Engineered Vascular Tissue Generated by Cellular Self-Assembly

Small diameter vascular grafts comprised entirely from cells and cell-derived extracellular matrix (ECM) have shown promise in clinical trials and may have potential advantages as in vitro vascular tissue models. A challenge with current cell-derived tissue engineering approaches is the length of time required to generate strong, robust tissue. There is a lack of alternative methods to rapidly assemble cells into a 3D format without the support of a scaffold. Toward the goal of engineering a new approach to rapidly synthesizing vascular tissue constructs entirely from cells, we have developed and characterized a strategy for creating cell-derived tissue rings by cellular self-assembly. The focus of this thesis was to develop the system to rapidly generate engineered tissue rings, and to evaluate their structural and functional properties. To generate tissue rings, rat smooth muscle cells (SMCs) were seeded into round-bottomed, ring-shaped agarose wells with varying inner post diameters (2, 4, and 6 mm). Within 24 hours of seeding, cells aggregated, contracted, and formed robust tissue that could be removed from their wells and handled. If kept in culture, the thickness of these tissue rings increased with time. Mechanical analysis of the tissue showed that it was stronger after only 8 days in culture than engineered tissues generated by other approaches (such as seeding cells in biopolymer gels) cultured and tested at similar time points. Histological staining of the tissue rings revealed high cell densities throughout, along with the presence of glycosaminoglycans and some collagen. We also found that we could use the tissue rings as building blocks to generate larger tubular structures. Briefly, tissue rings were removed from the agarose wells and transferred onto silicone tubing mandrels. Once the rings were placed in contact with each other on the mandrel, they were cultured to allow the rings to fuse together. We found that the ability of tissue rings to fuse decreased with increasing ring “pre-culture� duration, and that we were able to generate fully fused tissue tubes in as little as 8 days (with only one day of ring pre-culture and seven days of fusion). In the last section of this thesis, we established the feasibility of using primary human SMCs to generate self-assembled tissue rings, similar to the self-assembled rings generated with rat SMCs. Compared to the rat SMC rings, human SMC rings were stronger, stiffer and appeared to contain increased levels of collagen. These data showed that human SMCs are capable of self-assembling into tissue rings similar to rat SMCs, and may therefore be used to create engineered human vascular tissue. Overall, we have developed a platform technology that can be used to screen the effects of culture parameters on the structure, mechanics, and function of vascular tissue. We anticipate that through the use of this technology, we can further improve vascular grafts by better understanding factors which promote ECM synthesis and SMC contraction. We can use these results directly toward the generation of vascular grafts by fusing self-assembled cell rings together to form tissue tubes. These novel bioengineered vascular tissues may also serve as a method to produce in vitro models to help further our understanding of vascular diseases, as well as facilitate pre-clinical screening of vascular tissue responses to pharmacologic therapies

Propriétés supramoléculaires des cations diimidazolium disubstitués : des complexes d’inclusion en solution aux interactions à l’état cristallin et cristal liquide

