Tackling bioactive glass excessive in vitro bioreactivity: Preconditioning approaches for cell culture tests

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 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 b

a r t i c l e

i n f o

Article history: Received 20 March 2018 Received in revised form 8 May 2018 Accepted 12 May 2018 Available online xxxx Keywords: Bioactive glasses Bioactivity Cell culture In vitro pH

a b s t r a c t Bioactive glasses (BGs) are being increasingly considered for biomedical applications in bone and soft tissue replacement approaches thanks to their ability to form strong bonding with tissues. However, due to their high reactivity once in contact with water-based solutions BGs rapidly exchange ions with the surrounding environment leading in most cases to an undesired increase of the pH under static in vitro conditions (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 to limit this problem. This paper presents an overview of the different strategies that have been put forward to pre-treat BG samples to tackle the pH raise issue in order to enable cell biology studies. The paper also discusses the relevant criteria that determine the selection of the optimal pre-treatment depending on the BG composition and morphology (e.g. particles, scaffolds). 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 tissues once in contact with biological fluid. The enhanced interaction of BGs with the biological environment 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 determination 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 support 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. 2. 3. 4.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . Bioreactivity of bioactive glasses. . . . . . . . . . . . Preconditioning methods for bioactive glasses. Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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⇑ Corresponding author. E-mail address: [email protected] (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

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

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

1. Introduction In the last years the demand for new materials for bone replacement applications has gained continuous importance due to the increase of the average age of the population and the increasing number of surgical procedures [1]. In particular, bone defects above a critical size cannot be repaired by the self-healing of bone tissue and require an osteoconductive and osteoinductive device (scaffold) able to support the regeneration of the new tissue [2]. Although autografts are still considered the ‘gold standard’, they have many drawbacks such as limited availability and morbidity of the donor site [3]. Xenografts and allografts could be considered a valid alternative, however they have potential drawbacks such as relatively low rates of integration, risk of contamination, immune rejection or viral transmission from the donor [4]. For these reasons, engineered biomaterials are considered highly promising candidates for bone tissue regeneration [2,5–7]. 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 fabricating tissue engineering scaffolds [5,6,9–12]. The tissue bonding ability of BGs is based on the high surface reactivity of these materials in contact with aqueous environments. Moreover, a special advantage 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]. Moreover 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 osteoblast activity [10]. Such bioreactivity of BGs has been also considered 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 settings [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 stimulation of osteogenic differentiation or the upregulation of angiogenic growth factors [10,14,16,17]. However, one important issue to consider when BGs get in contact with biological fluids is the development of possible pHdependent 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 in vivo [6,12], it becomes highly relevant when testing BGs in vitro.

For this reason, in vitro studies to evaluate the behaviour of cells in contact with bioactive glasses must adopt some form of preconditioning of BG samples to limit such non-realistic pH changes. As an example, Fig. 1 shows the pH variation in simulated body fluid (SBF) containing glass powders (45S5 and 58S) with and without pre-conditioning treatment as a function of time [18]. It is shown that pre-conditioning clearly limits the pH excursion without affecting the hydroxyl-carbonate-apatite formation on the powder surfaces, as reported in ref. [18]. Over the years, laboratories around the world have developed a variety of methods and conditioning protocols to pre-treat BGs prior to cell biology studies. This review summarizes and discusses the different preconditioning methodologies put forward for in vitro cell culture characterization of BGs and offers suggestions to select the most efficient, cost effective and fastest method available. Here, we will consider silicate BGs, both sol-gel and met-derived, also discussing different BG morphologies, such as granules, pellets and porous scaffolds.

Fig. 1. pH variation of SBF containing bioactive glasses (45S5 and 58S composition) 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 tetrahedron, 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 leading to glass dissolution and release of ions (e.g. calcium and phosphate ions). A characteristic ion exchange at the glass/water interface occurs when silicate glasses come in contact with aqueous 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 diffusion 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 correct 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.

