Thesis

PREPARATION AND CHARACTERISATION OF
TRI-CALCIUM PHOSPHATE SCAFFOLDS WITH

TUNNEL-LIKE MACRO-PORES FOR BONE TISSUE
ENGINEERING
Wei Zheng
BE(Medical Engineering)
Principal Supervisor: Associate Professor Cheng Yan

Associate Supervisor: Associate Professor Clayton Adam
A Thesis submitted for Master of Engineering (Research)
Faculty of Built Environment and Engineering

Queensland University of Technology
2011
Key Words
Bioglass, bone formation, Compressive strength,
Hydroxyapatite
(HA),
Macro-tube Scaffold, MTT test, Phase transformation, Scanning electron

microscopy (SEM), Simulated body fluids (SBF), Sintering, Tissue engineering,
Tri-calcium phosphate (TCP).
Abstract
Calcium Phosphate ceramics have been widely used in tissue engineering due to
their excellent biocompatibility and biodegradability. In the physiological
environment, they are able to gradually degrade, absorbed and promote bone
growth. Ultimately, they are capable of replacing damaged bone with new tissue.
However, their low mechanical properties limit calcium phosphate ceramics in
load-bearing applications. To obtain sufficient mechanical properties as well as
high biocompatibility is one of the main focuses in biomaterials research.
Therefore, the current project focuses on the preparation and characterization of
porous tri-calcium phosphate (TCP) ceramic scaffolds. Hydroxapatite (HA) was
used as the raw material, and normal calcium phosphate bioglass was added to
adjust the ratio between calcium and phosphate. It was found that when 20%
bioglass was added to HA and sintered at 1400oC for 3 hours, the TCP scaffold
was obtained and this was confirmed by X-ray diffraction (XRD) analysis. Test
results have shown that by applying this method, TCP scaffolds have significantly
higher compressive strength (9.98MPa) than those made via TCP powder
(<3MPa). Moreover, in order to further increase the compressive strength of TCP
scaffolds, the samples were then coated with bioglass. For normal bioglass coated
TCP scaffold, compressive strength was 16.690.5MPa; the compressive strength
for single layer mesoporous bioglass coated scaffolds was 15.030.63MPa. In
addition, this project has also concentrated on sizes and shapes effects; it was
found that the cylinder scaffolds have more mechanical property than the club
ones. In addition, this project performed cell culture within scaffold to assess
biocompatibility. The cells were well distributed in the scaffold, and the
II
cytotoxicity test was performed by 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-diphenytetrazoliumromide (MTT) assay. The Alkaline Phosphatase (Alp) activity of
human bone marrow mesenchymal stem cell system (hBMSCs) seeded on
scaffold expressed higher in vitro than that in the positive control groups in
osteogenic medium, which indicated that the scaffolds were both osteoconductive
and osteoinductive, showing potential value in bone tissue engineering.
III
Table of Contents
KEY WORDS ............................................................................................... I
ABSTRACT .................................................................................................II
TABLE OF CONTENTS .......................................................................... IV
LISTS OF FIGURES ............................................................................. VIII
LISTS OF TABLES .................................................................................. XI
LISTS OF ABBREVIATIONS ................................................................ XII
GLOSSARY ............................................................................................ XIII
STATEMENT OF ORIGINAL AUTHORSHIP ................................... XV
ACKNOWLEDGEMENTS ................................................................... XVI
LISTS OF PUBLICATIONS ................................................................ XVII
CHAPTER 1 INTRODUCTION................................................................. 1
1.0 INTRODUCTION ............................................................................................... 1
1.1 BACKGROUND ................................................................................................ 1
1.2 RESEARCH GOALS ........................................................................................... 4
1.3 INNOVATIONS ................................................................................................. 4
CHAPTER 2 LITERATURE REVIEW .................................................... 6
2.0 INTRODUCTION ............................................................................................... 6
2.1 THE ROLE OF SCAFFOLDS IN TISSUE ENGINEERING ......................................... 7
2.2 SOLID FREEFORM FABRICATION TECHNIQUES (SFFT) ................................... 10
2.3 TARGET MECHANICAL PROPERTIES FOR BONE SCAFFOLDS ............................ 16
2.4 CHOICE OF BIOMATERIALS ............................................................................ 18
2.4.1 CaP bioceramics for bone replacement and repair ............................. 19
2.4.2 Surface coating ..................................................................................... 21
2.4.3 Composite mechanics ........................................................................... 23
IV
2.4.4 Bioglass ................................................................................................ 23

2.5 MATERIAL AND SCAFFOLD CHARACTERISTICS THAT INFLUENCE THE
MECHANICAL PROPERTIES OF CAPS.................................................................... 24
2.5.1 Porosity ................................................................................................ 24

2.5.2 Phase transformation ........................................................................... 25
2.5.3 Microstructure ..................................................................................... 26
2.5.4 Binder ................................................................................................... 26
2.6 TCP PREPARATION TECHNIQUE .................................................................... 27
2.7 APPLICATION OF BONE MARROW MESENCHYMAL CELLS IN BONE REPAIR ..... 29
2.8 COATING METHODS ...................................................................................... 31
2.8.1 Dip coating ........................................................................................... 31
2.8.2 Sol-gel method ...................................................................................... 31
2.9 SUMMARY .................................................................................................... 32
CHAPTER 3 METHODOLOGY AND SAMPLE CHARACTERIZATION
...................................................................................................................... 33
3.0 INTRODUCTION ............................................................................................. 33
3.1 METHODOLOGY............................................................................................ 34
3.1.1 Preparation of TCP ceramic ................................................................ 34
3.1.1.1 Preparation of hydroxyapatite powder .......................................... 34
3.1.1.2 Preparation of bioactive glass powder .......................................... 35
3.1.1.3 Preparation of HA/bioglass composite material ........................... 35
3.1.2 Fabrication of porous TCP scaffolds ................................................... 36
3.1.2.1 Fabrication of sacrificial moulds ................................................... 36
3.1.2.2 Coating ABS scaffold with wax .................................................... 38
3.1.2.3 Sintering of the ceramic-coated scaffold....................................... 38
3.1.2.4 Coating the TCP scaffolds with normal bioglass .......................... 40
3.1.2.5 Coating TCP scaffold with sol-gel mesoporous bioactive glass ... 40
3.2 SAMPLE CHARACTERIZATION ....................................................................... 40
3.2.1 XRD (X-ray diffraction) analysis ......................................................... 40
3.2.2 Total porosity of the calcium phosphate ceramic scaffold .................. 41
3.2.3 Shrinkage ............................................................................................. 41
V
3.2.4 Imaging the scaffold using SEM ........................................................... 41

3.2.5 Mechanical testing for scaffolds ........................................................... 42
3.2.6 Apatite-formation ability of microspheres in SBF................................ 42
3.2.7 Cell seeding and culture ....................................................................... 43
3.2.8 Observation of cell attachment on scaffolds using SEM ...................... 44
3.2.9 Cytotoxicity test by MTT assay ............................................................. 44
3.2.10 Alkaline phosphatase activity ............................................................. 45
3.2.11 Statistical analysis .............................................................................. 45
3.3 SUMMARY..................................................................................................... 46
CHAPTER 4 MECHANICAL AND BIOLOGICAL TESTING ........... 47
4.0 INTRODUCTION ............................................................................................. 47
4.1 CONFIRMATION OF TCP FORMATION ............................................................ 47
4.2 SHRINKAGE ................................................................................................... 49
4.3 MICROSTRUCTURE OF CERAMIC COMPOSITES ............................................... 51
4.3.1 10% bioglass addition .......................................................................... 51
4.4 MECHANICAL PROPERTIES ............................................................................ 58
4.5 Porosity and compressive strength of the uncoated TCP scaffold .......... 61
4.6 POROSITY OF THE COATED SCAFFOLD (NORMAL BIOGLASS).......................... 62
4.7 POROSITY OF THE COATED SCAFFOLD (MESOPOROUS BIOGLASS) .................. 64
4.8 MECHANICAL TESTING.................................................................................. 66
4.8.1 Shape and size effects ........................................................................... 66
4.8.2 Mechanical properties for bioglass coated scaffold............................. 70
4.9 APATITE-FORMATION ABILITY OF THE MACRO-TUBE SCAFFOLDS IN SBF ..... 71
4.10 CELL CULTURE............................................................................................ 72
4.10.1 SEM images of cells growth on uncoated scaffolds ........................... 72
4.10.2 SEM images of cell growth on bioglass coated scaffolds ................... 75
VI
4.10.3 Cell proliferation................................................................................ 76

4.10.4 Alp (Alkaline Phosphatase) activity ................................................... 77
4.11 SUMMARY .................................................................................................. 79
5.1 CONCLUSIONS ............................................................................................ 81
5.2 FUTURE WORK .............................................................................................. 84
REFERENCES ........................................................................................... 85
VII
Lists of Figures
Figure 1 Photograph of a scaffold produced by SFF techniques (machined
into a cylinder) .............................................................................. 11
Figure 2 The top row shows the rod diameter and the out-of-plane rod
spacing. The bottom row shows the in-place spacing. Cordell et.al [57]
...................................................................................................... 13
Figure 3 Dip coating processing [218] ................................................ 31
Figure 4 Flow chart showing the six main processing steps for fabricating
TCP scaffolds ............................................................................... 34
Figure 5 ABS macrotube scaffold templates (a) and schematics of in-plane
(top view of black box in (a)) (b) and out-of-plane (view of cross
section AA) geometry (c) ............................................................. 37
Figure 6 Sintering conditions............................................................... 39
Figure 7 (a) XRD pattern for a sintered porous calcium phosphate
(mainly-TCP) sample; (b) XRD patterns of -TCP starting powder
and sintered body [204] ................................................................ 49
Figure 8 Linear shrinkage of TCP with respect to sintering temperature
50
Figure 9 SEM images of the fracture surfaces of HA with 10% bioglass

addition at (a) 1200oC (b) 1300oC (c) 1400oC (d) The porosity of 10%
bioglass addition ceramic scaffold sintered at three temperatures. Error
bars represent mean SD for n=5. ............................................... 52
Figure 10 SEM images of the fracture surfaces of HA with 15% bioglass
VIII

bars represent mean SD for n=5................................................ 54
Figure 11 SEM images of the fracture surface of HA with 20% bioglass
bars represent mean SD for n=5................................................ 56
Figure 12 Compressive strength of 10% bioglass addition sintered at three
sintering temperatures. Error bars represent mean Standard
deviation(SD) for n=5 .................................................................. 58
sintering temperatures. Error bars represent mean SD for n=5 59
sintering temperatures. Error bars represent mean SD for n=5 60
Figure 15 SEM images of the fracture surfaces of the porous TCP showing
the presence of micro-pores: (a) made via sintering HA/bioglass and (b)
made via TCP powder [207] ........................................................ 61
Figure 16 SEM micrographs of a porous TCP scaffold with a normal
bioglass at layer 42X (a), 160X (b) and 3000X (c), as well as the surface
of the normal bioglass layer (d) ................................................... 63
Figure 17 SEM micrographs of a porous TCP scaffold with a mesoporous
bioglass layer 52X (a), and 200X (b) (a and b were single coatings);
1600X (c) 1600X(d) (c and d were double coatings) ................... 65
Figure 18 Compression stressstrain curves of the sintered porous scaffold
samples (a) made via pure TCP powder, (b) made via HA/Bioglass, (c)
IX
TCP made via HA/Bioglass and coated with two different bioglasses.
(Error bars represent mean SD for n=5) .................................... 69
Figure 19 (a) SEM image of the scaffold after soaking in SBF for 21 days (b)
EDS spectra of the surfaces of TCP ............................................. 72
Figure 20 SEM micrographs show the attachment of the cells on TCP
ceramic strut surface 800X (a) and 2000X (b) ............................. 74
Figure 21 SEM image of TCP ceramic scaffold immersed into DMEM
without cells.................................................................................. 74
Figure 22 SEM micrographs show the attachment of the cells on bioglass
coated TCP ceramic strut surface 2000X (a) and 2500X (b) ....... 75
Figure 23 MTT assay for proliferation of hBMSCs and hBMSCs combined
with TCP scaffolds at different incubation periods under the same
culture condition. Error bars represent mean SD for n=3 ......... 76
Figure 24 Alp activity of hBMSCs after 7 days, 14 days of culture, and the
novel TCP scaffolds significantly increased the Alp activity to higher
level compared to positive control group after 14 days (a); Alp activity
of hBMSCs after culturing for 7 days on TCP scaffold and TCP scaffold
coated with normal bioglass (b). Error bars represent mean SD for
n=3 ................................................................................................ 78
Lists of Tables
Table 1 Studies defining optimal pore size for bone generation ........... 9
Table 2 Summary of scaffold geometry, porosity and compressive properties
for the 3D porous scaffolds fabricated by SFFT .......................... 13
Table 3 Summary of mechanical properties and porosity of human bone 17
Table 4 Summary of advantages and disadvantages of each method for TCP
preparation.................................................................................... 28
Table 5 Sintering temperature and the bioglass additions ................... 36
Table 6 Parameters of the cube and cylinder ABS scaffold templates 38
Table 7 Ion concentrations of SBF and human blood plasma ............. 43
Table 8 XRD phase analysis data for composites of HA with the addition of
10, 15, and 20wt% bioglass.......................................................... 47
Table 9 Porosity and mechanical property for composites of HA with the
addition of 10, 15, 20wt% bioglass at three temperatures ........... 57
Table 10 Average porosity and compressive strength for the scaffolds67
XI
Lists of Abbreviations
ABS
Alp
BMPs
hBMSCs
CaPs
CMCs
Acrylontrile butadiene styrene.

