15

CHAPTER 3

LITERATURE REVIEW

The literature reviews presented in the thesis are classified into four
major domains namely, Bone science, Bone grafting, Bone substitute
materials and Hydroxyapatite.

3.1 BONE SCIENCE

Bone is a beautiful substance with an intricate structure. To

simplify quite a bit, the basic unit is the Haversian system, which is a hollow,
laminated rod of collagen and calcium phosphate; the hollow core is a nutrient
channel, the Haversian canal. Within the shaft of a long bone, many of these
Haversian systems are bundled together in parallel, forming a kind of bone
called compact bone which is optimized to handle compressive and bending
forces. Near the ends of the bones, where the stresses become more complex,
the Haversian systems splay out and branch to form a meshwork of cancellous
or spongy bone.

3.1.1 Introduction

Bone is one of the most important and fascinating bio mineralized

nano composite structures in nature. On a macroscopic scale, the bone
provides the structural support for our bodies, protects our internal organs,
provides a reservoir for the production and storage of hematopoietic and
mesenchymal stem cells (MSC) and serves as a storage site for calcium,
phosphate and other biologically relevant ionic species.
 16

It has become the focus of intensive study in the light of the rising
average population age and associated bone diseases. In recent years, it has
become increasingly clear that, in addition to the insights achieved in the
biological sciences and medicine, information on the nano scopic architecture
of the bone is needed to understand and predict bone fracture (Fratzl et al
2004).Bone consists mainly of mineralized collagen fibrils. The collagen
fibrils, being the main organic component in bone, are reinforced with nano
scale hydroxyapatite particles (Rosen et al 2002). This results in a mineral
reinforced protein fibril of 50100 nm diameters. These fibrils are the
elementary building block for the large variety of bones in the body. To
facilitate the function of the specific bone, they are arranged in several
possible patterns.

In addition to its biomedical significance, the bone has been used as

a model for many artificial bio-ceramic composites (Kikuchi et al 2004,
Zhang et al 2004). In many of these artificial composites, a combination of a
soft polymer matrix reinforced with stiff particles is used as an approximation
of the interaction between collagen and hydroxyapatite. Such materials are
based on the crystalpolymer interactions on the molecular and nano scopic
level. In this research, the aim is to present additional strength increasing
mechanisms in the bone that may add to the quality of the artificial bio-
mineral composites.

3.1.2 Hierarchical Structure of Bone

The histology of bone tissue offers a wealth of information to

describe the anatomy of the femur bone. The complicated structure of the
bone exhibits anisotropic mechanical stress with respect to direction; hence,
an introductory anatomical description on bone morphology is essential for
material scientists.
 17

Figure 3.1 The hierarchical structure of bone, from macro to nano-

assembly (Murugan and Ramakrishna 2005)

In terms of structure, the typical human femur (Figure 3.1) is

primarily composed of two separate types of bone; compact or cortical and
cancellous or spongy bone and is capable of accommodating several modes of
stress, from a stationary condition to brisk walking (Cowin et al 1987).
Trabecular bone generally resides at the ends of bones and is porous (50-
90%), lighter, and more energy absorbent than the compact bone, providing
effective cushioning for bone impact (Van der Meulen et al 2001). The
longevity of bone is directly and/or indirectly related to the bone composition
and morphology; a typical bone matrix is bundles of collagen fibers infiltrated
by a crystalline bio apatite mineral (40% by volume, 60% by weight),
which resembles with synthetic hydroxyapatite (Murugan and Ramakrishna
2005).

Figure 3.1 reveals the basic shape of a femur, which is strongly

influenced by cortical and trabecular bone. A tissue called the periosteum
covers the bone and brings in blood, the lymph vessels and the nerves. The
central cavity of the femur is filled with bone marrow, where stem cells give
rise to all the types of blood cells.
 18

As with all biological tissues, the cortical bone contains many

different structures that exist at many levels of scale (Sarkar et al 2008). It is
much denser than other types of bone and is found primarily in the shaft of
long bones, forming the outer shell around cancellous bone at the ends of
joints and around vertebrae. The cylindrical Haversian canal (~10 m to
~30 m in diameter) controls the transportation of blood, lymph vessels and
nerves within the cortical bone (Figure 3.2). Around the Haversian canal a
group of concentric circular lamellae are present and these the lamellae are
roughly 3 m to 7 m thick. Moreover, within and between lamellae are found
small hollow gaps (~10 m in diameter) that are interconnected by small
canaliculi (~1 m in diameter), usually running perpendicular to the
circumference of the Haversian systems (Sarkar et al 2008). These gaps are
called lacunae.

Figure 3.2 Typical compact bone with Haversian system (Aubin et al 1996)

Figure 3.3 illustrates that the cancellous bone has an

interconnecting porous architecture made up of curvilinear struts called
trabeculae. The trabecular bone appears at the ends of long bones and the
 19

vertebral body. Its basic first-level structure is the trabeculae (Cristofolini et al

1996). The trabecular bone contributes about 20% of the total skeletal mass
within the body, while the remaining 80% is contributed by the cortical bone.
However, the trabecular bone has a much greater surface area than the cortical
bone. The trabecular bone is more compliant than cortical bone and is
believed to distribute and dissipate the energy from articular contact loads.

