Gradient 3D printed PEEK with nano-coating facilitates rapid manufacturing with dual enhanced osteogenic and anti-bacterial property

Lingtong Bu; Xiang Wei; Yuxin Zhang; Qingyu Xu; Zixiang Han; Yinjun Chen; Jisi Zheng; Chi Yang

doi:10.1016/j.mtbio.2025.102432

. 2025 Oct 16;35:102432. doi: 10.1016/j.mtbio.2025.102432

Gradient 3D printed PEEK with nano-coating facilitates rapid manufacturing with dual enhanced osteogenic and anti-bacterial property

Lingtong Bu ^a,¹, Xiang Wei ^a,², Yuxin Zhang ^a,³, Qingyu Xu ^a, Zixiang Han ^a, Yinjun Chen ^b,^⁎, Jisi Zheng ^a,^⁎⁎, Chi Yang ^a,^⁎⁎⁎

PMCID: PMC12554190 PMID: 41146665

Abstract

Artificial prosthesis and implants in oral and maxillofacial surgery are in urgent need of innovation. Plenty of bioactive agents have been added to PEEK, whereas complex processing technique hinders clinical translation. To break through the limitation, this study first applied gradient 3D printing technique to incorporate hydroxyapatite and nano-Ag into the functional coating on surface of PEEK. The advantage of gradient 3D printing facilitates rapid manufacturing and customization with minimum cost, which presents a significant advance toward clinical application. Compared to traditional PEEK, the dual enhancement of osteogenesis and anti-bacterial property were testified in vitro and in vivo. In addition, the bio-mechanism behind was first revealed to bridge the gap between theory and practice. Hydroxyapatite activated mTOR signaling pathway to enhance osteogenesis, and nano-Ag showed prominence in FoxO signaling pathway to mediate anti-bacterial property, while triggered ROS reaction and activated PPARγ pathway, minorly hindering osteointegration process. The novel gradient 3D printed PEEK with nano-coating could be rapidly manufactured with micro-structural surface, bone-like elastic modulus and excellent osteogenesis and anti-bacterial property, which has great potential for clinical translation in the field of oral and maxillofacial surgery.

Keywords: Joint prosthesis, Hydroxyapatites, Nano-Ag, Osteogenesis, Anti-bacterial agent, Gradient 3D printing

Graphical abstract

Highlights

•
Gradient 3D printing formed a functional nano-layer with rapid manipulation.
•
Multi-omics revealed the bio-mechanism behind osteogenesis and anti-bacterial property.
•
Enhanced mechanical, osteogenic and anti-bacterial properties proved in vitro and in vivo.
•
A new generation of artificial joint prothesis and oral implantations was introduced.

1. Introduction

There exists a tremendous need for bone grafting in the field of oral and maxillofacial surgery, resulting from trauma, joint ankylosis and tumor resection [1,2]. Traditionally metallic materials exhibited stress shielding issue and faced the need for a second operation [3]. In addition, radiopacity made metallic materials less optimal for post-operative tracking, and long-term metal ion release tended to trigger a local biological response [[4], [5], [6]]. Thus, polymer-based artificial material with appropriate mechanical, osteogenic, anti-bacterial properties should be introduced [7].

PEEK (–C₆H₄–O–C₆H₄–O–C₆H₄–CO–)_n is a polyaromatic semicrystalline thermoplastic polymer with chemical stability, closer mechanical property to the natural bone, and radiolucency, which has been successfully used as long-term medical implants in spinal fusion, joint replacements and craniomaxillofacial surgeries [[7], [8], [9]]. However, the bio-inertness of PEEK hinders its direct integration with the surrounding bone tissue [2,8,[10], [11], [12]]. Thus, bioactive second phase reinforcing particles as HA (Ca₁₀(PO₄)₆(OH)₂) could help with improving osteogenesis. Moreover, nano-Ag as a broad-spectrum antimicrobial agent could participate release-killing and contact-killing activities [1], which was crucial to improve the osteointegration of prosthesis in contaminated defects [13]. However, complex traditional processing techniques such as hot pressing, physical mixing, aerosol deposition, chemical deposition, ion beam–assisted deposition, spin coating, and cold spraying impeded clinical translation [14,15].

Gradient 3D printing enables the formation of different composites within single construct, thus, functional agents such as hydroxyapatite and nano-Ag could be incorporated onto the surface of PEEK to optimize tissue integration [1], which breaks through the limitation of complex manufacturing process [16,17]. During medical applications, the control of costs is a key issue. Since the biochemical properties of artificial prosthesis are mostly dependent on the surface characteristics, this study provided self-designed gradient 3D printed HA-(AgNPs-)PEEK@PEEK, to maximize the plasticity of PEEK substrate, and minimize the consumption of hydroxyapatite and nano-Ag, while assuring dual enhanced osteogenesis and anti-bacterial properties. Furthermore, gradient 3D printing could form a uniform layer with increased thickness and higher concentration of HA compared to conventional coatings [[18], [19], [20], [21]], avoiding wear issues. The most noteworthy highlight is the facilitation of rapid manufacturing process and the capability of selective customization. Different gradient 3D printed customized surfaces could be applied in various clinical conditions, such as aseptic or contaminated defect. In conclusion, gradient 3D printing can realize multiple self-designed functional surfaces regarding complex clinical conditions with minimum cost and dual enhanced osteogenesis and anti-bacterial properties.

This study first applied gradient 3D printing to incorporate hydroxyapatite and nano-Ag into the functional coating on surface of PEEK. The advantage of gradient 3D printing facilitates rapid manufacturing with minimum cost and customization in various clinical conditions, which presents a significant advance toward clinical translation. This study proved enhanced mechanical, osteogenic and anti-bacterial properties in vitro and in vivo. In addition, the bio-mechanism behind was first revealed to bridge the gap between theory and practice. This novel gradient 3D printed PEEK with high-concentration HA and nano-Ag could be rapidly manufactured with micro-structural surface, bone-like elastic modulus and excellent osteogenesis and anti-bacterial property, which has great potential for clinical translation in the field of oral and maxillofacial surgery.

2. Results

2.1. Mechanical performance

2.1.1. Bending test

The average bending elasticity modulus of PEEK was 2.955 ± 0.033 GPa, and increased steadily to 5.090 ± 0.222, 5.313 ± 0.598, 6.373 ± 0.213 GPa as the percentage of HA increased to 10 %, 20 % and 40 %. 40 % HA-PEEK showed a significant improvement compared to PEEK (P = 0.0021). As 2 % AgNPs added to 40 % HA-PEEK, the bending elasticity modulus slightly dropped down to 6.023 ± 0.165 GPa, while still showed better mechanical property than PEEK.

The maximum bending strength was highest in 10 % HA-PEEK, which was 184.6 ± 5.158 MPa and significantly superior to PEEK, which was 122.2 ± 3.281 MPa (P = 0.0017). As the percentage of HA increased to 20 % and 40 %, the maximum bending strength reduced to 166.4 ± 3.781 MPa (P = 0.0497) and 136.1 ± 2.279 MPa, while still showed better mechanical property than PEEK. As 2 % AgNPs added to 40 % HA-PEEK, the maximum bending strength remained at 138.2 ± 11.08 MPa.

The total bending breakage elongation decreased significantly from 14.81 ± 1.313, 11.15 ± 3.641, 6.325 ± 0.978, 3.165 ± 0.370 (P = 0.0229 compared to PEEK), to 3.140 ± 0.484 mm (P = 0.0100 compared to PEEK) as the addition of HA increased from 0 %, 10 %, 20 %–40 % and the addition of 2 % nano-Ag. The corresponding stress-strain curves of scaffolds under bending process were displayed, with the slope referring to the bending elasticity modulus, and the plateau referring to the maximum bending strength (Fig. 1A).

Fig. 1 — Mechanical property of HA (0 %, 10 %, 20 %, 40 %)-AgNPs (2 %)-PEEK. A. Bending elasticity modulus, maximum bending strength, total bending breakage elongation and stress-strain bending curve (n = 6). B. Tensile elasticity modulus, maximum tensile strength, maximum tensile elongation and stress-strain tensile curve (n = 6). C. The finite element analysis model of condylar neck fracture fixed by modified PEEK, overall displacement of the mandibular bone and relative displacement between the fracture line. D. Stress distribution on screws, plate, the cortical bone and the cancellous bone.

2.1.2. Tensile test

The average tensile elasticity modulus of PEEK was 3.195 ± 0.177 GPa, and increased significantly to 3.695 ± 0.177 (P = 0.0036), 5.140 ± 0.144 (P = 0.0002), 5.703 ± 0.118 (P < 0.0001), and 6.348 ± 0.130 (P < 0.0001) GPa as the percentage of HA increased to 10 %, 20 %, 40 %, and 2 % nano-Ag added.

The maximum tensile strength was highest in 20 % HA-PEEK, which was 98.32 ± 5.802 MPa and superior to PEEK and 10 % HA-PEEK, which was 89.17 ± 6.639 and 95.35 ± 5.097 MPa without significance. However, as the percentage of HA increased to 40 %, the maximum tensile strength reduced to 80.71 ± 1.234 MPa without significance. As 2 % nano-Ag added, the maximum bending strength further dropped down to 72.96 ± 5.662 MPa without significance.

The maximum tensile elongation decreased gradually from 6.243 ± 0.206, 5.668 ± 0.249, 4.720 ± 0.447, 3.568 ± 0.219 (P = 0.0280 compared to PEEK), to 2.478 ± 0.153 % (P = 0.0069 compared to PEEK) as the addition of HA increased from 0 %, 10 %, 20 %–40 % and the addition of 2 % nana-Ag. The corresponding stress-strain curves of scaffolds under tensile process were displayed, with the slope referring to the tensile elasticity modulus, and the plateau referring to the maximum tensile strength (Fig. 1B).

2.1.3. Finite element analysis

The above mechanical data (Supplementary Table 1) were used in finite element analysis, in which a reconstructed mandibular condylar neck fracture model (Supplementary Fig. 1 and 1C) was fixed by HA (0 %, 10 %, 20 %, 40 %)-PEEK with/without the addition of nano-Ag (2 %) under the condition of maximum masticatory contraction (Supplementary Fig. 2, Supplementary Table 2).

