Aseptic loosening of orthopaedic implants is induced by wear particles generated from the polymeric and metallic components of the implants. Substantial evidence suggests that activation of Toll-like receptors (TLRs) may contribute to the biological activity of the wear particles. Although pathogen-associated molecular patterns (PAMPs) produced by Gram-positive bacteria are likely to be more common in patients with aseptic loosening, prior studies have focused on LPS, a TLR4-specific PAMP produced by Gram-negative bacteria. Here we show that both TLR2 and TLR4 contribute to the biological activity of titanium particles with adherent bacterial debris. In addition, lipoteichoic acid, a PAMP produced by Gram-positive bacteria that activates TLR2, can, like LPS, adhere to the particles and increase their biological activity, and the increased biological activity requires the presence of the cognate TLR. Moreover, three lines of evidence support the conclusion that TLR activation requires bacterially derived PAMPs and that endogenously produced alarmins are not sufficient. First, neither TLR2 nor TLR4 contribute to the activity of "endotoxin-free" particles as would be expected if alarmins are sufficient to activate the TLRs. Second, noncognate TLRs do not contribute to the activity of particles with adherent LPS or lipoteichoic acid as would be expected if alarmins are sufficient to activate the TLRs. Third, polymyxin B, which inactivates LPS, blocks the activity of particles with adherent LPS. These results support the hypothesis that PAMPs produced by low levels of bacterial colonization may contribute to aseptic loosening of orthopaedic implants.Osteoimmunology is a newly emerging field that refers to bi-directional interactions between the skeletal system and the immune system (1). One example of regulation of the skeletal system by the immune system is inflammatory osteolysis, which is the local bone loss induced by inflammation in response to pathogens or nonpathogenic stimuli (1). Inflammatory osteolysis in response to either pathogens or nonpathogenic stimuli can contribute to loosening of orthopaedic implants and the resultant need to replace the implant. For example, implant infection is a devastating complication that often necessitates implant removal and long term intravenous treatment with antibiotics (2). In contrast, "aseptic loosening" results from inflammation induced by polymeric and metallic wear particles derived from the implant components in the absence of any clinical signs of infection (3, 4). The primary events involved in this process include: phagocytosis of the wear particles by macrophages; activation of the macrophages to produce pro-inflammatory cytokines; stimulation of bone resorptive cytokines, such as RANKL, in response to the proinflammatory cytokines; stimulation of osteoclast differentiation by the resorptive cytokines; and local osteolysis caused by the increased number of osteoclasts (3-5).Subclinical levels of bacteria may contribute to wear particleinduced inflammation, and...