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DEVELOPMENT OF MELT ELECTROSPUN COMPOSITE SCAFFOLDS FOR BONE
REGENERATION
Jiongyu Ren BEng, MEng
Submitted in fulfilment of the requirements for the degree of
Doctor of Philosophy
Institute of Health and Biomedical Innovation
Faculty of Science and Engineering
Queensland University of Technology
2017
development of melt electrospun composite scaffolds for bone regeneration i
Keywords
Melt-electrospinning, hybrid melt-electrospinning, bioactive glass,
polycaprolactone, composite scaffold, bone tissue engineering, strontium-substituted
bioactive glass, bone histology
ii development of melt electrospun composite scaffolds for bone regeneration
Abstract
A scaffold based approach of tissue engineering can potentially circumvent the
issues associated with current bone grafting treatment for bone defect regeneration.
Polycaprolactone (PCL) is a resorbable polymer used extensively in bone tissue
engineering owing to good structural properties and processability. Strontium-
substituted bioactive glass (SrBG) has the ability to promote osteogenesis and may be
incorporated into scaffolds intended for bone repair. The incorporation of SrBG
particle filler phase into PCL matrix produced bioactive PCL/SrBG composite that
possesses good processability. Melt-electrospinning is an additive manufacturing
technique to produce porous scaffolds with high surface area to volume ratio. In this
PhD project, I contributed to three key processes of scaffold development including
scaffold fabrication, in vitro assessment of scaffolds and the optimization of endpoint
histological techniques for the ex vivo analysis of these scaffolds. In the first study,
PCL/SrBG (10 wt%) composite scaffolds were developed using the technique of melt-
electrospinning. The characterization of these scaffolds demonstrated their bioactivity
and favourable in vitro characteristics. Osteoblast-precursor cells cultured on
PCL/SrBG (10 wt%) scaffolds showed improved osteogenic properties indicated by
enhanced alkaline phosphatase (ALP) activity when compared to scaffolds made of
pure PCL. In order to enhance osteoblastic differentiation by the scaffolds, I increased
the percentage of SrBG particles up to 50 wt% in the PCL matrix in study 2. This was
made possible by a novel hybrid melt-electrospinning technique that was developed to
produce scaffolds with controlled fibre spacing and lay down pattern. The PCL/SrBG
(50 wt%) scaffolds showed significantly enhanced bioactivity compared to PCL/SrBG
(10 wt%) ones. In vitro assessment of PCL/SrBG (50 wt%) scaffolds indicated these
scaffolds were osteogenic in vitro as evidenced by the significantly enhanced alkaline
phosphatase activity compared to PCL control scaffolds. While the first and second
study were carried out to improve the fabrication of bioactive scaffolds as suitable
bone defect implants, the third study of this Ph.D. project was carried out in parallel to
improve the histological tools for the evaluation of future animal implantation
experiments with these PCL/SrBG composite scaffolds. Using samples from different
species used in typical biomaterial implantation experiments found in the literature, I
optimised all the essential techniques, resulting in detailed protocols for the
development of melt electrospun composite scaffolds for bone regeneration iii
standardized histological processing of animal native bone and tissue engineered bone
explants.
development of melt electrospun composite scaffolds for bone regeneration i
List of Publications
1. A Berner, M A Woodruff, C X F Lam, M T Arafat, S Saifzadeh, R Steck, J
Ren, M Nerlich, A K Ekaputra, I Gibson, D W Hutmacher, Biomimetic tubular
nanofiber mesh and platelet rich plasma-mediated delivery of growth factors for large
bone defect regeneration, Cell and Tissue Research, 2012
2. A Berner, J D Boerckel, S Saifzadeh, R Steck, J Ren, C Vaquette, J Qiyi
Zhang, M Nerlich, R E Guldberg, D W Hutmacher, M A Woodruff, Effects of
scaffolds architecture on bone healing, International Journal of Oral and Maxillofacial
Surgery, 2013
3. Jiongyu Ren, Keith A Blackwood, Amir Doustgani, Patrina P Poh, Roland
Steck, Molly M Stevens, Maria A Woodruff, Melt-electrospun polycaprolactone
strontium-substituted bioactive glass scaffolds for bone regeneration, Journal of
Biomedical Materials Research Part A, 2014
4. Nikola Ristovski, Nathalie Bock, Sam Liao, Sean K Powell, Jiongyu
Ren, Giles T S Kirby, Keith A Blackwood, Maria A Woodruff, Improved fabrication
of melt electrospun tissue engineering scaffolds using direct writing and advanced
electric field contorl, Biointerphases, 2015
5. K. A. Blackwood, N. Ristovski, S. Liao, N. Bock, J. Ren, G. T. S. Kirby, M.
M. Stevens, R. Steck, M. A. Woodruff, Improving Electrospun Fibre Stacking with
Direct Writing for Developing Scaffolds for Tissue Engineering for Non-load Bearing
Bone, IFMBE Proceedings, 2015
Papers in preparation
Jiongyu Ren, Keith A Blackwood, Seamus Tredinnick, Roland Steck, Giles T Kirby,
Christina Theodoropoulos, Flavia M Savi, Maria A Woodruff, Development of
optimised histological processes for analysis of large and complex bone and implants
Jiongyu Ren, Keith A Blackwood, Roland Steck, Molly M Stevens, Maria A
Woodruff, Developing Strontium-substituted Bioactive glass and Polycaprolactone
composite scaffolds for bone repair via hybrid electrospinning in a direct writing mode
ii development of melt electrospun composite scaffolds for bone regeneration
Table of Contents
Keywords .................................................................................................................................. i
Abstract .................................................................................................................................... ii
List of Publications .................................................................................................................... i
Papers in preparation ................................................................................................................. i
Table of Contents ..................................................................................................................... ii
List of Figures .......................................................................................................................... v
List of Tables ......................................................................................................................... viii
List of Abbreviations ............................................................................................................... ix
Statement of Original Authorship ........................................................................................... xi
Acknowledgements ................................................................................................................ xii
Chapter 1: Introduction ...................................................................................... 1
1.1 Overview ........................................................................................................................ 1
1.2 Purpose of research ........................................................................................................ 4
1.3 Significance of research ................................................................................................. 5
1.4 Thesis Outline ................................................................................................................ 6
Chapter 2: Research Hypothesis and Aims ....................................................... 9
2.1 Hypothesis ...................................................................................................................... 9
2.2 Research Aims ............................................................................................................... 9
Chapter 3: Literature review ............................................................................ 15
3.1 Bone Biology ............................................................................................................... 15 3.1.1 Structure of bone ................................................................................................ 15 3.1.2 Bone fracture healing ......................................................................................... 18
3.2 Current treatments for large bone defects .................................................................... 19 3.2.1 Bone grafting ..................................................................................................... 19
3.3 Bone Tissue Engineering ............................................................................................. 20 3.3.1 3D printing scaffolds ......................................................................................... 21 3.3.2 Tissue engineering scaffolds .............................................................................. 21 3.3.3 TE scaffolds specifications ................................................................................ 22
3.4 Scaffold materials ........................................................................................................ 23 3.4.1 Polymers ............................................................................................................ 23 3.4.2 Bioactive glass (BG) .......................................................................................... 25 3.4.3 Biodegradable polymer/bioactive glass composites .......................................... 28
3.5 Scaffold fabrication techniques .................................................................................... 30 3.5.1 History of electrospinning ................................................................................. 32 3.5.2 Basic electrospinning equipment ....................................................................... 32 3.5.3 Theory of electrospinning .................................................................................. 33 3.5.4 Process of electrospinning ................................................................................. 34 3.5.5 Melt-electrospinning .......................................................................................... 35 3.5.6 Hybrid electrospinning system .......................................................................... 36
development of melt electrospun composite scaffolds for bone regeneration iii
3.6 TE scaffolds assessment ...............................................................................................37 3.6.1 Histology ............................................................................................................38
3.7 Conclusion ....................................................................................................................42
Chapter 4: (Study 1) Fabrication and In vitro investigation of PCL, 10 wt% PCL/SrBG electrospun scaffolds for bone regeneration ...................................... 45
4.1 Introduction ..................................................................................................................45
4.2 Materials and Methods .................................................................................................47 4.2.1 Scaffold fabrication and characterisation ...........................................................47 4.2.2 Ion dissolution and precipitation ........................................................................48 4.2.3 In vitro studies ....................................................................................................49 4.2.4 Statistical analyses ..............................................................................................54
4.3 Results ..........................................................................................................................54 4.3.1 Characterization of PCL and PCL/SrBG scaffolds ............................................54 4.3.2 Ion dissolution and precipitation analysis ..........................................................55 4.3.3 In vitro studies ....................................................................................................57
4.4 Discussion .....................................................................................................................64
4.5 Conclusions of study 1..................................................................................................71
Chapter 5: (Study 2) Developing 50 wt% Strontium-substituted bioactive glass and Polycaprolactone composite scaffolds for bone repair via hybrid electrospinning in a direct writing mode ............................................................... 73
5.1 Introduction ..................................................................................................................73
5.2 Materials and Methods .................................................................................................75 5.2.1 PCL/SrBG composite preparation ......................................................................75 5.2.2 Scaffold fabrication ............................................................................................76 5.2.3 Scaffold characterisation ....................................................................................77 5.2.4 In vitro studies ....................................................................................................77 5.2.5 Statistical analyses ..............................................................................................81
5.3 Results ..........................................................................................................................82 5.3.1 Particle grinding and sizing ................................................................................82 5.3.2 Scaffold fabrication ............................................................................................82 5.3.3 Characterisation ..................................................................................................83 5.3.4 In vitro studies ....................................................................................................89
5.4 Discussion .....................................................................................................................94
5.5 Conclusion of study 2 .................................................................................................102
Chapter 6: (Study 3) Development of optimised histological processes for analysis of large and complex bone and implants ............................................... 105
6.1 Introduction ................................................................................................................105
6.2 Materials and Methods ...............................................................................................108 6.2.1 Bone tissues and pre-processing preparation....................................................108 6.2.2 Processing and embedding ...............................................................................109 6.2.3 Sectioning .........................................................................................................111 6.2.4 Staining .............................................................................................................114 6.2.5 Microscopy and image documentation .............................................................119
6.3 Results and Discussion ...............................................................................................119 6.3.1 Study Overview ................................................................................................119 6.3.2 Stain optimization and comparison ..................................................................120
iv development of melt electrospun composite scaffolds for bone regeneration
6.4 Conclusion of study 3 ................................................................................................ 133
6.5 Supplementary Figures .............................................................................................. 137
Chapter 7: Conclusions.................................................................................... 141
7.1 Research Summary .................................................................................................... 141
7.2 Summary of Chapter 4 (Study 1) ............................................................................... 142
7.3 Summary of Chapter 5 (study 2) ................................................................................ 143
7.4 Summary of Chapter 6 (Study 3) ............................................................................... 144
7.5 Limitations and recommendation for future work ..................................................... 145 7.5.1 Composite scaffold design ............................................................................... 145 7.5.2 In vivo investigation of composite scaffolds .................................................... 146
7.6 Concluding Remarks .................................................................................................. 148
References ............................................................................................................... 151
Appendices .............................................................................................................. 169
development of melt electrospun composite scaffolds for bone regeneration v
List of Figures
Figure 1.1 General development process of bone TE scaffolds................................... 3
Figure 1.2 Overview of PCL/SrBG scaffolds development in this PhD project. ........ 4
Figure 3.1 The seven hierarchy levels of bone structure, as demonstrated by Weiner and Wagner [18]. ............................................................................. 16
Figure 3.2 Schematic illustration of long bone and its micro-structure [23] ............. 17
Figure 3.3 The four stages of bone fracture regeneration [24] .................................. 19
Figure 3.4 Schematic overview of the scaffold-based approach to bone TE [12] ..... 22
Figure 3.5 The degradation and elimination pathway of PCL. .................................. 25
Figure 3.6 The effect of bioactive glass ion dissolution products on biological responses [49] .............................................................................................. 27
Figure 3.7 Schematic illustration of typical electrospinning setup (not to scale) [82] ............................................................................................................... 33
Figure 3.8 A schematic summary of electrospinning configurations for desired fibre alignments [87] .................................................................................... 34
Figure 4.1 Electrospun scaffolds light microscopy and microCT characterization. ........................................................................................... 55
Figure 4.2 EDX image of melt-electrospun scaffolds after 14 days incubation in α-MEM. ................................................................................................... 57
Figure 4.3 LIVE/DEAD staining of MC3T3 cells cultured on melt-electrospun scaffolds. ...................................................................................................... 58
Figure 4.4 SEM images of MC3T3 cells cultured on PCL (a)(b) and (c) and PCL/SrBG (d)(e) and (f) scaffolds. .............................................................. 59
Figure 4.5 Confocal laser scanning microscopy images of MC3T3 cells cultured on melt-electrospun PCL/SrBG (a) and PCL scaffolds (b) for 3 days. .......................................................................................................... 59
Figure 4.6 MTT metabolic activity assay of MC3T3 cells over 28 days culture....... 60
Figure 4.7 Normalised ALP activity of MC3T3 cells cultured on melt-electrospun PCL and PCL/SrBG scaffolds cultured in osteogenic and control media. .............................................................................................. 61
Figure 4.8 Alizarin red S staining of PCL and PCL/SrBG scaffolds cultured with MC3T3 cells in control and osteogenic media over 28 days. .............. 62
Figure 4.9 Gene expression of osteoblast markers ALP (a) and OCN (b), in all experimental groups over 28 days. .............................................................. 63
Figure 4.10 Van Gieson staining of PCL and PCL/SrBG scaffolds cultured with MC3T3 cells in control and osteogenic media over 28 days. .............. 64
Figure 5.1 SrBG particle size distribution before grinding, after grinding and after drying. .................................................................................................. 82
vi development of melt electrospun composite scaffolds for bone regeneration
Figure 5.2 SEM images of 50% PCL/SrBG (a-d) and PCL (e-h) scaffolds. .............. 84
Figure 5.3 Confocal laser scanning microscopy images of PCL/SrBG scaffolds stained by Alizarin red S. ............................................................................. 85
Figure 5.4 Backscattered SEM image of a PCL/SrBG scaffold. ............................... 86
Figure 5.5 Elemental concentrations of Ca2+ (a), PO43- (b), Si4+ (c), and Sr2+ (d)
of a-MEM media incubated with 50% PCL/SrBG and PCL scaffolds over 28 days as determined by ICP-MS testing. .......................................... 87
Figure 5.6 Example surface elemental compositions of 50% PCL/SrBG scaffolds after 3h (a), 6h (b), 1 day (c) and 2 days (d) in a-MEM media as determined by EDX. ..................................................................... 88
Figure 5.7 LIVE/DEAD staining of MC3T3 cells cultured on melt-electrospun scaffolds. ...................................................................................................... 90
Figure 5.8 SEM images of MC3T3 cells cultured on PCL (a) and (b) and PCL/SrBG (c) and (d) scaffolds. .................................................................. 91
Figure 5.9 Confocal laser scanning microscopy images of MC3T3 cells cultured on melt-electrospun ........................................................................ 91
Figure 5.10 MTT metabolic activity assay of MC3T3 cells over 28 days culture. .......................................................................................................... 92
Figure 5.11 Normalised ALP activity of MC3T3 cells cultured on melt-electrospun PCL and PCL/SrBG scaffolds cultured in osteogenic and control media. ............................................................................................... 93
Figure 5.12 Gene expression of osteoblast markers ALP (a) and OPN (b) as fold change to PCL control group in all experimental groups over 28 days. ............................................................................................................. 94
Figure 5.13 The direct comparison of Si4+ and Sr2+ ion concentration of PCL/SrBG (10wt%) scaffolds in study 1 and PCL/SrBG (50wt%) scaffolds in study 2. ................................................................................... 103
Figure 6.1 Schematic summary of study 3 design. .................................................. 109
Figure 6.2 An overview of resin block preparation and ground sectioning process. ....................................................................................................... 113
Figure 6.3 Comprehensive comparison of Goldner’s trichrome staining on resin ground sections and paraffin sections of bone of all four animal species. ....................................................................................................... 121
Figure 6.4 Specimen preparation and Goldner’s trichrome staining results of a tissue engineered sheep tibia with a 3 cm critical sized defect post mechanical testing. ..................................................................................... 125
Figure 6.5 Specimen preparation and staining results of sheep tibiae with scaffolds implanted into 3 cm critical sized defects. ................................. 129
Figure 6.6 Comparison of mouse paws prepared as resin ground sections, resin thin sections and paraffin sections stained with Goldner’s trichrome stain. ........................................................................................................... 130
development of melt electrospun composite scaffolds for bone regeneration vii
Figure 6.7 Overview and high magnification images of a whole mouse hind paw and a sheep tibia/femur with porous titanium implant prepared by ground sectioning and stained with Goldner’s trichrome. ......................... 132
Figure A.1 Goldner’s trichrome staining of resin embedded sheep tibia bone tissues around titanium implant, sections obtained by ground sectioning technique................................................................................... 169
Figure A.2 Goldner’s trichrome staining of resin embedded sheep tibia native bone tissues and regenerated bone tissues around implanted PCL scaffolds ..................................................................................................... 170
Figure A.3 Goldner’s trichrome staining of resin embedded sheep tibia bone tissues around Ti implant, sections obtained by ground sectioning technique [132]. ......................................................................................... 171
viii development of melt electrospun composite scaffolds for bone regeneration
List of Tables
Table 3.1 Properties of biodegradable polymers as bone scaffold materials [35,36] .......................................................................................................... 24
Table 3.2 Reaction stages of HCA layer formation on 45S5 BG [54] ....................... 26
Table 3.3 Comparison of different bone scaffold fabrication techniques [50,66] ..... 31
Table 3.4 Important parameters for electrospinning process [80] ............................. 34
Table 4.1 Parameters for melt-electrospun scaffold fabrication ................................ 48
Table 4.2 In vitro experimental groups of study 1. PCL/SrBG scaffolds (10 wt%) were studied in both growth media and osteogenic media and PCL scaffolds were studied as control. ........................................................ 49
Table 5.1 Parameters for 50 wt% PCL/SrBG and PCL scaffolds fabrication ............ 76
Table 5.2 In vitro experimental groups of study 2. PCL/SrBG scaffolds (50 wt%) were studied in both growth media and osteogenic media and PCL scaffolds were studied as control. ........................................................ 78
Table 5.3 Web of Science search results of ‘melt-electrospinning’ AND ‘polymer composite’ on 10/10/2016 ............................................................ 95
Table 6.1 Summary of embedding media and sectioning techniques in study 3. .... 107
Table 6.2 Preparation of Technovit 9100 New® solutions ....................................... 111
Table 6.3 Suitable sandpaper grits according to section thickness: a. final thickness=27 μm; b. final thickness=50 μm .............................................. 114
Table 6.4 Goldner’s trichrome staining solutions .................................................... 115
Table 6.5 Von Kossa/MacNeal’ tetrachrome staining solutions .............................. 117
Table 6.6 Summary of the advantages and disadvantages of commonly used media for histology embedding: paraffin, MMA resin and Technovit 9100 resin. .................................................................................................. 134
Table 6.7 Summary of the advantages and disadvantages of commonly used sectioning techniques for resin embedded specimens ................................ 136
development of melt electrospun composite scaffolds for bone regeneration ix
List of Abbreviations
Full name Abbreviation 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide MTT Additive manufacturing AM Alkaline phosphatase ALP Alpha-minimum essential medium α-MEM Bioactive glass BG Bone morphogenetic protein BMP Bovine serum albumin BSA Calcium Phosphate CaP Confocal laser scanning microscopy CLSM Dimethyl sulfoxide DMSO Double-stranded deoxyribonucleic acid dsDNA Energy-dispersive X-ray spectroscopy EDX Electron probe microanalyzer EPMA Backscattered electron BSE Extracellular matrix ECM Fetal bovine serum FBS Hydroxyapatite HA Inductively coupled plasma - optical emission spectrometer ICP-OES Inductively coupled plasma - mass spectrometer ICP-MS Mega Pascal Mpa Micro-computed tomography μCT Nano-sized bioactive glass nBG Osteocalcin OCN Osteopontin OPN Power of hydrogen pH Phosphate buffer solution PBS Poly(3-hydroxybutyarate) P(3HB) Poly(caprolactone-co-DL_lactide) P(CL-DLLA) Poly(lactic acid) PLA Poly(glycolic acid) PGA Poly(L-lactic-co-glycolic acid) PLGA Poly(L-lactide) PLLA Poly(trimethylene carbonate) TMC Polycaprolactone PCL Protein kinase PK Rapid prototyping RP Real-time-quantitative polymerase chain reaction RT-qPCR Scanning electron microscopy SEM Sodium hydroxide NaOH Strontium-substituted bioactive glass SrBG Hydroxycarbonate apatite HCA Three dimensional 3D Tissue engineering TE
x development of melt electrospun composite scaffolds for bone regeneration
Methyl methacrylate MMA Hematoxylin & Eosin H & E Von Willebrand factor vWF
development of melt electrospun composite scaffolds for bone regeneration xi
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.
Signature: QUT Verified Signature
Date: May 2017
xii development of melt electrospun composite scaffolds for bone regeneration
Acknowledgements
First and foremost I want to thank my supervisors, Associate Professor Maria A.
Woodruff, Dr. Roland Steck and Dr. Keith A. Blackwood. I could not have achieved
this major milestone in my career without their ongoing guidance and support. I am
especially grateful to A/Prof Woodruff for supporting me not only by providing a
research assistantship, but also academically through the rough road to finish this PhD
thesis. I acknowledge the professional help in editing this thesis from my supervisors
Dr. Roland Steck and A/Prof Mia Woodruff.
This thesis is also the result of support and help from dozens of remarkable
individuals at QUT. I would like to first thank my colleagues of the Biofabrication &
Tissue Morphology group and the Regenerative Medicine group who have contributed
to my PhD project. In particular, I thank Dr. Patrina Poh for sharing the knowledge
and techniques of in vitro assays and the support from CARF Histology Facility.
I acknowledge the following colleagues for their consultation: Dr
Henrietta Cathey (EPMA), Mr Tony Raftery (micronizing mill), Mr David Page
(Malvern Mastersizer), Emeritus Professor Graeme George and Dr John Colwell
(SrBG surface modification), Dr Charlotte Allen, Dr Sunny Hu and Mr. Mitchell De
Bruyn (ICP-MS), Dr Marie-Luise Wille (μCT), Dr Tong Li (AFM), Ms Rachel
Hancock (SEM), Dr. Christina Theodoropoulos and Dr Leonore de Boer (CLSM) and
Mr Shane Russell (ICP-OES).
I would like to acknowledge our collaborator professor Molly M. Stevens and
Dr. Anu Solanki from Imperial College London for providing bioactive glass that
made this project possible. Furthermore, I acknowledge Australian Research Council
for sponsoring this project (grant LP110200082 and LP100200084), and QUT APA
scholarship for the financial support.
Last but not least, I thank the support from my parents, parents-in-law, my wife
Xue Zhang, my daughter Ravenna Zirong Ren and my son Joshua Zitao Ren, I dedicate
this PhD thesis to you.
Chapter 1: Introduction 1
Chapter 1: Introduction
1.1 OVERVIEW
Musculoskeletal disorders have a significant socioeconomic impact worldwide.
They place a huge burden on the healthcare systems and community. Critical-sized
bone defects (a defect size which will not naturally heal) are one of the biggest
contributors, among these only a few patients will fully restore their limb function to
the level whereby they can return to previous employment [1]. In Australia, millions
of people are suffering from severe pain and disability caused by musculoskeletal
conditions and fatalities still occur due to ineffective available treatments. According
to Authoritative information and statistics to promote better health and wellbeing
(AIHW), 6.1 million Australians are affected by musculoskeletal conditions and
725,500 Australians are diagnosed with osteoporosis in 2011 to 2012 [2]. As a result,
musculoskeletal conditions have become a major burden to direct health expenditure
which accounted for $9153.7 million in 2012 [3], the fourth leading contributor
following cardiovascular diseases, oral health and mental disorder [4]. The figures
from the United States are even higher, reaching US$849 billion [5]. Besides the
economic expenditures, musculoskeletal conditions also affect the lives of patients and
place a significant burden on the community and health care services. Therefore,
researchers are actively seeking effective treatment for large bone defects in clinical
practice.
Current clinical treatments for critical bone defects rely on bone grafting,
including autografting (transplanting a patient’s own bone), allografting and
xenografting (transplanting bone from a donor or another species, respectively).
Although these procedures exhibited healing capacities, the bone regeneration results
have not always been satisfactory and these techniques have shown prominent
disadvantages such as disease transmission, immunogenic response, and
complications in bone harvesting surgeries. These complications have driven the
development of biomaterials which may ultimately substitute and improve on current
techniques. Over the last two decades, many bone substitute materials have been
evaluated with the intention of replacing autografts or allografts. The endeavours of
2 Chapter 1: Introduction
researchers over the years led to the emergence of a new field of research known as
Tissue Engineering (TE).
TE combines the knowledge of both engineering and the life sciences with the
aim to produce tissue-like substitutes for the purpose of restoring or improving the
natural tissue function. Researchers in the bone TE field have extensively investigated
using polymeric scaffolds as potential bone graft substitutes. Amongst the variety of
clinical grade scaffolds available to patients today, bioresorbable polymeric scaffolds
have drawn a lot of attention, as they have the potential to integrate with the host bone
and degrade away within a controllable time frame to be replaced by newly-formed
bone tissue. However, current research suggests that no single component material can
satisfy all the requirements of viable bone-substituting scaffolds, due to the large
number of characteristics required for a suitable tissue engineered material. Composite
materials composed of polymers and bioactive glass/ceramic particles have emerged
as highly promising candidates because the bulk materials combine the processability
of polymers with the bioactivity of particle filler phase. The fine particle filler phase
also enhances the mechanical stability of polymer matrix making the composite
material stronger than polymer only. Composite scaffolds of high porosity (>80%) are
rarely reported for the treatment of critical bone defects within medium to high load-
bearing sites, owing to a lack of strength and the fact that they are still in a developing
stage. When developing a tissue engineered scaffold for bone regeneration we need to
consider the properties of the materials. Polycaprolactone (PCL) has good mechanical
properties and processibility; strontium-substituted bioactive glass (SrBG) has the
ability to promote osteogenesis. The PCL/SrBG composite combines the advantages
of these two materials and therefore is superior to PCL or SrBG only.
Several tissue engineering approaches have been developed to fabricate porous
3D composite scaffolds with high pore interconnectivity. Among them,
electrospinning has recently gained substantial research attention. Electrospinning is a
versatile technique for the production of fibres ranging from the micro to nano scale
in diameter, and the scaffolds produced with these micro- or nanofibres are of
extremely high surface area to volume ratio. This technique is well established in the
field of tissue engineering due to the ease to set up and low cost to run. Many different
polymers can be spun into scaffolds with interconnective pores using electrospinning.
There are mainly two ways of preparing polymers for electrospinning: dissolving the
Chapter 1: Introduction 3
polymer in an organic solvent (solution- electrospinning) and melting the polymer
(melt-electrospinning). Melt- electrospinning overcomes technical restrictions of
solution-electrospinning governed by solvent accumulation and toxicity. In spite of the
potential benefits of melt spinning, it has gained less attention in the TE field compared
to solution- electrospinning.
The scaffolds are characterised and tested for their osteogenic potential in vitro
and in vivo after fabrication following a development process shown in Figure 1.1.
Figure 1.1 General development process of bone TE scaffolds. The process normally starts from fabricating the scaffolds which are rigorously tested in vitro and in vivo step by step before they are clinically relevant. Histology forms the last but critical chain of the assessment.
Generally, in vivo examination, which utilises pre-clinical animal models to
learn the progress of tissue regeneration once a scaffold is implanted in the body
system, is considered to be a true indicator of a TE scaffold’s performance. Histology
is one of the most important post-explant analytical methods in biology and medicine,
and it has been widely applied in bone tissue engineering for bone regeneration
assessment [6]. Histology consists of the microscopic analysis of two-dimensional
tissue sections via a sequential procedure of fixation, dehydration, clearing and
infiltration and embedding (referred here as “processing”); followed by sectioning and
staining [7]. Even though a variety of embedding and sectioning techniques have been
developed in the last two decades, it still remains a challenge to process samples from
bone healing experiments owing to the heterogeneity of the tissues formed during bone
regeneration [8]. Considering the number of animals that can be used in these studies
is also limited, the samples obtained from these in vivo studies are extremely precious.
Thus, it is imperative to select the right analytical techniques to obtain the most
information from limited specimen sections. In this PhD project, I have compared and
optimised the histological techniques for bone specimens embedded in paraffin,
4 Chapter 1: Introduction
Methyl Methacrylate (MMA) and Technovit 9100 NEW® resin. The outcome of this
study is the standardization of the advanced histological analysis for the future
assessment of bone tissue engineering scaffolds in vivo. Owing to the scaffold
development component of this thesis, it was deemed crucial to also develop advanced
histology techniques to enable the scaffold to be eventually assessed in vivo.
Figure 1.2 Overview of PCL/SrBG scaffolds development in this PhD project. Study 1 and study 2 aimed to produce scaffolds with good bioactivity that are suitable for in vivo implantation. Study 3 was carried out in parallel to provide an optimised method for ex vivo assessment of PCL/SrBG scaffolds.
1.2 PURPOSE OF RESEARCH
The purpose of this PhD project was to develop a PCL/SrBG composite scaffold
using the technique of melt-electrospinning, which may be an alternative treatment
option for healing bone defect compared to autografting. The PCL/SrBG composite
possesses good processibility and bioactivity, and was fabricated into porous scaffolds
via electrospinning. Following fabrication and characterisation, a series of in vitro tests
were carried out on these scaffolds to thoroughly investigate their bioactivity and
osteogenic capacity. I then investigated the possibility to increase the percentage of
SrBG content in the composite and even achieve ordered fibre alignment via a novel
hybrid electrospinning. In vitro tests were performed on the optimised scaffolds for
their bioactivity and osteogenic capacity. The aims of study 1 and study 2 in this study
were to produce PCL/SrBG composite scaffolds with sufficient osteogenic capacity to
Chapter 1: Introduction 5
induce bone differentiation when implanted in animal model. In parallel, this research
project also aims to advance the histological techniques for the analysis of complex
explants in our pre-clinical models as an initial aim was to implant these scaffolds as
part of this PhD, hence developing analysis technique in parallel was paramount. In
study 3 of this project, the histological methods for bone assessment were optimised
for the in vivo implanted PCL/SrBG scaffolds (Figure 1.2). The findings of this
research project not only built a basic understanding of the bioactivity and osteogenic
capacity of PCL/SrBG composite scaffold which may be suitable for potential clinical
applications, but also provide a viable tool for evaluating the bone regeneration
capacity of these scaffolds.
1.3 SIGNIFICANCE OF RESEARCH
Current clinical treatment for large bone defects relies on bone grafting:
autografting, allografting and xenografting. However, these techniques show
prominent disadvantages such as donor site morbidity, complications, and infections
and are far from satisfying the patients’ needs in many cases. Other strategies have
been introduced to facilitate bone defect healing such as by the addition of growth
factors. Growth factor treatments are commercially available such as bone
morphogenetic protein-2 (BMP-2) and BMP-7 for bone defect repair. Although BMPs
are strongly osteoinductive and effective [9], they do incur high in-hospital costs for
patients [10]. Even worse, in some cases, they cause undesirable heterotopic
ossification and raise safety concerns [11].
Additive manufacturing, also known as three-dimensional (3D) printing, is
widely employed in TE to produce 3D scaffolds with layer-by-layer deposition
instructed by anatomical and architectural information obtained by medical imaging
techniques (e.g. computed tomography and magnetic resonance imaging) [12]. 3D
printing techniques allow production of 3D structure of complex external shapes with
customized internal microstructures of reproducible porosity and interconnectivity
using biomaterials [13]. These features enable 3D printing to be used by tissue
engineers to fabricate tissue-analogous structures to provide patient-specific implants
for defect healing. Melt-electrospinning produces scaffolds like 3D printing but with
much smaller fibre diameter, which in turn means much higher surface area to volume
ratio. As potential bone graft substitutes, bioactive composite scaffolds may be used
in clinical intervention to circumvent issues associated with bone grafting. In
6 Chapter 1: Introduction
comparison to growth factors, the use of PCL/SrBG composite scaffolds can be
considered as a more economic and safe option [14]. Furthermore, these composite
scaffolds are made of biocompatible materials and they can be thoroughly sterilised,
therefore, the risk of inflammation and infection is minimised. The fact that the SrBG
binds actively to surrounding soft and hard tissues greatly reduced the chance of
fibrous encapsulation often observed in polymeric implants [15].
Ex vivo assessment of scaffolds using histological approaches is a key process
in the scaffold development. Histology is a traditional technique for the study of the
structure and composition of tissues at the microscopic level and is utilized to analyse
bone morphology in the TE field. Histology uses well-established sequential
procedures to visualise tissue morphology with cellular details. However, it remains
challenging to process bone due to the densely calcified components combined with
the large size of explants. In a parallel and complementary research project, I optimised
and compared common bone histological techniques to provide an optimal tool for
bone regeneration analysis. Our findings can also benefit researchers in the TE field to
pre-plan their histology for optimum results.
On the whole, PCL/SrBG composite scaffolds are promising cost-effective
substitutes to autografting for clinical treatment of bone defects. Combined with
advanced 3D printing, we move toward highly effective and personalised implants.
The findings of this project are expected to propel the production of synthetic bone
graft substitutes that can improve the patients’ quality of life, which is the ultimate
motivation of this PhD project.
1.4 THESIS OUTLINE
The overall aim of this PhD project is to develop a novel PCL/SrBG composite
scaffold via melt-electrospinning as a tissue engineering solution for non-load bearing
defects (such as skull defects), as such we will employ a multi-directional approach,
to both develop a complete scaffold and to develop and standardise end point analytical
assessment techniques (histology). The overall aim is divided into three detailed sub-
aims following the research hypothesis stated in chapter 2.
In chapter 3, I present an extensive literature review covering bone structure and
healing, clinical treatment for bone defects, bone tissue engineering, electrospinning
and biomaterials. This chapter helps to understand the impact of bone fracture situation
Chapter 1: Introduction 7
and the recent advancement in the tissue engineering field, which contextualize the
need of our research and how this PhD research can contribute to this research field.
Chapters 4 and 5 describe study outcomes of fabrication, characterization and in
vitro osteogenic capacity evaluation of the electrospun composite scaffolds. Chapter 6
describes the development of optimal histological tools for assessing calcified and
complex tissue engineering scaffold in pre-clinical models. The interrelated studies
reported in these chapters were completed and published or are in preparation for
submission.
Finally, the overall findings in chapter 4, 5 and 6 are collectively concluded and
discussed in chapter 7. The limitations of this PhD and recommended future work are
also discussed in this chapter.
Chapter 2: Research Hypothesis and Aims 9
Chapter 2: Research Hypothesis and Aims
2.1 HYPOTHESIS
We hypothesize that the bioactivity of PCL can be increased by incorporating
SrBG particles, and that these PCL/SrBG composites can be produced by melt-
electrospinning into porous scaffolds which are osteoinductive, osteoconductive,
capable of osteointegration and bioresorbable in a controlled rate.
