Philippine Journal of Science
153 No. 6A: 2165-2170, December 2024
ISSN 0031 - 7683
Date Received: 23 May 2024
RESEARCH NOTE
Assessment of the Decellularized
Extracellular Matrix (dECM) from the Skin
of Yellowfin Tuna (Thunnus albacares)
Chancy Louisse T. Barlisan1, Ronald P. Bual3,4*,
Kit Dominick Don Valle3, and Hernando P. Bacosa1,2
1Department of Biological Sciences, College of Science and Mathematics,
Mindanao State University–Iligan Institute of Technology (MSU-IIT),
Iligan, Lanao del Norte 9200 the Philippines
2Environmental Pollution and Innovation Laboratory,
Mindanao State University–Iligan Institute of Technology (MSU-IIT),
Iligan, Lanao del Norte 9200 the Philippines
3Center for Sustainable Polymers,
Mindanao State University–Iligan Institute of Technology (MSU-IIT),
Iligan, Lanao del Norte 9200 the Philippines
4Department of Chemical Engineering and Technology,
College of Engineering, Mindanao State University–Iligan Institute of Technology,
Iligan City 9200 the Philippines
Fish processing industries produce a wide range of by-products such as skin, bones, and viscera.
These wastes contain bioactive components and are a potential source of biomaterial scaffolds.
Thus, the utilization of the fish skin was employed for sustainable solutions to waste management
and biomedical material sourcing. The study aims to supplement the limited data on the
assessment of yellowfin tuna skin for developing decellularized extracellular matrix (dECM)
by evaluating the efficiency of a chemical decellularization method using 0.1% sodium dodecyl
sulfate (SDS). Moreover, the data indicated a reduction in cellular components in dECM, as
shown in the H&E staining images. The FTIR spectra confirmed the preservation of the collagen
triple helix structure. Similarly, SEM imaging revealed the preserved structural integrity of
the dECM. The DSC results highlighted the thermal stability of dECM tuna skin, crucial for
withstanding body temperature and inflammation during processing and transplantation. These
findings underscore the potential of dECM tuna skin as a sustainable biomaterial with diverse
biomedical applications, emphasizing its structural, functional, and thermal resilience. Further
research is necessary to optimize the decellularization process and evaluate the biocompatibility,
immunogenicity, mechanical properties, and in vivo applicability of the tuna skin-derived dECM
for comprehensive insights into its suitability for specific biomedical applications.
Keywords: decellularization, extracellular matrix, fish skin, yellowfin tuna
*Corresponding author: ronald.bual@msuiit.edu.ph
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Fish wastes such as skin, bones, and viscera are a good
source of protein, lipids, and minerals and a potential
source of collagen-rich biomaterial scaffolds (Atef et
al. 2020). Notably, fish skin – with its approximate 50%
collagen content – holds the potential as a valuable source
of collagen (Wibawa et al. 2015), which can be utilized
to produce scaffolds and carriers.
The application of fish skin in tissue engineering
represents considerable potential for substitutes for
compromised tissues, integrating three vital components:
cells, scaffolds, and biomechanical or biochemical signals
(Gilpin and Yang 2017). Decellularization creates natural
scaffolds mimicking ECM characteristics, offering
biodegradability and low immunogenicity, preserving
bioactive molecules, and fostering tissue growth and
repair (Lau et al. 2019). Conventionally, decellularization
methods include chemical, physical, and enzymatic
techniques, often combined for efficiency. Moreover, there
is a growing interest in decellularized tissues sourced from
marine origins due to reduced ethical constraints and a
lower risk of infection transmission.
Currently, limited studies are available regarding
the potential of yellowfin tuna skin for biomedical
applications. This study aims to assess the decellularized
extracellular matrix (dECM) yellowfin tuna skin by
evaluating the efficiency of a chemical decellularization
method using 0.1% SDS. In addition, characterization
tests provide vital information regarding its functional
properties, particularly in preserving the vital ECM
components of the yellowfin tuna skin, which provides
baseline data for more in-depth studies in the future.
Yellowfin tuna skins obtained from Tambo Market, Iligan
City, Lanao del Norte, the Philippines, were placed in an
ice box to maintain a cool temperature during transport
to the laboratory. The samples were washed several times
with distilled water and PBS to remove tissue residues. For
delipidation, the samples were immersed in 50% ethanol
for 1 h at 25 °C (Wibawa et al. 2015).
