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Journal of Biomaterials Science 22 (2011) 207223

brill.nl/jbs

Cellular Behavior on Epidermal Growth Factor (EGF)-Immobilized PCL/Gelatin Nanobrous Scaffolds


R. Seda T gl a , N. Merve Kazaro glu b , Bora Mav s c and Menem se Gm sderel o glu a,
a b c

Hacettepe University, Chemical Engineering Department, 06800, Beytepe, Ankara, Turkey Ba skent University, Biomedical Engineering Department, 06810, Ba glca, Ankara, Turkey Hacetepe University, Mechanical Engineering Department, 06800, Beytepe, Ankara, Turkey Received 5 October 2009; accepted 21 November 2009

Abstract Nano-scaled poly( -caprolactone) (PCL) and PCL/gelatin brous scaffolds with immobilized epidermal growth factor (EGF) were prepared for the purpose of wound-healing treatments. The tissue scaffolds were fabricated by electrospinning and the parameters that affect the electrospinning process were optimized. While the ber diameters were 488 114 nm and 663 107 nm for PCL and PCL/gelatin scaffolds, respectively, the porosities were calculated as 79% for PCL and 68% for PCL/gelatin scaffolds. Electrospun PCL and PCL/gelatin scaffolds were rst modied with 1,6-diaminohexane to introduce amino groups on their surfaces, then EGF was chemically conjugated to the surface of nanobers. The results obtained from Attenuated Total Reectance Fourier Transform Infrared (ATRFT-IR) spectroscopy and quantitative measurements showed that EGF was successfully immobilized on nanobrous scaffolds. L929 mouse broblastic cells were cultivated on both neat and EGF-immobilized PCL and PCL/gelatin scaffolds in order to investigate the effect of EGF on cell spreading and proliferation. According to the results, especially EGF-immobilized PCL/gelatin scaffolds exerted early cell spreading and superior and rapid proliferation compared to EGF-immobilized PCL scaffolds and neat PCL, PCL/gelatin scaffolds. Consequently, EGF-immobilized PCL/gelatin scaffolds could potentially be employed as novel scaffolds for skin tissueengineering applications. Koninklijke Brill NV, Leiden, 2011 Keywords Nanobers, polycaprolactone, gelatin, EGF, electrospinning, skin tissue engineering

1. Introduction Skin is composed of three layers, epidermis, dermis and subcutaneous fat, covering the entire external surface of the body and forming about 8% of the total body mass. Thus, skin provides an effective barrier against microbial invasion and has propTo whom correspondence should be addressed. Tel.: (90-312) 297-7447; Fax: (90-312) 299-2124; e-mail: menemse@hacettepe.edu.tr
Koninklijke Brill NV, Leiden, 2011
DOI:10.1163/092050609X12591500475424
*

