The long bones grow by the process of endochondral ossification, which occurs at the growth plate. This process is regulated by biological factors and mechanical factors. The biological factors which contribute to endochondral ossification process are genes, hormones, nutrients etc. The mechanical factor is the load acting on the bone. The major forces on the bone are due to joint contact load and muscle forces, which induce stresses in the bone. Carter and Wong proposed in a theory that cyclic or intermittent octahedral shear stress promotes the bone growth and cyclic or intermittent hydrostatic compressive stress inhibits the bone growth. Previously this theory has been used to predict the morphological development of long bones, but with studies using simplified femur and growth plate models. Furthermore, the Carter and Wong theory has a limitation that it does not intrinsically incorporate the resulting growth direction.In the first study, the importance of a subject-specific growth plate over a simplified growth plate has been studied, and growth has been simulated using two different growth direction models: Femoral neck shaft deformation direction and minimum shear stress direction. This study favors the minimum shear stress growth direction model, as it is less sensitive to applied boundary condition than the femoral neck shaft deformation direction model.The second study aims to understand how different muscle groups affect the bone growth tendency. Subject-specific femur and growth plate models of able-bodied children were used. The muscle forces and associated hip contact force from specific muscle groups were applied, and neck shaft angle and femoral anteversion growth tendencies were predicted. This study indicated a tendency for reduction of neck shaft angle and femoral anteversion. Hip abductor muscle forces contribute most, and hip adductor muscle forces least, to bone growth rate.Accurate prediction of bone growth tendency and knowledge of the influence of different muscle groups on bone growth tendency may help in better treatment planning for children at risk of developing bone deformity problems. / <p>QC 20151201</p>

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19223号 / 医博第4022号 / 新制||医||1010(附属図書館) / 32222 / 京都大学大学院医学研究科医学専攻 / (主査)教授 妻木 範行, 教授 開 祐司, 教授 松田 秀一 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(薬科学) / 甲第21047号 / 薬科博第90号 / 新制||薬科||10(附属図書館) / 京都大学大学院薬学研究科薬科学専攻 / (主査)教授 竹島 浩, 教授 中山 和久, 教授 金子 周司 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

BACKGROUND/OBJECTIVE: Converging evidence in recent years suggests growth chondrocytes, involved in the integral process of endochondral bone formation and fracture healing, exhibit a dynamic bioenergetic profile despite residing in the nutrient poor cartilaginous environment. Specifically, chondrocytes show an increased dependence on mitochondrial derived oxidative phosphorylation during differentiating, collagen product, but to a differing extent when mineralizing. Therefore, quantitative analysis of mitochondrial dynamics during these varying processes serves to corroborate existing metabolic studies and further elucidate the role of oxidative metabolism during the endochondral process. METHODS: The murine chondroprogenitor cell line ATDC5 was used, and groups were cultured in differentiating, collagen promoted, and mineralizing conditions. Fluorescence confocal 3D image acquisition and bioimaging analysis was used to quantify changes in mitochondrial volume and branch length per mitochondria along with organization and colocalization changes of the actin cytoskeleton to mitochondria in the various conditions over 21 days. RESULTS: We showed that chondrocyte differentiation resulted in significantly increased mitochondrial volume and fusion when compared to non-differentiating groups, and in collagen promoted groups, mitochondrial volume was significantly higher. Additionally, we showed that the process of mineralization resulted in a significant decrease in mitochondrial volume and branch length per mitochondria by day 21 of the experiment. Finally, colocalization analyses of the actin cytoskeleton to mitochondria showed significantly increased overlap in non-differentiating cells when compared to differentiating conditions. CONCLUSIONS: These findings suggest that collagen production is likely an energetically taxing process and mineralization does not heavily rely on oxidative metabolism. Furthermore, the actin cytoskeleton likely plays a role in mitochondrial remodeling that coincides with mitochondrial fission and fusion; increased fission is associated with actin accumulation to mitochondria and fusion is associated with actin disassociation from the outer mitochondrial membrane.

