Dedifferentiated chondrocytes reexpress the differentiated collagen phenotype when cultured in agarose gels

The differentiated phenotype of rabbit articular chondrocytes consists primarily of type II collagen and cartilage-specific proteoglycan. During serial monolayer culture this phenotype is lost and replaced by a complex collagen phenotype consisting predominately of type I collagen and a low level of proteoglycan synthesis. Such dedifferentiated chondrocytes reexpress the differentiated phenotype during suspension culture in firm gels of 0.5% low T_m agarose. Approximately 80% of the cells survive this transition from the flattened morphology of anchorage-dependent culture to the spherical morphology of anchorage-independent culture and then deposit characteristic proteoglycan matrix domains. The rates of proteoglycan and collagen synthesis return to those of primary chondrocytes. Using SDS-polyacrylamide gel electrophoresis of intact collagen chains and two-dimensional cyanogen bromide peptide mapping, we demonstrated a complete return to the differentiated collagen phenotype. These results emphasize the primary role of cell shape in the modulation of the chondrocyte phenotype and demonstrate a reversible system for the study of gene expression.     

Proteoglycan production by immortalized human chondrocyte cell lines cultured under conditions that promote expression of the differentiated phenotype

Large and small proteoglycans are essential components of articular cartilage. How to induce chondrocytes to repair damaged cartilage with normal ratios of matrix components after their loss due to degenerative joint disease has been a major research focus. We have developed immortalized human chondrocyte cell lines for examining the regulation of cartilage-specific matrix gene expression. However, the decreased synthesis and deposition of cartilage matrix associated with a rapid rate of proliferation has presented difficulties for further examination at the protein level. In these studies, proteoglycan synthesis was characterized in two chondrocyte cell lines, T/C-28a2 and tsT/AC62, derived, respectively, from juvenile costal and adult articular cartilage, under culture conditions that either promoted or decreased cell proliferation. Analysis of proteo[36S]glycans by Sepharose CL-4B chromatography and SDS-PAGE showed that the large proteoglycan aggrecan and the small, leucine-rich proteoglycans, decorin and biglycan, were produced under every culture condition studied. In monolayer cultures, a high initial cell density and conditions that promoted proliferation (presence of serum for T/C-28a2 cells or permissive temperature for the temperature-sensitive tsT/AC62 cells) favored cell survival and ratios of proteoglycans expected for differentiated chondrocytes. However, the tsT/AC62 cells produced more proteoglycans at the nonpermissive temperature. Culture of cells suspended in alginate resulted in a significant decrease in proteoglycan production in all culture conditions. While the tsT/AC62 cells continued to produce a larger amount of aggrecan than small proteoglycans, the T/C-28a2 cells lost the ability to produce significant amounts of aggrecan in alginate culture. In addition, our data indicate that immortalized chondrocytes may alter their ability to retain pericellular matrix under changing culture conditions, although the production of the individual matrix components does not change. These findings provide critical information that will assist in the development of a reproducible chondrocyte culture model for the study of regulation of proteoglycan biosynthesis in cartilage.     

Seeding density modulates migration and morphology of rabbit chondrocytes cultured in collagen gels

The cultures of rabbit chondrocytes embedded in collagen gels were conducted to investigate the cell behaviors and consequent architectures of cell aggregation in an early culture phase. The chondrocyte cells seeded at 1.0 x 10(5) cells/cm(3) underwent a transition to spindle-shaped morphology, and formed the loose aggregates with a starburst shape by means of possible migration and gathering. These aggregates accompanied the poor production of collagen type II, while the cells seeded at 1.6 x 10(6) cells/cm(3) exhibited active proliferation to form the dense aggregates rich in collagen type II. Stereoscopic observation was performed at 5 days to define the migrating cells in terms of a morphology-relating parameter of sphericity determined for individual cells in the gels. The frequency of migrating cells decreased with increasing seeding density, while the frequency of dividing cells showed the counter trend. The culture seeded at 1.0 x 10(5) cells/cm(3) gave the migrating cell frequency of 0.25, the value of which was 25 times higher than that at 1.6 x 10(6) cells/cm(3). In addition, the analysis of mRNA expression revealed that the chondrocyte cells seeded at 1.0 x 10(5) cells/cm(3) showed appreciable down-regulation in collagen type II relating to differentiation and up-regulation in matrix metalloproteinases relating to migration, as compared to the cells seeded at 1.6 x 10(6) cells/cm(3). These data supports the morphological analyses concerning the cell migration and aggregate formation in the cultures with varied seeding densities. It is concluded that the seeding density is an important factor to affect the cell behaviors and architecture of aggregates and thereby to modulate the quality of cultured cartilage.     

