Conexinas são proteínas que compõem as junções comunicantes do tipo gap, e a diminuição na sua expressão tem sido relacionada com diversas alterações fisiológicas, entre elas algumas síndromes, malformações genéticas, o aumento da proliferação celular e a carcinogênese. Dentre as isoformas das conexinas presentes nos tecidos animais, a Cx43 é a mais abundante e a mais estudada, tendo a sua importância relatada in vivo em camundongos que tiveram um dos alelos de Cx43 deletado (Cx43+/-), devido a morte desses animais logo após o nascimento devido a malformações cardíacas. Considerando o fato de esse gene ser expresso em dezenas de tipos celulares, tivemos como objetivo avaliar os outros tecidos em busca de anomalias ocorridas durante o desenvolvimento, e a possível interferência do background gentético. Para isso acompanhamos dia-a-dia o último terço gestacional de camundongos de background C57BL/6 e CD1, avaliando histologica e morfologicamente os fetos em busca de anomalias nos animais Cx43+/- e Cx43-/- em relação aos animais Cx43+/+. Exceto pelo tecido ósseo, não encontramos alterações nos órgãos que expressam esse gene, bem como alterações causadas pelo refluxo de sangue causado pela malformação da válvula tricúspide. Durante a osteogênese, por meio da avaliação das costelas e tíbia, percebemos um retardo no desenvolvimento, que se agrava conforme a deficiência do gene Cx43. Percebemos nitidamente que o processo de diferenciação celular ocorre de maneira menos eficiente, atrasando processos como deposição de colágeno e de matriz óssea. Conclui-se que a Cx43 é importante para o desenvolvimento ósseo na fase fetal em camundongos. / Connexins are proteins that compose the gap junctions, and the reduction in its expression has been related with diverse physiological alterations, like some syndromes, malformations, the increase of the cellular proliferation and carcinogenesis. Among isoforms of the connexins in animal cells, the Cx43 is the most abundant and studied, having its importance been shown up in alive mice that had one allele of Cx43 (Cx43+/-) deleted. REAUME et al. related that Cx43-/- mice presented cardiac malformation and died immediately after birth. Considering the fact that this gene is expressed in many cell types, we evaluate the possibility of other tissues also to present alterations during the fetal development. Due to this, we studied the mouse development initiating in 12.5 to 19.5 DE (embryologic day) and evaluated the histology of C57BL/6 and CD1 mice searching for anomalies of Cx43+/- and Cx43-/- mice in relation to the Cx43+/+ animals. We did not find alterations in the main organs that express Cx43, nor alterations due to blood out flow related to cardiac malformations. We only found significant difference was the bones; through the evaluation of the ribs and tibia. It has been observed a delay in the development, that was more important in Cx43 knockout mice. We observed clearly that the process of cellular differentiation occurs in less efficient way, delaying processes as deposition of collagen and bone matrix. In conclusion, this study showed that Cx43 is important for bone development in mice.

Meng, Fanbiao. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 114-138). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

The current clinical treatment for bone deficiencies in clinics includes autografts, allografts and bone graft substitutes. All these treatments, however, still have various limitations. Therefore, in this project, we aim to design, synthesise and characterise a new series of novel polymer networks (PNs) to promote bone formation and offer a new therapeutic solution. An innovative polymerisation technique was used to synthesise the novel polymer-based materials. Various lactone monomers were applied with layered double hydroxides (LDHs) as the initiator for the polymerisation. Copolymerisation was used to control the final product degradation rate, microstructure and biocompatibility. SEM and XRD were used to confirm the formation of the 3D polymer microstructure, PNs indicated interconnected fibrous microstructure (thickness of the fibre ranged from 50-150 nm). Up to 90% polymer yield was achieved using thermogravimetric analysis. Both 1H and 13C nuclear magnetic resonance (NMR) were used to understand the effects of monomer combinations in the polymerisation. The effects of the materials on osteosarcoma cell line Saos-2 were investigated. Representative material compositions and their determined mass concentrations were applied using osteogenic culture medium with Saos-2. The PNs have indicated enhanced osteogenesis using varied assays. A poly(lactide-co-caprolactone) PN (monomer mass ratio 1:1) at 6.25 mg/ml, in particular, resulted in enhanced alkaline phosphate activity not only in growth medium but also in osteogenic medium. Moreover, it indicated the highest bone nodule production in area percentage compared to the other PN samples using Saos-2. Mg-substituted calcified extracellular matrix was formed by the use of PN materials. Therefore, this novel PN could have the possibility to be used as an acellular scaffold or a sprinkle-on powder local to defect site to enhance bone formation in vivo. In addition, this novel technology could be used as a scaffold for tissue engineering.

