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Synthesis and Characterization of Bone Analogue Materials
Ivana Soten
A thesis submitted in confonnity wîth the requirements for the degree of Master of Science
Department of Chemistry University of Toronto
@Copyright by Ivana Soten 1998
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Synthetic analogues of bone are king actively pumied as materials for biomedical
applications in the field of bone replacement, augmentation and repair. Numerous stringent
criteria have to be met for a biomateriai to be considered as an acceptable bone implant,
including the ability to integrate into bone and not cause any deleterious side effects.
Different approaches to a bone implant materials are introduced.
A materials chemistry approach to synthesizing a new type of bone analogue material
is described. The strategy uivolves the spontaneous growtfi, under aqueous physiological pH
conditions, of an oriented hydroxyapatite film with micron dimension porosity, on the
surface of a layer of TiO, that has been sputter deposited on Ti metal. This procedure creates -
desirable a>-crystallized phases of hydroxyapatite (OHM) and octacalcium phosphate (OCP)
with prefened orientation respectively dong the [ûûl] and [101] directions. In an attempt to
mimic the hierarchical organic-inorganic composite construction of bone, a calcium
dodecylphosphate ester mesolamellar phase has been integrated into these oriented porous
films to create a CaDDP-OH.-OCP-Ti0,-Ti multilayer architechue. The nucleation and - growth processes, together with the characterization of these films, are investigated using a
multi-analytical approach based upon PXRD, SEM, TEM, SAED, XPS, FT-IR microscopy
and profiIornetry. The relationship of the synthetic calcium phosphate composite materials to
boae as well as other bone analogue materials is discussed.
1 wish to thank my supe~sor, Professor Geoffrey k Ozin for his encouragement,
enthusiasm, and belief in my abilities. 1 am t d y gratefui for his scientific guidance as weli as
his Wendship and support with al i the problems 1 had.
1 am thankful for technical assistance and stimdating discussions with Dr. Neil
Coombs, Dr. Douglas D. Perovic, Dr. Aieksandra Perovic and Dr. John Davies with various
aspects of the elecwn microscopy and electron diffraction midies, as well as the bone
biology relevant to this materials chemistry approach to the bone implant problem. The
expert advice of Dr. Srebri Petrov with the powder X-ray ciiffiaction measurements is also
gratefully acknowledged. The technical assistance of Ms. Sue Mamiche-Afara with the
sputter coating is deeply appreciated. 1 also wish to th& Dr. h e r Dag for FT-IR
microscopy, optical proflomehy results and most vaiuable discussions, and Dr. Deepa
Khushalani for the help with prwfreading the thesis. The expert direction of Dr. Natasha
Varaksa in the early stages of this projea proved to be extremely helpful.
Finally, 1 wish to thank my parents and my husband for their love and support.
TABLE OF CONTENTS
Abstract
Acknowieùgments
Table of Contents
List of Fi ires
C-R 1: General introduction
1 .O Thesis Org-tion
1.1 General Principles of BiomineralUation
1.2 Molecular Structure of Bone
1.2.1 Organic Bone Phase
1.2.2 Inorganic Bone Phase
1 -3. Biochemical Process of Bone Mineralization
1.4. Biomimetic Approach to Matenais Chemistry
1.4.1 Bone implant Materials
1 S. References
CHAPTER 2: Experimental Methods
2.1. Synthesis
2.1.1 Preparation of Calcium Phosphate on TiO, Surface
2.1.2. Preparation of CaDDPICaP Composite Material on the Surface
of TiO,
2.2 Instrumentation
2.2.1 Spuner Coating
2.22 Powder X-ray Diffraction
2.2.3 S d n g Electron Microscopy
2.2.4 X-ray Photoelectron Spectroscopy
2.2.5 Surface Roughness tester
2.2.6 Ion-Beam Milling
22.7 Transmission Eiectron Microscopy
2.2.8 FT-IR Microscopy
CHAPTER 3: Calcium Phosphate Based Bone Analogue Materials
3.1 Preamble
3.2 Background
3.2.1 Surfactant Science
3.3 Chemistry of Octacalcium Phosphate
3.4 Synthesis
35 Results and Discussion
35.1 Calcium Phosphate Growth on TiO, - Surface
35.2 CaDDP-CaP Composite on TiO, - Surface
3.6 Conclusion
3.7 References
Figure 1.1. Gibbs free energy of a cluster in relation to its size
Figure 1.2. SEM of the red abalone shell
Figure 13. Drawing of the hierarchical structure of bone
Figure 1.4. Structure of marine algae Emilianea Huxleyi
Fire 1.5. Drawing of the collagen sûucture
mre 1.6. Hydroxyapatite and fluoroapatite crystal structure
Figure 1.7. Schematic representation of boue mioerdhtion
Fire 2.1. Thickness of the calcium phosphate film obtained by RST technique
Fi ire 2.2. Illustration of an ion-beam milling apparatus
Figure 3.1. Critical micelle concentration, solubility and Krafft-point definition
F i i r e 3.2. Models of lyotropic liquid crystalline phases
Figure 33. Crystal structure of octacalcium phosphate
F i i r e 3.4. SEM images of samples after 7,22,30,48 and 72 hours growth tirne
Figure 35. TEM of sample after 22 hours growth t h e
Figure 3.6. PXRD patterns of sample on and scraped from the subsnate
Figure 3.7. Graph of thichess of the calcium phosphate film versus growth time
Figure 3.8. FT-IR microscopy spectra of samples after various growth time
Figure 3.9. Selected area diEraction patterns of calcium phosphate Nm
Figure 3.10. Representation of clean and hydroxylated surfaces of rutile
Figure 3.11. Oxygen 1s W S spectra of substrate prior and after growth periods
Figure 3.12. Calcium 2p XPS spectra of samples &ter different growth periods
Figure 3.13. PXRD patterns of Ti-Ti0,-OHAp - and Ti-Ti0;OHAp-CaDDP - F i 3.14. SEM of Ti-Ti0,-OHApCaDDP - F i r e 3.15. TEM of Ti-Ti0,-OHAp-CaDDP - F i 3.16. Illustration of the structure of Ti-Ti0,-OHAp-CaDDP -
Chapter One General Introduction
1.0 Thesis Organization
In this work a materiais chemistry approach to synthesizhg a new type of boae
analogue material is described. Formation of a synthetically prepared calcium phosphate-
calcium dodecylphosphate ester composite material is discussed as weU as the relationship of
the composite material to the hierarchical structure of bone.
Chapter 1 focuses on the principles of the biomineraluation process with an emphasis
on bone structure and bone formation. A materials chemistry approach to biomirnicry and
different bone implant materials is also discussed.
Chapter 2 presents the experimental details of the work presented as well as al1 the
characterization techniques employed.
Chapter 3 discusses the growth process of hydroxyapatite on a TiOl surface, the role
of the precursor phase octacalcium phosphate, substrate and solution conditions. The
formation of a synthetic bone analogue material is presented and the mode1 of interfaciai
mmplementarity between the inorganic and organic phase in the composite material is
proposed.
1.1 General Principles of Biominembation
Biomineralization is a process by which organisms form inorganic minerals via
intriate cellular activities. These activities provide the necessary conditions for mineral
nucleation and crystal growth.' The majority of the biominerals have calcium and iron as the
most common cations while phosphates are the most comrnon anions, followed by oxides
and carbonates, Table 1.1. The principal skeletal biominerals are calcium carbonates, calcium
1-2
Table 1.1. Types and functions of the main inorganic solids found in biologicai systemsl
phosphates and silica. The strongest carbonate and phosphate skeletons are well ordered
composites of organic polymer intenpersed with a mineral phase. A second and significant
hinction of biomineral deposits is that they act as a storage system from which ions may be
withdrawn during periods of physiological demand. It should be noted that crystais h a h g
unusual morphologies (fomed by living organisms) have been impossible to mimic in the
labarotory. Recently, however, several reports of synthetically prepared unusuai, nature-like
morphologies have emerged. Four main stages of biomineralization are most often
iatroduced in the literature'"
1. Preorganiration of organic components
2. InterfaCid molecular recognition
3. Vectorial (chernical) regdation
4. Cellular processing
1. Preorganization of organic components
This stage is considered to always ocnir f k t in ail known organisms. Preorganized
environment serves to control the formation of inorganic materials. Nature uses self-
assembly of enclosed protein cages and lipid vesicles or extended protein-polysaccharide
networks for confinement and physical shaping of the mineralization zone. VesicIes and
membranes are predominantly used in intracellular biornineralization while extended
networks dominate in extracellular biomineralization. The process is often confined to the
nanoscale but could be at the micron level as well, for example, in the preorganization of
polymeric collagen (elaborated in 1.2.1 .)
