marine drugs

Article
A Collagen Basketweave from the Giant Squid Mantle as a
Robust Scaffold for Tissue Engineering
Anastasia Frolova 1, * , Nadezhda Aksenova 2,3 , Ivan Novikov 4 , Aitsana Maslakova 5 , Elvira Gafarova 1 ,
Yuri Efremov 1,2 , Polina Bikmulina 1 , Vadim Elagin 6 , Elena Istranova 2 , Alexandr Kurkov 2 , Anatoly Shekhter 2 ,
Svetlana Kotova 2,3 , Elena Zagaynova 7 and Peter Timashev 1,2,3,8

1 World-Class Research Center “Digital Biodesign and Personalized Healthcare”, Sechenov First Moscow State
Medical University (Sechenov University), 8-2 Trubetskaya Street, 119991 Moscow, Russia;
[email protected] (E.G.); [email protected] (Y.E.);
[email protected] (P.B.); [email protected] (P.T.)
2 Institute for Regenerative Medicine, Sechenov First Moscow State Medical University (Sechenov University),
8-2 Trubetskaya Street, 119991 Moscow, Russia; [email protected] (N.A.);
[email protected] (E.I.); [email protected] (A.K.);
[email protected] (A.S.); [email protected] (S.K.)
3 N.N. Semenov Federal Research Center for Chemical Physics, RAS, 4 Kosygin Street, 119991 Moscow, Russia
4 Research Institute of Eye Diseases, 11 Rossolimo Street, 119021 Moscow, Russia; [email protected]
5 Faculty of Biology, Department of Human and Animal Physiology, M.V. Lomonosov Moscow State
University, 1-12 Leninskie Gory, 119991 Moscow, Russia; [email protected]
6 Institute of Experimental Oncology and Biomedical Technologies, Privolzhsky Research Medical University,
Minin and Pozharsky Square 10/1, 603950 Nizhny Novgorod, Russia; [email protected]
7



Institute of Experimental Oncology and Biomedical Technologies, National Research Lobachevsky State

 University of Nizhny Novgorod, Prospekt Gagarina (Gagarin Avenue) 23, 603950 Nizhny Novgorod, Russia;
Citation: Frolova, A.; Aksenova, N.;
[email protected]
8 Chemistry Department, M.V. Lomonosov Moscow State University, 1 Leninskie Gory, 119991 Moscow, Russia
Novikov, I.; Maslakova, A.; Gafarova,
* Correspondence: [email protected]
E.; Efremov, Y.; Bikmulina, P.; Elagin,
V.; Istranova, E.; Kurkov, A.; et al. A
Collagen Basketweave from the Giant
Abstract: The growing applications of tissue engineering technologies warrant the search and
Squid Mantle as a Robust Scaffold for development of biocompatible materials with an appropriate strength and elastic moduli. Here,
Tissue Engineering. Mar. Drugs 2021, we have extensively studied a collagenous membrane (GSCM) separated from the mantle of the
19, 679. https://doi.org/10.3390/ Giant squid Dosidicus Gigas in order to test its potential applicability in regenerative medicine. To
md19120679 establish the composition and structure of the studied material, we analyzed the GSCM by a variety
of techniques, including amino acid analysis, SDS-PAGE, and FTIR. It has been shown that collagen
Academic Editor: Sik Yoon is a main component of the GSCM. The morphology study by different microscopic techniques
from nano- to microscale revealed a peculiar packing of collagen fibers forming laminae oriented at
Received: 17 November 2021
60–90 degrees in respect to each other, which, in turn, formed layers with the thickness of several
Accepted: 20 November 2021
microns (a basketweave motif). The macro- and micromechanical studies showed high values of
Published: 29 November 2021
the Young’s modulus and tensile strength. No significant cytotoxicity of the studied material was
found by the cytotoxicity assay. Thus, the GSCM consists of a reinforced collagen network, has high
Publisher’s Note: MDPI stays neutral
mechanical characteristics, and is non-toxic, which makes it a good candidate for the creation of a
with regard to jurisdictional claims in
published maps and institutional affil-
scaffold material for tissue engineering.
iations.
Keywords: biomechanical properties; collagen membrane; AFM; SEM; tensile test; giant squid;
Dosidicus Gigas; jumbo squid; outer tunic; tissue engineering

Copyright: © 2021 by the authors.

Licensee MDPI, Basel, Switzerland.
This article is an open access article
1. Introduction
distributed under the terms and The basic objective of tissue engineering consists of obtaining such a scaffold material
conditions of the Creative Commons that would promote complete or at least partial regeneration of internal organs, skin,
Attribution (CC BY) license (https:// vascular, bone, cartilage, and other tissues. For a construct to successfully engraft in the
creativecommons.org/licenses/by/ body, its parameters ought to be similar to those of the region in which the construct
4.0/).

Mar. Drugs 2021, 19, 679. https://doi.org/10.3390/md19120679 https://www.mdpi.com/journal/marinedrugs

Mar. Drugs 2021, 19, 679 2 of 25

will function. The construct’s biocompatibility, biodegradability, as well as mechanical

properties determine its potential to substitute the corresponding live tissue in the body.
The interest to collagen-based materials is stipulated by the fact that collagen is
biocompatible with the recipients’ tissues, can biodegrade, is non-toxic, non-carcinogenic
and non-immunogenic, and combines many characteristics of synthetic polymers (strength,
stiffness, ability to form various supramolecular structures, etc.).
Currently, a plethora of pharmaceutical preparations and medical devices have been
created using collagen as a base [1].
Collagen is one of basic natural materials which have application in tissue engineer-
ing [2–6]. In many types of connective tissue, it is a fibrillar protein and the main component
responsible for the tissue integrity, shape, elasticity, and strength.
Connective tissue is present in all the organs and tissues in the body and comprises
~60–90% of their weight. Such collagenous structures as tendons [7,8] and ligaments [9]
have a certain structural hierarchy of collagen to withstand intensive mechanical loads
(extension and compression). The common idea about them has been that tendons and liga-
ments are structurally similar, if not identical [10]. Ligaments [10–12] and tendons [7,10,13]
consist of tightly packed parallel collagen fibers. Ligaments differ from tendons by the
predominance of elastic fibers; therefore, they are characterized by a lower strength but
higher flexibility as compared to tendons. The distinctions between them stem from the
connections they create; ligaments connect a bone with another bone, and tendons connect
a muscle with a bone. A number of studies are dedicated to the restoration of tendon
ruptures using different materials [14,15].
The skin is also an interesting and sophisticated collagen-based organ [16]. The skin
structure resembles a net consisting of differently oriented collagen fibers [17–19]. One of
the basic functions of the skin is to protect internal organs and tissues from mechanical
injuries. Skin as a material exhibits a viscoelastic behavior, and its mechanical response
to a stress involves both a viscous component related to energy dissipation and an elastic
component related to energy storage [20,21]. Collagen fibers comprise 75% of the skin
tissue dry weight [22], and it is those fibers that are responsible for the skin strength. The
skin’s mechanical properties are important for a number of applications, including surgery,
dermatology, forensic medicine, etc. [23]. The problem of skin replacement and search of
the appropriate materials has long been discussed (see, for example, a review [24]).
The anulus fibrosus, an outer fibrous ring of the intervertebral disc, is yet another
example of a collagen-based tissue that undergoes mechanical stresses of various directions
and has a corresponding collagen packing [25,26].
The knowledge of the mechanical characteristics of tissues and organs, and the con-
ditions in which they must function, help to create or select a material that would be
appropriate for their complete or partial replacement [27,28]. The basic mechanical proper-
ties include the strength, stiffness, viscosity, elasticity, plasticity, brittleness, etc.
Table 1 presents examples of tissues with a certain collagen packing related to their
mechanical properties and materials for tissue engineered constructs meant to replace such
tissues.
Mar. Drugs 2021, 19, 679 3 of 25

Table 1. Mechanical properties of some collagen-based tissues and materials for tissue engineered constructs.

