UNIVERSITI PUTRA MALAYSIA

HIGH INTERNAL PHASE EMULSION AS A REACTION MEDIUM FOR FABRICATION OF BRUSHITE CRYSTAL

LIM HONG NGEE FS 2009 44

HIGH INTERNAL PHASE EMULSION AS A REACTION MEDIUM FOR

FABRICATION OF BRUSHITE CRYSTAL

By

LIM HONG NGEE

Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia, in

Fulfilment of the Requirements for the Degree of Doctor of Philosophy

December 2009

To my husband for braving the many ups and downs with me during the trying times,

steadily and stoically. You are indeed my pillar of strength.

To Ma and Pa for your unceasing love, support and faith in me.

iii

Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfilment of

the requirement for the degree of Doctor of Philosophy

HIGH INTERNAL PHASE EMULSION AS A REACTION MEDIUM FOR

FABRICATION OF BRUSHITE CRYSTAL

By

LIM HONG NGEE

December 2009

Chair: Anuar Kassim, PhD

Faculty: Science

This present work was aimed at fabrication of porous brushite crystals using oil-in-water

high internal phase emulsion stabilized by synthesized palm-based nonionic surfactant

as a reaction medium. This research work was divided into four categories. The first part

of the work involved synthesizing palm-based nonionic surfactants. Palm oil derivatives,

lauryl, palmityl and stearyl alcohols as renewable resources, were ethoxylated with an

average of three, six (or eight or nine) and 100 moles of ethylene oxide. The critical

micelle concentration of the synthesized surfactants was found to decrease with

increasing ethylene oxide head groups due to intertwist amongst the head groups. This

phenomenon enhances surfactant-surfactant interaction rather than surfactant-solvent

interaction which increases the rate of micellization as proven by the Gibbs energy. The

increase in the surfactant tail length had minimal effect on micellization. The second

part of the work was to stabilize the high internal phase emulsion using the synthesized

surfactants. The oil phase was vegetable oil, namely olive and olein oils. These

iv

emulsions, with dispersed phase of more than 75 wt%, were easily prepared by one-pot

homogenization. Due to the high oil volume fraction, the oil droplets were no longer

spherical but were squeezed to take the shape of polyhedral. Light scattering results

showed that the droplet size increased with increasing ethylene oxide chain length. The

rheology of the emulsions was governed by droplet size and oil volume fraction. The

emulsions exhibited high stability as indicated by the rheological measurements even

after storage at 40oC for three months. The third part of the work was on the fabrication

of brushite crystals with high degree of porosity using the high internal phase emulsion

as a reaction medium. The porosity of the crystals was manifested by precursor

concentration, surfactant concentration, oil volume fraction, mixing method, mixing

time, aging temperature, precursor type, mode of recovery and surfactant head group.

Pore size of the brushite crystals was less than 5 µm. The mechanism for the formation

of porous brushite crystals was postulated schematically based on the small angle x-ray

scattering analysis. The fourth and final part of this work was related to the application

of the porous brushite crystals as drug delivery devices. Prior to the controlled release

study, the crystals were subjected to cytotoxicity test to ensure their compatibility with

synoviocytes, which are cells that line the knee joints of rabbits. The crystals were found

to enable cell growth for up to five days. Sodium ampicillin, a wide spectrum antibiotic,

was successfully loaded into the pores of the crystals and subsequently released in vitro

for 14 days. This work underlines the simplicity of using highly stable high internal

phase emulsion as a reaction medium for the fabrication of porous brushite crystals, in

which when loaded with drug, exhibited potential as localized bone treatment

demonstrated by the promising controlled release rate.