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erties that can protect against mechanical, chemical, osmotic, thermal and photo damage [1, 2]. Trauma to the skin, which can be caused by burns, venous ulcer, diabetic ulcer and acute injury, is categorized into several degrees [1]. A more serious trauma can lead to partial or complete damage to both dermal and subdermal tissue which are capable of regeneration, but unfortunately the body cannot adequately heal deep dermal injuries. In such cases, there are no remaining sources of cells for regeneration, except from the wound periphery. Complete re-epithelialization, therefore, takes a long time [35]. With regard to the continual demand for skinregeneration products, functional scaffolds, which should be able to mimic the structure and biological function of the native extracellular matrix (ECM) proteins, have been emphasized as skin grafts [6]. In recent years, there has been an increasing interest towards employing electrospinning for nano-scaled scaffold fabrication since nanobrous scaffolds have morphological and architectural features similar to that of the natural ECM in skin [7]. Moreover, in several studies it has also been suggested that mimicking of the structural dimensions of the ECM by small-sized bers promote the adhesion and proliferation of cells [2, 6, 8, 9]. Extensive research on engineered skin had been conducted on nanobrous polycaprolactone (PCL) which is among the most popular scaffold polymers approved by the US Food and Drug Administration [2, 6, 10]. However, a slow biodegradation rate (over 24 months) and hydrophobicity are the major drawbacks of this otherwise promising biopolymer [11, 12]. Gelatin, which is a natural biopolymer derived from collagen by controlled hydrolysis, has many merits, such as its biological origin, biodegradability, biocompatibility and commercial availability at relatively low cost. Previous studies have shown that the polymer blend of PCL/gelatin overcomes the shortcomings of natural and synthetic polymers, resulting in a new biomaterial with good biocompatibility and improved mechanical, physical and chemical properties [13] for wound healing [2] and nerve tissue-engineering applications [14]. On the other hand, surface modication has been recognized as a potential tool for enhancing the biocompatibility of the surface of engineered nanobers, since hydrophilic and protein-containing surfaces are maintained by immobilization of biomolecules such as gelatin, collagen and GlyArgGlyAspSer (GRGDS), known to promote cellular growth [6]. Current treatments for such ulcers or wounds include the administration of disinfectants followed by application of epidermal growth factor (EGF)-containing gels around the lesions [15]. EGF is a strong mitogen and stimulates mitosis through the regulation of intracellular calcium and pH [16]. Although a number of nanobrous matrices were prepared and investigated as skin substitutes, only a few studies are available to indicate immobilization of growth factors to the nanobrous scaffolds and their effects on the cellular behavior. Recently, Choi et al. [10] reported that EGF-immobilized PCLpoly(ethylene glycol) (PEG) nanobers helped the proliferating keratinocytes to maintain their original phenotypes during the wound-healing process and suggested as a novel wound-healing material. In our study, EGF was covalently immobilized on PCL

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and PCL/gelatin nanobers for in vitro feasibility of wound-healing treatments. Electrospinning of PCL and PCL/gelatin nanobers was followed by a two-step EGF immobilization procedure that employs aminolysis [17] and EGF conjugation [18]. Neat PCL, PCL/gelatin and EGF-immobilized scaffolds were characterized by FT-IR, scanning electron microscopy (SEM), porosity, contact angle measurements and degradation tests. Cell-culture studies were performed with L929 mouse broblast cells and evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assay, scanning electron microscopy (SEM) and uorescence microscopy in terms of cell proliferation and morphological observation. 2. Materials and Methods 2.1. Materials PCL (Mw = 8 104 ) and gelatin type A from porcine skin were all obtained from Sigma-Aldrich (Germany). Dichloromethane (DCM) (Merck, Germany) and N,N-dimethylformamide (DMF) (Sigma-Aldrich) were used as solvents for PCL. Hexauoropropanol (HFIP) (Sigma-Aldrich) was used as solvent for the PCLgelatin mixture. Epidermal growth factor (EGF) from mouse submaxillary glands (Cat. No. E4157), suberic acid bis(N -hydroxy-succinimide ester) (NHS), 2,2-dihydroxyindane-1,3-dione (ninhydrin), 1,6-diaminohexane and bovine serum albumin (BSA) were obtained from Sigma. For cell-culture studies, 3-[4,5dimethylthiazol-2-yl]-diphenyltetrazolium bromide (MTT), propidium iodide, Triton X-100 and glutaraldehyde were purchased from Sigma. Alexa Fluor 488 phalloidin was obtained from Molecular Probes (USA). All other solvents used in this study were of analytical grade. 2.2. Fabrication of PCL and PCL/Gelatin Nanobrous Scaffolds PCL polymer solution with a concentration of 12% (w/v) was prepared by dissolving PCL in DMF/DCM (50:50 (v/v)) [9]. PCL/gelatin solution with a ratio of (50:50 (v/v)) was prepared by dissolving PCL and gelatin in HFIP separately with a concentration of 5.4% (w/v) for each and then they were stirred for 2 days at room temperature [14]. PCL and PCL/gelatin nanober scaffolds were electrospun using an electrospinning setup. The electrospinning process was adjusted using an auxillary electrode in order to maintain a stable polymer jet which also helps to fabricate nanobers at high ow rates without using very high voltages. For the process of electrospinning, the polymer solution was placed in a 20 ml plastic syringe tted with a blunt-ended needle with a tip diameter of 0.9 mm. Electrospinning voltage was applied between the needle and the grounded collector (200 300 mm) at 25 kV for PCL solution and 12 kV for PCL/gelatin solution using a high voltage power supply (Gamma High-Voltage Research, USA). Above a critical voltage, a polymer jet was ejected from the needle tip towards a grounded collector which was laid with 0.2 mm aluminum foil. The needle is located at a distance of 350 mm from the ground collector. A syringe pump was used to feed the polymer solution to

