Thesis (Ph. D.)--Mechanical Engineering, Georgia Institute of Technology, 2008. / Barbara D. Boyan, Committee Member ; Ravi Bellamkonda, Committee Member ; Joseph Le Doux, Committee Member ; Andres J. Garcia, Committee Member ; Marc E. Levenston, Committee Chair.

Thesis (M.S.)--University of Alabama at Birmingham, 2007. / Title from first page of PDF file (viewed Oct. 30, 2007). Includes bibliographical references (p. 61-66).

Thesis (M.S.) -- Worcester Polytechnic Institute. / Keywords: histology; implant; biomaterials; MRI. Includes bibliographical references (p.80-86).

Thesis (M.S.)--California Polytechnic State University, 2009. / Mode of access: Internet. Title from PDF title page; viewed on November 19, 2009. Major professor: Dr. Kristen O'Halloran Cardinal. "Presented to the faculty of California Polytechnic State University, San Luis Obispo." "In partial fulfillment of the requirements for the degree [of] Master of Science in Biomedical Engineering." "August 2009." Includes bibliographical references (p. 143-158).

<p>The ends of long bones that articulate with respect to one another are lined with a crucial connective tissue called articular cartilage. This tissue plays an essential biomechanical function in synovial joints, as it serves to both dissipate load and lubricate articulating surfaces. Osteoarthritis is a painful and debilitating disease that drives the deterioration of articular cartilage. Like many chronic diseases, pro-inflammatory cytokines feature prominently in the onset and progression of osteoarthritis. Because cartilage lacks physiologic features critical for regeneration and self-repair, the development of effective strategies to create functional cartilage tissue substitutes remains a priority for the fields of tissue engineering and regenerative medicine. The overall objectives of this dissertation are to (1) develop a bioactive scaffold capable of mediating cell differentiation and formation of extracellular matrix that recapitulates native cartilage tissue and (2) to produce stem cells specifically tailored at the scale of the genome with the ability to resist inflammatory cues that normally lead to degeneration and pain. </p><p>Engineered replacements for musculoskeletal tissues generally require extensive ex vivo manipulation of stem cells to achieve controlled differentiation and phenotypic stability. By immobilizing lentivirus driving the expression of transforming growth factor-β3 to a highly structured, three dimensionally woven tissue engineering scaffold, we developed a technique for producing cell-instructive scaffolds that control human mesenchymal stem cell differentiation and possess biomechanical properties approximating those of native tissues. This work represents an important advance, as it establishes a method for generating constructs capable of restoring biological and mechanical function that may circumvent the need for ex vivo conditioning of engineered tissue substitutes.</p><p>Any functional cartilage tissue substitute must tolerate the inflammation intrinsic to an arthritic joint. Recently emerging tools from synthetic biology and genome engineering facilitate an unprecedented ability to modify how cells respond to their microenvironments. We exploited these developments to engineer cells that can evade signaling of the pro-inflammatory cytokine interleukin-1 (IL-1). Our study provides proof-of-principle evidence that cartilage derived from such engineered stem cells are resistant to IL-1-mediated degradation. </p><p>Extending on this work, we developed a synthetic biology strategy to further customize stem cells to combat inflammatory cues. We commandeered the highly responsive endogenous locus of the chemokine (C-C motif) ligand 2 gene in pluripotent stem cells to impart self-regulated, feedback-controlled production of biologic therapy. We demonstrated that repurposing of degradative signaling pathways induced by IL-1 and tumor necrosis factor toward transient production of cytokine antagonists enabled engineered cartilage tissue to withstand the action of inflammatory cytokines and to serve as a cell-based, auto-regulated drug delivery system.</p><p>In this work, we combine principles from synthetic biology, gene therapy, and functional tissue engineering to develop methods for generating constructs with biomimetic molecular and mechanical features of articular cartilage while precisely defining how cells respond to dysfunction in the body’s finely-tuned inflammatory systems. Moreover, our strategy for customizing intrinsic cellular signaling pathways in therapeutic stem cell populations opens innovative possibilities for controlled drug delivery to native tissues, which may provide safer and more effective treatments applicable to a wide variety of chronic diseases and may transform the landscape of regenerative medicine.</p> / Dissertation