Figure 3.3 Porous trabeculae bone with osteon bone (Aubin et al 1996)

The osteonal bone is the combination of concentric rings of bone

tissue and blood vessels, as described in Figure 3.3. The size and shape of
bones not only changes during growth, but bones are continuously being
remodeled in response to the stresses put on them throughout life. It is
important to note that the osteoclastic differentiation is usually balanced with
bone formation to preserve the bone architecture over many cycles of bone
replacement. Osteoblasts contain an organic bone matrix that is heavily cross-
linked with type-I collagen.
 20

The bone matrix is aligned along the lines of stress and osteoblasts
deposit the hydroxyapatite mineral into the gaps of the matrix, resisting
compression. According to Wolffs Law, osteocytes are responsible for the
growth and changes in the shape of the bone, where they function as
neurotransmitter cells and determine the response of the bone to its
mechanical environment. Hence, the study of the hierarchical organization of
the bone has two purposes: first, it provides a consistent way to compare the
properties of different tissues with respect to the morphology and the
constituents; second, it provides a consistent scheme for defining the levels
for computational analysis of the tissue micromechanics.

3.1.3 Functions of bone tissue

The skeleton, which consists mainly of bone tissue, forms a

supportive framework, giving shape and rigidity to the body.

The bone tissue forms a system of levers to which the voluntary

muscles are attached.

It serves to protect the soft and delicate organs of the body such
as the skull protecting the brain.

The red blood cells are manufactured in the red bone marrow,
which is situated in the spongy tissue at the ends of the long
bones.

The bone plays a part in homeostasis because it helps to

maintain a constant level of calcium in the blood.

The bone buffers the blood against excessive pH changes by

absorbing or releasing the alkaline salts.
 21

The bone tissue removes the heavy metals and other foreign
elements from the blood and thus reduces their effects on the
nervous and other tissues. It can later release these more slowly
for excretion.

3.2 BONE GRAFTING

Bone grafting is a surgical procedure by which a new bone or a

replacement material is placed into spaces between or around the broken bone
or holes in the bone defects to aid in healing. Bone grafting is used to repair
bone fractures that are extremely complex, pose a significant risk to the
patient, or fail to heal properly. Bone graft is also used to help the fusion
between vertebrae, correct deformities, or provide structural support for
fractures of the spine. In addition to fracture repair, bone graft is used to
repair defects in the bone caused by birth defects, traumatic injury, or surgery
for bone cancer.

3.2.1 Current Trend in Bone Grafting

When the normal physiologic reaction to fracture does not occur,

such as in fracture non unions or large-scale traumatic bone injury, surgical
intervention is warranted. Autografts and allografts represent current
strategies for surgical intervention and subsequent bone repair, but each
possesses limitations, such as donor-site morbidity with the use of auto graft
and the risk of disease transmission with the use of allograft. Synthetic bone-
graft substitutes, developed in an effort to overcome the inherent limitations
of autograft and allograft, represent an alternative strategy (Murugan and
Ramakrishna 2005).These synthetic graft substitutes, or matrices, are formed
from a variety of materials, including natural and synthetic polymers,
ceramics, and composites, that are designed to mimic the three-dimensional
 22

characteristics of auto graft tissue while maintaining the viablecell

populations.

Matrices also act as delivery vehicles for factors, antibiotics, and

chemotherapeutic agents, depending on the nature of the injury to be repaired.
This intersection of matrices, cells, and therapeutic molecules has collectively
been termed tissue engineering. Depending on the specific application of the
matrix, certain materials may be more or less well suited to the final structure;
these include polymers, ceramics, and composites of the two. Each category is
represented by matrices that can form either solid preformed structures or
injectable forms that harden in situ.

3.2.2 Auto Graft

Autogenous bone grafts are also called autografts; these types of

grafts are made from the patient's own bone, harvested from elsewhere in the
body. Typical harvest sites include the chin, jaw, bone of the lower leg (tibia),
hip (iliac crest) or the skull (cranium). Autogenous bone graft has traditionally
been considered the "gold standard" as a graft material because it is "live
bone" complete with the living cellular elements that enhance bone growth. A
potential downside of autogenous bone grafting, however, is that it involves a
second procedure to harvest the bone, which may be painful and not in some
patients' best interest, depending on their condition. It may not be a viable
option either in instances where the patient's overall bone quality and/or
density is poor, or when a large volume of graft material is required.

3.2.3 Allograft

The allogeneic bone, also called allograft, is bone derived from a

genetically unrelated member of the same species. It is typically non-vital
(dead) bone harvested from a cadaver, and then processed using a freeze-
 23

drying method that extracts all the water via a vacuum. An allogeneic bone
cannot produce new bone on its own; it is neither osteogenic (like autograft)
nor osteoinductive (like BMP). Rather, its primary mechanism of action is
that it is osteoconductive, and serves as a framework or scaffold over which
the bone from the surrounding bony walls can grow to fill the defect or void.

