2.1.2. Sample Surface Modification

After printing, the PEEK and CFR-PEEK disc samples for wettability, roughness, microstructure, and biological tests were divided into three groups: as-printed (untreated) group, polished group, and sandblasted group (*n* = 12 per group). The untreated group included the directly printed samples, without any surface treatment. For the polished and sandblasted groups, all the discs were manually polished with a series of SiC abrasive papers up to P4000 (Buehler, Lake Bluff, IL, USA) by a polisher (Buehler, Coventry, UK). Then, the samples of the sandblasted group were further modified using a sandblasting machine (P-G 400, Harnisch + Rieth, Winterbach, Germany) with 120 μm alumina (Al2O3) particles (Cobra, Renfert, Hilzingen, Germany) under the pressure of 0.1 MPa at a distance of 50 mm for 15 s.

All Ti disc surfaces were modified using the same processes as for PEEK and CFR-PEEK samples by a series of SiC abrasive papers (1200, 2500, 4000 grit, Buehler, Lake Bluff, IL, USA) by a polisher (Buehler, Coventry, UK). After polishing, all the samples were cleaned with deionized (DI) water by an ultrasonic cleaner (Sonorex Super RK102H, Bandelin, Berlin, Germany) to remove residual Al2O3 particles on the sample surfaces.

#### *2.2. Mechanical Properties Test*

Mechanical tests were carried out using an electro-hydraulic servo mechanical testing machine (CMT4304, MTS Corp., Eden Prairie, MN, USA) according to ISO standards. ISO 527-1: 2012 (Plastics—Determination of tensile properties), ISO 178: 2010 (Plastics—Determination of flexural properties), and ISO 604: 2002 (Plastics—Determination of compressive properties) were applied for the tensile, bending, and compressive tests, respectively [29–31]. Six samples were tested for each batch with a 1 mm/min testing speed, and the test was performed at an ambient temperature of 20 ◦C.

#### *2.3. Surface Characterization*

To determine the surface morphology, samples of PEEK and CFR-PEEK from the untreated, polished, and sandblasted groups (*n* = 2 per group) were sputtered with a 20 nm thick Au–Pd coating (SCD 050, Baltec, Lübeck, Germany) and characterized by a scanning electron microscope (SEM) (LEO 1430, Zeiss, Oberkochen, Germany) at 200× and 2000× magnification.

The surface topography of the discs (*n* = 6 per group) was analyzed by a profilometer (Perthometer Concept S6P, Mahr, Göttingen, Germany). For each sample, 121 profiles were measured over a 3 mm × 3 mm area. The arithmetic mean height (Sa) and root mean square height (Sq) were calculated based on these topographies by software (MountainsMap Universal 7.3, Digital Surf, Besançon, France).

The water contact angle (WCA) was measured at room temperature on six samples per group using a drop shape analyzer (DSA 10-MK 2, Kruess, Hamburg, Germany). Drops of 2 μL of distilled water were deposited on the respective disc surfaces using an automatic pipette. After 20 s wetting time, the contact angle at the air–water–substrate interface was quantified from the drop geometry using DSA software (version 1.90.0.11, Kruess, Hamburg, Germany).

#### *2.4. Biological Tests*

Biological tests consisted of an extract test and a direct contact test to analyze the cytotoxicity and of the investigation of cell attachment to the different samples (*n* = 9 per group). Two materials (PEEK and CFR-PEEK) with three different surfaces each (untreated, polished, and sandblasted) were tested. In each test, *n* = 3 samples were used for each surface modification. All tests were performed three times in independent experiments. Directly before the biological tests, samples were ultrasonically cleaned with DI water for 15 min and sterilized with 70% ethanol and 100% ethanol (15 min each). Subsequently, the samples were dried on filter paper in a sterile workbench (Lamin Air HB2472, Burgdorf, Switzerland).

### 2.4.1. Cell Culture

L929 fibroblasts (DSMZ GmbH, Braunschweig, Germany) were cultured in DMEM medium (21063-029, Gibco, Paisley, UK) containing 10% fetal bovine serum (FBS, Life Technologies, Carlsbad, CA, USA), 1% penicillin/streptomycin (15140-122, Life Technologies Co., Carlsbad, CA, USA), and 1% GlutaMAX (Life Technologies Co., Paisley, UK) in 75 cm<sup>2</sup> sterile cell culture flasks (Costar, Corning, Tewksbury, MA, USA). The cells were maintained in an incubator under a humidified atmosphere with 5% CO2 at 37 ◦C. The DMEM culture medium was renewed twice a week. When cells reached confluence, Trypsin (GIBCO, Paisley, UK) was used to detach cells from the bottom of flasks, and 1/10 of the total cells were transferred into a new flask.

