Fabrication and characterization of Ag- and Ga-doped mesoporous glass-coated scaffolds based on natural marine sponges with improved mechanical properties

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/jbm.a.37123 Fabrication and characterization of Ag- and Ga-doped mesoporous glass-coated scaffolds based on natural marine sponges with improved mechanical properties F. E. Ciraldo1, M. Arango-Ospina1, W. H. Goldmann2, A. M. Beltran3, R. Detsch1, A. Gruenewald1, J. A. Roether4, A. R. Boccaccini1,* 1Institute of Biomaterials, Department of Materials Science and Engineering, University of Erlangen-Nuremberg, Cauerstraße 6, 91058 Erlangen, Germany 2Institute of Biophysics, Department of Physics, University of Erlangen-Nuremberg, Henkestraße 91, 91052 Erlangen, Germany 3Department of Materials Science and Engineering. Escuela Politécnica Superior, Universidad de Sevilla. Virgen de África 7, 41011, Seville, Spain 4Institute of Polymer Materials, Department of Materials Science and Engineering, University of Erlangen- Nuremberg, Martenstraße 5, 91058 Erlangen, Germany * Correspondence: [email protected]; Tel.: +49-9131-852-8601 Abstract Natural marine sponges were used as sacrificial template for the fabrication of bioactive glassbased scaffolds. After sintering at 1050 ºC, the resulting samples were additionally coated with a sol silicate solution containing biologically active ions (Ag and Ga), well-known for their antibacterial properties in comparison with standard scaffolds made by PU foam templates. The produced scaffolds were characterized by superior mechanical properties (maximum compressive strength of 4 MPa) and total porosity of ~80%. Direct cell culture tests performed on the uncoated and coated samples showed positive results in terms of adhesion, proliferation, and differentiation of MC3T3-E1 cells. Moreover, vascular endothelial growth factor (VEGF) secretion from cells in contact with scaffold dissolution products was measured after 7 and 10 days of incubation, showing promising angiogenic results for bone tissue engineering applications. The antibacterial potential of the produced samples was assessed by performing agar diffusion tests against both Gram-positive and Gram-negative bacteria. KEYWORDS: bioactive glass; bone tissue engineering; scaffolds; ion delivery; VEGF; antibacterial This article is protected by copyright. All rights reserved. 1. Introduction Although bone has the capability to regenerate itself without forming a scar, it cannot heal in case of so-called critical size defects. Autografts are nowadays still considered “the gold standard” for bone grafting due to their biocompatibility [1]. Drawbacks of this approach include infection risk at the donor site, pain for the patient, prolonged hospitalization and rehabilitation periods as well as the extra operating time required for treating both the implant and donor sites [2–5]. A valid alternative is provided by tissue engineering (TE), which involves the support of damaged tissue regeneration by the use of engineered biomaterials called scaffolds. One of the main challenges of TE is the design of porous 3-D scaffolds to induce bone regeneration [6]. In the case of bone TE, bioactive materials able to promote bone regeneration have been in the focus of significant research efforts in the last 20+ years [6]. The most studied and used silicate biomaterial for bone defect filling is 45S5 bioactive glass (BG) (also known as Bioglass), which was developed in 1971 by Hench et al. [7]. Bioglass is a silicate glass with a nominal composition (in wt%) of 45% SiO2, 24.5% CaO, 24.5% Na2O, 6% P2O5, which is able to bond to both soft and hard tissue once in contact with biological fluids [8,9]. In several studies, it was demonstrated that dissolution products released from the bioactive glass surface enhance, on one side, the formation of hydroxyl-carbonate-apatite (HCA) similar to the mineral phase of bone and, on the other side, osteogenesis and angiogenesis [10–14]. Bioactive glasses are being used in bulk or particulate form in non-load bearing sites for dental and orthopedic applications [10,15]. On the other hand, porous 3-D BG scaffolds are not used in clinical applications yet. The main challenge is to fabricate 3-D scaffolds that combine high porosity and adequate mechanical strength and stability similar to natural bone. The first 3-D Bioglass-based scaffolds were developed by Chen et al in 2006 [16], who introduced the standard foam replica technique for the development of scaffolds with porosities > 90%, using polyurethane (PU) This article is protected by copyright. All rights reserved. foams