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Improved skin regeneration with acellular fish skin grafts

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Fish skin graft, a novel skin substitute, has seen a widespread clinical application since the approval for wound coverage by the FDA. Due to its properties in promoting wound healing and its efficient cost in manufacturing, fish skin grafts are a potential alternative to allograft and xenograft sources including, but not limited to cadaver and porcine grafts in a variety of applications. Additionally, fish skin grafts show promising results in treating diabetic foot ulcers (DFUs), venous leg ulcers (VLUs) and some evidence worthy of further exploration in treating a host of other acute and chronic wounds. Here we summarize the material and biological properties of fish skin grafts. How this graft compares to other prevailing skin substitutes on the market for different wound types was also explored. Fish skin grafts have many uses in clinical applications of the future.
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Engineered Regeneration 1 (2020) 95–101
Contents lists available at ScienceDirect
Engineered Regeneration
journal homepage: www.elsevier.com/locate/engreg
Improved skin regeneration with acellular sh skin grafts
Gabriella Fiakos
, Zeming Kuang , Evan Lo
Stony Brook University, Biomedical Engineering, 100 Nicolls Rd, Stony Brook, New York 11794, United States
Keywords:
Wound healing
Chronic wounds
Burn wounds
Skin xenografts
Omega-3 polyunsaturated fatty acid
Fish skin graft, a novel skin substitute, has seen a widespread clinical application since the approval for wound
coverage by the FDA. Due to its properties in promoting wound healing and its ecient cost in manufacturing,
sh skin grafts are a potential alternative to allograft and xenograft sources including, but not limited to cadaver
and porcine grafts in a variety of applications. Additionally, sh skin grafts show promising results in treating
diabetic foot ulcers (DFUs), venous leg ulcers (VLUs) and some evidence worthy of further exploration in treating
a host of other acute and chronic wounds. Here we summarize the material and biological properties of sh skin
grafts. How this graft compares to other prevailing skin substitutes on the market for dierent wound types was
also explored. Fish skin grafts have many uses in clinical applications of the future.
1. Introduction
1.1. Regenerative medicine in skin
As the largest organ in the human body, the skin contains a complex
structure. It plays a key role as the rst line of defense that acts as a
barrier to the outside environment, leaving it susceptible and vulnera-
ble. When the structural integrity of skin tissue is disrupted (torn, cut,
or punctured), pathogenic organisms and foreign bodies can easily enter
and contaminate the wound. If the wound site of a healthy individual
does not get infected, the skin is capable of healing without any addi-
tional treatment. However, if the site gets infected by microorganisms,
the healing process will easily get stuck in the inammatory phase or
biolms can develop that will delay cutaneous and subcutaneous wound
healing. There are some circumstances when a wound can remain in-
amed and become chronic, even if it is not from pathogenic infection,
including, but not limited to, disorders such as diabetes, a hypersensitiv-
ity reaction to the materials used for wound treatment, overexpression
on M1 macrophages, poorly controlled matrix metalloproteinases, and
broblast and keratinocyte senescence as well as phenotypic change.
Left untreated, chronic wounds can develop and lead to fatal complica-
tions [1] .
Regenerative medicine aims to focus on growing and replacing dam-
aged tissues and organs which can result in longer lasting, permanent
solutions [2] . The key strategy is to promote the growth and prolifer-
ation of host cells in the injury site and allow for faster healing times.
The body’s innate healing and repair mechanisms can be inuenced to
promote regeneration [2] . Skin grafts oer a promising and eective
solution for regenerative medicine in skin. By acting as a temporary
Corresponding author.
E-mail addresses: gabriella.akos@stonybrook.edu (G. Fiakos), zeming.kuang@stonybrook.edu (Z. Kuang), evan.lo@stonybrook.edu (E. Lo).
barrier as well as providing anti-microbial properties, skin grafts show
a capacity to promote healing eectively compared to traditional wound
dressings [3] .
1.2. Problems with existing skin grafts and unmet needs
Many types of non-allogeneic – non-cellular tissue-based thera-
pies exist, demonstrating key characteristics that promote wound heal-
ing. However, none have achieved all characteristics of an ideal skin-
replacement material. Since new wounds have to be created in order to
harvest skin grafts from donor sites in the case of an autograft, surgical
morbidity is a cause for concern [4] . Full-thickness cellular and/or tis-
sue based products (CTPs) using allografts is a prevailing dermatologic
repair option [4] . However, except for autograft, due to the large va-
riety of antibodies and antigens present inside the CTP, graft rejection
is another factor that causes adverse conditions to the recipient of the
CTP [5] . In a case study, a patient showed chronic antibody-mediated
allograft rejection 25 years after implantation [5] . Although allografts
are commonly used in dermatology, it is still not the ideal form of a skin
graft.
