Etude des phénomènes de mécanoactivation et de mécanotransduction des chondrocytes

NANCY1-SCD Medecine (545472101) / SudocSudocFranceF

Caractérisation des cellules souches mésenchymateuses du sang placentaire et de la gelée de Wharton

Les cellules souches suscitent de grands espoirs pour la thérapie cellulaire et l'ingénierie tissulaire. Les CSM du tissus foetaux (sang placentaire et gelée de Wharton du cordon ombilical), à l'origine d'épiblaste embryonnaire, sont considérées comme plus primitives que les CSM provenant de sources adultes. Les conditions de culture ayant un impact sur le comportement des cellules, dans notre étude, nous avons exploré l'effet de la concentration de l'oxygène sur l'expansion, l'immunophénotypage et la différenciation de ces cellules. L'objectif de ce travail est d'identifier la méthode optimale d'isolation des CSM issues de tissus foetaux. Compte tenu du faible taux de succès dans l'isolement des CSM extraites du sang placentaire, nous nous sommes dirigés vers les CSM-GW. Nous y avons déterminé in situ, les marqueurs spécifiques exprimés dans la gelée de Wharton et à la périphérie. Des études sur la morphologie, la cinétique de croissance, et sur l'expression phénotypique des marqueurs de surface, des CSM-GW, ont été effectuées sur une longue durée (7 passages) à différentes conditions de culture. Nous avons montré que la GW est composée d'une abondante matrice extracellulaire riche en collagènes et glycosaminoglycannes et que les cellules possèdent un phénotype variable selon leur localisation dans la gelée. Ce tissu est capable de fournir une quantité importante de CSM (6,7x105 Cs/cm de cordon) qui gardent une morphologie constante. Enfin, quel que soit le passage, la concentration de l'oxygène ne semble pas avoir d'effet sur le phénotype des cellules. En revanche, une faible teneur en oxygène durant l'expansion semble diminuer le temps de doublement des cellules, favoriser la chondrogénèse et inhiber la différenciation ostéogénique. Enfin, quelles que soient les conditions de culture, la différenciation adipogénique des CSM-GW semble difficile à obtenirStem cells are the hopes for cell therapy and tissue engineering. MSCs from fetal tissue (umbilical cord blood and WJ), which are a source of embryonic epiblast grow relatively faster comparing to other adult sources. The culture condition can affect cell behavior. In our study, we explored the effect of oxygen concentration on the expansion, immunophenotyping, and differentiation of these cells. The aim of this work is to identify the optimal method for isolation of MSCs derived from fetal tissue. Given the low rate of success in the isolation of MSCs from cord blood, we headed to WJ-MSCs. We have determined in siu, the specific markers expressed in the WJ and in the perivascular region. Studies on the morphology growth kinetics, and phenotypic expression of surface makers of MSCs isolated from WJ were made over a long period (7 passages) in different culture conditions. We have shown that WJ is composed of an abundant extracellular matrix rich in collagen and glycasominoglycans and have variable phenotype depending from their localization in the jelly. This tissue is able to provide a large amount of MSCs (6.7x105 Cs/cm of cord) that maintain a constant morphology. Finally, regardless of the passage, the oxygen concentration does not effect on the phenotype of the cells. In contrast, a low oxygen concentration during expansion appears to decrease the doubling time of MSCs, promote chondrogenesis and inhibit osteogenic differentiation. Finally, whatever the culture conditions, adipogenic differentiation of WJ-MSC seems difficult to obtainMETZ-SCD (574632105) / SudocNANCY1-Bib. numérique (543959902) / SudocNANCY2-Bibliotheque electronique (543959901) / SudocNANCY-INPL-Bib. électronique (545479901) / SudocSudocFranceF

Intérêt des cellules souches mésenchymateuses en ingénierie tissulaire du cartilage (effets des stimuli mécaniques et biochimiques)

