Synthesis and Turnover of Proteoglycans by Human and Bovine Adult Articular Chondrocytes Cultured in Alginate Beads

Häuselmann, H. J.; Aydelotte, Margaret B.; Schumacher, Barbara L.; Kuettner, Klaus E.; Gitelis, Steven; Thonar, Eugene J.‐M.A.

doi:10.1016/s0934-8832(11)80053-3

Cited by 340 publications

(263 citation statements)

References 27 publications

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Section: Introductionmentioning

confidence: 74%

“…In alginate and agarose cultures, human chondrocyte proliferation observed by Hauselmann et al [15] was lower for a 66-year-old donor versus a 14-year-old donor. Hauselmann et al [15] also reported that chondrocytes from the youngest donor synthesized aggrecan molecules with higher average molecular weights. In contrast, Verbruggen et al [38] did not observe any proliferation for 2-to 69-year-old human chondrocytes embedded in agarose, but an increase in donor age correlated with reduced aggrecan synthetic rate and a smaller proportion of large aggregates.…”

Section: Introductionmentioning

confidence: 74%

“…As performed by Hauselmann et al [15], incorporation in macromolecules was determined by subjecting labeling media, dissociative extracts (GuC1 extraction buffer) and solubilized residues to PD-10 columns (Amersham Biosciences, Piscataway, NJ, USA) to obtain the macromolecular fraction that was subjected to liquid scintillation (Fisher, Hampton, NK, USA).…”

Section: S-sulfate and ''C-l-proline Incorporationmentioning

confidence: 99%

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Aged bovine chondrocytes display a diminished capacity to produce a collagen-rich, mechanically functional cartilage extracellular matrix

et al. 2005

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Most fundamental studies in cartilage tissue engineering investigate the ability of chondrocytes from young animals to produce cartilaginous matrix under various conditions, while current clinical applications such as autologous chondrocyte implantation, use chondrocytes from donors that are decades past skeletal maturity. Previous investigations have suggested that several characteristics of primary chondrocytes are age-dependent but none have quantified cell proliferation, proteoglycan synthesis and accumulation, collagen synthesis and accumulation, compressive and tensile mechanical properties in order to examine the effects of donor age on all of these parameters. We enzymatically isolated primary bovine chondrocytes from fetal, young and aged animals and cultured these cells in agarose gels to assess the above-mentioned properties. We found that fetal and young (but still skeletally mature i.e. 18-month-old bovine) chondrocytes behaved similarly, while aged chondrocytes (5-to 7-year-old bovine) displayed diminished proliferation (-2x less), a slightly reduced proteoglycan accumulation per cell (-20%), and significantly less collagen accumulation per cell (-55%) compared to the younger cells. Histological observations and mechanical properties supported these findings, where a particularly significant reduction in tensile stiffness produced by aged chondrocytes compared to younger cells was observed. Our findings suggest that donor age is an important factor in determining the outcome and potential success when tissue-engineered cartilage is produced from articular chondrocytes. More specifically, primary chondrocytes from aged donors may not possess sufficient capacity to produce the extracellular matrix that is required for a mechanically resilient tissue.

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Section: Introductionmentioning

confidence: 74%

Section: Introductionmentioning

confidence: 74%

Section: S-sulfate and ''C-l-proline Incorporationmentioning

confidence: 99%

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Aged bovine chondrocytes display a diminished capacity to produce a collagen-rich, mechanically functional cartilage extracellular matrix

et al. 2005

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“…After isolation, chondrocytes were encapsulated in alginate beads at a density of 4 ϫ 10 6 cells/ml in a manner similar to the protocol described by Häuselmann et al (26). Briefly, cells were suspended in sterile 0.15M NaCl containing 1.2% medium-viscosity alginate (Sigma), then slowly pushed through a 22-gauge needle in a dropwise manner into a 0.1M CaCl 2 solution.…”

Section: Methodsmentioning

confidence: 99%

Relative messenger RNA expression profiling of collagenases and aggrecanases in human articular chondrocytes in vivo and in vitro

