Abstract Wear particles produced by joint replacements induce inflammatory responses that lead to periprosthetic osteolysis and aseptic loosening. However, the precise mechanisms driving wear particle-induced osteolysis are not fully understood. Recent evidence suggests that autophagy, a cellular degradation process, plays a significant role in this pathology. This study aimed to clarify the role of autophagy in mediating inflammation and osteolysis triggered by wear particles and to evaluate the therapeutic potential of zinc oxide nanoparticles (ZnO NPs). We incorporated ZnO into the prosthetic material itself, ensuring that the wear particles inherently carried ZnO, providing a targeted and sustained intervention. Our findings reveal that polymer wear particles induce excessive autophagic activity, which is closely associated with increased inflammation and osteolysis. We identified secretory autophagy as a key mechanism for IL-1β secretion, exacerbating osteolysis. Both in vitro and in vivo experiments demonstrated that ZnO-doped particles significantly inhibit autophagic overactivation, thereby reducing inflammation and osteolysis. In summary, this study establishes secretory autophagy as a critical mechanism in wear particle-induced osteolysis and highlights the potential of ZnO-doped prosthetic polymers for targeted, sustained mitigation of periprosthetic osteolysis. Keywords: Periprosthetic osteolysis, Aseptic loosening, Wear particle-induced inflammation, Secretory autophagy, Osteoclastogenesis, Macrophages Graphical abstract Image 1 [37]Open in a new tab 1. Introduction Periprosthetic osteolysis, a major cause of aseptic loosening in joint replacements, is primarily driven by the inflammatory response to wear particles generated from implant material[[38][1], [39][2], [40][3], [41][4], [42][5]]. The potential application of new polymers in artificial joints further complicates the mechanism of wear particle disease[[43][6], [44][7], [45][8]]. Polyetheretherketone (PEEK) has emerged as an alternative to conventional metallic material due to its excellent mechanical properties, biocompatibility, and radiolucency[[46][9], [47][10], [48][11], [49][12]]. Our previous clinical trial of PEEK-on-HXLPE knee arthroplasty demonstrated a satisfactory prognosis after two years, but concerns about long-term wear particle-induced osteolysis remain [[50]13]. With repetitive movements under loading, PEEK prostheses might inevitably generate wear debris in the joint microenvironment that triggers an innate immune response, leading to local inflammation and bone loss. Macrophages are key mediators in this process, responding to wear particles by secreting pro-inflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin (IL)-1, IL-6, and macrophage colony-stimulating factor (M-CSF) that promote osteoclastogenesis and bone resorption[[51][14], [52][15], [53][16]]. Multiple researches have indicated that macrophages also possess the differentiation potential of fully functional osteoclasts in response to inflammation triggered by wear particles [[54]17,[55]18]. Recent studies have also implicated autophagy, a cellular degradation and recycling mechanism, in the pathology of periprosthetic osteolysis [[56]19]. Overactivated autophagy in response to wear particles may exacerbate inflammation and osteolysis, while inhibiting autophagy has been shown to reduce these effects[[57][20], [58][21], [59][22]]. In addition to the classic role of autophagy in protein degradation, there is a growing body of evidence suggesting that the autophagy machinery promotes the unconventional secretion of leaderless proteins via a process called secretory autophagy [[60]23,[61]24]. During the disturbed homeostasis of the immune system induced by inflammation and infection, the autophagy machinery can redirect part of its cargo into exosomes for secretion, with the goal of actively providing information to neighboring cells and alerting immunity to emerging threats. Several inflammatory proteins such as IL-1β, IL-6, IL-18, and high mobility group box 1 (HMGB1) have been identified as targets of secretory autophagy. Interestingly, all of these inflammatory proteins are known to be involved in the periprosthetic tissues and stimulate osteoclastogenesis [[62]25,[63]26]. Thus, autophagy and its crucial role in the secretion of inflammatory cytokines could be a critical process in wear particle-induced osteolysis and aseptic loosening. Given the lack of approved therapies for periprosthetic osteolysis, developing biomaterials that minimize wear debris is crucial. Developing novel wear-resistant biomaterials that minimize wear debris formation may be a potential effective approach. Applying zinc oxide nanoparticles (ZnO NPs) in the composite material of prosthetic polymer to decrease wear debris shed light on the potential biological strategies to prevent aseptic loosening [[64]27,[65]28]. In our previous research, ZnO NPs (50 nm, 5 μg/mL) attenuated polymer wear particle-induced inflammation via regulation of the MEK-ERK-COX-2 axis and reduced bone tissue damage caused by wear debris-induced osteolysis [[66]29]. Several studies have indicated that ZnO