Type I collagen was manually extracted using a method explained in the following section. PET (Sky blue grade, Japan) and PDMS (Sylgard 184, Dow Corning, USA) were obtained. Tetrahydrofuran (THF), N, N-dimethylformamide (DMF), 1, 1,3,3,3 hexafluoro-2-isopropanol (HFIP), and Alamar blue were purchased from Sigma-Aldrich, Germany. Glutaraldehyde solution 25% (ready to use) was purchased from Panreac Applichem, Iran. DMEM/F12, PBS, FBS, and Trypsin-EDTA (0.25%) were purchased from Gibco, USA.
Type I collagen was extracted from mice tendons by a protocol explained in . Briefly, 12 rat tails were washed in 70% ethanol. The skin of each tail was removed with a razor, and the collagen fibers were extracted by pliers. The fibers were then rinsed three times with deionized water and placed on a magnetic stirrer with 0.2 percent acetic acid solution for 24 hours at 4°C. The solution was then centrifuged at 4°C for 30 minutes at 11200 RCF. The supernatant was then poured into flat containers and placed at -20°C. Then, frozen fibers were put in a freeze-dryer device (LYOQUEST-55, Telstartechnologies, Spain) for the solvent to evaporate.
Fabrication of nanofiber membrane
In this study, the electrospinning technique was used to prepare PET/PDMS/Collagen nanofiber membrane. The electrospinning device (FNM, Iran) includes two syringe pumps, two high-voltage power supplies, and a rotating collector which is covered by aluminum foil (Fig. 1A).
To construct the proposed nanofiber membrane, PET was dissolved in DMF/THF (1:1) solvent (25% wt) at 45°C in constant stirring conditions. Separately, PDMS/Curing agent (10:1) was added to DMF/THF (1:1) solvent (10% wt) on a magnetic stirrer at room temperature. Then, 5% wt collagen solution was prepared in HFIP solvent. When PET solvent reached transparency, it was left to cool down to room temperature and mixed with PDMS on a magnetic stirrer at room temperature. Then, PET/PDMS and collagen were loaded into two separate 5ml syringes with blunt 18G needles and placed inside the appropriate pumps. The distance between the needle of the syringe and the collector was maintained at 15 cm on each side and the pumping rates were set at 0.4 ml/h. The syringe loaded with PET/PDMS was connected to a power supply that provided 22 kV, and the other syringe that was loaded with collagen was connected to 20 kV. The speed of the roller collector was set to 90 rpm. The resulting woven nanofiber mat was placed inside a vacuum desiccator for 30 minutes and received Glutaraldehyde (25%) vapor to crosslink its surface.
Characterization of nanofibers
Scanning Electron Microscopic
Nanofiber samples were coated with gold nanoparticles by an ion sputtering coater (Polaris SCM-200, South Korea) to enhance electrical conductivity for imaging by scanning electron microscopy (SEM). Gold-coated nanofibers were then placed in a high-vacuum chamber and the images were taken at 20 kV (SNE-4500M, Korea). Nanofibers were evaluated for morphology, size, and size distribution. ImageJ and Origin were used for image visualization.
Atomic Force Microscopy
Atomic Force Microscopy (AFM, Nanosurf easy, Switzerland) analysis was carried out to determine the surface roughness and morphology of nanofibers.
The Brunauer, Emmett and Teller (BET) and Barrett-Joyner-Halenda (BJH)
The specific surface area of the nanofibers was determined by the BET method. Samples were dried in a clean dry degassed station (BEL SORP, Mini2, Japan) for 4 hours at 77℃ to eliminate humidity. Then, nitrogen (N2) gas adsorption and desorption data in the relative pressure in the range of 0.05 − 0.03 was graphed and the effective surface areas under the curve were calculated. The BJH model was then used to calculate pore size distributions. Similarly, surface porosity was determined using the BJH method. Diffuse reflectance spectrum was recorded using a UV-Visible spectrometer (Analytic Jena, Thuringia, Germany). Data was collected for nanofibers before and after crosslinking by glutaraldehyde vapor.
Contact angle measurement
To examine wettability, the angle at which water droplets became in contact with the surface of nanofibers was assessed. For this purpose, a five square centimeter nanofiber membrane was placed under a contact angle instrument (MehrTavNegar, Iran) equipped with a camera at 25℃. A small drop of deionized water (6µl) was carefully placed over the surface of the nanofiber with a pipette and images of the droplet and the surface of the nanofiber were taken at the interfaces (before and after crosslinking).
Fourier transform infrared spectroscopy analysis
Fourier transform infrared (FT-IR) spectroscopy analysis was conducted by an FT-IR analyzer (WQF-510A, China) to validate the structural constituents of the prepared PET, PDMS, and collagen nanofiber. Samples have been entirely mixed with KBr for analysis. KBr has been used as a carrier for the sample in the FT-IR analysis.
Mechanical strength properties
Universal Testing Machine (UTM, Roell z050, Zwick, German) was used to evaluate the mechanical tensile of the nanofibers before and after crosslinking at 2 mm/min and room temperature.
