Isolation et caractérisation des cellules souches gingivales (étude de leur potentiel multipotent)

Les capacités de cicatrisation de la gencive en font un modèle de régénération tissulaire naturelle. Ces capacités sont liées en grande partie à l activité des fibroblastes. Composante cellulaire principale du tissu conjonctif gingival, ils sont au cœur de la régulation des réponses inflammatoires et des processus de cicatrisation. Nous avons supposé que ce tissu pouvait contenir des cellules souches, pouvant expliquer en partie, ces capacités de réparation. Au cours de cette thèse, nous avons pu mettre en évidence la présence de cellules souches mésenchymateuses aux propriétés communes avec les cellules souches adultes dérivées des crêtes neurales. Ces cellules expriment des marqueurs spécifiques des cellules souches et des crêtes neurales. Par ailleurs, elles présentent des capacités d auto-renouvellement et de multipotence. Elles sont, en effet, capables de se différencier en adipocytes, ostéocytes et chondrocytes. Nous nous sommes plus particulièrement intéressés à la différenciation chondro/endochondrale. La culture des cellules, sous forme de sphères en suspension, a permis de mettre en évidence leurs capacités de différenciation en tissus cartilagineux et articulaires. Elles s organisent spontanément en plusieurs types cellulaires différents, générant notamment des chondrocytes hypertrophiques et des synoviocytes selon leur localisation au sein des sphères et du milieu de culture utilisé. Le comportement de ces cellules soumises à ces conditions a permis de montrer leurs facultés à reproduire, in vitro, des processus proches de ceux retrouvés au cours du développement. Ces résultats permettent une meilleure compréhension des phénomènes de différenciation des cellules souches adultes, ouvrant ainsi de nouvelles perspectives pour des applications en thérapie cellulaire articulaire et osseuse.The healing capacity of the gingiva makes it a model of natural tissue regeneration. These capabilities are largely related to the fibroblast activity. They are the main cellular component of the gingival connective tissue and they regulate inflammatory responses and healing process. We hypothesized that this tissue could contain stem cells, which could explain, in part, these repair capabilities. In this thesis, we were able to demonstrate the presence of mesenchymal stem cells with properties shared with the neural crest-derived adult stem cells. These cells express specific markers of stem cells and neural crest. Moreover, they do have the capacity to self-renew and multipotency. They are, indeed, able to differentiate into adipocytes, chondrocytes and osteocytes. We have particularly focused on the chondro / endochondral differentiation. When cultivated as micromasses cultures in suspension, cells were able to differentiate into cartilage and joint tissues. They organize themselves spontaneously into several different cell types, including hypertrophic chondrocytes and synoviocytes depending on their location within the micromasses and the culture medium used. The behavior of these cells under these conditions has shown their ability to replicate in vitro, close to those found during the development process. These results allow a better understanding of adult stem cells differentiation, opening new perspectives for applications in joint and bone cell therapy.PARIS5-Bibliotheque electronique (751069902) / SudocSudocFranceF

Effects of Aging and Cyclosporin A on Collagen Turnover in Human Gingiva

BACKGROUND: WE AIMED AT CHARACTERIZING THE AGING GINGIVA ANALYZING: i) collagen content and turnover in human gingival tissues and fibroblasts obtained from healthy young and aging subjects. ii) the effect of cyclosporin A administration in human cultured gingival fibroblasts obtained from aging compared to young subjects. METHODS: Morphological analysis was performed on haematoxylin-eosin and Sirius red stained paraffin-embedded gingival biopsies from young and aging healthy subjects. The expression of the main genes and proteins involved in collagen turnover were determined by real time PCR, dot blot and SDS-zymography on cultured young and aging gingival fibroblasts, and after cyclosporin A administration. RESULTS: Our results suggest that in healthy aged people, gingival connective tissue is characterized by a similar collagen content and turnover. Collagen turnover pathways are similarly affected by cyclosporin A treatment in young and aging gingival fibroblasts. CONCLUSIONS: Cyclosporin A administration affects gingival collagen turnover pathways in young and aging fibroblasts at the same extent, suggesting that during aging cyclosporin A administration is not related to relevant collagen turnover modifications