Some highly valued characteristics of skin replacement materials are
cost eectiveness, availability, long term function, conformation to ir-
regular wound, deep wound applications, low immunogenic response,
and high bioactivity. Sourcing cellular-based therapy materials from cer-
tain mammals in dierent cultures is also an issue to be considered. Few
materials can encapsulate all of these criteria, which means there is a
need to further develop new methods and materials in skin grafting tech-
nology. Acellular sh skin grafts serve as a potential candidate to ll this
gap in demand.
https://doi.org/10.1016/j.engreg.2020.09.002
Received 26 April 2020; Received in revised form 8 August 2020; Accepted 26 September 2020
2666-1381/© 2020 The Authors. Publishing Services by Elsevier B.V. on behalf of KeAi Communications Co. Ltd. This is an open access article under the CC
BY-NC-ND license ( http://creativecommons.org/licenses/by-nc-nd/4.0/ )
G. Fiakos, Z. Kuang and E. Lo Engineered Regeneration 1 (2020) 95–101
1.3. Decellularized fish skin and applications
Acellular sh skin is sourced from the Atlantic cod. They have very
little risk of transmitting viral diseases to humans, as compared to mam-
malian sourced skin cellular or tissue based product therapies (CTPs).
For this reason, the manufacturing process on sh skin is far less harsh
and can retain key bioactive features, such as omega-3-fatty acids and
collagen I structure [6] . This material is cost eective so it can be con-
verted into a usable resource and retains key biological features, com-
parably to other animal-derived skin technologies. Fish skin CTPs are a
source for facilitating wound healing, and are shown to possess antiviral
and antibacterial properties as well as faster healing times, as similarly
seen in other animal-derived skin graft materials [6] . In an acute wound
model sh skin is shown faster healing times than porcine intestinal sub-
mucosa and dehydrated amnion chorionic membrane [6] . It is also ca-
pable of 3-D cell ingrowth ability due to its porous scaold design [7] .
There are also no known cultural barriers to use of sh-derived skin CTPs
[6] . Acellular sh skin CTPs are shown to have improved wound healing
ability and a low-cost barrier, demonstrating vital characteristics for an
eective skin replacement material.
There are various applications where acellular piscine CTPs have
demonstrated some advantages. Chronic wounds that develop in pa-
tients with diabetes cannot overcome the inammatory response in
wound healing, so it is important to facilitate the completion of this
response [8] . Failure to overcome the inammatory response in chronic
wounds may lead to amputation if left untreated or insuciently treated
[8] . The anti-inammatory ability and promotion of cell ingrowth of
sh skin CTPs is shown to be successful in clinical cases [1] . Burn care
is another area of interest for application of sh skin. It is shown that
95% of burns occur in low-income countries, so further research must
be placed on developing inexpensive alternatives to burn care [9] . Due
to its antibacterial and antiviral properties as well as its acceleration of
3-D cell ingrowth, sh skin CTPss are applicable to severe burn victims
[10] . This material has also demonstrated widespread availability and
low processing requirements, keeping the cost of manufacturing low for
low-income patients. This material also has ecacy in wound care for
combat casualties. It is possible that sh skin CTPss can be used in a
battleeld scenario due to ease of storage and long shelf life. It might
potentially provide an eective physical protective barrier against in-
fection for severe wounds and create an better wound environment for
further treatments while a person is transported over extended periods
of time to a trauma treatment facility [11] . A skin-replacement material
that has antibacterial and antiviral properties, as well as an extended
shelf life is a potential candidate for combat care that exposes wounds
to a broad range of unideal conditions.
2. Material properties
2.1. Sourcing and manufacturing
Acellular Atlantic cod skin is a naturally derived biomaterial, so it
has some unique properties and manufacturing requirements when com-
pared to synthetic grafts or autografts. Acellular sh skin grafts are
xenografts originating from the Gadus morhua, or Atlantic cod. It is a
marine animal originating from cold water climates, of which are rich
in omega-3 polyunsaturated fatty acids in comparison to marine species
that have evolved to tropical climates [11] . As the structural and bi-
ological properties of natural biomaterials are dicult to standardize,
the question of quality control must be addressed. While it is possible
that factors such as age and gender of the donor animal may have an
eect on the structure and composition of the CTP, studies have shown
age does not aect its properties [12] . The FDA has specic regulations
regarding combination products that contain bioactive compounds. Pe-
riodic post-marketing reports must be submitted to conrm that there
are no unexpected or severely negative eects of this product [13] . This
helps ensure quality control for the product and avoid adverse eects.
There are some benecial characteristics of sh skin that makes the
manufacturing process and bioactivity of the material ecient and ben-
ecial to wound healing, respectively. Unlike most mammalian-derived
grafts, Atlantic cod skin CTPs do not require the use of highly reactive
chemicals and detergents, so the extracellular matrix retains most of
its biological and mechanical properties [14] . This is because it has no
measured risk of transmitting viruses nor prion diseases due to the vastly
dierent conditions that these pathogens must adapt to in a marine en-
vironment [11 , 15] . This puts piscine-derived CTPs at an advantage, as
most other xenografts on the market derived mostly of a collagen scaf-
fold missing the majority of its naturally present bioactive compounds
in processing [14] . Also, subjects treated with Atlantic cod skin CTPs
are unlikely to develop hypersensitivity [10] . Furthermore, this minimal
manufacturing process is cost-eective and eco-friendly [10] . The low-
cost of piscine CTPs is potentially useful in impoverished areas where
advanced wound care is inaccessible. Additionally, acellular sh skin
has an excellent shelf life, maintaining eectiveness for 3 years after
manufacturing [16] . The minimally destructive and ecient manufac-
turing process, low possibility for disease transfer and extensive shelf
life of acellular sh skin are excellent traits for a biomaterial, demon-
strating highly marketable characteristics that are eective in a clinical
setting.