2.2 RESEARCH AIMS
The overall aim of this PhD project was to develop melt-electrospinning as a
tissue engineering solution for non-load bearing defects, as such I employed a multi-
directional approach, to both develop the PCL/SrBG composite scaffold and
standardise end point histological analytical assessment criterion. This overall aim was
broken down to 3 specific aims that formed the basis for three interrelated studies
presented below.
(Study 1) To design, fabricate and characterise PCL/SrBG (10 wt%)
composite scaffolds for bone regeneration via melt-electrospinning, followed by
assessing the in vitro bone regeneration capacity of the composite scaffolds.
In study 1, I attempted to melt-electrospin porous scaffolds of PCL/SrBG
composite which combined the processibility of PCL and the osteogenic properties of
SrBG by mixing SrBG particles into PCL bulk. SrBG particles were ground to <38 μm
with mortar and then mixed into molten PCL by manual stirring until even distributed.
PCL/SrBG composites containing only 10 wt% of SrBG were fabricated into scaffolds
via melt-electrospinning due to the limitations posed by SrBG particle size and
composite mixing technique. These electrospun composite scaffolds were then
characterised and comprehensively assessed for their in vitro osteogenic properties.
Sections 1a – 1c describes the methodology undertaken in study 1.
1a: To optimise melt-electrospinning technique for the fabrication of PCL/BG
composite scaffolds and characterisation of these scaffolds
10 wt% PCL/SrBG composite was manually prepared by stirring the mixture of
SrBG particles and PCL bulk at 65 °C on a hot plate. The melt-electrospinning
10 Chapter 2: Research Hypothesis and Aims
parameters (i.e. temperature, voltage, polymer flow rate and collector to spinneret
distance) for the fabrication of PCL and PCL/SrBG composite scaffolds were
optimised for the built-in-house melt-electrospinning rig. The fabricated PCL/SrBG
composite scaffolds and control PCL scaffolds were characterised with micro-
computed tomography, light microscopy and scanning electron microscopy (SEM) for
their properties such as fibre diameter, surface topography and SrBG particle
distribution.
1b: In vitro bioactivity evaluation and comparison of both PCL and PCL/SrBG
scaffolds
Two mechanisms account for the bioactivity of SrBG: tissue bonding via the
formation of a hydroxycarbonate apatite (HCA) layer & ion dissolution of the SrBG
[16]. To observe the HCA layer formation and measure the ion concentration, the
scaffolds were immersed in serum-free culture media for up to 28 days incubated at
37 °C. At predetermined time points, the scaffolds were retrieved from the media for
SEM and energy-dispersive X-ray spectroscopy (EDX) analysis, while the remaining
media were analysed with Vista MPX Inductively Coupled Plasma - Optical Emission
Spectrometer (ICP-OES) to quantify the concentration of Ca2+, PO43-, Si4+ and Sr2+.
1c: Investigation of the in vitro osteogenic capacity of PCL and PCL/SrBG
scaffolds.
MC3T3-E1 cells, a mouse osteoblast precursor cell line, were seeded onto both
PCL and PCL/SrBG scaffolds and cultured in normal growth media and osteogenic
media (media supplemented with osteogenic factors). At predetermined time points,
the scaffolds with cells were retrieved and assessed with a series of qualitative and
quantitative assays. The cell morphology and attachment on scaffolds were assessed
with confocal laser scanning microscopy (CLSM) and SEM. The scaffolds
cytotoxicity were assessed with LIVE/DEAD and MTT (3-(4,5-Dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide) assays. Cell proliferation and osteoblastic
differentiation were quantified by PicoGreen assay normalised alkaline phosphatase
(ALP) assay and polymerase chain reaction (PCR) assay. In addition, the scaffolds
with cells were stained with Alizarin red S and Van Gieson' stain for the assessment
of matrix mineralisation and matrix formation, respectively.
Study 1 has been completed and published:
Chapter 2: Research Hypothesis and Aims 11
Ren, Jiongyu et al. 2014. “Melt-Electrospun Polycaprolactone Strontium-
Substituted Bioactive Glass Scaffolds for Bone Regeneration.” Journal of Biomedical
Materials Research - Part A 102(9):3140–53.
This study is presented in details in chapter 4.
(Study 2) To optimise the composite scaffold design and to further increase
SrBG filler percentage in PCL to enable greater osteoinductivity.
Here I aimed to improve bioactivity of PCL/SrBG composite by increasing the
percentage of SrBG in PCL. In study 1, we found that PCL/SrBG composite scaffolds
exhibited in vitro bioactivity and enhanced osteogenic capacity. However, these
composite scaffolds were not yet ideally osteogenic due to an insufficient amount of
SrBG in PCL (only 10% by weight). Also, the scaffolds were melt-electrospun into
circular sheets with random fibre layout. In order to improve the control of fibre
deposition and improve SrBG yield in the PCL/SrBG composite, the hardware design
of a new electrospinning rig was significantly upgraded including a motorised stage
and dual high voltage power packs, and the size of SrBG was reduced from ≤ 38 μm
to ≤ 6 μm (details reported in Chapter 5).
Our theory that increased SrBG in the composite may lead to enhanced
osteogenic capacity is based on the fact that polymer/BG composite materials
exhibited higher bioactivity when higher amount of BG presented in them, as indicated
by more rapid in vitro calcium phosphate layer formation [17].
2a: To optimise the preparation technique of PCL/BG composites with higher
weight percentage of SrBG filler phase
The SrBG particles were ground from 100 μm down to < 6 μm prior to composite
preparation with a micronizing mill to reduce the risk of needle blockage. In addition,
the reduced particle size also increased the total surface area of the SrBG particles.
The PCL/SrBG composites were prepared by incorporating fine SrBG particles into
the PCL bulk using solvent precipitation technique instead of manual stirring in order
to ensure even distribution of SrBG particles within PCL bulk. The SrBG filler phase
was incorporated into PCL bulk as high as 50 wt% for enhanced bioactivity of the
PCL/SrBG composite.
12 Chapter 2: Research Hypothesis and Aims
2b: To fabricate 50 wt% PCL/SrBG with controlled fibre alignment via a novel
hybrid melt-electrospinning technique in a direct writing mode, followed by scaffold
characterization
The 50 wt% PCL/SrBG composite scaffolds were fabricated using a novel
solvent assisted hybrid melt-electrospinning technique. This hybrid electrospinning
technique utilized solvent to reduce the viscosity of composite melt to facilitate
establishing a continuous and stable polymer jet that were deposited on the collector
in a 0/90 º cross hatch laydown pattern.
Light microscopy and scanning electron microscopy (SEM) were used to
characterise surface topography and fibre diameter. Mechanical stiffness of PCL/SrBG
fibres was assessed with atomic force microscopy. Inductively coupled plasma mass
spectrometry (ICP-MS) technique was used to analyse the elemental concentration of
specific ions in the dissolution cell culture of the composite scaffolds.
2c: Investigation of the in vitro osteogenic capacity of PCL and PCL/SrBG (50
wt%) scaffolds.
MC3T3-E1 cells were used again to study the effect of PCL/SrBG (50 wt%)
scaffolds when they were in contact with cells, the PCL scaffolds were studied as
control. The cells seeded on both type of scaffolds were cultured in normal growth
media and osteogenic media (media supplemented with osteogenic factors). Similar in
vitro assays with study 1 were carried out to examine the bioactivity and osteogenic
capacity of the PCL/SrBG (50 wt%) scaffolds and to make comparison to PCL/SrBG
(10 wt%) scaffolds produced in study 1. At predetermined time points, the scaffolds
with cells were retrieved and assessed with a series of qualitative and quantitative
assays. The cell morphology and attachment on scaffolds were assessed with CLSM
and SEM. The scaffolds cytotoxicity were assessed with LIVE/DEAD and MTT
assays. Cell proliferation and osteoblastic differentiation were quantified by
PicoGreen assay normalised ALP assay and PCR assay.
(Study 3) To optimise and standardize histology techniques for assessing
bone/TE bone
As a scaffold for bone, the composite implant is expected to contain both
mineralised and soft tissues in vivo, creating differentially dense interfaces. The
complex calcified tissues coupled with often large explant sizes make it challenging
Chapter 2: Research Hypothesis and Aims 13
for their histological assessment. In order to advance our pre-clinical models and post
explant analysis of PCL/SrBG scaffolds, we optimised and compared two most
common bone histological techniques: paraffin embedding (after decalcification) and
resin embedding in either methyl methacrylate (MMA) or Technovit 9100 NEW® in
study 3 in parallel scaffold fabrication. Four commonly used histological stains for
bone were investigated including Haematoxylin & Eosin (H&E), Goldner’s trichrome
staining, Von Kossa staining and immunohistochemistry (IHC). To draw a
comprehensive conclusion, I investigated tissues and implants from commonly used
preclinical models in TE: sheep, pig, rat and mouse.
3a: Optimisation of commonly used histological stains for resin embedded
specimens.
The bone specimens embedded in MMA and Technovit resin were sectioned via
Exakt cutting and grinding system, referred as ground sectioning, producing sections
of around 27 μm. Despite the above-mentioned stains were traditionally used, the
staining protocols were not readily applicable to the ground sections mainly due to the
hydrophobicity of resin. Therefore, firstly I optimised the staining techniques for resin
ground sections. The optimised staining protocols are presented in detail in chapter 6
which other researchers can easily follow.
3b: Comparison of H&E, Goldner’s trichrome staining, Von Kossa staining
and IHC staining techniques for the assessment of mineralised tissues originated
from sheep, pig, mouse and rat processed via paraffin and resin embedding and
sectioning routes
The ground sections of resin embedded specimens (MMA and Technovit) and
microtome sectioned paraffin embedded specimens were stained with H&E, Goldner’s
trichrome staining, Von Kossa staining and IHC stains, and compared accross all 4
species of sheep, pig, mouse and rat. Overall resin ground sectioning provided the
highest quality outcome in both complex (mouse paw) and large sections (sheep tibia).
Two disadvantages of ground sectioning are the relatively large specimen consumption
per section and long processing time. Paraffin sections showed good bone morphology
but poor hard/soft tissue interface preservation and no bone mineral information.
Goldner’s Trichrome staining on ground resin sections provides the best
differentiation between hard and soft tissue with clear cellular details. IHC worked
with paraffin microtome sections and ground sections of Technovit embedded
14 Chapter 2: Research Hypothesis and Aims
specimens. These results can guide researchers to preplan their histology for optimum
results.
Chapter 3: Literature review 15
Chapter 3: Literature review
3.1 BONE BIOLOGY
3.1.1 Structure of bone
Bone is a highly specialized organic/inorganic composite fabricated by nature.
As a complex connective tissue, bone consists of three components: the cellular
components, the hydrated extracellular organic matrix (mainly collagen,
approximately 35% dry weight), and the extracellular mineral phase (mainly
hydroxyapatite, 65% dry weight) [6,18,19]. The major component of the bone organic
extracellular matrix (ECM) is type I collagen, this accounts for approximately 90% of
the total volume of the ECM, with the rest formed from other minor collagens such as
type III and type V collagen and a variety of non-collagenous proteins, glycoproteins
and proteoglycans in relatively small quantities [6,20]. The molecular structure of
collagen is a stabilized left-hand superhelix fibril, and bundles of fibrils comprise
nano-size collagen fibres that provide bone with great tensile strength (a Young’s
modulus of 1-2 GPa and ultimate tensile strength of 50-1000 MPa) [6,18]. The
inorganic phase of bones’ ECM further enhances the stiffness and compressive
strength of the bone. The principle component of this organic phase is a calcium
phosphate based crystalline structure called hydroxyapatite (HA) with the chemical
formula of [Ca10(PO4)6(OH)2] (a Young’s modulus of 130 GPa and an ultimate tensile
strength of 100 MPa) [6,18]. The bonding between the organic matrix and inorganic
hydroxyapatite (HA) crystals makes bone a natural bio-composite material with
excellent mechanical properties. Weiner & Wagner provided an excellent overview of
bone linking its macro-structure and micro-structure by breaking down the bone
structure to seven levels of hierarchy (Figure 3.1) [18]. Nanoscopically, HA platelets
orient and align within self-assembled collagen fibrils, making the first level of bone
hierarchical structure. The mineralised collagen fibrils are the basic building block of
bone. They stack parallel as lamellae which are then arranged concentrically around a
central Haversian canal to form the basic unit of bone, Osteon. Finally, the macro-
structure of bone is formed by osteons: either densely pack into compact bone, or form
a trabecular network into cancellous bone [18,20].
16 Chapter 3: Literature review
Figure 3.1 The seven hierarchy levels of bone structure, as demonstrated by Weiner and Wagner [18]. Level 1 shows the two major components of bone: mineral particles (left) and collagen fibril (right). Level 2 shows a mineralized single collagen fibril which combines the two phases. These mineralized fibrils organize into arrays as shown in level 3 and level 4 and
Chapter 3: Literature review 17
eventually forms the basic unit of bone – osteon (level 5). Level 6 shows the two types of bones: the compact cortical bone and the porous spongy bone. Level 7 is an overview of a whole long bone.
Generally, bones are classified by their shape into several distinct types: long,
short, flat, irregular and sesamoid [21]. The typical structure of a long bone is shown
in Figure 3.2. When looking into their macroscopic structure, bones are not uniform,
but are composed of a hard outer layer, compact bone (or called cortical bone) with an
average density of 0.2 g/cm3 and an interior more porous part, trabecular bone (also
called cancellous or spongy bone) with an average density of 1.80 g/cm3 [20,22]. As
shown in Figure 3.2, both the inner and outer surfaces of the bone are covered by thin
vascular membrane-like layers: the outer membrane is named periosteum and the inner
membrane is called endosteum [23]. Both the periosteum and endosteum play essential
roles in bone growth and repair by providing a continuous supply of osteoprogenitor
cells or new osteoblasts [21].
Figure 3.2 Schematic illustration of long bone and its micro-structure [23]
The Haversian canal is essential for bone function as it contains nerve and blood
vessels. The blood vessels are responsible for nutrient supply and waste disposal for
the cells residing in the bone matrix [6,23]. Four types of cells populate the mineralised
bone matrix: osteoblasts, osteocytes, osteoclasts and bone lining cells [23]. Derived
from local osteoprogenitor cells, fully differentiated osteoblasts are responsible for
bone matrix regulation and membrane-associated alkaline phosphatase (ALP)
synthesis. Osteoblasts are considered differentiated into osteocytes once they are
surrounded by mineralized matrix or lacunae [6]. Due to the pivotal role of osteoblasts
18 Chapter 3: Literature review
in bone formation, the clinical success of implanted polymers in bone defects largely
depends on the attachment of osteoblasts to the material surface. Any biomaterial
which is to be used for the repair of a bone defect must be conductive to osteoblast cell
attachment, proliferation and differentiation in order for bone regeneration to occur.
Osteoclasts, on the contrary, are responsible for mineralized bone matrix resorption by
secreting acids and enzymes to degrade both organic and inorganic constituents of the
bone [23]. The synergistic actions of osteoblasts and osteoclasts are the basis of a
dynamic bone remodelling process whereby bone is constantly remodelled in order to
maintain its unique architecture throughout the life [6].
3.1.2 Bone fracture healing
Unlike the fibrous scar of the soft-tissue healing, bone has a remarkable self-
healing capacity characterised by its unique scarless repair. The repair process includes
four overlapping stages as shown in Figure 3.3 [24]. Briefly, the hematoma formation
stage (Figure 3.3a) is the first post-fracture stage which keeps the fracture area in an
enclosed environment. The hematoma is formed around the broken bone ends by blood
from adjacent structures due to disrupted blood vessels. Angiogenesis (formation of
new blood vessels) takes place when several types of cells are recruited to the defect
site to eventually form fibrocartilage at this stage. Simultaneously the periosteum
enables direct bone formation and an external callus is created as a result (Figure 3.3b);
Subsequently, a hard bony callus is formed from within through the mineralisation of
the internal callus (Figure 3.3c); Finally, the remodelling activity of the bone replaces
the hard callus with lamellar bone and also reduces the callus size at the fracture site
[24]. After these repair stages, the original biochemical and biomechanical properties
of the bone are fully restored as well as the vascular supply. It is reported that the
regenerated bone shows no difference to undamaged host bone tissue [25].
Chapter 3: Literature review 19
Figure 3.3 The four stages of bone fracture regeneration [24]
3.2 CURRENT TREATMENTS FOR LARGE BONE DEFECTS
Despite the exceptional regenerative capacity of bone, the healing of large
segmental defects that exceeds the body’s self-healing capacity (e.g. a gap over 2.5
times the bone radius in long bone defects [26]), such as those caused by congenital
disorder, traumatic injury and tumor removal, does not take place without surgical
intervention [27]. Bone reconstruction still remains a challenge in these conditions
[27]. This situation is referred to as a critical-size defect [23,27]. In critical-size bone
defects, it is necessary to implant grafting materials in order to bridge the gap. To
achieve positive regenerative results, the implanted material should ideally possess the
following properties: an osteoconductive structure, osteoinductivity, biocompatibility
and biodegradability. Favourably, the implant should provide structural support and
stability [9]. In the clinical practice, the treatments for large bone loss rely on bone
grafting, including autogenic (bone graft from patients own body), allogenic (bone
graft from a donor body) and xenogenic (bone graft from a different species to human)
bone grafting, to restore bone loss due to trauma or above mentioned conditions [9,27].
Every year, around 4 million bone grafting or bone substitutes surgeries are performed
worldwide, which again highlights the demand in the surgical demand as well as the
impact of critical-size bone defects [27].
3.2.1 Bone grafting
Among the bone grafting techniques, autografting, which is to graft bone tissue
from one site to another within an individual, is considered the ‘gold standard’ for
critical-size bone defect reconstruction [27,28]. Autografts are ideal for bone
reconstruction as they are not only osteoconductive but also osteoinductive [28]. It is
20 Chapter 3: Literature review
reported that the use of autografting has been successful in treating large bone defects
with no risk of disease transmission from a donor tissue (whereas allografting may
pose disease transmission problems) [27]. However, the disadvantages of autografting
are also prominent. Firstly, because the amount of donor bone is limited, it is often
difficult or sometimes impossible to harvest enough bone to graft in the situations of
large or multiple bone defects. Secondly, the harvesting procedure normally leads to
donor site morbidity and also a great pain for the patient. Thirdly, complications often
exist after the surgery such as infection, hematoma and in some cases pelvic instability
[28]. To overcome these difficulties, allograft (to transplant bone tissue from cadavers
or living donors) and xenografts (to transplant bone tissue from other species) have
been introduced to the clinical practice as alternatives to autograft. Allograft bone is
osteoconductive, it supports bone formation, and most importantly, the supply is much
less limited compared to autografting. Although it is possible to achieve large bone
defect structural restorations, the application in the clinical practice is limited due to
its poor osteogenic and osteoinductive capacity, large immunogenic response, limited
revascularization and the potential for disease transmission [29]. Additionally, results
revealed that newly formed bone with allograft procedure is not likely to incorporate
into the host [30]. Xenogenic graft is rarely used because of the high failure rate [29].
3.3 BONE TISSUE ENGINEERING
Since bone grafting is not able to consistently provide satisfactory results,
researchers have endeavoured to seek more effective alternatives. One avenue of
research has led to the emergence of the field of Tissue Engineering (TE). The term of
‘TE’ was first created in the mid-1980s but was not well established until late 1980’s
by a prominent research group in the US, who gave the first definition of tissue
engineering [31]. The definition of ‘TE’ developed with the evolution of the research
field, and was described by Robert Langer and Joseph P. Vacanti in their review paper
published in Science on May 14, 1993 [32]. According to their definition, tissue
engineering is ‘an interdisciplinary field that applies the principles of engineering and
the life sciences toward the development of biological substitutes that restore,
maintain, or improve tissue function’ [32]. Over the years, the research field of TE has
grown tremendously, especially after the development of three-dimensional polymeric
scaffolds for tissue or organ regeneration [33]. Not only does the field of TE hold great
promise for the future of medicine, but it has become a billion dollar industry and has
Chapter 3: Literature review 21
created almost 14,000 employment opportunities worldwide [34]. Additionally, the TE
industry shows an increase in growth lately as more and more TE products are being
trialled and applied to clinical practice, with a number of them already on the market
(e.g. INFUSE® Bone Graft (Medtronic) which is a bone graft material used in
treatments of certain types of spinal surgery, acute tibia fracture and oral maxillofacial
defects).
3.3.1 3D printing scaffolds
Since the first technique of producing solid three-dimensional (3D) structure was
introduced in the 1980s [13], a series of 3D printing techniques were introduced and
these techniques have been applied in a diversity of fields including TE [35,36].
Typical 3D printing, e.g. fused deposition modelling (FDM) [37], stereolithography
(SLA) [38], selective laser sintering (SLS) [39], selective laser melting (SLM), direct
metal deposition (DMD) and inkjet printing, can produce many designs of complex
geometries in a layer-by-layer deposition based on specific 3D models [35]. 3D
printing scaffolds have the advantages of accurately and precisely controlled pore size
and geometry over scaffolds manufactured with traditional techniques [37,38,40,41].
The features of 3D printing make them potential custom-made solutions for patients
by providing scaffolds that precisely match the defects using medical scanning data
injury site.
3.3.2 Tissue engineering scaffolds
As stated in the introduction, millions of people are affected by bone defect-
related diseases and there is a pressing need for better bone substitutes worldwide [42].
The bone TE field seeks to address the need by introducing engineered implants which
will eventually become integrated within the patients, thus providing potentially
permanent cures. Tissue engineered constructs, often termed as ‘scaffolds’, are 3D
structures that serve as temporary templates to guide cell attachment, proliferation,
differentiation and extracellular matrix (ECM) formation at defect sites. A typical
workflow for the proposed scaffold-based approaches to bone tissue engineering
involves 5 basic steps: i) 3D data acquisition through medical imaging techniques at
the time of diagnosis, such as computed tomography (CT) and magnetic resonance
imaging (MRI); ii) 3D model generation of the defect anatomy on computer by
converting the medical scan data with software such as computer-aided design (CAD);
iii) the 3D image of the final defect anatomy is then translated into machine language
22 Chapter 3: Literature review
to manufacture a biodegradable scaffold, usually made from a resorbable polymer; iv)
then seed the scaffold with cells and/or possibly supplement with bioactive molecules
(e.g., Bone morphogenetic proteins (BMPs)); v) finally the scaffold is implanted into
the defect site to induce and direct the growth of new bone [12,23]. The whole process
is illustrated in figure 3.4. Ideally, the cells will attach onto the scaffold, proliferate,
differentiate and produce bone matrix as the scaffold degrades, eventually, heal the
bone defect.
Figure 3.4 Schematic overview of the scaffold-based approach to bone TE [12]
3.3.3 TE scaffolds specifications
Fundamentally, bone TE promotes the body’s natural regenerative capacity in
defect repair with engineering principles, which is termed bone biomimetics [23]. For
this purpose, ideal synthetic bone scaffolds must address the following biomimetic
requirements. i) provide temporary mechanical support to the defect site and act as
substrate for bone deposition; ii) contain interconnective pores within the architecture
to allow for rapid vascularisation and bone growth and to prevent unwanted tissue
growth, mainly soft tissues, into to the wound bed; iii) be osteoconductive and
Chapter 3: Literature review 23
osteoinductive; iv) promote integration between host bone and regenerated bone; v)
be bioresorbable without toxic degradation products and degrade in a controlled
manner; vi) be capable of retaining bioactivity after sterilization and free of
inflammatory response; vii) potentially have controlled drug delivery capacity
[23,37,43]. To meet these requirements, it is critical for tissue engineers to select the
right material and proper processing technique for scaffold fabrication.
3.4 SCAFFOLD MATERIALS
The composition of scaffold materials plays a critical role in the overall success
of bone TE treatment because it is essential to mimic the biochemical and biophysical
properties of the native tissue. The ideal material must be non-toxic, biocompatible,
biodegradable, and must possess appropriate mechanical properties for load bearing
applications. Additionally, they should ideally be osteoinductive and osteoconductive
[23,43]. The development of synthetic bone scaffold materials has increased over the
years with a variety of candidates investigated, including metals, ceramics, polymers
and their composites. Due to their superior mechanical and osteogenic properties over
other materials, a paradigm shift has taken place towards the use of polymers,
bioceramics and their composites as the principle materials for bone scaffold
manufacturing [44].
3.4.1 Polymers
To date, both natural polymers and synthetic polymers have been investigated as
bone scaffold candidates. Although both types of polymers have been investigated for
TE, synthetic polymers are being more widely used because of their tunable
degradability, higher purity levels and better mechanical properties than natural
polymers [23]. Common synthetic polymers for bone repair include polyesters,
polydioxanone, poly (ethylene glycol) (PEG), poly (propylene fumarate) (PPF), poly
(orthoesters), polyanhydrides, and polyurethanes [23]. The properties of some
example polymers are summarized in Table 3.1.
24 Chapter 3: Literature review
Table 3.1 Properties of biodegradable polymers as bone scaffold materials [35,36] Polymer Thermal and Mechanical Properties Approx.
Degrade time (months)
Areas of application Melting
point (°C)
Glass Transition (°C)
Approx. Strength
Poly (lactide) 173-178 60-65 2.7GPa (Modulus)
6 to12 Orthopaedic Surgery Oral and Maxillofacial surgery
Poly (glycolide) 225-230 35-40 7.0GPa (Modulus)
>24 Orthopaedic surgery, General surgery, Sutures
Polydioxanone 58-63 -65-60 >24 Orthopaedic surgery General surgery Sutures
Poly (D,L-lactide-co-glycolide) Poly (D,L-lactide-co-glycolide) 85/15 Poly (D,L-lactide-co-glycolide) 82/18 Poly (D,L-lactide-co-glycolide) 1090
Amorphous 50-55 2.0GPa (Modulus)
5 to 6 Suture Drug Delivery Oral and Maxillofacial surgery, General surgery, Sutures, Drug delivery, Periodontal surgery
Polycaprolactone 58-63 -65-60 0.4 GPa (Modulus)
>24 Drug delivery, Sutures
Polycaprolactone (PCL) is a semi-crystalline polyester that belongs to the
family of aliphatic polyesters [45]. As shown in Table 3.1, PCL has a glass transition
and melting temperature of approximately -60 °C and 60 °C respectively [23,45]. It
stands out from other biodegradable polymers because of its exceptional biochemical
and physio-chemical properties [45]. From the Bone TE perspective, PCL has the
following advantages: (i) a low melting point meaning PCL has superior rheological
and viscoelastic properties which make the polymer easy to shape and manufacture
into a variety of scaffold shapes; (ii) PCL is biocompatible and non-toxic and has been
approved in a number of biomedical devices by the US Food and Drug Administration
(FDA); (iii) not only is PCL biodegradable, but it is also bioresorbable which means it
Chapter 3: Literature review 25
may resorb over time and be excreted through a citric acid cycle, resulting in total
elimination of PCL with no residual side effects (process shown in Figure 3.5) and (iv)
good solubility and blend-compatibility with other polymers such as cellulose
propionate, cellulose acetate butyrate, polylactic acid and polylactic acid-co-glycolic
acid [45].
Although PCL has the potential to be a strong candidate for a bone TE solution,
its application is limited mainly because of its hydrophobicity and lack of bioactivity
[33,46]. However, PCL scaffolds can be made more hydrophilic with sodium
hydroxide (NaOH) etching [47]. Like a typical polymer, PCL is considered ‘soft’ for
high load bearing applications because of its deficiency in the compressive modulus
[23]. Additionally, the long-term degradation (up to 3-4 years for homo-PCL to
completely resorb) and further prevent PCL from widespread applications in bone TE
[48]. Lastly, PCL is osteoconductive but not considered osteoinductive. In order to
overcome these shortcomings of PCL, we are seeking materials that can be
incorporated into the polymer bulk to render them more suitable. One approach is to
incorporate a high modulus micro and/or nano-scale bioactive material (e.g. bioactive
glass) to introduce bioactivity to PCL bulk and enhance its mechanical properties.
Figure 3.5 The degradation and elimination pathway of PCL. PCL formed hydrolytic intermediates 6-hydroxylcaproic acid and acetyl coenzyme A, which is eliminated from the body via the citric acid cycle [45]
3.4.2 Bioactive glass (BG)
BG is a subgroup of inorganic biomaterials based on silicate or phosphate
systems, which exhibit unique osteoinductive properties and show good bonding to
both bone and soft tissues in biological fluid [16,49]. Since Hench and his colleagues
developed the first bioactive glass in 1969, known as Bioglass® or 45S5 [48,50,51], a
26 Chapter 3: Literature review
variety of different bioactive glasses (BGs) have been developed and applied in bone
TE [16,52]. Bone bonding and ionic dissolution are the two mechanisms that account
for the bioactivity of BGs. The BGs bond strongly to bone tissue through a precipitated
layer of hydroxycarbonate apatite (HCA), which interacts with collagen matrix of bone
[53].
Mechanism of HCA layer formation
The formation of HCA is well understood, and Larry L. Hench revealed this
process of HCA precipitation on 45S5 BG (with a composition of 46.13 SiO2–2.60
P2O5–24.35 Na2O–26.91 CaO (mol%)) in 5 main stages shown in Table 3.2 [16,51].
Table 3.2 Reaction stages of HCA layer formation on 45S5 BG [54] Stages Reaction
1 Rapid ion exchange of Na+ or Ca2+ in BG with H+ from solution: Si – O – Na+ + H+ + OH- Si – OH+ + Na+ + OH-
2 Stage 1 leads to loss of silica in the form of Si(OH)4 and increase of pH and breaking of Si-O-Si bonds and form Si-OH (silanol bonds): Si-O-Si + H2O Si-OH + OH-Si
3 Si-OH groups condensate and repolymerise to SiO2-rich layer:
4 Migration of Ca2+ and PO43- groups to the surface through the SiO2-rich layer leads
to the formation of an amorphous CaO-P2O5 layer, followed by the growth of CaO-P2O5-rich film by incorporation of soluble calcium and phosphates from solution.
5 Crystallization of the amorphous CaO-P2O5 film by incorporation of OH-, CO32-,
or F- anions from solution to form a mixed hydroxyl, carbonate, fluorapatite layer.
Ionic dissolution
Once the HCA layer has formed, it absorbs proteins and then enhances cell
attachment, differentiation and bone matrix production [16]. Ionic dissolution products
through HCA are believed to be a key element in the osteogenesis of BGs, however,
the detailed mechanism of how BGs enhance osteogenic responses is still largely
unknown. Figure 3.6 shows the typical ions released from BGs and the biological
responses to these ions [49].
Chapter 3: Literature review 27
Figure 3.6 The effect of bioactive glass ion dissolution products on biological responses [49]
Strontium-substituted bioactive glass
Strontium ions have been found to be beneficial to patients suffering from
osteoporosis. Sr-doped products are already available on the market, for example, oral
administration of Sr-ranelate, under the trade name Protelos™, has been widely used
in osteoporotic patients and received satisfactory clinical outcomes [55,56]. In 2010,
Professor Molly Stevens’s research group developed a class of strontium-substituted
bioactive glasses (SrBG) by replacing 0-100% of the calcium components with
strontium in traditional 45S5 BG [57]. The resulting SrBGs showed enhanced
bioactivity compared to 45S5 BG [57,58]. As reported, a critical concentration of Sr
ions in the dissolution medium plays a significant role in enhancing bone cell activity
[49,57–59]. In their studies, SrBG not only promoted osteoblasts proliferation and
alkaline phosphatase (ALP) activity when directly applied to human osteosarcoma cell
line, Saos-2, but also inhibited osteoclasts differentiation and decreased tartrate
resistant acid phosphatase (TRAP) activity. This implicates that the SrBG not only
enhance new bone formation but also suppress bone resorption by osteoclasts, leading
to increased turnover of new bone regeneration. In a recent in vitro study using
mesenchymal stromal cells, SrBG was found to upregulate osteogenic genes starting
with BMP-2 which then led to a cascade of gene up-regulation including Runt-related
28 Chapter 3: Literature review
transcription factor 2 (Runx2), alkaline phosphatase (ALP), collagen type I (ColI) and
osteocalcin [60]. The molecular mechanisms regarding how the incorporation of Sr2+
promoted osteogenic differentiation were proposed as the activation of two cell
signalling pathways: the Ras_MAPK and Wnt-Catenin [60–62]. The advantages of
SrBG have made them potential candidates for a wide range of applications in
orthopaedic regenerative medicine. However, the innate brittle nature of BG makes it
very difficult to process it into useful 3D scaffold with adequate mechanical strength
and porosity [48], thus the application of BGs alone is limited in bone TE.
When looking at both PCL and SrBG, we can see that they have complementary
properties. PCL can be fabricated into 3D scaffolds with desired structure and adequate
mechanical strength, but it lacks bioactivity; while SrBG has great osteogenic capacity
but brittle in nature. Therefore, by incorporating SrBG micro/nano- particles into
PCL we believe that we can generate a superior composite material in which the
inorganic phase will act to improve the mechanical strength and the biological
properties of polymeric fibers, such as cell compatibility, osteogenic differentiation,
and bone matrix calcification [63]. Such composite materials will be detailed in the
following section in the context of bone regeneration.
3.4.3 Biodegradable polymer/bioactive glass composites
Facing a complex system like human bone, conventional single-component materials
are not capable of satisfying all the criteria for a bone replacement/regeneration
scaffold. To fulfil as many requirements as possible, composite systems of
biodegradable polymers and ceramics are being developed, and hold promise as a bone
TE scaffolds. In these composite systems, micro- or nano-scale ceramic particles are
incorporated into biodegradable polymers to take advantage of their processibility
[16]. The addition of an inorganic phase to bioresorbable polymers not only
counteracts their poor bioactivity, but also accelerates the polymer degradation rate by
increasing the hydrophilicity and water absorption of the normally hydrophobic
polymer matrix [16,50]. Furthermore, in addition to improving mechanical properties,
the cations released from BG filler phase buffers the local pH which may have been
affected by the acidic polymer degradation products [16]. Coating BG particles onto
polymer scaffold surface [64] or coating polymer onto BG scaffold surfaces [65] were
alternative methods to produce composite scaffolds. Because of the associated
disadvantages of the coating techniques such as clogging of pores with the scaffolds
Chapter 3: Literature review 29
and weak bonding between the organic and inorganic phases [50,66], this review
would not introduce these techniques extensively. To our knowledge, the number of
studies on polymer/bioactive glass composites as bone scaffolds is relatively limited.
In vitro studies carried out by Boccaccini et al. in 2003 confirmed that by filling their
poly(DL-lactic acid) foam scaffolds with BGs the osteogenic activity and
mineralization of human osteosarcoma cells (MG-63) were greatly promoted [67,68].
This result was in accordance with earlier studies conducted by Lu et al. [68], in which
porous composite scaffolds of polylactide-co-glycolide (PLAGA) and 45S5 bioactive
glass showed higher levels of Type I collagen synthesis and mineralization of human
osteosarcoma cells (SaOs-2). While most of the scaffolds used in these studies were
manufactured with traditional TE techniques (summaries in Table 3.3), the use of
additive manufacturing methods to produce 3D scaffolds has recently gained
momentum in the TE field.
