Engineered Regeneration 3 (2022) 217–231

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Engineered Regeneration
journal homepage: http://www.keaipublishing.com/en/journals/engineered-regeneration/

Development of fish collagen in tissue regeneration and drug delivery

Meison Furtado, Liang Chen, Zehao Chen, Ao Chen, Wenguo Cui∗
Department of Orthopaedics, Shanghai Key Laboratory for Prevention and Treatment of Bone and Joint Diseases, Shanghai Institute of Traumatology and Orthopaedics,
Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, P. R. China

a r t i c l e i n f o a b s t r a c t

Keywords: Tissues rely on collagen for structural and biological integrity, as well as for function and strength. Several
Collagen collagen sources have been investigated due to its wide variety of applications, such as collagen from cows and
Fish collagen pigs. However, mammalian-based collagen has been limited by diseases like bovine spongiform encephalopathy
Marine collagen
(BSE) and other religious limitations. Hence, fish collagen has caught the attention of the research community
Drug delivery
because it is easy to extract, has a high level of collagen content, excellent absorption properties, a low molecular
Tissue engineering
weight, biocompatibility, little risk of disease transmission from animals to humans, negligible environmental
contamination, and fewer ethical and religious concerns, posing as an ideal resource for product development.
This review focuses on the growing role of marine collagen in the advances of various biomedical applications,
such as drug delivery, tissue engineering, regeneration, and wound healing, which will be covered in depth.

1. Introduction
As a component of the connective tissue layer of the extracellular
matrix, collagen is an essential protein in the living body that forms the
structure of bones, cartilage, cartilage capsules, joints, tendons, skin,
hair, and nails [1] (Fig. 1). Tissues rely on collagen for structural and bi-
ological integrity, function, and strength [2,3]. Mammals have an abun-
dance of collagen in their connective tissues, roughly with their pro-
tein content ranging between 25% and 35%. The molecular weight of
collagen is about 300 kDa, diameter between 14 and 15 Å, and length
around 2800 Å. Generally, it consists of three polypeptide chains, known
as 𝛼 chains, arranged in triple-helical structures containing (Gly-X-Y)n
repeating sequences with Pro-usually located at X and Y. There are cur-
rently 28 different types of collagens, and these sequences contribute
to triple-helical conformation by limiting dihedral angles of the main
chain [4,5].
In biomedicine, collagen primarily serves as a drug carrier, tissue en-
gineering material, absorbable surgical suture, bone filling, osteogenic
material, hemostatic agent, enzyme immobiliser, and burn/wound
dressing [6–8]. Several sources of collagen have been investigated be-
cause of the wide variety of collagen applications, such as collagen from
cows and pigs. However, mammalian-based collagen has been limited
by diseases like bovine spongiform encephalopathy (BSE) and other re-
ligious beliefs [9].
One of the most crucial elements of biomaterial design is select-
ing a starting material similar to an existing one, as exemplified in
Fig. 2. Grasping the potential value of fish collagen as a biomaterial has
been a subject of intense research in biochemistry and biology in recent
years.
As a result of the fishing industry’s processing methods, a large ma-
jority of fish material is either discarded or turned into low-value fish oil
and meal. Despite this, discarded parts are full of valuable proteins, vita-
mins, and minerals. With the global population growth and consumption
leading to more fish waste being produced each year, commercial and
domestic waste account for a relatively significant amount of these by-
products. Furthermore, fish waste has detrimental effects on the marine
environment, contributes to pollution, emits unpleasant odours, and is
becoming an issue of public concern. It is of great importance to ex-
amine methods of reusing industrial wastes and by-products from the
fishing industry [10].
Moreover, significant overlooked merit of the seafood industry is that
by-products of marine life are a rich source of valuable protein (10–
25%) and lipids, attracting increasing attention to the notion of using
them as collagen sources [11]. The low molecular weights, simplicity of
extraction, high collagen content, ease of absorption, biocompatibility,
low risk of transmission of diseases from animals to humans, low en-
vironmental contamination, and no ethical or religious concerns make
them ideal for product development [12]. A general comparison of ma-
rine materials for collagen is shown in Table 1. To better understand
this article, a detailed organisation of the main topics can be followed
in Table 2.

∗
Corresponding author.
E-mail address: wgcui80@hotmail.com (W. Cui).

https://doi.org/10.1016/j.engreg.2022.05.002
Received 18 April 2022; Received in revised form 13 May 2022; Accepted 18 May 2022
Available online 21 May 2022
2666-1381/© 2022 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/)
M. Furtado, L. Chen, Z. Chen et al. Engineered Regeneration 3 (2022) 217–231

Fig. 1. Collagen distribution in the different parts of the

human body. Created with BioRender.com.

Fig. 2. Different potential marine sources

for collagen synthesis. Created with BioRen-
der.com.

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M. Furtado, L. Chen, Z. Chen et al. Engineered Regeneration 3 (2022) 217–231

Table 1
Applications and remarks of some of the most commonly used marine collagen sources.

Source species Collagen Application/Remarks Ref.

Sea urchin type I Mechanically resistant biomedical devices [119]

Eal fish type I Blue biomaterial for biomedical application 42 [120]
Squid type I and V No toxicity, used in 3D cell culture systems [121–123]
Tilapia Scales type I Osteoblastic differentiation, wound dressing, drug delivery [124,125]
Atlantic Codfish type I Tissue regeneration and engineering, Cosmetics, Orthopaedics [126]
Jellyfish (no umbrella) type II Can replace mammalian collagen [43]
C. Remiformis Type IV Suitable for biomedical applications, Orthopaedics, Dentistry, Drug delivery [127–129]
Marine Sponge Type IV Bone regeneration [130]
C. Tagi Jellyfish Type V and XI Drug delivery, Protein delivery [87,127]

Table 2
Main points of the review.

