Diplomová práce Hodnocení motorických dovedností dětí s OI Abstract The thesis deals with the assessment of motor skills and quality of life in children with osteogenesis imperfecta. The theoretical part summarizes aetiopathogenesis, clinical features, kinesiological abnormalities and the treatment strategies. A particular part describes therapeutic interventions during the various stages of childhood with an alignment to multidisciplinary cooperation. Furthermore, tests of motor skills and quality of life are presented. The practical part assesses the applicability of the BOT-2 for the evaluation of motor skills and PedsQL for the evaluation of the quality of life in children with OI. Moreover, the BOT-2 standardized test score evaluates and compares motor skills and quality of life in children with osteogenesis imperfecta to their peers. Contemporaneously, it investigates the effect of motor skills on the quality of life in children with OI. Based on the results the thesis confirms the hypothesis - worsened both gross motor skills and fine motor skills and the dependency of quality of life on the level of motor skills. The discussion part deals with the feasibility of BOT-2 and PedsQL for the assessment of children with OI, it comments the main limitations of children with OI, and based on up-to-date...

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Connective tissue growth factor (CTGF/CCN2) is axiomatically necessary for proper skeletal development and function. We need not look further than the studies that have been done to date utilizing mice genetically engineered to lack CTGF production. These CTGF null or knockout (KO) mice fail to form a normal murine skeleton and instead yield one littered with bony dysmorphisms, including incompetent craniofacial development, kinked limb bones, and misshapen ribs that are not conducive to proper respiratory function. As a result, the global lack of CTGF is incompatible with postnatal life. A closer look at several sites demonstrated defects in physiologic processes necessary for bone formation - angiogenesis, chondrogenesis, and osteogenesis. Therefore, the dogma in the CCN protein field to date has been that systemic ablation of CTGF production in vivo results in global defects in bone development. We believe this dogma is an oversimplification of the role of CTGF on skeletal development. Our initial impetus leading us to this belief was the gross identification of the specific skeletal sites malformed in CTGF KO mice, in particular the bones of the limbs. While in the lower limb of CTGF KO mice the tibiae and fibulae are misshapen, the adjacent femora and digits are phenotypically normal. The same is true for the upper limb, in which the radii and ulnae are phenotypically abnormal while the humeri and digits are normal. Therefore, we believe that the role of CTGF in skeletogenesis is site-specific such that its loss affects local skeletal patterning and/or mechanobiological cues resulting in the unique phenotype seen in CTGF KO mice. The research of this dissertation constitutes a comprehensive skeletal analysis of CTGF KO mice and in so doing we determined the extent and location of skeletal abnormalities. We found skeletal site-specific changes in growth plate organization, bone microarchitecture and shape and gene expression levels in CTGF KO compared to wild-type (WT) mice. Growth plate malformations included reduced proliferation zone and increased hypertrophic zone lengths. Appendicular skeletal sites demonstrated decreased metaphyseal trabecular bone, while having increased mid-diaphyseal bone and osteogenic expression markers. Axial skeletal analysis showed decreased bone in caudal vertebral bodies, mandibles, and parietal bones in CTGF KO mice, with decreased expression of osteogenic markers. Analysis of skull phenotypes demonstrated global and regional differences in CTGF KO skull shape resulting from allometric (size-based) and non-allometric shape changes. Localized differences in skull morphology included increased skull width and decreased skull length. We further continued the skeletal characterization of CTGF KO bones with an analysis of bone cell ultrastructure and matrix composition. These studies demonstrated that, while CTGF is not necessary for complete morphologic maturation of bone cells, global ablation results in ultrastructural features not commonly seen in WT bones. Our findings include drastically dilated rough endoplasmic reticulum (RER) in osteoblasts of the tibial diaphyseal region, comprising the phenotypic kink in CTGF KO mice and ultrastructural dysmorphologies of CTGF KO osteoclasts including multi-layered, membranous inclusions, decreased vacuolization and ruffled border extents, and disproportionately large clear zones. Lastly, FT-IR analysis demonstrated heterogeneity in CTGF KO bone composition. The results of this dissertation have revealed a more complex role for CTGF in osteogenesis and have identified potential mechanisms and future research directions to fully understand this intricate story. / Cell Biology