as sacrificial templates. In subsequent studies, it was shown that those scaffolds do not possess suitable mechanical properties for use in load-bearing clinical applications [6,17]. An alternative approach to fabricate BG-based scaffolds with increased mechanical strength is the reduction of the total porosity while maintaining pore interconnectivity and pore sizes adequate for new tissue ingrowth and vascularization. In the last years, porous BG-based scaffolds with higher mechanical properties have been produced by the foam replica method and a powder metallurgy approach [17,18], or using alternative sacrificial templates, e.g. marine natural sponges [6,19]. Samples fabricated with these techniques showed mechanical strength values comparable to those of trabecular bone (2-12 MPa). Mesoporous bioactive glasses (MBG) are gaining significant attention in the field of bone repair and drug delivery applications [20–23]. As a result of their large surface area, tailored mesoporosity and ordered pore structure, MBG show faster apatite mineralization compared to nonmesoporous bioactive glasses and offer the possibility to be loaded with drugs or biomolecules, i.e. introducing a local drug delivery function to the scaffold [20,24–27]. Different therapeutic ions have been incorporated into MBG during or after synthesis such as copper, lithium, boron, strontium, silver, cerium, and gallium because of their potential to promote the formation of new bone due to their stimulating effect on angiogenesis and osteogenesis [28–30]. In this work, sol-gel derived ordered mesoporous glasses doped with gallium or silver were employed to coat BG- based scaffolds applying the impregnation technique. Scaffolds were fabricated using 45S5 BG as the starting material and natural marine sponges as sacrificial templates. The scaffolds were coated using either Ag or Ga-doped mesoporous glass slurries. The ability of the produced glasses to form HCA on their surface was verified by immersing them in simulated body fluid [31]. The surface modifications of the scaffolds were evaluated using SEM and FTIR. The overall performance of the MBG-coated 3-D scaffolds in terms of biological activity and antibacterial effects was studied. 2. Materials and methods This article is protected by copyright. All rights reserved. 2.1 Synthesis of ordered mesoporous glasses Gallium- and silver-doped ordered mesoporous glasses (Ga-MBG and Ag-MBG) with a composition (in % mol.) of 78 SiO2-20 CaO-1.2 P2O5-0.8 AgO and 68.8 SiO2-20 CaO-1.2 P2O5- 10 Ga2O3, respectively, were produced by an evaporation induced self-assembly process (EISA). Briefly, Pluronic® F127 was dissolved in EtOH and HNO3 overnight. Glass precursors were added under continuous stirring as follows: tetraethyl orthosilicate (TEOS), triethyl phosphate (TEP), calcium nitrate (Ca(NO3)2·4H2O) and silver (AgNO3) or gallium nitrate (Ga(NO3)3), with an interval between each addition of 3 h. All reagents were used without further purification. Ga- MBG was characterized by means of scanning electron microscopy (SEM) (Zeiss, Oberkochen, Germany) in combination with energy dispersive spectroscopy (EDS) (Oxford Instruments, Abingdon, UK), which was used to qualitatively confirm the incorporation of gallium in the glass. The inner microstructure was investigated, using transmission electron microscopy (TEM) analysis operating at an acceleration voltage of 200 kV and small angle XRD (SAXRD). X-ray fluorescence (XRF) (Zetius, PANalytical) analysis was performed to confirm the gallium presence and to identify the chemical formulation of the glass. Fourier transformed infrared spectroscopy (FTIR) (4000-400 cm-1, 32 scans, Nicolet 6700, Thermo Scientific, Waltham, MA, USA) and SEM were used to analyze the samples after immersion in SBF. The used SBF solution was analyzed in terms of Ca, P, Si, Ag and Ga ion concentration, using inductively coupled plasma optical emission spectrometry. Ag-MBG has been characterized in a previous work [32]. 