Materials Letters 93 (2013) 6871

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Materials Letters
journal homepage: www.elsevier.com/locate/matlet

A simple recipe for direct writing complex 45S5 Bioglasss 3D scaffolds

Siamak Eqtesadi a, Azadeh Motealleh a, Pedro Miranda a,n, Alexandra Lemos b, M.F. Ferreira b Avito Rebelo b, Jose
a b

nica, Energe a Meca tica y de los Materiales, Universidad de Extremadura, Escuela de Ingenier as Industriales, Avenida de Elvas s/n, Badajoz 06006, Spain Departamento de Ingenier Department of Materials Engineering and Ceramics, University of Aveiro, CICECO, 3810-193 Aveiro, Portugal

a r t i c l e i n f o
Article history: Received 14 September 2012 Accepted 11 November 2012 Available online 20 November 2012 Keywords: 45S5 Bioactive glass Robocasting Direct-writing Carboxymethyl cellulose

a b s t r a c t
Direct-writing fabrication techniques, such as robocasting, have been ignored up to date for the production of scaffolds from 45S5 Bioglasss, probably due to difculties in the preparation of stable high solids loading colloidal suspensions (inks) from 45S5 Bioglasss powders using traditional approaches. Here, we report how these difculties can be overcome. Aqueous suspensions containing 45 vol% of 45S5 Bioglasss were successfully prepared using carboxymethyl cellulose (CMC) as single multifunctional (dispersant, binder, gelation agent) processing additive. Playing with the molecular weight (MW) and amount of CMC enabled tuning the rheological properties of the inks to meet the stringent requirements of robocasting. An incomplete surface coverage allowing bridging occulation to occur is the key to obtain highly performing inks. & 2012 Elsevier B.V. All rights reserved.

1. Introduction Bioactive glasses are seen with alacrity for their excellent potential in the repair and regeneration of bone defects, because of their ability to support the growth of bone cells [1,2]. Among numerous bioactive glass compositions proposed so far, the 45S5 Bioglasss developed by Hench [3], is the most comprehensively studied, exhibiting a long history of success in the clinical practice [4]. Most applications of bioactive glasses require the fabrication of 3-dimensional (3-D) porous scaffolds with tailored external geometries, pore volume fractions, pore sizes and controlled interconnectivity. However, most 45S5 Bioglasss scaffolds reported in the literature, were produced by conventional fabrications methods that offer limited control on the porous structure [5]. Novel solid freeform fabrication techniques such as robocasting (direct-writing) have been ignored for the production of scaffolds from this gold standard bioactive glass composition, probably due to difculties in the preparation of stable high solids loading colloidal suspensions from 45S5 Bioglasss powders. Indeed, there are only a couple of very recent articles reporting 45S5 Bioglasss scaffold fabrication by stereolithography (STL) [6] or selective laser sintering (SLS) [7], while most studies on the application of SFF techniques to bioactive glass scaffolds fabrication deal with other compositions [812]. Robocasting, an extrusion-based direct writing technique, utilizes high solids loaded paste-like colloidal suspensions (inks)

to build 3D structures through a layer-wise deposition of extruded cylinders [13]. Critical rheological parameters for the ink include its apparent viscosity and yield stress. The preparation of colloidal inks for robocasting usually include two steps: (i) the preparation of a low viscosity, yet concentrated suspension using, typically, anionic polyelectrolytes as dispersing agents; (ii) the induction of a drastic rheological change to turn the suspension into a pasty-like system with tuned elastic properties through a pH or temperature change, or the addition of salts or cationic additives [14]. An ink with appropriate rheological properties for robocasting must be able to ow through the nozzle (i.e. possess relatively low viscosity under stress) and have excellent shape retention capacity (i.e. high elastic modulus, G0 and high yield stress) upon deposition. The relatively high NaO2 content of 45S5 Bioglasss (24.5 wt%) produces leaching of Na ions into the dispersing solution and the consequent increase of pH and ionic strength strongly complicates 45S5 Bioglasss wet-processing. This was likely the main reason why less soluble bioactive glass compositions (13-93 and 6P53B), were adopted instead in some recent attempts to fabricate bioactive glass porous scaffolds by SFF methods [12,15]. The validity of this hypothesis is briey analyzed in this study, and an alternative means to tackle the problem of the preparation of 45S5 Bioglasss inks for robocasting is proposed and demonstrated.

