15 research outputs found
Identification des biomarqueurs précoces de la lésion tendineuse de fatigue dans le modèle équin
La fonctionnalité de l appareil locomoteur repose en grande partie sur l intégrité du tendon, essentiellement composé de fibres de collagènes de types I et III, dont la désorganisation participe à la tendinite de fatigue. Les microtraumatismes subis par les tendons des chevaux athlètes au cours d entraînements intensifs provoquent, à long terme, une altération des fibres collagéniques. Cette pathologie conduit le plus souvent à l arrêt prématuré de la carrière sportive du cheval. Cette étude vise à identifier les biomarqueurs précoces de la lésion tendineuse de fatigue chez le cheval. Des expérimentations in vivo tendant à mimer cette affection ont permis de mieux connaître la physiopathologie du tendon et de caractériser des marqueurs moléculaires des stades précoces de la tendinite. Deux modèles d induction de lésions, chirurgical et mixte , ont été développés afin se rapprocher du processus physiopathologique spontané. Après induction mécanique chirurgicale de lésions, des biopsies des tendons ont été réalisées et soumises à des mesures de l expression des gènes et des protéines extra- et intracellulaires, montrant une modification de toutes les molécules étudiées intervenant dans le remodelage de la MEC. Une surexpression des collagènes de types I et III et de la ténascine-C est démontrée dans le tendon lésé, ainsi qu une désorganisation progressive de la structure du tendon. L étude sur les facteurs de transcription a révélé que scléraxis semble être un marqueur plus tardif. Ces recherches nous ont permis d approfondir les connaissances sur la tendinite de fatigue in vivo chez le cheval, tant sur les aspects physiologiques que moléculaires et biochimiques. Elles s'inscrivent dans une démarche de développement d'une sonde moléculaire optique, spécifique d'un marqueur précoce de la tendinite, afin de détecter les lésions de la façon la plus précoce qui soit, avant l'acquisition de lésions irréparables, afin de développer des thérapies qui seront adaptées à l'avenir en clinique vétérinaire, et humaine à postériori. Ainsi, nos recherches ont permis de mettre en évidence de nouveaux marqueurs dans les conditions de tendinite de fatigue.The functionality of the musculoskeletal system is largely based on the integrity of the tendon, mainly composed of types I and III collagens fibrils, whose disorganization takes a great part in the fatigue tendinitis. Microtraumas exerted on the tendons of race horses during intensive training lead to long-term impairments resulting in fatigue tendinitis. This condition often leads to premature termination of the sport career of the horse. The goal of this study was to characterize early biomarkers of the fatigue tendon injury in horse. Experiments were designed to mimic this pathology by inducing fatigue tendinitis in vivo in horse through a surgery technique and the data led to a better knowledge of the pathophysiology of the tendon and to characterize molecular markers of tendinitis at early stages. Two models of induction of lesions, surgical and "mixed", have been developed to closer to the spontaneous pathophysiological process. After surgical induction of mechanical lesions, tendon biopsies were collected and were subjected to evaluation of gene and protein expressions of different extra-and intracellular proteins, showing a modification of the molecules studied involved in ECM remodeling. Overexpression of types I and III collagens and tenascin -C is observed in injured tendon and a progressive disorganization of the tendon structure. The study of transcription factors revealed that scleraxis seems to be a late tendinitis marker. Therefore, this research allowed us to extend our knowledge on the fatigue tendinitis in both physiological and pathological situations, through biochemical, molecular and cellular approaches. This study was carried out in order to develop an optical molecular marker probe relevant of the earliest stages of tendinitis, leading to the detection as early as possible micro-lesions before they become unrepairable, with the objective especially to promote appropriate therapies in veterinary or human clinic a posteriori. Thus, our research has highlighted new markers of the horse fatigue tendinitis.CAEN-BU Médecine pharmacie (141182102) / SudocSudocFranceF
Hypoxia Is a Critical Parameter for Chondrogenic Differentiation of Human Umbilical Cord Blood Mesenchymal Stem Cells in Type I/III Collagen Sponges
International audienc
RNA interference and BMP-2 stimulation allows equine chondrocytes redifferentiation in 3D-Hypoxia cell culture model: application for matrix-induced autologous chondrocyte implantation