2. Interfacial moleeular recognition
The second stage involves controlled nucleation of inorganic clusters. The
aforementioned preorganized organic phase has specinc sites on the surface that serve as
blueprints for site-directed inorganic nucleation. Assembly of mineral nuclei is governed by
electrostatic, structwal and stereochemical complementarity at the organic-inorganic
interface. The role of the organic surface is lowering of the activation energy of nucleation,
AG*. In addition to lowering the activation energy through the presence of heterogeneous
surfaces in the medium, organisms mntrol the local supersaturation levels. Nuclei will grow
into stable structures only if the energy released through the formation of bonds in the solid
Figurel.1. Gibbs fiee energy of a cluster in relation to ia size (r). a is the surface free energy, AG,, is the free energy change per mole associated with liquid-solid phase change, V, is the cluster molar volume, AG, is the free energy change of the cluster and r' is the critical radius of the cluster.'
Figure 1.2. SEM of the red abalone sheil exhibithg both the prismatic @) and nacre (n) section fomed from calcite and aragonite, re~pectively.~
state is greater than the energy required to maintain newly formed solid-liquid interface,
Figure 1.1.' Process of nucleation of inorganic crystals is considered as a transition state with
nuclei of different structure and orientation comprising a series of activated clusters that
differ in AG'. The fiequency by which they transform into stable entities depends on the
reaction trajecîory which is determined by molecular recognition process. Specific crystal
faces c m be preferentiaily nucleated by interfacial organic-inorganic complementarity thus
stabilizing the transition state.
3. Vectorid (chernical) regalation
This process is associated with crystal growth and termination. In the absence of
cellular intervention, crystals would grow within an organic host accordhg to the laws of
crystalluation. The resulting particles would be constrained in size but 4 t h nomal
morphology. As mentioned above, nature is able to produce various kinds of unique
morphologies. For instance, in the red abalone shell, calcite and aragonite polyrnorphs are
deposited at Mereut locations within the same material, Figure 1.2.' Chernical regulation
involves transport anaor reaction-rnediated processes. Transport mechanisms regulate levels
of supersaturation through facilitated ion flux, complexation-decumplexation switches, local
redox and pH modifications.' Reaction mechanisms influence the kinetics of surface-
mediated processes, such as cluster formation and expression of crystal habit. Control of
biomineralization at this level results in structural, compositional and morphological
specificity in the biomineral phase.
The f i a l stage in the process of biomineralization is the production of higher order
architectures, for example as found in bone, Figure 1.3: Bone architecture is built by starting
with the assembly of collagen and hydroxyapatite that further assemble into microfibrils and
then into osteons. At this level of hierarchy, cells and blood vessels are incorporated into
structure and by association of osteons the final structure of bone is obtained. It should be
noted that all of the stages of biomineralization mentioned are oontrolled at the cellular level,
that is, cells produce organic matrices initially, which assembles into various foms and exert
control over the growth of inorganic materials. Fuither cellular activities are responsible for
the production of mmplex, berarchical structures with unique shapes and functions.
Flgure 13. Drawing of the hierarchical structure of bone'
One of Natures many extraordinary structures that displays the different stages of the
biornineralizatioa process is the marine alga Emilianea Huxleyi, Figure 1.4.' The single-
celled organism is surrounded by a close-packed arrangement of calcite (CaCO,) scales
(coccoliths) organized dong the outer ce11 wall, Figure 1.4a. Individual units of the coccolith
have a characteristic species-specinc shape, Figure 1.4b. Electron diffraction patterns
recorded at different regions of individual units indicate that the complex shape is exhibited
by a well-ordered single crystal of calcite oriented dong specific crystallographic directions,
Figure 1.4b. The morphology of individual scales at the early stages of growth is tabular with
no curved edges and in addition they are oriented and aligned the same way as a mature
crystals, Figure 1.4~. Enclosed spaces provide control over the extent and location of
nucleation while ion pumps in membranes control the physicochemical conditions. In
particular, the organic surface influences the orientation of a particular face, namely [1210]
by means of epitaxial matching at the organic-inorganic interface. Fully formed coccoliths,
comprised of approximately 25 individual scales, are exocytosed nom the cells and assemble
F i i r e 1.4. Marine algae Emilianea Huxleyi: (a) SEM of the organism with calcite scales (coccoliths) organized dong the outer celi wall; (b) shape and crystallopphic directions of an individual scale: (c) SEM of the scales at the initd stage of development'
on the cell surface. Calcite crystals presumably carry on their surfaces organic moieties by
which they are recognized by functionalities in the outer rnembranous surface.
1.2 MoIecular Structure of Bone
12.1 Organic Bone Phase
Bone coasists of roughly 70% mineral embedded in an organic matrix consisting
largely of collagen. The collagen is the basic structural fiber in comective tissues and a l l of
tbem have similar structure at the fibrillar level. Differentiation in the hierarchical structure
takes place when these fibrils are arranged in a particular architecture, thus forming a tissue
for a unique function. The collagen matrix of mineralized bone tissues is formed almost
entirely of type 1 collagen which represents 70% of the organic constituents and 90% of
protein in bone. The characteristic feature of collagen is that of a chah which is wound into
triple helix to form rope like structure, roughly 300 MI by 1.5 nm, Figure 1.5.'
The collagen molecule has two unusual features. The a chah is so tightly coiled that
every third amino acid is glycine, the simplest amino acid and the only one without a c'de
chah that could fit into this helix. Also, collagen contains hydroxyproline and hydroxylysine
that are rarely found in other proteins. These hyckoxyl groups help stabilize the triple helix.
Once the collagen fibea are secreted from cells they become strengthened by cross-linking.
Lysine and hydroxylysine residues are deaminated to yield reactive aldehyde groups which
f o m covalent bonds between molecules. Five of these triple helices align longitudinally with
an overlap of approxirnately one quatter of the moiecule to fom a microfibril that is 4 nm in
diameter. Collagen fibril has a 67 nanometer pattern that arises fiom a "staggef' in the
assembly of adjacent molecules. M e r every fourth spacing there is only 32 nm of molecule
left to fit the next stagger. There are, therefore, 35 nm gaps between heads and tails of the
molecule. Electron micrographs of mineralized wmective tissue have shown that bone
crystallites ocnir at regular intervals dong the collagen fibers with the long c-axis oriented
paraIlel to fibriis. Crystals of hydroxyapatite occur in the gaps as weil as around the collagen
molecules."" It is assumed that coilagen promotes nucleation, however, collagen in some
tissues does not calcify. It is possible that some other mammolecules attach to mllagen and
convert it to the mineralized fonn.
Figure 15. Drawing of the coliagen str~cture.~
Table 13. Major noncollagenous proteins in bone'
Table 1 .2~ summarizes the major noncollagenous proteins to be found in bone.
Glycoproteins are providing calcium andor phosphate binding sites while s e d g as a
structural component as well. Osteopontin is being secreted by the cells in the early stages of
bone formation. This protein, together with bone proteoglycans, is highly sulfated. The d e
of osteopontin is prirnarily in the attachment of osteoblastic cells. Although it has calcium
binding sites, the role is primarily in the growth of the inorganic phase rather than in
nucleation which is assigned to bone sialoprotein.
13.2 Inorganic Bone Phase
Mineralized bone consists of approxirnately 70% hydroxyapatite. This is one of
several calcium phosphate phases listed in Table 1.3." The chernical formula of
hydroxyapatite (OHAp) is Ca,o(P04)6(OH)2 and it can crystallize in stoichiometric (s-OHAp,
Ca/P= 1.67) or nonstoichiometric (ns-OHAp, Cap* 1.67) form. Nonstoichiometric apatites
can be calcium nch (Cm1.67) or calcium deficient (CaP4.67). Aside nom
hydroxyapatite, bone consists of carbonated OHAp and fluoroapatite (FAp) in which OH'
ions are exchanged for CO? and Fions, respectively.
