No description available.

Submitted by Sandro Camargo (sandro.camargo@unipampa.edu.br) on 2015-05-07T22:56:27Z No. of bitstreams: 1 126110041.pdf: 1662239 bytes, checksum: 4c1751ff15692d571713c45fca07289c (MD5) / Made available in DSpace on 2015-05-07T22:56:27Z (GMT). No. of bitstreams: 1 126110041.pdf: 1662239 bytes, checksum: 4c1751ff15692d571713c45fca07289c (MD5) Previous issue date: 2014-03-24 / Células tronco são células indiferenciadas e multipotentes, capazes de se diferenciar nos mais diversos tipos celulares. São definidas por duas características principais, a autorrenovação e o potencial de diferenciação. Devido ao seu potencial de diferenciação, facilidade de isolamento e expansão em cultura, as células tronco mesenquimais (Mesenchymal Stem Cell – MSCs) são largamente utilizadas em testes para o tratamento de inúmeras doenças, como doenças cardíacas, mieloma e osteoartrite. O corticosteroide dexametasona é amplamente utilizado como indutor de diferenciação óssea em células tronco e está envolvido no aumento da atividade da enzima fosfatase alcalina (ALP). Há quatro genes que codificam a ALP: ALPL, ALPP, ALPPL2 e ALPI. A ALPL é um dos principais marcadores de diferenciação óssea promovida pelo tratamento com dexametasona, esse gene possui três diferentes variantes que são geradas por splicing alternativo. Porém, até hoje não se tem conhecimento de qual dessas variantes atua na deposição de matriz óssea. O objetivo do trabalho foi analisar, por PCR quantitativo (qPCR), a atividade das variantes de ALPL durante a diferenciação de MSCs tratadas com dexametasona. Os primers das três variantes do gene ALPL foram desenhados pelo nosso grupo de pesquisa. Para o experimento, aproximadamente 3x10 4 células por mL foram semeadas em placa de cultura. Para o tratamento utilizou-se dexametasona 10 -7 M, tendo como período de tratamento 0, 3 e 7 dias. Os resultados da qPCR mostraram que os primers desenhados possuem uma eficiência superior a 90% e R 2 = 1, sendo os primers da variante 2 mais eficiente que os primers da variante 1, e os primers de ambas as variantes mais eficientes que os primers de ALP atualmente utilizados. Os primers da ALPL/v3 não apresentaram amplificação. Nossa análise estatística indica que a diferença de expressão entre as células tratadas e os controles se mostrou relevante, apontando para um grande aumento de ALPL/v1 nas células tratadas. A expressão de ALPL/v2, apesar de significativa entre os controles e tratados, é baixa quando comparada à ALPL/v1. Os resultados obtidos neste trabalho indicam que a dexametasona, além de aumentar a atividade do promotor de ALPL, também dá preferência ao splicing alternativo que origina o mRNA da variante 1 do gene. / Stem cells are undifferentiated and pluripotent cells that are able to differentiate into several cell types. Stem cells are distinguished from other cell types by two important characteristics: self-renewal and differentiation potential. Due to their differentiation potential and easiness for isolate and growth in vitro, mesenchymal stem cells (MSCs) are widely used in trials for the treatment of numerous diseases such as heart disease, myeloma and osteoarthritis. The corticosteroid dexamethasone is broadly used to induce bone differentiation in stem cells and is involved in the increased activity of alkaline phosphatase (ALPL). There are four genes coding ALP: ALPL, ALPP, ALPPL2 and ALPI. The ALPL is one of the main markers for bone differentiation stimulated by dexamethasone. This gene has three different variants which are generated by alternative splicing. However, to date it is not known which of these variants act in the bone matrix deposition. In this context, the aim of this work was to analyze the activity of ALPL variants during the differentiation of MSCs treated with dexamethasone. Primers for the three ALPL variants were designed by our group. For the experiment, approximately 3x10 4 cells per ml were seeded in culture plate. For treatment, we used 10 -7 M dexamethasone for 0, 3 and 7 days. The qPCR results showed that the primers designed had an amplification efficiency higher than 90% and R 2 = 1; variant 2 (V2) primers was more efficient than the variant 1 (V1) primers and primers of both variants are more efficient than the currently used ALP primers. The primers of ALPL/v3 showed no amplification. Our statistical data showed that the differences in expression between treated and control cells were significant, indicating an expressive increase of ALPL/v1 in treated cells. The expression of ALPL/v2, although significant between controls and treated cells, was low when compared to ALPL/v1. The results of this study indicate that dexamethasone, besides increasing the activity of the ALPL promoter, gives preference to alternative splicing that originates the variant1 mRNA.

