1. Changes In Surface Microstructure

After argon (Ar) and oxygen (O₂) ionomer treatment, nanoscale spherical particles were formed on the surface of the PEEK samples (Figure 2).

Figure 2. SEM images of FFF 3D printed and polished PEEK before and after Ar and O₂ plasma treatment at 10,000× magnification.

The observed increases in the surface roughness parameters Sa and Sq were not statistically significant compared to the original samples, indicating that the plasma treatment improved the surface morphology at the microscopic level without significantly altering its macroscopic roughness (Figure. 3).

Figure. 3. 3D surface topology reconstruction.

2、Improvement Of Surface Hydrophilicity

The water contact angle (WCA) measurements of FFF-PEEK samples decreased from 90.4±7.4° to 35.7±13.0° after Ar treatment and 25.6±8.8° after O₂ treatment, and the WCA measurements of P-PEEK samples decreased from 89.5±2.5° to 41.1±7.3° after Ar treatment and 38.0±3.1° after O₂ treatment, and these changes were all were statistically significant (p<0.0001). As the storage time increased, although the water contact angle recovered, the measured values after 21 days were still significantly lower than the pre-treatment level, stabilizing below 60°, as shown in Figure 4.

Figure 4. Water contact angle (WCA) measurements of FFF 3D printed and polished PEEK before plasma treatment and at different time points (0, 1, 3, 7, 14 and 21 days).

3. Enhancement Of Cell Adhesion

After argon (Ar) and oxygen (O₂) plasma treatment, significant microstructural changes were observed on the surface of FFF-PEEK versus polished PEEK samples, especially the formation of spherical nanoparticles (shown in Figure 2). This treatment enhanced the adhesion of Saos-2 osteoblasts on the PEEK surface. after 4 h of incubation, the O₂ plasma-treated FFF-PEEK group exhibited a higher cell adhesion density compared to the untreated original samples (Figure 5).

Figure. 5. Qualitative and quantitative results of initial cell adhesion after 4 hours of incubation.

Continuing the incubation up to 24 h, the cells on the plasma-treated samples not only increased in number, but also had a more expanded morphology and an increased number of pseudopods (Figure. 6).

Figure 6.SEM images: qualitative and quantitative observation of Saos-2 osteoblast morphology before and after plasma treatment of FFF-PEEK and P-PEEK.

4. Enhanced Cellular Metabolic Activity

Plasma treatment increased the metabolic activity of Saos-2 osteoblasts on the FFF 3D printed PEEK surface (Figure. 7), and the O₂ plasma-treated FFF-PEEK samples showed an increase in cellular metabolic activity compared to untreated pristine samples during the incubation periods of days 3 and 5.

Fig. 7. Comparison of relative cell metabolic activity and proliferation of Saos-2 osteoblasts before and after plasma treatment of FFF-PEEK and P-PEEK samples.

5. Changes In Alkaline Phosphatase (ALP) Activity

The ALP activity of Saos-2 cells was slightly elevated after O₂ plasma treatment of the PEEK samples (Fig. 8).After 5 days of incubation, the ALP activity of the FFF-PEEK and P-PEEK groups treated with O₂ plasma increased slightly in comparison to the original samples, but this effect was not significant in the short term.

Figure 8. Alkaline phosphatase (ALP) activity of Saos-2 osteoblasts after 5 days of incubation of FFF 3D printed and polished PEEK samples after plasma surface treatment versus untreated. Data are expressed as mean ± standard deviation.

6. Promotion Of Mineralized Nodule Formation

The number of mineralized nodules of Saos-2 cells on PEEK samples treated with O₂ plasma was significantly increased (Fig. 9).After 21 days of incubation, the number of mineralized nodules formed by Saos-2 cells on FFF 3D printed and polished PEEK samples treated with O₂ plasma was significantly increased, suggesting an increase in the amount of calcium phosphate deposition, especially in the FFF-O₂-PEEK group. It was about 60% higher than the untreated FFF-PEEK group.

Figure 9. Qualitative and quantitative results of osteogenic differentiation after 21 days of culture.