We previously reported that the surface roughness of implants affected osteoconductivity after implantation [11] . PEEK plates (f6 mm) were polished by buffing using Al₂O₃ particles (particle size = 0.05 μm) and the surface roughness (Ra) was set as Ra/μm < 0.1. Samples were immersed in ~18 M (=mol L⁻¹) H₂SO₄, ~12 M HCl, ~13 M HNO₃, ~17 M CH₃COOH, or ~30 vol.% H₂O₂ for up to 1 d at 30˚C for the disconnection of the ether bond in PEEK, considering the chemical resistance of PEEK. The immersed samples were picked up from the solution and rinsed in distilled water, acetone, ethanol, or toluene, respectively, and dried in vacuum. After drying, the samples were subjected to the following second step for the hydrophilization. The samples were irradiated either with ultraviolet (UV, 172, 254, or 365 nm wavelength) radiation for up to 6 h at 20˚C, or with atmospheric plasma for up to 1 h. As a storing environment for the hydrophilicity of surface-treated samples, DW, phosphate buffered saline (PBS) (−), five times concentrated PBS (−) (´5 PBS (−)), and air were selected, respectively, and the hydrophilicity value of the samples was maintained. In addition, the WCA of the sample surface was controlled using the change in storing period and environment. WCA was measured using a 2-μL droplet of distilled water. The modified surfaces were analyzed using scanning electron microscopy (SEM), X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), and Fourier transform infrared spectroscopy (FTIR) in attenuated total reflection (ATR) mode.

In the protein adsorption testing, fibronectin and albumin were picked up as a protein that was either a cell-adhesive protein or not, respectively [12] . A fibronectin (~1.0 mg mL⁻¹) or albumin (~50 mg mL⁻¹) aqueous solution droplet (40 μL) was put on the surface of the WCA-controlled PEEK samples, respectively, and these were kept at 37˚C for up to 3 d. Then, they were rinsed out with ultrasonic cleaning in the distilled water and dried naturally. The amount of adsorbed protein was determined using FTIR in attenuated total reflection (ATR) mode. The protein content was evaluated from the peptide binding (1650 cm⁻¹) in the protein in the FTIR spectra. The content of the different three positions was measured for each samples using calibration curve, and the mean value was calculated.

All the animal studies were conducted in the laboratory at HAMRI Co., Ltd., Japan, of the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC).

1) Testing of osteoconductivity [13]

The sides of PEEK rods (2 mm in diameter, 5 mm in length) were polished by buffing using 0.05 μm Al₂O₃ particles. Hydrophilic PEEK rods (WCA = 85˚) were prepared in a combination of immersion in 16.2 M H₂SO₄ for 5 s at 30˚C and UV irradiation (172 nm) for 3 h. For the formation of the protein-adsorbed samples, hydrophilic PEEK samples were immersed in 1.0 mg mL⁻¹ fibronectin aqueous solution for 48 h at 37˚C. Next, they were rinsed with ultrasonic cleaning in the distilled water and dried naturally. All the surface-treated rod samples (n = 5) before and after protein adsorption were implanted in rat tibia for 14 d for evaluation of osteoconductivity. The samples were longitudinally sliced and stained with toluidine blue. The interface between the implant and the cortical bone, as well as the cancerous bone, was observed by optical microscopy. The sum of the linear bone contact with the implant surface was measured and was expressed as a percentage over the entire implant length (the bone-implant contact ratio, BIC) in the cancerous bone and in the cortical bone parts. Significant differences in BIC were analyzed statistically using the Tukey-Kramer method [14] .

2) Testing of anti-inflammatory properties

PEEK rods of 1 mm in diameter and 5 mm in length were used in the anti-inflammatory testing. The method of WCA control and fibronectin adsorption was the same as in the osteoconductivity testing. The surface-treated samples (n = 3) were implanted in the subcutaneous of the back of rats by inoculation using an injection needle and the rats were sacrificed at 14 d post implantation. Because serious inflammation, necrosis, suppuration, and bleeding were not detected around the surrounding tissue of implants by visual observation, tissue samples were fixed in 10% formalin, and embedded in methyl methacrylate (MMA) polymer. The samples were sliced and stained with toluidine blue. Optical microscope observation of the interface between the implanted and the surrounding tissue revealed whether inflammatory cells such as lymphocytes and neutrophils were present, as well as a new generation of vascular, and formation of a fibrous capsule film. The capsule film thickness of the eight positions was measured for each samples, and the mean value was expressed as an index of anti-inflammatory properties.

