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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) thermosensitive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-bglycerophosphate-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.The authors acknowledge the financial support to the Portuguese Foundation for Science and Technology (FCT) for the PhD fellowship to H. Sa-Lima (SFRH/BD/21779/2005) and for the Project PTDC/QUI/68804/2006; the European Union funded STREP Project HIPPOCRATES (NM3-CT-2003-505758); and the European NoE EXPERTISSUES (NMP3-CT-2004-500283)

Stimuli-responsive chitosan-starch injectable hydrogels combined with encapsulated adipose-derived stromal cells for articular cartilage regeneration