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.     

DIFFERENTIATION OF MESENCHYMAL LIMB BUD CELLS TO CHONDROCYTES IN ALGINATE BEADS

Manyin vitromodels of embryonic material used for the cultivation of chondrocytes yield mixed cultures consisting of chondrocytes and fibroblast-like cells. For the optimization of cartilage cell cultures, alginate, a semisolid medium, was employed to obtain pure chondrocyte cultures. Isolated mesenchymal cells from 12-day-old mouse limb buds were grown in alginate for up to 4 weeks. A sub-population of the cells differentiated to chondrocytes and exhibited a stable phenotype until the end of the culture period. After 3 to 4 days a cartilage-specific matrix started to develop. Fibroblast-like cells from this mixed culture did not survive; they became necrotic. When alginate was later on dissolved by chelating agents, only chondrocytes were isolated. During dissolution of alginate and centrifugation, chondrocytes did not lose their contact with their new matrix present on their surfaces. Cultivation of these chondrocytes or chondrones in mass culture yields a pure chondrocyte population. Immunoelectron microscopic investigations revealed collagen type II, fibronectin, decorin and chondroitin sulfate-proteoglycans in the chondrocyte capsules and in mass culture.     

bFGF influences human articular chondrocyte differentiation

BACKGROUND: The possible functional role of basic fibroblast growth factor (bFGF) in regulating the mitotic and metabolic activity of primary human articular chondrocytes was investigated. METHODS: [EF1]Chondrocytes were enzymatically isolated from femoral head cartilage, and were cultured in vitro in monolayer. bFGF-dependent cell proliferation, production of collagen type II and aggrecan were monitored 10 days after isolation. Furthermore, effect of bFGF on cell cycle, cell morphology, and mRNA expression of integrins and chondrogenic markers determined by real time PCR were analyzed. RESULTS: bFGF concentrations in supernatants of primary human articular chondrocytes peaked immediately after isolation and then declined. In a dose-dependent manner, bFGF enhanced cell amplification and viability. BFGF induced a decrease in the apoptotic cell population, while the number of proliferating cells remained unchanged. Supplementation of cell culture with bFGF reduced collagen type II mRNA by 49%, but increased expression of the integrin alpha(2) by 70%. bFGF did not significantly regulate the integrins alpha(1), alpha(5), alpha(10), alpha(v) and type I collagen. bFGF reduced the amount of collagen type II by 53%, which was correlated with diminished mRNA production. Monolayer cultured chondrocytes secreted significant amounts of aggrecan that decreased over time. Secretion of this cartilage-specific marker was further reduced by the addition of bFGF. DISCUSSION: These findings highlight the potential role of bFGF as an endogenous chondrocyte mediator that can enhance cell amplification and regulate cell differentiation.     

Synoviocyte Derived-Extracellular Matrix Enhances Human Articular Chondrocyte Proliferation and Maintains Re-Differentiation Capacity at Both Low and Atmospheric Oxygen Tensions

BackgroundCurrent tissue engineering methods are insufficient for total joint resurfacing, and chondrocytes undergo de-differentiation when expanded on tissue culture plastic. De-differentiated chondrocytes show poor re-differentiation in culture, giving reduced glycosaminoglycan (GAG) and collagen matrix accumulation. To address this, porcine synoviocyte-derived extracellular matrix and low (5%) oxygen tension were assessed for their ability to enhance human articular chondrocyte expansion and maintain re-differentiation potential.MethodsPorcine synoviocyte matrices were devitalized using 3 non-detergent methods. These devitalized synoviocyte matrices were compared against tissue culture plastic for their ability to support human chondrocyte expansion. Expansion was further compared at both low (5%), and atmospheric (20%) oxygen tension on all surfaces. Expanded cells then underwent chondrogenic re-differentiation in aggregate culture at both low and atmospheric oxygen tension. Aggregates were assessed for their GAG and collagen content both biochemically and histologically.ResultsHuman chondrocytes expanded twice as fast on devitalized synoviocyte matrix vs. tissue culture plastic, and cells retained their re-differentiation capacity for twice the number of population doublings. There was no significant difference in growth rate between low and atmospheric oxygen tension. There was significantly less collagen type I, collagen type II, aggrecan and more MMP13 expression in cells expanded on synoviocyte matrix vs. tissue culture plastic. There were also significant effects due to oxygen tension on gene expression, wherein there was greater collagen type I, collagen type II, SOX9 and less MMP13 expression on tissue culture plastic compared to synoviocyte matrix. There was a significant increase in GAG, but not collagen, accumulation in chondrocyte aggregates re-differentiated at low oxygen tension over that achieved in atmospheric oxygen conditions.ConclusionsSynoviocyte-derived matrix supports enhanced expansion of human chondrocytes such that the chondrocytes are maintained in a state from which they can re-differentiate into a cartilage phenotype after significantly more population doublings. Also, low oxygen tension supports GAG, but not collagen, accumulation. These findings are a step towards the production of a more functional, tissue engineered cartilage.     

