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CHAPTER 1

INTRODUCTION

1.1 GENERAL INTRODUCTION

In recent years, studies on biomaterials for bone tissue repair and

replacement have achieved great progress. Among them, calcium phosphates,

bioglass and glass-ceramics are well known bioactive materials due to their property

of better interlocking with bony tissues [1]. As a result, calcium phosphate based

materials have been extensively used in orthopaedic and dental applications [2].

Bioactive glasses (BG) have been used as a bone graft material to reconstruct or fill

bony defects and as a dental material to regenerate bone in periodontal pockets

[3-4]. BG is a non-resorbable and prominent biomaterial, due to its advantages of

forming a strong bond with living tissues, including bone and soft connective tissue

[5-7]. Bioactive glass family is composed of SiO2, Na2O, CaO and P2O5 [8].

Moreover, it has been shown that dissolution products of BG stimulate gene

expression in osteoblast cells [9] and angiogenesis [10]. The BG with collagen and

phosphatidylserine scaffolds promotes the matrix mineralization and differentiation

marker genes in both in vivo and in vitro [11]. In clinical trials, BG is effective as

an adjunct to conventional surgery in the treatment of intrabony defects as well as in

the treatment of dental extraction sites before dental implant placement, to

implement bone regeneration and to augment early fixation of the implant [12]. In

general, BG is considered as the material of choice for developing bioactive

composites for bone tissue engineering [13].

Tissue Engineering is ―an interdisciplinary field of research that applies

the principles of engineering and the life sciences towards the development of

biological substitutes that restore, maintain, or improve tissue function‖ [14-15].

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Over the recent years major focus is shifted towards the potential use of tissue

engineering principles for the development of new tissues that can be used in

replacement of diseased bone [16]. Bone regeneration research is needed to deal

with various clinical bone diseases such as bone infections, bone tumors and bone

loss by trauma.

Bone is the component of the skeletal system that is involved in body

protection, support, and locomotion [17]. Osteoblasts are called as mononucleate

bone-forming cells that descend from osteoprogenitor cells. They are located on the

surface of osteoid seams and make a protein mixture known as osteoid, which

mineralizes to become bone [18]. Osteoblast proliferation, differentiation and gene

expression are regulated by the major transcription factor called Runt-related

transcription factor 2 (Runx2) [19-21]. Runx2 is the predominant transcriptional

activator of osteoblast-associated genes, and its expression is essential for osteoblast

differentiation and bone formation [22-25]. The osteoblasts differentiate from

mesenchymal stem cells through osteoprogenitor cells and preosteoblasts and

ultimately into mature osteoblasts that synthesize collagenous and noncollagenous

extracellular bone matrix proteins and initiate and support matrix mineralization.

These processes are dependent on their expression of the Runx2 transcription factor.

Therefore, mechanisms which induce and modulate Runx2 expression and activity

are likely to play an important role in osteoblast differentiation and gene expression

[26-27]. The post transcriptional activities of the genes were regulated by small

endogenous molecule called micro RNA (miRNAs). It is single stranded RNAs

(∼23 nt), act as post transcriptional regulators thus, they are involved in various

biological and pathological processes. Altering the expression profile of miRNAs

can unexceptionally modulate the expression of proteins. In response to different

stimuli, the basal copy of miRNAs could increase or decrease, resulting alteration of

expression of their target genes. miRNAs mediated physiological process are in

huge extremities, because the mechanism of miRNAs action is quicker than the

mechanism of transcription of targets [28-29]. The external stimulations such as

environmental, physical and chemical factors indirectly perform the physiological

role through regulating the expression of numerous miRNAs [30-32]. Organic and

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inorganic materials which are used for tissue engineering have been documented for

the regulation of miRNAs expression during osteoblast differentiation [33-37]. So

the recent trend in bioceramic research is to overcome the limitations of bioceramics

and to improve their biological properties via exploring the unique advantages of

nanotechnology. Nanostructured ceramics represent an incomparable and promising

character for orthopaedic and dental implant formulations with better

osseointegrative properties [38-39]. By controlling the structural dimensions to nano

scale, the ossteoconductivity, sintering characters, solubility and mechanical

reliability of bioceramics can be improved [40-41]. These nanostructured

bioceramics are superior in their bioactivity compared with coarser crystals [42-43].

Hence, the present study was aimed to synthesis and characterization of

nano bioglass ceramic (nBGC) particles, followed by determination of their effect

on osteoblast cell proliferation and differentiation at molecular and cellular levels. In

addition, the molecular mechanism mediating the differentiation via post

transcriptional regulators (miRNA‘s) was also investigated.

1.2 OBJECTIVES OF THE STUDY

Hypothesis

It is hypothesized that Bioglass ceramic particles at nano scale may have

enhanced effect on bone formation.

Objectives

1. To synthesize and characterize nano bioglass ceramic particles.

2. To study the proliferative action mediated by nano bioglass ceramic

particles in osteoblastic cells.

3. To determine the differentiation process mediated by nano bioglass

ceramic particles at molecular and cellular level in osteoblastic cells.

4. To determine the regulation of nano bioglass ceramics particles

mediated osteoblast differentiation by microRNAs in osteoblast cells.

