Effective vascularization is crucial for almost any therapeutic tissue engineering concept. In this context, therapeutic angiogenesis attempts to enforce the natural process of blood vessel formation by provision of bioactive effectors. Along these lines, the aim of this work was to evaluate the potential of a modular hydrogel composed of the synthetic star-shaped poly(ethylene glycol) (starPEG) and the naturally occurring biopolymer heparin for the defined and orchestrated delivery of two major angiogenic growth factors, fibroblast growth factor-2 (FGF-2) and vascular endothelial growth factor (VEGF). While starPEG determines the structural properties of the gel materials, effective administration of both cytokines is based on their natural affinity to heparin, the highly charged polysaccharidic building block capable of reversibly binding various growth factors upon geometrically matching electrostatic interactions. Varying the molar ratio of starPEG to heparin upon network formation, different hydrogel types with distinct mechanical characteristics but constant heparin content could be produced. As heparin represents the basis for the growth factor interaction with the scaffolds, the matrices were found to bind and release FGF-2 or VEGF independently of the particular network stiffness and structural properties of the different gel types. Moreover, the material could be utilized for a modular delivery of growth factor combinations over a broad range of concentrations. To evaluate the general suitability for pro-angiogenic stimulation, the provision of FGF-2 and VEGF from starPEG-heparin hydrogels differing in their mechanical characteristics and biofunctionalization with adhesive peptides was studied using human endothelial cells, the cell type that forms the inner layer of any blood vessel. Results showed that the presence of the adhesion ligand was an essential requirement to mediate cell attachment and subsequent growth on the scaffolds. Apart from that, hydrogels with an intermediate stiffness showed beneficial effects on endothelial cell proliferation/survival while in parallel also the differentiation into elongated, pro-tubular structures could be promoted. While the delivery of FGF-2 was able to enhance cell growth, VEGF mainly initiated endothelial cell shape elongation. However, with a parallel administration of both growth factors, their beneficial effects could be combined to obtain high numbers of endothelial cells undergoing differentiation. Furthermore, besides the possibility of growing endothelial cells on top of the biofunctionalized hydrogels, the release of growth factors by starPEG-heparin matrices could be applied as a stimulus to attract the cells to migrate into the direction of the scaffolds. While FGF-2 and VEGF supported cell motility to a similar extent, their combined action was found to exert the strongest effect on endothelial cell migration. Based on the results of these in vitro experiments, matrices most effectively stimulating pro-angiogenic cellular responses were selected for in vivo studies applying the functionalized materials to the chorioallantoic membrane (CAM) of fertilized chicken eggs, an assay commonly used to evaluate the vascularization potential of biomaterials. In this assay, the delivery of FGF-2 and/or VEGF by starPEG-heparin hydrogels induced a substantial angiogenic response within the CAM system, while the combination of both growth factors tends to increase vascularization most effectively. In order to adjust the starPEG-heparin hydrogel system to the complex requirements of therapeutic angiogenesis, further options to specifically modulate the FGF-2 or VEGF release were explored. With the incorporation of enzymatically cleavable peptide linkers, not only the possibility for a cellular remodeling of the gel matrix could be permitted, but also the growth factor release was substantially enhanced upon network degradation. Moreover, with the gradual removal of FGF-2 and VEGF interaction sites from heparin upon selective desulfation, the binding of both growth factors to hydrogels composed out of starPEG and desulfated heparin was significantly reduced depending on the remaining sulfate content. Irrespective of the lower immobilized amounts of FGF-2 or VEGF, higher absolute quantities of both growth factors could be released and retained in the medium due to their decreased affinity to heparin, thereby enhancing the delivery efficiency of the scaffolds. Going beyond common concepts for triggered cytokine release, hydrogel-bound FGF-2 or VEGF could be effectively displaced from their heparin binding sites by an application of the competitive, highly-heparin affine molecule chitosan. As chitosan could be introduced at different time points, not only the amounts of delivered growth factor were enhanced, but also the FGF-2 or VEGF release kinetics could be specifically modulated. Taken together, starPEG-heparin hydrogels with independently adaptable physical and biomolecular composition were demonstrated to provide time-resolved multi-factor delivery of pro-angiogenic growth factors resulting in valuable new options for therapeutic angiogenesis.

