Bioactive and degradable hybridized nanofibers of gelatin ... - CiteSeerX

Bioactive and degradable hybridized nanofibers of gelatin–siloxane for bone regeneration Ju-Ha Song,1 Byung-Ho Yoon,1 Hyoun-Ee Kim,1 Hae-Won Kim2 1 School of Materials Science and Engineering, Seoul National University, Seoul 151-742, Korea 2 Department of Biomaterials Science, School of Dentistry, Dankook University, Cheonan 330-714, Korea Received 6 November 2006; accepted 13 December 2006 Published online 23 July 2007 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jbm.a.31330 Abstract: Organic–inorganic hybridized nanofibers constituted of gelatin and siloxane were generated by the electrospinning technique for use as bone regeneration matrices. The composition of the nanofibers selected was to be both degradable and bioactive. Precursors of gelatin and siloxane were dissolved in a modified acidic solvent composed of acetic acid, ethyl acetate, and distilled water. The hybridized nanofibers with various compositions (gelatin/siloxane ¼ 1/2, 1, and 2 by weight fraction) were successfully electrospun under the adjusted processing conditions. Compared to the pure gelatin nanofiber, the hybridized nanofibers showed improved chemical stability in a saline solution. This was attributed to the cross-linking

effect of the siloxane with the gelatin chains. Osteoblastic cells were observed to attach, spread, and populate actively on the hybridized nanofiber matrices. In particular, the cells on the hybridized nanofibers were recruited to elicit better osteoblastic activity (alkaline phosphatase) with respect to those on the pure gelatin. The newly-developed hybridized nanofiber is considered to be useful as a bone regeneration matrix, due to its nanofibrous structural trait as well as its degradability and bone cell activity. Ó 2007 Wiley Periodicals, Inc. J Biomed Mater Res 84A: 875–884, 2008

INTRODUCTION

conventional processing methods.6,7 Biomaterials with various compositions, including polymers, ceramics and their composites, have been developed in the form of nanofibers.1–6,8–11 Gelatin is a kind of natural biopolymer derived from collagen and has been widely used in the biomedical field, because of its merits, including its biological origin, biodegradability, hydrogel properties, and commercial availability at a relatively low cost.12 Recent studies have reported the electrospinning of gelatin using acidic solvents, however, the biodegradability of gelatin still remains a significant concern. Its polymeric composite with poly(e-caprolactone) has been suggested by Zhang et al. as a feasible approach that can produce polymeric nanofibers with reasonable degradability and cellular affinity.11 Although various polymeric composites have been developed comprising natural and synthetic polymers, to optimize their properties,13–15 the usefulness as bone regenerative matrices is still wanting, requiring improvements to be made in terms of their bioactivity, and bone forming ability. For bone regeneration applications, nanofibrous matrices with several compositions have been suggested, such as poly(caprolactone), poly(lactic acid), and collagen. However, these polymeric nanofibers normally do

Over the past few decades, significant level of research has been made in the development of tissue-regenerative biomaterials in terms of finding bioactive compositions and designing novel processings and methodologies. The recent advent of nanotechnology has also spurred the processing and design of materials for medical applications. The electrospinning process has recently gained widespread interest as it provides a simple and effective means of producing ultrafine fibers with diameters ranging from micrometers down to tens of nanometers.1,2 In the field of tissue engineering, this process is likely to play a crucial role in generating tissue regenerative matrices with a nanoscale fibrous structure.3–5 This nanofibrous structure is regarded as a promising architecture in the sense that the natural extracellular matrix exhibits a fibrous structure with diameters on the nanoscale, which is far smaller than that which can be achieved with Correspondence to: H.-W. Kim; e-mail: kimhw@dankook. ac.kr ' 2007 Wiley Periodicals, Inc.

