Clinical Hemorheology and Microcirculation - Volume 69, issue 1-2

Authors: Krüger-Genge, A. | Braune, S. | Walter, M. | Krengel, M. | Kratz, K. | Küpper, J.H. | Lendlein, A. | Jung, F.

Article Type: Research Article

Abstract: BACKGROUND: Physical and chemical characteristics of implant materials determine the fate of long-term cardiovascular devices. However, there is still a lack of fundamental understanding of the molecular mechanisms occurring in the material-tissue interphase. In a previous study, soft covalently crosslinked poly(n -butyl acrylate) networks (cPn BA) were introduced as sterilizable, non-toxic and immuno-compatible biomaterials with mechanical properties adjustable to blood vessels. Here we study the influence of different surface treatments in particular oxygen plasma modification and fibrinogen deposition as well as a combinatorial approach on the adhesion and viability of fibroblasts. MATERIAL AND METHODS: Two types of cPn …BA networks with Young’s moduli of 0.19±0.01 MPa (cPn BA04) and 1.02±0.01 MPa (cPn BA73) were synthesized and post-modified using oxygen plasma treatment (OPT) or fibrinogen coating (FIB) or a combination of both (OPT+FIB). The water contact angles of the differently post-treated cPn BAs were studied to monitor changes in the wettability of the polymer surfaces. Because of the key role of vascular fibroblasts in regeneration processes around implant materials, here we selected L929 fibroblasts as model cell type to explore morphology, viability, metabolic activity, cell membrane integrity as well as characteristics of the focal adhesions and cell cytoskeleton on the cPn BA surfaces. RESULTS: Compared to non-treated cPn BAs the advancing water-contact angles were found to be reduced after all surface modifications (p  < 0.05, each), while lowest values were observed after the combined surface treatment (OPT+FIB). The latter differed significantly from the single OPT and FIB. The number of adherent fibroblasts and their adherence behavior differed on both pristine cPn BA networks. The fibroblast density on cPn BA04 was 743±434 cells·mm-2 , was about 6.5 times higher than on cPn BA73 with 115±73 cells·mm-2 . On cPn BA04 about 20% of the cells were visible as very small, round and buckled cells while all other cells were in a migrating status. On cPn BA73, nearly 50% of fibroblasts were visible as very small, round and buckled cells. The surface functionalization either using oxygen plasma treatment or fibrinogen coating led to a significant increase of adherent fibroblasts, particularly the combination of both techniques, for both cPn BA networks. It is noteworthy to mention that the fibrinogen coating overruled the characteristics of the pristine surfaces; here, the fibroblast densities after seeding were identical for both cPn BA networks. Thus, the binding rather depended on the fibrinogen coating than on the substrate characteristics anymore. While the integrity of the fibroblasts membrane was comparable for both polymers, the MTS tests showed a decreased metabolic activity of the fibroblasts on cPn BA. CONCLUSION: The applied surface treatments of cPn BA successfully improved the adhesion of viable fibroblasts. Under resting conditions as well as after shearing the highest fibroblast densities were found on surfaces with combined post-treatment. Show more

Keywords: Biomaterial, poly(n-butyl acrylate), fibroblast, oxygen plasma, fibrinogen, cell adhesion, focal adhesion, actin cytoskeleton, viability

DOI: 10.3233/CH-189130

Citation: Clinical Hemorheology and Microcirculation, vol. 69, no. 1-2, pp. 305-316, 2018

Authors: Hiebl, B. | Ascher, L. | Luetzow, K. | Kratz, K. | Gruber, C. | Mrowietz, C. | Nehring, M.E. | Lendlein, A. | Franke, R.-P. | Jung, F.

Article Type: Research Article

Abstract: Laser tissue soldering (LTS) based on indocyanine green (ICG)-mediated heat-denaturation of proteins might be a promising alternative technique for micro-suturing, but up to now the problem of too weak shear strength of the solder welds in comparison to sutures is not solved. Earlier reports gave promising results showing that solder supported by carrier materials can enhance the cohesive strength of the liquid solder. In these studies, the solder was applied to the carriers by dip coating. Higher reliability of the connection between the solder and the carrier material is expected when the solder is bound covalently to the carrier material. …In the present study a poly(ether imide) (PEI) membrane served as carrier material and ICG-supplemented albumin as solder substrate. The latter was covalently coupled to the carrier membrane under physiological conditions to prevent structural protein changes. As laser source a diode continuous-wave laser emitting at 808 nm with intensities between 250 mW and 1500 mW was utilized. The albumin functionalized carrier membrane was placed onto the tunica media of explanted pig thoracic aortae forming an overlapping area of approximately 0.5×0.5 cm2 . All tests were performed in a dry state to prevent laser light absorption by water. Infrared spectroscopy, spectro-photometrical determination of the secondary and primary amine groups after acid orange II staining, contact angle measurements, and atomic force microscopy proved the successful functionalization of the PEI membrane with albumin. A laser power of 450 mW LTS could generate a membrane-blood vessel connection which was characterized by a shear strength of 0.08±0.002 MPa, corresponding to 15% of the tensile strength of the native blood vessel. Theoretically, an overlapping zone of 4.1 mm around the entire circumference of the blood vessel could have provided shear strength of the PEI membrane-blood vessel compound identical to the tensile strength of the native blood vessel. These in-vitro results confirmed the beneficial effects of solder reinforcement by carrier membranes, and suggest LTS with covalently bound solders on PEI substrates for further studies in animal models. Show more

Keywords: Laser tissue soldering, blood vessel anastomosis, laser welding, solder carrier, poly(ether imide) membrane

DOI: 10.3233/CH-189108

Citation: Clinical Hemorheology and Microcirculation, vol. 69, no. 1-2, pp. 317-326, 2018

Authors: Nowak, Elisabeth | Kammerer, Sarah | Küpper, Jan-Heiner

Article Type: Research Article

Abstract: BACKGROUND: Drug-induced liver injury (DILI) is the most common reason for withdrawal of anticancer drugs from the market. To prevent adverse side effects of drugs, it is important to investigate potential toxicity in vitro. However, outcome of cytotoxicity screenings can differ remarkably depending on the method used. OBJECTIVE: We aimed to compare XTT, ATP-based CellTiter-Glo® 2.0 and trypan blue exclusion (TBE) assays regarding their sensitivity in detecting acute cytotoxicity on HepG2 cells after incubation with the classical anticancer drugs Taxol and Imatinib or with the proteasome inhibitor MG-132. METHODS: HepG2 cells were treated for 48 h …and cell viability was analysed by XTT, CellTiter-Glo® 2.0 or TBE assay. RESULTS: All tested compounds showed a reduction of viability of HepG2 cells. However, assay results differed significantly: Both ATP-based and TBE assay showed concentration-dependent cytotoxic effects, but outcomes were less pronounced with TBE. In contrast, the widely used XTT assay did not detect any acute cytotoxicity of Taxol and Imatinib. CONCLUSIONS: Acute cytotoxic effects of tested compounds could be revealed. However, results were significantly different from each other with ATP assay being the most sensitive one under the conditions tested. Thus, acute cytotoxicity can be dramatically underestimated if only standard XTT test is used. Show more

Keywords: CellTiter-Glo®2.0, XTT, trypan blue exclusion, cytotoxicity, DILI, HepG2, Imatinib, MG-132, Taxol

DOI: 10.3233/CH-189120

Citation: Clinical Hemorheology and Microcirculation, vol. 69, no. 1-2, pp. 327-336, 2018