Human umbilical vein endothelial cells (HUVECs) were cultured in T-75 flasks with DMEM-F12 rich medium containing 10% FBS and 1% penicillin/streptomycin and incubated at 37℃ and 5% CO2. Cells were cultured once reached 90% confluence (about 3 days).
The viability of cells cultured on nanofibers was assessed by Alamar Blue based on a procedure explained in . Briefly, the nanofibers were cut into circles 1 cm in diameter, sterilized with 70% ethanol for 15 minutes, and then exposed to UV irradiation for 30 minutes. The nanofibers were then placed at the bottom of a 48 well plate where 10 mm autoclaved O-rings secured them in place. Then, 0.5 × 104 HUVECs cells in 250µl F12-enriched medium, 10% FBS, and 1% penicillin/streptomycin were poured on top of the nanofibers. Cell viability was assessed after 1, 3, and 5 days of culture. For that purpose, the culture medium in each well was aspirated and replaced by 250µl of complete culture medium containing 10% Alamar blue and incubated at 37°C and 5% CO2 for 4 hours. Then the plate was placed inside an ELISA microplate reader (BioTek, USA) and read at 570 and 630 nm.
Cell attachment by SEM
Cells were cultured in a 48 well culture plate and placed in an incubator at 37°C and 5% CO2 for 48 hours as explained in the last step. Then, the culture media was discarded, and the wells were rinsed with PBS. After that, the cells were fixed by adding a 1:1 solution of 2% glutaraldehyde and 2.5% paraformaldehyde and kept at 4℃ for 1 hour. The nanofibers were then exposed to 60, 70, 90, and 100% ethanol for 5 minutes to dehydrate. In the next step, the nanofibers were coated with gold nanoparticles as explained previously for SEM imaging and conformational cell attachment analysis.
Fabrication of the microfluidic device
A simple PDMS-based microfluidic device containing an upper and a lower channel was designed to house the nanofiber membrane (Fig. 1B). Once the masks were designed, soft lithography was used to fabricate the molds. In summary, SU8-2050 (Microchem, USA), a negative photoresist, was spread over a silicon wafer by a spin-coater (Microchem, Newton, MA, USA). Then UV light was lit through the mask over the wafer by a mask aligner (Danesh Equipping System, LSM5, Iran) allowing the SU8 to cross-link onto the surface of the wafer. Once the unbonded photoresist was washed away from the surface of the wafer, a negative imprint of the microchannel design was created. In this study, two silicon molds (upper and lower) were fabricated. To create the microfluidic device, PDMS was mixed with its curing agent (10:1) and debubbled inside a vacuum pump. The mixture was then poured inside the silicon molds and placed on a hot plate at 80°C for 1 hour. Once cast, designated inlets and outlets were created using a biopsy punch (diameter = 3 mm). Then, the surface of each casted PDMS was washed with isopropyl alcohol and acetone (Merck, Germany). After drying, the nanofiber membrane was fused between the upper and lower sections via an oxygen plasma treatment apparatus (Harrick Plasma, Ithaca, NY, USA) and a leak-free microfluidic device was formed. To sterilize, the device was rinsed with 70% ethanol and exposed to UV for 30 minutes.
Cell culture inside the microfluidic device
To ensure free fluid motion within the device, culture media was injected into the top channel. Then, the device was placed inside an incubator for 15 minutes. Then, yellow micropipette tips were left inside the input and the output ports. Two million cells (HUVEC) were diluted in 1 ml culture media. Then, 100µl of that solution was carefully injected inside the micropipette tip pinned to the channel input port, where gravitational and capillary forces allowed the gentle flow of cells inside the channel. Under a light microscope, the flow of cells towards the nanofiber membrane and the output port was monitored. For cells to sediment and attach to the nanofiber membrane, the device was placed face-up inside an incubator for 4 hours.
Acridine Orange and Propidium Iodide staining
To ensure whether the cells were able to survive inside the microfluidic device, acridine orange (AO) and propidium iodide (PI) were used. AO is a cationic dye that is able to penetrate the cell membrane and bind to the DNA of living cells where it produces green fluorescent light. PI on the other hand can only penetrate the nonintact membrane of dead cells and bind to the DNA whereby it generates an orange fluorescent light. Once the cells were sedimented inside the device, the device was kept inside an incubator for 24 hours. Then, 50µl of a 1:1 mixture of AO and PI with a concentration of 50µg/ml was injected into the input of the top channel and quickly placed under the fluorescence microscope. AO emits green fluorescence with the maximum wavelength at 526 nm (excitation 502 nm) where PI is excited at 488 nm and, with a relatively large Stokes shift, emits at a maximum wavelength of 617 nm (orange).
DAPI (4',6-diamidino-2-phenylindole) was used as a simple verification method to detect the presence of cells on the nanofiber membrane by staining the cell nucleus. Therefore, the microchannel was washed twice with PBS and then 50 µl of DAPI solution dissolved in deionized water (1 µg/mL) was injected into the input port and left for at least 1 minute. Then, the device was placed under fluorescence microscopy (Optika, IM-3, Italy) where DAPI was excited with ultraviolet light (358nm) and was detected through the blue/cyan filter.