Le projet PROMOCART (2007–2010) a été retenu suite à l’appel 2006 ANR-TecSan. Il a été conduit par le laboratoire « Biologie et ingénierie du cartilage » (Lyon) en partenariat avec les laboratoires « Matrice extracellulaire et pathologie » (Caen), l’institut de biologie de Lille, la société Symatèse biomatériaux (Chaponost) et le laboratoire des substituts cutanés des hospices civils de Lyon. Le cartilage est un tissu au potentiel de cicatrisation spontané très limité. La transplantation de chondrocytes autologues (TCA) est une technique mondialement utilisée pour le traitement de lésions limitées de cartilage articulaire. Cependant, cette approche implique une amplification des chondrocytes sur plastique, ce qui entraîne leur dédifférenciation. Un premier objectif de PROMOCART était de déterminer si la bone morphogenetic protein (BMP)-2 est capable de favoriser le maintien ou la restauration du phénotype des chondrocytes humains. Dans le but d’étendre la TCA à des lésions cartilagineuses plus importantes comme celles des arthroses débutantes, nous avons développé un procédé de reconstruction de cartilage humain dans des éponges de collagène en présence de BMP-2 et dans des conditions hypoxiques. Nous avons contrôlé la qualité du phénotype cellulaire par l’utilisation de nouveaux marqueurs du cartilage.

Project PROMOCART (2007-2010) was adopted following the call 2006 ANR-TecSan. It was led by the laboratory of Biology and Engineering of Cartilage (Lyon), and carried out in partnership with the laboratory of Extracellular Matrix and Pathology (Caen), Lille Institute of Biology, Symatese Biomateriaux company (Chaponost) and laboratory of Skin Substitutes (Lyon Hospital). Cartilage presents poor intrinsic healing capacity. Autologous chondrocyte implantation (ACI) is a worldwide used technique applied to focal defects of articular cartilage. However, it implies a step of cell amplification on plastic, which results in the loss of chondrocyte differentiation. The first objective of PROMOCART was to determine if bone morphogenetic protein (BMP)-2 could maintain or restore the differentiated phenotype of human chondrocytes. With the view of applying ACI to developing osteoarthritic lesions, we developed a new method of human cartilage reconstruction by using collagen sponges, BMP-2 and hypoxic culture conditions. We controlled the quality of the cellular phenotype by using new cartilage markers. (C) 2011 Elsevier Masson SAS. All rights reserved.

Regarding cartilage repair, tissue engineering is currently focusing on the use of adult mesenchymal stem cells (MSC) as an alternative to autologous chondrocytes. The potential of stem cells from various tissues to differentiate towards the chondrogenic phenotype has been investigated and it appears that the most common and studied sources are bone marrow (BM) and adipose tissue (AT) for historical and easy access reasons. In addition to three dimensional environment, the presence of member(s) of the transforming growth factor (TGF-β family and low oxygen tension have been reported to promote the in vitro differentiation of MSCs. Our work aimed at characterizing and comparing the degree of chondrogenic differentiation of MSCs isolated from BM and AT cultured in the same conditions. We also further aimed at and at determining whether hypoxia (2% oxygen) could affect the chondrogenic potential of AT-MSCs. Cells were first expanded in the presence of FGF-2, then harvested and centrifuged to allow formation of cell pellets, which were cultured in the presence of TGF-β3 and/or Bone Morphogenetic Protein-2 (BMP-2) and with 2 or 20% oxygen tension, for 24 days. Markers of the chondrocyte (COL2A1, AGC1, Sox9) and hypertrophic chondrocyte (COL10A1, MMP-13) were monitored by real-time PCR and/or by immunohistological staining. Our data show that BMP-2/TGF-β3 combination is the best culture condition to induce the chondrocyte phenotype in pellet cultures of BM and AT-MSCs. Particularly, a switch in the expression of the pre-chondrogenic type IIA form to the cartilage-specific type IIB form of COL2A1 was observed. A parallel increase in gene expression of COL10A1 and MMP-13 was also recorded. However when AT-MSCs were cultured in hypoxia, the expression of markers of hypertrophic chondrocytes decreased when BMP-2/TGF-β3 were present in the medium. Thus it seems that hypoxia participates to the control of AT-MSCs chondrogenesis. Altogether, these cellular model systems will help us to investigate further the potential of different adult stem cells for cartilage engineering.