Figure 2 shows photographs taken after immersion in several aqueous solutions and washing in distilled water. The immersion in 12 M HCl, 13 M HNO₃, 17 M CH₃COOH, and 30 vol.% H₂O₂ did not change the surface morphology of PEEK. The WCA values were 81˚, 84˚, 80˚, and 83˚, respectively, and they did not increase or decrease dramatically from the as-polished PEEK (~85˚). Immersion in H₂SO₄ had a clear effect on the surface appearance. In particular, 18 M H₂SO₄ attacked the surface and the color turned white from beige, and the Ra value increased from 0.05 to 0.44 μm. For immersion in 16.8 M H₂SO₄, SEM observation confirmed the attack, although discoloration and Ra change did not occur. For the immersion in less than 15 M, no changes were observed. Figure 3 shows FTIR spectra of samples after 18 M H₂SO₄ immersion (Figure 3(b)) and washing in DW (Figure 3(c)). Figure 3 indicates that the immersion in H₂SO₄ broke the

ether bond, the C-C single bond, and the C=C double bond, and increased the sulfo group. Because the subsequent washing process decreased the sulfo group, it was thought that the WCA value did not decrease after immersion in H₂SO₄, which in any case satisfied the first step for polymer hydrophilization, that is, the disconnection of the bonds.

As a second step, UV (172 nm) irradiation (>10 mW cm⁻², with 3 mm distance) and atmospheric plasma irradiation (500 W, 50 mm distance) were selected and applied on the solution-treated samples: immersion in 16.2 M H₂SO₄ for 5 s and rinsing in DW. With atmospheric plasma, the WCA value decreased rapidly and reached a constant WCA of ~10˚ in 10 min, regardless of the first step. FTIR showed that there was no change in the surface functional group by the plasma treatment (Figure 3(e)), and therefore it was thought that radicals might contribute to hydrophilization. However, the hydrophilicity formed by plasma irradiation, could not be maintained, and the WCA value rose rapidly in 1 d (Figure 4). In contrast, it took 1 - 2 h for the solution-treated samples to reach a constant WCA of ~20˚ under UV irradiation, and the Ra value was the same as after H₂SO₄ immersion (Ra = 0.05 μm). FTIR showed that the UV irradiation created a carboxyl group. By examination of UV irradiation under different partial pressure environments of CO₂ (Ar or CO₂ atmosphere), we determined that this carboxyl group was formed from CO₂ in air. The UV treatment on the as-polished sample gave a surface with ~40˚ and a longer irradiation time did not result in further reduction in WCA. The hydrophilicity could be kept for

7 d by storing in ´5 PBS (−) in the same manner as hydrophilic Ti and its alloy samples [15] . It is thought that ions in ´5 PBS (−), such as Na⁺, were adsorbed on the hydrophilic PEEK surface. Thus, in the following hydrophilization experiment, PEEK was immersed in 16.2 M H₂SO₄ for 5 s and rinsed in DW, and then irradiated with UV (172 nm) for more than 3 h in air. To maintain hydrophilicity, the surface-treated PEEK was stored in ´5 PBS (−).

Figure 5 shows the relationship between the BIC value of surface-treated PEEK and WCA compared with WCA-controlled Ti. The BIC value of as-polished PEEK was very low, and a fibrous capsule film formed over the entire surface in some samples. In contrast, hydrophilic PEEK had a high BIC value. This tendency agreed well with our results for surface-treated Ti [15] . The hydrophilization of PEEK dramatically improved the osteoconductivity, meaning that the osteoconductivity strongly depended on the surface property and not on the implant materials themselves. Based on our previous work [16] , we believed that the osteoconductivity was not directly affected by the WCA of the implants. In other words, it was thought that the WCA affected protein adsorption, which in turn controlled the osteoconductivity of the implants. The amount of adsorbed protein (fibronectin and albumin) with respect to the WCA value is shown in Figure 6(a) & Figure 6(b). Neither protein was adsorbed on the as-polished PEEK surface. In contrast, on surface-treated PEEK, the proteins were adsorbed,

depending on the WCA value. This tendency agreed well with our results for surface-treated Ti [15] . The BIC value of the fibronectin-adsorbed samples (n = 5) on the hydrophilic samples (n = 3) was also high, and the same level of hydrophilic sample, in spite that the fibronectin-adsorbed sample did not have a hydrophilic surface (WCA = 48˚). This also supports the notion that the osteoconductivity was not directly affected by the WCA of implants.

Figure 7 shows photographs inside of the stripped skin and optical micrographs of surface-treated PEEK samples. Serious inflammation and necrosis were not detected around the surrounding tissue of implants. A fibrous capsule film formed over the entire surface in all samples, and the inflammatory reaction occurred over the whole surface of all samples. The thickness of the capsule film was used as an index for the evaluation of anti-inflammatory properties. Figure 8 shows the thickness of the capsule film formed on the implants. On the

as-polished sample, a thick capsule film was formed, suggesting that the inflammation was strong, as reported [17] . However, the inflammation was weaker on the surface-treated PEEK, which was not related to the WCA value. In the implantation of the fibronectin-adsorbed PEEK samples on the hydrophilic surface in advance, the inflammation was not suppressed further. It is noteworthy that the surface treatment can suppress the inflammation without an anti-inflammatory drug, compared with as-polished PEEK.