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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.

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La morphogenèse crâniofaciale chez les vertébrés est un phénomène important, strictement régulé dans l’espace et dans le temps. Elle est basée sur une série complexe d'événements moléculaires et morphogénétiques qui implique un réseau interactionnel de gènes et de facteurs de transcriptions, tels les homéoboîtes. La crête neurale (CN) est au cœur de ce processus. Cette dernière fournit la principale source du mésenchyme crâniofacial. Cette population de cellules embryonnaires transitoires va subir une transition épithélio-mésenchymateuse et migrer en plusieurs vagues vers des sites prédéfinis puis se différencier en divers types cellulaires. La CN est à l’origine de plusieurs structures : une grande partie du squelette facial dont le maxillaire, la mandibule, l’os alvéolaire qui entoure les dents ainsi qu’une partie des tissus conjonctifs crâniofaciaux. Les cellules issues des CN sont pluripotentes et offrent un espoir en régénération osseuse et cartilagineuse. Ces caractéristiques ont généré un intérêt particulier des chercheurs pour les utiliser en thérapie cellulaire afin de réparer les défauts osseux des mâchoires. Parmi les tissus crâniofaciaux, nous avons choisi d’étudier plus avant la gencive et les cellules gingivales car leur accès est le plus facile et leurs capacités de différenciation autorisent l’observation d’autres phénotypes cellulaires.La gencive est un tissu kératinisé qui entoure les dents et recouvre l’os alvéolaire. Ce tissu est composé principalement de fibroblastes gingivaux (GFs). Parmi ces cellules, se trouvent des cellules souches gingivales (GSCs) caractérisées par leur auto-renouvellement et leur multipotence. Les GSCs sont facilement recueillies chez les patients adultes, elles montrent une plasticité importante et une activité immunomodulatrice qui en font un outil de choix pour la thérapie cellulaire. De plus, la biopsie se fait sans douleur et n’entraîne ni cicatrice ni problème fonctionnel.La première partie de mon travail de doctorat avait pour objectif d’évaluer le rôle de Msx1 dans la morphogenèse crâniofaciale et par la suite d’analyser l’os alvéolaire après une extraction dentaire afin d’analyser les mécanismes associés à ce processus et l’impact de Msx1 sur la cicatrisation osseuse.La deuxième partie de mon travail est axé sur la gencive et avait pour objectif de mettre en évidence l’origine embryologique des cellules souches orales, dont les GSCs, et de déterminer si elles proviennent des crêtes neurales, du mésoderme ou d’une mosaïque des deux. Enfin, pour appliquer nos connaissances sur l’origine embryologique des cellules souches gingivales, nous avons étudié le profil immunitaire des cellules dérivées des CN. Pour cela, nous avons déterminé la capacité phagocytaire des cellules souches gingivales murines dérivées des CN et comparé à des cellules de CN d’autres espèces vertébrées. / Craniofacial morphogenesis in vertebrates is an important phenomenon, strictly regulated in space and in time. It is based on a complex series of molecular and morphogenetic events involving an interactional network of genes and transcription factors, such as the homeobox. Neural crest (NC) is at the heart of this process. The latter provides the main source of craniofacial mesenchyme. This transient population of embryonic cells will undergo epithelial-mesenchymal transition and migrate in waves to predefined sites and to differentiate into various cell types. NC is the source of several structures: a large part of the facial skeleton including the maxillary, mandibular alveolar bone around the teeth as well as connective tissue in craniofacial portion. Cells from NC are pluripotent and offer hope for bone and cartilage regeneration. These characteristics have generated particular interest to researchers for use in cell therapy to repair bone defects of the jaw. Among the craniofacial tissues, we decided to further investigate the gums and gingival cells because their access is easier and differentiation capabilities allow observation of other cellular phenotypes.The gum is a keratinized tissue around the teeth and covers the alveolar bone. This tissue is composed mainly of populations of gingival fibroblasts (GFs). Among these populations, there are gingival stem cells (GSCs) characterized by their self-renewal and pluripotency. The GSCs are easily collected in adult patients, they show significant plasticity and immunomodulatory activity that make it a tool of choice for cell therapy. In addition, the biopsy is painless and involves neither scar nor functional problem.The first part of my PhD work was to evaluate the Msx1 role in craniofacial morphogenesis and subsequently analyse the alveolar bone after tooth extraction to analyse the mechanisms involved in this process and the impact of Msx1 on bone healing.The second part of my work focuses on the gingiva and was intended to highlight the embryological origin of oral stem cells, including GSCs and determine if they come from the neural crest, mesodermal or mosaic two. Finally, to apply our knowledge of the embryological origin of gum stem cells, we studied the immune profile derived NC cells. For this, we determined the phagocytic capacity gingival murine stem cells derived from CN and compared to cells of CN other vertebrate species.

