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DOI: 10.1002/adfm.200500750

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


By Hae-Won Kim,* Hyoun-Ee Kim, and Jonathan C. Knowles
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 by employing an electrospinning process based on a solgel precursor is reported for the first time. Under controlled processing conditions, continuous nanofibers have been generated successfully with variable diameters. The excellent bioactivity of the nanofiber is confirmed in vitro within a simulated body fluid by the rapid induction of bonelike minerals onto the nanofiber surface. The bone-marrow-derived cells are observed to attach and proliferate actively on the nanofiber mesh, and differentiate into osteoblastic cells with excellent osteogenic potential. The bioactive nanofibers have been further exploited in various forms, 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 nanofibrous matrix is regarded as a promising next-generation biomaterial in the bone-regeneration field.

1. Introduction
Materials for biomedical applications have been exploited to augment and regenerate human tissues that have been subjected to damage and diseases.[1,2] Over the last decade the demands on synthetic biomaterials have increased significantly, to the point where they are now indispensable because autologous surgery, regarded as the standard implantation technique, has limited material supplies and requires painful secondary operations. Moreover, the alternative allografts or xenografts have serious concerns associated with immunogenic responses.[3] In light of this, significant effort has been devoted to the area of biomaterials and tissue engineering, and this has brought about the development of several materials with clinical promise. Specifically for hard-tissue applications, such as the regeneration and repair of bones and teeth, several bioactive or bioinert materials have been used clinically.[4] Silica-based bio-

[*] Prof. H.-W. Kim Department of Dental Biomaterials, School of Dentistry Dankook University Cheonan 330-714 (Korea) E-mail: kimhw@dku.edu Prof. H.-E. Kim School of Materials Science and Engineering, Seoul National University Seoul 151-742 (Korea) Prof. J. C. Knowles Division of Biomaterials and Tissue Engineering, UCL Eastman Dental Institute London WC1X 8LD (UK) [**] The authors greatly appreciate Dr. Y. H. Koh for his constructive discussion and S. Y. Chae for her experimental assistance. This study was supported by a grant from the Korea Health 21 R & D Project, Ministry of Health and Welfare, Republic of Korea (A060125).

glasses constitute the essential part of such bioactive materials, having already been utilized in numerous orthopedic and dental applications.[5] Most in vivo studies on these bioglasses have confirmed their excellent biocompatibility with hard and even soft tissues. This is attributed mainly to their ability to form a bioactive layer at the interface in contact with living tissues, namely the hydroxycarbonate apatite (HCA) layer, which is equivalent to the mineral phase of human hard tissues.[6] Based on extensive research conducted in vitro and in vivo, bioactive glasses are considered as one of the most-promising biomaterials for the next generation.[6] Most studies in this field have focused on melt-derived glasses, either in the bulk or granular form. Fiber-type melt-derived glasses have been produced with diameters of hundreds to tens of micrometers, and these glass fibers reportedly have the potential to act as cell supporters for extracellular matrix production and tissue regeneration, with a mechanical strength superior to that of the equivalent bulk glasses.[7] When formulated with biodegradable polymers, the potential of this bioactive-glass fiber to act as a tissue-engineering scaffold should be further increased by adopting the shape flexibility of polymers while retaining optimized mechanical properties (toughness, strength, and elastic modulus) and without sacrificing its excellent bioactivity.[8] However, with the melt-spinning approach, the fiber diameter is limited to such micrometer-scale (ca. tens to hundreds of micrometers) because of the associated processing restrictions. More recently, a solgel approach was introduced in the production of bioactive silica glasses.[9,10] Based on the studies undertaken so far, these solgel glasses offer advantages over melt-derived glasses in several aspects. Firstly, the bioactivity of solgel glasses is maintained over a wider composition range (i.e., up to a higher silica content) than the melt-derived glasses.[10,11] Secondly, and more intriguingly, are the processing

