Development of novel human cellular models for neurotoxicity studies

Dissertation to obtain master degree in Genética Molecular e Biomedicina

Dynamics of Capillaria-hepatica-induced hepatic septal fibrosis in rats

INTRODUCTION: The pathogenesis of septal hepatic fibrosis, induced in rats by Capillaria hepatica infection, was studied with the aid of a large collection of stored paraffin blocks, representative of the different evolutive phases of fibrosis which appeared in 100% of infected rats. METHODS: Studies were conducted involving histology, immunohistochemistry, immunofluorescence and morphometric methods, in order to observe the dynamic behavior of the cellular and matrix components of fibrosis, over a one year period of evolution. RESULTS: Observation verified that septal fibrosis originates from several portal spaces simultaneously. Its origin and progression involve blood vessel proliferation (angiogenesis), multiplication of actin-positive cells (pericytes and myofibroblasts) and progressive collagen deposition. By the end of 4-5 months, a progressive decrease in all these components was observed, when signs of regression of septal fibrosis became more evident over time. CONCLUSIONS: Besides indicating the fundamental role played by angiogenesis in the pathogenesis of fibrosis, these morphological data concerning the dynamics of this C. hepatica experimental model proved to be adequate for future investigations regarding the functional aspects of fibrosis induction, progression and regression.

Age related changes in the elastic fiber system of the interfoveolar ligament

In order to evaluate age related changes of the elastic fiber system in the interfoveolar ligament, we studied the deep inguinal ring from 33 male cadavers aged from stillborn to 76 years. Selective and alternated staining methods for elastic fibers were performed to differentiate oxytalan, elaunin, and mature elastic fibers. We confirmed quantitative changes of the elastic fiber system with aging. There was a significant and progressive reduction of the oxytalan fibers (responsible for tissue resistance) and a significant increment in the mature elastic and elaunin fibers (responsible for tissue elasticity). Furthermore, there were structural changes in the thickness, shortness and curling of these mature elastic fibers. These changes induced loss of the elastic fiber function and loss of the interfoveolar ligament compliance. These factors predispose individuals to the development of indirect inguinal hernias that frequently emerge in adults and aged individuals, especially above the fifth decade.

Quantitative analysis of collagen and elastic fibers in the transversalis fascia in direct and indirect inguinal hernia

PURPOSE: Our previous studies demonstrated structural and quantitative age-related changes of the elastic fibers in transversalis fascia, which may play a role in inguinal hernia formation. To verify whether there were differences in the extracellular matrix between direct and indirect inguinal hernia, we studied the amount of collagen and elastic fibers in the transversalis fascia of 36 male patients with indirect inguinal hernia and 21 with direct inguinal hernia. MATERIAL AND METHODS: Transversalis fascia fragments were obtained during surgical intervention and underwent histological quantitative analysis of collagen by colorimetry and analysis of elastic fibers by histomorphometry. RESULTS: We demonstrated significantly lower amounts of collagen and higher amounts of elastic fibers in transversalis fascia from patients with direct inguinal hernia compared to indirect inguinal hernia patients. The transversalis fascia from direct inguinal hernia patients showed structural changes of the mature and elaunin elastic fibers, which are responsible for elasticity, and lower density of oxytalan elastic fibers, which are responsible for resistance. These changes promoted loss of resiliency of the transversalis fascia. CONCLUSION: These results improve our understanding of the participation of the extracellular matrix in the genesis of direct inguinal hernia, suggesting a relationship with genetic defects of the elastic fiber and collagen synthesis.

Analysis of outcome and cancer stem cells status in patients with resectable pancreatic adenocarcinoma

