carbon nanotube coatings on bioglass-based tissue engineering scaffolds

DOI: 10.1002/adfm.200600887

Carbon Nanotube Coatings on Bioglass-Based Tissue EngineeringScaffolds

By Aldo R. Boccaccini,* Florencia Chicatun, Johann Cho, Oana Bretcanu, Judith A. Roether,Saša Novak, and Qizhi Chen*

1. Introduction

Tissue engineering is a scientific field which involves the ap-plication of the principles and methods of engineering and lifesciences towards the fundamental understanding of structure-function relationships in normal and pathological mammaliantissues and the development of biological substitutes that re-store, maintain or improve tissue function.[1]

In order to define the anatomical shape of tissues and organs,a three dimensional porous matrix, known as scaffold, isneeded.[2,3] In tissue engineering strategies involving the use ofscaffolds, donor cells and growth factors are seeded or incorpo-rated into the scaffold, forming a construct with is then im-planted to induce and direct the growth of new tissue. The aimis for the cells to attach to the scaffold, then grow and replicate,differentiate and organize into normal healthy tissue as thescaffold degrades.

The state of the art in the area of tissue engineering scaffoldsis based on biodegradable matrices that try to mimic the struc-tural and functional properties of the native extra cellular ma-trix (ECM).[4] This approach is critical to promote cell-matrixinteraction and cell growth and differentiation in 3D biocom-patible structures. The biochemical surface composition as well

as surface texture, microporosity, pore size, density, connectiv-ity and 3D configuration all affect strongly the generation ofnew tissue. In addition, because the interactions between cellsand ECM involve nanoscale cues, texture and roughness on thenanoscale can directly influence cell attachment and func-tion.[5] Indeed nanotopography has been shown to positivelyaffect cell attachment and proliferation in comparison withconventional (micrometer-size) topographical features on bio-material surfaces.[6,7] There is therefore a need to develop ma-terials processing technologies to construct 3D scaffolds withcontrolled topography of the pore wall surfaces.

Chen et al.[2] recently reported the production of highly po-rous bioactive and biodegradable 45S5 Bioglass®-based foam-like scaffolds for bone tissue engineering. The macroporousstructure of the foams was shown to be very similar to that ofspongy bone with > 90 % open porosity. It was found thatwhen porosities were near 91 %, the compressive and bendingstrength values of the foams were in the range 0.3–0.4 and0.4–0.5 MPa, respectively. Moreover, the compressive strengthvalues of the foams were found to be in the range of values ofspongy bone[8] However, the Bioglass®-based foams fabricatedin the previous work[2] were not optimized in terms of their sur-face properties to enhance cell adhesion and growth. There isconsiderable evidence in the literature on the ability of Bio-glass® to support the growth and differentiation of osteoblastcells,[9–11] however only limited work has been carried out sofar on the biological performance of the new Bioglass®-basedfoams.[12] We hypothesise that cell adhesion and growth on theporous Bioglass® scaffolds will be improved by nanostructuringthe surface of the internal walls (struts) of the pores. An incres-ingly number of publications consider the beneficial effect ofnanostructured surfaces in supporting cell growth and prolif-eration.[13–15]

Adv. Funct. Mater. 2007, 17, 2815–2822 © 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 2815

–[*] Dr. A. R. Boccaccini, Dr. Q. Z. Chen, F. Chicatun, J. Cho,

Dr. O. Bretcanu, Dr. J. A. Roether, Dr. S. Novak[+]

Department of Materials, Imperial College LondonLondon SW7 2BP (UK)E-mail: [email protected]; [email protected]

[+] Permanent Address: Nanostructured Materials, J. Stefan Institute,Ljubljana, Slovenia.

The coating of highly porous Bioglass® based 3D scaffolds with multi-walled carbon nanotubes (CNT) was investigated. Foamlike Bioglass® scaffolds were fabricated by the replica technique and electrophoretic deposition was used to deposit homoge-neous layers of CNT throughout the scaffold pore structure. The optimal experimental conditions were determined to be:applied voltage 15 V and deposition time 20 minutes, utilizing a concentrated aqueous suspension of CNT with addition of asurfactant and iodine. The scaffold pore structure remained invariant after the CNT coating, as assessed by SEM. Theincorporation of CNTs induced a nanostructured internal surface of the pores which is thought to be beneficial for osteoblastcell attachment and proliferation. Bioactivity of the scaffolds was assessed by immersion studies in simulated body fluid (SBF)for periods of up to 2 weeks and the subsequent determination of hydroxyapatite (HA) formation. The presence of CNTs canenhance the bioactive behaviour of the scaffolds since CNTs can serve as template for the ordered formation of a nano-structured HA layers, which does not occur on uncoated Bioglass® surfaces.