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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) simulated body fluid (SBF), a solution containing similar ion concentration 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 necessary 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 preconditioning, 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 covered the BG surface and produced considerable extracellular matrix (ECM). When the treatment was stopped after 20 h, osteoblasts 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 supplemented 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 control 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 activity on BG samples conditioned by the two step procedure was analysed. 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, osteoblasts 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 biological 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 plateau at about 7.75, which was considered to be an optimal value for osteoblast adhesion [27]. The surface of the samples was covered by HCA crystals and the cells were anchored onto these agglomerates forming bridges between the HCA crystals.

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

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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-P2O5CaO-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 gellike 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 preconditioning 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 observations 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 therapeutic ion release ability, BGs are promising biomaterials for tissue 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 metabolism and function. Indeed, a fast increase of the pH can influence cell respiration mechanisms causing enzyme alteration and modification 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 concentration of Ca (of 88–109 ppm) is toxic for human osteoblasts and it

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 negative effects on cell viability, can be properly stabilized by this pretreatment. 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 medium. 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 synthesis 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 performed 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 consider that in vitro the effects of the release of alkali ions are magnified 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 negative 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 components (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

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

Material

Type of material

Glass composition

Preconditioning method*

Cells

CEL2 based scaffold produced by foam replica technique [27] Rods of 45S5 bioactive glass [29]

Porous scaffolds (porosity 48%) Bulk

45SiO2, 3P2O5, 26CaO, 7MgO, 15Na2O, 4K2O mol% 45SiO2, 24.5CaO, 24.5Na2O, 6P2O5 wt. %

SBF (1 week) + cell culture medium (24 h)

Human osteoblasts cell line (MG-63)

SBF (72 h)

45S5 bioactive glass based scaffold + PE [21]

Porous scaffolds (porosity 60–70%) Bulk

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

SBF (22 days) + a-MEM (48 h)

Human bone marrow derived mesenchymal stem cells (HBMSCs) MC3T3-E1 mouse pre-osteoblast cells

SBF (1 week)

Murine fibroblasts NIH-3 T3

Particles (1mm)

6P61: 61.1SiO2, 10.3Na2O, 2.8K2O, 12.6CaO, 8.9MgO, 6P2O5 wt.% 6P55: 54.5SiO2, 12Na2O, 4K2O, 15CaO, 8.5MgO, 6P2O5 wt.% 56.6SiO2, 1.7P2O5, 22.1CaO, 7.7MgO, 7.9K2O, 6Na2O wt. % 70SiO2, 30CaO mol%

SBF (2 weeks)

MC3T3-E1.4 mouse osteoblast-like cells

Unspecified cell culture medium (6h) DMEM + penicillin +streptomycin (72 h)

MC3T3-E1 line of mouse preosteobla-stic cells Human osteoblasts (HOBs)

60SiO2, 36CaO, 4P2O5 %mol

DMEM (24 h)

Human osteoblast cells (HOBs)

45SiO2, 60SiO2, 80SiO2, 45SiO2, 55SiO2,

24.5CaO, 24.5Na2O, 6P2O5 wt. % 36CaO, 4P2O5 %mol 16CaO, 4P2O5 %mol 24.5CaO, 24.5Na2O, 6P2O5 wt. % 40CaO, 5P2O5 %mol

Unspecified cell culture medium (72 h)

Murine Osteoblasts Murine Fibroblasts

Unspecified cell culture medium (12 h) Unspecified cell culture medium (1h)

Human osteoblast cell line (MG-63) Human osteoblast cell line (MG-63)

30CaO, 20Na2O, 50P2O5 %mol 30CaO, 19Na2O, 50P2O5, 1TiO2 %mol 30CaO, 17 Na2O, 50P2O5, 3TiO2 %mol 30CaO, 15Na2O, 50P2O5, 5TiO2 %mol 45SiO2, 24.5CaO, 24.5Na2O, 6P2O5 wt. %

Unspecified cell culture medium (24 h)

