Relationship between cell shape and type of collagen synthesised as chondrocytes lose their cartilage phenotype in culture
TL;DR: It is found that there is no strict correlation between cell morphology and type of collagen synthesised in cartilage colonies kept in monolayer culture at low density.
Abstract: WHEN chondrocytes from sternal or articular cartilage are kept in monolayer culture at low density, they eventually lose their cartilage phenotype1–4. Within four passages or approximately 1 month in culture they change from a polygonal or round to a flattened, amoeboid-like shape5–7, and instead of cartilage collagen (type II collagen8) they synthesise the genetically different type I collagen. It is not known whether there is a strict correlation between the occurrence of cell flattening and the change in collagen synthesis within individual cells. We have reported that preferentially flattened, fibroblast-like cells at the edge of cartilage colonies synthesise type I collagen, whereas round or polygonal chondrocytes generally synthesise type II collagen1–3. The change is nearly complete in a culture at a time when excessive flattening is observed4. Using an immunofluorescence double staining technique9,10, we have now found that there is no strict correlation between cell morphology and type of collagen synthesised.
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TL;DR: Using SDS-polyacrylamide gel electrophoresis of intact collagen chains and two-dimensional cyanogen bromide peptide mapping, this work demonstrated a complete return to the differentiated collagen phenotype and demonstrates a reversible system for the study of gene expression.
2,296 citations
TL;DR: Some biomaterials, which have been suggested to promote chondrogenesis and to have potentials for tissue engineering of articular cartilage, are reviewed and a new biomaterial, a chitosan-based polysaccharide hydrogel, is introduced and discussed in terms of the biocompatibility with chondrocytes.
1,868 citations
TL;DR: The focus of this Commentary will be on identifying and describing the fundamental features of 3D cell culture systems that influence cell structure, adhesion, mechanotransduction and signaling in response to soluble factors, which regulate overall cellular function in ways that depart dramatically from traditional 2D culture formats.
Abstract: Summary Much of our understanding of the biological mechanisms that underlie cellular functions, such as migration, differentiation and force-sensing has been garnered from studying cells cultured on two-dimensional (2D) glass or plastic surfaces. However, more recently the cell biology field has come to appreciate the dissimilarity between these flat surfaces and the topographically complex, three-dimensional (3D) extracellular environments in which cells routinely operate in vivo. This has spurred substantial efforts towards the development of in vitro 3D biomimetic environments and has encouraged much cross-disciplinary work among biologists, material scientists and tissue engineers. As we move towards more-physiological culture systems for studying fundamental cellular processes, it is crucial to define exactly which factors are operative in 3D microenvironments. Thus, the focus of this Commentary will be on identifying and describing the fundamental features of 3D cell culture systems that influence cell structure, adhesion, mechanotransduction and signaling in response to soluble factors, which – in turn – regulate overall cellular function in ways that depart dramatically from traditional 2D culture formats. Additionally, we will describe experimental scenarios in which 3D culture is particularly relevant, highlight recent advances in materials engineering for studying cell biology, and discuss examples where studying cells in a 3D context provided insights that would not have been observed in traditional 2D systems.
1,438 citations
Cites background from "Relationship between cell shape and..."
...Indeed, many cell types, when isolated from tissues and placed into planar cell culture, become progressively flatter, divide aberrantly and lose their differentiated phenotype (von der Mark et al., 1977; Petersen et al., 1992)....
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TL;DR: Current knowledge of the types of collagen and their distribution and biosynthesis is outlined, including fibronectin, which not only functions as an attachment protein, but also may play an important role in repair reactions.
