scholarly journals Selective emergence of differentiated chondrocytes during serum-free culture of cells derived from fetal rat calvaria.

1982 ◽  
Vol 92 (2) ◽  
pp. 493-504 ◽  
Author(s):  
L Rifas ◽  
J Uitto ◽  
V A Memoli ◽  
K E Kuettner ◽  
R W Henry ◽  
...  
Keyword(s):  
Primary Culture ◽  
Articular Chondrocytes ◽  
Type Ii Collagen ◽  
Progressive Increase ◽  
Ruthenium Red ◽  
Type I ◽  
Type Ii ◽  
Chondrocyte Phenotype ◽  
Rat Calvaria ◽  
Fetal Rat

Cells dispersed from the chondrocranial portions of fetal rat calvaria proliferated and performed specialized functions during primary culture in a chemically defined medium. Mature cultures were typified by multilayered clusters of redifferentiating cartilage cells. Flattened cells that lacked distinguishing features occupied areas between the clusters. Alkaline phosphate-enriched, ultrastructurally typical chondrocytes within the clusters were encased in a dense extracellular matrix that stained prominently for chondroitin sulfate proteoglycans. This matrix contained fibrils measuring 19 nm in diameter, which were associated with proteoglycan granules that preferentially bound ruthenium red. A progressive increase in the number of cells indicated the proliferation of certain elements in the primary culture. The cells in primary culture were biochemically as well as morphologically heterogeneous since they were found to synthesize type I and type II collagens. Homogeneous populations of redifferentiated chondrocytes were recovered as floating cells and were shown to express the chondrocyte phenotype in secondary culture. Subcultured cells synthesized type II collagen and its precursors almost exclusively and incorporated 35SO4 into proteoglycan monomer and aggregates to a greater degree than the cells in primary culture. The pattern of proteoglycan monomer and aggregate labeling resembled that of intact cartilage segments and bovine articular chondrocytes. Skin fibroblasts harvested from the same rat fetuses failed to proliferate when maintained under identical conditions. Hence, exogenous hormones, growth factors, and protein are not required for chondrocyte growth and maturation.

scholarly journals Maintenance of chondrocyte phenotype during expansion on PLLA microtopographies

2018 ◽  
Vol 9 ◽  
pp. 204173141878982 ◽  
Author(s):  
Elisa Costa ◽  
Cristina González-García ◽  
José Luis Gómez Ribelles ◽  
Manuel Salmerón-Sánchez
Keyword(s):  
Lactic Acid ◽  
Type I Collagen ◽  
Collagen Type ◽  
Articular Chondrocytes ◽  
Collagen Type I ◽  
Cell Phenotype ◽  
Type I ◽  
Type Ii ◽  
Collagen Type Ii ◽  
Chondrocyte Phenotype

Articular chondrocytes are difficult to grow, as they lose their characteristic phenotype following expansion on standard tissue culture plates. Here, we show that culturing them on surfaces of poly(L-lactic acid) of well-defined microtopography allows expansion and maintenance of characteristic chondrogenic markers. We investigated the dynamics of human chondrocyte dedifferentiation on the different poly(L-lactic acid) microtopographies by the expression of collagen type I, collagen type II and aggrecan at different culture times. When seeded on poly(L-lactic acid), chondrocytes maintained their characteristic hyaline phenotype up to 7 days, which allowed to expand the initial cell population approximately six times without cell dedifferentiation. Maintenance of cell phenotype was afterwards correlated to cell adhesion on the different substrates. Chondrocytes adhesion occurs via the α5 β1 integrin on poly(L-lactic acid), suggesting cell–fibronectin interactions. However, α2 β1 integrin is mainly expressed on the control substrate after 1 day of culture, and the characteristic chondrocytic markers are lost (collagen type II expression is overcome by the synthesis of collagen type I). Expanding chondrocytes on poly(L-lactic acid) might be an effective solution to prevent dedifferentiation and improving the number of cells needed for autologous chondrocyte transplantation.

scholarly journals Microfilament modification by dihydrocytochalasin B causes retinoic acid-modulated chondrocytes to reexpress the differentiated collagen phenotype without a change in shape.