The finite element analysis showed that the main deformation of the modified PEEK plates was bending (Supplementary Figure 3). The maximum mandibular bone displacements of HA (0 %, 10 %, 20 %, 40 %)-PEEK and 40 %HA-2 %Ag-PEEK were 0.904, 0.837, 0.834, 0.815, and 0.823 mm, among which 40 % HA-PEEK exhibited the best stability (Fig. 1C, Supplementary Fig. 4). The relative displacements between the fracture line of HA (0 %, 10 %, 20 %, 40 %)-PEEK and 40 %HA-2 %Ag-PEEK were 0.067, 0.044, 0.043, 0.036, and 0.039 mm, which again showed most stability in 40 % HA-PEEK (Supplementary Figure 5). The maximum strength on the screws made of HA (0 %, 10 %, 20 %, 40 %)-PEEK and 40 %HA-2 %AgNPs-PEEK were 40.75, 43.14, 42.96, 44.92, and 44.12 MPa, which were far less than the Rm strength (Fig. 1D, Supplementary Figure 6). The maximum strength on the plates made of HA (0 %, 10 %, 20 %, 40 %)-PEEK and 40 %HA-2 %AgNPs-PEEK were 64.14, 71.86, 71.40, 75.89, and 74.07 MPa, which were far less than the Rm strength (Fig. 1D, Supplementary Figure 7). The maximum strength on the cortical bone were 41.70, 43.21, 43.26, 43.88, and 43.58 MPa, for the cancellous bone, the maximum strength were 0.65, 0.78, 0.79, 0.86, and 0.82 MPa, which were far less than the Rm strength of the cortical and the cancellous bone (Fig. 1D; Supplementary Figs. 8 and 9). All data above concerning the maximum strength distribution on the implanted screws and plates, and the surrounding bone were exhibited in Supplementary Table 3.

2.1.4. Friction-wear test

For HA (40 % wt)-PEEK@PEEK, the coefficient of friction (COF) increased rapidly from ∼0.084 to ∼0.11 within the first 30 s and reached a relatively steady state around 0.125–0.13 (up to 3600 s), the COF remained stable with only minor fluctuations, indicating good wear resistance and surface compatibility. For HA(40 % wt)-AgNPs(2 % wt)-PEEK@PEEK, the coefficient of friction (COF) started at ∼0.086 and gradually stabilized within the range of 0.095–0.105 during the long-term sliding test (up to 3600 s) (Supplementary Figure 10).

Wear track morphology (Supplementary Figure 11) showed no large-scale cracks, surface peeling, or debris accumulation compared to the surface characteristic after gradient 3D printing (Fig. 2A).

Fig. 2 — Surface characteristics of PEEK, 40 %HA-PEEK, and 40 %HA-2 %AgNPs-PEEK. A. SEM images after additive manufacture showing tracks of gradient 3D printing. B. XRD functional groups showed featured peaks in 3 groups. C. EDS elements components and ratio showed success incorporation of HA and nano-Ag on the surface of PEEK matrix. D. Static water contact angle (n = 3) comparison among 3 groups showed significantly enhanced hydrophilicity for HA modified PEEK (P = 0.0036). E. SAXS curves of 3 groups demonstrated nano-scale homogeneity. F. AFM morphology and Ra values (n = 3) showed significantly increased surface roughness for HA-PEEK (P = 0.0045) and HA-AgNPs-PEEK (P = 0.0071).

40 % HA-PEEK and 40 % HA-2 % AgNPs-PEEK proved increased bending and tensile elasticity modulus, increased maximum bending strength, and finite element analysis proved safe even under the condition of lowered total breakage elongation. Moreover, increased HA percentage was assumed to display better osteointegration, while the brittleness prevented the compound with HA more than 40 % from additive manufacturing, thus 40 % HA-PEEK and 40 % HA-2 % AgNPs-PEEK were selected into in vitro and in vivo study.

2.2. Surface characteristics

2.2.1. Scanning electron microscope

As shown in Fig. 2A, pure PEEK scaffolds exhibited relatively smooth surface, while numerous evenly distributed HA and AgNPs particles and a porous morphology were observed on the surface of 40 % HA-PEEK and 40 % HA-2 % AgNPs-PEEK scaffolds with a concentric track of additive manufacturing.

As shown in Supplementary Figure 12, cross-sectional SEM observations exhibited a distinct functional layer with an approximate thickness of 100 μm for 40 % HA-PEEK and 40 % HA-2 % AgNPs-PEEK with a dense distribution of HA particles together with uniformly dispersed Ag nanoparticles.

2.2.2. X-ray diffraction

In Fig. 2B, PEEK exhibited peaks at 2θ = 18.8°, 20.8°, 22.8°, and 28.9°. After the addition of HA and AgNPs, the compound showed additional peaks at 2θ = 34.1°, 46.7°, and 49.5°, corresponding to HA (JCPDS 09-0432), and 38.1°, corresponding to AgNPs (JCPDS 04-0783). No secondary phase was observed.

2.2.3. Energy dispersive spectroscopy

The EDS spectrum showed that carbon (C, 85.41 % At) and oxygen (O, 14.56 % At) were the main components of PEEK. As for the 40 %HA-PEEK and 40 % HA-2 % Ag-PEEK, calcium (Ca, 7.41 % At), phosphorus (P, 4.39 % At), and silver (Ag, 0.23 % At) were added and uniformly distributed. EDS confirmed the successful incorporation of HA and AgNPs with a Ca/P ratio of 1.68. No significant peaks for contaminants were detected (Fig. 2C). As shown in Supplementary Fig. 12, cross-sectional EDS line-scanning demonstrated an evident enrichment of Ca, P, and Ag within this 100 μm functional layer, while gradually decreasing toward the bulk PEEK matrix, which was dominated by C and O signals.

2.2.4. Water contact angle

As shown in Fig. 2D, the static water contact angle of PEEK was 73.6° ± 4.26° and significantly reduced to 42.0° ± 2.64° for 40 % HA-PEEK (P = 0.0036 compared to PEEK). The static water contact angle of 40 % HA-2 %AgNPs-PEEK was 59.2° ± 3.88°, which was higher than 40 % HA-PEEK, while still exhibited a significant decrease compared with PEEK (P = 0.0488). Thus, the addition of HA significantly improved the hydrophilicity of PEEK substrate, whereas the further addition of nano-Ag slightly increased the water contact angle compared to HA-PEEK, while still showed significantly improved hydrophilicity compared to bare PEEK.

2.2.5. Small-angle X-ray scattering

In Fig. 2E, the SAXS curves of PEEK, 40 % HA-PEEK, and 40 % HA-2 % Ag-PEEK were all smooth without apparent peaks, indicating nanoscale homogenization.

2.2.6. AFM morphology

PEEK exhibited a relatively smooth surface with a low average roughness (Ra) of 19.97 ± 4.761 nm, HA-PEEK surface displayed significantly increased roughness values 68.2 ± 17.98 nm (P = 0.0045 compared to PEEK), and HA-AgNPs-PEEK showed moderately increased roughness values 63.83 ± 8.723 nm (P = 0.0071 compared to PEEK). Enhanced roughness is favorable for protein adsorption and cell adhesion (Fig. 2F).

2.2.7. Release behavior of Ag+; Ca; P; Ca/P in vitro

ICP-MS quantification revealed a time-dependent release of Ag⁺ from the HA-AgNPs-PEEK functional surface in PBS at 37 °C (Supplementary Figure 13). Mean Ag⁺ concentrations exhibited a roughly linear rise from 6H to 3D (6H: 2.08 ± 0.05 μg/L; 12H: 4.41 ± 0.13 μg/L; 1D: 7.09 ± 0.19 μg/L; 3D: 9.18 ± 0.23 μg/L). After 5D the release showed decelerated increase (5D: 10.40 ± 0.19 μg/L; 7D: 11.31 ± 0.14 μg/L; 14D: 11.86 ± 0.07 μg/L), indicating establishment of a near-steady release state.

ICP-MS quantification also revealed a time-dependent release of Ca and P from the HA-AgNPs-PEEK functional surface in PBS at 37 °C (Supplementary Figure 13). Mean Ca concentrations exhibited a roughly linear rise from 6H to 1D (6H: 106.553 ± 4.228 mg/L; 12H: 160.540 ± 6.608 mg/L; 1D: 187.023 ± 3.132 mg/L). After 1D the release showed decelerated increase (3D: 191.933 ± 3.464 mg/L 5D: 202.023 ± 3.707 mg/L; 7D: 207.087 ± 4.620 mg/L; 14D: 212.870 ± 3.312 mg/L), indicating establishment of a near-steady release state. Mean P concentrations exhibited a roughly linear rise from 6H to 12H (6H: 67.093 ± 2.192 mg/L; 12H: 110.732 ± 2.674 mg/L). After 12H the release showed decelerated increase (1D: 117.099 ± 2.328 mg/L 3D: 121.160 ± 1.497 mg/L 5D: 121.562 ± 2.012 mg/L; 7D: 124.247 ± 0.467 mg/L; 14D: 128.208 ± 2.134 mg/L), indicating establishment of a near-steady release state.

The Ca/P ratio maintained around 1.67 without severe fluctuation (6H: 1.449 ± 0.025; 12H: 1.589 ± 0.061 mg/L; 1D: 1.598 ± 0.047 mg/L 3D: 1.585 ± 0.048 mg/L 5D: 1.667 ± 0.032 mg/L; 7D: 1.660 ± 0.002 mg/L; 14D: 1.662 ± 0.052 mg/L) (Supplementary Figure 13). These results demonstrate a sustained and nearly stoichiometric release of Ca and P from the HA-AgNPs-PEEK surface, with the Ca/P ratio remaining close to the theoretical value of 1.67 throughout the immersion period.

MC3T3-E1 cells continued to proliferate over time at the highest Ag⁺ concentration tested (20 μg/mL) (Supplementary Figure 14). Since ICP-MS analysis revealed that the cumulative Ag⁺ release concentration after 2 weeks stabilized at approximately 11.86 ± 0.07 μg/L, Ag⁺ level remained within a safe range for MC3T3-E1 cells during the experimental period (2 weeks)

2.3. Adhesion and proliferation of osteoblast MC3T3-E1

2.3.1. SEM of MC3T3-E1 adhesion

Fig. 3A showed the morphology of MC3T3-E1 on the scaffolds of PEEK, 40 % HA-PEEK, 40 % HA-2 % AgNPs-PEEK after 1 and 3 days of culture via SEM. On PEEK, MC3T3-E1 exhibited a spherical morphology, while those on 40 % HA-PEEK and 40 % HA-2 % Ag-PEEK showed more polygonal shape with apparent filipodia and microfilaments.

