AYUSH MISHRA
Phosphate Glasses and Fibers as an Alternative to Silicate Bioactive Glasses
Tampere University Dissertations 120
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7KHRULJLQDOLW\RIWKLVWKHVLVKDVEHHQFKHFNHGXVLQJWKH7XUQLWLQ2ULJLQDOLW\&KHFN VHUYLFH &RS\ULJKW DXWKRU &RYHUGHVLJQ 5RLKX,QF ,6%1 SULQW ,6%1 SGI ,661 SULQW ,661 SGI KWWS XUQIL851 ,6%1 3XQD0XVWD2\± The field of bioactive glasses started with the discovery of the first bioactive glass composition 45S5 or Bioglass® in 1969. In the 1980s, the glass particulates of 45S5 depicted the ability to induce osteogenesis, as opposed to the then preva- lent bioinert metallic implants. Later, the dissolution products of 45S5, which was a silicate glass, were found to stimulate the genes in human osteoblasts and act as growth factors, providing signals to the cells. Over the years, other bioactive glass compositions such as 13-93 and BonAlive® (S53P4) were discovered, which were also silicate glasses. Even today, 45S5 remains the gold standard for bioactive glasses. However, in recent times, there has been a rise in the demand for more sophisticated implants, such as scaffolds made from polymer and glass, or only glass. Furthermore, bioactive glass fibers are also appealing to the re- searchers for mechanically reinforcing the scaffolds, owing to their better me- chanical properties than polymers. However, in this regard, the silicate glasses are known to pose serious challenges to the researchers, due to their incongruent dissolution mechanism and poor thermal processability. Moreover, for short term applications, ranging from a few weeks to a few months, the degradation rate of the commercially available silicate glasses is considered too slow. In light of the above facts, the main aims of this thesis are: (i) to develop new glasses within the phosphate glass family, which possess good thermal pro- cessability, have additional functionalities such as antimicrobial properties and invoke favorable cell response for developing novel biomaterials in future, and (ii) to develop glass fibers based on these compositions for advanced applications such as biosensing, and possessing decent mechanical properties for reinforcing composites. To accomplish these objectives, a new series of phosphate glasses doped with metallic ions such as Ag, Cu and Fe was developed based on a promising phos- phate glass composition studied earlier. These glasses were analyzed in terms of their structural, thermal and in-vitro dissolution properties. Furthermore, the antimicrobial properties of Ag and Cu glasses were also tested, relative to com- iii mercial silicate bioactive glass. Then, preliminary cell culture tests were con- ducted using glass discs, and glass extracts (glass dissolution products contain- ing cell culture medium). Moving forward, two approaches were taken simultane- ously to improve the cell response to these glasses. Firstly, the glasses were subjected to a series of surface treatments to improve protein adhesion at their surface, which in-turn should play a role in improving the cell attachment at their surface. In the second approach, a small addition of borate at the expense of phosphate was done to increase the glass’ resistance to aqueous dissolution. Cell culture tests were then performed using glass extracts obtained from the new borophosphate glasses, and cell viability and proliferation were assessed and cytotoxicity of the extracts obtained. Finally, newly developed single core and core-clad glass fibers were also studied in terms of their mechanical properties and their potential in biosensing. The newly developed phosphate glasses depicted a high degree of thermal pro- cessability and promising in-vitro dissolution characteristics. The Ag and Cu glasses presented antimicrobial properties even superior to the commercially available S53P4 in eliminating Staphylococcus epidermis. The surface treat- ments performed on the glass discs revealed an improvement in the protein ad- hesion, which in-turn vastly improved the cell attachment at their surface. In the other approach to improve cell growth and proliferation in the glass extracts, a small addition of borate at the expense of phosphate resulted in an improved cell response to the glass extracts, barring that of the Cu-doped glass. Indeed, the undiluted Cu extract exhibited a much higher cytotoxicity than all the other glass extracts, and hence was toxic to cell survival. In the study on phosphate glass fibers, the core-clad fiber was found to be promising for biosensing, owing to its ability to guide light effectively and maintain decent mechanical properties in-vitro (up to 4 weeks, and ~6 weeks in TRIS buffer solution respectively). In summary, phosphate glasses with favorable cell response were obtained by tailoring their composition and using suitable surface treatments. These glasses were found to possess favorable thermal properties for fiber drawing and scaffold sintering, and the fibers drawn from these glass compositions showed promise in biosensing. Furthermore, they possessed high mechanical strength, and in the future, they can be considered for reinforcing scaffolds. These new glasses and iv glass fibers should pave the way for a new family of bioresorbable implants for a wide array of biomedical applications in future. v vL Preface This study was carried out at Tampere University in the doctoral programme of the Fac- ulty of Medicine and Health Technology. The project was financially supported by the Academy of Finland, through the Academy Research Fellow Grant (#275427) and the Initial Funding for Research Cost (#284492). I would like to express my sincere gratitude to my supervisor, Assoc. Prof. Jonathan Massera for his guidance and support throughout the duration of my PhD project, and for giving me the opportunity to work under his supervision. I also wish to extend my gratitude to Prof. Minna Kellomäki and Dr. Miina Ojansivu, for their guidance and sug- gestions during my follow up group meetings. I would further like to thank Miina Ojansivu for helping me with cell culture experiments. I am also thankful to the pre-examiners of my thesis, Prof. Annelise Faivre and Assoc. Prof. Ifty Ahmed for their constructive criti- cism and feedback to help improve my thesis. I would like to further thank Prof. Delia Brauer and Prof. Johann Troles, and their research groups, for hosting me as a visiting researcher at their universities, and helping me with my research. I would like to acknowledge my co-authors, Jean Rocherulle, Laeticia Petit, Maria Pihl, Martin Andersson, Turkka Salminen, Frederic Desevedavy, Frederic Smektala, Susanna Miettinen, Sari Vanhatupa and Reija Autio for their contributions to my research and sharing their expertise with me. I am also thankful to the present and former members of our “Glass group”, especially Jenna Tainio and Amy Nommeots-Nomm for creating a nice work atmosphere during the years. I also extend my thanks to everyone at the “Bi- omaterials group”, including the laboratory and technical personnel Suvi Heinämäki and Heikki Liejumäki for all the help over the years. A big cheers goes out to Aleksi, Janne, Mart and Inari for all the lunch talks and shared experiences. I am also grateful to Anna- Maija Honkala and Sari Kalliokoski, the laboratory personnel at the Adult Stem Cells group at Arvo, for training and helping me with the cell culture tests. I am eternally grateful to my parents, and the rest of my family in India for their unending support. Thank you for not letting the long distance between us matter much during these years. Finally, my deepest gratitude goes out to my wife Priya, who has been my rock over the years, and the source of unconditional support. Cheers to my sister Amisha too, for sharing in this journey. Tampere, August 2019 Ayush Mishra vii vLLL Table of Contents ABSTRACT ...... III PREFACE ...... VII LIST OF PUBLICATIONS ...... XIII UNPUBLISHED MANUSCRIPTS ...... XV AUTHOR’S CONTRIBUTION ...... XVII LIST OF SYMBOLS ...... XIX LIST OF ABBREVIATIONS ...... XXI 1 INTRODUCTION ...... 1 2 LITERATURE REVIEW ...... 3 2.1 EVOLUTION OF BIOMATERIALS ...... 3 2.2 GLASS ...... 5 2.2.1 Bioactive Silicate glasses ...... 6 2.2.1.1 Structure ...... 6 2.2.1.2 Dissolution mechanism ...... 7 2.2.1.3 Bioactivity ...... 10 2.2.1.4 Limitations of bioactive silicate glasses ...... 10 2.2.2 Phosphate glasses ...... 11 2.2.2.1 Structure ...... 12 2.2.2.2 Dissolution mechanism ...... 13 2.2.2.3 Bioactivity ...... 14 2.3 MODIFYING GLASS PROPERTIES ...... 15 2.3.1 Doping with metal oxides ...... 16 2.3.2 Surface modification methods ...... 19 2.4 GLASS FIBER PROCESSING ...... 20 2.4.1 Fibers for reinforcement ...... 20 2.4.2 Biosensing ...... 21 3 AIMS OF THE STUDY...... 23 4 MATERIALS AND METHODS ...... 25 4.1 BULK GLASS ...... 25 4.1.1 Glass melting ...... 25 ix 4.1.2 Physical properties ...... 28 4.1.3 Thermal properties ...... 28 4.1.4 FTIR Analyses...... 29 4.1.5 Optical properties ...... 29 4.1.6 In-vitro dissolution ...... 29 4.1.7 Imaging and elementary analysis ...... 30 4.1.8 Antimicrobial tests ...... 30 4.2 SURFACE MODIFICATION ...... 31 4.2.1 Preparation of buffer solution ...... 31 4.2.2 Washing and silanization ...... 31 4.2.3 Contact angle measurements ...... 33 4.2.4 Zeta potential ...... 33 4.2.5 Protein adsorption and confocal microscopy ...... 33 4.3 CELL CULTURE TESTS ...... 34 4.3.1 Preparation of extracts ...... 34 4.3.2 Adipose stem cell isolation, expansion and culture ...... 34 4.3.3 Cell viability and proliferation ...... 35 4.3.4 Cytotoxicity assessment ...... 35 4.3.5 Statistical analyses ...... 36 4.4 GLASS FIBERS ...... 36 4.4.1 Core preforms ...... 36 4.4.2 Core-clad preforms ...... 36 4.4.3 Fiber drawing ...... 37 4.4.4 Refractive index measurements ...... 38 4.4.5 Immersion tests ...... 39 4.4.6 Mechanical testing ...... 39 4.4.7 Light loss ...... 39 5 RESULTS ...... 41 5.1 CHARACTERIZATION OF NEW PHOSPHATE GLASSES ...... 41 5.1.1 Physical properties ...... 41 5.1.2 Thermal properties ...... 42 5.1.3 Structural properties ...... 43 5.1.4 Optical properties ...... 46 5.1.5 In-vitro dissolution characteristics ...... 47 5.1.5.1 Change in pH ...... 48 5.1.5.2 Ion release ...... 48 x 5.1.5.3 Change in structure ...... 51 5.1.5.4 Precipitation of a surface layer ...... 52 5.1.6 Antimicrobial properties ...... 53 5.2 PRELIMINARY CELL CULTURE TESTS ...... 54 5.2.1 Cell culture on glass discs ...... 54 5.2.2 Cell culture in glass extracts ...... 57 5.3 IMPROVING CELL ATTACHMENT ON PHOSPHATE GLASSES ...... 59 5.3.1 Impact on structure due to washing and silanization ...... 60 5.3.2 Impact on contact angle due to washing and silanization ...... 63 5.3.3 Impact on surface charge due to washing and silanization ...... 64 5.3.4 Impact on protein adsorption due to washing and silanization ...... 64 5.3.5 Quantitative analysis of confocal microscopy images ...... 68 5.3.6 Impact of protein adsorption and silanization on cell attachment ...... 70 5.4 CHARACTERIZATION OF NEW BOROPHOSPHATE GLASSES ...... 71 5.4.1 Thermal properties ...... 71 5.4.2 In-vitro dissolution characteristics ...... 72 5.4.2.1 Change in pH ...... 72 5.4.2.2 Ion release ...... 72 5.4.2.3 Structural properties ...... 74 5.4.2.4 Surface analysis ...... 75 5.4.3 Cell growth and proliferation ...... 78 5.5 PHOSPHATE GLASS FIBERS FOR BIOSENSING ...... 82 5.5.1 Single core and core-clad fibers ...... 82 5.5.2 Mechanical strength and origin of fracture ...... 83 5.5.3 Biosensing ...... 85 6 DISCUSSION ...... 89 6.1 CHARACTERIZATION OF NEW PHOSPHATE GLASSES ...... 89 6.1.1 Impact of doping on glass properties ...... 90 6.1.2 Impact of doping on in-vitro dissolution characteristics ...... 91 6.2 PRELIMINARY CELL CULTURE TESTS ...... 94 6.3 IMPROVING CELL ATTACHMENT ON PHOSPHATE GLASSES ...... 94 6.3.1 Impact on structure ...... 95 6.3.2 Impact on contact angle ...... 96 6.3.3 Impact on surface charge ...... 96 6.3.4 Impact on protein adsorption ...... 97 6.3.5 Quantitative analysis of confocal microscopy images ...... 97 xi 6.3.6 Impact of protein adsorption on cell proliferation ...... 99 6.4 CHARACTERIZATION OF NEW BOROPHOSPHATE GLASSES ...... 100 6.4.1 Thermal properties ...... 100 6.4.2 In-vitro dissolution characteristics ...... 100 6.4.3 Cell growth and proliferation ...... 102 6.5 PHOSPHATE GLASS FIBERS FOR BIOSENSING ...... 104 6.5.1 Mechanical strength upon immersion ...... 104 6.5.2 Etched fibers for biosensing ...... 106 CONCLUSIONS ...... 108 BIBLIOGRAPHY ...... 111 PUBLICATIONS ...... 146 xii List of Publications I A. Mishra, J. Rocherulle, J. Massera, Ag-doped phosphate bioactive glasses: Thermal, structural and in-vitro dissolution properties, Biomed. Glas. 2 (1) (2016) 38–48. doi:10.1515/bglass-2016-0005 II A. Mishra, L. Petit, M. Pihl, M. Andersson, T. Salminen, J. Rocherullé, J. Massera, Thermal, structural and in vitro dissolution of antimicrobial copper- doped and slow resorbable iron-doped phosphate glasses, J. Mater. Sci. 52 (15) (2017) 8957–8972. doi:10.1007/s10853-017-0805-3 III A. Mishra, M. Ojansivu, R. Autio, S. Vanhatupa, S. Miettinen, J. Massera, In- vitro dissolution characteristics and human adipose stem cell response to novel borophosphate glasses, J. Biomed. Mater. Res. - Part A. 107 (9) (2019) 2099– 2114. doi:10.1002/jbm.a.36722 IV A. Mishra, F. Désévédavy, L. Petit, F. Smektala, J. Massera, Core-clad phosphate glass fibers for biosensing, Mater. Sci. Eng. C. 96 (2019) 458–465 doi:10.1016/j.msec.2018.11.038. xiii [LY Unpublished manuscripts I A. Mishra*, L. Azizi*, C. Palma*, N. B. Huynh, R. Rahikainen, P. Turkki, A. S. Ribiero, V. P. Hytönen, J. Massera “Surface modified phosphate glasses for improved protein adsorption and cell viability” (2019) *authors contributed equally. xv xvL Author’s contribution I. The author was responsible for writing the paper, planning the work and analyz- ing the data with co-authors. The author also contributed partly to the manufac- turing of the glasses used in this study. Further, the author was responsible for conducting all the experiments pertaining to the characterization of the glasses, except the SEM-EDS and ICP-OES analysis. II. The author was responsible for writing the paper, planning the work and analyz- ing the data with co-authors. The author also contributed partly to the manufac- turing of the glasses used in this study. Further, the author was responsible for conducting all the experiments pertaining to the characterization of the glasses, except the SEM-EDS and ICP-OES analysis, and the antimicrobial tests. III. The author was responsible for writing the paper, planning the work and analyz- ing the data with co-authors. The author was solely responsible for manufacturing the glasses used in this study. Further, the author was responsible for conducting all the experiments pertaining to the characterization of the glasses, except the SEM-EDS analysis. The author also performed the cell culture tests reported in this study. IV. The author was responsible for writing the paper, planning the work and analyz- ing the data with co-authors. Further, the author was responsible for conducting all the experiments pertaining to the in-vitro dissolution characteristics of the glass fibers, including the light loss experiments. The author also conducted the tests for obtaining the mechanical strength of the glass fibers. V. The author was responsible for writing the paper, planning the work and analyz- ing the data with co-authors. Further, the author was partly responsible for man- ufacturing and surface functionalization of the glasses. xvii xviiL List of Symbols η refractive index ρ Density μ Micro c Concentration m mili t Time x Dopant metal ion concentration D Diffusivity M Mol/l Tg Glass transition temperature Tx Onset of crystallization temperature ΔT Thermal processing window Tc Crystallization temperature xix xx List of Abbreviations APTS 3-Aminopropyltriethoxysilane BM Basic Medium BO Bonding oxygen CaP Calcium phosphate CD Classification determinant CFU Colony forming unit DMEM Dulbecco’s modified eagle medium DNA Deoxyribo nucleic acid DTA Differential thermal analysis EthD-1 Ethdium homodimer-1 FDA U.S. Food and Drugs Administration FTIR- Fourier-transform infrared spectroscopy-Attenuated total reflection ATR hASC Human adipose stem cells HCA Carbonated hydroxyapatite xxi HLA-DR Human leukocyte antigen – DR isotype ICP-OES Inductively coupled plasma - optical emission spectrometry L/D Live/dead LDH Lactase dehydrogenase MnOm Metal oxide NBO Non-bonding oxygen PDMS Polydimethylsiloxane PG Phosphate glasses PGF Phosphate glass fibers PLA Polylactic acid RNC Relative number of clusters RTF Relative total fluorescence S. epider- Staphylococcus epidermis mis SBF Simulated body fluid SEM Scanning electron microscopy SG Silicate glasses xxii TRIS tris(hydroxymethyl)aminomethane (IUPAC: 2-Amino-2-hydroxymethyl- propane-1,3-diol), or a buffer solution obtained using it UV-Vis Ultraviolet-visible Vm Molar volume WA Washed in acidic buffer solution WAS Washed in acidic buffer solution and silanized WB Washed in basic buffer solution WBS Washed in basic buffer solution and silanized WN Washed in neutral buffer solution WNS Washed in neutral buffer solution and silanized xxiii [[LY ,QWURGXFWLRQ %LRPDWHULDOVDUHGHILQHGDVWKHPDWHULDOVXVHGWRUHSODFHRUVXSSOHPHQWWKHIXQFWLRQVRI OLYLQJWLVVXHV>@7KHGLIIHUHQWNLQGVRIELRPDWHULDOVLQXVHWRGD\DUHD QDWXUDORUV\Q WKHWLF SRO\PHUV OLNH FROODJHQ RU 3/$ UHVSHFWLYHO\ E 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H[KLELWHGFRPSDUDEOHF\WRWR[LFLW\H[FHSW&X%ZKLFKGHSLFWHGWKHKLJKHVWF\WRWR[ LFLW\DWDOOWKHWLPHSRLQWV$PRQJWKHGLOXWHGH[WUDFWVWKH/'+YDOXHVZHUHDWWKHLUSHDN DW ' WKHQ GURSSHG DW ' DQG WKHQ URVH DJDLQ DW ' $W ' WKH GLOXWHG 6U%DQG&X%H[WUDFWVSUHVHQWHGDVOLJKWO\KLJKHUF\WRWR[LFLW\WKDQWKHFRQWURO ZKHUHDVWKDWRI&H%ZDVVOLJKWO\ORZHU$SDUWIURPWKHVHDOOWKHGLOXWHGH[WUDFWV SUHVHQWHGVLPLODUF\WRWR[LFLW\DWDJLYHQWLPHSRLQWLQGHSHQGHQWRIWKHGRSDQWPHWDOLRQ Undiluted 1:10 1:100 D3 D7 D14 D3 D7 D14 D3 D7 D14 Control Sr50-B2.5 Cu2-B2.5 Ce2-B2.5 Ag1-B2.5 Figure 5.30 Viability of hASC cultured on undiluted, 1:10 and 1:100 diluted glass extract media, analyzed by live/dead staining at 3, 7 and 14 days of culture. The scale bar corresponds to 500 μm (Publication III) 80 5 Undiluted 1:10 1:100 Ctrl * Sr50-B2.5 * 4 Cu2-B2.5 * Ce2-B2.5 * * 3 Ag1-B2.5 2 * * 1 Relative cellamount * * * 0 D-3 D-7 D-14 D-3 D-7 D-14 D-3 D-7 D-14 Time point (days) Figure 5.31 The proliferation of hASC cultured in a) undiluted, b) 1:10 diluted and c) 1:100 diluted glass extract media analyzed with CyQUANT Cell Proliferation Assay kit at 3, 7 and 14 days of culture. All the values presented are relative to the control at D-3. 