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.

Orientador: Rui Seabra Ferreira Junior / Resumo: Atualmente são muitos casos de pacientes que perdem estrutura óssea em acidentes ou reabsorção patológica. A bioengenharia óssea é um tratamento promissor que visa reconstruir estas estruturas sem a morbidade do enxerto autógeno. O tecido ósseo é um conjuntivo especializado com a função principal de proteção e sustentação dos tecidos moles, mas também é responsável pela produção de tipos celulares e homeostase de minerais. Sua reparação é complexa com diferentes tipos celulares e agentes quimiotáticos que funcionam de forma orquestrada até a reparação. As terapias celulares vêm sendo estudadas para promover a reparação de defeitos que o organismo por si não consegue resolver. Células menos especializadas como as células-tronco embrionárias (ESCs) possuem grande potencial terapêutico, mas são complicadas eticamente. Já as células-tronco mesenquimais (MSC) podem ser autólogas, o que minimiza o risco de imunogenicidade mas necessitam área doadora do paciente. Atualmente ainda não há consenso quanto ao uso de células tronco na terapia regenerativa pois há grandes variáveis como a melhor forma de aplicação, a quantidade correta e o melhor tipo celular para a regeneração óssea. As células produzem mediadores químicos no local enxertado, que segundo pesquisas recentes é o principal mecanismo de reparação tecidual. Estes mediadores são depositados em abundância no meio de cultura durante a cultura celular e usados na bioengenharia com a ajuda de scaffolds. Os biopolímeros de fibrina ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Chapter I present a review about bone repair and its biological events, bioengineering, cells, fibrin biopolymer as scaffold and the secretoma derived from cell culture. Many patients nowadays lose bone structure in accidents or pathological reabsorption. Bone bioengineering is a promising treatment that aims to reconstruct these structures without autogenous graft morbidity. Bone tissue is a specialized connective tissue specialized in protecting and supporting soft tissues, but it is also responsible for the production of cell types and mineral homeostasis. The bone healing is a complex process where different cell types and chemotactic agents work in an orchestrated way. The cell therapies can promote the repair of defects that the body cannot solve. Less specialized cells like embryonic stem cells (ESCs) have great therapeutic potential, but are ethically complicated. In contrast, mesenchymal stem cells (MSCs) may be autologous, which minimizes the risk of immunogenicity but requires a patient's donor area. Currently there is still no consensus regarding the use of stem cells in regenerative therapy, studies uses different methods, cells and biomaterials for bone regeneration. Recent researches advocate that paracrine secretions by cells are main mechanism of tissue repair. These mediators are deposited in abundance in the culture medium during cell culture. Fibrin biopolymers (BF) are natural biomaterials to the body and can function as drug delivery of growth factors, c... (Complete abstract click electronic access below) / Doutor

Le ranelate de strontium (RS) est un traitement anti-ostéoporotique agissant sur la formation osseuse via les ostéoblastes et la résorption osseuse via les ostéoclastes grâce au Calcium Sensing Receptor (CaSR). L’activité physique (EXE) est bien connue pour améliorer les propriétés osseuses. Dans ce travail, nous avons étudié ① l’effet de différentes activités physiques (tapis roulant et impact). Nous avons observé que 10 impacts par jour pendant 8 semaines à 45cm de hauteur ont eu des effets bénéfiques sur l’os (paramètres de microarchitecture et biomécanique, marqueurs du remodelage). ② L’étude du RS et de l’EXE sur l’os de rate ovariectomisée a montré que le RS a des effets comparables à l’EXE et que le RS+EXE ont des effets cumulatifs sur l’os (paramètres de microarchitecture et biomécanique, marqueurs du remodelage). ③ Enfin, une étude in vivo sur des MLO-Y4 a montré la présence des CaSR sur la membrane des ostéocytes et leur nombre est modulé en fonction de la concentration en RS. De plus, le RS a un effet sur la différenciation des CSM lors d’une différenciation ostéogénique en favorisant la différenciation ostéocytaire et elle est modulée par la concentration en RS. En conclusion, ce travail a démontré l’importance d’une pratique d’un exercice physique et du traitement du RS contre l’ostéoporose : maintien de la balance du remodelage osseux du côté de la formation. L’effet cumulatif du RS+EXE s’explique par le fait que le RS agit sur les ostéoblastes, ostéocytes et ostéoclastes via les CaSR et l’EXE sur les mécanorécepteurs des ostéocytes. / Strontium ranelate (SR) is an anti-osteoroporotic treatment acting on bone formation via osteoblasts and bone resorption via osteoclasts thanks to Calcium Sensing Receptor (CaSR). Physical activity is well known to improve bone properties. In this work, we studied ① different physical acticities (treadmill and impact). We observed that 10 impacts per day during 8 weeks from 45cm of height had beneficial effects on bone (microarchitecture and biomechanical parameters, bone remodeling markers). ② The study of SR and EXE effects on bone ovariectomized rats showed that RS had similar effects to EXE and SR+EXE had cumulative effects on bone (microarchitecture and biomechanical parameters, bone remodeling markers). ③ Finally, an in vivo study on MLO-Y4 showed CaSR presence on osteocyte with their number depending on SR concentration. Moreover, RS had positive effects on CSM differentiation in favor of osteocyte differentiation and it is modulated by SR concentration. In conclusion, this work has demonstrated the importance of taking physical exercise as well as SR treatment for osteoporosis: maintaining the bone remodeling in favor of bone formation. The cumulative effect of SR+EXE is explained by the fact the SR acts on osteoblasts, osteocytes and osteoclasts via CaSR and the EXE on osteocyte mechanoreceptors.

