Functionalization of a Ti-based alloy with synthesized recombinant fibronectin fragments to improve cellular response

According to a study of the European Commission, approximately one million hips are replaced by prostheses worldwide every year. The interaction of the human body with foreign materials that are subjected to alternating mechanical load in a highly corrosive environment still provides challenges. The main factors affecting prosthesis failure are stress shielding effect and poor osseointegration. In this thesis the problem of prosthesis failure has been approached from the material and from the osseointegration point of view trying to give a global solution to the problem. Niobium and hafnium, which are demonstrated to be totally biocompatible, were used to design a Ti-based alloy. The effect of the alloying elements regarding microstructure and elastic modulus was studied and the best composition was deeply characterized in terms of microstructure, elastic modulus, corrosion resistance and superficial energy. Recombinant fragments of fibronectin were synthesised spanning the cell attachment site and the heparin binding domain which are important for cell viability. These motifs were used to functionalise the surface of the TiNbHf alloy. Two tethering methods were studied: physisorption and silanisation. Silanisation was not used before to immobilise fibronectin recombinant fragments onto metallic substrates and in this thesis, its good performance was demonstrated. In vitro studies were made with each fragment and with different combinations of the fragments, which showed the importance of the heparin binding domain to obtain a cell response equivalent to that of fibronectin in terms of cell adhesion, proliferation and differentiation.De acuerdo con un estudio de la Comisión Europea, aproximadamente un millón de caderas son remplazadas por prótesis en el mundo anualmente. La interacción del cuerpo humano con materiales externos sujetos a una carga mecánica alternante en un medio altamente corrosivo todavía presenta ciertos desafíos. Los factores que contribuyen principalmente al fallo de una prótesis son el apantallamiento de cargas y la pobre osteointegracion. En la presente tesis el problema de la fallida de prótesis ha sido abordado desde el punto de vista del material y de la osteointegracion en un intento de dar una solución global al problema. El niobio y el hafnio, cuya total biocompatibilidad ha sido demostrada, se han utilizado para diseñar una aleación de titanio. El efecto de dichos aleantes respecto a la microestructura y el módulo elástico ha sido estudiado y la mejor composición ha sido profundamente caracterizada en términos de microestructura, módulo elástico, resistencia a la corrosión y energía superficial. Fragmentos recombinados de fibronectina han sido sintetizados abarcando la zona de adhesión celular y la unión de heparina, las cuales son esenciales para la viabilidad celular. Dichos motivos han sido utilizados para funcionalizar la superficie de la aleación TiNbHf. Dos métodos de unión diferentes han sido estudiados: fisisorción y silanización. La silanización es un método que no se ha utilizado hasta el momento para inmovilizar fragmentos de fibronectina sobre superficies metálicas y en la presente tesis su idoneidad ha sido demostrada. Finalmente, estudios celulares in vitro se han llevado a cabo con cada fragmento y con diferentes combinaciones de ambos, lo cual ha mostrado la importancia de la zona de unión de heparina para obtener una respuesta celular equivalente a la obtenida con la molécula de fibronectina en cuanto a adhesión celular, proliferación y diferenciación

Biomaterials Out of Thin Air: In Situ, On-Demand Printing of Advanced Biocomposites: A New Materials Design and Production Technique Using 3D-Printed Arrays of Bioengineered Cells

