Stimulation of bone resorption in an ex vivo mandible model

Central to the pathogenesis of periodontal disease is the recruitment and subsequent differentiation of osteoclasts leading to bone resorption. Osteoclasts are large multinuclear cells which are Tartrate Resistant Acid Phosphatse (TRAP) positive. Their differentiation may be mediated by inflammatory cytokines and pro-inflammatory mediators including bacterial lipopolysaccharide (LPS). Objectives: To investigate the effects of LPS stimulation on osteoclastogenesis, cellular proliferation and matrix resorption in an ex vivo murine mandible model. Methods: Trowel-type cultures of 1mm murine mandible slices from 10-12 week old male CD1 mice were maintained for 0 to 14 days at 37^oC, 5% CO₂ in air in either the presence or absence of 100ng/ml LPS. Following culture, slices were fixed and processed for histological examination and slice viability was confirmed using counts of periodontal ligament cell nuclei in representative sections. Osteoclasts were identified and counted via TRAP labelling. Resident osteoclastic response to stimulation was measured by quantifying the number of TRAP positive multinuclear cells following LPS treatment. Immunohistochemical examination for markers of cell proliferation (PCNA) and bone matrix markers (BSP, osteopontin) were performed in control and LPS treated cultures. Results: The number of periodontal ligament cell nuclei was significantly increased (p<0.05) following 7 days of LPS stimulation, while longer culture periods resulted in reduced cell numbers. No increase in cellular proliferation was discovered via PCNA immunopositivity (p>0.05) in any culture period. The number of resident TRAP positive multinuclear osteoclasts were consistently significantly increased (p<0.05) as a result of LPS treatment, while immunolabelling for BSP was significantly (p<0.05) reduced, suggesting increased bone resorption. Conclusion: LPS appears to induce pathophysiological bone metabolism in this model and may directly influence resident mandible cells and osteoclast differentiation. This model may be suitable for investigation of inflammatory bone destruction during periodontitis. (Supported by an NC3Rs research grant No. 77844)