Subglottic stenosis, malignancy and traumatic injury to the trachea require surgical resection. When defects are less than 50% of the tracheal length in adults and 1/3 in children, a circumferential resection and primary anastomosis affords excellent results. For longer lesions, on the other hand, there are no currently acceptable solutions leading to patients requiring permanent tracheostomies or palliative treatment. Tracheal replacement approaches with synthetic prosthesis and scaffolds have all led to inflammation, obstruction, mucous build-up and eventual restenosis. Tissue-engineering approaches using recipients’ own stem cells and biologic scaffolds derived from decellularized donor trachea have shown great promise. They have the potential to abrogate the need for immunosuppressive therapy. Our research focuses on three major limitations in this field including the structural integrity, the immunogenicity and the recellularization of donor tracheae. We compared three decellularization protocols, quantified and qualified the extracellular matrix (ECM) components and performed compliance measurements on large circumferential tracheal scaffolds following cyclical decellularization techniques and illustrated significant differences in ECM composition and resultant structural integrity of decellularized tracheal scaffolds depending on the protocol. In addition, we investigated the immunogenicity of decellularized and recellularized tracheal allografts at a protein level and in vitro and in vivo T cell proliferation. Decellularization is associated with a delay in leukocyte infiltration and recellularization promoted cartilage preservation and the recruitment of regulatory T cells. We described a dramatic increase of TGF-β1 in recellularized scaffolds. Moreover, we designed a dual-chamber bioreactor for recellularization of tracheal allografts. Our method allowed for dynamic perfusion seeding, confirmed adherence of two different cell types and achieved higher cell numbers and homogeneous structures compared to traditional static seeding methods. In summary, we have identified and addressed three major limitations for tissue-engineering of long-segment decellularized tracheal scaffolds relating to structural integrity, immunogenicity and recellularization techniques.

No description available.

Tissue engineering (TE) integrates methods of cells, engineering and materials to improve or replace biological functions of native tissues or organs. 3D printing technologies have been used in TE to produce different kinds of tissues. Based on review of the exiting 3D printing technologies used in TE, special requirements of fabricating soft scaffolds are identified. Soft scaffolds provide a microenvironment with biocompatibility for living cells proliferation. This research focuses on 3D printer design and printing parameters investigation for fabrication of soft scaffolds. A 3D printer is proposed for producing artificial soft scaffolds, with components of a pneumatic dispenser, a temperature controller and a multi-nozzle changing system. Relations of 3D printing parameters are investigated to improve the printing quality of soft scaffolds. It provides guidance for printing customized bio-materials with improved efficiency and quality. In the research, printing parameters are identified and classified based on existing research solutions. A deposition model is established to analyze the parameters relations. Quantitative criteria of parameters are proposed to evaluate the printing quality. A series of experiments including factors experiments and comparison tests are conducted to find effects of parameters and their interactions. A case study is conducted to verify the analytic solution of proposed models. This research confirms that the hydrogel concentration and nozzle diameters have significant effects on the filament diameter. Factor interactions are mainly embodied in between the concentration of hydrogel solutions and dispensing pressures. Besides filament diameters, the nozzle height and space also affect the printing accuracy significantly. An appropriate nozzle height is considered to be 1.4 times than the nozzle diameter, and a reasonable nozzle space is suggested from 2.0 to 2.5 times of the nozzle diameter.

The focus of this research is to advance the processing techniques of fabricating scaffolds for tissue engineering and to gain a better understanding of the scaffold properties and behaviours. To achieve these objectives, the fundamental properties of two widely used biomaterials, poly(lactide-co-glycolide acid) (PLGA), poly(ε-caprolactone) (PCL), and their composites with hydroxyapatite were examined. Though increasing the mechanical properties of the bulk polymers, the addition of hydroxyapatite did not affect the thermal and viscoelastic properties, suggesting little interactions may exist between the polymer and the particles. Interestingly, though the addition of the fillers increased the mechanical properties of the bulk materials, the particles worsened the mechanical properties of gas foamed/salt leached scaffolds possibly due to the struts of the porous structure having similar thicknesses as the particles. In such a case, the filler acted as stress raisers and decreased the properties of the struts. The viscoelasticity of the scaffolds was also not affected by the fillers but was affected by the testing environment. An aqueous environment caused the PLGA, but not PCL, to transition such that the porous structure was altered. These results suggest that PLGA may not be ideal for scaffolds for load bearing applications. For electrospinning, a parametric study was performed to control the scaffold morphology, but more importantly, a novel process to fabricate 3D electrospun scaffolds was developed. The novel technique exploited the plasticizing effect of pressurized carbon dioxide on the polymer such that multiple layers of the thin meshes can be sintered together without the use of heat. The process was optimized for adhering layers of PLGA and its composite with nano-hydroxyapatite, and these scaffolds have a high open-porosity and better mechanical properties compared to the gas foamed/salt leached scaffolds. Finally, a model was derived for the viscoelasticity of the bulk materials and their scaffolds by applying fractional calculus on the classical standard linear solid model based on a system of springs and dashpots. The model fitted the data, and correlations between the static mechanical properties and the fitting parameters were found such that by performing static mechanical tests, the viscoelastic behaviours can be approximated.

