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Running head:

Evaluation of Alkali-based Decellularization Protocol for Human Amniotic Membrane


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Evaluation of Alkali-based Decellularization Protocol for Human Amniotic Membrane


Decellularization Team
Technological Institute of the Philippines

ADVANCES ON DECELLULARIZATION METHODS

Table of Contents

Title Page.........1
Table of Contents.....2
Acknowledgment.....3
Abstract....4
Chapter I The Problem and its Background..........5
Background of the Study.....5
Objectives....7
Significance of the Study....8
Scope and Limitation...8
Chapter II Conceptual Framework.......9
Definition of Terms.9
Related Literature and Studies...11
Chapter III Methodology17
Appropriateness of Research Design.17
Pilot Study.17
Research Setting18
Instrumentation..18
Procedure...19
Ethical Considerations...20
Chapter IV - Presentation, Analysis, And Interpretation...21
Chapter V - Summary of Findings, Conclusions, And Recommendations...36
Summary36
Conclusion.37
Recommendations..39
Bibliography..40
Appendices43

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ACKNOWLEDGEMENT

This research is made possible through the help and support of everyone,
including our family, friends and faculties. We are grateful, first, to Our Lord for guiding
us and comforting us in finishing this research. We are also thankful to our instructor,
Engr. Jopeth Ramis, in supporting us, assisting us and imparting us his knowledge about
tissue engineering research.
We also expressed our gratitude to Chemical Engineering laboratory technicians
for their patience and for providing us laboratory apparatus we had used in the
experiment.
We are also thankful to the Lung Center of the Philippines and to UP Diliman
Chemical Engineering Department Laboratory for helping us and providing us apparatus
to obtain our results.
We are also grateful for the help of Safebirth Lying-in Clinic, East Avenue
Medical Center, and Dr. Jose Fabella Memorial Hospital for permitting us to get
placentas. Without them, this research was for nothing

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Abstract
Tissue engineering has shown advancement in the medical field in recent years.
One of the most promising biological materials is human amniotic membrane which is
used as a scaffold for cell proliferation and differentiation. The HAM undergoes the
decellularization process, where cells are being removed so as to grow only the desirable
cells. The study aims to determine the most suitable combination of procedures for the
decellularization process. The amnions harvested and pretreated with 0.25, 0.5, 0.75and
1M NaClO or PBS was decellularized by either soaking or swabbing using 0.25, 0.5, 0.75
and 1M NaOH. Cell count results showed that the combination of 0.25 NaClO
pretreatment and 0.75M NaOH soaking of amnion was the best procedure because it
removed the most number of cells.
After a series of experimentations, the optimum process was determined to be the
soaking of amnion pretreated with 0.25 NaClO with 0.75M NaOH because of significant
effect in removing the cells and preserve the growth factors in amnions as based on the
FTIR results. Also this method is cost-effective, could save time and resources.

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CHAPTER I
THE PROBLEM AND ITS BACKGROUND

1.1 Background of the Study


Tissue engineering is a field wherein principles of engineering and biological
sciences are interconnected such that biomaterials are created, developed and produced to
improve, replace, or maintain the function of a particular organ or tissue [1].
The basic concept of tissue engineering includes the main strategy of producing
engineered tissue constructs which involve a scaffold structure that provides an
architecture upon which the seeded cells can grow and develop into the desired organ or
tissue being combined with growth factors prior to implantation [2] . The scaffold
provides an initial foundation for the cells to grow and develop until the formation of an
extracellular matrix. It is also necessary to consider the composition, topography, and
architecture of the scaffold since it greatly affects and modify the cell behavior and the
tissue formation [3].
The choice for the source of the cells that are suitable to produce the desired
tissue is also an important factor. A way to solve this is to use cells taken from the patient
in order to perform an autologous implantation, which reduces the risk of immunological
responses such as rejections and viral infections [2]. However, the collection of certain
cell types and the unhealthy state of cells could be a problem. One proposed approach to
solving this is through the use of stem cells. These stem cells are pluripotent, which
indicates its ability to differentiate into a variety of specialized cell types.

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The scaffold structures, design, and manufacture also play an important role.
Scaffold materials for tissue engineering must be compatible to meet the nutritional and
biological needs of the specific cell population involved in the tissue formation. Some of
the materials used currently as potential scaffolds are injectable materials, scaffolds
customized with the use of growth factors, polymers made up of carbon dioxide
properties, and plasma modification [3]. These scaffold types provide pathways for the
production and manufacturing of highly complex matrices that emphasizes the
multidisciplinary approach of the tissue engineering field.
Creating

the

scaffold

material

involves

the

decellularization

process.

Decellularization can be defined as the process of degrading the resident cells of a tissue
or an organ in order to derive the extracellular matrix of the primary tissue so that it can
be used as a scaffold material for the formation of the fabricated novel tissue.
These biologically derived matrices can come from various methods, such as
using chemical agents, enzymes, detergents, and physical and miscellaneous agents like
temperature, force, pressure, non-thermal irreversible electroporation. The effectiveness
of these agents is dependent on many factors including the tissue cellularity, density, lipid
content, and thickness. The removal agent has the possibility of producing undesirable
effects on the composition of the extracellular matrix and must also be considered [4].
The application of decellularization agents is characterized into four categories,
namely, whole organ perfusion, pressure gradient, supercritical fluid, immersion and
agitation. Each of these applications depends on the properties of the tissue and the
agents used. The choice of decellularization methods can be successful with a complete

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knowledge and understanding of the advantages and disadvantages of each mechanism
[4].

