Children born with congenital heart valve defects require open-heart surgery to implant an artificial replacement valve. These valves are unable to grow with the developing child and need replacing every 5 years. Tissue engineered heart valves, capable of growing with the patient, would alleviate the need for repeat surgery. I hypothesize chitosan and collagen possess advantageous qualities as scaffolding for a tissue engineered heart valve. This study evaluated chitosan and collagen hydrogels as potential scaffold materials. Chitosan scaffolds had suitable pore size/distribution and scaffold strength; however, they were unable to sustain cell attachment or viability. Collagen gels were assessed for compaction, mechanical properties and expression of matrix metalloproteases in the presence or absence of biochemical and mechanical stimuli. Pressure increased the remodeling potential. This was augmented further in the presence of TGF-β. In conclusion, both materials have potential as scaffolding substrate in a tissue engineered heart valve.

The broad objective of this research was to develop transgenic poinsettia that express tryptophan decarboxylase (TDC) capable of protecting poinsettia against whitefly. An effective and efficient in vitro micro propagation and proliferation technique of poinsettia ‘Prestige Red’ was successfully developed in this study and this protocol can be used for potential development of transgenic poinsettia. Poinsettia ‘Prestige Red’ was successfully infected by Agrobacterium rhizogenes producing hairy roots at the site of infection. Investigations of more effective PGR concentrations are necessary in order to develop transgenic poinsettia through hairy roots. Stem disks of poinsettia ‘Eckespoint Pollys Pink’ developed into somatic embryos when they were transformed by A. tumefaciens harboring TDC. A. tumefaciens-mediated transformation of poinsettia through somatic embryogenesis is cultivar dependent. Additional research into more effective PGR combinations, antibiotic concentrations and antinecrosis chemicals is required in order to develop transgenic poinsettia harboring TDC through somatic embryogenesis using A. tumefaciens.

No description available.

No description available.

The immune response is a major barrier to the successful transplantation of organs and tissues required in the treatment of many human diseases. Although the field of tissue engineering was created to address the shortage of human organs and tissues, the immune response remains a substantial challenge, impeding the development of allogeneic biological substitutes to repair, replace and regenerate tissues. Specifically, the T cell mediated immune response initiated through the recognition of cell surface Major histocompatibility complex Class I (MHCI) molecules is the primary cause of acute allograft rejection. In nature, viruses have evolved many mechanisms to exploit weaknesses of the T cell response to evade detection. Viral mechanisms to modulate the MHCI molecule can be effectively applied to allogeneic cells in a tissue-engineered construct to evade detection by CD8+ Cytotoxic T Lympocytes (CTLs) and Natural Killer (NK) cells of the immune system. We demonstrate the successful application of a retroviral vector to over-express the Kaposi's sarcoma-associated herpesvirus (KSHV) immunomodulatory protein, MIR2, in human monocyte-like leukemia cells to differentially downregulate cell surface MHCI, ICAM-1 and B7-2 molecules. We also developed a novel flow cytometry-based cytotoxicity assay to demonstrate that this differential downregulation of immunoactive molecules has the functional effect of significantly reducing CTL-mediated cytotoxicity, without altering NK-mediated cytotoxicity. We believe that this approach provides a potential solution to circumvent the acute immune rejection of allografts in vivo, and can also lead to the development of "universal" donor cells for tissue engineering applications that will not require anti-rejection drugs. / Thesis / Master of Applied Science (MASc)

Yes

The field of tissue engineering has grown immensely since its inception in the late 1980s. However, currently commercialized tissue engineered products are simple in structure. This is due to a pre-clinical bottleneck in which complex tissues are unable to be fabricated. 3D bioprinting has become a versatile tool in engineering complex tissues and offers a solution to this bottleneck. Characterizing the mechanical properties of engineered tissue constructs provides powerful insight into the viability of engineered tissues for their desired application. Current methods of mechanical characterization of soft hydrogel materials used in tissue engineering destroy the sample and ignore the effect of 3D bioprinting on the overall mechanical properties of a construct. Herein, this work reports on the novel use of a non-destructive method of viscoelastic analysis to demonstrate the influence of 3D bioprinting strategy on mechanical properties of hydrogel tissue scaffolds. 3D bioprinting is demonstrated as a versatile tool with the ability to control mechanical and physical properties. Structure-function relationships are developed for common 3D bioprinting parameters such as printed fiber size, printed scaffold pattern, and bioink formulation. Further studies include effective real-time monitoring of crosslinking, and mechanical characterization of multi-material scaffolds. We envision this method of characterization opening a new wave of understanding and strategy in tissue engineering. / Graduate

