Fassini D , Duarte ARC , Reis RL , Silva TH .

321266 European Research Council

	Scheme 1. Schematic representation of the extraction procedure as described in Fassini et al. 2014 [37] (blue squared text, left side) and the new extraction procedure (black squared text, right side). The initial step is the same for both the treatments (broken line); different treatments with similar aims are put on the same line in order to highlight the similarities and differences between the two extraction procedures. The final results of the two protocols is showed in the double bounded squares. PBS = phosphate buffer saline; EDTA = ethylenediaminetetraacetic acid; DS = disaggregating solution.
	Figure 1. Image of a choanosome sample before (a) and during (b) the dialysis step.
	Figure 2. SDS-PAGE of ectosome (Ec) extract after one month (lane 1A, 1B) or two months (lane 2A, 2B) in DS stained with Coomassie R-250 (1A, 2A) or alcian blue (1B, 2B). A volume corresponding to 250 µg of material was added to each well.
	Figure 3. Effects of pepsin digestion on choanosome (Ch) collagen extract. (a) Images of the materials soon after the digestion/control treatment; and (b) meniscus details of the supernatants obtained after the removal of the insoluble component. Tubes legend: A = Ec digested; B = Ec control; C = Ch digested; D = Ch control.
	Figure 4. 7.5% (a,b) and 15% (c,d) sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) stained with R-250 Coomassie (a,c) and alcian blue (b,d) of digested and undigested samples. Lanes 1/5 = digested Ec; 2/6 = undigested Ec; 3/7 = digested Ch; 4/8 = undigested Ch. Lanes 1–4 = pellet; 5–8 = supernatant. Pellets obtained after centrifugation were resuspended in 800 µL of loading buffer and freeze-dried supernatants in 80 µL; 40 µL of the resulting dispersions were added in each well. Arrowhead = presumptive collagen; broken line rectangle = possible glycan.
	Figure 5. 10% Tris/borate/EDTA polyacrylamide gel electrophoresis (TBE-PAGE) of glycosaminoglycans (GAG) extracted from choanosome (Ch; lane 2) and ectosome (Ec; lane 3), after staining with alcian blue. A similar quantity (170 µg) of shark chondroitin sulfate was loaded into lane 1 as reference.
	Figure 6. FTIR spectra of Sigma bovine type I collagen (black); Ec (dark grey); Ch (light grey); Ch supernatant (red). Broken line rectangle indicate Amide A region. Absorbance values have been shifted to facilitate the reading.
	Figure 7. Flow curves of ectosome (Ec) and choanosome (Ch) subjected to increasing shear rates. (a) = Ec 14.4 mg/mL; (b) = Ch 9.6 mg/mL; (c) = Ch 8.2 mg/mL (Ch 9.6 mg/mL after centrifugation and resuspension). Square = viscosity; diamonds = shear stress. Values are expressed as the average of three repeated experiments; bars = standard deviation.
	Figure 8. Storage and loss modulus measurements of choanosome (Ch) and ectosome (Ec) materials. Changes in the storage (G′, closed symbols), and loss (G″, open symbols) moduli at increasing frequency (a,c) and loss tangent (b,d) of Ec 14.4 mg/mL (a,b) and Ch 9.6 mg/mL (c,d). Values are expressed as the average of three repeated experiments; bars = standard deviation.
	Figure 9. Ramp-up and ramp-down experiment showing the thixotropic property of the collagenous materials. Shear stress/rate (a) and viscosity/shear rate (b) curves of coanosome (Ch) 9.6 mg/mL in a ramp up/down experiment. Open grey symbols = ramp up; closed black symbols = ramp down. Lines connecting the points have been added, the points represent the average values of three different experiments. Standard deviations bars have been omitted to facilitate the reading of the graphs.
	Figure 10. Viscosity values (grey dots) of coanosome (Ch) 9.6 mg/mL in a three-step experiment. Black dots = shear rate. Values are expressed as the average of three repeated experiments; bars = standard deviation.

