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Hierarchically Structured Materials by Anodic Coagulation Casting of Fibrinogenic Alumina Suspensions Rolf Zehbe and Claudia Fleck TU Berlin, Institute of Materials Science & Technologies, Strasse des 17. Juni 135, Berlin 10623, Germany Anodic coagulation casting of fibrinogenic ceramic suspensions is a novel processing technology, which is based on the electri- cally induced transformation of the water soluble fibrinogen into the insoluble fibrin. Contrary to the direct coagulation casting (DCC) technology, green formation does not depend on a pH-shift and as the fibrin coagulate forms on an anode, it can be combined with the electrophoretic deposition (EPD) technology.In this study, the conversion of fibrinogen into fibrin is activated via electron transfer processes at an electrode material and is combined with the green formation of alumina by embedding the ceramic particles in the protein matrix. The focus of this work was to establish a technology to shape thin hierarchically structured ceramic films and thick porous mate- rials with a distinct pore structure. Film thickness and porosity were controlled by the applied voltage and the processing-time. The range of the established green bodies included two-dimen- sional and simple three-dimensional shapes including multilay- ered deposition and fiber coatings. Overall the process of anodic coagulation casting can be reported to be successful for all established ceramic shapes except multilayers, where delam- ination was observed. The deposited alumina ceramics were characterized using light microscopy, scanning electron micros- copy (SEM), atomic force microscopy (AFM), and synchrotron micro computed tomography (lCT), while the coagulation mechanism was studied using high-performance liquid chroma- tography (HPLC). I. Introduction D IRECT coagulation casting (DCC) and electrophoretic deposition (EPD) have been described as promising technologies to green form ceramics. DCC has originally been described by Gauckler et al. 13 This process is based on the solidification of an aqueous ceramic suspension due to the urease-mediated decomposition of urea. This technique has been described to deliver complex, near net shape three- dimensional (3D) green bodies, with good homogeneity and sinter densities of 95%99%. A different consolidation process is used in EPD, which has first been studied in ceramic powder deposition applications in 1940 by Hamaker et al. 4 EPD uses a DC voltage to attract ceramic particles from a suspension to a charged electrode and therefore requires particles with a high electrophoretic mobility. 5 Hence, as long as the attractive electrophoretic and electrostatic forces dominate the repulsive van der waals forces, a particle network is formed. 6 The prin- ciple mechanisms of the EPD process have been described in detail by Sarkar et al. 7 and more recently by Boccaccini et al. 8 reviewing the state of the art concerning EPD of biomaterials. In this regard, we ourselves have implemented an EPD process together with coworkers establishing a hier- archically structured multi-layered zirconia material with promising crack deflection properties, 9 furthermore, Moritz et al. 10 investigated the formation of porous zirconia layers via EPD. Finally, Besra et al. 11 reported on the possibility to reduce porosity by applying a square-wave pulse potential. While several workgroups, including our own, are cur- rently reinvestigating the vast potential of the EPD-process, only few report methodologies for the co-deposition of ceramics and polymers or proteins. In this regard, Pishbin et al. reported the co-deposition of bioglass with chitosan. 12 In this study, we present a novel processing technology, combining ceramic-shaping mechanisms of DCC and EPD, which is, however, not based on the urea-urease-mediated consolidation process. Instead, it uses a DC voltage to induce a molecular peptide cleavage in the water soluble protein fibrino- gen. This initiates the subsequent polymerization and results in the formation of the insoluble fibrin species. This process is well known from secondary hemostasis and involves a series of enzymes that catalyze different biochemical processes, which result in the thrombin-mediated removal of two pairs of fibri- nopeptides in the central domain of the fibrinogen-molecule. Subsequently, the electrically charged fibrinogen fragments are polymerized and a network of fibrin fibers is established. The exact locations of the molecular binding sites were identified by crystal structure studies on fibrinogen and fibrin. 13 Blood clotting has also been observed on anodic surfaces. Sawyer et al. 14 first studied this contact activation of fibrino- gen in an electrochemical experiment using platinum elec- trodes and observed that blood clotting only occurred on the anode, indicating an electron transfer at the interface of the anode and the fibrinogen molecule. Subsequently, Baursch- midt et al. 15 showed that an electron transfer from a germa- nium electrode to the fibrinogen molecule resulted in the irreversible formation of fibrin. Rzany et al. 16 suggested that the relaxation energy resulting from the oxidation of the amino acids at the electrode is sufficiently high to split pep- tide bonds. It was further stated that the peptide separation by electron transfer is not specific and peptides are also released at sites where thrombin is not biochemically active in the natural enzymatic process. We have adapted these voltage-mediated processes in the past not only to deposit cells on the anode part of patterned microelectrodes 17 but also to establish a biocompatible sub- strate of fibrin before or during cell deposition. 1820 This cell deposition is very similar to the EPD process for ceramic green formation. Therefore, we present a combined processing for the struc- tured deposition of fibrinogenic alumina suspensions. Depending on the electrode shape and material, complex two- dimensional (2D) and simple 3D structures have been realized as well as multi-layers and fibers. Thickness and porosity of the deposited layers were controlled by the deposition time and the applied voltage and were in the range of some milli- meters, while the porosity was in the range of 25% and 65%. We refer to this process as anodic coagulation casting. Our main aim regarding the results reported here is the validation L. Gauckler—contributing editor Manuscript No. 32370. Received November 27, 2012; approved March 18, 2013. Author to whom correspondence should be addressed. e-mail: [email protected] 1745 J. Am. Ceram. Soc., 96 [6] 1745–1750 (2013) DOI: 10.1111/jace.12344 © 2013 The American Ceramic Society J ournal

