Bioactive Glass Derived Scaffolds with Therapeutic Ion Releasing ...
Transcript of Bioactive Glass Derived Scaffolds with Therapeutic Ion Releasing ...
Bioactive Glass Derived Scaffolds with Therapeutic Ion
Releasing Capability for Bone Tissue Engineering
Dreidimensionale bioaktive Glasgerüste mit therapeutischer
Ionenfreisetzung zur Gewebezüchtung von Knochen
Der Technischen Fakultät
der Friedrich-Alexander-Universität Erlangen-Nürnberg
zur Erlangung des Grades
Doktor-Ingenieur
vorgelegt von
Herrn Dipl.-Ing. Alexander Hoppe
aus Kustanaj
Als Dissertation genehmigt
von der Technischen Fakultät
der Friedrich-Alexander-Universität Erlangen-Nürnberg
Tag der mündlichen Prüfung: 14.07.2014
Vorsitzende des Promotionsorgans: Prof. Dr.-Ing. habil. Marion Merklein
Gutachter: Prof. Dr. Aldo R. Boccaccini
Prof. Dr. Uwe Gbureck
Acknowledgment
Acknowledgment
My greatest gratitude and appreciation goes to my supervisor Prof. Boccaccini who
gave me the opportunity to start my PhD research at the newly formed Institute of
Biomaterials. I want to thank him for his support and advices during all stages of
my PhD and also for giving me the freedom to develop my own research ideas and
framing my entire PhD project. Exceptionally, I want to thank him for supporting
and enabling numerous research fellowships and workshops abroad including stays
in Turin, Montreal, São Carlos (Brazil) or Buenos Aires which have had huge
impact not only my scientific career but also on my private life.
I want to thank Dr. Julia Will who has been supporting me from the very beginning
of my scientific career by supervising my diploma thesis whose great outcome
encouraged me to stay in academia and to pursue a PhD. She has always succeeded
to motivate me in all my research efforts and was helping with any issue during my
PhD studies.
My gratitude extends to Dr. Rainer Detsch whom I thank for introducing me to the
“world of cell biology”, for all the fruitful discussions and proof reading of parts of
my thesis. I also thank him for persistently motivating me to finish my PhD and his
unlimited trust in my skills.
I am thankful to Alina Grünewald for teaching me practical skills for the work in
the cell lab and for always being helpful no matter what issue would come up. I also
thank her for creating an extremely nice working atmosphere with many enjoyable
hours in the cell lab.
I want to thank Tobias Zehnder, Bapi Sarker and Raquel Silva and the complete
“Henkestrasse-crew” for the great time sharing the office, the labs and the lunch
breaks while exploring the infinite number of culinary options the nearby Aldi
offered us.
I would like to thank all my fellow PhD students for the grate time in and outside of
the institute.
I thank Dr. Gerhard Frank for his tireless patience and his help with the
management of the institute’s projects and administration issues.
Acknowledgment
I am grateful to Heinz Mahler for his help with technical and computer related
problems.
I would like to express my gratitude to all bachelor and master student I was lucky
to supervise who all contributed to my PhD project: Alexander Kent, Vincent
Bürger, Lukas Weidenbacher, Harald Unterweger, Stefan Grimm, Tobias Reichel,
Florian Ruther and Katharina Rzepka.
All collaborators who contributed to the completion of my PhD work are gratefully
acknowledged, in particular: Eva Springer for countless SEM images, Robert
Meszaros and Prof. Wondraczek for the help with glass melting, Chris Stähli and
Prof. Nazhat for helping with glass structural and degradation studies, Stefan
Romeis and Jochen Schmidt for ICP measurements and fruitful discussion, Jonathan
Lao and Prof. Jallot for micro-PIXE-RBS measurements, Juan Catallini and Prof. V.
Mourino for capillary electrophoreses measurements, Prof. Janackovic and Bojan
Jokic for the collaboration on bioactive glass.
I want to express my deepest gratitude to my parents and my brother for their
support during the last years who never gave up their faith that I would finally
graduate.
Abstract
Abstract
Regarding the need for highly vascularised engineered bone constructs to regenerate
bone defects, loading biomaterials with angiogenic therapeutics, like metallic ions,
has emerged as promising approach to develop novel biomaterials for regenerative
medicine.
In the present work, bioactive silicate glasses based on 45S5 and 1393 compositions
containing the well-known angiogenic ions Cu and Co were fabricated and used for
templating 3D scaffolds by foam replica technique.
It was shown that bioactive glasses can be used as carrier for therapeutic metal ions
with controlled release kinetics tuneable via the glass composition. The degradation
and bioactivity studies revealed that incorporation of metallic ions does not impair
the bioactivity and that traces of metallic ions are incorporated in the calcium
phosphate layer formed on the scaffolds surface in contact with simulated body
fluid. Cu and Co ions in therapeutic range were released and cell culture assays
confirmed high compatibility of 45S5-Cu and 1393-Co derived particulate glasses
and corresponding scaffolds with MG-63 osteoblast-like cells, human bone marrow
derived stem cells (hBMSCs) as well as human dermal micro vascular endothelial
cells (hDMECs). Cellular response, however, was shown to be dependent on the
concentration of the metallic dopant. Furthermore, Cu ions were shown to stimulate
angiogenesis in vitro by enhanced VEGF (vascular-endothelial growth factor)
expression in hBMSCs likely due to the activation of the HIF-1 transcriptional
factor. In a co-culture study of hDMECs and hMSCs 1 wt% Cu containing 45S5
scaffolds also enhanced the expression of endothelial cell specific markers vWF and
VEGFR and stimulated the hDMECs towards formation of prevascular tube-like
structure indicating the overall angiogenic potential of Cu. Altogether, Cu or Co
doped BG scaffolds represent a new family of highly promising materials for
applications in bone regeneration.
Kurzzusammenfassung
Kurzzusammenfassung
Die Vaskularisierung von gezüchtetem Knochengewebe ist von entscheidender
Bedeutung für die klinische Applikation dieser Gewebekonstrukte und stellt hohe
Anforderungen an das als Trägerstruktur (Scaffold) eingesetzte Biomaterial.
Ein vielversprechender Ansatz ist dabei das Dotieren von anorganischen
Biomaterialien mit Spurenelementen mit therapeutischer, angiogener Wirkung um
die Vaskularisierung von Scaffolds zu stimulieren.
In der vorliegenden Arbeit wurden ausgehend von zwei bekannten
Zusammensetzung, 45S5 und 1393, Cu and Co dotierte bioaktive Gläser (BG)
synthetisiert und anschließend zur Herstellung von 3D Poröser Scaffolds verwendet.
Es konnte gezeigt werden, dass bioaktive Glas-Scaffolds als Trägerstrukturen zur
kontrollierten Freisetzung von Metallionen geeignet sind und die
Freisetzungskinetik über die Glaszusammensetzung gesteuert werden kann.
Bioaktivitäts- und Degradationsuntersuchungen in simulierter Körperflüssigkeit
ergaben, dass die dotierten Elemente keinen negativen Einfluss auf die Bioaktivität
haben und Cu bzw. Co in der Hydroxylapatitschicht auf der BG Oberfläche
substituiert sind. Außerdem konnten Cu und Co in einem als therapeutisch wirksam
geltenden Bereich freigesetzt werden, wobei Zelluntersuchungen hohe
Biokompatibilität der Cu und Co dotierten Scaffolds mit osteoblastähnlichen Zellen,
mesenchymalen Stammzellen (MSCs) sowie Endothelzellen (ECs) bestätigten.
Darüber hinaus, wurde deutlich, dass Cu2+
Ionen die Expression des angiogenen
Signalmoleküls VEGF in humanen Stammzellen signifikant erhöhen und in einer
Co-Kultur aus MSCs und ECs die Ausbildung tubulärer prevaskulärer Strukturen
durch ECs stimuliert. Dies bestätigt das angiogene Potential von Cu dotiertem BG.
Insgesamt stellen Cu and Co dotierte bioaktive Gläser aufgrund ihrer hohen
Bioaktivität und stimulierenden Wirkung auf relevante Zelltypen vielversprechende
Materialen für den Einsatz in der regenerativen Medizin dar.
Contents i
Contents
1 Introduction ..................................................................................................... 11
1.1 Motivation: General need for bone regeneration therapies ....................... 11
1.2 Aim of the work ......................................................................................... 13
2 Fundamentals ................................................................................................... 14
2.1 General aspects of bone tissue engineering ............................................... 14
2.1.1 Human bone ....................................................................................... 14
2.1.2 Concept of (bone) tissue engineering (TE) ........................................ 18
2.2 Biocompatibility and Biomaterials ............................................................ 23
2.3 Bioactive silicate glasses ........................................................................... 24
2.3.1 Glass production ................................................................................. 24
Sol-gel process ................................................................................ 24
Melt derived bioactive glasses ........................................................ 25
2.3.2 Structure and general properties of (silicate) bioactive glasses ......... 26
2.3.3 Acellular in vitro bioactivity .............................................................. 28
2.3.4 Cellular response to bioactive glasses (BG) ....................................... 33
2.4 Bioactive glass derived scaffolds (State of the art) .................................... 37
2.5 Metal ions and bioinorganics in silicate glasses ........................................ 41
2.5.1 Role of bioinorganics in bone metabolism ......................................... 41
2.5.2 Biological performance of metal ion containing glasses and
glass ceramics ................................................................................................. 45
3 Materials and Methods ................................................................................... 50
3.1 Glass fabrication ........................................................................................ 50
3.1.1 Cu-containing 45S5 ............................................................................ 50
3.1.2 Co containing 13-93 ........................................................................... 50
3.2 Preparation of the glass powder ................................................................. 51
3.3 Characterization techniques ....................................................................... 51
3.3.1 Scanning electron microscopy (SEM) / Energy dispersive X-ray
spectroscopy (EDS)......................................................................................... 51
3.3.2 Fourier transform infrared spectroscopy (FT-IR) ............................... 52
3.3.3 Raman spectroscopy ........................................................................... 52
3.3.4 Inductively coupled plasma atomic emission spectroscopy
(ICP-OES) ....................................................................................................... 52
3.3.5 Micro-Ion beam .................................................................................. 53
3.3.6 XRD ................................................................................................... 54
3.3.7 X-ray microtomography (µCT) .......................................................... 54
3.4 Scaffold fabrication .................................................................................... 54
3.5 Acellular bioactivity in simulated body fluid (SBF) ................................. 55
Contents ii
3.6 Compressive strength ................................................................................ 55
3.7 Cell tests .................................................................................................... 55
3.7.1 Cells and culture ................................................................................ 56
Osteoblast-like cell line MG-63 ....................................................... 56 Human bone-marrow mesenchymal stem cells (hBMSCs) ............. 57 Human dermal microvascular endothelial cells (hDMECs) ............ 57
3.7.2 Analytical methods ............................................................................ 57
Cell viability and cell proliferation .................................................. 57 Analysis of osteogenic and angiogenic markers / Gene
expression and protein release ......................................................... 58 Cell morphology and cell adhesion ................................................. 59
Evaluation techniques of 2D experiments with EC ......................... 60 Statistical analysis ............................................................................ 62
3.7.3 Powder cytotoxicity ........................................................................... 62
3.7.4 Evaluation of the scaffolds ................................................................ 63
Indirect study (2D) ........................................................................... 63 Direct study (3D) ............................................................................. 63 Co-culture of hBMSCs and ECs ...................................................... 63
3.8 In vivo study .............................................................................................. 64
4 Results and Discussion.................................................................................... 66
4.1 Cu containing 45S5 bioactive glasses ....................................................... 66
4.1.1 Glass properties ................................................................................. 66
Glass structure .................................................................................. 66
Thermal properties ........................................................................... 70
Role of CuO in 45S5 glass structure ................................................ 71
4.1.2 Scaffold properties ............................................................................. 74
Macro-Structure ............................................................................... 74
Mechanical properties ...................................................................... 76 Acellular bioactivity in SBF ............................................................ 77 Degradation of 45S5 derived scaffolds and Cu release ................... 90
4.1.4 In vitro cell response .......................................................................... 94
Powder cytotoxicity ......................................................................... 94 Cell attachment on 2D pellets .......................................................... 97 In vitro cell studies with 3D scaffolds ........................................... 100
4.1.5 In vivo evaluation ............................................................................. 112
4.2 Cobalt containing 13-93 based glasses .................................................... 115
4.2.1 Glass properties ............................................................................... 115
Structure and Thermal Properties .................................................. 115
The effect of Co on 1393 glass structure ....................................... 119
4.2.2 Scaffold properties ........................................................................... 120
Macrostructure ............................................................................... 120 Mechanical properties .................................................................... 122 Acellular Bioactivity in SBF .......................................................... 124
Contents iii
Degradation and ion release in SBF .............................................. 131
4.2.3 In vitro cell response ........................................................................ 137
Powder cytotoxicity ....................................................................... 137 Scaffolds ........................................................................................ 139
4.3 Evaluation of the compressive strength of the 45S5 and 1393 scaffolds
in the context of bone TE .................................................................................. 145
4.4 Degradation behaviour of 45S5 and 1393 glass scaffolds and their
suitability as carrier for therapeutic metal ions ................................................. 148
5 Summary ........................................................................................................ 152
6 Conclusion and Outlook ............................................................................... 154
7 References ...................................................................................................... 160
8 Appendix ........................................................................................................ 188
Introduction 11
1 Introduction
1.1 Motivation: General need for bone regeneration
therapies
Bone healing is a biologically optimised process and hence the majority of small
bone defects will heal spontaneously with minimal treatment. However, in certain
clinical situations an additional bone regeneration therapy is required: for example
in order to support compromised bone healing (possibly due to impaired blood
supply or infection) after fracture or to restore bone tissue loss due to osteoporosis,
malignant tumours or osteomyolitis.1 In this context, functional disorder and defects
of bone have become a severe global health care problem with major clinical and
socioeconomic impact.2, 3
The clinical demand for engineered bone tissue has been
growing in recent years in direct relation to the increasing human population and its
aging.4 The use of autologous material for bone regeneration is still considered as
the “gold standard” by the clinicians. However, limited availability and donor
morbidity are major drawbacks of autologous grafts5 while the use of allogenic
transplants bears the risk of cell-mediated immune response and pathogen transfer.
Hence, in last decades there has been large amount of work on developing new
biomaterials and therapy approaches for bone regeneration.
Tissue engineering (TE) is one of the approaches being investigated to tackle this
problem by regenerating bone tissue using a combined cell/material therapy.6, 7
In
common TE strategies a three-dimensional structure, termed “scaffold”, fabricated
from a suitable artificial or natural material and exhibiting high porosity and pore
interconnectivity is used.8-11
Ideally, these scaffolds should not only provide a
passive structural support for bone cells, but they also should favourably affect bone
formation by stimulating osteoblastic cell proliferation and differentiation.12
Additionally, vascularisation of the engineered bone construct is essential for
successful bone healing process and hence induction of vascularisation should be
considered when developing new biomaterials and bone regeneration therapies.
Several approaches have been successfully introduced to construct porous scaffolds
with desired porosity and appropriate mechanical performance from inorganic
Introduction 12
materials such as bioactive ceramics and glasses as well as from biodegradable
polymers and their composites.10, 11
Due to their chemical similarity to the inorganic
phase of bone calcium phosphates (CaP), like hydroxyapatite (HAp) or α- and β-
tricalciumphosphate (TCP) have been widely used as bone engineering
grafts/scaffolds.8, 9, 13, 14
These materials are bioactive, osteoconductive and are able
to bond directly to bone.15
However, the optimal degradation rate of CaP adapted to
the bone forming rate is difficult to achieve while also fabrication of pure CaP phase
may be challenging. Bioactive glasses (BGs), in turn, are considered 3rd
generation
biomaterials16
which have the ability to stimulate specific intrinsic cell responses
resulting in osteoinductive behaviour, ability to bond to soft tissue as well as to hard
tissue and also to form a strong bonding to bone via formation of a surface
carbonated hydroxyapatite layer (HCA) when exposed to biological fluid.9, 15
More
specifically, it was observed that ionic dissolution products from silicate based BGs
(e.g. Si, Ca, P) stimulate expression of osteogenic genes resulting in enhanced bone
formation.17
Overall, these unique features make BGs highly attractive biomaterials
for bone tissue engineering applications. Another advantage of BGs is that they are
biodegradable and may be used as carrier for metallic ions and bioinorganics.
Recently, progress has been made to enhance the biological impact of BGs by
incorporating specific metallic ions in silicate (or phosphate) glasses leading to
novel biomaterials with controlled release of inorganic or metallic species with
specific biological effect depending on the field of biomedical application.18-20
As
many trace elements such as Sr, Cu, Zn or Co present in the human body are known
for their anabolic effects in bone metabolism,21-23
these approaches for enhancing
bioactivity of scaffold materials are being investigated by introducing therapeutic
ions into the scaffold material. The subsequent release of these ions after exposure
to a physiological environment is believed to favourably affect the behaviour of
human cells and to enhance the bioactivity of the scaffolds related to both
osteogenesis and angiogenesis. Since too high concentrations of metallic ions are
known to be toxic24
a controlled release of these ions from a suitable (inorganic)
carrier system is required.19
Introduction 13
1.2 Aim of the work
The aim of this work is, therefore, to incorporate metallic ions (Cu and Co were
selected) in a bioactive silicate glasses and to fabricate 3D glass derived scaffolds
for bone tissue engineering applications. The hypothesis was that upon degradation
of the glass derived scaffolds the metallic ions are released in a controlled manner
into the physiological environment and are suggested to enhance the osteogenic and
angiogenic potential of the material. This approach should lead to a novel group of
inorganic materials with specific functionalities to be used in regenerative medicine.
Fig. 1 gives a scheme illustrating the basic idea of this work. Materials
characterisation of the starting glass and glass derived scaffolds was performed to
determine the effect of metal ion doping on the glass properties as wells as the
macro-/ microstructure and mechanical properties of the scaffolds. The effect of
metal ion doping on the mineralisation behaviour of the scaffold was assessed in an
acellular in vitro study by immersion in simulated body fluid (SBF) and evaluation
of the HAp forming ability. In vitro cell biology studies were performed in order to
assess the biocompatibility of the glasses and corresponding scaffold and their
osteogenic and angiogenic potential. Furthermore, the scaffolds angiogenic
potential was evaluated in an in vivo model.
Fig. 1: Schematic overview of the general idea of this work using bioactive glass derived
scaffolds as carriers for metallic ions. Upon degradation of the scaffolds the released metallic
ions exhibit osteogenic, angiogenic or antibacterial properties [graphical abstract from Hoppe
at al.19
]
Fundamentals 14
2 Fundamentals
2.1 General aspects of bone tissue engineering
2.1.1 Human bone
Bone formation (Ossification)
The bone formation process is known as ossification or osteogenesis. Basically, in
the developmental biology of bone two types of bone formation are discriminated:
intramembranous and endochondral ossification. The former one describes direct
bone formation process from mesenchymal stem cell via cell differentiation and
proliferation and which forms mandible, clavicle, and craniofacial bones.25
Endochondral ossification, in turn, involves cartilage tissue as precursor which is
converted to bone through cartilage degradation and deposition of bone matrix by
osteoblast cells.25
Bone forming cells, osteoblasts, arise from pluripotent mesenchymal stem cells
whereby the differentiation process is regulated by tissue-specific transcription
factors, e.g. Runx2, Cbfa1. After osteoblastic differentiation of the progenitor cells
osteoblasts undergo proliferation, matrix maturation and mineralisation: During
ossification osteoblasts (size ~20-30 μm) secrete an amorphous matrix the so-called
osteoid predominantly consisting of collagen I protein and other proteins like
osteopontin, osteocalcin and sialoprotein.25, 26
Further, osteoblasts facilitate the
mineralisation of the osteoid forming calcified hard tissue which is likely driven by
two mechanisms: i) formation of matrix vesicles that create microenvironment with
Ca and P enrichment in a ratio being optimal for crystallisation followed by
alignment and subsequent mineralisation of hydroxyapatite (the inorganic part of
bone) or ii) vesicle independent initialisation by components of the collagen
molecules.25, 27
In any case, osteoblasts play an critical role in bone mineralisation
as they both secrete collagen and form matrix vesicles. During matrix maturation
and mineralisation osteoblasts are entrapped in the mineralised matrix and become
osteocytes (the most abundant bone cell) which exhibit dendritic-like morphology
and are connected and communicate via gap junctions.28
Fundamentals 15
Fig. 2: Schematic illustration of bone formation /bone remodelling process including osteoblast
and osteoclast differentiation from mesenchymal and hematopoietic stem cell, respectively;
matured osteoblast become osteocytes and are entrapped in the bone matrix.29
Osteoblast proliferation and differentiation is regulated by different factors
including biophysical factors, e.g. mechanical loads or matrix deformation and
biological factors. The latter ones include growth factors, e.g. bone morphogeneic
proteins (BMPs) which are secreted by osteoprogenitor cells and mature osteoblast
and active osteoblastic differentiation in vitro.28
Other growth factors include FGF
(fibroblast growth factor) IGF (insulin growth factor) and PDGF (platelet-derived
growth factor) which may regulate osteoblastic differentiation and proliferation.28
Human bone is a dynamic, highly vascularised tissue with the ability to remodel
throughout the life by regulated activity of bone-forming (osteoblasts) and bone-
resorbing cells (osteoclasts). This process of bone remodelling fulfils two functions
in the human body: adaptation of the skeleton system to metabolic demands and
changes of mechanical stress. Bone remodelling describes the addition of new bone
on the preexisistent bone surface,25
whereby it is regulated by a variety of
systematic and local regulatory agents30
including growth factors, hormones and
stress actions.26, 31
When the remodelling process is in balance it involves
osteoblasts as bone forming cells and also equal participation of bone resorbing
cells, the osteoclast.32
Osteoclasts derive from hemopoetic stem cells and the differentiation process
requires cell-cell interactions with wither osteoblasts or osteoblast progenitor cells.
Fundamentals 16
Mature osteoclasts contain typically 6-8 (or even more) nuclei and are 20-100 µm in
size.32
After migration of the osteoclasts to the resorption site on the bone surface
osteoclast form a sealing zone with a “ruffled boarder” (actin ring). Inside this zone
hydroxyapatite crystals are dissolved are resorbed through creation of acidic
environment by targeted secretion of HCl through the ruffled border into the
resorption lacuna. Then proteolytic enzymes (like metalloproteasen, MMP, e.g.
MMP-9, MMP-13 and cathopsines) degrade the collageneous bone matrix.33
Osteoclasts resorb the bone by forming corrosion pits, the so-called Howship's
Lacunae.26
Bone macro and microstructure
Regarding the microstructure human bone has two forms: woven and lamellar,
whereas the former one is considered primary and immature bone which is resorbed
and turned into lamellar bone during adolescence.25
Lamellar bone is further
structurally organized into can cancellous (spongiosa) and dense cortical bone
(compacta). Cancellous bone is composed of irregular, sinuous, convolutions of
lamellae, whereby, the microstructure of cortical bone is composed of regular
cylindrically shaped lamellae.34
Compacta makes up to 80 % of the human hard tissue in the body. The main
structural unit of the compacta is the osteon; Fig. 3 shows the microscopic structure
of spongy and cortical bone. The osteon, or the haversion system is build of
lamellae (collagen fibres arranged in planes, 3-7 µm thick) forming a cylindrical
structure of about 200- 250 µm in diameter.34
In comparison, the woven form of
bone does not show well-arranged mineralised bone and has somewhat less pattern
which can be distinguished. In the centre of each osteon there are Haversian
channels containing blood vessels and nerves. The singe lamellae consist of
collagen fibrils (1 µm) which have plate-like nanosized hydroxyapatite crystals
within the discrete spaces between the collagen fibres.26, 34, 36
Bone structure is
highly anisotropic which results in broad variations in its mechanical properties. For
compact bone and the mechanical properties depend on porosity, mineralisation
level and organisation of the solid matrix.
Fundamentals 17
Fig. 3: Microscopic structure of human lamellar bone.35
For cancellous bone the differences in mechanical properties are usually much
broader and can vary by a factor of 2-5 depending on the origin of bone. Cancellous
bone is composed of bony trabecular struts which are preferentially oriented
according to the load.26
The anisotropic structure forms according to Wolff´s law
directing the orientation of the trabeculae. The trabecular struts (50-300 µm in
diameter) form a highly porous interconnected network in which they are comprised
in cellular structures.34
Cancellous bone makes up 20 % of the human bone and
creates an highly porous environment with porosity values of 50-90 %.37
The
mechanical properties of human bone are listed in Table 1.
Fundamentals 18
Table 1: Porosity P, compressive strength σC, flexural strength σF, fracture toughness KIC tensile
strength, σT and elastic modulus of human bone.38
P[%] σC [MPa] σF [MPa] KIC[MPam1/2
] E-modulus [GPa]
Cortical bone 5-10 100-150 135-193 2-12 10-20
Cancellous bone 50-90 2-12 10-20 0.1-0.8 0.1-5
2.1.2 Concept of (bone) tissue engineering (TE)
The so called “gold standard” for regeneration of critical size bone defects is the use
of autologous bone materials, usually extracted from healthy bone tissue in the
pelvic ridge of the patient. However, this process involves a complicated two-step
surgical procedure which often results in highly problematic donor morbidity.
Hence, to overcame this: i) synthetic osteostimulating biomaterials are being
investigated for use as bone grafts and ii) bone tissue engineering approaches have
been intensively investigated during the last two decades in order to establish a
robust bone regeneration strategy.
The general aim of tissue engineering (TE) is to find ways to restore and maintain
damaged tissues and organs.39
This approach combines cell biology techniques and
materials engineering whereby cultured cells (primary or isolated cell lines) are
coaxed to grow on bioactive degradable scaffolds which provides physical support
and chemical guidance to cell differentiation and their assembly into 3D tissue. Fig.
4 gives a schematic overview of single steps involved in the tissue engineering
process in vitro. After isolation of the cells from the patient and cultivating and
multiplying cells are seeded on a scaffold which can be loaded with drugs, growth
factors, nano particles, or metallic ions inorganic in order to stimulate the cells.
After proliferation and differentiation of the cells towards the required cell type, the
tissue construct is formed which can be re-transplanted into the patient’s bone
(defect) site.
Common approaches in TE include the use of an inorganic scaffold or organic
matrix with aim to provide an optimal environment for the cells to differentiate,
proliferate and communicate.
Fundamentals 19
Fig. 4: Basic concept of tissue engineering. Inorganic scaffolds based on bioactive glass can
serve as supportive porous 3D structure for cell proliferation and differentiation. Reprinted
from.40
From the materials point of view there is a need for an ideal scaffolds with
appropriate biological and mechanical properties being able to fulfil the tasks
mentioned above. The scaffold should maintain cell attachment, migration,
differentiation and proliferation.41
In order to achieve these goals an ideal scaffold,
specifically used for bone tissue engineering, should exhibit6, 42, 43
High biocompatibility
High bioactivity, the ability to bond to hard tissue
Surface texture and chemistry that enables adsorption of biological moieties
(e.g. proteins) and cell attachment
Interconnected pore system, high total porosity with hierarchical pore
distribution enabling cell migration (micropores >100 µm) but also blood
vessel ingrowth (macro pores >400 µm)
Fundamentals 20
Degradation rates that correspond to the formation of native (bone) tissue
and degradation products which are non-toxic and can be easily excreted by
the body (e.g. by respiratory or urinary systems)
Mechanical properties adapted to the specific tissue being replaced and
sufficient to replace bone tissue in load bearing sites. For biodegradable
materials a sufficient temporal mechanical stability during matching the
growth rate of the native tissue is desired
Vascularisation strategies of engineered bone constructs
Bone formation and regeneration is considered a complex multi-component
biological system which involves two major elements: firstly, recruitment,
differentiation of progenitor cells into osteoblasts and their proliferation and
migration as described in 2.1.1; and secondly, formation of new bold vessels,
(neovascularisation) to provide newly formed tissue with nutrition and
oxygenation.44
Neovascularisation, in turn, includes two components: i)
vasculogenesis, the in situ assembly of capillaries from undifferentiated endothelial
cells and ii) angiogenesis, the sprouting of capillaries from pre-existing blood
vessels.
Vasculature of the hard tissue plays a key role in bone remodelling through acting as
reservoir and conduit for bone cells, growth factors and providing key signals
involved in bone metabolism.45
Therefore, angiogenesis not only precedes
osteogenesis but it is also required for its occurrence. This is accomplished by a
combination of factors, which include adequate oxygen tension, compression
forces, nutrients, and secretion of growth factors.46
Considering this, the successful
clinical application of engineered bone constructs highly depends on a functional
vascularised network which might result in enhanced bone metabolism and bone
formation. Several strategies for induction of vascularisation in engineering bone
tissue have been proposed as shown in Fig. 5. These include for instance:46
- Delivery of angiogenic growth factors (GFs, e.g. VEGF, bFGF):
i) direct approach by local and sustained delivery from natural, organic or
inorganic matrices; In vivo instability of growth factors and controlled
release in critical doses are main challenges for GFs delivery approaches;
Fundamentals 21
ii) indirect approach by applying other growth factors (e.g. HIF-1, BMP-2,
or inorganic molecules acting as angiogenic agents, e.g. Cu, Co which, in
turn, would stimulate secretion of angiogenic factors.20, 46
Indirect approach
enables controlled VEGF release mediated by cells which is adapted to the
physiologically, locally needed conditions avoiding “overdose”.
- Micro fabrication of blood vessels and scaffolding: inclusion of network of
vascular geometry in a 3D scaffold using rapid prototyping, foam replica
and other scaffold fabrication techniques
- Micro surgical techniques: engineered (bone) tissue construct is connected
to host vascular system to induce vascularisation. Usually, the tissue graft is
prevascularised in vivo (e.g. in an AV-loop47
or so-called flap fabrication 48
)
before transplantation to the defect site and connected to local vasculature.
High vascularisation degrees and immediate blood perfusion can be
achieved; however, technical challenges and two surgical steps with donor
morbidity are major drawbacks.
- Pre vascularisation in vitro: i) endothelial cells (ECs) and
ii) co-cultures of ECs and osteoblast cells (OCs) are cultured in natural or
synthetic scaffold to form a prevascular network (micro capillary structures)
in vitro which will connect to the host vascular tissue implantation. Host
blood vessel only need to connect the outer region of the bone construct
which will anastomose to the existing blood vessels. Co-cultures of OC and
ECs can may improve vascularisation by forming a more stable and mature
prevascularised network.
Fundamentals 22
Fig. 5: Vascularisation strategies for engineering bone constructs as described by Santos and
Reis46
including applications of angiogenic growth factors (e.g. VEGF), microfabrication of
blood vessels, micro surgical techniques (e.g. AV-loop model prevascularisation), delivery of
mature or precursor endothelial cells (ECs) and co-cultures of ECs and osteoblast cells.
Modified after Santos and Reis.46
One other possibility for stimulating vascularisation of a scaffold materials would
also include the enhancement of angiogenic properties of the scaffold material.
Hence the research has been expanded towards investigations on the angiogenic
effects of biomaterials.46
One common approach includes the loading and
subsequent release of therapeutic inorganic ions (TII) into inorganic scaffold
materials. Potential TIIs are discussed in 2.5. The general requirements, however,
for a material used as a scaffold for (bone) tissue engineering are summarized in
next chapter.
Fundamentals 23
2.2 Biocompatibility and Biomaterials
A biomaterial is defined as a non-viable material used in a medical device and
intended to interact with host tissue whereas biocompatibility is described as the
“ability of a material to perform with an appropriate host response”.49
However,
more recently considering different implant systems and taking into account that a
biomaterial is always a part of a regenerative therapy D. F. Williams updated and
specified this definition by elaborating that
“Biocompatibility refers to the ability of a biomaterial to perform its desired
function with respect to a medical therapy [...] generating the most appropriate
beneficial cellular or tissue response [...].50
When applied to a biomaterial scaffold
used for bone tissue engineering one can state that such a scaffold should perform
as a “substrate that will support the appropriate (bone cell) activity, including the
facilitation of molecular and mechanical signalling systems, in order to optimise
(bone) tissue regeneration.”50
Regarding the reaction with the surrounding tissue biomaterials are classified as
bioinert, bioresorbable and bioactive. Bioinert materials, e.g. alumina, titania or
most of metallic compounds, do not cause any toxic response from their host, yet
they are known to induce a fibrous tissue, disallowing formation of tissue
bonding.51
Bioresorbable materials, such as tricalcium phosphate or biodegradable
polymers dissolve in physiological environment and can be used as temporary
implants which are resorbed by the body and replaced by the native tissue.
