Differentiation State Of Chondrocytes Research Articles

Correlation Analysis of SOX9, -5, and -6 as well as COL2A1 and Aggrecan Gene Expression of Collagen I Implant-Derived and Osteoarthritic Chondrocytes.

ObjectiveMatrix-assisted autologous chondrocyte implantation is frequently applied to replace damaged cartilage in order to support tissue regeneration or repair and to prevent progressive cartilage degradation and osteoarthritis. Its application, however, is limited to primary defects and contraindicated in the case of osteoarthritis that is partially ascribed to dedifferentiation and phenotype alterations of chondrocytes obtainable from patients’ biopsies. The differentiation state of chondrocytes is reflected at the level of structural gene (COL2A1, ACAN, COL1A1) and transcription factor (SOX9, 5, 6) expression.Methods/DesignWe determined the mRNA abundances of COL2A1, ACAN, and COL1A1as well as SOX9, -5, and -6 of freshly isolated and passaged collagen I implant–derived and osteoarthritic chondrocytes via reverse transcription–polymerase chain reaction. Moreover, we analyzed the correlation of structural and transcription factor gene expression. Thus, we were able to evaluate the impact of the mRNA levels of transcription factors on the expression of cartilage-specific structural genes.ResultsSignificant differences were obtained (1) for freshly isolated osteoarthritic versus collagen I implant–derived chondrocytes, (2) due to passaging of the respective cell sources, (3) for osteoarthritic versus nonosteoarthritic chondrocytes, and (4) for COL2A1 versus ACAN expression with respect to the coherence with SOX9, -5, and -6 transcript levels.ConclusionOur results might contribute to a better understanding of the transcriptional regulation of structural gene expression of chondrocytes with implications for their use in matrix-assisted autologous chondrocyte implantation.

Collagen II regulates chondroycte integrin expression profile and differentiation

Collagen II is the major fibril-forming collagen in cartilage. Complete absence of collagen II in mice is not compatible with life and in humans mutations in the COL2A1 gene lead to osteochondrodysplasias with diverse phenotypes. However, mechanistic studies on how chondrocytes respond to a lack of collagen II in their extracellular matrix are limited. Primary mouse chondrocytes were isolated from knee joints of newborn mice and transfected with siRNA targeting Col2α1 to suppress collagen II expression. The expression of integrin receptors and matrix proteins was investigated by RT-PCR and immunoblots. The localization of matrix components was evaluated by immunostaining. Signaling pathways and the differentiation state of chondrocytes was monitored by RT-PCR and flow cytometry. We demonstrate that in the absence of collagen II chondrocytes start to produce collagen I. Some binding partners of collagen II are partially lost from the matrix while other proteins, e.g. COMP, were still found associated with the newly formed collagen network. The lack of collagen II induced changes in the expression profile of integrins. Further, we detected alterations in the Indian hedgehog/parathyroid hormone-related protein (Ihh/PTHrP) pathway that were accompanied by changes in the differentiation state of chondrocytes. Collagen II seems not to be essential for chondrocyte survival in culture but it plays an important role in maintaining chondrocyte differentiation. We suggest that a crosstalk between extracellular matrix and cells via integrins and the Ihh/PTHrP pathway is involved in regulating the differentiation state of chondrocytes.

SOX9 exerts a bifunctional effect on type II collagen gene (COL2A1) expression in chondrocytes depending on the differentiation state.

As a key transcription factor in cartilage formation, SOX9 is a potent activator of type II collagen expression, a phenotypic marker of articular chondrocytes. This study was designed to determine the potential role of SOX9 on COL2A1 gene transcription during chondrocyte dedifferentiation, a characteristic feature of osteoarthritic cartilage that can be partially mimicked in vitro by subculturing primary chondrocytes. Constructs containing different regions from the promoter and the first intron of human COL2A1 gene were transfected in differentiated (primary) and dedifferentiated (passaged) rabbit articular chondrocytes (RAC), together with an expression vector containing or not the SOX9 cDNA. As expected, low levels of SOX9 overexpression were capable of enhancing COL2A1 gene transcription in both fully differentiated and slightly phenotypically altered chondrocytes, through the specific intronic enhancer. In contrast, when overexpressed at high levels, SOX9 induced an inhibition of COL2A1 gene expression, mediated by the -266 bp promoter region, whatever the differentiation state of chondrocytes. However, in the advanced stages of dedifferentiation, SOX9, independently of its expression level, depressed COL2A1 transcriptional activity through the -63 bp short promoter. Although SOX9 has a crucial role in chondrocyte differentiation, our findings indicate that this factor cannot restore the phenotype of osteoarthritic chondrocytes by itself.

