Barbara Seifert

Membranes for biohybrid liver support systems--investigations on hepatocyte attachment, morphology and growth.

The biological properties of four different membranes were studied regarding their possible application in biohybrid liver support systems. Two of them, one made of polyetherimide (PEI), and a second based on polyacrylonitrile-N-vinylpyrollidone co-polymer (P(AN-NVP)), were recently developed in our lab and studied for the first time. Together with pure polyacrylonitrile (PAN) membranes, the three preparations were characterised as ultra-filtration membranes. Their ability to support cell attachment, morphology, proliferation and function of human hepatoblastoma C3A cells was studied. The role of surface morphology for the interaction with hepatocytes was highlighted using a commercial, moderately wettable polyvinylidendifluoride (PVDF) membrane with micro-filtration properties. Comparative investigations showed strongest interaction of C3A cells with PAN membranes, as the focal adhesion contacts were more expressed and cell growth was also high. However, the functional activity in terms of albumin synthesis was reduced. Very similar results were obtained with the most hydrophobic PEI membrane. In contrast, the most hydrophilic membrane P(AN-NVP) was found to provoke stronger homotypic adhesion (E-cadherin expression) of C3A cells and less substratum attachment (focal adhesions), but enhanced albumin secretion. However, proliferation of C3A cells was lowered. Micro-porous PVDF membrane showed very good initial attachment, but the resulting cell material and cell-cell interaction were relatively poor developed. Among four membranes tested, PEI seems to be the most attractive membrane for biohybrid liver devices, as it provides good surface properties for hepatocytes interaction, but in addition it is highly thermostable, which would permit steam sterilisation. No simple relationship, however, between the wettability of the membranes and their ability to support hepatocyte adhesion and function was found in this study.

Cytocompatibility testing of cell culture modules fabricated from specific candidate biomaterials using injection molding

Most polymers used in clinical applications today are materials that have been developed originally for application areas other than biomedicine. Testing the cell- and tissue-compatibility of novel materials in vitro and in vivo is of key importance for the approval of medical devices and is regulated according to the Council Directive 93/42/EEC of the European communities concerning medical devices. In the standardized testing methods the testing sample is placed in commercially available cell culture plates, which are often made from polystyrene. Thus not only the testing sample itself influences cell behavior but also the culture vessel material. In order to exclude this influence, a new system for cell testing will be presented allowing a more precise and systematic investigation by preparing tailored inserts which are made of the testing material. Inserts prepared from polystyrene, polycarbonate and poly(ether imide) were tested for their cytotoxity and cell adherence. Furthermore a proof of principle concerning the preparation of inserts with a membrane-like surface structure and its surface modification was established. Physicochemical investigations revealed a similar morphology and showed to be very similar to the findings to analogous preparations and modifications of flat-sheet membranes.

Poly(ether imide) membranes: studies on the effect of surface modification and protein pre-adsorption on endothelial cell adhesion, growth and function.

Poly(ether imide) (PEI) membranes of which the surface was modified with carboxylic groups were tested in comparison to pure PEI and poly(ethylene terephtalate) (PET) for their ability to support attachment, growth and function of human umbilical vein endothelial cells (HUVEC) with respect to endothelization of the above materials. Flat sheet PEI membranes were modified by covalent binding of iminodiacetic acid (IDA) for different periods of time (1 to 30 min) to obtain surfaces with various content of carboxylic groups. In addition, fibronectin (FN) and fibrinogen (FNG) pre-adsorption on the various membranes were studied for their effect on HUVEC behaviour. The results show a decreased protein adsorption and HUVEC adhesion, growth and function in terms of prostacyclin production with an increase in carboxylic groups. Pre-adsorption of the membranes with FN or FNG promoted activity of HUVEC, which became superior to cells on PET. FN-coated membranes were found to be a better substrate for HUVEC adhesion and prostacyclin production, while on FNG-coated membranes cells grew better. Overall it can be concluded that PEI is a promising materials for endothelial cells immobilization as it is needed for improving the haemocompatibility of cardiovascular devices.

Automated image-based analysis of adherent thrombocytes on polymer surfaces

A dataset of 439 confocal laser scanning microscopic images was analyzed to investigate the potential of an image-based automated analysis for identifying and assessing adherent thrombocytes on polymer surfaces. Parameters for image optimization of glutardialdehyde induced fluorescence images were classified and data mining was performed using the Java image processing software ImageJ. Previously reported analysis required that each thrombocyte had to be identified interactively and outlined manually. Now, we were able to determine the number and area of adherent thrombocytes with high accuracy (spearman correlation coefficient r = 0.98 and r = 0.99) using a two-stage filter-set, including a rolling ball background subtraction- and a watershed segmentation-algorithm. Furthermore, we could proof a significant correlation between these parameters (spearman correlation coefficient r = 0.97), determining both as suitable predictors for the evaluation of material induced thrombogenicity. The here reported image-based automated analysis can be successfully applied to identify and measure adherent thrombocytes on polymer surfaces and, thus, might be successfully integrated in a high-throughput screening process to evaluate biomaterial hemocompatibility.

