cytotoxicity (DIN EN ISO 10993-5), antimicrobial activity (JIS ......with human tissue [DIN EN ISO...

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Kaufmännischer Vorstand und Sprecherin des

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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




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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





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1. Quality certificate





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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


Universitätsklinikum Jena

Erfurter Straße 35

D-07740 Jena

Germany

Study director: PD Dr. Uta-Christina Hipler





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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





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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.





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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





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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].





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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.





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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 +





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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)





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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





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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).





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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.





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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.





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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





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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





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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





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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





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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)





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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





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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





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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).





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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





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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





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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





Routinediagnostik

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





Routinediagnostik

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





Routinediagnostik

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





Routinediagnostik

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





Routinediagnostik

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

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