Magíster en ciencias médicas, mención biología celular / El Osteosarcoma (OS) es el tumor sólido maligno más frecuente en la infancia y la adolescencia, corresponde al 20% de todos los tumores óseos y al 5% de los cánceres pediátricos. Los tratamientos actuales logran tasas de sobrevida a 5 años de 75% en pacientes sin metástasis, mientras que el 80% de los pacientes con enfermedad metastásica recaen a pesar del tratamiento efectuado. Runx2 es un factor de transcripción maestro que regula el proceso de diferenciación osteogénica, aunque este factor también promueve progresión tumoral en células de cáncer de próstata y de mama. Recientemente, se demostró la amplificación del gen Runx2 en pacientes con OS, así como la expresión aumentada de la proteína Runx2 en líneas celulares de OS. En esta tesis se propuso la hipótesis que el factor de transcripción Runx2 promueve migración e invasión en líneas celulares de osteosarcoma. Así, el objetivo general del proyecto fue definir el rol de Runx2 en los procesos de progresión tumoral en osteosarcoma. Para estudiar el rol de Runx2 sobre parámetros de progresión tumoral en líneas celulares de osteosarcoma, se moduló la expresión de Runx2 y se estudió su efecto en migración e invasividad. Nuestros resultados muestran que la sobreexpresión de Runx2 mediante adenovirus produjo un aumento en la capacidad de migración e invasión de líneas celulares de OS con bajos niveles de Runx2. El silenciamiento de Runx2 mediante siRNA disminuyó la capacidad de migración e invasión en líneas celulares de OS. De esta forma, hemos demostrado por primera vez que Runx2 promueve progresión tumoral en líneas celulares de OS. / Osteosarcoma (OS) is the most common malignant solid tumor in childhood and adolescence and represents 20% of all bone tumors and 5% of childhood cancers. Survival rates after five years are at 75% in patients without metastases, while 80% of patients with metastatic disease relapse despite the treatment. Runx2 is a master transcription factor that regulates the osteogenic differentiation process. However, this factor also functions as tumor promoter in prostate cancer cells and breast cancer. Runx2 gene amplification in patients with OS, as well as increased expression of Runx2 protein in OS cell line have recently been demonstrated. To this project, we hypothesized that the transcription factor Runx2 promotes migration and invasion in OS cell lines. Thus, the general aim was to define the role of Runx2 in osteosarcoma tumor progression processes. To study the role of Runx2 on parameters involved in tumor progression of OS cell lines, we modulated the expression of Runx2 and studied the outcome of this modifications on cell migration and invasiveness. Our results demonstrated that overexpression of Runx2 by adenovirus in OS cell lines with low levels of Runx2 increased cell migration and invasion. Runx2 silencing by siRNA in OS cell lines decreased their ability to migrate and invade. Thus, we have demonstrated for the first time that Runx2 promotes tumor progression in OS cell lines.