v

Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai

memenuhi keperluan untuk ijazah Doktor Falsafah

EMULSI BERKEPEKATAN TINGGI SEBAGAI MEDIA TINDAK BALAS

UNTUK PEMBENTUKAN HABLUR BRUSHITE

Oleh

LIM HONG NGEE

Disember 2009

Pengerusi: Anuar bin Kassim, PhD

Fakulti: Sains

Kajian ini bertujuan untuk menghasilkan hablur brushite berliang menggunakan emulsi

berkepekatan tinggi minyak-dalam-air yang distabilkan oleh surfaktan nonionik

berasaskan sawit sebagai media tindak balas. Kajian ini dipecahkan kepada empat

bahagian. Bahagian pertama kajian melibatkan sintesis surfaktan nonionik berasaskan

sawit. Terbitan minyak sawit, iaitu alkohol lauril, palmitil and stearil sebagai sumber

guna semula, telah dietoksilasikan dengan purata tiga, enam (atau lapan atau sembilan)

dan 100 mol etilena oksida. Kepekatan misel kritikal surfaktan yang disintesiskan

menurun dengan peningkatan kumpulan kepala etilena oksida disebabkan pembelitan

antara kumpulan-kumpulan tersebut. Fenomena ini menambahkan interaksi surfaktan-

surfaktan berbanding dengan interaksi surfaktan-pelarut yang akan meningkatkan kadar

permiselan seperti yang dibuktikan oleh tenaga Gibbs. Peningkatan dalam panjang ekor

surfaktan memberikan kesan yang sedikit terhadap permiselan. Bahagian kedua kajian

merangkumi penstabilan emulsi berkepekatan tinggi menggunakan surfaktan yang

disintesiskan. Fasa minyak ialah minyak sayuran, khasnya minyak-minyak zaitun dan

vi

olein. Emulsi ini, dengan fasa tersebar melebihi 75 wt%, dapat disediakan dengan

mudah melalui penghomogenan satu-kali. Akibat daripada pecahan isipadu minyak yang

tinggi, titisan-titasan minyak tidak lagi berada dalam keadaan sfera tetapi dihimpitkan

kepada bentuk polihedral. Hasil penyerakan cahaya menunjukkan saiz titisan meningkat

dengan penambahan rantai panjang etilene oksida. Reologi emulsi dikawal oleh saiz

titisan dan pecahan isipadu minyak. Emulsi menonjolkan kestabilan yang tinggi

berdasarkan pengukuran reologi walaupun selepas penyimpanan pada 40oC selama tiga

bulan. Bahagian ketiga kajian ini adalah berkaitan dengan penghasilan hablur brushite

menggunakan emulsi berkepekatan tinggi sebagai media tindak balas. Keporosan hablur

dipengaruhi kepekatan bahan pemula, kepekatan surfaktan, pecahan isipadu minyak,

cara pencampuran, masa pencampuran, jenis bahan pemula, cara perolehan dan

kumpulan kepala surfaktan. Saiz liang hablur brushite adalah kurang daripada 5 µm.

Mekanisma pembentukan hablur brushite berliang dijangka secara skematik berdasarkan

analisis penyerakan sinar-X bersudut kecil. Bahagian keempat dan terakhir kajian ini

adalah berhubungan dengan penggunaan hablur brushite berliang sebagai alat

penghantaran ubat. Sebelum kajian kawalan perlepasan, ujian ketoksikan dijalankan

terhadap hablur tersebut untuk memastikan keserasiannya dengan sinoviosit, iaitu sel

yang melapik sendi lutut arnab. Hablur itu didapati menggalakan pertumbuhan sel

selama lima hari. Natrium ampisilin, antibiotik dengan spektrum yang luas, berjaya

dimasukkan ke dalam liang hablur dan seterusnya, dilepaskan in vitro selama 14 hari.

Kajian ini menyerlahkan kemudahan menggunakan emulsi berkepekatan tinggi sebagai

media tindak balas untuk pembentukan hablur brushite berliang, apabila dimasukkan

ubat, memaparkan potensi sebagai perubatan tulang setempat seperti yang ditunjukkan

oleh kadar kawalan perlepasan yang memuaskan.

vii

ACKNOWLEDGEMENTS

Sheer happiness. No words can better describe my feelings. My heart glows with pride

at the completion of my PhD thesis. Of course, my dream of getting this much sought

after degree will not be realized without the help and support of many people along the

arduous and winding but pleasant road.

I would like to thank MOSTI for granting the eSciencefund grant for this research work.

Also, I am very grateful to UPM for the award of GRF.

