Tracheal epithelium is organized along two axes: apicobasal, seen through apical ciliogenesis, and planar seen through organized ciliary beating, which moves mucus out of the airway. Diseased patients with affected ciliary motility have serious chronic respiratory infections. The standard method to construct epithelium is through air liquid interface culture which creates apicobasal polarization, not planar organization. Nanogrooved surface topography created in diffusible substrates for use in air liquid interface culture will induce planar organization of the cytoskeleton. We have created a nanogrooved gelatin device which allows basal nutrient diffusion. Multiple epithelial cells have been found to align in the direction of the nanogrooves in both sparse and confluent conditions. This device is also congruent with ALI culture as seen through formation of tight junctions and ciliogenesis. Thus, we have created nanogrooved surface topography in a diffusible substrate that induces planar alignment of epithelial cells and cytoskeleton.

The ability to generate cardiomyocytes from either embryonic stem cells or induced pluripotent stem cells provides an unprecedented opportunity to establish human in vitro models of cardiovascular disease as well as to develop platforms for the testing of novel cardiac therapeutics. We designed two different platforms, a biowire platform and post deflection platform, to generate engineered heart tissues (EHTs) to study a fundamental process in cardiomyocytes: hypertrophy. Both pathological and physiological hypertrophy was studied in order to garner a better understanding of each process. Physiological hypertrophy characteristics were observed using the biowire platform seen in improved myofibril alignment and downregulation of fetal genes. When electrical stimulation was added, a rate dependent effect on sarcomere maturation was observed by the increased frequency of I-bands and H-zones. Certain hallmark features of pathological hypertrophy, such as upregulation of brain natriuretic peptide and sarcomere structure breakdown, were recapitulated when EHTs were treated with isoproterenol.

Analogous to the extracellular matrix (ECM) of natural tissues, properties of a tissue engineering scaffold direct cell behavior and thus regenerated tissue properties. These include both physical properties (e.g. morphology and modulus) and chemical properties (e.g. hydrophobicity, hydration and bioactivity). Notably, recent studies suggest that scaffold properties (e.g. modulus) may be as potent as growth factors in terms of directing stem cell fate. Thus, 3D scaffolds possessing specific properties modified for optimal cell regeneration have the potential to regenerate native-like tissues. Photopolymerizable poly(ethylene glycol) diacrylate (PEG-DA)-based hydrogels are frequently used as scaffolds for tissue engineering. They are ideal for controlled studies of cell-material interactions due to their poor protein adsorption in the absence of adhesive ligands thereby making them “biological blank slates”. However, their range of physical and chemical properties is limited. Thus, hydrogel scaffolds which maintain the benefits of PEG-DA but possess a broader set of tunable properties would allow the establishment of predictive relationships between scaffold properties, cell behavior and regenerated tissue properties. Towards this goal, this work describes a series of unique hybrid inorganic-organic hydrogel scaffolds prepared using different solvents and also in the form of continuous gradients. Properties relevant to tissue regeneration were investigated including: swelling, morphology, modulus, degradation rates, bioactivity, cytocompatibility, and protein adhesion. These scaffolds were based on the incorporation of hydrophobic, bioactive and osteoinductive methacrylated star polydimethylsiloxane (PDMSstar-MA) [“inorganic component”] into hydrophilic PEG-DA [“organic component”]. The following parameters were varied: molecular weight (Mn) of PEG-DA (Mn = 3k & 6k g/mol) and PDMSstar-MA (Mn = 1.8k, 7k, 14k), ratio of PDMSstar-MA to PEG-DA (0:100 to 20:80), total macromer concentration (5 to 20 wt%) and utilizing either water or dichloromethane (DCM) fabrication solvent. The use of DCM produced solvent induced phase separation (SIPS) resulting in scaffolds with macroporous morphologies, enhanced modulus and a more homogenous distribution of the PDMSstar-MA component throughout. These hybrid hydrogel scaffolds were prepared in the form of continuous gradients such that a single scaffold contains spatially varied chemical and physical properties. Thus, cell-material interaction studies may be conducted more rapidly at different “zones” defined along the gradient. These gradients are also expected to benefit the regeneration of the osteochondral interface, an interfacial tissue that gradually transitions in tissue type. The final aspect of this work was focused on enhancing the osteogenic potential of PDMS via functionalization with amine and phosphonate. Both amine and phosphonate moieties have demonstrated bioactivity. Thus, it was expected that these properties will be enhanced for amine and phosphonate functionalized PDMS. The subsequent incorporation of these PDMS-based macromers into the previously described PEG-DA scaffold system is expected to be valuable for osteochondral tissue regeneration.