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benefits that the solgel approach can (c) (b) (a) provide, that is, the accessibility to systems requiring a scale-reduction for nanobiotechnology. The nanoscale formulation of the bioactive glasses can also open the way to the exploitation of new biomedical areas, such as biocatalysts, biomembranes, biosensors, and reinforcements. Most of all, the bioactive glass matrices and scaffolds with nanofibrous internal structure will unquestionably have a significant im1 m 1 m 1 m pact on the bone-tissue regeneration field.[2] (e) (d) Herein, we report the production of solgel-derived bioactive glass as a nanoscale fiber by means of an electrospinning (ES) technique. The ES process has recently gained significant attention as a methodology for synthesizing fibrous structures on the micro-/nanoscale.[12,13] (f) Compared to the conventional drawing techniques used for producing microscale fibers, the ES method allows the generation of much smaller diameters, i.e., the nanoscale fibers. ES is a simple and cost100 nm Energy (keV) effective process, and is thus utilized in diverse fields, such as electronics, optics, Figure 1. Analysis of the glass nanofibers after electrospinning and heat treatment at 700 C. and tissue engineering.[1214] Various types ac) SEM images of the nanofibers of different average diameters (630 nm, 220 nm, and 84 nm in of polymeric fibers have been produced sequence) with varying sol concentration (1, 0.5, and 0.25 M in sequence). d) TEM image of the nanofiber with dave = 84 nm. e) SAED pattern and f) EDS profile of the nanofiber in (d). with scales of 101000 nm.[1214] In morerecent studies, alkoxide-based solgel precursors were used for the production of metallic oxide fibers by the ES process.[15,16] As such, the sol and uniform fibers were created successfully under all condigel glass system described herein can also satisfy the solution tions. At concentrations of over 1 M, micrometer-scale fibers conditions for the ES process. To the best of our knowledge, were obtained. As the concentration decreased, the diameter this is the first report on the production of a bioactive glass of the fibers was reduced. However, it was difficult to preserve nanofiber, and includes examination of its excellent bioactivity the fiber shape at concentrations below approximately 0.25 M, and osteogenic cell responses. Based on its potential, we also since below this threshold discrete beads were produced extenexploited this nanofiber in various structures, such as bundled sively. After the heat-treatment process, the fiber diameters filaments, fibrous membranes, 3D scaffolds, and in a nanocomwere observed to be reduced by a factor of 23, due to the posite with a biopolymer, showing versatility for practical apburn-out of residual polymeric precursors and the consolidaplications in bone-tissue regeneration. tion of the glass network. The control of the nanofiber diameter made possible by the electrospinning process is effective to produce tissue-regeneration matrices with a pore size and fiber 2. Results and Discussion network tunable to the demands of specific applications. Transmission electron microscopy (TEM) observation of a fiber with an average diameter (dave) of 84 nm showed the fiber To obtain bioactive nanofibers, a glass sol of a composition that retains good bioactivity (here we chose 70 SiO225 CaO5 P2O5) clearly (Fig. 1d). No crystals were formed, as far as could be was prepared and electrospun under appropriate conditions; confirmed by the TEM selected area electron diffraction (SAED) pattern (Fig. 1e). The energy dispersive spectroscopy the ES was then followed by a thermal treatment. Depending (EDS) profile of the fiber revealed the glass composition on the processing conditions, the electrospun glass fibers pos(70 SiO225 CaO5 P2O5) quite well (Fig. 1f). sessed a range of diameters. Among the parameters, we observed that the sol concentration was the most dominant factor The heat-treated nanofibers were subsequently incubated in in controlling the diameter. In Figure 1ac, scanning electron a simulated body fluid to examine their bioactivity by determicroscopy (SEM) showing the morphologies of the nanofibers mining whether they induce the precipitation of bonelike with different average diameters (630 to 84 nm) at varying iniminerals on the surface. Data on the fibers obtained with the tial sol concentrations (1 to 0.25 M) are presented. Continuous 0.25 M sol (dave = 84 nm) are presented as a representative ex 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Adv. Funct. Mater. 2006, 16, 15291535