We have completed the proof of concept described in our Phase I proposal, a two-material array of nonstructural proteins. We created an implementation of each step in our technology concept and demonstrated its critical functionality. The biological chassis and printing hardware we created as part of this work can be re-used for future work by inserting a material coding region upstream of the fluorescent tag. Overall, we showed that our technology concept is sound. The mission benefit analyses, as described in our Phase I proposal, are complete and contained in this report. These calculations show that our technology can save hundreds of kilograms of upmass for a potential planetary human habit construction mission: the mass per habitat module can be reduced by approximately one third if the biomaterials are manufactured on Earth and included in the mission upmass, and the full 240 kg per module can be saved if the materials are derived entirely from in situ resources. Mass savings between these two extremes is expected for an actual mission, depending on the level of in situ resource extraction technology. We have shown that continued advancement of this technology concept for use in a space mission environment is justified. Our survey of future development pathways proved extremely informative in light of the lessons learned from our proof of concept work and mission scenario analyses. For example, we were able for the first time to distinguish between the levels of functionality provided by production of structural proteins, other polymers such as polysaccharides, and true organic-inorganic composites such as bone and mineralized shell. This new information represents a significant advance in formulating specific applications, and key enabling technologies, for our proposed concept. We surveyed potential collaborations with other projects and synergies with enabling technologies that are developing. We have received requests for collaboration from other institutions, including labs at Stanford University and Drexel University. We have also received visits from industry, including Organovo, a tissue engineering company, and Autodesk, a major 3D and materials design software company. Finally, we have been in touch with the team behind the 2013 NIAC Phase ll 'Super Ball Bot-Structures for Planetary Landing and Exploration' and are planning to develop our biomaterial printing technology with the goal of enabling tensegrity-based rovers such as theirs to use lighter, more robust materials. A smooth transition from TRL 2 to TRL 3 assumes that the implementations of the technology concept which demonstrate critical functionality are also pathways for future development; while this is the case for most hardware or software projects, the multidisciplinary nature of our project, particularly the biological aspect of it, means that this is not always true. For example, as part of this work we showed that although there are large number of known genetic parts that correspond to non-structural materials, this is not true for sequences for structural organic proteins, let alone biominerals. These realizations allowed us to further subdivide our concept into more detailed development areas, some of which are clearly established at TRL 3, others of which were newly identified sub-technologies moved from TRL 1 to TRL 2. Similarly, although a single feasibility /benefit analysis is sufficient for advancement from TRL 2 to TRL 3, not all potential benefits to a technology concept as broad in scope as ours are apparent at TRL 2. Both our future pathways survey and our proof of concept work highlighted that the true mass savings potential of our technology concept cannot be quantified without modification of existing materials modelling tools to take into account the possibility of positional materials properties customization. Therefore, we have simultaneously both advanced one potential set of applications of our technology concept from TRL 2 to TRL 3 and also identified a previously unknown set of applications and advanced it from TRL 1 to TRL 2. Overall, we have moved the original formulation of our concept forward from TRL 2 to TRL 3, and the expanded formulation of it presented in this document has been advanced from a combination of TRL 1 and early 1RL 2 to an overall late TRL 2. We have also identified the key areas necessary for both short-term and long-term advancement, and made recommendations for specific future work in the most promising directions. With future work on a 1-2 year timeframe to continue advancement to overall TRL 3, we will be well positioned to begin work on a specific space mission technology insertion path

Electrospinning for skin tissue engineering and drug-eluting antimicrobial biomaterials

