**1. Introduction**

Collagen is an ubiquitous high molecular weight fibrous protein occurring in both invertebrate and vertebrate organisms, existing in more than 20 different types depending on its role in distinct tissues [1,2]. Its polypeptide chains are organized in a unique structure, in which three α-helices are intertwined forming a characteristic right-handed triple helix. These peptides are rich in glycine, proline, and hydroxyproline amino acids, all being crucial for the formation of the helical configuration [3].

Due to its high biocompatibility and biodegradability, collagen finds a plethora of applications, primarily in the sectors of cosmetics, pharmaceuticals, and medical care products [4,5]. Additionally, gelatin, the denatured form of collagen obtained by its partial hydrolysis, is used as an additive in the food processing industry and in nutraceuticals [6]. Its intrinsic low immunogenicity renders this natural biopolymer an ideal material for bone grafting, tissue regeneration, and construction of artificial skin [7,8]. Collagen destined for industrial use originates mainly from bovine and porcine sources, via an acid hydrolysis-based procedure [9]. Incidences of allergic reactions and connective

tissue disorders, such as arthritis and lupus, as well as bovine spongiform encephalopathy and transmissible spongiform encephalopathy [10], have led to the reconsideration of cattle as the main source for collagen production. Furthermore, porcine collagen is prohibited for the Muslim and Jewish communities due to religious restrictions. Taking into account these two limitations, an alternative and safer source is currently actively sought.

Nowadays, collagen of marine origin as an alternative to mammalian sources is gaining ground, especially since the employment of recombinant technology is excluded due to its high cost [11,12]. Since collagen is the major constituent of the extracellular matrices of all metazoans, sponges are considered as one of the most promising sources [13–15]. Sponges, belonging in the phylum Porifera, composed of a mass of cells forming a porous skeleton made of organic (collagen fibers and/or spongin, especially in the case of the class Demospongiae) and inorganic (spicules) components, are the most primitive among multicellular animals (Metazoa) [16,17]. Marine sponges have been proven an inexhaustible source of secondary metabolites exhibiting diverse pharmacological properties [18–21]. In addition to these, macromolecules have gained interest since such biopolymers possess a wide range of bioactivities that can find applications in the biomedical sector. Collagen has been isolated from different marine sponges, e.g., *Spongia graminea*, *Microciona prolifera*, *Haliclona oculata* [22], *Hippospongia communis*, *Cacospongia scalaris* [23], *Geodia cydonium* [24] *Chondrosia reniformis* [25,26], and various *Ircinia* species [27], and in certain cases has shown high potency in tissue regeneration [28]. Although the importance of marine collagen has been recognized, only a few thorough investigations on marine sponges have so far been reported [25,27,29], probably due to its characteristic insolubility and mineralization, which cause difficulties in its isolation and characterization [30,31].

In the present study we report, for the first time, the isolation and characterization of collagens from the marine demosponges *Axinella cannabina* (Axenillidae) and *Suberites carnosus* (Suberitidae). By employing two different experimental approaches, the insoluble collagen (InSC), intercellular collagen (ICC), and spongin-like collagen (SlC) were obtained. The morphology of these collagens was analyzed by scanning electron microscopy (SEM), and their fibril formation and characteristic band periodicity was studied by transmission electron microscopy (TEM). Their secondary structure was evaluated based on their FT-IR spectra, while the amino acid composition of the ICCs was also determined. The thermal behavior of the ICCs was investigated by differential scanning calorimetry (DSC) and circular dichroism (CD) analyses.

#### **2. Results**

#### *2.1. Isolation of Sponge Collagen*

Two different procedures were used for the isolation of collagens from the demosponges *A. cannabina* and *S. carnosus*. The first method was initially introduced for the isolation of insoluble collagen (InSC) from *G. cydonium* [24] and *C. reniformis* [26] by employing an alkaline, both denaturing and reducing, homogenization buffer affording collagen in high yield. The second one utilizes a trypsin-containing extraction buffer, known to destroy the interfibrillar matrix and, therefore, releasing the collagen fibrils (ICC) [22,23]. After exhaustive water extraction, the remaining debris generally comprises the spongin/spongin-like collagen. In our case, since the specific sponges are deprived of spongin, the isolated samples are considered to contain spongin-like collagen (SlC).

The InSCs obtained by the application of the first method corresponded to 12.6% and 5.0% of the sponges' dry weight for *A. cannabina* and *S. carnosus*, respectively (Table 1). Application of the second method resulted in the isolation of ICC and SlC, leveling to 3.0% and 42.8% dry weight for *A. cannabina* and 1.9% and 21.8% dry weight for *S. carnosus*, respectively (Table 1). The percentages found for the ICC yield are in accordance with previously-reported results for *Hippospongia gossipina* [32].