Given these several approaches in creating a decellularized biological matrix, the


great challenge of the decellularization process is to decellularize without damaging and
disrupting the naturally occurring growth factors of the tissue and retain its extracellular
matrix components.
Tissue engineering is an emerging technology that can lead us into a new
milestone in clinical research. Yet, it still needs further progress and developments.
The proposed method of this research is the decellularization with the use of
alkaline treatment. The alkaline solution involved in this method is specifically sodium
hydroxide (NaOH). The main process we used was the swabbing method. Through the
gentle rubbing of the amniotic membrane with NaOH-soaked cotton tip until the blue
color stain get lost which indicates that the cells were removed. Compared to the other
treatment methods, this treatment also prevented any changes in the structure and patterns
of the membrane components because of its mild concentration.

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1.2 Objectives
1.2.1

General Objective
To produce a decellularized amnion tissue that is suited for creating a biologic

scaffold material.
1.2.2

Specific Objective

1. Compare decellularization methods of Human Amniotic Membrane in terms of


a. NaOH concentration used
i. 0.25M
ii. 0.50M
iii. 0.75M
iv. 1M
b. Reagent application method
i. Soaked
ii. Swabbed
2. Analyze the secondary protein structure changes on different protocols used for
decellularization.
3. Obtain optimized conditions of decellularizing Human Amniotic Membrane using
NaOH.

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1.3 Significance of the Study
The research would be of great help to the field of tissue engineering in view of
the fact that the proposed method for the decellularization of the amnion tissue could save
time, effort, and resources.
The success of the experiment will provide a way for producing a biological
material that may be used as an alternative for some medical treatment, such as using the
materials as a scaffold for cell proliferation in burnt parts of the body instead of patching
it with skin taken from another part of the body. Also, this study will pave the way to
production of organs, a breakthrough in the field of tissue engineering that will be useful
to the medical field, to individuals in need of organs and to the society in general.

1.4 Scope and Limitation


This study was conducted at two different institutions. The decellularization was
conducted at the Technological Institute of the Philippines, Quiapo Manila campus,
National Capital Region (NCR). The research laboratory room has an average occupancy
of 48 students where the experiments regarding the decellularization were performed.
The decellularized samples were brought and analyzed at the University of the
Philippines-Diliman, Department of Chemical Engineering.
The research focused only on the decellularization process of the amnion
tissue from the placenta of mothers that undergone natural birth. The research was
conducted from June 2015 and ended March 2016 at Technological Institute of the
Philippines-Manila. Tests were conducted in the Lung Center of the Philippines and

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University of the Philippines, Diliman. The research aims to produce an efficient yet cost
effective procedure in decellularizing amnions.
Sodium Hydroxide of 0.25, 0.50, 0.75 and 1M concentrations were used in both
soaking and swabbing the amnion where time of soaking, pressure and number of swabs
were varied to determine which provide the desirable results.

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CHAPTER II
CONCEPTUAL FRAMEWORK

Related Literature and Studies

Decellularization of tissues has been gaining attention as one of the most


important techniques for making artificial organs because it can preserve the complicated
3D structure of native tissues. This technique has been effective when applied to tissues
such as the heart, lung, blood vessels, cornea, and nerves. [5] Decellularization is defined
as the technology used to remove all parenchyma cells, myofilaments, endothelial cells
and other cellular components from the organ while retaining its three-dimension
architecture and vascular tree. The use of natural vasculature and rapid separation of
debris and waste fluid from organ or tissue significantly improves the quality of organ
scaffold for reconstitution of new organ/tissue. Decellularization is ideal for tissue
regeneration because it preserves the three-dimensional structure of the organ and the
extracellular matrix (ECM)the framework between the cellsthat are complex and
difficult to mimic. While current methods use specific ECM proteins to transform stem
cells into a particular cell type, scientists have found it difficult to imitate the natural
ECM. [6] When tissues are decellularized, the structure of the native ECM should be
preserved while sufficient cellular components should be removed. [5]

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The most effective agents for decellularization of each tissue and organ will
depend upon many factors, including the tissue's cellularity (e.g. liver vs. tendon), density
(e.g. dermis vs. adipose tissue), lipid content (e.g. brain vs. urinary bladder), and
thickness (e.g. dermis vs. pericardium). It should be understood that every cell removal
agent and method will alter ECM composition and cause some degree of ultrastructure
disruption. Minimization of these undesirable effects rather than complete avoidance is
the objective of decellularization.[4]

Various Decellularization Methods


Organ

Decellularization Method

Rat liver

Perfusion with detergents


(SDS, Triton X-100)

Porcine
liver

Mechanical perfusion
(electroporation)

Mouse heart
Porcine
trachea

Enzymatic, detergents, and


acids
Enzymatic (trypsin) non
enzymatic (EDTA),
detergent (Triton X-100)
and de-ionized water

Study Out come


Perfusing with SDS
removes most of
cells, damages the
ECM when treated
with Triton X-100
and removes 97%
of DNA
Most of the cells
are removed,
preserves the blood
vessels
Cells are removed
Cells are removed,
clear the cell debris

Limitation

SDS damages the


ECM

Disruption of
microfilament and
microtubule
Loss of cellmediated function
Disruption of
glycosaminoglycan,
reduce the laminin
and fibronectin

Various studies have been published to demonstrate the methodologies for whole
organ decellularization using different approaches but all have its limitations. These
includes Perfusion with detergents (SDS, Triton X-100); Mechanical perfusion

