Ollitrault D.,University of Caen Lower Normandy |
Legendre F.,University of Caen Lower Normandy |
Drougard C.,University of Caen Lower Normandy |
Briand M.,BIOTICLA |
And 12 more authors.
Tissue Engineering - Part C: Methods | Year: 2015
Osteoarthritis (OA) is an irreversible pathology that causes a decrease in articular cartilage thickness, leading finally to the complete degradation of the affected joint. The low spontaneous repair capacity of cartilage prevents any restoration of the joint surface, making OA a major public health issue. Here, we developed an innovative combination of treatment conditions to improve the human chondrocyte phenotype before autologous chondrocyte implantation. First, we seeded human dedifferentiated chondrocytes into a collagen sponge as a scaffold, cultured them in hypoxia in the presence of a bone morphogenetic protein (BMP), BMP-2, and transfected them with small interfering RNAs targeting two markers overexpressed in OA dedifferentiated chondrocytes, that is, type I collagen and/or HtrA1 serine protease. This strategy significantly decreased mRNA and protein expression of type I collagen and HtrA1, and led to an improvement in the chondrocyte phenotype index of differentiation. The effectiveness of our in vitro culture process was also demonstrated in the nude mouse model in vivo after subcutaneous implantation. We, thus, provide here a new protocol able to favor human hyaline chondrocyte phenotype in primarily dedifferentiated cells, both in vitro and in vivo. Our study also offers an innovative strategy for chondrocyte redifferentiation and opens new opportunities for developing therapeutic targets. Copyright 2015, Mary Ann Liebert, Inc.
Faraj K.A.,Radboud University Nijmegen |
Faraj K.A.,EMCM BV |
Faraj K.A.,Salahaddin University Erbil |
Brouwer K.M.,Radboud University Nijmegen |
And 10 more authors.
Tissue Engineering and Regenerative Medicine | Year: 2011
Ethylene oxide (EtO) gassing and β- and γ-irradiation are currently used for sterilising collagen scaffolds. During the process, scaffolds may undergo chemical and physical alterations that may compromise their structural integrity and functional characteristics. In this study, we compared the effects of EtO gassing, and β- and γ-irradiation at 15 and 25 kGy on type I collagen fibril-based scaffolds with and without crosslinking, and with and without heparin. Evaluation was performed using a wide range of biophysical, biochemical, morphological and biological parameters. EtO treatment, β-irradiation and γ-irradiation did not induce morphological changes, nor did they have an effect on the amount of primary amine groups, or the amount of heparin covalently attached to the scaffolds. Cytocompatibility was also not affected. Irradiation, however, did result in collagen degradation products, a decrease in collagen denaturation temperature, and an increase in proteolytic degradation, all in a dose dependent fashion. These parameters were hardly influenced by EtO treatment. Sterilisation methods had hardly any effect on tensile strength of crosslinked scaffolds, but -surprisingly- they increased the tensile strength of non-crosslinked scaffolds. In conclusion, a number of the collagen scaffold parameters were influenced by sterilisation, whereas others were not. Irradiation had a much larger effect than EtO. However, tensile strength and cytocompatibility, important in tissue engineering, were not negatively influenced by any of the methods. Therefore, aspects like costs, safety and practicality of use may be taken into account in the choice of sterilisation method.
Deciphering signaling mechanisms controling chondrocyte differentiation: Application to tissue engineering of cartilage: The ANR-TecSan PROMOCART project [Décryptage des signalisations moléculaires contrôlant la différenciation des chondrocytes: Retombées pour l'ingénierie tissulaire du cartilage: Le projet ANR-TecSan PROMOCART]
Claus S.,French National Center for Scientific Research |
Aubert-Foucher E.,French National Center for Scientific Research |
Perrier-Groult E.,French National Center for Scientific Research |
Bougault C.,French National Center for Scientific Research |
And 13 more authors.
IRBM | Year: 2011
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, Symatèse Biomatériaux 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. © 2011 Elsevier Masson SAS. All rights reserved.
