Development and characterization of niobium-releasing silicate bioactive glasses for tissue engineering applications

Accepted Manuscript Title: Development and Characterization of Niobium-Releasing Silicate Bioactive Glasses for Tissue Engineering Applications Authors: V. Miguez-Pacheco, D. de Ligny, J. Schmidt, R. Detsch, A.R. Boccaccini PII: DOI: Reference:

S0955-2219(17)30514-9 http://dx.doi.org/doi:10.1016/j.jeurceramsoc.2017.07.028 JECS 11385

To appear in:

Journal of the European Ceramic Society

Received date: Accepted date:

25-2-2017 25-7-2017

Please cite this article as: Miguez-Pacheco V, de Ligny D, Schmidt J, Detsch R, Boccaccini A.R.Development and Characterization of Niobium-Releasing Silicate Bioactive Glasses for Tissue Engineering Applications.Journal of The European Ceramic Society http://dx.doi.org/10.1016/j.jeurceramsoc.2017.07.028 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Development and Characterization of Niobium-Releasing Silicate Bioactive Glasses for Tissue Engineering Applications V. Miguez-Pacheco1, D. de Ligny2, J. Schmidt3, R. Detsch1, A. R. Boccaccini1* 1

Institute of Biomaterials, Department of Materials Science and Engineering, University of Erlangen-

Nuremberg, 91058 Erlangen, Germany 2

Institute of Glass and Ceramics, University of Erlangen-Nuremberg, 91058 Erlangen, Germany

3

Institute of Particle Technology (LFG), University of Erlangen-Nuremberg, 91058 Erlangen, Germany

(*) Corresponding author: [email protected]

ABSTRACT Novel niobium-containing bioactive glass formulations (Nb-BGs) were designed, produced and used to fabricate sintered glass-ceramic granules to examine their in vitro bioactivity and angiogenic potential. Nb-BGs were prepared by melting and quenching. Afterwards, the glasses were crushed and milled into fine powders. These powders were used to make aqueous slurries which were poured into molds, dried and sintered to produce pellets, from which granules of 0.5-0.85 mm in size were obtained. In vitro bioactivity was tested by immersing the granules in simulated body fluid for up to 14 days. Cell biology tests were carried out by indirect culture of bone marrow stromal cells (ST-2) with supernatants resulting from incubation of BG granules in cell culture medium. The effect of dissolution products from Nb-BGs on the secretion of vascular endothelial growth factor (VEGF) was assessed to characterize the angiogenic potential of the new Nb-containing BG compositions.

Keywords: Bioactive glasses; scaffolds; tissue engineering; Niobium; angiogenesis

1

INTRODUCTION

In terms of tissue replacement, materials that can be used off the shelf and can circumvent additional surgical procedures, possible immune reactions and donor tissue shortages are in high demand. Bone tissue engineering and replacement have been tackled using a number of approaches, usually involving the use of bioactive (osteoconductive, osteoinductive) materials 1. Some of these

approaches consider bioactive glasses (BGs), which through their surface reactivity enable bonding to bone and soft tissues

2–4

, and together with their ability to deliver therapeutic ions for enhancing

tissue repair mechanisms 5–7, have become very attractive platforms for bone tissue engineering

8, 9

.

Novel formulations of BGs are being continuously investigated after the discovery that BG dissolution products after implantation or in contact with relevant fluids, namely ions released from the surface of BG constructs, have profound effects on cell behaviour 7. In particular, biologically active ions released from BGs can have a therapeutic effect on the affected tissues, and have been shown to upregulate cellular processes of interest in tissue engineering, including important effects on vascularization according to in-vitro and in vivo studies

5, 7, 10

. Therefore, BGs offer an alternative to

promote an angiogenic response without resorting to the use of fragile biological molecules, namely growth factors11. Niobium (Nb) is a relatively unexplored metallic ion in tissue engineering but it may have relevance for bone regenerative applications. Nb has been reported to have a lower cytotoxicity than other metal ions

