3D printing technology is rapidly advancing and is being increasingly used for biological applications. The spatial control of 3D printing makes it especially attractive for fabricating 3D tissues and for studying the role of geometry in biology. We utilized two different types of 3D printing to engineer vascularized tissues with complex vascular architectures, to use engineered vasculature to treat ischemia, and to study directional endothelial cell migration on curved wave topography. To engineer 3D tissues, perfusable vascular networks must be embedded within the tissue to supply nutrients and oxygen to cells. 3D-printed sugar filaments have previously been used as a cytocompatible sacrificial template to rapidly cast vascular networks. We improved upon the 3D-printed sugar method and used it to fabricate complex vascular geometries that were not previously possible, such as a branched channel geometry, with controlled fluid flow through the channels. We also integrated an approach utilizing vascular self-assembly to generate thick tissues with dense, capillary-scale vessel networks. The vascularized tissues fabricated using 3D-printed sugar successfully integrated with a host vasculature upon implantation and restored perfusion in two different animal models of ischemia. Cell migration critical to numerous biological processes can be guided by surface topography. However, fabrication limitations constrain topography studies to geometries that may not adequately mimic physiological environments. Direct Laser Writing (DLW) provides the necessary 3D flexibility and control to create well-defined curved waveforms similar to those found in physiological settings, such as the lumen of blood vessels. We found that endothelial cells migrated fastest along square waves, intermediate along triangular waves, and slowest along sine waves and that directional cell migration on sine waves decreased at longer sinusoid wavelengths. Interestingly, inhibition of Rac1 decreased directional migration on 3D sine waves but not on 2D micropatterned lines, suggesting that cells may utilize different molecular pathways to sense curved topographies. Our study demonstrates that DLW can be employed to investigate directional migration on a wide array of surfaces with curvatures that are unattainable using conventional manufacturing techniques. / 2020-10-22T00:00:00Z

The thesis aims at investigating the connection between nucleus mechanical characteristics with pluripotency state and differentiation associated with altered cell gene expression levels. The project investigates the deformation characteristics of the cell nucleus during unconfined compression in a 3D cell-seeded agarose constructs. The studies report modification in the mechanical behaviour of the nucleus in different embryonic stem cell phenotypes based on various pluripotent states (naïve or primed states) or following triggering of early differentiation. A multi-scale model is also presented to simulate dynamic details of mechanical perturbation to cells during compression. The first chapter presents a review of the relevant literature to introduce current progress in the related research field and the second chapter describes the general methods used in the thesis including cell culture, agarose construct preparation, construct compression and microscopy recording. The third chapter presents findings of studies involving the application of compression to embryonic stem cells in naïve and primed sate within agarose scaffolds. A range of parameters relating to the relative cell/nucleus morphological modifications are recorded with analysis and discussion. Chapter four presents studies that investigate the early differentiation of embryonic stem cells from either the naïve and primed pluripotency, achieved by altering cell culture condition, and further reveals the nuclear mechanical characteristic changes. The fifth chapter describes a multi-scale model developed to simulating the 3D cell-seeded agarose compression reported in previous chapters. This model is also used to estimate cell mechanical parameters and show accurate deformation detail in different locations within the construct. A final discussion of the thesis is provided in chapter 6 with a plan for future work.