My gratitude and many thanks to my supervisor, Prof. Anuar for having the full

confidence in my research work. I truly appreciate your trust, support, guidance and

understanding throughout my research. Your graceful ways in handling matters make it

a breeze working with you.

To Prof. Ambar, thank you for guiding and advising me whenever I need help. I really

appreciate your generosity in allowing me to use your laboratory as and when I need it.

Work aside, your doses of jokes really help me to relief stress and relax my frowning

facial muscles.

viii

I would also like to thank Dr. Yeong for her generosity in permitting the use of

instruments at AOTD. Thanks also to Prof. Dzulkefly and Dr. Halim for their kind

advice. I would also like to thank Prof. Shahidan for his generosity in allowing me to

make full use of his laboratory and his valuable advice. I am also indebted to Dr.

Syahida and Prof. Fauziah for permitting the use of their laboratories.

To my counterparts at UKM, thank you very much for accepting my presence though “I

am from UPM”. And especially to Ina, I am most grateful for your precious time in

guiding and helping me. Also to my friends at UPM, thank you for always being there to

lend me a hand(s). I may not be at UPM most of the time but I am always well-informed

about the going-ons there. Special thanks to the laboratory technicians and assistants

from UKM, UPM and AOTD for your kind support.

To my friends who are threading the same path as I am, thank you for your

encouragement and support. Our frequent exchanges of news make me feel that I am not

alone whenever I feel downhearted.

Thank you Ma and Pa for believing in your daughter. Your unwavering support and love

lifted my spirits and confidence. Your generous help really reduces my physical and

mental burdens. To my sisters, thank you for having faith in me.

ix

To my loving husband, thank you for your solid support, help and advice for matters that

boggle my mind. You are indeed my beacon of hope, like the light at the end of a pitch

dark tunnel. Words can’t describe my gratitude towards your understanding, guidance,

patience, love and care whenever I am in a dire need of help and support. Without you,

my dream of earning this PhD degree would not materialize.

It may sound cliché but my baby is my inspiration. His smile is enough to make me

forget the darkest moment in my life. I love you and thank you for being a good baby

when Mama was rushing to complete her assignments.

This chapter of my life opens my eyes and heart and makes me believe that this world is

full of hope and kindness. The next chapter continues what promises to be an even more

exciting and enticing journey. I look forward to more colourful sparks awaiting me.

Life rocks!

x

I certify that a Thesis Examination Committee has met on 24 December 2009 to conduct

the final examination of Lim Hong Ngee on her thesis entitled "High Internal Phase

Emulsion as a Reaction Medium for Fabrication of Brushite Crystal" 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 Degree.

Members of the Thesis Examination Committee were as follows:

Md. Jelas Haron, PhD

Professor

Faculty of Science

Universiti Putra Malaysia

(Chairman)

Taufiq Yap Yun Hin, PhD

Professor

Faculty of Science

Universiti Putra Malaysia

(Internal Examiner)

Mahiran Basri, PhD

Professor

Centre of Foundation Studies for Agriculture Sciences

Universiti Putra Malaysia

(Internal Examiner)

Hamdan Suhaimi, PhD

Professor

Department of Chemical Sciences

Faculty of Science and Technology

Universiti Malaysia Terengganu

Malaysia

(External Examiner)

BUJANG BIN KIM HUAT, PhD

Professor and Deputy Dean

School of Graduate Studies

Universiti Putra Malaysia

Date:

xi

This thesis was submitted to the Senate of Universiti Putra Malaysia and has been

accepted as fulfilment of the requirement for the degree of Doctor of Philosophy. The

members of the Supervisory Committee were as follows:

Anuar Kassim, PhD

Professor

Faculty of Science

Universiti Putra Malaysia

(Chairman)

Dzulkefly Kuang Abdullah, PhD

Professor

Faculty of Science

Universiti Putra Malaysia

(Member)

Abdul Halim Abdullah, PhD

Associate Professor

Faculty of Science

Universiti Putra Malaysia

(Member)

Mohd. Ambar Yarmo, PhD

Professor

School of Chemical Sciences and Food Technology

Faculty of Science and Technology

Universiti Kebangsaan Malaysia

(Member)

Yeong Shoot Kian, PhD

Head

Advanced Oleochemicals Technology Division

Malaysian Palm Oil Board

(Member)

________________________________

HASANAH MOHD. GHAZALI, PhD

Professor and Dean

School of Graduate Studies

Universiti Putra Malaysia

Date: 11 February 2010

xii

DECLARATION

I declare that the thesis is my original work except for quotations and citations which

have been duly acknowledged. I also declare that it has not been previously, and is not

concurrently, submitted for any other degree at Universiti Putra Malaysia or at any other

institution.