12

and has been shown to stimulate mineralization in human osteoblast

populations 13. With this in mind and within our current research scope of exploring bioactive glass compositions incorporating novel biologically active ions, two niobium-containing formulations based on the 45S5 silicate bioactive glass composition3 (see Table 1 for compositions in wt%) were prepared in this study by the melting route. In the same fashion as the report of Lopes et al. 14, Nb2O5 was substituted for P2O5 to obtain niobium-containing silicate bioactive glasses (Nb-BGs). The in vitro bioactivity and cellular biocompatibility of these novel glasses were investigated. The granular morphology employed in this study was chosen in accordance to existing products in clinical use, such as BonAlive® 15, which consists of bioactive glass granules of the formulation S53P4 of different size ranges which are used to fill bone defects, and which have been shown to exhibit angiogenic effects in cell culture experiments 16. Given the interest in exploring new compositions of BGs with angiogenic effects7, 16–21, the cell biology study here was focused on assessing the release of vascular endothelial growth factor (VEGF) from multipotent stem cells exposed to BG dissolution products, as a first marker of the angiogenic in-vitro capability of Nb-BGs.

2 2.1

EXPERIMENTAL METHODS Materials

Sodium carbonate (Na2CO3 Merck, Germany), ≥99% purity, calcium phosphate (Ca3(PO4)2, SigmaAldrich, Germany), ≥98% purity, niobium pentoxide (Nb2O5, Merck, Germany), ≥99% purity, silicon oxide (SiO2, Merck, Germany), and calcium carbonate (CaCO3, Merck, Germany) were used to prepare the glasses. The as-received powders were weighed and mixed to obtain two different formulations of Nb-BGs of compositions shown in Table 1. The homogenized powders were melted in Pt crucibles at 1400 °C for 1 hour. The melt was then quenched rapidly in water. The frit was crushed

into a rough powder using a Jaw Crusher (Retsch, Germany) and subsequently ground into a fine powder using a zirconia planetary ball mill (Retsch, Germany) to obtain a fine powder of mean particle size in the range of 6-20µm. Commercially available bioactive glass powder of 45S5 composition of mean particle size < 5µm (Schott, Germany) was used as a base material to compare the properties of the Nb-BGs. 2.2

Granule preparation

An aqueous slurry containing 1g/ml BG powder and 0.01 mol/ml polyvinyl alcohol (PVA) was prepared, poured into silicone molds and allowed to dry overnight at 60°C. This particular slurry composition was chosen as it had been shown in preliminary experiments (not shown here) to be suitable for fabrication of BG-based scaffolds by the foam replica technique

22

. After de-molding,

samples were sintered at the characteristic temperatures of each glass in the range 930-1050°C, depending on Nb content. Sintered pellets were crushed using an agate mortar and pestle, and sieved to achieve the desired size range, namely 0.5-0.85mm. 2.3

In vitro bioactivity

The bioactivity of Nb-BGs was investigated by immersing sintered granules (size 0.5-0.85mm) in simulated body fluid (SBF) for up to 14 days and examining the deposition of hydroxyapatite (HA) on their surfaces. Samples were immersed at a ratio of 150mg of sample to 100ml of SBF, prepared according to Kokubo et al.

23

, and placed in an orbital shaker (KS 4000i control, IKA®) at 37°C and

90rpm. The volume of solution used was in agreement with existing literature

24

and it was not

exchanged throughout the duration of the study to examine the ionic release in solution. The glass structure and the formation of an apatite phase on the surface of samples after immersion in SBF were investigated using Fourier-transform infrared (FTIR) spectroscopy. To this end, KBr pellets containing 1wt% sample were prepared by pressing the fine powders to 12MPa in a hydraulic press. Scanning electron microscopy (SEM) images of the immersed granules before and after soaking in SBF were obtained to investigate the morphological changes on sample surfaces using a field-emission scanning electron microscope (FE-SEM; ULTRA plus, Carl-Zeiss, Oberkochen, Germany) at an operating voltage of 1kV. Elemental concentrations in SBF upon different time periods were measured by inductively coupled optical emission spectroscopy (ICP-OES) (Optima 8300, PerkinElmer, Germany). 2.4

Indirect cell culture

Bone marrow stromal cells (ST-2, of mouse origin) were placed in 24-well plates (at a count of 100,000 cells/well) and incubated for 48h with BG extracts obtained by pre-incubating glass granules in RPMI medium for 24h. Extracts were prepared at 10mg/ml (1 wt/vol% or henceforth referred to as

