Acta Biomaterialia xxx (2018) xxx–xxx

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Acta Biomaterialia
journal homepage: www.elsevier.com/locate/actabiomat

Review article

Tackling bioactive glass excessive in vitro bioreactivity: Preconditioning

approaches for cell culture tests
Francesca E. Ciraldo a, Elena Boccardi a, Virginia Melli b, Fabian Westhauser c, Aldo R. Boccaccini a,⇑
a
Institute of Biomaterials, Department of Materials Science and Engineering, University of Erlangen-Nuremberg, 91058 Erlangen, Germany
b
Department of Chemistry, Materials, and Chemical Engineering ‘G. Natta’. Politecnico di Milano, Piazza L. Da Vinci 32, 20131 Milano, Italy
c
Centre of Orthopaedics, Traumatology, and Spinal Cord Injury, Heidelberg University Hospital, 69118 Heidelberg, Germany

a r t i c l e i n f o a b s t r a c t

Article history: Bioactive glasses (BGs) are being increasingly considered for biomedical applications in bone and soft tis-
Received 20 March 2018 sue replacement approaches thanks to their ability to form strong bonding with tissues. However, due to
Received in revised form 8 May 2018 their high reactivity once in contact with water-based solutions BGs rapidly exchange ions with the sur-
Accepted 12 May 2018
rounding environment leading in most cases to an undesired increase of the pH under static in vitro con-
Available online xxxx
ditions (due to alkaline ion ‘‘burst release”), making difficult or even impossible to perform cell culture
studies. Several pre-conditioning treatments have been therefore proposed in laboratories worldwide
Keywords:
to limit this problem. This paper presents an overview of the different strategies that have been put for-
Bioactive glasses
Bioactivity
ward to pre-treat BG samples to tackle the pH raise issue in order to enable cell biology studies. The paper
Cell culture also discusses the relevant criteria that determine the selection of the optimal pre-treatment depending
In vitro on the BG composition and morphology (e.g. particles, scaffolds).
pH
Statement of Significance

Bioactive glasses (BGs), since their discovery in 1971 by L.L Hench, have been widely used for bone
replacement and repair, and, more recently, they are becoming highly attractive for bone and soft tissue
engineering applications. BGs have in fact the ability to form a strong bond with both hard and soft tis-
sues once in contact with biological fluid. The enhanced interaction of BGs with the biological environ-
ment is based on their significant surface bioreactivity. This surface effect of BGs is, on the other hand,
problematic for cell biology studies by standard (static) cell culture methods: an excessive bioreactivity
leads in most cases to a rapid and dramatic increase of the pH of the surrounding medium, which results
in cell death and makes cell culture tests on BG samples impossible. The BG research community has
been aware of this for many years and numerous pre-treatments have been proposed by different groups
worldwide to limit this problem. For the first time, we have reviewed in this paper the variety of surface
preconditioning treatments that have been put forward over the years, we provide a summary of such
pre-treatments used in laboratory practice, discussing and offering criteria that can be used for the deter-
mination of the optimal pre-treatment depending on BG composition and morphology of the sample
tested (bulk, particulate, scaffolds). The information and discussion provided in this review should sup-
port best research practice when testing bioactive glasses in cell culture.
Ó 2018 Acta Materialia Inc. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND
license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
2. Bioreactivity of bioactive glasses. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
3. Preconditioning methods for bioactive glasses. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
4. Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

⇑ Corresponding author.
E-mail address: aldo.boccaccini@ww.uni-erlangen.de (A.R. Boccaccini).

https://doi.org/10.1016/j.actbio.2018.05.019
1742-7061/Ó 2018 Acta Materialia Inc. Published by Elsevier Ltd.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Please cite this article in press as: F.E. Ciraldo et al., Tackling bioactive glass excessive in vitro bioreactivity: Preconditioning approaches for cell culture
tests, Acta Biomater. (2018), https://doi.org/10.1016/j.actbio.2018.05.019
2 F.E. Ciraldo et al. / Acta Biomaterialia xxx (2018) xxx–xxx

5. Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

1. Introduction For this reason, in vitro studies to evaluate the behaviour of cells in
contact with bioactive glasses must adopt some form of precondi-
In the last years the demand for new materials for bone replace- tioning of BG samples to limit such non-realistic pH changes. As an
ment applications has gained continuous importance due to the example, Fig. 1 shows the pH variation in simulated body fluid
increase of the average age of the population and the increasing (SBF) containing glass powders (45S5 and 58S) with and without
number of surgical procedures [1]. In particular, bone defects pre-conditioning treatment as a function of time [18]. It is shown
above a critical size cannot be repaired by the self-healing of bone that pre-conditioning clearly limits the pH excursion without
tissue and require an osteoconductive and osteoinductive device affecting the hydroxyl-carbonate-apatite formation on the powder
(scaffold) able to support the regeneration of the new tissue [2]. surfaces, as reported in ref. [18]. Over the years, laboratories
Although autografts are still considered the ‘gold standard’, they around the world have developed a variety of methods and condi-
have many drawbacks such as limited availability and morbidity tioning protocols to pre-treat BGs prior to cell biology studies.
of the donor site [3]. Xenografts and allografts could be considered This review summarizes and discusses the different precondi-
a valid alternative, however they have potential drawbacks such as tioning methodologies put forward for in vitro cell culture charac-
relatively low rates of integration, risk of contamination, immune terization of BGs and offers suggestions to select the most efficient,
rejection or viral transmission from the donor [4]. For these rea- cost effective and fastest method available. Here, we will consider
sons, engineered biomaterials are considered highly promising silicate BGs, both sol-gel and met-derived, also discussing different
candidates for bone tissue regeneration [2,5–7]. BG morphologies, such as granules, pellets and porous scaffolds.
Since first reported in 1971 [8], bioactive glasses (BGs) have
been extensively used in bone replacement and repair and, more
recently, tissue engineering applications due to their ability to
bond in vivo to tissues through the development of a biologically
equivalent hydroxyl-carbonate-apatite layer, similar to the mineral
phase of bone [5,6,9].
The first reported bioactive glass, known as 45S5 BioglassÒ,
with composition (wt. %): 45SiO2-24.5CaO-24.5Na2O-6P2O5, [8],
has been used as bulk material for the production of medical
devices for dental and orthopaedic applications, as particulate in
bone-filler defects, as coating on metallic implants and for fabricat-
ing tissue engineering scaffolds [5,6,9–12]. The tissue bonding abil-
ity of BGs is based on the high surface reactivity of these materials
in contact with aqueous environments. Moreover, a special advan-
tage of BGs is the possibility to tailor their chemical composition
by the incorporation of biologically active ions that elicit specific
cellular functions [13].
Calcium and phosphorous are the main components of the bone
mineral phase and the release of such ions from BGs is relevant in
the context of bone tissue engineering applications [9,11]. More-
over silicon, as dissolution product of BGs, is well known to
enhance the formation and calcification of the extracellular matrix
(ECM) and soluble silica has been shown to contribute to osteo-
blast activity [10]. Such bioreactivity of BGs has been also consid-
ered to be relevant for applications in contact with soft tissues [14].
New glass compositions and/or BGs doped with bioactive ions
are being increasingly investigated with the aim to provide the
most suitable glass composition for applications in different set-
tings [6,11,14,15]. The biological properties of these new glass
compositions have to be evaluated to assess their usability in the
respective fields of application. Hence, in vitro cell culture studies
are always used to analyse the interaction of BGs with cells and
to estimate their biological ability, for example regarding the stim-
ulation of osteogenic differentiation or the upregulation of angio-
genic growth factors [10,14,16,17].
However, one important issue to consider when BGs get in con-
tact with biological fluids is the development of possible pH-
dependent cytotoxicity due to significant changes in localized pH
due to an undesired high rate of ion exchange reactions that occur
upon interaction of the glass surface with cell culture medium,
leading to a burst release. While this effect is not usually observed
Fig. 1. pH variation of SBF containing bioactive glasses (45S5 and 58S composition)
in vivo [6,12], it becomes highly relevant when testing BGs in vitro. with and without pre-conditioning (adapted from Pryce et al. [18]).

