To understand fully cellular mechanisms during bone tissue repair and engineering, there is a need to develop reproducible three-dimensional organotypic culture models, whereby cells in their natural extracellular matrix can be manipulated. Limitations in current model systems do not allow for this integrated approach. This study aimed to develop and validate an ex vivo fractured rat mandible model, to investigate specific molecular and cellular processes involved in bone repair. Slices of mandible from 28-day-old male Wistar rats were cultured in Trowel-type cultures at the liquid-gas interface for up to 21 days. Maintenance of cell and tissue architecture and viability was shown within fractured mandible slices during all culture periods. Autoradiographic studies demonstrated that resident cells were actively synthesizing and secreting proteins, and cells of the osteoblast lineage were shown to survive throughout the culture periods. The model was responsive to exogenously added transforming growth factor-β1, with observed increases in cellular migration/proliferation and expression of bone matrix proteins. The ex vivo mandible model developed within this study may represent an ideal system for investigating specific processes of bone repair, as well as a promising alternative to in vivo testing of novel clinical therapeutics.