The Synthesis Of Nanosized Hydroxyapatite Powders

THE SYNTHESIS OF NANOSIZED HYDROXYAPATITE POWDERS

by

TAN SU ANNE

Thesis submitted in fulfillment of the

requirements

^{for the}

degree

of Masters of Science

April

²⁰⁰⁷

ACKNOWLEDGEMENTS

I would like to express my

gratitude

^{to Assoc. Professor Dr.} Luay Bakir Hussain for

proposing

^me this Master's position ^and also for his continuous

supervision

^and support ^as my main advisor, and Assoc. Professor Dr.

Hj.

^Ahmad Fauzi Bin Mohd.

Noor for his advice and support ^as my co-advisor. I wish also to thank Professor Dr.

Radzali Bin Othman for

financing

my research

through

^{his IRPA}

grant.

The technical staff of the School of Material and Mineral Resources

Engineering

and their help,

especially

^Madam Fong Lee Lee for all the technical know-how she had taught ^{me, are} very much

appreciated.

^A

special

thanks also to Assoc. Professor Dr.

Azizan Aziz, Dr. Ahmad Sadri Bin Ismail, ^{Professor Panchanan Pramanik} of the Indian Institute of Technology

(liT) Kharagpur,

^{Dr. Sunara Purdawaria} of Institut

Teknologi Bandung

(ITS), ^{Mr. Ian} Staton from the

University

of Sheffield and also Ms. Teoh Wah Tzu for encouraging ^{me in} my research,

helping

^{me with}

practical

^{work and for}

valuable discussions and advises.

Not

forgotten

^{are my}

deeply

supportive parents ^and

family,

my

boyfriend,

^Yew

Kuan Min, and friends for their prayer and support

throughout

my research. Most

important ^of all, ^I give ^{thanks to} God, ^{who made}

everything possible

^and provided ^me

with wisdom and grace ^to complete my

journey.

TABLE OF CONTENTS

Page ACKNOWLEDGEMENTS

TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES LIST OF PLATES LIST OF SYMBOLS LIST OF ABBREVIATION LIST OF APPENDICES

LIST OF PUBLICATIONS & SEMINARS ABSTRAK

ABSTRACT

ii iii vii viii

x

xi xii xiii xii xiv xvi

CHAPTER ONE: THE SYNTHESIS OF NANOSIZED HYDROXYAPATITE POWDERS

1.0 Introduction 1

1.1 Application ^of

hydroxyapatite

in various fields 1 1.2 Advantages of nanosized

hydroxyapatite

^overconventional 2

hydroxyapatite

1.3 Economic and social impact ^{of local}

production

^of

hydroxyapatite

⁴

1.4 Main

objective

of research 5

1.5 Approach ⁵

1..6 Scope of the thesis 7

CHAPTER TWO : BACKGROUND AND LITERATURE SURVEY

2.1

Composition

^{of bone 8}

2.1.1

Organic

phase of bone tissue 8

(a) Collagen

⁹

(b) Non-collagenous

protein ⁹

2.1..2 Mineral phase ^{of bone 9}

2.2 Bone cells 2.2.1 2.2.1 2.2.1

Osteoblasts

Osteocytes

Osteoclasts

11 11 11 12 1.3 13 14 14 15 16 17 17 20 23 24 24 25 25 26 26 27 27 2.3 Biological implants

2.3.1

Body's

reaction to implants (a) Toxic response

(b) Nearly

^inert

(c)

^Bioactive

(d)

Dissolution 2.4

Hydroxyapatite

2.4.1

Hydroxyapatite

^{as an}

implant

^material

2.4.2 Dense

hydroxyapatite

2.5 Different methods of

producing hydroxyapatite

and their merits 2.5.1 Solid-state reaction

2.5.2

Mechanosynthesis

2.5.3 Wet methods

(a) Hydrothermal techniques (b) Hydrolysis ^method

(c) ^Wet

precipitation

Calcium

hydroxide

^and orthophosphoric ^acid

ii Calcium nitrate and diammonium hydrogen

phosphate

2.6 The chemistry ^of

hydroxyapatite

²⁸

2.6.1 The formation of

hydroxyapatite

^from phosphate ^{and calcium 29}

salts

2.6.2 Ca/P ratio of hydroxyapatite ³⁰

2.6.3 Kinetics of hydroxyapatite precipitation

2.6.4 Thermal behavior of

hydroxyapatite

2.6.5 PH and zeta

potential

^of

hydroxyapatite

^{in an aqueus}

32 33 34

(a)

(b)

XRF results XRD results

98 100 4.3.3 Effect of calcination temperature 106

CHAPTER ^FIVE: SUMMARY AND CONCLUSION 5.1 Conclusions

5.2 Recommendation for future work

107 109

BIBLIOGRAPHY 110

APPENDICES

2.1

3.1

4.1

4.2 4.3

4.4

4.5

LIST OF TABLES

Calcium phosphate ^{salts thatare}biologically ^and chemically relevant to

hydroxyapatite ^{and its}production.

Summaryof variable parameters ^ofsynthesis ^thatare investigated ^{in this}

research

Acomparison ^ofaging temperature ^versusaverage particle size obtained from the particle ^sizer.

Average particlesize measurement by ^the particle^sizer.

Surface area and equivalent sphere diameter measurement from the BET method

Results fromXRF^- the CaJP ratio forsamples co-precipitated ^at temperatures^{40,60 and 80°C}

Resultsfrom XRF^- the CaJP ratio forsamples co-precipitated ^at pH 9, 10 and 11.