[1]) BG granule concentration and diluted with additional medium to produce extracts at 1 and 0.1mg/ml (0.1 and 0.01 wt/vol%, henceforth referred to as [0.1] and [0.1], respectively), designed in accordance to a previous similar study16. Culture medium was then removed from the culture wells and used to determine the release of vascular endothelial growth factor (VEGF) using an ELISA (Enzyme-Linked Immunosorbent Assay) kit (RayBiotech, USA). A WST assay was carried out on the plated cells to measure their viability post-incubation. 2.5

Statistical analysis

In vitro experiments were run in rounds of 6 for each BG composition and concentration. Results for cell viability measurements and VEGF release experiments are shown in bar charts representing the mean value ± standard deviation. The differences between each BG composition and concentration were evaluated by one-factor analysis of variance (ANOVA) with a level of statistical significance defined as p <0.001 (Origin 8.6, Origin Lab Corporations, USA).

3 3.1

RESULTS AND DISCUSSION BG bioactivity study

In depth information on the thermal behavior of the same BG compositions presented in this study has been reported by Lopes et al.

14

. It is worth noting that whilst the samples crystallized after

sintering, Nb-BG formulations failed to fully densify and sharply defined glass particles could be appreciated in SEM images of samples prior to immersion in SBF (see Fig 1a and c). In terms of bioactivity, the formation of cauliflower-like precipitates on the surface of all BG granules could be observed after immersion in SBF, and continued to be present for longer immersion times (Fig 1). This result qualitatively confirms that the substitution of Nb for P has not significantly impaired the intrinsic high bioactivity of 45S5 BG.

Figure 1- SEM images of BG granule surface before (a,c) and after 7 days immersion in SBF(b,d); Figs. 1a and 1b correspond to 0.5Nb-BG and 1c and 1d to 1.0Nb-BG

The formation of HA on the surface of BG samples upon immersion in SBF has been well established as a bioactivity marker 23, 24. Fig. 2 shows the FTIR spectra of 0.5 and 1.0Nb-BG granules before and after immersion in SBF for up to 14 days. Before soaking in SBF, the FTIR spectra points out to the structure of the sintered sample (0d), which shows two major bands at around 1020 and 930 cm-1, assigned to the Si-O-Si(b) and Si-NBO (non-bridging oxygen) bonds25, respectively, and bands at approximately 620 and 580 cm-1 showing the P-O bending mode26. The FTIR results show that the peak at 930 cm-1 eventually disappears (after 14 days), which could be related to the formation of a silica rich gel layer, depleted in alkali ions, resulting from leaching of Ca2+ and Na+ ions from the glass into solution and formation of a superficial Si-OH layer. At 7 days, the P-O double peak is seen to increase in intensity, more evidently in the 0.5Nb-BG spectra than in the 1.0Nb-BG one, which possibly indicates the presence of a crystalline HA phase 27. HA precipitation on the surface of P2O5-free BGs (as in the case with the 1.0Nb formulation) in contact with SBF has been explored before, and has been shown that whilst the rate of HA formation is reduced, this calcium phosphate layer is still present

28, 29

. This lower rate of HA formation is

reflected in the apparent lower intensity of relevant bands in the FTIR spectrum for SBF soaked

1.0Nb BG granules. In particular, the absence of a defined peak at around 930 cm-1 in the 1.0Nb spectrum (in comparison to both, 0.5Nb and 45S5 BG spectra) points to the possible absence of NBOs at the highest Nb concentration in the glass structure, i.e. 1.0Nb BG, in accordance with findings in other studies14, 30. A decrease in NBOs would also lead to a more ‘closed’ glass structure 31 thus causing a decrease in glass reactivity, reflected again in the slower HA precipitation on the 1.0Nb BG granules. Furthermore, the emergence of a small broad band (Si-O-Si stretching) at around 790cm-1 indicates the possible formation of a silica-rich layer. The emergence of a peak at approximately 1400cm-1 and a small band at 870 cm-1 that can be matched to the C-O stretching bond also indicates the precipitation of a carbonated HA layer; these peaks can be observed in natural carbonated HA or surface bonded carbonate compounds 32.

a)

b)

c)

Figure 2-FTIR spectra of a)45S5, b)0.5 and c)1.0Nb-BG granules immersed in SBF for up to 14d. The relevant peaks are discussed in the text.