2.2 Fabrication of bioactive glass-based scaffolds Melt-derived 45S5 bioactive glass powder (Schott Vitryxx®, Mainz, Germany) of nominal mean particle size < 2 μm was used to fabricate BG-based scaffolds and natural marine sponges “Spongia Agaricina” (SA) were used as sacrificial templates [6]. Marine sponges belong to the “Elephant Ears” family and were harvested in the Indo-Pacific Ocean (Pure Sponges, Solihull, UK). BG scaffolds were produced according to the foam replica technique described by Chen et al. in 2006 [16]. PVA with a ratio of 0.01 mol·L-1 was dissolved in deionized water at 80°C for and This article is protected by copyright. All rights reserved. then BG powder was added using a concentration of 40 wt%. After cutting the foams in cylindrical shapes (Ø=8 mm and h=8mm), they were dipped in the PVA/BG slurry for 10 min. Subsequently, the excess of slurry was manually removed and the coated foams were slowly dried at room temperature. The procedure was repeated a second time. Compressed air, as previously reported [6,32], was applied this time to eliminate the excess of slurry. After drying, a heat treatment was necessary to burn out the natural marine sponges and to obtain the sintered structure. A temperature of 400 °C for 1 h was used to remove the sacrificial template and of 1050 °C for 1 h to densify the structure. The resulting scaffolds were coated by Ga-MBG or Ag- MBG using the impregnation technique. 500 µL of the sol silicate solution was dropped onto the scaffold, the samples were then dried at room temperature for 24 h and then they underwent a second heat treatment at 700 °C for 3 h (Figure 1). Where required, samples were labelled SA_Ag-MBG and SA_Ga-MBG to refer to Ag-MBG and Ga-MBG coated scaffolds, respectively. 2.2.1 Characterization of coated scaffolds The ability of the coated scaffolds to form HCA on their surfaces was verified by immersing them in simulated body fluid [31]. The surface modifications of the scaffolds were evaluated using SEM and FTIR. Also, the inner microstructure was investigated using transmission electron microscopy (TEM) operating at an acceleration voltage of 200 kV. The mechanical properties of the obtained samples were also evaluated before and after immersion in SBF up to 28 days. An ion release study was performed by means of ICP-OES device. The overall biological performance of the MBG-coated 3-D scaffolds in terms of cell biology activity and antibacterial effects was evaluated. Indirect and direct cell culture tests were performed to prove the biocompatibility of the samples against MC3T3-E1 mouse derived pre-osteoblasts. Agar diffusion tests were carried out using both Gram-positive (S. carnosus) and Gram-negative (E. coli) bacteria. 2.3 Indirect cell culture This article is protected by copyright. All rights reserved. Indirect cell culture tests were carried out following the general guidance set by the International Standardization Organization (ISO 10993-5:2009). Samples were disinfected at 160 °C for 2h and placed in contact with cell culture medium for 24 h in a culture incubator at 37 °C and 5% CO2. The ratio mass/volume used was 0.1 g·mL-1 as recommended by the ISO standard. At the end of the incubation time, the supernatant was collected and diluted to obtain the following concentrations: 100%, 50%, and 25%, which were used for the test. MC3T3-E1 osteoblast likecells were placed in contact with the conditioned media and incubated for 48 h at 37 °C and 5% CO2. The influence of the different glasses on the cell viability was assessed using a WST-8 assay (Sigma-Aldrich, Germany). At the end of the cultivation period the activity of MC3T3-E1 cells was measured through the conversion of tetrazolium (WST-8) to formazan by intracellular enzymes. A solution containing 1 vol% WST reagent and 99 vol% α-MEM was prepared. The cell culture medium was removed, cells were washed with PBS and 300 µL of WST solution was added to each well. The cells were then incubated for 3 h at 37 °C and 5% CO2. At the end of the incubation time, 100 µL of the solution was taken from the samples and transferred into a 96-well plate. The absorbance was measured at 450 nm (Anthos, Germany). Results are presented as mean value and standard deviation of six replica of each sample type. All the samples were normalized to 0 wt/vol. 6% DMSO was used as the negative control. One-way analysis of variance (ANOVA) was used to evaluate the differences in analysis parameters between the different dilution concentrations and the different glass types. The level of significance was defined at p <0.05 (Origin 8.1, Origin Lab Corporations, USA). The significance levels were set as p <0.05=*, p <0.01=** and p <0.001=***. The Bonferroni test was used for comparison of the mean values. 2.4 Direct cell test Uncoated and coated scaffolds were tested using osteoblast-like cells. Both the cell viability and proliferation capability were investigated. Direct cell culture tests were performed to investigate cell behavior, once in contact with BG scaffolds. From the literature, it is well known that a pre- This article is protected by copyright. All rights reserved. conditioning treatment is necessary to avoid a rapid increase of pH, which will make in vitro cell culture tests difficult or even impossible [31]. In the present work, every scaffold was sterilized by means of dry heat at 160 °C for 3.5 h and pre-conditioned in 1 mL of cell culture medium in an incubator at 37 °C and 5% CO2 until the pH was lower than 8. The medium was changed every day to better simulate the physiological conditions, where a continuous wash-out of the ions takes place. 