Page

28

46

66

67 68

88

94

LIST OF FIGURES

Page

1'.1 Hierarchical levels of structural organization ^{in a human} long ^bone 10 2.1 Relationship between the timerequired ^forforming pure-phase 33

hydroxyapatite and the reaction temperature

2.2 Schematic representation ^ofzeta potential. 37

2.3 Plot of zeta potential ^versus pH showing the isoelectricpoint ^of 38 hydroxyapatite ^{and the} pH ^{valueswhere the}dispersion ^{would be} expected ^to

be stable.

3.1 Schematic representation ^{of the} setup ^oftheequipment ^{used in the}synthesis ⁴³

of hydroxyapatite powders

3.2 ^An x-ray ^is produced ^{when an atom is hit} by^a photon ^orx-ray 56 4.1 Graph^ofequivalent sphere ^{diameter, or}average particle ^{diameterversus 69}

reaction temperature ^forhydroxyapatite samples ^{reacted at} pHi^{O and} pH11.

4.2 Graph ^of equivalentsphere^{diameter, or}average particle^{diameter versus 70}

various reaction pH.

4.3 Graph ^of particle ^sizeversus aging duration forsamples aged ^{at room 74} temperature ^for24, 4B and 72 hours.

4..4 Particle size distribution forsample aged for 24 hours at room temperature ⁷⁶ 4.5 Particle size distribution forsampleaged for 4B hours at ^room temperature ⁷⁷

4.6 Particle size distribution forsample aged for 72 hours at room temperature. ⁷⁷

4.7 Graph^of Equivalent Sphere ^Diameterofhydroxyapatite particles ^versus 85 calcination temperature^forsamples ^{reacted at 40,60 and Booe.}

4.8 Graph^of Equivalent Sphere ^Diameterofhydroxyapatite particles ^{versus 86}

calcination temperature^for samples reacted underpH 9, ^{10and 11.}

4.9 Graph^ofcrystallite^{size versus} calcination temperature^for hydroxyapatite ⁹¹ synthesized ^attemperatures40, ^{60 and aooe.}

4.10 Graph ^of crystallite^{size versus} calcinationtemperature^forhydroxyapatite ⁹² synthesized ^at pH 9, ^{10 and 11.}

4.11 Graph of ealP ratioversus calcination temperature^forhydroxyapatite 94 calcined attemperatures 40,60 ^{and aooe.}

4.12 ^XRD spectra ^of hydroxyapatitesynthesized ^{at40°C and calcined atvarious} 95 temperatures: (a) ^uncalcined (b)^700°C (c)^aoooe (d) ^gOOoe (e)1^ooooe

(f)1100oe

4.13 ^XRD spectra ^ofhydroxyapatite synthesized ^atsooe and calcined atvarious 96 temperatures: (a) ^uncalcined (b) ^7000e (c) ^BOOoe (d) ^900De (e)1^ooo-c

(f)1100oe

4.14 ^XRD spectra^of hydroxyapatite synthesized ^{at aooe and}calcined at various 97 temperatures: (a) ^uncalcined (b) ^700°C (c) ^eco-c (d)^soo-c (e)10000e

(f)11000e

4.15 Graph^{ofCalP ratioversus}calcination temperature ^forhydroxyapatite^co- 99

precipitated^at pH9. ^{10 and 11.}

4.16 ^XRD spectra^of hydroxyapatitesynthesized ^atpH09 and calcined atvarious 103 temperatures: (a) ^uncalcined (b) ^700°C (c) ^800°C (d) ^900°C (e)1000°C

(f)1i00°C

4.17 ^XRD spectra ^ofhydroxyapatite synthesized ^at pHi^{O and calcined atvarious 104} temperatures: (a) ^uncalcined (b) ^700°C (c) ^800°C (d) ^900°C (e)i000°C

(f)1100°C

4.18 ^XRDspectra ^ofhydroxyapatite synthesized ^at pH^{11 and calcined atvarious} 105 temperatures: (a) ^uncalcined (b) ^700°C(c) ^800°C(d) ^900°C (e)1000°C

(f)ii00°C

LIST OF PLATES

Page

3.1 A photograph ^{of the} co-precipitation equipment set up within ^a fume 42

cupboard.

3.2 Maturation of

precipitation

^at temperature 90°C for 30 minutes

using

^{a 48} hotplate ^stirrer.

3.3

Aged precipitates

^{within the}

centrifuge

^{machine. 49}

4.1 FeSEM photograph ^ofa

hydroxyapatite

Sample ^A, aged for 24 hours 78 at room temperature, ^{viewed at} 50,000x magnification.

4.2 FeSEM

photograph

^{of a}

hydroxyapatite Sample

^A, viewed at 10000x 79

magnification.

4.3 FeSEM photograph ^ofa hydroxyapatite

Sample

B,

aged

for 48 hours 80 at room temperature, ^{viewed at 100000x}

magnification.

4.4 FeSEM

photograph

^{of a} hydroxyapatite Sample ^{B, viewed at 10000x 81}

magnification.

4.5 FeSEM photograph ^{of a}

hydroxyapatite Sample

^C,

aged

^{for 72 hours 82}

at room temperature, viewed at 100000x

magnification.

4.6 FeSEM

photograph

^ofa

hydroxyapatite sample

^{11RT72RT, viewed at 84}

50000x magnification.