Alkaline Phosphatase
Bone morphogenic proteins
Human bone marrow mesenchymal stem cell system
Calcium phosphate
Ceramicmatrix composites
CNT
Carbon nanotubes
DMEM
Dulbeccos Modified Eagle Medium
EDS
Energy dispersive spectroscopy
FCS
Fetal calf serum
MAPCs
Multipotent adult progenitor cells
MTT (3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-diphenytetrazoliumromide)
HA
Hydroxapatite
HSCs
Hematopoietic stem cells
PBS
Phosphate buffer saline
PLGA
PLLA
PS
PVC
pNPP
SFFT
SEM
SBF
TCP
Poly (lactic-co-glycolic acid)

Polylactic acid
Polystyrene
Polyvinyl chloride
pnitrophenylphosphate
Solid Freeform fabrication technique
Scanning electron microscopy
Simulated body fluids
Tri-calcium phosphate
TEOS
TEP
XRD
Tetraethyl orthosilicate
Triethyl phosphate
X-ray diffraction
XII
Glossary
Adipocytes: also known as lipocytes and fat cells, are the cells that primarily
compose adipose tissue, specialized in storing energy as fat.
Alkaline phosphatase (ALP, ALKP): a hydrolase enzyme responsible for
removing phosphate groups from many types of molecules, including nucleotides,
proteins, and alkaloids.
Biodegradability: describes the ability of the scaffold material to degrade in vivo.
Bioglass: a commercially available family of bioactive glasses, composed of SiO2,
Na2O, CaO and P2O5 in specific proportions.
Chondrocytes: the only cells found in cartilage.
Cytotoxicity is the quality of being toxic to cells.
Dexamethasone: a potent synthetic member of the glucocorticoid class of steroid
drugs.
Furnace: a device used for heating
Glutaraldehyde: an organic compound with the formula CH2(CH2CHO)2.
Hepatocyte: a cell of the main tissue of the liver.
Mesenchymal stem cells:
multipotent stem cells that can differentiate into a
variety of cell types, including: osteoblasts (bone cells), chondrocytes (cartilage

cells) and adipocytes (fat cells).
Mesoporous material is a material containing pores with diameters between 2
and 50 nm.
Neovascularization: a formation of functional micro-vascular networks with red
blood cell perfusion.
Osteoblasts: mononucleate cells that are responsible for bone formation
XIII
Osteoconductivity An ability of the scaffold to serve as a template for bone

formation by encouraging cells to adhere to the surface and to proliferate and
produce bone
Osteoinductivity of a scaffold refers to the ability to induce bone formation
without osteoinductive agents, such as bone morphogenic proteins (BMPs)
Osseointegration derives from the Greek osteon, bone, and the Latin integrare, to
make whole.
Phase transition: the transformation of a thermodynamic system from one phase
or state of matter to another
Scaffolding: a temporary structure used to support material in the construction or
repair of some large structures
Sintering: a method for making objects from powder, by heating the material in a
sintering furnace
Solid freeform fabrication techniques: a collection of techniques for
manufacturing solid objects by the sequential delivery of energy and/or material
to specified points in space to produce that solid.
Trypsinization: a process of using trypsin, a proteolytic enzyme which breaks
down proteins, to dissociate adherent cells from the vessel in which they are being
cultured.
Wollastonite is a calcium inosilicate mineral (CaSiO3) that may contain small
amounts of iron, magnesium, and manganese substituting for calcium.
XIV
Statement of Original Authorship
The work contained in this thesis has not been previously submitted to meet
requirements for an award at this or any other higher education institution. To the best
of my knowledge and belief, the thesis contains no material previously published or
written by another person except where due reference is made.
2011/7/13
XV
Acknowledgements
I would like to take this opportunity to express my gratitude and appreciation to
everyone that helped me in some way whilst completing this research. Special
recognition and thanks goes to my supervisor associate professor Cheng Yan for
his kind guidance, providing different solutions and inspiring motivation towards
my master thesis. He was also the person who suggested and introduced to me
how to work with my topic and how to enhance my skills. He has provided his
valuable time in discussing, going through my drafts, providing comments and
advising me on how to improve my work from time to time.
Also apart from my supervisor, I would like to thank some professional people
from different organization and institution for their suggestion, comments,
information, materials and other help. They are Simon Miao and Clayton Adam,
Greg Peterson, and Melissa Johnson.
XVI
Lists of Publications
1. Wei Zheng, Cheng Yan, Feng Lin, Wei Fan, Clayton Adam, AdeKunle
Oloyede. (2010) Preparation of porous tri-calcium phosphate ceramic scaffold
for bone tissue engineering. First International Conference on Cellular,
Molecular Biology, Biophysics and Bioengineering (CMBB 2010). 3: 300. Dec.
2010. Harbin, China
2. Feng Lin, Cheng Yan, Wei Zheng, Wei Fan, Clayton Adam, AdeKunle
Oloyede. (2010)Preparation of mesoporous bioglass coated zirconia scaffold
for bone tissue engineering. First International Conference on Cellular,
Molecular Biology, Biophysics and Bioengineering (CMBB 2010). 3: 330. Dec.
2010. Harbin, China
3.
Preparation and characterisation of strong and bioactive tri-calcium

phosphate scaffolds with tunnel-like macropores for bone tissue engineering,
in preparation
4.
Mechanical and biological properties of sol-gel derived zirconia scaffold

coated with mesoporous bioglass, in preparation
XVII
Chapter 1 Introduction
1.0 Introduction
In this chapter, the authors motivation for embarking upon research into the
specified area is introduced and explained. Background to the research topic is
presented first, followed by an introduction of the research goals and objectives of
this work.
1.1 Background
Tissue engineering applies methods from materials engineering and life sciences to
create artificial constructs for regeneration of new tissue [1]. Even though a range
of tissues has been studied, the translation of engineered tissues to clinical
applications has been limited. Bone tissue engineering has the potential to reach
millions annually through the repair of bone defects caused by disease, trauma or
congenital defects. In 2003 the potential market for tissue engineered products for
musculoskeletal applications totaled $23.8 billion in the USA and is expected to
rise to $39 billion by the year 2013 [2]. In 2004 alone there were $1.5million bone
graft procedures [3]. In the United States alone, at least eight million surgical
operations were carried out annually, requiring a total national healthcare cost
exceeding $400 billion per year [4, 5]. Autografts (from the patient) were
considered the gold standard for bone defect repair and allografts (from a donor)
were also commonly used. While multiple complications and risks were
associated with the use of both types of grafts [68], these remain appropriate
options for some simple and non-load-bearing defects that do not require a
significant amount of graft material (i.e. non-critical size defects). However, for
many defects the use of allogenic and autologous bone is not an option.
Researchers in bone tissue engineering are working to develop alternatives to
allogenic and autogolous bone grafts in order to address the growing needs of the
population, and much of the research is scaffold based. Thus the development of
interconnected porous scaffolds plays a significant role in bone tissue engineering,
especially in the restoration of large bone defects [9]. A three-dimensional (3D)
scaffold is usually used as an artificial matrix for bone regeneration and defects
repair or combined with cells and/or biologics, which are added to further
enhance bone regeneration [10]. However, the optimal scaffold recipe,
including target mechanical properties, is still very much under debate. There are
several characteristics that are considered to be essential for bone scaffolds, such
as biocompatibility, osteoconductivity and interconnected porosity. Other
considerations in bone scaffold design and optimization include biodegradability,
permeability and mechanical integrity.
Calcium phosphate ceramics have been extensively used to produce porous

scaffolds due to their bone-like chemical composition as well as excellent
biological
properties,
including
biocompatibility,
osteoconductivity
and
osteoinductivity [11]. In the calcium phosphate compounds, hydroxyapatite

(Ca10(PO4)6(OH)2, HA), the main inorganic component of natural bone, has been
widely investigated since calcium phosphates are recognized as ceramic materials
that simulate the composition and mineralogical structure of natural bone [12].
Nonetheless, because of the weak mechanical properties of HA ceramics, such
2
materials cannot be used as implant devices for replacing human bones [13]. It is
widely acknowledged that the incorporation of a ceramic reinforcement (i.e. fibers,
whiskers, platelets or particles) in a ceramic scaffold improves the mechanical
properties [14]. Thus, many studies have added some bioactive glass into the HA.
Bioglasses are popular biomaterials, because their compositions are similar to the
inorganic constituent of the mineral part of bone [15]. As a result, bioglass
addition greatly improves HAs compressive strength, largely due to improved
densification through the presence of a liquid phase during sintering [16]. When
dense HA ceramics are implanted, low resorption rates of HA hinder bone
ingrowth, resulting in chemical bonding only at the interface between the bone
and HA implant. This low biodegradability is the disadvantage of dense HA
ceramics, which limits the wide application of bulk HA [20, 21]. Compared to HA,
tri-calcium phosphate is generally considered as a resorbable bio-ceramic [20].
TCP ceramics display better biodegradability than HA and tend to be replaced by
bone as they degrade. Experimental studies have also showed that short sintering
time and limited glass addition such as 2.55wt%, can be used to control phase
transformation between HA and TCP [17-19]. It was reported that greater
amounts of phase transformation occurred with the addition of 5wt% bioglass [18].
However, to the authors knowledge there is no research been conducted to
investigate the processing parameters such as temperatures and addition of
bioglass into HA to obtain TCP. Therefore, in this project, the HA/bioglass
composite is sintered at high temperature to obtain the TCP ceramic, and the
novel macrotube scaffolds were designed to help the bone formation.
1.2 Research goals

The overall goal of this research is to create a macro-tube porous tri-calcium
phosphate scaffold through the use of composite hydroxyapatite and bioactive
glass, sintered at high temperature. The purpose of using macro-tubes is to
provide necessary oxygen and nutrients, which flow through the tube in order to
promote osteoblasts. The pores on the inner-walls of each macro-tube are used for
cell loading, ingrowth, and tissue-formation. The mechanical properties of
tri-calcium phosphate scaffolds depend on scaffold structure, the bioglass
additions and the sintering temperature. The biological properties are based on the
scaffolds structure and material properties.
1.3 Innovations
In
the
past
decade,
experiments
were
usually
performed
using
polyurethane-sponge [24-27] as a sacrificed scaffold, for the structure of the

sponge is fairly similar to trabecular bone. However, the high porosity sponge
ceramic scaffold has low mechanical property. As an improvement, the
innovations of this project were that the sacrificed scaffold would be designed as a
controlled macro-tube scaffold in order to control scaffolds porosity and
mechanical property. Furthermore, in past experiments [64, 66, 95], tri-calcium
phosphate ceramics were usually obtained by using more complex methods (see
section 2.6), however in this project, the TCP ceramic would be produced by
mixing HA and bioglass and sintering at high temperatures. It was known that
when HA reacts with bioglass at high temperatures, TCP and other phases
4
(depending on the bioglass) can be formed, which has a higher mechanical

property than conventional methods. Finally, the porous TCP ceramic scaffold
would be coated with bioglass to achieve greater mechanical property and
biocompatibility.
Chapter 2 Literature Review

2.0 Introduction
Chapter 2 reviews the literature relevant to this research field. This chapter is
divided into five components, (1) the importance of scaffolds in tissue engineering,
(2) scaffold design, (3) choice of materials, (4) current TCP preparation technique
and (5) cell culture. Through this chapter, the reader can understand the
importance of the role that porous ceramic scaffolds playing in tissue engineering.
They are required to be biocompatible, biodegradable and osteoinductive. The
scaffold can be fabricated using multiple techniques. However the solid freeform
fabrication technique (SFFT) is one of the most advanced one. By using this
technique, the porosity of the scaffold can be controlled, and consequently the
compressive strength of the scaffold can be controlled as well.
Various materials have been used for fabricating bone tissue engineering scaffolds.
Calcium- phosphate ceramics are the most common material used for the ceramic
scaffold, and polymers are commonly used for coating the ceramic scaffold.
However, there are some disadvantages for the polymer coating, thus the current
trend is towards the use of bioactive ceramic as the coating material. Moreover, in
recent studies, it was shown that in order to enhance the HA scaffolds
compressive strength, bioglass was added to HA. Some phases, including TCP
were formed when the composite material was sintered at high temperatures [22].
TCP is regarded as one of the most favorable bioactive materials for fabricating
porous ceramic scaffold [23]. Nevertheless, techniques for fabrication often vary.
In the TCP preparation section, some preparation techniques will be introduced,
6
and each techniques advantages and disadvantages will be listed. Unfortunately,

there have not been many studies on TCP fabrication using HA and bioglass
composites. The coating method will also be introduced at the end of this chapter.
2.1 The role of scaffolds in Tissue Engineering