Die Osteoarthrose ist eine degenerative Erkrankung der Gelenke. Sie zeichnet sich durch eine sukzessive Destruktion des Knorpels sowie der umliegenden Strukturen aus. Die Osteoarthrose ist die häufigste Gelenkerkrankung des Menschen und tritt insbesondere im hohen Lebensalter auf. Schätzungen zu Folge wird Osteoarthrose im Jahr 2020 zu der viert häufigsten Ursache für Erwerbsunfähigkeit aufsteigen. Neben dem Leidensdruck für den Patienten ist dies auch mit erheblichen Kosten für das Gesundheitssystem verbunden. Die natürliche Regeneration des avaskulären hyalinen Knorpels ist aufgrund der geringen Zelldichte und dem niedrigen Metabolismus der Zellen beschränkt. Es fehlt ein Perichondrium das den Knorpel stabilisiert und mit undifferenzierten Mesenchymzellen, als Quelle für frische Chondroblasten bzw. Chondrozyten, versorgt. Das fibrokartilaginäre Ersatzgewebe im erkrankten artikulären Knorpel beherbergt eine Population von Vorläuferzellen mit chondrogenen Eigenschaften. Die Herkunft dieser Zellen ist wenig erforscht. Es wird vermutet, dass diese durch Einsprossungen von Blutgefäßen aus dem subchondralen Knochen in die beschädigten Areale migrieren. Diese chondrogenen Progenitorzellen (CPCs) könnten sich als zugänglich für medikamentöse Therapien zur Stimulation ihres Reperaturpotentials erweisen. In dieser Arbeit sollten Coaktivatoren von dem chondrogenen Transkriptionsfaktor SOX9 oder Corepressoren von dem osteogenen Transkriptionsfaktor RUNX2 identifiziert werden. Dazu wurden CPCs verwendet, die durch siRNA-vermittelten Knockdown von RUNX2 in der Produktion von chondrogenen Faktoren stimuliert werden sollten. Auch der umgekehrte Versuch wurde durchgeführt. Durch den Knockdown von SOX9 sollten osteogene Faktoren stimuliert werden, die als Ziel inhibitorischer Therapien dienen könnten. Dabei konnte zunächst der antagonistische Zusammenhang zwischen SOX9 und RUNX2 in CPCs gezeigt werden. Der Knockdown von RUNX2 führte zu einer Hochregulation von SOX9 und damit zu einer Erhöhung des chondrogenen Potentials der Zellen. Umgekehrt führte der Knockdown von SOX9 zu einer Hochregulation von RUNX2 und damit zu einer Verringerung des chondrogenen Potentials der CPCs. Mithilfe massenspektrometrischer Analysen konnten aus Immunpräzipitationen und Pulldown-Isolaten von SOX9 und RUNX2 eine Vielzahl von Proteinen isoliert werden, die mit Signaltransduktionen und der Transkription in Verbindung stehen. Im Rahmen dieser Arbeit wurden einige der möglichen Coaktivatoren von SOX9 in CPCs überexprimiert und mögliche Corepressoren von SOX9 durch siRNA in ihrer Expression inhibiert. Die Überexpression von DDX5, HSPA8, RAB5C und YWHAE führte zu einer Zunahme der Genexpression von SOX9. Während HSPA8 auch eine Zunahme der Genexpression von RUNX2 bewirkte, führte YWHAE zu dessen Herabregulation und damit zu einer Erhöhung des chondrogenen Potentials von CPCs. Durch den Knockdown von LEMD2 und TMPO konnte ebenfalls eine Hochregulation von SOX9 erreicht werden. Ein Anzeichen für die Erhöhung des chondrogenen Potentials bot hierbei zusätzlich die erhöhte Expression der extrazellulären Bestandteile ACAN und die verminderte Expression von COL1A1. Darüber hinaus wurde zum ersten Mal die Interaktion zwischen SOX9 und DDX5 durch Coimmunpräzipitation untersucht. SOX9 konnte dabei copräzipitiert werden allerdings war der umgekehrte und damit der validierende Versuch nicht erfolgreich und bedarf weiterer Untersuchung, z. B. mithilfe einer anderen Methode wie Yeast-Two-Hybrid, um die tatsächliche Protein-Protein-Interaktion zu bestätigen bzw. um sie auszuschließen. Diese Ergebnisse zeigen, dass mithilfe der affnitätschromatographischen Aufreinigung von SOX9 sowie RUNX2 mögliche Interaktionspartner durch massenspektrometrische Analysen der isolierten Proteinkomplexe identifiziert werden können. Inwieweit diese Proteine direkt auf SOX9 einwirken oder Teil einer übergeordneten Transkriptionsmaschinerie sind muss in weiteren Studien untersucht werden, um die Frage zu beantworten, ob diese auch als potentielles Ziel für therapeutische Maßnahmen in Betracht kommen. Darüber hinaus muss die Frage geklärt werden, inwieweit sich die identifizierten Proteine auf die Expression von nachgeschalteten chondrogenen Faktoren auswirken. Diese Arbeiten könnten einen wichtigen Beitrag in der Entwicklung von neuen Therapeutika gegen Osteoarthrose leisten.