The growth plate resting zone consists of hyaline-like chondrocytes disbursed in a proteoglycan rich extracellular matrix. These cells give rise to the columns of the growth zone, consisting of progressively hypertrophic cells. Proliferation of resting zone chondrocytes induced by systemic and local stimuli is the driving force of longitudinal growth of long bones. Therefore, homeostasis of this cell population has great importance. Although the regulation of proliferation and differentiation of these cells has been well studied, little is known about the regulation of their apoptosis. We have previously shown that chelerythrine and tamoxifen induce apoptosis in resting zone chondrocytes in a nitric oxide (NO)-dependent pathway. In this study we explored two physiological apoptogens: inorganic phosphate (Pi) and 17β-estradiol (E₂). We found NO production is necessary in Pi-induced apoptosis. We also found that NO donors induced chondrocyte apoptosis by up-regulating p53 expression, Bax/Bcl-2 expression ratio and cytochrome C release from mitochondria, as well as caspase-3 activity, indicating that NO induces chondrocyte apoptosis in a mitochondrial pathway. Mitogen activated protein kinase (MAPK) activity was involved. A c-Jun N-terminal kinase (JNK) inhibitor, but not inhibitors of p38 or extracellular signal-regulated kinase (ERK1/2), was able to block NO-induced apoptosis, indicating that JNK is necessary in this pathway. Taken together, Pi elevates NO production, which leads to a mitochondrial apoptotic pathway dependent on JNK. On the other hand, although E₂caused apoptosis in resting zone chondrocytes in a dose-dependent manner, up-regulated p53 and Bax, and induced release of cytochrome C from the mitochondria, which indicated a mitochondrial apoptotic pathway, the apoptosis did not involve elevated nitric oxide production or MAPK as was found in Pi-induced apoptosis. This study elucidates the signaling pathway underlying Pi and E₂-induced chondrocyte apoptosis. It has important implications on understanding the development of mammalian growth plate. It also provides further information about the physiological functions of estrogen on longitudinal bone growth.

Growth plate injuries account for 15-30% of long bone fractures in children. About 10% of these result in significant growth disturbances due to formation of a boney bar. If not treated correctly, this can lead to life-lasting consequences of limb length inequalities and angular deformities. Current treatments for growth plate injuries include removal of boney bar and insertion of fat, silicone, bone cement, etc.. This treatment y is inadequate, leaving almost half of these patients with continued deformities. This dissertation reports characterization of a DNA–containing porous poly(lactic-co-glycolic acid) (PLGA) scaffold system, chondrogenesis using insulin-like growth factor I (IGF-I) plasmid-releasing scaffolds in vitro, and in vivo testing of IGF-I plasmid-releasing scaffolds to regenerate growth plate . Controlled release of naked and DNA complexed with polyethylenimine (PEI) was achieved from porous PLGA scaffolds. PEI affected release of complexes from PLGA scaffolds, as PEI:DNA complexes were released at a lower rate compared to naked DNA encapsulated in low molecular weight (LMW) and high molecular weight PLGA scaffolds, as well as hydrophilic and hydrophobic PLGA scaffolds. Hydrophilicity and molecular weight of PLGA affected the release profiles of both naked DNA and PEI:DNA complexes from the scaffolds, as evidenced by later peak DNA and PEI:DNA release with increasing hydrophilicity and molecular weight. LMW hydrophilic PLGA scaffolds supported growth and chondrogenic differentiation of mesenchymal multipotent D1 cells, chondrocytes, and bone marrow cells (BMCs) in vitro. Culturing BMCs on IGF-I plasmid-encapsulated scaffolds resulted in elevated expression of IGF-I compared to blank scaffolds. Removal of boney bar and implantation of IGF-I plasmid-releasing LMW PLGA scaffolds in a rabbit model of growth plate injury resulted in some improvement of leg angular deformity compared to no scaffold implantation. Histological analysis of the newly developed cartilage showed growth plate-like columnar arrangement of chondrocytes in a defect that received IGF-I plasmid encapsulated scaffold, although the level of organization of newly formed cartilage was inferior to that of native growth plate. This appears to be the first report of the regeneration of growth plate-like structure without the use of stem cells in an animal model of physeal injury.