Nano-sized BG particles or nanofibres have been reported to exhibit advantages over
their micro-scale counterparts. Compared with micro-sized particles of the same total
volume, BG nanoparticles have much larger surface area relative to their volume
which could contribute to improved bioactivity compared with micro-sized particles.
This was confirmed by S.K. Misra et al. [69] who investigated the in vitro activity,
protein adsorption capability and mechanical properties of both micro- and nano-sized
BG/poly (3hydroxybutyrate) composites (10 – 30 wt%). The results of these studies
indicated that the incorporation of nano-sized BGs not only reinforced the mechanical
stability of the composites, but also induced nanotopography on the scaffold surface
hence improved total protein adsorption and bioactivity of the composite [69–73].
Fabbri et al. reported that the mechanical properties of PCL/45S5 BG scaffolds (with
0 - 50 wt%), fabricated by a solid-liquid phase separation method, were enhanced with
higher amount of the BG loading in the scaffolds [74]. Further in vivo data were
obtained by Jo et al., which revealed good bone-forming ability of PCL/nano-BG
composite in a rat calvarial bone defect model [75]. Rowe et al. produced PCL/borate
bioactive glass membranes with up to 10 wt% BG loading using solution-
electrospinning technique [76]. These composite electrospun membranes were found
to support osteoblast attachment and proliferation.
30 Chapter 3: Literature review
PCL/SrBG composite scaffolds
Because of the favourable properties of PCL and SrBG as bone implant materials, the
PCL/SrBG composite material has been made into scaffolds and explored for their
bioactivity by other researchers [14,77]. The composite material of PCL/45S5 BG and
PCL/SrBG were produced into 3D scaffolds with BG loading of 10% [14] and 50%
[77] using the melt extrusion based additive manufacturing techniques, resulting in
scaffolds with fibre diameters of ~ 500 μm (estimated based on the SEM images). The
PCL/SrBG scaffolds with 10% SrBG loading showed in vitro bioactivity and had
higher compressive strength compared to PCL scaffolds. However, in vitro testing of
these scaffolds showed the PCL/SrBG (10%) scaffolds were not able to stimulate
osteoblast differentiation in the absence of osteogenic cytokines (typically ascorbic
acid, β-glycerophosphate, and dexamethasone).
In order to further enhance the bioactivity of the composite scaffolds, Poh et al. then
increased the SrBG loading to produce PCL/SrBG scaffolds with 50wt% SrBG loading
[77]. In vitro testing was performed on these scaffolds and the PCL/SrBG scaffolds
were found to be able to stimulate osteoblast differentiation even in the absence of
osteogenic supplement in the cell culture media. The osteogenic properties of the
PCL/SrBG scaffolds were indicated by the up-regulation of osteopontin (OPN) and
osteonectin (OCN), two genes expressed at different stages of osteoblast
differentiation [77]. The results of these studies proved that PCL/SrBG composite
materials had potential to fabricate the next generation bone scaffolds.
3.5 SCAFFOLD FABRICATION TECHNIQUES
There are several techniques available for the fabrication of 3D composite
scaffolds which feature high porosity and interconnectivity. Currently, the main
fabrication methods used in bone tissue engineering include: additive manufacturing,
electrospinning, thermally-induced phase separation (TIPS), solvent casting/particle
leaching, solid free-form fabrication, microsphere sintering and surface coating of
scaffold [12,50,78]. A summary of advantages and disadvantages of these individual
techniques can be found in Table 3.3 below.
Chapter 3: Literature review 31
Table 3.3 Comparison of different bone scaffold fabrication techniques [50,66]
Fabrication route Advantages Disadvantages
Thermally induced phase separation (TIPS)
High porosity (~95%)
Highly interconnected pore
structures
Anisotropic and tubular pores
possible
Control of structure and pore size by varying preparation conditions
Long time to sublime solvent
(48 hours)
Shrinkage issues
Small scale production
Use of organic solvents
Solvent casting/particle leaching
Controlled porosity
Controlled interconnectivity (if particles are sintered)
Structures generally
isotropic
Use of organic solvents electrospinning Process versatility and large surface
area to volume ratio
fully interconnected porous structure
Good mechanical properties
Small pore size can be
detrimental for the desired
cell infiltration into the inner
regions [79]
Limit in scaffold thickness
Solid free-form fabrication
Porous structure can be tailored to
host tissue
Protein and cell encapsulation
possible
Good interface with medical imaging
Resolution needs to be
improved to the micro-scale
Some methods use organic solvents
Rapid Prototyping fully interconnected channel network, controlled porosity and channel which mimic the microstructure of living tissue
Relatively expensive and complicated set-up
Microsphere sintering
Graded porosity structures possible
Controlled porosity
Can be fabricated into complex shapes
Interconnectivity is an issue
Use of organic solvents
Scaffold surface coating
Quick and easy Clogging of pores, sometimes organic solvents used, coating adhesion substrate can be too weak
Of these techniques, electrospinning has gained substantial attention with the
bone tissue engineering community in recent years; due to the relatively simple and
low-cost fabrication process, combined with the significant potential to construct bone
32 Chapter 3: Literature review
scaffolds that mimic native bone matrix [80,81]. To our knowledge, there has been
limited application of electrospun PCL/SrBG composite scaffolds. More details of
electrospinning are introduced in the following sections.
3.5.1 History of electrospinning
Similar to mechanically forced extrusion spinning in textile industries,
electrospinning employs electrostatic forces to produce polymer fibres which range in
diameter from tens of microns down to tens of nanometers [82,83]. First introduced
by Zeleny in 1914, this technique was further developed by Anton Formhals who
repeatedly patented electrospinning for the production of continuous fine fibres for the
textile industry between 1934 and 1944 [82,84]. Electrospinning was not used for
scaffold fabrication in tissue-engineering applications until 1995, when Doshi and
Reneker discovered the potential of using electrospun fibres for tissue engineering
[84,85]. Since then, there has been a strong growth in this area due to the simplicity
and uniqueness of electrospinning. This technique has the ability to produce 3D nano-
fibre scaffolds with large area-volume ratio and interconnected pores with spatial
orientation, which promote cell adherence, proliferation and differentiation [82].
3.5.2 Basic electrospinning equipment
As shown in Figure 3.8, a typical electrospinning setup consists of three major
components: a spinneret, a collector and a high-voltage power source [80,82].
Additionally, a syringe is directly connected to the spinneret as a reservoir for polymer
solution (or melt). To ensure a steady and controllable feed rate, a syringe pump is
employed to serve this purpose.
Chapter 3: Literature review 33
Figure 3.7 Schematic illustration of typical electrospinning setup (not to scale) [82]
3.5.3 Theory of electrospinning
In the electrospinning process, a high voltage/low current is applied to the
spinneret to create an electrically charged jet of polymer solution or melt. As the
electric field accumulates at the tip of the needle, a pendant droplet of polymer solution
at the spinneret becomes highly electrified which subsequently leads to the elongation
of the hemispherical surface of the droplet to form a shape known as the Taylor cone
[80,82]. The Taylor cone is the result of droplet surface tension and electrostatic force.
Further increasing the electric field will reach a critical value when the repulsive
electrostatic force exceeds the surface tension of the droplet and a fine jet of charged
polymer solution is ejected from the tip of the Taylor cone [80,82,86]. Subjected to
elongation and instabilities, this jet then undergoes a stretching and whipping process,
accompanied by rapid solvent evaporation from the polymer solution, leaving only
charged ultra-fine fibres. Polymer fibres tend to move away from each other due to the
mutual electrostatic repulsion resulting in limited layers of scaffold and a random coil
of nanofibres [80,82,84,86]. In order to obtain desired fibre alignments, various
collector set-ups have been developed over the years, including rotating drum
collectors. A variety of electrospinning configurations were summarised by Sahay et
al. as shown in Figure 3.9 [87].
34 Chapter 3: Literature review
Figure 3.8 A schematic summary of electrospinning configurations for desired fibre alignments [87]
3.5.4 Process of electrospinning
It is known that the electrospinning process is influenced by polymer solution
properties, process parameters and ambient conditions. From our standpoint, we can
manipulate the variables of this process to fabricate the desired scaffolds. Of these
parameters, the most controllable variables include the flow rate, electric field strength,
distance between spinneret and collector, needle tip design, and collector composition
and geometry [80,86,88]. The influence of individual parameters on the fibre
morphology is summarized in Table 3.4 below.
Table 3.4 Important parameters for electrospinning process [80] Process parameter Effect on fibre morphology
Viscosity/concentration • Low concentration/viscosities yielded defects in the form of beads and junctions; increasing concentration/viscosity reduced the defects
• Fiber diameters increased with increasing concentration/viscosity
Conductivity/solution charge density
• Increasing the conductivity aided in the production of uniform bead-free fibers
Chapter 3: Literature review 35
• Higher conductivities yielded smaller fibers in general (exceptions were PAA and polyamide-6)
Surface tension • No conclusive link established between surface tension and fiber morphology
Polymer molecular weight • Increasing molecular weight reduced the number of beads and droplets
Dipole moment and dielectric constant
• Successful spinning occurred in solvents with a high dielectric constant
Flow rate • Lower flow rates yielded fibers with smaller diameters
• High flow rates produced fibers that were not dry upon reaching the collector
Distance between tip and collector
• A minimum distance was required to obtain dried fibers
• At distances either too close or too far, beading was observed
Needle tip design • Using a coaxial, 2-caplillary spinneret, hollow fibers were produced
• Multiple needle tips were employed to increase throughput
Collector composition and geometry
• Smoother fibers resulted from metal collectors; more porous fiber structure was obtained using porous collectors
• Aligned fibers were obtained using a conductive frame, rotating drum, or a wheel-like bobbin collector
• Yarns and braided fibers were also obtained
Ambient parameters • Increased temperature caused a decrease in solution viscosity, resulting in smaller fibers
• Increasing humidity resulted in the appearance of circular pores on the fibres
Field strength/voltage • At too high voltage, beading was observed
• Correlation between voltage and fibre diameter was ambiguous
3.5.5 Melt-electrospinning
Generally, there are two ways to prepare liquid polymer: by dissolving the
polymer in a solvent or by melting the polymer. The solution-electrospinning approach
is well established and has been popular for scaffold manufacturing in TE field
compared to melt-electrospinning [86]. However, melt-electrospinning has revealed to
be a potentially more suitable technique for bone scaffold fabrication for the following
reasons. First of all, melt-electrospinning is solvent free while solution-
36 Chapter 3: Literature review
electrospinning depends on using an organic solution to dissolve the polymer. In most
cases these solvents are toxic, and solution-electrospinning has the potential for trace
harmful solvents remaining in the scaffold which may adversely affect cells and tissues
[89]. Secondly, the melt-electrospinning process is more controllable than solution-
electrospinning attributable to the nature of the process, resulting in better aligned and
thicker fibres. In the solution-electrospinning process, a rapid whipping jet generates
ultrathin fibres within a relatively large collection area often with chaotic deposition
[86,90,91], the subsequent solvent vaporization further decreases the fibre diameter
[80]. According to important results obtained by Mikos et al., larger fibre diameter
facilitates increased pore size and interconnectivity, which in turn promotes cell
infiltration which is an important consideration when we are attempting to make large
tissue engineered scaffolds [80,92]. Thirdly, polymer melts have higher viscosity and
lower conductivity compared to polymer solutions, and there is thus not a need for a
minimum collection distance for melt-electrospinning, which means it is feasible to
collect polymer jets in the stability region, enabling fabrication of scaffolds of ordered
layout. While for solution-electrospinning, it is really necessary to maintain the
distance between the spinneret and collector to allow solvent removal by evaporation,
leading to random meshes of polymer jets collected in the instability region [90]. These
unique properties of melt-electrospinning have enabled the emergence of a new
biofabrication technique: melt-electrospinning writing (MEW) [93]. MEW uses
additive manufacturing principles that allow the layer-by-layer fibre deposition like
FDM to fabricate scaffolds with specific designs, shapes, porosities and sizes [93,94].
MEW is realised by moving the collector in x, y and z coordinates according to pre-
programmed patterns allowing the polymer jet to deposit into coherent 3D structures,
which makes it possible to produce scaffolds tailored to patients’ specific defect size.
3.5.6 Hybrid electrospinning system
Although rarely investigated, several studies have reported scaffold fabrication
processed that combine usage of solvents and elevated temperature, which is termed
as hybrid systems of melt- and solution-electrospinning [83]. Different to other
‘hybrid’ systems which are simply to combine the electrospun fibres produced from
individual solution- and melt-electrospinning [95], these hybrid systems defined by
Hutmacher and Dalton actually merges the two electrospinning processes to dissolve
the polymers in solvents at elevated temperature. These ‘melt’ assisted solution-
Chapter 3: Literature review 37
electrospinning systems have shown better solvent removal during the process via
accelerated evaporation and improved polymer solubility in the solvents, and also
possibility to take advantage of physical transformations due to thermal changes [96].
Some polymers, such as polyethylene (PE), are not considered amenable to
electrospinning as it readily crystallises from solution due to its low solubility. In the
study by Givens et al., the authors successfully produced polyethylene microfibers via
conventional solution-electrospinning (2-5 wt%) by maintaining the electrospinning
temperature at 105-110 °C with an IR emitter system [97]. Yoshioka et al. then adopted
the same hybrid solution-electrospinning system with improved temperature control
to produce PE electrospun fibres in nano scale [98]. The hybrid electrospinning system
shows the capacity to overcome some of the limitations of materials selection
experienced with individual solution-/melt-electrospinning. These systems could
potentially further advance the TE field by expanding the spectrum of materials for
scaffolds production, including polymer/bioceramic composite biomaterials
conventionally not successful.
3.6 TE SCAFFOLDS ASSESSMENT
As a result of our aging population and decreased physical activity levels, the
annual market for bone grafts is in excess of $2.5 billion and is expected to further
grow at 7-8% every year [99]. This demand presents great needs and potential growth
for TE strategies. However, before translating the TE scaffolds into clinical
applications, a series of bio-capability tests must be undertaken in vitro and in vivo
[100]. In vitro testing uses isolated cells for the characterisation of biological activity
effects of bone TE scaffolds and for screening acute toxicity and cytocompatibility. In
vitro assays are considered the first stage testing and are popular as they also give
information regarding how well cells attach, proliferate and differentiate on the
scaffolds [101]. The information obtained from in vitro studies serves as a valuable
guide for the following in vivo tests and also minimise animal usage. However, in vitro
cell culture does not reflect the complex in vivo environments of implanted scaffolds,
and specifically for a bone implant. It lacks control of physiological loading which has
proven to be essential for bone development [101]. In comparison, in vivo testing uses
animal models to evaluate scaffolds in a reproducible approximation of the mechanical
and physiological clinical situation [100,101]. For this purpose, a number of animal
models have been established, ranging from small animals such as a mouse to large
38 Chapter 3: Literature review
animals such as sheep [100]. The small animal models have shown fast bone turnover
rate, cheaper maintenance and are easier to standardise, but they do not closely mimic
human bone architecture [100]. On the other hand, the large animal models have
shown a closer resemblance to human bone architecture, physiology and
biomechanical properties [100]. Their disadvantages include expensive maintenance
cost, lower bone turnover and difficulties in standardisation. Given their limitations,
these animal models are actively used in the TE field where appropriate [102–105].
For the accurate interpretation of these in vivo test results, comprehensive post
explantation analysis techniques are employed, including micro-computed
tomography, histology, scanning electron microscopy and mechanical testing
[105,106]. Among these ex vivo tests, histology is a key assessment to characterise
bone formation and mineralisation mechanisms including tissue, cellular and
molecular pattern and distribution within the bone matrix. In addition, histology also
provides information on the nature of soft tissues and scaffold/implant interface with
mineralised bone [105]. Immunohistochemistry (IHC) is a vital histological technique
that can further characterise osteogenic/angiogenic markers presented on the
regenerated bone tissue [104,107]. IHC demonstrates specific markers on the surface
of tissue through antibody-antigen interaction either directly or indirectly [108].
Commonly the antibody is conjugated a fluorescent chemical or an enzyme (normally
a peroxidase) that can convert a substrate to a visible dye, the antibody distribution is
assumed to reflect antigen location [108].
3.6.1 Histology
Histology, which is essential for the understanding of disciplines including
biology, medicine and veterinary medicine, is the scientific study of the fine detail of
cells and tissues to study their structure and composition at the microscopic level [6].
It provides microscopic analysis of two-dimensional thin tissue slice (called a ‘section’)
carefully prepared using special subsequent procedures of tissue fixation, dehydration,
infiltration and embedding, followed by sectioning and staining [109]. Because
biological tissues/TE scaffolds must be supported in a hard matrix to allow sufficiently
thin sections to be cut, the tissue dehydration and infiltration process are necessary to
remove and then replace water within tissues with a medium that solidifies to allow
thin sectioning [109]. Histology is a highly versatile technique, numerous stains are
available for specific tissue types, and it is used in routine medical analysis and is now
Chapter 3: Literature review 39
widely employed in tissue engineering research for the ex vivo analysis [105]. Besides
conventional stains, immunohistochemistry (IHC) and enzyme activity assays, such as
tartrate-resistant acid phosphatase (TRAP) and alkaline phosphatase (ALP) staining
can detect specific antigens (e.g. proteins) and enzymes providing a high degree of
details of tissue composition unmatched by other techniques [6,110].
Paraffin processing is the most routinely used tissue preparation technique for
histology [109]. In this process, the water content of the specimen is removed through
a series of dehydration steps, and then the specimen is impregnated with paraffin to
create a more homogeneous cutting profile. The paraffin embedded specimens are
sectioned via a microtome, producing sections between 2 and 10 microns in thickness
[109]. Due to the ease of use, high throughput and efficiency, long storage life, and
high quality final results, the paraffin route has become the most commonly used
preparation technique. A broad variety of tissue types and species have near identical
paraffin processing parameters, making it an extremely robust and versatile technique.
However, the sample sizes are limited by the embedding mould to 3.5×2.2×1 cm in
length, width and depth respectively.
Calcified tissues, such as bone and tooth, are an exception and are not readily
suited to paraffin processing [111]. The dense mineralised component of calcified
tissues makes them too hard for the standard sectioning process, mainly because of the
mismatch of hardness and density between bone tissue and paraffin embedding media,
leading to the dissatisfactory quality of the produced section. To tackle this problem a
variety of embedding and sectioning techniques designed specifically for calcified
tissues have been developed and used for over three decades [109,110,112,113]. These
techniques can be summarised into two distinct approaches: 1) decalcification of the
mineral component of the tissue, followed by paraffin processing; 2) and resin
embedding for fully calcified samples [111,114].
Paraffin approach
In order to section bone tissue with a standard paraffin microtome, the calcium
salts within the tissue must be removed by a process called ‘decalcification’ before
paraffin embedding [113]. Decalcification is a necessary step to produce high quality
paraffin sections of bone specimens [6,109]. Routine decalcification methods use
chemical reagents ranging from slow ‘gentle’ metal ion chelating agents such as
ethylenediaminetetraacetic acid (EDTA) [109,113,115] to ‘harsh’ rapid acting acid
40 Chapter 3: Literature review
solutions such as hydrochloric acid [116,117]. The time taken to decalcify bone
specimens varies significantly dependent upon the size of the sample being decalcified,
the type of decalcification solution and temperature [113,118]. Acid solutions and
higher temperatures yield more rapid decalcification, but this can come at the cost of
reduced preservation of tissue/cell structure and poor enzyme and epitope preservation
or even complete digestion of bone, leading to inferior quality final results [113].
Decalcification of tissue in EDTA at 4oC has been shown to be effective in preserving
structure and epitope, however, it is a slow process which can take weeks to complete
dependent upon sample size and initial mineral content [119]. The decalcification
process enables the production of paraffin sections of bone and other calcified tissues,
however, it has two major disadvantages: potentially poor preservation of bone matrix
structure [110] and the inability to assess mineralisation of the bone tissue due to the
removal of the calcified phase [113]. As such, alternative approaches have been
developed which negate the need to decalcify the bone specimens at all: resin
embedding.
Resin approach
This technique is the ‘gold standard’ for undecalcified bone and metallic implant
analysis. The procedures of resin embedding have much in common with routine
paraffin approach, including i) fixation, ii) dehydration, iii) clearing, iv) infiltration, v)
resin embedding and vi) sectioning [113]. However, compared to paraffin section
preparation, resin approaches do not require decalcification as resin as an embedding
media can better match the hardness of bone specimens than paraffin, which allows
for a greater preservation of tissue morphology as well as an assessment of the mineral
content of the bone tissue. Many resins have been used, ranging from araldites [120]
to acrylics [121] with methyl methacrylate (MMA) based resins being most commonly
used [110]. MMA has a long history of use in hard tissue research spanning almost 50
years [8,110,122]. One great drawback of MMA embedding media is the high
polymerisation temperatures (~80 °C) as a result of the exothermic reaction [121]. The
heat generated during the embedding process initiates the destruction of epitopes and
enzymes of bone tissues, therefore, MMA embedding is considered to be unsuitable
for specimens which require IHC or enzyme analysis [121]. Over the last few decades,
low temperature polymerizing resin have been developed to overcome this limitation
[8,110]. Technovit 9100 New® resin, a low temperature curing MMA-based resin, has
Chapter 3: Literature review 41
become commercially available since 2000 [110]. Technovit 9100 New® has the
capacity to polymerise in an environment as low as -20 °C allowing the preservation
of epitopes, which makes it suitable for IHC assessments [8,110,121,123]
Resin embedded specimens can be sectioned by a microtome [104,106,122,124–
128] using a heavy duty blade or a purpose built resin microtome, producing sections
approximately 8 to 10 microns thick. The other routine section technique is ground
sectioning where specimen resin blocks are cut thicker (200 microns) with a saw blade
and then ground down 80 to 30 microns prior to staining [111,129–135]. The ground
sectioning technique for resin sections is more robust than resin microtome sectioning,
as it also allows for sectioning of a wider variety of calcified tissue and implants
including metallic implants in cortical bone in situ [136]. This technique is able to
provide a detailed analysis of tissue-implant interactions unattainable by other classical
histological approaches as they require the removal of the implant prior to embedding
and sectioning [8,135]. One major drawback of the ground sectioning technique is the
large consumption of specimen, taking approximately 1 mm of specimen per produced
section compared to several microns in microtome sectioning. It’s also highly technical
and time-consuming.
Histological stains
When focusing on histology of bone, most of the routine histological stains have
been developed for paraffin embedded samples. The most commonly utilised ones are
haematoxylin and eosin (H&E) [114] and IHC [109]. While H&E is an effective and
simple general stain, it has limited capacity to differentiate mineralised hard tissues
from surrounding tissues by colour, which is not ideal for tissue engineers to determine
bone regeneration. In order to improve this situation, stains such as von Kossa silver
nitrate methods and Goldner’s trichrome staining were developed and employed [8].
Goldner’s trichrome is an effective staining technique employed in numerous in vivo
bone regeneration studies, this stain provides comprehensive detail of calcified tissues
(typically in green colour), with strong contrasting colours [104,106,121,124–
135,137–139] as well as decalcified ones (red) and connective tissues (orange) [140].
In this thesis, I sought to compare and contrast various histological methods for the
purpose of bone TE applications.
42 Chapter 3: Literature review
3.7 CONCLUSION
Over the last two decades, enormous effort has been made in developing the
ideal bone substitute materials and a broad variety of materials have been investigated
by researchers around the world. To date, composite materials comprising
biodegradable polymers and bioactive glasses are promising candidates, as they meet
most of the requirements for bone regenerative scaffolds. PCL/SrBG composites will
be the primary material for this research project due to their excellent individual
properties which complement each other when combined. These composite scaffolds
are potentially bioresorbable and bioactive and capable of supporting osteoblasts
adhesion, spreading and viability.
To make this composite material into a viable bone scaffold, optimal
manufacturing techniques are vital. Electrospinning has been established as an easy-
to-operate and low-cost scaffold fabrication technique, and its popularity with the bone
tissue engineering community rocketed in the past decade, demonstrated by a large
number of publications. By controlling the key parameters of the electrospinning
process, we can fabricate bone scaffolds with desired size, shape and mechanical
strength to fit in the defect site. As described above, melt-electrospinning provides us
with larger and thicker solvent-free scaffolds with better mechanical properties
compared to scaffolds produced using solution-electrospinning. In the context of this
project, as the scaffolds may eventually be applied in low load-bearing conditions, it
is necessary for them to be structurally sound with adequate mechanical strength.
Therefore, melt-electrospinning is the preferable scaffold fabrication technique for this
purpose.
By combining the advantages of the composite PCL/SrBG with the melt-
electrospinning process, we are able to fabricate promising bone scaffolds that are
osteoconductive and osteoinductive, and with appropriate mechanical properties.
Bone TE research demands rigorous characterisation methods to examine the
progress of bone regeneration in the defect sites via in vivo studies, which are pivotal
to provide a complete understanding of the bone healing capacity of the materials and
scaffolds. However, in vivo studies are considerably resource consuming. Considering
that numbers of animals that can be used in these studies are also limited, the samples
obtained from these in vivo studies are extremely precious, and the data achieved from
such samples need to be rigorous and relevant. It still remains a challenge to obtain
Chapter 3: Literature review 43
maximal information of bone specimens [8], thus, it is imperative to continuously
optimise and improve the current histological techniques to meet the demand of
processing more complex and large bone/implant explants, and to ensure that bone TE
strategies have every opportunity to reach the clinic to treat patient suffering tissue
loss.
Chapter 4: (Study 1) Fabrication and In vitro investigation of PCL, 10 wt% PCL/SrBG electrospun scaffolds for bone regeneration 45
Chapter 4: (Study 1) Fabrication and In vitro investigation of PCL, 10 wt% PCL/SrBG electrospun scaffolds for bone regeneration
This study has been completed and published.
Jiongyu Ren, et al. Melt-electrospun polycaprolactone strontium-substituted
bioactive glass scaffolds for bone regeneration. J. Biomed. Mater. Res. - Part A 102,
3140–3153 (2014)
4.1 INTRODUCTION
Electrospinning is a versatile technique for the production of fibres ranging from
the micro to nano scale in diameter [82]. Many different polymers may be electrospun
via two different techniques: dissolving the polymer in an organic solvent (solution-
electrospinning) [141], and melting the polymer (melt-electrospinning) [142]. Melt-
electrospinning overcomes technical restrictions governed by solvent accumulation
and possible toxicity to cells [90]. In spite of the potential benefits of melt-
electrospinning, it has not yet gained widespread attention in the field of tissue
engineering (TE) when compared to solution-electrospinning.
Bone TE aims to combine aspects of conventional biology, engineering,
medicine, and chemistry to produce functional scaffolds for clinical treatments of bone
loss [143]. Current treatments in clinical practice rely on bone grafting, the gold
standard of which is autografting which involves taking bone tissue from a donor site
on the same patient. This technique has prominent issues related to limited supply,
donor site morbidity and surgical complications [28] hence it is important to develop
alternative solutions and move towards off-the-shelf solutions for bone loss.
Developing TE scaffolds to be used for the treatment of bone defects presents
challenges, including those associated with the need for a porous structure to allow for
good cellular infiltration and vascular ingrowth, while still maintaining the required
mechanical strength to support the tissue [144]. These scaffolds are commonly
constructed from either synthetic or natural biodegradable polymeric materials.
46 Chapter 4: (Study 1) Fabrication and In vitro investigation of PCL, 10 wt% PCL/SrBG electrospun scaffolds for bone regeneration
Polycaprolactone (PCL) belongs to the family of aliphatic polyesters and is
becoming an increasingly popular FDA-approved biomaterial [45]. PCL can be easily
fabricated into a scaffold of almost any size and shape by a variety of fabrication
techniques, owing to its low melting point (60 °C), and superior rheological and
viscoelastic properties compared to other synthetic polymers. These properties make
melt-electrospinning of PCL easily achievable; in contrast, more commonly used
polyesters, such as poly(L) lactic acid (PLLA), require temperatures in excess of 200
°C for successful melt-electrospinning [145]. Although PCL has many desirable
features, making it a promising candidate for a bone scaffold material, it is limited by
its hydrophobicity which is not conducive to cell attachment [33,46] and lack of
osteoconductivity [146]. In order to improve the suitability of PCL scaffolds for bone
TE applications, composite systems comprising the biodegradable polymer matrix
combined with inorganic components are emerging as promising candidates. Early
research into developing composite scaffolds has focused upon improving the
osteoinductive potential of the polymeric component by the incorporation of a
biocompatible inorganic phase such as hydroxyapatite (HA), ceramics and bioactive
glasses [50,72,147].
Bioactive glasses (BGs) are a group of inorganic bioactive materials that have
been shown to form a strong bond with hard and soft tissues; a result of carbonate-
substituted hydroxyapatite layer formation at the interface [51,148]. The first
commercially available BG was introduced in the 1970s, named 45S5, (commercial
name, Bioglass®), with a chemical composition of SiO2-P2O5-Na2O-CaO. Since then,
a large variety of BGs based upon derivations of the 45S5 composition have been
developed and applied in bone TE [48,149,150].
Gentleman et al. have developed strontium-substituted BG (SrBG) by
substituting 0-100% of the calcium (Ca) component of the 45S5 formulation with
strontium (Sr) [58]. It has already been established that Sr ions may significantly
enhance bone regeneration [49,57,59], and Sr-dosed drugs such as Sr-ranelate,
marketed under the trade name Protelos™, are available on the market for treating
osteoporosis [55,56]. In vitro results have shown that SrBG not only promotes
osteoblast proliferation and alkaline phosphatase (ALP) activity when directly applied
to the human osteosarcoma cell line, Saos-2, but also inhibits osteoclast differentiation
Chapter 4: (Study 1) Fabrication and In vitro investigation of PCL, 10 wt% PCL/SrBG electrospun scaffolds for bone regeneration 47
and decreases tartrate resistant acid phosphatase (TRAP) activity in the mouse
monocyte cell line, RAW264.7 [56,58].
To our knowledge, the production of composite scaffolds comprising PCL and
SrBG utilising the technique of melt-electrospinning has not been achieved to-date.
Here I report for the first time the ability to produce composite scaffolds suitable for
bone TE applications using melt-electrospinning approaches. I show PCL/SrBG
scaffolds with interconnective pores, structural robustness, and the potential to
promote osteogenesis. In this initial study, I firstly optimised techniques for melt-
electrospinning PCL fibres with SrBG particles homogeneously dispersed throughout
the fibres. I furthermore assessed the scaffolds’ ability to support cell attachment and
proliferation in addition to the osteogenic potential of the composite scaffold in vitro.
4.2 MATERIALS AND METHODS
4.2.1 Scaffold fabrication and characterisation
PCL/SrBG composite preparation and scaffold fabrication
The 75% strontium-substituted bioactive glass (SrBG) was produced by Stevens
et al as described in [57,151]. Briefly, the composition of BG containing 46.13 SiO2 –
2.60 P2O5 – 24.35 Na2O – 26.91 (SrO:CaO) (mole %) where 75% of the calcium was
substituted with strontium was melted in a platinum crucible at 1400 °C for 90 min
and rapidly quenched into deionised water to form frits. SrBG frits were then ground
and sieved to yield particles < 38 μm in diameter. In order to obtain a homogeneous
distribution of SrBG within the Polycaprolactone (PCL, Capa 6400, Perstorp UK
Limited), PCL was first melted on a hotplate (~70 °C). SrBG particles (10% wt.) were
then added and manually mixed into the PCL melt for 20 mins before being loaded
into a 2 ml syringe. Air bubbles were removed from the composite melt after loading
into the syringe via placement of the syringe in a vacuum oven (-80 kPa for 30 mins
at 90 °C).
Both PCL and PCL/SrBG composite scaffolds were produced using the melt-
electrospinning technique as previously described [90], using the optimised
parameters (Table 4.1).
48 Chapter 4: (Study 1) Fabrication and In vitro investigation of PCL, 10 wt% PCL/SrBG electrospun scaffolds for bone regeneration
Table 4.1 Parameters for melt-electrospun scaffold fabrication Scaffold type Voltage Temperature Collection
distance Needle gauge
Flow rate
PCL 7 KV 80 °C 4 cm G21 20 μl/h
PCL/SrBG 7 KV 85 °C 6 cm G19 20 μl/h
Scaffold surface modification via sodium hydroxide (NaOH) etching was
performed to reduce the hydrophobicity of PCL, increase the surface roughness of the
fibres and to increase surface exposure of SrBG particles to facilitate enhanced cell
attachment [88]. As reported in the literature [47], scaffolds were etched with 5M
NaOH for 1 hour @ 37 °C and subsequently washed with dH2O until the supernatant
pH reached 7.
Scaffold characterization
Light microscopy PCL and PCL/SrBG scaffolds were weighed with an
AUW2200 analytical balance (Shimadzu, Canada); Light microscopy (Zeiss Axio
Imager A2, Germany) and a Quanta 200 scanning electron microscope (SEM, FEI
Australia, Australia) were used to assess morphology and diameter of the fibres were
assessed using ZEN blue software (Zeiss, Germany). Average fibre diameters were
determined by random selection of 6 scaffolds per group. A total of 42 fibres per group
were measured via the ZEN imaging software.
Micro-computed tomography (µCT) PCL and PCL/SrBG (n = 2) scaffolds
were scanned in the air in a micro-computed tomography scanner (µCT, 40Scanco
Medical, Brüttisellen, Switzerland), at an energy of 45 kVp and intensity of 177 µA
with 300 ms integration time. The scans were reconstructed to three-dimensional
datasets with an isotropic voxel size of 6 µm. The scans were segmented to visualise
bioglass particle distribution throughout the scaffolds. Next, the scans were analysed
with the scanner’s software using the distance transformation method to determine
interfibre spacing.
4.2.2 Ion dissolution and precipitation
PCL and PCL/SrBG scaffolds were submerged in alpha-Minimum Essential
Medium (α-MEM, Invitrogen, Australia) at a ratio of 5 g/L supplemented with 1%
(v/v) of penicillin-streptomycin (Invitrogen, Australia), incubated at 37 °C/5% CO2.
Chapter 4: (Study 1) Fabrication and In vitro investigation of PCL, 10 wt% PCL/SrBG electrospun scaffolds for bone regeneration 49
Samples of dissolution ion media were collected after 6 hours, 1, 2, 3, 7, 14, 21 and 28
days for elemental analysis.
Elemental concentrations of calcium (Ca), phosphorus (P), silicon (Si) and
strontium (Sr) were measured on a Vista MPX Inductively Coupled Plasma - Optical
Emission Spectrometer (ICP-OES, Varian, USA).
In order to identify elemental deposition on the surface of the scaffolds, energy-
dispersive X-ray spectroscopy (EDX) (Quanta 200, FEI, Australia) was used to scan
the surfaces of PCL and PCL/SrBG fibres.
4.2.3 In vitro studies
To examine the osteogenic potential of the PCL/SrBG composite scaffolds, cell
culture experiments which considered cell metabolic activity, proliferation, viability
and gene expression upon the scaffolds were undertaken using mouse osteoblast
precursor cell line, MC3T3-E1 (passage 9).