En cancérologie cervico-faciale, la couverture d’éléments nobles ou de pertes de substance profondes fait souvent appel à l’utilisation de lambeaux locaux, locorégionaux ou libres. La réalisation de lambeaux autologues nécessite de sacrifier une structure indemne au profit de la structure lésée. Lorsque la réalisation d’un lambeau n’est pas envisageable, l’utilisation de substitut dermique peut être une alternative.L’ingénierie tissulaire permet de cultiver presque tous les types cellulaires (cellules différenciées, cellules souches) seuls ou en association (pe peau totale reconstruite) avec des matrices telles que le collagène pour obtenir des cultures tridimensionnelles. Utilisées in vivo en tant que substrat dermique, les matrices de collagène vont servir de guide aux cellules de l’hôte qui vont la coloniser, synthétiser leur propre matrice extra-cellulaire et développer un réseau vasculaire.Les tissus reconstruits se comportent comme des greffes : leur nutrition se fait d’abord par imbibition à partir du site receveur, puis par recolonisation vasculaire à partir du lit de la greffe. L’épaisseur du tissu greffable est donc limitée.Une revue de la littérature sur la peau reconstruite par ingénierie tissulaire et des différentes stratégies de vascularisation d’un tissu reconstruit est présentée. Le but de notre travail était d’évaluer les capacités des substituts dermiques comme vecteur de cellules souches pour la régénération tissulaire in vivo et comme support au développement d’une neovascularisation à partir d’un vaisseau sanguin ouvrant la voie au lambeau microanastomosable reconstruit in vitro. / In head and neck cancer, coverage of noble elements or deep wounds often requires the use of local, locoregional or free flaps. Autologous flaps consist of transferring the patient’s own tissues, but require sacrifice of the healthy structure to replace the damaged structure. When performing a flap is not an option, the use of dermal substitute may be an alternative.Tissue engineering is a rapidly growing discipline comprising multiple fields of research. Almost all cell types can be cultured alone or in combination (e.g. reconstructed full-thickness skin) with matrices such as collagen to obtain three dimensional cultures. Collagen matrices, used in vivo as dermal substrates, are used to guide the growth of host cells (fibroblasts, endothelial cells, etc.) that colonize the matrix, synthesize their own extracellular matrix and develop a vascular network.Reconstructed tissues behave like tissue grafts: nutrition of these tissues is initially based on diffusion from the recipient site and then by vascular recolonization from the bed of the graft. The thickness of graftable tissue is therefore limited.A review of the literature on skin tissue engineering and current strategies to create vascularized tissue is presented. The aim of our study was to evaluate the capacity of dermal substitutes as a carrier of stem cells for tissue regeneration in vivo and as a support to the development of neovascularization from a blood vessel opening the way to flap suitable for microvascular transplantation reconstructed in vitro.

Cardiovascular disease (CVD) is broadly characterized by a loss of global function, exacerbated by a very limited ability for the heart to regenerate itself following injury. CVD remains the leading cause of death in the United States and the leading citation in hospital discharges. The overall concept of this dissertation is to investigate the use of biophysical signals that drive physiologic maturation of myocardium, and lead to its deterioration in disease. By incorporating biophysical signaling into cardiac tissue engineering methods, the aim is to generate high fidelity engineered platforms for cell delivery and maturation of surrogate muscle, while understanding the cues that lead to pathological cell fate in disease. The first part of this thesis describes the development of a composite scaffold, derived from human myocardium, to use as a delivery platform of mesenchymal stem cells to the heart. Through biochemical signaling, we are able to modulate MSC phenotype, and propose a mechanism through which angio- and arteriogenesis of the heart leading to global functional improvements, following myocardial infarction, may be attributed. We further demonstrate cardioprotection of host myocardium in a setting of acute injury by exploiting non-invasive radioimaging techniques. The mechanism through which we can attribute cell mobilization to the infarct bed is further explored in patient-derived myocardium, to understand how this pathway remains relevant in chronic heart failure. The second focus of the thesis is the use of electro-mechanical stimulation to generate high fidelity Engineered Heart Muscle (EHM). We report that electro-mechanical stimulation of EHM at near-physiologic frequency leads to development and maturation of Calcium handling and the T- tubular network, as well as improved functionality and positive force frequency relationship. Lastly, we return to human myocardium as platform understand regulation of cardiomyocyte function by the extracellular matrix. Here, we seek to understand how the ECM from different disease states (eg. non-diseased, ischemic, non-ischemic) affects cell phenotype. Specifically, can bona fide engineered myocardium successfully integrate and remodel diseased ECM? Using stem cell derived cardiomyocytes and patient-derived decellularized myocardium to generated engineered myocardium (hhEMs), we report that hhEMs mimic native myogenic expression patterns representative of their failing- and non-failing heart tissue.