1988 ◽  
Vol 106 (1) ◽  
pp. 161-170 ◽  
Author(s):  
P D Benya ◽  
P D Brown ◽  
S R Padilla
Keyword(s):  
Retinoic Acid ◽  
Cell Shape ◽  
Articular Chondrocytes ◽  
Type Ii Collagen ◽  
Phenotypic Change ◽  
Type Iii Collagen ◽  
Type I ◽  
Type Ii ◽  
The Stability ◽  
Rabbit Articular Chondrocytes

Primary monolayers of rabbit articular chondrocytes synthesize high levels of type II collagen and proteoglycan. This capacity was used as a marker for the expression of the differentiated phenotype. Such cells were treated with 1 microgram/ml retinoic acid (RA) for 10 d to produce a modulated collagen phenotype devoid of type II and consisting of predominantly type I trimer and type III collagen. After transfer to secondary culture in the presence of RA, the stability of the RA-modulated phenotype was investigated by culture in the absence of RA. Little reexpression of type II collagen synthesis occurred in this period unless cultures were treated with 3 X 10(-6) M dihydrocytochalasin B to modify microfilament structures. Reexpression of the differentiated phenotype began between days 6-8 and was essentially complete by day 14. Substantial reexpression occurred by day 8 without a detectable increase in cell rounding. Colony formation, characteristic of primary chondrocytes, was infrequent even after reexpression was complete. These data suggest that the integrity of microfilament cytoskeletal structures can be a source of regulatory signals that mechanistically appear to be more proximal to phenotypic change than the overt changes in cell shape that accompany reexpression of subculture-modulated chondrocytes in agarose culture.

Effect of seeding density on stability of the differentiated phenotype of pig articular chondrocytes in culture

1988 ◽  
Vol 89 (3) ◽  
pp. 373-378
Author(s):  
F.M. Watt
Keyword(s):  
Type I Collagen ◽  
Articular Chondrocytes ◽  
Type Ii Collagen ◽  
High Density ◽  
Seeding Density ◽  
Type I ◽  
Low Density ◽  
Keratan Sulphate ◽  
Chondrocyte Phenotype ◽  
Culture Cells

Articular chondrocytes are known to be phenotypically unstable in culture. One condition that has been reported to suppress dedifferentiation is cultivation at high density on tissue-culture plastic. The aim of the experiments described here was to study the effect of seeding density on chondrocyte proliferation and 35SO4 incorporation, and on the types of collagen and proteoglycan synthesized. I found that cells seeded at low or high density reached the same final density at confluence, and that 35SO4 incorporation, while initially higher (per cell) in high-density cultures, fell under both conditions, reaching the same low level after 3 weeks. The proportion of cells expressing keratan sulphate fell in low- but not high-density cultures and the decline was not prevented by inhibition of cell division. In all the cultures cells expressing keratan sulphate tended to have a rounded morphology. After 21 days in culture, chondrocytes grown at high density expressed predominantly large proteoglycans that aggregated with hyaluronic acid, whereas in low-density cultures a smaller, non-aggregating form was also present. By 21 days in culture cells at both high and low density were expressing type I collagen, although the high-density cells also had an extensive extracellular matrix of type II collagen. These observations support the conclusion that high seeding density stabilizes the chondrocyte phenotype to a greater extent than low seeding density. They also suggest that enhanced dedifferentiation at low density may be due to cell spreading, rather than to selective proliferation of a phenotypically unstable subpopulation of cells.

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.

