bioglass nanofibers

DOI: 10.1002/adfm.200500750

Production and Potential of Bioactive Glass Nanofibers as aNext-Generation Biomaterial**

By Hae-Won Kim,* Hyoun-Ee Kim, and Jonathan C. Knowles

1. Introduction

Materials for biomedical applications have been exploited toaugment and regenerate human tissues that have been sub-jected to damage and diseases.[1,2] Over the last decade the de-mands on synthetic biomaterials have increased significantly,to the point where they are now indispensable because autolo-gous surgery, regarded as the ‘standard implantation’ tech-nique, has limited material supplies and requires painful sec-ondary operations. Moreover, the alternative allografts orxenografts have serious concerns associated with immunogenicresponses.[3] In light of this, significant effort has been devotedto the area of biomaterials and tissue engineering, and this hasbrought about the development of several materials with clini-cal promise.

Specifically for hard-tissue applications, such as the regen-eration and repair of bones and teeth, several bioactive orbioinert materials have been used clinically.[4] Silica-based bio-

glasses constitute the essential part of such bioactive materials,having already been utilized in numerous orthopedic and den-tal applications.[5] Most in vivo studies on these bioglasses haveconfirmed their excellent biocompatibility with hard and evensoft tissues. This is attributed mainly to their ability to form abioactive layer at the interface in contact with living tissues,namely the hydroxycarbonate apatite (HCA) layer, which isequivalent to the mineral phase of human hard tissues.[6] Basedon extensive research conducted in vitro and in vivo, bioactiveglasses are considered as one of the most-promising biomateri-als for the “next generation”.[6]

Most studies in this field have focused on melt-derivedglasses, either in the bulk or granular form. Fiber-type melt-de-rived glasses have been produced with diameters of hundredsto tens of micrometers, and these glass fibers reportedly havethe potential to act as cell supporters for extracellular matrixproduction and tissue regeneration, with a mechanical strengthsuperior to that of the equivalent bulk glasses.[7] When formu-lated with biodegradable polymers, the potential of this bioac-tive-glass fiber to act as a tissue-engineering scaffold should befurther increased by adopting the shape flexibility of polymerswhile retaining optimized mechanical properties (toughness,strength, and elastic modulus) and without sacrificing its excel-lent bioactivity.[8] However, with the melt-spinning approach,the fiber diameter is limited to such micrometer-scale (ca. tensto hundreds of micrometers) because of the associated process-ing restrictions.

More recently, a sol–gel approach was introduced in the pro-duction of bioactive silica glasses.[9,10] Based on the studies un-dertaken so far, these sol–gel glasses offer advantages overmelt-derived glasses in several aspects. Firstly, the bioactivityof sol–gel glasses is maintained over a wider composition range(i.e., up to a higher silica content) than the melt-derivedglasses.[10,11] Secondly, and more intriguingly, are the processing

Adv. Funct. Mater. 2006, 16, 1529–1535 © 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 1529

–[*] Prof. H.-W. Kim

Department of Dental Biomaterials, School of DentistryDankook UniversityCheonan 330-714 (Korea)E-mail: [email protected]. H.-E. KimSchool of Materials Science and Engineering,Seoul National UniversitySeoul 151-742 (Korea)Prof. J. C. KnowlesDivision of Biomaterials and Tissue Engineering,UCL Eastman Dental InstituteLondon WC1X 8LD (UK)

[**] The authors greatly appreciate Dr. Y. H. Koh for his constructive dis-cussion and S. Y. Chae for her experimental assistance. This studywas supported by a grant from the Korea Health 21 R & D Project,Ministry of Health and Welfare, Republic of Korea (A060125).

Over the past decades, bioactive glass has played a central role in the bone regeneration field, due to its excellent bioactivity,osteoconductivity, and even osteoinductivity. Herein, exploitation of bioactive glass as a one-dimensional nanoscale fiber byemploying an electrospinning process based on a sol–gel precursor is reported for the first time. Under controlled processingconditions, continuous nanofibers have been generated successfully with variable diameters. The excellent bioactivity of thenanofiber is confirmed in vitro within a simulated body fluid by the rapid induction of bonelike minerals onto the nanofibersurface. The bone-marrow-derived cells are observed to attach and proliferate actively on the nanofiber mesh, and differentiateinto osteoblastic cells with excellent osteogenic potential. The bioactive nanofibers have been further exploited in variousforms, such as bundled filament, nanofibrous membrane, 3D macroporous scaffold, and nanocomposite with biopolymer,suggesting their versatility and potential applications in bone-tissue engineering. Based on this study, the bioactive nanofibrousmatrix is regarded as a promising next-generation biomaterial in the bone-regeneration field.