2.2. Mechanical properties
Acellular sh skin has a physical macrostructure that is advanta-
geous as a skin replacement material on both chronic and acute in-
juries. Similar to humans, Atlantic cod has three basic layers to its skin:
an epidermal layer, an intermediate layer and a basal epithelial layer
[17] . Furthermore, these layers have a similar structure to human skin
[6] . The average thickness of these tissues are 450 µm [6] . It has a
porous structure with an average of 16.7 holes per 100 µmˆ2 of sam-
ple [6] . Also, the matrix is mostly composed of type I collagen, which
eciently binds with pro-inammatory cytokines present in the human
body [10] . Furthermore, the collagen matrix has proline incorporated
within it, so it is physically and thermally stabilized [10] . This naturally
contributes to the long shelf life of piscine CTPs. Some other consider-
ations must be made when applying this CTPs. During the application
stage of treatment, the CTP must be pre-wet in a saline solution, and
it will shrink upon wetting [16] . Adjustments to the size of the tissue
based therapy must be made when tting a wound. Overall, acellular
piscine tissue based therapy has a similar macrostructure to human ep-
ithelia, so it is able to encourage the proliferation human cells within
it.
In addition to piscine tissue based therapy’s (CTP) similar
macrostructure to human skin, it also has a microstructure that is ideal
for penetration by native human cells and compounds, further con-
tributing to wound healing eciency. Pore size and geometry are some
of the most important factors when assessing a biomaterial for scaold
purposes, and is often the limiting factor in material ecacy [18] . The
average pore diameter of Atlantic cod skin is 16.1 µm [6] . It is important
to note that pore size varies signicantly but remains within the range of
epithelial tissue. It was found that pores must be smaller than 160 µm to
optimize the ingrowth and attachment of human skin broblasts [18] .
Also, broblasts best attach to scaolds with pore sizes between 50 µm
and 160 µm [18] . The pore size of decellularized sh matrix emulates
the size of pre-existing epithelial tissue, which is comparable to that of
human tissues. Furthermore, piscine CTP pores’ diameter never falls be-
low 1 µm [6] . This is an ideal environment for human broblast cells, as
their migration is inhibited by any pore that is less than 1 µm diameter
[18] . These properties demonstrate support for cellular ingrowth with
signicantly more penetration compared to human amnion/chorion
membrane [6] . The structure is ideal for the incorporation of human
broblasts within the matrix, so they populate the matrix quickly [10] .
In addition to the numerous benets that the physical composition
and structure of piscine CTP, there are many compounds naturally
96
G. Fiakos, Z. Kuang and E. Lo Engineered Regeneration 1 (2020) 95–101
present within the scaold that make it bioactive and further encourage
wound healing response, which will be explored in the succeeding
section.
2.3. Biological properties
The complex composition of acellular piscine CTPs contributes to
biocompatibility and bioresorbability. The FDA has found piscine CTPs
to be biocompatible and the fact that the collagen is sourced from
animals has little signicance on ecacy [7] . As the tissue matrix
is inltrated by host cells, the ECM is broken down and remodeled
by broblast activity [6] . The material also has a slow degradation
period, providing ample time for autologous cell inltration [6] .
This demonstrates the biocompatibility and the bioresorbable na-
ture of a collagen-based matrix. In a clinical study treating chronic
wounds, no patients had an allergic reaction to the CTP, which can
be attributed to the fact that parvalbumin, a common sh aller-
gen, is mainly present in the esh of sh, and not inside sh skin
[11] . Decellularized sh skin CTPs are an excellent candidate for
patients who may be prone to hypersensitive reactions to foreign
bodies.
Piscine CTPs have a unique lipid prole that provides an improved
wound-healing time. When comparing lipid contents with the com-
monly accepted tissue based therapy material, cadaver skin, sh skin is
found to be more potent in omega-3 fatty acids while cadaver skin has
more omega-6 fatty acids [12] . Approximately 19 ± 3.8% of the total
lipid content of piscine CTPs is omega-3 fatty acids, while the content
in mammalian grafts do not exceed 0.5 ± 0.3% [12] . Omega-3 fatty
acids are theoretically responsible for piscine CTPs anti-inammatory
and anti-microbial properties. However, this fatty acid content does
not increase proliferation of native tissue, but it increases migration
rate [12] . Furthermore, the presence of omega-3 promotes host-cell
as well as adipose stem-cell ingrowth [11] . The fast-wound healing
eects of omega-3 fatty acids are demonstrated when acellular sh
skin had a statistically signicant wound closure time when compared
to porcine small-intestinal derived matrices [6] . Further research
is necessary to determine if the presence of high concentrations of
omega-3 is a major factor in accelerating wound-healing in acellular
piscine CTPs due to its contributions to increased migration rates,
anti-inammatory, anti-microbial and anti-viral properties as clear
and thoroughly studied experiments in the subject are yet to be
seen.