3.2 Ion release study The ionic concentrations measured using ICP-OES, resulting from the dissolution of Nb-BG granules in solution at different immersion times, are shown in Fig. 3. Results for 45S5 BG are also presented for comparison. The zero time point indicates the ionic concentrations in plain SBF (0h). There is an increasing release of Si throughout the study, which indicates the dissolution of the silica network, as expected. This effect seems to stabilize after 7d (168h) immersion.

a) 45S5 BG 220 200 180

Concentration (ppm)

160 140 120 100 80 60 40 20 0 0

50

100

b)

150

200

250

300

350

250

300

350

Immersion time (h) 0.5Nb-BG 220 200 180

Concentration (ppm)

160 140 120 100 80 60 40 20 0 0

50

100

150

200

Immersion time (h)

c)

Si 251.611 Si 212.411 P 213.617 P 214.914 Ca 317.933

1.0Nb-BG 220 200

Concentration (ppm)

180 160 140 120 100 80 60 40 20 0 0

50

100

150

200

250

300

350

Immersion time (h)

Figure 3- Ion release from a) 45S5, b) 0.5Nb-BG and c) 1.0 Nb-BG granules in SBF for up to 14d, as measured by ICP-OES

It can be appreciated that Si ion release is slightly higher in Nb-BGs, with an increase in concentration of around 10ppm being measured. The calcium content in solution increases initially, stabilizing after 7d. Considerably higher Ca ion concentrations were measured for Nb-BG supernatants as compared

to the 45S5 BG reference. This increase can be explained by a disruption of Si-NBO bonds, resulting from the introduction of Nb ions into the glass network, and subsequent release of Ca2+ ions at the glass surface. In particular for the Nb-containing BGs, the measured Si concentrations were above the biologically relevant range (15-30ppm), whereas for 45S5 BG it remained within this range. In the case of Ca ions, all BG formulations eluted concentrations higher than its biologically relevant range (60-90 ppm) 7. For all BGs there was a marked decrease in the P ion concentration in solution after 1d incubation, which is related to the precipitation of a calcium phosphate (CaP) layer. P ion release remained largely unchanged for all 3 BG compositions throughout the experiments. Niobium ions could not be detected in the supernatant solutions by ICP-OES, possibly due to Nb ion levels that were well below the detection limit of this method. However, it is important to consider that the solubility of Nb2O5 at room temperature and neutral pH has been found to be extremely low (subppb) , only increasing at pH>1133. Thus, there is a possibility that Nb is indeed being released into the medium during the dissolution reactions, which occur upon immersion of the BGs in SBF, and forming insoluble complexes that cannot be detected using the aforementioned technique.

3.3 Cell biology studies WST assays, which can be directly correlated to cell viability in indirect culture with Nb-BG granules, showed statistically significant cytotoxic effects only at the highest concentrations of the Nbcontaining granules (Fig. 4). On the other hand, the dissolution products from the BGs investigated seemed to exert a significant stimulatory effect on cell populations incubated with intermediate concentrations ([0.1]) and the most dilute 1.0Nb exudate ([0.01]), denoted with asterisks, as compared to culture controls. However, modified culture medium at the highest dilution ([1]) for Nbcontaining BGs appeared to have a cytotoxic effect. These results are in accordance with the study by Prado da Silva et al. 34, where different compositions of niobium phosphate glasses were tested in indirect culture with fibroblast cells and it was found that an increase in BG exudate concentration in culture medium resulted in increased fibroblast cell death. In terms of the effects of Nb ion concentration in the conditioned media on cell proliferation, no detrimental effects were observed at lower concentrations compared to controls. It was previously reported by Obata et al. 13 that changes in niobium ion concentration alone had no effect on cellular proliferation of mouse osteoblast-like cells cultured in media containing different amounts of niobium ions.

250

45S5 0.5Nb 1.0Nb

***

***

Cell viability [%]

200

150

100

***

50

0 Control

[1]

[0.1]

[0.01]

BG granule concentration

Figure 4- Relative viability of ST-2 cells incubated with different concentrations of BG dissolution products. Asterisks indicate a significant increase in cell proliferation with respect to culture controls, *** denotes p<0.001 (Bonferroni’s post hoc test was used)

The results of the ELISA test indicate a stimulatory effect from the BG ionic dissolution products on fibroblast cells in terms of VEGF production. Specifically, there was a significant increase in VEGF release (p<0.001) in cells incubated with 1.0Nb at [0.01]. The effect of S53P4-BG dissolution products on VEGF release has been shown in previous studies using human fibroblasts (CD-18CO cells)

16

.