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benefits that the sol–gel approach canprovide, that is, the accessibility to systemsrequiring a scale-reduction for nanobio-technology. The nanoscale formulation ofthe bioactive glasses can also open theway to the exploitation of new biomedicalareas, such as biocatalysts, biomembranes,biosensors, and reinforcements. Most ofall, the bioactive glass matrices and scaf-folds with nanofibrous internal structurewill unquestionably have a significant im-pact on the bone-tissue regenerationfield.[2]

Herein, we report the production ofsol–gel-derived bioactive glass as a nano-scale fiber by means of an electrospinning(ES) technique. The ES process has re-cently gained significant attention as amethodology for synthesizing fibrousstructures on the micro-/nanoscale.[12,13]

Compared to the conventional drawingtechniques used for producing microscalefibers, the ES method allows the genera-tion of much smaller diameters, i.e., thenanoscale fibers. ES is a simple and cost-effective process, and is thus utilized in di-verse fields, such as electronics, optics,and tissue engineering.[12–14] Various typesof polymeric fibers have been producedwith scales of 10–1000 nm.[12–14] In more-recent studies, alkoxide-based sol–gel pre-cursors were used for the production ofmetallic oxide fibers by the ES process.[15,16] As such, the sol–gel glass system described herein can also satisfy the solutionconditions for the ES process. To the best of our knowledge,this is the first report on the production of a bioactive glassnanofiber, and includes examination of its excellent bioactivityand osteogenic cell responses. Based on its potential, we alsoexploited this nanofiber in various structures, such as bundledfilaments, fibrous membranes, 3D scaffolds, and in a nanocom-posite with a biopolymer, showing versatility for practical ap-plications in bone-tissue regeneration.

2. Results and Discussion

To obtain bioactive nanofibers, a glass sol of a composition thatretains good bioactivity (here we chose 70 SiO2·25 CaO·5 P2O5)was prepared and electrospun under appropriate conditions;the ES was then followed by a thermal treatment. Dependingon the processing conditions, the electrospun glass fibers pos-sessed a range of diameters. Among the parameters, we ob-served that the sol concentration was the most dominant factorin controlling the diameter. In Figure 1a–c, scanning electronmicroscopy (SEM) showing the morphologies of the nanofiberswith different average diameters (630 to 84 nm) at varying ini-tial sol concentrations (1 to 0.25 M) are presented. Continuous

and uniform fibers were created successfully under all condi-tions. At concentrations of over 1 M, micrometer-scale fiberswere obtained. As the concentration decreased, the diameterof the fibers was reduced. However, it was difficult to preservethe fiber shape at concentrations below approximately 0.25 M,since below this threshold discrete beads were produced exten-sively. After the heat-treatment process, the fiber diameterswere observed to be reduced by a factor of 2–3, due to theburn-out of residual polymeric precursors and the consolida-tion of the glass network. The control of the nanofiber diame-ter made possible by the electrospinning process is effective toproduce tissue-regeneration matrices with a pore size and fibernetwork tunable to the demands of specific applications.Transmission electron microscopy (TEM) observation of a fi-ber with an average diameter (dave) of 84 nm showed the fiberclearly (Fig. 1d). No crystals were formed, as far as could beconfirmed by the TEM selected area electron diffraction(SAED) pattern (Fig. 1e). The energy dispersive spectroscopy(EDS) profile of the fiber revealed the glass composition(70 SiO2·25 CaO·5 P2O5) quite well (Fig. 1f).

The heat-treated nanofibers were subsequently incubated ina simulated body fluid to examine their bioactivity by deter-mining whether they induce the precipitation of bonelikeminerals on the surface. Data on the fibers obtained with the0.25 M sol (dave = 84 nm) are presented as a representative ex-

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Figure 1. Analysis of the glass nanofibers after electrospinning and heat treatment at 700 °C.a–c) SEM images of the nanofibers of different average diameters (630 nm, 220 nm, and 84 nm insequence) with varying sol concentration (1, 0.5, and 0.25 M in sequence). d) TEM image of thenanofiber with dave = 84 nm. e) SAED pattern and f) EDS profile of the nanofiber in (d).

