The manufacturing of the OSTE+ samples started with a molding step where the samples were cured in a PDMS mold using UV-light, followed by a post exposure bake at 65 °C. Half of the OSTE+ samples were then incubated in MilliQ water (Millipore, Bedford, MA, USA) at room temperature for 7 days to allow non-bound constituents to leach out. These samples are called OSTE+H2O throughout this paper. All samples were then incubated in water at 37 °C for 72 h (in compliance with ISO standard 10993–12) and the extractions were used in two different assay systems: chemical analysis and cytotoxicity analysis (in compliance with ISO standard 10993–5). The whole process is shown schematically in Fig. 1.
The cytotoxicity tests were performed using MTT assays in compliance with ISO standard 10993–5, and all samples were extracted using ISO standard 10993–12.
Polymer fabrication
OSTE+
OSTE+ is produced in a two-step polymerization reaction as previously described [18]. First, a fast UV-initiated radical polymerization step results in a cross-linked network of some of the thiol groups and of all the ene groups in the allyls. Then a thermal anionic polymerization step results in a fully polymerized network with the unreacted thiol groups and the epoxy resin.
In the first polymerization step, UV light was used o activate Lucirin® TPO-L (BASF, Germany) that initiated cross-linking between the thiol, tris[2-(3-mercaptopropionyloxy)ethyl] isocyanurate (90 %, Sigma Aldrich, Germany), and the two allyls, trimethylolpropane diallyl ether (90 %, Sigma Aldrich) and 2,4,6-triallyloxy-1,3,5-triazine (97 %, Sigma Aldrich). In the second polymerization step, 1,5-diazabicyclo[4.3.0]non-5-ene (98 %, DBN, Sigma Aldrich) was used as the initiator for cross-linking between the thiol and the epoxy resin, D.E.N 341 Epoxy Novolac resin (Dow Chemicals, USA).
All ingredients were weighed according to a stoichiometric ratio of monomers of 1.5 : 0.47 : 0.53 : 0.5 for thiol, diallyl, triallyl and the epoxy resin, respectively. TPO-L and DBN were used at 0.2 %. Finally, 3 % acetone (Sigma Aldrich) was added to the mixture. All ingredients were mixed and degassed under vacuum for 5 min. The mixture was then poured into a pre-fabricated PDMS mold. The mold containing the mixture was then covered with a polycarbonate film and exposed to UV-light for 40 min using a Karl Suss MA4 mask aligner. The polycarbonate film was removed after the exposure and the OSTE+ pieces were hard-baked (to finalize the thiol-epoxy polymerization) in an oven at 65 °C overnight. The ratio of the different constituents results in an OSTE+ with a glass transition temperature of 39 °C [18].
Polyimide
Polyimide was selected as a reference material since it is a material used in neural probes today [8, 9, 11, 13, 20, 22]. Polyimide (Durimide 7505, FujiFilm, Belgium) was poured into a PDMS mold, followed by a 2 h soft-bake (to remove the solvents) at 85 °C. The mold with polyimide was exposed to UV-light for 40 min using a Karl Suss MA4 mask aligner. The polyimide was then hard-baked (cured) in an oven at 200 °C for 4 h. Finally, the polyimide pieces were rinsed in MilliQ water.
HDPE
A sheet of HDPE (Bay Plastics LTD, England) was cut into pieces using a milling machine. The pieces were cleaned with acetone (Sigma Aldrich) in an ultrasonic bath followed by a rinse in ethanol (Solveco, Sweden) and MilliQ water.
Polymer extraction
Extractions of polymer samples were obtained according to ISO standard 10993–12. The extraction was performed in cleaned borosilicate glass vials and water (LC-MS Ultra Chromasolv, Sigma Aldrich, for the chemical analysis and MilliQ water for the cytotoxicity analysis) was used as the extraction medium. The ratio of the surface area of the OSTE+, OSTE+H2O, polyimide or HDPE samples and the water was 3 cm2/ml. The samples were washed in MilliQ water before extraction. The vials were gently shaken at 37 °C for 72 h. When the extraction solution was used in cytotoxicity tests, the polymer samples were first sterilized using an autoclave at 121 °C and the extractions were performed with sterile water.
