*Trends in the Applications of Spongins*

The history of studies on the chemistry, molecular biology, biochemistry, and bioinspired materials science of spongins remains relevant today, partly due to the poorly understood basis of ecological disaster in the case of sponge diseases, but mostly due to recent progress in the direct applications of sponge skeletons as 3D spongin scaffolds in tissue engineering and biomimetics. Additionally, the marine ranching of bath sponges worldwide is a crucial factor in the adoption of spongins as renewable naturally prestructured proteinaceous scaffolds.

The spongin-based skeletons of bath sponges appear to possess a number of unique and useful properties, which had been exploited long before such scientific fields as tissue engineering and bioengineering were proposed. As reviewed by Szatkowski et al. [86], from the 18th century commercial bath sponges were valued in medicine due to their softness, high compressive strength, ability to retain shape, and high sorption rates. For these reasons, they were used as compression bandages for pressing open sinuses, in overcoming strictures of body passages (including the rectum), for dilation of the cervix uteri [86–90], and in the form of sponge tents applied in the uterus to expand the cavity and enable examination. More intriguingly, fragments of sponge skeleton were used as small prostheses in early "plastic surgery" [91]. Revolutionary results were obtained by Hamilton in 1881. In a paper entitled "*On sponge-grafting*" [92], he reported the following case. A woman underwent surgery for removal of a mammary tumor, during which a large area of skin was removed. The skin was replaced with a thin slice of an aseptic sponge skeleton, which ten days after the surgery was observed to be vascular, and three months later was covered with epithelial tissue (Figure 5).

**Figure 5.** Sketch of a fragment of spongin framework (**b**) surrounded by a great number of living cells (**a**,**c**) in a sponge-grafting application (adapted from [92]).

Today, spongin-based scaffolds are actively used in diverse applications related to tissue engineering. Positive results have been reported with human osteoprogenitor cells on the skeleton of *S. officinalis* [46], with osteoblast-like MG-63 cells growing on spongin from *Hymeniacidon sinapium* [93] and with mouse primarily osteoblasts on spongin from *Callyspongiidae* marine demosponges [49]. Recently, Nandi et al. [51] have proposed that the skeleton of the marine sponge *Biemna* sp.—alone and in combination with growth factors—is a promising biomaterial for bone repair and bone augmentation.

Besides applications in the biomedical field, spongin-based scaffolds have been successfully used as adsorbents of diverse dyes [94,95] and as supports for enzyme immobilization [96]. It was recently shown that spongins are thermostable up to 260 ◦C [86,97,98]. This property opens the door for applications of spongin-based scaffolds with 3D architecture in such novel scientific disciplines as Extreme Biomimetics [99], with the aim of developing novel advanced composite materials.

#### **3. Collagen IV and Related Proteins in Sponges**

It is now well established that collagens are key to the structural integrity and biomechanical properties of various tissues of Metazoans. One of them, the basement membrane-forming collagen IV, is extremely ancient. Collagen IV networks have a polygonal architecture that endows basement membranes (BMs) with a tensile strength sufficient to protect tissues from mechanical stress, in addition to serving as important regulators of the dynamic events associated with cell adhesion, signaling, and survival [100]. According to the modern view [101], only the presence of the collagen IV gene was precisely correlated with the emergence of BMs in animals. Thus, the triple helical collagen IV was required for the development of BMs.

BMs underlie the epithelia in Metazoa from sponges to humans [102]. Interestingly, until 1996, basement membrane structures and type IV collagen were known to be present in all multicellular animal species except sponges. In Porifera, BMs are associated with the basal surfaces of polarized epithelial cells [103]. After the first report on the identification of type IV collagenous sequences in the homoscleromorph sponge *Pseudocorticium jarrei* by cDNA and genomic DNA [103], this collagen has been found in diverse poriferans. For example, in corresponding transcriptome data from a calcareous sponge (*Sycon coactum*) and another homoscleromorph sponge (*Corticium candelabrum*), two new type IV collagen genes were found in each [104]. Homologs of important components

of basement membrane genes, including type IV collagen, have been found in the Demospongiae *Spongilla lacustris*, *Ircinia fasciculata*, and *Chondrilla nucula* [105]. The discovery of type IV collagen in Calcarea and Demospongiae is very important, because nowhere in this group has a BM-like structure been noted. The presence of type IV collagen in glass sponges (Hexactinellida) remains to be detected. Polyclonal antibodies have detected type I (but not type IV) collagen in the anchoring spicules of the *Hyalonema sieboldii* glass sponge [3].