Section 1 Section 2 Section 3 Section 4 Section 5

Titles Introduction Advantages Preparation of fish collagen General application Specific Other applications Conclusion Future
marine-based collagen Extraction methods application directions
characterization
Main points Remarks of different Preparation, extraction and Drug delivery Protein Tissue engineering Bone
marine sources recovery Different techniques delivery Gastric delivery repair Vascular tissue
for extraction Mineral delivery Hormone regeneration engineering
delivery Multifunctional Corneal tissue regeneration
effect and engineering

2. Advantages and characterisation
Tropocollagen, a collagen monomer, is a cylinder-shaped protein
with a diameter of 15 Å and a length of 3000 Å that is made up of three 𝛼
chains of polypeptide or peptide molecules having 100,000 Daltons each
[13]. Alpha chains of each collagen type have unique domain structures
that contribute to the differentiation of the collagen types [14]. Among
collagen’s components are glycine (33%), proline (12%), alanine (11%),
and hydroxyproline (10%), but it lacks the necessary amount of amino
acids like tryptophan and lysine [15]. Most of the collagen in the body
is type I, while type II, III, and IV are also common [16]. As a result of
their inherent biocompatibility, flexibility, and biodegradability, deriva-
tive biomaterials are suitable candidates for numerous functional appli-
cations. A potential advantage of these biopolymers is that thanks to
their amenable chemical properties, they can easily be combined with
synthetic compounds and greatly enhance their physiochemical capabil-
ities [17]. Collagen derived from porcine, bovine, and cattle sources is
the most abundant, commercially available and economically expensive
[18]. Genetically engineered bacteria can also be used to produce col-
lagen. Although this method makes a small amount of collagen, it leads
to a high cost of production [19].
Unlike animals’ collagen, being bovine and porcine collagen, recom-
binant collagen retains its triple helix (triple-helix configurations), an
essential characteristic of its biological properties. In this structure, in-
termolecular bonds link three chains together, resulting in rigidity and
poor solubility. Tropocollagen molecules interact to form collagen [20].
Regardless of its biocompatibility, amino acid composition and aquatic-
type collagen have similar properties to mammals’ collagen. The amino
acid glycine makes up about 30% of both collagen sources, while hy-
droxyproline makes up about 35% to 48% of total amino acid content
[21]. Its structure differs from mammalian collagen in terms of proline
concentration and hydroxyproline content resulting in lower viscosity
and thermal stability [22]. Among polypeptide chains, proline and hy-
droxyproline are present at the X and Y positions, respectively [23].
Fish collagen is one of the essential proteins used in food, cosmet-
ics, pharmaceuticals and biotechnology. It has superior water absorption
capacity, biocompatibility, biodegradability, porosity, and low immuno-
genicity. It is easy to process, has a lipid-free interface, a native ability
to combine with synthetic polymers, and nonexistent or limited ethi-
cal considerations or religious concerns [24]. Obtaining cheap collagen
may be possible through fish processing waste from the fishing indus-
try. The processing industries utilise approximately 25% of the total fish
weight, while the remaining 75% is considered waste [25]. Skin, bones,
and scale make up about 30% of this waste, which is high in collagen;
hence, it could be used for synthetic collagen. Moreover, it could reduce
the environmental impact of fish waste and increase the economic re-
turns of the fishing industry by converting waste into collagen [26,27].
Since marine collagen (proline and HYP compounds) contains a lit-
tle amino acid, its denaturation temperature is lower, rendering it chal-
lenging to handle since human body temperature causes it to denature
[28]. Compared to mammalian collagen (39–40 °C), fish collagen has a
lower denaturation temperature with a range between 25 and 30 °C for
most fish species and varies in composition, which restricts its applica-
tion in biomedical technology [29]. The collagens made from fish and
invertebrate sources, including the skin, bones, cartilage, and scales,
are more bioavailable and absorb up to 1.5 times better than bovine
or porcine collagen [30]. Their small particle size and low molecular
weight provide more rapid bloodstream circulation [31]. Furthermore,
marine-based collagens have a similar amino acid composition and bio-
compatibility to bovine and porcine collagen [32]. A general compari-
son between a marine and mammalian collagen is reflected in Table 3.
Collagen matrices can easily absorb wound exudates. Biodegradable gels
or sheets are formed on the wound surface to prevent dryness, promote
healing, and prevent mechanical trauma [33]. As a result of their cell ad-
hesion, high porosity, permeability, biocompatibility and low toxicity,
collagen from marine sponges, for example, provide adequate properties
for tissue regeneration [34].
The preparation of collagen sponges involves preparing aqueous so-
lutions and then lyophilising them. Variations in the freezing rate and
collagen concentration before lyophilisation can control the porosity of
sponges [35]. In collagen, glycine and hydroxyproline play a crucial role
in establishing the structure and maintaining its characteristics. Never-
theless, some differences in amino acid concentrations between aquatic
species have been reported. Researchers report that isolated collagen
from marine sponges (Chondrosia reniformis) contains 18.9 glycines and
40% hydroxyproline, respectively [36].
Above all, fish-derived collagen, compared to their original non-
hydrolysed form, also presents anti-hypertensive properties, wound
healing properties, and bioactive properties such as anti-inflammatory,
antimicrobial mechanisms, antioxidant and anti-ageing protection for
the skin [36]. From all the properties mentioned earlier of marine-
based collagen, researchers have become increasingly interested in ma-