2. Materials and methods Materials and reagents: The as received 45S5 Bioglasss [45% SiO2, 24.5% CaO, 24.5% Na2O and 6% P2O5 (wt%), (MO-SCI

Corresponding author. Tel.: 34 924289300x86735; fax: 34 924289300. E-mail addresses: pmiranda@unex.es (P. Miranda), jmf@ua.pt (J.M.F. Ferreira).

0167-577X/$ - see front matter & 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.matlet.2012.11.043

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Corporation (USA)] powder was attrition milled (Model 01-HD, Union Process, Akron, OH) in ethanol for 4 h down to 110 mm (Mastersizer 2000 MU, Malvern, UK), which is typically the optimal size range for colloidal processing. Dispersion of 45S5 Bioglasss powder in water was investigated in the presence of poly(methylvinyl ether) (Easysperse; ISP, Wayne, NJ) and CMC additives (CMC-35, Mw 35,000, and CMC n, 250, Mw 250,000, both from Lamberti Iberia s.a.u., Castello Spain). According to the supplier, the viscosities (Z) of 2 wt% aqueous solutions measured with a Brookeld LVT at 60 rpm and 25 1C, are Z 0.30.4 Pa s (CMC-35), and Z 13 Pa s (CMC-250). Characterization and processing techniques: Zeta potential (z) versus pH measurements (Zetasizer Nano ZS, Malvern, UK ) for the 45S5 Bioglasss particles were performed without/with added 1 wt% Easysperse or 2 wt% CMC additives, by adding drop by drop appropriate amounts of 0.1 M HCl to the suspension. The ow properties of colloidal inks prepared in a planetary mixer (ARE 250, Thinky Corp., Japan) were evaluated using a cone and plate (41, 40 mm, 150 mm gap) rheometer (Bohlin C-VOR Instruments, Worcestershire, UK) within the shear rate range of 0.1100 s 1. The inks, housed in a syringe, were extruded through a conical nozzle (D 250 mm) by a computer-controlled robocasting system (3D inks, Stillwater, OK, USA) at room temperature (RT) to fabricate 3D 45S5 Bioglasss structures. The position of the nozzle, immersed in an oil bath to prevent water evaporation, was driven by the control software (Robocad 3.0, 3D inks, Stillwater, OK) according to the set CAD model. The 3D constructs, dried for 24 h at room temperature, were thermal debinded in air at 450 1C for 2 h, using a heating rate of 1 1C min 1. Following the binder removal, the constructs were sintered in air for 1 h at 1000 1C, using a heating rate of 2 1C min 1. The compressive strength of resulting scaffolds was measured (AG-IS 10KN, Shimadzu Corp., Kyoto, Japan) at a constant crosshead speed (0.6 mm min 1) using nine (3 3 6 mm3) sintered samples. Total porosity of these samples was measured by weighting, and the pre-designed macroporosity was geometrically estimated using a mathematical calculus software from dimensional measurements (rod diameter and spacing, and layer height) performed on SEM micrographs.

Fig. 1. Evolution of zeta potential of 45S5 Bioglasss particles in absence and in the presence of indicated additives. Schematic diagrams of the proposed solid/liquid interface events explaining the results are included.

3. Results and discussion Fig. 1 illustrates the evolution with pH of z of 45S5 Bioglasss particles before and after the addition of Easysperse and CMCs. All curves tend to converge at a common isoelectric point (pHiep  1.5), which is close to the pHiep values reported for silica [16], and exhibit a continuous decrease up to pH  56 where the trend is reversed with a slight increase up to about pH 8, followed by a new sharp decrease within the more alkaline region. These z versus pH proles were reproducible with standard deviation values smaller than the size of plot markers. This evolution means that besides H and OH ions, cationic species, likely Ca2 and CaOH leached from the 45S5 Bioglasss, are also playing the role of potential determining ions, as schematized in Fig. 1, an hypothesis supported by the ndings of Nardin et al. [17]. Interestingly, the highest absolute z values were generally measured for bare particles. Also, the electrophoretic curves did not shift towards the acidic direction even in the presence of Easysperse, a typically anionic polyelectrolyte. This can be understood considering: (i) the low dissociation degrees of polyelectrolytes in the lowest pH regionCMC molecules are almost fully dissociated only at pH 4 5.5 [18]; (ii) the electrostatic repulsion between dissociated dispersant species and the negatively charged surface of the particles in the remaining pH range, which oppose adsorption. The apparent upward-shift of z-curves for all additives does not support extensive anionic surface adsorption.

