Objetivos: O objetivo desse estudo foi avaliar comparativamente o potencial osteogênico in vitro de células obtidas do ramo mandibular (RM, área doadora) e do seio maxilar enxertado com uma mistura de RM e osso bovino inorgânico (OBI), previamente à instalação de implantes de titânio (SM, sítio do seio maxilar enxertado). Material e Métodos: As células foram obtidas de três pacientes submetidos a procedimentos de aumento do soalho do seio maxilar com a proporção de 1:1 de RM e OBI (GenOx Inorg®). No momento da realização dos enxertos no seio maxilar e após 08 meses, antes da inserção dos implantes de titânio, fragmentos ósseos foram colhidos do RM e do SM, respectivamente, e submetidos à digestão enzimática com tripsina e colagenase para obtenção de células primárias. As células foram subcultivadas e crescidas sob condições osteogênicas por até 21 dias, tendo sido avaliados os seguintes parâmetros: proliferação/viabilidade celular, expressão gênica de marcadores osteoblásticos, atividade de fosfatase alcalina (ALP) e conteúdo de cálcio, por extração do vermelho de Alizarina. Culturas primárias derivadas do RM foram expostas ao GenOx Inorg® por 7 dias, quando se avaliou a atividade de ALP. Os resultados foram comparados por ANOVA two-way, seguido do teste de Tukey, ou pelo teste de Mann-Whitney. Resultados: Culturas do SM exibiram uma redução significante do potencial osteogênico se comparado ao de culturas do RM, com um aumento progressivo na proliferação celular associado a uma redução da expressão dos marcadores osteoblásticos, da atividade de ALP e do conteúdo de cálcio. A exposição do GenOx Inorg® às células primárias derivadas do RM inibiram a atividade de ALP. Conclusão: Esses resultados sugerem que o uso do GenOx Inorg® em associação a fragmentos do RM para aumento do soalho do seio maxilar inibe a diferenciação de células osteoblásticas no sítio de inserção de implantes de titânio após 8 meses de enxertia. / Objectives: This study aimed to comparatively evaluate the in vitro osteogenic potential of cells obtained from the mandibular ramus (MR, autogenous bone donor site) and from the maxillary sinus bone grafted with a mixture of anorganic bovine bone (ABB) and MR prior to titanium implant placement (MS, grafted implant site). Material and methods: Cells were obtained from three patients subjected to maxillary sinus floor augmentation with a 1:1 mixture of ABB (GenOx Inorg®) and MR. At the time of the sinus lift procedure and after 8 months, prior to implant placement, bone fragments were taken from MR and MS, respectively, and subjected to trypsin-collagenase digestion for primary cell culturing. Subcultured cells were grown under osteogenic condition for up 21 days and assayed for proliferation/viability, osteoblast marker mRNA levels, alkaline phosphatase (ALP) activity and calcium content/Alizarin red staining. ALP activity was also determined in primary explant cultures exposed to GenOx Inorg® (1:1 with MR) for 7 days. Data were compared using the two-way ANOVA followed by the Tukey test; otherwise, the Mann-Whitney test was used. Results: MS cultures exhibited a significantly lower osteogenic potential compared with MR cultures, with a progressive increase in cell proliferation together with a downregulation of osteoblast markers, reduced ALP activity and calcium content. Exposure of MR-derived primary cultures to GenOx Inorg® inhibited ALP activity. Conclusion: These results suggest that the use of GenOx Inorg® in combination with MR fragments for maxillary sinus floor augmentation inhibits the osteoblast cell differentiation at the implant site in the longterm.

