This thesis deals with the fixation of lower jaw with bone tissue defect using commercially produced fixator. Large defects of bone tissue are mainly caused due to the removal of bone tissue affected by tumor. These topics have been researched on the basis of the literature. Subsequently, stress strain analysis of the lower jaw with the applied fixation plate was performed. This analysis was solved by using computational modeling with variational approach, ie the finite element method. The work also describes in detail the procedure of creating model of geometry, model of material, model of boundary conditions and loads with subsequent solution of several computational models. The stress strain analysis was done for lower jaw with varying size of the removed bone tissue with applied reconstruction plate made of CP-Ti Grade 4 and for the lower jaw with the plate, which is made of -Ti-Mo. Finally, the influence of the mechanical properties of the callus during formation of new bone tissue (callus healing) on the stress and deformation of the solved system was evaluated.

This present thesis comprise two major parts both experimental and numerical study which have been conducted in four distinct steps as following: (1) Design, construction, and evaluation of control and hydrodynamic of a bioreactor system. (2) Visualization of fluid flow perfusion in the hollow fibre membrane bioreactor (HFMB) using a biomedical noninvasive imaging technique, i.e. positron emission tomography (PET). (3) Development of a mathematical model for analyzing a hybrid hollow fibre membrane bioreactor (hHFMB) and (4) Development of a dynamic and two-porous media model for analyzing the HFMB with the aid of computational fluid dynamics (CFD), specifically for bone tissue engineering application. The experimental part includes the steps 1 and 2. In the step 1, the flow perfusion bioreactor system has been designed and constructed. The experimental evaluations of hydrodynamic, and control were performed. In this system, mean pressure, mean flow rate, frequency and waveform of the pulsatile pressure and flow rate can be modulated and controlled over the time to simulate both physiological and non-physiological conditions. The temperature, dissolved oxygen, and pH can be controlled.This bioreactor system can be applied to a variety of scaffold configurations, geometries, and sizes as the cell/tissue culture chamber is adjustable in length.This system is autoclavable, and compatible with noninvasive medical imaging techniques. Designing of the inlet and outlet manifold of the bioreactor were performed according to data obtained from CFD simulation of the flow distribution to achieve high efficiencies in the uniformity of flow perfusion. In the second step, PET was proposed for the very first time and a small animal PET system was used to obtain new information about steady and pulsatile flow patterns in the HFMB for tissue engineering applications. The non-homogeneous tracer distribution, as found with PET imaging, implies the occurrence of non-efficient regions with respect to mass transfer. In steady inlet flow condition, a non-uniform distribution of radioactive tracer was obtained. In contrast, the pulsatile inlet flow generated more uniform perfusion than that of steady flow. Further, it was found that in the case of pulsatile flow, the accumulation of the tracer within the bioreactor was efficiently less than that of steady inlet flow at the same condition. Therefore, in one hand these findings have the potential to improve bioreactor design and in the other hand can explore a very important rout to employ PET in developing bioreactors for tissue engineering applications. The numerical part includes the step 3 and 4 in which the numerical study has been performed for 3-D bone tissue growth in HFMB as a case study for large-scale tissue culture. In the step 3, the feasibility of utilizing newly proposed hHFMB for the growth of mesenchymal stem cells (MSCs) to form bone tissue was investigated using numerical simulations. To this aim, a mathematical model using a CFD code was developed to optimize the design and operation parameters of hHFMB for the growth of MSCs. The volume averaging method was used to formulate mass balance for the nutrients and the cells in the porous extracapillary space (ECS) of the hHFMB. The cell-scaffold construct in the ECS of the hollow fibres and membrane wall were treated as porous medium. Cell volume fraction dependent porosity, permeability, and diffusivity of mass were used in the model. The simulations allowed the simultaneous prediction of nutrient distribution and nutrient-dependent cell volume fraction. In addition, this model was used to study the effects of the operating and design parameters on the nutrient distribution and cell growth within the bioreactor. The modeling results demonstrated that the fluid dynamics within the ECS and transport properties and uptake rates in hHFMB were sufficient to support MSCs required for clinical-scale bone tissue growth in vitro and enabled to solve nutrition difficulties because of high cell density and scaffold size. In the step 4, the new dynamic and two-porous media model has been used for analyzing the nutrient-dependent MSCs growth in order to form the bone tissue in the HFMB. In the present model, hollow fibre scaffold within the bioreactor was treated as a porous domain. The domain consists of the porous lumen region available for fluid flow and the porous ECS region, filled with collagen gel containing cells, for growing tissue mass. Furthermore, the contributions of several design and process parameters, which enhance the performance of the bioreactor, were studied. In addition, the dynamic evaluation of cell growth, oxygen and glucose distributions were quantitatively analyzed. The obtained information can be used for better designing of the bioreactor, determining of suitable operational conditions and scale up of the bioreactor for engineering of clinical-scale bone tissue.--Résumé abrégé par UMI.