Indiana University-Purdue University Indianapolis (IUPUI) / Osteogenesis imperfecta is a rare congenital disease commonly characterized by brittle bones caused by mutations in the genes encoding Type I collagen, the single most abundant protein produced by the body. The murine model (oim) exists as a natural mutation of this protein, converting its heterotrimeric structure of two Col1a1 molecules and a single Col1a2 molecule into homotrimers composed of only the former. This defect impacts bone mechanical integrity, greatly weakening their structure. Femurs from male wild type (WT), heterozygous (oim/+), and homozygous (oim/oim) mice, all at 12 weeks of age, were assessed using assays at multiple length scales with minimal sample processing to ensure a near-physiological state. Atomic force microscopy (AFM) demonstrated detectable differences in the organization of collagen at the nanometer scale that may partially attribute to alterations in material and structural behavior obtained through mechanical testing and reference point indentation (RPI). Changes in geometric and chemical structure through the use of µ-Computed Tomography and Raman spectroscopy respectively indicate a smaller, brittle phenotype caused by oim. Changes within the periodic D-spacing of collagen point towards a reduced mineral nucleation site, supported by reduced mineral crystallinity, resulting in altered material and structural behavior in oim/oim mice. Multi-scale analyses of this nature offer much in assessing how molecular changes can compound to create a degraded, brittle phenotype.

Os eventos celulares e extracelulares que ocorrem durante o processo de osseointegração do titânio (Ti) são bastante influenciados por suas propriedades de superfície, como morfologia, topografia e composição química. A modificação bioquímica da superfície do Ti consiste em imobilizar proteínas ou peptídeos nessa superfície com a finalidade de induzir respostas celulares e teciduais específicas na interface osso-implante que acelerem ou aumentem a osseointegração. O objetivo deste estudo foi avaliar o desenvolvimento do fenótipo osteoblástico em culturas de células crescidas sobre Ti revestido com colágeno tipo I. Para tanto, células osteoblásticas derivadas de fragmentos ósseos do processo alveolar de humanos foram cultivadas sobre discos de Ti usinados revestidos (Ti-col) ou não (Ti-usinado) com colágeno tipo I e foram avaliados os seguintes parâmetros: adesão, morfologia e proliferação celulares, síntese de proteína total, atividade de fosfatase alcalina (ALP), formação de matriz mineralizada, e expressão de genes marcadores do fenótipo osteoblástico por reação em cadeia da polimerase em tempo real (PCR em tempo real). O Ti-col alterou o crescimento e a expressão gênica das culturas e não teve efeito na adesão e morfologia celulares, síntese de proteína total, atividade de ALP e formação de matriz mineralizada comparado ao Ti-usinado. Esses resultados indicam que a superfície Ti-col pode favorecer um maior crescimento da cultura durante a fase proliferativa e um aumento e/ou aceleração da diferenciação, como indicado por alterações na expressão gênica de marcadores do fenótipo osteoblástico. Portanto, essa modificação de superfície pode ter um impacto nos processos de reparo e remodelação do tecido ósseo adjacente a implantes, favorecendo a ocorrência de maior formação óssea. / Cellular and extracellular events that occur during titanium (Ti) osseointegration process are highly influenced by its surface properties, such as morphology, topography and chemical composition. The objective of biochemical modification of Ti is to immobilize proteins or peptides on its surface in order to induce specific cellular and tissue responses at the boneimplant interface in order to accelerate or enhance osseointegration. The aim of this study was to evaluate the osteoblastic phenotype development in cells grown on collagen type I-coated Ti surface. Osteoblastic cells from human alveolar bone fragments were cultured on turned Ti either coated with collagen type I (col-Ti) or not (turned-Ti) and the following parameters were assessed: cell adhesion, morphology, and proliferation, total protein content, alkaline phosphatase (ALP) activity, bone-like formation and gene expression of osteoblastic markers by real-time polymerase chain reaction (real-time PCR). Col-Ti altered culture growth and gene expression of osteoblastic markers without affecting cell adhesion, morphology, protein synthesis, ALP activity, and matrix mineralization. These results demonstrated that col-Ti favours cell growth during the proliferative phase and osteoblastic differentiation, as demonstrated by changes in mRNA expression profile during the matrix mineralization phase, suggesting that this Ti surface modification may affect the processes of bone healing and remodelling.