Acellular sh skin has antimicrobial, anti-inammatory and anal-
gesic eects. Omega-3 polyunsaturated fatty acids increase signaling ac-
tivity for concluding the inammatory stage of wound healing, as well as
mediating subject pain and tissue remodeling [11] . In an animal model
study on thermal pain sensation, it was found that polyunsaturated fatty
acids inhibit neurosensory pathways by activating microglia [19] . These
properties reduce the need for analgesic medications and promote faster
wound healing for chronic wounds [17] . The omega-3 fatty acid content
found in this CTP is uniquely high in docosahexaenoic acid (DHA) and
eicosatetraenoic acid (EPA) in contrast to commonly used mammalian
grafts, with 10.7 ± 1.4% and 8.5 ± 1.1% of the total lipid content [11] .
The mechanism of action of PUFAs such as DHA and EPA are shown
in Fig. 1 . The eicosatetraenoic acid and docosahexaenoic acid modu-
late acute inammatory response and as antimicrobial agents [16 , 19] .
They diminish inammatory responses via the up-regulation of special-
ized pro-resolving mediators [6] . There are multiple contributing factors
to this CTP’s anti-inammatory response, due to the various metabolic
responses to the rich content of omega-3 fatty acids. Another contrib-
utor to the inammatory response is the binding of metalloproteases
to the CTP, which are known to prolong the wound healing process
in high concentrations, which rebalances its concentration and has an
anti-inammatory eect [20] . Acellular sh CTPs have some biologi-
cal properties that may warrant further research into its ecacy as a
cell-based wound treatment material.
Fig. 1. Mechanism of action for PUFAs such eicosatetraenoic acid and docosa-
hexaenoic acid Abbreviations: ECM: Extracellular matrix, ICM: Intracellular ma-
trix, NM: Nuclear matrix, EPA: Eicosatetraenoic acid, DHA: docosahexaenoic
acid, MEKK1: MAPK kinase 1, MEK1: MAPK kinases 1, JNK: c-Jun N-terminal
kinase, AP1: Activator protein 1, MMP1: Matrix metallopeptidase 1, IKK: IkB Ki-
nase, IkBa: NFkB Inhibitor a, COX-2: Cyclooxygenase-2, IL1-B: Interleukin 1-B.
[21] .
3. Applications
3.1. Fish skin tissue based therapy in diabetic lower extremity wounds
According to the Center for Disease Control’s national report, approx-
imately 34.2 million (1 in 10) Americans are aected by diabetes as of
2020. Along with the constant maintenance of glucose levels, diabetics
with uncontrolled and high glucose levels can develop complications
including major cardiovascular diseases, neuropathy, nephropathy, and
lower extremity wounds. In 2016, there were 130,000 reported cases of
lower extremity amputations by hospitals [22] . These lower extremity
amputations are a result of foot ulcers which costs patients a total of
$38.6 billion in the United States annually [23] .
Diabetics with persistently high levels of blood glucose levels
typically develop wound healing complications. Microcirculatory de-
ciencies in diabetics include a smaller capillary size, larger basement
membrane, thickening of arterial vessel wall. These microcirculatory
deciencies described can result in improper circulation in the ex-
tremities of body. Nutrients, oxygen, and immune cells have diculty
passing through the membrane and vessel walls for exchange with
surrounding cells. Neuropathy is also linked with microcirculatory
defects in motor, sensory, and autonomic neurons. These can lead to an
increased risk for trauma as well as infections due to high compressive
forces from motor neuro deformation [24] .
Unlike normal wounds, chronic wounds are persistent wounds that
do not heal in the proper stages and have a prolonged healing time of
over 3 months. The etiology behind chronic wounds can include a com-
plex variety of neuropathy, repeated trauma, mechanical deformation,
venous insuciency, and arterial disease. Wound healing can be char-
acterized into 4 stages: hemostasis, inammation, proliferation, and re-
modeling. Chronic wounds often remain stuck in the inammation stage
of wound healing which results in chronic inammation of the site. In
97
G. Fiakos, Z. Kuang and E. Lo Engineered Regeneration 1 (2020) 95–101
addition, chronic wounds can develop infections, drug-resistant micro-
bial biolms, and unresponsive epidermal cells [1] .
Diabetic foot ulcers are the most common form of chronic wounds
found in diabetic patients with uncontrolled blood glucose levels. These
ulcers typically have an increased secretion of matrix metalloproteinases
(MMPs) and decrease in tissue inhibitors of metalloproteinases (TIMPs)
which results in delayed healing. A large reduction in transforming
growth factor (TGF- 𝛽1) –an important cytokine needed for immune cell
activation –can also be observed in diabetic foot ulcers with increased
infections [25] . Chronic foot ulcers are at a higher risk of infection and
eventual amputation.
