The biodegradability and when functionalised biocompatibility of aliphatic polycarbonates (APCs) makes them an attractive class of materials for biomedical applications such as tissue engineering scaffolds and drug-delivery carriers. One route to accessing a wide-range of well-defined and functional APCs is the controlled ring-opening polymerisation (ROP) of cyclic carbonates. In turn, these would ideally be prepared by the direct coupling of CO2 with diols to give water as the only by-product. In this way, the combination of CO2 and sugar-derived diols draws upon two natural renewable building blocks for the construction of polycarbonates that are anticipated to show good biocompatibility properties. Chapter 2 develops a simple and mild alternative to the traditional use of phosgene derivatives for the synthesis of six-membered cyclic carbonates from 1,3-diols and CO2. DFT calculations highlighted the need to lower both the CO2-insertion and ring-closing kinetic barriers to cyclic carbonate formation. Organic superbase, 1,8- diazabicyclo[5.4.0]undec-7-ene (DBU) enabled the formation of carbonate species at 1 atm CO2 pressure whereas, the introduction of a leaving group strategy lowered the cyclisation barrier. Mechanistic considerations suggested a kinetic preference for ring- closing via a nucleophilic addition-elimination pathway rather than a SN2-like intramolecular cyclisation. Chapter 3 applies the procedure with CO2 to the preparation of a novel monomer from natural sugar, ᴅ-mannose. ROP was carried out via an organocatalytic approach and a preference for head-tail linkages in the polycarbonate backbone indicated by NMR spectroscopy and supported by DFT calculations. Chapter 4 utilises CO2 to invert the natural stereochemistry of sugars and create a thymidine-based monomer. The thermodynamic parameters of the ROP with 1,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD) catalyst are determined and the properties of the polycarbonates investigated to include preliminary cell attachment studies. Finally, chapter 5 details the synthesis of cyclic carbonates from 2- deoxy-ᴅ-ribose and the investigation into the different ROP behaviour of the α- and β- anomers. The ability to tune the polymer properties through copolymerisation with trimethylene carbonate (TMC) is also discussed.

Many congenital skeletal muscle disorders including muscular dystrophies are caused by genetic mutations that lead to a dysfunction in myocytes effectively binding to the extracellular matrix. This leads to a chronic and continuous cycle of breakdown and regeneration of muscle tissue, ultimately resulting in loss of muscle function and patient mortality. Such disorders lack effective clinical treatments and challenge researchers to develop new therapeutics. The current drug development process often yields ineffective therapeutics due to the lack of genetic homology between pre-clinical animal models and humans. In addition current engineered tissue models using human cells fail to properly emulate native muscle morphology and function due to necrotic tissue cores and an abundance undigested ECM protein. Thus, a more precise benchtop model of 3D engineered human muscle tissue could serve as a better platform for translation to a disease model and could better predict candidate drug efficacy during pre- clinical development. This work presents the methodology for generating a high-content system of contiguous skeletal muscle tissue constructs produced entirely from human cells by using a non-adhesive hydrogel micro-molding technique. Subsequent culture and mold modifications confirmed by morphological and contractile protein analysis improve tissue longevity and myocyte maturation. Finally, mechanical strength and contractile force measurements confirmed that such modulations resulted in skeletal muscle microtissues that were more mimetic of human muscle tissue. This cell self-assembly technique yielded tissues approximately 150um in diameter with cell densities approaching that of native muscle. Modifications including seeding pre-differentiated myoblasts and the addition of ECM producing fibroblasts improved both tissue formation efficiency and cell alignment. Further culture modifications including supplementation of the culture medium with 50ug/ml ascorbic acid and 100ng/ml Insulin-like growth factor-1 coupled with a mold redesign that allowed tissue to passively contract during maturation while still remaining anchored under tension further improved ECM production, myogenic differentiation, and long-term longevity in culture. Further confirmation of the culture improvements were demonstrated by increases in mechanical strength and contractile force production. In conclusion, this approach overcomes cell density limitations with exogenous ECM-based methods and provides a platform for producing 3D models of human skeletal muscle by making tissue entirely using cells. Future work will attempt to translate the methodology used for tissue generation and long-term culture to create benchtop models of disease models of skeletal muscle, streamlining pre- clinical benchtop testing to better predict candidate drug efficacy for skeletal muscle diseases and disorders along with elucidating side effects of non-target drugs.