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Hierarchically Structured Materials by Anodic Coagulation Castingof Fibrinogenic Alumina Suspensions

Rolf Zehbe† and Claudia Fleck

TU Berlin, Institute of Materials Science & Technologies, Strasse des 17. Juni 135, Berlin 10623, Germany

Anodic coagulation casting of fibrinogenic ceramic suspensions

is a novel processing technology, which is based on the electri-cally induced transformation of the water soluble fibrinogen

into the insoluble fibrin. Contrary to the direct coagulation

casting (DCC) technology, green formation does not depend on

a pH-shift and as the fibrin coagulate forms on an anode, itcan be combined with the electrophoretic deposition (EPD)

technology.In this study, the conversion of fibrinogen into fibrin

is activated via electron transfer processes at an electrode

material and is combined with the green formation of aluminaby embedding the ceramic particles in the protein matrix. The

focus of this work was to establish a technology to shape thin

hierarchically structured ceramic films and thick porous mate-

rials with a distinct pore structure. Film thickness and porositywere controlled by the applied voltage and the processing-time.

The range of the established green bodies included two-dimen-

sional and simple three-dimensional shapes including multilay-ered deposition and fiber coatings. Overall the process of

anodic coagulation casting can be reported to be successful for

all established ceramic shapes except multilayers, where delam-

ination was observed. The deposited alumina ceramics werecharacterized using light microscopy, scanning electron micros-

copy (SEM), atomic force microscopy (AFM), and synchrotron

micro computed tomography (lCT), while the coagulation

mechanism was studied using high-performance liquid chroma-tography (HPLC).

I. Introduction

DIRECT coagulation casting (DCC) and electrophoreticdeposition (EPD) have been described as promising

technologies to green form ceramics. DCC has originallybeen described by Gauckler et al.1–3 This process is based onthe solidification of an aqueous ceramic suspension due tothe urease-mediated decomposition of urea. This techniquehas been described to deliver complex, near net shape three-dimensional (3D) green bodies, with good homogeneity andsinter densities of 95%–99%.

A different consolidation process is used in EPD, whichhas first been studied in ceramic powder depositionapplications in 1940 by Hamaker et al.4 EPD uses a DCvoltage to attract ceramic particles from a suspension to acharged electrode and therefore requires particles with a highelectrophoretic mobility.5 Hence, as long as the attractiveelectrophoretic and electrostatic forces dominate the repulsivevan der waals forces, a particle network is formed.6 The prin-ciple mechanisms of the EPD process have been described indetail by Sarkar et al.7 and more recently by Boccacciniet al.8 reviewing the state of the art concerning EPD ofbiomaterials. In this regard, we ourselves have implemented

an EPD process together with coworkers establishing a hier-archically structured multi-layered zirconia material withpromising crack deflection properties,9 furthermore, Moritzet al.10 investigated the formation of porous zirconia layersvia EPD. Finally, Besra et al.11 reported on the possibility toreduce porosity by applying a square-wave pulse potential.

While several workgroups, including our own, are cur-rently reinvestigating the vast potential of the EPD-process,only few report methodologies for the co-deposition ofceramics and polymers or proteins. In this regard, Pishbinet al. reported the co-deposition of bioglass with chitosan.12

In this study, we present a novel processing technology,combining ceramic-shaping mechanisms of DCC and EPD,which is, however, not based on the urea-urease-mediatedconsolidation process. Instead, it uses a DC voltage to induce amolecular peptide cleavage in the water soluble protein fibrino-gen. This initiates the subsequent polymerization and results inthe formation of the insoluble fibrin species. This process iswell known from secondary hemostasis and involves a series ofenzymes that catalyze different biochemical processes, whichresult in the thrombin-mediated removal of two pairs of fibri-nopeptides in the central domain of the fibrinogen-molecule.Subsequently, the electrically charged fibrinogen fragments arepolymerized and a network of fibrin fibers is established. Theexact locations of the molecular binding sites were identified bycrystal structure studies on fibrinogen and fibrin.13

Blood clotting has also been observed on anodic surfaces.Sawyer et al.14 first studied this contact activation of fibrino-gen in an electrochemical experiment using platinum elec-trodes and observed that blood clotting only occurred on theanode, indicating an electron transfer at the interface of theanode and the fibrinogen molecule. Subsequently, Baursch-midt et al.15 showed that an electron transfer from a germa-nium electrode to the fibrinogen molecule resulted in theirreversible formation of fibrin. Rzany et al.16 suggested thatthe relaxation energy resulting from the oxidation of theamino acids at the electrode is sufficiently high to split pep-tide bonds. It was further stated that the peptide separationby electron transfer is not specific and peptides are alsoreleased at sites where thrombin is not biochemically activein the natural enzymatic process.

