cytotoxicity (din en iso 10993-5), antimicrobial activity (jis ......with human tissue [din en iso...
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Bachstraße 18 · 07743 Jena · Telefon 03641 93 00
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Universitätsklinikum Jena · Körperschaft des Öffentlichen Rechts
als Teilkörperschaft der Friedrich-Schiller-Universität Jena
Verwaltungsratsvorsitzender: Staatssekretär Markus Hoppe
Kaufmännischer Vorstand und Sprecherin des
Klinikumsvorstandes: Dr. Brunhilde Seidel-Kwem
Medizinischer Vorstand: PD Dr. Jens Maschmann
Wissenschaftlicher Vorstand: Prof. Dr. Klaus Benndorf
Klinik für Hautkrankheiten
Qualitäts-zertifiziert nach DIN EN ISO 9001:2008
Labor für In-vitro-Forschung und
Routinediagnostik
Universitätsklinikum Jena Klinik für Hautkrankheiten Erfurter Str. 35 07743 Jena
Herr Marco Hahn Geschäftsführer GREY Fashion UG Pappelallee 78/79 D-10437 Berlin
Direktor: Prof. Dr. med. Peter Elsner Laborleiter: PD Dr. Uta-Christina Hipler Telefon 03641 93 73 31 Telefax 03641 93 74 37 Ansprechpartner: Dr. Cornelia Wiegand Telefon 03641 93 75 84 Telefax 03641 93 74 37 web: http:// www.derma.uni-jena.de 22. Januar 2018
study report: 18-003
In vitro assessment of Vitadylan for
cytotoxicity (DIN EN ISO 10993-5),
antimicrobial activity (JIS L 1902), and
antioxidative capacity
page 2 of 33 CONFIDENTIAL – VERTRAULICH Study report 18-003 In vitro assessment of Vitadylan for cytotoxicity (DIN EN ISO 10993-5), antimicrobial activity (JIS L 1902), and antioxidative capacity
Klinik für Hautkrankheiten
Qualitäts-zertifiziert nach DIN EN ISO 9001:2008
Labor für In-vitro-Forschung und
Routinediagnostik
Index
page
1. Quality certificate 3
2. General information
2.1 Test items 4
2.2 Reference items 4
2.3 Sponsor 4
2.4 Test facility 4
2.5 Operating schedule 5
3. GLP and quality assurance statement 5
4. Summary 6
5. Background 7
6. Description of materials and test methods
6.1 Preparation of sample material 9
6.2 Cell culture of HaCaT keratinocytes 9
6.3 ATP bioluminescence assay 10
6.4 Determination of cellular protein content 10
6.5 Detection of cytotoxicity (LDH) 10
6.6 Determination of the antibacterial activity according to JIS L 1902 11
6.7 Determination of the antioxidant capacity against ROS 12
6.8 Determination of the antioxidant capacity against RNS 12
6.9 Measurement of vitamin A, C, and E content 13
6.10 Statistical analysis 13
7. Deviations from the study protocol 14
8. Archiving 14
9. Results and discussion 15
10. Appendix
10.1 Abbreviations 26
10.2 Tables and Figures 27
10.3 References 29
10.4 Measurement data 30
page 3 of 33 CONFIDENTIAL – VERTRAULICH Study report 18-003 In vitro assessment of Vitadylan for cytotoxicity (DIN EN ISO 10993-5), antimicrobial activity (JIS L 1902), and antioxidative capacity
Klinik für Hautkrankheiten
Qualitäts-zertifiziert nach DIN EN ISO 9001:2008
Labor für In-vitro-Forschung und
Routinediagnostik
1. Quality certificate
page 4 of 33 CONFIDENTIAL – VERTRAULICH Study report 18-003 In vitro assessment of Vitadylan for cytotoxicity (DIN EN ISO 10993-5), antimicrobial activity (JIS L 1902), and antioxidative capacity
Klinik für Hautkrankheiten
Qualitäts-zertifiziert nach DIN EN ISO 9001:2008
Labor für In-vitro-Forschung und
Routinediagnostik
2. General information
2.1 Test items
Vitadylan - 30% SeaCell, 30% Smartcell, 33% Modal, 7% Elasthan - (GREY Fashion UG)
2.2 Reference items
-
2.3 Sponsor
GREY Fashion UG smartfiber AG
Pappelallee 78/79 Im Weidig 12
D-10437 Berlin D-07407 Rudolstadt
Germany Germany
Person responsible: Marco Hahn Person responsible: Martina Finken
2.4 Test facility
Klinik für Hautkrankheiten
Universitätsklinikum Jena
Erfurter Straße 35
D-07740 Jena
Germany
Study director: PD Dr. Uta-Christina Hipler
page 5 of 33 CONFIDENTIAL – VERTRAULICH Study report 18-003 In vitro assessment of Vitadylan for cytotoxicity (DIN EN ISO 10993-5), antimicrobial activity (JIS L 1902), and antioxidative capacity
Klinik für Hautkrankheiten
Qualitäts-zertifiziert nach DIN EN ISO 9001:2008
Labor für In-vitro-Forschung und
Routinediagnostik
2.5 Operating schedule
Start of experiment: 2017-11-13
End of experiment: 2018-01-19
Date of final report: 2018-01-22
3. GLP and quality assurance statement
I assure that the Test facility complies with the Principles of Good Laboratory Practice.
Appropriate and technically valid Standard Operating Procedures are established for the
described tests. The Test facility is certified according to DIN EN ISO 9001:2008.
___________ _____________________________________
Date Study director: PD Dr. Uta-Christina Hipler
22.01.2018
page 6 of 33 CONFIDENTIAL – VERTRAULICH Study report 18-003 In vitro assessment of Vitadylan for cytotoxicity (DIN EN ISO 10993-5), antimicrobial activity (JIS L 1902), and antioxidative capacity
Klinik für Hautkrankheiten
Qualitäts-zertifiziert nach DIN EN ISO 9001:2008
Labor für In-vitro-Forschung und
Routinediagnostik
4. Summary
The purpose of the study was the determination of the influence of the textile Vitadylan on
viability and proliferation of human HaCaT keratinocytes in accordance to DIN EN ISO 10993-5.
In addition, the release of lactate dehydrogenase (LDH) by HaCaT cells after treatment with
sample extracts was analyzed to assess their cytotoxic potential. Furthermore, the antibacterial
activity of Vitadylan against Staphylococcus aureus and Klebsiella pneumoniae according to the
JIS L 1902:2008 was evaluated and the antioxidative capacity against free radicals such as
ROS (reactive oxygen species) and RNS (reactive nitrogen species) was investigated in vitro.