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the needle tip at a feed rate of 12 ml/h for PCL and 1.8 ml/h for PCL/gelatin solutions. The nanobers were collected to prepare scaffolds which have approximately 20 m thickness. The constructs were wetted with 70% ethanol and dried overnight under vacuum. 2.3. Immobilization of EGF on PCL and PCL/Gelatin Scaffolds PCL and PCL/gelatin scaffolds were modied with the growth factor EGF using a covalent immobilization technique which was described in our previous study [18]. Before immobilization of EGF on nanober scaffolds, an aminolysis procedure described elsewhere [17] was followed in order to incorporate amine groups into the surface of PCL in PCL and PCL/gelatin nanober scaffolds using 1,6hexanediamine (Sigma-Aldrich). Briey, PCL ad PCL/gelatin scaffolds, 15 mm in diameter, were immersed into a 10% (w/v) solution of 1,6-hexanediamine prepared in isopropanol for 3 h at room temperature, then they were washed in PBS ve times. After aminolysis of PCL and PCL/gelatin, scaffolds were rinsed with 70% ethanol and phosphate-buffered saline (PBS, pH 7.4), then soaked in 10 ml PBS containing 0.08 mmol NHS with gentle agitation for 4 h at room temperature. After washing with PBS ve times, the activated scaffolds were soaked into 10 g/ml EGF solutions in PBS and were shaken for 12 h at 4 C. EGF-immobilized scaffolds were washed with PBS and freeze-dried. 2.4. Characterization of Scaffolds 2.4.1. Scanning Electron Microscopy (SEM) Analysis Size, size distribution and surface structures of PCL and PCL/gelatin nanobers were examined by using a SEM (Zeiss Evo 50, Germany) after coating with a gold palladium layer. 2.4.2. Ninhydrin Assay A ninhydrin assay was employed to conrm the presence of amine groups on the surface of PCL and PCL/gelatin scaffolds (diameter 15 mm, thickness 20 m) treated with 1,6-hexanediamine as described previously [17]. Briey, a 1.0 M solution of ninhydrin in ethanol was prepared and each scaffold was immersed into this ninhydrin solution for 1 min. The scaffold was then transferred to a glass vial that was heated for 15 min at 70 C in a water bath. The ninhydrin-treated scaffold was then dissolved in 2 ml THF. A total of 2 ml isopropanol was added to the vial and mixed with the solution. The nal solution was placed in a silica cuvette and the absorbance measured at 560 nm using a UV-Vis Double Beam PC (Labomed, USA) spectrophotometer. A standard curve was prepared using solutions of known 1,6-hexanediamine concentrations in order to quantify amount of amine groups on PCL and PCL/gelatin scaffolds. 2.4.3. ATRFT-IR Analysis The surface chemical structures of the PCL and PCL/gelatin scaffolds were investigated using an ATRFT-IR spectrophotometer (Thermo Scientic, Nicolet 6700-

