Consensus guidelines for the use and interpretation of angiogenesis assays

The formation of new blood vessels, or angiogenesis, is a complex process that plays important roles in growth and development, tissue and organ regeneration, as well as numerous pathological conditions. Angiogenesis undergoes multiple discrete steps that can be individually evaluated and quantified by a large number of bioassays. These independent assessments hold advantages but also have limitations. This article describes in vivo, ex vivo, and in vitro bioassays that are available for the evaluation of angiogenesis and highlights critical aspects that are relevant for their execution and proper interpretation. As such, this collaborative work is the first edition of consensus guidelines on angiogenesis bioassays to serve for current and future reference

Development of Silk based Bio-polymeric Porous Matrices for Tissue Engineering Applications

Tissue engineering has emerged as a promising approach for the development of artificial body organs, repair, recover or improve tissue structures and functionality. 3D porous scaffolds possessing biomimicking properties are needed to support the neogenesis of tissues and mass transport of cells, nutrients and metabolic waste. Keeping this in view, the present dissertation work was undertaken for the development of SF based scaffolds with improved surface, mechanical and biological properties that can be used as artificial extracellular matrices for tissue regeneration. The silk fibroin was extracted from B. mori silk cocoon and the process was optimized by Response surface methodology using Box-Behnken Design. Porous SF and SF/PVA) blend scaffolds were prepared by salt leaching process and characterized for morphological (SEM), structural (XRD and FTIR), thermal (DSC and TGA) and mechanical (compressive strength) behaviour. The SF scaffolds were further modified with soluble eggshell membrane protein (SEP) with the aim of improving cell affinity for tissue regeneration. The pore size of the prepared SEP-SF and SEP-(SF/PVA) scaffold were in the range of 250-350µm and porosity of 90-93%. The measured compressive strength of SF and SF/PVA (50:50) scaffold were 279.8 ± 36.2 KPa and 235 ± 67.1 KPa respectively. The existence of soluble eggshell membrane protein on the scaffold surface, structural and thermal stability was confirmed by EDX, XRD, FTIR, DSC and TGA analysis. An increase in compressive strength of the prepared SF scaffolds was achieved by modification with SEP (321.5 ± 42.2 KPa for SEP-SF and247.5± 23.7 KPa for SEP-(SF/PVA) scaffolds). The cell culture study has indicated the significant improvement in cell adhesion and proliferation observed with hMSCs cultured on SF and SF/PVA scaffolds modified with SEP. The cyto-compatibility of the SEP conjugated SF scaffolds was confirmed by in-vivo animal model testing. This study has demonstrated that the biomimic property of SF scaffold can be enhanced by surface modification with SEP. Finally, it is concluded that the SEP conjugated SF/PVA (50:50) has the potential for use as artificial extra cellular matrix particularly for soft and other non-load bearing tissue engineering applications

Bioactive glass based fibre mats for wound healing

Wounds are one of the most prevailing healthcare problems. Two of the most common wound types, burn injuries and chronic ulcers, are imposing a great threat to the medical systems due to their high incidence, high morbidity, high cost of treatment, and high likelihood to induce further problems, such as infection, loss of sensory nodes, cell mutation, etc. Burn injuries usually have a damaged epidermis with excessive exudates that can become infected and cause severe inflammation while in the meantime, the sensory nodes, follicles, and glands are fully damaged. Diabetic ulcers, the most common type of chronic wounds, have unepithelialized epidermis that causes biofilm colonization, strong accumulation of cytokines and protease, and neuropathy from diabetes. The most important steps in the regeneration of both wounds are the regeneration of the ECM and vascular network, in order to ensure cell attachment and migration as well as the delivery of oxygen and nutrients to the wound site. This thesis describes the development of 3D ECM-mimic bioactive glass fibre mats as strategies for the regeneration of both injuries. Bioactive glasses (BGs) are biodegradable materials that have already been widely used as a synthetic bone graft material that can regenerate bone defects and as the active ingredient for remineralizing toothpastes, but now they are finding use as medical devices (scaffolds or matrices) for wound healing applications. BGs degrade into therapeutic ions that promote individual steps in the wound healing cascades, including hemostasis, antibacterial effect, anti-inflammation, appropriate cell proliferation, angiogenesis, re-epithelialization, and extracellular matrix production. BG fibre mats were obtained through an optimized sol-gel electrospinning approach, in which the electrospinning and material parameters were determined through experimentation. The fabricated fibre mats mimicked the extracellular matrix in the dermis layer and can be easily packed into wound defects. We synthesized zinc-delivering bioactive glass fibre mats as scaffolds for burn regeneration as the controlled release of zinc ions is thought to be antibacterial, and have anti-inflammation and follicle regeneration properties. In vitro studies confirmed the capabilities of the fabricated fibre mats in ECM deposition and angiogenesis. 3 mol% of Zn incorporation reduced the cytotoxic effect of BGs to fibroblasts and stimulated their expression of growth factors (PDGF, VEGF, bFGF, TGF-β, and HIF-1α) from fibroblasts and endothelial cells for ECM deposition and angiogenesis. Media conditioned with HDFs that had been precultured with BG dissolution products influenced the proliferation and migration of endothelial cells and their protein expression, which provides insights into understanding the mechanism of BGs in angiogenesis. We also synthesized borosilicate bioactive glass fibre mats through modified sol-gel electrospinning for diabetic ulcer regeneration. Boron was selected as it promotes epithelialization, tissue granulation, anti-inflammation, ECM deposition, and most importantly, angiogenesis. The incorporation of boron into the BG system stimulated high expressions of angiogenic factors (VEGF, bFGF, HIF-1α) from fibroblasts and promoted the proliferation and migration of endothelial cells. In vivo studies were conducted in order to investigate the efficacy of silicate and borosilicate BGs in healing diabetic ulcers and to understand the mechanism of them to individual phases of wound healing, including wound closure, epithelialization, anti-inflammation, angiogenesis, tissue granulation, and ECM deposition. The assays included imaging the wound areas through optical cameras, histology analysis and immunohistochemistry analysis. BGs were found to stimulate faster wound healing as they highly promoted all the above-mentioned healing steps. The incorporation of boron into the BG system and processing BGs to fibrous structure both impact the healing effect.Open Acces