Tissue/Material Treatment Tensile Test Data References

Young’s
Ultimate Tensile
Modulus
Strength, UTS (MPa)
(MPa)
Human Skin 27.2 ± 9.3 MPa 98.9 ± 97 MPa [23,29]
Reconstructed anterior cruciate Glutaraldehyde
26 MPa [30]
ligament (ACL) rabbit model cross-linked prostheses
Reconstructed anterior cruciate Carbodiimide
12 MPa [30]
ligament (ACL) rabbit model cross-linked prostheses
Reconstructed anterior cruciate
Sham-operated controls 49 ± 20 MPa [30]
ligament (ACL) rabbit model
Human patellar tendon 60–100 MPa 300–400 MPa [31]
Human native rotator cuff
11.5 ± 5 MPa 50–170 MPa [32]
tendon
TSPC seeded knitted
Control group Control group
silk–collagen sponge scaffold
5.9 ± 1 MPa; 44.3 ± 12.1 MPa;
for TSPC seeded [33]
TCPC group TCPC group
functional shoulder repair
8.3 ± 1.5 MPa 67.8 ± 14.6 MPa
rabbit model
Human Achilles tendon 40 ± 8 MPa 1600 ± 200 MPa [34]
Rabbit Achilles tendon 4.5 MPa 45 MPa [14]
Human fibrocartilage 10 MPa [26]
15,000 MPa
Human compact bone 0.03 MPa (Depending on type [35]
and size of the bones)
Human vaginal tissue 0.82–2.62 MPa [36]
Human cornea 3.81 ± 0.4 MPa [37]
DBP, decellularized bovine Along 23 MPa Along 120 MPa
[38]
pericardium Across 20 MPa Across 50 MPa
Normal human skin (NHS) 2.8 MPa [39]
ASC from bovine hide
0.4 MPa [39]
scaffolds by electrospinning
Un-crosslinked collagen film
10 ± 0.5 MPa [40]
from bovine tendon
Un-crosslinked collagen film
37.7 ± 4.5 MPa 1100 ± 100 MPa [41]
from (Coll type I)
Collagen films from rat tail
100 MPa 27 MPa [42]
(Coll type I)
Chitosan-AS collagen biofilms
33.5 ± 4 MPa [43]
from mantle D. gigas
Collagen fiber films from cattle Dry 17.25 ± 0.07 MPa
[44]
skin Wet 2.61 ± 0.05 MPa
Fresh (non-treated) pulmonary
0.5 ± 0.2 MPa [45]
heart valves pigs

In the view of creating tissue engineered constructs with predefined mechanical

properties, the mantle of squids attracts special attention, since these animals, living in
the deep under great pressure, must have a robust musculature and outer coating to
Mar. Drugs 2021, 19, 679 4 of 25

protect their internal organs. The morphological features of the squids’ mantle affect the
mechanisms of their locomotion [46].
Among many species of cephalopods, the Giant squid (Humboldt squid) represents
the most important object of fishing, which covers 30% of the world fishing volume and
about 4% of the entire world market of squids [47]. It is the biggest of the known mollusks.
This species of large predatory squids lives in the eastern Pacific region, along the Peru
coast at depths of 200 to 700 m. Its mantle can reach almost two meters in length, while its
lifespan is only about 2 years, since the squid dies upon spawning [48].
A number of publications have appeared recently on the use of marine collagen,
obtained, for example, from fish scales [49], mantle, fins, and tentacles of squids [50], and
even sea cucumbers [51]. Uriarte-Montoya et al. (2010) described a film for application in
the food and medical industries, prepared from collagen extracted from the mantle of the
Giant squid of the Dosidicus gigas species [43]. Adamowicz et al. (2021) conducted a study
on the use of the decellularized mantle of Loligo vulgaris squid in tissue engineering as a
material for the urethra reconstruction [52]. Collagen-based materials prepared from the
mantle of the Giant squid might also become a prospective carrier in tissue engineering,
however, no studies on this idea have been reported so far.
Oliveira et al. (2021) discuss the application directions and advantages of marine
collagen, as well as the need for the research in this area, aiming at strengthening this
biopolymer’s position on the world’s collagen market [53]. Physically, biochemically, and
spectroscopically, marine collagen is identical to mammalian collagen [54,55].
Application of mollusks for collagen production has other advantages, including
safety from Creutzfeldt–Jakob disease, which is associated with collagen obtained from
cattle, and no ethical or religious barriers.
In this study, our objective was to assess the possibility of using a material obtained
from the mantle of the Giant squid, Dosidicus gigas, for the tasks of regenerative medicine,
based on the data on its chemical composition, structural analysis, biomechanical properties,
and cytotoxicity. The studied material represented a collagenous membrane prepared from
the squid’s outer tunic (hereafter, Giant Squid Collagenous Membrane, GSCM).

2. Results
2.1. Collagen Is a Basic Component of the GSCM
2.1.1. Amino Acid Analysis
According to the amino acid analysis, the content of hydroxyproline (Hyp) in the
GSCM was 86.3 residues, proline (Pro)—91.3 residues per 1000 residues (Table 2). The Hyp
percentage in the studied specimen was 10.13 weight %.
The specimen also contained a large amount (330 per 1000 residues) of glycine (Gly).
The weight percentage of Gly was 22.18%. This finding is related to the fact that a molecule
of collagen consists of a triple helix formed by three polypeptide helical strands, and each
helical chain is formed by three-residue-long repeats, with glycine as one of the three
residues. Thus, the primary structure of collagen is characterized by a large content of
glycine. The high content of glutamic acid (Glu) in the specimen is explained by the
presence of proline (Pro) since Pro is synthesized from glutamic acid.
Mar. Drugs 2021, 19, 679 5 of 25

Table 2. Amino acid composition of the D. gigas CM.

Molecular Mass,
Name of Amino Acids Abbreviation Letter Code Residues per 1000 Residues w% *
g/mol
Alanine Ala A 89.094 86.2 6.87
Arginine Arg R 174.203 56.4 8.79
Aspartic acid Asp D 133.104 62.9 7.49
Cysteine Cys C 121.154 3.5 0.74
Glutamic acid Glu E 147.131 86.4 11.38
Glycine Gly G 75.067 330.0 22.18
Histidine His H 155.156 7.7 1.07
Hydroxyproline Hyp O 131.131 86.3 10.13
Hydroxylysine Hyl 162.187 10.3 1.5
Isoleucine Ile I 131.175 13.9 1.64
Leucine Leu L 131.175 29.5 3.47
Lysine Lys K 146.189 14.0 1.83
Methionine Met M 149.208 10.4 1.39
Phenylalanine Phe F 165.192 11.1 1.64
Proline Pro P 115.132 91.3 9.4
Serine Ser S 105.093 41.1 3.86
Threonine Thr T 119.119 27.9 2.97
Tyrosine Tyr Y 181.191 6.4 1.04
Valine Val V 117.148 24.9 2.61
Total 1000
Hyp/Hyl 8.4
* Percentage of amino acids to the mass of the test sample.

2.1.2. SDS-PAGE
The SDS-PAGE analysis showed four main bands in the studied GSCM (Figure 1).
Two bands had the molecular weights of 133.3 and 151.6 kDa, and they were assigned
to two α-chains of collagen, α1 and α2. The two high-molecular components, with the
weights of 295.7 kDa and 300 kDa, were identified as a β-chain consisting of two α-chains
and a γ-chain consisting of three α-chains, respectively.

