Collagen-immobilized patch for repairing small tympanic membrane

perforations: In vitro and in vivo assays

Mohammad Farhadi,1 Hamid Mirzadeh,2 Atefeh Solouk,2 Alimohamad Asghari,2
Maryam Jalessi,1 Hadi Ghanbari,1 Parin Yazdanifard1
1

ENT-Head and Neck Research Center and Department, Hazrat Rasoul Akram Hospital, Tehran University of Medical
Sciences, Tehran, Iran
2
Faculty of Polymer Engineering, Amirkabir University of Technology (Tehran Polytechnic), Tehran, Iran
Received 15 September 2011; accepted 6 October 2011
Published online 12 December 2011 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.a.33293
Abstract: Tympanic membrane (TM) perforation is still one
of the most common otology complications. New designs of
biomaterials, and lately tissue-engineered composites and
grafts, have thoroughly revolutionized the management of
TM perforation. In this study, we examined a biologically
modied collagen-immobilized polydimethyl siloxane patch
to repair TM perforation. In vitro potential of the aforementioned patch as a scaffold to support broblast cell growth
and adhesion was assessed. An in vivo assay of the patch for
initiating repair of TM perforations also was investigated.
In vitro assay showed that the patch has signicantly

increased cell adhesion and growth in comparison with

unmodied ones (p < 0.05). In vivo study also showed an
overall closure rate of TM perforation of 70% and an average
gain of 15.75 6 4.29 dB in air-bone gap. This study shows
that the preliminary in vivo evaluation of a modied siloxane patch in humans had promising results and is comparaC 2011 Wiley Periodicals,
ble to existing biomaterial patches. V
Inc. J Biomed Mater Res Part A: 100A: 549553, 2012.

Key Words: tympanic membrane perforation, myringoplasty,

collagen, polydimethyl siloxane

How to cite this article: Farhadi M, Mirzadeh H, Solouk A, Asghari A, Jalessi M, Ghanbari H, Yazdanifard P. 2012. Collagenimmobilized patch for repairing small tympanic membrane perforations: In vitro and in vivo assays. J Biomed Mater Res Part A
2012:100A:549553.

INTRODUCTION

Chronic tympanic membrane (TM) perforation due to otitis

media or trauma has long been regarded as the most common complication of ontological diseases. Longstanding TM
perforations may cause hearing loss and worsen middle ear
infection leading to chronic otitis media even if they are
small in size. Tympanoplasty is still the most effective
approach to repair TM perforations and subsequently repair
the sound-conducting mechanism. However, in recent decades, several studies have focused on type I tympanoplasty
reconstruction techniques using various graft materials
rather than expensive, complex, and high-risk operative
approaches with long hospitalization periods.1,2 Since 1878,
a variety of materials such as autologous and allografts,
paper patches, synthetic and bioabsorbable materials,
composite materials, and, lately, cell-seeded and tissueengineered grafts have been used for TM grafting.39 The
current concern is which composite materials and biomaterials are the best bases and scaffolds regarding high susceptibility to support cell adhesion, proliferation, and differentiation, as well as their ability to promote tissue repair
in vivo and consequently repair the perforation.6,1014 Poly-

dimethyl siloxane (PDMS) is one of these preferred biomaterial patches. PDMS-based elastomers seem to be a great
choice due to their physiological inertness, low toxicity and
modulus, and good mechanical properties.1416
Previously, we did some surface modications on PDMS
by creating acrylic acid (AAc) grafting using a two-step oxygen plasma treatment (TSPT) that was used to chemically
immobilize collagen type I via introduction of carboxylic
groups to the inert surface of PDMS with promising applications in ophthalmology.12,17,18 Despite the superior advantages of PDMS, it suffers from a lack of long-term biocompatibility due to low hydrophilicity and, consequently, a low
tendency for cell support. Conversely, it has been reported
that collagen has been used to prepare a suitable network
and base for cell adhesion.1416
In this study, collagen was immobilized onto the surface
of PDMS and the potential of this biologically modied
collagen-immobilized PDMS (CI-PDMS) as a tympanic patch
was evaluated both in vitro and in vivo. To the best of our
knowledge, there is no report in the literature dealing with
the potential application of CI-PDMS as a tympanic patch to
repair small TM perforations.

Correspondence to: H. Mirzadeh; e-mail: mirzadeh@aut.ac.ir

Contract grant sponsor: Iran National Science Foundation, Tehran, Iran (The Collagen-Immobilized Siloxane Patch Preparation)

C 2011 WILEY PERIODICALS, INC.

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549

plemented with 10% heat-inactivated fetal bovine serum

and 100 IU mL 1 streptomycin. A routine subculture was
used to maintain the cell line. The cells were incubated in a
humidied atmosphere of 5% CO2 at 37 C. After 1 week of
incubation, the monolayer was harvested by trypsinization.
The cell suspension of 4  106 cell mL 1 was prepared for
culture. The samples were placed into each well using a
multiwell plate with 5 mL of cell suspension and seeded,
keeping one well as a control without any sample, and then
maintained in an incubator for 48 h. The samples were
removed from the well, washed twice by phosphate buffer
saline solution, placed on a glass slide, xed with ethanol,
and then stained with Giemsa. The samples were examined
by a light microscope.