_________________

LIM HONG NGEE

Date: 22 February 2010

xiii

LIST OF TABLES

Table Page

2.1. Key physical properties of ethylene oxide

18

2.2. Naming and properties of fatty alcohols (Cognis, 1990)

19

2.3 HLB values and their typical properties

24

2.4. Relaxation times for materials

29

2.5. Emulsion types

38

2.6. Bone cells and their associated functions and origins

48

3.1. Properties of the palm-based nonionic surfactants

64

4.1. Composition of HIPE1 to 5

79

4.2. Light scattering measurements of HIPE1 to 5

83

4.3. Light scattering measurements of HIPE A to F measured fresh and after

three months of storage at an elevated temperature (40oC)

99

4.4. Rheological properties of HIPE A to F measured fresh and after three

months of storage at an elevated temperature (40oC)

103

6.1. Loading capacity (%) of drug onto brushite crystals

150

6.2. Cumulative release (%) of control and drug-loaded bulk brushite

153

xiv

LIST OF FIGURES

Figure Page

1.1. Basic chemical structure of a surface active molecule.

1

1.2. (a) Top part of the ethoxylation reactor comprising (A) inlets, (B) outlets,

(C) valves and (D) weigh to determine the amount of (E) ethylene oxide used

for ethoxylation. (b) Bottom part of the ethoxylation reactor consisting of (F)

cooler, (G) heater and (H) reaction chamber.

6

1.3. Preparation of porous brushite crystals using O/W HIPE as a reaction

medium.

8

1.4. Brushite crystal growth within the constraint of continuous aqueous phase:

A – oil droplets, B – continuous aqueous phase and C – surfactant layer.

8

1.5. Proliferating synoviocytes on a surface.

9

2.1. The illustration of an ethylene oxide ring.

17

2.2. Scheme of the automated reactor employed in ethoxylation. A =

Computer, B = Computer interface, C = On-off valve for feeding ethylene

oxide, D = Ethylene oxide tank, E = Pressure transducer, G = Exit for

withdrawing, H = Jacketed reactor, I = Freezing coil, L = Holed stirrer, M =

Magnedrive stirrer, N = Thermocouple (Di Serio et al., 1996).

21

2.3. Schematic representation of CMC formation.

25

2.4. Schematic explanation of Deborah Number.

29

2.5. Flow curves for Newtonian and non-Newtonian fluids.

30

2.6. Thixotropic area.

33

2.7. Rheological tests used in emulsion characterization.

34

2.8. Schematic representation of the breakdown processes in emulsions.

39

2.9. The dashed line shows the region of Brownian movement of particle c. All

other neighbours (c1, c2, c3) of particle b could occupy any arbitrary position

beside this region (Mishchuk et al., 2004).

42

xv

2.10. Lowering the energy barrier as a consequence of the combined

interaction of three particles: globule b under the influence of two neighboring

globules c1 and c2. The lines 1 and 2 represent the interaction between the

globules c1 and b in the absence of c2 and between c2 and b in absence of c1; Vd

is the energy barrier to be surmounted if b approaches c1 from infinity. It is

reduced to Vc if the simultaneous interaction with c1 and c2 is taken into

account. Line 3 is the sum of lines 1 and 2.

42

2.11. Synovium, the soft tissue that lines the non-cartilaginous surfaces within

joints with cavities.

49

3.1. Schematic diagram of the ring method.

66

3.2. GC chromatograms of (a) laureth, (b) palmiteth and (c) steareth and (d)

Brij 30.

