Type VI collagen expression is upregulated in the early events of chondrocyte differentiation (original) (raw)
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Changes in the expression of collagen genes show two stages in chondrocyte differentiation in vitro
The Journal of Cell Biology, 1988
This report deals with the quantitation of both mRNA and transcription activity of type I collagen gene and of three cartilage-specific collagens (types II, IX, and X) during in vitro differentiation of chick chondrocytes. Differentiation was obtained by transferal to suspension culture of dedifferentiated cells passaged for 3 wk as adherent cells. The type I collagen mRNA, highly represented in the dedifferentiated cells, rapidly decreased during chondrocyte differentiation. On the contrary, types II and IX collagen mRNAs sharply increased within the first week of suspension culture, peaked in the second week, and thereafter began to decrease. This decrease was particularly significant for type IX collagen mRNA. The level of type X collagen mRNA progressively increased during the course of the culture, reached its maximal value after 3-4 wk, and decreased only at a later stage of cell differentiation. As determined by in vitro run-off transcription assays, all these changes in coll...
Biochemistry, 1977
The radioactive collagens synthesized by the fourth subculture progeny of rabbit articular chondrocytes were extracted and purified after limited pepsin digestion by neutral and acid salt precipitation. In order to identify the different types of collagen present, denatured collagen chains were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 5% gels, electrophoretically eluted, and cleaved with cyanogen bromide, and the resultant peptides were fractionated by a new sodium dodecyl sulfate electrophoresis system (tris(hydroxymethyl)aminomethane-borate buffer, 15% gels). Comparison of these separate peptide profiles with those from alpha1(I) and alpha1(III) collagen chains permitted the unambiguous identification of these chains in the radioactive collagen synthesized by chondrocytes. Although cartilage slices predominantly synthesized alpha1(II) chains, only alpha1(I) chains were made by cells in fourth subculture. A large fraction of these alpha1(I) chains could not be accounted for by the presence of type I collagen. While in a native, triple-helical conformation, some of these extra alpha1(I) chains were completely separated from type I collagen by their solubility at pH 8.0 in 2.6 M NaCl and therefore identified as [alpha1(I)]3, type I trimer. In addition to type I collagen and type I trimer, these chondrocyte progeny also synthesized type III collagen and two new collagen chains, X and Y. Each collagen type was further characterized by carboxymethylcellulose chromatography and its distribution between the medium and the cell layer. These findings support the idea that cultured chondrocytes assume a collagen phenotype similar to that of their undifferentiated mesenchymal cell precursors.
Journal of Orthopaedic Research, 2014
For autologous chondrocyte transplantation, articular chondrocytes are harvested from cartilage tissue and expanded in vitro in monolayer culture. We aimed to characterize with a cellular resolution the synthesis of collagen type II (COL2) and collagen type I (COL1) during expansion in order to further understand why these cells lose the potential to form cartilage tissue when reintroduced into a microenvironment that supports chondrogenesis. During expansion for six passages, levels of transcripts encoding COL2 decreased to <0.1%, whereas transcript levels encoding COL1 increased 370-fold as compared to primary chondrocytes. Flow cytometry for intracellular proteins revealed that chondrocytes acquired a COL2/COL1-double positive phenotype during expansion, and the COL2 positive cells were able to enter the cell cycle. While the fraction of COL2 positive cells decreased from 70% to <2% in primary chondrocytes to passage six cells, the fraction of COL1 positive cells increased from <1% to >95%. In parallel to the decrease of the fraction of COL2 positive cells, the cells' potential to form cartilage-like tissue in pellet cultures steadily decreased. Intracellular staining for COL2 enables for characterization of chondrocyte lineage cells in more detail with a cellular resolution, and it may allow predicting the effectiveness of expanded chondrocytes to form cartilage-like tissue.
Proceedings of the National Academy of Sciences, 1976
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 t...
Effect of Collagen Type I or Type II on Chondrogenesis by Cultured Human Articular Chondrocytes
Tissue Engineering Part A, 2013
Introduction: Current cartilage repair procedures using autologous chondrocytes rely on a variety of carriers for implantation. Collagen types I and II are frequently used and valuable properties of both were shown earlier in vitro, although a preference for either was not demonstrated. Recently, however, fibrillar collagens were shown to promote cartilage degradation. The goal of this study was to evaluate the effects of collagen type I and type II coating on chondrogenic properties of in vitro cultured human chondrocytes, and to investigate if collagen-mediated cartilage degradation occurs. Methods: Human chondrocytes of eight healthy cartilage donors were isolated, expanded, and cultured on culture well inserts coated with either collagen type I, type II, or no coating (control). After 28 days of redifferentiation culture, safranin O and immunohistochemical staining for collagen types I, II, X, and Runx2/ Cbfa1 were performed and glycosaminoglycan (GAG) and DNA content and release were examined. Further, expression of collagen type I, type II, type X, MMP13, Runx2/Cbfa1, DDR2, a2 and b1 integrin were examined by reverse transcriptase-polymerase chain reaction. Results: The matrix, created by chondrocytes grown on collagen type I-and II-coated membranes, resembled cartilage more than when grown on noncoated membranes as reflected by histological scoring. Immunohistochemical staining did not differ between the conditions. GAG content as well as GAG/DNA were higher for collagen type II-coated cartilage constructs than control. GAG release was also higher on collagen type I-and IIcoated constructs. Expression of collagen type X was higher of chondrocytes grown on collagen type II compared to controls, but no collagen X protein could be demonstrated by immunohistochemistry. No effects of collagen coating on DDR2 nor MMP-13 gene expression were found. No differences were observed between collagen types I and II. Conclusion: Chondrocyte culture on collagen type I or II promotes more active matrix production and turnover. No significant differences between collagen types I and II were observed, nor were hypertrophic changes more evident in either condition. The use of collagen type I or II coating for in vitro models, thus, seems a sound basis for in vivo repair procedures.