<p>High-throughput processing technologies hold critical position in biomedical research. These include screening of cellular response based on phenotypic difference and production of homogeneous chemicals and biologicals for therapeutic applications. The rapid development of microfluidics technology has provided an efficient, controllable, economical and automatable processing platform for various applications. In particular, emulsion droplet gains a lot of attention due to its uniformity and ease of isolation, but the application of water-in-oil (W/O) single emulsion is hampered by the presence of the oil phase which is incompatible with aqueous phase manipulation and the difficulty in modifying the droplet environment.</p><p>This thesis presents the development of a double emulsion (DE) droplet platform in microfluidics and two applications: (1) high-throughput screening of synthetic gene and (2) production of multicellular spheroids with adjustable microenvironment for controlling stem cell differentiation and liver tissue engineering. Monodisperse DE droplets with controllable size and selective permeability across the oil shell were generated via two microfluidics devices after optimization of device design and flow rates. </p><p>Next, bacterial cells bearing synthetic genes constructed from an inkjet oligonucleotide synthesizer were encapsulated as single cells in DE droplets. Enrichment of fluorescent signals (~100 times) from the cells allowed quantification and selection of functionally-correct genes before and after error correction scheme was employed. Permeation of Isopropyl β-D-1-thiogalactopyranoside (IPTG) molecules from the external phase triggered target gene expression of the pET vector. Fluorescent signals from at least ~100 bacteria per droplet generated clearly distinguishable fluorescent signals that enabled droplets sorting through fluorescence-activated cell sorting (FACS) technique.</p><p>In addition, DE droplets promoted rapid aggregation of mammalian cells into single spheroid in 150 min. Size-tunable human mesenchymal stem cells (hMSC) spheroids could be extracted from the droplets and exhibited better differentiation potential than cells cultured in monolayer. The droplet environment could be altered by loading matrix molecules in it to create spheroid-encapsulated microgel. As an example, hMSC spheroid was encapsulated in alginate or alginate-RGD microgel and enhanced osteogenic differentiation was found in the latter case.</p><p>Lastly, the capability of forming spheroids in DE droplet was applied in liver tissue engineering, where single or co-culture hepatocyte spheroids were efficiently produced and encapsulated in microgel. The use of alginate-collagen microgel significantly improved the long-term function of the spheroid, in a manner similar to forming co-culture spheroids of hepatocytes and endothelial progenitor cells at a 5 to 1 ratio. The hepatocyte spheroid encapsulated in microgel could be useful for developing bioartificial liver or drug testing platform or applied directly for hepatocyte transplantation.</p> / Dissertation

Tendon plays an important role in regulating body locomotion and providing additional stability to the body. However, tendon is susceptible to injuries and the healing process could be devastating along with the several issues, namely adhesion formations, slow healing and failure at fixation sites, which have deferred the success of proper tendon healing via tendon tissue engineering. This dissertation thus aims to create a sustained dual-drug therapy to address these issues. For adhesion formation, naproxen sodium (NPS) has been shown to be able to avoid this symptom through inhibiting inflammation process.