Cell culture
Prior to experimentation, MC3T3 cells (sub-clone 14) [152] were cultured in
growth media: α-MEM cell culture media supplemented with 10% (v/v) foetal bovine
serum (FBS, Invitrogen, Australia) and 1% (v/v) penicillin-streptomycin at 37 °C/5%
CO2. Scaffolds were cut with a 6 mm biopsy punch and sterilised with 70% ethanol,
followed by UV irradiation for 20 min each side. MC3T3 cells were seeded at a density
of 4500 cells per scaffold (159 cells/mm2), after 24 hours culture, half the samples
were treated with osteogenic media (growth media supplemented with 10mM β-
glycerophosphate, 50 µg/ml ascorbic acid and 0.1mM dexamethasone (all from
Sigma)), the other half were cultured in growth media as a control. A total of four
different groups were studied as shown in Table 4.2 for the following experiments.
Table 4.2 In vitro experimental groups of study 1. PCL/SrBG scaffolds (10 wt%) were studied in both growth media and osteogenic media and PCL scaffolds were studied as control.
Group Scaffold type Culture media type n
1. PCL_C PCL Growth (control) 164
2. PCL_O PCL Osteogenic 164
3. PCL/SrBG_C PCL/SrBG Growth (control) 164
4. PCL/SrBG_O PCL/SrBG Osteogenic 164
50 Chapter 4: (Study 1) Fabrication and In vitro investigation of PCL, 10 wt% PCL/SrBG electrospun scaffolds for bone regeneration
LIVE/DEAD staining
A qualitative LIVE/DEAD assay was used to indicate both the cell viability and
cell distribution within the seeded scaffolds [153]. After days 1 and 7, samples were
moved into a fresh plate, cell culture media was removed and the scaffolds were
washed twice with phosphate buffered saline (PBS, Invitrogen). The scaffolds were
then incubated with 0.67 µg/ml fluorescein diacetate (FDA, Invitrogen) and 5 µg/ml
propidium iodide (PI, Invitrogen) for 5 min in the dark. The staining was visualized
using a Zeiss Axio M2 Imager (Zeiss, Germany) fluorescent microscope (at λ = 488
nm and λ = 568 nm excitation).
Assessment of cell attachment, morphology, and BG identification
Scanning Electron Microscopy (SEM) was used to qualitatively investigate
cellular attachment and morphology on the respective scaffolds. As described
previously [154], after days 3, 7, 14, 21 and 28, scaffolds were fixed in 3%
glutaraldehyde immediately following cell culture. Scaffolds collected from all time
points were washed in sodium cacodylate buffer (Sigma, Australia) and 1% osmium
tetroxides in cacodylate. Samples were then washed twice in ultrapure UHQ water
(Millipore Australia, Australia) and dehydrated in a graded ethanol followed by
Hexamethyldisilazane drying (all reagents were supplied by ProSciTech, Australia).
After sputter coating with gold, samples were visualized using a Quanta 200 SEM
(FEI, Australia).
Confocal Laser Scanning Microscopy (CLSM) [155] was used to visualise the
morphology of actin fibres and nuclei of MC3T3 cells on the scaffolds. Here we
introduce a new way to visualise the cell attachment onto SrBG particles by combining
actin staining and nuclei staining with Alizarin red S staining (to stain SrBG particles).
Briefly, after 3, 7, 14, 21, 28 days, cell culture media were removed and scaffolds were
transferred into fresh 48-well plates. After 2 careful washes with PBS supplemented
with 0.5 mM Mg2+, 0.9 mM Ca2+, scaffolds were fixed with 4% paraformaldehyde
(PFA, Sigma) solution for 30 min at room temperature. Samples were then washed
with PBS and permeabilized with 0.2% (v/v) Triton X-100 (Invitrogen)/PBS solution
for 5 min, followed by 2 washes with PBS. Samples were then incubated with 0.5%
Chapter 4: (Study 1) Fabrication and In vitro investigation of PCL, 10 wt% PCL/SrBG electrospun scaffolds for bone regeneration 51
(w/v) Bovine serum albumin (BSA, Sigma)/PBS for 10 min, followed by 0.5% (v/v)
BSA/PBS solution containing 0.8 U/ml Alexa Fluor® 488 Phalloidin (Invitrogen) and
5 µg/ml 4’,6-diamino-2-phenylindole (DAPI, Invitrogen). After 1 wash with MiliQ
water, the scaffolds were stained with Alizarin red S (pH 4.2) for 5 min and washed
with MiliQ water twice to remove excess stain. The scaffolds were then stored in PBS
until imaging. The PCL/SrBG scaffolds were visualized with Leica SP5 Confocal
microscope (Leica, Germany), images of identically treated PCL only scaffolds were
taken as controls.
Cell metabolic assay
MTT (3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide; Sigma,
Australia) is a cell metabolic activity assay which works on the principle of measuring
the absorbance of formazan, which is reduced from MTT by mitochondria in active
cells [58]. This was used to assess the metabolic activity of the cells when cultured on
the different scaffolds with and without the presence of osteogenic inducing media. At
days 1, 7, 14, 21 and 28, scaffolds (n = 6) were transferred into a fresh 48-well plate
and 500 µl of fresh media supplemented with 20 µl of MTT solution (5 mg/ml) was
added. Scaffolds were incubated (37 °C/5% CO2) for 4 hours, after which the media
was removed and 100 µl (D1, 7), 200 µl (D14), 400 µl (D21) or 500 µl (D28) dimethyl
sulfoxide (DMSO, Merck, Australia) was added to each well. The plates were then
covered with tinfoil and placed on an orbital shaker for 10 min before 100 µl of DMSO
eluant was taken from each well and transferred into fresh 96-well plates and
absorption at λ = 540 nm was measured. The obtained reading was multiplied with the
dilution factors of DMSO at all 5 time points respectively.
Alizarin red S staining (mineralisation)
Mineralisation of the cells on the scaffold was used as an indicator of
differentiation of the MC3T3 cells. I used a qualitative detection based on the selective
bonding between Alizarin red S and calcium salts [156]. After 7, 14, 21 and 28 days,
cell culture media was removed from the well and scaffolds (n = 6) were first washed
in PBS and then fixed with ice-cold methanol. Alizarin red S (Sigma, Australia) dye
solution (1 g of powder into 50 ml of distilled water) of 150 µl was added to each
scaffold. The pH of Alizarin red S solution was maintained at 4.15 for all time points.
52 Chapter 4: (Study 1) Fabrication and In vitro investigation of PCL, 10 wt% PCL/SrBG electrospun scaffolds for bone regeneration
Scaffolds were incubated for 5 min and then were washed repeatedly with MiliQ water
until the solution ran clear. Plates were scanned via a Canoscan 8600F flatbed scanner
(Cannon, US).
Cell differentiation
In order to compare the osteogenic potential of the cells cultured on PCL/SrBG
scaffolds to those on PCL scaffolds, I measured Alkaline Phosphatase (ALP) activities
of the cells adhere on each scaffold. ALP is a known early marker of osteoblast
differentiation and plays a key role in mineralisation [58]. In order to represent the
ALP activity as activity per cell, I normalized the ALP data with a total DNA
quantification technique (the PicoGreen assay) described hereafter.
For both assays, at the 7, 14, 21 and 28 day time points, cell culture media was
removed and scaffolds were washed twice with PBS and transferred into fresh 48-well
plates. 300 μl of 0.2 Triton-X/ ×1 TE buffer was added to each well and samples were
stored at -80 °C until further processing. Scaffolds were scraped with pipettes until
cells were adequately lysed, followed by sonication. Cell lysates were transferred into
1.7 ml Eppendorf tubes. From this point, lysates were assayed for DNA content and
ALP activity.
ALP assay
100 μl of cell lysate was transferred into a 96-well transparent microplate in
triplicates. 200 μl of para-Nitrophenyl phosphate (pNPP, Sigma, Australia) was added
into each well containing lysate or Triton-X/ ×1 TE buffer as blanks. The plates were
incubated in the dark for precisely 30 min at room temperature on a rocker plate. The
absorbance was read on a BIO-RAD multi-well plate reader at wavelength = 405 nm
(n = 6).
PicoGreen assay
PicoGreen is a cyanine dye that selectively binds dsDNA and sensitively detects
the DNA content which reflects cell numbers, as such, this assay may be used to
indicate cell proliferation [157]. Here I used PicoGreen to quantify the total amount of
DNA and then converted it to a cell number using a conversion factor of 8 pg of DNA
per MC3T3 cell [158]. The PicoGreen assay (Invitrogen, Australia) was performed
according to the manufacturer’s instructions. Briefly, samples were diluted into 40:60,
Chapter 4: (Study 1) Fabrication and In vitro investigation of PCL, 10 wt% PCL/SrBG electrospun scaffolds for bone regeneration 53
20:80 and 10:90 (lysate: ×1 TE buffer) in fresh 96-well plates. After the solution was
mixed, 100 μl of ×1 PicoGreen dye solution were added to each sample, making a total
volume of 200 μl in each well. The 96-well plates were covered with aluminium foil
and incubated at room temperature for precisely 5 min on a rocker plate. The plates
were then read by OPTIMA plate reader (BMG LABTECH, Australia) at excitation λ
= 480 nm and emission λ = 520 nm (n = 6).
The ALP absorbance obtained from the assay was normalised by dividing by the
cell number to give the value of ALP activity per cell. The cell number was obtained
from the DNA quantity via the PicoGreen assay.
Real-time quantitative polymerase chain reaction (RT-qPCR)
In order to obtain more detailed information about the osteogenic effect the
SrBG component of the scaffolds has upon the MC3T3 cells, RT-qPCR was used to
quantify the up-regulation of specific genes associated with early stage osteogenesis
[159].
After days 7, 14, 21 and 28, cell culture media was removed and scaffolds (n =
9) were transferred into fresh 48-well plates with 3 scaffolds per well. 300 μl of lysis
buffer containing 1% (v/v) mercaptoethanol (Sigma, Australia) and 7 μl of carrier
RNA (5 ng/μl) was added to each well. RNA was isolated with PureLink RNA Micro
Kit (Invitrogen, Australia) according to the manufacturer’s instructions.
The total quantity and purity of the extracted RNA were tested with a Nanodrop
Microvolume UV-Vis spectrophotometer (ThermoFisher Scientific, Australia). The
concentration of RNA was calculated to obtain a total of 1000 ng cDNA per sample.
The reverse transcription process was carried out with DyNAmo* cDNA Synthesis
Kit (Thermo Scientific, Australia) following manufacturer’s instructions.
RT-qPCR was performed on an ABI Prism 7000 Thermal Cycler (Applied
Biosystems, Australia) using SYBR Green as detection reagent following the same
procedure that has been reported previously [159]. The relative mRNA expressions of
ALP and Osteocalcin (OCN) were assayed and normalized against the house keeping
gene β-ACTIN. Each sample was analysed in triplicate. The mean cycle threshold (Ct)
of each target gene was normalized against Ct of β-ACTIN; the relative expression
calculated using the following formula: 2-(normalized average Cts) ×104.
54 Chapter 4: (Study 1) Fabrication and In vitro investigation of PCL, 10 wt% PCL/SrBG electrospun scaffolds for bone regeneration
Collagen staining in extracellular matrix (ECM)
Van Gieson’ stain (Dorn & Hart Microedge Inc, USA) was used to stain collagen
deposited on the scaffolds during cell culture. After 7, 14, 21 and 28 days, samples (n
= 3) were washed twice in PBS, transferred into a fresh 48-well plate and fixed in 10%
neutral buffered formalin (NBF, Sigma, Australia). Samples were then washed once
with PBS, followed by two washes with MiliQ water and then stained with 150 μl of
Van Gieson per scaffold for 20 min. Samples were rinsed with MiliQ water until the
solution ran clear. The plate was scanned and light microscope images were taken with
a Zeiss Axio Imager M2 microscope (Zeiss, Germany).
4.2.4 Statistical analyses
Statistical significance between groups in MTT, ALP and RT-qPCR assays was
assessed by Two-way ANOVA with a post-hoc Tukey test using IBM SPSS Statistics
Software (Version 19). A p value of less than 0.05 was considered statistically
significant.
4.3 RESULTS
4.3.1 Characterization of PCL and PCL/SrBG scaffolds
PCL and PCL/SrBG were successfully electrospun into fibrous mats. Light
microscopy indicated that the two scaffold types had similar morphologies (Figure
4.1); however, the PCL/SrBG fibres (46.1 +/- 16.6 µm) were slightly larger in diameter
than the PCL fibres (30.6 +/- 1.8 µm) (n = 42, p < 0.05). The µCT analysis determined
the average fibre spacing within the scaffold was 135.6 +/- 64.3 µm for the PCL fibres
and 222.1 +/- 113.6 µm for the PCL/SrBG ones. µCT analysis demonstrated that the
SrBG particles were homogeneously distributed throughout the PCL fibres (Figure
4.1f). No significant difference was detected between PCL scaffolds and PCL/SrBG
scaffolds in terms of weight (n = 70, p > 0.05).
Chapter 4: (Study 1) Fabrication and In vitro investigation of PCL, 10 wt% PCL/SrBG electrospun scaffolds for bone regeneration 55
Figure 4.1 Electrospun scaffolds light microscopy and microCT characterization. Light microscopy images of melt-electrospun PCL lower magnification (a) and higher magnification (c) and melt-electrospun PCL/SrBG lower magnification (b) and higher magnification (d), scale bars are as shown. μCT image of PCL scaffold shown as a control (e). PCL/SrBG scaffold showing the distribution of bioactive glass particles (highlighted in red) throughout the scaffold (f), a higher magnification image is shown in (h). Image (g) shows the SrBG particles only.
4.3.2 Ion dissolution and precipitation analysis
EDX analysis indicated the presence of elements Ca and P on the surface of
PCL/SrBG scaffold, and this calcium phosphate (CaP) layer was also visible under
SEM (Figure 4.2a, shows an example image that was taken after 14 days incubation in
56 Chapter 4: (Study 1) Fabrication and In vitro investigation of PCL, 10 wt% PCL/SrBG electrospun scaffolds for bone regeneration
α-MEM at 37oC). No Ca or P was found on the surface of the PCL only scaffolds at
the same time point (Figure 4.2b). The elemental concentrations of calcium,
phosphorus, silicon and strontium released from both PCL and PCL/SrBG scaffolds
into cell culture media are shown in Figure 4.2(c, d, e and f). In the PCL/SrBG group,
the calcium and phosphate concentrations (in the media) declined rapidly after 3 days
in cell culture, and then further decreased over the next 3 weeks. No significant change
in calcium and phosphate concentrations was observed in cell culture media exposed
to PCL only scaffolds. In the PCL/SrBG group, the concentrations of both silicon and
strontium increased rapidly after 3 days in cell culture and were stable after 14 days in
cell culture. No changes in silicon and strontium concentrations were observed in PCL
only groups.
Chapter 4: (Study 1) Fabrication and In vitro investigation of PCL, 10 wt% PCL/SrBG electrospun scaffolds for bone regeneration 57
Figure 4.2 EDX image of melt-electrospun scaffolds after 14 days incubation in α-MEM. (a) PCL/SrBG scaffolds showing a deposition of CaP (Calcium phosphate layer) over the surface of the fibres and (b) PCL scaffolds showing no CaP deposition on the surface of the fibres, scale bar as shown. Elemental analysis of the concentrations of Ca (c), P (d), Si (e) and Sr (f) in α-MEM over 28 days as determined by ICP-OES testing.
4.3.3 In vitro studies
Attachment and proliferation of MC3T3 cells on scaffolds
The LIVE/DEAD staining showed viable MC3T3 cells (as shown in green)
evenly distributed across both PCL and PCL/SrBG scaffolds after 1 day cell culture
with negligible red stained cells detected. After 7 days, the cell number had
significantly increased (Figure 4.3). High magnification images obtained by SEM
showed cells bridging to adjacent fibres on scaffolds in all four groups (Figure 4.4).
58 Chapter 4: (Study 1) Fabrication and In vitro investigation of PCL, 10 wt% PCL/SrBG electrospun scaffolds for bone regeneration
CLSM images showed numerous filopodia of MC3T3 cells attaching onto the SrBG
particles exposed on PCL/SrBG fibre surfaces (Figure 4.5a) whereas cells attached and
spread evenly on PCL fibres (Figure 4.5b). The MTT assay indicated that the cell
metabolic activity increased with length of cell culture time and significantly increased
after 7 days and 14 days in culture in all four experimental groups (Figure 4.6).
Figure 4.3 LIVE/DEAD staining of MC3T3 cells cultured on melt-electrospun scaffolds. (a) PCL and (b) PCL/SrBG are after 1 day culture, and (c) PCL and (d) PCL/SrBG are after 7 days culture. FDA (green fluorophor), indicates live cells, while PI (red fluorophor) indicates dead cells. Scale bars are as shown.
Chapter 4: (Study 1) Fabrication and In vitro investigation of PCL, 10 wt% PCL/SrBG electrospun scaffolds for bone regeneration 59
Figure 4.4 SEM images of MC3T3 cells cultured on PCL (a)(b) and (c) and PCL/SrBG (d)(e) and (f) scaffolds. Time points are: 3 days (a) and (d), 7 days (b) and (e), and 14 days (c) and (f). Scale bars are as shown.
Figure 4.5 Confocal laser scanning microscopy images of MC3T3 cells cultured on melt-electrospun PCL/SrBG (a) and PCL scaffolds (b) for 3 days. Green: Alexa Fluor 488 Phalloidin conjugates (actin), Blue: DAPI (nuclei), and Red: Alizarin red S (SrBG particles). Scale bars are as shown.
60 Chapter 4: (Study 1) Fabrication and In vitro investigation of PCL, 10 wt% PCL/SrBG electrospun scaffolds for bone regeneration
Figure 4.6 MTT metabolic activity assay of MC3T3 cells over 28 days culture (n=6). Error bars = ± SD of mean. * indicates significant increase in metabolic activity (p < 0.05).
ALP activity and mineralisation (Cell differentiation)
Normalized ALP activity (per cell) in all four experimental groups showed
increasing values with length of culture time and peaked on day 21 as presented in
Figure 4.7. The PCL/SrBG_O group showed significantly higher ALP activity when
compared to PCL_O group (p < 0.05) at both the 21 and 28 day time points (Figure
4.7). Scaffolds cultured in osteogenic media (PCL_O and PCL/SrBG_O groups)
showed significantly higher ALP activity compared to control groups (PCL_C and
PCL/SrBG_C groups) at all time points after 7 days. Alizarin red S staining indicated
more calcium mineralization (dark red) formed over the duration of cell culture and
the stain appeared darker in both PCL/SrBG groups compared to the PCL scaffolds
(Figure 4.8). Scaffolds cultured without cells (PCL Control and PCL/SrBG Control)
showed no staining and low levels of staining, respectively.
Chapter 4: (Study 1) Fabrication and In vitro investigation of PCL, 10 wt% PCL/SrBG electrospun scaffolds for bone regeneration 61
Figure 4.7 Normalised ALP activity of MC3T3 cells cultured on melt-electrospun PCL and PCL/SrBG scaffolds cultured in osteogenic and control media (n=4). ALP activity is divided by a total number of cells based on the DNA content obtained via PicoGreen assay. Error bars = ± SD of mean. * indicates significantly higher ALP activity per cell in the indicated group compared to the PCL_Control group of the time point (p < 0.05), † indicates significantly higher ALP activity per cell compared to the PCL_Osteogenic media group of the time point (p < 0.05).
62 Chapter 4: (Study 1) Fabrication and In vitro investigation of PCL, 10 wt% PCL/SrBG electrospun scaffolds for bone regeneration
Figure 4.8 Alizarin red S staining of PCL and PCL/SrBG scaffolds cultured with MC3T3 cells in control and osteogenic media over 28 days. Control scaffolds are unseeded but have been cultured for the same periods of time in culture media.
Expression of specific genes by MC3T3 cells on both scaffolds
Normalized RT-qPCR results indicated peak expression of ALP gene on day 21
(Figure 4.9a) and OCN gene on day 14 (Figure 4.9b). PCL/SrBG_O scaffolds showed
significantly up-regulated ALP expression levels after 7 and 21 days compared to
PCL_O. There was no significant difference in ALP expression between control
groups (PCL_C and PCL/SrBG_C). The results also showed significantly higher OCN
expression level in the PCL/SrBG_O groups compared to PCL_O after 14 days in cell
culture.
Chapter 4: (Study 1) Fabrication and In vitro investigation of PCL, 10 wt% PCL/SrBG electrospun scaffolds for bone regeneration 63
Figure 4.9 Gene expression of osteoblast markers ALP (a) and OCN (b), in all experimental groups over 28 days (n=9). Data represents mean + standard deviation. Error bars = ± SD of mean. * indicates a significant increase in gene expression compared to PCL_O (p < 0.05).
Extracellular matrix (ECM) formation
Van Gieson staining showed that the concentration of collagen increased with
length of time in culture in all scaffold groups (Figure 4.10). Among them, PCL_O
and PCL/SrBG_O showed higher levels of collagen formation compared to the PCL_C
and PCL/SrBG_C groups. Also, PCL/SrBG_C and PCL/SrBG_O groups showed
noticeably higher ECM formation compared to the PCL_C and PCL_O groups.
64 Chapter 4: (Study 1) Fabrication and In vitro investigation of PCL, 10 wt% PCL/SrBG electrospun scaffolds for bone regeneration
Figure 4.10 Van Gieson staining of PCL and PCL/SrBG scaffolds cultured with MC3T3 cells in control and osteogenic media over 28 days. Control scaffolds are unseeded but have been cultured for the same periods of time in culture media.
4.4 DISCUSSION
Organic polymer/inorganic particle composites have been fabricated into
scaffolds via the technique of solution-electrospinning [160–162] in the past, however,
the use of melt-electrospinning, which has prominent advantages over its solution
counterpart has gained little attention in the field of tissue engineering [83]. I was able
to successfully establish and optimise a technique for melt-electrospinning PCL/SrBG
composite fibre scaffolds, for the first time, which show promise for application in the
bone TE field. The complexity of melt-electrospinning PCL composite scaffolds
occurs due to the inclusion of the inorganic SrBG phase. Large clumps of SrBG can
interfere with the continuity of fibre production by blocking the nozzle of the spinneret,
causing the formation of beads, and an agglomeration of inorganic particles. A similar
situation also occurs when solution-electrospinning scaffolds comprising inorganic
and organic phases, and to address this problem in solution-electrospinning, a
surfactant can be introduced to ensure the homogeneous dispersion of the inorganic
Chapter 4: (Study 1) Fabrication and In vitro investigation of PCL, 10 wt% PCL/SrBG electrospun scaffolds for bone regeneration 65
particle phase throughout the organic polymer [163]. However, this introduces another
impurity to the scaffold which can potentially contribute to increased toxicity to cells
(in addition to the organic solvent). In this study, I circumvented these issues by
manually dispersing the SrBG particles into molten PCL bulk until a homogeneous
distribution was achieved via mechanical mixing. The SrBG particles maintained an
even dispersion during the entire electrospinning process (as demonstrated with μCT
scanning showing an even distribution of particles in Figure 4.1f) this is due to the
molten PCL (at ~60 °C) being sufficiently viscous to prevent any detectable
sedimentation effect occurring. Using this novel approach of composite fabrication by
melt-electrospinning, we were able to reproducibly manufacture a scaffold with
microfibers and with pore sizes large enough to allow for cellular infiltration and
nutrient diffusion [164]. A loading of 10 wt% SrBG particles were selected in this
study based on previously published literature on polymer/BG composite materials
[14,165–167] and on ongoing studies in our team [14].
The parameters used to produce PCL/SrBG scaffolds via melt-electrospinning
technique were explored based on the ones to produce PCL scaffolds due to lack of
previous experience in TE field. I aimed to produce PCL/SrBG composite scaffolds
with smooth fibre jets of random deposition (as that of PCL) scaffold by altering key
parameters of melt-electrospinning shown in Table 4.1. Heating temperature during
the electrospinning process is one of the essential parameters, the PCL/SrBG
composite fibre started to break to show a ‘spitting’ phenomenon when the
electrospinning temperature is too high and the composite was too viscous to
electrospin and blocked the needle when it was too low. This process to produce
continuous composite fibres to form melt-electrospun scaffolds was defined as
optimisation in study 1.
Previous work has determined that the fibre diameter of the melt-electrospun
PCL scaffolds (5–33 μm) is optimal for supporting cell culture [90,164]. Due to the
slightly different conditions required for successful production of melt-electrospun
PLC-SrBG scaffolds compared to melt-electrospun PCL ones, the PCL/SrBG fibres
(46.1 +/- 16.6 µm) were slightly larger than the PCL ones (30.6 +/- 1.8 µm) produced,
as were the inter-fibre spacing within the scaffold. One of the reasons for this
difference owed to the addition of the SrBG particles which has an effect on the
thermal properties and crystallinity of the PCL component [147], requiring higher
66 Chapter 4: (Study 1) Fabrication and In vitro investigation of PCL, 10 wt% PCL/SrBG electrospun scaffolds for bone regeneration
temperatures to obtain similar viscosity. The presence of occasional large SrBG
particles up to 38 microns in size created a few very large fibres in the PCL/SrBG
scaffolds, resulting in a greater variance in fibre diameter of PCL/SrBG than the PCL
alone. It is well established in electrospinning that fibre size is proportional to inter
fibre spacing, so it is to be expected that the slight increase in fibre size led to an
increase in inter fibre spacing of the scaffolds. However, both scaffold types had no
significant difference in measurable weight and had a similar gross morphology when
examined by light microscopy and µCT (Figure 4.1a and 4.1b, 4.1e and 4.1f).
The ability for PCL/SrBG scaffolds to promote osteogenesis is dependent upon
the SrBG particles leaching ions into the local environment; as previously discussed,
the Sr ion has been shown to increase osteogenesis by enhancing osteoblast metabolic
activity and ALP activity [58]. Strontium incorporated biomaterials have been
increasingly interesting as Sr ions have great affinity to bone tissue were found to be
able to stimulate bone formation and reducing bone resorption [168–170]. As a result,
Sr has been incorporated into bioactive ceramics including bioactive glass for
enhanced bioactivity [58,171–173].The SrBG with 75% Sr substitution used in this
study was provided by our collaborator – Prof. Molly Stevens’s group. At the
development stage of the SrBG, serial percentages (0-100%) of Sr substitution were
studied [57,58]. According to the results, even though the SrBG with 100% Sr
substitution showed best promotion of osteoblast activity and inhibition of osteoclast
activity of cells cultured on these materials compared to lower substitutions [58], the
high Sr content in the SrBG also inhibited osteoblast proliferation [57]. As a result, the
Sr75 SrBG was a balance between the efficacy and toxicity of Sr ions. I confirmed that
the PCL/SrBG composite scaffolds were releasing Si and Sr ions into the media,
therefore, the scaffolds offer potential to promote osteoblasts differentiation [58]. The
5g/L SrBG scaffold to media ratio was adopted from previous studies for easy
comparison [171,174]. The scaffolds were immersed in the media and the containers
were sealed to minimise the risk of bacterial and fungal contamination, and the media
were collected for analysis at predetermined time points. It was also observed that the
ion release rate was very slow for the initial three days, but then rapidly increased,
most likely due to surface hydration of the PCL fibres resulting in an increase in the
direct exposure of SrBG particles to the media. Additionally, the presence of SrBG
particles stimulated CaP deposition on the surface of scaffolds, detected by the
Chapter 4: (Study 1) Fabrication and In vitro investigation of PCL, 10 wt% PCL/SrBG electrospun scaffolds for bone regeneration 67
decrease of Ca and P ions in the media, and confirmed directly by EDX (Figure 4.2a).
According to the literature, the exposed SrBG particles may initiate the surface
mineralization by providing nucleation sites [175]. This would also explain why a CaP
layer was not present on PCL scaffolds subjected to the same conditions. In the bone
TE field, CaP coating is a popular approach to enhance bioactivity of polymeric
scaffolds by decreasing their surface hydrophobicity and thus creating scaffolds which
can better mimic the natural structure of bone ECM [102,176–180]. As a mineralized
surface is known to be favourable for cell attachment and migration, the inclusion of
SrBG particles in PCL fibres has provided a mechanism for the PCL/SrBG scaffolds
to present a higher degree of bioactivity than PCL scaffolds alone.
As expected, both PCL and PCL/SrBG scaffolds were capable of facilitating cell
attachment and proliferation, as shown using MC3T3 cells. After 1 day and 7 days
culture, negligible numbers of dead cells could be found on either scaffold type (<
10%), and live cells were evenly distributed across the scaffold, suggesting that
attachment and migration were not significantly altered by the incorporation of SrBG
to the PCL component, or the increase in fibre diameter (Figure 4.3). The cell viability
(over 90%) is high when compared to previous studies [181,182] and suggests that the
fibres encourage cell growth and proliferation. Quantitative results obtained by the
MTT assay suggest that the addition of SrBG particles did not have any significant
effect on the proliferation of the cells upon the scaffolds, and visualising the cells via
SEM showed that the fibres were acting as structural support for cells, with strong
attachment, and bridging of the larger pores in both scaffolds (Figure 4.4). It is
expected that these interconnective pores provide a large volume of space for cells to
proliferate and infiltrate, and allow nutrient transfer within the structure of the scaffold.
While cells could attach and proliferate on both scaffolds, a distinct difference between
the attachment of cells onto PCL and PCL/SrBG scaffolds was detected via CLSM
(Figure 4.5). The presence of SrBG particles directed cellular attachment to be more
localised around the particles, and this observation is in line with the established
literature, confirming the formation of a strong bond between SrBG and living tissue
[148]. This again supports the theory that the organic fibre/inorganic particle system
better mimics the natural structure of bone than polymers alone and can facilitate cell
attachment, proliferation and formation of appropriate tissue.
68 Chapter 4: (Study 1) Fabrication and In vitro investigation of PCL, 10 wt% PCL/SrBG electrospun scaffolds for bone regeneration
Higher mineralization was observed in the PCL/SrBG_O than the other three
groups as identified by the qualitative results of Alizarin red S staining. This suggests
that even though it has been established that SrBG has the capacity to enhance
mineralization of osteoblasts, this has not been directly observed without additional
osteogenic factors introduced into the media. It should be noted that there is
background staining present on the scaffolds with the inclusion of SrBG (control
scaffolds after 28 days of incubation in media) as the Alizarin red S dye binds to
calcium which is a component of the bioactive glass. However, by including negative
controls in our experiments (Figure 4.8) I illustrate that this staining level is
significantly lower than the groups with the cell inclusions, implying that the
mineralisation detected is more likely attributed to the cell response (mineralisation)
to the bioactive glass and/or osteogenic media. As shown in Figure 4.2, the precipitated
CaP layer could also contribute to the positive staining by Alizarin Red S due to the
nature of this stain. This explains why the PCL/SrBG scaffolds with cells showed
overall more intense staining compared to PCL control scaffolds. The effect of CaP
layer was more dominant at the early time point (day 7) when the cell ECM
mineralization level was low, which explains why some scaffolds in PCL/SrBG_C
group stained as intensely as the ones in PCL/SrBG_O group. In order to further
support the osteogenic effect of the SrBG component, the expression of two key genes
(ALP and OCN) which are expressed when osteoprogenitors cells commence
differentiation into osteoblasts [183,184], were investigated. Normalized ALP activity
confirmed up-regulated osteoblast differentiation on PCL/SrBG_O scaffolds
compared to PCL_O scaffolds alone, in the presence of osteogenic media after 21 days.
At earlier time points, the ALP activity per cell was not significantly different between
the two groups. However, after 3 weeks in culture, there was significantly higher ALP
activity in the PCL/SrBG_O group than in the PCL_O one. This increase of ALP
activity at 21 days is most likely induced by the presence of ions from the SrBG eluting
into the media (Si and Sr), as the ion dissolution studies show that relatively little
elution occurs for the first 3 days, followed by a more rapid release building up to 14
days, it is possible that this delayed release is responsible for postponing an observable
effect until 21 day culture. As Gentlemen et al. discussed previously[58], the increased
amount of Si and Sr ions released from PCL/SrBG scaffolds works synergistically to
better promote osteoblast differentiation compared to PCL scaffolds alone. The up-
regulation of the ALP gene, which we observed, correlates with the observed ALP
Chapter 4: (Study 1) Fabrication and In vitro investigation of PCL, 10 wt% PCL/SrBG electrospun scaffolds for bone regeneration 69
activity. The significantly higher OCN gene expression present in PCL/SrBG scaffolds
compared to the PCL ones after 14 days culture further suggests an additional
osteogenic capability of the composite scaffold compared to PCL scaffolds. However,
I found no such significant difference between groups without osteoinductive reagents
(PCL_C and PCL/SrBG_C) indicating that the SrBG present in the composite scaffold
alone was not sufficiently osteogenic to induce differentiation alone. Our data does
suggest that the incorporation of SrBG particles into the PCL scaffold can enhance the
osteogenic potential of cells when paired with osteogenic media to a higher degree
than the osteogenic media alone. A possible reason for the lack of osteogenic response
from PCL/SrBG scaffolds alone could be an insufficient amount of SrBG particles
present within the scaffold (10 wt%). Commonly, cytokines (typically ascorbic acid,
β-glycerophosphate and dexamethasone) are used for osteoblasts differentiation in
vitro[185]. However, in the previous study by Gentleman et al., osteogenesis was
observed when the human osteosarcoma cell line Saos-2 cells were cultured on SrBG
discs in standard culture media (RPMI 1640 media supplemented with 10% (v/v) foetal
bovine serum (FBS) and 2 mM L-glutamine) without any osteogenic reagents such as
ascorbic acid [58]. Based on these results, we decided to explore the osteogenic
capacity of PCL/SrBG scaffold without any other osteogenic factors. In Gentleman’s
study, cell culture media were treated with a total of 1.5 mg/ml of SrBG powder for
ion dissolution and found significantly enhanced osteoblasts proliferation and
differentiation [58]. In our experiment, the average weight of a PCL/SrBG scaffold
was 2.6 mg, with only 10% of the weight being SrBG particles, so there was an average
SrBG concentration of 0.325 mg/ml cell culture media available for ion dissolution,
with not all of the particles exposed at one time. Unsurprisingly, Sr2+ and Si4+
concentration in the PCL/SrBG composite scaffolds dissolution media was much
lower than those observed by Gentleman et al. when they investigated SrBG particles
alone, based on elemental analysis. In addition to the lower total SrBG present, the
PCL component acts as a physical barrier or coating inhibiting the burst release of the
ions from the SrBG particles present within the core of the PCL fibres. However, our
system has the advantage of facilitating a longer term sustainable ion release (releasing
ions as the PCL fibres degrade thus exposing the SrBG), an effect which is more
desirable for bone regeneration compared to the burst release observed of SrBG alone.