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1.3 OUTLINE OF THE STUDY

Fig. 1.1 Schematic brief representation of the present study

The present study was aimed to evaluate the action of nano Bioglass

Ceramic (nBGC) particles on osteoblast cells for bone formation. Hence, the study

was designed with four chapters dealing different aspects of possible events

mediated by nBGC particles on osteoblast cell proliferation and differentiation

processes. The chapter I involve synthesis of nBGC particles followed by

characterization i.e. size, elemental composition, functional group attachment and

phase analysis. The chapter II deals about the triggering of various intracellular

events towards proliferation of osteoblast cells by nBGC particles. The chapter III

deals about differentiation events at molecular and cellular levels in osteoblast cells

in response to nBGC treatment. Finally, the chapter IV deals with osteoblast

differentiation process mediated by nBGC particles via post-transcriptional

regulators such as microRNAs.

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1.4 REVIEW OF LITERATURE

1.4.1 Regenerative Medicine

Human organs maintain homeostasis, transfer information, generate

force, and adapt to changes in the environment. Over the past 2 decades, there have

been tremendous advances in the fields of stem cell biology and bioengineering, the

intersection of which offers increasing potential to achieve the goal of truly

regenerative therapies for myriad clinical pathologies [44]. Regenerative medicine

has set high expectations worldwide in the cure of human diseases, such as

Parkinson‘s disease, Alzheimer‘s disease, osteoporosis, spine injuries or cancer to

treat the regenerating diseased or damaged tissues [45-46]. Damaged organs can be

regenerated with the aid of various drugs, mechanical supports or transplantation.

Fundamentally the regenerative medicine refers to technology that could be used for

reconstructing damaged tissues or organs with various items and techniques

including cells, scaffolds, medical devices or gene therapy [47].

Fig. 1.2 Strategies of regenerative medicines [48]

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1.4.2 Tissue Engineering

Tissue engineering (TE) is a rapidly growing scientific area [49] that

aims to create, repair, and/or replace tissues and organs by using combinations of

cells, biomaterials, and/or biologically active molecules [50-51]. Basically the TE

provides permanent solutions to tissue damage and tissue loss to millions of people

each year [52-55]. When tissues or organs have been so severely diseased or lost by

various kind of pathological defects like cancer, congenital anomaly, trauma and

fracture, in order to treat the above defects the conventional pharmaceutical

treatments are no more applicable. So the artificial organs (including tissues) or

organ transplantation are the first choices to reconstruct the devastated tissues or

organs [56]. Artificial organs have been improved by remarkable advances in the

field of biomedical engineering in the past decades, but still need better

biocompatibility and biofunctionality. Problems in current organ transplantation

include shortage of donated organs and immune rejection, although

immunosuppressive therapy has recently much advanced [57]. TE is intensively

searching solutions that have the potential to reduce the complications related to

current treatment methods. Basically it is tightly associated with the field of

regenerative medicine. TE is based on the profound understanding of embryology,

tissue formation and regeneration and aims to growing new functional tissues rather

than building new spare parts. As mentioned above, it combines integral knowledge

from physicists, chemists, engineers, material scientists, biologists and physicians to

a comprehensive interdisciplinary approach [58]. The following things are the three

major pillars in the field of TE 1) isolated cells, (2) tissue inducing-substances, and

(3) matrices [53]. The classical TE strategy consists of isolating specific cells

through a biopsy from a patient, growing them on a biomimetic scaffold under

controlled culture conditions, delivering the resulting construct to the desired site in

the patient‘s body, and directing the new tissue formation [50-51]. Most of the

presently existing TE techniques rely on the use of macro structured porous

scaffolds, which act as supports for the initial cell attachment and subsequent tissue

formation, both in vitro and in vivo [59-61]. This kind of approach has been

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successful to a certain extent in producing relatively simple constructs relying on the

intrinsic natural capability of cells and tissues to self-regenerate, remodel, and adapt.

Fig. 1.3 Schematic representation of Basic Principles of Tissue Engineering [62]

1.4.3 Biomaterials

Biomaterials are used to make devices to replace a part or a function of

the body in safe, reliably economically, and physiologically acceptable manner.

A variety of devices and materials are used in the treatment of disease or injury. The

basic definition of biomaterials is that any material that is used to replace or restore

a body tissue and is continuously or intermittently in contact with body fluids [63].

In other words, a biomaterial is a non-toxic material that can be used to construct

artificial organs, rehabilitation devices or prostheses, and to replace natural body

tissues [64]. Biomaterial science encompasses elements of medicine, biology,

chemistry, tissue engineering and materials science [65-67]. The Comparison with

conventional materials the biomaterials offer better results in terms of cell adhesion,

spreading, proliferation, and differentiation. Various biomaterials are in use with

regard to their nature, origin, and site of application [68]. It can be made of a single

material or be a composite of several materials. They can be modified with chemicals or

biological agents, such as growth factors and adhesion peptides, in order to create

suitable environments for cells to attach, proliferate and differentiate [69].

