Construction and evaluation of sponge scaffolds from hyaluronic acid derivatives for potential cartilage regeneration Fabio Salvatore Palumbo, a Calogero Fiorica, a Giovanna Pitarresi, * ab Mario Giorgi, c Francesca Abramo, d Alessandro Gulino, e Mauro Di Stefano a and Gaetano Giammona af A two or one pot synthesis has been used for the reaction of hyaluronic acid (HA) with octadecylamine (C 18 ) and hydrazine (Hy). In both cases, the chemical derivatization involved primary hydroxyl groups of hyaluronic acid and not its carboxyl groups, whose presence is important for receptor interaction. In this way, Hy-HA-C 18 derivatives have been obtained with appropriate hydrophobic and hydrophilic character. Their ability to form homogeneous physical hydrogels has been evaluated as well as the possibility to obtain porous sponges through salt leaching technology. Sponges showing the highest porosity, potentially compatible with cell entrapment, have been characterized with regard to their physicochemical and biological properties. Swelling ability under simulated physiological conditions and stability in the absence or in the presence of hyaluronidase have been investigated. Bovine chondrocytes were viable in Hy-HA-C 18 sponges as determined with MTS assay and were able to produce collagen and glycosaminoglycans, as assessed by using Masson's trichrome and Alcian blue, respectively. Finally, in vivo degradation of Hy-HA-C 18 sponges has been confirmed after subcutaneous implantation in mice until 6 weeks. Introduction Cartilage damage affects many people in the world but unfor- tunately this tissue shows a reduced ability for self regeneration due to the absence of blood vessels and the scarce cell turnover. 1 Regenerative medicine is a promising strategy for the treatment of damaged cartilage, thanks to the use of three-dimensional scaffolds able to allow differentiation, growth and proliferation of chondrocytes. 2 Taking into account that hydrogels are networks with a high water content as well as the natural cartilaginous tissue, these systems are considered appropriate candidates as scaffolds for cartilage regeneration. Since components of the extracellular matrix (ECM) are able to interact with cells by means of surface receptors and to control their biological functions, several scaffolds are prepared using these natural materials or their derivatives. 3 In particular, hya- luronic acid (HA) is considered an optimal material to prepare scaffolds for the regeneration of cartilage. 4 However HA alone lacks appropriate hydrolytic resistance in vivo, therefore chem- ical modications involving HA crosslinking or graing to other molecules have been performed to improve its performances. Polymers such as polyesters (polylactic acid, poly(lactic-co-gly- colic) acid, polycaprolactone) or aliphatic chains have been linked to HA and resulting derivatives have been described for the production of scaffolds or nanoparticles. 5–7 In particular the graing with hydrophobic chains can be useful to confer appropriate hydrolytic resistance to HA based hydrogels applied for cartilage regeneration 8,9 as well as to improve the solubility in organic solvents thus allowing the use of appropriate tech- niques for preparing scaffolds with a controlled micro-archi- tecture. 5 As an example physical hydrogels, obtained through aqueous dispersions of HA-g-alkyl chain derivatives, have been proposed as biomaterials to release chondrocytes into full thickness articular defects. 10 Aqueous dispersions of such derivatives at 1% w/v have produced viscoelastic hydrogels that have been loaded into rat articular cartilage defects and tested for their property to improve cartilage repair. However, in such derivatives, aliphatic chains have been linked to the glucuronic carboxyl groups of HA through the formation of amide linkages. Since it is known that carboxyl functionalities are important for a Dipartimento di Scienze e Tecnologie Biologiche Chimiche e Farmaceutiche, Plesso di Chimica e Tecnologie Farmaceutiche, Universit` a degli Studi di Palermo, Via Archira 32, 90123, Palermo, Italy b IBIM-CNR, Via Ugo La Malfa 153, 90146 Palermo, Italy. E-mail: giovanna.pitarresi@ unipa.it c Department of Veterinary Sciences, Via Livornese (lato monte), San Piero a Grado, Pisa, 56122, Italy d Department of Veterinary Sciences, Viale delle Piagge 2, 56124 Pisa, Italy e Department of Health Science, Human Pathology Section, School of Medicine, Via del Vespro 129, 90127, Palermo, Italy f Institute of Biophysics at Palermo, Italian National Research Council, Via Ugo La Malfa 153, 90146 Palermo, Italy Cite this: J. Mater. Chem. B, 2014, 2, 3243 Received 24th February 2014 Accepted 12th March 2014 DOI: 10.1039/c4tb00311j www.rsc.org/MaterialsB This journal is © The Royal Society of Chemistry 2014 J. Mater. Chem. B, 2014, 2, 3243–3253 | 3243 Journal of Materials Chemistry B PAPER