2.2. Hydration and Thermal Properties of the GSCM

2.2.1. FTIR Spectroscopy
The IR spectrum of the GSCM (blue curve in Figure 2) shows bands at 876, 918, 939,
972, 1030, 1060, 1080, and 1119 cm−1 , which are characteristic of carbohydrate moieties
(CO stretching and COC stretching); an Amide III band at 1236 cm−1 (associated with CN
stretching and NH deformation); bands positioned at 1336 and 1451 cm−1 , attributable
to methylene vibrations (CH2 deformation and CH3 deformation); N–H in-plane bend
and the C–N stretching vibrations at 1540 cm−1 (Amid II). The polypeptide backbone CO
stretching vibration is found in the range of 1600–1700 cm−1 : bands at 1740 cm−1 due to
carbonyl vibrations, and the one 1630 cm−1 due to Amide I. The spectrum shows bands at
2878 and 2927 cm−1 assigned to aliphatic chains (CH stretching and CH3 stretching) an
Amide B band at 3073 cm−1 (NH stretching), and a broad band at 3500–3300 cm−1 related
to Amide A (NH stretching) and OH vibrations [56–60].
Mar. Drugs 2021, 19, 679 6 of 25

Figure 1. Evaluation of the GSCM collagen chains’ electrophoretic mobility in 8% PAAG under the
denaturing and reducing conditions. Collagen Type I (Coll 1) and Type II (Coll 2) were used as
collagen standards. SqM—collagen extracted from the GSCM. The high range protein ladder bands
are shown in kDa.

For comparison, the FTIR spectra of collagens Type I and Type II were examined [61].
The spectra of both samples are presented in Figure 2, and the band positions are presented
in Table S1. FTIR confirmed a similar triple helical structure with the secondary α-chain
structure for all three samples [56]. The IR spectra of the GSCM and collagen type II
differed slightly in regard to the bands at 1740, 2800–2930, and 3620–3690 cm−1 (associated
with OH stretching and H-bonding). From the general view of the spectra, one can assume
that the GSCM belongs to collagen Type II, but the increased intensity of bands at 2930
and 1740 cm−1 indicates that it rather belongs to a mixture of collagens Type I and Type II.
These results confirm the results of SDS-PAGE (see Section 2.1, Figure 1).
The position of the Amide I band in the GSCM spectrum is in agreement with the
literature data on the Amide I band in the spectra of oligopeptides containing Gly, Pro, and
Ala in various combinations, as well as the spectra of polyproline [60]. This is consistent
with the results of the study of the amino acid composition, demonstrating that the main
amino acids of the GSCM are Gly, Pro, Hyp, and Glu (see Section 2.1, Table 2).
Mar. Drugs 2021, 19, 679 7 of 25

Figure 2. FT-IR spectra of the GSCM is the blue curve; collagen Type I is the gray curve; collagen Type II is the red curve.

2.2.2. TGA/DSC Studies

A typical weight loss vs. temperature curve (a thermogravimetric, TG curve), as
well as a DSC curve, for the GSCM are displayed in Figure 3. In TG curves, there were
two temperatures at which the onset of the thermal degradation occurred. The DSC
curves showed two endothermic peaks. The broad endothermic peak in DSC curves in
the temperature range of 50–170 ◦ C is associated with thermal dehydration [62–64]. This
process was accompanied by a ~10% weight loss (the TG curve). The broad and multimodal
endothermic peak in the temperature range of 220–330 ◦ C is assigned to the collagen matrix
thermal denaturation and destruction. The latter process was accompanied by a ~65%
weight loss (TG curves). According to [64], for dehydrated collagen type I, the endothermic
peak of denaturation was observed at Tdn = 225 ◦ C. It can be assumed that, below the
temperature of Tdn ~225–235 ◦ C, the interchain hydrogen bonds rupture, dehydrated
collagens unfold, and amorphous polymers form. The second stage of destruction was
observed at Tdst > 235 ◦ C. In general, the GSCM TG and DSC curves were similar to those
for collagenous materials [62,64].

Figure 3. TG (blue) and DSC (red) curves for the GSCM.

2.2.3. Shrinkage Temperature

The shrinkage temperature of the GSCM was experimentally found at 58 ◦ C. The
swelling degree was measured as 102% in distilled water and as 176% in PBS. The much
higher degree of swelling in the PBS medium is due to the fact that ions present in the
saline facilitate hydration of collagen fibers.
Mar. Drugs 2021, 19, 679 8 of 25

2.3. Morphological Properties of the GSCM

2.3.1. Histological Studies
The histological studies of the GSCM cross-sections showed that the material had a
layered structure that consisted of 8–12 tightly packed “laminae” with the total thickness
of ~50–70 µm (Figure 4(A1,B1,C1,D1)). The thickness of each lamina was ~5–7 µm. When
stained with hematoxylin and eosin, the material of laminae had uniform eosinophilic
staining (Figure 4(A1)). However, the picrosirius red stain (Figure 4(B1)), especially, when
using phase contrast (Figure 4(C1)) and polarized light microscopy (Figure 4(D1)) showed
that in some regions the material had a fibrillar structure due to poorly visible small
collagen fibers oriented along the laminae. In the polarized light microscopy images, these
fibers produced a bright glow in the material, testifying the birefringence (anisotropy)
specific for oriented fibers in collagen.

Figure 4. Morphological and optical characteristics of the GSCM before and after the collagenase
treatment. (A1) As seen at a cross-section, the GSCM consists of parallel uniform pink (eosinophilic)
layers—“laminae”; (A2) lysis of the material with homogenization, loss of crisp contours, and
appearing purple (basophilic) regions; (B1) predominantly red staining of laminae with regions of
poorly visible fine-fibred structure; (B2) loss of the red and appearance of yellow (picrinophilic)
staining in most parts of the material with single loose and multidirectional red collagen fibers; (C1)
a somewhat more visible fibrillar structure of the material than that in (B1); (C2) scattered collagen
fibers among the picrinophilic material are more visible than they are in (B2); (D1) laminae produce
a bright yellow-green, yellow-orange, and orange-red glow due to the collagen fibers within their
structure; (D2) no material glow was noted; ×1000 (Scale bar = 50 µm).
Mar. Drugs 2021, 19, 679 9 of 25

2.3.2. Scanning Electronic Microscopy Studies (SEM)

The SEM studies revealed that the GSCM surface had a multilayered basketweave
structure, with laminae laid at different angles, which resembled a reinforcing mesh. The
angle between the laminae was ~60–90◦ (Figure 5a,b).

Figure 5. SEM-BSE images of the GSCM surface. (a) Native surface, and (b) a region inside a fracture
zone of the material (Scale bar = 100 µm).

The reinforcing layers consisting of laminae have a definite mutual layer-by-layer

orientation. Each layer represents a set of parallel laminae with the width of 38–50 µm and
thickness of 4.0–4.5 µm. In turn, each lamina consists of tightly packed parallel collagen
fibers longitudinally packed along the whole lamina length (Figure 6a,b). Besides, there is
a thin layer that covers the upper reinforcing layer with laminae (Figure 6c). This surface is
extremely stable chemically (it was not damaged by the sample preparation procedure)
and is formed by a randomly crossed motif of collagen fibers and fibrils.

Figure 6. Microtopography of the dried GSCM (SEM-SE). (a) the surface of a lamina comprising the reinforcing layer
(Scale bar = 10 µm), (b) the enlarged fragment of the lamina surface (Scale bar = 1 µm), and (c) the layer covering the GSCM
reinforcing layers (Scale bar = 20 µm).