A desnutrição é uma condição clínica decorrente da deficiência de um ou mais nutrientes essenciais para homeostase do organismo. Em crianças continua a ser um dos problemas mais graves de saúde pública no mundo, em virtude de sua grandeza e consequências desastrosas para o crescimento, desenvolvimento e sobrevivência, o acarretando graves comprometimentos na idade adulta. O osso é extremamente sensível a mudanças metabólicas, hormonais e nutricionais. Alterações internas e externas ao organismo podem interferir na aquisição de massa óssea máxima, levar a doenças osteometabólicas e afetar o crescimento de ossos longos. Uma alimentação adequada, com fornecimento de todos os nutrientes necessários ao organismo é crucial para o desenvolvimento do sistema esquelético. No entanto, os estudos de qualidade óssea e crescimento no desnutrido ainda são poucos na literatura. Sendo assim, o nosso objetivo foi avaliar os efeitos da restrição alimentar no tecido ósseo (trabecular e cortical) e na cartilagem de crescimento de ratos em crescimento. Neste estudo, a qualidade do tecido ósseo e da cartilagem de crescimento das tíbias foi analisada em ratos normais e desnutridos. Foram utilizados 72 ratos machos, com massa corpórea inicial de 70 a 80 gramas, que permaneceram alojados em gaiolas individuais e foram aleatoriamente divididos em 2 grupos experimentais com dois subgrupos cada, conforme o tempo de exposição à restrição alimentar: CON1: ratos normais, observados por 56 dias; RA1: ratos submetidos à restrição alimentar, observados por 56 dias; CON2: ratos normais, observados por 70 dias; RA1: ratos submetidos à restrição alimentar, observados por 70 dias. A desnutrição foi induzida por meio da restrição alimentar em 50%, ou seja, metade da quantidade ingerida pelos animais controles. Ao término do período experimental, após 56 e 70 dias, os ossos foram submetidos às análises macroscópica, microtomografia computadorizada (avaliação qualitativa e quantitativa da microestrutura óssea), densitometria óssea (DXA), microscopia óptica (caracterização dos tecidos), histomorfometria (quantificação de colágeno e volume trabecular), de expressão gênica e ensaio mecânico (avaliação da resistência mecânica). O nível de significância estatística foi de 5%. Embora a desnutrição não tenha resultado em alterações na expressão gênica, várias alterações fenotípicas foram observadas na cartilagem de crescimento (diminuição do volume; redução da área total, área de ossificação e espessura; alterações celulares nas zonas hipertrófica e proliferativa; tecido mais fraco na resistência ao cisalhamento), osso trabecular e cortical (redução no tamanho e massa óssea; menor densidade óssea; deterioração na microarquitetura trabecular e cortical e; menos trabéculas com menor deposição de colágeno). Concluiu-se que a restrição alimentar causou efeitos deletérios para a cartilagem de crescimento, osso trabecular e cortical, indicando interferência nos mecanismos de ossificação osteocondral e intramembranosa. / Clinical undernutrition is characterized by the depletion of one or more nutrients that are essential for human homeostasis. Undernutrition in children results in a serious health impact due to the impairment in growth and bone quality in adulthood. Several factors may interfere with the peak mass achieved by an individual and interfere in growth and osteometabolic disorders. An adequate nutritional intake is an essential factor for bone development. However, few studies have investigates the relation between bone quality and growth in malnourished subjects. Therefore, we aimed to study the effects of food restriction on the growth plate and bone tissue (trabecular and cortical) in growing rats. 72 male Wistar rats weighing approximately 70-80g were randomly assigned into two groups and, subsequently subdivided into two subgroups according to the experimental follow-up: CON1: control rats followed by 56 days; RA1: food restricted rats followed by 56 days; CON2: control rats followed by 70 days and, RA2: food restricted rats followed by 70 days. After weaning, food restricted rats were fed 50% of the ad libitum food intake. At the end of each experimental period, the rats were euthanized and tibias were analyzed by morphological measurements, micro-computed tomography (qualitative and quantitative analysis of bone microstructure), dual-energy X-ray absorptiometry (DXA), optical microscopy (tissue characterization), histomorphometry (trabeculae quantity and collagen deposition), gene expression and mechanical test (mechanical strength assessment). Statistical significance was set when p<0.05. Although undernutrition did not alter gene expression, several phenotypic changes were seen at the growth plate (i.e. decreased in growth plate volume, reduction in total growth plate area and thickness, decrease in area of proliferative, hyperthrofic and ossified zones and, decrease in hyperthrofic cells and lesser mechanical resistance), trabecular and cortical bone (i.e. reduction in bone size and mass, bone density, deterioration at the trabecular and cortical microstructure, reduction in trabeculae bone and collagen deposition) of growing rats subjected to food restriction. We concluded that food restriction caused detrimental effects on growth plate, trabecular bone and cortical shaft, evidencing deleterious changes in the mechanisms of osteochondral and intramembranous bone formation.

