UNIVERSITI PUTRA MALAYSIA DEVELOPMENT OF COCKLE …psasir.upm.edu.my/id/eprint/41860/1/FPV 2013 4R.pdfkalsium karbonat bersaiz mikron dan kalsium karbonat komersial. Dua belas arnab

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

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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

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DEVELOPMENT OF COCKLE SHELL-BASED NANOCOMPOSITE

BIOMATERIAL BONE PASTE AND ITS EFFECTIVENESS FOR BONE


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

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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

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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

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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


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

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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

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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.

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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

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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.

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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

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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.

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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.

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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


Prof. Dr.Mohamed Ali b Rajion

Internal Examiner

Department of Veterinary Pre-clinical Sciences



Assoc. Prof. Dr. Srihadi Agungpriyono

External Examiner

Department of Anatomy Physiology & Pharmacology


Bogor Agricultural University

16680 Jl. Agatis Kampus lpbDramaga

Indonesia

NORITAH OMAR, PhD

Associate Professor and Deputy Dean

School of Graduate Studies


Date: 26 June 2013

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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



(Chairman)

Mohd Zobir Bin Hussein, PhD

Professor

Department of Chemistry

Faculty of Science


(Member)

Noordin Mohamed Mustapha, PhD

Professor

Department of Veterinary Pathology and Microbiology



(Member)

LoqmanMohamadYusof, PhD

Senior Lecturer

Department of Veterinary Clinical Studies



(Member)

BUJANG BIN KIM HUAT, PhD

Professor and Dean

School of Graduate studies


Date:

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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

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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

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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


4.2.1 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


5.2.1 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

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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

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