We also studied the GSCM cross-section using SEM, which showed the layered struc-
ture, in agreement with the histological data. The SEM images (Figure 7a,b) demonstrate
that laminae change their angle in each layer, thus making a basketweave multilayered
collagen structure.
Mar. Drugs 2021, 19, 679 10 of 25

Mar. Drugs 2021, 19, x 10 of 25

Figure 7. A SEM-BSE image of the GSCM cross-section. (a,b) Two different regions (Scale bar = 10 µm).
2.3.3. Laser Scanning Microscopy (LSM) (Second Harmonics Generation Signal—SHG)
2.3.3.The
Laser Scanning
LSM studiesMicroscopy
revealed the(LSM)
SHG(Second Harmonics
signal from collagenGeneration
Type I and Signal—SHG)
Type II in the
sample. In consistency with SEM, it was found that collagen in the
The LSM studies revealed the SHG signal from collagen Type I and Type GSCM was bundled
II in the
into laminae
sample. with the with
In consistency widthSEM,
of about
it was60 µ m.that
found Laminae located
collagen in theat different
GSCM depths have
was bundled into
different, up to
laminae with theperpendicular,
width of aboutmutual
60 µm.orientation (the angle
Laminae located of packing
at different is ~60–90°).
depths Lam-
have different,
inae
up toconsist of longitudinally
perpendicular, positioned(the
mutual orientation parallel
anglecollagen fibers
of packing (Figure◦ 8a).
is ~60–90 At the surface
). Laminae consist
of
of some regions, bundles
longitudinally of collagen
positioned parallel in the form
collagen of cords
fibers (Figureare8a).
found (Figure
At the 8b).of
surface Similar
some
structures were observed
regions, bundles by in
of collagen SEM,
the as well
form of(Figure 7a,c).
cords are found (Figure 8b). Similar structures
were observed by SEM, as well (Figure 7a,c).

(a)

(b)
Figure 8. SHG
Figure 8. SHG images
images of
of the
the GSCM.
GSCM. (a) Collagen bundles
(a) Collagen bundles in
in the
the form
form of
of laminae
laminae at
at different
different depths;
depths; (b) collagen in
(b) collagen the
in the
form of cords at the sample’s surface (Scale bar = 100 µm). The SHG signal from collagen is marked by green.
form of cords at the sample’s surface (Scale bar = 100 µm). The SHG signal from collagen is marked by green.

2.3.4. Atomic-Force Microscopy (AFM)

The microrelief of the GSCM surface was visualized using AFM. As seen from Figure 9,
the GSCM surface has a fibrillar structure, with collagen fibers consisting of tightly packed
longitudinally oriented fibrils.
Mar. Drugs 2021, 19, 679 11 of 25

Figure 9. AFM topography of the GSCM with the sequential decrease of the scan size from the left to right image (increase
of resolution): (a) 100 × 100 µm; (b) 30 × 30 µm; (c) 10 × 10 µm; (d) 3 × 3 µm. The samples’ topography is presented using
the Peak Force Error for the better detail resolution.

For comparison, we obtained the topography of the outer tunic of another squid
species, B. magister, which has an essentially smaller size. As seen from Figure 10, the
collagen structure of the outer membrane of this squid species is similar to that of the GSCM,
with the corresponding scaling. The basketweave structure of both squids’ reinforcing
layers in the outer tunic is clearly visible in AFM images, which testifies the universal
character of this structure. Since laminae comprising the reinforcing layer in the GSCM
are rather wide (40–50 µm) and located at a certain angle relative to each other, AFM
cannot visualize the whole laminar motif of the GSCM, even at the largest available scan
size, 100 × 100 µm, so only one cell of the basketweave is seen (Figure 9a). However,
for the small squid, B. magister, this laminar motif is clearly visible at a 50 × 50 µm scan
(Figure 10a), since the B. magister has the proportionally smaller mantle and outer tunic
thickness (Table 3).

Figure 10. AFM topography of the collagenous membrane of a B. magister squid. From the left to right image (increase of
resolution): (a) 50 × 50 µm; (b) 10 × 10 µm; (c) 3 × 3 µm. The samples’ topography is presented using the Peak Force Error
for the better detail resolution.

With a higher resolution (a 3 × 3 µm scan, see more on the Figure 11), one can see
the characteristic striation of collagen fibrils (D-period). The D-period is equal to 67 nm,
although the experimentally obtained values depend on the sample hydration [65].
Mar. Drugs 2021, 19, 679 12 of 25

Figure 11. Molecular packing of collagen in the GSCM (a) AFM topography (Peak Force Error channel), scan size is 3 × 3
µm; (b,c) D period of an individual fibril longitudinal section (red line on the topography image). The section shows the
characteristic D-period of collagen ([66,67]).

2.4. Mechanical Properties of the GSCM

2.4.1. Uniaxial Stretching Tests
The uniaxial stretching tests with the final sample rupture showed that the GSCM
of D. gigas contained at least two basic directions of collagen fibers (Figure 12). The
selected directions of collagen bundles may lead to the complex dependency of the GSCM
mechanical properties on the deformation direction.

Figure 12. A uniaxial stretching test: (a)—start, (b)—end of test.

As seen from the results presented in Table 3, the studied samples of the GSCM of the
D. gigas species had a rather high tensile strength for a biological material. The Young’s
modulus of a dry sample was 1.5 ± 0.5 GPa, while, after 20-min-long hydration of the
material, its Young’s modulus drastically dropped to 20 ± 6 MPa. The ultimate tensile
strength of the hydrated sample also essentially decreased, however, the strain at rupture
grew (to almost 50%).
Mar. Drugs 2021, 19, 679 13 of 25

For comparison, we tested the collagenous membrane of the B. magister squid, since it
has a similar structure, as shown by AFM. The B. magister membrane demonstrated similar
mechanical properties as well. Its Young’s modulus was somewhat higher than that of
the GSCM, while the ultimate tensile strength and maximum elongation at rupture were
slightly lower (in the hydrated state). However, in general, the membrane from the B.
magister squid is more deformable due to its lower thickness (20 µm).

2.4.2. Micromechanical Properties Studied by AFM

As a result of the AFM-based nanoindentation studies at the micro- and nanoscale, the
Young’s modulus of the GSCM surface was measured as 4.1 ± 0.5 MPa. The corresponding
value for the B. magister squid was slightly higher, 6.1 ± 0.5 MPa. The observed difference
between the values at the macro- and microscale is related to the different packing and
thickness of collagen structures at different levels. However, the values belong to the same
order of magnitude.
Table 3. Mechanical properties of the GCSM and collagenous membranes from other squid species.

DML, T,µ W,µ E(w), UTS(w), Max ε(w), E(d), UTS(d), Max ε(d), E(w),
Type of Squid
cm m m MPa MPa % GPa MPa % MPa
Macromechanical Properties Micromechanical Properties
1500–
Dosidigus gigas 50–70 40–50 20 ± 6 20 ± 8 47 ± 9 1.5 ± 0.5 80 ± 20 20 ± 15 4.1 ± 0.5
2000
Loligo peale [68] 30–50 20–35 2–7 No data No data No data No data No data No data No data
Berryteuthis
25 20 4–7 54 ± 17 10 ± 3 27 ± 7 0.4 ± 0.2 28 ± 9 16 ± 5 6.5 ± 0.5
magister

DML—dorsal mantle length of squid; T—thickness of GSCM; W—width of lamina GSCM; E(w)—Young’s modulus of wet GSCM;
UTS(w)—ultimate tensile strength of wet GSCM; Max ε(w)—maximum elongation of wet GSCM; E(d)—Young’s modulus of dry GSCM;
UTS(d)—ultimate tensile strength of dry GSCM; Max ε(d)—maximum elongation of dry GSCM.