Tissue engineering has emerged as a promising alternative to conventional orthopaedic grafting therapies. The general paradigm for this approach, in which phenotype-specific cells and/or bioactive growth factors are integrated into polymeric matrices, has been successfully applied in recent years toward the development of bone, ligament, and cartilage tissues in vitro and in vivo. Despite these advances, an optimal cell source for skeletal tissue repair and regeneration has not been identified. Furthermore, the lack of robust, functional orthopaedic tissue interfaces, such as the bone-ligament enthesis, severely limits the integration and biological performance of engineered tissue substitutes. This works aims to address these limitations by spatially controlling the genetic modification and differentiation of fibroblasts into a mineralizing osteoblastic phenotype within three-dimensional polymeric matrices. The overall objective of this project was to investigate transcription factor-based gene therapy strategies for the differentiation of fibroblasts into a mineralizing cell source for orthopaedic tissue engineering applications. Our central hypothesis was that fibroblasts genetically engineered to express Runx2 via conventional and biomaterial-mediated ex vivo gene transfer approaches will differentiate into a mineralizing osteoblastic phenotype. We have demonstrated that a combination of retroviral Runx2 overexpression and glucocorticoid hormone treatment synergistically induces osteoblastic differentiation and biological mineral deposition in primary dermal fibroblasts cultured in monolayer. We report for the first time that glucocorticoids induce osteoblastic differentiation in this model system by modulating the phosphorylation state of a negative regulatory serine residue (Ser125) on Runx2 through an MKP-1-dependent mechanism. Furthermore, we utilized these Runx2-genetically engineered fibroblasts to create mineralized templates for bone repair in vitro and in vivo. Finally, we engineered a heterogeneous bone-soft tissue interface with a novel biomaterial-mediated gene transfer approach. Overall, these results are significant toward the ultimate goal of regenerating complex, higher-order orthopaedic grafting templates which mimic the cellular and microstructural characteristics of native tissue. Cellular therapies based on primary dermal fibroblasts would be particularly beneficial for patients with a compromised ability to recruit progenitors to the sight of injury as result of traumatic injury, radiation treatment, or osteodegenerative disease.

