Adenoviral transduction supports matrix expression of alginate cultured articular chondrocytes

The present study examines the effects of adenoviral (Ad) transduction of human primary chondrocyte on transgene expression and matrix production. Primary chondrocytes were isolated from healthy articular cartilage and from cartilage with mild osteoarthritis (OA), transduced with an Ad vector and either immediately cultured in alginate or expanded in monolayer before alginate culture. Proteoglycan production was measured using dimethylmethylene blue (DMMB) assay and matrix gene expression was quantified by real-time PCR. Viral infection of primary chondrocytes results in a stable long time transgene expression for up to 13 weeks. Ad transduction does not significantly alter gene expression and matrix production if chondrocytes are immediately embedded in alginate. However, if expanded prior to three dimension (3D) culture in alginate, chondrocytes produce not only more proteoglycans compared to non-transduced controls, but also display an increased anabolic and decreased catabolic activity compared to non-transduced controls. We therefore suggest that successful autologous chondrocyte transplantation (ACT) should combine adenoviral transduction of primary chondrocytes with expansion in monolayer followed by 3D culture. Future studies will be needed to investigate whether the subsequent matrix production can be further improved by using Ad vectors bearing genes encoding matrix proteins.     

Maturational differences in superficial and deep zone articular chondrocytes

To examine whether differences in chondrocytes from skeletally immature versus adult individuals are important in cartilage healing, repair, or tissue engineering, superficial zone chondrocytes (SZC, from within 100 μm of the articular surface) and deep zone chondrocytes (DZC, from 30%–45% of the deepest un-mineralized part of articular cartilage) were harvested from immature (1–4 months) and young adult (18–36 months) steers and compared. Cell size, matrix gene expression and protein levels, integrin levels, and chemotactic ability were measured in cells maintained in micromass culture for up to 7 days. Regardless of age, SZC were smaller, had a lower type II to type I collagen gene expression ratio, and higher gene expression of SZ proteins than their DZC counterparts. Regardless of zone, chondrocytes from immature steers had higher levels of Sox 9 and type II collagen gene expression. Over 7 days in culture, the SZC of immature steers had the highest rate of proliferation. Phenotypically, the SZC of immature and adult steers were more stable than their respective DZC. Cell surface α5 and α2 integrin subunit levels were higher in the SZC of immature than of adult steers, whereas β1 integrin subunit levels were similar. Both immature and adult SZC were capable of chemotaxis in response to fetal bovine serum or basic fibroblast growth factor. Our data indicate that articular chondrocytes vary in the different zones of cartilage and with the age of the donor. These differences may be important for cartilage growth, tissue engineering, and/or repair. This work was supported in part by the National Chapter of the Arthritis Foundation, the Ira DeCamp Fellowship for Musculoskeletal Research of the Hospital for Special Surgery, the Institute for Sports Medicine Research in New York, and the National Institute of Health grant AR045748 and was conducted in a facility constructed with support from the Research Facilities Improvement Program (grant no. C06-RR12538-01) of the National Center for Research Resources, National Institutes of Health.     

A proteomic approach for identification and localization of the pericellular components of chondrocytes

Although the pericellular matrix (PCM) plays a central role in the communication between chondrocytes and extracellular matrix, its composition is largely unknown. In this study, the PCM was investigated with a proteomic approach using chondrons, which are enzymatically isolated constructs including the chondrocyte and its surrounding PCM. Chondrons and chondrocytes alone were isolated from human articular cartilage. Proteins extracted from chondrons and chondrocytes were used for two-dimensional electrophoresis. Protein spots were quantitatively compared between chondron and chondrocyte gels. Cellular proteins, which had similar density between chondron and chondrocyte gels, did not proceed for analysis. Since chondrons only differ from chondrocytes in association of the PCM, protein spots in the chondron gels that had higher quantity than that in the chondrocyte gels were selected as candidates of the PCM components and processed for mass spectrometry. Among 15 identified peptides, several were fragments of the three type VI collagen chains (α-1, α-2, and α-3). Other identified PCM proteins included triosephosphate isomerase, transforming growth factor-β induced protein, peroxiredoxin-4, ADAM (A disintegrin and metalloproteinases) 28, and latent-transforming growth factor beta-binding protein-2. These PCM components were verified with immunohisto(cyto)chemistry for localization in the PCM region of articular cartilage. The abundance of type VI collagen in the PCM emphasizes its importance to the microenvironment of chondrocytes. Several proteins were localized in the PCM of chondrocytes for the first time and that warrants further investigation for their functions in cartilage biology.     

