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Martin-Luther-Universität Halle-Wittenberg, 06112 Halle (Saale)

PERMANON GmbH z. Hd. S. Krücken Winterstetten 53 88299 Leutkirch 04_2013

June 4 th, 2013

Studies on cyto compatibility and complement activation of a coating solution (PERMANON® Supershine Medicare) for bio-materials

Customer: Permanon GmbH, Winterstetten 53, D-88299 Leutkirch, S. Krücken Registration-No. 04_2013 Delivery Date: 28.01.2013 Immediate Packaging:PET Secondary Packaging:none Storage: dry and protected from light at 20-24°C Labeling: Supershine Medicare, UBA-No. 43180022 (German Federal Environmental Agency) Researcher: PD Dr. Bernhard Hiebl (FW Toxicology) Place of Research: Center for Basic Medical Research, Magdeburger Str. 18, 06112 Halle (Saale), laboratory is not accredited.

Summary Evaluation PERMANON® Supershine Medicare (UBA-No. 43180022) is a coating solution which according to manufacturer's specifications has the property of remaining stable within the temperature range of -40°C to +300°C and within the pH range of 1 to 10. This makes the solution interesting and suited for utilization in the area of medical products. In order to examine possible toxic effects of the coating solution a representative selection of materials, which are frequently used with medical products (soda-lime-silica glass, silicone, stainless steel, Nitinol und polystyrene), were coated with PERMANON® Supershine Medicare. Subsequently, extracts of the coated materials were produced in accordance with DIN EN ISO 10993, so that tests with cell line 3T3 (mice-fibroblasts) could be performed. The results showed that the coating does not have any influence on the morphology of the cells or the cell layer. Furthermore, the coating had no influence on the cell count (MTS-test) nor did it have an influence on complement activation (C5a). The latter was determined after an incubation period of the coated samples in human serum for 30 min at 37°C. The release of lactate dehydrogenase as a parameter for the integrity of the cellular membrane was found to be significantly smaller for incubation of cells in extracts of coated materials compared to incubation in extracts of uncoated materials. In conclusion, these results indicate that the coating solution tested is suitable for use in the medical field.

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The findings of the study were presented for the first time ever as part of the 32nd Annual Meeting of the German Society for Microcirculation and Haemorheology in Dresden.

Study Objective A concept currently underway in the field of research is the appropriate optimization of surface characteristics in order to enhance the biocompatibility of materials in clinical applications. It has proven to be an effective tool in this context to improve the interfacial compatibility of material surfaces through the application of a coating. As part of this study a coating solution (PERMANON® Supershine Medicare) was analyzed, which is based on monomolecular silicium. This coating substrate adheres to the material surface due to electrostatic interaction and is of interest for medical applications, especially due to its temporary resistance in the face of current disinfection and sterilization procedures (thermo-stable between -40°C and +300°C, chemically stable between pH 1 and pH 10). The aim of the study was to test the biocompatibility of this coating solution (PERMANON® Supershine) on a range of materials and test samples, which are frequently used as biocompatible material products (soda lime silica glass, silicone, stainless steel, Nitinol und polystyrene).

Material and Methods Sample preparation, Sterilization The test samples made of polystyrene were commercially purchased in a clean and sterilized condition (Greiner Bio-One). Prior to use, the other test samples (soda lime silica glass, silicone, stainless steel, and Nitinol) were initially immersed for 12 hours in absolute ethanol (undenatured) for the purpose of cleaning. This was followed by a cleaning procedure in an ultrasonic bath (35 kHZ, Elmasonic One) with absolute ethanol (undenatured) for 20 min. Thereupon, the test samples were rinsed with double distilled water three times (immersion in three separate beakers filled with double distilled water, 200ml/beaker), after this the test samples were cleaned again in an ultrasonic bath. After rinsing the test samples three times in double distilled water, they were dried at 37°C in a heating chamber (Binder FED) and then subjected to an autoclave treatment ( 121°C for 20 min, Varioklav 500) for the purpose of sterilization.

Coating Prior to producing the extract the test samples were dipped twice into PERMANON® Supershine Medicare (2,0 vol %) at intervals of 5 min, each for 10 min. The coating solution was sterilized by way of sterile filtration (0,45 µm pore size) prior to use.

Extract manufacturing The extracts were generated conforming to EN ISO 10993-12 by incubating the test samples in a serum supplemented cell culture medium (MEM + 10 vol % FCS, Biochrom; in each case 48 cm2 in 7 ml MEM) at a temperature of 37°C for 72 hours under permanent horizontal shaking (20 rpm, Julabo SW23).

