Bones have a large intrinsic capacity for repair and regeneration following an injury, however, an estimated 5-10% of nearly 8 million fractures that occur every year in the United States lead to nonunions. The process of bone regeneration is a complex trait that brings together different complements of molecular and cellular interactions to carry out its necessary mechanical functions. These interactions may be attributable to the effects of genetic variations that contribute to differences in bone morphology and fracture healing. This study was undertaken to determine how genetic variability that controls phenotypic qualities of bone affect rates and patterns of fracture healing. Three inbred strains of mice (A/J (AJ), C57BL/6J (B6), and C3H/HeJ (C3)) with known structural and biomechanical differences resulting from fetal bone development were examined. Transverse fractures were generated in the femur and healing traits were evaluated using quantitative real-time polymerase chain reaction (qRT-PCR), micro-computed tomography (μCT), biomechanical torsional testing, and cartilage contrast-enhanced micro-computed tomography (CECT). The temporal analysis of gene expression revealed that B6 had the longest duration of chondrocyte maturation and the greatest relative expression of osteogenic genes relative to either C3 or AJ. While AJ and C3 exhibited similar patterns of chondrogenesis, AJ initiated osteogenesis earlier than C3. These results suggest that compared to either AJ or B6, the C3 strain exhibited the least temporal coordination between the chondrogenic and osteogenic stages. Consistent with the relative patterns of RNA expression, μCT evaluations at day 21 post fracture showed that B6 had higher callus mineralization than AJ and C3. μCT, cartilage CECT, and biomechanical testing revealed less tissue mineralization and more cartilage near the fracture gap, which indicated a less developed bony bridge in C3 calluses at day 21 post fracture. The lack of large amounts of cartilage in calluses of all strains by day 21 indicated that all strains had initiated osteogenesis by this time. Taken together, these results showed that mice with different genetic backgrounds express different patterns of mobilization and renewal of skeletal stem cells with differing temporal progressions of chondrogenic and osteogenic differentiation. These data further show that these variations affect the phenotypic properties of fracture calluses during the process of fracture healing.

Made available in DSpace on 2015-05-14T16:53:10Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-08-11Bitstream added on 2015-05-14T16:59:19Z : No. of bitstreams: 1 000826475.pdf: 1230617 bytes, checksum: 9906192468cedb06e3bcaba8d01384f7 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Proposição: Tendo em vista a influência positiva do tratamento de superfície do implante no processo de reparo e/ou regeneração óssea peri-implantar, propusemos neste estudo caracterizar histologicamente o tecido ósseo neoformado em condições diferentes de superfícies de titânio. Material e Método: foram realizados procedimentos cirúrgicos em 75 ratos (Rattus norvegicus albinus,Wistar), divididos em 3 grupos de 25 animais, onde se obteve acesso ao terço superior da tíbia direita de cada rato para a posterior instalação dos implantes. O 1º grupo recebeu o implante de superfície polida (GRUPO POLIDO - GP), o 2° recebeu um implante com superfície usinada (GRUPO USINADO - GU) e o 3º grupo recebeu o implante de superfície tratada com ácido (GRUPO TRATADO - GT). No total foram 3 grupos, 25 implantes por grupo e 1 implante por animal. Nos períodos de 3, 7, 15, 21 e 40 dias pós-operatórios, os animais foram submetidos à eutanásia e com as amostras teciduais obtidas foram realizadas as análises histomorfométricas, por microscopia eletrônica de varredura (MEV) e Espectroscopia de Raios X por Dispersão em Energia (EDS). Resultados: O resultado das análises por MEV das superfícies demonstrou diferenças topográficas entre elas, sendo a superfície mais lisa e regular a do GP, seguida pela superfície do GU e, por fim, a superfície com maior rugosidade, a do GT. Comparando-se os valores de BIC (Bone to Implant Contact) dos três grupos para um mesmo período (medindo tanto osso em contato