Comparison of marker gene expression in chondrocytes from patients receiving autologous chondrocyte transplantation versus osteoarthritis patients

Currently, autologous chondrocyte transplantation (ACT) is used to treat traumatic cartilage damage or osteochondrosis dissecans, but not degenerative arthritis. Since substantial refinements in the isolation, expansion and transplantation of chondrocytes have been made in recent years, the treatment of early stage osteoarthritic lesions using ACT might now be feasible. In this study, we determined the gene expression patterns of osteoarthritic (OA) chondrocytes ex vivo after primary culture and subculture and compared these with healthy chondrocytes ex vivo and with articular chondrocytes expanded for treatment of patients by ACT. Gene expression profiles were determined using quantitative RT-PCR for type I, II and X collagen, aggrecan, IL-1β and activin-like kinase-1. Furthermore, we tested the capability of osteoarthritic chondrocytes to generate hyaline-like cartilage by implanting chondrocyte-seeded collagen scaffolds into immunodeficient (SCID) mice. OA chondrocytes ex vivo showed highly elevated levels of IL-1β mRNA, but type I and II collagen levels were comparable to those of healthy chondrocytes. After primary culture, IL-1β levels decreased to baseline levels, while the type II and type I collagen mRNA levels matched those found in chondrocytes used for ACT. OA chondrocytes generated type II collagen and proteoglycan-rich cartilage transplants in SCID mice. We conclude that after expansion under suitable conditions, the cartilage of OA patients contains cells that are not significantly different from those from healthy donors prepared for ACT. OA chondrocytes are also capable of producing a cartilage-like tissue in the in vivo SCID mouse model. Thus, such chondrocytes seem to fulfil the prerequisites for use in ACT treatment.     

CCN2 (Connective Tissue Growth Factor) is essential for extracellular matrix production and integrin signaling in chondrocytes

The matricellular protein CCN2 (Connective Tissue Growth Factor; CTGF) is an essential mediator of ECM composition, as revealed through analysis of Ccn2 deficient mice. These die at birth due to complications arising from impaired endochondral ossification. However, the mechanism(s) by which CCN2 mediates its effects in cartilage are unclear. We investigated these mechanisms using Ccn2 ^−/− chondrocytes. Expression of type II collagen and aggrecan were decreased in Ccn2 ^−/− chondrocytes, confirming a defect in ECM production. Ccn2 ^−/− chondrocytes also exhibited impaired DNA synthesis and reduced adhesion to fibronectin. This latter defect is associated with decreased expression of α5 integrin. Moreover, CCN2 can bind to integrin α5β1 in chondrocytes and can stimulate increased expression of integrin α5. Consistent with an essential role for CCN2 as a ligand for integrins, immunofluorescence and Western blot analysis revealed that levels of focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK)1/2 phosphorylation were reduced in Ccn2 ^−/− chondrocytes. These findings argue that CCN2 exerts major effects in chondrocytes through its ability to (1) regulate ECM production and integrin α5 expression, (2) engage integrins and (3) activate integrin-mediated signaling pathways.     