Tissue Engineering is a scientific discipline that aims to regenerate tissues and organs that are diseased, lost or congenitally absent. It encompasses the use of suitable synthetic equivalents of native extracellular matrix that may or may not be supplemented with cells or relevant growth factors. Such scaffolds are designed to reside at the site of implantation for a variable period of time during which they induce the regeneration of native tissue. During this time, they also provide a template for new cells to attach, infiltrate, differentiate into appropriate phenotype and eventually restore function of the concerned tissue. Among the factors that affect the outcome are the composition of scaffold, methods of fabrication, bulk properties of the scaffold and topography and architecture at the cellular level. Bone is unique in the body in that it is one of the few tissues capable of complete regeneration even in adults, as seen during fracture healing. However, certain conditions (non-union of fractures, congenital and acquired bone deficiencies) exist in which the regenerative capacities of bone are exceeded and appropriate intervention becomes necessary. Current treatment options include autologous bone grafts harvested from iliac crest or de-cellularized allografts or synthetic substitutes made from metals, ceramics and polymers. However these options have serious limitations: while autografts are limited in supply, necessitate second surgery and show inadequate vascularization, allografts can transmit viral infections. Metals, ceramics and polymers are in essence structural replacements without performing any biological function. Other problems associated with these synthetic materials include adverse immune reactions, corrosion, stress-shielding and secondary fractures due to inadequate osseo-integration. Bone tissue engineering is a specialized field of research that provides an alternative strategy to repair bone defects by exploiting the advances in engineering and better understanding of bone biology. Scaffold-based tissue engineering approach is a promising field that involves implantation of a biomaterial that is specifically matched in terms of biological and material properties to the tissue it replaces. This study explores the feasibility of using electrospinning as a potential fabrication strategy for bone tissue engineering applications, more specifically intended for cleft palate repair. This model represents a congenital deformity that affects both hard and soft tissues and presents unique challenges and opportunities. Among the challenges are: the need for the implant allow growth of the most complex areas of the facial skeleton, integrate and grow with the patient through adolescence, the ability of the implant to not interfere with vital functions including breathing and feeding. Further the implant should provide a flexible matrix that can effectively support erupting teeth. In spite of these extreme demands, maxilla is a non load-bearing membranous bone, a favorable consideration from materials engineering perspective. The present study is organized into three independent sections. The first section investigates developing strategies intended to improve the material properties of electrospun bone scaffold. Bone is composed of a high volume fraction (50%) of inorganic hydroxyapatite nanocrystals that is closely associated with collagen. The dispersal of brittle mineral is critical in not only strengthening the bone in compression but also contributes to the osteoconductivity of the matrix. Since loading of mineral in a bone scaffold is a serious limitation, we attempted to achieve improved loading of bone mineral by dual mineralization approach. We first incorporated nanocrystalline hydroxyapatite (nHA) directly into the scaffold by adding it to the electrospinning polymer solution. The second step involves inducing biomimetic mineralization of electrospun scaffolds by incubating them in simulated body fluid (SBF) for 2 weeks. The hypothesis was that the nanocrystalline hydroxyapatite seeded during electrospinning would act as sites for nucleation and further crystal growth when incubated in solution supersaturated with respect to calcium and phosphate ions. We tested this approach in two synthetic, biocompatible polymers-polydioxanone and poly (lactide: glycolide) and four formulations of SBF with differential loading of nHA (0-50% by wt. of polymer). A modified Alizarin Red S (ARS) staining that specifically binds to calcium was developed that allowed us to quantify the mineral content of 3D scaffold with great accuracy. Results indicated a unique combination of factors: PDO scaffolds containing 50% nHA incubated in 1x revised-SBF incubated under static conditions gave maximum mineralization over a period of two weeks. We then sought to exploit these findings to engineer a stiffer scaffold by stacking multiple layers together and cold welding them under high pressure. Electrospun scaffolds (1, 2 or 4 layered stacks) were either compressed before or after mineralizing treatment with SBF. After two weeks, scaffolds were analyzed for total mineral content and stiffness by uniaxial tensile testing. Results indicated while compression of multiple layers significantly increases the stiffness of scaffolds, it also had lower levels of mineralization partly due to increased density of fibers and loss of surface area due to fiber welding. However this can be offset to a reasonable degree by increasing the number of stacks and hence this strategy can be successfully adopted to improve the mechanical properties of electrospun scaffolds. The second section introduces a novel infrared imaging technique to quantify and characterize the biological activity of biomaterials, based on cell adhesion. Cells attach to the surface by the formation of focal contacts where multiple proteins including vinculin and talin assemble to signal critical processes like cell survival, migration, proliferation and differentiation. After allowing MG-63 osteoblasts to adhere to 2D biomaterial surface coated with extracellular matrix proteins (collagen, gelatin, fibronectin) cells were fixed and probed with antibodies for vinculin and talin. Secondary antibodies, tagged with infrared-sensitive fluorescent dyes, were used to quantify the molecules of interest. In addition, the kinetics of focal contact formation in these different substrates was followed. Successful quantification of focal contacts were made and further research revealed phosphorylation of vinculin at pY-822 as one potential mechanism for recruitment of vinculin to focal contacts. Hence it could represent a subset of vinculin and might serve as a specific molecular marker for focal contacts. As an extension, we evaluated the possibility of using such an assay to quantify 3D electrospun tissue engineering scaffolds. We fabricated scaffolds of graded biological activity by electrospinning blends of polydioxanone and collagen in different ratios. Vinculin and talin expressed by MG-63 cultured on these scaffolds for 24 hours were quantified in a similar manner. Results indicate that while talin does not show a significant difference in expression among different scaffolds, vinculin showed a positive correlation with increasing biological activity of scaffolds. In conclusion, we have identified vinculin as a reliable marker of focal contacts in 3D scaffolds while phosphovinculin (pY-822) was more specific to focal contacts in coated 2D substrates. In both instances, infrared imaging proved to be reliable in study of focal contacts. The third section aims to make the bone scaffolds osteoinductive- a property of a material to induce new bone formation even when implanted in subcutaneous and intramuscular heterotopic sites. Bone morphogenetic proteins (BMP) are potent cytokines that can induce migration, proliferation and differentiation of stem cells along osteoblastic lineage. The therapeutic efficacy of BMPs in the treatment of severe bone defects has been identified and is currently FDA approved for specific orthopedic applications. BMPs are clinically administered in a buffer form that not only makes the treatment expensive but less effective. Suitable delivery systems for BMP delivery have been an intense area of investigation. We rationalized electrospinning as a strategy to incorporate BMP within the scaffold and that would enable controlled release when implanted. One of the drawbacks of using electrospinning to deliver bioactive molecules is the potential denaturing effect and eventual loss of activity of BMPs. The final section of this dissertation tries to develop sensitive and relevant assays that could answer intriguing questions about solvent-protein interaction. We chose to use the BMP-2/7 heterodimer as the osteoinductive molecule of choice because of its superior potency compared to homodimer counterparts. We characterized the detection and quantification of BMP-2/7 using a slot blot technique. Further, we used a novel cell line (C2C12 BRA) to test the retention of activity of BMP-2/7 that has been exposed to organic solvents. Results indicate significant loss of activity when BMPs are exposed to organic solvents but complete recovery was possible by diluting the solvent with an aqueous buffer.