Periodate-treated, non-anticoagulant heparin-carrying polystyrene (NAC-HCPS) affects angiogenesis and inhibits subcutaneous induced tumour growth and metastasis to the lung

Periodate-treated, non-anticoagulant heparin-carrying polystyrene consists of about ten periodate-oxidized, alkaline-degraded low molecular weight-heparin chains linked to a polystyrene core and has a markedly lower anti-coagulant activity than heparin. In this study, we evaluated the effect of non-anticoagulant heparin-carrying polystyrene on tumour growth and metastasis. Non-anticoagulant heparin-carrying polystyrene has a higher activity to inhibit vascular endothelial growth factor-165-, fibroblast growth factor-2- or hepatocyte growth factor-induced human microvascular endothelial cell growth than heparin, ten periodate-oxidized-heparin and ten periodate-oxidized-low molecular weight-heparin, which is probably due to the heparin-clustering effect of non-anticoagulant heparin-carrying polystyrene. Non-anticoagulant heparin-carrying polystyrene inhibited human microvascular endothelial cell, B16 melanoma and Lewis lung cancer cell adhesion to Matrigel-coated plates. Non-anticoagulant heparin-carrying polystyrene also showed strong inhibitory activities in the tubular formation of endothelial cells on Matrigel and B16-melanoma and Lewis lung cancer cell invasion in a Matrigel-coated chamber assay. In vivo studies showed that growth of subcutaneous induced tumours and lung metastasis of B16-melanoma and Lewis lung cancer cells were more effectively inhibited by non-anticoagulant heparin-carrying polystyrene than ten periodate-oxidized-heparin and ten periodate-oxidized-low molecular weight-heparin. Furthermore, non-anticoagulant heparin-carrying polystyrene markedly reduced the number of CD34-positive vessels in subcutaneous Lewis lung cancer tumours, indicating a strong inhibition of angiogenesis. These results suggest that non-anticoagulant heparin-carrying polystyrene has an inhibitory activity on angiogenesis and tumour invasion and may be very useful in cancer therapy

Meta-Profiles of Gene Expression during Aging: Limited Similarities between Mouse and Human and an Unexpectedly Decreased Inflammatory Signature

Background: Skin aging is associated with intrinsic processes that compromise the structure of the extracellular matrix while promoting loss of functional and regenerative capacity. These processes are accompanied by a large-scale shift in gene expression, but underlying mechanisms are not understood and conservation of these mechanisms between humans and mice is uncertain. Results: We used genome-wide expression profiling to investigate the aging skin transcriptome. In humans, age-related shifts in gene expression were sex-specific. In females, aging increased expression of transcripts associated with T-cells, B-cells and dendritic cells, and decreased expression of genes in regions with elevated Zeb1, AP-2 and YY1 motif density. In males, however, these effects were contrasting or absent. When age-associated gene expression patterns in human skin were compared to those in tail skin from CB6F1 mice, overall human-mouse correspondence was weak. Moreover, inflammatory gene expression patterns were not induced with aging of mouse tail skin, and well-known aging biomarkers were in fact decreased (e.g., Clec7a, Lyz1 and Lyz2). These unexpected patterns and weak human-mouse correspondence may be due to decreased abundance of antigen presenting cells in mouse tail skin with age. Conclusions: Aging is generally associated with a pro-inflammatory state, but we have identified an exception to this pattern with aging of CB6F1 mouse tail skin. Aging therefore does not uniformly heighten inflammatory status across all mouse tissues. Furthermore, we identified both intercellular and intracellular mechanisms of transcriptome aging, including those that are sex- and species-specific