Les sels d’imidazolium ont un rôle important dans certaines protéines et acides nucléiques et ont été utilisés à de nombreuses reprises dans des assemblages supramoléculaires en raison de leurs propriétés uniques. Les sels de diimidazolium dérivés sont toutefois moins connus. Ils ont pour l’instant uniquement été utilisés comme des précurseurs de carbènes N-hétérocycliques. Ils sont donc à la base de plusieurs catalyseurs utilisés pour des réactions de couplage croisés mais leurs propriétés sont toutefois méconnues dans le cadre de la chimie supramoléculaire. Cette classe de composés a nottament attiré notre attention en raison de la facilité de modification de leurs propriétés physico-chimiques par modification de leur structure chimique. L’objectif général des travaux présentés dans cette thèse est l’étude des propriétés supramoléculaires des sels de diimidazolium disubstitués en solution (aqueuse ou organique), ainsi qu’en phase solide ou cristal-liquide. L’influence de l’espaceur entre les deux noyaux imidazolium ainsi que l’influence des substituants latéraux et des contre-ions a été étudiée. Dans un premier temps, les propriétés de complexation des sels de diimidazolium à des macrocycles sont étudiées. Les sels bromure sont étudiés en solution aqueuse avec plusieurs cyclodextrines et le cucurbit[7]uril, et les sels hexafluorophosphate sont étudiés en solution organique pour leur complexation avec l’éther couronne DB24C8 et un calix[4]arène. Cette nouvelle classe de composés a montré de très bonnes propriétés de complexation à ces différents macrocycles en solution et a également permis de contrôler différents assemblages supramoléculaires à l’interface air-eau. Dans un deuxième temps, l’étude des sels de phénylènediimidazolium a permis de modifier les propriétés de complexation en solution pour obtenir la formation de complexes multiples avec le cucurbit[7]util en solution aqueuse. Cette même famille de composés a également permis la formation de cristaux liquides ioniques lorsque les substituants sont des chaînes alkyles plus longues. La résolution de plusieurs structures cristallines de différents sels de diimidazolium a finalement permis de comprendre la nature des interactions intermoléculaires à l’état cristallin. La recherche présentée dans cette thèse a donc permis une étude détaillée des propriétés supramoléculaires des sels de diimidazolium dans tous les états de la matière qui leur sont accessibles.Imidazolium salts play an important role in different proteins and nucleic acids and have been used many times in supramolecular assemblies due to their unique properties. Diimidazolium salts derived from imidazolium salts are less known. To date, they have only been used as precursors for N-heterocyclic carbenes, which are used to catalyze various cross-coupling reactions. Their properties are not well known in supramolecular chemistry. This class of compounds attracted our attention because of the ease of tuning their properties by modifying their chemical structure. The main goal of the research presented in this thesis was to study the supramolecular properties of disubstituted diimidazolium salts in solution (aqueous or organic solution), in the solid state and in the liquid-crystalline state. The role of the spacer between the two imidazolium moieties, of the sidechains and of the counterions was studied. Firstly, the complexation between diimidazolium salts and various macrocycles was studied. Bromide salts were studied in aqueous solution with cyclodextrins and cucurbit[7]uril, while hexafluorophosphate salts were studied in organic solution with a DB24C8 crown ether and a calix[4]arene. This novel class of compounds showed very promising complexation properties with these macrocycles in solution and also allowed us to control the formation of various supramolecular assemblies at the air-water interface. Secondly, phenylenediimidazolium salts were studied and allowed the modification of the complexation properties in aqueous solution. Multiple complexes can be formed simultaneously with cucurbit[7]uril in aqueous solution. The same class of compounds also has the ability to yield ionic liquid crystals when the alkyl sidechains are long. The resolution of the crystalline structures of some synthesized diimidazolium salts allowed us to understand the nature of the intermolecular interactions in the solid state. The research presented in this thesis is a complete study of the supramolecular properties of diimidazolium salts in every accessible state of matter

Tailoring the architecture of 3d porous scaffolds made of plla for regenerative medicine

Nel presente lavoro, abbiamo investigato gli effetti delle variabili di processo, come la temperatura di raffreddamento (-30°C 0 Tc 0 +5°C), tempo di raffreddamento (2.5 h 0 tc 0 32 h), e la concentrazione del polimero (da 1.5 a 6.5 wt%), sulla microstruttura dei pori di scaffold tridimesionali in poly(L-lactic acid) (PLLA) ottenuti per dTIPS. Gli scaffold hanno mostrato zone dendritiche ad elevato ordine, alle quali spetta il compito di fungere da struttura guida per i vasi sanguigni in crescita. Gli stessi hanno mostrato porosità totale fino al 95%, ed un grado di interconnessione maggiore del 90%.Pori di poche decine di micron sono stati ottenuti con la maggiore tc, concentrazione di PLLA, e la minor Tc. Dall !"altro lato, alla minore tc e concentrazione di polimero, e mantenendo il sistema alla Tc appena al di sotto della temperatura di congelamento, siamo riusciti a spingere il diametro dei pori fino a 260 m