67

3.3. FTIR spectra of (a) laureth, (b) palmiteth and (c) steareth.

70

3.4. Surface tension isoterms at 25oC for the determination of CMC values (a)

laureth, (b) palmiteth and (c) steareth.

72

3.5. Schematic drawing of laureth-100 head groups intertwist with one another.

The initial head group is relatively smaller than the intertwisted (enlarged)

head group.

74

4.1. Droplet size distribution of HIPE1 to 5. Data points were presented in an

average of three replications.

83

4.2. Optical micrographs of (a) HIPE2 and (b) HIPE5 with oil droplets

separated from the parent emulsion upon water diffusion.

85

4.3. SAXS plot of intensity, I (q) as a function of scattering vector (q) of

HIPE1 to 5.

86

4.4. Guinier plot of ln I (q) as a function of q2 at very low angle and Guinier

fits (black lines) of HIPE1 to 5.

88

4.5. Plot of shear rate (s-1

) as a function of shear stress (Pa) of HIPE1 to 5.

89

4.6. Plot of viscosity (Pas) as a function of shear rate (s-1

) of HIPE1 to 5.

90

4.7. The circled region magnifies the sharp rise in the shear rate to indicate the

yield stress value determined from the intersection point of the two linear lines

(inset).

91

4.8. Plot of viscosity (Pas) as a function of shear stress (Pa) of HIPE1 to 5.

93

xvi

4.9. Plots of dynamic moduli as a function of average droplet size (µm) of

HIPE1 to 5. (■) refers to storage modulus, G’ (Pa) and (●) refers to loss

modulus, G” (Pa). Inset shows frequency dependence storage modulus of

HIPE1.

94

4.10. Plot of loss tangent as a function of frequency (s-1

) of HIPE1 to 5.

95

4.11. Plot of storage modulus, G’ (Pa) as a function of strain (%) of HIPE1 to

5.

96

4.12. Plot of storage modulus, G’ (Pa) versus strain (%) shows the intersection

point of two linear lines, which is the critical strain value.

96

4.13. Plot of critical strain, γc (%) as a function of droplet size (μm) of HIPE1

to 5.

97

4.14. Droplet size distribution of HIPE A to F. RT and 40oC refer to the fresh

HIPE samples and after storage at 40oC for three months, respectively. Data

points were presented in an average of three replications.

100

4.15. Average droplet size of HIPE A to F. RT and 40oC refer to the fresh

HIPE samples and after storage at 40oC for three months, respectively.

100

4.16 SAXS plot of intensity, I (q) as a function of scattering vector (q) of HIPE

A to F.

101

4.17. Guinier plot of ln I (q) as a function of q2 at very low angle and Guinier

fits (black lines) of HIPE A to F.

102

4.18. Flow curves of HIPE E measured fresh and after three-month storage at

40oC.

106

4.19. Dynamic moduli-frequency profile of fresh HIPE E.

107

5.1. Rheological measurements of HIPEs without (A) and with (B) the

presence of crystal growth. The linear line K shows the abrupt drop in

viscosity (b). The intersection point of the two linear lines for each profile of

sample A and B shows the critical strain value (d).

115

5.2. XRD patterns of (a) bulk brushite and (b) brushite crystals prepared with

0.50 M calcium ion and 0.30 M phosphate ion, 5.0 wt % surfactant

concentration and Ø = 0.80.

118

5.3. FTIR spectra of (a) brushite and (b) highlights of chemical bonding of

brushite crystals prepared with 0.50 M calcium ion and 0.30 M phosphate ion,

5.0 wt % surfactant concentration and Ø = 0.80.

119

xvii

5.4. SEM image of bulk brushite. 120

5.5. SEM images of brushite crystals prepared with calcium/phosphate molar

concentrations of (a) 0.50 M/0.30 M, (b) 0.30 M/0.18 M and (c) 0.10 M/0.06

M.

122

5.6. SEM images of brushite crystals prepared with HIPE stabilized by (a) 2.0

wt % and (b) 8.0 wt % surfactant concentrations.

125

5.7. SEM images of brushite crystals prepared with HIPE at (a) Ø = 0.75, (b) Ø

= 0.85 and (c) Ø = 0.90.