Human osteoblast cell line (MG-63)

Unspecified cell culture medium (2 weeks)

Human osteoblast cell line (MG-63)

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

Unspecified cell culture medium (48 h)

Human osteoblast cell line (MG-63)

70SiO2, 30CaO mol% ICIE16: 49.46SiO2, 36.6CaO, 6.6Na2O, 1.07P2O5, 6.6K2O %mol PSrBG: 44.5SiO2, 17.8CaO, 4Na2O, 4.5P2O5, 4K2O, 7.5MgO, 17.8SrO %mol 60SiO2, 36CaO, 4P2O5 %mol 53SiO2, 6Na2O, 12K2O, 5MgO, 20CaO, 4P2O5 % mol doped with 0.4, 0.8, 2% of CuO 58SiO2, 33CaO, 13MgO, 6P2O5 mol%

Serum free a-MEM (72 h) DMEM + penicillin + streptomycin (48 h)

Human osteoblast cell line (MG-63) Human osteoblast cell line (MG-63)

Unspecified cell culture medium (72 h) Unspecified cell culture medium (1h)

MC3T3-E1 cells pre-osteoblastic MC3T3-E1 cells

DMEM (24 h)

Human osteoblast cell line (MG-63)

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

a-MEM (24 h)

55SiO2, 26CaO, 13MgO, 6P2O5 mol% 46.1SiO2, 2.6P2O5, 24.4Na2O, 26.9CaO mol%

DMEM (24 h) TE (48 h) tissue culture medium (1 h) TE (48 h) + tissue culture medium (1 h) Tissue culture nedium (1 h)

Human bone marrow derived mesenchymal stem cells (HBMSCs) MG63 osteoblasts Osteoblasts Human osteoblast cell line (MG-63)

DMEM (24 h)

Human primary osteoblasts (HOBs)

Glasses belonging to the system SiO2-P2O5-CaO-MgOK2O-Na2O [28] Bioactive glass coating on Ti6Al4V [30]

13–93 bioactive glass fibers and scaffolds [37]

Bulk

70S30C sol–gel bioactive glass scaffolds [34]

Porous scaffolds (porosity 90%) Porous scaffolds (porosity 85–90%) Particles (90–150 mm)

58S sol–gel based scaffolds [38] 45S5 Bioglass [39] 58S sol–gel 77S sol–gel

P(3HB)/BioglassÒ composite [40] Chitosan-gelatin/nano bioactive glass scaffolds [41] Titanium dioxide doped phosphate based glass [42]

45S5 bioactive glass based scaffolds [43] 45S5 bioactive glass based scaffold produced via soft lithography [44] Sol-gel bioactive glass based scaffolds [45] Melt derived bioactive glasses [46]

Sol-gel based glass ceramic composites [47] Copper-doped silicate 13–93 bioactive glass scaffolds [48] Graphene nano 58S bioactive glass scaffolds [49]

Bulk Porous scaffolds (porosity not specified) Bulk

Porous scaffolds (porosity 78–92%) Porous scaffolds (porosity 78–92%) Fibers Porous scaffolds (porosity 75%)

Sol-gel glasses [51] Bioactive glass disks [33]

Bulk Porous scaffolds (porosity 85%) Porous scaffolds (porosity non specified) Porous scaffolds (porosity 90%) Bulk Bulk

Bulk 13–93 [52] 1–98 3–98

Bulk

45S5 bioactive glass monolithic [53]

Bulk

PDLLA + BioglassÒ scaffolds [50]

56.6SiO2, 1.7P2O5, 22.1CaO, 7.7MgO, 7.9K2O, 6Na2O wt.% 53SiO2, 2P2O5, 22CaO, 5MgO, 11K2O, 6Na2O, 1B2O3 wt. 55SiO2, 4P2O5, 22CaO, 5MgO, 9K2O, 4Na2O, 1B2O3 wt.% 45SiO2, 24.5CaO, 24.5Na2O, 6P2O5 wt. %

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(continued on next page)

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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

Table 1 Summary of pre-conditioning methods for different types of bioactive glasses used for carrying out cell biology tests.