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Smart Orbit (Diamond Crystal ATR), USA). The spectra of samples were taken at 5254000 cm1 wavelength with 4 cm1 resolution. 2.4.4. Quantitative Analysis of EGF-Immobilized Scaffolds The amount of immobilized EGF was determined by measuring the uorescence intensity of tryptophan group of EGF according to the procedure described before [18]. EGF assay was performed using a uorescence spectrophotometer (Cary Eclipse, Australia) at 280 nm excitation and 342 nm emission wavelengths as the intrinsic tryptophan uorescence. The amount of immobilized EGF was determined by subtracting EGF intensity remaining in solution at the end of the immobilization process from the initial amount of EGF in the immobilization solution. 2.4.5. Water Contact Angle Measurements The wettability of the nanobrous PCL and PCL/gelatin scaffolds was assessed by water contact angle values. Water contact angles were measured by sessile drop method at room temperature (Kruss DSA 100, Germany). 2.4.6. Porosity The thickness of the PCL and PCL/gelatin scaffolds was measured with the aid of a micrometer and their apparent density and porosity were estimated using equations (1) and (2) [2], respectively: Scaffold apparent density (g/cm3 ) Mass of scaffold (g) = , Scaffold thickness (cm) Scaffold area (cm2 ) Scaffold porosity (%) = 1

(1)

Scaffold apparent density (g/cm3 ) 100. (2) Bulk density (g/cm3 )

2.4.7. Biodegradability Degradation tests of PCL and PCL/gelatin scaffolds were carried out in cell-culture media. Incubations were performed at 37 C in a humidied incubator for 14 days. Dry weights of dehydrated scaffolds were measured on days 3, 5, 7 and 14 of incubation. Scaffold weights were then recorded and compared to their initial dry weight. Degradation was determined as the percentage of weight loss (WL ) using equation (3): WL (%) = W0 W W0 100, (3)

where W0 is initial weight and W is weight after degradation. Structural degradation of scaffolds were also examined through SEM images. 2.5. Cell-Culture Studies Cell-culture studies were carried out with L929 mouse broblasts. The L929 cell line was obtained from HUKUK Cell Line Collection (No. 92123004, Foot and Mouth Disease Institute, Ankara, Turkey). The cells were subcultured in asks

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using Dulbeccos modied Eagles medium (DMEM, Sigma) supplemented with 10% (v/v) fetal bovine serum (FBS, Sigma). The cells, maintained at 37 C in a humidied CO2 (5%) atmosphere (Heraus Instruments, Germany), were dissociated with 0.01% trypsin/10 mM EDTA (Sigma), centrifuged and resuspended in medium prior to cell seeding. 2.5.1. Cell Culture and Seeding Cell cultures were conducted in sterile 24-well tissue-culture polystyrene (TCPS) dishes in stationary conditions. Prior to cell-culture experiments, 24-well TCPS dishes were precoated with paralm and were soaked in 96% ethanol and placed under UV light for 30 min for sterilization. PCL and PCL/gelatin scaffolds were sterilized with 70% ethanol for 1 h and equilibrated in sterile Dulbeccos PBS (pH 7.4, 24 h). Then scaffolds were immersed in conditioning medium (DMEM supplemented with 10% (v/v) FBS) for 1.5 h prior to cell seeding. Cell suspension (1 ml) containing 1 105 cells was then added to each well to inoculate the cells on scaffolds. The medium was replenished every two days. 2.5.2. Cell Metabolic Activity L929 cells on scaffolds were quantitatively assessed with 3-[4,5-dimethylthiazol2-yl]-diphenyltetrazolium bromide (MTT) formazan at different times during the culture period. At selected time intervals, culture medium was aspirated and washed with 600 l pre-warmed PBS (pH 7.4). Of the pre-warmed culture medium supplemented with 60 l MTT solution (2.5 mg/ml MTT dissolved in PBS), 600 l was added to each sample which was incubated at 37 C for 3 h. After the incubation period, the medium was removed from each well and scaffolds were transferred to another 24-well Petri dish. HCl in isopropanol solution (400 l, 0.04 M) was added to each well to dissolve formazan crystals. The resulting solution in crystal violet color was taken out and centrifuged at 1.3 104 rpm for 2 min. Of the supernatant 200 l was used for measuring optical density spectrophotometrically at 570 nm with reference to 690 nm using a microplate reader (Asys UVM 340, Austria). MTT assay was also applied to the scaffolds without cells as control and the data was subtracted from measured values. 2.5.3. Microscopic Imaging of Cells on Nanobrous Scaffolds The morphology of cells on scaffolds was observed by a SEM (Zeiss Evo 50, Germany) on days 1, 2, 3, 5 and 7 of the incubation period. The scaffolds were gently washed with PBS and cells were xed with 2.5% (v/v) glutaraldehyde in 0.1 M Dulbeccos PBS (DPBS) for 1 h. Then the scaffolds were dehydrated in ethanol series and rinsed with hexamethyldisilazane. Cytoskeleton organization within scaffolds was assessed at the end of days 1, 2, 3 and 5 after cell seeding. Cells were rinsed twice with PBS, xed in 2.5% (v/v) glutaraldehyde in 0.1 M PBS (pH 7.4) for 10 min at 4 C and permeabilized in 0.1% buffered Triton X-100 for 5 min. Cell cytoskeletal lamentous actin (F-actin) was visualized by treating the cells with 2.5% (v/v) Alexa Fluor 488 phalloidin for 20 min. Cell nuclei were counterstained