2.3.2. Cell counting-8 kit-8 (CCK8) pathway

The OD₄₅₀ values of MC3T3-E1 cells on PEEK, 40 % HA-PEEK, 40 % HA-2 % Ag-PEEK samples all exhibited proliferation from day 1 to day 14 without cytotoxicity (Fig. 3B. PEEK modified by HA and AgNPs had a significantly positive influence on cells proliferation after D5 (p < 0.05).

2.3.3. AMPI staining and flow cytometry

AMPI staining (Fig. 3C and D) captured the live/dead statement of MC3T3-E1 cells on PEEK, 40 % HA-PEEK, and 40 % HA-2 % Ag-PEEK. Green fluorescence (Calcein AM: Ex/Em = 494/517 nm) referred to living cells, and red fluorescence (PI: Ex/Em = 535/617 nm) referred to dead cells. 96.53 % ± 4.234 % (P = 0.0034 compared to PEEK) living MC3T3-E1 cells were found on 40 % HA-PEEK and 98.15 % ± 3.584 % (P = 0.0022 compared to PEEK) on 40 % HA-2 % Ag-PEEK, while reduced to 86.03 % ± 6.224 % on PEEK.

Flow cytometry showed 88.41 % ± 1.228 % living cells on PEEK and rose up to 95.45 % ± 0.335 % (P = 0.0076) for 40 %HA-PEEK, and 98.11 % ± 0.246 % (P = 0.0047) for 40 %HA-2 %Ag-PEEK (Fig. 3E).

2.4. In vitro osteoinductivity

2.4.1. Real-time PCR

After culturing for 14 and 28 days, MC3T3-E1 exhibited enhanced BMP2, RUNX2, ALP, OPN, and OCN gene expressions on 40 %HA-PEEK and 40 %HA-2 %Ag-PEEK compared to PEEK (P < 0.05). Interestingly, MC3T3-E1 on 40 %HA-PEEK expressed more osteogenic genes compared to 40 %HA-2 %Ag-PEEK, which might be due to the slight cytotoxic effect of AgNPs (Fig. 4A).

Fig. 4 — Osteogenic related ALP activity, gene, protein, and antibodies expression of MC3T3-E1 cell on the surface of PEEK, 40 %HA-PEEK, and 40 %HA-2 %Ag-PEEK (n = 3). A. PCR showed significantly enhanced expression of osteogenic related genes in HA and nano-Ag modified PEEK. B. ALP activity showed early-stage osteogenic activity of MC3T3-E1 cell with significantly enhanced expression of osteogenic activities in HA and nano-Ag modified PEEK from D5. C. ALP staining of MC3T3-E1 cell early-stage osteogenic activity on 3 groups. D. Alizarin red staining exhibited the formation of calcium nodules. E. Immunofluorescence (IF) showed osteogenic antibodies (ALP, BMP2, RUNX2, OCN, OPN). F. IF quantification showed significantly enhanced osteogenic related antibody expressions in HA and nano-Ag modified PEEK. G. Western blot exhibited expressions of osteogenic proteins (ALP, OCN, BMP2, RUNX2, and OPN) and quantification showed significantly enhanced osteogenic related protein expressions in HA and nano-Ag modified PEEK. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

2.4.2. ALP staining and activity

As the early osteogenic differentiation marker of MC3T3-E1 cell, the ALP activity showed an up-regulation from 5 to 14 days. The highest ALP expression was detected in 40 %HA-PEEK (P = 0.0003 compared to PEEK), followed by 40 %HA-2 %Ag-PEEK (P = 0.0075 compared to PEEK) (Fig. 4B). More sites of ALP staining (blue dots) were found on the surface of 40 %HA-PEEK and 40 %HA-2 %Ag-PEEK on day 7 and 14, indicating higher MC3T3-E1 cell osteogenic performance (Fig. 4C).

2.4.3. Alizarin red staining

Alizarin red staining reflected the mineralization ability of MC3T3-E1 cells on the surface of PEEK, 40 %HA-PEEK and 40 %HA-2 %Ag-PEEK after 21 and 28 days of osteoinduction (Fig. 4D). Limited calcium nodule was observed on PEEK, while obvious calcium deposition was formed on the 40 %HA-PEEK and 40 %HA-2 %Ag-PEEK scaffolds, which indicated that the addition of HA and AgNPs could increase the mineralization ability of MC3T3-E1 cells.

2.4.4. Immunofluorescence

Immunofluorescence staining was performed to assess the expression of key osteogenic antibodies, including ALP, BMP2, OPN, OCN, and RUNX2. As shown in Fig. 4E, the fluorescence intensity of ALP, BMP2, and RUNX2 were notably more apparent in MC3T3-E1 cells cultured on 40 %HA-PEEK and 40 %HA-2 %Ag-PEEK compared to those on PEEK, suggesting an enhanced early-stage osteogenic antibody expression. For OPN and OCN, markers of the late-stage mineralization process, a similar expression pattern was observed. These results indicated that HA and AgNPs modification promoted osteogenic antibodies expressions.

Quantification analysis (Fig. 4F) showed significant improvements in osteogenic antibodies found in 40 %HA-PEEK and 40 %HA-2 %AgNPs-PEEK (P < 0.05) with 40 %HA-PEEK expressed more compared to 40 %HA-2 %AgNPs-PEEK.

2.4.5. Western blot

As shown in Fig. 4G, western blot analysis revealed distinct bands for ALP (∼57 kDa), BMP2 (∼45 kDa), OCN (∼11 kDa), OPN (∼60 kDa), RUNX2 (∼57–60 kDa) in all groups. The band intensity was higher in 40 %HA-PEEK and 40 %HA-2 %Ag-PEEK compared to PEEK. Quantification analysis confirmed these trends, with protein levels normalized to β-actin (∼42 kDa). Significant improvements in osteogenic proteins were found in 40 %HA-PEEK and 40 %HA-2 %Ag-PEEK (P < 0.05). Interestingly, 40 %HA-PEEK expressed more osteogenic proteins compared to 40 %HA-2 %Ag-PEEK, which agreed to rt-PCR in 2.4.1. These findings suggested that HA and nano-Ag modification enhanced osteogenic related protein expression, whereas nano-Ag exhibited minor cytotoxicity.

2.5. In vitro antibacterial activity

2.5.1. SEM morphology

The morphology of S. aureus and E. coli on the surface of PEEK, 40 %HA-PEEK, and 40 %HA-2 %Ag-PEEK were captured by SEM (Fig. 5A). Fewer S. aureus and E. coli were found on 40 %HA-2 %Ag-PEEK, and there were less interactions between bacteria.

2.5.2. Antibiofilm test

A clearly less biofilm was formed on 40 %HA-2 %AgNPs-PEEK. The surface of PEEK was totally covered, and nearly 3/4 of the surface area on 40 %HA-PEEK were occupied (Fig. 5B). The absorbance of biofilm for S. aureus at OD₅₇₀ reduced significantly from 2.68 (PEEK) to 0.94 (40 %HA-2 %Ag-PEEK) (P = 0.0037), and the absorbance of biofilm for E. coli reduced significantly from 2.57 (PEEK) to 0.68 (40 %HA-2 %Ag-PEEK) (P = 0.0022) (Fig. 5D).

2.5.3. Bacterial inhibition ring

40 %HA-2 %Ag-PEEK incubated with S. aureus and E. coli exhibited an obvious inhibition ring around the sample evenly on the agar plate, indicating the antibacterial activity released by AgNPs around the sample. On the contrary, S. aureus and E. coli attached directly to PEEK without spaces. For 40 %HA-PEEK, a tiny inhibition ring existed (Fig. 5C).

2.5.4. Bacterial dynamics curves

The real-time quantification of S. aureus and E. coli was measured at OD₆₀₀ with growth kinetic curves and showed that 40 %HA-2 %Ag-PEEK suppressed bacterial growth within 6 h and exhibited complete inhibition of logarithmic phase, which indicated both contact-killing and release-killing capacities (Fig. 5E).

2.5.5. Macrophage polarization

2.5.5.1. Rt-PCR of M1/M2 polarization related genes

The expressions of Arg1, IL-6, IL-10, TNF-α targeted genes were quantified by rt-PCR (Fig. 5F). Relative to PEEK, the expressions of IL-6 and TNF-α (related to M1 polarization) on 40 %HA-PEEK were down-regulated by 0.269 times (P = 0.0170) and 0.828 times (P = 0.0137), and 40 %HA-2 %Ag-PEEK further down-regulated IL-6 and TNF-α by 0.217 times (P = 0.0008) and 0.250 times (P < 0.0001). On the contrary, the expressions of Arg1 on 40 %HA-PEEK were up-regulated by 4.584 times (P = 0.0049), IL-10 remained the same with PEEK. 40 %HA-2 %Ag-PEEK further up-regulated Arg1 by 22.894 times (P < 0.0001), and the expression of IL-10 (related to M2 polarization) was also raised by 2.48 times (P = 0.032).

2.5.5.2. WB of M1/M2 polarization related proteins

The band intensity of Arg1 and IL-10 (related to M2 polarization) was significantly higher in 40 %HA-PEEK and 40 %HA-2 %Ag-PEEK compared to PEEK, especially in 40 %HA-2 %Ag-PEEK. On the contrary, the band intensity of IL-6 and TNF-α (related to M1 polarization) weakened in 40 %HA-PEEK and 40 %HA-2 %Ag-PEEK, while exhibiting the highest intensity in PEEK (Fig. 5G). Quantification analysis confirmed 40 %HA-2 %Ag-PEEK with up-regulated expression of IL-10 and Arg1 (P < 0.01), and down-regulated IL-6 and TNF-α (P < 0.01) compared to PEEK.