Submitted by Maria Anália Conceição (marianaliaconceicao@gmail.com) on 2016-06-27T18:14:05Z No. of bitstreams: 1 Publico-NOVO-19.pdf: 3346874 bytes, checksum: 17dd5440a6491e9f9e098ed990f9a67e (MD5) / Approved for entry into archive by Maria Anália Conceição (marianaliaconceicao@gmail.com) on 2016-06-27T18:14:55Z (GMT) No. of bitstreams: 1 Publico-NOVO-19.pdf: 3346874 bytes, checksum: 17dd5440a6491e9f9e098ed990f9a67e (MD5) / Made available in DSpace on 2016-06-27T18:14:55Z (GMT). No. of bitstreams: 1 Publico-NOVO-19.pdf: 3346874 bytes, checksum: 17dd5440a6491e9f9e098ed990f9a67e (MD5) Previous issue date: 2015 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Rede Ibero-Americana de Biofabricação / Introdução: A aplicação de dexametasona em cultura de células mesenquimais induz diferenciação osteoblástica, consequentemente formação de tecidos mineralizados. A Engenharia Tecidual propõe o desenvolvimento de estratégias terapêuticas direcionadas à regeneração funcional e estrutural de tecidos biológicos. Nesse sentido, a caracterização celular in vitro é fundamental para garantir o desenvolvimento destas técnicas. Objetivo: Avaliar o efeito da dexametasona administrada como medicação pré-operatória na cultura de células primárias de polpa dental humana. Métodos: Foram utilizadas células provenientes da polpa de terceiros molares. Essas foram distribuídas em dois grupos experimentais com dois protocolos de medicação pré-operatória utilizados em rotina odontológica, onde no protocolo B, o paciente ingeria 1 comprimido de dexametasona 1hora antes à cirurgia e no A não. A avaliação da proliferação, viabilidade e diferenciação, foram pelos testes Trypan Blue, MTT, Von Kossa e Alizarin Red respectivamente, e realizadas em intervalos fixados. Análise de variância de Friedman e t test foram aplicados, fixando em 95% de confiança. Resultados: As células pertencentes ao protocolo A atingiram pico de proliferação aos 21 dias de cultura enquanto as células do protocolo B em 14. Células do protocolo A foram estatisticamente mais viáveis aos 7 e 21 dias enquanto as do protocolo B, aos 14. Na análise de Von Kossa e Alizarin Red observou-se que as células pertencentes ao protocolo B formaram nódulos de calcificação desde 7 dias de cultura enquanto no A aos 14. Conclusão: A utilização da dexametasona como medicação pré-operatória em cirurgia de terceiros molares promove diferenciação celular precocemente, quando observada in vitro. / Introduction: The use of dexamethasone in mesenchymal cell culture induces osteoblastic differentiation and, consequently, formation of mineralized tissues. Tissue Engineering proposes the development of therapeutic strategies aiming at structural and functional regeneration of biological tissues. In this sense, cell characterization in vitro is critical to ensure the development of such techniques. Objective: To evaluate the effect of dexamethasone administered as preoperative medication in primary cell culture of human dental pulp. Methods: We used cells from the third molar pulp. These cells were divided into two experimental groups, each with two preoperative medication protocols used in dental routine and differentiated by the intake of dexamethasone in one of them. The assessment of proliferation, differentiation, and viability through Trypan Blue, MTT and von Kossa, and Alizarin Red tests, respectively, were held in fixed intervals. Friedman analysis of variance and t test were applied, and confidence interval was set at 95%. Results: Protocol A cells proliferation reached its peak on day 21 while protocol B cells proliferation reached its peak on day 14. Protocol A cells were statistically more viable between days 7 and 21 whereas protocol B cells viability was higher on day 14. Von Kossa and Alizarin Red analyses showed that calcified nodules formation occurred from the seventh day of cell culture in protocol B cells and on day 14 in protocol A cells. Conclusion: The use of dexamethasone as preoperative medication in third molar surgery promotes cell differentiation earlier, when observed in vitro. / FAPESP: 07/51227-4 / FAPESP: 08/57860-3 / CNPq: 573661/2008-1