Tissue engineering is a promising alternative strategy to produce artificial bone substitutes / however, the control of the cell organization and cell behavior to create fully functional 3-D constructs has not yet been achieved. To overcome these, activities have been concentrated on the development of multi-functional tissue engineering scaffolds capable of delivering the required bioactive agents to initiate and control cellular activities. The aim of this study was to prepare tissue engineered constructs composed of polymeric scaffolds seeded with mesenchymal stem cells (MSCs) carrying a nanoparticulate growth factor delivery system that would sequentially deliver the growth factors in order to mimic the natural bone healing process. To achieve this, BMP-2 and BMP-7, the osteogenic growth factors, were encapsulated in different polymeric nanocapsules (poly(lactic acid-co-glycolic acid) (PLGA) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV)) with different properties (degradation rates, crystallinity) and, therefore, different release rates to achieve the early release of BMP-2 followed by the release of BMP-7, as it is in nature. Initially, these nanoparticulate delivery systems were characterized and then the effect of single, simultaneous and sequential delivery of BMP-2 and BMP-7 from these delivery systems was studied in vitro using rat bone marrow MSCs. The effect of using these two growth factors in a sequential manner by mimicking their natural bioavailability timing was shown with maximized osteogenic activity results. BMP-2 loaded PLGA nanocapsules were subcutaneously implanted into Wistar rats and according to initial results, their biocompatibility as well as the positive effect of BMP-2 release on the formation of osteoclast-like cells was shown. To complete the construction of the bioactive scaffold, this nanoparticulate sequential delivery system was incorporated into two different types of polymeric systems / natural (chitosan) and synthetic (poly(&amp / #949 / -caprolactone) (PCL)). 3-D fibrous scaffolds were produced using these materials by wet spinning and 3-D plotting. Incorporation of nanocapsules into 3-D chitosan scaffolds was studied by two different methods: incorporation within and onto chitosan fibers. Incorporation into 3-D PCL scaffolds was achieved by coating the nanocapsules onto the fibers of the scaffolds in an alginate layer. With both scaffold systems, incorporation of nanocapsule populations capable of delivering BMP-2 and BMP-7 in single, simultaneous and sequential fashion was achieved. As with free nanocapsules, the positive effect of sequential delivery on the osteogenic differentiation of MSCs was shown with both scaffold systems, creating multi-functional scaffolds capable of inducing bone healing.

Surface modification techniques have played important roles in various aspects of modern technology. They have been employed to improve substrates by altering surface physicochemical properties. An ideal surface modifying technique would be a method that is applicable to any kind of materials prepared from a wide range of polymers and that can occur under mild reaction conditions. The work in this thesis has utilized four main concepts: I) the development of a ‘grafting-from’ technique by covalently growing polymer grafts from particle surfaces, II) the presence of steric and electrosteric forces due to long-range repulsive interactions between particles, III) a combined surface grafting and layer-by-layer approach to create polyelectrolyte multilayers (PEMs) on particle surfaces to fabricate strong and functional materials, and IV) the roles of hydrophilic polymer grafts and substrate geometry on surface degradation. A non-destructive surface grafting technique was developed and applied to polylactide (PLA) particle surfaces. Their successful modification was verified by observed changes to the surface chemistry, morphology and topography of the particles. To quantify the aggregation behavior of grafted and non-grafted particles, force interaction measurements were performed using colloidal probe atomic force microscopy (AFM). Long-range repulsive interactions were observed when symmetric systems, i.e., hydrophilic polymer grafts on two interacting surfaces, and asymmetric system were applied. Electrosteric forces were observed when the symmetric substrates interacted at pH 7.4. When PEMs were alternately assembled on the surface of poly(L-lactide) (PLLA) particles, the grafted surfaces played a dominated role in altering the surface chemistry and morphology of the particles. Three-dimensional scaffolds of surface grafted particle coated with PEMs demonstrated high mechanical performance that agreed well with the mechanical performance of cancellous bone. Nanomaterials were used to functionalize the scaffolds and further influence their physicochemical properties. For example, when magnetic nanoparticles were used to functionalize the scaffolds, a high electrical conductivity was imparted, which is important for bone tissue regeneration. Furthermore, the stability of the surface grafted particles was evaluated in phosphate buffered saline (PBS) solution. The nature of the hydrophilic polymer grafts and the geometry of the PLLA substrates played central roles in altering the surface properties of films and particles. After 10 days of PBS immersion, larger alterations in the surface morphology were observed on the film compared with microparticles grafted with poly(acrylic acid) (PAA). In contrast to the PAA-grafted substrates, the morphology of poly(acrylamide) (PAAm)-grafted substrates was not affected by PBS immersion. Additionally, PAAm-grafted microparticulate substrates encountered surface degradation more rapidly than PAAm-grafted film substrates. / <p>QC 20151002</p>

