2.4.2. Toxicity Evaluation

The toxicities of the PUSX nanofibers and films were evaluated by using direct contact based on ISO 10993-5. Briefly, cells were seeded evenly over the surface of each plate and incubated at 37 ◦C until the cells covered the whole surface. The samples were then placed on the cell layers in the center of the plates, and the culture medium was replaced. In order to determine the toxicity in accordance with Grade 0 (nontoxic) to Grade 4 (severe toxic) evaluation, Trypan blue was added after 24 h in each plate, and observed by morphological changes. This is the most obvious and direct way to reflect the impact of testing the materials on the cell [11].

#### *2.5. Statistical Analysis*

Significance in vitro biocompatibility and physical properties were statistically analyzed by a one-way analysis of variance (ANOVA) using R free software. Statistical significance was set at *p* < 0.05 to identify which groups were significantly different from the other groups.
