The current study characterizes the mechanical and biodegradation properties of a polycarbonate polyurethane (PU) electrospun nanofiber scaffold intended for use in the growth of a tissue engineered annulus fibrosus (AF) intervertebral disc component. Both the tensile strength and initial modulus of aligned scaffolds were higher than those of random scaffolds and remained unaffected during a 4 week biodegradation study, suggesting a surface-mediated degradation mechanism. The resulting degradation products were non-toxic. Confined compressive mechanical force of 1kPa, was applied at 1Hz to in vitro bovine AF tissue grown on the scaffolds to investigate the influence of mechanical force on AF tissue production, which was found to decrease significantly at 72 hours relative to 24 hours, independent of any effects from mechanical forces. Overall, the consistent rate of PU degradation, along with mechanical properties comparable to those of native AF tissue, and the absence of cytotoxic effects, make this polymer suitable for further investigation for use in tissue-engineering the AF.

This thesis describes a highly interdisciplinary approach to discern the differing impact of scaffold dimensionality and physical space restrictions on the behavior of single cells. Rolled-up nanotechnology is employed to fabricate three-dimensional (3D) SiO/SiO2 microtube geometries of varied diameter, that after a biofunctionalization step are shown to support the growth of U2OS and six different types of stem cells. Cell confinement quantifiable through the given microtube diameter is tolerated by U2OS cells through a remarkable elongation of the cell body and nucleus down to a certain threshold, while the integrity of the DNA is maintained. This confinement for NSPCs also leads to the approaching of the in vivo morphology, underlining the space-restrictive property of live tissue. The dimensionality of the cell culture scaffold however is identified as the major determiner of NSPC migration characteristics and leads to a morphologically distinct mesenchymal to amoeboid migration mode transition. The 3D microtube migration is characterized by exclusively filopodia protrusion formation, a higher dependence on actin polymerization and adopts aspects of in vivo-reported saltatory movement. The reported findings contribute to the determination of biomaterial scaffold design principles and advance our current understanding of how physical properties of the extracellular environment affect cell migration characteristics.

Cell culture has been a fundamental tool for the study of cell biology, tissue engineering, stem cell technology and biotechnology in general. It becomes more and more important to have a well-defined physiochemical microenvironment during cell culture. Conventional cell cultures employ expensive, manually controlled incubation equipment, making it difficult to maximize a cultures yield. Furthermore, previous studies use qualitative methods to assess cell culture proliferation that are inherently inaccurate and labour intensive, thereby increasing the cost of production. In addition, three dimensional cell culture, in scaffold, has been shown to provide more physiological relevant information as it mimic more accurate conditions that are similar to the physiological conditions of the human body compared with two dimension, which has special interest to regenerative medicine. Therefore, a portable and automated total-analysis-system (μTAS) was proposed with microenvironment control and quantitative analysis techniques to monitor cell proliferation and metabolic activity. The automated portable heating system was validated to be capable to maintain a stable physiochemical microenvironment, with little margin of error, for cellular substrate outside of conventional incubation. A standalone platform system was designed and fabricated with accurate temperature control by employing an optically transparent ITO-film with a large heating area. The transparency of the film is critical for continuous in-situ microscopic observation over long-term cell culture process. Previous studies have attempted to use ITO-film as a heating element, but were unable to distribute the heat evenly onto the microbioreactor platform. This nagging problem in the literature was improved through a novel film design. As a result, the ITO-film based heating system was evaluated and constructed successfully to serve as a heating element for long-term static cell culture with facilitated proliferation rate in gas-permeable PDMS microbioreactor outside of conventional incubation. In addition to maintaining a stable microenvironment, a non-invasive in-situ technology for monitoring cell viability and proliferation rate was constructed and developed based on bioimpedance spectroscopy (BIS). It was primarily focused on making decisions for structure and specification of proposed system-on a chip BIS measurement. The miniaturization of BIS system on microbioreactor platform was achieved by utilizing and integrating switching matrix array, impedance analyzer chip with reliable analogue-front-end circuitry. The realized system was verified with the DLD-1 cells and its monitored data were validated with conventional bioassays. Three dimensional cell cultures with scaffold is a key to the success of tissue engineering. Engineered cornea collagen scaffold may be feasible using re-seeding proper human cells onto a decellularized corneal scaffold. The quality of the scaffold and the interaction of the cells are critical to the key function (i.e transparency, haze and total transmittance) of final products. An integrated corneal collagen scaffold quality assessment system, via optical property inspection unit, was innovatively designed and realized with non-invasive and non-destructive characteristics. The H1299 cells were seeded onto inspected corneal scaffold and BIS system, which were realized in the previous chapter, were used to validate its applicability for 3D cell culture. The cell adhesion as an outcome at different scaffolds with different optical properties has revealed the importance of the microstructure of scaffold on the cell functions. The results showed the developed technologies can be used for the quality control of corneal scaffold and the fabricated μTAS not only enabled environmental control but, with BIS-based in-situ assay, it also facilitate the function (i.e adhesion) and viability monitoring with quantitative and qualitative analysis in 3D-alike cell culture. Additionally, by considering its low decontamination and cost-effective nature with compatibility for high-throughput screening applications, the fabricated and integrated systems has significant applications in tissue engineering.