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>

Fundamento: Fratura é ocorrência frequente em lesados medulares, em virtude da acentuada osteoporose, o que compromete o esqueleto como um todo e a consolidação óssea da fratura, em particular, o que resulta em calo ósseo de má qualidade, ou mesmo surgirem anomalias de consolidação. Assim, são importantes a busca e a avaliação de intervenções terapêuticas que possam melhorar o reparo ósseo nessa população. Objetivo: Avaliar os resultados da administração de alendronato de sódio, paratormônio e ultrassom de baixa intensidade na qualidade do calo ósseo de fraturas diafisárias do fêmur de ratos previamente submetidos à lesão medular. Material e Métodos: 75 ratos machos foram distribuídos em 05 grupos (n=15): (1) CON: ratos normais submetidos à fratura do fêmur; (2) LM: ratos com lesão medular e fratura; (3) LM+ALE: ratos com lesão medular, fratura e tratamento com alendronato de sódio; (4) LM+PTH: ratos com lesão medular, fratura e tratamento com paratormônio e (5) LM+US: ratos com lesão medular, fratura e tratamento com ultrassom. A medula espinhal foi completamente seccionada no nível da décima vértebra torácica (T10). No grupo controle (Sham) a medula foi apenas exposta, mas não seccionada. A fratura do fêmur foi realizada 10 dias após a produção da lesão medular. Sua localização foi na diáfise femoral, seguida de fixação com fio de aço no canal medular. A terapêutica em cada grupo foi aplicada durante as duas semanas seguintes à fratura. A avaliação da qualidade óssea e do calo foi feita por imagens de DEXA, microtomografia computadorizada (?CT), histologia, histomorfometria, imunoistoquímica (OPG, RANK e RANKL), resistência mecânica, PCR em tempo real para avaliar a expressão gênica e ELISA (OPG e IGF-1). Resultados: A lesão medular afetou gravemente a qualidade óssea com deterioração da microarquitetura óssea e redução da densidade mineral óssea, além de prejuízo da resistência mecânica. Houve redução da expressão dos genes de formação óssea e aumento da reabsorção, com alteração fenotípica. O processo de consolidação ocorreu predominantemente por ossificação intramembranosa nos animais paraplégicos. No entanto, a qualidade do calo foi pobre, em relação aos animais que não sofrem a lesão. Por outro lado, o uso de recursos terapêuticos como o alendronato de sódio, paratormônio e ultrassom de baixa intensidade foi capaz de recuperar muitas das propriedades microarquiteturais, celulares e mecânicas do osso não fraturado. De forma semelhante, osrecursos farmacológicos e mecânico utilizados neste estudo atuaram positivamente no processo de consolidação uma vez que houve aumento na densidade do calo, melhora da sua microarquitetura, e atividade celular, além do aumento na resistência aos esforços mecânicos. Em resumo, a lesão medular causou grave deterioração do tecido ósseo e modificações no processo de consolidação que, foram amenizadas e até normalizadas pela administração de alendronato de sódio, paratormônio e ultrassom. Conclusão: Os recursos terapêuticos acima citados foram benéficos para a recuperação do tecido ósseo intacto e para melhorar a qualidade do calo ósseo, após a deterioração causada pela lesão medular. / Background: Bone fracture is a frequent event in individuals with spinal cord injury (SCI) as a consequence of the marked reduction in bone mass and quality. SCI-induced bone loss may also induce changes in bone healing, leading to a poor callus formation and even non-unions. Therefore, the search for therapeutic interventions that may improve bone healing in this particular population may have a remarkable clinical importance. Purpose: To evaluate the influence of sodium alendronate, parathyroid hormone and low intensity ultrasound on the quality of fracture bone callus formation in SCI rats. Material and Methods: 75 male Wistar rats were randomly divided into five groups (n=15) (1) CON: control rats subjected to fracture, but not to spinal cord injury; (2) SCI: spinal cord injured rats and femur bone fracture; (3) SCI+ALE: spinal cord injured rats with bone fracture and treatment with sodium alendronate; (4) SCI+PTH: spinal cord injured rats with bone fracture and treatment with parathyroid hormone, and (5) SCI+LIPUS: spinal cord injured rats with bone fracture and treatment with low intensity ultrasound. A complete transection of the spinal cord was performed at the T10 level. In the control group the spinal cord was exposed but not sectioned. At 10 days post-injury (or Sham) a bone fracture was produced at the femoral shaft and fixed with an intramedullary pin. The ALE and PTH treatments began on day 1, and US began on day 3, after fracture and on day 14 all rats were euthanized. Non-fractured tibias and femur bone callus were analyzed by DXA, micro-computed tomography (?CT), histology, histomorphometry, immunohistochemistry (OPG, RANK and RANKL), mechanical test, real time PCR and ELISA (OPG and IGF-1). Results: SCI significantly impaired the bone quality, downregulated the osteoblastic-related gene expression and increased bone resorption, resulting in several phenotypic changes. SCI deteriorated the microarchitecture, reduced bone mass and weakened bone strength. Bone healing in SCI occurred predominantly by intramembranous ossification, leading to a poor callus formation. Conversely, the administration of alendronate, PTH and LIPUS were effective at ameliorating and even fully reestablish the bone microarchitecture, cells activity and mechanical strength. Similarly, both the pharmacological and mechanical therapeutics analyzed in this study were potentially efficient to increase bone callus density, to improve callus microstructure and metabolism, and to increase newly bone strength. In short,we revealed that SCI induced severe bone deterioration and changes in bone healing, which were ameliorated and even fully restored by the administration of alendronate, PTH, LIPUS and passive standing. Conclusions: The therapeutic approaches used in this study had great efficacy in restoring non-fractured bone integrity and with improvement of bone callus in paraplegic rats.