O agregado de trióxido mineral (MTA) tem-se demonstrado aplicável em diversas situações que exigem reparação dos tecidos dentais e periapicais. Contudo, desvantagens relacionadas ao seu elevado custo, propriedades físico-químicas e dificuldade de manuseio têm limitado sua utilização. Neste sentido, um novo cimento de aluminato de cálcio e aditivos (CAC+) foi desenvolvido para superar algumas características negativas do MTA, mantendo, no entanto, suas propriedades satisfatórias. O objetivo deste estudo foi avaliar a progressão de culturas de células osteogênicas e de células indiferenciadas da polpa dental (linhagem OD-21) expostas ao CAC+, utilizando MTA como controle, ou a preparações alternativas do CAC+, com maior conteúdo de cloreto de cálcio (CaCl2) e/ou com substituição do óxido de zinco por óxido de bismuto como radiopacificador. Para isso, células osteogênicas derivadas de calvárias de ratos ou células da linhagem OD-21 foram crescidas sobre Thermanox® por 24 h e expostas, por períodos de até 14 dias, a amostras dos diferentes materiais, posicionadas sobre Transwell®. Em células osteogênicas, apesar de a proximidade com as amostras de CAC+ e MTA inibir o crescimento celular, nas áreas periféricas das lamínulas, foram observados proliferação, viabilidade celular e expressão de marcadores-chave da diferenciação osteoblástica, que precederam a mineralização da matriz extracelular. Culturas expostas ao CAC+, comparativamente ao MTA, exibiram aumentos na viabilidade celular, atividade de fosfatase alcalina e na expressão de marcadores de diferenciação, o que não se repetiu para células OD-21. Além disso, demonstrou-se que os efeitos destes cimentos sobre a osteogênese in vitro variaram de acordo com o período de exposição das culturas, sendo mais favoráveis durante sua fase proliferativa. Entre as preparações de CAC+, verificou-se que o aumento no teor de CaCl2 promoveu maior disponibilização de Ca2+ no meio de cultura, o que correspondeu a maior diferenciação celular e formação de matriz mineralizada em culturas de células osteogênicas e OD-21, e à redução de efeitos negativos da adição de óxido de bismuto sobre osteoblastos. Conclui-se que o CAC+ favorece o desenvolvimento do fenótipo osteogênico, atuando principalmente em células em estágios iniciais de diferenciação e que a adição de CaCl2, independentemente do agente radiopacificador, potencializa os efeitos benéficos sobre células osteogênicas e favorece o desenvolvimento e diferenciação de células indiferenciadas derivadas do tecido pulpar, podendo ser considerado como material alternativo ao MTA. / Mineral trioxide aggregate (MTA) has been successfully applied in endodontic procedures in which dental and periapical tissue repair are required. However, some drawbacks of MTA as high cost, physicochemical properties and difficulty in handling have limited its use. In this context, a novel calcium aluminate cement plus additives (CAC+) has been developed to overcome some negative features of MTA. The aim of this study was to evaluate the progression of either osteogenic cell cultures or undifferentiated dental pulp-derived ones (OD-21 cell line) exposed to CAC+ or to alternative formulations of CAC+ with a higher content of calcium chloride (CaCl2) and/or replacement of zinc oxide by bismuth oxide as radiopacifier agent. Rat calvaria-derived cells or OD-21 cells were grown on Thermanox® coverslips for 24 h and exposed to samples of CAC+ or MTA (control) placed on Transwell® for periods of up to 14 days. In osteogenic cell cultures, the proximity to MTA or CAC+ samples inhibited cell growth, whereas at distance it was observed cell proliferation, cell viability and expression of differentition markers prior to mineralization of the extracellular matrix. Comparatively to MTA, osteogenic cell cultures exposed to CAC+ exhibited higher cell viability, alkaline phosphatase activity and expression of key osteoblast markers, contrary to what was observed for OD-21 cells. Furthermore, it was demonstrated that the effects of these cements on in vitro osteogenesis varied according to the timing of exposure, with a more favorable impact during the proliferative phase of cultures. Among the diverse formulations of CAC+, it was found that the increase in the CaCl2 content promoted greater availability of Ca2+ in the culture medium, which corresponded to higher cell differentiation and mineralized matrix formation in osteoblastic cell cultures and OD-21 cells, while reducing the negative effects of bismuth oxide on osteoblasts. In conclusion, CAC+ supported the acquisition of the osteogenic cell phenotype, mostly for cells in early stages of differentiation. Additionaly, the increase in the CaCl2 content, regardless of the radiopacifier agent, potentiates the beneficial effects on osteogenic cells and promotes the growth and differentiation of OD-21 cells, rendering CAC+ a potential alternative material to replace MTA in endodontic procedures.

Combinatorial techniques, which overcome limitations of actual models of material research permitting to effectively address this large amount of variables, are utilized in this work to prepare combinatorial libraries of the blend of the biodegradable polymers Poly(e-caprolactone) and Poly(lactic acid). These libraries present continuous composition and temperature gradients in an orthogonal fashion that permit to obtain multiple surface morphologies with controllable microstructures due to the blends low critical solution phase behavior (LCST). The goal of this study is to investigate the effect of surface morphology (surface chemical patterning and surface topography) on cell behavior. The varied surface topography of the libraries is used as a valuable tool that permits to assay the interaction between MC3T3-E1 cells and hundreds of different values of critical surface properties, namely, surface roughness and microstructure size. The outcome of this tool is a rapid screening of the effect of surface topography on cell behavior that is orders of magnitude faster than the standard 1-sample for 1 measurement techniques. The results obtained show that cells are very sensitive to surface topography, and that the final effect of surface properties on cell function is intimately related with the stage of the cell developmental process. Meaning that, for example, areas with optimal characteristics to elicit enhancement of cell attachment is not necessarily the same that promotes cell proliferation. This study imparts an improved understanding of an often neglected factor in biomaterials performance: surface morphology (particularly surface topography). The results provide a new insight into the importance of taking into consideration both chemistry and physical surface features for superior biomaterial design.

