Expression of the human chondrocyte phenotype in vitro

Aulthouse, Amy Lynn; Beck, Michael; Griffey, Edward; Sanford, Julie; Arden, Karen C.; Machado, Mirta A.; Horton, William A.

doi:10.1007/bf02623638

Cited by 171 publications

(111 citation statements)

References 28 publications

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“…Dedifferentiation is a reversible process characterized by the cessation of type II collagen mRNA and protein synthesis and the induction of type I collagen mRNA and protein synthesis. When articular chondrocytes cultured in monolayer are subsequently transferred to suspension culture in semisolid media they "redifferentiate" and express the collagen and proteoglycan network consistent with hyaline cartilage over a period of weeks (Aulthouse et al, 1989;Benya and Shaeffer, 1982;Binette et al, 1998;Watt and Dudhia, 1988). Because the precise molecular mechanisms involved in the regulation of articular chondrocyte dedifferentiation and redifferentiation remain to be elucidated, we used a subtractive hybridization approach to study the early molecular events in the initiation of articular chondrocyte redifferentiation.…”

Section: Discussionmentioning

confidence: 99%

Differential expression of multiple genes during articular chondrocyte redifferentiation

Haudenschild¹,

McPherson²,

Tubo³

et al. 2001

Anat. Rec.

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Articular chondrocytes undergo a rapid change in phenotype and gene expression, termed dedifferentiation, when isolated from cartilage tissue and cultured on tissue culture plastic. On the other hand, "redifferentiation" of articular chondrocytes in suspension culture is characterized by decreased cellular proliferation and the reinitiation of synthesis of hyaline articular cartilage extracellular matrix molecules. The molecular triggers for these events have yet to be defined. Subtracted cDNA libraries representing genes involved in the early events of adult human articular chondrocyte redifferentiation were generated from human articular chondrocytes that were first cultured in monolayer, and subsequently transferred to suspension culture at 10 6 cells/ml for redifferentiation. Differential regulation of genes involved in cellular organization, nuclear structure, cellular growth regulation, and extracellular matrix deposition and remodeling were observed within 48 hr of this transfer. Many of these genes had not been previously identified in the chondrocyte differentiation pathway and a number of the isolated cDNAs did not have homologies to sequences in the public data banks. Genes involved in IL-6 signal transduction including acute phase response factor (APRF), Mn superoxide dismutase, and IL-6 itself were up-regulated in suspension culture. Membrane glycoprotein gp130, a component of the IL-6 receptor, was down-regulated. Other genes involved in cell polarity, cell adherence, apoptosis, and possibly TGF-beta signaling were differentially regulated. The differential regulation of the cytokine connective tissue growth factor (CTGF) during the early stages of articular chondrocyte redifferentiation, decreasing within 48 hours of transfer to suspension culture, was particularly interesting given its reported role in the stimulation of cellular proliferation. CTGF was highly expressed in proliferative monolayer culture, and then greatly reduced by redifferentiation in standard high-density suspension culture. When articular chondrocytes were seeded in suspension at low-density (10 4 cells/ml), however, high levels of CTGF were observed along with increased levels of mature articular cartilage extracellular matrix protein RNAs, such as type II collagen and aggrecan. Although the role of CTGF in articular cartilage biology remains to be elucidated, the results described here demonstrate the potential utility of subtractive hybridization in understanding the process of articular chondrocyte redifferentiation. Anat Rec 263: [91][92][93][94][95][96][97][98] 2001.

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Section: Discussionmentioning

confidence: 99%

Differential expression of multiple genes during articular chondrocyte redifferentiation

Haudenschild¹,

McPherson²,

Tubo³

et al. 2001

Anat. Rec.

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“…Agarose, a linear polysaccharide extracted from marine red algae, is a thermosetting hydrogel that undergoes gelation in response to a reduction in temperature. Chondrocytes cultured in agarose will maintain their phenotype and synthesize near normal levels of collagen II and proteoglycan (Benya and Shaffer 1982;Aydelotte and Kuettner 1988;Aulthouse et al 1989). Over time, chondrocyte seeded agarose hydrogels have been shown to produce a functional extracellular matrix in free swelling culture (Buschmann et al 1992).…”

Section: Introductionmentioning

confidence: 99%

The effect of concentration, thermal history and cell seeding density on the initial mechanical properties of agarose hydrogels