Engineering epithelium with correct structure is essential for generating functional tissue. During tissue development, cells organize in defined patterns through cellular signalling. Artificial generation of the signalling that organizes cells within the tissue offers a novel approach for engineering tissues with appropriate structure. Planar cell polarity (PCP) is a cellular signalling pathway involved in the organization of epithelial cells. Our goal is to study the effect that co-culturing genetically distinct populations of epithelial cells, with variable levels of one of the core PCP proteins, has in epithelial cell sheet organization. MDCK cells transduced with a tagged PCP core protein (GFP-Vangl2) and wild type MDCK cells were co-cultured side-by-side. The effect of tight junction and cilia formation, and localization of the GFP-Vangl2 protein were evaluated. The results suggest that tight junction and cilia formation are not affected. On the other hand, the GFP-Vangl2 protein seems to be affected at some level.

The focus of this research is to advance the processing techniques of fabricating scaffolds for tissue engineering and to gain a better understanding of the scaffold properties and behaviours. To achieve these objectives, the fundamental properties of two widely used biomaterials, poly(lactide-co-glycolide acid) (PLGA), poly(ε-caprolactone) (PCL), and their composites with hydroxyapatite were examined. Though increasing the mechanical properties of the bulk polymers, the addition of hydroxyapatite did not affect the thermal and viscoelastic properties, suggesting little interactions may exist between the polymer and the particles. Interestingly, though the addition of the fillers increased the mechanical properties of the bulk materials, the particles worsened the mechanical properties of gas foamed/salt leached scaffolds possibly due to the struts of the porous structure having similar thicknesses as the particles. In such a case, the filler acted as stress raisers and decreased the properties of the struts. The viscoelasticity of the scaffolds was also not affected by the fillers but was affected by the testing environment. An aqueous environment caused the PLGA, but not PCL, to transition such that the porous structure was altered. These results suggest that PLGA may not be ideal for scaffolds for load bearing applications. For electrospinning, a parametric study was performed to control the scaffold morphology, but more importantly, a novel process to fabricate 3D electrospun scaffolds was developed. The novel technique exploited the plasticizing effect of pressurized carbon dioxide on the polymer such that multiple layers of the thin meshes can be sintered together without the use of heat. The process was optimized for adhering layers of PLGA and its composite with nano-hydroxyapatite, and these scaffolds have a high open-porosity and better mechanical properties compared to the gas foamed/salt leached scaffolds. Finally, a model was derived for the viscoelasticity of the bulk materials and their scaffolds by applying fractional calculus on the classical standard linear solid model based on a system of springs and dashpots. The model fitted the data, and correlations between the static mechanical properties and the fitting parameters were found such that by performing static mechanical tests, the viscoelastic behaviours can be approximated.

Treatment of cardiac diseases involves transplantation of donor hearts, since the damaged heart has limited self-regeneration potential. An alternative treatment option has emerged as engineered cardiac tissues, grown in vitro by cultivation of cardiac cells on biomaterials, have comparable properties to native myocardium and can be implanted for cardiac repair. Major current limitations are a viable cell source and adequate vascularization to support cell survival. In this thesis, two proangiogenic biomaterials, a scaffold and a hydrogel, were developed to achieve vascularization in vitro and in vivo for cardiac repair. Scaffold patches are suitable for repairing congestive heart failure or congenital malformations, while injectable biomaterials allow minimally-invasive treatment post-myocardial infarction (MI). In the first aim, a collagen scaffold with covalently immobilized vascular endothelial growth factor (VEGF) was developed, and improved cell mobilization, survival and proliferation when used for free wall repair in adult rats. This increased angiogenesis, which aided in retaining the biomaterial size to allow tissue growth. In the second aim, a collagen-chitosan hydrogel with encapsulated thymosin β4 (Tβ4) was developed to 1) recruit cells from the heart epicardium for repair post-MI in vivo, and 2) guide capillary outgrowths from arteries and veins to form oriented capillary structure for in vitro cardiac tissue engineering. Results showed that the encapsulation of Tβ4 into collagen-chitosan hydrogels led to cell outgrowths from rat or mouse cardiac explants in vitro. A portion of the recruited cells were CD31-positive endothelial cells (ECs) that formed tubes. The hydrogel was injected in vivo to increase vascularization and number of cardiomyocytes within the infarct area post-MI, which improved left ventricular wall thickness. Tβ4-hydrogel also promoted the outgrowth of capillaries from vascular explants that followed the direction of the hydrogel-coated grooves of a micropatterned polydimethylsiloxane (PDMS) substrate. These capillary outgrowths eventually formed a vascular bed for engineering vascularized cardiac tissues. This thesis presents two bioinstructive biomaterials with sustained and localized delivery of angiogenic molecules to be used for in situ cardiac repair based on improved vascularization. The use of cell-free bioactive materials overcomes limitations of cell isolation and expansion as required for cell therapies or implantation of engineered tissues.