Fish skin cellular/tissue based therapy (CTP) presents a portion of
a solution to diabetic wound care for the lower extremity. These CTPs,
like other tissue based therapies, can transition chronic wounds stalled
at the inammatory stage to the proliferation and remodeling stage for
faster wound healing. Acellular sh skin CTPs, and other products high
in collagen type I, have been reported to balance the enzymatic activi-
ties of MMPs by acting as an attachment site for breakdown of scaold
rather than developing host tissue and ECM components [26] . An in-
crease of pro-inammatory cytokines in chronic wounds are known to
delay wound healing and aect cellular processes. Bioactive lipid me-
diators such as omega-3 polyunsaturated fatty acids, EPA, and DHA are
shown to activate downstream anti-inammatory molecules. These lipid
mediators are found in abundance in sh skin CTPs which give it unique
anti-inammatory medical applications [21] . Modulators that increase
the expression of TGF- 𝛽1 were reported in sh skin CTPs, which was
shown to increase wound healing time [27] . Additionally, acellular sh
skin has favorable anti-microbial properties as described previously. The
initial application of the CTPs acts as a barrier against drug-resistant bac-
teria for at least 48 in optimum growth conditions [6] . Antimicrobial
peptides (AMP) are secreted from sh skin that defend against invading
pathogenic bacteria, viruses, fungi, and parasites [28] .
An Omega-3 wound matrix is a widely used FDA approved acellular
intact sh skin CTPs containing a variety of proteins and lipids such as
collagen, brin, omega-3, and proteoglycans. This sh skin CTP has been
applied to several studies featuring diabetic foot ulcers with promising
results. A clinical evaluation of acellular sh skin CTPs as a treatment
for diabetic foot wounds found an 84.9% median decrease in wound
area. Eight diabetic patients with postoperative diabetic foot wounds
from amputation were selected based on several criteria: wound size
between 0.5–30cm
2
, wound grade of 1 to 3, non-active infection, and
HbA1c < 12%. Fish skin CTPs were cut to cover the size of wound and
dressings were applied and changed weekly for six weeks.
Three patients were found to have 100% wound area reduction in
six weeks. In patients with chronic wounds ( > 3 months), slower healing
rates were observed at 41.1% and 41.2%. An overall trend of earlier and
faster healing times was observed for all subjects. Complete healing was
observed in 46% of patients at 5 months for transmetatarsal amputations
and 72% for digital amputations. In addition to a general reduction of
wound area, no pain, irritation, or odor was perceived as intolerable by
the patients [29] .
A retrospective, nonblinded evaluation of diabetic foot ulcer healing
through acellular skin CTP was conducted with 51 patients. The mean
ulcer duration prior to CTP application was 18 weeks which ts the
chronic criteria. A mean wound area reduction of 87.57% was observed
at 16 weeks. 60.34% of all wounds were fully healed at this mark as
well. A surface area reduction of > 90% was observed in 43 DFUs and
> 75% in 49 DFUs ( Table 1 ). Of the two wounds that saw no reduction
at 16, one healed at 24 weeks after 2 additional applications. The other
wound healed at 36 weeks after 3 additional debridement.
In a case study of a 56-year old male presenting with hemophilia
as well as type 2 diabetes had a history of chronic ulcerations on his
left foot. Prior to sh skin CTP application, worsening pain, swelling,
and redness was reported. X-rays showed degenerative changes to the
joints in the foot and multiple areas of subcutaneous air. A pathology
report noted acute and chronic inammation of the distal metatarsal
Table 1
Surface Area (SA) wound healing at 16 weeks [8] .
Wounds & Grafts N
Woun ds with 100% SA healed 35 (60.34%)
Woun ds with > 90% SA healed 43 (74.14%)
Woun ds with > 75% SA healed 49 (84.48%)
heads which indicates osteomyelitis. A trans-metatarsal amputation was
performed and the ulcer was removed. An acellular sh skin CTP was
applied 5 weeks post-surgery and followed up every 1–3 weeks for 6
more applications. At week 14, the open wound surface area was fully
reduced with no recurrence in the future [30] .
3.2. Fish skin tissue based therapy in burn care
In most industrialized countries, burn care is standardized and the
mortality of burn injuries have been dramatically reduced over the past
decades. An issue remains where 95% of all burn wounds still occur in
third world countries and emergent nations. More than 225,000 annual
deaths are resulted from burns in low and middle-income nations [31] .
In addition, the current burn care system is unlikely to be directly uti-
lized in low-income countries due to development gaps among nations
[9] . In order to promote the burn care system in under-developed coun-
tries, an economical and eective skin substitute is needed to improve
the lives of burn patients.
Before assessing the ecacy of piscine CTP in burn management, the
severity of burn is rst taken into consideration. Generally, burn wounds
are evaluated mainly based on three aspects. First, the type of burns is
classied into thermal, chemical, electrical, and radiation burns. Dier-
ent management strategies are selected depending on the cause of burns.