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ample in Figure 2. After incubation of the fiber mesh for 1 day, the surface became quite irregular with some elongated crystals being detected on the surface by TEM observation (Fig. 2a). When incubated for 3 days, the fiber morphology was changed more significantly, with numerous elongated crystals being generated over almost the entire surface (Fig. 2b). A high-magnification image of the fiber incubated for 1 day clearly 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 confirmed that they consisted of a poorly crystallized apatite with (112) strong and (002) weak rings (Fig. 2d). The EDS analysis of the apatite crystal area shows a higher concentration of Ca and P with respect to that of Si (Fig. 2e, compare data with Fig. 1f). The Ca/P ratio was approximately 1.56, being close to, but slightly lower than, the stoichiometry of pure hydroxyapatite [Ca10(PO4)6(OH)2)], 1.67, which has often been reported in the poorly crystallized or carbonated apatites produced by this kind of biomimetic process, and is more similar to that of bonelike minerals.[17] The ion-concentration change of the medium was monitored using inductively coupled plasma atomic emission spectroscopy (ICP-AES) after incubation of the nanofiber for periods of up to 7 days, as shown in Figure 3a. Initially the prepared medium contained 2.5 and 1 mM of Ca and P, respectively, and no Si. Within a short period of time, both the Ca and P concentrations decreased abruptly and then stabilized with increasing time, while the Si concentration increased initially and then leveled off. Only a slight increase in the Ca concentration was

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Figure 3. Change in a) ion concentration of medium and b) Fourier transform infrared (FTIR) spectroscopy of the nanofiber (dave = 84 nm) before and after incubation for 5 and 7 days. In (b), symbols indicate bands related to silicate (), phosphate (), and carbonate (diamond).

(a)

(b)

(c)

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200 nm

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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) magnification of (a). d) SAED pattern of the crystal in (c), and e) EDS profile of the crystal.

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observed in the first few hours. These changes in the ionic concentration illustrate the dissolution/precipitation process, i.e., the dissolution of Ca, P, and Si from the nanofiber, and the subsequent precipitation of CaP crystals from the medium, which became supersaturated by the dissolution of Ca and P. In detail, a series of reactions occurred, as proposed by Hench and co-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 structure to form silanol groups (SiOH), and through which the precipitation of Ca2+ and PO43 (and mostly CO3 also) occurs, followed by the crystallization of HCA.[6] Of special note, when compared to bulk glasses (melt-derived or solgel synthesized) wherein the increase of Ca and P concentrations (in the initial dissolution region) usually continues for several days to weeks, the nanofibers exhibited a more rapid initial drop (as short as a few days) in the Ca and P concentrations of the medium. This was mainly attributed to the large surface area afforded by the nanoscale fibers, which resulted in faster dissolution and supersaturation of the medium with respect to the HCA crystal nucleation. Specifically, the solgel derived nanofibers developed in this study possess around 23 orders of magnitude higher surface area than the conventional melt-derived glass fibers (diameters of approximately hundreds of micrometers) at an equivalent volume.[16] To confirm whether these nanoscaledriven surface properties influence the rate of the CaP induction on the surface, we performed a comparison test using a sintered solgel glass disk (with a composition same as the nanofiber) under equivalent medium conditions (see Experimental section for details). However, the glass disk layer could not induce the formation of CaP on the surface as rapidly as the nanofiber: the CaP ionic drop analyzed by ICP-AES was observed ca. 7 days after the incubation of the sintered glass disk. The crystals formed from the silica fibers were analyzed with Fourier transform infrared (FTIR) spectroscopy at different incubation times, as shown in Figure 3b. As the incubation time increased, the bands related to the silica glass (800, 930, 1080, and 1200 cm1) were attenuated, while those attributed to the phosphate groups (570, 605, 960, and 10301090 cm1) increased and carbonate bands (870 and 12001300 cm1) appeared, suggesting the formation of carbonate-substituted apatites (HCA), which is similar to the composition of bone mineral. These results concerning the crystal formation on the silicaglass nanofiber have much in common, for the most part, with the previous results obtained for melt-derived or solgel processed bulk glasses. However, the degree of bioactivity (the crystal formation) of the nanofibers in vitro appeared to be significantly enhanced by the nanoscale production process. As suggested above, the extremely large surface area of the nanofibers should accelerate the series of reactions taking place on the glass surface in contact with the medium. Therefore, when these silica glass nanofibers are used as bone substitutes, they could be expected to quickly provide a favorable environment owing to the rapid formation of bonelike minerals on the surface, thereby exhibiting excellent responses for adhesive proteins and cells and, consequently, resulting in improved bone

