scholarly journals Immunological and biochemical studies of collagen type transition during in vitro chondrogenesis of chick limb mesodermal cells

1977 ◽  
Vol 73 (3) ◽  
pp. 736-747 ◽  
Author(s):  
K Von Der Mark ◽  
H Von Der Mark
Keyword(s):  
Collagen Synthesis ◽  
Type I Collagen ◽  
Type Ii Collagen ◽  
Mesenchymal Cells ◽  
Gradual Transition ◽  
Type I ◽  
Type Ii ◽  
Total Collagen ◽  
Collagen Antibodies

This work describes an approach to monitor chondrogenesis of stage-24 chick limb mesodermal cells in vitro by analyzing the onset of type II collagen synthesis with carboxymethyl-cellulose chromatography, immunofluorescence, and radioimmunoassay. This procedure allowed specific and quantitative determination of chondrocytes in the presence of fibroblasts and myoblasts, both of which synthesize type I collagen. Chondrogenesis was studied in high-density cell preparations on tissue culture plastic dishes and on agar base. It was found that stage-24 limb mesenchymal cells initially synthesized only type I collagen. With the onset of chondrogenesis, a gradual transition to type II collagen synthesis was observed. In cell aggregates formed over agar, type II collagen synthesis started after 1 day in culture and reached levels of 80-90 percent of the total collagen synthesis at 6-8 days. At that time, the cells in the center of the aggregates had acquired the typical chondrocyte phenotype and stained only with type II collagen antibodies, whereas the peripheral cells had developed into a "perichondrium" and stained with type I and type II collagen antibodies. On plastic dishes plated with 5 X 10(6) cells per 35mm dish, cartilage nodules developed after 4-6 days, but the type II collagen synthesis only reached levels of 10-20 percent of the total collagen. The majority of the cells differentiated into fibroblasts and myoblasts and synthesized type I collagen. These studies demonstrate that analysis of cell specific types of collagen provides a useful method for detailing the specific events in the differentiation of mesenchymal cells in vitro.

scholarly journals Collagen gene expression during limb cartilage differentiation.

1986 ◽  
Vol 102 (4) ◽  
pp. 1151-1156 ◽  
Author(s):  
R A Kosher ◽  
W M Kulyk ◽  
S W Gay
Keyword(s):  
Gene Expression ◽  
Type I Collagen ◽  
Type Ii Collagen ◽  
Mesenchymal Cells ◽  
Type I ◽  
Collagen Gene ◽  
Type Ii ◽  
Collagen Mrna ◽  
Cytoplasmic Type

As limb mesenchymal cells differentiate into chondrocytes, they initiate the synthesis of type II collagen and cease synthesizing type I collagen. Changes in the cytoplasmic levels of type I and type II collagen mRNAs during the course of limb chondrogenesis in vivo and in vitro were examined using cloned cDNA probes. A striking increase in cytoplasmic type II collagen mRNA occurs coincident with the crucial condensation stage of chondrogenesis in vitro, in which prechondrogenic mesenchymal cells become closely juxtaposed before depositing a cartilage matrix. Thereafter, a continuous and progressive increase in the accumulation of cytoplasmic type II collagen mRNA occurs which parallels the progressive accumulation of cartilage matrix by cells. The onset of overt chondrogenesis, however, does not involve activation of the transcription of the type II collagen gene. Low levels of type II collagen mRNA are present in the cytoplasm of prechondrogenic mesenchymal cells at the earliest stages of limb development, well before the accumulation of detectable levels of type II collagen. Type I collagen gene expression during chondrogenesis is regulated, at least in part, at the translational level. Type I collagen mRNAs are present in the cytoplasm of differentiated chondrocytes, which have ceased synthesizing detectable amounts of type I collagen.