La myélofibrose primitive (MFP) est un néoplasme myéloprolifératif chromosome Philadelphie négatif rare, mais de pronostic sévère. Elle se caractérise par une prolifération clonale et une mobilisation des cellules souches et progéniteurs hématopoïétiques (CSH/PH) de la moelle osseuse vers la rate et le foie. Cette anomalie de l’hématopoïèse est associée à une pathologie du stroma (myélofibrose, ostéosclérose et néoangiogenèse). L’existence d’anomalies moléculaires de la CSH/PH telles que les mutations de Jak2, Mpl, TET2 ou CALR ne permet pas à elle seule d’expliquer la physiopathologie de la maladie. Les résultats obtenus dans le laboratoire suggèrent que le microenvironnement médullaire au sein des niches hématopoïétiques et en particulier les cellules stromales mésenchymateuses (CSM), participe vraisemblablement à cette dérégulation de l’hématopoïèse, favorisant le développement du clone pathologique. Cependant, aucune preuve tangible d’une altération des CSM médullaires n’a été jusqu’à présent apportée.Dans ce travail, nous avons isolé les CSM de la moelle de patients atteints de MFP et réalisé une caractérisation « complète » de ces cellules : prolifération, phénotype, soutien de l’hématopoïèse, sécrétome, transcriptome, miRNome et capacités de différenciation. Nos résultats ont permis de dégager un faisceau d’arguments en faveur d’une dérégulation de leur différenciation ostéoblastique (DOB). (i) Les cytokines BMP2, RANTES, PDGF, TGF-β1, VEGF et Il-6 sont significativement produites en plus grande quantité par ces cellules. (ii) L’étude du transcriptome a révélé une expression significativement différente d’un ensemble de gènes impliqués dans la DOB tels que RUNX2, DLX5, TWIST1 et NOGGIN. (iii) De nombreux micro-ARN, dont certains sont connus pour être impliqués dans la DOB comme miR-210 ou dans le nichage des cellules souches hématopoïétiques comme miR-34a, sont dérégulés à l’état basal et au cours de cette DOB. (iv) Enfin, l’étude de leurs capacités de différenciation ostéoblastique in vitro et in vivo chez la souris immunodéprimée est en faveur d’une augmentation de ces capacités. Nous avons étudié l’impact du TGF- β1 dans cette DOB. Nous avons mis en évidence que les CSM de malades présentent un état basal d'activation de la voie de signalisation pSmad significativement augmenté, confirmant l’expression endogène de TGF-β1. En utilisant des inhibiteurs spécifiques du récepteur de type I au TGF- β, nous avons montré l’implication de cette cytokine dans les altérations de la DOB. En conclusion, notre travail montre pour la première fois que les CSM des malades de MFP sont anormales et ce indépendamment de la stimulation par le clone hématopoïétique pathologique, suggérant la présence d'anomalies constitutives ou acquises. Ces anomalies impliquent deux acteurs majeurs de la pathologie : le TGF-β1 et l'ostéogenèse. / Primary myelofibrosis (PMF) is a Philadelphia-negative myeloproliferative neoplasm, rare but associated with a poor prognosis. Its features are a clonal proliferation and an egress of hematopoietic stem cells (HSC) from bone marrow to spleen. These abnormalities of hematopoiesis are in relation with a pathological stroma (myelofibrosis, osteosclerosis and neoangiogenesis). Molecular abnormalities present in HSC partially explain the physiopathology of the disease. Results from our lab suggest that the bone marrow micro-environnement, especially mesenchymal stromal cells (MSC), are involved in the deregulation of hematopoiesis, promoting the clonal cells. However, there is no strong evidence of bone marrow MSC alterations reported for now.In our study, we isolated MSC from bone marrow of patients suffering from PMF and performed a broad characterization: proliferation, phenotype, hematopoiesis supporting capacities, secretome, transcriptome and miRNome analysis. Our results highlight arguments in favor of a deregulation of their osteogenic capacities. (i) Cytokines NMP2, RANTES, PDGF, TGF-β1, VEGF and Il-6 were significantly overproduced by MSCs. (ii) Transcriptome analysis revealed a specific signature involving genes participating in osteogenic differentiation such as RUNX2, DLX5, TWIST1 and NOGGIN. (iii) Many micro-RNAs, some know to be involved in osteogenic differentiation regulation, as mir-34a, are deregulated in MSCs and in MSC-derived osteoblasts. (iv) Finally, study of their osteogenic potency in vitro and in vivo in nude mice showed an increasing of their osteogenic potency. We studied the impact of TGF-β1 in this process and showed that PMF MSCs showed a basal expression of Smad pathway significantly increased as compared to control. Using specific inhibitor of TGF-β1 receptor, we demonstrated the implication of this cytokine in the osteogenic impairment.To summarize, our work shows for the first time that MSCs from PMF patients are abnormal, independently from stimulation by clonal cells, suggesting intrinsic abnormalities. These abnormalities involve two main factor of the disease: TGF-β1 and osteogenesis.