Restoration of the differentiated functions of serially passaged chondrocytes using staurosporine

Summary Among the various directions explored in order to have a large number of differentiated articular chondrocytes easily available, the restoration of the differentiated properties after cell multiplication in monolayer has been proposed. It has been clearly shown that the synthesis of cartilage proteoglycans and type II collagen synthesis is coincident with the presence of a faint microfibrillar architecture but is absent in chondrocytes showing well-defined actin cables. Staurosporin, mainly described as a protein kinase C inhibitor, has also been shown to rapidly induce the disruption of the actin microfilaments. The purpose of this paper was to investigate whether properties of differentiated chondrocytes were reinitiated upon staurosporin treatment of serially passaged chondrocytes. Results showed, after staurosporine treatment of cells at Passage two for 5 d, complete suppression of type I and type III collagen synthesis and induction of type II collagen synthesis and of Alcian blue stainable matrix. Additionally, we showed that staurosporin restored metabolic responses that chondrocytes in primary culture exhibit upon interleukin-1β treatment (decrease of Alcian blue- positive cells, induction of expression of the 92 kDa gelatinase, nitric oxide production). We conclude that staurosporin is a potent redifferentiating agent of articular chondrocytes that have been subcultured up to Passage two for multiplication. Taking into account that the cellularity of cartilage is very low, staurosporine-treated chondrocytes could be useful as an alternative cellular model to evaluate pharmacotoxicological effects of drugs.     

IL-10 overexpression differentially affects cartilage matrix gene expression in response to TNF-α in human articular chondrocytes in vitro

Cartilage-specific extracellular matrix synthesis is the prerequisite for chondrocyte survival and cartilage function, but is affected by the pro-inflammatory cytokine TNF-α in arthritis. The aim of the present study was to characterize whether the immunoregulatory cytokine IL-10 might modulate cartilage matrix and cytokine expression in response to TNF-α. Primary human articular chondrocytes were treated with either recombinant IL-10, TNF-α or a combination of both (at 10 ng/mL each) or transduced with an adenoviral vector overexpressing human IL-10 and subsequently stimulated with 10 ng/ml TNF-α for 6 or 24 h. The effects of IL-10 on the cartilage-specific matrix proteins collagen type II, aggrecan, matrix-metalloproteinases (MMP)-3, -13 and pro-inflammatory cytokines were evaluated by real-time RT-PCR and immunohistochemistry. Transduced chondrocytes overexpressed high levels of IL-10 which significantly up-regulated collagen type II expression. TNF-α suppressed collagen type II and aggrecan, but increased MMP and cytokine expression in chondrocytes compared to the non-stimulated controls. The TNF-α mediated down-regulation of aggrecan expression was significantly antagonized by IL-10 overexpression, whereas the suppression of collagen type II was barely affected. The MMP-13 and IL-1β expression by TNF-α was slightly reduced by IL-10. These results suggest that IL-10 overexpression modulates some catabolic features of TNF-α in chondrocytes.     

Mechanical regulation of proteoglycan synthesis in normal and osteoarthritic human articular chondrocytes--roles for alpha5 and alphaVbeta5 integrins

The importance of biomechanical forces in regulating normal chondrocyte metabolism is well established and the mechanisms whereby mechanical forces are transduced into biochemical responses by chondrocytes are beginning to be understood. Previous studies have indicated that cyclical mechanical stimulation induces increased aggrecan gene expression in normal but not osteoarthritic chondrocytes in monolayer. It remains unclear, however, whether these effects on gene expression are associated with changes in proteoglycan production and whether any changes in proteoglycan expression is dependent on integrins or integrin associated proteins. Normal and osteoarthritic articular chondrocytes in monolayer were exposed to 0.33 Hz mechanical stimulation for 20 min in the absence or presence of function modifying anti-integrin antibodies. Following stimulation GAG and proteoglycan (PG) synthesis was assessed by DMMB assay and western blotting. Mechanical stimulation of normal chondrocytes resulted in increased GAG synthesis that was blocked by the presence of antibodies to alpha5 and alphaVbeta5 integrins and CD47. Electrophoretic patterns of PGs released from normal chondrocytes following mechanical stimulation showed an increase in newly-synthesized aggrecan that was not fragmented or degraded. Chondrocytes from osteoarthritic cartilage showed lower levels of GAG production when compared to normal chondrocytes and synthesis was not influenced by mechanical stimulation. These studies show that chondrocytes derived from normal and OA cartilage have different matrix production responses to mechanical stimulation and suggest previously unrecognised roles for alphaVbeta5 integrin in regulation of chondrocyte responses to biomechanical stimulation.     