It could be shown that the textile Vitadylan has a direct cytotoxic effect on human HaCaT
keratinocytes in vitro. HaCaT cells challenged with doses ≥ 0.1 g:mL died over an incubation
period of 24 hours. Extraction ratios of 0.05 and 0.02 g:mL decreased cell proliferation
significantly over time in this test. Only the concentration of 0.002 g:mL was well tolerated by
the HaCaT cells during the incubation period of 48 hours. Results obtained for 24-hour and 72-
hour extracts were found to be similar. However, it could be demonstrated that the textile
Vitadylan exhibits a strong antibacterial activity against Staphylococcus aureus and Klebsiella
pneumonia according to JIS L 1902:2008 (log-reduction > 3). Furthermore, Vitadylan was found
to be able to inhibit the formation of free radicals such as ROS (reactive oxygen species) and
RNS (reactive nitrogen species) in vitro. The textile showed a significant, dose-dependent
antioxidative capacity against ROS and RNS which probably can be attributed to the vitamins A,
C, and E detected in the textile Vitadylan.
page 7 of 33 CONFIDENTIAL – VERTRAULICH Study report 18-003 In vitro assessment of Vitadylan for cytotoxicity (DIN EN ISO 10993-5), antimicrobial activity (JIS L 1902), and antioxidative capacity
Klinik für Hautkrankheiten
Qualitäts-zertifiziert nach DIN EN ISO 9001:2008
Labor für In-vitro-Forschung und
Routinediagnostik
5. Background
Biocompatibility is the central request for materials and devices that come into direct contact
with human tissue [DIN EN ISO 10993-1]. The determination of toxic effects is part of the initial
evaluation process stipulated by ISO standards. In vitro-cytotoxicity is tested according to DIN
EN ISO 10993-5. Samples can be analysed in direct and indirect contact with cells,
respectively, as well as in form of extracts [DIN EN ISO 10993-5, DIN EN ISO 10993-12]. A
wide variety of cell lines are suitable for biocompatibility tests and are commonly used for in
vitro cytotoxicity testing such as fibroblasts from human skin, buccal mucosa, periodontal
membrane or embryonic lung, cultures of human keratinocytes and HaCaT cells, different
murine cell lines (C3H-L, Balb/c 3T3, L929, liver and spleen cell lines, others), and T-
lymphocytes from lymph nodes. Nevertheless, the general opinion is that toxicity tests in vitro
will be more convincing when performed with cells that are homologous with the human tissue
concerned. In accordance, appropriate cell lines for testing of local skin compatibility are human
dermal fibroblasts and epidermal keratinocytes [Wiegand & Hipler 2009, Wiegand & Hipler
2008]. Determination of cell proliferation and cell viability has become the key technology to
assess biocompatibility of materials like medical textiles or wound dressings in vitro, especially if
dressings contain active agents such as silver (Ag+) or zinc (Zn2+). Their increasing use led to
the growing concern that too much metal ions could be delivered into the tissue, resulting in
toxic effects on growing keratinocytes and fibroblasts [Wiegand et al. 2009, Ip et al. 2006]. Tests
that measure cellular growth are capable to express toxic effects via loss of proliferation
capacity or reduction of living cells. These negative effects can be quantified by methods that
determine cell death. A characteristic sign for necrotic cell death is the degradation of the cell
membrane and activation of the inflammatory response. Cell membrane damage leads to
release of LDH (lactate dehydrogenase) from the cytosol into the supernatant [Wiegand &
Hipler 2009, Wiegand & Hipler 2008].
Bacteria on the skin surface of patients with atopic dermatitis (AD) can amplify and/or
perpetuate a pro-inflammatory environment. AD is a chronic inflammatory disease characterized
by the impairment of the skin barrier function, increased oxidative cellular stress and bacterial
colonization. Patients with AD display an augmented susceptibility to cutaneous bacterial,
fungal and viral infections [Melnik 2006, Baker 2006]. Microbial flora of atopic skin exhibits
page 8 of 33 CONFIDENTIAL – VERTRAULICH Study report 18-003 In vitro assessment of Vitadylan for cytotoxicity (DIN EN ISO 10993-5), antimicrobial activity (JIS L 1902), and antioxidative capacity
Klinik für Hautkrankheiten
Qualitäts-zertifiziert nach DIN EN ISO 9001:2008
Labor für In-vitro-Forschung und
Routinediagnostik
noticeable differences in contrast to normal skin, e.g. more than 90% of patients with AD are
colonized with Staphylococcus aureus whereas it is found in less than 10% in healthy
individuals [Leyden et al. 1974]. Especially silver-coated textiles are known to significantly
reduce the numbers of Staphylococcus aureus, and have been shown to improve the symptoms
and decrease the severity of atopic dermatitis (AD) [Hipler et al. 2006, Gauger 2006, Gauger et
al. 2003].
The skin is the major interface between body and environment. It is the most versatile human
organ and plays a key role in protecting the body against environmental influences and
participates in the regulation of homeostasis, metabolic processes as well as immunological
reactions. Oxidative stress by free radicals accelerates skin aging and has been implicated in
dermatological diseases such as atopic dermatitis [Sezer et al. 2007, Briganti & Picardo 2003].
UV light induces the generation of free radicals in the cells; hence, the application of topical
antioxidants has been recommended [Masaki 2010, Maela-Azulay & Bagatin 2009]. The
antioxidant capacity (AOC) of soluble substances and other materials, e.g. fabrics, can be
monitored and quantified using in vitro tests. The several methods are based on different
reaction mechanisms and employ various radicals and substrates. Peroxyl radicals (ROO•) are
the most often used radicals for in vitro procedures as they present the key radical for auto
oxidation of lipids [Ou et al. 2001]. The ROS (reactive oxygen species) test determines the
inhibition of the Pholasin® oxidation by superoxide anions and other oxygen radicals. In
contrast, the RNS (reactive nitrogen species) test measures the efficacy of an antioxidant to
decrease the Pholasin® oxidation by peroxynitrite (ONOO-). Pholasin is a photo protein isolated
from the mollusc Pholas dactylus, which emits light in the presence of certain oxidants
(chemiluminescence). Previously these tests has been successfully used to determine the
antioxidant capacity of wound dressings [Wiegand et al. 2009, Wiegand et al. 2006, Schönfelder
et al. 2005] and textiles [Hipler & Wiegand 2011, Wiegand et al. 2010, Fluhr et al. 2010].
page 9 of 33 CONFIDENTIAL – VERTRAULICH Study report 18-003 In vitro assessment of Vitadylan for cytotoxicity (DIN EN ISO 10993-5), antimicrobial activity (JIS L 1902), and antioxidative capacity
Klinik für Hautkrankheiten
Qualitäts-zertifiziert nach DIN EN ISO 9001:2008
Labor für In-vitro-Forschung und
Routinediagnostik
6. Description of materials and test methods
6.1 Preparation of sample material
For cytotoxicity testing, extract of sample material was produced according to DIN EN ISO
10993-12 by incubating 10 g sample material with 50 mL Dulbecco’s modified Eagle medium
(DMEM, Lot LB02129P; BioConcept) without fetal calf serum (FCS) for 24 h and 72 h at 37°C,
respectively. The extract was sterile filtrated and supplemented with 10% FCS (fetal calf serum,
Lot P160509; PAN). Extract dilutions of the original extract (100% - extraction ratio 0.2 g:mL) in
DMEM were used for the in vitro assays: 75% - 0.15 g:mL, 50% - 0.1 g:mL, 25% - 0.05 g:mL,
10% - 0.02 g:mL und 1% - 0.002 g:mL.
