An Ex Vivo Culture System for Bone Fracture Repair

In order to advance current clinical treatments of bone tissue injury, the factors that are involved in regulating bone regeneration need to be further investigated. A lack of in vitro models and the limitations that are associated with in vivo modelling have hampered experimental investigation of bone regeneration. The development of an ex vivo organ culture system that can be used to investigate the influence of specific factors during bone regeneration would offer significant advantages. Objectives: To develop and characterise an ex vivo culture model of fractured slices of the rat mandible to study bone regeneration. Methods: Mandibles were dissected from 28 day old male Wistar rats, surrounding soft-tissue removed and 2mm transverse slices cut on a diamond-edged bone saw. Slices were fractured with a sterile scalpel blade prior to culture in DMEM containing 200mM glutamine, 10% heat inactivated FCS, 0.15mg/ml ascorbic acid and 1% antibiotics. Fractured mandibles were cultured for 7 and 14 days at 37^oC in 5% CO₂ in air. Cell and tissue viability around the fracture site was assessed with acridine orange/ethidium bromide vital dye staining and histology/histomorphometry. Expression of PCNA, to assess cell proliferation during culture, and osteopontin, to assess cellular behaviour adjacent to the fracture site during culture was examined by immunhistochemistry. Results: Fractured mandible slices exhibited good cell and tissue viability adjacent to the site of fracture at 7 and 14 days, with maintenance of cell phenotype and tissue architecture. Positive staining for PCNA and osteopontin was observed in cells of the periodontal ligament and cells of the bone marrow. Osteopontin staining was localised to cells adjacent to the site of fracture. Conclusions: This method provides suitable conditions for the culture of fractured rat mandible slices and may be a valuable tool in the study of bone regeneration. (Welsh Assembly Govt. CMO Research grant)