2.5.5.3. FACS of M1/M2 polarization related antibodies

As shown in Fig. 5H and I, Raw264.7 cultured on PEEK predominantly (71.71 %) expressed CD86⁺ antibodies, indicating a strong M1-polarized phenotype. In contrast, cells on 40 %HA-PEEK showed a shift toward the M2 phenotype, with 45.28 % CD206+ antibodies. Notably, 40 %HA-2 %Ag-PEEK exhibited a pronounced M2 polarization with 72.04 % CD206+ antibodies.

2.5.6. MIC/MBC vs S. aureus and E. coli

For S. aureus, the MIC was 1 μg/mL and the MBC was 5 μg/mL, while for E. coli, the MIC and MBC were 5 μg/mL and 10 μg/mL, respectively.

2.6. Bio-mechanism behind osteogenic and anti-bacterial properties

PCA revealed tight intra-group clustering and significant inter-group separation among groups PEEK, 40 %HA-PEEK, and 40 %HA-2 %AgNPs-PEEK (Supplementary Figure 15).Differential protein analysis (Supplementary Figure 16) revealed that 40 %HA-PEEK exhibited 78 up-regulated and 75 down-regulated proteins compared to PEEK, while 40 %HA-2 %Ag-PEEK showed 132 up-regulated and 81 down-regulated proteins relative to PEEK. Hierarchical clustering analysis (heatmap) (Supplementary Figure 17) confirmed conserved intra-group up/downregulation patterns and pronounced inter-group divergence.

Pathway analysis (Fig. 6A) highlighted that 40 %HA-PEEK significantly activated the mTOR signaling pathway compared to PEEK, promoting cell cycle progression and survival by integrating growth signals. 40 %HA-2 %AgNPs-PEEK retained robust mTOR activity while additionally upregulated the FoxO signaling pathway, which expressed antibacterial efficacy via autophagy-mediated pathogen clearance and phagocytic activation. Compared to 40 %HA-PEEK, 40 %HA-2 %AgNPs-PEEK showed significant upregulation of cytochrome P450, which might increase ROS reaction and inhibit osteogenesis signal. Moreover, AgNPs activated PPAR signaling pathway, transforming pre-osteoblast to adipose differentiation instead of osteointegration (Supplementary Figure 18).

Pfam enrichment analysis (Fig. 6B) revealed the up-regulation of stress protection related proteins, adhesion related proteins, metabolic regulation and electron transport related proteins expressed by S. aureus on the surface of HA-AgNPs-PEEK compared to bare PEEK. During AgNPs-mediated antibacterial processes, a variety of bacterial stress-responsive proteins were significantly upregulated, potentially modulating host immune responses. FimH promoted IL-10 expression and skewing macrophages toward an M2 phenotype. Transketolase, through upregulation of the pentose phosphate pathway, indirectly induced IL-10 and Arg-1 expression, favoring M2 polarization. Proteins such as FKBP-type peptidyl-prolyl cis-trans isomerase and CoA-ligase contributed to protein folding and lipid metabolic reprogramming, which might support IL-10 and Arg-1 expression, promoting M2-associated immunoregulation. Collectively, the upregulation of these proteins reflected a complex bacterial adaptation involving structural repair, metabolic reprogramming, and immunomodulatory strategies under AgNPs-induced stress (Supplementary Figure 19).

WB verification (Fig. 6C and D) confirmed the up-regulation of p70S6K and 4EBP1 by 40 %HA-PEEK, which were related to mTOR pathway, enhancing osteogenesis. In a loss-of-function test with rapamycin suppressing mTOR pathway on osteogenic differentiated MC3T3-E1 cells, the expression levels of p70S6K and 4EBP1 were markedly reduced in the rapamycin-treated group compared with the untreated group, and osteogenic markers RUNX2 and OCN showed decreased band intensity upon rapamycin treatment, indicating that mTOR inhibition partially suppressed osteogenic differentiation (Supplementary Figure 20). FoxO1 and SOD2, which were related to FoxO pathway, were up-regulated by 40 %HA-2 %AgNPs-PEEK, expressing anti-bacterial property. Meanwhile, PPARγ and NRF2, which represented ROS reaction, were also upregulated by 40 %HA-2 %AgNPs-PEEK. The above enhanced expression of PPARγ pathway and ROS reaction suppressed β-Catenin and P-Akt, hindering osteogenesis. This could explain why the osteogenesis of 40 %HA-2 %AgNPs-PEEK was slightly worse than 40 %HA-PEEK, while expressing anti-bacterial activity instead. The potential bio-mechanism behind the interaction of osteogenesis and anti-bacterial property were summarized in Fig. 6E.

2.7. In vivo osteogenic and anti-bacterial property

2.7.1. In vivo osteogenesis

PEEK, 40 %HA-PEEK, and 40 %HA-2 %AgNPs-PEEK were inserted into the rabbit femur defects (Fig. 7A–C). A more continuous layered bone was found in 40 %HA-PEEK and 40 %HA-2 %Ag-PEEK compared to the sham group and the PEEK group (Fig. 7E). The mean fibrous tissue thickness was 92.78 ± 10.22 μm for PEEK (95 % CI: 82.05–103.51 μm), 1.335 ± 0.846 μm for 40 %HA-PEEK (P < 0.0001 compared to PEEK), and 9.278 ± 5.324 μm for 40 %HA-2 %AgNPs-PEEK (P = 0.0004 compared to PEEK) (Supplementary Figure 21). A thicker layer of fiber formed around PEEK compared to 40 %HA-PEEK and 40 %HA-Ag-PEEK, where a relatively more direct attach between the implanted materials and the newly formed bone were found. There existed a slight difference between the interface of 40 %HA-PEEK and 40 %HA-2 %AgNPs-PEEK: a tiny gap showed in 40 %HA-2 %AgNPs-PEEK, whereas barely any gap formed in 40 %HA-PEEK even with new bone grown inside the implanted material (Fig. 7G).

The bone mineral density (BMD) around PEEK was 0.187 ± 0.010 g/cc, and raised significantly up to 0.265 ± 0.049 g/cc in 40 %HA-PEEK (P = 0.0066), and 0.263 ± 0.027 g/cc in 40 %HA-2 %AgNPs-PEEK (P = 0.0022). The trabecular number (Tb.N) rose significantly from 3.572 ± 0.881/mm in PEEK to 5.137 ± 0.1601/mm in 40 %HA-PEEK (P = 0.0012) and 5.187 ± 1.3231/mm in 40 %HA-2 %AgNPs-PEEK (P = 0.0067), while the trabecular pattern factor (Tb.Pf) remained without statistical significance. The percent bone volume (BV/TV) rose significantly from 33.317 ± 8.212 % in PEEK to 50.204 ± 3.131 % in 40 %HA-PEEK (P = 0.0003) and 49.821 ± 12.043 % in 40 %HA-2 %AgNPs-PEEK (P = 0.0007), likely, the bone surface density (BS/TV) rose significantly from 9.753 ± 2.8/mm in PEEK to 15.424 ± 1.873/mm in 40 %HA-PEEK (P = 0.0088) and 13.136 ± 4.738/mm in 40 %HA-2 %AgNPs-PEEK (P = 0.0243). On the contrary, the total porosity (Po) was reduce significantly from 73.663 ± 9.140 % to 49.644 ± 2.967 % in 40 %HA-PEEK (P = 0.0007) and 44.503 ± 10.603 % in 40 %HA-2 %Ag-PEEK (P = 0.0009) (Fig. 7I). A slightly osteogenic advantage over 40 %HA-PEEK to 40 %HA-2 %AgNPs-PEEK could be found without significance.

2.7.2. In vivo anti-bacterial property

PEEK, 40 %HA-PEEK, and 40 %HA-2 %AgNPs-PEEK were inserted into rabbit femur infectious defects (Fig. 7B–D). A more continuous layered bone was found in 40 %HA-2 %AgNPs-PEEK compared to the sham group, group PEEK, and group 40 %HA-PEEK (Fig. 7F). In the infectious defect model, the fibrous tissue was generally thicker compared to the defect model, with mean values of 142.3 ± 22.56 μm for PEEK (95 % CI: 118.6–166.0 μm), 16.32 ± 5.794 μm for 40 %HA-PEEK (P = 0.0007 compared to PEEK), and 2.244 ± 1.786 μm for 40 %HA-2 %Ag-PEEK (P < 0.0001 compared to PEEK) (Supplementary Figure 21). A thicker layer of fiber formed around PEEK compared to 40 %HA-PEEK and 40 %HA-AgNPs-PEEK, where a relatively more direct attach between the implanted materials and the newly formed bone were found (Fig. 7H). Different from the defect model, the tiny gap appeared in 40 %HA-PEEK, whereas barely any gap formed in 40 %HA-2 %AgNPs-PEEK even with new bone grown inside.

The BMD around PEEK was 0.192 ± 0.052 g/cc, and raised significantly up to 0.212 ± 0.033 g/cc in 40 %HA-PEEK (P = 0.0087), and 0.253 ± 0.032 g/cc in 40 %HA-2 %AgNPs-PEEK (P < 0.0001). The Tb.N rose significantly from 2.749 ± 1.409/mm in PEEK to 3.454 ± 1.379/mm in 40 %HA-PEEK (P = 0.0023) and 4.688 ± 1.1.909/mm in 40 %HA-2 %Ag-PEEK (P < 0.0001), and the Tb.Pf rose significantly from 4.364 ± 1.634/mm in PEEK to 6.365 ± 3.093/mm in 40 %HA-PEEK (P = 0.0079), while reduced significantly to 3.246 ± 1.219/mm in 40 %HA-2 %AgNPs-PEEK (P = 0.0033), which referred to a transformation of rod-shaped bone to lamellar bone. The BV/TV rose significantly from 27.360 ± 14.091 % in PEEK to 32.570 ± 13.679 % in 40 %HA-PEEK (P = 0.0476) and 47.208 ± 18.140 % in 40 %HA-2 %AgNPs-PEEK (P = 0.0009), likely, the BS/TV rose significantly from 8.161 ± 4.118/mm in PEEK to 11.323 ± 4.427/mm in 40 %HA-PEEK (P = 0.0359) and 13.699 ± 5.635/mm in 40 %HA-2 %AgNPs-PEEK (P = 0.0049). On the contrary, the Po was reduced significantly from 72.640 ± 14.091 % to 67.430 ± 13.679 % in 40 %HA-PEEK (P = 0.0288) and 52.791 ± 18.140 % in 40 %HA-2 %AgNPs-PEEK (P = 0.0005) (Fig. 7J).