L’objectif de la thèse est d’étayer l’activité de conception expérimentale avec l’utilisation d’un environnement informatique pour l’apprentissage humain (EIAH). La conception expérimentale correspond à une partie de la démarche d’investigation qui fait l’objet de nombreuses recherches, à la fois parce que cette activité favorise l’apprentissage mais aussi parce que c’est une tâche complexe à l’origine de difficultés identifiées.La situation que nous avons choisie est la mise en évidence de la fermentation alcoolique, thème abordé en classe de terminale scientifique de spécialité en sciences de la vie et de la terre. Les élèves doivent concevoir une expérience pour mettre en évidence ce métabolisme.La première étape de la thèse a consisté à effectuer une modélisation didactique des connaissances en jeu. Pour cela nous nous plaçons dans le cadre de la théorie anthropologique du didactique et plus précisément l’approche praxéologique (Bosch & Chevallard, 1999). Nous avons ainsi obtenu un premier résultat qui est la modélisation d’une praxéologie de référence à partir d’une analyse épistémologique des savoirs en jeu et des attentes institutionnelles.Afin d’aider les élèves dans cette activité à l’origine de difficultés, nous utilisons des supports d’étayage, portés par la plateforme informatique, LabBook. Cet EIAH structure des rapports expérimentaux, à l’aide de plusieurs outils (texte, tableur, dessin, protocole) mis à disposition des élèves. L’outil protocole « Copex », permet de pré structurer un protocole expérimental.La deuxième étape de la thèse a été de proposer un protocole pré structuré en étapes, actions et paramètres d’actions qui prend en charge les difficultés attendues, et de l’implémenter dans LabBook, en tenant compte des contraintes de l’EIAH.L’étape suivante a été de tester l’efficacité de la prise en charge de ces difficultés dans la situation proposée en classe. Pour cela nous avons ainsi réalisé deux expérimentations en classes de terminale scientifique dans trois lycées différents. Nous avons recueilli les productions des élèves ainsi que leur réponse à des questionnaires (pré-test et post-test).L’analyse des résultats a montré que l’activité proposée favorise les apprentissages des concepts en jeu, et fait évoluer les conceptions des élèves.Concernant la conception du protocole, la pré structuration proposée aide les élèves à produire des protocoles communicables et pertinents.A partir des praxéologies personnelles modélisées a priori nous avons mis en évidence la présence de praxéologies personnelles d’élèves.Les analyses effectuées ont permis de faire évoluer la situation proposée et de valider la proposition de pré structuration du protocole en étapes et en actions paramétrées.Enfin nous proposons des préconisations pour un diagnostic automatique des erreurs des élèves, dans le but de produire des rétroactions élaborées à partir du modèle praxéologique développé dans la thèse. / The thesis work involves a scaffold of the experimental design activity carried out by high school students in scientific activities using a TEL system (Technology Enhanced Learning).This type of activity promotes learning, but it is also a complex task that leads to difficulties identified in the literature. The situation highlights a specific cellular metabolism, the alcoholic fermentation, this topic being studied in high school biology classes. Students have to design an experimental procedure to highlight this metabolism.The first step of the thesis consisted in knowledge modelling for designing an experimental situation in biology. The framework used to model knowledge, is the Anthropological Theory of Didactics (ATD) and more precisely the praxeology model (Bosch & Chevallard, 1999). An epistemological analysis has been done in order to identify difficulties in this situation: difficulties related to knowledge and also to experimental procedure.A structured procedure was modelled into steps, actions and parameters, which takes into account difficulties identified a priori and implemented in a web environment named LabBook,.This TEL system offers fixed scaffolds in order to help students in an experimental design activity.The situation implemented in LabBook has been tested in three biology classes in high school, during two sets of experimentation. The analysis is based on the students’ productions and their answers to questionnaires (pre-test and post-test).The results analysis showed that the experimental design activity promotes learning and changes students’ conceptions. Regarding design experiment, the pre structuration helps students to produce relevant and communicable procedures.This reveals that students are able to use the pre-structured experimental procedure tool in LabBook.This is a requirement for students’ errors diagnosis in order to propose automatic personalised feedbacks. We make recommendations for such feedbacks based on the praxeology model developed in this thesis.

