Introduction and Aims: Regeneration of bone is an important goal in orthopaedic surgery. The repair of a critical skull defect is a model for investigating the efficacy of cell signalling factors and biomaterials in inducing new bone formation. We aim to investigate a 5mm critical skull defect in the mouse, as an in vivo tool for analysis of potential bone active factors that have been bio-prospected from dairy milk protein.

Method: Adult Swiss CD1 mice were divided into two groups. Each group contained animals treated with vehicle (n=11), milk protein (4mg, n=10) and TGF-β₁ (2μg, n=6). Under anaesthetic, a high-speed burr was used to create a five-mm craniotomy in the left parietal bone and a pre-cut collagen sponge with 20μl 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.

Results: The formal analysis of this study is underway at present. TGF-β1 has been shown in the literature to augment the healing of critical skull defects and is included in this study as a positive control. Our radiography results show significantly complete closure of the skull defect in TGF-β₁ group.

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.

Conclusions: 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 of osseous defects. The purified milk protein tested 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.

Paget’s disease of bone is a common disorder characterised by focal areas of increased bone resorption coupled to increased and disorganised bone formation. Pagetic osteoclasts have been studied extensively, however, due to the integral cross-talk between osteoclasts and osteoblasts, we propose that pagetic osteoblasts may also play a key role in the pathogenesis of Paget’s disease. Any phenotypic changes in the diseased osteoblasts are likely to result from alterations in the expression levels of specific genes. To determine any differences in expression between pagetic and non-pagetic osteoblasts and their precursors the gene expression profiles of RANK, RANKL, OPG, VEGF, IL-1beta, IL-6, MIP-1, TNF and M-CSF were investigated in primary cultures of human osteoblasts and in the osteoblast precursor population of bone marrow stromal cells. We present preliminary data of this study.

Trabecular bone explants were finely chopped, washed free of marrow and cellular debris then either snap frozen in liquid nitrogen or placed in flasks to culture outgrowth osteoblast-like cells. Mononuclear stromal cells from bone marrow were isolated and grown in culture flasks. RNA and conditioned media were collected from cultured osteoblasts and stromal cells at confluency. The innovative method of Real-Time PCR, the most accurate technique available at present to quantitatively measure gene expression, was used for the comparison of gene expression levels in our samples. 18S ribosomal RNA was used as an endogenous control to normalise the expression in the various samples.

RANK, MIP-1 and TNF were only detected in stromal cells whereas RANKL, OPG, VEGF, IL-1beta, IL-6 and M-CSF were detected in both osteoblasts and stromal cells. OPG displayed higher expression in osteoblasts while IL-1beta showed higher expression in stromal cells.

To date we have not seen any significant differences in gene expression between pagetic and non-pagetic subjects when comparing a small number of samples. A larger cohort is currently being investigated. We are also comparing levels of secreted proteins in the conditioned media from pagetic and non-pagetic cell cultures. This may lead to further candidate genes involved in the pathology of the pagetic lesion.

Introduction Paget’s disease of bone is a common disorder characterised by focal areas of increased bone resorption by osteoclasts and disorganised bone formation by osteoblasts. Because there is integral cross-talk between osteoclasts and osteoblasts during normal bone remodelling, we propose that Pagetic osteoblasts may also play a key role in the pathogenesis of Paget’s disease. Any phenotypic changes in the diseased osteoblasts are likely to result from alterations in the expression levels of specific genes.

Methods To determine any differences in expression between Pagetic and non-Pagetic osteoblasts and their precursors the gene expression profiles of RANK, RANKL, OPG, VEGF, IL-1beta, IL-6, MIP-1, TNF and M-CSF were investigated in primary cell cultures of human osteoblasts and in the osteoblast precursor population of bone marrow stromal cells. Trabecular bone explants were finely chopped, washed free of marrow and cellular debris then either snap frozen in liquid nitrogen or placed in flasks to culture outgrowth osteoblast-like cells. Mononuclear stromal cells from bone marrow were isolated and grown in culture flasks. RNA and conditioned media were collected from cultured osteoblasts and stromal cells at confluency. Real-Time PCR was used for the comparison of gene expression. 18S ribosomal RNA was used as an endogenous control to normalise the expression in the various samples.

Results RANK, MIP-1 and TNF were only detected in stromal cells whereas RANKL, OPG, VEGF, IL-1beta, IL-6 and M-CSF were detected in both osteoblasts and stromal cells. OPG displayed higher expression in osteoblasts while IL-1beta showed higher expression in stromal cells. To-date we have not seen any significant differences in gene expression between pagetic and non-pagetic subjects when comparing a small number of samples. A larger cohort is currently being investigated. Mutations in the sequestosome 1 gene have been showed to be associated with Paget’s disease. When a small number of Pagetic samples were sequenced for these mutations we found one out of seven patients (14%) to possess a known transition mutation at position 1215 in this gene.

Conclusions These results may further our understanding of the pathology of Paget’s disease.