Submitted by Johnny Rodrigues (johnnyrodrigues@ufcg.edu.br) on 2018-04-16T19:44:01Z No. of bitstreams: 1 WILLIAMS TELES BARBOSA - DISSERTAÇÃO PPG-CEMat 2014..pdf: 2309358 bytes, checksum: 60fe4478ffb44784348a9ae62c055d89 (MD5) / Made available in DSpace on 2018-04-16T19:44:01Z (GMT). No. of bitstreams: 1 WILLIAMS TELES BARBOSA - DISSERTAÇÃO PPG-CEMat 2014..pdf: 2309358 bytes, checksum: 60fe4478ffb44784348a9ae62c055d89 (MD5) Previous issue date: 2015-02-25 / Capes / Biocerâmicas porosas são utilizadas para fornecer local onde o tecido ósseo possa crescer e fixar o implante biologicamente. A hidroxiapatita [HA, Ca10(PO4)6(OH)2] é um fosfato de cálcio que tem recebido atenção considerável nas últimas duas décadas como material de implante. Devido à sua ocorrência natural no tecido ósseo, os fosfatos de cálcio possuem boas propriedades de biocompatibilidade e osteocondução, tornando-a um dos biomateriais mais promissores na fabricação de scaffolds para a engenharia de tecido ósseo. O objetivo do presente trabalho centrou-se no desenvolvimento e otimização de estruturas tridimensionais porosas a base de HA combinando o método Sol-Gel e a réplica da esponja de poliuretano (PU), permitindo uma interconectividade e distribuição variada dos poros. Os scaffolds desenvolvidos foram caracterizados pelas técnicas de Espectroscopia na Região do Infravermelho com Transformada de Fourier (FTIR), Difração de Raios X (DRX), Microscopia Eletrônica de Varredura (MEV), Espectroscopia por Energia Dispersiva de Raios X (EDS), Análise Termogravimétrica (TG), Porosidade, Ensaio de Compressão. Os resultados de FTIR apresentaram as bandas características da HA. A técnica de DRX revelou a presença da fase cristalina de HA (95%), como também em menor quantidade o α-Fosfato Tricálcico (2,5%). As análises por MEV revelaram scaffolds com poros interconectados com tamanhos de poros variando entre 50µm a 200μm e o EDS detectou a presença dos elementos químicos característicos da HA, como o Cálcio e o Fósforo. Os resultados de TG permitiram confirmar que as curvas de temperatura utilizadas no processo de sinterização, são eficientes para a queima da esponja, obtendo-se somente uma fase inorgânica de apatita. Os scaffolds apresentaram uma porosidade total de aproximadamente 75% e resistência à compressão variando de 3,13 a 4,86 MPa. Diante dos resultados obtidos foi possível produzir scaffolds de apatita através da metodologia Sol-Gel e combinação com a metodologia de replica de esponja porosa, com características que devem permitir a regeneração óssea. / Porous bioceramics are used to provide location where the bone tissue can grow and biologically fixing the implant. Hydroxyapatite [HA, Ca10(PO4)6(OH)2] is a calcium phosphate which has received considerable attention over the past two decades as an implant material. Due to its naturally occurring in bone tissue, the calcium phosphate has good biocompatibility and osteoconductive properties, making it one of the most promising biomaterials in the manufacture of scaffolds for bone tissue engineering. The objective of this work was the development and optimization of porous three-dimensional structures composed of HA, combining sol-gel method with the replica of a polyurethane foam, allowing interconnectivity and scattered distribution of pores. The developed scaffolds were characterized by Fourier Transform in the Infrared Region (FTIR), X-Ray Diffraction (XRD), Scanning Electron Microscopy (SEM), Energy Dispersive Spectroscopy (EDS), Thermogravimetric Analysis (TG), Porosity Tests and Compression Tests. The FTIR results showed the characteristic bands of the hydroxyapatite. The XRD technique revealed the presence of a crystalline phase belonging to hydroxyapatite (97,5%), and to a lesser extent the α-Tricalcium Phosphate (2,5%). Analysis by SEM revealed scaffolds with interconnected pores which had sizes ranging from 50μm to 200μm and EDS detected the presence of specific chemical elements of hydroxyapatite such as Calcium and Phosphorus. TG results allowed to confirm that the temperature curves used in the sintering process, is effective for burning of the sponge, yielding only an inorganic phase of apatite. The scaffolds showed a porosity of about 75% and compressive moduli ranging from 3.13 to 4.86 MPa. Based on these results, it was possible to produce scaffolds of HA by Sol-Gel method in combination with replica of a polyurethane foam, with attributes for bone regeneration.

