*4.6. FTIR*

FTIR analysis was performed on freeze-dried samples. Dried materials were mixed with potassium bromide (Sigma), crushed with a pestle and processed into a thin pellet using a hand press. Spectra were recorded from 32 scans with a resolution of 2 cm−<sup>1</sup> from 4000 to 700 cm−<sup>1</sup> using a Shimadzu IR Prestige 21 Infrared spectrometer (Shimatzu, Kyoto, Japan).

#### *4.7. Rheology*

The rheological properties of the collagenous materials obtained after the dialysis process were investigated by means of a Kinexus Prot rheometer (Malvern instruments, Malvern, UK). Steady-state flow measurements were carried out using a controlled-stress rheometer fitted with parallel plate geometry with a 10-mm diameter (PU20 SR1740 SS) and 1-mm gap. The torque amplitude was imposed by using a logarithmic ramp of shear rate, in a range of 0.1 to 100 s−1. Rheological measurements in oscillatory frequency sweep strain controlled were performed in using the same parallel plate geometry with a 1 mm gap. Gel rheological behavior with storage/loss moduli and cross-over analysis was assessed at a constant 1% strain in a frequency region from 0.01 to 100 Hz.

All experiments were performed at a controlled temperature of 37 ◦C and results represent the average of three measurements.

#### **5. Conclusions**

We described a new method to extract collagen from the marine sponge *C. reniformis* resulting in sticky collagenous hydrogels. The materials obtained from two sponge regions, ectosome and choanosome, have singular rheological properties, with the observed shear thinning behavior seeming to depend on the presence of collagen fibrils and other associated proteins/GAGs. The weak interaction between those macromolecules have shown to be reversible and induce a fast recovery to the rest state of the viscosity after manipulation, suggesting their possible use as an injectable hydrogel medium for biomedical applications. The material obtained should be, however, further investigated to completely characterize its content and to fully address the role of the soluble compound(s). The data presented in this study represent a detailed starting point for further investigations aimed at producing tailored hydrogels with different interesting properties through a biomimetic approach.

**Supplementary Materials:** The following are available online at www.mdpi.com/1660-3397/15/12/380/s1, Video S1: demonstration of the adhesive and self-healing properties of the collagenous materials obtained from the choanosome.

**Acknowledgments:** The authors gratefully acknowledge the financial support from the European Union Seventh Framework Programme (FP7/2007–2013) under grant agreement ERC-2012-ADG 20120216-321266 (ERC Advanced Grant project ComplexiTE), as well as from the European Regional Development Fund (ERDF) under the projects "Accelerating tissue engineering and personalized medicine discoveries by the integration of key enabling nanotechonologies, marine-derived biomaterials and stem cells" (NORTE-01-0145-FEDER-000021), supported by Norte Portugal Regional Operational Program (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, and 0687\_NOVOMAR\_1\_P, co-financed by transborder cooperation programme POCTEP. The authors are also thankful to the Area Marina Protetta Portofino (Italy) for permission to collect sponge specimens and to Daniela Candia (University of Milan, Italy) and Marco Giovine (University of Genoa, Italy) for the logistical support on the sponge sampling and immediate processing. We are grateful to the two anonymous referees for improving the quality of the present article.

**Author Contributions:** D.F., A.R.C.D. and T.H.S. conceived and designed the experiments; D.F. and A.R.C.D. performed the experiments; D.F., A.R.C.D. and T.H.S. analysed the data; R.L.R. and T.H.S. contributed in reagents/materials/analysis tools; D.F. wrote the draft of the manuscript and all the authors reviewed it to reach the final version.

**Conflicts of Interest:** The authors declare no conflict of interest.