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(electroporation); Enzymatic, detergents, and acids; Enzymatic (trypsin) non-enzymatic
(EDTA), detergent (Triton X-100) and de-ionized water. [6]
For perfusion with detergents (SDS, Triton X-100), Shupe, et al, developed a
method for the decellularization of whole rat livers by perfusion with increasing
concentrations of detergents. Rats received a lethal (100 mg/kg) dose of sodium
pentobarbital. The inferior vena cava (IVC) was cannulated, the portal vein severed and
the superior vena cava (SVC) was clamped as previously described.8 The liver was first
perfused with 100 mL PBS (pH 7.4) to clear blood from the organ. The liver was then
perfused with biological detergents to solubilize cell membranes. Isotonic solutions
(PBS) of 1, 2 and 3% (w/v) Triton X-100 (300 mL each) were perfused through the organ
by peristaltic pump at a flow rate of 5 mL/minute. This was immediately followed by
perfusion with 300 mL each of PBS containing 0.1% SDS (w/v). Detergent containing
solutions were cleared from the liver by perfusion with 300 mL PBS. Finally, 10 mL fetal
bovine serum was pumped into the organ. For experiments involving administration of
cells to the IDL, 106 cells were delivered to the organ through the IVC. The IVC and
SVC were then tied off and the liver was excised intact. All perfusion solutions (including
FBS) contained 1% antibiotic/mycotic (Invitrogen, Carlsbad, CA). Analysis of sections
from livers treated only with Triton X-100 indicated clearing of all cells. Subsequent
perfusion of the acellular organ with solution containing SDS resulted in the clearance of
all DNA and damages the ECM. [7]
Most of the cells were removed and blood vessels were preserved but there was
disruption of microfilament and microtubule from the experiment conducted by Sano, et
al. brief but intense electric pulses were applied to porcine livers while under active low-

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temperature cardio-emulation perfusion. Histological analysis and lesion measurements
were used to determine the effects of the pulses in decellularizing the livers as a first step
towards the development of extracellular scaffolds that may be used with stem cell
reseeding. A dynamic conductivity numerical model was developed to simulate the
treatment parameters used and determine an irreversible electroporation threshold. This
method is the first effort towards creating decellularized tissue scaffolds that could be
used for organ transplantation using N-TIRE. [8]
Mouse heart was decellularized using enzymatic, detergents and acids, according
to Lu et al, cells were removed but resulted to damage of the ECM proteins and the 3D
architecture were poorly maintained. They created heart constructs by repopulating
decellularized mouse hearts with human induced pluripotent stem cell-derived
multipotential cardiovascular progenitor cells. The seeded multipotential cardiovascular
progenitor showed that cells migrate, proliferate and differentiate in situ into
cardiomyocytes, smooth muscle cells, and endothelial cells to reconstruct the
decellularized hearts. After 20 days of perfusion, the engineered heart tissues exhibit
spontaneous contractions, generate mechanical force and are responsive to drugs. In
addition, they observe that heart extracellular matrix promoted cardiomyocyte
proliferation, differentiation and myofilament formation from the repopulated human
multipotential cardiovascular progenitor cells. [9]
Using Enzymatic (trypsin) non-enzymatic (EDTA), detergent (Triton X-100) and
deionized water to decellularized porcine trachea, cells were removed and cell debris
were cleared. However, this resulted in the disruption of glycosaminoglycan and reduced
the laminin and fibronectin of the organ. [6] [10]

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Whole organ decellularization represents a novel approach to create bio-artificial
natural 3D architecture. However, various methods have been used to remove the cells
and its components from different organ leaving its ECM and organ scaffold. But still
there is a need to find more appropriate decellularization agent further to improve the
quality organ scaffold. Several studies have demonstrated the use of various types of
chemical, biological and physical substance for whole organ decellularization. But which
is the best still needs to be found based on the organ architecture and its further
application. [6]
In evaluating decellularized matrices, various tests are conducted in order to see
the effectiveness of the method use in decellularizing. In reviewing decellularization,
three criteria for satisfactory decellularization were proposed, namely: <50 ng of doublestranded DNA (dsDNA) per mg of ECM (dry weight) and a lack of visible nuclear
material in tissue sections stained with 4,6-diamidino-2-phenylindole or haematoxylin
and eosin (H&E). The third criterion suggested was a < 200 bp DNA fragment length. [4]
A standard for tissue decellularization provides numerous benefits, including: (1)
allowing investigators and ECM product manufacturers to evaluate the effectiveness of a
protocol when reporting new decellularization techniques or describing products
composed of ECM derived from a decellularized tissue; (2) enabling congruous
comparison of different ECM products; (3) eliminating variations in cell and host
responses to ECM products caused by variations in residual DNA, thereby facilitating
interpretation and comparison of in vitro and in vivo results; and (4) promoting the rapid
and effective development of additional clinical applications for ECM products within the
field of regenerative medicine and tissue engineering. [4]

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In a study conducted by Syed et al, tubular Small intestine submucosa (SIS) was
produced and decellularized by the conventional peracetic acidagitation method.
Peracetic acid under agitation has been the standard decellularization protocol for
production of SIS sheets for a number of years [11] [12]. However, with the SIS in
tubular form, an alternative decellularization protocol was considered necessary to allow
for the tissue to be adequately decellularized. Perfusion decellularization protocols have
been successfully applied to a variety of tissues, and a large number of protocols and
agents exist [10], [4]. In the study, the two protocols selected have been used to
decellularize a variety of tissues, and these two were compared to peracetic acid under
agitation, along with an undecellularized control, to obtain an optimal decellularization
protocol for tubular SIS. [13]
The samples were prepared for different analysis including histology, scanning
electron microscopy, DNA quantification, Cell culture, and Biocompatibility assay. [13]
With verification of cellular removal completed, the effects of decellularization on
the mechanical and material properties of the remaining ECM scaffold are of interest.
Currently, there is no consensus regarding the effects of any individual decellularization
agent upon mechanical properties. Studies have shown that detergents disrupt collagen in
certain tissues, thereby decreasing the mechanical strength of the tissue, while the same
detergent may have no apparent effect on the collagen even in fairly similar tissues (e.g.,
tendon vs. ligament) [14], [15]. Another variable that can affect the mechanical properties
of a graft is the duration of exposure to decellularization agents. The specific mechanical
testing that should be performed is dependent on the intended clinical application [16].