Bioactive materials (which can be bioresorbable as well) can react with the local
tissue and form strong bonds to hard tissue (class B bioactivity). The discovery of
these complex surface reactions that allow for a strong bone bonding have made
glass a suitable implant material lead to the development of large amount of
bioactive materials including 45S5 Bioglass®
, glass-ceramics such as Ceravital®,
A/W glass ceramic, dense hydroxylapatite such as Durapatite® or Calcitite
®, as well
as composites such as polyethylene-Bioglass®, polysulfone-Bioglass
®, and
polyethylene-hydroxylapatite (Hapex®).
52 The physico-chemical reactions occurring
at the interface between a bioactive (glass) surfaces upon implantation in living
body leading to the tissue integration are summarized in 2.3.3. Moreover, bioactive
Fundamentals 24
glasses have been shown to stimulate human cells towards a specific cell response
by activating several genes related to osteogenesis and angiogenesis. This
osteoinductive behavior which promoted the development of so called 3rd
generation biomaterials53
is described in detail in section 2.3.4
2.3 Bioactive silicate glasses
Bioactive glasses (BG) such as 45S5 Bioglass®
(wt.%: 45SiO2-25CaO-25Na2O-
6P2O5) and other silicate based compositions represent an important group of
inorganic, bioactive biomaterials which have been widely used in regenerative
medicine.9 For an overview on the history, the properties and diverse applications of
bioactive glasses the reader is referred to articles by Hench53
and Jones54
. In this
section, however, some fundamental aspects on bioactive glasses fabrication, their
structure, general properties, further processing of BGs to scaffolds and their
biological performance are described.
2.3.1 Glass production
Sol-gel process
Low temperature techniques, like the sol-gel route offer an opportunity to produce
bioactive glasses. Hereby, the synthesis of an inorganic network is processed by
mixing organic precursor (e.g. metal alkoxides) in solution which is followed by
hydrolysis, gelation, and low-temperature firing. Silicate glass alkoxide precursors,
such as tetraethylorthosilicate (TEOS), undergo hydrolysis forming a colloidal
solution (sol). After polycondensation of silanol (Si–OH) groups, a silicate (–Si–O–
Si–) network is formed. While the gel is forming the viscosity of the system
increases as the network connectivity raises. Afterwards the gel is dried and
stabilised during a thermal process at round 600-800 °C.55, 56
Sol-gel derived glasses
exhibit mesoporous characteristics and have larger surface area than melt derived
glasses showing pores from 300 to 800 nm with total porosities of over 60%.57
By
choosing suitable precursor materials novel bioactive glass compositions containing
metal oxides can be produced. For instance, Zn containing sol-gel derived glass,58, 59
Fundamentals 25
and Mg-containing59
as well as Sr - glasses60
for biomedical applications have been
developed. However, long processing times, high costs of the precursor materials,
large shrinkage rates and residual carbon/hydroxyl are some of the disadvantages of
the sol gel process.61
Melt derived bioactive glasses
In the melt derived process conventional glass melting is used where desired
amounts of oxides, carbonates or phosphates are homogenized and melted in a
platinum crucible at 1350-1500 °C. The molten glass is then either poured into
graphite moulds in order to obtain solid glass blocks or quenched in water or oil
which results in a glass frit. Subsequent mechanical grinding (e.g. in a planetary
mill) can be applied to obtain glass powder which can be directly used as bone
defect filler material, as addition to polymer-BG composites or can be further
processed for fabrication of 3D scaffolds by sintering methods, as described in 2.4.
Melting is a flexible technique which allows the production of various different
glass compositions, simply by varying the number and proportion ratio of the raw
materials. Several melt-derived bioactive glass compositions have been used in
order to obtain metal oxide containing BGs including Sr-containing BG,62
Boron
derived BG,63
Co-BG64
and F-containing glasses.65, 66
The melt-derived route for
processing bioactive glasses also shows some disadvantages. For example it might
be difficult to achieve high purity glasses due to the high melting temperatures
involved (impurities from crucible material) and the subsequent use of grinding
steps which could lead to contamination with debris particles of the grinding media.
Moreover, the standard 45S5 Bioglass® obtained by the melting process tends to
crystallise during the sintering process forming a predominantly crystalline phase
(combeite)67
which may reduce the hydroxyapatite (HAp) conversion rat 68
and thus
affect the bioactivity of the material. Other modified bioactive glass compositions,
like “13-93” have been developed which show enhanced viscous flow and can be
densified without crystallisation.69
Despite these drawbacks mentioned above the
melting route was chosen for glass fabrication used in this study which exhibits
Fundamentals 26
advantages like easy controllable chemistry of the glass, low costs (regarding the
raw materials), short processing times and large outcome (amounts).
2.3.2 Structure and general properties of (silicate) bioactive glasses
Since Hench et al.70
has discovered the first bioactive silicate material, now called
45S5 Bioglass®
various different composition of bioactive glasses have been
developed including silicate melt derived glasses phosphate glasses, boro-silicate
glasses and sol-gel derived bioactive glasses.71
45S5 is a silicate glass based on
SiO2 as network former building the 3D structure in which Si is fourfold
coordinated to O. In the presence of Ca and Na cations acting as network modifiers
the 3D structure is disrupted and non-bridging oxygens (NBO) are created in the
glass network.
Common nomenclature describing the coordination of Si atom uses Qn
notation (Q
short for quaternary) where n is the number of bridging O bonds to a Si atom.72
NMR studies and network simulations have shown that 45S5 Bioglass® primarily
consists of chains and rings of Q2 (69%) with lower fractions of Q
3 (31%) providing
some cross linking.73
Even though it is well accepted that the 45S5 Bioglass®
structure is dominated by Q2 and Q
3 units a more precise model is given by
assuming a trinodal distributions with Q1, Q
2 and Q
3 units.
74-76 The exact
proportions of the Q units slightly vary between specific studies and simulation
models: one example is given by Pedone et al. who have shown based on MAS-
NMR studies that 45S5 Bioglass® matrix consists of 67.2 % Q
2 SiO2 tetrahedral
cross-linked by 22.3 % Q3 where the chains and rings are terminated by 10.5 %
Q1.77
Despite suggestions by molecular dynamic simulations77, 78
and spectroscopy
data74, 79
for the existence of Si-O-P bonds Pedone et al. showed in a combined
theoretical and experimental study that P is present in isolated orthophosphate
environment (QP0) charged balanced with cations without forming Si-O-P bonds.
80
Moreover, it has been confirmed that the cations are non-randomly distributed in the
glass compensation the NBOs from the Si species. Also the PO43-
species distract
the cations from the silica network for charge balancing. A fragment of the 3D
structure of 45S5 bioactive glass is given in Fig. 6.
Fundamentals 27
One of the key properties of 45S5 Bioglass® (and related bioactive glass
compositions) is their bioactive and osteostimulating behaviour which is described
in more detail in 2.3.3 and 2.3.4, respectively. One reason why 45S5 Bioglass®
lacks full commercial success compared to other bioactive ceramics like HAp or
TCP is its difficulty to be processed to foams or fibres.54
This is because 45S5 BG
tends to crystallise during high temperature treatment above 1000 °C. This leads to
poor densification of 45S5 solid bodies and to formation of micro cracks resulting
in low mechanical strength of 45S5 glass-ceramic. Overall the workability of 45S5
BG is quite poor.
As one alternative to 45S5 BG the so called 13-93 (wt%: 53SiO2-6Na2O-12K2O-
5MgO-20CaO-4P2O5) has been developed and has been approved for in vivo use in
Europe. Based on 45S5, 1393 has a comparatively higher SiO2 content and addition
network modifiers K2O and MgO. Furthermore, 1393 BG exhibits better processing
characteristics (larger process window with lower Tg and higher Tc) by viscous flow
sintering enabling densification of 1393 derived scaffolds without crystallisation.
Also processing of 1393 tapes or glass fibres is possible.71
Some of the key
parameters regarding the thermal behaviour of 45S5 and 1393 BGs are given in
Table 2.
Table 2: Material characteristics of 45S5 and 13-93 bioactive glass; glass transition point Tg,
crystallisation onset To, crystallisation peak Tc and melting point Tm. Temperatures are given in
°C.81, 82
Tg To Tc Tm
45S5 532 655 708 1180
13-93 606 714
In this work 45S5 and 1393 will be chosen as basic silicate systems for metal ion
doping. The density and some selected specific mechanical characteristics of these
two glasses are given in Table 3.
Fundamentals 28
Fig. 6: 3D structure model of 45S5 BG showing isolated PO4 tetrahedral, Q2 silica fragments
crosslinked with Q3 units and terminated Q
1 species. Na (green) and Ca (yellow) are charge
balancing the non-bridging oxygens.
Table 3: Density ρ, compressive strength σC, flexural strength σF, fracture toughness KIC tensile
strength, σT and elastic modulus of 45S5 and 1393 glass.
ρ [g cm-3
] σC [MPa] σF [MPa] KIC [MPam1/2
] σT [MPa] E-modulus [GPa]
45S5 2.7 500 30-60¥ 0.7-1.1
83 88±32
84*
4285#
3585
13-93 2.65 n.a. n.a. n.a. 440±15186**
n.a.
*for fibres of 165-310 µm;
**for fibres of 93-160 µm;
#annealed bulk;
¥tape cast and sintered
2.3.3 Acellular in vitro bioactivity
As mentioned above the ability of a biomaterial to bond to hard tissue is termed
“bioactivity”. The index of bioactivity (Ib) was proposed in order to quantify a
relative bioreactivity between different biomaterial which marks the time for more
than 50% of the interface to be bonded to bone in vivo.51
𝐼𝐵 =100
𝑡50% Eq. 1
The Ib has been shown to be the largest for 45S5 BG compared to other
bioceramics, e.g. alumina or hydroxyapatite.
Fundamentals 29
Silicate-based bioactive glasses exhibit a highly reactive surface capable of
releasing alkali and alkali earth ions into solution due to low SiO2 content in the
glass. BG show bioactive behavior in a broad range of different silicate glass
composition depending on the SiO2 content, as shown in Fig. 7.
Increasing the network modifier concentration increases the reactivity of the glass
surface up to the limit of a non-glass forming region at about 40 wt% SiO2. In fact,
the glass network is filled with so many cations that the reduced melting
temperature at the BG composition allows for a lower temperature glass production
with standard laboratory, with pure silica having a melting temperature of 1720
°C.87
The ternary diagram for bioactive glasses is depicted by Fig. 7 containing a constant
6 wt% of P2O5. A very high reactive region at low CaO composition does not bond
bone. A low reactivity region at low Na2O composition shows the A/W glass
ceramic. The region A has been reported as gene activating, an important step in the
ossification process.53
As indicated in the figure, this region has also the possibility
to bond to soft tissue,88
which is important for the complex mineralised tissue of
bone.
Fig. 7: Schematic compositional diagram for bioactive glasses at constant 6 wt% P2O5.13, 89
Fundamentals 30
Basically, the mechanism behind bioactive behaviour involves the formation of
carbonated hydroxyapatite (CHA) layer in the surface which is bonded to bone
tissue in vivo.
Table 4 shows the currently reaction events occurring ion BG surface in
physiological environment.9, 11, 90
The first 5 steps describe the physico-chemical
reaction on the surface of bioactive implant, whereas steps 6-11 show further
interaction of the surface with biological moieties and bone tissue formation:
(Step 1): First, a rapid diffusion driven (t0.5
dependent) exchange of sodium (Na+),
potassium (K+), or calcium (Ca
2+) ions from the glass with the proton ions from the
solution (H+ or H3O
+) occurs:
91, 92
𝑆𝑖 − 𝑂 − 𝑁𝑎+ + 𝐻+ + 𝑂𝐻− → 𝑆𝑖 − 𝑂𝐻+ +𝑁𝑎+(𝑎𝑞. ) + 𝑂𝐻− Eq. 2
The cationic alkali content is depleted to a depth > 0.5 pm.51
(Step 2): The second, though not sequential, step involves a loss of SiO2 in the form
of Si(OH)4 from the glass mediated by the hydrolysis of water. The produced
silanols form on the glass surface, exhibiting a t1.0
dependence.51
𝑆𝑖 − 𝑂 − 𝑆𝑖 + 𝐻2𝑂 → 𝑆𝑖 − 𝑂𝐻 + 𝐻𝑂 − 𝑆𝑖 Eq. 3
(Step 3): A low solubility product of the released orthosilicate induces a
condensation and repolymerisation of an amorphous silica-based (SiO2) layer on the
surface without the alkali and alkaline-earth cations present.
(Step 4): Ca2+
and PO43-
ions diffuse through the SiO2 layer forming a CaO-P2O5-
rich amorphous film that is continuing to grow by including ions from the
physiological solutions.
(Step 5): Finally, CHA crystallizes from the amorphous CaO-P2O5 film, the
amorphous silica thought to be acting as a nucleation site.51
Anions form solution
(OH-, CO3
2-, or F
-) can be substituted in the CHA crystals. Other calcium phosphate
Fundamentals 31
phases may co precipitate or other crystalline phases, like calcite, may also be
formed.93, 94
(Step 6): Adsorption of biological moieties in CGA layer. Protein adsorption is an
essential step occurring prior to cell adhesion. CHA is known to support adsorption
of certain proteins and hence enhance cell adhesion. Moreover, it has been
described in literature that an in vitro immersion of a BG surface lead to a
competitive effect of serum adsorption and the growth of the Ca-P rich amorphous
layer95
where a full hydroxyapatite layer did not develop after 3 or more days. It has
been suggest that “adsorbed serum proteins impede the nucleation and growth
reactions by which [calcium phosphate] would transform to carbonated apatite”,
indicating that proteins would quickly cover any BG-derived surface.
(Step 7-11): Action of osteoblast, formation and mineralization of bone matrix and
bone tissue formation.
Considering the network connectivity bioactive behavior of BGs is given for NC
values <2 whereas NC>2 results in non-bioactive behavior.96
Influences of glass
composition on HA forming abilities were investigated by Kim et al. 97
For SiO2
glass ranking from 50-70 mol %, the soaking time in SBF required for the
formation of HA varied from 0.5 days to 28 days. Hence the time of appearance of
the HAp layer can be used as an “bioactivity marker” in vitro to compare the
reactivity of different bioactive glass compositions.
Fundamentals 32
Table 4: Sequence of reactions induced by Bioglass® in physiological conditions.
9, 11, 90.
Time [h] Step Surface reaction stages
100
Bo
ne
form
atio
n
11 Mineralisation of matrix and bone growth
10 Generation of organic matrix
9 Differentiation of stem cells
20 8 Attachment of osteoblast stem cells
7 Action of macrophages
10 6 Adsorption of biological moeities in HCA layer
2
Ch
emo
-ph
ysi
cal
surf
ace
reac
tio
ns
5 Crystallization of hydroxyl carbonate apatite (HCA)
1 4 Migration of Ca
2+ and PO4
3- groups through the SiO2 layer and formaton
of amourphous calcium phoshpate (ACP). Further growth of ACP
through incorporation of Ca amd P-species from the solution
3 Polycondensation of SiOH + SiOH --> Si-O-Si+H2O
2 Network dissolution and formation of silanol (SiOH) bonds
1 Exchange of Na
+ with hydrogen ions from body fluids
Bioactive glass surface
Bioactivity assessment in vitro and degradation
In order to assess the bioactivity of biomaterials in vitro it has been proposes that
the formation of the CHA layer in simulated body fluid (SBF) is an indicator for in
vivo bioactivity.98
SBF attempts to mimic the physiological environment of the body
by simulating the inorganic part of the human blood regarding the ionic
concentration, ionic species, and the pH.99
Different recipes for SBF solution have
been proposed98, 100
in order to find the optimal conditions for assessing the
bioactive of materials in vitro. Despite the criticism of the “SBF-test” concerning
the correct prediction of the bioactivity101, 102
this test remains a widely used tool
used for assessing the mineralization potential of biomaterials in vitro.99
The formation of CHA on the BG surface in SBF can be used as a marker of
bioactivity. Hereby, on easily approachable method to quantify the bioactivity is to
detect the time point of appearance of the HCA layer which can be used to compare
the bioreactivity of newly developed glass compositions.
One other possibility to quantify bioactivity is by using activation energy to for
silicon release from the BG which follows an Arrhenius behaviour. It has been
shown that higher activation energies can be correlated to decreased bioactivity.103
Fundamentals 33
Taking this into account the rate of formation of CHA on 45S5 glass surfaces and
the dissolution behaviour of the glass by means of Si release should be considered
for characterization of the reactivity of BGs in physiological environment.
In first place the reaction scheme of a BG surface reaction (Table 4) in
physiological environment describes the formation of CHA leading to strong
interfacial bonding to bone. However, ionic dissolution products released from BG
have been shown to induce an intrinsic cell response influencing differentiation and
proliferation of cells involved in the bone formation process. These effects are
discussed in the following paragraph.
2.3.4 Cellular response to bioactive glasses (BG)
Beside acellular bioactivity BGs have been shown to stimulate bone cells via
upregulation of osteogenesis and angiogenesis related genes, hence enhancing bone
regeneration. Starting with the Xynos et al.17
investigation in 2001, the research
work has been focussing on the molecular interactions of ionic dissolution products
of BGs and their physiological environment in order to gain greater understanding
of these mechanisms and to be able to fabricate “smart” glasses with tailored
properties for specific tissue engineering applications, the so-called third generation
biomaterials.16, 104
One key finding from Xynos et al. studies in 2001 was that
Bioglass®
dissolution products are able to regulate gene expression in human
osteoblastic cells (HOC).17
Several genes known to play a role in osteoblast
metabolism, proliferation and cell-cell and matrix-cell adhesion were up-regulated
up to 5 fold when HOC were cultured in Bioglass® conditioned medium.
17 These
results were confirmed by Kaufmann et al.105
who observed expression of several
genes (osteocalcin, osteonectin, osteopontin) in osteoblast-like cells and increased
alkaline phosphatase (ALP) activity and collagen I formation. Related studies by
Jell at al.106
showed six and threefold upregulation of osteogenic markers, namely
bone sioloprotein (BSP) and ALP, in osteoblasts treated with BG conditioned
culture medium resulting in enhanced cell differentiation.
Beside 45S5 Bioglass, good cellular compatibility was also observed for 13-93 BG.
It has been shown that 1393 derived scaffolds support osteoblast growth and
differentiation in vitro as well as bone tissue formation in vivo.107-109
Similarly, 1393
Fundamentals 34
derived scaffolds have been shown to support growth and differentiation of
osteoblast-like cells (MC3T3-E1).110
The osteostimulating effect of the 13-93 BG,
where the defects filled with the glass showed high mRNA expression of genes for
both bone formation and bone resorption, thus affecting not only osteoblast but also
osteoclasts function and enhancing bone turn over.111
However, the glass particles
were injected along with bone morphogenic protein 2 (BMP-2), so that synergetic
effects of addition of bioactive glass and BMP-2 on bone formation were evaluated.
The osteogenic potential of the 1393 glass was also observed in vivo by implanting
particles in bone defects of rat tibia and by investigating gene expression at the
defect area.111
Osteogenic potential of bioactive glasses has been also confirmed for sol-gel
derived BGs. For example, sol-gel derived 77S (mol%: 80SiO2-16CaO-4P2O5)
bioactive glass has been shown to induce osteogenic differentiation of bone marrow
stromal cells into osteoblast-like cells and to promote cell mineralisation. 112
Furthermore, ionic dissolution products another sol-gel derived BG composition
(58S, mol%: 60SiO2-36CaO-4P2O5) resulted in cell activation through stimulation
of the proliferation of osteoblasts cells113
and upregulation of the expression of a
number of genes including IGF-I, gpl30 or MAPK3/ERK1.114
Additionally, it has
been observed that sol-gel derived phosphate-free glass 70S30C (mol%: 70SiO2-
30CaO) is able to enhance osteoblast maturation and differentiation as well as
production of bone-like minerals by osteoblasts indicating that P may be not
necessarily required for in vitro mineralization of the extracellular matrix.12
Beyond
that, a large number of additional studies have confirmed the osteogenic effects of
bioactive silicate glasses and their dissolution products which have been
comprehensively reviewed in literature.18, 115
Beside osteogenic potential also angiogenesis-related studies have been carried out
on BGs using endothelial cells and fibroblasts.116-121
Day et al.121
observed
increased neovascularisation into Bioglass®
coated polymer meshes after being
subcutaneously implanted in rats. Furthermore, Day116
showed that Bioglass®
stimulates the secretion of angiogenic growth factors from human fibroblasts cells,
providing further evidence that bioactive glass enhances angiogenesis. In this study,
fibroblast cells (CCD-18Co) cultured in Bioglass® particles containing medium
Fundamentals 35
secreted increased amounts of vascular endothelial growth factor (VEGF) and basic
fibroblast growth factor (bFGF). These and other studies on angiogenic effects of
bioactive glasses have been comprehensively reviewed confirming angiogenic
potential of BGs117
. However, it should be mentioned that the angiogenic effect of
bioactive glasses seems to be dependent on the morphology and the form in which
the BG is applied. The influence of the shape, morphology and size of bioactive
glass particles (which are for instance used as fillers in polymer-BG composites)
should be taken into account and need further investigation. It has been shown that
the angiogenic effect of bioactive glass seems to be more pronounced in bioactive
glass-based scaffolds (i.e. BG loaded collagen sponges,119
discs,122
meshes,121
tubes,123
and porous glass-ceramic scaffolds117, 124, 125
than in composite structures
incorporating and fully embedding bioactive glass particles in polymer matrices
such as microsphere composites126
or foams.127, 128
The large amount of results on the biological performance of bioactive glasses
discussed above confirms the hypotheses that “ionic dissolution products released
from bioactive glasses stimulate the genes of cells towards a path of regeneration
and self-repair” as discussed by Hench 129
. It is now well accepted that bioactive
silicate glasses are able to stimulate osteogenesis as well as angiogenesis and in
some cases exhibit antibacterial properties. Fig. 8 shows an overview of biological
response to bioactive glasses based on literature reports.
Fundamentals 36
Fig. 8: Schematic overview of biological responses to ionic dissolution products from bioactive
glass (BG). Additional release of therapeutic metal ions upon the degradation of the glass
matrix is proposed to enhance the biological performance of BGs. [From Hoppe et al.18
]
However, the exact mechanisms of interaction between the ionic products from
bioactive glasses and cells remain unclear. It has been discussed in literature that
critical concentrations of Si, Ca, and P are required for osteostimulating effects of
BGs. Based on Xynos et al. 17, 130, 131
studies, Hench 129
stated that eluted Ca and Si
concentrations of 60-88 ppm and 17-21 ppm, respectively, are critical for
upregulation of several osteogenic genes. The importance of Si was also
meantioned by Valerio et al. 132
who suggested that higher osteoblastic proliferation
and collagen secretion after treatment with BG60S dissolution products is related to
Si contact, since despite higher Ca concentration no increased osteoblast activity
was observed in presence of biphasic calcium phosphates (BCP) with no Si release.
Indeed, as described in 2.5.1 in more detail Si has been shown to be an essential
element for metabolic processes associated with the formation and calcification of
bone tissue 133, 134
and to be present in early stages of bone matrix calcification 134
.
Moreover, high Ca concentrations (of 88-109 ppm) have been shown to reduce
Saos-2 osteoblast proliferation when exposed to bioactive glass “MBG85” (mol%:
Fundamentals 37
85SiO2-10CaO-5P2O5) conditioned culture, whereas a Si concentration of 60 ppm
did not have any negative effect on the cell proliferation 135
. Despite high Ca
concentrations beeing toxic for human osteoblastic cells, Ca has been shown to
increase the glutamate release of osteoblast cells.136
Since glutamate signalling
pathways are known to play an important role in bone mechanosensitivity,137
the
extracellular Ca concentration must be considered as an important stimulating agent
in bone formation.
Altogether bioactive glasses offer various beneficial features making them
promising material for use in bone tissue replacement and bone regeneration
applications. In this context, BGs have been widely used as filler in polymer
composites,10
as bioactive coatings,138
in dentistry for alveolar augmentation 139
and
in the field of maxillofacial surgery.140
The use of BG to produce 3D porous
scaffold for tissue engineering in described in the following chapter.
2.4 Bioactive glass derived scaffolds (State of the art)
Various fabrication techniques have been described to produce 3D porous bioactive
glass and ceramic foams141
including foam replica68
, diverse rapid prototyping
methods,142, 143
freeze casting144
or freeze extrusion.145, 146
Table 5 provides an
overview of selected techniques currently used for fabrication of bioactive glass
foams and corresponding structural and mechanical properties. A more
comprehensive overview over fabrication methods of bioactive glass scaffolds and
bioglass-polymer composite foams can be found elsewhere.11
Clearly, all these
methods lead to different morphologies and structures of bioactive glass scaffolds.
Related to in vitro bioactivity in simulated body fluid (SBF), early studies involving
dense specimens indicated that crystallinity reduces the bioactivity of bioactive
glass.147
However, later studies focusing on highly porous scaffolds have shown that
the bioactive character remains for crystalline materials and the formation of HAp is
just delayed.68
Fundamentals 38
Table 5: Overview of different methods reported in literature for fabrication of bioactive glass
derived foams and their corresponding properties.
Fabrication
technique
Glass
composition
P [%] Pore size [µm] σc [MPa] Ref.
Foam replica “Fa-GC” 75 ~100 2
148
“13-93” 85 ± 2 ~100-500 11±1
69
“45S5”
∼90 510–720 0.3-0.4
68
Gel casting “ICIE”16
~80 ~380 1.9 149
Freeze extrusion “13-93“ ~50 Pore width:300 µm and
struts diameter 300 µm
~140 145, 146
“45S5” ~53 - -
150
“13-93” 55–60% 90–110 (pore width,
columnar);
20–30 (pore width,
lamellar)
25 ±3
(columnar)
10 ±2 (lamellar)
108
Direct ink „6P53B“ 60 500 μm (pores size),
100 μm (rod diameter)
136 ±22 151
Lithography “45S5” - - 0.33
152
P...relative Porosity; σc...compressive strength; “Fa-GC...(mol%: 50SiO2-18 CaO-9CaF2-Na2O-7K2O-6P2O5-3MgO);
“13-93”...(wt%: 53SiO2-6Na2O-12K2O-5MgO-20CaO-4P2O5); “45S5”...(wt.%: 45SiO2-24.5Na2O-24.5CaO-6-P2O5);
“ICIE”16…(mol%:49.5SiO2, 36.3CaO, 6.7Na2O, 1.1P2O5, 6.6K2O); „6P53B“...(wt%: 52.7SiO2-10.3Na2O-2.8K2O-
10.2MgO-18.0CaO-6P2O5)
Besides the now well established foam replica method to make bioactive glass
scaffolds, other techniques have been considered to fabricate 3D porous glass and
glass-ceramic scaffolds. Organic molecules, such as starch from rice, potato, or corn
grains for instance can be used to introduce porosity by swelling these molecules in
water.153
After the sintering process and burn out of the organic fillers, a highly
interconnected pore system remains which contains pores of size 84 μm and a pore
content of 40% vol., as reported for bioactive glass.153
Fibre-derived scaffolds have been also presented, which are based on the assembly
of bioactive glass fibres to produce porous structures. Melt derived glass fibres are
packed and bonded together in a ceramic mould using a continuous bead of silicone
adhesive110
or sintered together.154
Typically fibre based scaffolds show porosities of
40-60 vol% and compression strength values of 12-18 MPa, notably higher than
values achieved by the foam replica method, albeit at lower porosities.
Other techniques for fabricating glass foams include freeze casting144
and freeze
extrusion.145, 146
Camphene, ice or water and glycerol can be used as freezing
vehicles.150, 155
After mixing the glass powder with the relevant vehicles, the slurries
Fundamentals 39
are cast and frozen at temperatures between -20 and -70°C, followed by a sintering
process.
Freeze casted 13-93 scaffolds with oriented (lamellar and columnar) pores and
equivalent porosity of 55–60% were shown to have a compressive strength of 25±3
MPa, compressive modulus of 1.2 GPa and pore width of 90–110 µm for columnar
scaffolds, compared to values of 10 ± 2 MPa, 0.4 GPa, and 20–30 μm, respectively,
obtained for the lamellar scaffolds.108
Rapid prototyping techniques have also been
described for fabricating porous bioactive glass based scaffolds. Direct ink writing
for instance was used to develop bioactive glass (6P53B composition) scaffolds
exhibiting a compressive strength of 136 ± 22 MPa, which is comparable to the
value for cortical bone (100–150 MPa) with porosity of 60% 151
. In a recent study, a
method based on lithography-based additive manufacturing technologies (AMTs)
was applied to create 45S5 bioactive glass scaffolds152
resulting in scaffolds
showing biaxial strength and compressive strength of ~40 MPa and 0.33 MPa,
respectively.
Using sol-gel derived bioactive glass particles direct foaming methods can be
applied in order to fabricate porous scaffolds.42, 156
Jones et al.157
described sol gel
derived BG foams where the scaffolds are obtained by direct foaming of the sol
using Teepol as foaming agent. After a drying process the gelled foams are aged at
60 °C, dried at 130 °C and stabilized at 600 °C. In a further heat treatment process
the foams are densified at 800 °C. Fig. 2 shows a typical structure of a sol-gel
derived bioactive glass scaffold. By varying the amount of foaming agent the pore
size distribution and overall porosity can be tuned 157
. The mechanical strength of
sol-gel derived bioactive glass scaffolds is usually in the range of 0.3–2.3´MPa (in
compression) limiting their applications to non-load bearing tissue engineering
approaches. Another related technique involving sol-gel derived glasses has been
developed using sugar cane as a template.158
In this study, the foam replica (FR) technique will be used for scaffold fabrication.
The polymer foam replica method was introduced in 2006 to fabricate 3D “45S5”-
based scaffolds for bone tissue engineering and it is widely used since then.68, 69
Briefly, polyurethane (PU) foam used as sacrificial template is infiltrated with a
glass powder containing slurry which adheres to the PU foam surface. Afterwards
Fundamentals 40
the excess slurry is removed and the coated PU foam is dried and then densified in a
sintering step. The polyurethane template determines the macro structure of the
final glass or glass-ceramic foam-like scaffold. Typically glass foams made by the
FR method show total porosities of >85 vol% and pore sizes in the range 100 - 400
µm. The chemical composition and extent of crystallinity depends on the starting
glass powder composition used. While 45S5 Bioglass® derived scaffolds crystallize
during sintering and form silicate and phosphorous rich phases,67, 68
more recently
developed glasses like “13-93”, which contain larger amounts of alkali oxides,
remain amorphous without any crystallization during the densification heat
treatment.69, 159
The structure and chemistry of the scaffolds also determine the
scaffold’s in vitro bioactivity, mechanical properties and has also an effect on the
protein adsorption on scaffold surfaces. For example dense highly crystalline 45S5
Bioglass®
derived scaffolds have compressive strength in the range of 0.25-0.4 MPa
(Table 5), lying even below the lowest compressive strength values reported for
spongy bone. On the other hand for amorphous 13-93 bioactive glass derived
scaffolds compressive strength values up to 11 MPa have been reported.69
The
different values for the strength of these scaffolds might be related to the processing
conditions and the resulting structure of the scaffolds. 13-93 bioactive glass can be
densified in a viscous flow process which should lead to crack-free struts where
sintering is not impaired by the crystallization process, which occurs in 45S5 type
bioactive glass. Fu et al. 69
reported pore size values of 100-500 µm and a
compressive strength of 11 ± 1 MPa for 13-93 derived glass scaffolds made by foam
replica technique. The influence of chemical composition, fabrication method and
scaffold structure on the mechanical properties of bioactive glass derived foams has
been discussed in the literature.38
Fundamentals 41
2.5 Metal ions and bioinorganics in silicate glasses
2.5.1 Role of bioinorganics in bone metabolism
Metallic ions are essential in human metabolism and are also known to play a
critical role in osteogenesis and angiogenesis.22, 160
They have been considered
highly promising for the field of biomedicine.161
Single inorganic ions such as
Calcium (Ca).136, 137, 162, 163
, Phosphorous (P),164
Silicon (Si),133, 134, 165
Strontium
(Sr),30, 166-170
Zinc (Zn),31, 171
as well as Boron (B),172, 173
Vanadium (V),23, 174, 175
,
Cobalt (Co)176-179
and Magnesium (Mg)180-186
are known to be involved in the
bone metabolism and to play a physiological role in angiogenesis, growth and
mineralization of bone tissue. Table 6 gives an overview of biological responses to
single inorganic ions. In particular, metal ions are known to act as enzyme co-
factors and therefore influence signalling pathways and stimulate metabolic
effects occurring during tissue formation.22, 187
Considering these effects and the
fact that metallic ions are cheap, easy to process and are less risky compared to
gene therapy or growth factor based techniques metal ions attractive for use as
therapeutic agents in the fields of hard and soft tissue engineering.188
The
advantages of therapeutic inorganic molecules such as metallic ions for use as
stimulating agents for osteogenesis and angiogenesis have been comprehensively
reviewed in literature.18-20, 160, 188
In the following few key properties of selected
bioinorganics are highlighted demonstrating their importance in bone physiology.