For testing of the antioxidative capacity, samples were cut using 8 mm and 5 mm punch
biopsies (Stiefel Laboratorium GmbH, Germany) corresponding to 0.5 cm2 and 0.25 cm2,
respectively, and transferred to white 96-well plates (greiner bio-one, Germany).
6.2 Cell culture of HaCaT keratinocytes
The human HaCaT keratinocytes were a gift from Prof. Dr. N.E. Fusenig (DKFZ, Germany).
Human HaCaT keratinocytes were cultured in Dulbecco´s modified Eagle’s Medium (DMEM Lot
Lot LB02129P; BioConcept) supplemented with 1% antibiotic-antimycotic solution (10000 U/mL
penicillin, 10000 µg/mL streptomycin, 25 µg/mL amphotericin; Lot LA07722P; Pelobiotech) and
10% fetal calf serum (FCS; Lot P160509; PAN). The cells were cultured for 7 days in 75cm2 cell
culture flasks (Greiner bio-one, Germany) at 37°C and in humidified atmosphere containing 5%
CO2 atmosphere. For experiments, the cells were harvested through trypsin-EDTA (Lot
1768152; Gibco) treatment and seeded into 96-well plates (Greiner bio-one, Germany) at a
density of 30,000cells/cm2. After 48 hours, the culture medium was replaced by either fresh
DMEM (control) or extract dilutions as indicated. As cytotoxicity control Triton X-100 was used.
page 10 of 33 CONFIDENTIAL – VERTRAULICH Study report 18-003 In vitro assessment of Vitadylan for cytotoxicity (DIN EN ISO 10993-5), antimicrobial activity (JIS L 1902), and antioxidative capacity
Klinik für Hautkrankheiten
Qualitäts-zertifiziert nach DIN EN ISO 9001:2008
Labor für In-vitro-Forschung und
Routinediagnostik
6.3 ATP bioluminescence assay
Determination of cell proliferation was carried out on the basis of a luminometric ATPLiteTM-M
assay (Lot 69-17291, PerkinElmer) according to the manufacturer’s recommendations. In brief,
50µL lysis solution was added to the wells containing treated or control cells. Subsequently,
50µL substrate solution (luciferase/D-luciferin) was added to each well. After incubation, the
luminescence was measured using a microplate luminometer (LUMIstar Galaxy, BMG Labtech).
ATP concentrations were calculated on the basis of a standard curve.
6.4 Determination of cellular protein content
Determination of cell proliferation was carried out by measurement of total protein content. In
brief, cell culture medium was removed completely, and cells were washed two times with PBS.
Human HaCaT keratinocytes were lysed with 0.1% Triton-X 100 in PBS at 95°C for 10 min.
After cooling, the plates were stored at -20°C until testing. Total protein content was determined
by the Pierce BCA Protein Assay (Lot PL212737, Thermo Scientific Inc.) based on the biuret
reaction. The method combines the reduction of Cu2+ to Cu+ by protein in an alkaline solution
with the colorimetric detection of Cu+ using bichinoninic acid (BCA). The assay was performed
as recommended by the manufacturer using the microplate procedure protocol. Absorbance
was measured by means of the microplate photometer FLUOstar Galaxy (BMG Lab
Technologies GmbH). Protein concentrations were calculated on the basis of a standard curve.
6.5 Detection of cytotoxicity (LDH)
Determination of in vitro cytotoxicity was carried out by measurement of lactate dehydrogenase
(LDH) activity in cell culture supernatants. LDH is released by loss of membrane integrity from
cytosol to the cell culture medium. LDH activity was determined by the Cytotoxicity Detection Kit
(Lot 11644793001, Roche). In this assay, LDH activity is measured with an enzymatic test. LDH
catalyze the reaction from lactate to pyruvate together with the conversion of NAD+ to NADH +
page 11 of 33 CONFIDENTIAL – VERTRAULICH Study report 18-003 In vitro assessment of Vitadylan for cytotoxicity (DIN EN ISO 10993-5), antimicrobial activity (JIS L 1902), and antioxidative capacity
Klinik für Hautkrankheiten
Qualitäts-zertifiziert nach DIN EN ISO 9001:2008
Labor für In-vitro-Forschung und
Routinediagnostik
H+. A catalyst (diaphorase) mediates the conversion of a tetrazolium salt to formazan. The
absorption of the formazan dye can be measured at 490nm.
The assay was performed as recommended by the manufacturer using the microplate
procedure protocol. In brief, 100µL of cell culture supernatant were collected and transferred to
a new 96-well plate. 100µL tetrazolium dye solution was added and after incubation absorbance
was measured by means of the microplate photometer FLUOstar Galaxy (BMG Lab
Technologies GmbH). Cytotoxicity was calculated in comparison with positive control (Triton X-
100):
ODsample – ODcontrol
Cytotoxicity [%] = x 100 % ODpositive control - ODcontrol
6.6 Determination of the antibacterial activity according to JIS L 1902
The determination of antimicrobial activity was performed according to the internationally
recognized Japanese industrial standard (JIS L 1902:2008, „Testing method for antibacterial
activity of textiles“). Staphylococcus aureus ATCC 6538 and Klebsiella pneumoniae ATCC 4352
were purchased from the DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen,
Germany).
Caso-bouillon (Oxoid, U.K.; Lot. 23124) was inoculated with the test microbes and cultivated for
24 hours at 37°C under aerobic conditions. For experiments, 400 mg samples of the textile were
incubated with 200 µL of each test microbe suspension (approx. 105 cfu/mL) for 24 h at 37°C
under aerobic conditions. Samples of polyester material were used as growth control. For germ
number determination the incubated samples were extracted in 0.9% NaCl solution (Fresenius
Kabi Deutschland GmbH, Germany; Lot. 14HD24) with Tween 20 (Carl Roth GmbH, Germany;
Lot. 1005482700). Serial dilutions were plated on Columbia agar plates (Biomerieux, France;
Lot. 1005838550) and incubated for 24 hours at 37°C. Afterwards, colonies were counted,
cfu/mL (colony forming units per milliliter) and total cfu per sample were calculated, and growth
reduction compared to the starting value was determined according to the following equation:
growth reduction [log] = log (mean [cfu]control) – log (mean [cfu]sample)
page 12 of 33 CONFIDENTIAL – VERTRAULICH Study report 18-003 In vitro assessment of Vitadylan for cytotoxicity (DIN EN ISO 10993-5), antimicrobial activity (JIS L 1902), and antioxidative capacity
Klinik für Hautkrankheiten
Qualitäts-zertifiziert nach DIN EN ISO 9001:2008
Labor für In-vitro-Forschung und
Routinediagnostik
rating according to JIS L 1902:2008:
no antimicrobial activity = < 0.5 log microbial growth reduction
slight antimicrobial activity = 0.5 – 1 log microbial growth reduction
significant antimicrobial activity = >1 - ≤3 log microbial growth reduction
strong antimicrobial activity = >3 log microbial growth reduction
6.7 Determination of the antioxidant capacity against ROS
The capability of the textile to scavenge free radicals such as ROS (reactive oxygen species)
was assessed using the chemiluminescent ABEL Antioxidant Test Kits specific for superoxide
anion and other radicals containing Pholasin (Knight Scientific Limited, U.K.).