L'ingénierie tissulaire du cartilage permet de reconstruire un néo-cartilage réimplantable en utilisant des cellules telles que les chondrocytes et les cellules souches mésenchymateuses (CSMs) cultivés dans des biomatériaux tridimensionnels. Des facteurs mécaniques et biochimiques y jouent un rôle essentiel. Le but de nos travaux a été d'évaluer, dans un premier temps, l'influence de l'expansion des chondrocytes et des CSMs humains en monocouche sur l'évolution du phénotype cellulaire. Dans un deuxième temps, une étude concernant les réponses biologiques des chondrocytes et des CSMs exposés aux stimulis mécaniques (entrechoquement et compression dynamique intermittente) et biochimiques (TGF-pl et BMP-2) a été réalisée dans un hydrogel d'alginate/HA. Nos résultats ont montré une dédifférenciation notable des chondrocytes à partir du deuxième passage, alors que les CSMs ont maintenu leur phénotype indifférencié après 5 passages. Par ailleurs, nous avons mis en évidence que l'application de contraintes mécaniques ainsi que la stimulation de facteurs de croissance peuvent améliorer l'activité métabolique, la prolifération cellulaire, l'expression des gènes chondrogéniques ainsi que la synthèse de la matrice cartilagineuse. Cependant, ces effets sont étroitement liés aux paramètres mécaniques (durée, fréquence) et au type cellulaire. L'ensemble de ces travaux suggère que le contrôle des conditions de culture ainsi que l'application des contraintes mécaniques et biochimiques peuvent moduler le stade de différenciation chondrocytaire et donc la chondrogénèse.Cartilage tissue engineering could lead to reconstruct a re-implantable neo-cartilage by using cells such as chondrocytes and mesenchymal stem cells (MSCs) cultured in a scaffold. Different mechanical and biochemical factors play an essential role in this process as well. The aim of this study was to investigate firstly, the influence of monolayer expansion of human chondrocytes and MSCs on the cell phenotype. Secondly, the cellular responses of chondrocytes and MSCs to mechanical stimulations (agitation and intermittent dynamic compression) as well as to biochemical stimulations (TGF-pl and BMP-2) have been examined in an alginatelHA hydrogel. Our results showed that notable dedifferentiation of chondrocytes has occurred from passage 2, while MSCs have still kept their undifferentiated phenotype until passage 5. ln addition, the application of these mechanical strains and growth factors has been demonstrated to improve metabolic activity, cell proliferation, chondrogenic gene expression as well as synthesis of cartilaginous matrix. These effects are closely related to the mechanical parameters (duration, frequency) and the cell type.AlI of these results suggested that the control of culture conditions and the application of mechanical strains as well as growth factors could modulate the differentiation state and the chondrogenesis.NANCY1-SCD Medecine (545472101) / SudocSudocFranceF

Constitutive equations for Ca2+ -alginate gels

International audienc

Conception d'un hydrogel stratifié (application pour l'ingénierie du cartilage)

Le cartilage articulaire est composé de chondrocytes et d'une matrice extracellulaire organisés de manière stratifiée dans l'épaisseur du tissu. Ce tissu ne se régénère pas de manière efficace après une lésion. L'objectif de ce travail est de construire par pulvérisation des hydrogels à base d'alginate et de film multicouches de polyélectrolytes pour créer in vitro un néotissu pouvant combler des lésions de cartilage articulaire. La méthode a été validée en observant une bonne viabilité et une synthèse matricielle par les cellules, et de meilleures propriétés mécaniques des hydrogels pulvérisés à 0,9 bar par rapport au moulage. Après la pulvérisation de cellules souches mésenchymateuses, les résultats ont montré une bonne viabilité et une différenciation des cellules. Puis, des hydrogels bistratifiés ont été construits et cultivés jusqu'à 56 jours sans dissociation des couches et sans migration des cellules. Enfin, les hydrogels ont été fonctionnalisés en modifiant la composition des couches et en y appliquant des stimulations mécaniques. Les propriétés mécaniques des hydrogels varient en fonction de leur composition et sont meilleures pour ceux stratifiés. De plus, leur stimulation mécanique a permis de potentialiser l'effet du biomatériau sur la différenciation des cellules. En conclusion, cette étude montre que des cellules souches mésenchymateuses ensemencées dans un hydrogel bistratifié pulvérisé sont fonctionnelles en termes de différenciation chondrocytaire et de synthèse matricielle. Les propriétés mécaniques des hydrogels stratifiés ne sont pas altérées. De plus, la stimulation mécanique a potentialisé la différenciation des cellulesThe articular cartilage is composed of chondrocytes and of a specific extracellular matrix which are organized depth-dependently. The tissue did not have an efficient self-renewal of defects. The purpose of this study is to build up layer-by-layer a stratified hydrogel by alternating gels and multilayers polyelectrolytes film spraying, in order to obtain a neotissu in vitro to fill lesions. First, the process was validated by observing a good cells viability and matrix synthesis, and stronger mechanical behaviors of sprayed hydrogels compared to molded one. Secondly, after their spraying, mesenchymal stem cells still have a good viability and their differentiation potential. Then, bistratified scaffolds were built up and cultured up to 56 days without layers dissociation and without cells migration between layers. Finally, scaffolds were functionalized by changing biomaterial composition and by applying mechanicals stimulations. Results show us not only that the composition influences the mechanical behavior of the hydrogel, but that the stratification did not affect it. Furthermore, mechanicals stimulations improve stem cells differentiation in function of biomaterials compositions. In conclusion, this study proves not only that we are able to build up stratified scaffold seeded with mesenchymal stem cells which still have their differentiation capability and synthesize matrix, but that mechanical behaviors are improved after the biomaterial spraying and not alter by the stratification. Moreover, mechanical stimulation applied to the scaffold improves the differentiation of mesenchymal stem cells to a chondrogenic phenotypeNANCY1-Bib. numérique (543959902) / SudocSudocFranceF