Table 13. Different calcium phosphate phasesZS
F o i r e 1.6. Hydroxyapatite crystal structure. (a) oxygen coordination of columnar ca2+ (a) and linking of columns via PO, tetrahedra @). (b) projection of the fluoroapatite structure on to the basal plane (0001)'
The space group for OHAp is P& and for FAp P6Jm. The structure consists of
columns of Ca2+ ions spaced by one-haif of the c-axis parameter dong the three-fold axis.
Each of these CaZ* ions is comected to its neighboring CaZ' ions above and below by three
shared oxygen atoms that lie in the mirror plane; on one side there are three O(1) atoms and
on the other side three O(2) atorns at a distance of -2.4 Each calcium atom is also
coordinated by three M e r oxygens O(3) at a greater distance. Thus, the columnar calcium
is 9-fold coordinated by oxygen atoms, Figure 1.6a: Columns of c ~ Z ' ions with their
coordinathg oxygen atoms are iinked together by PO: tetrahedra in which three oxygen
atoms corne from one column and the fourth from the adjacent column, Figure 1.6a. The
result is a three dimensional network of PO,$ tetrahedra with enmeshed wlumnar Ca2+ ions
and channels passing through it, Figure 1.6b: The remaining ions, OH- and their adjacent
~ a " ions that are required to complete the structure are located in the channels. Calcium ions
fomi triangles rotated by 60" from each other about the c-axes at whose centers OH* (or F)
ions are located. In the FAp structure, fluorine ions are three fold coordinated by ~ a ' ' and al1
four atoms are in the same plane while in OHAp, the OH- groups lie above the plane. The
solubility of FAp is lower than that of OHAp and FAp can be formed by exchange of 08
ions or by dissolution of OHAp followed by direct precipitation of FAp or mixed F-OHAp.
Aside from being the main part of the bone structure, hydroxyapatite forms the enamel of the
teeth.
1 3 Biochemid frocess of Bone Mineralization
Bone is a dynamic tissue that undergoes remodeling throughout life. In the
remodeling process, new bone is laid down on the resorbed surface of the old one. Therefore,
bone tissue creates an interface with itself on a continual basis. There are two major types of
bone cells that are responsible for bone resorption and growth throughout Me. Osteoblasts
are cells that produce new bone and osteoclasts are cells that resorb the old one. In vitro
shidies of bone formation can be perfonned using a substrate immersed in ce11 culture
(assuming that the substrate can successfully play the role of the resorbed bone surface):
The sequence of bone formation starts with secretion of a collagen fiber-free organic
matrix which is cailed the cement line, of which the major components are osteopontin,
calcium, phosphorou and bone sialoprotein. The width of the cernent line is reported to Vary
from 0.2 - 5 p. Minerakation of this matrix occurs by the seeding of nanocrystalline
calcium phosphate. The generai appearance of the calcified globular accretion laid d o m on
the substrate by osteogenic cells are shown in Figure 1.7am6 Proteins in this organic matrix are
believed to provide binding sites for nucleation of calcium phosphate. It is important to
differentiate berneen this cell-mediated and protein dependent biological mineralization
phenomenon and that of spontaneously forming calcium phosphate layes. Early crystals are
1-2 nm in size as determined by field-emission transmission electron microscopy (FETEM).
They grow and form globular ametions approximately 1 pm in size, Figure 1.7b. The
mineralized interfacial layer is succeeded by a collagen fiber assembly where the collagen is
stiil uncalcified, Figure 1.7~. In the extracellular space, hy droxy lated p rocdagen is
assembled into fibrils by the introduction of cross-links. Collagen fiber assembly becomes
evident in the interfacial mineralized zone. This zone acts as an anchoring surface for the
collagen that becomes mineralized by two methods, narnely fiber mineralization and
extracollagenous matrix mineralization. The latter is described by random seeding of
crystallites in the noncollagenous proteins that are continually secreted by osteogenic cells.
These proteins rnay or may not be aîtached to coilagen fibers. Thus, whereas secretion of
coilagen continues, enveloprnent of the collagen fibers by the underlying mineralized matrk
ocnirs, Figure 1.7d.
Initiai organic matrix
Seeding of calcium phosphate crystallites
Clystal growth and collagen assembly - -
I . - p.--
Cdlagen calcification and matrix mineralization
Figure 1.7. Schematic representation of bone mineralization. (a) secretion of noncollagenous matrix. (b) calcium phosphate nucleation at protein active sites. (c) crystal growth and coliagen secretion and assembly. (d) coiiagen and rnatrix minemüzation6
1.4 Biomimetic Approach to Materials Chemistrg
Over the y e m , a huge interest has emerged in mimicking the chernical processes in
Narure. The complex processes observed in biomineralization are characterized by the ability
known as "synthesis with constnictiony' or 4?nolecular tectonics" that serve as inspiration for
innovations in chemistry, in particular inorganic materials che~nistry-'~ Biomimcry could
lead to materials with better mechanical performance and "smart" materials that respond to
changes in their environment.' Biological materials are capable of remodeling and structural
redesign in response to environmental stresses. For example, in bone, more materiai is
deposited in the areas of greater load. This is due to the fact that collagen is piezoelectric so
the osteocytes may be able io detea whether the bone is being bent or being loaded off-
center as might happen if a broken bone does not join properly in line. Interestingly, the very
opposite occurs in a passive inorganic system, for example a loaded spring wiU corrode much
more quickly then a non-loaded one, hence the development of self-correcthg structures is
very desirable.'
The drive for novel adhesives based on proteins used by mussels is driven by the
lack of adhesives that will cure in a wet environment, the poisonous nature of solvent
systems and the need for medical adhesived Soft materials are also of interest in
biomimicry, for medical use (artifïcid skin and artery) and robotics applications. Biological
ceramics, such as bone and nacre are some of the toughest materials known cornpared to
synthetic ceramic materials which are an order of magnitude lower in toughness. The main
problem at present seems to be control of the orientation of the ceramic phase. Therefore,
material scientists are constantly looking at the ways that Nature is able to exert control over
production of such materials.
1.4.1. Bone Implant Materials
Different approaches to the preparation of possible bone implant rnaterials have
been studied extensiveiy."" In certain studies metals or metal ailoys are used directiy as
implants,130r these are coated with bioactive materials, such as hydroxyapatite, to aid in
adhesion to bone." Although metals or meral alloys meet biomechanid requirements of
implants and some achieve measures of biocompatibility, they exhibit poor interfacial
bonding between the metallic surface and the surroundhg bone. Good biocompatibility of
certain metals, for example titanium, is thought to occur as a result of spontaneous formation
of a thin titanium oxide layer on the surface. This in tuni is responsible for the low corrosion
rate. in addition, titanium and titanium based alloys have been shown to be bioactive as
calcium phosphate is readily able to nucleate and form a thin film on these substrate~.'~
However, the fiims obtained in this way are not uniform and grow to a thickness of only ca.
3-4 nm even after a month of aging, thereby reducing their efficacy as bioactive materials. In
vivo experirnents have established that bone does not bind as weU to titanium metal as it does
to hydroxyapatite coated rnetal.'"l6 It has been suggested that if a titanium implant is initially
coated with a layer of hydroxyapatite then the layer has to be at least one millimeter thick
and also exhibit bulk properties in order for the bone to properly adhere to this surface.
Different methods for preparing surface coatings for implants have been explored
such as dip coating, chernical vapor deposition (CVD), electrophoretic deposition, and
plasma spray-coating being the most widely used. However, coating of the intemal cavities
of cornplex-shaped implant materials is not feasible and the stoichiornetry of the coated
phase is hard to contr01.~ Furthemore, porous bioactive coatings exhibit better bone ingrowth
properties and most of the above mentioned techniques are able to produce only dense,
ceramic materials. Lack of strong adhesion between coatings and metal substrates is an
additional problem.
Coilagen molecules as well as synthetic polyrnea have been extensively studied for
their potentiai as bone implants. Most promising materials included coliagen -
glycosaminoglycan copolymer comp~site,'~ poly (lactic-a-glycolic acid) biodegradable
foamdg and bone cements? Bone cements include poly (methyl methacrylate) or a
copolyrner of methyl methacrylate and styrene that are polymerized in situ. In addition,
various biomacromoledar controlled dmg delivery systems were investigated such as geiled
bicontinuous cubic phasesa and hydrogels."