Bau¹,

Gebhard²,

Haag³

et al. 2002

Arthritis & Rheumatism

370

322

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Objective. Osteoarthritic (OA) cartilage destruction depends on collagen-and aggrecan-degrading proteases such as collagenases (MMP-1 and MMP-13), stromelysin (MMP-3), MMP-14, as well as the so-called aggrecanases (ADAM-TS4 and ADAM-TS5). In this study, we tried to clarify whether these proteases are expressed in vivo in human normal and OA cartilage (and whether they are up-regulated or down-regulated during the disease process) and in interleukin-1␤ (IL-1␤)-stimulated chondrocytes in vitro.Methods. Quantitative polymerase chain reaction assays were developed and performed on RNA isolated directly from normal and degenerative cartilage tissue as well as from primary human articular chondrocytes cultured with and without IL-1␤.Results. In vivo, MMP-1 was detectable only at very low levels in any condition. MMP-13 expression was low in normal and early degenerative cartilage but was strongly up-regulated in late-stage OA specimens. MMP-1 and MMP-13 were expressed much higher in vitro than in vivo and were up-regulated by IL-1␤. Among all proteases, MMP-3 was by far the most strongly expressed, although it was strongly downregulated in late-stage OA specimens. Expression of MMP-3 was higher in vitro than in vivo and was up-regulated by IL-1␤. ADAM-TS5 and MMP-14 were expressed in all sample groups. Expression of ADAM-TS4 was very low in vivo and was induced in vitro after stimulation by IL-1␤.Conclusion. Our expression data clearly support MMP-13 as the major collagenase in OA cartilage. The most strongly expressed aggrecanase was ADAM-TS5. ADAM-TS4 was expressed only at a very low level in normal cartilage and was only slightly up-regulated in OA cartilage, casting doubt on this enzyme being the relevant aggrecanase of articular cartilage. Results of our study show that expression of many enzymes is significantly different in vitro and in vivo and suggest that IL-1␤ stimulation of articular chondrocytes might not be a good model for the matrix catabolism in OA cartilage.Osteoarthritic (OA) cartilage degeneration as well as cartilage destruction in rheumatoid arthritis depend, at least to a significant degree, on enzymatic degradation of matrix components. Two types of molecules, collagen fibrils and the proteoglycan aggrecan, represent the major targets in terms of enzymatic activity and functional loss of the cartilage matrix as a result of damage to these molecules. Whereas degradation of collagen fibrils leads to matrix instability with tissue swelling, degradation of proteoglycans leads to cartilage softening and loss of fixed charges (1), both of which are classic features of cartilage destruction.Catabolism of both types of molecules is supposed to involve different types of enzymes, largely from 2 protease families: matrix metalloproteinases (MMPs) and the "A disintegrin and metalloproteinases with thrombospondin type 1 motif" (ADAM-TS). The initial degradation of collagen fibrils (within the triple-helical region) depends on cleavage at the collagenase site, for which there exist 2 major candidate enzymes...

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“…The chondrocyte phenotype can be reexpressed in these cells by culturing them in suspension, in agarose, with alginate beads, or on a hydrogel substrate (17,19,25,28,(30)(31)(32)(33)(34)(35)(36). These changes in the biosynthetic profile of dedifferentiated chondrocytes resemble some of the phenotypic changes displayed by OA chondrocytes, and the matrix they produce is similar to that synthesized by chondroprogenitor cells (7)(8)(9)(10)(11)(12)(13)17,(37)(38)(39)(40).…”

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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Synthesis and Turnover of Proteoglycans by Human and Bovine Adult Articular Chondrocytes Cultured in Alginate Beads

Cited by 340 publications

References 27 publications

Aged bovine chondrocytes display a diminished capacity to produce a collagen-rich, mechanically functional cartilage extracellular matrix

Aged bovine chondrocytes display a diminished capacity to produce a collagen-rich, mechanically functional cartilage extracellular matrix

Relative messenger RNA expression profiling of collagenases and aggrecanases in human articular chondrocytes in vivo and in vitro

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

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