nanoparticles can affect macrophages' phagocytosis and autophagy while inhibiting macrophages’ activation stimulated with lipopolysaccharide (LPS) [[67]30]. In this study, we demonstrated that autophagy was stimulated in macrophages in the presence of wear debris, and secretory autophagy was involved in the pathogenesis of wear particle-induced periprosthetic osteolysis. Moreover, we modified the composition of PEEK-on-HXLPE prostheses by doping ZnO NPs into the polymer matrix. The wear debris generated by the ZnO-modified composite materials (PEEK-ZnO and PE-ZnO) interfered with the autophagy process and attenuated the inflammation and osteolysis in vitro and in vivo. [68]Scheme 1 illustrates the general mechanism of secretory autophagy in polymer wear debris-induced periprosthetic osteolysis and the protective effect of nanoscale ZnO doping to attenuate inflammation and osteolysis. Overall, our findings suggest that ZnO-modified materials reduce autophagy-related inflammation and osteolysis, highlighting secretory autophagy as a promising therapeutic target for mitigating periprosthetic osteolysis. Scheme 1. [69]Scheme 1 [70]Open in a new tab Graphical abstract of the current study. (A)Macrophages at the implant site and those migrating from distant locations phagocytose wear particles (such as PEEK and PE), leading to the secretion of inflammatory cytokines such as IL-1β. These cytokines then promote osteoclast activation and bone resorption, contributing to periprosthetic osteolysis. (B) In macrophages, wear particles cause lysosomal damage, leading to the activation of caspase, which converts pro-IL-1β into mature IL-1β (mIL-1β). This mIL-1β is then secreted extracellularly through secretory autophagy, mediated by TRIM16 and Sec22b. ZnO-containing wear particles inhibit this secretory autophagy pathway, thereby reducing the secretion of IL-1β. 2. Materials and methods 2.1. Material preparation and characterization Materials: PEEK (VICTREX Technology Centre, UK.); XLPE (Borealis, Austria); ZnO nanoparticles (MedChemExpress, USA) Preparation of wear particles:[71]Fig. 1A illustrates the detailed fabrication process. First, pure PEEK and HXLPE materials were fabricated using a compression molding process. To prepare the PEEK/ZnO composite material. PEEK powder and ZnO nanoparticles (10 wt%) were dried in a vacuum oven at 120 °C for 12 h. Next, they were ball-milled mixed in a planetary ball mill (Emax, Germany) at 25 °C and 400 rpm for 2 h. Finally, the PEEK/ZnO composite material was fabricated by compression molding. Similarly, the PE/ZnO composite material was fabricated using the same procedure. The fabricated materials were then applied to the wear experiment using UMT-3 (Bruker Corporation, USA). After the wear experiment, the samples were cleaned with acetone to remove and collect the generated wear particles. Wear particles from different material samples were donated as PEEK, PE, PEEK-ZnO, and PE-ZnO groups. Fig. 1. [72]Fig. 1 [73]Open in a new tab Fabrication and characterization of the wear debris. (A) Schematic illustration of materials preparation. The average tensile strength (B) for the PEEK, PEEK-ZnO, PE, and PE-ZnO groups was 79.20 MPa, 77.37 MPa, 35.63 MPa, and 32.11 MPa, respectively, while the average elastic modulus (C) was 3.901 GPa, 4.127 GPa, 1.642 GPa, and 1.868 GPa, respectively. (D) The stress-strain curves of different groups. (E) The average weight content distribution of the Zinc element in the PEEK-ZnO and PE-ZnO groups was 11.87 % and 8.705 %, respectively. Each bar represents the average of three independent samples. (F) SEM images of the fabricated materials. (G) SEM images of the wear particles. (H) EDS mapping and spectrum of different elements in different wear particles. FT-IR (I) and XRD (J) patterns of the wear particles. Broad XPS spectrum of the fully scanned region (K) and the (L) Zn 2p region of the wear particles. Characterization and mechanical property test: The morphology and element composition of the composite materials and wear particles were examined using a field emission scanning electron microscopy (FE-SEM, TESCAN MIRA LMS, Czech Republic) equipped with energy-dispersive X-ray spectrometry (EDS), X-ray photoelectron spectroscopy (XPS, Thermo Scientific K-Alpha, USA), FT-IR (Thermo Scientific Nicolet iS5, USA) as well as X-ray diffraction (XRD, Rigaku Ultima IV, Japan). To assess the mechanical properties of the pure PEEK, PE, and ZnO-doped composite materials, we analyzed the stress-stain curve of all samples using a universal testing machine (Instron 5567, USA). The values of elastic modulus and tensile strength were obtained according to the results of the stress-strain curve. Particle size analysis is achieved using a Malvern laser particle size analyzer. 