Key words: hybridized nanofiber; electrospinning; gelatin; siloxane; bone regeneration

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not have a sufficient level of bioactivity in terms of their apatite forming ability under biological conditions or the stimulation of osteogenic factors, as compared to bioactive inorganic materials such as calcium phosphate ceramics and bioactive glasses. In a more recent study, an elegant system was developed by Kim et al., wherein a gelatin and apatite coprecipitated sol was electrospun to produce a nanocomposite fiber with diameters of hundreds of nanometers.16 The nanocomposite nanofibers showed significantly improved osteoblastic responses with respect to the pure gelatin and were proposed to be useful for guided bone regeneration.16 Other nanofiber systems based on organic–inorganic composites have also been exploited for use as bone regeneration matrices, including the composites of poly(lactic acid) with hydroxyapatite and poly(caprolactone) with calcium carbonate.17,18 With the same goal in mind, we attempted to create a bone regenerative nanofibrous matrix that is bioactive and has an appropriate level of degradability. The composition selected was a gelatin–siloxane hybrid. In previous studies, the gelatin–siloxane scaffold system was reported to be bioactive, forming apatite crystals on its surface in a simulated body fluid.19,20 Hybrid materials derived from the integration of biopolymers with some inorganic species are known to be both biodegradable and bioactive. Moreover, siloxane was found to be effective in stabilizing the biodegradability of gelatin through the cross-linking of the gelatin chains.19 In the current study, we report the production of gelatin–siloxane hybridized nanofibers. In particular, a cosolvent containing ethyl acetate was newly composed to improve the electro-spinnability of the gelatin–siloxane hybrid sol. The possibility of electrospinning this hybrid sol to produce gelatin–siloxane nanofibers is described and the feasibility of using the hybridized nanofibers as bone regeneration matrices is addressed in terms of their chemical and biological properties.

MATERIALS AND METHODS Preparation of hybrid sols As the reagents of hybrid sols for electrospinning, gelatin (type B, from bovine, Sigma), 3-(glycidoxypropyl) trimethoxysilane (GPTMS) (98%, Aldrich), glacial acetic acid (99.99%, Aldrich), and ethyl acetate (anhydrous, 99.8%, Aldrich) were used. Calcium chloride (CaCl2) (anhydrous, 99.99%, Aldrich) was kept in an oven at 608C to avoid its hydration with the moisture in the air before usage. A gelatin solution (10 wt %) was prepared by dissolving the gelatin in a cosolvent made of acetic acid, ethyl acetate, and water at a weight ratio of 4.2:2.8:2. A series of hybrid Journal of Biomedical Materials Research Part A

TABLE I The Prepared Compositions and Characteristics of the Gelatin – Siloxane Hybrid Sols Used for Electrospinning Mass Fraction Hybrid I II II-I III III-I IV IV-I

fG/(GþS) fCa/(GþS) 1 0.33 0.33 0.50 0.50 0.67 0.67

0 0 0.25 0 0.25 0 0.25

Sol Feature

Aging Time

Yellow, transparent Yellow, transparent Yellow, translucent Yellow, transparent Yellow, translucent Yellow, transparent Yellow, translucent

* 10 h 12 – 24 h 12 – 24 h 12 – 24 h 12 – 24 h 18 – 30 h 18 – 24 h

sols made of gelatin and siloxane were then prepared as follows: The solvent composition for the hybrid sols was the same as that used for the gelatin solution. Various ratios of siloxane to gelatin were used, viz. 1:1, 2:1, and 1:2, as indicated in Table I, as fG ¼ 0.50, fG ¼ 0.33, and fG ¼ 0.67, respectively. Separately, CaCl2 (2.5 wt %) was also added to the hybrid sols to improve the bioactivity of the gelatin–siloxane nanofibers and then the solutions were homogenized completely. The prepared sols were then aged at 378C prior to electrospinning. Table I summarizes the composition and characteristics of the hybrid sols.

Electrospinning into nanofibers The prepared sols were loaded into a syringe (with a capacity of 5 mL and a needle diameter of 500 lm) and injected onto a metal collector under a high direct current field strength (12 kV/8 cm) at an injection rate of 0.10 mL/h. The electrospun samples were dried under vacuum for 2 days at 378C overnight to evaporate the remaining solvent.