Following the results of these studies the size range of 0.5-0.8mm for the BG granules was chosen for this study, as well as the concentrations of exudates following pre-incubation, given that they exhibited the greatest VEGF production. This is in accordance with a study by Day et al.

35

, where

there was a significant increase in fibroblasts grown on 45S5-BG coated cell culture plates at 0.01 wt/vol%, corresponding to the [0.01] concentration in this study. The present results indicate that much lower concentrations of BG particles are required to produce a significant increase in VEGF release if niobium ions are included in the BG composition. Although no niobium ions could be detected in the supernatants using ICP-OES (due to their concentrations being under the detection limit of the technique), it can be stated that under the conditions in which the in vitro experiments were carried out, there was a clear increase in VEGF released from cells in contact with the dissolution products of the Nb-containing BG granules at the lowest particle concentration. It is likely that the overall ionic release profile related to the novel Nb-BG formulations resulted in a synergic effect on the cell populations, which led to the measured increase in VEGF release.

45S5 0.5Nb 1.0Nb

VEGF release [%]

150

***

100

50

0 Control

[1]

[0.1]

[0.01]

BG granule concentration

Figure 5- VEGF release by bone marrow stromal cells in indirect culture with different BG granule compositions at different concentrations. Asterisks indicate a significant increase in cell proliferation with respect to culture controls, *** denotes p<0.001 (Bonferroni’s post hoc test was used)

4

CONCLUSIONS

Incorporation of Nb2O5 in 45S5 BG appears to have no detrimental effect on the in vitro bioactivity of the Nb-BGs investigated, as evidenced by the precipitation of apatite-like crystals on the surface of granules immersed in SBF. Whilst the initial results from the cell culture experiments on Nb-BGs point out to a concentration dependence in terms of cytotoxicity, it was observed that at lower glass concentrations, there seems to be a stimulatory effect on cells in terms of their proliferation. Under the experimental conditions tested in this study, it was observed that the resulting ionic cocktail in Nb-BG exudates produced a significant increase in the release of VEGF at lower BG granule concentrations. Thus, it can be concluded that an angiogenic response can be elicited in cells cultured in the presence of Nb-BG dissolution products, without needing to resort to additional growth factors. Future work should involve further dissolution studies to ascertain the release pattern of Nb species and in vitro cell experiments involving culture of Nb-BGs with osteoblast cells in order to investigate the effects of Nb-BGs in direct cell culture on cell attachment and proliferation.

5

ACKNOWLEDGMENTS

The authors would like to acknowledge the European Commission funding under the 7th Framework Programme (Marie Curie Initial Training Networks; grant number: 289958, Bioceramics for bone repair).

REFERENCES 1

A.R. Amini, C.T. Laurencin, and S.P. Nukavarapu, “Bone tissue engineering: recent advances and challenges.,” Crit. Rev. Biomed. Eng., 40 [5] 363–408 (2012).

2

L.L. Hench and D. Greenspan, “Interactions between Bioactive Glass and Collagen : A Review and New Perspectives,” J. Aust. Ceram. Soc., 49 1–40 (2013).

3

Hench LL, “Bioceramics,” J. Am. Ceram. Soc. , 81 1705–1728 (1998).

4

V. Miguez-Pacheco, L.L. Hench, and A.R. Boccaccini, “Bioactive glasses beyond bone and teeth: Emerging applications in contact with soft tissues.,” Acta Biomater., 13 1–15 (2014).

5

G. Jell and M.M. Stevens, “Gene activation by bioactive glasses.,” J. Mater. Sci. Mater. Med., 17 [11] 997–1002 (2006).

6

I.D. Xynos, A.J. Edgar, L.D. Buttery, L.L. Hench, and J.M. Polak, “Ionic products of bioactive glass dissolution increase proliferation of human osteoblasts and induce insulin-like growth factor II mRNA expression and protein synthesis.,” Biochem. Biophys. Res. Commun., 276 [2] 461–5 (2000).

