A Review of The Biological Response To Ionic Dissolution Products From Bioactive Glasses and Glass-Ceramics

Biomaterials 32 (2011) 2757e2774
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Biomaterials
journal homepage: www.elsevier.com/locate/biomaterials
Review
A review of the biological response to ionic dissolution products from bioactive

glasses and glass-ceramics
Alexander Hoppe, Nusret S. Gldal, Aldo R. Boccaccini*
Institute of Biomaterials, Department of Materials Science and Engineering, University of Erlangen-Nuremberg, Cauerstrasse 6, 91058 Erlangen, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 14 December 2010
Accepted 4 January 2011
Available online 2 February 2011
Several inorganic materials such as special compositions of silicate glasses, glass-ceramics and calcium
phosphates have been shown to be bioactive and resorbable and to exhibit appropriate mechanical
properties which make them suitable for bone tissue engineering applications. However, the exact
mechanism of interaction between the ionic dissolution products of such inorganic materials and human
cells are not fully understood, which has prompted considerable research work in the biomaterials
community during the last decade. This review comprehensively covers literature reports which have
investigated specically the effect of dissolution products of silicate bioactive glasses and glass-ceramics
in relation to osteogenesis and angiogenesis. Particularly, recent advances made in fabricating dense
biomaterials and scaffolds doped with trace elements (e.g. Zn, Sr, Mg, and Cu) and investigations on the
effect of these elements on the scaffold biological performance are summarized and discussed in detail.
Clearly, the biological response to articial materials depends on many parameters such as chemical
composition, topography, porosity and grain size. This review, however, focuses only on the ion release
kinetics of the materials and the specic effect of the released ionic dissolution products on human cell
behaviour, providing also a scope for future investigations and identifying specic research needs to
advance the eld. The biological performance of pure and doped silicate glasses, phosphate based glasses
with novel specic compositions as well as several other silicate based compounds are discussed in
detail. Cells investigated in the reviewed articles include human osteoblastic and osteoclastic cells as well
as endothelial cells and stem cells.
2011 Elsevier Ltd. All rights reserved.
Keywords:
Bone tissue engineering
Bioactive glasses
Bioactivity
Metal ion release
Cell proliferation
Osteoblasts
1. Introduction
The clinical demand on engineered bone tissue has been
growing in recent years in direct relation to the increasing of the
human population [1]. Tissue engineering (TE) is one of the
approaches being investigated to tackle this problem [2,3]. In
common TE strategies a three-dimensional structure, termed
scaffold, fabricated from a suitable articial or natural material
and exhibiting high porosity and pore interconnectivity is used
[4e7]. 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 [8]. Several attempts have been successfully
made to construct porous scaffolds with desired porosity and
appropriate mechanical performance from inorganic materials
such as bioactive ceramics and glasses, from biodegradable
* Corresponding author. Tel.: 49 44 207 594 6731.

E-mail address: aldo.boccaccini@ww.uni-erlangen.de (A.R. Boccaccini).
0142-9612/$ e see front matter 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biomaterials.2011.01.004
polymers and their composites [6,7]. Different fabrication techniques have been proposed including foam replication, sol-gel
foaming, electro-spinning or rapid prototyping methods [2,3]. Due
to their chemical similarity to the inorganic phase of bone, inorganic biomaterials such as calcium phosphates (CaP), e.g.
hydroxyapatite (HAp), a- and b-tricalciumphosphate (TCP), have
been more intensively investigated in respect to their possible
application as bone scaffolds [4,5,9,10]. These materials are bioactive, osteoconductive and are able to bond directly to bone [11].
Moreover, numerous in vitro and vivo studies have shown that HAp
[12e17] and related CaPs [18e21] support the adhesion, differentiation and proliferation of osteogenesis related cells (e.g. osteoblasts, mesenchymal stem cells). Additionally, it has been shown
that biphasic calcium phosphates (BCP), e.g. mixtures of HAp and
TCP, are osteinductive and induce gene expression in bone cells
[20,22,23]. Amongst the common CaPs, hydroxyapatite is the most
thermodynamically stable at physiological pH of 7.4 and it is known
to be bioactive but not bioresorbable [4,5]. The biodegradability of
CaPs depends on many parameters such as crystallinity, porosity,
chemical purity and surface roughness [4]. HAp will degrade faster
2758
A. Hoppe et al. / Biomaterials 32 (2011) 2757e2774
with increasing porosity, with increasing content of ion substitution in the crystal lattice and increasing content of second phase,
for example when used in BCP mixed with other different CaPs [4].
Bioactive glasses such as 45S5 Bioglass [5] and other compositions based on silicate or phosphate systems represent another
important group of inorganic, bioactive biomaterials used for scaffolds for bone tissue engineering [5,6,24,25]. Bioactive glasses (BG)
exhibit unique properties for this application showing osteoinductive behaviour, ability to bond to soft tissue as well as to hard tissue
and to form a carbonated hydroxyapatite layer (HCA) when exposed
to biological uid [5,11]. This layer is responsible for the strong
bonding between bioactive glasses and human bone [5,9,11]. More
recently, it was observed that ionic dissolution products from Bioglass (e.g. Si, Ca, P) and from other silicate based glasses stimulate
expression of several genes of osteoblastic cells [26]. Furthermore,
bioactive glasses were shown to stimulate angiogenesis in vitro and
in vivo [27e29], whilst possible antibacterial [30e38] and inammatory [39] effects of bioactive glasses have also been investigated.
A schematic overview of biological responses to ionic dissolution
products of bioactive glasses is given in Fig. 1.
Increasing evidence in the literature indicates that ionic dissolution products from inorganic materials are key to understand the
behaviour of these materials in vitro and in vivo, in the context of
tissue engineering applications. Since many trace elements such as
Sr, Cu, Zn or Co present in the human body are known for their
anabolic effects in bone metabolism [40e42], new approaches for
enhancing bioactivity of scaffold materials are being investigated by
introducing therapeutic ions into the scaffold material. As indicated
above, 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. Thus,
research efforts are devoted to incorporate these ions in different

CaPs, bioactive silicate glasses and phosphate glasses resulting in the
changed dissolution behaviour of these materials and their modied
(usually improved) biological performance. For example, in vitro and
in vivo studies have shown enhanced bioactivity of Si-doped HAp
[43,44], Sr-doped silicate glasses [45] or Mg-doped phosphate glasses
[46], just to mention only a few examples, whereas a comprehensive
treatment of this topic is presented in the next sections. Clearly, cell
response depends not only on chemical composition, but also on
surface roughness [47] porosity [48], topography, grain size and
crystallinity of the scaffolds [2,48]. However, this review will focus
specically on the biological response to ionic products coming from
the dissolution of different glass compositions based on silicate and
phosphate systems which is relevant in angiogenesis and osteogenesis applied to bone tissue engineering.
The aim of the review is therefore to summarise the information
available in the open literature on stimulation effects of currently
used inorganic biomaterials on osteogenesis and angiogenesis, with
emphasis on the specic effects of therapeutic ionic dissolution
products. Biological performance of pure and ion-doped bioactive
silicate glasses, glass-ceramics, phosphate based glasses containing
different metal oxides and other specic calcium and silica based
compounds will be discussed. Hereby different direct and indirect
methods have been applied in order to asses the cell response to
degradable biomaterials as schematically shown in Fig. 2.
2. Role of inorganic ions in bone metabolism
Human bone is a dynamic, highly vascularised tissue with the
ability to remodel throughout the life by regulated activity of boneforming (osteoblasts) and bone-resorbing cells (osteoclasts) [49].
The processes of bone formation and bone resorption (bone
Fig. 1. Overview of biological responses to ionic dissolution products of bioactive glasses.
2759
Fig. 2. Different methods for analysing the cellular response to degradable biomaterials.
remodelling) are regulated by a variety of systematic and local

