Role of extracellular matrix components and structure in new renal models in vitro

The extracellular matrix (ECM), a complex set of fibrillar proteins and proteoglycans, supports the renal parenchyma and provides biomechanical and biochemical cues critical for spatial-temporal patterning of cell development and acquisition of specialized functions. As in vitro models progress towards biomimicry, more attention is paid to reproducing ECM-mediated stimuli. ECM’s role in in vitro models of renal function and disease used to investigate kidney injury and regeneration is discussed. Availability, affordability, and lot-to-lot consistency are the main factors determining the selection of materials to recreate ECM in vitro. While simpler components can be synthesized in vitro, others must be isolated from animal or human tissues, either as single isolated components or as complex mixtures, such as Matrigel or decellularized formulations. Synthetic polymeric materials with dynamic and instructive capacities are also being explored for cell mechanical support to overcome the issues with natural products. ECM components can be used as simple 2D coatings or complex 3D scaffolds combining natural and synthetic materials. The goal is to recreate the biochemical signals provided by glycosaminoglycans and other signaling molecules, together with the stiffness, elasticity, segmentation, and dimensionality of the original kidney tissue, to support the specialized functions of glomerular, tubular, and vascular compartments. ECM mimicking also plays a central role in recent developments aiming to reproduce renal tissue in vitro or even in therapeutical strategies to regenerate renal function. Bioprinting of renal tubules, recellularization of kidney ECM scaffolds, and development of kidney organoids are examples. Future solutions will probably combine these technologies

Role of extracellular matrix components and structure in new renal models in vitro

The extracellular matrix (ECM), a complex set of fibrillar proteins and proteoglycans, supports the renal parenchyma and provides biomechanical and biochemical cues critical for spatial-temporal patterning of cell development and acquisition of specialized functions. As in vitro models progress towards biomimicry, more attention is paid to reproducing ECM-mediated stimuli. ECM’s role in in vitro models of renal function and disease used to investigate kidney injury and regeneration is discussed. Availability, affordability, and lot-to-lot consistency are the main factors determining the selection of materials to recreate ECM in vitro. While simpler components can be synthesized in vitro, others must be isolated from animal or human tissues, either as single isolated components or as complex mixtures, such as Matrigel or decellularized formulations. Synthetic polymeric materials with dynamic and instructive capacities are also being explored for cell mechanical support to overcome the issues with natural products. ECM components can be used as simple 2D coatings or complex 3D scaffolds combining natural and synthetic materials. The goal is to recreate the biochemical signals provided by glycosaminoglycans and other signaling molecules, together with the stiffness, elasticity, segmentation, and dimensionality of the original kidney tissue, to support the specialized functions of glomerular, tubular, and vascular compartments. ECM mimicking also plays a central role in recent developments aiming to reproduce renal tissue in vitro or even in therapeutical strategies to regenerate renal function. Bioprinting of renal tubules, recellularization of kidney ECM scaffolds, and development of kidney organoids are examples. Future solutions will probably combine these technologies

Recombinant carbohydrate-binding modules for biomedical applications Biocompatibility of polysaccharide-based materials