The decellularization of the fish skins described by Bual
et al. (2022) was done with minor modifications. The 50-g
tuna skins were immersed in 500 mL (n = 3) of 0.1% SDS
decellularizing agent for 2 d with continuous stirring at
300 rpm and 4 °C, and the SDS solution was then changed
every 24 h. The samples were extensively washed for 48
h. Consequently, the fish skins were stored at –80 °C in an
ultralow-temperature refrigerator (Haier, Qingdao, China)
for at least 24 h. Finally, a freeze dryer was utilized for the
lyophilization process to occur, and the fish skins were
freeze-dried at –55 °C for 24 h (Gyrozen, Gimpo, South
Korea) and were stored at 4 °C until needed.
The dECM tuna skin had undergone four characterization
analyses: hematoxylin and eosin staining (H&E) or
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Matrix from T. albacares Skin
visualization of cellular components, attenuated total
reflectance–Fourier transform infrared (ATR-FTIR) for
identification of distinctive chemical bonds, scanning
electron microscopy (SEM) for structural integrity, and
differential scanning calorimetry (DSC) for thermal
stability.
For H&E staining, the samples were fixed using buffered
10% formaldehyde for 72 h and were then coated with
paraffin wax. The paraffin blocks were cut using a
microtome (Slee CUT 4062, Nieder-Olm, Germany)
to obtain 0.4-µm-thick ribbons. The samples were then
mounted on glass slides and stained with hematoxylin
and eosin solution (Biognost®, Zagreb, Croatia) and were
assessed using a phase-contrast microscope (Olympus
CX43, Tokyo, Japan) to examine the decellularized
tissues. Hematoxylin stains the acid structures using bluepurple (DNA and nuclei), whereas eosin stains the basic
cell components in pink (proteins).
For the ATR-FTIR, the native and dECM fish skins were
placed on the spectrum plate of the QATR-10 single
reflection FTIR (Shimadzu, Kyoto, Japan). Infrared
rays with a wavelength range of 400–4000 cm−1 were
allowed to pass through the spectrum plate. The obtained
absorbance spectra were recorded using LabSolutions IR
software. Subsequently, the obtained absorbance spectra
were used to identify the samples' chemical bonds and
functional groups.
The microstructure of the surface and cross-section of the
native and dECM fish skins were assessed using SEM. The
samples were mounted and fixed in the sample holder on
a standard SEM sample holder using double-sided carbon
tape. After, it was then subjected to gold coating using
the sputtering unit (JEOL Smart Coater, Tokyo, Japan)
for 1 min. The coated samples were then placed into the
SEM specimen chamber, and their surface topology was
examined and photographed using scanning microscopy
(JSM-IT200, Tokyo, Japan). The test was conducted per
the manufacturer’s user manuals and in the Center for
Sustainable Polymers (CSP) Laboratory of MSU-IIT.
Lastly, the denaturation temperature of the native and
dECM fish skins was determined using a DSC 4000 (Perkin
Elmer, Waltham, MA, USA). Approximately 5 mg of the
fish skin samples were used and subjected to a heating
rate of 10 °C/min, and a temperature range of 30–300 °C.
The SDS, an ionic detergent, is usually employed in
decellularization methods due to its efficiency in cell
component removal from tissues (Gilpin and Yang 2017),
specifically in the solubilization of both cytoplasmic and
nuclear cellular membranes, as well as in the denaturation
of proteins through the disruption of the protein-protein
interactions (Kim et al. 2017). A change in the visual
appearance, particularly the loss of black pigments in the tuna
Philippine Journal of Science
Vol. 153 No. 6A, December 2024
Barlisan et al.: Decellularized Extracellular
Matrix from T. albacares Skin
Figure 1. Representative images of tuna skin before (a) and after decellularization (b), and H&E-stained cross-sections of native (c) and
dECM tuna skin (d) (scale: 1 bar = 100 µm).
skin, was observed after decellularization (Figure 1a and b).
Fish skin color is a critical marketable trait, making it essential
to consider decellularization protocols that effectively reduce
pigments. Reducing pigments enhances aesthetic appeal and
ensures consistency and quality in the final product, which
is crucial for both biomedical and consumer acceptability
and marketability. Likewise, a decellularization protocol that
maintains the structural integrity of the skin while effectively
minimizing pigmentation is highly beneficial.
