A Bottom-Up Assembly Of Vascularized Bioartificial Constructs Using Ecm Based Microscale Modules

Tissue engineering aims to create functional biological tissues to treat diseases and damaged organs. A primary goal is to fabricate a 3D construct that can promote cell-cell interaction, extra cellular matrix (ECM) deposition and tissue level organization. Accomplishing these prerequisites with the currently available conventional scaffolds and fabrication techniques still remains a challenge. To reproduce the full functionality there is a need to engineer tissue constructs that mimic the innate architecture and complexity of natural tissues. The limited ability to vascularize and perfuse thick, cell-laden tissue constructs has hindered efforts to engineer complex tissues and organs, including liver, heart and kidney. The emerging field of modular tissue engineering aims to address this limitation by fabricating constructs from the bottom up, with the objective of recreating native tissue architecture and promoting extensive vascularization. Here, we report the elements of a simple yet efficient method for fabricating vascularized tissue constructs by fusing biodegradable microcapsules with tunable interior environments. Parenchymal cells of various types, (i.e. trophoblasts, vascular smooth muscle cells, hepatocytes) were suspended in glycosaminoglycan (GAG) solutions (4%/1.5% chondroitin sulfate/carboxymethyl cellulose, or 1.5 wt% hyaluronan) and encapsulated by forming chitosan-GAG polyelectrolyte complex membranes around droplets of the cell suspension. The interior capsule environment could be further tuned by blending collagen with or suspending microcarriers in the GAG solution. These capsule modules were seeded externally with vascular endothelial cells (VEC), and subsequently fused into tissue constructs possessing VEC-lined, inter-capsule channels. The microcapsules supported high density growth achieving clinically significant cell densities. Fusion of the endothelialized capsules generated 3D constructs with an embedded network of interconnected channels that enabled long-term perfusion in-vitro and accelerated neovascularization in-vivo. A prototype, engineered liver tissue, formed by fusion of hepatocyte-containing capsules exhibited urea synthesis rates and albumin synthesis rates comparable to standard collagen sandwich hepatocyte cultures. Our modular approach has the potential to allow rapid assembly of liver constructs with clinically significant cell densities, uniform cell distribution, and endothelialized, perfusable channels

Synthetic and structural studies of Calix[4]pyrogallolarenes: towards biological applications

This work presented in this thesis details efforts towards understanding what factors control the formation of the myriad of architectures in calix[4]pyrogallolarenes, leading towards biological application. A variety of C-alkyl-calix[4]pyrogallolarenes have been synthesised and their solidstate and in-solution behaviour has been studied by diffusion NMR spectroscopy and single crystal X-ray diffraction. It has been found that calix nano-capsules can be formed by calix[4]pyrogallolarenes in polar protic solvents when the alkyl chain is substituted with bromine at its terminus. This is speculated to be due to formation of a dipolar interaction between the bromine atoms of adjacent calix[4]pyrogallolarene molecules. Calix[4]pyrogallolarenes with pendant chains bearing hydroxyl and cyano groups have been synthesised, and their behaviour in the solid state have been investigated by single crystal X-ray diffraction. They have been shown to form head-to-tail packing interactions in the solid state. As the hydroxyl functional group offers opportunity for further synthetic manipulations, in future investigations these molecules will provide a key intermediate in the synthesis of therapeutic delivery vectors. Preliminary investigation of cellular toxicity of the calixarenes has also been performed and indicates that this class of molecules does not appear to possess a high degree of toxicity towards dendritic cells and peripheral blood mononuclear cells which are the envisioned target of this delivery vector

Exploiting the layer-by-layer nanoarchitectonics for the fabrication of polymer capsules : A toolbox to provide multifunctional properties to target complex pathologies

Polymer capsules fabricated via the layer-by-layer (LbL) approach have attracted a great deal of attention for biomedical applications thanks to their tunable architecture. Compared to alternative methods, in which the precise control over the final properties of the systems is usually limited, the intrinsic versatility of the LbL approach allows the functionalization of all the constituents of the polymeric capsules following relatively simple protocols. In fact, the final properties of the capsules can be adjusted from the inner cavity to the outer layer through the polymeric shell, resulting in therapeutic, diagnostic, or theranostic (i.e., combination of therapeutic and diagnostic) agents that can be adapted to the particular characteristics of the patient and face the challenges encountered in complex pathologies. The biomedical industry demands novel biomaterials capable of targeting several mechanisms and/or cellular pathways simultaneously while being tracked by minimally invasive tech-niques, thus highlighting the need to shift from monofunctional to multifunctional polymer capsules. In the present review, those strategies that permit the advanced functionalization of polymer capsules are accordingly introduced. Each of the constituents of the capsule (i.e., cavity, multilayer membrane and outer layer) is thor-oughly analyzed and a final overview of the combination of all the strategies toward the fabrication of multi-functional capsules is presented. Special emphasis is given to the potential biomedical applications of these multifunctional capsules, including particular examples of the performed in vitro and in vivo validation studies. Finally, the challenges in the fabrication process and the future perspective for their safe translation into the clinic are summarized.Peer reviewe

Exploiting the layer-by-layer nanoarchitectonics for the fabrication of polymer capsules: A toolbox to provide multifunctional properties to target complex pathologies