We have adapted these voltage-mediated processes in thepast not only to deposit cells on the anode part of patternedmicroelectrodes17 but also to establish a biocompatible sub-strate of fibrin before or during cell deposition.18–20 This celldeposition is very similar to the EPD process for ceramicgreen formation.

Therefore, we present a combined processing for the struc-tured deposition of fibrinogenic alumina suspensions.Depending on the electrode shape and material, complex two-dimensional (2D) and simple 3D structures have been realizedas well as multi-layers and fibers. Thickness and porosity ofthe deposited layers were controlled by the deposition timeand the applied voltage and were in the range of some milli-meters, while the porosity was in the range of 25% and 65%.

We refer to this process as anodic coagulation casting. Ourmain aim regarding the results reported here is the validation

L. Gauckler—contributing editor

Manuscript No. 32370. Received November 27, 2012; approved March 18, 2013.†Author to whom correspondence should be addressed. e-mail: [email protected]

1745

J. Am. Ceram. Soc., 96 [6] 1745–1750 (2013)

DOI: 10.1111/jace.12344

© 2013 The American Ceramic Society

Journal

of this approach and of its feasibility to achieve different cera-mic shapes. Our investigations included high-performanceliquid chromatography (HPLC) to investigate the proteintransformation mechanism. Light microscopy, scanning elec-tron microscopy (SEM), and atomic force microscopy (AFM)were used in conjunction with synchrotron micro computedtomography (lCT) investigations to characterize the depos-ited ceramics, the porosity, and the deposition kinetics.

II. Experimental Procedure

(1) Electrode DesignAnodic coagulation casting requires sufficiently inert elec-trodes to avoid electrochemical corrosion of the electrodematerial. Precious metals like gold or platinum or the simi-larly nonreactive carbon are well suited electrode materials.Gold and carbon electrodes were utilized either as thin coat-ings (gold) on a ceramic substrate, as machined bulk material(graphite) to achieve 3D shapes or as carbon fibers toachieve continuous fiber coatings.

Figure. 1a displays the processing methodology for theestablishment of microstructured 2D shapes (1) and multilay-ers (2) on alumina substrates. The microstructured electrodeswere realized by inverse-inkjet-printing according to themethod described previously19 and were subsequently goldsputtered on 1.0 mm thick alumina foils with a resultingeffective electrode area of approximately 4 cm². The inverseelectrode structure was printed with black ink on the aluminafoil using a custom-adapted HP Deskjet 3520-inkjet printer.Afterward, samples were gold sputter coated in argon-plasmafor 4 minutes with a sputter current of ~25 mA using anEmitech K550 (Röntgenanalytik Messtechnik, Taunusstein,Germany) sputter coater. The inverse-printed and gold-coated alumina foil was then ultrasonicated in deinonizedwater with the addition of 10% isopropyl alcohol to removethe printing ink. The on-top gold layer remained, resulting inthe positive-electrode-structure. These electrodes were used asanodes with bulk gold cathodes.

Multilayered deposition was achieved by initial sputtercoating as described above on one side of the alumina sub-strate followed by anodic coagulation casting, air drying ofthe deposited layer, and repeated sputter coating and anodiccoagulation casting.

Figure. 1b displays a setup for the shaping of 3D struc-tures based on a conductive lost mold using a bulk graphite

electrode, which is burned out after the anodic coagulationcasting. The conductive lost mold was used as anode andwas placed inside a carbon vessel (cathode) with the fibrino-genic alumina suspension inside.

Figure. 1c shows the setup for the continuous coating ofcarbon fibers. The anodic carbon fiber was pulled through acarbon vessel (cathode) containing the fibrinogenic aluminasuspension at a speed of ~1.0 cm/min.

(2) Characterization of Fibrinogen TransformationThe anodic coagulation of the water-soluble fibrinogen andits transformation into its insoluble form was analyzedusing HPLC according to Kehl et al. and Ebert et al.18,21

comparing the electrically mediated coagulation with the throm-bin-mediated coagulation of a 2 wt% fibrinogen, 0.9 wt%NaCl-solution. The electrically coagulated sample was pre-pared from this solution under a voltage of 3.5 V on a planargraphite electrode for 30 minutes, the thrombin-coagulatedsample was prepared by adding 100 ll thrombin (8.8 units/ml) to 500 ll of the fibrinogen-solution.

For HPLC measurements, the resulting coagulate was dis-solved in 0.1% trifluoroacetic acid (ratio 1:1) for 5 minutesusing an ultrasonic bath followed by ultrafiltration.

(3) Anodic Coagulation CastingFibrinogenic alumina suspensions were prepared by adding1.0 wt% dispersant (Dispex A-40, BASF Switzerland AG,Basel, Switzerland), 1.0 wt% fibrinogen (fraction I frombovine plasma, MP Biomedicals, Eschwege, Germany), and60 wt% Al2O3 (AKP 50, Sumitomo Chemical Co., Ltd.,Osaka, Japan) to deionized water under constant stirring.Suspensions were kept stirring for 4 h at room temperaturefollowed by several short ultrasonication steps of 5 s. each.