ADVANCES ON DECELLULARIZATION METHODS


Definition of terms

Amnion- a thin membrane that surrounds the fetus during pregnancy. The amnion is the
inner of the two fetal membranes and it contains the amniotic fluid.

Cell counting- cell counting is any of various methods for the counting or similar
quantification of cells in the life sciences, including medical diagnosis and treatment. It is
an important subset of cytometry, with applications in research and clinical practice.

Chorion- the outermost of the two fetal membranes that surround the embryo. The
chorion develops villi (vascular finger-like projections) and develops into the placenta.

Decellularization- the process used in biomedical engineering to isolate the extracellular


matrix (ECM) of a tissue from its inhabiting cells, leaving an ECM scaffold of the
original tissue, which can be used in artificial organ and tissue regeneration.

Extracellular matrix (ECM) - substance produced by cells and secreted into the
environment in which the cells are embedded; contains collagen, proteoglycans,
glycosaminoglycans, and fluid; can influence the behavior of the cells.

Fourier Transform Infrared Spectroscopy (FTIR) Analysis- FTIR offers quantitative


and qualitative analysis for organic and inorganic samples. Fourier Transform Infrared

ADVANCES ON DECELLULARIZATION METHODS


Spectroscopy (FTIR) identifies chemical bonds in a molecule by producing an infrared
absorption spectrum.

Growth factor- any of a group of proteins that stimulate the growth of specific tissues.
Growth factors play an important role in promoting cellular differentiation and cell
division.

Placenta- A temporary organ that joins the mother and fetus, transferring oxygen and
nutrients from the mother to the fetus and permitting the release of carbon dioxide and
waste products from the fetus.

Scaffold- The chromosome structure consisting entirely of nonhistone proteins remaining


after all the DNA and histone proteins have been removed from a chromosome.

Swab- A wad of cotton, gauze, or other absorbent material attached to the end of a stick
or clamp, used to apply or remove a substance from a surface.

Trypan blue- is a vital stain used to selectively colour dead tissues or cells blue. It is a
diazo dye.

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CHAPTER III
METHODOLOGY
Procedure

On the processing procedure of separating placenta membrane (amniotic and


chorionic), the amniotic layer was cut apart and goes through decullarization. The
technique of decellularizing derived amnion was to remove cells and cellular debris. The
amnion is washed and soaked in phosphate-buffered saline (PBS) solution for 30
minutes. The membrane was cut into 2 x 2 inch per piece of specimen. The Petri dishes
were decontaminated using 95% ethyl alcohol and washed with PBS solution. The
amnion was then placed on the sanitized dish. For the first run of experiment, the settled
amnion on the dish will be soaked using 0.25 M, 0.5 M, 0.75 M and 1 M of Sodium
Hydroxide (NaOH) for about 10 minutes. After 10 minutes, the soaked amnion at
different molarity solution was placed on the sample container with specified labels. The
second run of experiment dealt with the swabbing of the amnion on the dish. The amnion
was swabbed using a sponge filled with 0.25 M, 0.5 M, 0.75 M and 1 M NaOH solution
at different trials. Both sides were swabbed 100 times. After swabbing, the specimens
were kept in the labeled sample containers. The specimens were taken to the Fourier
Transform Infrared Spectroscopy (FTIR) at the University of the Philippines - Diliman.
The amnion undergoes series of observations to be analyzed whether decellularized or
not. The results gathered concerning the present make-up of the amnion helped the
researchers examine qualitatively the current state of the amnion.

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CHAPTER IV

PRESENTATION, ANALYSIS, AND INTERPRETATION

In the present work, tissue decellularization was performed and confirmed prior to
use as scaffolds. Human amnion was harvested and decellularized and examined for cell
count and chemical composition before/after decellularization.

4. 1 Fourier transform infrared spectroscopy (FTIR)


The chemical composition analysis is performed through Fourier transform
infrared spectroscopy. FTIR spectroscopy is a measurement of wavelength and intensity
of the absorption of IR radiation by a sample. In infrared spectroscopy, IR radiation is
passed through a sample. Some of the infrared radiation is absorbed by the sample and
some of it is passed through (transmitted). The resulting spectrum represents the
molecular absorption and transmission, creating a molecular fingerprint of the sample.
FTIR is used to identify organic, polymeric, and in some cases, inorganic materials. IR
can provide a molecular fingerprint that can be used when comparing samples. If two
pure samples display the same IR spectrum it can be argued that they are the same
compound.
Infrared spectra of fresh and decellularized human amnion were obtained by using
Fourier-Transform Infrared Spectrometer in the Chemical Engineering Department
Laboratory of University of the Philippines-Diliman. Once the infrared spectrum has
been recorded, the next stage of this experimental technique is the interpretation of data.

