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International audience; Urothelium is a highly specialized epithelium covering the entire urinary excretory system. Tissue engineering of this urinary tract may allow to consider its reconstruction to perform in vitro studies or in vivo replacement. Therefore, the question of specific reconstruction of the urothelium arises in order to guarantee the neotissue’s ability to act as a barrier against highly cytotoxic urine. This literature review describes the different cell types and strategies available for this reconstruction. The non-reconstruction of urothelium relies on the colonization of a biomaterial by the adjacent healthy tissue but allows only incomplete reconstruction and fibrosis. The use of autologous urothelial cells requires preliminary surgery and has not been successful enough in humans. Research has therefore focused on the use of stem cells. Adipose Derived Stem Cells (ADSCs) and Bone Marrow Derived Stem Cells (BMSCs) allow the reconstruction of the smooth muscle layer, but have little effect on urothelium reconstruction. Urine Derived Stem Cells (UDSCs) and Bladder mesenchymal Stem Cells (BSCs) are very promising because they allow the achievement of a differentiated urothelium. Induced Pluripotent Stem Cells (IPSCs) and Embryonic Stem Cells (ESCs) can be differentiated towards urothelial phenotype but their use is restricted by ethics.

1- article; International audience; BACKGROUND: Biodegradable polymers used in tissue engineering applications, such as poly(ε-caprolactone) (PCL), are hydrophobic leading to a lack of favorable cell signalization and finally to a poor cell adhesion, proliferation and differentiation. To overcome this problem, scaffolds undergo generally a surface modification. OBJECTIVE: Our laboratory has demonstrated that the grafting of poly(sodium styrene sulfonate) (pNaSS) onto titanium or poly(ethylene terephthalate) surfaces, leads to a more specific protein adsorption and a better control of cell proliferation. The objective of this work is to develop, through a straightforward way, bioactive elastomeric PCL scaffolds by grafting pNaSS. METHODS: Porous elastomeric PCL scaffolds were developed using a particulate-leaching process. pNaSS was grafted into the scaffold by a "grafting from" technique. In vitro tests were carried out to assess cell adhesion and protein expression. RESULTS: pNaSS was grafted homogeneously onto PCL scaffolds without degrading the biodegradable polymer or the porous structure. The in vitro studies have shown that pNaSS grafted onto PCL improves the cell response with a better expression of collagen, fibronectin and integrin α1. CONCLUSIONS: The grafting of pNaSS onto biomaterial surfaces is a versatile method that can provide a new generation of biodegradable scaffolds which could be "biointegrable".

The extracellular matrix (ECM) has long been viewed primarily as an organized network of solid-phase ligands for integrin receptors. During degenerative processes, such as osteoarthritis, the ECM undergoes deterioration, resulting in its remodeling and in the release of some of its components. Matrilin-3 (MATN3) is an almost cartilage specific, pericellular protein acting in the assembly of the ECM of chondrocytes. In the past, MATN3 was found required for cartilage homeostasis, but also involved in osteoarthritis-related pro-catabolic functions. Here, to better understand the pathological and physiological functions of MATN3, its concentration as a circulating protein in articular fluids of human osteoarthritic patients was determined and its functions as a recombinant protein produced in human cells were investigated with particular emphasis on the physical state under which it is presented to chondrocytes. MATN3 down-regulated cartilage extracellular matrix (ECM) synthesis and up-regulated catabolism when administered as a soluble protein. When artificially immobilized, however, MATN3 induced chondrocyte adhesion via a α5β1 integrin-dependent mechanism, AKT activation and favored survival and ECM synthesis. Furthermore, MATN3 bound directly to isolated α5β1 integrin in vitro. TGFβ1 stimulation of chondrocytes allowed integration of exogenous MATN3 into their ECM and ECM-integrated MATN3 induced AKT phosphorylation and improved ECM synthesis and accumulation. In conclusion, the integration of MATN3 to the pericellular matrix of chondrocytes critically determines the direction toward which MATN3 regulates cartilage metabolism. These data explain how MATN3 plays either beneficial or detrimental functions in cartilage and highlight the important role played by the physical state of ECM molecules.