L’Ingénierie tissulaire (IT) est un domaine interdisciplinaire qui applique les principes de l'ingénierie et des sciences de la vie au développement de substituts biologiques afin de restaurer, maintenir ou améliorer la fonction tissulaire. Sa première application consiste à remplacer les tissus endommagés par des produits cellulaires artificiels. Une autre application de l’IT est basée sur la production des modèles en 2 et 3 dimensions (2D et 3D) pour des études biologiques et pharmacologiques in vitro. Ces modèles ou remplacements de tissus peuvent être fabriqués en utilisant des différentes méthodes de médecine, biologie, chimie, physique, informatique et mécanique, fournissant un micro-environnement spécifique avec différents types de cellules, facteurs de croissance et matrice. L'un des principaux défis de l'IT la pénétration cellulaire limitée dans les parties internes des biomatériaux poreux. Une faible viabilité cellulaire au centre du produit d'IT est la conséquence de la diffusion limitée d'oxygène et de nutriments du fait d’un réseau vasculaire insuffisant dans l'ensemble de la construction 3D. Le BioAssembage couche-par-couche est une nouvelle approche basée sur l'assemblage de petites constructions cellularisées permettant une distribution cellulaire homogène et une vascularisation plus efficace dans des produits d’IT.Notre hypothèse est que l'approche couche-par-couche est plus adaptée à la régénération osseuse que l'approche conventionnelle de l'IT. L'objectif principal de cette thèse était d'évaluer les avantages de l'approche couche-par-couche en utilisant des membranes de polymères imprimées en 3D et ensemencées avec des cellules primaires humaines. Nous avons évalué l'efficacité de la formation du réseau vasculaire in vivo dans toute la construction 3D en utilisant cette approche et en la comparant à l'approche conventionnelle basée sur l'ensemencement des cellules sur la surface des scaffolds massives. Il n'y avait pas de différence significative dans le nombre de vaisseaux sanguins formés en 3D au niveau des parties externes des constructions implantées en site souscutanée chez des souris. Mais dans les parties internes des implants qui n'étaient pas en contact direct avec un tissu hôte, nous avons pu observer une formation des vaisseaux sanguins statistiquement plus efficace lorsque l'approche du bio-assemblage couche-par-couche a été utilisée. Cette formation de réseau vasculaire était plus importante dans le cas de co-cultures que de mono-cultures.Il y avait plusieurs objectifs secondaires dans ce travail. Le premier était de fabriquer des constructions 3D cellularisées pour l'IT en utilisant des membranes d'acide polylactique (PLA) et des cellules primaires humaines : des cellules de stroma de moelle osseuse humaine (HBMSCs) isolées de la moelle osseuse et des cellules progénitrices endothéliales (EPCs) isolées du sang du cordon ombilical. Ensuite, nous avons comparé différentes technologies de fabrication des scaffolds: impression 3D directe à partir de poudre de PLA et impression par fil fondu en utilisant une imprimante commerciale et une autre fabriquée sur mesure. L'imprimante sur mesure a permis le plus haut niveau de résolution d'impression spécialement adaptée à la forme et la taille des pores. Par ailleurs, nous avons évalué différents systèmes de stabilisation pour l'assemblage couche par couche : l’utilisation de clips en PLA imprimés en 3D a fourni une stabilisation plus efficace pour empiler les membranes PLA couche par couche. Un autre avantage de ce système de stabilisation est qu'il peut être implanté avec des implants. Ensuite, nous avons observé une prolifération et une différenciation cellulaire plus efficaces lorsque le système de co-culture était utilisé, en comparaison avec des mono-cultures.L'approche du bioassemblage couche-par-couche semble être une solution appropriée pour une vascularisation efficace dans des structures 3D entières d'ingénierie tissulaire. / Tissue Engineering (TE) is “an interdisciplinary field that applies principles of engineering and the life sciences toward development of biological substitutes that restore, maintain, or improve tissue function”. The First application of TE is to replace damaged tissues by artificial cell-materials products of tissue engineering (TE). Another TE application is to produce 2 or 3 dimensional (2D and 3D) models for biological and pharmacological in vitro studies. These models or tissue replacements can be fabricated using a combination of different interdisciplinary methods of medicine, biology, chemistry, physics, informatics and mechanics, providing specific micro-environment with different cell types, growth factors and matrix.One of the major challenges of tissue engineering is related to limited cell penetration in the inner parts of porous biomaterials. Poor cell viability in the center