Indiana University School of Dentistry / Introduction: The goal of nonsurgical root canal therapy is to reduce the bacterial load within an infected root canal system, and the subsequent objective is to prevent or treat apical periodontitis. Clinical studies have shown more expedient healing of apical periodontitis treated with electromagnetic wave stimulation (EMS) as compared to apical periodontitis not treated with EMS. Stimulation of osteoblasts and growth factors has been shown when EMS was applied to rat calvaria, resulting in increased bone healing. Objective: The purpose of this vitro study was to evaluate the effects of EMS on the proliferation and differentiation of osteoblasts. Using primary neonatal calvaria osteoblast-lineage cells, the effects of different EMS regimens on proliferation, alkaline phosphatase (ALP) activity, and mineral deposition were determined. Materials and Methods: EMS regimen included currents of 0mA, 0.1mA, 1mA, and 10mA delivered for five consecutive 1s pulses per day for one, two, and three days. Cell proliferation was assayed after 1 or 2 days using an MTS assay. Alkaline phosphatase activity and mineral deposition were assayed after culturing the cells in osteogenic media containing ascorbic acid and -glycerol phosphate for 7 days. Comparisons were performed using analysis of variance, with a 5% significance level. Results: There was no statistically significant differences noted in MTS proliferation and mineral deposition between the experiment EMS treatment groups of 0.1, 1.0, and 10.0 mA compared to the control group of 0 mA current on calvaria-derived osteoblast. While there were no statistically significant differences noted in ALP activity in the 0.1, and 1.0 mA EMS groups, compared to 0 mA control, alkaline phosphatase activity was significantly increased in the 10 mA EMS group. Conclusion: There was no significant differences in MTS proliferation and mineral deposition of the EMS group compared to the control group. However, 10 mA EMS favored increased ALP activity suggesting EMS can promote matrix maturation by osteoblasts. Additional in vitro experimental studies, including different stem cell populations, culture duration and EMS treatment regimens are needed to understand the mechanism of action of EMS for future applications in regenerative endodontics.

O crescimento, desenvolvimento e manutenção do tecido ósseo são processos altamente regulados. Diversas proteínas como hormônios, fatores de crescimento e citocinas estão envolvidas nestes processos e exercem atividade direta sobre células osteoblástica e osteoclástica, atuando em sua diferenciação e ativação metabólica. O processo de regeneração óssea é iniciado por fatores estimuladores locais como as proteínas morfogenética óssea (BMP Bone Morphogenetic Proteins). As BMPs são um produto do metabolismo dos osteoblastos, odontoblastos e de várias células tumorais, sendo armazenadas na forma de concentrados no osso, dentina e em células neoplásicas do osteossarcoma e de certos tumores odontogênicos, tais como: fibroma cementificante, cementoblastoma benigno, dentinoma, fibroma odontogênico e odontoma. Esclarecer os mecanismos que controlam a remodelação óssea é uma questão bastante relevante. Nesse sentido, as células-tronco mesenquimais têm despertado grande interesse devido ao seu potencial envolvimento no processo de reparo tissular. A obtenção de osteoblastos funcionais a partir de células-tronco mesenquimais tem sido utilizada na engenharia de tecidos e terapia celular. Desse modo, no presente trabalho foi realizada uma análise proteômica das proteínas envolvidas nas diversas fases de diferenciação osteoblástica de células-tronco mesenquimais de medula óssea de rato Wistar e humana, no sentido de obter maiores informações sobre a diferenciação celular e a biologia do tecido ósseo. Células-tronco mesenquimais obtidas de medula óssea foram cultivadas em meio osteogênico por diferentes períodos para obter células em diversas fases da diferenciação osteoblástica. Para análise proteômica foram utilizadas ferramentas como a estratégia de shotgun proteomics e quantificação relativa (iTRAQ - Isobaric Tag for Relative and Absolute Quantitation) para separação de proteínas e a espectrometria de massas para a identificação e quantificação relativa de proteínas e peptídeos. Neste contexto, os nossos resultados nos levam a concluir que: as CTMs de medula óssea de rato Wistar expressam genes que estão envolvidos na diferenciação osteogênica quando estimuladas in vitro formando matriz óssea no período de 14 dias, ou seja, o fator estimulante no microambiente é de fundamental importância; as CTMs de medula óssea humana apresentaram resultados semelhantes com as CTMs de ratos em nível genômico durante a diferenciação osteogênica, entretanto quando estimuladas in vitro formaram a matriz óssea no período de 21 dias; utilizando duas