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Mishra, J. Rocherulle, J. Massera Biomedical Glasses 2 (1) (2016) 38–48 https://doi.org/10.1515/bglass-2016-0005 Publication reprinted with the permission of the copyright holders. Biomed. Glasses 2016; 2:38–48 Research Article Open Access A. Mishra, J. Rocherullé, and J. Massera* Ag-doped phosphate bioactive glasses: thermal, structural and in-vitro dissolution properties DOI 10.1515/bglass-2016-0005 Phosphate bioactive glasses (PBGs) arise as a poten- Received Feb 01, 2016; revised May 14, 2016; accepted Jun 04, 2016 tial substitute to the typical silicate glasses. The chemical resistance of phosphate glasses can be tailored to suit dif- Abstract: Ag doped-bioactive phosphate glasses were pro- ferent applications. The time required for complete degra- cessed by traditional melt quenching technique with the dation can be adjusted from hours to years [7]. PBG demon- concentration of Ag2O ranging from 0 to 5 mol%. The Ag strate bioactivity and are promising materials for use in doping led to the depolymerization of the phosphate net- bone repair and reconstruction [8]. Furthermore PBG are work which is accompanied by a decrease in the glass tran- known to possess thermal properties showing wider pro- sition temperature. The processing window represented cessing window as evidenced by the large number of by ΔT(ΔT=Tx-Tg) exhibited a maximum for glasses con- bioactive phosphate fibers studied in the past years [9, 10]. taining 2-3 mol% of Ag2O. An increase in Ag content in- Glasses within the 50P O -10Na O-(40-x)CaO-xSrO duced an increase in the glass dissolution rate. The pre- 2 5 2 composition exhibited a minimum in the dissolution when cipitation of a Sr-CaP layer at the surface of the glass par- half of the CaO was replaced with SrO [11]. It was found ticulates was found to occur at shorter immersion time for that the dissolution rate is dependent on the glass struc- the Ag containing glasses. The congruent dissolution and ture and a reduction in the phosphate chain length leads wide processing window of these Ag containing glasses to an increase in the chemical resistance. Furthermore, may be of great interest for scaffold manufacturing from the proliferation and growth of gingival fibroblasts cells sintering of glass powders with antimicrobial properties. increased with increasing the SrO content. Whereas the SrO-free glass led to cell death within 24h, the CaO-free glass showed a cell count similar to the one measured at 1 Introduction the surface of the glass S53P4 used as reference [12]. This is partially attributed to the decrease in initial dissolu- Since the discovery of the bioactive glass 45S5 (Bioglass ) tion rate when SrO is introduced in place of CaO in the by L.L. Hench [1] and S53P4 by Andersson et al. [2], glass. The effect of strontium ions both in the media andin much work has focused on tailoring the silicate bioac- the reactive layer also play an important role as discussed tive glass composition to enable fiber drawing or powder in [12]. However, despite those promising results, these sintering [3–5]. Studies show that typical silicate bioac- metaphosphate glasses present a rapid initial dissolution tive glasses have a crystallization kinetics that not only in- rate and late precipitation of a reactive layer disabling the hibits shaping at medium to high temperature but also re- cells to efficiently attach for the first 1-3 days of culture [12]. duce the glass’s bioactivity [3, 6]. This is in agreement with Salih et al. who showed that fast dissolving glasses did not allow for proper cell prolifera- tion [13]. Metal ions such as Silver (Ag), Copper (Cu) or Cobalt A. Mishra: Department of Electronics and Communications Engi- (Co) just to cite a few can be added to glasses in order to neering, Tampere University of Technology, Korkeakoulunkatu 3, FI-33720, Tampere, Finland give them unique properties or modify the existing physi- J. Rocherullé: Equipe Verres et Céramiques, UMR-CNRS 6226, Sci- cal or chemical properties for clinical applications. These ences Chimiques de Rennes, Université de Rennes I, F-35042 Rennes ions are known to exhibit antimicrobial properties for ex- Cedex, France ample [14–16]. Ag is a popular choice as a dopant for sev- *Corresponding Author: J. Massera: Department of Electron- eral biomedical devices owing to its low toxicity to human ics and Communications Engineering, Tampere University of cells and effectiveness against many types of microbial Technology, Korkeakoulunkatu 3, FI-33720, Tampere, Finland; BioMediTech, University of Tampere and Tampere University growth, even at low concentrations [17]. Ag ions in the form of Technology, Biokatu 10/FI-33520 Tampere, Finland; Email: of nitrate, oxide are commonly found in several healthcare [email protected] © 2016 A. Mishra et al., published by De Gruyter Open. This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivs 3.0 License. - 10.1515/bglass-2016-0005 Downloaded from PubFactory at 07/27/2016 07:13:29AM via free access Ag-doped phosphate bioactive glasses | 39 Table 1: Glasses nominal compositions. Glass P2O5 (mol%) CaO (mol%) SrO (mol%) Na2O (mol%) Ag2O (mol%) x=0 50 20 20 10 0 x=1 49.5 19.8 19.8 9.9 1 x=2 49 19.6 19.6 9.8 2 x=3 48.5 19.4 19.4 9.7 3 x=5 47.5 19 19 9.5 5 products, for e.g., Ag coated catheters, wound dressing for measured using a differential thermal analyzer (DTA). The example [17]. Ag based ointments have long been used to structure was assessed by Fourier transformed infrared treat wounds susceptible to bacterial infections [18]. When spectroscopy in attenuated total reflectance mode (FTIR- PBGs are doped with Ag, the metal atoms are assumed to ATR). The dissolution test was performed in TRIS buffer so- be incorporated in the structure [19]. As opposed to silicate lution in order to confirm the congruent dissolution of in- glasses, PBG are known to have, in general, a congruent vestigated glasses. The formation of a CaP layer at the sur- dissolution. Thus the rate of release of Ag ions, which de- face of the glasses upon immersion in TRIS was evidenced pends upon the dissolution of the glass itself, can then be by SEM/EDS and FTIR-ATR. Finally the ions released in so- controlled if the Ag ions are added in PBG. Ahmed et al. lution were quantified using ion chromatography and ICP. developed silver-doped phosphate bioactive glasses with substitution of Na2O for Ag2O up to 15 mol%. Maximum antimicrobial effect was found to occur in glasses with1to 2 Materials and Methods 5 mol% of Ag2O [14]. The structure and properties of the silver-doped glasses were also studied by the authors [20]. More recently, the investigation of silver-doped phosphate Glass preparation glasses with antimicrobial properties were also investi- Glasses with nominal composition [(Ag2O)x-(0.5P2O5 · gated for glasses with 65 and 70 mol% of P2O5 [21]. In this study an increase in the antimicrobial effect was seen with 0.2CaO·0.2SrO·0.1Na2O)100−x] where x = 0, 1, 2, 3 and increasing the Ag content while similar effect was found 5 mol% were prepared. The nominal compositions are re- on Ag-free glasses when increasing the phosphate con- ported in Table 1. The glasses were obtained by melting tent. However, in these studies, the phosphate content was in a silica crucible in air. Analytical grade CaCO3, SrCO3, 50 mol% or greater. The fast dissolution rate of glass in NH4H2PO4,Ag2O, NaPO3 and Na2SO4 were used to pre- pare the batch. Ca(PO3)2 and Sr(PO3)2 were first prepared the P2O5-CaO-Na2O family, with P2O5 content greater that ◦ 50 mol%, was found to inhibit growth and bone antigen by heating NH4H2PO4 and carbonates at 300 C during 2 hours to remove NH3,H2O and CO2 then maintained expression. On the other hand the glasses with slow solu- ◦ ◦ bility upregulated the proliferation of cells [13]. Based on 10 hours at 850 C and 750 C for the calcium and stron- the previous research on Sr-containing bioactive glasses tium samples, respectively. The batch was placed in a silica crucible and heated up to 1000 ◦C for 30 minutes. 1 wt% it appears that despite a P2O5 content of 50% gingival fi- broblast cells can attach and proliferate [12]. Thus it is of Na2SO4 was added to the batch to avoid the reduction of silver oxide into metallic silver. The melt was poured tremendous interest to study the impact of Ag doping on ◦ this glass composition and assess whether antimicrobial into a preheated brass mould and annealed at Tg+15 C for ◦ ◦ −1 Ag-doped strontium containing phosphate glasses can be 30 minutes, then cooled down to Tg-50 Cat1 C·min obtained. and annealed during one hour to lower the internal stress. In this paper we report on the effect of Ag doping on Finally, the furnace was turned off and the glass cooled the thermal, structural and in-vitro dissolution properties to room temperature before removal. After melting, the of the phosphate bioactive glass previously studied in [12]. glasses were analyzed with EDS and the composition was found to be in agreement with the nominal one, within the Ag2O was introduced relative to the entire base glass as opposed to the typical isomorphic substitution. The rea- accuracy of the measurement (1.5 wt%). Despite the melt- son for such doping was to maintain the same (Ca+Sr)/P ing in silica crucible and the addition of Na2SO4,noSior ratio in the glass while lowering the phosphate content. S were found in the analysis. The thermal properties as a function of Ag content were - 10.1515/bglass-2016-0005 Downloaded from PubFactory at 07/27/2016 07:13:29AM via free access 40 | A. Mishra et al. Physical properties Optical Properties The density of the glasses was measured using The UV-Vis absorption spectra for all the glasses were Archimedes principle with an accuracy of ±0.02 g/cm3. recorded in the range 200–1600 nm at room temperature Ethanol was used as immersion liquid and the measure- using a UV-3600 Plus UV-VIS-NIR Spectrophotometer Shi- ment was performed on a polished glass bulk. Molar vol- madzu. The glass samples used for this measurement were ume of the glasses was calculated using their density and 2 mm thick and optically polished. molecular weight. Young’s modulus (E) was measured by the ultrasonic velocity technique. This technique is based on time-of-flight measurements using the pulse-echo tech- In-vitro dissolution nique [22] with ±2 GPa as the accuracy value. Vickers mi- crohardness (Hv) was measured by the indentation tech- The glasses were crushed and sieved to obtain powder nique using a Matsuzawa Digital Microhardness Tester with size ranging from 125 to 200 μm. These particles were MXT 70 with a pyramid shaped diamond indenter. A load immersed in TRIS buffer solution and placed in an incu- of 0.4905 N was applied for 5 s for all the measurements bating shaker HT Infors Multitron at 37◦C, 100 rpm to ob- which were performed at room temperature on polished tain laminar flow mixing without moving the particles. surfaces. The displacement rate was the same on load- The mass to volume ratio was kept constant at 75 mg of ing and unloading. All the characteristics were averaged glass for 50 ml TRIS. Immersion test was conducted for over measurements on 10 indentations per sample. The up to 4 weeks. The pH of the solution was measured be- accuracy is considered as better than ±0.2 GPa. fore and after immersion using pH ion analyzer Mettler Toledo SevenMultimeter with accuracy better than ±0.02. The pH of the solutions containing glass was compared Thermal properties to a TRIS blank sample. The pH of the blank solution was found to remain unchanged throughout the testing Differential Thermal Analysis (DTA, Netzch JUPITER F1) period. Post immersion, the glass powder was recovered, of all the glasses was carried out at a heating rate of 10 washed with acetone and dried for FTIR-ATR and SEM- ◦ C/min, in a Pt crucible and with a flow of 50 ml/min2 ofN . EDS analysis. 10 ml of the TRIS solution containing glassy The Tg (glass transition temperature), Tx (crystallization powder was diluted in 90ml of ultra-pure water for ICP- temperature) and Tp (crystallization temperature) were as- OES measurement Inductively coupled plasma - Optical sessed from the obtained thermogram. Tg was determined emission spectrometer (ICP-OES; Optima 5300DV, Perkin as the inflection point of the endotherm obtained by taking Elmer) was employed to quantify the amount of P, Sr, Ca first derivative of the DTA curve with an accuracy of2 ◦C. and Na ions found in the TRIS solution after glass immer- The Tx was taken as the onset of the crystallization peak sion. The value obtained for each ions was compared to the and Tp as the maxima of the exotherm. All measurements value obtained for the blank samples (only TRIS). The TRIS were obtained with an accuracy better than ±3◦C samples used as blanks were analyzed to ensure that the concentration of P,Ca, Sr and Na was consistently 0, or un- der the detection limit, as no such ions should be present Structural Properties in TRIS buffer solutions. The IR absorption spectra for all glass powders before and after immersion in TRIS were recorded with Perkin Elmer Imaging and elementary analysis Spectrum One FTIR Spectrophotometer in Attenuated To- tal Reflectance (ATR) mode. All spectra were recorded in Scanning electron microscopy (SEM), using a JEOL JSM the range 600–1600 cm−1, corrected for Fresnel losses and 7100F apparatus, was used for high-resolution imaging of were normalized to the band with maximum intensity. the sample surfaces (Pressure: 10−4 Pa, accelerator volt- Each spectrum is an average of 8 scans and has a resolu- age: 20 kV). Energy Dispersive X-Ray Spectroscopy (EDS) tion of 1 cm−1. was used as the chemical microanalysis technique used in conjunction with SEM. The associated detector (EDS SDD X-Max 80mm2 Oxford Instruments AZtecEnergy) allows one to identify what particular elements are and their rela- tive atomic proportions. This powerful tool of the elemen- - 10.1515/bglass-2016-0005 Downloaded from PubFactory at 07/27/2016 07:13:29AM via free access Ag-doped phosphate bioactive glasses | 41 Table 2: Thermal and mechanical properties. ◦ ◦ ◦ ◦ Glass Tg(±3 C) Tx(±3 C) Tp(±3 C) ΔT=Tx-Tg (±6 C) HV (±0.2GPa) E (±2GPa) x =0 436 550 603 114 2.3 55 x =1 430 554 583 124 2.2 56 x =2 428 567 611 139 2.2 55 x =3 420 560 597 140 2.2 55 x =5 418 546 587 128 2.2 56 3 Results The glasses of investigation with composition [(Ag2O)x- (0.5P2O5 · 0.2CaO·0.2SrO·0.1Na2O)100−x]wherex= 0, 1, 2, 3 and 5 mol% were analyzed using EDS/SEM. All the compositions were found to be in agreement with the nominal one within the accuracy of the measurement. As already mentioned in the previous section, we assume that no Si was found despite using a silica crucible. Never- theless, the Sr Lα1 EDX peak located at 1806 keV can mask the Si K peak at 1740 keV,however the low melting temper- ature (1000◦C) and the short fining time used for the glass Figure 1: Density (ρ) and molar volume (Vm) of the investigated synthesis, allows to prevent the silica dissolution in phos- glasses. phate melts, as stated in previous studies conducted on phosphate glasses which do not contain Sr and for which, the chemical compositions were checked by EDS [23, 24]. Figure 1 presents the density (ρ) and molar volume (Vm) of the investigated glass as a function of Ag2O con- tent. With an increase in Ag2O, the density increases while the molar volume remains constant, within the accuracy of the measurements. The thermal properties of the investigated glasses are reported in Table 2 and the DTA traces are shown in Fig- ure 2. With an increase in Ag2O, the glass transition tem- perature Tg decreases whereas Tx and Tp exhibit a maxi- mum for the glass with x = 2 mol%. ΔT(ΔT=Tx-Tg), which is a gauge of glass’s resistance to crystallization, is also found to present a maximum for glasses with x = 2 and Figure 2: DTA thermogram of the investigated glasses. 3 mol%. It is interesting to note that the glass with x = 1 mol% possesses a sharper crystallization peak as com- pared to the other glasses under investigation. In addition, tal analysis can identify elements heavier than Be with a the Young’s modulus and the Vickers microhardness were 3 spatial resolution better than 1 μm , corresponding to a determined for all the glass samples. These values, sum- 2 smallest spot size of about 1 μm , and the accuracy is ±1% marized in Table 2, which do not exhibit any difference, for polished bulk target in this case where pure standards when considering the accuracy of the measurements, are are collected on site. 55 GPa and 2.2 GPa for the Young’s modulus and for the Vickers microhardness, respectively. The glass’s structural properties were assessed using FTIR-ATR and Raman spec- troscopy. The IR spectra of the glasses are shown in Fig- ure 3a. All spectra were normalized to the band located - 10.1515/bglass-2016-0005 Downloaded from PubFactory at 07/27/2016 07:13:29AM via free access 42 | A. Mishra et al. (a) Figure 4: Change in pH as a function of immersion time. and 672h of immersion, the particles were rinsed and dried for analyses using FTIR-ATR and EDS/SEM. The solution was diluted ten times for ion concentration analysis using ICP-OES. At each immersion time, the pH of the solution was measured and the change in pH (as compared to the pH of the blank TRIS solution) is presented in Figure 4. As the immersion time increases, the pH of the solutions contain- ing the newly prepared glasses decreased. One can notice that an increase in Ag2O leads to a stronger decrease in the solution pH. The ion concentration in solution was quantified us- (b) ing ICP-OES. The evolution over time of the Ca, Sr, Na, and P concentrations is presented in Figure 5a, b, c and d, re- Figure 3: FTIR-ATR a) and Raman b) spectra of the investigated spectively. With an increase of the immersion time, an in- glasses. crease in the Ca, Sr, Na and P concentration is seen. The release of ions is greater with an increase in Ag2O. This in- at ~880 cm−1. The spectra exhibit five absorption bands crease is seen despite the decrease in these ions in the base located around 1260, 1085, 880, 775 and 718 cm−1 and glass (Table 1), due to the Ag doping. two shoulders at ~1154 and 980 cm−1. With an increase The FTIR-ATR spectra of the glasses with silver con- in Ag2O, all bands remained unchanged in terms of inten- tent x = 0 a), x = 1 b), x = 3 c) x = 5 d) as a function sity and shape, except for the band at 1260 cm−1 which de- of immersion time are presented in Figure 6. For immer- creases in intensity. sion up to 72 h, no significant changes could be seen in Figure 3b presents the Raman spectra of the glasses. the FTIR-ATR spectra of all investigated glasses. With an All spectra were normalized to the band with maximum increase in immersion time, all glasses exhibit a decrease intensity peaking at ~1175 cm−1. The spectra exhibit four in intensity and shift to lower wavenumber of the bands bands and one shoulder which are all characteristics of located at 1260 and 775 cm−1. The main band, at 890 cm−1, the metaphosphate network. With an increase in Ag2O, all is also found to shift to lower wavenumber. The band at bands are found to shift to lower wavenumber. The bands 1085 cm−1, is found to decrease in the glass with x = 0, at 695 and 1020 cm−1 as well as the shoulder at 1090 cm−1 whereas, for all Ag-containing samples the band decrease are all found to increase in intensity as compared to the in intensity for immersion up to 336 h and then increases band at 1175 cm−1. in intensity and shift to higher wavenumber for longer im- Glass powder of each composition was immersed in mersion time. All spectra for the Ag-containing glasses ex- TRIS solution for up to 672 hours. After 0, 24, 72, 168, 336 hibit the appearance of new bands at 988 and 1030 cm−1. - 10.1515/bglass-2016-0005 Downloaded from PubFactory at 07/27/2016 07:13:29AM via free access Ag-doped phosphate bioactive glasses | 43 (a) (b) (c) (d) Figure 5: Ca a), Sr b), Na c) and P d) concentration in TRIS solution as a function of immersion time. Comparison of the spectra for all glasses when immersed pear clear that the layer thickness increases with increas- for 672 h is presented in Figure 6e. It is clearly seen that the ing Ag2O, whereas the layer composition was identical at rise of the peak at 1150 and the intensity of the new bands the surface of all materials −1 at 988 and 1030 cm are higher with increasing Ag2O. The SEM images of the glasses with x = 0 after 672 h of immersion and of the glasses with x = 3 and 5 mol% after 4 Discussion 336 h of immersion are presented in Figure 7a, 7b and 7c, respectively. At 168 h, no change in the glass surface com- The aim of this research is to better understand the impact position could be evidenced by EDS analysis. After 672 h of of Ag doping on the thermal, structural and dissolution of immersion, no layer could be seen (or only sparsely) at the a phosphate bioactive glass. surface of the glass with x = 0, while a reactive layer could Doping the glass Sr-50, which was found in the past be seen at the surface of the Ag-containing glasses already to be a promising bioactive glass [11, 12], with Ag led to after 336h of immersion. We noticed that its thickness grew an increase in the density. This was expected as Ag has a thicker with increasing the immersion time. From SEM im- larger mass than all the other elements present in the glass ages the layer grew up to 5 μm for the glass with 5 mol% composition. However, the addition of Ag does not signifi- Ag O. EDS analysis revealed that this layer was rich in 2 cantly change the molar volume indicating that the Ag acts Ca and P and contained a significant amount of Sr. It ap- - 10.1515/bglass-2016-0005 Downloaded from PubFactory at 07/27/2016 07:13:29AM via free access 44 | A. Mishra et al. (a) (b) (c) (d) (e) Figure 6: FTIR-ATR of the glass x =0a), x =1b), x =3c) and x =5d) as a function of immersion time and all glasses immersed for 672 h in TRIS e) - 10.1515/bglass-2016-0005 Downloaded from PubFactory at 07/27/2016 07:13:29AM via free access Ag-doped phosphate bioactive glasses | 45 (a) (b) (c) Figure 7: SEM images of