Einen Aspekt dieser Arbeit stellt die Analyse der Stimulation von Ratten-Calvaria-Osteoblasten (RCA) mit den beiden Wachstumsfaktoren TGF-b1 und BMP-4 während der Proliferations- sowie Differenzierungs- und Mineralisierungsphase dar. Hierfür soll die Phosphorylierung und Aktivierung von Erk1 und Erk2, sowie von Smad1 und Smad2 mit Hilfe eines Kinase-Aktivitätsassays sowie der Westernblot-Analyse untersucht werden. Im Rahmen dieser Arbeit soll weiterhin untersucht werden, welche Bedeutung die Wachstumsfaktoren TGF-b1 und BMP-4 auf die Aktivität der alkalischen Phosphatase (ALP), einem wichtigen Differenzierungsmarker in Osteoblasten, ausüben. Enzymatische Aktivitätsbestimmungen und zytochemische Färbung aktiver ALP sollen darüber Aufschluss geben. Weiterhin soll der Gehalt an ALP-mRNA durch PCR bestimmt werden. Ein weiteres wichtiges Ziel dieser Arbeit ist die Analyse der Bedeutung von Erk1, Erk2, Smad1 und Smad2 auf die Aktivität der ALP. Dafür sollen Inhibitoren eingesetzt werden. Die enzymatische Aktivitätsbestimmung soll darüber aufklären. Außerdem soll mit Hilfe von kurzen, doppelsträngigen RNA-Molekülen (siRNA) ein knock down der Kinasen herbeigeführt werden und dessen Auswirkung auf die Aktivität der ALP enzymatisch bestimmt werden. Dafür muss zunächst die Wirksamkeit der siRNA auf RNA-Ebene mittels PCR und auf Proteinebene mittels Westernblot-Analysen überprüft werden. Zusätzlich soll die Bedeutung der Wachstumsfaktoren und der Kinasen Erk1 und Erk2 auf die Mineralisierung der RCA analysiert werden. Dafür wird die Menge des zellassoziierten Kalziums und Phosphats experimentell bestimmt, wodurch der Mineralisationsgrad der Zellen wiedergegeben werden kann.

Αντικείμενο της παρούσας διατριβής, ήταν η διερεύνηση της διεπιφάνειας ανθρώπινων μεσεγχυματικών κυττάρων (Human Mesenchymal Stem Cells, hMSCs) -Νανοσωλήνων Άνθρακα Πολλαπλού Τοιχώματος (Multi Walled Carbon Nanotubes, MWCNTs) υπό στατικές και δυναμικές συνθήκες. Οι MWCNTs έχει αποδειχθεί ότι, έχουν μοναδικές ηλεκτρικές και φυσικές ιδιότητες, μηχανική αντοχή και χαμηλή πυκνότητα, χαρακτηριστικά που τους καθιστούν εξαιρετικά ελκυστικούς για το σχεδιασμό βιοϋλικών για ορθοπαιδικές εφαρμογές. Αρχικά, μελετήθηκε η βιοσυμβατότητα των hMSCs σε επιφάνειες MWCNTs, ως προς την κυτταροτοξικότητα, τη μορφολογία, τον πολλαπλασιασμό, τη διαφοροποίηση και την οργάνωση του κυτταροσκελετού. Το υπόστρωμα των MWCNTs ευνόησε την εξάπλωση των κυττάρων, προήγαγε τον πολλαπλασιασμό και προώθησε τη διαφοροποίηση των hMSCs σε οστεοβλάστες, όπως έδειξε η έκφραση αλκαλικής φωσφατάσης, οστεοποντίνης και οστεοκαλσίνης. Μελετήθηκε η γονιδιακή έκφραση των ιντεγκρινικών υποδοχέων, υπεύθυνων για την προσκόλληση των κυττάρων στους MWCNTs. Με την τεχνική του περιστρεφόμενου δίσκου, εκτιμήθηκε η δύναμη προσκόλλησης των hMSCs στους MWCNTs και η επίδραση της κάθε ιντεγκρίνης στη μεταβολή της δύναμης προσκόλλησης. Για τη διερεύνηση της απόκρισης των οστεοβλαστών στη μηχανική φόρτιση, τα προσκολλημένα κύτταρα στους MWCNTs καταπονήθηκαν για 3 και 24 ώρες, με σύστημα μηχανικής φόρτισης βασισμένο στην Αρχή Κάμψης Τεσσάρων Σημείων. Τα αποτελέσματα έδειξαν ότι, η φόρτιση επηρεάζει θετικά την έκφραση γονιδίων