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)

It would be a great advantage in reconstructive surgery to have an off-the-shelf biomaterial to promote regeneration and volume augmentation following soft tissue damage. With this long-term objective, human adipose tissue (fat) is an abundant and accessible source of extracellular matrix (ECM) for bioscaffold fabrication. The main goal of the current research project was to optimize the established 5-day detergent-free decellularization protocol developed by the Flynn group, by shortening it to a maximum of 3 days, while achieving comparable results in terms of cell and lipid extraction with preservation of the ECM. The effectiveness of the optimized protocol was assessed by examination of the decellularized adipose tissue (DAT) and its characteristic biological properties, including in vitro bioactivity assays with human adipose-derived stem cells (ASCs) to measure adipogenic potential, as well as in vivo testing of scaffold biocompatibility. In the optimized approach, the addition of mechanical processing steps including repeated pressing and centrifugation were shown to enhance cell extraction. Fibrous ultrastructure was observed under scanning electron microscopy (SEM) for the original and optimized protocols. The preservation of collagen fibres was assessed with picro-sirius red staining and confirmed by high hydroxyproline content. Enhanced preservation of glycosaminoglycans (GAGs) was determined for the optimized protocol. Residual DNA content was higher in the DAT scaffolds processed with the optimized protocol, including larger DNA fragments that were not typically observed in the samples treated with the original protocol, which incorporated additional enzymatic treatment stages with DNase, RNase and lipase. However, no residual nuclei were visualized through DAPI staining for both protocols. Enhanced removal of DNA was achieved with electron beam (e-beam) sterilization. E-beam sterilization caused some changes in the fine fibrous structure of the ECM, but did not negatively affect the adipo-conductive potential in vitro. In comparison to the original protocol, DAT produced via the optimized protocol exhibited similar adipo-conductive properties in vitro. The in vivo biocompatibility study over a 16 week period using an immunocompetent Wistar rat model showed promising results. DAT implants produced with the original and optimized protocols promoted adipogenesis and angiogenesis, gradually being remodelled to resemble mature adipose tissue. / Thesis (Master, Chemical Engineering) -- Queen's University, 2014-01-30 12:25:22.044

This study consists of two projects related to Tissue Engineering: Engineering biomimetic scaffolds for bone regeneration and ear reconstruction, and bioreactor design for ex-vivo bioengineered scaffold. The co-electrospinning method was used to produce composite membranes with different layers from gelatin and polycaprolactone (PCL) nanofibers, followed by paper-stacking cell seeded membranes to mimic the twisted plywood structure found in lobster cuticles. 3D laser scanner was used to capture the precise shape of a human ear model; and the negative molds were fabricated to compress scaffolds into the shape of human ear. Design for assembly (DFA) method was used to analyze and improve the design of the current bioreactor. A new design is proposed to ease operation, save time and increase the application efficiency. The proposed solution is evaluated in a virtual environment using 3D assembly modeling and simulation.

Modular tissue engineering involves assembling tissue constructs with integral vasculature from units containing adipose-derived mesenchymal stromal cells (adMSCs) and endothelial cells. Here, the effects of implant volume and adMSC density on the vascularization of modular tissues were explored. Both parameters affected the contributions of host- and graft-derived vessels, without affecting total vessel density. Increasing implant volume from 0.01 to 0.10 mL increased HIF1α expression and graft-derived vessel density, suggesting a role of hypoxia in graft-derived vessel formation. However, increasing adMSC density within small-volume implants did not increase HIF1α expression. Vascularization of small-volume implants of high (4.3•10^6 cells/mL) and low (1.0•10^6 cells/mL) adMSC densities was dominated by host vessel ingrowth at day 7. By increasing adMSC density, a high proportion of host-derived vessels was maintained to day 14, presumably via paracrine effects. Further dissection of the role of hypoxia in modular tissue engineering remains a promising avenue to pursue.

Modular tissue engineering involves assembling tissue constructs with integral vasculature from units containing adipose-derived mesenchymal stromal cells (adMSCs) and endothelial cells. Here, the effects of implant volume and adMSC density on the vascularization of modular tissues were explored. Both parameters affected the contributions of host- and graft-derived vessels, without affecting total vessel density. Increasing implant volume from 0.01 to 0.10 mL increased HIF1α expression and graft-derived vessel density, suggesting a role of hypoxia in graft-derived vessel formation. However, increasing adMSC density within small-volume implants did not increase HIF1α expression. Vascularization of small-volume implants of high (4.3•10^6 cells/mL) and low (1.0•10^6 cells/mL) adMSC densities was dominated by host vessel ingrowth at day 7. By increasing adMSC density, a high proportion of host-derived vessels was maintained to day 14, presumably via paracrine effects. Further dissection of the role of hypoxia in modular tissue engineering remains a promising avenue to pursue.