the glasses with x =0after 672 h of immersion a) and of the glasses with x =3b) and 5 c) after 336 h of immersion. It results the chemical balance: 0 2− 2− + 2Ag +SO4 2O +2Ag +SO2 Furthermore, to confirm that the use of Na2SO4 pre- vents formation of Ag nanoparticles, the UV-Vis spectra of the glasses were recorded and are presented in Figure 8. The absence of absorption band in the visible which could be a characteristic of the presence of Ag nanoparticles con- firms that all the Ag is present as positively charged ionsin the glasses under investigation. This is of particular inter- est since Ag+ ions have been found to possess antimicro- bial properties [19, 27]. The thermal properties of these glasses were recorded. With an increase in Ag2O, the Tg decreases whereas Tx,Tp Figure 8: UV-Vis absorption spectra of the glasses of investigation. and ΔT show a maximum for the glasses with 2 and 3 mol% of Ag2O. More interestingly the crystallization peak of the glass containing 1 mol% of Ag O was narrower than that as a monovalent cation in the phosphate vitreous network 2 of the other glasses (Fig. 2). This can be attributed to the with a similar ionic radius and similar positions than the change in crystallization mechanism of the glass induced other modifiers in the network. However the redox activity by the occurring of a small amount of silver particle during of a glass melt is definitely a function of the composition the reheating of the glass. It is well known that Ag can be and, at first sight, it is generally found that increasing ba- used in glass as nucleating agent [28]. It is also rather com- sicity favors the upper oxidation state for most redox cou- mon to see Ag0 formation upon heat treatment in glassy ples [25]. Compared to silicon oxide, phosphorus pentox- thin films or bulk [29, 30]. The decrease inT when Ag O ide is characterized by a lower value of the optical basic- g 2 increases may be explained by the lower cationic field ity, 0.45 and 0.33, respectively [26]. As a result, phosphate strength value (Z/r2) of silver (0.6) when compared to the glasses favor the lowest stable oxidation state, i.e. Ag0.To others modifiers cations, namely sodium (1.06), strontium avoid the presence of metallic silver droplets, the use of (1.56) and calcium (2.04), the coordination number being sodium sulfate as an oxidizing agent was necessary. The considered as 6 for the calculation. dissolution of the sulfate in the melt leads to the oxidation Contrary to the glass transition temperature which is of the metallic silver according to the following chemical strongly dependent of the cationic strength, it has been reactions: demonstrated that the mechanical properties clearly de- 1 SO2− O2− +SO + O pend on physical quantities related to the compactness or 4 2 2 2 the energy of cohesion of the vitreous network, such as those defined in [31]. The steadiness of the molar volume, 1 2Ag + O 2Ag+ +O2− whatever the silver oxide content, leads to the same com- 2 2 pactness and of the microhardness values. Regarding the - 10.1515/bglass-2016-0005 Downloaded from PubFactory at 07/27/2016 07:13:29AM via free access 46 | A. Mishra et al. Table 3: FTIR-ATR and Raman bands attribution. when Ag2O increases. The decrease in the absorption band located at 1260 cm−1 indicates a decrease in the network FTIR-ATR connectivity and an increasing amount of Q1 units. This is Wavenumber Attribution Reference further confirmed from the analysis of the Raman spectra −1 (cm ) of the glasses (Fig. 3b). The band at around 695 cm−1 cor- 718–775 νs P-O-P in metaphosphate [38] responds to symmetric vibration P-O-P in metaphosphate 2 880 νas P-O-P in Q [33, 34] type chains, the band at 1020 cm−1 to symmetric stretch 2− 1 1 −1 980 νs PO3 in Q [34–36] mode of NBO in Q units, and the ones at 1175 cm and 2− 1 2 −1 1085 νas PO3 in Q .PO2 in Q [38] 1250 cm to, respectively, symmetric and antisymmetric − 2 1154 νs PO2 in Q [34–36] vibrations of PO2 also in phosphate chains [38–40]. The − 2 −1 1260 νas PO2 in Q [34–36] shoulder at ~1090 cm corresponds to motion of termi- Raman nal oxygen bond vibration in phosphate chains [41]. The 695 P-O-P in chains [38–40] shift of all the bands toward lower wavenumber indicates 1 1020 νsNBO in Q [38–40] a weakening of the chemical bonds in the network when 1090 Terminal oxygen bond [38–40] Ag2O is added in the network, due to a network depolymer- 1175 νs PO2 chains [38–40] ization. The increase in intensity of the bands at 695 and −1 1250 νas PO2 chains [38–40] 1020 cm along with the increase in intensity of the shoul- 1320 P=O [38–40] der at 1090 cm−1 further confirm the increase inQ1 units and the shortening of the phosphate chain. This change in the glass structure induced by an increase in Ag2Oisin Young’s modulus, its variation, as a function of the glass agreement with the shift of the optical band gap toward composition, can be estimated with a good approximation higher wavelength when Ag2O increase (Fig. 8). The in- from the following relation: crease in the intensity of the shoulder at 1090 cm−1 along with the shift of the optical band gap indicates an increas- E =2 x G x C i i i i ing number of non-bridging terminal oxygens [42]. This is i i expected as the overall phosphate content decreases at the where Gi and Ci are the dissociation energy per unit vol- expense of the glass modifier elements [43]. Furthermore ume and the compacity factor of the oxyde i with the mo- the small shoulder at 1320 cm−1 related to P=O remains un- lar content xi, respectively. Details for the calculation of changed indicating that the amount of Q3 is similar in all these physical quantities are given in [31]. Using thermody- investigated samples. A summary of the band attribution namic data from [32], the calculation gives almost 56 GPa can be found in Table 3. for both the parent glass and the one with the highest sil- Typically an increase in Q1 units leads to an increase ver oxide content. As a consequence, a small amount of in the glass chemical durability [7, 44]. However, in the Ag2O doesn’t modify the mechanical properties of the par- studied glasses this seems not to be the case. As shown ent glass. in Fig. 4, it is clear that the decrease in the solution pH is All the absorption bands in the FTIR-ATR spectra steeper with an increase in the Ag content. Furthermore, (Fig. 3a) can be attributed to the phosphate glass network. the ICP-OES results show that with increasing the immer- −1 The main band at ~880 cm is attributed to P-O-P asym- sion time, the Ca, Na, Sr and P are being released in so- 2 metric stretching of bridging oxygen in Q units (νas P-O- lution. The increased dissolution rate with increasing Ag 2 −1 PQ) [33–35]. The shoulder centered at ~980 cm and content is clearly seen from the higher content of each ele- −1 the band peaking at 1085 cm correspond to the sym- ment in the solution. Except for Na, all the curves present a 2− 1 metric and asymmetric stretching vibration of PO3 in Q parabolic shape. It seems that the element release follows −1 units, respectively [34–36]. The band at 1085 cm can be at1/2 law which is typical for a diffusion controlled pro- 1 attributed to an overlap between PO3 Q terminal group cess. However, the parabolic shape of the ion release could 2 and PO2 Q groups in metaphosphate [37]. The shoulder also come from the saturation of the solution with those −1 −1 at 1154 cm and the absorption band at 1260 cm corre- ions leading to the precipitation of a reactive layer. The fi- − 2 spond to symmetric and asymmetric vibration of PO2 in Q nal Na concentration appears to be high, compared to the units, respectively [34–36]. The absorption 3 band located amount of the other ions, if a congruent dissolution is as- −1 at 718 and 775 cm are characteristic of P-O-P symmet- sumed [7]. Typically Na is the first element to leach out in ric stretching vibration in metaphosphate structure [38]. solution upon dissolution of phosphate glasses. Further- Only little change could be seen in the FTIR-ATR spectra more, no Na is usually found in the reactive layer precipi- - 10.1515/bglass-2016-0005 Downloaded from PubFactory at 07/27/2016 07:13:29AM via free access Ag-doped phosphate bioactive glasses | 47 I,II I,II Table 4: (M /P)layer /(M /P)core values with the corresponding clear sign of CaP precipitation (as shown by the arrow in standard deviations. Fig.7b and 7c) already happening after 332 h of immersion. As the dissolution rate increases with Ag2O, the layer thick- Na/P Ca/P Sr/P ness of the reactive layer was found to be thicker for the Parent glass 1.00 1.00 1.00 glass with higher Ag content. The EDS analysis confirmed x=2-2w 0.24 1.59 1.46 that the layer was enriched in Ca and Sr, while Na was pref- (0.03) (0.07) (0.10) erentially released in the solution. This is illustrated by the x=5-2w 0.32 1.31 1.25 different ratios between the MI,II/P values obtained for the (0.03) (0.09) (0.09) layer divided by those obtained for the core of the glass particles. These ratios are summarized in Table 4. Furthermore, when comparing the EDS composition tating at the surface of bioactive glasses [11]. The Ag con- of all reactive layers, at the surface of all investigated tent was not quantified by ICP as the noise to signal ratio glasses, a typical layer composition could be expressed was too high to draw any clear conclusion. as follow: 59±3%P, 24±4%Ca, 15±3%Sr, 1±1%Na, 0±1%Ag. The FTIR-ATR spectrum of the glasses was recorded at Even more the layer was found of be free of Na and Ag. In each immersion time point. From Fig. 6a, it is clear that at addition, the average value of the (Ca+Sr)/P ratio for the 168 h of immersion, structural modification occurs at the layer is 0.7±0.1 and is higher than those of the parent glass surface of the glasses. All glasses present a disruption of (0.4). Based on this ratio, as well as the position of the the Q2 units with a subsequent increase of the Q1 units. new absorption bands appearing in the FTIR-ATR spectra This is attributed to the first phase of phosphate bioac- one can assume that the layer forming is a Sr-substituted tive glass reaction in physiological medium [7, 11]. The calcium-phosphate chemically close to the dibasic cal- shift to lower wavenumber of the bands attributed to Q2 cium phosphate, as seen previously [11]. Moreover, the units also confirms a weakening of the bonds strength for layer composition was found to be independent of the Ag these structural units. The phosphate chains breakage is concentration. This can further explain the higher final found to proceed up to the longest immersion time. In a level of Na in solution compared to the other ions. Ag ap- first time, as explained by Bunker et al., it appears that the pears to behave similarly as Na, i.e. it is not incorporated glass dissolution is controlled by the rate at which the wa- in the reactive layer and thus Ag is expected to leach out ter diffuses at the surface of the glass. Once a full phos- at a similar rate than Na. Such layer was found to pro- phate chain is surrounded by OH group it is released in mote the adhesion and proliferation of human gingival fi- solution [7]. This explains that ICP results reveal a per- broblasts [12]. Furthermore, the controlled release of Ag as fect match between the ions released in solution and the monovalent ions may be of interest for its antimicrobial ion concentration of the parent glass (congruent dissolu- properties. tion). At longer immersion time P-O-P bond breakage oc- curs as seen by the change in Q2 to Q1 ratio in the FTIR- ATR spectra. However, while the glass with x=0 only show the disruption of the phosphate chains at the surface of the 5 Conclusion particles (Fig. 6a), the spectra of all Ag-containing glasses 1 (Fig. 6b–6d) present a sharp increase in Q units and ap- The addition of Ag2O in a phosphate bioactive glass, with −1 pearance of new bands at 988 and 1030 cm , after 673 h composition 0.5P2O5·0.2CaO·0.2SrO·0.1Na2O, leads to a of immersion. The new bands have been attributed to the glass with shorter phosphate chains and higher number formation of a CaP layer at the surface of the glass parti- of terminal non bridging oxygen, as evidenced by the in- 1 cles [11]. As shown in Fig. 6e, with an increase in Ag2O crease in Q units and the shift of the optical band gap to- the signal related to the CaP layer becomes stronger, as wards higher wavelength. Adding up to 3 mol% of Ag2O evidenced by the increasing intensity of the peaks at 988 was also found to increase the processing window. Despite and 1030 cm−1. This might be attributed to the faster dis- the decrease in the chain length, the decrease in network solution rate with increasing Ag2O, which leads to faster connectivity as evidenced by a progressive decrease in Tg saturation of the solution toward the reactive layer forma- when Ag2O increases, leads to a glass more prone to re- tion. The layer forming at the surface was analyzed via act in aqueous solution. The formation of a Sr-substituted EDS/SEM. While the layer could be sparsely seen at the calcium phosphate layer was found to occur sooner in the surface of the undoped glass (x = 0) even after the longest Ag-containing glass due to the increased dissolution rate. immersion time, the glasses with x = 3 and 5 mol% showed However the layer composition was found to be indepen- - 10.1515/bglass-2016-0005 Downloaded from PubFactory at 07/27/2016 07:13:29AM via free access 48 | A. Mishra et al. dent of the Ag content. Finally the Ag-containing glasses [18] H.S. Carr, T. Wlodkowski, H.S. Rosankranz: Silver sulfadiazine were found to dissolve in a congruent manner. Such ma- Che-mother 4 (1973), 585–587 terials could be employed for processing of implants with [19] S. P. Valappil, D. M. Pickup, D. L. Carroll, C. K. Hope, J. Pratten, R. J. Newport, M. E. Smith, M. Wilson, J. C. Knowles, Antimicr Ag high surface area to volume ratio, with subsequent antimi- and Chemo 51 (12) (2007), 4453–4461. crobial properties. [20] I. Ahmed, E.A. Abou-Neel, S.P. Valappil, S.N. Nazhat, D.M. Pickup, D. Carta, D.L. Caroll, R.J. Newport, M.E. Smith, J.C. Acknowledgement: The authors would like to acknowl- Knowles, Journal of Materials Science, 42 (2007) 9827–9835 edge the Academy of Finland for the financial support of [21] A.A. Ahmed, A.A. Ali, Doaa A.R. Mahmoud, A.M. El-Fiqi, Solid JM through the Academy Research Fellow and Initial Re- State Sciences, 13 (2011) 981–992 [22] G. V. Blessing, ASTM STP 1045, Philadelphia, PA, USA (1990) search Cost. 1045 [23] S. Chenu, J. Rocherullé, R. Lebullenger, O. Merdrignac, F. Cheviré, F. Tessier, H. Oudadesse, Journal of Non-Crystalline Solids 356 (2010) 87–92 References [24] S. Chenu, R. Lebullenger, J. Rocherullé, Journal of Material Sci- ence, 45 (2010) 6505–6510 [1] L.L. Hench, R.J. Splinter, W.C. Allen, T.K. Greenlee, J Biomed [25] C. Petitjean, P.J. Panteix, C. Rapin, M. Vilasi, R. Podor, Procedia Mater Res Symp, 334 (1971), 117–141 Materials Science, 7 (2014) 101–110 [2] O.H. Andersson, K. H. Karlsson, K. Kangasniemi, A. Yli-Urpo, [26] J.A. Duffy, J Non-Cry Sol 196 (1996), 145-50 Glastechnische Berichte 61(10) (1988), 300–305 [27] T. N. Kim, Q.L. Feng, J.O. Kim, J. Wu, H. Wang, G.C. Chen, F.Z. Cui, [3] S. Fagerlund, J. Massera, M. Hupa, L. Hupa, J Eur Ceram Soc 32 J Mater Sci Mater Med. 9(3): (1998) 129–134 11(8) (2012), 2731–2738 [28] S.D. Stookey, Ind. Eng. Chem, 51 (1959), 805–808 [4] E. Pirhonen E, H. Niiranen, T. Niemelä, M. Brink P. Törmälä, J [29] A.S. Kuznetsov, Ngo T. Cuong, V.K. Tikhomirov, M. Jivanescu, A. Biomed Mater Res B Appl Biomater 77(2) (2006), 227–33 Stesmans, L.F. Chibotaru, J.J. Velázquez, V.D. Rodríguez, D. Kir- [5] D. Groh, F. Döhler, D.S. Brauer, Acta Biomat., 10(10) (2014), ilenko, G. Van Tendeloo, V.V. Moshchalkov, Opt Mat, 34 (2012), 4465–4473 616–621 [6] J. Massera, S. Fagerlund, L. Hupa, M. Hupa, Journal of the Amer- [30] J. Massera, A. Martin, J. Choi, T. Anderson, L. Petit, M. Richard- ican Ceramic Society, 95 (2012), 607–613 son, Y. Obeng, K. Richardson, J Phy Chem Sol, 71 (2010), 1634– [7] B.C. Bunker, G.W. Arnold, J.A. Wilder, J. Non Crys Solids 64 (3) 1638 (1984), 291–316 [31] J. Rocherullé, C. Ecolivet, M.Poulain, P. Verdier, Y. Laurent, J. of [8] J. Clement, J.M. Manero, J.A. Planell, J Mater Sci: Mater Med 10 Non-Cryst. Solids 108 (1989) 187–193 (1999), 729-32. [32] D.D. Wagman, W.H. Evans, V.B. Parker, I. Halow, R.H. Schumm, [9] I. Ahmed, M. Lewis, I. Olsen, J.C. Knowles, Biomaterials 25 Selected values of chemical thermodynamic properties, NBS (2004), 501–507 technical notes, 270, 3–9, 1981 [10] J. Massera, Y.Shpotyuk, F. Sabatier, T. Jouan, C. B. Plédel, C. Roi- [33] P.Y. Shih, H.M. Shiu, Mater Chem Phy 106: (2007), 222–226. land, B. Bureau, L. Petit, N.G. Boetti, D. Milanese, L. Hupa, J Non [34] H. GaO, T. Tan, D. Wang, J of Contr Rel 96: (2004), 21–28. Crys Sol 425 (2015), 52–60 [35] Y.M. Moustafa, K. El-Egili, J Non-Crys Sol, 240: (1998), 144–153 [11] J. Massera, L. Petit, T. Cardinal, J. J. Videau, M. Hupa, L. Hupa, J [36] E.A. Abou Neel, W. Chrzanowski, D.M. Pickup, L.A. O’Deel, N.J. Mater Sci: Mater Med (24) (2013), 1407–1416 Mordan, R.J. Newport, M.E. Smith, J.C. Knowles, J Roy Soc Inter, [12] J. Massera, A. Kokkari, T. Narhi, L. Hupa, J Mater Sci: Mater Med 6: (2009), 435–446. 26 (2015), 196 [37] D. Ilieva, B. Jivov, G. Bogachev, C. Petkov, I. Penkov, Y.Dimitriev, [13] V. Salih, K. Franks, M. James, W. Hastings, C. Knowles, I. Olsen, J Non-Crys Sol, 283 (2001), 195–202. J Mater Sci: Mater Med 11 (2010) 615-620 [38] S. Lee, A. Obata, T. Kasuga, J Cer Soc Jap, 117 (2009), 935–938 [14] I Ahmed, D. Ready, M. Wilson, J.C. Knowles, Journal of Biomedi- [39] M.A. Karakassides, A. Saranti, I. Koutselas, J Non-Crys Sol 347 cal Research Part A, 79 (2006) 618–626 (2004), 69–79. [15] C. Wu, Y. Zhou, M. Xu, P. Han, L. Chen, J. Chang, Y. Xiao, Bioma- [40] A.G. Kalampounias, J Phy Chem Sol, 73 (2012), 148–153. terials, 34 (2013) 422–433 [41] R. C. Lucacel, A.O. Hulpus, V. Simon, I. Ardelean, J Non-Crys [16] G. Poongodi, P. Anandan, R.M. Kumar, R. Jayavel, Spectrochim- Sol,355: (2009), 425–429. ica Acta Part A: molecular and Biomolecular Spectroscopy, 148 [42] J. Massera, M. Vassallo-Breillot, B. Törngren, B. Glorieux, L. (2015) 237–243. Hupa, J Non-Crys Sol, 402 (2014), 28–35 [17] A.P. Adams, E.M. Santschi, M.A. Mellencamp, Vet. Surg. 28 [43] R.K. Brow, Journal of Non Crystalline Solids, 263–264 (2000), (1999), 219–225 1–28 [44] J. Massera, K. Bourhis, L. Petit, T. Cardinal, L. Hupa, M. Hupa, Journal of Physics and Chemistry of Solids, 74 (2013) 121–127. - 10.1515/bglass-2016-0005 Downloaded from PubFactory at 07/27/2016 07:13:29AM via free access PUBLICATION II Thermal, structural and in vitro dissolution of antimicrobial copper-doped and slow resorbable iron-doped phosphate glasses A. Mishra, L. Petit, M. Pihl, M. Andersson, T. Salminen, J. Rocherullé, J. Massera Journal of Material Science 52 (15) (2017) 8957–8972 https://doi.org/10.1007/s10853-017-0805-3 Publication reprinted with the permission of the copyright holders. PUBLICATION III In-vitro dissolution characteristics and human adipose stem cell response to novel borophosphate glasses A. Mishra, M. Ojansivu, R. Autio, S. Vanhatupa, S. Miettinen, J. Massera Journal of Biomedical Materials Research Part A 107 (9) (2019) 2099-2114 https://doi.org/10.1002/jbm.a.36722 Publication reprinted with the permission of the copyright holders. Received: 17 January 2019 Revised: 3 May 2019 Accepted: 7 May 2019 DOI: 10.1002/jbm.a.36722 ORIGINAL ARTICLE In-vitro dissolution characteristics and human adipose stem cell response to novel borophosphate glasses Ayush Mishra1 | Miina Ojansivu2 | Reija Autio3 | Sari Vanhatupa2 | Susanna Miettinen2,4 | Jonathan Massera1 1Laboratory of Biomaterials and Tissue Engineering, Faculty of Medicine and Health Abstract Technology and BioMediTech Institute, The main drawbacks of traditional silicate bioactive glasses are their narrow hot forming Tampere University, Tampere, Finland domain and noncongruent dissolution. In this article, we report on new borophosphate 2Adult Stem Cell Group, Faculty of Medicine and Health Technology and BioMediTech, glasses [xMnOm + (100 − x) (47.5P2O5 +2.5B2O3 +10Na2O + 20CaO + 20SrO)], MnOm Tampere University, Finland being CuO, Ag2O, and CeO2, having high thermal processability, hence suitable for fiber 3Faculty of Social Sciences and BioMediTech, Tampere University, Tampere, Finland drawing