Please cite this article in press as: F.E. Ciraldo et al., Tackling bioactive glass excessive in vitro bioreactivity: Preconditioning approaches for cell culture
tests, Acta Biomater. (2018), https://doi.org/10.1016/j.actbio.2018.05.019
 F.E. Ciraldo et al. / Acta Biomaterialia xxx (2018) xxx–xxx
2. Bioreactivity of bioactive glasses
The main building unit of silicate glasses is the SiO4 tetrahe-
dron, which can be connected to another SiO4 tetrahedron through
Si-O-Si bonding, commonly known as bridging oxygen atoms [19].
Network modifiers remodel the glass structure, transforming
bridging oxygen atoms into non-bridging oxygen atoms, leading
to a decrease of the network connectivity, defined as the number
of bridging atoms per network forming element, and resulting in
an increase in the BG dissolution rate. The open silicate network
allows water-based solutions to penetrate easily the network lead-
ing to glass dissolution and release of ions (e.g. calcium and phos-
phate ions). A characteristic ion exchange at the glass/water
interface occurs when silicate glasses come in contact with aque-
ous solutions, which has been discussed frequently in literature
[8,11,19]. Rapid exchange of Na+ and Ca+ with H+ or H3O+ from
the solution occurs leading to a fast and notable increase of the
pH of the solution due to the replacement of H+ ions by cations
[20].
The breaking of Si-O-Si bonds by the action of OH– groups
causes the dissolution of the network and the resulting release of
soluble silica into the solution in form of silicic acid [Si(OH)4].
The ionic dissolution rate from the glass generates an alkalization
of the surrounding environment, which affects cell metabolism
and function. If the dissolution rate of the glass is too rapid, the
ion concentration released can reach critical levels influencing cell
gene expression [13,18]. An increase of pH can have severe effects
on cell metabolism and function, and can furthermore influence
cell respiration provoking enzyme alteration and affecting the dif-
fusion of nutrients and gases to cells [21]. Good laboratory practice
advises to keep the pH of cell culture in a range between 7.2 and
7.4. This is because an excessive deviation from this pH value
may alter the transmembrane electric potential and hinder the cor-
rect function of Na/K and Ca ion pumps [22]. A mismatch in the
correct concentration of metabolic ions can then lead to protein
denaturation and, eventually, to cell death by either apoptosis or
necrosis [22]. Kaysinger et al. [23] investigated the effect of pH
on the activity of cultured human osteoblasts. Osteoblasts were
shown to be sensitive to extracellular pH in vitro. It was observed
that collagen synthesis and alkaline phosphatase activity, which
are related to the osteoblastic activity and cell proliferation,
increase with increasing pH in a range between 7.0 and 7.6. At
pH 7.8 this trend decreases, indicating that at high pH the activity
of osteoblasts is considerably suppressed. Furthermore, osteoclasts
Fig. 2. Different behavior of cells in response to BG samples of different
morphologies with or without pre-conditioning treatment.
Please cite this article in press as: F.E. Ciraldo et al., Tackling bioactive glass excessive in vitro bioreactivity: Preconditioning approaches for cell culture
tests, Acta Biomater. (2018), https://doi.org/10.1016/j.actbio.2018.05.019
3
that are of crucial relevance during the early phase of osseous
regeneration are triggered by acidic environments; consequently,
osteoclast function is limited by increasing pH [24]. These pH
effects on cell activity pose a challenge for the in vitro biological
characterization of BGs and therefore approaches have been put
forward and applied by laboratories around the world to tackle it.
3. Preconditioning methods for bioactive glasses
The easiest method used to pre-condition BG samples to avoid
reaching cytotoxic pH values for cell culture is the immersion of
the samples for a certain period of time in an aqueous solution
before carrying out cell biology studies. Four kinds of media have
normally been used in the context of bioactive glass dissolution
studies: i) TRIS buffer, an organic buffer solution [18,25], ii) simu-
lated body fluid (SBF), a solution containing similar ion concentra-
tion to that of human blood plasma [21,26–31], iii) Alpha
Minimum Essential Medium (a-MEM) and iv) Dulbecco’s Modified
Eagle’s Medium (DMEM), both being cell culture media containing
inorganic and biological organic components of blood plasma
(Fig. 2) [18].
In an early report, El Ghannam et al. [32] found that it is neces-
sary to condition BG samples (bulk disks, 10 mm diameter, 2 mm
thickness) to minimize the pH variation due to the release of alkali
elements into the tissue culture medium. Without pre-
conditioning, the dramatic increase of pH was shown to lead to
profound changes in cell function and activity. It was reported that
after 48 h of pre-conditioning in SBF, osteoblasts completely cov-
ered the BG surface and produced considerable extracellular
matrix (ECM). When the treatment was stopped after 20 h, osteo-
blasts attached to the BG surface but they created a limited amount
of ECM components. In a related work [33], authors investigated
the effect of BG disks’ surface modification on cellular activity.
Three different conditioning methods were used before seeding
