In the US alone, more than 500,000 coronary artery bypass procedures are performed annually. Tissue engineering shows the potential to construct functional grafts to overcome the limited availability of autologous saphenous veins, relatively poor elasticity and low compliance of synthetic materials (mainly Dacron and polytetrafluoroethylene). In order to meet the low modulus associate with myocyte differentiation, the high suture retention and an ultimate tensile strength (UTS) sufficient to withstand implantation and peak physiological stresses, we designed and characterized a multi-component scaffold comprised of polyurethane electrospun mesh layers bonded together by a fibrin hydrogel matrix. We have demonstrated this composite construct retains the high tensile strength and suture retention strength but displays a "J-shaped" mechanical response similar to that of native coronary artery. To improve our design, poly(ethylene glycol) diacrylate based hydrogel system was utilized as a blank slate to study the phenotypic regulation by cell material interactions. Fibrinogen, fibronectin, laminin and collagen type IV were incorporated into the hydrogel to mimic the stimuli from extracellular matrix (ECM) proteins. Surprisingly, no significant effect was detected on induction of smooth muscle cell (SMC) differentiation marker expression, activation of mitogen-activated protein (MAP) kinases pathway, or alteration of surface integrin expression profile. However, fibronectin showed repression of undesired phenotypes in SMC differentiation. In contrast to ECM proteins, glycosaminoglycans (GAGs) showed more influence on regulating SMC phenotype. By using a scaffold environment intended to be mimetic of early atherosclerosis, the impact of GAG identity on SMC foam cell formation was explored. We focused on chondroitin sulfate C (CSC), dermatan sulfate (DS), and an intermediate molecular weight hyaluronan (HA_IMW, ~400 kDa), the levels and/or distribution of which are significantly altered in atherosclerosis. CSC and DS hydrogels were associated with greater SMC phagocytosis of apolipoprotein B than HA_IMW gels. However, only SMCs in DS constructs maintained increased expression of adipocyte marker A-FABP relative to HA_IMW gels over 35 days of culture. Combined, our results suggested interesting roles for fibronectin and HA_IMW in repression of undesired phenotypes in SMC differentiation, which could give insights into rational design of novel biomaterials for vascular tissue engineering applications.

Our ability to analyze the microstructure of biological tissue in three dimensions (3D) has proven invaluable in modeling its functionality, and therefore providing a better understanding of the basic mechanisms of life. Volumetric imaging of tissue at the cellular level, using serial imaging of consecutive tissue sections, provides such ability to acquire microstructure in 3D. Three-dimensional light microscopy in biology can be broadly classified as using either optical sectioning or physical sectioning. Due to the inherent limitations on the depth resolution in optical sectioning, and the recent introduction of novel techniques, physical sectioning has become the sought-out method to obtain high-resolution volumetric tissue structure data. To meet this demand with increased processing speed in 3D biological imaging, this thesis provides an engineering study and formulation of the tissue sectioning process. The knife-edge scanning microscopy (KESM), a novel physical sectioning and imaging instrument developed in the Brain Networks Laboratory at Texas A&M University, has been used for the purpose of this study. However, the modes of characterizing chatter and its measurement are equally applicable to all current variants of 3D biological microscopy using physical sectioning. We focus on chatter in the physical sectioning process, principally characterizing it by its geometric and optical attributes. Some important nonlinear dynamical models of chatter in the sectioning process, drawn from the metal machining literature, are introduced and compared with observed measurements of chatter in the tissue cutting process. To understand the effects of the embedding polymer on tissue sectioning, we discuss methods to characterize the polymer material and present polymer measurements. Image processing techniques are introduced as a method to abate chatter artifacts in the volumetric data that has already been obtained. Ultra-precise machining techniques, using (1) free-form nanomachining and (2) an oscillating knife, are introduced as potential ways to acquire chatter-free higher-resolution volumetric data in less time. Finally, conclusions of our study and future work conclude the thesis. In this thesis, we conclude that to achieve ultrathin sectioning and high-resolution imaging, embedded plastic should be soft. To overcome the machining defects of soft plastics, we suggested free-form nanomachining and sectioning with an oscillating knife.