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

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 pericellular matrix formation by chondrocytes cultured in agarose gel on the viscoelastic properties of agarose gel matrix

The viscoelasticity of chondrocyte-seeded agarose gel (AGC0) and that of chondrocyte-seeded agarose gel after 21 days of cultivation (AGC3) were investigated. In AGC3, pericellular matrix (PCM)-like material around each chondrocyte was found to be constructed, which was confirmed by an optical micrograph in conjunction with toluidine blue staining. The relaxation modulus of each of the chondrocyte-agarose gel composite systems was measured by a non-constrained indentation method. Stress-strain curves for all of the specimens examined had a toe region followed by a linear region terminated by specimen fracture. The slope of the linear region of AGC0 was smaller than that of AG, while the SS curve of AGC0 was indistinguishable from that of AGC3. All of the relaxation curves studied were typical of gels, having a fast relaxation process up to 10³ s followed by a plateau. The relaxation modulus of AGC0 was smaller than that of agarose gel (AG), the decrement in relaxation modulus from AG to AGC0 being attributed to the seeding of chondrocytes that have a smaller modulus than that of agarose gels. However, the relaxation modulus of AGC3 was increased at the early viscoelastic region in particular, as compared with that of AGC0. The increments in the relaxation modulus in AGC3 were attributed to the PCM-like material produced by chondrocytes, where the produced material may provide crosslink points and reinforce the agarose gel.     

Stimulation by glucocorticoids of the differentiated phenotype of chondrocytes and the proliferation of rabbit costal chondrocytes in culture

Hydrocortisone stimulated glycosaminoglycan (GAG) synthesis, a characteristic of the cartilage phenotype, of rabbit costal chondrocytes in confluent quiescent culture, as judged by the incorporations of [35S]sulfate and [3H]glucosamine. Hydrocortisone also stimulated incorporation of [3H]serine into proteoglycan. The stimulation of GAG synthesis by hydrocortisone was dose-dependent and maximal at a physiological concentration of 10(-7) M. Hydrocortisone also stimulated GAG synthesis in cultures in the log-phase of growth. In this case, its maximal effect was observed at a concentration of 10(-6) M. The magnitude of the increase of GAG synthesis in response to hydrocortisone was larger in confluent culture than in log-phase cultures. Hydrocortisone stimulated DNA synthesis dose-dependently, and its effect was observable at a physiological concentration. However, no stimulation of DNA synthesis by hydrocortisone was observed in serum-free medium, in contrast to that of GAG synthesis. Hydrocortisone also increased protein synthesis and the cell number. Dexamethasone also stimulated the syntheses of both GAG and DNA. These results show that glucocorticoids stimulated both the differentiated phenotype of chondrocytes and the proliferation of rabbit costal chondrocytes in culture. Moreover, the effect of glucocorticoids was primarily on the differentiated phenotype of chondrocytes and its effect on proliferation was permissive.     