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Cell Culture The tests were conducted with 3T3 cells (immortalized fibroblasts of mice embryos). Cells were utilized for testing starting at a confluence of cell layer of 80% (stage of sub-confluence). Culture growth occurred at 37°C, 5% CO2 in a steam-saturated atmosphere with serum supplemented MEM (10 vol % FCS, Biochrom).

Evaluation of Cell Morphology The phenotype of the cell layer and of the cells was assigned on the basis of defined morphometric characteristics pursuant to the requirements of USP23-NF18 and of EN DIN ISO 10993-5 stages of cytotoxicity. Cytotoxicity Level

Interpretation

0

no cytotoxicity

1

slight cytotoxicity

2

mild cytotoxicity

3

moderate cytotoxicity

4

severe cytotoxicity

Changes in Cell Morphology · · · · · · · · · · · · ·

no changes in cell morphology thick cell layer no cell lysis round and weakly attached cells: < 20% cell layer virtually dense cell lysis occasionally recognizable (< 5%) round and weakly attached cells: < 50% few empty gaps in cell layer substantial cell lysis (< 50 %) round and weakly attached cells: < 70% a lot of empty areas in cell layer cell lysis voluminous (< 70 %) cell layer almost completely destroyed

LDH Test The evaluation of cell membrane integrity was determined by the extracellular activity of lactate dehydrogenase (cytotoxicity detection kit (LDH), Roche Applied Science). The test was carried out in accordance with the manufacturer's instructions.

MTS Test The MTS test (CellTiter 96® Aqueous Non-Radioactive Cell Proliferation Assay, Promega) determines the activity of mitochondrial and cytosolic dehydrogenases and makes it possible to draw conclusions on the cell count. The test was carried out in accordance with the manufacturer's instructions.

Statistics Data regarding the LDH and MTS test is given as average values ± standard deviation and has been analyzed with the Student's t-test or with the Chi-square test. Deviations with a p<0,05 were deemed as significant.

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Results Evaluation of the morphology of cells and cell layer respectively The cell layer showed no considerable variations between the coated and uncoated materials after cultivation of sample extracts for 48 hours. In each case the cell layer was within the range of highly confluent till dense and no changes in cell morphology and cell lysis could be recognized (cp. fig. 1). Consequently, there are no indications for any cytotoxic effects of the coating based on the cell and cell layer morphology (cytotoxicity level 0).

Polystyrene

silicone

V2A steel soda lime silica glass

Nitinol

uncoated

coated

Fig. 1: 3T3 cells 48 hours after cultivation in sample extract (72 hour extract); transmitted lightphase- contrast microscopy, primary magnification 20×.

LDH Test The extracellular lactate dehydrogenase activity was significantly less for those cells which had been cultured with extracts of coated samples compared to the corresponding activities of cells which had been cultured with extracts of uncoated samples (fig. 2, p<0,05). Accordingly, the LDH testing revealed no indications of any toxic effects of the coating solution. Cell Membrane Integrity (LDH Test) □ uncoated □ coated

Polystyrene

silicone

V2A steel

soda lime silica glass

Nitinol

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Fig. 2: Extracellular lactate dehydrogenase activity after cultivation of 3T3 cells with extracts of coated and uncoated samples; average values ± standard deviation, n = 8. The MTS test, which provides an indication for cell count, did not show any significant differences between coated and uncoated materials. (fig. 3). Accordingly, the MTS testing also revealed no indications of any toxic effects of the coating solution. The coating also showed no significant influence on complement activation (C5a, fig. 4).

Cell Count (MTS Test) □ uncoated □ coated

Polystyrene

silicone

V2A steel

soda lime silica glass

Nitinol

Fig. 3: Intracellular dehydrogenases activity to measure the cell count after cultivation of 3T3 cells with extracts of coated and uncoated samples; average values ± standard deviation, n = 8.

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□ after 30 min, coated

□ after 30 min, uncoated

□ after 3 hours, coated

□ after 3 hours, uncoated

Polystyrene

silicone

V2A steel

soda lime silica glass

Nitinol

Fig 4: C5a concentration after incubation of coated and uncoated samples in human serum for 30 min at 37°C; average values ± standard deviation, n = 3.

Halle, June 4th, 2013

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University of martin luther germany toxicity test bericht supershine 2013