Runx2 is a transcription factor required for bone formation and osteoblastic differentiation during normal development and is implicated in metastatic disease during breast cancer progression. Runx2 is highly expressed in many metastatic breast cancers and breast cancer cell lines Knockdown of Runx2 in various breast cancer cell lines restores epithelial characteristics and reduces proliferation, migration, and invasion. However, the role of Runx2 in breast cancer progression from early to late stages is not well understood. The MCF10A derived breast cancer progression model provides the opportunity to study the role of Runx2 in a series of cell lines that progress from nearly normal, with low Runx2 levels, to highly metastatic and aggressive, with much higher Runx2 levels. To address if removal of Runx2 affects gene expression and what pathways it may influence, specifically focused on breast cancer progression, we knocked down Runx2 using an shRNA lentivirus. Depletion of Runx2 inhibits the expression of mesenchymal markers including N-cadherin, Fibronectin, and Vimentin. Despite this finding, functional characteristics including proliferation, migration, and invasion were minimally affected. Possible reasons for the difference in results compared to other cell systems are discussed. As an alternative approach, we have generated stable, inducible cell lines using CRISPRi dCas9-KRAB to target Runx2 and in the future will investigate the effects of Runx2 knockdown in these cells.

Breast cancers frequently metastasise to the skeleton where they cause osteolytic bone destruction. Effective treatment of bone metastasis remains a considerable clinical challenge. In the UK around 70% of the 12,000 patients that die from breast cancer annually have bone metastasis. Whilst existing therapies provide some pain relief, by limiting the tumour-mediated bone degradation, bone metastases are presently incurable. There is therefore an urgent need to develop therapies to prevent bone metastatic breast cancer. The transcription factor complex Runx2/CBFβ is a key regulator of bone development and is aberrantly expressed in breast cancer, leading to up-regulation of bone metastasis-associated genes. Previous work has demonstrated that Runx2/CBFβ determines the invasive phenotype of metastatic breast cancer cells and is required for the expression of metastatic genes. The Runx2/CBFβ complex also has a role in normal breast gene expression, activating expression of the milk protein β-casein in response to hormones. However, little is known about the normal role of Runx2/CBFβ in breast cells. The overarching aim of this project was to determine the role of Runx2/CBFβ in metastasis and identify the target genes that determine the metastatic phenotype. In order to understand the role of Runx2/CBFβ in breast cancer, initial experiments were performed to determine the role of Runx2/CBFβ in normal breast cells. A 3D culture system was established to examine the role of Runx2/CBFβ in regulating gene expression in non-cancerous differentiated epithelial breast cells. Attempts were also made to determine the Runx2/CBFβ target genes after lactogenic hormone stimulation. Unfortunately siRNA knockdown of Runx2 was incompatible with hormonal stimulation. However, 3D cell culture of normal mammary gland cell line HC11 showed Runx2 was expressed throughout the development of mammary acini structures. In addition the expression of CBFβ was confirmed in these cells. Having established the 3D culture system, experiments were subsequently performed to examine the role of CBFβ in the metastatic breast cancer cell line MDA-MB-231. These experiments demonstrated that depletion of CBF has a remarkable effect on the phenotype of the cells, leading to the development of mammary acini structures normally formed by non-cancerous breast cancer cell lines. Thus, depletion of CBF results in a reversion to an epithelial phenotype, suggesting that CBF is required to maintain the epithelial to mesenchymal transition (EMT). RT-PCR analysis also revealed changes in the expression of EMT marker genes. We also demonstrated that the EMT reversion could be rescued by re-expressing an inducible form of CBFβ. These data suggest that CBFβ is required to maintain the mesenchymal phenotype of metastatic breast cancer cells. Finally, a microarray analysis of MDA-MB-231 cells was performed to identify Runx2/CBFβ target genes that might contribute to the mesenchymal phenotype. Cells depleted of CBFβ and grown in 3D revealed reduced expression of IL11. This is known to be involved in bone remodelling. Inspection of the IL11 promoter revealed potential DNA binding sites which confirmed binding to Runx2 using EMSA.