The relationship between type IV collagen and the so-called spongin short chain collagen (SSCC) [106] is still under investigation [101]. SSCC has been considered as ancestral to type IV collagen [107]. Like type IV collagen, SSCC also has NC1 domains which produce the globular heads particular to type IV collagen and which are required for assembly of the unique scaffold of the BM (see for review [104]). It is suggested that collagen IV and its spongin variant are primordial components of the extracellular microenvironment, where collagen IV especially was a key player in the evolution of epithelial tissues in Metazoa, including sponges, due to the transition to multicellularity [101].

Interestingly, collagen IV from the demosponge *Chondrosia reniformis* has recently been patented as a source of special membranes for biomedical applications [108]. The collagen was isolated with an extraction solution of 100 mM Tris-HCl, 10mM EDTA, 8 M urea, and 100 mM 2-mercaptoethanol, rendering the protein in the form of a precipitate. This was used for the development of stable and non-cytotoxic type IV collagen membranes, which can be applied in tissue engineering and regenerative medicine approaches for epithelial repair, regeneration, or replacement. The technology includes the re-epithelialization of any single and stratified epithelium, with emphasis on the skin.

#### **4. Fibrillar Collagens in the Mesohyl of Demosponges**

The mesohyl includes a noncellular colloidal mesoglea with embedded collagen fibers, spicules, and various cells, being as such a type of mesenchyme. It is currently debated whether the mesohyl and pinacoderm layers in sponges are true tissues [109]. Collagens serve several functions in sponges [27,106,110]. The formation of mesohyl certainly involves the activity of fine fibrils made of fibrillar collagen. The collagen fibrils both mediate cell–matrix interactions via membrane receptors and provide the structure of the extracellular matrix (ECM), a situation observed in vertebrates. The increase in the structural diversity of fibrillar collagen chains, their different forms of maturation, and interactions with other ECM components appeared during the process of evolution [111]. The diversity of sponges which contain high amounts of fibrillar collagen within their mesohyl has been described previously (see for review [110–112]).

Fibrillar bundles, formed by the association of several hundred collagen fibrils, have been observed in diverse species of *Tethya*, *Chondrosia*, *Chondrilla*, *Jaspis*, and *Suberites* (see for details [112,113]). The densely packed bundles of collagen fibrils are secreted exclusively by the highly polarized lophocyte cells [43,111]. These are actively moving cells, pulling behind them a bundle of regularly arranged collagen fibrils.

Another kind of collagen-producing cell has been discovered in the mesohyl of the demosponge *Suberites domuncula* [114,115], in which the expression of collagen genes is controlled by silicate and myotrophin [116]. SEM observations have revealed the complex collagen network surrounding the spicules within the mesohyl of adult specimens (Figure 6).

Collagen fibers have also been identified in the mesohyl of the demosponge *Haliclona rosea* [116]. Collagen has also been reported in the mesohyl of such Calcarea sponges as *Leucosolenia* sp. and *Leucandra* sp. [117].

Collagen fibril content is also high in the external asexual buds that occur in *Tethya lyncurium* [118]. Similarly, the buds of *T. sychellensis* contain a dense collagen matrix [119]. Buds consist of cellular masses that sprout out from the surface of adults and are able to develop into new functional individuals [119].

Recently, special attention has been focused on fibrillar collagens in the mesohyl of *C. reniformis*. This species is the only sponge which has been experimentally proven to contain a dynamic collagenous

mesohyl capable of stiffening upon being manipulated [120]. It was shown that the different physiological states recorded in laboratory experiments are expressions of the mechanical adaptability of the collagenous mesohyl of *C. reniformis*, and suggest that stiffness variability in this sponge is under cellular control [121].

**Figure 6.** SEM view through the collagenous mesohyl of the demosponge *S. domuncula*. Layers of collagen fibrils (**A,B**) are a result of the activity of the unique collagen-producing cells which are seen to line up along the surface of the spicules (**C**–**E**). The line of cells (**A**) can move from left to right along the spicule, depositing a rough, nanofibrillar collagenous layer in their wake (**C**) (see also [114]).