4.7 Enlarged ^FeSEM photograph ^ofSample ^{C 84}

4.8 SEM

micrograph

^of

hydroxyapatite

^{reacted at 40°C,}

aged

^{at room 88} temperature ^{for 24 hours} and calcined at 1000 oe, viewed at 3k x

magnification.

4.9 SEM micrograph ^of

hydroxyapatite

^{reacted at40°C,} aged ^{at room 89} temperature for 24 hours and calcined at 1000 °C, ^{viewed at 3.5 k x}

magnification.

Tl

A V p d

m

Ssp

%wtc.

%wt,.

LIST OF SYMBOLS

Reaction temperature Aging temperature

Calcination temperature Surface area ofa particle Particle volume

Particle density

Diameter ofa

particle

Particle mass

Specific ^{surface area} per unit ^mass

Weight

percentage of Calcium atoms in

hydroxyapatite

Weight percentage ^of Phosphate ^{atoms in}

hydroxyapatite

Page

41 47 50 66 66·

66 66 66 73 73 73

DCPD DCPA OCP ACP TCP

HAp

TTCP

TCPM DAP XRF XRD SEM BET DLS ESD FeSEM

LIST OF ABBREVIATION

Dicalcium phosphate dehydrate, Ca(HP04hH20

Dicalcium Phosphate Anhydrous, Ca(HP04)

Octacalcium Phosphate, CaaH2(P04)s,5H20 Amorphous^calcium phosphate, Ca3(P04h^.xH20

Tricalcium Phosphate, Ca3(P04)2 Hydroxyapatite, Ca1O(PO",)s(OHh

Tetracalcium Phosphate, Ca",P20a

Tetracalcium Phosphate monoxide, Ca4P207

Calcium Deficient Hydroxyapatite X-Ray Fluorescence

X-Ray Diffraction

Scanning ^Electron Microscope Brunauer, Emmett, ^Teller Dynamic Light Scattering EquivalentSphere ^Diameter

Field Emission Scanning ^Electron Microscopes

LIST OF APPENDICES

Appendix

¹

Composition

^ofchemicals used in the wet

precipitation

^method

Appendix

^{2 Molecular}

weights

^{ofelements and} molecules used in the calculation of the Ca/P ratio from XRF results.

Appendix

^{3 The} distribution of

particle

^size:

Graphs

plotted by ^the

particle

^sizer

Appendix

⁴ Results from the BET method

Appendix ^S

Crystallite

size from the XRD

LIST OF PUBLICATIONS & SEMINARS

1 S A Tan, ^{M N Ahmad} Fauzi., ^{B H}

Luay,

^{O Radzali.} (2004)

Synthesis

^{of Nano­}

sized

Hydroxyapatite.

Medical Journal of

Malaysia

^59,

Supplement

B, pp162-

163.

SINTESIS SERBUK HIDROKSIAPATIT BERSAIZ NANO

ABSTRAK

Partikel

hidroksiapatit

disintesis melalui kaedah

mendapan

^kimia dan dicirikan.

Ujikaji

ⁱⁿⁱ

mengkaji bagaimana parameter

^utama

seperti pH tindakbalas,

^serta

suhu

tindakbalas,

penuaan dan

pengkalsinan mempengaruhi

^saiz

partikel

^dan

ketulenan fasa

hidroksiapatit

yang dihasilkan. Matlamat ^utama adalah untuk menentukan keadaan

paling optimum

^untuk

menghasilkan hidroksiapatit

yang bersaiz nanometer dan berfasa

tulen,

^serta

mengalami aglomerasi

^yang

minimum

untulkdigunakan sebagai

^bahan

pengganti tetulang.

Suatu larutan 0.2 M

Ca(N03)2.4H20 (Kalsium

^Nitrat

Tetrahidrat)

^{dan larutan}

0.12 M

(NH4)2HP04 (Diamonium Hidrogen Fosfat) dimendapkan

^secara

serentak dalam kehadiran

dispersan K4P207

^dibawah

pelbagai pH,

^dan

pengkacauan

berterusan. Ini diikuti oleh proses penuaan

pada

^{suhu dan}

tempoh

yang

berbeza,

^dan

kemudiannya dikeringkan. Akhirnya, pengkalsinan

dilakukan

pada

^suhu yang

meningkat,

^{bermula dari 700}

hingga

^1000°C.

Pencirian

dijalankan dengan pembelauan

^sinar-X

(XRD)

^serta

pendarflour-X (XRF)

^untuk

penentuan fasa,

^manakala

pengukuran

^saiz

partikel

^dilakukan

melalui

SEM,

^kaedah

BET,

^serta

pengukur

^saiz

partikel.

Keputusan menunjukkan

^{bahawa kedua-dua}

komposisi

^dan

morfalagi hidroksiapatit dipengaruhi

^{oleh suhu}

tindakbalas,

^suhu penuaan,

pH

tindakbalas

serta suhu

pengkalsinan.

^{Suhu penuaan dan} tindakbalas yang

tinggi

membolehkan

pembentukan hidraksiapatit

^yang

cepat, tetapi juga

menghasilkan partikel

yang lebih

besar,

^{manakala suhu} penuaan dan tindakbalas yang rendah memerlukan

tempoh

tindakbalas yang lebih lama

tetapi menghasilkan partikel

yang lebih kecil.