Scaffolds for bone regeneration mainly serve an osteoconductive function, in
which new bone forms through creeping substitution from adjacent living bone
tissue [25]. Scaffolds can also act as three-dimensional vehicles for cell delivery
and tissue regeneration and to further enhance the regeneration process. A popular
approach is to expand bone progenitor stem cells in vitro and disperse them into
scaffolds to stimulate osteogenic differentiation, followed by implantation onto
the site of the bone defect. An ideal scaffold for bone tissue engineering would
have certain characteristics, which are high biocompatibility and biodegradability
[221]. HA is an example of a biodegradable and biocompatible scaffold material,
as approved by the US Food and Drug Administration [222]. Other ideal
characteristics are osteoinductivity (actively inducing bone formation) or
osteoconductivity (guiding and supporting bone regeneration) [26-27]. Bioglass
and calcium phosphate ceramics are typical osteoconductive materials.
Bone that is damaged by accident or degenerated as a result of age and disease
often needs to be reconstructed by surgical treatments. Therefore, synthetic
bone-graft substitutes are in great demand [28]. The relationship between scaffold
structure and tissue compatibility has been studied previously [29-33]. Optimal
scaffolds for cell loading, ingrowth, and tissue-formation are basic problems in
7
synthetic bone-graft applications. Cellular composites are then seen as consisting

of three main structural components: (1) cells that are organized into functional
units, (2) extracellular matrix, and (3) scaffold architecture [223-224]. This
architecture is increasingly believed to contribute significantly to the development
of specific biological functions in tissues and thought to provide appropriate
nutritional conditions and spatial organization for cell growth. The regeneration of
specific tissues aided by synthetic materials has been indicated to be dependent on
the pore size and porosity of the supporting three-dimensional structure [34]. A
large surface area is necessary for cell attachment and growth. A large pore
volume is also required to accommodate and subsequently deliver a cell mass
sufficient for tissue repair. Highly porous biomaterials are also desirable for the
easy diffusion of nutrients to and waste products from the implant and for
vascularization, which are important requirements for the regeneration [36]. The
surface area/volume ratio of porous materials depends on the density and average
diameter of the pores. Nevertheless, the diameter of cells in suspension dictates
the minimum pore size, which varies from one cell type to another. Depending on
the envisioned applications, pore size must be carefully controlled. The effect of
implant pore size on tissue regeneration is emphasized by experiments
demonstrating optimum pore size of 5m for neovascularization, 5-15m for
fiberblast ingrowth, close to 20m for the ingrowth of hepatocytes, 20-125 m for
regeneration of adult mammalian skin, 40-100m for osteoid ingrowth [35], and
100-350m for regeneration of bone [36] (See Table 1). Fibrovascular tissues
appear to require pores sizes greater than 500m for rapid vascularization and for
the survival of transplanted cells [37]. While the increase in porosity and pore size
8
would favor bone ingrowth and nutrition supply, increase in porosity and pore
size would consequently decrease the biomechanical strength [38].
Table 1 Studies defining optimal pore size for bone generation
Mineralize tissue
Scaffold pore size

Reference
Porosity (%)
(mm)
Klawitter et
al.[36]
ingrowth/comments
No tissue ingrowth
Type I: 26 m
33
(22weeks)
No bone ingrowth,
Type II: 1540 m
46.2
fibrous tissue ingrowth

(22weeks)
50 m of bone
Type III: 30100
ingrowth, osteoid and
m(80% pores <100
46.9
m)
fibrous 80% pores , 100

m tissue ingrowth
(22weeks)
20 m of bone ingrowth
Type IV: 50100
by 11 weeks and 500
m(63% pores <100
46.9
m)
m of ingrowth by 22
weeks, osteoid and
fibrous tissue ingrowth
600 m of bone
ingrowth by 11 weeks
Type V: 60100
48
m(37%<100 m)
and 1,500 m of
ingrowth by 22 weeks,
osteoid and fibrous
tissue ingrowth
Not statistically
Whang et al.[35]
<100 m
35.3
different from untreated

controls
Not statistically
<200 m
51
different from untreated

controls
Statistically significant
<350 m
73.9
more bone than all other

groups
It is rather challenging to find a porous structure with a large pore size and a
suitable mechanical strength. A number of fabrication techniques have been
developed over the years for manufacturing porous bioceramics. These include,
amongst others, the replication of polymer foams by ceramic dip coating [39-40]
or impregnation, foaming of aqueous ceramic powder suspensions [41-42],
pyrolysis of pre-ceramic precursors [43] and firing of ceramic powder compacts
[44-45]. However, none of these methods can completely satisfy all the necessary
requirements. For instance, a controlled level of interconnected porosity combines
with good compressive strength (the strength in the region of 2-14MPa with a
porosity >50%) [46].
Trabecular bone can also be referred as spongy bone since the structure of the
Trabecular bone is quite similar with the sponge. Hence, sponges made from
Polyurethane (PU) are widely used as a template for making ceramic scaffolds.
Although PU sponge has very high volumes of porosity and excellent
interconnectivity levels, the mechanical properties of the sponge ceramic is poor
[40], and the pores of the sponge cannot be controlled.
2.2 Solid freeform fabrication techniques (SFFT)

SFFT, such as fused deposition modeling, have been employed to fabricate highly
10
reproducible scaffolds with fully interconnected porous networks [47-48] as

shown in Fig. 1. Using digital data produced by an imaging source such as
computer tomography or magnetic resonance imaging enables accurate design of
the scaffold structure [48]. Solid freeform (SFF) manufacturing combined with
conventional foam scaffold fabrication procedures such as sponge coating may be
used to develop scaffolds with controlled micro and macro-porous structures
[49-53]. This enables scaffold properties such as pore size, pore fraction, strength,
modulus and permeability to be varied systematically.
Figure 1 Photograph of a scaffold produced by SFF techniques (machined into a

cylinder)
Scaffolds fabricated using SFF technique is generally reported to have better
mechanical properties than those fabricated by conventional ceramic processing
routes [54]. Such biomimetic internal architectures may prove valuable for
multi-tissue and structural tissue interface engineering. To the authors knowledge,
there is no literature available on degradable HA/bioactive glass made by SFF
Techniques. This technique has been only applied for composites containing
11
calcium phosphates as the bioactive phase [55-56]. For example, Xiong et al. [56]
fabricated composites of PLLA/TCP with porosities of up to 90% and mechanical
properties close to human cancellous bone by using low-temperature deposition
based on
a layer-by-layer manufacturing method of SFF fabrication
(computer-driven by 3D digital models). PLLA was dissolved in dioxane and TCP

powder mixed to prepare slurry, which was formed into frozen scaffolds, and
subsequently freeze-dried. Alternate parallel layers formed macro-pores (400m
diameter) and sublimation of the solvent during freeze-drying formed micropores
(5m diameter). Taboas et al. [55] produced PLA scaffolds with computationally
designed pores (500-800m wide channels) and solvent-derived local pores
(50-100m wide voids or 5-10m length plates). Indirect fabrication using casting
in SFF moulds provided enhanced control over scaffold shape, porosity and pore
architecture,
including
size,
geometry,
orientation,
branching
and
interconnectivity. One of the major disadvantages of this method is to increase

scaffold fabrication time compared with direct methods, a temporary mould must
be made first. The macro-pore size and volume fraction are controlled by the
combination of the rod diameter and spacing in and out of plane. Details of
macro-porous scaffolds from several independent studies are shown in Table 2.
12
Figure 2 The top row shows the rod diameter and the out-of-plane rod spacing.
The bottom row shows the in-place spacing. Cordell et.al [57]
Table 2 Summary of scaffold geometry, porosity and compressive properties for
the 3D porous scaffolds fabricated by SFFT
(*NR, not report by the author)
Reference
Cordell
Woodard
Dellinger
Miranda
[57]
[58]
[59]
[60]
Unit cell
geometry
(m)
Rod
394
394
415
415
570
570
220
220
252
252
315
315
280
280
100
100
359
359
315
315
270
270
80
80
diameter
Edge to edge
(out of
plane)
Edge to edge
(in plane)
13
Porosity
Calculated
(or stated)
28
50%
50%
41%
41%
28%
macro-pore
28%
28%
NR*
NR*
47
14
fraction
Micro-pore
1-3
1-10
2-15
2-8
2-8
1-30
0
size (m)
Strength in
Compressio
n
Compressive
Strength
7.8
8.3
101.5
53.9
302.3
10.4
(MPa)
Test Sample
Geometry
Test
Cylind
geometry
er
Height (mm)
8.1
Cylinder
Cylinder
Cube
4.5
10
The essential characteristics of scaffolds for using in bone generation and repair
are outlined below. Other characteristics that are usually considered in scaffold
design are also discussed. As no single study has addressed all these, many are
coupled and, therefore, difficult to assess.
1) Biocompatibility is among the most important scaffold characteristics. It is
defined broadly as the ability of a material or device to perform its intended
function, including an appropriate degradation profile, without eliciting any
undesirable local or systemic effects in that host [61]. Host responses, both
positive and negative, may include osteoblast/osteoclast response, prolonged
inflammation,
microvascular
changes,
fibrous
adsorption and endothelial proliferation [62-63].

14
encapsulation,
protein
2) Osteoconductivity is the ability of the scaffold to serve as a template for bone

formation by encouraging cells to adhere to the surface and to proliferate and
produce bone.
3) Interconnected porosity is required for nutrient and waste transport throughout
and for bone growth. The minimum pore size for bone formation has been
quoted by many as 100 m [64-69]. However, more recently researchers have
shown bone formation in interconnected micropores less than 10m in size in
scaffolds that contained both macroporosities (>100m) and microporosities
(<10m) [70-71].
4) Biodegradability describes the ability of the scaffold material to degrade in
vivo. CaPs degrade relatively slowly by physiochemical, cell mediated or
mechanical degradation mechanisms [72-73].
5) Bioactivity is the tendency of the material to form a chemical bond with the
host bone. For CaPs this is postulated to occur through material dissolution
and precipitation of a carbonated apatite that is more similar to the mineral
phase of bone. Some factors that affect bioactivity are composition,
crystallinity, grain size and impurities.
6) Osteoinductivity of a scaffold refers to the ability to induce bone formation
without osteoinductive agents, such as bone morphogenic proteins (BMPs).
7) Permeability is the ease with which fluid can flow through a porous material.
It depends on the pore size, fraction and fenestration, which are all dependent
upon the scaffold pore architecture.
15
8) Manufacturability refers to the ease and flexibility of scaffold fabrication.

Solid free-form fabrication techniques are a common fabrication route
[74-75].
9) Mechanical integrity is a broad term that encompasses all mechanical
properties from post-manufacture through to complete healing.
2.3 Target mechanical properties for bone scaffolds

The need for scaffolds that can be used to repair load-bearing bone defects, which
are often also large defects, is apparent. However, the specific mechanical
requirements of the scaffolds being studied for the repair of such defects,
beginning from implantation through to complete healing, are yet to be
established. The most appropriate materials and their corresponding mechanical
properties are still under debate. The target properties such as strength and elastic
modulus that have been explicitly stated or implied in the literature, span several
orders of magnitude and several different approaches have been taken with regard
to the design for specific mechanical properties. For example, a number of
literatures have stated that bone scaffold properties should match those of natural
bone [76-80]. Table 3 summarizes the compressive, flexural and tensile strengths,
elastic moduli and porosities for both cortical and cancellous bone for referencing
purpose. Another study took a different approach and optimized scaffold pore
architecture such that the scaffold stiffness matched the stiffness of native bone
initially and the stiffness of the regenerated bone matched that of native bone at
the end point [79]. In a different paper the authors stated simply that the
mechanical properties of the scaffold must be sufficient and not collapse during
16
handling and during the patients normal activities [81]. In contrast to these
studies, another argued that scaffold strength should be greater than the bone it
will replace [82]. The same authors indicated a need for fixation devices to shield
scaffolds from loading. These examples from the literature demonstrate the wide
range of target mechanical properties for bone scaffolds.
Table 3 Summary of mechanical properties and porosity of human bone
*NR, not reported by author.

A final strategy is to design scaffolds such that the mechanical properties of the
composite of bone and scaffold are within some percentage of the mechanical
properties of the host bone.
To achieve this, the initial mechanical properties of the scaffold should account
for the change in properties with degradation and the change with the expected
bone in-growth. It was discovered that a high order for some systems whose
degradation rates are sensitive to processing parameters and the in vivo
environment or simply are not well characterized. On the other hand, degradation
17
of CaPs can be of the order of months to years, depending on porosity,

crystallinity and grain size as shown in the study by Grynpas [90].
CaPs is an excellent candidates for use in bone replacement and repair. CaPs can
have strengths and stiffnesses that are similar to those of cortical and cancellous
bone in some forms [91-99, 81, 65]. The real limiting factor for CaPs in
load-bearing applications may be in the inherent brittleness of this class of
materials. Unfortunately, few studies in the literature report properties such as
fracture toughness, reliability (i.e. Weibull modulus), or energy to failure for CaPs
or composite scaffolds. CaPs usually degrade slowly and in a controlled manner.
The stiffness and strength of the CaP bone composite is reasonably stable overall.
However, the issue of brittle behavior has yet to be resolved.
2.4 Choice of biomaterials