abordagens proteômicas, foi possível identificar proteínas importantes que estão envolvidas no processo de diferenciação. Mas cabe salientar que, embora tenham sido detectados genes que parecem envolvidos no processo de diferenciação, isso não teve reflexo no proteoma dessas células nos períodos de 7 e 14 dias da indução de diferenciação à osteogênese, o que indica que a maior parte da funcionalidade dessas células quanto aos outros processos biológicos estão preservados, como por exemplo a proliferação celular permaneceu sem grandes alterações. Isso indica que manipulações de isolamento, cultivo e indução da diferenciação dessas células não afetaram o proteoma, com aspectos positivos para a utilização de células-tronco mesenquimais em terapia celular. Do ponto de vista metodológico, esse trabalho abre perspectivas da utilização de estratégias proteômicas baseadas na marcação por isóbaros em combinação com separação de proteínas por eletroforese unidimensional SDS-PAGE para a análise de amostras biologicamente complexas e de quantidades limitadas de obtenção como células-tronco mesenquimais. O estudo da expressão de proteínas durante as fases de diferenciação osteoblástica de células-tronco mesenquimais de medula óssea deve refletir seu estado funcional e contribuir para o entendimento das diversas vias envolvidas no processo de diferenciação. / The growth, development and maintenance of bone tissue are highly regulated processes. Several proteins such as hormones, growth factors and cytokines are actively involved in these processes and exert direct activity on osteoblastic and osteoclastic cells, acting in their differentiation and metabolic activation. The process of bone regeneration is initiated by local stimulating factors as bone morphogenetic proteins (BMP). BMPs are a product of the metabolism of osteoblasts, odontoblasts and various tumor cells and is stored in the form of concentrates in bone, dentin and neoplastic cells of osteosarcoma and certain odontogenic tumors such as fibroma cementifying, cementoblastoma benign dentinoma, odontogenic fibroma and odontoma. Clarify the mechanisms that control bone remodeling is a very relevant issue. Accordingly, the mesenchymal stem cells have attracted great interest because of its potential involvement in the process of tissue repair. Obtaining functional osteoblasts from mesenchymal stem cells has been used in tissue engineering and cell therapy. Thus, this present work performed a proteomic analysis of proteins involved in various stages of osteoblast differentiation of mesenchymal stem cells from bone marrow of Wistar rat and human, in order to obtain more information on the biology of cell differentiation and bone tissue. Mesenchymal stem cells obtained from bone marrow were cultured in osteogenic medium for different periods to obtain cells at different stages of osteoblast differentiation. For proteomics analysis tools were used as the strategy of shotgun proteomics and relative quantification (iTRAQ - isobaric Tag for Relative and Absolute quantitation) for protein separation and mass spectrometry to identify proteins. In this context, our results take us to conclude that the MSCs of Wistar rat bone marrow express genes that are involved in osteogenic differentiation in vitro when stimulated to form bone matrix during the 14 days, ie stimulating factor in the microenvironment is of fundamental importance, the MSCs from human bone marrow showed similar results with rat MSCs at the genomic level during osteogenic differentiation, however, when stimulated in vitro formed bone matrix within 21 days, using two proteomic approaches, we could identify proteins important that are involved in the process of differentiation. But it should be noted that although it has been identified genes that seem involved in the process of differentiation, it was not reflected in the proteome of these cells at 7 and 14 days after induction of the osteogenic differentiation, indicating that most of the functionality of these cells and other biological processes are preserved, such as cell proliferation remained without major changes. This indicates that manipulations of isolation, culture and induction of differentiation of these cells did not affect the proteome, with positive aspects to the use of mesenchymal stem cells in cell therapy. From the methodological point of view, this work opens up the use of proteomic strategies based on the score for isobars in combination with protein separation by electrophoresis, one-dimensional SDS-PAGE for the analysis of complex biological samples and limited quantities of production as mesenchymal stem cells. The study of protein expression during stages of osteoblast differentiation of mesenchymal stem cells from bone marrow should reflect their functional status and contribute to the understanding of pathways involved in the process of differentiation.