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1.4.4 Development of Biomaterials

Over the last 4 decades innovations in biomaterials and medical

technology have had a sustainable impact on the development of biopolymers,

titanium/stainless steel and ceramics utilized in medical devices and implants [70-

71]. This progress was primarily due to issues of biocompatibility and demands for

enhanced mechanical performance of permanent and non-permanent implants as

well as medical devices and artificial organs. In the 1980s there were more than a

hundreds of implants and devices in clinical use made from about 30 different

materials [72]. A common feature of most of the biomaterials used these days was

limited and its biological activity was also known as ―inertness‖. The underlying

principle of the biomaterials is to reduce the immune response to a minimum, not to

cause foreign body reactions and to prevent biological rejection [72-73]. The

evolution of biomaterials research and their clinical availability during the last 60

years, has shown three different generation demarcation [74]; bioinert materials

(first generation), bioactive and biodegradable materials (second generation), and

materials designed to stimulate specific cellular responses at the molecular level

(third generation). These three generations should not be interpreted as

chronological, but conceptual, since each generation represents an evolution on the

requirements and properties of the materials involved. The development of smart

biomaterials for tissue regeneration has become the focus of intense research

interest. More opportunities are available by the composite approach of combining

the biomaterials in the form of biopolymers and/or bioceramics either synthetic or

natural. Strategies to provide smart capabilities to the composite biomaterials

primarily seek to achieve matrices that are instructive/inductive to cells, or that

stimulate/trigger target cell responses that are crucial in the tissue regeneration

processes [75]. In the 21st century, the biomaterials community aims to develop

advanced medical devices and implants, to establish the techniques to meet these

requirements followed by to facilitate the treatment of older as well as younger

patient cohorts [76].

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1.4.5 Necessary for the Development of Biomaterials

Bone and joint degenerative and inflammatory problems such as bone

fractures, lower back pain, osteoporosis, scoliosis and other musculoskeletal defects

affects millions of people worldwide [77]. In fact, they account for half of all

chronic diseases in people over 50 years of age in developed countries. In addition,

it is predicted that the percentage of persons over 50 years of age affected by bone

diseases will double by 2020 [78]. The ideal bone-graft substitute should be

biocompatible, bioresorbable, osteoconductive, osteoinductive, structurally similar

to bone, easy to use, and cost-effective. Approximately 2.2 million bone graft

procedures are performed each year worldwide to repair bone defects in orthopedics

and oral and maxillofacial surgery with a yearly estimated costs of $2.5 billion [79],

besides the numerous permanent, temporary and biodegradable devices and

implantations. Therefore, orthopaedic biomaterials are meant to be implanted in the

human body as constituents of devices that are designed to perform certain

biological functions by substituting or repairing different tissues such as bone,

cartilage or ligaments and tendons, and even by guiding bone repair when necessary

[77]. The biomaterials which are used in the tissue engineering should possess great

commercialization aspect with low production cost. Fine modifications to the

biomaterials yield optimized or modified products, which possess improved

properties than the parent biomaterial [68].

These cell biological discoveries significantly affected the way of

biomaterials design and use. At the same time both clinical demands and patient

expectations continued to grow. Therefore, the development of cutting-edge

treatment strategies that alleviate or at least delay the need of implants will open up

new vistas in the field of tissue engineering for the treatment of various biological

defects in the system to improve the value of human health [80]. Biomaterials

undoubtedly improve the quality of life for an ever increasing number of people

each year with a vast range of applications an increasing demand for biomaterials

arises from an aging population with higher expectations regarding their quality of

life [74, 81].

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1.4.6 Treatment of Bone Defects

Large bone defects represent major clinical problems in the practice of

reconstructive orthopaedic and craniofacial surgery. In such situation, bone grafts

and/or bone substitutes are preferred [82-83]. Traditional therapeutic approaches in

treating large bone defects include bone grafts [83] and transplants [84] (autologous

– from the iliac bone or fibular grafts, allograft – fresh or frozen after cleaning, or

xeno-grafts). These grafts are supported by different fixtures, in hope that native

bone will bridge the gaps and a boney fusion will occur. Other options include

specialised implants that can serve as internal prosthesis (for example, tumour

prosthesis after large bone resection, due to bone tumours [85]. Among the

techniques which are used for treatment of bone defects the autologous bone

grafting is the gold standard for osteogenic bone replacement in osseous defects

[86-87]. Autologous bone grafts reliably fill substance deficits and induce bone

tissue formation at the defective site following transplantation. These grafts exhibit,

depending on donor site, size, shape and quality, some initial stability [88].

However, the clinical use of autologous osseous transplants is limited by a

considerable donor site morbidity that increases with the amount of harvested bone.

Bleeding, hematoma, infection, and chronic pain are common complications of bone

graft harvest [89-91]. Processed allogenic or xenogenic bone grafts are also

commonly used for repair of osseous defects when autologous transplantation is not

applicable [86, 92-94]. The use of allograft or xenografts prevents the problems

involved with donor site morbidity, and allows larger substitutes. However, since

they undergo sterilisation and purification, allografts and xenografts do not provide

osteoinductive signals, and do not have living cells. In addition, they also present the

potential risk of viral or bacterial infections and of an immune response of the host

tissue after implantation [95]. In addition, full integration of the graft is rare, ending

at most cases with only bone substitution at the ends of the grafts, leading to late

graft fracture, reported as high as 60% at 10 years [96]. The another treatment

procedure is that Syngraft (isograft), which is tissue or organ replacement between

genetically identical individuals such as identical twins (Monozygotic twins) and

cloned individual (which may present in future). The antigens are not foreign, so no

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rejection reaction occurs. Therefore, elaboration of appropriate bone replacement

materials would be of considerable importance to the osseous defects. Currently

bone construct created by tissue engineering principles using an appropriate scaffold

and cellular materials is considered as an ideal bone substitute [97-99].