In tissue engineering, many types of materials have been applied for bone repair
and regeneration; these biomaterials in general can be divided into several groups:
metals, ceramics, polymers, and composites [100]. Ideally, materials and scaffolds
utilized in tissue engineering should meet some prerequisites including
mechanical
properties
and
integration
with
host
[101-102],
enhanced
osteoinductive, osteoconductive properties and material biocompatibility [103]. In

addition, porosity, pore diameter, interconnectivity and microstructure should also
be considered for bone repair and regeneration as well [104]. Many studies have
demonstrated that hydroxyapatite (HA) or other calcium phosphate (CaP)
ceramics including tricalcium phosphate (TCP) and bioactive glasses can improve
18
the formation of a bone-like apatite layer on their surfaces [105]. The inorganic
ceramic phase can be combined with polymers or polymer precursors to produce
bioactive and biodegradable composites [106]. Therefore, the new strategies for
creating a CaP composition similar to bone apatite will be advantageous.
2.4.1 CaP bioceramics for bone replacement and repair
Calcium phosphate (CaP) is considered the most popular biomaterial for
exogenous bone grafts in bone reconstruction [107-109]. Calcium phosphates
were first considered for clinical application as a filler for bone defects in the
1920s and first incorporated in dentistry and orthopedics in the 1980s [33]. The
interest in CaP based ceramics for bone replacement and repair is well-deserved,
given that they have the requisite characteristics and many other attributes that
make them excellent candidates for such applications. CaPs are biocompatible,
have a composition and structure similar to the mineral phase of bone [91]. They
are osteoconductive [110-113] and they have been reported to be intrinsically
osteoinductive in some cases [110, 114, 64]. They are also bioactive.
The degradation products of CaPs are conducive to bone formation and strongly
linked to bioactivity; the dissolution process is followed by reprecipitation of a
carbonated apatite, which has a composition and chemical structure that is similar
to the mineral phase in bone [61, 76, 115]. The degradation rate for CaPs is
typically slow compared to that of many polymers [81] and even comparable with
bone growth [116]. This is advantageous because it addresses concerns about
balancing degradation and bone in-growth. A range of CaPs compositions have
been considered, hydroxyapatite (HA), with the chemical composition
Ca5(PO4)3OH and a calcium to phosphate ratio of 1.67 [117], and -tricalcium
19
phosphate (-TCP), with the chemical composition Ca3(PO4)2 and a calcium to

phosphate ratio of 1.5 [93]. Among these, HA is most commonly used in clinical
applications and has been used in bone cements for the repair of craniofacial
defects [118-119], for maxillary sinus floor augmentation [120] and in coatings
for hip replacements [121,122]. -TCP ceramics have been commercialized as
bioresorbable synthetic bone substitutes and are used in orthopedic and dental
applications [123], including augmentation of the alveolar ridge [124], sinus
reconstruction [125] and general bone reconstruction following injury or disease
[126]. Perhaps the most emphasized difference between the properties of HA and
-TCP is their relative degradation rates; HA is considered relatively
non-degrading while TCPs purportedly degrade readily [127-129], which has been
seen in previous studies. In Fang clinical report, -TCP could help the bone
replacement in animal experiments [133].
TCP has three polymorphs: low temperature phase, -TCP is presented below
1180C, while -TCP appears in the temperature range 1180-1400C and -TCP
is observed above 1470C [130-131]. The phase transformation is accompanied
by density changes for calcium phosphate materials. The density decreases with
phase transformation in the following sequence when heat-treated to higher
temperatures [132]
HA (3.14 g*cm-3) >-TCP (3.07 g*cm-3) >-TCP
(2.77 g*cm-3) >-TCP
(1)
Among the three allotropic forms of TCP, -TCP is the preferred as a bio-ceramic
on account of its mechanical strength, good tissue compatibility, and ability to
bond directly to tissue to regenerate bone without any intermediate connective
20
tissue. In addition, fast bone regeneration and proper bioresorption rate are other
additional attributes of -TCP [132]. It has been documented that the dissolution
rate of -TCP is 3-12 times faster than HA [133]. In vitro studies reveal that
-TCP exhibits a higher dissolution rate than -TCP [132-134]. The order of
relative solubility is
-TCP>-TCPHA[132,134]
(2)
Since tri-calcium phosphate exhibits higher solubility, it is expected that it can

potentially degrade upon implantation in the host and may be gradually replaced
by the newly formed regenerated bone.
2.4.2 Surface coating
A success of an implant also depends on its surface chemistry, which determines
the interactions at the implant material-tissue interface. To elicit desirable
material-host tissue interaction, the implant may need to be coated with suitable
coatings. Another important reason for a suitable surface coating is the wear of the
implant surface being in contact with the host tissues. For example, the hardness
of the bone leads to very heavy abrasion by fretting or direct wearing as soon as
the interfacial strains between the implant and the hard tissue occur. The final
reason for the coating is the coating material could improve the mechanical or
biological properties for the scaffold, and this reason is the most important for the
CaPs ceramic scaffold coating.
There are two main possibilities for kinds of the coating materials: polymer and
ceramic. As it was mentioned previously, CaPs make excellent candidates for
using in bone replacement, however, it has been reported that one of the limiting
factors for the CaP in load bearing applications is poor strength [117,135,136].
21
Polymer with excellent mechanical properties can improve CaPs scaffolds

strength, reaching to a level which is similar to the cancellous bone strength [137].
However, there are some disadvantages for the polymer coating. Poly
(lactic-co-glycolic acid), (also commonly known as PLGA) is a type of bioactive
polymer, which is used as the coating material and it has been approved by the US
Food and Drug Administration (FDA). Unfortunately, the chemical used to
dissolve PLGA may possess potential health and safety risks. When preparing
PLGA, it is usually required to be dissolved in dichloromethane (CH2Cl2)
solution first. Dichloromethane (CH2Cl2) is a toxic chemical and can
potentially damage the human nervous system. Recent studies have shown that
although the polymer coating can largely increase the compressive strength, it
decreases the osteoconductivity of the scaffold [138]. Another disadvantage is
that after applying polymer coating, the scaffold surface will become very
smooth. This could discourage cell growth as rough surfaces result in good
osseointegration as compared to smooth surfaces, it was shown that the number
of Alp (alkaline phosphatase) -positive cells on the rough surface was two to three
times higher than that on the smooth surface [139]. Another coating material is
bioactive ceramic. Many researchers have used bioactive ceramics (for
instance HA, bioglass) to improve scaffolds mechanical and biological
properties [140-146]. Normally, these kinds of scaffolds have high mechanical
properties, and the base materials for those scaffolds were bioinert ceramics or
metals. After the coating film is degraded, the base material will be connected
with the host-tissue, which can have detrimental effects on the bone
regeneration process. Therefore, bioactive ceramic base with bioactive
22
ceramic coating is the best way to improve scaffolds mechanical and

biological properties.
2.4.3 Composite mechanics
One of the main purposes of creating ceramicmatrix composites (CMCs) is to
improve the materials fracture toughness, or its ability to resist fracture when a
crack is present. Low strength in tension compared with compression is a major
problem with brittle ceramics. In tension, even the slightest flaw, such as a surface
or interior crack, internal pore, or grain corner, will amplify the applied stress.
Therefore, as the two go hand in hand, an improvement in toughness generally
leads to improvements in strength and stiffness as well [147]. The reinforcing
phase in a ceramic serves to prevent crack growth. This can be accomplished by a
number of methods, including deflecting crack tips, forming bridges across crack
faces, absorbing energy during pullout, and causing a redistribution of stresses in
regions adjacent to crack tips [148]. In addition to the reinforcing materials
mechanical properties, its morphology is critical.
2.4.4 Bioglass
Bioglass belongs to a family of bioactive glasses containing various proportions
of chemical constituents. When incorporated into a damaged part of the human
body, it is able to associate with bony structure, promoting repair and bringing it
to normal function. The difference of bioglass from traditional soda-lime glasses
is that the proportions of SiO2, Na2O, CaO and P2O5 are different. Bioglass has a
lower
amount
of
silica,
but
more
sodium
and
calcium,
and
high
calcium/phosphorus ratio. However, due to the weak mechanical strength of

23
bioglass, this material can only be used in applications subjected to a lower load.
Currently, its main use includes coating metal, increasing the biological
properties.
2.5 Material and scaffold characteristics that influence

the mechanical properties of CaPs
There are a number of main characteristics that influence the mechanical
properties of CaPs, as well as other ceramics. It is well known that the
characteristics that influence mechanical properties can be controlled by the
processing parameters [78], because this fabrication process requires high
temperatures and long heat treatments.
2.5.1 Porosity
Pore size, fraction, distribution and architecture have a strong influence on the
mechanical properties of CaPs and ceramics in general [64, 66, 95, 150-152]. One
of the most common representations of the relationship between porosity and
density is given by Eq. [153]
Total Porosity =1- M/ (V x )
(3)
In which M is weight of the sintered porous scaffold, V is volume of the sintered

sample porous scaffold,is the solid density of the ceramic. It is well accepted
that an increase in pore size leads to a decrease in strength. Pore geometry also
affects the strength [152] through the local stress concentration introduced by the
pores.
24
2.5.2 Phase transformation

The phases present in the CaPs bulk materials and scaffolds, determined by the
average composition as well as by the heat treatment [154], affect the mechanical
properties [155]. However, the trends are not well characterized and the
mechanisms are not well understood. The HA, TCP and bio-glass materials are
mainly consist of Ca, P, OH atoms, which are also the main constituents construct
main composition of human hard tissues. In general, sintering HA can lead to the
partial thermal decomposition of HA into tri-calcium phosphate (TCP). There are
two steps in the thermal decomposition i.e. dehydroxylation and decomposition.
Dehydroxylation to oxyhydroxyapatite proceeds at temperatures about 850C to
900C by the fully reversible reaction in accordance to equation 4 [156-157]:
Ca10 (PO4)6(OH)2 Ca10(PO4)6(OH)2-2xOx + xH2Ogas
(4)
The decomposition to TCP and tetracalcium phosphate occurs at temperatures

greater than 900C: According to the reaction given in equation 5 [156-157]:
Ca10 (PO4)6(OH)2 2Ca3(P04)2 (TCP)+Ca4P2O9+H2Ogas (5)
When bioglass is added to HA for the purpose of increasing HAs mechanical

properties, it is shown that HA can be reacted with bioglass at the critical
temperature, and this results in TCP and other phases (depends on the
composition in bioglass) [158-162]. For example, in Seema.ks experiment [22]
where P2O5, CaO, and Na2O were used for making bio-glass, and 10% bioglass
was added to HA at the temperature of 1250oC, the results showed the primary
25
ceramic present is hydroxyapatite, and -TCP (Ca3(PO4)2) as the secondary phase.

No other phases like -tricalcium phosphate (-TCP) and calcium oxide (CaO)
were detected. In another experiment, A.Carr [161] mixed P2O5, CaO, Na2O,
Al2O3, and B2O3 to fabricate bioglass. When 25% bioglass was added at the
temperature of 1200oC, the amount of HA occupation decreased from 75% to
30%, and 12.6% of -TCP was found in the final powder. The phases were
possibly some -Na2Ca4(PO4)2SiO4 and wollastonite [163]. Therefore, it can be
concluded that phase transformation is an important factor for fabricating calcium
phosphate ceramic, and the phases formed depends on three parameters: 1) the
quantity of bioglass added into HA, 2) sintering temperature, and 3) the
composition of bioglass.
2.5.3 Microstructure
Crystallinity and grain size also affect the mechanical behavior of CaPs. A more
prominent crystalline phase, lower pore fraction, and smaller grain size are all
associated with increased stiffness, compressive and tensile strengths, and fracture
toughness [81]. In general, the strength for ceramics is proportional to the inverse
square root of the grain size [150], similar to the relationship to metals.
2.5.4 Binder
When a low ceramic content is employed to increase the porosity, cracks are
prone to occur in the sample, because of shrinkage during sintering, which,
consequently, causes a severe reduction in strength. So in most of the previous
experiments, use of binder is a requisite for sintering ceramic. Polymers are
usually used (i.e. Polystyrene (PS), Polyvinyl acetate (PVA)), as the binder, since
26
it would be expected to improve the strength of the body, which, in turn, would
prevent it from cracking [157]. The presence of small pores formed by the
removal of the polymer on the ceramic during sintering, the polymer could also be
used for increasing ceramics porosity [164]. However, if polymer is over-added
into the ceramic, the mechanical properities would decrease instead. Thus, the
content of the binder in ceramic is one of the most important parameters. In
Yooks experiment, where a polystyrene (PS) polymer was used as the binder, he
pointed out that the compressive strength of the porous HA scaffolds was
significantly affected by the PS content, when increasing PS content from 0 to
20vol.%, the compressive strength of the sample was significantly increased.
However, a higher PS content of 30vol. % was observed to lead to a lower
compressive strength [165]. Safronovas report indicated that the presence of 0.25%
0.50% PVC (Polyvinyl chloride) strongly influences the mechanical properties
of the powder [166].
2.6 TCP preparation technique

TCP has been shown to be resorbable in vivo with new bone growth replacing the
implanted TCP [167]. This property imparts significant advantage onto TCP
compared to other biomedical materials that are not resorbed and replaced by
natural bone. Conventionally, -TCP powders are synthesized via a solid-state
process [168-171] and wet-chemical method [172-176], as described by the
following equations
Wet-chemical methods
27
Ca(NO3)2+2(NH4)2HPO4+2NH4OHCa3(PO4)2+6NH4NO3+2H2O
Ca(OH)2+H3PO4Ca3(PO4)2+2H2O
(6)
(7)
Solid-state process
Ca(H2PO4)2+2Ca(OH)2Ca3(PO4)2+4H2O (sintered at 900oC)
(8)
There are some other methods for fabricating TCP powder. Table 4 summaries
the advantage and disadvantage of each method.
Table 4 Summary of advantages and disadvantages of each method for TCP

preparation
Preparation
Average
techniques
particle sizes
Advantages
Takes a long time
Particle sizes are
Simple and low
Wet-chemical method
[172-176]
Disadvantages
0.64m
cost
not even
solid-state process
[170-171]
not even
0.1~1.0m
~1.0m
Other phases exist
Not easy to control
Should be
bigger
Simple and could