Embryologie des crêtes neurales et leur classification

La morphogenèse de la face est étudiée à la lumière des acquisitions les plus récentes de l'embryologie. La formation et la migration des crêtes neurales, ainsi que les moyens de l'étude de celles-ci sont largement décrits. La différenciation des cellules crestales et leur interaction avec les tissus environnants interviennent dans la genèse des principaux constituants de l'extrémité céphalique : non seulement la plupart des dérivés du mesenchyme, mais aussi les structures osseuses et cartilagineuses. Les cellules crestales jouent également un rôle essentiel dans l'embryogenèse du système nerveux. Dans une seconde partie, les anomalies de l'extrémité céphalique sont présentées en fonction de leur point de départ encéphalique. Une classification neurocristopathique est ainsi ébauchée, différenciant les neurocristopathies prosencéphaliques, intéressant le bourgeon naso-frontal, les neurocristopathies mésencéphaliques, intéressant les bourgeons maxillaires et mandibulaires, et les neurocristopathies rhombencéphaliques, intéressant la région cervico-thoracique

Traitement de l'anévrysme abdominal aortique par transplantation de fibroblastes gingivaux autologues (études in vitro)

MONTROUGE-BUFR Odontol.PARIS5 (920492101) / SudocSudocFranceF

Thérapie cellulaire de l'anévrisme aortique par le fibroblaste gingival (études ex vivo et in vivo)

L'anévrisme aortique abdominal s'accompagne d'une dégradation du réseau élastique et d'une augmentation des métalloprotéases. Nous avons essayé de transposer les qualités de réparations du fibroblaste gingival sur ces artères dans des modèles ex-vivo et in vivo. Un modèle de culture d'artère de lapin en gel de collagène est évalué puis utilisé en coculture avec des fibroblastes gingivaux pour évaluer l'effet de ces fibroblastes sur le remodelage artériel. Les fibroblastes gingivaux sont également cultivés avec des artères issues d'anévrismes aortiques humains. Enfin notre hypothèse est testée sur un modèle d'anévrisme chez le lapin où les cellules sont transférées par voie endoluminale. En coculture, les fibroblastes gingivaux inhibent la MMP-9 par une augmentation de son inhibiteur le TIMP-1. La même inhibition est présente dans des cocultures avec des artères anévrismales humaines. La MMP-7 est également inhibée par augmentation du TIMP-1 mais aussi au niveau transcriptionnel par une augmentation du TGF-pl. Ces cocultures permettent la préservation du réseau élastique artériel. Le transfert des fibroblastes dans des anévrismes créés chez le lapin entraîne la diminution de leurs diamètres et de la MMP-9. Ces résultats obtenus sur des modèles ex vivo et in vivo montrent la capacité des fibroblastes gingivaux à préserver le réseau élastique et à moduler l'activité de protéases impliquées dans la pathologie. La transplantation de fibroblastes gingivaux semble être une approche intéressante dans le traitement des anévrismes aortiques. Néanmoins des expériences complémentaires sont nécessaires pour confirmer nos résultats et comprendre comment le fibroblaste gingival influe positivement le remodelage.Aortic abdominal aneurysm is accompanied by a degradation of the elastic network and an increase of the metalloproteinases. We tried to transpose repair qualities of the gingival fibroblast on these arteries in ex-vivo and in vivo models. A culture model of rabbit artery in collagen gel is evaluated then used in coculture with gingival fibroblasts to evaluate the effect of these fibroblasts on arterial remodeling. The gingival fibroblasts are also cultivated with human aneurismal aortas. Finally our hypothesis is tested on an in vivo model of rabbit aneurism where the cells are transplanted into the lumen. In coculture, the gingival fibroblasts inhibit MMP-9 by an increase of its inhibitor: TIMP-1. Same inhibition is present in cocultures with human aneurismal aortas. The MMP-7 is also inhibited by increase in the TIMP-1 but also at a transcriptional level by an increase of TGF-pl. These cocultures allow the preservation of the arterial elastic network. The transfer of the fibroblasts in aneurisms created in rabbit reduced their diameters and the MMP-9. These results obtained on ex vivo and in vivo models show the capacity of the gingival fibroblasts to preserve the elastic network and to modulate the activity of proteases implied in pathology. The transplantation of gingival fibroblasts seems to be an interesting approach in the treatment of aortic aneurisms. Nevertheless complementary experiments are necessary to confirm our results and to understand how the gingival fibroblast influences remodeling.PARIS5-BU Méd.Cochin (751142101) / SudocSudocFranceF