Submitted by Maria Anália Conceição (marianaliaconceicao@gmail.com) on 2016-06-27T19:41:41Z No. of bitstreams: 1 Publico-NOVO-21.pdf: 2987989 bytes, checksum: fedc0b7588511ac9f1b74f18e86aed33 (MD5) / Approved for entry into archive by Maria Anália Conceição (marianaliaconceicao@gmail.com) on 2016-06-27T19:42:44Z (GMT) No. of bitstreams: 1 Publico-NOVO-21.pdf: 2987989 bytes, checksum: fedc0b7588511ac9f1b74f18e86aed33 (MD5) / Made available in DSpace on 2016-06-27T19:42:44Z (GMT). No. of bitstreams: 1 Publico-NOVO-21.pdf: 2987989 bytes, checksum: fedc0b7588511ac9f1b74f18e86aed33 (MD5) Previous issue date: 2015 / Agência Brasileira de Cooperação (ABC) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Rede Ibero-Americana de Biofabricação / Introdução: A Engenharia Tecidual (ET) é uma ciência multidisciplinar que visa produzir órgãos e partes humanas substitutas acometidas por lesões traumáticas, doenças degenerativas ou agenesias. Uma das suas etapas é a produção de arcabouços biocompatíveis para aplicação na Medicina Regenerativa. Estas estruturas são conhecidas como scaffolds, que apresentam macrogeometria semelhante ao tecido original, em textura e porosidade e direcionam o comportamento das células que serão semeadas. A recuperação da integridade anatômica e funcional de tecidos lesados garante a sobrevivência dos seres vivos e o tratamento de perdas extensas é desafiador. Objetivo: Avaliar a eficiência para desmineralizar e descelularizar dentes viabilizando-os como scaffolds naturais. Métodos: As amostras foram submetidas a um tratamento com soluções desmineralizadoras/descelularizadoras. Foram usadas 5 soluções: G1-Formol 10% controle, EDTA 28% para desmineralização nos quatro grupos; G2- hipoclorito de sódio 2,5%; e G3-peróxido de hidrogênio 9%; G4- hipoclorito de sódio 2,5% associado com detergente enzimático; G5- detergente enzimático associado a peróxido de hidrogênio 9%. A evolução da desmineralização e descelularização foi acompanhada durante 12 semanas, por meio de pesagem, técnicas analíticas MEV (Microscopia eletrônica de Varredura), fotografia e radiografia. As amostras foram pesadas a cada sete dias para controle da perda de mineral. Os resultados receberam análise estatística de variância de Friedman, Kruskal-Wallis, Resumo xix Teste do Quiquadrado e Teste exato de Fisher. Foi fixado em 0,05 ou 5% o nível de rejeição da hipótese de nulidade. Conclusão: O grupo 5 mostrouse microscopicamente a melhor solução, mesmo mantendo em 30% das amostras resíduos biológicos. / Tissue Engineering (TE) is a multidisciplinary science that aims to produce replacement organs and parts affected by trauma, degenerative diseases or agenesis. One of its goals is to produce biocompatible scaffolds for application in regenerative medicine. These structures are known as scaffolds, presenting three-dimensional shape similar to the original tissue in texture and porosity and directing the behavior of cells to be seeded. The recovery of anatomical and functional integrity of damaged tissues ensures the survival of living beings and the treatment of extensive losses is challenging. Objective: Get decelullarized and demineralized teeth to make them feasible as natural scaffolds. Methods: The samples will be subjected to a treatment with demineralizing/decelularizing solutions. 4 different solutions were used (14% EDTA, 2.5% sodium hypochlorite, hydrogen peroxide and 9% and one group of control (10% formaldehyde). The evolution of demineralization/decellularization was monitored for 90 days through the use of electron scanning microscopy, X-ray and photography. Samples’ weights were measured each seven days to control the mineral loss. Results were subjected to statistical analysis of variance, KruskalWallis test Wilcoxan and Chi-square Test. Was fixed at 0,05 or 5% rejection level of the null hypothesis. / FAPESP: 07/58856-7 / FAPESP: 07/59488-1 / FAPESP: 07/51227-4 / CNPq: 573661/2008-1 / FAPESP: 08/57860-3