7

A. Hoppe, N.S. Güldal, and A.R. Boccaccini, “A review of the biological response to ionic dissolution products from bioactive glasses and glass-ceramics,” Biomaterials, 32 [11] 2757– 2774 (2011).

8

M.N. Rahaman, D.E. Day, B.S. Bal, Q. Fu, S.B. Jung, L.F. Bonewald, and A.P. Tomsia, “Bioactive glass in tissue engineering.,” Acta Biomater., 7 [6] 2355–73 (2011).

9

L.-C. Gerhardt and A.R. Boccaccini, “Bioactive Glass and Glass-Ceramic Scaffolds for Bone Tissue Engineering,” Materials, 3 [7] 3867–3910 (2010).

10

A. Hoppe, V. Mouriño, and A.R. Boccaccini, “Therapeutic inorganic ions in bioactive glasses to enhance bone formation and beyond,” Biomater. Sci., 1 [3] 254–256 (2013).

11

A.A. Gorustovich, J.A. Roether, and A.R. Boccaccini, “Effect of bioactive glasses on angiogenesis: a review of in vitro and in vivo evidences.,” Tissue Eng. Part B. Rev., 16 [2] 199– 207 (2010).

12

A. Yamamoto, R. Honma, and M. Sumita, “Cytotoxicity evaluation of 43 metal salts using murine fibroblasts and osteoblastic cells.,” J. Biomed. Mater. Res., 39 [2] 331–40 (1998).

13

A. Obata, Y. Takahashi, T. Miyajima, K. Ueda, T. Narushima, and T. Kasuga, “Effects of Niobium

Ions Released from Calcium Phosphate Invert Glasses Containing Nb2O5 on Osteoblast-Like Cell Functions,” ACS Appl. Mater. Interfaces, 4 [10] 5684–90 (2012). 14

J.H. Lopes, A. Magalhães, I.O. Mazali, and C.A. Bertran, “Effect of Niobium Oxide on the Structure and Properties of Melt-Derived Bioactive Glasses,” J. Am. Ceram. Soc., 97 [12] 3843– 3852 (2014).

15

N.C. Lindfors, P. Hyvönen, M. Nyyssönen, M. Kirjavainen, J. Kankare, E. Gullichsen, and J. Salo, “Bioactive glass S53P4 as bone graft substitute in treatment of osteomyelitis.,” Bone, 47 [2] 212–8 (2010).

16

R. Detsch, P. Stoor, A. Grünewald, J.A. Roether, N.C. Lindfors, and A.R. Boccaccini, “Increase in VEGF secretion from human fibroblast cells by bioactive glass S53P4 to stimulate angiogenesis in bone.,” J. Biomed. Mater. Res. A, 102, 4055–4061 (2014).

17

S. Zhao, L. Li, H. Wang, Y. Zhang, X. Cheng, N. Zhou, M.N. Rahaman, Z. Liu, W. Huang, and C. Zhang, “Wound dressings composed of copper-doped borate bioactive glass microfibers stimulate angiogenesis and heal full-thickness skin defects in a rodent model,” Biomaterials 53, 379–91 (2015).

18

C. Wu, W. Fan, J. Chang, and Y. Xiao, “Mesoporous bioactive glass scaffolds for efficient delivery of vascular endothelial growth factor.,” J. Biomater. Appl., 28 [3] 367–74 (2013).

19

P. Balasubramanian, L. Hupa, B. Jokic, R. Detsch, A. Grünewald, and A.R. Boccaccini, “Angiogenic potential of boron-containing bioactive glasses: in vitro study,” J. Mater. Sci., in press (2017).

20

C. Wu, Y. Zhou, W. Fan, P. Han, J. Chang, J. Yuen, M. Zhang, Y. Xiao, "Hypoxia-mimicking mesoporous bioactive glass scaffolds with controllable cobalt ion release for bone tissue engineering", Biomaterials 33, 2076–2085 (2012).

21

S.N. Rath, A. Brandl, D. Hiller, A. Hoppe, U. Gbureck, R.E. Horch, A.R. Boccaccini, U. Kneser, et al., “Bioactive Copper-Doped Glass Scaffolds Can Stimulate Endothelial Cells in Co-Culture in Combination with Mesenchymal Stem Cells,” PLoS One, 9 [12] e113319 (2014).

