Spider silk is a material that both has impressive mechanical properties and is also environmentally friendly. Though there are limitless potential engineering applications for such materials, industrial production of spider silk has proven to be challenging. Farming silk from spiders, as is done with silkworms, is not a viable option for large-scale production of spider silk due to the venomous and predatory nature of spiders. Here, an attempt is made to express synthetic spider silk minifibroins heterologously in Escherichia coli, to purify the recombinant spidroins from cell lysate, and to spin them into artificial fibers through a biomimetic process. Silk minifibroins were designed to be similar to Major Ampullate Spidroin 1 from Latrodectus hesperus. Synthetic fibers were examined by scanning electron and light microscopy, and their mechanical properties were tested by a tensometer. Properties of synthetic silk were compared to those of native dragline silk from the same species from which their design was inspired, revealing synthetic silk fibers with lower breaking stress and breaking strain.

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Spinal cord injury causes the partial or total loss of the anatomical and functional continuity of the spinal cord tissue, leading to the damage of the organs controlled by nerves that branch off downstream the injury. This thesis analyses the mechanics of two possible treatments based on two different approaches: intraspinal microstimulation (ISMS) and tissue engineering. These two approaches have a common rationale, the delivery of electrical stimuli to the injured spinal cord. In the literature, the feasibility of the electrodes for ISMS is often limited to the analysis of stiffness. The mechanical validation of the device is then focused on the step after the in vivo implantation, considering the interplay with the surrounding tissue. In this work, the mechanical performance of an innovative intraspinal microstimulation device is evaluated thoroughly before the in vivo step, to avoid the waste of material, animals, and time. The study involves the characterisation of the single components (electrodes), prototypes, and possible failure mechanisms. A work on silk fibroin hydrogels for the regeneration of the spinal cord is also presented. Silk fibroin is a highly versatile material for biomedical purposes, and thus largely used in tissue engineering. Moreover, it has piezolectric properties subjected to micro and nanostructure. Given the proven benefits of electrical stimulation in the regeneration of the spinal cord after injury, different approaches studied in literature often require the use of external devices to generate electrical stimuli. This thesis aims to study the mechanical properties of silk fibroin hydrogels obtained by applying an electric field to silk fibroin solutions, to investigate the eventual increase of the microstructure orientation and consequent improvement of the piezoelectric effects of fibroin.

To mitigate climate change a proposed space-based geoengineering solutionis to screen off solar irradiance by placing a membrane in between the Earthand the Sun. The feasibility of such a project largely depends on minimizingthe mass of the shading screen and as an extension to the Sunshade projectthis thesis investigated how such a low-mass membrane could be designed.Because of the acting forces at location in space, minimizing the mass impliesthat the material ought to have a low reflection coefficient and surface densityand therefore the highly transparent material of artificial spider silk was chosenas the proposed material. The only possibility to block light is then byrefraction or diffraction and, since the presence of apertures might lower thesurface density, the structure of the suggested membrane is a grid patternof wires. Such a diffraction grating was investigated while applying twomethods. Method 1 optimized the dimensions of the structure to lower thetotal transmission on Earth when placed on the direct transmission axis ofthe membrane and method 2 tilted the membrane in order to place Earth ata diffraction minimum. This resulted in three suggested designs A, B, andC with surface densities varying from that of 0.00867 to 0.228 gm−2. Theresults were compared with two previous design proposals where the lowestareal density was 0.34g/m2, which is 3/2 to 40 times larger than the densitiesproposed in this paper. The reflectivities for A and B were 12.5 and 3.75 timeslarger than that of the smallest previously achieved reflectivity. The reflectivityof C could not be determined exactly but ought to have a reflectivity at leastas low as B at 3%, making it the most promising candidate for a membranedesign of the three.