Osteogênese Imperfeita (OI) é uma doença da formação do colágeno que leva à baixa densidade mineral óssea e consequentemente a fraturas relacionadas ou não aos mínimos traumas. Pacientes com OI requerem acompanhamento médico regular, cirurgia corretiva, terapia periódica de medicamentos e fisioterapia, bem como as práticas específicas de cuidados diários. Além disso, na ocorrência de fraturas necessitam de imobilização que causam importante desconforto e podem gerar limitações a curto e longo prazo. Objetivo: Avaliar a Qualidade de Vida (QV) de crianças, adolescentes e adultos com OI e seus cuidadores. Métodos: Estudo prospectivo transversal utilizando diferentes questionários para avaliação da QV, de acordo com a idade e condição médica. Em crianças e adolescentes foi utilizado o Pediatric Quality of Life Inventory (PedsQLTM), em adultos foi aplicado The 36-Item Short form Health Survey Questionnaire (SF-36) e em cuidadores foi utilizado WHOQOL-Bref. A classificação socioeconômica foi realizada utilizando o questionário desenvolvido pela Associação Brasileira de Empresas de Pesquisa (ABEP). Para classificação clínica da OI os participantes foram agrupados em forma leve (tipo I) ou f moderada/grave (tipos III-V). A dor e o número de fraturas foram avaliados por auto relato. Resultados: Na avaliação de crianças e adolescentes a amostra foi composta por 52 pacientes com OI (com idade entre 5-17 anos); Em relação ao tipo de OI, 26 (50%) do tipo I, 13 (25%) do tipo IV, 12 (23,1%) do tipo III, e 1 (1,9%) do tipo V. A menor média dos domínios avaliados foi no domínio físico (62,07 ± 2,9), sendo observado diferença significativa entre OI leve versus moderada/grave (69,23 ± 3,3 vs 54,56 ± 4,4; p= 0,01), e correlacionada positivamente com a mobilidade (r= 0,43, p= 0,01) e negativamente com a dor (r= -0,33, p= 0,025). Na avaliação dos adultos com diagnóstico de OI a amostra foi composta por 31 indivíduos, com média de idade de 32,84 anos (± 12,11), destes 24 (77,4%) eram do sexo feminino. Em relação ao tipo de OI, 24 (77,4%) apresentavam OI tipo I, 2 (6,4 %) tipo III, 2 (6,4%) tipo IV, 3 (9,8%) tipo V. Com relação à QV, o escore que apresentou menor média foi de capacidade funcional (41,9±12,3) e o que apresentou melhor média foi a vitalidade (53,3±9,3). Quando comparados pela condição física, forma moderada/grave versus forma leve, o domínio de capacidade funcional continuou com a menor média (30,91), não sendo observada diferença significativa entre os grupos, por condição física. A variável dor mostrou correlação negativa nos escores capacidade funcional (p=0,04 r=-0,3), aspectos sociais (p=0,02 r=-0,4) e aspectos emocionais (p=0,02 r=-0.4). Foi testada a correlação das variáveis classe econômica e número de fraturas em relação aos escores de QV estudados, não sendo encontrada significância estatística. Em relação à avaliação dos cuidadores foram incluídos 24 cuidadores de 27 pacientes com OI, 10 com OI tipo I, 4 com tipo III e 13 com tipo IV. Dezoito dos cuidadores eram mães, dois também apresentavam diagnóstico de OI, e 22 cuidavam de um indivíduo com OI, as demais cuidavam de dois ou mais. As médias dos domínios avaliados foram 14,59 (±3,29) para o domínio físico, 13,80 (±2,8) para o domínio psicológico, 15,19 (±3,7) para o domínio relações sociais, e 12,87 (±2,9) para o domínio ambiental; a pontuação QV total foi de 14,16. Os domínios de QV não diferiram significativamente de acordo com o tipo de OI ou com o número de fraturas. O nível socioeconômico não se correlacionou significativamente com os domínios de QV avaliados. Conclusões: De um modo geral podemos observar que a OI interfere na avaliação da QV, tanto na avaliação de crianças e adolescentes quanto em adultos, principalmente no dom físico, embora não avaliada por questionário específico, e, este domínio resultou em menores médias nos indivíduos com formas moderadas e graves de OI. Observamos que a dor foi frequentemente relatada e foi uma das variáveis que mais interferiu nos domínios avaliados, isto reforça a importância do manejo e acompanhamento clínico desses pacientes. Em relação aos cuidadores a QV mostrou-se prejudicada em decorrência da doença daquele que recebe o cuidado. Ressaltamos que estudos adicionais são necessários para confirmar estes resultados e determinar quais os fatores que mais influenciam os domínios de QV. / Osteogenesis Imperfecta (OI) is a congenital disorder of collagen biosynthesis which leads to low bone mineral density and consequently fractures related or not to minimum trauma. Patients are often requiring regular medical assistance, surgery, periodic drug therapy and physiotherapy, as well as specific practices for daily care activities. Moreover, immobilization leads to significant discomfort and physical limitations both at short and long term follow-up. Objective: To evaluate quality of life (QoL) of caregivers as well as children, adolescents and adults with OI. Methods: Cross-sectional study using different questionnaires to evaluate QoL, according to age and clinical data. The Pediatric Quality of Life Inventory (PedsQLTM), the 