Hydrogels are three dimensional, insoluble, porous and crosslinked polymer networks. Due to their high water content, they have great resemblance to natural tissues, and therefore, demonstrate high biocompatibility. The porous structure provides an aqueous environment for the cells and also allows influx of nutrients needed for cellular viability. In this study, a natural biodegradable material, agarose (Aga), was used and semi-interpenetrating networks (semi-IPN) were prepared with polymers having different charges, such as positively charged chitosan (Ch) and negatively charged alginate (Alg). Hydrogels were obtained by the thermal activation of agarose with the entrapment of Ch or Alg in the Aga hydrogel structures. Chemical composition of hydrogels were determined by ATR-FTIR examinations, mechanical properties of hydrogels were examined through compression tests, morphologies were confirmed by scanning electron microscopy (SEM) and confocal microscopy, thermal properties were evaluated by differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA). Moreover, swelling ratios, water contact angles and surface free energies (SFE) were determined. Cell proliferation and cell migration within these hydrogels were examined by using L929 fibroblast cell line. MTS assays were carried out to observe the cell proliferation on hydrogels. Confocal microscopy was used in order to examine the cell behavior such as cell attachment and cell migration towards the hydrogels. It was observed that addition of positively charged Ch into agarose increased the ultimate compressive strength (UCS), decreased elastic modulus (E), increased the thermal stability and hydrophobicity of the semi-IPN hydrogels. On the other hand, addition of negatively charged Alg into agarose decreased UCS, E, thermal stability and hydrophilicity. Cell-material interaction results showed that Aga hydrogels in tissue engineering applications was improved by adding different charged polyelectrolytes. Cell migration within Aga hydrogels was enhanced by adding Ch, and hindered by addition of Alg. Maximum cell proliferation and maximum penetration of the cells were obtained with the Ch/Aga hydrogels most probably due to attraction between the negatively charged cell surface and the positively charged Ch/Aga hydrogel surface. It was shown that cell interaction of agarose hydrogel scaffolds could be enhanced by introducing chitosan within the agarose hydrogels and obtained structures could be candidates for tissue engineering applications.

Submitted by Cláudia Bueno (claudiamoura18@gmail.com) on 2016-06-09T18:10:50Z No. of bitstreams: 2 Tese - Flávia Resende Martins da Costa - 2015.pdf: 3435054 bytes, checksum: 67b9295abbfceafe67eca1e096f71930 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2016-06-10T12:37:22Z (GMT) No. of bitstreams: 2 Tese - Flávia Resende Martins da Costa - 2015.pdf: 3435054 bytes, checksum: 67b9295abbfceafe67eca1e096f71930 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2016-06-10T12:37:22Z (GMT). No. of bitstreams: 2 Tese - Flávia Resende Martins da Costa - 2015.pdf: 3435054 bytes, checksum: 67b9295abbfceafe67eca1e096f71930 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2015-06-30 / Chitosan is a polysaccharide amino derivative of chitin, and constitutes most of the insects and crustaceans exoskeletons and fungal cell wall, thus being a low-cost, renewable and abundant, biodegradable natural product. Biological characteristics, biocompatibility and biodegradability allows various uses of this biomaterial in healthcare. The present study aimed to evaluate the hemostatic contact effect and tissue response to the implant of chitosan-alginate sponge in hepatic lesion in Swiss mice, comparing to freeze-dried hydrolyzed collagen sponges. The lesions were surgically induced. The hemostatic contact effects of chitosan and hydrolyzed collagen sponges were evaluated through its mechanical compression on hemorrhagic lesions, while the effects of his implants were observed from the incorporation of fragments of those sponges in liver failures. Gauze sponges were used as control for contact study. For the implants evaluation, liver segments removed to produce hepatic injury were used as controls. Average hemostasis times for chitosan and collagen treatments were 49 seconds, and one minute and 28 seconds for gauze treatments. Grossly, omental adhesions were observed on the implants and faster integration of collagen sponge at receiver tissue. Microscopic evaluation showed inflammatory reaction to both implanted materials and collagen synthesis stimulation. Average percentage of collagen from hepatic recipients segments of the chitosan-alginate sponge implant, and collagen hydrolyzate sponge implant increased significantly between 7 and 14 days postoperatively, pointing to a stimulating effect exerted by chitosan sponges for tissue repair. In conclusion, chitosan sponges have hemostatic action and stimulating effect on the synthesis of hepatic collagen fibers. / A quitosana é um polissacarídeo amino derivado da quitina. Constitui a maior parte dos exoesqueletos dos insetos, crustáceos e parede celular de fungos, sendo um produto natural, de baixo custo, renovável e biodegradável. Características como biocompatibilidade e biodegradabilidade possibilitam diversas utilizações deste biomaterial na área da saúde. Com o presente estudo objetivou-se avaliar o efeito hemostático ao contato e a reação tecidual ao implante de esponja de quitosana-alginato em lesão hepática em camundongos (linhagem Swiss) saudáveis, comparando-os aos de esponjas de colágeno hidrolisado liofilizadas. As lesões foram induzidas cirurgicamente. O acesso à cavidade abdominal foi feito por meio de incisão longitudinal mediana pré-retro-umbilical, seguida de localização e exposição do lobo hepático esquerdo. O defeito hepático foi realizado por incisão circular através da cápsula hepática com punch dermatológico de 5,0mm, centralizado na região exposta do lobo esquerdo. Os efeitos hemostáticos de contato das esponjas de quitosana e colágeno hidrolisado foram avaliados por meio de sua compressão sobre as lesões hemorrágicas, enquanto os efeitos de seu implante foram observados a partir da incorporação de fragmentos das referidas esponjas nos defeitos hepáticos produzidos. Esponjas de gaze foram usadas como controle para o estudo de contato. Para a avaliação dos implantes, os segmentos hepáticos retirados para a produção da falha hepática foram utilizados como controles. Os tempos médios para hemostasia nos tratamentos com quitosana e colágeno foram de 49 segundos e, nos tratamentos com gaze, um minuto e 28 segundos. À macroscopia observaram-se aderências omentais sobre os implantes e integração mais rápida da esponja de colágeno ao tecido receptor. A avaliação microscópica evidenciou reação inflamatória aos dois materiais implantados e estímulo à síntese de colágeno. A percentagem média de colágeno dos segmentos hepáticos receptores dos implantes de esponja de quitosana-alginato e de esponja de colágeno hidrolisado apresentou elevação significativa entre 7 e 14 dias pós-operatórios, permitindo inferir efeito estimulante exercido pelas esponjas de quitosana sobre a reparação tissular. Concluiu-se que esponjas de quitosana apresentam ação hemostática e efeito estimulante sobre a síntese de fibras colágenas hepáticas.