O osso requer que alguns de seus elementos detectem estímulos mecânicos e os transformem em sinais celulares adequados para que o tecido ósseo possa manter a eutrofia e adaptar-se às solicitações mecânicas a que é submetido. Assim, a estrutura óssea pode torna-se mais resistente quando a demanda mecânica aumenta e, da mesma forma, menos resistente em condições de hipoatividade como imobilizações, sequelas motoras, repouso prolongado ou em ambientes de microgravidade como as experimentadas por astronautas. Dessa interação podem resultar ossos com massa diminuída, o que caracteriza situações de osteopenia ou osteoporose. Uma das indicações do exercício de natação é estimular e manter o trofismo do tecido ósseo. No entanto, a relação entre natação e qualidade óssea em pessoas com osteopenia, não foi suficientemente estudada de forma a embasar recomendações clínicas. Assim, o objetivo desta pesquisa foi investigar a eficácia da natação na recuperação da qualidade óssea avaliada em tíbias de ratas com osteopenia induzida por suspensão dos membros posteriores. Foram utilizadas 50 ratas da raça Wistar divididas em cinco grupos experimentais (n=10/grupo); dois controles e três experimentais. Houve um grupo suspenso pela cauda durante 21 dias consecutivos (S) e seu controle que permaneceu em gaiolas comuns pelo mesmo período (CI). No grupo suspenso e treinado os animais permaneceram em suspensão durante 21 dias, e depois foram submetidos a 20 sessões de natação (ST). No grupo suspenso não treinado os animais foram suspensos por 21 dias e soltos em gaiolas comuns por 30 dias (SNT). No grupo controle II as ratas foram observadas em gaiolas comuns por 51 dias, sem intervenção (CII). A avaliação da qualidade óssea foi feita pela densitometria (DXA) e ensaio mecânico realizados na tíbia direita e histomorfometria da região metafisária proximal da tíbia esquerda. Para análise estatística todos os valores foram considerados diferentes significativamente em p<0,05. Os ensaios mecânicos mostraram que, para o grupo S os valores de força máxima (-14,03%, p=0,0003), rigidez (-21,68%, p=0,0055), DMO (-17,62%, p=0,019) e percentual de osso trabecular (-57,2%, p=0,0001), diminuíram significativamente em comparação com o grupo CI. A comparação das propriedades mecânicas simultâneas entre os grupos avaliados após 51 dias (STxCIIxSNT) mostrou diferença estatística significante entre os grupos para força máxima de (p=0,0014), rigidez (p=0,0010), DMO (p= 0,0095) e percentual de osso trabecular (p<0,0001). O grupo ST apresentou aumento significativo na força máxima (+10,23%, p<0,05), rigidez (+21,91%, p<0,001), DMO (+9,46%, p<0,05) e percentual de osso trabecular (+48,82%, p<0,001) em comparação com o grupo SNT. Na comparação dos grupos SNT e CII também houve diminuição significativa (-14,4%, p<0,05) para força máxima, rigidez (-25,21%, p<0,005), DMO (-13,34%, p<0,05) e percentual de osso trabecular (-52,06%, p<0,001). Finalmente, a comparação entre os grupos ST e CII não apresentou diferença estatística significante (p>0,05) para os valores de força máxima, rigidez, DMO e percentual de osso trabecular. Em relação à análise histológica qualitativa o grupo S apresentou ossos com características histológicas condizentes com osteopenia, com trabéculas ósseas bem mais delgadas em quantidade muito menor tanto na epífise quanto na metáfise, quando comparado ao grupo CI. O grupo suspenso apresentou ainda, diminuição na espessura do tecido ósseo compacto e redução significativa na quantidade de tecido esponjoso em sua porção mais interna. A espessura do tecido ósseo compacto se mostrou similar nos diferentes grupos (ST, SNT e CII). Todavia, a espessura e a quantidade de trabéculas do osso esponjoso diferiram expressivamente nos diferentes grupos. O grupo ST apresentou uma aparente maior quantidade de trabéculas ósseas, bastante delgadas, ao passo que o grupo CII apresentou, comparativamente, uma menor quantidade de trabéculas bem mais espessas. Comparado a estes últimos, o grupo SNT apresentou uma menor quantidade de trabéculas com delgada espessura. Com a suspensão por três semanas os parâmetros mostraram que houve deterioração da qualidade óssea expressa pela diminuição da densidade mineral óssea, enfraquecimento mecânico do osso e perda de massa óssea trabecular. A quantidade de osso trabecular na região metafisária foi o parâmetro mais afetado pela hipoatividade, mas também o elemento que apresentou a resposta mais rápida para a atividade motora. A conclusão foi que a natação reverteu mais rapidamente a deterioração osteopênica causada pela hipoatividade, com recuperação completa da qualidade óssea. / The bone is able to detect mechanical stimuli and to transform them into cell response so that the bone tissue can maintain its health and to adapt to different mechanical demands (mechanotransduction). Thus, the bone structure become more resistant when the mechanical solicitations are increased and, conversely, less resistant when in hypoactivity conditions as occurring during immobilizations, in locomotor sequela, prolonged bed rest or in a microgravity environment as experienced by astronauts. In general, hypoactivity leads the bone to develop a decreased and deteriorated mass, thus characterizing conditions of osteopenia or osteoporosis. Thus, physical activity such as swimming is indicated as one of the factors that contribute to the bone health. However, the relation between swimming and bone quality in individuals with osteopenia has not been sufficiently studied to fully support clinical recommendations. So, the aim of this study was to investigate the effectiveness of swimming in recovering bone quality evaluated in osteopenic tibias of rats that spent three weeks in hindlimb suspension. Fifty Wistar rats were used and divided into five groups (n = 10/group); two control and three experimental groups. There was a group suspended by the tail for 21 consecutive days (S) and its control kept in regular cages for the same period (CI). In the suspended and trained group, animals were suspended for 21 consecutive days and then underwent a 20 swimming sessions (ST). In the suspended, not trained group, animals were suspended for 21 days and released in regular cages for 30 days (SNT). In the control group II rats were kept in regular cages for 51 days without intervention (CII). The bone quality was assessed by densitometry (BMD), bending mechanical testing and histomorphometry. Statistical significance was set at p<0.05. Mechanical tests showed that for the suspended group there was a decrease of the maximum force (-14.03%, p = 0.0003), stiffness (-21.68%, p = 0.0055), BMD (-17.62 %, p = 0.019) and the percentage of trabecular bone (-57.2%, p = 0.0001) in comparison with the control group. Comparison among the groups after 51 days (STxCIIxSNT) showed statistically significant differences between groups for maximum force (p = 0.0014), stiffness (p = 0.0010), BMD (p = 0. 