com a região cortical quanto medular da tíbia), houve diferença estatisticamente significante (p<0,05) somente no período inicial de osseointegração (7 dias) entre os valores de GP e GT, onde p=0,0189 e a porcentagem de BIC foi de 70% e 94%, respectivamente. Conclusão: O processo de osseointegração dos implantes aconteceu com o mesmo padrão qualitativo tanto nas superfícies... / Purpose: Given the positive influence of the surface treatment of implants in the healing and/or peri-implant bone regeneration process, we proposed on this study histologically characterize the newly formed bone tissue under different titanium surfaces. Material and Methods: Surgical procedures were performed on 75 rats (Rattus norvegicus Albinus, Wistar) were divided into 3 groups of 25 animals, where we had access to the upper third of the right tibia of each rat for the subsequent installation of the implants. The first group received the implant polished surface (POLISHED GROUP - GP), the second received an implant with machined surface (MACHINED GROUP - GU) and the third group received the implant surface treated with acid (TREATED GROUP - GT). In total there were 3 groups, 25 implants per group and 1 implant per animal. In periods of 3, 7, 15, 21 e 40 postoperative day, the animals were euthanized and tissue samples obtained were submitted to the histomorphometric analyses by scanning electron microscopy (SEM) and were performed by Energy Dispersive X Ray Spectroscopy (EDS). Results: The results of SEM analysis of the surfaces showed topographical differences among them being the most smooth and regular surface of the GP, then the surface of the GU and, finally, with greater surface roughness, the GT. Comparing the BIC values of the three groups for the same period (measuring both cortical as trabecular region of the tibia), a statistically significant 17 difference (p <0.05) was found only in the initial period of osseointegration (7 days) between the GP and GT values, where p = 0.0189 and the BIC percentage was 70% and 94%, respectively. Conclusion: The process of osseointegration of implants happened with same quality standard in both polished surfaces (GP), as on machined (GU) and treated with acid subtraction (GT). In contact with the surface...

Orientador: Rita Cássia Menegati Dornelles / Coorientador: Poli Mara Spritzer / Banca: João Cesar Bedran de Castro / Banca: Roberta Okamoto / Banca: Keico Okino Nonaka / Banca: Marcelo Alves da Silva Mori / Resumo: A ação da ocitocina (OT) como regulador direto da massa óssea foi reportada em roedores jovens e este efeito anabólico foi atribuído à ação periférica deste hormônio. O objetivo deste estudo foi investigar a ação periférica de OT no processo de reparo alveolar de ratas Wistar de 12, 18 e 24 meses. Ratas de 12 meses com ciclo estral normal e ratas de 18 e 24 meses em diestro permanente receberam duas injeções intra-peritoniais (12 horas de intervalo) de salina (NaCl 0,15M - grupos controle) ou OT (134g/Kg - grupos tratados). Sete dias após, o incisivo direito foi extraído e as análises foram realizadas com 28 dias de reparo alveolar (35 dias após administração de salina ou OT). Os resultados plasmáticos de cálcio e fósforo não diferiram entre os grupos. Os marcadores bioquímicos sistêmicos de formação óssea, fosfatase alcalina (FAL) e osteocalcina (OC), não revelaram diferença significativa de valores na comparação entre grupos controle e tratado de 12 meses, porém esta diferença foi significativa para os dois marcadores quando os animais tratados de 18 e 24 meses foram comparados com os respectivos controles. A análise histomorfométrica e a reação de imunohistoquímica contra OC confirmaram estes resultados mostrando que o tratamento com OT, promoveu maior formação óssea nos animais de 18 e 24 meses. O marcador sistêmico de reabsorção óssea, fosfatase ácida resistente ao tartarato (TRAP) não foi estatisticamente diferente entre animais dos grupos controle e tratado de 12 e 18 meses, porém nos de 24 meses este valor foi significativamente menor nos animais tratados indicando diminuição de reabsorção por ação de OT nos animais desta idade. A imunomarcação para TRAP realizada nos cortes alveolares confirmou os... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The action of oxytocin (OT) as a direct regulator of bone mass has been reported in young rodents and this anabolic effect was attributed to the peripheral action of this hormone. The aim of this study was to investigate the peripheral action of OT in the process of alveolar repair of Wistar rats with 12, 18 and 24 months. Rats of 12 months with normal estrous cycle and rats with 18 and 24 months in permanent diestrus received two intra-peritoneal injections (12 hours apart) of saline (NaCl 0.15 M - control group) or OT (134  g / kg - treated groups). Seven days later, the right incisor was extracted and the analyzes were performed with 28 days of alveolar repair (35 days after administration of saline or OT). The plasma results of calcium and phosphorus did not differ between groups. The systemic biochemical bone formation markers, alkaline phosphatase (ALP) and osteocalcin (OC), revealed no significant difference of values in animals of 12 months, but this difference was significant for both markers when treated animals of 18 and 24 months were compared to controls. The histomorphometric analysis and immunohistochemical reaction against osteocalcin confirmed these results showing that treatment with OT, promoted greater bone formation in animals with 18 and 24 months. The systemic marker of bone resorption, tartrate-resistant acid phosphatase (TRAP) was not statistically different between animals of the control and treated groups of 12 and 18 months, but in 24 months the values were significantly lower in treated animals indicating decreased resorption action of OT in animals of this age. The immunostaining for TRAP performed on alveolar cuts confirmed the results of the results of systemic markers in groups of 12 and 24 months... (Complete abstract click electronic access below) / Doutor

Orientador: Maria José Hitomi Nagata / Banca: Michel Reis Messora / Banca: Mário Taba Júnior / Banca: Álvaro Francisco Bosco / Banca: Valdir Gouveia Garcia / Resumo: Este estudo avaliou a influência do Plasma Rico em Plaquetas (PRP) e da Fibrina Rica em Plaquetas (FRP) no reparo ósseo de defeitos de tamanho crítico (DTC) criados cirurgicamente em calvárias de ratos. 90 ratos foram aleatoriamente divididos em 3 grupos: C (controle), PRP e FRP. Um DTC de 5 mm de diâmetro foi criado na calvária de cada animal. No Grupo C, o defeito foi preenchido com coágulo sanguíneo somente. Nos grupos PRP e FRP, os defeitos foram preenchidos com PRP e FRP, respectivamente. Os animais foram eutanasiados aos 7, 15 ou 30 dias pós-operatórios. Análises histomorfométrica e imunoistoquímica foram realizadas. A Área de Osso Neoformado (AON) foi calculada como uma porcentagem da área total do defeito original. Imunomarcações do antígeno nuclear de proliferação celular (PCNA), fator de transcrição relacionado à Runt 2 (Runx2), proteína morfogenética óssea - 2 (BMP-2) e fosfatase alcalina específica do osso (BALP) foram realizadas. Células positivas para PCNA e Runx2 foram quantificadas e as imunomarcações para BMP-2 e BALP foram semi-quantificadas. Os dados foram estatisticamente analisados. Aos 7 dias, o Grupo PRP (10,74 ± 4,80%) apresentou AON significativamente maior do que os grupos C (0,86 ± 0,98%) e FRP (2,95 ± 2,37%). Aos 15 dias, os grupos C, PRP e FRP apresentaram AON similares (13,07 ± 4,48%; 14,19 ± 2,85%; 18,83 ± 6,71%, respectivamente). Aos 30 dias, os grupos PRP (32,75 ± 7,39%) e FRP (28,73 ± 10,67%) apresentaram AON significativamente maior do que o Grupo C(13,55 ± 4,62%). Não houve diferenças estatisticamente significativas entre os grupos PRP e FRP aos 30 dias. Aos 7 dias, os grupos PRP e FRP apresentaram número de células PCNA-positivas significativamente maior do que o Grupo C. Aos 15 dias, o Grupo FRP apresentou número de células... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: This study evaluated the influence of Platelet-Rich Plasma (PRP) and Platelet-Rich Fibrin (PRF) on bone healing in surgically created critical-size defects (CSD) in rat calvaria. 