Functional integration of bone with soft tissues such as tendon is essential for joint motion and musculoskeletal function. This is evident in the rotator cuff of the shoulder, which consists of four muscles and their associated tendons that connect the humerus and scapula. The cuff functions to stabilize the shoulder joint, and actively controls shoulder kinematics. Rotator cuff injuries often occur as a result of tendon avulsion at the tendon-bone interface, with more than 250,000 cuff repair surgeries performed annually in the United States. However, these procedures are associated with a high failure rate, as re-tears often occur due to the lack of biological fixation of the tendon to bone post-surgery. Instead of regenerating the tendon-bone interface, current repair techniques and augmentation grafts focus on improving the load bearing capability of the repaired rotator cuff. Biologically, the supraspinatus tendon inserts into bone via a biphasic fibrocartilaginous transition, exhibiting region-dependent changes in its compositional, structural and mechanical properties, which enables efficient load transfer from tendon to bone as well as multi-tissue homeostasis. Inspired by the native tendon-bone interface, we have designed and evaluated a biomimetic bilayer scaffold, comprised of electrospun poly (lactide-co-glycolide) (PLGA) nanofibers seamlessly integrated with PLGA-hydroxyapatite (HA) fibers, in order to engineer tendon-bone integration. The objective of this thesis is to explore the key design parameters that are critical for integrative tendon-bone repair using this biphasic scaffold as a model. Specifically, intrinsic to the scaffold, effects of fiber alignment, fiber diameter, mineral distribution, and polymer composition on integrative rotator cuff tendon-bone healing were evaluated in vivo using a rat model. Results indicated that an aligned, nanofiber-based scaffold with a distinct order of non-mineralized and mineralized regions will lead to insertion regeneration and integrative tendon-bone repair. Additional tissue engineering design parameters such as healing time and animal model were also tested. It was observed that the biphasic scaffold exhibited a stable long term response, as the mechanical properties of rat shoulders repaired by this scaffold remained comparable to that of the control at 20 weeks post-surgery. This scaffold was also evaluated in a large animal model (sheep), in which a clinically-relevant rotator cuff repair procedure was implemented with the biphasic scaffold. Results demonstrated the scaffold lead to integrative rotator cuff repair through the regeneration of the enthesis in both small and large animal models. In summary, through a series of in vivo studies, the work of this thesis has identified the critical tissue engineering parameters for integrative and functional rotator cuff tendon repair. More importantly, the design principles elucidated here are anticipated to have a broader impact in the field of tissue engineering, as they can be readily applied towards the regeneration of other soft-hard tissue interfaces.

Articular cartilage is a hydrated soft tissue with a fibrous solid matrix characterized by high porosity and low permeability. It is the bearing material of diarthrodial joints, permitting motion and transmitting loads with extraordinarily low friction. This function may be disrupted pathologically by osteoarthritis, a disease where cartilage becomes weakened and eroded. Osteoarthritis creates pain during normal activities like walking or grasping, thus diminishing quality of life. The disease affects nine percent of Americans and is one of the leading causes of disability worldwide. There is presently no cure or prevention for osteoarthritis, only palliative treatments designed to help patients manage pain and regain mobility. New such treatments are developed in part by advancing the science of cartilage mechanics, structure and function, and this dissertation presents novel contributions toward this effort: Chapters 2, 3, and 4 enhance our knowledge of the structure-function relationships critical to our understanding of cartilage friction and load support. Whereas most prior theoretical and experimental studies have focused on the analysis of small cylindrical explants, or idealized joint geometries such as cylindrical or spherical articular layers, these chapters describe novel investigations performed on whole articular layers of the shoulder and knee joints. Insights from these investigations have a direct impact on our formulation of design objectives in cartilage tissue engineering, whose purpose is to grow constructs that reproduce the functional properties of native cartilage. The studies presented in this chapter are critical to ongoing tissue engineering studies in our laboratory, which has pioneered the development of anatomically sized cartilage constructs. Finally, Chapter 5 describes the development of a novel clinical treatment for thumb osteoarthritis that uses bent osteochondral allografts (living bone and cartilage from human donors) to replace the eroded thumb trapezial articular layer with a healthy and thick articular layer from