Regenerative medicine is an interdisciplinary field which has advanced with the use of biotechnologies related to biomaterials, growth factors, and stem cells to replace or restore damaged cells, tissues, and organs. Among various therapeutic approaches, cell-based therapy is most challenging and exciting for both scientists and clinicians pursuing regenerative medicine. Specifically, stem cells, including mesenchymal stem cells and adipose-derived stem cells, are promising candidate cell types for cell-based therapy because they can differentiate into multiple cell types for tissue regeneration and stimulate other cells through neovascularization or paracrine signaling. Also, for effective treatment using stem cells, the tissue engineered constructs, such as bioactive degradable scaffolds, that provide the physical and chemical cues to guide their differentiation are incorporated with stem cells before implantation. Also, it was previously demonstrated that tissue-engineered matrices can promote tubulogenesis and differentiation of stem cells to vascular cell phenotypes. Hence, during tissue regeneration after stem cell therapy, there are numerous factors that need to be monitored. As a result, imaging-based stem cell tracking is essential to evaluate the distribution of stem cells as well as to monitor proliferation, differentiation, and interaction with the microenvironment. Therefore, there is a need for a stem cell imaging technique that is not only noninvasive, sensitive, and easy to operate, but also capable of quantitatively assessing stem cell behaviors in the long term with high spatial resolution. Therefore, the overall goal of this research is to demonstrate a novel imaging method capable of continuous in vitro assessment of stem cells as prepared with tissue engineered constructs and noninvasive longitudinal in vivo monitoring of stem cell behaviors and tissue regeneration after stem cell implantation. In order to accomplish this, gold nanoparticles are demonstrated as photoacoustic imaging contrasts to label stem cells. In addition, ultrasound and photoacoustic imaging was utilized to monitor stem cells and neovascularization in the injured rat tissue. Therefore, using these methods, tissue regeneration can be promoted and noninvasively monitored, resulting in a better understanding of the tissue repair mechanisms following tissue injury. / text

In recent years, significant success has been made in the field of regenerative medicine. Tissue engineering scaffolds have been developed to repair and replace different types of tissues. The overall goal of the current work was to develop scaffolds of native extracellular matrix components for soft tissue regeneration, more specifically, neural tissue engineering. To date, much research has been focused on developing a nerve guidance scaffold for its ability to fill and heal the gap between the damaged nerve ends. Such scaffolds are marked by several intrinsic properties including: (1) a biodegradable scaffold or conduit, consisting of native ECM components, with controlled internal microarchitecture; (2) support cells (such as Schwann cells) embedded in a soft support matrix; and (3) sustained release of bioactive factors. In the current dissertation, we have developed such scaffolds of native biomaterials including hyaluronic acid (HA) and collagen. HA is a nonsulphated, unbranched, high-molecular weight glycosaminoglycan which is ubiquitously secreted by cells in vivo and is a major component of extracellular matrix (ECM). High concentrations of HA are found in cartilage tissue, skin, vitreous humor, synovial fluid of joints and umbilical cord. HA is nonimmunogenic, enzymatically degradable, non-cell adhesive which makes HA an attractive material for biomedical research. Here we developed new photopolymerizable HA based materials for soft tissue repair application. First, we developed interpenetrating polymer networks (IPN) of HA and collagen with controlled structural and mechanical properties. The IPN hydrogels were enzymatically degradable, porous, viscoelastic and cytocompatible. These properties were dependent on the presence of crosslinked networks of collagen and GMHA and can be controlled by fine tuning the polymer ratio. We further developed these hydrogel constructs as three dimensional cellular constructs by encapsulating Schwann cells in IPN hydrogels. The hydrogel constructs supported cell viability, spreading, proliferation, and growth factor release from the encapsulated cells. Finally, we fabricated scaffolds of photopolymerizable HA with controlled microarchitecture and developed designer scaffolds for neural repair using layer-by-layer fabrication technique. Lastly, we developed HA hydrogels with unique anisotropic swelling behavior. We developed a dual-crosslinking technique in which a super-swelling chemically crosslinked hydrogel is patterned with low-swelling photocrosslinked regions. When this dual-crosslinked hydrogel is swelled it contorts into a new shape because of differential swelling among photopatterned regions. / text