Didapati

^bahawa

pH

tindakbalas

mempunyai pengaruh

yang

paling

^kuat

terhadap komposisi hidroksiapatit

yang

terhasil, dengan pH tertinggi

yang

diuji, pH11,

^memberikan

hidroksiapatit

yang

paling

^tulen.

Penggunaan ejen pengurai

^{membantu dalam}

pembentukan populasi

^saiz

partikel

berbentuk dwimod yang

kebanyakannya

^terdiri

daripada partikel primer

^dan

peratusan

yang kecil

partikel

sekunder yang berdiameter ^1-

2�m.

Kehabluran

hidroksiapatit

^bertambah

dengan

^suhu

pengkaisinan

yang

meningkat daripada

⁷⁰⁰

hingga

¹¹

OO°C,

^dan

begitu juga dengan

^saiz

partikel,

dan suatu

peningkatan

yang amat ketara

bagi

^saiz

partikel diperhati

^{untuk suhu}

pengkalsinan

^lebih

daripada

^1000°C.

Saiz kristalit

berkurang

^semasa

pengkalsinan pada

^{suhu 700°C}

kepada

kira-kira 2

hingga 3nm, tetapi

bertambah semula

dengan

^suhu

pengkalsinan

yang semakin

meningkat

^dan

jatuh

^sedikit

pada

^{suhu 900 dan} 1000°C. Suhu tindakbalas yang lebih

tinggi (aO°C)

^akan

menghasilkan

kristalit yang

besar,

berbanding dengan

suhu yang lebih

rendah,

^manakala

pH

yang ^lebih

tinggi

(dalam

^kes

ini, 11)

^akan

menghasilkan

kristalit yang kecil.

THE SYNTHESIS OF NANOSIZED HYDROXYAPATITE POWDERS

ABSTRACT

Nanometer sized

hydroxyapatite particles

^are

synthesized

^{via wet chemical}

precipitation

^and characterized. This research studies how

key parameters

^such

as the

pH

^of

synthesis,

^{and also the}

reaction, aging

^and calcination

temperatures

^{affect the size and}

phase purity

^{of the}

hydroxyapatite produced.

The main aim is to determine the

optimum

condition for

producing phase-pure,

nano-sized

hydroxyapatite

with minimum

agglomeration,

^{to be used as bone}

replacement

^material.

A 0.2 M solution of

Ca(N03)2.4H20 (Calcium

^Nitrate

Tetrahydrate)

^{and a 0.12}

M solution of

(NH4)2HP04 (Diammonium Hydrogen Phosphate)

^{are co­}

precipitated

^{in the} presence of a

dispersant. �P207 (Tetrapotassium Pyrophosphate Tetrabasic),

^{under various}

temperatures, pH

and continuous

stirring.

^{This is followed}

by aging

^{at various}

temperatures

^and

durations,

^and

after that

drying,

^and

finally

calcination at various

temperatures ranging

^from

700 to 1000°C. Characterization was carried out

using X-ray powder

diffraction and

X-ray fluoroscopy

^for

phase

identification while

particle sizing

^{was done via}

Scanning

^Electron

Microscopy (SEM),

^{the BET}

method,

^{and also a}

particle

sizer.

Results showed that both the

composition

^{and the}

morphology

^{of the}

hydroxyapatite

^{are affected}

by

the reaction

temperature,

^the

aging temperature,

the

pH

^{of the} reaction and also the calcination

temperature. High aging

^and

reaction

temperatures

allowed for

rapid

formation of

hydroxyapatite,

^{but also led}

to

larger particles,

^{while lower}

aging

^{and reaction}

temperatures required

^a

longer

reaction time but smaller

particles

^{are obtained.}

Meanwhile,

^the

pH

^{of the}

reaction has the most intense effect upon the

composition

^{of the}

hydroxyapatite,

with the

highest pH,

11,

yielding

^{the most}

phase-pure hydroxyapatite powder.

The usage of ^a

dispersant

assisted in the

production

^{of a bimodal}

population

^of

particle

^size

consisting mainly

^{of nanosized}

primary particles

about 20 to 150nm in

diameter,

^{and a minor}

percentage

^of

larger secondary particles

^about

1-2JJm

in diameter.

The

crystallinity

^{of the}

hydroxyapatite

increased when the calcination

temperatures

increased from 700 to 11

ao°c,

^{as did the}

particle

sizes, ^{and a}

sharp

increase of

particle

^{sizes from over 200nm to above} 1 JJm ^was observed at calcination

temperatures

^above 1000°C. The

crystallite

^sizes

dropped

upon calcination at 700° to about 2 to 3 ^nmI but increased with

increasing

calcination

temperature

^before

dipping slightly again

^{at 900 to 1000°C.}

Higher

^reaction

temperatures (80°C) produced larger crystallites,

^{while a}

high pH (in

^{this case}

11),

^{led tothe formation of}the smallest

crystallites.

CHAPTER 1

THE SYNTESIS OF NANOSIZED HYDROXYAPATITE POWDERS

1.0 Introduction

The

importance

^{of calcium}

hydroxyapatite

^{as a} biomaterial has

long

been realized and studied worldwide in the field of

orthopedics

^and

orthodontics due to its excellent

biocompatibility

^and

bioactivity.

^{With the}

chemical formula

Ca10 (P04)6(OH)2, hydroxyapatite

^is

chemically

^{similar to}

the mineral

component

of bones and hard tissues in

mammals,

^{and is a}

very attractive material to be used as a bone

replacement

^material.