0095) and the percentage of trabecular bone (p<0.0001). The suspended and trained group showed a significant increase in maximum force (+10.23%, p <0.05), stiffness (+21.91%, p<0.001), BMD (+9.46%, p<0.05) and the percentage of trabecular bone (+48.82%, p <0.001) compared to the suspended and not trained group. When the suspended but not trained group was compared to control CII, there was also a significant decrease (- 14.4%, p<0.05) for maximum force, stiffness (-25.21%, p<0.005), BMD (-13.34%, p<0.05) and the percentage of trabecular bone (-52.06%, p<0.001). Finally, the comparison between group suspended and trained ad its control and CII showed no statistically significant difference (p>0.05) for all the parameters. Considering the qualitative histological analysis, suspended group presented bones with histological characteristics consistent with osteopenia, with thinner trabecular bone and in much less quantity both in the epiphysis and in the metaphysis. The suspended and trained group had a higher quantity of trabecular bone, with thinner trabeculae, while the CII group showed a comparatively smaller amount of much thicker trabeculae. Compared to the latter, the suspended and not trained group had an apparent smaller amount of trabeculae with a thinner thickness. In conclusion, the three week suspension caused a marked deterioration of the tibia bone tissue that was completely reverted by swimming and partially reverted with spontaneous cage activities taking in consideration all the parameters analyzed.

A regeneração de defeitos ósseos continua sendo um grande desafio na área de Odontologia e Medicina. É bem estabelecido que células-tronco mesenquimais (CTMs) e osteoblastos (OBs) desempenham um papel crítico na osteogênese, tornando-se candidatos a utilização em procedimentos de terapia celular que visam otimizar o processo de reparação óssea. Porém, pouco se sabe sobre a interação entre CTMs e OBs, e a maioria dos estudos enfatiza o efeito dos OBs sobre CTMs, fazendo com que a influência das CTMs na atividade osteogênica dos OBs continue sendo uma questão desafiadora. Baseados em nossos estudos anteriores, formulamos a hipótese de que a terapia celular que fizesse uso de uma associação de CTMs e OBs poderia ser mais eficaz para o reparo do tecido ósseo do que essas células isoladamente, principalmente como resultado da estimulação de OBs por CTMs. Para tal, foi realizado estudo in vitro para avaliar os efeitos das CTMs sobre os OBs e in vivo para avaliar os efeitos dessas células, isoladamente e combinadas, sobre a reparação óssea. CTMs da medula óssea de rato foram cultivadas em meio de crescimento para manterem-se como CTMs ou em meio osteogênico para diferenciarem-se em OBs. Após alcançar a subconfluência, as células foram cultivadas in vitro em três diferentes condições: (1) co-cultura direta de CTMs e OBs usando três proporções celulares (1:1, 1:2 e 2:1), (2) co-cultura indireta de CTMs e OBs usando insertos e (3) OBs cultivados em meio condicionado por CTMs. Para avaliação das respostas celulares foram realizados ensaios de proliferação celular, atividade de fosfatase alcalina (ALP), formação de matriz mineralizada, expressão gênica de marcadores osteoblásticos, imunolocalização de sialoproteína óssea (BSP) e osteopontina (OPN) e migração celular. Para os experimentos in vivo, as células foram carreadas em esponja de colágeno através de vários ciclos de centrifugação. Após, defeitos ósseos em calvária de rato foram preenchidos com (1) esponja de colágeno sem células, (2) esponja de colágeno com CTMs, (3) esponja de colágeno com OBs e (4) esponja de colágeno com associação de CTMs e OBs. Para avaliação da reparação óssea in vivo após 4 semanas, foram realizadas análises histomorfométricas através de cortes histológicos e microtomografia computadorizada. Os dados foram comparados pelo teste de Kruskal-Wallis e, se necessário pelo teste de Mann-Whitney (p&le;0,05). Foi observado que CTMs têm efeito repressivo sobre a proliferação e as expressões fenotípicas e genotípicas de OBs (P&le;0,05). Em relação ao reparo dos defeitos ósseos, somente naqueles tratados com células observou-se formação óssea predominantemente como ilhotas isoladas e diferenças, principalmente qualitativas, entre os tipos celulares utilizados, com tendência de maior formação óssea em defeitos tratados com OBs em comparação ao uso de CTMs. Com base nos resultados obtidos, pôde-se concluir que as CTMs apresentam efeito inibitório sobre OBs e que a terapia celular com OBs parece ser mais eficaz no reparo do tecido ósseo. / The regeneration of bone defects remains a major challenge in the field of Dentistry and Medicine. It is well known that mesenchymal stem cells (MSCs) and osteoblasts (OBs) play critical roles in osteogenesis, making them promising alternatives to be employed in cell therapy procedures to enhance the process of bone regeneration. Studies about the crosstalk between MSCs and OBs are mainly focused on the effect of OBs on MSCs, thus how MSCs may affect OBs phenotype expression remains a challenging question. Based on our previous studies, we have hypothesized that cell therapy using a combination of MSCs and OBs could be more effective for the bone repair than these cells separately, mainly due to the stimulation of OBs by MSCs. For this, we carried out in vitro experiments to evaluate the effects of MSCs on the OBs and in vivo experiments to assess the effects of these cells either isolated or combined on bone repair. Rat bone marrow MSCs were cultured either in growth medium to keep MSCs features or in osteogenic medium to differentiate into OBs. After reaching