As in humans, osteoarthritis (OA) causes considerable economic loss to the equine industry. New hopes for cartilage repair have emerged with the matrix-associated autologous chondrocyte implantation (MACI). Nevertheless, its limitation is due to the dedifferentiation occurring during the chondrocyte amplification phase, leading to the loss of its capacity to produce a hyaline extracellular matrix (ECM). To enhance the MACI therapy efficiency, we have developed a strategy for chondrocyte redifferentiation, and demonstrated its feasibility in the equine model. Thus, to mimic the cartilage microenvironment, the equine dedifferentiated chondrocytes were cultured in type I/III collagen sponges for 7 days under hypoxia in the presence of BMP-2. In addition, chondrocytes were transfected by siRNA targeting Col1a1 and Htra1 mRNAs, which are overexpressed during dedifferentiation and OA. To investigate the quality of the neo-synthesized ECM, specific and atypical cartilage markers were evaluated by RT-qPCR and Western blot. Our results show that the combination of 3D hypoxia cell culture, BMP-2 (Bone morphogenetic protein-2), and RNA interference, increases the chondrocytes functional indexes (Col2a1/Col1a1, Acan/Col1a1), leading to an effective chondrocyte redifferentiation. These data represent a proof of concept for this process of application, in vitro, in the equine model, and will lead to the improvement of the MACI efficiency for cartilage tissue engineering therapy in preclinical/clinical trials, both in equine and human medicine
Enhanced chondrogenesis of bone marrow-derived stem cells by using a combinatory cell therapy strategy with BMP-2/TGF-β1, hypoxia, and COL1A1/HtrA1 siRNAs
International audienceMesenchymal stem cells (MSCs) hold promise for cartilage engineering. Here, we aimed to determine the best culture conditions to induce chondrogenesis of MSCs isolated from bone marrow (BM) of aged osteoarthritis (OA) patients. We showed that these BM-MSCs proliferate slowly, are not uniformly positive for stem cell markers, and maintain their multilineage potential throughout multiple passages. The chondrogenic lineage of BM-MSCs was induced in collagen scaffolds, under normoxia or hypoxia, by BMP-2 and/or TGF-β1. The best chondrogenic induction, with the least hypertrophic induction, was obtained with the combination of BMP-2 and TGF-β1 under hypoxia. Differentiated BM-MSCs were then transfected with siRNAs targeting two markers overexpressed in OA chondrocytes, type I collagen and/or HtrA1 protease. siRNAs significantly decreased mRNA and protein levels of type I collagen and HtrA1, resulting in a more typical chondrocyte phenotype, but with frequent calcification of the subcutaneously implanted constructs in a nude mouse model. Our 3D culture model with BMP-2/TGF-β1 and COL1A1/HtrA1 siRNAs was not effective in producing a cartilage-like matrix in vivo. Further optimization is needed to stabilize the chondrocyte phenotype of differentiated BM-MSCs. Nevertheless, this study offers the opportunity to develop a combinatory cellular therapy strategy for cartilage tissue engineering
Chondrogenic commitment of human umbilical cord blood-derived mesenchymal stem cells in collagen matrices for cartilage engineering
International audienceUmbilical cord blood (UCB) is a promising alternative source of mesenchymal stem cells (MSCs), because UCB-MSCs are abundant and harvesting them is a painless non-invasive procedure. Potential clinical applications of UCB-MSCs have been identified, but their ability for chondrogenic differentiation has not yet been fully evaluated. The aim of our work was to characterize and determine the chondrogenic differentiation potential of human UCB-MSCs (hUCB-MSCs) for cartilage tissue engineering using an approach combining 3D culture in type I/III collagen sponges and chondrogenic factors. Our results showed that UCB-MSCs have a high proliferative capacity. These cells differentiated easily into an osteoblast lineage but not into an adipocyte lineage. Furthermore, BMP-2 and TGF-β1 potentiated chondrogenic differentiation, as revealed by a strong increase in mature chondrocyte-specific mRNA (COL2A1, COL2B, ACAN) and protein (type II collagen) markers. Although growth factors increased the transcription of hypertrophic chondrocyte markers such as COL10A1 and MMP13, the cells present in the neo-tissue maintained their phenotype and did not progress to terminal differentiation and mineralization of the extracellular matrix after subcutaneous implantation in nude mice. Our study demonstrates that our culture model has efficient chondrogenic differentiation, and that hUCB-MSCs can be a reliable source for cartilage tissue engineering
Enhanced Hyaline Cartilage Matrix Synthesis in Collagen Sponge Scaffolds by Using siRNA to Stabilize Chondrocytes Phenotype Cultured with Bone Morphogenetic Protein-2 Under Hypoxia
International audienceCartilage 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
RNA Interference and BMP-2 Stimulation Allows Equine Chondrocytes Redifferentiation in 3D-Hypoxia Cell Culture Model: Application for Matrix-Induced Autologous Chondrocyte Implantation
Characterization and use of equine bone marrow mesenchymal stem cells in horse cartilage engineering
