Adult Human Articular Chondrocytes Research Articles

Adult human chondrocytes in alginate culture. Preservation of the phenotype for further use in transplantation models

Successful and cartilage-specific cultivation of chondrocytes requires a stable phenotype during the in vitro culture period. This is based on a differentiated extracellular matrix synthesis. The alginate system as a three-dimensional support is a useful system to culture chondrocytes and to analyze the biochemical processes in this system. Talar cartilage from the talocrural joints of 40 different donors were obtained through the Regional Organ Bank of Illinois within 24 hours of death. In 65% of the tissues the cartilage was classified as being undamaged. In these studies we were interested in the results of short-term culture over 14 days. Cell proliferation, total collagen content and total proteoglycan content were measured in the different matrix compartments and were visualized by histology and immunohistochemistry. Already after 7 days in culture the adult human chondroctes looked intact and formed a stable and cell-associated cartilage-specific extracellular matrix in the presence of 10% calf serum. This could be also demonstrated in the presence of IGF-I. With regards to the collagen content IGF-I at a concentration of 50 ng/ml seemed to induce an equal effect to 10% serum; with regards to the proteoglycan content IGF-I at a concentration of 20 ng/ml was equivalent. These encouraging preliminary results may lead to a new approach in tissue engineering for chondrocyte transplantation in combination with their extracellular matrix.

Modulation of keratan sulfate synthesis on lumican by the action of cytokines on human articular chondrocytes.

Adult human articular chondrocytes were used to investigate why keratan sulfate/polylactosamine chains are deficient on the lumican residing in the matrix of adult articular cartilage, whereas they are present on the lumican residing in the matrix of juvenile cartilage. Under serum-free conditions with either monolayer cultures, agarose cultures, or micromass cultures, the adult chondrocytes synthesized a form of lumican possessing keratan sulfate/polylactosamine chains. Thus, the adult chondrocytes are capable of producing a proteoglycan form of lumican and this appears to be the default synthesis preference. The micromass culture system proved useful for demonstrating that growth factors/cytokines present in the extracellular milieu are capable of influencing the structure of the keratan sulfate/polylactosamine chains on the secreted lumican. Of particular note was the ability of IL-1beta to promote the secretion of a form of lumican deficient in keratan sulfate/polylactosamine chains, whereas with bFGF, IGF-1 and TGFbeta keratan sulfate/polylactosamine chains were present, though their size or degree of substitution varied. Thus, growth factors/cytokines are able to modulate the molecular form of lumican. Furthermore, additional studies showed that this modulation was not due to the degradation of keratan sulfate/polylactosamine chains following proteoglycan secretion, but represented a direct effect on synthesis.

Expression of NG2/human melanoma proteoglycan in human adult articular chondrocytes

Objective: NG2 is a transmembrane chondroitin (CS) rich proteoglycan originally identified in rats. It has recently been shown to be identical to human melanoma proteoglycan (HMPG). In rats NG2 has a limited distribution in adult tissues, being expressed predominantly by neuronal and glial cells whereas during development it is also expressed in developing mesenchyme including cartilage. NG2/HMPG has putative roles in interactions between glial and melanoma cells with extracellular matrix (ECM) molecules. This study was undertaken to assess whether NG2/HMPG was expressed by normal and osteoarthritic human articular chondrocytes.Design: Cryostat sections of human fetal knee joints and normal and osteoarthritic articular cartilage were immunostained with antibodies against rat NG2 (N143.8) and HMPG (M28B5, 9.2.27). Immunoprecipitation and Western blotting was carried out on protein extracts of chondrocytes from normal and osteoarthritic cartilage. Immunofluorescence of NG2 and potential ligands was carried outin vitroon cells from normal and osteoarthritis cartilage.Results: Fetal and both normal and osteoarthritic adult cartilage showed strong immunoreactivity for NG2/HMPG. Western blotting showed a smeared component of molecular weight greater than 400 kDa and a faint band at 250 kDa which became predominant upon digestion with chondroitinase ABC. Immunofluorescence of chondrocytesin vitroshowed NG2 to be distributed in a punctate pattern without co-localization of actin or several ECM proteins including fibronectin and type VI collagen.Conclusion: NG2/HMPG is expressed by human fetal and adult chondrocytes and in adult articular chondrocytes the core protein is chondroitin sulfated. The function of this molecule in human articular cartilage remains to be defined.

