Prior fibroblastic BHK-21 cell experiments, spin-coated PIs (PI-2525 films and PI-2771 films cured at 200 °C and at 350 °C) and USPLD coatings (C3N4 and Al2O3) were characterized in terms of their roughness, wettability and zeta potential (Table 7). The surface roughness values obtained were all very low, i.e. the Ra values were below 1 nm. However, the Ra value for the PI-2525 was significantly higher than for other coatings (p < 0.001). The contact angles for water on both PI-2771 film types were significantly higher than for the PI-2525 (p < 0.001). The same tendency was also noticed when contact angles were measured for diiodomethane (results are shown in the original article III, Table 1) highlighting the more hydrophobic nature of the PI-2771 with significantly lower polar and dispersive surface energy values (p < 0.001). Somewhat surprisingly, the effect of the chosen curing protocol (200 °C vs. 350 °C) for the PI-2771 on roughness or on wettability was not statistically significant. The contact angles for water on C3N4 were in the same range as on PI-2525 film, evidence of a slightly hydrophobic thin film coating. In contrast, the Al2O3 coating was very similar to PI-2771 film with respect to its wettability properties. All of the surfaces were negatively charged, and the zeta potential values measured at a fixed pH of 7.4 were in a narrow range of -54.2 mV – -66.2 mV. The PI-2771-200 film followed by the PI-2525 film was the most negatively charged and their zeta potential values differed significantly from the all other test materials (p <
0.001).
Table 7: Average surface roughness (Ra), water contact angle and zeta potential values of tested polyimide and ceramic coatings. All of the materials were extremely smooth although the roughness of PI-2525 was significantly higher than that of other materials. The PI-2525 and carbon nitride (C3N4) were more hydrophilic compared to alumina (Al2O3) and both PI-2771 types. Zeta potential values were all in the same range by demonstrating the negative surface charge of these coatings.
Material Ra (nm) Contact angle (°) Zeta potential (mV) PI-2525 0.89 ± 0.13 * 69.5 ± 1.8 ## -59.9 ± 0.3 *
PI-2771-200 0.43 ± 0.15 95.1 ± 1.3 -66.2 ± 1.6 * PI-2771-350 0.39 ± 0.04 93.2 ± 0.8 -54.2 ± 0.1 C3N4 0.24 ± 0.05 # 70.8 ± 0.9 ## -55.1 ± 1.2 Al2O3 0.18 ± 0.03 # 94.0 ± 1.3 -56.6 ± 3.4
* p < 0.001, as compared to all other materials
# p < 0.001, as compared to all polyimides
## p < 0.001, as compared to all other materials, except of C3N4/PI-2525 The values are mean ± SD. One-way ANOVA was used to determine the statistical differences between materials.
In this study, the cytotoxicity of the insulator candidate materials was examined with in vitro tests conducted along to the guidelines of the international standard ISO-10993-5. Latex rubber and PE were used as positive and negative control material, respectively. The MTS assay defined the number of viable BHK-21 cells attached on the surfaces 24 h after cell-seeding. The results were normalized relative to the highest value, i.e., the value for the PE control (Fig. 19). The relative cell numbers in the different polyimide groups and C3N4 were at a level of 62-70 % of the PE controls. The number of cells attached to the Al2O3 surface was lower, i.e. 50 %, but still significantly higher than attained with the latex rubber control.
Fluorescence microscopy studies with live/dead staining probes revealed that all test materials were highly cytocompatible, in contrast to latex rubber, on which viable cells were rarely found, instead dead cells had most probably detached and were floating in the medium (illustrated in the original article III). Results from the quantitative analysis included the determination of the density of dead cells
(nuclei/mm2) and the surface area of live cells (µm2) is presented in the Table 8. The numbers of dead cells were very low on each surface, i.e., mean values ranged from 13 cells/mm2 to 48 cells/mm2. Even though the dead cell density was also minimal on the surface of latex rubber, the cytotoxic effect could be clearly seen when the surface area of viable cells was examined.
The total surface area of viable cells on latex rubber was significantly lower (p < 0.001) than that of the other test materials, being only around 2% of the corresponding values obtained for other surfaces. The values for C3N4 and Al2O3 were slightly but not significantly higher than that for PE control or PI materials.
Table 8: The density of the dead cells (nuclei/mm2) and the surface area of viable cells (µm2) on polyethylene (PE, negative control), different polyimide surfaces, alumina (Al2O3), carbon nitride (C3N4) and latex rubber (positive control) after a 24-hour cultivation period evaluated using a combination of two fluorescent probes and Image J software.
Material Density of dead cells
(1/mm2) Surface area of viable cells (µm2)
PE (n = 10) 13.9 ± 5.4 70400 ± 2900
PI-2525 (n = 13) 13.5 ± 5.5 71900 ± 8500
PI-2771-200 (n = 8) 18.8 ± 8.2 67300 ± 13200 PI-2771-350 (n = 8) 30.8 ± 8.8 63000 ± 5800 C3N4 (n = 13) 48.0 ± 10.3 83900 ± 11600 Al2O3 (n = 15) 47.4 ± 5.8 82600 ± 12300 Latex rubber (n = 8) 24.9 ± 8.6 1400 ± 320*
The values are the mean ± SEM. *p < 0.001 (One-way ANOVA).
SEM imaging was used to monitor cell morphology, adhesion and spreading on the test surfaces. The BHK-21 cells which had been cultivated for 24 h on each PI surface, Al2O3 and C3N4 exhibited a normal fibroblastic morphology, adhered well with filamentous extensions, and had started to form clusters (Fig. 20). Thus, all insulator materials were considered not to be toxic and equally good for allowing the adhesion and spreading of cultured fibroblasts.
0 20 40 60 80 100 120
PE PI2525 PI2771-200 PI2771-350 Alumina CN Latex
Relative cell number (%)
*
*
0 5 10 15 20
PE PI-2525 PI2771-200 PI2771-350 alumina CN Latex
Cell coverage (%)
*
PE PI-2525 PI-2771-200 PI-2771-350 Al2O3 C3N4 Latex PE PI-2525 PI-2771-200 PI-2771-350 Al2O3 C3N4 Latex
A
B
Figure 19: (a) Relative number of BHK-21 cells on polyethylene (PE, negative control), conventional polyimide 2525), two differently cured photosensitive polyimides (PI-2771-200 and PI-2771-350), alumina (Al2O3), carbon nitride (C3N4) and latex rubber (positive control). The data was obtained by MTS assay and normalized to the highest optical density value. (b) Surface area covered by BHK-21 cells at 24 h on the same test materials. The data was acquired from confocal laser scanning microscope images of the samples with live/dead-stained cells. All polyimide and ceramic coatings can be considered to be non-cytotoxic and they allowed adhesion and spreading of fibroblasts.
The error bars indicate standard errors of the means. *p < 0.001 (one-way ANOVA).
Figure 20: SEM images of BHK-21 cells cultured for 24 h on PI-2771-200 at two different magnifications. The cells exhibited a normal fibroblastic morphology, adhered well with filamentous extensions, and had even started to form clusters. Cells cultured on the other test materials, i.e. PI-2525, PI-2771-350, C3N4 and Al2O3, appeared to be very similar and thus, are not shown.