Every year, 400,000 coronary artery bypasses (CABG) are performed in the United States. However, one third of all patients who need a CABG cannot undergo the procedure because of the lack of suitable autologous blood vessels. Both synthetic and tissue engineered vascular grafts have been used clinically for vascular grafts or other surgical applications, but no small- diameter engineered vessels have yet been successfully used for CABG. The success of vascular tissue engineering is strongly dependent on being able to control tissue contractility and extracellular matrix (ECM) production to achieve balance between tissue strength and physiological function. Smooth muscle cells (SMCs), the main contributor of contractility in blood vessels, retain phenotypic plasticity, meaning they possess the ability to switch between a contractile and synthetic phenotype. In 2D culture, a number of biochemical and mechanical cues have been shown to promote the switch to a contractile phenotype in SMCs. However, achieving a stable contractile phenotype in 3D tissue has proven difficult. The work in this dissertation describes an investigation of how passive and dynamic environmental cues influence the smooth muscle phenotype. We studied the effects of substrate modulus in conjunction with changes in cell culture media composition on SMC phenotype in 2D and 3D cultures. Culturing SMCs in a low-serum culture medium resulted in an increase in SMC contractility in 2D cell culture but not in 3D cell-derived tissue. We found that, in SMCs cultured on soft substrates, the ability to modulate SMC phenotype in response to changes in media was diminished. Passively crosslinking the ECM of our cell-derived tissues with genipin resulted in modest increases in elastic modulus, though not enough to observe changes in SMC phenotype. Additionally, we investigated how dynamic cyclic mechanical stretch, in conjunction with cell culture medium, modified SMC contractility in cell and tissue cultures. SMCs increased contractile protein expression when exposed to dynamic stretch in 2D culture, even on soft substrates, which have previously been shown to inhibit phenotypic modulation. In 3D tissue rings, after mechanical stimulation, SMCs became more aligned, the tissue became tougher, and SMCs exhibited a measurable increase in contractile protein expression. In summary, we found that increasing substrate modulus, culturing in low serum cell culture medium, and imparting cyclic mechanical stretch can promote SMC differentiation and cellular alignment, and improve tissue mechanical properties. This information can be used to more accurately recapitulate vascular tissue for use in modeling or in the creation of tissue engineered blood vessels.

Every year over 20,000 [3] people die as a result of being in a fire. Although flames have the biggest visual impact, it is usually the smoke produced by the combustion of natural and synthetic materials that causes more damage and claims more lives. The main constituents of smoke, both the particulate matter as well as the hot and toxic gasses, are devastating to the tracheal and lung tissues. The damage caused to the lung and trachea by inhaling this smoke can increase a fire victim's susceptibility to infectious disease significantly [1]. Between 20% and 50% of people who suffer inhalation injury contract pneumonia due to the weakened status of their body's defenses [2] and between 4,800 and 6,400 [1] people die from either pneumonia or other complications. Despite the importance of the inner-lining of the trachea to a burn victim's health and survival, current treatments consist of keeping the patient in a clean environment, supplying fresh oxygen, keeping the airways open, and letting the patient's body heal itself [1]. This treatment is not so much an active healing mechanism; rather it is a passive means of allowing the body to repair itself. The main goal of this work is to develop a minimally invasive technique that will replace lost cells on the inside surface of the trachea as efficiently as possible, actively healing the patient's injury. Ideally, the patient would receive a single treatment and then make a complete recovery on his or her own. The main challenge lies in delivering an even layer of intact cells to the inner-surface of the trachea in such a manner that they will stay in place and will replace the damaged or missing tissue. The overall approach is to spray a suspension, composed of epithelial cells in an aqueous solution of Pluronic F-127 polymer, onto the trachea using a jet atomizer. Because Pluronic F-127 solutions can be liquids at room temperature but gels at body temperature, the role of the polymer will be to immobilize the cells onto the tracheal surface long enough for them to attach and grow.

Collagen implants are widely used in clinical practice and are an active area of research. The continued development of collagen-based implants often relies on histological techniques to fully evaluate the host response post implantation. These destructive, end-point analyses limit the rate that data can be obtained from samples. Magnetic resonance imaging has the potential to non-invasively monitor the remodeling of collagen scaffolds. In this study, scaffolds prepared from insoluble bovine collagen, with varied and predictable tissue responses were implanted in rats and evaluated using both histological and MRI techniques. Treatment of scaffolds with a carbodiimide crosslinker was found to slow the degradation and cellular infiltration into the scaffolds compared to uncrosslinked scaffolds. Angiogenesis was observed in core regions of crosslinked scaffolds, but not uncrosslinked scaffolds. Conjugation of chondroitin sulfate to the collagen scaffolds in combination with crosslinking improved both the cellular infiltration and angiogenesis compared to uncrosslinked and crosslinked scaffolds. Correlations between histology and MRI analyses showed that statistically significant relationships existed between cellular density, void area, T2 and apparent diffusion coefficient (ADC) measurements demonstrating that MRI is sensitive to specific remodeling parameters. Understanding the relationship between histology and MRI parameters may be used to help guide the interpretation of MRI data as well as to reliably detect changes within the implants using the MRI data alone, reducing the need for scaffold harvesting and destructive testing.

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.