Chronic or degenerative diseases affecting the lumbar spine, commonly referred to as low back pain (LPB), are a major cause of dysfunction, pain, and disability worldwide. According to the Global Burden of Disease (GBD) report of 2019, LPB affects over half a billion people, severely limiting their well-being and lifestyle. Unfortunately, these numbers have been steadily increasing over the last decade, with a rise of more than 15%, mainly due to demographic aging of the population, making it a significant socioeconomic global issue. When conservative treatments such as medications, drugs, and injections fail to alleviate the symptoms, surgical interventions become necessary. Spinal surgeries have become increasingly common and account for 40% of the top ten surgical procedures in the United States alone. As a result, the global market for spinal implants and medical orthopedic devices has been growing at a compound annual growth rate (CAGR) of 5.0% in the United States. Degenerative disc diseases, herniated intervertebral discs, and spondylolisthesis are among the most common problems requiring implant surgery, with lumbar interbody fusion cages or total disc replacements being the most common options. These surgical techniques often utilize a metal endplate or hollow cage as a load-bearing structure to ensure correct load transmission and biomechanical spinal functionality. Currently, endplates for total disc replacement are produced using subtractive manufacturing techniques from bulk biomedical-graded metal alloys like Ti-6Al-4V. The endplates are inserted between two adjacent vertebral bodies, where bone ingrowth and implant fusion are necessary. However, the elastic properties of bulk metals and bone tissue do not match, resulting in stress-shielding phenomena, implant loosening, or implant subsidence. These complications frequently necessitate surgical revision of the implant, which not only impacts the daily activities of the patients but also has a relevant economic impact. Therefore, researchers are exploring alternative design and manufacturing strategies to develop next-generation prosthetic devices that overcome these challenges. Metal additive manufacturing (MAM), particularly Laser-Powder Bed Fusion (L-PBF), has revolutionized the fabrication of specialized components with complex shapes, including architected cellular materials - a novel class of engineered materials with tunable mechanical properties. The biomedical field is a prime example of where lattice application has proved beneficial. MAM provides numerous advantages, including patient-specific customization, a vast design space, and reduced stress shielding. However, issues with structural integrity, lack of AM-specific norms, and the need for fine-tuning process optimizations are still hindering MAM's widespread adoption on the international market. An essential issue that requires resolution is the impact of process-induced flaws on the fatigue behavior of components made of L-PBF lattices. Despite a growing body of scientific literature on the fatigue behavior of lattice unit cells, little attention has been given to the function of fatigue at a millimetric scale, specifically the role of sub-unital lattice elements such as struts and junctions. As fatigue is highly localized, understanding primary fatigue behavior and fracture mechanisms at a strut scale may be critical to addressing the aforementioned problems. Moreover, designing proper prosthetic devices requires fulfilling both biomechanical and biological requirements, leading to a bottleneck in component quality. Proper tuning of osteointegration often requires large porosity and small strut dimensions, approaching the limits of industrial 3D printers. This increases the likelihood of manufacturing lattices with unconnected struts, drosses, parasitic masses, and severe deviations from the nominal as-designed geometries, leading to highly susceptible components under fatigue. To address these challenges, combined approaches with bone tissue engineering may be advantageous. Biopolymers from natural sources, such as silk fibroin and collagen derivatives (i.e., gelatin), are widely used for bone-filler applications due to their exceptional biological properties. These polymers can create highly interconnected biodegradable porous 3D scaffolds suitable for cell differentiation towards an osteogenic phenotype, such as in the form of foams. These foams can be embedded into metal lattice structures, resulting in a hybrid composite device that simultaneously fulfills the load-bearing, fatigue, and osteointegrative requirements that a spinal prosthetic device necessitates. This thesis work covers a range of topics mentioned above. Firstly, an introductory theoretical background is presented in Chapter I, followed by experimental findings which are presented in three different chapters. Chapter II is dedicated to the fatigue behavior of L-PBF Ti-6Al-4V sub-unital lattice elements in the form of miniaturized dog-bone specimens that mimic struts and nodes. This chapter is divided into four sections. The first section investigates the fatigue strength of strut-like specimens based on their building orientations at four different angles with respect to the printing job plate. Morphological features of the miniaturized specimens such as average and minimum cross-section, eccentricity, waviness, and surface texture are correlated with fatigue strength. The role of inner and surface defects, such as lack-of-fusion (LoF) and gas holes, is also considered to explain the main failure mechanisms. The impact of building orientation on the printing quality of the specimens is highlighted, with an increase in surface roughness and defectiveness as the printing angle decreases, resulting in a shorter fatigue life for miniaturized struts. In the second section, the fatigue effect is studied across different fatigue regimes. The role of the mean stress effect is assessed using the Haigh diagram, which reveals an increase in fatigue life moving towards compressive loading regimes. The effect of the printing angle is also investigated, showing different trends according to the different stress ratios, suggesting different fatigue failing mechanisms. The third section introduces strut-junction miniaturized specimens and evaluates their fatigue behavior according to building orientations. Horizontal specimens show an increased fatigue life compared to their thin strut counterparts, and different morphological outcomes are highlighted, including improved surface quality even at lower angles, possibly related to the node acting as an additional supporting structure. The fourth section presents a design-led compensation strategy for sub-unital lattice specimens, aimed at reducing as-designed/as-built deviations. This systematic decrease in geometrical mismatch suggests potential new design strategies for fatigue enhancement. In Chapter III, bone tissue engineering strategies are explored for the design of foam scaffolds as bio-fillers for lattice-based design. The feasibility of the polymer-metal composite is assessed, using an N2O-based gas foaming technique to fabricate silk fibroin and silk fibroin/gelatin porous scaffolds infilled into a cubic L-PBF Ti-6Al-4V lattice structure. The adhesion at the polymer/metal interface is assessed, with simultaneous electrowetting, showing promise for better and more intimate contact on the outermost surface of the lattice struts. A statistical-based analysis of the foam porosity is then carried out, aimed at optimization towards osteointegration improvement. Selected foams are biologically evaluated, revealing good cell adhesion and differentiation towards an osteogenic phenotype. Chapter IV reports on two different strategies for the design of a Ti-6AL-4V L-PBF lattice-based endplate for total disc replacement. The first strategy focuses on homogenized-based topology optimization, designing an octet-truss prosthetic device with a graded structure and a cell size suitable for polymeric infilling. The second strategy aims at optimizing octet-truss lattice components for fatigue, evaluating the optimal building orientation for the specimens. Experimental results reveal an improvement in the fatigue life of three-point bending test specimens, suggesting the potential of the proposed model. In Chapter V, the major takeaways of this thesis work are discussed, highlighting important advancements in understanding the fatigue behavior of lattice structures and the development of novel hybrid strategies for the design of biomedical devices, with a particular focus on spinal orthopedics. Future possible directions for research are also explored.