90 rats were randomly divided into 3 groups: C (control), PRP and PRF. A 5 mm diameter CSD was created in the calvarium of each animal. In Group C, the defect was filled by blood clot only. In groups PRP and PRF, the defects were filled with PRP and PRF, respectively. Animals were euthanized 7, 15 or 30 days postoperatively. Histomorphometric and immunohistochemical analyses were performed. Newly formed bone area (NFBA) was calculated as percentage of the total area of the original defect. Proliferating cell nuclear antigen (PCNA), Runt-related transcription factor 2 (Runx2) Bone morphogenetic protein 2 (BMP-2) and Bone Alkaline phosphatase (BALP) immunohistochemical staining were performed. PCNA-positive and Runx2-positive cells were quantified and BMP-2 and BALP immunostaining were semi-quantified. Data were statistically analyzed. At 7 days, Group PRP (10.74 ± 4.80) had significantly greater NFBA than groups C (0.86 ± 0.98) and PRF (2.95 ± 2.37). At 15 days, groups C, PRP and PRF presented similar amount of NFBA (13.07 ± 4.48; 14.19 ± 2.85; 18.83 ± 6.71, respectively). At 30 days, groups PRP (32.75 ± 7.39) and PRF (28.73 ± 10.67) presented significantly greater NFBA than Group C (13.55 ± 4.62) and no significant differences were observed between groups PRP and PRF. At 7 days, Groups PRP and PRF showed a significantly higher number of PCNA-positive cells than Group C. At 15 days, Group PRF had a significantly higher number of PCNA-positive cells than groups C and PRP; Group PRP had a significantly higher number of these cells than Group C... (Complete abstract click electronic access below) / Doutor

Orientador: Luana Marotta Reis de Vasconcellos / Co-orientador: Anderson de Oliveira Lobo / Banca: Carlos Alberto Alves Cairo / Banca: Yasmin Rodarte Carvalho / Banca: Cristina Pacheco Soares / Banca: Alexandre Luiz Souto Borges / Resumo: A necessidade da fabricação de novos biomateriais que possam, além de mimetizar o tecido ósseo, fornecer resistências mecânicas favoráveis próximas às do tecido ósseo natural têm despertado interesse de pesquisadores com o objetivo de melhorar a qualidade de vida de pessoas que sofreram algum tipo de lesão. Scaffolds de nanofibras poliméricas fabricados por eletrofiação apresentam características tridimensionais (3D) e poros interconectados que permitem a colonização de toda a superfície 3D por células com a consequente formação de tecidos. O scaffold de poli (butileno adipato-co-tereftalato) (PBAT) mostra-se um biomaterial promissor para regeneração óssea, porém tem sido pouco explorado até a data. Embora do uso da HA seja consagrado para uso biomédico, sua utilização em polímeros ainda é pouco estudada, principalmente em associação ao PBAT. Desta forma, o objetivo deste estudo foi avaliar a efetividade in vitro de scaffolds poliméricos (PBAT) com incorporação de nanopartículas de HA (nHAp) em diferentes concentrações, produzidos por eletrofiação, por meio da bioatividade celular e expressão gênica de osteoblast-like MG63. Células (MG63) foram cultivadas sobre scaffolds de PBAT; PBAT/3%nHAp e PBAT/5% nHAp e sem a presença dos mesmos (controle) e avaliadas pelos testes qualitativo (MEV) e quantitativo de adesão e proliferação celular (1 e 7 dias e aos 1, 3, 7, 14 e 21 dias, respectivamente), citotoxicidade celular (1, 3 e 7 dias), corante vermelho de alizarina e formação de mineralização (14 dias) e análise da expressão de genes relacionados à osteogênese por qRT-PCR aos 7, 14 e 21 dias de cultura celular. Os dados foram analisados estatisticamente por variância (ANOVA) e Tukey (p<0,05). Os scaffolds de PBAT e PBAT/nHAp não apresentaram efeito citotóxico e sua arquitetura tridimensional influenciou positivamente na adesão e proliferação celular, formação de matriz mineralizada bem... / Abstract: The need for the manufacture of new biomaterials that may, in addition to mimic to bone tissue, providing favorable mechanical strength close to natural bone have aroused the interest of researchers in order to improve the quality of life of people who have suffered some kind of injury. Scaffolds polymer nanofibers fabricated by electrospinning have three dimensional features (3D) and interconnected pores that allow the colonization of the entire 3D surface of cells with the consequent formation of tissue. Poly (butylene adipate-co-terephthalate) (PABT) scaffold showed to be a promising biomaterial for bone regeneration, however, has been underexplored to date. Although the use of HA is consecrated to biomedical use, their use in polymers is not well known, especially in association with