with cells: i) BG disks were immersed in TRIS buffer solution sup-
plemented with electrolytes typical of the interstitial fluid (TE) for
48 h, ii) BG disks were immersed in tissue culture medium (TCM)
for 1 h or iii) BG disks were immersed in TE for 48 h first and then
in TCM for 1 h. The two step conditioning method allowed the con-
trol of the pH, promoted the expression of osteoblasts and
enhanced the formation of a mineralized bone-like apatite layer
on the surface of the glass. The authors reported that cells seeded
on samples conditioned by the two step process colonized the
material and produced a significant amount of bone-like tissue
covering the entire sample. Moreover, the effect of different ratios
of volume of TCM (Vol) / surface area of BG disks (SA) on cell activ-
ity on BG samples conditioned by the two step procedure was anal-
ysed. It was observed that at a low Vol/SA ratio the pH rose up to 8
after 24 h and that a minimal amount of extracellular matrix was
developed. On the contrary, when the Vol/SA ratio was high, osteo-
blasts were able to produce a considerable amount of ECM on the
conditioned BG samples.
Vernè et al. [27] carried out cell biology tests on CEL2 (45SiO2,
3P2O5, 26CaO, 7MgO, 15Na2O, 4K2O mol%) BG-based scaffolds.
The samples were tested with and without pre-treatment in SBF
for a week, refreshing SBF every 2 days in order to mimic the bio-
logical environment. The immersion in SBF was followed by the
soaking of the samples in cell culture medium for 24 h. During
the pre-treatment in SBF, a considerable ion release was observed
which led to increase of pH. It reached in the first 2–3 days a value
of 8. After 1 week of soaking in SBF the pH variation reached a pla-
teau at about 7.75, which was considered to be an optimal value
for osteoblast adhesion [27]. The surface of the samples was cov-
ered by HCA crystals and the cells were anchored onto these
agglomerates forming bridges between the HCA crystals.
4 F.E. Ciraldo et al. / Acta Biomaterialia xxx (2018) xxx–xxx
In another study, Vernè et al. [28] produced and characterised
silica based bioactive glasses belonging to the system SiO2-P2O5-
CaO-MgO-K2O-Na2O produced by the traditional melt-quenching
route. Prior to the seeding with fibroblasts, samples (slices of 1
mm thickness and about 1 cm2 area) were pre-conditioned in
SBF for a week. It was observed that after 7 days of immersion in
SBF the surface of these materials was ‘‘more biocompatible”
because the release of ions, which could have had negative effects
on cell viability, had been already stabilized. Moreover, thanks to
the treatment in SBF, the surface of the samples developed a gel-
like layer that should be easily colonized by fibroblasts, known to
adhere better on hydrophilic surfaces [28]. The authors concluded
that, although the treatment in SBF can affect the glass properties
in terms of ion release kinetics and hydrophilicity, it can positively
influence cell viability and protein adsorption.
As mentioned above, another method used to pre-condition BG
samples is immersion in cell culture medium. Midha et al. [34]
investigated the dissolution of 70S30C (70SiO2-30CaO mol.%)
bioactive glass based foams in contact with cell culture medium.
The scaffolds were soaked in cell culture medium for 72 h. After
this period of time the pH was found to be 8.12 and further 72 h
of immersion was needed to reduce the pH to 7.5, a value in the
physiological conditions range. The pre-conditioned scaffolds were
implanted in a rat tibial defect model to assess their ability to be
used as template for bone tissue regeneration. Pre-conditioned
foams turned out to be promising and effective candidates to
regenerate rat tibial defects. Vascularized new bone replaced most
of the scaffolds that degraded into non-toxic by-products with no
evidence of foreign body reaction or inflammation. Moreover,
pre-conditioned samples developed a strong bond with bone.
Boccardi et al. [35] investigated the behaviour of cells in contact
with 45S5 BG derived scaffolds based on natural marine sponges.
Similar observations were made by Westhauser et al. [36] on the
same scaffold type. The authors corroborated the need of a pre-
conditioning treatment in order to limit the rapid increase of pH
over physiological conditions. Scaffolds were soaked in cell culture
medium and HEPES until the pH was lower than 8 and the medium
was refreshed every day. After 14 days of cell culture, SEM obser-
vations revealed clear evidence of cell adhesion and proliferation
on the surface of the scaffolds [35].
Table 1 presents a complete summary of pre-conditioning
methods for cell culture studies on BGs in different morphologies
that have been proposed and applied over the years.
4. Discussion
Thanks to their ability to bond to soft and hard tissues through
the formation of a biologically equivalent apatite surface and ther-
apeutic ion release ability, BGs are promising biomaterials for tis-
sue replacement, repair and regeneration [6,9,11–13]. When BGs
become in contact with water-based solutions, an ion exchange
at the material/solution interface takes place, leading to a fast
increase of the pH of the solution due to the replacement of H+ ions
by metal cations [20]. This phenomenon, often defined as ‘burst
release’, creates an alkaline environment that can affect cell meta-