2.4.1 Cell seeding MC3T3-E1 cells were chosen to perform the experiments. This cell line can differentiate into osteoblasts and thus the cells are relevant for the present study as they promote formation of calcified bone tissue. Mineral deposits have been identified as hydroxyapatite. They were cultured in high glucose DMEM supplemented with 10 vol.% of fetal bovine serum (FBS) and 1 vol.% of penicillin-streptomycin (PenStrep) and incubated at 37 °C in a humidified atmosphere (95%) containing 5% CO2. Trypsinization was used to collect cells once they had reached confluency of between 80 and 100%. Trypan blue exclusion method was used to count cells. A concentrated cell suspension in a drop-wise manner was chosen to seed cells directly onto the scaffolds. A droplet of 50 µL containing 80,000 cells was used. After this procedure, scaffolds were incubated at 37°C, in 5% CO2 for 45 min. Each well was then flooded with medium and cultured for 24 h, 3, 7, and 10 days. Cell viability was measured by means of WST-8 metabolism and lactate dehydrogenase (LDH) activity. VEGF release was measured after 7 and 10 days of incubation. Cell morphology was observed by SEM analysis. 2.4.2 VEGF production Production of Vascular Endothelial Growth Factor (VEGF), known to be a fundamental biomolecule for the regulation of angiogenesis, was quantitatively measured using an ELISA (Enzyme-linked Immunosorbent Assay) kit (RayBio® Mouse VEGF-A ELISA, Ray Biotech, Ins. Nocross, USA). This assay consists of a well plate coated with a specific antibody for mouse VEGF. Aliquots of the culture medium from the direct test were taken after 7 and 10 days and This article is protected by copyright. All rights reserved. transferred to the ELISA well plate. After 2.5 h of incubation, the wells were washed with the provided wash buffer and then incubated with the following reagents: a biotinylated antibody for 1 hour, a streptavidin solution for 45 minutes, and a one-step substrate reagent for 30 minutes and finally a stop solution. Between incubation steps, the samples were washed using the provided buffer. Once the stop solution was added, the absorbance at 450 nm was read immediately. The intensity of the colored solution is directly proportional to the concentration of VEGF secreted by the MC3T3-E1 cells in the presence of the samples. In addition a standard curve was created according to the manufacturer’s protocol to read out the unknown amount of VEGF in the samples. 2.4.3 LDH activity Lactate dehydrogenase (LDH) is an enzyme, which can be found inside cells and can be used to measure both the number of cells or their membrane integrity. Cell numbers can be measured by total cytoplasmic lactate dehydrogenase; the membrane integrity depends on the amount of cytoplasmic LDH released in the medium. The main reaction characteristic of the assay is the reduction of NAD by LDH, which leads to a reduced NADH. A stoichiometric reaction is used to convert the reduced NADH in a tetrazolium dye, a colored compound that can be detected spectrometrically. The amount of LDH is proportional to the cell number. Cells, which were attached to the scaffolds, were lysed after 1, 3, 7, and 10 days of incubation using a lysis buffer (0.1 wt.% Triton X, 20 mM TRIS, 1 mM MgCl2 and 0.1 mM ZnCl2) and centrifuged. A mastermix was prepared according to the manufacturer’s protocol (TOX-7, Sigma-Aldrich). 20 µL of dye solution was mixed with 20 µL of cofactor preparation and 20 µL of substrate solution. Afterwards, 140 µL of the supernatant was mixed with 60 µL of mastermix and incubated at room temperature for 30 minutes. During this incubation time, NADH is used to transform a tetrazolium salt to formazan. The reaction was then stopped by adding 300 µL of HCl 1 M. UV-VIS (Specord 40, Analytic Jena) spectroscopy was used to measure the absorbance at 490 nm and 690 nm. This article is protected by copyright. All rights reserved. 