Indian Journal of Forensic Medicine & Toxicology, April-June 2021, Vol. 15, No.

2 3379

Experimental Study of Collagen Density in Reconstruction of

Tracheal Defect Using Dried Amniotic Membrane

Sahudi Abdul Mujib1, Sonny Seputra2

1
SMF/Lab Teaching Staff Head and Neck Surgical Division – Department of Surgery, Dr. Soetomo Genetral
Hospital, Universitas Airlangga, Surabaya, Indonesia, 2 Participants in the Specialist Education Program of
Department of Surgery, Dr.Soetomo General Hospital, Universitas Airlangga, Surabaya, Indonesia

Abstract
Background: Current tracheal defect management is still controversial for surgeons because there are
no ideal materials to substitute trachea and many long-term complications occur in patients after tracheal
reconstruction. Prosthetic graft has the risks of chronic infection and post-reconstructive tracheal stenosis.
Dried Amniotic Membrane (DAM) expresses several endogenous matrix metalloproteinase (MMP) genes
that can degrade extracellular matrix. Therefore, DAM is expected to be able to degrade collagen formed
excessively during the wound healing phase, hence post-reconstruction stenosis will not occur. This study
aimed to evaluate the differences of collagen density between polypropylene mesh (only) and DAM-added
polypropylene mesh as graft in tracheal reconstruction.

Methods: The animals used in this study were 36 male white rabbits, which were divided into two
groups. Both groups were wounded in the trachea area. The defects from the first group were closed using
polypropylene mesh, while the defects from the second group were closed using DAM-added polypropylene
mesh. After 30 days, the collagen density of each group was assessed.

Results: In the first group there are 10 samples (55.56%) with collagen density score = 3, while in the second
group there are 16 samples (88.89%) with collagen density score = 2. The significance value using Mann-
Whitney U test for differences in the collagen density score in both groups is p = 0.407.

Conclusion: Tracheal reconstruction using polypropylene mesh along with DAM provides a lower collagen
density score than using polypropylene mesh alone as a graft, but it is not statistically significant.