Additionally, H&E staining was used to visualize the
cellular components before and after the decellularization
process. In the native sample, purple stains indicated
cellular components, while pink stains represented protein
structures (Figure 1c). In contrast, the visible reduction
in purple stains (Figure 1d) indicates the removal of
cellular components relative to the native sample. This
is consistent with the findings of Lau et al. (2019) and
Bual et al. (2022). When employing chemical treatments
like SDS for decellularization, it is crucial to consider the
specific tissue being treated. In this study, a 0.1% SDS
concentration effectively removed cellular components
while minimizing significant damage to the ECM.
As depicted in Figure 2, the absorption peaks of the amide
bond (A, B, I, II, and III) in both native and decellularized
tuna skin confirm the preservation of the vital ECM
Figure 2. ATR-FTIR spectra of the native and dCEM tuna skin.
component – collagen. Collagen contributes significantly
to the strength and elasticity of the skin due to its fibrous
structure and resistance to stretching. Additionally, it
plays a crucial role in reinforcing tissue development
(Göçer 2022).
The Amide A, with absorption peaks at 3400–3440 cm–1,
is associated with N-H stress vibration (Atef et al. 2020).
However, as observed in Figure 2, the absorption peaks for
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the native and dECM tuna skin were below the established
range of 3300–3292.49 cm–1, respectively. The shift of
amide A toward lower wavelengths is due to the shifting
of the hydrogen bonding in the peptide chain (Muyonga
et al. 2004) that is present in the dECM fish skins, similar
to the findings of Göçer et al. in 2022. The asymmetrical
-CH2 stretching vibration of Amide B in both native and
dECM tuna skin was observed at 2926 and 2924 cm–1,
respectively, falling within the defined range for Amide
B at 2920–2944 cm–1 (Heu et al. 2010).
Amide I, II, and III regions showed peaks within the
expected ranges: 1635.64, 1539.20, and 1236.37 cm–1,
respectively. The strong absorbance in the Amide I region
at 1600–1700 cm–1 suggests hydrogen bonding between
the carbonyl (C=O) and N–H groups, which may be
responsible for the triple helical structure (Zanaboni et al.
2000) and is generally applied to evaluate the secondary
structure of proteins (Muyonga et al. 2004). Amide II,
with an absorbance peak at 1550–1600 cm–1, is associated
with NH bending and CN stretching and is involved with
the triple helical structure of collagen (Heu et al. 2010),
along with Amide III at 1220–1320 cm–1, related to NH
bending and CN vibration (Atef et al. 2020).
In summary, the dECM tuna skin effectively retained the
collagen and ECM structure in the native skin, indicating
minimal alteration to the collagen's triple-helical structure.
This preservation is essential for maintaining the physical
properties necessary for various biomedical and consumer
Barlisan et al.: Decellularized Extracellular
Matrix from T. albacares Skin
applications, ensuring that the dECM skin can function
similarly to the native tissue.
The native inner surface image (Figure 3a) showed
a semi-rough and complex image with certain areas
of irregular loose filaments of the cellular debris. In
contrast, the dECM inner surface image (Figure 3d) had
a relatively smooth surface with areas of irregular loose
filaments. The outer surface (Figures 3b and e) had a
pattern of rigid collagen fibers, whereas the cross-section
image (Figures 3c and f) showed dense and compact
layers of collagen fibers. Furthermore, the SEM images
complemented the FTIR spectra (Figure 2), demonstrating
that the vital ECM components were preserved and the
decellularization protocol had minimal damaging impact
on the microstructure of the fish skin, thereby maintaining
its structural integrity.
Overall, the complex fibril form of collagen and relatively
well-distributed pore structures of dECM are associated
with a high ability to hold water and matrices for cell
proliferation suitable for biomedical applications (Mitra
et al. 2012; Tamilmozhi et al. 2013).
The first endothermal peak (30–100 °C) of the native and
dECM, which is associated with the thermal denaturation
temperatures of the collagen and the breaking of the triple
helix are 79.44 and 71.93 °C, respectively.
The variation in denaturation temperature observed
between the native and dECM tuna skin is attributed
Figure 3. Scanning electron microscope (SEM) images (scale: 1 bar = 50 µm).
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STATEMENT ON CONFLICT OF
INTEREST
The authors have no competing interests to declare that
are relevant to the content of this article.
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The authors would like to acknowledge the following
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