Type VI collagen expression is upregulated in the early events of chondrocyte differentiation

Development ◽  
1993 ◽  
Vol 117 (1) ◽  
pp. 245-251
Author(s):  
R. Quarto ◽  
B. Dozin ◽  
P. Bonaldo ◽  
R. Cancedda ◽  
A. Colombatti
Keyword(s):  
Suspension Culture ◽  
Type I Collagen ◽  
Type Ii Collagen ◽  
Type I ◽  
Type Ii ◽  
Type Vi Collagen ◽  
Full Spectrum ◽  
Collagen Expression ◽  
Hypertrophic Chondrocyte

Dedifferentiated chondrocytes cultured adherent to the substratum proliferate and synthesize large amounts of type I collagen but when transferred to suspension culture they decrease proliferation, resume the chondrogenic phenotype and the synthesis of type II collagen, and continue their maturation to hypertrophic chondrocyte (Castagnola et al., 1986, J. Cell Biol. 102, 2310–2317). In this report, we describe the developmentally regulated expression of type VI collagen in vitro in differentiating avian chondrocytes. Type VI collagen mRNA is barely detectable in dedifferentiated chondrocytes as long as the attachment to the substratum is maintained, but increases very rapidly upon passage of the cells into suspension culture reaching a peak after 48 hours and declining after 5–6 days of suspension culture. The first evidence of a rise in the mRNA steady-state levels is obtained already at 6 hours for the alpha 3(VI) chain. Immunoprecipitation of metabolically labeled cells with type VI collagen antibodies reveals that the early mRNA rise is paralleled by an increased secretion of type VI collagen in cell media. Induction of type VI collagen is not the consequence of trypsin treatment of dedifferentiated cells since exposure to the actin-disrupting drug cytochalasin or detachment of the cells by mechanical procedures has similar effects. In 13-day-old chicken embryo tibiae, where the full spectrum of the chondrogenic differentiation process is represented, expression of type VI collagen is restricted to the articular cartilage where chondrocytes developmental stage is comparable to stage I (high levels of type II collagen expression).(ABSTRACT TRUNCATED AT 250 WORDS)

Type V collagen synthesis and deposition by chicken embryo corneal fibroblasts in vitro

1989 ◽  
Vol 94 (2) ◽  
pp. 371-379
Author(s):  
J.S. McLaughlin ◽  
T.F. Linsenmayer ◽  
D.E. Birk
Keyword(s):  
Collagen Synthesis ◽  
Type I Collagen ◽  
Fibrillar Structure ◽  
Type I ◽  
Homogeneous Population ◽  
Type V Collagen ◽  
Total Collagen ◽  
Corneal Fibroblasts ◽  
Type V

Chick embryo corneal fibroblasts were grown in culture to study the processes whereby fibroblasts regulate the deposition and organization of the collagenous, secondary stroma. The effects of an existing type I collagen substratum, cell density, and serum concentration on type V collagen synthesis were investigated. Type V collagen represented approximately 20% of the total fibrillar collagen synthesized, regardless of whether the cells were subcultured, grown on untreated or collagen-coated plastic, grown under confluent or subconfluent conditions, or grown in the presence of low (0.1%) or high (10.0%) serum concentrations. The synthesis of type V collagen remained constant at 20% of the total collagen when cells were grown in 1.0% serum, even though total collagen synthesis increased nearly twofold when compared to total synthesis in 0.1% or 10.0% serum. Immunocytochemistry with anti-collagen, type-specific monoclonal antibodies revealed a homogeneous population of cells synthesizing types I and V collagen. The fibrils deposited by cells grown in a three-dimensional collagen matrix contained a helical epitope on the type V molecule that was inaccessible unless the fibrillar structure was disrupted, mimicking the situation in situ. The production in vitro of heterotypic fibrils, with a constant I/V ratio and molecular packing mimicking the natural stroma, offers opportunities for studying in more detail this important process, which is essential for optical transparency.

scholarly journals Thyroid hormone, insulin, and glucocorticoids are sufficient to support chondrocyte differentiation to hypertrophy: a serum-free analysis.