A critical challenge in the design of biomaterials for tissue engineering relies on the development of tissue-specific biomimetic scaffolds capable of replacing cell-matrix interactions required for the repair of injured tissues. Further, such biomaterials with the additional capacity to prevent bacteria contamination can resolve issues surrounding surgical prosthesis infection. Fibrous micro- and nanostructures are extensively researched in tissue engineering due to their intrinsic similarities to decellularised human tissues. Among the several fibre-forming processes, electrospinning has drawn much attention due to its ability to produce scaffolds that morphologically resemble the native extracellular matrix (ECM) of human tissues. Electrospinning is a versatile method that uses electrohydrodynamic principles to produce fibres with diameters ranging from microns to tens of nanometres. By varying the chemistry and morphology of the fibres, it is feasible to attain different physiological and mechanical responses. The wide array of raw natural and synthetic materials – including polymers and complex molecules – that can be used to electrospin fibres can resolve well-documented problems associated with the inferiority of synthetic biomaterials and the limitations of biological tissues. In this thesis, electrospinning is utilised to contribute to the engineering of advanced ECM-mimicking biomaterials. The work will focus on (1) improving the physicochemical and mechanical responses of skin substitutes and (2) preventing mesh-associated surgical site infection. The initial study of this thesis presents the design and construction of a nozzle-free electrospinning device, which is an economically viable method of scaling-up fibre production output. The equipment is then used to fabricate elastic skin-like composite nanofibres consisting of poly(vinylpyrrolidone) (PVP) and poly(glycerol sebacate) (PGS). The findings indicate that the mechanical properties of the electrospun mats could be tuned by varying the concentration of PGS and the molecular weight of PVP within the blends. Photocrosslinking the fibres prevented the rapid degradation of the composite mats due to the hydrophilic nature of PVP, making it feasible to assess the biological responses of the construct in vitro, displaying good viability and proliferation of human dermal fibroblasts. This study provides a different approach towards the development of skin substitutes, based on the fact that mechanical stimuli influence the ability of dermal cells to adapt and reconstruct the ECM at an injured site; being able to adjust the mechanics to those of different anatomical sites of the body can have a positive effect on the overall outcome of a healing wound. Synthetic biomaterials tend to present suboptimal cell growth and proliferation, with many studies linking this phenomenon to the hydrophobicity of such surfaces. This thesis continues with the development of a protocol for silk fibroin extraction from Bombyx mori cocoons, which achieved significantly increased yields of the protein in a third of the time required by the conventional molecular cut-off extraction approach. The extracted silk fibroin was then used to produce electrospun membranes consisting of poly(caprolactone) (PCL) blended with variant forms of PGS. The main aim of this work was the development of fibre mats with tuneable hydrophobicity/hydrophilicity properties, depending on the esterification degree and concentration of PGS within each composite. By altering the surface properties of the electrospun membranes, the trinary composite biomaterial presented improved fibroblast attachment behaviour and optimal growth in comparison to PCL-only fibrous mats. The study continued with the development of an ultralight-weight nanostructured bicomponent antimicrobial construct with a similar microstructure to biologic meshes, which preserved the required mechanical integrity of synthetic mesh materials. A core/shell nanofibrous structure was developed, consisting of nylon-6 in the core and chitosan/polyethylene oxide in the shell. The bicomponent fibre structure comprised a binary antimicrobial system incorporating 5-chloro-8-quinolinol in the chitosan-shell, with the sustained release of polyhexamethylene biguanide from the nylon-6 core of the fibres. The antimicrobial nanofibres were found to elicit a robust bactericidal response, in vitro, against the two most commonly occurring pathogenic bacteria in deep incisional surgical site infections; Staphylococcus aureus and Pseudomonas aeruginosa. The results of this study advocate that the bicomponent nanofibres developed can be a promising alternative to biologic meshes, employed for hernia repair today, due to similar architecture and mechanics, but at the same time capable of actively protecting the patient from subsequent mesh-associated infections, thus tackling this life-threatening postoperative complication. Overall, the work in this thesis has expanded upon the fields of skin tissue engineering and drug-eluting antimicrobial biomaterials, potentially guiding new areas of research

Chitosan/PEO blend films crosslinked by genipin as potential membranes for controlled drug release and protein separation

This study was conducted in order to develop novel chitosan/poly(ethylene oxide)(PEO) blend films crosslinked by genipin as potential membranes for potential medical applications, such as controlled drug carriers and separation of proteins from eleutherococcuss enticosus (ES). Genipin, a naturally occurring and non-toxic crosslinking reagent, was used to form chitosan and chitosan/PEO blend networks. Genipin is found in traditional Chinese medicine and extracted from Gardenia fruit. Importantly,it overcomes the problem of physiological toxicity inherent in the use of common synthetic chemicals as crosslinking agents. The miscibility and morphology of chitosan /PEO blends were examined by means of differential scanning calorimetry (DSC), Fourier transform infrared spectroscopy (FT-IR) and small angle X-ray scattering (SAXS). The experimental results indicate that the chitosan/PEO blends are miscible over the whole composition range. A phenomenon was observed for chitosan/LPEO (lower molecular weight PEO) blends: when the composition of chitosan was less than 60% by weight, the melting points increased as the composition of chitosan was increased. It is believed that there are strong molecular interactions between chitosan molecules and the molecules in the crystalline phase of LPEO in the chitosan ALPEO blends. FT-IR and UV spectra analysis of the crosslinking reaction of chitosan revealed that the hydroxyl groups (3400 cm-1) and the amide groups (1645 cm-) in chitosan participated actively in the reaction. SAXS results showed that a heterogeneous structure exists in the chitosan/HPEO (or /LPEO) blend networks. Crosslinking by genipin restricts crystallisation and leads to smaller crystals. The mechanical properties, the stability in water, the swelling behaviour and surface properties of the films were also investigated. (Continues...).EThOS - Electronic Theses Online ServiceGBUnited Kingdo