and sintering into scaffolds. Furthermore, the glasses dissolve congruently in 4Research, Development and Innovation simulated body fluid (SBF) and TRIS buffer solution, eventually leading to the precipita- Centre, Tampere University Hospital, Tampere, tion of a reactive layer. Human adipose stem cells (hASC) were cultured in media Finland enriched with glass extract at different dilutions, to investigate the optimal ion concen- Correspondence tration for cell survival. Cells grew in all the extracts, except in the undiluted Cu-doped Ayush Mishra, Laboratory of Biomaterials and Tissue Engineering, Faculty of Medicine and glass extract. At dilution 1:10, the lactate dehydrogenase (LDH) activity and cell prolifer- Health Technology and BioMediTech Institute, ation were comparable to the control, while at 1:100, the cells proliferated faster than Tampere University, Tampere, Finland. Email: [email protected] the control. Thus, the reference (undoped), Ag and Ce-doped glasses were found to be suitable for cell viability and proliferation. Cytotoxicity assessments using the LDH Funding information Buisness Finland; Competitive State Research assay indeed revealed the high cytotoxicity of the Cu extract. This raises questions Financing of the Expert Responsibility area of about the use of Cu in bioactive glasses and its optimal concentration as a dopant. Tampere University Hospital; Suomen Akatemia; Academy of Finland KEYWORDS bioactive glass, borophosphate glass, cell proliferation, cytotoxicity, in-vitro dissolution 1 | INTRODUCTION expected to degrade over time and eventually disappear from the site of implantation, it was reported that the commercial bioactive silicate Due to the aging of our society, there is a growing need for novel bioac- glass, S53P4, did not degrade completely in-vivo even 14 years post- tive materials that can be used as implants in the form of powders, scaf- surgery (Lindfors, Koski, Heikkilä, Mattila, & Aho, 2010). This phenome- folds, fibers, and so on. Since the discovery of Bioglass® by Hench, non, coupled with the narrow thermal processing window of the FDA Splinter, Allen, and Greenlee (1971), bioactive glasses have emerged as approved silicate glasses (Fagerlund, Massera, Hupa, & Hupa, 2012; a promising candidate for biomedical applications. Understandably, Massera, Hupa, & Hupa, 2012), forced the researchers to look at alter- most of the research on bioactive glasses since then has focused on sili- natives. Phosphate glasses (PGs) are completely biodegradable and have cate glasses, owing to their ability to promote cell growth, proliferation, a higher resistance to crystallization (Massera, Mayran, Rocherullé, & and differentiation (Ojansivu et al., 2015; Ojansivu et al., 2018). Part of Hupa, 2015). Additionally, they have a congruent dissolution, and the silicate glass bioactivity is assigned to their ability to precipitate a cal- degradation rate can be adjusted by varying the composition (Bunker, cium phosphate (hydroxyapatite) reactive layer. This layer has the same Arnold, & Wilder, 1984). PGs are known to have a good solubility composition and structure as the mineral phase of the bone, thereby toward metal ions enabling to dope the glass composition with ions enabling it to bond with the tissue. However, while the glass was having therapeutic relevance (Abou Neel, Ahmed, Pratten, Nazhat, & J Biomed Mater Res. 2019;107A:2099–2114. wileyonlinelibrary.com/journal/jbma © 2019 Wiley Periodicals, Inc. 2099 2100 MISHRA ET AL. Knowles, 2005; Mishra et al., 2017; Mishra, Rocherulle, & Massera, The cytotoxicity of the extracts on hASC was assessed using quantita- 2016; Mulligan, Wilson, & Knowles, 2003a; Mulligan, Wilson, & tive lactate dehydrogenase (LDH) measurement. Live/dead staining and Knowles, 2003b). The sustained and congruent degradation of PGs CyQUANT were performed at 3, 7, and 14 days to evaluate the influ- enables to deliver those therapeutic metal ions in a controlled manner ence of undiluted, 1:10 and 1:100 diluted extracts on cell viability and (Abou Neel et al., 2005). Consequently, the potential of Cu and Ag proliferation. doped PGs, for example, has been explored extensively in view of their antimicrobial properties (Abou Neel et al., 2005; Ahmed, Ali, 2 | MATERIALS AND METHODS Mahmoud, & El-Fiqi, 2011; Ahmed, Ready, Wilson, & Knowles, 2006; Mulligan et al., 2003a; Mulligan et al., 2003b; Wu et al., 2013). 2.1 | Glass melting In light of the above facts and observations, new PGs within the composition 50P2O5 + 10Na2O + (40 − x)CaO + xSrO (mol%) were Glasses within the composition xMnOm + (100 − x) (47.5P2O5 + investigated (Massera et al., 2013), and were found to support the 2.5B2O3 + 10Na2O + 20CaO + 20SrO) (mol%) with x = 0 for Sr47.5, attachment and proliferation of human gingival fibroblasts, except for 1forMnOm =Ag2SO4, and 2 for CuO and CeO2 were prepared using the glass with x = 0, which possessed the fastest dissolution rate of all a standard melting process in a silica crucible in air. x was chosen the glasses of investigation. The glass with x =20{50P2O5 +10Na2O+ such that the number of metallic ions was constant across all doped 20CaO + 20SrO (mol%)}, labelled Sr50, had the slowest dissolution borophosphate glasses. rate, and its dissolution products promoted proliferation of human gin- To prepare the glass, a batch consisting of Ca(PO3)2, Sr(PO3)2, gival fibroblasts (Massera, Kokkari, Närhi, & Hupa, 2015). However, Na(PO3), CaCO3, SrCO3,H3BO3, and Ag2SO4/CuO/CeO2 (depending when compared with known silicate bioactive glasses, their fast initial on the composition to be melted) was prepared. All the chemicals degradation rate and late reactive layer precipitation led to poor cell used were analytical grade. Ag2SO4 was used in order to prevent the 0 attachment for the initial 1–3 days of culture. PGs with a fast degrada- reduction of Ag ions to Ag . Ca(PO3)2 and Sr(PO3)2 were prepared tion rate have been shown to have poor attachment and proliferation beforehand using CaCO3, SrCO3, and NH4H2PO4. The carbonates of human osteoblasts by Salih et al. (2000). Based on previous results, were mixed with NH4H2PO4 in separate batches and heated to the glass Sr50 was further doped with Ag and Cu, and found to be 250 C to remove the NH3 and H2O, then to 650 and 850 Cto promising in terms of thermal, dissolution, and antimicrobial properties remove the CO2. The heating rate was kept constant at 1 C/min (Mishra et al., 2016; Mishra et al., 2017). The doped glasses possessed throughout the process, and the batch was kept for 12 hr at each a wide thermal processing window, a steady ion release in TRIS buffer temperature step. The glass batches were heated at 10 C/min to solution, and effectively eradicated Staphylococcus epidermidis (S. epi- 1,100 C and kept there for 30 min. The molten batch was cast into a dermis). However, the dissolution rate was further increased by the brass mold and annealed at Tg −40 C for 5 hr to relieve the internal presence of these metallic ions. When doped with Ce, the glasses stresses. Post melting, EDS analysis of the glasses was performed. No were found to have a wider thermal processing window, a steady ion Si from the silica crucible or S from the Ag2SO4 were evidenced in the release, albeit a slower degradation rate than Sr50 (Massera, Vassallo- glasses, within the accuracy of the measurement (1.5 mol%). Breillot, Törngren, Glorieux, & Hupa, 2014). The fast degradation of the metal-doped PGs led to human adipose stem cell death in a pre- 2.2 | Thermal properties liminary cell culture experiment. In response to the high degradation rate of the Ag and Cu doped Sr50 DTA thermograms of all the glasses were obtained using a SDTA, glasses, a new glass system with the composition xMnOm +(100− x) Netzsch Jupiter F1 (Selb, Germany). The glass powders were placed in (47.5P2O5 +2.5B2O3 +10Na2O + 20CaO + 20SrO) where x =1for Pt crucibles under 50 mL/min N2 flow and heated at 10 C/min. The Ag2O, and 2 for CeO2 and CuO, was developed in this study. This new Tg (glass transition temperature) was determined as the inflection glass system is derived from the Sr50 glass, where 2.5 mol% P2O5 is point of the first second order deviation in heat flow. The Tx (onset of replaced with B2O3 to stabilize the glass network. Hence, the new crystallization temperature) and the Tp (crystallization temperature) glasses are expected to have a reduced degradation rate, while were ascertained as the beginning and the maximum, respectively, of maintaining a high thermal processing window (Bingham, Hand, & the exothermic peak. The accuracy of the temperatures obtained was Forder, 2006; Harada, In, Takebe, & Morinaga, 2004; Karabulut, Yuce, estimated at ±3 C. The thermal processability window was calculated Bozdogan, Ertap, & Mammadov, 2011; Koudelka & Mošner, 2001; as ΔT = Tx − Tg, as done earlier in (Ahmed, Shaharuddin, Sharmin, Massera et al., 2011). In this article, we investigate these glasses in Furniss, & Rudd, 2015; Brauer, Brückner, Tylkowski, & Hupa, 2016; terms of their thermal properties and in-vitro dissolution in TRIS and Fagerlund & Hupa, 2016; Massera, Ahmed, Petit, Aallos, & Hupa, simulated body fluid (SBF). The Ca, Sr, and P-rich surface layer depos- 2014; Massera et al., 2010). ited at the surface of the glasses after immersion in TRIS buffer solution and SBF was evidenced with scanning electron microscopy-energy dis- 2.3 | In-vitro dissolution persive X-ray spectroscopy (SEM-EDS). Human adipose stem cells (hASC) were cultured up to 14 days in glass extracts obtained by Powders with particle size 125–250 μm were obtained by crushing enriching the culture media with the glass's dissolution by-products. and sieving. 75 mg of the glass powders were immersed in 50 mL of MISHRA ET AL. 2101 TRIS buffer solution or SBF, and placed in an incubating shaker HT 2.4 | Structural properties Infors Multitron (Bottmingen, Germany) at 37 C, 100 rpm to obtain a The glass particles recovered post immersion were analyzed with a laminar flow, as proposed earlier (Maçon et al., 2015). In this study, PerkinElmer Spectrum One FTIR Spectrophotometer (Waltham, MA) the protocol used by Maçon et al. (2015) was followed, thereby the in attenuated total reflectance (ATR) mode. The IR spectra in the mass of glass to volume ratio was maintained constant. While it is range of 650–1800 cm−1 were obtained and corrected for Fresnel known that the surface area to volume of solution ratio is more critical losses. All the spectra were normalized to the band having maximum when studying glass dissolution, as in (Massera & Hupa, 2014), here intensity. All the presented spectra have been obtained as an average the mass was maintained constant pertaining to the low variation in of 8 scans and have a resolution of 1 cm−1. glass density with adding the dopant. The density of the glasses mea- sured by the Archimedes principle were found to be 2.78 ± 0.02 g/cm3 (Sr47.5), and 2.96 ± 0.02 g/cm3 (for all the other glasses). The immer- 2.5 | SEM-EDS sion tests were conducted for up to 42 days in TRIS and 21 days in SEM/EDXA (Leo 1530 Gemini from Zeiss, Oberkochen, Germany and SBF. The SBF was prepared using the protocol described by Kokubo, EDXA from Vantage by Thermo Electron Corporation, Waltham, MA) Kushitani, Sakka, Kitsugi, and Yamamuro (1990), with a pH adjusted to was used to confirm the presence/absence of any surface layer pre- 7.40 at 37 C. The ion concentration of the SBF can be found in cipitated on the glass particles due to immersion in SBF, and to ana- Table 1. The pH of the immersion media with and without (“blank”) lyze the composition of the layer as well as the bulk of the particle. glass powders was measured using a pH ion analyzer Mettler Toledo The glass particles collected postimmersion were embedded in resin SevenMultimeter (Columbus, OH) with an accuracy of ±0.02. The pH and polished to reveal the particles cross-section. The accuracy of the of the blanks was not found to change throughout the duration of the elemental analysis is ±1.5 mol%, based on the measurement of five study, indicating that the immersion solution was stable across the areas in the samples (Table 2). The glass composition, postprocessing, length of the study. All samples were measured in triplicates and pH was also checked with regards to the nominal composition. The B values are presented as average of the three values. If the deviation content within the glass was not quantified by EDS due to the system between the values was <0.02, then the accuracy of the measurement of the pH meter was used. If the deviation was >0.02 then the devia- limitation. Therefore, the elemental composition obtained by EDS was tion between the triplicates was used. compared to the nominal glass compositions as if B was not present. Post immersion, 1 mL of the immersion media were recovered, Sr content seemed to be slightly lower and P slightly higher than the expected content, most likely due to a Sr deficient Sr(PO3)2. This was and diluted with distilled water containing 10% 1 M HNO3 by volume and stored at +4 C. These solutions were then analyzed with already reported in (Massera et al., 2013). Despite the use of silica inductively-coupled plasma––optical emission spectrometer (Agilent crucible, no Si was found in the EDS analysis of the investigated Technologies 5110 inductively coupled plasma-optical emission spec- glasses within the accuracy of the measurement. trometer (ICP-OES), Santa Clara, CA) to obtain the concentrations of Ca, Sr, P, Na, Ag, Cu, and Ce within the immersion solution. From the 2.6 | Preparation of PG extracts measured ion concentrations, the amount (in %) of each element dis- – μ solved from the glass was calculated. The accuracy of the measure- Glass powders with size 500 1,000 mwereobtainedbycrushingand ment was estimated at ±5% of the ICP-OES values, except when the sieving, and were used to prepare the extracts. Disinfection of the pow- deviation between the triplicates was greater than 5%. ders was carried out by first washing with deionized water, then twice After recovering the media, the solution containing particles was in 70% ethanol for 10 min, and subsequent drying for 2 hr at room filtered using filter paper with a pore size <5 μm. The particles were temperature. The powders were then immersed for 24 hr at 37 Cin then washed with acetone, allowed to dry for 30 min in air, and then the extraction medium prepared from Dulbecco's Modified Eagle stored for further analysis in a desiccator. Medium/Ham's Nutrient Mixture F-12 (DMEM/F-12 1:1; Life Tech- nologies, Gibco, Carlsbad, CA), which was supplemented with 1% TABLE 1 Ion concentrations in simulated body fluid (SBF; Kokubo antibiotics (100 U/mL penicillin and 0.1 mg/mL streptomycin; Lonza, et al., 1990) BioWhittaker, Verviers, Belgium) and 1% L-glutamine (GlutaMAX I; Life Ion Concentration in SBF (mM) Technologies, Gibco). The extraction was conducted in the cell culture Na+ 142.0 incubator at +37 C. The ratio of granules to the extraction medium was K+ 5.0 kept at 87.5 mg/mL for all compositions as reported in (Ojansivu et al., Mg2+ 1.5 2015). The extraction media was recovered via sterile filtering (0.45 μm), and human serum (Biowest, Nuaillé, France) was added to a Ca2+ 2.5 concentration of 5%. This mixture containing glass ions released from Cl− 148.8 − the powders is referred to as Basic Medium (BM) extract, and was HCO3 4.2 stored at +4 C. The control was prepared using the same recipe as HPO 2− 1.0 4 above, but without immersing the glass particles, and is called SO 2− 0.5 4 BM. Fresh extracts were prepared every 2 weeks, and no precipitation 2102 MISHRA ET AL. TABLE 2 Comparison of the surface After immersion in TRIS for 42 days layer composition with that of the bulk CaO (%) SrO (%) P2O5 (%) Na2O (%) CuO (%) CeO2 (%) Ag2O (%) obtained by EDX analysis (all indicated values are in mol%) Sr47.5 Surface 28.8 22.6 45.0 3.6 - - - Bulk (21.0) (17.2) (51.3) (10.5) - - - Cu-2 Surface 26.8 20.4 47.0 5.2 0.6 - - Bulk (21.9) (16.6) (52.1) (9.4) (0) - - Ce-2 Surface 27.1 ± 2.1 25.0 40.6 ± 1.6 1.2 - 6.0 - Bulk (20.6) (16.8) (50.0) (10.8) - (1.8) - Ag-1 Surface 23.7 18.9 48.9 7.7 - - 0.8 Bulk (21.9) (17.7) (49.7) (9.9) - - (0.8) After immersion in SBF for 21 days CaO (%) SrO (%) P2O5 (%) Na2O (%) CuO (%) MgO (%) Sr47.5 Surface 39.6 ± 1.8 9.7 42.0 4.2 - 4.5 Bulk (22.4) (17.0) (50.0) (10.6) - 0 Cu-2 Surface 45.2 ± 6.5 6.4 ± 3.4 39.2 ± 3.1 3.1 0.5 5.6 ± 1.6 Bulk (18.8) (17.9) (50.4) (10.9) (1.9) 0 was observed during the course of the cell-culture experiments. Diluted this was considered as the Day 0. The assay time points refer to the extracts were prepared by mixing 1 part glass extract with 9 parts BM corresponding time after Day 0. Fresh extract media was given to the and 99 parts BM, for 1:10 and 1:100 diluted extracts, respectively. cellstwiceaweek,andtheoldmediawascollectedandfrozenforLDH analysis. Control samples were grown in BM. The cell culture for both the donors was done separately. All the 2.7 | Adipose stem cell isolation, expansion, and analysis presented in this study, that is, Live/Dead, CyQUANT, and culture LDH were performed for both the donors at the same respective time The study was carried out in accordance with the Ethics Committee points. As the cells from both the donors behaved similarly with of the Pirkanmaa Hospital District, Tampere, Finland (R15161). The respect to time in culture and across the various glass compositions, hASC were isolated from subcutaneous white abdominal adipose tis- the CyQUANT and LDH results were combined. sue samples with a written informed consent of the donors. The donors were two women, aged 28 and 62 years. 2.8 | Cell viability The hASC were isolated using the protocol described in (Tirkkonen et al., 2012; Zuk et al., 2001). The hASC were then The cell viability was assessed qualitatively at Day 3, 7, and maintained in T75 Polystyrene flasks (Nunc, Roskilde, Denmark) in 14 (referred to as D-3, 7, 14, respectively) using live/dead staining BM. After primary cell culture the surface marker expression of hASC (Invitrogen, Life Technologies), as described in Tirkkonen et al. (2012). was analyzed by flow cytometry (fluorescence-activated cell sorting; The cells were incubated in a working solution containing 0.25 μM FACS, FACSAria®; BD Biosciences, Erembodegem, Belgium; Lindroos EthD-1 (staining dead cells red) and 0.5 μM Calcien-AM (staining live et al., 2009).The hASC used in this study, had a strong expression of cells green) for 30 min. The cells were imaged immediately afterward, CD73, CD90, and CD105, and negative or very low (<7%) expression using a fluorescence microscope (IX51, Olympus, Tokyo, Japan) of CD3, CD11a, CD14, CD19, CD34, CD45, CD54, CD80, CD86, and equipped with a fluorescence unit and a camera DP30BMW. HLA-DR. In addition, the cells used were in passage 1 (i.e., were sub- cultured only once after the isolation from adipose tissue). 2.9 | Cell proliferation The cell viability and proliferation analyses were conducted in 24-well plates (Nunc) with plating density ~4,200 cells/cm2 and in Cell proliferation was determined at D-3, 7, and 14 by analyzing the 48-well plates (Nunc) with plating density ~8,400 cells/cm2, respectively. DNA amount using CyQUANT Cell Proliferation Assay Kit (Invitrogen, The media was changed to the extract media 24 hr after the plating and Life Technologies), by following the manufacturers' protocol. At the MISHRA ET AL. 2103 chosen time points, the cells were lysed with 0.1% Triton X-100 lysis and/or scaffolds for biomedical applications. The biological response of buffer (Sigma-Aldrich) and then frozen (−70 C) to be analyzed at a hASC toward the extract obtained from these glasses was examined. later date. On the day of analysis, the plates were thawed at room Post processing, a visual inspection of the glasses revealed that temperature. Three parallel 20 μL samples of each lysate were pip- the Ag doped glass was transparent, while the Cu doped glass was etted to a 96 well plate (Nunc), and 180 μL of working solution pre- blue and the Ce doped glass yellow. UV-Vis spectroscopy (not shown pared from CyQUANT GR dye and cell lysis buffer was added to each here) confirmed the absence of an absorption band for the Ag doped parallel. Victor 1420 Multilabel counter (Wallac, Turku, Finland) was glass indicating that no or little Ag0 formed during the processing of used to measure the fluorescence at 480/520 nm. the glass, as in (Mishra et al., 2016). The UV-Vis spectra of the Cu doped glass revealed the presence of a broad absorption band at 850 nm correlated to Cu2+ ions as discussed in (Bae & Weinberg, 2.10 | Cytotoxicity assessment 1994; Mishra et al., 2017). While the UV-Vis spectra of the Ce doped Lactate dehydrogenase (LDH) Assay Kit (Calorimetric) from Abcam glass does not exhibit any absorption band, a strong shift of the (Cambridge, UK) was used to evaluate the cytotoxicity of the glass absorption band gap, to higher wavelength was noticed, pertaining to extracts at D-3, 7, and 14, using manufacturer's instructions. 