Qusous, Ala (2009) The role of DEL1 in chondrocyte expansion and mechanotransduction: an investigative study. PhD thesis, University of Westminster, School of Life Sciences.
Matrix-induced autologous chondrocyte implantation offers a potential cure for joint disease and is currently challenged by loss of differentiated phenotype upon culture. To investigate the mechanism of dedifferentiation, chondrocytes were cultured at low density (1x104cell/cm2) and subcultured on days 9 (P1), 14 (P2) and 21 (P3). A loss of a differentiated phenotype was observed in P1 with a reduction in sphericity from 0.72±0.02 to 0.52±0.03. Changes in cellular dimensions in response to 2D culture were additionally recorded with an increase and decrease in cell length and depth, respectively, yielding an increase in cell volume from 474.72±32.08 to 725.20±35.55μm3. Furthermore, the effect of 2D culture-induced dedifferentiation on mechanotransduction was investigated in response to a hyperosmotic challenge whereby regulatory volume increase (RVI) was only observed in 2D cultured chondrocytes with linear volume recovery rates in P1 and biphasic RVI in P2 and P3. Similarly, a REV5901-induced intracellular calcium rise via PLCβ and PKC was shown to be sensitive to extracellular sodium ([Na+]e) in freshly isolated and Gd3+ in 2D cultured chondrocytes. A 2.70±0.63-fold increase in type I collagen (col1) expression was observed in P1 chondrocytes, whereas a 1.58±0.43-fold increase in type II collagen (col2) was followed by a decline to baseline levels upon further culture. A transient rise in the chondrocytic transcription factor Sox9 and Developmental Endothelial Locus 1 (DEL1) was observed in P1 chondrocytes, suggesting the existence of a third phenotype termed ‘mesodifferentiated*’ and a potential role for DEL1 in chondrocyte dedifferentiation. DEL1 knockdown by RNA interference (RNAi) promoted a differentiated phenotype as characterised by a decrease in cell volume, reduced col1 expression, inhibition of RVI and elevated sensitivity to [Na+]e. DEL1 knockdown was shown to inhibit P1-associated Akt phosphorylation and the expression of the dedifferentiation transcription factor Twist, additionally reduced in the presence of R-Etodolac. The induction of Sox9 expression using Bone Morphogenetic Protein 2 and Twist knockdown using RNAi enhanced the expression of DEL1, suggesting DEL1 regulation of dedifferentiation by a feedback signal previously unreported.
|Item Type:||Thesis (PhD)|
|Research Community:||University of Westminster > Life Sciences, School of|
|Deposited On:||17 Aug 2010 09:50|
|Last Modified:||17 Aug 2010 09:50|
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