Aim of the study. - Cartilage has a limited capacity for healing after trauma. Autologous chondrocyte implantation is widely used for the treatment of patients with focal damage to articular cartilage. Chondrocytes are isolated from biopsy specimen, cultured in monolayers on plastic then transplanted over the cartilage defect. However, chondrocyte amplification on plastic triggers their dedifferentiation. This phenomenon is characterized by loss of expression of type II collagen, the most abundant cartilage protein. The challenge for autologous chondrocyte implantation is to provide patients with well-differentiated cells. The aim of the present study was to test the capability of bone morphogenetic protein (BMP)-2 to promote redifferentiation of human chondrocytes after their expansion on plastic.Materials and methods. - Chondrocytes extracted from nasal cartilage obtained after septoplasty were serially cultured in monolayers. After one, two or three passages, BMP-2 was added to the culture medium. The cellular phenotype was characterized at the gene level by using RT-PCR. The expression of genes coding for type II procollagen with the ratio of IIB/IIA forms, aggrecan, Sox9, osteocalcin and type I procollagen was monitored.Results. - Our results show that BMP-2 can stimulate chondrogenic expression of the chondrocytes amplified on plastic, without inducing osteogenic expression. However, this stimulatory effect decreases with the number of passages.Conclusion. - The efficiency of autologous chondrocyte implantation could be improved by using chondrocytes treated with BMP-2 during their in vitro preparation. (C) 2008 Elsevier Masson SAS. Tous droits reserves.

Le cartilage est un tissu nécessaire au fonctionnement articulaire. Il peut se dégrader à la suite de traumatismes ou au cours du vieillissement, mais il ne se répare pas, car il n’est ni vascularisé ni innervé. Actuellement, aucune des nombreuses thérapies développées ne permet de régénérer un cartilage suffisamment fonctionnel. Aussi, les espoirs se sont tournés vers la production de cartilage in vitro par ingénierie tissulaire. Les cellules souches mésenchymateuses adultes humaines (CSM), présentes dans plusieurs tissus, sont capables de se différencier in vitro en chondrocytes sous l’action d’inducteurs spécifiques. L’utilisation des membres de la superfamille du transforming growth factor comme la bone morphogenetic protein (BMP)-2 s’est révélée très prometteuse dans ce contexte. En particulier, la BMP-2 favorise l’expression du collagène IIB, isoforme du collagène de type II spécifique du cartilage. L’expression du collagène de type IIB est donc un bon indicateur de la différenciation des CSM en chondrocyte. Par ailleurs, ce facteur de croissance peut aussi induire une maturation osseuse et le contrôle différentiel des propriétés chondrogéniques et ostéogéniques de la BMP-2 est un point critique dans le cadre de l’obtention in vitro de cartilage à partir de CSM. Plusieurs stratégies pour restreindre l’action de la BMP-2 à la conversion chondrocytaire sont proposées dans cette revue. Très certainement, l’utilisation contrôlée de facteurs de croissance comme la BMP-2 en combinaison avec des biomatériaux devrait permettre d’utiliser avec succès les CSM pour la réparation du cartilage.