Hydroxyapatite

^{is also one} of few materials that are classed as

bioactive. When used as a

biological implant,

^{a bioactive material}

encourages ^a

direct,

interfacial bond between the material and the tissue

surrounding it, lending stability

^{to the}

implant

^and

improving

^clinical

success.

Thus,

^it is the best candidate for hard tissue

replacement

ⁱⁿ

terms of

compatibility (Muster, 1992,

^{Hench and}

Wilson, 1993).

1.1

Application

^of

hydroxyapatite

^{in various fields}

Synthetic apatites

^are

generally

used in bone

repair

^I

augmentation

and

substitution,

^{as well as}

coatings

^{on dental and}

orthopedic implants.

Among

^the innumerable

applications

^of

hydroxyapatite

^{are the}

reconstruction and

replacement

^of

damaged

^{bone or tooth zones in}

plastic

and dental surgery. Some

examples

^{are the}

augmentation

^{of alveolar}

ridge

for better denture fit in

orthopedic

surgery,

filling

ⁱⁿ

bony

^{defects in}

dental and

orthopedic

surgery, ^{such as} the

filling

^of

periodontal

^defects

with ceramic

powder

^of

hydroxyapatite

^{and as} extenders for

autogenous

bone ^or demineralized bone matrix

(Muster, 1992).

Metals coated with

hydroxyapatite

^{have been introduced as artificial}

bones. The

coating helps

^the

surrounding

^{tissue to bond}

firmly

^{with the}

implant

^while

retaining

^the

strength

of the metal

(Oonishi, 1991).

^Other

medical

applications

include the usage of

nanocrystalline hydroxyapatite

as a carrier material for local

delivery

of antibiotics and time-controlled

drug

^release in bone infections

(Rauschmann

^et

al., 2005,

^{Komlev et}

al., 2002).

Hydroxyapatite

^{also finds}

applications

^{in other} fields of industrial or

technological

^{interest as}

packing

^{media for column}

chromatography, catalysts,

gas ^sensors, host material for lasers and may also find

possible

uses in water

purification,

^fertilizer

production

^{and also as a dielectric}

coating.

^HA

specimens

^{sintered to}

transparency

may used ^as matched filters

(Hoepfner

^and

Case, 2003).

makes it easier to prepare materials with ^a uniform and dense structure, therefore

greatly increasing

^the

strength

of the sintered

body.

^As

coatings,

the

greater

^structural

integrity

^of

closely packed nanoparticles gives

^the

coatings improved

^adhesive and cohesive

properties

for many

applications.

Most

importantly, biological implants

^{made from nanosized}

hydroxyapatite

have been proven ^to exhibit enhanced osteoblast function

(better

^cell

adhesion,

accelerated

growth

^{and tissue}

deposition)

^{within the}

body (Webster

^et

al., 1999,

Balasundaram et

aL, 2006,

^{Webster et}

al., 2000).

Meanwhile,

^nanosized

hydroxyapatite

^{has found its own}

application

niche as carriers of antibiotics to reduce bacterial infections in the bone­

implant

interface, ^{as well as vectors} for selective

delivery

^of

drugs

^to

tarqeted

cells in the treatment of cancer for better effectiveness

compared

to

conventionally

administered

drugs.

In non-medical

applications,

^nanosized

particles

^{contribute to}

higher sensitivity

^{as sensors and a much}

higher reactivity

^as

catalysts.

Nanoparticles

have also been known to exhibit

properties

^far different from those of the same material but with

larger particles

^or

grain

^{sizes. This}

opens the door to unlimited

potential

^{for new}

applications yet

^{to be}

discovered.

Despite

^the

high potential,

very few

applications

^{have been}

developed

^{so far}

using nanoparticles.

^The

key

^{limitation is} the lack of

large

quantities

of nanosized

particles

^with

closely

^controlled

properties

^{at a cost}

that will allow scientists and

engineers

^to

comfortably explore

^new

practical applications

^{of these}

particles

^{as well the science of}

processing

these new materials. Therefore research should be focused on

developing

the science and

engineering

^{that would allow the}

development

^of

inexpensive technologies

for manufacture of

nanoparticles

^{that can be}

densified into

nanocompacts.

1.3 Economic and Social

impact

^{of local}

production

^of

h

yd

roxyapatite

Having glanced

^{at the vast}

potential

^{for this}

material,

^{we now take a}

look at the

impact

^{and aim of this} research, ^{which is}

primarily

^to

produce

nanosized

hydroxyapatite powders

^{which are} intended for

processing

^into

dense sintered material for

orthopedic implants.

In a

published

market survey

by

^Medtech

Insight,

the market for

orthopedic

biomaterials sales in the USA was found to exceed US$980 million in 2001. The number was

predicted

^{to increase to US$1.16 billion}

in 2002, ^and $3.32 ^billion

by

²⁰⁰⁶

(AZoM.com, 2002). Meanwhile, Malaysia

^is

reported

^{to have}

imported orthopedic (medical)

^and

orthodontic

(dental) implants amounting

^to

approximately US$6

^{million in}

further

fuelling

^research

activity

among the

engineering

^{and medical}

communities in our nation.

Consequently,

^more

people suffering

^{from bone}

loss due to

injuries

^{or diseases would have access to}

high quality orthopedic implants

^{at a low} cost,

improving

^the

quality

of lives for these

people.

