The name collagen designates a family of structural proteins of the extracellular matrix-forming supramolecular aggregates that involve lateral interactions between characteristic triple-helical domains. The cartilage collagen fibrils are made up of, in large part, type II collagen molecules. It has recently been demonstrated that cartilage collagen fibrils are actually mixtures of several collagen types, namely types II, IX, and XI. These three types are characteristic of cartilaginous tissues, although they have been observed at low levels in some other extracellular matrices. Type X collagen is also cartilage specific, but its expression is restricted to the hypertrophic chondrocyte. Type II collagen is a genetically distinct member of the fibril-forming collagens and the gene is present in a single copy in the genome. Type V collagen is distributed in various connective tissues is associated with type I collagen in heterotypic fibrils. Type XI collagen can form fibrils in vitro. The interactions between proteoglycans and type XI collagen are strongly inhibited by heparin.

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.

Among the existing repair strategies for cartilage injury, tissue engineering approach using biomaterials and chondrocytes offers hope for treatments. In this context, collagen-based biomaterials are good candidates as scaffolds for chondrocytes in cell transplantation procedures. These scaffolds are provided under different forms (gel or crosslinked sponge) made with either type I collagen or type I or type II atelocollagen molecules. The present study was undertaken to investigate how bovine articular chondrocytes sense and respond to differences in the structure and organization of these collagen scaffolds, over a 12-day culture period. When chondrocytes were seeded in the collagen scaffolds maintained in free-floating conditions, cells contracted gels to 40-60% and sponges to 15% of their original diameter. Real-time polymerase chain reaction analysis indicated that the chondrocyte phenotype, assessed notably by the ratio of COL2A1/COL1A2 mRNA and alpha10/alpha11 integrin subunit mRNA, was comparatively better sustained in type I collagen sponges when seeded at high cell density, also in type I atelocollagen gels. Besides, proteoglycan accumulation in the different scaffolds, as assessed by measuring the sulfated glycosaminoglycan content, was found be highest in type I collagen sponges seeded at high cell density. In addition, gene expression of matrix metalloproteinase-13 increased dramatically (up to 90-fold) in chondrocytes cultured in the different gels, whereas it remained stable in the sponges. Our data taken together reveal that type I collagen sponges seeded at high cell density represent a suitable material for tissue engineering of cartilage.

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.

Joint cartilage has a poor intrinsic ability to heal. Common surgical treatments for traumatic lesions, after debridement of the chondral defect, include stimulation of subchondral bone (microfracture), perichondrial or periosteal grafting, and mosaicplasty (osteochondral cylinder transplantation). Autologous chondrocyte transplantation (ACT) was the first application of cell therapy to orthopaedic surgery. Despite promising results, several groups have tested tissue engineering protocols based on ex vivo colonization of biodegradable polymer matrices that are subsequently transplanted to the target site. Tissue engineering as a treatment for osteoarthritis is even more challenging. Transplantation of genetically modified cells is an interesting concept, based on the production of therapeutic proteins directly at the target site.

Joint cartilage has a poor intrinsic ability to heal. Common surgical treatments for traumatic lesions, after debridement of the chondral defect, include stimulation of subchondral bone (microfracture), perichondrial or periosteal grafting, and mosaicplasty (osteochondral cylinder transplantation). Autologous chondrocyte transplantation (ACT) was the first application of cell therapy to orthopaedic surgery. Despite promising results, several groups have tested tissue engineering protocols based on ex vivo colonization of biodegradable polymer matrices that are subsequently transplanted to the target site. Tissue engineering as a treatment for osteoarthritis is even more challenging. Transplantation of genetically modified cells is an interesting concept, based on the production of therapeutic proteins directly at the target site.

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.

