As proteínas com potencial osteoindutor, com destaque a rhBMP-2, possuem amplos efeitos sobre o crescimento e a diferenciação celular. Este estudo, teve o objetivo avaliar a relação entre diferentes enxertos ósseos e a rhBMP-2 no reparo de defeitos ósseos críticos em calvárias de ratos. Foram utilizados 56 ratos Wistar albinos, machos, aproximadamente 250g, com um período de espera de 6 semanas desde a criação do defeito até o sacrifício, divididos em 8 grupos (n=7): enxerto autólogo (AUT); enxerto homólogo (HOM); enxerto heterólogo (HET); enxerto autólogo e 5 g rhBMP-2 (AUT BMP); enxerto homólogo e 5 g rhBMP-2 (HOM BMP); enxerto heterólogo e 5 g rhBMP-2 (HET BMP); 5 g rhBMP-2 (BMP) e defeito ósseo crítico apenas (DOC). Os resultados foram avaliados estatisticamente pelo teste ANOVA e pós-teste de TUKEY com múltiplas comparações (nível de significância de p<0,05), após o emprego de metodologias de análise histológica qualitativa, histomorfometria e micro CT. Na quantificação do volume ósseo neoformado, todos os grupos obtiveram melhores resultados que DOC com diferença significante. Os grupos HOM e HET apresentaram diferença significante de AUT BMP, HOM BMP e HET BMP que tiveram maior formação óssea enquanto que AUT apresentou diferença significante de HOM BMP e HET BMP. O grupo BMP não teve diferença significante em relação a AUT, HOM, HET, AUT BMP, HOM BMP e HET BMP. Os grupos AUT, HOM e HET não tiveram diferença significante entre si da mesma forma que os grupos AUT BMP, HOM BMP e HET BMP também não o tiveram. Quanto à quantificação de osteoclastos, não houve diferença significante entre os grupos; porém, HET BMP teve mais células osteoblásticas que DOC com diferença significante. Considerando os osteócitos, DOC e AUT apresentaram poucas células. Assim, DOC teve diferença significante de BMP, HET, HOM BMP e HET BMP e AUT teve diferença significante de BMP, HET, HOM BMP e HET BMP. Da mesma forma, AUT BMP teve diferença significante em relação a HET que apresentou mais células. Na quantificação de fibras colágenas, não houve diferença significante entre os grupos. A neoformação óssea progrediu das bordas para o centro do defeito e no grupo BMP o osso neoformado ocupou mais de 2/3 do defeito. No grupo DOC, foi constatada uma faixa estreita de tecido ósseo neoformado nas bordas e o restante do defeito foi preenchido por tecido conjuntivo. Nas imagens de micro CT, todos os grupos apresentaram maior formação óssea que o grupo DOC; sendo que AUT superou HOM e HET. Além disso, os diferentes enxertos associados à rhBMP-2 apresentaram maior formação óssea que os respectivos enxertos usados sem a proteína, com destaque para AUT BMP. Diante disso, foi possível concluir que a rhBMP-2 auxiliou o reparo ósseo quando administrada isoladamente ou associada aos diferentes enxertos ósseos, conferindo neste caso melhores resultados / Proteins with osteoinductive potential, particularly the rhBMP-2, have great effects on growth and cell differentiation. This study aimed to evaluate the relationship between different bone grafts and rhBMP-2 on the repair of critical bone defects in rat skulls. Fifty six Wistar rats, male, approximately 250g of body weight were used, sacrificed six weeks after surgical experiment, and divided into 8 groups(n=7): autograft (AUT); homograft (HOM); xenograft (HET); autograft and 5 g rhBMP-2 (AUT BMP); homograft and 5 g rhBMP-2 (HOM BMP); xenograft and 5 g rhBMP-2 (HET BMP); 5 g rhBMP-2 (BMP) and critical bone defect only (DOC). The results were analyzed statistically by ANOVA and Tukey post-test with multiple comparisons (p<0.05 of significance level), after the use of qualitative methodologies, micro CT morphology and histological analysis. According to the quantification of newly formed bone volume, all groups had better results than DOC group with a significant difference. The HOM and HET groups showed significant difference than AUT BMP, HOM BMP and HET BMP, that had higher bone formation, while AUT group was significantly different from HOM BMP and HET BMP. The BMP group had no significant difference in relation to AUT, HOM, HET, AUT BMP, HOM BMP and HET BMP. The AUT, HOM and HET groups had no significant difference between them, and also the groups AUT BMP, HOM BMP and HET BMP between them. Regarding the quantification of osteoclasts, there was no significant difference between groups; however, HET BMP had more osteoblastic cells than DOC group, with significant difference. For osteocytes quantification, AUT and DOC showed few cells. Thus, DOC had significant difference in relation to BMP, HET, HOM BMP and HET BMP; and AUT had significant difference in relation to BMP, HET, HOM BMP and HET BMP. Likewise, AUT BMP had significant difference compared to HET group, which showed more cells. Regarding to collagen fibers quantification, there was no significant difference between groups. New bone formation progressed from the edges to the center of the defect and for BMP group the newly formed bone occupied more than two thirds of the defect. DOC group showed a narrow band of newly formed bone at the edges and the remainder of the defect was filled by connective tissue. For micro CT analysis, all groups showed greater bone formation than the DOC group, being greater in AUT group than HOM and HET. Furthermore, the various grafts associated with rhBMP-2 showed greater bone formation than the isolated grafts, highlighting the AUT BMP. Therefore, it was concluded that rhBMP-2 helped bone repair when administered alone or associated with different bone grafts, giving better results when combined to the grafts