National audiencePURPOSE: Articular cartilage is of great importance for physiological mobility. The structure and the function of this tissue which is characterized by a poor self-repair capacity are frequently disrupted or damaged upon physical trauma or in degenerative osteoarthritis (OA). Given that musculoskeletal disorders are the leading cause of poor performance or early retirement of sports and race horses treating horses for these disorders is relevant. The proposed therapeutic approach which was first developed for human has the potential of being considered as a pre-clinical step for human medicine because the horse is recognized as an excellent model for the study of articular cartilage disorders in humans. This study aims to improve the Autologous Chondrocytes Implantation (ACI) technique by using Mesenchymal Stem cells (MSCs) from bone marrow (easy to collect in horse) as the cell source. Thus MSCs were first characterized before being cultured with chondrogenic conditions in order to find the combination that best enhances and stabilizes the characteristics of the chondrocyte phenotype in order to perform clinical trials in horse. METHODS: MSCs from equine bone marrow were isolated and expanded in monolayer cultures until 4 passages. These cells were characterized by analyzing their proliferative potential their pluripotency (with microenvironmental stimuli) and their capacity to express MSCs specific phenotypic markers defining the MSCs (flow cytometry). In parallel MSCs differentiation in chondrocytes was accomplished via a combinatory approach based on the association of 3D-culture in type I collagen sponges low oxygen tension with chondrogenic factors (BMP-2 TGF-β1) and RNA interference (siRNA to down-regulate type I collagen and HtrA1 protein expression). Finally an extensive analysis at gene and protein levels corresponding to differentiated dedifferentiated and hypertrophic chondrocyte phenotypes was performed. RESULTS: Our results show a very high proliferation potential of MSCs isolated from equine bone marrow. Furthermore the isolated cells satisfy the various criteria of stem cells definition (pluripotency expression of surface markers). In addition siRNAs targeting equine Col1a1 and Htra1 have been functionally validated. Finally we show that the BMP-2 and TGF-β1 combination strongly induces the differentiation of MSCs in chondrocytes. Thus the combined use of specific culture conditions defined within the laboratory with specific growth factors and siRNAs association leads to the in vitro synthesis of a hyaline type neo-cartilage by chondrocytes which present an optimal phenotypic index comparable to the one established with induction of specific differentiation of human MSCs in chondrocytes and close to the one of differentiated/healthy chondrocytes. CONCLUSIONS: These data represent a first step in the development of equine clinical trials which are planed - to better understand the reaction of cartilage tissue after a few weeks of intra-articular implantation and - to make the proof of concept in a large animal model. This approach will allow us to explore the criteria necessary to begin to consider the development of cell therapy for cartilage repair in humans
Characterization and use of Equine Bone Marrow Mesenchymal Stem Cells in Equine Cartilage Engineering. Study of their Hyaline Cartilage Forming Potential when Cultured under Hypoxia within a Biomaterial in the Presence of BMP-2 and TGF-Ăź1
International audienceArticular cartilage presents a poor capacity for self-repair. Its structure-function are frequently disrupted or damaged upon physical trauma or osteoarthritis in humans. Similar musculoskeletal disorders also affect horses and are the leading cause of poor performance or early retirement of sport- and racehorses. To develop a therapeutic solution for horses, we tested the autologous chondrocyte implantation technique developed on human bone marrow (BM) mesenchymal stem cells (MSCs) on horse BM-MSCs. This technique involves BM-MSC chondrogenesis using a combinatory approach based on the association of 3D-culture in collagen sponges, under hypoxia in the presence of chondrogenic factors (BMP-2 + TGF-β1) and siRNA to knockdown collagen I and HtrA1. Horse BM-MSCs were characterized before being cultured in chondrogenic conditions to find the best combination to enhance, stabilize, the chondrocyte phenotype. Our results show a very high proliferation of MSCs and these cells satisfy the criteria defining stem cells (pluripotency-surface markers expression). The combination of BMP-2 + TGF-β1 strongly induces the chondrogenic differentiation of MSCs and prevents HtrA1 expression. siRNAs targeting Col1a1 and Htra1 were functionally validated. Ultimately, the combined use of specific culture conditions defined here with specific growth factors and a Col1a1 siRNAs (50 nM) association leads to the in vitro synthesis of a hyaline-type neocartilage whose chondrocytes present an optimal phenotypic index similar to that of healthy, differentiated chondrocytes. Our results lead the way to setting up pre-clinical trials in horses to better understand the reaction of neocartilage substitute and to carry out a proof-of-concept of this therapeutic strategy on a large animal model
Research data supporting for "Shadow Technique Algorithm (STA) Sheds a New Light on Differential Interference Contrast (DIC) Microscopy"
<p>This folder contains data to support the paper entitled:</p>
<p>Shadow Technique Algorithm (STA) Sheds a New Light on Differential Interference Contrast (DIC) Microscopy<br>
Trinel D ,Vandame P, Hervieu M, Floquet E, Aumercier M, Biondi EG , Bodart JF and Spriet C*<br>
Analytical & Bioanalytical Techniques (2015)</p>
<p>Supplementary data and the STA macro for imageJ are at the folder root.<br>
Data used for the paper are available as test sample in the data folder.<br>
In each case, the data before and after STA are present.<br>
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