FIGURE 1. SEM cross-sectional micrograph of grafted collagen on the

CI-PDMS surface (1000). The thickness of the grafted layer is about
3 lm.

MATERIALS AND METHODS

The preparation of AAc grafts via modication of PDMS

patches using TSPT was according to our previously published works.17,18 In summary, the TSPT samples were pretreated with 60 W of oxygen plasma for the desired time.
Immersion of the plasma-treated PDMS in aqueous monomer solutions of AAc then was performed. The second step
was carried out with plasma copolymerization of preadsorbed reactive monomers on the surfaces of dried pretreated patches.
The modied patches (AAc grafted) then were utilized to
chemically immobilize collagen type I via introduction of the
carboxylic groups to the surface. The amount of immobilized
collagen was determined by Bradford protein assay, which
determined that the amount of immobilized collagen on the
surface was 32 lg/cm2.19 The thickness of the grafted layers
was observed using a scanning electron microscope (SEM),
Tescan Vega-II XMU (Tescan). The patches were mounted on
SEM stubs and coated by vapor deposition using a sputter
coater with a gold (Au) target to show conductance and
high-resolution imaging. The optimum parameters for SEM
imaging used an electron-accelerating voltage of 20 kV with
a working distance of about 16 mm. The presence and
thickness of the collagen-grafted layer was observed by
cross-sectional view of the SEM micrograph (Fig. 1).
In vitro assay
The CI-PDMS patch and untreated silicone patch as control
were immersed into sterilized saline for 12 h headed for
cell seeding. The mouse broblast cell (L929) was obtained
from our countrys National Cell Bank (Pasteur Institute).
The cells were maintained in medium consisting of Roswell
Park Memorial Institute (RPMI)-1640 growth medium, sup-

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FARHADI ET AL.

In vivo assay
Ten patients with longstanding TM perforations were admitted to the otology clinic and enrolled into the study after
considering the following inclusion criteria: presence of dry
central TM perforation not exceeding 5 mm in size; TM perforation present for at least 12 months; absence of ossicular
or mastoid pathology. The pure tone audiometry and
tympanometry along with otoscopic and endoscopic examinations were performed before and after the procedure.
Otoscopic and endoscopic ndings including perforation
size were documented by photography. All patients were
fully informed about the procedure and alternatives available such as the paper patch and conventional myringoplasty approaches, and informed consent was obtained from
all patients. This study was approved by our research centre
ethics committee at Tehran University of Medical Sciences.
The procedures were performed under topical anesthesia with 0.5% Tetracaine drop. Margins of perforations were
freshened microscopically under sterile condition. The graft
was tailored to be 2 mm larger than the perforation size in
each direction and used as an onlay graft with the treated
surface in contact with the ear drum. The graft was held in
place by small pieces of gel foam soaked in dexamethasone
(4 mg/cc). Oral antibiotic and decongestant were administered to the patients for 5 days. The postoperative visits
were on day 7, 14, 30, and 60. The follow up audiometry
was performed 2 months after the procedure.
RESULTS

Figure 2 shows optical photomicrographs of L-929 broblast cell behavior on the CI-PDMS patches and untreated
samples. It conrms that the number of attached cells on
the modied PDMS patches is signicantly higher than the
numbers on the nonmodied PDMS (p < 0.05).
CI-PDMS patches were used in 10 cases with TM perforations. Complete closure of the perforation was achieved in
a total of six out of 10 patients (60%) after one attempt,
which was documented with tympanometry. In one of the
four unsuccessful cases, regrafting was performed, which
led to complete closure after 1 month. Considering this
second attempt, overall closure rate reached 70%. Gradual
repair of TM perforation and successful regeneration of TM
are shown in Figures 3 and 4.

COLLAGEN-IMMOBILIZED PATCH FOR MYRINGOPLASTY

ORIGINAL ARTICLE

FIGURE 2. L929 cell adhesion, spreading, and growth after 48 h: (A) unmodied PDMS and (B) CI-PDMS (400). [Color gure can be viewed in
the online issue, which is available at wileyonlinelibrary.com.]