2.4.4 Osteocalcin production Osteocalcin, also known as bone gamma-carboxyglutamic acid-containing protein (BGLAP), is a non-collagenous protein hormone, which can be found in bone and dentin. Osteocalcin is secreted by osteoblasts, and it is involved in bone mineralization. Osteocalcin was quantitatively measured using an ELISA kit (Abbexa, Mouse Osteocalcin (BGLAP) ELISA Kit, Cambridge, UK). Absorbance measurements were used to determine the amount of osteocalcin present in the samples. The absorbance of both, the unknown sample and the standard solutions having known OC concentration, were measured. By means of indirect calculation, the unknown OC concentration was obtained. 2.5 Antibacterial test Antibacterial tests were carried out to verify the efficacy of silver and gallium ions against both Gram (+) and Gram (-) bacteria. Two strains were selected to perform antibacterial tests: Staphylococcus epidermidis as Gram (+) and Escherichia coli as Gram (-). Antibacterial agar diffusion tests were also performed using Staphylococcus Carnosus (Gram +). A solution containing bacteria was prepared by suspension in lysogeny broth (LB) medium and the optical density (O.D.) was adjusted (600 nm, Biphotometer Plus, Eppendorf AG, Hamburg, Germany) to obtain a value of 0.015, following a protocol established in our laboratory. 20 µL of the prepared suspension was pipetted and homogeneously spread onto a petri dish (10 cm diameter), previously covered with a layer of LB Agar. The scaffolds, uncoated and coated, were positioned on the agar and the culture was placed in an incubator at a temperature of 37°C and a high relative humidity (80%). After 24 h the formation of an inhibition zone around the samples was assessed. 3. Results 3.1 Characterization of Ga-MBG This article is protected by copyright. All rights reserved. The VEGF values were normalized with respect to the LDH ones to prove that VEGF secretion does not depend on the higher number of cells present on the scaffolds, but on the presence of the different therapeutic ions incorporated in the MBG coating (Ag or Ga). Results are shown in Figure 20. From the graph, it can be observed that after 7 days of culture, no significant differences exist between the uncoated 45S5 BG scaffolds and Ag-MBG and Ga-MBG coated scaffolds. However, after 10 days of incubation, VEGF expression values seem to be higher for the uncoated 45S5 BG and Ag-MBG coated scaffolds in comparison to the Ga-MBG-coated scaffolds. We hypothesize that Ag ions, released form the scaffolds surface, stimulate MC3T3-E1 cells to secrete higher amounts of VEGF compared to Ga-MBG-coated scaffolds. 3.4.4 Osteocalcin production Mineralized bone matrix and ECM components such as calcium phosphate and osteocalcin, produced by mature osteoblasts, are generally used as markers for assessing osteoblast differentiation at the later stage [38]. Moreover osteocalcin is deposited on the matrix but a small faction can be released [39]. In this study, osteocalcin release into the medium was measured and is reported in Figure 21. After one week, osteocalcin production of 0.4 ng/mL was observed across all experimental groups. After two weeks, production of osteocalcin slightly increased for all tested samples. For both time points, no significant difference was observed between samples indicating that the MBG coated scaffolds supported the late stage of osteoblast differentiation. 3.5 Antibacterial test Staphylococcus carnosus (Gram +) and Escherichia coli (Gram -) bacteria were used to perform antibacterial agar diffusion tests, following a procedure previously published [32]. An inhibition zone, where bacteria did not grow around the coated scaffolds, was visible in both cases (see Figure 22, for Ag-MBG coated scaffolds). Tests were also carried out for samples after up to 3 days of immersion in SBF to verify their antibacterial properties. Even after 72 hours of soaking This article is protected by copyright. All rights reserved. in SBF, it was possible to observe the presence of an inhibition zone, suggesting that silver was still present and could be still released from the coated scaffolds. Staphylococcus epidermidis (Gram (+)) and Escherichia coli (Gram (-)) bacteria were selected as strains for further testing. Results are reported in Figure 23. Also, in this case after 24 h of incubation, it was possible to observe an inhibition halo around the sample, where no bacteria are present. It is also apparent that around the reference (uncoated 45S5 BG scaffold), an inhibition zone is visible, probably due to the increase of pH that takes place once the BG based samples get in contact with water-based solutions. However, the inhibition zone was small and difficult to measure. This can probably be related to the fact that the surface of the scaffolds is not regular, and they do not adhere completely to the agar surface. 