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with 10 g/ml propidium iodide (Sigma) for 5 min. Samples were visualized using uorescence microscope (Olympus, USA). 2.6. Statistical Analysis All data are expressed as means standard deviation of a representative of three similar experiments carried out in triplicate. Statistical analysis was performed by one-way analysis of variance (ANOVA) in conjunction with Tukeys post hoc test for multiple comparisons using Graph-Pad Instant (GraphPad Software, USA) statistics program with signicance at P < 0.05. 3. Results and Discussion 3.1. Morphology and Properties of PCL and PCL/Gelatin Nanobrous Scaffolds SEM images of PCL and PCL/gelatin scaffolds are shown in Fig. 1. As can be seen in Fig. 1a and 1b, PCL and PCL/gelatin nanobers were randomly distributed and created interconnected structures. In the presence of gelatin the nanobers have a smooth morphology and monosize diameter due to the increased viscosity of the polymeric solution (Fig. 1b). The calculated diameters of PCL/gelatin nanobers and PCL nanobers were 663 107 nm and 488 114 nm, respectively. The results were in good correlation with previous studies [2, 9]. It is not easy to create well-dened pore sizes through the electrospinning technique because of the randomly deposited bers. However, the overall network architecture fabricated best mimics the natural ECM, and this provides an advantage in its application as tissueengineering scaffold. Such nanobrous scaffolds which would be used for skin reconstitution should largely be made porous in order to maintain cellular inltration and proliferation, as well as ensuring sufcient gas and nutrient exchange for wound healing. The preferred porosity of scaffolds used for cellular penetration should generally be in the range of 6090% [2]. The porosities of the electrospun PCL and PCL/gelatin nanobrous scaffolds in this study are estimated to be 79% and 68%, respectively. Moreover, previous studies on PCL/gelatin nanobers have also shown that biodegradability of gelatin during cell-culture provides easier opening of spaces for cell penetration to a deeper level of the scaffold [13]. Figure 1cf illustrates SEM images of PCL/gelatin nanobrous scaffolds during in vitro degradation of 14 days. As can be seen from Fig. 1cf, signicant morphological changes were observed on PCL/gelatin nanobers, indicating surface erosion of homogenously distributed gelatin from the scaffolds. In this study, in vitro degradability of PCL and PCL/gelatin scaffolds was investigated gravimetrically and results are shown in Fig. 2. As can be seen in Fig. 2, a rapid 20% mass loss of PCL/gelatin scaffolds was observed in the rst 5 days. After 7 days, degradability of PCL/gelatin scaffolds was decreased which may be due to the presence of more PCL in the remaining composition of the nanobers. However, PCL scaffolds showed no weight loss during 14 days of incubation (Fig. 2). According to the ATRFT-IR spectra of PCL scaffolds (Fig. 3a) and PCL/gelatin

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Figure 1. SEM images of (a) electrospun PCL and (b) PCL/gelatin nanobrous scaffolds. Morphology of PCL/gelatin scaffolds on (c) day 3, (d) day 5, (e) day 7 and (f) day 14 of degradation test. Magincations: 1 104 (a, b), 8780 (c, e, f) and 8770 (d).