προσκόλλησης και δεικτών διαφοροποίησης. Επιπρόσθετα, μελετήθηκε η συμπεριφορά των hMSCs ως προς την κυτταροτοξικότητα, τον πολλαπλασιασμό, τη διαφοροποίηση, την οργάνωση του κυτταροσκελετού και την έκφραση γονιδίων προσκόλλησης, σε τροποποιημένες επιφάνειες MWCNTs με υδροξυλομάδες, καρβοξυλομάδες και αμινομάδες. Τα αποτελέσματα έδειξαν ότι, η αμινοτροποποιημένη επιφάνεια ευνόησε σημαντικά την κυτταρική συμπεριφορά σε σύγκριση με τις άλλες δύο επιφάνειες. Τέλος, μελετήθηκε η επίδραση της τοπογραφίας με χρήση κάθετα ευθυγραμμισμένων MWCNTs, σε σύγκριση με τυχαία προσανατολισμένους MWCNTs. Η απόκριση των hMSCs στους κάθετα ευθυγραμμισμένους MWCNTs ήταν καλύτερη σε σύγκριση με τους τυχαία προσανατολισμένους, τόσο ως προς τον πολλαπλασιασμό και τη διαφοροποίηση, όσο και ως προς την οργάνωση του κυτταροσκελετού. Τα αποτελέσματα της διατριβής είναι υποσχόμενα για το μελλοντικό σχεδιασμό βιοϋλικών με MWCNTs, με τελικό σκοπό την εφαρμογή σε θεραπείες στις οποίες απαιτείται ανακατασκευή του οστού. / The aim of the present study was the investigation of the interface of human Mesenchymal Stem Cells (hMSCs) – Multiwalled Carbon Nanotubes (MWCNTs), under static and dynamic conditions. MWCNTs have been proven to obtain unique electric and physical properties, mechanical strength and low density, which render them highly attractive for the design of biomaterials for orthopaedic applications. Firstly, the biocompatibility of MWCNTs was studied, in terms of hMSCs cytotoxicity, morphology, proliferation, differentiation, cytoskeleton organization and toxicity. The substrate of MWCNTs favored cell spreading, increased proliferation and promoted cell differentiation, as measured by the expression of alkaline phosphatase, osteopontin and osteocalcin. The gene expression of integrin receptors responsible for cell attachment on MWCNTs was studied. Using the Spinning Disc Technique, the attachment strength of hMSCs on MWCNTs was evaluated, as well as the impact of each integrin to the alteration of attachment strength. In order to investigate the cell response to mechanical loading, the attached cells on MWCNTs were stressed for 3 and 24 hours, using a system for mechanical loading based on the 4-point bending principle. Results showed that loading positively induces the expression of genes associated with attachment and differentiation markers. Additionally, the cell behavior concerning proliferation, differentiation, cytoskeleton organization, apoptosis and gene expression associated with attachment, was studied on MWCNTs after surface modification with hydroxyl-, carboxyl-, and amino- groups. The findings indicated that the amino- modified surface significantly favored the cell behavior, compared to the other two surfaces. Lastly, the topography effect was studied using vertically aligned MWCNTs. Cell response was found better on the vertically compared to the randomly oriented, in terms of proliferation, differentiation and cytoskeleton organization. The findings of the study are promising for the future design of biomaterials of MWCNTs, aiming for application in therapies where bone reconstruction is demanded.

No description available.