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Carbon nanotubes (CNTs) have been a subject of extensiveresearch in the last 10 years.[16] Due to their impressive struc-tural, electrical and mechanical properties as well as their smallsize and mass, carbon nanotubes have become one of the mostpromising materials for future developments and have openeda new era in materials science and nanotechnology.[16,17]

In recent years, considerably efforts have been devoted toapply CNTs in the biological and medical fields.[18] There is al-ready available data suggesting that carbon nanotube contain-ing materials may be optimal for tissue engineering applica-tions.[5–7,15,19–26] This is due not only to their ability to simulatedimensions of proteins that comprise native tissue but also dueto their higher reactivity for interactions involved in the cellattachment mechanism. Several studies have been carried outon the interaction between CNTs and a variety of cells in-cluding osteoblasts and focusing on the biocompatibility ofCNTs.[15,21,22,26] These studies suggest the possibility of usingCNTs as an alternative material for the treatment of bonephatologies in combination with bone cells, with the potentialfor enhanced osteoblast proliferation and bone formation.

In addition, next-generation scaffolds could incorporatefurther functionalities to enhance neotissue formation. For in-stance, an electric field is known to stimulate the healing of var-ious tissues.[19] In the case of bone regeneration and fracturehealing the use of an electric field is based on the observationthat, when a bone is subjected to mechanical stresses, deforma-tion of bone is normally accompanied by an electrical signalbearing the strain characteristics.[27] Therefore, a conductivescaffold, e.g., incorporating carbon nanotubes, can potentiallybe used for stimulating cell growth and tissue regeneration byfacilitating the physioelectrical signal transfer.[21]

One of the requirements for TE scaffolds is their reliablemanufacturability in adequate (3D) sizes and shapes with therequired porosity and surface topography. This is still a de-manding challenge for biomaterials technologists,[3,28] which iscertainly exacerbated if scaffolds are based on nanomaterialssuch as carbon nanotubes due their intrinsic difficult process-ability.

Electrophoretic deposition (EPD) is a material processingtechnique based on the movement of charged particles in liquidsuspensions and their deposition on a substrate acting as elec-trode in the EPD cell.[29] EPD is a method involving low costs,short formation time, few substrate shape restrictions and sim-ple experimental equipment being useful to produce coatingsand films of homogeneus microstructure and controlled thick-ness on different substrates.[29,30] This processing technique isbeing increasingly considered for the production of nanostruc-tured coatings and layers on a variety of substrates for numer-ous applications, wich include wear and oxidation resistance,bioactive coatings for biomedical implants and devices as wellas functional coatings for photocatalytic, electronic, magneticand related applications.[31]

In this context, electrophoretic deposition (EPD) has beenshown to be a very convenient method to manipulate CNTsand to produce reliable CNTs assemblies and layers on planarsubstrates.[32–34] For example, Du et al.[32] deposited CNT onmetallic substrates by EPD using ethanol/acetone mixed sus-

pensions. Further studies were carried out by Thomas et al.[33]

where homogeneous deposition of CNT assemblies using aque-ous suspensions was accomplished on stainless steel substrates.A review of previous work on the EPD of CNT has been pub-lished recently.[34] However, no previous research has beenconducted focusing on production of CNTs coatings on non-conductive porous substrates by EPD, including scaffolds fortissue engineering applications.

The aim of the current study, therefore, is to explore the pos-sibility of using EPD to produce uniform deposits of CNTs onporous Bioglass®-based scaffolds intended for bone tissue engi-neering. The incorporation of CNTs into the scaffolds has anumber of purposes, such as to encourage cell adhesion andproliferation by inducing nanotopography, following literatureevidence,[15,21,22,26] to provide a crack inhibiting mechanism onthe scaffold surfaces, according to results presented in recentstudies[35] and to confer biosensing (electrical conduction)properties[15] while maintaining the bioactivity and intercon-nected porous network of the scaffold. Moreover, addition ofcarbon nanotubes to a biocompatible matrix has further advan-tages towards multifunctional (smart) scaffolds since CNTcould be used for targeted delivery of growth factors or drugs.