Keywords: Collagen density; Dried Amniotic Membrane; Tracheal reconstruction

Introduction surgeons. In cases of defects that require a small size

Tracheal reconstruction due to several pathological
abnormalities, such as trauma, neoplasia, and other
abnormalities is still a problem and challenge for
Corresponding Author
Sahudi Abdul Mujib
Head and Neck Surgical Division, Department of
Surgery, Faculty of Medicine, Universitas Airlangga/
Dr. Soetomo General Hospital, Jl. Mayjen Prof. Dr.
Moestopo 47, Surabaya, 60132, Indonesia
Tel: +62 813-3242-3302
E-mail: sahudisurg@yahoo.co.id
closure, primary anastomosis can be done. While in
the case of larger defects (more than 50% area around
the trachea), the surgeons often need to perform
tracheal reconstruction.1 Some techniques in tracheal
reconstruction are the use of stents, prosthesis or
implants, autograft, allograft, and tissue engineering.2 A
variety of prosthetic grafts and tissues have been used
to close the tracheal defect, but the success rate was still
low. Ischemic grafts, immunorejection of tissue grafts,
and stenosis in prosthetic grafts are the main reasons for
the failure.3,4
3380 Indian Journal of Forensic Medicine & Toxicology, April-June 2021, Vol. 15, No. 2
Dried Amnion Membrane (DAM) has long been
considered a potential source of scaffold material. DAM
has been proven to have many uses in the treatment of
burns, oral cavity, bladder and vaginal reconstruction,
tympanoplasty, arthroplasty, and many more.5 DAM
also has the advantage of reducing the formation of
scar tissue and inflammation, accelerating the healing
time of wounds, and functioning as a scaffold for cell
proliferation and differentiatio.6 Specifically, the human
amniotic membrane expresses several endogenous
matrix metalloproteinase (MMP) genes that function in
the degradation of extracellular matrix.7 This gave rise
to the idea that human DAM has unique characteristics
that make it suitable when added to polypropylene mesh
prosthetic graft. The addition of DAM in the prosthetic
graft is expected to reduce collagen density to decrease
the risk of post-reconstruction stenosis.
Based on the above thought, this study aimed to
compare collagen density in tracheal defects treated with
DAM-added polypropylene mesh and tracheal defect
treated with only polypropylene mesh.
Methods
Research Design
This research was an experimental laboratory study,
using a randomized control trial design conducted
in Animal Laboratory, Department of Biochemistry,
Faculty of Medicine, Universitas Airlangga. Thirty-
six New Zealand male white rabbits aged 6 to 9 weeks
with bodyweight between 1900-2500 grams were
obtained and the protocol was approved by Animal
Care and Use Committee from Faculty of Veterinary,
Universitas Airlangga, Indonesia (certificate number:
2.KE.072.04.2018). Rabbits were acclimatized for 7
days, then divided into control and treatment groups
and permuted block randomization was applied. In the
control group, the tracheal wounds of the rabbits were
covered with polypropylene mesh, while in the treatment
group, the wounds were covered with polypropylene
mesh added with dried amniotic membrane (DAM).
After 30 days, collagen density was examined in the
area of the surgical wound. This animal experiment had
complied with the ARRIVE guidelines.8
Manufacture of Dried Amniotic Membrane
(DAM)
Fresh amniotic membrane was taken from the
mother who had elective cesarean delivery by using
aseptic instruments and techniques. The amniotic
membrane was washed with saline containing 50 μg/
ml streptomycin, 50 μg/ml penicillin, and the inside part
was bluntly separated from the chorion. After that, the
amniotic membrane was immersed in the transport media
and stored at 40C before being extracted within the next
24 hours. Then the extracted amniotic membrane was
sent to the laboratory in a sterile tube with dry ice.
The amniotic membrane was dried on drying paper,
then weighed and measured in wet weight and volume.
After that, it was moved into a deep freezer for 24-48
hours then it was freeze-dried for 7-8 hours until the
drying is optimal. In the end, the amniotic membrane
was packed through laminar airflow and chemically
sterilized using gamma light radiation (25kGy). The
DAM used in this study is the freeze-dried amniotic
membrane that was available in tissue bank of Dr.
Soetomo Surabaya Hospital, Indonesia. DAM with a
thickness of 6 folds with a size of 0.6 x 0.6 cm2 was
used, based on the previous studies.
Sample Preparation and Surgical Procedure
A total of 36 New Zealand male white rabbits aged 6