regulatory agents [50] including growth factors, hormones and
stress actions [51,52]. In addition, single inorganic ions such as
Calcium (Ca) [53e56], Phosphorous (P) [57], Silicon (Si) [58e60],
Strontium (Sr) [50,61e65], Zinc (Zn) [51,66] as well as Boron (B)
[67,68], Vanadium (V) [42,49,69], Cobalt (Co) [70e73] and
Magnesium (Mg) [74e80] are known to be involved in the bone
metabolism and to play a physiological role in angiogenesis, growth
and mineralization of bone tissue. Table 1 gives a summary of
biological responses to single inorganic ions relevant in the context
of this review. In particular, metal ions act as enzyme co-factors and
therefore inuence signalling pathways and stimulate metabolic
effects occurring during tissue formation [41,81]. These effects
make metal ions attractive for use as therapeutic agents in the
elds of hard and soft tissue engineering [82].
Since Ca and P are the main components of biological apatite
(Ca10(PO4,CO3)6OH2), the inorganic phase of human bone, these
ions obviously play an essential role in bone formation and
resorption. Nevertheless it is important to know the specic
extracellular matrix concentration of these ions and the mechanism of their interaction with bone cells and their stimulating effect
on bone formation. This knowledge would allow fabricating
advanced scaffolds with tailored ion release kinetics and controlled
biological response in the relevant physiological environment. For
example, Ca is known to affect osteoblastic cells in vitro. Maeno
et al. [54] found that low (2e4 mmol) and medium (6e8 mmol) Ca
concentrations are suitable for osteoblast proliferation, differentiation and extracellular matrix (ECM) mineralization, respectively,
whereas higher Ca concentrations (>10 mmol) are cytotoxic.
Moreover, extracellular Ca plays an important role in bone

remodelling by directly activating intracellular mechanisms by
affecting Ca-sensing receptors in osteoblastic cells. For example, Ca
increases the expression of Insulin like growth factors IGF-I or IGFII, which regulate human osteoblast proliferation. These ndings
have been recently reviewed in the literature [55]. Similar results
were observed by Valerio et al. [56], who found that extracellular Ca
increases the glutamate release of osteoblast cells. Since glutamate
signalling pathways are known to play an important role for bone
mechanosensitivity [53], extracellular Ca concentration must be
considered as an important regulating agent in bone metabolism.
Inorganic Phosphate (P) was recently shown to stimulate expression of matrix Gla protein (MGP), a key regulator for bone formation, in osteoblastic cells when added (10 mmol) to cell culture
medium [57].
Beside Ca and P, bone apatite is substituted by many different
trace elements occurring in smaller concentration [40]. Hence,
several ions have been considered to be promising agents in
enhancing the bone-forming ability of implant materials and
scaffolds, which can be achieved by controlling the release of
specic ions during in vivo dissolution of the scaffold. These ions
include Si, Sr, Zn, Cu, Mg and B.
Si is known to be an essential element for metabolic processes
associated with the formation and calcication of bone tissue
[58,59]. High Si contents have been detected in early stages of
bone matrix calcication [59], whereby aqueous Si was shown to
be able to induce precipitation of hydroxyapatite, the inorganic
phase of human bone [83]. Additionally, dietary Si intake was
shown to increase the bone mineral density (BMD) in men and
2760
Table 1
Effect of selected metallic ions on human bone metabolism and angiogenesis:
summary of literature studies.
Ion
Biological response in vivo / in vitro
Reference
Si
essential for metabolic processes, formation

and calcication of bone tissue
dietary intake of Si increases bone mineral
density (BMD)
aqueous Si induces HAp precipitation
Si(OH)4 stimulates collagen I formation and
osteoblastic differentiation
favours osteoblast proliferation, differentiation
and extracellular matrix (ECM) mineralization
activates Ca-sensing receptors in osteoblast cells,
increases expression of growth factors, e.g.
IGF-I or IGF-II
stimulates expression of matrix la protein (MGP) a key
regulator in bone formation
shows anti-inammatory effect and stimulates bone
formation in vitro by activation protein synthesis in
osteoblasts
increases ATPase activity, regulates transcription
of osteoblastic differentiation genes, e.g. collagen I, ALP,
osteopontin and osteocalcin
stimulates new bone formation
increases bone cell adhesion and stability
(probably due to interactions with integrins)
shows benecial effects on bone cells and bone
formation in vivo
promising agent for treating osteoporosis
signicant amounts of cellular Cu are found in
human endothelial cells when undergoing angiogenesis
promotes synergetic stimulating effects on angiogenesis
when associated with angiogenic growth factor FGF-2
stimulates proliferation of human endothelial cells
induces differentiation of mesenchymal cells
towards the osteogenic lineage
stimulates RNA synthesis in broblast cells
dietary boron stimulates bone formation
[58,59]
Ca
P
Zn
Mg
Sr
Cu
[84]
[83]
[60]
[54]
[55,56]
[57]
[51]
[92]
[80]
[80,101]
[50,65]
[88]
[93]
[94]
[95]
[96]
[102,103]
[68]
premenopausal women [84]. Another study with Ca-decient rats

showed that Si supplementation caused positive effects on bone
mineral density by reducing bone resorption [85]. Moreover,
Nielsen et al. [86] suggested that Si has a biochemical function in
bone growth processes affecting bone collagen turn over and
sialic acid-containing ECM proteins like osteopontin. Moreover,
orthosilicate acid (Si(OH)4) at physiological concentration of
10 mmol has been shown to stimulate collagen I formation in
human osteoblast cells (HOC) and to stimulate osteoblastic
differentiation [60].
Because of the chemical analogy to Ca (same main group, similar
atomic radius, Ca2 e 1.0 A, Sr e 1.16 A), Sr can accumulate in bone
by exchanging with Ca in the hydroxyapatite crystal lattice [87].
The therapeutic potential of Sr in bone metabolism has been
investigated by Marie et al. [50,65], who showed that Sr exhibits
benecial effects on bone cells and bone formation in vivo. Sr has
also been shown to be a promising agent in treating osteoporosis
[88]. Additionally, Sr based drug such as strontium renalate
enhances bone healing indicated by increased callus resistance in
rat bones which has conrmed Sr as a promising agent in healing
bone fractures [89].
Zinc is also known to play an important role in bone metabolism
[51] and to have anti-inammatory effects [90]. Furthermore, Zn
stimulates bone formation in vitro by activating protein synthesis
in osteoblast cells and increasing ATPase activity in bone [51].
Moreover, Zn shows inhibitory effect on bone resorption inhibiting
the formation of osteoclast cells in mouse marrow cultures [51].
The regulatory effects of Zn on bone cells has suggested the
importance of Zn in gene expression [91]. More recently, zinc was
identied as regulation agent in transcription of osteoblastic

differentiation genes, such as collagen I, ALP, osteopontin and
osteocalcin [92]. 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 [92].
Cu has been shown to play a signicant role in angiogenesis
[93e95]. 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 [93]. Another recent study revealed that copper,
associated with angiogenesis growth factor FGF-2, promotes
synergetic stimulating effects on angiogenesis in vitro [94]. Moreover, Cu was shown to stimulate proliferation of human endothelial
cells [95] and to induce an increase in differentiation of mesenchymal stem cells (MSC) towards the osteogenic lineage [96]. Cu2
at a concentration of 106 mol l1 has been also shown to inhibit
osteoclast activity [97]. However, a possible positive effect of Cu on
bone metabolism is still not clearly demonstrated. Cashman et al.
[98] 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 [98]. Similarly, Lai and Yamaguchi [99]
have shown that supplementation with copper induced a signicant
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 [100].
Mg is essential to bone metabolism and it has been shown to
have stimulating effects on new bone formation [80]. Mg is suggested to interact with integrins of osteoblast cells which are
responsible for cell adhesion and stability [80,101]. Rude et al.
[75e77] observed that Mg depletion results in impaired bone
growth, increased bone resorption and loss in trabecular bone
underlining the signicant role that Mg plays in bone metabolism.
Boron is another element which can be considered as potential
stimulating agent for bone tissue engineering [102]. It was shown
that boron affects the RNA synthesis in broblast cells [103].
Moreover, dietary boron can stimulate bone formation, which was
observed by in vivo studies with rabbits [68]. Boron has been
shown also to increase vertebral resistance to crash force in rats as
observed after dietary intake of boron [104]. Gorustovich et al. [105]
have found that boron-decient diet affects alveolar bone modelling and remodelling in rats. Histological ndings have revealed
that B-deciency results in decreased osteogenic activity and
inactive surfaces indicating that B-deciency alters the dynamic
process of alveolar bone modelling and remodelling due to inhibition of bone formation.
In summary, all mentioned metallic ions have high potential to
be used as therapeutic agents released by resorbable scaffolds to
enhance osteogenesis and in some cases angiogenesis of scaffold
materials. Specic inorganic materials (i.e. bioactive glasses and
glass-ceramics) suitable for TE scaffolds and corresponding tissue
engineering approaches will be discussed in detail in the following
sections.
3. Silicate based glasses and glass-ceramics
3.1. Bioactive glasses based on SiO2eCaOeP2O5
Since Hench et al. [106] discovered the rst bioactive silicate
material, now called 45S5 Bioglass (wt%: 45SiO2e25CaOe25
Na2Oe6P2O5), with its unique biological properties, there has been
extensive research work on bioactive silicate glasses for biomedical
applications including bone tissue engineering. However, only