One of the recent most promising approaches to bone implant materials is the use of
composite organic-inorganic materials since they can be designed to be cornpositional,
structurai and functional analogues of bone. Different combinations of materials have been
used, the most well known examples being polymer-hydroxyapatite composites.'l Moreover,
because organic and polymeric structures are used as targeted dmg delivery systems, it is
easy to imagine how composite organic-inorgaaic bone analogue materials could facilitate
bone repair and overcome infection. With this concept in mind it has been suggested that
bone mimicry could be accomplished using a composite of a calcium phosphate-based
mesophase and hydroxyapatite7?
1.5. References
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4. J. Vinant, Strzictzual Biomateriuls; Princeton Univ. Press: New Jersey, 1990
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22. Shun-yan Wu, C. A. Steiner, MRli ProceedUigs, 1993,331,205-21 1
23. Tissue Engineering, M-RS. Bulletin, 1996,Z 1,18-65
24. G. k Ozin, N. Varaksa, N. Coombs, J. E. Davies, D. Perovic, M. Ziliox, J. Mater.
Ch., 1997,7(8), 1601- 1607
Chapter Two
Experimental Methods
2.1. Synthesis
2.1.1. Preparation of Calcium Phosphate on TiO, Surface
Titaaiwn foi1 was cut to a size of 10 x 8 mm, washed with ethanol and air dried.
The resdting clean substrates were sputter wated with titanium oxide using a Ti target in
a mixed oxygen-argon atrnosphere. For optimum results, sputtering was performed for six
houn and the thickness of the oxide layer produced was found to be CU. 900 A using a
quartz crystai microbalance.
The substrates prepared were placed on the bottom of polypropylene bottles in a
100 mi of solution with the following concentrations: 1.6 x 104 M CaCI2 and 7.6 x 10"
M KWO,. The pH of the resulting solution was close to physiological i.e. pH of 7.4, the
solutions were completely clear and no precipitation was observed throughout the
duration period of t&e experiment. The solutions were aged at 37 OC for periods of 7 to
120 hours. After different times, the substrates were removed from the soIutions carefully
washed with deionized water to remove any non-adsorbed electrolytes and dried under
arnbient conditions.
2.1.2. Preparation of CaDDPICaP Composite Material on the Surface of
TiO,
Mono-n-dodecylphosphate was transformed to the water soluble fom,
potassium dodecylphosphate ( W D P ) with a solution of potassium hydroxide in a molar
ratio of KOWDDP = 2/1. The concentration of W D P in water was 0.1 %. The Ti02
subsû-ate with hydroxyapatite grown on it was placed vertically in a beaker containing
&DDP by clamping it with custom-made tweezen. The nibswte was kept in the
solution for haif an hour after which t h e one molar equivalent of 0.01 M solution of
CaCl, was slowly added to form the CaDDP lamellar phase. The rate of addition of CaCI,
solution was lW15 min. The solution was stirred at a constant rate at 50°C for 24 hours.
Substrates were rinsed with copious amounts of water and air dned.
2.2, Instrumentation
2.2.1. Sputter Coating
Sputter coating of the titanium substrate was performed using a Perkin
Elmer 2400 sputtering apparatus. Titanium dioxide was produced using a titanium target
and mixed argon/oxygen amiosphere. Sputtering was performed for different periods of
time at lOOOV DC, using 12 mtorr pressure of 6 sccm argon gas and 6 sccm oxygen gas.
By adjusting the sputtering conditions, the deposited film could be arranged to be
crystalline rutile and/or anatase or amorphous titania. It tums out that the nature of the
titania film did not have a dramatic effea on the structure, orientation or composinon of
the hydroxyapatite overlayers.
23.2. Powder X-ray diffraction (PXRD)
Powder X-ray diffraction (PXRD) data were obtained on a Siemens D5000 diffractometer
using Ni filtered Cu K a radiation (h = 154178 A) and a Kevex 2005-22 solid state
detector. Typicd accelerating conditions were 50 kV and 35 mA. For strongiy diffracting
samples, the intensity of the 100 % peak was fbst checked by quick manuai sans of the
angles in the region of the strongest peak under lower accelerating conditions, typically
40 kV and 20 mk The step size was 0.02' and the step time was 1.0 seconds, with a s a u i
range of 1 to 45" (28). A slit size of 1 mm was used on the X-ray tube, and 1 mm and 0.2
mm for the detector slits. The latter was changed to 0.1 mm for low angle 28 diffraction
( 1 to 7 O ). Samples were prepared by gluing the substrates on the back of the sample
holder using iittle amounts of the silicon paste.
223. Scanning electron microscopy (SEM)
Scanning electron microscopy (SEM) images were obtained on a E O L 840 scanning
electron microscope using an accelerating voltage of 15 kV or les. Substrates were cut to
a small piece and using conducting carbon paste, mounted to the sample holder. AU the
samples were gold coated in order to produce a conducting surface layer and reduce
charging effects which were cornmon for most of the samples.
2.2.4. X-ray photoelectron spectroscopy (XPS)
X-ray photoelectron spectrosmpy P S ) was perfomed using a Laybold MAX 200 W S
apparatus. A non-monochromatized Mg K a X-ray source was used at 15 kV and 20 mA.
Al1 the spectra were collected under a vacuum of l e s than 1 x 1 0 ~ Ton. Satellite line
subtraction was performed as well as correction for charging effects by calibrating the
position of the carbon 1s peak at 285 eV.
23.5. Surface Roughness Tester
Thickness measurements were performed on WYKO RST Surface Roughness
tester. The principle of this optical profilornetric technique is based upon constructive
interference between light that is refiected fiom the surface and a reference beam, and has
a quoted vertical resolution of ca. 1 nrn. The RST is a vertically scanning interference
microscope system that operates with one of seveml interchangeable magnincation
objectives. Each objective contains an interferorneter, consisting of a reference mirror and
beamsplitter. Interference fringes are produced when light reflected off the reference
rnirror combines with light reflected off the sample. When short-coherence-length white
light is used, these interference f i g e s are present only over a very shaUow depth on the
surface. The surface is pronled by scanning v e r t i d y so that each point on the surface
produces an interference signal and than locating the exact vertical position where each
signal reaches its maximum amplitude.
The RST starts the measurement sequerce by focusing above the top of the
surface being profiled and quickly scanning downward At evenly spaced intervals during
the scan, frames of interference data imaged by the video camera are captured and
processed by high-speed digital signal processing hardware. As the system s a n s
downward, an interference signal for each point on the surface is formed. The system
uses a senes of advanced amputer algorithms to precisely locate the peak of the
interference signal for each point on the surface. Each point is thus processed in parallel,
allowing the RST to determine surface height differences quickly and with an ememely
high degree of accuracy.
The software package provided with the instrument allows for full system control,
surface calculation, graphic display and statistical analysis. Analyses that can be
performed include average roughness (Ra), mis roughness (Rq), maximum surface height
(Rp), maximum surface depth (Rv), maximum peak-to-valley height (Rt), average peak-
to-valley height (Rz), while plots include culor contour, 3D isometrk, histograms, dopes
and 2D subandyses. Figure 2.1. shows the obtained profiling measunnent result of a
calcium phosphate sample obtained
Vertical scanning interference mode was used with a tungsten-halogen lamp as a
light source and solid-state CCD detector. Magnifications of 10 times and 20 times were
used which corresponds to 422 x468 mm and 211 x 234 mm , respectively. Vertical
measurement range is 10 nm to 100 p with a resolution of 3 m. Objectives wuld be of
either 2.5X 10X, 20X, and 40X magnification.
Figare 2.1. Thiclaiess of the calcium phosphate gmwn for 4 days obtained by RST technique
2.2.6. Ion-Beam Milling
Cross sectional samples for TEM analysis were obtauied using an ion-beam
milling technique. In ion-beam thinning (milling), gas ions are accelexaîed to an energy
of severai keV and directed onto the specimen unda oblique incidence. Figure 22. is
schematic illustration of a . ion thinning apparatus. Two ion guns are arranged in a
vacuum chamber such that the specirnen is bombardecl h m both sides at the same t h e .