International audience; Introduction: We have taken advantage of the large screening capacity of a multiplex immunoassay to better define the respective contribution of articular versus systemic cytokines in experimental arthritis.Methods: We performed a follow up (from 7 hours to 14 days) multiplex analysis of 24 cytokines in synovial fluid and sera of rats developing Antigen-Induced Arthritis (AIA) and confronted their protein level changes with molecular, biochemical, histological and clinical events occurring in the course of the disease.Results: The time-scheduled findings in arthritic joints correlated with time-dependent changes of cytokine amounts in joint effusions but not with their blood levels. From seven hours after sensitization, high levels of chemokines (MCP-1, MIP1α, GRO/KC, RANTES, eotaxin) were found in synovial fluid of arthritic knees whereas perivascular infiltration occurred in the synovium; local release of inflammatory cytokines (IFNγ, IL-1β, IL-6) preceded the spreading of inflammation and resulted in progressive degradation of cartilage and bone. Finally a local overexpression of several cytokines/adipocytokines poorly described in arthritis (IL-13, IL-18, leptin) was observed.Conclusions: Distinct panels of cytokines were found in arthritic fluid during AIA, and the expected effect of mediators correlated well with changes occurring in joint tissues. Moreover, multiplex analysis could be helpful to identify new pathogenic mediators and to elucidate the mechanisms supporting the efficacy of putative targeted therapies.

International audience

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 x 10(6) 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.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 x 10(6) 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.

To establish a system for efficient direct in vivo gene targeting into rat joint, we have evaluated a strategy of gene transfer by means of the delivery of external electric pulses (EP) to the knee after intra-articular injection of a reporter gene (GFP). Rats were killed at various times after the electro gene-therapy to analyze GFP gene expression by immunohistochemistry. GFP staining was detected in the superficial, middle, and deep zones of the patellar cartilage at days 2 and 9, and thereafter only in the deep zone (months 1 and 2). The average percentage of GFP-positive cells was estimated at 30% both one and 2 months after the gene transfer. Moreover, no pathologic change caused by the EP was detected in the cartilage. The level and stability of the long-term GFP expression found in this study demonstrate the feasibility of a treatment of joint disorders (inflammatory or degenerative, focal or diffuse) using electric gene transfer.

One of the major issues with treatment of osteoarticular diseases is reaching cells or tissue inside the joint. Administration of an active compound by intravenous or per os requires elevated amount to be effective, and can initiate systemic and negative side effects. In this work we have evaluated a drug delivery system for hydrophilic molecule, which can be used for articular tissues. Polymeric nanoparticles of poly lactic-co-glycolic acid, labelled with a fluorescent dye were evaluated both in vitro and in vivo. These nanoparticles are covered with hyaluronic acid to favor particles-cells interaction. We demonstrated here that such particles are internalized after 8 hours of exposure in synoviocytes, chondrocytes and mesenchymal stem cells. Evaluation of cytotoxicity and inflammation revealed the neutrality of these particles for articular cell types. Finally, intraarticular injections of particles were performed in healthy rat joint and in pathological models. Histological analyses of extracellular matrix integrity and inflammatory status do not demonstrate any side-effect following particles injections. These healthy nano-device target primarily synovial cells, and their degradation inside cells does not provoke deleterious effects. This PLGA based drug delivery system would be used to safely deliver active molecules to the joint. The combination of these particles with various hydrophilic molecules (as nucleic acid, drugs,…) may allow their direct distribution in the joint, and lead to new therapeutics perspectives.; Un des écueils du traitement des pathologies ostéoarticulaires est l’adressage de molécules aux cellules et tissus qui constituent l’articulation. L’administration de principes actifs par voie sanguine ou orale nécessite l’apport de quantités importantes et peut causer des effets indésirables. Dans ces travaux, nous avons évalué un système d’adressage de molécules hydrophiles. Des particules de polymère d’acide lactique et glycolique, marquées par un traceur fluorescent ont été évaluées in vitro puis in vivo. Ces particules possèdent un recouvrement en acide hyaluronique pour améliorer les interactions avec les cellules. Les études réalisées ont permis de mettre en évidence l’internalisation des particules dans les synoviocytes, chondrocytes, et cellules souches mésenchymateuses après 8 heures d’exposition. Parallèlement, l’évaluation in vitro de différents paramètres de cytotoxicité et d’inflammation a permis de souligner la compatibilité de ces vecteurs avec les cellules articulaires. Enfin, les effets de l’injection intra-articulaire de particules ont été évalués chez le rat sain et différents modèles pathologiques. L’analyse histologique des articulations a démontré l’absence de dégradation de la matrice cartilagineuse et de réaction inflammatoire suite à l’injection de particules. Il a été également constaté un ciblage majoritaire des particules vers la membrane synoviale. Ces travaux permettent de valider l’utilisation de ce type de vecteur pour le ciblage de l’articulation et l’apport de principes actifs. L’association de ces particules à des molécules hydrophiles (acides nucléiques, médicaments,…), pourra permettre leur apport direct dans l’articulation et ouvrir de nouvelles perspectives thérapeutiques.