Fig. 1.4 Diagrammatic representation various methods used in treatment of

bone defects [100]

1.4.7 Bone Tissue Engineering

Bone is a remarkable organ playing key roles in critical functions of

human physiology including protection, movement and support of other critical

organs, blood production, mineral storage and homeostasis, blood pH regulation,

multiple progenitor cell (mesenchymal, hemopoietic) housing, and others. Bone is a

very forgiving tissue; it withstands multiple insults and can regenerate itself into a

healthy bone. One of the strengths of bone is its ability to build new osteones when

the native structure of the bone is injured [101]. Bone injuries and defects present a

significant clinical problem. The importance of bone becomes clear in the case of

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diseases such as osteogenesis imperfecta, osteoarthritis, osteomyelitis, and

osteoporosis in which bone does not function properly. These diseases along with

traumatic injury, orthopaedic surgeries (i.e., total joint arthroplasty, spine

arthrodesis, implant fixation, etc.) and primary tumor resection lead to or induce

bone defects or voids. In serious fractures and defects or in elderly patients,

complications such as mal-union or non-union are more common and prevent the

bone from healing naturally [102]. The clinical and economic impact of treatments

of bone defects is staggering [103].

For example, the number of total joint arthroplasties (TJAs) and revision

surgeries in the US has increased from 700,000 in 1998 to over 1.1 million in 2005.1

Medical expenses relating to fracture, reattachment, and replacement of hip and

knee joint was estimated to be over $20 billion (USD) in 2003, and predicted to

increase to over $74 (USD) billion by the year 2015 [104-105]. For a variety of

reasons (such as bone defect size, infection, and many others), injured or diseased

bone may not be capable of repairing itself by means of mechanical fixation alone

which results in a non-union. Concerns including the aforementioned defects of

bone and others promoting the utilization of autogenous cancellous bone grafts as

the gold standard treatment for critical-sized defects in bone have motivated the

development of a wide variety of sophisticated synthetic (tissue-engineered) bone

scaffolds in recent years. Advantages to utilizing synthetic bone scaffolds include:

the elimination of disease transmission risk, fewer surgical procedures, a reduced

risk of infection or immunogenicity, and the abundant availability of synthetic

scaffold materials [106].

The fundamental concept behind bone tissue engineering is to utilize the

body‘s natural biological response to tissue damage in conjunction with engineering

principles. As the role of cell signaling and subsequent functionality in tissue

engineering emerges with greater clarity, tissue engineers are developing

multifunctional bioactive scaffolds for the treatment of various bone defects in bone

tissue engineering [106]. Ideal biomaterials must be capable of presenting a

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physiochemical biomimetic environment while biodegrading as native tissue

integrates and actively promote or prevent desirable and undesirable physiological

responses [53, 106-108]. Multiple bone tissue engineering strategies such as cell

transplantation, acellular scaffolds, gene therapy, stem cell therapy, and growth factor

delivery have been applied to address the challenging requirements [82, 109-111]. In

practice, most of the bone tissue engineering approaches implement a combination

of these strategies. However, two primary tissue engineering strategies have

emerged as the most promising approaches [53].

They are as follows: Before implantation, mesenchymal stems cells

(MSCs) are isolated (typically from the patient), expanded ex vivo and seeded onto a

synthetic scaffold, allowed to produce extracellular matrix (ECM) on the scaffold in

controlled culture conditions, and finally implanted into the osseous defect or void

in the patient [112].

Fig. 1.5 Schematic representation of primary strategy used in treatment of

bone defects by using bone tissue engineering applications [112]

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Fig. 1.6 Schematic describing the second bone tissue engineering strategy

wherein biological molecules and pharmaceutical agents are

encapsulated in an acellular scaffold for release after implantation

[106]

1.4.8 Applications of Biomaterials

There can be no doubt that the most widely recognized applications of

biomaterials involve those situations where a tissue or organ has suffered from some

disease or condition that has resulted in pain, malfunction or structural degeneration,

and which can only be alleviated by the replacement or augmentation of the affected

part [113]. The Biomaterials are used in various fields in TE to treat many defects;

they are such as Heart values, Breast implants, Hip implants, Dental filling

materials, Blood vessel prosthesis and Contact lenses, etc., [114]. Biomaterials can

have a benign function, such as being used for a heart valve, or may be bioactive;

used for a more interactive purpose such as hydroxy‐apatite coated hip implants

(the Furlong Hip, by Joint Replacement Instrumentation Ltd, Sheffield is one such

example – such implants are lasting upwards of twenty years). It is also used every

day in dental applications, surgery, and drug delivery [115].

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Table 1.1 Some of the Biomaterials and its different applications [116]

S. No Biomaterials Applications

01 Silicone rubber Catheters, tubing

02 Dacron Vascular grafts

03 Cellulose Dialysis membrane

04 Poly( methyl methacrylate ) Intraocular lenses, bone cement

05 Polyurethanes Catheters, Pacemaker leads

06 Hydrogels Ophthalmological devices, Drug delivery

07 Stainless steel Orthopedic devices, stents

08 Titanium Orthopedic & Dental devices &

Wound dressing

The biomaterials have been incorporated into drug delivery systems and

used as carriers within packages for biologics, and incorporated into key

components of medical devices in broad applications from disposable tubing and

syringes to implantable devices for sustaining the life or restoring organ or limb

function. The application of biomaterials to medical device technology in nearly

every industrialized country is regulated according to the intended use of the product

incorporating the material and the relative risk of the use of the materials [117].