scaffolds (Fig. 3b) PCL-related stretching modes include 2943 (asymmetric CH2 stretching), 2866 (symmetric CH2 stretching), 1721 (C=O stretching), 1294 (CO and CC stretching) and 1240 cm1 (asymmetric COC stretching) bands. Common bands of protein appeared at approx. 1650 (amide I) and 1540 cm1 (amide II), corresponding to the stretching vibrations of C=O bond, and coupling of bending of NH bond and stretching of CN bonds, respectively. The amide I band at 1650 cm1 was attributable to both a random coil and -helix conformation of gelatin [14]. The detection of a step-wise decrease of the intensities of amide I (1646 cm1 ) and amide II (1537 cm1 ) bands during the incubation period clearly showed that gelatin was hydrolyzed and degraded to its amino acids (Fig. 3c).

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Figure 2. Degradation characteristics of PCL and PCL/gelatin nanobrous scaffolds. This gure is published in colour in the online edition of this journal, that can be accessed via http://www.brill.nl/jbs

Figure 3. ATRFT-IR spectra of (a) PCL and aminolyzed PCL, (b) PCL/gelatin and aminolyzed PCL/gelatin nanobrous scaffolds, (c) PCL/gelatin scaffolds before and after 3, 5, 7 and 14 days of degradation, (d) PCL scaffolds before and after 14 days of degradation. Arrows indicate amide I (1646 cm1 ) and amide II (1537 cm1 ) bands.

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Moreover, no signicant differences were detected at 2866 (COOH groups) and 2943 cm1 (OH groups) which should be expected because of the presence of degradation products of PCL after hydrolytic degradation. From these results we concluded that PCL scaffolds were not degraded during the 14 days of incubation (Fig. 3d). 3.2. Immobilization and Characterization of EGF on PCL and PCL/Gelatin Nanobrous Scaffolds Although nanobrous scaffolds mimic the ECM structure best, surface modication with certain growth factors motivates biocompatibility of the material and enhances interactions between the cells and scaffolds. Zhu et al. [19] modied solvent-cast PCL membranes by rst reacting the membranes with 1,6-hexamethylenediamine to introduce NH2 groups on their surface. Then, the aminolyzed PCL membranes were activated with glutaraldehyde, prior to being reacted with gelatin, chitosan or collagen [19]. Santiago et al. [17] utilized the method proposed by Zhu et al. [19] to immobilize three different peptides onto the surface of solvent-cast PCL disks. Mattanavee et al. [6] used the method proposed by Zhu et al. [19] in order to immobilize collagen, chitosan and GRGDS to PCL electrospun scaffolds. Choi et al. immobilized EGF on PEG-grafted PCL nanobers under carbodiimide functionalization [10]. In this study, EGF was covalently immobilized on PCL and PCL/gelatin nanobrous scaffolds according to the procedures described before [17, 18]. An immobilized EGF concentration of 10 g/ml was selected according to the literature of wound healing and epithelialization [20]. Figure 4 demonstrates the immobi-

Figure 4. Schematic representation of EGF immobilization on PCL nanobrous scaffolds. (a) aminolysis of PCL, (b) activation of PCL aminated surfaces, (c) EGF immobilization via activated PCL conjugation.

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Figure 5. Ninhydrin assay applied to neat and 1,6-hexanediamine treated PCL and PCL/gelatin scaffolds.

