Flow cytometric analysis of cell death and viability of osteoarthritic chondrocytes cultured in presence of apoptosis inhibitors for tissue engineering

Authors: Luminita TAKÁCS-BUIA1, Daniela BRATOSIN1*, Ana-Maria GHEORGHE1, Catalin IORDĂCHEL1, Nicolae EFIMOV2, Maria CALOIANU3, Jean MONTREUIL4

Affiliation: 1 National Institute of Biological Science Research and Development (INSB), Romania, 2 Hospital CFR 2, Bucharest, Romania, 3 University of Bucharest, Faculty of Biology, Romania, 4 Université de Lille 1, Laboratoire de Chimie Biologique, Villeneuve d’Ascq, France

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ABSTRACT. In autologous cell implantation (ACI), the autologous chondrocytes recovered from the patient are amplified in tissue culture prior to re-implantation. Recently, we demonstrated that a dual mechanism, apoptosis and replicative senescence, could be responsible for pathogenesis of osteoarthritis diseases. In some systems, inhibition of caspases can prevent apoptosis and may therefore have important therapeutic implications. To confirm this view, chondrocyte apoptosis was induced in vitro with Ca2+, a potential inducer of apoptosis in vivo, either in the presence or absence of the caspase inhibitor, Ac-DEVD-fmk or of leupeptin specific inhibitor of calpains. Cell viability and death were analyzed by flow cytometry. Our results provide that the progression of cartilage lesions is related to an apoptosis phenomenon and suggest the potential efficacy of caspase inhibitors. DEVD-cmk, however, was less efficient to prevent cell death as compared to leupeptin and the mixture of both had complementary effect and prevented all hallmark of apoptosis

Keywords: osteoarthritis, chondrocytes, apoptosis, flow-cytometry, autologous cell implantation, tissue engineering