2.7.3. Release behavior of Ag+ in rabbit femur defect model

The initial serum Ag⁺ concentration before implantation was 8.14 ± 1.16 μg/L (n = 5). After 14 days, the serum Ag⁺ level remained relatively stable at 8.18 ± 1.40 μg/L (n = 5) (Supplementary Figure 13). These results indicated that Ag⁺ was detectable in systemic circulation throughout the observation period with no significant fluctuations.

3. Discussion

Several studies have been conducted concerning the cooperation of HA or AgNPs into PEEK [1,16,18,[22], [23], [24], [25], [26], [27], [28], [29], [30], [31], [32], [33], [34], [35], [36], [37], [38], [39], [40], [41], [42], [43], [44], [45], [46], [47], [48], [49]]. The difference and advances of this study were concluded by three parts: 1. Gradient 3D printing technique formed a functional layer containing high-concentration HA and AgNPs on the surface of PEEK substrate, optimizing the plasticity of PEEK, lowering the cost, and successively introduced dual enhanced osteogenesis and anti-bacterial properties to bio-inert PEEK, which was crucial in clinical applications. 2. The novel technique of gradient 3D printing facilitated self-customized multi-functional surface design which could be suitable to various clinical conditions such as aseptic defect or contaminated defect, aligned with the real clinical requirements. 3. The absence of potential bio-mechanisms behind the enhanced osteogenesisgenerated by HA and the anti-bacterial property released by nano-Ag and how HA and nano-Ag interacted were revealed by omics and validation. In conclusion, an overall fundamental proof of enhanced mechanical, osteogenic and anti-bacterial properties both in vitro and in vivo were provided with potential bio-mechanisms revealed, and the gradient additive manufacturing process could facilitate multiple functional surfaces design, which opened insight to a new generation of artificial joint prosthesis and oral implantations in the field of oral and maxillofacial surgery.

According to Guo et al., HA could be deposited on the surface of PEEK via hot pressing, aerosol deposition, chemical deposition, ion beam–assisted deposition, spin coating, and cold spraying [2,33,[50], [51], [52], [53], [54]]. Aerosol deposition, ion beam–assisted deposition and cold spraying request sophisticated machines, and chemical deposition [11,13] and spin coating required more precise technical control, while hot-press molding and selective laser sintering showed limited customization. This study first accomplished the gradient 3D printing of PEEK incorporated by higher concentration of HA (40 % wt) and nano-Ag compared to previous studies where mostly 10–20 % wt were incorporated [36,37], and a balance point with the maximum amount of HA (higher expression of osteogenesis) and feasibility of gradient 3D printing was found. The greatest advantage of gradient 3D printing is the realization of multiple functional surfaces customization according to various clinical situations. In addition, gradient 3D printing could perfectly balance the cost and biological behavior by incorporating functional agents merely onto surface of PEEK, which would benefit the patients eventually. Moreover, recent evidence pointed out that some HA-coated PEEK implants generated wear debris that initiated peri-implant osteolysis and led to inflammation [18,19,55]. By gradient 3D printing, functional modification agents could be embedded into the surface layer, and wear tests proved stable surface characteristics under maximum occlusal force (Supplementary Figure 10).

Apart from easy manufacturing process and multiple surface customization, gradient 3D printing retained the benefits of a porous surface and enhanced mechanical characteristics. 3D printing process facilitated bony ingrowth [56], provided higher surface area, higher protein adsorption, and increased cell adhesion and proliferation (Supplementary Figure 11; Fig. 2F) [[57], [58], [59]], affecting subsequent signaling pathways and gene expression [60], which was demonstrated by Chen et al. [28] and Han et al. [61]. The HA content should be selected to balance the bioactivity, the mechanical properties, and the feasibility of gradient 3D printing. Since the percentage of HA needed to be highest to promote osteointegration as well maintaining the capability of additive manufacturing, while the compound became too fragile to endure 3D printing process once the percentage of HA reached 50 %. Thus, the percentage of 40 % HA-PEEK was the optimal choice. Song et al. and Kang et al. proved that appropriate mechanical properties were beneficial to the transfer of physiological load, and the stress stimulation of the scaffold on the bone tissue is conductive to bone growth [62,63]. Better mechanical property was proved in 40 % HA-PEEK [10,54,64], which agreed to this study. The mechanical parameters of the prosthesis generated by gradient 3D printing were proved safe in a mandibular condylar neck fracture fixation model (2.1.3).

The gradient 3D printed HA-(AgNPs-)PEEK@PEEK showed dual enhanced osteogenesis and anti-bacterial properties in vitro and in vivo. A major drawback of PEEK is its poor bioreactivity and low antibacterial properties, which have resulted in fusion failures [8,[65], [66], [67]]. Further studies in craniofacial surgery revealed a total complication rate of nearly 16 % with 9 % implant failure in cranioplasties with PEEK [68]. PEEK implants may also favor biofilm formation and bacterial adhesion [69], which increases the risk of infection around the implants [70,71]. Instead of anchoring to the surrounding bone, PEEK implants tend to become encapsulated by fibrous tissue and/or colonized by bacteria as a result of the foreign body reaction that occurs post-implantation [23]. In this study, significant improvements in biocompatibility, osteogenesis and anti-bacterial property generated from the surface of gradient 3D printed HA-(AgNPs)-PEEK@PEEK scaffolds were proved in vitro and in vivo.

Moreover, this study revealed the potential mechanism behind the enhanced osteogenesis and anti-bacterial properties by multiple omics and validation. Compared to PEEK, HA modified PEEK significantly upregulated the mTOR signaling pathway, which promoted osteogenesis. In addition, if HA and nano-Ag were incorporated together into PEEK, the mTOR signaling pathway maintained up-regulated, and the FoxO signaling pathway showed up-regulated as well, which elevated IL-10 secretion, Arg1 expression, while reduced TNF-α and IL-6/IL-10 ratio (2.5.5.2). This study might also have answered the question raised by Buck et al. [23] regarding the interactions between functional agents HA and nano-Ag: FoxO pathway up-regulated cytochrome P450, which indicated the enhancement of ROS reaction, inhibiting the expression of β-catenin and Akt and hindering downstream osteogenic regulation. Moreover, nano-Ag triggered PPAR signaling pathway, and induced adipose differentiation of MC3T3-E1 osteoblast. The above-mentioned bio-mechanisms revealed behind the osteogenic and anti-bacterial properties were merely in the association stage, and deeper mechanism verifications including loss-of-function controls needed to be carried out in future studies to verify causality.

Even though Nano-Ag displayed slight cytotoxicity, while still showing significant enhancement of osteogenesis and anti-bacterial properties compared to unmodified PEEK. These results agreed with Pryjmaková et al. [41]. In conclusion, in real clinical practice, 40 % HA-PEEK with optimal osteogenesis and cytocompatibility would be the best choice for aseptic defects. While for contaminated defects, the addition of nano-Ag would improve osteogenesis compared to 40 % HA-PEEK and unmodified PEEK. The surface characteristics of the biomaterials should be altered with the change of clinical requirements, which could be accomplished by gradient 3D printing, forming selective multiple functional surfaces regarding various clinical conditions.

Several limitations existed: 1. Contrary to the slight cytotoxicity nano-Ag displayed during osteogenesis, MC3T3 osteoblast proliferated more on HA-AgNPs-PEEK in early stage. The biomechanism behind this contradictory phenomenon should be studied in the future. 2. Long-term cyclic durability, pull-out test, and hematological assessments of the implants should be carried out in future studies to prove mechanical and biological safety in practical applications. 3. More in-depth study (loss-of-function validation) on the bio-mechanisms behind the anti-bacterial property of nano-Ag should be carried out.

4. Materials and methods

4.1. Fused filament fabrication (FFF) of HA-(Ag-)PEEK@PEEK composite

4.1.1. The origin of PEEK, HA, and AgNPs powder

Medical grade nano powder HA (<200 nm, Sigma-Aldrich, USA), 450 PF PEEK (50 mikron, GoodFellow, England), and AgNPs (50 nm, Imnano, China) were used as original materials to extrude filaments.

4.1.2. Ultrasonic blending

PEEK, HA and AgNPs were mixed ultrasonically [72]. 500 ml absolute ethyl alcohol (≥99.5 %, Merck, USA) was used as the liquid medium. A magnetic stirrer (MS7-H550-Pro, SCILOGEX, USA) was used to keep the absolute ethyl alcohol stirring at a rotate speed of 250 rpm with a magnet rotor. PEEK powder was poured into absolute ethyl alcohol and kept stirring for 10 min, then moved into an ultrasonic cell disruptor (XM-1000T, XIAO MEI CHAO SHENG, China) to disperse ultrasonically at a frequency of 65 % for 10 min. After that HA and/or AgNPs powder was added and repeated stirring and ultrasonic disperse. Finally, HA (10 %, 20 %, 40 % wt) - (Ag (2 % wt) -) PEEK ultrasonically blended powder in absolute ethyl alcohol were heated at 85 °C while kept stirring at 250 rpm until all absolute ethyl alcohol were evaporated.

4.1.3. Extrusion of filaments

The homogeneously mixed powders generated from ultrasonic blending were sieved via screen meshes (150 mesh, 100 mesh) successively to break the agglomeration. After that, they were dried in an electric thermostatic oven (DHG-9245A, NANBEI, China) at 160 °C for 5 h [73]. Those dehydrated mixed powders were extruded via a table extruder (Type-C, Wellzoom, China) at 380 °C to form HA(10 %, 20 %, 40 % wt)-(Ag(2 % wt)-)PEEK filaments with a diameter of 1.75 mm. Pure PEEK granules (Mv∼35,000, MACKLIN, China) were used between different mixed powders to clean up the extruder. The filaments were dried again at 160 °C for 5 h to be ready for gradient 3D printing.