lization process of EGF on PCL scaffolds. Briey, PCL was aminated with 1,6hexanediamine, then the amine end-group of PCL was activated with NHS to conjugate with the amino-terminal of EGF. Similarly, EGF was immobilized on PCL/gelatin scaffolds through the amine end-group of PCL/gelatin. The presence of amine groups on 1,6-hexanediamine treated PCL and PCL/gelatin scaffolds was conrmed with a ninhydrin assay. The quantied amount of amine groups (g per scaffold) on scaffolds treated with ninhydrin is shown in Fig. 5. The increase in the quantied amount of amine groups in both PCL and PCL/gelatin scaffolds clearly indicates the success of aminolysis (Fig. 5). ATRFT-IR was employed to conrm the presence of amine groups on the surface of 1,6-hexanediamine treated PCL and PCL/gelatin scaffolds (Fig. 3a and 3b). As seen in Fig. 3a, a peak can be seen clearly at 1550 cm1 , corresponding to the primary NH bending region (1500 1550 cm1 ) of PCL [21]. For PCL/gelatin scaffolds, the increase in the intensity of amide I and amide II bands clearly conrmed the successful coupling of amine groups (Fig. 3b). Quantitative results obtained from uorescence spectrophotometer were also supported the success of EGF immobilization. Results showed that the amount of immobilized EGF on PCL and PCL/gelatin scaffolds was 9.80 and 9.91 g per scaffold where immobilization efciency reaches 98% and 99% on saturation level, respectively. Despite the inherit biodegradability and biocompatibility of PCL, the actual utilization of this material is limited by its hydrophobicity [19]. The incorporation of gelatin improves the hydrophilicity of PCL/gelatin nanobrous scaffolds. This might be attributed to the amine and carboxylic functional groups in gelatin structure, whereas such functional groups are not present in PCL structure [14]. Table 1 shows the water contact angle values of PCL, aminated PCL, EGF-immobilized PCL, PCL/gelatin, aminated PCL/gelatin and EGF-immobilized PCL/gelatin scaffolds. As seen from Table 1, both gelatin and aminolysis improve hydrophilicity. Moreover, the contact angle values of EGF-immobilized PCL and PCL/gelatin scaffolds were found to be zero, since it is known that EGF increases the hydrophilicity of substrates [22].

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R. S. T gl et al. / Journal of Biomaterials Science 22 (2011) 207223 Table 1. Water contact angle values of nanobrous scaffolds Nanobrous scaffold PCL PCLamine PCLEGF PCL/gelatin PCL/gelatinamine PCL/gelatinEGF Water contact angle () 133.0 0.2 121.6 1.6 0 125.0 1.7 86.7 2.4 0

3.3. Effect of EGF Immobilization on Cell Culture 3.3.1. Cell Proliferation on PCL and PCL/Gelatin Nanobrous Scaffolds L929 broblastic cell proliferation on EGF-immobilized or neat PCL and PCL/gelatin scaffolds was evaluated by MTT assay on days 1, 2, 3 and 5 (Fig. 6). TCPS was used as control. As can be seen from Fig. 6, both EGF immobilized and neat PCL and PCL/gelatin scaffolds provided efcient support for proliferation of L929 cells during the incubation period. Although cells on EGF-immobilized PCL scaffolds better supported proliferation of cells compared to cells on EGF-immobilized PCL/gelatin scaffolds for the rst 2 days, after day 2 EGF-immobilized PCL and PCL/gelatin scaffolds provided similar effects on L929 cells. EGF-immobilized PCL and PCL/gelatin scaffolds highly supported cell proliferation compared to neat scaffolds during the rst 5 days of the incubation period (P < 0.001). Moreover, on day 5 of incubation, proliferation of L929 cells on EGF-immobilized PCL and PCL/gelatin scaffolds were as good as they did on TCPS. Results clearly indicated superior effect of EGF on cell proliferation on PCL and PCL/gelatin nanobrous scaffolds. T gl and Gm sderelio glu modied chitosan scaffolds by immobilization of EGF and reported signicant improvements in the ability of cell proliferation [18]. Karakeili et al. successfully described the necessity of EGF immobilization on chitosan membranes for L929 broblastic cell proliferation [16]. With a different aspect, Mattanavee et al. indicated the inferior L929 cell proliferation on neat PCL and modied PCL brous scaffolds by collagen, chitosan and GRGDS compared to TCPS because of lower hydrophilic nature of scaffolds compared to TCPS [6]. In our study presented here, immobilization of EGF improved the poor ability of PCL and PCL/gelatin to support the cell proliferation with respect to low hydrophilicity of PCL and PCL/gelatin nanobrous scaffolds. The increased number of cells as a result of accelerated cell growth on EGF-immobilized scaffolds can also be attributed to the high mitogenic activity due to presence of EGF. It was known that, there is an effective interaction with EGF receptors of adhered cells without any steric hindrance and high mitogenic activity is persistent as the immobilization of EGF is thought to inhibit the internalization and down-regulation. EGF binds to the cell surface receptor and forms complexes that are then internalized and decom-

