Changes in type of collagen synthesized as clones of chick chondrocytes grow and eventually lose division capacity.

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 Ial(II)H) to a mixture of Type I collagen (chain composition [al(I) MATERIALS AND METHODS Materials. F-10 medium containing twice the usual concentrations of amino acids and pyruvate (F-10 2 X), trypsin (2.5%), bovine-serum albumin (Fraction V), fetal calf serum, Ca++_, and Mg++-free saline, and glutamine were obtained from the Grand Island Biological Co. The radioactive precursor [2-3H]glycine (6.9 Ci/mmol) was obtained from the New England Nuclear Corp. Carrier Type I and Type II chick collagens were prepared as described previously (6). Ascorbic acid, f3-aminopropionitrile fumarate, and BrdUrd were purchased from the Sigma Chemical Co.Cell Culture and Cloning Procedures. Chondrocytes isolated from the sterna of 13-day chick embryos were grown for 4-5 days without feeding in medium F-10 2 X plus 10% fetal calf serum (vol/vol) and 1% bovine-serum albumin (wt/vol) as described previously (7). To isolate clones, the cells selected as "floaters" (4) were first centrifuged from the medium and incubated for 10 min at 370 in Ca++-and Mg++-free saline containing 0.06% trypsin. After centrifugation, cells were resuspended in F-10 2 X and a finely-drawn glass micropipette was used to withdraw a single cell, which was placed within a drop of medium located at the center of a 60 mm tissue culture dish (Falcon Plastics) (4, 8). Several dishes were prepared and each dish was incubated at 370 for 4-6 hr until all cells attached. Medium (3 ml) was added to dishes and the morphology and growth characteristics of each clone were observed daily. Only those clones that grew rapidly and initially possessed the distinct polygonal morphology of chondrocytes were retained. By these criteria, successful clones were obtained from 20-30% of all single cells. Occasionally clones of fibroblasts were observed (5/208 chondrocyte clones) and these were also retained. After about 3 weeks, cells in the chondrocyte clones were enveloped with matrix and some of the cells began to float away from the central mass of chondrocytes and to form secondary colonies. At this time the cells were dissociated with 0.1% trypsin in Ca++-and Mg++-free saline for 45 min. At the first subculture cells were replated into two or more 60 mm tissue culture dishes at 50,000/ml, and at subsequent subcultures were replated into 100 mm dishes at 100,000/ml. When a high cell density was achieved after each subculture, the cells in one or more of the dishes were incubated for 24 hr with [2-3H]glycine (50-100 ,uCi/ml) in the presence of (3-aminopropionitrile (100 ug/ml) and ascorbic acid (50 ,g/ml) in order to label newly-synthesized collagen.Isolation of Collagen from Cell Cultures. The procedures used to i...

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

“…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).…”

Permanently proliferating rat vascular smooth muscle cell with maintained expression of smooth muscle characteristics, including actin of the vascular smooth muscle type.

“…Another frequently found phenomenon is the expression of structures and chemical compounds in the cultured cell that are not found in the cell in situ. Typical examples include the formation of collagen type I in chondrocytes (24) and of intermediate-sized filaments of the vimentin type in cultured cells from epithelial, neuronal, and muscle origin (e.g., see references 2, 11, and 12).…”

Influence of extracellular pyrophosphate on the synthesis of collagen by chondrocytes

“…Similar results have also been obtained with CaC12 [5], suggesting that the local increase in calcium or pyrophosphate ions around the chondrocytes stimulates them to produce type I collagen. The clones of chick embryonic chondrocytes switch their synthesis of collagen from type II to type I and type I trimer, as a result of aging in culture [4]. One chondrocyte, therefore, has the capability of synthesizing more than one type of collagen molecule.…”

“…The cartilage undergoing osteoarthritic changes, as well as normal aged cartilage, produces type I collagen (composed of two al (I) and one a2 chains) in addition to the tissue-specific type II collagen [1][2][3]. Chick-embryonic chondrocytes, when subcultured to senescence, synthesize type I collagen and type I trimer rather than type II collagen [4]. The chondrocytes from normal rabbitarticular cartilage switch their phenotypic expression to type I collagen when isolated from the tissue and maintained in monolayer culture [5][6][7].…”