Le développement et l'homéostasie des os requièrent l'orchestration spatio-temporelle d'un grand nombre de signaux moléculaires. Ces signaux entraînent l'activation ou l'inhibition de différents facteurs de transcription, lesquels sont en mesure de contrôler la prolifération et la différenciation des ostéoblastes et des chondrocytes. L'intégrité de ces différents mécanismes se doit d'être maintenu tout au long de la vie. Ainsi, une anomalie dans l'un de ces mécanismes conduit à l'apparition de pathologies osseuses et métaboliques telles qu’une hypophosphatémie, l'ostéoporose ou l'ostéoarthrite (OA). Afin d'en apprendre davantage sur la biologie osseuse, le projet décrit dans cette thèse a pour objectif de caractériser de nouveaux mécanismes de régulation transcriptionnelle pour deux gènes importants dans le développement des os et le maintien de leur intégrité. Il s’agit du Paired-like Homeodomain Transcription Factor 1 (PITX1) et du Phosphate-regulating gene with homology to endopeptidase on the X chromosome (PHEX). Le premier mécanisme présenté dans cette thèse concerne la régulation transcriptionnelle du gène PITX1, un facteur de transcription à homéodomaine nécessaire, notamment, au développement des os des membres inférieurs et au maintien de l'intégrité du cartilage articulaire chez l'adulte. Ainsi, dans les chondrocytes articulaires, on note que l'expression de PITX1 est assurée par le recrutement du facteur de transcription E2F1 à deux éléments de réponse présents dans la région proximale du promoteur de PITX1. Aussi, dans les chondrocytes articulaires de patients souffrant d'OA, dans lesquels l'expression de PITX1 est fortement diminuée, un mécanisme de répression transcriptionnelle, lequel implique la protéine multifonctionnelle Prohibitin (PHB1), semble être activé. En effet, dans ces chondroytes, on note une forte accumulation nucléaire de PHB1 comparativement aux chondrocytes articulaires de sujets sains. Le second mécanisme présenté dans cette thèse concerne la répression transcriptionnelle de PHEX, la peptidase mutée dans le syndrome d'hypophosphatémie lié au chromosome X (X-Linked Hypophosphatemia, XLH), lequel se caractérise par une hypophosphatémie et une ostéomalacie. Le traitement d'ostéoblastes à la Parathyroid hormone-related protein (PTHrP) permet d’observer la répression de PHEX. Afin de caractériser le mécanisme responsable de cette répression, des expériences de gènes rapporteurs ont révélé la présence de deux éléments de réponse pour le répresseur transcriptionnel E4BP4 dans le promoteur de PHEX. La suppression de l'expression d'E4BP4 par l'utilisation d'ARN d'interférence a permis de valider que ce facteur de transcription est responsable de la répression de PHEX suite au traitement d'ostéoblastes à la PTHrP. En somme ces nouveaux mécanismes de régulation transcriptionnelle permettent de mieux comprendre la régulation de l'expression de PITX1 et de PHEX. Aussi, cette nouvelle implication de PHB1 dans la pathogenèse de l'OA offre de nouvelles possibilités de traitement et pourrait servir pour le diagnostic précoce de cette pathologie. Enfin, la caractérisation d'E4BP4 en tant que médiateur pour la répression de PHEX par la PTHrP suggère que ce répresseur transcriptionnel pourrait être impliqué dans le contrôle de la minéralisation des os et des niveaux de phosphate sanguin. / Bone development and homeostasis need a large amount of molecular signals to be finely regulated in time and space. These signals lead to the activation or to the inhibition of different transcription factors, which are implicated in the control of osteoblast and chondrocyte proliferation and differentiation. The integrity of these mechanisms is required in order to have a healthy life. Indeed, if one of these mechanisms is dysfunctional, different diseases could develop such as hypophosphatemia, osteoporosis and osteoarthritis (OA). In order to contribute to the comprehension of bone biology, the present thesis describes new mechanisms for the transcriptional regulation of two genes implicated in bone development and regulation: PITX1 (Paired-like Homeodomain Transcription Factor 1) and PHEX (Phosphate-regulating gene with homology to endopeptidase on the X chromosome). The first mechanism described in this thesis relates to the transcriptional regulation of PITX1, a gene that encodes for a member of the homeobox family of transcription factors. PITX1 is required in bone development of inferior members and in the maintenance of the articular cartilage integrity in adults. Thereby, we showed that in articular chondrocytes, the expression of PITX1 is activated after the transcription factor E2F1 was recruited at two response elements in the proximal region of its promoter. Moreover, in articular chondrocytes from OA patients, we observed that the expression of PITX1 is strongly decreased. We proposed that the mechanism responsible for this repression requires the multitask protein Prohibitin (PHB1), which is strongly accumulated in OA chondrocyte nuclei, but not in chondrocyte nuclei from healthy individuals. The second mechanism described in this thesis reports a transcriptional mechanism by which PHEX, the gene that encodes for the peptidase mutated in the syndrome X-Linked Hypophosphatemia (XLH)and characterized by hypophosphatemia and osteomalecia, is repressed. We showed that the treatment of osteoblasts with the Parathyroid hormone-related protein (PTHrP) induced a decrease in PHEX expression. In order to characterize the mechanism responsible for this repression, we performed gene reporter experiments and identified two response elements for the transcription factor E4BP4 in the PHEX promoter. The downregulation of E4BP4 by siRNA led to the validation that this repressor decreased the expression of PHEX in osteoblasts after their treatment with PTHrP. In conclusion, the new transcriptional mechanisms presented in this thesis allow a better understanding of PITX1 and PHEX expression. Moreover, the potential role of PHB1 in the establishment of OA presents many interesting possibilities regarding the treatment and diagnosis of this disease. Finally, the characterization of E4BP4 as a mediator of PHEX repression by the PTHrP suggests that E4BP4 could be implicated in the control of bone mineralization and phosphate levels in the blood.