36-Item Short Form Health Survey Questionnaire (SF-36) and WHOQOL-Bref were used for QoL assessment in children/adolescents, adults and caregivers, respectively. The socioeconomic classification was assessed using the Brazilian Association of Research Companies (ABEP) survey. OI was classified as mild (type I) or moderate to severe forms (III-V types). Pain and number of fractures were assessed by self-report questionnaires. Results: The children and adolescents sample consisted of 52 patients with OI (aged 5-17 years); twenty-six (50%) of type I, 13 (25%) of type IV, 12 (23.1%) of type III, and 1 (1.9%) of type V. Physical domain reached lowest means (62.07 ± 2.9), with significant differences between mild versus moderate to severe OI (69.23 ± 3.3 vs 54.56 ± 4.4, p = 0, 01), being positively correlated with mobility (r = 0.43, p = 0.01) and negatively with pain (r = -0.33, p = 0.025). The adults sample consisted of 31 OI subjects with a mean age of 32.84 years (± 12.11), of these 24 (77.4%) were female. Twenty-four (77.4%) had type I, 2 (6.4%) type III, 2 (6.4%) type IV and 3 (9.8%) type V OI. Functional capacity reached the lowest score (41.9 ± 12.3) and vitality the highest (53.3 ± 9.3). No significant difference between severity groups were observed in the functional capacity domain when compared for physical condition. Pain showed negative correlation with the functional capacity scores (p = 0.04 r = -0.3), social aspects (p = 0.02 r = -0.4) and emotional aspects (p = 0.02 r = -0.4). Economic class and number of fractures did not reached statistical significance when tested with QoL scores. Twenty-four caregivers of 27 patients were included, 10 with type I, 4 with type III and 13 with type IV OI. Eighteen caregivers were mothers, two diagnosed with OI, 22 took care of single patients, and the remainder took care of two or more. The means were 14.59 (± 3.29) for the physical domain, 13.80 (± 2.8) for the psychological domain, 15.19 (± 3.7) for the social relationships domain, and 12.87 (± 2.9) for the environmental domain; the total score QoL was 14.16. QoL domains did not differ significantly according to the type of OI or the number of fractures. The socioeconomic classification did not correlate with domains assessing QoL Conclusions: OI interferes in the assessment of QoL, both in the evaluation of children, adolescents and adults, especially in the physical component. Although not evaluated by specific questionnaires, the study shows lower means in individuals with moderate and severe forms of OI. Pain was commonly reported and it was the main factor influencing the domains. This reinforces the importance of management and clinical monitoring of these patients. Regarding caregivers, QoL assessment proved to be impaired due to the disease of their dependents. We emphasize that further studies are needed to confirm these results and to determine which factors most influence in QoL domains.

前言：間充質幹細胞容易擴增並且能分化為成骨細胞、軟骨細胞和脂肪細胞，並且能對炎症、感染和損傷做出反應，並且遷移到相應的組織部位。這些特性使間充質幹細胞成為骨骼組織工程學中非常重要的細胞來源。外周血間充質幹細胞是一種存在於血液中的間充質幹細胞，而主要的間充質幹細胞存在與骨髓中，被稱之為骨髓間充質幹細胞。在我們實驗室之前的研究中通過DNA微陣列發現外周血間充質幹細胞中很多基因的表達與骨髓間充質幹細胞有很大區別。這其中的一些基因可能參與調控間充質幹細胞的分化和歸巢，我們從中挑選了三個變化比較明顯的基因--CRBP1, N-cadherin和 SOX11做進一步研究。本研究的目的在於研究CRBP1, N-cadherin和 SOX11在骨髓間充質幹細胞分化和遷移中的作用及相關機理。 / 方法：培養的骨髓間充質幹細胞來源於6-8周大小的SD大鼠。細胞的表型經過多分化潛能測試（成骨分化，成脂分化和成軟骨分化）和流式細胞儀檢驗。克隆大鼠的CRBP1, N-cadherin和SOX11基因到慢病毒載體。而且還設計了針對CRBP1和 N-cadherin的shRNA及非特異性對照shRNA。慢病毒由暫態轉染293FT細胞產生。細胞遷移實驗採用了BD Falcon的細胞遷移系統（cell culture insert）。實驗採用了定量PCR、免疫共沉澱、western雜交和雙螢光報告檢驗。對於體內實驗，細胞經感染帶有不同基因的病毒後，種植到Si-TCP材料並移植到裸鼠皮下。8周後，收集樣品進行組織學和免疫組織學分析。最後，我們建立了大鼠的股骨開放式骨折模型，並在4天后將SOX11基因修飾的間充質幹細胞通過心臟注射打到大鼠體內。4周後，收集股骨骨折樣品並進行microCT、力學測試和組織學分析。 / 結果：CRBP1過表達能夠促進骨髓間充質幹細胞的成骨分化潛能，並能抑制其成脂分化。進一步的機理研究表明CRBP1可以通過與RXRα的蛋白相互作用抑制RXRα誘導的β-catenin降解，從而維持β-catenin和磷酸化-ERK1/2在較高的水準，導致間充質幹細胞成骨能力增強；N-cadherin過表達可以促進間充質幹細胞的遷移，但是卻通過下調β-catenin和磷酸化ERK1/2抑制其成骨分化。過表達SOX11可以通過增強BMP信號通路促進三系分化。SOX11還可以通過啟動CXCR4的表達來促進細胞遷移。最後，在大鼠的股骨開放骨折模型上通過系統注射，我們證明穩定過表達SOX11的間充質幹細胞遷移到骨折部位的數量明顯增加。這些細胞到達骨折部位以後可以起始骨痂的鈣化，促進骨折的修復。 / 結論：本研究證明CRBP1, N-cadherin 和SOX11具有調節骨髓間充質幹細胞遷移和/或分化的功能。這些基因也許會成為幹細胞治療的新靶點。系統注射SOX11基因修飾的骨髓間充質幹細胞對於骨折修復可能具有較好的療效。本研究初步研究了CRBP1, N-cadherin 和SOX11在間充質幹細胞中的作用，為探討以間充質幹細胞為基礎的組織工程的某些新臨床應用提供了一些線索。 / Introduction: Mesenchymal stem cells (MSCs) can be easily harvested, expanded, and have the capability of differentiating into osteoblasts, chondrocytes and adipocytes, and they can home to various tissues in response to stimuli such as inflammation, infection and injuries. MSCs are therefore valuable cell source for musculoskeletal tissue engineering. Peripheral blood-derived MSCs (PB-MSCs) are one kind of MSCs that reside in peripheral blood, whereas the main source of MSCs is bone marrow-derived MSCs (BM-MSCs). In our previous study, we found many genes were differentially expressed in the PB-MSCs compared to their counterpart BM-MSCs demonstrated by microarray analysis, among which the effects of CRBP1, SOX11 and N-cadherin on MSCs in terms of migration and differentiation are studied. / Methods: BM-MSCs and PB-MSCs were cultured from 6-8 weeks SD