As BMPs (Bone Morphogenetic Proteins) são membros da superfamília de proteínas TGF-β (Transforming Growth Factor β ), regulam o crescimento e diferenciação de vários tipos celulares em diversos tecidos, e algumas delas desempenham um papel crítico na diferenciação de células de origem mesenquimal em osteoblastos. Particularmente, rhBMP2 e rhBMP7, promovem osteoindução tanto \"in vitro\" como \"in vivo,\" sendo, ambas as proteínas utilizadas terapeuticamente em Ortopedia/Odontologia para reparo ósseo. A expressão diferencial de genes durante a osteodiferenciação de células C2C12 induzida por rhBMP2 e rhBMP7, foi analisada através de microarranjos de DNA, selecionando 31 genes, dos quais 24 foram validados por qPCR, 13 dos quais são relacionados à transcrição, quatro associados a algumas vias de sinalização celular e sete associados à matriz extracelular. Análise funcional destes genes permitirá conhecer, com maiores detalhes, os eventos moleculares que ocorrem durante a diferenciação osteoblástica de células C2C12 induzida por rhBMPs. Em paralelo, foi perseguida a super-expressão de rhBMP2 e rhBMP7 em células HEK293T, demonstrando-se a atividade de rhBMP7, induzindo osteodiferenciação \"in vitro\" e formação de osso \"in vivo\", demonstrando a viabilidade do objetivo de se produzir estas proteínas para futura aplicação como biofármacos no Brasil. / The BMPs (Bone Morphogenetic Proteins) are members of the TGF-β (Transforming Growth Factor β) superfamily of proteins, regulate growth and differentiation of various cell types in various tissues, and some play a critical role in differentiation of mesenchymal cells into osteoblasts. Particularly, rhBMP2 and rhBMP7, promote osteoinduction \"in vitro\" and \"in vivo\" and both proteins are used therapeutically in Orthopedics and Dentistry. The differential expression of genes during osteodifferentiation induced by rhBMP2 and rhBMP7 in C2C12 cells was analyzed through DNA microarrays, allowing the selection of 31 genes, of which 24 were validated by qPCR, 13 of which are related to transcription, four associated with cell signaling pathways and seven are associated with the extracellular matrix. Subsequent functional analysis of these genes should reveal more details on the molecular events which take place during C2C12 cells osteoblastic differentiation induced by rhBMPs In paralel, rhBMPs 2 and 7 were overexpressed in HEK293T cells and BMP7 activity to induce osteodifferentiation \"in vitro\" and bone formation \"in vivo\" was demonstrated, reinforcing the viability of our objective to produce these proteins for future application as biopharmaceuticals in Brazil.

