**1. Introduction**

The basic objective of tissue engineering consists of obtaining such a scaffold material that would promote complete or at least partial regeneration of internal organs, skin, vascular, bone, cartilage, and other tissues. For a construct to successfully engraft in the body, its parameters ought to be similar to those of the region in which the construct

**Citation:** Frolova, A.; Aksenova, N.; Novikov, I.; Maslakova, A.; Gafarova, E.; Efremov, Y.; Bikmulina, P.; Elagin, V.; Istranova, E.; Kurkov, A.; et al. A Collagen Basketweave from the Giant Squid Mantle as a Robust Scaffold for Tissue Engineering. *Mar. Drugs* **2021**, *19*, 679. https://doi.org/10.3390/ md19120679

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Academic Editor: Sik Yoon

Received: 17 November 2021 Accepted: 20 November 2021 Published: 29 November 2021

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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 engineering [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 ligaments 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 ye<sup>t</sup> 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 conditions 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 properties 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.


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

> 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 grea<sup>t</sup> pressure, must have a robust musculature and outer coating to

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).
