Supporting videos attached / The nanoscale structure of fully dense human cortical bone is explored using advanced transmission electron microscopy (TEM) techniques. Studies of fully dense cortical bone are rare because of the sample preparation challenges. In this work, cryogenic ion milling is compared favourably with traditional ultramicrotoming methods because of the clearer imaging results and better preservation of biological structures in the ion milled samples. Ion milled samples were prepared parallel, perpendicular and at a 45 degree angle to the long axis of a human femur. The samples are cooled with liquid nitrogen while being milled to prevent heating damage to the bone structure. Bright-field and dark-field imaging show that mineral mainly occurs as 65 nm wide, 5 nm thick mineral structures, external to the collagen fibrils, and with the long axis of the mineral running parallel to the fibrils. In samples cut parallel to the long axis of the bone, the mineral structures have their c-axes aligned with the collagen fibril long axis. In these sections the mineral structures extend up to 200 nm and are grouped into stripe-like bunches, 53 nm apart. Samples cut perpendicular to the long axis of the bone show open areas roughly 45 nm in diameter. These open areas are assumed to be the location of collagen fibrils within the structure and are tangentially surrounded by 65 nm wide, 5 nm thick mineral structures. On average, there are 22 nm of mineral structures between adjoining collagen fibrils. Samples cut at 45 degrees to the long axis of the bone confirm that the open structure seen in the perpendicular section is not an artefact of sample preparation. By tilting the sample, the 45 degree sample shows the structure of both the parallel and perpendicular sections. The parallel structure strongly resembles images of embryonic bone and other mineralized tissues seen in the literature, so the perpendicular open structure is not caused by sample preparation. An examination of ultramicrotoming’s effect on mineral structure size compared with that of ion milling shows that the mineral structures in ion milled samples are twice as long as in ultramicrotomed samples, indicating that bone mineral may be damaged by the forces applied to the complex composite structure existing in fully dense cortical bone. Using energy dispersive X-ray spectroscopy (EDXS) results and a simplified model of the locations of mineral within the collagen/mineral framework, a calculation of the percentage of external mineral was performed. The result showed that 80+_ 6 % of the mineral in fully dense cortical bone must be external to the collagen fibrils to obtain the EDXS results. Finally, Z-contrast tomography, based on the use of high angle annular darkfield (HAADF) imaging, was used to prepared tomographic reconstructions of the external mineral in fully dense cortical bone. Unlike bright-field tomography, the Z-contrast technique allows examination of crystalline materials as the contrast in HAADF images is mass-thickness dependent instead of diffraction based. These reconstructions again showed the mineral tangentially surrounding 50 nm diameter cylindrical holes, assumed to be the location of collagen fibrils in all directions. This work shows the importance of mineral that is external to the collagen fibrils to the nanoscale structure of fully dense cortical bone. / Thesis / Doctor of Philosophy (PhD)

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As osteotomias mandibulares são procedimentos consagrados para o tratamento de prognatismo e para outros reposicionamentos cirúrgicos mandibulares. As técnicas mais utilizadas são: a osteotomia sagital de mandíbula (OSM) e a osteotomia vertical de ramo mandibular (OVRM), cada técnica tem suas indicações e limitações. A OVRM é considerada efetiva devido ao sucesso clínico e análise imaginológica, entretanto não existem estudos que descrevam seu processo de reparação óssea, pois nesta técnica o osso mandibular fica sobreposto num contato cortical sobre cortical. O objetivo deste trabalho foi avaliar a reparação óssea cortical-cortical da técnica de osteomia vertical de ramo mandibular em mandíbulas de coelho. Após aprovação do Comitê de Ética e Pesquisa em Animais da Faculdade de Odontologia da Universidade de São Paulo (Protocolo: 014/2014) foram utilizados 12 animais, divididos em quatro grupos de três. Os animais foram submetidos a osteotomia em -L? invertido no ângulo mandibular bilateralmente, este fragmento ósseo