Estudos sugerem que a cafeína atua sobre o osso promovendo aumento da excreção de cálcio e inibição da proliferação de osteoblastos, aumentando o risco de fraturas, osteoporose e doença periodontal. Os efeitos da cafeína sobre o tecido ósseo dificultam a aplicação de implantes dentários devido à presença de grandes defeitos ósseos ou volume ósseo insuficiente. Vários métodos são propostos para a regeneração de defeitos ósseos, entre eles, o uso de diferentes tipos de enxertos, os quais demonstram capacidade em promover a formação óssea A despeito das desvantagens, o osso autógeno ainda é considerado a referência padrão como enxerto ósseo, devido ao seu potencial osteogênico, osteoindutor e osteocondutor. A engenharia tecidual óssea tem sido utilizada como uma estratégia para a regeneração óssea. As células tronco mesenquimais são consideradas multipotentes e podem replicar como células indiferenciadas, possuindo potencial para se diferenciarem em linhagens de osso, cartilagem, gordura e cartilagem. O objetivo deste estudo foi quantificar histomorfometricamente a reparação óssea pelo enxerto de uma associação de osso autógeno obtido da calota craniana e células osteoblásticas em defeitos ósseos produzidos pela extração dental de ratos submetidos à administração diária de cafeína. Os animais foram divididos em: Controle (c), osso autógeno (oa) e osso autógeno + células osteoblásticas (oa+co) e receberam injeções diárias intraperitonealmente de 30 mg/kg/dia de cafeína durante trinta dias, os homólogos receberam de solução salina. Os ratos foram sacrificados nos períodos de 7, 21 e 42 dias pós-cirurgia e as amostras teciduais foram processadas para a obtenção de secções finas (5 m) e coradas com HE. Através de um sistema de análise de imagens se estimou a fração de volume de osso, conjuntivo e coágulo, no defeito ósseo. Os resultados histológicos e histométricos mostraram que nos animais sob tratamento com cafeína houve uma menor formação óssea estatisticamente significante a 1%, e um retardo na reabsorção do coágulo sanguíneo quando comparado aos alvéolos dos animais sob tratamento com soro fisiológico. A análise qualitativa do fragmento de osso autógeno isoladamente ou associado às células osteoblásticas mostrou uma osteointegração progressiva e sem reação de corpo estranho nos animais tratados com soro fisiológico e, as células implantadas não propiciaram reações imunogênicas nem a formação tumoral, possibilitando um aumento (25%) na reparação óssea dos animais tratados com a cafeína. Conclui-se que o enxerto/implante das células osteoblásticas associadas ao osso autógeno da calota craniana foi capaz de compensar, nos períodos tardios, os efeitos deletérios da cafeína na reparação óssea alveolar. / Studies suggest that caffeine acts on the bone for increasing the excretion of calcium and inhibition of osteoblasts proliferation, increasing the risk of fractures, osteoporosis and periodontal disease. The effects of caffeine on bone difficult the application of dental implants due to large bone defects and insufficient bone volume. Several methods are proposed for the regeneration of bone defects, including the use of different types of grafts, which show ability to promote bone formation. Despite the disadvantages, the autogenous bone is still considered the gold standard as bone graft because the potential osteogenic, osteoinductive and osteoconductive. The bone tissue engineering has been used as a strategy for bone regeneration. The mesenchymal stem cells are considered multipotent and can replicate as undifferentiated cells, with potential to differentiate into lineages of bone, cartilage, fat and cartilage. This study aimed to quantify histomorphometrycally bone repair by grafts of a combination of autogenous bone obtained from the skull and osteoblastic cells in bone defects produced by dental extraction in rats subjected to daily administration of caffeine. The animals were divided into: Control (c), autogenous bone (ab) and autogenous bone + osteoblastic cells (ab + oc) and received daily injections intraperitoneally of 30 mg/kg/day of caffeine for thirty days, the counterparts received saline solution. The rats were sacrificed at times of 7th, 21st and 42nd days post-surgery and tissue samples were processed to obtain thin sections (5 m) and stained with HE. Through an image analysis system was estimated the fraction of volume of bone, collagen and blood clot in the bone defect. The histological and histometric results showed that in animals under treatment with caffeine had a lower bone formation statistically significant at 1%, and a delay in the resorption of blood clots when compared to the alveoli of animals under treatment with saline. The qualitative analysis of the fragment of autogenous bone alone or associated with osteoblastic cells showed a progressive osteointegration and no foreign body reaction in animals treated with saline, and implanted the cells not provided immunogenic reactions or tumor formation, allowing an increase (25%) on bone repair in animals treated with caffeine. It was concluded that the graft/implant of osteoblastic cells associated with autogenous bone from the skull was able to compensate in later periods, the deleterious effects of caffeine on alveolar boné repair.