Characterization The morphology of the electrospun nanofibers was examined by means of field-emission scanning electron microscopy (FESEM, JEOL, Japan). The diameter of the nanofibers was measured from 30 different, arbitrarily selected samples and averaged. The chemical analysis of the electrospun hybrids was conducted using a Fourier transform infrared (FTIR) spectrometer (Nicolet Magma 550 series II, Midac, USA) in the wavelength range between 4000 and 400 cm1 at a resolution of 1 cm1 with an average of 64 scans per sample. As a reference, the monomers of GPTMS were polymerized through gelation at 378C for 10 days. To confirm the stability of the hybrids, the nanofibers of gelatin and the hybrids were soaked in phosphate buffered saline (PBS) solution at 378C. After 7 days, the fibers were washed gently with distilled water and observed using FESEM. The incorporation of Ca within the hybridized nanofibers was observed using a Ca staining assay. The hybridized nanofibers (fG ¼ 0.50) with or without Ca were soaked in 0.5% Alizarin red S/PBS (no calcium addition) for 10 min at room temperature and washed with PBS.

GELATIN–SILOXANE HYBRIDIZED NANOFIBER FOR BONE REGENERATION

In vitro cellular assay The specimens used for the cell tests were prepared by electrospinning the sols on cover glasses (diameter of 12 mm), which were mounted on a metal collector. The final thickness of the nanofibers was measured to be *10– 20 lm. In particular, the gelatin nanofiber was cross-linked using 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide for 3 h, followed by washing fully with distilled water and drying. Murine-derived preosteoblast MC3T3-E1 (ATCC, CRL2593) cells were cultured in regular culturing media consisting of a-modified minimum essential medium (a-MEM; Join Bio Innovation, Korea) supplemented with 10% fatal bovine serum (FBS; GIBCO, USA) and 1% antibiotic/ antimycotic (GIBCO, USA) in a humidified atmosphere containing 5% CO2 at 378C. The cells were plated on the specimens at a density of 1.5 3 104 cells/cm2 and cultured in an osteogenic medium (the regular culturing media plus 10 mM b-glycerol phosphate and 50 lg/mL L-ascorbic acid). After harvesting the cells at 3 days, the cell viability was measured as the mitochondrial NADH/NADPHdependant dehydrogenase activity, using a cell proliferation assay kit (CellTiter 96 Aqueous One Solution, Promega). The culturing medium was removed and 100 lL of MTS solution in 1 mL of culture media was added to each well and left to stand for 3 h. Finally, the colorimetric measurement of a 200 lL sample of each solution was performed on a spectrophotometer at 490 nm. The cell morphology at 1 and 3 days was observed with confocal laser scanning microscopy (CLSM, LSM 510 NLO, Zeiss) and SEM. For the observation by CLSM, the cells on the samples were washed twice with PBS, fixed in 3.7% formaldehyde solution in PBS for 10 min at room temperature, and dyed with Alexa Fluor1 546 phalloidin (Molecular Probes, Eugene, Oregon) and ProLong1 Gold antifade reagent with DAPI (Molecular Probes, Eugene, Oregon). For SEM, the samples were prepared by fixing with glutaraldehyde (2.5%), dehydrating with a graded series of ethanols (75, 90, 95, and 100%), critical point drying, and gold coating. For the assessment of the alkaline phosphatase (ALP) activity, the cells were cultured for 10 days. After harvesting the cells, the cell pellets were resuspended by vortexing them in 150 lL of 0.1% Triton X-100 and further disrupted by means of cyclic freezing/thawing processes. The cell lysates were quantified using a protein assay kit (BioRad, Hercules, CA) and assayed colorimetrically for their ALP activity using p-nitrophenyl phosphate as a substrate (ALP yellow liquid substrate for ELISA, Sigma, St. Louis, MO) and measured at 405 nm using a microplate reader. The statistical analysis was performed using the oneway analysis of variance (ANOVA) and significance was considered at p < 0.05.

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water. In particular, the addition of ethyl acetate improved the spinnability of the solution and decreased its acidity, as was previously reported in the case of the electrospinning of gelatin.21 When using conventional acidic solvents without ethyl acetate, it was difficult to preserve the nanofibrous structure of the gelatin–siloxane hybrid electrospun product, owing to the prolonged gelation period of the sols. For the successful electrospinning of the hybrid sols, the optimal composition of the cosolvent was determined to be a ratio of acetic acid to ethyl acetate of 3:2 by weight with 20 wt % distilled water. Although the gelatin and siloxane mixtures were initially unclear, they became transparent with prolonged aging, and the aging time increased as the ratio of gelatin to siloxane increased. The solubility limit of gelatin in the optimal cosolvent was observed to be *11 wt % and the siloxane was more soluble than gelatin in the cosolvent. As a result, the solubility limit of the hybrid sols was found to be *20 wt %. When CaCl2 was added to the hybrid sols that were then aged, the solutions became translucent without any precipitates being observed. The weight fraction of CaCl2 with respect to gelatin and siloxane was required to be less than 0.25, because above this fraction precipitation started to occur. The aging