Si is an essential element for metabolic processes and is also associated with the
formation and calcification of hard tissue.133, 134
High Si contents have been
detected in early stages of bone matrix calcification,134
while aqueous Si was
shown to be able to induce hydroxyapatite precipitation, the inorganic phase of
human bone.189
Furthermore, dietary Si intake was shown to increase the bone
mineral density (BMD) in men and premenopausal women.190
In a in vivo study
with Ca-deficient rats showed that Si supplementation caused positive effects on
bone mineral density by reducing bone resorption.191
Regarding its physiological
role Nielsen et al.192
suggested that Si has a biochemical function in bone growth
Fundamentals 42
processes affecting bone collagen turnover and sialic acid-containing extracellular
matrix (ECM) proteins like osteopontin. Moreover, orthosilicate acid (Si(OH)4) at
physiological concentration of 10 µmol has been shown to stimulate collagen I
formation in human osteoblast cells (HOC) and to stimulate osteoblastic
differentiation.165
Table 6: Effect of metallic ions on human bone metabolism and angiogenesis.
Ion Biological response in vivo / in vitro
Si - essential for metabolic processes, formation and calcification of bone tissue
133, 134
- dietary intake of Si increases bone mineral density (BMD) 190
- aqueous Si induces HAp precipitation189
- Si(OH)4 stimulates collagen I formation and osteoblastic differentiation165
Ca - favours osteoblast proliferation, differentiation and extracellular matrix (ECM)
mineralization 162
- activates Ca-sensing receptors in osteoblast cells, increases expression of growth factors,
e.g. IGF-I or IGF-II136, 163
P - stimulates expression of matrix la protein (MGP) a key regulator in bone formation
164
Zn - shows anti-inflammatory effect and stimulates bone formation in vitro by activation
protein synthesis in osteoblasts31
- increases ATPase activity, regulates transcription of osteoblastic differentiation genes,
e.g. collagen I, ALP, osteopontin and osteocalcin193
Mg - stimulates new bone formation
186
- increases bone cell adhesion (probably due to interactions with integrins) 186, 194
Sr - shows beneficial effects on bone cells and bone formation in vivo
30, 170
- promising agent for treating osteoporosis195
Cu - significant amounts of cellular Cu are found in human endothelial cells when undergoing
angiogenesis196
- promotes synergetic stimulating effects on angiogenesis when associated with angiogenic
growth factor FGF-2197
- stimulates proliferation of human endothelial cells198
- induces differentiation of mesenchymal cells towards the osteogenic lineage199
Co - mimics hypoxia hence stabilises HIF-1 factor
200
-In vivo: CoCl2 pre-treated BMSCs induced higher degree of vascularisation and enhanced
osteogenesis within the implants179
B - stimulates RNA synthesis in fibroblast cells
201, 202
- dietary boron stimulates bone formation173
Mg is another essential element for bone metabolism and it has been shown to
have stimulating effects on new bone formation.180-186
Mg is suggested to interact
with integrins of osteoblast cells which are responsible for cell adhesion and
stability.186, 194
Rude et al. 181-183
observed that Mg depletion results in impaired
Fundamentals 43
bone growth, increased bone resorption and loss in trabecular bone underlining
the significant role that Mg plays in bone metabolism.
Because of the chemical analogy to Ca (same main group, similar atomic radius,
Ca2+
- 1.0 A, Sr – 1.16 A), Sr can accumulate in bone by exchanging with Ca in
the hydroxyapatite crystal lattice.203
The therapeutic potential of Sr in bone
metabolism has been investigated by Marie et al., who showed that Sr exhibits
beneficial effects on bone cells and bone formation in vivo.30, 170
Sr has also been
shown to be a promising agent in treating osteoporosis.195
Additionally, Sr based
drug such as strontium renalate enhances bone healing indicated by increased
callus resistance in rat bones which has confirmed Sr as a promising agent in
healing bone fractures.204
Zinc is also known to play an important role in bone metabolism 31
and to have
anti-inflammatory effects.205
Furthermore, Zn stimulates bone formation in vitro
by activating protein synthesis in osteoblast cells and increasing ATPase activity
in bone 31
. Moreover, Zn shows inhibitory effect on bone resorption inhibiting the
formation of osteoclast cells in mouse marrow cultures.31
The regulatory effects of
Zn on bone cells has suggested the importance of Zn in gene expression 206
. More
recently, zinc was identified as regulation agent in transcription of osteoblastic
differentiation genes, such as collagen I, ALP, osteopontin and osteocalcin.193
It
was assumed that Zn can be considered a Runx2 stimulating agent being able to
directly stimulate bone formation through increasing Runx2-targeted osteoblast
differentiation gene transcription.193
Cu has been shown to play a significant role in angiogenesis.196-198
For example,
remarkable distributions of cellular Cu have been found in human endothelial
cells when they were induced to undergo angiogenesis revealing the importance of
the ion as angiogenic agent.196
Another recent study revealed that copper,
associated with angiogenesis growth factor FGF-2, promotes synergetic
stimulating effects on angiogenesis in vitro.197
Moreover, Cu was shown to
stimulate proliferation of human endothelial cells 198
and to induce an increase in
differentiation of mesenchymal stem cells (MSC) towards the osteogenic lineage
199. Cu
2+ at a concentration of 10
-6 mol l
-1 has been also shown to inhibit osteoclast
activity.207
. However, a possible positive effect of Cu on bone metabolism is still
Fundamentals 44
not clearly demonstrated. Cashman et al.208
for instance found that copper
supplements over a period of 4 weeks do not show any effect on biochemical
markers of bone formation or bone resorption.208
Similarly, Lai and Yamaguchi 209
have shown that supplementation with copper induced a significant decrease in
bone tissue of rats showing no anabolic effects on bone formation in vivo and in
vitro and, additionally, it was shown that Cu reduces anabolic effects of Zn. Other
researchers have found that dietary copper depletion causes a reduction of bone
mineral density (BMD), although no biological markers for bone formation and
bone resorption were explicitly affected by Cu.210
Cobalt ions have been considered as important ions in the context of bone
physiology176-179
since Co is a known to induce hypoxia conditions and to
stabilize HIF-1.211, 212
Hypoxia conditions, in turn, are known to activate several
pro-regenerative processes in human body213
via regulation of the hypoxia-
inducible factor 1 (HIF-1). HIF-1 activation has been shown to result in
accelerated bone ingrowth whereby the HIF-1a pathway has been identified being
critical for angiogenesis and skeleton regeneration214
and are important for
development of angiogenesis, stem cell differentiation and fracture repair.64, 215
Furthermore, HIF-1 is stabilized under hypoxic conditions and regulates several
genetic pathways relevant for skeleton repair.216
Hence Co-releasing bioactive glass has been proposed as hypoxia mimicking
material217
to be used for artificial stabilization of HIF-1. These potential benefits
of Co2+
ions were further tested in vitro and in vivo. For instance, Cobalt was
shown promote angiogenesis via activation of hypoxia inducible factor 1 (HIF-1)
in a rat remnant kidney in vivo model when Co was applied by subcutaneous
injections.179
Also in a rat bladder in vivo model Co was shown to enhance
hypoxia response, cell growth and angiogenesis indicated by stimulated
expression of HIF-1α and vascular endothelial growth factor (VEGF).176
Furthermore, bone marrow derived stem cells (BMSCs) pre-treated with CoCl2
induced higher degree of vascularisation and enhanced osteogenesis within
collagen scaffolds implanted in vivo.200
Fundamentals 45
The use of metallic ions incorporated in a silicate glass system has been widely
investigated. In the following chapter relevant studies on the biological
performance of metal ion containing bioactive glasses are highlighted.
2.5.2 Biological performance of metal ion containing glasses and
glass ceramics
In order to enhance the bioactivity of bioactive glasses towards a specific
biological response in relevant physiological environments many approaches have
been investigated incorporating various metal ions in the silicate network. The
main goal is to increase the stimulating effects of bioactive glasses on
osteogenesis, angiogenesis and the promotion of antibacterial properties.
The incorporation of selected metal ions into silicate matrices has resulted in
enhanced bone formation and angiogenesis,18
whereby phosphate based glasses
can also be used as effective carriers for TII.218
These effects have been shown for
various silicate systems including Co-BG,64, 219
Zn-BG,220-224
Cu-BG,224-226
Sr-
BG,62, 227-230
Mg-BG,225, 231-235
, Ag-BG,148, 236-241
Ce-BG242
as well as B-BG222, 243-
245 and F-BG.
66, 246 Comprehensive reviews on the biological impact of such novel
silicate and also phosphate based glass compositions can be found in literature.18,
247 In the following few selected glass systems and their relevance for bone tissue
engineering applications.
Diverse silicate glasses based on CaO–MgO–SiO2 with different additive agents
(B2O3, CaF2, Na2O and P2O5) have been shown to exhibit appropriate level of
acellular bioactivity proved through standard bioactivity testing by soaking the
material in simulated body fluid, according to Kokubo et al.15, 99
, in order to prove
the formation of surface HCA layer.248
In the study of Agathopoulos et al. 248
, for
example, it was observed that increasing amount of phosphates favours the
deposition of HCA while increasing contents of CaO and SiO2 inhibit its
deposition. However no biological data through in vitro cell test were provided for
the glass compositions investigated. Moreover, Vrouwenvelder et al.249
reported
on the osteoblast behaviour on 45S5 bioactive glass doped with iron, titanium,
fluorine and boron giving (to our knowledge) the first overview of the biological
response to ion doped bioactive glasses. It was shown that Ti-BG exhibited higher
Fundamentals 46
proliferation and osteoblast expression probably due to more controlled release of
the ionic glass products, whereas doping with B, Fe and F resulted in lower
osteoblast activity compared to undoped control 45S5 bioactive glass.
Moreover, HAp formation was delayed through the incorporation of CoO, ZnO
and MgO as it was revealed by SBF studies,64
, which is, in fact, desirable for soft
tissue engineering applications, e.g. cartilage regeneration.
In the following paragraphs different studies on particular ions and their effect on
cell behaviour are summarized and the results discussed regarding their relevance
for bone tissue engineering.
Bioactive silicate glasses doped with zinc have been shown to enhance acellular
formation of calcium phosphate layer on the BG surface after soaking BG
substrates in biological fluids (Dulbecco's Modified Eagle's Medium, DMEM)250
and in simulated body fluid (SBF),251, 252
which is a fundamental requirement for
bioactive glasses to bond to bone.9 Independently of the acellular behaviour
investigations, in vitro cell culture studies have shown the anti-inflammatory
effect of Zn-doped bioactive sol-gel derived 58S glass.224
In this study, murine
macrophage cells (RAW 264.7) stimulated with lippopolysaccharide (LPS)
endotoxin were cultivated in DMEM treated with BG suspension (1mg ml-1
) and
glass ionic dissolution products, respectively, resulting in decreased secreted
amount of tumor necrosis factor TNF-α and interleukin-1 which indicated the
inflammatory effect on the cells. Glass compositions with 5mol-% and 11mol-%
Zn did not have any therapeutic effects oncells since the TNF-α and IL-1 contents
were not significantly decreased after treatment with the BG suspension. On the
other hand the pre-treatment of cells with bioactive glasses resulted in a
significantly decreased amount of TNF and IL-1 indicating a prophylactic anti-
inflammatory effect of the Zn-doped glasses compared to the undoped control
glasses. However, cell treatments with ionic dissolution products of the same glass
did not show any positive anti-inflammatory effects of the Zn-doped glasses
compared to undoped control glass, which might be related to the high
concentrations of Zn (up to 16 ppm) released into the cell medium being toxic for
cells. Other studies have also shown that Zn concentrations in the range of 2-8
ppm can cause damage in human osteoblasts via oxidative stress.253
Similar
Fundamentals 47
results were observed for endothelial cells seeded on wells in the presence of Zn-
doped glass slabs revealing that bioactive glasses doped with 20 wt.% Zn increase
adhesion of cells but decrease the proliferation rate probably due to toxic Zn
concentration of 2.7 ppm released into the culture medium, whereas 5Zn-BG
promoted well cell adhesion as well as high cell proliferation of endothelial cells
at Zn concentrations of 1.1 ppm.220
However, results on cell adhesion related to
the undoped glass composition, indicated that all glass samples, undoped 58S,
5%Zn-58S and 20%Zn-58S, exhibited poorer cell adhesion and proliferation than
the silica control samples.
Additionally, Zn-releasing bioactive glass scaffolds have been investigated in
relation to their possible osteogenic effects.58, 222, 252, 254, 255
It has been shown that
sol-gel derived glasses containing 5 mol% ZnO resulted in increased ALP activity
and increased osteoblast proliferation 58
indicating the possible stimulating effect
of Zn-BG on osteoblast cells.
In related research, Oki et al.255
observed that Zn containing bioactive glass (5
mol%) increased AP activity of human fetal osteoblastic cells (hFOB 1.19) when
seeded on glass discs in relation to polystyrene control. However, these results
were not compared to un-doped glass samples, so that no definitive statement on
the stimulatory effect of Zn can be made based on the available experimental
evidence. More recently, Haimi et al.222
investigated the effects of Zn-doped BG
scaffolds on human adipose stem cells proliferation and osteogenic differentiation
revealing no significant effect of Zn-doping on cell activity when cells were
seeded on the Zn-doped BG scaffolds. The authors suggested that the possible
stimulatory effect of Zn is inhibited through the decreased degradation profile of
the bioactive glass caused by the Zn addition. Similar results were observed by
Lusvardi et al.252
, who found that Zn-doping provides no significant effect on
adhesion and proliferation of osteoblast-like cells (MC3T3-E1) compared to un-
doped control glasses, probably due to the slowed degradation of the BG caused
by Zn addition resulting in a negligible amount of Zn ions released. However, the
degradation kinetics was investigated only in SBF (release of 4.6 ppm after 70
days) and no cell cultures treated with ionic dissolution products of the Zn doped
Fundamentals 48
glasses were carried out missing a specific investigation of possible anabolic
effect of Zn. Despite the fact that in vitro studies with systematic supplements of
Sr-doped glasses have been shown to exhibit enhanced acellular bioactivity and to
release critical concentrations of Sr ions in the range of 1-5 ppm into the
dissolution medium.229, 256
Moreover, Gentleman et al. 227
have shown that ion
release from Sr-doped silicate glasses enhances bone cell activity. They
investigated the in vitro behaviour of SiO2-P2O5-Na2O-CaO silicate glass in which
Ca was systematically replaced by Sr up to 100%. In this study, treatment with
ionic dissolution products of Sr-doped glasses (ion release in culture medium in
the range of 5-23 ppm) resulted in enhanced osteoblast (Saos-2 cell line) activity
and inhibited osteoclasts differentiation. Moreover, these Sr-doped glasses
promoted osteoblast proliferation and ALP activity when directly applied in
contact with cells as solid BG-discs. Similarly, it has been shown that Sr doped
BGs produce increased proliferation of rat calvaria osteoblastic cells and enhance
cell differentiation and ALP activity.60
In vivo studies have also confirmed the
high biocompatibility of Sr-containing 45S5 Bioglass® expressed through strong
bonding to bone via HCA layer without any inflammatory affects, though no
differences of the in vivo behaviour compared to undoped Bioglass® control were
observed 228
.
Different approaches have been made in order to incorporate Cu in an inorganic
scaffold material, including Cu addition in calcium phosphates,257, 258
silicate
glasses,226
phosphate based glasses259, 260
and in polymer coatings on bioactive
glass scaffolds.261
Cu release from calcium phosphates was shown to enhance
angiogenesis257
whereas Cu-containing meso-porous bioactive glass scaffolds
were reported to stimulate angiogenesis as well as osteogenesis.226
The direct
incorporation of Cu ions into 45S5 Bioglass® scaffolds has not been investigated
to date and will be part of this work.
Cobalt-containing inorganic biomaterials have been also poropsed for use in
regenerative medicine. For example, cobalt-containing β-tri-calcium phosphate
ceramic (β-TCP) have been shown to stimulate VEGF expression of BMSCs and
to enhance the formation of network structure of human umbilical vein
endothelial cells (HUVECs) compared with pure TCP.262
Co-containing glasses
49
co-doped with Zn and Mg 64
have been fabricated in order to create “hypoxia-
mimicking” (low pressure environment) biomaterials to be used in bone tissue
engineering. Creating hypoxia conditions is suggested to be a strategy for
activating pro- and anti-angiogenic genes.263
Melt derived Co-containing BGs
have been reported in literature which a have been introduced as suitable carrier
for Co2+
ions.64
. From these glasses Co concentrations in the range of 10-14 ppm
were released in physiological medium, which are believed to be within the
biologically active limits. Sol-gel Co-containing bioactive glasses have been
reported by Wu et al. who used sol-gel derived BG derived scaffolds by foam
replica technique as Co releasing platform.219
However, no Co containing 13-93
bioactive glass (1393-Co) has been presented so far. Also no Co-containing highly
porous scaffolds with sufficient mechanical strength have been reported in
literature. Hence, on of the goals of this study was to fabricate Co-releasing
mechanically robust 3D scaffolds based on 13-93 bioactive glass suitable for
potential applications in regenerative medicine.
Most of the ion-doped bioactive glasses reported in literature have been shown to
exhibit sufficient levels of acellular bioactivity indicated through the formation of
carbonated hydroxyapatite layer after soaking in SBF18
which is an essential
requirement of biomaterials to form a strong bonding to bone.9 Moreover, doping
with different metal ions seems to be a promising approach to enhance the
biocompatibility of the glasses and to stimulate cell proliferation, as it was shown
for a few specific novel glass compositions discussed above.
In this work Cu and Co were chosen as therapeutic ions to be incorporated in BG
glass derived scaffold.
Materials and Methods 50
3 Materials and Methods
3.1 Glass fabrication
3.1.1 Cu-containing 45S51
Melt derived 45S5 bioactive glasses with different CuO contents (45S5-Cu) were
produced by mixing analytical grade silicon oxide (SiO2, Merck, Darmstadt,
Germany), sodium carbonate (Na2CO3, Merck, Darmstadt, Germany), calcium
carbonate (CaCO3, Merck, Darmstadt, Germany), tri-calcium phosphate (Ca3(PO4)2,
Sigma-Aldrich, Germany) and basic Cu carbonate (CuCO3*Cu(OH)2, Sigma
Aldrich, Germany). The raw materials were well homogenized and melted in a
platinum crucible at 1450 °C for 45 min (Furnace LHT 3 KW, C295 Control Unit,
Nabertherm, Germany). The nominal compositions of the glasses investigated in
this study are given in Table 7.
Table 7: Nominal 45S5 Bioglass® derived glass compositions with different Cu contents.
Glass Composition in wt%
SiO2 Na2O P2O5 CaO CuO
45S5 45 24.5 6 24.5 -
45S5-0.1Cu 45 24.5 6 24.4 0.1
45S5-1Cu 45 24.5 6 23.5 1
45S5-2.5Cu 45 24.5 6 22 2.5
3.1.2 Co containing 13-932
Co-containing bioactive glasses based on the 1393 composition (1393-Co) were
fabricated using melt-derived technique. Silicic-acid (pure anhydrated, Riedel de
Haën, Germany), Sodium-carbonate (Riedel de Haën, Germany), Potassium-
carbonate (Riedel de Haën, Germany), Sodium-dihydrogenphosphate (Riedel de
1 Melting of the 45S5-Cu glass series was carried out at the Institute of Glass and Ceramics (Glass group, Prof. Wondraczek) under assistance of R. Meszaros 2 Melting of the 1393-Co glasses was carried out by B, Jokic and Prof. Jankovic at Universtiy of Belgrade
Materials and Methods 51
Haën, Germany), Magnesium-carbonate (Lachner, Czech Republic), Calcium-
carbonate (Lachner, Czech Republic), Cobalt-nitrate (Carlo Erba-Italy) calculated to
the glass composition. All chemicals were p.a. grade. Melting was performed in a
platinum pot at 1300 oC for 2h and rapidly cooled by quenching in water. The glass
compositions are given in Table 8.
Table 8: 13-93 derived glass compositions with varying amounts of CoO (Composition in wt%).
Glass SiO2 Na2O K2O P2O5 MgO CaO CoO
1393 53.0 6.0 12 4 5 20 -
1393-1Co 53.0 6.0 12.0 4.0 5.0 19.0 1.0
1393-5Co 53.0 6.0 12.0 4.0 5.0 15.0 5.0
1393-10Co 53.0 6.0 12.0 4.0 5.0 10.0 10.0
3.2 Preparation of the glass powder
Then, the glass was fritted, pre-milled in a jaw crusher and milled in a planetary
mill to the final particles size of d50 =5 µm. Fritted glass was then pre-milled in a
jaw crasher (BB51, Retsch, Germany) and milled in a planetary mill using ZrO2 jar
and ZrO2 milling balls (PM100, Retsch, Germany) to a final particle size of d50~5
um.
3.3 Characterization techniques
3.3.1 Scanning electron microscopy (SEM) / Energy dispersive X-
ray spectroscopy (EDS)
For SEM analyses the scaffolds were fixed on a sample holder with a dry
conductive adhesive (Leit-C, Fluka Analytical). Before analyzing, the scaffolds
were sputtered with gold. Images were taken with a FEI-Quanta 200 scanning
electron microscope at an operation voltage of 20 kV.
Materials and Methods 52
3.3.2 Fourier transform infrared spectroscopy (FT-IR)
The glass structural properties and the formation of an apatite-like phase on the
scaffold surface were investigated by FT-IR (Impact 420 Nicolet, Thermo
Scientific, Waltham, US). For that the glass or ground scaffolds were pressed into
potassium bromide (KBr) pellets. The pellets were made by mixing 1 mg sample
and 300 mg of KBr (Spectroscopy grade, Merck, Darmstadt, Germany). Spectra
were collected between 2000 and 400 cm-1
with a resolution of 4 cm-1
. In order to
compare the intensities of single bands all spectra were normalized to the silica
band at ~1040 cm-1
.
3.3.3 Raman spectroscopy
Raman spectra (LabRAM HR – Evolution, Horiba Scientific, Germany) of the glass
powders were obtained using a diode pumped solid state laser (YAG. = 532 nm).
Hole, slit and grating were set to 1000 µm, 100 µm, and 600 gr/mm, respectively.
Data acquisition time was 5s and averaging was performed over 20 measurements.
The spectra were normalized to the intensity of the band centered at 630 cm-1
.
3.3.4 Inductively coupled plasma atomic emission spectroscopy
(ICP-OES)
Static conditions3
Ion release from scaffolds soaked in SBF was quantified using an inductively
coupled plasma - optical emission spectrophotometer (ICP-OES) (Thermo
Scientific iCAP 6500). Values were calibrated against certified standards serially
diluted with SBF to 100, 10, 1 and 0.1 and ppm. (4% nitric acid was added to all
samples and standards).
Quasi-dynamic conditions4
Ion release (Cu, Si) under quasi-dynamic conditions from scaffolds soaked in SBF
was measured using the ICP-OES Optima 8300 (Perkin Elmer, USA). Five point
3 ICP measurements of ions released under static conditions were carried out by C. Stähli at McGill University, Montreal, Canada 4 ICP measurements under quasi-dynamic conditions were carried out by S. Romeis and J. Schmidt at the Institute of Particle Technology, University of Erlangen-Nuremberg
Materials and Methods 53
calibrations (25; 10; 5; 1; 0.1 ppm) were performed by diluting certified standards
(Carl Roth, Germany) with SBF. Given errors are estimated by linear regression
analysis. The samples were measured in triplicate and mean values with standard
deviation were derived. For scaffold digestions a of 2 ml hydrofluoric acid (48 %,
supra purity, Roth Chemicals, Germany), 2 ml nitric acid (69 %, supra purity, Roth
Chemicals, Germany), and 4 ml hydrochloric acid (35 %, supra purity, Roth
Chemicals, Germany) was used. After the microwave procedure 2 g of boric acid
(>99.8 %, Roth Chemicals, Germany) was added and the total volume was set to
250 ml.
3.3.5 Micro-Ion beam5
Analyses of the glass derived scaffolds/biological fluids interface were carried out
using nuclear microprobes at CENBG (Centre d’Études Nucléaires de Bordeaux-
Gradignan, France).264
PIXE-RBS analyses were performed on the nanobeam line
with a proton scanning micro-beam of 3 MeV energy and 60 pA in intensity. The
beam diameter was nearly 1 µm. An 80 mm2 Si(Li) detector was used for X-ray
detection, orientated at 135° with respect to the incident beam axis and equipped
with a beryllium window 12 µm thick. An aluminium funny filter of 100 µm in
thickness with a hole of 2 mm in diameter was added on the detector. The software
SUPAVISIO was used to define the different regions of interest with the use of
masks. These masks isolate the spectra corresponding to the region of interest in
order to calculate the elemental composition in that region. Quantification of PIXE
spectra was done using GUPIX software.265
The data was calibrated against NIST
standard reference glass materials. Relating to RBS, a silicon particle detector
placed at 135° from the incident beam axis provided the number of protons that
interacted with the sample. Data were treated with the SIMNRA code.29 The
thickness of reaction layers formed during immersion in SBF was determined using
ImageJ software. Three measurements at different spots were taken per chemical
map and the mean value and standard deviation were derived from 3 chemical
maps.
5 PIXE-RBS analysis was carried out by J. Lao and E. Jallot, fClermont Université, Université
Blaise Pascal
Materials and Methods 54
3.3.6 XRD
X-ray diffraction analysis was performed using a D8 ADVANCE X-ray
diffractometer (Bruker, Madison, US) in a 2Theta range from 20-80°. BG powders
were dispersed in ethanol. Then, the solution was dripped on off-axis cut, low
background silicon wafers (Bruker AXS, Germany). BG derived scaffolds were
powdered and prepared in the same way.
3.3.7 X-ray microtomography (µCT)6
µCT analysis was performed with a SkyScan 1172 (SkyScan, Kontich, Belgium).
Briefly, 45S5 glass-ceramic scaffolds were analyzed through a 360° flat-field
corrected scan at 40 kV and 250 µA, and then reconstructed (NRecon software,
SkyScan) with a beam hardening correction of 10, a ring artifact correction of 20
and an “auto” misalignment correction. The 2D analysis (software CTAn, SkyScan)
of reconstructed microCT transverse cross-sections of 45S5 glass-ceramic scaffolds
was carried out using a greyscale intensity range of 16 to 255 (8 bit images) in order
to remove background noise. 3D reconstruction and visualization of the scaffold
microstructure was achieved using CTVol software, Skyscan.
3.4 Scaffold fabrication
BG derived scaffolds were produced using the foam replica technique as
schematically shown in Fig. 9.68
Briefly, polyurethane foam (45 ppi, Recticel, UK)
was immersed in slurry containing 60 wt% BG-particles and 1.1 wt% PVA as
binder. The green bodies were dried at 60 °C for 24 h and sintered at 1050 °C for 2
h for glass series 45S5, 45S5-0.1Cu and 45S5-1Cu and 1000 °C for 45S5-2.5Cu
glass composition. The 1393-Co glass derived scaffolds were densified at 700 °C,
680 °C, and 670 °C for 1393. 1393-1Co and 1393-5Co, respectively.
Also, multiple coatings were applied by dip-coating the sintered foam and
centrifugation in order to remove the excess slurry.
6 µCT analysis was performed by B. Marelli at McGill Universtiy, Montreal, Canada
Materials and Methods 55
Fig. 9: Scheme of the foam replica technique used for scaffold fabrication.
3.5 Acellular bioactivity in simulated body fluid (SBF)
In order to assess the in vitro bioactivity, the scaffolds (5x5x5 mm3) were immersed
in 50ml simulated body fluid (SBF) for 1, 3 7, 14 and 21 days. SBF was produced
according to the protocol by Kokubo et al.99
After immersion, the samples were
gently washed with deionized water, dehydrated with acetone and dried at 60 °C for
12 h. Analysis of CHA formation was performed with SEM, FT-IR and micro-Ion
beam techniques.
3.6 Compressive strength
Compressive strength of the scaffolds (5x5x5 mm3) was measured using a tensile
testing machine (Z050, Zwick Roell, Germany). The testing speed was 10 mm min-
1. In order to assure homogenous loading of the scaffolds a polymeric rubber
interlayer was placed between the sample and the steel plates. 10 samples were
measured per scaffolds series and the standard deviation was derived.
3.7 Cell tests
In vitro cell investigations were carried out in order to assess the biocompatibility of
the developed Cu and Co containing bioactive glasses. Different cell types were
used in order to evaluate specific cell response analysing the cytotoxicity,
osteogenic differentiation and angiogenic potential. Hereby, 2D and 3D experiments
were conducted using glass powders, dense pellets and 3D scaffolds. Table 9 gives
Materials and Methods 56
an overview of cell types materials modifications used in the cell culture
experiments.
Table 9: Overview of the cell types and material modifications used for biocompatibility
assessment.
Cell type
Osteoblast-like
cells (MG-63)
Human bone marrow
derved stem cells
(hBMSC)
Human dermal
microvascular
endothelial cells
(hDMECs)
Co-culture of
hBMSC and
hDMECs
Aim Cytotoxicity Osteogenic
differentiation Angiogenic potential
Assessment Cell viability
Cell morphology
Cell vitality
Gene expression
Cell vitality, formation of tube-like
structures, VEGF release
Powder , - - -
2D pellets - - -
3D scaffolds
direct , - -
indirect - ,
...45S5-Cu, ...1393-Co
3.7.1 Cells and culture
Osteoblast-like cell line MG-63
MG-63 osteoblast-like cells Human osteosarcoma cell line (Sigma-Aldrich,
Germany) were used cultured at 37 °C in a humidified atmosphere of 95 % air and 5
% CO2, in DMEM (Dulbecco’s modified Eagle’s medium, Gibco, Germany)
containing 10 vol% fetal bovine serum (FBS, Sigma-Aldrich, Germany) and 1 vol%
penicillin/streptomycin (Sigma-Aldrich, Germany). Cells were grown for 48 hours
to confluence in 75 cm2 culture flasks (Nunc, Denmark), washed with phosphate
buffered saline (PBS) and harvested using Trypsin (Sigma, Germany). Cells were
counted by a hemocytometer (Roth, Germany) and diluted to a final concentration
of 100.000 cells ml-1
(if not specified otherwise).
Materials and Methods 57
Human bone-marrow mesenchymal stem cells (hBMSCs)
Human BMSCs were (at passage two) were purchased from PromoCell GmbH,
Heidelberg, Germany. They were cultured in flasks (COSTAR, Cambridge, USA)
by using MSC growth medium with supplemented cytokines as specified by the
manufacturer (PromoCell GmbH, Heidelberg, Germany), in an incubator with a
humidified atmosphere maintained at 37 ºC and 5% CO2. The media were changed
twice weekly. At 80-90% confluency, cells were trypsinized (Trypsin/EDTA, PAA,
Pasching, Austria) and sub-cultured further at 80% confluence until passage five.
For all the experimental protocols cells at passage 5 cells were used. For the 2D and
3D cell experiments the BMSCs were cultured in basal culture media
(DMEM/Ham’s F-12 (1:1) with 10% FCS, 2 mg l-1
of L-glutamine (Biochrom AG,
Berlin, Germany).
Human dermal microvascular endothelial cells (hDMECs)
hDMCs and the specialized endothelial cell growth media (EGM MV2) were
obtained from PromoCell GmbH, Heidelberg, Germany.
3.7.2 Analytical methods
Cell viability and cell proliferation
Mitochondrial activity.
The mitochondrial activity was assessed applying a WST-8 assay (Sigma-Aldrich).
After the time point of interest the culture media was removed from the respective
well culture plate and the cells were washed with PBS. After addition of the WST-8
reagents (1vol% in DMEM) in each well, the plates were incubated for 1.5 h.
Subsequently, the supernatant of all samples was transferred to a 98-well plate (100
µl in each well) to measure the absorbance at 450 nm and 650 nm with an ELISA-
Reader (Perkin Elmer, Multilabel Reader Enspire 2300, Germany).
Materials and Methods 58
LDH-activity
Lactate dehydrogenase (LDH-) activity provides a measurement of the amount of
attached cells on the samples. A commercially available LDH-activity quantification
kit (TOX7, Sigma-Aldrich) was used to quantify cell number by the LDH enzyme
activity in the cell lysate. Cells were washed with PBS and lysed with lysis buffer
for 30 min (1 mL/well). Lysates were centrifuged (5 min, 2500 rpm) and 100 µL
from the supernatant solutions were transferred to a 1 mL cuvettes. 100 µl of
master-mix containing equal amounts of substrate solution, dye solution, and
cofactor solution for LDH assay were added to each cuvette. After 30 min reaction
in the dark and the reaction was stopped with 300 ml 1M HCl per cuvette. The dye
concentration was measured on a spectrophotometer (Specord 40, Jena Analytik,
Germany) at 490 nm.