RESUMO: Actualmente, a única possibilidade de cura para doentes com adenocarcinoma do pâncreas (PDAC) é a ressecção cirúrgica, no início deste estudo, perguntamo-nos se os predictores clínico-patológicos clássicos de prognostico poderiam ser validados em uma grande cohort de doentes com cancro do pâncreas ressecável e se outros predictores clínicos poderiam ter um papel na decisão de que doentes beneficiariam de ressecção cirúrgica. No capítulo 2, observamos que até 30% dos doentes morrem no primeiro ano após a ressecção cirúrgica, pelo que o nosso objectivo foi determinar factores pré-operatórios que se correlacionam com mortalidade precoce após ressecação cirúrgica com recurso a um instrumento estatisticamente validado, o Charlson-Age Comorbidity Index (CACI), determinamos que um CACI score superior a 4 foi preditivo de internamentos prolongados (p <0,001), complicações pós-operatórias (p = 0,042), e mortalidade em 1 ano pós- ressecção cirúrgica (p <0,001). Um CACI superior a 6 triplicou a mortalidade no primeiro ano pós-cirurgia e estes doentes têm menos de 50% de probabilidade de estarem vivos um ano após a cirurgia. No capítulo 3, o nosso objectivo foi identificar uma proteína de superfície que se correlacionasse estatisticamente com o prognostico de doentes com adenocarcinoma do pâncreas e permitisse a distinção de subgrupos de doentes de acordo com as suas diferenças moleculares, perguntamo-nos ainda se essa proteína poderia ser um marcador de células-estaminais. No nosso trabalho anterior observamos que as células tumorais na circulação sanguínea apresentavam genes com características bifenotípica epitelial e mesenquimal, enriquecimento para genes de células estaminais (ALDH1A1 / ALDH1A2 e KLF4), e uma super-expressão de genes da matriz extracelular (colagénios, SPARC, e DCN) normalmente identificados no estroma de PDAC. Após a avaliação dos tumores primários com RNA-ISH, muitos dos genes identificados, foram encontrados co-localizando em uma sub-população de células na região basal dos ductos pancreáticos malignos. Além disso, observamos que estas células expressam o marcador SV2A neuroendócrino, e o marcador de células estaminais ALDH1A1/2. Em comparação com tumores negativos para SV2, os doentes com tumores SV2 positivos apresentaram níveis mais baixos de CA 19-9 (69% vs. 52%, p = 0,012), tumores maiores (> 4 cm, 23% vs. 10%, p = 0,0430), menor invasão de gânglios linfáticos (69% vs. 86%, p = 0,005) e tumores mais diferenciados (69% vs. 57%, p = 0,047). A presença de SV2A foi associada com uma sobrevida livre de doença mais longa (HR: 0,49 p = 0,009) bem como melhor sobrevida global (HR: 0,54 p = 0,018). Em conjunto, esta informação aponta para dois subtipos diferentes de adenocarcinoma do pâncreas, e estes subtipos co-relacionam estatisticamente com o prognostico de doentes, sendo este subgrupo definido pela presença do clone celular SV2A / ALDH1A1/2 positivo com características neuroendócrinas. No Capítulo 4, a expressão de SV2A no cancro do pâncreas foi validado em linhas celulares primárias. Demonstramos a heterogeneidade do adenocarcinoma do pâncreas de acordo com características clonais neuroendócrinas. Ao comparar as linhas celulares expressando SV2 com linhas celulares negativas, verificamos que as linhas celulares SV2+ eram mais diferenciadas, diferindo de linhas celulares SV2 negativas no que respeita a mutação KRAS, proliferação e a resposta à quimioterapia. No capítulo 5, perguntamo-nos se o clone celular SV2 positivo poderia explicar a resistência a quimioterapia observada em doentes. Observamos um aumento absoluto de clones celulares expressando SV2A, em múltiplas linhas de evidência - doentes, linhas de células primárias e xenotransplantes. Embora, tenhamos sido capazes de demonstrar que o adenocarcinoma do pâncreas é uma doença heterogénea, consideramos que a caracterização genética destes clones celulares expressando SV2A é de elevada importância. Pretendemos colmatar esta limitação com as seguintes estratégias: Após o tratamento com quimioterapia neoadjuvante na nossa coorte, realizamos microdissecação a laser das amostras primarias em parafina, de forma a analisar mutações genéticas observadas no adenocarcinoma pancreático; em segundo lugar, pretendemos determinar consequências de knockdown da expressão de SV2A em nossas linhas celulares seguindo-se o tratamento com gemicitabina para determinação do papel funcional de SV2A; finalmente, uma vez que os nossos esforços anteriores com um promotor - repórter e SmartFlare ™ falharam, o próximo passo será realizar RNA-ISH PrimeFlow™ seguido de FACS e RNA-seq para caracterização deste clone celular. Em conjunto, conseguimos provar com várias linhas de evidência, que o adenocarcinoma pancreático é uma doença heterogénea, definido por um clone de células que expressam SV2A, com características neuroendócrinas. A presença deste clone no tecido de doentes correlaciona-se estatisticamente com o prognostico da doença, incluindo sobrevida livre de doença e sobrevida global. Juntamente com padrões de proliferação e co-expressão de ALDH1A1/2, este clone parece apresentar um comportamento de células estaminais e está associado a resistência a quimioterapia, uma vez que a sua expressão aumenta após agressão química, quer em doentes, quer em linhas de células primárias.