Collagen-based biomaterials and cartilage engineering. Application to osteochondral defects.

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.

Activation by IL-1 of bovine articular chondrocytes in culture within a 3D collagen-based scaffold. An in vitro model to address the effect of compounds with therapeutic potential in osteoarthritis.

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)-1beta (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 Ikappa-Balpha 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 10(7) chondrocytes showing a minimal cell proliferation, a maximal sulphated glycosaminoglycan (sGAG) deposition and a high expression of COL2A1, aggrecan and the alpha10 integrin sub-unit and low expression of COL1A2 and the alpha11 integrin sub-unit. In the presence of 1 ng/ml IL-1beta, 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.

[Cartilage tissue engineering: state-of-the-art and future approaches]. / Ingénierie tissulaire du cartilage : état des lieux et perspectives.

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.

Optimization of dynamic culture conditions: effects on biosynthetic activities of chondrocytes grown in collagen sponges.

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.

Cartilage repair: surgical techniques and tissue engineering using polysaccharide- and collagen-based biomaterials.

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.

Influence of medium composition, static and stirred conditions on the proliferation of and matrix protein expression of bovine articular chondrocytes cultured in a 3-D collagen scaffold.

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.

Differential gene expression analysis in a rabbit model of osteoarthritis induced by anterior cruciate ligament (ACL) section.

Osteoarthritis (OA) is the most common of all joint diseases to affect mankind and is characterized by the degradation of articular cartilage. The low availability of normal and pathologic human cartilage and the inability to study the early stages of the disease in humans has led to the development of numerous animal models of OA. The aim of our study was to establish gene expression profiles during the progression of a rabbit model of OA induced by anterior cruciate ligament (ACL) section. Semiquantitative RT-PCR was used to follow expression of several relevant molecules (type II and X collagens, aggrecan, osteonectin, betaig-h3, BiP, TIMP-1, MMP-1, -3, -13, aggrecanase-1, -2) during development of OA in articular cartilage. In parallel, we monitored the activities of collagenase, caseinase, phospholipase A2 and glycosyltransferases (xylosyl-, galactosyl-, glucuronyl- and N-acetyl-galactosaminyl-transferase). Novel cDNA clones for rabbit type X collagen, aggrecanase-1 and -2, osteonectin and BiP were constructed to obtain species-specific primers. Ours result show that MMP-13 (collagenase-3) gene expression increased dramatically early after ACL surgery and remained high thereafter. An increase in MMP-1 (collagenase-1) and MMP-3 expression was also noted with an absence of variation for TIMP-1 expression. In addition, the global MMPs activities paralleled the MMP gene expression. These data together characterize at the molecular level the evolution of OA in this rabbit model. Furthermore, we have undertaken a search for identifying differentially expressed genes in normal and OA cartilage in this model, by differential display RT-PCR. We present here preliminary results with the determination of the best technical conditions to obtain reproducible electrophoresis patterns of differential display RT-PCR.

Matrix metalloproteinase-1, -3, -13 and aggrecanase-1 and -2 are differentially expressed in experimental osteoarthritis.

The aim of this study was to characterize the cellular phenotypes of articular cartilage and meniscus in rabbits with experimentally induced osteoarthritis (OA), by histological and molecular biological techniques. OA was induced by severing the anterior cruciate ligament of the knee and rabbits were killed 2, 4 or 9 weeks following surgery. Our histological observations show a progressive destruction of extracellular matrix in both tissues. To determine whether these morphological changes could be related to alterations in the regulation of gene expression for a subset of relevant molecules, levels of mRNA for proteinases and one inhibitor (MMP-1, -3 and -13, aggrecanase-1 and -2 and TIMP-1), matrix molecules and one chaperone (type II and X collagens, aggrecan, osteonectin, betaig-h3 and BiP) were assessed by reverse transcription-polymerase chain reaction. Our results indicate that for most markers expression profiles were similar in both tissues. In particular, matrix protein gene expression remained stable or varied little during progression of OA, suggesting a poor repair capacity of the tissues. MMP gene expression increased rapidly whereas aggrecanase gene expression remained stable. These findings suggest that differential regulation of mRNA levels of MMP-1, -3 and -13 on the one hand and aggrecanase-1 and -2 on the other, occurs during OA.