of engineered tissue is a consequence of limited oxygen and nutrients diffusion due to insufficient vascular network within the entire construct. Layer-by-layer (LBL) BioAssembly is a new approach based on assembly of small cellularized constructs that may lead to homogenous cell distribution and more efficient three dimensional vascularization of large tissue engineering constructs.Our hypothesis is that LBL Bioassembly approach is more suitable for bone regeneration than conventional tissue engineering approach. The primary objective of this thesis was to evaluate the advantages of LBL Bioassembly approach using 3D-printed polymer membranes seeded with human primary cells. We have evaluated the efficiency of vascular network formation in vivo within entire 3D tissue engineering construct using LBL bioassembly approach and comparing it to the conventional approach based on seeding of cells on the surface of massive 3D scaffolds. There was no significant difference in number of formed blood vessels in 3D at the outer parts of constructs implanted subcutaneously in mice 8 weeks post-implantation. But in the inner parts of implants which were not in direct contact with a host tissue, we could observe statistically more blood vessel formation when LBL bioassembly approach was used. This vascular network formation was more important in the case of co-cultures than mono-vultures of HBMSCs.There were several secondary objectives in this work. The first was to fabricate cellularized 3D constructs for bone tissue engineering using poly(lactic) acid (PLA) membranes and human primary cells: human bone marrow stroma cells (HBMSCs) isolated from the bone marrow, and endothelial progenitor cells (EPCs) isolated from the umbilical cord blood. Then, we have compared different Additive manufacturing technologies to fabricate scaffolds: direct 3D printing (3DP) starting from PLA powder dissolved in chloroform and fused deposition modelling (FDM) using a commercial or a custom-made printer with different resolutions.The custom-made printer equipped with 100 μm nozzle allowed the highest level of printing resolution concerning pores shape and size. In the meantime we evaluated different stabilization systems for layer-by-layer assembling of PLA membranes with human primary cells: the use of 3D printed PLA clips provided the most efficient stabilization to stack PLA membranes in 3D. Another advantage of this stabilization system is that it could be implanted together with LBL constructs. Then we investigated the most suitable cell culture system for such constructs and we observed more efficient cell proliferation and differentiation when co-culture system is used, comparing to mono-cultures.LBL bioassembly approach seems to be suitable solution for efficient vascularization within entire large 3D tissue engineering constructs especially when co-cultures of mesenchymal and endothelial cells are used.

Made available in DSpace on 2016-06-02T20:18:11Z (GMT). No. of bitstreams: 1 2456.pdf: 6548607 bytes, checksum: 486173aee7443e35a2910af043a12256 (MD5) Previous issue date: 2009-04-03 / Universidade Federal de Sao Carlos / Bioactive materials have the ability to bond and to integrate with bone tissue by forming a biologically active bonelike apatite layer, which has chemical and structural properties equivalent to the mineral phase of living bone. This process is determined by chemical reactions, whose products also influence the attachment, the proliferation, the differentiation and the mineralizing capacity of bone cells. Cellular responses contribute to the bioactive behavior, which is known for being higher in glass materials. However, as low mechanical properties are also inherent characteristics of glasses, researchers from Federal University of Sao Carlos were stimulated to develop nucleation and growth thermal treatments for the obtainment of the Biosilicate®, a fully-crystallized bioactive glassceramic of the quaternary system P2O5-Na2O-CaO-SiO2. Although a high in vitro osteogenic potential of this novel glass-ceramic has been previously demonstrated, its in vivo effects have not been investigated yet. To contribute to this knowledge, two studies were developed. The first one aimed to investigate the in vivo biological performance of Biosilicate® in bone defects of rat tibias, by means of hystomorphometric and biomechanical analyses 20 days after the surgical procedure. This study revealed that the fully-crystallized Biosilicate® has good bone-forming and bone-bonding properties. Hence, the second study aimed to compare the kinetics of the bone reactions to two different granulometric distributions of this novel glass-ceramic. Although they were both efficient for bone formation, smaller-sized particles of Biosilicate® showed partial reabsortion, which was accompanied by a more pronounced osteogenic activity