----------------------------- ABSTRACT: Currently, the only chance of cure for patients with pancreatic adenocarcinoma is surgical resection, at the beginning of my thesis studies, we asked if the classical clinicopathologic predictors of outcome could be validated in a large cohort of patients with early stage pancreatic cancer and if other clinical predictors could have a role on deciding which patients would benefit from surgery. In chapter 2, we found that up to 30% of patients die within the first year after curative intent surgery for pancreatic adenocarcinoma. We aimed at determining pre-operative factors that would correlate with early mortality following resection for pancreatic cancer using a statistically validated tool, the Charlson-Age Comorbidity Index (CACI). We found that a CACI score greater than 4 was predictive of increased length of stay (p<0.001), post-operative complications (p=0.042), and mortality within 1-year of pancreatic resection (p<0.001). A CACI score of 6 or greater increased 3-fold the odds of death within the first year. Patients with a high CACI score have less than 50% likelihood of being alive 1 year after surgery. In chapter 3 we aimed at identifying a surface protein that correlates with patient’s outcome and distinguishes sub-groups of patients according to their molecular differences and if this protein could be a cancer stem cell marker. The most abundant class of circulating tumor cells identified in our previous work was found to have biphenotypic features of epithelial to mesenchymal transition, enrichment for stem-cell associated genes (ALDH1A1/ALDH1A2 and KLF4), and an overexpression of extracellular matrix genes (Collagens, SPARC, and DCN) normally found in the stromal microenvironment of PDAC primary tumors. Upon evaluation of matched primary tumors with RNA-ISH, many of the genes identified were found to co-localize in a sub-population of cells at the basal region of malignant pancreatic ducts. In addition, these cells expressed the neuroendocrine marker SV2A, and the stem cell marker ALDH1A1/2. Compared to SV2 negative tumors, patients with SV2 positive tumors were more likely to present with lower CA 19-9 (69% vs. 52%, p = 0.012), bigger tumors (size > 4 cm, 23% vs. 10%, p= 0.0430), less nodal involvement (69% vs. 86%, p = 0.005) and lower histologic grade (69% vs. 57%, p = 0.047). The presence of SV2A expressing cells was associated with an improved disease free survival (HR: 0.49 p=0.009) and overall survival (HR: 0.54 p=0.018) and correlated linearly with ALDH1A2. Together, this information points to two different sub-types of pancreatic adenocarcinoma, and these sub-types correlated with patients’ outcome and were defined by the presence of a SV2A/ ALDH1A1/2 expressing clone with neuroendocrine features. In Chapter 4, SV2A expression in cancer was validated in primary cell lines. We were able to demonstrate pancreatic adenocarcinoma heterogeneity according to neuroendocrine clonal features. When comparing SV2 expressing cell lines with SV2 negative cell lines, we found that SV2+ cell lines were more differentiated and differ from SV2 negative cell lines regarding KRAS mutation, proliferation and response to chemotherapy. In Chapter 5 we aimed at determining if this SV2 positive clone could explain chemoresistance observed in patients. We found an absolute increase in SV2A expressing cells, with multiple lines of evidence, in patients, primary cell lines and xenografts. Although, we have been able to show evidence that pancreatic adenocarcinoma is a heterogeneous disease, our findings warrant further investigation. To further characterize SV2A expressing clones after treatment with neoadjuvant chemotherapy in our cohort, we have performed laser capture microdissection of the paraffin embedded tissue in this study and will analyze the tissue for known genetic mutations in pancreatic adenocarcinoma; secondly, we want to know what will happen after knocking down SV2A expression in our cell lines followed by treatment with gemcitabine to determine if SV2A is functionally important; finally, since our previous efforts with a promoter – reporter and SmartFlare™ have failed, we will utilize a novel PrimeFlow™ RNA-ISH assay followed by FACS and RNA sequencing to further characterize this cellular clone. Overall our data proves, with multiple lines of evidence, that pancreatic adenocarcinoma is a heterogeneous disease, defined by a clone of SV2A expressing cells, with neuroendocrine features. The presence of this clone in patients’ tissue correlates with patient’s disease free survival and overall survival. Together with patterns of proliferation and ALDH1A1/2 co-expression, this clone seems to present a stem-cell-like behavior and is associated with chemoresistance, since it increases after chemotherapy, both in patients and primary cell lines.