A regeneração óssea alveolar tem sido um dos principais alvos de estudo na odontologia, tanto humana como veterinária, principalmente na implantodontia e nas cirurgias periodontais e buco-maxilo-faciais. Em função disto, este trabalho foi realizado com o objetivo de avaliar a regeneração óssea alveolar, utilizando como enxerto osso liofilizado e células-tronco mesenquimais (MSCs), oriundas da polpa dentária de um doador macho para enxerto alogênico. Foram utilizados 57 coelhas, Nova Zelândia, sendo um coelho doador das MSCs, distribuídos em sete grupos: controle (G1), osso liofilizado (G2), Matrigel (G3), Matrigel e MSC (G4), osso liofilizado e Matrigel (G5), Osso liofilizado, Matrigel e MSC (G6) e somente MSC (G7). Após a exodontia do incisivo inferior esquerdo, o alvéolo recebia o implante de acordo com cada grupo e avaliados em sete dias. As amostras foram coletadas para análise microscópica, desmineralizadas e não desmineralizadas, PCR, além de terem sido submetidas à análise radiográfica, a qual também era realizada no pré e no pós-operatório imediato. Macroscopicamente, foi observado espessamento do ramo mandibular dos animais dos grupos que receberam Matrigel e aceleração do crescimento dos dentes incisivos remanescentes nos animais que receberam terapia celular. Na análise microscópica, constatou-se que, todos os grupos que receberam como enxerto o osso liofilizado, o tempo de regeneração foi menor, embora o grupo controle tenha apresentado melhor organização na regeneração óssea, sendo que o tratamento com Matrigel resultou ainda em uma reação inflamatória exacerbada, dado este confirmado também nas amostras não desmineralizadas. As radiografias periapicais também apontaram que os grupos que foram tratados com osso liofilizado apresentavam maior área de radiopacidade, sugerindo aceleração do processo de regeneração. Porém, o teste de PCR não detectou a presença do cromossomo Y do doador nas fêmeas receptoras das MSCs. Os resultados sugerem que o uso da terapia celular diminui o tempo de regeneração óssea alveolar e, quando aliada ao osso liofilizado, acelera este processo. Entretanto, decorridos sete dias da aplicação do Matrigel, houve aumento da espessura do ramo mandibular no alvéolo onde foi aplicado, necessitando maior tempo de avaliação para melhor elucidar seu uso clínico. / The alveolar bone regeneration has been a major focus of study in dentistry, both human and veterinary medicine, especially in implant and periodontal surgery and in the bucco-maxillo facial. Because of this, this study was to evaluate alveolar bone regeneration, using lyophilized bone and implant as mesenchymal stem cells (MSCs) derived from the dental pulp of a male donor for allogeneic graft. We used 57 female New Zealand rabbits, one rabbit MSCs from donor, divided into seven groups: control (G1), lyophilized bone (G2), Matrigel (G3), Matrigel and MSC (G4), lyophilized bone and Matrigel(G5), lyophilized bone, MSC and Matrigel (G6) and MSC only (G7). After extraction of the left lower incisor, the socket receiving the implant according to each group and evaluated in seven days. The samples were collected for microscopic analysis, demineralized and non-demineralized, PCR, and they have been subjected to X-ray analysis, which was also held in pre-and postoperatively. Grossly, there was thickening of the mandibular branch of animals that received and accelerate growth of the incisor teeth remaining in the Matrigel animals that received cell therapy. Under microscopic analysis, we found that all groups that received the bone graft as lyophilized, regeneration time was lower, although the control group had a better organization in bone regeneration, and treatment with still resulted in a Matrigel exaggerated inflammatory response, since this is also confirmed in samples not demineralized. The periapical radiographs also showed that the groups were treated with lyophilized bone had a greater area of radiopacity, suggesting acceleration of the regeneration process. However, the PCR test failed to detect the presence of Y chromosome in female recipients of the donor of MSCs. The results suggest that the use of cell therapy reduces the duration and alveolar bone regeneration when combined with lyophilized bone, accelerates this process. However, Matrigel, there was increased thickness after seven days of applying of the mandibular alveolus in which it was applied, requiring longer evaluation to elucidate its clinical use.

Submitted by Jaqueline Bastos (keca78@yahoo.com.br) on 2016-04-26T17:00:34Z No. of bitstreams: 1 TESE JAQUELINE S BASTOS - finalizando 180316.pdf: 13526973 bytes, checksum: d40891f9f50c56b18b4ee564f6453a39 (MD5) / Approved for entry into archive by Felipe Augusto Arakaki (arakaki@reitoria.unesp.br) on 2016-04-28T17:50:25Z (GMT) No. of bitstreams: 1 bastos_js_dr_guara.pdf: 13526973 bytes, checksum: d40891f9f50c56b18b4ee564f6453a39 (MD5) / Made available in DSpace on 2016-04-28T17:50:25Z (GMT). No. of bitstreams: 1 bastos_js_dr_guara.pdf: 13526973 bytes, checksum: d40891f9f50c56b18b4ee564f6453a39 (MD5) Previous issue date: 2016-01-20 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O objetivo desse estudo in vivo foi verificar a resposta óssea de scaffolds porosos revestidos processados pela técnica de Sinterização Direta de Metais a laser (DMLS) associado à terapia de ultrassom de baixa intensidade. Os scaffolds foram processados empregando a técnica DMLS e tratados termicamente a 1000°C por 24 horas. Três tipos de tratamento de superfície foram avaliados: Alcalino, biomimético e imobilização de alendronato de sódio. Para o tratamento alcalino, as amostras foram imersas na solução de NaOH (5M) a 60ºC por 24 horas. O tratamento biomimético consistiu na imersão dos scaffolds em solução SBF (SimulatedBodyFluid) enquanto a imobilização do alendronato foi realizada a partir da imersão dos scaffolds em uma solução formada por SBF e medicamento durante 5 dias. As superfícies dos scaffolds foram avaliadas para cada etapa empregando microscopia eletrônica de varredura (MEV) e análise por difração de raios-X. Os scaffolds foram implantados na tíbia direita de 85 ratos machos da raça wistar com idade média de 12 semanas. A microtomografia computadorizada (µCT) e análise histológica foram realizadas para avaliar o reparo ósseo no defeito. As micrografias das superfícies obtidas mostraram mudanças no aspecto da superfície e composição química de acordo com o tratamento. O tratamento biomimético promoveu o crescimento da apatita sobre a superfície enquanto a imobilização com alendronato suprimiu sua formação. As imagens