Bottom-Up Assembly of Silica and Bioactive Glass Supraparticles with

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Bottom-up Assembly of Silica and Bioactive Glass Supraparticles with Tunable Hierarchical Porosity Steffen Egly, Christina Froehlich, Stefanie Vogel, Alina Gruenewald, Junwei Wang, Rainer Detsch, Aldo R. Boccaccini, and Nicolas Vogel Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b03904 • Publication Date (Web): 08 Jan 2018 Downloaded from http://pubs.acs.org on January 8, 2018

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Bottom-up Assembly of Silica and Bioactive Glass Supraparticles with Tunable Hierarchical Porosity Steffen Egly1, Christina Fröhlich1, Stefanie Vogel1,2, Alina Gruenewald2, Junwei Wang1, Rainer Detsch2, Aldo R. Boccaccini2,3*, and Nicolas Vogel1* 1

Institute of Particle Technology, Friedrich-Alexander University Erlangen-Nürnberg,

Cauerstrasse 4, 91058 Erlangen, Germany 2

Institute of Biomaterials, Friedrich-Alexander University Erlangen-Nürnberg, Cauerstrasse 6,

91058 Erlangen, Germany

KEYWORDS: colloids, bioactive materials, self-assembly, hierarchy, porosity

We investigate the formation of spherical supraparticles with controlled and tunable porosity at the nanometer and micrometer scale using self-organization of a binary mixture of small (nanometer scale) oxide colloidal particles with large (micrometer scale) polystyrene particles in the confinement of an emulsion droplet. The external confinement determines the final, spherical structure of the hybrid assembly, while the small particles form the matrix material. The large particles act as templating porogens to create micropores after combustion at elevated temperatures. We control the pore sizes at the micrometer scale by variation of the size of the coassembled polystyrene microspheres and produce supraparticles from both silica and calciumcontaining CaO/SiO2 particles. Although porous supraparticles are obtained in both cases, we find that the presence of calcium ions substantially complicates the fabrication process since the

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increased ionic strengths of the dispersion compromises the colloidal stability during the assembly process. We minimize these stability issues via the addition of a steric stabilizing agent and by mixing bioactive and silica colloidal particles. We investigate the interaction of the porous particles with bone marrow stromal cells and find an increase in cell attachment with increasing pore sizes of the self-assembled supraparticles.

Introduction Colloidal particles can self-organize in regular superstructures, enabling the fabrication of materials with precise, nanoscale structuration in an experimentally simple, fast and parallel process.1-5 Polymeric colloidal particles can also be used as templates to yield materials and structures with defined and precisely tunable porosity in the nano- or micrometer after thermal combustion of the templating particles.6-8 Nanostructuration resulting from self-assembly processes and the generation of regular pore structures significantly influences the properties of a material and may lead to entirely novel properties not observed in the unstructured bulk material.2,3,9 The creation of such structures in experimentally simple and low cost approaches has therefore found applications in a wide range of technologies, including photovoltaic devices,10 antireflective11-13 and liquid-repellent coatings,11,14,15 the design of materials exhibiting structural coloration,2,9,16 and the control of cell-surface interactions.17-20 An additional level of complexity of self-assembled materials can be achieved by hierarchical structuration, i.e. by creating defined structural features at multiple length scales. This is, for example, achieved by providing colloidal building blocks with different dimensions that


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subsequently self-organize in binary superstructures.21-23 Additionally, external confinements can be applied to guide and control the assembly process. A simple yet interesting way to provide external confinements is the emulsification of colloidal dispersions. The self-organization of colloidal particles within an emulsion droplet yields powder particles with an internal structure defined by the constituent building blocks.24 Such supraparticles have been used in optical applications, including pigment technology,25-28 sensors,29 magnetically switchable colorants,30 and as color-coded substrates for biomaterials evaluation.18,31 Similar to the analogous structures in thin films, polymer colloidal particles can be used as porogens, yielding powder particles with defined nanoscale porosities when mixed with an inorganic matrix material.21,27,32-34 A more complex class of colloidal particles are bioactive nanoparticles.35,36,37 Such particles can be synthesized by sol-gel chemistry based on the silica Stöber process38,39 and consist of silica with a high content of biologically relevant ions, like calcium, boron or sodium. These ions are slowly released into the aqueous environment of the bioactive oxide particle, where they can influence cellular behavior and cell-material interactions.40-42 Bioactive glasses are well-known for their osteoinductive behavior as well as their ability to form a carbonated hydroxyapatite layer when exposed to biological fluids, which is the reason for the extraordinary strong bonding between bioactive glass and human bone.35,40 Furthermore, it has been shown that bioactive glasses stimulate gene expression and stimulate angiogenesis.43-45 Antibacterial and inflammatory effects have also been observed.40 Besides coatings on implant surfaces,46,47 bone tissue engineering approaches employ bioactive glasses deposited on porous templates, for example polymer foams, to create materials with micron-scale porosity mimicking the natural bone structure.48,49 The presence of porosity of the biomaterial is crucial for cell ingrowth and diffusion of oxygen, nutrients and metabolic products