Ellen Lai Ping Chan. / Thesis (Ph.D)--Chinese University of Hong Kong, 1990. / Bibliography: leaves 238-272. / Chapter CHAPTER I --- LITERATURE REVIEW / Chapter 1. --- History --- p.1 / Chapter 2. --- Species Distribution of BAT --- p.3 / Chapter 3. --- Distribution of BAT --- p.4 / Chapter 4. --- Structure of BAT --- p.4 / Chapter 4.1. --- Macroscopic Appearance --- p.4 / Chapter 4.1.1. --- Innervation --- p.4 / Chapter 4.1.2. --- Blood supply --- p.5 / Chapter 4.2. --- Microscopic Structure of BAT --- p.6 / Chapter 4.3. --- Difference Between Brown Fat and White Fat --- p.9 / Chapter 5. --- Composition of BAT --- p.11 / Chapter 6. --- The Mechanisms of Brown Adipose Tissue Thermogenesis --- p.12 / Chapter 6.1. --- Factors Influencing Proton Transport by UCP --- p.16 / Chapter 6.2. --- Postulated Sequence of Events during Thermogenesis --- p.18 / Chapter 7. --- Measurements of thermogenic Capacity of BAT --- p.21 / Chapter 8. --- Age-related Differences in BAT --- p.28 / Chapter 9. --- Non-shivering Thermogenesis and BAT --- p.32 / Chapter 9.1. --- Changes In BAT During Cold Acclimation --- p.35 / Chapter 9.1.1. --- GDP Binding --- p.35 / Chapter 9.1.2. --- Concentration of UCP --- p.37 / Chapter 9.1.3. --- Metabolic changes in BAT during Cold Acclimation --- p.39 / Chapter 10. --- Diet-induced Thermogenesis and BAT --- p.41 / Chapter 10.1. --- Mechanism of DIT --- p.42 / Chapter 10.2. --- Controversies in DIT --- p.44 / Chapter 10.3. --- Nutritional Factors Inducing DIT --- p.46 / Chapter 10.4. --- DIT in Man --- p.47 / Chapter 10.5. --- Neuroendocrine Control of BAT in DIT --- p.48 / Chapter 10.6. --- Effects of Fasting in BAT --- p.51 / Chapter 11. --- Obesity and BAT --- p.53 / Chapter 11.1. --- NST and DIT in Obese Animals --- p.58 / Chapter 11.2. --- Regulation of BAT in Obese Animals --- p.59 / Chapter 11.2.1. --- Sympathetic Nervous System in Obese Animals --- p.59 / Chapter 11.2.2. --- Corticosteriods in Obese Animals --- p.61 / Chapter 11.2.3. --- Adrenergic Receptors in Obese Animals --- p.63 / Chapter 11.2.4. --- Insulin in Obese Animals --- p.64 / Chapter 12. --- Pregnancy and Lactation and BAT --- p.67 / Chapter 12.1. --- Energy Balance During Pregnancy and Lactation --- p.67 / Chapter 12.2. --- Some Metabolic Changes During X Pregnancy and Lactation --- p.68 / Chapter 12.3. --- Role of BAT in Pregnancy and Lactation --- p.70 / Chapter 12.4. --- Mechanism of Regulation of Thermogenesis during Pregnancy and Lactation --- p.71 / Chapter 13. --- Factors Controlling the Thermogenesis --- p.75 / Chapter 13.1. --- Sympathetic Nervous Control --- p.75 / Chapter 13.1.1. --- Studies of Administration of Noradrenaline --- p.75 / Chapter 13.1.2. --- Control of the Fuel Supply to BAT by Sympathetic Nervous System --- p.77 / Chapter 13.1.3. --- Sympathetic denervation --- p.78 / Chapter 13.2. --- Hormonal Control --- p.79 / Chapter 13.2.1. --- Thyroid Hormone --- p.79 / Chapter 13.2.2. --- Insulin --- p.81 / Chapter 13.2.3. --- Pituitary Hormones --- p.83 / Chapter 13.2.4. --- Glucocorticoids --- p.83 / Chapter 13.2.5. --- Corticotropin-Releasing Factor --- p.85 / Chapter 14. --- Aims of the Study --- p.87 / Chapter CHAPTER II --- ISOLATION AND PURIFICATION OF UCP AND DEVELOPMENT OF AN ENZYME LINKED IMMUNOSORBENT ASSAY FOR UCP / Chapter 1. --- INTRODUCTION --- p.88 / Chapter 2. --- MATERIALS AND METHODS --- p.89 / Chapter 2.1. --- Animals --- p.89 / Chapter 2.2. --- Collection of BAT --- p.89 / Chapter 2.3. --- Isolation of Mitochondria --- p.90 / Chapter 2.4. --- Electron Microscopy (EM) of Isolated BAT Mitochondria --- p.92 / Chapter 2.5. --- Measurement of Protein and Cytochrome C Oxidase Activity --- p.94 / Chapter 2.5.1. --- Measurement of Protein Concentration --- p.94 / Chapter 2.5.2. --- Measurement of Cytochrome C Oxidase Activity --- p.99 / Chapter 2.6. --- GDP Binding Assay of BAT Mitochondria --- p.101 / Chapter 2.6.1. --- GDP Binding Assay of Mitochondria by Centrifugation Method --- p.103 / Chapter 2.6.2. --- GDP: Binding Activity by Equilibrium Dialysis --- p.106 / Chapter 2.6.3. --- GDP Binding by Microfiltration Method --- p.108 / Chapter 2.7. --- Experiments Designed for Validation of GDP Binding Assay --- p.109 / Chapter 2.7.1. --- GDP Binding Activity in BAT Mitochondria after Noradrenaline Treatment --- p.109 / Chapter 2.7.2. --- GDP Binding Activity in BAT Mitochondria after Cold Acclimation and Noradrenaline Treatment --- p.110 / Chapter 2.7.3. --- Effect of Food