Constitutive myc expression impairs hypertrophy and calcification in cartilage.

The myc oncogene is expressed by proliferating quail embryo chondrocytes (QEC) grown as adherent cells and is repressed in QEC maintained in suspension culture. To investigate the interference of myc expression during chondrocyte differentiation, QEC were infected with a retrovirus carrying the v-myc oncogene (QEC-v-myc). Uninfected or helper virus-infected QEC were used as control. In adherent culture, QEC-v-myc displayed a chondrocytic phenotype and synthesized type II collagen and Ch21 protein, while control chondrocytes synthesized type I and type II collagen with no Ch21 protein detected as long as the attachment to the plastic was kept. In suspension culture, QEC-v-myc readily aggregated and within 1 week the cell aggregates released small single cells; still they secreted only type II collagen and Ch21 protein. In the same conditions control cell aggregates released hypertrophic chondrocytes producing type II and type X collagens and Ch21 protein. In the appropriate culture conditions, QEC-v-myc reconstituted a tissue defined as nonhypertrophic, noncalcifying cartilage by the high cellularity, the low levels of alkaline phosphatase enzymatic activity, and the absence of type X collagen synthesis and of calcium deposition. We conclude that the constitutive expression of the v-myc oncogene keeps chondrocytes in stage I (active proliferation and synthesis of type II collagen) and prevents these cells from reconstituting hypertrophic calcifying cartilage.     

Expression of the human chondrocyte phenotype in vitro

Summary We report a culture scheme in which human epiphyseal chondrocytes lose their differentiated phenotype in monolayer and subsequently reexpress the phenotype in an agarose gel. The scheme is based on a method using rabbit chondrocytes. Culture in monolayer allowed small quantities of cells to be amplified and provided a starting point to study expression of the differentiated human chondrocyte phenotype. The cells cultured in monolayer produced type I procollagen, fibronectin, and small noncartilaginous proteoglycans. Subsequent culture in agarose was associated with the acquisition of typical chondrocyte ultrastructural features and the synthesis of type II collagen and cartilage-specific proteoglycans. The switch from the nonchondrocyte to the differented chondrocyte phenotype occurred under these conditions between 1 and 2 wk of agarose culture and was not necessarily homogeneous throughout a culture. This culture technique will facilitate direct investigation of human disorders of cartilage that have been addressed in the past by alternative approaches. This research is supported in part by research grants from the National Institutes of Health, (HD 20691) Bethesda, MD, and Shriners of North America (15953).     

Proteoglycan and collagen synthesis are correlated with actin organization in dedifferentiating chondrocytes.

The dedifferentiation of chondrocytes in culture is classically associated with a transition from a rounded to a spread morphology. However, the loss of chondroitin sulfate proteoglycan (CSPG) and type II collagen gene expression (markers of the differentiated chondrocyte) does not occur for all polygonal or fibroblast-like cells at the same stage of culture. Furthermore, it has been demonstrated that retinoic acid-dedifferentiated chondrocytes can reexpress type II collagen if treated by the microfilament disruptive drug dihydrocytochalasin B, without a return to the spherical shape. In the present study, we have investigated by fluorescent double-staining whether the synthesis of both CSPG and type II collagen by dedifferentiating chick chondrocytes in low density cultures is dependent on a type of actin organization. We report that the synthesis of CSPG and type II collagen synthesis is coincident with the presence of a faint microfibrillar actin architecture but is absent in chondrocytes showing well defined actin cables. This correlation was observed independently of the shapes exhibited by the cells. Moreover, type I collagen (marker of the dedifferentiated chondrocyte) is synthesized mainly in cells showing large actin cables. This study, performed in the absence of drugs, suggests that actin organization, rather than changes in cell shape, is involved in modulating the chondrogenic phenotype in vitro.     