rats. The phenotypes of MSCs were characterized by tri-lineage (adipo-, osteo- and chondrogenic) differentiation and flow cytometry analysis. The genes encoding rat CRBP1, SOX11 and N-cadherin were cloned into lentiviral vectors respectively. shRNAs targeting CRBP1, N-cadherin, and one nonspecific shRNA were designed. Pseudo-lentivirus was produced by transient transfection of 293FT cells. Cell migration was examined using transwell insert culture system. Quantitative RT-PCR, CO-IP, western blot and dual-luciferase assay were employed in the studies. For in vivo study, MSCs transduced with different genes were seeded on Si-TCP scaffolds and implanted subcutaneously in nude mice. 8 weeks later, the samples were collected for histological and immunohistological analysis. Finally, an open femoral fracture model was established in 8-week old SD rats, SOX11-modified MSCs were injected at four days after fracture. At 4-week after MSCs injection, the femurs were collected for microCT, mechanical test and histological analysis. / Results: For CRBP1gene, our results showed that CRBP1 overexpression promoted osteogenic differentiation of BM-MSCs, while inhibited their adipogenic differentiation. We demonstrated that CRBP1 promoted osteogenic differentiation by inhibiting RXRα-induced β-catenin degradation through physical interactions, and maintaining β-catenin and pERK1/2 at higher levels. For N-cadherin gene, we found that N-cadherin overexpression promoted MSCs migration, and suppressed osteogenic potential of MSCs through inhibiting ERK and β-catenin signaling pathways. For SOX11 gene, we demonstrated that SOX11 overexpression enhanced the adipo-, osteo- and chondrogenic differentiation of BM-MSCs, through enhancing BMP signaling pathways. The migration capacity of BM-MSCs was also enhanced when Sox-11 was overexpressed, through activating CXCR4 expression. Finally, in the open femur fracture model we demonstrated that a larger number of SOX11-overexpressing BM-MSCs migrated to the fracture site, initiated earlier callus ossification and improved bone fracture healing quality. / Conclusions: This study demonstrated that CRBP1, N-cadherin and SOX11 gene can regulate the migration and/or differentiation potentials of BM-MSCs. These genes may become new therapeutic targets in stem cell therapy applications. Systemic administration of genetically modified SOX11-overexpressing BM-MSCs may be useful in promoting fracture healing. Overall, this study defined some unknown functions of CRBP1, N-cadherin and SOX11 in MSCs and shed the lights on some novel therapeutic implications for MSCs-based tissue engineering. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Xu, Liangliang. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 128-144). / Abstract also in Chinese. / Declaration --- p.i / Abstract --- p.ii / 摘要 --- p.v / Acknowledgements --- p.vii / Chapter 1 --- p.1 / Introduction --- p.1 / Chapter 1.1 --- Mesenchymal stem cells --- p.2 / Chapter 1.1.1 --- Characteristics of mesenchymal stem cells --- p.2 / Chapter 1.1.2 --- Bone marrow- and peripheral blood-derived MSCs --- p.4 / Chapter 1.1.3 --- Other tissue-derived MSCs --- p.5 / Chapter 1.2 --- Adipogenesis of MSCs --- p.6 / Chapter 1.3 --- Chondrogenesis of MSCs --- p.7 / Chapter 1.4 --- Osteogenesis of MSCs --- p.8 / Chapter 1.4.1 --- Regulators of osteogenesis --- p.9 / Chapter 1.4.2 --- Stratergies for improving bone tissue engineering --- p.11 / Chapter 1.5 --- Signaling pathways involved in osteogenesis --- p.13 / Chapter 1.5.1 --- ERK signaling pathway --- p.14 / Chapter 1.5.2 --- Wnt signaling pathway --- p.15 / Chapter 1.5.3 --- BMP signaling pathway --- p.17 / Chapter 1.6 --- Migration of MSCs --- p.20 / Chapter 1.7 --- Fracture healing --- p.22 / Chapter 1.8 --- Clinical application of MSCs --- p.23 / Chapter 1.8.1 --- BM-MSCs vs. PB-MSCs --- p.24 / Chapter 1.8.2 --- Autologous vs. Allogeneic MSCs transplantation --- p.25 / Chapter 1.9 --- Scope of the present study --- p.26 / Chapter 1.9.1 --- CRBP1 --- p.26 / Chapter 1.9.2 --- N-cadherin --- p.27 / Chapter 1.9.3 --- SOX11 --- p.27 / Chapter 1.10 --- Experimental scheme --- p.29 / Chapter 2 --- p.31 / Comparison between PB-MSCs and BM-MSCs --- p.31 / Chapter 2.1 --- Chapter introduction --- p.32 / Chapter 2.2 --- Materials and methods --- p.33 / Chapter 2.2.1 --- Cell culture --- p.33 / Chapter 2.2.2 --- Flow cytometry --- p.33 / Chapter 2.2.3 --- Adipogenic differentiation --- p.34 / Chapter 2.2.4 --- Osteogenic differentiation --- p.34 / Chapter 2.2.5 --- RNA Extraction and Real-time PCR --- p.34 / Chapter 2.3 --- Results --- p.35 / Chapter 2.3.1 --- Morphology of PB-MSCs --- p.35 / Chapter 2.3.2 --- Cellular surface markers of BM-MSCs and PB-MSCs --- p.36 / Chapter 2.3.3 --- Multi-differentiation potential of BM-MSCs and PB-MSCs --- p.38 / Chapter 2.3.4 --- Target genes expression in BM-MSCs and PB-MSCs --- p.39 / Chapter 2.4 --- Discussion and future work --- p.40 / Chapter 3 --- p.41 / Role of CRBP1 in Differentiation and Migration of MSCs --- p.41 / Chapter 3.1 --- Chapter introduction --- p.42 / Chapter 3.2 --- Materials and methods --- p.46 / Chapter 3.2.1 --- Chemicals --- p.46 / Chapter 3.2.2 --- Isolation and culture of BM-MSCs --- p.46 / Chapter 3.2.3 --- RNA Extraction and Real-time PCR --- p.47 / Chapter 3.2.4 --- Plasmid construction, transfection, production of lentivirus and infection --- p.48 / Chapter 3.2.5 --- Osteogenic differentiation --- p.50 / Chapter 3.2.6 --- Adipogenic differentiation --- p.50 / Chapter 3.2.7 --- Western blot --- p.51 / Chapter 3.2.8 --- Immunofluorescence labeling and fluorescence microscopy --- p.52 / Chapter 3.2.9 --- Cell migration assay --- p.52 / Chapter 3.2.10 --- Ectopic bone formation assay --- p.52 / Chapter 3.2.11 --- Statistical analysis --- p.53 / Chapter 3.3 --- Results --- p.53 / Chapter 3.3.1 --- Transducing BM-MSCs with lentivirus carrying CRBP1 or shRNAs --- p.53 / Chapter 3.3.2 --- CRBP1 accelerates osteogenesis of BM-MSCs via enhancing ERK1/2 and β-catenin pathways --- p.56 / Chapter 3.3.3 --- CRBP1 stabilizes β-catenin by inhibiting RXRα-induced degradation --- p.58 / Chapter 3.3.4 --- CRBP1 inhibits adipogenesis of BM-MSCs --- p.61 / Chapter 3.3.5 --- CRBP1 overexpression has no effect on MSCs migration potential --- p.63 / Chapter 3.3.6 --- CRBP1 promotes ectopic bone formation in vivo --- p.64 / Chapter 3.4 --- Discussion --- p.66 / Chapter 3.5 --- Future work --- p.73 / Chapter 4 --- p.74 / Role of N-cadherin in Differentiation and Migration of MSCs --- p.74 / Chapter 4.1 --- Chapter introduction --- p.75 / Chapter 4.2 --- Materials and methods --- p.78 / Chapter 4.2.1 --- Chemicals --- p.78 / Chapter 4.2.2 --- Isolation and culture of BM-MSCs --- p.78 / Chapter 4.2.3 --- Plasmid construction, transfection, production of lentivirus and infection --- p.79 / Chapter 4.2.4 --- Osteogenic differentiation and ALP activity assay --- p.81 / Chapter 4.2.5 --- Western blot --- p.81 / Chapter 4.2.6 --- Ectopic bone formation assay --- p.82 / Chapter 4.2.7 --- Statistical analysis --- p.82 / Chapter 4.3 --- Results --- p.83 / Chapter 4.3.1 --- Expression of N-cadherin during osteogenesis in MSCs --- p.83 / Chapter 4.3.2 --- N-cadherin overexpression inhibits osteogenesis through suppressing β-catein and ERK1/2 signaling pathways --- p.84 / Chapter 4.3.3 --- N-cadherin silencing increases osteogenesis through enhancing β-catenin and ERK1/2 signaling pathways --- p.86 / Chapter 4.3.4 --- N-cadherin promotes migration of MSCs --- p.87 / Chapter 4.3.5 --- Cellular surface markers of SV40-immortalized MSCs --- p.89 / Chapter 4.3.6 --- N-cadherin inhibits ectopic bone formation in vivo --- p.89 / Chapter 4.4 --- Discussion --- p.91 / Chapter 4.5 --- Future work --- p.94 / Chapter 5 --- p.96 / Role of SOX11 in Differentiation and Migration of MSCs --- p.96 / Chapter 5.1 --- Chapter introduction --- p.97 / Chapter 5.2 --- Materials and methods --- p.105 / Chapter 5.2.1 --- Plasmid construction, transfection, production of lentivirus and infection --- p.105 / Chapter 5.2.2 --- Cell culture --- p.106 / Chapter 5.2.3 --- Luciferase reporter gene assay --- p.106 / Chapter 5.2.4 --- Osteogenic differentiation and ALP activity assay --- p.106 / Chapter 5.2.5 --- Adipogenic differentiation --- p.107 / Chapter 5.2.5 --- Chondrogenic diffferentiation --- p.107 / Chapter 5.2.6 --- Western blot --- p.108 / Chapter 5.2.7 --- RNA Extraction and Real-time PCR --- p.108 / Chapter 5.2.8 --- Cell migration --- p.110 / Chapter 5.2.9 --- Ectopic bone formation --- p.110 / Chapter 5.2.10 --- Fracture healing model and analysis --- p.111 / Chapter 5.2.11 --- Statistical Analysis --- p.112 / Chapter 5.3 --- Results --- p.112 / Chapter 5.3.1 --- SOX11 is upregulated during osteogenesis of BM-MSCs --- p.112 / Chapter 5.3.2 --- SOX11 promotes adipogenesis in BM-MSCs --- p.113 / Chapter 5.3.3 --- SOX11 promotes migration of BM-MSCs --- p.114 / Chapter 5.3.4 --- SOX11 promotes osteogenesis in BM-MSCs --- p.115 / Chapter 5.3.5 --- SOX11 promotes chondrogenesis of MSCs --- p.117 / Chapter 5.3.6 --- Mechanisms of how SOX11 regulates differentiation and migration of MSCs --- p.118 / Chapter 5.3.7 --- SOX11-modified MSCs promote bone fracture healing in an open femur fracture rat model --- p.122 / Chapter 5.4 --- Discussion --- p.126 / Chapter 5.5 --- Future work --- p.131 / Appendix --- p.153

Orientador: Luana Marotta Reis de Vasconcellos / Banca: Yasmin Rodarte Carvalho / Banca: Renata Falchete do Prado / Resumo: Atualmente, as pesquisas em Implantodontia enfatizam modificações na superfície dos implantes, visando melhorar a osseointegração. Nesse sentido, o presente estudo teve como objetivo avaliar a influência da porosidade, bem como do tratamento biomimético (TB), na osteogênese in vivo em implantes de titânio. Foram confeccionados, por meio da metalurgia do pó, implantes de titânio puro grau 2, que foram divididos em seis grupos: a) G1: denso; b) G2: 40% de porosidade; c) G3: 50% de porosidade; d) G4: G1 + TB; e) G5: G2 + TB; f) G6: G3 + TB. Inicialmente, as amostras foram caracterizadas por análise metalográfica e por espectroscopia por energia dispersiva (EDS). Em seguida, os implantes foram inseridos nas