Restriction on Cold Acclimated Rats --- p.110 / Chapter 2.7.4. --- GDP Binding Activity of BAT Mitochondria of Rats of Different Ages --- p.111 / Chapter 2.8. --- Isolation and Purification of UCP --- p.111 / Chapter 2.9. --- Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) --- p.115 / Chapter 2.10. --- Methods for Raising Anti-Rat-UCP Antibody and the Characterization of Antiserum --- p.120 / Chapter 2.10.1. --- Raising Rabbit Anti-Rat-UCP Antibody --- p.120 / Chapter 2.10.2. --- Western Blot Analysis For Cross Reactivity Study --- p.120 / Chapter 2.10.3. --- Immuno-Autoradiographic Method for Detection of Specificity of Rabbit Anti-Rat UCP Antiserum --- p.121 / Chapter 2.11. --- Enzyme Linked Immunosorbent Assay For UCP --- p.124 / Chapter 2.12. --- Experiment Designed to Validate the ELISA --- p.129 / Chapter 2.13. --- Statistical Analysis --- p.129 / Chapter 3. --- RESULTS --- p.130 / Chapter 3.1. --- Electron Microscopy of Isolated BAT Mitochondria --- p.130 / Chapter 3.2. --- GDP Binding Assay of BAT Mitochondria --- p.130 / Chapter 3.3. --- Experiments Designed for Validation of GDP Binding Assay --- p.133 / Chapter 3.3.1. --- GDP Binding Activity of BAT Mitochondria after Noradrenaline Injection --- p.133 / Chapter 3.3.2. --- GDP Binding Activity of BAT Mitochondria after Cold Acclimation and Noradrenaline Treatment --- p.136 / Chapter 3.3.3. --- Effects of Food Restriction on Cold Acclimated Rats --- p.136 / Chapter 3.3.4. --- GDP Binding Activity of BAT Mitochondria from Rats of Different Ages --- p.140 / Chapter 3.4. --- Isolation and Purification of UCP --- p.140 / Chapter 3.4.1. --- Results of SDS-PAGE --- p.143 / Chapter 3.4.2. --- Results of GDP Binding Activity --- p.149 / Chapter 3.5. --- Rabbit Anti-rat-UCP Antibody and the Characterization of Antiserum --- p.151 / Chapter 3.5.1. --- Immuno-autoradiography for Specificity of Rabbit Anti-rat-UCP Antiserum --- p.153 / Chapter 3.5.1.1. --- Cross-reactivity of the Rabbit Anti-rat-UCP Antiserum to Mitochondrial Proteins of BAT and from other Tissues --- p.153 / Chapter 3.5.1.2. --- Cross-reactivity of the Rabbit Anti-rat-UCP Antiserum to BAT Mitochondrial Protein from Different Rodent Species --- p.156 / Chapter 3.5.1.3. --- Dose Response of Rabbit Anti-rat-UCP Antiserum to UCP --- p.159 / Chapter 3.6. --- ELISA of UCP --- p.161 / Chapter 3.6.1. --- Determination of Maximum Amount of UCP Binding on Microtitre Plate --- p.161 / Chapter 3.6.2. --- Antibody Dilution Curve --- p.161 / Chapter 3.6.3. --- Incubation Time for Enzyme-Substrate Reaction --- p.163 / Chapter 3.6.4. --- Competitive ELISA --- p.163 / Chapter 3.6.5. --- Precision of ELISA --- p.167 / Chapter 3.7. --- Experiment Designed for Validation of ELISA by Measuring UCP in Cold Acclimated Rats --- p.170 / Chapter 4. --- DISCUSSION --- p.172 / Chapter 4.1. --- GDP Binding Assay of BAT Mitochondria --- p.172 / Chapter 4.2. --- Isolation and Purification of UCP --- p.176 / Chapter 4.3. --- Development and Evaluation of ELISA --- p.178 / Chapter CHAPTER III --- CHANGES IN BAT DURING PREGNANCY AND LACTATION AND ROLE OF PROLACTIN / Chapter 1. --- INTRODUCTION --- p.184 / Chapter 2. --- MATERIALS AND METHODS --- p.187 / Chapter 2.1. --- Animal --- p.187 / Chapter 2.2. --- Experimental Designs --- p.187 / Chapter 2.2.1. --- "Effects of Pregnancy, Lactation and Post Weaning on BAT" --- p.187 / Chapter 2.2.2. --- Effect of Metoclopramide on BAT --- p.188 / Chapter 2.2.3. --- Effect of Metoclopramide and Bromocriptine on BAT --- p.188 / Chapter 2.2.4. --- Effect of PRL Injection on BAT --- p.189 / Chapter 2.2.5. --- Continuous infusion of PRL --- p.189 / Chapter 2.6.6. --- Measurements of BAT Parameters --- p.191 / Chapter 2.2.7. --- RIA of serrum PRL --- p.191 / Chapter 2.2.8. --- PRL Receptors in BAT --- p.197 / Chapter 2.4. --- Statistical Analysis --- p.201 / Chapter 3. --- RESULTS --- p.202 / Chapter 3.1. --- Effects of Pregnancy and Lactation --- p.202 / Chapter 3.1.1. --- Food Consumption and Body Weight --- p.202 / Chapter 3.1.2. --- BAT --- p.205 / Chapter 3.1.3. --- Serum PRL level --- p.209 / Chapter 3.2. --- Effects of PRL njection --- p.213 / Chapter 3.3. --- Effects of Continuous Infusion of PRL on BAT --- p.213 / Chapter 3.4. --- Effects of Metoclopramide on BAT --- p.216 / Chapter 3.5. --- Effects of Bromocriptine and Metoclopramide on BAT --- p.216 / Chapter 3.6. --- PRL Receptor in BAT --- p.219 / Chapter 4. --- DISCUSSION --- p.223 / GENERAL CONCLUSION --- p.236