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H.-W. Kim et al./Bioactive Glass Nanofibers as a Next-Generation Biomaterial

ample in Figure 2. After incubation of the fiber mesh for 1 day,the surface became quite irregular with some elongated crys-tals being detected on the surface by TEM observation(Fig. 2a). When incubated for 3 days, the fiber morphologywas changed more significantly, with numerous elongated crys-tals being generated over almost the entire surface (Fig. 2b). Ahigh-magnification image of the fiber incubated for 1 dayclearly shows the formation of crystals (contrast by dark area)on the fiber surface with sizes of a few to tens of nanometers(Fig. 2c). The SAED pattern of the crystals in Figure 2c con-firmed that they consisted of a poorly crystallized apatite with(112) strong and (002) weak rings (Fig. 2d). The EDS analysisof the apatite crystal area shows a higher concentration of Caand P with respect to that of Si (Fig. 2e, compare data withFig. 1f). The Ca/P ratio was approximately 1.56, being close to,but slightly lower than, the stoichiometry of pure hydroxyapa-tite [Ca10(PO4)6(OH)2)], 1.67, which has often been reportedin the poorly crystallized or carbonated apatites produced bythis kind of biomimetic process, and is more similar to that ofbonelike minerals.[17]

The ion-concentration change of the medium was monitoredusing inductively coupled plasma atomic emission spectroscopy(ICP-AES) after incubation of the nanofiber for periods of upto 7 days, as shown in Figure 3a. Initially the prepared mediumcontained 2.5 and 1 mM of Ca and P, respectively, and no Si.Within a short period of time, both the Ca and P concentra-tions decreased abruptly and then stabilized with increasingtime, while the Si concentration increased initially and then le-veled off. Only a slight increase in the Ca concentration was

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Figure 2. Bioactivity analysis of the glass nanofiber (dave = 84 nm) after incubation in a simulated body fluid: TEM image for a) 1 and b) 3 days, c) magni-fication of (a). d) SAED pattern of the crystal in (c), and e) EDS profile of the crystal.

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Figure 3. Change in a) ion concentration of medium and b) Fourier trans-form infrared (FTIR) spectroscopy of the nanofiber (dave = 84 nm) beforeand after incubation for 5 and 7 days. In (b), symbols indicate bandsrelated to silicate (�), phosphate (�), and carbonate (diamond).

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observed in the first few hours. These changes in the ionic con-centration illustrate the dissolution/precipitation process, i.e.,the dissolution of Ca, P, and Si from the nanofiber, and the sub-sequent precipitation of Ca–P crystals from the medium, whichbecame supersaturated by the dissolution of Ca and P. In de-tail, a series of reactions occurred, as proposed by Hench andco-workers: the exchange of alkali ions, such as Ca2+ and Na+

(Ca2+ in this study) with H3O+, the attack of hydroxyl ions(OH–) present in the medium through the silica network struc-ture to form silanol groups (Si–OH), and through which theprecipitation of Ca2+ and PO4

3– (and mostly CO3– also) occurs,followed by the crystallization of HCA.[6] Of special note, whencompared to bulk glasses (melt-derived or sol–gel synthesized)wherein the increase of Ca and P concentrations (in the initialdissolution region) usually continues for several days to weeks,the nanofibers exhibited a more rapid initial drop (as short as afew days) in the Ca and P concentrations of the medium. Thiswas mainly attributed to the large surface area afforded by thenanoscale fibers, which resulted in faster dissolution and super-saturation of the medium with respect to the HCA crystal nu-cleation. Specifically, the sol–gel derived nanofibers developedin this study possess around 2–3 orders of magnitude highersurface area than the conventional melt-derived glass fibers(diameters of approximately hundreds of micrometers) at anequivalent volume.[16] To confirm whether these nanoscale-driven surface properties influence the rate of the CaP induc-tion on the surface, we performed a comparison test using asintered sol–gel glass disk (with a composition same as the nano-fiber) under equivalent medium conditions (see Experimentalsection for details). However, the glass disk layer could not in-duce the formation of CaP on the surface as rapidly as the nano-fiber: the Ca–P ionic drop analyzed by ICP-AES was observedca. 7 days after the incubation of the sintered glass disk.