Tese de Doutoramento em Engenharia Química e Biológica - Ramo do Conhecimento Tecnologia MicrobianaThe development of biomaterials for medical applications envisages the design of three-dimensional structures – the scaffolds. These structures, mimicking the biological structures and interacting with the surrounding tissues through biomolecular recognition, elicit cellular responses mediated by specific interactions. Among the different scaffolds used in biomedicine, the materials based on polysaccharides present promising characteristics, due to their biocompatibility, hydrophilicity, degradability and appropriate mechanical properties, allowing for a favorable controlled interaction with living systems. Recombinant proteins are widely used in the biomedical field, namely in the fuctionalization of biomaterials. It is well established that Carbohydrate-Binding Modules (CBMs) present in several glycanases are structural and functionally independent of the catalytic module; therefore, their application as fusion partners may be exploited, contributing to protein expression, solubilization, purification, and finally for the functionalization of polysaccharide-based materials. This is the main subject of this thesis: the evaluation of the potential of CBMs as tools for the improvement of the biocompatibility of polysaccharides. One of the molecules often used to improve cells adhesion is the peptide Arg-Gly-Asp (RGD). The RGD sequence, present in several proteins of 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. Several polysaccharide-based materials have been produced recently at the DEB-UM laboratories, namely dextrin based hydrogels and bacterial cellulose scaffolds. In this study, recombinant proteins containing a CBM with starch affinity were fused to the bioactive molecule RGD, using recombinant DNA technology, in order to functionalize dextrin-based hydrogels. The general introduction of this thesis is presented in chapter 1 and includes a bibliographic revision of: 1) the applications of polysaccharides as biomedical biomaterials (this revision is restricted to the dextrin and bacterial cellulose (BC) based materials, the ones that were used in this work); 2) the strategies available for the production of recombinant proteins, using bacterial systems; and 3) a state of the art on the CBMs and their applications. The chapter 2 describes the development of a methodology for the expression and purification of the recombinant protein CBM-RGD, which has a CBM from the human protein laforin fused to a RGD sequence. Different commercial heterologous Escherichia coli expression systems (pET 29a, pET 25b and pGEXT41) were used in order to obtain high levels of soluble protein. Despite the use of the periplasmatic secretion approach (pET25) or the fusion of CBM with enhancing solubility tag (GST), the recombinant proteins were always obtained in the insoluble fraction. The utilization of CHAPS and arginine allowed the protein solubilization and purification, but not the production of functional protein with starch binding ability. Using the pET29a vector, the recombinant proteins were obtained in inclusion bodies (IB). After solubilization and refolding, the CBM was recovered and showed starch affinity. This is the first report on the expression of the functional CBM from the human protein laforin. The chapter 3 describes the production of recombinant proteins containing a bacterial CBM, which belongs to an α- amylase from Bacillus sp. TS-23. This protein, like the laforin CBM, also has starch affinity, being designated a Starch- Binding Module (SBM). The recombinant SBM and RGD-SBM proteins were cloned, expressed, purified and tested in vitro. 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. 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. The fusion protein RGD-SBM provides an efficient way to functionalize the dextrin-based hydrogel, improving the interaction with cells. The characterization of dextrin-vinyl acrylate (dextrin-VA) and dextrin-hydroxyethylmethacrylate (dextrin-HEMA) hydrogels was presented in a previous study carried out at the DEB-UM laboratories. In this work (chapter 4) the in vivo biocompatibility and degradability of these hydrogels are reported. The histological analysis of subcutaneous implants of these hydrogels, featuring inflammatory and resorption events in mice, was carried out over a period of 16 weeks. While dextrin-HEMA hydrogel was quickly and completely degraded and reabsorbed, dextrin-VA degradation occurred slowly, apparently through an erosion controlled process. A thin fibrous capsule was observed 16 weeks post-implantation, surrounding the non-degradable hydrogel. In the case of the degradable material, only a mild inflammatory reaction was observed, with few foamy macrophages being detected around the implant. This reaction was followed by complete resorption, with no signs of capsule formation or fibrosis associated with the implants. Altogether, these results strongly suggest that the dextrin hydrogels are fully biocompatible, since no toxicity on the tissues surrounding the implants was found. Moreover, it may be speculated that a controlled degradation rate of the hydrogels may be obtained, using dextrin with grafted HEMA and VA in different proportions. Chapter 5 presents the evaluation of Bacterial Cellulose – NanoFibers (BC-NFs) nanotoxicology. BC is a promising material for biomedical applications, namely due its biocompatibility. Although BC has been shown to be neither cytotoxic nor genotoxic, the properties of isolated BC-NFs on cells and tissues has never been analysed. Considering the toxicity associated to other fibre-shaped nanoparticles, it seems crucial to evaluate the toxicity associated to the BC-NFs. The results from single cell gel electrophoresis (also known as comet assay) and the Salmonella reversion assay showed that NFs, produced from BC by a combination of acid and ultrasonic treatment, are not genotoxic under the conditions tested. A proliferation assay using fibroblasts and CHO cells reveals a slight reduction in the proliferation rate, although no modification in the cell morphology is observed. Overall, this work reports the successful expression and isolation of the atypical human CBM, from the protein laforin. It provides a contribution to the development of a strategy based on the use of CBMs as tools for the modification of the surface properties of biomaterials, improving the interaction with cells. Finally, this work characterizes biocompatibility aspects of biomaterials currently under development at DEB-UM laboratories.O desenvolvimento de biomateriais para aplicações biomédicas