Articular cartilage has a limited capacity for self-repair after trauma. Besides the conventional surgical techniques for repairing such defects, treatments involve implantation of autologous cells in suspension or within a variety of cell carrying scaffolds such as hyaluronic acid, alginate, agarose/alginate, fibrin or collagen. For the repair of full-thickness osteochondral defects, tissue engineers started to design single- or bi-phased scaffold constructs often containing hydroxyapatite-collagen composites, usually used as a bone substitute. The purpose of this study was to compare the behavior of bovine chondrocytes cultured in collagen-based scaffolds containing or not hydroxyapatite and cross-linked following two different methods. Calf chondrocytes seeded within Hemotèse and Collapat II sponges (SYMATESE biomaterials), chemically cross-linked with glutaraldehyde or EDC/NHS, were maintained up to one month in culture. The cells exhibited a similar behavior in the four scaffolds regarding proliferation level, deposition of glycosaminoglycans in the scaffolds and gene expression of types I, II and X collagens, aggrecan, MMP-1, -13 and the integrin subunits alpha10 and alpha11.

Articular cartilage is essential for the motion of the skeleton. However, this tissue is unable to spontaneously repair once injured, since it is avascular and aneural. Numerous repair strategies are developed, but they do not lead to a functional tissue and research into cartilage repair focuses now on tissue engineering technics. Adult mesenchymal stem cells (MSC), present in various tissues, have the potential to differentiate into chondrocytes in vitro in response to specific growth factors. The members of the transforming growth factor P, among them the bone morphogenetic protein (BMP)-2, appear very promising inducers in this context. BMP-2 favours chondrogenic expression, in particular expression of type IlB collagen, the cartilage-specific isoform of this collagen. Therefore, collagen type IIB is a good indicator of the differentiation state of MSC. However, since BMP-2 has also osteogenic properties, it is critical to differentially control chondrogenic and osteogenic properties of BMP-2 when used with MSC. Strategies for this control are presented in this review. Most likely, this is the combination of growth factors such as BMP-2 with biomaterials that will lead to the successful use of MSC for cartilage repair. (C) 2007 Elsevier Masson SAS. Tous droits reserves.

Purpose: To determine the effects of pharmacologically relevant concentrations of Rhein, the metabolite of Diacerhein, on cell proliferation rate of both human chondrocytes and synoviocytes.Methods: Human articular chondrocytes and synoviocytes were obtained from the knee or hankle of osteoarthritic patients.They were isolated by enzymatic procedure and cultured as monolayers in DMEM + 10% FCS, either in normal atmosphere (21% O 2 for synoviocytes) or in hypoxia (5% O 2 for chondrocytes), with 10 -6 , 10 -5 , and 10 -4 M Rhein or the corresponding concentrations of its vehicle DMSO.[ 3 H] thymidine incorporation was used to determine Rhein proliferative effects at 24 h, 48 h and one week incubation times.Cytotoxicity of the drug was also assayed with a non radioactive assay kit.Nuclear extracts were prepared and used to detect variation of cell cycle proteins by Western blotting.Moreover, Rhein effects on apoptosis were investigated through measurement of caspase activity.Results: Rhein inhibited the proliferation rate of both chondrocytes and synoviocytes in a dose-dependent manner, with significant decrease of thymidine labeling for 10 -5 M Rhein (by approximately two-fold) and for 10 -4 M Rhein (around 5-6 fold).In the same experimental conditions, no cytotoxicity of the drug was observed as compared to its vehicle DMSO.Western blots showed that Rhein modulates expression of cell cycle proteins: Rhein at 10 -4 M decreased p27 expression, but not that p21, nor cyclin D1 expression.Conclusions: Previous results indicated that Rhein may reduce the IL-1β deleterious effects on osteoarthritis cartilage through inhibition of the expression of degrading enzymes.Here we showed that Rhein could inhibit proliferation of both synoviocytes and chondrocytes.Thus, by its antiproliferative properties, this drug may decrease the development of the inflammatory synovial tissue that accompanies both OA and RA joint pathologies.Both its anti-catabolic and anti-proliferative effects may explain its value for the treatment of joint diseases.