A descoberta das células tronco adultas tornou possível a sua aplicação clínica em protocolos de medicina regenerativa e de engenharia de tecidos para a reparação de órgãos danificados ou pouco funcionais. Neste sentido, são conhecidas muitas fontes diferentes de células tronco adultas, entre elas o tecido epitelial, o tecido muscular e a medula óssea. Recentemente, o tecido adiposo adulto foi reconhecido como uma fonte alternativa, acessível e rica em células tronco mesenquimais (MSCs), também denominadas de células tronco derivadas do tecido adiposo (hADSCs). As hADSCs são as células tronco adultas que apresentam a maior plasticidade, e são facilmente isoladas pela característica de aderência a substratos plásticos. Sob condições específicas de cultivo, as hADSCs podem diferenciar-se em células precursoras osteoblásticas. Dentre as proteínas necessárias para a diferenciação das hADSCs, o papel do complexo minichromosome maintenance (MCM) não é muito bem conhecido. A proteína MCM2 é parte do complexo MCM, fazendo parte do complexo pré-replicativo (pre-RC). Em células proliferativas, o gene MCM2 apresenta alta expressão, sendo que o seu produto gênico está relacionado com os mecanismos de reparação de DNA. Sendo assim, analisamos as mudanças na expressão do gene MCM2 durante a osteodiferenciação das hADSCs em diferentes meios de cultura, contendo BMP-4 ou OM. Os genes relacionados com a osteodiferenciação, como osteocalcina (OC), osteopontina (OP) e fosfatase alcalina (ALP) foram analisados por RT-PCR, por PCR em tempo real e citoquímica. Os dados obtidos pelas técnicas citoquímicas e de biologia molecular indicaram uma diminuição da expressão do gene MCM2 durante a osteodiferenciação, que pode estar relacionada com danos ao DNA ou senescência nas hADSCs. / The discovery of adult stem cells is a promise field in regenerative medicine and tissue engineering. There are many sources of adult stem cells among which of whom are the skin, muscle and bone marrow. Currently, the adipose tissue has been an alternative source of mesenchymal stem cells (MSCs), termed human adipose-derived stem cells (hADSCs). The MSCs are the most plastic adult stem cells known until now and are easily isolated by adherence in plastic substrates. Under specifics conditions of culture hADSCs can be differentiated in osteoblast cell precursors. Among proteins needed for hADSCs differentiation, the roles of minichromosome maintenance (MCM) complex remains poorly understood. The protein complex MCM2-7 is part of DNA pre-replicative complex (pre-RC), being abundant in proliferating cells and involved in DNA repair mechanisms. In this sense, the Mcm2 is essential for the activity of the MCM complex. Thus, we analysed MCM2 gene expression changes during osteoinduction by culturing hADSCs in different media containing BMP-4 or in osteogenic medium. Genes related to osteodifferentiation, like osteocalcin (OC), osteopontin (OP) and alkaline phosphatase (ALP) were analysed by molecular and cytochemistry approaches during osteodifferentiation. Interestingly, we observed a decrease in the MCM2 gene level osteoinduction of hADSCs in osteogenic medium, which could be related to an increase in genome damage and senescence in hADSCs.