obtidas na microtomografia mostraram elevado valor de densidade óssea para o último grupo. No entanto, análises histológicas mostraram a formação de cápsula fibrosa em torno dos scaffolds a qual foi minimizada usando ultrassom pulsado de baixa intensidade. No entanto, mais estudos precisam ser realizados para avaliar a influência da geometria dos scaffolds na incorporação de medicamentos. / The objective of this in vivo study was to verify the bone response of coated Ti6Al4V porous scaffolds processed by Direct Metal Laser Sintering (DMLS) technique associated to low intensity pulsed ultrasound therapy. Scaffolds were processed by using Direct Metal Laser Sintering technique (DMLS) and heat treated at 1000 °C for 24 hours. Three types of surface treatments were evaluated: alkaline, biomimetic and sodium alendronate immobilization. For alkaline treatment, samples were immersed in a NaOH (5M) solution at 60ºC for 24 hours. Biomimetic treatment consisted in the immersion of the scaffolds into Simulated Body Fluid solution while for sodium alendronato immobilization the scaffolds were immersed in the solution formed by SBF plus drug during 5 days. The scaffolds surfaces were evaluated after each step employing SEM (Scanning Electron Microscopy)and X-rays diffraction analysis(XRD). Scaffolds were implanted into right tibia of 85 male Wistar rats with average age of 12 weeks. X-rays micro-computed tomography (µCT) and histological analysis were carried out to evaluate the bone repair on the defect. Micrographs analysis showed that the aspect of the surfaces and chemical composition changed according treatment. Biomimetic treatment promoted the growth of the apatite on the surface; in contrast the immobilization of alendronate suppressed apatite formation. Micro CT images showed higher value of bone density for the last group. However, histological analysis showed the formation of encapsulation fibrous around the scaffolds. This formation was minimized by using low intensity pulsed ultrasound technique, however, more studies can be carried out to evaluate the influence of scaffolds geometry in the drug incorporation.

A regeneração óssea alveolar tem sido um dos principais alvos de estudo na odontologia, tanto humana como veterinária, principalmente na implantodontia e nas cirurgias periodontais e buco-maxilo-faciais. Em função disto, este trabalho foi realizado com o objetivo de avaliar a regeneração óssea alveolar, utilizando como enxerto osso liofilizado e células-tronco mesenquimais (MSCs), oriundas da polpa dentária de um doador macho para enxerto alogênico. Foram utilizados 57 coelhas, Nova Zelândia, sendo um coelho doador das MSCs, distribuídos em sete grupos: controle (G1), osso liofilizado (G2), Matrigel (G3), Matrigel e MSC (G4), osso liofilizado e Matrigel (G5), Osso liofilizado, Matrigel e MSC (G6) e somente MSC (G7). Após a exodontia do incisivo inferior esquerdo, o alvéolo recebia o implante de acordo com cada grupo e avaliados em sete dias. As amostras foram coletadas para análise microscópica, desmineralizadas e não desmineralizadas, PCR, além de terem sido submetidas à análise radiográfica, a qual também era realizada no pré e no pós-operatório imediato. Macroscopicamente, foi observado espessamento do ramo mandibular dos animais dos grupos que receberam Matrigel e aceleração do crescimento dos dentes incisivos remanescentes nos animais que receberam terapia celular. Na análise microscópica, constatou-se que, todos os grupos que receberam como enxerto o osso liofilizado, o tempo de regeneração foi menor, embora o grupo controle tenha apresentado melhor organização na regeneração óssea, sendo que o tratamento com Matrigel resultou ainda em uma reação inflamatória exacerbada, dado este confirmado também nas amostras não desmineralizadas. As radiografias periapicais também apontaram que os grupos que foram tratados com osso liofilizado apresentavam maior área de radiopacidade, sugerindo aceleração do processo de regeneração. Porém, o teste de PCR não detectou a presença do cromossomo Y do doador nas fêmeas receptoras das MSCs. Os resultados sugerem que o uso da terapia celular diminui o tempo de regeneração óssea alveolar e, quando aliada ao osso liofilizado, acelera este processo. Entretanto, decorridos sete dias da aplicação do Matrigel, houve aumento da espessura do ramo mandibular no alvéolo onde foi aplicado, necessitando maior tempo de avaliação para melhor elucidar seu uso clínico. / The alveolar bone regeneration has been a major focus of study in dentistry, both human and veterinary medicine, especially in implant and periodontal surgery and in the bucco-maxillo facial. Because of this, this study was to evaluate alveolar bone regeneration, using lyophilized bone and implant as mesenchymal stem cells (MSCs) derived from the dental pulp of a male donor for allogeneic graft. We used 57 female New Zealand rabbits, one rabbit MSCs from donor, divided into seven groups: control (G1), lyophilized bone (G2), Matrigel (G3), Matrigel and MSC (G4), lyophilized bone and Matrigel(G5), lyophilized bone, MSC and Matrigel (G6) and MSC only (G7). After extraction of the left lower incisor, the socket receiving the implant according to each group and evaluated in seven days. The samples were collected for microscopic analysis, demineralized and non-demineralized, PCR, and they have been subjected to X-ray analysis, which was also held in pre-and postoperatively. Grossly, there was thickening of the mandibular branch of animals that received and accelerate growth of the incisor teeth remaining in the Matrigel animals that received cell therapy. Under microscopic analysis, we found that all groups that received the bone graft as lyophilized, regeneration time was lower, although the control group had a better organization in bone regeneration, and treatment with still resulted in a Matrigel exaggerated inflammatory response, since this is also confirmed in samples not demineralized. The periapical radiographs also showed that the groups were treated with lyophilized bone had a greater area of radiopacity, suggesting acceleration of the regeneration process. However, the PCR test failed to detect the presence of Y chromosome in female recipients of the donor of MSCs. The results suggest that the use of cell therapy reduces the duration and alveolar bone regeneration when combined with lyophilized bone, accelerates this process. However, Matrigel, there was increased thickness after seven days of applying of the mandibular alveolus in which it was applied, requiring longer evaluation to elucidate its clinical use.