L’ostéoarthrose (OA) est une pathologie de forte incidence affectant les articulations. Elle est caractérisée principalement par une dégradation du cartilage articulaire, un déséquilibre au niveau du remodelage osseux et une sclérose de l’os sous-chondral. L’étiologie de cette pathologie reste encore méconnue, cependant il semble de plus en plus que tous les tissus composant l’articulation soient affectés dans cette pathologie. L’importance du rôle de l’os dans le développement de l’OA est incontestable et représente donc une cible thérapeuthique intéressante. Des études effectuées par tomodensitométrie ont démontré une structure et une organisation anormales du tissu osseux des patients OA. Parallèlement, les cultures primaires d’ostéoblastes (Ob) humains OA issus de l’os sous-chondral démontrent un phénotype altéré et une faible minéralisation in vitro. La signalisation Wnt, essentielle dans l’embryogenèse, a montré avoir un rôle clé dans la régulation de l’ostéogenèse en régulant notamment la différenciation terminale des Ob. Le facteur de croissance transformant-β1 (TGF-β1), un facteur agissant notamment sur la prolifération et sur le début de la différenciation des Ob, est surexprimé par les Ob OA et pourrait moduler cette signalisation. Aussi, deux populations de patients OA peuvent être différenciées in vitro par la production de prostaglandines E2 (PGE2) par leurs Ob et les PGE2, dans une étude sur le cancer colorectal, ont montré moduler la signalisation Wnt. Notre hypothèse de travail est que l’activation de la voie de signalisation Wnt/β-caténine est diminuée dans les Ob OA. Cette diminution est responsable de la sous-minéralisation et de l’altération du phénotype des Ob humains OA. Par ailleurs, DKK2, dont l’expression est contrôlée par TGF-β1, est responsable de la diminution de l’activité Wnt/β-caténine et les PGE2 peuvent en partie corriger cette situation. L’objectif général de cette étude est d’une part, de démontrer le rôle de TGF-β1, DKK2 et de PGE2 sur l’altération de la signalisation Wnt/β-caténine et d’autre part, de démontrer le lien entre TGF-β1 et DKK2 et l’effet de ces derniers sur le phénotype des Ob. Dans cette étude on a montré que la signalisation canonique Wnt est altérée dans les Ob OA et que cela était responsable de l’altération du phénotype des Ob OA. On a montré, parmi les acteurs de la signalisation Wnt, que l’expression de l’antagoniste Dickkopf-1 (DKK1) était relativement similaire entre les Ob OA et normaux contrairement à celle de l’antagoniste DKK2 qui était augmentée et à celle de l’agoniste Wnt7B qui était diminuée dans les Ob OA. On a également montré que les PGE2 pouvaient potentialiser l’activité de la signalisation Wnt dans les Ob OA. L’inhibition de DKK2 a permis d’augmenter l’activité de la signalisation Wnt et de corriger le phénotype anormal ainsi que d’augmenter la minéralisation des Ob OA. L’inhibition de TGF-β1, un facteur aussi surexprimé dans les Ob OA, a également permis la correction du phénotype et l’augmentation de la minéralisation dans les Ob OA. L’inhibition de TGF-β1 a aussi menée à l’inhibition de DKK2. Le contraire ne fût pas observé démontrant ainsi la régulation de DKK2 par TGF-β1. En conclusion, la signalisation canonique Wnt est diminuée dans les Ob OA et cela est dû au niveau élevé de DKK2 dans ces Ob. TGF-β1 régule positivement DKK2 et donc la surexpression de TGF-β1 entraîne celle de DKK2 ce qui a pour conséquences d’altérer le phénotype des Ob. Les PGE2 ont aussi montré pouvoir potentialiser l’activité de la signalisation Wnt et auraient donc un rôle positif. Ensemble, ces données suggèrent que ces altérations au niveau des Ob OA pourraient être responsables de la structure osseuse anormale observée chez les patients OA. / Osteoarthritis (OA) is a disease affecting joints and it has a very strong incidence in the population. It is characterized by articular cartilage degradation, an abnormal bone remodelling cycle and subchondral bone sclerosis. The aetiology of this disease is still unknown although OA is now considered as a joint disease involving all the tissues of the joint. The importance of bone in OA pathogenesis is now considered as fundamental and thus bone represent an interesting target for treatments. Tomodensitometric studies have shown an abnormal structure and organisation of the bone tissue of OA patients. In parallel, primary human OA osteoblast (Ob) cultures grown from the tibiofemoral subchondral bone show an abnormal phenotype and a reduced mineralization in vitro. The Wnt signalling pathway, known primarily for its important role in embryogenesis, is of utmost importance in osteogenesis and it has been shown to regulate terminal Ob differentiation. Transforming growth factor-β1 (TGF-β1), known