RESULTS Hybrid sols Gelatin and siloxane were dissolved in the cosolvent made of acetic acid, ethyl acetate, and distilled

Figure 1. SEM morphologies of the gelatin–siloxane hybridized nanofibers electrospun with different sol compositions: (A) fG ¼ 0.50 with 7.5 wt % gelatin, (B) fG ¼ 0.50 with 10 wt % gelatin, (C) fG ¼ 0.67 with 6.7 wt % gelatin, (D) fG ¼ 0.67 with 10 wt % gelatin, (E) fG ¼ 0.33 with 5 wt % gelatin, and (F) fG ¼ 0.33 with 6.7 wt % gelatin. Journal of Biomedical Materials Research Part A

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Figure 2. Diameter of the gelatin–siloxane hybridized nanofibers as represented with respect to the material concentration at different compositions.

time for the sols also had to be not more than 2 days, as otherwise the sols underwent gelation.

Nanofiber generation

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shown in Figure 1. In the case of the composition in which the gelatin and siloxane were present at an equivalent weight (gelatin:siloxane ¼ 1:1, fG ¼ 0.5), the nanofibers were generated well without any beads being formed [Fig. 1(A)]. When using a higher concentration of these two materials with the same composition, a similar nanofiber morphology was observed, while the diameter was increased [Fig. 1(B)]. This successful generation of nanofibers was similarly observed when the fraction of gelatin exceeded that of siloxane (gelatin:siloxane ¼ 2:1, fG ¼ 0.67) [Fig. 1(C,D)]. However, when the fraction of siloxane exceeded that of gelatin (gelatin:siloxane ¼ 1:2, fG ¼ 0.33), the morphology of the nanofibers somewhat collapsed [Fig. 1(E,F)]. Figure 2 shows the diameter of the nanofibers as represented with respect to the concentration of materials at each composition. As the concentration of the materials increased, the diameter of the fibers increased. At the same concentration of the materials, the composition with a higher gelatin fraction produced nanofibers with a larger diameter. Consequently, it was possible to generate gelatin–siloxane hybridized fibers with average diameters ranging from 40 to 670 nm by adjusting the composition and material concentration used in the electrospinning process.

The typical electron morphologies of the electrospun nanofibers of the gelatin–siloxane hybrids are

Figure 3. FTIR spectra of the electrospun nanofibers with different compositions : (A) pure gelatin, (B) fG ¼ 0.67, (C) fG ¼ 0.50, (D) fG ¼ 0.33. As references, GPTMS before (E) and after gelation (F) are included. (*; OH str, &; CH2 str, 3; CH3 str, ~; epoxide, !; C O C str, 3; CH3 rock, l; Si O Si str). Journal of Biomedical Materials Research Part A

Figure 4. SEM morphologies of the nanofibers before (A, C, E) and after (B, E, F) the immersion test in PBS for 7 days at 378C: The compositions of the nanofibers are (A, B) pure gelatin, (C, D) fG ¼ 0.50, and (E, F) fG ¼ 0.33.


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Figure 5. (A) SEM morphologies of the nanofibers containing Ca and (B) Ca-stained image of the nanofibers. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Characteristics of hybridized nanofibers The FTIR absorption spectra shown in Figure 3 represent the chemical bonding status of the gelatin– siloxane hybrids nanofibers. As a reference, the spectrum of pure gelatin is shown in Figure 3(A). The spectra of the silane (GPTMS) monomer and its homopolymer after gelation are also given in Figure 3(E,F), respectively. The spectrum of the gelatin nanofiber shows the general gelatin bands including the N H stretching at *3310 cm1 for the amide A, C H stretching at *3063 cm1 for the amide B, C¼ ¼O stretching at 1600–1700 cm1 for the amide I, N H deformation at 1500–1550 cm1 for the amide II, and NH deformation at 1200–1300 cm1 for the amide III band.22 The GPTMS silane monomer has four characteristic peaks at 2840 cm1 (methoxy group), 1192 and 914 cm1 (epoxy group), and 825 cm1 (methyl group) [Fig. 3(E)]. After the polymerization of the silane, OH stretching bands and SiOSi