PBAT. The aim of this study was evaluating in vitro effectiveness of polymeric (PABT) scaffolds with incorporated HA (nHAp) nanoparticles, obtained by electrospinning, through cellular bioactivity and osteoblast-like MG63 gene expression. MG63 cells were grown on PABT; PABT/3%nHAp and PABT/5%nHAp scaffolds and without their presence (control), and evaluated by qualitative (MEV) and quantitative tests of cell adhesion and proliferation (1 and 7 days and at 1, 3, 7, 14 and 21 days, respectively), cell cytotoxicity (1, 3 and 7 days), alizarin red dye and mineralization formation (14 days) and expression of genes related to osteogenesis by qRT-PCR to 7, 14 and 21 days of cell culture. Data were statistically analyzed by variance (ANOVA) and Tukey test (p<0.05). The PBAT and PBAT/nHAp scaffolds showed no cytotoxic effect and its three-dimensional architecture influenced positively in the cell adhesion and proliferation, mineralized matrix formation as well as in some periods the expression of genes ALP, Col I, Runx2, OC and OPN in relation the control group. The osteoconductive and osteoinductive effect ofnHAp promoted better cellular response in scaffolds of ... / Doutor

Orientador: Idelmo Rangel Garcia Júnior / Banca: Roberta Okamoto / Banca: Ana Lúcia Alvares Capelozza / Resumo: Proposição: Tendo em vista a influência positiva do tratamento de superfície do implante no processo de reparo e/ou regeneração óssea peri-implantar, propusemos neste estudo caracterizar histologicamente o tecido ósseo neoformado em condições diferentes de superfícies de titânio. Material e Método: foram realizados procedimentos cirúrgicos em 75 ratos (Rattus norvegicus albinus,Wistar), divididos em 3 grupos de 25 animais, onde se obteve acesso ao terço superior da tíbia direita de cada rato para a posterior instalação dos implantes. O 1º grupo recebeu o implante de superfície polida (GRUPO POLIDO - GP), o 2° recebeu um implante com superfície usinada (GRUPO USINADO - GU) e o 3º grupo recebeu o implante de superfície tratada com ácido (GRUPO TRATADO - GT). No total foram 3 grupos, 25 implantes por grupo e 1 implante por animal. Nos períodos de 3, 7, 15, 21 e 40 dias pós-operatórios, os animais foram submetidos à eutanásia e com as amostras teciduais obtidas foram realizadas as análises histomorfométricas, por microscopia eletrônica de varredura (MEV) e Espectroscopia de Raios X por Dispersão em Energia (EDS). Resultados: O resultado das análises por MEV das superfícies demonstrou diferenças topográficas entre elas, sendo a superfície mais lisa e regular a do GP, seguida pela superfície do GU e, por fim, a superfície com maior rugosidade, a do GT. Comparando-se os valores de BIC (Bone to Implant Contact) dos três grupos para um mesmo período (medindo tanto osso em contato com a região cortical quanto medular da tíbia), houve diferença estatisticamente significante (p<0,05) somente no período inicial de osseointegração (7 dias) entre os valores de GP e GT, onde p=0,0189 e a porcentagem de BIC foi de 70% e 94%, respectivamente. Conclusão: O processo de osseointegração dos implantes aconteceu com o mesmo padrão qualitativo tanto nas superfícies... / Abstract: Purpose: Given the positive influence of the surface treatment of implants in the healing and/or peri-implant bone regeneration process, we proposed on this study histologically characterize the newly formed bone tissue under different titanium surfaces. Material and Methods: Surgical procedures were performed on 75 rats (Rattus norvegicus Albinus, Wistar) were divided into 3 groups of 25 animals, where we had access to the upper third of the right tibia of each rat for the subsequent installation of the implants. The first group received the implant polished surface (POLISHED GROUP - GP), the second received an implant with machined surface (MACHINED GROUP - GU) and the third group received the implant surface treated with acid (TREATED GROUP - GT). In total there were 3 groups, 25 implants per group and 1 implant per animal. In periods of 3, 7, 15, 21 e 40 postoperative day, the animals were euthanized and tissue samples obtained were submitted to the histomorphometric analyses by scanning electron microscopy (SEM) and were performed by Energy Dispersive X Ray Spectroscopy (EDS). Results: The results of SEM analysis of the surfaces showed topographical differences among them being the most smooth and regular surface of the GP, then the surface of the GU and, finally, with greater surface roughness, the GT. Comparing the BIC values of the three groups for the same period (measuring both cortical as trabecular region of the tibia), a statistically significant 17 difference (p <0.05) was found only in the initial period of osseointegration (7 days) between the GP and GT values, where p = 0.0189 and the BIC percentage was 70% and 94%, respectively. Conclusion: The process of osseointegration of implants happened with same quality standard in both polished surfaces (GP), as on machined (GU) and treated with acid subtraction (GT). In contact with the surface... / Mestre

Orientadores: Marcio Zaffalon Casati, Sergio de Toledo / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-08T14:46:02Z (GMT). No. of bitstreams: 1 Casarin_RenatoCorreaViana_M.pdf: 9678286 bytes, checksum: 95c6838ca5a99289bdbad65404d1cd7e (MD5) Previous issue date: 2007 / Resumo: O objetivo do presente estudo foi avaliar a resposta clínica das lesões de bifurcação proximais tratadas com proteínas derivadas da matriz do esmalte (EMD). Foram selecionados 24 pacientes com pelo menos uma lesão de bifurcação proximal classe II apresentando profundidade de sondagem (PS) = 5 mm e presença de sangramento a sondagem. As furcas foram divididas aleatoriamente em 2 grupos: Grupo controle (n=20) ¿ acesso para raspagem e alisamento radicular e aplicação de EDTA 24% (PrefGel®); Grupo Teste (n=20) ¿ acesso para raspagem e alisamento radicular + aplicação de EDTA 24% + aplicação das EMD (Emdogain®). Os pacientes foram avaliados quanto ao Índice de Placa (IP) e Sangramento à Sondagem (SS), Profundidade de Sondagem (PS), Posição da Margem Gengival (PMG), Nível Clínico de Inserção Vertical e Horizontal Relativo (NICVR e NICHR, respectivamente), Nível Ósseo Vertical e Horizontal (NOV e NOH) e fechamento da lesão. As avaliações foram realizadas antes do tratamento, 2, 4 e 6 meses após. Aos 6 meses de acompanhamento o ganho de NICVR dos grupos controle e teste foram de 0,63 e 0,74 mm, enquanto o ganho de NICHR foram 1,13 e 1,64 mm, ambos sem diferença entre os grupos. O ganho de NOV e NOH para os grupos controle e teste foram de 0,98 e 0,85 mm e 0,94 e 1,08 mm respectivamente, também sem diferença estatística entre os grupos. Na avaliação do número de bifurcações fechadas aos seis meses, no grupo teste houve fechamento de 4 lesões, enquanto no grupo controle não ocorreu nenhum fechamento (p<0,05). Pode-se concluir que, embora os defeitos de bifurcação proximais tenham apresentado ganhos semelhantes de NICVR NICHR, NOV e NOH, o uso do EMD possibilitou um maior fechamento completo das bifurcações que o acesso para raspagem e alisamento radicular e biomodificação radicular com EDTA 24% / Abstract: The aim of the present study was to evaluate the clinical response of proximal furcations treated with enamel matrix derived proteins (EMD). Twenty four patients with at least one class II proximal furcation involvements presenting probing depth (PD) = 5 mm and bleeding on probing were selected. The patients were randomly assigned to: Control Group (n=20) ¿ Open Flap Debridement (OFD) + EDTA 24% conditioning (PrefGel®); Test Group (n=20) ¿ OFD + EDTA 24% conditioning + EMD application (Emdogain®). Plaque Index (PI), Bleeding on Probing (BOP), Probing Depth (PD), Gingival Margin Position (GMP), Relative Vertical and Horizontal Clinical Attachment Level (RVCAL and RHCAL, respectively), Vertical and Horizontal Bone Level (VBL and HBL) and Furcation Closure were evaluated immediately before and 2, 4 and 6 months after the surgeries. At 6th month the gain of RVCAL of control and test group were 0.63 e 0.74 mm, while the RHCAL gain were 1.13 e 1.64 mm, both without statistical difference between the groups. The VBL and HBL gain of control and test group were 0.98 and 0.85 mm and 0.94 and 1.08 mm respectively, also without statistical difference. Four closed furcations were observed at 6th month in the test group, while no closed furcations was observed at control group (p<0.05). It could be conclude that, although the proximal furcations showed similar gains of RVCAL, RHCAL, VBL and HBL at 6th month, the EMD application promote a superior, but unpredictable, frequency of closed furcations than that found with open flap debridement and EDTA 24% conditioning / Mestrado / Periodontia / Mestre em Clínica Odontológica