20 μLof the presence of Ce4+ ions leading to the yellow coloration as dis- the previously collected cell media was pipetted in duplicates, sup- cussed in Blinkova, Vakhidov, Islamov, Nuritdinov, and Khaidarova plemented with 30 μL/well of LDH assay buffer. Fifty micro liter per (1994); Massera, Vassallo-Breillot, et al. (2014). well of working solution was then added immediately prior to the Their thermal properties are reported in Table 3. While no change in analysis, and the absorbance at 450 nm at 20 min was measured after the glass transition temperature was recorded for the Ag and Cu doped adding the working solution. All the values were normalized to the glasses, Ce doping led to a slight increase in Tg. Furthermore, while the number of cells (obtained from CyQUANT measurements). onset of crystallization (Tx)andcrystallizationpeak(Tp)remained unchanged when doping the glass with Ag, doping with Ce and Cu led to an increase in both characteristic temperatures. All the glasses pre- 2.11 | Statistical analyses sent a thermal processing window (ΔT = Tx − Tg), ΔT>100C. The DTA SPSS Statistics version 25 (IBM, Armonk, NY) was used to perform thermograms are presented as supplementary information Figure S1. statistical analyses. The CyQUANT and LDH results are presented as FTIR-ATR was performed on the glasses to evaluate the changes mean and standard deviation (SD). The number of biological replicates in the structure due to doping. Figure 1 exhibits the IR spectra of the was n = 6 (3 per cell line) for CyQUANT and n = 4 (2 per cell line) for LDH, with three measurements done per condition for each analysis. The significance of the impact of diluted and undiluted glass extracts was assessed with Mann–Whitney test. The Mann–Whitney test is a nonparametric test which evaluates the difference between non-normally distributed data samples. Furthermore, to control the familywise error-rate, Bonferroni corrections were made based on the number of meaningful comparisons. For both the CyQUANT and LDH analyses, the number of meaningful comparisons was 39. The results were considered to be statistically different when the adjusted p-value after Bonferroni corrections was <0.05. 3 | RESULTS In this study, the in-vitro dissolution characteristics of Cu, Ag, and Ce doped borophosphate glasses were obtained. Their thermal processabil- FIGURE 1 FTIR-ATR spectra of the glasses under investigation ity was also assessed as these glasses are intended to be used as fibers before immersion TABLE 3 Thermal properties of the Glass transition Onset of Crystallization Thermal processing investigated glasses temperature (Tg) crystallization temperature (Tc) window (ΔT = Tx – Tp) Glass (±3 C) (Tx) (±3 C) (±3 C) (±6 C) Sr47.5 450 C 616 C 660 C 166 C Cu-2 448 C 630 C 671 C 182 C Ce-2 463 C 627 C 665 C 163 C Ag-1 452 C 617 C 659 C 165 C 2104 MISHRA ET AL. glasses under investigation. All the spectra were normalized to the The initial ion release was fast and linear, then slowed down after the band at 890 cm−1, which has the maximum intensity. Five absorption 7 day time point. In Figure 3f), the release of Ce from Ce-2 nearly sat- bands can be observed at 1260, 1085, 980, 890 cm−1 and a broad urated around the 14 day time point. A more steady release of Cu band in the 700–800 cm−1 region. All the bands can be attributed to from Cu-2 can be observed in Figure 3g). To further understand the the PG network. The band having the maximum intensity at 890 cm−1 ion release behavior, the fraction of ions released from the glass into 2 is attributed to P–O–P asymmetric stretching in Q units (νas P–O–P the TRIS buffer solution as a function of immersion time was calcu- Q2) (Gao, Tan, & Wang, 2004; Moustafa & El-Egili, 1998; Shih & Shiu, lated, relative to the total amount of ions which would be present in 2007). The bands at 980 and at 1085 cm−1 correspond to the sym- the solution if the glass particles dissolved completely (presented in 2− 1 metric and asymmetric stretching vibration of PO3 in Q units, Figure 4). The fraction of Ca, Sr, and P ions released at all the time respectively (Abou Neel et al., 2009; Gao et al., 2004; Moustafa & El- points was similar within the error of measurement, for all the glasses. Egili, 1998). In addition, the band at 1085 cm−1 is attributed to the The B and Na ion release was slightly higher than the Ca, Sr, and P 1 2 overlap between PO3 Q terminal group and PO2 Q groups in meta- ions at the long immersion time points such as 28, 35, and 42 days. At phosphate glass structure (Ilieva et al., 2001). The band at 1260 cm−1 42 days, Cu-2 had the highest fraction of ions dissolved among the − 2 relate to the symmetric and asymmetric vibration of PO2 in Q units glasses under investigation. Exceptionally, after 42 days, Na release (Abou Neel et al., 2009; Gao et al., 2004; Moustafa & El-Egili, 1998). was highest in the case of the Ce-2. The broad absorption band located in the 700–800 cm−1 region cor- In SBF (Figure 5), the Ca concentration and therefore release were responds to P–O–P symmetric stretching vibration in metaphosphate similar within the error of measurement for all the glasses over time. structure (Lee, Obata, & Kasuga, 2009). The spectra present little Ce-2 exhibited a lower release of each ion (other than Ca), as com- observable changes as a result of doping except for a small decrease pared to the other glasses. Sr47.5, Cu-2, and Ag-1 released similar in the absorption band at 1260 cm−1 in the case of the glass doped amounts of B, Sr, and P ions in the solution. Additionally, a slight with Ce, while it slightly increased when doping with Cu and decline in the amount of Ca, Sr, and P ions, in solution, is evidenced Ag. Furthermore, this band shifts from 1,250 cm−1 for the Sr47.5, Ag- after 7 days. The Cu ion release from the Cu-2 glass was linear up to 1 and Cu-2 to 1,245 cm−1 for the Ce-2 glass. the 7 day time point, and then saturated up to 21 days. Ag could not The in-vitro dissolution properties of the glasses under investigation be quantified in the SBF or TRIS owing to a very high noise to signal were evaluated prior to the cell culture tests. The glass particles were ratio, and further investigation is needed to understand the underlying immersed in TRIS buffer solution from 1 to 42 days, and in SBF from phenomenon of Ag release from PG in TRIS and SBF. Similarly, no Ce 8 hr to 21 days. Figure 2 presents the change in pH, ΔpH, as a function was quantified upon dissolution of the Ce-2 glass despite being of immersion time in TRIS buffer solution and SBF. For the first 21 days evidenced upon dissolution in TRIS. of immersion in either media, no change in pH were recorded within Figure 6a–d present the IR spectra of the Sr47.5, Cu-2, Ce-2, and the accuracy of measurement. However, at longer immersion time in Ag-1 glasses, respectively, as a function of immersion time in TRIS TRIS, a small decrease in pH was recorded for Cu-2 and Ag-1. buffer solution. The changes in the spectra were similar for all the Figure 3 depicts the ion concentrations in the immersion solution glasses. A decrease in the intensity and a shift toward lower as a function of immersion time in TRIS buffer solution. In Figure 3 wavenumbers of the band at 1,260 cm−1, can be observed as a function (a) B, (b) Ca, (c) Sr, (d) P, (e) Na, all the immersion solutions show a sim- of immersion time. In the case of the Ce and Ag doped glasses an ilar ion concentration increase, throughout the duration of immersion. increase in a shoulder located at 1,154 cm−1 and assigned to symmetric FIGURE 2 Change in pH of the (a) TRIS buffer solution and (b) SBF upon immersion of glass particles as a function of immersion time MISHRA ET AL. 2105 FIGURE 3 (a) B, (b) Ca, (c) Sr, (d) P, (e) Na, (f) Ce and (g) Cu concentration in TRIS buffer solution as a function of immersion time of the glass particles − 2 vibration in PO2 in Q units can be seen (Abou Neel et al., 2009; Gao obtained with EDX analysis, and are presented in Table 2. As B could et al., 2004). New bands appeared at 990 and 1,032 cm−1 for all the not be analyzed with the EDX, all the concentrations presented have glasses. Figure 7a–d depict the IR spectra of the Sr47.5, Cu-2, Ce-2, been corrected for the absence of B. The CaO concentration increased and Ag-1 glasses, respectively, with respect to the immersion time in at the surface of all glasses. This increase is even greater at the surface SBF. As in the case of immersion in TRIS buffer solution, similar of the Sr47.5 and Cu-2 immersed in SBF. The SrO content increased changes in the spectra may be observed, except for the Ce-2 glass. Fur- slightly at the surface, upon immersion in TRIS, except in the case of thermore, in all Figures 6 and 7a,b,d, an absorption band in the Ce-2. However, the Sr content decreased at the surface of the Sr47.5 −1 1,500–1,650 cm region can be seen, typically assigned to OH vibra- and Cu-2 immersed in SBF. The P2O5 concentration decreased at the tion (Queiroz, Santos, Monteiro, & Prado da Silva, 2003). The only dif- surface of all glasses, except Ce-2, irrespective of the immersion solu- ference between the spectra recorded post TRIS immersion and post tion. It is noteworthy that the decline in the P2O5 concentration is more SBF immersion lay in the time required to evidence the appearance of pronounced upon immersion in SBF. The Na concentration decreased at the new bands. the surface of all the glasses, with the largest decrease in case of Ce-2. The SEM images of the glass particles before and after immersion To evaluate the cell response to these new glasses, hASC were for 6 weeks (6 W) and 3 weeks (3 W) in TRIS and SBF, respectively, are cultured in glass extracts. The cell viability and proliferation of hASC presented in Figure 8. A surface layer can be observed in all the condi- were examined in glass extracts at different dilutions, to adjudge the tions except Ag-1 and Ce-2 in SBF. The composition of the observable optimal ion concentrations for both cell viability and added functional- surface layer and the bulk glass upon immersion in TRIS and SBF were ity, potentially imparted by the metal ions. 2106 MISHRA ET AL. FIGURE 4 Fraction of the constituent ions leached into TRIS buffer solution after 42 days of immersion (a) Sr47.5, (b) cu-2, (c) Ce-2, (d) Ag-1 The undiluted extracts were analyzed with ICP-OES, and the ion Consequently, LDH assay kit was used to quantitatively determine concentrations obtained are presented in Table 4. Cu-2 exhibits the the cytotoxicity of the undiluted, 1:10 and 1:100 extracts. Figure 11 highest ion concentrations among all the glasses, while Ce-2 the low- depicts the cytotoxicity of all the glass extracts, normalized to the cell est. Ag was also released in the extract media, contrary to its absence amounts. Among the undiluted extracts, all the glasses exhibited simi- in the TRIS and SBF during in-vitro dissolution tests. The survival of lar cytotoxicity level at all the time points, except Cu-2 which had a hASC was evaluated at D-3, 7, and 14 (3, 7, and 14 days respectively) consistently higher cytotoxicity than the other extracts and the con- via live/dead staining in the undiluted, 1:10 diluted and 1:100 diluted trol. For the 1:10 and 1:100 diluted extracts, the LDH values were glass extracts (Figure 9). Based on this staining, high amount of green highest at D-3, then decreased at D-7 and then increased again at D- (live) cells could be seen in all condition, except for the undiluted Cu-2 14. At D-7, the 1:10 diluted Sr47.5 and Cu-2 extracts exhibited extract. Almost no red (dead) cells were observed in all other condi- slightly higher cytotoxicity than the control, whereas Ce-2 a bit lower. tions. In some cases, the images at D-14 seemed to exhibit lower den- Other than that, all the diluted extracts presented the same cytotoxic- sity of cells than D-7. To ascertain the above observations, CyQUANT ity level at a given time point, independent of the doped metal ion. cell proliferation assay was also performed at the same time points (Figure 10). For all the extracts except the undiluted Cu-2, the cell 4 | DISCUSSION amount increased from D-3 to D-7 and remained constant from D-7 to D-14 with an increase in the time in culture. The undiluted Cu-2 The goal of this study was to develop new glasses doped with metal extract presented a significantly lower cell amount at all the time ions for added functionality, which possess a wide thermal processing points. The undiluted Ag-1 extract exhibited a slightly lower cell window, and support cell viability and proliferation. From Table 3, all amount than the control, Sr47.5 and Ce-2 at all the time points, but the glasses exhibit a thermal processing window (ΔT)wellover100 C, much higher than the Cu-2. For the 1:10 dilution, Ce-2 presented the indicating their high degree of thermal processability. The glass doped highest cell amount at D-14 and for the 1:100 dilutions, the cell with Cu appeared to have the wider hot forming domain of all amount was independent of the composition and increased over time processed glasses. Also, these glasses had a higher thermal processing in culture for all the extracts. window than those investigated earlier (Massera, Vassallo-Breillot, MISHRA ET AL. 2107 FIGURE 5 (a) B, (b) ca, (c) Sr, (d) P, and (e) Cu concentration in SBF as a function of immersion time of the glass particles et al., 2014; Mishra et al., 2016, 2017), which may be attributed to par- Q1 units, is in agreement with structural changes reported on Ag and tial substitution of P2O5 with B2O3. The improvement in the thermal Cu doped PGs (Mishra et al., 2016, 2017). While these findings are in processing window of PGs as a result of boron addition has been agreement with previous studies on MnOm doped PGs, one should reported in the past (Bingham et al., 2006; Harada et al., 2004; keep in mind that in this study boron was also introduced. The pres- Karabulut et al., 2011; Koudelka & Mošner, 2001; Massera et al., 2011). ence of boron will lead to BO3 and BO4 structural units, which in turn, In Figure 1, the IR spectra of the glasses before immersion are will affect the phosphate structural units by either phosphate dispro- presented. The decrease in the 1,260 cm−1 band intensity and the portionation and/or B–O–P linkages. A more thorough structural shift to lower wavenumber, in the case of the Ce-2 samples, is attrib- study would be required to fully grasp the changes in structure due to utable to a decrease in the network connectivity, weakening of the metal ion doping and will be the scope of future research. Q2 units and an increase in the amount of Q1 units. This is in agree- The change in pH (ΔpH) of the TRIS buffer solution and SBF as a ment with structural changes reported on PGs by Massera, Vassallo- function of immersion time is presented in Figure 2a,b), respectively. In- Breillot, et al. (2014). In contrast, the intensity of this band increased vitro dissolution tests were performed in TRIS buffer solution, in order in the case of Ag-1 and Cu-2, indicating a decrease in the amount of to judge the dissolution mechanism of the glasses of investigation, while 2108 MISHRA ET AL. FIGURE 6 FTIR change in the structure of (a) Sr47.5, (b) Cu-2, (c) Ce-2, (d) Ag-1 as a function of immersion time in TRIS buffer solution FIGURE 7 FTIR change in the structure of (a) Sr47.5, (b) Cu-2, (c) Ce-2, (d) Ag-1 as a function of immersion time in SBF MISHRA ET AL. 2109 FIGURE 8 SEM images of the glass particles: Before immersion and after immersion in TRIS and SBF solution for 42 and 21 days, respectively TABLE 4 Ion concentrations in the Ca Sr P B Cu Ce Ag undiluted extract media of the glasses under investigation (mg/L) Extract media 43 ± 2 0 37 ± 2 0 0 0 0 Sr47.5 79 ± 4 40 ± 2 111 ± 6 1 ± 0.05 0 0 0 Cu-2 107 ± 5 51 ± 3 141 ± 7 1 ± 0.05 2 ± 0.1 0 0 Ce-2 47 ± 2 22 ± 1 66 ± 3 1 ± 0.05 0 0 0 Ag-1 55 ± 3 27 ± 1 83 ± 4 1 ± 0.05 0 0 2 ± 0.1 immersion in SBF was done to assess if a reactive layer would form due were much less pronounced compared to the glasses investigated in to super-saturation of the solution. The in-vitro dissolution also aimed (Massera, Vassallo-Breillot, et al., 2014; Mishra et al., 2016, 2017). This at evaluating the change in the immersion solution pH which could later is attributed to the B2O3 substitution for P2O5, which leads to enhanced impact the cell viability. When immersed in TRIS and SBF, no significant hydrolytic resistance (Massera et al., 2015; Sharmin et al., 2013). change in the pH were observed for up to 21 days. Therefore, the solu- The ICP analysis of the TRIS solutions recovered after immersion tions remained at a physiological pH. A small decrease in pH at longer (Figure 3), revealed that all the glasses show similar ion release behavior immersion times for Cu-2 and Ag-1 in TRIS, was expected due to the overall. The ion release profiles in TRIS buffer solution are in agreement high release of phosphorous ion (Ahmed, Lewis, Olsen, & Knowles, with previously reported data (Mishra et al., 2016, 2017). Figure 4 2004; Massera et al., 2013), upon dissolution of PG. However, the depicts the fraction of ions released from the glasses upon immersion change in pH remained minor and should not affect the cells adversely. in TRIS buffer solution. Overall, the glasses presented a congruent dis- The slightly lower pH recorded upon immersion (for over 21 days) of solution, as evidenced by the similar release of the constituent ions at the Cu-2 and Ag-1 was consistent with their higher solubility, as already least up to 7 days. Furthermore, the change in glass composition does reported in (Mishra et al., 2016, 2017). However, the changes in pH not seem to significantly impact the glass dissolution rate. At longer 2110 MISHRA ET AL. FIGURE 9 Viability of hASC cultured on undiluted, 1:10 and 1:100 diluted glass extract media, analyzed by live/dead staining at 3, 7, and 14 days of culture. The scale bar corresponds to 500 μm FIGURE 10 The proliferation of hASC cultured in (a) undiluted, (b) 1:10 diluted, and (c) 1:100 diluted glass extract media analyzed with CyQUANT cell proliferation assay kit at 3, 7, and 14 days of culture. All the values presented are relative to the control at D-3. The number of biological replicates was n =6. The level of significance is set at p < .05. *implies that p < .05 between the indicated extract and control (Ctrl) at the same time point. The reported values correspond to the combined results from the two donors immersion times, the release of the ions appeared to be parabolic which solution is already thermodynamically prone to form apatite crystals may imply the formation of a diffusion barrier. (Bohner & Lemaitre, 2009). Furthermore, while all the glasses were From Figure 5, the different ion concentrations in SBF compared to found to release ions at similar speed upon immersion in TRIS TRIS, may be attributed to the significantly higher amount of ions pre- (Figure 4), here the glass containing Ce demonstrated significantly sent in the SBF. The SBF closely mimics the inorganic phase of the lower ion release kinetics. This might be correlated to the higher hydro- blood plasma, and provides a favorable model for evaluating a materials' lytic resistance of the Ce doped PGs, as reported earlier (Massera, bioactivity by measuring the materials' ability to induce apatite forma- Vassallo-Breillot, et al., 2014). However, if the Ce would stabilize the tion at its surface. As Ca is already present in SBF, it leads the solution glass network toward aqueous dissolution, similar trend would be seen to reach super-saturation at earlier time points compared to that in upon immersion in TRIS and SBF. Therefore, it is also possible that Ce TRIS. Indeed, the decline in Ca, Sr, and P measured upon dissolution of readily precipitated CePO4 crystals as seen in Ce doped silicate glasses PG for longer than 7 days in SBF is often assigned to the precipitation (Leonelli, Lusvardi, Malavasi, Menabue, & Tonelli, 2003). of a reactive layer (Massera et al., 2013; Mishra et al., 2016). The pres- In Figures 6 and 7, the IR spectra of all the glasses present similar ence of Sr in Ca-P reactive layer has been discussed previously changes as a function of immersion in TRIS and SBF, respectively. (Massera et al., 2013). However, one should keep in mind that the SBF With an increase in immersion time, the decrease in the intensity and MISHRA ET AL. 2111 FIGURE 11 LDH activity in the (a) undiluted, (b) 1:10 diluted, and (c) 1:100 diluted glass extract media normalized with the corresponding CyQUANT assay values. The values are presented relative to the control at D-3. The number of biological replicates was n = 4. The level of significance is set at p < .05. *implies that p < .05 between the indicated extract and control (Ctrl) at the same time point. The reported values correspond to the combined results from the two donors a shift toward lower wavenumbers (−14 cm−1) of the band at layer and (b) lead to a lower dissolution rate of this glass in SBF, while it 1260 cm−1, indicates the progressive increase in the amount of Q1 is not greatly impacted in TRIS. The precipitation of the reactive layer units at the expense of Q2 units, due to de-polymerization of the in TRIS buffer solution is also in agreement with the ICP data (Figure 5). phosphate network. The shift to lower wavenumbers of the band at The higher release of B and Na than Sr, Ca, and P after 7 days can be 1260 cm−1, attributed to Q2 units, represents the weakening of their attributed to the precipitation of the reactive layer. It should be men- inherent bond strengths. These changes are characteristic of the early tioned, that in the case of Sr47.5 and Cu-2 the reactive layer is thicker stages of glass dissolution (Bunker et al., 1984; Massera et al., 2013). upon immersion in SBF than in TRIS solution. The new bands at 990 and 1,032 cm−1 for all glasses along with the The new glasses, developed in this study, were found to possess a increase in the shoulder at 1154 cm−1 can be assigned to the precipi- slower dissolution rate than their counterparts studied earlier in tation of a Ca-P layer, as dicalcium phosphate dehydrate, possibly par- (Massera, Vassallo-Breillot, et al., 2014; Mishra et al., 2016, 2017). This tially substituted with Sr, at their surface as explained in (Mishra et al., should have a positive impact on the cell viability and proliferation. 