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In this study, the researchers focused on the interpretation of presence or absence of
specific functional groups functional groups and secondary structure of polypeptides and
proteins of the amnion. The polypeptide and protein repeat units give rise to nine
characteristic IR absorption bands, namely, amide A, B, and I-VII. In here, we will limit
the analysis of the sample in determining the frequencies of amide I band. The observed
amide I band contours of proteins or polypeptides consist of overlapping component
bands, representing -helices, -sheets, turns and random structures. In this work, all
spectra presented were measured by FTIR spectrometer. PeakFit and IRPal software were
used for data acquisition and analysis.

4.2 Infrared Spectral Interpretation


IR is the most useful in providing information about the presence or absence of
specific functional groups. Mid-Infrared Region is use for the determination of molecular
structure of each samples.
The mid-infrared spectrum (4000400 cm1) can be approximately divided into
four regions and the nature of a group frequency may generally be determined by the
region in which it is located. The regions are generalized as follows: the XH stretching
region (40002500 cm1), the triple-bond region (25002000 cm1), the double-bond
region (20001500 cm1) and the fingerprint region (1500600 cm1).
The fundamental vibrations in the 40002500 cm1 region are generally due to O
H, CH and NH stretching. Triple-bond stretching absorptions fall in the 25002000
cm1 region because of the high force constants of the bonds. CC bonds absorb between
2300 and 2050 cm1, while the nitrile group (CN) occurs between 2300 and 2200 cm 1.

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The principal bands in the 20001500 cm1 region are due to C=C and C=O stretching.
Many vibrations are not so well behaved and may vary by hundreds of wavenumbers,
even for similar molecules. This applies to fingerprint region (1500600 cm1).
4.3 Results
Fresh amnion
Figure 1. FTIR spectra of fresh amnion. The graph shows a spectrum in absorption mode

Figure 2. The graph shows the second derivative of the fresh amnion spectra which is in
transmission mode.

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(a)

(b)

ADVANCES ON DECELLULARIZATION METHODS


(c)

(d)

Table 1. Secondary derivative of FTIR spectra of (a) Raw Amnion (b) 0.25M (c) 0.50M (d) 1M
(Swabbed)

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Table 1.Compound groups obtained from decellularized amnion usimg NaOH by swabbing.

NaOH (Swabbed)
Fresh Amnion

Group Frequency
Wavenumber (cm-1)
3679-3577
3478-3329
3313-3236
3214-3135
2983-2892
2872-2810
1774-1702
1684-1588
1577-1496
1462-1417
1405-1337
1258-1185

Fresh Amnion

0.25M

0.50M

Peak
3620
3381
3264
3191
2928
2836
1738
1639
1536
1442
1369
1222

Functional Gro

Free Hydroxyl Alc


Phenols O-H
1, 2 Amines, Amid
Carboxylic Acids
Carboxylic Acids
Carboxylic Acids
Carboxylic Acids
C=O
1 Amines N-H
Nitro Compounds
Aromatics C-C
Alkanes C-H
Amine C-N

3626-3581
3208-3079
3066-2928
2918-2844
1829-1678
1614-1565
1520-1459

3616
3149
3007
2890
1698
1596
1497

Free Hydroxyl Alc


Phenols O-H
Carboxylic Acids
Aromatics C-H
Carboxylic Acids
C=O
1 Amines N-H
Nitro Compounds

3634-3588
3230-3065
3065-2958
2236-2167
1845-1678
1611-1562
1536-1464

3615
3145
3017
2206
1706
1596
1505

Free Hydroxyl Alc


Phenols O-H
Carboxylic Acids
Aromatics C-H
Alkynes C=C
C=O
1 Amines N-H
Nitro Compounds

3628-3586
3209-3060
3022-2970
1862-1678
1605-1529

3616
3132
3010
1706
1594

Free Hydroxyl Alc


Phenols O-H
Carboxylic Acids
Aromatics C-H
C=O
1 Amines N-H

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(a)

0.75M

3216-3087
3083-2949
1755-1684
1613-1559
1526-1455
1368-1326
1305-1264
1222-1155

3154
3008
1706
1597
1496
1363
1292
1199

Carboxylic Acids
Aromatics C-H
C=O
1 Amines N-H
Nitro Compounds
Alkanes C-H
Amine C-N
Amine C-N

1M

3216-3087
3083-2949
1755-1684
1613-1559
1526-1455
1368-1326
1305-1264
1222-1155

3154
3008
1706
1597
1496
1363
1292
1199

Carboxylic Acids
Aromatics C-H
C=O
1 Amines N-H
Nitro Compounds
Alkanes C-H
Amine C-N
Amine C-N

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(b)

(c)

Table 2. Secondary derivative of FTIR spectra of (a) Raw Amnion (b) 0.25M (c) 0.50M (d) 1M
(Soaked)

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Table 2.Compound groups obtained from decellularized amnion using NaOH by soaking.
NaOH (Swabbed)

Group Frequency
Wavenumber (cm-1)

Fresh Amnion
3679-3577
3478-3329
3313-3236
3214-3135
2983-2892
2872-2810
1774-1702
1684-1588
1577-1496
1462-1417
1405-1337
1258-1185
Fresh Amnion

0.25M

0.50M

Peak
3620
3381
3264
3191
2928
2836
1738
1639
1536
1442
1369
1222

Functional Grou

Free Hydroxyl Alco


Phenols O-H
1, 2 Amines, Amide
Carboxylic Acids O
Carboxylic Acids O
Carboxylic Acids O
Carboxylic Acids O
C=O
1 Amines N-H
Nitro Compounds N
Aromatics C-C
Alkanes C-H
Amine C-N