Temperature dependent optical properties of lipid tissue verify critical information of tissue dynamics which can be applied to tissue treatment and diagnosis of various pathological features. Current methods of treating lipid rich tissues via heating are associated with post operation complications. Recent studies shows potential of lipid rich tissue removal by cooling. For monitoring cooling procedure and physical and chemical changes in lipid tissue, temperature dependent optical properties in subzero cooling need to be verified. This study designed heat transfer system estimating heat flux by cooling and programmed codes for image and data processing to obtain refractive indices of rodent subcutaneous lipid tissue. Phase transition of lipid tissue was observed and finally verified temperature dependent refractive index coefficient of lipid tissue from 24°C to -10°C. / text

There exists a need for a biocompatible polymer system of appropriate degradation properties for use in the production of tissue-engineered cartilage replacement implants. The implant consists of a layer of cartilage grown using autogenous chondrocyte cells on a porous calcium phosphate base for anchoring in situ. This implant would serve to improve the current treatments for wear and age-related degradation of articular cartilage. Pilot dissolution studies of the biodegradable polymers Polyvinyl Alcohol (PVA), Polycaprolactone (PCL), and Polyethylene Glycol (PEG), provided strong evidence supporting the use of PVA and PEG, not PCL, in film preparation. Results indicate that the dissolution of PVA rapidly exceeds that required for this application while the dissolution of PCL is not fast enough. The results of a literature review indicate that PEG dissolves faster than PCL, but not PVA. Consequently, a co-polymer hydrogel film of PVA and PEG, to fully degrade in 10 hours, was prepared to serve as a support for the in vitro seeding of cartilage-producing chondrocyte cells onto the artificial bone scaffold base. In preparing the film, the concentration of the PVA and PEG stock solutions, the composition of PVA and PEG (by mass % ratio) in the film, and the thickness of the film were defined to be the design variables. The degradation properties of the film are hypothesized to be influenced by the design variables, such that the degradation rates can be engineered by manipulating these parameters. A full factorial DOE was applied to determine the significance of the design variables and their interaction on the degradation rate. To determine degradation rate, in vitro dissolution studies of the hydrogel film were conducted in Earle’s balanced salt solution at 37oC. Upon optimizing the degradation rate, it was theoretically determined that an optimized film of 50wt% PVA, 50wt% PEG, and thickness of 3mm dissolves by 88.19 % in 10 hours. Validation testing indicated that the optimized film was prematurely perforated at approximately 22 minutes of immersion in EBS at room temperature suggesting failure by bulk dissolution, which was later confirmed through investigation and identification of a heterogeneous, multi-phase microstructure under transmitting light microscope. / Thesis (Master, Mechanical and Materials Engineering) -- Queen's University, 2008-05-01 14:28:06.935 / Octane Orthobiologics & Ontario Centres of Excellence (OCE)