One of the major issues with treatment of osteoarticular diseases is reaching cells or tissue inside the joint. Administration of an active compound by intravenous or per os requires elevated amount to be effective, and can initiate systemic and negative side effects. In this work we have evaluated a drug delivery system for hydrophilic molecule, which can be used for articular tissues. Polymeric nanoparticles of poly lactic-co-glycolic acid, labelled with a fluorescent dye were evaluated both in vitro and in vivo. These nanoparticles are covered with hyaluronic acid to favor particles-cells interaction. We demonstrated here that such particles are internalized after 8 hours of exposure in synoviocytes, chondrocytes and mesenchymal stem cells. Evaluation of cytotoxicity and inflammation revealed the neutrality of these particles for articular cell types. Finally, intraarticular injections of particles were performed in healthy rat joint and in pathological models. Histological analyses of extracellular matrix integrity and inflammatory status do not demonstrate any side-effect following particles injections. These healthy nano-device target primarily synovial cells, and their degradation inside cells does not provoke deleterious effects. This PLGA based drug delivery system would be used to safely deliver active molecules to the joint. The combination of these particles with various hydrophilic molecules (as nucleic acid, drugs,…) may allow their direct distribution in the joint, and lead to new therapeutics perspectives., Un des écueils du traitement des pathologies ostéoarticulaires est l’adressage de molécules aux cellules et tissus qui constituent l’articulation. L’administration de principes actifs par voie sanguine ou orale nécessite l’apport de quantités importantes et peut causer des effets indésirables. Dans ces travaux, nous avons évalué un système d’adressage de molécules hydrophiles. Des particules de polymère d’acide lactique et glycolique, marquées par un traceur fluorescent ont été évaluées in vitro puis in vivo. Ces particules possèdent un recouvrement en acide hyaluronique pour améliorer les interactions avec les cellules. Les études réalisées ont permis de mettre en évidence l’internalisation des particules dans les synoviocytes, chondrocytes, et cellules souches mésenchymateuses après 8 heures d’exposition. Parallèlement, l’évaluation in vitro de différents paramètres de cytotoxicité et d’inflammation a permis de souligner la compatibilité de ces vecteurs avec les cellules articulaires. Enfin, les effets de l’injection intra-articulaire de particules ont été évalués chez le rat sain et différents modèles pathologiques. L’analyse histologique des articulations a démontré l’absence de dégradation de la matrice cartilagineuse et de réaction inflammatoire suite à l’injection de particules. Il a été également constaté un ciblage majoritaire des particules vers la membrane synoviale. Ces travaux permettent de valider l’utilisation de ce type de vecteur pour le ciblage de l’articulation et l’apport de principes actifs. L’association de ces particules à des molécules hydrophiles (acides nucléiques, médicaments,…), pourra permettre leur apport direct dans l’articulation et ouvrir de nouvelles perspectives thérapeutiques.