centra-se no desenho de estruturas tri-dimensionais – scaffolds – capazes de mimetizar as funções biológicas e interagir com os tecidos envolventes, através do reconhecimento biomolecular. Entre os diferentes materiais usados para produzir scaffolds, os constituídos por polissacarídeos (como é o caso dos hidrogeis de dextrino e os materiais de celulose bacteriana - BC) apresentam características promissoras devido à sua biocompatibilidade, hidrofilicidade, degradabilidade e propriedades mecânicas, permitindo a sua utilização biomédica. As proteínas recombinantes são amplamente usadas em biomedicina, nomeadamente na funcionalização de diversos biomateriais. Sabe-se que os módulos de ligação a carbohidratos (CBMs), presentes em várias glicanases, são estrutural e funcionalmente independentes do domínio catalítico. Assim, a sua utilização em proteínas de fusão tem sido explorada, com o propósito de facilitar ou aumentar a expressão, solubilidade e purificação das proteínas. Uma das moléculas frequentemente usada para melhorar a adesão celular é o péptido Arg-Gly-Asp (RGD). Esta sequência, presente em diversas proteínas da matriz extra-celular, é um ligando para adesão celular mediada por integrinas, sendo reconhecido como o principal grupo funcional na adesão celular. Nos últimos anos, foram produzidos nos laboratórios do DEB-UM diversos materiais à base de polissacarídeos, nomeadamente hidrogeis de dextrino. Neste trabalho, usando tecnologia de DNA recombinante, foram produzidas proteínas bi-funcionais constituídas por um CBM (com afinidade para o amido) fundido com a molécula bio-activa RGD, com o propósito de os funcionalizar. Pretende-se assim melhorar a interacção do material com as células, favorecendo a adesão celular pela interacção com a molécula RGD que por sua vez está ligado ao material através do CBM. Na Introdução geral desta tese (capítulo 1) apresenta-se: 1) uma revisão sobre biomateriais baseados em polissacarídeos (em particular dos hidrogels de dextrino e das nanofibras (NFs) de celulose bacteriana); 2) as estratégias usadas para produzir as proteínas recombinantes em sistemas de expressão bacterianos; 3) e uma revisão sobre os CBMs e as suas aplicações. O segundo capítulo descreve a metodologia desenvolvida para a expressão e purificação da proteína de fusão CBMRGD, pertencendo este CBM à proteína humana laforina. Foram utilizados diferentes sistemas comerciais para expressão heteróloga em Escherichia coli (pET 29a, pET 25b e pGEXT41), com o intuito de obter elevados níveis de proteína solúvel. Os sistemas de expressão que permitem a secreção das proteínas para o espaço periplasmático (pET25) ou a fusão com a GST (pGEXT4 1), um tag que potencia a solubilidade das proteínas, conduziram à obtenção de proteínas insolúveis. A adição de CHAPS e arginina ao tampão de lise, embora resultando num aumento da solubilidade, não permitiu a obtenção de proteína funcional, isto é, com afinidade para o amido. Usando o vector pET29a, a proteína foi obtida em corpos de inclusão que, depois de solubilizados e submetidos ao processo de refolding, permitiram obter proteína funcional com afinidade para o amido. Este é o primeiro relato da expressão funcional deste CBM humano. No capítulo 3 descreve-se a produção de proteínas de fusão contendo um CBM bacteriano, da α-amilase do Bacillus sp. TS-23. Este CBM também apresenta afinidade para o amido, sendo por isso designado por SBM (Starch-binding module). As proteínas recombinantes SBM e RGD-SBM foram produzidas usando um sistema de expressão de E. coli. O seu efeito na adesão, spreading e proliferação celular foi avaliado in vitro, usando fibroblastos de embrião de rato 3T3. Os resultados mostraram que o tratamento do hidrogel de dextrino com RGD-SBM melhorou a adesão celular em mais de 30%. Para além disso, só na presença da proteína foi possível observar as células alongadas na sua superfície. Assim, a proteína de fusão revelou-se eficiente para funcionalizar o hidrogéis de dextrino. A caracterização dos hidrogéis de dextrino-vinil acrilato (dextrino-VA) e dextrino-hidroxietilmetacrilato (dextrino-HEMA) foi objecto de estudo em trabalhos anteriores, também desenvolvidos no DEB-UM. Neste trabalho (capítulo 4) apresentam-se os resultados da caracterização de biocompatibilidade e degradação destes hidrogéis in vivo. A análise histológica de implantes subcutâneos em ratinhos permitiu estudar os eventos de reabsorção e a resposta inflamatória. De acordo com os resultados, a degradação e reabsorção dos géis de dextrino-HEMA ocorre rapidamente; a degradação dos géis de dextrino-VA é mais lenta, devendo-se principalmente a processos de erosão. Após 16 semanas, foi observada uma fina cápsula fibrosa a rodear o implante não degradável. No caso do gel degradável, observou-se uma resposta inflamatória de baixa intensidade, sendo detectados alguns macrófagos com material fagocitado a envolver o implante. Esta reacção foi seguida pela completa reabsorção do material, não havendo sinais de formação de qualquer cápsula fibrosa. Estes resultados sugerem que os hidrogéis de dextrino são biocompatíveis, uma vez que não foram detectados sinais de toxicidade nos tecidos que envolviam o material. Os resultados sugerem também que é possível obter hidrogéis com velocidades de degradação controlada, usando dextrino substituído com HEMA e VA em diferentes proporções. O capítulo 5 apresenta o estudo da nanotoxicidade de NFs de celulose bacteriana. A BC apresenta grandes potencialidades para aplicações biomédicas, sendo descrita como um material não citotóxico ou genotóxico. No entanto, o efeito das NFs, isoladas por tratamento ácido e ultrasons, nas células e nos tecidos não foi descrito. Considerando a toxicidade associada a outros nanomateriais com forma de agulha, o estudo da nanotoxicidade destas fibras torna-se crucial. Os resultados obtidos no ensaio cometa e de reversão da Salmonella mostraram que as NFs produzidas a partir da BC, não são genotóxicas na condições utilizadas. Para além disso, os resultados obtidos nos ensaios de proliferação celular usando fibroblastos e células CHO mostraram que, apesar de uma ligeira redução na proliferação, não são detectadas diferenças morfológicas. Em resumo, este trabalho descreve, pela primeira vez, a expressão funcional do CBM atípico da proteína humana laforina. Este trabalho também contribui para o desenvolvimento de ferramentas que utilizam os CBMs recombinantes para a modificação das propriedades da superfície de materiais. Por último, são caracterizados aspectos da biocompatibilidade de materiais que estão a ser desenvolvidos nos laboratórios do DEB-UM.Fundação para a Ciência e a Tecnologia (FCT

