RESUMEN
The bovine patella model has been used extensively for studying important structure-function aspects of articular cartilage, including its degeneration. However, the degeneration seen in this model has, to our knowledge, never been adequately compared with human osteoarthritis (OA). In this study, bovine patellae displaying normal to severely degenerate states were compared with human tissue displaying intact cartilage to severe OA. Comparisons of normal and OA features were made with histological scoring, morphometric measurements, and qualitative observations. Differential interference contrast microscopy was used to image early OA changes in the articular cartilage matrix and to investigate whether this method provided comparable quality of visualisation of key structural features with standard histology. The intact bovine cartilage was found to be similar to healthy human cartilage and the degenerate bovine cartilage resembled the human OA tissues with regard to structural disruption, cellularity changes, and staining loss. The extent of degeneration in the bovine tissues matched the mild to moderate range of human OA tissues; however, no bovine samples exhibited late-stage OA. Additionally, in both bovine and human tissues, cartilage degeneration was accompanied by calcified cartilage thickening, tidemark duplication, and the advancement of the cement line by protrusions of bony spicules into the calcified cartilage. This comparison of degeneration in the bovine and human tissues suggests a common pathway for the progression of OA and thus the bovine patella is proposed to be an appropriate model for investigating the structural changes associated with early OA.
Asunto(s)
Osteoartritis de la Rodilla/patología , Rótula , Animales , Cartílago Articular , Bovinos , Modelos Animales de Enfermedad , Femenino , HumanosRESUMEN
Mechanical loading is essential for the health and homeostasis of articular cartilage, although the fundamental mechanotransduction pathways are unclear. Previous studies have demonstrated that cyclic compression up-regulates proteoglycan synthesis via an intracellular Ca(2+) signalling pathway, mediated by the release of ATP. However, the mechanism(s) of ATP release has not been elucidated. The present study examines expression of the putative mechanosensitive ATP-release channel, connexin 43 and whether it is expressed on the chondrocyte primary cilium, which acts as a mechanosensor in a variety of other cell types. In addition the study characterized the expression of a range of purine receptors through which ATP may activate downstream signalling events controlling cell function. Bovine articular chondrocytes were isolated by sequential enzyme digestion and seeded in agarose constructs. To verify the presence of functional hemichannels, Lucifer yellow (LY) uptake into viable cells was quantified following treatment with a hemichannel agonist (EGTA) and antagonist (flufenamic acid). LY uptake was observed in 45% of chondrocytes, increasing to 83% following EGTA treatment (P < 0.001). Treatment with the hemichannel blocker, flufenamic acid, significantly decreased LY uptake to less than 5% with and without EGTA. Immunofluorescence and confocal microscopy confirmed the presence of primary cilia and the expression of connexin 43. Approximately 50% of bovine chondrocyte primary cilia were decorated with connexin 43. Human chondrocytes in situ within cartilage explants also expressed connexin 43 hemichannels. However, expression was confined to the upper 200 microm of the tissue closest to the articular surface. Immunofluorescence revealed the expression of a range of P2X and P2Y receptor subtypes within human articular cartilage. In conclusion, the expression of functional hemichannels by articular chondrocytes may represent the mechanism through which mechanical loading activates ATP release as part of a purinergic mechanotransduction pathway. Furthermore, the expression of connexin 43 on the chondrocyte primary cilium suggests the possible involvement of the cilium in this pathway.
Asunto(s)
Cartílago Articular/química , Condrocitos/química , Conexina 43/análisis , Mecanorreceptores/fisiología , Receptores Purinérgicos P2/análisis , Animales , Cartílago Articular/metabolismo , Bovinos , Condrocitos/metabolismo , Cilios/química , Cilios/fisiología , Citoplasma/química , Citoplasma/metabolismo , Femenino , Técnica del Anticuerpo Fluorescente , Humanos , Isoquinolinas , Masculino , Mecanotransducción Celular , Microscopía Confocal , Persona de Mediana Edad , Receptores Purinérgicos P2X2 , Receptores Purinérgicos P2X4 , Receptores Purinérgicos P2X7 , Receptores Purinérgicos P2Y1 , Receptores Purinérgicos P2Y2 , Coloración y EtiquetadoRESUMEN
Primary cilia are highly conserved organelles found on almost all eukaryotic cells. Tg737(orpk) (orpk) mice carry a hypomorphic mutation in the Tg737 gene resulting in the loss of polaris, a protein essential for ciliogenesis. Orpk mice have an array of skeletal patterning defects and show stunted growth after birth, suggesting defects in appositional and endochondral development. This study investigated the association between orpk tibial long bone growth and chondrocyte primary cilia expression using histomorphometric and immunohistochemical analysis. Wild-type chondrocytes throughout the developing epiphysis and growth plate expressed primary cilia, which showed a specific orientation away from the articular surface in the first 7-10 cell layers. In orpk mice, primary cilia were identified on very few cells and were significantly shorter. Orpk chondrocytes also showed significant increases in cytoplasmic tubulin, a likely result of failed ciliary assembly. The growth plates of orpk mice were significantly smaller in length and width, with marked changes in cellular organization in the presumptive articular cartilage, proliferative and hypertrophic zones. Cell density at the articular surface and in the hypertrophic zone was significantly altered, suggesting defects in both appositional and endochondral growth. In addition, orpk hypertrophic chondrocytes showed re-organization of the F-actin network into stress fibres and failed to fully undergo hypertrophy, while there was a marked reduction in type X collagen sequestration. These data suggest that failure to form a functional primary cilium affects chondrocyte differentiation and results in delayed chondrocyte hypertrophy within the orpk growth plate.