[EN] Polymer capsules fabricated via the layer-by-layer (LbL) approach have attracted a great deal of attention for biomedical applications thanks to their tunable architecture. Compared to alternative methods, in which the precise control over the final properties of the systems is usually limited, the intrinsic versatility of the LbL approach allows the functionalization of all the constituents of the polymeric capsules following relatively simple protocols. In fact, the final properties of the capsules can be adjusted from the inner cavity to the outer layer through the polymeric shell, resulting in therapeutic, diagnostic, or theranostic (i.e., combination of therapeutic and diagnostic) agents that can be adapted to the particular characteristics of the patient and face the challenges encountered in complex pathologies. The biomedical industry demands novel biomaterials capable of targeting several mechanisms and/or cellular pathways simultaneously while being tracked by minimally invasive techniques, thus highlighting the need to shift from monofunctional to multifunctional polymer capsules. In the present review, those strategies that permit the advanced functionalization of polymer capsules are accordingly introduced. Each of the constituents of the capsule (i.e., cavity, multilayer membrane and outer layer) is thoroughly analyzed and a final overview of the combination of all the strategies toward the fabrication of multifunctional capsules is presented. Special emphasis is given to the potential biomedical applications of these multifunctional capsules, including particular examples of the performed in vitro and in vivo validation studies. Finally, the challenges in the fabrication process and the future perspective for their safe translation into the clinic are summarized.The authors are thankful for funds from the Basque Government, Department of Education (IT-927-16 and PIBA_2021_1_0048)

Novel Carbon Nanomaterials: Synthesis and Applications

Carbon nanotubes (CNTs) hold exceptional promise for an array of applications in numerous disciplines. Discovered in 1991 by Sumio Iijima, CNTs have been synthesized in a myriad of varieties including single-walled (SWNTs), multiwalled (MWNTs), and chemically doped, resulting in unique electronic and physical properties, as well as an innate biocompatibility towards biological organisms. Such attractive properties have been demonstrated in CNTs' implementation in applications such as electronic sensors, drug delivery vehicles, and reinforcements in composite materials. Moreover, by chemical doping, carbon nanomaterial hybrids have been formed with intrinsic morphological and physical properties differing from their un-doped counterparts.Despite successful execution in these areas, reports have been generated indicating degrees of cytotoxicity induced by carbon nanotubes. Specifically, CNTs have demonstrated asbestos-like pathogenicity, resulting in pro-inflammatory response and granuloma formation. Additionally, because CNTs are composed of sp2 hybridized carbon atoms, they possess an inherent resiliency toward degradation. In my Ph.D. studies, we have addressed multiple facets of the growing field of carbon nanomaterials. We have synthesized a nitrogen-doped carbon nanomaterial using the process of chemical vapor deposition, which we called nitrogen-doped carbon nanotube cups. These doped nanostructures can be conceptualized as stacked cups, which can be separated into individual cups through mechanical grinding. Characterized through a variety of microscopic and spectroscopic methods, we observed that the presence of nitrogen functionalities on the cups' open basal rims allows for cross-linkage, forming nanocapsules capable of encapsulating a desired cargo. We also hypothesize that the presence of nitrogen functionalities will increase their biocompatibility for drug delivery and imaging applications. To address the realm of "nanotoxicity", we have performed investigations of benign degradation methods, specifically enzymatic catalysis, to oxidize carbon nanomaterials. We have observed that incubation of carbon nanomaterials with a peroxidase-based enzyme (such as horseradish peroxidase) with low levels of H2O2 (40 µM) results in the oxidation of carbon nanomaterials to CO2 gas over ten days at room temperature. Subsequent research has elucidated the nature of interactions between carbon nanotubes and enzymatic cofactors. These studies, thus, elucidate a benign approach to degrade carbon nanomaterials, typically oxidized under harsh acidic conditions or thermal oxidation in air

Characterising the neck motor system of the blowfly

Flying insects use visual, mechanosensory, and proprioceptive information to control their movements, both when on the ground and when airborne. Exploiting visual information for motor control is significantly simplified if the eyes remain aligned with the external horizon. In fast flying insects, head rotations relative to the body enable gaze stabilisation during highspeed manoeuvres or externally caused attitude changes due to turbulent air. Previous behavioural studies into gaze stabilisation suffered from the dynamic properties of the supplying sensor systems and those of the neck motor system being convolved. Specifically, stabilisation of the head in Dipteran flies responding to induced thorax roll involves feed forward information from the mechanosensory halteres, as well as feedback information from the visual systems. To fully understand the functional design of the blowfly gaze stabilisation system as a whole, the neck motor system needs to be investigated independently. Through X-ray micro-computed tomography (μCT), high resolution 3D data has become available, and using staining techniques developed in collaboration with the Natural History Museum London, detailed anatomical data can be extracted. This resulted in a full 3- dimensional anatomical representation of the 21 neck muscle pairs and neighbouring cuticula structures which comprise the blowfly neck motor system. Currently, on the work presented in my PhD thesis, μCT data are being used to infer function from structure by creating a biomechanical model of the neck motor system. This effort aims to determine the specific function of each muscle individually, and is likely to inform the design of artificial gaze stabilisation systems. Any such design would incorporate both sensory and motor systems as well as the control architecture converting sensor signals into motor commands under the given physical constraints of the system as a whole.Open Acces

Proceedings of the EAA Spatial Audio Signal Processing symposium: SASP 2019

International audienc