bolism and function. Indeed, a fast increase of the pH can influence
cell respiration mechanisms causing enzyme alteration and modi-
fication of the diffusion of nutrients and gases to cells [21]. On the
other hand, the (controlled) release of dissolution products of BGs
is one of the key advantageous properties of BGs to positively affect
cell behaviour [10,13]. Based on early studies of Xynos et al. [10],
Hench and co-workers proved that Ca and Si concentrations of
60–80 ppm and 17–21 ppm, respectively, are required to enhance
osteoblast cell activity [59]. On the other hand, a high concentra-
tion of Ca (of 88–109 ppm) is toxic for human osteoblasts and it
Please cite this article in press as: F.E. Ciraldo et al., Tackling bioactive glass excessive in vitro bioreactivity: Preconditioning approaches for cell culture
tests, Acta Biomater. (2018), https://doi.org/10.1016/j.actbio.2018.05.019
has been shown to reduce Saos-2 osteoblast proliferation [13,59].
Different methods have been investigated in order to prevent or
limit the alkalization of the environment in cell culture studies.
Dissolution studies of bioactive glasses in different media have
been carried out as pre-conditioning step, as summarized in
Table 1. The analysis of the available data reveals that immersion
of bioactive glasses in SBF, refreshing the medium every 2–3 days
in order to better mimic the physiological conditions, leads to a
‘‘more biocompatible” surface for cell culture studies
[27,28,32,33]. The high rate of release of ions, which can have neg-
ative effects on cell viability, can be properly stabilized by this pre-
treatment. The pH reaches values of 8 in the first 2 days and it
then stabilizes after 1 week at pH 7.75, being an optimal value
for osteoblasts adhesion [27]. Similar results were obtained in
studies [34] performed soaking the samples in cell culture med-
ium. After a week of immersion in DMEM the pH was found to
be 7.2, a value in the physiological range.
A recent approach proposed by Li et al. [22] consists in the syn-
thesis of a ‘‘pH neutral” calcium phosphosilicate bioactive glass
that does not require pre-conditioning prior cell culture tests. A
sol–gel glass, named PSC-pH (composition in mol. %: 54.2SiO2,
35CaO, 10.8P2O5), was prepared and its cell compatibility was
tested using a preosteoblast cell line (MC3T3-E1) without any
pre-conditioning. 45S5 and S70C30 BGs were used as reference.
PSC-pH showed better cell proliferation compared to 45S5 and
S70C30 BGs. Already after 1 day of culture cells adhered well on
the surface of the sample and after 3 days the presence of filopodia
and spread cells was observed. Dissolution tests were also per-
formed and PSC-pH glass showed a stable pH, suggesting that a
higher content of phosphorous helps to stabilize the pH.
It should be taken into account that while glass dissolution
in vitro takes place in a static medium, glass corrosion in vivo takes
place in dynamic conditions resulting in a continuous dilution of
the ions leached from the material [33]. It is thus important to con-
sider that in vitro the effects of the release of alkali ions are magni-
fied because of the accumulation of ions in the sample vicinity and
the consequent excessive pH increase in the medium [33].
The specific effects of the increase of ion (e.g. Ca+) concentration
and rise of pH on cell behaviour, in this case, cannot be decoupled.
Therefore, future studies should focus on these two possible nega-
tive effects, separately. This aspect is especially important when
considering BGs with designed compositions that aim at releasing
biologically active ions, which can also contribute to the change of
pH when released.
Considering the results of the literature search carried out
(Table 1), it becomes clear that it is challenging to establish which
pre-treatment method (in SBF or cell culture medium) is most
effective to pre-condition BG samples for cell culture studies. Both
media have advantages and disadvantages (Table 2). On the one
hand, SBF has the advantage of being low cost and to have an ionic
concentration similar to that of human plasma, on the other hand
SBF only contains inorganic components without proteins, amino
acids or enzymes, which are obviously always present in biological
fluids. Cell culture medium, on the contrary, does not contain all
inorganic components of blood plasma, but it has the organic com-
ponents (e.g. proteins, amino acids, enzymes); on the other hand
cell culture medium has the advantage of being the same medium
used for growing and culturing cells. An alternative medium for
pre-treatment BGs could be the use of SBF enriched with suitable
organic compounds, e.g. proteins, enzymes [60,61]; however, such
media have not been yet investigated to condition BG samples. As
mentioned above, it is important to consider that physiological
processes occur in dynamic conditions in which a continuous
wash-out of the ions released from the material takes place. Such
dynamic fluid condition likely avoids a rapid and drastic
fluctuation of pH. In this context, the importance of changing the
 Table 1
tests, Acta Biomater. (2018), https://doi.org/10.1016/j.actbio.2018.05.019
Please cite this article in press as: F.E. Ciraldo et al., Tackling bioactive glass excessive in vitro bioreactivity: Preconditioning approaches for cell culture