A descoberta das células tronco adultas tornou possível a sua aplicação clínica em protocolos de medicina regenerativa e de engenharia de tecidos para a reparação de órgãos danificados ou pouco funcionais. Neste sentido, são conhecidas muitas fontes diferentes de células tronco adultas, entre elas o tecido epitelial, o tecido muscular e a medula óssea. Recentemente, o tecido adiposo adulto foi reconhecido como uma fonte alternativa, acessível e rica em células tronco mesenquimais (MSCs), também denominadas de células tronco derivadas do tecido adiposo (hADSCs). As hADSCs são as células tronco adultas que apresentam a maior plasticidade, e são facilmente isoladas pela característica de aderência a substratos plásticos. Sob condições específicas de cultivo, as hADSCs podem diferenciar-se em células precursoras osteoblásticas. Dentre as proteínas necessárias para a diferenciação das hADSCs, o papel do complexo minichromosome maintenance (MCM) não é muito bem conhecido. A proteína MCM2 é parte do complexo MCM, fazendo parte do complexo pré-replicativo (pre-RC). Em células proliferativas, o gene MCM2 apresenta alta expressão, sendo que o seu produto gênico está relacionado com os mecanismos de reparação de DNA. Sendo assim, analisamos as mudanças na expressão do gene MCM2 durante a osteodiferenciação das hADSCs em diferentes meios de cultura, contendo BMP-4 ou OM. Os genes relacionados com a osteodiferenciação, como osteocalcina (OC), osteopontina (OP) e fosfatase alcalina (ALP) foram analisados por RT-PCR, por PCR em tempo real e citoquímica. Os dados obtidos pelas técnicas citoquímicas e de biologia molecular indicaram uma diminuição da expressão do gene MCM2 durante a osteodiferenciação, que pode estar relacionada com danos ao DNA ou senescência nas hADSCs. / The discovery of adult stem cells is a promise field in regenerative medicine and tissue engineering. There are many sources of adult stem cells among which of whom are the skin, muscle and bone marrow. Currently, the adipose tissue has been an alternative source of mesenchymal stem cells (MSCs), termed human adipose-derived stem cells (hADSCs). The MSCs are the most plastic adult stem cells known until now and are easily isolated by adherence in plastic substrates. Under specifics conditions of culture hADSCs can be differentiated in osteoblast cell precursors. Among proteins needed for hADSCs differentiation, the roles of minichromosome maintenance (MCM) complex remains poorly understood. The protein complex MCM2-7 is part of DNA pre-replicative complex (pre-RC), being abundant in proliferating cells and involved in DNA repair mechanisms. In this sense, the Mcm2 is essential for the activity of the MCM complex. Thus, we analysed MCM2 gene expression changes during osteoinduction by culturing hADSCs in different media containing BMP-4 or in osteogenic medium. Genes related to osteodifferentiation, like osteocalcin (OC), osteopontin (OP) and alkaline phosphatase (ALP) were analysed by molecular and cytochemistry approaches during osteodifferentiation. Interestingly, we observed a decrease in the MCM2 gene level osteoinduction of hADSCs in osteogenic medium, which could be related to an increase in genome damage and senescence in hADSCs.

