*3.3. Cytotoxicity*

Cell metabolic activity is expressed as a percentage of the mean OD value of cells cultured with extracts of the negative control (Ti), as displayed in Figure 6i,j. The data showed high cell viability in the cultures treated with 1:1, 1:3, and 1:10 extract concentrations of both tested materials, PEEK and CFR-PEEK, independent of the respective surface treatment (PEEK: untreated: 98 ± 23%, 106 ± 17%, 97 ± 22%; polished: 105 ± 25%, 106 ± 16%, 102 ± 15%; sandblasted: 106 ± 33%, 114 ± 27%, 98 ± 37%; CFR-PEEK: untreated: 98 ± 20%, 100 ± 28%, 97 ± 23%; polished: 102 ± 27%, 97 ± 31%, 105 ± 25%; sandblasted: 99 ± 38%, 100 ± 33%, 96 ± 23%). All extracts of PEEK and CFR-PEEK samples showed no toxicity after 24 h incubation. Cell viability in all cultures was significantly above the 70% level regarded as toxicity threshold according to ISO 10993-5 [32]. The results were confirmed by morphology analysis as seen in Figure 6a–h. Cells in the 100% extracts of the PEEK and CFR-PEEK groups exhibited a similar appearance as the negative control (Ti) group with distinct fibroblastic

profiles. On the contrary, a large number of dead cells appeared in the positive group with a cell survival rate of 1 ± 2%.

**Figure 6.** Cytotoxicity tests of L929 cells of PEEK and CFR-PEEK 100% extracts: (**a**) untreated PEEK; (**b**) polished PEEK; (**c**) sandblasted PEEK; (**d**) untreated CFR-PEEK; (**e**) polished CFR-PEEK; (**f**) sandblasted CFR-PEEK; (**g**) negative control (Ti); (**h**) positive control (Cu). (**i**,**j**) shows the quantitative result of the CCK-8 test in the culture media with different extract concentrations of PEEK and CFR-PEEK. The data are presented as means ± standard deviation. UP: untreated PEEK; PP: polished PEEK; SP: sandblasted PEEK; NC: negative control; PC: positive control; UCP: untreated CFR-PEEK; PCP: polished CFR-PEEK; SCP: sandblasted CFR-PEEK. The dotted line indicates the toxicity threshold of 70% cell viability according to ISO 10993-5.