The siliceous or calcareous sponges are characterized by a large number of inorganic spicules, which, in *Haliclona* and *Microciona*, are bound together by spongin [22]. In order to remove the expected siliceous spicules in the InSC, the samples were treated with an HF solution for 20 min at

room temperature to obtain spicule-free insoluble collagen (SF-InSC). The spicules accounted for 32% and 49% (*w*/*w*) of the sponges' InSCs from *A. cannabina* and *S. carnosus*, respectively.


**Table 1.** Collagen composition (*w*/*w* %) <sup>1</sup> of the sponges *A. cannabina* and *S. carnosus*.

<sup>1</sup> Data are presented as the percent of sponge dry weight.

### *2.2. Examination of Surface Morphology*

The collagenous nature of the isolated materials was investigated by SEM and TEM. Overall, the microscopically-observed structures (Figure 1) were similar to those already reported for collagen isolated from other sponges [25–27,29]. Figure 1A,E show the microstructure of the InSCs from *A. cannabina* and *S. carnosus*, respectively, as observed by SEM analysis. Smoothly wrinkled and folded sheets were evident. Additionally, the SEM pictures revealed that both sponges possess significant amount of spicules embedded in the very thin and soft sheet-like collagenous structure [22]. After removal of the spicules, the SF-InSCs appeared more as an amorphous matrix, while TEM depicted (Figure 1J,N) the collagenous material as appearing transparent, resembling those obtained before treatment with HF (Figure 1I,M). The complete removal of spicules was also confirmed by SEM (data not shown), where no silicate spicules were observed.

In the case of the SlCs, analogous structures were visible. In particular, siliceous spicules, known to support the sponges and provide defense against predation, were also detected (Figure 1D,H). On the other hand, ICCs presented the typical striation and sheet-like appearance of collagen fibers (Figure 1B,C,F,G), which conclusively proved the collagenous nature of the materials. Specifically, the ICCs from both sponges were observed as threads of various diameters along with the collagen sheets which is a combination of several collagen fibrils and fibers that are bundled together to form a fibril network and a dense pleated sheet-like structure. Sheets were smoothly wrinkled and folded, and appeared as very thin and soft (Figure 1C,G). Pleating of the sheets was visible at a magnification of 5000×.

The collagenous nature of the ICCs of *A. cannabina* and *S. carnosus* was further proved by TEM studies (Figure 1K,O). The obtained micrographs revealed the existence of filaments composed of striated collagen fibrils with repeated band periodicity, a characteristic feature of collagens, as observed earlier for sponge collagen fibrils [16]. Collagen fibrils were organized into bundles, while fibrils became aligned laterally in an ordered way, or curled into bundles consisting of up to 20 fibrils [24]. The individual fibrils displayed a visible, regular transverse banding pattern of about 300 Å periodicity (313 and 288 Å for *A. cannabina* and *S. carnosus*, respectively). These banding patterns are in accordance with the one reported for collagen fibrils isolated from *C. reniformis* [25,26]. The bundles revealed remarkable uniformity in the diameter of their constitutive fibrils (Table 2) with an average of 187 and 199 Å for *A. cannabina* and *S. carnosus*, respectively, in accordance with previously-reported data for other sponges [25,29].

The recorded TEM micrographs for the InSCs (Figure 1I,M) and the SlCs (Figure 1L,P) samples did not present a characteristic pattern. However, in the case of the SlC isolated from *S. carnosus* an area with clearly-striated collagen was detected (Figure 1P insert), most likely due to the nature of the preparation, composed of a mixture of ICC and SlC, also previously reported by Gross and coworkers [22]. The lack of a clear banding pattern might be attributed to the isolation, under the described conditions, of dominating collagenous structures presenting common characteristics with basement membrane (type IV) collagen. Transparent sheets of collagenous material were also previously observed for irciniid collagens, attributed to the non-fibrillar basement-type resembling collagens [27].

**Figure 1.** SEM micrographs of insoluble collagen (InSC; **A**,**E**), intercellular collagen (ICC; **B**,**C**,**F**,**G**), and spongin-like collagen (SlC; **D**,**H**) from *A. cannabina* (row 1) and *S. carnosus* (row 2), respectively. TEM micrographs of insoluble collagen before (InSC; **I**,**M**) and after (SF-InSC; **J**,**N**) spicule removal, intercellular collagen (ICC; **K**,**O**) and spongin-like collagen (SlC; **L**,**P**) from *A. cannabina* (row 3) and *S. carnosus* (row 4), respectively.

**Table 2.** Morphological characteristics (presented as means ± S.E.) of insoluble collagen (InSC), intracellular collagen (ICC) and spongin-like collagen (SlC) isolated from *A. cannabina* and *S. carnosus*.


1,2,3,4 Data denoted by the same superscript are not significantly different (*p* > 0.05).