more recently, starting with the Xynos et al. investigation in 2001
[26], the research work has been focussing on the molecular
interactions of ionic dissolution products of bioactive glasses (BG)
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 specic tissue engineering applications, the so-called third generation biomaterials
[24,107]. In this context ionic dissolution products of silicon based
bioactive glasses in the SiO2eCaOeNa2OeP2O5 system have been
shown to change the intracellular ionic concentrations and therefore to mediate cell metabolism. Silver et al. [108] observed higher
2761
Ca concentration in osteoblast cells and enhanced ATP generation

after adding 45S5 Bioglass to the culture medium, whereas other
study showed that an another bioactive glass composition (mol%:
50SiO2e16CaOe6P2O5e5K2Oe2Al2O3e1MgO) led to increased
cellular concentration of phosphorous and sulphur, which are
reecting the phosphorylation state and protein content of the
cells, respectively [109]. Moreover, a large number of studies have
shown the osteogenic effects of bioactive silicate glasses and their
dissolution products [26,47,56,108,110e135], as summarized in
Table 2. One key nding from Xynos et al. [26] studies in 2001 was
that Bioglass dissolution products are able to regulate gene
expression in osteoblastic human cells (HOC). Several genes known
Table 2
Biological responses to ionic dissolution products of silicate based bioactive glasses.
Glass composition
by mol%
45S5
46.1SiO2e24.3Na2Oe
27.0CaOe2.6P2O5
Ions
released
Si
Ca
P
Na
Conc.*[ppm]
Result
Remarks, Conclusions
Ref.
16.5
88.3
30.4
2937.0
16.58
88.35
30.45
2938.0
55.48
72.70
23.45
3341.66
Ionic dissolution products directly stimulated

the up-regulation of most genes in HOC (up to
vefold) known for their relevant role in bone
metabolism
Which released ion

responsible is unclear
[26]
Osteoblast number increased to 150%.

Increase in osteoblast number implies increase
in cell proliferation.
Ionic products of 45S5 Bioglass

may increase proliferation rate.
[134]
Si concentration shortens osteoblast growth cycle

and promotes the proliferation.
Exact molecular mechanism

should be claried.
[239]
Upregulation of BSP and ALP in FOB cells
Ionic concentration in culture

medium is not known
[123]
Better proliferation on amorphous

BG is due to slower degradation of
sintered partially crystallized BG
[114]
Osteogenic supplements effect on gene

expression proling and mineralization.
[129]
[110]
50 % increase in osteoblast proliferation

Increased collagen production
No glass dissolution studies on the BG

were performed; ion conc. in culture
medium is unknown
[130]
Extracellular Ca2+ concentration, via bioglass dissolution,

increases the mobilization of intracellular Ca2+
in osteoblast cells
[56]
Good attachment, proliferation and nodule formation

of osteoblast
[117]
Good osteoblast attachment on BG scaffold

Nodules formation and mineralization after 10 days of
seeding (without supplements of mineralization agents)
Higher Si concentrations (> 120 ppm) of ions lead to
apoptosis
Si concentration is likely the key factor

for mediating mineralization and nodule
formation and cell death
[120]
No statistically signicant effect on proliferation of HOC
Author suggests higher Si content in

medium to achieve stimulating effects
[115]
Upregulation of several genes in HOC, e.g. gp130,

MAPK3/ERK1, MAPKAPK2, and IGF-I.
First study to conrm gene expression

of sol-gel derived bioactive glass
[116]
100% increase in osteoblast proliferation
[111]
Stimulated differentiation of bone marrow cells into

osteoblast cells in BG (77S as well as control 45S5)
conditioned medium
77S BG caused inhibition of osteoclast cell formation
Dissolution kinetics and ion release are

not investigated
[136]
14.5 e 19.4
47.5 e 49.9
24.6 e 23.6
4983.8 e 4895.8
MBG 85
85SiO2e10CaO
e5P2O5
BG60S
59.9SiO2e38.4CaO
e1.7P2O5
S520
52SiO2e21Na2Oe7K2O
e18CaOe2P2O5
59.2
64.0
16.9
58.06
70.07
20.02
30
90
10
47.7
0
67.63
58S
60SiO2e36CaOe4P2O5
77S
80SiO2-16CaO-4P2O5
50.02
95.8
26.4
50.2
95.8
26.4
203.11
47.06
6.88
-
Increased cell proliferation (MG 63) on BG pellets

(2D) compared to control (Thermanox) amongst
which non-crystallized BG shows best results
Signicantly higher cell proliferation on BG-based
scaffolds (3D) compared to control
BG extracts create an extracellular environment for
support of osteoblast phenotype expression
Increased osteoblast differentiation and enhanced
ECM deposition and mineralization
Increased osteocalcin (OCN) and collagen I synthesis
High viability of osteoblasts (Saos-2), murine broblasts
(L929) and murine lymphocytes (SR.D10 T) treated with
BG extracts
But decreased osteoblast and broblast proliferation
*) absolute ion concentration in the cell culture medium after treatment with BG
2762
to play a role in osteoblast metabolism, proliferation and cellecell

and matrix-cell adhesion were up-regulated up to 5 fold [26] when
HOC were cultured in Bioglass conditioned medium. Kaufmann
et al. [126] conrmed these results observing 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. [123] 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.
Similar results have been observed for sol-gel derived bioactive
glasses. For example sol-gel derived 77S (mol%: 80SiO2e16CaOe
4P2O5) bioactive glass has been shown to induce osteogenic
differentiation of bone marrow stromal cells into osteoblast-like
cells and to promote cell mineralization [136]. In addition studies
on 58S (mol%: 60SiO2e36CaOe4P2O5) bioactive glasses have
shown that treatment with their ionic dissolution products results
in cell activation through stimulation of the proliferation of osteoblasts cells [111] and upregulation of the expression of a number
of genes including IGF-I, gpl30 or MAPK3/ERK1 [116]. Previous
work (up to 2005) on the osteogenic stimulating effects of bioactive
silicate glasses has been already reviewed [124].
Additionally, molecular effects of bioactive silicate glasses were
observed in vivo by implanting 13-93 (Vivoxid) glass particles
(wt.%: 53SiO2e6Na2Oe12K2Oe5MgOe20CaOe4P2O5) in bone
defects of rat tibia and by investigating gene expression at the
defect area [132]. In agreement with previous results, the study
revealed an osteoinductive effect of bioactive glass 13-93, where
the defects lled 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 [132]. 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. Other studies have conrmed the stimulating
ability of bioactive glass extracts on osteogenesis, which are
believed to be related to gene expression as previously observed,
showing enhanced proliferation of human osteoblast cells (HOC)
and murine osteoblast cells [111,130] and increased collagen I
formation in osteoblastic cells [113,130]. It has been shown that
Bioglass conditioned culture media with Si concentrations of 15
and 20 mg l1 promoted higher metabolic activity, expression of the
Cbfa1 factor and enhanced formation of mineralized bone nodules
in primary fetal osteoblasts [129]. 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 [125].
Since it has become clear that successful clinical application of
scaffolds in bone tissue engineering highly depends on a functional
vascularised network, relevant research is being expanded to
include investigations on the angiogenic effects of biomaterials
[137]. 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 [138]. Thus, functional vascularisation of the scaffold
implants would result in enhanced bone metabolism and bone
formation. For this reason several studies have been carried out in
order to investigate the potential stimulating effects of bioactive
glasses on angiogenesis, using both endothelial cells and broblasts. [27e29,118,139,140]. An early study showing the angiogenic
effect of Bioglass was provided by Day et al. [140] revealing
increased neovascularization into Bioglass coated polymer
meshes after being subcutaneously implanted in rats. In further
investigations Day [27] showed that Bioglass stimulates the