Cartilage proteoglycan aggregate and fibronectin can modulate the expression of type X collagen by embryonic chick chondrocytes cultured in collagen gels

Chick embryo sternal chondrocytes from the caudal and cephalic regions were cultured within type I collagen gels and type I collagen/proteoglycan aggregate composite gels in normal serum. Caudal region chondrocytes were also cultured within type I collagen gels in the presence of fibronectindepleted serum. There was a marked stimulation of type X collagen synthesis by the caudal region chondrocytes after 9 days in the presence of fibronectin-depleted serum and after 14 days in the presence of proteoglycan aggregate. These results provide evidence for the ability of chondrocytes from a zone of permanent cartilage to synthesise type X collagen and for the involvement of extracellular matrix components in the control of type X collagen gene expression.     

Effect of relaxin on the phenotype of collagens synthesized by cultured rabbit chondrocytes

The effect of porcine relaxin on rabbit articular and growth plate chondrocytes in primary culture was investigated by measurement of total collagen production and analysis of the phenotypes of newly synthesized collagen chains. A 24-h treatment of monolayer articular and multilayer growth plate chondrocytes with 2 micrograms per ml relaxin had no effect on total DNA and did not significantly modify the amount of [3H]proline-labelled collagen chains secreted by the cells. However, polyacrylamide gel electrophoresis demonstrated relevant modifications in relaxin treated chondrocytes. A significant increase was observed in the proportion of type III collagen and in the intensity of the band corresponding to alpha 2I chains. Two-dimensional peptide mapping of CNBr-cleaved molecules indicated that the band that was identified as alpha 1II on monodimensional gels contained a significant proportion of alpha 1I collagen chains, as demonstrated by the presence of alpha 1I cyanogen bromide-digested peptides. The intensity of this band was increased by relaxin treatment. Furthermore, total RNA analysis by slot blot and Northern blot techniques showed a dose-dependent stimulation of alpha 1I and alpha 1III mRNA levels after incubation with increased relaxin concentrations, but no change in the amount of alpha 1II mRNA. These results suggested that when added to cartilage cells in vitro, relaxin modulated the expression of type I, type II and type III collagen genes by amplifying the dedifferentiation process.     

Effect of nitrogen-rich cell culture surfaces on type X collagen expression by bovine growth plate chondrocytes

Background  

Recent evidence indicates that osteoarthritis (OA) may be a systemic disease since mesenchymal stem cells (MSCs) from OA patients express type X collagen, a marker of late stage chondrocyte hypertrophy (associated with endochondral ossification). We recently showed that the expression of type X collagen was suppressed when MSCs from OA patients were cultured on nitrogen (N)-rich plasma polymer layers, which we call "PPE:N" (N-doped plasma-polymerized ethylene, containing up to 36 atomic percentage (at.%) of N.     

Cytoskeleton and adhesion patterns of cultured chick embryo chondrocytes during cell spreading and Rous sarcoma virus transformation

The cytoskeleton and the adhesion complex of chick embryo chondrocytes maintained in vitro have been studied by fluorescence and interference reflection microscopy during the process of cell spreading. The pattern of actin-containing microfilaments and the distribution of vinculin speckles on adhesion plaques have been found to change as a function of the culture time. Newly plated chondrocytes adhere to the substratum mostly around a peripheral ring-like region and show a complex tridimensional array of microfilaments. When chondrocytes flatten, they develop stress fibres and show a diffuse system of vinculin-containing adhesion plaques scattered over the entire ventral side of the cells. Upon infection with Rous sarcoma virus (RSV) chondrocytes display one or more actin-containing ruffles located on the dorsal side similar to the 'actin flowers' earlier described in other cell types. These structures have been found to accumulate vinculin too. In chondrocytes infected with two td-ts mutants of RSV, 'actin flowers' have been found to persist at the restrictive temperature. At this temperature, however, in the majority of cells, stress fibres and adhesion plaques reappear.     

