Effects of fibrin micromorphology on neurite growth from dorsal root ganglia cultured in three-dimensional fibrin gels.

The effect of fibrin matrix micromorphology on neurite growth was investigated by measuring the length of neurites growing in three-dimensional fibrin gels with well characterized micromorphologies. Dorsal root ganglia (DRGs) from 7-day chick embryos were entrapped and cultured in gels made from varying concentrations of fibrinogen (5-15 mg/mL) or calcium (2-10 mM). The length of growing neurites was measured with light videomicroscopy, and the number and diameter of fibrin fiber bundles were measured from scanning electron micrographs. An increase in fibrinogen concentration caused a decrease in the average fiber bundle thickness, an increase in the number of fiber bundles, and a marked decrease in neurite length. Gels made with different calcium concentrations had a similar range of variation in fibrin fiber bundle number or diameter, but these variations had little effect on neurite and associated nonneuronal cell outgrowth. These results provide insights into the process of neurite advance within fibrin and may be useful in the design of fibrin-based materials used for peripheral nerve regeneration. Furthermore, this study provides the first detailed experimental data on the micromorphology of fibrin matrices made from more than 5 mg/mL of fibrinogen and indicates that existing kinetic models of fibrin polymerization do not accurately predict fibrin structure at these higher concentrations.

Micropatterning gradients and controlling surface densities of photoactivatable biomolecules on self-assembled monolayers of oligo(ethylene glycol) alkanethiolates.

BACKGROUND: Bioactive molecules that are covalently immobilized in patterns on surfaces have previously been used to control or study cell behavior such as adhesion, spreading, movement or differentiation. Photoimmobilization techniques can be used, however, to control not only the spatial pattern of molecular immobilization, termed the micropattern, but also the surface density of the molecules--a characteristic that has not been previously exploited. RESULTS: Oligopeptides containing the bioactive Arg-Gly-Asp cell-adhesion sequence were immobilized upon self-assembled monolayers of an oligo(ethylene glycol) alkanethiolate in patterns that were visualized and quantified by autoradiography. The amount and pattern of immobilized peptide were controlled by manipulating the exposure of the sample to a UV lamp or a laser beam. Patterns of peptides, including a density gradient, were used to control the location and number of adherent cells and also the cell shape. CONCLUSIONS: A photoimmobilization technique for decorating surfaces with micropatterns that consist of variable densities of bioactive molecules is described. The efficacy of the patterns for controlling cell adhesion and shape has been demonstrated. This technique is useful for the study of cell behavior on micropatterns.

Formation of microscale gradients of protein using heterobifunctional photolinkers.

Gradients of biological molecules on a microscale have been postulated to elicit cellular responses, such as migration. However, it has been difficult to prepare such gradients for experimental testing. A means for producing such gradients has been developed using a heterobifunctional photolinking agent with laser light activation. The photolinking agent synthesized includes an N-hydroxysuccinimide group and a photoreactive benzophenone (BP) separated by a tetraethylene glycol (TEG) spacer. The presence of the tetraethylene glycol spacer renders the photolinker hydrophilic, a desirable trait for conjugation in aqueous solutions. The linker was then conjugated to R-phycoerythrin (R-PE), a fluorescent protein. The resulting photolinker-R-phycoerythrin conjugate (BP-TEG-PE) was then immobilized onto a polystyrene surface by laser irradiation on a motorized stage. By varying exposure time of the sample to the beam, the amount of BP-TEG-PE immobilized on the surface was changed over an order of magnitude over a distance of 250 microns. This method can be applied to prepare gradients of proteins that elicit biological responses, such as extracellular matrix proteins or growth factors, and to study the biological effects of such gradients.

Platelet adhesion to polyurethane blended with polytetramethylene oxide.

The surface and blood compatibility characteristics of Pellethane polyurethane blended with 1% or 5% (w/w) polytetramethylene oxide (PTMO) were evaluated. Analysis by X-ray photoelectron spectroscopy indicated that blending of PTMO caused an increased amount of amide wax, a processing agent present in Pellethane, to be expressed on the surface of the blended films in vacuo. Dynamic contact angle measurements in water, however, showed that PTMO was preferentially expressed on the blend film surfaces in water. The two lower molecular weight species, PTMO and amide wax, were thus capable of reorienting, depending on the environmental conditions. An in vitro assay of platelet adherence and thrombosis showed that polyurethane blended with 5% PTMO had about two-thirds fewer adherent platelets compared to unblended polyurethane and that a blend containing 1% PTMO was intermediate in platelet adherence. Measurements of albumin adsorption from binary solution with fibrinogen indicated that PTMO blends did not preferentially adsorb albumin compared to unblended polyurethane. (c) 1996 John Wiley & Sons, Inc.

Effects of fibinolysis on neurite growth from dorsal root ganglia cultured in two- and three-dimensional fibrin gels.

The mechanism of neurite penetration of three-dimensional fibrin matrices was investigated by culturing embryonic chick dorsal root ganglia (DRGs) within fibrin gels, upon fibrin gels, and upon laminin. The length of neurites within three-dimensional matrices of fibrin was decreased in a concentration-dependent manner by agents that inhibited plasmin, e.g. aprotinin, or that inhibited plasminogen activation, e.g., epsilon-aminocaproic acid (EACA), or plasminogen antiserum. In contrast, such agents increased the length of neurites growing out from DRGs cultured upon two-dimensional substrates of fibrin and had no effect on the length of neurites growing out from DRGs cultured upon laminin. Visualization of neurites within three-dimensional fibrin matrices demonstrated that the distance between fibrin strands was much smaller than the diameter of neurites. All these data were consistent with the hypothesis that fibrinolysis localized to the region of the neurite tip is an important mechanism for neurite penetration of a physical barrier of fibrin strands arranged in a three-dimensional matrix.