Integrins mediate the effects of quinolones and magnesium deficiency on cultured rat chondrocytes.

Chondrocyte-matrix interaction is mediated by a series of adhesion molecules. Both alpha and beta integrin subunits are involved and govern crucial functions of cell adhesion and signal transduction. These molecules modulate proliferation and differentiation, thus establishing cartilage integrity. We studied the influence of magnesium deficiency and quinolone antibiotics (which form chelate complexes with divalent cations) on chondrocytes in vitro in order to assess the role of Mg2+ ions in integrin function and to establish cellular changes mediated via integrin signal transduction. Mg2(+)-free medium and quinolone supplementation was found to decrease chondrocyte attachment to collagen type II-coated coverslips. Adhesion and growth of chondrocytes were reduced in the respective medium. Organisation of cytoskeletal fibers (vimentin) was changed and formation of stress fibers (f-actin) was disturbed. Additionally, rates of cell proliferation declined. These results indicate that quinolone-magnesium complex formation is important for chondrotoxicity of these substances. Cell-matrix detachment and morphological alterations described in vitro may explain the lesions observed in articular cartilage after quinolone administration in vivo. The attachment assay described could serve as a simple test to establish the susceptibility of chondrocytes of different species to different quinolones in use or new ones to be introduced.     

Development and phenotypic characterization of a high density in vitro model of auricular chondrocytes with applications in reconstructive plastic surgery

Cultivation of phenotypically stable auricular chondrocytes will have applications in autologous chondrocyte transplantation and reconstructive surgery of cartilage. Chondrocytes grown in monolayer culture rapidly dedifferentiate assuming a fibroblast-like morphology and lose their cartilage-specific pattern of gene expression. Three-dimensional high-density culture models mimic more closely the in vivo conditions of cartilage. Therefore, this study was undertaken to test whether the high-density cultures might serve as a suitable model system to acquire phenotypically and functionally differentiated auricular chondrocytes from porcine cartilage. Freshly isolated porcine auricular chondrocytes were cultured for 7 passages in monolayer culture. From each passage (passage 0 and 1-7) cells were introduced to high-density cultures and examined by transmission electron microscopy. Western blotting was used to analyse the expression of cartilage-specific markers, such as collagen type II and cartilage specific proteoglycan, fibronectin, cell adhesion and signal transduction receptor beta1-integrin, matrix metalloproteinases (MMP-9, MMP-13), cyclo-oxygenase (COX)-2 and the apoptosis commitment marker, activated caspase-3. When dedifferentiated auricular chondrocytes from monolayer passages 0-4 were cultured in high-density culture, they recovered their chondrocytic phenotype and formed cartilage nodules surrounded by fibroblast-like cells and synthesised collagen type II, proteoglycans, fibronectin and beta1-integrins. However, chondrocytes from monolayer passages 5-7 did not redifferentiate to chondrocytes even when transferred to high-density culture, and did not synthesize a chondrocyte-specific extracellular matrix. Instead, they produced increasing amounts of MMP-9, MMP-13, COX-2, activated caspase-3 and underwent apoptosis. Three-dimensional high-density cultures may therefore be used to obtain sufficient quantities of fully differentiated auricular chondrocytes for autologous chondrocyte transplantation and reconstructive plastic surgery.     

