The purpose of this study is to determine the safety and efficacy of local administration of lentivirus-mediated small interfering RNA (siRNA) targeting tumor necrosis factor-a (TNF-a) in a modified murine air pouch model of wear debris-induced osteolysis, which causes aseptic loosening of prosthesis. Air pouches were established on the back of BALB/c mice, followed by the surgical introduction of a section of calvaria from a syngeneic mouse donor, and all pouches were stimulated by ultrahigh molecular weight polyethylene (UHMWPE) particles. Pouches were divided into 3 groups randomly, and lentivirus-mediated siRNA targeting TNF-a, lentivirus-mediated missense (MS) siRNA and saline were injected into pouches respectively. The lentiviral vector was designed to express the enhanced green fluorescent protein (EGFP) as a marker gene. Pouch membranes and lavage fluid were harvested at 7 and 14 days post transfection, and assayed for markers of osteolysis using histological, molecular, immunological techniques and Xenogen IVIS 50 vivo bioluminescent assay system. Xenogen IVIS 50 vivo image revealed strong expression of EGFP localized in pouch areas and no expression in other parts of mice both in TNF-a siRNA-treated and MS siRNA-treated pouches for up to 2 weeks, indicating stable transfection of lentivirus-mediated siRNA, while no expression of EGFP was found in untreated pouches. Realtime-PCR and ELISA revealed a significant reduction of TNF-a both at the mRNA and protein level in TNF-a siRNA-treated pouch membranes and lavage fluid compared to MS siRNA-treated and untreated pouches for up to 2 weeks, whereas TNF-a level in peripheral blood, liver, spleen, kidney, lung and brain remained invariant. The gene therapy also resulted in prominent diminution of IL-6, Cathepsin K, RANK and RANKL expression. Less inflammatory responses (thinner pouch membrane and decreased cellular infiltration) and less bone collagen loss were observed in TNF-a siRNA-treated pouches. Tartrate-resistant acid phosphatase (TRAP) staining showed prominently less osteoclastogenesis in TNF-a siRNA-treated pouches. Efficient local delivery of lentivirus-mediated siRNA targeting TNF-a into modified murine air pouch inhibits debris-induced osteolysis with no systemic adverse effects, and might be an excellent therapeutic candidate to prevent or retard the osteolysis response to wear debris that contributes to the pathology of aseptic loosening.