Factors influencing isolation of functional pancreatic rat islets.

Yields and function of isolated islets vary considerably in spite of the introduction of new or improved methods for isolation. In most studies, these variations have been attributed to inadequacies of the applied collagenase preparations. However, when we retrospectively analyzed our rat islet isolations, we found large variations in yield and function in spite of application of identical collagenase sources. Therefore, in the present study, we determined the effect of rat donor strain, the source of inhibition of proteolytic activity (by bovine serum albumin), and the culture conditions on yield and function. AO rats showed a twofold higher islet yield than Wistar and Lewis rats. However, a higher yield was not associated with a higher response on glucose load since this response was more pronounced with Lewis islets than with Wistar and AO islets. Rats with a higher weight donate more islets but have a lower insulin secretory capacity. Islet yield and function also vary with application of different sources of bovine serum albumin during digestion. Moreover, the culture conditions influence the functional survival of isolated rat islets. CMRL1066 preserves the insulin secretory capacity of rat islets better than RPMI1640. Finally, the number of islets surviving the culture is higher when 4 instead of 12 and 24 islets were applied per square centimeter. Our observations indicate that strain and weight of the rat donor, the source of bovine serum albumin, and the culture conditions of islets are pertinent factors in efficacious isolation of islets.

Plasma-treated polystyrene surfaces: model surfaces for studying cell-biomaterial interactions.

Biocompatibility of biomaterials relates, amongst others, to the absence of adverse cellular reactions and modulation of cell adhesion and subsequent responses. With respect to tissue-engineering applications, most materials need to evoke cell adhesion and spreading, while potentially displaying differential cell function. Adhesion has frequently been studied in a controlled fashion, using adhesion-supporting and -inhibiting substrata. The aim of this study is to create a panel of related materials with gradually changing surface characteristics in order to sustain similar individual cell adhesion and spreading, yet different cell population behaviour. A series of polystyrene materials was created with increasing oxygen surface incorporation and, concurrently, decreasing water-contact angles. Individual cells adhered and spread on all surfaces whilst showing well-developed focal adhesions and stress fibres. Cell populations demonstrated a decreased growth on surfaces with lower wettability. The biochemical activity of cell populations was not influenced by the surface treatment, but cell proliferation on surfaces increased with increasing oxygen incorporation. Furthermore, surface coverage with assembled fibronectin matrix was higher on the substrata with higher wettability. Finally, the expression of the adhesion-related proteins cadherin-5, focal adhesion kinase and RhoA was increased on surfaces with higher wettability. Further explorations of the cell biological basis of the observed differential behaviour will give more detailed answers on the rules governing cell-material interactions.

Platelet adhesion and activation on a shielded plasma gradient prepared on polyethylene.

Contact of blood with foreign materials evokes thrombogenic effects to an extent determined partly by the wettability of the biomaterials surface. Tools to study blood response towards a variation in materials wettability with minimal variation in chemistry are "gradient surfaces". However, most gradients have been prepared by diffusion or density immersion techniques, which results in a limited gradient range. Through glow discharge with partial shielding, gradients on polymers were prepared over a length of 5 cm, which facilitated studies to platelet adhesion on separate gradient sections. On polyethylene, advancing water contact angles varied from 90 degrees to 40 degrees, with a hysteresis of 30 degrees. ESCA indicated an increasing incorporation of oxygen towards the hydrophilic end. To examine the role of materials wettability on the activation of adhering platelets, sections of shielded plasma gradients were incubated in anticoagulated whole human blood. Fewer platelets adhered to the hydrophobic end, but those platelets were more activated than those on the hydrophilic end, as judged from their morphology and exposure of GpIIb-IIIa complex. However, partly related to the increased binding of platelets, the clotting activation after platelet deposition was highest on the hydrophilic end. Concluding, this new technique results in a large gradient range, which facilitates studies of formed blood elements in relation to the wettability. Platelets are more activated on hydrophobic polyethylene, while on moderate hydrophilic polyethylene more platelet adhesion and activation of the clotting system occurs.

Influence of abciximab on the adhesion of platelets on a shielded plasma gradient prepared on polyethylene.

INTRODUCTION: Thrombotic effects of biomaterial implants are mediated merely through activation of the platelet glycoprotein IIb-IIIa (GpIIb-IIIa) receptor. Consequently, platelet GpIIb-IIIa receptor inhibitors are successfully used during stent implantation procedures to prevent thrombosis. However, currently a new generation of stents contains surface coating, which changes the surface to more hydrophobic or hydrophilic. This change markedly affects the interaction of platelets and may influence the efficiency of GpIIb-IIIa inhibitors. MATERIALS AND METHODS: To study the influence of the wettability of biomaterials on the effectiveness of abciximab, 5-cm polyethylene gradients with contact angles of 100 degrees to 40 degrees were made by means of glow discharge. Fresh whole blood with or without abciximab was recirculated over this gradient. RESULTS: Inhibition of platelet adhesion by abciximab was maximal, but not complete, on the hydrophobic and moderate hydrophobic part of the gradient, with contact angles of 55 degrees to 90 degrees. Percentage inhibition by abciximab was maximal around 60 degrees. CONCLUSIONS: Intermediate hydrophobicity of currently applied stent materials, such as stainless steel, seems optimal in combination with abciximab. However, on hydrophobic and particularly on hydrophilic materials, abciximab is less effective.