Steven L. Goodman

Three-dimensional extracellular matrix textured biomaterials

Clinical and experimental investigations have reported that manufactured surface topographies have significant effects on cell adhesion and tissue integration. However, essentially all previously examined topographies bear little relation to cell adhesion substrates found in biological tissues. In vivo, many cells are adherent to extracellular matrices (ECM), which have an extremely complex 3-D topography in the micrometre to nanometre range. In addition, many studies indicate that micro- and nano-scale mechanical stresses generated by cell-matrix adhesion have significant effects on cellular phenotypic behaviour. In this report we describe methodology for the fabrication of topographic replicas of the subendothelial ECM topography with a biomedical polyurethane. Using three-dimensional high resolution scanning electron microscopy, accurate replication of subendothelial ECM topography from the macroscopic to the macromolecular scale is demonstrated. Bovine aortic endothelial cells cultured on the ECM replicas spread more rapidly and had a three-dimensional appearance and spread areas at confluence which appeared more like endothelial cells in native arteries, compared with cells cultured on untextured control surfaces. Since the fabrication process may be used with many different types of materials, including polymers of synthetic and biological origin, these biomimetic ECM-textured surfaces may find both research and clinical applications.

Platelet interaction with pyrolytic carbon heart-valve leaflets

Although the newest generation of mechanical heart-valve prosthetics constructed either partially or wholly of lowtemperature isotropic pyrolytic carbon (LTIC) have significantly reduced thromboembolic complications compared with early-generation mechanical valves (e.g., Starr-Edwards), thromboembolism remains an important clinical complication. In the present study, high-resolution, lowvoltage scanning electron microscopy (HR-LV-SEM) was used to examine the structure and platelet interaction properties of LTIC valve leaflets manufactured by both Carbo Medics, Inc. and by St. Jude Medical, Inc. Valve leaflets from both manufacturers, prepared and polished exactly as used in clinical heart valves, had similar surface energetics and elemental composition. Examination with LV-SEM revealed a rough and complex three-dimensional surface structure with nanometer- to micron-size features. In vitro adhesion of human platelets on the LTIC materials and Formvar were evaluated in the presence of 1 mg/mL albumin. Platelet-surface activation, as evaluated by shape change, spread area, and deposition, was extremely extensive on the LTIC materials compared with the Formvar positive control material. LTIC-adherent platelets were extremely thin, and closely followed the rough LTIC contours, greatly limiting their visibility with conventional SEM. These observations demonstrate that LTIC surfaces can extensively activate platelets even in the presence of albumin, thereby suggesting that platelet interactions with pyrolytic carbon may have a significant role in mechanical-valve thromboembolism.

Surface modification of poly(tetrafluoroethylene) with benzophenone and sodium hydride by ultraviolet irradiation

Poly(tetrafluoroethylene) (PTFE) films were surface modified in a solution of benzophenone and sodium hydride in dry dimethylformamide by ultraviolet (UV) light irradiation. The extent of surface modification was characterized after durations of UV light irradiation from 5-20 min at temperatures from 19-60°C. The modified films were analyzed by electron spectroscopy for chemical analysis, diffuse reflectance ultraviolet-visible light spectroscopy, attenuated total reflection Fourier transform infrared spectroscopy, dynamic contact angle measurement, and low-voltage scanning electron microscopy. PTFE surfaces produced by this modification demonstrated extensive defluorination, oxygen incorporation, surface unsaturation, and reduction in both advancing and receding dynamic water contact angles in a manner that was more extensive at long durations of irradiation and at high temperatures. Morphological damage depended upon treatment conditions, but extensive surface modification could be obtained without substantial morphological damage to PTFE films. Control experiments indicated that the surface modification proceeded by photoexcitation of either diphenyl ketyl radical anion or benzhydrol anion, the products of reaction of benzophenone with sodium hydride.

Preferential adsorption of plasma proteins onto apolar polyurethane microdomains

Abstract Adsorbed proteins mediate interactions between synthetic biomaterials and physiologic environments. Segmented polyurethane elastomers are extensively used in biomedical devices such as catheters, blood pump bladders, and vascular grafts. Microphase separation in these materials has been correlated with biocompatibility, however, whether separate microdomains exist at aqueous polyurethane surfaces is unknown. Using high-voltage transmission and low-voltage scanning electron microscopy, 10–30 nm soft segment microdomains were imaged at the surface of a hydrated polybutadiene-polyurethane, within the nanometer level surface sensitivity of the instrumentation. Subsequently, two different colloidal gold labeling techniques were used to determine if the distribution of fibronectin, fibrinogen, and albumin, adsorbed from single protein solutions, were influenced by the microdomain morphology. Both immunogold labeling and a novel technique of labeling with unconjugated colloidal gold, which provides very high spatial precision, showed that all three proteins preferentially adsorb to the apolar polybutadiene soft segment microdomains. Thus, protein adsorption is influenced by surface microenvironments of comparable size to the protein itself. This suggests that the phase-separated microstructure of polyurethanes may organize the biological-material interface.

The effects of substrate-adsorbed albumin on platelet spreading

Adsorbed albumin appears to passivate nearly all materials, minimizing platelet adhesion and thrombus formation. Since in vitro platelet spreading can be an indicator of in vivo reactivity leading to thrombosis, and as in vitro platelet adhesion investigations are routinely done in the presence of bovine or human serum albumin (BSA or HSA), we examined the influence of albumin on platelet reactivity to material substrates. Platelet spreading was examined subsequent to adherence onto several related polyurethanes, and to Formvar, in the presence of bulk albumin concentrations sufficient to form an adsorbed monolayer or a multilayer. No other exogenous proteins were present. The spreading behavior of adherent platelets was analyzed using generalized linear interactive modeling (GLIM). The models showed that the polymer type always influenced platelet responses, irrespective of the albumin concentration. In many experiments, platelet behavior could be adequately modeled without including the effects of albumin. Thus, the polymer type appeared to be the primary determinant of platelet shape-change with adsorbed albumin producing a secondary effect. Additionally, somewhat different effects on spreading were observed with HSA and BSA, suggesting qualitatively different interactions between human platelets and HSA, than with BSA, which is commonly used in platelet preparations.

Platelet‐polymer interactions: Morphologic and intracellular free calcium studies of individual human platelets

Light and electron microscopic methods were used to correlate changes in platelet morphology with levels of internal free calcium in platelets adhering to Formvar, Pellethane, and 14% SO3 Pellethane. Free calcium levels were elevated when platelets initially activated in response to contact with the polymer substrates. With buffer containing 1 mM CA2+, representative of in vivo plasma calcium levels, platelets activating on the sulfonated substrate exhibited significantly higher intracellular free calcium levels compared to those on Formvar and Pellethane. Furthermore, the intracellular free calcium remained elevated in these platelets which failed to spread normally on the sulfonated substrate. In contrast, the platelets adherent on Formvar and Pellethane achieved normal fully spread morphologies with correspondingly low or resting calcium levels. Our results indicate that the addition of the sulfonated group to Pellethane affected internal platelet calcium regulation to cause an abnormal spreading response. The nonviable platelet morphologies observed on sulfonated Pellethane compare to other dead cell morphologies caused by abnormally elevated levels of intracellular free Ca2+.