4.1.4. Gradient 3D printing

Various sample models were fabricated: spindle for tensile test, rectangle for bending test, round (φ13 mm∗1.1 mm and φ32 mm∗1.1 mm) for in vitro studies, and cylinder (φ5 mm∗7 mm) for in vivo studies. Those shapes were designed and converted into STL data, which could be imported into Matter Control (Version 2.19.1.10116, Matter Hackers, USA) and read by the 3D printer. Filaments were processed through a 3D printer (INTAMSYS FUNMAT HT) with nozzle temperature of 400 °C, bed temperature of 150 °C, chamber temperature of 90 °C, layer thickness of 0.1 mm, brim of 2 mm and printing speed of 10 mm/s. The diameter of the nozzle was 0.4 mm. 10 % PVP (mol wt 10,000, SIGMA-ALDRICH, USA) was used as the bounding between the printer bed and the first layer of PEEK. After the PEEK substrate reached 1 mm height, attention should be paid to the maintenance of nozzle temperature (400 °C), bed temperature (150 °C) and chamber temperature (150 °C) to prevent detachment of the PEEK substrate from the print bed. In addition, the X and Y axis of the print endpoint should not be changed to ensure perfect combination of the functional layer and the substrate, but the Z axis should increase 1 mm to adapt with the height of PEEK substrate. Then, modified filaments containing various percentage of HA and nano-Ag were fused with nozzle temperature of 420 °C, bed temperature of 150 °C, chamber temperature of 90 °C and formed the 0.1 mm functional surface layer, accomplishing gradient printing. The diameter of the nozzle was changed to 0.8 mm to avoid blocking, since the viscosity of fused modified filaments was significantly higher than fused PEEK. Moreover, layer thickness remained at 0.1 mm, and the brim was turned down to ensure tight connection of the functional surface and the PEEK substrate. The printing speed was adjusted much slower from 10 mm/s to 2 mm/s to ensure consistency.

4.2. Mechanical test

Samples were ultrasonically cleaned in acetone (99+%, Acros Organics™, USA), absolute ethanol (≥99.5 %, EMPARTA® ACS reagent, Germany), and double-distilled water sequentially for 30 min each. After that, the samples were autoclaved (GI54DP, ZEALWAY, China) at 120 °C for 20 min and dried at 60 °C for 1 h. Each group had 6 repetitions for each test.

Thickness, width, and length of each spindle or rectangle sample were measured 3 times by a digital caliper (DELIXI, France) and inputted into TestXpert software (version III, Switzerland), which was used to catch the real-time stress and strain data, as well as calculate tensile parameters: E-modulus (GPa), maximum tensile strength (Rm, MPa), maximum total elongation (Agt, %); and bending parameters: Eb-modulus (kN/mm²), maximum bending strength (σbb, N/mm²), total break elongation (fbf-Break, mm). Every group had 6 repetitions.

4.2.1. Tensile test

The tensile property of each spindle sample composed of HA(10 %, 20 %, 40 % wt)-(Ag(2 % wt)-)PEEK was tested by Zwick/Roell tensile testing machine (Z20, Germany) according to GB/T 228-2002 standard.

4.2.2. Bending test

The bending property of each rectangle sample composed of HA(10 %, 20 %, 40 % wt)-(Ag(2 % wt)-)PEEK was tested by Zwick/Roell bending machine (Z100, Germany) according to GB/T 14452-93 standard.

4.2.3. Finite element analysis

A mandibular condylar neck fracture model fixed by HA and AgNPs modified PEEK compound plates and screws was established by Mimics Research 21.0 (Leuven, Belgium) and imported into Geomagic Wrap Software (3D-SYSTEM, USA) to get reverse processed and preprocessed by Hypermesh Finite Element Mesh Module Software (HyperWorks, USA), detailly described in previous works [74,75]. The mechanical parameters of each fixation materials were calculated as 4.2.1 and 4.2.2. The General Finite Element Analysis Software ABAQUS (DASSAULT, France) was used as both the solver and the processor. Finally, the stress and displacement with the fixation of HA(10 %, 20 %, 40 % wt)-(AgNPs(2 % wt)-)PEEK under maximum occlusal force were analyzed.

4.2.4. Friction-wear test

The friction and wear behavior of the samples was evaluated using a ball-on-disk tribometer (Bruker, CETR, UMT-2, USA) in phosphate buffer solution (PBS) at 37 °C. Samples are fixed securely on the stage, and an alumina ball with 6 mm of diameter was loaded onto the sample surface as the counterpart. Rotational sliding tests were conducted with a load of 30 N [74,75] at a frequency of 1 Hz and 3 mm of stroke length, simulating the loading conditions around the condyle. The wear duration was 60 min.

4.3. In vitro osteogenesis and anti-bacterial property

4.3.1. Samples preparation

Round samples were divided into 3 groups: PEEK, HA(40 % wt)-PEEK, and HA(40 % wt)-Ag(2 % wt)-PEEK. The samples were ultrasonically cleaned in acetone (99+%, Acros Organics™, USA), absolute ethanol (≥99.5 %, EMPARTA® ACS reagent, Germany), and double-distilled water sequentially for 30 min each. After that, the samples were autoclaved (GI54DP, ZEALWAY, China) at 120 °C for 20 min and dried at 60 °C for 1 h. Each group had 3 repetitions for each test.

4.3.2. Surface characteristics

4.3.2.1. Scanning electron microscope

SEM images of both the surface and cross-sectional morphologies were captured. A thin layer of Au coating was applied, then the morphologies were characterized via scanning electron microscope (GeminiSEM 300, ZEISS, Germany) at an acceleration voltage of 5 kV in low vacuum mode.

4.3.2.2. Energy dispersive spectroscopy

An energy-dispersive spectrometer (X-act one, OXFORD INSTRUMENTS, England) was used to detect the chemical elements distribution both on the surface and across the cross-section of the samples. Elemental distribution along the cross-sectional line was recorded and analyzed using the instrument's software to generate compositional profiles, allowing evaluation of material gradients.

4.3.2.3. X-Ray diffraction

X-Ray diffraction was manipulated by Rigaku Ultima IV to study the crystal structure on the surface of the samples. Jade software (6.5, Materials Data, USA) was used to analyze crystal composition.

4.3.2.4. Water contact angle

Static water contact angle on the surface of the samples was carried out to evaluate the surface hydrophilia. At room temperature, a 2 μL deionized water droplet was dropped onto the sample surface and allowed to stabilize for 10 s. Images of the droplet profile were captured with a CCD camera, and the static water contact angle was calculated by a goniometer connected to the camera.

4.3.2.5. Small-angle X-ray scattering

Small-angle X-ray scattering (SAXS) was carried out on the Xeuss 2.0 apparatus (Xenocs) of LLB installed in the SWAXS-Lab (Saclay, France) to test the homogeneity within the samples. SAXS measurements were performed with an X-ray wavelength of 1.24 Å (10 keV) and a sample-to-detector distance of 2.0 m, covering a q-range of 0.05–3.0 nm⁻¹ (q = 4πsinθ/λ) [76].

4.3.2.6. AFM characterization

Surface topography and roughness of the samples after gradient 3D printing were analyzed using an atomic force microscope (Bruker Dimension Icon, Germany). Scan areas of 5 × 5 μm² were performed to achieve surface roughness (Ra) and three-dimensional surface morphology.

4.3.2.7. ICP-MS time-related courses for Ag⁺; Ca; P; Ca/P release in vitro

Each HA-AgNPs-PEEK@PEEK disc was immersed in 10 mL phosphate-buffered saline (PBS, pH 7.4) at 37 °C in a shaking incubator (∼80 rpm) to ensure homogeneous mixing. Aliquots of 5 mL of PBS were collected at 6h, 12h, 1d, 3d, 5d, 7d, 14d. Each aliquot was immediately acidified by ∼2 % HNO₃ to stabilize Ag⁺, Ca, P prior to analysis. The concentration of Ag⁺, Ca, P and the Ca/P ratio were detected by inductively coupled plasma mass spectrometry (Agilent 8800 triple-quadrupole spectrometer, Agilent Technologies, Santa Clara, CA, USA). The arithmetical means and standard deviations are calculated, and the results are exported as concentration–time curves and a corresponding numerical dataset.

The MC3T3-E1 toxicity of nano-Ag with varying concentrations (0.01, 0.1, 0.5, 1, 5, 10, 20 μg/mL) was revealed by CCK8 pathway for 1, 3, 5, 7 and 14 days (4.3.3.2). Results are expressed as means ± deviations.

4.3.3. Osteoblast adhesion and proliferation

Preosteoblast MC3T3-E1 (P7) was acquired from Chinese Academy of Sciences Cell Bank, seeded on the samples and cultured in the α-minimum essential medium (α-MEM) with 10 % fetal bovine serum (FBS) at 37 °C in a humidified atmosphere of 5 % CO₂ furnished by an incubator. The culture medium was refreshed every 2 days. Each group included 3 repetitions per observation time.

4.3.3.1. MC3T3-E1 cell adhesion

MC3T3-E1 cells on samples of 3 groups were fixed with 4 % paraformaldehyde after 1- and 3-days culturing and serially dehydrated with gradient ethanol solutions. The cells attached to the samples were observed by SEM (GeminiSEM 300, ZEISS, Germany).

4.3.3.2. Cell counting-8 kit-8 (CCK8) pathway

MC3T3-E1 cells on samples of 3 groups were proliferated for 1, 3, 5, 7 and 14 days, at each point attached cells incubated for 30 min in the CCK-8 solution (comprising of αMEM and 10 v/v% CCK-8). The absorbance of supernatant was measured by a microplate reader at OD₄₅₀ to qualitatively evaluate cell proliferation.

4.3.3.3. AMPI staining and flow cytometry

MC3T3-E1 cells cultivated on samples of 3 groups after 3 days’ incubation were stained by AMPI and observed by Zeiss fluorescence microscope (Axiocam, Carl Zeiss AG, Germany) to visually evaluate the cell viability. The percentage of live and dead cells were calculated by ultra-high speed flow cytometer (BeckMan, moflo astrios, USA) at OD₅₃₀ and OD₆₁₇, and analyzed by CytExpert (2.3.1.22, Beckman Coulter, Inc.).

4.3.4. Osteogenesis

MC3T3-E1 cells were seeded onto samples of 3 groups. Each group included 3 repetitions per observation time. After MC3T3-E1 cells proliferated and reached the density of 80–90 % in each well, MC3T3-E1 Cell Osteogenic Induction Differentiation Kit (MUXMT-90021, Cyagen, USA) was used to replace α-MEM and initiated osteogenic differentiation, and the induction medium was refreshed every 2 days.