Traumas nas asas de aves são muito comuns, causando lesões em tecidos moles e ossos, e tendem, muitas vezes a causar desvascularização da região, o que implica em complicação no tratamento além de facilitar a instalação de processo infeccioso. Com o objetivo de estudar possível vascularização via enxerto muscular pediculado, foram utilizados 12 pombos, divididos em dois grupos iguais (E e S), para a avaliação do uso de enxerto pediculado de músculo peitoral como técnica de revascularização de defeito criado em porção distal de bíceps braquial. As asas contralaterais de todos os animais foram utilizadas para a avaliação termográfica, sendo este grupo de n=12 denominado C. As asas contralaterais de dois animais foram utilizadas como controle anatômico para a avaliação histomorfométrica, grupo este denominado A. Realizaram-se avaliações clínica, termográfica, angiográfica e histomorfométrica para quantificação e qualificação da vascularização, e determinação da viabilidade desta técnica, em até vinte e um dias. A técnica causou pouca morbidade, exceto em dois animais do grupo E que apresentaram necrose parcial do enxerto, porém isto não resultou em falha da técnica. As avaliações termográficas demonstraram que imediatamente após a cirurgia, tanto leito doador, quanto leito receptor apresentaram redução da temperatura, porém esta temperatura aumentou gradativamente, sendo que no grupo E, a temperatura, aos sete dias foi mais baixa, quando compaprada ao grupo S e ao grupo A, e aos catorze dias não houve diferença significativa entre os grupos. A avaliação angiográfica post mortem determinou patência de vasos principais e secundários em todos os animais, comprovando tratar-se de técnica que causa baixa mortificação do tecido no sítio doador, e na asa. A avaliação histomorfométrica incluiu dados de área total de vasos por amostra, área total de tecido, diâmetro médio dos vasos, número absoluto de vasos, e percentagem de tecido ocupada por vasos. Tal avaliação demonstrou aumento importante da vascularização dos dois grupos avaliados, sendo que o único parâmetro que apresentou diferença entre os dois, foi o número de vasos, aumentado no grupo E. Conclui-se que o enxero apresenta neovascularização importante, e provavelmente cria ambiente adequado para promover cicatrização de lesão pobremente vacularizadas em aves. / Trauma in avian wings is very common, causing lesions in soft tissue and bones, and they tend to, many times, cause avascularization of the region, which aggravates treatment and allows for the installation of infectious processes. Twelve pigeons, divided in two equal groups (E and S) were used for the evaluation of the use of a pectoral muscle flap as revascularization technique for a distal biceps brachii defect. Contralateral wings os all animals were used for termographic evaluation, and this group with n=12 was called C. Contraletarl wings of two animals were used as anatomic control for the histomorphometric evaluation, and this group was called A. Clinical, termographic, angiographic and histomorphometric evaluations were made to quantify and qualify the vascularization, and determine the viability of this technique. The technique caused little morbidity, except for two animals in group E, who had partial necrosis of the grafts, but that didnt result in failure of the technique. Termography showed that after surgery, both, donor and receptor sites had a reduced temperature, but it increased gradually. Group E showed a lower temperature at seven days, when compared to group S and to group C, and at fourteen days, there was no significant difference between groups. Post mortem angiografic evaluation determined patency of main and secondary vessels in all animals, proving to be a low morbidity technique for the donor site and for the wing. Histomorphometric evaluation includes total area of vessels and of soft tissue, mean vessel diameter, total vessel number and percentage of soft tissue covered by vessels. This evaluation showed and important increase of vascularization of both groups, and only total number of vessels was different among groups, being bigger in group E. We conclude that the graft shows important neovascularization, and probably creates adequate environment for the healing of little vascular bird wounds.