Fibrinogenic alumina suspensions were solidified in con-tact with the electrodes applying voltages in the range of1.5 V to 6.0 V for periods of up to 30 min (see table I for adetailed presentation of the processing parameters).

In general, solutions were processed at room temperatureand were stirred with a magnetic stir bar during anodic coag-ulation casting. Afterward, the samples were rinsed in etha-nol, water and dispersant was removed, followed by air

(a)

(b) (c)

Fig. 1. Schematic representation of the different processingmethodologies for anodic coagulation casting: (a) microstructured (i)and multilayered (ii) deposition on thin gold on alumina samples; (b)three-dimensional shaping on conductive lost mold graphiteelectrodes; (c) continuous fiber coating on conductive carbon fibers.

Table I. Processing Parameters

Dispex

(wt%)

Fibrinogen

(wt%)

Voltage

(V)

Processing

time (min)

HPLC – 2 3.5 30Microstructureddeposition(Einstein)

1 1 2.03.0

30

Microstructureddeposition (Japan)

1 1 2.0 30

Microstructureddeposition(TU Berlin)

1 1 2.0 30

Continuousfiber coating

1 1 2.0 1.0 cm/min

Multilayeredceramic deposition

1 1 2.0 30

Conductivelost mold(three-dimensionalshaping)

1 1 3.5 30

Kinetics anddeposition analysis

1 1 1.52.02.53.03.56.0

1015202530

1746 Journal of the American Ceramic Society—Zehbe and Fleck Vol. 96, No. 6

drying under laminar air flow. For multilayered depositionaccording to Fig. 1a-(i), the samples were repeatedly sputtercoated after each step of anodic coagulation casting.

The deposition kinetics of the formed fibrin-ceramic layerswere studied as a function of voltage (with a constantprocessing time of 30 min) and as a function of processingtime (with constant voltage of 6 V) in an experimental setupwith a square-shaped gold electrode and an effectiveelectrode area of 1.0 cm². The layer thicknesses were mea-sured using a digital caliper and a Leica DM4000M (LeicaMicrosystems GmbH, Wetzlar, Germany) light microscope.

Finally, samples were heated to 1200°C at 1 K/min toallow for complete burnout of organic compounds includingfibrinogen. Furthermore, this step was used to burn out thecarbon electrodes. After this calcination step, samples wereheated up to the sintering temperature of 1650°C for 2 h at aheating rate of 5 K/min, which effectively removed persistentcarbon or the thin (approximately 50 nm) sputtered goldlayer, which was used as electrode for several samples.

(4) Scanning Electron MicroscopyScanning electron microscopy was used for structural charac-terization of the deposited ceramic layer. Specimens weremounted on aluminium sample holders (∅: 12 mm) with self-adhesive carbon pads followed by carbon sputter coating inargon atmosphere. SEM images were recorded as Tiff-filesusing a Philips XL 20 (FEI Company, Eindhoven, Nether-lands) scanning electron microscope at accelerating voltagesbetween 10 kV and 20 kV.

(5) Atomic Force MicroscopyAtomic force microscopy analysis was carried out using aNanosurf easyScan 2 AFM (Nanosurf AG, Liestal, Switzer-land) system with non contact measurement mode cantilevers(NCLR) similar to the methodology described previously.22,23

Briefly, analysis was performed on an inversely printed andmicrostructured sample prepared as described in section 2.1.

The region of interest was a square-shaped area of20 lm 9 20 lm with a moderate topography in the range of4 lm. Atomic force microscopy analysis was done on theraw data showing the 3D-topography and on the deriveddata (calculating the difference between two successive datapoints), which gives a better visual representation in 2D.

The scope of these measurements was to obtain informa-tion on the topography and growth of the deposited ceramicon a micrometer scale.

(6) Synchrotron lCTTwo sintered ceramic structures made by the conducting lostmold technique were characterized via synchrotron l-CTimaging at the BAMline (BAM, Federal Institute for Materi-als Research and Testing) at BESSY Berlin. The overall exper-imental setup has been described elsewhere.24,25 The resolutionof the lCT-data and the corresponding voxels was 3.6 lm andwas determined geometrically in projection by displacing a testobject of known geometry. Samples were rotated in steps of180°/1200 and were exposed to the beam for 0.4 s–0.6 s perrecorded projection image. The beam energy was set to20 keV. Reconstructed data processing was performed on anApple iMac Quad-Core i7 (2.93 GHz, 12 Gbyte RAM AppleInc., Cupertino, CA) using ImageJ (64 bit), and VGStudio-MAX (Volumegraphics Heidelberg, Germany).

III. Results

(1) Characterization of the Fibrinogen TransformationResults from HPLC analysis are displayed in Fig. 2, showingthe comparison between thrombin coagulated and electricallycoagulated fibrinogen. Confirming previous results,19 the

most relevant differences have been found for retention timesof ~20 min, when the thrombin-coagulated sample featurestwo peaks that indicate the cleavage of the fibrinopeptides Aand B. In contrast, the electrically coagulated sample doesnot show these peaks —indicating a crosslinking mechanism,which does not involve this specific cleavage.

