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 (<6 mm) estes materiais geralmente apresentam mau desempenho, devido a formação de hiperplasia íntima anastomótica e trombogenicidade superficial. A produção de vasos sanguíneos funcionais por meio de técnicas de engenharia de tecidos já é uma realidade, no entanto, devido aos elevados custos e longo tempo de produção, o desenvolvimento de novos materiais adequados para a substituição de vasos sanguíneos de pequeno diâmetro é ainda necessário. O objectivo geral desta tese foi o melhoramento da matriz de celulose bacteriana (CB) para o seu potencial uso como substituto de pequenos vasos sanguíneos. Entre as estratégias desenvolvidas ao longo dos anos destinadas à modificação de materiais usados como substitutos vasculares, o pré-revestimento com o tripeptídeo Arg-Gly-Asp (RGD) tem melhorado a endotelialização, reduzindo assim a trombogenicidade dos biomateriais. Neste trabalho, proteínas recombinantes bifuncionais, contendo um módulo de ligação à celulose (Cellulose – Binding Module – CBM) – do celulossoma da bactéria Clostridium thermocellum - e sequências conhecidas por promover a adesão de células (RGD, GRGDY) foram clonadas e expressas com sucesso na bactéria Escherichia coli, sendo posteriormente purificadas e usadas no revestimento de fibras de CB. Os CBMs adsorvem fortemente e especificamente à celulose, assim apresentam-se como uma ferramenta útil para direccionar de maneira simples e específica a sequência RGD (ou outros péptidos bioactivos) às superfícies de celulose. Nesta tese, os efeitos sobre a adesão celular/biocompatibilidade das proteínas quiméricas produzidas foram estudados usando culturas de fibroblastos de ratinhos (3T3) e células microvasculares humanas (HMEC). Os resultados obtidos demonstraram que as proteínas bifuncionais foram capazes de aumentar significativamente a adesão e o alongamento de fibroblastos e HMECs, além de promover uma distribuição uniforme das células sobre a matriz de celulose. A presença do RGD estimulou a formação antecipada de estruturas tipo-capilares em HMECs, porém diminuiu a invasão destas células na CB. A hemocompatibilidade da CB nativa e RGD-modificada também foi estudada. Os resultados mostraram que uma quantidade significativa de proteínas plasmáticas adsorvem às fibras da CB, sendo que a albumina apresentou uma maior afinidade pela CB do que a γ-globulina ou fibrinogênio e que estas mesmas proteínas quando adsorvidas à celulose parecem não sofrer grandes alterações conformacionais. A presença do RGD na superfície da CB nãoendotelializada aumentou a adesão de plaquetas, porém quando este material foi revestido com células endoteliais, a adesão de plaquetas foi fortemente inibida. Uma vez que a proteína recombinante contém um CBM bacteriano, a biocompatibilidade da CB nativa e a tratada com RGD-CBM - para analisar se a presença desta proteína é capaz de originar alguma reação imunológica - foi investigada através de estudos in vivo, em ovelhas. Os implantes foram avaliados quanto à reação inflamatória, invasão celular e angiogênese. Os resultados histológicos mostraram que a CB provoca uma resposta biológica tipicamente observada em implantes que apresentam uma grande relação superfície x volume. Uma semana após a implantação a presença de um infiltrado inflamatório sugeriu uma reação inflamatória aguda/subaguda que progrediu para uma inflamação crónica, porém limitada ao local do implante, e associada à proliferação de pequenos vasos sanguíneos. A presença de células gigantes foi observada em períodos tardios e uma cápsula fibrosa delgada estava presente ao redor do implante. Não houve diferença significativa no grau de inflamação entre a CB tratada com RGD-CBM e a nativa. Nesta tese, tubos de CB com pequenos diâmetros foram produzidos e suas propriedades mecânicas avaliadas. De modo geral, este trabalho relata a funcionalização bem sucedida de matrizes de celulose bacteriana através do uso de peptídeos de adesão ligados a um CBM, resultando em uma melhor hemocompatibilidade e assim, aumentando seu potencial como substitutos de vasos sanguíneos.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES – Brasil