In the four unsuccessful cases, displacement of graft

occurred due to fungal external otitis in one case, eustachian tube dysfunction and acute upper respiratory tract
infection in one case, and unknown causes of otorrhea in
two cases (Table I).
The fungal external otitis resolved with antifungal otic
drops and the patient was regrafted 1 month later. Tympanometry conrmed complete closure of the perforation
2 months after the second attempt.
We encountered no other complications such as hematoma, laceration, or hemorrhage. As the only performed procedure was freshening of the perforation margin, there was
no pain for the patients. Procedure time was less than
15 min in all the cases.
The audiometric results were compared before and
2 months after the procedure and revealed an average gain
of 15.75 6 4.29 dB in air-bone gap.
DISCUSSION

In the past century, a wide range of biomaterials have been

used to make more suitable grafts for TM repair and sound
conduction mechanism restoration. Although the closure
rate of these grafts such as autografts, allografts, synthetic,
and biomaterial grafts have been reported of up to 8090%,

none yet has superiority for repair of all types of perforations due to its own disadvantages.3,610,2023 For instance,
TM closure rate of autologous grafts has been reported of
about 8097% for temporalis fascia, 7197% for perichondrium and cartilage, and 86% for fat, which are the most
commonly used autografts followed by vein, dura mater,
periostum, subcutaneous tissue, and skin.1,8,20,2427 However, preparation of these autologous grafts needs an extra
surgical procedure that imposes surgical and anesthetic
risks to the patients along with longer procedure times and
subsequent scar tissue on the skin.
Allografts like acellular dermis or dura with acceptable
closure rates of 7895% also have their own disadvantages
such as requiring decellularizing procedures to reduce immunogenicity and screening for a wide range of viral or
other contagious infections.8,28
Synthetic paper patches have less closure rates than
allografts and autografts, about 6668%, but are favorable
due to their uncomplicated grafting procedure with no need
for general anesthesia, less procedure time, pain, and infection rate.7,20,28
Currently, scientists are more interested in biomaterials
such as silk, chitosan, and collagen-modied polymers for
use as TM patches. As the in vitro studies revealed, they

FIGURE 3. Closure of perforation with gradual extrusion of the graft: (A) Preoperative and (B) postoperative. [Color gure can be viewed in the
online issue, which is available at wileyonlinelibrary.com.]

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551

have every single aforementioned advantages of all types

of grafts along with high recorded closure rates of 93%
for silk and 89.5% for chitosan and high ability to
initiate the patients own broblasts for growth and
proliferation.7,23,25,29
In addition, these materials are comparable with each
other due to their production process difculties and costs
along with their ability to support broblast adhesion and
proliferation and subsequent complete repair of the TM perforation.3,69,2023
In this study, CI-PDMS patches were valuated both
in vitro and in vivo. To the best of our knowledge, there is
no report in the literature concerning an in vivo study evaluating CI-PDMS as a TM patch in humans.
Figure 1 clearly indicated that the grafted layer was restricted to the surface region of the patch with a thickness
of about 3 lm. The cell survival and growth on the bottom
of the wells with spindle-shaped morphology also was
observed, which was a sign of no toxic intergradient in CIPDMS and also its biocompatibility. Results showed that cell
adhesion was remarkably higher on CI-PDMS patches compared to cells that were in touch with untreated ones
(Fig. 2). It suggested that collagen, as main component of
extracellular matrix proteins, was benecial to the attachment of broblast cells.30 Other than adhesion, growth and
proliferation of cells toward our patch, its good manipulation properties, easy adhesion to TM surfaces, full transparency, which could let surgeons observe the edges of the
margins during surgery, and allowing the possibility of
following up the healing processes of the perforations were
other advantages of CI-PDMS patches. This in vitro evaluation showed the biocompatibility of CI-PDMS and its suitability as a substrate for cell growth.30
In in vivo study, the overall complete closure of small
TM perforations in seven out of 10 cases (70%) and an
average gain of 15.75 6 4.29 dB in air-bone gap were
observed. It shows that CI-PDMS can serve as a suitable
substrate, allowing the patients broblasts to grow and
proliferate on the graft.

FIGURE 4. Complete closure of perforation; the graft is on the canal

wall. [Color gure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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FARHADI ET AL.

TABLE I. In Vivo Assay: Outcome of CI-PDMS Grafting

Characteristics
Closure rate
One attempt
Overall rate
Air-bone gap gain in audiometrya
Procedure time
Complications
Otorrhea
Hematoma
Laceration
Hemorrhage
Otitis extern
Otomycosis

6/10 (60%)
7/10 (70%)
15.75 6 4.29 dB
<15 min in all cases
3/10 (30%)
0
0
0
1 (10%)
0

a
The mean air-bone gap gain between before and after procedure
pure tone audiometry.

The CI-PDMS showed a closure rate of 70%, which

seems to be acceptable in comparison to other biomaterials
such as silk (93%) and chitosan (89.5%) for the rst clinical
attempt considering the small number of cases with no
adjustment for the other demonstrated predictive factors of
successful closure of TM perforations (e.g., patients age,
perforation size and location, onlay or underlay approaches,
mucosal inammation, and concomitant contralateral ear
pathology).7,21,27,3133
In conclusion, the rst attempt of in vivo evaluation of
CI-PDMS as a TM patch in human ear seems to be effective
and practical for TM repair. Further clinical studies with
larger numbers of patients and adjustment of other relevant
factors such as different methods of grafting to indicate
whether CI-PDMS has the potential to compete with other
existing biomaterials such as silk to repair TM perforations
is required.
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