An, Collagen interactions: Drug design and delivery. 2016, Pubmed

An, Collagen interactions: Drug design and delivery. 2016, Pubmed
Antoine, Review of collagen I hydrogels for bioengineered tissue microenvironments: characterization of mechanics, structure, and transport. 2014, Pubmed
Aouacheria, Insights into early extracellular matrix evolution: spongin short chain collagen-related proteins are homologous to basement membrane type IV collagens and form a novel family widely distributed in invertebrates. 2006, Pubmed
Bavestrello, Body Polarity and Mineral Selectivity in the Demosponge Chondrosia reniformis. 1998, Pubmed
Bentkover, The biology of facial fillers. 2009, Pubmed
Berthod, Collagen synthesis by fibroblasts cultured within a collagen sponge. 1993, Pubmed
Berthod, Optimization of thickness, pore size and mechanical properties of a biomaterial designed for deep burn coverage. 1994, Pubmed
Cen, Collagen tissue engineering: development of novel biomaterials and applications. 2008, Pubmed
Chandran, Structural mechanism for alteration of collagen gel mechanics by glutaraldehyde crosslinking. 2012, Pubmed
Czirók, Extracellular matrix dynamics during vertebrate axis formation. 2004, Pubmed
Dewavrin, Tuning the architecture of three-dimensional collagen hydrogels by physiological macromolecular crowding. 2014, Pubmed
Di Lullo, Mapping the ligand-binding sites and disease-associated mutations on the most abundant protein in the human, type I collagen. 2002, Pubmed
Ehrlich, First evidence of chitin as a component of the skeletal fibers of marine sponges. Part I. Verongidae (demospongia: Porifera). 2007, Pubmed
Exposito, The fibrillar collagen family. 2010, Pubmed , Echinobase
Fassini, The reaction of the sponge Chondrosia reniformis to mechanical stimulation is mediated by the outer epithelium and the release of stiffening factor(s). 2014, Pubmed
Fernandes-Silva, Porous hydrogels from shark skin collagen crosslinked under dense carbon dioxide atmosphere. 2013, Pubmed
Garrone, Fine structure and physiocochemical studies on the collagen of the marine sponge Chondrosia reniformis nardo. 1975, Pubmed
Gelse, Collagens--structure, function, and biosynthesis. 2003, Pubmed
Ghobril, The chemistry and engineering of polymeric hydrogel adhesives for wound closure: a tutorial. 2015, Pubmed
Gough, Cytotoxicity of glutaraldehyde crosslinked collagen/poly(vinyl alcohol) films is by the mechanism of apoptosis. 2002, Pubmed
Heinemann, Ultrastructural studies on the collagen of the marine sponge Chondrosia reniformis Nardo. 2007, Pubmed
Imhoff, Solubilization and characterization of Chondrosia reniformis sponge collagen. 1983, Pubmed
Junqueira, Biology of collagen-proteoglycan interaction. 1983, Pubmed
Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T4. 1970, Pubmed
Lai, Effect of concentration and temperature on the rheological behavior of collagen solution. 2008, Pubmed
Lee, Biomedical applications of collagen. 2001, Pubmed
Lynn, Antigenicity and immunogenicity of collagen. 2004, Pubmed
Matsumura, Disaggregation of connective tissue: preparation of fibrous components from sea cucumber body wall and calf skin. 1973, Pubmed , Echinobase
Müller, A new three-dimensional model of the organization of proteoglycans and collagen fibrils in the human corneal stroma. 2004, Pubmed
Nandini, Novel 70-kDa chondroitin sulfate/dermatan sulfate hybrid chains with a unique heterogeneous sulfation pattern from shark skin, which exhibit neuritogenic activity and binding activities for growth factors and neurotrophic factors. 2005, Pubmed
Orban, Crosslinking of collagen gels by transglutaminase. 2004, Pubmed
Osborne, Investigation into the tensile properties of collagen/chondroitin-6-sulphate gels: the effect of crosslinking agents and diamines. 1998, Pubmed
Pallela, Biochemical and biophysical characterization of collagens of marine sponge, Ircinia fusca (Porifera: Demospongiae: Irciniidae). 2011, Pubmed
Pozzolini, Molecular Cloning, Characterization, and Expression Analysis of a Prolyl 4-Hydroxylase from the Marine Sponge Chondrosia reniformis. 2015, Pubmed
Riesgo, The analysis of eight transcriptomes from all poriferan classes reveals surprising genetic complexity in sponges. 2014, Pubmed
Schuppan, Collagens in the liver extracellular matrix bind hepatocyte growth factor. 1998, Pubmed
Silva, Marine origin collagens and its potential applications. 2014, Pubmed
Silva, Following the enzymatic digestion of chondroitin sulfate by a simple GPC analysis. 2015, Pubmed
Silva Júnior, Effect of papain-based gel on type I collagen--spectroscopy applied for microstructural analysis. 2015, Pubmed
Sundararaghavan, Genipin-induced changes in collagen gels: correlation of mechanical properties to fluorescence. 2008, Pubmed
Swatschek, Marine sponge collagen: isolation, characterization and effects on the skin parameters surface-pH, moisture and sebum. 2002, Pubmed
Tronci, Triple-helical collagen hydrogels via covalent aromatic functionalization with 1,3-Phenylenediacetic acid. 2013, Pubmed
Uriz, Siliceous spicules and skeleton frameworks in sponges: origin, diversity, ultrastructural patterns, and biological functions. 2003, Pubmed
Walters, Strategies for directing the structure and function of three-dimensional collagen biomaterials across length scales. 2014, Pubmed
Weadock, Physical crosslinking of collagen fibers: comparison of ultraviolet irradiation and dehydrothermal treatment. 1995, Pubmed
Wilkie, Mechanical adaptability of a sponge extracellular matrix: Evidence for cellular control of mesohyl stiffness in Chondrosia reniformis Nardo. 2006, Pubmed
Wollensak, Riboflavin/ultraviolet-a-induced collagen crosslinking for the treatment of keratoconus. 2003, Pubmed
Xu, Effect of marine collagen peptides on long bone development in growing rats. 2010, Pubmed
Zeugolis, Cross-linking of extruded collagen fibers--a biomimetic three-dimensional scaffold for tissue engineering applications. 2009, Pubmed
Zhu, Design properties of hydrogel tissue-engineering scaffolds. 2011, Pubmed