1992 ◽  
Vol 119 (4) ◽  
pp. 989-995 ◽  
Author(s):  
R Quarto ◽  
G Campanile ◽  
R Cancedda ◽  
B Dozin
Keyword(s):  
Collagen Synthesis ◽  
Type I Collagen ◽  
Type Ii Collagen ◽  
Hypertrophic Chondrocytes ◽  
Type I ◽  
Type Ii ◽  
Type X Collagen ◽  
Serum Free ◽  
Low Levels ◽  
Complete Cell

Chondrocytes from chicken embryo tibia can be maintained in culture as adherent cells in Coon's modified Ham's F-12 medium supplemented with 10% FCS. In this condition, they dedifferentiate, losing type II collagen expression in favor of type I collagen synthesis. Their differentiation to hypertrophy can be obtained by transferring them to suspension culture. Differentiation is evidenced by the shift from type I to type II and type IX collagen synthesis and the following predominant expression of type X collagen, all markers of specific stages of the differentiation process. To identify the factors required for differentiation, we developed a serum-free culture system where only the addition of triiodothyronine (T3; 10(-11) M), insulin (60 ng/ml), and dexamethasone (10(-9) M) to the F-12 medium was sufficient to obtain hypertrophic chondrocytes. In this hormonal context, chondrocytes display the same changes in the pattern of protein synthesis as described above. For proper and complete cell maturation, T3 and insulin concentrations cannot be modified. Insulin cannot be substituted by insulin-like growth factor-I, but dexamethasone concentration can be decreased to 10(-12) M without chondrogenesis being impaired. In the latter case, the expression of type X collagen and its mRNA are inversely proportional to dexamethasone concentration. When ascorbic acid is added to the hormone-supplemented medium, differentiating chondrocytes organize their matrix leading to a cartilage-like structure with hypertrophic chondrocytes embedded in lacunae. However, this structure does not present detectable calcification, at variance with control cultures maintained in FCS. Accordingly, in the presence of the hormone mixture, the differentiating chondrocytes have low levels of alkaline phosphatase activity. This report indicates that T3 and insulin are primary factors involved in the onset and progression of chondrogenesis, while dexamethasone supports cell viability and modulates some differentiated functions.

scholarly journals Synthesis of cartilage matrix by mammalian chondrocytes in vitro. III. Effects of ascorbate.

1984 ◽  
Vol 99 (6) ◽  
pp. 1960-1969 ◽  
Author(s):  
J C Daniel ◽  
B U Pauli ◽  
K E Kuettner
Keyword(s):  
Extracellular Matrix ◽  
Type I Collagen ◽  
Phenotypic Expression ◽  
Type Ii Collagen ◽  
Ruthenium Red ◽  
Type I ◽  
Type Ii ◽  
Morphological Examination ◽  
Associated Matrix

Chondrocytes isolated from bovine articular cartilage were plated at high density and grown in the presence or absence of ascorbate. Collagen and proteoglycans, the major matrix macromolecules synthesized by these cells, were isolated at times during the course of the culture period and characterized. In both control and ascorbate-treated cultures, type II collagen and cartilage proteoglycans accumulated in the cell-associated matrix. Control cells secreted proteoglycans and type II collagen into the medium, whereas with time in culture, ascorbate-treated cells secreted an increasing proportion of types I and III collagens into the medium. The ascorbate-treated cells did not incorporate type I collagen into the cell-associated matrix, but continued to accumulate type II collagen in this compartment. Upon removal of ascorbate, the cells ceased to synthesize type I collagen. Morphological examination of ascorbate-treated and control chondrocyte culture revealed that both collagen and proteoglycans were deposited into the extracellular matrix. The ascorbate-treated cells accumulated a more extensive matrix that was rich in collagen fibrils and ruthenium red-positive proteoglycans. This study demonstrated that although ascorbate facilitates the formation of an extracellular matrix in chondrocyte cultures, it can also cause a reversible alteration in the phenotypic expression of those cells in vitro.

scholarly journals Efficacy of collagen and alginate hydrogels for the prevention of rat chondrocyte dedifferentiation