3.2.4 Xenograft

Similar to the allograft bone, xenogenic bone is non-vital bone

derived from another species, usually a cow. Because the potential for
immune rejection and contamination by viral proteins is higher in bovine
bone than in human cadaver bone, xenograft material is processed at very
high temperatures (600-1,000 oC). Xenograft's mechanism of action is similar
to that of allograft. It serves as an osteoconductive framework on which the
bone from the surrounding area can grow to fill the void.

3.2.5 Alloplastic

Alloplastic grafts are inert, manmade synthetic materials. The

modern artificial joint replacement procedure uses metal alloplastic grafts. For
bone replacement a man-made material that mimics the natural bone is used.
Most often this is a form of calcium phosphate. Depending on how it is made,
it may be "resorbable" or "non-resorbable". That is, the human body may or
may not replace the alloplastic graft with the natural bone. In those cases
where it is not replaced it acts as a lattice or scaffold upon which natural bone
is built. In either case, the end result is to create enough bone for the
placement of dental implants.

3.2.6 Market survey

In 2000, the worldwide use of bone grafts was estimated to be

about 1 million, of which about 15% of the surgery is used synthetic bone
 24

grafts. It was also suggested that the future growth largely attributed to tissue-
engineered composites, i.e., composites containing osteogenic cells and
growth factors (Murugan and Ramakrishna 2005).

Now, more than 500,000 bone graft procedures are performed in

the US each year and approximately 2.2 million worldwide. The estimated
cost of these procedures approaches $2.5 billion per year.

The Brazilian, Indian, and Chinese (BIC) market for orthopedic

biomaterials, consisting of bone graft substitutes (BGS) is expanding as
procedure volumes and disposable incomes continue to grow in these
countries. Procedure growth is being driven primarily by the aging population
and the associated increase in age-related ailments, growing awareness of and
improved accessibility to treatment, as well as the expanding medical tourism
industry. Although improving, accessibility to medical treatment and
reimbursement issues continue to inhibit procedure volumes and market
potential to some extent; patients in rural areas do not have access to the
higher-quality medical care that is focused on urban areas. The presence of
alternative and more affordable traditional treatments such as ayurveda, yoga
and naturopathy, Unani, Siddha, and homeopathy in India, and traditional
Chinese medicine, such as tai chi or acupuncture will also continue to limit
the markets potential, especially in the bone grafting market. Nevertheless, as
the respective governments in these growing economies continue to invest in
health care, the accessibility and reimbursement issues will become less
inhibitive. Moreover, as the discretionary income for the average citizen
continues to rise, there will be an increasing shift toward BGS surgeries.

3.3 BONE SUBSTITUTE MATERIALS

Various bone grafts and bone substitute materials are given in

Table 3.1. These include
 25

(a) Harvested bone grafts and graft substitutes

Bone grafts, endogenous or exogenous, are often essential to

provide support, fill voids, and enhance biologic repair of skeletal defects due
to traumatic or non-traumatic origin. The limitations of the use of endogenous
bone substance involve additional surgery; often resulting donor site
morbidity and limited availability whereas the allograft has been encountered
with the risk of disease transmission and immunogenicity. Therefore, there is
a growing need for the synthesis of allograft bone substitute used alone or in
combination with other materials (e.g., Allogro [AlloSource, Centennial,
Colo], Opteform [Exactech, Inc, Gainesville, Fla], Grafton [BioHorizons,
Birmingham, Ala], OrthoBlast [IsoTisOrthoBiologics, Irvine, Calif]).

(b) Growth factor-based bone graft substitutes

Natural and recombinant growth factors used alone or in

combination with other materials such as transforming growth factor-beta
(TGF-beta), platelet-derived growth factor (PDGF), fibroblast growth factor
(FGF), and bone morphogenetic protein (BMP).

(c) Cell-based bone graft substitutes

The use of cells to generate new tissue alone or are seeded onto a
support matrix (e.g., Mesenchimal stem cells).

(d) Ceramic-based bone graft substitutes

These include calcium phosphate, calcium sulphate, and bioglass

used alone or in combination (e.g., OsteoGraf [DENTSPLY
FriadentCeraMed, Lakewood, Colo], Norian SRS [Synthes, Inc, West
Chester, Pa], ProOsteon [Interpore Cross International, Irvine, Calif],
Osteoset [Wright Medical Technology, Inc, Arlington, Tenn]).
 26

(e) Polymer-based bone graft substitutes

Degradable and non-degradable polymers are used alone or in

combination with other materials (e.g., Cortoss [Orthovita, Inc, Malvern, Pa],
open porosity polylactic acid polymer [OPLA], Immix [Osteobiologics, Inc,
San Antonio, Tex]).

(f) Miscellaneous

Various unconventional marine biomaterials are also in use as bone

graft substitute which includes coral, chitosan, sponge skeleton etc.