Objective: To determine the best protocol for the preparation of a tissue-engineered cartilage to investigate the potential anti-arthritic and/or anti-osteoarthritic effects of drugs.Methods: Calf articular chondrocytes, seeded in collagen sponges were grown in culture for up to 1 month. At day 14 cultures received interleukin (IL)-1 beta (ranging from 0.1 to 20 ng/ml) for 1 to 3 days. Analyses of gene expression for extracellular matrix proteins, collagen-binding integrins, matrix metalloproteinases (MMPs), aggrecanases, TIMPs, IL-1Ra and I kappa-B alpha were carried out using real-time polymerase chain reaction (PCR). Metalloproteinase activities were analysed in the culture medium using both zymography and fluorogenic peptide substrates.Results: We selected a culture for 15 or 17 days with collagen sponges seeded with 107 chondrocytes showing a minimal cell proliferation, a maximal sulphated glycosaminoglycan (sGAG) deposition and a high expression of COL2A1, aggrecan and the alpha 10 integrin sub-unit and low expression of COLA2 and the alpha 11 integrin sub-unit. In the presence of 1 ng/ml IL-1 beta, we observed at day 15 up-regulations of 450-fold for MMP-1, 60-fold for MMP-13, 54-fold for ADAMTS-4 and MMP-3 and 10-fold for ADAMTS-5 and IL-1Ra. Down-regulations of 2.5-fold for COL2A1 and aggrecan were observed only at day 17. At the protein level a dose-dependent increase of total MMP-1 and MMP-13 was noted with less than 15% in the active form.Conclusions: This in vitro model of chondrocyte culture in three dimensional (3D) seems well adapted to investigate the responses of these cells to inflammatory cytokines and to evaluate the potential anti-inflammatory effects of drugs. (C) 2006 OsteoArthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

Application of mechanical stimulation, using dynamic bioreactors, is considered an effective strategy to enhance cellular behavior in load-bearing tissues. In this study, two types of perfusion mode (direct and free flow) are investigated in terms of the biosynthetic activities of chondrocytes grown in collagen sponges by assessment of cell proliferation rate, matrix production, and tissue morphology. Effects of the duration of preculture and dynamic conditioning are further determined. Our results have demonstrated that both bovine and human-derived chondrocytes demonstrate a dose-dependent response to flow rate (0-1 mL/min) in terms of cell number and glycosaminoglycan (GAG) content. This may reflect the weak adhesion of cells to the sponge scaffolds and the immature state of the constructs even after 3 weeks of proliferative culture. Our studies define an optimal flow rate between 0.1 and 0.3 mL/min for direct perfusion and free flow bioreactors. Using fresh bovine chondrocytes and a lower flow rate of 0.1 mL/min, a comparison was made between free flow system and direct perfusion system. In the free flow bioreactor, no cell loss was observed and higher GAG production was measured compared with static cultured controls. However, as with direct perfusion, the enhancement effect of free flow perfusion was strongly dependent on the maturation and organization of the constructs before the stimulation. To address the maturation of the matrix, preculture periods were varied before mechanical conditioning. An increase in culture duration of 18 days before mechanical conditioning resulted in enhanced GAG production compared with controls. Interestingly, additional enhancement was found in specimens that were further subjected to a prolonged duration of perfusion (63% increase after an additional 4 days of perfusion) after prematuration. The free flow system has an advantage over the direct perfusion system, especially when using sponge scaffolds, which have lower mechanical properties; however, mass transfer of nutrients is still more optimal throughout the scaffolds in a direct perfusion system as demonstrated by histological analysis.