The crystals formed from the silica fibers were analyzed withFourier transform infrared (FTIR) spectroscopy at differentincubation times, as shown in Figure 3b. As the incubation timeincreased, the bands related to the silica glass (800, 930, 1080,and 1200 cm–1) were attenuated, while those attributed tothe phosphate groups (570, 605, 960, and 1030–1090 cm–1)increased and carbonate bands (870 and 1200–1300 cm–1) ap-peared, suggesting the formation of carbonate-substituted apa-tites (HCA), which is similar to the composition of bone miner-al.

These results concerning the crystal formation on the silica-glass nanofiber have much in common, for the most part, withthe previous results obtained for melt-derived or sol–gel pro-cessed bulk glasses. However, the degree of bioactivity (thecrystal formation) of the nanofibers in vitro appeared to be sig-nificantly enhanced by the nanoscale production process. Assuggested above, the extremely large surface area of the nano-fibers should accelerate the series of reactions taking place onthe glass surface in contact with the medium. Therefore, whenthese silica glass nanofibers are used as bone substitutes, theycould be expected to quickly provide a favorable environmentowing to the rapid formation of bonelike minerals on the sur-face, thereby exhibiting excellent responses for adhesive pro-teins and cells and, consequently, resulting in improved bone

formation.[2] However, this postulation, established from in vi-tro conditions, warrants further experiments in vivo when con-sidering the dynamic fluidic effect as well as the mediation ofproteins in the series of reactions.[18]

The biocompatibility of the glass nanofibers was furtherassessed by their in vitro cellular responses. We used bone-marrow-derived stem cells (BMSCs) to examine the osteogenicpotential of our newly developed bioactive nanofiber. Thenanofibrous meshes with an average diameter of 220 nm wereused for the cellular test. From electron microscopy images,the cells on the nanofiber mesh with 5 days of culturing wereobserved to grow favorably (shown in Fig. 4a). On closer ex-amination, the cells spread actively on the nanofibrous surfacewith numerous cytoplasmic extensions, typical of the osteoblas-tic cellular growth (Fig. 4b). The grown cells were stained toelicit alkaline phosphatase (ALP) enzyme, which is known tobe expressed in the differentiation of osteogenic cells and thusis regarded as an important marker for osteogenic potentialand bone-forming ability. As references, data on the sinteredsol–gel glass disk (same composition as the glass nanofiber)and the degradable biopolymer (polycaprolactone, PCL) in ananofibrous form are also provided. The optical image pre-sented of the glass nanofiber (Fig. 4c1) shows a thicker violetstaining of ALP with respect to that on the glass disk (Fig. 4c2),and the difference was clearer when compared to that on thePCL nanofiber (Fig. 4c3). The ALP level, as well as the viabili-ty of the cells cultured on the three different samples, wasquantified as summarized in Figure 4d. The cells grown on thebioactive glass composition (both nanofiber and disk forms)were more viable than those on the PCL nanofiber, while onlya slight difference was observed between the two glass types.However, the expression of ALP on the nanofiber was higherthan that on the glass disk (particularly significant at day 5).Based on these cellular results, we confirm that the nanofibrousglass favors the attachment and growth of the BMSCs and in-duces them to differentiate into osteoblastic cells. Moreover,this osteogenic potential of the nanofiber glass was observed tobe stimulated significantly to levels similar to or even higherthan those on the relatively flat surfaces with equivalent com-position (glass sintered disk), suggesting the morphologicallydriven (surface associated) properties of the nanofiber, such asnanoscale-roughened morphology, larger surface area, andhigher ionic release, play constructive roles in the osteogenicpotential. In this study, we used the glass nanofiber with anaverage diameter of 220 nm, based on the consideration that itis representative of other-sized nanofibers, as we observed sim-ilar in vitro apatite-forming behavior among all the nanofibers.However, further study is still warranted regarding the effectof diameter of the nanofiber glass on the cellular responses.

Although more extensive works are required as to such ef-fects on the stimulation of osteogenic potential, the presentfinding drives us to underscore the potential of the nanofiberwhen considering the perspectives of manipulation and versa-tility of the nanofibrous form, which is useful in a tissue-engi-neering matrix (demonstrated in the following Fig. 5). Clearly,the osteogenic potential of our developed nanofiber wasmostly endowed by the bioactive sol–gel glass composition, as


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was demonstrated by the above cellular data in its comparisonwith the degradable but bio-inactive polymeric nanofiber PCL.This bioactive glass nanofiber preserves the beneficial aspectsof not only the bioactive composition with high osteogenic po-tential but also the morphological merits permitting use as atissue-regeneration matrix like the degradable polymer nanofi-bers. With its excellent properties for the recruitment of stemcells, the glass nanofiber holds great promise in the tissue-engi-neering field. However, this postulation, established in vitro,should be substantiated with further in vivo animal studies onthe bone-formation ability and the mechanical stability. Never-theless, the present in vitro observations, including the rapidinduction of bonelike minerals and the osteogenic cellular re-sponses, suggest great potential in the hard-tissue-regenerationfield.