Metabolic activity
Each week, the cell-seeded scaffolds or the cells in 12-well plates were analyzed by
alamarBlue® (Biosource Int., Camarillo, CA, USA) assay, according to
manufacturer’s protocol. The absorbance of the reduced dye was measured by a
plate reader (SPECTRAmax 190, Molecular Devices, Sunnyvale, CA, USA) and
was calculated according to manufacturer’s instructions.
Analysis of osteogenic and angiogenic markers / Gene expression and protein
release
Specific Alkaline phosphatase (ALP) activity
ALP is a membrane-bound metalloenzyme which catalyzes the hydrolysis of
phosphomonoesters at an alkaline pH. For determining the osteoblastic activity of
the MG-63, ALP was analysed by measuring the specific enzyme activity. The cells
were lysed with a cell lysis buffer containing 20 mM TRIS buffered solution
(Merck) with 0.1 wt% Triton X-100 (Sigma, Germany), containing 1mM MgCl2
(Merck) and 0.1 mM ZnCl2 (Merck). The cell lysate was incubated with a reacting
solution containing 0.1M Tris solution, 2 mM MgCl2 and 9 mM p-Nitrophenyl-
phosphate. After incubation absorption was measured at 405 nm using a
Materials and Methods 59
spectrometer (Specord 40) after 200 min of incubation. The specific activity was
calculated with respect to the protein concentration of the cell lysates. The protein
content of the cell lysates was determined by means of a commercial kit based on
Bradford assay (Sigma).
Real time RT-PCR
Total RNA was isolated from the 2D cultured MSCs (n=4) using TRIzol Reagent
(Invitrogen, Carlsbad, CA, US) followed by RNeasy Mini Kit (Qiagen, Hilden,
Germany) as described elsewhere.266
Total RNA was converted to cDNA
(QuantiTect reverse transcription kit, Qiagen, Hilden, Germany). The amount of
cDNA corresponding to 20 ng of total RNA was then analyzed by semi-quantitative
real-time PCR (iQ SYBR green, Bio-Rad, Munich, Germany) for selected genes
with primers as shown in Appendix Fig. A 2 (CFX 96 real time systems, Bio-Rad,
Munich, Germany). The gene expressions were normalized to internal GAPDH
expression, and the relative fold change was expressed by comparing to that of
45S5 samples.
Cell morphology and cell adhesion
SEM analysis of the cells
After cell culture experiments the scaffolds were washed with PBS, fixed with a
solution containing 3 vol.% glutaraldehyde (Sigma, Germany) and 3 vol.%
paraformaldehyde (Sigma, Germany) in 0.2 M sodium cacodylate buffer (pH 7.4)
and finally rinsed three times with PBS For SEM analysis (ESEM, Quanta 200, FEI,
Netherlands). All samples were dehydrated in a graded ethanol series (30, 50, 75,
90, 95 and 99.8 vol.%). Samples were maintained at 99.8 vol.% ethanol and critical-
point dried (Leica, EM CPD 300). Prior to SEM examination the samples were
sputtered with gold.
Materials and Methods 60
Actin staining
In order to observe and to quantify the cell attachment on the BG samples labelling
of the cell skeleton (Actin labelling) was performed and the spreading behaviour of
the cells was analysed using fluorescence microscopy (Axio Scope.A1, Leica,
Germany). Actin staining was performed using a commercially available dye kit
(Alexa Fluor® 488 Phalloidin, Life Technologies GmbH, Darmstadt, Germany) at 5
units/ml concentration to see the cell spreading on the bio-active glass scaffold. The
nuclei of the cells are counterstained by 300 nM DAPI (SelectFX®, Life
Technologies GmbH, Darmstadt, Germany).
Vybrant™ cell-labeling
To analyse the adherent growth and distribution of cells on the BG scaffold
samples, commercially available Vybrant™ cell-labeling solution (Molecular
Probes, The Netherlands) was used. After 48 hours of incubation, cell culture
medium was removed and staining solution (5 μl dye labelling solution to 1 ml of
growth medium) was added and incubated for 15 min. Afterwards the solution was
removed, the samples were washed with PBS (phosphate buffered saline, Gibco)
and cells on the BG samples were fixed by 3.7 vol.% paraformaldehyde. The
samples were prepared for confocal scanning laser microscopy (CSLM, Leica TCS
SP5 II, Germany) to analyse cell morphology and distribution.
Evaluation techniques of 2D experiments with EC
Flow Cytometry analysis (FACS)
The human endothelial cell surface markers (Cluster of differentiation, CD31; von
Willebrand factor, vWF, Vascular endothelial growth factor 2, VEGFR2) were
stained at 5x104 cells for each antigen. CD31 is a known platelet adhesion protein
which is expressed at cell connecting junctions and is found in early and mature
endothelial cells and is known to be involved in activation of integrins. CD31 was
stained by mouse anti-human CD31-Biotin followed by a second staining step with
Streptavidin PerCP-eFluor® 710 (both from eBioscience, San Diego, CA, USA).
Materials and Methods 61
vWF is an important platelet adhesion factor which is also used as a marker for any
pathological dysfunctions of endothelial cells as increased release of vWF from
endothelial cells is related to endothelia cells damage. vWF was stained by sheep
anti-human vWF-FITC (Abcam, Cambridge, UK). VEGFR2, an important
signalling molecule for endothelial cell mitogenesis and migration, was stained by
mouse anti-human CD309 (VEGFR2)-Alexa Fluor® 647 (Biolegend, San Diego,
CA, USA). All staining steps were performed on ice for 30 minutes in dark. Stained
cells were analyzed by FACS-Calibur (BD Biosciences, San Diego, CA, USA) and
Cell Quest software (Beckton Dickinson, Heidelberg, Germany). Data
quantifications were perfomred by FlowJo software (Tree Star, Inc., Ashland, OR,
USA).
Matrigel™ sprouting assay
For analysis of capillary tube formation, 75 µl of Matrigel™ (Becton Dickinson,
Heidelberg, Germany), an extracellular mouse sarcoma matrix known to produce
pro-angiogenetic stimulus both in vitro and in vivo, was pipetted into each well of a
96-well plate (Falcon, Heidelberg, Germany) and incubated at 37°C for 60 minutes.
HDMECs were harvested at week 1 or week 2 and suspended at 50,000 cells per
150 µl of EGM MV2 media. 150 µl of this media were added to the Matrigel coated
96-well plates and incubated for 24 h at 37°C. Capillary tube formation on Matrigel
was observed with a light microscope (DMIL, Leica, Germany) and images were
processed with Leica application suite software (Leica GmbH, Wetzlar, Germany).
LDL (Low density lipoprotein)-staining
At end time points, the media were removed and the cells were washed once with
PBS to remove non-adherent cells. The wells were incubated with 2.5 µg/ml ac-
LDL-Alexa Fluor®
488 (Life Technologies GmbH, Darmstadt, Germany) for 4 h at
37°C. Afterwards cells were washed twice with PBS and further analysed by a
fluorescence microscope.
Materials and Methods 62
VEGF quantification ELISA
At each time point, the cultured media were collected from 3 different samples,
pooled together, labeled and frozen at -20°C. At the end of all experiments, the
frozen media were thawed overnight at 4°C and the VEGF content of the media was
quantified by using an ELISA kit (Thermo Fisher Scientific GmbH, Schwerte,
Germany).
Statistical analysis
The statistical significant of the results was evaluated by one-way analysis of
variance (ANOVA). The level of the statistical significance was defined at p < 0.05
(Origin 8.1G, OriginLab Corporations, USA).
3.7.3 Powder cytotoxicity
Initial cytotoxicity tests of the glasses fabricated were assessed by culturing BG
powders in direct contact with MG-63 cells. The BG powder was dispersed in
DMEM and ultra-sounded for 5 min to break agglomerates. MG-63 cells were
cultured in direct contact with BG particles at concentrations from 0.1 to 1000 µg
ml-1
) in 600 µl DMEM for 48 hours. As positive control cells cultured without BG
particles, as negative control ZnO was used.
Cell distribution and morphology were evaluated using phase contrast light
microscopy (LM, Nikon Eclipse TE 2000-U, Japan). Cells viability was assessed
through WST-8 test (mitochondrial activity) and LDH assay (cell number) after 48h
of incubation. From these the LC50 value (lethal concentration of the used particles
where the activity of the cells is reduced to 50%) was derived.
Materials and Methods 63
3.7.4 Evaluation of the scaffolds7
Indirect study (2D)
In the indirect setup the cells were seeded on the bottom of the cell culture well
exposed to the ionic dissolution products released from the scaffold which was
placed in a permeable insert as shown in Fig. 10. Indirect studies were performed
with hBMSCs. Cell morphology was detected by light microscopy, ALP activity,
VEGF and Runx2 expression was analysed with PCR technique.
Fig. 10: Indirect setup for cell culture studies with scaffolds.
Direct study (3D)
Additionally, in order to test the interaction of cells and the scaffold surface the cells
were directly seeded on the BG derived scaffolds. MG-63 cells and hBMSCs were
used. Before cell seeding the scaffolds were preconditioned in DMEM for 5d. The
cells attachment and growth was evaluated with SEM, Actin staining, Live staining
(Calcein/Vybrant). Further, ALP activity was detected.
Fig. 11: Direct setup for cell culture studies with scaffolds.
Co-culture of hBMSCs and ECs
A co-culture model of hDMECs and hBMSCs was applied to 45S5-Cu derived
scaffolds as shown in Fig. 11. hBMSCc were seeded directly on the scaffolds and
7 Cell studies with hMBSCs were carried out in collaboration with D. Hiller and S. Rath, U. Kneser and R. Horch, Plastic and Handsurgery Department, University Clinic Erlangen
Materials and Methods 64
the constructs where placed into permeable membranes. hDMECS were seeded in a
concentration of 100.000 cells/ml and on the well bottom being exposed to ionic
dissolution products released from the scaffods as well as effects of the hBMSCs.
Cell morphology of the hDMECS was observed with light microscopy. Further,
HDMSCs specifically the LDL uptake and FACS analysis of endothelial markers
vWF, VEGFR and CD31 was performed.
Fig. 12: Set-up for the co-culture experiment with hBMSc and hDMECSs using permeable
insert containing the scaffold seeded with hBMSCs.8
3.8 In vivo study
In vivo evaluation of the angiogenic potential of Cu-doped glass scaffolds was
carried out at the Department for Hand plastic surgery, University of Erlangen9.
Six male Lewis rats were used and the experiments were carried out in compliance
with the animal care committee of the FAU and the government of Mittelfranken.
45S5-1Cu BG derived scaffolds were chosen as most promising candidates for in
vivo study, to prove their angiogenic potential in relevant model in addition to in
vitro results. Fabricated scaffold were crushed into small pieces with a diameter of ~
2-3 mm in order to create granulate like units. A Teflon chamber with an inner
diameter of 10 mm and a height 10 mm was used. The half of the chamber was first
filled scaffold granules and fibrin gel with a fibrinogen concentration of 10mg ml-1
and thrombin concentration of I.U ml-1
. Afterwards the AV loop was placed on the
BG/fibrin gel matrix and the chamber was completely filled with BG granules/fibrin
gel. The construct was, then, implanted subcutaneously and sutured to the
musculature of the rats. The arrangement of the AV-loop and the BG granules in the
8 Cell studies with hDMECs were carried out in collaboration with A. Brandl and O. Bleizifffer, U. Kneser and R. Horch, Plastic and Handsurgery Department, University Clinic Erlangen 9 In vivo studies were carried out by Ulrike Rothensteiner from the group by Dr. A. Arkudas, Dr. U. Kneser and Prof. Dr. Horch
Materials and Methods 65
teflon chamber are shown in Fig. 13. After 3 weeks of implantation the constructs
were explanted and were investigated by means of microCT, histological and
morphometrical analysis. 3D reconstruction of the vessel sprouting, vascular
density as wells as vessel number and vessel density were derived were derived.
Fig. 13: In vivo setup for assessing the angiogenesis of 45S5 and 45S5-1Cu BG scaffolds. Firstly,
the teflon chamber is filled half with BG granules and the AV-loop is placed (a). Afterwards the
chamber is fully filled with BG granules and implanted subcutaneously (b).
Results and Discussion 66
4 Results and Discussion
In this chapter the properties of different bioactive glass compositions and the
respective glass-derived scaffolds are described. The influence of metal ion doping
on the glass structure and glass properties is discussed. Further the structural and
mechanical properties of the glass derived scaffolds are shown. The apatite forming
ability as indicator for acellular bioactivity is assed in simulated body fluid and the
physic-chemical reactions are monitored in detail by SEM, FT-IR and RBS-PIXE
techniques. Moreover, the ion release kinetics from the BG scaffolds is observed
and the suitability of glass derived scaffolds as “carrier” of therapeutic metal ions is
discussed. The cellular biocompatibility is analysed in relevant cell culture studies
and also an in vivo model is applied.
4.1 Cu containing 45S5 bioactive glasses
4.1.1 Glass properties
In this section the glass structure of the synthesised Cu containing glasses is
discussed on the basis of FT-IR, Raman and NMR-analyses and is related to glass
thermal properties derived from DSC measurements.
Glass structure
Fig. 14 shows the FT-IR spectra of as-fabricated glass powder. The bands at ~ 500
cm-1
and ~1080 cm-1
can be assigned to Si-O-Si stretching and Si-O-Si bending
modes, respectively.267
The peak at 1460 cm-1
is related to carbonate species
adsorbed on the BG surface.268
No major differences for Cu-containing glasses can
be observed compared to undoped reference 45S5 glass indicating that the main
structure of the glass network remains unchanged. However, the intensity of the
non-bridging oxygen, Si-ONBO peak (~930 cm-1
), is decreasing with increasing Cu-
content in the 45S5 glass.
Similar observations can be made from Raman analysis, Fig. 15. The bands at ~620
cm-1
and 1060 cm-1
can be assigned to the rocking and stretching mode of Si-O-Si
Results and Discussion 67
band, respectively. While no major differences are observed for Cu-containing
glasses the band at 860 cm-1
which is assigned to non-bridging oxygen-silica band
Si-O2NBO is reduced in intensity for the 45S5-2.5Cu. Also the Si-O band at 1060 cm-
1 increases in intensity for the glasses with higher Cu content (>1%).
Fig. 14: FT-IR spectra of as-fabricated Cu-doped 45S5 glasses. A. Hoppe et al. J. Mater. Chem.
B 1 (2013), p. 5659. - Reproduced by permission of The Royal Society of Chemistry.
Fig. 15: Raman spectra of as-fabricated Cu-doped 45S5 glasses. A. Hoppe et al. J. Mater.
Chem. B 1 (2013), p. 5659. - Reproduced by permission of The Royal Society of Chemistry.
Results and Discussion 68
Fig. 16 shows the UV-VIS spectra of the glasses. For reference 45S5 glass a
continuous transmission in the range from 300 to 2000 nm is observed. Cu
containing glasses 45S5-0.1Cu, 45S5-1Cu and 45S5-2.5Cu, however, show an
absorption band at 800 nm which can be assigned to the presence of Cu2+
in the
glass network.269, 270
Fig. 16: UV-VIS spectra of 45S5-Cu BGs showing an absorption band at ~800 nm which is
assigned to Cu2+
. A. Hoppe et al. J. Mater. Chem. B 1 (2013), p. 5659. - Reproduced by
permission of The Royal Society of Chemistry.
For 45S5-0.1Cu no NMR measurements were performed since the Cu content is too
low and its influence on the 45S5 glass network structure cannot be resolved by
NMR. Fig. 17 shows the 29
Si MAS NMR spectra of the 45S5and 45S5-2.5Cu
glasses. The effect of Cu on 45S5 silicate glass structure is discussed on the basis of
45S5-2.5Cu data only as the NMR spectra for 45S5-1Cu (data not shown) and
45S5-2.5Cu show almost exactly the same characteristics and are completely
overlapped. An asymmetrical peak is observed for both glasses with the overall
peak position of the Si-related signal slightly shifted to negative values for 45S5-
2.5Cu glasses by 0.75 ppm. The NMR results show a broadening of the 29
Si peak.
Compared to reference 45S5 sample the FWHM (full width at half maximum) of
the Si signal for 45S5-2.5 Cu increased to 13.8 ppm (+ 1ppm).
Results and Discussion 69
Table 10: Distribution of Qn units calculated from the peak area of the deconvoluted
29Si NMR
signal and network connectivity NC for the 45S5 and 45S5-2.5Cu samples. A. Hoppe et al. J.
Mater. Chem. B 1 (2013), p. 5659. - Reproduced by permission of The Royal Society of
Chemistry.
45S5 45S5-2.5Cu
Q1 26.9% 12.7%
Q2 39.5% 47.6%
Q3 33.1% 38.9%
NC* 1.99 2.17
Commonly the structure of silicate glasses is described by quantifying the Qn
distribution (a Q species is a Si atom with n bridging oxygen atoms). Hence, for
detailed analysis of the NMR results for the 29
Si NMR signal of 45S5 and 45S5-2.5
Cu the curves were fitted and peak deconvolution applying Gauss profiles was
performed, Fig. 17. The glass structure of both glasses is dominated by Q2 with
smaller fractions of Q1 and Q
3 species as depicted in Fig. 17. For 45S5-2.5Cu a
slightly larger fractions of Q2 and Q
3 species is observed at the expense of Q
1. The
fractions of Qn and resulting network connectivity (NC) can be calculated from the
peak area. The exact values for the Qn distribution obtained for 45S5 and 45S5-
2.5Cu and corresponding values for the network connectivity (NC) are given in
Table 10. NC was calculated according to:
𝑁𝐶 = 1𝑄1 + 2𝑄2 + 3𝑄3 Eq. 4
Results and Discussion 70
Fig. 17: MAS NMR 29
Si analysis for 45S5 and 45S5-2.5Cu glass samples. Gauss fit and peak
deconvolution show the distribution of the Qn species in the glass network. A. Hoppe et al. J.
Mater. Chem. B 1 (2013), p. 5659. - Reproduced by permission of The Royal Society of
Chemistry.
Thermal properties
Fig. 18 shows the DSC curves for the 45S5-Cu glasses as fabricated. The
characteristic temperatures of glass transition Tg, crystallization onset and maximum
Tc and Tp and melting temperature Tm are given in Table 11. Both glasses show two
crystallisation events described by Tc1, Tc2 and Tp1, Tp2, respectively. Compared to
the 45S5 reference, for 45S5-2.5Cu a decrease of Tg, Tc1, Tp1 and Tm is observed
while Tc2 increases and Tp2 remains constant. For the lower Cu concentrations,
however, no significant differences in the DSC diagram for the characteristic
Results and Discussion 71
temperatures were observed. The origin of the crystalline phases occurring during
the sintering of 45S5 bioactive glass is discussed in 4.1.2.
Table 11: Characteristic features (temperatures in °C) of the Cu-containing glasses observed
with DSC.
Glass Tg Tc1 Tc1-Tg Tp1 Tc2 Tp2 Tm
45S5 522 592 70 682 756 908 1105
45S5-0.1Cu 526 611 85 696 - - 1110
45S5-1Cu 519 610 91 698 783 832 1112
45S5-2.5Cu 496 610 114 645 806 908 1044
Fig. 18: DSC analysis of 45S5-Cu glasses showing the glass transition point and two
crystallisation events at ~650-700 °C and 850 °C, respectively. A. Hoppe et al. J. Mater. Chem.
B 1 (2013), p. 5659. - Reproduced by permission of The Royal Society of Chemistry.
Role of CuO in 45S5 glass structure
The 45S5-1Cu and 45S5-2.5Cu glasses show almost the same characteristics.
Hence, 45S5-2.5Cu glass is used for deeper discussion of the effect of Cu doping on
the structure of 45S5 silicate glass.
FT-IR analysis, Raman spectroscopy, NMR and UV-VIS spectroscopy were applied
in order to investigate the glass structure and the effect of Cu-doping. It was
confirmed through UV-VIS spectroscopy that Cu is present as Cu2+
in the glass
Results and Discussion 72
structure which has been also proposed for soda silicate glasses containing CuO
showing Cu2+
valence state of Cu.271
Cu2+
(as well as Cu+) predominantly work as
network modifiers and are incorporated in the glass work matrix in octahedral
coordination.272
According to Abdrakhmanov et al.273
Cu2+
is surrounded by two
non-bridging oxygens (NBO) in order to achieve electro neutrality. Since no major
differences were observed in FT-IR and Raman spectroscopy the Q2 SiO2 network
of the 45S5 glass seems to be dominant and is not significantly affected by the Cu-
doping. Basically, according to FT-IR and Raman analyses a decreased intensity of
the Si-ONBO bond with increasing Cu content and at the same time increased
intensity of asymmetric Si-O vibration mode for glass with high Cu contents in the
glass was observed.
Si-NMR is a well-established experimental technique for characterising the
structure of silicate glasses which allows detailed insight about the distribution of
Qn species. As described in 2.3.2 the best descriptive model describing the structure
of 45S5 BG is achieved by assuming a trinodal distribution of Q1, Q
2 and Q
3 silica
species. According to this for the experimental NMR data the best fit of the 29
Si
peak (R2=0.99) was achieved by applying a ternary peak deconvolution, Fig. 17.
Concerning the 29
Si MAS-NMR analysis a shift of the peak maximum was observed
for 45S5-2.5Cu compared to 45S5 control. According to Elgayar et al.274
the
negative shift of the maximum to lower frequencies of 0.75 ppm between the 45S5
and 45S5-2.5Cu is likely caused by changing the cation in the glass structure which
is accomplished with an increase of the Q2 species in the glass network neutralized
with Cu2+
ions. Furthermore, the increased line width of the 29
Si peak indicate more
structurally disordered Q2 and Q
3 species neutralized by Cu
2+.274
This might be due
to the larger ion radius of Cu2+
replacing Ca2+
.
The higher fractions of Q2 and Q
3 species and hence the resulting higher network
connectivity in 45S5-2.5Cu samples suggests a repolymerisation effect caused by
introducing CuO into the glass network. Cu are also bigger ion, so the SiO2 network
is disrupted, a higher amount of silica chains Q2 are present and also there is
indication that the chains are repolymerised which results in higher Q3 fractions and
less terminating Q1
species. This is likely due to the more covalent character of the
Cu-O bond compared to Ca-O bond allowing repolymerisation of Si-NBOs.
Results and Discussion 73
Accordingly, FT-IR and Raman spectra revealed decreased intensity of the NBO
peak which is consistent with higher Q2 and Q
3 fraction and thus lower amounts of
free NBO in the glass network.
Fig. 19: Model of the Cu-containing 45S5 BG structure. Cu2+
(blue) replace Ca2+
ions (yellow)
as network modifiers.
Even though the main glass structure remains similar consisting mainly of Q2 and
Q3 units, the NMR results show higher Q
2 and Q
3 fractions for the 45S5-2.5Cu glass
which indicates higher silicate network distortion. The weakening of the glass
matrix is confirmed by the DSC measurement which revealed a decrease in glass
transition temperature for Cu containing 45S5 glasses. Incorporation of Cu into the
45S5 glass matrix was found to reduce the Tg of 45S5 glass and to stabilize the
amorphous phase during sintering.
The ionicity, iG, of the Me-O bonds within the glass structure can be used as
explanation of the decrease of Tg. With increasing Cu content in the glass the Tg
decreases likely due to the more covalent character of the Cu-O (iG=0.617) bond
compared to Ca-O (iG=0.707) bond resulting in higher degree of structure relaxation
and hence lower glass transition temperature.275
During heat treatment the 45S5 glass tends to crystallise and two crystalline phases,
combeite and silicorhenanite, form which have been described in literature for this
glass.276, 277
Results and Discussion 74
4.1.2 Scaffold properties
Macro-Structure
Scaffold macro and microstructure was analysed with SEM and µCT. The porosity
was derived by the Archimedes method.
Fig. 20a-d show the macrostructure of 45S5-Cu derived scaffolds. High pore
interconnectivity as well as pore sizes of ~ 200-300 µm were observed for all glass
compositions. Porosity values of 92 %, 91 %, 92 % and 93% and were observed for
45S5, 45S5-0.1Cu, 45S5-1Cu and 45S5-2.5Cu respectively. Fig. 20e) shows
additionally high magnifications of a 45S5 derived strut showing a hollow strut as it
is typical for polyurethane foam derived scaffolds. Furthermore, Fig. 20f shows the
3D reconstruction of a 45S5 derived scaffold from µCT data confirming the
interconnected pore system. The total porosity was calculated to be 93.5 ±2.0 % and
the strut thickness of ~67 µm was derived. The average pore size was 314 ± 87 m.
99.1±0.4 % of the total porosity was open indicating a completely interconnected
pore system. High porosity values and interconnected pore system of the scaffold
should enable vascularisation and tissue ingrowth when applied as engineered bone
construct. Vascularisation has been shown to be enhanced in scaffolds with pores >
250 um. 278
. However, high interconnectivity is considered even more important for
blood vessel and tissue ingrowth.43, 279
Hence, 45S5 derived scaffolds fulfil these
requirements.
Results and Discussion 75
Fig. 20: SEM images of 45S5-Cu derived scaffolds: a) 45S5, b) 45S%-0.1Cu, c) 45S5-1Cu and
d) 45S5-2.5Cu. e) typical hollow strut of 45S5 derived scaffolds and f) microCT reconstruction
of a 45S5 derived scaffold. A. Hoppe et al. J. Mater. Chem. B 1 (2013), p. 5659. - Adapted by
permission of The Royal Society of Chemistry.
Fig. 21 shows the XRD analysis of the 45S5-Cu derived scaffolds after sintering.
Two crystalline phases were observed after the heat treatment of the glass: a sodium
calcium silicate phase (combeite, Na2Ca2Si3O9) and silicorhenanite
(Na2Ca4(PO4)2SiO4) which have been shown to occur during high temperature
treatment of Bioglass®.67, 276, 280
Basically, no qualitative differences among the Cu-
containing and 45S5 reference were observed. All glasses show high crystallinity
after sintering. However, Rietveld analysis was performed in order to quantify the
crystalline phases. Since the sensitivity of the XRD technique is limited only 45S5
with the highest copper content (45S5-2.5Cu) was investigated in order to
qualitatively evaluate the role of copper during the crystallisation of 45S5 glass. The
Results and Discussion 76
amount of the amorphous phase was higher for 45S5-2.5 Cu compared to 45S5
reference. The content for the amorphous phase was calculated to 3.5 ±2.2 and 10.5
±2.8 % for 45S5 and 45S5-2.5Cu, respectively.
Fig. 21: XRD analysis of 45S5-Cu derived scaffolds showing two crystalline phases forming
during heat treatment. A. Hoppe et al. J. Mater. Chem. B 1 (2013), p. 5659. - Reproduced by
permission of The Royal Society of Chemistry.
Mechanical properties
For the 45S5-Cu glass series no differences in the compressive strength was
observed between the different glass compositions (data not shown) likely due to
the low amount of CuO presented in the glass. Fig. 22 summarises the mechanical
properties of the 45S5 derived scaffolds for multiple coatings regimes. Fig. 22a and
Fig. 22b shows show the mean values and typical stress-strain curves, respectively,
for 45S5 after multiple coatings with BG slurry were applied to the polyurethane
template. Maximal values of 0.16 ±0.07 MPa were observed for 45S5 scaffolds
after application of 3rd
coating with BG slurry.
Results and Discussion 77
Fig. 22: Mechanical properties of the 45S5 BG derived scaffolds: a) mean values calculated
from 10 measurements per scaffold; b) selected typical stress-displacement curves.
The enhancement of the compressive strength through multiple coating of the foam
is not significant and the overall mechanical strength of the scaffolds is rather poor.
The values observed here are in agreement with literature reports where
compressive strength values of 0.3-0.4 MPa (but with lower porosity compared to
scaffolds prepared in this work) were observed for 45S5 derived scaffolds by foam
replica technique. For clinical application, however, the mechanical properties of
the scaffolds should also be considered. The mechanical properties of the 45S5
derived scaffolds are discussed in the context of bone tissue engineering in 0.
Acellular bioactivity in SBF
Despite recent critical discussion in the literature regarding the suitability of SBF
studies for predicting the bioactivity,101, 102
this well-known and established
technique was employed in this work since the objective was to establish a close
comparison with non-doped 45S5 Bioglass® which have been largely investigated
by the SBF test as well as in vitro cell culture studies in the last decades.
The reaction stages of 45S5 bioactive glass derived scaffolds, as documented by
SEM, are summarised in Fig. 23. Fig. 23a shows the typical hollow macrostructure
of the initial 45S5 BG scaffolds strut. After 1d of immersion in SBF, Fig. 23b, the
surface is homogenously covered with calcium phosphate (CaP) precipitates as
indicated through their typical morphology. A higher magnification images of the
cross-section of a scaffold strut, Fig. 23c-d reveal a more detailed view on the
Results and Discussion 78
reaction stages. Fig. 23c shows a porous structure of roughly 8-10 µm which is
covered by a thinner dense layer (roughly estimated to be ~1-2 µm). These two
layer s are likely to be identified as a silica rich layer and CaP layer, respectively.
However, at higher magnification according to Fig. 23d no clear discrimination of
the silica and the CaP layer can be made. The CaP enrichment on the BG scaffold
surface is further indicated through small round-shaped particles marked as ACP
precipitates in Fig. 23 c). After 3 d in SBF, the phosphate layer continues to grow
reaching 3-5 µm after 3d (Fig. 23d). After 7 days only minor further growth of the
CaP layer was observed which remained roughly at 5 µm (Fig. 23e). Furthermore,
the inner region of the scaffold (inner part of the struts) shows signs of dissolution
turning into a porous silica structure with the modifier ions Ca and Na leached out.
After 7 days the entire scaffold strut seems to have transformed into pure silica
phase.
However, the analysis of the reaction layers on 45S5 bioactive glass scaffold and
identifying surface reaction layer with SEM is rather speculative. Hence, FT-IR
analysis and micro-PIXE-RBS analysis are presented in the following. Fig. 24
shows the FT-IR spectra of a 45S5 BG derived scaffold after immersion in SBF for
1d, 3d, and 7d. As-fabricated 45S5 scaffold shows bands at 629 cm-1
, 575 cm-1
and
530 cm-1
which can be attributed to apatite-like crystalline phase as the P-O bending
modes are located at 580 and 620 cm-1
.281
This crystalline phase corresponds to the
XRD results showing silicoarhenite (Na2Ca4(PO4)2SiO4) phase which is
isostructural to apatite.277
The bands at 450 and 930 cm-1
can be assigned to
symmetric Si-O-Si vibrations and to non-bridging oxygen Si-ONBO, respectively.267,
282 In contrast to the amorphous glass powder (Fig. 14) the broad silicate band at
~1020 cm-1
is split into two bands which are related to the vibrations of isolated Si
tetrahedral.67
Results and Discussion 79
Fig. 23: Reaction stages of HAp formation on crystallized 45S5-0.1Cu derived scaffold: a)
initial strut; b) scaffold after 1 day in SBF; c) and d) scaffold after 1 day in SBF at higher
magnification showing the formation of CaP-SixOy layer and amorphous calcium phosphate
(ACP) precipitates; e) CHA formation and f) further growth of carbonated hydroxyapatite
(CHA) on BG scaffold surface. A. Hoppe et al. J. Mater. Chem. B 1 (2013), p. 5659. -
Reproduced by permission of The Royal Society of Chemistry.
During treatment in SBF, the following changes in the FT-IR spectra occur:
- (after 1d): the P-O bands resulting from the crystalline, rhenanite-like phase
in the BG scaffold transform to a broad peak at 600 cm-1
which is typical for
amorphous calcium phosphate phase (ACP).283
The sharp peak of the Si-O-
Si band at 450 cm-1
is reduced in intensity and becomes broader indicating
the formation of a phosphate phase as O-P-O (bending mode) band absorbs
in this region.284
The shoulder at 960 cm-1
can be attributed to HPO42-
units
incorporated in the calcium phosphate phase.284
Furthermore, a broad peak
appears at 800 cm-1
which corresponds to silica.91
Results and Discussion 80
- (after 3d): with increasing immersion time in SBFa a sharp double band was
detected at 560 cm-1
and 600 cm-1
which is assigned to the vibration modes
of the PO43-
groups284
typically observed in crystalline HAp
- (after 7d): the HAp bands increase in intensity and, additionally, CO32-
band
at 870 cm-1
appears which are observed for natural carbonated
hydroxyapatite (CHA)284
but, however, could also be attributed to surface
bonded carbonate compounds.91
Also, double peaks at 1430 and 1500 cm-1
are observed which can be assigned to v3
(CO32-
) of carbonate ions
incorporated in CHA.284
Fig. 24: Evolution of FT-IR spectra of 45S5 derived scaffold during immersion in SBF for 1d,
3d and 7d. A. Hoppe et al. J. Mater. Chem. B 1 (2013), p. 5659. - Reproduced by permission of
The Royal Society of Chemistry.