within the period of time studied. Since positive results were obtained, the search for scaffolds that could serve as supports for the guided bone regeneration had started. A third study preliminarily evaluated cell culture and cocultures in porous structures made of Biosilicate® and of other chemical compositions that were specifically developed for this purpose. The findings suggest that, when in adjusted conditions, the scaffolds can create favorable cellular responses for bone tissue engineering purposes. Taken togheter, these studies point to a promising potential and provide directives for the use of Biosilicate® in bone regenerative processes. / Materiais bioativos possuem a capacidade de se ligar ao tecido ósseo por meio da formação de uma interface apatítica que apresenta similaridade química e estrutural com a fase mineral dos ossos. Esse processo ocorre devido a uma série de reações químicas, cujos produtos também influenciam a adesão, a proliferação, a diferenciação e a capacidade de mineralização da matriz pelas células ósseas. As respostas celulares contribuem para o comportamento bioativo, que é conhecido por ter maiores índices em materiais vítreos. No entanto, como baixas propriedades mecânicas também são características inerentes aos vidros, pesquisadores da Universidade Federal de São Carlos foram estimulados a empregarem nucleação e tratamentos térmicos especiais para o desenvolvimento do Biosilicato®, uma vitrocerâmica biotiva, totalmente cristalina, pertencente ao sistema quaternário P2O5-Na2O-CaO-SiO2. Embora um elevado potencial osteogênico in vitro tenha sido demonstrado para esta vitrocerâmica, seus efeitos in vivo ainda não são conhecidos. Para auxiliar este entendimento, foram desenvolvidos dois estudos. O primeiro teve como objetivo investigar o desempenho biológico in vivo do Biosilicato® particulado em defeitos ósseos em tíbias de ratos, por meio de análises histomorfométricas e biomecânicas 20 dias após o procedimento cirúrgico. Este estudo evidenciou que o Biosilicato® parece favorecer a formação óssea in vivo e o estabelecimento de fortes ligações com o tecido neoformado. Com isso, o objetivo do segundo estudo foi comparar a cinética das reações ósseas frente a duas diferentes distribuições granulométricas desta nova vitrocerâmica. Embora ambas tenham sido eficientes para a formação óssea, as partículas de Biosilicato® com menores diâmetros demonstraram reabsorção parcial no período estudado, que foi acompanhada de uma maior atividade osteogênica. Com os resultados positivos obtidos nestas investigações, iniciou-se uma busca para o desenvolvimento de matrizes porosas que pudessem servir de suporte para a regeneração guiada do tecido ósseo. Um terceiro estudo preliminarmente avaliou monoculturas e coculturas celulares em matrizes porosas de Biosilicato® e de outras novas composições químicas desenvolvidas especificamente para este propósito. Os achados sugerem que, quando em condições adequadas, as matrizes avaliadas podem produzir respostas celulares favoráveis ao seu emprego na engenharia do tecido ósseo. Estes estudos, de maneira conjunta, apontam para um potencial promissor e fornecem diretrizes para o emprego do Biosilicato® no favorecimento de processos regenerativos ósseos.

Made available in DSpace on 2016-06-02T19:02:42Z (GMT). No. of bitstreams: 1 5491.pdf: 1920804 bytes, checksum: 6c1ae0832cc23b3e3e7f4ac2965acd1d (MD5) Previous issue date: 2013-06-14 / Universidade Federal de Sao Carlos / The DM is a chronic metabolic disorder characterized by a deficiency in the secretion or action of insulin, leading to a series of physiological changes that determine changes in normal operation of various organs and tissues, among which bone tissue is affected, leading to bone fragility. In this context, several treatments have been shown to accelerate bone metabolism. The Resistance Exercise (ER) is highly recommended for diabetics and among its beneficial effects promotes increased bone mineral density. The low-level laser therapy (LLLT) is able to stimulate the activity of osteoblasts, as well as increase the biomechanical properties of bone. However, its effects on bone metabolism in diabetic animals are not completely understood, and its action associated with the ER. Therefore, this study aimed to investigate the action of a protocol and resistance exercises LLLT applied individually or in combination on bone metabolism in diabetic rats. Fifty male Wistar rats were randomly divided into 5 experimental groups (N = 10): non-diabetic control group (CG), diabetic control (GD), diabetic group irradiated with laser (GL), trained diabetic group (TG) and trained group laser and diabetic (GTL). In the first study we evaluated the effects of LLLT on bone diabetic in three groups: GC, GD and GL. The GL was subjected to laser irradiation Ga-Al-As, 808 nm, 100 mW, 3.57 