To each sample the respective assay solutions were added. In brief, 25 µL assay buffer (Lot.
BA252A B1 150317), 50 µL Pholasin solution (Lot. AA171A B3 150309) as well as 100 µL
solution A (Lot. JA512A A1 140820) were added. Then 25 µL of solution B (Lot. KA612A B3
130701) were injected to each well immediately before measurement. A control without sample
was run with each assay. The measurement of luminescence was carried out using the
LUMIstar Galaxy plate reader (BMG Labtech GmbH, Germany).
The antioxidant capacity of a sample is expressed as percent reduction of peak luminescence
as follows:
[(peak-control) – (peak-sample)] x 100
% inhibition =
(peak-control)
6.8 Determination of the antioxidant capacity against RNS
The capability of the textile to scavenge free radicals such as RNS (reactive nitrogen species)
was assessed using the chemiluminescent ABEL Antioxidant Test Kits specific for peroxynitrite
anion containing Pholasin (Knight Scientific Limited, U.K.).
To each sample the respective assay solutions were added. In brief, 100 µL assay buffer (Lot.
TA 512A B2 150506) as well as 50 µL Pholasin solution (Lot. AA 173A A2 150228) were
page 13 of 33 CONFIDENTIAL – VERTRAULICH Study report 18-003 In vitro assessment of Vitadylan for cytotoxicity (DIN EN ISO 10993-5), antimicrobial activity (JIS L 1902), and antioxidative capacity
Klinik für Hautkrankheiten
Qualitäts-zertifiziert nach DIN EN ISO 9001:2008
Labor für In-vitro-Forschung und
Routinediagnostik
added. Then 50 µL of SIN-1 solution (Lot. UA713B B3 140820) were injected to each well prior
to the measurement. A control without sample was run with each assay. The measurement of
luminescence was carried out using the LUMIstar Galaxy plate reader (BMG Labtech GmbH,
Germany).
The antioxidant capacity of a sample is expressed as percent reduction of peak luminescence
as follows:
[(peak-control) – (peak-sample)] x 100
% inhibition =
(peak-control)
6.9 Measurement of vitamin A, C, and E content
2 g of the sample material were grinded and took up in 20 mL extraction medium. For extraction
media, n-hexane (vitamins A and E) and phosphoric acid (vitamin C) were used respectively.
Measurement of α-tocopheryl acetate was carried out using fluorescent HPLC (Merck) at 292
and 330 nm at 35°C. Retinol was analyzed by standard HPLC (Merck) at 450 nm at 13°C.
Ascorbic acid content was determined by UV-Vis spectrometry using the method of Al-Duais et
al. [Al-Duais et al. 2009]. Results were calculated as mg / 100 g sample.
6.10 Statistical analysis
Experiments and measurements for cytotoxicity testing were performed in duplicate each.
Measurements for JIS L 1902 were performed in triplicate. For testing of the antioxidative
capacity, experiments were performed in duplicate and measurements were performed in
triplicate. All values are expressed as means ± SD (standard deviation). One-way analysis of
variance was carried out to determine statistical significances (Microsoft® Excel 2000).
Differences are considered statistically significant at a level of p < 0.05. Asterisks indicate
significant deviations from the control ( p < 0.05; p < 0.01; p < 0.001).
page 14 of 33 CONFIDENTIAL – VERTRAULICH Study report 18-003 In vitro assessment of Vitadylan for cytotoxicity (DIN EN ISO 10993-5), antimicrobial activity (JIS L 1902), and antioxidative capacity
Klinik für Hautkrankheiten
Qualitäts-zertifiziert nach DIN EN ISO 9001:2008
Labor für In-vitro-Forschung und
Routinediagnostik
7. Deviations from the study protocol
There were no deviations from the study protocol.
8. Archiving
The following records will be stored in the archives of the Klinik für Hautkrankheiten,
Universitätsklinikum Jena according to the GLP regulations:
A copy of the final report, the study plan and a documentation of all raw data generated during
the conduct of the study will be stored for at least 4 years after completion of the study.
Unused test items and reference items are stored for at least 12 month after completion of the
study.
Materials and samples that are unstable may be disposed of before that time and without
sponsor’s prior consent.
Records and reports of the maintenance and calibration of apparatus, validation documentation
for computerized systems and the historical file of all Standard Operating Procedures (SOPs)
are stored in accordance with the appropriate authorities.
page 15 of 33 CONFIDENTIAL – VERTRAULICH Study report 18-003 In vitro assessment of Vitadylan for cytotoxicity (DIN EN ISO 10993-5), antimicrobial activity (JIS L 1902), and antioxidative capacity
Klinik für Hautkrankheiten
Qualitäts-zertifiziert nach DIN EN ISO 9001:2008
Labor für In-vitro-Forschung und
Routinediagnostik
9. Results and discussion
24-hour and 72-hour extracts of the textile Vitadylan exhibited a noticeable effect on the HaCaT
cells over the time course of 48 hours (figures 1 and 2). A direct negative effect on cell viability
was observed for an extraction ratio of 0.2 g:mL. In lower doses (< 0.2 and ≥ 0.1 g:mL), cells
died over an incubation period of 24 hours. Extraction ratios of 0.05 and 0.02 g:mL decreased
cell proliferation significantly over time. Only the concentration of 0.002 g:mL was well tolerated
in the test. Results obtained for 24-hour and 72-hour extracts were similar in the luminometric
ATP assay.
Photometric measurement of the total cellular protein content confirmed the effects of the 24-
hour and 72-hour extracts of Vitadylan on viability and proliferation of HaCaT cells (figures 3
and 4). However, it was found that the protein assay is considerably less sensitive compared to
the ATP measurement.