Modular soft pneumatic actuator system design for compliance matching

The future of robotics is personal. Never before has technology been as pervasive as it is today, with advanced mobile electronics hardware and multi-level network connectivity pushing âsmartâ devices deeper into our daily lives through home automation systems, virtual assistants, and wearable activity monitoring. As the suite of personal technology around us continues to grow in this way, augmenting and offloading the burden of routine activities of daily living, the notion that this trend will extend to robotics seems inevitable. Transitioning robots from their current principal domain of industrial factory settings to domestic, workplace, or public environments is not simply a matter of relocation or reprogramming, however. The key differences between âtraditionalâ types of robots and those which would best serve personal, proximal, human interactive applications demand a new approach to their design. Chief among these are requirements for safety, adaptability, reliability, reconfigurability, and to a more practical extent, usability. These properties frame the context and objectives of my thesis work, which seeks to provide solutions and answers to not only how these features might be achieved in personal robotic systems, but as well what benefits they can afford. I approach the investigation of these questions from a perspective of compliance matching of hardware systems to their applications, by providing methods to achieve mechanical attributes complimentary to their environment and end-use. These features are fundamental to the burgeoning field of Soft Robotics, wherein flexible, compliant materials are used as the basis for the structure, actuation, sensing, and control of complete robotic systems. Combined with pressurized air as a power source, soft pneumatic actuator (SPA) based systems offers new and novel methods of exploiting the intrinsic compliance of soft material components in robotic systems. While this strategy seems to answer many of the needs for human-safe robotic applications, it also brings new questions and challenges: What are the needs and applications personal robots may best serve? Are soft pneumatic actuators capable of these tasks, or âusefulâ work output and performance? How can SPA based systems be applied to provide complex functionality needed for operation in diverse, real-world environments? What are the theoretical and practical challenges in implementing scalable, multiple degrees of freedom systems, and how can they be overcome? I present solutions to these problems in my thesis work, elucidated through scientific design, testing and evaluation of robotic prototypes which leverage and demonstrate three key features: 1) Intrinsic compliance: provided by passive elastic and flexible component material properties, 2) Extrinsic compliance: rendered through high number of independent, controllable degrees of freedom, and 3) Complementary design: exhibited by modular, plug and play architectures which combine both attributes to achieve compliant systems. Through these core projects and others listed below I have been engaged in soft robotic technology, its application, and solutions to the challenges which are critical to providing a path forward within the soft robotics field, as well as for the future of personal robotics as a whole toward creating a better society

NASA technology utilization survey on composite materials

NASA and NASA-funded contractor contributions to the field of composite materials are surveyed. Existing and potential non-aerospace applications of the newer composite materials are emphasized. Economic factors for selection of a composite for a particular application are weight savings, performance (high strength, high elastic modulus, low coefficient of expansion, heat resistance, corrosion resistance,), longer service life, and reduced maintenance. Applications for composites in agriculture, chemical and petrochemical industries, construction, consumer goods, machinery, power generation and distribution, transportation, biomedicine, and safety are presented. With the continuing trend toward further cost reductions, composites warrant consideration in a wide range of non-aerospace applications. Composite materials discussed include filamentary reinforced materials, laminates, multiphase alloys, solid multiphase lubricants, and multiphase ceramics. New processes developed to aid in fabrication of composites are given

Cosmographia Metallica

In his book The Living Rock, historian A.J. Wilson writes, “The history of metals is the history of civilization”. From its beginnings in simple toolmaking, to modern-day machinery and weapons, the development of metallurgy ranks second only to agriculture in its impact on human society; to be sure, civilization as we know it would not exist without the discovery and use of metals. Modern man is familiar with dozens of metals, some common, some precious, some useful, some deadly. It is remarkable then to consider that, until the late Middle Ages, there were only seven metals known in their elemental form: gold, silver, copper, iron, lead, tin and mercury. For thousands of years, until isolated zinc was recognized in the fifteenth century, mankind would rely on what we now refer to as the seven classical metals. Through a survey of their historical, scientific, cultural, utilitarian and mythological characteristics, the seven classical metals are investigated. From this research, common threads emerge that suggest a spirit or essence of each metal, expressed as the metal’s imperative. The concept of the imperative embodies more than the metal’s physical characteristics, symbolism and history; it suggests that each metal is compelled to be considered and used in a particular way. It is an idea that metals are not simply passive materials, but possessing of an active and influential force. The metal imperatives, projected onto a barren site in the Sudbury area, are used as generators for the design of seven metal spaces. On this expansive landscape, the interventions communicate the natures of gold, silver, copper, iron, lead, tin and mercury against a backdrop of earth and sky

