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The role of complement activation by artificial surfaces relative to inflammatory response is not well understood. This study was performed to evaluate the inflammatory cell recruitment, distribution, and ex vivo metabolic activation of surfaces with different plasma protein adsorption and complement activation properties in vitro. The implants were (1) pure gold (reference), (2) albumin-precoated (3) IgG-precoated gold, and (4) 3-mercapto-1, 2-propanediol [mercaptoglycerol (MG)] and (5) glutathione (GSH) immobilized to gold. The implant disks were inserted subcutaneously in rats for 24 h, and the number of inflammatory cells that were recruited to the implant adjacent to the surrounding fluid phase (exudate) and the surfaces were quantified by DNA measurements. The oxidative burst was analyzed ex vivo using spontaneous and phorbol myristate acetate (PMA)-stimulated, luminol-enhanced chemiluminescence (CL). The in vitro surface-induced anti-rat C3 binding was evaluated by ellipsometry and antibody techniques after plasma incubations for 1 and 30 min. The ellipsometric results showed that immobilized mercaptoglycerol and IgG-coated, but not the immobilized glutathione or the reference Au, bound anti-C3. The in vivo results revealed that the largest amount of cells was associated with the IgG-coated surfaces, followed by immobilized GSH and MG, albumin-coated, and gold surfaces, respectively. No spontaneous ex vivo luminol-enhanced CL was recorded from the cells irrespective of surface functionality or localization. A down-regulation of surface-associated and exudate leukocyte CL was observed ex vivo, irrespective of surface functionality. The results do not indicate a clear relationship between the degree of complement activation in vitro and leukocyte recruitment and adhesion in vivo for differently functionalized surfaces.

It is believed that adsorbed blood or plasma components, such as water, peptides, carbohydrates and proteins, determine key events in the concomitant inflammatory tissue response close to implants. The aim of the present study was to develop a procedure for the collection and analysis of minor amounts of proteins bound to solid metal implant surfaces. The combination of a sodium dodecyl sulfate washing method coupled with a polyacylamide gel electrophoretic protein separation technique (SDS-PAGE), Western blot and image analysis enabled the desorption, identification and semiquantification of specific proteins. The analyzed proteins were albumin, immunoglobulin G, fibrinogen and fibronectin. Concentration procedures of proteins were not required with this method despite the small area of the test surfaces. The plasma proteins were adsorbed to pure gold and hydroxylated and methylated gold surfaces, which elicit different tissue responses in vivo and plasma protein adsorption patterns in vitro. The image analysis revealed that the pure gold surfaces adsorbed the largest amount of total and specific proteins. This is in accordance with previous ellipsometry/antibody experiments in vitro. Further, the principles described for the protein analysis can be applied on implant surfaces ex vivo.