FTIR-analysis did not reveal any effect of Cu on the CHA formation. Fig. 25 shows
the FTIR-spectra for 45S5-Cu derived scaffolds after 3d. Clear formation of CHA
was detected after 3 days of immersion in SBF independently of Cu content in the
glasses.
Considering the time point of appearance of the CHA layer as marker for bioactivity
one can conclude that the bioactivity of the 45S5 derived scaffolds is not impeded
Results and Discussion 81
by the incorporation of Cu. In order to gain more detailed information about the
nature of the CHA layer formed on Cu.
Ion beam method allows a chemical analysis with an excellent sensitivity of several
ppm due to very good signal to background ratio. Compared to other techniques like
SEM/EDS, micro-PIXE-RBS method allows an improvement of the sensitivity up
to 3 orders of magnitude. This is a great advantage to study the distribution and the
role of relevant bone trace elements. In addition to other conventional methods
PIXE-RBS analysis enables to detect the local chemical composition of the reaction
surface layer.
Fig. 25: FT-IR spectra of Cu-containing scaffolds after 3d of immersion in SBF compared to
reference 45S5 glass. A. Hoppe et al. J. Mater. Chem. B 1 (2013), p. 5659. - Reproduced by
permission of The Royal Society of Chemistry.
Chemical maps were acquired for the distribution of Si, Ca, P, (Na is not shown for
simplicity) and Cu in the fabricated Cu-containing scaffolds. 45S5-0.1Cu serves as
representative example for the element distributions in the inner part of the scaffold
and the periphery layer and for the elemental evolution during immersion in SBF.
The glass composition with the lowest Cu amount was also chosen in order to
confirm high sensitivity of the micro ion beam technique enabling monitoring the
evolution of low concentrations of trace elements during the reaction of a bioactive
Results and Discussion 82
glass scaffold. Fig. 26 shows typical multichemical elemental maps for Si, Ca, P,
and Cu for an as-fabricated 45S5-0.1Cu cross section (Na is not shown for
simplicity). All elements are homogenously distributed.
Fig. 26: Element distribution of a 45S5-0.1Cu scaffold cross-section in the as-fabricated stage
derived from micro-PIXE-RBS measurements. A. Hoppe et al. J. Mater. Chem. B 1 (2013), p.
5659. - Reproduced by permission of The Royal Society of Chemistry.
Fig. 27-Fig. 29 are representative elemental maps of the scaffolds after immersion
in SBF for different time periods. In correlation to SEM/FT-IR results, during
immersion in SBF the following different regions can be distinguished from the
chemical maps:
i) primary bioactive glass network
ii) silica rich layer on the surface of the scaffolds (clearly distinguishable after 1d)
iii) Ca-P rich layer in the scaffold periphery
For the areas i) and iii) additionally the elemental concentrations were quantified in
these two distinct regions of interests. The elemental evolution for all 45S5-Cu
glass derived scaffolds as function of immersion time in SBF is shown in Fig. 30
and Fig. 31 for the inner region (i) and the periphery region (iii), respectively.
Results and Discussion 83
Fig. 27: Element distribution of a 45S5-0.1Cu scaffold after 1 day in SBF monitoring the
formation of a calcium phosphate layer. A. Hoppe et al. J. Mater. Chem. B 1 (2013), p. 5659. -
Reproduced by permission of The Royal Society of Chemistry.
Fig. 28: Element distribution of a 45S5-0.1Cu scaffolds after 3 days in SBF monitoring the
formation of a calcium phosphate layer. A. Hoppe et al. J. Mater. Chem. B 1 (2013), p. 5659. -
Reproduced by permission of The Royal Society of Chemistry.
Results and Discussion 84
After 1 day, a silica rich layer is detected at the scaffold surface and Ca and P
enrichment is observed as shown in Fig. 27. The silicon content increased from 21
wt% in the inner part of the scaffold to 33 wt% in the silicon enriched layer (12.0 ±
0.2 µm). The Ca/P ratio for the (iii) periphery layer was determined as 1.3 ± 0.1 µm.
The P layer is rather distinct whereas Ca traces can be also found in the Si-rich
layer. This corresponds to the SEM observation where no clearly distinguishable
CaP but rather a mixed Ca-P-SiO2 layer was detected.
After 3d, Fig. 28, a CaP layer was observed on the pore surface with a Ca/P ratio of
1.88 ± 0.06, Fig. 31. The layer thickness was determined to 10.5 ±0.9 µm. The inner
parts of the BG scaffolds showed further dissolution and depletion in Ca and P. At
the same time the relative amount of silicon present within the glass matrix
increases since Ca, P and Na are leached out. After further immersion in SBF for 7d
no significant changes occured: the Ca-P rich layer thickness remains nearly
constant (10.9 ±1.6 µm), Fig. 29, while the Ca/P ratio slightly increases to 1.9 ±0.1.
Fig. 29: Element distribution of a 45S5-0.1 scaffold after 7 days in SBF showing further growth
the calcium phosphate layer. A. Hoppe et al. J. Mater. Chem. B 1 (2013), p. 5659. - Reproduced
by permission of The Royal Society of Chemistry.
Results and Discussion 85
Elemental concentrations for all 45S5-Cu derived scaffolds were calculated in two
areas, the inner area (i) of a strut and the outer layer (surface periphery (iii)).
Hereby, the outer layer was defined as the area of interest once the Ca and P rich
peripheral layer has formed. The evolution of the element concentration during
immersion in SBF is shown in Fig. 30 and Fig. 31 for the inner part and the
peripheral layer, respectively. Si release from the periphery was slowed down for
the 45S5 and 45S5-0.1Cu scaffolds as compared to 45S5-1Cu and 45S5-2.5Cu
indicating slower surface degradation kinetics of these glasses. Accordingly, Ca and
P enrichment is also slower for 45S5 and 45S5-0.1Cu. With higher Cu contents in
the glass (≥ 1wt.-%) Ca and P seem to diffuse faster through the silica layer and are
faster accumulated in the scaffold periphery.
Fig. 30: Evolution if elements in the inner region of the 45S5-Cu derived scaffolds as function
of immersion time in SBF. Lines are for eyes guidance only. A. Hoppe et al. J. Mater. Chem. B
1 (2013), p. 5659. - Reproduced by permission of The Royal Society of Chemistry.
Results and Discussion 86
However, the evolution of Ca and P in the inner region of the scaffolds, Fig. 30, is
not significantly different among the glasses indicating that the global dissolution of
the glass is independent of the Cu content in the glass.
The kinetics for Na release from the scaffolds was found to be similar for all 45S5-
Cu derived scaffolds. However, for higher Cu contents (45S5-1Cu and 45S5-2.5Cu)
small amounts of Na can be detected even after 7d of immersion in SBF (in the
inner part of the scaffold), whereby no Na is detectable for 45S5 reference and
45S5-0.1Cu.
Fig. 31: Evolution if elements in the periphery layer of the 45S5-Cu derived scaffolds as
function of immersion time in SBF. Lines are for eyes guidance only. A. Hoppe et al. J. Mater.
Chem. B 1 (2013), p. 5659. - Reproduced by permission of The Royal Society of Chemistry.
Results and Discussion 87
Altogether the mechanism for apatite formation on 45S5-Cu derived scaffolds can
be described as follows:
Basically, the present observations confirm the reaction mechanisms of
(amorphous) bioactive 45S5 glass as originally proposed by Hench et al.9 and
Kokubo.285
For Cu containing 45S5 glasses, the relevant physico-chemical reactions
were shown exemplary for the 45S5-0.1Cu composition. However, since no
significant differences in the reaction within the Cu-glass series were detected by
FT-IR (Fig. 25) it is possible to assume that the proposed model is valid also for the
other Cu-containing glass composition.
(1d-3 d): Initial reaction of the BG surface and release of Na+ and Ca
2+ from the
scaffold periphery in an exchange reacting with H+ from the solution according to
Eq. 5.13, 286
. Free Si-NBO are protonated forming silanols groups.
𝑆𝑖 − 𝑂−𝑁𝑎+ + 𝐻2𝑂 → 𝑆𝑖 − 𝑂𝐻 + 𝑂𝐻− + 𝑁𝑎+ Eq. 5
Soluble Si species are released upon breakage of Si-O bonds by hydrolysis in the
surface region of the glass:13, 287
𝑆𝑖 − 𝑂 − 𝑆𝑖 + 𝐻2𝑂 → 𝑆𝑖 − 𝑂𝐻 + 𝐻𝑂 − 𝑆𝑖 Eq. 6
The formation of silanols is triggered by both reactions Eq.5 and Eq.6.288
A SiO2 layer is formed upon condensation of SiOH groups on the BG surface:
2𝑆𝑖𝑂𝐻𝑐𝑜𝑛𝑑.→ 𝑆𝑖 − 𝑂 − 𝑆𝑖 + 𝐻2𝑂 Eq. 7
From SEM images and from micro-PIXE-RBS analysis the thickness of the SiO2
layer was estimated to approx. 8-10 µm. The combination of the two methods
enables a reasonable estimation. The formation of silica-rich region (ii) was indicted
by FT-IR measurements, Fig. 24, and also confirmed in the elemental maps which
revealed increase of Si content. At the same time a thin ACP layer (iii) is formed (2-
3 µm) through migration of Ca and P ions to the BG surface.13
Indeed, the inner
side of BG scaffold (i) is depleted in P, as shown by ion beam measurements.
Results and Discussion 88
Interestingly, the inner part of the scaffold is not depleted in Ca. The scaffold
peripheral layer (ii), however, shows depletion in Ca content with a slight increase
in Ca concentration in the region (iii) on top of the Si-enrichment, Fig. 27. From the
ion beam measurements a Ca/P ratio of 1.28 was calculated for this specific region
of interest (iii) which corresponds to typical values of ACP.54
However, as shown in
Fig. 23c-d the silica layer and the ACP layer cannot be clearly distinguished from
the SEM figures. Also ion beam analysis shown in Fig. 27 reveals that Ca is also
found in the (ii) region which is described previously as silica-rich layer. Hence, it
can be concluded, that instead of a distinct ACP layer on top of the silica gel as
proposed by Hench et al.13
more likely a mixed layer of CaP+SixOy is formed on the
scaffolds surface after immersion for 1d in biological fluid as described by Aguiar et
al.289
for bioactive glasses.
(3d-7d) of immersion in SBF, a CHA layer was formed as confirmed by FT-IR
measurements, Fig. 25. Also from micro-PIXE-RBS analysis a Ca/P ratio of 1.85
(after 3d) and 1.77 after 7d was determined which are closer to stoichiometric
apatite (Ca/P~ 1.67) confirming the crystallisation of ACP to CHA. Furthermore,
traces of Cu are incorporated in the CHA layer.
Altogether, the combination of FT-IR, SEM and micro-PIXE-RBS results give an
comprehensive picture of physico-chemical reaction on a 45S5-Cu derived scaffolds
during immersion in SBF. The analysis performed confirmed enhanced apatite
forming ability of the 45S5 derived scaffolds after 3d of immersion in SBF. Despite
some reports in literature on the potential negative impact of crystallinity on the in
vitro bioactivity of BG derived (sintered) scaffolds,147
in this work high bioactivity
was observed which is comparable to those of amorphous bioactive glass products
widely reported in literature.30, 35
Similar rapid CHA formation was also found on
micron sized 45S5 bioactive glass particles after 3 d in SBF has been shown.94
Despite some critical considerations related to the “bioactivity test” by SBF
immersion102
this method can still be considered a relevant technique for assessing
the reactivity of BGs in physiological environment. Particularly, the “SBF test”
serves as an internal control for new developed Cu-containing bioactive glasses in
order to compare the reactivity to the undoped 45S5 bioactive glass as reference.
Results and Discussion 89
Moreover, in this work for the first time the reaction stages of a highly crystalline
45S5 derived (glass-ceramic) scaffold in simulated body fluid were observed in
high detail using the micro-PIXE-RBS technique. On top of FT-IR measurements
and SEM observations revealing the reaction on the BG scaffold surface the micro
ion beam method enabled detailed evaluation of elements distributed and evolution
in the 45S5 scaffold. Moreover, the chemical composition of the CHA layer was
derived. Detailed insight into the reaction scheme of 45S5 derived scaffolds was
gained. On top of the conventional methods thickness layer and details of the
composition of the reaction layers formed on the BG scaffold surface was obtained.
In particular for metal ion containing bioactive glass derived scaffolds the
distribution of the dopant element in the BG matrix is of great interest which cannot
be assed with conventional spectroscopic methods.
Additionally, it was confirmed that Cu inclusion in the 45S5 BG does not have any
negative impact on the CHA forming ability of the 45S5 bioactive glasses was
observed. Formation of CHA was observed after 3 days of immersion in SBF for all
glasses investigated,Fig. 25. It was shown that a homogenous CHA layer was
formed covering the BG scaffolds surface completely after 3d in SBF.
These observations are important for better understanding of the physico-chemical
reactions occurring on 45S5 Bioglass® derived scaffolds in physiological fluids.
Since chemistry of the materials surface strongly affects cell adhesion and cell
proliferation (particularly calcium phosphates)290
it is important to consider the
changes of the surface chemistry when testing BG scaffolds in vitro. CaPs for
instance are known to influence osteoblast cell attachment and differentiation.
Hence, the transformation of the silicate bioactive glass to a CaP phase is an
important parameter dictating the biological performance of BG derived scaffolds.
Interestingly, the chemical maps derived from micro-PIXE-RBS analyses revealed
that traces of Cu are also incorporated into the HAp layer formed on the BG
scaffolds surface. Thus, this effect should also be considered as parameter affecting
cell response beside the ionic dissolution products from the BG scaffold. Also, the
kinetics of the transformation of BG to CaP should determine the bone ingrowth
kinetics of BG scaffolds in vivo, which has been shown for ingrowth of 13-93 based
scaffolds.71
Results and Discussion 90
Degradation of 45S5 derived scaffolds and Cu release
Two different conditions were used in order to assess the degradation behaviour of
the BG derived scaffolds as it has been shown that degradation behaviour of
bioactive glasses and glass derived scaffolds depend on immersion condition
whether static or dynamic.291
Fig. 32 shows the Cu and Si release from the BG
derived scaffolds when immersed in SBF for a period up to 21d under static
condition. The error bars indicate the standard deviation derived from 3 samples
measured. Depending on the glass composition Cu ions in the range from 0.04 up to
3.4 ppm were released. For all glasses, a rapid increase in Cu release was observed
in the initial stage of degradation reaching a plateau after 7d with ~0.04 ppm, ~0.4
ppm and ~3.4 ppm Cu released from 45S5-0.1Cu, 45S5-1Cu and 45S5-2.5Cu
scaffolds, respectively.
Similar trend was observed for Si released from the 45S5-Cu derived scaffolds
under static conditions. Burst release of Si was detected in the first 7d of reaction in
SBF reaching a plateau at Si levels of ~30-35 ppm.
Depending on the glass composition Cu concentrations in the range from 0.03 up to
3.5 ppm were released in SBF under static conditions. These values were reached
after 7d immersion in SBF and remained constant during further reaction in SBF.
Also, the Si values reach a plateau after 7d immersion in SBF which indicates a
decrease in degradation of the scaffolds due to saturation of the solution. Being a
silicate based glass the release of silicon can be used as marker for degradation of
45S5 derived scaffolds. Since no further increase in Si concentration in SBF was
observed after 7d it can be concluded that the degradation of the scaffolds is
inhibited due to saturation of the solution. This behaviour corresponds to literature
reports which have shown that amorphous BG scaffolds (mol%: 70SiO2-30CaO)
reach a degradation stop after 3d in SBF under static conditions due to saturation of
the solution in Si.291
Variations in Si release from different glasses are detected for
1d and 3d of immersion in SBF, while no significant difference was observed for an
immersion time after longer than 7d. However, the effect of Cu is inconsistent for
this data: 45S5-2.5Cu and reference 45S5 derived scaffolds show highest Si values
for initial stage of immersion (1-3d). Also, micro-PIXE-RBS derived data in Fig. 31
show that 45S5-1Cu and 45S5-2.5Cu exhibit faster Si release from the periphery of
Results and Discussion 91
the scaffold indicating enhanced surface reactivity for BG with higher Cu contents
(≥ 1wt%). Altogether it seems that Cu-addition to the 45S5 matrix enhances the
initial surface reactivity of the scaffold while after longer reaction time no effect of
Cu is visible.
Fig. 32: Cu and Si release from 45S5-Cu derived scaffolds in SBF under static conditions.
Fig. 33 shows the Cu and Si release under quasi-dynamic conditions. For all
glasses, the overall released (cumulative) Cu concentrations were found to be higher
compared to the release rates under static conditions.
For 45S5-0.1Cu, 45S5-1Cu and 45S5-2.5Cu glasses, highest Cu levels of ~0.6 ppm,
~2.8 ppm and ~4.6 ppm, respectively, were detected. This was expected as under
quasi-dynamic conditions the concentration gradient and, thus, the ion diffusion is
enhanced. Cu concentrations reached a plateau after 14 days of immersions in SBF.
Accordingly, Si concentrations in the SBF were continuously increasing until 14
days of immersion reaching a plateau suggesting that the overall degradation of the
scaffolds is decelerated. In the same way, Si concentration increased during the first
days of immersion in SBF reaching a saturation level after 7d whereas Si values in
the range of 30-40 ppm depending on the glass composition were obtained.
Considering the standard deviation within the triplicate sample no major differences
in Si release is observed for different glass composition after 7 days of immersion in
Results and Discussion 92
SBF as the mean Si values is in the range between 30 ppm and 50 ppm. Solely, in
the initial state of reaction higher values of Si were observed for 45S5 and 45S5-
2.5Cu scaffolds after 1 and 3 days in SBF. Among the Cu-doped glasses the final Si
levels decreased released decreased with higher Cu-content. This might be a result
of higher network connectivity of the silica network in the glass caused by Cu
incorporation which inhibits the release of soluble silica.
Under quasi-dynamic conditions higher absolute Cu as well Si levels were detected
for all glasses indicating overall enhanced degradation of the scaffolds under quasi-
dynamic conditions compared to static setup. These observations should be
considered for in vitro experiment of bioactive glasses.
In literature reports silicate based glass derived scaffolds were shown to follow an
linear degradation profile when cultured in SBF up to 7 days under quasi-dynamic
conditions similar to the parameters used in this work.291
However, after 14 days the
degradation slowed down and Si as well as Cu values reach constant constant
levels. Furthermore, with increasing Cu-content in the glass lower Si values are
released from the scaffolds. Considering the NMR results this is likely due to higher
Q2 and Q
3 fractions in the glass network resulting in higher SiO2 network stability
and less amount of soluble silica being released.
Fig. 33: Cu and Si release from 45S5-Cu derived scaffolds in SBF under quasi-static conditions.
A. Hoppe et al. J. Mater. Chem. B 1 (2013), p. 5659. - Reproduced by permission of The Royal
Society of Chemistry.
Results and Discussion 93
After 21 days under both, static and dynamic conditions no significant mass gain
was observed for all 45S5-Cu derived scaffolds. Despite high release rates of
soluble silica the material loss is seemingly compensated with the mass gain
through the formation of hydroxyapatite on the 45S5 scaffolds surface.
Cu ions exhibit dose-dependent effects on human cells. For example, 50 ppm of
CuSO4 (313 µM) were shown to be optimal for stimulation of tube-like structures
formed by ECs when exposed to a Cu concentration range from 0 ppm to 100
ppm.197
Similarly, Wu et al. showed that Cu levels from 60.4 to 152.7 ppm favor
angiogenesis and osteogenesis via expression of osteogenic markers (ALP, OPN,
OCN) and secretion and expression of the angiogenic marker VEGF in human bone
marrow derived stem cells (hBMSCs).226
However, also lower Cu concentrations of
4 ppm released from phosphate glasses (similar to the Cu release ranges observed in
our study) were favorable for HUVEC cells via down regulating apoptosis.260
Furthermore, related values of 1.6 ppm to 8 ppm of CuSO4 were reported to
significantly increase the VEGF expression in keratinocytes.292
In this work Cu
levels from 0.3 ppm to 4.6 ppm were released in SBF from Cu-containing 45S5
bioactive glass derived scaffolds depending on the culturing conditions, hence it can
be concluded that 45S5-Cu bioactive glass might be a potential material for bone
tissue engineering applications where both osteogenic and angiogenic properties are
required.
Results and Discussion 94
4.1.4 In vitro cell response
Theoretically the included Cu into the 45S5 Bioglass could affect cell behaviour in
two ways: Firstly, Cu ions released from the 45S5 Cu derived scaffold might
enhance vascularisation as Cu2+
ions are well-known angiogenic agents. As
indicated above, the Cu levels released into physiological environment are within
the therapeutic range according to literature reports for Cu2+
ions.293
Since ionic
dissolution products have been shown to stimulate cells on molecular level towards
osteogenic differentiation18
the Cu doping is expected to impart additional
angiogenic functionalities and enhance the overall biological activity of the 45S5
BG derived scaffolds. Secondly, Cu was incorporated in the CHA layer formed on
the scaffold surface after immersion in SBF, which might influence the attachment
and growth of relevant cell types used in regenerative medicine.290
Metal ion doped
hydroxyapatite, for instance, has been shown to influence osteoblast adhesion and
differentiation.294
The assessment of such biological effects of the incorporation of
Cu in the basic 45S5-Bioglass® material is described in this section.
Powder cytotoxicity
Cu in high dosage might be toxic to human cells and organism and therefore
cytotoxicity of Cu-containing glasses was investigated. Commercial use of
bioactive glass products also includes applications of particulate glass, such as
powder or granula (BonAlive® or NOVABONE
®). Hence in the first place the
cytotoxicity of the particulate 45S5-Cu powder was assessed. Fig. 34 shows light
microscopy evaluation of MG-63 cell morphology after 48 h of incubation with
45S5 bioactive glass particles at different concentrations. The cells show a slightly
elongated triangle morphology which is typically observed for healthy MG-63 cells.
Even at BG particle concentrations of 1 mg ml-1
no negative effects on cell growth
were observed. It seems that the particles sediment during the culturing time and the
cell grow on the top of the particles.
For comparison of the Cu containing glasses the cell morphology of MG-63 cells
cultured with 45S5-Cu containing samples at 100 and 1000 µg ml-1
is shown in Fig.
35. Similarly, no toxic effects are seen for the Cu samples for particle
Results and Discussion 95
concentrations up to 100 µg ml-1
. For 1000 µg ml-1
, however, less cells were visible
indicating possible negative effect of the BG particles at 1000 µg ml-1
. Particularly,
for the 45S5-2.5Cu lees cells are visible compared to 45S5-Cu glasses with lower
Cu contents.
.
Fig. 34: Light microscope of MG-63 after 48h of incubation in drect ccontact with 45S5 BG
particles at different concentrations.
Fig. 35: Light microscope images of MG-63 after 48h of incubation in direct contact with 45S5-
Cu BG particles at 100 µg/ml and 1000 µg/ml.
In order to gain more detailed analysis of the cell viability the mitochondrial
activity and the cell number were derived. Fig. 36 shows the mitochondrial activity
and the cell number indicated by LDH activity of osteoblast-like cells after
Results and Discussion 96
cultivation time of 48 h as function of BG concentration in the cell culture medium.
All Cu containing 45S5 BGs show high cell mitochondrial activity of > 50% of the
reference control for the entire concentration range investigated. Also the cell
number remains nearly constant for all particle concentrations tested. This indicated
that 45S5-Cu particles do not show any toxic effects on the cells during the first 48
h of incubation. Moreover, statistically significant enhancement of the
mitochondrial activity of cells in contact with 45S5-0.1Cu and 45S5-1Cu for
particles concentration between 0.1 and 100 µg ml-1
were observed compared to
plain 45S5 reference. At 1000 µg ml-1
for the Cu containing glasses a reduction of
the mitochondrial activity to 70 % (for 45S5-0.1Cu and 45S5-1Cu) and ~50 % (for
45S5-2.5Cu) was observed while remaining at ~100 % for the reference 45S5.
Hence, even though the cells remain at high viability higher than 50 % this indicates
possible cytotoxic effects of the 45S5-Cu particles at too high concentrations.
However, this effect could be also assigned to the alkaline pH shift due BG
dissolution when applied at a critical concentration ≥1000 µg ml-1
.
These observations confirm good biocompatibility of Cu-containing 45S5 glass
powders and beyond that stimulating effect on MG-63 cells when applied at
concentration range of 0.1-100 µg ml-1
. In particular, 45S5-0.1Cu and 45S5-1Cu
glass particles enhanced the mitochondrial activity of MG-63 cells compared to the
undoped 45S5 reference.
Fig. 36: Mitochondrial activity and cell number (LDH activity) as function of particle
concentration for 45S5-Cu glass series. *p<0.5 and **p<0.001 compared to 0 µg/ml reference.
Results and Discussion 97
It has been shown in literature that Cu can stimulate the cell activity and
proliferation of osteoblastic cells.258
Accordingly, 45S5-0.1Cu and 45S51Cu glass
samples showed enhanced MG-63 cell activity compared to 45S5 reference.
In literature it has reported that hat BG particles of ~100 µm did not have any
significant effect on osteoblastic cell proliferation and metabolic activity.295
On the
other hand nano-sized bioactive glass particles can be cytotoxic as shown by means
of reduced cell activity of mesenchymal stem cells when cultured with BG particles
of ~70 nm at a concentration of 0-200 µg/ml (similar range as tested in this
work).296
The BG glass particles used in this study are ~ 6 µm and were shown to
stimulate the activity of osteoblast like cells indicating good biocompatibility of
micron-sized BG particles.
These findings are important for designing of new in vitro studies incorporating BG
particles. Further, these fundamental studies confirm the biocompatibility of the
45S5-Cu glasses which is a first step in the evaluation of biomaterials for use in
clinical applications. Indeed, commercial use bioactive glasses also involve the
application of particulate bioactive glass (Novabone, BoneAlive).
Cell attachment on 2D pellets
Fig. 37 shows the attachment and growth of MG-63 cells on 45S5-Cu derived
pellets after culturing for 48 hours. A dense cell layer was observed on the 45S5,
45S5-0.1Cu and 45S5-1Cu samples. The cells are widely spread indicating high
compatibility of the samples surface towards MG-63 cells. On 45S5-0.1Cu and
45S5-1Cu even multilayer growth of the cells was observed. However, on the 45S5-
2.5Cu samples no cells were detected indicating possible cytotoxicity of the high
Cu concentration in the 45S5-2.5Cu samples. These results are in agreement with
the cytotoxicity tests carried out on powdered 45S5-Cu glasses which indicated
possible cytotoxic effects of 45S5-2.5Cu glass.
Results and Discussion 98
Fig. 37: Fluorescence microscopy of cytoskeleton (red) and cell nucleus (green) staining of
osteoblast-like cells seeded on 45S5-Cu derived pellets for 48h. No cells were observed on 45S5-
2.5Cu.
One well accepted explanation for copper-induced cytotoxicity is related to the
formation of reactive oxygen species (ROS) by Cu ions the via Fenton reaction.297
with the consequence of peroxidative damage of membrane lipids.298
In order to test
this hypothesis a western dot-blot analysis was performed with HOS cells seeded on
45S5-Cu pellets and the formation of 4-hydroxynonenal (HNE), one of the best
known and well-studied products of lipid peroxidation.10
A detailed description of
the experiment is given elsewhere.299
Fig. 38 shows the HNE formation for 2D pellets of Cu-containing 45S5 glasses. For
pure 45S5 (reference material) the cell growth was associated with low HNE
formation which slightly increased from after 7d and 14d of cell culture. With
10
The immuno-blot analysis of the HNE formation was carried out in collaboration with L.
Milkovic and Prof. N. Zarkovic, Laboratory for Oxidative Stress, Rudjer Boskovic Institute, Bijenicka 54, 10000 Zagreb, Croatia. These results are also part of the PhD thesis of L. Milkovic entitled “Beneficial effects of lipid peroxidation in the bone cell growth on bioactive glass - New perspectives in tissue engineering and regenerative medicine”
Results and Discussion 99
addition of Cu higher values of HNE were detected whereby the strongest
enhancement was observed for 45S5-1Cu and 45S5-2.5Cu samples.
HNE is an important signaling molecule involved in various cellular processes
including cell proliferation and differentiation.300, 301
However, HNE acts in
concentration-depended manner and hence high HNE levels can also correspond to
cell death.302
This dese-dependent role is in accordance to the results shown in this
study: while for 45S5, 45S5-0.1Cu and 45S5-1Cu the enhanced HNE formation and
(hence peroxidation) are correlated to cell growth, for 45S5-2.5Cu the enhanced
HNE formation is related to cytotoxicity.
From these results it can be concluded lipid peroxidation is involved in the
interaction between 45S5 BG and osteoblast-like cells and that the effect of copper
is dose-depending and is correlated to the formation of lipid peroxidation products.
These correlations should be further explored in future studies in order to get more
information on the mechanism of the interaction of BG and human cells.
Fig. 38: Immuno-blot analyses of the HNE-protein adducts formation in osteoblast-like cells
(HOS) cells after 3, 7 and 14 days. The results are expressed as nmol of HNE-protein
adducts/mg of protein. Enhancement of the lipid peroxidation was in particular pronounced
after 1d (*** p<0.0001) and 14d (** p<0.01) for the cells grown on 45S5-2.5Cu samples.*p<0.05
Results and Discussion 100
In vitro cell studies with 3D scaffolds
Response of osteoblast-like cells (MG-63)
From the powder cytotoxicity and the 2D cell attachment studies it was concluded
that 45S5-2.5Cu BG seems to be cytotoxic and, hence, this composition was not
further considered for biological investigations. For the 3D studies with MG-63
cells only 45S5, 45S5-0.1Cu and 45S5-1Cu were tested.
Good cell attachment and growth of osteoblast-like cells was observed with direct
seeding of the cell on the 3D scaffolds. Fig. 39 shows the attachment and growth of
osteoblast-like cells on 45S5-Cu derived scaffolds. Similar to 2D experiments no
signs of cytotoxicity of Cu was observed. The cell can attach and proliferate on the
scaffolds surface. MG-63 are an established cell culture model for assessing
osteoblast-like behaviour of cell in the context of bone tissue engineering.303
In
particular the attachment of human osteoblast cells can be well monitored by using
the MG-63 cell line as they show similar integrin profile as human osteoblasts. Also
it is a widely used cell model to test therapeutic agents and cytocompatibility testing
of materials.303
Hence, it can be concluded that 45S5-Cu derived scaffolds are
suitable for attachment and growth of osteoblast-like cells. Combined with the
results from the studies with powders and dense pellets in general a good
biocompatibility of 45S5-Cu scaffolds was observed. Hence, Cu levels up to 4 ppm
as shown in the degradation studies are not toxic to osteoblast-like cells up to a
period of 21 days. Also independently of the BG morphology applied as particulate,
dense pellets or porous scaffolds 45S5-Cu BG is biocompatible in vitro.
Results and Discussion 101
Fig. 39: SEM images of osteoblast-like cells cultured for 21d on 3D scaffolds at different
magnifications: 45S5 (a, b), 45S5-0.1Cu (c, d) and 45S5-1Cu (e, f).
For more detailed evaluation of the cell response to 45S5-Cu derived scaffolds a
more clinically relevant cell type, human bone marrow derived stem cells
(hBMSCs) were used which allow a more accurate evaluation of osteogenic and
angiogenic behaviour of cell.303
Response of hBMSCs to ionic dissolution products of 45S5-Cu BG derived scaffolds
(2D cell culture)
Fig. 40 shows the cell morphology of hBMSCs after exposure to ionic dissolution
products from 45S5-Cu derived scaffolds for 3 weeks. No cytotoxic effects are
indicated since typical elongated morphology of the hBMSCs was observed.
Accordingly, high metabolic activity of the hBMSCs given by AlamarBlue (AB)
Results and Discussion 102
reduction was detected as shown in Fig. 41a. For all time points investigated high
AlamarBlue reduction and hence high metabolic activity compared to reference
(hBMSCs only) was observed for hBMSCs when exposed to ionic dissolution
products. This indicates that ions released from the scaffolds do not show any toxic
effects on cells for time period up to 4 weeks. Moreover, after 4 weeks 45S5.1Cu
samples seems to stimulate the metabolism of the cells as indicated by higher AB
reduction compared to plain 45S5 glass. Even though the enhancement is not
statistically significant there is a tendency for Cu to stimulate hBMSCs in 2D
indirect culture model.