This diploma thesis deals with encapsulation of natural extracts. In the theoretical part the methods of encapsulation, materials for particle preparation, as well as application of encapsulation techniques in food industry were described. In experimental part selected natural extracts of propolis, green barley and probiotics were characterized. There substances were encapsulated into alginate and chitosan. In the total of 25 types of prepared particles long-term stability in some model physiological conditions as well as in four different model foods was evaluated. Additionally, stability of selected particles in several real milk-based products was followed too. The stability of particles was determined spectroptohometrically. In natural extract a content of polyphenols, proteins, chlorophylls, as well as total antioxidant activity were analysed. To analysis of probiotics optical and fluorescence microscopy were used. In propolis and green barley antimicrobial activity was tested too. Moreover, in the sample of propolis also cytotoxic assay was applied. Agar-chitosan was chosen as the best shell material for propolis due to its optimal stability in model physiological conditions as well as model foods. Liposomes were evaluated as unstable and were not recommended for further application. As the suitable shell material for powdered green barley starch-alginate (rate 1:4) and agar-chitosan were proposed, while the second one showed better stability for released proteins. Agar-chitosan shell material was usable for fresh green barley too. For probiotics encapsulation alginate or alginate-starch were chosen because of their porosity and possibility of nutrients diffusion. In real foods the best results were reached with application of probiotic particles into milk. Coencapsulation of powdered barley and probiotics did not confirm inhibition of culture growth. Neither the antimicrobial effect of propolis and barley nor the cytotoxic effect of propolis were confirmed.

Rupture of the anterior cruciate ligament (ACL) is one of the most common ligamentous injuries of the knee. Post rupture, the ACL does not heal on itself due to poor vasculature and hence surgical intervention is required to treat the ACL. Current surgical management of ACL rupture consists of reconstruction with autografts or allografts. However, the limitations associated with these grafts have prompted interest in tissue engineered solutions that combine cells, scaffolds and stimuli to facilitate ACL regeneration. This thesis describes a ligament tissue engineering strategy that involves incorporating biomolecules within fibers-based electrospun meshes which mimics the extra-cellular matrix microarchitecture of ligament. However, challenges exist with incorporation of biomolecules. Therefore, the goal of this research project was to develop two techniques to incorporate biomolecules within electrospun meshes: (1) co-axially electrospinning fibers that support surface-grafting of biomolecules, and (2) co-axially electrospinning fibers decorated with biomolecule-loaded microspheres. In the first approach, chitosan was co-axially electrospun on the shell side of poly caprolactone (PCL) and arginine-glycine-aspartate (RGD) was attached to the electrospun meshes. Bone marrow stromal cells (BMSCs) attached, spread and proliferated on these meshes. In the second approach, fluorescein isothiocyanate labelled bovine serum albumin (FITC-BSA) loaded chitosan-alginate (CS-AL) microspheres were fabricated. The effects of cation to alginate ratio, type of alginate and concentration of CaCl2 on microsphere size, FITC-BSA loading and release were systematically evaluated. The CS-AL microspheres were then incorporated into the sheath phase of co-axially electrospun meshes to achieve microsphere-decorated fiber composite meshes. The results from these model study suggest that both approaches are tractable for incorporating biomolecules within fibers-based electrospun meshes. Both these approaches provide platform for future studies that can focus on ligament-relevant biomolecules such as FGF-2 and GDF-5. / Master of Science

In theoretical part of this diploma thesis the basic properties of antioxidants, especially anthocyanins and phenolic compounds are described, as well as the basic features and principles of free radicals formation. The theoretical part also describes some possible ways of encapsulation of antioxidants into polysaccharide and lipid particles. In the experimental part basic characteristics of extracts from selected lyophilized fruits and vegetables (carrots, apples and mixed berries) are described. As group parameters of plant extracts the total antioxidant activity, content of flavonoids and phenolics, carotenoids, anthocyanins and ascorbic acid were determined. In experimental part also various encapsulation techniques were tested, encapsulation effectiveness of each technique was evaluated and the stability and size of the created particles were determined. As the best encapsulation method in terms of encapsulation efficiency in most of samples/parameters ethanol injection was found. On the other hand the particles prepared by ethanol injection are relatively unstable in terms of zeta potential, which is followed by their tendency to aggregate. As the most stable particles prepared by thin layer evaporation (TLE) and reverse phase thin layer evaporation (RP-TLE) were evaluated. Particles prepared by TLE, RP-TLE and chitosan-alginate particles exhibited a negative charge, while particles prepared by ethanol injection stayed uncharged and chitosan particles have a positive charge.