Methylene bridged anthracene polymer (PMAn) was synthesized via Friedel-Crafts alkylation reaction [26, 27]. Weight average molecular weight (M
w) of the resultant polymer by gel permeation chromatography (GPC) was 6700 with M
of 1.7. α-Minimum Essential Medium (α-MEM), Fetal Bovine Serum (FBS), antibiotics (Penicillin/Streptomycin), Phosphate-Buffered Saline (PBS, pH 7.4), Trypsin/EDTA (0.05 %), and Tryphan blue (0.4 %) were purchased from Gibco (Invitrogen, USA). Albumin (5 %) was purchased from Green cross corporation (Korea). Bone Marrow (BM)-derived Mesenchymal Stem Cells (MSCs), on patient compliance, were used for this study. A frozen stock of MSCs was provided by Cell Therapy Center, Severance hospital (from University of Yonsei, Seoul, Korea) at passage 3. Other chemicals and solvents were purchased from Aldrich.
1H spectrum was determined on a Bruker ARX-300 spectrometer. The average molecular weight of the polymer was characterized by a gel permeation chromatography (GPC) (model: Waters R-401 ALC/GPC) with THF as an eluent and polystyrene standard for calibration. Fluorescence spectra were obtained with a luminescence spectrometer (PerkinElmer, Model LS55) under excitation at 370 nm. The polymer films were illuminated with a UV lamp (Rolence Enterprise, Inc., Taiwan, power: 13.05 mW/cm2), model POWERARC UV 100. The surface wettability was investigated by a water (DI) drop contact angle measurement using Contact Angle Meter-CAM 101 model (KSV Instruments Ltd, FINLAND). AFM analysis was carried out in room temperature with a Dimension 3100 SPM equipped with Nanoscope IVa devised by Digital Instruments from Santa Barbara, CA. The fluorescent patterns such as were imaged under Olympus-BX51 fluorescence microscope with WB – dichroic mirror DM500, excitation filter BP450-480 and barrier filter BA515. The optical MSCs patterns were obtained from Olympus inverted research microscope model IX71. To detect cell patterns more in detail, MSCs were observed with field emission-scanning electron microscope (HITACHI S-800, Tokyo, Japan) and the picture was taken by scanning microscope image analysis system (ESCAN-4000, Bummi Universe, Tokyo, Japan)
The preparation of PMAn film and PMAn patterned substrates
PMAn films having average thickness of 185 nm were prepared by spin coating of chloroform solution of polymers (1 wt %) at 1200 rpm for 15 s and then dried under room temperature for solvent removal. These pristine PMAn films, without pattern, were used for the spectroscopic measurements, contact angle measurement, and the proliferation assay of MSCs. For fluorescent pattern formation, the PMAn films were illuminated with a high-intensity UV lamp (13.05 mW/cm2) through a photomask. These PMAn patterns were used for the AFM and MSCs pattern.
The analyses of PMAn surface with contact angle and AFM measurements
Surface wettability of the PMAn films through photo-reaction was investigated by water (DI) drop contact angle measurement. PMAn films were illuminated by high intensity UV source for 1, 3, 5, 10 mins. For surface morphology experiments, the polymer films on silicon wafer were illuminated by a high-intensity UV source for 15 min through a 10 μm line pattered photomask. AFM analyses were carried out at room temperature with a Dimension 3100 SPM equipped with Nanoscope IVa devised by Digital Instruments from Santa Barbara, CA. The AFM tip was oscillated at its resonance frequency (75 kHz). Next, the tip was lifted with fixed distance above the sample surface and scanned at that constant height with a voltage applied.
Cell culture and proliferation assay on PMAn film substrates
MSCs were thawed, placed at a density of about 10,000 cells/cm2 in 15 mL medium (α-MEM supplemented with 10 % FBS, 100 U/mL penicillin and 100 μg/mL streptomycin) in a 75 cm flask (Nunc, Denmark), at 37 °C in 5 % humidified CO2. Medium was replaced with fresh α-MEM with 10 % FBS, 100 U/mL penicillin and 100 μg/mL streptomycin every 3 or 4 days and cells were grown to 90–95 % of confluence over about 3–7 days. Once the cells reached confluence, they were detached using 0.05 % Trypsin/EDTA and replaced for further expansion. The MSCs in this study were between passage of 4 and 7.
To investigate the proliferation of MSCs on PMAn film substrates, two PMAn films were prepared on glass substrates by spin coating. One of the films was exposed to UV source for 5 min and the other film was not exposed to UV. The prepared PMAn film substrates were washed with α-MEM supplemented with 10 % FBS, 100 U/mL penicillin and 100 ug/mL streptomycin. Cells (from passage 4) were harvested by treating a solution of 0.05 % Trypsin/EDTA for 3–5 min at 37 °C, placed at 2,000 cells/cm2 in unexposed PMAn substrate and UV-exposed PMAn substrate. Cells (from passage 4) were harvested also on a tissue culture polystyrene as a reference experiment. Each well was dispensed with 3 mL α-MEM medium and changed once after 2 days. Cultures were maintained for 5 days and 7 days and then harvested for cell counting, respectively. This proliferation assay was examined at three different times. The growth rates for MSCs on the sample after 5 days and 7 days of culture were determined by counting the number of cells with a hemacytometer after tryphan blue staining in a counting chamber.
Cell attachment to micro-patterned PMAn substrate
For cell patterning studies, the PMAn pattern on a glass substrate (1.6 cm × 2.8 cm) was prepared by exposing the pristine PMAn film on UV light for 5 min. The micro-patterned substrates were placed in six well plate (Nunc, Denmark) containing culture medium, α-MEM supplemented with 10 % FBS, 100 U/mL penicillin and 100 μg/mL streptomycin. MSCs were detached from the cell culture substrates by trypsinization. The cells at a concentration of 4,000 cells/cm2 were seeded on a patterned substrate in six well plate (Nunc, Denmark) and maintained under culture condition for 2 days at 37 °C in 5 % humidified CO2.