Fig. 40: LM images of the morphology of hBMSCs during exposure to ionic dissolution
products from 45S5-Cu derived scaffolds.
Fig. 41b shows the ALP activity of the hBMSCs in the 2D culture experiment. For
all sample groups significant ALP values were detected. However, no differences
for 45S5-Cu were observed compared to plain 45S5 and also the control (hBMSCs
only). In a similar way, the osteogenic marker RUNX2 was expressed after 2 weeks,
Results and Discussion 103
Fig. 41d (normalized to their Glyceraldehyde 3-phosphate dehydrogenase
(GAPDH) expression). However, again no significant differences among the 45S5-
Cu derived scaffold groups were observed. Altogether no significant enhancement
of Cu on the expression of osteogenic markers ALP and Runx2 were observed even
though osteogenic potential of Cu has been presented in literature. For example, Cu
has been shown to stimulate MSCs towards the osteogenic cell line199
and has been
shown to upregulate expression of osteogenesis-related genes, e.g. alkaline
phosphatase (ALP), osteopontin (OPN) and osteocalcin (OCN).226
Fig. 41: Evaluation of hBMSCs during exposure to ionic dissolution products from 45S5-Cu
derived scaffolds: (a) metabolic activity by AlamarBlue dye reduction, (b) alkaline phosphatase
activity, (c) relative VEGF and (d) Runx2 expression related to hBMSCs group after 2 weeks. *
statistically significant for p<0.05 compared to all groups.
Interestingly, in this work no significant effect of Cu on osteogenic differentiation
of hBMSCs was observed. This might be due to the fact that in contrast to this work
in the study by Rodríguez et al.199
a osteoinduction medium was used which is
usually essential in order to induce osteogenic differentiation of MSCs in in vitro.
Furthermore, it should be taken into account that plain 45S5 Bioglass is known to
Results and Discussion 104
induce osteogenic differentiation of MSCs which has been extensively described in
literature.18, 115
Hence it is likely that the Cu ion levels released from the 45S5-Cu
derived scaffolds did not excess the osteogenic ability of the plain 45S5 BG.
However, the expression of the angiogenic marker VEGF was enhanced for
hBMSCs seeded in contact with 45S5-1Cu scaffolds, Fig. 41c. VEGF expression
was 12 fold, which is 6 fold higher compared to hBMSCs only control and 3fold
stronger than 45S5 and 45S5-0.1Cu. Our observations are in very good accordance
with the well-known role of Cu in the VEGF signalling path ways as described in
literature: Cu ions were shown to stabilize and to upregulate hypoxia-inducible
factor 1 (HIF-1)304, 305
which, in turn, regulates the VEGF expression in MSCs.306
Hence, these results confirm that Cu released from a bioactive 45S5 glass scaffolds
show angiogenic potential in vitro. Since the cell response does not only depend on
the ionic dissolution products released from the scaffolds but also on parameters
like surface chemistry, roughness and pore curvature the biocompatibility 45SS5-Cu
derived scaffolds with hBMSCs was additionally tested in a 3D culture model
whereby the cells were directly seeded on the scaffolds.
Response of hBMSCs in direct contact (3D) with 45S5-Cu BG derived scaffolds
Similar to indirect studies for the hBMSCs seeded directly on the scaffold surface
no signs of cytotoxicity were found as indicated by high values of AB reduction,
Fig. 42. A rapid increase in AlamarBlue reduction was observed after 2 weeks for
all sample groups remaining at this level up to weeks of culture. All samples show
high AlamarBlue reduction values which indicate high metabolic activity and hence
high vitality of the cells. Even though no significant differences among the 45S5-Cu
glass scaffold series were observed the results show that high viability of hBMSCs
is maintained over a period up to 4 weeks and that no signs of toxicity due to Cu
presence were observed. Also the overall metabolic activity was significantly
enhanced after weeks (for all samples) compared to day 1 indicating high
proliferation ability of the cells when seeded on Cu-derived scaffolds.
Similar to the indirect 2D assay no significant differences in ALP activity was
observed among the 45S5-Cu scaffolds. However, all scaffolds induced ALP
Results and Discussion 105
activity indicating osteogenic potential of 45S5 BG even though no additional
osteogenic stimulating effect of Cu was detected.
Fig. 42: Evaluation of hBMSCs in direct contact with 45S5-Cu derived scaffolds: (a) metabolic
activity by AlamarBlue dye reduction, (b) alkaline phosphatase (ALP) activity.
Evaluation of co-culture of hDMECs and hBMSCs
Fig. 43 shows the morphology of the ECs observed with a light microscope when
cultured for 2 weeks in presence of BG scaffold/hBMSCs constructs. For all
constructs the ECs show no signs of toxicity, the cells remain vital showing typical
“cobble stone” morphology. However, under the effect of 45S5-1Cu/hBMSC
construct, the cells show endothelial tube formation as indicated in Fig. 43e (dashed
circles).
Further viability and functionality characterisation of the HDMSCs was done by
LDL uptake assay. The LDL uptake was positive for all groups tested as shown in
Fig. 44a-e indicating high functionality of the ECs. Being a viability marker the
high LDL uptake confirms the high vitality of the ECs. In addition to light
microscopic analysis this confirms that the hDMECs retain their phenotype when
cultured in indirect contact. Again, only for the group cultured in presence of 45S5-
1Cu/hBMSC construct a formation of tube-like structures by the HDMSCs was
observed, Fig. 44e-f.
Results and Discussion 106
Fig. 43: LM pictures of hDMECs cultured for 2 weeks in the presence of 45S5-Cu/hBMSCs
constructs: a) control ECs only, b) 45S5 c) 45S5-1Cu, d) 45S5/hBMSCs, and e) 45S5-
1Cu/hBMSCs. Only combination of 45S5-1Cu and hBMSCs stimulated hDMSCs towards
formation of tube-like structures.
Fig. 44: FLM analysis of the LDL uptake of hDMECs cultured for 2 weeks in the presence of
45S5-Cu/hBMSCs constructs: A) control ECs only, (B) 45S5 (C) 45S5-1Cu, (D) 45S5/hBMSCs,
and E) 45S5-1Cu/hBMSCs. Only the combination of 45S5-1Cu and hBMSCs leads to
formation of tube-like structures. LDL uptake is directly correlated with light-microscopy
images of the same well (F).
Furthermore, the endothelial phenotype and the ability of the ECs to form tube-like
structure was assessed by seeding the cells on Matrigel (after being trypsinysed).
The results of light-microscopic evaluation are shown in Fig. 45. Basically, for all
groups the hDMECs nicely grow on the Matrigel forming tubular structures. Even
though no major differences among the tested constructs were observed it is visible
Results and Discussion 107
that for the 45S5-1Cu/hBMSCs group a more dense tubular structures were formed
indicating higher functionality and viability of the hDMECs.
Fig. 45: LM images of trypsinised ECs seeded for 2 weeks on matrigel for assessment of tube
formation. A) control ECs only, (B) 45S5 (C) 45S5-1Cu, (D) 45S5+hBMSCs, and E) 45S5-
1Cu+hBMSCs. Similar pictures were observed also at week 1.
In order to analyse the detailed EC phenotype flow cytometric analyses (FACS) was
performed identifying vWF (Von Willebrand factor), CD31 and VEGFR2 in
hDMECs. Fig. 46 summarises the results of the ES phenotype analyses by FACS.
Generally, the EC phenotype is better retained in all samples with presence of
hBMSCs compared to non-hBMSCs groups.
Additionally, Cu seems to have a stimulating effect on HDMSCs: for the
45S5/hBMSCs and 45S5-1Cu/hBMSCs 60.3% and 95.1 %, respectively, were in
presence of 45S5-1Cu/hBMSCs (47%) compared to 45S5/hBMSCs (16.5 %).
The CD31 marker was also expresses in the hDMECs even though no significant
difference between the presence of 45S5-1Cu/hBMSCs (98%) and 45S5/hBMSCs
(97.3%). This indicates that the 45S5-1Cu/hBMSCs constructs enhanced the
expression of relevant specific endothelial markers which can be used to
characterise the phenotype of endothelial cells. Usually high expressions of CD31,
vWF and VEGFR2 are correlated with physiological endothelial phenotype while
under pathological conditions the expression of these markers can be inhibited.
Results and Discussion 108
According to Fig. 46 addition of Cu to the 45S5 BG enhanced the expression of the
endothelial markers indicating its angiogenic potential.
The VEGF release in culture medium is shown in Fig. 47. After 2 weeks
significantly higher VEGF release was observed for the constructs containing
hBMSCs. After 4 weeks the VEGF values further increase for the constructs
containing BG scaffolds and hBMSCs, while remaining at lower values at ~pg/ml
for the constructs without stem cells. Interestingly, the constructs with plain
45S5/hBMSCs showed higher VEGF release compared to 45S5-1Cu/hBMSCs
construct despite the enhanced expression of VEGF in hBMSCs when cultured in
contact with 45S5-1Cu alone as shown in Fig. 41.
Fig. 46: FACS analysis for vWF antigen, VEGFR2 and CD 31 on surface of hDMECs after 2
weeks of culture. For the green curves the number of positive markers is given in %.
Results and Discussion 109
Fig. 47: VEGF release in culture medium from hDMECS cultured indirectly with BG
scaffold+hBMSCs constructs.
Altogether three main observations can be made regarding the effect of Cu on
mesenchymal stem cells and endothelial cells
i) Cu ions stimulate VEGF expression in hBMSCs
ii) Higher VEGF release is observed for hDMECs co-cultured with
hBMSCs seeded on a 45S5 BG scaffolds. But is not enhanced when Cu
is added.
iii) Enhanced endothelial phenotype is observed when hDMECs are exposed
to specifically Cu-containing 45S5-1Cu/hBMSCs construct and are
stimulated to form tube-like structures
Firstly, Cu ions stimulate hBMSCs to express VEGF and thus activate relevant
angiogenesis related pathways. Furthermore, the hBMSCs seeded on a 45S5 derived
scaffold secrete VEGF which stimulates ECs as enhanced EC phenotype was
observed in presence of MSCs indicated by larger numbers of cells being positive
for vWF, CD31, and VEGF2R. However, the enhanced release of VEGF was
observed for both constructs with and without Cu. Nevertheless, the only for 45S5-
1Cu/hBMSCs constructs an enhanced functionality and stimulation of ECs was
observed indicated by increased activity of EC markers and formation of tube-like
structures by the hDMECs confirming their angiogenic activity.
Results and Discussion 110
VEGF plays a critical role in angiogenic process as one of the main transcription
mediation blood vessel development.307
Cu, in turn, induces VEGF expression not
only in MSCs but also in ECs like keratocytes213
and cardiomyocites.308
It is
important to mention that Cu has been shown not only to induce VEGF expression
when applied in excessive concentrations (related to physiological values). When
applied at physiological values (5 µM, ~ 0.132 pm), however, it was shown that Cu
is essential for VEGF expression.293
Furthermore, it is known that Cu is not only an
essential co-participant in angiogenesis but can be angiogenic itself.198
McAulan et
al., for example, showed that Cu ions induced neovascularisation in an in vivo rabbit
cornea pocket assay.309
There is also direct evidence of Cu action in increased flap
survival in an in vivo rat model rats due to enhanced VEGF expression. This action
is again due to effects of Cu on nearby cells in the random flap in inducing VEGF.
The mechanisms behind the stimulating effect of Cu ions are usually related to
activation of several transcriptional factors, as for example HIF-1 that is crucial for
VEGF expression.293
Cu ions have been shown to stabilise HIF-1 even under
normoxic conditions and hence activate HIF-1 related pathways.304
However, considering that high VEGF amounts were found also for 45S5/hBMSCs
constructs this indicates that there must be synergetic effects of Cu and the presence
of hMSCs leading to stimulation of ECs. Indeed Cu plays a versatile role in the
physiology of angiogenesis. It is likely that Cu induced expression of some other
angiogenic factors besides VEGF as it is known that expression if VEGF is not the
only possible action of Cu in angiogenesis.293
For example expression off FGF-1
and FGF-2 factors known for regulation of blood vessel functions are also known to
be mediated by Cu as well as fibronectin and angiogenin.293
Indeed, it is known that Cu has direct angiogenic effects in vitro and in vivo.
It has already been reported that VEGF expression in ECs293
can be induced by
copper ions and this property may be exploited to accelerate dermal wound
healing.310
Furthermore, there is evidence in literate that Cu can directly stimulate
ECs. Cu ions are known to regulate endothelial cell proliferation and migration this
having a direct impact on the process of angiogenesis and vascularisation.293
Li et
al., for instance, showed that Cu enhances the proliferation of human umbilical vein
endothelial cells (HUVECs).311
Similarly, Hu showed that application of CuSO4 in
Results and Discussion 111
the range of 1-500 µM CuSO4 significantly increased the proliferation of
HUVECs.198
Similar results were observed with 3D printed scaffolds loaded with
CuSO4 and VEGF and FGF-2 growth factor.257
It was shown that Cu combined with
growth factor exhibits a stimulating synergetic effect on angiogenesis in vivo
indicated through formation of tube-like structures and collagen deposition.197
Based on the experimental observation and literature reports the proposed
mechanism for Cu acting in the hBMSCs/hDMECs co-culture model is
schematically shown in Fig. 48.
Fig. 48: Scheme of the mechanism of Cu involved in the angiogenic pathways in a co-culture of
hBMSCs and hDMECs. Cu2+
activate the HIF-1 transcription factor which mediations
expression of VEGF and other angiogenic factors which, in turn, activate signalling pathways
in hDMECs mediating cell migration, proliferation and cell survival. Additionally, Cu ions
might directly stimulate endothelial cells as shown in literature.
The use of co-culture models of osteoblast cells and endothelial cells has been
extensively investigated in the context of bone tissue engineering in the last two
Results and Discussion 112
decades.312, 313
The general idea involves the induction of osteogenic hBMSCs
differentiation while in the same time the ECs are stimulated towards formation if
tube-like prevascular structure for providing nutrition and oxygen for osteoblast
cells. In turn, hBMSCs can be used as vesicles for growth factor delivery of
angiogenic factor in order to stimulate the ECs. However, usually additional
amounts of VEGF are supplemented to the cell culture to provide strong initial
stimulation.313
Thus, by stimulating hBMSCs towards higher release of VEGF, which in turn
stimulated ECs, the presented 45S5-Cu scaffold act as an “indirect” angiogenic
growth factor delivery system.46
This indirect approach is advantageous since it
enables controlled VEGF release mediated by cells which is adapted to the
physiologically, local needed conditions avoiding growth factor “overdose”.
It was observed that 45S5 combined with hBMSCs can be used as indirect system
for releasing VEGF as stimulating factor for hDMECs leading to enhanced
angiogenesis. Additionally, incorporation of Cu leads to enhanced expression of
VEGF in hMBSCs hence seemingly activating signalling pathways related to
angiogenesis. Altogether, the combination of 45S5-1Cu scaffolds with hBMSCs has
a stimulating effect on ECs to form tube-like structures and hence can be considered
as a promising biomaterial-cell approach in bone tissue engineering.
45S5-1Cu derived scaffolds showed enhanced expression in MSCs and hence were
chosen as the most promising candidate for the in vivo study.
4.1.5 In vivo evaluation
Angiogenic potential of Cu-containing bioactive glass has been assessed in an AV-
loop model. After 3 weeks the explanted constructs were analysed by means of
vessel density and total vessel cross area, vessel length as well as vessel radius. Fig.
49 shows the microCT reconstruction of the blood vessels in the 45S5 and 45S5-
1Cu constructs. It is clearly visible that both 45S5 and 45S5-1Cu scaffolds support
intrinsic vascularisation as indicated through micro vessel sprouting originating
from the AV-loop. However, no statistically significant stimulation effect of copper
could be shown. The quantification of the microCT data revealed that for 45S5
more vessels with larger radius were formed, whereas for the 45S5-1Cu samples
Results and Discussion 113
small radius vessels were present, Fig. 50. It seems that for 45S5-1Cu higher
amount of small radius vessels was formed whereas almost no blood vessel with a
radius below 10 µm were detected. Furthermore, the vessel density was found to be
higher in 45S5-1Cu containing constructs, being 0.16±0.18 mm-2
compared to
0.07±0.05 mm-2
. However, only a trend of Cu stimulating the formation of blood
vessel especially in the initial stage resulting in higher number of small radius
vessels was observed. In this work it was shown that Cu stimulated angiogenesis in
vitro but, however, this effect could not be confirmed in vivo even though there is
also evidence in literature indicating that Cu directly stimulated vascularisation in
vivo.257, 310, 314
This might be due to angiogenic potential of plain 45S5 derived
scaffolds is it was shown in the AV-loop model.315
Hence, the amount released from
the 45S5-1Cu scaffolds under in vivo conditions might not be sufficient enough to
exceed the angiogenic potential of 45S5 BG scaffolds. Also the 3 weeks of
investigation might be to short shown full potential of Cu in the present in vivo
model. Indeed, the formation of small radius vessels was enhanced for the 45S5-
1Cu scaffolds compared to 45S5 reference which might result in a more dense
vascularisation after further maturation.
Fig. 49: MicroCT reconstruction showing the intrinsic vascularisation induced in the AV model
for a) 45S5 and b) 45S5-1Cu scaffolds. Small vessel sprouting originates from the AV loop.
Results and Discussion 114
Fig. 50: MicroCT derived quantification of the vessel sprouting in the AV-loop model.
The results also indicate that for in vivo studies even higher Cu concentrations
might be considered biocompatible as 45S5-1Cu did will not impair the angiogenic
potential of 45S5 scaffolds. Higher amounts of Cu doping to 45S5 BG is, hence,
suggested for further investigations. Also constructs seeded with hBMSCs could be
used in in vivo model. This should enhance the angiogenic effect as it has been
shown that combination of 45S5 and cells implanted in vivo result in enhanced
vascularisation.316
Overall, it was shown that 45S5derived scaffolds support intrinsic vascularisation as
confirmed in the AV loop model in rats. Hence, 45S5 derived scaffolds are potential
materials to be used in prevascularisation in vivo models.315
Furthermore, the results
indicate that 45S5-1Cu show a tendency to improve the intrinsic vascularisation.
Even though the results are not statistically significant 45S5-1Cu might be a
candidate to be used in bone regeneration applications where enhanced
angiogenesis is required.
Results and Discussion 115
4.2 Cobalt containing 13-93 based glasses
As described in chapter 4.1.2 45S5 BG undergoes crystallisation during sintering
leading to unsatisfied densification ad formation of micro cracks. Hence, 1393 glass
was employed as possible alternative scaffold material and its suitability as carrier
for therapeutic inorganic ions whilst showing appropriate mechanical performance
was assessed.
4.2.1 Glass properties
Structure and Thermal Properties
Fig. 51 shows the FT-IR spectra of Co-containing glasses in the as-fabricated state.
The main bands appearing in the FT-IR spectra are summarised in Table 12.
The intense band at ~1050 cm-1
are attributed to asymmetric stretching vibrations of
the Si-O bond becomes sharper with increasing Co content in the glass.
Furthermore, for 1393-5Co and 1393-10Co additional peak at ~1105 cm-1
and a
weak shoulder at ~1205 cm-1
were detected which are typically observed for silicate
glasses with higher SiO2 content and vitreous silica, respectively.317
With increasing
Co concentration in the glass the Si-NBO (non-bridging oxygen) peak (at ~950 cm-
1)91
is reduced in intensity and is slightly shifted to lower wave numbers. Also, the
intensity of the bending mode of the Si-O band at ~790 cm-1
is increasing with
higher Co content. Also, for 1393-5Co and 1393-10 an additional absorption peak
was observed at ~990 cm-1
which, according to literature, might be attributed to Si-
O-Co bonds as it was shown for metal containing mesoporous alumino-silicates
(zeolithes).318
Hence, the FT-IR analysis gives indication that for concentrations ≥
5wt% Co may be entering the glass network by forming Si-O-Co bonds resulting in
higher polymerized SiO2 network.
Raman spectra (normalised to the Si-O(r) peak) of the 1393-Co glasses are shown
in Fig. 52 with the main bands occurring summarised in Table 12.
Results and Discussion 116
According to literature the wide absorption band at ~1100 cm-1
can be assigned to
asymmetric stretching mode of Si-O-Si group, whereas the bands at ~610 cm-1
and
~930 cm-1
can be attributed to Si-O-Si rocking vibrations and Si-NBO bond,
respectively.319, 320
Fig. 51: FT-IR spectra of as fabricated 1393-Co glasses.
Fig. 52: Raman spectra of as fabricated 1393-Co glasses.
Results and Discussion 117
The Band at~450 cm-1
is likely assigned to defect lines of vitreous silica typically
observed for silicate ring structures.321
For 393-1Co and 1393-5Co a new broad
peak is evolving at 750 cm-1
which is assigned to the bending mode of Si-O-Si.319
Likely, this band appears due to the longer length of the Co-O bond (192 pm)
compared with the Si-O bond (177 pm) causing stronger bending vibrations of the
Si-O bond.
Table 12: Main absorption bands in the FT-IR spectra observed for as fabricated 1393-Co
glasses. Reprinted with permission from A. Hoppe at al, ACS Appl. Mater. Interfaces 6 (2014),
p. 2865. Copyright (2014) American Chemical Society.
Assignment FT-IR [cm-1
] Raman [cm-1
] Remarks
Symmetric
stretching,
νsym(Si-O-Si)
~490-500 ~450 νsym(Si-O-Si) at ~450 cm-1
is observed
for pure silica. 282
For bioactive
glasses the band is shifted to 500 cm-
1.91
In the Raman spectra this band is
assigned to silica ring structures.321
Asymmetric
stretching,
νasym(Si-O-Si)
1000-1300
~1040/1140*
~1220**
~1060-1080319
In the FT-IR spectra317
the most
intense Si-O band is at ~1040 cm-1
.
With Co doping additional bands at
~1140 cm-1
and ~1220 cm-1
appear
which are typically seen in vitreous
silica. In the Raman spectra319
the
peak maximum is shifted towards
lower frequencies for 1393-5Co and
1393-10Co glasses.
Bending mode
δ(Si-O-Si)
Peak at ~75091
Broad band at
~750319
Intensity is increasing with higher Co
content in the glass.
Rocking
vibrations of Si-
O-Si
n.a. ~620319
--
SiONBO Shoulder at
~930
Peak at ~940 In the FT-IR spectra the SiONBO is
decreasing in intensity and is shifted
to lower frequencies with
incorporation of 5wt% and 10wt%
Co.91
,282
Si-O-Co 992 Appears in the FT-IR spectra of 1393-
5Co and 1393-10Co glass and is
attributed to Si-O-Co bonding.318
*TO1; **TO2; TO=transverse optical groups of Si-O-Si bonds. 317
Results and Discussion 118
Thermal properties
Fig. 53 shows the DSC diagrams for the 1393-Co glass powders as fabricated. The
characteristic features glass transition point Tg and the crystallization onset To
derived from the DSC measurement are summarized in Table 13.
Table 13: Glass transition point Tg and crystallisation onset To (in °C) of the Co-containing
glasses derived from DSC measurements. Reprinted with permission from A. Hoppe at al, ACS
Appl. Mater. Interfaces 6 (2014), p. 2865. Copyright (2014) American Chemical Society.
Glass 1393 1393-1Co 1393-5Co 1393-10Co
Tg 626 621 606 587
To 750 848 -* -*
*Outside of the observed temperature range.
Tg decreased continuously with increasing Co2+
content in the glass. For 1393,
1393-1Co, 1393-5Co and 13-93-10Co Tg values of 626 °C, 621 °C, 606 °C and 587
°C, respectively, were determined. In turn the Tc, onset is increasing with higher Co
content: For 1393-1Co the To increased from 750 for 1393 to 848 °C for 1393-1Co,
whereas for Co contents >1 wt% the To was not detected as it was outside of the
measured temperature range. The process window for the glass fabrication is larger
for Co containing glass, and Co is stabilizing the amorphous glass state which
indicated better processing properties of the Co-containing 1393 glasses.
Results and Discussion 119
Fig. 53: DSC curves for the 1393-Co glass series showing decrease in Tg with increasing Co
content in the glass. Reprinted with permission from A. Hoppe at al, ACS Appl. Mater.
Interfaces 6 (2014), p. 2865. Copyright (2014) American Chemical Society.
The effect of Co on 1393 glass structure
The effect of Co incorporation on the glass structure and its properties can be
discussed on the basis of FT-IR as well as Raman spectroscopy and correlated to the
glass thermal behaviour as derived from DSC measurement. Co is known to be an
intermediate oxide and, thus, can enter the silicate network or act as modifying
oxide.64
As the Tg decreases with Co incorporation it is likely that Co is entering the
glass network creating Si-O-Co bonds replacing the stronger Si-O-Si bonds.64
Thus,
the glass network becomes weaker and the Tg decreases.64
This observation is in
agreement with FT-IR results which indicated the formation of additional Si-O-Co
bonds in the glass network as a result of Co substitution. However, according to FT-
IR analysis, Fig. 51, the formation of Si-O-Co bonds is evident only for Co contents
higher than 5wt%. Hence, it can be concluded that Co plays a concentration-
dependent role in the glass network acting as network modifier at 1wt% and
network former at CoO concentrations of 5wt% and higher. This corresponds to
literature reports which also showed the concentration dependent behaviour of CoO
in silicate glasses.64
At 1wt% CoO is likely acting as network modifier replacing
Ca2+
ions, while no significant effect on the structure or thermal behaviour was
observed by FT-IR/Raman spectroscopy as well as DSC analysis. A lower Tg is
Results and Discussion 120
associated with weaker bonds within the glass network and should therefore result
in enhanced in vitro degradation of the glass scaffolds which will be discussed in
3.3.
Fig. 54: Struture of 1393 glass with 1wt% CoO (left) and >5% wt% CoO (right). Depending on
the concentration CoO oxide acts as network modifier replacing Ca2+
ions (left) or as network
former by entering the network and forming Si-O-Co bonds (right).
4.2.2 Scaffold properties
Macrostructure
Fig. 55 shows the macrostructure of the 1393-Co glass derived scaffolds. Scaffolds
were prepared using the 1393, 1393-1Co and 1393-5Co glasses. For the highest Co
content of 10wt% the glass could not be processed to a powder of suitable particle
size and scaffolds, respectively. No major differences between the glass
compositions were observed. All scaffolds show completely interconnected pore
systems with pore diameters of ~ 200-400 µm. The porosity of the scaffolds after a
2nd
coating was 91%, 90% and 89% for 1393, 1393-1Co and 1393-5Co samples,
respectively, confirming that the main macrostructural features are independent of
the glass composition. Fig. 55d-e additionally show representative higher
magnification images of a strut of a 13-93 sample: smooth and dense scaffold
surface was observed and almost fully densified struts were found.
Furthermore, a µCT reconstruction of a 1393 scaffold is given in Fig. 55f
confirming the interconnected pore system of the scaffolds (98% of the total
porosity was confirmed to be open porosity). From microCT analysis a total
Results and Discussion 121
porosity of 92% and average strut thickness of ~74 um were derived. These data are
in good agreement with microscopic analyses and porosity results obtained from
Archimedes measurements.
High porosity values and interconnected pore system of the scaffold should enable
vascularisation and tissue ingrowth when applied as engineered bone construct.
Vascularisation has been shown to be enhanced in scaffolds with pores > 250 µm 278
and also high interconnectivity is considered even a highly important factor for
blood vessel and tissue ingrowth.43, 279
Thus, the 1393-Co derived scaffold meet the
macrostructural requirements for use as bone tissue engineering scaffolds. In Fig.
3d) a 1393 scaffold strut at higher magnification is shown revealing dense struts and
smooth scaffold surface without presence of cracks. This is in agreement with
literature reports which showed that 13-93 bioactive glass can be densified by
viscous flow sintering avoiding crystallization.69
Results and Discussion 122
Fig. 55: SEM images of the scaffold macrostructure for (a) 1393, (b) 1393-1Co and (c) 1393-
5Co glass compositions; (d) and (e) show higher magnification of a strut of a 1393 derived with
dense structure and smooth surface; (f) µCT reconstruction of 1393 derived scaffold. Adapted
with permission from A. Hoppe at al, ACS Appl. Mater. Interfaces 6 (2014), p. 2865. Copyright
(2014) American Chemical Society.
XRD analysis of the scaffolds revealed that the scaffolds remain in the amorphous
state; no crystalline phases were detected (see appendix, Fig. A 3). This is in
agreement with DSC results shown above which revealed crystallisation onset
temperature of Tc, onset > 750 °C being below the sintering temperature thus
precluding crystallisation during the densification process.
Mechanical properties
Fig. 56 shows the compressive strength, σc, values measured for the 1393-Co glass
scaffolds series and typical stress-way curves (Fig. 56b). A catastrophic failure of
the scaffolds was observed at a given maximal stress (marked with * in Fig. 56b)
followed by rapid decrease of the stress. The further increase in the stress is
correlated to compression of remaining scaffolds struts.68
However, the
measurement was stopped shortly after the scaffold failure.
For the 1393-Co derived glass scaffolds with high compressive strength values of
2.3±0.4 MPa, 2.3±0.5 MPa and 4.2±0.6 MPa for 1393, 1393-1Co and 1393-5Co,
respectively, were measured. For 1393-5Co the compressive strength is higher (~4
MPa) compared to 1393 and 1393-1Co scaffolds which is likely due to the
influence of Co on the sintering behaviour of the glass. According to DSC
measurement the Tg of the 1393-5Co glass is reduced hence improving the viscous
flow sintering of the scaffolds and leading to higher strength. However, for
comprehensive evaluation of the mechanical strength the porosity of the scaffolds
has to be taken into account. A detailed discussion of the mechanical performance
of the 1393 derived scaffolds in the context of bone tissue engineering is given in 0.
Results and Discussion 123
Fig. 56: Mechanical properties of the 1393-Co glass derived scaffolds (double coated): a) mean
values calculated from 10 measurements per scaffold group; b) selected typical stress-strain
curved. Adapted with permission from A. Hoppe at al, ACS Appl. Mater. Interfaces 6 (2014), p.
2865. Copyright (2014) American Chemical Society.
Results and Discussion 124
Acellular Bioactivity in SBF
HAp formation
First, the reaction stages occurring on the scaffolds surface during reaction in SBF
are shown exemplary for the plain 13-93 scaffolds and are discussed on the basis of
SEM, FT-IR and micro-PIXE-RBS analysis. Further on, the effect of Co
incorporation in the glass on the hydroxyapatite forming ability and degradation of
the glass scaffolds is presented and discussed based. Fig. 57 shows the SEM images
of 1393 derived scaffolds after immersion in SBF for 1d (a, b), 3d (c, d) and 7d (e,
f), respectively. After 1d first indications of surface reaction were observed on the
scaffold surface visible as a thin reaction layer formed on the scaffolds surface.
However, the inner glass matrix remained intact without any signs of dissolution.
After 3d of immersion the reaction three distinct areas phases can be distinguished
as indicated in Fig. 57d: light grey area showing the inner BG network, a dark grey
layer (SiO2) and thin layer on top of it (CaP). This distinction was made based on
the EDS analysis, as shown in Table 14. The thickness of each layer was estimated
from the SEM figures to 2.4±1.7 µm and 0.61±0.04 um for SiO2 and CaP layers,
respectively.
Table 14: EDS derived elemental concentration (at%) of the reaction phases formed on 1393
scaffolds after 3d in SBF.
Mg K Si P Ca Ca/P
BG 2.79 4.74 20.30 1.34 6.71 5.00
SiO2 0.63 1.23 26.21 1.32 2.02 1.53
CaP 1.21 0.93 14.87 4.87 6.80 1.40
The SiO2 reaction region is quite inhomogeneous and hence the estimation of the
thickness is given with a high standard deviation. In fact, SEM analysis provides
only rough estimations; more reliable data on the dimensions of the reactions layers
is given by PIXE-RBS analysis further below. After 7 days further growth of the
CaP layer occurred: the formation of the typical morphology of hydroxyapatite was
observed. However, again no clearly visible dissolution of the inner region of the
scaffold strut was observed with SEM, Fig 5f.
Results and Discussion 125
Fig. 57: SEM analysis of 1393-Co derived scaffolds after immersion in SBF for 1d (a, b), 3d (c,
d) and 7 d (e, f). Reprinted with permission from A. Hoppe at al, ACS Appl. Mater. Interfaces 6
(2014), p. 2865. Copyright (2014) American Chemical Society.
PIXE-RBS derived chemical maps
In order to identify the origin of the reaction phases formed on the BG scaffolds
concentrations maps derived from ion beam measurements which give a more
precise picture of the reactions taking place at the interface scaffold/fluid. The Ion
beam analysis has been shown to be a powerful technique for identifying the
reactions on bioactive glass / fluid interface.322
In the as-fabricated state P, Si, Ca and Mg and Co (Na and K are not shown for
simplicity) are homogenously distributed (see supplementary data).