2. Results and Discussion

2.1. Preparation of the Scaffolds

SEM images in Figure 1 show the interconnected, macropo-rous structure of the scaffolds prepared with two different poresizes of polyurethane foams. The selection of two different

2816 www.afm-journal.de © 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Adv. Funct. Mater. 2007, 17, 2815–2822

(b)

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Figure 1. SEM images showing the interconnected porous structure ofBioglass® scaffolds prepared by using polyurethane foams with a) 45 ppiand b) 60 ppi.

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A. R. Boccaccini et al./Carbon Nanotube Coatings on Bioglass-Based Tissue Engineering Scaffolds

foam templates was to demonstrate the feasibility of themethod to produce suitable scaffolds with different poremorphologies. The choice of porosity and pore size will ulti-mately depend on the site of application in the body for boneengineering applications. The foams produced are very similarto spongy bone in terms of their pore structure. As reportedelsewhere,[2,8] the highly porous (porosity: > 90 %, cell diame-ter: 510–720 lm) scaffolds are in fact made of a partial crystal-lized glass, e.g., a glass-ceramic microstructure, which conferssome mechanical competence to the scaffolds despite the highporosity. When sintered at a temperature > 1000 °C, the nearlyfull densification achieved and the fine crystals of Na2Ca2Si3O9

present in the pore struts are responsible for the adequate com-pression strength of the scaffold.[8] The optimization of the in-ternal surface topography of the pore network by applying aCNT coating has been the primary objective of the present in-vestigation.

2.2. Zeta-Potential Analysis

In order to determine suitable conditions for the carbonnanotubes deposition on the bioglass, the surface charge ofboth materials was analysed. The pH related zeta-potential inaqueous suspensions is presented in Figure 2. At the naturalpH of the CNT suspension, i.e., 7.5, the zeta potential of CNTwas determined to be –17 mV, while on the contrary, the bio-

glass is positively charged below pH 9.5, where isoelectric pointappears. This implies that due to the opposite surface chargesat pH 7.5, the CNTs moving in the electric field toward the an-ode will be attracted by the bioglass scaffold placed betweenthe electrodes in EPD cell.

2.3. EPD of CNT on Bioglass® Scaffolds Using a DilutedSuspension

Two series of EPD experiments were carried out. One set ofexperiments involved constant deposition time and different

applied voltages in the range 10–55 V. In the second experi-ments series, the deposition time was varied in the range4–20 minutes, while the applied voltage was kept constant atvalues between 10 and 55 V. Experiments were carried outwith dilute or concentrated suspensions, as shown in Table 1.

Typical examples are shown below to document the effect ofthe different variables investigated and to determine the opti-mal conditions.

Figure 3a and b are SEM images of a bioactive glass scaffoldcoated with CNTs by EPD using a voltage of 55 V and deposi-tion time of 4 minutes, using the diluted suspension (Table 1).It can be seen from the SEM images that the scaffold is onlypartially coated with CNTs. This can be due to the low concen-tration of CNTs in suspension or the relatively low depositiontime.

2.4. EPD of CNT on Bioglass® Scaffolds Using a ConcentratedSuspension

In order to enhance the homogeneity of the CNT coating, aconcentrated suspension was prepared (suspension nr. 2 in Ta-ble 1). EPD using the concentrated suspension was conductedat constant voltage values in the range 15–30 V, with deposi-tion times ranging from 4 to 20 minutes and electrode separa-tion of 1 cm. When the applied voltage was below 15 V, CNTscould not be deposited successfully. This behavior may be dueto the insufficient electric field force acting on the CNTs. Itwas also found that if the voltage applied was above 30 V gasevolution occurred, due to water electrolysis. The evolved gasinterfers during the coating deposition producing an heteroge-neous film, as discussed in the literature.[36]

The best results in terms of homogeneity of the CNT depositmicrostructure and uniform CNT infiltration of the pore net-work were achieved using the concentrated suspension. Theimages in Figure 3c and d reveal a uniformly coated scaffold aswell as a homogeneous coating of the pore walls (struts) in themicro-scale. According to SEM observations, the optimal ex-perimental conditions for EPD were found to be: applied volt-age of 15 V and deposition time of 20 minutes. Figure 3c and dillustrate qualitatively that the scaffold pore structure re-mained invariant after electrophoretic deposition of CNTs, in-dicating that carbon nanotubes or agglomeration of CNTs didnot block the pores. The present investigation has thus demon-strated for the first time the high versatility of EPD to developCNTs coatings on highly porous Bioglass® scaffolds. Furtherquantitative characterization of the mechanical and functionalproperties of the scaffolds, such as compressive strength and

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Table 1. Composition of the suspensions for EPD.