subconfluence, cells were grown in vitro in three different conditions: (1) direct coculture of MSCs and OBs using three cell proportions (1:1, 1:2 and 2:1), (2) indirect coculture of MSCs and OBs using transwell porous filters and (3) OBs cultured in MSCs conditioned medium. Cell responses were evaluated by assaying cell proliferation, alkaline phosphatase activity (ALP), mineralized matrix formation, gene expression of osteoblast markers, immunolocalization of bone sialoprotein (BSP) and osteopontin (OPN), and cell migration. For in vivo experiments, cells were seeded into collagen sponge by a centrifugation method. After, calvarial defects were implanted with (1) collagen sponge without cells, (2) collagen sponge with MSCs, (3) collagen sponge with OBs, and (4) collagen sponge and association of MSCs with OBs. To evaluate bone repair at the end of 4 weeks, histomorphometric analyzes were carried out using histological slides and micro-computed tomography. Data were compared by Kruskal-Wallis test and, if appropriated, by Mann-Whtiney test (p&le;0.05). It was observed that MSCs repressed proliferation, phenotypic and genotypic expressions of OBs (P&le;0.05). Bone formation was observed only in cell treated defects as isolated islets and qualitative differences were noticed among cell types, with a tendency of more bone formation in OBs treated defects compared with MSCs ones. Based on these results, we can conclude that MSCs exhibited inhibitory effect on OBs and that cell therapy with OBs seemed to be more effective for bone repair.

Cette thèse se concentre sur la caractérisation ultrasonore de l’interphase os-implant. Cette région est une zone de transition où a lieu le processus d’ostéointégration (i.e. le processus de guérison du tissu entourant l’implant). Donc, cette interphase a un rôle crucial dans l’ancrage à long-terme de l’implant, puisqu’elle dépend de la quantité ainsi que la qualité du tissu osseux environnant. Ensuite, en plus d’être un milieu complexe en remodelage continu, l’os néoformé présente une nature multi échelle et qui évolue dans le temps. Toutes ces motivations rendent la caractérisation de l’interphase os-implant critique et difficile. Dans ce contexte, les méthodes ultrasonores sont largement utilisées aujourd’hui dans le domaine clinique pour leur capacité de donner des informations sur les propriétés biomécaniques du tissu osseux. Compte tenu de ces éléments, dans le but de caractériser les propriétés mécaniques et microstructurales de l’interphase os-implant à travers des méthodes ultrasonores, il est important de développer et valider des modèles mécaniques ainsi que de méthodes de traitement du signal. A cause de la complexité du problème, afin de décrire avec précision le tissu environnant à l’implant, il est d’abord essentiel une modélisation fiable du tissu osseux. Pour cela, on étudie l’interaction entre une onde ultrasonore et le tissu osseux, en considérant aussi les effets dues à la microstructure. Pour ce faire, un modèle continu généralisé a été utilisé. Dans ce contexte, un test de transmission/réflexion réalisé sur un échantillon poroélastique immergé dans un fluide a renforcé la fiabilité du modèle. Les champs de pression réfléchi et transmis sont influencés par les paramètres de la microstructure. De plus, les résultats issus de l’analyse de dispersion sont en accord avec ceux observés dans les expériences pour les échantillons poroélastiques. Après, le problème a été compliqué en considérant une interphase qui se situe entre l’os et l’implant. Ainsi, on peut gérer la complexité ajoutée par la présence du tissu néoformé. Comme on l’a déjà mentionné, une difficulté additionnelle est représentée par le fait que l’interphase est un milieu hétérogène, un mélange de phases solides et fluides dont les propriétés évoluent avec le temps. Donc, afin de modéliser l’interaction des ondes ultrasonores avec une interphase, on a considéré dans le modèle une couche très fine avec des propriétés élastiques et inertielles. En partant de ça, on a étudié les effets des propriétés de réflexion d’une transition entre un milieu homogène et un milieu microstructuré. De même, il a aussi été étudié la caractérisation du milieu via des techniques avances de traitement du signal. En particulier, la réponse dynamique due à l’excitation ultrasonore du système os-implant a été analysée à travers une approche multifractale. Une première analyse basée sur les coefficients des ondelettes a montré une signature multifractale pour les signaux dérivants des simulations et aussi des expériences. Ensuite, une étude de sensibilité a aussi montré que la variation des paramètres tels que la fréquence centrale et la densité de l’os trabéculaire ne contribue pas à un changement dans la réponse. L’originalité réside dans le fait qu’il s’agit d’un des premiers efforts d’exploiter l’approche multifractale dans la propagation ultrasonore dans un milieu hétérogène / This thesis focus on the ultrasonic characterization of bone-implant interphase. This region is a transition zone where the osteointegration process (i.e. the healing process of the tissues surrounding the implant) takes place. Thus, this interphase is of crucial importance in the long-term anchorage of the implant, since it depends on the quantity and quality of the surrounding bone tissue. However, other than being a complex medium in constant remodeling, the newly formed bone presents a multiscale and time evolving nature. All these reasons make the characterization of the bone-implant interphase critical and difficult. In this context, ultrasound methods are nowadays widely used in the clinic field because of their ability to give information about the biomechanical properties of bone tissue. On this basis, with the aim of characterizing the mechanical and microstructural properties of the bone-implant interphase by ultrasound methods, it is important to develop and validate mechanical models and signal processing methods. Due to the complexity of the problem, in order to precisely describe the bone tissue surrounding the implant, first an accurate modelling of bone tissue is essential. Thus, the interaction between an ultrasonic wave and bone tissue has been investigated by also taking into account the effects dues to the microstructure. To do