1.4 Main

objective

of research

The main

objective

^of this research is to

study

^the effect of several parameters ^of

synthesis,

^{such as the reaction}

temperature, aging

temperature ^and

duration,

^{as well as the} calcination

temperature,

upon ^the

phase-purity

^and

particle

^{size ofthe}

hydroxyapatite produced.

The results will be used to select the most suitable

parameters

towards

producing

^a

phase-pure, nanoparticulate hydroxyapatite.

1.5

Approach

The method used in this

experiment

^{is wet}

precipitation

^{due to the}

low

production

^cost

involved, easily

^available

equipment

^{and also}

comparatively easily adjustable parameters.

^The

key

^to

obtaining

^nano

powders

^with this method is the

manipulation

of the reaction kinetics so as to encourage

particle

^{nucleation over}

particle growth.

The ^raw materials used are calcium nitrate

tetrahydrate [Ca(N03)2.4H20]

^{and diammonium}

hydrogen phosphate [(NH4)2HP04].

The calcium solution and

phosphate

^{solution are}

co-precipitated

^under

constant and

vigorous stirring (level

^{4 on}

magnetic stirrer)

^{at various}

temperatures

and various

pH,

^and then allowed to mature under various

temperatures

^{and durations.}

Finally,

^{it is}

washed, dried,

^and

ground loosely

before calcination under

temperatures ranging

^{from 700 to 11OO°C.}

The

powders

^obtained

using

this method are characterized

using

^x­

ray diffraction for

phase identification,

determination of

phase purity

^and

also crystallite size;

^x-ray

fluoroscopy

for chemical

analysis

^{to determine}

the elemental content of the

powder

^{and the} ratio of calcium to

phosphate

of the

powder; scanning

^electron

microscope

^for

particle

^{size and}

morphology; particle

^{sizer to measure the} distribution of

particle sizes,

^and

finally

^{the BET to measure surface area and the}

equivalent sphere

diameter.

A

comparison

^{is then made to} compare the effect of the various

changes

ⁱⁿ

temperature

upon the characteristics of the

powder

^{obtained in}

order to determine the best

parameters

^{to be used to}

synthesize

^the

hydroxyapatite powder.

1.6

Scope

of the thesis

Chapter

^{two contains}

background

information on the

composition

^of

bone and the role

played by hydroxyapatite

^{in its structure. Also discussed}

here, ^{is the}

potential

^of

hydroxyapatite

^{as an}

implant material,

^its

characteristics,

^{various methods of}

synthesis

^of

hydroxyapatite

^{and also}

previous

^{works other} researchers have done on this same material.

Chapter

^{three describes the}

co-precipitation

process,

including

^the

equipment

^{and material}

preparation.

^The

synthesis parameters

^{to be}

investigated

and the characterization of the

synthetic hydroxyapatite

^are

also described here. In

Chapter

Four, ^the results obtained from the tests

are

presented

and correlated with the variable

parameters

^{that were tested.}

Finally,

ⁱⁿ

Chapter Five,

^{a conclusion is drawn about the}

synthesis

^and

properties

^{and an outline is drawn on the}

optimum parameters

^{to be} chosen for future

synthesis

^of

hydroxyapatite powders

^{with the}

targeted

properties.

CHAPTER2

BACKGROUND AND LITERATURE SURVEY

2.1

Composition

^{of bone}

The skeletal

system

^is

comprised

of "bone

organs"

^{- individual}

bones, ^each

having

^a distinctive size and

shape. Meanwhile,

^{each bone}

organ is made up of bone

tissue,

^{which is our main interest} here, ^{and also}

cartilage tissue,

^otherconnective tissue and ^nerves.

Bone tissue is a

composite

^{made of 60 to 70%} mineral substances and 30 to 40%

organic

^{tissues. This ratio holds true}

although

^the

properties

^{of a bone} vary from

point

^to

point,

^{and the ratio of the}

substances in the skeleton differ from one bone organ ^to another. The

major

^{role of bone is to serve as a}

support

^{for the}

body's

^muscles,

protection

^forsome

parts

^ofthe

body,

^{and also a reserve of calcium for the}

body (Krajewski

^and

Ravaglioli, 1991).

2.1.1

Organic phase

^of bone tissue

The

organic phase

of bone tissue consists of

mainly collagen

^and

non-collagenous protein,

^{as well as small amounts of}

polysaccharides

^and

(a) Collagen

Over 90% of the

composition

^of

organic

bone tissue is made up of

collagen. Collagen,

^{which can} be considered as the matrix of

bone,

^{is in}

the form of small micro-fibers. The

deposition

^of

collagen

^as osteoid is the first

step

in the process of bone formation.

(b) Non-Collagenous protein.

There are two

major non-collagenous proteins

^{found in bone,}

namely

osteocalcin and osteonectin.

They

^{are believed to be}

synthesized by specific bone-forming

cells called osteoblasts. Osteocalcin has a

strong affinity

^for

hydroxyapatite

^and may constitute up ^to 2% of vertebrae bone

protein. Osteonectin,

^{on the other}

hand,

^{has a}

strong affinity

^{for both}

hydroxyapatite

^and

collagen.

^Other

non-collagenous

^bone

proteins present

in small amounts include

osteopontin,

^some

proteoglycans,

^bone

morphogenetic protein

and bone-derived

growth

^factors.