stretching bands were additionally observed, along with a significant reduction in the intensity of the band at 825 cm1 (methyl group) [Fig. 3(F)]. In the spectrum of the gelatin–siloxane hybrid nanofibers [Fig. 3(B–D)], the SiOSi stretching bands were observed in the 1100–1000 cm1 region, indicating the occurrence of siloxane gelation in the hybridized nanofibers and the possible existence of chemical bonding between gelatin and siloxane.19 The water stability of the electrospun nanofibers was evaluated by soaking the nanofiber meshes in PBS at 378C for 7 days and observing the morphological change. Figure 4 shows the SEM morphology of the nanofibers before and after the soaking test. The gelatin nanofiber completely disappeared with soaking [Fig. 4(B)]. As the fraction of siloxane increased, the stability of the nanofibers was observed to increase. At the composition fG ¼ 0.50, the fiber morphology slightly changed but the nanofibrous form was almost preserved [Fig. 4(D)]. At the Journal of Biomedical Materials Research Part A

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Figure 6. MC3T3-E1 cell morphologies on the nanofibrous matrices after culturing for 1 day: (A) cross-linked gelatin, (B) fG ¼ 0.50, (C) fG ¼ 0.50 containing Ca, and (D) fG ¼ 0.67 containing Ca. The cells were dyed with Alexa-546 (red) and DAPI (blue). [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

composition fG ¼ 0.33, the initial nanofiber morphology was almost completely maintained [Fig. 4(F)].

with Ca ions and causes the color of the specimen containing Ca to turn red.23

Calcium incorporated nanofibers

Osteoblastic responses

Calcium was added to the hybridized nanofibers to improve their biocompatibility in the biological environment.20 When CaCl2 was added to the pure gelatin solution, the gelatin was precipitated along with CaCl2 and it was difficult to electrospin the solution. However, the hybrid sols containing Ca ions were stable, without any precipitation occurring, and were easily electrospun into nanofibers. Figure 5(A) shows the hybridized nanofibers containing CaCl2. The fiber morphology was very similar to that of the hybridized nanofibers free of Ca. The incorporation of Ca into the hybridized nanofibers was confirmed by means of a Ca-staining assay. As shown in Figure 5(B), the color transition of Alizarin red S in the Ca-added nanofiber indicates the existence of Ca ions, because Alizarin red S reacts

The in vitro biocompatibility of the hybridized nanofibers was assessed in terms of the proliferation and differentiation of murine-derived MC3T3-E1 cells on the nanofibrous substrates. The sample group consisted of the cross-linked gelatin nanofiber and hybridized nanofibers with three different compositions (fG ¼ 0.50 free of Ca, fG ¼ 0.50 containing Ca, and fG ¼ 0.67 containing Ca). The cell adhesion behavior on the nanofibers was evaluated using a fluorescent image colored in red (actin) and blue (nucleus). As shown in Figure 6, the cells were observed to adhere and spread actively on all of the nanofiber substrates. After culturing for 3 days, the typical cell growth morphology was observed with SEM and CLSM. In Figure 7(A), the images of the cells on the hybri-

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Figure 7. (A) Cell morphologies observed by SEM (left) and CLSM (right) after culturing for 3 days on the nanofibrous matrix with a composition of fG ¼ 0.50 containing Ca and (B) cell proliferation on each substrate after culturing for 3 days as quantified by the MTS assay. A statistically significant difference (ANOVA, p < 0.05) was observed on the hybridized nanofiber with a composition of fG ¼ 0.50 containing Ca with respect to the pure gelatin nanofiber. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

dized nanofiber (fG ¼ 0.50 with Ca) are shown as a representative example. Although it was difficult to distinguish the cells from the material surface by means of the SEM image (left-side), the fluorescent image revealed the presence of a number of cells grown on the substrate (right-side). The surface of the nanofibrous substrate was almost completely covered with cells, which were in intimate contact with each other, with extensive cytoskeletal expansions. This cell growth morphology was similarly observed in the other samples (not shown here). The viability of the cells at day 3 was measured using the MTS assay,23 as shown in Figure 7(B). The data were represented with respect to the control tissue culture plastic. The cell proliferation on the