Chemical analysis
LC-MS analysis
To obtain standards for the LC-MS analysis, an exact amount of each chemical used for the production of OSTE+ was dissolved in methanol (Sigma Aldrich) overnight (except acetone and D.E.N 431). The following day the solutions were diluted to a concentration of 500 μg/ml using 50 % MilliQ water and 50 % methanol. This was followed by a second dilution step to 50 μg/ml using MilliQ water. Acetone was not tested since it is not detectable in the LC-MS system used; D.E.N 431 was not tested since it is not water-soluble. To simulate the UV-lithography process of the fabrication of OSTE+, all standards were exposed to UV-light using a Karl Suss MA4 mask aligner for 40 min. All standards, extractions (described above) of OSTE+, extractions of OSTE+H2O as well as blank samples, were analyzed using LC-MS.
A LC-MS system (QStar XL, Sciex, together with an Agilent 1100 LC system) was used with an Acquity CHS C18 1.7 μm 2.1x50 mm column and a sample volume of 5 μl. The mass spectrometer was configured to scan 120–1000 Da at 2 scans/s. Acetonitrile (LC-MS, Chromasolv, Sigma Aldrich) and MilliQ water were used at a flow rate of 250 μl/min. The concentration of acetonitrile in the mobile phase was: starting at 5 % for 1 min, ramp to 95 % over 3 min, hold for 2 min, ramp down to 5 % in 0.1 min.
GC-MS analysis
To obtain standards for the GC-MS analysis, an exact amount of all constituents (except acetone) was dissolved in toluene (Sigma Aldrich and VWR). To simulate the UV-lithography process of the fabrication of OSTE+, all samples were exposed to UV-light for 40 min using a Karl Suss MA4 mask aligner. 400 μl of the OSTE+ extractions or OSTE+H2O extractions (described above) was pipetted to a new vial containing 1 ml of toluene. The toluene extraction samples were mixed using a vortex for 40 h, followed by centrifugation for 1 min. To ensure that all chemicals in the extracts were transferred into the organic phase, 2 samples of each extraction were made acidic by adding 50 μl of 0.1 M HCl (VWR) and 2 samples of each extraction were made basic by adding 50 μl of 0.1 M NaOH (VWR) just before the addition of toluene to each extraction. The extracts spiked with HCl are referred to as OSTE+/HCl and OSTE+H2O/HCl throughout the paper and the extracts spiked with NaOH are referred to as OSTE+/NaOH and OSTE+H2O/NaOH throughout the paper. All samples and extracts together with a sample of pure toluene were analyzed using GC-MS. Acetone was not tested since it is used as a cleaning step in the GC-MS analysis.
All analyses were performed on a gas chromatograph (Agilent 6890) equipped with an injector in split mode at 250 °C, equipped with an auto sampler (sample volume of 5 μl) and a mass spectrometry detector (Agilent 5973-N). The column used in the system was a HP-5 ms fused silica capillary column (5 % phenyl-methylpolysiloxane) of 30 m × 0.25 mm with a phase thickness of 0.25 μm. The temperature program of the column was: 50 °C, hold for 1 min, ramp to 300 °C over 10 min, hold for 15 min. The carrier gas used was helium at a flow rate of 1.2 ml/min. The mass spectrometer was run in either scan mode (30–600 Da with 2.6 scan/s) or in selected ion monitoring (SIM) mode, using mass to charge ratios (m/z) of 70, 81, 82, 99, 123, 125, 147, 187, 197, 254, 321 together with a dwell time of 20 ms.
Scanning electron microscopy analysis of OSTE+
Four OSTE+ pieces were cut into two halves. One half was put into MilliQ water for 7 days and then each pair was studied by scanning electron microscopy (SEM, SU 8010, Hitachi, Japan) to see if 7 days of incubation in water affected the surface of OSTE+.
Cytotoxicity analysis
Four different materials were tested in the MTT assay: OSTE+, OSTE+H2O, polyimide and HDPE. Polyimide was selected since it is regarded as biocompatible and there are several studies that can be used for comparison. HDPE is a biocompatible material that sometimes is used as a negative control in different cell viability assays [22, 25, 26].
Cell culture
The mouse fibroblast cell line L929 was purchased from American Type Culture Collection (ATCC, LGC standards AB, Borås, Sweden) and was grown as a monolayer culture in complete cell culture medium, i.e. RPMI-1640 medium supplemented with 10 % fetal bovine serum (FBS), 100 U/ml penicillin, 100 μg/ml streptomycin and 1 mmol/l L-glutamine, at 37 °C in 5 % CO2 in air. The cells were sub-cultured twice every week at a density of 12 500 cells/cm2.