Expression of a stable articular cartilage phenotype without evidence of hypertrophy by adult human articular chondrocytes in vitro.

Chondrocytes that were isolated from adult human articular cartilage changed phenotype during monolayer tissue culture, as characterized by a fibroblastic morphology and cellular proliferation. Increased proliferation was accompanied by downregulation of the cartilage-specific extracellular matrix proteoglycan, aggrecan, by cessation of type-II collagen expression, and by upregulation of type-I collagen and versican. This phenomenon observed in monolayer was reversible after the transfer of cells to a suspension culture system. The transfer of chondrocytes to suspension culture in alginate beads resulted in the rapid upregulation of aggrecan and type-II collagen and the downregulation of expression of versican and type-I collagen. Type-X collagen and osteopontin, markers of chondrocyte hypertrophy and commitment to endochondral ossification, were not expressed by adult articular chondrocytes cultured in alginate, even after 5 months. In contrast, type-X collagen was expressed within 2 weeks in a population of cells derived from a fetal growth plate. The inability of adult articular chondrocytes to express markers of chondrocyte hypertrophy has underscored the fundamental distinction between the differentiation pathways that lead to articular cartilage or to bone. Adult articular chondrocytes expressed only hyaline articular cartilage markers without evidence of hypertrophy.

Synergistic Action of Transforming Growth Factor-β and Insulin-like Growth Factor-I Induces Expression of Type II Collagen and Aggrecan Genes in Adult Human Articular Chondrocytes

Reexpression of aggrecan and type II collagen genes in dedifferentiated adult human articular chondrocytes (AHAC) in suspension culture varied widely depending on the specific lot of bovine serum used to supplement the culture medium. Some lots of serum provided strong induction of aggrecan and type II collagen expression by AHAC while others did not stimulate significant production of these hyaline cartilage extracellular matrix molecules even following several weeks in culture. Addition of 50 ng/ml insulin-like growth factor-I (IGF-I) to a deficient serum lot significantly enhanced its ability to induce aggrecan and type II collagen mRNA. Given this observation, IGF-I and other growth factors were tested in defined serum-free media for their effects on the expression of these genes. Neither IGF-I nor insulin nor transforming growth factor β (TGF-β) alone stimulated induction of aggrecan or type II collagen production by dedifferentiated AHAC. However, TGF-β1 or TGF-β2 combined with IGF-I or insulin provided a strong induction as demonstrated by RNase protection and immunohistochemical assays. Interestingly, type I collagen, previously shown to be downregulated in serum supplemented suspension cultures of articular chondrocytes, persisted for up to 12 weeks in AHAC cultured in defined medium supplemented with TGF-β and IGF-I.

Growth factor responsiveness of human articular chondrocytes in aging and development

To examine growth factor responses of human articular chondrocytes in aging and development. We have previously established a growth factor response profile for adult human articular chondrocytes and shown that transforming growth factor beta (TGF beta) is the most potent mitogen among a variety of factors tested. Chondrocytes were isolated from human articular cartilage obtained from donors ages 11-83 years and tested in primary culture for proliferative responses to serum and recombinant preparations of the major chondrocyte growth factors. Proliferation was measured by 3H-thymidine incorporation and cell counting. Skeletal maturity of the young donors was determined by radiographic assessment of Risser's index. Chondrocytes showed a continuous age-related decline in the proliferative response to serum. Analysis of recombinant growth factors showed that with increasing donor age, there was a decrease in the levels of DNA synthesis in response to all factors tested. In chondrocytes from adult donors, there was no change in the relative potencies of the different growth factors. The decrease in the levels of DNA synthesis as measured by 3H-thymidine incorporation corresponded to a reduced rate of in vitro cell replication with increasing donor age. In addition to the quantitative changes in the proliferative responses of chondrocytes with increasing age, there was a qualitative change in the pattern of growth factor responses during development. Cells from young donors (ages 10-20) responded better to platelet-derived growth factor, AA chain homodimer (PDGF-AA) than to TGF beta 1, while the inverse pattern was seen in cells from adult donors. This decrease in the response to PDGF-AA was significantly correlated with increasing skeletal maturity. Chondrocyte growth factor responsiveness shows qualitative changes during development, and after skeletal maturity, there is a profound decline in the levels of DNA synthesis and cell replication in response to the known chondrocyte growth factors.