secretion of angiogenic growth factors from human broblasts
cells, providing further evidence that bioactive glass enhances
angiogenesis. In the study of Day [27], broblast cells (CCD-18Co)
cultured in Bioglass particles containing medium secreted
increased amounts of vascular endothelial growth factor (VEGF)
and basic broblast growth factor (bFGF). Subsequent culturing of
endothelial cells in conditioned medium obtained from Bioglass
stimulated broblast growth resulting in increased endothelial cell
proliferation and formation of anastomosed networks of cell
tubules thus indicating enhanced angiogenesis. The studies of Day
et al. [27,140] were extended by Leu and Leach [29,139], who found
similar results. These and other studies on angiogenic effects of
bioactive glasses have been comprehensively reviewed recently
[28]. In recent investigations, Deb et al. [118] performed coculturing of osteoblast and endothelial cells showing increased
proliferation of both, human osteoblast cells and human umbilical
vein endothelial cells (HUVEC) seeded on Bioglass based scaffolds
compared to HAp scaffolds.
The large amount of results on the biological performance of
bioactive glasses discussed above conrms the hypotheses that
ionic dissolution products released from bioactive glasses stimulate the genes of cells towards a path of regeneration and selfrepair as recently discussed by Hench [141]. It is now well accepted
that bioactive silicate glasses are able to stimulate ostegenesis as
well as angiogenesis and therefore they exhibit unique properties
which make them highly attractive for bone tissue engineering
applications. However, it remains unclear which ionic products
(and in which concentration) are responsible for cell activation and
which are the exact mechanisms of interaction between the ionic
products and cells. In fact, the importance of single dissolution
products from bioactive glasses is still controversially discussed.
Based on Xynos et al. [26,134,135] studies, Hench [141] stated that
eluted Ca and Si concentrations of 60e88 ppm and 17e21 ppm,
respectively, are critical for upregulation of several osteogenic
genes. Valerio et al. [131] 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 BCP (no Si release). Indeed, as mentioned in the
previous section, Si has been shown to be an essential element for
metabolic processes associated with the formation and calcication
of bone tissue [58,59] and to be present in early stages of bone
matrix calcication [59]. Moreover, high Ca concentrations (of
88e109 ppm) have been shown to reduce Saos-2 osteoblast
proliferation when exposed to bioactive glass MBG 85 (mol%:
85SiO2e10CaOe5P2O5) conditioned culture, whereas a Si concentration of 60 ppm did not have any negative effect on the cell
proliferation [110]. Despite the fact that a too high Ca concentration
is toxic for human osteoblastic cells, Ca has been shown to increase
the glutamate release of osteoblast cells [56]. Since glutamate signalling pathways are known to play an important role in bone
mechanosensitivity [53], the extracellular Ca concentration must
be considered as an important stimulating agent in bone formation.
Phosphor has also been suggested to be essential for calcium
phosphate deposition and extracellular matrix mineralization,
whereby higher inorganic Phosphor (P) concentrations in osteoblast culture medium resulted in more rapid mineral deposition
[142]. Elevated phosphate levels have been shown to increase Nrf2
expression in osteoblastic cells (MC3T3-E1) when treated with
10 mmol phosphate [143]. More recently, P has been shown to
upregulate Glvr-1 and Glvr-2 in murine odontoblast-like cells
correlated with ERK1/2 phosphorylation and CaP crystallisation
[144]. However, this effect requires the presence of Ca indicating
that both, P and Ca, are essential in signalling pathways occurring in
Table 3
Biological response to ion-substituted silicate based bioactive glasses.
Glass composition (mol%)U
U
if not otherwise specidied
6.4SiO2-1.1P2O5-26.4Na2O-23.1SrO
45S5.6Sr
45SiO2e24.5Na2Oe18.5CaOe6P2O5-6SrO
Ion(s)
released
Si
Ca
P
Sr
Conc.* [ppm]
Result
Remarks
Ref.
w
w
w
w
increased osteoblast (Saos2) activity (increased proliferation

and ALP activity) after treatment with BG extracts
reduced osteoclast activity (RAW264.7)
[45]
good bone bonding ability in vivo after 30 days implantation

in rat tibia bone marrow
increased proliferation of rat calvaria osteoblastic cells
enhanced the cell differentiation and ALP activity
No signicant difference in bioactivity

compared to undoped glass was observed
Sr release into the culture medium is not
investigated and remains unknown
21.2
85.3
19.37
0.31
increased proliferation and differentiation of osteoblast cells
No dissolution studies in cell culture

medium were performed; thus ionic
concentration remains unknown.
[148]
Zn doping results in increased surface area

favors HAP formation and growth
More investigations on different

concentrations of Zn in a cellular
medium are required to evaluate specic
effect of Zn on cells
[151]
15
28
1.4
2.7
higher Zn content (20%) decreases cell (Endothelial cells)

proliferation rate
Zn content in culture medium is

suggested to be cytotoxic
[146]
Cell response depends on the form BG is

applied, e.g. as supsension, glass extract
[159]
20
30
5
80
(wt.%): 58.8SiO2-22.3CaO-P2O5-7.9SrO
64SiO2-26CaO-P2O5-5ZnO
(wt.%): 73.7SiO2-25.3-CaO-1ZnO
74SiO2-21.2CaO-4.8ZnO
Si
Ca
P
Zn
[165]
67SiO2-5P2O5-17CaO-11ZnO/CuO
Zn
Cu
w13
w58-23
Reduced TNF production and Il10 following pretreatment with

doped BG compared to undoped control indicating
prophylactic inammatory effect of the Zn and Cu containing
glasses
However, no therapeutic effect after post treatment with Zn
and Cu glasses detected, likely due to toxic Cu and Zn levels
released into the culture medium
(Zn Sr)- BG
40SiO2-0CaO-28SrO-32ZnO
Si
Ca
Zn
Sr
no inammatory response In vivo with juveline rats

showed immediate bone formation response due to higher cell
viability
Further in vivo studies are planned
[150]
Up to 99.8 % reduction of bacteria (Staphylococcus aureus,

Escherichia coli) adhesion and growth
Ag surface doping has no effect on acellular in vitro bioactivity.
[181]
Ag-BG
57SiO2-34CaO-6Na2O-3Al2O3
surface is doped with Ag
Ag-BG (S70C30)
70SiO2-28CaO-2Ag2O
45S5.2B
wt%: 45SiO2-24.5CaO-24.5-Na2O-P2O52B2O3
Si
Ca
Ag
B
71
155
1
-
13-93B2
wt%: 36B2O3-18SiO2-22CaO-6Na2O8MgO-8K2O-2P2O5-
0.65 mmol
HCaCaF2
46.2-SiO2-24.3 Na2O, (26.9x)CaO2.6P2O5-xCaF2
(x 0, 5, 10, 15)
15.4 (for x5)

30.4(for x10)
40.9(for x15)
after 24 h
(wt.%): 45SiO2-24.5CaO-6P2O5-24.5Na2O5TiO2/Fe2O3/B2O3
43SiO2-14.5-CaO-5.77P2O5-23.64Na2O13.1CaF2
Si
Ca
Na
P
No cytotoxic effect on human epidermal keratinocytes

Larger area and increased thickness of neoformed bone tissue
around B-BG compared to undoped control indicating
enhanced bone formation
Boron concentration above 0.65 mmol inhibit the growth and
proliferation of BMSCs and MLO-A5 cells
Fluoride release causes oxidative damage of MG-73 cells
indicated through:
Increased extracellular LDH (index of cytotoxicity) levels and
accumulation of intracellular MDA (index of lipoperoxidation) and
Release of ROS (indicator of stress) and inhibition of PPP
(signal of cell response to oxidative stress) activity
_
Increased
osteoblast proliferation and osteoblast expression
on Ti-BG
B-BG, Fe-BG and F-Bg exhibit lower osteoblast proliferation
and activity
Different treatments to the silver ion

release may cause different cellular
responses.
Increased rate of bone formation can be
due to the enhanced degradation of
bioglass after B substitution to Si.
Threshold Boron concentration of 0.65
mmol is achieved through deluting BG
extracts indicating a too high B2O3
amount in the BG composition
37SiO2-18CaO-4P2O5-18Na2O-20ZnO
[164]
[180]
[191]
[188]
[195]
[198]
2763
[133]
bone metabolism. Even though phosphate-free sol-gel derived

bioactive glasses have been shown to induce extracellular matrix
mineralization even without presence of P [125], inorganic phosphates should be considered important regulating agents in bone
metabolism.
Taking all these facts into account one can state that one of the
challenges for developing scaffold materials for bone regeneration
is to achieve controlled dissolution and ion release kinetics making
sure that required critical concentrations of certain ions are
released into the physiological environment. Future in vitro studies
should therefore explicitly investigate in greater detail ion release
kinetics and the respective concentrations in cultured medium to
make results comparable within the biomaterials scientic
community, which will help to identify the specic role of single
dissolution products of bioactive glasses and inorganic materials in
general.
Enhanced expression is most likely due

to the dissoluted ions.
Only Si conc. signicantly higher than
control medium
[173]
Mg containing glass samples are not

compared to undoped standard bioactive
glass ceramics
In vitro results are not compared to Mgfree glass, so that effect of Mg doping on
cell behavior remains unclear
[171]
2764
6P53-b (LBL)
(wt.%): 52.7-SiO2-10.3Na2O-18CaO-6P2O52.8K2O-10.2MgO-)
*) absolute ion concentration in the cell culture medium after treatment with BG
Enhanced stimulation of osteogenic markers expression

including Collagen I, ECM formation, ALP activity and osteocalcin
No differences between the experimental composition and pure
Bioglass control were detected
33.4 3.8
4458 186
298.3 34.9
57.1 2.8
32.7 5.4
32.6 9.3
64SiO2-26CaO-5P2O5-5MgO
45.98SiO2-43.30CaO-10.72MgO
Mg
Si
Ca
P
Ca
Mg
Si
Na
K
Ca
Mg
P
Increased viability and proliferation of osteoblast seeded on

glass wafers compared to control (pure culture medium).
increased ALP activity after 7 days culturing
Increased proliferation and differentiation of hFOB cells
seeded on glass samples compared to PS control
No toxic effects with L929 cells
3.2. Ion-doped bioactive silicate based glasses