INTRODUCTION: The surface properties of biomaterials have a direct in vitro and in vivo influence on a cell's morphology, migration, orientation and protein synthesis 1,2 . We propose to study the protein expression of rat osteoblasts and human fibroblasts on two different Nickel- Titanium (NiTi) surfaces. The NiTi samples were mechanically polished to obtain two kinds of surface roughness (Ra=0.07 µm for "NiTi 2400" and Ra=0.15 µm for "NiTi 400"). RT-PCR was performed to evaluate the expression of type I collagen and fibronectin for both osteoblasts and fibroblasts; and osteocalcin, osteopontin and osteonectin for osteoblasts. METHODS: Cell Culture: Osteoblasts were obtained after collagenase digestion of neonatal rat calvaria. Fibroblasts were obtained from biopsies of clinically healthy human gingiva. RT-PCR : Total RNA was isolated from cells after 7 days of culture on substrates using the "Qiagen RNeasy" kit. First-Strand cDNA synthesis was performed by RT with 2 µg of total RNA using the Superscript II enzyme with oligo dT12-18

Lesions of the articular cartilage have a large variety of causes among which traumatic damage, osteoarthritis and osteochondritis dissecans are the most frequent. Returning damaged cartilage in articular joints back to a functionally normal state has been a major challenge for orthopaedic surgeons. This interest results in large part because cartilage defects cannot adequately heal themselves. Current techniques used in orthopaedic practice to repair cartilage give variable and unpredictable results. Bone marrow stimulation techniques such as abrasion arthroplasty, drilling and microfracture produce mostly fibrocartilage. Autologous osteochondral transplant systems (mosaicplasty) have shown encouraging results. Autologous chondrocyte transplantation has led to a hyaline articular cartilage repair but little is known about the predictability and reliability of the procedure. The rapidly emerging field of tissue engineering promises creation of viable substitutes for failing cartilage tissue. Current tissue engineering approaches are mainly focused on the restoration of pathologically altered tissue structure based on the transplantation of cells in combination with supportive matrices and molecules. Among natural and synthetic matrices, collagen and polysaccharidic biomaterials have been extensively used with promising results. Recently, interest has switched to the use of mesenchymal stem cells instead of chondrocytes. Tissue engineering offers the possibility to treat localised cartilage lesions. Genetic engineering techniques using genetically modified chondrocytes offer also the opportunity to treat diffuse cartilage lesions occurring in osteoarthritis or inflammatory joint diseases. Electroporation is specially a reliable and inexpensive technique that shares with electrochemotherapy an ability to target the chondrocytes despite the barrier effect of the extracellular matrix without viral vectors. The authors review recent research achievements and highlight the potential clinical applications of new technologies in the treatment of patients with cartilage injuries.

This study evaluated the in vitro behaviour of bovine chondrocytes seeded in collagen gels, promising recently reported scaffolds for the treatment of full-thickness cartilage defects. To determine how chondrocytes respond to a collagen gel environment, 2×106 chondrocytes isolated from fetal, calf and adult bovine cartilage were seeded within type I collagen gels and grown for 12 days in both attached and floating (detached from the culture dish after polymerisation) conditions. Monolayer cultures were performed in parallel. All chondrocytes contracted floating gels to 55% of the initial size, by day 12. Contraction was dependent on initial cell density and inhibited by the presence of dihydrocytochalasin B as previously observed with fibroblasts. Gene expression was determined using conventional and real-time PCR. The chondrocyte phenotype was better maintained in floating gels compared to attached gels and monolayers. This was demonstrated by comparing the ratio of COL2A1/ COL1A2 mRNA and also of alpha10/alpha11 integrin mRNA. A strong up-regulation of MMP13 expression was measured at day 12 in floating gels. The composition of cartilage-like tissue obtained by growing chondrocytes in a collagen gel varied depending on the floating or attached conditions and initial cell density. It is thus important to consider these parameters when using this culture system in order to prepare a well-defined implant for cartilage repair.

