HIGH-PURITY RESIDENT TISSUE MACROPHAGE ISOLATES FROM HUMAN SYNOVIUM AND PERIPROSTHETIC TISSUES USING IMMUNOMAGNETIC TECHNIQUES

Destruction of periarticular and periprosthetic bone by activated macr ophages, a process often termed ''macrophage mediated osteolysis,'' is recognized as a leading mechanism of aseptic arthroplasty failure. To develop effective interventional approaches and increase the longevit y of implanted joint prostheses, the pathobiology of activated human-s ynovium-derived macrophages needs to be better characterized. The firs t step toward achieving this research objective is the acquisition of pure populations of macrophages from human synovial tissue. A simple, fast, and highly efficient method for isolating a relatively pure popu lation of macrophages from periprosthetic tissue received from either primary or secondary arthroplasty is presented. This technique uses mu rine monoclonal antibodies (IgG) that recognize a phagocyte-specific m arker, CD68, for primary binding, and sheep anti-murine IgG antibodies bound to polystyrene-coated magnetic microspheres for secondary bindi ng. While the primary antibody specifically labels CD68-positive phago cytes in the digestion of synovial and periprosthetic tissue, the seco ndary antibody bound to polystyrene-coated iron oxide beads facilitate s the removal of CD68-positive cells from CD68-negative cells by ancho ring the former with a magnet. This protocol requires centrifugation o nly in the washing steps, which reduces the frequency of cell death an d altered cell morphology. The patient population includes three prima ry and eight revision arthroplasties. The tissue macrophage isolation protocol yielded on average 4 x 10(5) cells/g tissue, of which 91% wer e viable nonspecific esterase positive macrophages. The experimental r esults suggest that immunomagnetic beads coupled to anti-CD68 enable t he isolation of a purified population of resident tissue macrophages s uitable for further biologic characterization.