In order to enhance the bioactivity of bioactive glasses towards
a specic 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. These approaches consider diverse bioactive glass
compositions including Co-BG [145], Zn-BG [146e161], Cu-BG
[159,162], Sr-BG [45,150,156,163e169], (Zn Sr)-BG [150,156,170],
Mg-BG [133,161,162,171e175], Ag-BG [176e184], Ce-BG [185] as
well as B-BG [154,186e194] and F-BG [195,196]. Table 3 gives
a representative overview of silicate based BGs containing specic
metal ions and the respective biological response to their ionic
dissolution products.
Diverse silicate glasses based on CaOeMgOeSiO2 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 uid, according to Kokubo et al. [11,197], in order to prove the
formation of surface HCA layer [186]. In the study of Agathopoulos
et al. [186], 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. [198]
reported on the osteoblast behaviour on 45S5 bioactive glass
doped with iron, titanium, uorine and boron giving (to our
knowledge) the rst overview of the biological response to iondoped bioactive glasses. It was shown that Ti-BG exhibited higher
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.
Cobalt containing glasses co-doped with Zn and Mg [145] have
been fabricated in order to create hypoxia-mimicking (low
oxygen 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 [199]. Co
ions (known for their ability to mimic hypoxia) have been introduced to bioactive glass and it has been shown that Co ions are
released in proportional manner to the CoO content (incorporated
into the BG network) into TRIS solution used as dissolution medium
[145]. Co2 concentrations in the range of 10e14 ppm in the
medium were measured, which are believed to be within the biologically active limits. Moreover, HAp formation was delayed
through the incorporation of CoO, ZnO and MgO as it was revealed
Table 4
Biological response to phosphate based bioactive glasses.
Glass composition by mol%
Ions released
Phosphate glasses based on P2O5eCaOeNa2O (PBG)
ACaO-(55-A)Na2O-45P2O5
A: 30 e 48
Ca
P
Na
50P2O5-(50-X)CaO-XNa2O-
Biological response
More soluble glasses with less than 20% CaO inhibit growth and bone
antigen expression in osteoblasts (MG63 and HOS)
Less soluble glasses increase cell proliferation and expression of BSP,
ON and FN osteogenic factors
PBGs containing less than 40% CaO do not support cell adhesion and
growth of human osteosarcoma cells (MG-63)
Human oral osteoblasts (HOB) and broblasts show well spread
morphology on higher calcium containing glasses.
All PBG investigated inhibit cell adhesion and proliferation and
increase cell death of hBMSC and HFOB 1.19 compared to TCP and
HA control
Remarks
Ref.
[210]
No dissolition were performed; ion release kinetic

and ion concentration in culture medium remains
unknwown
[211]
[212]
Cell behavior differs when applied directly to the

materials or to the materials extracts
[213]
[215]
[214]
No comparison to control samples and undoped glass,

role of Zn not specically investigated
[218]
Cytotoxicity of Mg-free PGG is likely due to higher

dissolution rates
[46]
PBG containing different metal oxides
*)
Ti-PBG
44.5P2O5e44.5CaOe6Na2Oe5TiO2
Ca
P
Na
Ti
60.1 0.06
32.3 0.003
3350.7 0.6
not detected
Ti-PBG
27.5CaO-33 P2O5-22.5-Na2O-9.5MgO5.5TiO2
Ca
P
Na
Mg
Ti-PBG
44.5CaO-44.5P2O5-6Na2O-5TiO2
Ca
P
Na
Ti
4.1 0.36
5.4 0.12
90.3 1.84
Not detected
Zn-PBG
48CaOe45P2O5e5Na2Oe2ZnO
Mg-PBG
40P2O5-25CaO-ANa2O-BMgO
A: 5 e 35
B: 0 e 30
Ca
P
Na
Mg
No toxic effect of the glass extracts, but however slightly toxic cell
response when seeded directly on glass disc (toxic effect lower on Ti
doped glass)
better human skin broblast cell adhesion on Ti-free glass (more soluble)
Proliferation of MC3T3-E1.4 osteoblast like cells do not changed for
polished samples for all glasses investigated.
MC3T3-E1.4 osteoblast proliferation is higher on porous glass samples
with lower solubility, namely BG containing 5.5 mol% TiO2.
However for all samples (porous and non-porous) the proliferation rate
was always lower than the positive control TCP control
Non-cytotoxic effects on osteoblast cells (SAOS-2) of glass extracts
and of the glass scaffolds in the direct testing are detected
Cells directly seeded on the glass scaffold exhibit higher viability
(after 3 days of culturing) compared to control medium
High degree of adhesion and spreading of mouse-derived MC3T3-E1
cells on scaffold surface
No HCA formation after soaking in SBF for 28 days indicating
insufcient acellular bioactivity
Decrease in dissolution rate is observed with increasing MgO content
Cytocompatibility of Mg-PBG comparable to TCP control; Mg-free
PBG shows higher cytotoxicity
XCaO-(50-X)Na2O-50XP2O5
X: 30 e 48
Conc.* [ppm]
absolute ion concentration in the cell culture medium after treatment with BG
2765
2766
by SBF studies [145], 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.
3.2.1. ZinceBG
Bioactive silicate glasses doped with Zinc (Zn-BG) have been
shown to enhance acellular formation of calcium phosphate layer
on the BG surface after soaking BG substrates in biological uids
(Dulbeccos Modied Eagles Medium, DMEM) [151] and in simulated body uid (SBF) [149,155], which is a fundamental requirement for bioactive glasses to bond to bone [5]. Similarly, Zn and Fe
containing glass compositions have been shown to increase acellular bioactivity with increasing the combined Zn-Fe content [158].
However, the results on the acellular bioactivity of Zn-doped BG
are not consistent. Aina et al. [146], for example, have shown that
Zn content reduces the overall leaching activity of the glass
inhibiting the formation of the HCA layer on its surface. Haimi
et al. [154] obtained similar results showing a delayed formation of
HCA which is related to the slower degradation prole of the Zndoped bioactive glasses. The analysis of the limited literature
available indicates that further specic studies on the relationship
between microstructure and physicochemical properties of Zndoped BG are needed to determine with accuracy the inuence of
Zn-doping on the dissolution behaviour of bioactive glasses in
order to control the ion release kinetics and the biological
performance.
Independently of the acellular behaviour investigations, in vitro
cell culture studies have shown the anti-inammatory effect of Zndoped bioactive sol-gel derived 58S glass [159]. In this study,
murine macrophage cells (RAW 264.7) stimulated with lippopolysaccharide (LPS) endotoxin were cultivated in DMEM treated with
BG suspension (1 mg ml1) and glass ionic dissolution products,
respectively, resulting in decreased secreted amount of tumor
necrosis factor TNF-a and interleukin-1 which indicated the
inammatory effect on the cells. Glass compositions with 5mol-%
and 11mol-% Zn did not have any therapeutic effects on cells since
the TNF-a and IL-1 contents were not signicantly decreased after
treatment with the BG suspension. On the other hand the pretreatment of cells with bioactive glasses resulted in a signicantly
decreased amount of TNF and IL-1 indicating a prophylactic antiinammatory 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-inammatory 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 2e8 ppm can cause damage in
human osteoblasts via oxidative stress [147]. Similar 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 [146]. 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
[148,153e155,157]. It has been shown that sol-gel derived glasses
containing 5 mol% ZnO resulted in increased ALP activity and
increased osteoblast proliferation [148] indicating the possible

