Journal of Biomaterials and Nanobiotechnology, 2011, 2, 234-238
doi:10.4236/jbnb.2011.23029 Published Online July 2011 (http://www.SciRP.org/journal/jbnb)
A Study of the Cytotoxicity of a New Nonwoven
Polymeric Fibrous Bandaging Material in Vitro
Alexander M. Dygai1, Ludmila M. Ogorodova2, Sergey G. Psakhie3, Yuri P. Belsky1, Natalia V. Belska1,
Marina G. Danilets1*, Anastasia A. Ligatcheva1, Alexey A. Сhurin1
1Research Institute of Pharmacology, Siberian Branch of the Russian Academy of Medical Sciences, Tomsk, Russia; 2Siberian State
Medical University, Tomsk, Russia; 3Institute of Strength Physics and Materials Science, Siberian Branch of Russian Academy of
Sciences, Tomsk, Russia.
Received March 24th, 2011; revised April 25th 2011; accepted May 10th, 2011.
ABSTRACT
Traditionally used cotton-based bandaging materials have several disadvantages which can be overcome by using an-
other fabric structure – nonwoven fabric. Moreover, these materials are more spongeous which increases their sorption
capacity. The new bandaging material developed by the Institute of Physics of Strength and Material Science of the
Siberian Branch of the Russian Academy of Sciences has even better sorption capacity with improved sorption proper-
ties. Its sorption capacity has been increased by means of an additional introduction of porous aluminum hydrate parti-
cles into fabric. It is important that a bandaging material has a good biocompatibility and does not have any cytotoxic
effect on cells and tissues. Here it is present results of the study of the material’s direct contact and indirect cytotoxicity
assays in comparison with cotton gauze. It have found that in the direct contact of nonwoven polymeric fibrous ban-
daging material (NPFBM) with cells for 24 hours of cultivation no changes in cell morphology take place, nor does the
amount of dead cells increase. These conclusions have been made by means of both a visual examination and an МТТ
assay. The NPFBM extract did not have any cytotoxic effect on the tested cells either. The obtained results allow us to
make a conclusion that the NPFBM complies with the international standard ISO 10993-5, which is applied to medical
goods, and can from now on be applied in the treatment of infected wounds in clinical practice.
Keywords: Cytotoxicity, Bandaging Materials, Development of Biocompatible Biomaterials
1. Introduction
Modern bandaging materials must meet certain require-
ments, including good absorbing and sorption capacity in
order to absorb and keep exudates, capture and securely
isolate pathogenic microorganisms and, at the same time,
to prevent microorganisms from getting into the wound
[1]. Gauze has been used mostly in local wound care
until now, mainly because of its low price and accessibil-
ity [2]. There is a variety of products that control proc-
esses of wound healing and which are produced with use
of polymers such as hydrocolloids, alginate, chitin and so
on [3-5]. Some of products possess an antimicrobial pro-
perties and contain antiseptics (iodine, polyhexamethyl-
ene biguanide, silver) [5,6]. These dressings vary by
compounds content of antiseptics and dressing compo-
nent such as nylon, mesh, hydrocolloid or methylcellu-
lose. Silver as antimicrobial component is most occurring
metal of wound dressings materials. In our opinion alu-
minium hydrate is such antimicrobial component in a
dressing material which we investigated.
Nonwoven fabric of 1 - 3 μm diameter has a more de-
veloped surface and a more porous structure than materi-
als of thicker fiber, which renders this fabric the ability to
absorb quicker and keep the absorbed liquid better. We
have produced a nonwoven polymeric fibrous bandaging
material (NPFBM). This material can absorb and retain
the absorbed liquid due to an additional introduction of
porous particles of aluminum hydrate into the fabric.
Moreover, the aluminum hydrate particles produced from
electroexplosive aluminium nanopowders have a high
sorption capacity due to their hydrophilic properties and
a high specific surface area. In the course of previously
conducted research, we have been discovered the
NPFBM’s wound healing and antibacterial properties. In
this research, the non-organic nanofibers of aluminium
oxide-hydroxide phases were applied on polymeric ace-
tylcellulose fibers. It was discovered that this material re-

A Study of the Cytotoxicity of a New Nonwoven Polymeric Fibrous Bandaging Material in Vitro 235
duces the wound healing period and significantly accel-
erates sanitation of infected injuries, influencing posi-
tively the angiogenesis process, making positive influ-
ence on regeneration processes in damaged tissues [7]. It
hadn’t toxic effect on experimental animals [7]. In this
connection, the purpose of the current research is the
examination of compliance of the studied material with
international standards for medical goods and investiga-
tion of new bandaging material cytotoxicity using in vi-
tro methods [8].