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Table 3
Basic summary of marine and mammalian collagen in terms of their properties.
Properties Marine collagen
Water absorption 1.5x higher
Biocompatibility Similarly high
Aminoacidic composition Lower
Viscosity/ thermal stability Lower due to lower concentration of proline concentration and hydroxyproline content
Denaturation Lower (25–30 °C)
Flexibility Higher
Molecular weight Lower due to small molecular size
Biodegradability Similarly high
Porosity Similarly high
Rigidity Higher due to its triple helix
Solubility Lower due to its triple helix
rine collagen’s anti-inflammatory properties, which have broad applica-
tions in biomedical research. Scientists have developed tissue engineer-
ing scaffolds that have anti-inflammatory properties based on the anti-
inflammatory properties of marine collagen. Pathological changes and
the development of osteoarthritis (OA) clinical symptoms are influenced
by the sustained release of pro-inflammatory cytokines by macrophage
M1 cells. In addition to reducing macrophage migration, marine colla-
gen can inhibit the production of inflammatory cytokines. This finding
strongly suggests that marine collagen could be used in the treatment of
OA [37].
By administering collagen peptides from skin Salmo Salar and Tilapia
Nilotica skin cells in an animal wound model, it is possible to upregu-
late NOD2 and BD14 activity. Additionally, collagen peptides increased
wound angiogenesis and collagen deposition in the wound tissue. There-
fore reducing the number of cytokines and providing better control over
the inflammatory reaction [38]. The collagen hydrolysate obtained from
marine collagen also exhibits excellent anti-inflammatory properties.
According to another study, the collagen hydrolysate from squid skin
showed a significant ability to reduce levels of oxidative stress prod-
ucts, which aggravate inflammation [39]. The first type II collagen to
be isolated from squid cartilage was separated by Mei et al. The effects
of collagen type II on macrophage chemotaxis and inflammation were
investigated. The type II collagen enhanced TCPTP activity, resulting
in significant reductions in inflammatory cytokines and dephosphoryla-
tion of p-STAT1 in macrophages [38]. An overall generalization of the
transformation process from marine sources into collagen with various
properties and its possible applications are illustrated in Fig. 3.
3. Preparation of fish collagen
Waste fish can be converted into feed, biodiesel, biogas, dentifrice,
natural pigments, natural soil fertiliser, and used in enzyme isolation,
moisture stabilization in foods, and lastly, as a source of fish collagen
that is sustainable [40]. Various fish by-products, such as scales, fish-
bone, and skins, resulting in tremendous amounts of waste (50–70 per-
cent of the original raw materials) from fish processing factories and
fish shops, can be used to produce fish collagen [41] (Fig. 4). The pro-
cess of isolating collagen from marine sources, such as fish by-products,
can be broken down into three primary steps: preparation, extraction,
and recovery [2]. Fabrication processes significantly influence the prop-
erties of marine collagen-based constructs [42]. A general preparation
method for marine collagen and the different extraction techniques be-
tween acid-soluble collagen (ASC) and pepsin soluble collagen (PSC) are
shown in Fig. 5.
3.1. Preparation
The preparation of fish parts includes cleaning, separation from the
animal, and sizing or grinding into powder before a chemical pretreat-
ment removes non-collagenous proteins, fats, and pigments [43,44].
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Engineered Regeneration 3 (2022) 217–231
mammalian collagen
Lower
Similarly high
Higher
Higher
Higher (39–40 °C)
Lower
Relatively higher
Similarly high
Similarly high
Lower
Higher
The triple helix structure of marine collagen (MC) renders it insolu-
ble in cold water. Therefore, it must be subjected to specific treatment
with dilute acids or bases capable of breaking the crosslinks prior to ex-
traction. In collagenous tissues of older animals, more cross-linkers are
present, resulting in a more challenging time solubilising them than in
the collagenous tissues of younger animals. In addition, starved fish tend
to produce more collagen than well-fed fish [45]. Hence, the methods
used to obtain collagen-based macromolecules vary depending on the
collagen source.
Collagen has poor solubility in acetic acid in invertebrates that con-
tain a lot of water, such as jellyfish; therefore, its extraction capacity
is enhanced through homogenisation or freeze-drying when such con-
ditions occur [2]. Sea urchin collagen cannot be soluble in acid using
the traditional acid solvent extraction method. Using SDS-based decel-
lularising solution combined with hypotonic solution, Sugni et al. ex-
tracted intact collagen fibrils from sea urchins [46]. However, to facili-
tate acid action, these compounds must also be reduced in size where,
for instance, jellyfish have arms separated from umbrellas, as mesoglea
generally have exumbrellas and subumbrellas [43].
3.1.1. Non-collagenous proteins removal
In most cases, non-collagenous proteins and pigments can be re-
moved by using sodium hydroxide (NaOH) [9], and the use of its di-
luted form has been the most common alkaline method because it does
not affect the functionality of collagen. In contrast, an increase in NaOH
concentration can result in significant collagen loss [47]. According to
Skierka et al.’s study, NaCl can be used to remove non-collagen proteins
from cod skin, but it is not as effective as that of NaOH [48]. As for acidic
and enzymatic approaches, pepsin is overall widely preferred [49,50].
3.1.2. Pretreatment and fat removal
Pretreatment with an acidic solution cleaves non-covalent interac-
tions within and between molecules. In contrast, pretreatment with an
alkaline solution removes pigments and non-collagenous proteins with-
out altering collagen structure [44]. Alcohols such as butyl‑alcohol or
ethanol effectively remove fats and pigments, as are oxygen peroxide
solutions [9].
3.1.3. Demineralisation
Collagen is extracted after non-collagen proteins are removed, dem-
ineralisation is conducted, and by-products are defatted. The appropri-
ate conditions must be established to get collagen’s best properties and
functionality [51]. It was found that HCl, citric acid, and ethylenedi-
aminetetraacetic acid (EDTA) were effective in demineralising scales
and bones with approximately 90% efficiency. Due to the possible loss
of collagen caused by these acids, chemical agents must be carefully
selected [52].
3.2. Extraction
As for the extraction phase, a topic discussed in further detail
throughout this review, acid and pepsin solutions are widely used for
M. Furtado, L. Chen, Z. Chen et al. Engineered Regeneration 3 (2022) 217–231

Fig. 3. Overall illustration of the transformation process of marine collagen sources into multi properties collagen and its various material adaptations for different
applications. Created with BioRender.com.

Fig. 4. SEM analysis of ASC from Larimichthys crocea. a, b, c and d: the ASC from bones, skins, scales and muscles before fibres, × 1000 magnification; and e, f, g
and h: the ASC from bones, skins, scales and muscles after fibres, × 10 000 magnification. Reproduced with copyright permission [118].