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Figure 6. MTT results showing the proliferation trends of L929 cells on neat and EGF-immobilized PCL and PCL/gelatin scaffolds. TCPS was used as control. P < 0.01, P < 0.001, statistically signicant differences, n = 3. PCL and PCL/gelatin are control group through their own EGF immobilized ones, ... P < 0.001. TCPS is control group.

posed in the cell. As a result of decomposition the stimulating effect of the growth factor molecule on cell metabolism is reduced. This phenomenon known as downregulation is thought to be inhibited by using immobilized growth factor molecules instead of native molecules [16]. 3.3.2. Morphology of Cultured Cells Figures 7 and 8 present SEM images of L929 cells cultured on PCL and PCL/gelatin nanobrous scaffolds, respectively. As seen from Fig. 7a,b and 8a,b, at day 1 of culture cells on PCL and PCL/gelatin were still round, while those seeded on EGF-immobilized scaffolds were already spreading. During the rst 3 days of cell culture, cells on EGF-immobilized PCL and PCL/gelatin scaffolds rapidly spread on scaffolds, indicating the increase in cell proliferation due to existence of EGF. After 5 days, the difference of cell spreading between EGF-immobilized and neat scaffolds decreased and scaffolds were completely covered with cells and ECM proteins secreted by cells. Figure 9 demonstrates the cytoskeleton organization of cells on scaffolds. Similar to the SEM images, starting from the rst day of incubation, the morphology of L929 cells on EGF-immobilized PCL and PCL/gelatin were broblastic, while neat scaffolds were still round. Moreover, spreading was rapid and homogenous throughout the EGF-immobilized scaffolds. During recovery from severe burns and wounds, proliferation of broblasts around lesions widely occurs, subsequently resulting in contraction and deformation of the original skin [23]. In the early stages of the wound-healing process, one of the important parameters to consider is the migration of cells to wound area as soon as possible. By this way, wound area is covered rapidly and the risk of trace formation on the area is decreased [24]. Choi et al. clearly reported that EGF was required only at the initial stage of the wound repair [10]. In our study, cell proliferation was signicantly en-

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Figure 7. SEM images of L929 cells on PCL and EGF-immobilized PCL scaffolds during 7 days of incubation. Magnications: 500.

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Figure 8. SEM images of L929 cells on PCL/gelatin and EGF-immobilized PCL/gelatin scaffolds during 7 days of culture. Magnications: 1000 (ag), 2500 (h) and 500 (i, j).

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Figure 9. Fluorescence microscopy images of L929 cells on (a) PCL, (b) PCLEGF, (c) PCL/gelatin and (d) PCL/gelatinEGF scaffolds during 3 days of incubation. Magnications: 20 and 40. Green and red areas indicate F-actin and DNA of L929 cells, respectively. This gure is published in colour in the online edition of this journal, that can be accessed via http://www.brill.nl/jbs

hanced by EGF immobilization on nanobrous scaffolds which were suggested as smart biomaterials for skin tissue-engineering applications, especially for the demand of the wound-healing processes. 4. Conclusions Immobilization of EGF on PCL and PCL/gelatin nanobrous scaffolds was carried out in this study for their use in wound-healing treatments. PCL/gelatin nanobers altered the low hydrophilicity and slow degradation of PCL nanobers. Results conrmed that EGF can be successfully immobilized to PCL and PCL/gelatin scaffolds in a two-step process, in which surface aminolysis was followed by conjugation of EGF. Cell-culture studies clearly concluded that EGF had a superior effect on cell proliferation on nanobrous scaffolds which was supported with SEM and uorescence microscope images of cells on scaffolds. With this information providing an initial basis, furthermore, EGF immobilization on nanobrous scaffolds can be evaluated for dermal reconstitution. References
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