Filamin A is a well-characterised cytoskeletal protein which regulates cell shape and migration by cross-linking with actin. Filamin A mutations cause a number of human developmental disorders, many of which exhibit skeletal dysplasia. However, the molecular mechanisms by which Filamin A affects skeletal development are unknown. The transcription factor Runx2 is a master regulator of osteoblast and chondrocyte differentiation. Data presented in this thesis show that Filamin A forms a complex with Runx2 in osteoblastic cell lines. Moreover, it is demonstrated that Filamin A is present in the nucleus in several cell lines, including those of osteoblastic origin. The data presented show that the Filamin A/Runx2 complex suppresses the expression of the gene encoding the matrix-degrading enzyme, matrix metalloproteinase-13 (MMP-13), which is an important osteoblastic differentiation marker. ChIP assays were employed to demonstrate that endogenously expressed Filamin A associates with the promoter of the MMP-13 gene. In addition, Filamin A is not only located in the nucleus but also in the nucleolus, an important nuclear compartment involved in ribosomal RNA (rRNA) transcription. Ribosomal DNA promoter-driven reporter assays, Filamin A-knockdown experiments and exogenous Filamin A transfections demonstrated that Filamin A and Runx2 can repress ribosomal gene expression activity. Importantly, Filamin A is recruited to the human ribosomal DNA promoter, suggesting its direct involvement in the regulation of rRNA transcription. These findings reveal a novel role of Filamin A in the direct regulation of ribosomal gene expression. Finally, by using microarray technology, changes in gene expression were identified when Filamin A was downregulated. Some of the differentially expressed genes were known orchestrators of bone development. The data presented in this thesis strengthen the link between Filamin A and bone development and provide a molecular rationale for how Filamin A, acting as a regulator of gene expression, might influence osteoblastic differentiation.

O objetivo deste estudo foi avaliar a resposta dos tecidos perirradiculares após perfuração de furca intencional e selamento com Biodentine (BD), agregado trióxido mineral (MTA) ou guta percha. Foram utilizados pré-molares de 3 cães, num total de 30 dentes, distribuídos em 3 grupos: experimental BD (n= 14), controle negativo (MTA) (n= 10) e controle positivo (guta percha) (n= 6), por um período de 120 dias. Radiograficamente foi analisada a área correspondente à perfuração de furca. Na análise histopatológica qualitativa foi avaliada a presença, ou não de tecido mineralizado no local da perfuração de furca e adjacências. Na análise histopatológica semi-quantitativa foram atribuídos escores para os parâmetros: presença ou ausência de tecido mineralizado, intensidade do processo inflamatório e reabsorção dos tecidos mineralizados. Na análise histopatológica quantitativa foi medida a espessura de tecido mineralizado na área de perfuração de furca. Foram realizados ensaios de imuno-histoquímica para os marcadores de mineralização: RANKL e osteoprotegerina (OPG). Ensaio de imunofluorescência indireta avaliou a expressão Runx-2 para a síntese de proteínas de mineralização. Os dados foram avaliados pelos testes qui-quadrado, teste exato de Fisher e teste de Mann Whitney, utilizando o programa estatístico Graph Pad Prism 6.0. Os grupos foram comparados entre sí pelo Teste de Kruskal Wallis com pós-teste de Dunn. O nível de significância adotado para todas as análises foi de 5%. Na análise radiográfica a (BD) apresentou melhor desempenho em relação ao MTA, em todos os aspectos analisados. Histologicamente, tanto o MTA quanto a BD induziram a formação de tecido mineralizado, quando comparado à guta percha, que não induziu a formação de tecido mineralizado (p<.001). O selamento completo das perfurações de furca foi mais frequente com o MTA, que