However, in order to enhance osteogenic potential of this composite PLC/SrBG
scaffold sufficiently to elicit an effect in in vivo bone repair, it will most likely be
70 Chapter 4: (Study 1) Fabrication and In vitro investigation of PCL, 10 wt% PCL/SrBG electrospun scaffolds for bone regeneration
necessary to increase the weight percentage of the SrBG component which would
involve higher loading during the melt-electrospinning process, which is a difficult
undertaking owing to complexities in continuous polymer fibre production when
higher concentrations of particles are present. One future approach would be to surface
coat the fibres with additional SrBG. Another approach to increase the rate of release
of the SrBG ions is to etch the PCL/SrBG scaffold for longer with NaOH, this is a
procedure which is already employed with PCL scaffolds in order to increase the
hydrophobicity of the scaffold, and the technique can be used to expose a higher
amount of SrBG to the media. I undertook surface etching treatment to allow for
maximum SrBG particle exposure and reached a critical time limit of 12 hours at which
time the etching was leading to particles “falling” out of the polymer matrix, therefore
it is important to have a trade-off between etching time and not losing important SrBG,
and also not to degrade the PCL matrix to an extent where the fibres may become
mechanically compromised, in this study I opted for one hour etching as I observed a
greater number of particles exposed at this time point with no particles falling out of
the PCL matrix.
In natural bone, collagen-based extracellular matrix (ECM) is essential for the
structural support of cells and mineral contents [6]. As such, I looked into the amount
of collagen deposited by cells onto the scaffolds in the experimental groups and found
that cells on the composite scaffolds appeared to produce a greater amount of ECM
than the PCL scaffolds alone (Figure 4.10). This was a qualitative observation which
suggests that SrBG may have other stimulatory effects, in addition to enhancing
mineralization.
In addition to electrospinning, there are many other techniques available in TE
to incorporate an inorganic phase such as SrBG to a polymeric scaffold. For example,
BG coating of polymers is another common approach [50] which is relatively easy and
quick method for the incorporation of BG particles onto the polymeric scaffolds
surface. In comparison to melt-electrospinning, this technique facilitates the maximum
contact of bioglass to cells initially possible; however, this approach can potentially
reduce the interconnective porosity of scaffolds by clogging the spaces between fibres
with bioglass particles. Additionally, the technique requires the use of organic solvents
involved in the coating process which can pose potential toxic effects to living cells
and tissues if they are not efficiently removed prior to cell exposure. It would be
Chapter 4: (Study 1) Fabrication and In vitro investigation of PCL, 10 wt% PCL/SrBG electrospun scaffolds for bone regeneration 71
desirable to produce a sustained release of ions from a bone TE scaffold, which cannot
be easily achieved from BG coating alone due to the burst release nature of ions from
BG particles which are highly exposed on the surface of the composite [186]. The
technique of melt-electrospinning enables an even distribution of BG particles
embedded (and exposed through etching) throughout the scaffold fibres which is
suitable for long-term sustainable ion release. Approaches to increase the percentage
of SrBG in the polymer bulk, combined with surface coating approaches should be
investigated to exploit the potential these highly porous melt-electrospun scaffolds
show in the field of bone TE.
4.5 CONCLUSIONS OF STUDY 1
An organic/inorganic composite biomaterial was produced for the first time by
incorporating SrBG particles into PCL bulk and melt-electrospinning to form highly
porous fibrous sheets. The composite scaffolds produced facilitated cellular
attachment and proliferation, and possessed enhanced osteogenic potential compared
to PCL scaffolds alone. ALP activity of MC3T3 cells cultured on these composite
scaffolds was enhanced and increased osteoblast differentiation was observed through
up-regulation of gene expression (ALP and OCN) in vitro. The composite scaffolds
also enhanced collagen deposition. These results showing enhanced osteogenesis
support similar studies using SrBG particles alone. The simple processibility of PCL
allows it to provide a potential platform for delivering the SrBG component and melt-
electrospinning has demonstrated promise as a fabrication technique which can
produce PCL/SrBG scaffolds which are highly porous, and these results show potential
for the technique to be further developed for the application of designing a versatile
biomaterial composite for the bone tissue engineering arena.
The 10 wt% PCL/SrBG scaffolds showed enhanced osteogenic capacity in
osteogenic media. However, their practical application is limited mainly due to the
lack of osteoinductivity, which means the composite scaffolds are not yet the ideal
solution for bone defect repair. In addition, another limitation is that the structure of
the scaffolds produced using melt-electrospinning in this study presented in a random
mesh layout instead of controlled size and shapes. Therefore in the subsequent study
2, I planned to further enhance the bioactivity of the PCL/SrBG scaffolds by increasing
the proportion of SrBG contents in the composite material and optimise the melt-
72 Chapter 4: (Study 1) Fabrication and In vitro investigation of PCL, 10 wt% PCL/SrBG electrospun scaffolds for bone regeneration
electrospinning process to produce PCL/SrBG composite scaffolds with enhanced
bioactivity (ideally making the scaffolds osteoinductive) and controlled fibre
deposition.
Chapter 5: (Study 2) Developing 50 wt% Strontium-substituted bioactive glass and Polycaprolactone composite scaffolds for bone repair via hybrid electrospinning in a direct writing mode 73
Chapter 5: (Study 2) Developing 50 wt% Strontium-substituted bioactive glass and Polycaprolactone composite scaffolds for bone repair via hybrid electrospinning in a direct writing mode
In study 1, I produced 10 wt% PCL/SrBG scaffolds which showed enhanced
osteogenic capacity compared to PCL only scaffolds but the results indicated that there
was not enough SrBG filler phase to make the composite with better osteogenic
properties. In this study, in order to further enhance the bioactivity of composite
scaffolds, I increased the weight percentage of SrBG particles in the PCL bulk. To
minimize the risk of needle blockage and obtain a homogeneous composite, I reduced
the SrBG particle size by milling and improved PCL/SrBG composite preparation
techniques. I then optimised the melt-electrospinning technique to produce PCL/SrBG
composite scaffolds with higher SrBG contents in a direct writing mode to obtain
scaffolds with ordered layer-by-layer fibre deposition. These new PCL/SrBG scaffolds
with increased SrBG, which was up to 50 wt% of PCL, were investigated in the same
manner as the scaffolds in Chapter 4 with an in vitro cell study for their osteogenic
properties. The results of this study of PCL/SrBG (50 wt%) scaffolds can be used to
determine whether these improved scaffolds are a potential patient-specific solution
for bone defect healing.
5.1 INTRODUCTION
Bone has a remarkable self-healing capacity to repair itself scarlessly after injury
[24]. However, this capacity can be impeded when the large area of bone loss exceeds
its regenerative capacity [26]. While the classic bone-grafting clinical treatment for
large bone defects renders suboptimal results, the scaffold-based tissue engineering
approach offers an off-the-shelf alternative [26]. Recently a paradigm shift has
occurred towards tissue engineering smart biomaterials with patient specific structures
[12,44,166]. These ‘intelligent’ bone scaffolds are expected to function as a temporary
74 Chapter 5: (Study 2) Developing 50 wt% Strontium-substituted bioactive glass and Polycaprolactone composite scaffolds for bone repair via hybrid electrospinning in a direct writing mode
matrix to allow for cell recruitment from nearby host tissues, cell proliferation and cell
differentiation towards osseous tissue, while maintaining the mechanical support to
the defect site [23]. In spite of the recent advance in biomaterials and fabrication
techniques in TE field, it is still challenging to produce fully functioning scaffolds for
bone regeneration.
Materials selection and structural design are two key research focuses towards
the development of ideal scaffolds for bone. Synthetic materials including polymers
and bioactive glass (BG) are common for bone scaffold fabrication [50,70]. Among
the synthetic polymers, polycaprolactone (PCL), which is an FDA-approved aliphatic
polyester, has become a promising candidate of bone scaffold material due to its
favourable rheological and viscoelastic properties [45]. Bioactive glasses (BGs) are a
sub-category of bioactive ceramics that bond with host tissues via the formation of a
biologically active hydroxyl carbonate apatite (HCA) layer on the surface [16]. The
ions released from BGs make them superior bone forming materials to other
bioceramics by stimulating osteogenic differentiation [16,50]. Since Larry Hench
developed the first BG - Bioglass®, known as 45S5, a variety of BGs have been
introduced over the years [16]. Strontium-substituted BG is one of the derivatives that
modified the 45S5 BG by substituting 0-100% of the calcium component of the 45S5
formulation with strontium [57], and it has shown superior osteogenic capacity to 45S5
BG [58].
In isolation, polymer alone or bioactive glass alone is considered suboptimal for
bone defect healing. However the polymer/BG composite materials, which exploit the
processability of polymers and bioactivity of BG filler phase, have been introduced to
TE as a new family of bioactive materials [50]. Previously, polymer/BG composite
scaffolds have been developed for TE purposes. However, these scaffolds were mainly
produced with traditional manufacturing techniques (thermally induced phase
separation [187], solvent casting [188], microsphere sintering [68]), which are limited
to produce scaffolds with simple geometries and uncontrolled internal architectures.
Alternatively, melt based additive manufacturing techniques have been employed to
produce scaffolds with desired geometry and controlled internal architecture [14,189].
Among these techniques, melt-electrospinning is a versatile fibre-based technique for
scaffold production with smaller fibre diameter and thus larger surface area to volume
ratio compared to melt extrusion. Recent developments the direct writing melt-
Chapter 5: (Study 2) Developing 50 wt% Strontium-substituted bioactive glass and Polycaprolactone composite scaffolds for bone repair via hybrid electrospinning in a direct writing mode 75
electrospinning techniques make use of the stable and predictable fibre deposition to
fabricate scaffolds in a layer-by-layer manner [93,94]. However, the range of candidate
materials that can be used for melt-electrospinning is restricted by their viscoelastic
properties [190] and therefore melt-electrospun polymer/BG composite scaffolds are
rarely reported. In order to minimise this limitation, hybrid electrospinning systems,
which combine the processes of individual solution- and melt-electrospinning, have
been proposed but with limited application [86,97,98].
In this study, I introduced a novel solution-assisted hybrid melt-electrospinning
system for the production of PCL/SrBG composite scaffolds with increased SrBG
filler phase (50% in PCL) compared to our previous study (chapter 4) [191]. I report
for the first time, obtaining continuous and stable composite fibres producing 50%
PCL/SrBG composite scaffolds in a layer-by-layer manner with 0/90° cross-hatched
deposition. I furthermore characterised the scaffolds and assessed their bioactivity in
vitro. The in vitro study results indicated that the PCL/SrBG scaffolds showed
enhanced osteogenic capacity compared to PCL ones.
5.2 MATERIALS AND METHODS
5.2.1 PCL/SrBG composite preparation
SrBG with the composition of 46.13 SiO2 – 2.60 P2O5 – 24.35 Na2O – 26.91
(SrO:CaO) (mole %) where 75% of the calcium was substituted with strontium was
prepared as previously described [57,191]. SrBG frits were ground and sieved to yield
particles 20 μm to 100 μm in diameter and then provided to us by our collaborator
Professor Molly Stevens’ group at Imperial College London, UK.
Particle size optimisation
In order to obtain homogeneous distribution, reduce the risk of needle blockage
and to increase the surface area of SrBG particles, the SrBG particles were ground with
a micronizing mill (McCrone Microscopes & Accessories, USA). Briefly, 2.5g of
SrBG particles were loaded into a plastic chamber filled with zirconia beads and then
wetted with 13ml of absolute ethanol. A series of incremental grinding times (from
30s to 8h) were used to obtain the optimal grinding time, particle sizing was performed
at each time point with a Malvern Mastersizer 3000 (Malvern, UK). The particles were
traditionally dried in the oven at 60 °C but aggregation was seen to occur indicated by
increased particle size. To minimise particle aggregation, I used freeze drying instead
76 Chapter 5: (Study 2) Developing 50 wt% Strontium-substituted bioactive glass and Polycaprolactone composite scaffolds for bone repair via hybrid electrospinning in a direct writing mode
of oven drying, to ensure particle size distribution remained in the same range as
ground.
PCL/SrBG composite preparation
The ground SrBG particles were incorporated into the PCL bulk by fast
precipitation into excess ethanol [14,192]. Briefly, 10% (w/v) PCL solution was
prepared by dissolving 2g of PCL pellets (CAPA 6500, Perstorp, UK) into 20 ml of
chloroform (MERCK, Millipore, Australia) at room temperature. Next, 1g of SrBG
particles were added into PCL solution for a composite of 50 wt% PCL/SrBG. The
composite solution was homogenised by constant stirring for 8 h with 2 x 2min
ultrasonication in between. The homogeneous mixture was then precipitated into a 10-
fold excess of 100% ethanol (MERCK, Millipore, Australia). The 50 wt% PCL/SrBG
composite was then collected and air dried in the fumehood to evaporate the solvents.
5.2.2 Scaffold fabrication
Both PCL/SrBG composite and PCL control scaffolds were fabricated with the
electrospinning rig built in house at QUT. The outstanding features of this rig include
positive/negative dual power packs and motorised stage that allows layer-by-layer
fibre deposition [193]. PCL/SrBG composite scaffolds were fabricated using a novel
hybrid melt-electrospinning technique in the direct write mode. PCL control scaffolds
were fabricated using direct writing melt-electrospinning (link to video:
https://www.youtube.com/watch?v=EzhMCzn8C80). Briefly, every 1g of composite
PCL/SrBG composite was mixed into 1 mL of chloroform in a 2 mL syringe. The
syringe was then inserted into a water jacket installed in the rig and the temperature
was maintained at 60 °C by a water bath. The PCL scaffolds were melt-electrospun at
80 °C. All scaffolds were fabricated with a 90° cross-hatched laydown pattern and
fibre spacing of 1mm (links to videos: https://www.youtube.com/watch?v=5ZB-
PCpw8qU and https://www.youtube.com/watch?v=nlRtngXuoJE). The optimised
electrospinning parameters are summarised in Table 5.1.
Table 5.1 Parameters for 50 wt% PCL/SrBG and PCL scaffolds fabrication Scaffold type
Voltage Temperature
Collection distance
Needle gauge
Flow rate
Feed speed
PCL 3.5/-3.2 KV 80 °C 1 cm G20 45 μl/h 800 mm/min PCL/SrBG 3.5/-3.2 KV 60 °C 0.5 cm G20 95 μl/h 800 mm/min
Chapter 5: (Study 2) Developing 50 wt% Strontium-substituted bioactive glass and Polycaprolactone composite scaffolds for bone repair via hybrid electrospinning in a direct writing mode 77
5.2.3 Scaffold characterisation
Microscopy
A light microscope with ZEN blue software (Zeiss Axio Imager M2, Germany)
and a Sigma field emission scanning electron microscope (FESEM, Zeiss, Germany)
were used to examine scaffold surface morphology and fibre diameter.
Confocal microscopy
PCL/SrBG and PCL scaffolds were stained for 5 min with Alizarin red S (Sigma,
Australia) dye solution (1 g of powder into 50 ml of distilled water) 150 µl was added
to each scaffold. The scaffolds were washed repeatedly with MiliQ water until the
solution ran clear. The scaffolds were imaged with a Nikon A1R confocal microscope
(Nikon, Australia).
Backscattered SEM imaging
The PCL/SrBG scaffolds were embedded in epoxy resin and ground down until
the scaffolds fibres were exposed to the resin surface. The embedded scaffolds were
imaged with SEM (JEOL JXA 8530F Hyperprobe, USA) to detect backscattered
electron (BSE) for the qualitative analysis of the elemental composition on the surface
of the PCL/SrBG scaffolds.
Ion dissolution and precipitation
PCL/SrBG and PCL scaffolds were incubated at 37 °C/5% CO2 in alpha-
Minimum Essential Medium (α-MEM, Invitrogen, Australia) supplemented with 1%
(v/v) of penicillin-streptomycin (Invitrogen, Australia) at w/v of 5g/L. The media were
collected at 3, 6 hours, 1, 2, 3, 7, 14, 21 and 28 days for further elemental analysis.
The ion concentration of calcium (Ca2+), phosphate (PO43-), silicon (Si4+) and
strontium (Sr2+) in α-MEM was assessed with an Agilent 8800 Inductively Coupled
Plasma Mass Spectrometer (ICP-MS, Agilent, USA). The scaffolds retrieved from the
media were air-dried under vacuum for 48 hours and scanned with energy-dispersive
X-ray spectroscopy (EDX) (Sigma FESEM, Zeiss, Germany) to identify elemental
deposition on the surface of fibres.
5.2.4 In vitro studies
To examine the osteogenic potential of the PCL/SrBG composite scaffolds with
50 wt% SrBG loading and to compare their bioactivity to the scaffolds produced in
study 1, in vitro cell culture experiments similar to were carried out using mouse
78 Chapter 5: (Study 2) Developing 50 wt% Strontium-substituted bioactive glass and Polycaprolactone composite scaffolds for bone repair via hybrid electrospinning in a direct writing mode
osteoblast precursor cell line, MC3T3-E1 (passage 9). A series of in vitro assays were
used to assess cell viability and metabolic activity, proliferation, osteoblast
differentiation and gene expression.
Cell culture
Prior to experimentation, MC3T3 cells (sub-clone 14) [152] were cultured for
expansion in growth media: α-MEM cell culture media supplemented with 10% (v/v)
foetal bovine serum (FBS, Invitrogen, Australia) and 1% (v/v) penicillin-streptomycin
at 37 °C/5% CO2. Scaffolds (both types) were etched in 5M NaOH for 15min at 37 °C
to enhance initial cell attachment and cut with a 6 mm biopsy punch. The scaffolds
were sterilised by 30min immersion in 70% ethanol and air dried in the fume hood,
followed by UV irradiation for 20 min each side. The PCL/SrBG (50 wt%) scaffolds
were conditioned in α-MEM supplemented with 1% (v/v) penicillin-streptomycin 1
week prior to cell seeding. MC3T3 cells were seeded at a density of 50,000 cells per
scaffold, and after 24 hours culture, half the samples were treated with osteogenic
media (growth media supplemented with 10mM β-glycerophosphate, 50 µg/ml
ascorbic acid and 0.1mM dexamethasone (all osteogenic supplements were supplied
by Sigma)), the other half were cultured in growth media as a control. A total of four
different groups were studied as shown in Table 5.2 for the subsequent experiments.
Table 5.2 In vitro experimental groups of study 2. PCL/SrBG scaffolds (50 wt%) were studied in both growth media and osteogenic media and PCL scaffolds were studied as control.
Group Scaffold type Culture media type
n
1. PCL_C PCL Growth (control) 125
2. PCL_O PCL Osteogenic 125
3. PCL/SrBG_C PCL/SrBG Growth (control) 125
4. PCL/SrBG_O PCL/SrBG Osteogenic 125
LIVE/DEAD staining
To determine both the cell viability on the scaffolds and their distribution,
LIVE/DEAD assay was carried out as described in study 1. Briefly, after days 1 and
7, cell culture media were removed and the scaffolds were moved into a fresh plate,
and the scaffolds were washed twice with PBS. The scaffolds were then incubated in
solution containing 0.67 µg/ml fluorescein diacetate (FDA, Invitrogen) and 5 µg/ml
propidium iodide (PI, Invitrogen) for 5 min in the dark. The stained samples were
Chapter 5: (Study 2) Developing 50 wt% Strontium-substituted bioactive glass and Polycaprolactone composite scaffolds for bone repair via hybrid electrospinning in a direct writing mode 79
visualized using a Zeiss Axio M2 Imager (Zeiss, Germany) fluorescent microscope (at
λ = 488 nm and λ = 568 nm excitation).
Assessment of cell attachment, morphology, and BG identification
Scanning Electron Microscopy (SEM) was used to examine cellular
attachment and morphology on the respective scaffolds. After 3, 7, 14, 21 and 28 days
in cell culture, the scaffolds were fixed and processed for imaging as described in study
1. Briefly, scaffolds were fixed in 3% glutaraldehyde immediately following cell
culture, and these scaffolds were washed in sodium cacodylate buffer (Sigma,
Australia) and 1% osmium tetroxides in cacodylate. After 2 washes in ultrapure UHQ
water (Millipore Australia, Australia), the scaffolds were dehydrated in ascending
grades of ethanol followed by Hexamethyldisilazane drying (all reagents were
supplied by ProSciTech, Australia). The sputter gold-coated scaffolds were visualized
using a Sigma field emission scanning electron microscope (FESEM, Zeiss,
Germany).
Confocal Laser Scanning Microscopy (CLSM) [155] was used to visualise the
morphology of actin fibres in green and nuclei of MC3T3 cells in blue on the scaffolds
and their interaction with the SrBG particles stained red by Alizarin red (Sigma,
Australia). As described in study 1, after 3, 7, 14, 21, 28 days, cell culture media were
removed and scaffolds were transferred into a fresh 48-well plate. Scaffolds were fixed
with 4% paraformaldehyde (PFA, Sigma) solution for 30 min at room temperature
after 2 careful washes with PBS. Samples were then washed with PBS and
permeabilized with 0.2% (v/v) Triton X-100/PBS solution for 5 min. Followed by 2
washes with PBS, samples were then incubated with 0.5% (w/v) Bovine serum
albumin (BSA, Sigma)/PBS for 10 min. The samples were stained by 0.8 U/ml Alexa
Fluor® 488 Phalloidin (Invitrogen) and 5 µg/ml DAPI in 0.5% (v/v) BSA/PBS
solution. After 1 wash with MiliQ water, the scaffolds were stained with Alizarin red
S (pH 4.2) for 5 min and washed with MiliQ water twice to remove excess stain. The
scaffolds were then stored in PBS until imaging. The PCL/SrBG scaffolds were
visualized with Leica SP5 Confocal microscope (Leica, Germany), images of
identically treated PCL only scaffolds were taken as controls.
80 Chapter 5: (Study 2) Developing 50 wt% Strontium-substituted bioactive glass and Polycaprolactone composite scaffolds for bone repair via hybrid electrospinning in a direct writing mode
Cell metabolic assay
MTT (3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide; Sigma,
Australia) assay, a cell metabolic activity assay, measures the absorbance of formazan,
reduced from MTT by mitochondria in active cells [58]. The absorbance value reflects
the metabolic activity of the cells when cultured on the scaffolds in the four experiment
groups. Briefly, on days 1, 7, 14, 21 and 28, scaffolds (n = 4) were transferred into a
fresh 48-well plate containing 500 µl of fresh media supplemented with 20 µl of MTT
solution (5 mg/ml) each well. Scaffolds were incubated (37 °C/5% CO2) for 4 hours
in the MTT supplemented media, after which the media was removed and 100 µl (D1,
7), 200 µl (D14), 400 µl (D21) or 500 µl (D28) dimethyl sulfoxide (DMSO, Merck,
Australia) was added to each well. The plates were then covered with tinfoil and placed
on an orbital shaker for 10 min. After mixing, 100 µl of DMSO eluant from each well
was transferred into fresh 96-well plates and absorption at λ = 540 nm was measured.
The obtained reading was multiplied with the dilution factors of DMSO at all time
points respectively.
Cell osteoblastic differentiation and DNA quantification assays
In order to investigate the osteogenic potential of the PCL/SrBG scaffolds and
compare the osteoblastic differentiation of cells cultured on the composite scaffolds
and on PCL scaffolds, I measured Alkaline Phosphatase (ALP) activities of the cells
adhere scaffolds in 4 experimental groups at predetermined time points. ALP protein
was selected as it is a known marker of osteoblast differentiation and plays a key role
in mineralisation [58]. The ALP assay was coupled with a PicoGreen assay which
quantifies the DNA concentration. The DNA concentration can then be used to
calculate the cell number. Therefore I normalized the ALP activity by the cell number
to find the actual potential of osteoblastic differentiation of the MC3T3 cells.
For both assays, at the 7, 14, 21 and 28 day time points, cell culture media was
removed and scaffolds were washed twice with PBS and transferred into sterile 1.5ml
Eppendorf tubes containing 500 μl of 0.2 Triton-X/ ×1 TE buffer each. The collected
samples were stored at -80 °C until further processing. At the day of assays, the tubes
containing scaffolds were vortex for 6 × 30s each to lyse the cells off the scaffolds,
and the cell lysates were transferred into fresh 1.5 ml Eppendorf tubes. From this point,
Chapter 5: (Study 2) Developing 50 wt% Strontium-substituted bioactive glass and Polycaprolactone composite scaffolds for bone repair via hybrid electrospinning in a direct writing mode 81
lysates were assayed for DNA content and ALP activity as described in section 4.2.3
of study 1.
Real-time quantitative polymerase chain reaction (RT-qPCR)
In order to further investigate the regulation of genes related to osteogenic
differentiation by the incorporation of SrBG component, the RT-qPCR was used to
quantify the relative expression of specific genes associated with early stage of
osteogenesis [159].
After days 7, 14, 21 and 28, cell culture media was removed and scaffolds (n =
9) were transferred into fresh sterile 1.5ml Eppendorf tubes containing 700μl Trizol
Reagent® (Invitrogen) with 3 scaffolds per tube. RNA was isolated according to the
manufacturer’s instructions.
The total quantity and purity of the extracted RNA were tested with a Nanodrop
Microvolume UV-Vis spectrophotometer (ThermoFisher Scientific, Australia). The
concentration of RNA was calculated to obtain a total of 500 ng cDNA per sample.
The reverse transcription process was carried out with DyNAmoTM cDNA Synthesis
Kit (Thermo Scientific, Australia) following manufacturer’s instructions.
RT-qPCR was performed on a 7500 Fast Real-Time PCR Systems (Applied
Biosystems ®, Australia) using SYBR Green as detection reagent following the same
procedure that has been reported previously [159]. The relative mRNA expressions of
ALP and Osteopontin (OPN) were assayed and normalized against the house keeping
gene β-ACTIN. Each sample was analysed in triplicate. The mean cycle threshold (Ct)
of each target gene was normalized against Ct of β-ACTIN; the relative expression
calculated using the following formula: 2-(normalized average Cts) ×104.
5.2.5 Statistical analyses
Statistical analysis was performed on results of all 4 groups at all time points
generated by MTT, ALP and RT-qPCR assays using two-way ANOVA with a post-
hoc Tukey test using IBM SPSS Statistics Software (Version 19). A p value of less
than 0.05 was considered statistically significant.
82 Chapter 5: (Study 2) Developing 50 wt% Strontium-substituted bioactive glass and Polycaprolactone composite scaffolds for bone repair via hybrid electrospinning in a direct writing mode
5.3 RESULTS
5.3.1 Particle grinding and sizing
By comparing the SrBG particle size obtained from incremental series of
grinding times, 8 hours of grinding with the micronizing mill was determined to be the
optimal grinding time, where 90% of SrBG particles by volume (Dv90) were reduced
from 109 μm to < 6 μm. (The full experimental data can be found in Appendix B) The
freeze drying took place at -20 °C for 3 days and the dried particles did not show
significant aggregation of particles as shown in Figure 5.1.
Figure 5.1 SrBG particle size distribution before grinding, after grinding and after drying. The SrBG particles were ground by micronizing mill and size distribution and their size distribution was detected by Malvern Mastersizer 3000. As shown in the graph, the peak of particle size (green line) reduced from around 100 μm down to less than 1 μm after grinding (blue line). With the optimised drying method, the dried particles showed minimum agglomeration (red line).
5.3.2 Scaffold fabrication
After a series of optimization, the electrospinning parameters used for the
composite scaffold fabrication are shown in Table 5.1. PCL and PCL/SrBG composite
were successfully electrospun into 30×30×1.5 mm fibrous scaffolds, and both type of
0
2
4
6
8
10
12
14
0.01 0.1 1 10 100 1000 10000
Volu
me
Den
sity
(%)
Particle size (μm)
SrBG 8 hrs grinding
SrBG 8 hrs grinding-freeze driedSrBG before grinding
Chapter 5: (Study 2) Developing 50 wt% Strontium-substituted bioactive glass and Polycaprolactone composite scaffolds for bone repair via hybrid electrospinning in a direct writing mode 83
scaffolds were cut into cylinders of 6 mm in diameter with a biopsy punch for further
analysis.
5.3.3 Characterisation
Scaffold morphology
Both PCL and PCL/SrBG scaffolds had similar morphology and showed 0/90°
cross-hatched laydown pattern under SEM. The PCL/SrBG scaffold showed a rough
surface with ‘brush’ like structures (Figure 5.2a, 5.2b, 5.2c, and 5.2d), while PCL
scaffolds showed smooth surface (Figure 5.2e, 5.2f, 5.2g, and 5.2h). The PCL/SrBG
fibres (75±21 μm) were larger in diameter to PCL ones (28±4 μm).
84 Chapter 5: (Study 2) Developing 50 wt% Strontium-substituted bioactive glass and Polycaprolactone composite scaffolds for bone repair via hybrid electrospinning in a direct writing mode
Figure 5.2 SEM images of 50% PCL/SrBG (a-d) and PCL (e-h) scaffolds. Both PCL and PCL/SrBG scaffolds showed 0/90° cross-hatched deposition of electrospun fibres. With the zoomed-in images shown in c and d, surface of PCL/SrBG scaffolds were rough with mini fibre-like objects possible produced during scaffold fabrication. In comparison, g and h showed a smooth surface of PCL scaffolds.
PCL
/SrB
G
PCL
Chapter 5: (Study 2) Developing 50 wt% Strontium-substituted bioactive glass and Polycaprolactone composite scaffolds for bone repair via hybrid electrospinning in a direct writing mode 85
SrBG particle distribution
As shown in Figure 5.3, the SrBG particles were stained in red by Alizarin red
as described in section 5.2.3 under a confocal microscope. These particles were found
all across the PCL/SrBG scaffold fibres. PCL scaffolds stained by the same technique
are not shown here as no fluorescence was detected by the confocal microscope. The
distribution was further assessed by a backscattered SEM, (Figure 5.4), Figure 5.4a
was an overview of a cross section of PCL/SrBG scaffold, and it showed a
homogeneous distribution of SrBG particles (bright spots) across the PCL fibres (dark
grey regions) in the whole scaffold. The elemental composition of the bright particles
were confirmed by the Sr elemental mapping, which indicated the areas with bright
spots matched the Sr rich areas as shown in Figure 5.4c and d.
Figure 5.3 Confocal laser scanning microscopy images of PCL/SrBG scaffolds stained by Alizarin red S. Red colour: SrBG particles. The composite scaffold fibres fluorescence red as Alizarin red bides to calcium in the SrBG components. The PCL scaffolds was stained with the identical conditions and as there were not biding sites for Alizarin red, the PCL fibres remained invisible under confocal microscope. Thus the images of stained PCL scaffolds were not included here.
86 Chapter 5: (Study 2) Developing 50 wt% Strontium-substituted bioactive glass and Polycaprolactone composite scaffolds for bone repair via hybrid electrospinning in a direct writing mode
Figure 5.4 Backscattered SEM image of a PCL/SrBG scaffold. (a) an overview of the scaffold and inset (b) shows a zoomed in the central region of the scaffold. The bright spots are mineral contents and in this case the SrBG particles, whereas the grey areas indicate the contrast PCL material (b), and a zoomed-in image of the arrow-point area (c). To confirm this, a Sr element mapping was performed and (d) shows the distribution of Sr in the scaffold in green, which matches the area of bright spots. Overall, the distribution of SrBG components indicate a high loading and homogeneous distribution of the inorganic filler phase with the PCL fibres.
Ion dissolution
The elemental concentrations of Ca2+, PO43-, Si4+ and Sr2+ released from both
PCL and PCL/SrBG scaffolds into α-MEM media are shown in Figure 5.5(a–d). In the
media exposed to PCL/SrBG scaffolds, the Ca2+ and concentration (in the media)
decreased rapidly after 3 hours in cell culture, and then further decreased over the next
4 weeks. PO43- concentration of the PCL/SrBG scaffold dissolution media also
decreased over the period of 4 weeks. No significant change in Ca2+ and PO43-
concentrations was observed in cell culture media exposed to PCL scaffolds. In the
PCL/SrBG scaffold dissolution media, the concentration of Si4+ showed a sharp
increase after 3 hours and 6 hours in the media and became stable after 14 days in cell
culture. The Sr2+ concentration increased slowly in the first 2 days and increased
rapidly after 3 days and 14 days in the media. No change in Si4+ and Sr2+ concentrations
were observed in PCL scaffold dissolution media.
Chapter 5: (Study 2) Developing 50 wt% Strontium-substituted bioactive glass and Polycaprolactone composite scaffolds for bone repair via hybrid electrospinning in a direct writing mode 87
Figure 5.5 Elemental concentrations of Ca2+ (a), PO4
3- (b), Si4+ (c), and Sr2+ (d) of a-MEM media incubated with 50% PCL/SrBG and PCL scaffolds over 28 days as determined by ICP-MS testing. The blue lines show the elemental concentration of PCL scaffolds dissolution media, whereas the red lines show the concentration of elements in PCL/SrBG scaffolds dissolution media. Overall, the Ca and P elements showed decreased concentration over time in the dissolution media of PCL/SrBG scaffolds and it remained constant in PCL scaffolds dissolution media (a and b). The Sr and Si elements had increased concentration in the dissolution media of PCL/SrBG scaffolds, and showed no increase in the PCL control samples (c and d).
CaP layer precipitation
The EDX analysis detected the presence of Ca and P elements on the surface of
PCL/SrBG fibres after 3 hours of scaffold immersion in α-MEM, which indicates the
formation of a CaP layer (Figure 5.6a). The CaP precipitation was not detected on PCL
surface and example EDX scanning results after 7 and 14 days in α-MEM shown in
Figure 5.6e and f respectively.
88 Chapter 5: (Study 2) Developing 50 wt% Strontium-substituted bioactive glass and Polycaprolactone composite scaffolds for bone repair via hybrid electrospinning in a direct writing mode
Figure 5.6 Example surface elemental compositions of 50% PCL/SrBG scaffolds after 3h (a), 6h (b), 1 day (c) and 2 days (d) in a-MEM media as determined by EDX. The increase of Ca/Sr and P/Sr ratio indicated the continuous formation of CaP layer at composite scaffold surface. The results of PCL scaffolds were shown as control after 7 days (e) and 14 days (f).
PCL
/SrB
G
PCL
Chapter 5: (Study 2) Developing 50 wt% Strontium-substituted bioactive glass and Polycaprolactone composite scaffolds for bone repair via hybrid electrospinning in a direct writing mode 89
5.3.4 In vitro studies
Attachment and proliferation of MC3T3 cells on scaffolds
The LIVE/DEAD staining showed viable MC3T3 cells (as shown in green)
evenly distributed across both PCL and PCL/SrBG scaffolds after 1 day cell culture
with negligible red stained cells detected. After 7 days, the cell number had
significantly increased (Figure 5.7). High magnification SEM images showed initial
attachment of cells on to both types of scaffolds (Figure 5.8a and c), and Figure 5.8b
and d showed a large number of cells covering scaffolds fibres and bridging to adjacent
fibres on scaffolds in all four groups. CLSM images showed numerous filopodia of
MC3T3 cells attaching onto the SrBG particles exposed on PCL/SrBG fibre surfaces
(Figure 5.9a) whereas cells attached and spread evenly on PCL fibres (Figure 5.9b).
The increase of MTT absorbance quantitatively indicated that PCL/SrBG composite
scaffolds were not cytotoxic by showing that the cell metabolic activity increased with
length of cell culture time in all four experimental groups (Figure 5.10). MTT results
showed a trend of cell metabolic activity in all experimental groups except PCL_O, in
which the activity peaked on day 21.
90 Chapter 5: (Study 2) Developing 50 wt% Strontium-substituted bioactive glass and Polycaprolactone composite scaffolds for bone repair via hybrid electrospinning in a direct writing mode
Figure 5.7 LIVE/DEAD staining of MC3T3 cells cultured on melt-electrospun scaffolds. (a) PCL and (b) PCL/SrBG are after 1 day culture, and (c) PCL and (d) PCL/SrBG are after 7 days culture. FDA (green fluorophor), indicates live cells, while PI (red fluorophor) indicates dead cells.