La gencive correspond à un modèle de régénération naturelle grâce notamment à sa capacité de cicatrisation « ad integrum ». Ce phénomène est permis par sa composition en fibroblastes gingivaux. Ces cellules, composante cellulaire principale du tissu conjonctif gingival, sont au cœur de la régulation des réponses inflammatoires et de la cicatrisation. Ce tissu contient, comme d’autres tissus mésenchymateux, des cellules souches ; qui expliquent en partie ces capacités de régénération. De plus, comme le tissu gingival est abondant et facilement accessible, l’utilisation de ces cellules souches pourraient être d’un intérêt prometteur en thérapie cellulaire ou pour de la modélisation in vitro. Au cours de cette thèse, nous avons pu montrer que les Cellules Souches dérivées de la Gencive Humaine (CSGH) possèdent des propriétés communes avec les cellules souches adultes dérivées des crêtes neurales. Ces cellules peuvent être qualifiées de « souche » par leur capacité d’auto-renouvèlement, d’adhésion au plastique et de multipotence. Premièrement, nous avons montré que la méthode ainsi que les produits de culture utilisés pour l’isolation des fibroblastes gingivaux in vitro à partir de biopsies de gencive avait une influence sur les cellules obtenues. Dans un second temps, une analyse clonale in vitro de populations de fibroblastes gingivaux a permis de montrer que les fibroblastes gingivaux sont composés de sous-populations qui expriment des marqueurs spécifiques des cellules souches et des crêtes neurales. Outre leur origine embryologique, l’étude de leur multipotence a aussi été caractérisée après expansion et en fonction des additifs utilisés. Pour finir, deux exemples d’utilisation de ces cellules comme modèle d’étude de la biocompatibilité de biomatériaux in vitro ont été développés; imitant la muqueuse buccale ainsi que les réactions dentaires (réparatrices et réactionnaire). / Gingiva is a natural regeneration model thanks to its "ad integrum" healing capability. Gingival fibroblasts are the main actors of this property. These cells, the main cellular component of the gingival connective tissue, regulate the inflammatory responses and healing process. This tissue contains, like many others, mesenchymal stem cells; which also partly explain these regenerative abilities. Moreover, as the gingiva is abundant and easily accessible, the use of these stem cells may interest cell therapy or in vitro model tissues responses. In this work, we demonstrated that Stem Cells Derived from Human Gingiva (SCHG) have common properties with neural crest adult stem cells. These cells can be called "stem cells" for their ability to self-renew, adhere to plastic and to differentiate. First, we have shown that the method and the culture products used for isolation of gingival fibroblasts from gingival biopsy had an influence on the obtained cells. Secondly, an analysis of in vitro clonal populations of gingival fibroblasts has shown that gingival fibroblasts are composed of subpopulations that express specific markers of stem cells and neural crests. In addition to their embryological origin, the study of their multipotency was also characterized after expansion and depending on the used additives. Finally, two examples of using these cells and dental pulp stem cells as a model to study the in vitro biocompatibility of biomaterials have been developed, mimicking oral mucosa or dentin reactions (reparative or reactional).