cross-linked gelatin nanofiber was similar to that on the control. The hybridized nanofibers showed better cell proliferation levels than the gelatin nanofiber. A significant difference with respect to the gelatin nanofiber (p < 0.05) was observed only in the case of the hybridized nanofiber with the composition fG ¼ 0.50 containing Ca. The osteoblastic differentiation of the MC3T3-E1 cells was evaluated by measuring the ALP activity at 10 days. The ALP was better expressed by the cells on the hybridized nanofibers than those on the gelatin nanofiber (Fig. 8). A significant difference with respect to the gelatin nanofiber (p < 0.05) was observed in the case of the Ca-containing hybridized nanofibers (both fG ¼ 0.50 and 0.67). Journal of Biomedical Materials Research Part A

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Figure 8. ALP activity for MC3T3-E1 cells after 10 days of culturing on the nanofibrous matrices. A statistically significant difference (ANOVA, p < 0.05) was observed on the hybridized nanofibers containing Ca with respect to the pure gelatin nanofiber.

DISCUSSION The electrospinning technique has come to be regarded as a facile tool to produce nanofibrous matrices that are applicable in the field of tissue regeneration.3,4 The morphological features of the nanofiber make it highly attractive for use as a substrate to populate and support tissue cells. Since the extracellular matrices of connective tissues mainly exhibit a fibrous network of collageneous proteins with a size of tens to hundreds of nanometers, the electrospun nanofibrous substrates at least mimic the morphological traits of the tissues.6–8 This study utilized the electrospinning method to generate bone regenerative matrices with a nanofiber structure, whose composition is bioactive and degradable. The composition selected was gelatin– siloxane hybrid, because previous studies have shown the excellent in vitro bioactivity of this composition, such as its apatite forming ability in a simulated body fluid and bone cell responses.20 For the hybrids to be electrospun into nanofibers, various fabrication conditions such as the type of solvent and the equipment parameters need to be satisfied. In particular, the conventional acid solvents used in the electrospinning of gelatin were not appropriate for the electrospinning of the hybridized sols. As a result, we used a newly-developed cosolvent made of ethyl acetate in concert with acetic acid and water. Previously, we observed that the addition of ethyl acetate to the acidic solvent was highly effective in improving the electro-spinnability and chemical stability of gelatin by reducing the pH.21 In the case of the gelatin–siloxane hybridized composition, the Journal of Biomedical Materials Research Part A

addition of ethyl acetate was observed to be even more beneficial for the creation of well-shaped nanofibers. Gelatin–siloxane hybridized nanofibers were successfully produced with average diameters in the range of 42–667 nm, and this was significantly larger than the diameter of the pure gelatin nanofiber (47– 145 nm),21 suggesting that the addition of siloxane increased the diameter of the fibers remarkably. As the solubility of gelatin in the cosolvent was limited to *11 wt %, the siloxane-containing solution is considered to modify the rheological properties of the solution and thicken the fibers. As a result, the hybridized nanofibers could be obtained with diameters in a broader range, extending from tens to hundreds of nanometers. The effective role of the siloxane added into the gelatin composition was observed in the chemical stability of the product. As illustrated in Figure 4, the pure gelatin nanofiber, even if cross-linked with EDC, which is one the well-known cross-linking agents, was observed to be degraded rapidly in a saline solution. In practice, this degradability of gelatin is often regarded as one of the major problems involved in using gelatin nanofibers as cell supportive matrices.6,21 However, the gelatin–siloxane hybridized nanofibers, even without the external crosslinking process, showed sufficient chemical stability, and their stability increased with increasing siloxane fraction. It was reported that the addition of siloxane to natural polymers including gelatin improved the structural stability of the polymers by bridging the chains of polymers through the reaction between the active groups in the polymer (NH2 or COOH) with siloxane, leading to the formation of polymersiloxane grafts.24–26 This cross-linking effect of siloxane within the gelatin matrix is highly beneficial in the sense that the hybridized composition does not need any secondary cross-linking process and can be securely used as a substrate to recruit cellular processes. Moreover, it is further anticipated that the in situ cross-linking can be highly efficient in the case of the nanofiber system wherein drugs or bioactive molecules can be loaded for therapeutic treatments because during the cross-linking process the stability and activity of the incorporated molecules easily weaken. The active role of the siloxane hybridized in the gelatin was reflected on the osteoblastic responses. The hybridized matrices were observed to support the adhesion and spreading of osteoblastic cells, as manifested by the well-developed cytoskeletal processes along the nanofibrous networks at day 1, as well as the population of the cells within the matrix, as confirmed by the full coverage of the surface by the cells at day 3. In particular, we incorporated Ca within the hybridized nanofibers to improve the