stimulating effect of Zn-BG on osteoblast cells.
In related research, Oki et al. [157] 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
undoped glass samples, so that no denitive statement on the
stimulatory effect of Zn can be made based on the available
experimental evidence. More recently, Haimi et al. [154] investigated the effects of Zn-doped BG scaffolds on human adipose stem
cells proliferation and osteogenic differentiation revealing no
signicant 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 prole of the bioactive glass caused by the
Zn addition. Similar results were observed by Lusvardi et al. [155],
who found that Zn-doping provides no signicant effect on adhesion and proliferation of osteoblast-like cells (MC3T3-E1)
compared to undoped 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 glasses were carried out missing a specic investigation of possible anabolic effect of Zn. Despite the fact that in vitro
studies with systematic supplements of Zn ions showed osteogenic
effect of Zn (as previously described), to our knowledge no clear
data has been presented to date to conrm in a denite way the
osteogenic effect of Zn released as ionic dissolution product from
Zn-doped bioactive silicate glasses.
3.2.2. Strontium-BG
Sr-doped glasses have been shown to exhibit enhanced acellular
bioactivity and to release critical concentrations of Sr ions in the
range of 1e5 ppm into the dissolution medium [166,167]. Moreover, Gentleman et al. [45] have shown that ion release from Srdoped silicate glasses enhances bone cell activity. They investigated
the in vitro behaviour of SiO2eP2O5eNa2OeCaO 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 5e23 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 [165]. In vivo studies have also conrmed the high
biocompatibility of Sr-containing 45S5 Bioglass expressed
through strong bonding to bone via HCA layer without any
inammatory affects, though no differences of the in vivo behaviour compared to undoped Bioglass control were observed [164].
(Zn Sr)-co-doping has been also applied to create novel
bioactive glass compositions. For example, Murphy et al. [156]
showed that SiO2eCaOeNa2OeSrOeZnO2 glasses release Zn and
Sr concentrations in the 3e18 ppm and 0e3500 ppm ranges,
respectively, having therapeutic potential with capacity for
controlled release of Zn and Sr ions. Moreover, another study by
Murphy et al. [170] ovserved higher viability of mouse broblast
cells (L929) when ionic extracts of these ZneSr doped glasses were
applied to the cell culture medium. However, no specic stimulating effect of the a certain ion released in to the culture medium
could be identied and the authors suggest that the improved
biocompatibility of the Sr and Zn containing glasses compared to
Novabone is likely due to synergetic effects of all ions released
from the glass as dissolution products. Boyd et al. [150] observed

that 0.4SiO2-0.32ZnO-0.28SrO (mol%) glass exhibits higher cell
viability compared to 0.4SiO2-0.32ZnO-0.28CaO and 0.4SiO20.32ZnO-0.14CaO-0.14SrO glasses as well as to Novabone
(commercial Bioglass product) control, indicating the positive
effects of the Sr-doping. However, no dissolution studies were
performed and it was only assumed from the authors that the lower
cytotoxicity of the Sr-doped glass was due to higher chemical
stability resulting in a lower amount of Sr ions released into the
culture medium.
3.2.3. Magnesium-BG
Bioactive glasses doped with Mg (Mg-BG) have been shown to
have improved dissolution behaviour due to the Mg effect disrupting the silica network [172]. On the other hand these glasses
exhibit decreased crystallisation of the HAp layer on the BG surface
[172]. Varanasi et al. [133] observed enhanced expression of
collagen I, ALP, Runx2 and osteocalcin osteogenic markers after
treatment of the cell culture medium with Mg-BG-dissolution
products indicating their signicant effect on the osteoblast
differentiation. However, Mg concentration in culture medium did
not change signicantly after treating with BG, so that the osteogenic effect of this particular BG could be related to increased levels
of Ca and Si. Other studies have shown an increased osteoblast
proliferation and differentiation [171] as well as enhanced ALP
activity in human fetal osteoblastic cells (hFOB 1.19) [173] and
enhanced expression of osteogenic markers including OP, OC, ON in
human bone derived cells (HBDC) [200]. These ndings conrm the
good biocompatibility of BGs with MgO addition. However, Mgdoped samples have not been compared to undoped control
glasses; thus, it remains unclear whether or not Mg released from
these bioactive glasses has a specic osteogenic effect.
3.2.4. Flouride-BG (F-BG)
CaF2 has also been considered as promising doping agent to
enhance the biocompatibility of bioactive glasses, since CaF2 is
known to inhibit the formation of alveolar cavities [201] and to
provide higher acidic resistance of enamel by substituting OH sites
in dental apatite [202]. Fluorides are also known for stimulating
effects on osteoblast cells when applied at moderate concentrations (25e500 ng ml1), whereas higher concentrations (>500 ng
ml1) suppress osteoblast activity [203]. However, CaF2 doped
bioactive glasses have been shown to be cytotoxic and to promote
oxidative cell damage as indicated by increased lactate dehydrogenase (LDH, indicator of cytoxicity) formation and accumulated
extracellular malonyldialdehyde (index of lipoperoxidation) when
investigated in contact with osteoblasts (MG 63) [195]. Only acellular bioactivity studies on F-doped bioactive glasses showed that
incorporation of F in silicate glasses results in a smaller pH raise
(which is favourable for the cells) and favoured formation of
ourapatite (FAp), which is chemically more stable than HAp and
thus a very promising material for dental applications [196].
3.2.5. Boron-BG
Boron doped 45S5 Bioglass (B-BG) materials with different
boron contents have been shown to exhibit increased acellular
bioactive behaviour as the rate of HAp formation on the surface
during soaking in P-rich solution increases [187]. However, with
increasing B-content the proliferation of osteoblastic like cells
decreased, with B-concentration of 1.5 mmol in the culture
medium having inhibiting effect on cell proliferation. Similar
results were found by Fu et al. [188] who observed accelerated HAp
formation of boron doped BG but inhibited cell proliferation with
increasing B-content in culture medium. In the study of Fu et al.
[188] a boron concentration of 0.65 mmol in the cell culture
2767
medium was shown to be the threshold concentration, whereas B

values below 0.65 mmol supported the proliferation of preosteocytic MLO-A5 cells indicating good biocompatibility of the
boron doped glass. Moreover, a Boron concentration above
0.65 mmol was shown to decrease the growth and proliferation
rate of bone marrow cells (BMSc). However, the dissolution products of the processed BG with a Boron oxide content of 36 mol%
were diluted up to a ratio of 1:8 indicating that the B2O3 content in
the BGs investigated was too high [188]. Clear evidences of
enhanced biological performance of boron doped BG have been
observed in vivo by Gorustovich et al. [189,191], who found
a signicant increase of quantity of bone tissue formed around
Boron-BG particles after implantation in wist rats indicating
simulating effects of boron doped bioactive glasses on osteogenesis.
However, more in vitro studies on boron modied bioactive glasses
are needed to clearly identify the effect of these glasses on human
cells and to conrm the potential simulating effect of boron, as
discussed previously (Section 2).
3.2.6. Silver-BG
Since Ag ions are known for their antibacterial effects
[204e206] Ag-doped silicate glasses (Ag-BG) have been developed
to create bioactive glasses exhibiting inhibitory effects on bacterial
growth using different techniques including sol-gel [176e180] and
ion exchange processes [181e184]. It has been conrmed that Ag
ions with concentrations in the range of 0.05e0.20 mg ml1
released as dissolution products from Ag-doped BGs inhibit the
growth of different bacterial strains (Escherichia coli, Pseudomonas
aeruginosa, Staphylococcus aureus) [176]. Moreover, an ion exchange
process has been applied to glass-ceramic scaffolds in order to
fabricate Ag-containing porous structures with antibacterial properties for bone tissue engineering [183]. Thus, Ag-doped BGs are
considered highly promising materials for tissue engineering
applications with anti-inammatory properties that are attractive
for wound healing applications as well.
All ion-doped bioactive glasses discussed above have been
shown to exhibit sufcient levels of acellular bioactivity indicated
through the formation of carbonated hydroxapapatite layer after
soaking in SBF which is an essential requirement of biomaterials to
form a strong bonding to bone [5]. 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 specic novel glass compositions discussed
above. However, more quantitative experimental data on the biological response of relevant cells to ion-doped bioactive glasses are
needed to conrm the specic role of the ions released as dissolution products from those silicate systems in the context of bone
tissue engineering.
4. Phosphate based glasses
4.1. Basic compositions: (CaO)-(Na2O)-(P2O5)
Phosphate based glasses (PBG) have gained high interest as
bone lling material and for fabricating scaffolds for bone tissue
engineering because of their high solubility, which can be tailored
by varying the composition [207], and their chemical similarity to
the inorganic phase of human bone [208]. Moreover, early studies
on phosphate glasses have shown that they are non toxic with
regard to macrophage cells causing low level of activation and
promoting adequate support for osteoblastic cell responses [209].
Ionic dissolution products of highly soluble phosphate glasses have
been shown to affect osteoblastic cell behaviour in a dissolution
rate depending manner. Salih et al. [210] observed that glass
compositions with lower CaO content, and therefore lower
2768
dissolution rate, up-regulated the proliferation of osteoblast cells