Progenitor cells in auricular cartilage demonstrate promising cartilage regenerative potential in 3D hydrogel culture

The reconstruction of auricular deformities is a very challenging surgical procedure that could benefit from a tissue engineering approach. Nevertheless, a major obstacle is presented by the acquisition of sufficient amounts of autologous cells to create a cartilage construct the size of the human ear. Extensively expanded chondrocytes are unable to retain their phenotype, while bone marrow-derived mesenchymal stromal cells (MSC) show endochondral terminal differentiation by formation of a calcified matrix. The identification of tissue-specific progenitor cells in auricular cartilage, which can be expanded to high numbers without loss of cartilage phenotype, has great prospects for cartilage regeneration of larger constructs. This study investigates the largely unexplored potential of auricular progenitor cells for cartilage tissue engineering in 3D hydrogels

Developing active biomaterials for implantable devices: platforms to investigate capacitive charge based control of biofouling

Implantable devices, in particular biosensors, have clear utility within medicine, but face a hurdle to long-term function due to adsorption of biomolecules (biofouling) and subsequent immune re- sponse to implants, the foreign body response (FBR). Strategies to control this immune reaction have included material selection, drug release and, more recently, engineered surface properties. The increasing use of embedded electronics within many classes of implanted devices presents an opportunity to exploit electromagnetic phenomena at the device surface to mitigate biofouling and FBR. Such active biomaterials would allow dynamic modification of the apparent material properties of an implanted device. A hypothesis was developed that biological interaction with a biomaterial surface can be altered by capacitive charging. A platform was constructed to test this and related hypotheses around cell and protein surface interactions in vitro and adapted into a second platform for initial characterisa- tion work on an early in vivo model using chick eggs. These platforms were designed to be easy to fabricate and to provide multiple electrical connections into a substrate in contact with biological solutions or tissue. Electrodes were fabricated from fluoropolymer coated tantalum pentoxide, a high-κ dielectric, and compared against adjacent, identically coated, silicon dioxide regions. Cells from the MDA- MB-231 cancer cell line were cultured on these regions under electrical stimulation. A voltage de- pendent reduction of cell attachment and spreading was detected on capacitively charged surfaces compared to uncharged controls. The tentative results, suggest capacitively charged surfaces hold promise as active biomaterials. A second cell type MCF-7 did not reproduce the effect, implying a more coherent understanding is required of the mechanisms behind cell surface interactions on these surfaces. Multiple independent bioelectrochemical cell-surface interactions were observed using the plat- form and several quantification techniques were successfully employed. It is therefore argued that the platform may have wide applicability as a future research tool