Bacterial cellulose : production and applications

Bacterial cellulose (BC), excreted by Gluconacetobacter xylinus, is a unique nanofibrilar biopolymer with a wide range of applications in human and veterinary medicine, odonthology, pharmaceutical industry, biotechnological, food and paper industry. The major research activities of our research group include the following headlines: - Surface-modification of BC matrices and BC whiskers for the design of novel functional BC nanocomposite systems. This domain includes the surface-activation of BC with CBMs (Carbohydrate Binding Modules) conjugated with bioactive peptides for biomedical applications.[1] CEB-UM has already shown that the adsorption of CBM-RGD (the minimal essential cell adhesion recognition motifs) onto BC improves its ability to adsorb fibroblasts.[2] Also, research on the use of BC tubes as new guides for neuronal growth (CAPES, 3989/05-4) and, for the first time, on assessing the in vivo cytotoxicity of BC nanofibers (SFRH/BD/18418/2004), is on course. - Design of novel BC structures with tailored microporosity, for biomedical applications (SFRH/BD/48759/2008). - Engineering of electro-conductive and electro-active BC scaffolds with potential applications in neuronal growth. The embedded polymeric directionally of the BC nanofibers is expected to exhibit shear piezoelectricity which, coupled with a high in situ moldability, thrusts a promising future for novel BC-based materials such as lightweight, biodegradable electro-actives, biosensors and flexible electric displays, with a tailored oriented stiffness and strength. - Exploring the large-scale fermentation of BC. A novel bioreactor, based on a surfaceculture method was designed. A simple and low-cost piece of equipment is capable of direct nebulization of a high volume of dispersed and microparticulated subtrate over the growing bacteria. The developed system may reveal to be an interesting economic solution for the large-scale production of BC

Production of recombinant carbohydrate-binding modules fused to RGD : functional studies using bacterial cellulose

The attachment of cells to biomedical materials can be improved by using adhesion molecules, present in the extracellular matrix substances, such as fibronectin, vitronectin, or laminin. In many cases, Arg-Gly-Asp (RGD) was found to be the major functional amino acid sequence responsible for cellular adhesion. In the present study, a method for producing chimerics proteins, RGDCBM, with functions similar to fibronectin, which contains a cellulose-binding module (CBM), was developed. The CBM used was from the cellulosoma of the bacteria Clostridium thermocellum. The genes encoding these CBM-containing chimeric proteins were cloned, and the protein expressed and purified. Bacterial cellulose (BC) secreted by Gluconacetobacter xylinus was produced. Polystyrene surfaces and bacterial cellulose sheets where ‘‘coated’’ with these RGD-containing proteins, and then used in adhesion/biocompatibility tests, using a mouse embryo fibroblasts culture. The results showed that the proteins containing the RGD or GRGDY sequence were able to improve the adhesion of the fibroblast on the polystyrene plate, furthermore proteins containing the RGD sequence were more effective than the proteins containing the GRGDY sequence. Preliminary adhesion studies of fibroblast cultures on cellulose sheets, functionalized with the recombinant proteins, showed positive effects on the adhesion and proliferation of the cells. The results demonstrated that the proteins containing the RGD sequence were able to increase significantly the adhesion of fibroblast to BC surfaces when compared with the controls (cellulose treated with the CBM or buffer). The results also demonstrated that the protein containing one RGD sequence have a stronger effect than the protein containing two RGDs

Development of a strategy to functionalize a dextrin-based hydrogel for animal cell cultures using a starch-binding module fused to RGD sequence