A regeneração óssea alveolar tem sido um dos principais alvos de estudo na odontologia, tanto humana como veterinária, principalmente na implantodontia e nas cirurgias periodontais e buco-maxilo-faciais. Em função disto, este trabalho foi realizado com o objetivo de avaliar a regeneração óssea alveolar, utilizando como enxerto osso liofilizado e células-tronco mesenquimais (MSCs), oriundas da polpa dentária de um doador macho para enxerto alogênico. Foram utilizados 57 coelhas, Nova Zelândia, sendo um coelho doador das MSCs, distribuídos em sete grupos: controle (G1), osso liofilizado (G2), Matrigel (G3), Matrigel e MSC (G4), osso liofilizado e Matrigel (G5), Osso liofilizado, Matrigel e MSC (G6) e somente MSC (G7). Após a exodontia do incisivo inferior esquerdo, o alvéolo recebia o implante de acordo com cada grupo e avaliados em sete dias. As amostras foram coletadas para análise microscópica, desmineralizadas e não desmineralizadas, PCR, além de terem sido submetidas à análise radiográfica, a qual também era realizada no pré e no pós-operatório imediato. Macroscopicamente, foi observado espessamento do ramo mandibular dos animais dos grupos que receberam Matrigel e aceleração do crescimento dos dentes incisivos remanescentes nos animais que receberam terapia celular. Na análise microscópica, constatou-se que, todos os grupos que receberam como enxerto o osso liofilizado, o tempo de regeneração foi menor, embora o grupo controle tenha apresentado melhor organização na regeneração óssea, sendo que o tratamento com Matrigel resultou ainda em uma reação inflamatória exacerbada, dado este confirmado também nas amostras não desmineralizadas. As radiografias periapicais também apontaram que os grupos que foram tratados com osso liofilizado apresentavam maior área de radiopacidade, sugerindo aceleração do processo de regeneração. Porém, o teste de PCR não detectou a presença do cromossomo Y do doador nas fêmeas receptoras das MSCs. Os resultados sugerem que o uso da terapia celular diminui o tempo de regeneração óssea alveolar e, quando aliada ao osso liofilizado, acelera este processo. Entretanto, decorridos sete dias da aplicação do Matrigel, houve aumento da espessura do ramo mandibular no alvéolo onde foi aplicado, necessitando maior tempo de avaliação para melhor elucidar seu uso clínico. / The alveolar bone regeneration has been a major focus of study in dentistry, both human and veterinary medicine, especially in implant and periodontal surgery and in the bucco-maxillo facial. Because of this, this study was to evaluate alveolar bone regeneration, using lyophilized bone and implant as mesenchymal stem cells (MSCs) derived from the dental pulp of a male donor for allogeneic graft. We used 57 female New Zealand rabbits, one rabbit MSCs from donor, divided into seven groups: control (G1), lyophilized bone (G2), Matrigel (G3), Matrigel and MSC (G4), lyophilized bone and Matrigel(G5), lyophilized bone, MSC and Matrigel (G6) and MSC only (G7). After extraction of the left lower incisor, the socket receiving the implant according to each group and evaluated in seven days. The samples were collected for microscopic analysis, demineralized and non-demineralized, PCR, and they have been subjected to X-ray analysis, which was also held in pre-and postoperatively. Grossly, there was thickening of the mandibular branch of animals that received and accelerate growth of the incisor teeth remaining in the Matrigel animals that received cell therapy. Under microscopic analysis, we found that all groups that received the bone graft as lyophilized, regeneration time was lower, although the control group had a better organization in bone regeneration, and treatment with still resulted in a Matrigel exaggerated inflammatory response, since this is also confirmed in samples not demineralized. The periapical radiographs also showed that the groups were treated with lyophilized bone had a greater area of radiopacity, suggesting acceleration of the regeneration process. However, the PCR test failed to detect the presence of Y chromosome in female recipients of the donor of MSCs. The results suggest that the use of cell therapy reduces the duration and alveolar bone regeneration when combined with lyophilized bone, accelerates this process. However, Matrigel, there was increased thickness after seven days of applying of the mandibular alveolus in which it was applied, requiring longer evaluation to elucidate its clinical use.