A subnutrição tem sido ao longo dos anos um dos maiores problemas de saúde pública do mundo, que acomete principalmente crianças de países em desenvolvimento. Estima-se que em Moçambique na África Oriental, cerca de 40% das crianças são acometidas pela desnutrição crônica que resulta dentre outras afecções em baixa estatura e diminuição da capacidade física. Este estudo visa avaliar a estrutura óssea de ratos wistar submetidos à dieta básica da população de Moçambique (DM) reproduzida em laboratório. Para isso a DM foi oferecida aos animais em fase pré e pós-natal durante 42 dias. Para alguns desses animais foi fornecida a DM acrescida de 20% de caseína (DMC) subsequentemente os grupos subnutrido de Moçambique (SM) e nutrido de Moçambique (NM). Foi formado também um grupo denominado renutrido (RM) composto por animais que receberão a DMC a partir do 22&ordm; dia e até completarem 42 dias de vida. Após o desmame foram mensurados os parâmetros metabólicos da ingestão alimentar e hídrica, excreção de urina e fezes. A tíbia e o úmero do lado direito foram macerados quimicamente a fim de verificar as medidas ósseas. Esses ossos do lado esquerdo foram processados com técnicas rotineiras histológicas e corados de forma a evidenciar as estruturas cartilagineas. Os dados obtidos foram submetidos a análise de variância paramétrica seguida por comparações múltiplas pelo método de Tukey, com nível de significância p&lt;0,05. Os resultados apontam que a Subnutrição é capaz de modificar a espessura da CA e da LE e os componentes de sua MEC. Embora a inserção de caseína na dieta dos animais SM, tenha reestabelecido parâmetros da osteometria, na LE e na CA a melhora apresentada, não foi o suficiente para atingir seus parâmetros normais apresentados no grupo NM / Undernutrition has been over the years one of the major public health problems worldwide, which affects mainly children in developing countries. It is estimated that in Mozambique in East Africa, about 40% of children are affected by chronic undernutrition resulting from other conditions in stature and diminished physical capacity. This study aims to evaluate the bone structure of Wistar rats submitted to the staple diet of the population of Mozambique (DM) reproduced in the laboratory. To this DM was given to the animals in pre and postnatal 42 days. For some of these animals was provided to DM plus 20% casein (DMC) subsequently the undernourished groups of Mozambique (SM) and nursed Mozambique (NM). Also it has formed a group called renutrido (RM) consists of animals that receive the DMC from the 22nd day until completing 42 days of life. After weaning metabolic parameters were measured food and water intake, urine output and feces. The tibia and the humerus on the right side were chemically macerated in order to verify the bone measurements. These bones on the left side were processed with routine histological techniques, and stained to show the form cartilage structures. The data were subjected to parametric analysis of variance followed by multiple comparisons by the Tukey method, with significance level of p <0.05. The results show that the Malnutrition is able to modify the thickness of the CA and LE and the components of its MEC. Although the inclusion of casein in the diet of animals SM, has reestablished osteometria parameters in LE and CA improvement presented, was not enough to achieve their normal parameters presented in NM group

Esta pesquisa teve como objetivo avaliar o desempenho e o desenvolvimento da discondroplasia tibial (DT) em frangos de corte de 1 a 21 dias. Foram utilizados 440 pintinhos machos de um dia, provenientes de matrizes com 60 a 62 semanas de idade, distribuídos em delineamento inteiramente casualisado em esquema fatorial 3x3+2, resultando em 11 tratamentos com quatro repetições de 10 aves cada. Os fatores estudados foram: linhagem da ave (Ross 308, Cobb 500 e Hybro), níveis e fontes de vitamina D (1250UI D3/kg sem 25-(OH)D3; 1250UI D3/kg com 69mg 25-(OH)D3/ton e 3000UI D3/kg com 69mg 25-(OH)D3/ton) e dois tratamentos controles com níveis de cálcio e fósforo segundo Rostagno et al. (2005) com 3000UI D3/kg sem 25-(OH)D3 e 3000UI D3/kg com 69mg 25-(OH)D3/ton de ração. Foram avaliadas características de desempenho e ósseas. No período de 1 a 21 dias, os resultados indicaram que o ganho de peso, conversão alimentar e as concentrações de cálcio e fósforo nas tíbias não foram influenciadas pelos tratamentos e que o consumo de ração foi superior para os tratamentos que compóem o fatorial. A resistência óssea também não foi influenciada pelos tratamentos e a análise histológica não evidenciou lesões características de DT. Conclui-se que, nas condições experimentais da presente pesquisa, o desenvolvimento