Feasibility of a rat femoral-defect model for immediate perioperative and delayed post-implantation implant-associated bone infections

Abstract. Background: Implant-associated bone infections (IAIs) represent a major clinical challenge, causing implant failure, prolonged morbidity, and costly revision surgeries. Infection risk is highest in the immediate perioperative period, but delayed post-implantation contamination, occurring weeks after surgery in a partially healed host, represents an equally important and underappreciated clinical scenario. Bacteria rapidly colonize implant surfaces and form biofilms that resist both antibiotics and host immune responses, making prevention critical. Well-characterized small-animal models that capture both early and delayed post-implantation infections are therefore essential to develop and test antibacterial implants and coatings. Methods: We developed a rat femoral-segmental-defect model stabilized with a polyether-ether-ketone plate and a 3D-printed porous titanium implant. Three studies were performed using a total of 28 animals (3 excluded due to early humane endpoints; n=25 in final analysis): (i) early inoculation during surgery with planktonic Staphylococcus aureus (ATCC 49230) at 104 or 106 CFU (colony-forming units) per rat, (ii) delayed inoculation 28 d after surgery with planktonic S. aureus at 104 or 106 CFU per rat, and (iii) delayed inoculation with 108 CFU per rat delivered as planktonic or ruptured-biofilm inoculum. Controls received phosphate-buffered saline at implantation. The primary endpoint was infection at day 14, quantified by CFU enumeration from homogenized bone and from a sonicated implant, plate, and screws. Micro-computed tomography (micro-CT) was used to visualize fixation and hardware position. Results: Early inoculation with 104 or 106 CFU per rat produced consistent infections across bone, implants, and screws. The same doses given 28 d later yielded low and inconsistent colonization. Escalation to 108 CFU per rat in the delayed setting produced consistent infections; the ruptured-biofilm inoculum generated higher implant-associated bacterial burdens than planktonic suspensions, while bone burdens were consistently high in both arms. Conclusions: This femoral-implant rat model demonstrates feasibility for studying both immediate perioperative and delayed post-implantation infections, providing a platform for preclinical evaluation of anti-infection strategies. Timing, inoculum magnitude, and bacterial state critically determine infection establishment.