Type III secretion is a virulence system utilized by several clinically important Gram-negative pathogens. Computational methods have been used to develop two classes of type III secretion inhibitors, the salicylidene acylhydrazides and the acetylated salicylanilides. For these classes of compounds, quantitative structure-activity relationship models have been constructed with data from focused libraries obtained by statistical molecular design. The models have been validated and shown to provide useful predictions of untested compounds belonging to these classes. Scaffold hopping of the salicylidene acylhydrazides have resulted in a number of synthetic targets that might mimic the scaffold of the compounds. The synthesis of two libraries of analogs to two of these scaffolds and the biological evaluation of them is presented.

The focus of this thesis was to design a scaffold for in vitro culture that would mimic the structure of the native ligament in order to influence primary ligament cells towards the production of ligament-specific tissue. A major part of this project was material selection and subsequent testing to determine if the chosen materials were suitable for the scaffold design. A 20:80 (CL:DLLA) poly(ε-caprolactone-co-D,L-lactide) copolymer (PCLDLLA) was synthesized and electrospun with sub-cellular fibre diameters. The fibres were manufactured into aligned arrays to mimic the collagen fibrils of the ligament. To enhance cell and protein adhesion properties, the PCLDLLA polymer surface was modified using a base catalyzed etching technique. A photocrosslinked methacrylated glycol chitosan (M-GC) hydrogel was used to deliver encapsulated ligament cells to the biomimetic scaffold and mimic the hydrated proteoglycan matrix portion of the ligament. The scaffolds were cultured in vitro for a 4 week period and characterized using immunohistochemistry to identify and localize ligament specific proteins produced within the scaffolds. Cell culture results indicated that the M-GC hydrogel was an effective method of delivering viable cells evenly throughout the biomimetic scaffold. Compared to the unmodified PCLDLLA surfaces, the base-etched electrospun PCLDLLA fibre surfaces increased cell adhesion and acted as new tissue growth guides in the biomimetic scaffold. The biomimetic scaffolds produced and accumulated ligament specific proteins: collagens type I and III. The biomimetic scaffold design was determined to be a viable alternative to the current designs of ligament tissue engineering scaffolds. / Thesis (Master, Chemical Engineering) -- Queen's University, 2011-06-22 10:46:12.291

Repair of bone defects is a major challenge in orthopaedic surgery. Current bone graft treatments, including autografts, allografts and xenografts, have many limitations making it necessary to develop a biomaterial to be a bone graft substitute. One such biomaterial is bioactive resorbable silica-calcium phosphate nanocomposite (SCPC). SCPC was processed using a 3D rapid prototyping technique and sintered at different temperatures to create porous scaffolds. SEM analyses and mercury intrusion porosimetry showed SCPC to be highly porous with micro- and nanopores. BET analysis indicated that SCPC had high surface area. Mechanical testing demonstrated that SCPC had a compressive strength similar to trabecular bone. Analysis of different thermal treatment temperatures indicated as the temperature was increased, the porosity decreased and the mechanical strength increased. When loaded with rhBMP-2 (SCPC-rhBMP-2), SCPC provided a sustained release profile of rhBMP-2 for 14 days. This was shown to be a greater release than hydroxyapatite (HA)-rhBMP-2. After immersion in SBF, ICP analyses showed the calcium concentration of SBF dropped drastically after one day of immersion. In conjunction, FTIR showed the formation of a hydroxyapatite layer on the SCPC surface and was confirmed by SEM. SCPC thermally treated at 850 ??C demonstrated the greatest dissolution/precipitation reactions when immersed in SBF. Processing the SCPC-rhBMP-2 hybrid using a rapid prototyping technique allowed for an exact replica of the rabbit ulna to be fabricated. This was implanted into a 10 mm segmental defect in the rabbit ulna. CT scans during the healing of the defect showed intimate union between SCPC-rhBMP-2 and the bone and about 65% healing of the defect after 4 weeks. Rabbits were euthanized after 12 and 16 weeks. Digital images show almost complete healing of the defect after 16 weeks. Torsional testing of the ulna after 12 weeks demonstrated restoration of maximum torque and angle at failure. Histological evaluation after 12 weeks showed the regenerated bone has all the morphological characteristics of mature bone. Through in-vitro and in-vivo testing, it can be recommended that the porous bioactive SCPC can serve as a successful delivery system for biological growth factors and serve as an alternative to autologous bone grafting.