The Persistence in the Synthesis of Type H Proteoglycan and Type II Collagen by Chondrocytes Cultured in the Presence of Chick Embryo Extract1

The effect of chick embryo extract on the phenotypic expression of differentiated chondrocytes has been studied in consideration of the fact that these cells are well characterized by certain specific cell products, such as type H proteochondroitin sulfate and type II collagen. In this study, we utilized floating chondrocytes derived from chick embryonic sterna, which can be cultured in suspension with no apparent change in the type of cell products for at least a period of eight weeks, as described in a previous paper (1). In the presence of chick embryo extract in the medium, the floating chondrocytes became attached to the bottom of the culture dish, and the attached cells took on a fibroblast-like appearance. Biochemical analyses of the proteochondroitin sulfate and collagen synthesized by the attached cells revealed that if the culture medium was renewed everyday, the cells having a fibroblast-like appearance continued to synthesize type H proteochondroitin sulfate and type II collagen. When however, the medium was replaced every other day, the synthesis of both proteochondroitin sulfate and collagen by the attached cells switched from the chondrocyte type to the fibroblast type, i.e. the synthesis of type M proteochondroitin sulfate and type I collagen, with little change in the fibroblast-like appearance. The results show that the morphological features of chondrocytes are not necessarily associated with the biochemical properties of these cells, and further suggest that, in chick embryo extract, there is no modulator capable of acting directly on the chondrocytes to bring about phenotypic changes with respect to the synthesis of collagen and proteoglycans.     

Reduced chondrocyte proliferation and chondrodysplasia in mice lacking the integrin-linked kinase in chondrocytes

Chondrocyte proliferation and differentiation requires their attachment to the collagen type II-rich matrix of developing bone. This interaction is mediated by integrins and their cytoplasmic effectors, such as the integrin-linked kinase (ILK). To elucidate the molecular mechanisms whereby integrins control these processes, we have specifically inactivated the ILK gene in growth plate chondrocytes using the Cre-lox methodology. Mice carrying an ILK allele flanked by loxP sites (ILK-fl) were crossed to transgenic mice expressing the Cre recombinase under the control of the collagen type II promoter. Inactivation of both copies of the ILK-fl allele lead to a chondrodysplasia characterized by a disorganized growth plate and to dwarfism. Expression of chondrocyte differentiation markers such as collagen type II, collagen type X, Indian hedgehog and the PTH-PTHrP receptor was normal in ILK-deficient growth plates. In contrast, chondrocyte proliferation, assessed by BrdU or proliferating cell nuclear antigen labeling, was markedly reduced in the mutant growth plates. Cell-based assays showed that integrin-mediated adhesion of primary cultures of chondrocytes from mutant animals to collagen type II was impaired. ILK inactivation in chondrocytes resulted in reduced cyclin D1 expression, and this most likely explains the defect in chondrocyte proliferation observed when ILK is inactivated in growth plate cells.     