Innovative Strategies in Tissue Engineering

In spite of intensive investments and investigations carried out in the last decade, many aspects of the stem cell physiology, technology and regulation remain to be fully defined. After the enthusiasm that characterized the first decade of the discovery that when given the right cue, stem cells could repair all the different tissues in the body; it is now time to start a serious and coordinated action to define how to govern the stem cell potential and to exploit it for clinical applications. This can be achieved only with shared research programs involving investigators from all over the world and making the results available to all.The Disputationes Workshop series (http://disputationes.info) is an international initiative aimed at disseminating stem cell related cutting edge knowledge among scientists, healthcare workers, students and policy makers. The present book gathers together some of the ideas discussed during the third and fourth Disputationes Workshops held in Florence (Italy) and Aalborg (Denmark), respectively. The aim of this book is to preserve those ideas in order to contribute to the general discussion on organ repair and to bolster a fundamental scientific and technological leap forwards the treatment of otherwise incurable diseases

Innovative Strategies in Tissue Engineering

In spite of intensive investments and investigations carried out in the last decade, many aspects of the stem cell physiology, technology and regulation remain to be fully defined. After the enthusiasm that characterized the first decade of the discovery that when given the right cue, stem cells could repair all the different tissues in the body; it is now time to start a serious and coordinated action to define how to govern the stem cell potential and to exploit it for clinical applications. This can be achieved only with shared research programs involving investigators from all over the world and making the results available to all.The Disputationes Workshop series (http://disputationes.info) is an international initiative aimed at disseminating stem cell related cutting edge knowledge among scientists, healthcare workers, students and policy makers. The present book gathers together some of the ideas discussed during the third and fourth Disputationes Workshops held in Florence (Italy) and Aalborg (Denmark), respectively. The aim of this book is to preserve those ideas in order to contribute to the general discussion on organ repair and to bolster a fundamental scientific and technological leap forwards the treatment of otherwise incurable diseases

Electrospun Nanofibers for Biomedical Applications

Electrospinning is a versatile and effective technique widely used to manufacture nanofibrous structures from a diversity of materials (synthetic, natural or inorganic). The electrospun nanofibrous meshes’ composition, morphology, porosity, and surface functionality support the development of advanced solutions for many biomedical applications. The Special Issue on “Electrospun Nanofibers for Biomedical Applications” assembles a set of original and highly-innovative contributions showcasing advanced devices and therapies based on or involving electrospun meshes. It comprises 13 original research papers covering topics that span from biomaterial scaffolds’ structure and functionalization, nanocomposites, antibacterial nanofibrous systems, wound dressings, monitoring devices, electrical stimulation, bone tissue engineering to first-in-human clinical trials. This publication also includes four review papers focused on drug delivery and tissue engineering applications