However, adsorption cannot be completely ruled out because some masking effects might be simultaneously occurring: for example, the enhanced hydrodynamic hindrance associated to adsorption can reduce the electrophoretic mobility of the particles, or the shear-plane could be displaced to larger distances where z is smaller. The low maximum solid loadings (28.1, 29.2, 31.5 and 33.1 vol%) achieved with Easysperse (0.5, 1.0, 1.5, 2.0 wt%), although in accordance with values reported in literature for 45S5 Bioglasss [19], are unsuitable for robocasting; meaning that typical anionic polyelectrolytes will not enable accomplishing the purposes of the present work. The main reason behind this might be the formation of a thick adlayer with the polyelectrolyte chains stretching into the solution due to the repulsive electrostatic interactions. In contrast, the less accentuated anionic character of CMC molecules facilitates adsorption [20], and thus, CMC enabled increasing the solids loading up to 45 vol%. CMC has also the ability to play different roles (dispersant, binder, thickening/gelation agent) depending on MW which enabled varying the ow/elastic properties of the colloidal inks. Fig. 2 shows the shear elastic (storage) modulus, G0 , versus shear stress, t, for 45 vol% suspensions with 0, 1 and 2 wt% of CMC-35 or CMC-250. Linear viscoelastic region plateaus are followed by sudden drops of G0 at a given yield stress (ty) values where the gel networks start disrupting. The proles reveal: (i) low G0 values and high curve roughness in the absence of CMC, indicative of low consistency and poor ow properties of the suspension; (ii) an increase of ink stiffness by 12 orders of magnitude with added CMC, with G0 and ty being strongly dependent on the amount and MW of CMC; (iii) CMC-35 acts as dispersing/gelling agent adsorbed in an airy-like conguration, enhancing the gel network stiffness upon increasing the added amount to 2 wt%; (iv) The longer CMC-250 chains induce a stronger thickening effect especially at the lower concentration used (1 wt%). This is consistent with and incomplete surface coverage that would enable bridging occulation to occur, a hypothesis that is under investigation. These results suggested that the simple addition of 1 wt% of CMC-250 would yield optimum shape retention performance to the ink for robocasting, since it exhibits the highest G0 and ty, while its relatively low viscosity (800200 Pa s at 1100 s 1 shear rates, data not shown) warrants its owability. Indeed, Fig. 3(a) shows 3D scaffolds with up to 60 layers produced by robocasting using such an ink. The cross-sectional cut through

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Fig. 2. Loglog plot of the shear storage modulus versus shear stress for 45 vol% 45S5 Bioglasss suspensions with indicated amounts of CMC-35 or CMC-250 additives.

structurestotal porosity 63 7 3%, pre-designed macroporosity 52 7 2% was 13 7 1 MPa, which is far superior to previously reported values on SFF 45S5 Bioglasss scaffolds (0.33 MPa, 50% porosity [6]). While the level of porosity of these samples is somewhat lower than typical values reported for 45S5 Bioglasss scaffolds fabricated by foam replication [21], similar and even lower values of porosity have been demonstrated to be more than enough for promoting bone ingrowth and regeneration in robocast calcium phosphate scaffolds [22,23]. This is probably due to the large interconnection sizes and the unique combination of macro- and microporosity typically achieved in robocast structures [23]. In any case, higher porosities can be easily attained by increasing the inter-rod spacing within each deposition layer. Besides, the good mechanical performance exhibited by these samples sintered at high temperature, and thus fully crystallized (veried by XRD, not shown), suggests that it should be possible to reduce the sintering temperature, in an attempt to preserve the glassy phase, retaining yet some signicant mechanical integrity. Studies are currently underway to verify this assertion.

4. Conclusions The intrinsic aqueous processing difculties of 45S5 Bioglasss could be overcome using CMC of high MW as single but multifunctional additive. This simple and versatile recipe will enable the fabrication of scaffolds with customized external geometry and optimized pore architecture, paving the way for the use of this bioactive material in a broader range of tissue engineering applications (patient specic reconstructions, structures with porosity gradients mimicking cortical-cancellous natural bone structure, mildly load-bearing applications etc.).

Acknowledgments This study was supported by the Junta de Extremadura and FEDER Funds under Grant IB10006. The support of DEMac/ CICECO, University of Aveiro, is highly appreciated. References
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Fig. 3. 3D porous scaffolds produced by robocasting from a 45S5 Bioglasss ink with 1 wt% CMC-250 additive: (a) optical image of different scaffolds geometries and (b) SEM micrograph of a lattice cross-section demonstrating excellent shape retention after sintering.

a tetragonal lattice sintered at 1000 1C for 1 h shown in Fig. 3(b) evidences that the rods maintain their cylindrical shape during the multilayer deposition process and posterior sintering. The compressive strength measured in such porous

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