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Many marine organisms such as sandcastle worms, barnacles and mussels, produce natural adhesives to attach to wet surfaces in aqueous tidal environments. In mussels, the adhesion is possible through the secretion of a protein-based water-resistant glue, composed of a mixture of proteins called mussel adhesive proteins (MAPs) or mussel foot proteins (mfps), that allow anchoring to almost any kind of surface in wet conditions [1]. The proteins confined to adhesive plaques are mfp-2, -3, -4, -5, and -6. All these proteins contain an atypically high concentration of the catecholic amino acid 3,4- dihydroxy-l-phenylalanine (DOPA), obtained by the post-translational enzymatic hydroxylation of tyrosine (Tyr) [2]. DOPA is the key molecule allowing the underwater mussel adhesion to different surfaces through the formation of irreversible covalent and reversible noncovalent bonds [3]. However, growing evidences show that the presence of DOPA is not a sufficient condition to generate strong underwater adhesion. A synergistic effect of catechols and adjacent lysine in bioadhesion of mussels has been suggested. The pair structure of Tyr/DOPA with basic residues of lysine, rather than the post-translational modification of Tyr to DOPA, should be responsible for the strong binding ability of mussel adhesive proteins [4]. We have recently fine-tuned the expression of the recombinant Pvfp5β protein in Escherichia coli [5]. Furthermore, the structural characterization showed that the produced protein was correctly folded as a β-rich protein with precise pairing of the sulfur bridges. We have also demonstrated that surfaces coated with recombinant Pvfp5β are not toxic, and improve cell adhesion, proliferation and spreading of NIH-3T3 and HeLa cell lines [5]. Thus, adhesive protein/peptide coatings and efficient exposure of bioactive domains might improve the biological properties of scaffold materials. Hydrogels, a unique class of polymeric materials, are three-dimensional porous networks constituted by polymeric chains crosslinked by chemical and/or physical bonds. They exhibit remarkable structure-derived properties, including high surface area, stimuli-responsiveness, inherent flexibility, controllable mechanical strength, and excellent biocompatibility. They are capable of absorbing and retaining large amounts of water in their networks and can extensively swell without dissolution due to the presence of crosslinks, that are at the basis of the network structure, and maintain macroscopic integrity [6]. k-carrageenan (kCar) is a naturally occurring polysaccharide extracted from marine red algae (Rhodophyceae). Chemically it consists of an alternating linear chain of (1->3)-β-D-galactose-4SO3- - (1->4)-3,6, anhydro-α-D-galactose. Due to its half-ester sulphate moieties, it is a strong anionic polymer and resembles the natural glycosaminoglycans (GAGs), which are an important component of the connective tissue. kCar is biocompatible, biodegradable, non-toxic, and gel-forming. Thus, kCar has been investigated for a wide range of biomedical puropses, such as wound dressing [7] and tissue engineering [8] applications. The generally accepted model of the gelling process of carrageenan solutions involves a coil-to-helix transition, followed, in the presence of certain cations, by aggregation of double helices to form a stiff, extended network [9]. Blending kCar with polyvinyl alcohol (PVA) allows obtaining composite scaffolds with higher, interconnected porosity, high swelling degree, improved toughness and degradability. PVA can be chemically crosslinked, with glutaraldehyde, or physically crosslinked by inducing the formation of crystalline domains via freeze-thawing [10]. In the present study, we used the crosslinking-agent free, freeze–thaw approach for the fabrication of PVA/kCar composite hydrogels as 3D scaffolds for cell culture, whose adhesion was enhanced by simple adsorption of cationic recombinant Pvfp5β protein. The physico-chemical properties of the PVA/kCar hydrogel (morphology, swelling and degradation, water absorption, mechanical strength) were evaluated. Furthermore, biocompatibility, adhesion, proliferation and morphology of the cells grown in presence of PVA/kCar-Pvfp5β hydrogel were investigated. All gathered