Does making method of alginate hydrogel influence the chondrogenic differentiation of human mesenchymal stem cells?

To overcome cartilage injury, strategies have been developed in the last few years based on tissue engineering to rebuild the defects. Cartilage engineering is principally based on three main biological factors: cells (native cells (chondrocytes) or a more primitive ones as mesenchymal stem cells), scaffolds and functionalization factors (growth factors, mechanical stimulation and/or hypoxia). Cartilage tissue engineering strategies generally result in homogeneous tissue structures with little resemblance to native zonal organization of articular cartilage. The main objective of our work concerns the buildup of complex biomaterials aimed at reconstructing biological tissue with three dimensional cells construction for mimicking cartilage architecture. Our strategy is based on structures formation by simple and progressive spraying of mixed alginate hydrogel and human mesenchymal stem cells (hMSC). In this work, the comportment of cells and more precisely their chondrogenic differentiation potential is compared to a traditional making process: the mold. We report here that spraying method allowed to product a scaffold with hMSC that confer a favorable environment for neocartilage construction.This work was supported by the "Lorraine region" grant. N. Jessel is indebted to CHU de Nancy, Hôpital Central, orthopedic surgery (Contrat d’interface INSERM-CHU).Not peer reviewe

Mesenchymal stem cells derived from Wharton's jelly: comparative phenotype analysis between tissue and in vitro expansion.

International audienceMesenchymal stem cells (MSCs) are useful multipotent stem cells that are found in many tissues. While MSCs can usually be isolated from adults via bone marrow aspiration (BM-MSCs), MSCs derived from the discarded umbilical cord, more precisely from Wharton's jelly (WJ), offer a low-cost and pain-free collection method of MSCs that may be cryogenically stored, and are considered extremely favorable for tissue engineering purpose. The aim of this study was to analyze the harvested number of cells per centimeter of human umbilical cord (UC) and carry out the phenotype of these WJ-MSCs after explant or enzymatic methods. Fresh UCs were obtained from full-term births, and processed within 6 hours from partum to obtain the WJ-MSCs. UC sections were analyzed in confocal microscopy to analyze cells phenotype in situ. Others UC components were treated either by enzymatic method or by explant method to obtain isolated cells and to analyze cells phenotype until the end of the first passage. We have successfully generated MSCs from UC by using explant and enzymatic methods. Using microscopy confocal, we identified the expression of some MSCs markers in situ of Wharton's jelly tissue as well as in perivascular region. Our comparative study, between explant and enzymatic digestion, indicated, that WJ expressed most of MSCs markers in both conditions, but a remarkable variation of cell phenotype expression was distinguished after primary culture comparing to directly isolated cells by enzymatic digestion. We also studied the expression of CD271, which showed to be weakly expressed in situ on fresh fragment of WJ

