No description available.

In coronary artery bypass surgery the autologous saphenous vein is the most commonly used vascular graft. However, in a growing number of patients this vein is not available due to disease or availability. To date, there are no commercially available vascular grafts to replace the autologous saphenous vein. Nevertheless, it is widely accepted that a successful small diameter blood vessel alternative will be found using a tissue engineering approach. A photo-cross-linked biodegradable elastomer of acrylated star-poly(ε-caprolactone-co-D,L-lactide) (ASCP) has recently been developed. The elastomer possesses many desirable properties, such as manufacturability and mechanical properties, making it an interesting scaffolding material candidate for this application. To test the feasibility of the ASCP elastomer as a scaffolding material, a porous scaffold with 90% porosity was constructed using paraffin microbeads combined with an emulsion of ASCP prepolymer and water. Native arterial mechanical properties were matched with an 1800 Da ASCP elastomeric scaffold (ELAS 1800) having 85% porosity. In vitro degradation of scaffolds prepared with two different ASCP Mn (1800 and 4500 Da) was investigated for 8 weeks. Bulk hydrolysis was the mode of degradation regardless of configuration, with the porous scaffold degrading slower than the nonporous control. In addition, the ELAS 4500 scaffold also degraded faster than the ELAS 1800 scaffold with the same porosity. In order to promote the cellular response to this potential vascular scaffold, the surface of the elastomer was modified to enhance bovine coronary artery smooth muscle cell (SMC) attachment and proliferation. Base etching the surface was not as effective as adding a small peptide sequence Gly-Arg-Gly-Asp-Ser (GRGDS) known to enhance cell adhesion. The surface modifications did not change SMC phenotype as all surfaces expressed the contractile marker proteins smooth muscle α-actin and h-caldesmon. The SMCs also expressed these marker proteins when seeded on porous scaffolds. Finally, it was possible to integrate the porous scaffold into a biomimetic blood vessel design. With this initial testing, it appears that the ASCP elastomer is a feasible scaffolding material for small diameter blood vessel tissue engineering. Nevertheless, more detailed testing of mechanical properties and cell behaviour must be conducted to ascertain that the ASCP elastomer and the proposed biomimetic blood vessel design can be appropriate replacements for the autologous saphenous vein. / Thesis (Master, Chemical Engineering) -- Queen's University, 2007-11-18 20:27:30.635

Tissue engineering holds great promise for developing functional tissue to repair damaged articular cartilage. However, cartilaginous tissues formed in vitro typically possess poor mechanical properties in comparison to native tissue due to the inability of chondrocytes to accumulate adequate amounts of extracellular matrix (ECM). While mechanical stimuli can enhance the properties of engineered cartilage, it may be more efficient to harness the underlying mechanotransduction pathways responsible. Substantial evidence suggests that the mechanotransduction signaling cascade is initiated by rapid adenosine 5’-triphosphate (ATP) release from chondrocytes (purinergic receptor pathway). Thus, the purpose of this study was to investigate the effects of exogenous ATP supplementation on the biochemical and mechanical properties of tissue engineered cartilage. Primary bovine articular chondrocytes, seeded on MilliporeTM filters, were grown in the presence of 0, 62.5 or 250 µM ATP for a period of four weeks. Both anabolic and catabolic effects were examined and a therapeutic dose range of ATP was determined. ATP stimulation (62.5 - 250 µM) enhanced ECM synthesis by 23 - 43% and long-term collagen accumulation by 16 - 26%, in a dose-dependent manner; however, long-term proteoglycan accumulation decreased as a result of 250 µM ATP. In addition, ATP supplementation significantly improved the mechanical properties of the developed tissues (5- to 6.5-fold increase in tissue stiffness). Interestingly, high doses of ATP (250 µM) also elicited a 2-fold increase in MMP-13 gene expression, 39% increase in MMP-13 activity, and 54% more extracellular inorganic pyrophosphate (ePPi) – an ATP degradation product. Dose-dependent increases in MMP-13 activity suggested that catabolic effects were occurring alongside anabolic effects, which initiated the investigation of a therapeutic dose of ATP. Doses of 31.25 µM and 125 µM ATP were added to cartilaginous tissues and investigated in terms of MMP-13 activity and ECM synthesis. Tissues supplemented with 62.5 – 125 µM ATP exhibited a balance between anabolic and catabolic effects. By harnessing the purinergic receptor pathway, anabolic effects of mechanical stimuli were achieved in the absence of externally applied forces. Understanding how the catabolic effects of ATP are manifested would be valuable in order to further maximize the therapeutic potential of ATP stimulation. / Thesis (Master, Mechanical and Materials Engineering) -- Queen's University, 2010-09-22 14:08:28.114

No description available.

