Design
An in vitro contrast observational experiment regarding nerve tissue engineering.
Time and setting
Experiments were performed at the Laboratory of Electrospinning, Materials and Textile Engineering College and Laboratory of Cell Culture, Medical College of Jiaxing University, China between March 2010 and July 2011.
Materials
Pyrrole monomer (Guoyao Chemical Reagent Co., Ltd., Shanghai, China) was distilled under reduced pressure and stored below 0°C. PLLA granules were purchased from Jiaxing Haobang Science and Technology Development Corporation, China.
Methods
Preparation of PLLA micro/nano fibers by electrospinning
The preparation of PLLA micro/nano fibers by electrospinning was as described previously[21]. In brief, PLLA granules were dissolved in dichloromethane using a magnetic stirrer for at least 5 hours to prepare a 12% (w/v) PLLA solution. The solution was held in a syringe for electrospinning. A piece of flat aluminum foil was placed 13 cm below the tip of the needle, and used to collect the PLLA micro/nano fibers. The voltage for electrospinning was 12 kV. The spun micro/nano fibers were left in the aluminum foil for 24 hours to allow the volatilization of dichloromethane to complete. The fibers were then peeled off from the aluminum foil.
Preparation of PLLA/APS-doped PPy composite micro/nano fibrous scaffolds
The thickness of the PLLA fibrous scaffold was approximately 0.45 mm. PPy was introduced using in situ polymerization on the surface of the PLLA multi-porous fibrous scaffold. Typically, 0.5 g of pyrrole was dissolved in 40 mL of deionized water while stirring at 0°C. A dimension of 45 mm × 35 mm PLLA multi-porous fibrous scaffold was immersed into the above solution followed by ultrasonication for 30 minutes to allow the scaffold to be saturated with the pyrrole solution. APS (1.22 g) in 40 mL of deionized water at 0°C was added dropwise to the above mixture with ultrasonic aided dispersion. The polymerization process proceeded for 5 hours without any dispersion. A thin, black PPy coating thus formed on the porous surface and was anchored to the polymer substrate. The pyrrole monomer was oxidized and doped with APS. The scaffold was then washed three times with 80 mL of deionized water and acetone (in sequence) and incubated in 40 mL deionized water for 24 hours to remove any unreacted pyrrole monomers. Finally, the fibrous scaffold was dried under vacuum and stored for usage.
Characterization of fibrous scaffolds
The morphologies of PLLA and PLLA/APS doped-PPy composite fibrous scaffolds were observed using the Hitachi JSM-5510 scanning electron microscope (Tokyo, Japan). Conductivities of PLLA/PPy composite fibrous scaffolds were measured using the four-probe method (ST512-SZT-2A; Zhongxiyuanda Science and Technology Co., Ltd., Beijing, China). X-ray photon spectroscopy was used to characterize the surface compositions of PLLA/PPy composite fibrous scaffolds. High resolution spectra of elements were obtained using a Kratos AXIS Ultra XPS system (Shimadzu, Tokyo, Japan).
PC12 cell culture
Rat PC12 cells (Cell Bank of Chinese Academy of Science in Shanghai, China) were cultured with RPMI 1640 culture solution (Promega) containing 10% (v/v) fetal calf serum and 100 U/mL penicillin and streptomycin, in an incubator containing 5% CO2 at 37°C for 24 hours. Using 0.05% (w/v) trypsin-0.01% (v/v) ethylenediaminetetraacetic acid solution, the PC12 cells were detached from the culture flasks. Following two washes, PC12 cells were resuspended in RPMI 1640 culture solution at 1.6 × 106 cells/mL and were seeded onto the surface of the test scaffolds at a density of 2.4 × 104 cells/cm2.