A degradable/polar/hydrophobic/ionic (D-PHI) polyurethane with properties intended to promote tissue regeneration in a small diameter peripheral artery vascular graft was evaluated for cell biocompatibility and growth. Films were cast in polypropylene 96 well plates for monocyte/endothelial cell (EC) co-culture in vitro studies and porous scaffold discs were implanted in an in vivo subcutaneous mouse model. After 7 days in culture the co-culture demonstrated cell adhesion and growth, low esterase activity (a measure of degradative potential and cell activation), no detectable release of pro-inflammatory cytokine (tumour necrosis factor -α) but measurable anti-inflammatory interleukin (IL)-10. The EC and the co-culture expressed the EC biomarker CD31, whereas the monocyte monoculture did not. Cytokine array analysis of the in vivo characterization of D-PH supported an anti-inflammatory phenotype of cells at the site of the implant. Levels of IL-6 significantly decreased over time while IL-10 was significantly higher at 6 weeks post implant. TNF-α levels did not change significantly from 24 hours onwards, however the trend was towards lesser amounts following the initial time point. Histological analysis of the explanted scaffolds showed excellent tissue ingrowth and vascularization. A live/dead stain showed that the cells infiltrating the scaffolds were viable. Both the in vitro and in vivo results of this thesis indicate that D-PHI is a good candidate material for tissue engineering a peripheral artery vascular graft.

Tissue engineering the annulus fibrosus (AF) for use in a functional intervertebral disc replacement is a promising alternative to current treatments of degenerative disc disease. Polycarbonate urethane (PU) scaffolds have demonstrated the ability to support AF cell attachment and matrix synthesis and are suitable for tissue engineering the AF. The present study investigates the effects of the physical and biochemical environment on AF cells grown on aligned nanofibrous PU scaffolds. First, the effect of dynamic spinner flask culture and fibronectin pre-coating on tissue formation was analyzed and then the role of scaffold fibre tension on annulus fibrosus cells was examined using a tailored culture system. The results of these studies demonstrated that AF cells are sensitive to differences in biochemical cues at the scaffold surface and their physical environment and respond by altering their cellular responses and, potentially by manipulating their microenvironments, including the physical characteristics of the PU-ADO scaffolds.

Tissue engineering the annulus fibrosus (AF) for use in a functional intervertebral disc replacement is a promising alternative to current treatments of degenerative disc disease. Polycarbonate urethane (PU) scaffolds have demonstrated the ability to support AF cell attachment and matrix synthesis and are suitable for tissue engineering the AF. The present study investigates the effects of the physical and biochemical environment on AF cells grown on aligned nanofibrous PU scaffolds. First, the effect of dynamic spinner flask culture and fibronectin pre-coating on tissue formation was analyzed and then the role of scaffold fibre tension on annulus fibrosus cells was examined using a tailored culture system. The results of these studies demonstrated that AF cells are sensitive to differences in biochemical cues at the scaffold surface and their physical environment and respond by altering their cellular responses and, potentially by manipulating their microenvironments, including the physical characteristics of the PU-ADO scaffolds.

In cardiovascular disease the repair response is insufficient to restore blood flow, leading to the death of muscle and loss of tissue function. Therefore, strategies to augment the endogenous cell response and its effects may help improve tissue recovery and function. In this study we explored the use of tissue-engineered collagen matrices for augmenting endogenous regenerative processes after myocardial infarction. Treatment with the sLeX-collagen matrix reduced inflammation and apoptosis and had a positive regenerative effect on the infarcted mouse heart, through improved vascular density and possibly enhanced cardiomyogenesis. Additionally, we investigated the effects of cryopreservation on generating circulating angiogenic cells (CACs) from peripheral blood mononuclear cells (PBMCs), as a potential source of stem cells that could be used in combination with our collagen scaffold. Our findings show that despite PBMCs experiencing phenotypic changes after cryopreservation, they may still be used to generate the same therapeutic CACs as freshly procured PBMCs.