2016; NIST, 2019). Ce-2, however, did not exhibit the appearance of Upon ICP analysis of the undiluted glass extracts (Table 4), Cu-2 new bands, indicating that the reactive layer precipitation was either presented the highest dissolution rate in the extract media as indi- delayed or a new form of reactive layer precipitated and did not cated by the high concentrations for all the constituent ions, and Ce-2 attach to the glass surface. The overall changes to the spectra as a the lowest. Also, Ag-1 seemed to have a slower dissolution rate than function of immersion time were also smaller for Ce-2 which could be the reference glass Sr47.5. Notably, the release of Ag may be supported by either the delayed formation the Ca-P reactive layer or observed in the extract media, which is probably due to a higher glass the formation of CePO4 crystals. amount to media ratio than in the dissolution tests. The release of Cu The formation of the reactive layer was further confirmed in was also evidenced, whereas Ce was not released into the extract Figure 8. A surface layer can be observed in all the conditions across media. It is worth noting here that DMEM, which is a major compo- both the immersion media, except Ag-1 and Ce-2 in SBF. This is in dis- nent of cell culture medium in this study, has a smaller Ca2+ concen- − agreement with the IR spectra of the Ag-1 glass after immersion for tration compared to SBF and a high concentration of HCO3 21 days in SBF, which does show the formation of new bands. This (Rohanová et al., 2014). Hence, the ion release profiles were expected may be due to poor attachment of the surface layer, leading to detach- to be different than in the SBF, justifying the need for quantifying ion ment before the SEM measurement or to a surface layer forming only release from glass when comparing data from different immersion in limited areas. However, the absence of the surface layer in case of media. Furthermore, while all three solution are, to some extend buff- Ce-2 in SBF is consistent with its IR spectra (Massera, Vassallo-Breillot, ered, their buffering capacities are significantly different. This could et al., 2014). From EDX analysis (Table 2) of the particles after immer- also explain some of the discrepancy between the dissolution rat- sion, it can be observed that Ce is incorporated in the surface layer, as e/mechanism evidenced here. Finally, from the Ce release point of the Ce concentration increases, compared to the nominal value. This view it appears that the glass dissolution/reaction is more closely explains the saturation in Ce concentration after immersion for 42 days related to the one seen in SBF rather than in TRIS buffer solution. in TRIS. In line with this observation, an earlier study (Leonelli et al., During cell culture with hASC, cell viability in different dilutions of 2003) reported the formation of a mixed phase of Ce2O3-CePO4 in the glass extracts was ascertained at D-3, 7 and 14 with the help of live/ surface layer, upon dissolution of Ce containing phospho-silicate dead (L/D) staining (Figure 9). Cells were found to be viable in all the glasses in SBF, which might (a) limit the formation of a Ca-P reactive conditions, with no observable dead cells, except for the undiluted Cu-2 2112 MISHRA ET AL. extract. Some images at D-14 depict less cells than the ones at D-7, but precipitation of CePO4 crystals in phosphorus rich solution. The change this could be due to peeling-off of the cell layer owing to very high cell in dissolution mechanism and kinetics was further confirmed when pre- amounts. Furthermore, the morphology of the cells remained unchanged paring the extract. The Cu-2 glass leached more ions, while the Ag-1 across the conditions and time points (Figure S2). leached out less ions than the reference glass. No Ce could be detected From cell proliferation analysis in Figure 10, other than undiluted in the extract of the Ce-doped glass supporting the probable precipita- Cu-2, all the extracts exhibit an increase in the cell amount from D-3 tion of CePO4 crystals in solution with high P content. to D-7. The cell amounts remained constant from D-7 to D-14, per- In hASC culture up to 14 days, the undiluted and diluted extracts haps due to reaching 100% confluence at D-7. The low cell amounts of these glasses were found to support cell viability and proliferation, observed in the case of Cu-2 can be attributed to the high concentra- except the undiluted Cu-2 extract. The Ag-doped extract, undiluted tion of the dissolution products in the extract, and/or the presence of was found to be more cytotoxic than the control and the Sr47.5 and Cu ion in the media. However, it appears that release of up to Ce-2 at 3 days of culture, but at the same level later on. The higher 79 mg/L of Ca, 40 mg/L of Sr, and 111 mg/L of P do not negatively LDH activity could be due to the relatively higher ion concentrations affect the cells behavior as seen by the ion concentration of the in the extracts resulting from higher dissolution rate, or excess Cu ion Sr47.5 glass extract (Table 4). In the case of the Ag-1 glass extract, concentration. Thus, despite the popularity of Cu as a dopant for bio- despite an increase in the cell number as a function of culture time, a active glasses, the dopant concentration needs to be optimized for lower amount of cells than in the control, were recorded. This can every glass system. On the other hand, the reference Sr47.5, Ag-1, hardly be attributable to the Ca, Sr, P, and B concentration which are and especially Ce-2 show great promise as biomaterials, and could lower than for the Sr47.5 glass extract, and therefore could be pave way for new glasses and glass fibers for new biomedical applica- assigned to the presence of Ag ions in the solution. In 1:10 diluted tions. Ce doped glass fibers have been shown earlier to be antimicro- glass extracts, the cell amount increased over time in culture for all bial, and are currently being researched by the authors for their the extracts, and Ce-2 exhibited the highest cell amount at D-14. potential in biosensing. However, this cannot be assigned to the Ce as no Ce was found in the extract, maybe due to the precipitation of CePO4 during the extract preparation. Among the 1:100 diluted glass extracts, cell ACKNOWLEDGMENTS amounts increased over time in culture, and were also independent of The authors would like to acknowledge the support of Academy of the glass composition. Finland for the financial support of Dr. Jonathan Massera and Ayush LDH is released from dead cells and, therefore, a higher LDH is Mishra through the Academy Research Fellow and Initial Research indicative of higher cytotoxicity. Consequently, upon LDH analysis of Cost. This study was partly supported by the Academy of Finland, the undiluted and diluted glass extracts, the undiluted Cu-2 exhibited Business Finland and Competitive State Research Financing of the a higher cytotoxicity than the other extracts at all the time points. Expert Responsibility area of Tampere University Hospital. The authors Among the diluted extracts, all the extracts at the same dilution pre- would also like to thank Anna-Maija Honkala and Sari Kalliokoski for sent similar cytotoxicity at a given time point, which was due to the the technical assistance. lower metal ion concentrations in the diluted extracts as compared to the undiluted ones. Additionally, the increased LDH of diluted extracts at D-14 can be attributed to the high cell amount at this time point. CONFLICT OF INTEREST There are no conflict to declare. 5 | CONCLUSION ORCID In this article, we investigated the undoped and Ag, Cu, and Ce doped glasses within the system xMnOm +(100− x)(47.5P2O5 +2.5B2O3 + Ayush Mishra https://orcid.org/0000-0002-2916-8427 10Na2O + 20CaO + 20SrO) (mol%), to assess the impact of low metal ion doping on the in-vitro dissolution, thermal, and structural proper- REFERENCES ties. The data can also be compared to previously reported glasses with similar doping within the phosphate matrix. These new glasses were Abou Neel, E. A., Ahmed, I., Pratten, J., Nazhat, S. N., & Knowles, J. C. (2005). Characterisation of antibacterial copper releasing degradable found to have a wider thermal processing window compared to their phosphate glass fibres. Biomaterials, 26, 2247–2254. predecessors. The in-vitro dissolution study in TRIS and SBF solution Abou Neel, E. A., Chrzanowski, W., Pickup, D. M., O'Dell, L. A., revealed that the choice of the immersion media used was of para- Mordan, N. J., Newport, R. J., … Knowles, J. C. (2009). Structure and mount importance. All glasses appeared to have similar dissolution properties of strontium-doped phosphate-based glasses. Journal of the – kinetics in TRIS buffer solution with precipitation of a Ca-P reactive Royal Society Interface, 6, 435 LP 446 Retrieved from http://rsif. royalsocietypublishing.org/content/6/34/435.abstract layer for the Ag and Cu doped glasses, and Ce containing Ca-P layer for Ahmed, A. A., Ali, A. A., Mahmoud, D. A. R., & El-Fiqi, A. M. (2011). Study the Ce doped glass. The Ce doped glasses had a lower dissolution rate on the preparation and properties of silver-doped phosphate in SBF compared to the other glasses. This was attributed to the antibacterial glasses (part I). Solid State Sci. Elsevier Masson SAS, 13, MISHRA ET AL. 2113 981–992 Retrieved from https://doi.org/10.1016/j.solidstatesciences. Lee, S., Obata, A., & Kasuga, T. (2009). Ion release from SrO-CaO- 2011.02.004 TiO2-P2O5 glasses in Tris buffer solution. Journal of the Ceramic Soci- Ahmed, I., Lewis, M., Olsen, I., & Knowles, J. C. (2004). Phosphate glasses ety of Japan, 117, 935–938. for tissue engineering: Part 2. Processing and characterisation of a Leonelli, C., Lusvardi, G., Malavasi, G., Menabue, L. Ã., & Tonelli, M. (2003). ternary-based P2O5-CaO-Na2O glass fiber system. Biomaterials, 25, Synthesis and characterization of cerium doped glasses and in vitro 501–507. evaluation of bioactivity.Pdf. Journal of Non-Crystalline Solids, 316, Ahmed, I., Ready, D., Wilson, M., & Knowles, J. C. (2006). Antimicrobial 198–216. effect of silver-doped phosphate-based glasses. Journal of Biomedial Lindfors, N. C., Koski, I., Heikkilä, J. T., Mattila, K., & Aho, A. J. (2010). A Materials Research Part A, 79, 618–626. prospective randomized 14-year follow-up study of bioactive glass Ahmed, I., Shaharuddin, S. S., Sharmin, N., Furniss, D., & Rudd, C. (2015). and autogenous bone as bone graft substitutes in benign bone tumors. Core/clad phosphate glass fibres containing iron and/or titanium. Bio- Journal of Biomedical Materials Research Part B, 94, 157–164. medical Glasses, 1,20–30. Lindroos, B., Boucher, S., Chase, L., Kuokkanen, H., Huhtala, H., Bae, B.-S., & Weinberg, M. C. (1994). Optical absorption of copper phos- Haataja, R., … Miettinen, S. (2009). Serum-free, xeno-free culture phate glasses in the visible spectrum. Journal of Non-Crystalline Solids, media maintain the proliferation rate and multipotentiality of adipose 168, 223–231 Retrieved from https://linkinghub.elsevier.com/ stem cells in vitro. Cytotherapy, 11, 958–972. retrieve/pii/0022309394903336 Maçon, A. L. B., Kim, T. B., Valliant, E. M., Goetschius, K., Brow, R. K., Bingham, P. A., Hand, R. J., & Forder, S. D. (2006). Doping of iron phos- Day, D. E., … Jones, J. R. (2015). A unified in vitro evaluation for phate glasses with Al2O3, SiO2 or B2O3 for improved thermal stabil- apatite-forming ability of bioactive glasses and their variants. Journal ity. Materials Research Bulletin, 41, 1622–1630. of Materials Science. Materials in Medicine, 26,1–10. Blinkova, G. B., Vakhidov, S. A., Islamov, A. K., Nuritdinov, I., & Massera, J., Ahmed, I., Petit, L., Aallos, V., & Hupa, L. (2014). Phosphate- Khaidarova, K. A. (1994). On the nature of yellow coloring in cerium- based glass fiber vs. bulk glass: Change in fiber optical response to containing silica glasses. Glass Physics and Chemistry, 20, 283–286. probe in vitro glass reactivity. Materials Science and Engineering: C, 37, Bohner, M., & Lemaitre, J. (2009). Can bioactivity be tested in vitro with 251–257 Retrieved from https://linkinghub.elsevier.com/retrieve/pii/ SBF solution? Biomaterials, 30, 2175–2179 Retrieved from http:// S0928493114000290 www.ncbi.nlm.nih.gov/pubmed/19176246 Massera, J., Claireaux, C., Lehtonen, T., Tuominen, J., Hupa, L., & Hupa, M. Brauer, D. S., Brückner, R., Tylkowski, M., & Hupa, L. (2016). Sodium-free (2011). Control of the thermal properties of slow bioresorbable glasses mixed alkali bioactive glasses. Biomedical Glasses, 2,99–110 Retrieved by boron addition. Journal of Non-Crystalline Solids, 357, 3623–3630 from https://www.degruyter.com/view/j/bglass.2016.2.issue-1/bglass- Retrieved from https://doi.org/10.1016/j.jnoncrysol.2011.06.037 2016-0012/bglass-2016-0012.xml Massera, J., Haldeman, A., Milanese, D., Gebavi, H., Ferraris, M., Foy, P., … Bunker, B. C., Arnold, G. W., & Wilder, J. A. (1984). Phosphate glass disso- Richardson, K. (2010). Processing and characterization of core-clad lution in aqueous solutions. Journal of Non-Crystalline Solids, 64, tellurite glass preforms and fibers fabricated by rotational casting. 291–316. Optical Materials, 32, 582–588 Retrieved from https://doi.org/10. Fagerlund, S., & Hupa, L. (2016). Chapter 1. Melt-derived bioactive silicate 1016/j.optmat.2009.12.003 glasses. In A. R. Boccaccini, D. S. Brauer, & L. Hupa (Eds.), Bioactive Massera, J., & Hupa, L. (2014). Influence of SrO substitution for CaO on Glasses: Fundamentals, Technology, and Applications. Cambridge: Royal the properties of bioactive glass S53P4. Journal of Materials Science. Society of Chemistry Retrieved from http://ebook.rsc.org/?DOI=10. Materials in Medicine, 25, 657–668 Retrieved from http://www.ncbi. 1039/9781782622017 nlm.nih.gov/pubmed/24338267 Fagerlund, S., Massera, J., Hupa, M., & Hupa, L. (2012). T-T-T behaviour of Massera, J., Hupa, L., & Hupa, M. (2012). Influence of the partial substitu- bioactive glasses 1-98 and 13-93. Journal of the European Ceramic Soci- tion of CaO with MgO on the thermal properties and in vitro reactivity ety, 32, 2731–2738. of the bioactive glass S53P4. J Non Cryst Solids. Elsevier B.V., 358, Gao, H., Tan, T., & Wang, D. (2004). Effect of composition on the release 2701–2707 Retrieved from http://www.sciencedirect.com/science/ kinetics of phosphate controlled release glasses in aqueous medium. article/pii/S0022309312003754 Journal of Controlled Release, 96,21–28. Massera, J., Kokkari, A., Närhi, T., & Hupa, L. (2015). The influence of SrO Harada,T.,In,H.,Takebe,H.,&Morinaga,K.(2004).EffectofB2O3 and CaO in silicate and phosphate bioactive glasses on human gingival addition on the thermal stability of barium phosphate glasses for fibroblasts. Journal of Materials Science. Materials in Medicine, 26, 196. optical fiber devices. Journal of the American Ceramic Society, 87, Massera, J., Mayran, M., Rocherullé, J., & Hupa, L. (2015). Crystallization 408–411. behavior of phosphate glasses and its impact on the glasses' bioactiv- Hench, L. L., Splinter, R. J., Allen, W. C., & Greenlee, T. K. (1971). Bonding ity. Journal of Materials Science, 50, 3091–3102 Retrieved from mechanisms at the interface of ceramic prosthetic materials. Journal of https://doi.org/10.1007/s10853-015-8869-4 Biomedical Materials Research, 5, 117–141. Massera, J., Petit, L., Cardinal, T., Videau, J. J., Hupa, M., & Hupa, L. (2013). Ilieva, D., Jivov, B., Bogachev, G., Petkov, C., Penkov, I., & Dimitriev, Y. Thermal properties and surface reactivity in simulated body fluid of (2001). Infrared and Raman spectra of Ga2O3-P2O5glasses. Journal of new strontium ion-containing phosphate glasses. Journal of Materials Non-Crystalline Solids, 283, 195–202. Science. Materials in Medicine, 24, 1407–1416. Karabulut, M., Yuce, B., Bozdogan, O., Ertap, H., & Mammadov, G. M. Massera, J., Shpotyuk, Y., Sabatier, F., Jouan, T., Boussard-Plédel, C., (2011). Effect of boron addition on the structure and properties of Roiland, C., … Hupa, L. (2015). Processing and characterization of iron phosphate glasses. Journal of Non-Crystalline Solids, 357, novel borophosphate glasses and fibers for medical applications. Jour- 1455–1462 Retrieved from https://doi.org/10.1016/j.jnoncrysol. nal of Non-Crystalline Solids, 425,52–60 Retrieved from https://doi. 2010.11.023 org/10.1016/j.jnoncrysol.2015.05.028 Kokubo, T., Kushitani, H., Sakka, S., Kitsugi, T., & Yamamuro, T. (1990). Massera, J., Vassallo-Breillot, M., Törngren, B., Glorieux, B., & Hupa, L. Solutions able to reproduce in vivo surface-structure changes in bioac- (2014). Effect of CeO2 doping on thermal, optical, structural and tive glass-ceramic A-W3. Journal of Biomedical Materials Research, 24, in vitro properties of a phosphate based bioactive glass. J Non Cryst 721–734. Solids. Elsevier B.V., 402,28–35 Retrieved from https://doi.org/10. Koudelka, L., & Mošner, P. (2001). Study of the structure and properties of 1016/j.jnoncrysol.2014.05.006 Pb-Zn borophosphate glasses. Journal of Non-Crystalline Solids, 293, Mishra, A., Petit, L., Pihl, M., Andersson, M., Salminen, T., Rocherullé, J., & 635–641. Massera, J. (2017). Thermal, structural and in vitro dissolution of 2114 MISHRA ET AL. antimicrobial copper-doped and slow resorbable iron-doped phos- soluble glasses. Journal of Materials Science. Materials in Medicine, 11, phate glasses. Journal of Materials Science, 52, 8957–8972. 