To date, the reconstructive approach addressing chronic non-healing wounds, deep tissue damage, and severe wound defects relies upon avascular dermal grafts and autologous flap techniques. Such flaps are limited by donor site availability and morbidity, while current dermal grafts rely upon host cellular invasion for neovascularization and incorporation. These products fail to include an inherent vascular network and the supporting cells necessary to ensure adequate incorporation and graft survival beyond the most optimal wound beds. Herein, we fabricate a pre-vascularized full-thickness cellularized skin equivalent containing a three-dimensional vascularized network of interconnected macro and microchannels lined with vascular cells, within a collagen neodermis populated with fibroblasts, and an epidermis comprised of human keratinocytes capable of providing whole tissue perfusion. Previously, our lab has employed a sacrificial microfiber technique to develop tissue-engineered scaffolds with an inherent hierarchical network of microvessels, which recapitulates the organization of an arteriole, venule, and capillary bed. Utilizing a type-I collagen hydrogel matrix, vascular cells were seeded within pre-fabricated channels and allowed to proliferate to generate an endothelialized microvasculature. These collagen scaffolds were subsequently anastomosed into rat models to demonstrate the clinical feasibility of such approach. The present study aims to more closely recapitulate the in vivo structure of human skin via the incorporation of vital epidermal and dermal components of native skin into a biocompatible construct containing a complex hierarchical vasculature, which may be anastomosed using standard microsurgical techniques and immediately perfused. Pluronic F127 was used as the sacrificial material: 1.5 mm diameter “U” shaped macrofibers and 100-500 µm-interwoven microfibers were heat extruded and then embedded within type-I collagen into which Cyan Fluorescent Protein (CFP)-tagged human placental pericytes and human foreskin fibroblasts (HFF1) had been encapsulated. Following pluronic sacrifice, resultant channels were intraluminally seeded with Red Fluorescent Protein (RFP)-tagged human aortic smooth muscle cells, Green Fluorescent Protein (GFP)-tagged human umbilical vein endothelial cells, and topically seeded with human epidermal keratinocytes (HEK). Construct microstructure was analyzed using multiphoton microscopy (MPM) after 7, 14 and 28 days of culture. Additionally, after 14 and 28 days of culture, endothelial cells were extracted from the construct using collagenase digestion and Real Time (RT)-qPCR performed to analyze expression of markers of angiogenesis and maturation of the vascular network. MPM demonstrated a hierarchical vascular network containing macro and microvessels lined by endothelial and smooth muscle cells, supported by perivascular pericytes, all in appropriate microanatomic arrangement. Neodermal HFF1 proliferated throughout the observation period and the HEK neoepidermis developed into a stratified epidermis along the superior aspect of the construct. Angiogenic sprouting from the nascent vascular network into neovessel like structures was noted. RT- qPCR revealed relative expression of Jagged1, Dll4, Ve-Cadherin, and CD31. We have successfully fabricated a novel tissue-engineered pre-vascularized full thickness skin flap, which recapitulates the inherent hierarchical vasculature found within human skin and is suitable for in vivo perfusion. We provide the platform for an on- demand, geometrically tunable tissue engineered skin equivalent with an anastomosable vascular network. This tissue-engineered skin flap holds the potential to transform reconstructive surgical practice by eliminating the consequences of donor site morbidity, and enabling rationally designed, patient-specific flaps for each unique wound environment and anatomic location. / 2017-06-16T00:00:00Z