2018 ◽  
Vol 9 ◽  
pp. 204173141880243 ◽  
Author(s):  
Guang-Zhen Jin ◽  
Hae-Won Kim
Keyword(s):  
Tissue Engineering ◽  
Type I Collagen ◽  
Cartilage Tissue Engineering ◽  
Collagen Gel ◽  
Type Ii Collagen ◽  
Cartilage Tissue ◽  
Type I ◽  
Type Ii ◽  
Chondrogenic Phenotype

Dedifferentiation of chondrocytes remains a major problem in cartilage tissue engineering. The development of hydrogels that can preserve chondrogenic phenotype and prevent chondrocyte dedifferentiation is a meaningful strategy to solve dedifferentiation problem of chondrocytes. In the present study, three gels were prepared (alginate gel (Alg gel), type I collagen gel (Col gel), and their combination gel (Alg/Col gel)), and the in vitro efficacy of chondrocytes culture while preserving their phenotypes was investigated. While Col gel became substantially contracted with time, the cells encapsulated in Alg gel preserved the shape over the culture period of 14 days. The mechanical and cell-associated contraction behaviors of Alg/Col gel were similar to those of Alg. The cells in Alg and Alg/Col gels exhibited round morphology, whereas those in Col gel became elongated (i.e. fibroblast-like) during cultures. The cells proliferated with time in all gels with the highest proliferation being attained in Col gel. The expression of chondrogenic genes, including SOX9, type II collagen, and aggrecan, was significantly up-regulated in Alg/Col gel and Col gel, particularly in Col gel. However, the chondrocyte dedifferentiation markers, type I collagen and alkaline phosphatase ( ALP), were also expressed at significant levels in Col gel, which being contrasted with the events in Alg and Alg/Col gels. The current results suggest the cells cultured in hydrogels can express chondrocyte dedifferentiation markers as well as chondrocyte markers, which draws attention to choose proper hydrogels for chondrocyte-based cartilage tissue engineering.

scholarly journals Type II Collagen from Cartilage of Acipenser baerii Promotes Wound Healing in Human Dermal Fibroblasts and in Mouse Skin

Marine Drugs ◽  
2020 ◽  
Vol 18 (10) ◽  
pp. 511
Author(s):  
Ching-Shu Lai ◽  
Chun-Wei Tu ◽  
Hsing-Chun Kuo ◽  
Pei-Pei Sun ◽  
Mei-Ling Tsai
Keyword(s):  
Wound Healing ◽  
Type I Collagen ◽  
Type Ii Collagen ◽  
Dermal Fibroblasts ◽  
Type I ◽  
Type Ii ◽  
Acipenser Baerii ◽  
Skin Collagen

Type II collagen is an important component of cartilage; however, little is known about its effect on skin wound healing. In this study, type II collagen was extracted from the cartilage of Acipenser baerii and its effect on in vitro and in vivo wound healing was compared to type I collagen derived from tilapia skin. Sturgeon cartilage collagen (SCC) was composed of α1 chains and with a thermal denaturation (Td) at 22.5 and melting temperature (Tm) at 72.5 °C. Coating SCC potentiated proliferation, migration, and invasion of human dermal fibroblast adult (HDFa) cells. Furthermore, SCC upregulated the gene expression of extracellular matrix (ECM) components (col Iα1, col IIIα1, elastin, and Has2) and epithelial-mesenchymal transition (EMT) molecules (N-cadherin, Snail, and MMP-1) in HDFa. Pretreatment with Akt and mitogen-activated protein kinase (MAPK) inhibitors significantly attenuated the HDFa invasion caused by SCC. In mice, the application of SCC on dorsal wounds effectively facilitated wound healing as evidenced by 40–59% wound contraction, whereas the untreated wounds were 18%. We observed that SCC reduced inflammation, promoted granulation, tissue formation, and ECM deposition, as well as re-epithelialization in skin wounds. In addition, SCC markedly upregulated the production of growth factors in the dermis, and dermal and subcutaneous white adipose tissue; in contrast, the administration of tilapia skin collagen (TSC) characterized by typical type I collagen was mainly expressed in the epidermis. Collectively, these findings indicate SCC accelerated wound healing by targeting fibroblast in vitro and in vivo.

scholarly journals Pericellular coat of chick embryo chondrocytes: structural role of hyaluronate.