The effect of mechanical load on integrin subunits α5 and β1 in chondrocytes from mature and immature cartilage explants

Articular cartilage is subjected to cyclic compressive stresses during joint loading. There is increasing experimental evidence that this loading is essential for the chondrocytes to maintain the functionality of the cartilage extracellular matrix (ECM) and that members of the integrin family of transmembrane receptors may play an important role in signal mechanotransduction between the ECM and chondrocytes. Of particular interest are the integrin subunits 5 and 1, which are known to form the receptor for fibronectin, an important ECM protein, and to be involved in mechanotransduction as well as in the regulation of cytokine production. In this study, we measured the amounts of the integrin subunits 5 and 1 in chondrocytes from young (immature) and adult (mature) bovine articular cartilage explants which were subjected to a continuously applied cyclic compressive stress of 1 MPa for 6 and 24 h. The integrin content per chondrocyte was measured immediately after load cessation by flow cytometry following matrix digestion to release the cells. We found that a mechanical stress induced an increase in the number of integrin subunit 5 in immature and mature cartilage but not in the integrin subunit 1 content. The integrin contents were greatest after 6 h of loading and returned to control levels after 24 h of unloading. The results of this study supply further experimental evidence that chondrocytes respond to changes in their mechanical environment and that the integrin 51 may act as a mechanical signal transducer between the chondrocyte and the ECM for the modulation of cellular physiology.This work was supported by NIH grant AR45748 to PAT and the HSS MacArthur Cartilage Fund.     

Prostaglandin biosynthesis by lapine articular chondrocytes in culture

Secondary monolayer and spinner cultures of rabbit articular chondrocytes released into the culture medium prostaglandins the synthesis of which was inhibited by sodium meclofenamate. The prostaglandins measured by radioimmunoassay were, in order of decreasing abundance, prostaglandin E₂, 6-oxoprostaglandin F_1α, (the stable metabolite of prostacyclin) and prostaglandin F_2α. Several lines of evidence indicated that chondrocytes synthesize little if any thromboxane B₂ (the stable metabolite of thromboxane A₂). The presence of prostaglandins was confirmed by radiometric thin-layer chromatography of extracts of culture media incubated with [³H]arachidonic acid-labeled cells. In monolayer culture, chondrocytes synthesized immunoreactive prostaglandins in serum-free as well as serum-containing medium. Monolayer chondrocytes produced higher levels of prostaglandin E₂ relative to 6-oxo-prostaglandin F_1α than did spinner cells, but the latter synthesized more total prostaglandins. The identity of endogenous prostaglandins as well as those synthesized in short-term culture by rabbit cartilage slices was compared to those produced by chondrocytes in long-term culture. Chondrocytes synthesized all of the prosta-glandins found in articular cartilage. Minimal quantities of thromboxane B₂ were detected in cartilage. A higher percentage of 6-oxo-prostaglandin F_1α relative to other prostaglandins was found in cartilage than in either monolayer or spinner chondrocyte cultures. These results demonstrate that articular chondrocytes synthesize prostaglandins and prostacyclin. These prostaglandins may exert significant physiological effects on cartilage, since exogenous prosta-glandins depress chondrocyte sulfated-proteoglycan synthesis and may even promote proteoglycan degradation.     

Type II and VI collagen in nasal and articular cartilage and the effect of IL-1α on the distribution of these collagens

The distribution of type II and VI collagen was immunocytochemically investigated in bovine articular and nasal cartilage. Cartilage explants were used either fresh or cultured for up to 4 weeks with or without interleukin 1α (IL-1α). Sections of the explants were incubated with antibodies for both types of collagen. Microscopic analyses revealed that type II collagen was preferentially localized in the interchondron matrix whereas type VI collagen was primarily found in the direct vicinity of the chondrocytes. Treatment of the sections with hyaluronidase greatly enhanced the signal for both types of collagen. Also in sections of explants cultured with IL-1α a higher level of labeling of the collagens was found. This was apparent without any pre-treatment with hyaluronidase. Under the influence of IL-1α the area positive for type VI collagen that surrounded the chondrocytes broadened. Although the two collagens in both types of cartilage were distributed similarly, a remarkable difference was the higher degree of staining of type VI collagen in articular cartilage. Concomitantly we noted that digestion of this type of cartilage hardly occurred in the presence of IL-1α whereas nasal cartilage was almost completely degraded within 18 days of culture. Since type VI collagen is known to be relatively resistant to proteolysis we speculate that the higher level of type VI collagen in articular cartilage is important in protecting cartilage from digestion.     