Development of natural-based hydrogel particles using a biomimetic methodology

Tese de doutoramento em BioengenhariaSuperhydrophobic (SH) surfaces have been greatly explored in the biomedical field. Such surfaces are inspired by the repellent properties of natural structures. The most well known example is the lotus leaf, which has the capacity to repeal the water droplets due to the presence of micro/nano topographical features and the low surface energy. One particular application of artificial SH surfaces is their employment as supports where liquid droplets of aqueous-based polymeric solutions are dispensed, acquiring an almost spherical shape. Through application of one or multiple hardening steps solid particles are obtained in a fast way. Using this methodology, a variety of functional natural-based hydrogel spherical systems encapsulating cells and/or drugs are proposed in this thesis. The strategy of the first proposed system is the incorporation of cyclodextrins (CDs) in the dextranmethacrylate (Dex-MA) microgels networks in order to improve their loading efficiency for hydrophobic drugs. The formation of inclusion complexes between CDs and dexamethasone, when the CDs were copolymerized, increased significantly the amount of drug in the Dex-MA particles, when compared with formulations without or with freely dispersed CDs. Chitosan (Chi), unless it has been modified, is soluble in acidic media, which turn it incompatible with encapsulation of cells or pH sensitive molecules. The second system proposed envisaged a mild Chi-based system with two sequential hardening steps, where dexamethasone or fibroblast-like cells were successfully entrapped. SH surfaces methodology was also used to co-encapsulate cells and proteins into hydrogel particles without compromise the cell viability and protein activity. Collagen combined with platelet lysates were used to obtain easy-to-handle spherical formulations being capable to act as in situ growth factors release system as well as reservoirs of human adipose derived stem cells, for applications in skin regeneration. Mesenchymal stem cells derived from bone marrow and fibronectin were also encapsulated inside alginate spheres and the system was studied for bone regeneration. The control of the release of bioactive agents may be achieved by adjusting the chemistry and physical parameters such as particles architecture. Multicompartmentalized systems have emerged and are envisioned to be the next area of development due to the possibility to confines various bioactive agents exhibiting a variety of release kinetics. Taking advantage of the simplicity of the SH surfaces methodology, core/shell and multilayered particles composed by Dex-MA and alginate were efficiently prepared, with cells or drugs encapsulated into individual compartments. The developed work shows that a wide variety of particles useful for biomedical application, ranging from homogeneous spherical matrices to compartmentalized systems, could be obtained under mild conditions and in a fast way, using SH surfaces.Superfícies superhidrofóbicas (SH) têm vindo a ser exploradas no campo biomédico. Estas superfícies são inspiradas nas propriedades repelentes de estruturas naturais. O exemplo mais conhecido é a folha de lótus, com a sua capacidade de repelir gotas de água devido à presença de micro/nano estruturas e baixa energia de superfície. Uma aplicação particular das superfícies SH artificiais é a sua utilização como suportes onde gotas de soluções poliméricas de base aquosa são dispensadas, adquirindo uma forma quase esférica. Através da aplicação de um ou múltiplos passos de solidificação, partículas sólidas são obtidas de uma forma rápida. Usando esta metodologia, uma variedade de hidrogéis funcionais de base natural, encapsulando células e/ou fármacos, são propostos nesta tese. A estratégia do primeiro sistema proposto é a incorporação de ciclodextrinas (CDs) em microgéis de dextrano-metacrilatado (Dex-MA), com o intuito de melhorar a sua capacidade de carga para fármacos hidrofóbicos. A formação de complexos de inclusão entre CDs e dexametasona, quando as CDS estavam co-polimerizadas, aumentou significativamente a quantidade de fármaco no interior das partículas, quando comparadas com formulações sem, ou com CDs livres. O quitosano, a não ser que esteja modificado, é solúvel em meios ácidos, o que o torna incompatível com o encapsulamento de células ou moléculas sensíveis ao pH. O segundo sistema proposto teve como objectivo a produção de sistemas não agressivos, através de dois passos sequenciais de gelificação do quitosano, onde dexametasona e/ou fibroblastos foram encapsuladas com sucesso. A metodologia das superfícies SH foi também usada para o co-encapsulamento de células e proteínas em hidrogéis sem comprometer a viabilidade das células nem a atividade das proteínas. Colagénio combinado com lisados de plaquetas foram usados para obter formulações fáceis de manusear, sendo estas capazes de atuar como sistema de libertação de factores de crescimento in situ e também como reservatório de células estaminais derivadas do tecido adiposo, para a regeneração de pele. Em outro sistema apresentado, células estaminais mesenquimais e fibronectina foram encapsulados em esferas de alginato e o sistema foi estudado para regeneração óssea. O controlo da libertação de agentes bioativos pode ser conseguido através do ajuste da química e de parâmetros físicos tal como a arquitetura das partículas. O potencial dos sistemas multicompartimentalizados tem emergido devido à possibilidade de encapsulamento de vários agentes bioativos exibindo diferentes cinéticas de libertação. Aproveitando a simplicidade da metodologia das superfícies SH, partículas com uma ou mais camadas, compostas por Dex-MA e alginato foram eficientemente preparadas, encapsulando células e fármacos em compartimentos individuais. Os trabalhos desenvolvidos mostram que uma grande variedade de partículas úteis para aplicações biomédicas, desde matrizes esféricas homogéneas até sistemas compartimentalizados, podem ser obtidos em condições não agressivas e de uma forma rápida, usando superfícies SH.Fundação para a Ciência e Tecnologoa (FCT) SFRH/BD/71395/2010 e PTDC/CTM-BIO/1814/2012