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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].
2
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
3
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.
4
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.
5
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]
6
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
7
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].
8
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].
9
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].
10
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
11
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
12
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
13
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]
14
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].
15
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]
16
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.
17
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
18
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].
19
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
20
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
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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
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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
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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
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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]