DMEM (72 h) 70S30C sol–gel derived scaffolds [58]

45S5 bioactive scaffolds based on natural marine sponges [35] 45S5 BioglassÒ derived scaffolds coated with organic – inorganic hybrids containing graphene [56] Bioactive glass/polymer composite scaffolds [57]

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. *

DMEM (12 h)

45SiO2, 3P2O5, 26CaO, 7MgO, 15Na2O, 4K2O mol% 70SiO2, 30CaO mol%

Human primary osteoblasts (HOBs)

Human osteoblast cell line (MG-63) Unspecified cell culture medium (48 h) 45SiO2, 24.5CaO, 24.5Na2O, 6P2O5 wt. %

Human osteoblast cell line (MG-63)

Saos-2 cells Cell culture medium + HEPES (1 week) 45SiO2, 24.5CaO, 24.5Na2O, 6P2O5 wt. %

Human osteoblast cell line (MG-63) Tissue culture medium (48 h)

Bulk

45S5 bioactive glass with different P content (0. 3, 6, 12%) [54] Sol–gel derived 45S5 BioglassÒ – ceramic scaffolds [55]

Porous scaffolds (porosity 85%) Porous scaffolds (porosity 68%) Porous scaffolds (porosity 90%) Porous scaffolds (porosity 67–58%) Porous scaffolds (porosity 91%)

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

DMEM (24 h)

Type of material Material

Table 1 (continued)

Glass composition

Preconditioning method*

Human osteoblast cell line (MG-63)

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

Cells

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Table 2 Score Sheet for the pretreatment of BG-scaffolds prior to the use in cell culture, recommended by the authors based on the literature analysis. Morphology Powder Monolithic, bulk Low porous (<30%) Highly porous (>31%)

Points 1 1 5 10

BG-Type <20% Na2O >21% Na2O

Points 5 10

Medium/Buffer TRIS SBF a-MEM DMEM

Points 1 1 2 2

Culture type Highly dynamic Low dynamic Static

Points 1 2 10

Points <10 11–20 21–24 >25

Recommended time of pre-incubation 1h 24 h 48–72 h >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.

Table 3 Example of a comparison between the pre-treatment period used in literature and the one suggested by the score sheet approach (Table 2).

Reilly et al. [29] Midha et al. [34] Boccardi et al. [35] Balamurugan et al. [51]

Study

Score-sheet

Pretreatment period

Pretreatment period

Points

72 h 72 h 1 week 48 h

48–72 h 72 h 72 h 24 h

22 27 32 18

pre-conditioning medium during the test period must be highlighted in order to simulate, as close as possible, the actual physiological conditions. The use of dynamic tests is therefore an important alternative to test BGs [62–64]. 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 pre-treatment period compared with BGs with a lower Na2O content due to the rapid exchange of Na+ ions with H+ or H3O+ ions from the solution. In addition, the morphology of the sample (e.g. porosity, surface area) plays an important role in determining the duration of the pre-treatment. Bulk or monolithic materials seem to require a shorter period of pre-conditioning, most probably do to the slower 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. Especially when considering porous materials (scaffolds), the ratio of BG sample volume to medium volume must be carefully considered because of the direct correlation between the amount of solid phase and the pH variation in a defined volume of medium [36]. On the contrary, very porous materials need a longer period of 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

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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 preconditioning [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 parameters. 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 parameters was then compared to this value: for example, if a parameter was considered to have a 10 times stronger impact on the pretreatment 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 culture 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 pretreatment period used in each study corresponds with the pretreatment 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 protocols 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 preconditioning treatment is required before seeding cells on bioactive glass samples (powder, scaffolds) in order to prevent the alkalization of the surrounding environment that can provoke serious damage to the cells. Without pre-conditioning, the pH of the solution 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, highlighting 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

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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