tíbias de dezesseis coelhos da raça Nova Zelândia, para avaliar a reparação óssea peri-implantar, por meio de análise histológica e histomorfométrica. Como padronização, os implantes G1, G2 e G3 foram inseridos na tíbia direita e G4, G5 e G6 na esquerda. Quatro coelhos, selecionados aleatoriamente, foram submetidos à eutanásia, 01, 02, 04 e 08 semanas após a cirurgia. Os dados da análise metalográfica foram submetidos ao teste de Kruskal-Wallis e a análise histomorfométrica foi avaliada pelos testes ANOVA e Tukey. Os resultados da metalografia confirmaram porosidade de 40 e 50%, e revelaram poros com tamanho médio de 370 μm e interconexão entre os mesmos. Na análise por EDS foi observada a presença de íons Ca e P, caracterizando o sucesso do TB. Na análise histológica observou-se neoformação óssea, inclusive para o interior dos poros, independente do tipo de implante ou do tempo. Na análise histomorfométrica, verificou-se maior neoformação óssea em G5 e G6, e menor em G1, sendo que ambas as condições mostraram diferença estaticamente significante em relação aos demais grupos. Concluiu-se que o TB associado à porosidade exerce influência positiva na neoformação óssea. / Abstract: Researches in implantodontology have been recently focusing on implant surface modifications aiming to improve osseointegration. Thus, the purpose of the present study was to evaluate in vivo the influence of titanium implants surface porosity and surface biomimetic treatment (BT) on osteogenesis. Grade 2 pure titanium implants were fabricated by means of powder metallurgy and divided into six groups: a) G1: dense; b) G2: porosity of 40%; c) G3: porosity of 50%; d) G4: G1 + BT; e) G5: G2 + BT; and f) G6: G3 + BT. The samples were initially characterized by metallographic analysis and energy-dispersive spectroscopy (EDS). The implants were then placed into the tibia of sixteen New Zealand rabbits in order to evaluate the peri-implant bone repair by means of histological and histomorphometric analyses. As standardization, implants from groups G1, G2 and G3 were inserted into right tibias; while G4, G5 and G6 into left ones. Four randomly chosen rabbits underwent euthanasia 01, 02, 04 and 8 weeks after surgery. The results from metallographic analysis were analyzed by Kruskal-Wallis test and the histomorphometric results by ANOVA and Tukey tests. Metallographic results confirmed porosity between 40 and 50%, and revealed mean pore size of 370 μm and interconnection among them. EDS analysis detected the presence of Ca and P ions, confirming BT success. Bone neoformation, including into the pores, was detected by the histological analysis regardless the type of implant or evaluated period. By the histomorphometric analysis, bone neoformation was statistically greater for G5 and G6 and statistically lesser for G1 in comparison to the other groups. It was concluded that BT associated with porosity exerts positive influence on bone neoformation. / Mestre

Orientador: Maria Aparecida Neves Jardini / Banca: Carlos Alberto Alves Cairo / Banca: Caio Gorgulho Zanet / Banca: Luana Marotta Reis de Vasconcellos / Banca: Fernando Vagner Raldi / Resumo: Alterações nas propriedades físicas e químicas na superfície do titânio (Ti) buscam acelerar e melhorar a osseointegração. Além disso, a criação de substratos porosos, além de beneficiar fenômenos celulares, propiciaria uma maior resistência mecânica na interface osseointegrada devido ao crescimento de tecido ósseo para o interior dos poros. O objetivo deste estudo foi avaliar a influência da porosidade de amostras de Ti na osteogênese in vitro em amostras de titânio. Foram confeccionadas por metalurgia do pó, discos de titânio puro grau 2 com 12 mm de diâmetro e 3 mm de altura, que foram divididos em três grupos: a) G1: controle - titânio denso; b) G2: 30% de porosidade e poros com 300 μm; c) G3: 40% de porosidade e poros com 300 μm. Inicialmente, a superfície de 03 espécimes de cada grupo foi caracterizada por meio de analise metalográfica, visando confirmar a quantidade, área, morfologia e interligação dos poros. Para a realização da pesquisa, células osteogênicas obtidas da calvária de ratos recém-nascidos foram cultivadas sobre as amostras de cada grupo e avaliadas quanto à adesão celular, após 24 horas, proliferação e viabilidade celular, após 7, 10 e 14 dias. A diferenciação celular foi avaliada através da mensuração do conteúdo de proteína total, atividade da fosfatase alcalina e formação de matriz nodular mineralizada nos períodos de 7, 10 e 14 dias. Os resultados obtidos com os testes ANOVA e Tukey (5%) indicaram que houve adesão celular em todas as superfícies e que esta não interferiu nos resultados. As superfícies porosas (G2 e G3) favoreceram a proliferação celular quando comparadas as superfícies densas. O conteúdo de proteína total e a formação de matriz mineralizada foram significantemente maiores nas amostras de G3. Entretanto, não houve diferença estatística nos valores da atividade da fosfatase alcalina... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Changes in physical and chemical properties on the surface of titanium seek to accelerate and improve osseointegration. Furthermore, the creation of porous substrates, provide greater strength in the osseointegrated interface due to the bone ingrowth. The aim of this study was to evaluate the influence of porosity on osteogenesis in vitro in samples of titanium. Were made by the powder metallurgy, discs of pure titanium grade 2 with 12 mm diameter and 3 mm in height, which were divided into three groups: a) G1: control - titanium machined b) G2: 30% porosity and pore with 300 μm c) G3: 40% of porosity and pore size of 300 μm. Initially, the area of 03 specimens of each group was characterized by metallography analysis, to confirm the quantity, area, shape and interconnection of pores. To conduct the study, osteogenic cells derived from calvaria of newborn rats were cultured on the samples of each group and evaluated for adhesion, after 24 hours, proliferation and cell viability after 3, 7 and 10 days. The cell differentiation was assessed by measuring the total protein content, alkaline phosphatase activity and bone-like nodule formation at 7, 10 and 14 days. The results obtained with ANOVA and Tukey tests (5%) indicated that there was cell adhesion on all surfaces and it does not interfere in the results. The porous surfaces (G2 and G3) provide better results in cell proliferation when compared to smooth surfaces. The content of total protein and the formation of bonelike nodules were significantly higher in samples from G3. However, there was no statistical difference in the values of alkaline phosphatase activity between the experimental groups. These results suggest a correlation between the surface topography of the samples and the phenomena of adherence, proliferation and cell differentiation. / Doutor

Orientador: Luiz Gonzaga Gandini Júnior / Banca: Ana Claudia Moreira Melo / Banca: Ary dos Santos Pinto / Resumo: O objetivo deste trabalho foi avaliar, por meio de modelos de estudo, os efeitos da Distração Osteogênica Médio-Sagital da Mandíbula (DOMM) no arco mandibular de pacientes que utilizaram aparelhos dento-suportados. A amostra consistiu de 14 pacientes (5 do gênero masculino e 9 do gênero feminino) com idades variando de 13 a 19 anos e média de idade de 15,74 ± 1,89 anos no início do tratamento. Modelos de estudo foram obtidos de cada paciente no início do tratamento (T1), três meses após a DOMM (T2) e no momento da remoção dos aparelhos fixos (T3). Os dados foram submetidos à Análise de Variância e teste complementar de Duncan e demonstraram que todas as dimensões transversais aumentaram com a DOMM, o comprimento do arco não sofreu alteração estatisticamente significante, o perímetro do arco aumentou e o Índice de Irregularidade diminuiu consideravelmente, refletindo a correção do apinhamento ântero-inferior. Os dentes posteriores apresentaram maior expansão das cúspides do que das cervicais, indicando uma possível inclinação causada pelo uso de aparelho dento-suportado. Todas a medidas mostraram algum grau de recidiva, que variou de 2,8% a 8,84%. Os achados desta pesquisa permitiram concluir que a DOMM é uma alternativa eficaz para o tratamento do apinhamento associado à deficiência transversal de mandíbula. / Abstract: The purpose of this study was to evaluate the effects of Mandibular Symphyseal Distraction Osteogenesis (MSDO) with a tooth-borne distraction device, analysed in dental casts. The sample included 14 patients (5 males and 9 females) between 13 and 19 years of age, with a mean age of 15,74 ± 1,89 years at the start of treatment. Dental casts were obtained at the start of treatment (T1), three months after distraction (T2) and at the end of treatment, when fixed appliances were removed (T3). The data were analysed statistically using ANOVA and Duncan's test and showed that all transverse widths increased, arch length was maintained, arch perimeter increased significantly and Irregularity Index decreased, showing the correction of crowding. Molars and premolars had a disproportionate pattern of distraction, characterized by greater cusps expansion than cervical one. This pattern might be caused by the use of a tooth-borne distraction device. All the measurements demonstrated evidence of relapse, that varied from 2,8% to 8,84%. The findings of this study indicate that MSDO is an efficient treatment alternative for dental crowding and mandibular transverse deficiency. / Mestre

