Eur Arch Otorhinolaryngol (2013) 270:8186

DOI 10.1007/s00405-012-1934-6

OTOLOGY

Biocompatibility evaluation of cigarette and carbon papers used

in repair of traumatic tympanic membrane perforations:
experimental study
Emine Elif Altuntas Zeynep Sumer

Received: 26 July 2011 / Accepted: 12 January 2012 / Published online: 24 January 2012
Springer-Verlag 2012

Abstract 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-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT Test)
test and scanning electron microscopy (SEM). In this study,
time-dependent cytotoxic effects of cigarette and carbon
papers used in repairing small traumatic TM perforations
were investigated in vitro by using MTT test. And also
adhesion and spreading of cells over disk surface were
observed by SEM. 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 (P [ 0.05). In SEM studies, it was observed that
cells started to proliferate over disk surface as a result of 48-h
incubation, and SEM revealed that the cell proliferation over
cigarette paper was more compared to the one over carbon
paper. We believe that this is the first study where biocompatibility and adhesion features of carbon and cigarette paper
have been studied by using L929 fibroblast cell culture. As a
result, biocompatibility of cigarette paper and also whether
cigarette paper was superior to carbon paper in cell
E. E. Altuntas
Department of Ear Nose and Throat Education and Research
Hospital, Faculty of Medicine, University of Cumhuriyet,
Sivas, Turkey
E. E. Altuntas (&)
Department of Otorhinolaryngology, Cumhuriyet University
School of Medicine, Sivas 58140, Turkey
e-mail: ealtunta@yahoo.com
Z. Sumer
Department of Microbiology, Faculty of Medicine,
University of Cumhuriyet, Sivas, Turkey
e-mail: drzeynepsumer@gmail.com

attachment and biocompatibility were studied. It was found,

by MTT test and SEM test, that cigarette paper had a higher
biocompatibility and cell attachment, and thus cigarette
paper should be the patch to be preferred in cases where TM
perforations are repaired by paper-patch method.
Keywords Tympanic membrane
Traumatic perforation

Paper patches
Biocompatibility
MTT test
Scanning
electron microscopy

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.

Materials and methods

Cytotoxicity assessment of the paper patches
In this study, time-dependent cytotoxic effects of cigarette
and carbon papers used in repairing small traumatic TM
perforations were investigated in vitro by using MTT
test that measures mitochondrial succinic dehydrogenase
activity.

Eur Arch Otorhinolaryngol (2013) 270:8186

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

Preparation of study materials

The paper patches were made from cigarette and carbon
papers in the outpatient clinic and sterilized with ethylene
oxide gas.
Cigarette and carbon papers were cut into 1 9 1 cm2
pieces and put into sterile tubes. Then, 5 cc Dulbeccos
modified Eagles medium (DMEM) w/o phenol red was
added into the tubes, and leakage products were collected
after incubation at ?37C for 24 and 48 h.

Paper samples (1 9 1 cm2) were placed into cell culture

plates, and 1 ml (*1 9 105 cell/ml) of cell suspension was
added and kept at incubation for 24 and 48 h at ?38C in a
5% CO2 incubator. After incubation, samples were placed
into phosphate buffer containing 5% glutaraldehyde and
sent to Erciyes University Kosgeb Tekmer center. At this
center, adhesion and spreading of cells over disk surface
were observed by SEM [12].
Statistical analysis

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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SPSS 14.0 programs (SPSS Inc., Chicago, IL, USA) were

used to record and evaluate the results obtained. Mann
Whitney U test was used to evaluate the data, and error
level was set as 0.05.

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).

Eur Arch Otorhinolaryngol (2013) 270:8186

83

Fig. 1 Image of adhesion and spreading of cells over disk surface at

the end of 24 h in SEM evaluation: a Cigarette paper 5009
magnification, b Cigarette paper 2.00 KX magnification, c Cigarette

paper 3.50 KX d Carbon paper 5009 magnification, e Carbon paper

2.00 KX magnification, f Crabon paper 3.50 KX

Fig. 2 Image of spreading of cells over disk surface at the end of

48-h incubation in SEM evaluation: a Cigarette paper 500 9
magnification, b Cigarette paper 2.00 KX magnification, c Cigarette

paper 3.50 KX d Carbon paper 5009 magnification, e Carbon paper

2.00 KX magnification, f Carbon paper 3.50 KX

It was observed that cells started to proliferate over disk

surface as a result of 48-h incubation, and SEM revealed
that the cell proliferation over cigarette paper was more
compared to the one over carbon paper (Fig. 2).

