The goal of this proof-of-concept study was the fabrication of porous silk fibroin (SF) microspheres which could be used as cell culture carriers under very mild processing conditions. The SF solution was differentiated into droplets which were induced by a syringe needle in the high-voltage electrostatic field. They were collected and frozen in liquid nitrogen and water in droplets formed ice crystals which sublimated during lyophilization and a great quantity of mi cropores shaped in SF microspheres. Finally, the microspheres were treated in ethanol so as to transfer the molecular conformation into β-sheet and then they were insoluble in water. SF particles were spherical in shape with diameters in the range of 208.4 μm to 727.3 μm, while the pore size on the surface altered from 0.3 μm to 10.7 μm. In vitro, the per formances of SF microspheres were assessed by culturing L-929 fibroblasts cells. Cells were observed to be tightly ad hered and fully extended; also a large number of connections were established between cells. After 5-day culture, it could be observed under a confocal laser scanning microscope that the porous microenvironment offered by SF parti cles accelerated proliferation of cells significantly. Furthermore, porous SF particles with smaller diameters (200 - 300 μm) might promote cell growth better. These new porous SF microspheres hold a great potential for cell culture carriers and issue engineering scaffolds.
Amplification of seed cells is the basis of tissue engineering, while microcarrier technology has realized the extensive culture of animal cells. The higher specific surface area of microspheres could provide adequate adherent place for cells so as to be conducive to cell adhesion and proliferation. The current research about microcarriers focuses on polysaccharides, including cellulose, chitosan, hyaluronic acid, alginate, dextran and starch, as well as on proteins such as collagen, gelatin, elastin, albumin and silk fibroin [1,2].
Silk fibroin is a natural, fibrous protein with excellent biocompatibility and mechanical property [3,4]. SF materials can support the attachment, proliferation, and differentiation of primary cells and cell lines [5-7], and is easily prepared as films [
The preparation methods of SF microspheres mainly include protein denaturation method, emulsificationcuring method, spray-drying method, template method, and high-voltage electrostatic technology. Desolvation technique was implemented for the preparation SF particles using dimethyl sulfoxide (DMSO) as desolvating agent [
SF materials with porous structure on surface are more helpful for cellular pseudopods extension, intercellular signal transmission, and extracellular matrix deposition [
Silk fibroin aqueous solutions were prepared as previously described [
SF spheres were prepared by using high pressure electrostatic generator (DW-P503-4ACCD, Dongwen High Voltage Power Plant, Tianjin, China) and micro-injection pump (WZS50F2, Zhejiang University Medical Instrument Co., Ltd, Zhejiang, China). A nozzle with diameter of 0.7 mm was linked with the syringe and the whole were fixed on the pump. The distance between the needle and the collection box was settled 100 mm. The 3 wt% SF solution in injector was differentiated into droplets under a high-voltage electrostatic field. The produced droplets were continuously collected and frozen in a liquid nitrogen bath (
microspheres which were insoluble in water could be obtained after lyophilized again followed after steeping into deionized water for 3 days to remove residual ethanol.
The surface and cross-section of SF microspheres were examined morphologically by scanning electron microscopy (SEM, Hitachi S-4700, Japan). The particle size of SF spheres was analyzed on the basis of SEM images with the Nano Measurer analysis software (Department of Chemistry, Fudan University, Shanghai, China). For each particle preparation we determined the average of the equivalent circular diameter of a total of 100 spheres.
Untreated and treated with ethanol SF spheres were cut into powder with radii less than 40 μm, the samples were mixed with KBr and compressed to KBr disks. FTIR spectroscopy was performed with a Nicolet 5700 FT-IR (Nicolet Company, USA). The wave number ranged from 400 to 4000 cm−1.
Murine fibroblasts cell line L-929 (Basic Medicine and Life Science Academy of Soochow University, Suzhou, China) was chosen to evaluate the influence of SF spheres with different particle sizes on cell adhesion and proliferation. 3 mg various SF spheres were put in a 24- well tissue culture plate (TCP, Corning Inc., USA), and then rinsed with tri-distilled water for 7 days followed by sterilization with γ-ray irradiation. SF spheres were soaked with serum-containing medium for 30 min before the fibroblasts were seeded so as to increase cell adhesion. Murine fibroblasts at a density of 1 × 105 cells per well were seeded onto the SF spheres in 24-well plates and blank culture plates for control experiments. The medium used was 90% Dulbecco’s modified Eagle’s medium (DMEM), 9.0% bull serum albumin (BSA), and 1.0% Streptomgein/Ampicillin (purchased from Sibas Biotechnology Co., Ltd., Shanghai, China). The cellseeded SF spheres were incubated at 37˚C in 5% CO2 atmosphere, and the medium in the well was replaced with fresh medium every other day. After 5 days in culture on microspheres, cells were fixed with 2.5% glutaraldehyde and then incubated overnight at 4˚C. The fixed microsphere cultures were washed twice with phosphate-buffered saline (PBS), froze at −80˚C for 2 h, and freeze-dried for 36 h. Dry samples were platinumcoated in vacuum and examined by SEM.