Argon is the gas most commonly used for the ion guns. A glow discharge is ignited when
a high voltage is applied between anode and cathode and a beam of particles is emitted
fiom the hole in the cathode. At a fixed gun voltage, the particle flux is controlled by
changing the flow rate of the gas.
Figure 2.2. Illustration of an ion-beam milling apparatus
To create a symmetrical dimple under ion bombardment, the specimen holder is
rotated about an axis normal to the specimen. Selection of the thinning parameters
represents a compromise between the highest possible thinning rate and an acceptable
damage level due to ion bombardment. Since ion d i n g is more than one order of
magnitude slower than chernical or electrochemicai polishing, the a h is generally to
achieve highest possible thinning rates. The thinning rate is diredy proportionai to the
flux of particles incident on the specimen. In order to maximize this flux, ion guns must
be aligned so that the center of the beam strikes the center of the specimen. Given a
constant flux of parcicles, the sputtering rate increases linearly with ion energy up to
about 10 keV. At high energies, the ions penetrate the specimen more deeply, causing ion
implantation and damage by way of specimen heaMg and irradiation damage. in practice,
smaller angles of incidence (about 10-15") of the beam are used to reduce the damage.
Momentum transfer and hence the sputtering rate are also dependent on the mas ratio
between the gas atoms in the incident beam and the target atoms. Both variables are
maximum when the masses are equai.
23.7. Transmission Electron Microscopy (TEM)
Transmission elecûon microscopy images were obtained on a JEOL 2010F field-
emmision electron microscope operating at 200 kV by Dr. Alehancira Perovic. Minimum
probe diameter was about 0.4 nm. Analytical techniques employed were high resolution
phase contrast irnaging and selected area electron diffraction. The samples were
embedded in a TAAB epoxy ma&, cured at 60 O C for 24 hours and sectioned using an
ion-beam milling technique. The thin sections were then mounted on either carbon
coated, plastic coated or 400 mesh size 3.05 mm copper support grids.
22.8. ET-IR Microscopy
FT-IR Microscopy is one of the primary methods for determining the rnolecular
composition of smdl amounts of material or small areas of large objects. It allows for
choosing an exact part of the sample to be analyzed. In a microscope measurement the IR
radiation is sent through a tiny opening so that the effects of diffraction must be
considered. Spatial resolution is the very important performance criterion and excellence
is achieved in the instrument used in this sîudy by the use of Spectra-Tech IR-Plan
laboratory microscope equip ped wi th Targeting and Redundant Aperturing . The sample
under consideration has two phases the centrai area of interest and a swounding matrix.
A conventional FT-IR microscope lacks a targeting aperture, that is an aperture between
the source and the sample. This results in a large percentage of the IR radiation passing
through the ma& resulting in spectral impdty due to the scattered radiation from the
matrix. In a Spectra-Tech microscope a targeting aperture is employed which restricts the
IR radiation to a much smaller area, greatly reducing scatter from the matrix. The second
redundant aperture further eliminates scatter fiom the matrix.
The Spectra-Tech microscope provides 15 mm of working distance , has a 100
Pm pinhole that determines the area to be analyzed and transmitted or reflected
illumination settings. For d l the sarnples, diffuse reflectance spectroscopy was used.
When infrared radiation is directed ont0 the surface of the solid sample, specular
reflectance or diffuse reflectance can occur. The specular component is the radiation
which reflects directly off the sample surface (it is the energy not absorbed by the
sample). Difhise reflectance is the radiation which penetrates into the sample and then
emerges. A diffuse reflectance accessory is designed so the difhisely reflected energy is
optimized and the specular component minimized. The optics collect the scattered
radiation and direct it to the hfiared detector. Several -ors exert a sigainant ifluence
over bandshape and relative/absolute intensity and these include rehctive index of the
sample ma&, particle size, sample homogeneity and concentration. Reffactive index
effects result in spedar refiectance contributions. The bandwidth decrease and relative
intensities are changed as particle size decreases. Peak intensities are changed if the
sample is not a homogenous and with high concentrations of the sampies, there is a
dramatic increase in spenilar contribution to the spectral data.
Chapter Three
Calcium Phosphate Based Bone Analogue Materials
Synthetic analogues of bone are king actively pursued as materials for biomedical
applications in the field of bone replacement, augmentation and repair, as discussed in
section 1 Al. Numerous stringent criteria have to be met for a biomaterial to be considered as
an acceptable bone implant, including the ability to integrate into bone and not cause any
deleterious side effects. Before discussing possible implant materials and their expected
response in the body environment, one has to take into account not ody the hierarchical
structure of bone but also the mode of bone formation and remodeiing thmughout life.
Kimwledge of the underlying principles of such biornineral reamstructruction chemistry
could lead to the production of novel, self-organizing structures, able to remodel and
optimize in response to the way that they are being used. in particular, a bone analogue
material could be synthesized with new and/or enhanced bioactivity and biocompatibility
characteristics that is capable of succesfil integration and remodeling with bone itself. One
of the most promising approaches to bone implant materials is the use of composite organic-
inorganic materials since they can be designed to be compositional, structural and functional
analogues of bone. Moreover, because organic and polymenc structures are used as a targeted
dmg delivery systems, it is easy to imagine how composite inorganic-organic bone analogue
materials could fatilitate bone repair and overcome infection.
In this chapter, a materials chemistry approach to synthesinng a new type of bone
analogue material is described. The strategy involves the spontaneous growth, under aqueous
physiological pH conditions, of an oriented hydroxyapatite film with micron dimension
porosity, on the surface of a Layer of TiO, that has been sputter deposited on Ti metal. The
results indicate strong adhesion and rapid growth of calcium phosphate on sputtered TiOt
surface within hours, rendering them as a viable option for bone implant matenals. Bath
oriented octacalcium phosphate and hydroxyapatite phases are found to CO-crystallize as a
macroporous nIm indicating an epitaxial growtb process. It is suggested that OCP acts as a
prewsor nucleation phase at the TiO, surface, that hydrolyzes to hydroxyapatite and
continues to grow epitaxially. In an attempt to rnimic the hierarchical organic-inorganic
composite construction of bone, a dc ium dodecylphosphate ester rnesolamellar phase has
been integrated into these oriented porous nIms to create a CaDDP-OHApOCP-TiQ-Ti
multi-layer architechne. A multi-analytical approach is employed to follow the growth
process and characterize the composition and structure of the calcium phosphate based films
on the titania-titanium substrate. The relationship of the spthetic calcium phosphate
composite materials to bone as weli as other bone analogue materials is discussed.
It should be noted that understanding underlying principles of self-assembling
organic structures is a necessary pre-requisite for an appreciation of the hierarchical organic-
inorganic stmctures that are produced either in the laboratory or by Nature. In addition,
possible precursors to the formation of inorganic bone phase have to be taken into account
w hen discussing bone analogue/implant matenals.
33. Background
3.2.1. Surfactant Science
Surfactants or surfaceactive agents are amphiphilic molecules that function
predominantly at the interface between two separate phases. Amphiphilic nature denotes that
they are comprised of two dissimilar groups namely, a lyophobic (solvent repelling) and a
lyophilic (solvent attracting) rnoiety. When water is used as a solvent, these are referred to as
the hydrophobie and hydrophilic groups. Thus, there are two parts with extremely different
solubility properties withui one molede. Depending on the nature of the polar functionality,
surfactants are divided into three groups : cationic, anionic and nonionic. Surfactant behavior
in a solvent is characterized by enhanced adsorption at surfaces/interfaces due to the
distortion of the solvent phase." Lyophobic group causes a distortion of the solvent liquid,
increasing the overail energy of the system. For the present purposes, behavior of surfactant
moledes in water will be discussed. Water molecules form inter-molecular hydrogen bonds
which leads to a teaahedral network with a relatively low density. A large number of ailowed
positions of the hydrogen atoms due to the tetrahedral surroundings maintaincd by hydrogen
bonds is responsible for the large entropy of the phase. The presence of hydrophobic groups
cause water molecules to rearrange and form cage structures around the solute.' This process
is costly in entropy since the water is more ordered. Therefore, surfactants either adsorb at the
surfaces/interfaces or they aggregate into structures such as micelles, liquid crystals or they
crystaliize out from solution. In the case of micelle and liquid crystal formation, water
molecules are repelled h m the hydrophobic part of the amphiphile. This entropy driven
process lowen the energy of the systern and favoa an increase in solubility. It should be
noted that micelles only form above a so called critical micelle concentration, "cmc".