2.5. Cytotoxicity and Biodegradability of the GSCM

2.5.1. Viability Test
To assess the potential GSCM cytotoxicity, cell viability and proliferation assays were
performed. The MSC primary culture was chosen because MSCs are commonly applied
in tissue engineering [69–71] and were shown to be more sensitive to toxic agents than
3T3 or L929 cell lines [72–74]. MSCs seeded at a concentration of 5000 cells per well and
exposed to the GSCM extracts at any dilution showed neither reduction in the cell viability
nor a decrease in the proliferation rate (Figure 13A). In contrast, both of the assays showed
a significant drop (to 20% of the cell viability compared to the control cells) in the cell
viability in the presence of SDS at a concentration of 0.05 mg/mL and higher (Figure 13B).
Hence, the GSCM does not contain any cytotoxic compounds that could be released during
cultivation. The adhesive properties of the GSCM were also shown to be appropriate—
MSCs successfully adhered to GSCM films, remained viable during 3 days of cultivation,
and proliferated on them. The metabolic activity of cells cultured on the surface of the
GSCM was slightly higher than that of the monolayer control (Figure 13C). However,
proliferation of collagen-cultivated cells was inhibited in comparison to the monolayer
cell culture grown on culture plastic, probably due to the different mechanical properties
of the surface. The Live/Dead assay of the GSCM revealed normal MSC spindle-shaped
morphology and outnumbering living cells relative to the dead ones (Figure 13D–G).
Overall, despite the decreased proliferation rates of cells, the GSCM was shown to maintain
the normal cell metabolic activity, proliferation capacity, and morphology both by the
extraction and contact cytotoxicity test.
Mar. Drugs 2021, 19, 679 14 of 25

Figure 13. (A)—Elution test: AlamarBlue cytotoxicity assay and PicoGreen DNA assay of the GSCM extracts, 3 days of
MSCs’ cultivation, 5000 cells per well. (B)—AlamarBlue cytotoxicity assay and PicoGreen DNA assay of SDS (positive
control), 3 days of MSCs cultivation, 5000 cells per well. (C)—AlamarBlue contact cytotoxicity assay and PicoGreen DNA
assay of cells adhered to culture plastic (2D control) or GSCM, 3 days of cell cultivation, 20,000 cells per well. * p < 0.05
relative to other groups. (D–G)—Live/Dead cell viability assay with nuclei staining (Hoechst, blue); live cells are stained
with Calcein AM (green), and dead are stained with propidium iodide (red). At 7 days of cultivation, laser scanning confocal
microscopy, scale bar is 100 µm.

2.5.2. Resistance to Collagenase

The sensitivity to collagenase was studied in order to estimate the biodegradability of
the GSCM. The collagenase cleavage study showed that in 6 h the GSCM was digested by
85 ± 5% from the initial weight.
The histological study of the GSCM samples treated with collagenase showed signs of
their destruction in the form of the loss of the typical structure, as well as changes in the tinc-
torial and optical properties of the laminar material (see Section 2.3, Figure 4(A2,B2,C2,D2)).
These signs included homogenization with lysis and appearing basophilic (Figure 4(A2))
and picrinophilic regions (Figure 4(B2)), as well as loosening and loss of orientation of colla-
gen fibers (Figure 4(C2)) with the disappeared anisotropy (Figure 4(D2)). At the same time,
in the picrosirius red-stained samples, the remaining material represented a homogenic
picrinophilic mass, in which few chaotically located destroyed collagen fibers were seen.

2.5.3. LAL Test

To further assess the GSCM biosafety, we tested its pyrogenicity. The most common
pyrogens are endotoxins derived from the cell walls of gram-negative bacteria. The LAL
test is commonly applied to assess their concentration and is one of the two assays rec-
ognized by the U.S. Pharmacopeia (USP) for medical devices. For the GSCM extract, we
revealed that the endotoxin level was 0.28 EU/mL, which does not exceed the concentration
permitted (0.5 EU/mL) [75,76]. Therefore, the GSCM did not contain endotoxins able to
induce a notable pyrogenic reaction. We also performed preliminary in vivo testing of
GSCM samples implanted in rats (see Supplementary Information). It showed that the
intact GSCM was still poorly compatible with the host tissues and caused notable inflam-
matory reaction. However, the GSCM treatment with supercritical carbon dioxide before
implantation solved this problem, reducing the inflammatory reaction to only insignificant.
Mar. Drugs 2021, 19, 679 15 of 25

3. Discussion
The results of the biochemical and structural studies confirm that collagen is a basic
component of the GSCM material. The amino acid analysis showed a high content of Hyp,
which is known as a detector for the presence of collagen [77]. Its weight percentage in
the samples was 10.13%, while the content of Pro and Hyp in the extracted collagen from
the GSCM was 10.9% and 2.8%, respectively [43]. The presence of Cys might indicate that
the GSCM possibly contains traces of elastin [78]. Gauza-Włodarczyk et al. (2017) found a
similar amino acid composition for bone collagen in [79].
The comparative SDS-PAGE analysis of the GSCM with collagen Type I and Type II
revealed the similarity of the GSCM collagen to collagen Type I, based on the characteristic
bands. Nam et al. (2008), in the study [80], described collagen extracted from a squid’s skin
and compared its physicochemical properties with those of collagen prepared from bovine
tendons. The similarity between the two was found, and the squid collagen was classified
as Type I.
FTIR demonstrated the presence of collagen Type II, also, in the GSCM. The DSC
study showed that the GSCM collagen behaved similarly to both collagen types. The
characteristic shrinkage temperature also confirmed the collagenous nature of the GSCM.
The extensive morphology study, including histology, SEM, LSM, and AFM, showed
the presence of ordered collagen structures at various levels of organization. From the
ultrastructure of fibrils to fibers and fiber bundles, they are characterized by tight packing,
orientation, and formation of a basketweave from larger collagen units, laminae. Such a
sophisticated arrangement of collagen structures is apparently related to the mechanical
properties of the GSCM, such as high strength and Young’s modulus.
Based on the SEM study, we have deduced a possible concept of the collagen arrange-
ment in the studied material, displayed in Figure 14. The arrows in Figure 14 indicate
which SEM-revealed feature corresponds to each component of the schematic structure.
The structure and packing of laminae revealed by SEM are confirmed by the other structural
techniques.
We have not found any published studies on the structure of the collagenous mem-
brane from the Giant squid of the D. gigas species, based either on SEM or on any other
visualization technique. However, the squid mantle is known to consist of three layers:
muscle fibers and two collagenous membranes surrounding them (outer and inner tunic).
There is one literature source in which Otwell et al. (1980) presented a sketch of the Loligo
peale squid mantle with the specifics of all the three layers, as well as the corresponding
SEM images [68].
The structural information, especially the unique architecture of collagen fibers in the
GSCM, is of special importance in regard to its mechanical properties. The SEM, AFM,
and LSM data show that the collagen laminae are arranged in a basketweave manner. We
also have studied the structure of the same part from another squid species, B. magister.
This small squid is easily available as a food product. Its mantle was separated from the
muscle layer and studied with AFM. The AFM studies demonstrated a similar structure of
the outer tunic for both squid species, despite a significant difference in their sizes. The
characteristic features of the GSCM are repeated in the outer tunic of B. magister at a smaller
scale. It is the structure that was observed in [68] for the Loligo peale species. In spite of
essential differences in sizes, these squid species have similar morphological and structural
features, as well as comparable mechanical characteristics (Table 3).
Mar. Drugs 2021, 19, 679 16 of 25

Figure 14. A possible concept of the arrangement of collagen fibers in the GSCM based on the SEM
findings.

As the basic component of the squid mantle, collagen is related to the mechanism
of the animal’s locomotion. The collagenous membrane of the cephalopod has a bas-
ketweave structure that must work as a reinforcing frame in the squid’s body, providing
the appropriate strength and stiffness and allowing it to function at high depths.
Indeed, the data of the mechanical tests show rather high values of the tensile strength
and Young’s modulus for a biological collagen-based material [41,42,81]. A high value of
strain at rupture is also notable. The GSCM mechanical characteristics at the microlevel
measured by AFM are also high, which is associated with the tight collagen packing in the
material in the form of a basketweave revealed by the microscopical visualization (SEM,
LSM, ASM, histological staining). These findings are very important from the viewpoint of
the potential GSCM applications in regenerative medicine.
A surgical material must have a good compatibility with the host organism tissues.
Our cell experiments with gingival MSC and AlamarBlue, Live/Dead, and PicoGreen
assays, as well as the LAL test and preliminary in vivo studies, have demonstrated that the
GSCM does not exhibit any cytotoxic properties that testify its good biocompatibility.
The collagenase digestion experiment has additionally confirmed the collagenous
nature of the material and proven that it can undergo almost complete destruction in vitro
in as soon as 6 h. After the treatment, a non-collagenous amorphous component is left,
which binds to picric acid and hematoxylin, but it does not bind to picrosirius red and does
not show birefringence. Most likely, this component consists of glycoproteins that bind
collagen fibers together, thus providing their corresponding orientation and packing in
each layer-lamina and also binding together laminae themselves. However, the presence of
this non-collagenous component does not prevent the enzymatic action on collagen fibers
in the material that may lead to its biodegradation in vivo.
Thus, the collagen nature, basketweave layered structure, good mechanical properties,
absence of cytotoxicity, and ability to biodegrade make the GSCM a prospective candidate
for tissue engineering applications.