In Vitro Evaluation of Elastic Multiblock Co-polymers as a Scaffold Material for Reconstruction of Blood Vessels

There is a need to create cell- and histocompatible implant materials, which might temporarily replace the mechanical function of a native tissue for regenerative therapies. To match the elastic behavior of the native tissue two different multiblock co-polymers were investigated: PDC, consisting of poly(p-dioxanone) (PPDO)/poly(ε-caprolactone) (PCL), and PDD, based on PPDO/poly((adipinate-alt-1,4-butanediol)-co-(adipinate-alt-ethylene glycol)-co-adipinate-alt-diethylene glycol) (Diorez). PDC is capable of a shapememory effect. Both multiblock co-polymers show an improved elasticity compared to materials applied in established vascular prosthesis. PDD is softer than PDC at 20°C, while PDC maintains its elasticity at 37°C. Thermodynamic characteristics indicate a more polar surface of PDD. Low cell adhesion was found on surfaces with low molar free energy of hysteresis (ΔG) derived from contact angle measurements in wetting and dewetting mode and high cell adhesion on high-ΔG surfaces. An increasing content of PCL in PDC improved cell adhesion and spreading of human umbilical vein endothelial cells. The prothrombotic potential of PDD is higher than PDC. Finally, it is concluded that PDC is a promising material for vascular tissue engineering because of its improved elastic properties, as well as balanced prothrombotic and anti-thrombotic properties with endothelial cells.

Elastic multiblock copolymers for vascular regeneration: Protein adsorption and hemocompatibility

Hemocompatibility of elastic multiblock copolymers PDC, based on poly(p-dioxanone) (PPDO)/poly(ε-caprolactone)-segments, capable of a shape-memory effect, and PDD, based on PPDO/poly((adipinate-alt-1,4-butanediol)-co-(adipinate-alt-ethylene glycol)-co-adipinate-alt-diethylene glycol)-segments, was studied in order to assess their suitability for an application aiming at blood vessels regeneration. The results were compared with polypropylene (PP) which is a widely used blood-contacting material for devices as blood oxygenators and dialysis tubes. Protein adsorption studies showed diverse blood plasma proteins in a relatively high amount on both elastic polymers compared to the poor amount of plasma proteins adsorbed on PP. Study of the coagulation system revealed high thrombin formation on PDC and no difference in plasma kallikrein activation between elastic multiblock copolymers and the reference PP. Activation of complement system was higher for PDC followed by PDD and lower for PP. However, platelet adhesion and activation were hardly suppressed on the multiblock copolymers compared to the PP surface, where the number of adhered platelets and the activation rate were significant. The present results reveal that the tested multiblock copolymers with improved elastic properties and shape-memory capability (PDC) show low thrombogenicity and are promising candidates for vascular tissue engineering.

Novel Polymer Blends for the Preparation of Membranes for Biohybrid Liver Systems

It was found previously that membranes based on co-polymers of acrylonitrile (AN) and 2-acrylamido-2-methyl-propansulfonic acid (AMPS) greatly stimulated the functionality and survival of primary hepatocytes. In those studies, however, the pure AN–AMPS co-polymer had poor membrane-forming properties, resulting in quite dense rubber-like membranes. Hence, membranes with required permeability and optimal biocompatibility were obtained by blending the AN–AMPS co-polymer with poly(acrylonitrile) homopolymer (PAN). The amount of PAN (P) and AN–AMPS (A) in the blend was varied from pure PAN (P/A-100/0) over P/A-75/25 and P/A-50/50 to pure AN–AMPS co-polymer (P/A-0/100). A gradual decrease of molecular cut-off of membranes with increase of AMPS concentration was found, which allows tailoring membrane permeability as necessary. C3A hepatoblastoma cells were applied as a widely accepted cellular model for assessment of hepatocyte behaviour by attachment, viability, growth and metabolic activity. It was found that the blend P/A-50/50, which possessed an optimal permeability for biohybrid liver systems, supported also the attachment, growth and function of C3A cells in terms of fibronectin synthesis and P-450 isoenzyme activity. Hence, blend membranes based on a one to one mixture of PAN and AN–AMPS combine sufficient permeability with the desired cellular compatibility for application in bioreactors for liver replacement.