Developmental Dynamics, 2000
The assembly and resorption of the extracellular matrix in the physis of the growth plate are poorly understood. By examining isolated fetal growth plate chondrocytes in culture and using immunochemical methods we show that type II collagen, proteoglycan aggrecan, and type IX collagen are assembled into a matrix that is initially enriched in type II collagen over proteoglycan and type IX collagen. When compared to the content of the COL2 domain in the ␣ 1 (IX) chain it is evident that the majority ( 90%) of type IX molecules lack the NC4 domain unlike in articular cartilage. During matrix assembly the molar ratio of type II/COL2 of ␣ 1 (IX) varied from 25:1 to 2.5:1. Following expression of the hypertrophic phenotype (initiation of type X collagen synthesis) there are parallel changes in both collagen and proteoglycan contents (inversely related to collagenase cleavage of type II collagen). The NC4 domain is then selectively, rapidly and irreversibly removed as mineralization is initiated, leaving the ␣ 1 (IX) chain COL2 domain. Subsequently as mineralization progresses type II and type IX collagen (COL2 domain), but not the proteoglycan aggrecan, are resorbed coincident with a markedly increased cleavage of type II collagen by collagenase as mineral is deposited in the matrix. This study, therefore reveals a carefully orchestrated series of events in matrix assembly and resorption that prepares the extracellular matrix for mineralization.
FEBS Letters, 1989
Cell cultures were nuttated from eptphyseal cartrlages, dtaphyseal penosteum, and muscle of 16-week human fetuses Total RNAs tsolated from these cultures were analyzed for the levels of mRNAs for major fibnllar collagens, two proteoglycan core protems and osteonectm In standard monolayer cultures the dtfferenttated chondrocyte phenotype was replaced by a dedtfferenttated one the mRNA levels of carttlage-specrfic type II collagen decreased upon subculturmg, whtle those of types I and III collagen, and the core proteins Increased When the cells were transferred to grow m agarose, redtfferenttatton (reappearance of type II collagen mRNA) occurred Ftbroblasts grown from penosteum and muscle were found to contam mRNAs for types I and III collagen and proteoglycan cores When these cells were transferred to agarose they acquired a shape mdtstmgutshable from chondrocytes, but no type II collagen mRNA was observed Cartrlage, Chondrocyte, Collagen, Proteoglycan
Type X collagen synthesis during in vitro development of chick embryo tibial chondrocytes
The Journal of Cell Biology, 1986
In the developing chick embryo tibia type X collagen is synthesized by chondrocytes from regions of hypertrophy and not by chondrocytes from other regions (Capasso, O., G. Tajana, and R. Cancedda, 1984, Mol. Cell. Biol. 4:1163-1168; Schmid, T. M., and T. F. Linsenmayer, 1985, Dev. Biol. 107:375-381). To investigate further the relationship between differentiation of endochondral chondrocytes and type X collagen synthesis we have developed a novel culture system for chondrocytes from 29-31-stage chick embryo tibiae. At the beginning of the culture these chondrocytes are small and synthesize type II and not type X collagen, but when grown on agarose-coated dishes they further differentiate into hypertrophic chondrocytes that synthesize type X collagen. The synthesis of type X collagen has been monitored in cultured cells by analysis of labeled collagens and in vitro translation of mRNAs. When the freshly dissociated chondrocytes are plated in anchorage-permissive dishes, most of the c...
Hypertrophic chondrocytes undergo further differentiation in culture
Journal of Cell Biology, 1992
Conditions have been defined for promoting growth and differentiation of hypertrophic chondrocytes obtained in culture starting from chick embryo tibiae. Hypertrophic chondrocytes, grown in suspension culture as described (Castagnola P., G. Moro, F. Descalzi Cancedda, and R. Cancedda. 1986. J. Cell Biol. 102 :2310-2317), when they reached the stage of single cells, were transferred to substrate-dependent culture conditions in the presence of ascorbic acid. Cells showed a change in morphology, became more elongated and flattened, expressed alkaline phosphatase, and eventually mineralized. Type II and X collagen synthesis was halted and replaced by type I collagen synthesis. In addition the cells started to produce and to secrete in large amount a protein with an apparent molecular mass of 82 KD in reducing conditions and 63 KD in unreducing conditions. This protein is soluble in acidic solutions, does not contain oNG bone organogenesis occurs in the embryo by endochondral ossification from undifferentiated mesenchyme. During the early stages of development, mesenchymal cells in the limb buds condense to form a core of differentiated chondrocytes ; osteogenesis starts at the periphery ofthe cartilage core, which is subsequently invaded by blood vessels and replaced by bone marrow and trabecular bone. After birth, similar events take place in the long bone growth plate and at the bone fracture sites. Bone formation and remodeling have been extensively investigated, starting from pioneering work describing the morphological and biochemical changes occurring during early bone formation to more recent studies aimed at the elucidations of the cellular and molecular mechanisms involved (7, 22, 34). It is widely agreed that cells present in a continuous collar surrounding, but separated from the cartilage rudiment, give rise to osteoblasts, i.e., cells responsible for the synthesis and mineralization of the osteoid extracellular matrix. In the past, occasionally and recently more frequently, it has been postulated thatgrowth platehypertrophic chondrocytes might also contribute to the formation of a bone matrix, since in some organ culturesthese cells start to express bone markers. During culture of mouse mandibular condyles, the expression of type I collagen, osteonectin, alkaline phosphatase, osteopontin, and osteocalcin by mature chondrocytes was detected by in situ hybridization (38). A morphological study