1. University of Minho School of Engineering Uma Escola a Reinventar o Futuro – Semana da Escola de Engenharia - 24 a 27 de Outubro de 2011 Main goal To develop a new “hydrogel” for application in the cartilage tissue engineering field through the self-assembly of nanoparticles loaded with growth factors enabled throught the use of platelets lysates (PL) in two aways : (i) source of growth factors and (ii) as connecting hydrogel matrix. Introduction The cartilage tissue engineering (TE) strategy described in this work relies on the combination of natural polymer-based nanoparticles (NPs), produced from the complexation of chitosan (CH) with chondroitin sulfate (CS), for the incorporation and sustained release of bioactive agents, namely platelet lysate (PL). The CH/CS complex mimics the extracellular matrix (ECM) interactions and PL is an autologous source of a cocktail of GFs acting on tissue healing and repair. When used at determined concentrations, the PL-releasing NPs can assemble in a simple and quick mode to form a three- dimensional (3D) stable hydrogel while in suspension with human Adipose derived Stem Cells (hASCs), following a mild centrifugation, allowing the cells to be entrapped in this enriched 3D environment. VITOR E. SANTO*, ELENA G. POPA Supervisors: Rui L. Reis, João F. Mano and Manuela E. Gomes * v.espirito.santo@dep.uminho.pt ASSEMBLY OF PLATELET LYSATE-LOADED NANOPARTICLES AS A NEW HYDROGEL SYSTEM FOR CARTILAGE TISSUE ENGINEERING PL-adsorbed CH/CS NPs hASCs (isolated from human lipoaspirates) Spin down Materials & Methods Formulations: (i) hASCs pellets; (ii) assembled empty NPs + hASCs; (iii) assembled PL-loaded NPs + hASCs. Characterization: (i) In vitro GF release; (ii)Histology: Hematoxylin & Eosin (H&E), alcian blue and safranin-O; (iii) real time Polymerase Chain Reaction (rt-PCR) for evaluation of collagen type II and type I gene expression. + CH in acetic acid CS in distilled water Centrifugation and resuspension PL Adsorption Polyelectrolyte Complexation + + + + + + + + - - - - - - - + + + + - - - - + + + + + + + + + - - - - - - - + + + + - - - - + 1. Nanoparticles Production and Platelet Lysate Adsorption 2. Assembly of PL-loaded Nanoparticles for hASCs entrapment 3. In vitro culture under chondrogenic stimulus hASCs+ PL loaded NPs hASCs+ empty NPs hASCs pellets 100µm H&E Alcian Blue Safranin-O Results and Discussion 2. Histological Characterization 3. Real time PCR – chondrogenic gene expression Collagen type II Collagen type I The GF release profile for PDGF-BB and TGF-β1 is characterized by a strong burst release at day 1, followed by a more controlled delivery during the remaining days. After day 7, the GF release was undetectable. 1. In vitro GF release The H&E staining reveals that after 28 days, the stability of the PL-loaded NPs hydrogel is higher in comparison with the empty NPs. Moreover, we can observe the formation of cartilage ECM comparable with the positive control. The scaffolds loaded with PL showed a stronger upregulation of col II in comparison with col I, showing the formation of hyaline articular cartilage ECM. Materials and Methods Results and Discussion The presence of PLs in the 3D matrix enhances the in vitro chondrogenic diferentiation of hASCs. PLs have a multifunctional role as a connective/stability agent for the matrix and as a GFs supplier for chondrogenic differentiation, creating an effective hydrogel network for cartilage TE.. Conclusions Acknowledgments The authors thank the FCT grants (SFRH/BD/39486/2007 and SFRH/BD/64070/2009), the European projects (NMP3-CT-2004-500283 and NMP4-SL-2009-229292), IPS and Hospital da Prelada.