A descoberta das células tronco adultas tornou possível a sua aplicação clínica em protocolos de medicina regenerativa e de engenharia de tecidos para a reparação de órgãos danificados ou pouco funcionais. Neste sentido, são conhecidas muitas fontes diferentes de células tronco adultas, entre elas o tecido epitelial, o tecido muscular e a medula óssea. Recentemente, o tecido adiposo adulto foi reconhecido como uma fonte alternativa, acessível e rica em células tronco mesenquimais (MSCs), também denominadas de células tronco derivadas do tecido adiposo (hADSCs). As hADSCs são as células tronco adultas que apresentam a maior plasticidade, e são facilmente isoladas pela característica de aderência a substratos plásticos. Sob condições específicas de cultivo, as hADSCs podem diferenciar-se em células precursoras osteoblásticas. Dentre as proteínas necessárias para a diferenciação das hADSCs, o papel do complexo minichromosome maintenance (MCM) não é muito bem conhecido. A proteína MCM2 é parte do complexo MCM, fazendo parte do complexo pré-replicativo (pre-RC). Em células proliferativas, o gene MCM2 apresenta alta expressão, sendo que o seu produto gênico está relacionado com os mecanismos de reparação de DNA. Sendo assim, analisamos as mudanças na expressão do gene MCM2 durante a osteodiferenciação das hADSCs em diferentes meios de cultura, contendo BMP-4 ou OM. Os genes relacionados com a osteodiferenciação, como osteocalcina (OC), osteopontina (OP) e fosfatase alcalina (ALP) foram analisados por RT-PCR, por PCR em tempo real e citoquímica. Os dados obtidos pelas técnicas citoquímicas e de biologia molecular indicaram uma diminuição da expressão do gene MCM2 durante a osteodiferenciação, que pode estar relacionada com danos ao DNA ou senescência nas hADSCs. / The discovery of adult stem cells is a promise field in regenerative medicine and tissue engineering. There are many sources of adult stem cells among which of whom are the skin, muscle and bone marrow. Currently, the adipose tissue has been an alternative source of mesenchymal stem cells (MSCs), termed human adipose-derived stem cells (hADSCs). The MSCs are the most plastic adult stem cells known until now and are easily isolated by adherence in plastic substrates. Under specifics conditions of culture hADSCs can be differentiated in osteoblast cell precursors. Among proteins needed for hADSCs differentiation, the roles of minichromosome maintenance (MCM) complex remains poorly understood. The protein complex MCM2-7 is part of DNA pre-replicative complex (pre-RC), being abundant in proliferating cells and involved in DNA repair mechanisms. In this sense, the Mcm2 is essential for the activity of the MCM complex. Thus, we analysed MCM2 gene expression changes during osteoinduction by culturing hADSCs in different media containing BMP-4 or in osteogenic medium. Genes related to osteodifferentiation, like osteocalcin (OC), osteopontin (OP) and alkaline phosphatase (ALP) were analysed by molecular and cytochemistry approaches during osteodifferentiation. Interestingly, we observed a decrease in the MCM2 gene level osteoinduction of hADSCs in osteogenic medium, which could be related to an increase in genome damage and senescence in hADSCs.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The aim of this study was to assess and compare the bone mineral density (BMD): total and regional (legs, spine, pelvis and arms) of athletes from four sports: volleyball (VA), soccer (SA), swimming (SA) and cycling (CA) and a control group (CG). The sample was selected conveniently, was composed by 51 subjects: 10 SA (21,28 ± 4,43 years, 74,75 ± 6,63 kg and 1,78 ± 0,06 m), 11 VA (20,10 ± 1,34 years, 81,27 ± 8,89 kg and 1,86 ± 0,07 m), 10 SA (22,2 ± 3,92 years, 73,61 ± 16,55 kg; 1,76 ± 0,08 m), 10 CA (29,06 ± 1,45 years, 71,00 ± 11,31 kg, 1,71 ± 0,08 m) and 10 subjects who formed the CG (24,59 ± 2,75 years, 72,85 ± 10,52 kg, 1,73 ± 0,05 m). The BMD, total and regional levels were measured and compared using ANOVA and post hoc LSD beyond the ANCOVA to lessen the effects of intervening variables, with the level of p <0,05. Mean value for BMD total of SA was 1,31 ± 0,09, BMD of the legs of that group was 1,55 ± 0,09, BMD of the arms had mean score of 0,92 ± 0,10, BMD of the spine and BMD of the pelvis was 1,21 ± 0,13 and 1,47 ± 0,12, for VA, the results of BMD total, BMD leg, BMD of the arms, spine and pelvis were respectively 1,35 ± 0,08, 1,58 ± 0,11, 1,00 ± 0,07, 1,25 ± 0,14, 1,45 ± 0,21 for SA the average results for the BMD total BMD of the legs, BMD arms, BMD of the spine and pelvis, were respectively 1,21 ± 0,05, 