2.1.2 Mineral Phase of Bone

The mineral

phase

of bone tissue contains calcium

phosphate,

^of

which the

major

^{form is}

hydroxyapatite,

and the less

abundant,

^calcium

carbonate. Small amounts of

magnesium, fluoride, carbonate, citrate, potassium

and other ions are also found in the mineral

phase

^{of bone.}

Hydroxyapatite

^{is a}

crystalline

^{substance which}

comprises calcium,

phosphate

^and

hydroxyl

^{ions. It}

provides

^{stiffness to the bone}

by

way of

the

incorporation

^of

hydroxyapatite crystals,

ⁱⁿ the form of needles and

plates,

^{into the} osteoid matrix

template.

Figure

1.1 shows the structure and location of the

apatite

^mineral

crystals

within the human

long

bone. These

crystals

^are

deposited parallel

to the

collagen

^{fibers so that the}

larger

dimensions of the

crystals

^are

along

^the

long

^axis of the fiber and reinforce the

collagen

similar to the way

glass

^{fibers reinforce}

plastic

^{matrices in}

fiberglass composites (Suchanek

^and

Yoshimura,

1998, ^{Woods and} Ellis,

1994).

Nutrient artery- In

tramedulla;.;;,;ry::--...,;:

cavity

Articular

cartilage

rm �-- �

/ t���

Osteon

._

_

if

^{Collagen nbcrs}

".

/�� \

1.:__{, Concentric}

:;_"\"")'"')..,.e�)

^....

�-:

lamella ^'

Haversian (3-⁷ Ilm) Apatite

canal mineralcrystals

(20-40 ^nm long) Micro- ^toUltra-

Ultra- tomolecular Line of

Macro-

Figure

^1.1: Hierarchical levels of structural

organization

^{in a human}

long

bone

(Park, 2000).

2.2 Bone cells

There are three distinct cell

populations directly

involved with bone tissue and are derived from the bone marrow

cavity.

These bone cells are

osteoblasts,

osteocytes

^and osteoclasts ^- each has its own role to

play

ⁱⁿ

the process of

formation,

maintenance and removal of bone mineral.

2.2.1 Osteoblasts

Osteoblasts ^are

specialized bone-forming cells, belonging

^{to the}

more

general category

^of

fibroblasts,

^{which are cells}

typical

^{of connective}

tissue of any organ

(Krajewski

^and

Ravaglioli, 1991). During

^their

developmental

^{and maturation}

stages, they actively synthesize

^many

proteins

^{and secrete the matrix} framework of

bone,

^{and thus create the} conditions ^for mineralization. The matrix is

initially

^{laid down as}

unmineralized

type

^I

collagen,

the osteoid. Osteoblasts are rich in alkaline

phosphatase

^and

osteocalcin,

^{both of which} may have ^a role in the mineralization of the osteoid matrix.

2.2.2

Osteocytes

Osteocytes

^{are found within the bone itself.}

They

^{are, in}

fact,

mature bone cells derived from osteoblasts. As the

collagen

^framework

becomes

mineralized,

^some osteoblasts become

trapped

^{in the}

newly­

formed osteoid matrix. These

remaining cells,

^{now known as}

osteocytes,

reside in the bone matrix ^as

long

^as the bone is functional. Juvenile

osteocytes

^{or those}

trapped

^{in the} osteoid for

only

^{a short}

time,

^{are able to}

participate

^{in bone}

deposition

^and

probably

^{some calcium}

deposition.

Older

osteocytes

^{do not have this}

ability,

and function

mainly

^to

provide

the bone tissue around them with a

supply

of nutrients from the marrow

cavity through communicating

^{channels known as} canaliculi. When

osteocytes

^{are cut} off from blood

supply, they

^die

(Woods

^and

Ellis, 1994, Krajewski

^and

Ravaglioli, 1991).

2.2.3 Osteoclasts

Lastly,

mineralized bone tissue is

degraded by specialized giant

cells known as osteoclasts. These multinucleated cells

playa

role in the

resorption

^{of bone mineral}

by dissolving

^the mineralized bone-matrix

extracellularly

^and

releasing

the calcium ions into circulation. It is

thought

that

TGF�

^{and bone}

morphogenic protein

^{are also} released this

time,

promoting

^{bone formation}

(Woods

^and

Ellis, 1994, Krajewski

^and

Ravaglioli, 1991).

2.3

Biologicallmplants

Implants

^are

basically

^{devices made} from inanimate materials that

are

placed

^{in the}

body; they

may be made from one or more biomaterial which is

intentionally placed

^{within the}

body,

^and

designed

^to remain there for a substantial

period

^{of time.}

Meanwhile,

^a biomaterial is an

object

^able

to

replace

^an

original living part

^{of the}

body.

Although implants

^{may be} utilized for medical reasons such as the

replacement

of diseased ^or

degenerated

^{tissues to}

improve

^the

quality

^of

life,

they

^{are also}

employed

^{for aesthetic} purposes such as breast

augmentation

^{and other forms of}

plastic

surgery.

Important

ⁱⁿ

surgical implants,

however, ^{is the host's} response to the biomaterial

given by surrounding

^tissue

(Krajewski

^and

Ravaglioli, 1991).

2.3.1

Body's

^{reaction to}

implants

When a

foreign

material is

implanted

^{into the}

body,

^{the host tissue}

responds

^{in a number of} ways ^at the

tissue-implant

^{interface. The}

response

given

is influenced

by

many

factors, induding

tissue conditions such ^as age,

health,

^blood

condition,

^{as well as}

implant properties

^{such as}

composition, present phases,

^surface

morphology

^and the mechanical fit of the

implant.