The usefulness of the bioactive nanofiber was further dem-onstrated by utilizing it in 3D structures that could find practi-cal applications as cell-supporting matrices and tissue-engi-neering scaffolds. We formulated the nanofibers as various 3Dmatrices using our laboratory-developed techniques, as shownin Figure 5. As one example, a macroscale filament was pro-duced after aligning the nanofibers at a high mandrel windingspeed (ca. 2000 mm s–1) followed by bundling and compactingthem (Fig. 5a). This longitudinally aligned filament with an in-ternal nanofibrous structure can be specifically applied to sup-port the cell ingrowth of aligned tissues such as muscles andnerves. The texture designing of the filament for specific partsremains another area for development. A nanofibrous mem-brane (Fig. 5b) was produced by means of stacking the nanofi-brous sheets and warm-pressing followed by heat treatment.


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Figure 4. Bone-marrow derived osteoblastic cell responses to the bioactive glass nanofiber: Electron microscopy images of the cells on the nanofibrousmesh at low (a) and high (b) magnification with 5 days of culturing. c1–c3) alkaline phosphatase (ALP) staining of the cells grown on the bioactive glassnanofiber (c1) and on other comparison samples for 5 days: sol–gel glass sintered disk with the same composition as glass nanofiber (c2) and PCL elec-trospun nanofiber (c3); ALP was enzymatically stained in violet. d) Quantification of the cellular assays, represented by mean ± one standard deviation(in brackets), on the samples: cell viability assessed by MTT method at 2 and 5 days and ALP level assessed by an enzymatic reaction at 5 and 10 days.Statistical significance for the glass nanofiber was considered with respect to the glass disk (+p < 0.05) and PCL nanofibers (*p < 0.05, ** p < 0.01) byANOVA.

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The nanofibers were observed to maintain their initial fibrousstructure and form a nanoporous 3D structure. Potential applica-tions for this nanofibrous membrane are believed to includewound healing and guided bone/cartilage tissue regeneration,wherein polymeric matrices are still dominantly used. A 3D mac-roporous scaffold, characterized by a microfilament frameworkwith a nanofibrous internal structure, was also created to allowmacropores within the nanofibrous structure (Fig. 5c). Here, anegative-molding technique was used, where the microfilamentsof nanofibers were organized with carbon filaments and thenheat-treated to produce a 3D open-channeled network with con-trolled pore size (ca. 500 lm) and porosity (ca. 50 %). This mac-roporous design should facilitate vasculisation and cell ingrowthmore effectively through macroscale open channels. Of particularinterest is the fact that the nanofibrous surface can provide favor-able surroundings for the initial protein adhesion and cellular re-sponses, which is typically dissimilar to conventional 3D scaffoldswith a dense surface.[19] These 3D matrices designed with bioac-tive nanofibers can have their mechanical properties optimizedwhen hybridized with biopolymers. We infiltrated a biodegrad-able polymer (5 % poly(D,L-lactic acid) (PLA) solution dissolvedin tetrahydrofuran) into the interspacings of the glass nanofiber

network (Fig. 5d). The image shows a well-constructed nanocomposite comprisingthe dense nanofibrous network and thepolymeric filler. The glass nanofibers wereobserved to be dispersed well and distrib-uted uniformly in the PLA matrix.Further macroporous design of the nano-composite is expected to be achievable byutilizing diverse scaffolding techniques.This nanocomposite approach, employingthe nanofibrous bioactive inorganic inconjunction with biodegradable polymer,should have good prospects in the bone-regeneration field because of the combi-natorial benefits, such as shape flexibilityand moldability, mechanical propertiestunable to the bone matrix, and the bioac-tivity and degradability. Further evalua-tions on the in vivo tissue responses andmechanical properties of the nanocom-posite are currently underway.