Legendre F.,Laboratoire Microenvironnement Cellulaire et Pathologies MILPAT EA 4652 |
Ollitrault D.,Laboratoire Microenvironnement Cellulaire et Pathologies MILPAT EA 4652 |
Hervieu M.,Laboratoire Microenvironnement Cellulaire et Pathologies MILPAT EA 4652 |
Bauge C.,Laboratoire Microenvironnement Cellulaire et Pathologies MILPAT EA 4652 |
And 7 more authors.
Tissue Engineering - Part C: Methods | Year: 2013
Cartilage healing by tissue engineering is an alternative strategy to reconstitute functional tissue after trauma or age-related degeneration. However, chondrocytes, the major player in cartilage homeostasis, do not self-regenerate efficiently and lose their phenotype during osteoarthritis. This process is called dedifferentiation and also occurs during the first expansion step of autologous chondrocyte implantation (ACI). To ensure successful ACI therapy, chondrocytes must be differentiated and capable of synthesizing hyaline cartilage matrix molecules. We therefore developed a safe procedure for redifferentiating human chondrocytes by combining appropriate physicochemical factors: hypoxic conditions, collagen scaffolds, chondrogenic factors (bone morphogenetic protein-2 [BMP-2], and insulin-like growth factor I [IGF-I]) and RNA interference targeting the COL1A1 gene. Redifferentiation of dedifferentiated chondrocytes was evaluated using gene/protein analyses to identify the chondrocyte phenotypic profile. In our conditions, under BMP-2 treatment, redifferentiated and metabolically active chondrocytes synthesized a hyaline-like cartilage matrix characterized by type IIB collagen and aggrecan molecules without any sign of hypertrophy or osteogenesis. In contrast, IGF-I increased both specific and noncharacteristic markers (collagens I and X) of chondrocytes. The specific increase in COL2A1 gene expression observed in the BMP-2 treatment was shown to involve the specific enhancer region of COL2A1 that binds the trans-activators Sox9/L-Sox5/Sox6 and Sp1, which are associated with a decrease in the trans-inhibitors of COL2A1, c-Krox, and p65 subunit of NF-kappaB. Our procedure in which BMP-2 treatment under hypoxia is associated with a COL1A1 siRNA, significantly increased the differentiation index of chondrocytes, and should offer the opportunity to develop new ACI-based therapies in humans. © Copyright 2013, Mary Ann Liebert, Inc.
Claus S.,University of Lyon |
Mayer N.,University of Lyon |
Aubert-Foucher E.,University of Lyon |
Chajra H.,Symatese Biomateriaux |
And 6 more authors.
Tissue Engineering - Part C: Methods | Year: 2012
Objective: Articular cartilage has a poor capacity for spontaneous repair. Tissue engineering approaches using biomaterials and chondrocytes offer hope for treatments. Our goal was to test whether collagen sponges could be used as scaffolds for reconstruction of cartilage with human articular chondrocytes. We investigated the effects on the nature and abundance of cartilage matrix produced of sequential addition of chosen soluble factors during cell amplification on plastic and cultivation in collagen scaffolds. Design: Isolated human articular chondrocytes were amplified for two passages with or without a cocktail of fibroblast growth factor (FGF)-2 and insulin (FI). The cells were then cultured in collagen sponges with or without a cocktail of bone morphogenetic protein (BMP)-2, insulin, and triiodothyronine (BIT). The constructs were cultivated for 36 days in vitro or for another 6-week period in a nude mouse-based contained-defect organ culture model. Gene expression was analyzed using polymerase chain reaction, and protein production was analyzed using Western-blotting and immunohistochemistry. Results: Dedifferentiation of chondrocytes occured during cell expansion on plastic, and FI stimulated this dedifferentiation. We found that addition of BIT could trigger chondrocyte redifferentiation and cartilage-characteristic matrix production in the collagen sponges. The presence of FI during cell expansion increased the chondrocyte responsiveness to BIT. © 2012 Mary Ann Liebert, Inc.