4. Discussion It is well-known that bone defects above a critical size cannot heal themselves. They need osteoinductive and osteo-conductive support to promote and support the regeneration of the new tissue [38]. Bioactive ceramics have been studied for many years and are considered promising candidates in bone tissue engineering applications [10]. In particular, bioactive glasses have been used in bone replacement and tissue engineering applications due to their ability to bond to host tissue and to release ions that promote bone growth [9]. In this work, BG based scaffolds were fabricated by the foam replica method from 45S5 BG using natural marine sponges as sacrificial templates. The scaffolds were then impregnated with a silicate sol solution to obtain ion doped MBG coatings (Ag-MBG and Ga-MBG). A heat treatment followed the coating procedure to remove the structure directing agent and nitrates. Based on previous studies, one single impregnation was used for the coating of BG scaffolds [41]. In fact, Boccardi found that the increasing number of impregnation cycles led to an overflow of sol-gel glass [41]. Uncoated and coated scaffolds were characterized in terms of morphology, mechanical properties, and bioactivity once in contact with biological fluids. E. coli This article is protected by copyright. All rights reserved. SEM observation confirmed the presence of the MBG coatings and SBF immersion tests for up to 7 days showed the ability of the scaffolds to form HCA on their surfaces. As a consequence of immersion in SBF, the porosity of the scaffolds increased over time, reaching a value of 90% after 28 days of immersion. These results are in good agreement with those found in previous studies [41]. Scaffolds were also tested in terms of mechanical properties before and after immersion in SBF. The as-fabricated samples showed a maximum compressive strength of 4±1 MPa, a value in the range of those found for spongy bone (2-12 MPa). The achievement of improved mechanical properties, as already discussed in previous studies [42], is the result of the lower porosity of the natural marine sponges in comparison to the polyurethane sponges traditionally used to make this type of scaffolds [16]. Natural marine sponges are also characterized by pores in the range <200 µm, which are important for adhesion, migration, and proliferation of cells on the scaffold [43]. These pores in fact allow biological fluids to reach the inner core of the porous scaffold, and thus integration with the surrounding tissue is facilitated [44]. Macropores in the 200-500 µm range, beneficial for osteointegration and neovascularization [45,46], were also part of the structure of the scaffolds. Mechanical properties decreased after 1 week of immersion in SBF (reaching a value of 1.0±0.6 MPa) due to the degradation of the BG scaffolds in contact with water-based solutions. The ion release capability of the coated BG-based scaffolds was also tested. From the ion release profiles, it was possible to observe that the release of Si, Ca, and P ions takes place immediately for both scaffold types. The decrease of P concentration over time suggested the formation of a P- containing layer on the surface of the scaffolds during soaking in SBF. The therapeutic ions (Ag and Ga) were also detected. The release profile of Ag did not increase linearly potentially due to the deposit of insoluble AgCl, while the Ga ion release profile increased continuously, showing a controlled and sustained release. Agar diffusion tests were performed against Gram-positive and Gram-negative bacteria and showed promising results against both strains. A significant inhibition zone was observed for both bacterial strains after 24 h of incubation. Tests were also carried out for samples up to 3 days after immersion in SBF to ensure their antibacterial This article is protected by copyright. All rights reserved. properties. Even after 3 days of soaking in SBF the antibacterial properties of the coated scaffolds was retained. Furthermore, cytocompatibility tests were performed. Indirect and direct cell culture tests were carried out according to the general guidance of the International Standardization Organization (ISO 10993-5-2009). The extracted media from Ag- and Ga-MBG coated scaffolds did not show toxicity or negative effects on cell viability. Direct cell culture tests were performed after the scaffold had been subjected to a pre-conditioning treatment, which was carried out to avoid a rapid increase of pH during cell culture experiments [33]. To better mimic the physiological condition, the medium was changed every day. Previous studies have shown that refreshing the medium every 2-3 days leads to a “more