Dental and orthopaedic implants are currently the solutions of choice for teeth and joint replacements with success rates continually improving, but they still have undesirable failure rates in patients who are compromised by disease or age, and who in many cases are the ones most in need. The success of titanium (Ti) implants depends on their ability to osseointegrate with the surrounding bone and this, in turn, is greatly dependent on the surface characteristics of the device. Advancements in surface analysis and surface modification techniques have improved the biological performance of metallic implants by mimicking the hierarchical structure of bone associated with regular bone remodeling. In this process, damaged bone is resorbed by osteoclasts, which produce resorption lacunae containing high microroughness generated after mineral dissolution under the ruffled border, as well as superimposed nanoscale features created by the collagen fibers left at the surface. Indeed, increasing Ti surface roughness at the micro and sub-microscale level has been shown to increase osteoblast differentiation in vitro, increase bone-to-implant contact in vivo, and accelerate healing times clinically. Recently, the clinical application of surface nanomodification of implants has been evaluated. Still, most clinically-available devices remain smooth at the nanoscale and fundamental questions remain to be elucidated about the effect of nanoroughness on the initial response of osteoblast lineage cells. Another property that could be used to control osteoblast development and the process of osseointegration is the electrical surface charge of implants. The presence of endogenous electrical signals in bone has been implicated in the processes of bone remodeling and repair. The existence of these native signals has prompted the use of external electrical stimulation to enhance bone growth in cases of fractures with delayed union or nonunion, with several in vitro and in vivo reports confirming its beneficial effects on bone formation. However, the use of electrical stimulation on Ti implants to enhance osseointegration is less understood, in part because of the lack of in vitro models that truly represent the in vivo environment. In addition, an aspect that has not been thoroughly examined is the electrical implication of implant corrosion and its effect on the surrounding tissue. Implants are exposed to extreme conditions in the body such as high pH during inflammation, and cyclic loads. These circumstances may lead to corrosion events that generate large electrochemical currents and potentials, and may cause abnormal cell and tissue responses that could be partly responsible for complications such as aseptic loosening of implants. Consequently, Ti implants with tailored surface characteristics such as nanotopography and electrical polarization, could promote bone healing and osseointegration to ensure successful outcomes for patients by mimicking the biological environment of bone without the use of systemic drugs. The objective of this thesis is to understand how surface nanostructural and electrical characteristics of Ti and Ti alloy surfaces may affect osteoblast lineage cell response in vitro for normal tissue regeneration and repair. Our central hypothesis is that combined micro/nanostructured surfaces, as well as direct stimulation of Ti surfaces with fixed direct current (DC) potentials, can enhance osteoblast differentiation.

O crescimento, desenvolvimento e manutenção do tecido ósseo são processos altamente regulados. Diversas proteínas como hormônios, fatores de crescimento e citocinas estão envolvidas nestes processos e exercem atividade direta sobre células osteoblástica e osteoclástica, atuando em sua diferenciação e ativação metabólica. O processo de regeneração óssea é iniciado por fatores estimuladores locais como as proteínas morfogenética óssea (BMP Bone Morphogenetic Proteins). As BMPs são um produto do metabolismo dos osteoblastos, odontoblastos e de várias células tumorais, sendo armazenadas na forma de concentrados no osso, dentina e em células neoplásicas do osteossarcoma e de certos tumores odontogênicos, tais como: fibroma cementificante, cementoblastoma benigno, dentinoma, fibroma odontogênico e odontoma. Esclarecer os mecanismos que controlam a remodelação óssea é uma questão bastante relevante. Nesse sentido, as células-tronco mesenquimais têm despertado grande interesse devido ao seu potencial envolvimento no processo de reparo tissular. A obtenção de osteoblastos funcionais a partir de células-tronco mesenquimais tem sido utilizada na engenharia de tecidos e terapia celular. Desse modo, no presente trabalho foi realizada uma análise proteômica das proteínas envolvidas nas diversas fases de diferenciação osteoblástica de células-tronco mesenquimais de medula óssea de rato Wistar e humana, no sentido de obter maiores informações sobre a diferenciação celular e a biologia do tecido ósseo. Células-tronco mesenquimais obtidas de medula óssea foram cultivadas em meio osteogênico por diferentes períodos para obter células em diversas fases da diferenciação osteoblástica. Para análise proteômica foram utilizadas ferramentas como a estratégia de shotgun proteomics e quantificação relativa (iTRAQ - Isobaric Tag for Relative and Absolute Quantitation) para separação de proteínas e a espectrometria de massas para a identificação e quantificação relativa de proteínas e peptídeos. Neste