Effects of matrix macromolecules on chondrocyte gene expression: synthesis of a low molecular weight collagen species by cells cultured within collagen gels

Chick-embryo sternal chondrocytes have been cultured within three- dimensional collagen gels as part of a study concerned with the effects of extracellular matrix macromolecules on chondrocyte gene expression. Data are presented indicating that chondrocytes cultured within such a collagenous environment synthesize significantly more of an hitherto unidentified, low molecular weight collagen species than do cells grown on plastic tissue-culture dishes in the conventional manner. This low molecular weight collagen species contains noncollagenous domains (as indicated by its decreased molecular size after mild pepsin digestion), is distinct from the known collagen types (as judged by CNBr peptide analysis), and forms part of the insoluble collagenous matrix produced by the chondrocytes. Cells growing within the gel tend to form colonies consisting of a linear array of cells reminiscent of the cellular organization in growth cartilage.     

Comparison of inhibition by a tumor promoter (12-O-tetradecanoylphorbol-13-acetate) of expression of the differentiated phenotype of chondrocytes in rabbit costal chondrocytes in culture with inhibition by retinoic acid

Both retinoids and the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) inhibit expression of the differentiated phenotype by rabbit costal chondrocytes in culture, as judged by morphological changes and decreased sulfation of glycosaminoglycans (GAG). However, the inhibition of the differentiated phenotype of chondrocytes in TPA-treated cells is restored by parathyroid hormone (PTH), while the inhibition by retinoids is not [Takigawa et al. (1982) Mol. Cell. Biochem. 42, 145-153; Takigawa et al. (1983) Cell Differ. 13, 283-291]. In the present study, we examined the difference between TPA-treated chondrocytes and retinoic acid-treated chondrocytes to determine the mechanism of the restoration of the differentiated phenotype in de-differentiated cells treated with TPA. PTH increased the activity of ornithine decarboxylase [ODC; EC 4.1.1.17], a rate limiting enzyme of polyamine biosynthesis, and proteoglycan synthesis in chondrocytes pretreated with TPA as well as in normal chondrocytes. The maximal stimulations of ODC activity and GAG synthesis were observed 4 h and 24-36 h, respectively, after addition of PTH. The dose-response curve for ODC induction by PTH was parallel to that of PTH-stimulated proteoglycan synthesis both in TPA-treated chondrocytes and in normal chondrocytes. PTH also increased the intracellular cyclic AMP level after 2 min in TPA-treated cells as in normal cells. Addition of dibutyryl cyclic AMP (DBcAMP) induced ODC and restored proteoglycan synthesis in TPA-treated cells. The dose-response curve for induction of ODC by DBcAMP was parallel to that of DBcAMP-stimulated proteoglycan synthesis in both TPA-treated chondrocytes and normal chondrocytes. On the other hand, the increases by PTH in the intracellular cyclic AMP level, ODC activity, and proteoglycan synthesis were inhibited in chondrocytes pretreated with a combination of TPA and retinoic acid as well as in those pretreated with retinoic acid alone. TPA stimulated the syntheses of DNA and RNA in chondrocytes but did not increase the cyclic AMP level or ODC activity. PTH and DBcAMP inhibited the syntheses of DNA and RNA both in TPA-treated cells and in normal cells. These results suggest that ODC induction mediated by elevation of cyclic AMP plays an important role in re-differentiation of de-differentiated cells pretreated with these agents.     

The morphology of mesangial cells cultured at high density and in collagen gels

Primary mesangial cells (rat) from monolayer cultures of the 6th to 12th passage and permanent SV40 Mes13 cells were grown at high density in organoid culture at the medium/air interphase. After adaptation to the in vitro conditions, both mesangial cell types developed after 7 days a synthesis apparatus (endoplasmic reticulum, Golgi apparatus) and produced matrix which consisted of Lamina densa-like material, collagenous fibrils and filaments. Unspecific contacts, gap junctions and adhesion belts could be demonstrated in the contact areas. Additionally, some cells exhibited thick bundles of actin filaments. A close resemblance of the mesangial cells in high density culture to those in vivo can, therefore, be stated. Hence, they differentiated with regard to their matrix formation, contraction and contact behaviour and can therefore be used for experimental studies within a short culture period of 7 days. Cell aggregates in monolayer culture and in cultures in collagen gels had not differentiated at this stage.     