Summary of pre-conditioning methods for different types of bioactive glasses used for carrying out cell biology tests.

Material Type of material Glass composition Preconditioning method* Cells

CEL2 based scaffold produced by foam replica technique Porous scaffolds 45SiO2, 3P2O5, 26CaO, 7MgO, 15Na2O, 4K2O SBF (1 week) + cell culture medium (24 h) Human osteoblasts cell line (MG-63)
[27] (porosity 48%) mol%
Rods of 45S5 bioactive glass [29] Bulk 45SiO2, 24.5CaO, 24.5Na2O, 6P2O5 wt. % SBF (72 h) Human bone marrow derived
mesenchymal stem cells (HBMSCs)
45S5 bioactive glass based scaffold + PE [21] Porous scaffolds 45SiO2, 24.5CaO, 24.5Na2O, 6P2O5 wt. % SBF (22 days) + a-MEM (48 h) MC3T3-E1 mouse pre-osteoblast cells
(porosity 60–70%)
Glasses belonging to the system SiO2-P2O5-CaO-MgO- Bulk SBF (1 week) Murine fibroblasts NIH-3 T3
K2O-Na2O [28]
Bioactive glass coating on Ti6Al4V [30] Particles (1mm) 6P61: 61.1SiO2, 10.3Na2O, 2.8K2O, 12.6CaO, SBF (2 weeks) MC3T3-E1.4 mouse osteoblast-like
8.9MgO, 6P2O5 wt.% cells
6P55: 54.5SiO2, 12Na2O, 4K2O, 15CaO, 8.5MgO,
6P2O5 wt.%
13–93 bioactive glass fibers and scaffolds [37] Bulk 56.6SiO2, 1.7P2O5, 22.1CaO, 7.7MgO, 7.9K2O, Unspecified cell culture medium (6h) MC3T3-E1 line of mouse pre-
6Na2O wt. % osteobla-stic cells
70S30C sol–gel bioactive glass scaffolds [34] Porous scaffolds 70SiO2, 30CaO mol% DMEM + penicillin +streptomycin (72 h) Human osteoblasts (HOBs)
(porosity 90%)
58S sol–gel based scaffolds [38] Porous scaffolds 60SiO2, 36CaO, 4P2O5 %mol DMEM (24 h) Human osteoblast cells (HOBs)

F.E. Ciraldo et al. / Acta Biomaterialia xxx (2018) xxx–xxx

(porosity 85–90%)
45S5 Bioglass [39] 58S sol–gel 77S sol–gel Particles (90–150 mm) 45SiO2, 24.5CaO, 24.5Na2O, 6P2O5 wt. % Unspecified cell culture medium (72 h) Murine Osteoblasts Murine
60SiO2, 36CaO, 4P2O5 %mol Fibroblasts
80SiO2, 16CaO, 4P2O5 %mol
P(3HB)/BioglassÒ composite [40] Bulk 45SiO2, 24.5CaO, 24.5Na2O, 6P2O5 wt. % Unspecified cell culture medium (12 h) Human osteoblast cell line (MG-63)
Chitosan-gelatin/nano bioactive glass scaffolds [41] Porous scaffolds 55SiO2, 40CaO, 5P2O5 %mol Unspecified cell culture medium (1h) Human osteoblast cell line (MG-63)
(porosity not specified)
Titanium dioxide doped phosphate based glass [42] Bulk 30CaO, 20Na2O, 50P2O5 %mol Unspecified cell culture medium (24 h) Human osteoblast cell line (MG-63)
30CaO, 19Na2O, 50P2O5, 1TiO2 %mol
30CaO, 17 Na2O, 50P2O5, 3TiO2 %mol
30CaO, 15Na2O, 50P2O5, 5TiO2 %mol
45S5 bioactive glass based scaffolds [43] Porous scaffolds 45SiO2, 24.5CaO, 24.5Na2O, 6P2O5 wt. % Unspecified cell culture medium (2 weeks) Human osteoblast cell line (MG-63)
(porosity 78–92%)
45S5 bioactive glass based scaffold produced via soft Porous scaffolds 45SiO2, 24.5CaO, 24.5Na2O, 6P2O5 wt. % Unspecified cell culture medium (48 h) Human osteoblast cell line (MG-63)
lithography [44] (porosity 78–92%)
Sol-gel bioactive glass based scaffolds [45] Fibers 70SiO2, 30CaO mol% Serum free a-MEM (72 h) Human osteoblast cell line (MG-63)
Melt derived bioactive glasses [46] Porous scaffolds ICIE16: 49.46SiO2, 36.6CaO, 6.6Na2O, 1.07P2O5, DMEM + penicillin + streptomycin (48 h) Human osteoblast cell line (MG-63)
(porosity 75%) 6.6K2O %mol
PSrBG: 44.5SiO2, 17.8CaO, 4Na2O, 4.5P2O5,
4K2O, 7.5MgO, 17.8SrO %mol
Sol-gel based glass ceramic composites [47] Bulk 60SiO2, 36CaO, 4P2O5 %mol Unspecified cell culture medium (72 h) MC3T3-E1 cells
Copper-doped silicate 13–93 bioactive glass scaffolds Porous scaffolds 53SiO2, 6Na2O, 12K2O, 5MgO, 20CaO, 4P2O5 % Unspecified cell culture medium (1h) pre-osteoblastic MC3T3-E1 cells
[48] (porosity 85%) mol doped with 0.4, 0.8, 2% of CuO
Graphene nano 58S bioactive glass scaffolds [49] Porous scaffolds 58SiO2, 33CaO, 13MgO, 6P2O5 mol% DMEM (24 h) Human osteoblast cell line (MG-63)
(porosity non
specified)
PDLLA + BioglassÒ scaffolds [50] Porous scaffolds 45SiO2, 24.5CaO, 24.5Na2O, 6P2O5 wt. % a-MEM (24 h) Human bone marrow derived
(porosity 90%) mesenchymal stem cells (HBMSCs)
Sol-gel glasses [51] Bulk 55SiO2, 26CaO, 13MgO, 6P2O5 mol% DMEM (24 h) MG63 osteoblasts
Bioactive glass disks [33] Bulk 46.1SiO2, 2.6P2O5, 24.4Na2O, 26.9CaO mol% TE (48 h) tissue culture medium (1 h) TE (48 Osteoblasts
h) + tissue culture medium (1 h)
Bulk 13–93 [52] 1–98 3–98 Bulk 56.6SiO2, 1.7P2O5, 22.1CaO, 7.7MgO, 7.9K2O, Tissue culture nedium (1 h) Human osteoblast cell line (MG-63)
6Na2O wt.%
53SiO2, 2P2O5, 22CaO, 5MgO, 11K2O, 6Na2O,
1B2O3 wt.
55SiO2, 4P2O5, 22CaO, 5MgO, 9K2O, 4Na2O,
1B2O3 wt.%
45S5 bioactive glass monolithic [53] Bulk 45SiO2, 24.5CaO, 24.5Na2O, 6P2O5 wt. % DMEM (24 h) Human primary osteoblasts (HOBs)