Table 3.1 Bone grafts and bone substitutes (Nandi et al 2010)

Properties of
Class Description Examples
action
Osteoconductive
Autograft
Used alone Osteoinductive
based
Osteogenic
Allegro,
Allograft bone used
Allograft based Orthoblast, Osteoconductive
alone or in combination
Grafton Osteoinductive
with other materials

Natural and Both

recombinant growth osteoconductive
Factor based TGF- , PDGF,
factors used alone or in and osteoinductive
FGF, BMP
combination with other with carrier
materials materials
Cells used to generate
steoconductive
Cell based new tissue alone or Mesenchymal
with carrier
seeded onto a support stem cells
materials
matrix
 27

Table 3.1 (Continued)

Properties of
Class Description Examples
action
Includes calcium
steoconductive
phosphate, bio inert
Osteograft, Limited
ceramics like alumina,
Ceramic based Osteoset, osteoinductive
titania, zirconia,
Nova Bone when mixed with
calcium sulfate, and
bone marrow
bioactive glass used
alone or in combination
Includes degradable and
steoconductive
non degradable
Polymer based Cortoss, Bioresorbable in
polymers used alone
OPLA, Immix degradable
and in combination with
polymer
other materials
Miscellaneous Coral HA granules, ProOsteon Osteoconductive
blocks and composite Bioresorbable

3.3.1 Essential Factors for an Ideal Bone Graft

The ideal bone-graft substitute is biocompatible, bioresorbable,

osteoconductive, osteoinductive, structurally similar to bone, easy to use, and
cost-effective. Within these parameters a growing number of bone graft
alternatives are commercially available for orthopedic applications, including
the reconstruction of cavitary bone deficiency and augmentation in situations
of segmental bone loss and spine fusion. They are variable in their
composition and their claimed mechanisms of action.

Biocompatibility is a general term used to describe the adaptability

of a material for exposure to the body or body-fluids. Biocompatible materials
are generally non-inflammatory, non-toxic, non-carcinogenic and non-
immunogenic, or have other suitable physical properties. The specific
meaning of biocompatibility is dependent on a particular application or
 28

circumstance. In fact, there are no completely biocompatible materials.

However, the continuous success of many medical devices and bone implants
is contingent upon the successful interaction of the biocompatible materials
and various body tissues.

Traditionally, metallic materials such as stainless steel, Ti alloys

and cobaltchromium alloys have been widely used as bone implants in
orthopedic applications (Healy and Ducheyne 1996, Noort 1987, Zhuang and
Langer 1989). The use of materials stiffer than bone tissue may lead to
mechanical mismatch problems (e.g., stress shielding) between the implant
and the adjacent bone tissue, where the integrity of the bone/implant interface
may be compromised due to the resorption of bone tissue. On the other hand,
most polymers, by themselves, do not possess sufficient mechanical
properties to bear physiological loads. In order to minimize the stress-
shielding effect while maximizing the resistance to fractures, a polymer
matrix based material is a possible alternative. Ceramic reinforced (e.g. HAp)
polymer composites for different load-bearing orthopedic applications have
been significantly developed (Hasegawaa et al 2006, Itokawa et al 2007).

3.3.2 Osteoconductive

The main role of osteoconductive grafts is to serve as a structural

framework through which the host bone infiltrates and regenerate a new bone
tissue. Autogenous bone, allogeneic bone, HAp, and collagen are best
examples of osteoconductive bone grafts.

3.3.3 Osteoinductive

Osteoinductive grafts are capable of inducing differentiation of

undifferentiated stem cells into osteogenic cells or to induce stem cells to
proliferate.
 29

3.3.4 Osteogenic

Osteogenes is defined as the ability to produce new bone and is

determined by the presence of osteoprogenitor cells and osteogenic precursor
cells in the area. Both fresh autografts and bone marrow cells contain
osteogenic cells.

3.3.5 Bio Active Ceramics

Synthetic ceramic materials based on calcium phosphates (CaP)

particularly in the composition of tri calcium phosphate [TCP: Ca 3(PO4)2] and
hydroxyapatite have extensively been studied and clinically used. The
biomaterials research field has been focusing on the synthesis of these
ceramics for three decades to applications in orthopedics and dentistry (Liou
et al 2003). Their use is proposed because they exhibit biological affinity and
activity to surrounding host tissues when implanted. Furthermore, according
to the literature, calcium phosphates are widely used in medicine and oral
biology due to the apatite like structure of the enamel, dentin and bones,
usually called "hard tissues".

Calcium phosphate ceramics like hydroxyapatite, tricalcium

phosphate and tetra calcium phosphate, alumina, zirconia, silica based glasses
or bioactive glasses and also pyrolitic carbons, generally termed as
bioceramics, have been used as bone substitutes (Mastrogiacomo et al 2006,
Rush 2005, Giannoudis et al 2005).These materials range from inert to
bioactive based on their biological activity as they can remain unchanged,
dissolve or actively take part in physiological processes to enhance bone
tissue formation. Bioactive ceramics provide a suitable substrate for the
attachment of the cells followed by osteogenic differentiation directly on the
surface of the ceramic, which results in bone bonding (Hajime and
Caplan1999).
 30

Several studies indicated that growth hormones can stimulate bone

growth, collagen synthesis and can affect repair in vivo (Burg et al 2000).
Scaffolds have been developed containing osteoinductive proteins with and
without osteogenic cells utilizing various polymers, ceramics and polymer-
ceramic composite towards bone tissue engineering. Ceramics are brittle and
not suitable for load bearing applications in the case of orthopedic implants.
Numerous studies are being reported on polymer-ceramic composites for
improving the mechanical properties to match with the natural load bearing
tissues.