4.3.4.1. ALP activity and alizarin red staining

The ALP activity was calculated on 1, 3, 5, 7, 14 days after osteoinduction using the alkaline phosphatase (AKP/ALP) test kit (A059-2-2, Nanjing Jiancheng Bioengineering Research Institute, China). RIPA (P0013B, Beyotime, China) was used to lyse the cell, and centrifuged with 12,000 rpm at 4 °C. OD520 of the supernatant was read by a microplate reader. For normalization, a BCA protein assay kit (P0012S, Beyotime, China) was used to calculate the total protein concentration by measuring OD₅₇₀. The ALP levels were normalized to the total protein content and described as μM/mg total proteins.

For ALP and alizarin staining, the samples with MC3T3-E1 cells osteoinducted for 7 and 14 days were dyed by alkaline phosphatase staining solution (G1480, Beijing Solaibao Technology Co., LTD, China). Later, the formation of calcium nodule was reflected by alizarin red staining on 21 and 28 days after osteoinduction. The stained specimens were pictured by EPSON scan (Perfection 610, America).

4.3.4.2. Real-time PCR

The expressions of osteogenesis-related genes were quantitatively analyzed by rt-PCR on day 14 and 28. RNAfast200 (Hefei Feijie Biotechnology Co., LTD, China) was used to extract the total RNA, and cDNA was traverse-transcribed using PrimeScript™ RT Master Mix (TaKaRa, Japan) with a thermal cycler (LightCycler, Roche, Switzerland). Forward and reverse primers of BMP-2, Runx2, OPN, OCN and OSX were introduced into cDNA, and LightCycler480 System (LightCycler, Roche, Switzerland) was used for the polymerase chain reaction. β-actin acted as the house-keeping gene for normalization. LightCycler 96 (1.1.0.1320, Roche, Switzerland) was used for analysis.

4.3.4.3. Western blot

The expressions of osteogenesis-related protein were captured by western blot on day 28. The extraction of total proteins was described in 4.3.4.1. Protein suspensions were mixed with 5X Protein sample loading buffer (Shanghai Yase Biomedical Technology Co., LTD, China) and heated at 95 °C for 5 min. Proteins were separated on SDS-PAGE at 90 V for 50 min and transferred to a PVDF membrane at 100V for 50 min (Bio-Rad, USA). Membranes were incubated with the primary antibody (OPN, OCN, RUNX2, BMP2, ALP, β-actin, Affinity, China) overnight at 4 °C. Then incubated with HRP-conjugated goat anti-rabbit IgG(H + L) (Proteintech, USA) for 1 h RT. The protein bands were illuminated by ECL and captured with chemiluminescence detection system (Bio-Rad, U.S.A).

4.3.4.4. Immunofluorescence

The expression of osteogenesis-related protein was also revealed by immunofluorescent staining on day 28. MC3T3-E1 cells were fixed by 4 % paraformaldehyde for 30 min at room temperature RT and then permeabilized with 0.1 % Triton X-100 for 10 min, while non-specific binding was blocked by 5 % BSA for 1 h. Samples were incubated with the primary antibody (OPN, OCN, RUNX2, BMP2, ALP, Affinity, China) overnight at 4 °C and then incubated with IFKine™ Green Donkey Anti-Rabbit IgG (Abbkine, China) for 1 h RT. Cell nuclei were stained by DAPI (1 μg/mL, Beyotime, China) for 1 min. Zeiss fluorescence microscope was used to capture images of the nuclei (blue) and the antibodies expressions in cytoplasm (green), and merged by Photoshop (21.0, San Jose, California, USA)

4.3.4.5. Bio-mechanism of osteogenesis

Proteomic analysis was used to reveal the mechanism behind early osteogenesis of 3 groups on day 7 after osteoinduction. Protein extraction and quantification were described as 4.3.4.1. Proteins were digested by trypsin (Sequencing Grade Modified, Promega, USA) at 37 °C for 4 h, followed by Nano-LC-MS/MS analysis. Separation of the peptide fragment was conducted on VanquishNeo (Thermo Fisher, USA) coupled to an EASY-Spray™ HPLC system (150 μm × 15 cm, Thermo, USA). DIA (Data Independent Acquisition) was accomplished by Astral (Thermo, USA) according to UniProt database (Supplementary Figure 22).

Metabolomics figured out the intracellular metabolites of MC3T3-E1 cells on day 14 after osteoinduction. Intracellular metabolites were extracted with 50 % methanol (Honeywell, USA)/acetonitrile (Merck, Germany) (v/v), and centrifuged at 25,000×g for 15 min at 4 °C. Supernatants were lyophilized (Labconco FreeZone, USA), reconstituted in 50 % methanol, and analyzed via Q Exactive HF Mass Spectrometer (Thermo Fisher Scientific, USA). Data were processed using Compound Discoverer 3.3 (Thermo Fisher Scientific, USA) with BGI Metabolome Database (Supplementary Figure 23).

4.3.5. In vitro antibacterial activity evaluation

S. aureus and E. coli were cultivated in Luria-Bertani (LB) broth at 37 °C. No antibiotics were added during antibacterial tests to avoid any confounding effects. The antibacterial property of AgNPs released from group HA(40 % wt)-AgNPs(2 % wt)-PEEK was compared to group PEEK and HA(40 % wt)-PEEK. Each group had 3 repetitions for each time point.

4.3.5.1. Bacterial inhibition ring

The bacteria suspension (10⁸ CFU/ml, 100 μL) was introduced to the LB agar culture medium, then the samples of 3 groups were put on the center of the agar plates and incubated for 24 h at 37 °C. The formation of the inhibition ring proved the antibacterial property.

4.3.5.2. Bacterial dynamics curves

Bacterial dynamics curves were used to quantitatively compare the antibacterial efficacy among 3 groups. The samples were cultured with an initial bacterial concentration of 10⁴ CFU/ml 2, 6, 12, 16, 24 h after, OD₆₀₀ of the bacterial suspension was measured by a microplate reader according to the National Standard of China GB/T 4789.2 protocol.

4.3.5.3. Antibiofilm test

Samples of 3 groups were exposed to bacterial suspension (10⁸ CFU/ml) for 7 days, then stained with 1 % (w/v) crystal violet RT for 15 min to reveal the attached biofilm, and the absorbance of the eluates were read at OD₅₇₀.

4.3.5.4. SEM observation

Samples of 3 groups were incubated with a bacteria concentration of 10⁷ CFU/ml and cultured at 37 °C for 24 h. S. aureus and E. coli were fixed with 2.5 % glutaraldehyde and then serially dehydrated. The dried samples were sputter coated with platinum and the morphology of S. aureus and E. coli was observed by SEM (GeminiSEM 300, ZEISS, Germany).

4.3.5.5. MIC/MBC vs S. aureus and E. coli

5.0 μL of S. aureus and E. coli suspension (2.5 × 10⁵ CFU/mL) were added separately to 1 mL of broth medium. Various concentrations (0.01, 0.1, 0.5, 1, 5, 10, 20 μg/mL) of AgNPs suspension were added to the bacterial culture medium and cultures in the shaking incubator (37 °C) for 24 h. Then, OD₆₀₀ of the bacterial suspension was measured by a microplate reader. The inhibition rate of S. aureus and E. coli were calculated by absorbance value. The MBC value of AgNPs was examined from the MIC dilutions. MIC dilutions are cultured in the agar plates and incubated at 37 °C for 24 h. The minimum concentration of AgNPs which exhibit barely any S. aureus and E. coli proliferation was documented as the MBC.

4.3.5.6. Macrophage polarization

Mouse macrophage cell line Raw264.7 (Chinese Academy of Sciences Cell Bank) was cultured in DMEM (Gibco, U.S.A) supplemented with 10 % FBS at 37 °C in a humidified atmosphere of 5 % CO₂ furnished by an incubator on the surface of PEEK, 40 % HA-PEEK, and 40 % HA-2 % Ag-PEEK for 3 days. Raw264.7 co-cultured with substrates were acquired and the post-stimulation and polarization were validated via qPCR on D2 and WB on D4 (M1 markers: TNF-α, IL-6; M2 markers: Arg1, IL-10) with the same procedure described in 4.3.4.3. and 4.3.4.4., and flow separation on D3 (CD86 for M1; CD206 for M2) as described in 4.3.3.3.

4.3.5.7. Mechanism of anti-bacterial property

The procedure of proteomic analysis was described in 4.3.4.5. The experimental groups were S. aureus and E. coli co-cultured with HA-Ag-PEEK, and the control groups were S. aureus and E. coli cultured in LB. DIA was accomplished by Astral according to UniProt database.

4.4. In vivo osteointegration and anti-bacterial property

4.4.1. Samples and S. aureus suspension preparation

Sample size justification was calculated by power analysis (G∗Power 3.1.9.7). A total amount of 76 New Zealand white rabbits weighed around 3 kg were enrolled, in which 36 rabbits were allocated to the defect-only model, and the other 36 rabbits were allocated to the femur defect infectious model. The rest 4 rabbits were defined as the sham group with simple defect and no implantation. The experimental groups each consisted of PEEK, HA(40 % wt)-PEEK, and HA(40 % wt)-Ag(2 % wt)-PEEK cylinder samples (φ5mm∗8 mm). Rabbits were randomly allocated into experimental groups (defect only and infectious) using a computer-generated randomization sequence (Random.org). Group allocation was performed by an independent investigator (Xu Q) to ensure unbiased distribution.

Cylinder samples of PEEK, HA(40 % wt)-PEEK, and HA(40 % wt)-AgNPs(2 % wt)-PEEK were 3D printed as described in 4.1.4. and ultrasonically sterilized as 4.3.1. All the animal experiments were approved by the Animal Ethical Committee at the Ninth People's Hospital affiliated to Shanghai Jiaotong University School of Medicine (JGLL-2022071101).

S. aureus were incubated in 100 ml liquid LB and amplified to 5∗10⁷ CFU. 35 ml of S. aureus suspension were centrifuged at 3000 g for 8 min, and sediment were resuspended by 10 ml PBS to reach 1.75∗10⁸ CFU and stored at 4 °C.

