Dedifferentiated chondrocytes reexpress the differentiated collagen phenotype when cultured in agarose gels

https://core.ac.uk/download/pdf/2756387.pdf

Collagens—structure, function, and biosynthesis

Application of chitosan-based polysaccharide biomaterials in cartilage tissue engineering : a review

In recent years, the basement membrane (BM)--a specialized form of extracellular matrix (ECM)--has been recognized as an important regulator of cell behaviour, rather than just a structural feature of tissues. The BM mediates tissue compartmentalization and sends signals to epithelial cells about the external microenvironment. The BM is also an important structural and functional component of blood vessels, constituting an extracellular microenvironment sensor for endothelial cells and pericytes. Vascular BM components have recently been found to be involved in the regulation of tumour angiogenesis, making them attractive candidate targets for potential cancer therapies.

Basement membranes: structure, assembly and role in tumour angiogenesis

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.

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Deconstructing the third dimension – how 3D culture microenvironments alter cellular cues

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.

Relationship between cell shape and type of collagen synthesised as chondrocytes lose their cartilage phenotype in culture

Collagen VI is a large, disulfide-bonded protein complex which is widely distributed in connective tissue The constituent polypeptide chains (Mr = 110,000-140,000) consist of collagenous and noncollagenous segments, are degraded to chains of about half the size when collagen VI is solubilized by pepsin, and assemble to a unique pattern of oligomers As revealed by electron microscopy, the triple-stranded protomer consists of a triple helix 105 nm in length flanked on each side by globular domains of similar size (diameter about 7 nm) Protomers are assembled to dimers by an antiparallel staggered alignment of triple-helical segments This leads to inner regions, 75 nm in length, of two slightly supercoiled triple helices flanked by globular domains At both sides 30-nm-long outer triple-helical segments emerge that are terminated by globules Tetramers are formed from laterally aligned dimers that cross with their outer triple-helical segments in a scissors-like fashion The same structures, except with much smaller globular domains, are found in pepsin-treated collagen VI Disulfide-linked collagen VI produced by cultured fibroblasts has a size similar to that of genuine collagen VI found in tissue extracts Larger forms of collagen VI are assembled from tetramers by end-to-end aggregation which because of an overlap of the outer segments brings all globular domains close together This arrangement predicts microfibrillar structures in tissues with a periodicity of 100-110 nm and a diameter of 5-10 nm Structures consistent with this proposal were indeed found by immunoelectron microscopy of placenta and aorta using the ferritin technique Large, lateral aggregates of collagen VI microfibrils may in addition exist in cell cultures and tissues ("zebra collagen," "Luse bodies") and are presumably maintained by contacts between globular domains

Structure and macromolecular organization of type VI collagen.

Origin of collagen types I, III, and V in cultures of avian skeletal muscle.

A procedure was developed for purifying the globular domain NC1 of basement membrane collagen from collagenase digests of a variety of tissues. The globule (Mr = 170,000) is a hexameric structure originating from two collagen IV molecules that are cross-linked at their COOH-terminal ends. Dissociation into subunits derived from alpha 1(IV) and alpha 2(IV) chains occurs at a pH below 4 and after denaturation (8 M urea). The subunits obtained include monomers (Mr = 28,000) and two different dimers (Da,Db) which are connected by disulfide bonds (Db) and/or nonreducible bonds (Da). Almost perfect reconstitution to hexamers is obtained in neutral buffer with mixtures of the subunits or purified dimers but not with purified monomers. Stabilization by dimer formation and other physical data suggest conformationally distinct segments within the subunits, which is also supported by a repeating subdomain structure deduced from cDNA sequences. Monocline crystals of NC1 give a sufficiently detailed X-ray diffraction pattern that should permit elucidation of the three-dimensional structure of the hexamer. Antibodies raised against the globular domain react with all subunits and mainly recognize epitopes stabilized by internal disulfide bridges and/or the hexameric assembly. Immunoprecipitation tests with these antibodies demonstrated a slightly larger subunit size of NC1 in PYS-2 cell culture and the rapid release of precursor-specific segments prior to secretion from the cells. Autoantibodies against mouse tumor NC1 were produced in mice and were detected both in the blood and as tissue-bound forms (kidney, lung). The autoantibody response is accompanied by certain pathological alterations mimicking Goodpasture's syndrome. The possible relationship between the two diseases is substantiated by reaction of Goodpasture antisera with the globular domain obtained from various tissue sources.

Structure and biology of the globular domain of basement membrane type IV collagen.

The present study examined the effect of long-term, moderate physical exercise on trabecular bone volume (TBV), calcium content,3H-proline uptake, and the activities of alkaline and acid phosphatases in lumbar vertebrae of aging and senescent mice. It became apparent that if physical activity starts at an early stage of life, i.e., prior to middle age and is extended until old age, it exerts beneficial effects on trabecular bone mass and mineralization. Such a positive effect is not obtained if the training program is initiated after middle age. The training-induced reduction in bone loss was accompanied by a significant decrease in acid phosphatase activity whereas no changes took place with regard to the activity of alkaline phosphatase. Long-term physical exercise also enhanced the uptake of3H-proline by lining cells along the bone trabecules. In spite of its moderate nature, the endured training program served as a stress factor for the involved animals, a fact that was manifested by an increase in the serum levels of corticosterone. Thus, it seems that whereas young animals respond favorably to such a stimulatory stress, older animals lose this ability of adaptation.

Helga von der Mark

Papers

Relationship between cell shape and type of collagen synthesised as chondrocytes lose their cartilage phenotype in culture

Structure and macromolecular organization of type VI collagen.

Origin of collagen types I, III, and V in cultures of avian skeletal muscle.

Structure and biology of the globular domain of basement membrane type IV collagen.

Long-term physical exercise retards trabecular bone loss in lumbar vertebrae of aging female mice