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during tissue formation around and/or inside the scaffold.50 The deposition of bioactive glass on polymeric foams yields highly porous materials with interconnected pores. However, the resulting porous substrate is characterized by a random (micro-)structure. If the porosity of the bioactive ceramic could be controlled, new ways to improve cell-material interaction based on highly defined and “designed” porosity would become available. In this context, it has been recognized that a hierarchical organization of bioactive materials with defined structural features spanning multiple length scales58 critically affects the performance in a range of biomedical applications including the regeneration of bone- and cartilage tissue.51 Here, we explore a self-assembly approach to create porous particles via a bottom up approach. This approach enables us to precisely control the porosity of the material at the micro- and nanoscale. While the nanoscale porosity emerges directly from the interstitial sites of the assembled colloidal particles, hierarchical pores are created by co-assembling micron-scale polymer colloidal particles as porogens with the nanoscopic colloidal particles. In proof-ofprinciple experiments, we demonstrate that our created porous particles enhance cellular adhesion based on solely structural motifs, opening up a potential alternative avenue towards scaffold fabrication in bone tissue engineering.

Experimental Section Materials: tetraethylorthosilicate, ammonium hydroxide solution (28-30 %), ethanol, calcium nitrate tetrahydrate, deuterium oxide and poly vinylpyrrolidone were bought from Sigma Aldrich unless otherwise stated. Fluorescence dyes used for cell imaging were purchased from Life


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Technologies. Polystyrene microspheres with diameters of 10µm, 20µm and 30µm were purchased from Sigma Aldrich. Scanning electron microscopy images were taken on a Zeiss Ultra microscope. Confocal microscopy was performed on a Lyca confocal microscope (model TCS SP5 II). Inductively coupled plasma-optical emission spectroscopy (PerkinElmer) was performed in liquid phase after digestion of a precise amount of silica or CaO/SiO2 particles with 1ml hydrofluoric acid.

Synthesis of silica particles: Silica particles were synthesized via the Stöber process. In brief, to a solution of tetraethylorthosilicate (3,75g) and ethanol (37g), a mixture of water (19g), ethanol (12g) and ammonium hydroxide solution (6,75g) was added at room temperature under vigorous stirring. The stirring speed was adjusted to 600 rpm and stirred for 16 h to complete the sol-gel reaction. The resulting colloid dispersion was purified by repeated centrifugation. The properties of the colloidal particles were characterized by SEM image analysis and zeta potential measurements (Malvern instruments; 1mmol KCL background electrolyte), as shown in Supplementary Table 1.

Synthesis of CaO/SiO2 particles: The calcium containing oxidic colloidal particles were synthesized by a modified Stöber reaction and adopted from literature.38,39 Similar to the synthesis of pure silica particles, to a solution of tetraethylorthosilicate (3,75g) and ethanol (37g), a mixture of water (19g), ethanol (12g) and ammonium hydroxide solution (6,75g) was added at room temperature under vigorous stirring. To this solution, a mixture of water (6,2g), ethanol (15,8g) and calcium nitrate tetrahydrate (4,25g) was added after different time intervals specified


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in Figure 3. The mixture was stirred for 16h and purified by repeated centrifugation. The properties of the colloidal particles were characterized by SEM image analysis and zeta potential measurements, as shown in Supplementary Table 1. Supraparticle fabrication: A droplet-based microfluidic device was fabricated following a protocol from literature.28 A master featuring the inverse of the cross-junction device was fabricated by photolithography and replicated with PDMS to yield the actual device. The PDMS device structure was bonded to glass by a brief activation with oxygen plasma (10s, 30W power). Inlets were punched into the device using a 1mm biopsy punch. The channels within the device were hydrophobized using commercially available AquaPel solution. The cross-junction had a lateral diameter of 200µm and is shown in Supplementary Figure 5. The device was operated with an aqueous colloidal dispersion containing all colloidal species as specified above as the inner phase and a fluorinated oil (3M Novec 7500) as the outer phase. The flow rates were 100µl/h (inner phase) and 200µl/h (outer phase). The emulsion droplets were collected in a hydrophobized glass vial and dried in an oven at 70°C or a hotplate.