Expression of cartilage-specific molecules is retained on long-term culture of human articular chondrocytes

Normal human adult articular chondrocytes were used to determine how the chondrocyte phenotype is modulated by culture conditions following long-term culture. We report here for the first time that human articular chondrocytes have a lifespan in the range of 34-37 population doublings. While chondrocytes cultured as monolayers displayed a fibroblastoid morphology and grew faster, those cultured as suspensions over agarose adopted a round morphology and formed clusters of cells reminiscent of chondrocyte differentiation in intact cartilage, with little or no DNA synthesis. These morphologies were independent of the age of the culture. Despite, these morphological differences, however, chondrocytes expressed markers at mRNA and protein levels characteristic of cartilage: namely, types II and IX collagens and the large aggregating proteoglycans, aggrecan, versican and link protein, but not syndecan, under both culture conditions. However, they also expressed type I collagen alpha 1(I) and alpha 2(I) chains. It has been suggested that expression of collagen alpha 1(I) by chondrocytes cultured as monolayers is a marker of the loss of the chondrocyte phenotype. However, we show here, using reverse transcriptase/polymerase chain reaction, that normal fresh intact human articular cartilage expresses collagen alpha 1(I). The data show that following long-term culture human articular chondrocytes retain their differentiated characteristics and that cell shape does not correlate with the expression of the chondrocyte phenotype. It is proposed that loss of the chondrocyte phenotype is marked by the loss of one or more cartilage-specific molecules rather than by the appearance of non-cartilage-specific molecules.

Growth factor responsiveness of human articular chondrocytes: Distinct profiles in primary chondrocytes, subcultured chondrocytes, and fibroblasts

The objectives of this study were to establish a growth factor response profile for adult human articular chondrocytes, to determine whether this is unique for chondrocytes or influenced by the differentiation status of the cells, and to characterize growth factor interactions. It is shown that transforming growth factor-beta (TGF-beta) is the most potent mitogen among a variety of factors tested. All three isoforms of TGF-beta caused similar dose-dependent increases in chondrocyte proliferation. Other members of the TGF-beta family, including bone morphogenetic protein 2B (BMP2B), activin, and inhibin, did not detectably increase chondrocyte proliferation. Platelet-derived growth factor-AA (PDGF-AA), basic fibroblast growth factor (bFGF), and insulin-like growth factor 1 (IGF-1) also stimulated proliferation but were less effective than TGF-beta. In contrast to findings with other cell types, the effects of TGF-beta on chondrocyte proliferation were not dependent on the endogenous production of PDGF. The cytokines Interleukin 1 (IL-1) and tumor necrosis factor-alpha (TNF-alpha) gave no stimulation, but IL-1 inhibited chondrocyte proliferation induced by TGF-beta or serum. This response profile was characteristic for primary chondrocytes from human adults and distinct from subcultured (dedifferentiated) chondrocytes or skin fibroblasts. The latter preferentially responded to PDGF, and IL-1 caused greater increases in proliferation than TGF-beta. In summary, these results describe growth factor responses that are characteristic for chondrocytes and provide a basis for the analysis of changes in chondrocyte growth proliferation that occur in aging and tissue injury.