MechanicalChemical
Synthesis
Particle sizes are
Hydrothermal
[177]
Particle sizes are
Good crystalline
0.13~0.14m
be completed in a
conducted in strict
short time, also the
conditions
particles sizes are
[178]
even
28
Should be done in
manually
Particle sizes are
Sol-gel processing
Nano size
[179]
50nm
[180]
small
Average particle
Chemical Synthesis
sizes are smaller
Easily introduce
other impurities
Easily introduce
other impurities
2.7 Application of bone marrow mesenchymal cells in

bone repair
Bone marrow cells consist of progenitor cells: the hematopoietic cell system and
non-hematopoietic human bone marrow mesenchymal stem cell system (hBMSCs)
[181-182]. Hematopoietic stem cells (HSCs) in bone marrow are the reservoir of
various blood cells, such as erythrocytes, leukocytes, macrophages or platelets
[183-184]. This mixture of multipotent progenitor cells can differentiate into
various other mesodermal cells, such as osteoblasts, chondrocytes, fibroblasts or
adipocytes [185]. When these cells are cultured in vitro, hBMSCs quickly adhere
and can be easily separated from the nonadherent hematopoietic cells by repeated
washing. hBMSCs form colonies and are therefore defined as Colony Forming
UnitsFibroblasts (CFU-Fs), each colony derives from a single proliferating
progenitor [186]. An approach to distinguish the subsets of hBMSCs with the
most active replication and individual differentiation potential would certainly be
very important for both theoretical and applicative reasons. Some laboratories
have developed monoclonal antibodies in order to identify one or more markers
suitable for hBMSCs identification and sorting [187-189]. As a mixture of
29
multipotent cells, bone marrow cells have the latent ability to differentiate into
different cell lineages depending on the cell culture environment, a fact that makes
applying bone marrow cells as a cell source for various tissue engineering
applications possible, especially in bone tissue engineering. As multipotent adult
progenitor cells (MAPCs) and a pool of cells are committed to various lineages
and stages of differentiation, the hBMSCs contain potentially osteogenic colonies.
These colonies can be referred to as skeletal stem cells meaning they are
ancestors to mesodermal supportive tissues such as bone, cartilage, fibrous tissues,
and so on [190]. These skeletal stem cells, however, stay in different stages of
differentiation. Some are tripotential and are able to differentiate into osteoblasts,
chondrocytes, and adipocytes in vitro; some are only able to bipotentially
differentiate into a chondrogenic-osteogenic phenotype, whereas the others
differentiable only into osteogenic clones. Interestingly, over a prolonged time
cultured in vitro, the differentiation potential is reduced and the tripotential clones
progressively lose adipogenic, then chondrogenic phenotypes, and finally
osteogenic potential, suggesting a preferential commitment of the progenitors
towards the osteogenic phenotype [191]. Many studies have shown the osteogenic
abilities of hBMSCs, both in vitro and in vivo, and applied it to bone defect
regeneration [192]. It is undoubted that under certain conditions, hBMSCs are
able to differentiate into osteogenic progenitor cells and finally osteoblasts,
thereby able to regenerate tissue of bone defects by direct orthotopic placement in
conjunction with appropriate scaffolds, most commonly those containing
hydroxyapatite/tricalcium phosphate (HA/TCP).
30
2.8 Coating methods

2.8.1 Dip coating
Dip coating refers to the immersing of a substrate into a tank containing coating
material, removing the piece from the tank, and allowing it to drain. The coated
piece can then be dried by force-drying or baking. It is an easy and economic way
to coat scaffolds. Fig.3 shows the dip coating processing.
Figure 3 Dip coating processing [218]

2.8.2 Sol-gel method
Many different coating methods have been used to combine mechanical and
biological durability of coating films for the best performance of the implants.
Lately, the sol-gel method has come to prominence because of its significant
advantages, such as low crystallization temperature, enhanced substrate adhesion,
low cost, controllable microstructure, and excellent chemical homogeneity due to
atomic level mixing [142,193,194]. The milder thin film synthesis conditions of
sol-gel methods eliminate most of the defects that originate during plasma
spraying and lead to better structural integrity [195].
31
2.9 Summary
The main conclusions of this chapter are that porous ceramic scaffolds play an
important role in tissue engineering. For scaffold manufacture, solid freeform
fabrication technique is one of the most advanced methods in the world, so the
controlled porous scaffold could be fabricated by using this technique. Due to
some disadvantages using the polymer coating, calcium phosphate ceramics
could preferring be used for both base material and coating material. Furthermore,
many studies report that TCP could be formed by sintering HA and bioglass
composite, thus porous TCP scaffolds could be fabricated by this novel technique.
32
Chapter 3 Methodology and Sample

characterization
3.0 Introduction
Chapter 3 was focused on the materials and methodology of the project. HA and
bioglass were used as the raw materials to fabricate TCP. In this study, SFFT was
firstly used to fabricate a sacrificial scaffold. The sacrificial scaffold was then
coated with HA and bioglass composite slurry, the TCP scaffold was obtained
when the scaffold was sintered at 1400oC for 3hrs. In order to further increase the
scaffold's compressive strength, the porous TCP scaffolds were then coated with
bioglass. Results from the in vitro biological tests performed in this research will
be presented in Chapter 4. The sample characterization techniques used were
XRD analysis, total porosity of TCP scaffold, imaging of the scaffold by scanning
electron microscopy, mechanical testing and in vitro biological test (including
image of the cell, MTT test and Alkaline phosphatase activity). This study
examined effect of the macro-tube scaffold size on the mechanical properties of
tri-calcium phosphate scaffolds. Fig.4 is a flow chart illustrating the fabrication
route for the porous TCP scaffolds, and the corresponding mechanical and
biological tests conducted on each scaffold.
33
Figure 4 Flow chart showing the six main processing steps for fabricating TCP
scaffolds
3.1 Methodology
3.1.1 Preparation of TCP ceramic
3.1.1.1 Preparation of hydroxyapatite powder
Synthetic hydroxyapatite was prepared by reaction of orthophosphoric acid in
aqueous solution and calcium hydroxide [196], according to the following
equation:
34
6H3PO4(aq)+10Ca(OH)2(aq)Ca10(PO4)6(OH)2(s)+18H2O(l)
(9)
The Ca(OH)2 (>=95%) and H3PO4 (85%) were both supplied by in Sigma-Aldrich,
AUS. The reaction was carried out at 85oC, with the pH maintained above 9.5 by
the addition of ammonia solution. X-ray diffractometry (XRD) indicated that
when sintered Synthetic hydroxyapatite in air at temperatures between 900oC and
1350oC, there was no secondary phase and was stable up to at least 1350oC.
3.1.1.2 Preparation of bioactive glass powder
The method of preparation of bioglass followed previous publications [197].
Typically, 33.5g of tetraethyl orthosilicate (TEOS, 98%), 7g of Ca(NO3)2.4H2O,
3.65g of triethyl phosphate (TEP, 99.8%) and 5g of 37% HCl were dissolved in
300 g of ethanol (Si/Ca/P 80:15:5, molar ratio) and stirred at room temperature for
1 day. After the composite powders were completely dry, they were milled
(8000M Mixer/Mill) to a fine powder. Finally, the powders were sintered at 700oC
for 5 hours.
3.1.1.3 Preparation of HA/bioglass composite material
HA/bioglass composite powders containing 10, 15 and 20 wt% glass, were
prepared by wet ball-milling for 1 hour. The resulting paste was then dried at
100oC for 24 hours, then the calcined ceramic pieces were milled using a ball mill
under a wet condition for 2 hours. Finally, polyvinyl alcohol and 3 drops of a
dispersant were added to produce the final slurry.
After the ceramic was
completely dry, it was slowly sintered to the controlled temperature for 2 hours
and furnace cooled. The sintering temperature and bioglass additions were
detailed in Table 5. The powder was then examined by X-ray diffraction (XRD),
35
it was found that 20% bioglass additions at the 1400oC for 2h, HA/bioglass were
completely transferred to calcium phosphate, mainly -TCP, little -TCP. And
these results will be observed and discussed in Chapter 4.
Table 5 Sintering temperature and the bioglass additions
3.1.2 Fabrication of porous TCP scaffolds

3.1.2.1 Fabrication of sacrificial moulds
In this study, a scaffold template with macro-tubes was prepared using a 3D
printer which can produce a 3D structure with designed architectures. The
commonly used material for the 3D printing was acrylontrile butadiene styrene
(ABS). Fig.5 shows the scaffold of different sizes and shapes prepared by 3D
printer. Table 6 summarizes all details of the scaffolds template design. The ABS
scaffolds were cooled in the water after removal from the 3D printer, and then
dried for 24 hours.
36
Figure 5 ABS macrotube scaffold templates (a) and schematics of in-plane (top
view of black box in (a)) (b) and out-of-plane (view of cross section AA)
geometry (c)
37
Table 6 Parameters of the cube and cylinder ABS scaffold templates

rods
Spacing
diameter(mm
between
rods(mm)
676
1.5
15
169
1.5
15
56.25
1.5
15
1.5
15
1.5
15
Top
Shapes of the scaffold
Cube
Cylinder
area(mm2)
Height(m
two
m)
3.1.2.2 Coating ABS scaffold with wax

In this project, wax was used to take precautions against ceramic scaffold cracking.
Basically, during the sintering process, the wax was burned away and provided
enough space for ABS expansion. The wax (SigmaAldrich, AUS) was prepared
for the ABS scaffold. The wax was heated in an oven at 80oC for 10 minutes.
After the wax was fully melted, the ABS scaffold was dip coated with liquid wax.
This process should be completed quickly since wax can easily become solidified
in room temperature, and it may block the gaps. After coating, the scaffold was
kept at room temperature for at least 12 hours.
3.1.2.3 Sintering of the ceramic-coated scaffold
During the sintering process there were several parameters involved that need to
be optimized: the concentration of slurry, the maximum temperature, and the
dwelling time at the maximum temperature. The heating rate can also affect the
final density and phase purity of the material. Higher ramp rates can give a higher
final density, but a rate greater than 10oC/min has been shown to result in
decomposition [198]. In this step, two heating rates were used for sintering the
38
scaffold. The sintering was carried out in an electric tube furnace, using a heating
rate of 1oC/min up to sintering temperature, which was held for 3 hours, followed
by cooling rate 5oC/min to room temperature. The reason for setting 1oC/min as
the heating rate was that the ABS would expand during the melting process, and if
the heating rate was too high, more cracks may be created. There were five stages
in the schedule (Fig.6), including:
(1) Heating from room temperature to 700oC with a heating rate 1oC/min to
prevent the mould from cracking (the ABS and other organic additives will be
burned out at this step);
(2) Holding this temperature (700oC) for 3hours;
(3) Increasing the temperature from 700oC to 1400oC at a rate of 5oC/min;
(4) Holding this temperature for 3 hours;
(5) Cooling the furnace down to room temperature at a rate of 5oC/min. The
HA/Bioglass scaffolds were then removed from the furnace after it had cooled
down.
4
3
5
Time (hrs)
Figure 6 Sintering conditions
39
3.1.2.4 Coating the TCP scaffolds with normal bioglass

The sintered TCP scaffold was immersed into the normal bioglass slurry, and then
the vacuum pump was used to allow full infiltration. The soaked scaffolds were
then placed in a centrifuge running at 700rpm for 30sec to remove the excess
bioglass solution from the scaffolds. The scaffold was then removed from the
centrifuge tube and left to dry in a fume hood overnight. After the scaffolds were
completely dry, they were sintered at 700oC for 5hours to obtain the normal
bioglass coated TCP scaffold.
3.1.2.5 Coating TCP scaffold with sol-gel mesoporous bioactive glass
To prepare mesoporous bioglass scaffold required P123 (EO20PO70EO20) is
required to create nano pores [197]. Thus, 20g of P123, 33.5g of tetraethyl
orthosilicate (TEOS, 98%), 7g of Ca(NO3)2.4H2O, 3.65g of triethyl phosphate
(TEP, 99.8%) and 5 g of 37% HCl were dissolved in 300 g of ethanol (Si/Ca/P
80:15:5, molar ratio) and stirred at room temperature for 1day and the sol-gel
bioglass solution was prepared. Then the prepared TCP scaffold was put into this
sol-gel and centrifuged at 700rpm for 30 sec. After the scaffolds were completely
dry and sintered at 700oC for 5hours, the mesoporous bioglass coated TCP
scaffold was obtained.
3.2 Sample characterization

3.2.1 XRD (X-ray diffraction) analysis
XRD was used to identify the crystallographic phases of the reaction products
such as the TCP powder. For the XRD analysis, the samples were ground into fine
powders and powder was mounted in a specimen holder for the diffractometer
40
(6000 Shimadzu). Cu K1 ray (=1.5406 ) scanning was conducted using a 2

angle of from 20to 45. The scan rate and the step size were 2.0min1 and 0.02,
respectively.
3.2.2 Total porosity of the calcium phosphate ceramic scaffold
The pores in the calcium phosphate ceramic scaffold which were made by the HA
and the macro-tubes in the scaffold were responsible for the total porosity. In this
study, the total porosity of the sintered calcium phosphate ceramic scaffolds was
determined using the following equations [153]:
Total Porosity =1- M/ (V x )
(10)
Where
M= mass of the sintered sample
V= volume of the sintered sample
= Density of the sintered ceramic is 3.07 g/cm3
Specimen bulk density () was measured by application of Archimedes' principle.
The dimensions and the weight of each sample were measured and recorded using
a vernier calliper and an electronic balance.
3.2.3 Shrinkage
The shrinkage was determined using the following equations
Shrinkage=(O-F)/O
(11)
O= the original length of the scaffold

