In vitro model of vascular healing in the presence of biomaterials

The objective of this thesis research was to elucidate the influence of biomaterial-induced activation of platelets or leukocytes on endothelial cell (EC) or smooth muscle cell (SMC) phenotype, as well as EC/SMC cross-talk in co-culture systems. Understanding these complex interactions will help provide design rules for stents or vascular grafts. Specifically, model biomaterial beads were used to pretreat whole blood, or isolated blood cells (platelets, monocytes, or neutrophils (PMNs)). The model biomaterials used were polystyrene (PS), or PS beads grafted with 3-kDa polyethylene glycol (PEG) with hydroxyl (PS-PEG-OH) or amine (PS-PEG-NH2) terminal groups. These beads were chosen for their controllable, different surface chemistries wherein the PEG modifications are relevant to biomaterials science. Bead form allows for more uniform cell contact, and at 45mum, beads were non-phagocytosable, so as to avoid complications of this response. The biomaterial-pretreated blood cells were then applied to EC or SMC monocultures, or the EC surface of EC/SMC co-cultures. Two divergent co-culture models were developed and characterized with different SMC phenotypes, to determine the impact of SMC phenotype on EC interactions with biomaterial-pretreated blood cells. This work was initially inspired because it was found that biomaterial-pretreated whole blood or isolated monocytes (but not PMNs or platelets) induced a human umbilical vein EC (HUVEC) proinflammatory phenotype (based on upregulation of ICAM-1, E-selectin, and VCAM-1 expression, as well as increased secretion of IL-1, IL-8, and MCP-1) upon static culture (Lester and Babensee, 2003). It was hypothesized that biomaterial contact by leukocytes affects downstream healing processes in part through altering EC behavior, or SMC behavior in sites of EC denudation.; Preliminary work began by pretreating whole blood with biomaterial beads (PS, PS-PEG-OH, or PS-PEG-NH2) at two bead concentrations (5.4 x 104 or 54 x 104 beads/ml) and assessing HUVEC procoagulant phenotype following static culture with biomaterial-pretreated whole blood. Assessment was done by flow cytometry for procoagulant (tissue factor, phosphotidylserine exposure) and anti-coagulant (thrombomodulin, ATPDase) markers. However, it was found that untreated whole blood (no biomaterial or exogenous activator) alone induced a HUVEC procoagulant phenotype. This result made determining the effect of biomaterial-contact by whole blood indiscernible. Based on this, it was determined that to study HUVEC procoagulant phenotype in static culture, isolated blood cells must be used to avoid excessive background activation. In addition, the culture system used was thoroughly examined and further optimized to ensure the ability to see differences between samples.; Before analyzing the effect of biomaterial-pretreated monocytes, PMNs, or platelets on HUVEC procoagulant phenotype, blood cell isolation techniques were first perfected. Purity of isolations were checked, and flow cytometry was performed to examine blood cell activation and microparticle formation due to biomaterial contact or controls. It was found that all bead types tested resulted in monocyte, PMN, and platelet activation, as well as monocyte and PMN microparticle formation. However, only polystyrene induced platelet microparticle formation. Flow cytometry for pro- and anti-coagulant markers, a functional tissue factor assay, and a plasma recalcification clotting time assay were used to examine HUVEC phenotype following static incubation with biomaterial-pretreated monocytes, PMNs, or platelets. It was found that static co-culture with biomaterial-pretreated monocytes or PMNs (but not platelets) differentially induced a HUVEC procoagulant phenotype. This induced phenotype was primarily contact dependent, as the bead-adherent, intact, and microparticle fractions of monocytes and PMNs more strongly induced a HUVEC procoagulant phenotype upon static culture than the solubl...