O avanço da ciência regenerativa está se comprometendo com a busca de novas opções terapêuticas no combate as diversas doenças e disfunções orgânicas. Nessas avançadas linhas de pesquisas, as células-tronco são um símbolo dessa evolução. A variedade da aplicação deste novo e experimental método de tratamento está sendo utilizado, pela bioengenharia, para reparar tecidos e órgãos lesados. Defeitos ósseos extensos ocorrem após diversos tipos de injúrias ao esqueleto facial, como traumas faciais, ressecções por lesões agressivas e malformações congênitas. Essas sequelas são tratadas, preferencialmente, através da reconstrução utilizando enxertos ósseos de origem autógena. Entretanto, às desvantagens proporcionadas pela obtenção do tecido ósseo autógeno, lançaram um dos maiores desafios, da bioengenharia que é a busca pelo aprimoramento dos substitutos ósseos. Nesse caminho do aprimoramento dos biomateriais a adição de células-tronco mesenquimais representam a possibilidade da criação de um sinergismo, entre as células e o arcabouço, para otimizar a osteogênese. Nessa linha, esse estudo propõe-se a avaliar o reparo ósseo em estudo experimental, em modelo animal, através do tratamento de defeito ósseos criados em fêmures de ratos. A amostra dessa pesquisa foi composta de 96 ratos albinos da espécie Rattus novergicus albinus, linhagem SHR (Spontaneously Hypertensive Rats), isogênicos. Os animais foram divididos aleatoriamente em quatro grupos de acordo com o tipo de tratamento (Grupo I: α – TCP + ADSCs; Grupo II: α – TCP + ADSCs/ENDO; Grupo III: α – TCP; Grupo IV). A cultura de células tronco tiveram como tecido de origem o tecido adiposo da região abdominal e as células endoteliais formam coletadas da medula óssea. A peças foram avaliadas em 03 períodos de tempo diferentes (07, 14 e 21 dias). A histomorfometria avaliou a área de neoformação óssea dos defeitos bem como a imunohistoquímica com marcação para a proteína VEGF avaliou a eficácia da adição das células endoteliais. Os resultados demonstraram, através dos testes estatísticos que houve uma diferença estatisticamente significante, no reparo ósseo, favorecendo os tratamentos dos defeitos que utilizaram a terapia celular e a vascularização foi também otimizada no grupo que foi tratado com a adição das células endoteliais. Dessa forma conclui-se que nesse modelo de estudo a utilização das ADSCS foram capazes de otimizar o reparo ósseo. / The regenerative medicine has been searching for a new therapeutic options to manage diseases and also organic dysfunctions. The advanced research fields, has enrolled the stem cells to achieve the upgrade in the new treatment objectives. The bioengineering is application of this new and experimental, treatment method to repair damaged tissues and organs using the stem cells, includes the possibility to acelerating and improving the bone repair process. The autogenous bone graft has been considered the gold standart graft material to reconstruction the bone defects, fundamentally because of the osteogenic potential. However, the harvest disadvantages autogenous bone tissue leads to the search for bone substitutes improvement. In this field a promising alternative has been proposed by tissue engineering. The totipotent cells, also called mesenchymal stem cells, which has the cellular plasticity ability, is associated with biomaterials, creating a synergy, between these cells and the scaffold, to optimize the osteogenesis. The tissue engineering application to tissue repair has been extensively researched with the aim of proposing more reliable and more efficient clinical methods. Although the effects of the use of adult stem cells are well known in bone marrow transplants, in some areas, such as bone repair, there is still lack of scientific data. This research was conducted, in animal model, to assessment bone repair in created femural bone defects treated with mesenchymal stem cells. 96 animals (Rattus novergicus albinus - Spontaneously Hypertensive Rats) were randomly divided into four groups (Group I: α - TCP + ADSCs, Group II: α - TCP + ADSCs/Endo, and Group III: α - TCP; (07, 14 and 21 days). The histological sections were stained in H&E and the histomorphometry was used to evaluated the new bone formation area in the defects and also the immunohistochemical expression of VEGF was analysed. Our results suggest that the combination of ADSCs and the scaffold was able to enhance the bone repair in this study model.