Bioactive ceramic materials like tricalcium phosphates,

hydroxyapatite and bio glass gained significant importance as scaffolds for
bone tissue engineering in recent times. These scaffolds can either induce the
formation of bone from the surrounding tissue or can act as a carrier or guide
for enhanced bone regeneration by cell migration, proliferation and
differentiation (Ramay and Zhang 2003, Ramay and Zhang 2004, Zhang and
Zhang 2002). The main issue regarding the applicability of bioceramics in the
filed of bone tissue engineering is its degree of biodegradability (resorption)
along with its poor mechanical strength. Nanotechnology can play an
important role in this regard to develop porous bioceramics with high
mechanical strength and enhanced bioactivity and resorbability.

The group of bioceramics includes hydroxyapatites, which, due to

their specific properties, are widely used in biotechnology. These compounds
exist in the skeletons of human and animal bodies. A range of advantages of
implants, which contain, among other things, hydroxyapatite, results also
from the level of their porosity.

An appropriate ceramic-polymer composite could be an acceptable

alternative material for use as the prosthetic bone in terms of accommodating
growth and maintaining the necessary physical properties. The similarities
 31

between the elastic modulus, compressive strength and tensile strength of the
human cancellous bone and HAp-bioactive ceramic-reinforced polymer
composite suggests the latter as a probable candidate for orthopedic implants
which may bear physiological levels of load.

3.3.6 Nano Composites for Bone Grafting

Nano composites could play a pivotal role in bone grafting as a new

class of bone graft material, which uses a combination of several nano scale
bone graft materials and/or in conjunction with osteoinductive growth factors
and osteogenic cellular components. The term nano composite can be defined
as a heterogeneous combination of two or more materials in which at least
one of those materials should be on a nanometer-scale.

Nano crystalline HAp promotes osteoblasts cells adhesion,

differentiation, and proliferation, osteointegration and deposition of calcium
containing minerals on its surface better than microcrystalline HA; thus
enhancing the formation of new bone tissue within a short period (Murugan
and Ramakrishna 2005). Current trends in HAp-based nano composites for
bone grafting applications are summarized in Table 3.2 (Murugan and
Ramakrishna 2005). Nano composites can be made, conventionally, by
blending or mixing a heterogeneous combination of two or more materials,
differing in morphology or composition in which at least one of the materials
should be on nano scale. Blending is a technique used to produce a composite
product with specific characteristics by combining at least two materials. A
novel way of fabricating nano composite bone grafts using strategies found in
nature has recently received much attention and is perceived to be beneficial
over conventional methods.

The term biomimetic process can be defined as a micro structural

processing technique that either mimics or inspires the biological mechanism,
 32

in part or whole (Green et al 2002). Nucleation and growth of HAp

crystallites onto collagen in a controlled fashion is perceived to be beneficial
to enhance the properties of nano composites. This process partly mimics the
biological phenomenon and provides a good system for bone regeneration
with enhanced osteoconductivity.

Table 3.2 HAp based nano composites (Murugan and Ramakrishna 2005)

Tissue engineered bone scaffolds of

HAp based nano composites
nHAp and its composites
Nanocomposite Methods of Bonetissue Cells/growth
systems Preperation scaffolds factors studied
HAp/collagen Precipitation nHAp Osteoblast-like cells
nHAp Mesenchymal
HAp/collagen Biomimetics
stem cells
nHAp Intended as tissue
HAp/collagen Biomimetics
scaffold
HAp/collagen Biomimetics nHAp Cell carrier
HAp/collagen/
Biomimetics nHAp Angiogenic factors
PLA
HA/collagen/
Biomimetics nHAp/collagen Mesenchymal cells
alignate
nHAp/collagen/
HAp/chitosan Precipitation Osteoblasts
PLA
nHAp/gelatin/
HAp/gelatin Biomimetics rhBMP-2
PLA
nHAp/collagen/
HAp/silk fibroin Mechano chemical Fibroblasts/osteoblasts
alginate
nHAp/collagen
HAp/PLA Solvent-casting osteoblasts
rhBMP-2

3.3.7 Applications of Bio composite Material in Medical Industry

Human bone and tissue are essentially composite materials with

anisotropic properties. The anisotropy of the elastic properties of the
 33

biological tissues has to be considered in the design criterion for implants

made from composite biomaterials. The solution to this is a new porous
resorbable ceramic-polymer bio composite, with morphology and a
mechanical resistance similar to those of natural cancellous bone. Moreover
surgeons can easily cut the graft directly in the surgery room to adapt its
shape to the defect. Since they offer both low elastic modulus and high
strength, they have been proposed for several orthopedic applications. Also by
controlling the percentage of the reinforcing and continuous phase the
properties and design of the implant can be tailored to suit the mechanical and
physiological conditions of the host tissues. Moreover problems of corrosion
and release of allergenic metal ions, such as nickel or chromium, are totally
eliminated.