Modulation of the secretome of hBMSCs by tailoring the macromolecular gradient in hydrogels to generate tissue-to-tissue interfaces

Tissue-to-tissue interfaces are commonly present in all tissues exhibiting structural, biological and chemical gradients serving a wide range of physiological functions. These interfaces are responsible for mediation of load transfer between two adjacent tissues. They are also important structures in sustaining the cellular communications to retain tissueâ s functional integration and homeostasis. [1] All cells have the capacity to sense and respond to physical and chemical stimulus and when cultured in three-dimensional (3D) environments they tend to perform their function better than in two-dimensional (2D) environments. Spatial and temporal 3D gradient hydrogels better resemble the natural environment of cells in mimicking their extracellular matrix. [2] In this study we hypothesize that differential functional properties can be engineered by modulation of macromolecule gradients in a cell seeded threedimensional hydrogel system. Specifically, differential paracrine secretory profiles can be engineered using human Bone Marrow Stem Cells (hBMSCâ s). Hence, the specific objectives of this study are to: assemble the macromolecular gradient hydrogels to evaluate the suitablity for hBMSCâ s encapsulation by cellular viability and biofunctionality by assessing the paracrine secretion of hBMSCâ s over time. The gradient hydrogels solutions were prepared by blend of macromolecules in one solution such as hyaluronic (HA) acid and collagen (Col) at different ratios. The gradient hydrogels were fabricated into cylindrical silicon moulds with higher ratio solutions assembled at the bottom of the mould and adding the two solutions consecutively on top of each other. The labelling of the macromolecules was performed to confirm the gradient through fluorescence microscopy. Additionally, AFM was conducted to assess the gradient hydrogels stiffness. Gradient hydrogels characterization was performed by HA and Col degradation assay, degree of crosslinking and stability. hBMSCâ s at P3 were encapsulated into each batch solution at 106 cells/ml solution and gradient hydrogels were produced as previously described. The hBMSCâ s were observed under confocal microscopy to assess viability by Live/Dead® staining. Cellular behaviour concerning proliferation and matrix deposition was also performed. Secretory cytokine measurement for pro-inflammatory and angiogenesis factors was carried out using ELISA. At genomic level, qPCR was carried out. The 3D gradient hydrogels platform made of different macromolecules showed to be a suitable environment for hBMSCâ s. The hBMSCâ s gradient hydrogels supported high cell survival and exhibited biofunctionality. Besides, the 3D gradient hydrogels demonstrated differentially secretion of pro-inflammatory and angiogenic factors by the encapsulated hBMSCâ s. References: 1. Mikos, AG. et al., Engineering complex tissues. Tissue Engineering 12,3307, 2006 2. Phillips, JE. et al., Proc Natl Acad Sci USA, 26:12170-5, 2008

Organic-inorganic nanostructured multilayers for calcium phosphate biomineralization

The weak fixation of biomaterials within the bone structure is one of the major reasons of implants failures. Calcium phosphate (CaP) coatings are used in bone tissue engineering to improve implant osseointegration by enhancing cellular adhesion, proliferation and differentiation, leading to a tight and stable junction between implant and host bone. It has also been observed that materials compatible with bone tissue either have a CaP coating or develop such a calcified surface upon implantation. Thus, the development of bioactive coatings becomes essential for further improvement of integration with the surrounding tissue. However, most of current applied CaP coatings methods (e.g. physical vapor deposition), cannot be applied to complex shapes and porous implants, provide poor structural control over the coating and prevent incorporation of bioactive organic compounds (e.g. antibiotics, growth factors) because of the used harsh processing conditions. Layer-by-layer (LbL) is a versatile technology that permits the building-up of multilayered polyelectrolyte films in mild conditions based on the alternate adsorption of cationic and anionic elements that can integrate bioactive compounds. As it is recognized in natureâ s biomineralization process the presence of an organic template to induce mineral deposition, this work investigate a ion based biomimetic method where all the process is based on LbL methodology made of weak natural-origin polyelectrolytes. A nanostructured multilayer component, with 5 or 10 bilayers, was produced initially using chitosan and chondroitin sulphate polyelectrolyte biopolymers, which possess similarities with the extracellular matrix and good biocompatibility. The multilayers are then rinsed with a sequential passing of solutions containing Ca2+ and PO43- ions. The formation of CaP over the polyelectrolyte multilayers was confirmed by QCM-D, SEM and EDX. The outcomes show that 10 polyelectrolyte bilayer condition behaved as a  better site for initiating the formation of CaP as the precipitation occur at earlier stages than in 5 polyelectrolyte bilayers one. This denotes that higher number of bilayers could hold the CaP crystals more efficiently. This work achieved uniform coatings that can be applied to any surface with access to the liquid media in a low-temperature method, which potentiates the manufacture of effective bioactive biomaterials with great potential in orthopedic applications.