and expression of bone sioloprotein (BSP), osteonectin (ON),
bronectin (FN) genes, whereas glasses with higher CaO content
(and higher dissolution rate) caused inhibition of osteoblastic cell
growth and bone antigen expression. Similar results were obtained
by Bitar et al. [211] who found that phosphate based glasses containing at least 46 mol% CaO do not produce any toxic effect on cells,
but support attachment and proliferation of MG63 cells and human
oral osteoblasts (HOB). However, studies carried out by Skelton
et al. [212] revealed that the presence of PBGs with varying
amounts of Na2O in the culture medium reduced the adhesion and
proliferation and increased the death of human bone marrow
stromal cells (hBMSC) and human felt osteoblast (HFOB 1.19)
compared to TCP and HAp control.
In order to tailor the solubility of phosphate glasses several
oxides such as TiO2 [213e215], Al2O3 [216], B2O3 [217], ZnO [218],
MgO [46,219,220], SrO [221], CuO [222] and Fe2O3 [219,223] have
been investigated as additives incorporated into the phosphate
based glass compositions in order to stabilize the glass network and
to control the degradation rate. The biological performance of the
resulting phosphate based glass compositions is discussed in the
following section. Table 4 gives an overview of the biological
response to phosphate based bioactive glasses containing different
metal oxides.
4.2. Variant compositions
Clearly, the varying solubility of bioactive phosphate glasses
affects their biological performance since, as mentioned above,
ionic dissolution products are known to stimulate angiogenesis and
osteogenesis in vivo and in vitro. For example, addition of TiO2 has
been shown to reduce the solubility of the P2O5eCaOeNa2OeTiO2
system and to affect the cellular response of this glass [213]. The
addition of 5 mol% TiO2 resulted in a slower degradation of the
glass. Additionally, the more soluble glass exhibited a higher toxic
effect on human broblast cells than the undoped glass (when
applied as glass extracts), but, surprisingly, an increased cell
attachment was observed when cells were directly seeded on the
glass samples. Another study by Navarro et al. [214] conrmed the
non-cytotoxicity of the same glass composition by obtaining positive results from both cell treatment with glass extracts and direct
cell seeding on the glass samples. Brauer et al. [215] investigated
the inuence of SiO2 and TiO2 doping on the degradation behaviour
and the biological performance of P2O5eCaOeMgOeNa2O glass.
The authors reported that addition of TiO2 decreased the solubility
of the glass in water and SBF, while SiO2 doping resulted in
increased dissolution rates. However, the cell culture test revealed
that proliferation of MC3T3-E1.4 osteoblast-like cells did not
change for dense samples of all glasses investigated. The cell
proliferation on porous samples, however, was changed by the
varying chemistry of the glasses fabricated. It was shown that cell
proliferation was higher on glass samples with lower solubility,
namely PBG containing 5.7 mol% TiO2. However, for the cells seeded
on all samples, porous and non-porous, the proliferation rate was
always lower than the one on the positive control material (Tissue
Culture Polysterene, TCPS).
Addition of MgO to P2O5eCaOeNa2O glasses has also been
shown to decrease the degradation rate and thus to inuence the
cell response [46]. Compared to TCP positive control, PBG compositions with O mol% MgO caused the lowest anabolic activity of
osteoblast cells, while MgO contents of 10, 20 and 30 mol% resulted
in osteoblast activity at similar levels as the positive control. Leonardi et al. [220] investigated phosphate based glasses with SiO2,
MgO and K2O additions observing good cell adhesion and viability
of hMSCs seeded on the glass samples though it was lower
compared to the positive TCP control. Moreover, gene expression

levels of collagen and osteocalcin, presented in early osteogenic cell
differentiation, were higher compared to TCP indicating stimulating effects of the glass dissolution products on osteogenesis.
However, cell culture tests were carried out using the direct contact
method and the concentration of the ions released into the culture
medium were not measured. Abou Neel et al. [221e223] extensively reported on degradation kinetics of phosphate glasses
modied with several oxides including Fe2O3, CuO or SrO and their
biological performance. It has been shown that adding 1e5 mol%
Fe2O3 to P2O5eCaOeNa2O glass bres reduced the degradation rate
of the material in water [223]. Inclusion of 1e10 mol% CuO [222]
resulted in decreased glass degradation but, on the other hand, it
was shown to increase the amount of Cu2 ions released due to the
increased surface area to volume ratio. The release of Cu2 ions has
been shown to have antibacterial effects reducing the number of
Staphylococcus epidermis cultured on the glass bres, whereby the
addition of 10 mol% CuO exhibited the strongest effect amongst the
samples tested. Doping with 1e5 mol% SrO has been shown to
increase the degradation rate of the glass and to exhibit better
cellular response related to HOS viability compared to Sr-free
samples [221]. However, the cellular response was still lower than
that on the positive control (not specied in the study).
Pickup et al. [224] reported on Ga-doped PBG containing molar
fractions of Ga2O3 in the range of 0.01e0.05. The Gallium doping
resulted in an increased stability of the glass network, which is
relevant for potential use of Ga-PBG materials as delivery system of
antibacterial Ga3 ions for biomedical applications. In fact, recent
studies by Mourino et al. [225] on Ga-alignate coated bioactive
glass scaffolds have conrmed the antibacterial characteristics of
Ga3 ions.
5. Other calcium and silicate based compounds
Apart from bioactive silicate and phosphate based glass
compositions discussed above numerous other inorganic materials
and their dissolution products are being considered as promising
materials for bone tissue engineering applications, including
sulphates [226], silicate ceramics [160,227e230], xerogels [231]
and cements [232]. Relevant studies are summarized and discussed in this section.
Mesoporous silica xerogels (SiO2eCaOeP2O5, SXC) with varying
amounts of Ca (0, 5, 10 and 15), for example, have been studied in
order to investigate the role of calcium on murine osteoblast cell
(MC3T3-E1) behaviour [231]. It has been shown that samples
containing small amounts of Ca (5 wt.%) can stimulate cell differentiation indicated through increased ALP activity, whereas higher
Ca contents tend to decrease ALP stimulation levels. Moreover, the
nitric oxide (NO) levels of cultured cells has been shown to increase
with increasing Ca content of the silica xerogels [231], which can be
of interest for biomedical applications, as NO is known to play
a signicant role in several physiological processes including
neuronal communication, neurotransmission, inammation as well
as angiogenesis, stem cell proliferation and differentiation [233].
Several approaches have been put forward to use synthetic materials, including metallic and silica nano-particles, polymeric particles and carbon nanotubes (CNT), as NO release agents for
biomedical applications as recently reviewed in the literature [233].
Shin et al. [234,235] and Hetric et al. [236] for instance have
synthesized and characterized NO-containing silica nano-particles
and have shown that NO release from these scaffolds induces
strong antibacterial effects against Pseudomonas Aeruginosa, while
being not toxic to mouse broblast cells (L929) [236].
Further it has been shown that Zn-doped calcium sulphate
cements exhibit higher cell viability and increased ALP activity
when applied to the culture medium of human osteosarcoma cells