A implantação de materiais vítreos e vitrocerâmicos bioativos representa estratégia terapêutica importante para se promover a formação de matriz extracelular mineralizada em defeitos ósseos críticos. Quando expostos a fluidos biológicos, estes biomateriais sofrem alterações químicas e topográficas de superfície que afetam as interações de células com sua superfície, reduzindo o espraiamento celular e alterando o padrão de marcação de proteínas do citoesqueleto. O objetivo deste estudo foi avaliar se as alterações no padrão de marcação para as proteínas citoesqueléticas actina e tubulina observadas in vitro em células osteogênicas sobre superfícies do vidro Bioglass® 45S5 e da vitrocerâmica Biosilicato®, são decorrentes de redução quantitativa na expressão do RNAm e das proteínas correspondentes. Células osteogênicas foram obtidas a partir da digestão enzimática de calvárias de ratos Wistar recémnascidos e plaqueadas sobre superfícies de Bioglass® 45S5, Biosilicato® e borosilicato (controle bioinerte) para a avaliação dos seguintes parâmetros: 1) detecção de actina e tubulina por microscopia de fluorescência; 2) expressão de RNAm para actina e tubulina por reação em cadeia da polimerase em tempo real (Real time PCR); 3) quantificação de actina e tubulina por ensaio imunoenzimático direto (ELISA), e 4) análise da morfologia celular por microscopia eletrônica de varredura (MEV). Aos 3 e 7 dias, células crescidas sobre borosilicato exibiam padrões de marcação para actina e tubulina típicos de células aderidas e espraiadas sobre substratos planos in vitro, enquanto que sobre Bioglass® 45S5 e Biosilicato® as células apresentavam áreas circulares destituídas de marcação para essas proteínas. Nos mesmos períodos, culturas crescidas sobre os materiais bioativos apresentavam alterações significantes da expressão de RNAm para actina e tubulina, embora fossem observadas apenas discretas variações na quantidade das proteínas correspondentes em relação ao borosilicato. Além disso, apenas para culturas crescidas sobre borosilicato observava-se correlação positiva entre RNAm e proteína e correspondência entre as observações por epifluorescência e os dados quantitativos. Aos 3 dias, imagens de MEV revelaram células aderidas e espraiadas sobre os materiais bioativos, parcial ou totalmente recobertas por acúmulos de material de aspecto semelhante ao da topografia do substrato, por vezes impedindo a visualização dos limites celulares. Com base nos resultados obtidos, conclui-se que as superfícies bioativas de Bioglass® 45S5 e Biosilicato® afetam a expressão de RNAm para actina e tubulina, mas não de proteína. Assim, as alterações nos padrões de marcação por fluorescência para essas proteínas devem ser atribuídas, pelo menos em parte, a acúmulos de material sobre as células, possivelmente decorrentes das reações de superfície a que estão submetidos Bioglass® 45S5 e Biosilicato® quando em contato com fluidos biológicos. / Bioactive glasses and glass-ceramics have been successfully applied in various therapeutic strategies to promote the formation of mineralized matrix in bone defects. The exposure of these materials to biological fluids results in chemical and topographical modifications that may affect the interactions of cells with the biomaterial surface, with potential effects on cytoskeletal protein expression and/or organization and cell spreading. The aim of the present study was to evaluate whether changes in the labelling pattern for the cytoskeletal proteins actin and tubulin in osteogenic cells cultured on bioactive Bioglass® 45S5 and Biosilicate® are due to altered mRNA and protein expression levels. Osteogenic cells were obtained by enzymatic digestion of newborn Wistar rat calvarial bone and plated on Bioglass® 45S5, Biosilicate® and borosilicate (bioinert control) for periods of up to 7 days. The following parameters were assayed: i) qualitative epifluorescence analysis of actin and tubulin distribution; ii) quantitative mRNA expression for actin and tubulin by real time polymerase chain reaction (real time PCR); iii) quantitative actin and tubulin expression by enzymelinked immunoabsorbent assay (ELISA), and iv) qualitative analysis of cell morphology by scanning electron microscopy (SEM). At days 3 and 7, cells grown on borosilicate showed typical actin and tubulin labeling patterns of adherent and spread cells on flat, rigid substrates, whereas those on Bioglass® 45S5 and Biosilicate® showed dark areas devoid of fluorescent signals for the cytoskeletal proteins. At the same time points, cultures grown on the bioactive materials showed significant changes in mRNA expression for