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M. Furtado, L. Chen, Z. Chen et al.
solubilising collagen; hence the terms acid-soluble collagen (ASC) and
pepsin-soluble collagen (PSC). For MC extraction, acetic acid is the most
popular dilute acid, although acids like citric acid, HCl and lactic acid
can also provide good results.
3.3. Recovery
The recovery step requires the precipitation of collagen, which is
generally accomplished by adding NaCl to the final concentration of
Tris–HCl (pH 7.5). Acid and Pepsin soluble collagen can be obtained
by centrifuging the precipitate, dissolving in acetic acid, dialysing, and
freeze-drying [10,50,53–55].
3.4. Yields
The yield of collagen from insoluble components can be increased
by hydrolysing them with enzymes to safely remove collagen from the
target tissue by cleaving the non-helical parts called telopeptides [56].
Certain enzymes have been employed during this process, including pan-
creatin, trypsin, fucine, papain, bromelain, and pepsin, with marine by-
products primarily made of the latter [49,50]. Apart from telopeptides,
the NaOH treatment method can also remove antigenicity from non-
collagenous proteins, cells, and cell remnants [57]. Although used on
codfish skin, the conventional acid solubilisation method yielded little
collagen, while acetic and lactic acids produced more significant quanti-
ties [58]. According to Skierka and Sadowska, acetic and lactic acids ren-
der higher collagen extraction yields than hydrochloric and citric acids
[48]. Solubilised collagen is then added to sodium chloride (NaCl), cen-
trifuged, and salted as the final step. The resulting precipitate is dialysed
and then freeze-dried.
4. Extraction methods
Collagen extraction has always been held to high efficiency and ef-
fectiveness [16]. In light of the different compositions and structures
of extracellular matrix (ECM) in other animal regions, suitable prepa-
ration processes must be used to extract collagen from marine animals.
The adjacent name for collagen derives from the method of extraction.
It’s referred to as acid-soluble collagen (ASC) when extracted with an
acid solution, and it’s called pepsin-soluble collagen (PSC) or atelocolla-
gen when extracted with pepsin. Generally, proteolytic procedures are
used after removing ASCs [56]. To remove collagen soluble in acid, a
strongly acidic solution is used, and hydroxychloric, acetic, citric, and
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Engineered Regeneration 3 (2022) 217–231
Fig. 5. General preparation method for ma-
rine collagen. 1. Selection of fish parts as colla-
gen source; 2. Acid treatment or Pepsin treat-
ment; 3. Purification steps; 4. Dialysis and
freeze-drying; 5. Acid-soluble collagen (ASC)
and Pepsin soluble collagen (PSC). Created
with BioRender.com.
lactic acids have been traditionally used for extracting collagen. A study
by Skierka et al. shows an improved performance when organic acids
are used, including lactic acid and acetic [48]. A triple helix structure
gives collagen poor solubility in cold water because of solid crosslinks.
Heat can increase water solubility; however, it is necessary to use either
diluted acids or bases to break crosslinks in the animal tissue before ex-
traction. Marine by-products like skin scales, bones, and fish fins can
be extracted using the mentioned methods to isolate collagen soluble in
acetic acid [51,59,60].
4.1. A general method for extraction
The following methods are typically used to obtain collagen from
aquatic by-products:
1) Extraction with acids and alkalis: A combination of acids and alkalis
extracts non-collagenous proteins from the prepared skin by mix-
ing it with NaOH for two hours. A mildly basic pH is obtained by
washing the mixture with tap water. A one-hour demineralisation
process with HCl is subsequently administered to the deproteinised
skin. This residue is then thoroughly washed with tap water until the
water becomes neutral or mildly basic in pH. The demineralised skin
is then swollen by stirring for 15 min with acetic acid mixed into the
skin. To end the process, the swollen skin is thoroughly washed with
tap water until the pH of the wash water becomes neutral or barely
acidic. In various research studies, distilled water was mixed with
swollen skin at different temperatures (45–75 °C) for varying peri-
ods (6–12 h). Collagen was then obtained by filtering the mixtures
[61].
2) Extractions with acid and enzymes: Bones and scales are soaked in
NaOH for 24–48 h with stirring. After filtering and rinsing with dis-
tilled water until a neutral pH is achieved, the alkali-insoluble com-
ponents are removed. The insoluble components are extracted over
3–6 days using acetic acid or acetic acid containing pepsin. After cen-
trifuging the solution, the supernatants are salted again by adding
NaCl overnight, centrifuging the residues and then dissolving them
in acetic acid. A dialyse of the resulting solution is performed against
acetic acid to obtain collagen [62].
Collagen is hydrolysed, then dissolved in acetic acid and dialysed
[63], and the following centrifugation, the residue is re-dissolved in
acetic acid. To achieve the final product, the extract is dialysed in acetic
acid and lyophilised [64].
M. Furtado, L. Chen, Z. Chen et al.
4.2. Other methodologies for extraction
Although the technique used above is affordable and straightfor-
ward, these methods are time-consuming, and bulky raw materials and
excessive solvents must yield better. Using a one-step extraction, under
mild conditions, for short times (3 h), Silva et al. present an advanced
method for gelatin extraction from the sponge Chondrosia reniformis
[65]. Using carbon dioxide pressurised water is the basis of this method.
This optimization study involved the usage of high-pressure vessels (up
to 10 bar) with 37 °C, carbon dioxide pressure, and constant agitation of
300 rpm for 3 h while collagen from marine sponges was used. This sys-
tem makes it possible to extract more than 50% collagen from a sample,
improving more than 30% on extraction yield. Compared to traditional
acids/enzymes, this approach has a higher yield. In addition, it appears
to be a simple and inexpensive method that is environmentally sustain-
able.
In a study by Kim et al., ultrasound collagen extraction improved
performance over traditional methods, even when acetic acid concentra-
tions were lower [66]. According to other studies, ultrasound extraction
can cause hydrogen bonds to break between collagen chains, damaging
the collagen proteins and the enzyme used to isolate them [67]. There
is also evidence that continuous use of ultrasound leads to a rise in tem-
perature, which causes collagen to denaturalise.
An extrusion-hydro extraction method was proposed by Huang et al.
for collagen extraction from tilapia scales (Oreochromis spp.). Three ma-
jor stages are involved in the process: pre-conditioning with citric or
acetic acid, extrusion (360 rpm with 135 °C), and hydro extraction (25
and 50 °C). Extraction of collagen is made possible by combining heat
and mechanical force. The collagen isolated from this process had type
I properties and could make foods, cosmetics, and medicines [68].
5. Universal application
The major component of the ECM, collagen, is a protein with a fi-
brous structure and varies in appearance according to the tissues. Among
its many applications is the formation of bone (Type I), cartilage (Type
II), and blood vessel walls (Type III), as well as presenting an exciting
ability to delay the natural aging process of the human tissue [69]. This
protein is non-cytotoxic, biocompatible, and biodegradable. However,
it has low elasticity and mechanical strength, poor dimensional stability
due to swelling in vivo, and the ability to crosslink to tailor its me-
chanical, degradation, and water-uptake properties [70]. Hemostasis,
inflammation, proliferation, and remodeling require collagen as a struc-
tural support scaffold or base for new and progressive tissue growth. If
collagen is absent, new tissue cannot form [71]. Aside from tissue engi-
neering, it can also be used to manufacture artificial blood vessels and
valves, skin replacement, and bone substitutes [72].
Collagen from mammalian sources has many applications, includ-
ing delivering drug-based formulations such as mini-pellets and tablets,