bone cell activity, since Ca ions were previously shown to enhance the in vitro apatite forming ability within a simulated body fluid when added to the gelatin–siloxane gel matrices.19,20 The addition of Ca to the gelatin–siloxane composition was found to have little effect on the electro-spinnability. However, the bone cell responses were significantly altered by the incorporation of Ca. The viability of the cells assessed at day 3 was better on the hybridized nanofibers than on the pure gelatin equivalent and was even better on the Ca-incorporated one. Of special note was that the ALP activity at day 10 was significantly higher on the Ca-incorporated nanofibers than on the pure gelatin nanofiber. The enzyme ALP, excreted in the cell membrane, is known to be intimately involved in the osteoblast differentiation process.27 Moreover, it regulates the phosphate metabolism and the formation of bone minerals. As such, ALP is regarded as a useful indicator of osteoblastic differentiation and bone forming ability. Therefore, the higher ALP activity of the cells on the hybridized composition containing Ca strongly suggests that this composition is better at encouraging the cells to differentiate into bone-associated cells with bone forming ability. Currently, more in-depth studies on the hybridized nanofibers are under way, particularly cellular assays to examine the osteogenic potential of the compositions, as well as animal tests to find clinical applications for them, such as in guided bone regeneration membranes and tissue engineering matrices.

CONCLUSIONS Gelatin–siloxane hybridized nanofibers were newlydeveloped using the electrospinning method. A cosolvent composed of acetic acid, ethyl acetate, and distilled water was used to enable the hybrid sols to be electrospun into nanofibers. The hybridized nanofibers exhibited better chemical stability than the pure gelatin nanofiber, because of the cross-linking role of the siloxane in the gelatin chains. Osteoblastic cells were shown to attach and populate well on the hybridized nanofibers. Moreover, the hybridized nanofibers stimulated the cells to elicit osteoblastic activity at levels even higher than those observed in the case of the pure gelatin nanofiber. The gelatin– siloxane hybridized nanofibers are considered to be useful as bone regenerative matrices.

References 1. Langer R, Tirrell DA. Designing materials for biology and medicine. Nature 2004;428:487–492.