615–620. Mishra, A., Rocherulle, J., & Massera, J. (2016). Ag-doped phosphate bioac- Sharmin, N., Hasan, M. S., Parsons, A. J., Furniss, D., Scotchford, C. A., tive glasses: Thermal, structural and in-vitro dissolution properties. Ahmed, I., & Rudd, C. D. (2013). Effect of boron addition on the ther- Biomedical Glasses, 2,38–48. mal, degradation, and cytocompatibility properties of phosphate-based Moustafa, Y. M., & El-Egili, K. (1998). Infrared spectra of sodium phos- glasses. BioMed Research International, 2013,1–12. phate glasses. Journal of Non-Crystalline Solids, 240, 144–153. Shih, P. Y., & Shiu, H. M. (2007). Properties and structural investigations of Mulligan, A. M., Wilson, M., & Knowles, J. C. (2003a). Effect of increasing UV-transmitting vitreous strontium zinc metaphosphate. Materials silver content in phosphate-based glasses on biofilms of streptococcus Chemistry and Physics, 106, 222–226. sanguis. Journal of Biomedical Materials Research Part A, 67, 401–412. Tirkkonen, L., Haimi, S., Huttunen, S., Wolff, J., Pirhonen, E., Mulligan, A. M., Wilson, M., & Knowles, J. C. (2003b). Effect of increasing Sándor, G. K., & Miettinen, S. (2012). Osteogenic medium is superior copper content in phosphate-based glasses on biofilms of streptococ- to growth factors in differentiation of human adipose stem cells cus sanguis. Biomaterials, 24, 1797–1807. towards boneforming cells in 3D culture. European Cells & Materials, NIST, NIST chemistry WebBook, SRD 69. 2019. Retrieved from https:// 25, 144–158. webbook.nist.gov/cgi/cbook.cgi?ID=B6009796&Mask=80#IR-Spec Wu, C., Zhou, Y., Xu, M., Han, P., Chen, L., Chang, J., & Xiao, Y. (2013). Ojansivu, M., Mishra, A., Vanhatupa, S., Juntunen, M., Larionova, A., Copper-containing mesoporous bioactive glass scaffolds with Massera, J., & Miettinen, S. (2018). The effect of S53P4-based borosilicate multifunctional properties of angiogenesis capacity, osteostimulation glasses and glass dissolution products on the osteogenic commitment of and antibacterial activity. Biomaterials, 34, 422–433. human adipose stem cells. Boccaccini a editor. PLoS One, 13, e0202740 Zuk, P. A., Zhu, M., Mizuno, H., Huang, J., Futrell, J. W., Katz, A. J., … Retrieved from https://dx.plos.org/10.1371/journal.pone.0202740 Hedrick, M. H. (2001). Multilineage cells from human adipose tissue: Ojansivu, M., Vanhatupa, S., Björkvik, L., Häkkänen, H., Kellomäki, M., Implications for cell-based therapies. Tissue Engineering, 7, 211–228. Autio, R., … Miettinen, S. (2015). Bioactive glass ions as strong enhancers of osteogenic differentiation in human adipose stem cells. SUPPORTING INFORMATION Acta Biomaterialia, 21, 190–203 Retrieved from https://doi.org/10. 1016/j.actbio.2015.04.017 Additional supporting information may be found online in the Queiroz, A. C., Santos, J. D., Monteiro, F. J., & Prado da Silva, M. H. (2003). Supporting Information section at the end of this article. Dissolution studies of hydroxyapatite and glass-reinforced hydroxyap- atite ceramics. Materials Characterization, 50, 197–202 Retrieved from https://linkinghub.elsevier.com/retrieve/pii/S1044580303000925 Rohanová, D., Boccaccini, A. R., Horkavcová, D., Bozděchová, P., How to cite this article: Mishra A, Ojansivu M, Autio R, Bezdicka, P., & Častorálová, M. (2014). Is non-buffered DMEM solu- Vanhatupa S, Miettinen S, Massera J. In-vitro dissolution tion a suitable medium for in vitro bioactivity tests? Journal of Mate- characteristics and human adipose stem cell response to novel rials Chemistry B, 2, 5068–5076. borophosphate glasses. JBiomedMaterRes. 2019;107A: Salih, V., Franks, K., James, M., Hastings, G. W., Knowles, J. C., & Olsen, I. – (2000). Development of soluble glasses for biomedical use part II: The 2099 2114. https://doi.org/10.1002/jbm.a.36722 biological response of human osteoblast cell lines to phosphate-based PUBLICATION IV Core-clad phosphate glass fibers for biosensing A. Mishra, F. Désévédavy, L. Petit, F. Smektala, J. Massera Journal of Material Science and Engineering C 96 (2019) 458–465 https://doi.org/10.1016/j.msec.2018.11.038 Publication reprinted with the permission of the copyright holders. 0DWHULDOV6FLHQFH (QJLQHHULQJ& ² Contents lists available at ScienceDirect Materials Science & Engineering C journal homepage: www.elsevier.com/locate/msec ☆ Core-clad phosphate glass fibers for biosensing ⁎ A. Mishraa, F. Désévédavyb, L. Petitc, F. Smektalab, J. Masseraa, a Faculty of Medicine and Health Technology, Tampere University, Korkeakoulunkatu 10, FI-33720 Tampere, Finland b Laboratoire Interdisciplinaire Carnot de Bourgogne, UMR 6303 CNRS-Université de Bourgogne Franche-Comté, 9 Av. A. Savary, 21078 Dijon, France c Laboratory of Photonics, Tampere University, Korkeakoulunkatu 10, FI-33720 Tampere, Finland ABSTRACT Recently, a phosphate glass with composition 20 CaO-20 SrO-10 Na2O-50 P2O5 (mol%) was found to have good potential as a biomaterial and to possess thermal properties suitable for fiber drawing. This study opened the path towards the development of fully bioresorbable fibers promising for biosensing. In the past, this phosphate glass with CeO2 was found to increase the refractive index and the glass stability. Therefore, a new SrO-containing glass was prepared with 1 mol% of CeO2 and core fibers were drawn from it. A core-clad fiber was also processed, where the core was a Ce-doped glass and the clad undoped, to allow for total internal reflection. The mechanical properties of the core and core-clad fibers are discussed as a function of immersion time in TRIS-buffer solution. Finally, a sensing region was created, in the core-clad fiber, by etching the cladding using phosphoric acid. Then, the change in light transmission, upon immersion in TRIS-buffer solution, was quantified to assess the potential use of the novel core-clad fiber as a biosensor. Upon immersion in TRIS, the core-clad fiber was found to guide light effectively and to maintain a tensile strength of ~150–200 MPa up to 6 weeks in TRIS, clearly showing that this fiber has potential as a biosensing device. 1. Introduction infrared (UV–Vis/NIR) range, and their refractive index is comparable to the commercially available silicate glass fibers [17–19]. Phosphate glasses (PGs) have emerged as good alternatives to the Glass with composition 50P2O5-20SrO-20CaO-10Na2O (mol%), re- traditional silicate glasses for biomedical applications. Silicate glasses ferred to as Sr50 has favorable degradation characteristics for potential have long been the popular choice as bioactive glasses among re- use in biomedical applications as reported in [20]. When Sr50 is doped searchers, since the discovery of 45S5 by Hench et al. [1]. However, the with CeO2 (from 1 to 7 mol%), the glass network becomes more cross- processability of silicate glasses into scaffold, fibers etc. is hindered due linked and therefore more resistant to dissolution delaying the pre- to their crystallization kinetics [2,3]. Meanwhile, phosphate glasses cipitation of a reactive surface layer [7]. The CeO2 doping was done have emerged as promising candidates as bioactive materials due to such that the (Ca + Sr)/P remained the same than in Sr50, thereby not their unique properties: they are completely biodegradable, and their affecting its ability to form a Ca-Sr-P layer at the surface. The resistance composition can be adjusted to attain desirable degradation rates (from to crystallization, of these CeO2 doped glasses, increased compared to weeks to years) [4]. Moreover, phosphate glasses possess excellent the Sr50 [7]. Furthermore, the addition of CeO2 leads to an increase in thermal properties enabling fiber drawing without adverse crystal- the refractive index, allowing the drawing of core-clad fiber from Sr50 lization [4–7]. A detailed study of the potential use and benefits of PGs, (Clad) and a Ce-doped-Sr50 composition (core) having suitable Δn in the medical field, can be found in [8]. (where Δn = ncore −nclad is positive) for total internal reflection. In this Glass fibers have been researched extensively both in the medical study, core and core-clad preforms were drawn into fibers to determine and optical research fields. Degradable bioactive glass fibers can be their suitability for biosensing. Core fibers were drawn from the Sr50 used in composites to improve the mechanical properties of scaffolds glass and Ce-1 glass with the [0.99 (50P2O5-20SrO-20CaO- and of other biomedical devices [9]. Similar to the bulk glasses, phos- 10Na2O)–1CeO2 (mol%)] composition, with a diameter of (140 ± 10) phate glass fibers have been found to be particularly suitable not only μm and (110 ± 10) μm respectively. For the core-clad fiber, Ce-1 glass for bone repair and reconstruction but also in soft tissue engineering was chosen as the core, and Sr50 as the cladding, and fibers were drawn applications [10–14]. From an optics perspective, the potential of glass with a diameter of (140 ± 10) μm from preform produced using a fibers as biosensors has been investigated in detail in [15]. Their po- homemade rotational caster. tential in tracking glass dissolution is discussed in [16]. Additionally, In this paper, we investigated the effect of in-vitro dissolution of the phosphate fibers exhibit high transparency in the UV–Visible/Near fibers on their mechanical and optical properties. In the core-clad ☆ This work will lay the base on using optical properties to probe protein and/or cell attachment at the surface of a fiber. ⁎ Corresponding author. E-mail address: jonathan.massera@tut.fi (J. Massera). https://doi.org/10.1016/j.msec.2018.11.038 Received 16 January 2018; Received in revised form 14 November 2018; Accepted 24 November 2018 $YDLODEOHRQOLQH1RYHPEHU (OVHYLHU%9$OOULJKWVUHVHUYHG A. Mishra et al. 0DWHULDOV6FLHQFH (QJLQHHULQJ& ² fibers, a sensing region was produced by selectively etching the clad- ding, revealing the core of the fiber. Then, light loss of the etched core- clad fibers was measured as a function of immersion time in TRIS. The mechanical properties of the etched core-clad fibers, were also ascer- tained. Additionally, the impact of immersion in TRIS buffer on the mechanical properties of the fibers was obtained. 2. Materials and methods 2.1. Preform preparation and fiber drawing 2.1.1. Core preforms The core preforms with the composition [50P2O5-20SrO-20CaO- 10Na2O] (labeled as Sr50) and [0.99 (50P2O5-20SrO-20CaO- 10Na2O)–1CeO2 (mol%)] (labeled as Ce-1) were prepared by melting batches of 45 g comprising of Ca(PO3)2, Sr(PO3)2,NaPO3 (and CeO2 for Ce-1 composition) at 1100 °C (heating rate 10 °C/min) for 30 min. Ca (PO3)2 and Sr(PO3)2 were prepared beforehand by heating NH4H2PO4 with CaCO3 and SrCO3 respectively, in separate batches. The batch was heated to 250 °C for 12 h, then to 650 °C for 12 h and finally to 850 °C −1 for 12 h at 1 °C·min to remove CO2,NH3 and H2O. After melting, the molten batch was cast into a 10 cm long pre-heated (350 °C) brass mold having a diameter of 12 mm, and annealed at 15 °C below their re- spective glass transition temperature for 15 h, to obtain a mechanically stable preform. Annealing was performed below Tg to decrease the risk of nuclei formation. 2.1.2. Core-clad preform The core-clad preform, with core Ce-1 and clad Sr50, was prepared using a homemade rotational caster. The clad composition was first poured in a pre-heated brass mold (350 °C), which is then spun at 1000 rpm for 10 s. The spinning results in the formation of a hollow cylinder in the mold. Then the core composition is poured slowly inside the hollow cladding. The preform was then annealed similarly as for the core preforms. The process was optimized as in [21] to guarantee a constant Øclad/Øcore, before and after drawing. The small CeO2 addition to the glass composition did not lead to a significant change in thermal Fig. 1. thermal profiles of the drawing tower furnace. The zero mm position expansion coefficient and all the processed core-clad preforms were free corresponds to the top of the heating element. of cracks or any defects. 2.2. Optical properties 2.1.3. Fiber drawing Fibers were drawn using the “rod” method in a specially designed A fully automated Metricon, model 2010 prism coupled re- single zone drawing tower furnace. To prevent nucleation or crystal- fractometer was used to measure the refractive index of 2 mm thick lization, the temperature profiles of this furnace were precisely mapped glass samples of bulk core and cladding glass samples at 1060 nm. The beforehand, and the dwell time in the zones was controlled prior to and accuracy of the measurement was ± 0.0001. The samples were polished during the drawing. Thermal profiles for different setting temperatures with SiC polishing paper (600, 800, 1200, 2400 and 4000 grit) on both were performed by means of a thermocouple attached to the preform faces. motion cane. After waiting for the furnace to stabilize to the set tem- perature, the thermocouple was gradually moved down into the fur- 2.3. Immersion tests nace. In the meantime the thermocouple temperature and position were registered, as presented in Fig. 1. The drawing temperature was 645 °C Immersion tests were carried out by immersing the fibers in TRIS for all the fibers under an inert He gas laminar flow of 2.5 l·min−1. This buffer solution for up to 6 wks. For each immersion time point, 10 fibers temperature corresponded to a set temperature of 770 °C (Fig. 1). The of length 15 cm were entirely immersed in 50 ml of TRIS, and were high thermal conductivity KHe of helium allowed to minimize the dwell placed in an incubating shaker HT Infors Multitron at 37 °C, 100 rpm to time before drop formation and during drawing, KHe = −5 −1 −1 −1 −5 −1 −1 obtain laminar flow mixing without moving the fibers. 33,63.10 cal·sec ·cm .°C,KAr = 4,06.10 cal·sec ·cm . −1 −5 −1 −1 −1 °C ,Kair = 5.68.10 cal·sec ·cm .°C [22]. The drawing tem- perature was determined based on the glass transition temperature (Tg) 2.4. Mechanical testing and the furnace thermal profile. The glass rod was placed into the furnace and the temperature gradually increased above Tg until the Mechanical tests were performed, in tension (static), on the core and formation of a drop. During the drawing, the temperature was adjusted core-clad fiber prior to and after immersion in TRIS. Post immersion, based on readings from the tension measurement gauge. The fiber was the core and core-clad fibers were carefully collected, rinsed with then fixed on a rotary drum, while the fiber diameter and drawing acetone and dried in air, overnight, in an incubator at 37 °C. The spe- tension were controlled by a computer system based on LabView soft- cimens were then maintained in desiccator before testing. Mechanical ware. strength measurements were carried out at ambient temperature using A. Mishra et al. 0DWHULDOV6FLHQFH (QJLQHHULQJ& ² Instron 4411 Materials Testing Machine, with a grip distance of 50 mm, a load cell of 500 N, and a crosshead speed of 30 mm·min−1. The grips were covered with a rubber mat to avoid slipping of the fiber during the test and damage caused by the metallic grips. The tensile strength and the Young's modulus were calculated as the mean of 10 test fibers. Despite the low number of specimens, the Weibull modulus was esti- mated as reported in [23–24]. 2.5. Light loss The fibers' optical losses were measured using the cut back tech- nique. The signal from the FTIR spectrometer transmitted through a 75 μm long piece of fiber (L1) was recorded (I1), using a nitrogen cooled InSb detector. Then, without moving the input end, the fiber was cut (cleaved) and the output fiber signal was recorded (I2). From the measurement of the cut length corresponding to L1-L2, the signals I1 and I2, the losses were calculated by using the following equation: 10 I Losses(dB/m) = × log2 . LL12 I1 The same measurement was repeated several times to increase ac- curacy. The light transmission, as a function of immersion time, was mea- Fig. 2. Optical microscope image of the core-clad fiber cross section. sured in the wavelength range of 200–1000 nm. A 50 cm long fiber, was connected on one end to a light source and at the other end to a confirmed by the optical image of the core-clad fiber cross-section, spectrometer. The fibers were cleaved and coupled using FC connector presented in Fig. 2. The step index difference between the core and the (Thorlabs). The lamp position was adjusted to maximize the light in- clad can be seen on the micrograph by the bright coloration of the core tensity at the fiber output. A 5 cm portion (sensing region) of the fiber compared to that of the cladding [25]. The clad diameter was 140 μm as was immersed in TRIS. For the illumination, a broadband white light targeted, with a core diameter of 75 μm. source (Edmund BDS130) was used, and a ThorLabs Compact The attenuation spectra of the core fibers are shown in Fig. 3.The Spectrometer CCS200 was used for collecting the light. The light ac- optical losses of the Sr50 and Ce-1 core fibers were around 20 and cumulation was set to 7.5 ms to maximize the signal. The light intensity 10 dB/m, respectively. The lower optical losses in the Ce-1 fiber com- recorded at 680 nm was tracked as a function of immersion time. The pared to that of Sr50 fiber was attributed to an increase in the network signal was normalized to 1 prior to immersion and only the relative cross-linking due to the doping with CeO . Indeed, most of the phos- intensity is reported. The TRIS solution was refreshed each week in 2 phate glasses used as laser are within the metaphosphate structure and order to avoid significant evaporation of the solution. Evaporation contain large amount of Al O .TheaimofAl3+ ions in the glass is to would increase the surface area to volume of the solution ratio, leading 2 3 increase the connectivity between the phosphate chains that in turn to a change in the dissolution rate. reduce the light loss [26]. The core-clad fiber was found to have similar light losses than the Ce-1 core fiber at 1.55 μm. 2.6. Microscopy The core fibers (Sr50 and Ce-1) and the core-clad fiber were im- mersed in TRIS buffer solution for up to 42 days. The change in the The core-clad fibers were collected post mechanical tests for ima- fibers' mechanical properties (namely tensile strength, Young's modulus ging. They were imaged along their length using optical microscope and Weibull modulus) as a function of days in TRIS are reported in Olympus BH2-UMA to evidence the deposition of a surface layer after Fig. 4. Prior to immersion (t = 0), the Sr50 and Ce-1 core fibers exhibit immersion in TRIS. The optical microscope was also used to measure similar tensile strength at ~550 MPa indicating that the preform the initial and post-immersion fiber diameter. The fibers cross-section, post fracture was also visualized using Zeiss ULTRAplus scanning 40 electron microscope (SEM). FTIR Sr50 Diode 1.55 µm FTIR Ce-1 3. Results and discussion Diode 1.55 µm 30 To guide the light successfully, a core-clad fiber should be drawn using glasses with specific refractive index (n) to allow total internal reflection of the light: the ncore should be greater than nclad. Here, the 20 glasses under investigation were [50P2O5-20SrO-20CaO-10Na2O] (la- beled as Sr50) and [0.99 (50P2O5-20SrO-20CaO-10Na2O) – 1CeO2 (mol %)] (labeled as Ce-1) with a refractive index at 1060 nm of 1.5261 and (dB/m) Attenuation 10 1.5345, respectively. Core fibers were drawn from Sr50 and Ce-1 glasses with a diameter of (~140 ± 10) and (~110 ± 10) μm, re- spectively. A core-clad fiber was drawn using Sr50 glass as the clad and the Ce- 0 1 as the core, with a total diameter of (~140 ± 10) μm. This fiber had 1,0 1,1 1,2 1,3 1,4 1,5 1,6 a numerical aperture NA of 0.16, which is similar to the NA of some of λ (µm) our earlier studied fibers [21]. The preparation of fiber with such large NA guarantees the proper light guiding through the core of the fiber, as Fig. 3. Attenuation of the single core fibers, Sr50 and Ce-1. A. Mishra et al. 0DWHULDOV6FLHQFH (QJLQHHULQJ& ² a) b) 1600 140 Single core Sr50 Single core Sr50 1400 Single core Ce-1 120 Single core Ce-1 Core Clad Core Clad 1200 100 1000 80 800 60 600 40 400 Tensile strength (MPa) Young's modulus (GPa) 20 200 0 0 0 7 14 21 28 35 42 0 7 14 21 28 35 42 Immersion time (days) Immersion time (days) c) 14 Single core Sr50 Single core Ce-1 12 Core Clad 10 8 6 Weibull modulus Weibull 4 2 0 0 7 14 21 28 35 42 Immersion time (days) Fig. 4. a) Tensile strength, b) Young's modulus and c) Weibull modulus of single core and core-clad fibers immersed for up to 42 days in TRIS buffer solution. processing and fiber drawing of these two glass compositions lead to mirror and the hackles, as evidenced in [32]. Fig. 5b does not show the