(continued on next page)

5
6 F.E. Ciraldo et al. / Acta Biomaterialia xxx (2018) xxx–xxx

Table 2

The ratio between material and medium used for pre-conditioning is not reported because it was not described in most of the papers. It is a common practice to use an amount of medium able to completely cover the sample.
Score Sheet for the pretreatment of BG-scaffolds prior to the use in cell culture,
Human osteoblast cell line (MG-63)

Human osteoblast cell line (MG-63)

Human osteoblast cell line (MG-63)

Human osteoblast cell line (MG-63)

Human primary osteoblasts (HOBs)

recommended by the authors based on the literature analysis.

Morphology Points
Powder 1
Monolithic, bulk 1
Low porous (<30%) 5
Highly porous (>31%) 10
BG-Type Points
<20% Na2O 5
Saos-2 cells

>21% Na2O 10
Medium/Buffer Points
Cells

TRIS 1
SBF 1
a-MEM 2
DMEM 2
Unspecified cell culture medium (48 h)
Cell culture medium + HEPES (1 week)

Culture type Points

Highly dynamic 1
Low dynamic 2
Tissue culture medium (48 h)

Static 10
Points Recommended time of pre-incubation
Preconditioning method*

<10 1h
11–20 24 h
21–24 48–72 h
>25 >72 h
DMEM (24 h)

DMEM (12 h)

DMEM (72 h)

In static cultures, daily medium change required. Incubation at 37 °C. For porous
scaffolds, normalization to scaffold volume and medium volume may be necessary
as well as increased pretreatment periods.
45SiO2, 3P2O5, 26CaO, 7MgO, 15Na2O, 4K2O

Table 3
45SiO2, 24.5CaO, 24.5Na2O, 6P2O5 wt. %

45SiO2, 24.5CaO, 24.5Na2O, 6P2O5 wt. %

45SiO2, 24.5CaO, 24.5Na2O, 6P2O5 wt. %

Example of a comparison between the pre-treatment period used in literature and the
one suggested by the score sheet approach (Table 2).

Study Score-sheet
Pretreatment Pretreatment Points
period period
Reilly et al. [29] 72 h 48–72 h 22
70SiO2, 30CaO mol%
Glass composition

Midha et al. [34] 72 h 72 h 27

Boccardi et al. [35] 1 week 72 h 32
Balamurugan et al. 48 h 24 h 18
[51]
mol%

pre-conditioning medium during the test period must be high-

lighted in order to simulate, as close as possible, the actual
physiological conditions. The use of dynamic tests is therefore an
(porosity 67–58%)
Type of material

Porous scaffolds

Porous scaffolds

Porous scaffolds

Porous scaffolds

Porous scaffolds

important alternative to test BGs [62–64].

(porosity 85%)

(porosity 68%)

(porosity 91%)
(porosity 90%)

Moreover, the duration of the pre-conditioning treatment can

change depending on the sample’s composition and morphology.
BGs with a high content of Na2O (above 10%) require a longer
Bulk

pre-treatment period compared with BGs with a lower Na2O con-

tent due to the rapid exchange of Na+ ions with H+ or H3O+ ions
45S5 bioactive glass with different P content (0. 3, 6, 12%)

45S5 bioactive scaffolds based on natural marine sponges

Sol–gel derived 45S5 BioglassÒ – ceramic scaffolds [55]

45S5 BioglassÒ derived scaffolds coated with organic –

from the solution.

In addition, the morphology of the sample (e.g. porosity, surface
Bioactive glass/polymer composite scaffolds [57]

area) plays an important role in determining the duration of the

inorganic hybrids containing graphene [56]

pre-treatment. Bulk or monolithic materials seem to require a

shorter period of pre-conditioning, most probably do to the slower
70S30C sol–gel derived scaffolds [58]

rate of exchange of ions once in contact with the aqueous solution.