Fabrication of scaffold is the key for bone tissue engineering, which is commonly regarded as the most potential route for repairing bone defects. Previously, porous ceramic scaffolds were fabricated through a variety of traditional methods, like moulding and casting, but most of them cannot produce customised tissue-engineered scaffolds. Therefore, 3D printing methods are gaining more attention and are currently being explored and developed to make scaffolds with acceptable biocompatibility. With the considerable development of bone tissue engineering, the bioactivity of scaffolds is becoming increasingly demanded, which leads to new methods and techniques to produce highly biomimetic bone scaffolds. In this study, a new fabrication process to optimise the structures of scaffolds was developed, and intensive researches were performed on the porous scaffolds to confirm their advantages in biological performance. Specifically, by combination of motor assisted extrusion deposition and gas-foaming (graphite as the porogen) technique, hierarchically porous scaffolds with improved microstructures, i.e. multi-scaled pores from nanometre to millimetre (nm-μm-mm), was successfully developed. In this thesis, the optimal content of porogen for scaffolds was studied in terms of compressive strength and in-rod porosities. The most concerned physicochemical properties of scaffolds were carefully examined and the results revealed that such scaffolds exhibit excellent physicochemical properties owing to hierarchically porous structures. Due to additional in-rod micropores and increased specific surface area, along with better hydrophilicity, hierarchically porous scaffolds exerted complete superiority in biological activity, including promoting cellular proliferation of osteoblasts, adhesion and spreading status, as well as the ability to induce cellular differentiation.