A thin layer of calcified cartilage at the native cartilage-to-bone junction facilitates integration between deep zone articular cartilage and subchondral bone, while maintaining the integrity of the two distinct tissue regions. Regeneration of this interface remains a significant clinical challenge for long-term and functional cartilage repair. The strategy for osteochondral interface formation discussed in this thesis focuses on the design and optimization of a biomimetic scaffold for stable calcified cartilage formation. The ideal interface scaffold supports chondrocyte biosynthesis and the formation of calcified cartilage with physiologically-relevant mechanical properties. Furthermore, the interface scaffold allows for osteointegration and the maintenance of the calcified cartilage matrix. It is hypothesized that ceramic presence and zonal chondrocyte interactions regulate cell biosynthesis and mineralization, and these cell-matrix and cell-cell interactions are essential for calcified cartilage formation and maintenance. Biomimetic design parameters for an interface scaffold were determined by characterizing the native interface in terms of mineral and matrix distribution. A composite hydrogel-hydroxyapatite scaffold was then designed to support formation of a functional calcified cartilage matrix. The hydrogel phase maintains the chondrocyte phenotype and allows for incorporation of ceramic particles, while the biomimetic ceramic phase is osteointegrative and decreases the need for cell-mediated mineralization. This scaffold was optimized <italic>in vitro</italic> based on hydrogel type, chondrocyte population, and ceramic particle size. The collective findings from these cell-ceramic interaction studies determined that hypertrophic chondrocytes, cultured in the presence of micron-sized hydroxyapatite particles, exhibit enhanced hypertrophy and matrix deposition. Scaffold ceramic dose and seeding density were also optimized for promoting calcified cartilage formation <italic>in vitro</italic>. In order to implement the scaffold for integrative cartilage repair, a scaffold was designed to regenerate both uncalcified and calcified cartilage on a bilayered hydrogel scaffold. Furthermore, a polymer-ceramic nanofiber component was added to augment the original design for <italic>in vivo</italic> implementation. The hydrogel-nanofiber composite scaffold was evaluated <italic>in vivo</italic> and found to support mineralization and osteointegration within the bone region while preventing endochondral ossification within the repair tissue. Finally, inspired by the stratified organization of zonal chondrocyte populations above the calcified cartilage interface, the layered hydrogel model was used to determine the role of zonal chondrocyte organization on calcified cartilage stability. This thesis collectively explores cell-ceramic and cell-cell interactions, and their ramifications for calcified cartilage formation and maintenance. Specifically, ceramic presence promotes the deposition of a calcified cartilage matrix by hypertrophic chondrocytes in a dose-dependent manner, and furthermore, communication between surface zone and deep zone chondrocyte populations suppresses mineralization within articular cartilage above the calcified cartilage interface. It is anticipated that the scaffold design strategy developed in this thesis can also be applied to the regeneration of other complex interfaces where there are transitions from soft-to-hard tissue.