Ionic surfactants in an aqueous solvent dissociate completely at low concentrations.
Without the shielding action of nearby counterions an aggregation of amphiphilic ions wodd
be impossible because of the stmng repulsive electrostatic forces between neighboring
headgroups. In general, the behavior of ionic surfactants is dependent on the length of the
hydrophobic tail, the nature of the head group, the valency of the counterion and the solution
conditions. It is often observed that the solubility of ionic surfactants in water solutions
undergo a sharp, discontinuous increase at some characteristic temperature, referred to as the
K r a temperature, T,. Below that temperature, the solubiiity of the surfactant is determined
by the crystd lanice energy and heat of hydration of the system. This temperature is defined
by the intersection of the crnc and the solubility curves in temperature versw concentration
diagrams, Figure 3.1 .'
The rnicellar structure is the initial aggregate observed upon increasing the
concentration (above crnc). For ionic surfactants, the system has to be above T, as weli. Upon
further increase in concentration of solute, larger aggregate structure are formed such as
cylindncal micelles and liquid crystal phases. The latter can form hexagonal, lamellar and
cubic structures. Starting h m an isotmpic solution of anisometric micelles the first step to
achieve a long-range orientational order is to let certain preferred axes of rod-üke micelles
concentration
3.1. Critical micelle concentration, solubility and definition of Kmfft-point
anange parallel to each other in time, ~igure3.k' The resulting phase is called "nematic".
With increasing order rod-like aggregates cari anange in a two-dimensional hexagonal lattice,
Figure 3.2b.' Then, a long-range positional order occurs in addition to the orientational order
of the rods. If the amphiphile builds up paralle1 anay of aggregates then a lamellar phase may
be fonned, Figure 3.2c.' Aliphatic chahs can be in a disordered liquid-like or solid-like a- trans conformation. The lamellae can be built Born bilayen of monomen and interdigitation
of molecules in the bilayer may occur. Cubic lattices are built of globular micelles possessing
long-range orientational order ( in addition to positional order), Figure 3.2d.'
Geornetric shape constraints imposed by the individual amphiphiles restricts their
packing into aggregates of different structures. Two opposing major forces, acting mainly in
the water-amphiphile interfacial region are considered to govem the self-assernbiy of
amphiphiles into liquid crystal structures. The hydrophobic attraction causes the molecdes to
associate whereas the hydrophilic ionic or stenc repulsion of the headgroups has the opposite
effect of keeping them in contact with water. The former tends to decrease whereas the latter
tends to increase the interfacial area per molecule. These two opposing forces lead to the
Figure 3.2. Models of lyotropic liquid crystalline phases: (a) nematic order of rod-like micelles; @) hexagonal array of aggregates; (c) lamellar amy; (d) cubic armogement of spherical aggregates
concept of an optimal area per headgroup at which the total interaction energy per amphiphile
molecule is minimum. Geometric packing properties of different amphiphiles are expressed
in terms of the 'packhg parameter" VI& where v= chain volume, a,,= head group ma, le=
chain length, which determines the type of aggregate that forms.
33. Chemistry of Octacaicium Phosphate
The presence of HPO:- groups in bone apatites has suggested that octacalcium
phosphate (OCP) is a likely precursor to hydroxyapatite (OHAp) formation in bone. In
fact, spontaneous hydrolysis of OCP has been observed to result in the formation of
nonstoichiometric OHAp (ns-OHAp)." Nonstoichiometric apatite cm exist in three forms:
OCP-OHAp intergrowths, imperfectly hydrolyzed OCP, and OHAp formed directly
without a precursor.' Since the third possibility is not probable under physioiogical
conditions, the presence of nonstoichiometric apatites in the early stage of bone formation
as well as in some mature ones has suggested an OCP as a precursor. OCP serves to
establish the final morphology, composition, solubility, and interfacial energy of apatitic
materials, as well as controlling the nucleation and growth of OHA~: These properties result
from the f a a that OCP and OHAp have similar crystal structures and are able to epitaxially
grow together. As a consequence of this relationship, a prime consideration in the preparation
and performance of a bone implant materid is the selection of a surface that is able to induce
nucleation of OCP.
The structure of OCP is shown in Figure 3.3: Every second layer is the same as in
the hydroxyapatite (OHAp) lattice and the layers sandwiched in between are the "ydrated
layers". OCP and OHAp phases are able to grow together to form interlayered mixtures
without a concomitant large increase in interfacial energy. Interlayered mixtures, depending
on the thickness of the layes c m give two kinds of X-ray diffraction patterns. When the
layers are relatively thick, the two kinds of layen ciifha independently, givhg the
appearence of a physical mixture of the two salts. When the layers are thin and randomly
variable in thichess, the dm peaks of OCP interact with those of OHAp ousing the
3-7
variable in thickness, the d, peaks of OCP interact with those of OHAp causing the
positions of the mmbined peaks to shift with tbe Ca/P ratio of the interlayered ~rystals.~
Figure 33. Projection onto the (001) plane of the structure of OCP; the unit cells of
OCP (lower left) and the apatite (upper right) are shown
In addition, due to the similarity in the crystal structures, the diffraction patterns of the two
phases are very simüar (Appeodix A). Therefore, if the characteristic d,, peak of OCP is
absent (due to preferential orientation, for example) misassignment is often made in that
presence of nonstoichiometric apatite is assumed instead of mixtures of OCP and OHAp.
An important feature of the morphology of OCP is that it alrnost invariably occurs
as platy (100) crystals. In the cases where OCP acts as a precursor for the formation of
apatite, the latter crysmllizes in a plate-like fashion even though hexagonal needles are to be
expected. Therefore, OCP serves as a template for the growth of OHAp. The platy nature of
OCP expresses itself variously in the fom of platelets, elongated blades, nbbons, f h s y
sheers or broad plates, frequently clustered in the f o m of rosettes.
Except under conditions of very high pressures, OCP is thermodynamically less
stable than OHAp and would not f o m were it not for its ability to grow rapidly? This
kinetic advantage is probably the main reason why OCP frequently foms as the initial phase
instead of OHAp and undergoes spontaneous hydrolysis to the more stable phase OH&. As
a matter of experience, it appears that the hydrolysis results in the formation of non-
stoichiometric apatite that contains impurhies and defects." The imperfectly hydrolyzed
OCP *in be divided into two types: crystals formed under conditions where growth of OCP
is not as fast as its hydrolysis so that the product contains mostly defective OHAp and very
little OCP or the crystals formed when growth of OCP is fast in cornparison to the
hydrolysis. In the latter case, a substantial amount of OCP could be present and its
subsequent hydrolysis would produce highly non-stoichiornetric hydroxyapatite. The
following equations, (1) and (2) respectively, represent proposed models for hydrolysis
reactions in the presence and absence of a caZ+ source:
It can be seen nom equation (2) that the hydrolysis process in the absence of calcium ions
involves l o s of phosphate ions fiom the crystal and the =action may cease after
approximately 50% of the phosphate has been released by the crystal. This is an additional to
the effects that result in a formation of a non-stoichiometric apatites.
Refer to Chapter 2, Section 2.1.
3.5. Results and Discussion
3.5.1. Calcium Phosphate Growth on TiO, Surface
Figure 3.4. displays SEM images of samples obtained after 7,22,30 and 48 hours of
growth. During the early stages (7 hours) srna11 1 pm sized aggregates are observed, which
upon increased growth time sente as nucleation sites for subsequent growth of larger plate-
like crystds. The crystal plates appear oriented perpendicular to the substrate and upon
further growth increase in size and coalesce to fom "rose-like" structures. The entire
substrate surface is unifody covered within approximately 30 hours. Lri addition, the
coating does not have the texture of a dense calcium phosphate film but instead displays a
porous structure with an average pore size of approximately 1-4 p. As remarked in Chapter
1, section 1.4.1, a porous apatitic structure is advantageous in certain bone implant situations.
It should be noted that OCP crystds and not OHAp are commonly associated with a plate-
based rosette morphology?
