24 In brief, 96-well microtitre plates learn more were coated with fixed F. nucleatum (optical density 580 nm = 0·3) and blocked with 1% bovine serum albumin. Sera from infected mice collected on killing were serially diluted in PBS as indicated and 100 μl was added to each well. After incubation and washing, specific immunoglobulin G (IgG) subclasses were

detected with biotinylated rabbit anti-mouse IgG1 or IgG2a (BD Biosciences PharMingen, San Diego, CA). Wells were then incubated with streptavidin-conjugated horseradish peroxidase (Invitrogen), after which substrate and chromogen were added, and absorbance was read on an enzyme-linked immunsorbent assay (ELISA) plate reader (Dynatech, Chantilly, VT). Significance of differences was calculated by two–way analysis of variance with Bonferroni post-test (bone loss determinations), or by two-tailed t-test. Graph-Pad Prism (Graph Pad Software, LaJolla, CA) software was used for statistical calculations. Wild-type and OPN-deficient mice (both males and females at 5–12 weeks of age) on a 129 (S1, S7) mixed background were subjected to dental pulp exposure, and infected with a mixture of four human endodontic pathogens including P. intermedia, Cell Cycle inhibitor S. intermedius, F. nucleatum and P. micros. Three weeks after infection, mice were killed, and the infected mandibles were removed, fixed and analysed by microCT as described.7Figure 1

shows that bone loss associated with these endodontic infections was significantly higher in OPN−/− mice than in WT animals. The area of radiolucency in unexposed mice was minimal (average 0·07 mm2); it was not different between WT and OPN−/− mice – this radiolucent area represents the normal periodontal ligament that anchors teeth to the underlying bony structure. Following pulp exposure and infection, the area of bone loss averaged 0·18 mm2 Florfenicol in WT mice, but was 55% higher in OPN−/− animals (0·28 mm2, Fig. 1b). When corrected for the radiolucent area observed in unexposed areas, the extent of bone loss in OPN−/− mice was more than twice that seen in WT mice. This result was confirmed

in an independent experiment (data not shown). Bone loss was also estimated in histological sections as described in Materials and methods. These measurements confirmed the bone loss observed by microCT 21 days after infection, and the significantly increased bone loss occurring in the OPN-deficient mice (Fig. 1c). At 3 days after infection, there was a significant amount of bone loss adjacent to the infected pulp chamber, with many osteoclasts apparent (data not shown). However, the extent of bone loss at this time-point was not different between WT and OPN-deficient animals. The bone loss in infected animals was secondary to the inflammatory infiltration occurring in response to bacterial infection. This inflammatory response was quantified in haematoxylin & eosin-stained decalcified sections of infected mandibles at 21 days after infection (Fig. 2).