F= the final length of the scaffold
3.2.4 Imaging the scaffold using SEM
Scanning electron microscopy (SEM) was used to determine the pore size
41
distribution in the calcium phosphate ceramic macro-tubes. The scaffold was

sectioned with a knife in the longitudinal and transverse planes to show the best
overview of the porous structure to verify the pore homogeneity and
interconnectivity. The scaffold samples were mounted onto aluminium stubs with
carbon tape and coated with gold using a sputter Coater (BioRad SC500) and
imaged using a FEI QUANTA 200 scanning microscope.
3.2.5 Mechanical testing for scaffolds
The compressive strength was main parameter for the mechanical testing. An
Instron 3000 mechanical tester with 10kN load cells was used for the compression
mechanical tests. The crosshead speed was set at 0.5mm/min, and the load was
applied until the scaffold was crushed completely. Rubber pads (1mm thick) were
placed on the top and bottom surfaces of the sample to ensure an evenly
distributed load on the sample. Five samples of each type were tested for
mechanical properties.
3.2.6 Apatite-formation ability of microspheres in SBF
The SBF (simulated body fluids) solution was prepared according to the
procedure described by Kokubo [199] and Table 7 shows the ion concentrations
of the SBF solution and human blood plasma.
42
Table 7 Ion concentrations of SBF and human blood plasma
TCP scaffold was soaked in SBF at 37C for 21 days with refreshing SBF every
week, and the ratio of the sample weight to the SBF volume (mg/ml) as 3:5. After
soaking, samples were removed from the SBF, gently washed with deionized
water, dried at room temperature, and characterized by SEM.
3.2.7 Cell seeding and culture
Human bone marrow was sourced from patients in Orthopaedic Department of
Prince Charles Hospital with informed consent and ethics approval from the
Ethics Committee of Queensland University of Technology. The human bone
marrow stromal cells (hBMSCs) for this study were isolated by density gradient
centrifugation over Lymphoprep (Axis-shield PoC AS, Oslo, Norway) according
to the manufacturers protocol. The hBMSCs were seeded in culture flasks in
Dulbeccos Modified Eagle Medium (DMEM; Invitrogen Pty Ltd., Mt Waverley,
VIC, Australia) containing 10% fetal calf serum (FCS; InVitro Technology, Noble
Park, VIC, Australia) and 1% penicillin/streptomycin (Invitrogen) at 37C, 5%
CO2. The medium was changed twice weekly to wash out all non-adherent cells.
After the cells reached 80% confluence, the cells were trypsinized and
43
re-suspended in DMEM. 1105 cells were seeded onto each scaffold and cultured
for one week in DMEM at 37C, 5% CO2.
3.2.8 Observation of cell attachment on scaffolds using SEM
The cell culture medium in each well was pipetted out, and immediately replaced
with phosphate buffered saline (PBS). The rinsing was repeated three times for
each sample, and then the scaffolds were fixed with a 3% glutaraldehyde solution.
The scaffolds were then processed by two changes of cacodylate buffer for
20mins each; and then soaking them in an osmium tetroxide solution for 1 hour,
finally they were dehydrated through a series of ethanol solutions with graded
concentrations, followed by two changes of 100% amyl acetate for 15mins each.
The scaffolds were then dried using a supercritical point dryer before observation
using SEM.
3.2.9 Cytotoxicity test by MTT assay
To evaluate hBMSCs proliferation with the existence of different materials,
hBMSCs were seeded at a density of 1104 cells/well into 24-well plate and
incubated for 4 hours. 20mg of TCP was added to the culture plate. Cells were
then incubated at 37C in 5% CO2 for 1, 3 and 6 days. Then, 40L of 0.5 mg/ml
MTT solution (Sigma, Aldrich) was added in each well and incubated for 4 hours
at 37C. The reaction was terminated by the addition of 100L dimethyl sulfoxide.
The absorbance of the formazan was read at 495 nm using an Enzyme-linked
immunosorbent assay (ELISA) plate reader (Bio-Rad Laboratories, Pty., Ltd,
Gladesville, New South Wales, Australia). The MTT assay was to assess cell
viability and growth based upon the conversion of MTT to formazan. Results
44
were expressed as absorbance readings from each well. For the control, hBMSCs
proliferation in normal cell culture media was evaluated by the same procedure.
3.2.10 Alkaline phosphatase activity
Hunman bone marrow mesenchymal stem cell system (hBMSCs) were
respectively seeding into 6-well cell culture plates and TCP scaffolds for the
induction of hBMSCS at a number of 1104 cells in the osteogenic medium
(DMEM supplemented with 10% FBS, 100nM dexamethasone, 50 mg/ml
ascorbic acid, and 10mM b-glycerophosphate). Cells were cultured in osteogenic
differentiation media for 7, 14 days. Then alkaline phosphatase (Alp) activity was
determined using pNPP assay (p-nitrophenyl phosphate liquid substrate,
Sigma-Aldrich). Briefly, hBMSCs were washed with PBS, then were lysed in
0.5ml PBS containing 0.1M glycine, 1 mM MgCl2 and 0.05% Triton X-100 for
10min at 4C. The lysate was incubated with pnitrophenylphosphate (pNPP)
solution at 37oC for 30mins, and then subjected to a spectrophotometer on which
the absorbance at 405 nm was measured and recorded to indicate Alp activity
[200].
3.2.11 Statistical analysis
Three completely independent experiments for cell culture and induction were
performed for every assay and the results were expressed as means standard
deviations. Statistical significance was calculated using one-way analysis of
variance (one-way ANOVA). Comparison between the two means was performed
using Turkey test and the significance was determined by p<0.05
45
3.3 Summary
In this chapter, the materials and methodology used were discussed. HA and
bioglass were used as the raw material to fabricate the TCP and SFFT was used
for fabricate the sacrificial scaffold template from ABS. TCP scaffold was
obtained after sintered HA/Bioglass composite at 1400oC for 3 hours. In order to
further increase the scaffolds mechanical property, the porous TCP scaffolds
were then coated with bioglass. Several biological tests were done. Sample
characterizations, such as XRD analysis, total porosity of TCP scaffold, imaging
of the scaffold by using scanning electron microscopy, mechanical testing and
biological testing (including SBF, intro cell culture using hBMSCs , MTT test and
Alkaline phosphatase activity) were also completed.
46
Chapter 4 Mechanical and biological

testing
4.0 Introduction
This chapter presents the results of the mechanical and biological tests and
discusses these in light of the suitability of scaffolds for application in bone tissue
engineering. All porosity and compressive strength for each HA/bioglass ceramic
scaffold was shown in Table 9.
4.1 Confirmation of TCP formation

In all cases, reaction occurred between the HA and the bioglass addition, resulting
in a reduction of HA content, and the formation of additional phases such as
-TCP and -TCP. The XRD results were summarized in Table 8.
Table 8 XRD phase analysis data for composites of HA with the addition of 10,
15, and 20wt% bioglass
Bioglass(wt%)
Tsinter(oC)
HA (wt%)
TCP (wt%)
10
1200
53.3
29.1
1300
42.6
23.2
1400
26.2
33.5
1200
37.3
50.6
1300
20.6
63.2
1400
N.P*
87.2
15
47
20
1200
26.7
60.1
1300
12.4
75.3
1400
N.P*
100
*N.P not present
The data presented in Table 8 shows that at the same content of bioglass, with
increasing temperature, the content of the HA was decreased, and the weight
percentage of TCP was increased; at the same temperature, the weight percentage
of TCP was increased when more bioglass was added to HA. When 20% bioglass
was added to HA sintered at 1400oC, there was no HA phase present, which
meant that the HA and bioglass were totally transformed to TCP crystalline phase.
The phases formed in the scaffolds during the sintering process depended on two
factors, i.e. the sintering time and the sintering temperature. When the temperature
was above 1200C, HA became unstable and could potentially eliminate OH
groups to form decomposed products of additional phases such as -TCP and
-TCP. When the temperature was above 1400oC, -TCP phase was formed.
After the temperature was decreased, most of the -TCP was transformed to
-TCP [201-202]. Miao et. al., suggested that some -TCP phase could be
retained due to the elastic strain constraint from the surrounding matrix [203]. In
addition, according to the HA decomposition equations shown above, there were a
number of undesirable phases detected during the sintering process. It was then
decided to add bioglass in order to adjust the Ca/P ratio, thus ensuring
HA/bioglass would be completely transformed into TCP. As a result, the phases
achieved in the sample should contain both -TCP and -TCP (mainly -TCP).
This was confirmed by the XRD analysis (Fig 7a). The XRD patterns in Fig. 7a
48
mostly matched with the pure -TCP patterns in Fig 7b, thus, it was believed that
the TCP scaffold made via HA/bioglass was successfully obtained.
(a)
(o )
(b)
Figure 7 (a) XRD pattern for a sintered porous calcium phosphate (mainly-TCP)
sample; (b) XRD patterns of -TCP starting powder and sintered body [204]
4.2 Shrinkage
Porous structures were characterized for their physical, mechanical and biological
properties to understand the influence of porosity parameters. Shrinkage was
49
measured as a part of physical property measurement to determine the sintering

behavior of these ceramics. None uniform shrinkage tends to cause cracking and
warping in the final products. Moreover, controlled shrinkage information can be
utilized in the design stage to make necessary design modifications. Shrinkage
during sintering is dependent on sintering temperature and the densification.
Shrinkage also varies as a function of volume fraction porosity in these samples in
which higher volume fraction porosity showed a lower amount of shrinkage [205].
For the porous TCP samples, linear shrinkage varied from 27% to 30%, and the
shrinkage variations were primarily due to the variations in the temperature. One
study indicated that, in a HA specimen, shrinkage began at about 800oC, from the
initially expanded state, and a discontinuity was observed at 1160 oC [206]. Linear
shrinkage in percentage with respect to sintering temperature was shown in Fig.8.
Linear shrinkage(%)
The results indicated that TCP sintered at 1400oC exhibited highest shrinkage.
30
29
28
27
26
25
1200
1300
Temperature(oC)
1400
Figure 8 Linear shrinkage of TCP with respect to sintering temperature

50
4.3 Microstructure of ceramic composites

4.3.1 10% bioglass addition
Fig. 8 shows SEM images of HA with 10% bioglass addition at different
temperatures. With increasing sintering temperature (from 1200oC (Fig. 9a) to
1400oC (Fig. 9c)), increased amounts of pores were formed in the scaffold,
resulting in increased porosity values (Fig. 9d) from 61.151.3% to 70.230.8%.
The possible reason for this was that the higher temperature and higher shrinkage
caused the formation of some micro-cracks and pores, as well as large grains.
51
72
(d)
70
Porosity(%)
68
66
64
62
60
1200
1300
1400
Temperature(oC)
Figure 9 SEM images of the fracture surfaces of HA with 10% bioglass addition
at (a) 1200oC (b) 1300oC (c) 1400oC (d) The porosity of 10% bioglass addition
ceramic scaffold sintered at three temperatures. Error bars represent mean SD
for n=5.
52

The samples containing 15w% bioglass, sintered at 1200oC (a), 1300oC (b),
1400oC(c) are shown in Fig. 10, and the pictures show a high degree of fine
interconnecting micro-porosity.
53
75
(d)
Porosity(%)
72
69
66
63
Temperature(oC)
60
1200
1300
1400
Figure 10 SEM images of the fracture surfaces of HA with 15% bioglass addition
for n=5
Fig. 10d indicates that higher sintering temperature results in increased porosity.
As mentioned before, the HA and bioglass would react at high temperature, and
some other phases may formed. Consequently, in this condition, beside HA and
54
TCP, some other phases existed in the scaffold and influenced its structure and
porosity. It could also be noted that, at the lowest sintering temperature of 1200oC,
the structure was unusual, as some crystal-like features were formed, and this was
because a locally dense glassy matrix was formed on the samples.
Fig. 11shows the change in fracture surface for the 20wt% bioglass series between
1200oC (Fig.11a), 1300oC (Fig.11b) and 1400oC (Fig.11c). Different from the
10wt% and 15wt% bioglass additions, in this condition, the porosity of the
samples was decreased with increasing temperature, and the amounts of pores
were reduced.
55
70
(d)
Porosity(%)
68
66
64
Temperature (oC)
62
1200
1300
1400
Figure 11 SEM images of the fracture surface of HA with 20% bioglass addition
for n=5
At the sintering temperature of 1300oC, there was an evidence of structural
deterioration at the surface of the specimens, possibly due to localized melting of
the composite. Fracture surfaces for higher sintering temperatures were totally
different; the samples sintered at 1400oC showed a film covering the surface, and
connecting all the grains and this obviously decreased the amount of the pores as
well as the pore sizes.
56
Table 9 Porosity and mechanical property for composites of HA with the addition
of 10, 15, 20wt% bioglass at three temperatures
Bioglass(wt%)
10
15
20
Compressive
Tsinter(oC)
Porosity (%)
1200
61.151.3%
2.70.3
1300
63.051.2%
2.150.4
1400
70.230.8%
1.830.3
1200
60.630.4
2.970.5
1300
68.471.23%
1.750.2
1400
72.641.2%
1.690.2
1200
67.080.4%
1.920.8
1300
64.420.6%
1.871.1
1400
62.991%
9.980.6
strength (MPa)
57
4.4 Mechanical properties

Variations of the compressive strength depending on sintering temperature are
shown in Fig.12. The series containing 10 wt% bioglass sintered at 1200oC,
1300oC, 1400oC produced cracks on the specimens upon sintering. The pore size
was greater at higher temperature, and the cracking was more severe at the higher
temperature, thus the compressive strength was decreased from 2.70.3MPa at
1200oC to 1.830.3MPa at 1400oC.
3
2.5
Compressive
Strength(MPa)
1.5
1
0.5
0
1200
1300
1400
Temperature(oC)
sintering temperatures. Error bars represent mean Standard deviation(SD) for
n=5
58