Cartilage-specific 5' end of chick alpha 2(I) collagen mRNAs

Chondrocytes grown in suspension contain both type I and type II collagen mRNAs, yet synthesize only type II collagen. The inability of chondrocytes to synthesize the alpha 2 subunit of type I collagen, alpha 2(I), results from a severely reduced translation elongation rate (Bennett, V.D., and Adams, S.L. (1987) J. Biol. Chem. 262, 14806-14814). Furthermore, the alpha 2(I) collagen mRNAs from chondrocytes are translated inefficiently in vitro and appear slightly smaller than those from other cells (Focht, R.J., and Adams, S.L. (1984) Mol. Cell. Biol. 4, 1843-1852). These observations suggest that the reduced translation elongation rate may be due to an intrinsic property of the mRNAs. In this report we demonstrate that the alpha 2(I) collagen mRNAs from suspended chondrocytes are 120 bases shorter than those from other cells, and that the first 94 bases of the chondrocyte mRNAs differ from the corresponding region of the calvaria mRNAs. The unique 5' end of the chondrocyte alpha 2(I) collagen mRNAs accounts for their smaller size and may be responsible for the translation elongation defect. Interestingly, the alpha 2(I) collagen mRNAs from chondrocytes grown in monolayer, rather than in suspension, no longer display the cartilage-specific 5' end, suggesting that cell shape and/or adhesion may modulate the structure of the 5' end of the chondrocyte alpha 2(I) collagen mRNAs.     

Integrins alpha(6A)beta 1 and alpha(6B)beta 1 promote different stages of chondrogenic cell differentiation

The differentiation of chondrocytes and of several other cell types is associated with a switch from the alpha(6B) to the alpha(6A) isoform of the laminin alpha(6)beta(1) integrin receptor. To define whether this event plays a functional role in cell differentiation, we used an in vitro model system that allows chick chondrogenic cells to remain undifferentiated when cultured in monolayer and to differentiate into chondrocytes when grown in suspension culture. We report that: (i) upon over-expression of the human alpha(6B), adherent chondrogenic cells differentiate to stage I chondrocytes (i.e. increased type II collagen, reduced type I collagen, fibronectin, alpha(5)beta(1) and growth rate, loss of fibroblast morphology); (ii) the expression of type II collagen requires the activation of p38 MAP kinase; (iii) the over-expression of alpha(6A) induces an incomplete differentiation to stage I chondrocytes, whereas no differentiation was observed in alpha(5) and mock-transfected control cells; (iv) a prevalence of the alpha(6A) subunit is necessary to stabilize the differentiated phenotype when cells are transferred to suspension culture. Altogether, these results indicate a functional role for the alpha(6B) to alpha(6A) switch in chondrocyte differentiation; the former promotes chondrocyte differentiation, and the latter is necessary in stabilizing the differentiated phenotype.     

Wnt signaling is involved in human articular chondrocyte de-differentiation in vitro

Osteoarthritis is the most prevalent form of arthritis in the world. Certain signaling pathways, such as the wnt pathway, are involved in cartilage pathology. Osteoarthritic chondrocytes undergo morphological and biochemical changes that lead to chondrocyte de-differentiation. We investigated whether the Wnt pathway is involved in de-differentiation of human articular chondrocytes in vitro. Human articular chondrocytes were cultured for four passages in the presence or absence of IL-1 in monolayer or micromass culture. Changes in cell morphology were monitored by light microscopy. Protein and gene expression of chondrocyte markers and Wnt pathway components were determined by Western blotting and qPCR after culture. After culturing for four passages, chondrocytes exhibited a fibroblast-like morphology. Collagen type II and aggrecan protein and gene expression decreased, while collagen type I, matrix metalloproteinase 13, and nitric oxide synthase expressions increased. Wnt molecule expression profiles changed; Wnt5a protein expression, the Wnt target gene, c-jun, and in Wnt pathway regulator, sFRP4 increased. Treatment with IL-1 caused chondrocyte morphology to become more filament-like. This change in morphology was accompanied by extinction of col II expression and increased col I, MMP13 and eNOS expression. Changes in expression of the Wnt pathway components also were observed. Wnt7a decreased significantly, while Wnt5a, LRP5, β-catenin and c-jun expressions increased. Culture of human articular chondrocytes with or without IL-1 not only induced chondrocyte de-differentiation, but also changed the expression profiles of Wnt components, which suggests that the Wnt pathway is involved in chondrocyte de-differentiation in vitro.     