Fig. 1.7 Applications of Biomaterials in various forms in the field of Tissue

Engineering [117]

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1.4.9 Bioceramics and Glasses

Over the last decade efforts have been made to develop bone implant

materials composed of hydroxyapatite and related calcium phosphate compounds

because their crystalline and chemical composition are closely allied to the mineral

component of bone [118]. Metabolic processes with the bioactive ceramics are very

similar to the processes in natural bones. This is impossible to any kind of organic

matters and majority of inorganic materials [119-120]. Ceramic materials like

calcium phosphates are suitable as bone substitutes due to their biocompatible,

bioactive, biodegradable, and osteoconductive properties [212-123]. Several

inorganic materials such as special compositions of silicate glasses, glass-ceramics

and calcium phosphates have been shown to be bioactive and resorbable and to

exhibit appropriate mechanical properties which make them suitable for bone tissue

engineering applications [124-126]. The first generation of biomaterials has been

formed by inert ceramics. From the chemical point of view, two well-known

examples are zirconia and alumina. They are primarily used bioceramic to fabricate

femoral heads [127-129]. The clinical goal when using ceramic biomaterials, as is

the case with any biomaterial, is to replace lost tissue or organ structure and/or

function. The rationale for using ceramics in medicine and dentistry was initially

based on the relative biological inertness of ceramic materials compared with

metals. However, in the past two decades, this emphasis has shifted more toward the

use of bioactive ceramics, materials that not only elicit normal tissue formation but

may also form an intimate bond with bone tissue. In other words, the ceramic,

usually resorbable (i.e., a greater degree of bioactivity than surface-reactive

materials), facilitates the delivery and function of a biological agent (i.e., cells,

proteins, and/or genes) with an end goal of eventually regenerating a full volume of

functional tissue [130]. Most recently, bioceramics have been utilized in conjunction

more biological therapies. Basically five main ceramic materials are used in

musculoskeletal reconstruction/regeneration they are, carbon [131-132] alumina

(Al2O3) [133-137] zirconia (ZrO2) [138-139] bioactive glasses and glass ceramics

[140-145] and calcium-phosphates [146-149]. Alumina, zirconia, and carbon are

considered bioinert, whereas glasses and calcium phosphates are bioactive ceramics.

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In medical applications, these materials are provided in the following formats:

powder, porous pieces, dense pieces, injectable mixtures and coatings. They have

excellent features in terms of biocompatibility and bioactivity [150]. It is due to their

chemical similarity to the inorganic phase of bone, inorganic biomaterials such as

calcium phosphates (CaP), e.g. hydroxyapatite (HAp), α and β-tricalciumphosphate

(TCP), have been more intensively investigated in respect to their possible

application as bone scaffolds [151-154]. Moreover, numerous in vitro and in vivo

studies have shown that Hap [155-158] and related CaPs [159-162] promotes cell

adhesion, proliferation and differentiation of osteogenesis related cells (e.g.

osteoblasts, mesenchymal stem cells) [93].

Ceramic implants for osteogenesis are based mainly on HA, since this is

the inorganic component of bone [163]. In clinical applications the bioactive

calcium phosphate ceramics, such as hydroxyapatite (HA) and tricalcium phosphate

(TCP/Ca3(PO4)2), are mainly used as bone substituting materials [164]. Currently,

they are subject of intensive investigations assessing their suitability for tissue

engineering applications. The calcium-to-phosphate ratio of these ceramics closely

resembles the mineral phase of bone, which is considered to account for their

osteoconductive features [165].They provide a high surface chemistry that facilitates

proteins adsorption for better cell adhesion there by it display an osteoinductive

potential materials [166-168]. Most types of ceramics are inherently hard and brittle

materials with higher elastic moduli compared to bone. Traditional ceramics

provides high compressive but low tensile strength. Alumina (Al2O3) and zirconia

(ZrO2) are non-bioactive ceramics and are covered by a non-adherent fibrous layer

at the interface after implantation. In orthopaedics they are mainly used as artificial

femoral heads or acetabular liners due to their excellent mechanical strength and

durability in conjunction with low friction and wear coefficients [169]. These

features make them also suitable for applications in dentistry, where they are mainly

used for crown and bridge restoration [170]. In neutral conditions HA is almost

insoluble and the delay the process of in vivo degradation is mainly mediated by

cellular resorption mechanisms. Biphasic calcium phosphate (BCP) scaffolds are

composed of variable amounts of HA and TCPs aiming to compensate for the

undesired properties of each individual material. The mechanical characteristics of

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such a composite ceramic can be improved by increasing the percentage of HA. An

increase of the β-TCP content on the other hand leads to a higher degradation rate

and ion release [171]. Calcium phosphate cements (CPC) are multi-component

systems consisting of an inorganic phase and an aqueous solution. The suitable

compounds of CPCs are dicalcium phosphate (DCP), dicalcium phosphate dihydrate

(DCPD), tetracalcium-phosphate (TTCP), amorphous calcium phosphate (ACP),

calcium deficient HA, carbonate HA, α-TCP, β-TCP or octacalcium phosphate

(OCP). The paste or injectible cement is freely moldable and hardens in situ without

significant heat development. Other types of ceramics used in bone repair include

porous calcium meta phosphate [Ca (PO3)2] blocks (pore size 200 µm) that were

used for culturing rat marrow stromal cells ex vivo and for ectopic bone formation in

athymic mice [172] and natural coral scaffolds molded into the shape of a human

mandibular condyle with pore sizes 150–220 µm and 36% porosity that were seeded

with rabbit marrow mesenchymal cells and induced ectopic bone formation in nude

mice [173]. Combinations of ceramics also have been explored: porous biphasic

ceramic (hydroxyapatite—tricalcium phosphate) with 50% porosity and100–150 µm

pore sizes have been shown to heal femoral defects in dogs [174]. In general,

ceramic biomaterials are able to form bone apatite-like material or carbonate

hydroxyapatite on their surfaces, enhancing their osseointegration. These materials

are also able to bind and concentrate cytokines, as is the case of natural bone [175].