Figure 3.4. SEM images of samples grom after (a) 7 hours; @) 22 hours growth time
Figure 3.4. SEM images of the samples grown aher (c) 30 hours; (d) 48 hours growth tirne
.S. Cross :ts the int
Figure 3.4. (e) SEM image of sample after 72 hours growth t h e
#owth time; das te film
Cross-sectional TEM imaging studies of these films reveded direct contact of the
calcium phosphate crystals with the T i 0 substrate, Figure 35. The crystals appear oriented
with the long c-axes perpendicular to the substrate, consistent with the PXRD and SAED
results (see below). The integrity of the resulting nIms, irrespective of the growth time, was
maintained even &ter exîensive washing and the films have excellent shelf-Me. It required
the application of considerable force with a razor blade to even remove part of the film from
the substrate, indicating an impressive strength of adhesion.
In order to identify the phase(s) formed, PXRD studies were conducted on the
obtained film samples. The resuits are displayed in Figure 3.6. The PXRD pattern in Figure
3.6a is of the film on the Ti02 surface after 72 hours growth t h e . This pattern was observed
after 30 hours and before this period nothing was discemible as the N m was insufficiently
thick to observe a diffraction pattern. The peaks are well indexed to a hydroxyapatite phase
and the high relative intensity of the 002 reflection indicates preferential orientation of the
crystds dong the (001) plane (Appendix A). If the sample contained OCP oriented with the
(001) plane parallel to the surface then the most intense reflection from the (100) set of planes
would not be seen. When the sample was scraped from the surface and ground, a peak at 18.8
A was observed that is assigned to the 100 reflection of OCP, Figure 3.6b. Aside from this
low angle PXRD peak, the remaining peaks are indexable to a hydroxyapatite and oot an
octacalcium phosphate phase. From these results it can be concluded that the material which
forms on the TiO, surface most likely consists of an integrown mixture of OCP and OHAp,
the latter being the predominant phase and the former a minor one.
The thickness of the calcium phosphate films grown on the TiOl surface as a
funaion of growth M i e is s h o w in Figure 3.7. The values were obtained by surface opticd
profïlometry, (refer to Chapter 2, Section 225). During the first 22 hours, the surface is not
completely covered and the growth rate is determined primarily by the slow nucleation step.
Upon increasing the growth t h e , the rate appears to linearly increase and suggests that the
growth rate is no longer controlled by the nucleation rate on the substrate but is instead
18-98 A = (100) OCP 825 A = (100) OHAp 3.43 A = (002) O H A ~ 3.17A=(102)OHAp 2.81 A = (21 1) OHAp 2.77 A = (1 12) 0HAp 2.71 A = (300) 0HAp
Figure 3.6. PXRD patterns of (a) film on the TiOz surface after 72 hours growth tirne; (b) film scraped fiom the substrate and ground into randody oriented powder.
O 20 40 60 80 100 1 20 140
time (hours)
Figure 3.7. Thickness of calcium phosphate films as a function of growth time obtained by surface pronlometry; emor bars too srnall to be seen
dependent upon the production of the earlier forrned hydroxyapatite crystals.
In order to further elucidate the role of OCP and OHAp in the formation of these
films, time dependent Fï-IR microscopy studies were performed. It should be noted that the
FT-IR spectra of hydroxyapatite have two ranges of interest: v, (680-710 cm-')' v, (1000-
1200 cm").' Slight changes in the phosphate ion environment are readily observed by the
splitting of the degeneracy of the v, vibration." This region can be further subdivided into
two components: 1000-1085 and 1085-1200 an-' where the lower mmponent is generally
more intense. In stoichiometric apatites (s-OHAp) vibrations anse from symmetric v, and
antisymmetric v, P-O stretching modes in the region 950-1200 cm-' and v, antisymmeaic P-
O bending modes at 680-710 cm". In nonstoichiometric apatites (ns-OHM), the presence of
HPo,'' groups and vacaacies distort the pattern of P-O stretching modes. These give rise to
additional bands which have been extensively assigned and tabulated in the literat~re."~'~
- 7 hours - 22 hours - 30 hours - 48 hours - 72 hours 96 hours
8 0 0 900 1000 1100 1200 1 3 0 0
W ave numbers (cm-')
Figure 3.8. FT-IR mimscopy spectra of calcium phosphate films after different growth t h e
In addition, the OCP phase gives rise to IR spectra that are quite distinct h m s-OHAp and
ns-OHAp." The presence of bands at 830, 910 and 1200 cm-' are diagnostic of the OCP
phase.
Figure 3.8 displays the FT-IR microscopy spectra of the nIms grown on titania. It
can be seen that after 7 hours, characteristic bands of ~0,'- ions are present in the sample.
This signals the presence of a calcium deficient ns-OHAp phase. in addition, characteristic
peaks from OCP are also present, specifîcally at 867 an", 917 cm-' and 1200 cm-'. These
bands were however, only observed after 48 hours of growth time. It shouid be noted that
under the synthesis conditions used to grow these nIms, OCP is commody believed to be a
precursor to OHAp. The observance of OCP only after 48 hours implies that at earlier h e s
in the growth process, the amount of OCP present was insufncient to be detected by this FT-
IR microsmpy technique. It may be suggested that the absence of OCP is likely due to i a
hydrolysis to OHAp. As was mentioned for Figure 3.7, initially, the growth rate of the film is
slow. During this induction t h e OCP is able to convert to OHAp and hence rnay account for
it not being detected by FT-IR. However, once the growth rate increases (after 30 hours), the
rate of hydrolysis of OCP to OHAp is comparably slower and there exists the likelihood of
formuig an intergrown OCP/OHAp mixture a s recognized by the presence and subsequent
increase of OCP P-O vibrational modes in the FT'-IR specaum. This proposa1 receives
support from selected area electron diffraction studies of the films as described below.
Electron diffraction (ED) patterns obtained are displayed in Figure 3.9. These were
obtained using a small aperture size in order to ensure that the cross sectional area examined
belonged to a selected small region of one crystal and was approximately 0.1 pm in size. This
was also done to prevent contributions from streak and ring distortions of the ED patterns
arising from the expected mosaicity of the samples. The patterns obtained were indexed to
the hexagonal lanice of hydroxyapatite viewed dong the 4210, and c1213> zones, Figure
3.9a. Electron diffraction of the triclinic lattice down the OCP <010> zone was also
observed, Figure 3.9b. It should be noted that on scanning numerous areas of the films, the
F i r e 39. Selected Area Electron Diffraction (SAED) of (a) <1210> zone of hydroxyapatite; (b) <01b zone of octacalcium phosphate
rnajority of the patterns obtained belonged to the OHA. phase and the minonty were for
the OCP phase. The zones observed mnfïmed the presence of preferred orientation of both
phases. This is because the electron beam was parallel to the substrate which suggested that
the zones observed contained ody the information regarding planes that were parallel to the
sub~trate. Therefore, the (001) and (101) planes for both OHAp and OCP phases are parael
to the substrate as they are contained within the zones observed (Appendix B). In addition,
the OCP electron diffraction patterns were obtained from crystals mostly on the edges and
top parts of the hyàroxyapatite crystals that were the furthest away fiom the substrate. This
further confirmed the m'-IR resdts that the presence of OCP could only be detected after
prolonged growth times.
In order to further determine the interaction between the substrate and the calcium
phosphate film, W S studies were performed. Table 3.1 lists the atornic percentages and
binding energies of the representative elements. The presence of both calcium and
phosphorus was confinned after only one hour of deposition t h e with a ratio of Ca/P-111.
This can be explained by the fact that at pH = 7.5, the surface of Ti02 is known to be
hydroxylated and Br@nsted acid and base hydroxyl sites exist at the surface that cm aa as
nucleation/anchoring centen for calcium and phosphate ions.