4. Materials and Methods

4.1. Material
In this study, we used a commercial material—Aksolagen membrane—provided by
the Akses Swiss company (Zug, Switzerland). Aksolagen membrane is a specially treated
GSCM of Dosidicus gigas. The squid mantle consists of several layers (Figure 15), with the
Mar. Drugs 2021, 19, 679 17 of 25

central muscle layer surrounded by two collagenous membranes (outer and inner tunics);
the GSCM represents the outer tunic of the mantle.

Figure 15. The D. gigas mantle and its inner structure.

We also studied the structure of the collagenous membrane of another squid species,
a small squid B. magister. A frozen squid B. magister was purchased in a supermarket,
thawed, and the collagenous membrane (outer tunic) was mechanically separated from the
muscle layer of the mantle.
The thickness of the GSCM of D. gigas measured with a micrometer (a 5–10 N load)
was 50 ± 5 µm, and the thickness of the B. magister membrane was 25 ± 5 µm.

4.2. Amino Acid Analysis

To study the GSCM composition, we conducted the amino acid analysis. The analysis
was performed by ion-exchange chromatography, with the post column derivatization,
using an L-8800 amino acid analyzer (Hitachi, Ltd., Tokyo, Japan) with a steel Hitachi
Ion-Exchange Column 2622SC(PH) (Hitachi, Ltd., Tokyo, Japan) 4.6 × 80 mm. The column
temperature was 57 ◦ C, the flow rate was 0.4 mL/min, the charge volume was 50 µL,
and the elution regime involved a stepwise gradient of eluents A (AAA PH-1 Buffer—
AN0-8706 Merck Hitachi, Tokyo, Japan), B (AAA PH-2 Buffer—AN0-8707 Merck Hitachi,
Tokyo, Japan), C (AAA PH-3 Buffer—AN0-8708 Merck Hitachi, Tokyo, Japan), D (AAA
PH-4 Buffer—AN0-8709 Merck Hitachi, Tokyo, Japan), and E (0.2 M NaOH solution). As a
calibration mixture, standard concentrated amino acid mixtures in ampoules were used
(Amino Acid Standard Sigma Aldrich, St. Louis, MI, USA).
To prepare the studied solution, a dry sample was placed in a molybdenum glass
ampoule, and 0.3 mL of a freshly prepared hydrolyzing mixture (concentrated hydrochloric
and trifluoroacetic acids in a 2:1 ratio with the addition of 0.1% β-mercaptoethanol Sigma
Aldrich, St. Louis, MI, USA) was added. The sample was frozen, and the ampoule was
evacuated and sealed. The hydrolysis was conducted at 155 ◦ C for 1 h. After the hydrolysis,
the ampoule was cooled, opened, and the content was quantitatively transferred (0.1 mL
of water twice) in a plastic 1.5 mL tube, then the hydrolyzing mixture was completely
removed with a CentriVap vacuum concentrator (Labconco corporation, Kansas, MO, USA)
at 50 ◦ C. The residual acids were removed by repeating twice the procedure of evaporation
of small water portions (0.1 mL) added to the dry residue at 50 ◦ C. Then, 0.1 N HCl was
added to the dry residue, the mixture was centrifugated, and 0.1 N HCl was added to the
supernatant in a 10:1 ratio.

4.3. Collagen Molecular Weight Estimation (SDS-PAGE)

Following the collagen extraction, the protein concentration was evaluated by the
gravimetric analysis. The sample was ≈100-fold concentrated by ultrafiltration on a
Microcon Centrifugal filter unit with a 10 kDa molecular cut-off (MRCPRT010, Millipore,
Burlington, MA, USA) to obtain the final collagen at 10 mg/mL. Collagen from GSCM
Mar. Drugs 2021, 19, 679 18 of 25

and Type I collagen from the cattle dermis were isolated using a protocol described in [82],
while Type II collagen was isolated from the tracheal cartilage by a protocol described
in [83] omitting the use of pepsin. An amount of 10 µg of the proteins were diluted
with an SDS-loading buffer supplemented with 100 mM DTT (20710, SERVA, Heidelberg,
Germany) and heated at 95 ◦ C for 5 min. The samples were resolved by denaturing
polyacrylamide gel electrophoresis in 5% stacking and 8% separating gel using a Mini-
PROTEAN Electrophoresis System (Bio-Rad, Hercules, CA, USA). The electrophoresis
running conditions were as follows: at 15 mA, until samples reached the separating gel,
then at 30 mA until the front reached 0.5 cm from the gel edge. The gel was stained
with Coomassie Blue R-250 (35051, SERVA, Heidelberg, Germany) and scanned with a
ChemiDoc Imaging System (Bio-Rad, Hercules, CA, USA). The molecular weights of the
visual bands were calculated in the ImageLab software against the molecular weight
standards (Spectra Multicolor High Range Protein Ladder, SM1851, Fermentas, Waltham,
MA, USA).

4.4. IR-Spectroscopy
The FTIR analysis of the initial components was carried out using a Spectrum Two
FT-IR Spectrometer (PerkinElmer, Waltham, MA, USA) in the Attenuated Total Reflectance
(ATR) mode. The spectrometer features were as follows: a high-performance, room-
temperature LiTaO3 MIR detector, a standard optical system with KBr windows for the
data collection over a spectral range of 8300–350 cm−1 at a resolution of 0.5 cm−1 . All the
spectra were initially collected in the ATR mode and converted into the IR transmittance
mode. The spectra of collagens were normalized using the intensity of the Amid I band as
the internal standard.

4.5. Differential Scanning Calorimetry (DSC)

Differential scanning calorimetry (DSC) measurements were performed using an STA
6000 simultaneous thermal analyzer (PerkinElmer, Waltham, MA, USA). Samples for DSC
experiments (about 10 mg) were encapsulated in standard PerkinElmer pans and heated in
a nitrogen medium at a gas flow rate of 20 mL/min and a linear heating rate of 10 ◦ C/min.

4.6. Shrinkage Temperature

A sample with the sizes of 3 × 15 mm was placed in a special calibrated glass tube
and immersed in a vessel filled with distilled water. The vessel was placed in a water bath.
The water bath was heated from room temperature to the moment of the sample shrinkage
(~60 ◦ C). The shrinkage temperature was determined as the temperature at which the
beginning of the sample shrinkage was detected. The experiment was repeated thrice.

4.7. Histological Study

Intact and collagenase-treated fragments of the GSCM of D. gigas were fixed in a 10%
solution of neutral formalin, and 4 µm-thick histological sections were prepared using a
standard technique.
The prepared sections were stained with hematoxylin and eosin and picrosirius red to
reveal the collagen composition. The prepared slides were studied by optical (bright-field,
phase contrast and polarized light) microscopy, and the images were captured with a
LEICA DM4000 B LED microscope equipped with a LEICA DFC7000 T digital camera,
using the LAS V4.8 software (Leica Microsystems, Heerbrugg, Switserland).