2. Materials and Methods
2.1. Sample Preparation and Its Characteristics
The nonwoven polymeric fibrous bandaging material
(NPFBM) is produced on the basis of nonwoven mi-
crofibrous fabric made of a biologically inert poly-
mer—cellulose acetate—by means of electroforming in
the Institute of Physics of Strength and Material Science,
the Siberian Branch of the Russian Academy of Sciences,
Tomsk. The fine particles of aluminum hydrate, derived
from electroexplosive aluminium nanopowders 0.2 - 5.0
nm in size, with specific surface area 100 - 250 m2/g and
50% - 95% porosity, are fixed on microfibers.
2.2. Cells and Cell Culture
Two mouse cell lines (Р-815, L-929) and one human cell
line (K-562) were used. The cells were cultured in a cul-
ture medium containing RPMI 1640 (Sigma), 10% fetal
bovine serum (Hyclone), 2 mM L-glutamine (Sigma), 10
mM HEPES (Serva), 0.05 mM 2-mercaptoethanol (Fluka)
and 50 μg/ml gentamycin (Sigma).
2.3. Direct Contact Cytotoxicity Assay [8]
The cell culture incubation was carried out in the direct
contact with the NPFBM. Samples were compared with
nulls and with positive and negative controls. As the
positive control, we used 0.1% and 1% phenol solution (a
highly purified material distilled with argon). We used
cotton gauze as the negative control (Marketing Au-
thorization Ministry of Public Health of Russia No.
01012005/12161-05; State Standard 1172-93). Cells
were incubated in a 24-well plate at a density of 2 × 105
cells/ml, at 37˚C (Costar, Corning Incorporated, Corning,
NY) for 24 h in a 5% CO2 humidified incubator (Sanyo).
The studied sample of the NPFBM was put in each well
so that it took 10% of the well bottom. At the end of the
incubation, the cells were collected and used for quanti-
tative and qualitative assessment of the NPFBM’s cyto-
toxity. For the qualitative assessment, it was used a vis-
ual cell examination with supravital staining (0.1% try-
pan blue). We counted the number of viable and dead
cells as well as the cells with deviations from the normal
morphology (vacuolization, nuclear disintegration, cy-
tolysis, and membrane integrity) by means of a light mi-
croscope. The results are presented as a percentage.
Moreover, the L929 cells were photographed using an
Olympus IX50 microscope, and the morphological
changes indicating cytotoxicity and cell growth charac-
teristics were evaluated. For the quantitative assessment
of the NPFBM effect on cells, a colorimetric assay (MTT
assay) was used as described earlier [9]. Briefly, 0.1 ml
of cell suspension were transferred to 96-well flatbot-
tomed tissue culture plates (Costar, Corning Incorporated,
Corning, NY), MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-
diphenyltetrazolium bromide, Serva) was added to each
well in a final concentration of 200 μg/ml. Cells were
incubated at 37˚C in 5% CO2 humidified incubator for 4
h. After that, MTT solution was removed and the insolu-
ble formazan crystals were dissolved in 0.1 ml dime-
thylsulfoxide (DMSO, Sigma), and the absorbance was
then determined at 550 nm, using a microplate reader
(LabSystems). The results are presented in optical-density
units.
2.4. Indirect Cytotoxicity Assay [8]
To assess cytotoxity in the indirect contact, material ex-
tracts were prepared in the following way: the NPFBM
(test sample) and cotton gauze (the negative control,
Marketing Authorization Ministry of Public Health of
Russia No. 01012005/12161-05; State Standard 1172-93)
were placed into aseptic conditions in a culture medium
with a material area (cm2) and culture medium (ml) ratio
of 6/1, and incubated at 37˚C in 5%.
CO2 humidified incubator for 72 h. After that, extrac-
tions were collected and plated 0.1 ml each in 96-well
flat-bottomed tissue culture plates (Costar, Corning In-
corporated, Corning, NY), containing cells at a density of
2 × 105 cells/well in 0.1ml of the culture medium. Cells
were cultured at 37˚C for 24 h in a 5% CO2 humidified
incubator (Sanyo). As the null control, we used a culture
medium which was influenced by the same conditions
and procedures as the material extracts. For the qualita-
tive assessment, we used a visual cell examination with
supravital staining (0.1% trypan blue), as well as the di-
rect contact. For the quantitative assessment of the ex-
tracts effect, 4 hours before the end of incubation, MTT
was added to each well (200 µg/ml), and an MTT assay
was carried out, as well as in direct contact.