Second, a treatment is selected depending on the degree of burns. When
the dermis layer of skin (serious second-degree burns) or the fat layer
(third-degree burns) is aected by burns, wound dressing is required
to protect the injured area from microbes and to sustain a moist envi-
ronment. Lastly, an evaluation is made with the Wallace rule of nines
to estimate the total surface area that is aected by the burn [32] . In
smaller wound sizes, autografts are the best choice. However, in larger
wounds where large portions of skin can’t be taken from the patient,
temporary wound coverage using allografts or xenografts is ideal.
Autografts have high biocompatibility and can be left on wound
injuries permanently without rejection. However, the limitations of
autograft are the creation of new wounds and its availability. Skin
meshing is one way to cover large wounds, but healing eciency is
reduced [36] . Hence, the size and severity of burns must be taken
into consideration when using autograft [33] . When autologous skin
is not the best choice, other skin substitutes are needed for temporary
coverage to avoid complications [16] . Allograft, skin taken from donors
of the same species (mainly cadaver skin), is frequently used for
large burns as temporary coverage. Through lyophilization followed
by gamma irradiation in cadaveric skin, antigenic parts of grafts is
removed, and no immunogenic response is expected after skin meshing
[34] . Nonetheless, the major concern about cadaver skin is the risk of
transmission of diseases and infective agents that may still occur after
viral disease screenings and standardized sterilization [35] . The quality
control and manufacturing procedures required for allografts are
considerably high [36] . Xenografts have been used in previous studies
as temporary skin substitutes for burns. Genetically modied porcine
skin graft has been investigated in terms of its eciency and toxicity
in comparison with allograft. The alpha-1,3-galactosyl transferase
knockout porcine shows similar functions to cadaver allogeneic skin
and involves no hyperacute rejection, which occurs when wild-type
porcine skin was grafted onto humans. However, only small wounds
were tested with the porcine skin [37] . More investigation is needed for
large-scale burn injuries. Also, since viral inactivation is required by the
98
G. Fiakos, Z. Kuang and E. Lo Engineered Regeneration 1 (2020) 95–101
FDA, all the biological components that improve wound healing will be
removed from porcine graft. A skin substitute that is economical, easily
accessible, and eective in wound dressing without any viral and prion
transmission risk is highly needed. The Integra
R Dermal Regeneration
Template (DRT) is an option to fulll the need. The implantation of
DRT, a bilayer material composed of a crosslinked bovine collagen and
shark chondroitin-6-sulfate dermal replacement layer and a silicone
epidermal substitute, is the standard of care for many burn centers
[38] . Within 2–3 weeks after implantation, the silicone layer will be
replaced by an ultrathin autograft and healing time of the autograft
is approximately 8–25 days [39] . Low incidence of infection and high
take rate had been reported at DRT treated sites [38 , 39] . Overall, DRT
is a safe and ecient skin substitute in treating burn wounds; however,
healing time for large-scale burn wounds can be further accelerated by
using a novel type of skin substitute. As mentioned, the application of
Atlantic cod skin CTP, a regulator of inammation, in burn patient is
nontoxic and can accelerate stem cell ingrowth [7 , 40] .
Before the discussion about Atlantic cod skin CTP, another existing
sh skin CTP showing promising results in treating both supercial and
deep partial-thickness burn is Nile Tilapia Fish Skin. A report for the
phase II randomized controlled trial of Nile Tilapia skin graft was pub-
lished January 2020. The ecacy of Nile Tilapia skin was tested in 62
participants in terms of healing time, number of dressing changes during
treatment and pain intensity assessed via the Visual Analogue Scale and
the Clinical Global Impression Scale-Improvement. Results of the study
show that less dressing changes were required for patients using Nile
Tilapia skin than for patients under other treatments when there was
no signicant change in their re-epithelialization time. Also, signicant
pain reduction was reported by inpatients treated with Nile Tilapia skin
[41] . Nile Tilapia Fish Skin is an outstanding candidate among skin sub-
stitutes in treating partial thickness burns due to its ecacy and reduced
price; however, larger scale studies are needed before it is ocially used
in burn cares.
In 2018, Stone et al. used Atlantic cod sh skin CTP as a temporary
cover for full thickness 5 ×5 cm burn wounds on anesthetized Yorkshire
pigs for seven days before split thickness skin grafts were applied. The
outcomes, measured in terms of contraction rates, trans-epidermal water
loss, hydration levels, and blood perfusion levels, of burn injuries treated
with sh skin CTPs are similar to outcomes of burn wounds treated with
cadaver skin. In addition to its function as a temporary wound cover,
sh skin CTP also shows the ability to protect split thickness skin CTP
from desiccation and shearing in the study without transmitting any dis-
ease causative agent [36] . Beside the ecacy of piscine CTP, it is also an
excellent skin substitute to be used in large burn wounds. Kjartansson
et al. compared the performance of meshed/intact sh skin CTP with
cadaver skin and regular wound dressing (Allevyn) in a full thickness
porcine burn model. Increased healing was shown in wounds covered
with 3:1 meshed split thickness skin graft after the removal of sh skin
compared with that of wounds covered with the same split thickness
grafts after allografts (cadaver skin) or Allevyn [42] . In short, sh skin
CTP can be an ecient skin substitute in burn injuries, and it can aug-
ment the healing of burn injuries even after meshed.