Cryopreservation of Cell Sheets of Adipose Stem Cells: Limitations and Successes

Cell Sheets of hASCs (hASCs-CS) have been previously proposed for wound healing applications(1, 2) and despite the concern for production time reduction, the possibility of having these hASCs-CS off-the-shelf is appealing. The goal of this work was to define a cryopreservation methodology allowing to preserve cells viability and the properties CS matrix. hASCs-CS obtained from three different donors were created in UP-cell thermoresponsive dishes(Nunc, Germany) as previously reported(1,2). Different cryopreservation conditions were considered: i)FBS plus DMSO(5% and10%); ii)0.4M of Trehalose plus DMSO (5% and 10%); iii)cryosolution PLL (Akron Biotech, USA); and iv)vitrification. The cryopreservation effect was first assessed for cellular viability by flow cytometry using 7-AAD, and after dissociating the hASCs-CS with collagenase and trypsin-EDTA 0.25%. The expression (RT-PCR) and deposition (western blot and immunocytochemistry) of collagen type I, laminin and fibronectin, and the organization (TEM) of the extracellular matrix was further assessed before and after hASCs-CS cryopreservation to determine a potential effect of the method over matrix composition and integrity. The obtained results confirmed that cell viability is affected by the cryopreservation methodology, as shown before for different CS(3). Interestingly, the matrix properties were not significantly altered and the typical cell sheetâ s easiness of manipulation for transplantation was not lost.

Skin-derived cell-sheets as powerful tools to engineer skin analogues

Cell/cell-extracellular matrix (ECM) dynamic interactions appear to have a major role in regulating communication through soluble signaling, directing cell binding and activating substrates that participate in the highly organized wound healing process. Moreover, these interactions are also crucial for in vitro mimicking cutaneous physiology. Herein we explore cell sheet (CS) engineering to create cellular constructs formed by keratinocytes (hKC), fibroblasts (hDFB) and dermal microvascular endothelial cells (hDMEC), to target skin wound healing but also the in vitro recreation of relevant models. Taking advantage of temperature-responsive culture surfaces, which allow harvesting cultured cells as intact sheets along with the deposited native ECM, varied combinations of homotypic and heterotypic three-dimensional (3-D) CS-based constructs were developed. Constructs combining one CS of keratinocytes as an epidermis-like layer plus a vascularized dermis composed by hDFB and hDMECs were assembled as skin analogues for advancing in vitro testing. Simultaneously both hKC and hDMEC were shown to significantly contribute to the re-epithelialization of full-thickness mice skin wounds by promoting an early epithelial coverage, while hDMEC significantly lead to increased vessels density, incorporating the neovasculature. Thus, although determined by the cellular nature of the constructs, these outcomes demonstrated that CS engineering appear as an unique technology that open the possibility to create numerous combinations of 3D constructs to target defective wound healing as well as the construction of in vitro models to further mimic cutaneous functions crucial for drug screening and cosmetic testing assays.

Bacterial cellulose as a novel substrate for retinal pigment epithelium cells transplantation in age-related macular degeneration

Tese de Doutoramento em Engenharia Química e Biológica.

Evidence for inter- and intra-species biofilm formation variability among a small group of coagulase-negative staphylococci

Coagulase-negative staphylococci (CoNS) are common bacterial colonisers of the human skin. They are often involved in nosocomial infections due to biofilm formation in indwelling medical devices. While biofilm formation has been extensively studied in Staphylococcus epidermidis, little is known regarding other CoNS species. Here, biofilms from six different CoNS species were characterised in terms of biofilm composition and architecture. Interestingly, the ability to form a thick biofilm was not associated with any particular species, and high variability on biofilm accumulation was found within the same species. Cell viability assays also revealed different proportions of live and dead cells within biofilms formed by different species, although this parameter was particularly similar at the intra-species level. On the other hand, biofilm disruption assays demonstrated important inter- and intra-species differences regarding extracellular matrix composition. Lastly, confocal laser scanning microscopy (CLSM) experiments confirmed this variability, highlighting important differences and common features of CoNS biofilms. We hypothesised that the biofilm formation heterogeneity observed was rather associated with biofilm matrix composition than with cells themselves. Additionally, our results indicate that polysaccharides, DNA and proteins are fundamental pieces in the process of CoNS biofilm formation.

