Osteopontin functions as an opsonin and facilitates phagocytosis by macrophages of hydroxyapatite-coated microspheres: Implications for bone wound healing

Osteopontin (OPN) is a secreted protein abundant in mineralized tissue extracellular matrices and bodily fluids. Previously we have shown that mineralized debris at surgical wound sites in bone and teeth are coated by macrophage-derived OPN and phagocytosed. Here, we have performed opsonophagocytosis assays to determine whether OPN acts as an opsonin and facilitates phagocytosis by macrophages of protein- and hydroxyapatite mineral-coated microspheres. Moreover, we have examined the opsonization effects of monomer OPN versus OPN polymerized (crosslinked) by tissue transglutaminase 2. Murine macrophages J774A.1 were exposed to polystyrene-latex microspheres having different surface chemistries (non-ionic, aldehyde amidine, carboxyl and aliphatic amine) which were coated with either serum albumin, immunoglobulin, monomer OPN or polymer OPN. Similar experiments with the same protein coatings were performed using hydroxyapatite-covered microspheres. Internalization of microspheres by phagocytosis into macrophages was confirmed by co-localization with the (phago)lysosomal markers lysosome-associated membrane protein-1 (Lamp-1) and LysoTracker, and by light microscopy and transmission electron microscopy after serial sectioning of plastic/resin-embedded cells containing microspheres. OPN significantly increased phagocytosis of both microspheres and hydroxyapatite-covered microspheres compared to negative controls (albumin-coated and uncoated microspheres), with phagocytic indices similar to, or greater than, those of the positive control (IgG-coated). The effect of OPN and hydroxyapatite on microsphere phagocytosis was synergistic. Polymer OPN further enhanced the phagocytosis of aliphatic amine and aldehyde amidine microspheres. Taken together, these results indicate that OPN is an effective opsonin able to facilitate particle uptake (including mineralized particles) by macrophages.