Currently, organ transplant procedures are insufficient to address the needs of the number of patients that suffer of organ failure related disease. In the United States alone, only around 19% of the patients are able to get an organ transplant surgery and 25% die while waiting for a suitable donor. Tissue engineering (TE) has emerged as an alternative to organ transplant; thus, the aim of the present study was to validate a poly(ethylene glycol) diacrylate (PEG-DA) hydrogel system as a model for material scaffolding in TE applications. This work explores the influence of scaffold material properties on cell behavior. Specifically, scaffold modulus, mesh size, and biochemical stimuli were characterized and their influence on cell response was analyzed at the biochemical, histological and microenvironmental levels. Three different TE targets were evaluated: vocal fold restoration, vascular grafts and osteochondral applications. Vocal fold fibroblast (VFF) phenotype and extracellular matrix (ECM) production were impacted by initial scaffold mesh size and modulus. The results showed increasing levels of SM-α-actin and collagen production with decreasing initial mesh size/increasing initial modulus, which indicated that VFFs were induced to take an undesirable myofibroblast-like phenotype. In addition, it was possible to preserve VFF phenotype in long-term cultured hydrogels containing high molecular weight hyaluronan (HAHMW). On the other hand, regarding vascular graft applications, smooth muscle cell (SMC) phenotype was enhanced by increasing scaffold mesh size and modulus. Finally, the effect of scaffold inorganic content (siloxane) on rat osteoblasts and mouse mesenchymal stem cells was evaluated. Interestingly, the impact of inorganic content on cell differentiation seemed to be highly dependent on the initial cell state. Specifically, mature osteoblasts underwent transdifferentiation into chondrocyte-like cells with increasing inorganic content. However, Mesenchymal stem cells appeared to be preferentially driven toward osteoblast-like cells with an associated increase in osteocalcin and collagen type I production.

Cell-based treatments for insulin-dependent diabetes (IDD) may provide more physiologic regulation of blood glucose levels than daily insulin injections, thereby reducing the occurrence of secondary complication associated with IDD. An autologous cell source is especially attractive for regulatory and ethical reasons and for circumventing the need for immunosuppression, which is currently standard for islet transplantation. Our approach focuses on using adult non-β-cells engineered for physiologic insulin secretion. Specifically, we utilize enteroendocrine L-cells, which naturally exhibit regulated secretion of GLP-1 in response to physiologic stimuli, and upon genetic engineering, co-secrete insulin in a regulated manner. The overall goal of this project is to develop a tissue engineered pancreatic substitute based on a recombinant enteroendocrine cell line and test the efficacy of the pancreatic substitute by implantation into diabetic mice. The specific aims of this thesis were to (1) to modify murine L-cells for regulated insulin secretion and evaluate the insulin secretion properties of the recombinant cells; (2) to incorporate insulin-secreting L-cells into an implantable construct containing small intestinal submucosa (SIS) and to evaluate insulin secretion from the construct in vitro; and (3) to test the efficacy of the tissue engineered pancreatic substitute in vivo by implanting it intraperitoneally in mice made diabetic by streptozotocin. Thus, this proposal takes a tissue engineered pancreatic substitute for IDD from in vitro development to in vivo testing.