3626-3581
3208-3079
3066-2928
2918-2844
1829-1678
1614-1565
1520-1459

3616
3149
3007
2890
1698
1596
1497

Free Hydroxyl Alco


Phenols O-H
Carboxylic Acids O
Aromatics C-H
Carboxylic Acids O
C=O
1 Amines N-H
Nitro Compounds N

3634-3588
3230-3065
3065-2958
2236-2167
1845-1678
1611-1562
1536-1464

3615
3145
3017
2206
1706
1596
1505

Free Hydroxyl Alco


Phenols O-H
Carboxylic Acids O
Aromatics C-H
Alkynes C=C
C=O
1 Amines N-H
Nitro Compounds N

3628-3586
3209-3060
3022-2970
1862-1678
1605-1529

3616
3132
3010
1706
1594

Free Hydroxyl Alco


Phenols O-H
Carboxylic Acids O
Aromatics C-H
C=O
1 Amines N-H

ADVANCES ON DECELLULARIZATION METHODS


0.75M

3216-3087
3083-2949
1755-1684
1613-1559
1526-1455
1368-1326
1305-1264
1222-1155

3154
3008
1706
1597
1496
1363
1292
1199

Carboxylic Acids O
Aromatics C-H
C=O
1 Amines N-H
Nitro Compounds N
Alkanes C-H
Amine C-N
Amine C-N

1M

3216-3087
3083-2949
1755-1684
1613-1559
1526-1455
1368-1326
1305-1264
1222-1155

3154
3008
1706
1597
1496
1363
1292
1199

Carboxylic Acids O
Aromatics C-H
C=O
1 Amines N-H
Nitro Compounds N
Alkanes C-H
Amine C-N
Amine C-N

For the FTIR result of fresh amnion, organic compounds were identified from the
ranges of peaks observed from the graph. The graph reveals no IR bands in the triplebond region (25002000 cm1). Organic compounds from XH stretching region (4000
2500 cm1), the double-bond region (20001500 cm1) and the fingerprint region (1500
600 cm1) were determined. The fundamental vibrations in the 40002500 cm1 region
are generally due to OH, CH and NH stretching. The principal bands in the 2000
1500 cm1 region are due to C=C and C=O stretching. 12201020 cm1 is due to Aliphatic
CN stretching.
Comparing the graph of swabbed decellularized amnion using 0.25M NaOH,
some IR bands are missing, indicating the change in chemical composition of the tissue.
In the XH stretching region (40002500 cm1), 1, 2 Amines, Amides N-H and
Carboxylic Acids O-H are missing. All the IR bands in the fingerprint region (1500600

ADVANCES ON DECELLULARIZATION METHODS


cm1) disappeared. However, in the double-bond region (20001500 cm 1) all the IR
bands remained.
Evaluating the graph swabbing using 0.5M NaOH, the results are almost the same
from swabbing using 0.25M NaOH. In the XH stretching region (40002500 cm1), 1,
2 Amines, Amides N-H and Carboxylic Acids O-H are also missing. All the IR bands in
the fingerprint region (1500600 cm1) disappeared. Still, in the double-bond region
(20001500 cm1) all the IR bands remained.
By assessing the graph of fresh amnion and swabbed decellularized amnion using
0.75 NaOH, the XH stretching region (40002500 cm1), 1, 2 Amines, Amides N-H
and Carboxylic Acids O-H are missing. All the IR bands in the fingerprint region (1500
600 cm1) disappeared. Also, there is an absence of Nitro Compounds N-O in the doublebond region (20001500 cm1)
Swabbing using 1M NaOH some IR bands are missing, indicating the change in
chemical composition of the tissue. In the XH stretching region (40002500 cm1), Free
Hydroxyl Alcohols, Phenols O-H, 1, 2 Amines, Amides N-H and Carboxylic Acids O-H
are missing. In the fingerprint region (1500600 cm1), Aromatics C-C disappeared. On the
other hand, in the double-bond region (20001500 cm1) all the IR bands remained.
Comparing the graph of soaked decellularized amnion using 0.25 NaOH, some IR
bands are missing, indicating the change in chemical composition of the tissue. In the X
H stretching region (40002500 cm1), 1, 2 Amines, Amides N-H and Free Hydroxyl
Alcohols, Phenols O-H are missing.

There are also changes in the fingerprint region

(1500600 cm1). Though, in the double-bond region (20001500 cm 1) all the IR bands
remained.

ADVANCES ON DECELLULARIZATION METHODS


Various changes can be observed from the graph of fresh amnion and soaked
decellularized amnion using 0.75 NaOH. Appearance of IR bands in the triple-bond
region (25002000 cm1) can be observed. In the XH stretching region (40002500
cm1), 1, 2 Amines, Amides N-H and Free Hydroxyl Alcohols, Phenols O-H are missing.
Alkanes C-H in the fingerprint region (1500600 cm1) disappeared but there are some

functional groups that turn up. Moreover, there is an another Nitro Compounds N-O
appeared in the double-bond region (20001500 cm1)
Evaluating the graph of fresh amnion and decellularized amnion soaked using 1M
NaOH, the results are almost the same. In the XH stretching region (40002500 cm1),
1, 2 Amines, Amides N-H is missing. Amine C-N in the fingerprint region (1500600
cm1) is not present. Nevertheless, in the double-bond region (20001500 cm 1) all the IR
bands remained.