Osteoporosis and low bone mass are devastating and costly diseases affecting over half the US population over 50-years old. Peak bone mass is reached around age 30 and begins to decline in the following years, leading to a multi-decade disease. While there are several treatment options, including both ant-resorptive agents -- preventing bone loss -- and anabolic agents -- promoting bone formation -- they are inadequate due to either the limited scope of approval or decreased patient compliance from the perceived high risk of complications. Fracture risk increases significantly in osteopenic individuals with vertebral, hip, wrist, and pelvic fractures being among the most common. While there is a significant annual cost of osteoporotic fractures – on the order of $25 billion annually – the increased rate of mortality after osteoporotic fracture is halting. Risk of mortality one year after hip fracture ranges from 20% to 40%. Therefore, there is a clinical and economic need to develop the next-generation of bone saving therapeutics. Bone has the innate ability to respond to mechanical loading -- forming new bone to accommodate increased mechanical loads, and resorbing bone when there is a period of low mechanical loading or disuse. If we can determine how mechanical loads are detected on the cellular level, we can open up a new avenue for drug development. The osteocyte, a terminally differentiated cell embedded in the bone mineral matrix, is accepted as a key bone mechanotransducer -- detecting mechanical loading and translating it into biochemical signals promoting bone formation. There are several hypothesized models of how the osteocyte detects mechanically loading – fluid flow through the canalicular environment stimulates the osteocyte's dendritic processes; fluid flow results in deformation of the primary cilium within the lacunar cavity; or matrix deformation is directly transduced through integrin attachments at the lacunar wall. The primary cilium is a solitary antenna-like organelle that forms a distinct signaling domain with a unique protein pool, which makes it an attractive therapeutic target. In this thesis, we seek to unravel the role of the primary cilium in bone mechanotransduction in order to open new avenues for drug development. We examine if the osteocyte primary cilium contributes to load-induced bone formation, determine if adenylyl cyclase 3 (AC3) -- a cAMP catalyzing enzyme that localizes to the primary cilium -- contributes to osteocyte mechanotransduction, and investigate if the primary cilium coordinates actin adaptation in response to mechanical loading. Using a Cre-lox system to knockout Ift88, a gene encoding a critical protein for cilia formation, we find that load-induced bone formation is dependent on whether one or two alleles of Ift88 are present globally, but not if they are only deleted in the osteocyte cell population. We also find that knocking down AC3 mRNA expression leads to an increased response to mechanical stimulation and altered primary cilia length, likely through decreased cAMP production. Finally, we determine that inhibiting primary cilia formation dysregulates actin adaptation to mechanical loading and prevents the actin-dependent mechanoresponse of Taz, a transcriptional co-regulator. This action is likely through alterations in the expression of actomyosin components and in the activation of focal adhesion kinases. Together, this work demonstrates that the primary cilium plays a role in load-induced bone formation, but this effect is not localized to the osteocyte cell population. We also show that adenylyl cyclases play a role in osteocyte mechanobiology, and that whole cell mechanosensitivity may be determined through the primary cilium in its function regulating the actin cytoskeleton.