(G-292) [226]. Hereby samples with 0.74 wt.% Zn showed the best
result whereas higher Zn concentrations (>w2 wt.% Zn) resulted in
decreased cell viability indicating toxic behaviour of these samples.
Dicalcium silicate coatings [229] have been also used to
generate cell cultures containing critical Ca, Si and P concentrations
and to stimulate cell activity in vitro. Sun et al. [229] have shown
that culture medium with ionic products of CaSiO4 coatings can
stimulate osteoblast-like cells (MG 63) towards an enhanced
transition from the G1 to the S stage and from G2 to the M stage of
the cell cycle, respectively, indicating stimulated cell proliferation.
Sol-gel derived hardystonite ceramics (Zn stabilised calcium
silicate, Ca2ZnSiO7) have also been considered as promising materials for bone tissue engineering [227,228]. These materials have
been shown to increase the proliferation rate of MSCs when
exposed to Ca2ZnSiO7 immersed culture medium [227] and to
induce osteogenic differentiation of MSCs [227]. Moreover, hardystonite ceramics have been shown to support cell attachment,
increase cell proliferation and differentiation and to stimulate
expression of ALP, osteocalcin and collagen I when in contact with
HOB cells [228].
Zreiqat et al. [160] reported on the biological response of Srdoped hardystonite ceramic based scaffolds. It has been shown that
Sr-doped Hardystonite (Sr-HT, Sr-Ca2ZnSiO7) showed acellular
bioactivity indicated by HAp formation in SBF compared to HT
samples without Sr substitution (Ca2ZnSiO7) which did not show
HAp precipitations. Moreover, Sr-Ca2ZnSiO7 scaffolds have been
shown to increase the proliferation rate of HOB cells seeded on the
scaffolds compared to pure Ca2ZnSiO8 samples. In vitro studies on
the differentiation of HOB cells revealed that although the ALP
activity of cells seeded on Sr-HT and HT were below the TCP control,
Sr-HT samples showed signicant upregulation of osteocalcin (an
osteoblast maturation marker) compared to HT, b-TCP and tissue
culture plastic (TCP) control. In vivo studies conrmed the high
biocompatibility of pure and Sr-Hardystonite scaffolds revealing
increased osteoconductivity compared to b-TCP control, indicated
through greater bone in-growth into the scaffolds after 3 and 6
weeks of implantation in tibia bone defects in rats [160].
Strontium silicate powders (SrSiO3) have also been developed in
order to create novel resorbable materials for bone regeneration
[230]. SrSiO3 compounds, synthesized through chemical precipitation methods, have been evaluated in terms of acellular bioactivity and their effect on mouse broblast cells (L929) and rabbit
bone marrow stem cells (rMSC). SrSiO3 powders were shown to
form carbonated hydroxyapatite layer after 7 days of immersion in
SBF, which is an indication of sufcient acellular bioactivity.
Moreover, SrSiO3 powders showed no cytotoxicity when their ionic
extracts were applied to the L929 cell culture and they even
increased the cell proliferation of rMSCs. In this context Sr
concentrations in the range of 6.25e50 mg have been shown to
exhibit benecial effects on cellular response, whereas higher Sr
contents showed deleterious effects on cells.
6. Discussion
The control of metallic ion release from inorganic scaffolds is
being considered as an attractive approach to enhance the biological capability of scaffolds for bone tissue engineering. The present
review has discussed comprehensively reports in the open literature which have investigated specically ion release effects on
bioactivity from bioactive glasses and glass-ceramics.
Ionic dissolution products from bioactive silicate glasses have
been shown to stimulate osteogenesis and angiogenesis via activation of several genes associated with bone formation and vascularisation, as schematically shown in Fig. 1, making bioactive
2769
glasses (including also phosphate glasses) a group of materials of

choice for tissue repair and bone tissue engineering applications.
Moreover, new approaches have been put forward to advance the
eld of bioactive glass based scaffolds through introducing active
metal ions into the glass network in order to use the therapeutic
effects of these ions and to improve the biological performance of
the materials towards a specic host response. Indeed, the dissolution of ion-doped bioactive glasses has been shown to result in
controlled release of critical concentrations of metal ions showing
in some cases increased osteoblast activity (Sr-BG) or benecial
anti-inammatory effects (Zn-BG). However, more experimental
evidences are necessary to conrm the therapeutic effects of single
biologically active metal ions released as dissolution products from
bioactive glasses. Hereby, the accurate evaluation of the results is
hindered by the fact that the biological performance of the material
depends on whether it is applied as dense substrate, porous scaffolds, particle suspension or ionic extract [153] and whether cell are
seeded directly on the material or are applied to liquid extracts
containing the dissolution products [159,213,228]. Clearly, applying
glass extracts to the cells does not consider factors like surface
roughness, surface energy, surface electric charge, substrate stiffness as well as pore curvature and pore size [237,238] which all are
known to inuence the cell behaviour. Moreover, adding high
amounts of metal oxides to the glass network results in overall
changed dissolution behaviour of the glass, making hard to
compare the results to those on unmodied control glass [146,154].
Several different methods, schematically shown in Fig. 2, are being
applied by investigators worldwide to investigate the biological
response to biomaterials regarding their ionic dissolution products
leading to a large amount of experimental data which are difcult
to compare. The development of standardised methods for testing
the biological response to ionic dissolution products of inorganic
materials is suggested to improve the quantitative evaluation of the
experimental results and to gain a more complete understanding of
the cell response to these biomaterials.
7. Conclusions
A comprehensive analysis of the available literature on the effect
of ionic dissolution products of inorganic bioactive glasses and
glass-ceramics (silicate and phosphate systems) in the context of
bone tissue engineering has been presented. Taking into account
the experimental evidence summarized in this review and the
discussion above, a signicant challenge for developing scaffolds
for bone regeneration based on these inorganic materials is to
achieve controlled ion dissolution and release kinetics ensuring
that required critical concentrations of certain ions are released
into the physiological environment in a predetermined manner.
Future in vitro studies should therefore explicitly investigate the
release kinetics of specic ions (and combination of ions) and their
respective concentrations in cultured medium in order to make
results comparable among different materials. A systematic
approach like this will help to identify the specic role of single
dissolution products of bioactive glasses (and inorganic materials in
general). Fabricating polymer/glass-ceramic scaffolds has been
approached to achieve controlled dissolution behaviour of the
composite scaffold and the desired ion release proles [6].
However, in this case the effect of the degrading polymer matrix on
the biological performance of the composite material must be
considered, as there will be a combined effect of polymer and
inorganic material degradation kinetics on cellular behaviour.
The greater understanding of molecular mechanisms and
chemical pathways dictating the interaction between materials and
cells will allow constructing tissue specic scaffolds with tailored
chemistry, surface topography, porosity and ion release kinetics,
2770
which will induce the desired biological effect in the physiological

environment. In this regard, in future material-based approaches,
bioactive glasses and glass-ceramics can serve as valid biomaterial
platforms for the delivery of tailored concentrations of predetermined ions for targeted cellular or tissue responses.
Appendix
Figures with essential colour discrimination. Certain gures in
this article, particularly Figs. 1and 2 are difcult to interpret in black
and white. The full colour images can be found in the online
version, at doi:10.1016/j.biomaterials.2011.01.004.
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A Review of The Biological Response To Ionic Dissolution Products From Bioactive Glasses and Glass-Ceramics

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Biomaterials 32 (2011) 2757e2774

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A review of the biological response to ionic dissolution products from bioactive

* Corresponding author. Tel.: 49 44 207 594 6731.

A. Hoppe et al. / Biomaterials 32 (2011) 2757e2774

research efforts are devoted to incorporate these ions in different

Fig. 1. Overview of biological responses to ionic dissolution products of bioactive glasses.

A. Hoppe et al. / Biomaterials 32 (2011) 2757e2774

remodelling) are regulated by a variety of systematic and local

Moreover, extracellular Ca plays an important role in bone

A. Hoppe et al. / Biomaterials 32 (2011) 2757e2774

Biological response in vivo / in vitro

essential for metabolic processes, formation

premenopausal women [84]. Another study with Ca-decient rats

identied as regulation agent in transcription of osteoblastic

A. Hoppe et al. / Biomaterials 32 (2011) 2757e2774

applications including bone tissue engineering. However, only

Ca concentration in osteoblast cells and enhanced ATP generation

Ionic dissolution products directly stimulated

Which released ion

Osteoblast number increased to 150%.

Ionic products of 45S5 Bioglass

Si concentration shortens osteoblast growth cycle

Exact molecular mechanism

Upregulation of BSP and ALP in FOB cells

Ionic concentration in culture

Better proliferation on amorphous

Osteogenic supplements effect on gene

50 % increase in osteoblast proliferation

No glass dissolution studies on the BG

Extracellular Ca2+ concentration, via bioglass dissolution,

Good attachment, proliferation and nodule formation

Good osteoblast attachment on BG scaffold

Si concentration is likely the key factor

No statistically signicant effect on proliferation of HOC

Author suggests higher Si content in

Upregulation of several genes in HOC, e.g. gp130,

First study to conrm gene expression

100% increase in osteoblast proliferation

Stimulated differentiation of bone marrow cells into

Dissolution kinetics and ion release are

Increased cell proliferation (MG 63) on BG pellets

A. Hoppe et al. / Biomaterials 32 (2011) 2757e2774

to play a role in osteoblast metabolism, proliferation and cellecell

investigations Day [27] showed that Bioglass stimulates the

increased osteoblast (Saos2) activity (increased proliferation

good bone bonding ability in vivo after 30 days implantation

No signicant difference in bioactivity

increased proliferation and differentiation of osteoblast cells

No dissolution studies in cell culture

Zn doping results in increased surface area

More investigations on different

higher Zn content (20%) decreases cell (Endothelial cells)

Zn content in culture medium is

Cell response depends on the form BG is

Reduced TNF production and Il10 following pretreatment with

no inammatory response In vivo with juveline rats

Further in vivo studies are planned

Up to 99.8 % reduction of bacteria (Staphylococcus aureus,

15.4 (for x5)

No cytotoxic effect on human epidermal keratinocytes

Different treatments to the silver ion

A. Hoppe et al. / Biomaterials 32 (2011) 2757e2774