#### 2.4.2. Test for In Vitro Cytotoxicity

The in vitro cytotoxicity test of PEEK and CFR-PEEK was performed by an extract method based on ISO 10993-5 [32]. Extracts were derived from soaking the samples with DMEM cell culture medium for 24 h at 37 ◦C. The ratio between the sample surface area and extraction vehicle volume was 3 cm2/mL. In the meantime, the cells were precultured for 24 h. The seeding concentration of L929 cells was 30,000 cells/cm<sup>2</sup> in 200 μL DMEM medium per well in a 96-well plate (Cellstar 655180, Greiner Bio-One, Frickenhausen, Germany). After 24 h, the culture medium was removed from the cells and replaced by 150 μL extracts obtained from the respective sample groups. Three concentrations of each extract were tested: (a) undiluted (150 μL extracts), (b) 1:3 diluted with medium (50 μL extracts + 100 μL medium), and (c) 1:10 diluted (15 μL extracts + 135 μL medium). Ti samples were used as the negative control, and copper (Cu) samples were used as the positive control. After culturing for an additional 24 h, the extracts in all groups were replaced by 100 μL fresh DMEM medium to avoid artifacts in the following assay caused by blue color in the Cu extracts. The cytotoxicity was quantitatively analyzed by CCK-8 assay (Dojindo Molecular Technologies, Inc., Rockville, MD, USA). The volume of CCK-8 solution added to each test well was 10 μL. After incubating for 2 h, the optical density (OD) value was measured by a microplate ELISA reader (Tecan F50, Tecan Austria, Groedig, Austria) at 450 nm wavelength. The metabolic activity of L929 cells in the different test groups in comparison to the negative control was calculated according to the following formula:

$$\text{Cell metabolism activity (\%)} = \left(\text{OD}\_{\text{t}} - \text{OD}\_{\text{b}}\right) / \left(\text{OD}\_{\text{nc}} - \text{OD}\_{\text{b}}\right) 100\%,\tag{1}$$

where the OD value is the absorbance value of the respective test group (ODt), blank control group (ODb), and negative control group (ODnc).

#### 2.4.3. Cell Adhesion and Spreading

L929 cells were seeded on PEEK, CFR-PEEK, and Ti samples in 12-well plates (REF 3512, Costar, Kennebunk, ME, USA) with a density of 30,000 cells/cm<sup>2</sup> and incubated in 2.4 mL DMEM medium at 37 ◦C and 5% CO2. After incubation for 24 h, cell adhesion was terminated by rinsing with Hank's balanced salt solution (HBSS, Biochrom AG, Berlin, Germany). Adhering cells were vital stained for 10 min in a solution of 25 μg/mL fluorescein diacetate (FDA) and 1.25 μg/mL ethidium bromide (EB) (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) in HBSS. For each sample, a minimum of six typical surface areas of every magnification (25×, 100×, 200×, and 400×) was documented by an Optishot-2 fluorescence microscope (Nikon, Tokyo, Japan) equipped with a digital camera (550D, Canon, Tokyo, Japan). Cell adhesion and spreading were assessed by measuring the density of the vital-stained cells (cells/cm2) and the mean area of sample surface covered by cells (% of Ti) using a photo editing software (ImageJ, v1.8.0, National Institutes of Health, Bethesda, MD, USA).

#### *2.5. Statistical Analysis*

SPSS Version 21 (SPSS INC, Chicago, IL, USA) was used for analyzing the data. Shapiro–Wilk and Levene tests were applied to assess the assumptions of data normal distribution and homogeneity of variances. The results of the mechanical properties of each parameter were tested using the Student's *t*-test of unpaired data with equal variance. One-way analysis of variance (ANOVA) was used for the cell density, and cell adhesion and spreading followed by Tukey post-hoc test (α = 0.05). The contact angle and roughness data were analyzed by Kruskal–Wallis analysis (α = 0.05) for the disobedience of the data normality or homogeneity of variances.