similar density of surface defects (Fig. 4a). However, the core-clad fi- hackles as clearly as in Fig. 5a but can clearly be seen in Fig. 5d. In all bers exhibit lower tensile strength compared to the core fibers. This can cases, even for the core-clad fibers, the surface fracture origin was lo- be attributed to the manufacturing process; for example, coefficient of cated at the cladding surface. No core-clad fibers were found to break at thermal expansion mismatch between the core and the clad could cause the core-clad interface. Therefore, the low mechanical properties of the a drop in the mechanical properties [27–29]. This is in agreement with core-clad fiber seen in Fig. 4, did not come from defects at the core-clad [30]: Ahmed et al. reported on the processing of core-clad fibers ob- interface. tained via extrusion. In their study, a glass with composition 50P2O5- As seen in Fig. 4a, upon immersion for 2 days, an increase in tensile 16CaO-5Na2O-24MgO-5Fe2O3 (P50Fe5) was extruded together with a strength of all the fibers was observed. Such increase, upon immersion, glass with composition 45P2O5-16CaO-10Na2O-24MgO-5TiO2 (P45Ti5) was also evidenced in [33] and was due to the etching of the fiber to obtain fiber with 39–45 μm diameter. These core-clad fibers ex- surface, which reduced the amount of surface defects. At longer im- hibited an average tensile strength of 302 ± 73 MPa and mersion time, a decrease in tensile strength of the Sr50 fiber was 236 ± 53 MPa when the glass P50Fe5 was used as clad (P45Ti5 as measured due to the corrosion of the fiber [34]. However, the tensile core) and core (P45Ti5 as clad) respectively. Sharmin et al. reported a strength of the Ce-1 fiber started to decrease only after 21 days in TRIS. tensile strength of ~526 ± 110 MPa for a fiber drawn from the P45Fe5 The delay in the decrease of the Ce-1 fiber's mechanical properties may composition with diameter of ~20 μm [31]. This clearly indicated that be attributed to the presence of Ce in the Sr50 glass network, which was the processing of core-clad fibers might negatively influence the me- found to drastically reduce the glass reactivity in aqueous solution chanical properties of the fiber. The origin of the fibers' fracture was (both dissolution and precipitation of a reactive layer) as explained in investigated using optical microscopy. The core and core-clad fibers [7]. The core-clad fiber exhibited similar changes in the mechanical were inspected and two examples of the cross-section of fibers are given properties than the Sr50 core fiber. However, the increase in tensile in Fig. 5: (a) presents the optical microscope image of the core fiber (Ce- strength of the core-clad fiber during the first two days of immersion 1) and (b) the core-clad fiber cross-section while (c) presents the SEM was lower compared to the Sr50 core fiber. This might be related to a micrographs of the core fiber and (d) the core-clad fibers cross section high density of large defects at the core-clad fiber surface, which cannot post-fracture. From Fig. 5, one can see the surface fracture origin, the be efficiently etched. It should be pointed out that all the fibers A. Mishra et al. 0DWHULDOV6FLHQFH (QJLQHHULQJ& ² a) b) Fracture origin c) d) Fracture origin Fig. 5. Optical microscope and SEM micrograph image of the single core (Ce-1) a), c) and core-clad fiber b), d) cross section post tensile-test. exhibited a similar tensile strength of about 150 MPa after 42 days in Immersion of fibers is also known to affect the probability of early TRIS. fracture and therefore the scatter in brittle fracture strength, as defined Fig. 4b presents the Young's modulus of the investigated fibers as a by the Weibull modulus [39]. Weibull modulus of the fibers as a function of immersion time in TRIS. Initially (at t = 0), the core-clad function of immersion time is presented in Fig. 4c. A high modulus and the Sr50 core fibers exhibit similar Young's modulus. The Ce-1 core indicates low scatter in brittle fracture strength and, thus, a higher fiber exhibits a larger Young's modulus probably due to the increase in strength reproducibility. Both the core-clad and the Ce-1 fibers ex- network connectivity when adding CeO2 in the glass network [7]. While hibited an initial rise in the Weibull modulus which then drops at the fibers in [33,35] were reported to maintain their Young's modulus, longer immersion times in TRIS. The initial increase in the Weibull upon immersion in buffer solution, the investigated fibers exhibited a modulus was expected, as the etching of the fiber is expected to remove Young's modulus which increased initially and then decreased upon the largest surface flaws decreasing then the probability of a fiber to immersion in TRIS. Surprisingly, the Young's modulus of the fibers in- undergo premature breakage. This, in turn, reduces the scatter in brittle creased during the first few days in TRIS. The Young's modulus for Sr50 fracture strength. The decrease in the Weibull modulus of fibers im- and the core-clad fibers exhibited a maximum after 7 days in TRIS while mersed for longer immersion time indicated that the surface flaws that of the Ce-1 fiber exhibited a maximum during 21 days in TRIS due started to increase, leading to fibers with early fracture (lower tensile to its slower reactivity. A similar increase in the tensile modulus post- stress) and higher scatter in brittle fracture. annealing of the fibers was reported in [33] and was assigned to In the past, it was demonstrated that the changes in the optical structural changes. It is also accepted that the mechanical properties of properties of a core bioresorbable bioactive glass fibers, upon immer- phosphate glasses are a function of compositional parameters (dis- sion in SBF, could give information regarding the state of degradation sociation energy and packing density) and the total bonding energy [16]. To reveal a sensing region, the clad of the core-clad fiber was [36–37]. Despite the dissolution of these glasses being considered as etched away. Etching was performed using 1 M phosphoric acid. Fig. 6 congruent, the surface of the fiber is expected to undergo alkaline and exhibits the reduction in diameter of the core-clad fiber as a function of alkaline earth depletion as reported in [38]. The change in the fiber etching time. The fiber diameter decreased almost linearly at a rate of surface composition can lead to an initial increase in the Young's 4.4 μm·h−1. Therefore, the clad was suspected to be completely etched modulus. The drop in the modulus was then related to pitting and local away after 14.5 h. Fig. 7 presents the optical images of the cross-section defect formation upon selective dissolution of areas with higher re- of the partially etched fiber after 13 h of immersion in 1 M H3PO4, activity. showing near complete removal of the clad. A. Mishra et al. 0DWHULDOV6FLHQFH (QJLQHHULQJ& ² 200 a) 180 350 160 III y = 137 - 4.4 * x 140 300 R2= 0.97 m) μ 120 250 100 I 80 Diameter ( Diameter 200 60 II 40 150 20 100 0 Tensile Strength (MPa) Tensile Strength 02468101214 Etching time (h) 50 Fig. 6. Change in diameter of the core-clad fiber as a function of immersion 0 time in 1 M H3PO4. 012345678910 Specimen b) 75 μm Fig. 8. Fig. 7. Cross section of the core-clad fiber after 13 h of etching. a) Tensile strength of etched fibers on the basis of the region where fracture occurs, b) the different regions of fracture in the etched fiber. Etching of the fiber was suspected to have an effect on the fiber's mechanical properties as reported in [40]. Fig. 8a, depicts the tensile 1.0 strength of 10 etched core-clad fibers (etching time was 14.5 h to fully remove the cladding). The fibers were not all fractured in the etched region. Fig. 8b presents a schematic of the location of the fracture 0.8 origin. Out of the 10 fibers, four broke at the interface between the etched and un-etched region (Region I), four broke in the etched region 0.6 (Region II) and two broke in the un-etched part (Region III). The average tensile strength was ~225 MPa, ~150 MPa and ~350 MPa, in Region I, II and III, respectively (Fig. 8a). This clearly shows that the 0.4 Relative intensity etching of the fibers led to a decrease in the fiber mechanical properties due to an increase in flaw density, formation of surface pits, a decrease 0.2 in the fiber diameter and/or an increase in the size of surface flaws. Indeed, mild etching, obtained using dissolution of the fiber in TRIS initially increased the mechanical properties (such as tensile or bending 0.0 0 7 14 21 28 35 42 strength), while at longer dissolution time the mechanical properties Time (days) decreased most likely due to an increase in the fiber roughness and/or due to the precipitation of a reactive calcium phosphate layer at the Fig. 9. Relative intensity of the output light at 680 nm through the core-clad fibers' surface [40]. It is also interesting to point out that the low tensile fiber as a function of immersion time in TRIS. strength of the etched fibers are similar to that of fibers immersed for A. Mishra et al. 0DWHULDOV6FLHQFH (QJLQHHULQJ& ² a) b) c) d) Fig. 10. Microscope images of the etched part of the core-clad fibers immersed for a) 7 days, b) 21 days and c) 42 days in TRIS and SEM image of the fiber surface at 42 days of immersion d). At 21 days surface layer starts to form (red circle). At 42 days thicker and more homogeneous reactive layer. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) extended period of time in TRIS. This supported our hypothesis that output light at 680 nm of the core-clad fiber can be observed upon upon significant surface etching, the increase in surface roughness and immersion in TRIS. Fig. 10 presents the optical micrographs of the surface flaws dictate the fiber's failure. etched fiber upon immersion for a) 7 days, b) 21 days and c) 42 days as The sensing region (5 cm) was immersed in TRIS buffer solution for well as the SEM image of the fiber surface upon immersion for 42 days. up to 42 days. Fig. 9 shows the light loss through the etched portion of a The rapid decrease in light intensity, during the first 14 days, was at- core-clad fiber. A progressive reduction in the relative intensity of the tributed to the fast initial dissolution of the glass in aqueous solutions A. Mishra et al. 0DWHULDOV6FLHQFH (QJLQHHULQJ& ² and a decrease in the fiber diameter. At longer immersion time, it is functional materials: a perspective on phosphate-based glasses, J. Mater. Chem. 19 clear from Fig. 10(b, c and d) that a thin CaP reactive layer precipitated (2009) 690–701. [9] R. Colquhoun, K.E. Tanner, Mechanical behaviour of degradable phosphate glass at the surface of the fiber, the thickness of which grew overtime leading fibres and composites-a review, Biomed. Mater. 11 (2015) 014105. to further increase in the light losses as discussed in [16]. When com- [10] J. Clement, J.M. Manero, J.A. Planell, Analysis of the structural changes of a pared to our former study in [7], the layer precipitated at a slower rate phosphate glass during its dissolution in simulated body fluid, J. Mater. Sci. Mater. Med. 10 (1999) 729–732. when immersed in TRIS than in SBF, as SBF solution is known to be [11] J.C. Knowles, Phosphate based glasses for biomedical applications, J. Mater. Chem. thermodynamically unstable and to lead to rapid precipitation of a 13 (2003) 2395–2401. reactive layer [41]. It is interesting to point out that, from SEM analysis [12] M.A. De Diego, N.J. Coleman, L.L. Hench, Tensile properties of bioactive fibers for the reactive layer was fine and not well attached to the glass surface as tissue engineering applications, J. Biomed. Mater. Res. A 53 (2000) 199–203. [13] J. Choueka, J.L. Charvert, H. Alexander, Y.H. Oh, G. Joseph, N.C. Blumenthal, already shown in [20] confirming the role of the reactive layer for- W.C. LaCourse, Effect of annealing temperature on the degradation of reinforcing mation on the decreased mechanical properties reported here. fibers for absorbable implants, J. Biomed. Mater. Res. 29 (1995) 1309–1315. [14] I. Ahmed, M. Lewis, I. Olsen, J.C. Knowles, Phosphate glasses for tissue engineering: part 1. Processing and characterisation of a ternary-based P2O5-CaO-Na2O glass 4. Conclusions system, Biomaterials 25 (2004) 491–499. [15] B. Gholamzadeh, H. Nobovati, Fiber Optic Sensors, 18 World of Academy of Core (Sr50 and Ce-1) and core (Ce-1)-clad (Sr50) phosphate fibers Science, Engineering Technology, 2008, pp. 297–307. [16] J. Massera, I. Ahmed, L. Petit, V. Aallos, L. Hupa, Phosphate-based glass fiber vs. were successfully drawn into fibers. The tensile strength of the core-clad bulk glass: change in fiber optical response to probe in vitro glass reactivity, Mater. fibers was lower compared to that of the core and was assigned to an Sci. Eng. C 37 (2014) 251–257. increase in the density of the core-clad fiber surface defects surface of [17] V.P. Gapontsev, S.M. Matitsin, A.A. Isineev, V.B. Kravchenko, Erbium glass lasers and their applications, Opt. Laser Technol. 14 (4) (1982) 189–196. the fiber. Upon immersion in TRIS for 2 days, all the fibers exhibited an [18] S. Jiang, M.J. Myers, N. Peyghambarian, Er3+ doped phosphate glasses and lasers, increase in mechanical properties (tensile strength, Young's modulus J. Non-Cryst. Solids 239 (1998) 143–148. and Weibull modulus) due to the etching of the fibers' surface and a [19] D. Pugliese, N.G. Boetti, J. Lousteau, E. Ceci-Ginistrelli, E. Bertone, F. Geobaldo, D. Milanese, Concentration quenching in an Er-doped phosphate glass for compact subsequent decrease in the density of surface defects. At longer im- optical lasers and amplifiers, J. Alloys Compd. 657 (2016) 678–683. mersion, the mechanical properties of the fibers dropped due to the [20] J. Massera, A. Kokkari, T. Narhi, L. Hupa, The influence of SrO and CaO in silicate dissolution/reaction of the glass with the aqueous media. The drop in and phosphate bioactive glasses on human gingival fibroblasts, J. Mater. Sci. Mater. mechanical properties of the fibers was delayed by the presence of Med. 26 (2015) 196. [21] J. Massera, A. Haldeman, D. Milanese, H. Gebavi, M. Ferraris, P. Foy, W. Hawkins, CeO2. The change in the mechanical properties of the core-clad fibers J. Ballato, R. Stolen, L. Petit, K. Richardson, Processing and characterization of followed a similar trend than the Sr50 core fiber. With this study, we core–clad tellurite glass preforms and fibers fabricated by rotational casting, Opt. show that the change in mechanical properties upon immersion in TRIS Mater. 32 (2010) 582–588. [22] M. Curie, A. Lepape, Conductibilité thermique des gaz rares, J. Phys. Radium 2 is dictated by the cladding surface reaction with the medium rather (1931) 392–397. than the fiber geometry. This core-clad fiber exhibited good light [23] E. Pirhonen, L. Moimas, M. Brink, Mechanical properties of bioactive glass 9–93 transmission and the change in light transmission was assessed upon fibres, Acta Biomater. 2 (2006) 103–107. [24] E. Pirhonen, H. Niiranen, T. Niemelä, M. Brink, P. Törmälä, Manufacturing, me- immersion in TRIS. The clad of the core-clad fiber was etched in chanical characterization, and in vitro performance of bioactive glass 13–93 fibers, phosphoric acid to reveal the core. Such process led to a decrease of the J Biomed Mater Res B Appl Biomater 77B (2006) 227–233. fiber mechanical properties. Upon immersion, for 10 days in TRIS, a [25] N. Syam Prasad, J. Wang, R.K. Pattnaik, H. Jain, J. Toulouse, Preform fabrication and drawing of KNbO3 modified tellurite glass fibers, J. Non-Cryst. Solids 352 drop of 50% of the initial light intensity was recorded. (2006) 519–523. Owing to the ability to guide light effectively and to maintain de- [26] J.H. Campbell, T.I. Suratwala, Nd-doped phosphate glasses for high-energy/high- cent tensile strength in vitro (~ 150–200 MPa after 6 wks in TRIS), this peak-power lasers, J. Non-Cryst. Solids 263–264 (2000) 318–341. [27] G.W. Sherer, A.R. Cooper, Thermal stresses in clad-glass fibers, J. Am. Ceram. Soc. fiber looks promising for future use as a biosensing device in vivo. 63 (1980) 346–347. [28] D.A. Krohn, A.R. Cooper, Strengthening of glass fibers: I, cladding, J. Am. Ceram. Acknowledgement Soc. 52 (1969) 661–664. [29] U.C. Paek, C.R. Kurkijan, Calculation of cooling rate and induced stresses in drawing of optical fibers, J. Am. Ceram. Soc. 58 (1975) 330–335. The authors would like to acknowledge the financial support of the [30] I. Ahmed, S.S. Shaharuddin, N. Sharmin, D. Furniss, C. Rudd, Core/clad phosphate Academy of Finland through the Academy Research Fellow grant glass fibres containing iron and/or titanium, Biomed. Glasses 1 (2015) 20–30. (275427), the Initial and follow-on funding for research costs (284492 [31] N. Sharmin, C.D. Rudd, A.J. Parsons, I. Ahmed, Structure, viscosity and fibre drawing properties of phosphate-based glasses: effect of boron and iron oxide ad- and 312634) and Competitive Funding to Strengthen University dition, J. Mater. Sci. 51 (2016) 7523–7535. Research Profiles (310359). [32] P.K. Gupta, Fractography of fiberglass, in: R.C. Bradt, R.E. Tressler (Eds.), Fractography of Glass, Springer, Boston MA, 1994, pp. 185–206. [33] S. Cozien-Cazuc, A. Parsons, G. Walker, I. Jones, C. Rudd, Effects of aqueous aging References on the mechanical properties of P40Na20Ca16Mg24 phosphate glass fibres, J. Mater. Sci. 43 (14) (2008) 4834–4839. [1] L.L. Hench, R.J. Splinter, W.C. Allen, T.K. Greenlee, Bonding mechanisms at the [34] R. Colquhoun, K.E. Tanner, Mechanical behaviour of degradable phosphate glass interface of ceramic prosthetic materials, J. Biomed. Mater. Res. 2 (1) (1971) fibres and composites—a review, Biomed. Mater. 11 (2016) 014105. 117–141. [35] P. Haque, I. Ahmed, A. Parsons, R. Felfel, G. Walker, C. Rudd, Degradation prop- [2] S. Fagerlund, J. Massera, L. Hupa, M. Hupa, T-T-T behaviour of bioactive glasses erties and microstructural analysis of 40P2O5–24MgO–16CaO–16Na2O–4Fe2O3 1–98 and 13–93, J. Eur. Ceram. Soc. 32 (11) (2012) 2731–2738. phosphate glass fibres, J. Non-Cryst. Solids 375 (2013) 99–109. [3] J. Massera, S. Fagerlund, L. Hupa, M. Hupa, Crystallization behavior of the com- [36] S. Inaba, S. Fujino, K. Morinaga, Young's modulus and compositional parameters of mercial bioactive glasses 45S5 and S53P4, J. Am. Ceram. Soc. 95 (2012) 607–613. oxide glasses, J. Am. Ceram. Soc. 82 (1999) 3501–3507. [4] B.C. Bunker, G.W. Arnold, J.A. Wilder, Phosphate glass dissolution in aqueous so- [37] L.G. Baikova, V.P. Pukh, Y.K. Fedorov, A.B. Sinani, L.V. Tikhonova, M.F. Kireenko, lutions, J. Non-Cryst. Solids 64 (3) (1984) 291–316. Mechanical properties of phosphate glasses as a function of the total bonding energy [5] A. Mishra, J. Rocherullé, J. Massera, Ag-doped phosphate bioactive glasses: per unit volume of glass, Glas. Phys. Chem. 34 (2008) 126–131. thermal, structural and in-vitro dissolution properties, Biomed. Glasses 2 (2016) [38] J. Massera, A. Mishra, S. Guastella, S. Ferraris, E. Verné, Surface functionalization 38–48. of phosphate-based bioactive glasses with 3-aminopropyltriethoxysilane (APTS), [6] J. Massera, Y. Shpotyuk, F. Sabatier, T. Jouan, C. Boussard-Plédel, C. Roiland, Biomed. Glasses 2 (2016) 51–62. B. Bureau, L. Petit, N. Boetti, D. Milanese, L. Hupa, Processing and characterization [39] W. Weibull, A statistical distribution of wide applicability, J. Appl. Mech. 18 (1951) of novel borophosphate glasses and fibers for medical applications, J. Non-Cryst. 293–297. Solids 425 (2015) 52–60. [40] C.R. Kurkijan, M.J. Matthewson, J.M. Rooney, Effects of heat treatment and HF etching on the strength of silica lightguides, Proc. SPIE Int. Soc. Opt. Eng. 5465 [7] J. Massera, M. Vassallo-Breillot, B. Törngren, B. Glorieux, L. Hupa, Effect of CeO2 doping on thermal, optical, structural and in vitro properties of a phosphate based (September 2004). bioactive glass, J. Non-Cryst. Solids 402 (2014) 28–35. [41] M. Bohner, J. Lemaitre, Can bioactivity be tested in vitro with SBF solution? [8] E.A. Abou Neel, D.M. Pickup, S.P. Valappil, R.J. Newport, J.C. Knowles, Bioactive Biomaterials 30 (2009) 2175–2179. UNPUBLISHED MANUSCRIPT Surface modified phosphate glasses for improved protein adsorption and cell viability A. Mishra*, L. Azizi*, C. Palma*, N. B. Huynh, R. Rahikainen, P. Turkki, A. S. Ribiero, V. P. Hytönen, J. Massera *authors contributed equally