Figure 3.4 Hip Replacement Figure 3.5 Linear Osteoblast

(Aubin and Liau1996) (Aubin and Liau1996)

The composite provides high fracture toughness and high resistance

against fatigue failure. These bio composites are highly compatible with
modern diagnostic methods, such as computed tomography (CT) and
magnetic resonance imaging (MRI) as they show very low X Ray scattering
and their magnetic susceptibility is very close to that of the human tissue. To
 34

top it all they are lightweight. For some applications as in dental implants,
biopolymers offer a better aesthetic characteristic. The cost of production of
these implants is low and the production process is highly sophisticated. Bio
composites are used for hard tissue applications, including prosthetic socket,
dental post, external fixator, bone plate, orthodontic arch wire, orthodontic
bracket, total hip replacement (Figure 3.4) and linear osteoblasts (Figure 3.5),
and composite screws and pins. An example of the use of bio composites in
clinical application is cages for spinal fusion. Benefit for patients is a faster
bone healing, no risk of pathogen transfer compared to allograft, faster and
cheaper surgery and less pain compared to auto graft.

3.4 HYDROXYAPATITE

The inorganic phase of our bone is apatite. Apatite is the term of a

very abundant mineral in the earths crust, with Ca 10(PO4)6(OH)2, (more
commonly known as Hydroxyapatite (HAp)) as general formula (Ahoki et al
1994). Its structure has the special ability to accommodate several different
ions in its three sub lattices (Eliott1994, Vallet-Reget al 2008). Bone apatites
can be considered as basic calcium phosphates. Biological apatites are formed
in living bodies through a bio mineralization process where cells and proteins
are involved.

3.4.1 Structure and Properties

Hydroxyapatite is chemically similar to the mineral component of

bones and hard tissues in mammals. It is one of the materials that are classed
as bioactive, meaning that it will support bone in growth and osseointegration
when used in orthopedic, dental and maxillofacial applications. The chemical
nature of hydroxyapatite lends itself to substitution, meaning that it is not
uncommon for non-stoichiometric hydroxyapatites to exist. The most
common substitutions involve carbonate, fluoride and chloride substitutions
 35

for hydroxyl groups, while defects can also exist resulting in deficient
hydroxyapatites. The ability to integrate in bone structures and support bone
in growth, without breaking down or dissolving i.e. it is bioactive.

Hydroxyapatite is a thermally unstable compound, decomposing at

temperatures from about 800-1200C depending on its stoichiometry.
Generally speaking dense hydroxyapatite does not have the mechanical
strength to enable it to succeed in long term load bearing applications.
Hydroxyapatite is a class of calcium phosphate-based bio ceramic, frequently
used as a bone graft substitute owing to its chemical and structural similarity
with natural bone mineral (Jarcho1981, De Groot and Ducheyne1981). The
stoichiometric HAp has a chemical composition of Ca10(PO4)6(OH)2 with
Ca/P ratio of 1.67. The HAp derived either from natural sources or from
synthetic sources is regarded as bioactive substance, since it forms a strong
chemical bond with the host bone tissue, and hence it is recognized as a good
bone graft material.

HAp is not only bioactive but also osteoconductive, non-toxic, non-

immunogenic, and its structure is crystallographic ally similar to that of bone
mineral with adequate amount of carbonate substitution. A compilation of
physiochemical, mechanical, and biological properties of HAp are given in
Table 3.3which makes HAp an appropriate bone graft material. Possible
clinical uses of HAp range from augmenting atrophic alveolar ridges to
repairing long bone defects, ununited bone fractures, middle ear prostheses,
spinal fusions, cranioplasty, craniofacial repair, and vertebral fusions. On the
other hand, it has also been used in dental surgery, bio molecular delivery,
and drug delivery. Many companies have commercially developed and traded
HAp for clinical use.

HAp has a unique capability of binding to the natural bone through

biochemical bonding, which promotes the interaction between the host bone
 36

and grafted material. Although nano-HAp is an excellent bone graft material,

its inherent low fracture toughness has limited its use in certain orthopedic
applications, in particular heavy load-bearing implantation. The fracture
toughness of HAp is about 1.0 MPa m1/2, which is very low compared to the
natural bone (212 MPa m1/2) and the Weibull modulus is also sufficiently
low that depromotes the reliability of HAp for heavily loaded implants. The
Weibull modulus of HAp is in the range between 5 and 18, which means that
HAp behaves as a typical brittle material; therefore it is used only in low
weight-bearing orthopedic applications, e.g., as a bone defect filler, coating-
agent on metallic bio implants, bio molecular delivery, and drug delivery.

In order to improve reliability, it is necessary to introduce some

biocompatible reinforcement agents or matrix materials. However, the
introduction of foreign materials may lead to a decrease of reliability of HAp;
thereby choosing the reinforcement agents or matrix materials is of great
importance in making composites. Recently, composites of nHAp with
natural polymers, in particular collagen, are preferred to improve the
reliability of nHAp. Collagen is a natural extra cellular matrix (ECM) material
available in human bone tissue; thus choosing collagen as a matrix material
could enhance the composites reliability.

