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Culture Documents
DOI 10.1007/s00405-012-1934-6
OTOLOGY
Received: 26 July 2011 / Accepted: 12 January 2012 / Published online: 24 January 2012
Springer-Verlag 2012
Introduction
Tympanic membrane (TM) serves as a key component of
the tympano-ossicular system for sound transmission. TM
perforation is a common problem in otology practice and
can result from various causes such as trauma, infections of
the middle ear, and malignant tumors of the ear [1]. TM
perforation is a frequent cause of conductive hearing loss
and increases the risk for chronic infection. Rapid treatment for acute TM perforations is vital to enable the
growth of a new TM with normal histological characteristics [2]. Controversies of how best to treat fresh tympanic
membrane perforations have always existed. While some
otolaryngologists prefer the paper-patch method, others
prefer modified myringoplasty.
High rates of spontaneous healing following conservative management of traumatic TM perforation are reported
in many studies [35]. Spontaneous healing of larger perforations is more difficult, and surgery is required if
insufficient healing occurs. However, surgery involves
higher costs and surgical risks. Therefore, the use of substances that facilitate membrane regeneration has recently
been considered as an alternative to the surgical repair of
TM perforation [1, 2, 68].
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The purposes of this study were to investigate the biocompatibility of two different paper patches (carbon and
cigarette papers) and compare the adhesion and proliferation features of L929 fibroblast cells by using 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide
(MTT Test) test and scanning electron microscopy (SEM).
We think that to choose the more compatible patch at
cellular level among these two paper patches would
increase the efficiency and success of the treatment for the
traumatic TM perforations. To our knowledge, this is the
first study on comparing efficacy of two different paper
patches at cellular level with the MTT test and SEM.
In determining toxic effect by MTT, the leakage products solution was prepared by adding 10% FBS, 1% Lglutamine, and 1% penicillinstreptomycin into phenol
redfree DMEM as indicated in ISO 2009 protocol
10993-5 [911].
Collected leakage products were kept frozen at -20C
until cytotoxicity test. The cells grown in flasks were
detached from the surface as 1 9 105 cell/ml, transferred
to 96-well cell culture plates (100 ll/well), and incubated
for 24 h at 37C in a 5% CO2 incubator.
Plate bottoms were covered with cell, media were
removed, leakage products (100er ll) were placed, and
incubation was conducted for 24 h at 37C in a 5% CO2
incubator. After incubation, 5 mg/ml MTT solution (10 ll)
was added, incubation was conducted at 37C for 4 h and
in %5 CO2 incubator for 24 h, and 100 ll DMSO was
added. When formazan crystals were fully dissolved, microplates were shaken gently for 10 min at vertex until the
color changed from yellow to blue/purple. Optical density
of formazan was determined by ELISA [EL 312 microplate, Bio-Tek, Biokinetics Reader] reader at 490 nm
wavelength.
Scanning electron microscopy (SEM) evaluation
Cell culture
L929 mouse fibroblast cell culture was used in this study.
Preparation of the media used
The media used in cell culture were prepared by adding
1 ml of penicillinstreptomycin [PAA cat no., P11-010; lot
no., P01009-1013] and 10 ml of fetal bovine serum (FBS)
[Biological industries lot no., 715442] into 89 ml DMEM
[PAA cat no., E 15-806; lot no., E 80609-2477].
MTT test
By this test, toxic effects occurring as a result of direct
contact of leakage products obtained from tested materials
were determined.
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Results
Cytotoxicity test carried out by MTT analysis on leakage
products collected from two types of paper patches at the
end of 24 and 48 h revealed no cytotoxicity, and there was
no statistically significant difference between the patches
(P [ 0.05).
In SEM studies, it was observed that cells adhered to
and even started to spread over disk surfaces at the end of
24 h in both of the paper patches (Fig. 1).
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Discussion
In socioeconomically developing countries such as Turkey,
cigarette and carbon paper patches are commonly used in
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Conclusion
Moreover, this is the first study where biocompatibility of
carbon paper, which is not much preferred in developed
countries but a commonly used alternative to cigarette
paper in our Turkey in repair of TM perforations, is
studied.
As a result, biocompatibility of cigarette paper and also
whether cigarette paper was superior to carbon paper in cell
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86
None.
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