From our previous research,19 attenuated total reflectanceFourier transform infrared spectroscopy (ATR/FTIR) con-firmed the data from the literature26–28 indicating that theamid I-band between 1700 cm�1 and 1550 cm�1 is crucial forthe analysis of the transformation from fibrinogen into fibrinand was evaluated using the method developed by Byleret al.29 The spectra of the electrically coagulated fibrinogenand the non coagulated fibrinogen are almost identical, exceptfor an additional peak between 1550 cm�1 and 1600 cm�1,which could be identified through peak deconvolution andwhich is missing in the electrically coagulated sample.

(2) Anodic Coagulation CastingThe gold-based microstructured electrodes on the alumina-foil substrates needed not to be de-molded as the sputtergold was removed during subsequent sintering via evapora-tion/diffusion processes.

The deposition kinetics of the fibrin-ceramic layer clearlyshow an increase with increasing voltage as can be seen inFig. 3a. while an asymptotic behavior can be observed forlonger processing times for a suspension containing 1.0%fibrinogen being coagulated at a voltage of 6 V for up to30 min (Fig. 3b).

Furthermore, the applied voltage strongly influences thedeposition rate and the layer density. It is limited by the min-imal voltage necessary for the electron transfer from theanode to the fibrinogen molecule that we determined to be~0.8 V. This value is in good agreement with the resultsobtained in other investigations.16 Raising the voltage abovethe anode specific electrolysis voltage for water resulted in amore porous green body due to gas bubble inclusions.

The pore morphology has been investigated quantitativelyusing synchrotron lCT data (Figs. 3c–e) from a sintered,rectangular “conductive lost mould” sample. Besides somesmall bubbles trapped at the interface to the electrode(Figs. 3d and e), a hierarchical pore-channel structure hasbeen formed extending up to the surface of the depositedceramic. This correlation is represented as an increasingporosity as function of the distance to the electrode surfacein Fig. 3c. Furthermore, the cross sections of the larger andelongated pores become more non circular with increasingdistance to the electrode. The porosity increases from ~25%

Fig. 2. High-performance liquid chromatography HPLC analysisand comparison of thrombin coagulated fibrinogen and electricallycoagulated fibrinogen showing the absence of specific fibrinopeptidecleavage in the electrically coagulated sample.

June 2013 Anodic Coagulation Casting 1747

at the interface side to the electrode to ~65% for the maxi-mum distance from the electrode (650 lm). The mean poros-ity is 57.6% with a non open, channel-like pore structure,similar to the one described by Moritz et al.10 for zirconia.

In summary, deposition of thick layers in the range of sev-eral millimeters generally requires higher voltages and longerprocessing times in the range of several minutes. However,higher voltages favor electrolysis and entrapment of largerelectrolysis bubbles in the formed ceramic.

Therefore, in water-based suspensions, thin layers withnear net-shaped green bodies are advantageously obtainedusing low voltages applied over limited processing times,while thick porous structures can be obtained with reduced

near net shape using high voltages and long processing times.Therefore several different processing strategies for ceramicshaping were developed as follows.

(A) Microstructured Deposition: To achieve micro-structured deposition, an adaptation of a previouslydeveloped inverse inkjet printing technology was used result-ing in conducting gold on ceramic foil electrodes. The nearnet shape ability is demonstrated in Fig. 4a for a depositionvoltage of 2 V applied over 30 min, displaying the depositedceramic in shape of Albert Einstein. Only areas that areanodically charged are covered with ceramic particles as canbe seen for the jaw part in Fig. 4a, where one particularregion was not contacted and therefore was not covered. For

(a) (b)

(d)(c)

(e)

Fig. 3. Influence of voltage (a) and processing time (b) on the deposition thickness. Quantitative volumetric analysis (c) of one sample (d)regarding the circularity and the porosity with varying sample thickness (distance from substrate). A representative cross-sectional view is shownin (e).

(a)

(b) (d) (e)

(c)

Fig. 4. Microstructured deposition on inversely inkjet printed samples. Exemplarily, the shape of Albert Einstein was used for a structuredelectrode with a processing time of 30 min at a voltage of 2 V (a) and 3 V (b). The impact of deposition voltage on the porosity of the achievedceramic layer is clearly visible. Atomic force microscopy AFM analysis of a ceramic sample showing the terrace-like structure of the layertopography (after sintering): (c) three-dimensional topographic representation, (d) derivative image of the full area and (e) higher magnifiedimage showing the terrace-like structure.

1748 Journal of the American Ceramic Society—Zehbe and Fleck Vol. 96, No. 6

a higher deposition voltage (Fig. 4b, 3 V), the effect ofincreasing porosity and non net-shape layer formation aredisplayed. Here, the facial details are barely visible, and thecoagulated ceramic features larger pores. Below 1.0 V, nodeposition has been observed (not displayed).

To investigate the layer formation on a micrometer scale,further investigations were performed on the deposited shapeof Albert Einstein. SEM observations (data not shown) of thelayer after deposition, but before sintering shows a particulatestructure with no hint at the time dependent deposition. Inves-tigation of the topography after sintering using AFM (Fig. 4c–e) on the other hand clearly shows a terrace-like structure,which obviously is a result of the deposition kinetics and thesubsequent buildup of the formed ceramic layer.