Thyroid hormone treatment of cultured chondrocytes mimics in vivo stimulation of collagen X mRNA by increasing BMP 4 expression

During endochondral bone formation, chondrocytes undergo terminal differentiation, during which the rate of proliferation decreases, cells become hypertrophic, and the extracellular matrix is altered by production of collagen X, as well as proteins required for matrix mineralization. This maturation process is responsible for most longitudinal bone growth, both during embryonic development and in postnatal long bone growth plates. Among the major signaling molecules implicated in regulation of this process are the positive regulators thyroid hormone (T3) and bone morphogenetic proteins (BMPs). Both T3 and BMPs are essential for endochondral bone formation and cannot compensate for each other, suggesting interaction of the two signaling pathways. We have analyzed the temporal and spatial expression patterns of numerous genes believed to play a role in chondrocyte maturation. Our results show that T3 stimulates collagen X gene expression in cultured chondrocytres with kinetics and magnitude similar to those observed in vivo. Stimulation of collagen X gene expression by T3 occurs only after a significant delay, implying that this hormone may act indirectly. We show further that T3 rapidly stimulates production of BMP 4, concomitant with a decrease in the BMP inhibitor Noggin, potentially resulting in a net increase in BMP signaling. Finally, inhibition of BMP signaling with exogenous Noggin prevents T3 stimulation of collagen X expression, indicating that BMP signaling is essential for this process. These data position thyroid hormone at the top of a T3/BMP cascade, potentially explaining why both pathways are essential for chondrocyte maturation. J. Cell. Physiol. 219: 595–605, 2009. © 2009 Wiley‐Liss, Inc.     

Cytoskeleton and differentiation: effects of cytochalasin B and colchicine on expression of the differentiated phenotype of rabbit costal chondrocytes in culture

Cytochalasin B changed the shape of cultured rabbit costal chondrocytes from polygonal to nearly spherical and stimulated glycosaminoglycan synthesis, which is a differentiated phenotype of chondrocytes, whereas colchicine changed them from polygonal to flattened and inhibited glycosaminoglycan synthesis. These morphological changes occurred parallel with the changes in glycosaminoglycan synthesis. Induction of ornithine decarboxylase by parathyroid hormone, which is a good marker of differentiated chondrocytes, was markedly potentiated in the spherical cells which had been pretreated with cytochalasin B, whereas pretreatment with colchicine inhibited the induction of the enzyme. Both cytochalasin B and colchicine inhibited DNA synthesis. The inhibitions were observed after the appearance of changes in the morphology of the cells and glycosaminoglycan synthesis. These findings suggest that intactness of microtubules and disruption of microfilaments are involved in regulating the expression of the differentiated phenotype of chondrocytes in culture.     

Maintenance of differentiated phenotype of articular chondrocytes by protein kinase C and extracellular signal-regulated protein kinase.

The differentiated phenotype of chondrocyte is rapidly lost during in vitro culture by a process designated "dedifferentiation." In this study, we investigate the roles of protein kinase C (PKC) and extracellular signal-regulated protein kinase (ERK) in the maintenance of the differentiated chondrocyte phenotype. Chondrocytes isolated from rabbit articular cartilage underwent dedifferentiation upon serial monolayer culture with cessation of type II collagen expression and proteoglycan synthesis, which was reversed by culturing dedifferentiated cells in alginate gel. The expression pattern of PKC alpha was essentially the same as that of type II collagen during de- and redifferentiation, in that expression was decreased during dedifferentiation and increased during redifferentiation. In contrast to PKC alpha, ERK activity increased 15-fold during dedifferentiation. This enhanced activity was terminated during redifferentiation. Down-regulation of PKC alpha in passage 0 chondrocytes resulted in dedifferentiation. However, overexpression of PKC alpha did not affect type II collagen levels, suggesting that PKC alpha expression is not sufficient to maintain the differentiated phenotype. However, inhibition of ERK by PD98059 enhanced type II collagen expression and proteoglycan synthesis in passage 0 cells, retarded dedifferentiation during monolayer cultures, and reversed dedifferentiation caused by down-regulation of PKC. Unlike PKC-dependent ERK regulation of chondrogenesis, PKC and ERK independently modulated chondrocyte dedifferentiation, as confirmed by observations that PKC down-regulation and ERK inhibition did not alter ERK phosphorylation and PKC expression, respectively. In addition, expression of N-cadherin, alpha-catenin, and beta-catenin, which are oppositely regulated to type II collagen during phenotype alterations, were modulated by PKC and ERK during chondrogenesis but not dedifferentiation, supporting distinct mechanisms for the regulation of chondrocyte differentiation and maintenance of differentiated phenotype by these two protein kinases.     