The proliferation of cells on SF microspheres was observed by confocal scanning laser microscope (CLSM, TCS-SP2, Leica Company, German). L-929 cells on various SF microspheres and culture plates were labeled with CM-DiI fluorescent dye, then were observed by CLSM when incubated for 1, 3, and 5 days. Cultures on SF particles were selected for cell counting on days 1, 3, 5, 7, and 9 in vitro by cell counting chamber after they were washed twice with PBS and digested for 20 min with trypsin.
The methyl thiazolyl tetrazolium (MTT) assay was used to measure the cell viability. After 1, 3, 5, 7, and 9 days, 200 μl MTT dye solution (5 mg/ml in phosphate buffer at pH 7.4) was added into each well. After 4 h of incubation at 37˚C and 5% CO2, the medium was removed and formazan crystals were solubilized in HClIsopropanol overnight. The optical density (OD) of formazan was measured on a Synergy HT (BIO-TEK) microplate reader at 490 nm. Data were presented as means ± SD. Statistical comparisons were performed using ANOVA, and differences at P < 0.05 were considered statistically significant.
Process parameters included SF solution concentrations, electrostatic voltage, solution flow rate, inner diameter of needle, and collection distance, while electrostatic voltage and solution flow rate had deeper influence on particle size. Four groups of (a), (b), (c), and (d) microspheres with various diameters were obtained by changing electrostatic voltage and solution flow rate (
From
The SEM images (
tion was below ice point, ice nucleus was emerged due to heat exchange. Because of supercooling, the chemical potential of water in unstable SF solution phase was higher than that of ice nucleus so that ice nucleus grew into large ice particles [
FTIR spectra (
L-929 cells were seeded on SF spheres of groups (a), (b), (c), and (d) at a density of 1 × 105 cells per well in 1.0 ml of growth medium and allowed to attach for 4 h at 37˚C with 5% CO2 atmosphere in an incubator.
The SEM observation of L-929 cells cultured on SF particles with different sizes showed that cells on spheres were of high density and represented normal morphology (
Growth of L-929 cells on SF spheres was examined by CLSM. SF particles appeared black while CM-labeled cells presented red under CLSM; the single red point was clear which revealed that cells were labeled well (
In the early days of cell culture (1 - 5 d), cellular adhesion rate of SF spheres was lower than that of cell culture plates due to cell inoculation (
was also favorable for cellular pseudopods extension, intercellular and cell-surroundings signal transmission, and extracellular matrix deposition. During culture process, cells on various SF particles manifested different growth state. As time increased, the number of cells on smaller SF particles (208.4 μm, 306.2 μm) was obviously more than that on larger ones (505.7 μm, 727.3 μm) owing to the larger specific surface area and more pores of smaller spheres.
The quantity of viable L-929 cells on SF microspheres with various sizes was estimated using the MTT assay (
The fabrication of SF microspheres with controllable sizes combining high-voltage electrostatic field differentiating and freeze-drying technique was described, and the structure of SF particles was induced by the ethanol treatment. Murine fibroblasts L-929 could adhere tightly on the surface of porous SF microspheres with diverse diameters as the larger specific surface area of spheres offered adequate adherent place for cellular vast proliferation. The existence of plentiful pores further supplied
better microenvironment for cellular pseudopods extension, and cell-external signal transmission. Particles of smaller size were more conducive to cellular adhesion and proliferation. Therefore, such porous SF microspheres have a potential application as cell culture carriers.
This work was supported by the National Nature Science Foundation of China (30970714), the College Natural Science Research Project of Jiangsu Province (12KJA- 430003), the Key Program in Medical Science Research of Military (No.BWS11C061) and the Priority Academic Program Development of Jiangsu Higher Education Institutions.