Chondrocyte proliferation in a new culture system

Objective:  This study has aimed to study different culture systems that might stimulate an increase in cell proliferation of normal and osteoarthritis chondrocytes from articular cartilage in rat model.
Material and Methods:  Three culture systems using chondrocytes embedded in alginate beads were tested: chondrocytes cultured in Dulbecco's modified Eagle's medium (DMEM) as control, a co-culture system consisting of a monolayer of de-differentiated chondrocytes as a source of mitotic factors, and an enriched medium containing culture medium obtained from a monolayer of chondrocytes and DMEM. Normal and osteoarthritis chondrocytes were stained with 5-carboxyfluorescein diacetate succinimidyl ester and were cultured in each of the three systems. After 5 days of culture cell, proliferation was detected by flow cytometry. Chondrocyte phenotype was confirmed by collagen type II and MMP-3 expression. To determine possible molecules released into the medium by the cultured chondrocyte monolayer and which would probably be involved in cell proliferation, a study of mRNA and expression of transforming growth factor-β1 (TGF-β1), fibroblastic growth factor-2 (FGF-2), epidermal growth factor (EGF), platelet derived growth factor-A (PDGF-A) and insulin-like growth factor-1 (IGF-1) proteins was conducted.
Results and Conclusions:  Chondrocytes in the co-culture system or in enriched medium showed an increase in proliferation; only when osteoarthritis chondrocytes were cultured in enriched medium would they display a statistically significant increase in their proliferation rate and in their viability. When chondrocytes from the monolayer were analysed, differential mRNA expression of TGF-β1 and IGF-1 was found during all passages, which suggests that these two growth factors might be involved in chondrocyte proliferation.     

Regulation of α5 and αV Integrin Expression by GDF-5 and BMP-7 in Chondrocyte Differentiation and Osteoarthritis

The Integrin β1 family is the major receptors of the Extracellular matrix (ECM), and the synthesis and degradation balance of ECM is seriously disrupted during Osteoarthritis (OA). In this scenario, integrins modify their pattern expression and regulate chondrocyte differen-tiation in the articular cartilage. Members of the Transforming growth factor beta (Tgf-β) Su-perfamily, such as Growth differentiation factor 5 (Gdf-5) and Bone morphogenetic protein 7 (Bmp-7), play a key role in joint formation and could regulate the integrin expression during chondrocyte differentiation and osteoarthritis progression in an experimental OA rat model. Decrease of α5 integrin expression in articular cartilage was related with chondrocyte dedif-ferentiation during OA progression, while increase of α1, α2, and α3 integrin expression was related with fibrous areas in articular cartilage during OA. Hypertrophic chondrocytes expressedαV integrin and was increased in the articular cartilage of rats with OA. Integrin expression during chondrocyte differentiation was also analyzed in a micromass culture system of mouse embryo mesenchymal cells, micromass cultures was treated with Gdf-5 or Bmp-7 for 4 and 6 days, respectively. Gdf-5 induced the expression of theα5 sub-unit, while Bmp-7 induced the expression of the αV sub-unit. This suggests a switch in signaling for prehypertrophic chondrocyte differentiation towards hypertrophy, where Gdf-5 could maintain the articular chondrocyte phenotype and Bmp-7 would induce hypertrophy. Decrease of Ihh expression during late stages of OA in rat model suggest that the ossification in OA rat knees and endochondral ossification could be activated by Bmp-7 and αV integrin in absence of Ihh. Thus, chondrocyte phenotype in articular cartilage is similar to prehypetrophic chondrocyte in growth plate, and is preserved due to the presence of Indian hedgehog (Ihh), Gdf-5 and α5 integrin to maintain articular cartilage and prevent hy-pertrophy.     