Modulation of sulfated proteoglycan synthesis and collagen gene expression by chondrocytes grown in the presence of bFGF alone or combined with IGF1

Prepubertal rabbit epiphyseal chondrocytes were grown in high density primary culture for 3 d. They were then incubated for 3 additional d in serum-free culture medium to which bFGF (1-50 ng/ml) was added. During the last 24 h incubation period, either IGF1 (1-80 ng/ml) or Insulin (1-5 micrograms/ml) was added to the culture medium. Chondrocyte DNA was significantly augmented with the increasing concentration of bFGF used, thus confirming its mitogenic effect on chondrocytes. On the other hand, bFGF was also shown to modulate the phenotypic expression of the chondrocytes. The 35S-sulfate incorporation into newly synthesized proteoglycans by the cultured cells decreased in a dose-dependent manner with bFGF concentration used. In addition, chondrocyte collagen gene expression was also shown to be modulated by bFGF. Total RNA extracted from the cultured cells was analyzed by dot blot and Northern blot with cDNA probes encoding for alpha 1 II and alpha 1 I procollagen chains. A significant lower level of type II collagen mRNA, the marker of chondrocytic phenotype, was observed when cells were grown in the presence of bFGF while the level of type I mRNA remained unchanged. When IGF1 or a high concentration of insulin was added to the cells during the last 24 h of incubation with bFGF, sulfated proteoglycan synthesis, as well as collagen type II mRNA level, were significantly stimulated when compared with chondrocytes incubated with bFGF alone. In conclusion, in the present experimental conditions, bFGF appears to be a growth promoting agent for chondrocytes in vitro with dedifferentiating action on chondrocyte phenotype. IGF1 or insulin used at a high concentration can prevent the dedifferentiating effect of bFGF without inhibiting its stimulating effect on chondrocyte DNA synthesis.     

Monoclonal antibody 11-fibrau: a useful marker to characterize chondrocyte differentiation stage

The aim of this study was to determine the feasibility of discriminating between differentiated and dedifferentiated chondrocytes by using the Mab 11-fibrau. Mab 11-fibrau did not bind to differentiated chondrocytes in cartilage of human knee joint, auricle, or nasal septum. During monolayer culture, when cells dedifferentiate, the number of 11-fibrau positive cells gradually increased and reached up to 100% after 4 passages. When differentiated chondrocytes were cultured in alginate, most (90--95%) of the cells remained 11-fibrau negative, in accordance with previous studies demonstrating that differentiated chondrocytes cultured in alginate keep their phenotype. Dedifferentiated (11-fibrau positive) cells were subjected to different redifferentiation regimes. As a well-known fact, cultures in alginate in medium where FCS was replaced by IGF1 and TGF beta 2 results in increased collagen type II formation, indicative for redifferentiation. However, the cells remained 11-fibrau positive, suggesting they are not (yet) fully redifferentiated. On the other hand, when dedifferentiated cells (after 4 passages in monolayer culture) were seeded in a biomaterial and implanted subcutaneously in a nude mouse, the newly formed cartilage matrix contained collagen type II and the 11-fibrau staining on the cells had disappeared. Our results indicate that 11-fibrau may be a reliable and sensitive marker of chondrocyte phenotype.     

Dedifferentiated chondrocytes cultured in alginate beads: Restoration of the differentiated phenotype and of the metabolic responses to Interleukin-1β

Chondrocytes cultivated in monolayer rapidly divide and lose their morphological and biochemical characteristics, whereas they maintain their phenotype for long periods of time when they are cultivated in alginate beads. Because cartilage has a low cellularity and is difficult to obtain in large quantities, the number of available cells often becomes a limiting factor in studies of chondrocyte biology. Therefore, we explored the possibility of restoring the differentiated properties of chondrocytes by cultivating them in alginate beads after two multiplication passages in monolayer. This resulted in the reexpression of the two main markers of differentiated chondrocytes: Aggrecan and type II collagen gene expression was strongly reinduced from day 4 after alginate inclusion and paralleled protein expression. However, 2 weeks were necessary for total suppression of type I and III collagen synthesis, indicators of a modulated phenotype. Interleukin-1β, a cytokine that is present in the synovial fluid of rheumatoid arthritis patients, induces many metabolic changes on the chondrocyte biology. Compared with cells in primary culture, the production of nitric oxide and 92-kDa gelatinase in response to interleukin-1β was impaired in cells at passage 2 in monolayer but was fully recovered after their culture in alginate beads for 2 weeks. This suggests that the effects of interleukin-1β on cartilage depend on the differentiation state of chondrocytes. This makes the culture in alginate beads a relevant model for the study of chondrocyte biology in the presence of interleukin-1β and other mediators of cartilage destruction in rheumatoid arthritis and osteoarthrosis. J. Cell. Physiol. 176:303–313, 1998. © 1998 Wiley-Liss, Inc.     