1.4.10 Bioglass Ceramic Particles

The last two decades have seen a dramatic growth in the field of tissue

engineering. These efforts have resulted in cell-based regeneration of individual

tissues such as skin [176-179] bone [180-182] and cartilage [183-184]. In a general

sense, a bioactive material has been defined as a material that has been designed to

induce specific biological activity [185]. In a more narrow sense, a bioactive

material has been defined as a material that undergoes specific surface reactions,

when implanted into the body, leading to the formation of an HA-like layer that is

responsible for the formation of a firm bond with hard and soft tissues [186]. The

ability of a material to form an HA-like surface layer when immersed in a simulated

body fluid (SBF) in vitro is often taken as an indication of its bioactivity [187].

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In 1969 Hench and colleagues in Florida established a specific

compositional range of soda lime phosposilicate glasses that did not become

surrounded by fibrous (scar) tissue when implanted and instead bonded intimately to

bone [188-189]. This bone bonding melt derived glass was trademarked as Bioglass

45S5 R (45% SiO2, 24.5% Na2O, 24.5% CaO and 6% P2O2 (wt%)), and generated a

family of melt derived and sol-gel derived glasses collectively known as bioactive

glasses and have been widely used as bone void fillers in clinical settings. Since

1969 Bioglass 45S5 has obtained FDA approval for middle ear prosthesis (1985)

and endosseous ridge maintenance implants (1986) [189-191].

Bioactive glass mainly consists of sodium, calcium, silicon and

phosphorous oxides in various proportions. The bioactivities of the bioglasses were

mediated by the presence of a hydrated silicate-rich layer, which forms when

coming into contact with human fluids. This layer has catalytic effects on the

deposition of HA, which in turn leads to a stable bond between glass and bone

[192]. These bioglass formulations show a higher osteogenic potential when

compared to HA alone [193]. Studies have shown that bioactive glass scaffolds

completely dissolve within 6 months. However, the brittleness and low fracture

toughness of bioglass hampers its application for load-bearing applications. Since

the discovery of Bioglass was initiated by Hench, followed by various kinds of

glasses, glass ceramics and sintered ceramics have been found to make bond with

living bone. The use of ceramics in the replacement of bone has been well

documented [194-201]. Both the non-degradable and degradable types show

excellent tissue compatibility. Bioglass, a bone-bonding ceramic, apparently

facilitates ankylosis at biomaterial- bone interfaces, and there is much interest in its

potential uses in the health sciences [202-203].

1.4.11 Bone Tissue Engineering with Mesenchymal Stem Cells

Stem cells are undifferentiated cells characterized by self-renewal and

multipotential differentiation. Stem cell self-renewal is the consequence of cell

division that takes place within the microenvironment in which stem cells reside

(niche). Within the niche the stem cell number is maintained constant by balancing

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quiescent and activated cells [204-206]. The term stem cell refers to a cell or

population of undifferentiated cells that has extensive proliferative capacity with the

ability to self-renewal and differentiate into offspring, or daughter cells, that form

different lineage cells [207-209]. The term MSC was coined by Caplan in 1991

[210], usually refers to bone marrow-derived cells. MSCs were first described by

Friedenstein and colleagues as plastic-adherent cells derived from bone marrow that

exhibited a fibroblast-like morphology and were inherently osteogenic in addition to

their supportive role for other bone marrow cells [211-212]. Subsequently, these

cells were found to possess impressive capacity to differentiate into multiple

mesenchymal tissue types, including bone, cartilage, and muscle [213-214].

MSC are the principal source driving the regeneration of mesenchymal

tissues. Basically MSC populations can be obtained from various sites such as bone

marrow (as a gold standard), bone trabeculae, adipose tissue, and ligaments, which

show great promise for regenerative strategies [215-218]. This obvious

heterogeneity of MSC populations, which is often target of criticism when

presenting in vitro work investigating such cell preparations, may well be necessary

for biological success in a complex of tissue regeneration process [219]. Naturally,

the cellular part of tissue regeneration is initiated by a phase of transient

amplification of a precursor pool followed by the phase of differentiation and tissue

formation [220]. Materials used for regenerative applications should take into

account the phases compounding the regenerative process. These phases include

MSC recruitment to the site of injury, transient precursor amplification, tissue

formation and remodelling. Premature material induced cell differentiation has to be

avoided and the scaffold structure and porosity should allow ingrowth and

vascularization within an appropriate time frame [221]. In essence, biomaterials for

use in a healthy organism with manageable sites of tissue regeneration do not

require a multitude of intelligent features as long as they do not impede the sequence

of regeneration related events. However, materials can also be tailored to stimulate

and enhance a single component of the regenerative process, e.g. stem cell

amplification, lineage-specific differentiation or angiogenesis aiding to overcome

intrinsic/extrinsic deficits [222-223].