this, a generalized continuum modelling has been used. In this context, a transmission/reflection test performed on a poroelastic sample dipped in a fluid enhanced the reliability of the model. The reflected and transmitted pressure fields result to be affected by the microstructure parameters and the results coming from the dispersion analysis are in agreement with those observed in experiments for poroelastic specimens. Then, the problem has been complicated by considering the interphase taking place between the bone and the implant. In this way, we could handle the complexity added by the presence of the newly formed tissue. As already said, the fact that this interphase is a heterogeneous medium, a mixture of both solid and fluid phases whose properties evolve with time is an additional difficulty. Thus, in order to model the interaction of ultrasonic waves with this interphase, a thin layer with elastic and inertial properties has been considered in the model. The effects on the reflection properties of a transition between a homogeneous and a microstructured continuum have been investigated.Therefore, the characterization of the medium also via advanced signal processing techniques is investigated. In particular, the dynamic response due to the ultrasonic excitation of the bone-implant system is analyzed through the multifractal approach. A first analysis based on the wavelet coefficients pointed out a multifractal signature for the signals from both simulations and experiences. Then, a sensitivity study has also shown that the variation of parameters such as central frequency and trabecular bone density does not lead to a change in the response. The originality lies in the fact that it is one of the early efforts to exploit the multifractal approach in the ultrasonic propagation inside a heterogeneous medium

A Pseudoartrose Congênita da Tíbia (PCT) é uma das doenças mais desafiantes da ortopedia pediátrica pela dificuldade em obter a união óssea e, quando esta ocorre, em mantê-la. É uma doença muito rara, difícil de tratar devido à sua falta de conhecimento sobre a patogênese. As Células estromais mesenquimais multipotentes (CMM) podem desempenhar um papel na patogênese do PCT, possivelmente devido à falha da diferenciação osteogênica. O estudo das CMM pode ajudar a compreender a patogênese da doença e desenvolver novas estratégias terapêuticas baseados no uso desta célula no futuro próximo. Frente ao exposto, este trabalho teve como objetivo a análise das características biológicas das CMM isoladas de diferentes regiões anatômicas de medula óssea de pacientes com PCT. Para isto, amostras de medula óssea foram coletas a partir de locais afetados e não afetadas pela doença: Crista ilíaca do membro não afetada (CINA), crista ilíaca do membro afetada (CIA), tíbia não afetada (TNA), e tíbia afetada (TA). O numero de pacientes incluídos no estudo foi três: PCT1, PCT2 e PCT3. Os resultados mostraram que todas as células isoladas de pacientes com PCT apresentavam características compatíveis com as CMM. A taxa de formação de unidades formadoras de colônias das células da TA tanto no PCT2 quanto no PCT3 foi significativamente menor em relação às células da TNA e CINA respectivamente (p<0.05). A quantidade de células positivas para o marcador CD146 foi menor nas células da TA do PCT1 e PCT2, A análise estatística mostrou que não há uma diferença significativa. Este marcador esta relacionado com a capacidade multipotente e formação óssea in vivo. No PCT1 observou-se que formação de matriz mineralizada das CMM isoladas da CIA foi significativamente maior em relação a TA. Além disso, as células da TA do PCT1 observou-se um uma secreção significativa de alguns citocinas envolvidas no processo de formação óssea, como CCL2, CCL3, CCL4, TNA-alfa, PDGF-BB, e GM-CSF. A alteração destas citocinas pode levar a situações complicadas como o caso de não consolidação óssea. Com os resultados obtidos, se há demonstrado que as CMM da tíbia afetada tenta formar osso, mas no local da lesão é insuficiente, por tal motivo é preciso realizar estudos focados no mecanismo molecular. / Congenital pseudoarthrosis of the tibia (CPT) is one of the most challenging orthopedic diseases because of the difficulty in obtaining bone union and, when it happens, in maintaining it. It is a rare disease, difficult-to-treat due to the lack of knowledge about to pathogenesis. Multipotent mesenchymal stromal cells (MSC) may play a role in the pathogenesis of PCT, possibly due to a failure in the osteogenic differentiation. Studying these cells can help to better understand the pathogenesis of the disease and develop new therapeutic strategies based on the use of MSC in the near future. In view of the above, this work had the objective of analyzing the biological characteristics of CMM isolated from different anatomic regions of bone marrow of patients with PCT. For this, bone marrow samples were collected from sites affected and unaffected by the disease: unaffected limb iliac crest (CINA), affected limb iliac crest (CIA), unaffected tibia (TNA), and affected tibia (TA). The number of patients included in the study was three: PCT1, PCT2 and PCT3. The results showed that all cells isolated from PCT patients had characteristics compatible with CMM. The rate of formation of colonyforming units of TA cells in both PCT2 and PCT3 was significantly lower in TNA and CINA cells respectively (p <0.05). The amount of cells positive for the CD146 marker was lower in the TA cells of PCT1 and PCT2. Statistical analysis showed no significant difference. This marker is related to the multipotent capacity and bone formation in vivo. In PCT1 it was observed that the formation of mineralized matrix of CMCs isolated from CIA was significantly higher in relation to AT. In addition, PCT1 TA cells showed a significant secretion of some cytokines involved in the bone formation process, such as CCL2, CCL3, CCL4, TNA-alpha, PDGF-BB, and GM-CSF. The alteration of these cytokines can lead to complicated situations such as the case of non-consolidation of bone. With the results obtained, if the CMM of the affected tibia has been shown to try to form bone, but at the site of the lesion is insufficient, it is necessary to carry out studies focused on the molecular mechanism.

