Preparation and characterization of biomimetic silk fibroin/chitosan composite nanofibers by electrospinning for osteoblasts culture
© Chen et al; licensee Springer. 2012
Received: 30 November 2011
Accepted: 6 March 2012
Published: 6 March 2012
In this study, we have successfully fabricated electrospun bead-free silk fibroin [SF]/chitosan [CS] composite nanofibers [NFs] covering the whole range of CS content (0%, 25%, 50%, 75%, and 100%). SF/CS spinning solutions were prepared in a mixed solvent system of trifluoroacetic acid [TFA] and dichloromethane. The morphology of the NFs was observed by scanning electron microscope, and the average fiber diameter ranges from 215 to 478 nm. Confocal laser scanning microscopy confirms the uniform distribution of SF and CS within the composite NFs. To increase biocompatibility and preserve nanostructure when seeded with cells in culture medium, NFs were treated with an ethanol/ammonia aqueous solution to remove residual TFA and to change SF protein conformation. After the chemical treatment, SF/CS NFs could maintain the original structure for up to 54 days in culture medium. Properties of pristine and chemically treated SF/CS NFs were investigated by Fourier transform infrared spectroscopy [FT-IR], X-ray diffraction [XRD], and thermogravimetry/differential scanning calorimetry [TG/DSC]. Shift of absorption peaks in FT-IR spectra confirms the conformation change of SF from random coil to β-sheet by the action of ethanol, which is also consistent with the SF crystalline diffraction patterns measured by XRD. From TG/DSC analysis, the decomposition temperature peaks due to salt formation from TFA and protonated amines disappeared after chemical treatment, indicating complete removal of TFA by binding with ammonium ions during the treatment. This was also confirmed with the disappearance of F1s peak in X-ray photoelectron spectroscopy spectra and disappearance of TFA salt peaks in FT-IR spectra. The composite NFs could support the growth and osteogenic differentiation of human fetal osteoblastic [hFOB] cells, but each component in the composite NF shows distinct effect on cell behavior. SF promotes hFOB proliferation while CS enhances hFOB differentiation. The composite SF/CS NFs will be suitable for bone tissue engineering applications by choosing a suitable blend composition.
PACS: 87.85.jf; 87.85.Rs; 68.37.Hk.
Many polymers, including synthetic or natural ones, have been electrospun into nanofibers with diameters ranging from tens of nanometers to a few micrometers. Because of their intriguing characteristics such as large surface area, high porosity, and biomimetic of the structure and function of the natural extracellular matrices [ECMs] of native tissue, electrospun polymeric nanofibers [NFs] have found great interest in tissue engineering as scaffolding materials [1–3]. Natural ECMs in the body are mainly composed of two types of extracellular polymers, proteoglycans and fibrous proteins with fiber diameters ranging from 50 to 150 nm, depending on the tissue type . Specifically, ECMs in bone have many protein fibers (e.g., collagen fibers and elastin fibers) and proteoglycans consisting of a protein core covalently bound to long chains of sulfated glycosaminoglycan [GAG] disaccharides (e.g., chondroitin sulfate and hyaluronic acid). Fabricating biomimetic NFs intended for tissue engineering applications should therefore be considered not only from the structural characteristics of the NF but also from its composition, such as combining suitable ECM components like proteins and polysaccharides to fabricate composite NFs .
Silk emitted by the silkworm consists of two main proteins, sericin and silk fibroin [SF], fibroin being the structural center of the silk and sericin being the sticky material surrounding it. The SF protein mainly consists of the recurrent amino acid sequence (Gly-Ser-Gly-Ala-Gly-Ala) n . Among the biodegradable and biocompatible polymers, SF was extensively studied as one of the candidate materials for biomedical applications because it has several distinctive biological properties including good biocompatibility, biodegradability, and minimal inflammatory reaction . Various methods have been proposed to process SF into different kinds of scaffolds such as films, gels, sponges, and non-woven mats for biomedical applications , including NFs by electrospinning [8, 9]. The SF NFs have been used to culture with chondrocytes, osteoblasts, and mesenchymal stem cells and reported to enhance cell proliferation and attachment [10–12]. Chitosan [CS] is a GAG-like linear polysaccharide composed of glucosamine and N-acetyl glucosamine linked in a β(1→4) manner and is deacetylated from chitin, the second most abundant natural biopolymer in the world . This natural biomaterial is found in the shells of crustaceans and is with antibacterial, biocompatible, and biodegradable properties . It is widely used for biomedical applications, such as wound dressings, drug delivery carriers, and tissue engineering scaffolds . The CS NFs have also been produced by electrospinning .