(B) Multilayered Ceramic Deposition: By anodiccoagulation casting (2 V, 30 min) on a gold sputter-coatedalumina substrate, subsequent drying of the deposited layerand repeating the process of gold sputter coating and anodiccoagulation casting (2 V, 30 min), a two-layer alumina cera-mic has been prepared.

The cross-sectional view of this layered ceramic after sin-tering (Fig. 5) shows significant delamination of parts of thelayers (Fig. 5a and b). The first layer has large pores extend-ing up to 100 lm into the material, which are due to trappedelectrolysis gases. Furthermore, the first 10–50 lm show asignificant microporosity resulting in a weak interface (seeFig. 6a). The next deposited layer appears delaminated dueto necessary resputtering as can be seen in Fig. 5b.

(C) Continuous Fiber Coating: Using conductive car-bon fibers as electrodes for the continuous deposition ofceramics, both wound and linear hollow channel structureshave been established. As displayed in Fig. 6a–c, the formedceramic layer accurately follows single-fiber filaments, whichwere completely burnt out during sintering in oxidizingatmosphere.

Both SEM (Fig. 6a) and synchrotron l-CT imaging(Fig. 6b and c) show the near net shape-forming abilitydown to single-fiber filaments.

(D) Conductive Lost Mold: Using bulk graphiteanodes as substrates for the anodic coagulation castingenabled a lost mold equivalent technique in which the anodeis used to consolidate the ceramic and the protein and isafterwards removed via burnout.

Two graphite electrodes featuring a rectangular shape(Fig. 6d and e) and a round shape (Fig. 6f) were used at a volt-age of 3.5 V (30 minutes processing time) resulting in an alu-mina-layer with approximately 750 lm thickness. Both sampleswere imaged via synchrotron l-CT with an overall experimentalsetup according to.30 Synchrotron l-CT imaging allowed a

complete volumetric representation of the pore structure ofthese specimens. Both samples feature a radial pore structurewith a dense interface at the contact zone to the electrode fol-lowed by larger bubbles due to inclusions of trapped electrolysisgases. They have a high amount of radial pores which is best vis-ible for the rectangular sample (fig. 6d and e).

IV. Discussion

We applied for the first time an electrically mediated proteinconsolidation mechanism to ceramic processing by anodiccoagulation of fibrinogenic alumina suspensions. By variationof the applied voltage and the processing-time, the shape, thethickness, and the porosity of the ceramic material were easilyadjustable. In this regard, ceramic shaping has been achievedas microstructured coating on flat ceramic substrates, as mul-tilayer, as simple 3D hollow objects (bar and rod) and as con-tinuous fiber coatings. Although the process still needsoptimization, all processing methods used were successful andshow promising results. Only for the multilayer, the result wasnot optimal yet because of layer delamination. We assumethat the thin sputter-coated gold layer prevented fusion of thetwo deposited layers due to a too slow evaporation of thisgold layer and the consequent gas formation during sintering.Regarding layered deposition, we have achieved much betterresults in a recent study with zirconia were layer delaminationdid not occur and we were able to achieve mm-sized speci-mens with a high number of subsequent layers.31

Quantitative results have been obtained linking the kineticsof the ceramic deposition to the processing time and appliedvoltage, while a more detailed analysis on the deposited mate-rial morphology has been obtained via Synchrotron l-CT elab-orating on the pore structure of porously deposited 3D hollowshapes. Atomic force microscopy analysis was interpreted in

(a)

(b)

Fig. 5. Multilayered deposition (two layers) in scanning electronmicroscope SEM imaging showing larger bubble inclusions near theinterface to the electrode and delamination (a). Delamination is alsoclearly visible at the interface to the second layer (b).

(a)

(d)

(e)

(f)

(b)

(c)

Fig. 6. Scanning electron microscopic (a) and tomographicrepresentations (b, c) of a continuously deposited carbon fiber afterburnout. Deposition on single carbon fiber filaments is well visible inSEM, while a thin channel like structure can be seen in synchrotron-lCT. (d–f) Samples achieved via conductive lost mold technique.Rectangular-shaped material (d, e) and round-shaped material (f)with pores perpendicular to the surface are shown.

June 2013 Anodic Coagulation Casting 1749

regard of layer formation on a submicrometer scale, indicatinga terrace-like formation parallel to the surface of the electrode.

The fibrinogen-fibrin-transformation itself has been stud-ied using HPLC and ATR/FTIR and was found to bebased on unspecific peptide cleavage followed by fibrinogenpolymerization. Especially the unspecific peptide cleavagepresents an important difference compared to the blood-coagulation cascade in biological systems. These new find-ings support previous results that hinted to differences inthe chemical bonds built up in fibrinogen on the one handand the electrically coagulated fibrinogen on the otherhand.19 Other investigators reported a minimal voltage of0.8 V16 necessary for the transformation of fibrinogen intofibrin at a SnO2-electrode versus Ag/AgCl. Below this volt-age, no protein-ceramic layer was formed. In this study wewere able to trigger anodic coagulation at a minimal voltageof 1.0 V on gold electrodes. According to the model ofRzany, a direct contact between the electrode and the pro-tein is required for the electron transfer mechanism, result-ing in the formation of a thin, fibrin layer with thethickness restricted to the size range of single molecules.However, in our experiments the deposited layers weremuch thicker. One possible explanation could be the con-ductivity of the ion load of the electrolyte.