Purpose: Damaged cartilage has very limited potential for self-repair. Tissue bioengineering offers an interesting alternative for repair of cartilage injury caused by joint trauma or osteochondritis dessicans. The purpose of this work was to use primary chondrocytes cultivated in vitro on collagen gel to produce a neocartilage which can be reimplanted. Material and methods: Chondrocytes were extracted by enzymatic digestion from calf feet harvested from animals aged less than six months. Two million cells were seeded on collagen gels in multiple-well plates and covered with culture medium (1 ml). Type I collagen was acquired from ground calf skin used at a concentration of 1.25 mg/ml. The culture medium was a v/v mixture of RPMI 1640 and NCTC 109. This mixture was supplemented with 10% foetal calf serum, 100 U/ml penicillin, and 250 ng/ml amphotericin B. Cell proliferation was assess fluorometrically and synthesis of glycosaminoglycans (sGAG) by colorimetric assay. Histological study (safranine O) and immunohistochemistry tests (type I and II collagen) were performed to monitor synthesis of matrix components. Expression of genes coding for certain matrix proteins (collagen Ia 2 and 1, II, X, agrecan and MMP13) was studied using RT-PCR. Results: The chondrocyte phenotype was preserved. Type II collagen as well as agrecan was expressed and expression of type I collagen did not increase during the culture. Progressive synthesis of sGAG was observed as was moderate cell proliferation. Cell distribution within the gel was apparently homogeneous. The chondrocytes retained their round shape throughout the study. Type II collagen deposits were visible on day 9 in peripheral cells in areas of high-cell density, then progressed with time. Discussion: Our in vitro results show that three-dimensional cultures of chondrocytes using a collagen gel can produce construction of an extracellular matrix with preservation of chondrocyte phenotype during the culture period. Conclusion: The collagen matrix offers an environment favouring the formation of a functional artificial cartilage by chondrocytes and opens promising perspectives for repairing damaged cartilage.

During endochondral ossification, type I collagen is synthesized by osteoblasts together with some hypertrophic chondrocytes. Type I collagen has also been reported to be progressively synthesized in degenerative joints. Because Matrix Metalloproteinase-13 (MMP-13) plays an active role in remodeling cartilage in fetal development and osteoarthritic cartilage, we investigated whether type I collagen could activate MMP-13 expression in chondrocytes. We used a well-established chondrocytic cell line (MC615) and we found that MMP-13 expression was induced in MC615 cells cultured in type I collagen gel. We also found that alpha1beta1 integrin, a major collagen receptor, was expressed by MC615 cells and we further assessed the role of alpha1beta1 integrin in conducting MMP-13 expression. Induction of MMP-13 expression by collagen was potently and synergistically inhibited by blocking antibodies against alpha1 and beta1 integrin subunits, indicating that alpha1beta1 integrin mediates the MMP-13-inducing cellular signal generated by three-dimensional type I collagen. We also determined that activities of tyrosine kinase and ERK and JNK MAP kinases were required for this collagen-induced MMP-13 expression. Interestingly, bone morphogenetic protein (BMP)-2 opposed this induction, an effect that may be related to a role of BMP-2 in the maintenance of cartilage matrix.

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.

A spectrometric assay for the determination of concentration of para-sulphonato-calix[n]arenes and their derivatives has been developed using dimethylmethylene blue (DMMB) as a probe. Interaction with para-sulphonato-calix[n]arenes leads to a metachromatic shift in the spectrum of DMMB with appearance of a peak at 536 nm and diminution of the spectral intensity of the peaks at 594 and 649 nm. The method shows good linearity in the concentration range 0–6 μg/ml for para-sulphonato-calix[n]arenes.

The effect of 0.1-10 /tM retinoic acid (RA) on foetal bovine chondrocytes was investigated in high-density cultures (0.6 x 106 cells/cm2). After 5 days of culture in ascorbate-free medium, control chondrocytes presented a typical rounded shape and synthesized type II, IX, XI and III collagens. After RA treatment on days 2-5 of culture, the cells exhibited a fibroblast-like shape and decreased synthesis of total protein (48 %) and pepsinresistant proteins (60 %) as determined by [35S]methionine labelling. Addition of RA was not followed by the expression of type I collagen, but induced quantitative changes in the synthesis of cartilage-specific collagens (II, IX and XI) as measured by direct autoradiography of the corresponding bands after SDS/PAGE. The main change was in type II collagen synthesis, with a 80% decrease in the cell-layer fraction and a 89% decrease in culture-

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.

In osteoarthritic cartilage, chondrocytes are able to present heterogeneous cellular reactions with expression and synthesis of the (pro)collagen types characteristic of prechondrocytes (type IIA), hypertrophic chondrocytes (type X), as well as differentiated (types IIB, IX, XI, VI) and dedifferentiated (types I, III) chondrocytes. The expression of type IIA procollagen in human osteoarthritic cartilage support the assumption that OA chondrocytes reverse their phenotype towards a chondroprogenitor phenotype. Recently, we have shown that dedifferentiation of mouse chondrocytes induced by subculture was associated with the alternative splicing of type II procollagen pre-mRNA with a switch from the IIB to the IIA form. In this context, we demonstrated that BMP-2 favours expression of type IIB whereas TGF-beta1 potentiates expression of type IIA induced by subculture. These data reveal the specific capability of BMP-2 to reverse the program of chondrocyte dedifferentiation. This interesting feature needs to be tested with human chondrocytes since cell amplification is required for the currently used autologous chondrocyte transplantation.

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.