PC12 cell viability and proliferation on selected scaffolds
Following a 4-day culture, PC12 cells were detached from the test scaffolds after treatment with a 0.05% (w/v) trypsin-0.01% (v/v) ethylenediaminetetraacetic acid solution for 10 minutes at 37°C. Following enzyme treatment, the cells were washed twice with RPMI 1640 culture solution and centrifuged for 10 minutes at 3 000 r/min. The obtained centrifuged pellet was resuspended in 1 mL of culture solution and was used to determine cell viability using the MTT test. Briefly, 50 µL MTT (5 mg/mL in PBS; Promega) was added to each well of the plate and incubated at 37 ± 1°C for 4 hours, followed by the addition of 150 µL of dimethyl sulfoxide. The plate was gently agitated until the formazan precipitate was dissolved, and the absorbance was measured at 490 nm with an ElX-800 Microelisa reader (Bio-Tek Inc., Winooski, VT, USA). Data were reported as mean ± SD of four independent experiments. Noncoated PLLA micro/nano fibrous scaffolds were used as references. Four independent experiments were performed. The relative growth rate was calculated as follows:
Relative growth rate = (average absorbance value at 490 nm of the sample/absorbance value at 490 nm of the control) × 100%.
Electrical stimulation of PLLA/APS doped-PPy micro/nano fibrous scaffolds
The PLLA/APS doped-PPy micro/nano fibrous scaffolds with conductivities between 1.08–2.56 × 10-1 s/cm were used in this experiment. A sample scaffold 2.5 cm × 3 cm in size was fixed at the bottom of a homemade electrical cell culture plate. Two opposite edges of the scaffold were in tight contact with two flat platinum electrodes connected to a low voltage electrical power source as well as to a computerized monitoring system (self-made). To avoid direct contact between the electrodes and the culture medium, medical grade silicone grease (Promega) was used to isolate electrodes from the culture medium. Under this configuration, there was no measurable current between the two electrodes when a PLLA/APS doped-PPy micro/nano fibrous scaffold was installed. The surface area of the test scaffold exposed to the cell culture medium was 2 cm × 2 cm. Following their assembly into the electrical cell culture plates, the scaffolds were sterilized by extensive ultraviolet irradiation and alcohol prewashing, followed by extensive washing in sterilized culture medium. The PC12 cells were seeded onto the test scaffolds and cultured in RPMI 1640 medium containing 5% (v/v) fetal bovine serum without nerve growth factor, in 100 U/mL penicillin and 25 µg/mL streptomycin. The culture medium was changed daily. Before electrical stimulation, the PC12 cells (2.4 × 104 cells/cm2) were grown for 24 hours to allow settling and adhesion. Electrical stimulation was carried out in two phases. In the first phase, twenty culture plates were divided into five groups. Fixing the electrical stimulation time at 2 days, the plates of each group were applied with currents set at 0, 5, 10, 15, and 20 µA. In the second phase, sixteen culture plates were divided into four groups; the plates of each group were applied with the same current of 10 µA, and the electrical stimulation time was 1, 2, 3, 4 days. The temperature in the culture medium was measured and found to remain at 37 ± 1°C.
Assessment of cellular morphology
After several days of culture, half of the tested scaffolds were removed from the culture wells carefully. The morphological changes of PC12 cells detached from the fibrous scaffolds were observed under an inverted light microscope (Olympus, Tokyo, Japan) and photographed. Axon/neurite length was measured as the linear distance between the cell junction and the tip of an axon/neurite[22]. In addition, the percentage of PC12 cells with neurites and the numbers of neurites per cell (for cells that expressed at least one neurite) were calculated. More than 600 PC12 cells were analyzed for each condition. For PC12 cells, data was collected for neurite lengths greater than 5 µm. More than 100 PC12 cells were analyzed for each condition. Cellular morphologies on another half of the tested fibrous scaffolds were visualized using scanning electron microscopy. The samples were treated as previously described[11]. In brief, the fixed cells were dehydrated using increasing ethanol/water concentrations. Samples were dehydrated with hexamethyl disilazane (Sigma, St. Louis, MO, USA) and dried in air overnight. Gold was coated on the sample using a sputter coater. Scanning electron microscopy images were obtained with the Stereo Scan 260 Scanning Electron Microscope (Hitachi).
Statistical analysis
All data presented were expressed as mean ± SD. SPSS 17.0 statistical software (SPSS, Chicago, IL, USA) was used for statistical analysis of experimental data. One-way analysis of variance was carried out to compare the means of different data sets, and a value of P < 0.05 was considered statistically significant.
.jpg)
