Wolf, Michael F2010-03-312010-03-312009-12https://hdl.handle.net/11299/59993University of Minnesota M.S. thesis. December 2009. Major: Biomedical Engineering. Advisors: Richard Bianco and Paul A Iaizzo. 1 computer file (PDF); viii, 64 pages.Finding an effective small diameter synthetic vascular graft has been the focus of research for over three decades. A major direction has been on a means to establish a functional endothelial cell (EC) lining on common graft materials. Unfortunately, a simple operating room (OR) method has proven to be neither obvious to knowledgeable researchers nor feasible for standard hospital staff. In the space of the host of variables considered by researchers, the goal of this work was to see if a more practicable procedure might be identified using a series of factorial experimental design evaluations on certain process-simplifying and material-alteration concepts. These concepts were micro-geometric surface modification, g-force cell seeding, and graft pretreatment using a particular fraction of autologous blood. For this, clinical ePTFE graft material was surface-modified into ‘brushed’ ePTFE (bePTFE) to make the material more receptive to cell uptake and adhesion; seeding graft materials with ECs was achieved using high gforce from axial centrifugation; and, a graft pretreatment with autologous platelet poor plasma was used to generate an autologous fibrin-platelet-poor layer (aFPPL) on the graft surface. The goal of this approach was to rapidly identify, assess, and optimize the most minimal-impact conditions applicable in an operating room that positively influence formation of a healthy autologous cell lining. Bench top studies revealed the bePTFE material to show an increased uptake and retention of ECs compared to ePTFE (P<0.05). In addition, independent of material, the use of high rpm g-force seeding compared to seeding under slow rotation resulted in significantly more cells taken up by the materials iv under high g-force (P<0.05). The cells also appeared to be retained under simulated pulsatile flow conditions. Five day tissue culture experiments then showed that an aFPPL layer applied to the graft surface prior to g-force cell seeding was a substantial growth matrix for seeded cells. A follow-up in vivo 24 factorial experimental design study on graft material, EC seeding via g-force, pretreatment generating an aFPPL , and graft orientation indicated the following trends and significant observations: (1) bePTFE was associated with a trend in improved patency (P<0.07), reduced histological evidence of thrombosis (P<0.08), and reduced luminal red discolorations (P<0.0001), (2) EC seeding was shown to be associated with a trend in improved patency (P<0.08), a reduction in surface discoloration (P<0.0004), and an increased midgraft endothelium (P<0.04) assessed by scanning electron microscopy (SEM). Histology scores also revealed EC seeding to be associated with more neointimal development (P < 0.08), more graft cellularity (P < 0.001), and higher macrophage infiltration (P < 0.007); and (3) graft pretreatment with autologous PPP to generate an aFPPL on the graft surface showed a significant impact on the surface discoloration (P<0.009) and extent of endothelial cell coverage (P<0.01) as assessed by SEM. This work demonstrates that through a multiparameter screening study approach a number of potential significant improvements towards a practicable one-step OR-compatible vascular graft endothelialization technique were identified.en-USSmall diameter synthetic vascular grafFunctional endothelial cell (EC)Clinical ePTFE graft materialSeeding graft materialsEndothelial cell (EC)Biomedical EngineeringThesis or Dissertation