Dieter Paul

MORPHOLOGICAL EVIDENCE FOR A DIFFERENT FIBRONECTIN RECEPTOR ORGANIZATION AND FUNCTION DURING FIBROBLAST ADHESION ON HYDROPHILIC AND HYDROPHOBIC GLASS SUBSTRATA

A polyclonal antibody against the beta 1 subunit of the fibronectin (FN) receptor was used to mimic the early events of integrin receptor functioning to study the initial cellular processes during the organization of FN matrix on biomaterials. Hydrophilic glass and hydrophobic octadecylsilane (ODS) surfaces have been applied as models for different biocompatible materials. By immunofluorescence we could demonstrate that FN receptors organize on the dorsal cell surface of adhering fibroblasts in a specific linear pattern along with actin filaments, but only if the cells were attached to hydrophilic glass. In contrast, FN receptors were not reorganized on hydrophobic octadecylsilane (ODS). In parallel experiments, FN matrix formation after 72 h of incubation on the same substrata has been analyzed microscopically, and quantified by cell ELISA, in order to be further correlated with the integrin receptor functioning in contact with the biomaterials. It was found that FN structuring and the amount of FN matrix have been significantly diminished on ODS that was related to the observed changes in integrin receptor functioning. To learn more about the mechanism of this phenomenon, desorption of 125I-FN from these substrata was studied and found to be significantly decreased on hydrophobic ODS. As a consequence, FN receptor (function) might be arrested on the ventral cell surface, thus the important role of beta 1 integrins in the positional organization of the FN matrix may be disturbed. In light of these facts, antibody-induced clustering of FN receptor can be considered as a useful model for studying the early steps of FN matrix formation on biomaterials.

Remodeling of fibrinogen by endothelial cells in dependence on fibronectin matrix assembly. Effect of substratum wettability

The endothelization of cardiovascular implants is desirable to improve their blood compatibility. The capacity of the endothelial cells to attach, migrate, proliferate and function on the implant surface depends on the presence of matrix proteins such as fibronectin (FN) and fibrinogen (FNG). In this study, we show that the deposition of fibrinogen into extracellular matrix-like structures by human umbilical vein endothelial cells (HUVEC) is dependent on FN matrix formation. We found further that the process of organization of both adsorbed and soluble FN and FNG is dependent on the wettability of materials since it was observed only on a hydrophilic and not on a hydrophobic model surface. β3 integrin was involved in the process of cell attachment to adsorbed FNG, while the mechanism of FNG fibrillogenesis required the activity of the β1 integrin. Studies of EC morphology showed the predominant peripheral organization of actin filaments and the formation of distinct leading and trailing cell edges suggesting a motile phenotype of cells when they are seeded on FNG. In summary, we concluded that adsorbed fibrinogen may enhance the motility of HUVEC and that soluble FNG requires FN matrix assembly to be organized in fibrilar structures.

Fibrinogen adsorption and platelet interactions on polymer membranes

The hemocompatibility of four different wettable polymer membranes, namely Cuprophan (CE), polyether-polycarbonate (PC-PE), polysulfone (PSU), and polyetherimide (PEI), was investigated with respect to flbrinogen (Fng) adsorption and platelet adhesion/activation. In order to estimate the polar and dispersion components of the surface free energy, contact angles using water/vapor and water/n-hexadecane systems were measured. Adsorption of flbrinogen was studied using fluorescence-labeled protein. The adsorption isotherms showed that the amount and the affinity of adsorbed Fng increased with decreasing surface wettability of the membranes, which correlates with the dispersion and polar components of the surface free energy. The conformational changes of adsorbed Fng were detected by measuring the difference between monoclonal antibody binding to the conformation-sensitive epitope in the D-domain and the binding of polyclonal anti-Fng antibody. The anticipated conformational/orientational changes were greater for PEI and PSU membranes (the least wettable membranes) and negligible for the more wettable PC-PE and CE membranes. In addition, a possible relationship with the degree of platelet activation was found, showing negligible platelet adhesion on PC-PE and CE, but high platelet adhesion on PEI and PSU. Furthermore, platelets were spread to a large extent on PEI, while the formation of aggregates was observed on PSU. This may correspond to the anticipated differences in the conformational state of Fng on both membranes.