actin and tubulin, although only slight differences in the amount of actin and tubulin were detected compared with borosilicate. Moreover, a positive correlation between mRNA and protein expression levels as well as a correspondence between epifluorescence imaging and the quantitative data were only detected for cultures grown on borosilicate. SEM analysis revealed that cells cultured on bioactive surfaces were partly or totally covered with material accumulations, whose characteristics resembled the ones for the substrate topography, and which, in some cases, prevented the visualization of the cell limits. In conclusion, Bioglass® 45S5 and Biosilicate® affect actin and tubulin mRNA levels, but not the corresponding protein expression, in osteogenic cell cultures. Thus, the observed changes in the labeling pattern for these proteins should be attributed, at least in part, to the accumulation of materials on the cell surface, likely due to substrate reactions that take place on Bioglass® 45S5 and Biosilicate® when exposed to the cell culture medium.

A expansão craniana com o uso de molas tem demonstrado eficácia no tratamento das anormalidades craniofaciais, tais como as craniossinostoses. A ação expansora exercida pelas molas tem sido observada tanto quando colocadas entre as margens parietais dos ossos do crânio, como quando lateralmente à sutura sagital, principalmente nas escafocefalias. No presente estudo foi criado um modelo experimental com coelhos, e feita uma avaliação descritiva do comportamento da calota craniana e das suturas sob ação de molas. Foram utilizados 13 coelhos Nova Zelândia com quatro semanas de vida, divididos em quatro grupos: grupo I, foram implantados no crânio marcadores de amálgama para controle; no grupo II, marcadores de amálgama e osteotomia da sutura sagital; no grupo III, marcadores de amálgama, osteotomia da sutura sagital e colocação de uma mola expansora na região interparietal e, no grupo IV, marcadores de amálgama, craniotomia parassagital linear com colocação da mola. Os animais foram sacrificados com duas, quatro, oito e doze semanas. Foi realizado controle radiológico com avaliação do afastamento dos marcadores de amálgama, da variação dos ângulos cefalométricos e das medidas da base do crânio, bem como um estudo histopatológico da região de colocação das molas. Nos grupos com o uso de molas a separação dos bordos da craniotomia foi maior do que naqueles sem a utilização de mola. Houve ossificação em todos os grupos, com maior rapidez no grupo II. O crescimento ósseo deu-se a partir dos bordos e da profundidade. Não foram observadas diferenças significativas no padrão histopatológico da regeneração óssea entre os grupos com colocação de mola na região interparietal e parassagital. O modelo experimental com coelhos se mostrou adequado às análises propostas pelo estudo. Concluiu-se que houve osteogênese por distração nos grupos III e IV e que apresentaram uma expansão craniana semelhantes. / Spring-mediated skull expansion has proven to be effective in the treatment of craniofacial abnormalities, such as craniosynostosis. The use of springs in cranial expansion has been studied both in the sagittal and in parasagittal regions, especially in scaphocephaly. A rabbit model was used in the present study to analyze the effects of springs on the cranial vault and sutures. Thirteen 4-week-old New Zealand rabbits were used and divided into 4 groups: group I, amalgam markers were used as control; in group II, amalgam markers and osteotomy of the sagittal suture; in group III, amalgam markers and osteotomy of the sagittal suture with implant of an expansible spring in the interparietal region and in group IV, amalgam markers and linear parasagittal craniectomy with springs. Animals were sacrificed after 2, 4 and 12 weeks. Radiological control with assessment of the amalgam markers, variation of cephalometric angles and cranial base measurements, as well as the histopathological analysis of the spring implant area were carried out. In the groups using springs distraction of the craniectomy borders was greater than in those that did not use springs. Ossification was observed in all of the groups and was faster in group II. Bone growth started from the borders and depth. There were no significant differences in the histopathological pattern of bone regeneration between the groups with spring implant in the interparietal and parasagittal region. The rabbit model proved to be adequate for the analysis proposed by the study. It was concluded that there was osteogenesis caused by distraction in groups III and IV, with similar skull expansion rates.

