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The nature of the multinucleated cells involved in the resorption processes occurring inside macroporous calcium-phosphate biomaterials grafted into rabbit bone was studied using light microscopy, histomorphometric analysis, enzymatic detection of tartrate-resistant acid phosphatase (TRAP) activity, scanning, and electron microscopy. Samples were taken at days 7, 14, and 21 after implantation. As early as day 7, osteogenesis and resorption were observed at the surface of the biomaterials, inside the macropores. Resorption of both newly formed bone and calcium-phosphate biomaterials was associated with two types of multinucleated cells. Giant multinucleated cells were found only at the surface of the biomaterials; they showed a large number of nuclei, were TRAP negative, developed no ruffled border, and contained numerous vacuoles with large accumulation of mineral crystals from the biomaterials. Osteoclasts exhibited TRAP positivity and well-defined ruffled border. They were observed at the surface of both newly formed bone and biomaterials, around the implant, and inside the macropores. In contract with the biomaterials, infoldings of their ruffled border were observed between the mineral crystals, deeply inside the microporosity. The microporosity of the biomaterials (i.e., the noncrystalline spaces inside the biomaterials) increased underneath this type of cell as compared with underneath giant cells or to the depth of the biomaterials. These observations demonstrate that macroporous calcium-phosphate biomaterials implanted in bone elicit osteogenesis and the recruitment of a double multinucleated cell population having resorbing activity: giant multinucleated cells that resorb biomaterials and osteoclasts that resorb newly formed bone and biomaterials.

The surface topography of a substratum has been shown to influence the growth and morphology of cells in culture. In this study, human osteoblast-like cells (Saos-2) were cultured on two types of xenogenic biomaterials obtained from bovine bone. Both biomaterials were similar in architectural organization and surface topography, but they differed in matrix components. The first one was characterized by preservation of the mineralized collagen matrix, and the second by complete deproteinization which only preserved the mineral phase. Cells cultured at the surface of both biomaterials were observed using scanning electron microscopy. The beta 1-integrin subunit, known to bind cell and collagen, is the major integrin of the osteoblast. It was localized using immunogold in transmission electron microscopy. At the surface of the collagen-containing matrix, cells exhibited an elongated shape and oriented axis parallel to the underlying collagen bundles. The beta 1-integrin subunit was localized at the outer surface of cells, in close association with collagen and at the contact points between cells and biomaterials. In contrast, at the surface of the single mineral matrix, cells were round shaped with random disposition. Gold particles were found around the cells with no specific relation to the biomaterial. These results strongly suggest that the chemical nature of the surface of a bone biomaterial directly influences adhesion process, shape, and spatial organization of cultured osteoblastic cells.

Two hydroxyapatite ceramics, synthesized by sintering from bovine bone and from a mixture of phosphate tricalcium and natural hydroxyapatite, were implanted in bone sites in rabbits. From day 7 after implantation, osteoblast-like cells were visible with thin layers of new bone on both biomaterials. Histomorphometry showed progressive increase in volume and surface of newly formed bone. Signs of cell-dependent resorption were visible at the surface of biomaterials and newly formed bone. There was a progressive decrease in relative volume and trabecular thickness of the biomaterials. Resorption of biomaterials appears to involve two cell types: multinucleated giant cells and osteoclast-like cells. The multinucleated giant cells observed had neither tartrate resistant acid phosphatase activity (TRAP) nor a ruffled border. Vesicles and vacuoles containing crystals observed in these cells suggest phagocytosis of biomaterials. The number of these cells decreased after day 14 following implantation. The osteoclast-like cells were TRAP positive. The structured modification and the TRAP activity demonstrated in the subjacent biomaterial suggest that the dissolution of the implant may be associated to an extracellular enzymatic activity of these cells. Electron microscopy revealed a clear zone and cytoplasmic membrane infolding in these cells, suggesting a ruffled border differentiation. The number of these cells increased with delay after implantation. It was concluded that the implantation of calcium phosphate ceramics in bone leads to new bone formation as well as to resorption of the biomaterials. The mechanism of resorption appears to associate crystal endocytosis by multinucleated giant cells and more classical resorption by osteoclast-like cells.