controlling transdermal and biological delivery material, and collagen-
based sponges for wound healing [3]. Various factors contribute to col-
lagen degradation, including aging, ultraviolet light exposure, and to-
bacco use. To improve regenerative tissue applications, it is essential to
find new sources of collagen that prevent wrinkles, stiff joints, sagging
skin, dry skin, and so on [73].
Marine collagen is used in several fields, and different processes
can be followed depending on the precise application and formula-
tion desired. Marine collagens can be processed using similar process-
ing methodologies as mammal collagens. However, due to some differ-
ences in their properties, they may require some tweaking for further
improvements. Meanwhile, over the past few years, the idea of "sustain-
able development" has emerged as one of the essential elements of green
economy initiatives. Environmental and economic concerns dictate the
need to use eco-friendly alternatives to exploit natural resources glob-
ally. Therefore, the European Commission approved "Blue Growth" as a
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Engineered Regeneration 3 (2022) 217–231
long-term strategy to support sustainable developments in the maritime
and marine sectors [28].
The collagen from marine fish is used in a wide range of biomedical
applications, and among numerous studies, bone tissue engineering has
been the gold standard [74]. A recent survey by Hoyer et al. created
bone-regenerating scaffolds using salmon skin collagen and hydroxyap-
atite in a biomimetic mineralisation process [75].
Its good absorption characteristics enhanced this specimen’s me-
chanical properties and interconnected pores, which are essential for
Mesenchymal Stem Cell (hMSC) adhesion and proliferation, contribut-
ing to osteogenic differentiation [75]. In the paper published by Na-
gai and co-workers [76] for periodontal regeneration applications, the
extracted collagen from salmon was crosslinked with carbodiimide
hydrochloride (EDC) 1-ethyl-3-(3-dimethyl aminopropyl) and dehy-
drothermal treatment (DHT). Terada et al. [77], with the help of col-
lagen extracted from tilapia scales, developed chitosan-collagen com-
posite scaffolds for oral mucosa regeneration.
To evaluate the stability and delivery of drugs to the dermis, the
researchers developed microparticles made from collagen from the ma-
rine Sponge C. Reniformis and incorporated them into hydrogels [78].
Furthermore, composites of chitosan, hydroxyapatite, and collagen from
Ircinia Fusca were prepared using lyophilisation methods, the result of
which showed that osteoblast-like MG-63 cells could adhere and multi-
ply [79].
6. Specific delivery applications
In the past few decades, collagen has been considered a significant
material in the field of biochemistry, especially when it comes to devel-
oping new ways to synthesise biomaterials with stunning capabilities for
use in medicine. Similarly, marine collagen can follow the same trends
as expressed in Fig. 6.
6.1. Drug delivery
As the name implies, a drug delivery system is a vehicle for adminis-
tering pharmaceuticals to patients to achieve therapeutic effects. Many
approaches to drug delivery have been demonstrated throughout hu-
man and animal history, including dose titration, individualizing drug
therapy, therapeutic drug monitoring, controlled drug release, slow-
release, and targeted release [80]. Drug delivery by large-sized materials
presents several challenges, including instability, in vivo poor bioavail-
ability, poor solubility, poor body absorption, and potential side effects.
New delivery systems that allow drugs to be targeted to specific parts of
the body might be an option to solve these problems. An increasing body
of research is currently being conducted on nanoparticles and micropar-
ticles as drug delivery systems (Fig. 7). The controlled drug release and
delivery of nanotechnology-based formulations have proven immensely
successful for advanced medicine/drug formulations [81]. This section
aims to examine how the properties of marine collagen can be used to
administer drugs by acting as a matrix. These properties include fluidity,
ease of handling, and biocompatibility.
The properties of collagen films and gels synthesised from eel fish
(Evenchelys macroura) skin using ASC and PSC have been tested with
several drugs, such as ampicillin and tetracycline. By acting as carriers
for antibacterial and antifungal drugs, these collagen gels and films are
good candidates for use in drug delivery systems [82].
The collagen derived from marine sponges (Porifera, Dictyoceratida)
made polymeric wound healing films with l -cysteine hydrochloride.
Studies in vitro showed that cysteine released from the biopolymer is
slower than the pure drug at wound sites. The combined healing abili-
ties of cysteine and the benefit of using a biocompatible carrier capable
of absorbing excess wound exudate while releasing therapeutic agents
make the system ideal for sea-derived collagen and bio-based drug de-
livery [83]. As a result of its glycosaminoglycan content, sponge skele-
M. Furtado, L. Chen, Z. Chen et al.
tal scaffolds can function as bioactive-biomimetic carriers that speed
wound healing.
To make allopurinol more bioavailable in humans, Nguyen et al.
[84] developed a pH-sensitive hydrogel from fish scale collagen and
carrageenan. In simulated body fluids, this hydrogel allowed for a 1.5
to 6.7-fold slower release of drug from the carrier than in the control
sample, thus increasing its bioactivity and strengthening physical prop-
erties. Researchers have also developed a low-temperature press method
in which hydrogel microneedles were prepared from crosslinked colla-
gen in fish scales, and ferrous gluconate was applied when they were
dissolved in phosphate buffer solution (PBS); they swelled and released
34.5% of their drug load within 24 h them [85].
Studies have shown that modifying collagen structure results in sus-
tained drug release and the duration of drug presence in the body.
Presently, it is mainly used to form composite microspheres, such as
collagen–polylactic acid microspheres, collagen–hydroxyapatite micro-
spheres, collagen–chitosan microspheres, and so forth [84,86]. The fact
remains that fish collagen and other marine collagen play a vital role in
the development of better methods of drug delivery.
6.2. Protein delivery
By emulsifying gelation-solvent extraction, collagen microparticles
(CMPs) were made using collagen from the coral Catostylus Tagi; this
jellyfish collagen was used as the polymeric matrix to generate micro-
sized collagen particles. Through collagen matrix crosslinking, therapeu-
tic proteins are entrapped in the system and controlled to release with
retained biological activity. Microparticles derived from fish collagen
have been studied in vitro and show promising potential for the con-
trolled release of a therapeutic protein. Some researchers have created
a dressing based on marine collagen as a topical drug delivery system
[87].
224
Engineered Regeneration 3 (2022) 217–231
Fig. 6. Application of collagen-based materials in vascu-
lar, corneal, skin, wound, bone and cartilage tissue en-
gineering (TE) and drug delivery. Created with BioRen-
der.com.
Using a polymer matrix made from jellyfish collagen for protein de-
livery displayed the potential of marine collagen for controlled drug
release, as shown by Calejo et al. [87]. Dinescu et al. demonstrated
cell viability, proliferation, and biochemical properties [88]. PLGA mi-
crospheres formulated with bFGF-loaded collagen, chitosan, and chon-
droitin sulfate scaffolds were used in a scaffold-controlled release system
for skin tissue engineering (Fig. 8) MC-based loaded platforms had cus-
tomisable protein release rates dependent on collagen: chitosan ratio
present in the scaffold, and showed decent biocompatibility and poten-
tial in controlled release delivery systems, wound healing, and tissue
regeneration [89].
Using a marine sponge as a biocompatible carrier, an innovative for-
mulation for wound exudate absorption was developed, including l -
cysteine hydrochloride, a sulfur amino acid with wound healing proper-
ties. Due to its glycosaminoglycan content, the natural sponge skeletal
scaffold may act as a bioactive-biomimetic carrier to facilitate wound