ICP-OES measurements: The release of ions from both silica and silica/calcium oxide colloidal particles was investigated over time as a function of the annealing temperature. Colloidal particles were synthesized as described above and purified by centrifugation and replacement of the supernatant with fresh, ultrapure water to remove unreacted precursor material. The colloids were then immediately spray dried to produce a dry powder with defined aggregate size (the size of the aggregate was 10±5µm) and to avoid additional leakage of ions before the release experiments. The produced powders were sintered at different temperatures. 50 mg of the


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respective powders were weighted into a plastic vial (Falcon tubes, 15ml) to avoid contamination with silica. 10ml of ultrapure water was added and the suspension was left to sit at ambient condition for a defined time (as specified in the data of Fig. 4). For the determination of the released ions, the supernatant was removed and measured with the ICP-OES instrument (Perkin Elmer, Optima 8300), using 1400W power and the following flow conditions: Plasma flow 10l/min, Auxiliary gas flow: 0.2l/min, nebulizer gas flow: 0.6l/min, Sample flow: 1.5ml/min. Cell culture: The self-assembled particles were dropcasted onto cover slips and calcined at 500°C to remove the templating polystyrene particles and sterilized with hot air (2h, 160°C, L3/11, Nabertherm). 100,000 Mice ST2 stromal cells (DSMZ, Braunschweig, Germany) per milliliter culture medium (RPMI 1640, Gibco with 10% fetal bovine serum and 1 % penicillin streptomycin) were seeded for 24h at 37°C, 95% humidity and 5 % CO2. For fluorescence imaging, the cells were stained with 4µl of Calcein (Life Technologies) and 1µl Propidium Iodide (Life Technologies) for 45min at 37°C. For the statistical evaluation of the cell colonization on the different particles, we drop casted a defined amount of particles (10µl of the dispersion fabricated by microfluidics, solid concentration approx. 0.1%) onto a preheated substrate (60°C) to achieve a random deposition of individual particles on the substrate. We then cultured the cells as described above and used SEM and confocal imaging to investigate the colonization behavior. For all samples types, 100 particles were randomly chosen and the number of cells attached to their surface was evaluated. The water soluble tetrazolium (WST) assay was used to determine the LC50 value of silica/calcium oxide supraparticles. The particles were incubated with ST-2 cell in the range of 01000 µg/ml for 48 hours. After cell cultivation, the cell culture medium was removed and samples were washed with 0.5 mL phosphate buffered saline (PBS). Afterwards, 0.25 mL WST


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medium (containing 1 vol% of WST reagent (Cell Counting Kit-8, Sigma) and 99 vol% of DMEM medium) was added and incubated for 2 h. After incubation, 0.1 mL of the supernatant was transferred to a 96-well culture plate and spectrometically measured using a microplate reader (PHOmo, anthos Mikrosysteme GmbH, Germany) at 450 nm. The data shown in Fig. 5 was obtained by averaging over 6 individual measurements of the cell toxicity at all concentrations. For SEM investigations, the cells were fixated with a solution of glutaraldehyde (0.1%) and formaldehyde (2%) followed by a stepwise exchange of the aqueous medium to ethanol and critical point drying (EM CPD300; Leica). Confocal microscopy: All images were taken on a Leica TCS SP5 II confocal microscope using a HeNe laser with wavelengths of 543 nm and 633 nm. All samples were imaged in aqueous solution using an inverted setup and a 20x objective lens. Living cells were stained with calcein (life technologies; 4µl/ml medium) and dead cells were stained with propidium iodide (life technologies; 1 µl/ml medium). Staining was performed by incubating the samples for 45min at 37°C. Subsequently, the medium was removed and the cells were fixated with paraformaldehyde (3,7%) and stored in PBS buffer at 6°C until imaging. Results and Discussion We created particles with hierarchical porosity via the self-assembly of colloidal particles in the confinement of an emulsion droplet (Figure 1a). Using droplet-based microfluidics we emulsified an aqueous mixture of nanoscale silica and micron-sized polystyrene particles in a continuous fluorocarbon oil phase (Fig. 1.b).28,52 The cross-junction device was fabricated from polydimethylsiloxane (PDMS) by soft lithography and enabled the formation of monodispersed