2.5. Data Analysis
Statistical analysis was performed using nonparametric
Mann—Whitney test. Values in tables are presented as a
mean (arithmetic mean) and standard deviation.

A Study of the Cytotoxicity of a New Nonwoven Polymeric Fibrous Bandaging Material in Vitro 236
3. Results
3.1. Direct Contact Cytotoxicity
The visual examination has shown that the NPFBM cul-
tivation in a direct contact with cells does not cause any
increase of the amount of dead cells of the studied lines
(Table 1). Phenol displays a dose-dependent cytotoxicity
action. A direct contact of the NPFBM with cells for 24
hours (Table 2) did not cause any increase of the per-
centage of cells with deviations from the normal mor-
phology (vacuolization, nuclear disintegration, cytolysis,
and membrane integrity).
Phenol solutions caused significant changes in cells
morphology, the percentage of modified cells P-815 was
increasing along with the increasing of phenol concentra-
tion. The image of L929 cells after a 24-hour cultivation
with the NPFBM and phenol solutions are represented in
Figure 1.
The NPFBM (Figure 1(b)) showed a good biocom-
patibility, i.e. there was no cytotoxicity in relation to
L929 cells, when compared with intact, control cell cul-
ture (Figure 1(a)). As shown in Figure 1, phenol 0.1%
(Figure 1(с)) had a moderate toxic effect, while phenol
1% appeared moderately cytotoxic, causing changes in
the cell morphology and the decrease of the density of
viable cells (Figure 1(d)).
According to the MTT assay results, the NPFBM and
cotton gauze after a direct contact with cells for 24 hours
did not have any cytotoxic effect (Table 3). Phenol solu-
tions had a significant cytotoxic effect in both the applied
concentrations.
3.2. Indirect Cytotoxicity
The results of studying the NPFBM extracts effect on
cell viability are presented in Table 4. The NPFBM ex-
tract did not have any cytotoxic effect on any cell lines in
all the studied dilutions (1/2, 1/4, 1/8, 1/16). The cotton
gauze extract decreased the amount of viable P-815 cells
in ¼ dilutions. Phenol solutions decreased the percentage
Table 1. Cell viability (%) after a direct contact with the
NPFBM.
Cell line
Treatment
P-815 K-562
Medium alone (null control) 94.03 ± 10.77 97.88 ± 0.55
Cotton gauze (negative control) 93.78 ± 11.58 97.61 ± 0.71
NPFBM 96.98 ± 13.25 96.82 ± 1.57
Phenol 0.1% (positive control) 33.81 ± 12.62* 13.90 ± 1.46*
Phenol 1% (positive control) 7.75 ± 12.04* 7.06 ± 2.34*
Values are the mean ± S.D. (N = 4), *p < 0.05 versus null control.
Table 2. The number of cells with changes in cellular mor-
phology (%) after a direct contact with NPFBM.
Cell line
Treatment
P-815 K-562
Medium alone (null control) 19.37 ± 14.60 7.68 ± 1.47
Cotton gauze (negative control)23.73 ± 12.83 7.99 ± 2.25
NPFBM 16.57 ± 12.22 6.22 ± 1.61
Phenol 0.1% (positive control) 74.26 ± 13.69* 91.61 ± 3.27*
Phenol 1% (positive control) 97.35 ± 11.33* 98.33 ± 1.67*
Values are the mean ± S.D. (N = 4), *p < 0.05 versus null control.
(a) (b)
(c) (d)
Figure 1. The biocompatibility of the NPFBM in the L929
cells culture (direct contact), no staining, magnification 140 ×.
(a) Control cell culture; (b) NPFBM, an NPFBM fragment
and aluminum hydrate particles released from the NPFBM
are shown in the lower part of the picture; (c) Phenol
solution 0.1% and (d) Phenol solution 1%.
Table 3. The cytotoxicity of the NPFBM assessed by MTT
assays (optical density).
Cell line
Treatment
P-815 K-562
Medium alone (null control) 550 ± 17 395 ± 29
Cotton gauze (negative control) 493 ± 25 393 ± 11
NPFBM 517 ± 41 401 ± 16
Phenol 0.1% (positive control) 142 ± 15* 108 ± 6*
Phenol 1% (positive control) 83 ± 4* 90 ± 4*
Values are the mean ± S.D. (N = 5), * p < 0.05 versus null control.