Stroke is now a well-recognised risk factor for hip fracture. The aim of this study was to elucidate the pathophysiological mechanisms by which hip bone loss occurs in hemiplegia and to test the efficacy of a novel pharmaceutical strategy for preserving bone in stroke patients. Patients who were admitted acutely with a first-ever stroke and who remained unable to walk one week later were studied prospectively for 12 months, with a series of bone mineral density measurements of the hips (dual energy X-ray absorptiometry) in the context of a randomised controlled trial. Untreated patients (n=13) experienced a decline in bone mineral density at the hemiplegic hip that was rapid, with the greatest losses in the trochanteric region of the affected side. This bone loss was prevented by the administration of a single 4 mg dose of the intravenous bisphosphonate, zoledronate (n=14) within 35 days of stroke onset. Computed tomography of the hips in 8 untreated patients more than a year after stroke confirmed that the greatest difference between sides was in the trochanteric region. Serum vitamin D measurements in 44 patients with acute stroke were substantially lower than healthy elderly controls, with 77% of patients in the insufficient range, suggesting that vitamin D insufficiency preceded stroke. Histomorphometric analysis of iliac bone biopsies from hemiplegic patients 10 weeks following stroke showed normal erosion parameters, but a striking decrease in the surface extent of osteoid when compared with healthy reference values. Unexpectedly, treatment with zoledronate was associated with a significantly higher osteoid surface compared with placebo treated subjects in cancellous, endocortical and cortical bone. Sclerostin, a newly discovered osteocyte-derived protein was studied using immunohistochemical staining of the bone biopsies. Sclerostin is known to be an inhibitor of active osteoblasts, which led to the hypothesis that in stroke, the proportion of osteocytes expressing sclerostin would be inversely associated with the surface extent of bone formation. Histological analysis revealed widespread expression of sclerostin in osteocytes and their canaliculi in all subjects. However, examining individual osteocytes in relation to bone forming surfaces revealed that newly embedded osteocytes did not express sclerostin until after primary mineralisation. It is proposed that this precise pattern and timing of sclerostin expression by osteocytes allows bone formation to continue locally (during remodelling), but prevents excessive new bone formation elsewhere, as seen in the single gene disorder sclerosteosis.

La vascularisation joue un rôle important dans la biologie de l’os. Les souris génétiquement modifiées sont devenues le modèle de choix dans ce champ de recherche ; cependant les outils permettant son exploration structurelle (réseau capillaire) et fonctionnelle (perfusion) dans ce modèle animal, demeurent imparfaits. Notre travail a consisté en une adaptation chez la souris de la technique d’histomorphométrie quantitative vasculaire des os longs, fondée sur le remplissage du réseau par du sulfate de baryum, précédemment développée chez le rat. Nous avons, par ailleurs, mis au point une technique reproductible de mesure de la perfusion osseuse dans le tibia en utilisant le Laser Doppler. En termes d’exploration structurelle, et grâce à une imagerie par microtomographie 3D (Synchrotron ou Nano scanner) nous montrons la supériorité du sulfate de baryum sur le Microfil®, (produit associant silicone et plomb) et sa compatibilité avec les colorations histologiques usuelles et les marquages immuno histochimiques de la paroi vasculaire. Nous établissons la reproductibilité de la technique et nous proposons une nomenclature. En termes de perfusion, après avoir optimisé le protocole en limitant les facteurs de variabilité, nous montrons que le Laser Doppler permet de mesurer de façon reproductible une perfusion strictement osseuse dans le tibia de souris. Sous réserve de recourir { des groupes d’animaux de taille suffisante, (n=15), il devient possible de réaliser des comparaisons intergroupes. Ces deux approches combinées et intégrées, menées sur des souris C57BL/6 et 129sv/Cd1 nous ont permis de montrer (1) qu’il n’y a pas de corrélation entre paramètres de perfusion et de densité vasculaire, (2) que lors du vieillissement chez les mâles, la cinétique d’évolution de la vascularisation osseuse diffère selon le fond génétique, et que la perte osseuse n’est pas accompagnée d’une diminution de la perfusion dans ce modèle, (3) que l’ovariectomie (OVX) induit une diminution de la vascularisation et de la perfusion osseuses qui précède la perte osseuse. Enfin, notre étude des effets vasculaires osseux structurels et fonctionnels de la parathormone 1-84 a montré qu’ils dépendent, à l’instar de ses effets sur la masse osseuse, de son mode d’administration, intermittent ou continu et qu’ils ne sont pas impactés par l’OVX / Vascularisation plays a major role in bone biology. Genetically modified mice became the most favorite model in this research field; however, the