ADVANCES ON DECELLULARIZATION METHODS

4. 4 FTIR Analysis of Protein Secondary Structure


Infrared spectroscopy is one of the oldest and well established experimental
techniques for the analysis of secondary structure of polypeptides and proteins. FTIR
spectroscopy is a measurement of wavelength and intensity of the absorption of IR
radiation by a sample. The IR spectral data of high polymers are usually interpreted in
terms of the vibrations of a structural repeat unit. The polypeptide and protein repeat units
give rise to nine characteristic IR absorption bands, namely, amide A, B, and IVII. Of
these, the amide I and II bands are the two most prominent vibrational bands of the
protein backbone. The most sensitive spectral region to the protein secondary structural
components is the amide I band (17001600 cm1), which is due almost entirely to the
C=O stretch vibrations of the peptide linkages (approximately 80%). The frequencies of
the amide I band components are found to be correlated closely to the each secondary
structural element of the proteins.
Strictly speaking, the observed amide I band contours of proteins or polypeptides
consist of overlapping component bands, representing -helices, -sheets, turns and
random structures. A component centered between approximately 1658 and 1650 cm 1
has been assigned to the -helix, which is consistent with both theoretical calculation and
the observation of bands in the spectra of -helical proteins. More than one -component
has been observed in the spectra of many -sheet proteins. Bands in the regions of
16401620 cm1 and 16951690 cm1 have been assigned to -sheet by many authors.
Theoretical calculation of -sheets also predicts an IR active mode between

ADVANCES ON DECELLULARIZATION METHODS


approximately 1695 and 1670 cm1. These -components are often complicated by the
presence of more than one band above 1670 cm 1. The unordered conformation (usually
referred to as random coil) is usually associated with the IR band between 1640 and 1648
cm1.
In this work, Fourier self-deconvolution (FSD) procedure was applied in order to
analyze the secondary proteins of fresh amnion and compare it with the decellularized
amnion. IR in Amide I was the main focus in this study, -helices, -sheets, turns and
random structures were the proteins that were identified in this study.
4.5 Results for Secondary Structure of Amnion

Fresh amnion
Figure 3. Second derivative infrared spectrum of fresh amnion. The deconvolution was
carried out by PeakFit software.

ADVANCES ON DECELLULARIZATION METHODS

Figure 4. Tabulated results of the cell counting for different amnions pretreated with (a) PhosphateBuffered Saline solution, (b) 0.25M NaClO solution, (c) 0.50M NaClO solution, and (d) 0.75M NaClO

Figure 4 above shows the number of cells retained in the amnion after various
decellularization processes. The blue asterisk at the top of each data bar indicates the
significant difference of the number of retained cells in that set of trial to the fresh
amnion sample while the black bar indicates the significant difference between the two
data bar under it. Significant difference is expressed in p-value. Comparison of the
figures show that the best process in terms of cells removed is the soaking of the amnion
in 0.75 molar sodium hydroxide pre-treated with 0.25 molar sodium chlorate having a pvalue of 0.0204. Amnions with the same pretreatment but swabbed instead with 0.75
molar NaOH showed also a significant decrease in the number of cell compared with

ADVANCES ON DECELLULARIZATION METHODS


fresh amnions. Other decellularized amnion soaked in 0.75 molar NaOH but with
different pretreatment also such as 0.75 M NaCIO and PBS also showed showed
relatively low number of cells remaining however the best results are of those pretreated
with 0.25 M NaCIO.

ADVANCES ON DECELLULARIZATION METHODS


CHAPTER V
SUMMARY OF FINDINGS, CONCLUSIONS, AND RECOMMENDATIONS
Summary of Findings
This study was conducted for the purpose of producing a decellularized amnion
tissue that will be suited for the creation of biologic material. Conforming to this, the
study also aims to review the characteristics of the membrane after decellularization
compared to the fresh amnion. The amnions were processed and pretreated with varying
concentrations of sodium hypochlorite solution (NaClO) and phosphate-buffered saline
solution (PBS). After the decellularization, the amnions undergone cell counting and
Fourier Transform Infrared Spectroscopy (FTIR) to assess the aftereffects of the NaOH
used. The remaining cells from each amnion was counted using ImageJ software and was
presented using Graphpad Prism 6. Most of the samples produced relative low amounts
of cells remaining. The amnion pretreated with 0.25M NaClO solution then soaked with
0.75M NaOH solution showed the best results having 0.0204 p-value. However, other
samples from different pretreatment also showed significant difference between the cells
remaining compared to the fresh amnion. With regard to this, the FTIR results of every
sample verified the best result.
The chemical composition analysis is performed through Fourier transform
infrared spectroscopy. IR can provide a molecular fingerprint that can be used when
comparing samples. If two pure samples display the same IR spectrum it can be argued
that they are the same compound.

ADVANCES ON DECELLULARIZATION METHODS


In this work the researchers compare the result of FTIR test for fresh amnion and
decellularized amnion. Two methods of decellularizing were introduced in this study,
swabbing and soaking. Using NaOH solution, prepared in various concentration, 0. 25M,
0.5M, 0.75M and 1M, each sample were tested and analyzed.
The mechanical structure of the Extra Cellular Matrix (ECM) depends on the
integrity of the tertiary structure of the decellularized human amniotic membrane. This
tertiary structure depends on the functional group identified during the Fourier Transform
Infrared Spectroscopy. The more functional groups present the stronger the mechanical
structure of the HAM. The preservation of the integrity of the ECM allows for a better
cell proliferation for tissue engineering application.
Conclusion
Decellulariation was done to denude the membrane of the cells it contains. It is
found that 0.25M of hypochlorite solution was able to remove most of the cells in
comparison to that of NaClO solutions of different molarity and to PBS. However, most
often than not, there were significant differences found in the two modes of
decellularization, swabbing and soaking. With varying factors such as time for soaking,
force exerted in swabbing and the number of swabs, it can be resolved that the tensile
strength of the amnion varies greatly not only with the pretreatment but also with the
methods. Given all the data for cell counts, it is shown that pretreatment with 0.25M
NaClO solution then followed by 10-minute soaking to the 0.75M NaOH removes most
of the cells embedded in the amniotic membrane. Based from the FTIR results and
examination of fresh amnion and decellularized amnion under a Fourier-Transform
Infrared Spectrometer it was found that the results of samples decellularized by soaking

