Local application in bone and controlled release of C3 proteins or C3-transporters from orthopaedic implant surfaces could be achieved by the use of biocompatible carriers such as resorbable polymers or hydrogels. Cell culture materials were from TPP. Dulbecco��s Modified Eagle��s 1142090-23-0 medium was from LGC Standards GmbH and alpha?minimal essential medium from Biochrom. Foetal calf serum and L?glutamine were from PAA Laboratories GmbH. Hoechst 33342, penicillin?streptomycin, Alexa 488-coupled goat anti-rabbit antibody and Alexa 594-coupled phalloidin were from Invitrogen. Murine recombinant receptor activator of nuclear factor?kB ligand and biotinylated NAD+ was from R&D Systems GmbH. Acid Phosphatase, Leukocyte Kit for osteoclast staining and Triton X-100 were from Sigma-Aldrich. Complete? protease inhibitor and streptavidin-peroxidase were from Roche. Page Ruler pre-stained Protein ladder? was from Fermentas. Anti-C2IN and anti-C3bot1E174Q antisera were raised in rabbits from Pineda. Enhanced chemiluminescence system was obtained from Millipore. Alexa-488 coupled antibody and phalloidin-Alexa 594 coupled conjugate were purchased from Invitrogen. The recombinant proteins C3lim, C3bot1, C3bot1E174Q, C2INC3lim, C2I and C2IIa were expressed as GST-tagged proteins in E.coli BL21 and purified by affinity chromatography as described previously. To assess the influence of C3-treatment on osteoclastformation, C3 was administered to RAW 264.7 cells during their differentiation to osteoclasts from day on only to analyze timedependent effects more closely. After 5 days of culture in 96 well plates, differentiated osteoclasts were stained with the Acid Phosphatase, Leukocyte Kit according to the manufacturer��s instructions to verify the presence of tartrateresistant acid phosphatase, an enzyme specific for the monocyte/macrophage lineage. Briefly, incubation with a TRAP substrate resulted in a red staining. TRAP-positive cells with at least three nuclei were counted as osteoclasts at magnification in order to quantitatively determine the BI 2536 biological activity formation of multi?nucleated cells. Images of the stained cells were taken with a Leica DMI6000 B microscope and a DFC420 C camera.z. According to the World Health Organization, cutaneous and visceral
