Densely crosslinked polymer networks of poly(ethylene glycol) in trimethylolpropane triacrylate for cell-adhesion-resistant surfaces.

Densely crosslinked semi-interpenetrating polymer networks (semi-IPNs) of poly(ethylene glycol) (PEG) were synthesized by photopolymerizing a melt of PEG of various molecular weights and end-group functionalities in neat trimethylolpropane triacrylate (TMPTA). Increasing the molecular weight of PEG in the matrix from 1000 to 100,000 g/mol reduced the advancing and receding contact angles, contact angle hysteresis, and adsorption of human fibrinogen and bovine serum albumin. Crosslinked TMPTA homonetworks supported human fibroblast adhesion in vitro, whereas the resistance to cell adhesion of the semi-IPNs depended upon PEG molecular weight: Lower molecular weight PEG reduced the number of adherent cells; higher molecular weight PEG further reduced and eliminated cell adhesion, as did networks containing acrylate-functionalized PEG. A polymer system incorporated with PEG throughout a hydrophobic, densely crosslinked matrix, rather than as a blend or surface treatment, may be particularly useful for limiting biologic interactions when bulk material properties must be independent of the solvent environment and where surface abrasion may occur.

Polymer networks with grafted cell adhesion peptides for highly biospecific cell adhesive substrates.

Polymer networks of poly(ethylene glycol) (PEG) in densely cross-linked matrices of acrylic acid (AA) and trimethylolpropane triacrylate were synthesized as biospecific cell adhesive substrates. Networks grafted with synthetic adhesion peptides produced substrates to investigate long-term, receptor-mediated cell/surface interactions, without nonspecific protein adsorption producing spurious adhesion signals. PEG rendered the networks very resistant to cell adhesion in vitro, and AA provided reactive carboxyl moieties for N-terminal grafting of peptides. Networks with higher mass fractions of AA had greater background cell adhesion, which diminished with higher mass fractions of PEG such that complete resistance to cell adhesion could be obtained. Networks grafted with inactive control peptides (GRGES or no peptide) remained completely cell nonadhesive in the presence of serum or even when preincubated with adhesion proteins, while networks grafted with bioadhesive peptides (GRGDS, GYIGSRY, or GREDVY) supported morphologically complete fibroblast adhesion. The amount of AA in the network readily controlled the amount of incorporated peptide. These networks may be suitable as analytical tools specifically to investigate long-term cell/substrate interactions in the presence of serum, yet without non-specific protein adsorption producing adhesion signals other than those immobilized for study.

Multifunctional poly(ethylene glycol) semi-interpenetrating polymer networks as highly selective adhesive substrates for bioadhesive peptide grafting.

Novel artificial extracellular matrices were synthesized in the form of semi-interpenetrating polymer networks containing copolymers of poly(ethylene glycol) and acrylic acid (PEG-co-AA) grafted with synthetic bioadhesive peptides onto exposed carboxylic acid moieties. These substrates were very resistant to cell adhesion, but when they were grafted with adhesive peptides they were highly biospecific in their ability to support cell adhesion. Extensive preadsorption of adhesive proteins or peptides did not render these materials cell adhesive; yet covalent grafting of adhesive peptides did render these materials highly cell adhesive even in the absence of serum proteins. Polymer networks containing immobilized PEG-co-AA were grafted with peptides at densities of 475 +/- 40 pmol/cm(2). Polymer networks containing immobilized PEG-co-AA N-terminally grafted with GRGDS supported cell adhesion efficiencies of 42 +/- 4% 4 h after seeding and became confluent after 12 h. These cells displayed cell spreading and cytoskeletal grafted with inactive control peptides (GRDGS, GRGES, or no peptide) supported cell adhesion efficiencies of 0 +/- 0%, even when challenged with high seeding densities (to 100,000 cell/cm(2)) over 14 days. These polymer networks are suitable substrates to investigate in vitro cell-surface interactions in the presence of serum proteins without nonspecific protein adsorption adhesion signals other than those immobilized for study.

Local modulation of intracellular calcium levels near a single-cell wound in human endothelial monolayers.

An endothelial cell monolayer with a single mechanically lysed cell was used as a model to examine the extent, kinetics, and nature of local calcium mobilization in the neighborhood of a wound. Individual endothelial cells from confluent monolayers were mechanically lysed with a minutien needle coupled to a micromanipulator while producing no observable mechanical trauma to the neighboring cells. Changes in calcium levels in individual cells surrounding the wound site were monitored by epifluorescence microphotometry with the calcium-sensitive fluorophore indo-1. Individual cells adjacent to the wound site showed a substantial increase in their intracellular calcium levels, almost as high as the calcium levels attained by ionophore controls. The magnitude of intracellular calcium mobilization in confluent monolayers decreased with distance from the wound site, and those cells located at a radius greater than seven cells from the wound site showed no change in their calcium levels. Thus, lysis of a single cell resulted in calcium mobilization in approximately 200 neighboring cells. The time necessary for intracellular calcium to reach maximum levels also increased with distance from the wound site. Calcium mobilization was partly intracellular and was inhibited by disrupting cell-cell coupling or by increasing gap junction resistance by heptanol. This mobilization was greatly attenuated in subconfluent endothelial monolayers, and it was not observed in fibroblasts or smooth muscle cells; furthermore, the effect was defective in monolayers intentionally contaminated with smooth muscle cells. This study examines the extent and possible mechanisms of local endothelial activation near a microscopic endothelial wound.

Endothelial cell-selective materials for tissue engineering in the vascular graft via a new receptor.

We have found a novel adhesion receptor on the human endothelial cell for the peptide sequence Arg-Glu-Asp-Val (REDV), which is present in the III-CS domain of human plasma fibronectin, with a dissociation constant of 2.2 x 10(-6) M and 5.8 x 10(6) sites/cell. When a synthetic peptide containing this sequence was immobilized on otherwise cell nonadhesive substrates, endothelial cells attached and spread but fibroblasts, vascular smooth muscle cells, and platelets did not. Endothelial monolayers on REDV were nonthrombogenic: endothelial cells attached and spread upon other receptor-binding domains of fibronectin and laminin, but with lesser degrees of specificity or with a loss of nonthrombogenicity. This approach may provide a basis for a tissue engineered vascular graft where endothelial cell attachment is desired, but not the attachment of other blood vessel wall cells and blood platelets.

The effects of RPM and recycle on separation efficiency in a clinical blood cell centrifuge.

A COBE blood cell centrifuge, model 2997 with a single stage channel, was modified to allow computer controlled sampling, and to allow recycle of red blood cells (RBCs) and plasma streams using bovine whole blood. The effects of recycle of the packed RBC and plasma product streams, and of the centrifuge RPM on platelet and white blood cell (WBC) separation efficiencies were quantified using a central composite factorial experimental design. These data were then fit using second order models. Both the model for the WBC separation efficiency and the model for the platelet separation efficiency predict that RPM has the greatest effect on separation efficiency and that RBC and plasma recycle have detrimental effects at moderate to low RPM, but have negligible impact on separation efficiency at high RPM.