Native and DPPA cross-linked collagen sponges seeded with fetal bovine epiphyseal chondrocytes used for cartilage tissue engineering.

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.

Mechanical loading regulates protease production by fibroblasts in three-dimensional collagen substrates.

Mechanical loading is important in tissue formation and remodelling, notably in wound repair. The aim of this study was to measure the effects of controlled loading on the release of extracellular matrix protease activities by fibroblasts. Fibroblast populated collagen lattices were subjected to external cyclical loads through a computer controlled unit incorporated into a culture system, a tensioning-Culture Force Monitor. Cyclical loading was compared to untensioned and statically loaded gels (tethered endogenous contraction). Overall changes in a range of protease activities were monitored (chiefly by zymography) as measures of the cyto-mechanical response to these loads. Under static load, 2.5- and 13-fold more matrix metalloproteinase-2 was produced than matrix metalloproteinase-9, at 24 and 48 hours. Total matrix metalloproteinase-9 increased 37 fold on cyclical loading. Total matrix metalloproteinase-3 and urokinase plasminogen activator activities were dramatically reduced on cyclical loading while tissue type plasminogen activator activity was increased. Comparison with cell responses on stiffer substrates (collagen sponges) identified similar matrix metalloproteinase responses to load, but at much reduced levels (4-6 fold matrix metalloproteinase-9 stimulation on loading), showing the importance of matrix compliance to this mechano-response. In conclusion, physiological mechanical loading of fibroblasts in three dimensional collagen lattices elicited complex and substantial changes in matrix modifying proteases. These changes suggest that cells switch between expression of comparable protease activities mainly influencing cell-matrix interactions associated with migration or more generalized extracellular matrix remodelling.

Comparative analysis of different collagen-based biomaterials as scaffolds for long-term culture of human fibroblasts.

Biodegradable scaffolds, along with cells, are important components of most tissue-engineered constructs. In the study, there is a comparison of the behaviour of human fibroblasts cultured for up to six weeks in four different collagen-based three-dimensional matrices, in the form of sponges composed of pure native type I collagen (control), of collagen-GAG-chitosan (CGC) and of collagen cross-linked by two concentrations of diphenylphosphorylazide (DPPA-2 and DPPA-3). Variations in size and weight of the sponges, as well as fibroblast growth and migration, and total protein and collagen synthesis, are determined with time in culture. Owing to their low thermal stability, the partial denaturation and dissolution of the control sponges after incubation at 37 degrees C lead to considerable contraction and low cell proliferation. CGC sponges, stabilised by ionic interactions between the different components, show, after six weeks, limited contraction (20%) and weight increase (10% when seeded) and high cell growth (threefold increase). Similar results are obtained with weakly, cross-linked (DPPA-2) collagen sponges. Highly cross-linked (DPPA-3) sponges do not contract, whereas weight gain and cell proliferation are no different from those found with CGC and DPPA-2 sponges. Similar levels of total protein and collagen synthesis are shown for fibroblasts seeded in different matrices, with a slight general decrease (twofold) after three weeks, a much lower value than that observed with fibroblasts in culture within a contracted collagen gel (sixfold). Furthermore, the fraction of neo-synthesised collagen deposited in the sponges after six weeks represents more than 60% of the total, compared with only 10% obtained with fibroblasts in monolayer culture or 30% within a collagen gel. These results indicate that the matrices, particularly the CGC and DPPA-2 sponges, provide excellent supports for fibroblast growth and the formation of dermal and skin equivalents.

Collagen-based biomaterials as 3D scaffold for cell cultures: applications for tissue engineering and gene therapy.

Many substances are used in the production of biomaterials: metals (titanium), ceramics (alumina), synthetic polymers (polyurethanes, silicones, polyglycolic acid (PGA), polylactic acid (PLA), copolymers of lactic and glycolic acids (PLGA), polyanhydrides, polyorthoesters) and natural polymers (chitosan, glycosaminoglycans, collagen). With the rapid development in tissue engineering, these different biomaterials have been used as three-dimensional scaffolds and cell transplant devices. The principal biochemical and biological characteristics of the collagen-based biomaterials are presented, including their interactions with cells (fibroblasts), distinct from those of synthetic polymers, and their potential use in gene therapy through the formation of neo-organs or organoids.

