An Ex-Vivo Murine Mandible Culture Model for Inflammatory Bone Destruction

Inflammatory bone destruction is central to the pathogenesis of diseases such as periodontitis involving complex osteoclastic resorption and loss of bone matrix. Currently experimental modelling of such processes is hampered by a lack of suitable in vitro models. Objectives: To develop a reproducible ex vivo murine mandible slice culture model to examine osteoclast behaviour and the influence of inflammation on bone metabolism during periodontal disease. Methods: Hemi-mandibles dissected from 10 – 12 week old male CD1 mice were sliced into 1mm transverse sections using a diamond-edged bone saw and maintained in Trowel-type or submerged cultures at 37^oC in 5% CO₂ in air for up to 14 days. Tissue viability and maintenance of cell/tissue morphology was assessed by acridine orange/ethidium bromide staining, histology, and periodontal ligament (PDL) cell nuclei quantification. Resident osteoclasts were identified and counted via TRAP labelling, and markers of proliferation, bone matrix proteins and osteoblasts (alkaline phosphatase, BSP, Collagen Type I, Osteocalcin, Osteopontin, PCNA) were detected by immunohistohemistry. Results: Viability was confirmed throughout the culture period and cell/tissue architecture preserved under all culture conditions. PDL cell counts indicated that numbers of cells reduced significantly (p<0.01) at day 14 in submerged cultures. However, in Trowel cultures, an initial significant (p<0.05) reduction of cells was observed at day 7 but by day 14 cell number was similar to initial levels. Cellular proliferation and expression of bone matrix proteins were also maintained in all cultures without significant alteration (p>0.05). Osteoclast and osteoblast cells were sustained throughout culture with no significant reduction in cell number (p>0.05). Conclusion: This method provides suitable conditions for the culture of murine mandible slices and is a viable model for the study of bone pathophysiology. Further work will refine the model to investigate effects of inflammation and bone destruction. (SYT Supported by an NC3Rs research grant No.77844)