Chapter 5: (Study 2) Developing 50 wt% Strontium-substituted bioactive glass and Polycaprolactone composite scaffolds for bone repair via hybrid electrospinning in a direct writing mode 91
Figure 5.8 SEM images of MC3T3 cells cultured on PCL (a) and (b) and PCL/SrBG (c) and (d) scaffolds. Time points are: 3 days (a) and (c) (MC3T3 cells indicated by the yellow arrows), and 7 days (b) and (d) (MC3T3 cell sheets indicated by yellow arrows).
Figure 5.9 Confocal laser scanning microscopy images of MC3T3 cells cultured on melt-electrospun PCL/SrBG (a) and PCL scaffolds (b) for 3 days. Green: Alexa Fluor 488 Phalloidin conjugates (actin), Blue: DAPI (nuclei), and Red: Alizarin red S (SrBG particles).
92 Chapter 5: (Study 2) Developing 50 wt% Strontium-substituted bioactive glass and Polycaprolactone composite scaffolds for bone repair via hybrid electrospinning in a direct writing mode
Figure 5.10 MTT metabolic activity assay of MC3T3 cells over 28 days culture (n=4). Error bars = ± SD of mean. * indicates significant increase in metabolic activity (p < 0.05).
ALP activity and mineralisation (Cell differentiation)
Normalized ALP activity (per cell) in all four experimental groups showed that
scaffolds cultured in osteogenic media (PCL_O and PCL/SrBG_O groups) showed an
overall higher ALP activity compared to control groups without osteogenic media
(PCL_C and PCL/SrBG_C groups) (Figure 5.11). More importantly, cells cultured on
PCL/SrBG scaffolds showed higher ALP activity compared to those on PCL ones
regardless of culture media type. At each time point, there is a clear trend of ALP
activity increase: PCL_C < PCL/SrBG_C < PCL_O < PCL/SrBG_O groups, except
day 21 where the PCL_O group showed lower ALP activity than PCL/SrBG_C group.
After 21 days of cell culture, the cells on PCL/SrBG scaffolds showed significantly
higher ALP activity when compared to PCL scaffolds in control media (p < 0.05).
After 28 days of cell culture, the PCL/SrBG_O group showed significantly higher ALP
activity when compared to PCL_O group and PCL/SrBG_C group (p < 0.05) (Figure
5.11).
Chapter 5: (Study 2) Developing 50 wt% Strontium-substituted bioactive glass and Polycaprolactone composite scaffolds for bone repair via hybrid electrospinning in a direct writing mode 93
Figure 5.11 Normalised ALP activity of MC3T3 cells cultured on melt-electrospun PCL and PCL/SrBG scaffolds cultured in osteogenic and control media (n=4). ALP activity is divided by a total number of cells based on the DNA content obtained via PicoGreen assay. Error bars = SD of mean. * indicates significant difference (p < 0.05) between PCL/SrBG_C and PCL_C group at day 21 and PCL/SrBG_O and PCL_O group at day 28. ** indicates significant difference (p < 0.05) between PCL/SrBG_O and PCL/SrBG_C at day 28.
Expression of specific genes by MC3T3 cells on both scaffolds
The mRNA expression of ALP and OPN (osteogenic genes) were obtained by
RT-qPCR technique. The calculated relative expression of both genes in all 4 groups
at all time points were normalized by the ALP and OPN gene relative expression of
PCL_C group on day 7 to obtain the fold change of both genes respectively. Overall,
the fold change of ALP gene of PCL/SrBG_O group was higher than the other groups
over the cell culture period (Figure 5.12a). At day 21 time point, the ALP mRNA
expressed by cells in the PCL/SrBG_C group was significantly higher than the PCL_C
group; the ALP mRNA expression of PCL/SrBG_O group was significantly higher
than the PCL_O group and the PCL/SrBG_C group (Figure 5.12a). At 28 time points,
ALP mRNA expression of cells in the PCL/SrBG_C group was higher than the PCL_C
group. The RT-PCR results also showed significantly higher OPN expression level in
the PCL/SrBG_O group compared to PCL_O and PCL_C groups after 28 days in cell
culture (Figure 5.12b).
94 Chapter 5: (Study 2) Developing 50 wt% Strontium-substituted bioactive glass and Polycaprolactone composite scaffolds for bone repair via hybrid electrospinning in a direct writing mode
Figure 5.12 Gene expression of osteoblast markers ALP (a) and OPN (b) as fold change to PCL control group in all experimental groups over 28 days (n=9). Data represents mean + standard deviation. Error bars = SD of mean. (a) * indicates significant difference (p < 0.05) of ALP mRNA expression between PCL/SrBG_C and PCL_C group at day 21 and PCL/SrBG_O and PCL_O group at day 21. (b) * indicates significant difference (p < 0.05) of OPN mRNA expression between PCL/SrBG_O and PCL_O group at day 28. ** indicates significant difference (p < 0.05) of OPN mRNA expression between PCL/SrBG_O and PCL_C at day 28 and (p < 0.05).
5.4 DISCUSSION
A number of composite materials of bioactive glass/biodegradable polymer
combination have emerged in the field of tissue engineering [194]. This new family of
bioactive materials presented in this thesis, which exploits the processability of
polymers with the stiffness, strength and bioactive character of the bioactive glass
fillers, are being increasingly applied, ranging from structural implants to TE scaffolds,
instead of the traditional single-material constructs [50]. Additive manufacturing
(AM) techniques are taking the place of traditional methods in 3D composite scaffold
fabrication as the AM scaffolds have the advantages of high porosity, interconnectivity
and tailorable size and shape [12]. Electrospinning was not technically considered as
an AM technique until the recent emergence of melt-electrospinning in a direct-writing
mode, which uses the same principle as that of fused deposition modelling (FDM) to
produce 3D scaffolds with much smaller fibre size [93,94]. Several polymeric
materials have been successfully produced into scaffolds via melt-electrospinning
technique. However, melt-electrospinning of polymer/inorganic particle composite is
rarely reported (As summarized in Table 5.3, a Web of Science search for ‘melt-
electrospinning’ AND ‘polymer composite’ generated 10 results on 10/10/2016 and
Chapter 5: (Study 2) Developing 50 wt% Strontium-substituted bioactive glass and Polycaprolactone composite scaffolds for bone repair via hybrid electrospinning in a direct writing mode 95
only 2 of them as indicated by * were studies of polymer/inorganic composite
materials). This is mainly due to the fact the composite scaffold production via melt-
electrospinning has been proven an extremely difficult technique. Several factors
contribute to this situation. Firstly, the incorporation of inorganic filler phase greatly
changes the viscoelastic properties of polymers, which disturbs fibre elongation
between the spinneret/needle and the collector. This phenomenon can be explained by
studies where both strength and elongation at break of PCL composite were found to
decrease significantly with increased filler phase [195,196]. More importantly, the
incorporated inorganic filler phase acts as nucleation agents in the PCL matrix and
noticeably reduce its crystallization half time [197]. These material characterizations
match our experimental observation where the melt-electrospun PCL composite fibres
either broke between the needle tip and collector or solidified before they can attach
to the collector or existing fibres.
Table 5.3 Web of Science search results of ‘melt-electrospinning’ AND ‘polymer composite’ on 10/10/2016
Authors Composite material Year
1. Brown et al. Review paper 2016
2. Lee et al. PCL of different molecular weight 2016
3. Li et al. N/A 2016
4. Kim et al. Silk fibroin/PCL 2015
5. Ren et al. * PCL/SrBG 2014
6. Cao et al. * Multiwalled carbon nanotube/polypropylene 2014
7. Cao et al. Styrene-Acryllonitrile/Siotactic Polypropylene 2013
8. Yoon et al. Nano-/microfibrous poly(L- lactic acid) 2013
9. Carroll et al. Poly(ethylene oxide)/water 2008
10. McCann et al. TiO2-PVP/octadecane 2006
Previously, hybrid solution-electrospinning has been reported in studies where
an elevated temperature was used to assist solution-electrospinning by improving
polymer solubility in the solvents, which were essentially solution-electrospinning
[83,97,98]. In this project, we introduced the concept of hybrid melt-electrospinning
96 Chapter 5: (Study 2) Developing 50 wt% Strontium-substituted bioactive glass and Polycaprolactone composite scaffolds for bone repair via hybrid electrospinning in a direct writing mode
to successfully to circumvent the problems associated with using PCL and SrBG
particles and produce continuous fibres of PCL/SrBG composite. Different to the
reported hybrid system which was essentially solution-electrospinning, the hybrid
melt-electrospinning developed in this thesis used a minimal amount of solvent to
assist the electrospinning of polymer composite at melt temperature, which was a melt-
electrospinning process assisted by the solvent. Several key parameters play important
roles in this process: heating temperature, needle tip to collector distance, feeding
speed of the collector, voltage and flow rate of polymer composite. Most of these
parameters interact with each other and changing one individual parameter would
affect the whole scaffold production. The amount of chloroform was optimised to
enable a stable supply of composite material coming out from the electrospinning
needle and the heating temperature was kept at 60 ºC which is below the boiling point
of chloroform to avoid fast drying. Adding more chloroform to the composite would
dissolve electrospun fibres on the collector resulting in a non-porous ‘sheet’ structure
while adding less would not allow a continuous flow of composite fibre jet. The
feeding speed of collecting stage had to match the flow rate of polymer composite so
that straight fibres could be obtained. This speed also correlated with the needle tip to
collector distance and voltage. Longer collection distance would result in unstable
fibres and shorter distance would increase the risk of ‘arcing’. The additional reasons
of using minimum chloroform was to minimize its cytotoxic effect and possible impact
of solvent on the SrBG component during the electrospinning process. In this study, I
optimised the melt electrospinning process to produce smooth composite electrospun
fibres and to stack these fibres into ordered structure via machine control of collector
movement. Overall, this technique exhibited demonstrable advantages of not only
preventing breakage of PCL/SrBG composite jets but also maintaining their stability,
enabling the production of composite scaffolds in a direct writing mode, making it
suitable for 3D printing of scaffolds. These features are crucial to forming PCL/SrBG
composites into 3D electrospun scaffolds with controlled and custom layout and
anatomical precision.
As indicated by the SEM images (Figure 5.2), the PCL/SrBG scaffold fibres
were continuous and followed the machine code (G code) to form a cross-hatch
laydown pattern. However, the PCL/SrBG composite fibres (d=75±21 μm) were larger
in diameter compared to PCL ones (d=28±4 μm). This may have resulted from a
Chapter 5: (Study 2) Developing 50 wt% Strontium-substituted bioactive glass and Polycaprolactone composite scaffolds for bone repair via hybrid electrospinning in a direct writing mode 97
relatively short needle tip to collector distance, not allowing enough space for fibre
elongation. The short collection distance was adopted for depositing straight fibres in
the stability jet region by avoiding ‘whipping’ in the instability region. The addition
of SrBG particles increased the stiffness of PCL fibres mainly due to two reasons: the
composite scaffolds have thicker fibres; and the SrBG filler phase reinforced the fibre
and thus the whole matrix. In addition, the PCL/SrBG fibres were covered by ‘brush’
structures which are approximately 5 μm in length and 0.4 μm in width. These
individual brushes are actually short submicron fibres produced during scaffold
fabrication process due to rapid solvent evaporation and static charge at the surface of
fibres [198]. As there was only minimum amount of chloroform and the viscoelastic
properties of 50% PCL/SrBG composite, the jets solidified before full fibres could be
formed. Such multi-level structures of composite scaffold could potentially provide
increased surface area and additional attachment sites for cells.
The SrBG particles were homogeneously distributed along the scaffold fibres as
observed by confocal microscopy, indicating the composite preparation method to be
effective (Figure 5.3). However, only the surface SrBG particles were shown by this
technique due to the limited penetration of Alizarin red dye into PCL/SrBG composite
fibres. To observe the SrBG particles distribution further deep within the composite
fibres, SEM in the BSE mode was employed to investigate the composite scaffold.
Because the BSE signal intensity is closely related to the atomic number of elements,
BSE images can be used to provide information of elemental distribution in the
specimen [199]. BSE has also been used to identify mineral contents of biological
specimens including bone and teeth [199,200]. The PCL/SrBG scaffolds were
embedded in resin and ground to creat a cross section which exposed SrBG particles
inside the PCL fibres. The cross sections of PCL/SrBG scaffolds were surface scanned
to qualitatively identify SrBG particle distribution as they are brighter than PCL matrix
(Figure 5.4). A Sr mapping was applied on the zoomed-in areas of bright spots to
confirm they were SrBG particles which homogeneously distributed across the whole
scaffold (Figure 5.4). The large areas of SrBG particles indicated high loading of SrBG
filler phase in the composite scaffolds, and it is essential to assess whether the
increased SrBG content could enhance the bioactivity of these scaffolds.
The bioactivity of PCL/SrBG scaffolds relies on the SrBG filler phase, through
1) the formation of CaP layer which plays an important role in cell/tissue attachment
98 Chapter 5: (Study 2) Developing 50 wt% Strontium-substituted bioactive glass and Polycaprolactone composite scaffolds for bone repair via hybrid electrospinning in a direct writing mode
to scaffolds on the surface and 2) Si4+ and Sr2+ ion dissolution into the surrounding
environment that synergistically stimulates osteoblast differentiation [58]. Therefore I
found it essential to examine the formation of the surface CaP layer precipitation and
Si4+ and Sr2+ dissolution of the composite scaffolds. In order to determine the
bioactivity, the composite scaffolds were carefully characterised by EDX for surface
CaP deposition and by ICP-MS for ion dissolution α-MEM media. The elemental
concentration PCL/SrBG scaffolds in α-MEM media during a period of 4 weeks
showed a similar pattern to that of bare SrBG particles [58]. The sharp decrease of Ca
and P concentration after 3 h immersion of PCL/SrBG scaffolds (Figure 5.6a and b)
indicated a fast formation of a CaP layer, which was detected on scaffold surface by
EDX analysis (Figure 5.7). Compared to our previous study on 10% PCL/SrBG
scaffolds [191], the formation of CaP layer on 50% PCL/SrBG scaffolds was more
rapid attributed to the increased amount of SrBG within the scaffold. The increase of
Ca and P elements correlated well with the decrease of Ca and P elements in the media
shown in Figure 5.6a and 5.6b. Further studies of CaP precipitation of SrBG should be
carried out in the future as it is reported that the Sr can potentially substitute Ca in the
CaP precipitation process. Additionally, the elemental concentration of metal ions
such as Mg, Sr and Mn could negatively affect the formation of CaP layer [201], which
should also be studied to determine the optimal concentration of Sr2+ in the media.
Furthermore, Sr and Si concentration in the PCL/SrBG dissolution media increased
over the incubation period and the peak values of which were comparable to that of
bare SrBG particles [58]. In the study of SrBG particles with 50% and 100% Sr
substitution [58], the ion dissolution showed a burst release and the concentration of
Sr peaked after 60 min of incubation at around 50 ppm and 85 ppm respectively.
Unfortunately, the ion release profile of SrBG with 75% Sr substitution was not found
in published studies but it is projected to be between the peak values of 100% and 50%
SrBG. According to this analysis, the ion dissolution of Sr showed a burst release of
elements and peaks were reached after 120 min of incubation. In this study, the
concentration of Sr and Si increased sharply after 2 days and 7 days of incubation. This
2-stage increase of ion release may result from the partial degradation of the PCL layer
on the surface due to the high alkaline environment near the surface. The degradation
of PCL then leads to the exposure of more SrBG particles, which released more Si and
Sr ions into the media. When compared to a recent study on PCL/SrBG composite
scaffold [77], the electrospun PCL/SrBG scaffolds in this project released Sr and Si
Chapter 5: (Study 2) Developing 50 wt% Strontium-substituted bioactive glass and Polycaprolactone composite scaffolds for bone repair via hybrid electrospinning in a direct writing mode 99
into the cell culture media with sufficient concentration to potentially stimulate
osteoblast differentiation. Based on the SrBG composition [57], approximately 5.3
wt% of total the amount of Sr element and 5.5 wt% of Si element within the composite
scaffolds were calculated to have released into the media after 28 days. Therefore, the
PCL/SrBG scaffolds had the potential to enable a long-term sustainable ion dissolution
which is desirable for bone regeneration. To confirm the effect of increased SrBG
contents, an in vitro cell study was carried out following the scaffold characterization.
After being fabricated, the PCL/SrBG scaffolds were etched with 5M NaOH for
15 min to enhance surface hydrophilicity and to maximize the exposure of SrBG
particles on the scaffold surface. The etch time in this study was shorter compared to
the 1 hour NaOH etching in study 1 to maintain the structural integrity of the
PCL/SrBG scaffolds. In the PCL/SrBG (50 wt%) scaffolds there were a significant
increase of SrBG filler phase (from 10 wt% to 50 wt%) and removing too much PCL
from the PCL/SrBG fibres would weaken the bonding between fibres at the
intersection of the cross-hatch structure. The PCL scaffolds did not have this concern
but were etched in the same condition as control. As reported previously, an initial pH
increase was observed when the PCL/SrBG scaffolds were immersed in cell culture
media [77], which may pose cytotoxic effect on the seeded MC3T3 cells. The pH
increase was due to the rapid ion exchange of Na+, Sr2+ or Ca2+ in SrBG with H+ from
the cell culture media, and the pH would stop increasing when the Si-OH groups
condensate and repolymerise on the scaffold surface [54]. Therefore, the sterile
PCL/SrBG scaffolds were conditioned in serum-free media for 1 week prior to cell
seeding. The conditioned scaffolds were sterilized again by UV radiation immediately
before cell seeding. Again, it was not necessary for the PCL scaffolds but they were
processed with the same conditions as control.
The LIVE/DEAD assay found only negligible dead cells (red) on both
PCL/SrBG and PCL scaffolds at day 1 and day 7 time points. The live cells (green)
were evenly distributed across the scaffold, which suggested that the increased amount
of SrBG content did not have cytotoxic effects on cells. The stain at the day 7 time
point showed a large number of cells on both types of scaffolds, suggesting that
attachment and migration of MC3T3 cells were not significantly altered by the
incorporation of SrBG to the PCL component, or the increase in fibre diameter (Figure
5.7). The SEM images of cells on PCL/SrBG and PCL scaffolds were in line with the
100 Chapter 5: (Study 2) Developing 50 wt% Strontium-substituted bioactive glass and Polycaprolactone composite scaffolds for bone repair via hybrid electrospinning in a direct writing mode
LIVE/DEAD assay results, showing that the scaffold fibres were covered by the
attached cells (Figure 5.8). The active attempt to bridge adjacent scaffold fibres by
MC3T3 cells was observed after 7 days in cell culture at the intersection of the cross-
hatch structures (Figure 5.8b and d). These observations were quantitatively confirmed
by the MTT assays results showing the metabolic activity of cells increased over the
cell culture period among all 4 groups, which suggested that the high loading of SrBG
particles did not have any significant negative effect on the proliferation of the cells
upon the scaffolds. The PCL_O group showed a peak of MTT activity at day 21 time
point due to an increase of cell population on those scaffolds. This could be explained
that the majority of the cells were at the growth/proliferation stage of the osteoblast
development [202]. As the following osteoblasts differentiation took place, the MTT
showed a decrease at the day 28 time point. The interaction between cells and SrBG
particles were again detected via CLSM (Figure 5.9), and cells were found to attach
and well-stretched on the PCL/SrBG scaffold surface with a large amount of SrBG
particles. This further proved that the SrBG filler phase was not cytotoxic to the
attached MC3T3 cells.
Alizarin red S stain, the most commonly used in vitro assays to determine ECM
mineralization [156], was not used in this study as the large amount of SrBG contents
could be stained and interfere with the results. ALP assay was once again used to
quantify the extent of osteoblast differentiation between the experiment groups. As
mentioned earlier, ALP is a known early marker for osteoblast differentiation, the ALP
activity can be used to determine extent of osteoblast differentiation and mineralization
[203]. To exclude the effect of difference in cell numbers on total ALP activity across
all experimental groups, the ALP activity was normalized by the cell numbers
calculated from the PicoGreen DNA quantitation assay. The normalized ALP activity
results indicated that both osteogenic media and the PCL/SrBG scaffolds had
significant impact on the level of ALP activity. More specifically, the cells cultured on
PCL/SrBG scaffolds showed higher ALP activity compared to those on PCL ones
irrespective of culture media types at detection points over the culture period; cells
cultured in osteogenic media showed higher ALP activity compared to those in non-
osteogenic (growth) media (Figure 5.11). Notably, after 21 days in the control growth
media groups, the significantly higher ALP activity of cells on PCL/SrBG scaffolds
compared to cells on PCL scaffolds indicated that the incorporation of 50 wt% SrBG
Chapter 5: (Study 2) Developing 50 wt% Strontium-substituted bioactive glass and Polycaprolactone composite scaffolds for bone repair via hybrid electrospinning in a direct writing mode 101
made the composite scaffolds osteogenic in vitro without osteogenic cytokines. The
normalized ALP activity of PCL_O group at day 21 time point was lower than that of
the day 14 time point because the increase of cell population which matched the cell
metabolic activity assay result. After 28 days, the ALP activity of cells on PCL/SrBG
scaffold in osteogenic media was significantly higher compared to both PCL_O and
PCL/SrBG_C groups, which indicated that in addition to SrBG components, the
osteogenic media was still playing an important role in the osteoblastic differentiation
of MC3T3 cells. When comparing with study 1 (10% SrBG), the increase of SrBG
loading from 10 wt% to 50wt % in the PCL/SrBG composite scaffolds led to drastic
increase of Si4+ and Sr2+ ion concentration: peak concentration of Si4+ almost doubled
from 32.74ppm to 57.57ppm; peak concentration of Sr2+ increased from 55.4ppm to
74.34ppm (Figure 5.5). The increased SrBG led to sufficient Si4+ and Sr2+ ions
available for cells cultured on the PCL/SrBG scaffolds and the synergistic function of
these two ions was key in promoting osteoblastic differentiation as reported previously
[58]. More specifically, the Si4+ showed a more rapid release reaching the peak value
after 14 days of immersion in cell culture media compared to Sr2+ which peaked after
28 days of dissolution (Figure 5.5). This unsynchronized ion release combined with
the fact that the media were changed every two days had contributed to the significant
effect of the PCL/SrBG scaffolds was only observed towards the end of the culture
period.
In order to further support the osteogenic effect of the PCL/SrBG scaffolds, the
expression of ALP gene was investigated (Figure 5.12a). Overall, the ALP gene
expression followed a similar trend of ALP activity where the PCL/SrBG_O showed
the highest ALP expression over the culture period and both scaffold type and culture
media type played important roles in the expression of ALP gene. Notably at the day
21 time point, the ALP gene expression was significantly higher when cells were
cultured on the PCL/SrBG scaffolds group compared to the cells on PCL scaffolds in
both growth control media and media supplemented with osteogenic reagents (ascorbic
acid, β-glycerophosphate and dexamethasone). These discoveries agreed well with
ALP activity at the same time point, which further confirmed the osteogenic capacity
of the PCL/SrBG composite scaffolds, and are in line with the findings of previous
studies by Poh et al.[77] and Gentleman et al.[58]. Osteopontin (OPN), a downstream
gene associated with osteoblast development, was also assessed for its mRNA
102 Chapter 5: (Study 2) Developing 50 wt% Strontium-substituted bioactive glass and Polycaprolactone composite scaffolds for bone repair via hybrid electrospinning in a direct writing mode
expression in the 4 experimental groups (Figure 5.12b). At day 21 and 28 time points,
upregulation of OPN genes was observed in PCL/SrBG_C and PCL/SrBG_O groups
compared to PCL_C and PCL_O groups respectively. At day 28, the PCL/SrBG_O
group had significantly higher OPN expression compared to PCL_O and PCL_C
groups, which indicated the osteogenic effect of SrBG components of the composite
scaffolds. The OPN expression was also much higher in the PCL/SrBG_C group when
compared to PCL_C group at day 28 (the mean value of PCL/SrBG_C group was over
800 times of the PCL_C group), however, the difference was not statistically
significant. The enhanced ALP activity and upregulation of ALP and OPN genes
indicated that the increased SrBG components did contribute to the osteoinductivity of
the PCL/SrBG composite scaffolds.
5.5 CONCLUSION OF STUDY 2
In order to produce PCL/SrBG composite scaffolds with sufficient SrBG filler
phase, a hybrid melt-electrospinning technique was developed and reported for the
first time in this study. 50% PCL/SrBG composites were successfully fabricated with
this electrospinning technique in a direct writing mode into controlled porosity and
laydown pattern. The SrBG particles were found evenly distributed across the scaffold
fibres. The in vitro bioactivity of the PCL/SrBG scaffolds was characterised and the
concentration of Sr and Si elements in the cell culture media increased over the
incubation period and the peak value of which were comparable to that of bare SrBG
particles. The high concentration of Sr and Si and the sustained release of these
elements indicate that the PCL/SrBG scaffolds can potentially stimulate osteoblast
differentiation. The precipitation of CaP which facilitates cell attachment was
evidenced on the PCL/SrBG scaffold surface, and Ca and P were found to increase
over the incubation time. These results show the hybrid melt-electrospinning technique
is promising for the fabrication of a large variety of polymer/inorganic particle
composite. The PCL/SrBG (50 wt%) scaffolds demonstrated enhanced in vitro
bioactivity compared to PCL/SrBG of 10 wt% SrBG loading (figure 5.13). The
subsequent in vitro cell study on the new scaffolds proved the PCL/SrBG (50 wt%)
scaffolds presented minimum cytotoxicity and showed the ability to support MC3T3
cell attachment and proliferation using LIVE/DEAD assay, MTT assay, SEM and
CLSM imaging techniques. More importantly, the findings in this study also
quantitatively validated the osteogenic capacity of PCL/SrBG scaffolds to stimulate
Chapter 5: (Study 2) Developing 50 wt% Strontium-substituted bioactive glass and Polycaprolactone composite scaffolds for bone repair via hybrid electrospinning in a direct writing mode 103
osteoblast differentiation without osteogenic supplement in the cell culture media.
These properties of PCL/SrBG composite scaffolds thus make them potential tissue
engineered bone substitutes for treating human bone defects.
Figure 5.13 The direct comparison of Si4+ and Sr2+ ion concentration of PCL/SrBG (10wt%) scaffolds in study 1 and PCL/SrBG (50wt%) scaffolds in study 2. The figures indicated increased Si4+ and Sr2+ ion concentration (red lines) of the PCL/SrBG (50wt%) scaffolds dissolution media compared to their concentration in PCL/SrBG (10wt%) scaffolds dissolution media (green lines). The ion concentration of PCL scaffolds dissolution media was examined as controls (blue lines).
Chapter 6: (Study 3) Development of optimised histological processes for analysis of large and complex bone and implants 105
Chapter 6: (Study 3) Development of optimised histological processes for analysis of large and complex bone and implants
A typical scaffold development process includes scaffold fabrication, in vitro
assessment and in vivo assessment with histology being one of the key techniques for
scaffold explant analysis. This PhD project aims to develop a PCL/SrBG composite
scaffold through melt-electrospinning and assess the osteogenic capacity of the
scaffold using in vitro assays and examine their in vivo osteogenesis via animal
implantation followed by explantation analysis using histology. This histology
optimisation study was run in parallel to study 1 and 2 aiming to develop the optimal
histological techniques for the analysis of PCL/SrBG scaffolds at the time of their
implantation. The histological technique optimization was carried out with four animal
species to make sure it would cover the intended animal models for PCL/SrBG
scaffold implantation and also lead to a comprehensive histological study of bone
tissues. Owing to time restrains we did not implant the scaffolds but these will be
implanted in the future and can be assessed using the optimised histology methods
developed in this chapter.
6.1 INTRODUCTION
In recent years, the tissue engineering (TE) research has grown tremendously,
especially with the development of three-dimensional scaffolds for tissue or organ
regeneration [32]. To be able to translate TE scaffolds into clinical applications, a
series of bioactivity tests must be undertaken in vitro and in vivo [100]. Currently, it is
agreed that pre-clinical animal models reflect the complex clinical situation and
provide a real understanding of the tissue regenerative capacity of the
implants/scaffolds [100,101]. However, these in vivo studies are considerably resource
and time intensive. In addition, considering the limited numbers of animals that can be
ethically approved to use, the specimens obtained from these in vivo studies are
extremely precious. Therefore, it is imperative to employ the most appropriate
assessment techniques to obtain maximum information from extremely limited
106 Chapter 6: (Study 3) Development of optimised histological processes for analysis of large and complex bone and implants
resources. For bone tissue engineers, histology is one of the key ex vivo assessment
techniques to characterise bone formation and mineralization mechanisms including
cellular pattern and distribution within the bone matrix [105].
Histology is traditionally an analytical method in biology and medicine for the
structure and composition of tissues at the microscopic level [6]. It is also widely
employed in bone TE research as a primary method of investigation, as well as a
validation of other analytical techniques, such as conventional x-rays [204] and micro-
computed tomography imaging [205]. By processing tissue explants into thin sections
on the micron scale through fixation, embedding, sectioning and staining steps,
histology provides microscopic analysis of “two-dimensional” tissue sections [109].
Histology techniques are highly versatile with numerous stains available for specific
tissue types. Despite the recent advances in histological techniques, it remains a
challenge to process mineralized tissues such as bone and teeth due to their
significantly greater hardness and the heterogeneity of the tissues in healing bones, and
requires the use of different embedding materials than paraffin which remains the most
widely used embedding material for soft tissues [8].
In order to section bulk bone explants, two approaches have been employed
which aim to match the hardness of bone to that of the embedding media: i) to soften
bone specimens by decalcification (by reagents such as ethylenediaminetetraacetic
acid (EDTA)) for paraffin embedding; and ii) to use hard embedding materials such as
plastic/resin for non-decalcified bones [109]. The embedding media and sectioning
techniques for producing bone tissue sections are summarized in table 6.1. Paraffin
embedded specimens are routinely sectioned by a rotary microtome to yield good bone
morphology, enzyme, and immunohistochemistry results through standard histological
stains [6,109]. This approach, however, can lead to poor preservation of bone structure
[110] and often loses information on bone regeneration and mineralization due to the
decalcification process. Decalcification of bone is also a time-consuming process that
can take several months. Other prominent disadvantages of paraffin embedding
approaches include limited specimen size and inability to process metallic implants.
Chapter 6: (Study 3) Development of optimised histological processes for analysis of large and complex bone and implants 107
Table 6.1 Summary of embedding media and sectioning techniques in study 3. Embedding media Paraffin MMA resin Technovit 9100 resin
Bone tissue state Decalcified Non-decalcified Non-decalcified
Sectioning
technique
Paraffin
microtome
Sledge
microtome
Ground
sectioning
Sledge
microtome
Ground
sectioning
Section naming
and thickness
Paraffin
sections ~ 5
μm
Resin thin
sections ~
10 μm
Resin ground
sections ~ 20 to
50 μm
Resin thin
sections ~
10 μm
Resin ground
sections ~ 20 to
50 μm
As bone tissue engineering research advances towards larger and more complex
tissues, the traditional paraffin embedding approach can no longer provide optimal
results, especially at the scaffold/tissue or soft/hard tissue interfaces. Plastic
embedding, most commonly methyl methacrylate (MMA) resin, overcomes the
shortfalls with paraffin approach and causes minimum disruption of tissue and cellular
morphology. The MMA embedding approach has been providing quality
morphological results in hard tissue research for nearly 50 years [8,110,122].
However, MMA sections are not appropriate for protein markers detection techniques
like IHC owing to destruction of enzymes and antigen epitopes during the exothermic
polymerization process (reaction temperature up to 80 °C) [121]. To overcome this
disadvantage of conventional MMA, low temperature polymerizing resin was later
introduced [8,110]. The Technovit 9100 New® resin polymerizes at a temperature as
low as -20 °C [8,110,121,123], which allows the preservation of epitopes that are
suitable for IHC examination. Both MMA and Technovit 9100 New® resin embedded
tissues can be sectioned with ground sectioning technique and heavy-duty sledge
microtomes, in the same way as traditional MMA [109].
Haematoxylin and eosin (H&E) stain and IHC are the most routine histological
stains that demonstrate general tissue morphology and osteogenic/angiogenic markers
respectively. Other more specific stains differentiate mineralized bone from
surrounding tissues, including von Kossa silver nitrate methods and Goldner’s
trichrome stains [109]. Goldner’s trichrome stain being one of the most popular
staining technique that has been employed in numerous in vivo bone regeneration
studies [8-26].
108 Chapter 6: (Study 3) Development of optimised histological processes for analysis of large and complex bone and implants
With all of these histological techniques available to TE researchers, the
selection of techniques is, however, largely based on individual experience and can
lead to suboptimal results or irreversible loss of information if explanted specimens
are not appropriately and reproducibly processed. Over the years, different research
groups have been publishing results of histological stains of bone specimens using
their own variation of standard operating protocols. Without agreed standards with
respect to what constitutes successful histological staining, it is difficult to
communicate and compare the progress of bone mineralization based on the different
staining results from various research groups. As mentioned above, Goldner’s
trichrome has been one of most important histological stains for bone specimens, and
images of bone/bone implant sections stained by this method from the literatures have
demonstrated inconsistent and uneven stains of mineralized bone and surrounding
connective tissues [125,131,132,206]. Some of the exemplary images can be found in
Appendix A.
Presently, no published data exists to directly compare the staining results of
bone specimens embedded in paraffin, MMA, and Technovit 9100 NEW®. In this
study, we are therefore filling in this gap by optimizing and comparing the staining
techniques for the assessment of mineralized tissue processed via above mentioned
embedding and sectioning approaches using various common animal tissues including
sheep tibia, pig fibula, rat and mouse legs and mouse paws. The full panels of
microscopic images of stains are provided with detailed operating procedures, which
are expected to standardize the histological techniques for bone specimen analysis.
Here I also demonstrate in fine details of the methodology to divide and distribute
large tissue-engineering bone specimens for both resin and paraffin histology for their
comprehensive analysis. The outcome of this study can be used as a guide to
researchers in bone TE field for their pre-planning of histological analysis based on
specific research aims by providing a series of optimised staining methods which are
optimised for different animal species.
6.2 MATERIALS AND METHODS
6.2.1 Bone tissues and pre-processing preparation
In this study, I utilised bone tissues across four animal species: sheep tibiae
[207], pig fibulae [208], mouse paws and legs [209], rat legs [102], as well as, tissue
Chapter 6: (Study 3) Development of optimised histological processes for analysis of large and complex bone and implants 109
engineered sheep tibiae with polymeric and titanium implants. The use of bone tissues
and bone explants were approved by Queensland University of Technology Animal
Ethics Committee (ethics approval numbers: 1400000776 and 1000001139).