Buckley

Thorpe

O’Brien

et al. 2009

Journal of the Mechanical Behavior of Biomedical Materials

136

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Agarose hydrogels are commonly used for cartilage tissue engineering studies and to provide a three dimensional environment to investigate cellular mechanobiology.Interpreting the results of such studies requires accurate quantification of the mechanical properties of the hydrogel. There is significant variation in the reported mechanical properties of agarose hydrogels, and little is reported on the influence of factors associated with mixing these hydrogels with cell suspensions on their initial mechanical properties. The objective of this study was to determine the influence of agarose concentration, the cooling rate during gelation, the thermal history following gelation and the cell seeding density on the initial mechanical properties of agarose hydrogels. The average ramp modulus of 2% agarose gel in tension was 24.9 kPa (±1.7, n=10), compared with 55.6 kPa (±0.5, n=10) in compression. The average tensile equilibrium modulus was 39.7 kPa (±5.7, n=6), significantly higher than the compressive equilibrium modulus of 14.2 kPa (±1.6, n=10). The equilibrium and dynamic compressive modulus of agarose hydrogels were observed to reduce if maintained at 37ºC following gelation compared to samples maintained at room temperature. Depending on the methodology used to encapsulate chondrocytes within agarose hydrogels, the equilibrium compressive modulus was found to be significantly higher for acellular 2% agarose gels compared to 2% agarose gels seeded at approximately 40×10 6 cells/mL. These results have important implications for interpreting the results of chondrocyte mechanobiology studies in agarose hydrogels.3

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“…Studies that have examined the plasticity of the chondrocyte phenotype from many different species have consistently shown that culture of these cells in monolayers on plastic substrata for prolonged periods or upon repeated passages leads to the loss of their spherical shape and to the acquisition of an elongated fibroblast-like morphology (7,(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28). These morphologic alterations are accompanied by profound biochemical changes, including loss of the cartilage-specific phenotype, as evidenced by an arrest of the synthesis of the cartilage-specific collagens (types II, IX, and XI) and proteoglycans (aggrecan), initiation of synthesis of the interstitial collagens (types I, III, and V), and increase in the synthesis of fibroblasttype proteoglycans (versican) at the expense of aggrecan (7,17,(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29).…”

mentioning

confidence: 99%

Assessment of the gene expression profile of differentiated and dedifferentiated human fetal chondrocytes by microarray analysis

Stokes¹,

Liu²,

Coimbra³

et al. 2002

Arthritis & Rheumatism

152

106

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Objective. To study the changes in patterns of gene expression exhibited by human chondrocytes as they dedifferentiate into fibroblastic cells in culture in order to better understand the mechanisms that control this process and its relationship to the phenotypic changes that occur in chondrocytes during the development of osteoarthritis (OA).Methods. Human fetal epiphyseal chondrocytes (HFCs) were cultured either on poly-(2-hydroxyethyl methacrylate)-coated plates (differentiated HFC cultures) or in plastic tissue culture flasks as monolayers (dedifferentiated HFC cultures). Following 11 days of culture under either condition, poly(A؉) RNA was isolated from the two cell populations and subjected to a gene expression analysis using a microarray containing ϳ5,000 known human genes and ϳ3,000 expressed sequence tags (ESTs).Results. A >2-fold difference in the expression of 62 known genes and 6 ESTs was observed between the two cell types. The differences in expression of several of the genes detected by the microarray hybridization were confirmed by Northern analyses. Two transcription factor genes, TWIST and HIF-1␣, and a cellular adhesion protein gene, cadherin 11, were markedly regulated in response to differentiation and dedifferentiation. Expression of these genes was also detected in adult normal and OA cartilage and chondrocytes. Analysis of the gene expression profile of HFCs revealed a complex pattern of gene expression, including many genes not yet reported to be expressed by chondrocytes.Conclusion. Chondrocytes in monolayer become dedifferentiated, acquiring a fibroblast-like appearance and changing their pattern of gene expression from one of expression of chondrocyte-specific genes to one that resembles a fibroblastic or chondroprogenitor-like pattern. Changes in gene expression associated with the process of dedifferentiation of HFCs in vitro were observed in a wide variety of genes, including genes encoding extracellular matrix proteins, transcription factors, and growth factors. At least 3 of the genes that were regulated in response to dedifferentiation were also found to be expressed in adult normal and OA articular cartilage and chondrocytes.

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Expression of the human chondrocyte phenotype in vitro

Cited by 171 publications

References 28 publications

Differential expression of multiple genes during articular chondrocyte redifferentiation

Differential expression of multiple genes during articular chondrocyte redifferentiation

The effect of concentration, thermal history and cell seeding density on the initial mechanical properties of agarose hydrogels

Assessment of the gene expression profile of differentiated and dedifferentiated human fetal chondrocytes by microarray analysis

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