Interest in chemical and physical modifications of culture conditions and composition, as a way to improve engineered cartilage, has grown over the last decade. To address some of these aspects, articular bovine chondrocytes seeded in collagen sponges (2.3x10(6) cells/cm(3), whose growth and metabolism have been previously reported) were grown under static or stirred conditions (orbital shaker at 30 rpm), in either 10% FCS-supplemented or serum-free media (1% ITS+1mM cysteine). Under stirred conditions, we observed a 2-fold increase in both cell proliferation and sulphated glycosaminoglycan deposition after 1 month of culture, compared to static conditions, and after 3 months, a more homogeneous distribution of both cells and neomatrix in the constructs. During the first month of culture, the substitution of FCS by ITS led to low cell proliferation and poor neomatrix deposition but, after 2 months a steep increase was observed with ITS for these two parameters that reached, after 3 months the levels observed with FCS. Aggrecan was the more abundant component at both gene and protein levels, whereas the collagenous network formed was looser than with FCS. In conclusion, the use of these simple culture conditions should improve, in long-term culture, the quality of the cartilage construct.

Lesions of articular cartilage have a large variety of causes among which traumatic damage, osteoarthritis and osteochondritis dissecans are the most frequent. Replacement of articular defects in joints has assumed greater importance in recent years. This interest results in large part because cartilage defects cannot adequately heal themselves. Many techniques have been suggested over the last 30 years, but none allows the regeneration of the damaged cartilage, i.e. its replacement by a strictly identical tissue. In the first generation of techniques, relief of pain was the main concern, which could be provided by techniques in which cartilage was replaced by fibrocartilage. Disappointing results led investigators to focus on more appropriate bioregenerative approaches using transplantation of autologous cells into the lesion. Unfortunately, none of these approaches has provided a perfect final solution to the problem. The latest generation of techniques, currently in the developmental or preclinical stages, involve biomaterials for the repair of chondral or osteochondral lesions. Many of these scaffolds are designed to be seeded with chondrocytes or progenitor cells. Among natural and synthetic polymers, collagen- and polysaccharide-based biomaterials have been extensively used. For both these supports, studies have shown that chondrocytes maintain their phenotype when cultured in three dimensions. In both types of culture, a glycosaminoglycan-rich deposit is formed on the surface and in the inner region of the cultured cartilage, and type II collagen synthesis is also observed. Dynamic conditions can also improve the composition of such three-dimensional constructs. Many improvements are still required, however, in a number of key aspects that so far have received only scant attention. These aspects include: adhesion/integration of the graft with the adjacent native cartilage, cell-seeding with genetically-modified cell populations, biomaterials that can be implanted without open joint surgery and combined therapies, aimed at disease modification, pain relief and reduction of inflammation.

This report completes a previous study on the growth and metabolism of fetal bovine epiphyseal chondrocytes cultured, within native or cross-linked collagen sponges carried out without the addition of fresh ascorbate. At low initial cell density (2.3×106 cells/cm3) cell proliferation and a low matrix deposition were observed, whereas at high initial cell density (2.3×107 cells/cm3) there was an absence of cell proliferation, but the deposition of a cartilage-like matrix was measured. In both cases, only traces of type I collagen (marker of chondrocyte dedifferentiation) were detected. In this report, we observed, after 1 month in culture with ascorbate, in both type of scaffolds and initial cell densities, an increase in cell proliferation (2-fold) and in expression of genes encoding for collagen types I, II, X and MMP-2 and -13, but no change in the level of matrix deposition (collagen and GAG). With regard to the proteins present, the main differences with or without ascorbate concerned the increase of neosynthesised type I collagen (up to 35% of the total collagen deposited in the sponge) and of the MMP-2 active form. In conclusion, these results show that ascorbate is an important factor to consider when preparing cartilage constructs for its action on chondrocyte phenotype modulation and proliferation.