Objetivo: Avaliar as modificações estruturais do pericárdio bovino após hidrólise seletiva de grupos carboxiamidas em carboxílicos e resposta biológica em defeitos ósseos reparados com as matrizes de colágeno aniônico. Material e métodos: Amostras das matrizes modificadas foram comparadas com amostras de pericárdio bovino nativo através de microscopia óptica e de microscopia eletrônica de varredura. A resposta biológica foi avaliada implantando fragmentos das matrizes em trepanações criadas em tíbias de ratos. O reparo dos defeitos foi estudado 3, 7, 15 e 30 dias pós-implantação usando-se microscopia óptica. Resultados: O processo de hidrólise seletiva de grupos carboxamidas em carboxílicos removeu completamente as células intersticiais do pericárdio bovino e transformou a estrutura compacta e fibrilar do colágeno nativo em uma estrutura esponjiforme. Além disso, foi observado que as matrizes modificadas permitiram o reparo das trepanações ósseas com a formação de osso trabecular, sem induzir nenhuma reação adversa. Conclusões: A hidrólise utilizada aplicada ao pericárdio bovino produz uma matriz acelular com poros distribuídos na sua estrutura. Essas matrizes são biocompatíveis, não induzem reação inflamatória e permitem o reparo de trepanações ósseas experimentalmente induzidas. / Objective: Evaluate the structural modifications in the bovine pericardium after selective hydrolysis of carboxiamides groups into carboxilic and the biological response in bone defects repaired using the anionic collagen matrices. Material and methods: Samples of the modified matrices were compared to samples of native bovine pericardium using light microscopy and scanning electron microscopy. The biological response were evaluated implanting fragments of the matrices into defects made in rat tibias. The repair of the defects were evaluated 3, 7, 15, and 30 days after implantation using light microscopy. Results: The process of selective hydrolysis of carboxiamides groups into carboxilic completely removed the interstitial cells of the bovine pericardium and turned the compact and fibrilar structure of native collagen into a sponge-like structure. Besides, it was observed that the modified matrices allowed the repair of the bone defects through the formation of cancellous bones, without any side reaction. Conclusions: The used hydrolysis applied to bovine pericardium produces a acellular matrix with pores distributed along its structure. These matrices are biocompatible, they do not induce any inflammatory reaction and allow the repair of experimentally induced bone defects.

Conexinas são proteínas que compõem as junções comunicantes do tipo gap, e a diminuição na sua expressão tem sido relacionada com diversas alterações fisiológicas, entre elas algumas síndromes, malformações genéticas, o aumento da proliferação celular e a carcinogênese. Dentre as isoformas das conexinas presentes nos tecidos animais, a Cx43 é a mais abundante e a mais estudada, tendo a sua importância relatada in vivo em camundongos que tiveram um dos alelos de Cx43 deletado (Cx43+/-), devido a morte desses animais logo após o nascimento devido a malformações cardíacas. Considerando o fato de esse gene ser expresso em dezenas de tipos celulares, tivemos como objetivo avaliar os outros tecidos em busca de anomalias ocorridas durante o desenvolvimento, e a possível interferência do background gentético. Para isso acompanhamos dia-a-dia o último terço gestacional de camundongos de background C57BL/6 e CD1, avaliando histologica e morfologicamente os fetos em busca de anomalias nos animais Cx43+/- e Cx43-/- em relação aos animais Cx43+/+. Exceto pelo tecido ósseo, não encontramos alterações nos órgãos que expressam esse gene, bem como alterações causadas pelo refluxo de sangue causado pela malformação da válvula tricúspide. Durante a osteogênese, por meio da avaliação das costelas e tíbia, percebemos um retardo no desenvolvimento, que se agrava conforme a deficiência do gene Cx43. Percebemos nitidamente que o processo de diferenciação celular ocorre de maneira menos eficiente, atrasando processos como deposição de colágeno e de matriz óssea. Conclui-se que a Cx43 é importante para o desenvolvimento ósseo na fase fetal em camundongos. / Connexins are proteins that compose the gap junctions, and the reduction in its expression has been related with diverse physiological alterations, like some syndromes, malformations, the increase of the cellular proliferation and carcinogenesis. Among isoforms of the connexins in animal cells, the Cx43 is the most abundant and studied, having its importance been shown up in alive mice that had one allele of Cx43 (Cx43+/-) deleted. REAUME et al. related that Cx43-/- mice presented cardiac malformation and died immediately after birth. Considering the fact that this gene is expressed in many cell types, we evaluate the possibility of other tissues also to present alterations during the fetal development. Due to this, we studied the mouse development initiating in 12.5 to 19.5 DE (embryologic day) and evaluated the histology of C57BL/6 and CD1 mice searching for anomalies of Cx43+/- and Cx43-/- mice in relation to the Cx43+/+ animals. We did not find alterations in the main organs that express Cx43, nor alterations due to blood out flow related to cardiac malformations. We only found significant difference was the bones; through the evaluation of the ribs and tibia. It has been observed a delay in the development, that was more important in Cx43 knockout mice. We observed clearly that the process of cellular differentiation occurs in less efficient way, delaying processes as deposition of collagen and bone matrix. In conclusion, this study showed that Cx43 is important for bone development in mice.