The study design is summarized in Figure 6.1. Prior to fixation, samples were
resected to remove excess soft tissue. Triplicate 10 mm thick transverse sections were
cut for sheep tibia, 5 mm transverse sections for pig fibulae, 3 mm thick for mouse and
rat legs were cut using an EXAKT 310 Diamond Band Saw (EXAKT Apparatebau
GmbH & Co.KG, Norderstedt, Germany). Four mouse paws were kept as an entire
sample measuring between 1.4 - 1.9 cm. The tissue engineered bone explants were cut
as illustrated in Figure 6.4. The specimens were fixed in 10% neutral buffered formalin
(10:1 volume of fixative to tissue) for 24 h in sealed containers. To completely remove
the fixative solution, the bone specimens were thoroughly washed in deionized water
(DI water) for 4 h prior to further processing. Bone specimen triplicates were divided
equally into three groups for embedding in three media according to histological
methodology: paraffin, Methyl Methacrylate (MMA) and Technovit 9100 New®.
Figure 6.1 Schematic summary of study 3 design. The animal tissues were divided into three identical parts and embedded in paraffin, MMA and Technovit 9100 resin respectively. The obtained sections of these tissues were then stained by H&E, Von Kossa, Goldner’s trichrome and IHC techniques. The staining results were then compared.
6.2.2 Processing and embedding
Paraffin. Bone tissues were decalcified in 10% EDTA (Ajax Finechem,
Australia) at 4 °C for 14 weeks to ensure complete demineralization and the endpoint
110 Chapter 6: (Study 3) Development of optimised histological processes for analysis of large and complex bone and implants
was carefully determined by probing the tissues with a sharp needle. EDTA solution
was refreshed weekly. The bone specimens were briefly rinsed with DI water to
remove excess of EDTA solution, and then placed in embedding cassettes (Techno
Plas, South Australia) for processing in an ExcelsiorTM ES Tissue Processor (Thermo
Scientific, Australia), prior to embedding in molten paraffin wax at 60 °C (Thermo
Shandon Histocentre 3 Embedding Station, Thermo Scientific, Australia).
Resin. Specimens processed for MMA and Technovit resin were dehydrated for
3 weeks through graded series of ascending concentrations of ethanol. Specimens were
then degreased in 2 changes of xylene for 8 h at RT to remove ethanol residual and
facilitate resin penetration.
Methyl Methacrylate (MMA) Following degreasing with xylene, the specimens
were immersed in MMA infiltration solution (MMA supplemented with 3%
polyethylene glycol (PEG)) of 20 times of volume to that of tissues and kept for 1-2
weeks at RT: sheep tibiae, pig fibulae and mouse paws for 2 weeks and rat and mouse
legs for 1 week. The base molds were prepared during the infiltration by polymerizing
approximately 1 cm of MMA embedding solution (MMA+3% PEG+0.3% di(4-tert-
butylcyclohexyl) peroxydicarbonate Perkadox, Akzo Nobel Polymer Chemicals LLC)
at the bottom of embedding containers. Specimens were then transferred into the base
molds and covered by MMA embedding solution to 1-2 cm above the top of
specimens. The embedded specimens were vacuumed at -70 KPa for 2-5 min to
remove oxygen and polymerized at RT for 3-5 days.
Technovit 9100 New® Following degreasing with xylene, the allocated
specimens for Technovit 9100 New® methodology were processed and embedded in
the low-temperature embedding system Technovit 9100 New® (Heraeus Kulzer
GmbH, Germany). Pre-infiltration, infiltration, and embedding solutions were
prepared according to Table 6.1, using Technovit 9100 NEW® basic solution
destabilized by filtering through chromatography column loaded with 50 g of
aluminium oxide. All bone specimens were immersed in pre-infiltration solution at 4
°C for 1 week. Following pre-infiltration, the specimens were transferred into the
infiltration solution and vacuumed at -70 KPa for 2-5 min to facilitate infiltration. The
specimens were stored in infiltration solution at 4 °C for 1 week. The pre-infiltration
and infiltration solutions were 20 times of volume to that of tissues being processed.
The infiltrated specimens were finally transferred into plastic embedding moulds,
Chapter 6: (Study 3) Development of optimised histological processes for analysis of large and complex bone and implants 111
filled with fresh embedding solution (a mixture of 9 parts of stock solution A and 1
part of stock solution B) and vacuumed at -70 KPa for 2-5 min to remove oxygen. The
polymerization took place at -20 °C for 5-7 days. Once polymerized, the resin blocks
were transferred to 4 °C for 2 h, before storage at RT.
Table 6.2 Preparation of Technovit 9100 New® solutions
Solutions Basic solution
PMMA powder Hardener 1 Hardener 2 Regulator Storage
Pre-infiltration 200 ml 1 g 4 °C Infiltration 250 ml 20 g 1 g 4 °C Stock solution A* 500 ml 80 g 3 g 4 °C Stock solution B* 50 ml 4 ml 2 ml 4 °C
* Solution A and Solution B are polymerization solutions which must be mixed up in the ratio of 9A:1B immediately prior to use.
6.2.3 Sectioning
Paraffin microtomy. Five-micron transverse tissue sections were generated on
a Leica RM2235 rotary microtome (Leica Biosystems, Nussloch Germany). The
ribbons of sections were floated on a 40 °C water bath to unfold and collected onto
polylysine-coated microscope slides (Thermo Scientific, Australia). To improve
sectioning quality, the paraffin blocks were cooled on ice for 5 min after every 3-4
ribbons. The slides were oven dried at 60 °C for 16 h.
Resin sledge microtomy. Tissue engineered sheep tibiae embedded in either
MMA or Technovit 9100 were sectioned with a sledge microtome (Polycut-S,
Reichert-Jung, International Medical Equipment, USA) using a tungsten carbide blade
at a section thickness of 8 μm. The resin blocks were moistened constantly with ethanol
(70% for MMA and 30% for Technovit) to improve section quality. Sections were then
flattened with 95% ethanol onto gelatin-coated microscope slides, covered with
polyethylene film and compressed between bibulous paper sheets to remove excess
ethanol. Finally, the slides were stacked in a metal slide holder under compression and
dried in an oven at 60 °C for 3-4 days. The resin sections obtained by the sledge
microtome are referred to as resin thin sections in this paper to distinguish them from
resin ground sections, which are much thicker.
Resin ground sectioning. As described previously [111,210], this technique
involves two major steps: cutting a section from the polymerized resin block and
grinding the section to an appropriate thickness. Methyl methacrylate and Technovit
112 Chapter 6: (Study 3) Development of optimised histological processes for analysis of large and complex bone and implants
polymerized resin blocks were shaped and trimmed of excess resin, leaving a border
of resin 5 mm from the specimen, using an Exakt 310 diamond band saw (EXAKT
Advanced Technologies GmbH, Germany). An overview of the process for resin block
preparation before sectioning is schematically shown in Figure 6.2. Blocks were glued
to a 50 x 100 x 2mm acrylic slide (A) with cold curing resin Technovit 4000 system
(Technovit powder mixed in Technovit syrup I and syrup II, EXAKT Advanced
Technologies GmbH, Germany) using the Exakt vacuum system 401 (EXAKT
Advanced Technologies GmbH, Germany). The to-be-sectioned surface of resin block
was polished with 800 grit sandpaper to a smooth surface, parallel to slide A with the
Exakt 400 CS microgrinding system (EXAKT Advanced Technologies GmbH,
Germany). The resin block surface was then degreased with ethanol (100% ethanol for
MMA and 30% ethanol for Technovit embedded blocks) and affixed onto a second
acrylic slide (B) (dimension: 50 × 100 × 2 mm) with Technovit 7210 VLC photo curing
adhesive (EXAKT Advanced Technologies GmbH, Germany).
Chapter 6: (Study 3) Development of optimised histological processes for analysis of large and complex bone and implants 113
Figure 6.2 An overview of resin block preparation and ground sectioning process. The tissues embedded in resin are processed following the direction of arrows for the preparation of double-slide ‘sandwich’ structure. The double-slide prepared resin blocks are cut by the saw along the black line and processed following the direction of arrows for the production of resin ground sections. The resin ground sections are then stained by various histological staining techniques as detailed in section 6.2.4.
The overview of cutting and grinding of the prepared sandwiches structure is
shown in Figure 6.2. The mounted resin block was then cut with the Exakt 310
diamond band saw producing a section 100 μm – 200 μm thick. To yield the desired
thickness, the amount of tissue and resin to be removed was pre-selected with the AW
110 electronic measuring and control System (EXAKT Advanced Technologies
GmbH, Germany) and posteriorly ground using an Exakt 400 CS microgrinding
system (EXAKT Advanced Technologies GmbH, Germany) guided by a sequence of
finer abrasive papers summarized in Table 6.3. For sheep tibiae, pig fibulae, rat legs,
114 Chapter 6: (Study 3) Development of optimised histological processes for analysis of large and complex bone and implants
mouse paws, and legs bones sections were ground down to ~27 μm while tissue-
engineered sheep tibiae sections were ground down to a final thickness of ~50 μm. The
thickness measurement was carried out with a micrometer (Mitutoyo, USA). After the
desired section thickness was reached, specimens were air dried at RT prior to staining.
Table 6.3 Suitable sandpaper grits according to section thickness: a. final thickness=27 μm; b. final thickness=50 μm
a Thickness of Sample Grit of Paper Amount to be removed
250 microns P800 130 microns 120 microns P1000 40 microns 80 microns P1200 40 microns 40 microns P2500 10 microns 30 microns P4000 3 microns
Final section 27 microns
b
Thickness of Sample Grit of Paper Amount to be removed 250 microns P800 97 microns 153 microns P1000 40 microns 103 microns P1200 40 microns 63 microns P2500 10 microns 53 microns P4000 3 microns
Final section 50 microns
6.2.4 Staining
The majority of conventional histological stains have been developed for
paraffin sections. Here, I describe optimised and standardized staining protocols for
resin thin and ground sections.
Haematoxylin & Eosin (H&E) staining
H&E is one the principle stains in histology that allows a general overview of
the histological sections. The nuclei of cells are stained blue by hematoxylin and the
eosin stains the cytoplasm pink [109].
Paraffin. The sections were deparaffinized with 2 changes of xylene (8 min
each) and rehydrated with descending concentrations of ethanol (100%, 90% and 70%,
2 min each), and placed in DI water for 5 min prior to staining. The sections were
Chapter 6: (Study 3) Development of optimised histological processes for analysis of large and complex bone and implants 115
stained with Mayer’s haematoxylin (CliniPure, Grale HDS, Australia) for 10 min at
RT, followed by 10 min blueing under warm running tap water. The sections were then
stained with eosin (CliniPure, Grale HDS, Australia) for 10 s at RT. Following
staining, the sections were dehydrated in ascending concentrations of ethanol and
mounted with Eukitt mounting media (Sigma, Australia).
Resin. The staining protocol for resin sections was optimised during this PhD
project. Both MMA and Technovit ground sections were surface etched by immersion
in xylene for 20 min followed by immersion in 100% ethanol for 20 min. Following
rehydration with a descending concentration of ethanol (90% and 70%, 5 min each),
the sections were placed in DI water for 5 min prior to staining. The resin ground
sections were stained with Weigert’s haematoxylin for 15 min, followed by 10 min
blueing under warm running tap water, differentiated in 1% acetic acid (2 × 1 min)
(Merck, Australia), and then stained with eosin for 30 s. The sections were then air-
dried, cleared in xylene and mounted with Eukitt mounting media (Sigma, Australia).
Goldner’s trichrome staining
Goldner’s trichrome staining is a commonly used method in bone histology,
allowing tissue differentiation based on specific colours binding to different tissue and
cell structures providing colour difference for each tissue structure as well as by
morphological identification [109].
Goldner’s trichrome staining procedures were optimised based on existing
standard operating procedures in our lab. The solutions for Goldner’s trichrome
staining are listed in Table 6.4.
Table 6.4 Goldner’s trichrome staining solutions Solutions Recipe
Weigert’s iron hematoxylin A 10g haematoxylin monohydrate (Merck, Australia)
dissolved in 1000 mL 95% ethanol
Weigert’s iron hematoxylin B 5.8 g ferric chloride (Merck, Australia) and 5 mL
hydrochloric acids dissolved in 500 mL DI water
Acid Fuchsin Ponceau A 6g Ponceau Xylidine (Merck, Australia) dissolved in
600 mL DI water
Acid Fuchsin Ponceau B 2g Acid Fuchsin (Merck, Australia) dissolved in 600
mL DI water
116 Chapter 6: (Study 3) Development of optimised histological processes for analysis of large and complex bone and implants
Tungstophosphoric acid–orange G 10g orange G (Merck, Australia) and 20g
tungstophosphoric acid (Merck, Australia) dissolved
in 500 mL DI water
Light green (SF yellowish) 1g light green SF yellowish (Merck, Australia) and
1mL acetic acid (Merck, Australia) dissolved in 500
mL DI water
Paraffin. 5 μm sections were deparaffinised with 2 changes of xylene (8 min
each) and rehydrated with a descending concentration of ethanol (100%, 90% and
70%, 2 min each), and placed in DI water 5 min prior to staining. The sections were
placed in Weigert’s iron haematoxylin solution for 25 min and blued under warm water
for 10 min. The sections were then immersed in acid Fuchsin-Ponceau working
solution for 10 min, rinsed in fresh 1% acetic acid (2 × 30 s) before being placed in
tungstophosphoric acid - orange G solution for 20 min. Following another 2 × 30 s
rinse in 1% acetic acid, the sections were immersed in the light green solution for 10
minutes. Subsequently, the sections were rinsed in 1% acetic acid (2 × 30 s) and then
rinsed in DI water for 2 min. The stained sections were dehydrated in ascending
concentrations of ethanol (70% and 100%, 2 × 30 s), cleared in xylene (2 × 6 min),
and finally mounted with Eukitt mounting media.
Resin. Resin thin sections (8 μm) were deplasticized in acetone (2 × 20 min),
rehydrated with descending concentrations of ethanol (100%, 90% and 70%, 5 min
each), and placed in DI water for 5 min. The resin ground sections were surface etched
by immersion in xylene and 100% ethanol for 20 min each to facilitate dye infiltration.
Following rehydration with a descending ethanol series (90% and 70 %, 5 min each),
the resin ground sections were placed in DI water for 5 min prior to staining. Both
resin thin and ground sections were placed in Weigert’s iron haematoxylin solution
for 25 min and blued under warm tap water for 10 min. These sections were immersed
in acid Fuchsin-Ponceau working solution for 10 min, rinsed in fresh 1% acetic acid
(2 × 30 s) before being placed in tungstophosphoric acid - orange G solution for 20
min. Following another 2 × 30 s rinse in 1% acetic acid, the sections were immersed
in the light green solution for 10 min (resin thin sections) and 15 min (resin ground
sections). Subsequently, the sections were rinsed in 1% acetic acid (2 × 30 s) and then
rinsed in DI water for 2 min. (Some resin ground sections need extended immersion
Chapter 6: (Study 3) Development of optimised histological processes for analysis of large and complex bone and implants 117
in light green solution to stain the mineralized bone sufficiently.) The stained resin
thin sections were dehydrated in ascending concentrations of ethanol (70% and 100%,
2 × 30 s), while the resin ground sections were air dried in the dark for ~1 h. Both
sections were cleared in xylene (2 × 6 min for resin thin sections and 2 x 30 s for resin
ground sections) and finally mounted with Eukitt mounting media.
Von Kossa/MacNeal’s tetrachrome staining
The Von kossa stain is intended for histological visualization of calcium
deposition in sections. When the tissue sections are treated with a silver nitrate solution,
the silver ions are deposited by replacing calcium, following a developing stage, the
silver ions are reduced to metallic silver, which are visualised as a black colour [109].
Von Kossa staining was optimised based on previously described protocols
using solutions shown in Table 6.5 [211–213].
Table 6.5 Von Kossa/MacNeal’ tetrachrome staining solutions Solutions Recipe
5% Silver nitrate 20g Silver Nitrate dissolved in 400 ml DI water, filtered before use
5% sodium carbonate-
formaldehyde
22.5g sodium carbonate dissolved in the mixture of 337.5 mL of DI water
and 112.5 mL of 37% formaldehyde
Farmer’s Diminisher 2g potassium ferricyanide and 40g sodium dissolved in thiosulfate in 420
mL of DI water
Paraffin. Sections were deparaffinised and rehydrated, as previously described.
The sections were immersed in 5% silver nitrate solution for 5 min in the dark.
Following incubation, sections were rinsed in DI water (3 × 1 min) and immersed in a
5% sodium carbonate-formaldehyde solution for 2 min. Following, DI water rinse (2
× 1 min), sections were then submerged in fresh Farmer’s Diminisher solution for 30
s. Sections were then washed under running tap water for 20 min and counterstained
for 10 min with MacNeal’s tetrachrome solution (Dorn and Hart Microedge Inc., US).
After counterstaining, the sections were rinsed with DI water (4 × 30 s), then
dehydrated with ascending concentrations of ethanol (70%, 90% and 100%, 30 s each)
and mounted with Eukitt mounting media.
118 Chapter 6: (Study 3) Development of optimised histological processes for analysis of large and complex bone and implants
Resin. Thin sections were deplasticized in acetone (2 × 20 min), and rehydrated
with a descending ethanol series (100%, 90% and 70%, 5 min each) and placed in DI
water 5 min prior to staining. Resin ground sections were brought into DI water for 5
min prior to staining. Following incubation, sections (both thin and ground) were
rinsed in DI water (3 × 1 min) and immersed in a 5% sodium carbonate-formaldehyde
solution for 2 min. Followed by DI water rinse (2 × 1 min), sections were submerged
in fresh Farmer’s Diminisher solution for 30 s. Sections were then washed under
running tap water for 20 min and counterstained for 10 min with MacNeal’s
tetrachrome solution. After counterstaining, the resin thin sections were rinsed with
DI water (4 × 30 s), then dehydrated with ascending grades of ethanol (70%, 90% and
100%, 30 s each) and mounted with Eukitt mounting media. The resin ground sections
were air dried in the dark, cleared in xylene and mounted.
Immunohistochemistry (IHC)
IHC is used in histology to detect proteins of interest in cells of a tissue section
by exploiting the principle of specific binding between antibodies and antigens [109].
In bone tissue engineering (TE) research, IHC is used to locate specific osteogenic
and/or angiogenic markers on the regenerated bone, which provides information on
for example tissue regeneration progress and formation of new blood vessels. The
information provided by IHC is essential for the assessment of TE strategies.
Paraffin. The sections were deparaffinised with 2 changes of xylene (8 min
each) and rehydrated with descending concentrations of ethanol (100%, 90%, 70%
and 50%, 2 min each), and placed in DI water 5 min prior to staining.
Resin. Ground resin sections were deplasticized in three changes of 2-Methoxyl
Ethylacetate (2-MEA) for 48h as suggested in the literature [214] and rehydrated into
DI water.
Tissue sections were processed following standard IHC procedures, as
previously described [1, 5, 10-13]. In brief, both resin and paraffin sections were
circled by delimiting pen (DAKO, Australia) and slides placed in 50 mM Tris–HCl
buffer (pH 7.4). The endogenous peroxidase activity was blocked by 3% H2O2 in Tris–
HCl for 20 min. After washes (3 x 2 min) with Tris buffer (pH 7.4), sections were
incubated with proteinase K (DAKO, Botany, Australia) for 20 min for antigen
retrieval. The sections were incubated with 2% bovine serum albumin (BSA) (Sigma,
Chapter 6: (Study 3) Development of optimised histological processes for analysis of large and complex bone and implants 119
Sydney, Australia) in a humidified chamber at RT for 40 min to block non-specific
binding sites. The sections were incubated overnight at 4 °C with the following
osteogenic markers (diluted in 2% BSA): type I collagen (ColI, 2 μg/ml polyclonal
rabbit, #ab34710, Abcam, UK), osteocalcin (OC, 2 μg/ml polyclonal rabbit, #ab93876,
Abcam, UK) and an endothelial marker von Willebrand Factor (vWF, ready-to-use,
polyclonal rabbit anti human, DAKO Australia). To exclude false positive staining as
a result of rabbit IgG binding non-specifically to Fc receptors, two types of negative
controls were used: rabbit primary antibody isotype control (0.5 μg/ml, #086199,
Invitrogen, Australia) and 2% BSA negative control. Sections were incubated with the
specific antibodies or control solutions in a humidified chamber at 4 °C overnight.
Following 3 × 2 min washes in Tris-HCl buffer (pH 7.4), sections were incubated with
peroxidase-labelled dextran polymer conjugated to goat anti-mouse and anti-rabbit
immunoglobulins (EnVision+ Dual Link System Peroxidase, DAKO) at RT in
humidified chambers for 60 min. The sections were incubated for 10 s with a liquid
3,3-diaminobenzidine (DAB)-based system (DAKO, Australia) for colour
development. All sections were dehydrated in ascending grades of ethanol and
mounted with Eukitt mounting media.
6.2.5 Microscopy and image documentation
Images of histologically stained specimen were acquired and analysed using a
Zeiss Axio Imager M2 light microscope equipped with a scanning stage, Zeiss
AxioCam Mrc digital camera and Zeiss ZEN blue software (Zeiss, Oberkochen,
Germany).
6.3 RESULTS AND DISCUSSION
6.3.1 Study Overview
To ensure the PCL/SrBG composite scaffolds developed in this PhD project
could be effectively evaluated with the endpoint histological analysis, study 3
comprehensively optimised and standardized histological techniques using four
animal species. Following the optimization, I applied the histological processes to
tissue engineered bone specimens. Three most frequently used animal models in bone
tissue engineering research were studied: rat (38%), mouse (13%) and sheep (11%),
accounting for 62% of the total usage [105]. Pig bones were also included in this study
because of its close similarity in composition and biology with human [101]. For
120 Chapter 6: (Study 3) Development of optimised histological processes for analysis of large and complex bone and implants
histological standardization and comparison, the native bone specimens were
embedded in the representative embedding materials (paraffin, MMA, and Technovit
9100 NEW®) commonly employed in bone histological studies. Other types of
embedding media such as Technovit 7100 (Glycol Methacrylate resin) were not
explored here as they do not provide additional features. Microtome sectioning of non-
demineralized mature cortical bones was found challenging with the existing system
in our lab, therefore, the Exakt ground sectioning system was employed as the main
technique for resin section production. Sledge microtomes were used to section tissue
engineered bone and these sections are referred as resin thin sections (relative to resin
ground sections which are much thicker) in this paper. Four important stains for bone
histology were selected for comparison in this project: H&E for general tissue
morphology; von Kossa for mineralized bone and Goldner’s trichrome for hard/soft
tissue discrimination; and IHC for specific protein markers.
6.3.2 Stain optimization and comparison
Goldner’s trichrome stain is widely used in bone histology because of its ability
to sharply discriminate mature bone matrix to immature new bone matrix and
surrounding soft tissues without losing cellular information [215]. Because of its
excellent cell staining, Goldner’s trichrome is also considered more valuable than other
stains in pathology [109]. However to our knowledge, the currently published staining
protocols provided either sub-optimal or non-repeatable staining results for the resin
ground sections of non-decalcified bone specimens. Therefore here I present our
optimised staining procedure for application of Goldner’s trichrome stain to resin
ground sections of the 4 different species of bone. This stain was proven to be
reproducible across all four animal species providing consistent results, the
representative images of which are presented in Figure 6.3. Overall, good tissue
discrimination was found on resin ground sections where the mature bone matrix
stains green, immature bone matrix stains red, while the surrounding soft tissue stain
orange (Figure 6.3a, b, d, e, g, h, j, k). The cells are visible both in the bone matrix
(osteocytes) and surrounding soft tissues where the nuclei stain dark blue. Osteoid was
observed as a dark red line along the edges of mineralized bone matrix. The osteoblasts
can be found along the osteoid. Additionally, chondrocytes staining is shown as bright
yellow-orange with dark blue nuclei which are clearly visible on the articular joints of
mouse paws (Figure 6.3g, h and i). No significant difference of staining was found
Chapter 6: (Study 3) Development of optimised histological processes for analysis of large and complex bone and implants 121
between specimens embedded in MMA and Technovit 9100 NEW®. In comparison,
such details were lost on decalcified paraffin sections (Figure 6.3c, f, i, l), where the
bone, connective tissue and cartilage all stain green with nuclei staining dark blue since
bone mineral contents were completely removed prior to embedding. Across species,
little variance was observed in the staining specificity. The tissue discrimination and
cellular information envisaged by the Goldner’s trichrome stain are essential to
researchers who want to understand bone regeneration and mineralization progress and
the tissue/scaffold interface.
Figure 6.3 Comprehensive comparison of Goldner’s trichrome staining on resin ground sections and paraffin sections of bone of all four animal species. For the ease of comparison, the images are categorized by both animal species: (a,b,c) sheep tibiae, (d,e,f) pig fibulae, (g,h,i) mouse paws, and (j,k,l) rat leg bone; and types of embedding media: (a,d,g,j) MMA, (b,e,h,k) Technovit 9100 New®, and (c,f,i,l) paraffin. The resin ground sections are ~27 µm
122 Chapter 6: (Study 3) Development of optimised histological processes for analysis of large and complex bone and implants
in thickness, and paraffin sections are 5 µm in thickness. Inserts show overall images of the entire stained tissue. In (a,d,g,j) MMA and (b,e,h,k) Technovit 9100 NEW® ground sections, mineralized bone stains green, connective tissue and cartilage stain orange, osteoid stains red and cell nuclei stain dark blue. While in paraffin sections (c,f,i,l), bone, connective tissue and cartilage stain green, with nuclei staining dark blue. Scale bar = 25 µm
Von Kossa/MacNeal’s tetrachrome stain plays an important role in bone
research and the staining results indicated the discrimination of mineralized bone
(brown/black) and connective tissue (blue) with nuclei (dark blue) on resin ground
sections (Supplementary Figure 6.2). The location of mineralized tissue shown by von
Kossa stain agreed with that shown by Goldner’s trichrome stain in the identical tissue
cut. The colour of mineralized bone was not as densely black as that of resin thin
sections (Figure 6.5b3). This owes to the silver ions only being able to access the
surface layer of the mineralized bone matrix during impregnation as the bottom of
sections were glued to the slide, the existence of resin around the tissue sections further
restricted silver ion penetration and deposition on bone matrix. The cellular details on
the mineralized bone matrix were hardly visible for both thin and ground resin sections
as the whole matrix were covered by black metallic silver. There was no significant
difference in staining between specimens embedded in MMA and Technovit 9100
NEW®. In comparison, the decalcified paraffin sections did not take up any silver ion
and remained pale as there were no calcium ions to be replaced by silver ions on the
decalcified tissues.
In addition to the bone-specific stains, I also examined and compared the most
frequently employed histological stain: the haematoxylin & eosin (H&E) stain on resin
thin and ground sections. I used an existing H&E staining protocol [111] for resin
ground sections and found significant background staining of Mayer’s haematoxylin
on mineralized bone matrix. I hypothesized that it was due to the hemalum of Mayer’s
hematoxylin also binding to the anion groups in the mineral content. I then introduced
differentiating steps using 1% acetic acid to minimize hematoxylin stain on the
mineralized matrix. However, the differentiation decolorized the nuclei stain thus
affected the contrast between cell nuclei and cytoplasm. Mayer’s hematoxylin is an
aluminium modified hematoxylin solution and when exposed to acid it’s rapidly
removed from tissue sections because the aluminium hematoxylin dye-lake formation
fails to happen due to lack of OH− ions [216]. Therefore, I used Weigert’s iron
Chapter 6: (Study 3) Development of optimised histological processes for analysis of large and complex bone and implants 123
haematoxylin instead of Mayer’s haematoxylin for its resistance to acidic
differentiating solutions. Different to Mayer’s hematoxylin, Weigert’s iron
hematoxylin uses acidified ferric chloride as the mordant (chemicals combined with
hematoxylin) which can sustain acids [216]. The staining results showed improvement
of contrast between cell nuclei and cytoplasm on resin ground sections but were still
suboptimal compared to H&E staining of paraffin sections (Supplementary Figure
6.1).
Another common histological stain is immunohistochemistry (IHC). Here I used
a published method to deplasticize the resin ground sections [214] and stained both
resin ground sections and paraffin sections using the protocol established in our lab. I
examined two osteogenic markers: collagen type I (ColI) and osteocalcin (OC) and an
angiogenic marker: von Willebrand factor (vWF). Both Technovit 9100 NEW® and
paraffin sections showed immunoreactivity and stained positive for all three antibodies
(Supplementary Figure 6.3). Because of the exothermal nature of MMA
polymerization, the surface epitopes of antigens were destroyed by the high
temperature (80 °C), and as such, specimens embedded in MMA were not
immunoreactive for any of these antibodies and the results were not included in this
thesis. To overcome these difficulties of resin embedded specimens, Technovit 9100
NEW® was designed to preserve the immunoreactivity of specimens by polymerizing
at under-freezing temperature thus prevented the damage of antigens, making it
possible to examine specific markers of undecalcified bone tissues with IHC [110].
Similar staining intensity was found between resin and paraffin sections for ColI
(Supplementary Figure 6.3a1-a8) and OC (Supplementary Figure 6.3b1-b8) across all
4 animal species. More intense staining was found in Technovit 9100 NEW® sections
incubated with vWF antibodies compared to paraffin sections possibly due to greater
tissue thickness of resin ground sections, which provided a better understanding of
blood vessels morphology and their localization within the bone matrix
(Supplementary Figure 6.3ca1-c8). For bone tissue engineers, the immunolabelling of
Technovit 9100 NEW® sections can be valuable in providing the progress of bone
regeneration and mineralization, and more importantly the development and ingrowth
of blood vessels within the bone defect.
Even though paraffin embedded bone specimens do not preserve mineral
contents of bone, researchers have found ways to obtain genetic information from
124 Chapter 6: (Study 3) Development of optimised histological processes for analysis of large and complex bone and implants
sections of paraffin-embedded tissues. This process involves cell/tissue isolation and
RNA extraction from the isolated tissues. Laser microdissectioning (LMD) technique,
also known as Laser Capture Microdissection (LCM), is a contact- and contamination-
free method for isolating specific single cells or entire areas of tissue from a wide
variety of tissue samples [217]. LMD allows researchers to pick specific area of
interest or specific tissue/cell types within the paraffin section. Several methods have
been developed to extract RNA from the isolated sections, which allows subsequent
analysis of selected tissue/cells using further molecular biological methods such as
PCR and real-time PCR [218]. The quantitative data obtained from the combination of
LMD and subsequent techniques provide important complementary information to the
qualitative histological results. The fact that these techniques allow retrospective
biomarker studies of paraffin sections have made them popular in a large number of
research fields [219–221].
Ground sectioning vs resin microtome sectioning
As shown in Figure 6.4, a slab of tissue engineered sheep tibia was embedded in
Technovit 9100 NEW® and sectioned with both sledge microtome and Exakt cutting
and grinding system (Figure 6.4). The resin thin section showed noticeable 17%
compression in size compared to the original specimen, while resin ground sections
showed no such effect (Figure 6.4c and d). By comparing the sections stained with the
optimised Goldner’s trichrome technique, it is obvious that the resin ground section
not only better preserved the tissue morphology than the resin thin section, but also it
showed better differentiation of mineralized bone and soft tissues (Figure 6.4e). High
magnification images of stained resin ground section showed intact osteon structure
and Haversian canal (Figure 6.4g). In comparison, the resin thin section displayed
poorer overall morphology with shredded mineralized matrix (Figure 6.4f). High
magnification images showed compressed osteon structure and altered soft tissue
arrangement, also wrinkles were observed along the cutting direction which explains
the overall shortening of specimen size (Figure 6.4h). This difference of bone tissue
morphology was attributed to the different mechanical force applied to the embedded
tissue from microtome blade during sectioning and bandsaw cutting followed by
grinder polishing.
Chapter 6: (Study 3) Development of optimised histological processes for analysis of large and complex bone and implants 125
Figure 6.4 Specimen preparation and Goldner’s trichrome staining results of a tissue engineered sheep tibia with a 3 cm critical sized defect post mechanical testing. (a) Schematic illustration of sampling of tissue engineered sheep tibia bone. Note the tissue-engineered bone in the whole defect area and host bone on both opposing ends. (b) a 2 mm thick slab cut along the sagittal plane as depicted in the image via an Exakt cutting system. The specimen was then embedded in Technovit 9100 NEW® embedding media. The resin block was sectioned using a sledge microtome to generate (d) resin thin sections of ~10 μm, and (c) a resin ground section of ~50 μm with the Exakt cutting and grinding system. (d) The resin thin sections have undergone extensive shrinkage to a length of 4 cm, compared to (b) the original specimen before embedding (4.8 cm). Both resin ground and thin sections were stained with Goldner’s trichrome (e and f respectively). (g) High magnification images of resin ground section show Haversian canal (blood vessels in orange) and intact osteon structure. (h) High magnification images of thin section show Haversian canal (blood vessels in purple) and osteon structure at the identical location to that of the resin ground section. Noticeable alteration of Haversian
126 Chapter 6: (Study 3) Development of optimised histological processes for analysis of large and complex bone and implants
canal structure including blood vessel location and ruffling in the bone matrix was observed compared to the resin ground section. Scale bar = 50 µm Resin microtomy creates tissue sections differently to ground sectioning. In ground
sectioning, as shown in Figure 6.2, resin embedded bone tissues were cut by a
diamond-edged saw and then carefully polished down to desired thickness by
sandpapers. The grinding process used a series of fine silicon carbide sandpapers
(P800 to P4000) with constant oscillation by the Exakt grinder to ensure a consistent
production of tissue sections without compression, folding or displacement tissue
components. The sledge microtome used a tungsten carbide blade with a plane-shaped
edge (profile D microtome knife) [222]. This type of microtome blade was extremely
hard and designed to cut hard materials such as resin to produce undecalcified bone
sections. However, the edge of the blade contributed to friction between the edge of
the blade and resin block [222]. The friction as the knife edge passed through the resin
block may have what caused compression of sections. The compression force can
cause plastic deformation of the embedded tissue and lead to shortened sections in the
cutting direction.
Cutting and distribution of TE bone specimens for paraffin and resin
histology
When investigating the bone regenerative capacity of a TE scaffold in vivo, it is
imperative to make the best use of limited bone tissue for the maximal amount of data.
I examine these bone explants in the order of: 1) micro-Computed Tomography (μCT)
analysis, 2) mechanical testing and lastly 3) histological analysis. To ensure a
comprehensive histomorphometry and immunohistochemistry (IHC) study of
specimens, the same tissue engineered bone was sampled for both paraffin and resin
histology. If we consider a 3-cm defect sheep tibia model for example (figure 6.5a),
the bone explants were cut as depicted in Figure 6.5b. The ~2 mm thick central bone
slabs around 4 cm in length were processed for resin embedding (Figure 6.5c) and the
remaining bone specimens were cut into 1.5 cm long for paraffin embedding (P1, P2,
and P3). Good tissue discrimination was obtained from resin ground sections stained
by Goldner’s trichrome and resin thin sections stained by von Kossa/MacNeal’s
tetrachrome stain (Figure 6.5d and e). The areas of mineralized bone indicated by
Goldner’s trichrome (green colour) closely match that shown by Von
Chapter 6: (Study 3) Development of optimised histological processes for analysis of large and complex bone and implants 127
Kossa/MacNeal’s tetrachrome staining (black colour). The resin sections provide the
overview of the entire 3 cm bone defect site with ~0.5 cm host bone on both proximal
and distal end, which helps to understand the new bone integration with the host bone
at the scaffold/host bone interface. With the overview, the regenerated bone can be
quantified with OsteoMeasure system for detailed histomorphometric variables [223]
such as bone volume which are used to compare and validate the μCT analysis. Again,
the resin thin sections showed obvious compression along the cutting direction of
microtome blade, more significantly at defect sites with less regenerated bone (Figure
6.5e). The defect site with tissue engineered bone contributes to the compression as
only a small degree of compression was measured from the host bone area (by
comparing the black dashed lines to red dashed lines).
