Biomed. Glasses 2016; 2:63–71 Research Article Open Access Barbara Pföss*, Miriam Höner, Monika Wirth, Andreas Bührig-Polaczek, Horst Fischer, and Reinhard Conradt Structuring of bioactive glass surfaces at the micrometer scale by direct casting intended to influence cell response** DOI 10.1515/bglass-2016-0008 Received Aug 12, 2016; revised Oct 29, 2016; accepted Oct 31, 2016 1 Introduction Abstract: Defect-free bioactive glass surfaces with a Large bone defects cannot be healed by the body itself. grooved microstructure at the low micrometer scale were Since the availability of autologous material is limited, achieved by a mold casting process. The process was synthetic materials offer a promising solution for bone applied to the well-known glass compositions 45S5 and replacement applications [1]. In this respect, bioactive 13–93. Such microstructured surfaces may exhibit espe- glasses exhibit excellent properties: They are cytocompat- cially favorable conditions for bone cell orientation and ible, bind quickly to bone and can stimulate bone regener- growth. The aim of the study was to assess the parameter ation [2]. range for a successful casting process and thus to produce Cellular behavior is crucially influenced not only by samples suitable to investigate the interaction between the chemistry of the underlying substrate but by the sur- structured surfaces and relevant cells. Viscous flow in its face topography and mechanical properties as well. Sur- temperature dependence and thermal analysis were ana- face structures can increase the bone–to–implant con- lyzed to identify a suitable process window and to design tact and thus the fixation in the host bone [3]. It has a manageable time-temperature process scheme. Coun- been shown that cells react to surface structures from the teracting effects such as formation of chill ripples, mold nanometer to the micrometer scale [4, 5]. Furthermore it sticking and build-up of permanent thermal stress in the is well known that cell adhesion and even differentiation glass had to be overcome. A platinum gold alloy was cho- of cells can be influenced by surface structures in the low sen as mold material with the mold surface bearing the micrometer range [6–8]. Parallel groove–ridge structures mother shape of the microstructure to be imprinted on promote contact guided alignment of different cell types the glass surface. First experiments studying the behavior with the groove depth having more impact as the grooves of osteoblast-like cells, seeded on these microstructured become deeper [7, 9–11]. More than 5 µm in depth are glass surfaces revealed excellent viability and an orienta- needed to sufficiently guide the cells [7, 10, 12]. In addi- tion of the cells along the microgrooves. The presented re- tion, McBeath et al. and Kumar et al. have shown that sults show that direct casting is a suitable process to pro- elongated stem cells rather differentiate into osteoblasts duce defined microstructures on bioactive glass surfaces. whereas rounded cells become adipogenic cells [13, 14]. Photolithography and etching methods enabled the Keywords: casting; surface topography; groove structure; possibility to manufacture surface topographies at the mi- cell guidance crometer scale [15–18]. A wide variety of materials have thus become available to study the phenomenon of con- tact guidance [19, 20]. Still, due to their strong crystalliza- *Corresponding Author: Barbara Pföss: Institute of Mineral Engi- tion tendency, bioactive glasses are mostly ineligible for a neering, RWTH Aachen University, 52064 Aachen, Germany; Email: [email protected]; contributed equally to this work Miriam Höner: Dental Materials and Biomaterials Research, RWTH Aachen University Hospital, 52074 Aachen, Germany; contributed Reinhard Conradt: Institute of Mineral Engineering, RWTH Aachen equally to this work University, 52064 Aachen, Germany Monika Wirth, Andreas Bührig-Polaczek: Foundry Institute, ** Special Section: Larry L. Hench Memorial Symposium on Bioac- RWTH Aachen University, 52072 Aachen, Germany tive Glasses at the Annual Meeting of the Glass & Optical Materials Di- Horst Fischer: Dental Materials and Biomaterials Research, RWTH vision (GOMD) of the American Ceramic Society, 22nd–26th May 2016, Aachen University Hospital, 52074 Aachen, Germany Madison, Wisconsin, USA © 2016 B. Pföss et al., published by De Gruyter Open. This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivs 3.0 License. Bereitgestellt von | Universitätsbibliothek der RWTH Aachen Angemeldet Heruntergeladen am | 11.12.17 11:09 64 Ë B. Pföss et al. surface finish after manufacturing and therefore they are Combining the outstanding properties of bioactive rarely available for cell culture experiments regarding cell