another joint such as the knee. This highly promising treatment strategy overcomes the limitation of size mismatch between donor and recipient which had relegated osteochondral allograft surgery to a niche treatment. Like other fibrous tissues, cartilage exhibits tension-compression nonlinearity, meaning it can be 100 times stiffer in tension than in compression. Tension-compression nonlinearity allows compressive physiologic joint loads to be supported by tensile stress within the collagen fibers and elevated fluid pressure, effectively shielding the solid matrix from compressive load. According to theory, fluid load support derives directly from tension-compression nonlinearity. Fluid load support is also a dominant mechanism of cartilage lubrication. Because cartilage is 80 to 90% water, most of the contact traction on the porous cartilage surface takes the form of hydrostatic fluid pressure. Friction forces only occur upon solid-on-solid contact, so cartilage friction is nearly negligible, even for joint contact forces that may routinely exceed three or four times the body’s total weight. The dependence of friction on fluid load support is demonstrated by experiments that simultaneously measure interstitial fluid pressure and friction - a transient rise in friction occurs as pressure subsides and fluid drains from the tissue. These structure-function relationships have been identified over decades of research, mostly through small cartilage explant studies, which have supported hypothesized mechanisms under non-physiologic conditions. Therefore, in situ studies utilizing intact, naturally-congruent articular surfaces under physiologic loading conditions would significantly extend and validate these principles. For example, friction may rise nearly 100-fold after only 1 hour in cartilage explant experiments, yet there is no evidence that normal daily activities spanning 16 hours or more lead to cartilage damage. Can fluid load support sustain low friction under these relatively harsh conditions? To date, no study has examined this question, so Chapter 2 of this work addresses the hypothesis that the friction coefficient of diarthrodial joints can remain low over a full day of loading at physiologic speeds and load magnitudes. Another question that may be uniquely addressed by an in situ analysis is: What is the complete state of stress within naturally-congruent cartilage layers? A primary hypothesis for the initiation and progression of osteoarthritis is that the state of stress within articular cartilage may exceed a threshold beyond which the tissue is unable to repair itself. Since the complete stress tensor within a material is immeasurable, techniques such as finite element analysis must be used to examine the state of stress. Additionally, a theoretical framework such as mixture theory may be used to examine the stresses in the fluid and solid constituents of the tissue separately, making it possible to test theories of solid matrix damage. Chapter 3 of this work uses this strategy to examine the hypothesis that physiologic solid matrix stresses within anatomically-shaped, biphasic, tension-compression nonlinear cartilage layers are primarily tensile, despite the fact that the articular layers are loaded in compression. The proteoglycan content of articular cartilage gives the tissue an osmotic swelling pressure that is resisted by tensile stresses in the collagen fibrils, even in the absence of external loads. This charge effect may be additionally incorporated into a mixture theory finite element analysis to examine the role of osmotic swelling on the solid matrix stresses in a physiologic, in situ analysis. This capability has only been developed recently and is explored for the first time in Chapter 4. The final part of this work translates basic cartilage science into a clinical therapy for thumb joint osteoarthritis, a common site for this disease. The current gold-standard treatment for thumb joint osteoarthritis replaces the trapezium bone with a soft-tissue tendon autograft, relieving pain but significantly weakening hand strength. Living osteochondral allograft transplantation may provide a relatively straightforward treatment alternative, though this procedure has not been used for the thumb due to the inadequate availability of suitable allografts. The ideal allograft would have a relatively thick articular layer to provide sufficient compliance for promoting joint congruence with the mating metacarpal surface, and surface curvatures that match the saddle-shaped anatomy of the distal trapezial articular surface to reproduce the normal joint motions. A potential solution that would provide suitable trapezium osteochondral allografts for patients involves precisely machining and bending allografts from a lower extremity joint with thicker cartilage, such as the distal femoral surface of the knee, to match the shape and curvature of the trapezium. Such bent osteochondral allografts would provide all the desired benefits of the ideal arthroplasty. Chapter 5 outlines the development of this novel technology, including proof of concept and feasibility demonstrations, business strategy and market analysis.