Defeitos ósseos assumem importância na crescente prevalência de condições crônicas de saúde, agravando-se conforme o envelhecimento da população. O tratamento convencional baseia-se no transplante autólogo e abordagens extremamente invasivas. Uma proposta promissora é a obtenção de tecidos saudáveis em laboratórios utilizando suportes tridimensionais porosos (scaffolds) que atuarão como arcabouço para o crescimento e diferenciação de células tronco mesenquimais (CTMs) podendo ser otimizados para proporcionar adequada vascularização, uma importante característica na regeneração óssea. CTMs apresentam potencial de diferenciação, imunoregulação e angiogênese. O pico proteico F1 do soro do látex da seringueira Hevea brasiliensis apresenta importante atividade angiogênica e cicatrizante. O objetivo deste trabalho foi investigar a influência de scaffolds de policaprolactona (PCL) colonizados com CTMs na osteogênese experimental in vitro e in vivo (xenotransplante), a segurança e a influência do pico F1 do látex em cultura de CTMs aplicadas no scaffold de PCL e PCL reforçado com diferentes concentrações de grafeno (PCL/grafeno) na proliferação e diferenciação osteogênica. Para isso, as CTMs foram isoladas do tecido adiposo humano (ADSCs), caracterizadas por imunofenotipagem e diferenciação in vitro. Scaffolds de PCL, produzidos por técnica de manufatura aditiva, foram avaliados quanto ao potencial de adesão/viabilidade celular (ensaio MTT), diferenciação osteogênica (vermelho de alizarina) e potencial in vivo de osteointegração e osteoindução em defeito crítico de calvária avaliados por histologia e imunoistoquímica. O pico F1 do látex, em diferentes concentrações, foi avaliado em cultura de ADSCs e fibroblastos 3T3 quanto a citotoxicidade (MTT), potencial proliferativo (timidina-tritiada), migratório (scratch assay) e indução osteogênica (fosfatase alcalina). Scaffolds de PCL foram reforçados com grafeno (PCL/grafeno), revestidos com pico F1 (adsorção), avaliados quanto a viabilidade/proliferação celular (Alamar blue) e diferenciação osteogênica (fosfatase alcalina e vermelho de alizarina). A imunofenotipagem das ADSCs demonstrou baixa percentagem para marcadores negativos, alta para os positivos e diferenciação in vitro, comprovando o sucesso do isolamento e manutenção das ADSCs. O scaffold de PCL apresentou não-toxicidade e diferenciação osteogênica induzida pelo meio de cultura. Scaffolds de PCL, pré-colonizado e não colonizado com ADSCs, foram implantados em defeito crítico de calvária de ratos. O grupo que recebeu scaffolds com ADSCs humanas proporcionou melhor formação óssea no animal, com participação direta e indireta das ADSCs neste processo. Nos ensaios in vitro com o pico F1 (cultura 2D), observou-se estímulo proliferativo para as concentrações de 0.00001% e 0.0001%, além de maior percentagem de migração celular para as concentrações de 0.001%, 0.0001% e 0.00001%, diferentes do controle. Os scaffolds de PCL/grafeno demonstraram estimulo proliferativo quando colonizados por ADSCs e este estímulo foi ainda maior em scaffolds revestidos com F1, principalmente na concentração de 0.75% de grafeno. Embora o pico F1 não tenha potencializado a diferenciação osteogênico em cultura 2D, este estímulo foi observado em scaffolds revestidos com F1 com superior atividade da fosfatase alcalina. Este trabalho demonstrou sucesso no emprego de ADSCs humanas e scaffolds in vitro e in vivo (transplante xenogênico) para regeneração óssea, além de apresentar dois promissores produtos para engenharia tecidual como os scaffolds com reforço de grafeno em baixas concentrações e o pico proteico F1 na proliferação e diferenciação celular. / The increment of life expectancy and frequency of chronic diseases in the population has led to an increasing incidence of chronic bone defects. Conventional treatment is based on autologous transplantation, which depends on extremely invasive approaches. A promising proposal is to obtain healthy tissues in laboratories using porous three-dimensional matriz (scaffolds), which enable cellular growth and differentiation of mesenchymal stem cells (MSCs). Scaffolds can be optimized to provide adequate vascularization, a critical event to bone regeneration. MSCs have potential for differentiation, immunoregulation and angiogenesis. F1 natural latex protein from Hevea brasiliensis rubber tree presents important angiogenic and healing activity. The objective of this work was to investigate the influence of pre-colonized polycaprolactone (PCL) scaffolds on experimental in vitro and in vivo osteogenesis (xenotransplantation) and also the safety and influence of F1 protein on MSCs seeded on PCL and PCL reinforced with different concentrations of graphene scaffolds (PCL/graphene) in cell proliferation and osteogenic differentiation. MSCs were isolated from human adipose tissue (ADSCs), characterized by positive and negative markers and also in vitro differentiation. PCL Scaffolds, produced by an additive manufacturing technique, were evaluated for cell adhesion/viability potential (MTT assay), osteogenic differentiation (alizarin red) and in vivo xenogenic grafting potential for osteointegration and osteoinduction evaluated by histology and immunohistochemistry. F1 latex protein, prepared in different concentrations, was evaluated in contact with ADSCs and 3T3 fibroblasts culture in vitro regarding to cytotoxicity (MTT), proliferative potential (tritiated thymidine), migratory (scratch assay) and osteogenic induction (alkaline phosphatase). PCL scaffolds were reinforced with graphene (PCL/graphene scaffolds), coated with F1 protein (adsorption) and evaluated for cell viability/proliferation assay (Alamar blue) and osteogenic differentiation (alkaline phosphatase and alizarin red). ADSCs showed low percentage for negative markers, high percentage for positive markers and differentiation properties in vitro, providing enough information on the successful isolation and maintenance of human ADSCs. PCL scaffolds showed non-toxicity activity and osteogenic differentiation induced by culture medium. PCL scaffolds, pre-colonized and non-colonized with ADSCs, were implanted in a critical calvarial defect in rats. The group of rats which received scaffolds with ADSCs to treat the bone defect had improved bone formation with direct and indirect participation of ADSCs to the bone repair process. To in vitro assays with F1 (2D culture model), proliferative stimulus was observed to F1 0.00001% and 0.0001% samples, in addition to a higher percentage of cell migration to 0.001% and 0.0001%, different from control. PCL/graphene scaffolds demonstrated proliferative stimulation when colonized by ADSCs and this stimulus was even higher to F1 coated PCL/graphene scaffolds, mainly to 0.75% graphene. Although F1 have not enhanced osteogenic differentiation on 2D cell culture model, the stimulus was observed to F1 coated scaffolds with higher alkaline phosphatase activity. This work demonstrated success in the use of ADSCs and scaffolds for bone regeneration and presented two promising products to be applied in tissue engineering field, such as, scaffolds with graphene reinforcement at low concentration and F1 latex protein to improve cell proliferation and differentiation.