Assessment of a protease inhibitor peptide for anti-ageing

Ageing and skin exposure to UV radiation induces production and activation of matrix metalloproteinases (MMPs) and human neutrophil elastase (HNE). These enzymes are known to break down the extracellular matrix (ECM) which leads to wrinkle formation. Here, we demonstrated the potential of a solid-in-oil nanodispersion containing a competitive inhibitor peptide of HNE mixed with hyaluronic acid (HA), displaying 158 nm of mean diameter, to protect the skin against the ageing effects. Western blot analysis demonstrated that activation of MMP-1 in fibroblasts by HNE treatment is inhibited by the solid-in-oil nanodispersion containing the peptide and HA. The results clearly demonstrate that solid-in-oil nanodispersion containing the HNE inhibitor peptide is a promising strategy for anti-ageing effects. This effect can be seen particularly by ECM regulation by affecting fibroblasts. The formulation also enhances the formation of thicker bundles of actin filaments.

Enhancement of adhesion and promotion of osteogenic differentiation of human adipose stem cells by poled electroactive poly(vinylidene fluoride)

Poly(vinylidene fluoride) (PVDF) is a biocompatible material with excellent electroactive properties. Non-electroactive α-PVDF and electroactive β-PVDF were used to investigate the substrate polarization and polarity influence on the focal adhesion size and number as well as on human adipose stem cells (hASCs) differentiation. hASCs were cultured on different PVDF surfaces adsorbed with fibronectin and focal adhesion size and number, total adhesion area, cell size, cell aspect ratio and focal adhesion density were estimated using cells expressing EGFP-vinculin. Osteogenic differentiation was also determined using a quantitative alkaline phosphatase assay. The surface charge of the poled PVDF films (positive or negative) influenced the hydrophobicity of the samples, leading to variations in the conformation of adsorbed extracellular matrix (ECM) proteins, which ultimately modulated the stem cell adhesion on the films and induced their osteogenic differentiation.

Multiphasic, multistructured and hierarchical strategies for cartilage regeneration

Cartilage tissue is a complex nonlinear, viscoelastic, anisotropic, and multiphasic material with a very low coefficient of friction, which allows to withstand millions of cycles of joint loading over decades of wear. Upon damage, cartilage tissue has a low self-reparative capacity due to the lack of neural connections, vascularization, and a latent pool of stem/chondroprogenitor cells. Therefore, the healing of articular cartilage defects remains a significant clinical challenge, affecting millions of people worldwide. A plethora of biomaterials have been proposed to fabricate devices for cartilage regeneration, assuming a wide range of forms and structures, such as sponges, hydrogels, capsules, fibers, and microparticles. In common, the fabricated devices were designed taking in consideration that to fully achieve the regeneration of functional cartilage it is mandatory a well-orchestrated interplay of biomechanical properties, unique hierarchical structures, extracellular matrix (ECM), and bioactive factors. In fact, the main challenge in cartilage tissue engineering is to design an engineered device able to mimic the highly organized zonal architecture of articular cartilage, specifically its spatiomechanical properties and ECM composition, while inducing chondrogenesis, either by the proliferation of chondrocytes or by stimulating the chondrogenic differentiation  of stem/chondro-progenitor cells. In this chapter we present the recent advances in the development of innovative and complex biomaterials that fulfill the required structural key elements for cartilage regeneration. In particular, multiphasic, multiscale, multilayered, and hierarchical strategies composed by single or multiple biomaterials combined in a welldefined structure will be addressed. Those strategies include biomimetic scaffolds mimicking the structure of articular cartilage or engineered scaffolds as models of research to fully understand the biological mechanisms that influence the regeneration of cartilage tissue.

Synthesis of new peptide derivatives that self-assemble into nanostructured hydrogels for biomedical applications

Tese de Doutoramento em Ciências (área de especialização em Química)