W/cm2, 0.028 cm2, 120J/cm2, 33s, for 18 sessions, on alternate days for 6 weeks. As the GL results showed increased cortical area and RUNX-2 immunoreactivity increased compared to GD. Furthermore, LLLT produced a significant increase in the strength of fracture, and bone mineral density (BMD and BMC), compared with DG. Therefore LLLT stimulated bone formation, reducing osteopenia animals. The second study evaluated the effects associated with ER LLLT in diabetic animals from group 4: GC, GD, GT and GTL. The ER consisted of climbing, load tied to the tail of the animal, and these loads were increased weekly throughout the training sessions, the GTL at the end of each session ER animals were irradiated with laser Ga-Al-As. Performed 6 weeks, 3 times per week, totaling 18 sessions. The GT and GTL showed increased cortical area, BMD and biomechanical properties. The BMC, fracture strength and stiffness were higher in GTL over the GT. Furthermore, immunohistochemical analysis showed that GT and GTL immunoassayed for RUNX-2 increased relative to GD. Already RANK-L immunoreactivity was moderated at GD and week on the others experimental groups. In conclusion, resistance exercise promoted osteoblast activation, with the increase in the biomechanical properties and BMD. The combination of exercise and LLLT, promoted the osteogenic potential additional effect of ER performed alone. Consequently, these data highlight the potential of exercise in the treatment of bone loss due to DM. Further studies should be conducted to provide additional information on the effects of LLLT as adjuvant therapy resistance exercise. / A Diabetes Mellitus (DM) é uma doença metabólica crônica, caracterizada pela deficiência na secreção ou ação da insulina, levando a uma série de modificações fisiológicas, que determinam alterações do funcionamento normal de diversos órgãos e tecidos, dentre os quais, o tecido ósseo é afetado, levando à fragilidade óssea. Neste contexto, alguns tratamentos têm demonstrado melhorar o metabolismo ósseo. O Exercício Resistido (ER) é recomendado para indivíduos diabéticos e dentre seus efeitos benéficos promove o aumento da densidade mineral óssea. A terapia laser de baixa intensidade (LLLT) é capaz de estimular a atividade dos osteoblastos, e aumentar as propriedades biomecânicas ósseas. No entanto, seus efeitos sobre o metabolismo ósseo de animais diabéticos não estão completamente esclarecidos, bem como sua ação associado ao ER. Diante disso, este estudo teve o objetivo de investigar os efeitos de um protocolo de exercicios resistidos e da LLLT, aplicados individualmente ou em associação no metabolismo ósseo de ratos diabéticos. Cinquenta ratos Wistar machos foram distribuídos aleatoriamente em 5 grupos experimentais (N=10 cada grupo): Grupo Controle não diabético (GC), grupo controle diabético (GD), grupo Irradiado com Laser diabético (GL), grupo treinado diabético (GT) e grupo treinado e laser diabético (GTL). No primeiro estudo foram avaliados os efeitos da LLLT no osso diabético em 3 grupos: GC, GD e GL. O GL foi submetido à irradiação laser Ga-Al-As, 808 nm, 100 mW, 3,57W/cm2, 0,028cm2, 120J/cm2, 33s, durante 18 sessões, em dias alternados, por 6 semanas. Como resultados o GL mostrou aumento da área cortical e imunoexpressão de RUNX-2 aumentada em comparação o GD. Além disso, a LLLT produziu um aumento significativo na força de fratura, densidade e conteúdo mineral ósseo (DMO e CMO), em comparação com GD. Portanto a LLLT estimulou a formação óssea, reduzindo a osteopenia dos animais. O segundo estudo avaliou os efeitos da LLLT associado ao ER em animais diabéticos, a partir de 4 grupos: GC, GD, GT e GTL. O ER consistiu em escaladas, com carga atrelada à cauda dos animais, e estas cargas foram aumentadas semanalmente ao longo das sessões de treinamento, no GTL ao final de cada sessão de ER os animais foram irradiados com laser Ga-Al-As. Realizados durante 6 semanas, 3 vezes por semana, totalizando 18 sessões. O GT e GTL mostraram aumento da área cortical, DMO e propriedades biomecânicas. O CMO, a força de fratura e rigidez foram maiores no grupo GTL em relação ao GT. Ainda, a análise imunohistoquímica revelou que GT e GTL apresentaram imunoexpressão de RUNX-2, aumentada em relação à GD. Já a imunoexpressão de RANK-L foi moderada no GD e fraca nos demais grupos experimentais. Como conclusão, o exercício resistido promoveu ativação osteoblástica, com o aumento nas propriedades biomecânicas e na densidade mineral óssea. A associação de exercícios físicos e LLLT, promoveu efeito adicional ao potencial osteogênico do ER realizado isoladamente. Consequentemente, estes dados evidenciam o potencial do exercício físico no tratamento da perda óssea devido à DM. Outros estudos devem ser realizados para fornecer informações adicionais sobre os efeitos da LLLT como terapia coadjuvante ao exercício resistido.