2.2. In vitro analysis of wear particle-induced cellular responses Cells: RAW264.7 cells were obtained from ATCC (USA). The detailed procedure of the bone marrow-derived macrophages (BMDMs) extraction is illustrated in Supporting Information. Reagents: Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were purchased from Sigma-Aldrich (USA). Cytokines, including RANKL, LPS, and M-CSF, were purchased from MedChemExpress (USA). Chloroquine, Torin1, phalloidin-FITC, and Tartrate-Resistant Acid Phosphatase (TRAP) stain kit was purchased from Sigma-Aldrich (USA). ELISA kits (IL-1β, TNF-α) were purchased from Elab Sciences (China). Reverse transcription (RT) reagents and SYBR Green were purchased from TaKaRa (Japan). Cell counting kit 8 (CCK-8) was purchased from Dojindo (Japan). Antibodies: NFATc1 (Proteintech 66,963–1), CTSK (Santa Cruz sc-48353), TRAP (Abcam ab52750); LC3B (Sigma L7543); LC3B (MBL PM036); IL-1β (Abcam ab9722); TNF-α (Abcam, ab183218); Sec22b (Abcam ab181076); β-actin (Abcam ab8226); TRIM16 (Santa Cruz sc-79770); ATG16L1 (MBL PM040); WIPI2 (Abcam ab105459); FIP200 (Abcam ab313620); Goat Anti-Rabbit IgG Cy3 (Abcam ab6939); Goat Anti-Mouse IgG FITC (Abcam ab6785); Cell viability assay: Cell viability assay was performed by CCK-8 assay. RAW264.7 cells were cultured in 96-well microplates overnight (100 μL, 5000 cells per well). Then, the culture media were replaced with 100 μL of fresh media containing PBS or wear particles with gradient concentrations. After 12 h or 24 h of treatment, the cells were rinsed with PBS thrice, and the CCK-8 working solution was added. After 1.5 h of co-incubation, the absorbance at a wavelength of 450 nm was determined with a microplate reader (BioTek, USA). Real-time PCR (RT-PCR): RNA was extracted by TRIzol reagent, followed by reverse transcription reactions employing the PrimeScript RT kit (Takara, China). The housekeeping gene β-actin expression levels were utilized to standardize the expression levels of the genes of interest. The primers for real-time PCR are available in [74]Table S1 of the Supporting Information. ELISA assay: According to the manufacturer's recommendations (Elab Sciences, China), ELISA was utilized to detect IL-1β and TNF-α levels in cell culture supernatants and tissue homogenate. Western Blot Analysis: Briefly, after treatment, total protein was extracted using RIPA lysis buffer supplemented with 0.1 % phenylmethylsulfonyl fluoride (PMSF). The lysates were then centrifuged at 15,000×g for 15 min at 4 °C, and the supernatants were carefully collected. Protein concentration was determined using a BCA protein assay kit following the manufacturer's instructions. Subsequently, proteins were denatured by heating at 100 °C for 10 min. Equal amounts of protein from each sample were loaded onto 10 % sodium dodecyl sulfate-polyacrylamide gels for separation by electrophoresis (SDS-PAGE). After electrophoresis, the proteins were transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were then blocked with 5 % skimmed milk in TBST (Tris-buffered saline with 0.1 % Tween 20) for 1 h to prevent nonspecific binding. Following the blocking step, the membranes were incubated overnight at 4 °C with primary antibodies diluted in 5 % (w/v) skimmed milk in TBST. After washing, the membranes were incubated with HRP-conjugated secondary antibodies for 1.5 h at room temperature. Protein bands were visualized using an enhanced chemiluminescence (ECL) reagent and detected with a Bio-Rad imaging system. The primary antibodies used in this study were as follows: NFATc1 (66,963–1, Proteintech, 1:3000), CTSK (sc-48353, Santa Cruz, 1:500), TRAP (ab52750, Abcam, 1:5000), β-actin (ab8226, Abcam, 1:1000), SQSTM1 (H00008878-M01, Abnova, 1:1000), and LC3 (L7543, Sigma-Aldrich, 1:1000). Transwell culture and osteoclast differentiation assay: To evaluate the interaction between wear particle-challenged macrophages and BMDMs, we designed a double-chamber transwell (0.4 μm) system ([75]Fig. 4A). F-actin and TRAP staining were performed after 7 days of co-culturing. Bone slice absorption assay was performed after 10 days of co-culturing. The detailed culture condition and procedure of osteoclast differentiation and assessment are illustrated in Supplementary Material. Fig. 4. [76]Fig. 4 [77]Open in a new tab Autophagy is triggered and mediates the secretion of IL-1β upon stimulation of wear particles. (A, B) WB and immunofluorescence results of autophagy-related proteins in RAW264.7 cells challenged with different wear particles (scale bar: 20 μm). (C) Quantification of LC3-II level in WB. (D) Quantification of LC3 immunofluorescence intensity. (E) Concentrations of IL-1β and pro-IL-1β in culture supernatants of RAW264.7 cells stimulated with wear particles for 12 h in the absence or presence of 3-methyladenine (3-MA) (5 mM). (F) Immunofluorescence staining on RAW264.7 cells and 3D reconstruction for IL-1β (red), LC3 (green), and DAPI (blue) (scale bar: 5 μm). Yellow spots indicate co-localization. (G) 3D co-localization analysis was conducted using the IMARIS coloc plugin. (H) Co-localization analysis of red channel (IL-1β) and green channel (LC3) conducted by FIJI software. (I) Colocalization line tracing analysis from images in (F). Gray arrows indicate the region of red-green overlap. (J) Pearson's colocalization coefficient for IL-1β and LC3. Sample size: n = 3 per group. All experiments were performed with n = 3 independent biological replicates. (ns indicates P > 0.05, *** indicates P < 0.001, and **** indicates P < 0.0001). (For interpretation of the references to colour