4.8. Scanning Electron Microscopy (SEM)

The GSCM structure was visualized using an EVO LS10 scanning electron microscope
(Carl Zeiss Microscopy GmbH Jena, Germany). Two techniques for sample preparation
and visualization were used.
The first protocol allowed general images of the samples in the most native state.
Naturally dried samples were attached to the microscope stage with a special carbon
Mar. Drugs 2021, 19, 679 19 of 25

adhesive tape. The observations were conducted in the low vacuum regime (EP, 70 Pa)
at the accelerating voltage of 20 kV and the current of 94 pA per sample. A detector for
back-scattered electrons (BSE) was used. The images were obtained with the resolutions of
473.1 nm/px and 508.8 nm/px. To achieve a satisfactory resolution during back-scattered
electrons observations, a working distance of 4.5 mm was used.
For detailed evaluation of the structure, samples were fixed in neutral glutaric alde-
hyde, dehydrated (battery of alcohols from 20% to 97% and acetone), dried bypassing
the critical point of CO2, , and coated with an Au-Pd alloy. The so-prepared samples were
attached to the microscope stage providing the charge outflow from the coated surface.
The observations were conducted in the high vacuum regime at the accelerating voltage of
21 kV and the sample current of 19 pA. The microtopography images were obtained using
the detector for secondary electrons (SE). The 3072 × 2304 px images were captured with
the resolutions of 89.89 nm/px and 2697 nm/px.

4.9. Laser Scanning Microscopy (Second Harmonics Generation, SHG Signal)

The study was performed using a LSM 880 NLO laser scanning microscope (Carl Zeiss
Microscopy GmbH Jena, Germany) equipped with a tunable Ti:Sa MaiTai HP laser (Spectra-
Physics, Milpitas, CA, USA) with a pulse duration of less than 100 fs. The wavelength of
800 nm was used for the study, and the registration of the SHG signal was performed in
the range of 370–420 nm. The power of the probing radiation was about 9 mW. The images
were obtained using an oil immersion objective with the 40× magnification and numerical
aperture of 1.3. The field of view was 212 × 212 µm, and the resolution of images was
1024 × 1024 pixels. A series of images (z-stack) was acquired from the sample surface
into the depth with the step of 9 µm, the orientation being parallel to the surface. For the
convenience of perception, the acquired images were presented in the green palette.

4.10. Atomic Force Microscopy (AFM)

The morphological AFM studies of the surface were performed using an atomic
force microscope (BioScope Resolve, Bruker, Billerica, MA, USA) combined with an Axio
Observer inverted optical microscope (Carl Zeiss Microscopy GmbH Jena, Germany). A
ScanAsyst Air cantilever (Bruker, Billerica, MA, USA) was used with a nominal spring
constant k = 0.4 N/m and a nominal tip radius r = 2 nm, and scanning was performed on
air in the PeakForce QNM regime. The collagen structures’ periodicity was estimated with
the Section function of the NanoScope Analysis v1.9 software (Bruker, Billerica, MA, USA).

4.11. Uniaxial Stretching Test

The uniaxial stretching tests for dry and hydrated samples were conducted using a
Mach-1 v500c mechanical tester (Biomomentum, Laval, QC, Canada). For the hydration,
samples were immersed in distilled water for 20 min. The measurements were also
performed in distilled water. Dumbbell-shaped fragments of the dry and hydrated GSCM
were cut both in the tangential and radial directions in respect to the whole material area
(with the circle diameter of 30 cm). The working area of the fragments had the length of
15 mm and width of 5 mm. The dry material thickness was 45 µm, while the thickness of
the hydrated material was 60 µm. Before the test, the mechanical tester was calibrated using
a standard sample provided by the manufacturer. Both ends of the experimental sample
were tightly gripped in the clamps followed by gradual elongation at room temperature
(25 ◦ C) at a constant rate of 0.1 mm/s until rupture. The mechanical parameters were
calculated from the stress-strain curves according to the manufacturer’s protocol. The data
were averaged over 3 or more tests.

4.12. Micromechanics by AFM

The mechanical properties of the samples’ surface were studied in fluid (distilled
water) at room temperature (25 ◦ C), after 20 min of hydration, using an atomic force
microscope (BioScope Resolve, Bruker, Billerica, MA, USA). The sample micromechanics
Mar. Drugs 2021, 19, 679 20 of 25

was obtained in the regime of nanoindentation over a preset map of 50 × 50 µm with

the 32 × 32 pixels resolution, as described in [84]. A ScanAsyst Fluid cantilever (Bruker,
Billerica, MA, USA) with a nominal spring constant of 0.95 N/m and a nominal tip radius
of 50 nm was precalibrated using a standard titanium sample. The deflection sensitivity
was calibrated in the same conditions using a sapphire standard sample. The data were
processed using the NanoScope Analysis v1.9 software(Bruker, Billerica, MA, USA) and
averaged over 12 measurements.

4.13. In Vitro Cytotoxicity Assays

The biocompatibility and cytotoxicity tests were performed using the primary culture
of mesenchymal stromal cells (MSCs) isolated from human gingival mucosa as described
in [85]. The cells were cultivated in the medium that contained Dulbecco’s Modified
Eagle’s Medium (DMEM)/F12 (1:1, Biolot, St. Petersburg, Russia), 10% fetal calf serum
(HyClone, Logan, UT, USA), L-glutamine (5 mg/mL, Gibco, Gaithersburg, MD, USA),
insulin–transferrin–sodium selenite (1:100, Biolot, St. Petersburg, Russia), bFGF (20 ng/mL,
ProSpec, Rehovot, Israel), and gentamycin (50 µg/mL, Paneco, Moscow, Russia). Isolated
cells were routinely checked with a SH800S microfluidic flow cytometer (Sony Biotechnol-
ogy, San Jose, CA, USA) for the presence of mesenchymal surface markers (CD90, CD73,
CD105) and absence of hematopoietic and endothelial markers (CD45, CD34, CD11b, CD19
and HLA-DR), according to [86]. The cells were cultivated in the standard conditions of
37 ◦ C and 5% CO2 .
The cytotoxicity was analyzed via the elution and contact tests. In the first case,
the extracts of the GSCM were prepared according to recommendations of ISO 10993-12.
Briefly, 5000 cells per well of a 96-well plate were seeded 24 h before adding the extracts. To
prepare extracts, GSCM films were incubated in the culture medium for 24 h at 37 ◦ C. The
thickness of a film was less than 0.5 mm, and, therefore, in accordance with ISO 10993-12,
the required sample’s area was to be treated in a volume of 1 mL is 6 cm2 . Cells were
exposed to the maximum concentration of the extract (6 cm2 /mL) and its serial twofold
dilutions. We used serial two-fold dilutions of 1.5 mg/mL sodium dodecyl sulphate (SDS)
in a standard culture medium as a positive control. Cells cultivated in the standard culture
medium were applied as a negative control. After 24 h of cultivation with the extract, SDS,
or culture medium, the cell viability was assessed either with the AlamarBlue cell viability
reagent (Invitrogen, Waltham, MA, USA) or with the Quant-iT PicoGreen kit (Invitrogen,
Waltham, MA, USA). For the AlamarBlue metabolic activity assay, the cell culture medium
was replaced with a 10% reagent solution and incubated for 2 h. Then, the fluorescence of
samples was measured using a Victor Nivo spectrofluorometer (PerkinElmer, Waltham,
MA, USA) at a 530 nm excitation wavelength and a 590 nm emission wavelength. The
DNA amount was evaluated with the PicoGreen assay after 3 freeze-thaw cycles aimed at
releasing DNA, following the manufacturer’s instructions. The samples’ fluorescence was
estimated with the spectrofluorometer at a 480-nm excitation wavelength and a 520-nm
emission wavelength.
For the contact cytotoxicity, 20,000 cells were seeded on a surface of the 1 cm2 GSCM
films and cultivated for 3 days. Cells seeded on the culture plastic (monolayer culture)
served as a control. Afterwards, the metabolic activity and DNA amount were measured
as described above.
The morphology and viability of the cells seeded on the GSCM was visualized with the
Live/Dead assay. Briefly, live cells were stained with calcein-AM (Sigma-Aldrich, St. Lois,
MO, USA), dead cells were stained with propidium iodide (Thermofisher, Waltham,
MA, USA), and nuclei were stained with Hoechst 33,258 (Thermofisher, Waltham, MA,
USA). The images were obtained by laser confocal scanning microscopy using a LSM
880 instrument with Airyscan (Carl Zeiss Microscopy GmbH Jena, Germany).
All the samples were triplicated (plate wells for extract cytotoxicity and film samples
for the contact cytotoxicity and Live/Dead assay).
Mar. Drugs 2021, 19, 679 21 of 25