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electrical conductivity, is in progress. The possible activation ofcrack inhibiting mechanisms, such as those typical in fiber rein-forced composites, e.g., crack deflection, crack bridging, and fi-ber pullout, which have been considered to occur in CNT con-taining composites,[35] will be investigated.

2.5. Bioactivity Studies

The formation of an HA layer on the surface of the CNTcoated scaffolds upon immersion in simulated body fluid (SBF)was confirmed by SEM observations, as shown in Figure 4aand b for scaffolds soaked in SBF for 7 and 14 days, respective-ly. The formation of discrete HA crystals is observed after 7days (Fig. 4a) while a uniform layer of HA developed after 14days in SBF, as shown in Figure 4b. HA crystals can be recog-nized by their well-known globular, cauliflower shape, as illus-trated in Figure 5, which is a high magnification SEM image ofa CNT coated scaffold after 14 days of soaking in SBF. It wasalso found that the thickness of the precipitated HA layer on

the surface of the scaffolds increased with increasing immer-sion time in SBF, both in the uncoated and CNTs coated scaf-folds. After 14 days in SBF, CNTs remained completely em-bedded in the HA layer, being difficult to distinguish the twophases, as shown in Figure 5.

The small cracks noticed on the surface of the samples (seeFigs. 4b and 5), are characteristic of a dried silica-gel rich layer.Considering the well-known mechanism of precipitation of HAon bioactive glass surfaces upon immersion in SBF,[37] in thefirst stage, a silica-gel layer is formed due to the ionic exchangebetween the sample’s surface and the solution. The HA crystalsnucleate and growth on the top of this silica-rich layer. On re-moving the samples from the SBF solution, the silica-gel layerdries and cracks form on the surface.

X-ray diffraction (XRD) patterns of CNTs coated samplesbefore and after soaking in SBF are shown in Figure 6. Forcomparison, XRD patters of CNT free Bioglass® scaffolds be-fore and after immersion in SBF for similar periods of time areshown in Figure 7. The diffraction lines of pure hydroxyapatite


(c)

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(d)

Figure 3. SEM images of a Bioglass® scaffold coated with CNTs by EPD at 55 V for 4 min using the dilute suspension: a) low and b) high magnificationimages showing partial covering of the pore surfaces by CNT as well as coated with CNTs by EPD at 15 V for 20 min using the concentrated suspension:c) low and d) high magnification images, showing complete coating of the struts by CNT.

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(HA) (pattern 00-009-0432) are also shown. All samples haveXRD patterns characteristic of a glass-ceramic structure. Thecrystalline phase of the as-fabricated scaffolds was identified asa mixed sodium calcium silicate (Na2Ca2Si3O9), in agreementwith previous results.[2] The intensity of the carbon diffractionlines is too low and cannot be separated from the instrumentbackground. After soaking in SBF, broad HA diffraction peakscan be observed on the XRD patterns of all scaffolds, with orwithout CNT coating. The intensity of these peaks is very low,due to the small quantity of this phase formed on the poroussamples. Results from EDX analysis confirmed the presence ofCa and P in a ratio close to that in stoichiometric HA (1.67) inthe sample soaked for 14 days. After immersion in SBF, allsamples (CNT coated and uncoated) showed continuous de-crease in the Na and Si contents and concurrent increase in Caand P fractions (as determined by EDX), approaching thecomposition of HA.

