Browsing by Subject "Tissue Engineering"
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Item The effect of peroneal nerve relocation on skeletal muscle regeneration within an extracellular matrix seeded with mesenchymal stem cell populations derived from bone marrow and adipose tissue(2009-08) Tierney, Matthew Timothy; Farrar, Roger P.; Suggs, Laura J.Despite the normally robust regenerative capacity of muscle tissue, extensive soft tissue damage often results in a functional loss that cannot be restored using classic reconstruction techniques. Although implanted biomaterials are capable of mechanically transmitting force generated from the remaining tissue, cellular repopulation, reinnervation and revascularization of the injured area is necessary to achieve complete functional restoration. Using an in vivo tissue engineering model, a 1.0 x 1.0 cm portion of the lateral gastrocnemius (LGAS) of Lewis rats was removed and replaced with a muscle-derived extracellular matrix (ECM). Constructs were seeded with bone marrow-derived (BMSCs) or adipose-derived stem cells (ADSCs) and the peroneal nerve was relocated over the implanted ECM. Creation of the defect resulted in a functional impairment of the LGAS, only capable of producing 85.1 ± 4.1% of the force generated in the contralateral LGAS following ECM implantation. A significant increase in specific tension (SPo) was seen in all groups following the nerve relocation procedure when compared to their corresponding cellular treatment without nerve relocation (p < 0.05). Histological quantification revealed significant increases in cellular content and blood vessel density in the top and bottom regions of ECM implants seeded with BMSCs (p < 0.05). The nerve relocation procedure significantly increased these same variables within the middle region of the ECM when compared to all groups lacking this treatment (p < 0.05). The presence of regenerating myofibers was immunofluorescently confirmed using antibodies against desmin, myosin heavy chain and laminin, while their developmental state was substantiated by the presence of central nuclei. These data corroborate a therapeutic effect of BMSCs on skeletal muscle regeneration within the ECM implant that was not seen following ADSC injection. Furthermore, the nerve relocation procedure stimulated an increased cellular and vascular growth within the middle region of the construct, likely the cause of improved functional output.Item Inorganic-Organic Hydrogel Scaffolds for Tissue Engineering(2013-07-11) Bailey, BrennanAnalogous 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.Item Systematic Investigation of Hydrogel Material Properties on Cell Responses for Vocal Fold and Vascular Graft Tissue Engineering(2010-01-14) Bulick, AllenThe research presented here deals with synthetic materials for application in tissue engineering, primarily poly(ethylene glycol) (PEG) and poly(dimethyl siloxane)star (PDMS)star. Tissue engineering seeks to repair or replace damaged tissue through implantation of cell encapsulated in an artificial scaffold. Cell differentiation and extracellular matrix (ECM) deposition can be influenced through a wide variety of in vitro culture techniques including biochemical stimuli, cell-cell interactions, mechanical conditioning and scaffold physical properties. In order to systematically optimize in vitro conditions for tissue engineering experiments, the individual effects of these different components must be studied. PEG hydrogels are a suitable scaffold for this because of their biocompatibility and biological "blank slate" nature. This dissertation presents data investigating: the effects of glycosaminoglycans (GAGs) as biochemical stimuli on pig vocal fold fibroblasts (PVFfs); the effects of mechanical conditioning and cell-cell interactions on smooth muscle cells (SMCs); and the effects of scaffold physical properties on SMCs. Results show that GAGs influence PVFf behavior and are an important component in scaffold design. Hyaluronic acid (HA) formulations showed similar production in collagen I and III as well as reduced levels of smooth muscle a-actin (SMa-actin), while chondroitin sulfate (CSC) and heparin sulfate showed enriched collagen III environments with enhanced expression of SMa-actin. A physiological flow system was developed to give comprehensive control over in vitro mechanical conditioning on TEVGs. Experiments performed on SMCs involved creating multi-layered TEVGs to mimic natural vascular tissue. Constructs subjected to mechanical conditioning with an endothelial cell (EC) layer showed enhanced expression of SMC differentiation markers calponin h1 and myocardin and enhanced deposition of elastin. Consistent with other studies, EC presence diminished overall collagen production and collagen I, specifically. Novel PDMSstar-PEG hydrogels were studied to investigate the effects of inorganic content on mesenchymal stem cell differentiation for use in TEVGs. Results agree with previous observations showing that a ratio of 5:95 PDMSstar: PEG by weight enhances SMC differentiation markers; however, statistically significant conclusions could not be made. By studying and optimizing in vitro culture conditions including scaffold properties, mechanical conditioning and multi-layered cell-cell interactions, TEVGs can be designed to maximize SMC differentiation and ECM production.