SP3 Represses the SP1-mediated Transactivation of the HumanCOL2A1 Gene in Primary and De-differentiated Chondrocytes

Sp1 and Sp3 effects on the transcription of the human alpha1(II) procollagen gene (COL2A1) were investigated in both differentiated and de-differentiated rabbit articular chondrocytes. Transient transfection with constructs of deleted COL2A1 promoter sequences driving the luciferase reporter gene revealed that the region spanning -266 to +121 base pairs showed Sp1-enhancing effects, whatever the differentiation state. In contrast, Sp3 did not influence COL2A1 gene transcription. Concomitant overexpression of the two Sp proteins demonstrated that Sp3 blocked the Sp1 induction of COL2A1 promoter activity. Moreover, inhibition of Sp1/Sp3 binding to their target DNA sequence decreased both COL2A1 gene transcription and Sp1-enhancing effects. DNase I footprinting and gel retardation assays revealed that Sp1 and Sp3 bind specifically to cis-sequences of the COL2A1 gene promoter whereby they exert their transcriptional effects. Sp1 and Sp3 levels were found to be reduced in de-differentiated chondrocytes, as revealed by DNA-binding and immunochemical study. Sp1 specifically activated collagen neosynthesis whatever the differentiation state of chondrocytes, suggesting that this factor exerts a major role in the expression of collagen type II. However, our data indicate that type II collagen-specific expression in chondrocytes depend on both the Sp1/Sp3 ratio and cooperation of Sp1 with other transcription factors, the amounts of which are also modulated by phenotype alteration.

Dedifferentiated chondrocytes cultured in alginate beads: restoration of the differentiated phenotype and of the metabolic responses to interleukin-1beta.

Chondrocytes cultivated in monolayer rapidly divide and lose their morphological and biochemical characteristics, whereas they maintain their phenotype for long periods of time when they are cultivated in alginate beads. Because cartilage has a low cellularity and is difficult to obtain in large quantities, the number of available cells often becomes a limiting factor in studies of chondrocyte biology. Therefore, we explored the possibility of restoring the differentiated properties of chondrocytes by cultivating them in alginate beads after two multiplication passages in monolayer. This resulted in the reexpression of the two main markers of differentiated chondrocytes: Aggrecan and type II collagen gene expression was strongly reinduced from day 4 after alginate inclusion and paralleled protein expression. However, 2 weeks were necessary for total suppression of type I and III collagen synthesis, indicators of a modulated phenotype. Interleukin-1beta, a cytokine that is present in the synovial fluid of rheumatoid arthritis patients, induces many metabolic changes on the chondrocyte biology. Compared with cells in primary culture, the production of nitric oxide and 92-kDa gelatinase in response to interleukin-1beta was impaired in cells at passage 2 in monolayer but was fully recovered after their culture in alginate beads for 2 weeks. This suggests that the effects of interleukin-1beta on cartilage depend on the differentiation state of chondrocytes. This makes the culture in alginate beads a relevant model for the study of chondrocyte biology in the presence of interleukin-1beta and other mediators of cartilage destruction in rheumatoid arthritis and osteoarthrosis.

Synthesis of human cartilage using organotypic cell culture.

The limited supply of fresh autologous cartilage tissue for use in reconstructive surgery necessitates the use of vital banked allografts. A feasible in vitro production of cartilage tissue composed of living cells requires the use of modern tissue culture techniques retaining the phenotypic characteristics of chondrocytes. With this purpose in mind, human chondrocytes were isolated and cultured using different culture procedures: monolayer, suspension and agar gel. The differentiation state of chondrocytes as well as proteoglycan and collagen syntheses were assessed by histochemical and immunohistochemical methods. Whereas chondrocytes in monolayer displayed an unstable phenotype and tended to dedifferentiate, in three-dimensional culture the chondrocytes remained morphologically, phenotypically and functionally differentiated. Furthermore, an accumulation of matrix products pericellularly was observed in the agar gel. The results suggest that three-dimensional cultures in agar gel may allow the in vitro production of bioartificial cartilage for transplantation.