Regeneration of bone is an important goal in orthopaedic surgery, such as in augmentation of fracture healing, spinal fusion and filling of osseous defects. The repair of a critical skull defect is a well-established model for investigating the efficacy of cell signalling factors and biomaterials in inducing new bone formation. We aimed to investigate a 5-mm critical skull defect in the mouse, as an in vivo tool for analysis of potential bone active factors that have been bioprospected from dairy milk protein. Adult Swiss CD1 mice were divided into 2 groups. Each group contained animals treated with vehicle (n=11), milk protein (4mg, n=10) and TGF-b1 (2μg, n=6). Under anaesthetic a high-speed burr was used to create a 5-mm craniotomy in the left parietal bone and a precut collagen sponge with 20ml of the test factor inserted. Fluorochrome labels were administered to facilitate quantitative histological analysis of the defect. The animals were sacrificed on days 14 and 28 and the calvariae excised and fixed. The defects were assessed for percent closure using radiography, transillumination and histology. The formal analysis of this study is underway at present. Preliminary work in our laboratory with this milk protein has shown it to be a novel bone active factor. In vivo, local injection above the calvariae in adult mice resulted in significant increase in bone area and dynamic histomorphometric indices of bone formation. In vitro, the protein is anabolic, an effect that is consequent upon its potent proliferative and anti-apoptotic actions in osteoblasts, and its ability to inhibit osteoclastogenesis. TGF-b1 has been shown in the literature to augment the healing of critical skull defects and is included in this study as a positive control. We believe the critical skull defect in the mouse may be a useful means to assess the role of potential bone active factors in wound healing. The purified milk protein used in this study may have a physiological role in bone growth and a potential therapeutic application in bone regeneration. We await formal analysis of the specimens to further elucidate this statement. Further experiments will be required to determine whether it provides results that are reproducible and/or comparable to other models of fracture repair.