The main question that this thesis addresses is what is the optimal way of producing tissue grown in the peritoneal cavity around a foreign body for its use as a vascular graft? It is known that a foreign body implanted into the peritoneal cavity induces an inflammatory response with cells recruited from within the peritoneal cavity to encapsulate the foreign body. Over the course of two to three weeks these cells produce an organised matrix and differentiate to become myofibroblasts. Tubes of these ‗tissue capsules‘ have been transplanted into the arterial vasculature in several animal models where the tissue capsule differentiates into an arterial structure. This structure consists of a layer of smooth muscle-like cells, adventitia of dense connective tissue including vasa-vasorum and an endothelial layer of flattened mesothelial cells. In order to determine whether the tissue would further differentiate ex vivo in response to mechanical stimulus an in-vitro bioreactor system was built to house tissue capsules produced in a variety of animal models. This bioreactor system could house 4 tissue capsules under physiological conditions including standard pulse rates, pressures and temperatures experienced by an artery. Boiled blood clot (BBC) scaffolds were implanted into the peritoneal cavity of rats to produce tissue capsules. After two weeks of development in the peritoneal cavity, tissue capsules were harvested and implanted into the bioreactor. Tissue capsules grafted into the bioreactor were subjected to mechanical force for a range of time-points, pressure, pulse and flow rates. When analysing tissue immunohistochemically, elastin, myosin, αSMA and desmin were detected. This staining was not consistent across all samples and only present in small parts of some tissue tested. Western analysis did not show any expression of αSMA or myosin. Finally the morphology of the tissue also resembled that of tissue previously implanted into the arterial circulation, but development of mechanical properties were not to the extent that would make the tissue useful as a vascular graft. The bioreactor system was thus modified to be able to house tissue for a period of 3 weeks. This system successfully housed tissue capsules under mechanical force in physiological ranges. Next, a range of materials were tested for their ability to be included into the peritoneal implant device used for the large animal model. Elasteon 80A did not produce any cellular growth or peritoneal pathology in all implanted samples (n = 4). Cloisite, a pro-inflammatory material produced large tissue capsule development over a 2 week implant period in 25% of samples however this tissue was heavily adhered to the greater omentum and dependent on its vascular supply. This data suggested that Elasteon could be used to coat the outer surface of a peritoneal implant device to decrease the rate of peritoneal adhesions. Three devices were designed and fabricated for their use in generating tissue for the modified Mitrofanoff procedure which requires a length of tissue to be implanted between the umbilicus and the bladder as a fistula. In all three cases no implantable material was produced that could be used for this procedure. To modify the device that could be used to produce tissue for any surgical application, a range of devices was produced and the animal model was changed to pigs. Materials incorporated into these devices include Dexon mesh and polyethylene. These devices also did not produce any tissue that could possibly be used as a vascular graft. A novel material, polymer BD347 was then produced for use in developing tissue within the interior of the device to provide greater growth and mechanical properties for developing a vascular graft. In toxicological studies, the replacement rate of cells was unaffected after seven days of incubation of fibroblasts at confluence with the polymer. A range of mechanical properties from pig vasculature was gained so that a sheet of polymer with similar properties to that of a vascular graft could be made. This polymer was fabricated as a tube and implanted into the peritoneal cavity of rats. The implanted polymer remained free-floating with a capsule of tissue in 78% of cases. A device was designed that has the ability to impart a physiological pulsation force on the developing tissue capsule in the peritoneal cavity using a sheep model. When two devices were implanted for a period of 10 days in each animal these devices produced no complications for the animal. Upon harvest all devices were free of adhesion and did not cause any peritoneal or dermal infection. In 100% of cases this device produced tissue that was thick and consistent along the length of the implant. The quality of tissue differed greatly macroscopically between tissue produced around pulsing and non-pulsing scaffolds, but microscopically the structure of both tissues was not significantly different. Approximately 90% of cells in this tissue stained positively for CD45. Tissue in pulsing devices produced a higher amount of vimentin expression in CD45 positive staining cells than tissue in non-pulsing devices. Mechanical properties of tissue in pulsed devices were also much greater than tissue in non-pulsed devices. Two of the pulsed tissues were grafted into the carotid artery of sheep as arterial patches. In one animal tissue lasted a period of 1 week before it ruptured. In the second animal tissue lasted a period of 2 weeks at which time the animal was sacrificed. In this sheep a layer of endothelial cells had migrated to populate areas of the tissue patch. Pulsation of the implant device enhanced the development of tissue capsule in the peritoneal cavity towards arterial properties. These studies provide information on the materials and designs required to produce peritoneal-derived tissue capsules that can be used in a range of surgical applications. These studies also provide information on how this tissue responds to mechanical force and provides an in vitro system for testing this tissue. This work in this thesis has produced a device that is in the stage of pre-clinical development to be used as a potential therapy for cardiovascular disease. This device is a novel development from previous devices used for generating tissue capsules for engraftment and is a significant contribution to work in developing a replacement artery.

Thesis (M. Sc.)--University of Sydney, 2005. / Title from title screen (viewed 21 May 2008). Submitted in fulfilment of the requirements for the degree of Master of Science to the Unit for the History and Philosophy of Science and Centre for Values, Ethics and Law in Medicine. Includes bibliographical references. Also available in print form.