Stimuli-responsive chitosan-starch injectable hydrogels combined with encapsulated adipose-derived stromal cells for articular cartilage regeneration† Helena S a-Lima, * ab Sofia G. Caridade, ab Joa ˜o F. Mano ab and Rui L. Reis ab Received 1st March 2010, Accepted 20th May 2010 DOI: 10.1039/c0sm00041h Tissue engineering strategies have been showing promising early results in articular cartilage lesions repair. Hydrogels based on natural origin polymers as chitosan glycerol-phosphate (CGP) thermo- sensitive formulation that can be implanted in a minimal invasive manner, represent a great promise as injectable scaffold choice for cartilage tissue engineering, but it lacks in mechanical properties. A different formulation, from which a firm texture gels results is, therefore, desirable. In this work we first aim to investigate the suitability of CGP to produce an injectable thermosensitive, pH-dependent solution, when combined with increasing concentrations of starch: 0.5% (I), 1% (II), and 1.5% (III). The data collected from the rheological measurements showed that the addition of starch to the CGP did not alter the transition temperature and confirmed the heating inducing gelation of all solutions, supporting the ability of these novel formulations to be applied as minimal invasive systems. The evaluation of the dynamic mechanical analysis of the hydrogels showed an increase in the storage modulus within increasing starch concentration, clearly demonstrating that best viscoelastic properties were obtained with the novel chitosan-starch based solution. The incorporation of starch also improved the degradation profile. All materials showed to be biocompatible through the cytotoxicity screening in vitro. These data suggested the potential of novel thermo-responsive chitosan-starch hydrogels to be used as injectable vehicles for cell delivery in cartilage tissue engineering applications. In a second phase, the potential of chitosan-b-glycerophosphate (CGP) and chitosan-b- glycerophosphate-1% starch (CST) hydrogels to induce chondrocytic differentiation and cartilage matrix accumulation were evaluated, as well as the influence of starch in the chondrogenesis of encapsulated adipose derived stromal (ADSC) cells. The ADSC were homogeneously encapsulated, remained viable, proliferated, and maintained the expression of typical chondrogenic markers genes, and deposited cartilage ECM molecules. Improved results were obtained within the novel CST constructs. The overall data suggest that chitosan-b-glycerophosphate-starch hydrogels could be considered for chondrogenic differentiation of adipose derived stromal cells for cartilage-engineered regeneration using minimal invasive techniques. Introduction Articular cartilage defects resulting from injury or osteochondral diseases may lead to degenerative arthritis and affect the daily living activities. The incidence of cartilage pathologies is increasing each year, and consequently, more clinical procedures to repair the damaged tissue are being executed. Recent advances in therapeutic strategies have been showing encouraging results and are now beginning to shape clinical practice. The regenera- tion of cartilaginous tissue of better quality is due to the continuous improvements in tissue engineering and cell-based therapies and their different elements, by combining chondro- genic cells, biomaterial scaffolds, and suitable culture conditions. 1–3 The selection of an appropriate scaffold is of extremely importance, since it will act as a temporary three-dimensional (3D) support for cell growth, differentiation and production of extracellular matrix (ECM), providing the environment for the chondrocytic phenotype maintenance or restoring, and thus playing an important role in the new tissue formation. 4,5 There are a number of biomaterial options as a cell-carrying scaffold for cartilage tissue engineering applications, which can be natural, synthetic, or a combination of both. Many of these are smart hydrogels that could potentially be injected throughout a minimal invasive transplantation. 6–10 Additionally these water swellable networks exhibit higher water contents, and macro- molecular structure similar to native tissue, making them an ideal vehicle to transfer cells to articular defects. 11,12 Chitosan (C) is an aminopolysaccharide derived from partial depolymerisation and deacetylation of chitin found in the shells of crustaceans, and has been widely suggested for many different a 3B’s Research Group – Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, Zona Industrial da Gandra S. Cl  audio de Barco, 4806-909 Caldas das Taipas, Guimara˜es, Portugal. E-mail: hlima@dep.uminho.pt b IBB – Institute for Biotechnology and Bioengineering, PT Government AssociatedLaboratory, Guimara˜es, Portugal † This paper is part of a joint Soft Matter and Journal of Materials Chemistry themed issue on Tissue Engineering. Guest editors: Molly Stevens and Ali Khademhosseini. 5184 | Soft Matter , 2010, 6, 5184–5195 This journal is ª The Royal Society of Chemistry 2010 PAPER www.rsc.org/softmatter | Soft Matter