Chondrogenic induction of mesenchymal stromal/stem cells from Wharton's jelly embedded in alginate hydrogel and without added growth factor: an alternative stem cell source for cartilage tissue engineering

Background: Due to their intrinsic properties, stem cells are promising tools for new developments in tissue engineering and particularly for cartilage tissue regeneration. Although mesenchymal stromal/stem cells from bone marrow (BM-MSC) have long been the most used stem cell source in cartilage tissue engineering, they have certain limits. Thanks to their properties such as low immunogenicity and particularly chondrogenic differentiation potential, mesenchymal stromal/stem cells from Wharton's jelly (WJ-MSC) promise to be an interesting source of MSC for cartilage tissue engineering. Methods: In this study, we propose to evaluate chondrogenic potential of WJ-MSC embedded in alginate/hyaluronic acid hydrogel over 28 days. Hydrogels were constructed by the original spraying method. Our main objective was to evaluate chondrogenic differentiation of WJ-MSC on three-dimensional scaffolds, without adding growth factors, at transcript and protein levels. We compared the results to those obtained from standard BM-MSC. Results: After 3 days of culture, WJ-MSC seemed to be adapted to their new three-dimensional environment without any detectable damage. From day 14 and up to 28 days, the proportion of WJ-MSC CD73(+), CD90(+), CD105(+) and CD166(+) decreased significantly compared to monolayer marker expression. Moreover, WJ MSC and BM MSC showed different phenotype profiles. After 28 days of scaffold culture, our results showed strong upregulation of cartilage-specific transcript expression. WJ-MSC exhibited greater type II collagen synthesis than BM-MSC at both transcript and protein levels. Furthermore, our work highlighted a relevant result showing that WJ-MSC expressed Runx2 and type X collagen at lower levels than BM-MSC. Conclusions: Once seeded in the hydrogel scaffold, WJ-MSC and BM-MSC have different profiles of chondrogenic differentiation at both the phenotypic level and matrix synthesis. After 4 weeks, WJ-MSC, embedded in a three-dimensional environment, were able to adapt to their environment and express specific cartilage-related genes and matrix proteins. Today, WJ-MSC represent a real alternative source of stem cells for cartilage tissue engineering.peer-reviewe

Cellules souches et nouvelle loi de bioéthique française

International audienceLe sommaire de ce numéro http://www.john-libbey-eurotext.fr/fr/ revues/medecine/hma/sommaire.md?type= text.html Montrouge, le 14/08/2012 Loïc Reppel Vous trouverez ci-après le tirétiré`tiréà part de votre article au formatélectroniqueformat´formatélectronique (pdf) : Cellules souches et nouvelle loi de bioéthique française paru dans Hématologie, 2012, Volume 18, Numéro 3 John Libbey Eurotext Ce tirétiré`tiréà part numérique vous est délivré pour votre propre usage et ne peutêtrepeutêtre transmisàtransmis`transmisà des tiers qu'` a des fins de recherches personnelles ou scientifiques. En aucun cas, il ne doit faire l'objet d'une distribution ou d'une utilisation promotionnelle, commerciale ou publicitaire. Tous droits de reproduction, d'adaptation, de traduction et de diffusion réservés pour tous pays. Résumé. En juillet 2011, la loi de bioéthique française a fait l'objet d'une révision apportant, dans le domaine des cellules souches, quelques évolutions par rapport à la loi précédente. Concernant les cellules souches embryonnaires (CSE), la nouvelle loi de bioéthique confirme la possibilité d'effectuer des recherches sur l'embryon et sur les CSE uniquement sur dérogations strictement encadrées et délivrées par l'Agence de la biomédecine. En revanche, l'avancée majeure de cette loi repose, à présent, sur un encadrement spécifique des cellules souches hématopoïétiques du sang placentaire en vue d'une utilisation thérapeutique ou scientifique. Ce dernier n'est plus considéré comme un déchet opératoire mais bien comme une source à part entière de cellules souches hématopoïétiques au même titre que la moelle osseuse et le sang périphérique. Abstract. In July 2011, the French bioethics law was the subject of a revision bringing few remarkable changes compared to the previous law, especially in the field of stem cells. Regarding Embryonic stem cells (ESC), the new law confirms the possibility to carry out research on the embryo and ESC exclusively after obtention of a derogation strictly regulated and delivered by the French Biomedicine Agency. On the other hand, the main advance of this law concerns a specific regulating of cord blood hematopoietic stem cells for therapeutic or scientific uses. This collection site is no longer considered as a medical waste but as a source of hematopoietic stem cells as well as bone marrow and peripheral blood