A crescente demanda clínica para regeneração óssea tem dirigido esforços significativos para o desenvolvimento de novos biomateriais, incluindo aqueles aplicados em terapias baseadas em engenharia de tecidos. Neste contexto, os biovidros são considerados uma boa alternativa mas as suas propriedades mecânicas têm limitado a sua aplicação. Para melhorar tais propriedades sem afetar a biocompatibilidade, um novo material vitrocerâmico bioativo do sistema P2O5-Na2O-CaO-SiO2, chamado Biosilicato® com duas fases cristalinas (BioS-2P) foi desenvolvido. No entanto, os efeitos da adição das fases cristalinas sobre o comportamento biológico do BioS-2P ainda não foram estudados. Assim, os objetivos deste estudo foram investigar a capacidade do BioS-2P em induzir, in vitro, a diferenciação osteoblástica de células-tronco mesenquimais (CTMs); a capacidade do BioS-2P em aumentar, in vitro, a atividade dos osteoblastos em fase inicial de diferenciação (OBs) e osteoblastos da linhagem UMR-106 (UMRs); e a capacidade do BioS-2P em conduzir e induzir a neoformação óssea, in vivo, associado ou não a células. Células derivadas da medula óssea obtidas de fêmures de ratos foram cultivadas em meio de crescimento para obtenção de CTMs ou em meio osteogênico para obtenção de OBs. Essas células e UMRs foram cultivadas sobre discos de BioS-2P, Bioglass® 45S5 (45S5) e plástico de cultura (Controle) e utilizadas nas avaliações in vitro. Para as avaliações in vivo, defeitos de 5 mm criados em calotas de ratos foram implantados somente com arcabouços de BioS-2P ou com arcabouços de BioS-2P associados às CTMs ou aos OBs. Os dados foram comparados por teste não paramétrico de Kruskal-Wallis seguido pelo teste de Student Newman-Keuls, e o nível de significância adotado foi de 5%. As CTMs foram caracterizadas por apresentarem alta porcentagem de células expressando os marcadores de superfície CD29 e CD90 e baixa porcentagem expressando CD31, CD34, CD45 e CD106. A diferenciação osteoblástica das CTMs foi confirmada pela expressão dos genes marcadores da diferenciação osteoblástica fosfatase alcalina (ALP), runt-related transcriptor factor-2 (RUNX2), sialoproteína óssea (BSP) e osteocalcina (OC). CTMs cultivadas sobre discos de BioS-2P em meio não-osteogênico apresentaram diminuição da proliferação e aumento da atividade de ALP e da expressão dos genes marcadores da diferenciação osteoblástica ALP, RUNX2, osterix (OSX), proteína óssea morfogenética-4 (BMP-4), osteopontina (OPN) e OC, comprovando seu potencial osteoindutor similar ao 45S5. O BioS-2P foi capaz de aumentar a atividade de OBs e UMRs de maneira similar àqueles cultivados sobre o 45S5. OBs apresentaram diminuição na proliferação e aumento da atividade da ALP e da expressão dos genes marcadores da diferenciação osteoblástica RUNX2, OSX, BMP-4, OPN e OC. A análise em larga escala da expressão de mais de 23.000 genes mostrou que o BioS-2P induziu a sobre-expressão de genes envolvidos no aumento da atividade osteoblástica e a repressão de genes envolvidos na diminuição dessa atividade, em comparação com o Controle. Ao menos em parte, esse aumento da atividade osteoblástica foi atribuído à modulação das vias de sinalização proteíno-quinases ativadas por mitógenos (MAPK) e Wnt Canônica, e à modulação da expressão de microRNAs. UMRs crescidos sobre o BioS-2P corroboraram esses achados, pela capacidade em formar matriz mineralizada e por apresentarem aumento na expressão das proteínas ALP, RUNX2, dentin matrix protein-1 (DMP-1) e OPN. Arcabouços de BioS-2P (5 mm de diâmetro e 2 mm de altura com porosidade de 76 ± 5% e com tamanhos de poros variando entre 100 e 800 µm) implantados em defeitos na calota de ratos estimularam a formação de tecido ósseo, que ocorreu tanto na periferia como no interior dos defeitos e em íntimo contato com o material. A morfometria por microtomografia computadorizada não evidenciou qualquer diferença entre os parâmetros volume ósseo, volume ósseo/volume total, superfície óssea, superfície/volume ósseo, número de trabéculas, separação trabecular e espessura trabecular, avaliados na 4a, 8a e 12a semanas de implantação. As CTMs e os OBs foram carreados para os arcabouços de BioS- 2P (com eficiência de 90% e 81%, respectivamente) e essas células permaneceram nos defeitos por 14 dias. A combinação de arcabouços de BioS-2P com CTMs ou OBs, implantados por 8 semanas, resultou no mesmo padrão de formação óssea daquele observado para o arcabouço sem células. No entanto, essa combinação não resultou em aumento na quantidade de osso formado. Os resultados evidenciaram a capacidade