to act on proliferation and on early phases of the differentiation of Ob, is overexpressed by OA Ob and could also modulate the Wnt signalling activity. Furthermore, two populations of OA patients can be discriminated in vitro by the production of prostaglandins E2 (PGE2) of their Ob. In a study on colorectal cancer, PGE2 was shown to modulate the canonical Wnt signalling pathway. Our hypothesis is that canonical Wnt signalling is diminished in OA Ob. This reduction is responsible for the poor mineralization and the abnormal phenotype of human OA Ob. Furthermore, DKK2, overexpressed in OA Ob and whose expression is controlled by TGF-β1, is responsible for the diminution of Wnt signalling activity, and PGE2 can partly correct this situation. The general goal of this study is twofold: 1) to demonstrate the role of TGF-β1, DKK2 and of PGE2 in the Wnt signalling activity; 2) to demonstrate the link between TGF-β1 and DKK2 and their effect on the abnormal OA Ob phenotype. We have shown in this study that the canonical Wnt signalling pathway is altered in OA Ob and that this was responsible for the altered phenotype observed in OA Ob. Also, we have shown that among the mediators of the Wnt signalling pathway, the expression of antagonist Dickkopf-1 (DKK1) was similar between OA and normal Ob. In contrast, the antagonist DKK2 was overexpressed and the expression of the agonist Wnt7B was low in OA Ob. Moreover, PGE2 increased Wnt signalling activity in OA Ob. The inhibition of DKK2 expression also increased Wnt signalling activity and corrected the abnormal phenotype along with increasing the mineralization of OA Ob. The inhibition of TGF-β1 expression, also overexpressed in OA Ob, also resulted in the correction of the phenotype and increased the mineralization of OA Ob. Inhibiting TGF-β1 expression also led to DKK2 inhibition. As the contrary was not observed, this demonstrated that TGF-β1 could regulate DKK2 expression. To conclude, Wnt signalling is reduced in OA Ob and this is due to elevated DKK2 levels in these cells. High levels of TGF-β1 in OA Ob increased DKK2 expression which could be responsible, at least partially, for their altered phenotype. PGE2 was shown to also increase Wnt signalling activity in OA Ob. Taken together these data suggest that such alterations in OA Ob could be responsible for the abnormal bone structure observed in OA patients.

Healthy bone is bombarded with many different mechanical strain derived signals during normal daily activities. One of these signals is present as a direct connective tissue strain on the cells. However, there is also the presence of an electrically charged streaming potential during this straining. The electrical potential is created from the movement of charged fluid through the small bone porosities. To date, little focus has been applied to elucidating the possible synergistic effects of these two stimulants. The aim of this project was to evaluate the effects of mechanical strain and indirect electrical stimulation upon the development of bone forming osteoblast cells and any possible synergistic effects of the two stimulants. This aim was achieved by using a novel device, designed and developed with the capability of creating a cell substrate surface strain along with an exogenous electrical stimulant individually or at the same time. Proliferation and differentiation were determined as a measure of cellular development. The indirect electrical stimulation was achieved through the use of a pulsed electromagnetic field (PEMF) while the mechanical strain was produced from dynamic stretching of a deformable cell substrate. Strain and strain rate were modelled from recent studies proposing that relatively high frequency, low strain osteogenic mechanical stimulants are more indicative of what healthy bone would be experiencing during normal activities. The PEMF signal mimicked a clinically available bone growth stimulator signal. Results showed a PEMF stimulus on monolayers of SaOS-2 and MG-63 osteoblast-like cells leads to a depression in proliferation. A concomitant increase in alkaline phosphatase production was also observed for the SaOS-2 cultures, but not for the MG-63 cell line. It was hypothesised that this was due to the MG-63's lack of phenotypic maturity compared to the SaOS-2 cells. Mechanical strain of the cell substrate alone, at a relatively high frequency (5Hz) but small strain, did not significantly effect either cell proliferation or differentiation for the MG-63 cells. However, when the electrical and mechanical stimulants were combined a significant increase in cellular differentiation occurred with MG-63 cultures, revealing a possible synergistic effect of these two stimulants on the development of bone cells.