P38 mitogen‐activated protein kinase promotes dedifferentiation of primary articular chondrocytes in monolayer culture

Articular cartilage is an avascular tissue with poor regenerative capacity following injury, a contributing factor to joint degenerative disease. Cell‐based therapies for cartilage tissue regeneration have rapidly advanced; however, expansion of autologous chondrocytes in vitro using standard methods causes ‘dedifferentiation’ into fibroblastic cells. Mitogen‐activated protein kinase (MAPK) signalling is crucial for chondrocyte metabolism and matrix production, and changes in MAPK signals can affect the phenotype of cultured cells. We investigated the effects of inhibition of MAPK signalling on chondrocyte dedifferentiation during monolayer culture. Blockade of extracellular signal‐regulated kinase (ERK) and c‐Jun N‐terminal kinase (JNK) signalling caused a significant increase in cartilage gene expression, however, also caused up‐regulation of fibrotic gene expression. Inhibition of p38 MAPK (p38) caused a significant up‐regulation of collagen type II while suppressing collagen type I expression. P38 inhibition also resulted in consistently more organized secretion of collagen type II protein deposits on cell culture surfaces. Follow‐on pellet culture of treated cells revealed that MAPK inhibition reduced cell migration from the pellet. ERK and JNK inhibition caused more collagen type I accumulation in pellets versus controls while p38 inhibition strongly promoted collagen type II accumulation with no effect on collagen type I. Blockade of all three MAPKs caused increased GAG content in pellets. These results indicate a role for MAPK signalling in chondrocyte phenotype loss during monolayer culture, with a strong contribution from p38 signalling. Thus, blockade of p38 enhances chondrocyte phenotype in monolayer culture and may promote more efficient cartilage tissue regeneration for cell‐based therapies.     

Inhibition of terminal differentiation and matrix calcification in cultured avian growth plate chondrocytes by Rous sarcoma virus transformation

Endochondral bone formation involves the progression of epiphyseal growth plate chondrocytes through a sequence of developmental stages which include proliferation, differentiation, hypertrophy, and matrix calcification. To study this highly coordinated process, we infected growth plate chondrocytes with Rous sarcoma virus (RSV) and studied the effects of RSV transformation on cell proliferation, differentiation, matrix synthesis, and mineralization. The RSV-transformed chondrocytes exhibited a distinct bipolar, fibroblast-like morphology, while the mock-infected chondrocytes had a typical polygonal morphology. The RSV-transformed chondrocytes actively synthesized extracellular matrix proteins consisting mainly of type I collagen and fibronectin. RSV-transformed cells produced much less type X collagen than was produced by mock-transformed cells. There also was a significant reduction of proteoglycan levels secreted in both the cell-matrix layer and culture media from RSV-transformed chondrocytes. RSV-transformed chondrocytes expressed two- to- threefold more matrix metalloproteinase, while expressing only one-half to one-third of the alkaline phosphatase activity of mock infected cells. Finally, RSV-transformed chondrocytes failed to calcify the extracellular matrix, while mock-transformed cells deposited high levels of calcium and phosphate into their extracellular matrix. These results collectively indicate that RSV transformation disrupts the preprogrammed differentiation pattern of growth plate chondrocytes and inhibit chondrocyte terminal differentiation and mineralization. They also suggest that the expression of extracellular matrix proteins, type II and type X collagens, and the cartilage proteoglycans are important for chondrocyte terminal differentiation and matrix calcification. J. Cell. Biochem. 69:453–462, 1998. © 1998 Wiley-Liss, Inc.