formation.[2] However, this postulation, established from in vitro conditions, warrants further experiments in vivo when considering the dynamic fluidic effect as well as the mediation of proteins in the series of reactions.[18] The biocompatibility of the glass nanofibers was further assessed by their in vitro cellular responses. We used bonemarrow-derived stem cells (BMSCs) to examine the osteogenic potential of our newly developed bioactive nanofiber. The nanofibrous meshes with an average diameter of 220 nm were used for the cellular test. From electron microscopy images, the cells on the nanofiber mesh with 5 days of culturing were observed to grow favorably (shown in Fig. 4a). On closer examination, the cells spread actively on the nanofibrous surface with numerous cytoplasmic extensions, typical of the osteoblastic cellular growth (Fig. 4b). The grown cells were stained to elicit alkaline phosphatase (ALP) enzyme, which is known to be expressed in the differentiation of osteogenic cells and thus is regarded as an important marker for osteogenic potential and bone-forming ability. As references, data on the sintered solgel glass disk (same composition as the glass nanofiber) and the degradable biopolymer (polycaprolactone, PCL) in a nanofibrous form are also provided. The optical image presented of the glass nanofiber (Fig. 4c1) shows a thicker violet staining of ALP with respect to that on the glass disk (Fig. 4c2), and the difference was clearer when compared to that on the PCL nanofiber (Fig. 4c3). The ALP level, as well as the viability of the cells cultured on the three different samples, was quantified as summarized in Figure 4d. The cells grown on the bioactive glass composition (both nanofiber and disk forms) were more viable than those on the PCL nanofiber, while only a slight difference was observed between the two glass types. However, the expression of ALP on the nanofiber was higher than that on the glass disk (particularly significant at day 5). Based on these cellular results, we confirm that the nanofibrous glass favors the attachment and growth of the BMSCs and induces them to differentiate into osteoblastic cells. Moreover, this osteogenic potential of the nanofiber glass was observed to be stimulated significantly to levels similar to or even higher than those on the relatively flat surfaces with equivalent composition (glass sintered disk), suggesting the morphologically driven (surface associated) properties of the nanofiber, such as nanoscale-roughened morphology, larger surface area, and higher ionic release, play constructive roles in the osteogenic potential. In this study, we used the glass nanofiber with an average diameter of 220 nm, based on the consideration that it is representative of other-sized nanofibers, as we observed similar in vitro apatite-forming behavior among all the nanofibers. However, further study is still warranted regarding the effect of diameter of the nanofiber glass on the cellular responses. Although more extensive works are required as to such effects on the stimulation of osteogenic potential, the present finding drives us to underscore the potential of the nanofiber when considering the perspectives of manipulation and versatility of the nanofibrous form, which is useful in a tissue-engineering matrix (demonstrated in the following Fig. 5). Clearly, the osteogenic potential of our developed nanofiber was mostly endowed by the bioactive solgel glass composition, as

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(a)

(b)

50 m

10 m

(c1)

(c2)

(c3)

(d)

