Universiti Putra Malaysia
Date:
© COPYRIG
HT UPM
xiii
DECLARATION
I hereby declare that this thesis is my original work except for quotations and
citations that have been duly acknowledged. I also declare that it has not been
previously and also not concurrently submitted for any other degree at Universiti
Putra Malaysia or other institutions.
KH. NURUL ISLAM
Date: 24 /04/ 2013
© COPYRIG
HT UPM
xiv
TABLE OF CONTENTS
Page
DEDICATION ii
ABSTRACT iii
ABSTRAK vi
ACKNOWLEGEMENTS ix
APPROVAL xi
DECLARATION xiii
LIST OF TABLES xvii
LIST OF FIGURES xviii
LIST OF ABBREVIATIONS xxiv
CHAPTER
1. GENERAL INTRODUCTION 1
1.1 Background 1
1.2 Problems Identification
1.3 Hypothesis
1.4 Objectives
7
7
7
2. LITERATURE REVIEW 8
2.1 Bones 8
2.2 Bone Biomaterials 9
2.3 Functions of Bones 10
2.3.1 Mechanical 10
2.3.2 Synthetic 10
2.3.3 Metabolic 11
2.4 Mechanical Properties 11
2.5 Macroscopic Structure of Bone 12
2.5.1 Compact Bone 12
2.5.2 Trabecular Bone 12
2.6 Cellular Structure 14
2.7 Bone Formation 15
2.7.1 Intramembranous and Endochondral Ossification 16
2.8 Molecular structure 20
2.8.1 Matrix 20
2.8.2 Inorganic 20
2.8.3 Organic 21
2.9 Significance of Bone 24
2.10 Clinical Need for Bone Regeneration 25
2.11 Biomaterials for Bone Treatment 26
2.11.1 Bioactive Inorganic Materials 27
2.11.2 Polymers 29
2.11.3 Composite Materials 29
2.12 The Chitosan 30
2.13 The Cockle (Anadaragranosa) Shells 32
2.14 The Calcium Carbonate 34
2.15 Dodecyl Dimethyl Betaine (BS-12) 38
© COPYRIG
HT UPM
xv
2.16 The Calcium Carbonate Nanoparticles 39
2.17 The Pastes 42
2.18 The In Vivo Studies 45
3. SYNTHESIS AND CHARACTERIZATION OF NANO
CALCIUM CARBONATE FROM COCKLE SHELLS
(ANADARAGRANOSA)
50
3.1 Introduction 50
3.2 Materials and Methods 53
3.2.1 Preparation of Cockle Shell Powders 53
3.2.2 Characterization of Cockle Shell Powders 54
3.2.3 Synthesis of Nano Calcium Carbonate 57
3.2.4 Charaterization of Nano Calcium Carbonate 58
3.3 Results 60
3.3.1 Characterization of Calcium Carbonate and its
Polymorphs
60
3.3.2 Synthesis of Nano Calcium Carbonate 65
3.4 Discussion 82
4. DEVELOPMENT AND CHARACTERISATION OF PASTES
FROM COCKLE SHELL-BASED NANO CALCIUM
CARBONATE
88
4.1 Introduction 88
4.2 Materials and Methods 92
4.2.1 Materials 92
4.2.2 The Method for the Development of Pastes 93
4.2.3 Characterization of the Developed Pastes 93
4.3 Results 98
4.3.1 The Surface Morphologies of the Developed Pastes 98
4.3.2 The Elemental Analysis of the Developed Pastes 104
4.3.3 The FT-IR Spectrum Observation of the Developed
Pastes
108
4.3.4 The XRD Patterns of the Developed Pastes 112
4.3.5 The TGA Curves of the Developed Pastes 116
4.3.6 Estimation of Calcium Ions Dissolution of the
Developed Pastes by Inductively Coupled Plasma-
Optical Emission Spectrometry
118
4.3.7 Determination of Physiological Stability of the
Developed Pastes in the Phosphate Buffer Saline
Medium (PBS)
122
4.4 Discussion 125
5. IN VIVO EVALUATION OF NANOCOMPOSITE
BIOMATERIAL BONE PASTE IN A RABBIT MODEL
135
5.1 Introduction 135
5.2 Materials and Methods 137
5.2.1 Animals 137
5.2.2 Experimental Design 138
5.2.3 Anesthetizing the Rabbits and Preparation for Surgery 139
5.2.4 The Pastes 139
© COPYRIG
HT UPM
xvi
5.2.5 Surgical Procedure and Implantation 140
5.2.6 Post-Operative Care and Follow Up Procedure 142
5.2.7 Post Implantation Examination 142
5.2.8 Statistical Data Analysis 146
5.3 Results 146
5.3.1 Radiolographic Examination 146
5.3.2 Quantification of the Radiographic Images 155
5.3.3 Biochemical Analysis 156
5.3.4 Gross Examination 162
5.3.5 Histological Examination 165
5.4 Discussion
177
6. GENERAL DISCUSSION, CONCLUSION AND SUGGESSIONS 183
REFERENCES 195
APPENDICES 212
BIODATA OF STUDENT 218
LIST OF PUBLICATIONS 219