to 9 weeks with bodyweight between 1900-2500 grams
were acclimatized for 7 days. Then the randomization
was done by permuted block randomization into two
groups, namely the treatment group of 18 rabbits and
the control group of 18 rabbits. After being acclimatized
for 7 days, the rabbits were given treatment under the
influence of general anesthesia using intramuscular
ketamine at a dose of 20-40 mg/kg body weight. The
front neck’s fur of each rabbit was shaved and disinfected
using povidone iodine 10%. All rabbits from both groups
(control and treatment groups) were wounded on the 8th
ring of trachea an area of 0.6 x 0.6 cm2 using a scalpel
number 15. In the control group, tracheal defect was
closed with a 0.6 x 0.6 cm2 size polypropylene mesh
with simple interrupted sutures using 6-0 nylon thread.
In the treatment group, tracheal defect was closed with
a 0.6 x 0.6 cm2 polypropylene mesh added with DAM.
The area of DAM was of 0.6 x 0.6 cm2 and consisted of
6 layers, 3 layers above the polypropylene mesh and 3
 Indian Journal of Forensic Medicine & Toxicology, April-June 2021, Vol. 15, No. 2
layers under the polypropylene mesh. DAM was added
and then sewn with simple interrupted stitches 6-0 nylon
thread. (Figure 4.3). Daily antibiotics (penicillin 50,000
units/kg) intramuscularly were given after the surgery
for five days.
Specimens were taken from the area of 1 cm
cranio-caudal tracheal surgery involving healthy (0.5
cm) and pathological (0.5 cm) visible tissue, 0.5 cm
medio-lateral width, covering the entire thickness of the
trachea. Then the specimens were put in a 10% neutral
buffered formalin solution and sent for paraffin blocks
and histopathological examination.
Collagen Density Evaluation
Histopathology evaluation was performed using
Masson’s trichrome staining and collagen density was
assessed with a binocular light microscope (Olympus
CX 14) with 100x magnification for 1 field of view.
Collagen density was assessed using scoring:
Score 0: There are no collagen fibers in the wound
area
Score 1: The density of collagen fibers in the wound
area is low
Score 2: The density of collagen fibers in the wound
area is moderate
Score 3: The density of collagen fibers in the wound
area is tight
Score 4: The density of collagen fibers in the wound
area is very tight
Statistical Analysis
The data taken was in the form of ordinal data and
the control group was compared with the treatment
group using Mann-Whitney U nonparametric test.
Results
Collagen Density
3381
Collagen density examination was evaluated on the
30th day after surgery. Collagen density was assessed
based on the collagen density score. The highest score
in the control group (polypropylene mesh) was a score
of 3 with 10 samples (55.56%), followed by a score of
1 with 4 samples (22.22%), a score of 2 as many as 2
samples (11.11%), and a score of 0 as much as 2 samples
(11.11%). While the highest score in the treatment group
(DAM-added polypropylene mesh) was a score of 2 with
16 samples (88.89%), followed by a score of 3 with 2
samples (11.11%) (Table 1).
The lowest collagen density score (score 0) with
Masson’s trichrome staining at 100x magnification is
shown in Figure 2, score 1 is shown in Figure 3, score 2
is shown in Figure 4, and score 3 is shown in Figure 5.
In this study, the collagen density difference test
described by the collagen density score was carried
out using the Mann-Whitney U test because the type of
collagen density score data is ordinal data. The result
of the Mann-Whitney U test was p> 0.05 (0.407),
indicating that the difference of collagen density in the
control group (polypropylene mesh only) compared to
the treatment group (DAM-added polypropylene mesh)
was not statistically significant. Based on these results
it was concluded that the DAM-added polypropylene
mesh group had a lower collagen density than the
polypropylene mesh group, but it was not significantly
different.
In this study, the characteristics of the research
sample could influence the results of the study.
Therefore, the study sample was made as homogeneous
as possible to avoid bias. The experimental animals were
selected from the same place (Pusat Veterinaria Farma)
and given the same food. The statistical tests T-test on
body weight and the Mann-Whitney U test on the age
of rabbits gave the results that the weight and age of the
rabbits, although varied but still homogeneous, so there
was no bias from differences in body weight and age of
the rabbits.
3382 Indian Journal of Forensic Medicine & Toxicology, April-June 2021, Vol. 15, No. 2

