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<sup>−</sup>1, which are characteristic of carbohydrate moieties (CO stretching and COC stretching); an Amide III band at 1236 cm<sup>−</sup><sup>1</sup> (associated with CN stretching and NH deformation); bands positioned at 1336 and 1451 cm<sup>−</sup>1, attributable to methylene vibrations (CH2 deformation and CH3 deformation); N–H in-plane bend and the C–N stretching vibrations at 1540 cm<sup>−</sup><sup>1</sup> (Amid II). The polypeptide backbone CO stretching vibration is found in the range of 1600–1700 cm<sup>−</sup>1: bands at 1740 cm<sup>−</sup><sup>1</sup> due to carbonyl vibrations, and the one 1630 cm<sup>−</sup><sup>1</sup> due to Amide I. The spectrum shows bands at 2878 and 2927 cm<sup>−</sup><sup>1</sup> assigned to aliphatic chains (CH stretching and CH3 stretching) an Amide B band at 3073 cm<sup>−</sup><sup>1</sup> (NH stretching), and a broad band at 3500–3300 cm<sup>−</sup><sup>1</sup> related to Amide A (NH stretching) and OH vibrations [56–60].

**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<sup>−</sup><sup>1</sup> (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<sup>−</sup><sup>1</sup> 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 agreemen<sup>t</sup> 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).

**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.

### *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).

### 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 structure, in agreemen<sup>t</sup> 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.

**Figure 7.** A SEM-BSE image of the GSCM cross-section. (**<sup>a</sup>**,**b**) Two different regions (Scale bar = 10 μm).

2.3.3. Laser Scanning Microscopy (LSM) (Second Harmonics Generation Signal—SHG)

The LSM studies revealed the SHG signal from collagen Type I and Type II in the sample. In consistency with SEM, it was found that collagen in the GSCM was bundled into laminae with the width of about 60 μm. Laminae located at different depths have different, up to perpendicular, mutual orientation (the angle of packing is ~60–90◦). Laminae consist of longitudinally positioned parallel collagen fibers (Figure 8a). At the surface of some regions, bundles of collagen in the form of cords are found (Figure 8b). Similar structures were observed by SEM, as well (Figure 7a,c).

**Figure 8.** SHG images of the GSCM. (**a**) Collagen bundles in the form of laminae at different depths; (**b**) collagen in the 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.

**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].

**Figure 11.** Molecular packing of collagen in the GSCM (**a**) AFM topography (Peak Force Error channel), scan size is 3 × 3 μm; (**b**,**<sup>c</sup>**) 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%).

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—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.

**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 tinctorial 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 collagen 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 recognized 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 inflammatory reaction. However, the GSCM treatment with supercritical carbon dioxide before implantation solved this problem, reducing the inflammatory reaction to only insignificant.