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1.4.12 Nanotechnology

All biological and man-made systems have the first level of organization

at the nanoscale (such as a nanocrystals, nanotubes or nanobiomotors), where their

fundamental properties and functions are defined. The goal of nanotechnology might

be described by the ability to assemble molecules into objects, hierarchically along

several length scales, and to disassemble objects into molecules [224]. Since Drexler

(1986) who introduce the term nanotechnologies and the development of the first

critical nanotechnology roadmaps [226-227], the deployment of nanotechnologies

has become clearer. The prefix ―nano‖ is derived from the Greek word ―nanos‖

meaning ―dwarf‖. Nanotechnology involves the manipulation and application of

engineered particles or systems that have at least one dimension less than 100

nanometers (nm) in length. The term ―nanoparticles‖ applies to engineered particles

(such as metal oxides, carbon nanotubes, fullerenes, ceramics, etc.), for

nanomedicine research and applications [228]. Nanomedicine involves utilization of

nanotechnology for the benefit of human health and well-being. The applications of

nanotechnology in various sectors of therapeutics have revolutionized the field of

medicine. Used for diagnostics, therapeutics and as biomedical tools for research

[229].

Nanotechnology is being applied extensively to provide targeted drug

therapy, diagnostics, tissue regeneration, cell culture, biosensors and other tools in

the field of molecular biology. Various nanotechnology platforms like fullerenes,

nanotubes, quantum dots, nanopores, dendrimers, liposomes, magnetic nanoprobes

and radio controlled nanoparticles are being developed [230]. It provides the tools

and technology platforms for the investigation and transformation of biological

systems, and biology offers inspiration models and bio-assembled components to

nanotechnology [231].

Growing exploration of nanotechnology has resulted in the identification

of many unique properties of nanomaterials such as enhanced magnetic, catalytic,

optical, electrical, and mechanical properties when compared to conventional

formulations of the same material [232-235]. These materials are increasingly being

22

used for commercial purposes such as fillers, opacifiers, catalysts, water filtration,

semiconductors, cosmetics, microelectronics etc., leading to direct and indirect

exposure in humans [236]. Apart from the use of nanomaterials in consumer

products, numerous applications are being reported in the biomedical field,

especially as drug-delivery agents, biosensors or imaging contrast agents [232-233].

The applications pertaining to medicine involve deliberate direct ingestion or

injection of nanoparticles into the body. Also for imaging and drug delivery are

often intentionally coated with biomolecules such as DNA, proteins, and

monoclonal antibodies to target specific cells [237-238].

1.4.13 Nanotechnology in Bone Tissue Engineering

Nano-biotechnology is defined as a field that applies the nano-scale

principles and techniques to understand and transform bio-systems (living or

non-living) and which uses biological principles and materials to create new devices

and systems integrated from the nano-scale. The integration of nanotechnology with

biotechnology, as well as with infotechnology and cognitive science, is expected to

accelerate in the next decade [239-240]. Nanotechnologists have become involved in

regenerative medicine via creation of biomaterials and nanostructures with potential

clinical implications. Their aim is to develop systems that can mimic, reinforce or

even create in vivo tissue repair strategies [241]. Despite substantial progress, the

construction of structures able to provide the suitable physical and biological

properties of the bone still presents challenges. Bone is comprised of hierarchically

arranged collagen fibrils, hydroxyapatite and proteoglycans [242]. To mimic the

natural bone nano-composite architecture, novel biomaterials and nanofabrication

techniques are currently being employed and many different nanostructures have

already been designed and tested [128]. For example Titanium, as a biocompatible

material, has been used to enhance implant incorporation in bone for dental,

craniofacial, and orthopedic applications. Studies have demonstrated that nano-

porous titanium dioxide (TiO2) surface modification alters nano-scale topography

improving soft tissue attachment on titanium implants surface [243-244]. Also the

uses of nano-porous TiO2 surface-modified implants, in a human dental clinical

23

study, showed that TiO2 thin film increased adherence in early healing of the human

oral mucosa and reduced marginal bone resorption [245].

Nanostructured implant surfaces are also known to enhance osteoblast

activity. In vivo, these nanostructures have also demonstrated a higher percentage of

bone contact without producing any inflammatory response. These results point to

the importance of specific nano-morphologies in controlling tissue integration [246].

Clinical therapies implying the use of nanotechnology in bone regeneration are still

in the beginning stages. Considering that hydroxyapatite is one of the major

components in the bone matrix, synthetic nano-crystalline HA has been used to

construct scaffold for bone substitutes. Recently, the bone healing ability of a

nano-composite (DBSint®), approved for clinical use [247]. The field of

nanotechnology is advancing quickly. This interdisciplinary approach is leading to a

rapid expansion and development in the fabrication of biomimetic scaffolds for

tissue engineering. Many studies have been conducted in the search for appropriate

materials to create a scaffold that may play an active role in the regeneration process

instead of simply being a cell carrier or tissue template. The advantages of nano-

materials as therapeutic and diagnostic tools are vast, due to design flexibility, small

sizes, large surface-to-volume ratio, and ease of surface modification [241].

1.4.14 Skeletal System/Bone

Skeletal tissue is exposed to mechanical forces throughout a vertebrate‘s

life span, and bone mass is adjusted in response by either absorbing existing skeletal

material or synthesizing new bone in a site-specific manner [248]. The human

skeleton is split into two main sections: the axial skeleton (comprising the head and

the trunk) and the appendicular skeleton (the limbs). Of the 206 bones which

comprise the skeletal system, there are four different types: long bones of the limbs

(e.g. tibia, femur and humerus), short bones (phalanx), flat bones (skull, scapula, and

mandible) and irregular bones (the vertebrae) [249]. Within all the aforementioned

bones are two basic types of bone tissue (in differing ratios): trabecular

(cancellous/spongy) bone and cortical (compact) bone [250].

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Bone is a major source of inorganic ions, actively participating in

calcium homeostasis in the body. There is increasing evidence that the central

control of development and renewal of the skeleton is more sophisticated than

previously appreciated [25, 251-254].