Introduction.Le fibroblaste gingival (FG) est la cellule majoritaire de la gencive. Cette dernière fait face constamment aux agressions physico-chimiques, infectieuses et thermiques. L'une des caractéristiques de la gencive est sa réparation quasi-parfaite suite à une lésion ponctuelle. Ce n'est pas le cas pour d'autres tissus comme la paroi aortique. L'anévrisme aortique (AA) est un affaiblissement de la paroi aortique provoqué par une sécrétion exhaustive de métalloprotéases (MMPs) et en particulier de MMP-9. Il en résulte une dilatation de l'artère. Dans un modèle d'anévrisme de lapin, Durand et al (2012) avait montré que le FG pouvait ralentir, voire réparer un anévrisme. Dans notre étude, nous avons mis en place un modèle de coculture FG/AA d'origine humaine.Chez l'homme, la localisation de la pathologie peut être au niveau abdominal (Anévrisme Aortique Abdominale : AAA) ou thoracique (Anévrisme Aortique Thoracique : AAT). Etant donné que leur étiologie sont différentes, nous avons souhaité savoir s'il existait des différences selon les lésions. Cela nous permettrait en effet de mieux appréhender la prise en charge. Nous avons réalisé une étude comparative histo et physiopathologique entre les AAA et AAT. L'une des différences soulevée, est la présence d'un facteur infectieux au niveau des AAA. C'est un élément à prendre en compte pour une thérapie cellulaire et ainsi nous avons mis en culture des FG en présence de LPS, une endotoxine bactérienne.De plus pour approfondir notre travail sur l'utilisation du FG dans la thérapie cellulaire, nous avons initié une étude sur la plasticité de la sous-population souche des FG en étudiant, notamment leur orientation en cellules vasculaires (cellules endothéliales).Résultats/discussionLe FG, grâce à sa secrétion de TIMP-1, contribue à l'inhibition de la MMP-9 anévrismale. La sécrétion de MMP-9 est plus importante dans les lésions avec athérome (AAA) que celles sans athérome (AAT dans notre étude). Ceci est en corrélation avec la dégradation qui est plus importante dans les AAA que dans les AAT. La MMP-9 est une protéine sécrétée entre autre par les cellules inflammatoires. Une inflammation est présente dans les AAA et pas dans les lésions thoraciques. Ceci pourrait expliquer la différence de sécrétion de MMP-9 et donc de dégradation. Concernant l'origine de cette inflammation, nous avons recherché une cause infectieuse. Porphyromonas gingivalis (Pg) qui est une bactérie importante dans le développement de la parodontite (maladie inflammatoire des tissus de soutien de la dent) a été détectée dans les AAA. Une relation pathologique existerait entre la parodontite et l'AAA mais l'étude devrait être plus poussée pour connaître le mécanisme physiopathologique de ce phénomène. Toutefois, en ce qui concerne la thérapie cellulaire, le LPS qui est une endotoxine du Pg, n'affecte pas la capacité du FG à secréter du TIMP-1.En plus de la possibilité du FG à neutraliser la MMP-9 anévrismale, nous avons souhaité savoir si le FG avait des compétences de différentiation en cellule vasculaire. Un début d'exploration de la plasticité cellulaire de la souche multipotente de FG en cellule endothéliale, donnent des résultats préliminaires encourageants.Conclusion. Le FG pourrait être une cellule prometteuse pour une thérapie cellulaire de l'anévrisme aortique mais des explorations plus poussées sont encore nécessaires pour une telle application. / IntroductionGingival fibroblast (GF) is the main cell in gingiva which is constantly facing infectious, thermal and physico-chemical attacks. When a lesion occurs, the repair of gingiva is almost perfect. It is not the case for other tissues as the aortic wall. The aortic aneurysm (AA) is a pathologic expansion of aorta due to a weakening of the wall with an exhaustive secretion of metalloproteinases (MMPs) and particularly of MMP-9. In an aneurysm rabbit model, Durand and al (2012) have showed that GF could slow down or repair the aneurysm. In our study, we have established a co-culture model of human GF and human AA.For human, the location of the aortic disease may be at abdominal level (Abdominal Aortic Aneurysm: AAA) and thoracic level (Thoracic Aortic Aneurysm: TAA). Since the aetiologies are different, we wondered if histo and physiopathologic differences would existe between the both. It is impotant to know that for better supporting the disease. One of the difference between AAA and TAA is the presence of an infectious factor in AAA. This is an element to consider for cell therapy, so we studied the behavior of GF in presence of an endotoxin, the LPS.In addition, to further our work on the use of GF in cell therapy, we have initiated a study of the plasticity of the GF multipotente subpopulation including the differentiation into vascular cells (endothelial cell in particular).Results/DiscussionThanks to its TIMP-1 secretion, GF could contribute to the inhibition of MMP-9 activity in aneurysm. The secretion of MMP-9 in AA with atheroma (AAA) is highter than in TAA (without atheroma in our study). It is correlated to the degradation of AAA which is more important than the degradation of TAA. Inflammatory cells may secrete MMP-9. Inflammation is present in AAA and not in TAA. This, could explain the highter secretion of MMP-9 in abdominal lesion and also the degradation which is more important in AAA than in TAA. As for the origin of this inflammation, we researched an infectious factor. We isolated Porphyromonas gingivalis (Pg) in AAA, which might trigger or aggravate inflammation. This is an important bacterium in the development of periodontitis (inflammatory disease of the tissues supporting the tooth). A pathological relationship may exist between periodontitis and the AAA. The study should be further to know the pathophysiology of AAA related to Pg. But as regards the cell therapy, LPS, which is an endotoxin of Pg would not affect the secretion of TIMP-1 by the GF.In addition to its abilities to inhibate MMP-9 in aneurysm, we wondered if GF would be able to differentiate into vascular cell. An early exploration of GF multipotent subpopulation plasticity reveals a possible opportunity to go further in a the cell therapy.Conclusion.GF might be a promising cell for treating aortic aneurysm but further explorations are still necessary for its application.