healing [90].
6.3. Gastric delivery
Chondroitin reniformis collagen was used as the basis for a gastro-
resistant delayed-release tablet coating, and it was shown that the
tablets were resistant to exposure to HCl. The novel coating dis-
played outstanding mechanical characteristics and storage stability for
delayed-release tablets and disintegrated completely in pH 6.8 phos-
phate buffer solution, showing its applicability for these delayed-release
tablets [91]. Chondrosia reniformis collagen nanoparticles were pre-
pared through controlled alkaline hydrolysis and used to deliver 17𝛽-
estradiol-hemihydrate via transdermal injection for hormone replace-
ment therapy [92]. Langasco et al. concluded that sea sponge colla-
gen nanoparticles were promising carriers for transdermal drug deliv-
ery. They allowed stable estradiol release compared to commercial gels
M. Furtado, L. Chen, Z. Chen et al.
and increased estradiol absorption considerably. Studies using collagen
sponges with l -cysteine hydrochloride have also shown that the drug
will release in a slow, steady manner in vitro studies [90].
6.4. Mineral delivery
Guo et al. [93] developed simple methods to obtain marine collagen
peptides (MCP) derived from marine scales and calcium alginate for en-
capsulating calcium with a core-shell MCP chelated calcium/calcium
alginate nanoparticle, about 400 nm in diameter. These findings sug-
gest that MCP-based calcium chelated nanoparticles promote calcium
absorption, prevent calcium deficiency, and increase femoral bone min-
eral density and calcium content in rats, which means that they might
make suitable carriers for calcium supplementation.
6.5. Hormone delivery
Recently, researchers have reported using collagen nanoparticles de-
rived from marine sources for dermal drug delivery. Chondrosia reni-
formis sponge collagen was emulsified and crosslinked with glutaralde-
hyde to form hydrogels. After being hydrolysed in alkaline media,
collagen nanoparticles were formed. After adsorption, 17𝛽 -estradiol
hemihydrate was added to nanoparticles, and product testing demon-
strated its efficacy in postmenopausal patients. According to the re-
search, sponge collagen nanoparticles could prolong estradiol delivery
and increase estradiol absorption. In the future, transdermal delivery
could be used for hormone replacement therapy and other medicines
[92]. Hydrophilic scaffolds are also made using marine collagen glyco-
proteins (FSCP) that are conjugated with poly(3-hydroxybutyrate-co-4-
225
Engineered Regeneration 3 (2022) 217–231
Fig. 7. Mechanisms for controlled release of
drugs using different types of nanocarriers. Re-
produced with permission [81].
hydroxybutyrate) (P(3HB-co-4HB)) by aminolysis [94]. The P (3HB-co-
4HB) / FSCP scaffolds may facilitate fibroblast proliferation and cellular
proliferation with increased hydrophilic properties and provide a poten-
tial wound healing and drug delivery system.
6.6. Multifunctional effect
Dressings made from marine collagen can simultaneously be utilised
as wound dressings and drug carriers. For example, curcumin is
loaded into marine collagen and hydroxymethyl cellulose nanogels. A
prolonged-release profile and a significant increase in contraction were
observed when the nanogel was infused with curcumin [95].
Injections of a chitosan/marine collagen gel containing atelocolla-
gen obtained from new scales, bones, and skin of chum salmons (On-
corhynchus keta) were given to rats subcutaneously. As a result, infil-
tration of inflammatory cells and tumor necrosis factor-𝛼 (TNF-𝛼) pro-
duction were well controlled. Moreover, the injected gel was replaced
by fibroblasts and ECM, which means this composite gel could serve as
a carrier for tissue fillers and drugs [96].
7. Other applications
An overall advantage of collagen is that it can be combined and
changed into many different shapes or with different properties to bet-
ter adapt to certain conditions. Initially, marine collagen can be pro-
cessed into macroscopic materials, such as bulk hydrogels. Several mi-
cro/nanomaterials derived from marine collagen have also been devel-
oped. Nanofibers derived from marine collagen can, for example, be
produced using electrospinning technology.
M. Furtado, L. Chen, Z. Chen et al.
Fig. 8. Fish collagen-based scaffold containing PLGA microspheres for controlled growth factor delivery in skin tissue engineering. Reproduced with permission
[89]. Copyright 2015, Elsevier.
7.1. Tissue engineering
Currently, fish-derived extracts used as nutritional supplements have
not been extensively evaluated for their potential to support skin repair
[97].
Tissue engineering can be accomplished in two ways. Biomaterials
alone, which promote tissue regeneration, and biomaterials with growth
factors and conducive cells to enhance tissue regeneration are possible
[7].
Using the sea urchin as a source of native collagen in collagen-based
skin-like scaffolds (CBSS) for skin injury treatment, Cinzia and her team
propose the usage of sea urchin food waste to produce bilayer (2D + 3D)
CBSS. A process based on the extraction of fibrillar glycosaminoglycan
(GAG)-rich collagen from sea urchin gonad waste. The properties of the
samples were studied, including microstructure, mechanical stability,
water permeability, and ability to exclude bacteria and facilitate fibrob-
last growth. According to the results, the 2D collagen membrane reduces
water evaporation and protein diffusion and acts as a barrier against
bacterial invasion, mimicking the epidermal layer in function. Three-
dimensional collagen scaffolds, similar to the dermal layer in structure
and function, mimic the dermis mechanically, are biocompatible in vitro
and are efficient at infiltrating fibroblasts. In light of these properties,
CBSS derived from sea urchins could prove to be an advantageous, eco-
friendly, and economically viable biomaterial for tissue regeneration
[98]. To form collagen sponges with hemostatic properties, 1-ethyl-3-
(3-dimethyl aminopropyl) carbodiimide (EDC) is crosslinked with col-
lagen type I from the jellyfish R esculentum. Studies on animals have
shown that sponges made from EDC collagen can absorb blood quickly
because they have a high water absorption capacity and promote blood
cell aggregation at the fresh wound site [99].
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Engineered Regeneration 3 (2022) 217–231
7.2. Bone repair
In medical surgery, bone replacement is a significant concern result-
ing from injury/accident, trauma, advanced age, osteolysis, and joint
replacements. A typical way to deal with that is through autografts
and bone harvesting, both associated with complications. Physiologi-
cal functions of human hard tissue can be restored by using marine
collagen as a biomaterial [100]. It is now possible to treat cartilage
regeneration under abnormal circumstances using a variety of tech-
niques, such as advanced scaffolds, engineered growth factors, and mul-
tiple types of cells [101]. For the purpose of clinical translation, sev-
eral clinical studies have been conducted to investigate how PLGA-
based scaffolds affect bone defect healing due to their porosity, me-
chanical properties, and other characteristics [102]. Various combina-
tions of scaffolds may be used to repair a variety of tissues. It is under-
stood that Low-concentration fish collagen peptides upregulate the ex-
pression of osteoblasts proliferation, differentiation and mineralisation
through mitogen-activated protein kinase and Smad cascades [103].
Bone marrow stem cells (BMSCs) can be differentiated by developing bi-
layer fish collagen scaffolds with different components and porous sizes
[104](Fig. 9).
Biomaterials based on collagen from marine sponges of the class Hex-
actinellida appear to be particularly effective for bone repair. Besides
having silicon, calcium, and spongin as parts of their spicules, these
sponges also can regenerate themselves. Their main functions are to
support the organism during growth and defend it from predators. In
animal studies, collagen extracted from jellyfish, such as Rhopilema es-
culentum, has displayed a similar chondrogenic phenotype to human
collagen I and may replace conventional porcine collagen I scaffold for
cartilage regeneration [56].
M. Furtado, L. Chen, Z. Chen et al. Engineered Regeneration 3 (2022) 217–231