contexto, os nossos resultados nos levam a concluir que: as CTMs de medula óssea de rato Wistar expressam genes que estão envolvidos na diferenciação osteogênica quando estimuladas in vitro formando matriz óssea no período de 14 dias, ou seja, o fator estimulante no microambiente é de fundamental importância; as CTMs de medula óssea humana apresentaram resultados semelhantes com as CTMs de ratos em nível genômico durante a diferenciação osteogênica, entretanto quando estimuladas in vitro formaram a matriz óssea no período de 21 dias; utilizando duas abordagens proteômicas, foi possível identificar proteínas importantes que estão envolvidas no processo de diferenciação. Mas cabe salientar que, embora tenham sido detectados genes que parecem envolvidos no processo de diferenciação, isso não teve reflexo no proteoma dessas células nos períodos de 7 e 14 dias da indução de diferenciação à osteogênese, o que indica que a maior parte da funcionalidade dessas células quanto aos outros processos biológicos estão preservados, como por exemplo a proliferação celular permaneceu sem grandes alterações. Isso indica que manipulações de isolamento, cultivo e indução da diferenciação dessas células não afetaram o proteoma, com aspectos positivos para a utilização de células-tronco mesenquimais em terapia celular. Do ponto de vista metodológico, esse trabalho abre perspectivas da utilização de estratégias proteômicas baseadas na marcação por isóbaros em combinação com separação de proteínas por eletroforese unidimensional SDS-PAGE para a análise de amostras biologicamente complexas e de quantidades limitadas de obtenção como células-tronco mesenquimais. O estudo da expressão de proteínas durante as fases de diferenciação osteoblástica de células-tronco mesenquimais de medula óssea deve refletir seu estado funcional e contribuir para o entendimento das diversas vias envolvidas no processo de diferenciação. / The growth, development and maintenance of bone tissue are highly regulated processes. Several proteins such as hormones, growth factors and cytokines are actively involved in these processes and exert direct activity on osteoblastic and osteoclastic cells, acting in their differentiation and metabolic activation. The process of bone regeneration is initiated by local stimulating factors as bone morphogenetic proteins (BMP). BMPs are a product of the metabolism of osteoblasts, odontoblasts and various tumor cells and is stored in the form of concentrates in bone, dentin and neoplastic cells of osteosarcoma and certain odontogenic tumors such as fibroma cementifying, cementoblastoma benign dentinoma, odontogenic fibroma and odontoma. Clarify the mechanisms that control bone remodeling is a very relevant issue. Accordingly, the mesenchymal stem cells have attracted great interest because of its potential involvement in the process of tissue repair. Obtaining functional osteoblasts from mesenchymal stem cells has been used in tissue engineering and cell therapy. Thus, this present work performed a proteomic analysis of proteins involved in various stages of osteoblast differentiation of mesenchymal stem cells from bone marrow of Wistar rat and human, in order to obtain more information on the biology of cell differentiation and bone tissue. Mesenchymal stem cells obtained from bone marrow were cultured in osteogenic medium for different periods to obtain cells at different stages of osteoblast differentiation. For proteomics analysis tools were used as the strategy of shotgun proteomics and relative quantification (iTRAQ - isobaric Tag for Relative and Absolute quantitation) for protein separation and mass spectrometry to identify proteins. In this context, our results take us to conclude that the MSCs of Wistar rat bone marrow express genes that are involved in osteogenic differentiation in vitro when stimulated to form bone matrix during the 14 days, ie stimulating factor in the microenvironment is of fundamental importance, the MSCs from human bone marrow showed similar results with rat MSCs at the genomic level during osteogenic differentiation, however, when stimulated in vitro formed bone matrix within 21 days, using two proteomic approaches, we could identify proteins important that are involved in the process of differentiation. But it should be noted that although it has been identified genes that seem involved in the process of differentiation, it was not reflected in the proteome of these cells at 7 and 14 days after induction of the osteogenic differentiation, indicating that most of the functionality of these cells and other biological processes are preserved, such as cell proliferation remained without major changes. This indicates that manipulations of isolation, culture and induction of differentiation of these cells did not affect the proteome, with positive aspects to the use of mesenchymal stem cells in cell therapy. From the methodological point of view, this work opens up the use of proteomic strategies based on the score for isobars in combination with protein separation by electrophoresis, one-dimensional SDS-PAGE for the analysis of complex biological samples and limited quantities of production as mesenchymal stem cells. The study of protein expression during stages of osteoblast differentiation of mesenchymal stem cells from bone marrow should reflect their functional status and contribute to the understanding of pathways involved in the process of differentiation.