4.14. Resistance to Collagenase

The susceptibility to proteolytic degradation was studied in a Collagenase A (from
C histolyticum) solution. Approximately 4 mg (dry weight in triplicates) of the sample
were weighed. To the weighed samples, 0.5 mL aliquots of a 2.5 mg/mL Collagenase A
solution in the Tris buffer (50 mmol/L, pH 7.5) containing 10 mmol/L calcium chloride
and 0.02 mg/mL sodium azide (Paneco, Moscow, Russia) were added. The samples were
incubated at 37 ◦ C for 6 h. Then, the samples were centrifuged at 605 g (3000 RPM) for
90 s (a MiniSpin microcentrifuge by Eppendorf Corporation, Hamburg, Germany). We
used a low rotation speed and a short time of centrifugation in order to better preserve
the structure integrity for the following histological analysis. Then, the material was
washed from the residual collagenase with distilled water. The precipitate was carefully
transferred using a micropipette to a coverslip for the following drying in an oven at 50 ◦ C
for 20 h. Then, the dry residue was weighed using a WXTE ultramicrobalance (Mettler
Toledo GmbH Urdorf, Switzerland). Finally, the weight loss was calculated by a paired
comparison before and after the treatment.

4.15. LAL Test

The GSCM film was cut into 5*5-mm pieces under aseptic conditions. The extracts
were prepared in 1 mL of endotoxin-free water by continuous shaking for 24 h at 50 ◦ C. The
endotoxin concentrations were measured using the Chromogenic Endotoxin Quantitation
Kit (Thermo Fisher Scientific, Waltham, MA, USA) in accordance with the manufacturer’s
instruction. Briefly, we mixed 50 µL of the extract or the endotoxin standard dilution (0.1,
0.25, 0.5, 0.1 U/mL) and 50 µL of endotoxin-specific Limulus Amebocyte Lysate (LAL)
reagent in a well of a 96-well plate. The mixture was incubated for 10 min at 37 ◦ C and
then 100 µL of the chromogenic substrate was added and incubated for 6 min at 37 ◦ C. The
reaction was inhibited by adding 100 µL of 25% acetic acid. The absorbance was measured
at a wavelength of 405 nm using a microplate Victor Nivo spectrofluorometer (PerkinElmer,
Waltham, MA, USA). The minimal detection level of the kit used was 0.1 EU/mL (EU—unit
of measurement for endotoxin activity).

5. Conclusions
The literature analysis shows that the GSCM material has been very poorly studied,
and no application in tissue engineering has been discussed so far. The results of our
studies on the GSCM composition, structure, mechanical characteristics, cytotoxicity, and
biodegradability testify that the GSCM of D. gigas is characterized by a high tensile strength
and elasticity, along with a peculiar basketweave collagen structure and biocompatibility
that allows the assumption that this material may be applicable in a number of tissue engi-
neering fields (e.g., wound care materials, scaffolds for restoration of the musculoskeletal
system, repair of hernias and the prolapse of pelvic organs, dental membranes, and other
applications requiring good mechanical properties and slow degradation of the implanted
material).
Upon the comparison with other squid species (in particular, B.magister), one may
conclude that the GSCM structure is represented by a typical reinforcing mesh consisting
of collagen structures and providing the high strength and Young’s modulus. However,
since the Giant squid D. gigas has a large size of the mantle and, respectively, a large lateral
size of the GSCM, this material is more advantageous from the processing viewpoint.

Supplementary Materials: The following are available online at https://www.mdpi.com/article/

10.3390/md19120679/s1, Table S1: Band positions for the GSCM, collagen Type I and II; Figure S1:
Morphological and optical characteristics of the implanted intact GSCM; Figure S2: Morphological
and optical characteristics of the implanted GSCM after the scCO2 -treatment.
Author Contributions: Conceptualization, P.T. and S.K.; methodology, S.K., A.F. and E.I.; validation,
P.T. and S.K.; formal analysis, A.M., E.G., P.B., V.E. and E.I.; investigation, A.F., N.A., I.N., A.M., E.G.,
Y.E., P.B., V.E. and A.K.; resources, A.F., N.A., I.N., A.M., E.G., Y.E., P.B., V.E., A.K., S.K., E.I., A.S.,
Mar. Drugs 2021, 19, 679 22 of 25

E.Z. and P.T.; data curation, A.F., S.K., E.I., A.S., E.Z. and P.T.; writing—original draft preparation,
A.F., N.A., I.N., A.M., E.G., Y.E., P.B., V.E., A.K., S.K. and A.S.; writing—review and editing, A.F., A.S.,
S.K., E.I., E.Z. and P.T.; visualization, A.F., N.A., I.N., V.E., A.K. and A.S.; supervision, P.T.; project
administration, P.T.; funding acquisition, P.T. All authors have read and agreed to the published
version of the manuscript.
Funding: The study was financially supported by the Russian Science Foundation (18-15-00401).
Data Availability Statement: All the data supporting the conclusions of this article are included in
this article.
Acknowledgments: The study was carried out using the unique scientific facility Transgenebank.
The authors thank Anton Murashko (Moscow State University, Faculty of Physics) for drawing the
graphical abstract. The authors are thankful to Anastasia Shpichka for her kind assistance with the
cell experiments.
Conflicts of Interest: The authors declare no conflict of interest.

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Doku Mühendisliği için Sağlam Bir İskele Olarak Dev
Kalamar Mantosundan Bir Kolajen Sepet Örgüsü
Bu makale Anastasia Frolova , Nadezhda Aksenova , Ivan Novikov ,
Aitsana Maslakova ve ekip arkadaşları tarafından yazılmıştır.
Bu makale doku mühendisliği teknolojilerinin artan uygulamaları,
uygun bir mukavemet ve elastik modüle sahip biyouyumlu
malzemelerin araştırılmasını ve geliştirilmesini garanti eder. Burada,
rejeneratif tıpta potansiyel uygulanabilirliğini test etmek için Dev
kalamar Dosidicus Gigas'ın mantosundan ayrılmış bir kolajen zar
(GSCM) üzerinde kapsamlı bir şekilde çalışmışlar.Daha sonra GSCM'yi
amino asit analizi, SDS-PAGE ve FTIR gibi tekniklerle test
etmişlerdir.Kolajenin GSCM’nin ana bileşeni olduğu
belirtilmiş.Kolajen, doku mühendisliğinde uygulaması olan temel
doğal malzemelerden biridir. Birçok bağ dokusu türünde, bir fibriler
proteindir ve doku bütünlüğünden, şeklinden, elastikiyetinden ve
gücünden sorumlu ana bileşendir. Önceden tanımlanmış mekanik
özelliklere sahip doku mühendisliği yapılarının yaratılması açısından,
kalamarların mantosu özel dikkat çekiyor, çünkü derinlerde büyük
baskı altında yaşayan bu hayvanlar, iç organlarını korumak için
sağlam bir kas yapısına ve dış kaplamaya sahip olmalıdır.
Kalamarların mantosunun morfolojik özellikleri, hareketlerinin
mekanizmalarını etkiler.Sonuç olarak kolajen,GSCM’nin temel
bileşenidir.