Composite NFs are promising as tissue engineering scaffolds by blending protein and polysaccharide in the spinning solution. However, a well-designed processing condition must be found beforehand to fabricate bead-free nano-sized fibers from the blend spinning solution during the electrospinning step. Previously, collagen/CS NFs have been prepared and used as wound dressings and tissue engineering scaffolds [17, 18]. Both endothelial cells and smooth muscle cells proliferated well on these composite NFs. SF/chitin NFs were found to enhance keratinocytes and fibroblasts attachment and spreading . SF/CS NFs have also been fabricated, but the maximum CS content in the composite can only reach 30 wt.% . Application of this composite scaffold for bone tissue engineering applications will be promising as CS is beneficial for osteogenic differentiation only if the CS content can be raised.
In this study, electrospun SF/CS composite NFs covering the whole range of CS content from 0 to 100 wt.% were successfully fabricated using a trifluoroacetic acid [TFA]/dichloromethane [DCM] mixed solvent system. To make the NFs suitable for bone tissue engineering applications, a new chemical treatment method using ethanol/ammonia was developed to completely remove harmful TFA residues from the NFs and make the NFs insoluble in culture medium. The chemically treated NFs were shown to promote the proliferation and osteogenic differentiation of human fetal osteoblastic [hFOB] cells by the action of the SF and CS component in the NFs, respectively.
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 . 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 . 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.
Results and discussion
Preparation of electrospun SF/CS nanofibers
Fiber diameters and pore sizes of SF/CS nanofibrous membranes
Composition of nanofibers
399 ± 184
0.98 ± 0.21
75% SF/25% CS
215 ± 95
1.23 ± 0.39
50% SF/50% CS
447 ± 168
0.71 ± 0.18
25% SF/75% CS
239 ± 133
0.73 ± 0.11
317 ± 109
0.52 ± 0.10
Analysis of SF/CS nanofibrous membranes
DSC analysis reveals unique features for pristine (Figure 5C) and treated (Figure 5D) NFs of different CS contents. In a recent study, a strong exothermic peak at 305°C and a strong endothermic peak at 280°C were reported for pure CS and SF, respectively [28, 29]. Compared with DTG, DSC analysis shows that only the NH3+-CF3COO- salt in CS leads to a new peak with the disappearance of the exothermic peak at 305°C for CS and appearance of a new endothermic peak at 222°C (Figure 5C). For pristine SF/CS composite NFs, two endothermic peaks due to SF and TFA-CS appear (Figure 5C). After adding ammonia, the endothermic peak of TFA-CS at 222°C changed back to the strong CS exothermic peak at 298°C by completely removing TFA from NFs with ammonium ions (Figure 5D). Also, the strong endothermic peak of SF located at 278°C shifts to a higher temperature at 282°C after the protein structure being transformed into the β-sheet crystallization by ethanol (Figure 5D) . Interestingly, the DSC curve of treated 50% SF/50% CS composite NFs shows a strong exothermic peak similar to that of CS instead of the endothermic peak associated with pure SF (Figure 5D).
Culture of hFOB on SF/CS composite nanofibers
Composite SF/CS NFs covering the whole range of CS content can be prepared by electrospinning. Post-treatment with ammonia/ethanol solution can totally eliminate harmful TFA residue from the NF and render it insoluble in cell culture medium and useful as tissue engineering scaffold. hFOB cells could proliferate and differentiate on the NFs. Each component in the composite NF shows distinct effect on cell behavior, with SF promoting cell proliferation and CS enhancing osteogenic differentiation. By choosing a suitable blend of each component in the composite, 50% SF/50% CS NFs will be a promising candidate scaffold for bone tissue engineering.
Financial support from National Science Council of the Republic of China and Chang Gung Memorial Hospital are highly appreciated.
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