127

5.8. Illustration of ideal oil droplet arrangements of various oil volume

fractions.

128

Figure 5.9. SEM images of brushite crystals prepared by (a) stirred and aged

for seven days at 25oC, (b) homogenized for 30 minutes and aged for seven

days 25oC, and (c) homogenized for 30 minutes and aged for seven days at

40oC.

130

5.10. SEM images of brushite crystals prepared with calcium

nitrate/ammonium dihydrogen phosphate molar concentration of 0.50 M/0.30

M and recovered by (a) washing with ethanol and water and (b) direct

calcination.

132

5.11. Schematic of brushite crystal growth in the continuous aqueous film of

HIPE. The blank areas signify the pores after removal of the organic matters.

133

5.12. SEM images of brushite crystals prepared with HIPE stabilized by 5.0 wt

% (a) laureth-3, (b) laureth-6 and (c) laureth-100.

135

5.13. Schematic of micelles produced from surfactant with (a) short

polyoxyethylene chain length, laureth-3 and (b) long polyoxyethylene chain

length, laureth-100. X+ and Y

- represent calcium and phosphate ions,

respectively.

137

6.1. Calibration curve of sodium ampicillin.

144

6.2. Experimental set-up of controlled release study.

146

6.3. The morphology of the synoviocytes adhering and spreading on the

brushite crystals on the (a) first day, (b) third day and (c) fifth day.

148

6.4. Cell viability on the brushite crystals. Control consisted of only

synoviocytes in the well. Error bars represent means ± standard deviation for n

= 3.

149

xviii

6.5. (a) The insignificant loading of drug onto the surface of the bulk brushite.

(b) Brushite crystals prepared with 0.50 M calcium ion and 0.30 M phosphate

ion, 5.0 wt% surfactant concentration and Ø = 0.80, loaded with sodium

ampicillin. Inset shows that the drug penetrated the pores of the brushite

crystals.

151

6.6. Cumulative release profile of sodium ampicillin from brushite crystals

prepared with (a) 0.10 M calcium ion and 0.06 M phosphate ion, (b) 0.30 M

calcium ion and 0.18 M phosphate ion, and (c) 0.50 M calcium ion and 0.30 M

phosphate ion.

152

6.7. The cumulative release profile follows a second-order reaction for brushite

crystals prepared with (a) 0.10 M calcium ion and 0.06 M phosphate ion, (b)

0.30 M calcium ion and 0.18 M phosphate ion, and (c) 0.50 M calcium ion and

0.30 M phosphate ion.

154

xix

LIST OF ABBREVIATIONS

BMP Bone morphogenetic proteins

CMC Critical micelle concentration

CPCs Calcium phosphate cements

D Deborah Number

DCPD Bicalcium phosphate dehydrate or brushite

DSD Droplet size distribution

EO Ethylene oxide

FID Flame ionization detector

FTIR Fourier Transformed Infrared Spectroscopy

GC Gas chromatography

HA Hydroxyapatite

HIPE High internal phase emulsion

HLB Hydrophilie-lipophile balance

LVR Linear viscoelastic region

MTX Methotrexate

NMR Nuclear magnetic resonance

O/W Oil-in-water

OCP Octacalcium phosphate

PBS Phosphate buffer solution

PMMA Poly(methyl-methacrylate)

