4 Results and Discussion 55
4.7 Cytotoxicity and Cell Proliferation on Non-Functional and Functional Two-
Functional Two-Dimensional Polyphosphazene Films
Upon successful synthesis of the base and functional polymers using the new one-pot room temperature synthesis method, it was necessary to evaluate the interaction of the materials with cells to ensure their cytocompatibility. For this, two-dimensional films of the polymers were coated onto the bottom of the wells of a 96-well tissue culture plate, then cytoxicity and cell proliferation were analyzed using MTT assays. A mouse fibroblast cell line (NIH-3T3) was used to evaluate the effect of the polymers on cell viability and metabolic activity over a time period of five days. Figure 4.23 shows the metabolic activity of the cells normalized to TCPS at day 1. As can be observed from the overall comparison of the two graphs in Figure 4.23, preconditioning the materials overnight in HBSS leads to an increase in initial cell viability (higher absorbances at day 1 with preconditioning), although it does not change the fact that with and without preconditioning cells are capable of proliferating on the materials over the entire 5-day span (metabolic activity persists until day 5 on the materials with and without preconditioning). It is also clear from the confocal microscopy images of Figure 4.25
that the cells on the unconditioned materials (Panels A, C, E, G, I, K, and M) are still viable based on their elongated morphology with long filamentous F-actin protrusions (green). Preconditioning is common practice in cell studies with biomaterials as it acts as a buffering period in which the materials are washed of small molecules on their surface that can disrupt proper interaction of the cells with the materials themselves.50, 51, 53, 94, 114 Moreover, the preconditioning period “wets” the surface of the materials allowing them to interact properly with the aqueous cell suspensions, rather than seeding the cells on the dry films, which may suppress cell-material interaction and lead to diminished cell
attachment, and thus reduced viability, on the materials. Therefore, all statistical analysis of comparisons between materials in terms of cell viability and proliferation were conducted for the preconditioned films. At Day 1, both of the phenylalanine-based polyphosphazenes (PP-F100 and PP-F90E*10) showed significantly higher (p<0.001)
metabolic activity of fibroblasts in comparison to the TCPS positive control. At Day 3, the PP-F90E*10 material continues to exhibit significantly higher (p<0.01) metabolic
activity of the cells in comparison with the TCPS control. By Day 5, it seems that cell proliferation begins to plateau, which may be due to crowding of the fibroblasts, and the PP-F90E*10 material performs on par with the TCPS control in terms of metabolic activity.
For the PP-F100 material, the metabolic activity of the fibroblasts in comparison to TCPS
was not significant by Day 3. Taken together, these indicate that the phenylalanine-based polyphosphazenes synthesized via the new 1P RT method are suitable as biomaterials and should be explored further. For the remainder of the materials, it should be noted that despite having initial metabolic activity lower than the TCPS controls, these materials are also non-cytotoxic. This is shown in Figure 4.24, where it is clear that the metabolic activity is increasing from Day 1 to Day 5 for all of the functional and non-functional polymers, despite lower initial activity at Day 1, indicating that the cells are thriving on these materials. The decreased Day 1 activity levels may be due to decreased affinity of the cells to attach to the materials initially, although once they have successfully attached they are capable of surviving. The confocal images of the preconditioned materials in
Figure 4.25 (Panels, B, D, F, H, J, L, and N) also shows that the cells are morphologically healthy and spreading well on the materials.
Figure 4.23: Fibroblast metabolic activity as determined by MTT assay grouped by day to compare metabolic activity levels between materials and TCPS. Cells were seeded at 8,000 cells/well on 2-D polyphosphazene films (PP-A100, PP-F100, PP-M100,
PP-A90E*10, PP-F90E*10, and PP-M90E*10) that were either unconditioned or
preconditioned in HBSS at room temperature overnight. The cells were cultured for 1, 3, and 5 days with 5 replicates for each. The data are presented as the mean ± standard deviation and are normalized to TCPS at Day 1. (** = p<0.01, *** = p<0.001)
Figure 4.24: Fibroblast metabolic activity as determined by MTT assay grouped by material to show non-cytotoxicity from day 1 to day 5. Cells were seeded at 8,000 cells/well on 2-D polyphosphazene films (PP-A100, PP-F100, PP-M100, PP-A90E*10, PP-
F90E*10, and PP-M90E*10) that were preconditioned in HBSS at room temperature
The data are presented as the mean ± standard deviation and are normalized to TCPS at Day 1.
Figure 4.25: Confocal microscopy images showing the morphology and spreading of fibroblasts on the unconditioned and preconditioned polyphosphazene materials after 3 days of culture. The cells were seeded at a density of 30,000 cells/well on the polymer coated glass coverslips in a 24-well tissue culture plate. Untreated glass cover slips were used as a positive control in these experiments. The fibroblasts were stained for F-actin (green) and nuclei (blue). Scale bar = 20 μm (panel B).