Assessment of LDH release did not reflect results on the decrease of cell viability. While 24-
hour and 72-hour extracts of the textile Vitadylan did increase the release of LDH and
cytotoxicity, respectively (figure 5), it was less than expected. This could mean that the cells die
by another mechanism than necrosis or that the substances extracted from the textile interact
with the assay components.
page 16 of 33 CONFIDENTIAL – VERTRAULICH Study report 18-003 In vitro assessment of Vitadylan for cytotoxicity (DIN EN ISO 10993-5), antimicrobial activity (JIS L 1902), and antioxidative capacity
Klinik für Hautkrankheiten
Qualitäts-zertifiziert nach DIN EN ISO 9001:2008
Labor für In-vitro-Forschung und
Routinediagnostik
Figure 1: Influence of the 24-hour extract of the textile Vitadylan on the viability and proliferation of
HaCaT keratinocytes. Determination of the number of viable cells was done by measurement of the
cellular ATP content (A) and the number of viable cells (in [%]) was determined corresponding to the
untreated medium control (B). (For data see table A.1 in the appendix.)
A
B
page 17 of 33 CONFIDENTIAL – VERTRAULICH Study report 18-003 In vitro assessment of Vitadylan for cytotoxicity (DIN EN ISO 10993-5), antimicrobial activity (JIS L 1902), and antioxidative capacity
Klinik für Hautkrankheiten
Qualitäts-zertifiziert nach DIN EN ISO 9001:2008
Labor für In-vitro-Forschung und
Routinediagnostik
Figure 2: Influence of the 72-hour extract of the textile Vitadylan on the viability and proliferation of
HaCaT keratinocytes. Determination of the number of viable cells was done by measurement of the
cellular ATP content (A) and the number of viable cells (in [%]) was determined corresponding to the
untreated medium control (B). (For data see table A.2 in the appendix.)
A
B
page 18 of 33 CONFIDENTIAL – VERTRAULICH Study report 18-003 In vitro assessment of Vitadylan for cytotoxicity (DIN EN ISO 10993-5), antimicrobial activity (JIS L 1902), and antioxidative capacity
Klinik für Hautkrankheiten
Qualitäts-zertifiziert nach DIN EN ISO 9001:2008
Labor für In-vitro-Forschung und
Routinediagnostik
Figure 3: Influence of the 24-hour extract of the textile Vitadylan on HaCaT keratinocytes. Determination
of the cell number was done by measurement of the cellular protein content (A) and the number of cells
(in [%]) was determined corresponding to the untreated medium control (B). (For data see table A.1 in the
appendix.)
A
B
page 19 of 33 CONFIDENTIAL – VERTRAULICH Study report 18-003 In vitro assessment of Vitadylan for cytotoxicity (DIN EN ISO 10993-5), antimicrobial activity (JIS L 1902), and antioxidative capacity
Klinik für Hautkrankheiten
Qualitäts-zertifiziert nach DIN EN ISO 9001:2008
Labor für In-vitro-Forschung und
Routinediagnostik
Figure 4: Influence of the 72-hour extract of the textile Vitadylan on HaCaT keratinocytes. Determination
of the cell number was done by measurement of the cellular protein content (A) and the number of cells
(in [%]) was determined corresponding to the untreated medium control (B). (For data see table A.2 in the
appendix.)
A
B
page 20 of 33 CONFIDENTIAL – VERTRAULICH Study report 18-003 In vitro assessment of Vitadylan for cytotoxicity (DIN EN ISO 10993-5), antimicrobial activity (JIS L 1902), and antioxidative capacity
Klinik für Hautkrankheiten
Qualitäts-zertifiziert nach DIN EN ISO 9001:2008
Labor für In-vitro-Forschung und
Routinediagnostik
Figure 5: Cytotoxicity of the 24-hour (A) and the 72-hour (B) extract of the textile Vitadylan against
HaCaT keratinocytes. Determination was done by photometric measurement of the LDH release. (For
data see tables A.1 and A.2 in the appendix.)
A
B
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The textile Vitadylan was able to significantly reduce the growth of Staphylococcus aureus
(table 1, figure 6A). The antibacterial effect achieved could be rated as a strong antibacterial
activity according to JIS L 1902:2008 (table 1, figure 6B). Vitadylan further accomplished a
significant decrease of the growth of Klebsiella pneumonia (table 1, figure 7A). In accordance,
the Vitadylan also achieved a strong antibacterial activity against Klebsiella pneumonia
according to JIS L 1902:2008 (table 1, figure 7B).
Table 1: Antibacterial activity of the textile Vitadylan tested against Staphylococcus aureus and Klebsiella pneumoniae (for data see tables A.3 and A.4 in the appendix)
Staphylococus aureus Klebsiella pneumoniae
Vitadylan strong
(75.8% inhibition)
strong
(65.0% inhibition)
page 22 of 33 CONFIDENTIAL – VERTRAULICH Study report 18-003 In vitro assessment of Vitadylan for cytotoxicity (DIN EN ISO 10993-5), antimicrobial activity (JIS L 1902), and antioxidative capacity
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Figure 6: Growth of Staphylococcus aureus under the influence of the textile Vitadylan over 24 hours (A) and the reduction of bacterial growth achieved in [log cfu] according to JIS L 1902 (B). (For data see table A.3 in the appendix.)
A
B
page 23 of 33 CONFIDENTIAL – VERTRAULICH Study report 18-003 In vitro assessment of Vitadylan for cytotoxicity (DIN EN ISO 10993-5), antimicrobial activity (JIS L 1902), and antioxidative capacity
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Figure 7: Growth of Klebsiella pneumoniae under the influence of the textile Vitadylan over 24 hours (A) and the reduction of bacterial growth achieved in [log cfu] according to JIS L 1902 (B). (For data see table A.4 in the appendix.)
A
B
page 24 of 33 CONFIDENTIAL – VERTRAULICH Study report 18-003 In vitro assessment of Vitadylan for cytotoxicity (DIN EN ISO 10993-5), antimicrobial activity (JIS L 1902), and antioxidative capacity
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Vitadylan was able to significantly inhibit the formation of free reactive oxygen species in a
dose-dependent manner (figure 8). Furthermore, it significantly reduced the formation of
reactive nitrogen species (figure 9).
Figure 8: Inhibition of ROS formation (for data see table A.5 in the appendix).
Figure 9: Inhibition of RNS formation (for data see table A.6 in the appendix).
page 25 of 33 CONFIDENTIAL – VERTRAULICH Study report 18-003 In vitro assessment of Vitadylan for cytotoxicity (DIN EN ISO 10993-5), antimicrobial activity (JIS L 1902), and antioxidative capacity
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The vitamins A, C, and E were detected in the Vitadylan sample material (table 2). It can be
assumed that the amounts of vitamin C and E found in the textile are responsible for the good
antioxidative capacity observed.
