ABSTRACT
Tight-skin (TSK) mice are commonly used as an animal model to study the pathogenesis of Marfan syndrome (MFS), but little is known of their skeletal phenotype and in particular of the development of the spinal deformities, common in MFS. Here we examined growth of the axial skeletons of TSK and wild-type(B6) mice during their period of rapid growth. The whole bodies of mice, 4-12 weeks of age, were scanned after sacrifice, by micro-computed tomography (microCT). We reconstructed three-dimensional models of the spine and ribs, and measured vertebral body heights and rib lengths using the Mac-based image-processing software "OsiriX". Although the TSK mice were smaller than the B6 mice at 4 weeks, they experienced an early growth spurt and by 8 weeks the height, but not the width, of the vertebral body was significantly greater in the TSK mice than the B6 mice. Measurement of the angles of scoliotic and kyphotic curves post-mortem in the mice was problematic, hence we measured changes that develop in skeletal elements in these disorders. As a marker of kyphosis, we measured anterior wedging of the vertebral bodies; as a marker for scoliosis we measured asymmetries in rib length. We found, unlike in the B6 mice where the pattern was diffuse, wedging in TSK mice was directly related to spinal level and peaked steeply at the thoracolumbar junction. There was also significant asymmetry in length of the ribs in the TSK mice, but not in the B6 mice. The TSK mice thus appear to exhibit spinal deformities seen in MFS and could be a useful model for gaining understanding of the mechanisms of development of scoliosis and kyphosis in this disorder.
ABSTRACT
INTRODUCTION: Development of cell therapies for repairing the intervertebral disc is limited by the lack of a source of healthy human disc cells. Stem cells, particularly mesenchymal stem cells, are seen as a potential source but differentiation strategies are limited by the lack of specific markers that can distinguish disc cells from articular chondrocytes. METHODS: We searched for markers using the differential in-gel electrophoresis proteomic technology to compare proteins of bovine nucleus pulposus cells, phenotypically similar to mature human nucleus cells, with those of bovine articular chondrocytes. In the cohort of the differentially expressed proteins identified by mass spectrometry, cytokeratin 8 (CK8) was further validated by immunostaining of freshly isolated cells and frozen tissue sections using monoclonal antibodies. RESULTS: We identified a set of 14 differentially expressed proteins. Immunohistochemistry showed that only a subset of cells (approximately 10%) was positive for one of these proteins, CK8, an intermediate filament protein present in epithelial but not mesenchymal cells. In tissue sections, CK8-positive cells were seen in all discs examined and appeared as small isolated clusters surrounded by gelatinous matrix. Notochordal nucleus pulposus cells from pig, phenotypically similar to human infant nucleus pulposus cells, were all CK8-positive. The mesenchymal intermediate filament protein vimentin was present in all bovine and porcine nucleus pulposus cells. CONCLUSIONS: The notochordal cell population is reported to disappear from the nucleus pulposus of bovine discs before birth and from human discs in childhood. However our finding of the co-expression of vimentin and CK8 in small isolated clusters of the bovine nucleus pulposus cells indicates that a subpopulation of notochordal-like cells remains in the mature bovine disc. This finding agrees with reports in the literature on co-expression of cytokeratins and vimentin in adult human discs. As notochordal cells produce factors that promote matrix production, the CK8-positive subpopulation could have important implications for activity and survival of the nucleus pulposus, and should be considered in development of cell therapies for disc repair. In addition, the finding of differential expression of proteins in the cell population of nucleus pulposus has implications with regard to the search for specific markers.
