**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 arrangemen<sup>t</sup> 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 arrangement 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 membrane 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).

**Figure 14.** A possible concept of the arrangemen<sup>t</sup> 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 basketweave 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**