The ability to generate cardiomyocytes from either embryonic stem cells or induced pluripotent stem cells provides an unprecedented opportunity to establish human in vitro models of cardiovascular disease as well as to develop platforms for the testing of novel cardiac therapeutics. We designed two different platforms, a biowire platform and post deflection platform, to generate engineered heart tissues (EHTs) to study a fundamental process in cardiomyocytes: hypertrophy. Both pathological and physiological hypertrophy was studied in order to garner a better understanding of each process. Physiological hypertrophy characteristics were observed using the biowire platform seen in improved myofibril alignment and downregulation of fetal genes. When electrical stimulation was added, a rate dependent effect on sarcomere maturation was observed by the increased frequency of I-bands and H-zones. Certain hallmark features of pathological hypertrophy, such as upregulation of brain natriuretic peptide and sarcomere structure breakdown, were recapitulated when EHTs were treated with isoproterenol.

<p>The development of a functional tissue-engineered human skeletal muscle model in vitro would provide an excellent platform on which to study the process of myogenesis, various musculoskeletal disease states, and drugs and therapies for muscle toxicity. We developed a protocol to culture human skeletal muscle bundles in a fibrin hydrogel under static conditions capable of exerting active contractions. Additionally, we demonstrated the use of joint miR-133a and miR-696 inhibition for acceleration of muscle differentiation, elevation of active contractile force amplitudes, and increasing Type II myofiber formation in vitro. </p><p>The global hypothesis that motivated this research was that joint inhibition of miR-133a and miR-696 in isolated primary human skeletal myoblasts would lead to accelerated differentiation of tissue-engineered muscle constructs with higher proportion of Type I myofibers and that are capable of significantly increased active contractile forces when subjected to electrical stimulus. The proposed research tested the following specific hypotheses: (1) that HSkM would require different culture conditions than those optimal for C2C12 culture (8% equine serum in differentiation medium on uncoated substrates), as measured by miR expression, (2) that joint inhibition of miR-133a and miR-696 would result in 2D human skeletal muscle cultures with accelerated differentiation and increased Type I muscle fibers compared to control and individual inhibition of each miR, as measured by protein and gene expression, (3) that joint inhibition of miR-133a and miR-696 in this functional 3D human skeletal muscle model would result in active contraction significantly higher than control and individual inhibition by each miR, as measured by isometric force testing, and finally (4) that specific co-culture conditions could support a lamellar co-culture model in 3D of human cord blood-derived endothelial cells (hCB-ECs) and HSkM capable of active contraction, as measured by isometric force testing and immunofluorescence. </p><p>Major results of the dissertation are as follows. Culture conditions of 100 &#956;g/mL growth factor reduced-Matrigel-coated substrates and 2% equine serum in differentiation medium were identified to improve human skeletal myoblast culture, compared to conditions optimal for C2C12 cell culture (uncoated substrates and 8% equine serum media). Liposomal transfection of human skeletal myoblasts with anti-miR-133a and anti-miR-696 led to increased protein presence of sarcomeric alpha-actinin and PGC-1alpha when cells were cultured in 2D for 2 weeks. Presence of mitochondria and distribution of fiber type did not change with miR transfection in a 2D culture. Joint inhibition also resulted in increased PPARGC1A gene expression after 2 weeks of 2D culture. For muscle bundles in 3D, results suggest there exists a myoblast seeding density threshold for the production of functional muscle. 5 x 106 myoblasts/mL did not produce active contraction, while 10 x 106 myoblasts/mL and above were successful. Of the seeding densities studied, 15 x 106 myoblasts/mL resulted in constructs that exerted the highest twitch and tetanus forces. Engineering of human skeletal muscle from transfected cells led to significant increases in force amplitude in joint inhibition compared to negative control (transfection with scrambled miR sequence). Joint inhibition in myoblasts seeded into 3D constructs led to decreased presence of slow myosin heavy chain and increased fast myosin heavy chain. Finally, co-culture of functional human skeletal muscle with human cord blood-derived endothelial cells is possible in 3.3% FBS in DMEM culture conditions, with significant increases in force amplitudes at 48 and 96 hours of co-culture.</p> / Dissertation