ADVANCES ON DECELLULARIZATION METHODS


method shows almost similar results to the fresh amnion indicating that there is a minimal
changes in the chemical composition of the amnion. In addition to that, samples of
amnion decellularized by swabbing method clearly shows that the chemical composition
of the amnion were altered, for a lot of functional groups from the mid- infrared region
were missing. In compliance to that, the concentration that shows almost similar results
in terms of its chemical composition to the fresh amnion is using 1 M NaOH,
decellularized by soaking method.

Recommendations

With the data gathered and interpreted, the researchers arrived at certain
recommendations. The amnion pretreated with

0.25M NaClO is recommended to be

soaked for more than 10 minutes using 0.75M NaOH solution. This would ensure better
denuding of the amnion. For future researchers, they are encouraged to venture on lower
concentrations of alkaline solution to be used. Additional experimentations on the
potentials of amnion as a biomaterial must be performed, meanwhile the process that was
formulated by this experiment establishes to be effective in preserving the growth factors
in amnion. Lastly, products from this experiment may be used to produce electro-spun
nanofibers.

ADVANCES ON DECELLULARIZATION METHODS


BIBLIOGRAPHY

1.

Langer, R. and J. Vacanti, Tissue engineering. Science, 1993. 260(5110): p. 920926.

2.

Stock, U.A. and J.P. Vacanti, Tissue engineering: current state and prospects.
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3.

Howard, D., et al., Tissue engineering: strategies, stem cells and scaffolds.
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4.

Crapo, P.M., T.W. Gilbert, and S.F. Badylak, An overview of tissue and whole
organ decellularization processes. Biomaterials, 2011. 32(12): p. 3233-3243.

5.

Ishino, N. and T. Fujisato, Decellularization of porcine carotid by the recipients


serum and evaluation of its biocompatibility using a rat autograft model. Journal
of Artificial Organs, 2015. 18(2): p. 136-142.

6.

Khan, A., et al., Repopulation of decellularized whole organ scaffold using stem
cells: an emerging technology for the development of neo-organ. Journal of
Artificial Organs, 2014. 17(4): p. 291-300.

7.

Shupe, T., et al., Method for the decellularization of intact rat liver.
Organogenesis, 2010. 6(2): p. 134-136.

8.

Sano, M., et al., Towards the creation of decellularized organ constructs using
irreversible electroporation and active mechanical perfusion. BioMedical
Engineering OnLine, 2010. 9(1): p. 83.

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9.

Lu, T.Y., et al., Repopulation of decellularized mouse heart with human induced
pluripotent stem cell-derived cardiovascular progenitor cells. Nat Commun,
2013. 4: p. 2307.

10.

Gilbert, T.W., T.L. Sellaro, and S.F. Badylak, Decellularization of tissues and
organs. Biomaterials, 2006. 27(19): p. 3675-83.

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Badylak, S.F., et al., The use of xenogeneic small intestinal submucosa as a


biomaterial for Achille's tendon repair in a dog model. Journal of Biomedical
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Hodde, J.P., et al., Retention of Endohelial Cell Adherence to Porcine- Derived


Extracellular Matrix after Disinfection and Sterilization. 2004. 8(2): p. 225-234.

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Syed, O., et al., Evaluation of decellularization protocols for production of


tubular small intestine submucosa scaffolds for use in oesophageal tissue
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Woods, T. and P.F. Gratzer, Effectiveness of three extraction techniques in the


development of a decellularized bone-anterior cruciate ligament-bone graft.
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Cartmell, J.S. and M.G. Dunn, Effect of chemical treatments on tendon cellularity
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Badylak, S.F., D.O. Freytes, and T.W. Gilbert, Extracellular matrix as a


biological scaffold material: Structure and function. Acta Biomater, 2009. 5(1): p.
1-13.

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APPENDICES

Fig. 1: The students are cleaning the laboratory room.

Fig. 2: Ms. Remy Magbiray orienting the students about the basics of Aseptic Procedures.
Fig. 3: Students wearing their PPEs together with Engr. Jopeth Ramis demonstrating the
proper aseptic procedures for the experiment.

ADVANCES ON DECELLULARIZATION METHODS

Fig. 4: Demonstrating on the separation of Amnion-Chorion from the placenta.

Fig. 5: Separation of Amnion-Chorion from the placenta.

ADVANCES ON DECELLULARIZATION METHODS

Fig. 6: Stripping of amnion-chrion from the placenta and readying for


decellularization.

Fig. 7: Cutting of Amnion-Chorion by 2 x 2 inches

ADVANCES ON DECELLULARIZATION METHODS

Fig. 8(a)

Fig. 8 (b)
Fig. 8

M of
Trypan

(a) and (b) Decellularization of


amnion using
0.25M, 0.5M,
0.75M, and 1
NaOH and
blue.

ADVANCES ON DECELLULARIZATION METHODS

Fig.

9(a)
With
trypan
blue

Fig. 9 (b)
Without trypan blue
Fig. 9: (a) and
(b) Decellularize amnion membrane.

Fig. 10: Readied decellularized amnion for testing.