Figure 3.6 Typical Crystal structure of HAp (Currey1990)

 37

Table 3.3 Physicochemical, mechanical and biological properties of HAp

(Murugan and Ramakrishna 2005a)

Properties Experimental data

Chemical composition Ca10(PO4)6(OH)2
Ca/P molar 1.67
Crystal system Hexagonal
Space group P63/m
Cell dimensions (A ) a = b = 9.42, c = 6.88
Density (g/cm3) 3.16
Relative density (%) 9599.5
Fracture toughness (MPa m1/2) 0.71.2
Decomposition temperature (_C) >1000
Melting point (_C) 1614
Dielectric constant 7.4010.47
Thermal conductivity (W/cm K) 0.013
Biocompatibility High
Bioactivity High
Biodegradation Low
Cellular-compatibility High
Osteoconduction Nil
Osteoinduction Nil

3.4.2 Hydroxyapatite and Its Composites

As per the literature survey, HAp has a long history of use as a

biomaterial. In 1951, a synthetic HAp was prepared by Ray and Ward
(DeGroot 1980), suitable for bone defects. They implanted HAp into
surgically created defects in the long bones, iliac wings of dogs, the skulls of
cats and monkeys and obtained affirmative results. Schwartzwalder and
Somers (1963) studied the slurry infiltration process for making porous
ceramics. In this process poly urethane (PU) foam was infiltrated with
 38

ceramic slurry and the body was compressed by passing it through a set of
rollers to remove the excess slurry. In this manner the slurry remained coated
on the PU struts and open pore channels were left in between. The coated PU
preform was then dried, followed by the burn out of the PU and sintering at
higher temperature. The foams produced were reticulated foams with porosity
within the range 75-90%. Since then many investigations on HAp were
carried out and tested both in animals and in humans. In the 1970s, Aoki and
Kato (1973), De Groot (1980) and Jarcho (1976) pioneered multi-shapeable
HAp suitable for clinical orthopedics.

Kwon et al (2002) successfully fabricated porous bioceramics with

variable porosity, using the PU sponge technique. Porosity was controlled by
the number of coatings on the sponge struts. When a porous body was
produced by a single coating, the porosity was 90% and the pores were
completely interconnected. When the sintered body was coated five times
after the porous network had been made, the porosity decreased to 65%. The
compressive strength was strongly dependent on the porosity and weakly
dependent on the type of ceramics, HAp, TCP, or HAp/TCP composite. At
the 65% porosity level, the strength was 3 MPa. The TCP exhibited the
highest dissolution rate in a Ringers solution and HAp had the lowest rate.
The biphasic HAp/TCP showed an intermediate dissolution rate. The
biodegradation of calcium phosphate ceramics could be controlled by simply
adjusting the amount of HAp or TCP in the ceramic.

Thijs et al (2003) studied a novel technique to produce macro

porous ceramics using seeds and peas as sacrificial core materials. The first
step in this technique was to coat the seeds, peas with wetting ceramic slurry
that undergoes gelation. The coated seeds and peas were consolidated by
packing them in a container and infiltrating them with ceramic slurry which
underwent gelation. The compacts thus obtained were subjected to the
 39

conventional steps of drying, binder burn out and sintering. The resulting
bodies had greater than 90% porosity with pore size determined by the size of
the seeds and peas.

Itatani et al (2003) studied the preparation of porous

hydroxyapatite ceramics from hollow spherical agglomerates using a foaming
agent of hydrogen per oxide (H2O2)and observed that by changing the
concentration of H2O2 solution from 0 to 20 mass%, the total porosity of the
HAp compact fired at 10000C for 5 hrs could be changed linearly from 61.2 to
71.7%. The HAp compact exhibited pore sizes with maximum porosity
(71.7%) at around 0.7 m, 5-100 m and 100-200 m.

Deville et al (2006) studied the freeze casting of porous

hydroxyapatite scaffolds and observed that by varying the freezing rate of
slurry as well as slurry concentration, porosities in the range 40-60% could be
achieved. The pores were open and unidirectional and exhibited a lamellar
morphology. The processed scaffolds exhibited high compressive strength up
to 145 MPa.

Huang and Miao (2006) studied the HAp-based composite

scaffolds that have a unique macro porous structure and special struts of a
polymer/ceramic interpenetrating composite. A novel combination of PU
foam method and hydrogen H2O2 foaming method is used to fabricate the
macro porous HAp scaffolds. It is found that the HAp scaffolds fabricated by
the combined method show high porosities of 6165% and proper macro pore
sizes of 200600 m. The PLGA infiltration improved the compressive
strengths of the scaffolds from 1.5to 5.8 MPa.

Sopyan et al (2007) studied porous hydroxyapatite for artificial

bone applications. The preparation of HAp porous bodies via polymeric
sponge method was reported; the samples of which were prepared using sol
 40

gel method-derived HAp powders and commercial HAp powders showed a

considerable compressive strength ranging from 1.3 to 10.5 MPa for the
increased apparent density from 1.27 to 2.01 g/cm3. This was higher than the
0.555 MPa compressive strength obtained for the apparent densities of
0.0397 0.783 g/cm3, as reported by Ramay and Zhang (2003a). It was also
shown that the homogeneity of slurry and the heating rate affected porosity
and density of the porous bodies, in turn influencing the compressive strength.

Potoczek (2008) studied the gel casting of hydroxyapatite foams

using agarose as gelling agent and the rheological behavior of the
suspensions. The viscosity of the slurries could be adjusted by agarose
concentration and HAp solid loading. These parameters were essential in
tailoring the porosity as well as the cell and window sizes of the resulted Hap
foams. Depending on HAp solid loading (2429 vol.%) and agarose
concentration (1.11.5 wt.% with regard to water) in the starting slurry, the
mean cell size ranged from 130 to 380 m, while the mean window size varied
from 37 to 104 m. Depending on the porosity range (7392%) related with
this parameter, the compressive strength of HAp foams was found to be in the
range 0.85.9 MPa.