Cordia trichotoma (Vell.) Arrab. ex Steud. species of the Boraginaceae family is considered a priority for logging native tree plantations in southern Brazil, in addition to have an important hole in environmental restoration of degraded areas. However, despite the importance of this species, there are no published reports regarding irrigation and use of water retaining polymers in the production of seedlings, as well as the influence of shading levels and excess soil moisture in early growth in the field, based on morphological and physiological variables. This study was divided into three chapters: Chapter I aimed to define, based on morphological and physiological variables which irrigation blade is indicated, whether or not combined with water retaining polymer doses added to the substrate, in the production of C. trichotoma seedlings, aiming to reduce water consumption. The physiological variables of chlorophyll a fluorescence showed themselves important for behavioral studies of seedlings growth in nurseries. A suitable growth of seedlings in the nursery was obtained with irrigation blades of 4-8-8 mm day-1, alternating it every 60 days, with the possible use of a smaller blade at the start of growth, thereby reducing the consumption of water. The use of a continuous 4 mm day-1 blade in the production of seedlings is possible, since associated with a dose of 3 g L-1 water retaining polymer to the substrate. In Chapter II we evaluated the initial growth for C. trichotoma seedlings in the field under different levels of shading, considering morphological and physiological variables. The aerial part of the plant had the growth negatively influenced in full sun condition. The species responds with great plasticity to different light intensities. However, the shading of 18 and 50% provides favorable growth and physiological values for the early development of the species. The purpose of Chapter III was to identify the effect of excess soil moisture on survival and early growth of C. trichotoma seedlings after field planting, based on morphological and physiological characteristics. The higher content of micropores and clay in successive soil layers (0 to 20 cm) is indicative of the undesirable condition for the survival and early growth of seedlings of C. trichotoma. The species is intolerant to environments with excess soil moisture and the planting is not recommended in plain fields, with drainage problems. / A espécie Cordia trichotoma (Vell.) Arráb. ex Steud. da família Boraginaceae é considerada uma das arbóreas nativas madeireiras prioritárias para plantios florestais na região sul do Brasil, além de ter importância na recuperação ambiental de áreas alteradas. No entanto, apesar da importância desta espécie, não existe na literatura estudos com relação à irrigação e utilização de polímeros hidroretentores na produção de mudas, assim como a influência de níveis de sombreamento e do excesso de umidade do solo no crescimento inicial a campo, com base em variáveis morfofisiológicas. O presente trabalho foi dividido em três capítulos, no Capítulo I objetivou-se definir, com base em variáveis morfofisiológicas, qual lâmina de irrigação é indicada, combinada ou não com doses de polímero hidroretentor adicionadas ao substrato, na produção de mudas de C. trichotoma, visando à redução no consumo de água. As variáveis fisiológicas da fluorescência da clorofila a demonstraram ser importantes para estudos do comportamento do crescimento de mudas em viveiros. Um crescimento adequado das mudas em viveiro foi obtido, com lâminas de irrigação de 4-8-8 mm dia-1, alternadas a cada 60 dias, sendo possível a utilização de uma menor lâmina no início do crescimento, dessa forma reduzindo o consumo de água. A utilização da lâmina de 4 mm dia-1 contínua na produção de mudas é possível, desde que associada com a adição da dose de 3 g L-1 de polímero hidroretentor ao substrato. No Capítulo II foi avaliado o crescimento inicial a campo de mudas de C. trichotoma, sob diferentes níveis de sombreamento, considerando variáveis morfológicas e fisiológicas. A parte aérea das plantas teve o crescimento influenciado negativamente na condição de pleno sol. A espécie responde com muita plasticidade às diferentes intensidades luminosas. Porém, os sombreamentos de 18 e 50% proporcionam maior crescimento e valores fisiológicos favoráveis ao desenvolvimento inicial da espécie. Identificar o efeito do excesso de umidade do solo na sobrevivência e no crescimento inicial de mudas de C. trichotoma, após plantio a campo, com base em características morfofisiológicas foi o objetivo do Capítulo III. O maior teor de microporos e de argila em camadas sucessivas do solo (0 a 20 cm) é indicativo da condição indesejável para a sobrevivência e crescimento inicial de mudas de C. trichotoma. A espécie é intolerante a ambientes com excesso de umidade no solo, não sendo recomendado o plantio em locais planos, com problemas de drenagem.