tools allowing bone vessel analysis on both structural (capillary network) and functional aspects (perfusion) in this animal model remain unsatisfactory. Our work consisted in adapting in mice the quantitative histomorphometry technique allowing the analysis of long bone vascularization, based on the infusion of barium sulfate, which was previously developed in the rat. In addition, we set up a reproducible Laser Doppler-Based technique for measurement of mouse tibia perfusion. In terms of structural analyses, and thank to 3D micro-Tomography imaging (Synchrotron or Nano scanner), we show the superiority of barium sulfate on a lead/silicon-Based contrast product (Microfil®)and its compatibility with common staining used in histology and with immuno-Histochemsitry of the vessel wall. We establish the reproducibility of the technique and propose a nomenclature. In terms of perfusion, we show, after having optimized the protocol by limiting the factors of variability, that Laser Doppler allows to measure, in a reproducible way, perfusion signals specific to bone. As long as the animal group size is appropriate (n=15), it becomes possible to carry out intergroup comparisons. These combined and integrated approaches, carried out on C57BL/6 and 129sv/Cd1 mice allowed us to show that (1) there is no correlation between perfusion and vascular density parameters (2) during ageing in males, the kinetics of bone vascularisation evolution differ according to the genetic background and that bone loss was not associated with decrease in perfusion in this model (3) that ovariectomy (OVX) induces a decrease in both bone vessel density and perfusion which precedes bone loss. Finally, we studied the structural and functional vascular effects of 1-84 parathyroid hormone and show that, as for its effects on bone mass, they depend on its mode of administration, intermittent or continuous and that they are not affected by OVX

Orientador: Hirasilva Borba / Resumo: O objetivo deste estudo foi avaliar a qualidade física, química da carne e a histomorfometria das fibras musculares de peito amadeirado (PM), e submeter estas ao processo de maturação e produção de hambúrgures e linguiça frescal, com o intuito de obter uma aplicação industrial desta carne. O capítulo 1 aborda considerações gerais e revisão de literatura sobre o tema proposto. No capítulo 2 foram avaliadas as caracteristícas físicas, químicas e histológicas da carne de peito de frangos de corte acometidos pela miopatia PM. No capítulo 3 foram avaliados os possíveis efeitos da maturação por até sete dias sobre a qualidade da carne de peito de frangos de corte acometidos pelos diferentes graus de severidade da miopatia PM. No capítulo 4 foram avaliados a qualidade de linguiças tipo frescal fabricadas com carne de peito de frangos acometidos pelos diferentes graus de severidade da miopatia PM armazenadas por 28 dias. E no capítulo 5 foram avaliados a qualidade de hambúrgueres de frangos acometidos pelos diferentes graus de severidade da miopatia PM armazenados por 120 dias. As amostras cruas de frangos acometidos pelo grau severo da miopatia PM possuem qualidade física inferior comparada com a carne de frango normal, o que pode ser prejudicial ao processamento da carne. As carnes de grau severo da miopatia PM possui níveis superiores de docosa-hexaenoico e ácido araquidônico, além de menor teor de gordura que peitos normais. As análises histomorfométricas mostram que a miodegener... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The objective of this study was to evaluate the physical, chemical and meat quality and histomorphometry of the woody breast muscle fibers, and to submit them to the process of maturing and production of hamburgers and fresh sausages, in order to obtain an industrial application of this meat. Chapter 1 discusses general considerations and literature review on the proposed theme. In chapter 2 the physical, chemical and histological characteristics of the breast meat of broilers affected by PM myopathy were evaluated. In Chapter 3, the possible effects of maturation for up to seven days on the quality of breast meat of broilers affected by the different degrees of severity of PM myopathy were evaluated. Chapter 4 evaluated the quality of fresh sausages made with chicken breast meat affected by the different degrees of severity of PM myopathy stored for 28 days. And in Chapter 5, the quality of chicken burgers affected by different degrees of severity of PM myopathy stored for 120 days was evaluated. Raw chicken samples affected by the severe degree of PM myopathy have lower physical quality compared to normal chicken meat, which can be detrimental to meat processing. The severe meats of PM myopathy have higher levels of docosahexaenoic and arachidonic acid, and lower fat content than normal breasts. The histomorphometric analyzes show that the myocardial disease caused by PM myopathy presents muscle fiber hypertrophy as compensatory mechanism in the moderate and severe degrees ... (Complete abstract click electronic access below) / Doutor

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