Development of structures based on bacterial cellulose for the production of vascular prostheses

Tese de doutoramento em Engenharia BiomédicaCardiovascular disease is the leading cause of mortality in Western countries. For the reconstruction of arteries with large caliber currently available synthetic grafts offer a reasonable solution and proven clinical efficacy. However, for small sized (<6 mm) grafts these materials generally give poor performance, due to anastomotic intimal hyperplasia and surface thrombogenicity. The production of functional blood vessels by tissue engineering techniques is already possible, however due to the associated costs and lengthy production, the development of new materials appropriated for small diameter blood vessel replacements is still required. This thesis is a contribute for the improvement of bacterial cellulose for small blood vessel replacements. Among the strategies developed over the years to modify materials for vascular devices, pre-coating with the tripeptide Arg-Gly-Asp (RGD) improves endothelialization thus lowering thrombogenicity. In this work, bifunctional recombinant proteins, with a Cellulose- Binding Module – CBM, from the cellulosome of Clostridium thermocellum - and cell binding sequences - RGD, GRGDY – were successfully cloned and expressed in the bacteria Escherichia coli. These RGD-containing cellulose-binding proteins were purified and used to coat bacterial cellulose fibres. Bacterial cellulose (BC) secreted by Gluconacetobacter xylinus is a material with unique properties and promising biomedical applications. CBMs adsorbs specifically and tightly on cellulose. Thus, they are a useful tool to address the fused RGD sequence (or other bioactive peptides) to the cellulose surface, in a specific and simple way. In this thesis the effects of chimeric proteins containing a CBM fused to adhesion peptides on the cell adhesion/biocompatibility properties were studied using mouse embryo fibroblasts (3T3) and human microvascular endothelial cells (HMEC) cultures. The results obtained demonstrated that the recombinant proteins containing adhesion sequences were able to significantly increase the attachment and spreading of fibroblasts and HMECs to BC surfaces, specially the RGD sequence. The results also showed that the RGD decreased the ingrowth of the HMEC cells through the BC and stimulated the early formation of cordlike structures by these endothelial cells. The blood compatibility of native and RGD-modified BC was also studied. The clotting times (aPTT, PT, FT and PRT) and whole blood clotting results demonstrate the hemocompatibility of BC. A significant amount of plasma protein adsorbed to BC fibres, albumin presenting a higher BC affinity than γ-globulin or fibrinogen. According to analysis carried out by intrinsic tryptophan fluorescence, the BC adsorbed albumin, fibrinogen and γ-globulin do not undergo major conformational modifications. Although the presence of the adhesion peptide on bare-BC surface increases the platelet adhesion, when the material was cultured with human microvascular endothelial cells a confluent cell layer was readily formed, inhibiting the adhesion of platelets. Once the recombinant protein contains a bacterial CBM, the biocompatibility of native and RGD-CBM treated BC – to analyze whether the presence of the recombinant protein gives rise to any immunologic reaction – was investigated through in vivo studies in sheep. The fate of long term subcutaneous BC implants - 32 weeks - was analysed. Histological results showed that BC trigger a biological response typically observed for high surface-to-volume implants. After 1 week of implantation the presence of an inflammatory infiltrate suggests an acute/subacute inflammatory reaction that advance to a chronic inflammation confined to the implantation site and associated to the proliferation of small blood vessels. The presence of giant cells was observed at latter periods (16 and 32 weeks) and a narrow fibrous capsule was present surrounding the implant. BC tubes with small diameter (3mm ID) were produced and its mechanical properties evaluated. Overall, this work reports the successful functionalization of bacterial cellulose scaffolds with a CBM fused to adhesion peptides, leading to improved blood compatibility and increasing its potential use as blood vessels replacement.As doenças cardiovasculares estão entre as principais causas de morte em países ocidentais. Para a reconstrução de artérias de grande calibre, os enxertos sintéticos actualmente disponíveis oferecem uma solução razoável e com eficácia clínica comprovada. No entanto, como enxertos de pequeno calibre (