biocompatible” surface for cell culture studies [45,47,48]. Considering the possibility that during the pre-conditioning treatment therapeutic ions can be leaked from the system in this study, EDS analysis was performed on pretreated samples. EDS indicated that the therapeutic ions were still present in the coating. In addition, the differences in cell viability found between the BG compositions with different ion doping were considered an indirect evidence of the presence of the ions. As the ions are the only major difference between samples, variations in cell culture outputs are most probably caused by the effect that the ions have on MC3T3-E1 cells. Cell viability was found to be low after 24 h of culture. These results can have two possible explanations: (i) after 24 h cells did not have enough time to grow; (ii) it might be possible that part of the cells seeded into the scaffold flowed out of the sample and were lost, when the scaffolds were transferred on a new plate for the WST-8 assay. However, after 3 days of incubation, cells started to proliferate and after 7 days they completely colonized the scaffolds. The production of VEGF was also quantitatively measured. The expression of VEGF was found to be higher when cells were placed in contact with Ga- MBG-coated scaffolds compared to uncoated 45S5 BG scaffolds. Results of VEGF measurement were in agreement with those found for the WST-8 assay. Moreover, LDH was measured after 7 and 10 days of incubation. No significant differences were observed between the two incubation times, suggesting that cells after 7 days stopped proliferating and started to differentiate. This This article is protected by copyright. All rights reserved. hypothesis was confirmed by quantitatively measuring osteocalcin production. The coated scaffolds seemed to support the late stage of osteoblast differentiation. The differentiation of pre-osteoblast cells on Ga-MBGs has also been studied by Gomez-Cerezo et al.[49] , who measured the ALP activity after 7 days in presence of 1 mg/mL MBGs. The authors obtained similar results to those found in this work. A higher ALP phosphatase activity was expressed by MC3T3-E1 cells in presence of MBGs containing gallium in comparison to the Ga-free analogues [49]. The higher ALP activity of pre-osteoblasts in contact with Ga-MBGs indicates that Ga ions provide biological signals to stimulate cell differentiation toward bone forming cells [49]. Matkovic [50] and Bockman [51] et al. found that the level of plasma alkaline phosphatase, a marker for bone formation, and the bone calcium content in gallium nitrate-treated rats can increase in the presence of gallium. On the contrary, the incorporation of Ag+ in mesoporous bioactive glasses seems to have little influence on cell differentiation. Similar results were obtained by Zhu et al. [52], who investigate the biological behavior of silver containing mesoporous 58S bioactive glass. The results obtained in this work showed that ion-doped MBGscoated scaffolds have potential for applications in bone tissue engineering with specific ions, in the present case Ag and Ga, having different biological effects. 5. Conclusions Natural marine sponges were selected as sacrificial templates for the fabrication of bioactivebased scaffolds for bone tissue engineering. BG scaffolds were produced by the foam replica technique and successfully coated with a silicate sol solution to form Ag- or Ga-doped MBG coatings. The samples were characterized by relatively high mechanical properties (~4 MPa), high bioactivity and antibacterial properties. Moreover, the coated scaffolds showed good biocompatibility against MC3T3-E1 pre-osteoblastic cells thus representing a new family of antibacterial scaffolds for bone tissue engineering. Acknowledgments This article is protected by copyright. All rights reserved. This research was carried out within the EU Horizon 2020 framework project COACH (ITNETN, Grant agreement 642557). W. H. Goldmann thanks the German Science Foundation (DFG Go598) for financial support. The authors thank Astrid Mainka (FAU) for technical advice. The authors would like to thank the TEM facilities of CITIUS-US. TEM analysis was funded by the Grant P2017/837-A.M.Beltrán from University of Seville (Spain). The authors thank Prof. Enrica Vernè and Dr Marta Miola (Politecnico di Torino, Italy) for the technical support and the help with the ICP analyses. References [1] E. Gómez-Barrena, P. Rosset, D. Lozano, J. Stanovici, C. Ermthaller, F. Gerhard. Bone fracture healing: cell therapy in delayed unions and nonunions. 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