Asunto(s)
Cartílago Articular/metabolismo , Condrocitos/metabolismo , Cilios/metabolismo , Citoesqueleto/metabolismo , Regulación de la Expresión Génica , Placa de Crecimiento/metabolismo , Proteínas Supresoras de Tumor/fisiología , Actinas/metabolismo , Animales , Proliferación Celular , Colágeno Tipo X/metabolismo , Matriz Extracelular/metabolismo , Ratones , Ratones Transgénicos , Tubulina (Proteína)/metabolismo , Proteínas Supresoras de Tumor/genéticaRESUMEN
Meckel syndrome (MKS) is an inherited autosomal recessive hepatorenal fibrocystic syndrome, caused by mutations in TMEM67, characterized by occipital encephalocoele, renal cysts, hepatic fibrosis, and polydactyly. Here we describe an ovine model of MKS, with kidney and liver abnormalities, without polydactyly or occipital encephalocoele. Homozygous missense p.(Ile681Asn; Ile687Ser) mutations identified in ovine TMEM67 were pathogenic in zebrafish phenotype rescue assays. Meckelin protein was expressed in affected and unaffected kidney epithelial cells by immunoblotting, and in primary cilia of lamb kidney cyst epithelial cells by immunofluorescence. In contrast to primary cilia of relatively consistent length and morphology in unaffected kidney cells, those of affected cyst-lining cells displayed a range of short and extremely long cilia, as well as abnormal morphologies, such as bulbous regions along the axoneme. Putative cilia fragments were also consistently located within the cyst luminal contents. The abnormal ciliary phenotype was further confirmed in cultured interstitial fibroblasts from affected kidneys. These primary cilia dysmorphologies and length control defects were significantly greater in affected cells compared to unaffected controls. In conclusion, we describe abnormalities involving primary cilia length and morphology in the first reported example of a large animal model of MKS, in which we have identified TMEM67 mutations.
Asunto(s)
Anomalías Múltiples/genética , Síndrome de Dandy-Walker/genética , Síndrome Hepatorrenal/genética , Proteínas de la Membrana/genética , Mutación/genética , Quiste Pancreático/genética , Anomalías Múltiples/patología , Sustitución de Aminoácidos , Animales , Secuencia de Bases , Cromosomas de los Mamíferos/genética , Cilios/patología , Síndrome de Dandy-Walker/patología , Modelos Animales de Enfermedad , Células Epiteliales/metabolismo , Sitios Genéticos , Aparato de Golgi/metabolismo , Síndrome Hepatorrenal/patología , Homocigoto , Riñón/patología , Proteínas de la Membrana/química , Mutación Missense/genética , Quiste Pancreático/patología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ovinos , Pez CebraRESUMEN
Osteoarthritis (OA) is a common joint disease characterized by articular cartilage degeneration. The etiology of OA is unknown. Because several previous studies have shown that primary cilia play critical roles in joint development, this study examined the incidence and morphology of primary cilia in chondrocytes during joint degeneration in a naturally occurring bovine model of OA. Primary cilia were detected using antibodies to acetylated alpha-tubulin in normal cartilage as well as in mild and severe OA tissue. In normal cartilage, cilia number and length were lowest in the superficial zone and increased with distance from the articular surface. In OA tissue, the incidence and length of cilia increased at the eroding articulating surface, resulting in an overall increased proportion of cilia. This is the first study to show that primary cilia are present on chondrocytes throughout OA progression and that the overall percentage of ciliated cells within the degenerating cartilage increases with OA severity.
Asunto(s)
Cartílago Articular/patología , Condrocitos/patología , Condrocitos/ultraestructura , Cilios/patología , Osteoartritis de la Rodilla/patología , Animales , Bovinos , Condrocitos/metabolismo , Modelos Animales de Enfermedad , Matriz Extracelular/patología , Humanos , Microscopía Fluorescente , Rótula/patología , Índice de Severidad de la Enfermedad , Tubulina (Proteína)/metabolismoRESUMEN
Hyaline cartilage chondrocytes express one primary cilium per cell, but its function remains unknown. We examined the ultrastructure of chick embryo sternal chondrocyte cilia and their interaction with extracellular matrix molecules by transmission electron microscopy (TEM) and, for the first time, double-tilt electron tomography. Ciliary bending was also examined by confocal immunohistochemistry. Tomography and TEM showed the ciliary axoneme to interdigitate amongst collagen fibres and condensed proteoglycans. TEM also revealed the presence of electron-opaque particles in the proximal axoneme which may represent intraciliary-transport (ICT) particles. We observed a wide range of ciliary bending patterns. Some conformed to a heavy elastica model associated with shear stress. Others were acutely deformed, suggesting ciliary deflection by collagen fibres and proteoglycans with which the cilia make contact. We conclude that mechanical forces transmitted through these matrix macromolecules bend the primary cilium, identifying it as a potential mechanosensor involved in skeletal patterning and growth.