Porosity due to electrolysis and gas inclusions had beenobserved on some coagulated samples, which could bedesired for special applications. In this regard, porosity is aprerequisite for tissue ingrowths in biomaterials applicationsbut also in separation processes and guided matter transport.

During the anodic coagulation casting process, the proteinis finally removed via burnout during sintering; therefore, amost promising future development is envisioned in a meth-odology to achieve in situ formation of the ceramic togetherwith the protein in a functional biologically active material.In this regard, calcium phosphates would be possible candi-dates for anodic coagulation casting as they can be depositedvia electrochemical means.

V. Conclusion

The proposed anodic coagulation casting combining the for-mation of a protein structure and a ceramic-shaping technol-ogy offers a technology to shape both 2D and 3D ceramics.Here, we have achieved fine-structured 2D thin layers, por-ous bulk 3D materials, and ceramic fibers.

Currently, a drawback is regarded in the final burnout ofthe protein (which in this study served as ceramic binder ingreen formation). Nevertheless, future work is directed intocombining fibrin synthesis with biomimetic in situ calciumphosphate formation delivering a candidate for hierarchicalstructuring of a porous biomaterial.

Acknowledgment

This work is dedicated to my daughter Hanna Elisabeth Margret Zehbe (bornSeptember 20th 2012) and to Helmut Schubert who suddenly and unexpectedlypassed away on March 14th 2012. Furthermore, the authors thank DanielR€osch for support in the characterization of the fibrinogen-fibrin transforma-tion and Astrid Haibel for support in tomographic data reconstruction. Wegratefully acknowledge the financial support of the DFG (Deutsche Fors-chungsgemeinschaft) for our project within SPP1420 (http://spp1420.mpikg.mpg.de/projects/hierarchy-of-microstructural-features-as-the-origin-of-fracture-resistance-in-dentine-and-ceramic-composites).

References

1T. Graule, W. Si, F. Baader, and L. Gauckler, “Direct Coagulation Casting(DCC): Fundamentals of a new Forming Process for Ceramics,” Ceram.Trans., 51 [45] 7–61 (1994).

2W. Si, T. J. Graule, F. H. Baader, and L. J. Gauckler, “Direct CoagulationCasting of Silicon Carbide Components,” J. Am. Ceram. Soc., 82 [5] 1129–36(1999).

3E. Tervoort, T. A. Tervoort, and L. J. Gauckler, “Chemical Aspects ofDirect Coagulation Casting of Alumina Suspensions,” J. Am. Ceram. Soc., 87[8] 1530–5 (2008).

4H. C. Hamaker, “Formation of a Deposit by Electrophoresis,” Trans.Faraday Soc., 35 [27] 9–87 (1940).

5R. Moreno, “The Role of Slip Additives in Tape-Casting Technology. I:Solvents and Dispersants,” Am. Ceram. Soc. Bull., 71 [10] 1521–31 (1992).

6R. Moreno and B. Ferrari, “Advanced Ceramics via EPD of Aqueous Slur-ries,” Am. Ceram. Soc. Bull., 79 [1] 44–8 (2000).

7P. Sarkar and P. S. Nicholson, “Electrophoretic Deposition (EPD): Mecha-nisms, Kinetics, and Application to Ceramics,” J. Am. Ceram. Soc., 79 [8]1987–2002 (1996).

8A. R. Boccaccini, S. Keim, R. Ma, Y. Li, and I. Zhitomirsky, “Electropho-retic Deposition of Biomaterials,” J. R. Soc. Interface, 7 [Suppl. 5] S581–613(2010).

9C. Mochales, S. Frank, R. Zehbe, T. Traykova, C. Fleckenstein, A. Maer-ten, C. Fleck, and W. D. M€uller, “Tetragonal and Cubic Zirconia Multilay-ered Ceramic Constructs Created by EPD,” J. Phys. Chem. B, 117 [6], 1694–701 (2013).

10K. Moritz and T. Moritz, “ZrO2 Ceramics With Aligned Pore Structureby EPD and Their Characterisation by X-ray Computed Tomography,”J. Eur. Ceram. Soc., 30 [5] 1203–9 (2010).

11L. Besra, T. Uchikoshi, T. S. Suzuki, and Y. Sakka, “Bubble-Free Aque-ous Electrophoretic Deposition (EPD) by Pulse-Potential Application,” J. Am.Ceram. Soc., 91 [10] 3154–9 (2008).

12F. Pishbin, A. Simchi, M. P. Ryan, and A. R. Boccaccini, “ElectrophoreticDeposition of Chitosan/45S5 Bioglass� Composite Coatings for OrthopaedicApplications,” Surf. Coat. Technol., 205 [23–24] 5260–8 (2011).

13R. F. Doolittle, Z. Yang, and I. Mochalkin, “Crystal Structure Studies onFibrinogen and Fibrin,” Ann. N. Y. Acad. Sci., 936 [1] 31–43 (2001).