22

Q.Z. Chen, I.D. Thompson, and A.R. Boccaccini, “45S5 Bioglass-derived glass-ceramic scaffolds for bone tissue engineering.,” Biomaterials, 27 [11] 2414–25 (2006).

23

T. Kokubo and H. Takadama, “How useful is SBF in predicting in vivo bone bioactivity?,” Biomaterials, 27 [15] 2907–15 (2006).

24

A.L.B. Maçon, T.B. Kim, E.M. Valliant, K. Goetschius, R.K. Brow, D.E. Day, A. Hoppe, A.R. Boccaccini, et al., “A unified in vitro evaluation for apatite-forming ability of bioactive glasses and their variants.,” J. Mater. Sci. Mater. Med., 26 [2] 115 (2015).

25

L. Lefebvre, J. Chevalier, L. Gremillard, R. Zenati, G. Thollet, D. Bernache-Assolant, and A. Govin, “Structural transformations of bioactive glass 45S5 with thermal treatments,” Acta Mater., 55 [10] 3305–3313 (2007).

26

G.A. Stanciu, I. Sandulescu, B. Savu, S.G. Stanciu, K.M. Paraskevopoulos, X. Chatzistavrou, E. Kontonasaki, and P. Koidis, “Investigation of the Hydroxyapatite Growth on Bioactive Glass Surface,” J. Biomed. Pharm. Eng., 1 [1] 34–39 (2007).

27

I. Rehman and W. Bonfield, “Characterization of hydroxyapatite and carbonated apatite by photo acoustic FTIR spectroscopy.,” J. Mater. Sci. Mater. Med., 8 [1] 1–4 (1997).

28

Y. Ebisawa, T. Kokubo, K. Ohura, and T. Yamamuro, “Bioactivity of CaO�SiO2-based glasses:in vitro evaluation,” J. Mater. Sci. Mater. Med., 1 [4] 239–244 (1990).

29

B. Cabal, L. Alou, F. Cafini, R. Couceiro, D. Sevillano, L. Esteban-Tejeda, F. Guitián, R. Torrecillas, et al., “A New Biocompatible and Antibacterial Phosphate Free Glass-Ceramic for Medical Applications,” Sci. Rep., 4 45–55 (2014).

30

A. Aronne, V.N. Sigaev, B. Champagnon, E. Fanelli, V. Califano, L.Z. Usmanova, and P. Pernice, “The origin of nanostructuring in potassium niobiosilicate glasses by Raman and FTIR spectroscopy,” J. Non. Cryst. Solids, 351 [46] 3610–3618 (2005).

31

D.S. Brauer, “Bioactive glasses—structure and properties.,” Angew. Chem. Int. Ed. Engl., 54 [14] 4160–81 (2015).

32

K. Zhang, H. Yan, D.C. Bell, A. Stein, and L.F. Francis, “Effects of materials parameters on mineralization and degradation of sol-gel bioactive glasses with 3D-ordered macroporous structures,” J. Biomed. Mater. Res., 66A [4] 860–869 (2003).

33

C. Peiffert, C. Nguyen-Trung, D.A. Palmer, J.P. Laval, and E. Giffaut, “Solubility of B-Nb2O5 and the Hydrolysis of Niobium(V) in Aqueous Solution as a Function of Temperature and Ionic Strength,” J. Solution Chem., 39 [2] 197–218 (2010).

34

M.H. Prado da Silva, C.M. Ramirez, J.M. Granjeiro, and A.M. Rossi, “In Vitro Assessment of New Niobium Phosphate Glasses and Glass Ceramics,” Key Eng. Mater., 361–363 229–232 (2008).

35

R.M. Day, A.R. Boccaccini, S. Shurey, J. a Roether, A. Forbes, L.L. Hench, and S.M. Gabe, “Assessment of polyglycolic acid mesh and bioactive glass for soft-tissue engineering scaffolds.,” Biomaterials, 25 [27] 5857–66 (2004).

Table 1- 45S5 BG and Nb-BG compositions in mol% Na2O

CaO

SiO2

P2O5

Nb2O5

45S5

24.35

26.91

46.13

1.00

0.00

0.5Nb-BG

24.35

26.91

46.13

0.50

0.50

1.0Nb-BG

24.35

26.91

46.13

0.00

1.00