In 2019, Alam et al. at the Burn centre of the Queen Elizabeth Hos-
pital Birmingham used sh skin CTP as wound dressing on ten burn
casualties in both their donor sites and burn sites. The sh skin CTP was
soaked in saline and then applied to both the donor sites and burn sites
with dry gauze as a secondary dressing. Seven days after split thick-
ness skin CTPs were harvested, dressing was changed for the rst time
and every three days afterward until healing. In the donor site study, no
signs or symptoms of infection and adverse reactions was shown. All the
patients reached 100% re-epithelialization within 16 days and low pain
scores (1–4 out of 10) were reported by the patients. In the burn site
study, patients including a burn in the thigh caused by cooking-oil and
a ame injury on a hand were treated with sh skin. Feedbacks from the
patients showed that no discomfort by the sh skin except for the “sh
smell ”of the product and there was an immediate analgesic eect after
dressing. All wounds completed epithelialization within two weeks. In
comparison with other wound dressing products, reduced healing time
is shown when Kerecis is applied within this study [16] . Since Alam’s
study was a pilot case series, larger scale experiments must be conducted
to further investigate the eectiveness of sh skin CTP in burn injuries.
However, through combining the results from previous literatures using
sh skin CTP in porcine models and the results of this pilot case study,
sh skin CTP is a potentially safe, economic and eective skin substitute
in burn surgery for temporary coverage of wounds.
3.3. Fish skin tissue based therapy in combat casualty care
Combatants are subjected to severe thermal burns due to a higher
risk of exposure to rearms, incendiary weapons, and explosives. Due
to a lack of medical care, 88% of battleeld deaths happen before casual-
ties can arrive at any medical treatment facility [43] . The Joint Trauma
System and the Committee on Tactical Combat Casualty Care has pro-
vided guidelines for battleeld trauma care and re-resistant combat
shirts are provided to avoid burns resulting in an increase casualty sur-
vival. However, there are body parts that cannot be covered by these
shirts and the guideline focuses more on hypothermia prevention for
extensive burns with little detail on wound dressing and debridement.
Therefore, in terms of wound care on the battleeld, improved wound
cares are needed to improve casualties’ survivability before any com-
prehensive treatment is available.
As mentioned in the burn care section, cadaver skin and porcine skin
are frequently used skin substitutes in wound dressing. In addition to the
risk of viral disease transmission in cadaver, the preservation involved
with cadaver skin past 14 days is dicult [44] . Cryopreservation is an
option for extending storage life [45] . However, the viability of skin
decreases dramatically within two days at room temperature and is dif-
cult to maintain on the battleeld [46] . While short shelf life and cold
storage issues prevent the use of cadaver skin on the battleeld, decellu-
larized sh skin CTPs are able to be stored at room temperature for three
years [47] . In terms of its long shelf life at room temperature, decellu-
larized sh skin matrix is a functional skin substitute on the battleeld.
Another advantage of sh skin CTPs, over other skin graft options, is
that it is bioresorbable. Yang et al. conducted a study to evaluate the ef-
fectiveness of sh skin CTP in wound closure. In Yang’s study, moistened
sh skin CTPs were directly applied to wounds of 18 patients. Without
including any surgical procedure, grafts were attached to the wound by
applying surgical adhesive and surgical strips. Secondary dressing was
used to preserve moisture and the grafts were reapplied weekly on top of
the previous graft. Wound surface area and wound depth were dramati-
cally reduced (40% and 48% respectively) over a ve-week period after
dressing and the sh skin grafts were absorbed within ten days without
any complications shown [17] . This is an ideal property for battleeld
and combat applications as wounds don’t require constant care and re-
moval.
In a multicenter report demonstrating the uses of sh skin CTP in
complicated wounds, 25 patients with complicated wounds were treated
and achieved full wound healing at the end of treatment. Seven patients
with complicated wounds (including bone exposure and amputation)
were treated with sh skin CTPs covered by silicone foam dressings.
Even though wound healing of some patients was impaired before treat-
ment, complete wound closure was achieved for all the patients within
41 weeks. In addition, pain level reduction was reported by all patients.
Wounds of 10 patients (vascular wounds) were dressed by sh skin CTPs
with polyurethane foam dressing on top. The secondary dressing was
changed every two to three days while the sh skin CTPs were held in
place. All sh skin CTPs were absorbed within seven days and no compli-
cation was shown until completed wound closure. Improved broblast
activity and early granulation were seen in all ve patients with compli-
cated wounds after the sh skin CTPs were applied with polyurethane
foam dressing or negative pressure wound therapy [47] . To sum up,
wound healing of all the patients within the study was impaired due
99
G. Fiakos, Z. Kuang and E. Lo Engineered Regeneration 1 (2020) 95–101
to dierent complicated wound conditions and patients were all sub-
jected to the risk of amputation or re-amputation, a common issue on
the battleeld. All the wounds reached complete closure and the e-
cacy of sh skin CTP in complicated wounds was shown. In addition,
no surgical procedure is required, making it easy to apply on the bat-
tleeld. With surgical adhesive and a secondary dressing, sh skin CTP
can be applied to wounds and it will be absorbed within seven days. Ef-
cacy, ease of use, and bioresorbable are key features needed in austere
environments.