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Spider silk has extraordinary mechanical properties, displaying high tensile strength, elasticity, and toughness. Given the high performance of natural fibers, one of the long-term goals of the silk community is to manufacture large-scale synthetic spider silk. This process requires vast quantities of recombinant proteins for wet-spinning applications. Attempts to synthesize large amounts of native size recombinant spidroins in diverse cell types have been unsuccessful. In these studies, we design and express recombinant miniature black widow (Latrodectus hesperus) MaSp1 spidroins in bacteria that incorporate the NTD and CTD, along with varying numbers of codon-optimized internal block repeats. Following spidroin overexpression, we perform quantitative analysis of the bacterial proteome to identify proteins associated with spidroin synthesis. Nano-liquid chromatography with tandem mass spectrometry (nLC-MS/MS) reveals a list of molecular targets that are differentially expressed after enforced mini-spidroin production. This list included proteins involved in energy management, proteostasis, translation, cell wall biosynthesis and oxidative stress. Collectively, this study unveils new bacterial genes to target by genetic engineering to overcome bottlenecks that throttle spidroin overexpression in microorganisms.

Sticky prey capture threads are produced by many members of the spider Infraorder Araneomorphae. Cribellar threads are plesiomorphic for this clade, and adhesive threads are apomorphic. The surface of cribellar thread is formed of thousands of fine fibrils. Basal araneomorphs produce cylindrical fibrils, whereas more derived members produce fibrils with nodes. Cribellar fibrils snag and hold rough surfaces, but other forces are required to explain their adherence to smooth surfaces. Threads of Hypochilus pococki (Hypochilidae) that are formed of non-noded fibrils hold to a smooth acetate surface with the same force under low and high humidities. In contrast, threads of Hyptiotes cavatus and Uloborus glomosus (Uloboridae) that are formed of noded fibrils hold with greater forces to the same surface at intermediate and high humidities. Threads spun by eight species representing seven genera and four families with noded fibrils absorb water, while that of two families, represented by one species each with smooth fibrils, repel water, indicating increase hygroscopisity associated with the presence of nodes. Additionally, equations describing van der Waals and hygroscopic forces can predict the observed stickiness of these threads. This model supports the hypothesis that van der Waals forces allow non-noded cribellar fibrils to adhere to smooth surfaces, whereas noded fibrils employ van der Waals forces at low humidities and add hygroscopic forces at higher humidities. Thus, there appear to have been two major events in the evolution of spider prey capture thread: the addition of hydrophilic nodes to the fibrils of cribellar threads and the replacement of cribellar fibrils by glycoprotein glue. / Master of Science

Tissue engineering (TE) is an interdisciplinary field, in continuous evolution, that possesses as main goal the creation of efficient systems for tissues and organs healing and regeneration. For bone, TE strategies are typically based on the combined use of scaffolds, cells, and bioactive molecules. Different materials were successfully studied and proposed for the fabrication of scaffolds. Among them, silk fibroin (SF) was evaluated as particularly promising for different TE applications, especially for bone tissue regeneration. Silk fibroin, a natural protein forming the structural core of silk filaments, holds biocompatibility, mechanical properties and biodegradation rate suitable for applications in bone regeneration. However, in the past, SF has shown some limitations, especially in terms of bioactivity and effective differentiating ability of hMSCs in regenerating bone tissue. In this work, we wanted to demonstrate that SF, properly processed, chemically modified, and conjugated with selected bioactive species, can be used to prepare different systems: a functionalised scaffold; a bioresorbable material with mineralization ability; an implantable immunomodulatory material. The experimental activities performed and the deep investigation of the properties of the SF-based systems prepared, led to promising results, indicating that SF could be a flexible and powerful platform for the realization of different therapeutic tools. For some of the SF-based systems described in this dissertation, further studies are needed to assess the biological activity of the materials prepared.