This body of work is concerned with the genetics of craniofacial morphology and specifically with that of the cranial sutures which form fibrous articulations between the calvarial bones. The premature fusion of these sutures, known as craniosynostosis, is a common developmental abnormality and has been extensively utilised here as a tool through which to study the genetics of suture morphogenesis and craniofacial diversity. Investigations began with a search for polymorphisms associated with normal variation in human craniofacial characteristics. Denaturing High-Performance Liquid chromatography was used to identify polymorphisms in two genes causative for craniosynostosis by analysing DNA from a large cohort of individuals from four ethnogeographic populations. A single nucleotide polymorphism in fibroblast growth factor receptor 1 was identified as being associated with variation in the cephalic index, a common measure of cranial shape. To further, and specifically, investigate the molecular processes of suture morphogenesis gene expression was compared between unfused and prematurely fusing/fused suture tissues isolated from patients with craniosynostosis. Two approaches, both utilising Affymetrix gene expression microarrays, were used to identify genes differentially expressed during premature suture fusion. The first was a novel method which utilised the observation that explant cells from both fused and unfused suture tissue, cultured in minimal medium, produce a gene expression profile characteristic of minimally differentiated osteoblastic cells. Consequently, gene expression was compared between prematurely fused suture tissues and their corresponding in vitro de-differentiated cells. In addition to those genes known to be involved in suture morphogenesis, a large number of novel genes were identified which were up-regulated in the differentiated in vivo state and are thus implicated in premature suture fusion and in vivo osteoblast differentiation. The second microarray study involved an extensive analysis of 16 suture tissues and compared gene expression between unfused (n=9) and fusing/fused sutures (n=7). Again, both known genes and a substantially large number of novel genes were identified as being differentially expressed. Some of these novel genes included retinol binding protein 4 (RBP4), glypican 3 (GPC3), C1q tumour necrosis factor 3 (C1QTNF3), and WNT inhibitory factor 1 (WIF1). The known functions of these genes are suggestive of potential roles in suture morphogenesis. Realtime quantitative RT PCR (QRT-PCR) was used to verify the differential expression patterns observed for 11 genes and Western blot analysis and confocal microscopy was used to investigate the protein expression for 3 genes of interest. RBP4 was found to be localised on the ectocranial surface of unfused sutures and in cells lining the osteogenic fronts while GPC3 was localised to suture mesenchyme of unfused sutures. A comparison between each unfused suture (coronal, sagittal, metopic, and lambdoid) demonstrated that gene expression profiles are suture-specific which, based on the identification of differentially expressed genes, suggests possible molecular bases for the differential timing of normal fusion and the response of each suture to different craniosynostosis mutations. One observation of particular interest was the presence of cartilage in unfused lambdoid sutures, suggesting a role for chondrogenesis in posterior skull sutures which have generally been thought to develop by intramembranous ossification without a cartilage precursor. Finally, the effects of common media supplements used in in vitro experiments to stimulate differentiation of calvarial suture-derived cells were investigated with respect to their ability to induce in vivo-like gene expression. The response to standard differentiation medium (ascorbic acid + β-glycerophosphate) with and without dexamethasone was measured by both mineralisation and matrix formation assays and QRT-PCR of genes identified in the above described microarray studies. Both media induced collagen matrix and bone nodule formation indicative of differentiating osteoblasts. However, the genes expression profiles induced by both media differed and neither recapitulated the levels and profiles of gene expression observed in vivo for cells isolated from both fused and unfused suture tissues. This study has implications for translating results from in vitro work to the in vivo situation. Significantly, the dedifferentiation microarray study identified differentially expressed genes whose products may be considered candidates as more appropriate osteogenic supplements that may be used during in vitro experiments to better induce in vivo-like osteoblast differentiation. This study has made a substantial contribution to the identification of novel genes and pathways involved in controlling human suture morphogenesis and craniofacial diversity. The results from this research will stimulate new areas of inquiry which will one day aid in the development of better diagnostics and therapeutics for craniosynostosis, and other craniofacial and more general skeletal abnormalities.