induziu a deposição de tecido mineralizado com área e espessura maiores. Tanto as amostras seladas com BD, quanto com MTA, não apresentaram reabsorção óssea em área de furca, apresentaram poucas células inflamatórias e maior intensidade do imunomarcador RUNX2 quando comparadas com a guta percha. A OPG esteve presente em amostras seladas com BD e com MTA. Embora o MTA tenha apresentado maior frequência de selamento completo e maior área e espessura de tecido mineralizado recém-formado, a BD também apresentou bons resultados histopatológicos e pode ser considerada como um adequado material de selamento de perfuração de furca. / The objective of this study was to evaluate the periradicular tissue response after intentional furcation and sealing with Biodentine (BD), mineral trioxide aggregate (MTA) or gutta percha. Pre-molars of 3 dogs were used, in a total of 30 teeth, distributed in 3 groups: experimental BD (n = 14), negative control (MTA) (n = 10) and positive control (gutta percha), for a period of 120 days. The area corresponding to furcation was analyzed radiographically. In the qualitative histopathological analysis, the presence or not of mineralized tissue at the furcation site and adjacent areas was evaluated. In the semi-quantitative histopathological analysis, scores were assigned to the parameters: presence or absence of mineralized tissue, intensity of the inflammatory process and reabsorption of mineralized tissues. In the quantitative histopathological analysis the thickness of mineralized tissue in the furcation area was measured. Immunohistochemical assays were performed for the mineralization markers: RANKL and osteoprotegerin (OPG). Indirect immunofluorescence assay evaluated RUNX-2 expression for the synthesis of mineralization proteins. Data were evaluated by chi-square test, Fisher exact test and Mann Whitney test using the Graph Pad Prism 6.0 statistical software. The groups were compared by the Kruskal Wallis test with Dunn\'s post-test. The level of significance adopted for all analyzes was 5%. In the radiographic analysis the (BD) presented better performance in relation to the MTA, in all aspects analyzed. Histologically, both MTA and BD induced the formation of mineralized tissue when compared to gutta percha, which did not induce the formation of mineralized tissue (p <.001). The complete sealing of furcation holes was more frequent with the MTA, which induced the deposition of mineralized tissue with a larger and thickness area. Both the BD and MTA sealed samples did not show bone resorption in the furcation area, showed few inflammatory cells and a greater intensity of the RUNX2 immunomarker when compared to the gutta percha. OPG was present in samples sealed with BD and with MTA. Although the MTA presented higher frequency of complete sealing and greater area and thickness of newly formed mineralized tissue, BD also presented good histopathological results and can be considered as a suitable furcation perforation sealing material.

O objetivo deste estudo foi avaliar a resposta dos tecidos perirradiculares após perfuração de furca intencional e selamento com Biodentine (BD), agregado trióxido mineral (MTA) ou guta percha. Foram utilizados pré-molares de 3 cães, num total de 30 dentes, distribuídos em 3 grupos: experimental BD (n= 14), controle negativo (MTA) (n= 10) e controle positivo (guta percha) (n= 6), por um período de 120 dias. Radiograficamente foi analisada a área correspondente à perfuração de furca. Na análise histopatológica qualitativa foi avaliada a presença, ou não de tecido mineralizado no local da perfuração de furca e adjacências. Na análise histopatológica semi-quantitativa foram atribuídos escores para os parâmetros: presença ou ausência de tecido mineralizado, intensidade do processo inflamatório e reabsorção dos tecidos mineralizados. Na análise histopatológica quantitativa foi medida a espessura de tecido mineralizado na