A Engenharia de Tecidos tem como objetivo desenvolver alternativas para o tratamento de doenças degenerativas e regeneração de tecidos lesionados. O princípio básico da Engenharia de Tecidos é promover o crescimento de células sobre um substrato. Esse substrato deve reproduzir a matriz extracelular, influenciando a diferenciação e as funções celulares. Para isso, neste trabalho, nanopartículas poliméricas de poli(3-hidroxibutirato-co- 3-hidroxivalerato) (PHBHV), com diferentes diâmetros hidrodinâmicos, foram sintetizadas pelo método de emulsão-evaporação do solvente. A concentração do surfactante, a velocidade de agitação durante a emulsificação e o tempo de agitação foram variados a fim de se analisar a influência desses parâmetros no diâmetro hidrodinâmico, índice de polidispersidade e estabilidade coloidal das emulsões. Os parâmetros que mais influenciaram no diâmetro hidrodinâmico e no índice de dispersidade foram a concentração de surfactante e a amplitude de sonicação da emulsão, respectivamente. Após a obtenção das dispersões com estabilidade coloidal, realizou-se a modificação química da superfície dessas partículas utilizando os aminosilanos, 3-aminopropiltrimetoxisilano (APTMS) e 3- aminopropiltrietoxisilano (APTES). Essa modificação química na superfície das nanopartículas teve como objetivo a mimetização da matriz extracelular, permitindo a adesão e proliferação das células. As partículas modificadas foram caracterizadas por espalhamento de luz dinâmico (DLS), microscopia de força atômica (AFM), calorimetria exploratória diferencial (DSC), Espectroscopia de Infravermelho por Transformada de Fourier (FTIR) e Ressonância Magnética Nuclear de Hidrogênio (RMN 1H). Os resultados de FTIR mostraram o aparecimento de pico referente ao grupo amino, que foi indicativo da modificação química da superfície das nanopartículas. Os resultados de RMN 1H mostraram o sinal dos grupos CH3 do silano APTMS no espectro das nanopartículas modificadas por APTMS, e nas partículas modificadas por APTES, foi possível identificar o sinal referente aos grupos CH2 do APTES. A partir desses resultados comprovou-se a modificação química da superfície das nanopartículas pelos aminosilanos. / Tissue engineering aims to develop alternatives to treat damaged tissues by promoting tissue regeneration. The basic principle of Tissue Engineering is to promote the growth of cells on a substrate. This substrate must reproduce the extracellular matrix, influencing particular cell functions and differentiation fate. For this purpose, at the present work, polymeric nanoparticles of poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBHV) with different hydrodynamic diameters were synthesized by emulsion-solvent evaporation technique. The concentration of the surfactant, stirring speed during emulsification and stirring time were varied in order to analyze the influence of these parameters on hydrodynamic diameter, polydispersity and colloidal stability. The parameters that most influenced the hydrodynamic diameter and polydispersity index were the variation in the surfactant concentration and the variation of emulsion sonication amplitude, respectively. After that, the nanoparticles had their surface modified by 3-aminopropyltrimethoxysilane (APTMS) and 3- aminopropyltriethoxysilane (APTES). The aim of this chemical modification was to mimic the extracellular matrix, allowing the adhesion and proliferation of cells. The modified particles were characterized by dynamic light scattering (DLS), atomic force microscopy (AFM), differential scanning calorimetry (DSC), Fourier Transform Infrared Spectroscopy (FTIR) and proton nuclear magnetic resonance (1H NMR). The FTIR results showed a peak of the amino group, which was an indicative of the nanoparticles surface chemical modification. 1H NMR results showed the signal of the CH3 groups of the APTMS silane in the spectrum of the APTMS-modified nanoparticles and in the APTES-modified particles it was possible to identify signal relating to the CH2 groups of the APTES. From these results, it was verified the nanoparticles surface chemical modification by the aminosilanes.