Submitted by Maria Medeiros (maria.dilva1@ufcg.edu.br) on 2018-06-29T13:53:39Z No. of bitstreams: 1 GREYCE YANE HONORATO SAMPAIO - TESE (PPGCEMat) 206.pdf: 8427492 bytes, checksum: a008b21b874e092dc5037d0778bb1057 (MD5) / Made available in DSpace on 2018-06-29T13:53:39Z (GMT). No. of bitstreams: 1 GREYCE YANE HONORATO SAMPAIO - TESE (PPGCEMat) 206.pdf: 8427492 bytes, checksum: a008b21b874e092dc5037d0778bb1057 (MD5) Previous issue date: 2016-02-29 / Capes / A policaprolactona (PCL), utilizada como matriz biodegradável em combinação à nanohidroxiapatita (nHA), intrinsecamente bioativa, é uma alternativa promissora para a produção de matrizes tridimensionais (3D) porosas, visando a regeneração do tecido ósseo. No entanto, rotas de processamento visando otimizar a interação entre esses materiais devem ser investigadas. O objetivo deste trabalho foi desenvolver matrizes 3D de PCL/nHA estruturadas por esferas, via agregação por aquecimento a baixas temperaturas. Para isto, foram produzidas nHA por precipitação química via úmida, utilizando o Ca(NO3)2.4H2O e o (NH4)2HPO4 como precursores, conduzido à 80 °C (pH>10). Estas foram caracterizadas por difração de Raios X (DRX), espectroscopia na região no infravermelho com transformada de Fourier (FTIR) e microscopia eletrônica de transmissão (MET). A superfície das nHA foi modificada com ácido esteárico (AE), verificada pelos resultados de FTIR, MET e monitoramento de estabilidade. Esferas de PCL e PCL/nHA foram produzidas pelo método de emulsão simples com evaporação do solvente, o diclorometano, em 02 etapas, para obtenção de diâmetros entre 10-150 µm e maiores que 800 µm. Foram investigadas as influências da concentração de PCL, da adição de nHA com e sem AE, da concentração de álcool polivinílico e da velocidade de emulsificação. O material foi caracterizado por DRX, FTIR, microscopia eletrônica de varredura (MEV), calorimetria exploratória diferencial (DSC) e análise termogravimétrica (ATG), sendo calculado também o rendimento. Matrizes 3D porosas de PCL e de PCL/nHA foram produzidas e analisadas por microscopia ótica (MO), MEV e ensaios in vitro de bioatividade e de adesão celular. nHA deficientes em cálcio, em formato de haste (~47 nm x ~8 nm) e com superfície específica de 90,1 m2/g, foram obtidas. Esferas de PCL e PCL/nHA foram produzidas com diâmetros entre 2-3000 µm, densas ou porosas, contendo nHA distribuídas interna e externamente. Matrizes 3D foram obtidas a partir da agregação das esferas produzidas, estruturadas entre si a partir da formação de pescoços entre esferas adjacentes, com hierarquia de poros medindo até 1500 µm. As matrizes apresentaram capacidade bioativa e satisfatória adesão e desenvolvimento celular. Os resultados sugerem a aplicação vantajosa desses dispositivos para a regeneração de tecidos ósseos. / Polycaprolactone (PCL) have been largely used as a biodegradable matrix. This polymer in a combination with bioactive calcium deficient nanohydroxyapatite (CD-nHA) represent promising materials to be applied for bone tissue engineering. Nevertheless, processing routes to optimize the interaction between these materials should be investigated. Thus, the aim of this work was to develop PCL/nHA spherebased scaffolds structured by sinterization at low temperatures. For this, nHA were produced by wet chemical precipitation method, using the precursors Ca(NO3)2.4H2O and (NH4)2HPO4, conducted at 80 °C (pH>10). The powder was characterized by Xray diffraction (XRD), fourier transform infrared spectroscopy (FTIR) and transmission electron microscopy (TEM). nHA surface was modified with stearic acid (SA), verified with FTIR and TEM analysis and stability control in dichloromethane (DCM). PCL and PCL/nHA spheres were produced by oil-in-water and solid-in-oil-in-water emulsion solvent evaporation method. This production occurred in 02 steps, to obtain diameters ranging 10-150 µm and higher than 800 µm. The follow parameters were investigated: PCL concentration, nHA content with and without AE, polyvinyl alcohol concentration (stabilizing agent) and emulsification stirring. The material was characterized by DRX, FTIR, scanning electron microscopy (SEM), differential scanning calorimetry (DSC) and thermogravimetric analysis (ATG), and the percent yield was calculated. PCL and PCL/nHA porous scaffolds were produced and characterized by SEM, optical microscopy (MO), and in vitro bioactivity and cell adhesion assays. Calcium deficient nHA, shaped as rods (~47 nm x ~8 nm), with a specific surface of 90,1 m2/g was obtained. PCL and PCL/nHA spheres were produced with diameters ranging 2-3000 µm, dense or porous, containing nHA effectively distributed internally and at the surface. Scaffolds were successfully obtained by sintering the spheres at low temperatures. It was observed connections shaped as necks in adjacent spheres, and a hierarchical porous architecture with pores measuring up to 1500 µm. It was demonstrated satisfactory bioactivities capacity, cellular adhesion, and cellular development. Our results suggested the advantageous applicability of these scaffolds to bone tissue regeneration.