Mechanical stimulations on human bone marrow mesenchymal stem cells enhance cells differentiation in a three-dimensional layered scaffold

Scaffolds laden with stem cells are a promising approach for articular cartilage repair. Investigations have shown that implantation of artificial matrices, growth factors or chondrocytes can stimulate cartilage formation, but no existing strategies apply mechanical stimulation on stratified scaffolds to mimic the cartilage environment. The purpose of this study was to adapt a spraying method for stratified cartilage engineering and to stimulate the biosubstitute. Human mesenchymal stem cells from bone marrow were seeded in an alginate (Alg)/hyaluronic acid (HA) or Alg/hydroxyapatite (Hap) gel to direct cartilage and hypertrophic cartilage/subchondral bone differentiation, respectively, in different layers within a single scaffold. Homogeneous or composite stratified scaffolds were cultured for 28 days and cell viability and differentiation were assessed. The heterogeneous scaffold was stimulated daily. The mechanical behaviour of the stratified scaffolds were investigated by plane-strain compression tests. Results showed that the spraying process did not affect cell viability. Moreover, cell differentiation driven by the microenvironment was increased with loading: in the layer with Alg/HA, a specific extracellular matrix of cartilage, composed of glycosaminoglycans and type II collagen was observed, and in the Alg/Hap layer more collagen X was detected. Hap seemed to drive cells to a hypertrophic chondrocytic phenotype and increased mechanical resistance of the scaffold. In conclusion, mechanical stimulations will allow for the production of a stratified biosubstitute, laden with human mesenchymal stem cells from bone marrow, which is capable in vivo to mimic all depths of chondral defects, thanks to an efficient combination of stem cells, biomaterial compositions and mechanical loading.Scaffolds laden with stem cells are a promising approach for articular cartilage repair. Investigations have shown that implantation of artificial matrices, growth factors or chondrocytes can stimulate cartilage formation, but no existing strategies apply mechanical stimulation on stratified scaffolds to mimic the cartilage environment. The purpose of this study was to adapt a spraying method for stratified cartilage engineering and to stimulate the biosubstitute. Human mesenchymal stem cells from bone marrow were seeded in an alginate (Alg)/hyaluronic acid (HA) or Alg/hydroxyapatite (Hap) gel to direct cartilage and hypertrophic cartilage/subchondral bone differentiation, respectively, in different layers within a single scaffold. Homogeneous or composite stratified scaffolds were cultured for 28 days and cell viability and differentiation were assessed. The heterogeneous scaffold was stimulated daily. The mechanical behaviour of the stratified scaffolds were investigated by plane-strain compression tests. Results showed that the spraying process did not affect cell viability. Moreover, cell differentiation driven by the microenvironment was increased with loading: in the layer with Alg/HA, a specific extracellular matrix of cartilage, composed of glycosaminoglycans and type II collagen was observed, and in the Alg/Hap layer more collagen X was detected. Hap seemed to drive cells to a hypertrophic chondrocytic phenotype and increased mechanical resistance of the scaffold. In conclusion, mechanical stimulations will allow for the production of a stratified biosubstitute, laden with human mesenchymal stem cells from bone marrow, which is capable in vivo to mimic all depths of chondral defects, thanks to an efficient combination of stem cells, biomaterial compositions and mechanical loading.This work was supported by the project ANR06‐BLAN‐0197‐01/CartilSpray from the Agence Nationale de la Recherche, the Fondation Avenir, and the PIR‐CNRS vieillissement, longévité. The authors would like to thank Science Applications Industries (Lyon, France) for providing the hydroxyapatite, and L. O’Sullivan and H. Allison for reviewing the article. Confocal microscopy pictures were obtained thanks to the plate‐forme imagerie cellulaire IBISA (FR CNRS ‐ UL ‐ CHU 3209). (D. Dumas, S. Hupont).peer-reviewe