Table 2: Vitamin A, C, and E content of Vitadylan as measured by HPLC and UV-Vis spectrometry.
retinol equivalents
(vitamin A)
[mg/100g]
ascorbic acid
(vitamin C)
[mg/100g]
α-tocopheryl acetate
(vitamin E)
[mg/100g]
Vitadylan 1.30 0.03 2.70 0.03 1.53 0.19
page 26 of 33 CONFIDENTIAL – VERTRAULICH Study report 18-003 In vitro assessment of Vitadylan for cytotoxicity (DIN EN ISO 10993-5), antimicrobial activity (JIS L 1902), and antioxidative capacity
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10. Appendix
10.1 Abbreviations
cm centimeters
cm2 square centimeters
°C grad Celsius
DMEM Dulbecco’s modified Eagle Medium
g Gramms
h hour
IL-6 interleukin 6
IL-8 interleukin 8
LDH lactate dehydrogenase
M molar
mL milliliters
µL microliters
mM millimolar
mm millimeters
% percent
OD optical density
PBS phosphate buffered saline
RNS reactive nitrogen species
ROS reactive oxygen species
SD standard deviation
TSB tryptic soy broth
page 27 of 33 CONFIDENTIAL – VERTRAULICH Study report 18-003 In vitro assessment of Vitadylan for cytotoxicity (DIN EN ISO 10993-5), antimicrobial activity (JIS L 1902), and antioxidative capacity
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10.2 Tables and Figures
page
Figure 1: Influence of the 24-hour extract of the textile Vitadylan on the viability and
proliferation of HaCaT keratinocytes. Determination of the number of viable cells was done
by measurement of the cellular ATP content (A) and the number of viable cells (in [%]) was
determined corresponding to the untreated medium control (B). (For data see table A.1 in
the appendix.)
16
Figure 2: Influence of the 72-hour extract of the textile Vitadylan on the viability and
proliferation of HaCaT keratinocytes. Determination of the number of viable cells was done
by measurement of the cellular ATP content (A) and the number of viable cells (in [%]) was
determined corresponding to the untreated medium control (B). (For data see table A.2 in
the appendix.)
17
Figure 3: Influence of the 24-hour extract of the textile Vitadylan on HaCaT keratinocytes.
Determination of the cell number was done by measurement of the cellular protein content
(A) and the number of cells (in [%]) was determined corresponding to the untreated
medium control (B). (For data see table A.1 in the appendix.)
18
Figure 4: Influence of the 72-hour extract of the textile Vitadylan on HaCaT keratinocytes.
Determination of the cell number was done by measurement of the cellular protein content
(A) and the number of cells (in [%]) was determined corresponding to the untreated
medium control (B). (For data see table A.2 in the appendix.)
19
Figure 5: Cytotoxicity of the 24-hour (A) and the 72-hour (B) extract of the textile Vitadylan
against HaCaT keratinocytes. Determination was done by photometric measurement of
the LDH release. (For data see tables A.1 and A.2 in the appendix.)
20
Figure 6: Growth of Staphylococcus aureus under the influence of the textile Vitadylan
over 24 hours (A) and the reduction of bacterial growth achieved in [log cfu] according to
JIS L 1902 (B). (For data see table A.3 in the appendix.)
22
Figure 7: Growth of Klebsiella pneumoniae under the influence of the textile Vitadylan
over 24 hours (A) and the reduction of bacterial growth achieved in [log cfu] according to
JIS L 1902 (B). (For data see table A.4 in the appendix.)
23
Figure 8: Inhibition of ROS formation (for data see table A.5 in the appendix). 24
Figure 9: Inhibition of RNS formation (for data see table A.6 in the appendix). 24
Table 1: Antibacterial activity of the textile Vitadylan tested against Staphylococcus aureus
and Klebsiella pneumoniae (for data see tables A.3 and A.4 in the appendix)
21
Table 2: Vitamin A, C, and E content of Vitadylan as measured by HPLC and UV-Vis
spectrometry.
25
Table A.1: Influence of the 24-hour extract of the textile Vitadylan on HaCaT keratinocytes
after 1, 24, and 48h. Determination of viable cells by luminometric measurement of the
cellular ATP content, assessment of total amount of cells was performed by photometric
measurement of the total protein content, and evaluation of cytotoxicity by photometric
30
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measurement of the LDH release (n = 4).
Table A.2: Influence of the 72-hour extract of the textile Vitadylan on HaCaT keratinocytes
after 1, 24, and 48h. Determination of viable cells by luminometric measurement of the
cellular ATP content, assessment of total amount of cells was performed by photometric
measurement of the total protein content, and evaluation of cytotoxicity by photometric
measurement of the LDH release (n = 4).
31
Table A.3: Influence of the textile Vitadylan on the growth of Staphylococcus aureus
(n=3).
32
Table A.4: Influence of the textile Vitadylan on the growth of Klebsiella pneumonia (n=3) 32
Table A.5: Reduction of ROS formation by the textile Vitadylan (n=6). 33
Table A.6: Reduction of RNS formation by the textile Vitadylan (n=6). 33
page 29 of 33 CONFIDENTIAL – VERTRAULICH Study report 18-003 In vitro assessment of Vitadylan for cytotoxicity (DIN EN ISO 10993-5), antimicrobial activity (JIS L 1902), and antioxidative capacity