Figure 1. Rabbit’s treatment procedure. (A) The rabbit’s neck area was shaved clean. (B) Tissue dissection
was performed until the trachea appeared. (C) The tracheal defect was made of size 0.6 x 0.6 cm2 by taking
the entire thickness of the trachea. (D) Polypropylene mesh and DAM sized 0.6 x 0.6 cm2 that had been put
together were added and sutured to cover the tracheal defect with 4 stitches of simple interrupted sutures
using 6-0 nylon thread (treatment group). (E) Polypropylene mesh was sutured to cover the tracheal defect
with 4 stitches of simple interrupted suture using 6-0 nylon thread (control group). (F) The skin was sutured
with simple interrupted sutures using 3-0 nylon thread.

Figure 2. Score 0 of collagen density. Masson’s trichrome staining (100x magnification).

 Indian Journal of Forensic Medicine & Toxicology, April-June 2021, Vol. 15, No. 2 3383

Figure 3. Score 1 of collagen density. Masson’s trichrome staining (100x magnification).

Figure 4. Score 2 of collagen density. Masson’s trichrome staining (100x magnification).

3384 Indian Journal of Forensic Medicine & Toxicology, April-June 2021, Vol. 15, No. 2

Figure 5. Score 3 of collagen density. Masson’s trichrome staining (100x magnification).

Table 1. Collagen density score

Score 0 Score 1 Score 2 Score 3 Mann-Whitney U Test

2 4 2 10
Control
(11.11%) (22.22%) (11.11%) (55.56%)
p = 0,407
0 0 16 2
Treatment
(0%) (0%) (88.89%) (11.11%)