área de perfuração de furca. Foram realizados ensaios de imuno-histoquímica para os marcadores de mineralização: RANKL e osteoprotegerina (OPG). Ensaio de imunofluorescência indireta avaliou a expressão Runx-2 para a síntese de proteínas de mineralização. Os dados foram avaliados pelos testes qui-quadrado, teste exato de Fisher e teste de Mann Whitney, utilizando o programa estatístico Graph Pad Prism 6.0. Os grupos foram comparados entre sí pelo Teste de Kruskal Wallis com pós-teste de Dunn. O nível de significância adotado para todas as análises foi de 5%. Na análise radiográfica a (BD) apresentou melhor desempenho em relação ao MTA, em todos os aspectos analisados. Histologicamente, tanto o MTA quanto a BD induziram a formação de tecido mineralizado, quando comparado à guta percha, que não induziu a formação de tecido mineralizado (p<.001). O selamento completo das perfurações de furca foi mais frequente com o MTA, que induziu a deposição de tecido mineralizado com área e espessura maiores. Tanto as amostras seladas com BD, quanto com MTA, não apresentaram reabsorção óssea em área de furca, apresentaram poucas células inflamatórias e maior intensidade do imunomarcador RUNX2 quando comparadas com a guta percha. A OPG esteve presente em amostras seladas com BD e com MTA. Embora o MTA tenha apresentado maior frequência de selamento completo e maior área e espessura de tecido mineralizado recém-formado, a BD também apresentou bons resultados histopatológicos e pode ser considerada como um adequado material de selamento de perfuração de furca. / The objective of this study was to evaluate the periradicular tissue response after intentional furcation and sealing with Biodentine (BD), mineral trioxide aggregate (MTA) or gutta percha. Pre-molars of 3 dogs were used, in a total of 30 teeth, distributed in 3 groups: experimental BD (n = 14), negative control (MTA) (n = 10) and positive control (gutta percha), for a period of 120 days. The area corresponding to furcation was analyzed radiographically. In the qualitative histopathological analysis, the presence or not of mineralized tissue at the furcation site and adjacent areas was evaluated. In the semi-quantitative histopathological analysis, scores were assigned to the parameters: presence or absence of mineralized tissue, intensity of the inflammatory process and reabsorption of mineralized tissues. In the quantitative histopathological analysis the thickness of mineralized tissue in the furcation area was measured. Immunohistochemical assays were performed for the mineralization markers: RANKL and osteoprotegerin (OPG). Indirect immunofluorescence assay evaluated RUNX-2 expression for the synthesis of mineralization proteins. Data were evaluated by chi-square test, Fisher exact test and Mann Whitney test using the Graph Pad Prism 6.0 statistical software. The groups were compared by the Kruskal Wallis test with Dunn\'s post-test. The level of significance adopted for all analyzes was 5%. In the radiographic analysis the (BD) presented better performance in relation to the MTA, in all aspects analyzed. Histologically, both MTA and BD induced the formation of mineralized tissue when compared to gutta percha, which did not induce the formation of mineralized tissue (p <.001). The complete sealing of furcation holes was more frequent with the MTA, which induced the deposition of mineralized tissue with a larger and thickness area. Both the BD and MTA sealed samples did not show bone resorption in the furcation area, showed few inflammatory cells and a greater intensity of the RUNX2 immunomarker when compared to the gutta percha. OPG was present in samples sealed with BD and with MTA. Although the MTA presented higher frequency of complete sealing and greater area and thickness of newly formed mineralized tissue, BD also presented good histopathological results and can be considered as a suitable furcation perforation sealing material.