The results thus confirm that the presence of CNTs does notimpair the ability of the bioactive glass-ceramic surface to in-duce formation of HA, hence the novel CNT coated scaffoldsare bioactive. Bioglass® is a well known bioactive materialwhich promotes attachment, growth and proliferation of osteo-blast cells, as it has been widely demonstrated in the literature(e.g., [37]), via the formation of a hydroxyapatite layer on thematerial surface and by direct gene activation supported byBioglass® dissolution products.[38] However, the effect of nano-structured surfaces of bioactive glass scaffolds on cell attach-ment and proliferation has not been studied in detail. UsingCNT arrays it should be possible to induce the formation of anordered nanostructured HA layer as discussed next.,

Most previous work on biomineralisation of biomaterial sur-faces, including all work carried out on bioactive glass surfaces(e.g., [37]), has involved the deposition of of calcium phosphatecrystals on a surface rich in negatively charged groups, includ-ing hydrated silica or macromolecules.[39–41] However such pre-vious approaches have not provided control over shape, sizeand orientation of crystals. The use of carbon nanotubes with


(a)

(b)

Figure 4. SEM images of CNT coated Bioglass® scaffolds after soaking inSBF for a) 7 days and b) 14 days showing HA formation

Figure 5. High magnification SEM images of a CNT coated Bioglass® scaf-fold after soaking in SBF for 14 days showing CNTs embedded in a nano-structured HA layer.

10 20 30 40 50 60 70

14 days SBF

7 days SBF

0 day SBF

2 θθθθ (°)

10 20 30 40 50 60 700

20406080

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I (a

.u.)

HA 00-009-0432

I (%

)

2 θθθθ (°)

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10 20 30 40 50 60 70

14 days SBF

7 days SBF

0 day SBF

2 θθθθ (°)

10 20 30 40 50 60 700

20406080

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I (a

.u.)

HA 00-009-0432

I (%

)

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Figure 6. XRD patterns of CNT coated Bioglass® scaffolds before andafter soaking in SBF. Stoichiometric hydroxyapatite (HA) diffraction lines(pattern 00-009-0432) are also shown [� = HA; � = (Na2Ca2Si3O9)].

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negatively charged surfaces such as those investigated in thisstudy, should promote mineralization by their enhancedability to nucleate HA on their surfaces, as found by otherauthors.[20,42,43] In particular, the presence of COOH groups onmultiwalled carbon nanotubes has been discussed to be rele-vant in inducing the precipitation of nanocrystals of HA fromSBF in so-called biomimetic methods.[20,44] The large surfacearea of the CNT assembly enables the interaction of ions inSBF and carboxyl groups, which controls the crystal growththus leading to a nanostructured apatite morphology.

Previous studies have only considered CNT assemblies onflat surfaces.[20,42–44] In our 3D scaffolds, CNTs were seen to becompletely embedded in the HA layer demonstrating thatCNTs can act as a template for nucleation and growth of theHA nanosized crystals (Fig. 5). This feature of our novel com-posite scaffolds could improve their performance in bone tissueengineering applications and the concept of addition of CNTby an electrophoretic deposition technique can be extended toother substrates used in tissue engineering. As mentionedabove, many clinical applications of biomaterials depend ontheir surface morphology, topography and texture, and nano-structures have been shown to improve cell adhesion and pro-liferation.[25,26] Adhesion of cells such as osteoblasts is a crucialprerequisite to subsequent cell functions such as synthesis of

extracellular matrix proteins and forma-tion of mineral deposits. The CNT EPDcoating on the Bioglass® porous structurecan be efficiently used as a nano-matrixfor the nucleation and growth of HA withcontrolled morphology upon immersionin SBF. Thus, it is possible to design highlyporous bioactive scaffolds which provide,in addition to the required 3D porosity,[2]

a suitable surface morphology for specificcell adhesion and proliferation via theformed CNT/HA layers. As in all otherbiomedical applications suggested forCNTs, at some point the scaffold will be-come in surface contact with living tissue,hence biocompatibility between CNTsand host cells must be investigated.[45]

This is particularly important if carbonnanotubes are not eliminated through nor-mal physiological functions. To date, onlyfew investigations have focused on CNT-cell interactions and certainly more re-search is urgently required in this fieldif CNT are to be seriously consideredfor biomedical applications in the near fu-ture.