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droplets (Figure 1d,e). As particles, we used silica colloids with a diameter of 300 nm and micron-sized polystyrene colloids with diameters varying between 10µm and 30µm. Both colloidal particles were negatively charged and remained colloidally stable in the emulsion droplets without aggregating (Figure 1d). In the emulsion droplet used as template for the resulting spherical powder particle, the small silica particles and the larger polystyrene particles self-organized into a mixed assembly structure upon removal of water from the emulsion droplet.21 Water from the inner phase of the emulsion was removed by placing the assembly onto a hot plate at 90°C under gentle stirring. In the course of the drying process, the concentration of polymer particles continuously increases as a result of the shrinkage of the droplet. Eventually, this concentration increase brings the polymer particles into contact. The smaller silica particles continue to be present in the liquid bridges surrounding the microspheres and, upon solidification, subsequently organize themselves around the polymer microspheres.21 The resulting morphology of the supraparticles supports this established picture and shows small particles filling the interstitial sites connecting the large particles, which themselves are touching and thus provide interconnected micropores after calcination. We found that stirring and convection induced from anisotropic heating on a hot plate created more uniform particles compared to drying in an oven (Supplementary Figure 1), presumably because of a more homogeneous particle distribution by convection-induced rotations of the emulsion droplets. To further increase homogeneity, we minimized effects of gravity leading to sedimentation of the substantially heavier polystyrene microspheres and thus resulting in the formation of irregular assembly structures by matching the density of the aqueous phase to the density of polystyrene (d=1.05 g/cm3) using a mixture of water and heavy water.53


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The composite crystal was subsequently calcined to remove the polymer particles, creating an inorganic powder particle with micron-sized pores resulting from the templating polymer particles and nanoscale pores resulting from the interstitial sites of the close-packed silica colloids. The volume ratio between silica and polystyrene was optimized to 15 vol-% (SiO2/PS), which showed an ideal balance between pore morphology and mechanical stability in the resulting particle. In agreement with previous reports, lower silica concentration led to smaller volumes (Supplementary Figure 2) without changing the pore opening as the contact angle between inner phase and outer phase remains unaltered.21 Figure 1e-g exemplarily shows scanning electron microscopy (SEM) images of the spherical assembly structure formed by 300nm SiO2 colloids and 10µm polystyrene microspheres. A homogeneous distribution of the microspheres within the spherical supraparticle is visible (Fig. 1e). The silica particles form a homogeneous matrix that fills the interstitial sites of the large spheres. After calcination, a micron-scale porosity is created by removal of the templating microspheres (Fig. 1f). The high order of the assembled silica colloidal particles and the interconnected nature of the micropores are clearly visible in the high magnification SEM image shown in Fig. 1g. Figure 2 shows representative SEM images of supraparticles consisting of pure silica particles and porous silica-based powder particles with varying dimensions of the micron-scale pores, created by changing the diameter of the co-assembled polystyrene microspheres from 10µm to 30µm. In all cases, the interconnectivity of the pores is visible. The microfluidic process design enables the fabrication of spherical supraparticles with narrow particle size distribution and


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uniform pore diameters, as can be seen in the low magnification image of Figure 2e showing microporous particles template from 10µm microspheres and from quantitative image analysis shown in Supplementary Table 1.

Figure 1. Fabrication of powders with hierarchical porosity. a) Schematic illustration of the formation process using self-organization of bidisperse colloidal particles. b-d) Optical micrographs showing the fabrication via droplet-based microfluidics. b) Droplet formation in the microfluidic device. c,d) Monodispersed droplets of the colloidal dispersion in a continuous oil


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phase formed in the microfluidic device. e-g) Scanning electron micrographs of the resulting hierarchical supraparticles. e) Composite supraparticle of polystyrene microspheres and silica colloidal particles after self-organization upon drying of the emulsion droplet. f) Porous supraparticle after removal of the polystyrene microspheres by calcination. g) Close-up of the porous supraparticle showing pore connectivity and the matrix composed of silica colloidal particles.

Figure 2. Control of micropore structure in self-organized powder particles prepared from selforganization of silica colloids (d=300nm) and polystyrene (PS) microspheres of varying diameters. a) No polymer microparticles. b-d) addition of polymer microspheres with a diameter of 10µm (b), 20µm (c) and 30µm (d). e) Low magnification image of the samples with 10 µm pores showing the uniform size of the prepared, microporous supraparticles.