Oral Submucous Fibrosis (OSF) is a chronic inflammatory disease resulting in progressive fibrosis of the oral tissues that can cause difficulty in chewing, swallowing, speaking, and mouth opening. Epidemiological studies have shown that OSF is a precancerous condition and 2-8% of the OSF patients develop squamous cell carcinoma. This disease affects 0.5% of the population in the Indian subcontinent and is now a growing public health issue in many parts of the world. Habit of chewing betel quid has been proposed as an important etiological factor in the development of this disease and is coline, a principle alkaloid of areca nut is considered as major causative factor for OSF development. But the exact molecular mechanism of OSF pathogenesis is not known. Therefore, we set the following objectives for this study: 1) Gene expression profiling of OSF using microarray. 2) Role of areca nut constituents in OSF pathogenesis. 3) Effect of areca nut on epithelial and fibroblast cells. In order to delineate the possible molecular mechanism of OSF pathogenesis, we took microarray approach and identified differentially regulated genes in ten OSF tissues against eight pooled normals using whole human genome oligonucleotide arrays. Microarray results revealed differential expression of 5288 genes (p≤0.05 and Fold change≥1.5), among them 2884 were up-regulated and 2404 were down-regulated. Validation employing quantitative real-time PCR and immunohistochemistry confirmed up-regulation of transforming growth factor-β1 (TGF-β1), TGFBI, THBS1, SPP1, TIG1 and down-regulation of bone morphogenic protein 7 (BMP7), C4orf7 and ALOX12 in OSF tissues. Furthermore, activation of TGF-β pathway was evident in OSF tissues as demonstrated by p-SMAD2 strong immunoreactivity. Analysis of IHC data showed that in all the normal tissues and in 70% of the OSF tissues the expression of TGF-β and BMP7 are inversely correlated. In good correlation, treatment of keratinocytes (HaCaT) by TGF-βdown-regulated BMP7, while BMP7 expression could not be detected in fibroblast cells. Hence, the imbalance between TGF-βand BMP7 signalling, which are positive and negative modulators of extracellular matrix production, respectively may trigger the manifestation of OSF. We also studied the regulation few genes (CTGF, TGM2 and THBS1) identified in OSF microarray in response to TGF-βand arecoline. TGF-βwas able to induce all the above genes in both HaCaT and hGF cells but arecoline could only induce TGM2 in hGF and THBS1 in HaCaT. Therefore TGF-βpathway came out to be the most important pathway in OSF microarray and subsequent validations. But areca nut constituents responsible for TGF-βpathway activation and the source (epithelial or fibroblast cells) through which it activates TGF-βare not known. In an attempt to understand the role of areca nut and its constituents in inducing TGF-βsignalling in epithelial cells, we performed microarray on epithelial cells (HaCaT) treated with areca nut water extract. Surprisingly, 64% of the differentially regulated genes by areca nut water extract matched with TGF-βinduced gene expression profile. To find out areca nut induced genes through TGF-β, epithelial cells were treated with areca nut in presence of ALK5 (TβRI) inhibitor. Out of 64% differentially induced genes, 57% genes induced by areca nut got compromised in presence of ALK5 and 7% were independently induced by areca nut, highlighting the effect of areca nut via TGF-β. Accordingly, areca nut treatment induced both p-SMAD2 and TGF-βdownstream targets TGFBI, TGM2, TMEPAI and THBS1 in HaCaT cells. One possible mechanism of TGF-βsignalling induction by areca nut could be via induced ligand (TGF-β2) and its activator (THBS1). Induction of TGF-β2 ligand by areca nut was shown at both RNA (Real Time) and protein (ELISA) levels. To find out areca nut components responsible for inducing TGF-β signalling, areca nut fractionation was performed which gave three fractions namely, Ethyl acetate (polyphenol), water supernatant (alkaloids) and Dichloromethane (impurity). Out of these; polyphenol and alkaloid fractions