1,32 ± 0,11, 0,93 ± 0,07, 1,12 ± 0,13 and 1,25 ± 0,11, for CA the BMD total, legs, arms, spine and pelvis were respectively 1,26 ± 0,15, 1,43 ± 0,22, 1,11 ± 0,38, 1,13 ± 0,11 and 1,27 ± 0,19 for control group, the average performance of the same BMD were respectively 1,29 ± 0,09, 1,47 ± 0,14, 1,00 ± 0,10, 1,17 ± 0,13 and 1,30 ± 0,17.The BMD of the legs of VA and SA was greater than that of SA, the VA also showed superiority of this BMD than the CA. This same BMD in SA was lower than the CG. Thus, it can be concluded that VA and SA can present, at least with respect to the BMD of the lower limbs, the greater osteogenic response and possible protection against bone loss in old age, while athletes in sports with less participation of gravitational force or impact, particularly swimmers may not experience significant stress to promote osteogenic responses. / O objetivo desse estudo foi verificar e comparar a Densidade Mineral Óssea (DMO): total e regional (pernas, coluna, pelve e braços) de atletas de quatro modalidades esportivas: voleibol (AV), futebol de campo (AF), natação (AN) e ciclismo (AC) e, um grupo controle (GC). A amostra deste estudo foi selecionada convenientemente, foi composta por 51 sujeitos: 10 AF, (21,28±4,43 anos, 74,75±6,63 Kg e 1,78±0,06 m), 11 AV (20,10±1,34 anos, 81,27±8,89 Kg e 1,86±0,07 m), 10 AN (22,2±3,92 anos; 73,61±16,55 Kg; 1,76±0,08 m), 10 AC (29,06±1,45 anos; 71,00±11,31 Kg; 1,71±0,08 m) e 10 sujeitos que formaram o GC (24,59±2,75 anos; 72,85±10,52 Kg; 1,73±0,05 m). As DMO, total e regional, foram medidas e a comparação de cada uma, entre os grupos, foi feita utilizando a ANOVA e teste post hoc LSD além da ANCOVA para diminuir os efeitos das variáveis intervenientes, com o nível de significância de p< 0,05. O valor médio para a DMO total dos AF foi de 1,31±0,09, a DMO das pernas desse mesmo grupo foi de 1,55±0,09, a DMO dos braços apresentou resultado médio de 0,92±0,10, a DMO da coluna 1,21±0,13 e a DMO da pelve 1,47±0,12; para os AV, os resultados da DMO total, DMO das pernas, DMO dos braços, da coluna e da pelve foram, respectivamente 1,35±0,08, 1,58±0,11, 1,00±0,07, 1,25±0,14, 1,45±0,21; para os AN os resultados médios para as DMO total, DMO das pernas, DMO dos braços, DMO da coluna e da pelve, foram respectivamente 1,21±0,05, 1,32±0,11, 0,93±0,07, 1,12±0,13 e 1,25±0,11; para os AC as DMO total, das pernas, dos braços da coluna e da pelve foram, respectivamente 1,26±0,15, 1,43±0,22, 1,11±0,38, 1,13±0,11 e 1,27±0,19; para o GC os resultados médios das mesmas DMO foram, respectivamente 1,29±0,09, 1,47±0,14, 1,00±0,10, 1,17±0,13 e 1,30±0,17. A DMO das pernas dos AV e dos AF foi superior que a dos AN, os AV ainda apresentaram superioridade dessa mesma DMO que a dos AC. Essa mesma DMO nos AN foi menor que a do GC. Dessa forma, pode-se concluir que AV e AF podem apresentar, ao menos no que diz respeito à DMO dos membros inferiores, maior resposta osteogênica e possível proteção contra as perdas ósseas em idades avançadas, enquanto que atletas de modalidades esportivas com menor participação da força gravitacional ou impacto, particularmente atletas de natação podem não experimentar estresses significativos para promoção de respostas osteogênicas. Palavras-chave: osteogênese, saúde óssea, resposta osteogênica, exercício físico

Problems with musculoskeletal system, such as of developmental disorders, fractures or damage of the bone by age, inflammatory or tumor diseases, are still increasing in orthopaedics. Sometimes the bone tissue is not capable to completely regenerate to exert its physiological function in the organism. For this reason, using the bone replacements is necessary and common nowadays. Despite of an intensive research and testing of a wide range of the potential biomaterials and their combinations, the usage of metal materials for construction of the bone implants, still remains to be the gold standard. Ti-6Al-4V alloy is one of the commercialy used metal materials, which is known for the high mechanical and chemical resistance and a good biocompatibility. For a good biological response of the patient's organism for the bone implant, is an ability of osteointegration into the surrounding bone tissue, the key. This ability can be influenced in the case of the metals, by their surface structure. As it is known from earlier studies, the surface topography of the material is very important for the adhesion and proliferation of the bone cells, which are able to discriminate, very sensitively, between various stages of the material surface roughness. For this reason we have focused on studying of an influence...