Abstract: It has long been appreciated that, for many types of cells, attachment to a substrate is required for their replication. Collagen substrates enhance the growth (64, 70, 109, 238) as well as the differentiation (40, 69, 85, 86, 146, 161, 184), of many cells in culture above that observed with other substrates such as plastic and glass. As discussed in this report, many cultured cells including fibroblasts, myoblasts, hepatocytes, chondrocytes, and certain epithelial cells are thought not to bind directly to the collagen substrate or to the plastic surface of culture dishes (31, 73, 74, 76, 77, 89, 93, 113, 164, 220) . Instead, extracellular glycoproteins bind the cells to the substrate. One of these attachment proteins, fibronectin, has been extensively studied (99, 151, 166, 227, 228, 244) . Fibronectin is produced by fibroblasts (13, 197, 246) and endothelial cells (102, 140) as well as some other cell types . It is also present in high concentrations in blood and serum (152) . In culture, serum fibronectin, as well as that produced by the cells, can bind to both the collagen substrates and the tissue culture plastic surface and mediate the attachment of cells (73, 113, 164, 242) . Circulating fibronectin participates in a variety of reactions important to wound healing, including the adhesion and spreading of platelets on collagen (10, 14, 75, 100, 204), binding to the fibrin clot (74, 150, 198) and to collagen (37, 46, 94, 103, 119), promoting opsonization reactions by phagocytic cells (18, 94, 201), and promoting fibroblast migration (6, 61) . Thus, fibronectin not only functions as an attachment protein, but also may play an important role in repair reactions . In the case of certain differentiated cells, such as chondrocytes (89) and epithelial cells (159, 220), glycoproteins different from fibronectin are active in attachment. The components of these extracellular matrices of fibroblasts, chondrocytes, and epithelial cells differ with the cell type, and they require separate attachment proteins to provide additional specificity to the interaction ofthe cell with its matrix. Alterations in cellsubstratum interactions are observed in differentiating cells and after spontaneous transformation of cells or exposure to oncogenic agents (99, 228) . This review will briefly outline our current knowledge of the types of collagen and their distribution and biosynthesis. Then, the interaction of cells with colREVIEW
1,382 citations
TL;DR: Stem cell-based tissue engineering using 3D silk fibroin scaffolds has expanded the use of silk-based biomaterials as promising scaffolds for engineering a range of skeletal tissues like bone, ligament, and cartilage, as well as connective tissues like skin.
893 citations
Cites background from "Relationship between cell shape and..."
...In the 3D cultivation environment created by the highly porous aqueous-derived silk fibroin scaffolds, within 3 weeks the majority of MSCs were embedded in lacunae-like spaces and acquired a spherical morphology, which has been found to be essential for the synthesis of ECM components related to cartilage tissue [168]....
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References
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TL;DR: Specific antibodies against skin and bone collagen and cartilage collagen are prepared for the study of differential collagen synthesis during development of the chick embryo by immunofluorescence.
364 citations
TL;DR: Analysis of collagen type at each successive subculture until the time of cellular senescence has shown that a change in synthesis occurs from the cartilage-specific Type II collagen to a mixture of Type I collagen and the Type I trimer.
Abstract: Clones of embryonic chick chondrocytes have been isolated and collagen biosynthesis has been followed as the clones grow and eventually lose division capacity. Analysis of collagen type at each successive subculture until the time of cellular senescence has shown that a change in synthesis occurs from the cartilage-specific Type II collagen (chain composition [alpha1(II)]3) to a mixture of Type I collagen (chain composition [alpha1(I)2alpha2) and the Type I trimer (chain composition[alpha1(I)]3). The results demonstrate unequivocally that the expression of the chick chondrocyte phenotype is unstable in vitro, and that previous experiments with mass cultures of chondrocytes cannot be accounted for by overgrowth of fibroblasts. Since similar morphological changes and a similar "switching" in collagen biosynthesis have been observed after growth of chondrocytes for a few days in 5-bromo-2'-deoxyuridine, it is proposed that growth in this analog accelerates those changes that eventually lead to cellular senescence.
351 citations
295 citations
TL;DR: If the progeny of a single, genetically programmed chondrocyte may or may not synthesize chondroitin sulfate, then extragenic sites in the cytoplasm or cell surface must influence the decision as to which cluster of "luxur" molecules the cell will synthesize.
Abstract: A single, functional, mitotically quiescent chondrocyte may be induced to reenter the mitotic cyde, and produce a progeny of over 1011 cells. Sessile, adherent, polygonal cells deposit matrix, whereas amoeboid, dispersed, flattened fibroblastic cells do not. The prior synthetic history of a cell is of greater importance in determining whether the characteristic chondrogenic phenotype will be expressed, rather than growth in "permissive" or "nonpermissive" medium. Clonal conditions select for stem-like cells, some of whose progeny may become polygonal chondrocytes. The retention of the characteristic chondrogenic phenotype in vitro is favored by pruning the dedifferentiated chondrocytes which arise in these cultures. Dedifferentiated chondrocytes interfere with the deposition and synthesis of chondroitin sulfate by neighboring functional chondrocytes. Possible mechanisms are proposed to explain this type of cell-cell or cell exudate interference. If the progeny of a single, genetically programmed chondrocyte may or may not synthesize chondroitin sulfate, then extragenic sites in the cytoplasm or cell surface must influence the decision as to which cluster of "luxur" molecules the cell will synthesize.
216 citations
TL;DR: Specific antibodies against types I and III collagens and procollagens were used to localize these proteins in cultured human cells and indicate that the same cell makes both proteins.
Abstract: Specific antibodies against types I and III collagens and procollagens were used to localize these proteins in cultured human cells. These studies indicate that the same cell makes both proteins. No type III procollagen synthesis was observed in cells from two patients with two patients with the Ehlers-Danlos type IV syndrome.
205 citations