Figure 4. Bone-marrow derived osteoblastic cell responses to the bioactive glass nanofiber: Electron microscopy images of the cells on the nanofibrous mesh at low (a) and high (b) magnification with 5 days of culturing. c1c3) alkaline phosphatase (ALP) staining of the cells grown on the bioactive glass nanofiber (c1) and on other comparison samples for 5 days: solgel glass sintered disk with the same composition as glass nanofiber (c2) and PCL electrospun 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) by ANOVA.

was demonstrated by the above cellular data in its comparison with the degradable but bio-inactive polymeric nanofiber PCL. This bioactive glass nanofiber preserves the beneficial aspects of not only the bioactive composition with high osteogenic potential but also the morphological merits permitting use as a tissue-regeneration matrix like the degradable polymer nanofibers. With its excellent properties for the recruitment of stem cells, the glass nanofiber holds great promise in the tissue-engineering field. However, this postulation, established in vitro, should be substantiated with further in vivo animal studies on the bone-formation ability and the mechanical stability. Nevertheless, the present in vitro observations, including the rapid induction of bonelike minerals and the osteogenic cellular responses, suggest great potential in the hard-tissue-regeneration field.

The usefulness of the bioactive nanofiber was further demonstrated by utilizing it in 3D structures that could find practical applications as cell-supporting matrices and tissue-engineering scaffolds. We formulated the nanofibers as various 3D matrices using our laboratory-developed techniques, as shown in Figure 5. As one example, a macroscale filament was produced after aligning the nanofibers at a high mandrel winding speed (ca. 2000 mm s1) followed by bundling and compacting them (Fig. 5a). This longitudinally aligned filament with an internal nanofibrous structure can be specifically applied to support the cell ingrowth of aligned tissues such as muscles and nerves. The texture designing of the filament for specific parts remains another area for development. A nanofibrous membrane (Fig. 5b) was produced by means of stacking the nanofibrous sheets and warm-pressing followed by heat treatment.

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(a)

(b)

1 m

20 m

10 m

(c)

(d)

network (Fig. 5d). The image shows a wellconstructed nanocomposite comprising the dense nanofibrous network and the polymeric filler. The glass nanofibers were observed to be dispersed well and distributed uniformly in the PLA matrix. Further macroporous design of the nanocomposite is expected to be achievable by utilizing diverse scaffolding techniques. This nanocomposite approach, employing the nanofibrous bioactive inorganic in conjunction with biodegradable polymer, should have good prospects in the boneregeneration field because of the combinatorial benefits, such as shape flexibility and moldability, mechanical properties tunable to the bone matrix, and the bioactivity and degradability. Further evaluations on the in vivo tissue responses and mechanical properties of the nanocomposite are currently underway.