Discussion the ideal material for tracheal defect reconstruction.2

Tracheal defect reconstruction is considered to be
optimally successful when it fulfills 3 components: the
tracheal lumen must be kept open properly (not stenosis),
the inside of the trachea is protected by the epithelium,
and the trachea has good vascularity.9 Reconstruction of
tracheal defects using polypropylene mesh was carried
out by Furneaux in 1973 on a 7-year old Dachshund dog.
Based on the results of this study, complications were
obtained in the form of cough 2 weeks after surgery.10
In another publication, Haykal et al. reviewed the results
of several studies and concluded that the closure of
tracheal defects using prosthetic grafts in the form of
polypropylene mesh and tissue grafts still showed post-
reconstruction stenosis complication and therefore is not
According to Fernandes et al, DAM was first
used as a skin transplant in 1910. In its development,
DAM was found to have many uses, such as treating
burns, reconstruction of the oral cavity, bladder, and
vagina, tympanoplasty, arthroplasty, and many more.11
According to Diaz-Prado et al, DAM has advantages
including its ability to reduce scar tissue formation and
inflammation, its ability to accelerate healing time, and
its function as a scaffold for cells’ proliferation and
differentiation.6 Moreover, according to Niknejad et al,
besides its anti-inflammatory effect, amniotic membrane
has low or no immunogenicity. Amniotic membrane is
also able to stimulate wound healing and epithelialization.
Its anti-inflammatory effect works by suppressing pro-
 Indian Journal of Forensic Medicine & Toxicology, April-June 2021, Vol. 15, No. 2
inflammatory cytokines IL-1α and IL-β. It also produces
a number of natural matrix metalloproteinases (MMP)
for extracellular matrix degradation.5
Wound healing in the trachea follows the general
wound healing phases, starting from the hemostasis
phase to the maturation phase. Collagen synthesis begins
on the 3rd day after injury and progresses rapidly around
the 2nd to 4th week. Collagen synthesis is controlled by
collagenase and other factors that destroy collagen.12
Meanwhile, according to Velnar, collagen remodeling
during the maturation phase depends on the progress
of collagen synthesis and the presence of collagen
degradation. Collagenases and metalloproteinases in
the wound remove excess collagen while the newly
synthesized collagen remains. During remodeling,
collagen becomes better organized.13
In this study, collagen density evaluation was
carried out on the 30th day after treatment, when
sufficient collagen was expected to be formed and
collagen remodeling was expected to occur in the
maturation phase. The collagen density was evaluated
histopathologically using Masson’s trichrome staining.
According to Suh et al., in their study, a prosthetic
graft on the trachea in the form of polypropylene mesh
stimulates the formation of granulation tissue and
fibrosis, causing post-reconstruction tracheal stenosis.4
Meanwhile, Fortunato and Menon showed that amniotic
membrane expresses several MMPs which are proven
to cause premature rupture of membranes in obstetric
cases. Amniotic membrane expresses MMP-1 and-13
(collagenases),7 therefore it is expected that the addition
of DAM to polypropylene mesh will reduce collagen
density in reconstructed tracheal defects.
In this study, the collagen density score in the DAM-
added polypropylene mesh group was found lower than
the collagen density score in the polypropylene mesh
(only) group. However, in the statistical test using
the Mann-Whitney U test, the difference between the
two groups was not statistically significant. So it was
concluded that tracheal reconstruction with DAM-added
polypropylene mesh had lower collagen density than
reconstruction with polypropylene mesh alone, but it
was not significantly different. This may occur due to
the time factor for evaluating the collagen density. In this
study, an evaluation of collagen density was carried out
3385
on the 30th day after reconstruction, whereas according
to Orsted et al, the wound remodeling phase occurred
on days 21 to 2 years after reconstruction. Collagen
degradation by collagenase will be controlled by TIMP
(Tissue Inhibitor of Metalloproteases) until it reaches an
equilibrium point.14
In humans, the incidence of post-tracheal
reconstruction stenosis occurs from 6 months to 3
years after tracheal reconstruction.15 Therefore, further
collagen degradation can still occur after 30 days after
reconstruction. The reduction in collagen density could
be statistically more significant at evaluation after 30
days post-reconstruction.
Ethical Clearance: Taken drom Animal Care and
Use Committee from Faculty of Veterinary, Universitas
Airlangga, Indonesia.
Source of Funding: Self-funding.
Conflict of Interest: Nil.
References
1. Ten Hallers EJ, Rakhorst G, Marres HA, et al.
Animal models for tracheal research. Biomaterials.
2004;25(9):1533-43.
2. Haykal S, Salna M, Waddell TK, Hofer SO.
Advances in tracheal reconstruction. Plast Reconstr
Surg Glob Open. 2014;2(7):e178.
3. Shi H, Xu Z, Qin X, Zhao X, Lu D. Experimental
Study of Replacing Circumferential Tracheal
Defects With New Prosthesis. Ann Thorac Surg.
2005;79(2):672–6.
4. Suh SW, Kim J, Baek CH, Han J, Kim H.
Replacement of a tracheal defect with autogenous
mucosa lined tracheal prosthesis made from
polypropylene mesh. ASAIO J. 2001;47(5):496-
500.
5. Niknejad H, Peirovi H, Jorjani M, Ahmadiani
A, Ghanavi J, Seifalian AM. Properties of the
amniotic membrane for potential use in tissue
engineering. Eur Cell Mater. 2008;15:88-99.
6. Díaz-Prado S, Muiños-López E, Hermida-Gómez T,
et al. Human amniotic membrane as an alternative
source of stem cells for regenerative medicine.
Differentiation. 2011;81(3):162-71.
7. Fortunato SJ, Menon R. Screening of novel
matrix metalloproteinases (MMPs) in human
3386 Indian Journal of Forensic Medicine & Toxicology, April-June 2021, Vol. 15, No. 2
fetal membranes. J Assist Reprod
2002;19(10):483-6.
8. Kilkenny C, Browne W, Cuthill IC, Emerson
M, Altman DG; NC3Rs Reporting Guidelines
Working Group. Animal research: reporting in
vivo experiments: the ARRIVE guidelines. Br J
Pharmacol. 2010;160(7):1577-9.
9. Janssen LM, van Osch GJ, Li JP, et al. Tracheal
reconstruction: mucosal survival on porous
titanium. Arch Otolaryngol Head Neck Surg.
2009;135(5):472-8.
10. Furneaux RW. Tracheal reconstruction with knitted
polypropylene mesh in a Dachshund dog. J Small
Anim Pract. 1973;14(10):619-24.
11. Fernandes M, Sridhar MS, Sangwan VS, Rao GN.
Amniotic membrane transplantation for ocular
surface reconstruction. Cornea. 2005;24(6):643-
53.
Genet. 12. Werner S, Grose R. Regulation of wound healing
by growth factors and cytokines. Physiol Rev.
2003;83(3):835-70.
13. Velnar T, Bailey T, Smrkolj V. The wound healing
process: an overview of the cellular and molecular
mechanisms. J Int Med Res. 2009;37(5):1528-42.
14. Orsted H, Keast D, Forest-Lalande L, Megie MF.
Basic Principles of Wound Healing. Wound Care
Canada. 2011;9(2):4-12.
15. Sarper A, Ayten A, Eser I, Ozbudak O, Demircan
A. Tracheal stenosis aftertracheostomy or
intubation: review with special regard to cause and
management. Tex Heart Inst J. 2005;32(2):154-8.