Fig. 9. Fish collagen bilayer scaffolds in tissue engineering for osteochondral regeneration of bone marrow stem cells. Reproduced with permission [104]. Copyright
2020, Elsevier.

Fig. 10. Fish scale-derived collagen patch promotes blood and lymphatic vessels in vivo. Reproduced with Permission [85]. Copyright 2017, Elsevier.

7.3. Vascular tissue engineering and regeneration
As patients with vascular access requirements grow because of ail-
ments including cardiac disease, peripheral arterial disease, and is-
chemia, there is a crucial need to develop vascular grafts or artificial
blood vessels and help regulate vasculature development (Fig. 10). Re-
cent years have seen rapid advancements in vascular tissue engineering.
According to subsequent reviews, MC, a widespread and well-accepted
biomedical material, has shown acceptable properties for vascular tissue
engineering.
Lyophilising and cold pressing snakehead fish scale collagen and
then crosslinking it with the bifunctional epoxy compound 1,4-
butanediol diglycidyl ether (BDE), Wang et al. [85] synthesised a snake-
head fish scale-derived collagen patch. A positive charge was first
applied via methylation to reduce collagen’s carboxylic acid groups
through partial esterification. This positive charge reduced negatively
charged proteins from adhering to the cell membrane and could improve
cell interaction [105,106]. Furthermore, methylated collagen has been
reported to be a water-soluble drug carrier [107,108]. To strengthen its
Physico-chemical properties, the methylated collagen was crosslinked

227
M. Furtado, L. Chen, Z. Chen et al. Engineered Regeneration 3 (2022) 217–231

Fig. 11. Induction of stromal wound and application of CV with FG A Hessburg–Barron vacuum trephine was used to cut and score an 8-mm circle into the rabbit
cornea (A, B), followed by manual keratectomy of the trephined area utilizing a crescent knife (C, D). The fibrin component of Tisseal adhesive was first applied
to the injured corneal surface (E) and spread to cover the entire region of the wound for reconstruction (F). A CV patch supported by a nylon ring was applied to
the injured area, and the supporting ring was removed, leaving the CV membrane on the recipient cornea (G). A final layer of the thrombin component of Tisseal
adhesive was applied to the edge and surface of the cornea (H). CV, Collagen vitrigel; FG, fibrin glue. Reproduced with permission [115]. Copyright 2014, John
Wiley and Sons.

