Comparative effects of five bisphosphonates on apoptosis of macrophage cells in vitro M.F. Moreau a , C. Guillet b , P. Massin a,c , S. Chevalier b,d , H. Gascan d , M.F. Basle ´ a , D. Chappard a, * a INSERM, EMI 0335, LHEA, Faculte ´ de Me ´decine, 49045 Angers Ce ´dex, France b Service Commun de Cytome ´trie et d’Analyse Nucle ´otidique (SCCAN), Ba ˆ timent Monte ´clair, 4, rue Larrey, 49033 Angers Ce ´dex, France c De ´partement de Chirurgie Orthope ´dique, CHU d’Angers, 49033 Angers Ce ´dex, France d INSERM, U 564, Ba ˆ timent Monte ´clair, 4, rue Larrey, 49033 Angers Ce ´ dex, France 1. Introduction Migration of polyethylene wear debris is recognized to induce periprosthetic bone loss after total hip arthroplasty. Particles are generated in the joint by daily joint motion that provoke erosion of the polymer and they can migrate far from the area where they are generated [1]. Particles are then phagocytosed by macrophages that release large amount of inflammatory cytokines such as TNFa, IL-6 or IL-1 [2]. These cytokines induce a local fibrotic process associated with osteoclastogenesis biochemical pharmacology 73 (2007) 718–723 article info Article history: Received 27 July 2006 Accepted 21 September 2006 Keywords: Bisphosphonates Apoptosis Necrosis Macrophage Flow cytometry abstract Bisphosphonates (BPs) inhibits bone resorption by reducing osteoclastic activity; they induce osteoclast apoptosis. Pathophysiology of prostheses loosening is complex and implies an inflammatory reaction secondary to the phagocytosis of wear debris by macro- phages with a secondary increased bone resorption by osteoclasts. BPs inhibit proliferation and cause cell death in macrophages by induction of apoptosis. We have used mouse macrophage-like J774.1 cells to evaluate the effects of five BPs. J774A.1 cells were cultured in a standard culture medium for 2-days. BPs (alendronate, pamidronate, etidronate, risedronate, zoledronic acid) were added in the medium at con- centration of 10 À6 to 10 À4 M during 3 days. Cells were studied by fluorescence microscopy after staining with the fluorescent dye Hoescht H33342 and the percentage of apoptotic cells was determined on 300 nuclei. Cells were analyzed by flow cytofluorometry after staining with annexin V-FITC (for counting apoptotic cells) and propidium iodide (for necrotic/late- apoptotic cells) on 2000 cells. Etidronate did not cause significant apoptosis or necrosis, at any concentration. Alen- dronate and pamidronate caused apoptosis and death only at very high concentration [10 À4 M]. On the contrary, apoptotic and necrotic cells were evidenced with risedronate or zoledronic acid at lower concentrations. These effects were dose-dependant and occurred when concentration reached [10 À5 M]. The number of apoptotic cells was higher with zoledronic acid and then with risedronate. Cytofluorometry appeared superior to cytologic analysis in the investigation of macrophage apoptosis, since necrotic cells loose contact with the glass slides and are not identifiable in cytological counts. Some amino-BPs appear to induce apoptosis in macrophages. # 2006 Elsevier Inc. All rights reserved. * Corresponding author. Tel.: +33 241 73 58 64; fax: +33 241 73 58 88. E-mail address: daniel.chappard@univ-angers.fr (D. Chappard). available at www.sciencedirect.com journal homepage: www.elsevier.com/locate/biochempharm 0006-2952/$ – see front matter # 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bcp.2006.09.031