Structural aspects of fish skin collagen which forms ordered arrays via liquid crystalline states.

The ability of acid-soluble type I collagen extracts from Soleidae flat fish to form ordered arrays in condensed phases has been compared with data for calf skin collagen. Liquid crystalline assemblies in vitro are optimized by preliminary treatment of the molecular population with ultrasounds. This treatment requires the stability of the fish collagen triple helicity to be controlled by X-ray diffraction and differential scanning calorimetry and the effect of sonication to be evaluated by viscosity measurements and gel electrophoresis. The collagen solution in concentrations of at least 40 mg ml(-1) showed in polarized light microscopy birefringent patterns typical of precholesteric phases indicating long-range order within the fluid collagen phase. Ultrastructural data, obtained after stabilization of the liquid crystalline collagen into a gelated matrix, showed that neutralized acid-soluble fish collagen forms cross-striated fibrils, typical of type I collagen, following sine wave-like undulations in precholesteric domains. These ordered geometries, approximating in vivo situations, give interesting mechanical properties to the material.

Acellular mineral deposition in collagen-based biomaterials incubated in cell culture media.

Rapid developments in tissue engineering have renewed interest in biodegradable three-dimensional structures such as collagen-based biomaterials. Collagen matrices seeded in vitro with fibroblasts, osteoblasts, and chondrocytes can form tissues resembling skin, bone, and cartilage that could be used as functional substitutes for damaged tissues. Collagen is associated with both dystrophic calcification of collagenous implants and bone mineralization. We report here the calcification properties of collagen sponges incubated in cell-free media. Mineral deposited in sponges was identified by X-ray and electron diffraction, Fourier transform infrared spectroscopy, and the molar ratio of calcium:phosphorus (Ca:P) as a poorly crystalline apatite similar to bone. The degree of calcification increased with length of incubation and the Ca and P content of the media, with 10-15% Ca (dry weight) after 21 days' incubation in media containing 1.6-3 mM Ca and a Ca x P molar product of 2-3 mM(2), but only 2% Ca after incubation in medium with 1.33 mM Ca and a 1.7 mM(2) Ca x P molar product. Mineral deposition was completely inhibited in sponges that were washed extensively and initially contained less than 0.01% P. Readdition of phosphate in these sponges and subsequent freeze drying and sterilization restore their mineralization capacity, suggesting that collagen per se cannot initiate calcification and that the inorganic phosphate content associated with the collagen preparation process is in the solid state a potential nucleator. Addition of chondroitin 4-sulfate to the sponges partially or totally inhibited mineral deposition, even though 80-90% of the compound was released within 24 hours. These results indicate that acellular calcification of collagen-based biomaterials can occur under the culture conditions currently used in tissue engineering.

Mouse fibroblasts in long-term culture within collagen three-dimensional scaffolds: influence of crosslinking with diphenylphosphorylazide on matrix reorganization, growth, and biosynthetic and proteolytic activities.

With the rapid development of tissue engineering and gene therapy, collagen-based biomaterials frequently are used as cell transplant devices. In this study we determined the behavior of mouse fibroblasts cultured for up to 6 weeks in control sponges treated by severe dehydration and used commercially as hemostatic agents and in two sponges (DPPA 2 and 3) crosslinked by diphenylphosphorylazide, a method developed in our laboratory. Growth capacity, biosynthetic and proteolytic activities, and matrix reorganization were followed over time in cultures and compared with similar data for fibroblasts in monolayer culture on plastic and in floating or attached collagen gels. Control sponges with and without seeded mouse fibroblasts showed rapid partial denaturation or contraction, weight loss, and severe calcification (13-18% Ca) after 6 weeks. In contrast, the crosslinked sponges showed only slightly decreased size and weight, and the calcification was inhibited (0.2% Ca) in the presence of cells. Mouse fibroblasts seeded on the crosslinked sponge surface at 50,000-200,000 cells/cm(2) progressively penetrated the matrix and proliferated to give the same constant cell density after 3 weeks (around 600,000 cells/sponge). A specific, two- to threefold decrease in collagen synthesis was observed between 1 and 3 or 6 weeks, due mainly to a decrease in the fraction secreted into the medium (25-30% instead of 45-50%). No collagenase 3 activity was detected in the culture medium under any condition or time whereas 25% gelatinase A was found by gelatin zymography to be in an active form in cultures within sponges as compared with less than 10% in monolayers and more than 50% in floating collagen gel. A small amount of gelatinase B was observed after 1 week in sponge cultures and was completely absent thereafter. These results show that the biosynthetic and proteolytic behavior of mouse fibroblasts cultured in crosslinked collagen scaffolds is different from that in monolayers or in floating collagen gels and more similar to that previously described in attached collagen gels.

