Aims. Aseptic loosening is a leading cause of uncemented arthroplasty failure, often accompanied by fibrotic tissue at the bone-implant interface. A biological target, neutrophil extracellular traps (NETs), was investigated as a crucial connection between the innate immune system’s response to injury, fibrotic tissue development, and proper bone healing. Prevalence of NETs in peri-implant fibrotic tissue from aseptic loosening patients was assessed. A murine model of osseointegration failure was used to test the hypothesis that inhibition (through Pad4-/- mice that display defects in peptidyl arginine deiminase 4 (PAD4), an essential protein required for NETs) or resolution (via DNase 1 treatment, an enzyme that degrades the cytotoxic DNA matrix) of NETs can prevent osseointegration failure and formation of peri-implant fibrotic tissue. Methods. Patient peri-implant fibrotic tissue was analyzed for NETs biomarkers. To enhance osseointegration in loose implant conditions, an innate immune system pathway (NETs) was either inhibited (Pad4-/- mice) or resolved with a pharmacological agent (DNase 1) in a murine model of osseointegration failure. Results. NETs biomarkers were identified in peri-implant fibrotic tissue collected from aseptic loosening patients and at the bone-implant interface in a murine model of osseointegration failure. Inhibition (Pad4-/-) or resolution (DNase 1) of NETs improved osseointegration and reduced fibrotic tissue despite loose implant conditions in mice. Conclusion. This study identifies a biological target (NETs) for potential noninvasive treatments of aseptic loosening by discovering a novel connection between the innate immune system and post-injury bone remodelling caused by implant loosening. By inhibiting or resolving NETs in an osseointegration failure murine model, fibrotic tissue encapsulation around an implant is reduced and osseointegration is enhanced, despite loose implant conditions. Cite this article: Bone Joint J 2021;103-B(7 Supple B):135–144

Introduction. Patients with aseptic loosening, a cause of failure in uncemented total joint arthroplasty (TJA), often present with fibrous tissue at the bone-implant interface. 1. In this study, we characterize the presence of neutrophil extracellular traps (NETs) in the intramedullary fibrotic membrane of aseptic loosening patients. We further explore the role of NETs, mediated by peptidyl arginine deiminase (PAD4), in peri-implant fibrosis and osseointegration failure through a murine model of unstable tibial implantation. 2–4. Methods. Peri-implant membrane was retrieved from five patients during total hip revision surgery and analyzed for the presence of NETs (citH3+ with extracellular DNA) via immunofluorescence. A Ti-6Al-4V implant was inserted in an oversized drill-hole in the right proximal tibia of 8-week-old C57BL/6J and PAD4 knockout mice (n=3 per group). Fourteen days later, all mice were euthanized, and implanted tibias were dissected. Fibrosis and osseointegration at the bone-implant interface were assessed by micro-computed tomography (microCT) and hematoxylin and eosin (H&E) staining. H&E samples were scored blindly by the investigator and another observer for signs of poor (score=0) to excellent osseointegration (score=3) using a rubric established in our lab. Results. NETs were found in peri-implant membrane collected from aseptic loosening patients (Figure 1a) and at the bone-implant interface in a murine model (Figure 1b). Unstable implants in wild type mice failed to osseointegrate, indicated by presence of fibroblast-like cells (dashed arrow), immature bone matrix (Figure 1c), low bone volume fraction (BV/TV) and bone surface area (BS) (Figure 1e). Unstable implants in PAD4. −/−. mice showed signs of good osseointegration such as mature trabeculae (solid arrow) (Figure 1d), higher BV/TV (p<0.10) and BS (p<0.05) (Figure 1f). Histological osseointegration scoring indicated wildtype mice exhibited an average score of 0.83 and PAD4. −/−. exhibited an average score of 2.5 (p<0.05, weighted Cohen's kappa = 0.714) (Figure 1g). Conclusion. NETs were characterized in fibrotic tissue in both aseptic loosening patients and in a murine model of unstable tibial implantation. NET inhibition was able to successfully prevent peri-implant fibrosis and osseointegration failure, leading the way for a potential novel non-invasive therapeutic approach for the treatment of aseptic loosening. For any figures, tables, or references, please contact the authors directly