Bone undergoes constant turnover throughout life and has the capacity to regenerate itself. However, the repair of critical size defects, caused by bone diseases such as osteoporosis, can be more problematic. Therefore, there is a clinical need for a bone graft substitute that can be used at sites of surgical intervention to enhance bone regeneration. Poly(vinylphosphonic acid-co-acrylic acid) (PVPA-co-AA) has recently been identified as a potential candidate for use in bone tissue scaffolds. It is hypothesised that PVPA-co-AA can mimic the action of bisphosphonates – a class of drugs used in the treatment of osteoporosis – by binding to calcium ions from bone mineral surfaces. In this way, bisphosphonates can affect bone turnover by increasing the activity of osteoblasts and reducing osteoclast activity. Although PVPA-co-AA has been shown to improve bone formation, the mechanism of action has so far not been fully elucidated. Therefore, this work aims to understand the effect of copolymer composition on the properties of PVPA-co-AA, and thus to determine its effect on osteoblast adhesion and proliferation. PVPA-co-AA copolymers have been synthesised with a range of monomer feed ratios. It was found that a VPA content of 30 mol % led to the greatest calcium binding affinity of the copolymer and is thus expected to lead to enhanced bone formation and mineralisation of the matrix produced by osteoblast cells. The release profile of PVPA-co-AA from electrospun PCL scaffolds was investigated. It was shown that all of the PVPA-co-AA was released into aqueous media within 8 h of immersion. It was also found that the calcium chelation from osteogenic differentiation media significantly increased within the first 8 h. Therefore, it was concluded that PVPA-co-AA is released from the scaffolds, where it can then bind to calcium ions from the bone mineral surface to promote mineralisation, thus acting as a mimic of non-collagenous proteins, which are present in the extracellular matrix (ECM) of bone. Hydrogels of PVPA-co-AA have been produced and the effect of monomer feed ratio (0-50 mol % VPA) on the properties of the gels was explored. It was found that an increase in VPA content led to greater hydrogel swelling and increased porosities. Hydrogels that contained 30 and 50 mol % VPA were shown to have similar morphologies to the native ECM of bone. Rheological testing showed that hydrogels with higher VPA contents were more flexible and could be deformed to a large extent without permanent deformation of their structure. An increase in osteoblast adhesion and proliferation was observed for hydrogels with 30 and 50 mol % VPA content as well as superior cell spreading. Osteoblast cell metabolic activity also increased as a function of VPA content in the hydrogels. This work indicates that hydrogels of PVPA-co-AA, with VPA contents of 30 or 50 mol %, are ideal for use as bone tissue scaffolds. Furthermore, the mechanical and cell adhesion properties of the gels can be tuned by altering the copolymer composition. Finally, composite hydrogels of PVPA-co-AA and hydroxyapatite (HA) have been produced and investigated for their ability to remove fluoride ions from groundwater. It was found that the fluoride uptake ability of PVPA-HA hydrogels was significantly enhanced when compared with HA powder alone. Furthermore, the fluoride uptake was dependent on many factors, including pH, contact time and the presence of competing ions. It was possible to regenerate the hydrogel to remove the fluoride ions, and thus it was shown that the material can be used a number of times with only a slight reduction in its fluoride uptake capacity.

<p>Validerat; 20130918 (global_studentproject_submitter)</p>

Pre-clinical studies are a necessary step in the process of material and drug testing. For this, high-throughput monolayer cell cultures are conducted followed by in vivo animal experiments. However, animal use is ethically questionable and in many cases yields misleading results. In vitro three dimensional (3D) tissue engineered (TE) structures have been shown to better represent in vivo tissue morphology and biochemical pathways than monolayer cultures and are less ethically questionable than animal models. Therefore, an in vitro biomimetic musculoskeletal junction (MSKjct) is required as a more relevant pre-clinical testbed. This thesis describes the steps taken to co-culture 3D TE skeletal muscle and bone models as a material testbed and towards an in vitro MSKjct.