As fissuras lábiopalatinas consistem em anormalidades craniofaciais congênitas comuns quando comparada as demais anomalias, que podem acometer o lábio, rebordo alveolar, palatos duro e mole, resultantes da falta de nivelamento dos processos nasais mediais entre si, e destes com os processos maxilares laterais. O tratamento deve ser realizado por meio de uma equipe multidisciplinar de especialistas. Um dos meios auxiliares mais importantes no diagnóstico da malformação são os exames de imagem, como as radiografias intra e extra bucais e as tomografias computadorizadas, para uma melhor avaliação morfológica prévia das resultando em melhorias no diagnóstico, planejamento e manejo cirúrgico dos casos, prevendo melhor prognóstico e resultado final mais satisfatório. Esta pesquisa avaliou através de radiografias periapicais de fissuras transforame incisivo unilateral a dimensão das fissuras no pré-operatório e o nível de formação óssea no pós operatório de enxerto ósseo alveolar autógeno de crista ilíaca, objetivando investigar o tamanho crítico do defeito maxilar e o nível de formação óssea, levando em consideração fatores que também influenciam na previsibilidade do resultado como a idade, presença do canino não irrompido e a ortodontia pós enxerto, para que se possa oferecer condutas terapêuticas mais adequadas. Pacientes com menos de 16 anos de idade tem seis vezes mais chances para formação de um septo ósseo intermediário com altura próximo do normal. Resultados favoráveis também foram encontrados nos casos em que o canino não havia irrompido, com 16 vezes maior a capacidade de preenchimento ósseo do defeito em até 75%. Indivíduos com início da ortodontia no período ideal, 60 a 90 dias depois do enxerto ósseo, apresentaram uma formação óssea do tipo I e II com mais de 50% de formação óssea no defeito. A altura, largura apical e o gênero da fissura alveolar são fatores que não influenciam na formação óssea. / Cleft lip and palate are common congenital craniofacial abnormalities in comparison with other anomalies. They may affect the lip, alveolar ridge and the soft and hard palate resulting from the lack of leveling of the medial nasal processes between themselves and the lateral maxilla process. The etiology is well defined: heredity involving genetic and environmental processes with a worldwide incidence of 1:700 births. The treatment should be performed by a multidisciplinary team of experts.. One of the most important exams to support the malformation diagnosis are imaging exams, such as intra/extra oral radiographs and computed tomography because they may help in pre-surgical measuring, resulting in improvement in planning and surgical management, thereby providing a good prognosis and outcome. The present research will evaluate the cleft size in the preoperative period and the bony formation after autogenous alveolar bone graft surgery of the iliac crest through periapical radiographs. The main objective is to investigate the critical size of the maxillary defect and the level of bony formation, considering factors that could also affect the final result, such as age, presence of retained canine after orthodontic grafting treatment, and finally, to suggest standard therapeutic approaches. Patients younger than 16 years old has 6 six time more chances to form a intermediary bony septum, with a height close to normal. Good results also were found in cases of unerupted canine, with bony defect filling increased until 16 times in 75%. Patients with orthodontics treatment started in the ideal period (60-90 days after bone grafting), presented a Type I and II bone formation, with more than 50% of bone defect repaired. In conclusion, the height, width and type of alveolar fissure are factors that not influenced in bone formation.