Objective Adenosine acting at A2AR mediates the anti-inflammatory effects of methotrexate (MTX) in models of inflammation. (n=5) or ZM241385 1μM (A2AR antagonist) (n=5)) were injected to MTX-treated mice at the site daily for AM251 14 days. XenoLight Rediject Bone Probe was injected IV and fluorescence of calvaria measured (IVIS) to assay bone formation. microCT and immunostaining for osteoclast and osteoblast markers were performed. Results Implantation of put on particles induced bone pitting and thinning as exposed by microCT and MTX treatment markedly reduced osteolysis an effect abrogated by treatment with the A2AR antagonist ZM241385. Implantation of UHMWPE reduced new bone formation and MTX treatment restored fresh bone formation an effect completely reversed by treatment with ZM241385. Histological examination of particle-exposed calvarias proven that MTX prevented build up of an inflammatory infiltrate at the site of particle implantation improved the number of osteoblasts and reduced the number of osteoclasts at the site of inflammation an effect reversed by treatment with ZM241385. Summary MTX reduces inflammatory osteolysis indirectly via activation of A2AR and may represent a novel approach to enhance orthopedic implant survival delaying or removing the need for revision arthroplasty surgery. INTRODUCTION Inflammation is definitely a common cause of bone damage whether in response to put on particles at the site of a bone AM251 or joint prosthesis or as a result of inflammatory arthritis. Following joint replacement there is a predictable failure rate of hip and knee prostheses of approximately 1%/yr although younger age is an important risk element for early prosthesis failure requiring revision arthroplasty . The most common cause for prosthesis failure AM251 is definitely peri-implant osteolysis leading to loosening of the prosthesis. In recent studies we have demonstrated inside a murine model of put on particle-induced osteolysis that activation of adenosine A2A receptors (A2AR) markedly reduces inflammation and bone damage  and diminishes the number of osteoclasts present at sites of inflammatory osteolysis . Since its authorization in the late 1980s for the treatment of rheumatoid arthritis (RA)  methotrexate (MTX) remains the anchor drug for the treatment of RA. Indeed all other therapeutic agents for this disease including the newer biologic response modifiers are measured against MTX. Due to the different doses used (low doses for AM251 RA and high doses to target malignant cells) the mechanisms by which MTX suppresses swelling differ greatly from those mechanisms that target malignant diseases [4 5 MTX suppresses swelling via advertising adenosine release into the extracellular space. MTX is definitely taken up by cells where it is becomes polyglutamated. The metabolic action most potently inhibited by MTX-polyglutamate is definitely AICAR (5-aminoimidazole-4-carboxamide ribonucleotide) transformylase leading to an increase in intracellular AICAR levels [4 5 AICAR is a competitive inhibitor of AMP deaminase that results most likely in build up of adenine nucleotides extracellularly which are converted to adenosine from the combined action of nucleoside triphosphate phosphohydrolase and AWS ecto-5′Nucleotidase . Adenosine accumulates and suppresses swelling via A2AR A3R or both receptors on stimulated inflammatory cells to inhibit cytokine production and diminish swelling . Prospective studies in which ingestion of caffeine a non-selective adenosine receptor antagonist reduces the anti-inflammatory effects of MTX and the effects of oral methotrexate administration on vasodilation in individuals with RA provide support for this hypothesis . Because many if not most of MTX’s anti-inflammatory effects have been ascribed to enhanced adenosine release into the extracellular space we postulated that an agent that AM251 raises extracellular adenosine at inflamed sites such as methotrexate might reduce inflammatory osteolysis due to put on particle exposure. METHODS Methotrexate treatment MTX (Hospira Inc IL. USA) 1mg/kg was administered ip to 20 mice on a weekly basis starting 2 weeks prior to surgery and continuing until sacrifice. Control mice (n=10) were injected with.