Discussion
In socioeconomically developing countries such as Turkey,
cigarette and carbon paper patches are commonly used in

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the repair of traumatic tympanic membrane perforations

and small tympanic membrane perforations occurring as a
result of various reasons. In the literature, there are human
and animal studies comparing paper patches and other
alternative techniques and materials in the treatment for
tympanic membrane perforations. That paper patch is a
quick, inexpensive, and highly successful treatment with
minimal identifies potential complications and is accepted
by most patients and promotes early healing. That is the
common view in all these studies. However, to our
knowledge, there have been no studies comparing efficacy
of two different paper patches at cellular level. Our study is
the first on this.
Studies conducted in recent years show that mostly
cell culture test method is used in determining cytotoxicity and evaluating fibroblast attachment [13, 14]. Cell
culture test methods are preferred as they are not
affected by individual factors and allow for repeatability,
standardization of study conditions and parametric comparisons between materials [15]. Thus, the effect of two
different paper-patch materials on cytotoxicity and
fibroblast attachment was evaluated in vitro using L929
mouse fibroblast cell culture in this study.
Berthold [16] introduced the first surgical cure of TM
perforations in 1878 using full-thickness skin autografts
from the forearm. Heermann [17] performed successful
autografts for closure of TM perforations using temporalis
fascias in 1961. Currently, an autologous autograft
including muscle fascia or perichondrium is generally used
for treatment for TM perforations, with a success rate
between 88 and 97% in most studies [18, 19].
Several methods and techniques that could easily be
performed in outpatient clinics have been introduced as
alternatives to the surgical repair of TM perforations.
Several agents or factors, such as hyaluronic acid, epidermal growth factor (EGF), bFGF or pentoxifylline (Trental),
have been tried experimentally to promote the healing of
TM perforations [20, 21]. Controversies of how best to
treat fresh tympanic membrane perforations have always
existed. While some otolaryngologists prefer the paperpatch method, others prefer modified myringoplasty.
Blake [22] introduced the simple technique of a paperpatch graft that guides the migrating epithelium to a scaffold from the borders of the perforation. This technique is
still practiced for acute and traumatic perforations even
though the paper patch has the disadvantage of nonbiocompatibility, nontransparency, nonflexibility, and advantage much as ease of detachment and nonresistance to
infection [23, 24]. Kristensen et al. [23] and Chun et al.
[24] stated that paper patch had a disadvantage of nonbiocompatibility; however, our literature search did not
reveal any cell culture studies on biocompatibility of paper
batch. We believe that this is the first study where

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Eur Arch Otorhinolaryngol (2013) 270:8186

biocompatibility and adhesion features have been studied

by using L929 fibroblast cell culture.
Kim et al. [25] developed a new artificial eardrum patch
for healing TM perforations using water-insoluble chitosan
and investigated mechanical properties, biocompatibility,
and healing effects of this chitosan patch scaffold. Chitosan
patch scaffold was more effective than spontaneous healing
to repair traumatic TM perforations and SEM findings of
regenerated eardrums using chitosan patch scaffolds
showed thinner, smoother, and more compact tissues than
spontaneously healed eardrums. On the other hand, the
paper patch showed a very irregular and rough surface with
pores in this study. But in our study, when SEM images
were evaluated, it was observed that there was more
fibroblast attachment to cigarette paper than carbon paper.
We believe that this could be related to the fact that cigarette paper has a better biocompatibility and smoother
surface than the carbon paper.
Kim et al. [26] investigated the effects of a thin silk
patch, in comparison with the commonly used paper patch,
with regard to the healing of large TM perforations and
the subsequent healing time in rats. In this study, novel
tissue-engineered artificial eardrums were fabricated from
water-soluble chitosan, which is known to be a good
wound-healing biomaterial. The characteristics, cytotoxicity, and healing effects of several water-soluble chitosan
patches made using various concentrations of water-soluble
chitosan and glycerol were investigated. In Kim et al.s
[26] study, the silk patches seemed to have provided firmer
and longer-lasting support than paper patches during the
healing of acute TM perforations. Perforations spontaneously closed in 13 of 20 ears (65%) in controls and in 27 of
40 ears (67.5%) treated with paper patches, whereas in the
group treated with silk patches, perforations closed in 37 of
40 ears (92.5%). Both paper and silk patches improved the
healing rate compared with that in untreated ears. Furthermore, the healing times of the silk-patch and paperpatch groups were significantly different. In vivo studies
showed that the water-soluble chitosan patches were more
effective than spontaneous healing for the repair of traumatic TM perforations. They suggested that silk patches
may prove to be effective for repairing acute TM perforations in human patients in an outpatient clinical setting.
Spandow et al. [27] investigated two alternative methods of dressings for closure of the TM perforations. In this
study, an adhesive-coated hydrocolloid material was
compared with a conventional dressing of vaselineimpregnated rice paper patch after de-epithelialization of
the perforation border, and no significant difference was
demonstrated between the two types of dressings.
Camnitz and Bost [28] presented 50 consecutive
traumatic perforations seen over a 3-year period and
initially treated with paper tape patches applied in the

Eur Arch Otorhinolaryngol (2013) 270:8186

office under local anesthesia. The success rate was 92%.