Implantable Electrodes for Upper Limb Prosthetic Control

This thesis describes a study investigating implantable interfaces with muscles and peripheral nerves. Current prostheses for upper limb amputees do not provide intuitive control over hand, wrist and elbow motion. By implanting electrodes for recording and stimulating onto muscles and into nerves in the amputation stump a greater number of control signals may be made available, signals which will be used to control dextrous hand movements. An implantable epimysial interface was developed using a bone-anchored device to hard-wire signals across the skin barrier. In a single ovine model pilot study the bone-anchor was implanted transtibially and the epimysial electrode was place superficially to m. peroneus teritus. Physiological signals were obtained over 12 weeks during treadmill walking. The external connector on the bone-anchor failed at 12 weeks, correlating with a drop in signal quality in an otherwise robust interface integrated with bone and skin tissue. The ovine bone-anchor model was repeated in 6 sheep for 19 weeks, with epimysial recordings made regularly. Increasing signal quality was seen during the study and was significantly greater from implanted electrodes compared with skin surface electrodes at 19 weeks (p = 0.016). Some complications with skin-implant integration were observed in proximally located implants. Crosstalk between muscles was assessed using pre-terminal nerve stimulation, and was found to be dependent upon muscle location and innervation. The ovine m. peroneus teritus model was used to assess recovery following targeted muscle reinnervation. Muscle signal recovery was observed approximately one month after surgery correlating with the start of functional recovery (assessed by force plate analysis). These studies indicate that a suitably modified bone-anchored device may be suitable for signal transmission in human patients, providing a stable, long-term solution to both prosthesis attachment and control. The potential of nerve interfaces for prosthetic control was investigated. The microchannel neural interface (MNI) was chosen because it overcomes limitations with other neural microarray designs: signal strength; cross-talk, and the locations of Nodes of Ranvier. MNIs confine regenerating nerves to small, ∼ 100 µm diameter, insulating tubes, this increases the length within which nerve signals can be recorded and amplifies the recorded signals. However, in vivo MNIs can become occluded by fibrosis that reduces or prevents axon regeneration. Two in vitro studies of neurocompatibility were carried out to investigate strategies for improving axon regeneration within microchannels. The first in vitro study compared the effect of different adsorbed endoneurial basement membrane proteins on PC-12 cell neurite extension on silicone substrates. The optimal protein coating concentrations for poly-D-lysine, collagen-IV and laminin-2,(-4) were determined. The optimal concentrations were compared with mixtures of basement membrane proteins, the effect of mixture coating order and constitution were investigated. It was found that endoneurial BM proteins significantly enhance neurite outgrowth compared with controls. Two coatings were suggested as most suited for improving neural regeneration within microchannels: a single layer coating of 10 µg/cm2 collagen-IV; and a mixed coating of 10 µg/cm2 collagen-IV, 1 µg/cm2 laminin-2,(-4), and 0.175 µg/cm2 nidogen-1. The second in vitro study investigated the effect of grooved, roughened and multi-scale silicone surfaces on on PC-12 cell neurite extension. Deeper, narrower grooves were shown to increase the extent of neurite alignment, while resulting in fewer, longer, neurites. Roughening surfaces was shown to increase the amount of protein (collagen-IV) which adsorbed from solution and increase the number of neurites each cell extended. Surfaces with multiscale topographies synergistically increased the number and length of neurites and guided neurite growth along the groove direction. MNIs were manufactured for in vivo testing. These MNIs were used to determine the effect of adsorbed endoneurial basement membrane proteins on nerve regeneration in vivo, but the multiscale topographies were not applied during manufacturing. Four alternative manufacturing methods were investigated and iterative improvements were made to create a stacked interface with multiple microchannel layers. Microchannel layers were created by laser patterning silicone and metal foil components, followed by plasma bonding to create a 3-dimensional structure with 150 µm deep, 200 µm wide microchannels. Electrode impedances of 27.2 ± 19.8 kΩ at 1kHz were achieved by DC etching. The method overcomes some current limitations on electrode connectivity and microchannel sealing, and may improve recording capabilities over single layer designs by increasing the ratio of electrodes to microchannels. Manufactured MNIs were tested in a rat sciatic nerve transection model. Following implantation nerves were allowed to regenerate for one and two months. First, suture and fibrin glue were compared as MNI fixation methods for one month, the nerve regenerated within the fibrin glue, outside the interface lumen, therefore sutures were chosen as a long term fixation method. The influence of endoneurial basement membrane protein coatings, identified previously, on nerve regeneration with MNIs was investigated. Nerves regenerated through the MNIs over two months and began to reinnervate the distal limb. Improvements in the sciatic function index were observed over two months, with no significant differences between protein coated and control interfaces. Some weak histological evidence for the use of protein coatings was found, with axon diameters increased distal to protein coated MNIs. Electromyographic and electroneurographic recordings demonstrated similar signal amplitudes to previous studies. In order to bring the research described in this thesis to clinical practice further engineering improvements to the design and manufacture of electrodes, which utilise materials or coatings to enhance neurocompatibility, is required. Avenues for further research are discussed and additional experiments and investigations are described. By combining developments in implantable muscle and nerve interfaces with surgical techniques and improvements in neurocompatibility the promise of upper limb prosthetic control may be realised