A literatura atual aponta o exercício físico como uma das estratégias nãofarmacológicas mais utilizadas no tratamento e prevenção de condições que acometem o tecido ósseo. Ademais, estudos indicam que a suplementação de creatina pode exercer efeitos positivos sobre o ganho de massa óssea. Sendo assim, o objetivo deste estudo foi investigar os efeitos preventivos do exercício físico associado à suplementação de creatina na perda de massa óssea em ratas ovariectomizadas. Diante disso, sessenta e cinco ratas da linhagem Wistar foram pareadas pelo peso corporal e dividas aleatoriamente em cinco grupos, sendo eles: 1) ratas falso-operadas (SHAM); 2) ratas ovariectomizadas (OVX), sedentárias e suplementadas com placebo (PL); 3) ratas OVX, sedentárias e suplementadas com creatina (CR); 4) ratas OVX, treinadas e suplementadas com placebo (PL+TR) e 5) ratas OVX, treinadas e suplementadas com creatina (CR+TR). Os animais foram submetidos a um protocolo de treinamento físico em declive em esteira rolante e suplementados com creatina diariamente por meio de gavagem esofágica. Foi realizada a avaliação de densitometria óssea para a obtenção dos parâmetros ósseos de conteúdo mineral ósseo (CMO) e densidade mineral óssea (DMO) de corpo total e regional, assim como a composição corporal nos períodos pré e pósintervenção. Além disso, foi removido o fêmur direito para a análise biomecânica. Após a intervenção, o grupo PL+TR apresentou maiores valores de CMO e DMO em comparação ao grupo PL (p=0,004 e p=0,020, respectivamente), ao passo que o grupo CR+TR experimentou maiores incrementos para o CMO e tendência ao aumento da DMO em comparação ao grupo CR (p=0,011 e p=0,064). A análise biomecânica do fêmur demonstrou que ambos os grupos treinados (PL+TR e CR+TR) apresentaram valores de força máxima significantemente maiores em relação aos grupos SHAM (p=0,024 e p=0,020, respectivamente), PL (p<0,001 e p<0,001) e CR (p=0,002 e p=0,002). Com relação à rigidez do fêmur, observou-se que o grupo SHAM não apresentou diferença significante quando comparado à ambos os grupos treinados (p=0,973 vs. PL+TR e p=0,998 vs. CR+TR), entretanto, apresentou diferença significante em relação aos grupos sedentários (p=0,048 vs. PL e p=0,024 vs. CR), ainda para esse parâmetro, o grupo PL apresentou diferença significante em relação ao grupo PL+TR (p=0,009), assim como o grupo CR foi significantemente diferente em relação ao grupo CR+TR (p=0,043). Não houve diferenças significantes entre os grupos PL e CR e entre os grupos PL+TR e CR+TR ao longo do estudo. Dessa forma, concluímos que a suplementação de creatina não apresentou efeitos isolados, nem aditivos, quando combinada ao treinamento físico, porém, o exercício físico promoveu efeitos positivos sobre o tecido ósseo, enfatizando, portanto, o seu papel terapêutico ímpar em atenuar a perda de massa óssea / The current literature indicates exercise training as one of the most used nonpharmacological strategies in the treatment and prevention of conditions that affect the bone tissue. Moreover, studies indicate that creatine supplementation may exert positive effects on bone mass gain. Thus, the aim of this study was to investigate the preventive effects of exercise training associated with creatine supplementation on bone loss in ovariectomized rats. Thus, sixty-five female Wistar rats were matched by body weight and randomly assigned into five experimental groups, as follows: 1) shammed (SHAM); 2) ovariectomized (OVX), sedentary and placebo-supplemented rats (PL); 3) OVX, sedentary and creatine-supplemented rats (CR); 4) OVX, trained and placebo-supplemented rats (PL+TR) and 5) OVX rats, trained and creatinesupplemented rats (CR+TR). The animals were submitted to a downhill running training protocol performed on a treadmill and supplemented with creatine on daily basis via gavage. Bone density were evaluated pre and post-intervention to obtain bone mineral content (BMC) and bone mineral density (BMD) from whole body and regional area, as well as body composition. Right femur was removed to biomechanical assessment. After the intervention, PL+TR group had higher BMC and BMD compared to the PL group (p=0.004 and p=0.020, respectively), while the CR+TR group experienced greater increases in BMC and tended to increase BMD compared to the CR group (p=0.011 and p=0.064, respectively). Biomechanical assessment demonstrated significantly higher femur maximum strength of both trained groups (PL+TR and CR+TR) compared to SHAM group (p=0.024 and p=0.020, respectively), PL group (p<0.001 and p<0.001) and CR group (p=0.002 and p=0.002). With respect to femur stiffness, no significant difference was observed from the SHAM group compared to both trained groups (p=0.973 vs. PL+TR and p=0.998 vs. CR+TR), however, significant difference was observed when compared to sedentary groups (p=0.048 vs. PL and p=0.024 vs. CR), moreover, significant difference was observed when the PL group was compared to PL+TR group (p=0.009), as well as the CR group was significantly different compared to the CR+TR group (p=0.043). There were no significant differences between PL and CR groups and between PL+TR and CR+TR groups along the study. Thus, we conclude that creatine supplementation showed no isolated, nor additive effects when combined with exercise training, however, exercise training promoted positive effects on bone tissue, thus emphasizing its unique therapeutic role in attenuating the loss of bone mass