3. Conclusions

Bioactive nanofibers were producedsuccessfully by employing an electrospin-ning technique using a glass sol–gel pre-cursor with a bioactive composition. Thebiomedical usefulness of bioactive glasses,as has been established over recent de-cades, was imparted onto the nanofibrousstructure. We observed that the nanofiberpossessed excellent bioactivity and osteo-genic potential in vitro. Moreover, possi-

ble formulations of the bioactive nanofibers, such as aligned fil-aments, nanofibrous membranes, macroporous scaffolds, and innanocomposites with biopolymers, were provided for practicalapplications. Based on the present findings, the bioactive glassnanofiber developed herein is proposed as one of the mostpromising next-generation biomaterials.

4. Experimental

Sol Preparation and Electrospinning: The glass composition used inthis study (70 SiO2·25 CaO·5 P2O5) was chosen based on those of pre-viously developed bulk glasses which exhibit bioactivity in vitro, andthe sol–gel processing conditions were modified from other reports[10,11]. The glass precursors were added at appropriate ratios (tetra-ethyl orthosilicate, calcium nitrate, and triethyl phosphate added in se-quence with a 6 h interval between each addition, all from Aldrich) inan ethanol/water solution containing 2 % HCl (1 N) used for catalysis.The sol mixture was stirred for 24 h and aged without stirring at 25 °Cfor 24 h followed by a further 48 h at 40 °C. The acidic catalyst was nec-essary in order to produce a clear sol. Prior to electrospinning (ES),polyvinylbutyral (PVB, from Aldrich) dissolved in ethanol at 10 % wasadded to the sol at an equivalent volume to adjust the rheological prop-erties of the sol, so that they would be fit for the fiber generation dur-ing ES. 2 mL of the solution was loaded in a syringe and injected into a


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Figure 5. 3D structured matrices of the electrospun glass nanofibers for tissue regeneration. a) Fil-ament: electrospun nanofibers were aligned and bundled into a microfilament. b) Membrane: elec-trospun sheets were stacked and pressed gently (surface (upper) and cross-section view (lower)).c) 3D macroporous scaffold: ready-made filaments were architectured using a negative-mold tech-nique and then heat-treated; enlarged in inset. d) Polymer-filled nanocomposite: heat-treatedfibrous mesh was filled with biodegradable polymer PLA; enlarged in inset.

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rotating mandrel for 10 min at high voltage under controlled conditions(voltage: 12 kV, distance: 8 cm, and injection rate: 0.05 mL min–1). Theelectrospun fibers were subsequently heat-treated at 700 °C for 3 h inair at a heating and cooling rate of 1 and 5 °C min–1, respectively. Theheat-treatment temperature was determined to be high enough toeliminate organic sources and nitrates completely but low enough toavoid crystallization [10,11]. As a reference for the biological tests,we also prepared disk-type pallets of the sol–gel composition(12 mm × 1 mm thickness) after crushing the gelled product into pow-ders, molding them in a metal die, and partial sintering at 700 °C for3 h. As another reference material, a degradable biopolymer, polyca-prolactone (PCL, Sigma), was prepared in a nanofibrous matrix (aver-age fiber diameter ∼ 860 nm) by electrospinning using a solvent (tetra-hydrofuran/N,N-dimethylformamide = 3:2) under adjusted conditions(voltage: 12 kV, distance: 8 cm, and injection rate: 0.05 mL min–1).

Bioactivity Test: The heat-treated glass nanofibers were subjected toa simulated body fluid (containing similar ion concentrations to a bodyplasma), in order to form bonelike mineral HCA crystals on the glasssurface due to a dissolution/precipitation process [20]. This final stepwas carried out in an effort to 1) provide a preliminary assessment ofthe bioactivity of the glass nanofibers in vitro, since the HCA forma-tion has been regarded as the key phenomenon in explaining the bio-compatibility of bioactive materials [2], and 2) utilize the HCA-sur-face-modified glass nanofibers directly. 10 mg of fiber were incubatedin 10 mL of medium for up to 7 days without refreshing. The glass diskprepared as a reference was also tested under the same conditions. Atpredetermined time points, the change in the ion concentration of themedium was monitored with inductively-coupled plasma atomic-emis-sion spectroscopy (ICP-AES, Shimadzu), and the change in the chemi-cal bonding structure of the fiber was analyzed with Fourier transforminfrared (FTIR, System 2000, Perkin-Elmer) spectroscopy. The fiber di-ameter was measured from the SEM image in 20 different sections, andaveraged. The morphology of the fibers was characterized with field-emission scanning electron microscopy (FESEM, JSM6330F, JEOL),and transmission electron microscopy (TEM, CM20, Philips). The com-position and crystal pattern were also analyzed from the TEM opera-tion.