4.4.2. Establishment of the rabbit femur defect (infection) model

General anesthesia with a combination of 1.5 cc of 2 % Xylazine HCL and 0.5 cc of Tiletamine HCL was applied. Legs were shaved and disinfected with iodophor disinfectants. An incision with 3 cm long was made to dissect the subcutaneous tissue layer by layer until the femur condyle was exposed. Afterwards, a cylindrical hole (φ5 mm∗7 mm) perpendicular to the long axis of the femur was drilled by Straumann Dynamic System (Switzerland). Then samples of 3 groups were implanted and sutured layer by layer. After surgery, the rabbits were housed in separate ventilated cages with general feeding. Antibiotics (cephalosporin) were given for 3 days to prevent severe infection Postoperatively, butorphanol (0.2–0.4 mg/kg was administered for 2–3 days, with optional meloxicam (0.2–0.3 mg/kg) for anti-inflammatory support. For rabbit femur infection model, 5 μl of 1.75∗10⁸ CFU S. aureus suspension prepared in 4.4.1. were injected into the defect by a micro syringe before samples implantation. Then the same procedure described above was manipulated.

Surgeries on the rabbit femur were performed by a designated surgical team (Bu L, Han Z, Zheng J, Wei X), while micro-CT and histology staining were conducted by a blinded investigator (Zhang Y).

4.4.3. ICP-MS time-related courses for Ag⁺ release in vivo

5 New Zealand white rabbits weighed around 3 kg underwent general anesthesia with acombination of 1.5 cc of 2 % Xylazine HCL and 0.5 cc of Tiletamine HCL. Left legs were shaved and disinfected. An incision with 3 cm long was made to dissect the subcutaneous tissue until the femur condyle was exposed. Afterwards, a cylindrical hole (φ5 mm∗7 mm) perpendicular to the long axis of the femur was drilled by Straumann Dynamic System (Switzerland). Then samples of HA-AgNPs-PEEK@PEEK are implanted and sutured layer by layer.

After surgery, the rabbits were housed in separate ventilated cages with general feeding. 14 days after the rabbit femur implantation, 10 ml blood samples were collected from the marginal ear vein of rabbits. Blood collected were clot in serum collection tubes and centrifuged to obtain 3∼4 ml serum. The concentration of Ag⁺ was detected by inductively coupled plasma mass spectrometry (Agilent 8800 triple-quadrupole spectrometer, Agilent Technologies, Santa Clara, CA, USA) equipped with an autosampler.

4.4.4. Specimen acquirements, micro-CT and immumohistochemical staining

1 and 3 months after implantation, the rabbits were sacrificed, the femur along with the implants were acquired and fixed in 4 % paraformaldehyde.

X-ray (Faxitron MX-20, 20 kV, 30 s exposure), micro-CT (Bruker Skyscan 1276, 50 kV/200 μA, 9 μm resolution), and CT reconstruction (Materialise Mimics 21.0, Belgium) were conducted for each specimen. The bone integration result was evaluated by BV/TV (%), BS/TV (mm⁻¹), Po (%), BMD, Tb.Pf, and Tb.N. Thresholds for micro-CT analysis was 80∼255 HU with a cylindrical region surrounding the defect site (φ5.5 mm, height 7.5 mm) selected as the volume of interest (VOI). VENUS Micro CT (VNC-102) was used to capture CT data, Cruiser software for data acquisition, and Avatar 3 for visualization. A bone phantom (QRM-Micro-CT-HA D10, MHA-476) with predefined BMD values was used for calibration.

3 femur specimens of each group were dehydrated and embedded in PMMA and cut into 150 μm thick sections perpendicular to the bone using a Leica SP1600 saw microtome and polished to a final thickness of about 50 μm. Van Gieson staining (VG) was used to visualize the fiber tissue surrounding the implants, and hematoxylin-eosin staining (HE) was used to colorize the bone tissue. The images were captured by a fluorescence microscope. The thickness of fibrous tissue was measured on histological sections using ImageJ software at five evenly distributed points along the implant interface.

4.5. Statistical analysis

Statistical analysis was accomplished by GraghPad Prism (Version 9.0.0, GraghPad Software, LLC). Each group of each experiment included at least 3-6 repetitions, and all experiments were repeated 3 times by a same investigator (Bu L) to reach the average trend. All the data were expressed as means ± SD. Shapiro-Wilk/Kolmogorov-Smirnov test was used to verify gaussian distribution of each data set and statistically significant differences among groups were measured vy RM two-way ANOVA with Geisser-Greenhouse correction and Dunnett's multiple comparisons. The differences were considered statistically significant at ∗p < 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001, ∗∗∗∗p ≤ 0.0001.

5. Conclusion

The gradient 3D printed PEEK scaffold modified by nano-coating with micro-structured surface and bone-like elastic modulus could promote osteogenesis and anti-bacterial property in vitro and in vivo. The novel manufacture technique with selective multiple functional surfaces customization promotes potential clinical translation, which could be a good candidate for artificial joint replacement and bone implants in the field of oral and maxillofacial surgery.

CRediT authorship contribution statement

Lingtong Bu: Writing – original draft, Software, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Xiang Wei: Writing – original draft, Visualization, Validation, Supervision, Methodology, Data curation. Yuxin Zhang: Writing – original draft, Visualization, Validation, Supervision, Software, Methodology, Data curation. Qingyu Xu: Software, Resources. Zixiang Han: Investigation. Yinjun Chen: Writing – review & editing, Supervision, Resources, Project administration, Funding acquisition, Data curation. Jisi Zheng: Writing – review & editing, Supervision, Resources, Project administration, Funding acquisition. Chi Yang: Writing – review & editing, Validation, Supervision, Project administration, Methodology, Funding acquisition.

Funding

This study was supported by the National Key R&D Program of China (2023YFC2509100); Science and Technology Commission of Shanghai Municipality (23Y31900400); National Natural Science Foundation of China (82370984); Science and Technology Commission of Shanghai Municipality (22S31903400); Shanghai Jiao Tong University School of Medicine Affiliated Ninth People's Hospital (2022hbyjxys-zjs); Fundamental Research Funds for the Central Universities (project number YG2025QNA23); Fundamental research program funding of Ninth People's Hospital affiliated to Shanghai Jiao Tong university School of Medicine (JYZZ224).

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

Not applicable.

Footnotes

^{Appendix A}

Supplementary data to this article can be found online at https://doi.org/10.1016/j.mtbio.2025.102432.

Contributor Information

Lingtong Bu, Email: bulingtong97@163.com.

Xiang Wei, Email: weiflying@hotmail.com.

Yuxin Zhang, Email: yuxinzhang0129@163.com.

Yinjun Chen, Email: yj.chen@dhu.edu.cn.

Jisi Zheng, Email: 237111641@qq.com.

Chi Yang, Email: yang_chi63@163.com.

Appendix A. Supplementary data

The following is the Supplementary data to this article:

Multimedia component 1

mmc1.docx^{(6.9MB, docx)}

Data availability

Data will be made available on request.

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PERMALINK

Gradient 3D printed PEEK with nano-coating facilitates rapid manufacturing with dual enhanced osteogenic and anti-bacterial property

Lingtong Bu

Xiang Wei

Yuxin Zhang

Qingyu Xu

Zixiang Han

Yinjun Chen

Jisi Zheng

Chi Yang

Abstract

Graphical abstract

Highlights

1. Introduction

2. Results

2.1. Mechanical performance

2.1.1. Bending test

Fig. 1.

2.1.2. Tensile test

2.1.3. Finite element analysis

2.1.4. Friction-wear test

Fig. 2.

2.2. Surface characteristics

2.2.1. Scanning electron microscope

2.2.2. X-ray diffraction

2.2.3. Energy dispersive spectroscopy

2.2.4. Water contact angle

2.2.5. Small-angle X-ray scattering

2.2.6. AFM morphology

2.2.7. Release behavior of Ag+; Ca; P; Ca/P in vitro

2.3. Adhesion and proliferation of osteoblast MC3T3-E1

2.3.1. SEM of MC3T3-E1 adhesion

Fig. 3.

2.3.2. Cell counting-8 kit-8 (CCK8) pathway

2.3.3. AMPI staining and flow cytometry

2.4. In vitro osteoinductivity

2.4.1. Real-time PCR

Fig. 4.

2.4.2. ALP staining and activity

2.4.3. Alizarin red staining

2.4.4. Immunofluorescence

2.4.5. Western blot

2.5. In vitro antibacterial activity

2.5.1. SEM morphology

Fig. 5.

2.5.2. Antibiofilm test

2.5.3. Bacterial inhibition ring

2.5.4. Bacterial dynamics curves

2.5.5. Macrophage polarization

2.5.5.1. Rt-PCR of M1/M2 polarization related genes

2.5.5.2. WB of M1/M2 polarization related proteins

2.5.5.3. FACS of M1/M2 polarization related antibodies

2.5.6. MIC/MBC vs S. aureus and E. coli

2.6. Bio-mechanism behind osteogenic and anti-bacterial properties

Fig. 6.

2.7. In vivo osteogenic and anti-bacterial property

2.7.1. In vivo osteogenesis

Fig. 7.

2.7.2. In vivo anti-bacterial property

2.7.3. Release behavior of Ag+ in rabbit femur defect model

3. Discussion

4. Materials and methods

4.1. Fused filament fabrication (FFF) of HA-(Ag-)PEEK@PEEK composite

4.1.1. The origin of PEEK, HA, and AgNPs powder

4.1.2. Ultrasonic blending

4.1.3. Extrusion of filaments

4.1.4. Gradient 3D printing

4.2. Mechanical test

4.2.1. Tensile test

4.2.2. Bending test

4.2.3. Finite element analysis

4.2.4. Friction-wear test

4.3. In vitro osteogenesis and anti-bacterial property

4.3.1. Samples preparation

4.3.2. Surface characteristics

4.3.2.1. Scanning electron microscope

4.3.2.2. Energy dispersive spectroscopy

4.3.2.3. X-Ray diffraction

4.3.2.4. Water contact angle

4.3.2.5. Small-angle X-ray scattering

4.3.2.7. ICP-MS time-related courses for Ag⁺; Ca; P; Ca/P release in vitro

4.4.3. ICP-MS time-related courses for Ag⁺ release in vivo