Fig.13 indicates that the higher the sintering temperature, the lower the
compressive strength. The values were decreased from 2.970.5MPa (1200oC) to
1.690.2MPa (1400oC). In the graph, the compressive strength between 1300oC
and 1400oC were similar, but much higher compressive strength was shown at
1200oC. Compared to the previous study with 10% bioglass addition, the trends
were the same i.e. with the increase of the temperature, the compressive strength
is decreased.
Compressive
Strength(MPa)
0
1200
1300
1400
Temperature(oC)

sintering temperatures. Error bars represent mean SD for n=5
Compression test results of 20wt% bioglass composites at 1400oC were
substantially higher strength values compared to those of the 1200oC and 1300oC
sintered bodies (Fig.14). While strengths have been observed at 9.980.6MPa for
59
20wt% bioglass addition sintered at 1400oC, there were interesting values of

1.920.8MPa at 1200oC, and 1.871.1MPa at 1300oC as can be seen in Table 9.
These materials had strengths that were influenced by the sintering temperature,
with the exception in the trend compared to the other two bioglass additions.
10
Compressive
Strength(MPa)
8
6
4
2
0
1200
1300
1400
Temperature(oC)

sintering temperatures. Error bars represent mean SD for n=5
From Fig.12-14, it could be easily found that at 10% and 15% bioglass additions,
the values of compressive strength were decreased with increased temperature.
However, 20% bioglass addition was different from the others, i.e. the higher
temperature, the higher compressive strength. At 1400oC the values reached the
highest which was 9.98MPa and compressive modulus was 1.24GPa (higher than
the Trabecular bones compressive modulus), it was because all the HA and
bioglass were transformed to TCP.
60
4.5 Porosity and compressive strength of the uncoated TCP scaffold

The total porosity for uncoated TCP scaffolds was approximately 62.99%1.9%,
because the macro-tubes were mainly responsible for the porosity. Under the same
condition (i.e. temperature), with increase of the ceramics porosity, the
compressive strength would decrease. For the same template ABS scaffold, the
scaffold made from the TCP powder did not provide sufficient mechanical
stability due to higher porosity.
Figure 15 SEM images of the fracture surfaces of the porous TCP showing the
presence of micro-pores: (a) made via sintering HA/bioglass and (b) made via
TCP powder [207]
61
The pore morphology and distribution of macroporous TCP scaffold can be

visualized by the SEM photomicrographs presented in Fig. 15. It could be
observed that some interconnected macropores ranging from 5m to50m were
present in the 3D hybrid scaffold, and these pores may be produced by foaming a
mixture of PVA aqueous solution and water vapor. Compared with the hybrid
scaffold, the TCP powder scaffold (Fig.15 b) had more pores and the pore size
distrobution between 15m-600m [28]. Such a difference was because the
melting point for the bioglass was 700oC, and when the temperature reached
700oC, the bioglass transferred from the solid to the fluid, and filled some pores in
the scaffold, i.e. the bioglass acted binder, connecting all particles and resulting in
increasing of the scaffolds compressive strength.
4.6 Porosity of the coated scaffold (normal bioglass)

The sintered porous TCP scaffold mechanical properties nearly match those of
real bone but the still not strong enough, and this was due to the high porosity and
the intrinsically low mechanical strength of the calcium phosphates. One way to
strengthen and toughen the porous TCP was to coat the porous struts with a
bioactive ceramic. Thus, bioglass was used again in the project, and there were
two kinds of bioglass used (i.e. normal bioglass and sol-gel mesoporous bioglass).
62
Figure 16 SEM micrographs of a porous TCP scaffold with a normal bioglass at

layer 42X (a), 160X (b) and 3000X (c), as well as the surface of the normal
bioglass layer (d)
Normal bioglass slurry was used to infiltrate and coat the sintered porous TCP, as
shown in Fig.16 and the porosity of the TCP scaffold which coated with normal
bioglass was 59%3%, a little lower than the TCP scaffold (63%). The
micro-pores in the coated scaffolds (Fig.16b) were less than in uncoated scaffold
(Fig.15a). Also it was observed that the TCP scaffolds had more crack-like defects
on and within the ceramic struts than those coated with bioglass.
Several factors affected the coating process, namely, the viscosity of the slurry,
63
and the particle sizes of the bioactive glass. The coating quality was also
controlled by the details involved in the dipping coating process. Some bioglass
had become a layer covering the struts. Some bioactive glass particles had stayed
on the surface of the bioglass layer (Fig.16d).
4.7 Porosity of the coated scaffold (mesoporous bioglass)

The total porosity of mesoporous bioglass coated scaffold was 61%2%. The
macroporosity of the scaffolds after infiltration and coating with the bioglass was
slightly decreased due to the observed thin layer coating present on the strut
surfaces.
64
Figure 17 SEM micrographs of a porous TCP scaffold with a mesoporous

bioglass layer 52X (a), and 200X (b) (a and b were single coatings); 1600X (c)
1600X(d) (c and d were double coatings)
In a similar study [208], Jun et al. had shown an about 2% reduction in porosity as
a result of the bioglass coating on the ceramic scaffolds. Different from the
normal bioglass, the mesoporous bioglass needed to be coated first, and then
sintered. Fig. 17a shows that TCP scaffold was coated with mesoporous bioglass
and it was found that the mesoporous bioglass still did not cover the entire
scaffold; the reason for this was the viscosity of the meso-bioglass as well as the
sintering temperature. This problem was resolved in this study by a secondary
coating as well as sintering process, Fig.17c shows that bioglass was laid over the
scaffold. After the initial coating process, the TCP sample was coated again
(double coating) and had gone through the sintering process. It was discovered
that each piece of bioglass had been connected with each other. Fig.17d shows the
surface of the meso-porous bioglass, it was observed that some nano-size pores
were present in the meso-porous bioglass. The significance of the mesoporous
65
bioglass was that the nano-size pores imparted the scaffold with better
biocompatibility [217].
4.8 Mechanical testing

4.8.1 Shape and size effects
The sizes and shapes of porous scaffolds could influence their mechanical
strength, permeability and the presence of structure defects. Table 10 shows the
mechanical strength and porosity of TCP scaffolds prepared with different ABS
templates (Error bars represent mean SD for n=5). In Table 10, it was found that
that scaffolds with similar sizes (i.e. length and height), cylindrical structure have
higher mechanical strength than cube ones. In a recent study, Alsayed reported
that for comparison between compressive cube strength and compressive cylinder
strength, cube strength= 0.8*cylinder strength [220]. In this project, results
showed that compressive strength for cube scaffold was 9.98MPa (mid-size), and
for cylinder strength was 12.13MPa. The compressive strength of the cubic
scaffold was approximately 80% of the cylindrical scaffold compressive strength,
which was very similar to the results obtained from the literature.
In these kinds of scaffolds, macro-tubes were mainly responsible for the porosity,
and the average porosity of the scaffolds ranged from 40%0.5% to 72%1.3%.
The larger surface area, the higher scaffold porosity, and thus both favour cell
adhesion to the scaffold and promote bone tissue regeneration. As the surface area
was increasing, more cracks and pores were observed. This would be mainly
attributed to the difference in thermal expansion coefficient between the ABS
66
template and the TCP. As a result, the large scaffold exhibits an increasingly
brittle response towards the end. For a decrease of surface area from 676 to
56.25mm2, the compressive strength increased from 1.520.65 to 14.250.3MPa.
For the purpose of comparison, in this work the scaffold was also made via TCP
powder directly by using 169mm2 top area ABS templates (cube). A higher
porosity was observed in these scaffolds (66.211.20%) resulting in a lower
mechanical strength (2.81.3MPa) (Fig.18a) and the compressive modulus for this
scaffold was 0.021GPa.
Table 10 Average porosity and compressive strength for the scaffolds

Compressive
Compressive
Strength(MPa)
Strength(MPa)
(HA/bioglass)
(TCP powder)
Shapes of the
Top area
Average
scaffold
(mm2)
porosity (%)
Cube
676
72.401.3
1.520.65
169
63.341.5
9.981.40 (Fig.17b)
56.25
40.650.5
14.250.3
169
64.260.7
12.131.2
56.25
41.130.8
17.680.2
Cylinder*
2.81.3MPa
(Fig.17a)
*In this project, all scaffolds were fabricated as cube and the top area was 169mm2, the
cylinder and other sizes of scaffolds just for comparison in this chapter.
The difference in morphology between the scaffolds made via sintering

HA/bioglass and those via TCP powder was shown in Fig.15. The micropores in
HA/bioglass TCP scaffold were less than those in TCP powder scaffold, which
was because the bioglass was melted at 700oC, and the melted bioglass could fill
up some pores and cracks. In addition, when the temperature was increased to
67
1400oC, the bioglass would react with HA and form a continuous layer of TCP on
the surface of the scaffold, resulting in increase of the compressive strength.
Compressive Strength(Mpa)
3
2.5
2
1.5
1
0.5
0
0
20
40
60
Compressive Strain(%)
(a)
12
10
8
6
4
2
0
0
20
40
Compression strain(%)
(b)
68
80
18
Normal BG
15
Meso-porous
BG
12
9
6
3
0
0
10
20
30
40
Compression strain(%)
(c)
Figure 18 Compression stressstrain curves of the sintered porous scaffold
samples (a) made via pure TCP powder, (b) made via HA/Bioglass, (c) TCP made
via HA/Bioglass and coated with two different bioglasses. (Error bars represent
mean SD for n=5)
According to data from Table 10, for the cubic shape of TCP scaffold samples, in
terms of the average porosity, the scaffold sample with the top area of 676 mm2
was higher than the scaffold sample with the top area of 56.25mm2. However, the
compressive strength had decreased significantly as the top area of the scaffold
sample increased. The key objective was to achieve a harmonious balance
between the average porosity and the compressive strength. Based on this theory,
it was believed that the scaffold sample with the top area of 169mm2 was the best
result achieved among the three sample sizes.
69
4.8.2 Mechanical properties for bioglass coated scaffold

Fig.18b shows a stress-strain curve of the scaffold sample made via sintering of
HA/Bioglass. A higher compressive strength, i.e., 9.981.4MPa was observed. On
the other hand, Fig.18c observes a stress-strain curve of the sintered porous
HA/Bioglass interpenetrating composite that was infiltrated with the bioglass.
Two different kinds of bioglasses were used for enhancing scaffolds mechanical
and biological properties. The compressive strength for normal bioglass coated
scaffold was 16.690.5MPa, and for single mesoporous bioglass coated scaffold
was 15.030.63MPa, the mechanical data were comparable to those of spongy
bones, which show a compressive strength of 2-20MPa [209-210], respectively.
Thus it could be said that scaffold after bioglass coating, the compressive strength
was increased about 40%, which was almost identical to results found in Jun et
als report [24]. According to Jun et als report [24], HA scaffold was coated with
apatite-wollastonite glass (i.e. a type of bioglass), and compressive strength was
increased from 0.58MPa to 0.95MPa, also nearly increased 40%. When the TCP
scaffold for the secondary coating, its porosity decreased, and its compressive
strength was measured to be about 22MPa. The strength of the scaffold was
remarkably increased by coating scaffold for twice instead of the single coating.
This enhanced compressive strength was attributed not only to the elimination of
the defects present on the scaffold but also to reinforcement the scaffold by using
a strong glass ceramic phase as the framework (each piece of bioglass had been
connected with each other(Fig.17c)).
70
4.9 Apatite-formation ability of the macro-tube scaffolds

in SBF
SEM images of the scaffold after soaking in SBF solution for 21 days were shown
in Fig.19a. At a high magnification, it was noted that a rough deposit layer was
formed on the TCP scaffold, and some crystal clusters were also found on the
layer surface. To determine the chemical composition of the crystal deposits, the
surfaces of the scaffold were further characterized by EDS (Energy dispersive
spectroscopy). The EDS spectra of the TCP scaffold after incubation in SBF
solution for 21 days were shown in Fig. 19b.
71
Figure 19 (a) SEM image of the scaffold after soaking in SBF for 21 days (b)
EDS spectra of the surfaces of TCP
The HA granules showed high peaks of P, Ca, and the atom ratio of Ca to P was
about 1.65, which was close to that of carbonated apatite. These characteristics
could be found in HA. Since HA was capable of stimulating the osteo-induction
of stem cells in vivo with some ability of osteo-conductivity [211], it was believed
that these TCP scaffolds were highly bioactive.
4.10 Cell culture

4.10.1 SEM images of cells growth on uncoated scaffolds
Bone marrow stromal stem cells were seeded into the scaffolds by adding drops of
the cell suspension. As a result, the cells were homogeneously seeded across the
surface of the scaffolds. The penetration of the cells into the scaffolds was
evaluated using the cross-sections of the scaffolds. SEM imaging (Fig. 20) has
revealed that the cells spread and adhere well on the surface. Interestingly, it was
72
noted that a significant amount of HA granules were formed on the scaffold

surfaces and distributed among the cells, which was advantageous as far as
bioactivity was concerned. In general, HA granules can be formed in a simulated
body fluid (SBF) solution when a biomaterial has good bioactivity (Fig. 20a).
However, in this experiment, the HA granules were also formed when the TCP
scaffold was immersed into the DMEM solution. There were two possible reasons.
The first one was the solution contained bovine serum, which possessed some
ions; at the same time some P and Ca ions could be slowly released into the
solution from TCP, thus the solution in the medium was similar to a SBF solution.
Naturally, some HA granules were formed on the surface of the scaffold after 9
days (Fig. 20a). Fig.21 shows that the formation of HA granules (confirm by EDS)
when living cells were not present, indicating the formation of HA was
independent of the cell environment.
73
Figure 20 SEM micrographs show the attachment of the cells on TCP ceramic
strut surface 800X (a) and 2000X (b)
Figure 21 SEM image of TCP ceramic scaffold immersed into DMEM without
cells
74
4.10.2 SEM images of cell growth on bioglass coated scaffolds
Figure 22 SEM micrographs show the attachment of the cells on bioglass coated
TCP ceramic strut surface 2000X (a) and 2500X (b)
The bone marrow stromal stem cells were well spread on the surface of bioglass
coated TCP ceramic. This coated scaffold was different from the non-coated
scaffold, because no HA granules were found in the coated scaffold as shown in
Fig. 22. This occurs because more bioglass in the medium means more Ca and P
irons found in the medium, thus having negative impact on the balance in SBF.
75
4.10.3 Cell proliferation