1984 ◽  
Vol 99 (6) ◽  
pp. 2114-2122 ◽  
Author(s):  
R L Goldberg ◽  
B P Toole
Keyword(s):  
Cell Surface ◽  
Chondroitin Sulfate ◽  
Type I Collagen ◽  
Type Ii Collagen ◽  
Enzyme Linked Immunosorbent Assay ◽  
Type I ◽  
Type Ii ◽  
Culture Dish ◽  
Tissue Culture Dish

Chondrocytes produce large pericellular coats in vitro that can be visualized by the exclusion of particles, e.g., fixed erythrocytes, and that are removed by treatment with Streptomyces hyaluronidase, which is specific for hyaluronate. In this study, we examined the kinetics of formation of these coats and the relationship of hyaluronate and proteoglycan to coat structure. Chondrocytes were isolated from chick tibia cartilage by collagenase-trypsin digestion and were characterized by their morphology and by their synthesis of both type II collagen and high molecular weight proteoglycans. The degree of spreading of the chondrocytes and the size of the coats were quantitated at various times subsequent to seeding by tracing phase-contrast photomicrographs of the cultures. After seeding, the chondrocytes attached themselves to the tissue culture dish and exhibited coats within 4 h. The coats reached a maximum size after 3-4 d and subsequently decreased over the next 2-3 d. Subcultured chondrocytes produced a large coat only if passaged before 4 d. Both primary and first passage cells, with or without coats, produced type II collagen but not type I collagen as determined by enzyme-linked immunosorbent assay. Treatment with Streptomyces hyaluronidase (1.0 mU/ml, 15 min), which completely removed the coat, released 58% of the chondroitin sulfate but only 9% of the proteins associated with the cell surface. The proteins released by hyaluronidase were not digestible by bacterial collagenase. Monensin and cycloheximide (0.01-10 microM, 48 h) caused a dose-dependent decrease in coat size that was linearly correlated to synthesis of cell surface hyaluronate (r = 0.98) but not chondroitin sulfate (r = 0.2). We conclude that the coat surrounding chondrocytes is dependent on hyaluronate for its structure and that hyaluronate retains a large proportion of the proteoglycan in the coat.

scholarly journals Synthesis of cartilage matrix by mammalian chondrocytes in vitro. II. Maintenance of collagen and proteoglycan phenotype.

1982 ◽  
Vol 93 (3) ◽  
pp. 751-757 ◽  
Author(s):  
K E Kuettner ◽  
V A Memoli ◽  
B U Pauli ◽  
N C Wrobel ◽  
E J Thonar ◽  
...  
Keyword(s):  
Type I Collagen ◽  
Articular Chondrocytes ◽  
Type Ii Collagen ◽  
Type I ◽  
Type Ii ◽  
Roller Bottle ◽  
The Media ◽  
Bovine Articular Chondrocytes ◽  
Associated Matrix

The in vitro phenotype of bovine articular chondrocytes is described. Chondrocytes plated at high density in roller-bottle and dish cultures were maintained in vitro. The major matrix macromolecules, collagen and proteoglycan, synthesized by these cells were characterized during the course of the culture period. The chondrocytes synthesized mainly Type II collagen, which was found predominantly in the cell-associated matrix. The media contained a mixture of Type II and Type III collagens. Type I collagen was detectable in neither the medium nor the cell-associated matrix. The proteoglycan monomers found in media and cell-associated matrix had the same hydrodynamic sizes as monomers synthesized by cartilage slices or those extracted from adult articular cartilage. The majority of proteoglycans synthesized by the cells were found in high molecular weight aggregates which were readily recovered from the media and were extractable from cell-associated matrix with low ionic strength buffers. The results demonstrate the long-term in vitro phenotypic stability of the bovine articular chondrocytes. The advantages of the in vitro system as a model for studying the effects of external agents, such as drugs and vitamins, are discussed.

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