Synthesis and Turnover of Proteoglycans by Human and Bovine Adult Articular Chondrocytes Cultured in Alginate Beads

Human and bovine adult articular chondrocytes cultured in alginate beads or agarose gel retain their spherical shape and typical chondrocytic appearance for at least 5 weeks. Aggrecan is always the major population of proteoglycans (PGs) synthesized; its size varies depending upon the age of the cartilage from which the cells are derived but it is not influenced by the culture system used. Studies of human chondrocytes cultured in alginate showed that the majority of the newly-synthesized aggrecan molecules are rapidly incorporated into aggregates which can be extracted from the gel in their native form. About one-third of the aggregates formed during a 4-hour period of incubation in the presence of [35S]-sulfate are packed at high concentration in the cell-associated matrix; the remainder is present in areas of the gel further removed from the cells. Aggrecan appears to be turned over more slowly by human than by bovine cells in both culture systems; in both species, the rate of turnover is slower in alginate than in agarose. Decorin is also synthesized in small amounts but it is rapidly lost from the agarose or alginate gel.

Detection of mRNA for the transforming growth factor β family in human articular chondrocytes by the polymerase chain reaction

The Transforming Growth Factor-beta (TGF beta) family of polypeptides elicits diverse biological actions on a wide range of cell types. There are known to be several isoforms of TGF beta coded for by different genes, with possibly differential expression and potencies. We have demonstrated that there is constitutive expression of three forms of transforming growth factor beta in adult human articular chondrocytes. The presence of 10% fetal calf serum in the culture medium may influence expression. The addition of transforming growth factor beta or interleukin 1 beta to the culture medium does not appear to consistently influence the expression of TGF beta by the cells.

97 HUMAN FETAL AND ADULT CHONDROCYTES: CLONAL GROWTH PROMOTION BY INSULIN LIKE GROWTH FACTORS I AND II, INSULIN AND GROWTH HORMONE

A direct assay for cellular growth was used. It measures the capacity of isolated chondrocytes to proliferate in a clonal manner starting from a single cell suspension in a semisolid system (0.8% methylcellulose, BM Whissler Medium, 5% heat inactivated serum, 1000 inserted chondrocytes/ml). This assay was used to measure the clonal proliferation of human fetal (20th week of pregnancy, n=10) and adult articular chondrocytes 20yrs, n= 10) in response to insulin like growth factors I and II (IGF I/II), biosynthetic human insulin (BHI) and human growth hormone (hGh). Both BHI (100 ng/ml) and hGh (100 ng/ml) did not stimulate fetal and adult chondrocytes to proliferate in a clonal manner. IGF I (25 ng/ml) stimulated clonal growth of fetal chordrocytes (60 ± 12 colonies/1000 inserted cells; m ± 1 SD), however IGF II (25 ng/ml) was significantly more effective (116 ± 12 colonies/1000 inserted cells; p < 0.05). In contrast IGF I was more effective in adult chondrocytes (40 ± 5 colonies/1000 inserted cells) than IGF II (24 ± 2 colonies/1000 inserted cells; p < 0.05).

Sulfate incorporation by articular chondrocytes in monolayer culture.

AbstractArticular chondrocytes in primary monolayers or subcultures synthesize sulfated macromolecules, presumably mucopolysaccharides. Intracellular 35SO4 incorporation by postnatal rabbit chondrocytes reached a steady level by 24 hr of incubation; the nondialyzable extracellular (centrifuged medium + trypsin digest) counts increased over 72 hr, when they were up to 100 times the intracellular value. Cell for cell, radiosulfate counts in each fraction were higher in chondrocytes than fibroblasts. Data from a 3‐year‐old rabbit were comparable to those from recently weaned animals. Secretion of sulfated mucopolysaccharide was also found in cultures of adult human and bovine articular chondrocytes. Although Ham's F12 medium is advantageous in establishing a primary culture of chondrocytes, sulfate incorporation was greater when Dulbecco‐Eagle medium, with or without supplemental L‐proline, was used for subcultures. Ascorbic acid (5 mg/100 ml) added to an already confluent monolayer had no consistent effect.