Fig. 1.8 Hierarchical structure of human cortical and compact bone [255]

It is a remarkable organ playing key roles in critical functions in human

physiology including protection, movement and support of other critical organs,

blood production, mineral storage and homeostasis, blood pH regulation, multiple

progenitor cell (mesenchymal, hemopoietic) housing, and others. . In addition, bone

contributes to the mineral homeostasis of the body and participates in endocrine

regulation of energy metabolism [256]. It is majorly 30% organic, 90% of which is

Type I collagen and 70% of inorganic components [257]. It is a dynamic, highly

25

vascularized tissue with the unique capacity to heal and remodel without leaving a

scar [258-259]. It provides mechanical stability to the skeleton that is needed for

load bearing, locomotion and protection of internal organs. Furthermore bone serves

as a mineral reservoir and has the capacity to rapidly mobilize mineral stores if

needed for homeostasis of the calcium blood level [260].

It is mainly composed of four different cell types. They are, Osteoblasts,

osteoclasts, and bone lining cells are present on bone surfaces, whereas osteocytes

permeate the mineralized interior. Osteoblasts, osteocytes, and bone-lining cells

originate from local osteoprogenitor cells, whereas osteoclasts arise from the fusion

of mononuclear precursors, which originate in the various hemopoietic tissues [261].

The formation of bone is prolonged, strictly regulated process that takes place

during embryonic development, growth; remodelling and fracture repair [262]. It is

characterized by a sequence of events starting from the commitment of

osteoprogenitor cells and their differentiation into pre-osteoblasts and then into

mature osteoblasts whose function is to synthesize the bone matrix that becomes

progressively mineralized [263]. During development, two distinct mechanisms

determine how bone is formed. Most of the skeleton is crafted by endochondral

ossification, a process whereby an initial cartilage structure creates a backbone for

osteoblasts to invade and secrete a bony matrix. Intramembranous bone is formed de

novo from mesenchymal condensations that differentiate into mature osteoblasts to

construct bones of the skull [264].

1.4.15 Bone Cells

Osteogenic cells are found both on the surface of bone, and in the

lacunae of the bone matrix [249]. There are four main cell types of bone: OBs (the

bone forming cells), OCs (the bone resorbing cells) osteocytes and lining cells [265].

1.4.16 Bone Lining Cells

Bone lining cells are flat, elongated and inactive cells that cover bone

surfaces that are undergoing neither bone formation nor resorption. Because these

26

cells are inactive, they have few cytoplasmic organelles. It can be precursors for

OBs [261].

1.4.17 Osteoblast

Osteoblasts have a very important role in creating and maintaining

skeletal architecture; these cells are responsible for the deposition of bone matrix

and for osteoclasts regulation. Osteoblasts are mononuclear, not terminally

differentiated, specialized cells [266]. As they differentiate they acquire the ability

to secrete bone matrix [267]. Ultimately, some osteoblasts become trapped in their

own bone matrix giving rise to osteocytes [268].

Osteoblasts derive from pluripotent mesenchymal stem cells [210, 213,

269], which prior to osteoblast commitment can also differentiate into other

mesenchymal cells lineages such as fibroblasts, chondrocytes, myoblasts and bone

marrow stromal cells including adipocytes, depending on the activated signaling

transcription pathways [270-271].

1.4.18 Osteocyte

Osteocytes are senile OBs which are no longer free to move about the

bone surface or divide; they are embedded within the osteoid matrix they

synthesized. Their cytoplasmic processes allow them to communicate with other

osteocytes and also to activate OBs [249].

1.4.19 Osteoclast

Osteoclast is derived from the haemotopoietic stem cells of the

macrophage/monocyte lineage. They are giant multi-nucleated cells which line the

bone forming surface of bone tissue. They resorb bone; the process lasts

approximately 10 days and is closely followed by the deposition and mineralization

of a new matrix which lasts up to 3 months, thus the remodelling process maintains

a constant bone mass. Osteoclastic action can be stimulated by numerous factors

including PTH, Vit D, Prostaglandins, Cortisol, Interleukins (IL) and tumour

27

necrosis factors TNF). It is inhibited by Estrogen, Androgen, Cortisol, TGF-β and

Nitric Oxide (NO) [265]

Fig. 1.9 The origins and locations of cells present in bone [272]

1.4.20 Bone Remodelling

Bone remodeling is the continuous process by which old bone is

removed by bone-resorbing cells, the osteoclasts, and replaced by new bone

synthesized by bone forming cells, the osteoblasts. During adult life bone

remodeling primarily serves the purposes of regulating calcium homeostasis and

repairing bone micro-fractures resulting from mechanical loading. Knowing that the

skeleton is completely remodeled every ten years, microfracture repair prevents its

excessive ageing by preventing the accumulation of old bone [265, 273-275].

Remodeling responds also to functional demands of the mechanical

loading. As a result, bone is added where needed and removed where it is not

required. The bone remodeling cycle involves a series of highly regulated steps that

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depend on the interactions of two cell lineages, the mesenchymal osteoblastic

lineage and the hematopoietic osteoclastic lineage. The balance between bone

resorption and bone deposition is determined by the activities of these two principle

cell types, namely, osteoclasts and osteoblasts. Osteoblasts and osteoclasts, coupled

together via, paracrine cell signaling, are referred to as bone remodeling units [276-

278].

Fig. 1.10 Schematic representation of Bone Remodeling process in skeletal

development [279]