For any potential application of the hierarchical particles as building blocks in tissue engineering, it is crucial to investigate the biocompatibility of the particles and the influence of porosity on


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the interactions with tissue cells. As proof of principle experiments, we seeded mesenchymal stromal cells (ST2-cells), a cell line known for the ability to differentiate into osteoblasts54 onto a substrate coated with the prepared porous particles. Figure 3a-d shows representative confocal images of the powder particle samples with different porosities after cell incubation for 24h. After 24h of incubation, the cells were stained with calcein and propidium iodide indicating viable and dead cell, respectively. The cells showed high viability (green) and a negligible amount of dead cells (red). The dye used to detect dead cells (propidium iodide) adsorbed onto the porous particle surface, enabling us to visualize the particles in the confocal images (red color). All images showed adhesion between cells and particle surface without inducing cell death, supporting our quantitative investigations on toxicity shown below. In vitro results of powder particles with porosities of 20µm and 30µm showed cells within the particle interior, indicating sufficiently large pores to enable cellular migration into the porous particles (Figure 3c,d). The presence of cells within the prepared particles featuring 20µm pores is further evident in scanning electron microscopy (SEM) images obtained after critical point drying of the samples (Fig. 3f). In contrast, samples without porosity and with 10µm porosities exclusively showed cells at the surface of the particles, as evidenced by confocal microscopy (Fig. 3a,b) and SEM (Fig. 3e). In the powder particles with 10µm porosities, cells spanning the pore openings without entering are clearly visible (Fig. 3e). To quantify the adhesive capabilities of the different porous particles, we evaluated the number of adherent cells on approx. 100 particles (Fig. 3g) and found an increase in cellular adhesion with increasing porosities. The reference sample consisting of a self-assembled spherical particle consisting of 300nm silica colloids without micron-sized pores (as depicted in Fig. 2a) showed adherent cells on 45% of all investigated particles while 55% of the particles did not feature any


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cells. With increasing pore sizes, the number of particles with adhering cells increases to 65% (10µm pores), 70% (20µm) and 92% (30µm). These results corroborate with the results of Zinger et al.55 that compared cellular attachment on micron-scale cavities on a planar surface and found that cells tended to cover cavities with a size of 10µm and smaller while migrating into the cavities for larger pore sizes.


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Figure 3. Interaction of porous silica supraparticles with ST2 cells. a-d) Confocal images showing fluorescently labelled ST2 cells (green) interacting with porous particles with different micropore dimensions. e,f) Scanning electron microscopy images of ST2 cells in contact with


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microporous particles taken after critical point drying. g) Quantification of ST2 cell colonization of porous particles with different diameters.

Next, we applied the same confined self-organization strategy to a composite oxide particle system. We chose a binary mixture with a targeted composition of 70% silica and 30% calcium oxide as simple example of an oxide with enhanced biological activity caused by a release of calcium and silicon ions from the material.40 We synthesized colloidal building blocks via a modified Stöber process adopted from literature.38,39 In brief, a solution of calcium nitrate was added to the classical, ammoniacatalysted Stöber silica synthesis56 using tetraethylorthosilicate as the silica-network forming agent. The addition of calcium nitrate strongly affected the sol-gel reaction. If calcium ions were added directly at the start of the reaction, no colloidal particles were formed. Rather, large agglomerates were found in the reaction, indicating an instable dispersion (Supplementary Figure 4). We attribute this behavior to a reduction of electrostatic stabilization by the presence of calcium ions which causes aggregation of the freshly formed colloidal particles.5 With increasing reaction time before addition of calcium ions, stable dispersions with homogeneous colloidal particles and narrow size distributions were observed (Supplementary Figure 4). However, the integration efficiency of calcium ions into the silica network was reduced with increasing delay between the start of the reaction and the addition of calcium nitrate solution. We investigated the final calcium content via inductively-coupled plasma – optical emission spectrometry (ICP-OES)57 after purification of the synthesized particles by centrifugation and exchange of supernatant three times to remove non-incorporated calcium ions. Figure 4a shows