de DT não foi observado. / This research was carried out to evaluate the performance and the development of tibial dyschondroplasia (TD) in broilers from 1 to 21 days. Four hundred forty day-old male chickens, from broiler breeders aged 60-62 weeks, were randomly distributed in a 3x3+2 factorial arrangement, resulting in 11 treatments with four replicates of 10 birds each. The factors evaluated were: bird strains (Ross 308, Cobb 500, and Hybro), levels and sources of vitamin D (1250UI D3/kg without 25-(OH)D3; 1250UI D3/kg with 69mg 25-(OH)D3/ton, and 3000UI D3/kg with 69mg 25-(OH)D3/ton), and two control treatments containing calcium and phosphorus levels according to Rostagno et al. (2005) with 3000UI D3/kg without 25-(OH)D3 and 3000UI D3/kg with 69mg 25-(OH)D3/ton of feed. Performance and bone characteristics were evaluated. From 1 to 21 days, the results showed that weight gain, feed conversion, and bone calcium and phosphorus concentrations were not influenced by the treatments, however, feed intake was higher for factorial than control treatments. Bone breaking resistance was not influenced by the treatments and there were no typical lesions of TD. In conclusion, it was not observed TD development in broilers from 1 to 21 days according to experimental procedures of this research.

A subnutrição tem sido ao longo dos anos um dos maiores problemas de saúde pública do mundo, que acomete principalmente crianças de países em desenvolvimento. Estima-se que em Moçambique na África Oriental, cerca de 40% das crianças são acometidas pela desnutrição crônica que resulta dentre outras afecções em baixa estatura e diminuição da capacidade física. Este estudo visa avaliar a estrutura óssea de ratos wistar submetidos à dieta básica da população de Moçambique (DM) reproduzida em laboratório. Para isso a DM foi oferecida aos animais em fase pré e pós-natal durante 42 dias. Para alguns desses animais foi fornecida a DM acrescida de 20% de caseína (DMC) subsequentemente os grupos subnutrido de Moçambique (SM) e nutrido de Moçambique (NM). Foi formado também um grupo denominado renutrido (RM) composto por animais que receberão a DMC a partir do 22&ordm; dia e até completarem 42 dias de vida. Após o desmame foram mensurados os parâmetros metabólicos da ingestão alimentar e hídrica, excreção de urina e fezes. A tíbia e o úmero do lado direito foram macerados quimicamente a fim de verificar as medidas ósseas. Esses ossos do lado esquerdo foram processados com técnicas rotineiras histológicas e corados de forma a evidenciar as estruturas cartilagineas. Os dados obtidos foram submetidos a análise de variância paramétrica seguida por comparações múltiplas pelo método de Tukey, com nível de significância p&lt;0,05. Os resultados apontam que a Subnutrição é capaz de modificar a espessura da CA e da LE e os componentes de sua MEC. Embora a inserção de caseína na dieta dos animais SM, tenha reestabelecido parâmetros da osteometria, na LE e na CA a melhora apresentada, não foi o suficiente para atingir seus parâmetros normais apresentados no grupo NM / Undernutrition has been over the years one of the major public health problems worldwide, which affects mainly children in developing countries. It is estimated that in Mozambique in East Africa, about 40% of children are affected by chronic undernutrition resulting from other conditions in stature and diminished physical capacity. This study aims to evaluate the bone structure of Wistar rats submitted to the staple diet of the population of Mozambique (DM) reproduced in the laboratory. To this DM was given to the animals in pre and postnatal 42 days. For some of these animals was provided to DM plus 20% casein (DMC) subsequently the undernourished groups of Mozambique (SM) and nursed Mozambique (NM). Also it has formed a group called renutrido (RM) consists of animals that receive the DMC from the 22nd day until completing 42 days of life. After weaning metabolic parameters were measured food and water intake, urine output and feces. The tibia and the humerus on the right side were chemically macerated in order to verify the bone measurements. These bones on the left side were processed with routine histological techniques, and stained to show the form cartilage structures. The data were subjected to parametric analysis of variance followed by multiple comparisons by the Tukey method, with significance level of p <0.05. The results show that the Malnutrition is able to modify the thickness of the CA and LE and the components of its MEC. Although the inclusion of casein in the diet of animals SM, has reestablished osteometria parameters in LE and CA improvement presented, was not enough to achieve their normal parameters presented in NM group