Preparation of SF/CS blend solution
Bombyx mori silk fibers were treated twice with 0.5% (w/w) NaHCO3 solution at 70°C for 30 min and then rinsed with 70°C distilled water to remove sericin. Degummed silk was dissolved in a mix solvent system of CaCl2/CH3CH2OH/H2O (mole ratio, 1:2:8) at 70°C for 6 h and filtered to get a SF solution. After dialysis in a cellulose dialysis tubing (MWCO = 50,000) against distilled water for 5 days with water change every 12 h, the SF solution was lyophilized to obtain regenerated SF sponges. Chitosan (MW = 1 × 105, degree of deacetylation = 98%) was purchased from Fluka (Buchs, Switzerland). CS and SF solutions were prepared in a mixed solvent system of TFA/DCM (weight ratio = 7:3) at concentrations of 8 and 12.5 wt.%, respectively. CS/SF blend solutions with different SF/CS weight ratio (75:25, 50:50, and 25:75) were prepared in the same solvent system at 12, 10, and 9 wt.% (combined weight of CS and SF), respectively.
Preparation of SF/CS nanofibers by electrospinning and chemical treatment
The system for electrospinning includes a glass syringe, a 22-gauge stainless-steel needle, a syringe pump (KD Scientific Co., Holliston, MA, USA), a high-voltage power supply (Glassman, High Bridge, NJ, USA), and an aluminum foil as the collector [21
]. The distance between the needle tip and the collector was 16 cm. The syringe was mounted on the syringe pump, and spinning solution was drawn horizontally from the needle tip with an electrostatic force generated from the high voltage applied between the tip and the grounded collector. The applied voltage and flow rate were controlled at 18 kV and 0.3 ml/h, respectively. For chemical treatment, the SF/CS composite NFs were neutralized, crystallized, and insolubilized by immersing in 7% (v/v
) ammonia solution/75% (v/v
) ethanol aqueous solution for 30 min at room temperature.
Measurement and characterization
The morphology of the NFs was observed by a scanning electron microscope [SEM] (Hitachi S3000N, Hitachi, Ltd., Chiyoda, Tokyo, Japan). The diameters were calculated by measuring at least 100 fibers from 10 images at random using ImageJ. Chemical analysis was carried out with Fourier-transform infrared spectroscopy using a Horiba FT-730 spectrometer (Horiba, Ltd., Kyoto, Japan) over a wavenumber range between 600 and 2,000 cm-1 with a resolution of 2 cm-1. X-ray photoelectron spectroscopy [XPS] was performed with a Physical Electronics PHI 1600 ESCA spectrometer (Physical Electronics, Inc., Chanhassen, MN, USA) equipped with a spherical capacitor analyzer and a multi-channel detector. The X-ray source was generated with a magnesium anode at 15 kV and 400 W. The pressure in the analysis chamber was set below 2 ×10-6 Pa. X-ray diffraction [XRD] patterns were recorded on a Siemens D5005 X-ray diffractometer (Bruker AXS, Karlsruhe, Germany) composed of a CuKα source, a quartz monochrometer, and a geniometric plate at a scanning speed of 2° min-1 from 10° to 55°. Thermogravimetry/differential scanning calorimetry [TG/DSC] analysis was conducted with a Netzsch STA 449F1 (Netzsch Instruments, Inc., Burlington, MA, USA) from 25°C to 800°C at 10°C/min. Pore size of the nanofibrous membrane was measured by capillary flow porometry (PMI CFP-1100-AI, Porous Materials, Inc., Ithaca, NY, USA) using a wetting agent of 21 dynes/cm surface tension from four samples with three independent measurements for each sample.
Fluorescein isothiocyanate [FITC]-labeled chitosan (FITC-CS) and rhodamine [Rd]-labeled silk fibroin (Rd-SF) were used to prepare electrospun NF under the same processing condition to detect the presence of each component in the NF [17
]. The image of FITC-CS and Rd-SF was obtained from confocal laser scanning microscopy (Zeiss LSM 510, Carl Zeiss AG, Oberkochen, Germany) where FITC-CS fluoresces green and Rd-SF fluoresces red. The excitation wavelengths for FITC and rhodamine are 495 and 54 nm, respectively. The emission wavelengths for FITC and rhodamine are 525 and 576 nm, respectively.
Cell culture and analysis
Nanofibrous membranes were cut into disk shapes with 1.4-cm diameter, sterilized with 75% ethanol overnight, and rinsed three times with phosphate buffer saline before placing in 24-well culture plates (Nunc, Rochester, NY, USA). Human fetal osteoblastic cells (ATCC CRL-11372) were obtained from the American Type Culture Collection (Arlington, VA, USA), and cells at passage numbers 4 to 6 were used. An aliquot of 0.1 ml hFOB cells (4 × 105 cells/ml) was seeded onto the surface of the pre-wetted membrane in each well. Cell seeded scaffolds were incubated at 37°C for 4 h to allow cell adhesion, and the membrane was transferred to a new well with the addition of 1.5-ml culture medium (Dulbecco's modified Eagle medium [DMEM]/F12 (1:1) supplemented with 10% (v/v) fetal bovine serum, 1% (v/v) antibiotic-antimyotic) into each well. Cell culture was carried out at 37°C in a humidified 5% CO2 incubator with medium change every 3 days.
The viable cell number of hFOB at 0, 7, 14, 21, and 28 days was determined by (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) [MTS] assays using the CellTiter 96 AQueous One Solution kit from Promega (Promega Life Sciences, Madison, WI, USA). The kit contains a novel tetrazolium salt which interacts with metabolically active cells to produce a soluble formosan dye. Colorimetric measurement of the formazan product was performed at 492 nm using an enzyme-linked immunosorbent assay [ELISA] plate reader (BioTek Synergy HT, Winooski, VT, USA). Alkaline phosphatase [ALP] activity of hFOB was measured using alkaline phosphate yellow liquid substrate system for ELISA (Sigma-Aldrich Corporation, St. Louis, MO, USA). Nanofibrous membranes with cells were washed with phosphate buffer saline and immersed in the lysis buffer. After centrifugation, the supernatant was collected and reacted with the substrate p-nitrophenyl phosphate for 30 min, and optical density of the colored product was measured with an ELISA reader at 405 nm. Cell growth and ALP activity were determined from five samples with four independent measurements for each sample.
Cell viability was also assessed by LIVE/DEAD Viability/Cytotoxicity Assay kit (Invitrogen, Carlsbad, CA, USA) which provides two molecular probes, calcein AM and ethidium homodimer-1 [EthD-1], to simultaneously determine the existence of live (green) and dead (red) cells. Cells were stained with 2 mM EthD-1 and 4 mM calcein AM and imaged under a confocal laser scanning microscope (Zeiss LSM 510). The excitation wavelengths for calcein AM and EthD-1 are 494 and 528 nm, respectively. The emission wavelengths for calcein AM and EthD-1 are 517 and 617 nm, respectively. For SEM observations, cell/scaffold samples were fixed in 3% glutaraldehyde, dehydrated through a graded series of ethanol soaks, and dried in a critical point dryer (Balzer CPD 030, Bal-Tec AG, Liechtenstein, Germany).
All quantitative data were expressed as mean ± standard deviation. Statistical analysis was performed using the one-way ANOVA LSD test to determine significant differences. A value of p < 0.05 was considered statistically significant.