Several approaches can be used to functionalize biomaterials, such as hydrogels, for biomedical applications. One of the molecules often used to improve cells adhesion is the peptide Arg-Gly-Asp (RGD). The RGD sequence, present in several proteins from the extra-cellular matrix (ECM), is a ligand for integrin-mediated cell adhesion; this sequence was recognized as a major functional group responsible for cellular adhesion. In this work a bi-functional recombinant protein, containing a starch binding module (SBM) and RGD sequence was used to functionalize a dextrin-based hydrogel. The SBM, which belongs to an α-amylase from Bacillus sp. TS-23, has starch (and dextrin, depolymerized starch) affinity, acting as a binding molecule to adsorb the RGD sequence to the hydrogel surface. Results The recombinant proteins SBM and RGD-SBM were cloned, expressed, purified and tested in in vitro assays. The evaluation of cell attachment, spreading and proliferation on the dextrin-based hydrogel surface activated with recombinant proteins were performed using mouse embryo fibroblasts 3T3. A polystyrene cell culture plate was used as control. The results showed that the RGD-SBM recombinant protein improved, by more than 30%, the adhesion of fibroblasts to dextrin-based hydrogel. In fact, cell spreading on the hydrogel surface was observed only in the presence of the RGD-SBM. Conclusion The fusion protein RGD-SBM provides an efficient way to functionalize the dextrin-based hydrogel. Many proteins in nature that hold a RGD sequence are not cell adhesive, probably due to the conformation/accessibility of the peptide. We therefore emphasise the successful expression of a bi-functional protein with potential for different applications.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Brazil)Fundação para a Ciência e a Tecnologia (FCT

Improving bacterial cellulose for blood vessel replacement: functionalization with a chimeric protein containing a cellulose-binding module and an adhesion peptide

Chimeric proteins containing a cellulose-binding module (CBM) and an adhesion peptide (RGD or GRGDY) were produced and used to improve the adhesion of human microvascular endothelial cells (HMEC) to bacterial cellulose (BC). The effect of these proteins on the HMEC–BC interaction was studied. The results obtained demonstrated that recombinant proteins containing adhesion sequences were able to significantly increase the attachment of HMEC to BC surfaces, especially the RGD sequence. The images obtained by scanning electron microscopy showed that the cells on the RGD-treated BC present a more elongated morphology 48 h after cell seeding. The results also showed that RGD decreased the in-growth of HMEC cells through the BC and stimulated the early formation of cord-like structures by these endothelial cells. Thus, the use of recombinant proteins containing a CBM domain, with high affinity and specificity for cellulose surfaces allows control of the interaction of this material with cells. CBM may be combined with virtually any biologically active protein for the modification of cellulose-based materials, for in vitro or in vivo applications.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Brazil)Fundação para a Ciência e a Tecnologia (FCT

Hemocompatibility study of bacterial cellulose

Introduction: Vascular grafts must gather various complex attributes, like good mechanical properties, post-implantation healing response without any immunological reaction and no induction of blood coagulation. Over the years, many strategies were developed to modify materials for vascular devices. One strategy involves pre-coating with the tripeptide Arg-Gly- Asp (RGD), which improves endothelialization, thus lowering thrombogenicity. In the present work, the hemocompatibility of native and RGD-modified bacterial cellulose (BC) was studied. Despite being a promising material for vascular replacements, a comprehensive characterization of the BC-blood interaction, namely in the presence of RGD peptide, has not been performed to date. Methods: Blood from healthy donors was placed in contact with native or recombinant RGD-treated BC and parameters related to a materials hemocompatibility were determined. These included adsorption of plasma proteins, clotting times, whole blood coagulation time, plasma recalcification profiles, platelet adhesion and hemolysis. Results: The clotting times (aPTT, PT, FT and PRT) and whole blood clotting results demonstrate the good hemocompatibility of BC. A significant amount of plasma protein adsorbed to BC fibres, presenting albumin a higher BC affinity than gamma-globulin or fibrinogen. According to analysis carried out by intrinsic tryptophan fluorescence, BC-adsorbed plasma proteins tested 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. Conclusion: Generally, our data demonstrates that both native and RGD-modified BCs may be classified as hemocompatible materials, since they showed to be nonhemolytic and the whole blood coagulation studies show that the results are comparable to those produced by currently available materials for blood replacements. Acknowledgements.FCT project PTDC/EBB-EBI/112170/2009