Type X collagen expression in osteoarthritic and rheumatoid articular cartilage

Type X collagen is a short chain, non-fibrilforming collagen synthesized primarily by hypertrophic chondrocytes in the growth plate of fetal cartilage. Previously, we have also identified type X collagen in the extracellular matrix of fibrillated, osteoarthritic but not in normal articular cartilage using biochemical and immunohistochemical techniques (von der Mark et al. 1992 a). Here we compare the expression of type X with types I and II collagen in normal and degenerate human articular cartilage by in situ hybridization. Signals for cytoplasmic α1(X) collagen mRNA were not detectable in sections of healthy adult articular cartilage, but few specimens of osteoarthritic articular cartilage showed moderate expression of type X collagen in deep zones, but not in the upper fibrillated zone where type X collagen was detected by immunofluorescence. This apparent discrepancy may be explained by the relatively short phases of type X collagen gene activity in osteoarthritis and the short mRNA half-life compared with the longer half-life of the type X collagen protein. At sites of newly formed osteophytic and repair cartilage, α1(X) mRNA was strongly expressed in hypertrophic cells, marking the areas of endochondral bone formation. As in hypertrophic chondrocytes in the proliferative zone of fetal cartilage, type X collagen expression was also associated with strong type II collagen expression.     

Tissue engineering strategies for cartilage generation--micromass and three dimensional cultures using human chondrocytes and a continuous cell line

Utilizing ATDC5 murine chondrogenic cells and human articular chondrocytes, this study sought to develop facile, reproducible three-dimensional models of cartilage generation with the application of tissue engineering strategies, involving biodegradable poly(glycolic acid) scaffolds and rotating wall bioreactors, and micromass pellet cultures. Chondrogenic differentiation, assessed by histology, immunohistochemistry, and gene expression analysis, in ATDC5 and articular chondrocyte pellets was evident by the presence of distinct chondrocytes, expressing Sox-9, aggrecan, and type II collagen, in lacunae embedded in a cartilaginous matrix of type II collagen and proteoglycans. Tissue engineered explants of ATDC5 cells were reminiscent of cartilaginous structures composed of numerous chondrocytes, staining for typical chondrocytic proteins, in lacunae embedded in a matrix of type II collagen and proteoglycans. In comparison, articular chondrocyte explants exhibited areas of Sox-9, aggrecan, and type II collagen-expressing cells growing on fleece, and discrete islands of chondrocytic cells embedded in a cartilaginous matrix.     

Anionic glycoconjugates from differentiated and dedifferentiated cultures of bovine articular chondrocytes: Modulation by TGF-β

Summary Primary, high density bovine articular chondrocyte (BAC) cultures, stimulated with transforming growth factor-β-1, elaborated a high molecular weight anionic glycoconjugate, kDa 540, which does not contain glycosaminoglycan chains (Chan and Anastassiades, 1996). The effect of exogenously added transforming growth factor-β-1 on the elaboration of the high molecular weight glycoconjugate and of proteoglycans was studied during dedifferentiation of the chondrocytes, utilizing a serial subculture technique under anchorage-dependent conditions, up to four subcultures. The high molecular weight glycoconjugate was detected in the media of all growth-factor-stimulated chondrocyte subcultures, as well as stimulated primary cultures, but not in unstimulated primary cultures or subcultures. By contrast, a large proteoglycan, was only secreted by primary cultures and first subcultures, whether treated with transforming growth factor-β-1 or untreated. This proteoglycan contained mostly chondroitin sulfate chains, whose hydrodynamic size was increased by the addition of transforming growth factor-β-1. Further, the pattern of the proteoglycans appearing in the media of subcultures 2–4 was influenced by the addition of transforming growth factor-β-1, so that while these control subcultures elaborated both the large and small chondroitin sulfate proteoglycans, the equivalent stimulated subcultures elaborated only intermediate sized chondroitin sulfate proteoglycan(s). These results suggest that while dedifferentiation of articular chondrocytes, achieved by subculturing, strongly modulates the effect of exogenously added transforming growth factor-β-1 on the type of proteoglycan elaborated, the process of dedifferentiation does not influence the transforming-growth-factor-β-dependent synthesis of the high molecular weight anionic glycoconjugate.