These responses ^can be

categorized

^{into four}

general

groups ^of

implant-tissue

response, that is:

toxic,

^almost

inert,

bioactive and dissolution.

(a)

Toxic response

Some

implant

materials may ^{cause a toxic} response that kills ^cells

In the

surrounding

^{tissues or} release chemicals that can

migrate

^within

tissue fluids and cause

damage

^{to the}

patient.

For obvious _reasons,

implants

^that elicit such a response ^{from the}

body's

^{tissues are avoided}

(Hench

^and

Wilson, 1993).

(b) Nearly

^inert

The most common response ^{of tissues to an}

implant

is formation of

a non-adherent fibrous

capsule

^{around the}

implant

^{as a}

protective rnechanism

^to isolate the

implant

from the host.

The thickness of the fibrous

layer depends

^on the various tissue and

implant

conditions mentioned earlier.

Biologically inactive, nearly

^inert

ceramics like alumina or zirconia will elicit ^a thin fibrous

capsule

^{at their} interface. ^{while metals and most}

polymers

^{may cause a total}

encapsulation

^{of the}

implant

within the fibrous

layer.

The motion and fit between the

implant

^{and host is also} very

important.

^{Even a}

slight

movement at the interface between

implant

^and

implant loosening

very

quickly

^and

consequent

clinical failure of the said

implant

^{in the form of}

implant

^or host fracture.

Thus, ^{for an}

implant

^{made of inert}

materials,

^a

tight

mechanical fit with the host tissue is of utmost

importance

^{in order to}

prevent

interfacial

movement and

subsequent

^{clinical failure}

(Hench

^and

Wilson, 1993).

(c)

^Bioactive

A bioactive material is able to form a direct bond across the interface between

implant

and tissue. This bond is similar to the

type

^of

interface that is formed when natural tissues

repair themselves,

^{and the}

same bond now

prevents

interface motion between the

implant

^{and the}

host tissue.

In order for an

implant

^to

perform optimally,

^its

properties,

^{such as a}

controlled rate of chemical

reactivity, morphology

^and

phase composition

need to be

carefully engineered according

^{to its function and rate of}

bonding

^{to the} host tissue. Even a small

change

ⁱⁿ

composition

^can

change

^the

properties

^{of a} bioceramic from

nearly

^{inert to} resorbable to bioactive. Unlike resorbable

materials,

chemical reactions

only

^{occur atthe}

surface while the rest of the

implant

^remains

largely

^unaffected

(Hench

and

Wilson, 1993).

(d)

Dissolution

Resorbable materials

gradually degrade

^as

they

^{dissolve or resorb}

and are

replaced by

^the

surrounding

host tissues. Instead of

replacing

^the

tissues,

the material encourages the

regeneration

^{of tissues to take their}

place.

^This

requires

^{a much}

higher

^{rate of}

change

^{at the material's}

interface

compared

^{to a} bioactive material.

There _are,

however,

^{a few issues or} criteria that need to be taken into consideration in order to

successfully implement

^resorbable

implants.

Firstly,

the constituents of the material must be

metabolically acceptable, meaning

^{that the} material needs to be of a

composition

^{that can be broken}

down

chemically by body

^{fluids or}

digested easily by macrophages.

^The

degradation products

^{must be} non-toxic chemical

compounds

^{that can be}

easily disposed

^{of without}

damaging

^the

surrounding

^{cells or}

harming

^the

host's health.

Besides

that.

^the

resorption

^{rate of the material must also match the}

repair

^{rate of the}

body

^{tissues even as} the material

provides

^{a sufficient}

mechanical

strength

^to

support

^the host tissue while the

regeneration

^of

tissues takes

place (Hench

^and

Wilson, 1993).

2.4

Hydroxyapatite

2.4.1

Hydroxyapatite

^{as an}

implant

^material

Hydroxyapatite

^{is a}

representative

^{member of a}

large family

^of

compounds

^known

generally

^as

apatites.

^In

spite

^{of a}

large variety

^of

compositions, apatites crystallize

^{in the}

hexagonal system,

^and

particularly

in the monoclinic

system (P63/m

space

group) (Brown

^and

Constantz, 1994, Muster, 1992).

Chemical

analyses

^have shown that calcium and

phosphate

^{are the}

principal

constituents of

enamel,

^{dentin and bone while the}

X-ray

diffraction

patterns

^{of the} latter three are similar to those of mineral

apatites.

^{The conclusion}

gained

^{from these tests was that the}

inorganic phases

^{of bone and teeth are}

basically

^calcium

hydroxyapatite,

^{of which}

the ideal formula is

given

^as

Ca10(P04)6(OHh (Brown

^and

Constantz, 1994).

Its

similarity

^{to the}

inorganic phases

^of bone and teeth makes

hydroxyapatite

^a very attractive candidate for ^an

implant

material. More than that

however, hydroxyapatite

^{is also}

bioactive, meaning

^{it is neither}

toxic nor

resorbable,

^{and is}

capable

^of

bonding directly

^{with the}

bony

tissue in the

body. Upon implantation, hydroxyapatite

^{remains at the site}

while

positively influencing

^{bone formation and thus}

speeding

recovery.

(Muster, 1992). Having

^{such useful}

properties,

^{it is no} wonder that

hydroxyapatite

^{has been used in many}

applications

^{and in} many forms such as porous

blocks,

^dense

blocks, powder,

^{foam and}

coatings.

Hydroxyapatite

^{is often used as}