Fig. 58 - Fig. 60 show PIXE-RBS derived elemental maps for 13-93 scaffolds after
1d, 3d and 7d, respectively. These results can be directly correlated to the SEM
Results and Discussion 126
observations shown in Fig. 57. Basically, according to the elemental maps again
three distinct regions during reaction in SBF can be defined:
i) Inner region of the glass scaffolds (BG)
ii) Silica-rich layer (SiO2)
iii) Surface periphery (CaP layer)
Additionally, for the regions of interest i) and iii), the inner regions of the glass
scaffolds and the scaffold periphery, respectively, the quantitative elemental
concentration were derived which can be found in the appendix (Fig. A 4 and Fig. A
5).
Interestingly, the inner part of the scaffolds did not show any signs of degradation:
the elemental concentration in the inner region of the scaffolds remained constant
for immersion times up to 7 days for all glasses investigated. Hence, it is likely that
the degradation of 1393 derived scaffolds occurs preferably at the scaffolds
interface with separate reactions stages as discussed below.
After 1 day of immersion in SBF signs if initial surface reaction were observed: a
thin reaction layer of 2.5 ±0.4 µm on was formed the pore surface (Fig. 58) which is
depleted in Ca, P and Mg indicating fast release of these elements from the scaffold
surface.
After 3 days (Fig. 59) a silica rich layer with a thickness of roughly 5.9 ±0.5 µm is
formed on the scaffolds surface. This value is slightly higher compared to the value
of 2.4±1.7 µm observed with SEM (Fig. 57). Considering the high deviation of the
surface thickness as observed with SEM we conclude that a SiO2 layer with a
thickness ranging between 1-6 µm is formed on 1393 derived scaffold after 3 days
of reaction in SBF. On top of it a Ca and P enriched layer (3.1±0.2 µm) was
detected as already indicated by SEM /EDS analysis. This layer results from
diffusion of Ca and P-species from the glass network. It is clearly visible from the
chemical maps that this Si-rich layer acts as diffusion barrier as Ca is accumulated
in the region right before the silica layer.
Results and Discussion 127
Fig. 58: Elemental distribution in the cross-section of a 1393 scaffolds after 1 days. Adapted
with permission from A. Hoppe at al, ACS Appl. Mater. Interfaces 6 (2014), p. 2865. Copyright
(2014) American Chemical Society.
Fig. 59: Elemental distribution in the cross-section of a 1393 scaffolds after 3 days. Adapted
with permission from A. Hoppe at al, ACS Appl. Mater. Interfaces 6 (2014), p. 2865. Copyright
(2014) American Chemical Society.
Results and Discussion 128
Fig. 60: Elemental distribution in the cross-section of a 1393 scaffolds after 7 days. Adapted
with permission from A. Hoppe at al, ACS Appl. Mater. Interfaces 6 (2014), p. 2865. Copyright
(2014) American Chemical Society.
After 7 days (Fig. 60) the glass surface is further dissolving; the Si-rich layer is
expanded to 18.0±1.7 µm. Also the CaP layer continues to grow reaching 16.0±0.2
µm. It is worth noticing that the traces of Mg are incorporated in the CaP layer
which is typical for biomimetic hydroxyapatite formed upon reaction in body
fluids.100, 323
Effect of Co
The effect of Co on the bioactivity of the 1393-1Co and 1393-5Co scaffolds is
shown in Fig. 61 and Fig. 63, respectively. In contrast to reference 1393 glass
scaffold it is noticeable from the chemical maps that for 1393-1Co samples no
distinct CaP layer is formed after 7d in SBF. Even though P enrichment in the
surface is clearly detectable, a rather mixed Si-rich CaP layer with traces of
incorporated Co is observed.
For 1393-5Co scaffolds, however, a clearly distinguishable CaP layer was formed
on top of the SiO2 layer. Interestingly, Co is incorporated into the CaP layer. Co ions
appear to diffuse through the SiO2 layer (which itself is depleted in Co) and are
substituted in the CaP layer.
Results and Discussion 129
One can speculate that the change of the chemistry of the CaP layer will have an
impact on cellular response. For instance, Co incorporated in calcium phosphates
has been shown to increase osteoclast proliferation and overall mineral
resorption.177
Fig. 61: Elemental distribution in the cross-section of a 1393-1Co scaffolds after 7 days.
Reprinted with permission from A. Hoppe at al, ACS Appl. Mater. Interfaces 6 (2014), p. 2865.
Copyright (2014) American Chemical Society.
In order to prove the formation of carbonate hydroxyapatite FT-IR analysis was
performed for the 1393-Co scaffolds after 7 days in SBF as depicted in Fig. 62
The formation of carbonated hydroxyapatite can be monitored by the appearance of
the triply generated (ν3) bending modes of the O-P-O bands of CHA at 554 cm-1
and
Results and Discussion 130
602 cm-1
as detected for 1393 and 1393-1Co scaffolds, respectively.284
However, for
1393-5Co sample a broad band at ~600 cm-1
was observed which is typical for
amorphous CaP.283
Fig. 62: FT-IR spectra of 1393-Co derived scaffolds after 7d of immersion in SBF. At 5wt% Co
inclusion in the glass the crystallisation of CHA seems to be impeded. Adapted with permission
from A. Hoppe at al, ACS Appl. Mater. Interfaces 6 (2014), p. 2865. Copyright (2014) American
Chemical Society.
Based on the SEM/EDS and PIXE-RBS results following scheme can be proposed
for the physico-chemical reactions occurring on the 1393-Co bioactive glass
scaffold surface during immersion in SBF which are comparable to the reactions
described for 45S5-Cu derived scaffolds as shown in 4.1.2. However the kinetics of
the surface reactions also differ.
-1d: initial surface reaction and release of Na, K, Ca and Mg from the scaffold
periphery in an exchange reacting with H+ from the solution, e.g.: according to Eq.
5.13, 286
- 1-3d: Release of soluble Si species upon breakage of Si-O bonds by hydrolysis in
the surface region of the glass and formation of silanol groups triggered by the
reactions Eq. 5 and Eq. 6. In further reaction, surface SiO2 layer is formed upon
recondensation of SiOH groups according to Eq 7.
-7d: Formation of amorphous calcium phosphate (ACP) through migration of Ca2+
and PO43-
groups to the surface through the SiO2-rich layer. Crystallization of the
Results and Discussion 131
ACP into carbonated hydroxyapatite (CHA) film by incorporation of OH-, CO3
2-,
Mg2+
. Also Co2+
ions diffuse through the SiO2 layer and are incorporated into the
CHA.324
In this work, however, too high Co-content (and release) lead to inhibition
of CaP layer crystallization remaining in its amorphous state.
Fig. 63: Elemental distribution in the cross-section of a 1393-5Co scaffolds after 7 days.
Reprinted with permission from A. Hoppe at al, ACS Appl. Mater. Interfaces 6 (2014), p. 2865.
Copyright (2014) American Chemical Society.
Degradation and ion release in SBF
Under static conditions
The Co and Si release from the 1393-Co glasses under static conditions is shown in
Fig. 65. From the 1393-1Co scaffolds 0.6±0.1 ppm Co are released after 1 d of
Results and Discussion 132
immersion. However, during further immersion in SBF only slight increase in Co
was observed with maximum Co levels of 0.8±0.03 ppm after 21d of immersion.
For 1393-5Co scaffold an initial burst release of Co was detected during first 7d
days reaching 8.3±0.7 ppm. After 14 d the Co concentration drops to 5.4±0.07 ppm
and increases again to 7.3±0.6 pm after 21 d in SBF. The drop of the Co levels
might be due to precipitations of Co in insoluble calcium phosphate phases. Also
the saturated state of the SBF solution might lead to reincorporation of Co in the
glass structure or the surface calcium phosphate layer. It is known that
hydroxyapatite can act is cation exchanger an to incorporate metallic ions like Zn,
Cu, Co in it structure.325
Similar trend was observed for Su release showing burst
release in the first 7 days of immersion reaching levels of ~ 50 ppm independent
from the Co content in the glass. The Si concentration remains nearly constant for
further immersion in SBF. A little decrease in Si concentration is observed after 21
days of immersion. However, this is likely due to the static experiment conditions
leading to precipitation of insoluble salts incorporating Si. Overall, under static
conditions a plateau level of Co and Si release is reached after 7 days of immersion
in SBF.
Fig. 64: Co and Si release from 1393-Co glass scaffolds in SBF under static conditions.
Si release can be considered as marker for the glass degradation, so it can be
concluded that the glass degradation is retarded after 7 days due to the saturation of
Results and Discussion 133
the solution in Si. Also, according to FT-IR, SEM and PIXE-RBS data, as discussed
in 4.2.2 , after 7 days a hydroxyapatite layer is formed on the 1393 scaffold surface
which is acting as diffusion barrier. Additionally, the saturation of the solution
decreases the diffusion rate of Si due to lower concentration gradient.
Under quasi-dynamic condition
Si and Co concentrations released from the 1393-Co derived scaffolds under quasi-
dynamic conditions (SBF solutions was changed frequently) are sown in Fig. 65.
Generally, higher Co and Si concentrations were released from 1391-Co derived
scaffolds under quasi-dynamic conditions compared to static conditions. Also the
kinetics of the ion release differ which are described in the following:
The global degradation of the scaffolds can be tracked using the Si release being the
main marker of the glass network dissolution. Two different regions can be
distinguished in the Si release profile of the 1393 scaffolds immersion under quasi-
dynamic conditions: i) initial release of high Si levels reaching a peak during the
first three days of the reaction and ii) drop of Si release to lower release rates. For
1393 glass this initial peak is reached after 3 days, Fig. 65, with maximal Si levels
of 11.3±1.7 ppm and further decrease of absolute Si levels to ~0-1 ppm. The drop of
the Si release rates corresponds to the formation of a CaP layer that acts as diffusion
barrier leading to decreased Si release. Similarly, for the 1393-5Co scaffolds
peaking Si values were observed after 3 days. However, the Si concentration of 18.8
±2.2 ppm was significantly higher compared to 1393 glass which can be explained
with the weaker glass structure caused through Co substitution as discussed in 4.2.1.
For the 1393-1Co samples, in turn, the Si peak is delayed, occurring after 14 days of
immersion in SBF. Maximal absolute Si concentrations of 18.3±1.0 ppm were
released after 14d in SBF.
The exceptional behaviour of the 1393-1Co glass scaffolds might be explained with
with PIXE-RBS results which showed that in contrast to 1393 and 1393-5Co 1393-
1Co scaffolds did not show the formation of a distinct CaP layer until the longest
time period investigated (7 days in SBF). Hence, the passivating CaP layer is likely
to form at a later stage thus retarding the Si release peak from the 1393-1Co
samples.
Results and Discussion 134
Co is continuously released from the 1393-1Co and 1393-5Co scaffolds reaching
maximal cumulative values of 1.73±0.03 ppm and 11.4±0.2 ppm, respectively.
Fig. 65: Co and Si release from 1393-Co glass scaffolds in SBF under quasi-dynamic
conditions. Adapted with permission from A. Hoppe at al, ACS Appl. Mater. Interfaces 6
(2014), p. 2865. Copyright (2014) American Chemical Society.
Regarding the absolute values released after each time point of measurement the
highest Co releasing rated occurs in the first 3d of immersion. It is most evident for
the 1393-5Co samples which showed a Co peak of ~4 ppm after 3d of immersion in
SBF. After 7 days of immersion times the Co concentration dropped to ~1 ppm
remaining at this level for further immersion until 21 days. This is likely due to the
formation of a CaP layer on the scaffold surface after 3d as it was observed from
SEM and PIXE-RBS analysis. This layer likely acts as a diffusion barrier leading to
the drop of Co released into SBF.
After 21d in SBF the change in the scaffold mass was +4.3±0.6 %, -18.7±0.6 % and
-39.6±0.6 for 1393, 1393-1Co and 1393-5Co scaffolds, respectively. The slight
mass gain in the 1393 samples is likely due to the formation of the calcium
Results and Discussion 135
phosphate layer on the scaffold surface, while at the same time the 1393 scaffold
showed the lowest degradation rates as shown by to ICP OES measurements. After
7d of immersion in SBF the Si release dropped to values close to 0 ppm due the
formation of CaP layer acting as a diffusion barrier. Accordingly, the cumulative
values of Si released in SBF quickly reached an almost constant plateau at ~30
ppm. Hence, it can be assumed that the dissolution of 1393 derived scaffolds was
nearly completely impeded after 7d of reaction in SBF. In contrast, for both cobalt
containing scaffolds1393-1Co and 1393-5Co a continuous release of Si was
detected over a reaction time range of 21d. Even though for these samples also a
drop in absolute Si values released was observed the Si release rates remained at
significant levels of ~1.5-3 ppm/d. Thus, the mass loss of the 1393-1Co and 1393-
5Co is assigned to significantly higher Si release and degradation rates of these
scaffolds. After the quasi-dynamic degradation study the scaffolds were digested
and the fractions of the remaining oxides were analysed. The relative concentrations
of the elements are given in Table 15
Table 15 normalised to the SiO2 content. The relative glass compositions after the
degradations study reflects the dissolution behaviour of the scaffolds.
Table 15: Oxide contents normalized to silica in the glass remaining after the degradation
study.
Glass CaO P2O5 NaO MgO CoO
1393 0.45±0.02 0.51±0.06 0.14±0.00 0.05±0.02 -
1393-1Co 0.37±0.03 0.44±0.04 0.14±0.02 0.04±0.02 0.02±0.00
1393-5Co 0.08±0.02 0.08±0.02 0.06±0.01 0.02±0.01 0.03±0.01
For Co-containing samples higher loss in Ca and P is observed compared to 1393
glass which corresponds to higher Si release rates in SBF. Accordingly, Na and Mg
loss is highest for 1393-5Co scaffolds. For Co-containing samples a residual
amount of CoO of 2 wt% and 3 wt% were observed for 1393-1Co and 1393-5Co
samples, respectively.
The results on the degradation behaviour of the 1393-Co glass indicate that the
1393 glass matrix is suitable for controlled release of Co ions in physiological
Results and Discussion 136
environment. For example, Azevedo et al.64
reported Co values of ~13 ppm released
from melt-derived glasses in TRIS buffer after 21 days without refreshing the
solution. Also, Wu et al.219
have shown controlled release of maximum Co levels of
~ 20 ppm from Co containing sol-gel derived scaffolds. Since too high Co
concentrations can be toxic a controlled release mechanism is essential for
applications of such Co-releasing constructs. The results presented here showed that
13-93 glass derived scaffolds can be used for controlled release of Co2+
ions with
release rates (after the initial burst release) of 0.3 ppm/d and 0.1 ppm/d depending
on the glass composition.
The Co concentrations observed in this work are comparable to the values reported
to be within therapeutically active range. For example 50 µM, 100 µM, 200 µM of
CoCl2, which correspond to 12 ppm, 24 ppm and 48 ppm, respectively, stimulated
migration, proliferation, and tubule-like structure formation of umbilical cord
blood-derived CD133(+) cells, hence indicating angiogenic potential of Co inducing
hypoxic conditions.326
Others confirmed treating human microvascular endothelial
cells (HMEC-1) with 12 ppm CoCl2 resulted in binding of HIF-1 hence mediating
transcriptional responses to hypoxia.327
However, Co levels higher than 10 ppm
have been indicated to be cytotoxic as treatment of osteoblast-like cells with 10
ppm of Co2+
resulted in 40% decrease in cell number.328
Similarly, Wu et al.219
observed that Co2+
concentration of ~20 ppm reduced the viability of bone marrow
derived stem cells (BMSCs) even though they did not cause significant cytotoxicity.
Hence, the release of ~2 ppm from 1393-1Co may be considered non-critical
regarding potential cytotoxicity whereas ~12 ppm released from 1393-5Co might be
at the upper edge of the therapeutic, non-toxic range.
Altogether, the addition of Co into the 1393 glass network results in the weakening
of the glass structure leading to faster dissolution of the glass (under quasi-dynamic
conditions). 1393 bioactive glass composition is considered less reactive than 45S5
BG due its high SiO2 content. Hence, through adding CoO in to the glass network it
is possible to enhance the reactivity and the degradation rate of 1393 glass leading
to improved bioactivity. So, degradation of the scaffolds can be adjusted by
tailoring the composition of the glass hence enabling controlled release of ionic
species. CoO acts as network modifier and network former depending on its
Results and Discussion 137
concentration in the glass. That way, the 1393 glass matrix might be used as carrier
for controlled release of other therapeutic ions like ZnO or MgO also known to act
as intermediate oxides in silicate glasses.329
4.2.3 In vitro cell response
The potential cytotoxicity of Co ions released from metal alloys used as implant
materials in hip replacement therapies is known problem in the current medicine
which is related to systemic intoxication of the body through long term Co (and Cr)
release as results of wear debris and corrosion.330-332
However, as mentioned in
2.5.1 Co ions are also known to enhance angiogenesis via inducing hypoxic
conditions. Using 13-93 glass derived scaffolds it is possible to control the Co
release which can be adjusted within a therapeutic range. In order to test this
hypothesis n this section the biocompatibility of the 1393-Co glass powders and
glass derived scaffolds is presented and discussed. Firstly, powder cytotoxicity of
the particulate BGs is assessed in direct contact with MG-63 cells. Secondly, the
biocompatibility of the 1393-Co scaffolds is evaluated regarding the response of
MG-63 cells directly seeded on the scaffolds and of hDMECs exposed to ionic
dissolution products of the 1393-Co derived scaffolds.
Powder cytotoxicity
The cell morphology of the osteoblast-like cells after cultivation in direct contact
with 1393-Co glass particles for 48h is shown in Fig. 66. No signs of cytotoxicity
can be observed for 100 mg ml-1
particle concentrations: the cells exhibit slightly
elongated triangle shaped morphology typically found for MG-63 cells. However,
for 1000 µg ml-1
fewer cells seem to be present even though the cell number is
difficult to observe as at 1000 µg ml-1
the BG particles cover a large area of the well
plate.
Results and Discussion 138
Fig. 66: Light microscopy images of the MG-63 morphology cultured with 1393-Co glass
particles for 48h.
Fig. 67 shows the mitochondrial activity and cell number of osteoblast-like cells
derived from WST and LDH assays, respectively. For 1393 reference glass the
mitochondrial activity of the cells remains nearly constant for 0.1 and 10 µg ml-1
but is increased for concentrations of 10 and 100 µg ml-1
, while the cell number
stays constant for 0.1-1000 µg ml-1
range.
Compared to the plain 1393 reference, 1393-1Co shows statically significant higher
mitochondrial activity of MG-63 observed in the range from 0.1 µg ml-1
to 100 µg
ml-1
. The increase in the cell activity is statistically even higher (p<0.01) when
compared to 1393 reference. At the same time the cell number remains constant for
0.1 – 10 µg ml-1
whereas for higher particle concentrations a slightly reduced cell
number was observed. This indicated that even though the cell number is slightly
reduced or remains constant the cells show a higher mitochondrial activity detected
by the WST assay stimulated by the 1393-1Co glass particles.
For 1393-5Co glass particles, however, a clear reduction in cell activity and cell
number was observed for particle concentrations > 10 µg ml-1
indicating potential
cytotoxic effect of 1393-5Co glass. Altogether the 1393 glass particle at
concentrations in the range of 0.1-100 µg ml-1
showed good biocompatibility with
osteoblast-like cells with enhanced cell activity with 1393-1Co particulate glass and
indication of cytotoxicity of 1393-5Co glass.
Results and Discussion 139
Fig. 67: Mitochondrial activity (WST assay) and cell number (LDH assay) of osteoblast-like
cells seeded for 48 h in direct contact with particulate 1393-Co glasses. *p<0.05; **p<0.01
compared to 0 µg/ml reference.
Scaffolds
Fig. 68 shows the mitochondrial activity of MG-63 cells seeded directly on the
scaffolds. 1393-1Co did not show any cytotoxic effects. In fact after an initial drop
after 7d the cell activity was significantly increased for 1393-1Co samples
compared to 1393 reference. However, 1393-5Co samples show signs of
cytotoxicity from the beginning: after 3 days the cell activity dropped to 20% of the
reference value and further decreased to ~5% after 7d and 14d indicating that
almost no living cells were grown on the scaffolds.
Fig. 69 shows osteoblast-like cells grown on 1393-Co derived scaffolds after 14d
observed with SEM and confocal laser scan microscopy. For 1393 and 1393-1Co
god cell adhesion and cell spreading was observed. The cells fully cover the
scaffold strut and grow following the pore curvature. It has been shown in literature
that cell prefer the growth along the inner side of the pores in case of large pores.333
However, on the 1393-5Co scaffolds no spread cells were observed indicating
cytotoxic effects of the1393-5Co glass scaffold. These observations were confirmed
through WST measurements, as shown in Fig. 68.
Results and Discussion 140
Fig. 68: Mitochondrial activity of osteoblast-like cells seeded on 1393-Co scaffolds for 3d, 7d,
and 14d.
The toxic effect of 1393-5Co is likely due to the high concentration of Co ions
releases. Wu et al.219
for instance showed that 22 ppm caused a reduction of cell
vitality of human stem cells after culturing on Co containing BG scaffolds for 1
week. Even though in this study no significant cytotoxicity was observed, high Co
concentration seems to have a negative effect on the vitality of stem cells. Further in
the study of Wu et al.219
only short term effects were considered for a culturing time
of 1 week. Indeed it has been shown in literature that the potential cytotoxicity of
Co is not only dose but also time dependent.328
This might explain the cytotoxicity
of the 1393-5Co scaffolds even though the 12 ppm Co released are within non-toxic
ranges reported for Co ions.328
The cytotoxic effect of Co on MG-63 cells may be
related to oxidative stress caused by Co2+
ions leading to oxidation of proteins.328,
334
Regarding the response of MG-63 cells it can be summarised that 1393 and 1393-
1Co scaffolds show no signs of cytotoxicity when applied as powder and also as 3D
scaffolds. Furthermore, 1393-1Co scaffolds show slight stimulating effects on
mitochondrial activity of MG-63 cells when in contact with particulate glass at
concentrations from 0.1-100 µg ml-1
and seeded on the scaffolds. 1393-5Co glass
powder and corresponding scaffolds, however, show signs of cytotoxicity.
Results and Discussion 141
Fig. 69: SEM images (left) and confocal laser scan images (right) of Vybrant live staining (red)
of MG-63 cells cultured for 14d on 1393 (a-b), 1393-1Co (c, d) and 1393-5Co scaffolds.
Response of hDMECs
Fig. 70 shows the light microscopy images of hDMECs culture for 2 weeks exposed
to the ionic dissolution products from 1393-Co series. The 1393 control shows and
1393-1Co scaffolds show good biocompatibility: the cell morphology is retained
while for 1393-1Co indications of tube-like formation of the cells are visible. 1393-
5Co scaffolds, however, seem to be toxic. The morphology of the hDMECs was
impaired when seeded in contact with 1393-5Co scaffolds. These observations were
confirmed through the cell number analysis: the cell number increased continuously
for the hDMECs reference from initial number of 100.000 cells to 160.000 after
1week and 250.00 after 2 weeks of culture. For 1393 samples the cell number
Results and Discussion 142
increased to comparable values of 150.000 cells after 1 week and subsequent slight
reduce to 140.000 cells after 2 weeks.
Fig. 70: LM images of hDMECs when seeded for 2 weeks in contact with dissolution products
from 1393, 1393-1Co and 1393-5Co scaffold. hDMECs only served as control.
For 1393-1Co a slight reduction of the cell number from 100.000 to 90.000 and
50.000 after 1 week and 2 weeks, respectively, was observed. For 1393-5Co,
however, already after 1 week no living cells could be detected. These results were
confirmed by LDL uptake analysis which showed high LDL uptake of the hDEMCs
reference, 1393 and 1393-1Co while no LDL uptake was observed for 1393-5Co
samples, Fig. 71. Furthermore, FACS analysis revealed pronounced expression of
vWF, CD31 and VEGFR endothelial cell markers in hDMECS control and 1393
and 1393-1Co samples confirming high functionality and retained endothelial
phenotype of the hDMECs, Fig. 72.
Results and Discussion 143
Altogether, 1393 and 1393-1Co scaffolds show good biocompatibitly towards
endothelial cells while 1393-5Co is cytotoxic. As dissolution products from the
scaffolds were investigated in an indirect cell culture set up it can be concluded that
the release of 2 ppm Co from 1393-1Co samples is within compatible range for
endothelial cells. However, the release of ~ 10 ppm Co2+
from 1393-5Co scaffold
caused significant cytotoxicity.
Fig. 71: Florescence images of the LDL uptake of the hDMECs reference (cells only) and in
presence of 1393-1Co and 1393-5Co scaffolds.
In conclusion of 1393-1Co glass and respective scaffolds are not toxic to the
HDMSCs which show high LDL uptake. Also after culturing with 1393-1Co
hDMSCs have been shown to be positive for CD31, vWF and VEGFR-2
confirming the vital phenotype of endothelial cells.
Results and Discussion 144
Fig. 72: FACS analysis of endothelial markers expressed in hDMECs cells after 1 week of
culture in presence of 1393-Co scaffolds.
Altogether, good cytocompatibility was confirmed for 1393 and 1393-1Co scaffold
regarding the response of osteoblast-like cells as well as endothelial cells. However,
1393-5Co seems to have a cytotoxic effect on osteoblast-like cells as well as
hDMECs and thus might be not suitable for tissue engineering applications.
Obviously, the release of 12 ppm exceeds the therapeutic range of Co2+
ions
becoming toxic to the cells.
However, 1393-1Co glass derived scaffolds might be potential candidates for use as
hypoxia mimicking biomaterials for bone tissue engineering applications even
though a significant stimulating effect on endothelial cells was not confirmed in this
study. Also, there is convincing evidence in literature reporting stimulating effects
of Co on angiogenesis and bone tissue regeneration processes.179, 200, 326
The
stimulating effect of Co on angiogenesis, however, is suggested to be tested in a co-
culture of ECs and MSCs.
Results and Discussion 145
4.3 Evaluation of the compressive strength of the 45S5 and
1393 scaffolds in the context of bone TE
In order to evaluate the mechanical properties of the bioactive glass scaffolds the
influence of the porosity has to be considered. The theoretical strength σtheo of
cellular ceramics with open cells can be estimated by the model of Gibson and
Ashby:335
𝜎𝑡ℎ𝑒𝑜
𝜎𝑓𝑠= 𝐶 (
𝜌𝑓𝑜𝑎𝑚
𝜌𝑠𝑜𝑙𝑖𝑑)
3
2∙1+(
𝑡𝑖𝑡)2
√1−(𝑡𝑖𝑡)2 Eq. 8
= 𝐶(1 − 𝑃)3/2 ∙1+(𝑡𝑖/𝑡)
2
√1−(𝑡𝑖/𝑡)2 Eq. 9
where σfs, is the modulus of rupture of the struts of the foam, C is constant of
proportionality (for brittle ceramics it C=0.2), ρfoam is density of the foam, ρsolid is
the density of the solid ceramic, P is the total porosity of the foam and ti/t is the
ratio of the central void size of the strut to the strut diameter.
From theoretical calculations it can be assumed that for brittle materials σfs =1.1σts
with σts being the tensile strength of the bulk material. With σts of 42 MPa for
annealed 45S5 Bioglass®
and ti/t ratio of 0.5 (derived from Fig. 20) the theoretical
values for compressive strength of 45S5 BG derived scaffolds were calculated as
depicted in Fig. 73. For comparison also literature values for standard HAp bio
ceramic derived scaffolds by foam replica technique are given.
Considering the effect of porosity the values obtained for 45S5 derived scaffolds are
above the values reported for HAp scaffolds also fabricated with foam replica
method. Hence, 45S5 derived scaffolds exhibit comparable compressive strength as
porous scaffolds based on the standard HAp bioactive ceramic. In fact, in order to
match the lower limits of the compressive strength of cancellous bone (2MPa) with
of 45S5 derived scaffolds porosity values < 71% would be required. This, however,
might not be sufficient to meet the requirements for bone tissue engineering
scaffolds regarding porosity needed for cell and blood vessel ingrowth.
Results and Discussion 146
Fig. 73: Theoretical values for the compressive strength of open-cell 45S5 BG derived scaffolds
and the experimental values obtained in this work after for multiple coated scaffolds. For
comparison reported values for HAp foam made by replica technique were taken from
literature.336, 337
In other studies it has been reported that higher values for σc for bioactive glass
derived scaffolds can be achieved by modifying the regime of the foam replica
technique resulting in σc values up to 3 MPa.338, 339
However, in those studies the
porosity of the scaffolds were ≤ 70% which is not comparable to porosity levels of
the scaffolds fabricated in this work (~90 %).
Despite the limitations of relatively low mechanical strength of Bioglass®-derived
scaffolds fabricated by the foam replica method, this material remains an important
system in the context of bone tissue engineering due to its high bioactivity and
ability to actively stimulate cells towards osteogenic differentiation via up-
regulation of genes expression which results in enhanced bone regeneration.129
Furthermore, according to literature reports one can assume that in vivo culturing
will increase the mechanical strength and toughness of such scaffolds due to tissue
ingrowth and HAp formation on their surfaces thus leading to formation of a
“biocomposite” as for example shown for hydroxyapatite scaffolds.340
In order to overcome the drawback of limited mechanical stability of 45S5 scaffolds
1393 bioactive glass composition was alternatively investigated in this work. As
shown in the results section (4.2.2) for 13-93 derived scaffolds compressive
strength values of >2 MPa were achieved. The modulus of rapture of a strut is
Results and Discussion 147
defined as the maximums stress at failure of the cell wall material. Given the
average strut thickness of ~ 100 µm the tensile strength of 1393 fibers of 440 MPa
(diameter of 93-160 µm) was assumed for σfs.86
According to Fig. 55 the ratio ti/t
was estimated to be between 0 and 0.5. Fig. 74 shows the theoretical strength and
experimental values observed in this work for 1393-Co glass derived scaffolds. Also
compressive strength values reported by Fu et al. for 1393 glass derived scaffolds
made by foam replica technique are given for comparison.69
The experimental
values observed in this work follow the curve for the compressive strength as
predicted by Eq. 9. It is worth noticing that by changing the foam replica regime the
compressive strength of the scaffolds can be enhanced while high porosity of 89-
91% is maintained.
Fig. 74: Theoretical values for the compressive strength of open-cell 1393 derived scaffolds and
the experimental values obtained in this work. Scaffolds with porosities lower than 92% were
made by double-coating. Grey shadowed area marks the region of human spongy bone. For
comparison literature values reported by Fu et al.69
are given. Adapted with permission from
A. Hoppe at al, ACS Appl. Mater. Interfaces 6 (2014), p. 2865. Copyright (2014) American
Chemical Society.
Taking the differences in porosity into account the compressive strength of the
present scaffolds are comparable with values reported by Fu et al.69
for 1393
derived scaffolds even though those values are slightly above theoretical strength.
This might be due to the fact that for theoretical calculation of the strength a
Results and Discussion 148
constant modulus of rupture is assumed; in praxis, however, the rapture modulus is
not a constant but varies following the Weibull distribution.335
Also the strength
values reported for dense 1393 material (which is taken for calculations of the
porous foams) are given in literature with a high standard deviation (440±120 MPa)
which allows only rough estimation. Altogether, the σc values obtained for the 1393
derived scaffolds correspond to the lower limits of human spongy bone which could
enable their use for regeneration of moderate load-bearing bone defects.38
4.4 Degradation behaviour of 45S5 and 1393 glass
scaffolds and their suitability as carrier for therapeutic
metal ions
The detailed surface reactions of 45S5 and 1393 BG derived scaffolds in SBF are
described in sections 4.1.2 and 4.2.2, respectively. Despite the different kinetics of
these surface physico-chemical reactions no major differences were observed
between these two glass systems in their overall bioactive character. The surface
reactions of a bioactive glass surface are schematically given in Fig. 75. Basically,
the surface reactions follow the scheme as proposed by Hench13
with the formation
of a SiO2 layer, the precipitation of an amorphous calcium phosphate and
crystallisation of carbonated hydroxyapatite being the main steps, Fig. 75. Hereby,
one important finding concerning the metal ion containing glasses was that metal
ions released from the glass network are incorporated in the carbonated
hydroxyapatite layer on the bioactive glass surface. This model is valid for the
surface reactions of both 45S5 and 1393 BG surfaces. Despite some minor
differences the reaction scheme showed in Fig. 75 is identical for both glass systems
describing the local physical-chemical reactions at the BG scaffold/fluid interface.
