RESUMEN
The re-differentiation capacities of human articular and chick embryo sternal chondrocytes were evaluated by culture on HYAFF-11 and its sulphate derivative, HYAFF-11-S, polymers derived from the benzyl esterification of hyaluronate. Initial results showed that the HYAFF-11-S material promoted the highest rate of chondrocyte proliferation. RNA isolated from human and chick embryo chondrocytes cultured in Petri dishes, HYAFF-11 or HYAFF-11-S were subjected to semi-quantitative RT-PCR analyses. Human collagen types I, II, X, human Sox9 and aggrecan, chick collagen types I, II, IX and X were analysed. Results showed that human collagen type II mRNA expression was upregulated on HYAFF-11 biomaterials. In particular, a high level of collagen type IIB expression was associated with three-dimensional culture conditions, and the HYAFF-11 material was the most supportive for human collagen type X mRNA expression. Human Sox9 mRNA levels were constantly maintained in monolayer cell culture conditions over a period of 21 days, while these were upregulated when chondrocytes were cultured on HYAFF-11 and HYAFF-11S. Furthermore, chick collagen type IIA and IIB mRNA expression was detected after only 7 days of HYAFF-11 culture. Chick collagen type IX mRNA expression decreased in scaffold cultures over time. Histochemical staining performed in engineered cartilage revealed the presence of a de novo synthesized glycosaminoglycan-rich extracellular matrix; immunohistochemistry confirmed the deposition of collagen type II. This study showed that the three-dimensional HYAFF-11 culture system is both an effective chondrocyte delivery system for the treatment of articular cartilage defects, and an excellent in vitro model for studying cartilage differentiation.
Asunto(s)
Materiales Biocompatibles/química , Condrocitos/citología , Expresión Génica , Ácido Hialurónico/análogos & derivados , Ácido Hialurónico/química , Animales , Diferenciación Celular , División Celular , Células Cultivadas , Embrión de Pollo/metabolismo , Condrocitos/metabolismo , Colágeno Tipo I/química , Colágeno Tipo II/química , Colágeno Tipo X/química , Matriz Extracelular/metabolismo , Glicosaminoglicanos/metabolismo , Proteínas del Grupo de Alta Movilidad/metabolismo , Humanos , Inmunohistoquímica , Polímeros/química , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción SOX9 , Factores de Tiempo , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
Collagen VI is a main extracellular matrix protein whose mutation is linked to myopathic diseases. In myoblasts and other cell types, collagen VI gene transcription peaks during cell-cycle exit that precedes differentiation, upon serum withdrawal or confluence. To get insight into this transcriptional regulation, we characterized a growth arrest responsive region (GARR) in the Col6a1 promoter responsible for this effect. In this work, we identify sterol regulatory element binding protein (SREBP) as a GARR binding protein and provide evidence that SREBP contributes to induction of Col6a1 transcription in serum free conditions. Furthermore, our data unveil a previously unexpected link between extracellular matrix production and LDL signaling.