3. Conclusions
Bioactive nanofibers were produced successfully by employing an electrospin1 m 1 m 10 m 1 mm ning technique using a glass solgel precursor with a bioactive composition. The Figure 5. 3D structured matrices of the electrospun glass nanofibers for tissue regeneration. a) Filbiomedical usefulness of bioactive glasses, ament: electrospun nanofibers were aligned and bundled into a microfilament. b) Membrane: elecas has been established over recent detrospun sheets were stacked and pressed gently (surface (upper) and cross-section view (lower)). cades, was imparted onto the nanofibrous c) 3D macroporous scaffold: ready-made filaments were architectured using a negative-mold techstructure. We observed that the nanofiber nique and then heat-treated; enlarged in inset. d) Polymer-filled nanocomposite: heat-treated fibrous mesh was filled with biodegradable polymer PLA; enlarged in inset. possessed excellent bioactivity and osteogenic potential in vitro. Moreover, possible formulations of the bioactive nanofibers, such as aligned filThe nanofibers were observed to maintain their initial fibrous aments, nanofibrous membranes, macroporous scaffolds, and in structure and form a nanoporous 3D structure. Potential applications for this nanofibrous membrane are believed to include nanocomposites with biopolymers, were provided for practical wound healing and guided bone/cartilage tissue regeneration, applications. Based on the present findings, the bioactive glass wherein polymeric matrices are still dominantly used. A 3D macnanofiber developed herein is proposed as one of the most promising next-generation biomaterials. roporous scaffold, characterized by a microfilament framework with a nanofibrous internal structure, was also created to allow macropores within the nanofibrous structure (Fig. 5c). Here, a 4. Experimental negative-molding technique was used, where the microfilaments of nanofibers were organized with carbon filaments and then Sol Preparation and Electrospinning: The glass composition used in heat-treated to produce a 3D open-channeled network with conthis study (70 SiO225 CaO5 P2O5) was chosen based on those of pretrolled pore size (ca. 500 lm) and porosity (ca. 50 %). This macviously developed bulk glasses which exhibit bioactivity in vitro, and roporous design should facilitate vasculisation and cell ingrowth the solgel processing conditions were modified from other reports [10,11]. The glass precursors were added at appropriate ratios (tetramore effectively through macroscale open channels. Of particular ethyl orthosilicate, calcium nitrate, and triethyl phosphate added in seinterest is the fact that the nanofibrous surface can provide favorquence with a 6 h interval between each addition, all from Aldrich) in able surroundings for the initial protein adhesion and cellular rean ethanol/water solution containing 2 % HCl (1 N) used for catalysis. sponses, which is typically dissimilar to conventional 3D scaffolds The sol mixture was stirred for 24 h and aged without stirring at 25 C for 24 h followed by a further 48 h at 40 C. The acidic catalyst was necwith a dense surface.[19] These 3D matrices designed with bioacessary in order to produce a clear sol. Prior to electrospinning (ES), tive nanofibers can have their mechanical properties optimized polyvinylbutyral (PVB, from Aldrich) dissolved in ethanol at 10 % was when hybridized with biopolymers. We infiltrated a biodegradadded to the sol at an equivalent volume to adjust the rheological propable polymer (5 % poly(D,L-lactic acid) (PLA) solution dissolved erties of the sol, so that they would be fit for the fiber generation during ES. 2 mL of the solution was loaded in a syringe and injected into a in tetrahydrofuran) into the interspacings of the glass nanofiber 1534 www.afm-journal.de