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10.3 References
Al-Duais M, Hohbain J, Werner S, Böhm V, Jetschke G. Eur Food Res Technol 2009;
228:813-821
Baker BS. Clin Exp Dermatol 2006; 144:1-9
Briganti S, Picardo M. JEADV 2003; 17:663-9
DIN EN ISO 10993-1 April 2010 Biologische Beurteilung von Medizinprodukten Teil 1:
Beurteilung und Prüfung im Rahmen eines Risikomanagementsystems
DIN EN ISO 10993-5 Juni 2007 Biologische Beurteilung von Medizinprodukten Teil 5: Prüfung
auf in-vitro-Zytotoxizität
DIN EN ISO 10993-12 Februar 2008 Biologische Beurteilung von Medizinprodukten Teil 12:
Probenvorbereitung und Referenzmaterialien
Fluhr JW, Breternitz M, Kowatzki D, Bauer A, Bossert J, Elsner P, Hipler UC. Exp Dermatol
2010; 19:e9-15
Gauger A. Curr Probl Dermatol 2006; 33:152-164
Gauger A, Mempel M, Schekatz A, Schäfer T, Ring J, Abeck D. Dermatology 2003; 207:15-
21
Hipler UC, Elsner P, Fluhr JW. J Biomed Mater Res Part B 2006; 77B:156-163
Ip M, Lui SL, Poon VKM, Lung I, Burd A. J Med Microbiol 2006; 55:59-63
Leyden JJ, Marples RR, Kligman AM. Br J Dermatol 1974; 90:525-530
Manela-Azulay M, Bagatin E. Clinics Dermatol 2009; 27:469-474
Masaki H. J Dermatol Sci 2010; 58:85-90
Melnik B. JDDG 2006; 2:114-123
Ou BX, Hampsch-Woodill M, Prior RL. J Agricultur Food Chem 2001; 49:4619-26
Schönfelder U, Abel M, Wiegand C, Klemm D, Elsner P, Hipler U-C. Biomaterials 2005; 26:
6664-6673
Sezer E, Ozugurlu F, Ozyurt H, Sahin S, Etikan I. Clin Exp Dermatol 2007; 32:430-4
Wiegand C, Elsner P, Hipler UC, Klemm D. Cellulose 2006; 13:689-696
Wiegand C, Fluhr JW, Elsner P, Hipler UC. in Cellulose: Structure and properties, derivatives
and industrial uses, eds. A. Lejeune & T. Deprez, Nova Science Publishers, 2010
Wiegand C, Heinze T, Hipler UC. Wound Rep Reg. 2009; 17:511–21
Wiegand C, Hipler UC. Skin Pharmacol Physiol 2009; 22:74-82
Wiegand C, Hipler UC. GMS Krankhaushyg Interdiszip 2008 3(1):Doc12
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10.4 Measurement data
Table A.1: Influence of the 24-hour extract of the textile Vitadylan on HaCaT keratinocytes after
1, 24, and 48h. Determination of viable cells by luminometric measurement of the cellular ATP
content, assessment of total amount of cells was performed by photometric measurement of the
total protein content, and evaluation of cytotoxicity by photometric measurement of the LDH
release (n = 4). negative
control (medium)
positive control
(triton X-100)
extraction ratio [g:mL]
0.002 0.02 0.05 0.1 0.15 0.2
1h ATP [nM] 15862.26 1940.26 16568.16 15273.09 14329.11 13791.35 12581.97 10038.61
SD 887.68 656.68 144.65 353.01 77.35 255.31 588.84 327.68
viable cellsl [%] 100.00 12.23 104.45 96.29 90.33 86.94 79.32 63.29
SD 5.60 4.14 0.91 2.23 0.49 1.61 3.71 2.07
p-value - 0.0001 0.1798 0.2705 0.0104 0.0019 0.0001 0.0001
protein [µg/mL] 51.88 -1.06 60.95 57.17 52.91 49.51 47.38 47.23
SD 7.39 2.32 3.01 5.35 0.45 1.42 1.49 2.74
cell number [%] 100.00 -2.04 117.49 110.19 101.98 95.44 91.33 91.03
SD 14.25 4.47 5.81 10.31 0.86 2.73 2.87 5.29
p-value - 0.0001 0.0568 0.2722 0.8329 0.5753 0.2974 0.2922
LDH [mOD] 486.40 702.00 463.75 465.75 453.75 449.25 438.25 446.75
SD 4.59 1.00 5.40 6.42 10.59 11.88 8.50 6.65
cytotoxicity[%] 0.00 100.00 -10.51 -9.58 -15.14 -17.23 -22.33 -18.39
SD 2.13 0.46 2.51 2.98 4.91 5.51 3.94 3.08
p-value - 0.0001 0.0005 0.0016 0.0009 0.0008 0.0001 0.0001
24h ATP [nM] 31548.67 1383.29 34145.98 25159.59 20619.16 57.77 -158.50 -30.89
SD 1170.77 328.53 990.82 415.26 917.34 28.33 13.67 18.19
viable cellsl [%] 100.00 4.38 108.23 79.75 65.36 0.18 -0.50 -0.10
SD 3.71 1.04 3.14 1.32 2.91 0.09 0.04 0.06
p-value - 0.0001 0.0060 0.0001 0.0001 0.0001 0.0001 0.0001
protein [µg/mL] 97.02 -2.59 96.27 73.08 64.19 19.47 27.22 19.09
SD 4.31 2.77 5.94 4.09 5.34 8.27 6.30 2.19
cell number [%] 100.00 -2.67 99.22 75.32 66.16 20.07 28.06 19.68
SD 4.45 2.86 6.12 4.21 5.51 8.52 6.49 2.26
p-value - 0.0001 0.8240 0.0001 0.0001 0.0001 0.0001 0.0001
LDH [mOD] 432.40 1564.00 415.25 500.00 522.75 946.25 918.25 782.50
SD 8.06 73.65 3.77 6.96 8.20 8.95 26.82 32.48
cytotoxicity[%] 0.00 100.00 -1.52 5.97 7.98 45.41 42.93 30.94
SD 0.71 6.51 0.33 0.62 0.72 0.79 2.37 2.87
p-value - 0.0001 0.0105 0.0001 0.0001 0.0001 0.0001 0.0001
48h ATP [nM] 58338.08 1733.92 66131.15 39016.18 25286.79 26.30 -195.80 62.64
SD 2453.30 435.95 7068.48 3421.07 1148.83 59.04 22.31 40.79
viable cellsl [%] 100.00 2.97 113.36 66.88 43.35 0.05 -0.34 0.11
SD 4.21 0.75 12.12 5.86 1.97 0.10 0.04 0.07
p-value - 0.0001 0.0286 0.0001 0.0001 0.0001 0.0001 0.0001
protein [µg/mL] 139.33 0.50 150.73 82.81 62.21 9.19 11.12 11.87
SD 9.41 1.31 16.34 11.10 3.12 1.85 2.34 2.94
cell number [%] 100.00 0.36 108.18 59.44 44.65 6.60 7.98 8.52
SD 6.75 0.94 11.73 7.97 2.24 1.33 1.68 2.11
p-value - 0.0001 0.1927 0.0001 0.0001 0.0001 0.0001 0.0001
LDH [mOD] 379.80 1912.67 352.75 491.75 561.50 939.75 845.00 632.75
SD 12.98 35.24 8.84 19.65 16.50 29.84 20.27 25.46
cytotoxicity[%] 0.00 100.00 -1.76 7.30 11.85 36.53 30.35 16.50
SD 0.85 2.30 0.58 1.28 1.08 1.95 1.32 1.66