Mesenchymale Stammzellen können sich durch den Einfluss verschiedener Transkriptionsfaktor zu Osteoblasten, Adipozyten, Chondrozyten oder Myoblasten differenzieren. Während sie sich unter Runx2-Einfluss entlang der osteoblastären Linie differenzieren, entwickeln sie sich bei vorliegendem PPARγ entlang des adipogenen Differenzierungswegs. Das Gleichgewicht zwischen beiden Faktoren und ihr Zusammenspiel stellen einen wichtigen Bereich in der Osteoporoseforschung dar. In dieser Dissertation wurde durch Runx2-Suppression bzw. Runx2-Überexpression die Rolle dieses Faktors in pHOB und SCP1-Zellen erfasst und die Interaktion zwischen Runx2 und PPARγ untersucht. Der Runx2-Knockdown’ erfolgte mittels RNA-Interferenz, die Runx2-Überexpression durch ein Runx2 exprimierendes Plasmid. In RT-PCRs wurden mRNA-Messungen durchgeführt. Die Proteinbestimmung erfolgte im ‚Westernblot’. Der funktionelle Einfluss der Runx2-Überexpression auf die PPARγ-Transkription wurde durch Kotransfektion des an Luziferase gekoppelten PPARg-Promotorgens erfasst. Die funktionelle Aktivität des PPARg-Proteins wurde durch die Transfektion des an Luziferase gekoppelten PPRE-Gens gemessen. Promotoraktivität und Funktionalität der Proteine wurden in Luziferase-Reportergenassays erfasst. Unter basalen Kulturbedingungen differenzierten sich pHOB osteogen. Durch zweimalige siRunx2-Transfektion gelang auf mRNA-Ebene eine suffiziente Runx2-Suppression über 29 Tage auf durchschnittlich 10,1%. Neben einer Steigerung der PPARγ-mRNA nach sieben Tagen konnte darunter auch eine Suppression der osteogenen Differenzierungsmarker OC und AP beobachtet werden. Ein ‚Rescue’ der supprimierten Runx2-Genexpression konnte durch osteogene Stimulation nicht erreicht werden. In den Runx2-/PPARγ-Interaktionsversuchen wurden SCP1-Zellen adipogen stimuliert, um die PPARγ2-mRNA und PPARγ-Promotoraktivität zu erhöhen. Darunter konnte ebenfalls eine gesteigerte Funktionalität des PPARγ-Proteins beobachtet werden. Durch Runx2-Überexpression wurde in SCP1-Zellen die PPARγ-Promotoraktivität und somit der Beginn der mRNA-Synthese gehemmt. Die PPARγ2-mRNA hingegen blieb unbeeinflusst. Die zentrale Rolle des Runx2 in der osteogenen Differenzierung scheint durch den Einfluss auf die osteogenen Marker OC und AP in pHOB bestätigt zu werden. Auch der Einfluss auf die adipogene Differenzierung erfolgt über Runx2. Im Rahmen dieser Dissertation konnte erstmalig die Hemmung des PPARγ-Promotors durch Runx2 beschrieben werden. Hierdurch werden die PPARγ-Transkription und somit voraussichtlich die Interaktion zwischen Adipogenese und Osteogenese beeinflusst.

Bone tissue engineering strategies are currently being developed as alternative mechanisms to address the clinical demand for bioactive and biomechanical graft material. To date, these efforts have been largely restricted by inadequate supply of committed osteoprogenitor cells and loss of osteoblastic phenotype expression following in vitro culture and expansion. The objective of this thesis research was to address the cell sourcing limitations of tissue-engineered bone grafts through constitutive and sustained overexpression of the osteoblast-specific transcriptional activator Runx2/Cbfa1 in osteogenic marrow-derived stromal cells using retroviral gene delivery. Runx2 overexpression enhanced expression of multiple osteoblastic genes proteins and, more importantly, significantly up-regulated matrix mineralization in both monolayer culture and following cell seeding in 3-D polymeric scaffolds. To evaluate in vivo performance, Runx2-expressing cells were seeded into 3-D constructs and implanted both subcutaneously and in a critical size craniotomy bone defect model. Notably, in vitro pre-culture of Runx2-transduced cell-seeded constructs prior to implantation significantly enhanced their capacity to form mineralized tissue in the subcutaneous space and induce new bone formation in the critical size defect model compared to control cells. The described series of analyses provided a novel combination of tissue and genetic engineering techniques toward the development of a Runx2-modified stromal cell/polymeric scaffold composite tissue-engineered bone graft substitute.

京都大学 / 新制・論文博士 / 博士(医学) / 乙第13462号 / 論医博第2249号 / 新制||医||1055(附属図書館) / (主査)教授 萩原 正敏, 教授 松田 秀一, 教授 齊藤 博英 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM