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Original Review Article DOI: 10.26479/2020.0603.02
UTILIZATION OF FISH COLLAGEN IN PHARMACEUTICAL AND
BIOMEDICAL INDUSTRIES: WASTE TO WEALTH CREATION
Priyanka Kulkarni, Mithun Maniyar
SVERI’s College of Pharmacy, Pandharpur, Maharashtra, 413 304, India.
ABSTRACT: Collagen is having enormous applications in biomedical industries while it’s use is
predominantly limited because of its high cost. With ever-increasing requirement in pharmaceutical
industries, exploration of different sources and optimization of the extraction conditions of collagen
has attracted the attention of researchers in the last decade. The most abundant sources of collagen
are mammalian sources which are having major drawbacks. So, the industrial use of collagen
obtained from non-mammalian sources is growing in importance. However, compared to
mammalian sources, fish waste can be utilized as cost-effective alternative to produce collagen.
Around 50-80% part of fish is discarded as a waste which contains high concentration of collagen.
Fish collagen has multiple advantages over mammalian collagen and hence can be a promising
alternative for it. This review summarizes the information of collagen, various sources and
biomedical applications of collagen.
Keywords: Collagen; Biomedical; Pharmaceutical; Fish waste.
Article History: Received: March 22, 2020; Revised: April 14, 2020; Accepted: May 01, 2020.
Corresponding Author: Dr. Mithun Maniyar*
SVERI’s College of Pharmacy, Pandharpur, Maharashtra, 413 304, India.
1. INTRODUCTION
With enormous growth in biomedical sector in past decades, a novel field like research and
development of biomaterials is getting very good attention nowadays. With massive growth in the
development of science especially in medical fields and with increasing demands due to injuries and
diseases; need of newer biomaterials is rising. In current scenario, a sharp incline is observed for the
use of biodegradable materials in biomedical industries. Collagen has been potentially dominating
in the fields of biodegradable materials. Collagen and its structurally similar form gelatin have been
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exhaustively exploited in biomedical, cosmetic as well as food industries. It has been predominantly
utilized in tissue engineering scaffolds for the synthesis of artificial skin, artificial bone, artificial
tendon and artificial cartilage [1]. Collagen is the most abundant protein in mammals representing
nearly 30% of total proteins in the animal body. Collagen is the constitutive protein, having a crucial
role in the formation of the building block of almost all tissues, organs and hence the complete body.
Collagen has been considered as an excellent biomaterial for the development of tissue engineering
constructs and wound dressing systems due to its high direct cell adhesion ability, low antigenicity,
and exceptional biocompatibility. Collagens are reported to be processed into various forms such as
films, injectable solutions, composites, fleeces, sheets, scaffolds, tubes, sponges, membranes, and
dispersions [2]. Most imperative applications of collagen and gelatin lies in pharmaceutical
industries while collagen/gelatin mediated controlled release drug delivery system is getting
attention nowadays. In this review we discuss the structure of collagen, its sources and various
biomedical applications.
Collagen and its Structure
Collagen is the complex constituent of extracellular matrix (ECM) and most abundant fibrous
structural protein in all higher animals [3, 4]. It is mostly found in fibrous tissues such as tendon,
ligament and skin in the form of elongated fibrils and is also abundant in cartilage, intervertebral
disc, blood vessels, bone, and cornea [2]. A three-dimensional structure of collagen was proposed
by Ramchandran as Madras model using fibre diffraction pattern of kangaroo tail tendon [5]. This
model states that, collagen is a coiled coil conformation formed by the three polypeptide chains
(Figure 1). This coiling results in a unique tertiary structure which is the most prominent feature of
the collagen molecule called “triple helix”. Due to this structural complexity, collagen is very rigid
in nature [6].
Figure 1: Collagen type I chemical structure. (a) Sequence of amino acids - primary structure,
(b) left-handed helix - secondary structure; right-handed triple-helix - tertiary structure and
(c) staggered - quaternary structure [7].
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Collagen is formed of three identical or non-identical polypeptide chains; each of which composed
of nearly 1000 amino acids. The unique arrangement of amino acids helps to stabilize the structure
of collagen. Gly-X-Y is the most repeating unit in the collagen sequence. Glycine being the smallest
amino acid is repeated at every third location in the order, while 35% of the non-glycine positions
are occupied by proline at X position and 10% by the hydroxyproline at Y position [8]. Table 1
describes the abundance of amino acid in collagen from different sources such as rat, bovine and
codfish [9].
Table 1: Amino acid composition of collagen obtained from the skin of codfish, rat and
bovine commercial collagen (per 1000 residues) [9]
Amino Acid
Rat
Bovine
Codfish
Alanine
111.16
102.04
91.48
Arginine
42.24
32.86
30.45
Aspartic acid
45.32
36.65
38.82
Cystein
0.99
1.24
1.28
Glutamic acid
73.33
59.43
56.08
Glycine
333.18
296.44
266.12
Histidine
3.61
3.11
5.01
Hydroxylysine
9.33
8.86
6.65
Hydroxyproline
96.06
78.35
39.6
Isoleucine
7.48
6.74
5.61
Leucine
23.29
17.5
16.51
Lysine
27.07
22.2
19.62
Methionine
8.03
7.81
15.04
N-isobutylglycine
12.7
14.33
13.75
Phenylalanine
14.62
11.58
12.7
Proline
109.21
89.89
62.69
Serine
42.74
32.03
53.87
Threonine
18.79
13.2
16.89
Tyrosine
3.76
1.48
2.25
Valine
17.08
12.86
12.02
Sources of Collagen
In vertebrates, around twenty-eight diverse types of collagen have been identified composed of
forty-six distinct polypeptide chains. Most abundant collagens are of type I, II, and III which
provides the scaffolding and guide cells to migrate, proliferate and differentiate [2]. Gelatin shares
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the same physicochemical characteristic as that of collagen, being different form of the same
macromolecule obtained by partial hydrolysis of collagen. Most of the times the collagen and gelatin
used in the industrial products are obtained from mammalian sources (bovine and porcine) whereas;
production of collagen and gelatin from the fish waste has received considerable attention in recent
years [10]. Collagen can be obtained from animals and the most common sources are bovine, porcine,
fish and human collagen. Other terrestrial sources comprise from rat tail tendons, chicken, frog skin,
equine tendon, bird’s feet, sheep skin, duck feet, alligator skin and kangaroo tail. [11]. Collagen
from mammalian sources (bovine and porcine) are majorly utilized most of the times. The above-
mentioned sources are cost-effective and easily available, but after prolonged use and concrete
characterization, collagen from these sources tended to be allergenic and responsible for the risk of
transmissible diseases like foot/mouth disease, bovine spongiform encephalopathy (mad cow
disease), ovine and caprine scrapie and other zoonoses. Another reason for limiting the use of these
sources due to certain religious practices which forbid the use of bovine and porcine products. [10].
On the other hand, marine collagen is a reasonable source which is considered as GRAS (Generally
recognized as safe) by the FDA [12]. Fish is one of the most-traded food commodities in global
market. Marked growth in fisheries and aquaculture sector was observed which was around 154
million tonnes in 2007 and increased up to 171 million tonnes in 2014. India's worldwide share in
fishery industry is increasing day by day. India stands at 6th position worldwide in case of fish
captures from marine sources and it is at 2nd position in fish captures from inland sources as per the
report of Food and Agriculture Organization [13]. Huge amount of fish production and its
consumption results in the generation of waste in equal quantities as that of final product. Various
products are developed from fish waste such as enzymes, collagen peptides and fertilizers [14]. On
the other hand, collagen is the most important product which can be extracted from fish waste [10,
15, 16].
Collagen Extraction
Extraction of collagen from fish waste consist of numerous steps. Fish waste is subjected to various
pre-treatments followed by extraction method. Before pre-treatments fish waste is segregated such
as bone, scale, skin and swim-bladder. The pre-treatment is given to the segregated fish waste to
remove the impurities as well as to increase the quality of extracted collagen. Fish waste contains
numerous contaminants such as lipids, non-collagenous proteins, pigments, calcium and other
inorganic materials which can be removed in the pretreatment methods. There are number of
methods reported for the extraction of collagen which are classified depending on the different
methods used such as salt-soluble collagen (SSC), acid soluble collagen (ASC), and enzyme soluble
collagen (ESC). The properties of collagen and its recovery are directly affected by the collagen
extraction method. Therefore, all procedures must be performed at low temperature (~4 °C) to avoid
the degradation of collagen [17]. Methods of collagen extraction from fish waste are summarized in
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figure 2.
Figure 2. Methods for extraction of fish waste collagen
Collagen Biomaterials
Collagen has been considered as an excellent biomaterial due to its low antigenicity, high direct cell
adhesion ability and exceptional biocompatibility. Collagen-based biomaterials can be utilized in
two forms based on the degree of their purification: decellularized collagen matrices that maintain
the original ECM structure and tissue properties; and more refined scaffolds prepared via extraction,
purification, and polymerization of collagen with other biomaterials [18]. Extraction and
purification of collagen from natural tissues is carried out in various ways. As solubility of collagen
is very low due to its covalent cross-links mediated inert nature; it is insoluble in organic solvents
but can dissolve in aqueous solutions, depending on the extent of cross-linking [19]. Collagen based
biomaterials are particularly beneficial in wound healing since their wet strength permits their
suturing to soft tissue and delivers a template for new tissue growth. Depending on the degree of
cross-linking, the collagen biomaterials are degraded by collagenases into peptide and amino acids
in 3–6 weeks, and the implant is then replaced by native type I collagen produced by fibroblasts.
For medical applications, collagens are reported to be administered in numerous forms (Figure 3).
scaffolds [20], fleeces [21], composites [22], dispersions [23], sponges [24], sheets [25], tubes [26],
membranes [27], injectable solutions [28] and films [16].
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Figure 3. Medical applications of collagen
Scaffolds: Campbell et al. [20] developed collagen scaffolds that have been used to develop
analogues of complex tissues in vitro. Anisotropic scaffolds supported the enhanced migration of an
invasive breast cancer cell line MDA-MB-231 with an altered spatial distribution of proliferative
cells in contrast to invasive MDA-MB-468 and non-invasive MCF-7 cells lines. The provision of
controlled architecture in this system may act both to increase assay robustness and as a tuneable
parameter to capture detection of a migrated population within a set time, with consequences for
primary tumor migration analysis.
Fleeces: Zirk et al. [21] studied the effectiveness of collagen fleeces to prevent the post-operative
bleedings. No standard protocol in prevention of bleeding events has met general acceptance among
surgeons yet while this study gave very good results suggesting effectiveness of collagen fleeces.
Sufficiently performed local hemostyptic measures, like the application of collagen fleeces in
combination with atraumatic surgery, bears a great potential for preventing heavy bleeding events
in hemostatic compromised patients, regardless of their anticoagulant therapy.
Composites: Fu et al. [22] studied the collagen-chitosan composite which were used as the cell
matrix for smooth muscle cells, fibroblasts and BMSCs. The seeded cells retained their biological
activity after being cultured in vitro and seeded into the collagen-chitosan composite material.
Formed composite can be potentially exploited for biomedical applications as well as in novel field
of 3-D bioprinting.
Dispersions: Artery buckling has been proposed as a possible cause for artery tortuosity associated
with various vascular diseases. Since microstructure of arterial wall changes with aging and diseases,
it is essential to establish the relationship between microscopic wall structure and artery buckling
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behavior. Mottahedi and Han [23] developed arterial buckling equations to incorporate the two-
layered wall structure with dispersed collagen fiber distribution. Parametric studies showed that with
increasing fiber dispersion parameter, the predicted critical buckling pressure increases. These
results validate the microstructure-based model equations for artery buckling and set a base for
further studies to predict the stability of arteries due to microstructural changes associated with
vascular diseases and aging.
Sponges: Konstantelias et al. [24] carried out a systematic search for the effectiveness of
gentamicin-collagen sponges (GCS) for the prevention of surgical site infections (SSIs). They stated
that gentamicin-collagen sponges were associated with a lower risk of SSIs suggesting further high-
quality randomized studies which are needed to confirm the benefit of GCS for lowering mortality
rates.
Sheets: Peripheral nervous system injuries result in a decreased quality of life, and generally require
surgical intervention for repair. Currently, the gold standard of nerve autografting, based on the use
of host tissue such as sensory nerves is suboptimal as it results in donor-site loss of function and
requires a secondary surgery. Nerve guidance conduits fabricated from natural polymers such as
collagen are a common alternative to bridge nerve defects. Alberti et al. [25] stated that collagen
sheets support directional nerve growth and may be of use as a substrate for the fabrication of nerve
guidance conduits.
Tubes: Fujimaki et al. [26] developed a new scaffold material - oriented collagen tubes (OCT) and
evaluated the potential of OCTs combined with basic fibroblast growth factor (bFGF) to repair of a
15 mm sciatic nerve defect in rats. Their findings demonstrated that OCT alone or in combination
with bFGF accelerates nerve repair in a large peripheral nerve defect in rats.
Membranes: Nakahara et al. [27] assessed the impact of collagen membrane application and
cortical bone perforations in periosteal distraction osteogenesis. Collagen membrane were found to
be beneficial where cortical bone perforations have more impact on the osteogenic process.
Injectable solutions: Recently, stimuli-responsive nanocomposite-derived hydrogels have gained
prominence in tissue engineering because they can be applied as injectable scaffolds in bone and
cartilage repair. Due to the great potential of these systems, Moreira et al. [28] aimed to synthesize
and characterize novel thermosensitive chitosan-based composites, chemically modified with
collagen and reinforced by bioactive glass nanoparticles (BG) on the development of injectable
nanohybrids for regenerative medicine applications. The results demonstrated that the addition of
collagen and bioactive glass increases the mechanical properties after the gelation process. The
addition of collagen increased the stiffness by 95%. Injectable nanohybrids demonstrated no toxic
effect on the human osteosarcoma cell culture (SAOS) and kidney cells line of human embryo (HEK
293T). So, formed nanohybrids have the promising potential to be used as thermo-responsive
biomaterials for bone-tissue bio-applications.
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Films: Bhuimbar et al. [16] extracted collagen from skin of Centrolophus niger and used for the
preparation of collagen-chitosan film. Films incorporated with 5% pomegranate peel extract
declined solubility in water remarkably and showed antibacterial effect against food borne
pathogens. So, this formed film can be potentially exploited for numerous pharmaceutical and food
applications.
2. CONCLUSION
Collagen plays a crucial role in many pre-operative and post-operative surgical procedures. Intrinsic
biodegradability by endogenous collagenases and higher biocompatibility make externally
administered collagen ideal for use in biomedical applications. For several decades, dermatological
defects have been treated with subcutaneous injections of collagen solutions. This application is a
commercial success, particularly in the area of plastic and reconstructive surgery. Threat of allergic
reactions and cost of collagen from mammalian sources are the two main reasons drastically limiting
their use in biomedical applications. Collagen from fish sources are better alternative to mammalian
sources which can overcome both the limitations of mammalian collagen. Large quantities of fish
processing waste which act as a serious environmental pollutant are compelling source of collagen.
Reports on the use of fish collagen in the preparation of pharmaceutical products are scanty. So,
future research must be directed to utilize collagen from fish waste in various biomedical
applications.
ETHICS APPROVAL AND CONSENT TO PARTICIPATE
Not applicable.
HUMAN AND ANIMAL RIGHTS
No Animals/Humans were used for studies that are base of this research.
CONSENT FOR PUBLICATION
Not applicable.
AVAILABILITY OF DATA AND MATERIALS
The authors confirm that the data supporting the findings of this research are available within the
article.
FUNDING
None.
ACKNOWLEDGMENTS
The authors would like to thank Dr. Prashant Bhagwat for critically reviewing the manuscript.
CONFLICT OF INTEREST
The authors have no conflicts of interest to declare.
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