Cellular Response Assay: To observe the biocompatibility of thenanofiber, in vitro cellular responses were examined using rat bone-marrow derived mesenchymal stem cells (BMSCs). Bone marrow washarvested from the adult rats (age 4–8 weeks, Korean). The whole mar-row was flushed with a minimum essential medium (a-MEM) from theexcised proximal and distal epiphyses of femora and tibiae using a sy-ringe. After centrifugation of the marrow at 1000 g, the supernatantwas collected and suspended again in a-MEM. The homogeneouslysuspended cells were maintained in a culture flask containing growthmedium (a-MEM, 2 mM glutamine, 100 U mL–1 penicillin, and100 mg mL–1 streptomycin), supplemented with 15 % fetal calf serum(FCS) under a humidified atmosphere of 5 % CO2 at 37 °C. After1 day of incubation, the cells were washed with phosphate-buffered sa-line (PBS) to discard nonadherent cells and then cultured further forup to 7 days to reach near confluence. For accurate measurement ofthe cellular tests, the glass nanofiber was prepared by supporting it on abioinert zirconia sintered disk. Glass nanofibers prepared with an aver-age diameter of 220 nm were chosen as a representative example. Forthe purpose of comparison, the sol–gel glass disk pallet and PCL nano-fibrous matrix (preparation methods described above section in detail)were also prepared. The maintained cells were harvested with trypsin–EDTA (EDTA= ethylenediaminetetraacetic acid) and plated onto thethree types of samples at a seeding density of 5 × 104 cell cm–2. At thistime of culturing, the medium was supplemented with 50 lg mL–1 so-dium ascorbate, 10 mM sodium b-glycerol phosphate, and 10 nM dexa-methasone in order to induce osteoblastic differentiation. The culturemedium was changed every three days.

At the culturing periods of 2 and 5 days, the cell viability was as-sessed by means of an MTT method [21]. The cell-growth morphologywas observed with SEM after fixing the cells with 2.5 % glutaraldehyde,dehydrating them with a graded series of ethanols (70, 90, and 100 %),and gold coating. The osteogenic potential of the cells was assessed bydetecting the alkaline phosphatase (ALP), which is an important osteo-

genic differentiation marker. The cells cultured on the samples for5 days were treated enzymatically to stain the ALP and reveal it in vio-let. Moreover, the ALP expression level was quantified following ourprevious protocol [21]. At each culturing period, the cell layers weredetached by treatment with 0.1 % Triton X-100, and the cell pelletswere disrupted via cyclic freezing/thawing processes. Aliquots of thesamples normalized to the total protein content were enzymatically re-acted with p-nitrophenyl phosphate (p-NPP) substrate following themanufacturer’s instruction (Sigma ALP kit 104). The quantity of p-ni-trophenol (p-NP) produced was measured at an absorbance of 410 nmusing a spectrophotometer. The cellular tests were performed on fivereplicate samples and data were compared using one-way ANOVAanalysis with statistical significance at p < 0.05 and p < 0.01.

3D Structure Formulation: For the fabrication of the nanofiber in 3Dstructured matrices (bundled filament, nanofibrous membrane, macro-porous scaffold, and nanocomposite with biopolymer), several labora-tory-developed techniques were utilized. A filament was produced bybundling the electrospun fiber mesh and compacting to a diameter of∼ 300 nm, and followed by a heat-treatment at 700 °C for 3 h. For theproduction of fibrous membrane, the electrospun nanofiber mesh wasstacked layer-by-layer and warm-pressed ∼ 80 °C, and then heat-treatedunder the same conditions. The 3D macroporous scaffolding was per-formed using the nanofibrous filament (diameter of ∼ 600 nm) and adesigned carbon mold as a support material, by means of aligning andstacking them, and then followed by a thermal treatment. To produce ananofiber–biopolymer nanocomposite, a biodegradable polymerpoly(D,L-lactic acid) (PLA, Aldrich) solution (5 % in tetrahydrofuran)was filled within the stacked nanofibrous mesh (heat-treated), homoge-nized by vortexing gently, and then dried and warm-pressed (120 °C).The ratio of nanofiber to PLA was adjusted to 0.5 by weight.

Received: October 28, 2005Final version: December 14, 2005

Published online: June 27, 2006

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