Enhanced cell accumulation and collagen processing by keratocytes cultured under agarose and in media containing IGF-I,TGF-β or PDGF

We previously showed an agarose overlay on keratocytes cultured in media containing pharmacological levels of insulin enhanced collagen processing and collagen fibril formation. In this study, we compared collagen processing by keratocytes cultured in media containing physiological levels of IGF-I, TGF-β, FGF-2, and PDGF in standard and in agarose overlay cultures. Pepsin digestion/SDS PAGE was used to determine the levels of procollagen secreted into the media and the collagen content of the ECM associated with the cell layer. Distribution of collagen type I and fibronectin in the ECM of the agarose cultures was determined by immunoflorescence. Collagen fibril and keratocyte morphology was evaluated by electron microscopy. The agarose overlay significantly enhanced the cell number in the IGF-I, TGF-β and PDGF treated cultures by 2–3 fold. The overlay also significantly enhanced the processing of procollagen to collagen fibrils from 29% in standard cultures to 63–68% in agarose cultures for the IGF-I and PDGF cultures, and from 66% in standard culture to 85% in agarose culture for the TGF-β cultures. Cell accumulation and collagen processing was not enhanced by agarose overlay of the FGF-2 treated cultures. Collagen type I and fibronectin were more uniformly distributed and the collagen fibrils smaller in the ECM of the TGF-β treated cultures. Keratocytes in the FGF-2 treated cultures were in close cell contact with few collagen fibrils while IGF-I, TGF-β, and PDGF cultures had an extensive ECM with abundant collagen fibrils. The results of this study indicate that the agarose overlay enhances collagen fibril assembly and cell accumulation by keratocytes when both collagen synthesis and cell proliferation are stimulated.     

Restoration by parathyroid hormone and dibutyryl cyclic AMP of expression of the differentiated phenotype of chondrocytes inhibited by a tumor promoter, 12-0-tetradecanoylphorbol-13-acetate

12-0-Tetradecanoylphorbol-13-acetate (TPA) inhibited expression of the differentiated phenotype of chondrocytes in rabbit costal chondrocytes in culture. TPA transformed typical polygonal chondrocytes into multilayered, fibroblastic cells and also inhibited the rate of [35S]sulfate incorporation into glycosaminoglycan (GAG), a differentiated phenotype of chondrocytes. These changes were apparent within 24 h and reached a plateau at 48 h after the addition of TPA. Phorbol didecanoate and phorbol dibenzoate also inhibited sulfation of GAG, even though the effect was weaker than that of TPA. Phorbol diacetate and 4-0-methyl TPA did not inhibit sulfation of GAG. Addition of parathyroid hormone (PTH) or dibutyryl cyclic AMP simultaneously with TPA overcame the inhibition caused by TPA. PTH and dibutyryl cyclic AMP also reversed the inhibition and stimulated expression of the differentiated phenotype of chondrocytes even in de-differentiated cells which had been pretreated for 3 days with TPA. These findings suggest that cyclic AMP plays an important role in the restoration of the differentiated phenotype of chondrocytes in TPA-treated chondrocytes, and that the TPA-treated cells retain some of the differentiated phenotype of the original cells, such as responsiveness to PTH.