As BMPs (Bone Morphogenetic Proteins) são membros da superfamília de proteínas TGF-β (Transforming Growth Factor β ), regulam o crescimento e diferenciação de vários tipos celulares em diversos tecidos, e algumas delas desempenham um papel crítico na diferenciação de células de origem mesenquimal em osteoblastos. Particularmente, rhBMP2 e rhBMP7, promovem osteoindução tanto \"in vitro\" como \"in vivo,\" sendo, ambas as proteínas utilizadas terapeuticamente em Ortopedia/Odontologia para reparo ósseo. A expressão diferencial de genes durante a osteodiferenciação de células C2C12 induzida por rhBMP2 e rhBMP7, foi analisada através de microarranjos de DNA, selecionando 31 genes, dos quais 24 foram validados por qPCR, 13 dos quais são relacionados à transcrição, quatro associados a algumas vias de sinalização celular e sete associados à matriz extracelular. Análise funcional destes genes permitirá conhecer, com maiores detalhes, os eventos moleculares que ocorrem durante a diferenciação osteoblástica de células C2C12 induzida por rhBMPs. Em paralelo, foi perseguida a super-expressão de rhBMP2 e rhBMP7 em células HEK293T, demonstrando-se a atividade de rhBMP7, induzindo osteodiferenciação \"in vitro\" e formação de osso \"in vivo\", demonstrando a viabilidade do objetivo de se produzir estas proteínas para futura aplicação como biofármacos no Brasil. / The BMPs (Bone Morphogenetic Proteins) are members of the TGF-β (Transforming Growth Factor β) superfamily of proteins, regulate growth and differentiation of various cell types in various tissues, and some play a critical role in differentiation of mesenchymal cells into osteoblasts. Particularly, rhBMP2 and rhBMP7, promote osteoinduction \"in vitro\" and \"in vivo\" and both proteins are used therapeutically in Orthopedics and Dentistry. The differential expression of genes during osteodifferentiation induced by rhBMP2 and rhBMP7 in C2C12 cells was analyzed through DNA microarrays, allowing the selection of 31 genes, of which 24 were validated by qPCR, 13 of which are related to transcription, four associated with cell signaling pathways and seven are associated with the extracellular matrix. Subsequent functional analysis of these genes should reveal more details on the molecular events which take place during C2C12 cells osteoblastic differentiation induced by rhBMPs In paralel, rhBMPs 2 and 7 were overexpressed in HEK293T cells and BMP7 activity to induce osteodifferentiation \"in vitro\" and bone formation \"in vivo\" was demonstrated, reinforcing the viability of our objective to produce these proteins for future application as biopharmaceuticals in Brazil.

No description available.

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.

La cicatrisation de défauts osseux permet tout au plus une réparation de l'os et dans peu de cas, une régénération ad integrum. Le développement de biomatériaux issus de l'ingénierie tissulaire en vue d'une régénération osseuse est donc un enjeu majeur. Le but de cette étude a été d'évaluer si des peptides GXXPG issus de l'élastine sont capables de favoriser la différenciation ostéoblastique de cellules mésenchymateuses dérivées de la moelle osseuse humaine (CMMO) ainsi que la formation de la matrice osseuse et sa minéralisation. Pour y répondre, nous avons utilisés les lattis de collagène de type I (COL1). La contraction de lattis « flottants » (LF) stimule l'expression de marqueurs de l'ostéoblaste (Runx-2, BSP…) par les CMMO ainsi que la minéralisation de la matrice osseuse. Cette différenciation ostéoblastique est aussi associée à l'activation de la cascade MT1-MMP/MMP-2/MMP-13. Nous montrons ensuite que les peptides GXXPG stimulent de façon dose-dépendante l'expression de marqueurs ostéoblastiques comme Runx-2 via S-Gal. Sur « coating » de COL1, ils stimulent la différenciation ostéoblastique des CMMO, la formation de la matrice osseuse et sa minéralisation. Enfin, dans des conditions « inflammatoires » créées par l'ajout de plasminogène (Plg) exogène, ces peptides conservent une activité ostéogénique sous contraintes mécaniques ou non. Plg seul induit également la différenciation ostéoblastique. Bien que les peptides GXXPG stimulent la production d'enzymes à activité collagénolytique (MT1-MMP, MMP-1), la lyse des LF n'est pas significative. En conclusion, les peptides GXXPG apparaissent comme des biomolécules pharmacologiques prometteuses pour la régénération osseuse. / Bone healing leads in only a few cases to an ad integrum regeneration, but most often to an incomplete tissue repair. Thus, the development of new biomaterials from tissue engineering in order to promote bone regeneration is a major goal. The purpose of our study was to evaluate if GXXPG peptides, derived from elastin, are able to favor human bone marrow mesenchymal cells (HBMC) to mature osteoblasts and bone matrix formation and mineralization.To this end, we used type I collagen (COL1) lattices. Floating lattice (LF) contraction stimulates osteoblasts markers expression (Runx-2, BSP…) by HMBC and bone matrix mineralization. Osteoblast differentiation is also associated to MT1-MMP/MMP-2/MMP-13 proteolytic cascade activation. We then showed that GXXPG peptides stimulate osteoblast markers like Runx-2 in a dose-dependent manner, an effect which involves S-Gal receptor. On a type I collagen coating model, these peptides also promote CMMO differentiation into osteoblast, bone matrix formation and mineralization. Finally, under “inflammatory” conditions, which can be catalyzed by plasminogen (Plg) supplementation, these peptides keep their ability to induce osteogenic responses in HBMC, even under mechanical stress. Plg alone is also able to promote osteoblast differentiation. Although GXXPG peptides stimulate collagenolytic enzymes (MT1-MMP, MMP-1) production, collagen degradation in LF is not significant. To conclude, GXXPG peptides appear as promising pharmacological biomolecules in bone regeneration.

A major therapeutic challenge in musculoskeletal regenerative medicine is how to effectively replenish bone tissue lost due to pathological conditions such as fracture, osteoporosis, or rheumatoid arthritis. Mesenchymal stem cells are currently investigated for applications in bone-tissue engineering and human bone marrow-derived mesenchymal stem cells (hMSCs) could be a promising source for generation of tissue-engineered bone. However, the therapeutic potential of MSCs has not been fully exploited due to a lack of knowledge regarding the identity, nature, and differentiation of hMSCs. Epigenetic mechanisms regulating the chromatin structure as well as specific gene transcription are crucial in determination of stem cell differentiation. With the aim to systematically identify epigenetic factors that modulate MSC differentiation, the work in this thesis encompasses an approach to identify epigenetic mechanisms underlying, initiating, and promoting osteoblast differentiation, and the investigation of individual epigenetic modulators. Various osteogenic inducers were validated for differentiation of MSCs and an assay allowing assessment of differentiation outcome was developed. This assay was subsequently employed in knockdown experiments with lentiviral short hairpin RNAs and inhibitor screens with small molecules targeting putative druggable epigenetic modulator classes. This approach identified around 100 epigenetic modulator candidates involved in osteoblast differentiation, of these candidates approximately 2/3 downregulated and 1/3 upregulated alkaline phosphatase (ALP) activity. Serving as a proof-of-concept, orthogonal validation experiments employing locked nucleic acid (LNA) knockdown were performed to validate a subset of candidates. Two identified target genes were selected for further investigation. Bromodomain-containing protein 4 (BRD4) was identified as one component of epigenetic regulation; its inhibition led to a decrease in ALP expression, downregulation of key osteoblast transcription factors Runx2 and Osterix, as well as impaired bone matrix formation. Knockdown of lysine (K)-specific demethylase 1A (KDM1A/LSD1) upregulated ALP activity and treatment with a small molecule inhibitor targeting KDM1A led to an increase in ALP, RUNX2, and bone sialoprotein expression. Intriguingly, in a transgenic mouse model overexpressing Kdm1a a decrease in bone volume and bone mineral density was observed, thus supporting the hypothesis that KDM1A is a central regulator of osteoblast differentiation.