A Study of the Cytotoxicity of a New Nonwoven Polymeric Fibrous Bandaging Material in Vitro 237
Table 4. Effects of the NPFBM extracts on cell viability
(%).
Cell line
Treatment
P-815 K-562
Medium alone (null control) 98.73 ± 0.58 97.64 ± 0.66
Cotton gauze extract 1/2 diluted 96.71 ± 0.65 95.83 ± 0.83
Cotton gauze extract 1/4 diluted 95.67 ± 0.26* 98.33 ± 0.83
Cotton gauze extract 1/8 diluted 96.61 ± 0.83 97.08 ± 0.62
Cotton gauze extract 1/16 diluted 97.61 ± 0.65 ND
NPFBM extract 1/2 diluted 97.17 ± 1.15 95.63 ± 0.68
NPFBM extract 1/4 diluted 96.25 ± 1.68 97.44 ± 0.52
NPFBM extract 1/8 diluted 97.72 ± 0.69 97.08 ± 1.25
NPFBM extract 1/16 diluted 97.45 ± 0.53 ND
Phenol 0.1% (positive control) 44.95 ± 11.31* 38.59 ± 7.45*
Phenol 1% (positive control) 14.01 ± 5.34* 4.46 ± 0.95*
Values are the mean ± S.D. (N = 4), ND – value not determined, *p < 0.05
versus null control.
of P-815 and K-562 viable cells, depending on the dos-
age.
The results of studying the extracts effect on cellular
morphology are presented in Table 5. The morphology
of cells cultivated with different dilutions of the studied
NPFBM extracts and cotton gauze did not change. Phe-
nol solutions increased the percentage of deviated cellu-
lar morphology.
The results of studying cytotoxic effects of the
NPFBM extracts in MTT assays are presented in Table
6.
The NPFBM and cotton gauze extracts did not have
any cytotoxic effect on P-815 and К-562 line cells. Sta-
tistically significant decrease in P-815 cell viability pre-
sented in Table 4 probably was not a true toxic effect. It
proves by absence of changes in other dilution. The cot-
ton gauze extract in ½ and ¼ dilution had a cytotoxic
effect on L-929 line cells. Phenol solutions had a clearly
marked cytotoxic effect on P-815 and К-562 line cells
both in high and in low concentrations.
4. Discussion
We have shown that the direct contact of the NPFBM
with cells during a 24-hour cultivation period did not
cause any changes in cellular morphology and did not
cause any increase of the amount of dead cells upon a
visual examination. МТТ assays did not reveal any cyto-
toxic effect of the NPFBM either. The cotton gauze used
as negative control showed the same results. Therefore,
Table 5. Effects of NPFBM extracts on the number of cell
with changes in morphology (%) (Х ± m).
Cell line
Treatment
P-815 K-562
Medium alone (null control) 11.61 ± 0.81 8.23 ± 1.36
Cotton gauze extract 1/2 diluted 8.61 ± 1.46 10.29 ± 2.23
Cotton gauze extract 1/4 diluted 9.33 ± 1.05 7.84 ± 0.63
Cotton gauze extract 1/8 diluted 6.48 ± 2.18 10.00 ± 1.67
Cotton gauze extract 1/16 diluted 7.66 ± 1.69 ND
NPFBM extract 1/2 diluted 10.83 ± 1.20 10.83 ± 2.50
NPFBM extract 1/4 diluted 11.33 ± 2.01 10.42 ± 1.46
NPFBM extract 1/8 diluted 8.12 ± 1.26 7.92 ± 2.71
NPFBM extract 1/16 diluted 6.11 ± 0.34 ND
Phenol 0.1% (positive control) 67.72 ± 9.66* 70.47 ± 4.23*
Phenol 1% (positive control) 88.52 ± 5.74* 98.79 ± 1.21*
Values are the mean ± S.D. (N = 4), ND – value not determined, *p < 0.05
versus null control.
Table 6. Cytotoxicity of NPFBM extracts assessed by MTT
assays (optical density).