with BDE, as the standard chemical cross-linkers, such as glutaralde-
hyde, can be cytotoxic and bioincompatible [109]. The present study
demonstrated that collagen derived from fish scales is a good scaffold-
ing material for vascular repair and other biomedical applications, with
good integration of collagen patches into surrounding tissues, blood ves-
sels, and lymphatic vessels [110].
The collagen isolated from the tilapia scales (Oreochromis niloticus)
upregulates bone sialoprotein expression in rat odontoblast-like cells
(MDPC-23) and stimulates matrix mineralisation. A dental application
of fish collagen for dentin-pulp regeneration is proposed, with effects
comparable to those obtained with porcine skin [111].
For the last decade, regenerative dentistry has emerged as a new
field based on developing a new concept of the reparative paradigm in
dental tissue engineering [112].
7.4. Corneal tissue regeneration and engineering
Blindness due to corneal damage is one of the most common forms of
blindness globally [113]. Corneal transplantation (keratoplasty) is the
only option when other treatments do not successfully restore vision.
Among solid tissue transplants, corneal transplants are the most popu-
lar. The biggest challenge is the severe lack of corneal donor tissue in
this field. As corneal repair materials, various biomaterials have been
tested, and they have proven to be biocompatible and epithelizable.
However, some studies have examined corneal stromal wound healing
during scarring and transparency improvement [114]. Researchers have
successfully used collagen-based biomaterials for corneal repair and scar
prevention [115].
Freeze-dried MC scaffolds formed from a fish scale had better me-
chanical, physical, and microbial properties. These characteristics sug-
gest that MC could be a viable biomaterial candidate for corneal regen-
eration or transplantation. In the absence of donor corneas, MC-based
corneal implants have shown to be biocompatible, capable of light trans-
mission, and have fair and reasonable scattering values, suggesting their
application to corneal replacements [116] as expressed in Fig. 11.
8. Conclusion
Sea-based collagen is rapidly becoming the preferred choice due to
its unique properties to mammalian collagen. It is free from religious
restrictions and has a very low risk of disease transmission, high bio-
compatibility, and low molecular weight, all of its easy absorption.
The abundance of MC in living tissue has made it a promising nat-
ural biomaterial for the biomedical industry due to its similarities to
human collagen. Aside from its water-solubility, safety, and low man-
ufacturing costs, this biomaterial is also biodegradable, antimicrobial,
and functional, making it suitable for tissue engineering and drug deliv-
ery subjects. Further, MC’s flexibility allows for the fabrication of flexi-
ble substrates suitable for scaffolding tissue regeneration or transporting
biomolecules. A combination of its physical and biochemical properties
makes it an ideal scaffold for tissue engineering applications, which in-
cludes bone, cartilage, skin, vascular, dental, and corneal tissues, and
therapeutic application, such as illness associated with metabolic disor-
ders like obesity and diabetes and generally as potential universal drug
carrier. As a biomaterial for various medical applications, MC is an ex-
citing prospect.
A systematised review of this topic is relatively new in the scientific
community. Only a few cases have MC products been studied in human
clinical trials. There have been positive results from some of these trials,
but most have been focused on neutraceuticals. MC scaffolds have also
been studied for therapeutic purposes in a limited number of clinical
trials in humans. Moreover, these systematic reviews may be skewed
by the language bias because only literature based on the English lan-
guage was examined. Research involving comprehensive and complete
examination of fish and other marine-derived collagen products has to
be conducted to assess their therapeutic potential in various medical

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M. Furtado, L. Chen, Z. Chen et al.
disciplines, including the delivery of drugs and tissue engineering for
regenerative medicine and surgery.
9. Future directions
Fish collagen is being used for tissue engineering applications and
wound dressing systems. However, it has yet to be extensively explored
for applications in drug delivery. Fish collagen nanoparticles and micro-
particles are emerging as promising components for drug delivery sys-
tems using polymeric matrices. Fish collagen, despite its health benefits,
has several drawbacks, including its low amino acid content, low me-
chanical strength, melting point, and biomechanical stiffness, as well as
its rapid degradation.
Fish collagen research may contribute to innovation in other biomed-
ical research projects, combining the resources of various realities to
provide superior toolkits for the medical market.
Considering the remaining drawbacks of MC and the requirements
for future development, some vital topics need to be further discussed
and addressed:
1 Further studies focusing on micro/nanomaterials could lead to a
higher diffusion rate and a more controlled and time flexible drug
release. Microspheres and nanoparticles possess an improved bio-
vailability, drug encapsulation capacity, and the release rate to be
adjusted accordingly to the ratio of collagen. Interdisciplinary re-
search, particularly microfluidics, has yet to be explored regarding
how to prepare microfluidic collagen materials from marine sources.
2 Few experiments were done on hydrogel production based on ma-
rine collagen but with little specification on overcoming the poor
physical properties, sensitive enzymatic degradation, and substan-
tial shrinkage during cell culture tendency, limiting its potential ap-
plication in bone tissue engineering. However, a combination with
different polymers or active inorganic substances could bring to the
surface the high water content, easy mass transfer and diffusion, cell
proliferation and differentiation abilities of the hydrogel.
3 Marine collagen and collagen, in general, suffer mainly for their me-
chanical strength and poor chemical properties, restricting their use
in advanced manufacturing. As an example, bioinks for 3D printing
require a combination and balance of viscosity and extrusion proper-
ties. Therefore, targeting these aspects would make marine collagen
a much more usable material that could be applied in many different
areas.
4 Resulting from the triple-helical structure of the collagen triple-helix,
the sponge collagen matrix can exhibit reduced stability in vivo. In
addition, denatured collagen matrixes have been observed to induce
better cellular responses than native collagen matrixes, suggesting
that the partially denatured structure might contribute to tissue re-
generation [117]. These findings could help to play a role in the
exploration of artificial organs and regenerative medicine.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
This work was supported by grants from the National Key Research
and Development Program of China (2020YFA0908200).
Supplementary materials
Supplementary material associated with this article can be found, in
the online version, at doi:10.1016/j.engreg.2022.05.002.
229
Engineered Regeneration 3 (2022) 217–231
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