Interactions between periprosthetic cells and prosthetic wear debris have been recognized as an important event in the development of osteolysis and aseptic loosening. Although the ability of wear debris to activate pro-inflammatory macrophage signaling has been documented, the full repertoire of macrophage responses to wear particles has not been established. Here, we examined the involvement of alternative macrophage activation and defective osteogenic signaling in osteolysis. Using real-time RT-PCR analysis of periprosthetic soft tissue from osteolysis patients, we detected elevated levels of expression of alternative macrophage activation markers (CHIT1, CCL18), chemokines (IL8, MIP1 a) and markers of osteoclast precursor cell differentiation and multinucleation (Cathepsin K, TRAP, DC-STAMP) relative to osteoarthritis controls. The presence of cathepsin K positive multinuclear cells was confirmed by immunohistochemistry. Reduced expression levels of the osteogenic signaling components BMP4 and FGF18 were detected. Expression levels of TNF-a, IL-6, and RANKL were unchanged, while the anti-osteoclastogenic cytokine OPG was reduced in osteolysis patients, resulting in elevated RANKL:OPG ratios. In vitro studies confirmed the role of particulate debris in alternative macrophage activation and inhibition of osteogenic signaling. Taken together, these results suggest involvement in osteolysis of alternative macrophage activation, accompanied by elevated levels of various chemokines. Increased recruitment and maturation of osteoclast precursors is also observed, as is reduced osteogenesis. These findings provide new insights into the molecular pathogenesis of osteolysis, and identify new potential candidate markers for disease progression and therapeutic targeting. ß

Titanium inhibits both interferon-gamma and interleukin-6 signaling in human osteoclast precursor cells, whereas polymethylmethacrylate bone cement inhibits only the latter. Wear particle inhibition of interleukin-6 specifically involves the activation of p38 mitogen-activated protein kinase and is accompanied by substantial induction of SOCS3, an inhibitor of interleukin-6 signaling. In contrast, titanium inhibition of interferon-gamma signaling is not dependent on mitogen-activated protein kinase activation and is accompanied by only modest induction of the interferon-gamma inhibitor SOCS1.

The ability of prosthetic wear debris to induce pro-inflammatory responses in macrophages is widely appreciated, but little is known about the molecular mechanisms involved in particle recognition. Specifically, the nature of the cell surface receptors that interact with wear debris is poorly understood. Elucidating the identities of these receptors and how they interact with different types of wear debris are critical to understanding how wear debris initiates periprosthetic osteolysis. We examined the involvement of opsonization, complement receptor 3 (CR3), and scavenger receptor A (SRA), in responses to polymethylmethacrylate (PMMA) and titanium wear particles. Serum dependence of pro-inflammatory responses to PMMA and titanium was tested, and serum proteins that adhered to these two types of particles were identified. Several serum proteins, including known opsonins such as C3bi and fibronectin, adhered to PMMA but not titanium, and serum was required for pro-inflammatory signaling induced by PMMA, but not by titanium. Phagocytosis of PMMA and titanium by macrophages was demonstrated by flow cytometry. Blocking CR3 specifically inhibited phagocytosis of PMMA by macrophages, whereas blocking SRA specifically inhibited titanium uptake. Direct involvement of CR3 and SRA in cell-particle interaction was assessed by expression of these receptors in nonphagocytic HEK293 cells. CR3 specifically induced cell binding to PMMA particles and adhesion to PMMA-coated plates, while SRA specifically induced binding to titanium particles and adhesion to titanium-coated plates. Taken together, these results suggest involvement of opsonization, complement, and integrin receptors, including CR3 and fibronectin receptors, in PMMA action, and an involvement of scavenger receptors in responses to titanium. ß

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