Understanding the molecular mechanisms leading embryonic mesoblastic stem cells to be recruited towards a specific lineage and thereafter to develop the corresponding differentiation programme towards its endstage is a major challenge of today’s biology. These processes clearly depend on both autonomous genetic programmes and environmental signals. The characterization of the respective contributing factors is of interest in both fundamental and medical research. The availability of pluripotent and inducible progenitor cell lines would greatly facilitate the identification of the autocrine and paracrine signals involved in the recruitment towards a given lineage and in the implementation of a complete differentiation programme. Multipotential embryonic stem cells (ES) and embryonal carcinoma cells (EC) which are able to differentiate into derivatives of the three germ layers have provided in vitro differentiation model systems 1,2 . However, molecular studies using these systems remain difficult because of the heterogeneity of differentiation and of the short life span of the differentiated daughter cells. In an attempt to isolate lineage progenitor cell lines, we have transfected multipotential embryonal carcinoma cells with an immortalizing plasmid, pK4 which contains the early genes of SV40 under the control of the promoter of the E1A adenovirus, a virus known to be active early in mouse development 3 . Upon induction of differentiation of the multipotential cells, neuroectodermal, endodermal or mesodermal precursor cell lines were immortalized. The low level of constitutive expression of the SV40 T antigen promotes immortalization of the committed progenitors while still allowing

Collagen-based biomaterials in the form of sponges (bovine type I collagen, both native and cross-linked by treatment with diphenylphosphorylazide, noted control and DPPA sponges respectively) were tested as three-dimensional scaffolds to support chondrocyte proliferation with maintenance of the phenotype in order to form neocartilage. Control and DPPA sponges were initially seeded with 10(6) or 10(7) foetal bovine epiphyseal chondrocytes and maintained for 4 weeks in culture under static conditions in RPMI/NCTC medium with 10% FCS and without addition of fresh ascorbic acid. Both supports were always present during the study and a partial decrease in size and weight was detected only with control sponges, both seeded and unseeded. Cell proliferation was only noted in the 10(6) cells-seeded sponges (4-fold increase after 4 weeks of culture). Specific cartilage collagens (types II and XI) were deposited in the matrix throughout the culture and traces of type I collagen were noticed only in the culture medium after 2-3 weeks and 4 weeks in the case of 10(6) and 10(7) cells-seeded sponges, respectively. Glycosaminoglycans accumulated in the matrix, up to 1.8 and 9.8% of total dry weight after one month with both seeding conditions, which was much lower than in the natural tissue. In the 10(7) cells-seeded sponges, mineral deposition, observed with unseeded sponges, was significantly decreased (2- to 3-fold). These in vitro results indicate that both collagen matrices can support the development of tissue engineered cartilage.

Phenotypic expression of chondrocytes can be modulated in vitro by changing the culture technique and by agents such vitamins and growth factors. We studied the effects of ascorbic acid, retinoic acid (0.5 and 10 microM), and dihydrocytochalasin B (3, 10, 20 microM DHCB), separately or in combination (ascorbic acid + retinoic acid or ascorbic acid + DHCB), on the induction of maturation of fetal bovine epiphyseal chondrocytes grown for up to 4 weeks at high density in medium containing 10% fetal calf serum and the various agents. In the absence of any agent or with retinoic acid or DHCB alone, the metabolic activity of the cells remained very low after day 6, with no induction of type I or X collagen synthesis nor increase in alkaline phosphatase activity. Chondrocytes treated with fresh ascorbic acid showed active protein synthesis associated with expression of types I and X after 6 and 13 days, respectively. This maturation was not accompanied by obvious hypertrophy of the cells or high alkaline phosphatase activity. Addition of retinoic acid to the ascorbic acid-treated cultures decreased the level of type II collagen synthesis and delayed the induction of types I and X collagen, which were present only after 30 days. A striking increase in alkaline phosphatase activity (15-20-fold) was observed in the presence of both ascorbic acid and the highest dose of retinoic acid (10 microM). DHCB was also a potent inhibitor of the maturation induced by treatment with ascorbic acid, as the chondrocytes maintained their rounded shape and synthesized type II collagen without induction of type I or X collagen. The pattern of protein secretion was compared under all culture conditions by two-dimensional gel electrophoresis. The different regulations of chondrocyte differentiation by ascorbic acid, retinoic acid, and DHCB were confirmed by the important qualitative and quantitative changes in the pattern of secreted proteins observed by two-dimensional gel electrophoresis along the study.