Healing usually occurred within 23 weeks. They contended that this method of treatment was quick, inexpensive and highly successful, identified potential
complications, was accepted by most patients, and promoted early healing.
Kim et al. [29] reported a new healing method of TM
perforation by using three-dimensional (3D) porous chitosan scaffolds (3D chitosan scaffolds) as an alternative
method to surgical treatment or paper-patch graft. Kim
et al. [29] used various 3D chitosan scaffolds in this study,
and the structural characteristics, mechanical property, in
vitro biocompatibility, and healing effects of the 3D
chitosan scaffolds as an artificial TM in in vivo animal
studies were investigated. In in vivo animal studies, 3D
chitosan scaffold were able to migrate through the pores
and surfaces of TM cells, thus leading to more effective
TM regeneration than paper-patch technique. As a result,
these researchers stated they believed that the 3D chitosan
scaffold technique may be an optimal healing method used
in lieu of surgical tympanoplasty in certain cases to heal
perforated TMs.
Park et al. [6] investigated the effects of the steri-strips
patch (3 M) on the treatment for large TM perforations. In
total, 87 patients were analyzed in this study. The closure
rates of the perforations in the steri-strips patching, paper
patching, and observation groups were 93.3, 84.2, and
78.9%, respectively. There was no statistically significant
difference in tympanic membrane closure rate between the
3 groups. Overall, the steri-strips and paper patching
reduced the healing time compared with the observation
group, and steri-strips technique decreased the need for
repeat procedures in patients with large traumatic TM
perforations. However, the occurrence of otorrhea was
significantly more common in patients treated with the
steri-strips technique.
Kim et al. [2] investigated the effects of repairing large
TM perforations in rats with a thin silk patch compared
with the commonly used paper patch. The mean perforation
closure times were significantly shorter in silk-patch- and
paper-patch-treated ears than in the control animals. They
suggested that the silk patch might prove to be an effective
material for repairing TM perforations in human patients in
an outpatient clinical setting.
Golz et al. [30] designed a study to investigate results of
paper-patch myringoplasty in patients with chronic perforations in 2003. As paper patching is technically simple,
time saving, safe to perform, cost-effective, suitable as an
outpatient procedure, and has a good success rate, it should
be tried in perforations smaller than 5 mm before a patient
is referred for surgery. The closure ratio of paper patches
was 50% in cases of chronic perforations, showing the
limitation of this technique.

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Dursun et al. [31] investigated the effectiveness of

paper-patch, fat, and perichondrium myringoplasty in the
treatment for chronic tympanic membrane perforations
smaller than 3 mm. Closure rates of perforations in the
paper-patch, fat, and perichondrium myringoplasty groups
were 66.7, 86.7, and 86.7%, respectively. There were no
statistically significant differences in tympanic membrane
closure rates between techniques with regard to size.
Weber et al. [32] investigated the tissue-engineered
calcium alginate patches in the repair of chronic chinchilla tympanic membrane perforations. In the chinchilla
model, the alginate grafts demonstrated significantly
improved healing rates over both the untreated control
group (spontaneous repair) and the paper-patch group;
nine of 13 healed in the alginate group versus two of
nine healed in the paper-patch group versus one of 11
healed in the control group. They stated that calcium
alginate tympanic membrane perforation patches offered
a significant advantage in the repair of chronic perforations over traditional techniques in the chinchilla perforation model and might offer attractive opportunities in
the clinical setting.
Imamoglu et al. [8] investigated the recovery rate in
paper-patch and fat-plug myringoplasty in rats. They found
that the recovery rate for small perforations was 94.7% in
fat-plug myringoplasty, 94.4% in paper-patch myringoplasty, and 66.6% in control group (left for spontaneous
healing). The recovery rates in large perforations were
52.9, 56.2, and 26.6%, respectively. On the other hand, in
larger perforations, paper-patch or fat-plug myringoplasty
had not been found effective. As a result, these researchers
stated they believed that fat-plug or paper-patch myringoplasty could be suggested for reconstruction of small and
dry perforations of the tympanic membrane due to significant operational advantages.
In our study, when the differences between biocompatibility of cigarette and carbon papers and ability of L929
fibroblast cells to adhere to the surfaces of these paper
patches were evaluated, it was observed that cigarette
paper cells attached and spread better than carbon paper
cells. Moreover, it was studied whether these paper patches
had cytotoxic features.

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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Eur Arch Otorhinolaryngol (2013) 270:8186

attachment and biocompatibility were studied. It was

found, by MTT test and SEM test, that cigarette paper had
a higher biocompatibility and cell attachment, and thus
cigarette paper should be the patch to be preferred in cases
where TM perforations are repaired by paper-patch
method.
Acknowledgments The authors wish to warmly thank Professor
Haldun Sumer for her critical appraisal of the manuscript and statistical analysis.
Conflict of Interest

None.

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