In vitro grown cartilage is functionally inferior to native tissue, and improvements in its quality should be attempted so it can be used therapeutically. In these studies we investigated the effects of cell shape on tissue quality through alteration of substrate geometry and application of mechanical stimuli. Articular chondrocytes were isolated and cultured on the surface Ti-6Al-4V substrates with various geometries. When cultured on fully porous titanium alloy substrates, chondrocyte spreading was enhanced over those grown on substrates with solid bases. Chondrocytes which remained round did not synthesize significant amounts of matrix and were thus unable to form cartilaginous tissue. In contrast, chondrocytes which were directed to spread to a limited amount, resulting in a polygonal morphology, accumulated significantly more matrix molecules and in time formed cartilage-like tissue. Computational fluid dynamics analyses demonstrated that cells on fully porous substrates experience time-dependent shear stresses that differ from those experienced by cells on substrates with solid bases where media flow-through is restricted. Integrin-blocking experiments revealed that integrins are important regulators of cell shape, and appeared to influence the accumulation of collagen and proteoglycans by chondrocytes. Furthermore, compressive mechanical stimulation induced a rapid, transient increase in chondrocyte spreading by 10 minutes, followed by a retraction to pre-stimulated size within 6 hours. This has been shown to be associated with increased accumulation of newly synthesized proteoglycans. Blocking the α5β1 integrin, or its β1 subunit, inhibited cell spreading and resulted in a partial inhibition of compression-induced increases in matrix accumulation, thereby substantiating the role of β1 integrins in this process. These results suggest that both fluid induced shear forces and compressive forces regulate chondrocyte matrix accumulation by altering cell morphology, which is mediated by integrins. Identifying the molecular mechanisms that influence chondrocyte shape and thus tissue formation may ultimately lead to the development of a tissue that more closely resembles native articular cartilage.

Whole-organ perfusion decellularization was performed with rat hearts on a modified chromatography apparatus. Analysis of the flow properties and effluent material over time provided insights into the decellularization process, and allowed non-destructive testing of perfused cardiac tissue. Decellularized matrices were stored for up to 1 year at -80°C and then conditioned to remove residual detergent and cryoprotectant. Tissue was reseeded with canine blood outgrowth endothelial cells (BOECs) and cultured in an autoclavable closed-circuit bubble-free reactor. The entire process was considered in the context of eventual scale-up in equipment design, the use of disposable components, and extracellular matrix (ECM) product storage. Tissue patch substrates for cell growth were studied for cytotoxic effects towards process development. Decellularization protocols were compared. Extracellular matrix derived coatings and gels were investigated as process assays and potential cell delivery vehicles. Peracetic acid and UV disinfection were tested. Micronized ECM carriers were developed for scalable culture, with considerations to carrier morphology, cell attachment, and egress. Micronized ECM carriers were tested with a novel in vitro assay to simulate the support of adherent cells for gene-modified cell therapy.

Allograft tissue is used to reconstruct cardiac birth defects but induces an immune response resulting in allo-sensitization. Decellularization reduces the immune response, however, acellular vascular tissue is thrombogenic. In-vitro endothelialization may attenuate thrombogenicity. Here we offer our work, which determines a novel method of endothelial cell attachment using Arginine-Glycine-Aspartic Acid (RGD) peptides. We show that an RGD-FITC peptide can be bound to a decellularized ovine cardiac scaffold. RGD modification increases HUVEC cell adhesion to the surface at 3 days of static incubation in-vitro compared to decellularized tissue alone. Repetition using a decellularized human scaffold shows similar results. Cleavage of the potentially immunogenic FITC label retains our RGD peptide. In summary, we determine that decellularized allografts show enhanced HUVEC cell adhesion when modified with an RGD peptide under static conditions. This may increase cell retention in-vivo leading to a decellularized cardiac allograft repopulated with functional autologous cells from the recipient. / Experimental Surgery

Thesis (Ph.D.)--Drexel University, 2007. / Includes abstract and vita. Includes bibliographical references (leaves 204-213).

Thesis (Ph.D.)--Tufts University, 1999. / Submitted to the Dept. of Chemical Engineering. Includes bibliographical references. Access restricted to members of the Tufts University community. Also available via the World Wide Web;

Universiẗat, Diss.--Jena, 2008.