Cell line
Treatment
P-815 K-562 L-929
Medium alone (null control) 891 ± 35 455 ± 29713 ± 29
Cotton gauze extract 1/2 diluted 783 ± 50 558 ± 45545 ± 65*
Cotton gauze extract 1/4 diluted 841 ± 16 492 ± 32577 ± 27*
Cotton gauze extract 1/8 diluted 858 ± 27 450 ± 13667 ± 23
Cotton gauze extract 1/16 diluted992 ± 27 ND 656 ± 32
NPFBM extract 1/2 diluted 981 ± 101 433 ± 26688 ± 49
NPFBM extract 1/4 diluted 1009 ± 49 454 ± 41762 ± 74
NPFBM extract 1/8 diluted 988 ± 35 401 ± 19667 ± 35
NPFBM extract 1/16 diluted 986 ± 28 ND 684 ± 34
Phenol 0.1% (positive control) 142 ± 15* 163 ± 17*ND
Phenol 1% (positive control) 83 ± 4* 97 ± 5*ND
Values are the mean ± S.D. (N = 4), ND – value not determined, *p < 0.05
versus null control.
the NPFBM has the same biological safety as the cotton
gauze used widely for injured tissues protection. The
NPFBM extract did not have any cytotoxic effect on the
tested cells either. However, cotton gauze extract in high
concentrations could have a harmful effect on tested cells,
although it did not influence their morphology much.

A Study of the Cytotoxicity of a New Nonwoven Polymeric Fibrous Bandaging Material in Vitro 238
This may be as a result of technological peculiarities of
cotton gauze processing which is carried out with the use
of some chemical agents. Residual quantities of these
agents could cause the negative effects observed. The new
bandaging material NPFBM does not have this kind of
drawback. In conclusion, the new nonwoven polymeric
fibrous bandaging material has a good biocompatibility
and does not have any cytotoxic effect after a direct con-
tact with the cell culture. Unlike cotton gauze, the
NPFBM does not contain any additives that could have
an adverse effect on the cell culture through extractive
additives. The obtained results allow us to draw a con-
clusion that the NPFBM complies with the ISO 10993-5,
which is applied to medical goods, and can from now on
be applied to treating infected wounds in clinical prac-
tice.
5. Conclusions
The present work demonstrates the results of study of the
NPFBM’s direct contact and indirect cytotoxicity assays
in comparison with cotton gauze according to the main
principles of international standard ISO 10993-5. It has
been shown that the NPFBM extract had not any cyto-
toxic effect on the tested cells. Thus, these NPFBM pro-
duced with the use of nanotechnology may be used for
health care purpose like safe and nontoxic wound healing
bandaging material.
REFERENCES
[1] T. D. Turner, “Hospital Usage of Absorbent Dressings,”
Pharmaceutical Journal, Vol. 222, 1979, pp. 421-424.
[2] V. J. Jones, “The Use of Gauze: Will It Ever Change?”
International Wound Journal, Vol. 3, No. 2, 2006, pp.
79-88.doi:10.1111/j.1742-4801.2006.00215.x
[3] X. Wang, Y. Yan and R. Zhang “A Comparison of Chi-
tosan and Collagen Sponges as Hemostatic Dressings,”
Journal of Bioactive and Compatible Polymers, Vol. 21,
No. 1, 2006, pp. 39-54.doi:10.1177/0883911506060201
[4] G. Chaby, P. Senet, M. Vaneau, et al. “Dressings for
Acute and Chronic Wounds,” Archives of Dermatology,
Vol. 143, No. 10, 2007, pp. 1297-1304.
doi:10.1001/archderm.143.10.1297
[5] T. S. Stashak, E. Farstvedt and A. Othic “Update on
Wound Dressings: Indications and Best Use,” Clinical
Techniques in Equine Practice, Vol. 3, No. 2, 2004, pp.
148-163. doi:10.1053/j.ctep.2004.08.006
[6] M. Ip, S. L. Lui, V. Poon, et al., “Antimicrobial Activities
of Silver Dressings: An in Vitro Comparison,” Journal of
Medical Microbiology, Vol. 55, No. 1, 2006, pp. 59-63.
doi:10.1099/jmm.0.46124-0
[7] A. M. Dygaj, M. I. Lerner, V. V. Novitsky, L. M. Ogo-
rodova, S. G. Psakhie and A. A. Churin “The Nonwoven
Material of Medical Appointment Possessing Wound
Healing, Antibacterial and Antiviral Activity and Bandag-
ing Material on its Basis,” Patent (RU) N 2397781 from
27.08.2010, (in Russian).
[8] ISO 10993-5, “Biological Evaluation of Medical Devices,
Part 5: Tests for Cytotoxicity, in Vitro Methods,” Interna-
tional Standardization Organisation, Geneva, 1992.
[9] T. R. Mosmann, “Rapid Colorimetric Assay for Cellular
Growth and Survival: Application to Proliferation and
Cytotoxicity Assays,” Journal of Immunological Methods,
Vol. 65, No. 1-2, 1983, pp. 55-63.
doi:10.1016/0022-1759(83)90303-4