Mice deleted for cell division cycle 73 gene develop parathyroid and uterine tumours: model for the hyperparathyroidism-jaw tumour syndrome.

The hyperparathyroidism-jaw tumour (HPT-JT) syndrome is an autosomal dominant disorder characterized by occurrence of parathyroid tumours, often atypical adenomas and carcinomas, ossifying jaw fibromas, renal tumours and uterine benign and malignant neoplasms. HPT-JT is caused by mutations of the cell division cycle 73 (CDC73) gene, located on chromosome 1q31.2 and encodes a 531 amino acid protein, parafibromin. To facilitate in vivo studies of Cdc73 in tumourigenesis we generated conventional (Cdc73+/-) and conditional parathyroid-specific (Cdc73+/L/PTH-Cre and Cdc73L/L/PTH-Cre) mouse models. Mice were aged to 18-21 months and studied for survival, tumour development and proliferation, and serum biochemistry, and compared to age-matched wild-type (Cdc73+/+ and Cdc73+/+/PTH-Cre) littermates. Survival of Cdc73+/- mice, when compared to Cdc73+/+ mice was reduced (Cdc73+/-=80%; Cdc73+/+=90% at 18 months of age, P<0.05). Cdc73+/-, Cdc73+/L/PTH-Cre and Cdc73L/L/PTH-Cre mice developed parathyroid tumours, which had nuclear pleomorphism, fibrous septation and increased galectin-3 expression, consistent with atypical parathyroid adenomas, from 9 months of age. Parathyroid tumours in Cdc73+/-, Cdc73+/L/PTH-Cre and Cdc73L/L/PTH-Cre mice had significantly increased proliferation, with rates >fourfold higher than that in parathyroid glands of wild-type littermates (P<0.0001). Cdc73+/-, Cdc73+/L/PTH-Cre and Cdc73L/L/PTH-Cre mice had higher mean serum calcium concentrations than wild-type littermates, and Cdc73+/- mice also had increased mean serum parathyroid hormone (PTH) concentrations. Parathyroid tumour development, and elevations in serum calcium and PTH, were similar in males and females. Cdc73+/- mice did not develop bone or renal tumours but female Cdc73+/- mice, at 18 months of age, had uterine neoplasms comprising squamous metaplasia, adenofibroma and adenomyoma. Uterine neoplasms, myometria and jaw bones of Cdc73+/- mice had increased proliferation rates that were 2-fold higher than in Cdc73+/+ mice (P<0.05). Thus, our studies, which have established mouse models for parathyroid tumours and uterine neoplasms that develop in the HPT-JT syndrome, provide in vivo models for future studies of these tumours.

Distraction osteogenesis in the Cbfa-1+/- mouse.

Distraction osteogenesis involves division of a bone and gradually pulling the bone ends apart. This delivers mechanical stimulation to mesenchymal cells in the distraction gap, where new bone is regenerated predominantly by intramembranous ossification. The transcription factor Cbfa1 has been reported to be essential for the differentiation of mesenchymal cells to osteoblasts. In homozygous Cbfa1 knockout mice, both intramembranous and endochondral ossification mechanisms are blocked and no bone formation occurs. In heterozygous Cbfa1 knockout mice, only the cranial bones and the clavicles, which form through intramembranous ossification, fail to develop properly as in the human condition of cleidocranialdysostosis. It has been suggested, therefore, that intramembranous ossification is affected by the absence of one of the paired Cbfa1 genes. We have assessed the potential for intramembranous ossification following distraction osteogenesis in heterozygous Cbfa1 knockout mice. Fourteen skeletally mature male heterozygous mice were used, together with 10 wild-type controls. The tibia was distracted by 0.25 mm twice a day (0.5 mm/day) for 10 days using the half-ring type fixator. Nine mice were kept for a further 28 days to observe the consolidation phase. In four out of five mice of the heterozygous group and in all three wild-type mice, bony fusion within the distraction gap was observed on radiographs. Histological findings were almost the same in the two groups at various stages of the procedure and intramembranous ossification was predominant in both the groups. Despite the inhibition of intramembranous ossification during the foetal development of Cbfa1+/- mice, distraction osteogenesis was as successful as in control mice.

Vascularity in a new model of atrophic nonunion.

Our aim was to develop a clinically relevant model of atrophic nonunion in the rat to test the hypothesis that the vessel density of atrophic nonunion reaches that of normal healing bone, but at a later time-point. Atrophic nonunion is usually attributed to impaired blood supply and is poorly understood. We determined the number of blood vessels at the site of an osteotomy using immunolocalisation techniques in both normally healing bones and in atrophic nonunion. At one week after operation there were significantly fewer blood vessels in the nonunion group than in the healing group. By eight weeks, the number in the atrophic nonunion group had reached the same level as that in the healing group. Our findings suggest that the number of blood vessels in atrophic nonunion reaches the same level as that in healing bone, but at a later time-point. Diminished vascularity within the first three weeks, but not at a later time-point, may prevent fractures from uniting.

Human atrophic fracture non-unions are not avascular.

A small proportion of fractures progress to non-union. Non-unions are routinely classified into two groups either hypertrophic or atrophic according to their radiological appearance. It is a common preconception that non-unions with a hypertrophic appearance on X-ray are biologically active and vascular with potential to heal given the correct stable environment. Atrophic non-unions are considered to be avascular and inert and will not heal even under the correct stable environment. Non-unions are either infected or aseptic. In the present study, we tested the hypothesis that aseptic atrophic non-unions are less vascular than aseptic hypertrophic non-unions and healing fractures. Biopsies were taken from the fracture gap of patients with healing fractures, hypertrophic non-unions and atrophic non-unions. A dual labelling technique was used with antibodies against CD31 (JC70) and Collagen IV. Blood vessels were quantified using a Chalkley point eyepiece graticule. There was no statistically significant difference in the median vessel count between the three fracture groups. These findings do not support the hypothesis that established atrophic fracture non-union are less vascular than hypertrophic non-unions or healing fractures.