The present study analyzes the capacity of collagen (coll)/sulfated glycosaminoglycan (sGAG)-based surface coatings containing bioactive glass nanoparticles (BGN) in promoting the osteogenic differentiation of human mesenchymal stroma cells (hMSC). Physicochemical characteristics of these coatings and their effects on proliferation and osteogenic differentiation of hMSC were investigated. BGN were stably incorporated into the artificial extracellular matrices (aECM). Oscillatory rheology showed predominantly elastic, gel-like properties of the coatings. The complex viscosity increased depending on the GAG component and was further elevated by adding BGN. BGN-containing aECM showed a release of silicon ions as well as an uptake of calcium ions. hMSC were able to proliferate on coll and coll/sGAG coatings, while cellular growth was delayed on aECM containing BGN. However, a stimulating effect of BGN on ALP activity and calcium deposition was shown. Furthermore, a synergistic effect of sGAG and BGN was found for some donors. Our findings demonstrated the promising potential of aECM and BGN combinations in promoting bone regeneration. Still, future work is required to further optimize the BGN/aECM combination for increasing its combined osteogenic effect.

Longitudinal growth of long bone occurs at growth plates by a process called endochondral ossification. Endochondral ossification is affected by both biological and mechanical factors. This thesis focuses on the mechanical modulation of femoral bone growth occurring at the proximal growth plate, using mechanobiological theories reported in the literature. Finite element analysis was used to simulate bone growth. The first study analyzed the effect of subject-specific growth plate geometry over simplified growth plate geometry in numerical prediction of bone growth tendency. Subject-specific femur finite element model was constructed from magnetic resonance images of one able- bodied child. Gait kinematics and kinetics were acquired from motion analysis and analyzed further in musculoskeletal modelling to determine muscle and joint contact forces. These were used to determine loading on the femur in finite element analysis. The growth rate was computed based on a mechanobiological theory proposed by Carter and Wong, and a growth model in the principal stress direction was introduced. Our findings support the use of subject- specific geometry and of the principal stress growth direction in prediction of bone growth. The second study aimed to illustrate how different muscle groups’ activation during gait affects proximal femoral growth tendency in able-bodied children. Subject-specific femur models were used. Gait kinematics and kinetics were acquired for 3 able-bodied children, and muscle and joint contact forces were determined, similar to the first study. The contribution of different muscle groups to hip contact force was also determined. Finite element analysis was performed to compute the specific growth rate and growth direction due to individual muscle groups. The simulated growth model indicated that gait loading tends to reduce neck shaft angle and femoral anteversion during growth. The muscle groups that contributes most and least to growth rate were hip abductors and hip adductors, respectively. All muscle groups’ activation tended to reduce the neck shaft and femoral anteversion angles, except hip extensors and adductors which showed a tendency to increase the femoral anteversion. The third study’s aim was to understand the influence of different physical activities on proximal femoral growth tendency. Hip contact force orientation was varied to represent reported forces from a number of physical activities. The findings of this study showed that all studied physical activities tend to reduce the neck shaft angle and anteversion, which corresponds to the femur’s natural course during normal growth. The aim of the fourth study was to study the hypothesis that loading in the absence of physical activity, i.e. static loading, can have an adverse effect on bone growth. A subject-specific model was used and growth plate was modeled as a poroelastic material in finite element analysis. Prendergast’s indicators for bone growth was used to analyse the bone growth behavior. The results showed that tendency of bone growth rate decreases over a long duration of static loading. The study also showed that static sitting is less detrimental than static standing for predicted cartilage-to-bone differentiation likelihood, due to the lower magnitude of hip contact force. The prediction of growth using finite element analysis on experimental gait data and person- specific femur geometry, based on mechanobiological theories of bone growth, offers a biomechanical foundation for better understanding and prediction of bone growth-related deformity problems in growing children. It can ultimately help in treatment planning or physical activity guidelines in children at risk at developing a femur or hip deformity. / <p>QC 20170616</p>