Results and Discussion 149
Fig. 75: Scheme summarising the surface reactions on a BG derived scaffolds based on the
experimental results given in 4.1.2 and 4.2.2 (from left to right): formation of silanol groups
through protonation of free Si-NBOs and breakage of Si-O-Si by hydrolysis; formation of a
silica layer upon condensation of the silanols and formation of amorphous calcium phosphate,
ACP (or rather mixed layer of SiO-xCaP); crystallisation of ACP to carbonated hydroxyapatite
(CHA) which is enriched in metallic ions Me2+
, e.g. Cu2+
or Co2+
.
However, the global degradation process of these two BG systems seems to differ.
For 45S5 BG derived scaffolds the surface reaction and the degradation is
accompanied by release of soluble silica occurs not only from the surface area but
also from the inner region of the glass network. Clearly, dissolution signs in the
inner region of 45S5 derived scaffolds were observed by SEM. Also micro-PIXE-
RBS measurements clearly indicated the chemical changes in the inner regions of
the scaffolds during immersion in SBF. Furthermore, despite high levels of Si
released, no significant mass loss of the scaffolds was observed for 45S5-Cu series
which indicates that significant amount of silica was released from the inner regions
and the mass loss caused by the Si release was compensated with the mass gain of
the CaP layer. The mechanism for the global degradation of the 45S5 BG scaffold is
shown in Fig. 76.
Results and Discussion 150
Fig. 76: Reaction scheme of a 45S5 BG derived scaffold during immersion in SBF: a) initial
strut of the scaffold; b) first surface reaction and leaching of ions in the scaffold periphery; c)
formation of SiO2 layer; d) formation of CaP layer on the scaffold surface and dissolution of
the glass from the inner region; e) CaP continues to growth and the scaffold further dissolves
from inner region.
Tilloca summarised that the 45S5 bioactive glass network can be easily penetrated
by water molecules in the inner region which is due high fragmentation of the
network and the high hydrophilicity of Ca and Na cations which results in high H2O
affinity.287
This allows easy penetration and migration of H2O molecules without
the need of breakage of silanol groups that bears high amounts of energy. These
effects cause the high reactivity of 45S5 scaffolds.
For 1393 derived scaffolds, in turn, no dissolution signs of the inner region of the
scaffolds were observed. The loss of soluble silica occurs solely in the surface
region of the glass. Also with addition of Co to the 1393 matrix the glass
degradation was enhanced which was monitored by significant mass loss and high
Si release rates. The scheme of the 1393 derived scaffolds is shown in Fig. 77. In
contrast to 45S5 the degradation takes place from the surface of the scaffolds with
corresponding weight loss, while the inner region remains intact. This can be
explained with the network connectivity of the BG:287
45S5 has lower amount of
SiO2 hence, lower network connectivity which enables the migration of water
molecules deep in the glass network. 1393 glass, on the other hand, has a higher
amount of SiO2 (53 wt%). And hence higher network connectivity. Thus, the
migration of the eater molecules reaches only the periphery region. And the
dissolution takes place from the surface region of the scaffold. The reactivity of
BGs with higher network connectivity (which is the case for the 1393 BG with
151
higher SiO2 content) is reduced since the hydrolysis of silicate units with three or
more bridging oxygens (BOs) bears an excessive amount of energy costs.76
Fig. 77: Reaction scheme of a 1393 BG derived scaffold during immersion in SBF: a) initial
strut of the scaffold; b) first surface reaction and leaching of ions in the scaffold periphery; c)
formation of SiO2 layer; d) formation of CaP layer on the scaffold surface; e) the scaffold
degrades through material loss via dissolution of the surface region.
These are interesting findings as the degradation behaviour dictates the suitability of
the glass network to be used as carrier for therapeutic ions and as a biomaterial in
clinical application. From the finding in this work it can be stated that 45S5 derived
scaffolds remain in its original shape and the degradation occurs in the inner part of
the scaffolds via leaching of cations and soluble Si species. In praxis this means that
45S5 derived material will be integrated in the newly forming bone tissue being
converted to calcium phosphate phase while a core of SiO2 network will remain. In
contrast, 1393 BG derived scaffolds with enhanced degradation achieved through
modification with CoO, for instance, would be resorbed and metabolised by the
body within few weeks.
Summary 152
5 Summary
In this work novel Cu and Co containing bioactive glass compositions based on
45S5 (45S5-Cu) and 1393 BG (1393-Co) have been successfully synthesized and
characterised in terms of glass structure and glass properties. Furthermore, 3D
porous scaffolds have been fabricated using the foam replica technique and their
porosity, acellular bioactivity in simulated body fluid (SBF), degradation and ion
release kinetics as well as the biocompatibility in vitro and in vivo was assessed.
Cu was successfully incorporated as Cu2+
acting as network modifier in 45S5
bioactive glass network which resulted in a decrease of glass transition and lower
melting onset temperature, hence enlarging the process window for this glass.
Highly porous 45S5S-Cu scaffolds with interconnected pore system and total
porosity of ~90 % were fabricated by foam replica technique. Compressive strength
values of ~0.2 MPa were tested which are relatively low but sufficient for the
handling of the scaffold in tissue engineering applications. High acellular in vitro
bioreactivity of the Cu-containing scaffolds was confirmed through SBF test
showing rapid formation of carbonated hydroxyapatite (CHA) layer on the scaffold
surface after 3d without any negative impact of Cu doping on the bioactivity of
45S5 BG scaffolds. Further, detailed micro-PIXE-RBS analysis revealed that traces
of Cu were incorporated in the CHA layer. Degradation studies in SBF showed that
Cu levels from 0.3 to 4.5 ppm are released from 45S5-Cu scaffolds depending on
culturing conditions which are within the therapeutic ranges reported for Cu ions
(see 4.1.4). Cell culture assays confirmed the high compatibility of 45S5-Cu
particulate glasses and corresponding scaffolds with MG-63 osteoblast-like cells,
human bone marrow derived stem cells (hBMSCs) as well as human dermal micro
vascular endothelial cells (hDMECs). Furthermore, a significantly enhanced VEGF
(vascular-endothelial growth factor) expression was found in hBMSCs induced by
Cu ions released from the scaffold likely due to the activation of the HIF-1
transcriptional factor. In a co-culture study of hDMECs and hMSCs 1 wt% Cu
containing 45S5 scaffolds were shown to enhance the expression of endothelial cell
specific markers vFW and VEGFR and to stimulate hDMECs towards formation of
prevascular tube-like structure which is an indication of the overall angiogenic
Summary 153
potential of Cu. The AV-loop in vivo model in the rat showed that 45S5 derived
scaffolds support intrinsic vascularisation through micro vessel sprouting from the
initial AV loop. Qualitatively, 45S5-Cu derived scaffolds showed higher blood
vessel density and higher number of vessels than plain 45S5-derived scaffolds even
though the effect of Cu was not statistically significant.
Complementary to the 45S5 BG, a 1393 BG composition containing CoO was
fabricated. CoO was shown to act in a concentrations depending manner in the glass
network acting as network modifier for concentration of 1 wt% and entering the
glass network by forming Si-O-Co bonds for concentrations ≥5 wt% Co. Inclusion
of Co weakened the glass network due to replacing of the Si-O bonds by weaker Si-
O-Co bonds as indicated by decreased glass transition temperature with increasing
CoO content in the glass. Using the foam replica technique also highly porous
scaffolds with porosities of 89-92 % depending on the foam replica regime were
achieved. 1393-Co glass derived scaffolds revealed relatively high compressive
strength values of > 2MPa, which correspond to the lower boundaries of strength
values of human cancellous bone. Further, acellular in vitro studies revealed rapid
transformation of the 1393 scaffolds surface to CHA after 7d in SBF indicating high
bioactivity even though it is retarded compared to 45S5 derived scaffolds. Similar
to observations with 45S5-Cu derived scaffolds, Co is incorporated in the surface
calcium phosphate layer, as shown from micro-PIXE-RBS analysis. Moreover,
when applied at high concentrations (≥ 5wt% CoO in the glass) Co ions seem to
inhibit the crystallisation of the calcium phosphate surface layer remaining in the
amorphous state. Degradation studies in SBF revealed that Co ions in the range 0.6-
11.4 ppm are released depending on the glass composition and culturing conditions
which is in the therapeutic range (see 4.2.3). In vitro cell assays showed that 1393
and 1393 with 1 wt% CoO glass derived scaffold exhibit high cell compatibly with
a tendency of 1393-1Co glass to stimulate MG-63 cells and endothelial cells.
However, 1393-5Co was shown to be cytotoxic with Co2+
concentration of ~11 ppm
seemingly exceeding the physiologically vital level. Overall, the results of this
research project showed that 45S5 and 13-93 bioactive glass derived scaffolds
represent a new promising family of scaffold for bone regeneration which serve as
inorganic carriers for controlled release of therapeutic metal ions.
Conclusion and Outlook 154
6 Conclusion and Outlook
Applications of metal ion doped glasses in bone tissue engineering and beyond
The aim of this work was to produce robust inorganic scaffolds based on bioactive
glasses with the capability of controlled ion release to enhance bone formation and
angiogenesis. Bioactive glass systems of 45S5 and 1393, two well-known silicate
compositions, were shown to be suitable inorganic materials for incorporation of
biologically active metallic ions, e.g. Cu and Co. These metal ion containing glasses
are suitable carrier for the controlled release of such therapeutic metal ions because
the final ions concentration and release kinetics can be tailored by the glass
composition. Due to high flexibility of the glass melting process a wide variety of
different bioactive metallic ions and other bioinorganics can be incorporated
underlining the high potential of silicate bioactive glasses in the field of
biomaterials and regenerative medicine. Incorporation of osteogenic and angiogenic
agents for example is a promising strategy to enhance the impact of bioactive
glasses for bone tissue engineering applications and to enhance bone regeneration.
Various novel applications of ions for specific applications are possible. Osteogenic
agents like Li or Ga which have not been extensively investigated so far are
promising candidates to be substituted in a BG matrix. Also antibacterial agents
like, Zn, Ga or Ag can be incorporated in glass matrix creating multifunctional
bioactive glasses. Since inorganic therapeutics show many advantages160
this might
have a huge impact on the design and applications of novel osteogenic and
angiogenic materials. Inorganic therapeutics may be considered cheaper and safer
alternatives compared gene therapies or applications of growth factors which have
some drawbacks, such as uncontrolled release, degradation and tendency to diffuse
from the inflammatory site the as well as formation of malign tissue.341
Hence, the
use of therapeutic inorganics might give a cost-effective and safe solution for
enhancing the biological impact of biomaterials in regenerative medicine. Also
related to processing bioinorganics are advantageous since they can be processed at
high temperature using classic technologies of ceramic processing.
Conclusion and Outlook 155
Besides BTE, various other potential fields of medicine can be explored by using
novel metal ion containing bioactive glasses including nerve guidance conducts342,
343 and cancer treatment.
344-349 Ferromagnetic BG particles, for example, can be
used for hyperthermia cancer treatment.350
Hereby, BGs also provide
osteoconductive properties and hence can combine cancer treatment and bone
regeneration in one procedure. Also as guided drug-release systems or in tissue
engineering magnetic F2O3 containing BG particles can be additionally
functionalised with drugs and then specifically targeted to the site of action.
Furthermore, magnetic BG particles can be used as biocompatible targets (as
alternative to potentially toxic iron oxide particles) for placing inside cells for
guiding the cells into particulate shapes and geometries, e.g. assembling endothelial
cells in a 3D vessel-like structure for engineering of vascularised tissue constructs.
Furthermore, ZnO2 and CeO2 which are known to be involved in peripheral nerve
regeneration351, 352
and as neuroprotective agent353
, respectively, can be proposed as
therapeutic agents to be released from BGs used in nerve guidance constructs.
Degradation and reactivity of BG scaffolds in biological fluids
As presented in this work, the kinetics of the ion release from both 45S5 and 1393
show a burst release in the first 7 days of degradation in SBF, followed by further
continuous release over a period of 21d. This profile comes close to a “drug
delivery system” where an initial therapeutic effect is desired through a high “drug”
release in the beginning followed by a continuous release in the so-called
therapeutic zone in the 2-3 weeks following the (bone) defect treatment or
regeneration. Hence, 45S5 and 1393 BGs can be considered clinically relevant
“drug-release” carriers. However, the ion release capability in vivo may be different
to the in vitro situation and, hence, such in vivo studies remain a task for future
investigation. Indeed, there is a clinical need for such carriers of inorganic
therapeutics.
Also the degradation behaviour in SBF of the 45S5 and 1393 glass scaffolds was
assessed in detail and the results were compared revealing major differences in the
degradation mechanisms of these two silicate glass systems: while 45S5 scaffolds
degrade via leaching of cations and release of soluble silica from the inner regions
Conclusion and Outlook 156
of the scaffolds, with the mass loss compensated by calcium phosphate formation
on the surface, the degradation of 1393 scaffolds occurs in the surface region
without signs of dissolution of the inner parts. These observations should help in
designing novel glass compositions based on the 45S5 and 1393 glasses and
respective scaffolds predicting their degradation and ion release kinetics.
Detailed analysis of physico-chemical reactions occurred at the scaffolds /
biological fluid interface showed that Me2+
ions released from the BG matrix are
incorporated in the calcium phosphate layer formed during reaction in biological
fluids. This is an important finding for evaluation of the mineralization behavior and
biological performance of metal ion doped BG derived scaffolds regarding the
material-cell interaction which is known to be strongly influenced by materials
surface chemistry.354
Also this provides new aspects for understanding of
mechanism behind the materials cells interaction which is not only influenced by
ionic dissolution products but also by the surface chemistry. This implies also the
possibility to specifically modify the chemistry of a BGs and respective scaffolds.
Indeed, there is a wide field of biomaterials research which deals with doping of
calcium phosphates with bioinorganics.355
Biological impacts of metal ion containing BGs
Cu ions released from 45S5-Cu BG derived scaffolds were shown to enhance
VEGF expression in mesenchymal stem cells (MSCs) hence activating angiogenesis
related signalling pathways. In a co-culture model with endothelial cells (ECs) and
MSCs Cu simulated formation of tube-like prevascular structures was observed
indicating stimulated vascularisation. Since sufficient vascularisation is essential for
successful clinical application of engineered bone constructs the new developed Cu
containing 45S5 BG derived scaffolds are promising materials for applications in
regenerative medicine including bone tissue engineering and wound healing.
This work revealed some interesting results regarding the role of copper in
angiogenesis. The stimulating effect of Cu on the VEGF expression in stem cells is
described in literature. However, only the combination of 45S5 bioactive glass, Cu
ions and a co-culturing with stem cells seems to lead to stimulation of endothelial
cells. Hence, advanced cell culture studies are further needed to fully unveil the
Conclusion and Outlook 157
detailed intracellular mechanisms and the role of Cu (and other trace elements) in
cell-cell interaction and cell gene expression. Knowing the effect of Cu on a
specific type of cells and combination of those will allow designing bone
engineering constructs with tissue specific functionalities.
Even though a specific stimulating effect on endothelial cells of Co could not be
detected, the 1393-Co glass and its scaffolds remain interesting materials which
should be considered as potential candidate for use as hypoxia mimicking materials
in bone tissue regeneration applications as it is known that HIF-1 stabilised by
hypoxia is one of major transcription factors for regenerative process in bone
defects and wound healing. The use of co-cultures and alternative experimental cell
setups is suggested in order to obtain more information about the hypoxia effect
caused by Co ions indicated in literature.
45S5 vs. 1393 glass compositions
In accordance to literature 45S5 BG derived scaffolds showed high Si release rates
and hence a rapid degradation as well as fast in vitro mineralisation in simulated
body fluid making them promising materials bone tissue engineering applications.
However, 45S5 BG shows some drawbacks as it is difficult to process due to its
tendency to crystallise during high temperature treatment which results in poor
densification and micro-crack formation and hence in poor mechanical properties of
the scaffolds (~0.2-0.3 MPa compressive strength). Hence, depending on the
applications 45S5 based glasses are suggested where high reactivity and rapid ion
release is needed. 1393 BG derived scaffolds, in turn, can be fully densified without
crystallisation. 1393-Co derived scaffolds exhibiting compressive strength > 2 MPa
with high porosities were fabricated which enables the application of such scaffolds
for regeneration of cancellous bone defects.
Considering the lower reactivity of the 1393 glass compared to 45S5 and, thus,
reduced acellular bioactivity in SBF, the inclusion of therapeutic ions in the 1393
glass matrix would give a combination of the good processing ability of this glass
and enhanced biological performance of this system. In this work, introducing CoO
as intermediate oxide in the glass network enhanced the reactivity, degradation and
ion release kinetics of the 1393 glass matrix. In similar way, other therapeutic ions
Conclusion and Outlook 158
like Zn, also known to act as intermediate oxide in glass formation, could be used
for design of novel bioactive glasses with enhanced degradation and cellular
response.356
For improvement of the mechanical performance of the BG derived scaffolds
fabrication of polymer-BG composite10
is one common approach. Polymer coated
scaffolds show enhanced toughness leading to scaffolds with resistance to crack
propagation. Indeed, the brittleness of 3D BG derived scaffolds is a limiting factor
in their application due to the surgeon´s need to be able cut the scaffolds to the right
the right shape and size.54
Drawbacks of a polymer infiltration of BG derived
scaffolds might be the loss of the (at least temporary) bioactive properties and
inhibition of the degradation and ion release. Hence, future studies on polymer/BG
composite materials should also consider the effects of the polymer coating on the
ion release kinetics of bioactive glasses.
Bioactive glasses in medicine
Generally, glasses are a very versatile group of materials which can be easily shaped
and processed enabling a wide range of potential applications in the field of
biomedical engineering. Applications of particulate bioactive glass, glass fibres, 3D
scaffolds, glass coatings and processing of BG / polymer composites offer a wide
range of application opportunities for novel bioactive glass composition with
specific functionalities. BGs are known for their ability to promote scaffold
mineralisation when applied as inorganic filler in an organic matrix mimicking the
extracellular matrix (ECM) as for example shown for collagen derived scaffold.357
Bioactive glass nanoparticles have been shown to have enhanced biological
properties and to improve the mechanical properties of BG/polymer composites
thus making them promising biomaterials for biomedical applications.358
Hence,
applying a top down approach by mechanical comminution359
to metal ion
containing glasses it is possible to explore a new range of bioactive glasses with
advantageous nano-scaled features and advanced glass chemistry with therapeutic
ion release capability.
Beside traditional application fields of bioactive glasses in hard tissue engineering
(bone and teeth), novel areas of biomedical applications are emerging including
Conclusion and Outlook 159
cancer treatment, ophthalmology, wound healing and nerve guidance repair.
Creating novel BG compositions containing specific therapeutic ions adapted to a
certain application with specific functionalities will help to advance these fields, in
particular considering the interaction of BGs and soft tissue. In this work, foam
replica technique was used for scaffold fabrication. However, other techniques, like
rapid prototyping, e.g. additive manufacturing using UV-light lithography or
robocasting will allow smart combinations of novel BG compositions and creation
of defined patient customized biomaterials and devices.
References 160
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Appendix 188
8 Appendix
Fig. A 1: pH evolution of 45S5 bioactive glass derived scaffolds during immersion in different
solutions.
Fig. A 2: The primers used for real time RT-PCR analysis.
Appendix 189
Fig. A 3: XRD graphs of 1393-Co derived scaffolds confirming the amorphous state.
Fig. A 4: Elemental evolution in the inner region of the 1393-Co derived scaffolds during immersion
in SBF.
Appendix 190
Fig. A 5: Elemental evolution in the periphery layer of the 1393-Co derived scaffolds during
immersion in SBF.
Curriculum vitae
Alexander Hoppe
Henkestrasse91 91052 Erlangen +49 91318525525 [email protected]
Citizenship: German Date/place of birth: 1st July 1984 in Kustanaj/Kasachstan
Education
November 2009
(on-going)
PhD research (Institute of Biomaterials, University of Erlangen-Nuremberg) Topic: Bioactive glass based scaffolds with therapeutic ion release for bone tissue engineering Supervisor: Prof. Aldo R. Boccaccini
2004-2009
Study of Materials Science and Engineering (University of Erlangen-Nuremberg) completed with diploma degree (grade 1.3=”very good”) Thesis: Metal Ion doped biomimetic hydroxyapatite coatings Supervisor: Prof. P. Greil
2001-2004 Secondary education leading to Abitur (A levels); grade: 1.2 (=very good)
Main Research Activities / Expertise
Fabrication and characterisation of biomaterials
Synthesis and structural characterisation of melt-derived bioactive glasses and fabrication of 3D glass derived scaffolds
Compositional design of (bioactive) glasses
In vitro bioactivity and biomineralisation studies in biological fluids Materials surface characterisation (roughness, topography, wettability)
Biomimetic hydroxyapatite coatings; surface functionalisation Development of bioactive glass/polymer composites Characterisation of nano-scaled bioactive glass particles Degradation and ion release assessment of degradable (bio)materials
Cell biology / Cell-material interactions
Cell culture studies with osteoblast-like cells (MG-63, HOS), mesenchymal stem cells, osteoclasts (RAWs 264.7), endothelial cells (HDMECs)
Biocompatibility assessment of biomaterials (cell viability, cell attachment and cell morphology characterisation); Live/dead cell fluorescence staining
Osteogenic differentiation of stem cells (ALP activity, osteogenic gene-expression, ECM mineralisation)
In vitro angiogenesis assay with endothelial cells (cell viability,
vessel-like tube formation, expression of angiogenic marker
Characterisation techniques
Materials: X-ray diffraction (XRD), Fourier-Transform Infrared and Raman Spectroscopy (FT-IR), Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES), Scanning electron microscopy (SEM)+Energy Dispersive Spectroscopy (EDS), X-ray Photoelectron spectroscopy (XPS), Nuclear magnetic resonance spectroscopy (NMR) Cell biology: Gene expression (RT-PCR), Laser Confocal Fluorescence Microscopy (LCFM), SEM, Protein assay (Bradford), Viability (WST-8, AlamarBlue), ELISA assay (VEGF), DNA quantification (PicoGreen, BrdU assay)
International Experience / Fellowships / Grants
Mar 2014 Short Term Scientific Mission (STSM) within a COST (European Cooperation in Science and Technology) action (Project NAMABIO) at 3B's Research Group, University of Minho, Braga, Portugal Supervisor: Prof. Rui Reis
Nov-Dec 2012 and Nov-Dec 2013
Research stay at the University of Buenos Aires, Argentina in the framework of the project: Design and development of novel matrices for tissue engineering and/or drug delivery for pathologies of high social and economic impact Supervisor: Prof. Viviana Murino
2012-2014 (on-going)
Project Assistant for the bilateral research exchange programme with Rudjer Boskovic Institute, Zagreb Coratia, Prof. Neven Zarkovic Research topic: Effect of novel Cu-doped 45S5 bioactive glass and lipid peroxidation products on bone regeneration
Mar 2012 Travel grant to the First São Carlos School of Advanced Studies in Materials Science and Engineering (SanCAS–MSE), São Carlos, Brazil
Aug-Nov 2011
Research stay at McGill University, Montreal, Canada in the framework of the Quebec-Bavarian project Mesenchymal stem cell seeded nano-composite constructs for bone tissue engineering Supervisor: Prof. Showan N. Nazhat
Oct-Nov 2010 KMM-VIN Research Fellowship at Polytechnic University of Turin Topic: Glass-ceramic scaffolds with antibacterial capability for bone tissue engineering Supervisor: Dr. Enrica Verne
Teaching and Supervising
Teaching 2011-2014 (summer terms) 2013 (summer term)
Tutorial lecture on Materials for Biomedical Engineering within the Masters programme Life Science Engineering
Teaching assistant in undergraduate laboratory courses on physical optics
Supervising activities (selected)
Florian Ruther, Bachelor thesis, 2013: Metal ion containing bioactive glass derived 3D scaffolds with enhanced mechanical strength
Stefan Grimm, Master thesis 2011: Fabrication and characterization of 3D highly porous scaffolds with enhanced biocompatibility based on ion doped glasses
Vincent Bürger, Master thesis 2010: Effects of residual surface stresses on the bioactivity of bioactive silicate glasses (Winner of the 2011 Oldfield Award)
Alexander Kent, Bachelor thesis 2010: Fabrication and characterisation of highly porous three-dimensional bioactive glass-based scaffolds for bone tissue engineering
Language and other skills
Language German (native speaker), Russian (native speaker), English (fluent), Spanish (intermediate)
Memberships Member of the American Ceramic Society (ACerS), Member of the German Biomaterials Society (DGBM)
Reviewer activity Reviewer in international peer to peer journals: Materials Letters, Acta Biomaterialia, Surface and Coating Technology, Journal of American Ceramic Society, Colloids and Surfaces B: Biointerfaces, NANO
Computer Scientific software (Origin, ChemBioDraw, EndNote), Graphic software (Photoshop, CorelDraw), MS Office (Word, Excel, Powerpoint)
Other interests I am interested in playing guitar. I also enjoy various kinds of sports including squash, basketball, running, racing bicycle and trekking.
Publications
Publications in peer-reviewed journals Hoppe A, Boccaccini A. R. On the degradation of bioactive glass scaffolds. 2014 (in preparation) Hoppe A, Brandl A, Bleiziffer O, Jokic B, Janackovic D, Boccaccini A R. In vitro cell response to Co-containing 1393 bioactive glass. 2014 (submitted). Hoppe A, Jokic B, Janackovic D, Fey T, Greil P, Romeis S, et al. Cobalt-Releasing 1393 Bioactive Glass-Derived Scaffolds for Bone Tissue Engineering Applications. ACS Appl Mater Interfaces. 2014;6(4):2865-77. Hoppe A, Sarker B, Detsch R, Hild N, Mohn D, Stark WJ, et al. In vitro reactivity of Sr-containing bioactive glass (type 1393) nanoparticles. J Non-Cryst Solids. 2014;387: 41-6. Hoppe A, Will J, Detsch R, Boccaccini AR, Greil P. Formation and in vitro biocompatibility of biomimetic hydroxyapatite coatings on chemically treated carbon substrates. J Biomed Mater Res, Part A. 2014;102: 193-203. Hoppe A, Meszaros R, Stähli C, Romeis S, Schmidt J, Peukert W, et al. In vitro reactivity of Cu doped 45S5 Bioglass® derived scaffolds for bone tissue engineering. J Mater Chem B. 2013;1: 5659-74. Hoppe A, Mourino V, Boccaccini AR. Therapeutic inorganic ions in bioactive glasses to enhance bone formation and beyond. Biomater. Sci. 2013;1:254.
Hoppe A, Mačković M, Detsch R, Mohn D, Stark WJ, Spiecker E, et al. Bioactive glass (type 45S5) nanoparticles: in vitro reactivity on nanoscale and biocompatibility. J Nanopart Res. 2012;14: 1-22. Hoppe A, Güldal NS, Boccaccini AR. A review of the biological response to ionic dissolution products from bioactive glasses and glass-ceramics. Biomaterials. 2011;32: 2757-74. Rath S N, Brandl A, Hiller D, Hoppe A, Gbureck U, Horch R E, Boccaccini A R, Kneser U; Copper doped 45S5 bioactive glass scaffolds stimulate endothelial cells in a co-culture with mesenchymal stem cells. Biomaterials. Acta Biomater 2014 (submitted). Papageorgiou GZ, Papageorgiou DG, Chrissafis K, Bikiaris D, Will J, Hoppe A, et al. Crystallization and melting behavior of poly(butylene succinate) nanocomposites containing silica-nanotubes and strontium hydroxyapatite nanorods. Ind Eng Chem Res. 2014;53(2):678-92. Grigoriadou I, Nianias N, Hoppe A, Terzopoulou Z, Bikiaris D, Will J, et al. Evaluation of silica-nanotubes and strontium hydroxyapatite nanorods as appropriate nanoadditives for poly(butylene succinate) biodegradable polyester for biomedical applications. Composites Part B: Engineering. 2014;60: 49-59. Milkovic L, Hoppe A, Detsch R, Boccaccini AR, Zarkovic N. Effects of Cu-doped 45S5 bioactive glass on the lipid peroxidation-associated growth of human osteoblast-like cells in vitro. J Biomed Mater Res, Part A. 2013. Romeis S, Hoppe A, Eisermann C, Schneider N, Boccaccini AR, Schmidt J, et al. Enhancing In Vitro Bioactivity of Melt-Derived 45S5 Bioglass® by Comminution in a Stirred Media Mill. J Am Ceram Soc. 2014;97: 150-6. Arkudas A, Balzer A, Buehrer G, Arnold I, Hoppe A, Detsch R, et al. Evaluation of angiogenesis of bioactive glass in the arteriovenous loop model. Tissue Eng, Part C. 2013;19: 479-86. Strobel LA, Hild N, Mohn D, Stark WJ, Hoppe A, Gbureck U, et al. Novel strontium-doped bioactive glass nanoparticles enhance proliferation and osteogenic differentiation of human bone marrow stromal cells. J Nanopart Res. 2013;15: 1-9. Strobel LA, Rath SN, Hoppe A, Beier JP, Arkudas A, Boccaccini AR, et al. Influence of leptin on osteogenic differentiation of human marrow stromal cells (hMSC) and modulation of BMP-2-mediated osteoinduction. J Tissue Eng Regen Med. 2012;6: 271-. Cabal B, Malpartida F, Torrecillas R, Hoppe A, Boccaccini AR, Moya JS. The Development of Bioactive Glass-Ceramic Substrates with Biocide Activity. Adv Eng Mater. 2011;13(12):B462-B6. Will J, Hoppe A, Müller FA, Raya CT, Fernández JM, Greil P. Bioactivation of biomorphous silicon carbide bone implants. Acta Biomater. 2010;6: 4488-94 Book chapters A. Hoppe A, AR. Boccaccini. 7 - Bioactive glass foams for tissue engineering applications. In: Netti PA, editor. Biomedical Foams for Tissue Engineering Applications: Woodhead Publishing; 2014. p. 191-212. A. Hoppe, A. R. Boccaccini. Biological response to ionic dissolution products from bioactive glasses and glass ceramics. In S. Deb, editor. Karger Frontiers in Biology: Biomaterials in Regenerative Medicine and Dentistry. Basel: Karger. (submitted) Presentations in international conferences (selected) A. Hoppe, A. Brandl, O. Bleiziffer, D. Janackovic and A. R. Boccaccini. Cobalt releasing bioactive glass (type 13-93) derived scaffolds for bone tissue engineering applications. Oral presentation. Annual meeting of the German Biomaterials Society Sep 2013, Erlangen, Germany. A. Hoppe. 3D scaffolds for bone tissue engineering based on Cu-releasing 45S5 Bioglass®. Invited oral presentation at the Symposium on “Bioceramics as Carrier” Jun 2013, Medical Center, University of Freiburg, Germany. A. Hoppe, J. Will, R. Detsch, A.R. Boccaccini und P. Greil. Formation and biocompatibility of biomimetic hydroxyapatite on chemically treated carbon substrates. Poster presentation. Annual meeting of the German Biomaterials Society Nov 2012, Hamburg Germany.
A. Hoppe, D. Hiller, S. Narayan Rath, A. Arkudas, U. Kneser, A. R. Boccaccini. In vitro and in vivo studies of Cu-doped 45S5 bioactive glass derived scaffolds. Oral presentation. 3rd International Conference "Strategies in Tissue Engineering" May 2012, Würzburg Germany. A. Hoppe, T. Reichel, R. Detsch, A. Lenhart, A. R. Boccaccini. Ion doped bioactive glass derived scaffolds for bone tissue engineering. Poster presentation. 3rd International Conference "Strategies in Tissue Engineering" May 2012, Würzburg Germany A. R. Boccaccini, A. Hoppe. Cellular response to ionic dissolution products from bioactive glasses and glass ceramics. Invited oral presentation. Meeting of the American Ceramic Society Jan 2012, Daytona Beach (FL), USA. A. Hoppe, D. Hiller, U. Kneser, A. R. Boccaccini Novel scaffolds made from metallic ion doped bioactive glasses: fabrication and characterization. Oral presentation in the young researcher forum. Annual meeting of the American Ceramic Society Jan 2012, Daytona Beach (FL) USA A. Hoppe, D. Hiller, S. Narayan Rath, U. Kneser, A. R. Boccaccini Novel Cu-doped bioactive glass (45S5) derived scaffolds for bone tissue engineering. Oral presentation, Annual meeting of the American Ceramic Society Jan 2012, Daytona Beach (FL) USA A. Hoppe, M. Miola, E. Verné, A.R. Boccaccini. Novel Zn-doped bioactive glasses developed by ion-exchange. Poster presentation, EUROMAT Sep 2011, Montpellier, France A. Boccaccini, A. Hoppe. Oral presentation, The biological effect of ionic dissolution products from bioactive glasses, EUROMAT Sep 2011, Montpellier, France