An illustration of a clean and hydroxylated (110) Ti02 surface" is shown in Figure
3.10. Such a TiO, surface has 2- and 3-coordinate bridging oxygens and 5- and 6-coordinate
titanium. M e r exposure to water the surface becumes hydroxylated and two different types
of OH groups exist on the surface. One is denoted as acidic, the other as basic and these are
distinguishable by means of XPS as seen in Figure 3.11a and wnfirmed by reference to the
published ~iterature.'~"
\
-
4
6 Cri in
6 - m iA
- * Cr)
2 - 2 TP Cc,
Clean Surface
2-fold 3-fold 6-fold Ti bndging O bridging O 1 5-fold Ti
I I
hydroxyl groups hydroxyl groups i l
Hydro~lated Surface
acidic basic
Figure 3.10. Representation of clem and hydroxylated (110) surfaces of rutile
Binding Energy (eV) 7 Hours
Binding Energy (eV)
L -535 -530 -525
Bmding Energy (eV) -536 -532 -538 -524 -520
Binding Energy (eV)
Figure 3.11. XPS spectra of O 1s (a) TiO, sinface before immersion in calcium phosphate solution; (b) T i 4 sinface at different growth tunes
Figure 3.12. XPS spectra of Ca 2p core electrons after different growth periods
Quantitative WS studies of the nucleation and growth of these films shows that the atomic
concentrations of Ca and P do not change in the first three hours after which they increase
slightly with the . The h t 3 hours can therefore be considered as an induction-nucleation
period, followed for the next 30 hours by a nucleation-crystal growth period. Note the
disappearance of the titanium signal (Table 3.1.) indicahg cornplete average.
The oxygen 1s binding energy spectra are shown in Figure 3.11b. During the first
three hours, the ratio of the bulk to surface oxygen signal decreases due to the higher degree
of hydration of TiOl in the aqueous solution and du, because of the presence of phosphate
ions. Also after this tirne differential charging of the substrate and film is apparent. There are
two sets of signals ffom equivalent atorns, one fiom atoms close to the surface that are not
charging and the other from atoms hirther from the surface that are charging. Signals from
oxygen in hydroxyapatite overlap with oxygens from the substrate surface. This charging is
therefore indicative of the formation of the calcium phosphate crystals. M e r 48 hours of
growth time the surface is completely covered and the W S signal originates solely ffom the
calcium phosphate. Two peaks are observed with binding energies at 531 eV which is the
reported value for oxygen associated with the phosphate group in hydroxyapatite and the
second at 533 eV which is characteristic of oxygen in the HPO,~' group and intercrystalline
water.lC16 The same charging effect is seen with the calcium and phosphorus signals. Figure
3.12. is showing representative XPS spectra of Ca 2p core electmns. The binding energies of
Ca 2p and P 1s at 347.3 eV and 133.3 eV respecîively, are in agreement with the values
reported for hydroxyapatite. "16 The ratio of Ca/P oscillates around the value of 1.3 in
samples obtained after the nucleation penod was over (longer than 3 hours) which points to
the presence of a ns-OHM. This is in agreement with the FT-IR microscopy resdts
presented above.
In order to m e r prove that OCP is a prerequisite for the growth of the obtained
OHAp phase, controi experiments were perfonned. When the conditions of the synthesis
were changed in a way so as to favor OHAp nucleation rather then OCP such as, higher pH,
higher temperature and the presence of fluoride, no crystal growth was obsenred even a€ter
five days. W S spectra showed an interesting result whereby the presence of calcium and
phosphorous was in fact observed, albeit trace arnountso and in a 111 ratio. These values
however, did not change upon increased growth tirne. Conversely, when a lower pH (< 7)
was used which would ailow for nucleation of OCP, again film formation was not observed.
XPS spectra showed the absence of calcium or phosphorus on the substrate surface.
Taken together, these resdts cm be explained by the faa that in the case of
favorable OHAp nucleation, the surface of the substrate has both acidic and basic
aaivation/ancho~g sites to accommodate both calcium and phosphate species but the
inability of OCP to nucleate prevented any crystd growth. On the other hand, with a lower
pH, OCP could nucleate but the surface was mainly acidic which prevented calcium and
phosphorous from simul taneously binding to the surface. It is concluded therefore that the
presence of OCP as a precursor phase and the pH of the initial solution are both pivotai
factors that have to be controlled for the successful growth of porous, oriented and thick
crystalline hydroxyapatite films on the surface of TiO, on Ti.
35.2 CaDDP-Cap Composite on TiO, Surface
In Figure 3.13 PXRD diffraction patterns of Ti-TiO-OHAp and Ti-TiO-OHAp-
CaDDP films are displayed. It is evident that the phosphate ester surfactant forms a CaDDP
lamellar phase, with interlayer d-spacings of 40 A and 37 A under the experimental
conditions, and that its presence does not advenely affect the growth of the OHAp mineral
phase. The two Werent interlayer d-spacings probably correspond to distinct degrees of
interdigitation or tilting of the hydrophobic tails of the surfactant in the bilayer. The intensity
of the characteristic peaks of hydroxyapatite did not decrease upon formation of the CaDDP
which implies that no dissolution occurred during the hlm growih process.
A SEM micrograph of the calcium dodecylphosphate-hydroxyapatite (CaDDP-
OHAp) sample after one day growth tirne is s h o w in Figure 3.14. It can be seen that the
CaDDP phase is coating the füm of porous oriented hydroxyapatite crystals and that it is
without CaDDP
w
Figure 3.13. PXRD diffraction patterns of Ti-TiO-OHAp (top) and Ti-TiO-OH@-CaDDP @onom)
effectively following the contour surfaces of the crystals. The thickness of the CaDDP
lameliar phase varies £rom approximately 1 to 5 W. Figure 3.15 displays a TEM micrograph
of the same sample. It can be seen that the surface of the growing CaDDP lamellar phase is
onented parallel to the OHAp mineral long c-axes and moreover is organized in close contact
with the crystals. This micrograph is representative of the sample as a whole. It should be
noted that the same type of CO-assembly phenornenon was observed previously with
powdered foms of the CaDDP-OHAp composite material." The authors of this earlier work
proposed a model which could rationalize the formation and preferred orientation of the
composite material. The model assumed stereochernical, geometrical and
Figure 3.14. SEM micrographs of the calcium dodecy lphosphate-h ydroxy apatite composite film
Figure 3.15. Crosssectional TEM micrographs of the calcium dodecylphosphate- hydroxyapatite composite film
charge mmplementarity between the calcium and phosphate ester sites in the surface region
of the CaDDP 1ameUa.r phase and calcium and phosphate sites in the [O011 face of the
hydroxyapatite crystal lattice, with some degree of protonation of the interfacial phosphate
groups. Since hydroxyapatite fomed on TiO, is a nonstoichiometric HPO,~ containing
phase, it is expected that some of these HPO:- groups wiII also be located in the s d a c e
regions, particulariy because the source of the phosphate used is K-O, and the pH of the
solution was 7.4. This provides credence for the previously proposed mode1 of the CDDP-
OHAp chernical composite material." An illustration of the architecture of the CDDP-
OHAp-TiO-Ti composite Nm established in this study is shown in Figure 3.16.
Figure 3.16. Illustration of the architecture of CaDDP-OHAp-Ti0,-Ti composite material
The andogy between the construction of this purely synthetic multi-layer calcium
phosphate-based organic-inorganic composite film and the hierarchical structure of bone
grown in vitru on a planar substrate h m a bone cultue medium, can be appreciated by
cornparison of Figures 1.7. and Figure 3.16. Although the a d constituents and building
niles are not the same, the design and assembly phciples of the films have aspects in
common.
Fast growth of oriented hydroxyapatite crystalline films on a TiO, surface occurs as
a consequence of precursor OCP nucleation on distinct Brmsted acidic and basic anchoring
sites of the substrate and its in-situ hydrolysis to the more thermodynamically stable OHAp
phase. Porosity, nonstoichiometry and the rate of the OHAp film formation under
physiologicai conditions on the sputter deposited TiO, surface render this materiai as a viable
mating for bone implant materials. The presence of HPO,~ groups are found to be crucial not
only for ensuring chemical compatibility with the bone mineml phase but aiso as a key
cornponent for facilitating the formation of an organic-inorganic composite material. The
synthesis protocol of growing the OHAp and CaDDP phases separately, provides a hi@ level
of control over the thiclmess and composition of each component. It also allows for
alternation of these phases in a rnulti-layer composite construction, as well as a means for
incorporatiodrelease of bioactive substances fiom the hydrophobie region of the CaDDP
lamellar phase, that could improve the behaviour of this type of material in a body
environment.
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Appen& A: PXRD patterns of OCP (top) and OHAp @ottom)
Appendb B: Simulated e l emn diffraction patterns for [O101 zone of OCP (top) and
[1210] zone of OHAp
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