For example, rods of 45S5 bioactive glass studied by Reily et al. [29]
or based on 13–93, 1–98 and 3–98 BGs investigated by Itälä et al.
[52] were pre-conditioned for 3 days and 1 h, respectively. Espe-
cially when considering porous materials (scaffolds), the ratio of
BG sample volume to medium volume must be carefully consid-
ered because of the direct correlation between the amount of solid
Table 1 (continued)

phase and the pH variation in a defined volume of medium [36].

On the contrary, very porous materials need a longer period of
Material

[54]

[35]

incubation due to the rapid exchange of ions with the solution

and the consequent alkalization of the surrounding environment.
*

Bellucci et al. [21] investigated the pH variation which resulted

Please cite this article in press as: F.E. Ciraldo et al., Tackling bioactive glass excessive in vitro bioreactivity: Preconditioning approaches for cell culture
tests, Acta Biomater. (2018), https://doi.org/10.1016/j.actbio.2018.05.019
 F.E. Ciraldo et al. / Acta Biomaterialia xxx (2018) xxx–xxx
when soaking 45S5 BG derived scaffolds (porosity >70%) in SBF and
found that 22 days of pre-treatment are necessary to stabilize the
in vitro pH near the physiological value (7.4). Similar results were
obtained by Detsch et al. [43] who studied the biocompatibility of
45S5 BG based scaffolds. In this study, cultivation of two weeks in
cell culture medium was performed before starting the cell
seeding.
Alternatively, BGs free of sodium and with a high phosphorous
content can be used in cell culture experiments without pre-
conditioning [22], as mentioned above.
Table 2 summarizes the recommended pre-incubation times
depending on composition and morphology of the BG samples
investigated. Considering in detail the information in the literature,
it becomes apparent that the morphology, BG type, medium, and
cell culture type used, are the most important variables to define
adequate pre-treatment periods. The scores reported in Table 2
are given following an empirical evaluation of their effect on the
change of pH. Values (points) between 1 and 10 were given on
the basis of the different effects (weight) of the respective param-
eters. The rating system shown in Table 2 was designed to consider
the weight of the influence of the specific parameters: those
parameters with the lowest influence on pretreatment periods
were set with a value of 1. The level of influence of other parame-
ters was then compared to this value: for example, if a parameter
was considered to have a 10 times stronger impact on the pretreat-
ment time, the value for this feature was 10.
As an example, for the parameter ‘‘BG composition”, it has been
observed that BGs containing a high content of Na2O (>21 wt%)
require a pre-treatment time that is roughly double than for
glasses with low Na2O concentrations. In general, authors agree
that the type of pre-treatment medium does not affect significantly
the incubation period. However media with buffering capacity can
slightly reduce the incubation time as also shown in Table 2.
Indeed, the points assigned are very close to each other and would
not influence too much the overall score. Moreover, a dynamic cul-
ture usually shortens the pre-treatment period when compared to
a static culture, since more frequent medium exchanges increase
the buffer capacity of the experimental system.
To prove the proposed score points concept, publications listed
in Table 1 were taken randomly into account and the score sheet
reported in Table 2 was applied. Table 3 shows that the pre-
treatment period used in each study corresponds with the pre-
treatment period suggested by the score sheet.
The guideline proposed is meant to represent the minimum
time required for the pre-treatment of BGs in cell culture studies
using the listed conditions. However, there are laboratory proto-
cols used (e.g. frequency of medium refreshment) that are not
always described in published papers but may have a significant
effect in determining the incubation time.
5. Conclusions
An extensive review of the literature highlights that a pre-
conditioning treatment is required before seeding cells on bioac-
tive glass samples (powder, scaffolds) in order to prevent the alka-
lization of the surrounding environment that can provoke serious
damage to the cells. Without pre-conditioning, the pH of the solu-
tion rises over physiological values causing changes in cell function
and activity. This review paper has summarized the different
methodologies proposed for the pre-treatment of different types
of BG samples for cell culture tests according to literature, high-
lighting the important parameters that must be considered to
design pre-conditioning tests, mainly using SBF or cell culture
medium. It is expected that by assessing the results published
and the analysis provided in this paper, researchers will be able
Please cite this article in press as: F.E. Ciraldo et al., Tackling bioactive glass excessive in vitro bioreactivity: Preconditioning approaches for cell culture
tests, Acta Biomater. (2018), https://doi.org/10.1016/j.actbio.2018.05.019
7
to make an informed decision when considering their pre-
treatment protocol for future cell culture studies on BGs. An effort
to normalize the pre-treatment protocols for different BG sample
types should be also considered in future by the BG research
community.
Acknowledgments
This research was carried out within the EU Horizon 2020
framework project COACH (ITN-ETN, Grant agreement 642557).
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