14P. N. Sawyer, W. H. Brattain, and P. J. Boddy, “Electrochemical criteriain the choice of materials used in vascular prosthesis”; pp. 337–48 in Biophysi-cal Mechanism in Vascular Hemostasis and Intravascular Thrombosis, Edited byP. N. Sawyer (Editor). Appleton-Century-Crofts, New York, 1965.

15P. Baurschmidt and M. Schaldach, “The Electrochemical Aspects of theThrombogenicity of a Material,” J. Bioeng., 1 [1] 261–78 (1977).

16A. Rzany and M. Schaldach, “Physical Properties of AntithrombogenicMaterials-An Electronic Model of Contact Activation,” Prog. Biomed. Res., 1[4] 59–70 (1999).

17F. Schmidt, M. Kuhbacher, U. Gross, A. Kyriakopoulos, H. Schubert,and R. Zehbe, “From 2D Slices to 3D Volumes: Image Based Reconstructionand Morphological Characterization of Hippocampal Cells on Charged andUncharged Surfaces Using FIB/SEM Serial Sectioning,” Ultramicroscopy, 111[4] 259–66 (2011).

18M. Kehl, F. Lottspeich, and A. Henschen, “Analysis of Human Fibrino-peptides by High-Performance Liquid Chromatography,” Hoppe-Seyler’s Z.Physiol. Chem., 362 [12] 1661 (1981).

19R. Zehbe, U. Gross, C. Knabe, R. J. Radlanski, and H. Schubert, “Ano-dic Cell-Protein Deposition on Inverse Inkjet Printed Micro Structured GoldSurfaces,” Biosens. Bioelectron., 22 [7] 1493–500 (2007).

20R. Zehbe, U. Gross, and H. Schubert, “Inverse Inkjet Printed Gold MicroElectrodes for the Structured Deposition of Epithelial Cells and Fibrin,”Biomol. Eng., 24 [5] 537–42 (2007).

21R. F. Ebert and W. R. Bell, “Assay of Human Fibrinopeptides by High-Performance Liquid Chromatography,” Anal. Biochem., 148 [1] 70–8 (1985).

22B. Watzer, R. Zehbe, S. Halstenberg, C. J. Kirkpatrick, and C. Broch-hausen, “Stability of Prostaglandin E 2 (PGE 2) Embedded in Poly-d, l-Lac-tide-co-Glycolide Microspheres: A pre-Conditioning Approach for TissueEngineering Applications,” J. Mater. Sci. Mater. Med., 20 [6] 1357–65(2009).

23R. Zehbe, B. Watzer, R. Grupp, S. Halstenberg, H. Riesemeier, C. J.Kirkpatrick, H. Schubert, and C. Brochhausen, “Tomographic and Topo-graphic Investigation of Poly-D, L-Lactide-Co-Glycolide Microspheres LoadedWith Prostaglandine E2 for Extended Drug Release Applications,” Adv.Mater. Res., 89 [68] 7–91 (2010).

24R. Zehbe, A. Haibel, H. Riesemeier, U. Gross, C. J. Kirkpatrick, H.Schubert, and C. Brochhausen, “Going Beyond Histology. SynchrotronMicro-Computed Tomography as a Methodology for Biological Tissue Char-acterization: From Tissue Morphology to Individual Cells,” J. R. Soc. Inter-face, 7 [42] 49–59 (2010).

25R. Zehbe, H. Riesemeier, C. J. Kirkpatrick, and C. Brochhausen, “Imag-ing of Articular Cartilage-Data Matching Using X-ray Tomography, SEM,FIB Slicing and Conventional Histology,” Micron, 43 [10] 1060–7, (2012).

26L. Boulkanz, N. Balcar, and M. H. Baron, “FT-IR Analysis for StructuralCharacterization of Albumin Adsorbed on the Reversed-Phase Support RP-C6,” Appl. Spectrosc., 49 [12] 1737–46 (1995).

27C. E. Giacomelli, M. G. E. G. Bremer, and W. Norde, “ATR-FTIR Studyof IgG Adsorbed on Different Silica Surfaces,” J. Colloid Interface Sci., 220[1] 13–23 (1999).

28S. Servagent-Noinville, M. Revault, H. Quiquampoix, and M. H. Baron,“Conformational Changes of Bovine Serum Albumin Induced by Adsorptionon Different Clay Surfaces: FTIR Analysis,” J. Colloid Interface Sci., 221 [2]273–83 (2000).

29D. M. Byler and H. Susi, “Examination of the Secondary Structure ofProteins by Deconvolved FTIR Spectra,” Biopolymers, 25 [3] 469–87 (2004).

30R. Zehbe, A. Haibel, F. Schmidt, H. Riesemeier, C. J. Kirkpatrick, H.Schubert, and C. Brochhausen, “High Resolution X-Ray Tomography-3DImaging for Tissue Engineering Applications”; pp. 337–58 in Tissue Engineer-ing, Chapter 17, Edited by D. Eberli. InTech North America, Manhattan, NY,2010.

31R. Zehbe and C. Fleck, “Hierarchically Structured Biomaterials for TissueEngineering,” J. Tissue Sci. Eng., 3 [3] 1–3 (2012). h

1750 Journal of the American Ceramic Society—Zehbe and Fleck Vol. 96, No. 6