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促红细胞生成素（EPO）和EPO受体（EPOR）是调节红细胞生成所必需的细胞因子。越来越多证据表明，EPO/EPOR在非造血器官包括心脏、大脑和骨骼的发育和再生中发挥重要作用。但目前人们对EPO/EPOR在骨骼发育和再生中的机制知之甚少。最近一些研究表明，系统性或局部注射EPO可促进骨形成。但是EPO/EPOR在骨骼发育和再生中的作用机制尚不明确。 / 实验发现EPO/EPOR在生长板的前肥大软骨和肥大软骨区富集，且可促进软骨细胞增殖。但利用siRNA技术将软骨细胞EPOR沉默后，软骨细胞增殖会受抑制。随后，我们用阿利新蓝对软骨细胞外基质中的的蛋白聚糖进行染色，发现EPO可促进软骨细胞分化。此外软骨细胞标志基因包括SOX9、SOX5、SOX6、2型胶原蛋白和蛋白聚糖表达上调。实验还发现EPO可促进MSCs增殖。同时EPO可促进上述软骨细胞标志基因的表达增加。当基因沉默EPOR或使用EPO封闭肽后，软骨细胞标志基因的表达降低。以上数据表明， EPO促进软骨细胞增殖和分化的功能至少部分通过其同源受体EPOR介导。 / 我们还探讨了在低氧环境下，EPO对软骨细胞的作用。在低氧环境中， EPO/EPOR和HIF-1α mRNA和蛋白质表达水平均上调，且EPO促进软骨祖细胞集落形成。此结果提示EPO在低氧条件下介导软骨细胞增殖。同时在软骨细胞中标，EPO可激活JAK2和STAT3磷酸化，该结果表明JAK/STAT信号介导软骨细胞的生物学功能。 / 体外内皮细胞出芽实验发现EPO明显促进跖骨表面内皮细胞出芽。在小鼠骨折处局部注射EPO 14天后μCT血管成像发现，EPO可促进骨折处血管生成。体外与体内试验结果同时证实EPO可促进血管生成。 / 在骨折术后第7天和14天，藏红O染色发现EPO促进软骨骨痂形成。在术后第28天，X光和μCT扫描发现，三维重建和定量分析显示EPO促进骨形成，且伴随着骨量和骨面积的增加，以及骨生物力学特性的改善。以上结果表明， EPO可有效促进骨折修复。 / 综上所述， EPO/ EPOR信号调控软骨细胞和MSCs增殖及其软骨细胞分化。EPO还调控软骨细胞在低氧环境下的生物学特性。EPO/ EPOR信号分子有助于骨愈合过程中血管生成和骨形成。因此，EPO/EPOR可作为一个新的治疗靶点促进骨骼修复与再生。 / Erythropoietin (EPO) and EPO receptor (EPOR) are essential cytokine signals regulating erythropoiesis. Growing evidences suggest that EPO/EPOR signaling involves in the development and regeneration of non-hematopoietic organs including heart, brain and bone, et al. Several recent studies indicate that administration of EPO locally or systemically promotes bone formation. However, the underlying mechanisms of EPO/EPOR in skeletal development and regeneration remain unknown. / Our results show that EPO and EPOR are abundantly expressed in the pre-hypertrophic and hypertrophic zone of the growth plates. The proliferation rate of chondro-progenitors is increased following EPO treatment Alcian blue staining for extracellular matrix proteoglycan indicates that EPO promotes the differentiation of chondrocytes. This is accompanied by up-regulated chondrogenic marker genes including SOX9, SOX5, SOX6, type 2 collagen and aggrecan. In a parallel study, the proliferation rate of MSCs is increased following EPO treatment. The mRNA expression of above chondrogenic marker genes is also up-regulated. These effects are eliminated following knockdown of EPOR in chondrocytes by siRNA or treatment with EPO block peptide. These findings indicate that EPO promotes the proliferation and differentiation of primary chondrocytes at least partially mediated by its cognate receptor EPOR. / We next examined the role of EPO in chondrocytes under hypoxia. The mRNA and protein levels of EPO/EPOR and HIF-1α are up-regulated under hypoxia. EPO also enhances the colony forming efficiency of chondro-progenitors under hypoxia. This result suggests that EPO may serve as a mediator to regulate proliferation of chondro-progenitors under hypoxic condition. In addition, we show that EPO up-regulates the phosphorylation states of Janus kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3) in chondrocytes, suggesting that the function of EPO in chondrocytes is mediated through JAK/STAT signaling. / To address the function of EPO in angiogenesis, we performed metatarsal endothelial sprouting assay. The endothelial sprouting is significantly enhanced in metatarsals treated with EPO. This coincide with our in vivo data that local delivery of EPO increases vascularity of the healing bone at day 14 post-fracture in mice as indicated by micro-CT angiography analysis. / Interestingly, in the mouse fracture model, EPO promotes cartilaginous callus formation at days 7 and 14 post-surgery. This results in accelerated osteogenesis at day 28 post-surgery indexed by the radiographical scoring and micro-CT analysis characterized by increased bone volume and bone surface. This is accompanied by improved biomechanical properties of the healing bone. These results indicate that administration of EPO may serve as an efficient therapy to facilitate bone regenerartion. / In conclusion, EPO/EPOR signal regulates of the proliferation and differentiation of chondrocytes and MSCs to promote chondrogenesis. EPO may also function as a positive mediator in chondrocytes in response to low oxygen tension during chondrogenesis. EPO/EPOR signaling also contributes to angiogenesis and osteogenesis during bone healing. Therefore, EPO/EPOR may serve as a novel therapeutic target to promote skeletal regeneration. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Wan, Lin. / Thesis (Ph.D.) Chinese University of Hong Kong, 2014. / Includes bibliographical references (leaves 130-152). / Abstracts also in Chinese. / Wan, Lin.