Engineering of L-Plastin Peptide-Loaded Biodegradable Nanoparticles for Sustained Delivery and Suppression of Osteoclast Function In Vitro
Resorption assay using dentine matrix and immunoblotting analyses for resorption markers. Analysis of the effects of transduced TAT-LPL peptides (P1 and P3) and nanoparticles (NP1 and NP3) on resorption by osteoclasts using dentine slices. (a) Osteoclasts were cultured on dentine slices for 10–12h in the presence of TNF-α and indicated peptides. Osteoclasts untreated with any peptide but treated with TNF-α were used as controls (-). Pits were scanned in a Bio-Rad confocal microscopy. Scale bar- 25μm. Resorbed areas are seen as dark areas. (b) and (c) Statistic measurements of the pit area are provided as a graph: p < 0.001 versus respective controls (P3 or NP3). The resorbed pit areas (8-10 pits/slice) were quantified, and data were compiled from three dentine slices per treatment (b). The data showed in the graph is the mean ± SD of one experiment performed. Pit area measurements are also given as scatterplots for the number of pits scanned (c). Experiments were repeated three times with three different osteoclast preparations. (d) Immunoblotting analyses: equal amount of lysate proteins (15μg) made from osteoclasts untreated or treated with peptides in the presence of TNF-α and bone particles for 12-14h was used for immunoblotting analyses with TRAP (top panel) and cathepsin K (Cath K; middle panel) antibodies. Immunoblotting with a GAPDH antibody was used as a loading control (bottom panel). Experiments were repeated twice.