information demonstrates the great potential of PVA/kCar-Pvfp5β scaffolds for tissue engineering applications. Prospects for the future PVA/kCar formulations are also being evaluated as bioinks for 3D bioprinting, a key enabling technology for the manufacture of complex tissue structures to mimic native organs and tissues. The bioprinting involves layer by layer deposition of cells-laden biomaterials in a predetermined structural architecture to generate functional tissues or organs. They provide structure for the bioprinted tissue and support and nutrients for the cells, creating an environment in which the cells can survive, grow, and proliferate. The research will then explore the influence of Pvfp5β on 3D inkjet bioprinted PVA/kCar scaffolds integrated with human adipose stem-cell spheroids (SASCs) in terms of cell survival and differentiation and scaffold colonization. References (max. 10 references) [1] Y. He, C. Sun, F. Jiang, B. Yang, J. Li, C. Zhong, L. Zheng, H. Ding. Lipids as integral components in mussel adhesion. Soft Matter, 14:7145–7154, 2018 [2] W. Zhang, H. Yang, F. Liu, T. Chen, G. Hu, D. Guo, Q. Hou, X. Wu, Y. Su, J. Wang. Molecular interactions between DOPA and sur- faces with different functional groups: a chemical force microscopy study. RSC Adv., 7:32518 –32527, 2017 [3] S.M. Kelly, T.J. Jess, Price N.C. How to study proteins by circular dichroism. Biochim. Biophys. Acta, 1751: 119–139 [4] X Ou, B Xue, Y Lao, Y Wutthinitikornkit, R Tian, A Zou, L Yang, W Wang, Y Cao, Jingyuan Li. Structure and sequence features of mussel adhesive protein lead to its salt-tolerant adhesion ability. Sci. Adv. 6: eabb7620, 2020 [5] R. Santonocito, F. Venturella, F. Dal Piaz, M.A. Morando, A. Provenzano, E. Rao, M.A. Costa, D. Bulone, P.L. San Biagio, D. Giacomazza, A. Sicorello, C. Alfano, R. Passantino, A. Pastore. Recombinant mussel protein Pvfp-5β: A potential tissue biohadesive. J. Biol. Chem., 294:12826:12835, 2019 [6] G.M. Kavanagh, S.B. Ross-Murphy. Rheological characterization of polymer gels. Prog. Polym. Sci., 23:533-562, 1998 [7] L.A. Ditta, E. rao, F. Provenzano, J. Lozano Sanchez, R. Santonocito, R. Passantino, M.A. Costa, M.A. Sabatino, C. Dispenza, D. Giacomazza, P.L. San Biagio, R. Lapasin. Agarose/kCarrageenan-based hydrogel film enriched with natural plant extracts for the treatment of cutaneous wounds. Int. J. Biol. Macromol., 164:2818-2830 [8] S.M. Mihaila, A.K. Gaharwar, R.L. Reis, A.P. Marques, M.E. Gomes, A. Kademhosseini. Photocrosslinkable kappa-carrageenan hydrogels for tissue engineering applications. Advanced Healthcare Materials, 2:895-907, 2013 [9] L. Du, T. Brenner, J. Xie, S. Matsukawa. A study on phase separation behavior in kappa/iota carrageenan mixtures by micro DSC, rheological measurements and simulating water and cations migration between phases. Food Hydrocolloids, 55:8188, 2016 [10] S.R. Stauffer, N.A: Peppast. Poly(vinyl ancohol) hydrogels prepared by freezing-thawing cyclic processing. Polymer, 33:3932-3936, 1992

Polymeric hydrogels are increasingly considered as scaffolds for tissue engineering due to their extraordinary resemblance with the extracellular matrix (ECM) of many tissues. As cell adhesion is a key factor in regulating important cell functions, hydrogel scaffolds are often functionalized or loaded with a variety of bioactive molecules that can promote adhesion. Interesting biomimetic approaches exploit the properties of mussel-inspired recombinant adhesive proteins. In this work, we prepared hydrogel scaffolds with a 50%w mixture of k-carrageenan (kC) and polyvinyl alcohol (PVA), by a two-step physical gelation process, and we coated them with Perna viridis foot protein-5 beta (Pvfp5 beta). The mechanical and morphological properties of hydrogels were investigated both after conditioning with typical cell culture media and also after coating with the Pvfp5 beta. The protein resulted strongly adsorbed onto the surface of the hydrogel and also able to penetrate in its interiors to a certain depth, mainly interacting with the kC component of the scaffold as resulted from the confocal analysis. Mouse embryonic fibroblasts NIH-3T3 were seeded on top of the hydrogels and cultured up to two weeks. The role of Pvfp5 beta in promoting cell adhesion, spreading and colonization of the scaffold was demonstrated.