Covalently immobilized thrombomodulin inhibits coagulation and complement activation of artificial surfaces in vitro.

Thrombomodulin (TM) serves as the endothelial cell receptor for thrombin and alters its characteristics from pro- to anticoagulant. Additionally, it promotes the formation of activated protein C. We evaluated the conservation of the overall outcome of these functions in recombinant TM linked to artificial surfaces by incubation with human whole blood in vitro. TM was covalently immobilized through poly(ethylene glycol) (PEG) spacers onto thin films of poly(octadecene alt maleic anhydride) covering planar glass substrates. TM binding to the polymer films was achieved after active ester formation at the carboxylic acid terminus of the PEG spacers and thoroughly characterized by HPLC-based amino acid analysis, immunofluorescence and ellipsometry. TM-coated samples were incubated for 3h with freshly drawn whole human blood anticoagulated with heparin (5IU/ml) using in-house developed incubation systems. The substantially reduced activation of blood coagulation (TAT) for TM-coated samples correlates well with the degree of contact activation (bradykinin and FXIIa formation) while no significant effects were observed for the platelet activation (PF4). Further, complement activation (C5a levels), was strongly diminished at the TM-containing surfaces. We conclude that the suggested method for preparation of TM immobilization may serve to prepare model substrates for studies on TM interactions but similarly provides a promising coating strategy for blood contacting medical devices.

In vitro blood compatibility of polymeric biomaterials through covalent immobilization of an amidine derivative.

We present a surface coating with anticoagulant characteristics showing significantly reduced coagulation activation. The synthesis of a monomeric conjugate containing a benzamidine moiety was carried out and its inhibitory activity against human thrombin, the key enzyme of the blood coagulation cascade, was determined using a chromogenic assay. Based on that, low-thrombogenic interfaces were prepared by covalent attachment of this low-molecular weight thrombin inhibitor on poly(octadecene-alt-maleic anhydride) copolymer thin films and characterized using ellipsometry, XPS and dynamic contact angle measurements. The in vitro hemocompatibility tests using freshly drawn human whole blood showed, in agreement with the SEM images, that a PO-MA film modified with a benzamidine moiety using a PEG spacer decreased the activation of coagulation, platelets and the complement system. The decreased protein adsorption, in addition to the specific inhibition of thrombin, effectively enhanced the short-term hemocompatibility characteristics.

Design and evaluation of novel blood incubation systems for in vitro hemocompatibility assessment of planar solid surfaces.

Success in the development of hemocompatible biomaterials depends on adequate equipment and procedures for standardized analysis of blood-materials interactions in vitro. In view of the limited standard of knowledge on that important aspect, two novel incubation systems were designed, built, and evaluated for the in vitro assessment of the hemocompatibility of planar solid surfaces: A screening setup was introduced for the comparison of up to 12 different samples. A perfusion setup was developed to model the directed blood flow in the vascular system during incubation by a recirculation circuit, allowing the variation of the wall shear rate at the sample surface. The incubation procedures utilized freshly drawn, heparinized whole human blood. Hemocompatibility in terms of selected aspects of coagulation, thrombogenicity, and immune responses was quantified through plasma levels of characteristic molecules (immunoassays), cell counting, and analysis of adherent cells and fibrin formation (scanning electron microscopy), respectively. Prevention of blood-air contact and mechanical stress, constant temperature and blood pH during incubation, and the suitable choice of reference materials were found to be crucial for reliable testing. Considering those requirements, screening and perfusion system both provided sensitive discrimination between a given set of planar solid surfaces. In conclusion, the suggested methods for an in vitro hemocompatibility assessment permit versatile, sensitive, and efficient analysis of important blood-material interactions despite the unavoidable variability of blood characteristics in different experiments.

Fibrillar collagen assembled in the presence of glycosaminoglycans to constitute bioartificial stem cell niches in vitro.

Fibrillar collagen was reconstituted from mixtures of monomeric tropocollagen and heparin or hyaluronic acid, respectively. Turbidity measurements were utilized to follow the fibrillar assembly and demonstrated the influence of the concentration of the glycosaminoglycan on the maximum optical densities. Thin film coatings of maleic anhydride copolymers were utilized for the covalent immobilization of the fibrillar assemblies to solid supports. Quantification of surface-bound collagen was accomplished by ellipsometry and HPLC-based amino acid analysis indicating that less collagen was immobilized in the presence of the glycosaminoglycans. SEM and AFM revealed various sizes and shapes of the immobilized fibrillar assemblies if collagen fibrils were prepared in the presence of heparin or hyaluronic acid. Human hematopoietic stem cells (HSCs) were cultivated on the surface-bound collagen fibrils and the migration of adherent cells was studied by time-lapse microscopy. Migration rates on fibrillar structures were significantly lower then on tropocollagen indicating a more intimate contact of HSCs to the fibrillar substrates.