Polymer extractions for cell culture
The extracts were obtained as described above and kept at 4 °C for a maximum time of 24 h before use in the cytotoxicity assay with L929 cells. The extracts were diluted 1:2 in 2x concentrated RPMI-1640 medium supplemented with 20 % FBS, 200 U/ml penicillin, 200 μg/ml streptomycin and 2 mmol/l L-glutamine, i.e. a final extract concentration of 50 % in 1x concentrated RPMI-1640 containing 10 % FBS, 100 U/ml penicillin, 100 μg/ml streptomycin and 1 mmol/l L-glutamine, before addition to cell cultures Further dilutions of the 50 % concentrated polymer extracts were done in 1x concentrated RPMI-1640 medium supplemented with 10 % FBS, 100 U/ml penicillin, 100 μg/ml streptomycin and 1 mmol/l L-glutamine.
MTT assay – extract testing
L929 cells were seeded in complete cell culture medium in 96-well plates (2000 cells/well in 180 μl medium) and were then incubated at 37 °C in 5 % CO2 in humidified air for 24 h. The medium was then removed and changed to medium containing the different extracts at concentrations ranging from 6.25–50 % medium containing 2.5 % dimethyl sulfoxide (DMSO: positive control) or control medium (complete cell culture medium with 0 % extract). The cells were then kept at 37 °C in 5 % CO2 in humidified air for 72 h before addition of MTT to a final concentration of 0.5 mg/ml. The 96-well plates were incubated with MTT at 37 °C for 1 h before removal of the medium, and the cells with blue formazan crystals were then dissolved in DMSO. The following spectrometry reading at 540 nm was done using a SPECTRAmax M2 instrument (Molecular Devices, Sunnyvale, CA, USA) and analyzed using SoftMax® Pro v. 4.6 (Molecular Devices). A number of 3–6 biological replicates (i.e. the testing of independent extracts of OSTE+, OSTE+H2O, polyimide and HDPE) were used for all the treatments of the L929 cells and a number of 6 technical replicates (i.e. number of n) from each extract were used for each biological replicate. After correcting all absorbance values for background, the percent of control was calculated as absorbance units in the presence of test compound as percentage of that in control. The results of the MTT assay are resumed to reflect the cell number, thus, 50 % percent MTT reduction of control implies that there were 50 % less cells at that treatment concentration [27].
MTT assay - individual chemicals
Each chemical used in the fabrication (except acetone), 1 or 0.1 mg, was added to a sterile flask and 1000 μl sterile water was added. Further dilutions were made using sterile water. D.E.N 431 was not soluble in water and after shaking the sample and letting it stand for a while, only the water solution was used in the test. L929 cells were seeded in complete medium in 96-well plates (2000–3000 cells/well in 180 μl medium) and the plates were incubated in a CO2 incubator for 24 h before addition of the chemicals. Indicated concentrations of the chemicals were added to the wells and the cells were incubated for 72 h at 37 °C in 5 % CO2 in humidified air. The dose response was evaluated using MTT as described above.
Phase contrast and fluorescence microscopy
L929 cells were seeded in 12-well plates containing sterile glass coverslips, whereupon they were incubated at 37 °C in 5 % CO2 in humidified air for 24 h. The growth medium was then removed and changed to growth medium without extract or containing the different polymer extracts (at a concentration of 50 %, obtained as described above). The cells were then incubated for 72 h before they were photographed with an Olympus CKX41 inverted phase contrast microscope (Olympus Corporation, Tokyo, Japan) equipped with an Olympus SC30 camera (Olympus Corporation) then analyzed using the software cellSense Standard version 1.4 (Olympus Corporation). The cells were then fixed in 3.7 % paraformaldehyde for 15 min and washed with phosphate-buffered saline (PBS). The fixed cells were stained with Alexa Fluor 647-conjugated phalloidin in PBS containing 1 % bovine serum albumin and 1 % Tween-20 for 1 h and in PBS containing 1 μg/ml bisbenzimide for 1.5 min to visualize the cytoskeleton and nuclei, respectively. The glass coverslips were mounted on glass slides using Mowiol 4–88 and left in a refrigerator overnight. The samples were imaged with a LSM510 confocal laser scanning microscope (Carl Zeiss Microscopy GmbH, Oberkochen, Germany) equipped with a Hamamatsu R6357 photomultiplier (Hamamatsu Photonics K.K., Hamamatsu, Japan). A 405 nm diode-pumped solid-state laser was used to excite the bisbenzimide and a band-pass filter of 420–480 nm was used for the emission. The Alexa Fluor 647-conjugated phalloidin was excited with a 633 nm HeNe ion laser and a 650 nm long-pass filter was used for the emission. The cells were visualized by z-stacks composed of single optical planes in high magnification and the images were analyzed with the software ZEN 2012 (black edition) version 8.0 (Carl Zeiss Microscopy GmbH).