Altered vitronectin receptor (αv integrin) function in fibroblasts adhering on hydrophobic glass

Function of integrins is crucial for adhesion, movement, proliferation, and survival of cells. In a recent study we found impaired fibronectin receptor function on hydrophobic substrata (G. Altankov et al. J Biomater Sci Polym Edn 1997;8:712-740). Here, we have studied the distribution and function of the vitronectin receptor (alphav integrin) in fibroblasts adhering on hydrophilic glass and hydrophobic octadecyl glass (ODS). The morphology of fibroblasts and the organization of actin cytoskeleton were studied and found to be altered on ODS, where the cells did not spread and possessed condensed actin. Pretreatment of the surfaces with serum or pure vitronectin improved cell morphology on both substrata, resulting in the development of longitudinal actin stress fibers. It was found with biotinylated vitronectin that comparable quantities of vitronectin were adsorbed from single vitronectin solutions or serum on glass and on hydrophobic ODS. The organization of the vitronectin receptors on the ventral cell surface was investigated in permeabilized cells showing normal focal adhesions in fibroblasts plated on glass but none of these structures on ODS. The distribution of alphav integrin on the dorsal cell surface was studied on nonpermeabilized living cells after antibody tagging. While fibroblasts adhering on plain or serum-treated glass developed a linear organization of alphav integrin, cells on plain and serum-treated ODS were not able to reorganize the vitronectin receptor. Studies on signal transduction with antiphosphotyrosine antibodies revealed co-localization of alphav integrin and phosphotyrosine in focal adhesions on glass and serum-treated glass. However, signaling was almost absent on plain ODS and weak on serum-treated ODS. It was concluded that alterations in vitronectin receptor function on the ventral cell surface caused by the hydrophobic material surface inhibit signal transfer and subsequent intracellular events that are important for the organization and function of integrins.

Contact activation of plasmatic coagulation on polymeric membranes measured by the activity of kallikrein in heparinized plasma

Kallikrein is involved in the generation of bradykinin during extracorporal circulation, that is believed to play an important role in cases of anaphylactic shock during hemodialysis. Therefore, a method for the assessment of kallikrein generation was developed, based on the chromogenic substrate S-2302. Comparison of kallikrein-like activity on glass using citrate or heparinized plasma demonstrated enhanced activity in the presence of heparin. The applicability of the assay, and the time course of kallikrein generation was demonstrated with glass and cuprophan. Membranes based on pure polyacrylonitrile, or its copolymers differing in their content of acrylic acid, 2-hydroxyethyl acrylate, and allylsulphonate were investigated with respect to kallikrein-like activity, and physicochemical surface properties. It was found that high content in 2-hydroxyethyl acrylate, and acrylic acid caused a substantial activation of the contact system while low content in allylsulphonate (less than 2 mol%) did not result in enhanced kallikrein-like activity. The activating materials were characterized to be highly wettable, and had the most negative zeta potentials.

Functionality of MDCK kidney tubular cells on flat polymer membranes for biohybrid kidney

The prerequisite for the development of a biohybrid artificial kidney, is a substrate for confluent growth of renal cells forming an epithelial monolayer without any leaks. Conventional cell culture supports cannot be adapted for this purpose, because they lack adequate mechanical properties and thermal stability. From two suitable materials, polysulfone and polyacrylonitrile, two permeable polymeric membranes have been produced that were, according to ISO 10993-5, not cytotoxic. Cloned Madin Darby Canine Kidney (MDCK) cells (an established renal cell line) were cultured on the surface of the plastic materials, and on conventional cell culture supports. With all materials, assays of mitochondrial and lactate dyhydrogenases exhibited similar proliferation and the viability of the MDCK cells. Transmission electron microscopy showed the expression of a normal morphology of kidney tubular cells. Perfect barrier function, consequent on the formation of intercellular junctions in a confluent tight epithelium, was visualized in electron micrographs, and quantified by measurement of the transepithelial resistance. The uniformity of the cells grown was demonstrated in samples by electron microscopy and in the whole epithelium by intravital impedance analysis. It was concluded that polymeric membranes produced from polysulfone or polyacrylonitrile are appropriate substrates in the design of biohybrid kidney devices.

Formation of porous bilayer hollow fibre membranes

Support membranes in bioartificial organs contact blood or plasma on one-side and adhesion dependent cells on the other side. Since membranes for biomedical applications, such as for haemodialysis, are optimised for blood contact and membranes for biotechnological applications for cell contact, there are no membranes available addressing the requirements of artificial organ technology. One approach is the preparation of porous bilayer membranes with a wall consisting of two chemical different polymer layers. Results of the preparation of such membrane types using triple spinnerets and a wet phase inversion process are shown here. It is demonstrated that one of the most important parameter is the structural integrity of the membrane wall at the interface between both layers. A new spinneret construction is presented where the membrane forming polymer solutions are layered in the spinneret before extrusion. As a result porous bilayer hollow fibre membranes with a high structural integrity could be manufactured using different composed polysulfone (PSu) polymer solutions for model investigation.

A new module arrangement for plasmapheresis

On-line plasmapheresis using microporous membranes for filtration normally requires two external circulations (double treatment) before the detoxified plasma can be returned to the patient. The duomodule, a new filter arrangement developed by our group, integrates both steps in one equipment module using only one external circuit. Separations of aqueous polyethylene glycol (PEG) solutions as well as human plasma were carried out using the duomodule arrangement. The results revealed a considerable decrease of higher molecular substances in the feed solutions and a significant increase of these components in the permeate solution accumulated in the external compartment of the module. In conclusion, the duomodule arrangement seems to be an useful tool for the therapeutic apheresis.