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the decrease of incorporated calcium ions with increasing time span between the start of the reaction and the addition of the calcium nitrate solution. For all consecutive experiments, we chose a calcium nitrate addition time of 20 min after start of the sol-gel reaction as a compromise between colloidal stability and high calcium content (18%) of the resulting particles. SEM analysis of the resulting particles, shown as inset in Fig. 4a, indicated high uniformity of the formed calcium-containing silica colloidal particles. The formation of supraparticles from the calcium-containing silica particles proved significantly more difficult compared to the case of pure silica particles. With the pure calcium containing CaO/SiO2 colloidal particles, no homogeneous co-assembly with micron-scale polystyrene particles was observed. Instead, inhomogeneous distribution of the large particles (or, pores after calcination) was found (Fig. 4d). Importantly, in comparison to pure silica particles, no ordering of the colloidal matrix was achieved and the macropores were not connected, as can be seen in a high magnification SEM image shown in Fig. 4e. We attribute this behavior to a decrease in colloidal stability, leading to premature aggregation of the colloidal particles during the assembly process. This aggregation prevents the large microparticles from coming into close contact and, additionally, the hinders the assembly of the small particles into densely-packed structures with high order. We attribute the decreased colloidal stability to the trademark property of bioactive particles, namely the release of ions into the aqueous environment. In the course of the assembly process, water is continuously removed from the aqueous inner phase containing the colloidal particles, leading to an increase in concentration of all constituent components of the phase. Hence, even small amounts of calcium ions released from the particles will lead to an increasingly high concentration of Ca2+ during assembly, which will eventually compromise the electrostatic stabilization of the particles by screening of surface charges.5 Zeta potential


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measurements indicate a decreased colloidal stability. As shown in Supplementary Table 1, the pure SiO2 particles synthesized by the Stöber process had a zeta potential of -36.9±1.0 mV, while the Cao/SiO2 particles showed a decreased value of -26.8±0.7 mV. To mitigate the problem of reduced colloidal stability, we added 10 wt% of polyvinyl pyrrolidine, a non-ionic, steric stabilizer to the aqueous dispersion containing the SiO2/CaO composite particles. Steric stabilization is not affected by ion concentration and should thus increase colloidal stability in the course of the drying process. The resulting supraparticles, shown exemplarily in Fig. 4f, featured a more uniform appearance and interconnected pores, indicating decreased agglomeration (or, increased colloidal stability) of the colloidal particles. To further improve the assembly properties, we also mixed pure silica with bioactive ceramic colloidal particles in a ratio of 1:1. Using this mixture, it was possible to reliably fabricate supraparticles without and with macropores by the addition of micron-scale polystyrene particles (Fig. 4g,h) due to the overall lower concentration of released calcium ions. A high magnification SEM image, shown in Fig. 4i demonstrates the uniform assembly of the colloidal particles around the pore opening. We investigated the ion release kinetics of the supraparticles formed from SiO2 as well as SiO2/CaO colloidal particles as a function of the annealing temperature, following the hypothesis that with increasing annealing temperature, the sol-gel network will increasingly solidify and therefore retain the ions more efficiently. We used inductively-coupled plasma - optical emission spectroscopy (ICE-OES) to determine the concentration of calcium and silicon released into the aqueous matrix. We removed unreacted reactants (containing free silicon and calcium species) by centrifugation and replacement of the supernatant with ultrapure water directly after the colloid synthesis. This procedure ensure that released ions originated from the solid particles and were not initially present in the water phase. We then used spray drying to convert the aqueous


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dispersions into defined powder particles with sizes of 10±5µm to provide uniform samples and comparable release profiles. We annealed these dried samples at different temperatures and investigated the release of calcium and silicon over time after redispersing the powder into ultrapure water. Figure 4b,c show that the release profile of both calcium (Fig. 4b) and silicon (Fig. 4c) was very sensitive to the annealing temperature. The fastest release was found for samples without any temperature treatment, while calcination at 800°C significantly reduced the ion release over time. These results demonstrate that the release profile of the prepared supraparticles can be controlled by a simple sintering protocol. We also determined the silicon release of pure SiO2 particles (Fig. 4c). Such particles also showed an annealing-temperature dependent release of soluble silica species, however, at a much reduced concentration compared to the SiO2/CaO composite particles. These results further underline the facilitated decomposition of the composite particles as a result of the disturbed network formation reactions by the incorporated calcium ions.


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Figure 4. Bioactive powder particles with hierarchical porosities. a) Measured calcium content in the synthesized CaO/SiO2 oxide colloidal particles as a function of the time of calcium addition after start of the sol-gel reaction. The inset shows colloidal particles synthesized with calcium ions added 20 min after start of the reaction. b) Calcium release of the supraparticles over time as a function of the annealing temperature. c) Silicon release of the supraparticles over time as a function of the annealing temperature. Filled symbols are measurements on CaO/SiO2 particles, open symbols are pure SiO2 particles. d-e) SEM micrographs of self-assembled porous particles fabricated with 10 µm PS microspheres and CaO/SiO2 colloids without further modification. f) Resulting porous particles fabricated with 10 µm PS microspheres and CaO/SiO2 colloids stabilized with 10 wt.-% polyvinylpyrrolidone (PVP). g-h) Self-assembled particles


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from a 1:1 mixture of SiO2 and CaO/SiO2 colloidal particles without (g) and with addition of 10 µm PS microspheres (h,i).