A insuficiência cardíaca é a doença crônica com maior impacto na sobrevida e qualidade de vida dos pacientes. Apesar do constante desenvolvimento de novas estratégias de tratamento, esta doença continua atingindo altos índices de mortalidade. O coração adulto tem capacidade de regeneração limitada e há grande evidência experimental de que os transplantes de células-tronco poderiam ser uma abordagem eficiente na recuperação do miocárdio lesado. Contudo, a maioria dos estudos são realizados em cardiomiopatias isquêmicas, existindo poucos estudos na cardiomiopatia dilatada (CMD). Em função disto, este trabalho foi realizado com o objetivo de avaliar a regeneração do miocárdio em coelhos com CMD induzida pela doxorrubicina, por meio do uso de células-tronco mesenquimais (MSC) obtidas de tecido adiposo, associadas ou não com plasma rico em plaquetas (PRP). Foram utilizadas 40 coelhas, Nova Zelândia, e um coelho macho doador das MSCs derivadas do tecido adiposo. As coelhas foram divididas em dois grupos: CMD induzida pela doxorrubicina e o grupo saudável. Cada grupo foi subdividido conforme o tratamento recebido: solução fisiológica, MCSs, PRP e MSCs associadas ao PRP. Os subgrupos receberam o tratamento por injeção diretamente no miocárdio no ventrículo esquerdo mediante toracoscopia vídeo assistida. Os coelhos foram avaliados por exames de ecocardiograma, eletrocardiograma, troponina I, no dia da chegada, após a indução da CMD e 15 dias após o recebimento das terapias. Nesta última avaliação, foi realizada a eutanásia e coletado o coração para análise histológica. Foi observado que após a indução, a troponina I se elevou, o segmento QRS visto no eletrocardiograma, aumentou e, no ecocardiograma, as frações de ejeção (FE) e encurtamento (FS) diminuíram e o diâmetro sistólico do ventrículo esquerdo (VEs) aumentou, em todos os animais avaliados. Após os tratamentos, o subgrupo MSCs obtiveram os melhores resultados em todas as análises citadas. Houve menor elevação da troponina I, o segmento QRS diminuiu, as FS e FE aumentaram e o VEs diminuiu. No exame histopatógico, analisado pela coloração de hematoxicilina-eosina, constatou-se que o subgrupo MSCs apresentou menos lesões, e nos subgrupos MSCs associadas com PRP, solução fisiológica e PRP as lesões aumentaram gradualmente, respectivamente. Os resultados sugerem que o uso das MSCs melhoraram a função cardíaca em coelhos com cardiomiopatia dilatada e que há necessidade de mais estudos no uso de PRP no miocárdio. / Heart failure is a chronic disease with major impact on survival and quality of patient’s life. Despite the constant development of new treatment strategies, this disease still affects high mortality rates. The adult heart has limited ability to regenerate and there is experimental evidence that large transplants of stem cells could be an effective approach in the recovery of injured myocardium. However, most studies are performed in ischemic cardiomyopathy, there are few studies in dilated cardiomyopathy. Because of this, this study aimed at evaluating the regeneration of the myocardium in rabbits with dilated cardiomyopathy induced by doxorubicin through the use of mesenchymal stem cell (MSC) derived from adipose tissue, associated or not with platelet-rich plasma (PRP). 40 New Zealand rabbits were utilized and a male rabbit donor MSCs derived from adipose tissue. The rabbits were divided into two groups: dilated cardiomyopathy doxorubicin-induced and the healthy group. Each group was divided according to treatment received: saline, MSCs, PRP and MSCs associated with PRP. The subgroups receiving treatment through an injection directly into the myocardium of the left ventricle through video-assisted thoracoscopy. The rabbits were evaluated by echocardiogram, electrocardiogram, troponin I, on the day of arrival, after induction of dilated cardiomyopathy and 15 days after receipt of therapies. This last evaluation, euthanasia was performed and the hearts collected for histological analysis. It was observed that after induction the troponin I increased, the QRS segment, seen on the electrocardiogram, increased, and, in echocardiography, the ejection and shortening fractions decreased, and left ventricular systolic diameter increased in all animals evaluated. After treatments, the subgroup MSCs have the best results in all tests cited. There was a lower elevation of troponin I, decreased QRS segment, the ejection and shortening fractions increased and left ventricular systolic diameter decreased. On examination histologic, analyzed by hematoxylin-eosin staining, the subgroup found that MSCs had fewer injuries, and in the subgroups MSCs associated with PRP, PRP and saline lesions gradually increased, respectively. The results suggest that the use of MSCs improved cardiac function in rabbits with dilated cardiomyopathy and that there is need for more studies on the use of PRP in the myocardium.