do BioS-2P em induzir a diferenciação osteoblástica de CTMs e estimular a atividade osteoblástica de OBs, o que resultaria na neoformação óssea observada in vivo. No entanto, a combinação de BioS-2P com CTMs e OBs não foi capaz de aumentar a formação óssea e induzir o reparo dos defeitos ósseos. / The increasing clinical demand for bone regeneration has driven significant efforts to develop new biomaterials including those for tissue engineeringbased therapies. In this context, bioglasses emerges as a good alternative, but their use has been limited mainly due their poor mechanical properties. To improve these mechanical properties without affecting biocompatibility, a novel bioactive glass-ceramic of the P2O5-Na2O-CaO-SiO2 system, named Biosilicate® with two cristallyne phases (BioS-2P) was developed. However, the effects of these two phases on BioS- 2P biological behavior have not yet been evaluated. Thus, the aims of this study were to investigate the BioS-2P capability of inducing in vitro mesenquimal stem cell differentiation (MSC) towards osteoblasts; the BioS-2P capability to increase in vitro activity of osteoblasts derived from rat bone marrow at early stages of differentiation (OBs) and osteoblasts from rat cell line UMR- 106 (UMRs); and the BioS-2P capability to drive and induce bone formation in vivo, associated or not with cells. Bone marrow cells harvested from rat femurs were cultured either in growth media to obtain MSCs or in osteogenic media to obtain OBs. MSCs, OBs and UMRs were cultured on discs of BioS-2P, Bioglass® 45S5 (45S5) and tissue culture polystyrene (Control). For in vivo evaluations, 5-mm rat calvarial surgical defects were filled with BioS-2P with or without MSCs or OBs. Data were compared by non-parametric Kruskal-Wallis test followed by Student Newman- Keuls test and the significance level was set at 5%. MSCs were characterized by presenting high percentage of CD29 and CD90 surface markers and low percentage of CD31, CD34, CD45 and CD106 surface markers. Osteoblastic differentiation of MSCs was detected by gene expression of bone markers alkaline phosphatase (ALP), runt-related transcritption factor 2 (RUNX2), bone sialoprotein (BSP) and osteocalcin (OC). MSCs cultured on Bios-2P discs under non-osteogenic conditions exhibited a decrease on cell proliferation and an increase on ALP activity and gene expression of bone markers ALP, RUNX2, osterix (OSX), bone morphogenetic protein-4 (BMP-4), osteopontin (OPN) and OC, confirming its osteoinductive potential similar to 45S5. Also, BioS-2P increased the OBs and UMRs activity, similar to 45S5. OBs cultured on Bios-2P discs presented a decrease in cell proliferation and an increase on ALP activity and gene expression of bone markers RUNX2, OSX, BMP-4, OPN and OC. The large-scale analysis of over 23,000 genes showed that the BioS-2P induced overexpression of genes positively related to osteoblastic activity and repression of genes negatively related with its activity, compared with control. At least in part, the increase on OBs activity was associated to the modulation of two main signaling pathways, the mitogen activated protein kinases (MAPK) and the Canonical Wnt, and the modulation of microRNAs expression. These findings were corroborated by UMRs grown on BioS-2P, which produced mineralized matrix and exhibited increased expression of the ALP, RUNX2, dentin matrix protein-1 (DMP-1) and OPN proteins, than on control. BioS-2P scaffolds (5 mm diameter and 2 mm heigh, presenting 76 ± 5% of total porosity, with poros size ranging from 100 to 800 µm) implanted in calvarial defects promoted new bone formation in close contatc to BioS-2P, both on periphery and in the center of the defect. The computed microtomography morphometry showed no difference between the evaluated parameters bone volume, bone volume / total volume, bone surface, surface / bone volume, number of trabeculae, trabecular separation and trabecular thickness, measured at 4, 8 and 12 weeks. MSCs and OBs were seeded into the scaffold (with efficiency of incorporation 90% e 81%, respectively) and they remained on the defects for 14 days. After 8 weeks, the same pattern of bone formation was observed, however, the combination of BioS-2P with cells did not increase the amount of new bone. The results showed the BioS-2P ability to induce osteoblastic differentiation of MSCs and to stimulate osteoblastic activity, resulting in new bone formation in vivo. However, the combination of BioS-2P with MSCs and OBs was not able to increase bone formation and induce the repair of bone defects.