Guo et al (2009) studied biocompatibility and osteogenicity of

degradable Ca-deficient hydroxyapatite (CaD HAp) scaffolds from calcium
phosphate cement for bone tissue engineering by a particle leaching method.
The morphology, porosity and mechanical strength as well as degradation of
the scaffolds were characterized. The results indicated that the CaD HAp
scaffolds with a porosity of 81% showed open macro pores with pore sizes of
400500 m.

The impact of nano-HAp has also been extensively reviewed with

regard to recently developed manufacturing techniques (Murugan and
Ramakrishna 2005a). Some of the prominent processing methods for
 41

manufacturing nano-HAp include solid state (Ota and Itwashita 1998), wet
chemical (Murugan and Ramakrishna 2005b, Murugan and Ramakrishna
2004), hydrothermal (Zhang and Consalves 1997, Ioku et al 2002), mechano
chemical (Nakamura 2001), pH shock wave (Koumoulidis et al 2001), and
microwave processing (Yang et al 2002). HAp can also be processed from
animal bone (Murugan et al 2002, Murugan et al 2003,) and coral exo-
skeleton (Murugan et al 2002a, Murugan et al 2004a), but on a micro scale.

Maria et al (1999) indicated that the fracture toughness and

hardness of glass reinforced HAp composites was shown to be dependent on
the combined effect of its porosity and the secondary phases present in the
microstructure.

Li et al (2002) studied the effect of TiO2 incorporation with the

HAp coating and demonstrated that the shear strength slightly increased
with an increase of TiO2 content in the coatings. But, the Youngs
modulus decreased when the TiO2 content reached 20 volume % and only
small increase of fracture toughness (0.67MPa m1/2) was revealed for the
20 volume %.

Akira Chiba et al (2003) found that the addition of Al 2O3 with the
nHAp had no effect on the Vickers hardness when the composites were
sintered. This was due to the decrease in the relative density. Simultaneously,
they noticed the improvement in mechanical properties such as the
compressive strength with the addition of Al2O3. But the fracture toughness
varied linearly with Al2O3 content and the value 3.0MPa m1/2 was three times
that of HAp monolithic material. This is due to the large resistance to crack
propagation by the addition of Al2O3.Rajaa et al (2008) showed that the
fracture toughness of ceria stabilized zirconia-alumina nano composites have
a value of 8.8 MPa m1/2 for medical applications.
 42

Simone et al (2009) have reported the fracture toughness of newly

developed HAp/calcium silicate bioactive ceramics measured on notched
specimens, without any annealing. The obtained toughness value was 11.8
MPam1/2. The flexure strength of the composite materials was found to be
much higher than that of HAp alone. These results confirmed that composites
of HAp and Ca2SiO4 are promising for the development of load-bearing
ceramic bone substitutes.

During the past few years, significant research effort has been
devoted to nanostructure processing of HAp and its composites in order to
obtain ultra-fine structures with physical, mechanical, chemical, and
biological properties better than their micro scale counterparts and similar to
natural bone mineral. It has also been proven that the nano HAp, compared to
conventional micro HAp, promotes osteoblast adhesion, differentiation and
proliferation, osteointegration, and deposition of calcium-containing minerals
on its surface, which leads to the enhanced formation of new bone tissue
within a short period (Webster et al 2000).

3.4.3 Applications of Hydroxyapatite

HAp is often added to orthopedic devices to induce

osteoconductivity or bone-bonding ability (Vitoret al 2005). Few, if any,
synthetic polymers are osteoconductive. Hydroxyapatite, because of its
similar chemical structure to the inorganic composition of the human bone, is
often used in bone reconstruction. Hydroxyapatite has also been evaluated as
a reinforcing agent with polymers such as HDPE (Deb 1996, Huang et al
1997) PLLA, PMMA, PHB homo polymer, starch, and polyester-ether to
form bioactive compounds. In addition to increasing the modulus, apatites
have biocompatibility with several cell types such as osteoblasts, osteoclasts,
fibroblasts, and periodontal ligament cells that are found in calcified tissues.
 43

Coatings of hydroxyapatite are often applied to metallic implants

(most commonly titanium/titanium alloys and stainless steel) to alter the
surface properties. Without the coating, the body would see a foreign body
and work in such a way as to isolate it from the surrounding tissues. To date,
the only commercially accepted method of applying hydroxyapatite coatings
to metallic implants is plasma spraying.

Hydroxyapatite may be employed in forms such as powders, porous

blocks or beads to fill bone defects or voids. These may arise when large
sections of bone have had to be removed (e.g. bone cancers) or when bone
augmentations are required (e.g. maxillofacial reconstructions or dental
applications). The bone filler will provide a scaffold and encourage the rapid
filling of the void by naturally forming the bone and provide an alternative to
bone grafts. It will also become part of the bone structure and will reduce
healing times compared to the situation, if no bone filler was used.