SAXS Small angle x-ray scattering

SEM Scanning electron microscopy

xx

TCP Tricalcium phosphate

UV Ultraviolet

W/O Water-in-oil

XRD X-ray diffractometry

ζ Stress

ζ0 Critical stress

ε Strain

γc Critical strain

δ Phase angle

Ø Volume fraction

A Absorbance

a Proportionality constant

c Concentration

Ec Cohesive energy

ΔGo

mic Standard free energy of micellization

G’ Storage modulus

G” Loss modulus

G* Dynamic modulus

k Constant

l Pathlength

Mhg Molecular weight of the hydrophilic head group

Ms Total molecular weight of the surfactant

Mw Molecular weight

xxi

R Universal gas constant

R Aggregates diameter

Rg Radius of gyration

T Absolute temperature

Vc Energy barrier for three particles

Vd Energy barrier for two particles

TABLE OF CONTENT

Page

ABSTRACT iii

ABSTRAK v

ACKNOWLEDGEMENTS vii

APPROVAL x

DECLARATION xii

LIST OF TABLES xiii

LIST OF FIGURES xiv

LIST OF ABBREVIATIONS xix

CHAPTER

1 INTRODUCTION

1.1 Grounds for Research 1

1.2 Research Objectives 4

1.3 Research Approach 5

1.4 Structure of This Thesis 10

2 LITERATURE REVIEW

2.1 Surfactants 11

2.2 A Brief History on Nonionic Surfactants Development 12

2.3 Nonionic surfactants 13

2.4 Ethylene Oxide Condensation 14

2.5 Reactants 16

2.5.1 Ethylene Oxide 16

2.5.2 Fatty Alcohols 18

2.6 Product 19

2.6.1 Synthesis of Fatty Alcohol Ethoxylates 19

2.6.2 Fatty Alcohol Ethoxylates 21

2.7 Hydrophile Lipophile Balance 23

2.8 Critical Micelle Concentration 24

2.9 Rheology 27

2.9.1 Definition 27

2.9.2 Characterization of Materials 28

2.9.3 Rheological Characteristics 30

2.9.4 Rheological Measurements 33

2.10 Emulsions 37

2.11 High Internal Phase Emulsions 39

2.12 Rheology of High Internal Phase Emulsions 43

2.13 Applications of High Internal Phase Emulsions 44

2.14 Skeletal Tissues 46

2.15 Cellular Components 48

2.16 Periarticular Soft Tissue 49

2.17 Skeletal Tissues Repair 50

2.18 Calcium Phosphates 52

2.19 Brushite 53

2.20 Importance of Porosity 54

2.21 Calcium Phosphate Coatings 56

2.22 Delivery Devices 57

3 SYNTHESIS AND CHARACTERIZATION OF

NONIONIC SURFACTANTS BY ETHOXYLATION

3.1 Introduction 62

3.2 Experimental 63

3.2.1 Ethoxylation of Nonionic Palm-Based Surfactant 63

3.2.2 Characterization 64

3.3 Results and Discussion 66

3.4 Conclusion 75

4 PREPARATION AND CHARACTERIZATION OF OIL-

IN-WATER HIGH INTERNAL PHASE EMULSION

STABILIZED BY LAURETH

4.1 Introduction 76

4.2 Experimental 78

4.2.1 Preparation of High Internal Phase Emulsions 78

4.2.2 Characterization 79

4.3 Results and Discussion 82

4.3.1 Olive oil/Laureth/Water High Internal Phase

Emulsion

82

4.3.2 A Comparison between Olive Oil/Laureth/Water

and Olein Oil/Laureth/Water High Internal Phase

Emulsions

98

4.4 Conclusion 107

5 PREPARATION AND CHARACTERIZATION OF

CALCIUM PHOSPHATES USING NONIONIC

SURFACTANT BASED HIGH INTERNAL PHASE

EMULSION

5.1 Introduction 109

5.2 Experimental 110

5.2.1 Synthesis of Calcium Phosphates using High

Internal Phase Emulsion

110

5.2.2 Characterization 112

5.3 Results and Discussion 112

5.3.1 Rheological Properties 112

5.3.2 Crystallinity 117

5.3.3 Chemical Bonding 118

5.3.4 Morphology 120

5.4 Conclusions 138

6 APPLICATIONS OF BRUSHITE CRYSTALS

6.1 Introduction 140

6.2 Experimental 141

6.2.1 Cytotoxicity Test 141

6.2.2 Controlled Release Studies 143

6.3 Results and Discussion 146

6.3.1 Cell Proliferation 146

6.3.2 Drug Delivery 149

6.4 Conclusion 156

7 SUMMARY, GENERAL CONCLUSION AND

RECOMMENDATIONS FOR FUTURE RESEARCH

7.1 Summary 157

7.2 General Conclusion 159

7.3 Recommendations for Future Research 160

REFERENCES 163

BIODATA OF STUDENT 187

LIST OF PUBLICATIONS 189