Doxycycline Inhibits Collagen Synthesis by Differentiated Articular Chondrocytes

1998 ◽  
Vol 12 (1) ◽  
pp. 63-67 ◽  
Author(s):  
J.M. Tekoppele ◽  
B. Beekman ◽  
N. Verzijl ◽  
J.L. Koopman ◽  
J. Degroot ◽  
...  
Keyword(s):  
Collagen Synthesis ◽  
Articular Chondrocytes ◽  
Cruciate Ligament ◽  
Type Ii Collagen ◽  
Transcriptional Level ◽  
Mrna Levels ◽  
Type Ii ◽  
Chondrocyte Phenotype ◽  
Experimental Osteoarthritis

Doxycycline (DOX) profoundly inhibited collagen synthesis by differentiated articular chondrocytes. At 25 μM, the rate of collagen synthesis was suppressed by more than 50% without affecting cell proliferation (DNA levels) and general protein synthesis (35S-Met and 35S-Cys incorporation). Steady-state mRNA levels of type II collagen were also reduced, indicating that DOX may have an effect at the transcriptional level of type II collagen. The IC50 value of DOX to downregulate collagen synthesis (17 μM) is close to DOX levels attained in vivo (< 10 μM), and it is more than ten-fold lower than the IC50 values to inhibit the activity of most matrix metalloproteinases (MMPs). As such, these findings support the hypothesis that the reduced severity of OA observed in the dog anterior cruciate ligament model resulting from prophylactic treatment with DOX may involve mechanisms other than MMP inhibition alone. Our findings suggest that prevention of changes in the chondrocyte phenotype may be involved in the beneficial effect of doxycycline in experimental osteoarthritis. for differentiated chondrocytes in early stages of osteoarthritis exhibit elevated collagen synthesis.

Comparative Phenotypic Analysis of Articular Chondrocytes Cultured within Type I or Type II Collagen Scaffolds

2009 ◽  
Vol 15 (6) ◽  
pp. 1233-1245 ◽  
Author(s):  
Anne-Marie Freyria ◽  
Marie-Claire Ronzière ◽  
Delphine Cortial ◽  
Laurent Galois ◽  
Daniel Hartmann ◽  
...  
Keyword(s):  
Articular Chondrocytes ◽  
Type Ii Collagen ◽  
Type I ◽  
Type Ii ◽  
Collagen Scaffolds ◽  
Phenotypic Analysis

scholarly journals PTH decreases in vitro human cartilage regeneration without affecting hypertrophic differentiation

2019 ◽  
Author(s):  
Marijn Rutgers ◽  
Frances Bach ◽  
Luciënne Vonk ◽  
Mattie van Rijen ◽  
Vanessa Akrum ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Parathyroid Hormone ◽  
Articular Chondrocytes ◽  
Type Ii Collagen ◽  
Human Articular Chondrocytes ◽  
Type I ◽  
Human Articular Cartilage ◽  
Type Ii ◽  
Collagen Deposition ◽  
Healthy Human

AbstractRegenerated cartilage formed after Autologous Chondrocyte Implantation may be of suboptimal quality due to postulated hypertrophic changes. Parathyroid hormone-related peptide, containing the parathyroid hormone sequence (PTHrP 1-34), enhances cartilage growth during development and inhibits hypertrophic differentiation of mesenchymal stromal cells (MSCs) and growth plate chondrocytes. This study aims to determine whether human articular chondrocytes respond correspondingly. Healthy human articular cartilage-derived chondrocytes (n=6 donors) were cultured on type II collagen-coated transwells with/without 0.1 or 1.0 μM PTH from day 0, 9, or 21 until the end of culture (day 28). Extracellular matrix production, (pre)hypertrophy and PTH signaling were assessed by RT-qPCR and/or immunohistochemistry for collagen type I, II, X, RUNX2, MMP13, PTHR1 and IHH and by determining glycosaminoglycan production and DNA content. The Bern score assessed cartilage quality by histology. Regardless of the concentration and initiation of supplementation, PTH treatment significantly decreased DNA and glycosaminoglycan content and reduced the Bern score compared with controls. Type I collagen deposition was increased, whereas PTHR1 expression and type II collagen deposition were decreased by PTH supplementation. Expression of the (pre)hypertrophic markers MMP13, RUNX2, IHH and type X collagen were not affected by PTH. In conclusion, PTH supplementation to healthy human articular chondrocytes did not affect hypertrophic differentiation, but negatively influenced cartilage quality, the tissues’ extracellular matrix and cell content. Although PTH may be an effective inhibitor of hypertrophic differentiation in MSC-based cartilage repair, care may be warranted in applying accessory PTH treatment due to its effects on articular chondrocytes.

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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