Regulation of growth, protein synthesis, and maturation of fetal bovine epiphyseal chondrocytes grown in high-density culture in the presence of ascorbic acid, retinoic acid, and dihydrocytochalasin B.

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.

Lineage-dependent collagen expression and assembly during osteogenic or chondrogenic differentiation of a mesoblastic cell line.

The mesoblastic clone, C1, behaves as a tripotential progenitor able to self-renew and to differentiate toward osteogenesis, chondrogenesis, or adipogenesis in response to specific inducers. In this study, expression and deposition by the C1 cells of essential components of the extracellular matrix, collagens type I, II, III, V, XI, VI, IX, and X were followed along the osteogenic and chondrogenic pathways, through biochemical, immunochemical, and electron microscopy analyses. Implementation of each program involves profiles of collagen synthesis and matrix assembly close to those documented in vivo. Depending on the applied inducers, cells adopt a defined identity and, controls acting at transcriptional and posttranslational levels adapt the set of deposited collagens to one particular cell fate. Osteogenic C1 cells selectively build a type I collagen matrix also containing type III, V, and XI collagens but selectively exclude type II collagen. Chondrogenic C1 cells first elaborate a type II collagen network and then acquire hypertrophic chondrocyte properties while assembling a type X collagen matrix as in the growth plate. This study provides an example of how a mesoblastic cell line can develop, in vitro, each of its genetic programs up to terminal differentiation. Intrinsic factors and time-dependent cell-matrix interactions might, as in vivo, underline the implementation of an entire differentiation program.

A recurrent valpha17/vbeta10 TCR-expressing T cell clone is involved in the pathogenicity of collagen-induced arthritis in DBA/1 mice.

Collagen-induced arthritis (CIA) is an experimental model that mimics clinical and histological features of rheumatoid arthritis. In this disease, a crucial role in initiating the pathological changes has been assigned to T lymphocytes expressing the Th1 phenotype. Aiming at identifying type II collagen (CII)-specific T cells involved in CIA, T cell clones were generated in vitro from the lymph nodes (LN) of CII-immunized DBA / 1 mice. In three independent experiments, we repeatedly isolated CD4(+) Th1 clones recognizing the immunodominant epitope in the CB11 fragment of bovine CII and expressing a unique alpha betaTCR produced by the rearrangement of Valpha17/Jalpha20 and Vbeta10/Dbeta1.1/Jbeta2.5 gene segments. By reverse transcriptase-PCR, we demonstrated the presence of mRNA transcripts specific for the beta complementary-determining region 3 of this clonotype in the LN of the majority (73%) of mice with CIA whereas it was never detected in control animals. When transferred to CII-immunized DBA/1 mice, this recurrent Th1 clone augmented the incidence, aggravated significantly the clinical signs of CIA and greatly enhanced the anti-CII antibody response. Altogether, these results provide evidence that a CD4(+) Th1 clone belonging to the public arm of the response toward the immunodominant epitope of CII is involved in the cascade of events leading to CIA.

Isolation, cloning, and expression of a new murine zinc finger encoding gene.

With the aim of identifying genes involved in cartilage differentiation, we have used a subtractive hybridization strategy with cDNAs from a chondrocytic cell line (MC615) and mRNAs from a mesenchymal precursor cell line (10T1/2). We have isolated a cDNA clone representing a novel mouse gene. The predicted 368-amino acid protein, designated ZF-12, contains four C(2)H(2)-type zinc finger motifs and one region homologous to the LeR domain, a finger-associated structural domain. ZF-12 mRNAs are expressed during embryonic development and in different organs in adult, including rib cartilage. These data suggest that ZF-12 might play an important role not only in cartilage differentiation, but also in basic cellular processes.