The fabrication of colloidal particles with comparable sizes of around 200 nm, using a similar sol-gel chemistry enabled us to directly compare the toxicity of both types of particles (SiO2 vs CaO/SiO2). For the in vitro toxicity tests, we used the spray-dried aggregates from the ICP-OES investigations with an annealing temperature of 800°C. We performed a water soluble tetrazolium (WST) assay to determine the LC50 concentration, the concentration at which 50% of the cell viability is decreased. Addressing the cell viability after 48 hours of growth we observed that ST-2 cells showed increased viability in contact with SiO2/CaO between the concentrations of 0.1 and 10 µg/ml, while the LC

50

value for this material was approximately 343 µg/ml. For

SiO2, we no stimulation effect was observed and the LC50 value for this material was approximately 164 µg/ml. These results indicate that SiO2 was more cytotoxic compared to SiO2/CaO. The stimulation effect on cell viability of the novel SiO2/Ca supraparticles is in agreement with reports on conventional SiO2/CaO systems. For example, Dörfler et al. measured an increased cell proliferation and ALP activity in human osteoblasts in contact with bioactive glass flakes in the range of 0.1-200 µg/ml without any toxic effect.59 In another study, the cytocompatibility of micro- and nano-sized bioactive glass particles on human osteoblasts was compared. 60 The authors found that the cytocompatibility of all materials were the range of 0.1200 µg/ml. These values are comparable to the results of the WST tests of our supraparticles, indicating that the formulation of the primary particles into superstructures does not affect the cytocompatibility of the material.


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Besides the mentioned increased cell proliferation which has been reported in literature for bioactive glasses,59 the binary CaO/SiO2 particles seemingly show an increase the cytocompatibility as well. This finding is in agreement with a study in literature on sol-gel derived CaO containing silica networks on TST3 mouse cells, where the authors report an enhanced viability with the presence of CaO as well.61 However, to this point, the fundamental reason for the observed effect remains unclear. From our experiments, we note that besides the increase in calcium concentration in the cell medium, the amount of dissolved silica also changes significantly, which may further influence cell viability and proliferation.

Figure 5. In vitro toxicity measurement of silica and silica/calcium oxide supraparticles determined by the water soluble tetrazolium tests. The determined LC50 concentration is specified in the lower left part of the figure.

Finally, we tested the interactions of the produced CaO/SiO2-based porous particles with ST2 cells (Fig. 6a-c) in a proof-of-principle experiment to determine biocompatibility and cell-surface interactions. We compared ceramic particles consisting of a 50% mixture of silica and bioglass particles with particles of the same composition but featuring 10 µm macropores and


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investigated their interactions with ST2 cells. Cell-particle interactions were analyzed via confocal microscopy (Fig. 6a) and SEM imaging after critical point drying (Fig. 6b). We then compared the cellular adhesion of porous and non-porous particles by determining the number of adsorbed cells on approx. 100 particles. Similar to the experiments with pure silica supraparticles, we found an increase in cell-particle interactions for particles with 10 µm macropores as compared to the non-porous reference sample (Fig. 6c). The comparison between pure SiO2 particles to calcium-releasing CaO/SiO2 composite particles with similar microscale porosity (10 µm) showed a slight increase in cell adhesion for the bioactive particle system: SiO2-based porous particles with 10 µm pores were colonized with cells in 61 % of all tested particles while the 76 % of all CaO/SiO2 particles showed cellular adhesion, possibly as a result of the stimulating effect of the bioactive components.

Figure 6. Interaction of porous self-organized bioactive powder particles with ST2 cells. a,b) Confocal microscopy image (a) and scanning electron microscopy image (b) of cells colonializing porous particles assembled from 10 µm PS microspheres and a 1:1 mixture of SiO2


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and CaO/SiO2 colloidal particles. c) Quantification of ST2 cell colonization of self-assembled SiO2 – CaO/SiO2 supraparticles with and without additional microscale porosity.

