Regulation of type-II collagen gene expression during human chondrocyte de-differentiation and recovery of chondrocyte-specific phenotype in culture involves Sry-type high-mobility-group box (SOX) transcription factors

Abstract

During ex vivo growth as monolayer cultures, chondrocytes proliferate and undergo a process of de-differentiation. This process involves a change in morphology and a change from expression of chondrocyte-specific genes to that of genes that are normally expressed in fibroblasts. Transfer of the monolayer chondrocyte culture to three-dimensional culture systems induces the cells to re-acquire a chondrocyte-specific phenotype and produce a cartilaginous-like tissue in vitro. We investigated mechanisms involved in the control of the de-differentiation and re-differentiation process in vitro. De-differentiated chondrocytes re-acquired their chondrocyte-specific phenotype when cultured on poly-(2-hydroxyethyl methacrylate) (polyHEMA) as assayed by morphology, reverse transcriptase PCR of chondrocyte-specific mRNA, Western-blot analysis and chondrocyte-specific promoter activity. Essentially, full recovery of the chondrocyte-specific phenotype was observed when cells that had been cultured for 4 weeks on plastic were transferred to culture on polyHEMA. However, after subsequent passages on plastic, the phenotype recovery was incomplete or did not occur. The activity of a gene reporter construct containing the promoter and enhancer from the human type-II collagen gene (COL2A1) was modulated by the culture conditions, so that its transcriptional activity was repressed in monolayer cultures and rescued to some extent when the cells were switched to polyHEMA cultures. The binding of Sry-type high-mobility-group box (SOX) transcription factors to the enhancer region was modulated by the culture conditions, as were the mRNA levels for SOX9. A transfected human type-II collagen reporter construct was activated in de-differentiated cells by ectopic expression of SOX transcription factors. These results underscore the overt change in phenotype that occurs when chondrocytes are cultured as monolayers on tissue-culture plastic substrata.

Human platelet releasates combined with polyglycolic acid scaffold promote chondrocyte differentiation and phenotypic maintenance

“…In fact, throughout the experiment, in our cell model (5% PRPr) TGF-β is present at a concentration that is consistent with the one required in chondrocyte differentiation studies (5 ng/mL) and almost 5-fold higher than in control cultures (5% FCS). The transcription factor sox9 plays a critical role in cartilage development by initiating chondrogenesis, and the increased transcription of sox9 has been shown to be associated with the chondrocytic differentiation process (Stokes et al 2001). Moreover, sox9 over-expression may delay the subsequent maturation process, leading to chondrocyte hypertrophy through the inhibition of the Runx2 transcription factor, the main activator of hypertrophic chondrocyte differentiation (Zhou et al 2006;Yamashita et al 2009).…”

Thrombospondins: from structure to therapeutics

“…This approach has been employed because human chondrocytes are more likely to mimic the in vivo condition. However, human chondrocytes are a terminally differentiated cell type and rapidly lose the chondrocyte phenotype when cultured in monolayer [66,67]. To overcome this problem, chondrocytes are cultured either in alginate beads or in nonadherent 3-D conditions [16,43,49,57].…”

Dual delivery for stem cell differentiation using dexamethasone and bFGF in/on polymeric microspheres as a cell carrier for nucleus pulposus regeneration

“…Therefore, PLGA microspheres may also be suitable as injectable scaffolds for NP tissue engineering. However, conventional cell culture methodologies that use PLGA microspheres have been hampered by the wellknown behavior of NP cells, chondrocytes or rMSCs cultured on PLGA microspheres, which undergo a quick loss of the cell function, cartilage-specific phenotype and become fibroblastic [18][19][20]. In order to synthesize disc- Error bars showed the mean ± SD (n = 3).…”

“…Due to its established safety in humans, poly-(lactic-co-glycolic acid) (PLGA) family as a histocompatibility synthetic polymer has been widely applied in tissue engineering [17]. However, intervertebral disc cells or mesenchymal stem cells (MSCs) cultured on PLGA microspheres, using conventional cell culture methodologies, often undergo a quick loss of their cartilage-specific phenotype and become fibroblastic [18][19][20]. Therefore, several studies have tried the application of specific growth factors and drugs that were fabricated in the PLGA microspheres in NP tissue differentiation [21][22][23][24].…”