were found to be responsible for the induction of TGF-β signalling and its downstream targets. Upon treatment with purified components, catechin and tannin of polyphenol fraction and arecoline, arecaidine and guvacine of alkaloid fraction were found to be responsible for inducing TGF-β signalling, as seen by increased appearance of phopho-SMAD2 in HaCaT cells. Areca nut treatment on human gingival fibroblast cells (hGF) did not induce TGF-β signalling, highlighting that the source of TGF-β induction by areca nut could possibly be the epithelium. Further treatment of areca nut along with TGF-β on hGF cells potentiated TGF-β effect both in terms of TGF-β downstream targets like TGFBI, TGM2, TMEPAI, COL1A1 etc and activation of fibroblast by inducing α-SMA. Increasing concentration of areca nut is cytotoxic on HaCaT cells and pro-proliferative on hGF cells. This could provide a possible explanation for epithelial atrophy and proliferating fibroblast cells in connective tissue of OSF patients. Further exploration on HaCaT cell cytotoxicity by areca nut suggests the involvement of Reactive Oxygen Species (ROS) as a key molecule induced by areca nut. Compromising ROS generation by NAC (N-Acetyl-L-Cysteine) led to reversal of Sub-G1 peak induced by areca nut in HaCaT cells. This highlighted that cell death caused by areca nut could be ROS mediated. Areca nut treatment on hGF cells did not induce ROS generation, leading to no cytotoxicity on these cells. A possible explanation of this differential ROS generation can be due to dose dependent suppression of Catalase activity by areca nut in HaCaT cells but not in hGF cells. We also compared cytotoxicity of areca nut with all the alkaloids and found a good match with arecoline as both of them induce ROS, apoptotic ladder formation, annexin V positivity, suppression of Catalase activity and the cell death induced by them was compromised by NAC. The above results indicated that arecoline could be a mediator of areca nut water extract cytotoxicity on HaCaT cells. Betel nut chewer’s oral epithelium gets regularly exposed to areca nut and hence this exposure could be cytotoxic to oral epithelial cells too. We performed Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) in normal and OSF tissues. Our data showed 62.5% of OSF patients having significant percentage of epithelial cells with TUNEL positivity (Labeling index = 2-60%) compared to all normal tissues that were TUNEL negative. TUNEL positivity was predominantly seen in the upper keratin and supra basal layer of the epithelium. We also studied proliferation status of OSF epithelium and observed that 3-17% (LI) of epithelial cells in all normal tissues showed Ki-67 positivity in the germinal layer of epithelium. However, 65% of the OSF patients showed staining for Ki-67 (LI=.2-58%) in their epithelium. Also analysis of TUNEL positive and Ki-67 positive sections indicated that OSF patients with high TUNEL positivity have high Ki-67 labeling index, but stains in the supra basal or keratin layer (TUNEL) and basal layer (Ki-67) of epithelium respectively. This induced proliferation of epithelial cells could be the result of heavy apoptosis in the outer epithelium. But as these patients are regularly exposed to areca nut, this increased proliferation may not be able to cope up with the heavy apoptosis induced by areca nut, leading to atrophied epithelium. To understand the germinal status of OSF atrophied epithelium we performed staining for OCT4 in OSF tissues. To our surprise there were no OCT4 positive nuclei in the epithelium of 53% of OSF patients but a regular spread of OCT4 positivity has been seen in the epithelium of normal subjects. In conclusion, this thesis highlights the involvement of TGF-β pathway in OSF patho-physiology. In addition, activation of TGF-β pathway by areca nut constituents has been demonstrated. Moreover, the atrophied epithelium of OSF appears to be a consequence of apoptosis and stem cell deprivation. Taken together, areca nut perhaps causes atrophy of the epithelium and activates TGF-β pathway that may lead to manifestation of OSF.