obtido foi deslizado anteriormente e fixado com parafuso sobre a basal mandibular anterior, num dos lados de um animal de cada grupo foi realizada somente a osteotomia, sendo este utilizado como controle cirúrgico; um dos grupos foi submetido a processo de infiltração de marcadores fluorcromáticos. Os animais foram divididos em G1 (eutanasiados em 15 dias), G2 (eutanasiados em 30 dias), G3 (eutanasiados em 45 dias) e G4 (protocolo de marcadores fluorocromáticos e eutanasiados com 56 dias). Após a realização dos procedimentos cirúrgicos, os animais foram mantidos em biotério padrão, nos tempos determinados foram eutanasiados, as mandíbulas foram dissecadas, tomografadas em tomógrafo de feixe cônico e analisadas sob microscopia de fluorescência. Na análise tomográfica observou-se que existe correlação entre os valores de densidades nas áreas operadas e na região do controle, e que há correlação significativa entre as áreas de sobreposição óssea e onde foi realizado somente a osteotomia (controle cirúrgico). Com relação a análise de microscopia de fluorescência, observa-se que as proporções do marcador alizarina são superiores a calceína no osso neoformado, tanto na área operada, quanto na região onde foi feita somente a osteotomia (controle cirúrgico). Concluímos que houve consolidação óssea quando da sobreposição cortical-cortical, e que os valores de densidades tomográficas quando da sobreposição óssea ou não, são similares, e que há maior formação óssea no primeiro mês no processo de reparação. / Mandibular osteotomy is a well-established intervention for the treatment of prognathism and other conditions requiring mandibular repositioning. The most commonly used osteotomy techniques are intraoral vertical ramus osteotomy (IVRO) and sagittal split ramus osteotomy (SSRO), each of which has particular indications and limitations. Although IVRO is considered effective owing to observations of clinical success and imaging analyses, there are no studies describing the bone repair processes that follow IVRO because, in this technique, the mandibular bone is overlaid at a cortical-cortical contact site. The purpose of this study was to evaluate cortical-cortical bone repair in rabbit mandibles following IVRO. After approval of the Ethics and Research Committee on Animals of the Faculty of Dentistry of the University of São Paulo (Protocol: 014/2014), 12 rabbits, divided into four groups of three, were used. The four groups, G1, G2, G3, and G4, were euthanized 15 days, 30 days, 45 days, and 56 days post-IVRO; the animals in G4 were also infused with fluorochemical markers. All 12 animals were submitted to a bilateral inverted \"L\" osteotomy at the mandibular angle. On one side, the released bone fragment was advanced anteriorly and fixed with a screw onto the anterior mandibular basal bone; on the other side, only the osteotomy was performed to serve as a within-animal control site. After the operations, the animals were kept in a standard room until they were euthanized at the group-designated time points. Upon euthanasia, the mandibles were dissected, imaged digitally by conical beam tomography, and analyzed under fluorescence microscopy. The tomographic analysis results revealed a correlation between density values in the operated IVRO sites of bone overlap and the contralateral osteotomy-only surgical control sites. Fluorescence microscopy analysis revealed the presence of elevated alizarin marker (above calcein levels) in the neoformed bone, both in the operated side and in the osteotomy-only control side. Bone growth was greater at the 15-day and 30-day time points than at the later time points. In conclusion, bone consolidation occurred at cortical-cortical overlap sites, tomographic densities were similar between bone overlapping and nonoverlapping (control) sides, bone formation was maximal during the first month of repair.

Basic circuits, which form the building blocks of the brain, have been identiffied in recent literature. We propose to treat these basic circuits as "stochastic generators" whose instances serve to wire a portion of the mouse brain. Very much in the same manner as genes generate proteins by providing templates for their construction, we view the catalog of basic circuits as providing templates for wiring up the neurons of the brain. This thesis work involves a) deffining a framework for the stochastic generation of brain networks, b) generation of sample networks from the basic circuits, and c) visualization of the generated networks.