3. Conclusions

This work has successfully produceduniform CNTs deposits on highly porousbioactive and biodegradable 45S5 Bio-

glass®-derived glass-ceramic scaffolds, intended for bone tissueengineering, by the electrophoretic deposition technique. Thenegatively charged CNTs enforced by the electric field movedtowards the anode through the Bioglass® scaffold, where theydeposited due to the opposite net surface charge. The optimalexperimental conditions were determined to be: applied volt-age 15 V and deposition time 20 minutes, utilizing a concen-trated CNT suspension in water. The scaffolds pore structureremained invariant after the CNT coating, as assessed by SEM.The incorporation of CNTs into bioactive glass scaffolds hasthe aim of stimulating osteoblast cell attachment and prolifera-tion by providing a nanostructured surface of the pore walls, aswell as conferring biosensing properties to the scaffold by add-ing an electrical conduction function. By studying the forma-tion of hydroxyapatite crystals on the surface of the CNT coat-ed Bioglass® scaffolds upon immersion in SBF, it wasconfirmed that the bioactivity of the scaffolds was not impairedby the presence of CNTs. The results suggest that CNTs can in-duce the ordered formation of a nanostructured CNT/HAcomposite layer when scaffolds are in contact with biologicalrelevant media. The electrophoretic deposition techniqueshould have general applicability for the fabrication of CNTcoated substrates for biomedical applications. For the particu-lar scaffolds developed here, in vitro and in vivo biological tests


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Figure 7. XRD patterns of CNT free Bioglass® scaffolds before and after soaking in SBF for up to14 days. Stoichiometric hydroxyapatite (HA) diffraction lines (pattern 00-009-0432) are also shown[� = HA; ∇ = (Na2Ca2Si3O9)].

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are necessary to investigate the combined effect of the CNTscoating and Bioglass® substrate on osteoblast cell adhesion andproliferation.

4. Experimental

Scaffold Fabrication: Highly porous foam-like Bioglass® based scaf-folds were prepared by using the replication technique, as described indetail elsewhere [2]. Briefly, the method involves coating a polymerfoam (e.g., polyurethane) with a bioactive glass (Bioglass®) slurry. Thepolymer foam, having the desired pore structure, simply serves as a sa-crificial template for the glass coating. The polymer template is im-mersed in the slurry, which subsequently infiltrates the structure andglass particles adhere to the surfaces of the polymer. Excess slurry issqueezed out leaving a more or less homogeneous coating on the foamstruts. After drying, the polymer is slowly burned out in order to mini-mise damage to the glass coating. Once the polymer has been removed,the glass structure is sintered to a desired density. The process repli-cates the macrostructure of the starting sacrificial polymer foam, andresults in a rather distinctive and well-defined microstructure withinthe struts.

Our method involved the preparation of a slurry, mixing 3 g ofpoly(D,L-lactic acid) (PDLLA), 100 mL of dimethylcarbonate (DMC)and 40 wt % of 45S5 Bioglass® powder (mean particle size < 5 lm).This procedure was carried out under vigorous stirring using a mag-netic stirrer for 1 h. A 45S5 Bioglass® powder (composition in wt %.45 % SiO2, 24.5 % Na2O, 24.5 % CaO and 6 % P2O5 [37]) was used inas-received condition. Two different kinds of polyester-based polyure-thane foams with 45 ppi and 60 ppi (pores per inch) obtained from Re-ticel (Corby, UK), were used as the sacrificial templates. The foamswere cut into prismatic geometry of dimensions 10 × 10 × 10 mm3 andimmersed in the slurry for 15 min. After coating with Bioglass® thescaffolds were dried at room temperature for ∼ 12 h. Finally, the poly-mer was slowly burned out by heating the samples in air at 550 °C for1 h (heating rate: 2 °C min–1). Subsequently, the foams were sintered at1100 °C for 3 h using a heating rate of 2 °C min–1.

Carbon Nanotube Suspension Preparation: The CNTs suspensionwas prepared by adding to an aqueous solution of multi-walled carbonnanotubes of commercial origin, Triton X-100 as an ionic surfactantand iodine 99.999 % (Aldrich Chemical Company Inc) as a charger.The high dispersion efficiency of Triton X-100 is due to its chemicalstructure, where the aromatic group is responsible for the strong hydro-phobic interaction between Triton X-100 and the CNTs. It is believedthat the p-like stacking of the benzene rings of the surfactant onto thesurface of graphite increases the binding and surface coating of surfac-tant molecules onto graphite significantly [46]. In order to enhance par-ticle charging in the solution, iodine was added to the suspension. Oneof the reasons why iodine works as a charger is its ability to form com-plex with water [47].

The resulting suspension was sonicated for 4–5 h to helpthe particles to overcome the attractive van der Waalsforces. Finally, the suspension was centrifuged for 15 min at3000 rpm to remove the large CNTs agglomerations andavoid their deposition during EPD. After centrifugation, thesupernatant suspension was carefully extracted from thecentrifuge tube and the remaining suspension was placed ina glass recipient for EPD.

EPD of CNT on Bioglass® Scaffolds: As the bioglassitself is not conductive, an adapted EPD technique wasused. A schematic diagram of the EPD cell used in thisinvestigation is shown in Figure 8. The electrodes usedwere made of stainless steel 316L foil with dimensions of1.5 cm × 1.5 cm × 0.02 cm. In order to achieve a uniformCNTs coating throughout the 3D porous structure, the Bio-glass® scaffolds were placed inside a copper wire cage be-tween the two electrodes next to the anode were the deposi-tion should take place due to its negative net surface charge.

(see Fig. 2). The electrodes were then connected to a DC power supply.Two different CNT suspensions were used in order to achieve CNTsdeposition on the scaffolds, as shown in Table 1. EPD was carried outby setting a constant voltage in the range 10–55 V, with deposition timeranging between 4 and 20 min, and electrode separation of 2 cm. Afterthe EPD process, the copper wire frame was carefully and slowly with-drawn from the EPD cell in order to avoid any influence of a drag forcebetween the suspension and the deposited wet CNTs film. Finally, thesamples were dried slowly at room temperature in normal air. Prior toany future biological test, the scaffolds should be submitted to a ther-mal treatment to eliminate possible residuals of iodine and organics. Atreatment of 10 min at 320 °C in argon atmosphere is sufficient, as de-scribed in a previous study [48].

Characterisation Techniques: Net-surface charge at the carbon nano-tubes and at the bioglass scaffold in water was analyzed by measuringthe zeta-potential, using ZetaProbe device, Colloidal Dynamics, USA.The surface charge at the scaffold was estimated by analyzing a suspen-sion of the Bioglass® particles as a function of pH. The pH of thenaturally alkaline suspension was adjusted by hydrochloric acid.

Field emission gun scanning electron microscopy (FEG-SEM)(LEO1535) was used to examine the morphological and textural fea-tures of the scaffolds, before and after EPD. Comparison of the differ-ent EPD tests results led to the determination of the optimal experi-mental conditions to produce electrophoretically deposited CNT filmsof best quality in terms of nano and microstructure homogeneity, uni-formity and degree of CNTs infiltration throughout the porous struc-ture and adherence to the scaffold.

Bioactivity Tests: Bioactivity tests were carried out using the stan-dard in vitro procedure described by Kokubo et al. [49]. Selected sam-ples of Bioglass®-based glass-ceramic scaffolds, uncoated and coatedwith C-nanotubes, of about 5 × 5 × 5 mm3, were immersed in 10 mL ofacellular simulated body fluid (SBF) in conical flasks. The pH of theSBF solution was buffered at pH 7.3 before soaking the samples. Theconical flasks were placed in an incubator at a controlled temperatureof 37 °C. The SBF solution was refreshed twice a week, to simulate thebody fluid circulation. The samples were removed from the SBF solu-tion after 7 and 14 days. The experiments were carried out in triplicate.After extraction from the SBF fluid, the samples were gently rinsedwith deionised water, and left to dry at room temperature. After dryingin air atmosphere, the samples were observed by SEM in order to as-sess their bioactivity by detecting the formation of an hydroxyapatitelayer on their surfaces. A field emission gun (FEG) SEM LEO1535was used to examine the morphology of the composite scaffolds afterthe immersion in SBF. Samples were gold coated and observed at anaccelerating voltage of 15 kV. EDX spectra (Ka line) of scaffold sur-face areas of 1 × 1 lm2 were recorded at 20 kV in the field emissiongun SEM (FEG-SEM). The spectra were processed by “INCA” (Ox-ford instruments) software, using standard reference spectra. X-ray dif-fraction (XRD) patterns of samples before and after soaking in SBFwere obtained using a Philips diffractometer with Cu Ka radiation,using a step of 0.04° (2h) and a fixed counting time of 2 s per step. The


Figure 8. Schematic diagram of the electrophoretic deposition cell used for obtainingCNTs coating on Bioglass® scaffolds.

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samples were ground to powder and about 0.1 g was used for thesemeasurements. The XRD patterns of powder samples were collectedfor the 2h range 10–70°, at 40 kV and 40 mA. The crystalline phaseidentification was performed by using the “X’Pert HighScore” programapplying the PCPDFWIN database.

Received: September 27, 2006Revised: February 5, 2007

Published online: August 17, 2007–

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