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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 min1). The electrospun fibers were subsequently heat-treated at 700 C for 3 h in air at a heating and cooling rate of 1 and 5 C min1, respectively. The heat-treatment temperature was determined to be high enough to eliminate organic sources and nitrates completely but low enough to avoid crystallization [10,11]. As a reference for the biological tests, we also prepared disk-type pallets of the solgel composition (12 mm 1 mm thickness) after crushing the gelled product into powders, molding them in a metal die, and partial sintering at 700 C for 3 h. As another reference material, a degradable biopolymer, polycaprolactone (PCL, Sigma), was prepared in a nanofibrous matrix (average fiber diameter 860 nm) by electrospinning using a solvent (tetrahydrofuran/N,N-dimethylformamide = 3:2) under adjusted conditions (voltage: 12 kV, distance: 8 cm, and injection rate: 0.05 mL min1). Bioactivity Test: The heat-treated glass nanofibers were subjected to a simulated body fluid (containing similar ion concentrations to a body plasma), in order to form bonelike mineral HCA crystals on the glass surface due to a dissolution/precipitation process [20]. This final step was carried out in an effort to 1) provide a preliminary assessment of the bioactivity of the glass nanofibers in vitro, since the HCA formation has been regarded as the key phenomenon in explaining the biocompatibility of bioactive materials [2], and 2) utilize the HCA-surface-modified glass nanofibers directly. 10 mg of fiber were incubated in 10 mL of medium for up to 7 days without refreshing. The glass disk prepared as a reference was also tested under the same conditions. At predetermined time points, the change in the ion concentration of the medium was monitored with inductively-coupled plasma atomic-emission spectroscopy (ICP-AES, Shimadzu), and the change in the chemical bonding structure of the fiber was analyzed with Fourier transform infrared (FTIR, System 2000, Perkin-Elmer) spectroscopy. The fiber diameter was measured from the SEM image in 20 different sections, and averaged. The morphology of the fibers was characterized with fieldemission scanning electron microscopy (FESEM, JSM6330F, JEOL), and transmission electron microscopy (TEM, CM20, Philips). The composition and crystal pattern were also analyzed from the TEM operation. Cellular Response Assay: To observe the biocompatibility of the nanofiber, in vitro cellular responses were examined using rat bonemarrow derived mesenchymal stem cells (BMSCs). Bone marrow was harvested from the adult rats (age 48 weeks, Korean). The whole marrow was flushed with a minimum essential medium (a-MEM) from the excised proximal and distal epiphyses of femora and tibiae using a syringe. After centrifugation of the marrow at 1000 g, the supernatant was collected and suspended again in a-MEM. The homogeneously suspended cells were maintained in a culture flask containing growth medium (a-MEM, 2 mM glutamine, 100 U mL1 penicillin, and 100 mg mL1 streptomycin), supplemented with 15 % fetal calf serum (FCS) under a humidified atmosphere of 5 % CO2 at 37 C. After 1 day of incubation, the cells were washed with phosphate-buffered saline (PBS) to discard nonadherent cells and then cultured further for up to 7 days to reach near confluence. For accurate measurement of the cellular tests, the glass nanofiber was prepared by supporting it on a bioinert zirconia sintered disk. Glass nanofibers prepared with an average diameter of 220 nm were chosen as a representative example. For the purpose of comparison, the solgel glass disk pallet and PCL nanofibrous 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 the three types of samples at a seeding density of 5 104 cell cm2. At this time of culturing, the medium was supplemented with 50 lg mL1 sodium ascorbate, 10 mM sodium b-glycerol phosphate, and 10 nM dexamethasone in order to induce osteoblastic differentiation. The culture medium was changed every three days. At the culturing periods of 2 and 5 days, the cell viability was assessed by means of an MTT method [21]. The cell-growth morphology was 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 by detecting the alkaline phosphatase (ALP), which is an important osteo-

genic differentiation marker. The cells cultured on the samples for 5 days were treated enzymatically to stain the ALP and reveal it in violet. Moreover, the ALP expression level was quantified following our previous protocol [21]. At each culturing period, the cell layers were detached by treatment with 0.1 % Triton X-100, and the cell pellets were disrupted via cyclic freezing/thawing processes. Aliquots of the samples normalized to the total protein content were enzymatically reacted with p-nitrophenyl phosphate (p-NPP) substrate following the manufacturers instruction (Sigma ALP kit 104). The quantity of p-nitrophenol (p-NP) produced was measured at an absorbance of 410 nm using a spectrophotometer. The cellular tests were performed on five replicate samples and data were compared using one-way ANOVA analysis with statistical significance at p < 0.05 and p < 0.01. 3D Structure Formulation: For the fabrication of the nanofiber in 3D structured matrices (bundled filament, nanofibrous membrane, macroporous scaffold, and nanocomposite with biopolymer), several laboratory-developed techniques were utilized. A filament was produced by bundling 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 the production of fibrous membrane, the electrospun nanofiber mesh was stacked layer-by-layer and warm-pressed 80 C, and then heat-treated under the same conditions. The 3D macroporous scaffolding was performed using the nanofibrous filament (diameter of 600 nm) and a designed carbon mold as a support material, by means of aligning and stacking them, and then followed by a thermal treatment. To produce a nanofiberbiopolymer nanocomposite, a biodegradable polymer poly(D,L-lactic acid) (PLA, Aldrich) solution (5 % in tetrahydrofuran) was filled within the stacked nanofibrous mesh (heat-treated), homogenized 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, 2005 Final version: December 14, 2005 Published online: June 27, 2006

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