Compared to resin sections, the overall size of paraffin sections is limited by
standard embedding, sectioning and staining techniques. Even though oversized
specimens may be embedded in paraffin using custom-made large moulds, it is
challenging to obtain paraffin sections from these oversized paraffin blocks. Therefore
large specimens are regularly cut and divided into multiple parts to fit into the standard
embedding moulds for paraffin embedding. It is normally challenging to provide the
overview of such large specimens with a single paraffin section, but by placing
together the stained sections of all three parts (P1, P2, and P3) we can get an overview
of the whole specimen (Figure 6.5f). P1 shows the scaffold/proximal host bone
interface (Figure 6.5f-I), P3 shows the scaffold/distal host bone interface (Figure 6.5f-
III), while P2 shows the cross section of bone tissue within the scaffold (Figure 6.5f-
II).
128 Chapter 6: (Study 3) Development of optimised histological processes for analysis of large and complex bone and implants
Chapter 6: (Study 3) Development of optimised histological processes for analysis of large and complex bone and implants 129
Figure 6.5 Specimen preparation and staining results of sheep tibiae with scaffolds implanted into 3 cm critical sized defects. (a) A schematic of a sheep tibia with a 3 cm scaffold in the defect. (b) Schematic illustration of how sheep tibia bone was cut and divided for both paraffin and resin embedding. (c) A 2 mm thick bone slab was cut from each tibia along the sagittal plane via an Exakt cutting system. The bone slabs were embedded in Technovit 9100 NEW® embedding media and produced into resin ground sections (~50 μm) by the Exakt cutting and grinding system, and into resin thin sections (~10 μm) by a sledge microtome. (d) The resin ground sections were stained with Goldner’s trichrome and (e) resin thin sections stained with Von Kossa/MacNeal’s tetrachrome staining. The areas of mineralized bone indicated by Goldner’s trichrome (green colour) closely match that shown by Von Kossa/MacNeal’s tetrachrome staining (black colour), however, the resin thin sections show obvious compression as a result of sledge microtome sectioning. The remaining bone specimens were cut into 3 slices (P1, P2, and P3) approximately 1.5 cm long for decalcification followed by paraffin embedding, and 5 μm sections were produced along cutting planes (I, II and III) as highlighted in (b). (f) Sections were stained with H&E staining and the images were put collated for an overview of bone tissue [224]
Staining of complex tissues (mouse paw)
On most occasions, bone fractures happen in a complex tissue environment often
associated with other tissue types such as cartilage, muscle, and skin. As a result, tissue
engineers are interested in the knowledge of structure and interfaces of such complex
tissues to enable better understanding of bone regeneration [225]. Mouse paws have
complex tissue types including bone, muscle, cartilage at joints and various connective
tissues. I used these samples to assess different sectioning approaches for complex
tissue to ascertain the most optimised approach for future bone TE histology strategies
(Figure 6.6). Goldner’s trichrome stain was used to discriminate these tissues on the
non-demineralized resin ground section (Figure 6.6a): bone (green), connective tissue
(orange), muscle (red) and joint cartilage (bright yellow). Similarly, the resin thin
section of the same paw had an identical colour differentiation between tissue types
(Figure 6.6b). However, the resin thin section showed distorted knuckle joint structure
(Figure 6.6b2) and shredded mineralized bone matrix (Figure 6.6b4). The connective
tissue and muscle was comparable in quality to that of resin ground section (Figure
6.6b3). The paraffin section had well preserved bone and joint morphology, but poor
hard/soft tissue interface, as shown by the clearly detached skin and muscle from the
bone. The paraffin section stained by Goldner’s trichrome staining did not provide
optimal tissue discrimination compared to H&E stained section (Figure 6.6d).
130 Chapter 6: (Study 3) Development of optimised histological processes for analysis of large and complex bone and implants
Figure 6.6 Comparison of mouse paws prepared as resin ground sections, resin thin sections and paraffin sections stained with Goldner’s trichrome stain. Comparative images of (1) an
Resin ground section
Resin thin section
Paraffin section
Paraffin section
Goldner’s trichrome
Goldner’s trichrome
Goldner’s trichrome
H&E
Chapter 6: (Study 3) Development of optimised histological processes for analysis of large and complex bone and implants 131
overview, (2) knuckle joint, (3) muscle tissue and (4) metatarsal inter connective space show that the different sectioning techniques preserve tissue morphology differently. (a) The resin ground section of mouse paw embedded in methyl methacrylate (MMA) resin shows well preserved morphology of (a4) mineralized bone, (a2) joint cartilage and (a3) soft tissues including muscle, while (b) the sledge microtomed resin thin sections exhibit poor (b4) bone and (b2) joint morphology, although (b3) soft tissue is well preserved. (c) While the paraffin sections show well preserved (c4) bone and (c2) joint morphology, (c3) the soft tissue regions are poorly preserved. This figure highlights the versatile nature that ground sectioning has in the superior tissue morphology preservation of a complex hard/soft tissue environment as demonstrated in a mouse hind paw. Scale bar 1 = 2,000 µm, Scale bar 2-4 = 100 µm
Versatility
In our experience, the technique combination of resin ground sectioning and
Goldner’s trichrome staining provides the optimal bone tissue morphology
preservation and discrimination. These techniques are versatile and applicable to a
wide range of bone explant types. Figure 6.7a1 shows the sharp contrast overview
image of the mouse paw, and the tissue details of various types of a mouse paw
embedded in Technovit 9100 NEW® can be viewed with high magnification
microscope images (Figure 6.7a2 – a6), including digits, metatarsal joint, osteon
structure, muscle and blood vessels. Metallic implants are popular in bone tissue
engineering for their excellent mechanical properties, however they present a great
challenge for histological processing [6]. Our optimised techniques were applied to
bone defect containing a titanium implant to provide bone/implant contact information
without compromising the scaffold structure or tissue morphology (Figure 6.7b). The
porous titanium scaffolds were produced and implanted into sheep tibia by Seamus
Tredinnick et al. to investigate osteointegration of these scaffolds. The high
magnification images of tissue scaffold interface provide the information of bone
contact and osteointegration (Figure 6.7b2, b3 and b4).
132 Chapter 6: (Study 3) Development of optimised histological processes for analysis of large and complex bone and implants
Figure 6.7 Overview and high magnification images of a whole mouse hind paw and a sheep tibia/femur with porous titanium implant prepared by ground sectioning and stained with Goldner’s trichrome. Goldner’s Trichrome staining of the resin ground section produced high quality images, preserving the structure and providing high resolution detail. Both specimens were embedded in Technovit 9100 NEW® and ground sectioned to ~50 μm (b) with the Exakt cutting and grinding system. (a1) The morphology of the entire mouse paw (~27 μm) is shown, along with (a2) details of the nail, (a3) metatarsal joint, (a4) osteon detail, (a5) muscle structure, and (a6) blood vessel. (b1) The overall image shows the morphology of bone tissue integrated with the titanium implant. (b2 - b4) The high magnification images provide details of hard and soft tissue interactions around the metal implant. This figure highlights the high quality sections and supporting microscopic images obtainable from ground sectioning. Both overall tissue morphology and fine structures and details are preserved. Ground sectioning is the only histological technique capable of looking at metallic
Chapter 6: (Study 3) Development of optimised histological processes for analysis of large and complex bone and implants 133
implant/hard and soft tissue interfaces, to date. Scale bars, a1 b1 = 2,000 µm, a2 = 50 µm, a3-a6 = 25 µm, b2 = 100 µm, b3-b4 = 100 µm
6.4 CONCLUSION OF STUDY 3
Study 3 aimed to develop the optimal endpoint assessment for the planned
implantation of PCL/SrBG scaffolds produced in previous studies. In this study, I
optimised and standardized essential techniques for histological processing of animal
native bone and tissue engineered bone explants. Once the PCL/SrBG scaffolds are
implanted in the future, they can be assessed using these optimised histological
methods developed in this study.
Study 3 was part of my PhD project and was inevitably constrained by time and
resources, preventing us from including the whole spectrum of animal species and
histological stains used in bone TE. Therefore, I standardized and compared
histological processing on identical bone specimens of four most frequently utilized
animal species in bone TE in vivo research. The optimised histological techniques have
been successfully applied on bone specimens with TE scaffolds implanted in projects
conducted by our colleagues and collaborators. I believe that the present study is a
major step towards optimal explant assessment by providing repeatable histological
processing techniques and clear results that researchers can use as a guide to their
experiment planning. As I have mentioned, different processing routes require
different specimen preparation that can be irreversible. For example, paraffin
embedding and sectioning require complete decalcification of bone specimen, which
eliminates the possibility of sharp discrimination using mineral specific stains.
In summary, the main lessons learned for tissue engineers and bone histologists
have been summarized in table 6.6 (different embedding media) and 6.7 (different
sectioning techniques for resin embedded specimens) and are summarized as follows.
Our staining results indicate that each embedding and sectioning technique has its
unique strength and disadvantages and should be employed based on a case-by-case
basis for a specific purpose. As the most routinely used histological technique, paraffin
embedding and sectioning is robust and easy to use. It has the advantages of high
throughput and efficiency, long storage life, and it provides well preserved bone
morphology and high quality H&E staining and IHC results. However, its limitations
134 Chapter 6: (Study 3) Development of optimised histological processes for analysis of large and complex bone and implants
including small size, poor preservation of hard/soft tissue interfaces, the inability of
mineralized tissue discrimination due to complete mineral removal and slow
decalcification processes have rendered paraffin processing suboptimal for bone
explant assessment in tissue engineering. For researchers who are only interested in
the general morphology or have no access to specialised equipment for resin
embedding and sectioning, the paraffin approach is recommended for adequate results.
Resin embedding largely overcomes the limitations associated with paraffin approach
and the end results depend on sectioning techniques. The resin sledge microtome
sectioning is efficient in slide production and the best bone mineral discrimination by
von Kossa stain was observed on resin thin sections. However, the resin microtome
technique is found suboptimal at preserving the hard tissue morphology and hard/soft
tissue interface and a significant compression effect is observed. This technique is also
unreliable in cutting mature cortical bone specimens. On the other hand, the resin
ground sectioning technique provides the best bone tissue/explant morphology
especially in complex and large sized specimens. Goldner’s trichrome stain offers a
sharp contrast to tissue types by colour and maximizes the data output of a single
section. The combination of ground sectioning and Goldner’s trichrome techniques
brings the user the best overview of morphology with good cellular details. Little
difference in morphological staining results was observed between MMA and
Technovit 9100 NEW® resin ground sections, and immunolabelling was successful
only on Technovit 9100 NEW® ground sections. Two noticeable disadvantages of
ground sectioning techniques are the relatively large amount of specimen consumption
per section (owing to the grinding phase) and requirement of specialized equipment
and resources.
Table 6.6 Summary of the advantages and disadvantages of commonly used media for histology embedding: paraffin, MMA resin and Technovit 9100 resin.
Embedding
media
Advantages Disadvantages
Paraffin • robust and reliable, efficient in
tissue section production which is
• poor hard/soft tissue interface
• no preservation of minerals
Chapter 6: (Study 3) Development of optimised histological processes for analysis of large and complex bone and implants 135
critical for TE implants small in
size
• paraffin sections are ideal for H&E
staining and IHC which provide
high quality cellular details, matrix
morphology, specific markers of
regenerated bone on TE scaffolds
• automated processing, staining and
slides scanning (optional)
• relatively cheap to process
• limited specimen size
• not suitable for metal implants
• Collapsing or damage of
scaffold explant observed due
to melting or dissolving of
scaffolds structures
MMA resin • preserves mineral contents of
regenerated bone on scaffold
implants
• allows bone specific stains that
provide differentiation of hard/soft
tissues, which is crucial for the
assessment of osteogenic capacity
of scaffold implants
• allows oversized specimens to be
processed with integrity. This is
especially important for scaffold
implantation experiments in large
animal models (such as sheep and
pig)
• time consuming and laborious
• low level of automation
• damage to antigens thus IHC
not possible
Technovit
9100 resin
• preserves mineral contents of
regenerated bone on scaffold
implants
• allows bone specific stains that
provide differentiation of hard/soft
• time consuming and laborious
• low level of automation
• relatively expensive
136 Chapter 6: (Study 3) Development of optimised histological processes for analysis of large and complex bone and implants
tissues, which is crucial for the
assessment of osteogenic capacity
of scaffold implants
• allows oversized specimens to be
processed with integrity. This is
especially important for scaffold
implantation experiments in large
animal models (such as sheep and
pig)
• preserves the antigens and enables
IHC analysis
Table 6.7 Summary of the advantages and disadvantages of commonly used sectioning techniques for resin embedded specimens
Sectioning
technique
Advantages Disadvantages
Sledge
microtome • efficient in section
production
• shrinking effect
• poor preservation of hard tissue
morphology
• can alter scaffold implant
morphology leading to
complications for quantitative
analysis
• time consuming
Ground
sectioning • best in tissue morphology
(hard and soft) preservation
• consumes relatively large
volumes of the specimen
(specimen depletion), which is
not desirable for scaffold implant
small in size
Chapter 6: (Study 3) Development of optimised histological processes for analysis of large and complex bone and implants 137
• scaffold morphology
maintained as pre-implant
conditions
• can cut extremely hard
materials (specimens with
metal implants)
• very time consuming
The results of the present study will serve as a standard for testing the
performance of histological techniques for bone and as a guide to researchers for their
bone histological analysis.
6.5 SUPPLEMENTARY FIGURES
138 Chapter 6: (Study 3) Development of optimised histological processes for analysis of large and complex bone and implants
Supplementary Figure 6.1 Comprehensive comparison of H & E staining on bone sections prepared by different embedding techniques of all four animal species. The resin ground sections are ~27 µm in thickness, and paraffin sections are 5 µm in thickness. Cell nuclei are stained dark purple/blue, while cytoplasm is stained pink. Inserts show overall images of the entire stained tissue. For the ease of comparison, the images are categorized by both animal species: (a,b,c) sheep tibiae, (d,e,f) pig fibulae, (g,h,i) mouse paws, and (j,k,l) rat leg boned; and types of embedding media: (a,d,g,j) MMA, (b,e,h,k) Technovit 9100 New®, and (c,f,i,l) paraffin sections. Scale bar = 25 μm
Chapter 6: (Study 3) Development of optimised histological processes for analysis of large and complex bone and implants 139
Supplementary Figure 6.2 Comprehensive comparison of Von Kossa/MacNeal’s tetrachrome staining on bone sections prepared by different embedding techniques of all four animal species. The resin ground sections are ~27 µm in thickness, and paraffin sections are 5 µm in thickness. Mineralized bone tissue stains dark brown/black while soft tissue stains light blue and cell nuclei stain dark blue. Inserts show overall images of the entire stained tissue. For the ease of comparison, the images are categorized by both animal species: (a,b,c) sheep tibiae, (d,e,f) pig fibulae, (g,h,i) mouse paws, and (j,k,l) rat leg boned; and types of embedding media: (a,d,g,j) MMA, (b,e,h,k) Technovit 9100 New®, and (c,f,i,l) paraffin sections. Scale bar = 25 µm
140 Chapter 6: (Study 3) Development of optimised histological processes for analysis of large and complex bone and implants
Supplementary Figure 6.3 Comprehensive comparison of Immunohistochemistry on sections of bone embedded in Technovit 9100 New® and paraffin for all four animal species. The resin ground sections are ~27 µm in thickness, and paraffin sections are 5 µm in thickness. (a1-a8) Collagen type I (2μg/ml polyclonal rabbit), (b1-b8) osteocalcin (2μg/ml polyclonal rabbit), (c1-c8) Von Willebrand factor (vWF) (ready-to-use, polyclonal rabbit anti human). (d1-d8) Negative controls for immunohistochemistry using rabbit primary antibody isotype control (0.5μg/ml)
141
Chapter 7: Conclusions
7.1 RESEARCH SUMMARY
Nationally and globally, musculoskeletal disorders place a significant burden on
the healthcare system and overall economy, and in Australia, they account for $15
billion annual direct health expenditure [4,42]. The clinical treatment for these
conditions relies on bone grafting: autografting (donor tissue from the patient) and
allografting (donor tissue from another person). Despite these treatment options all
exhibiting bone defect healing capacity, they also have shown prominent
disadvantages [28]. The need for better treatment has driven researchers to develop
bone graft substitute and led to the emergence of tissue engineering field [31].
Scaffold-based tissue engineering approach investigates the use of biomaterials
(polymers, bioceramics etc.) as potential bone graft substitutes, potentially
overcoming many of the issues associated with autologous and allogeneic bone
grafting. In general, the development of scaffolds involves three stages (Figure 1.1),
which starts with scaffold fabrication and characterization (stage 1), followed by in
vitro cell assays (stage 2) and in vivo implantation with rigorous tissue explant
assessment (stage 3) to evaluate the efficacy and viability of scaffolds. Histology is
considered as one of the essential explants analytical methods for TE scaffolds.
In this PhD project, I hypothesized that by incorporating strontium-substituted
bioactive glass (SrBG) particles into polycaprolactone (PCL) I could increase the
bioactivity of PCL and produce porous scaffolds. To develop the composite scaffold,
I started with stage 1 – scaffold fabrication via melt-electrospinning technique and
characterization, following the successful production of PCL/SrBG (10 wt%) I worked
on stage 2 of in vitro assessment, to find out the osteoblast-like cells attachment,
proliferation, and osteoblast differentiation on the composite scaffolds compared to
PCL only control scaffolds; and in parallel, I optimised histological techniques for
bone specimen examination for the optimal assessment of intended in vivo
implantation of these composite scaffolds.
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7.2 SUMMARY OF CHAPTER 4 (STUDY 1)
Chapter title: Fabrication and in vitro investigation of PCL, 10 wt% PCL/SrBG
electrospun scaffolds for bone regeneration
Chapter 4 demonstrated for the first time the development of a PCL/SrBG
composite scaffold incorporating 10% (by weight) of SrBG particles into PCL bulk,
produced by the technique of melt-electrospinning. The PCL/SrBG composite
scaffolds were reproducibly manufactured with an interconnected porous structure.
MicroCT analysis showed the homogeneous distribution of SrBG particles throughout
the scaffold matrix. In vitro bioactivity of the composite scaffolds was investigated
with ICP-OES and EDX techniques. Quantitative analysis of ions dissolution in cell
culture media and evidence of CaP layer precipitation on scaffold surface indicated
similar bioactivity to that of bare SrBG particles.
MC3T3 cells were cultured on these scaffolds in two groups: normal cell culture
media and osteogenic media (normal culture media supplemented with osteogenic
factors). A series of in vitro assays were carried out. The PCL/SrBG scaffolds were
demonstrated to be non-cytotoxic in vitro. Ions present in the SrBG component were
shown to dissolve into cell culture media and promoted precipitation of a calcium
phosphate layer on the scaffold surface which in turn led to noticeably enhanced
alkaline phosphatase (ALP) activity in MC3T3-E1 cells compared to PCL only
scaffolds. Furthermore, up-regulation of ALP and OCN genes was also observed.
These results indicated PCL/SrBG composite scaffolds could enhance osteoblasts
differentiation in vitro. The composite scaffolds were also found to enhance collagen
deposition. These results suggested that melt-electrospun PCL/SrBG composite
scaffolds could noticeably enhance bone differentiation compared to PCL ones, and
the PCL/SrBG scaffolds showed potential to become effective bone graft substitutes.
However, the PCL/SrBG scaffolds did not show such effect when exposed to normal
cell culture media mainly due to insufficient bioactivity. Therefore, it was imperative
for me to increase the SrBG weight percentage in PCL to render an enhanced
bioactivity and thus bone differentiation of the PCL/SrBG scaffolds, which led to the
study 2.
143
7.3 SUMMARY OF CHAPTER 5 (STUDY 2)
Chapter title: Developing 50 wt% Strontium-substituted bioactive glass and
Polycaprolactone composite scaffolds for bone repair via hybrid electrospinning in a
direct writing mode
Following the research on 10% PCL/SrBG scaffolds in study 1, I actively
investigated the way of increasing the weight percentage of SrBG particles loaded in
the composite scaffolds in order to further enhance their bioactivity. In chapter 5, I
successfully fabricated PCL/SrBG scaffolds containing 50 wt% of SrBG with
controlled porosity and structure via melt-electrospinning in a direct writing mode. In
order to achieve this, I optimised the whole process including SrBG particle sizing,
PCL/SrBG composite preparation and the electrospinning technique. Firstly, the SrBG
particle size was reduced from over 100 μm to < 6 μm to minimise the risk of needle
blockage, which also led to increased surface area of these particles. The solvent
precipitation technique was adopted for the 50 wt% PCL/SrBG composite preparation
to ensure a homogeneous distribution of particles within the PCL matrix.
The production of the composite scaffolds was very challenging and in respond
I developed a novel hybrid melt-electrospinning system for the PCL/SrBG scaffold
production. This hybrid technique facilitated composite fibre continuity and fibre jets
stability for the precise control of electrospun fibres deposition. Compared to
PCL/SrBG (10 wt%) scaffold in study 1, the PCL/SrBG (50 wt%) scaffolds showed
greatly enhanced in vitro bioactivity indicated by the faster formation of CaP layer
only after 3 hours in serum-free media. More importantly, the concentration of Sr and
Si ions released from the PCL/SrBG (50 wt%) scaffolds also increased and the peak
values were comparable to that of bare SrBG particles. As a control, PCL only
scaffolds did not show CaP formation or Sr and Si ion dissolution.
In vitro tests were performed on these PCL/SrBG (50 wt%) scaffolds to evaluate
the osteogenic capacity of these scaffolds. Same as in study 1, MC3T3 cells were
cultured on PCL/SrBG and PCL scaffolds in both osteogenic media and growth control
media. A series of in vitro assays were carried out for the assessment of cell attachment,
proliferation and osteoblastic differentiation in all experimental conditions. Based on
LIVE/DEAD assay, SEM, CLSM and MTT assay results, the PCL/SrBG (50 wt%)
scaffolds were not cytotoxic and had the ability to support cell attachment and
proliferation. Notably, the significant increase of ALP activity of cells cultured on
144
PCL/SrBG scaffolds in growth media compared to their counterpart on PCL scaffolds
indicated the PCL/SrBG were able to induce osteoblast differentiation without
osteogenic supplement in the media, which made the composite scaffolds osteogenic
in vitro. Subsequent gene expression analysis showed upregulation of ALP and OPN
genes confirmed the osteogenic capacity of these PCL/SrBG (50 wt%) scaffolds.
Previous studies have shown that composite scaffolds were implanted for in vivo
investigation without in vitro confirmation of the osteogenic capacity of these
scaffolds [160]. Other studies investigate the osteogenic capacity of the scaffolds using
in vitro cell investigation prior to in vivo implantation using only osteogenic media
[75]. In this study, I made a step further and investigated the osteogenic potentials of
the PCL/SrBG composite scaffolds without osteogenic media in vitro, so we could
understand the real efficacy of these scaffolds to enhance osteogenesis. The findings
of this study indicated that these PCL/SrBG scaffolds with increased SrBG loading
were suitable for next stage of in vivo implantation.
7.4 SUMMARY OF CHAPTER 6 (STUDY 3)
Chapter title: Development of optimised histological processes for analysis of
large and complex bone and implants
As an essential endpoint tool for TE scaffold ex vivo assessment, histology has
become a key stage of TE scaffold development. Therefore in parallel to the
PCL/SrBG composite scaffold fabrication and in vitro evaluation in study 1 and 2, I
conducted study 3 to develop the optimised histological techniques for the ex vivo
assessment of the composite scaffolds developed in this project. To include all the
animal species in which the PCL/SrBG scaffolds were likely to be implanted, I
designed a comprehensive study to histologically process four most frequently utilized
animal species in bone tissue engineering in vivo research: sheep, pig, rat, and mouse.
In this study, I standardized and compared histological processing on identical bone
specimens embedded in paraffin, MMA resin, and Technovit 9100 NEW® resin.
Paraffin sections were obtained by microtomy and resin sections by sledge microtome
and ground sectioning technique. Common histological stains and bone specific stains
including H&E, Von Kossa, Goldner’s trichrome staining and IHC were performed on
all these sections. The stained sections were compared across all animal species to
reveal the difference between different processing techniques and to identify the
optimal histological techniques for bone analysis. The histological techniques
145
standardized in this study were applicable to mineralized tissues such as bone and
teeth, as well as regenerated bone via TE scaffold implantation. With the optimised
histological analysing techniques, we were poised to analyse the PCL/SrBG scaffolds
once they were explanted. Even though the PCL/SrBG composite scaffolds could not
be implanted due to time constrains of my PhD, I believe that the present study was a
major step towards optimal explant assessment by providing repeatable histological
processing techniques and detailed corresponding results that researchers can use as a
guide in their experiment planning.
7.5 LIMITATIONS AND RECOMMENDATION FOR FUTURE WORK
The PCL/SrBG (10 wt%) composites were fabricated into scaffolds via melt-
electrospinning technique for the first time and showed enhanced osteogenic capacity.
Further effort was made to fabricate scaffolds with increased SrBG filler phase and
resulted in PCL/SrBG (50 wt%) scaffolds via a novel hybrid melt-electrospinning
technique. The PCL/SrBG (50 wt%) scaffolds showed greatly enhanced in vitro
bioactivity compared to the PCL/SrBG (10 wt%) scaffolds. These PCL/SrBG scaffolds
were found osteogenic in vitro. However, more work needs to be done to optimise the
scaffold design and determine the osteoinductive capacity of these scaffolds through
in vivo implantation. The limitations and recommended future work of this PhD project
are discussed in the following sections.
7.5.1 Composite scaffold design
In order to obtain straight electrospun fibres, a short needle tip to collector
distance was used in study 2 during the 50% PCL/SrBG scaffolds fabrication, resulting
in larger fibre diameters compared to PCL ones. A series of in vitro assessment can be
done to determine the optimum fibre diameter. In situations where straight fibres are
not imperative, this collection distance may be increased for reduced fibre diameters.
In this project, I reduced the SrBG particle size by grinding for improved fibre
continuity but I could not modify the irregular shape of the particles and their sizes
were not uniform. Ideally, spherical nano-sized SrBG particles, which provide a much
higher surface area to volume ratio compared to the current micron-sized particles,
would be used in future studies to further improve the electrospun fibre production. As
we know, the particle size, size distribution and particle filler percentage within
composite play an important role in the mechanical properties of the PCL/SrBG
146
composite scaffolds. The enhanced 50 wt% SrBG filler phase in the PCL/SrBG
scaffolds developed in this PhD project was a trade-off of bioactivity (resulted from
high SrBG loading) and the mechanical integrity of the scaffolds. Further increasing
the SrBG filler phase may boost the bioactivity of PCL/SrBG composite, however, the
increased amount of SrBG particles will probably make the PCL/SrBG scaffold brittle.
In this PhD project, the PCL/SrBG (50 wt%) scaffolds were fabricated in a 0/90
degrees cross-hatch structure with a fibre space of 1mm. With the motorized stage and
aid of G code, other scaffold lay-down pattern can be explored to obtain different
scaffold structure such as 0/60/120 degree lay-down pattern. Shorter fibre spacing can
be adopted for reduced pore sizes which would facilitate bridging of scaffold pores by
seeded cells. Additionally, a rotating mandrel collector can be used to make porous
tubes out of the PCL/SrBG composites. During the hybrid electrospinning process, the
impact of SrBG incubation in organic solvents can be further investigated using bare
SrBG particles. The SrBG particles will be weighed prior and after immersion in
chloroform for a pre-determined time at 60 °C to check if there will be any possible
dissolution or degradation. The incubated SrBG particles can also be analysed using
ICP-MS technique, and the results of elemental concentration will be compared with
the non-immersed SrBG. These studies will help to determine whether the
characteristics of SrBG would alter during the scaffold fabrication process.
Furthermore, to understand the interaction of elemental concentrations (Ca, P,
Si and Sr) and cell activities, both osteogenic and control cell culture media in future
in vitro studies should be collected and analysed with ICP-MS technique. More
specifically, each scaffold for cell culture should be weighed and time interval and
volume of media change in each tissue culture plate well should be predetermined.
Triplicates of media at each time point should be collected and their ion concentration
measured.
7.5.2 In vivo investigation of composite scaffolds
Before these composite scaffolds may be used as bone graft substitutes in clinical
treatment for bone defects, they must be rigorously tested under in vivo conditions.
The composite scaffolds are recommended to be implanted into rat cranial defects for
the assessment of their osteogenic capacity. Post-operative assessment for bone
explants includes microcomputed tomography (μCT), mechanical testing and bone
147
histology, all of which are well optimised. The detailed plan is described in the
following sections.
Critical defect production and scaffold implantation via animal surgery
Twelve skeletally mature male Wistar rats will be used in three groups: 1)
PCL/SrBG scaffolds group; 2) PCL scaffolds and 3) empty control group. All rats will
be operated under general anesthesia. Two bone defects of 5mm in diameter will be
created in full thickness per calvaria. The animals will be sacrificed, by CO2 inhalation,
12 weeks post surgery and bone explants will be collected for further analysis using
techniques similar to previous study described by Berner et al. (2013) [107].
Subcutaneous injections of fluorochrome labelling (Tetracycline hydrochloride,
Alizarin complexone and calcein) will be administered at defined time points after the
surgery to identify bone mineral deposition rate [226].
μCT analysis
A Micro-CT 40 scanner (SCANCO Medical, Bassersdorf, Switzerland) will be
used to quantify the mineralization within the defects, presented as bone volume in
mm3.
Mechanical testing
An Instron Micro tester 5848 will be used to perform non-destructive micro-
compression tests on the calvaria defects to evaluate the mechanical stiffness of
regenerated bone in the defect.
Histological analysis
Following mechanical testing, the bone explant specimens will be fixed in 4%
paraformaldehyde in PBS for 48 h. These specimens will be divided for both paraffin
and resin embedding with five defects per group. For paraffin embedding, the
specimens will be decalcified in 10% Ethylenediaminetetraacetic acid (EDTA) for 3-
4 weeks. The 5-μm paraffin sections produced by microtome will be used for
histomorphometry and immunohistochemistry (IHC) analysis. The specimens
intended for resin embedding will be dehydrated and embedded in Technovit 9100
NEW®. Resin sections will be produced by a sledge microtome (Polycut-S, Reichert-
Jung, International Medical Equipment, USA) at the thickness of 6 μm. The resin
sections will be stained with Goldner’s trichrome and von Kossa/MacNeal’s
tetrachrome stain to identify new bone formation.
148
7.6 CONCLUDING REMARKS
With the increasing demand for autografts in the clinical setting, tissue engineers
are developing TE scaffolds as promising substitutes for bone graft materials.
Developing bioactive materials and improving fabrication techniques for scaffold
production are the research focus for viable scaffolds in bone TE. In the attempt to
improve the bioactivity of polymers, the use of polymer/BG composite biomaterial has
shown superior osteoconductive and even osteoinductive properties to single-
component polymers. The bioactive composite scaffolds have shown potential to be
used for the purpose of bone repair without cells or growth factors.
There has been a paradigm shift towards produce TE scaffolds using additive
manufacturing (AM) techniques to fabricate scaffolds with desired structure and
porosity. The AM scaffolds fabrication techniques or also known as 3D printing
produce scaffolds with layer-by-layer fibre deposition, and the 3D anatomically
precise implants are promising as future personalised medicine. Melt-extrusion based
techniques such as FDM have been used to produce porous composite scaffolds with
controllable size, shape and porosity. FDM scaffolds normally consist of fibres around
500 μm in diameter. In comparison, melt-electrospinning technique can produce
scaffolds fibres down to micron level which lead to better structural resolution and
higher surface area to volume ratio than the FDM scaffolds. The recent advances in
melt-electrospinning have enabled researchers to fabricate scaffolds with desired
structure and porosity. However, there are limited publications on composite scaffold
fabrication with this technique due to the difficulties associated with the change of
viscoelastic properties of polymers when inorganic phase was incorporated.
This PhD project aims to bridge this knowledge gap by advancing the melt-
electrospinning technique for the production of polymer/BG composite scaffolds. In
this PhD project, the PCL/SrBG composite scaffold with up to 50 wt% SrBG loading
was successfully fabricated. These composite scaffolds were bioactive and osteogenic
in vitro, which provided more options for the future off-the-shelf and patient specific
solutions for bone defect treatment. Another contribution of this project is the
advancing of the melt-electrospinning technique for the production of composite
scaffolds with aligned fibre deposition. This novel technique opens up the possibility
to fabricate PCL/SrBG scaffolds with different SrBG loading, or even the production
of PCL scaffolds incorporated with a whole different array of inorganic materials for
149
specific purposes. Lastly, in pursuance of preparing optimal histological assessment
tools for the planned in vivo implantation experiments, this PhD project also provided
recommendations, general guide and optimised operating protocols for the endpoint
assessment of bone tissues/scaffold explant for researchers in bone TE field.
In conclusion, this PhD project contributes to the overall tissue engineering
research towards effective and patient-specific treatment of tissue loss of human body
using synthetic scaffolds. However, being the first to fabricate the melt-electrospun
PCL/SrBG scaffolds has made the body of work in this PhD project an indispensable
link in understanding of future cell-free treatment for bone defects with fully synthetic
bioactive scaffolds.
References 151
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Appendices 169
Appendices
Appendix A
Figure A.1 Goldner’s trichrome staining of resin embedded sheep tibia bone tissues around titanium implant, sections obtained by ground sectioning technique [206]. The mineralized bone which should be green based on the staining protocol shows ‘patchy’ stains of green and red, thus weekending the differentiation between hard and soft tissues.
170 Appendices
Figure A.2 Goldner’s trichrome staining of resin embedded sheep tibia native bone tissues and regenerated bone tissues around implanted PCL scaffolds , sections obtained by ground sectioning technique [125]. The images c and d showed inconsistent bone staining mixing green and red colours.
Appendices 171
Figure A.3 Goldner’s trichrome staining of resin embedded sheep tibia bone tissues around Ti implant, sections obtained by ground sectioning technique [131]. Comparing image C and D, the fibrous tissue and bone tissue both stained red, which presents poor contrast between these two tissue types.
172 Appendices
Appendix B
SrBG particle grinding and sizing experiment
The bioactive glass particles were ground for 30 seconds, 1 minutes, 2 minutes, 4 minutes, 6 minutes, 8 minutes, 10 minutes, 12 minutes, 16 minutes, 20 minutes, 24 minutes, 30 minutes, 36 minutes, 42 minutes, 48 minutes, 54 minutes, 60 minutes, 66 minutes, 72 minutes, 84 minutes, 96 minutes and 108 minutes, 2 hours, 4 hours, 6 hours and 8 hours. Their size distribution was measured after each time point. The example results are shown in the figure below.
Volu
me
dens
ity (%
)
Particle size (μm)
