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 50–100 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 crystal–polymer 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 Wolff’s 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 (600-1,000oC). 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 market’s 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)

Class Description ExamplesProperties of

action

Autograftbased

Used aloneOsteoconductiveOsteoinductiveOsteogenic

Allograft based Allograft bone usedalone or in combinationwith other materials

Allegro,Orthoblast,Grafton

OsteoconductiveOsteoinductive

Factor based

Natural andrecombinant growthfactors used alone or incombination with othermaterials

TGF- , PDGF,FGF, BMP

Bothosteoconductiveand osteoinductivewith carriermaterials

Cell basedCells used to generatenew tissue alone orseeded onto a supportmatrix

Mesenchymalstem cells

steoconductivewith carriermaterials

27

Table 3.1 (Continued)

Class Description Examples Properties ofaction

Ceramic based

Includes calciumphosphate, bio inertceramics like alumina,titania, zirconia,calcium sulfate, andbioactive glass usedalone or in combination

Osteograft,Osteoset,Nova Bone

steoconductiveLimitedosteoinductivewhen mixed withbone marrow

Polymer based

Includes degradable andnon degradablepolymers used aloneand in combination withother materials

Cortoss,OPLA, Immix

steoconductiveBioresorbable indegradablepolymer

Miscellaneous Coral HA granules,blocks and composite

ProOsteon OsteoconductiveBioresorbable

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 cobalt–chromium 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: Ca3(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)

HAp based nano composites Tissue engineered bone scaffolds ofnHAp and its composites

Nanocompositesystems

Methods ofPreperation

Bonetissuescaffolds

Cells/growthfactors studied

HAp/collagen Precipitation nHAp Osteoblast-like cells

HAp/collagen BiomimeticsnHAp Mesenchymal

stem cells

HAp/collagen BiomimeticsnHAp Intended as tissue

scaffoldHAp/collagen Biomimetics nHAp Cell carrierHAp/collagen/

PLA Biomimetics nHAp Angiogenic factors

HA/collagen/alignate

Biomimetics nHAp/collagen Mesenchymal cells

HAp/chitosan Precipitation nHAp/collagen/PLA

Osteoblasts

HAp/gelatin BiomimeticsnHAp/gelatin/

PLArhBMP-2

HAp/silk fibroin Mechano chemicalnHAp/collagen/

alginateFibroblasts/osteoblasts

HAp/PLA Solvent-casting nHAp/collagenrhBMP-2 osteoblasts

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

(Aubin and Liau1996)

Figure 3.5 Linear Osteoblast

(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 earth’s crust, with Ca10(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-Regíet 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 800-1200°C 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 (2–12 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 composite’s 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 dataChemical composition Ca10(PO4)6(OH)2

Ca/P molar 1.67Crystal system HexagonalSpace group P63/mCell dimensions (A ) a = b = 9.42, c = 6.88Density (g/cm3) 3.16Relative density (%) 95–99.5Fracture toughness (MPa m1/2) 0.7–1.2Decomposition temperature (_C) >1000Melting point (_C) 1614Dielectric constant 7.40–10.47Thermal conductivity (W/cm K) 0.013Biocompatibility HighBioactivity HighBiodegradation LowCellular-compatibility HighOsteoconduction NilOsteoinduction 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 Ringer’s 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 61–65% and proper macro pore

sizes of 200–600 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

0.55–5 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 (24–29 vol.%) and agarose

concentration (1.1–1.5 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 (73–92%) related with

this parameter, the compressive strength of HAp foams was found to be in the

range 0.8–5.9 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

400–500 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 Young’s

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 Al2O3 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.