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2. Dankers PYW, Harmsen MC, Brouwer LA, Van Luyn MJA, Meijer EW. A modular and supramolecular approach to bioactive scaffolds for tissue engineering. Nat Mater 2005;4: 568–574. 3. Chronakis IS. Novel nanocomposites and nanoceramics based on polymer nanofibers using electrospinning process—A review. J Mater Proc Tech 2005;167:283–293. 4. Huang ZM, Zhang YZ, Kotaki M, Ramakrishna S. A review on polymer nanofibers by electrospinning and their applications in nanocomposites. Comp Sci Tech 2003;63:2223– 2253. 5. Kidoaki S, Kwon IK, Matsuda T. Mesoscopic spatial designs of nano- and microfiber meshes for tissue-engineering matrix and scaffold based on newly devised multilayering and mixing electrospinning techniques. Biomaterials 2005;26:37–46. 6. Li MY, Mondrinos MJ, Gandhi MR, Ko FK, Weiss AS, Lelkes PI. Electrospun protein fibers as matrices for tissue engineering. Biomaterials 2005;26:5999–6008. 7. Stevens MM, George JH. Exploring and engineering the cell surface interface. Science 2005;310:1135–1138. 8. Matthews JA, Wnek GE, Simpson DG, Bowlin GL. Electrospinning of collagen nanofibers. Biomacromole 2002;3:232– 238. 9. Ohkawa K, Cha D, Kim H, Nishida A, Yamamoto H. Electrospinning of chitosan. Macromol Rapid Comm 2004;25: 1600–1605. 10. Liu HQ, Hsieh YL. Ultrafine fibrous cellulose membranes from electrospinning of cellulose acetate. J Poly Sci Part B: Polymer Phy 2002;40:2119–2129. 11. Zhang YZ, Ouyang HW, Lim CT, Ramakrishna S, Huang ZM. Electrospinning of gelatin fibers and gelatin/PCL composite fibrous scaffolds. J Biomed Mater Res B 2005;72B:156– 165. 12. Choi YS, Hong SR, Lee YM, Song KW, Park MH, Nam YS. Studies on gelatin-containing artificial skin: II. Preparation and characterization of cross-linked gelatin-hyaluronate sponge. J Biomed Mater Res 1999;48:631–639. 13. Li MY, Guo Y, Wei Y, MacDiarmid AG, Lelkes PI. Electrospinning polyaniline-contained gelatin nanofibers for tissue engineering applications. Biomaterials 2006;27:2705– 2715. 14. Zhu YB, Gao CY, Liu XY, Shen JC. Surface modification of polycaprolactone membrane via aminolysis and biomacromolecule immobilization for promoting cytocompatibility of human endothelial cells. Biomacromole 2002;3:1312–1319. 15. Ma ZW, He W, Yong T, Ramakrishna S. Grafting of gelatin on electrospun poly(caprolactone) nanofibers to improve endothelial cell spreading and proliferation and to control cell orientation. Tissue Eng 2005;11:1149–1158. 16. Kim HW, Song JH, Kim HE. Nanofiber generation of gelatinhydroxyapatite biomimetics for guided tissue regeneration. Adv Funct Mater 2005;15:1988–1994. 17. Kim HW, Lee HH, Knowles JC. Electrospinning biomedical nanocomposite fibers of hydroxyapatite/poly(lactic acid) for bone regeneration. J Biomed Mater Res A 2007;79A:643–649. 18. Fujihara K, Kotaki M, Ramakrishna S. Guided bone regeneration membrane made of polycaprolactone/calcium carbonate composite nano-fibers. Biomaterials 2005;26:4139– 4147. 19. Ren L, Tsuru K, Hayakawa S, Osaka A. Synthesis and characterization of gelatin-siloxane hybrids derived through sol-gel procedure. J Sol-Gel Sci Tech 2001;21:115–121. 20. Ren L, Tsuru K, Hayakawa S, Osaka A. Novel approach to fabricate porous gelatin-siloxane hybrids for bone tissue engineering. Biomaterials 2002;23:4765–4773. 21. Song JH, Kim HE, Kim HW. Production of electrospun gelatin nanofiber by water-based co-solvent approach. J Mater Sci Mater Med. In press.


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22. Payne KJ, Veis A. Infrared spectroscopy of collagen and collagen-like polypeptide. Biopolymer 1975;14:937–957. 23. Ikeuchi M, Dohi Y, Ohgushi H, Noshi T, Horiuchi K, Yamamoto T, Sugimura M, Ito A. In vitro bone formation by human marrow cell culture on the surface of zinc-releasing calcium phosphate ceramics. Bioceramics 2000;192-1:503–506. 24. Shirosaki Y, Tsuru K, Hayakwa S, Osaka A, Lopes MA, Santos JD, Fernandes MH. In vitro cytocompatability of MG63 cells on chitosan-organosiloxane hybrid membranes. Biomaterials 2005;26:485–493.


SONG ET AL.

25. Liang WJ, Hsieh SJ, Hsu CY, Chen WF, Kuo PL. Microstructure and protonic conductivity of H3PO4-doped polyethylenimine-siloxane chemically covalently organic-inorganic hybrids. J Poly Sci B Poly Phy 2006;44:2135–2144. 26. Shchipunov YA, Karpenko TY. Hybrid polysaccharide-silica nanocomposites prepared by the sol-gel technique. Langmuir 2004;20:3882–3887. 27. Ozawa S, Kasugai S. Evaluation of implant materials (hydroxyapatite, glass-ceramics, titanium) in rat bone marrow stromal cell culture. Biomaterials 1996;17:23–29.