Engineering epithelium with correct structure is essential for generating functional tissue. During tissue development, cells organize in defined patterns through cellular signalling. Artificial generation of the signalling that organizes cells within the tissue offers a novel approach for engineering tissues with appropriate structure. Planar cell polarity (PCP) is a cellular signalling pathway involved in the organization of epithelial cells. Our goal is to study the effect that co-culturing genetically distinct populations of epithelial cells, with variable levels of one of the core PCP proteins, has in epithelial cell sheet organization. MDCK cells transduced with a tagged PCP core protein (GFP-Vangl2) and wild type MDCK cells were co-cultured side-by-side. The effect of tight junction and cilia formation, and localization of the GFP-Vangl2 protein were evaluated. The results suggest that tight junction and cilia formation are not affected. On the other hand, the GFP-Vangl2 protein seems to be affected at some level.

Knee injuries are a major cause of orthopedic disabilities in the United States. Current reconstruction techniques for torn anterior cruciate ligaments (ACL) require extensive surgery and long physical rehabilitation times since the tissue does not heal upon injury. A common ACL injury occurs where the gap at the rupture site remains open after injury and fails to heal, which can lead to premature osteoarthritis and disability. Hydrogels are a popular material used for tissue engineering applications due to their ability to retain water and good biocompatibility. Previous work has shown that hydrogels can be made through the mixed-mode reaction of radically crosslinked thiol groups and acrylate end groups. This project explores mixed-mode oligo[poly(ethylene glycol) fumarate] (OPF)-based hydrogels as alternate carriers for regeneration of partial tear ligament defects. The main purpose of this project was to determine the degradative properties of and cell response to thiol-PEG-thiol (PEG-diSH), a novel hydrogel material. The swelling and degradative properties of hydrogels containing three components OPF, PEG-diacrylate (PEG-DA), and PEG-diSH were characterized by their fold swelling. In addition, cell viability, morphology changes, proliferation and collagen production were analyzed in tri-ratio hydrogels with and without the presence of RGD over three weeks. Results showed that the hydrogels containing PEG-diSH demonstrated significantly larger fold swelling and promoted cell clustering (as shown by increased area of clusters), probably due to the larger mesh size and possibly due to the presence of free thiol functional groups present in the network from the mixed-mode reaction. However, an increase in cell number was not found in these gels up to eight days, suggesting that cell migration may play a role in the appearance of clusters. Additionally, increased cell spreading in response to RGD was observed inside gels containing PEG-diSH; no spreading was seen in the non PEG-diSH gels (± RGD), possibly because the mesh size was too small to allow for clustering or spreading within the matrix. Results from this work suggest that the presence of PEG-diSH could promote cell-cell contact within the clusters which could be useful in systems where direct contact promotes tissue formation or cell differentiation.

Fabrication of Tissue Engineering Scaffolds Using Stereolithography Benita M. Comeau 226 Pages Directed by Dr. Clifford L. Henderson New methods and materials for the fabrication of hierarchically structured, 3D tissue scaffolds using stereolithography (SL) are presented. The ability to chemically modify selected areas on a scaffold is one way to direct cell growth in deliberate patterns; which is necessary for the engineering of complex, functioning tissues. SL will allow for the building of complex 3D structures with well defined geometries, and a second level of order is created by subsequent modification of chemical groups via catalyzing a de-protection event through exposure to another wavelength of light. The investigated system utilizes an acid-catalyzed de-protection event to change the surface chemistry of an SL-made polymer, analogous to conventional chemically amplified photoresists. The chemical modification alters the surface energy, affecting how proteins interact with the material. This allows selective areas to be more favorable towards cell adhesion. The results of this work include the identification of cytocompatible photo-acid generators that are necessary for the acid-catalyzed de-protection, the demonstration that traditional photolithographic materials may be used for cell patterning, quartz crystal microbalance studies which illuminate why these patterning methods work, the design and performance of a mirror array based stereolithographic apparatus capable of multi-wavelength exposures, and the synthesis and formulation of a novel stereolithographic resin for use in this system. The findings suggest that this system has great potential for use in cell and tissue studies, and possibilities for future use and research are discussed.