Two of the main complications of battleeld wounds are sepsis,
mainly caused by bloodstream infection on the battleeld, and impaired
wound healing due to chronic inammation [48] . Omega-3 fatty acids,
found in abundance in sh skin, act as an antibacterial as well as con-
tains anti-inammatory properties [49 , 50] . Additionally, omega-3 fatty
acids have been shown to simulate cell ingrowth in grafts allowing for
improved wound environment [6] . Reduction in wound complications,
increased graft survival, and improved wound healing are needed in
combat wound casualty - which sh skin CTPs oer the ideal solution
to.
In comparison to other frequently used grafts, sh skin CTPs shows
the potential to become an eective and ecient wound dressing ma-
terial on the battleeld. First, sh skin CTP can be stored in room tem-
perature with a long shelf life. Second, the graft can be resorbed by the
wound bed and no surgery procedure is required for piscine CTP dress-
ings. Third, sh skin CTP is eective in wound covering and ecient
in promoting wound healing. All these characteristics of sh skin CTP
are highly valuable in the battleeld. Casualties can obtain better cov-
erage for their wounds before arriving at a medical treatment facility
and increased survivability with less adverse complications.
Conclusion
Decellularized sh skin is a xenograft that requires very little manu-
facturing before being usable in a clinical setting. This minimal man-
ufacturing requirement makes the product more aordable and con-
tains some unique properties. The structure is very similar to human
skin where the scaolding is primarily composed of collagen I and
this scaolding is extremely porous. Furthermore, this material is anti-
inammatory, antimicrobial, antiviral, hypoallergenic and has analgesic
eects. This is thought to be largely due to the retention of naturally de-
rived compounds within the collagen scaold, including an extremely
high content of omega-3 fatty acids like EPA and DHA. Piscine CTPs are
an aordable option for wound care that has a comparable ecacy to
commonly used and accepted natural CTP materials.
Fish skin CTPs have shown promising uses in a variety of applications
that complement the native healing mechanisms and immune defense.
Chronic diabetic foot ulcers are a pressing issue for those living with di-
abetes that deal with delayed wound healing due to uncontrolled blood
sugar levels [22] . Fish skin can progress these chronic wounds past the
inammatory stage and to the proliferation and remodeling stages of
wound healing. Several clinical studies have shown the accelerated heal-
ing time on diabetic foot ulcers in patients. With 84% of patients seeing
signicant wound healing of 75% or more, sh skin CTPs have seen
widespread success in clinical uses. In addition, an ongoing clinical trial
(NCT04133493) aims to evaluate the eect of Kerecis Omega3 Wound
in treating diabetic foot ulcers in compare with standard of care and the
study is expected to complete in January 30, 2021 [51] . Similarly, burns
wounds can be treated by sh skin CTPs which acts as a natural barrier
while promoting wound healing on the aected area. In human trails,
complete re-epithelization with little to no signs of infection or adverse
eects were observed in all subjects treated the FDA approved sh skin
CTP [16] . Fish skin CTPs have been shown to treat both surface and
deep level burn wounds as well as prevent bacterial infections, which
is commonplace in the battleeld where sterile conditions are dicult
[52] . These results have the potential to translate into a practical and
eective wound care treatment in combat casualty. Its ease of storage at
room temperature makes it an easy tool to bring to battle where proper
temperature control is not necessary.
As promising as sh skin CTPs appear to be, there are a few lim-
itations that should be addressed. Not many of the studies using sh
skin CTP as a tool for accelerating wound healing was tested against a
control group to show ecacy. While sh skins CTPs are promising, it
is quite possible that a subcutaneous injection of the bioactives found
will be sucient rather than harvesting sh skin. The resulting healed
wound of sh skin CTPs often appear discolored and has scar tissue for-
mation. For cosmetic purposes, sh skin CTPs may not heal the wound
to its original state.
A future direction for studying sh skin CTP properties would be to
examine its eect on the age and gender of the patient, as well as me-
chanical testing on its elastic modulus. Additionally, sh skin CTPs can
be used in the veterinary eld for burn wounds in other animals includ-
ing dogs and cats. Deployment of sh skin CTPs for third world countries
as a cheap and eective alternative to cadaver grafts would be promis-
ing. While sh skin CTPs are currently being used to study near-surface
level wounds, the future applications may not be limited to this aspect.
With high levels of collagen and proteoglycans, sh skin CTPs have po-
tential to be used in assisting tendon procedures. The decellularized CTP
may be used to wrap an injured tendon to enhance healing.
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