p-value - 0.0001 0.0164 0.0001 0.0001 0.0001 0.0001 0.0001
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Table A.2: Influence of the 72-hour extract of the textile Vitadylan on HaCaT keratinocytes after
1, 24, and 48h. Determination of viable cells by luminometric measurement of the cellular ATP
content, assessment of total amount of cells was performed by photometric measurement of the
total protein content, and evaluation of cytotoxicity by photometric measurement of the LDH
release (n = 4). negative
control (medium)
positive control
(triton X-100)
extraction ratio [g:mL]
0.002 0.02 0.05 0.1 0.15 0.2
1h ATP [nM] 15862.26 1940.26 17067.47 14908.09 14477.33 13613.02 12320.73 9951.53
SD 887.68 656.68 520.23 360.27 419.59 262.46 588.70 422.19
viable cellsl [%] 100.00 12.23 107.60 93.98 91.27 85.82 77.67 62.74
SD 5.60 4.14 3.28 2.27 2.65 1.65 3.71 2.66
p-value - 0.0001 0.0450 0.0886 0.0223 0.0011 0.0001 0.0001
protein [µg/mL] 51.88 -1.06 50.30 50.78 45.65 47.54 47.23 43.99
SD 7.39 2.32 3.17 2.01 1.95 1.88 3.44 1.27
cell number [%] 100.00 -2.04 96.96 97.87 87.99 91.64 91.03 84.79
SD 14.25 4.47 6.12 3.88 3.75 3.62 6.62 2.45
p-value - 0.0001 0.7171 0.7943 0.1612 0.3164 0.3005 0.0819
LDH [mOD] 486.40 702.00 477.25 474.00 456.75 456.75 443.25 443.75
SD 4.59 1.00 6.18 25.02 14.79 8.87 12.44 11.97
cytotoxicity[%] 0.00 100.00 -4.24 -5.75 -13.75 -13.75 -20.01 -19.78
SD 2.13 0.46 2.87 11.60 6.86 4.11 5.77 5.55
p-value - 0.0001 0.0595 0.3702 0.0073 0.0007 0.0004 0.0003
24h ATP [nM] 31548.67 1383.29 35041.30 24904.79 20729.37 122.82 -185.43 -179.21
SD 1170.77 328.53 515.08 646.37 267.45 69.77 20.91 78.63
viable cellsl [%] 100.00 4.38 111.07 78.94 65.71 0.39 -0.59 -0.57
SD 3.71 1.04 1.63 2.05 0.85 0.22 0.07 0.25
p-value - 0.0001 0.0004 0.0001 0.0001 0.0001 0.0001 0.0001
protein [µg/mL] 97.02 -2.59 99.73 72.70 59.75 5.91 10.96 13.22
SD 4.31 2.77 2.06 2.67 3.45 1.15 0.74 3.75
cell number [%] 100.00 -2.67 102.79 74.93 61.58 6.10 11.30 13.62
SD 4.45 2.86 2.12 2.75 3.56 1.19 0.76 3.87
p-value - 0.0001 0.3026 0.0001 0.0001 0.0001 0.0001 0.0001
LDH [mOD] 432.40 1564.00 434.75 510.50 530.75 941.50 882.00 759.75
SD 8.06 73.65 9.01 7.16 12.87 7.30 16.99 23.51
cytotoxicity[%] 0.00 100.00 0.21 6.90 8.69 44.99 39.73 28.93
SD 0.71 6.51 0.80 0.63 1.14 0.64 1.50 2.08
p-value - 0.0001 0.7269 0.0001 0.0001 0.0001 0.0001 0.0001
48h ATP [nM] 58338.08 1733.92 68052.39 38981.67 25284.52 68.09 -265.74 -233.95
SD 2453.30 435.95 3141.14 2727.65 427.70 17.59 41.92 130.44
viable cellsl [%] 100.00 2.97 116.65 66.82 43.34 0.12 -0.46 -0.40
SD 4.21 0.75 5.38 4.68 0.73 0.03 0.07 0.22
p-value - 0.0001 0.0003 0.0001 0.0001 0.0001 0.0001 0.0001
protein [µg/mL] 139.33 0.50 157.07 81.23 56.10 8.58 9.95 10.57
SD 9.41 1.31 18.77 9.28 1.46 2.72 3.49 3.37
cell number [%] 100.00 0.36 112.73 58.30 40.26 6.15 7.14 7.58
SD 6.75 0.94 13.47 6.66 1.05 1.95 2.50 2.42
p-value - 0.0001 0.0941 0.0001 0.0001 0.0001 0.0001 0.0001
LDH [mOD] 379.80 1912.67 369.00 483.75 586.00 934.75 821.00 626.00
SD 12.98 35.24 8.15 19.51 37.66 7.50 27.94 24.90
cytotoxicity[%] 0.00 100.00 -0.70 6.78 13.45 36.20 28.78 16.06
SD 0.85 2.30 0.53 1.27 2.46 0.49 1.82 1.62
p-value - 0.0001 0.2416 0.0001 0.0001 0.0001 0.0001 0.0001
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Table A.3: Influence of the textile Vitadylan on the growth of Staphylococcus aureus (n=3).
bacterial growth after 24h reduction of bacterial growth
[cfu] [log cfu] [%] [log cfu] [%]
growth control (polyester) 4.80E+06 6.68 98.85 0.08 -1.33
3.90E+06 6.59 97.52 0.17 6.11
1.01E+07 7.00 103.63 -0.25 -3.63
mean 6.27E+06 6.76 100.00 0.00 0.38
SE 1.58E+06 0.10 1.52 0.10 2.40
Vitadylan
4.00E+02 2.60 38.50 4.16 61.50
1.00E+00 0.00 0.00 6.76 100.00
2.00E+02 2.30 34.04 4.46 65.96
mean 2.00E+02 1.63 24.18 5.12 75.82
SE 9.40E+01 0.67 9.93 0.67 9.93
p-value - - - 0.0035 0.0038
Table A.4: Influence of the textile Vitadylan on the growth of Klebsiella pneumoniae (n=3).
bacterial growth after 24h reduction of bacterial growth
[cfu] [log cfu] [%] [log cfu] [%]
growth control (polyester) 1.16E+08 8.06 102.19 -0.17 -2.19
3.43E+07 7.53 95.48 0.36 4.52
1.19E+08 8.08 102.33 -0.18 -2.33
mean 8.98E+07 7.89 100.00 0.00 0.00
SE 2.27E+07 0.15 1.85 0.15 1.85
Vitadylan
1.00E+02 2.00 25.34 5.89 74.66
1.90E+03 3.28 41.55 4.61 58.45
1.00E+03 3.00 38.02 4.89 61.98
mean 1.00E+03 2.76 34.97 5.13 65.03
SE 4.24E+02 0.32 4.02 0.32 4.02
p-value - - - 0.0003 0.0003
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Table A.5: Reduction of ROS formation by the textile Vitadylan (n=6). sample size 0.25 cm
2 0.5 cm
2
control [%] 2.13 2.13
0.00 0.00
0.00 0.00
0.00 0.00
0.00 0.00
1.24 1.24
mean 0.56 0.56
SD 0.92 0.92
Vitadylan [%] 26.53 90.13
38.48 68.05
35.26 74.73
40.95 70.64
46.54 86.08
28.50 89.18
mean 36.05 79.80
SD 7.59 9.82
p-value 0.0001 0.0001
Table A.6: Reduction of RNS formation by the textile Vitadylan (n=6). sample size 0.25 cm
2 0.5 cm
2
control [%] 1.29 1.29
0.00 0.00
0.75 0.75
3.01 3.01
0.00 0.00
0.00 0.00
mean 0.84 0.84
SD 1.19 1.19
Vitadylan [%] 57.39 87.19
45.42 84.80
47.89 80.28
37.54 76.03
37.59 65.46
45.65 91.82
mean 45.25 80.93
SD 7.38 9.34
p-value 0.0001 0.0001