Conclusions In conclusion, we demonstrated the fabrication of hierarchical supraparticles composed of silica and bioactive oxide particles with uniform and tunable pore sizes in the nano- and micrometer range. We fabricated these particles using self-organization of a binary mixture of small (nanometer scale) oxide particles with large (micrometer scale) polystyrene particles in the confinement of an emulsion droplet. The external confinement determines the final, spherical structure of the hybrid assembly, while the small particles form the matrix material and the large particles act as templating porogens to create micropore structures after calcination. We optimized the assembly conditions for both silica and CaO/SiO2 colloidal particles by controlling electrostatic and steric interactions, volume fractions and drying protocols. Using CaO/SiO2 colloids for the formation of self-assembled supraparticles, the release of calcium ions requires the addition of a steric stabilizer to overcome aggregation during the assembly process. In proofof-principle experiments, we showed that the produced porous supraparticles are biocompatible to high particle concentrations and that the interaction of cells with such structures strongly depends on the porosity at the microscale. Larger pores lead to an increased colonization with tissue cells. Substituting pure SiO2 for CaO/SiO2 colloidal particles showed an increase in biocompatibility, biostimulating behavior and an increase in cellular adhesion. Our results indicate a possible pathway to create porous materials for bone tissue engineering in a bottom up approach. We anticipate that the reported system may open up avenues for powder-based


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additive manufacturing processes of porous scaffolds for customized tissue engineering applications. ASSOCIATED CONTENT Supporting Information. Additional information on drying conditions of colloid-containing emulsion droplets, influence of silica content, incorporation of nanoscale porosity, effect of calcium addition to synthesize bioactive nanoparticles and the design of the microfluidic device is available as Supporting Information.

AUTHOR INFORMATION Corresponding Author * Corresponding authors N. Vogel and A. R. Boccaccini; emails: [email protected], [email protected]

Author Contributions N.V., S.V. and A.B. designed the experiments, S.E., C.F., A.G., J.W. and S.V. conducted the experiments. C.F., determined the ion release profile. A.G., C.F. and R.D. conducted the cellculture experiments. N.V. supervised and coordinated the project. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.


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ACKNOWLEDGMENT The authors acknowledge financial support by the Deutsche Forschungsgemeinschaft (DFG) within the Cluster of Excellence “Engineering of Advanced Materials”(project EXC 315) (Bridge Funding). N.V. and A.B. acknowledge support of Interdisciplinary Center for Functional Particle System (FPS) at FAU Erlangen. S.V. acknowledges support from the DFG Return Fellowship. The authors thank Paula Hoppe for support with the ICP measurements. REFERENCES (1) Li, F.; Josephson, D. P.; Stein, A., Colloidal Assembly: The Road from Particles to Colloidal Molecules and Crystals, Angew. Chem.-Int. Edit. 2011, 50, 360-388. (2) von Freymann, G.; Kitaev, V.; Lotschz, B. V.; Ozin, G. A., Bottom-up assembly of photonic crystals, Chem. Soc. Rev. 2013, 42, 2528-2554. (3) Kraus, T.; Brodoceanu, D.; Pazos-Perez, N.; Fery, A., Colloidal Surface Assemblies: Nanotechnology Meets Bioinspiration, Adv. Funct. Mater. 2013, 23, 4529-4541. (4) Vogel, N.; Weiss, C. K.; Landfester, K., From soft to hard: the generation of functional and complex colloidal monolayers for nanolithography, Soft Matter 2012, 8, 40444061. (5) Vogel, N.; Retsch, M.; Fustin, C.-A.; del Campo, A.; Jonas, U., Advances in Colloidal Assembly: The Design of Structure and Hierarchy in Two and Three Dimensions, Chem. Rev. 2015, 115, 6265-6311. (6) Jones, M. R.; Osberg, K. D.; Macfarlane, R. J.; Langille, M. R.; Mirkin, C. A., Templated Techniques for the Synthesis and Assembly of Plasmonic Nanostructures, Chem. Rev. 2011, 111, 3736-3827. (7) Stein, A.; Wilson, B. E.; Rudisill, S. G., Design and functionality of colloidalcrystal-templated materials-chemical applications of inverse opals, Chem. Soc. Rev. 2013, 42, 2763-2803. (8) Phillips, K. R.; England, G. T.; Sunny, S.; Shirman, E.; Shirman, T.; Vogel, N.; Aizenberg, J., A colloidoscope of colloid-based porous materials and their uses, Chem. Soc. Rev. 2016, 45, 281-322. (9) Dumanli, A. G.; Savin, T., Recent advances in the biomimicry of structural colours, Chem. Soc. Rev. 2016, 45, 6698-6724


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Bottom-Up Assembly of Silica and Bioactive Glass Supraparticles with

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