MTT assay is an important method to evaluate the cytotoxicity of the scaffold and
its slowly released components in an aqueous environment. Fig. 23 shows the
result of MTT assay for proliferation status of hBMSCs as the negative control
group and as the experimental group of hBMSCs combining with TCP scaffold
during 9 days after the same culture condition.
Absorbance at 495nm
1.2
0.9
BMSCs
BMSCs in TCP
scaffold
0.6
0.3
0
1
94
Time/(days)
Figure 23 MTT assay for proliferation of hBMSCs and hBMSCs combined with
TCP scaffolds at different incubation periods under the same culture condition.
Error bars represent mean SD for n=3
Cell proliferation increases with the culture time in both groups, but the cell
growth rate of the experimental group was much higher the negative control
groups at 1, 3 and 6 days respectively. At 3, 6 and 9 days, the cell proliferation
76
between the negative control group and the experimental group has no significant
difference. Thus, the TCP scaffold was proven non-cytotoxic and has good
biocompatibility in vitro.
4.10.4 Alp (Alkaline Phosphatase) activity
To study the bioactivity of the TCP scaffold, the Alp activity was measured. As
one of the cell membrane associated enzymes, Alp is known to be closely
associated with osteoblast differentiation. Alp regulates phosphate metabolism
and locally down-regulates inhibitors of apatite crystal growth [219]. Therefore, it
is used as a marker that appears early during osteoblast differentiation. A high Alp
activity was detected in positive TCP scaffold groups in both day 7 and 14 in
comparison to pure hBMSCs differentiation in osteogenic medium (Fig. 24a).
Alp Activity
(OD/min/mg protein)
3.5
BMSCs induction
3
2.5
BMSCs induction with TCP

scaffold
1.5
1
0.5
0
7 days
14 days
Culture time (days)
(a)
77
Alp Activity
(OD/min/mg protein)
3
2.5
2
1.5
1
0.5
0
TCP scaffold
(b)
TCP scaffold coated with normal

bioglass
Figure 24 Alp activity of hBMSCs after 7 days, 14 days of culture, and the novel
TCP scaffolds significantly increased the Alp activity to higher level compared to
positive control group after 14 days (a); Alp activity of hBMSCs after culturing
for 7 days on TCP scaffold and TCP scaffold coated with normal bioglass (b).
Error bars represent mean SD for n=3
Bioactivity is thought to be an important issue in the chemical interactions
between the implanting materials and the bone tissue, and ultimately affects the in
vivo success of the bone grafting materials [212]. In this study, compared to the
positive differentiation group, the new scaffold has an enhanced effect on Alp
activity of hBMSCs after 14 days, which can be attributed to the higher
concentration of Ca and Si ions that were slowly released from bioglass, and more
stable pH environment [213] bioglass led to in cell culture medium than the
general in vitro environment. The proved reasons were that bioglass incorporated
78
in the TCP scaffolds can release Ca and Si ions which can stimulate cell response
[214-215]. The differentiation characteristics of the cells were evaluated by
measuring the Alp activity of hBMSCs after culturing 7 days. The cells on TCP
scaffold coated with bioglass expressed Alp activity at a significantly higher level
than those TCP scaffold, as shown in Fig.24b. The higher Alp activity of the cells
cultured on the scaffold coated with bioglass compared with that the cultured on
TCP scaffold strongly suggested that the bioglass coating on the TCP scaffold is
effective in improving the bioactivity of the porous scaffold.
4.11 Summary
A 3D macrotube porous TCP scaffold was successfully synthesized by the
sintering HA and 20% bioglass at 1400oC. The novel scaffold exhibits macro-tube
(0.8mm) and as well as a hierarchical structure with interconnected macropores.
The resultant scaffold exhibits good mechanical properties (i.e. 9.98MPa for
compressive strength and 1.24GPa for compressive modulus: cube, 169 mm2
top-area) that was significantly influenced by the bioglass melting at 1400oC and
the fluid bioglass covering on the surface of the scaffold. The mechanical
properties of the obtained TCP scaffold had values that were still not sufficient for
load-bearing applications, in order to further improve scaffolds mechanical and
biological properties, the TCP scaffold was further coated with bioglass, and
compressive strength was largely enhanced (more than 15MPa). These kinds of
scaffolds could be considered for the implantation in bone defects for the bone
formation. Also, the cells were well spread in the scaffold, and the MTT test
showed that there was no toxicity in this material. Alp activities tests indicated
79
that TCP scaffold has good bioactivity and improved bioactivity when the TCP
scaffold was coated with bioglass. This new class of material combines macrotube
scaffold possessing excellent physicochemical, biological properties, hence
indicating their potential application for bone tissue engineering. This scaffold
solves some existing problems in clinical grafts by providing strength and
controlled released bioactive molecules to prompt hBMSCs osteogenesis.
80
Chapter 5 Conclusions and future work

5.1Conclusions
The objective of this research was to develop a macro-tube porous tri-calcium
phosphate (TCP) scaffold through the use of composite HA (Hydroxyapatite) and
bioactive glass sintered at high temperatures. To achieve this objective, a number
of key steps were taken throughout the project. The first key step was to develop
and manufacture an acrylonitrile butadiene styrene (ABS) sacrificed scaffold
using solid freeform fabrication techniques (SFFT) as a template. SFFT has been
identified as an advanced technique for controlling porous scaffold morphology.
The second key step was to fabricate TCP scaffold by sintering 80% HA and 20%
bioglass at 1400oC for three hours. Following that, appropriate coating materials
for coating the TCP scaffold were selected. Poly (lactic-co-glycolic acid) (also
commonly known as PLGA) and calcium phosphate ceramics were initially
chosen. Unfortunately, it was discovered that the chemical used to dissolve PLGA
may possess potential health and safety risks. In addition, after applying polymer
coating, the scaffold surface will become smooth, which can discourage cell
growth. Therefore, calcium phosphate ceramics were the most favorable materials
to be used for both base and coating purposes. Cell culture was also conducted to
study the bioactivity of the TCP scaffold.
A number of conclusions can be drawn from this project:
The method of manufacturing TCP was explored throughout the project. TCP
81
was made by mixing two basic ingredients: HA and Bioglass together. By

varying the amount of HA and bioglass added into the mixture and altering
the sintering temperature, it was discovered that the most favorable method of
making TCP was when 20wt% bioglass was added into the mixture and the
sintering temperature was 1400oC.
In terms of the size and the shape of the scaffold and their associated effect
on both mechanical properties and porosity, it was discovered that as the top
area of the scaffolds increased, their mechanical properties were becoming
lower. The effect on the porosity was also studied. It was found that the
scaffolds with larger top areas had higher porosity. Taking both factors into
account, it was concluded that scaffolds with mid-size top areas were the best
candidate. This conclusion was supported by the relevant tests conducted
throughout the project. For instance, the compressive strength of the scaffold
with cubic cross-section area had a high reading of 9.98MPa, and high
compressive modulus reading of 1.24GPa. The porosity was measured to be
63%, which was also relatively high. The shape of the scaffold had also
impacted on the compressive strength of the scaffold. With identical size of
the top area, the cylindrical scaffolds had higher compressive strength then
the cubic scaffold.
In order to further improve the scaffolds mechanical properties, TCP

scaffold was coated with bioglass. Scaffolds compressive strength was
largely improved from 9.98MPa to above 15MPa. Two types of bioglass
(normal and single mesoporous bioglass) were used for enhancing the
82
mechanical properties of the TCP scaffold. It was discovered that the TCP
scaffold coated with normal bioglass had slight higher compressive strength
(16.690.5MPa) then the one coated with single mesoporous bioglass
(15.030.63MPa). These results were comparable with those of spongy bones.
Therefore, these scaffolds could be potentially used as implantation materials
for bone formation.
In terms of analyzing the bioactivity of the TCP scaffold, the first major study
was to evaluate the cytotoxicity of the TCP scaffold and its slowly released
components in an aqueous environment. By using MTT assay, the TCP
scaffold was proven to be non-cytotoxic. It would also have good
biocompatibility in vitro.
The second major study was to determine the bioactivity of the TCP scaffold.
This was based on the study of osteoblast differentiation in the TCP scaffold.
The Alp activity of human bone marrow mesenchymal stem cell systems
(hBMSCs) after culturing seven days and fourteen days were measured as
Alp is known to be closely associated with osteoblast differentiation. It was
discovered that hBMSCs induction with TCP scaffold had higher Alp activity
after seven days as well as after fourteen days. The higher Alp activity of the
cells cultured on the scaffold coated with bioglass compared with that the
cultured on TCP scaffold strongly suggested that bioglass coating on the TCP
scaffold was effective in improving the bioactivity of the porous scaffold.
83
5.2 Future work

It is recommended that the finding of this study be utilized to help the bone
formation. In future work, there are three aspects which can be improved. In terms
of fabrication method, as mentioned before, some cracks were created in the
scaffold due to the fact that ABS polymer expanded at high temperature, thus, the
ABS polymer could be instead replaced by other materials. In the mechanical part,
carbon nanotubes (CNT) could be considered as the fiber reinforcement within the
ceramic matrix for the scaffold fabricating. CNT have aroused increasing interest
due to their remarkable tensile strength, high resilience, flexibility and other
unique structural, mechanical, electrical and physicochemical properties [214,
215]. Attempts have been made to develop advanced engineering materials with
improved or novel properties through the incorporation of CNT in selected
matrices such as polymers, metals and ceramic. It is expected that the inclusion of
CNT in a ceramic matrix will produce composites possessing high stiffness and
improved mechanical properties compared with a single phase ceramic material.
According to the latest paper [216], the results showed that CoCl2 pre-treated
hBMSCs induced higher degree of vascularization and enhanced osteogenesis
within the implants in both ectopic and orthotopic areas, thus, in the future work
the CoCl2 could be added into the bioglass coating and the Co ions can be
released in the body help the blood and bone formation.
84
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PREPARATION AND CHARACTERISATION OF

TRI-CALCIUM PHOSPHATE SCAFFOLDS WITH

Principal Supervisor: Associate Professor Cheng Yan

A Thesis submitted for Master of Engineering (Research)

Faculty of Built Environment and Engineering

Macro-tube Scaffold, MTT test, Phase transformation, Scanning electron

2.4.4 Bioglass ................................................................................................ 23

2.5.1 Porosity ................................................................................................ 24

3.2.4 Imaging the scaffold using SEM ........................................................... 41

4.10.3 Cell proliferation................................................................................ 76

Figure 9 SEM images of the fracture surfaces of HA with 10% bioglass

bioglass addition ceramic scaffold sintered at three temperatures. Error

Acrylontrile butadiene styrene.

Poly (lactic-co-glycolic acid)

multipotent stem cells that can differentiate into a

variety of cell types, including: osteoblasts (bone cells), chondrocytes (cartilage

Osteoconductivity An ability of the scaffold to serve as a template for bone

Statement of Original Authorship

of my knowledge and belief, the thesis contains no material previously published or

written by another person except where due reference is made.

Preparation and characterisation of strong and bioactive tri-calcium

Mechanical and biological properties of sol-gel derived zirconia scaffold

Calcium phosphate ceramics have been extensively used to produce porous

osteoinductivity [11]. In the calcium phosphate compounds, hydroxyapatite

1.2 Research goals

polyurethane-sponge [24-27] as a sacrificed scaffold, for the structure of the

(depending on the bioglass) can be formed, which has a higher mechanical

Chapter 2 Literature Review

and each techniques advantages and disadvantages will be listed. Unfortunately,

2.1 The role of scaffolds in Tissue Engineering

synthetic bone-graft applications. Cellular composites are then seen as consisting

Scaffold pore size

Type II: 1540 m

fibrous tissue ingrowth

Type III: 30100

ingrowth, osteoid and

m(80% pores <100

fibrous 80% pores , 100

Type IV: 50100

by 11 weeks and 500

m(63% pores <100

different from untreated

different from untreated

more bone than all other

2.2 Solid freeform fabrication techniques (SFFT)

reproducible scaffolds with fully interconnected porous networks [47-48] as

Figure 1 Photograph of a scaffold produced by SFF techniques (machined into a

a layer-by-layer manufacturing method of SFF fabrication

(computer-driven by 3D digital models). PLLA was dissolved in dioxane and TCP

interconnectivity. One of the major disadvantages of this method is to increase

adsorption and endothelial proliferation [62-63].

2) Osteoconductivity is the ability of the scaffold to serve as a template for bone

8) Manufacturability refers to the ease and flexibility of scaffold fabrication.

2.3 Target mechanical properties for bone scaffolds

Table 3 Summary of mechanical properties and porosity of human bone

*NR, not reported by author.

of CaPs can be of the order of months to years, depending on porosity,

2.4 Choice of biomaterials

osteoinductive, osteoconductive properties and material biocompatibility [103]. In

phosphate (-TCP), with the chemical composition Ca3(PO4)2 and a calcium to

Since tri-calcium phosphate exhibits higher solubility, it is expected that it can

Polymer with excellent mechanical properties can improve CaPs scaffolds

ceramic coating is the best way to improve scaffolds mechanical and

calcium/phosphorus ratio. However, due to the weak mechanical strength of