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As osteotomias mandibulares são procedimentos consagrados para o tratamento de prognatismo e para outros reposicionamentos cirúrgicos mandibulares. As técnicas mais utilizadas são: a osteotomia sagital de mandíbula (OSM) e a osteotomia vertical de ramo mandibular (OVRM), cada técnica tem suas indicações e limitações. A OVRM é considerada efetiva devido ao sucesso clínico e análise imaginológica, entretanto não existem estudos que descrevam seu processo de reparação óssea, pois nesta técnica o osso mandibular fica sobreposto num contato cortical sobre cortical. O objetivo deste trabalho foi avaliar a reparação óssea cortical-cortical da técnica de osteomia vertical de ramo mandibular em mandíbulas de coelho. Após aprovação do Comitê de Ética e Pesquisa em Animais da Faculdade de Odontologia da Universidade de São Paulo (Protocolo: 014/2014) foram utilizados 12 animais, divididos em quatro grupos de três. Os animais foram submetidos a osteotomia em -L? invertido no ângulo mandibular bilateralmente, este fragmento ósseo obtido foi deslizado anteriormente e fixado com parafuso sobre a basal mandibular anterior, num dos lados de um animal de cada grupo foi realizada somente a osteotomia, sendo este utilizado como controle cirúrgico; um dos grupos foi submetido a processo de infiltração de marcadores fluorcromáticos. Os animais foram divididos em G1 (eutanasiados em 15 dias), G2 (eutanasiados em 30 dias), G3 (eutanasiados em 45 dias) e G4 (protocolo de marcadores fluorocromáticos e eutanasiados com 56 dias). Após a realização dos procedimentos cirúrgicos, os animais foram mantidos em biotério padrão, nos tempos determinados foram eutanasiados, as mandíbulas foram dissecadas, tomografadas em tomógrafo de feixe cônico e analisadas sob microscopia de fluorescência. Na análise tomográfica observou-se que existe correlação entre os valores de densidades nas áreas operadas e na região do controle, e que há correlação significativa entre as áreas de sobreposição óssea e onde foi realizado somente a osteotomia (controle cirúrgico). Com relação a análise de microscopia de fluorescência, observa-se que as proporções do marcador alizarina são superiores a calceína no osso neoformado, tanto na área operada, quanto na região onde foi feita somente a osteotomia (controle cirúrgico). Concluímos que houve consolidação óssea quando da sobreposição cortical-cortical, e que os valores de densidades tomográficas quando da sobreposição óssea ou não, são similares, e que há maior formação óssea no primeiro mês no processo de reparação. / Mandibular osteotomy is a well-established intervention for the treatment of prognathism and other conditions requiring mandibular repositioning. The most commonly used osteotomy techniques are intraoral vertical ramus osteotomy (IVRO) and sagittal split ramus osteotomy (SSRO), each of which has particular indications and limitations. Although IVRO is considered effective owing to observations of clinical success and imaging analyses, there are no studies describing the bone repair processes that follow IVRO because, in this technique, the mandibular bone is overlaid at a cortical-cortical contact site. The purpose of this study was to evaluate cortical-cortical bone repair in rabbit mandibles following IVRO. After approval of the Ethics and Research Committee on Animals of the Faculty of Dentistry of the University of São Paulo (Protocol: 014/2014), 12 rabbits, divided into four groups of three, were used. The four groups, G1, G2, G3, and G4, were euthanized 15 days, 30 days, 45 days, and 56 days post-IVRO; the animals in G4 were also infused with fluorochemical markers. All 12 animals were submitted to a bilateral inverted \"L\" osteotomy at the mandibular angle. On one side, the released bone fragment was advanced anteriorly and fixed with a screw onto the anterior mandibular basal bone; on the other side, only the osteotomy was performed to serve as a within-animal control site. After the operations, the animals were kept in a standard room until they were euthanized at the group-designated time points. Upon euthanasia, the mandibles were dissected, imaged digitally by conical beam tomography, and analyzed under fluorescence microscopy. The tomographic analysis results revealed a correlation between density values in the operated IVRO sites of bone overlap and the contralateral osteotomy-only surgical control sites. Fluorescence microscopy analysis revealed the presence of elevated alizarin marker (above calcein levels) in the neoformed bone, both in the operated side and in the osteotomy-only control side. Bone growth was greater at the 15-day and 30-day time points than at the later time points. In conclusion, bone consolidation occurred at cortical-cortical overlap sites, tomographic densities were similar between bone overlapping and nonoverlapping (control) sides, bone formation was maximal during the first month of repair.