glass with those of a triggered surface topography is very behavior e.g. guidance phenomena on structured surfaces. promising and holds potential to further improve bone re- Itälä et al. manufactured micro-rough bioglass sur- placement applications. The presented results show that faces by chemical etching and found an improved os- a tailored casting process can be used to realize the de- teoblast attachment compared to polished control speci- manded glass surfaces. mens [21]. Following in vivo experiments even showed a higher amount of incorporated new bone in comparison to smooth implants [22]. Porous structures were realized 2 Methods by lithography-based additive manufacturing as well, cre- ating crystallized bioglass samples [23]. Efforts have also PtAu5 sheet metal with 8 mm thickness was used as sub- been made to structure bioglass, showing alignment of strate material, micrometer sized grooves were applied osteoblast-like MG-63 cells. But as the samples were sin- by a solid state ultrashort pulsed laser (Timebandwidth tered, the tested material became a glass-ceramic [24]. Sur- Duetto, Zurich, Switzerland). The applied groove depth to face structures on amorphous bioactive glasses have not be imprinted on the glass was 15 µm, the groove width been realized before. 30 µm and the ridge width 10 µm. To choose an appropriate testing pattern we took into Two bioactive glasses namely 45S5 [27] and 13–93 [28] consideration the results of above mentioned former stud- were synthesized via melt–quench technique. Batches of ies, where many cell types react to grooved substrates in the pure chemical components: Fused silica (Aachener the low micrometer range, mesenchymal stem cell size, as Quarz–Glas Technologie Heinrich, Aachen, Germany), well as the requirement that a surface structure on bioac- CaHPO4 (Merck, Darmstadt, Germany), CaCO3, Na2CO3 tive glass has to endure the different states of corrosion to (both AppliChem, Darmstadt, Germany) for 45S5, and ad- provide the intended biophysical cue throughout the for- ditionally MgO (VWR Chemicals, Darmstadt, Germany) mation of different layers after all. Moreover, when consid- and K2CO3 (Merck, Darmstadt, Germany) for 13–93, were ering the average viscosity of a glass melt, our calculations ∘ homogenized and then melted at 1400 C for 2 hours in a and experiments indicated that surface topographies at platinum crucible. Batch sizes yielding 300 g of glass were the scale of a few 10 µm suitable for casting. Finally a par- prepared and the melts were fritted into water. XRF and allel groove pattern of 30 µm groove, 10 µm ridge width, XRD analysis were carried out to ensure the correct com- and 15 µm height was applied to bioactive glasses in this position and amorphous nature of the material implying study. the described melting conditions. The glass frit was sieved A few mold materials were preliminary discussed and to different designated particle size fractions from 63 µm tested with minor success, as we decided to stay with plat- to 1 mm and provided for the following casting procedure inum gold. Platinum alloys are commonly used in combi- respectively. nation with glass melts and batches, as chemical reactions 15 g of glass granules were remelted in a small plat- are minimal up to high temperatures. Platinum gold is fur- inum crucible for 40 minutes at a temperature level Tcast ther known to be only poorly wetted by oxide glass melts, and then cast. The PtAu5 substrate bearing the microstruc- hence a favorable demolding behavior is expected. ture was preheated at a temperature T . To ensure the Discontinuous casting does not belong to the major mold correct mold temperature, the mold was stored in a sepa- forming methods for mass glass production, but it is typi- rate furnace wherein the casting was performed. The tem- cally used to manufacture bulk glass samples for a variety perature of the melt as well as the mold were varied in a se- of standard testing methods in glass science, or in the field ries of experiments. Demolding of the solidified glass was of glass art. accomplished by turning the substrate upside down and Microstructured components are for example fabri- carefully pounding from the back. The samples were then cated in the field of Micro-optics by using hot embossing annealed for 12 hours at a temperature level 20 K above the of borosilicate glass [25, 26]. Surface features of some mi- glass transition temperature Tg, and cooled down to room crometers are standard requirements for this application. temperature within 10 hours. Hot embossing requires heating the glass samples up to Polished bioactive glass 45S5 and 13–93 samples to be a temperature level sufficiently high enough for forming, used as control for cell culture experiments