Poster Presentations / Osteoarthritis and Cartilage 18, Supplement 2 (2010) S45–S256 S85 and desalted with home-made Poros R2 columns, injected and separated in a nanoLC system, mixed with α-cyano and deposited in a MALDI plate using an automatic MALDI spotter. The MS run for each fraction was analyzed in an ABI 4800 MALDI-TOF/TOF instrument and fragmentation was done using a CID system. Results: 87 identified proteins were related with the chondrogenic differ- entiation process from a total of 209 proteins. Some of the proteins related with chondrogenesis obtained with this method were: Name Swissprot Accession # Type Day Collagen alpha-1 P02452 Collagen 4, 7 Collagen alpha-2 P08123 Collagen 4, 7 Decorin P07585 Proteoglycan 4, 7 Calreticulin P27797 Chaperone 4, 7 Collagen alpha-3 P12111 Collagen 14, 28 Lumican P51884 Proteoglycan 7, 14, 28 Fibulin P23142 Glycoprotein 14, 28 Biglycan P21810 Proteoglycan 46 Nidogen P14543 Glycoprotein 46 Conclusions: 1. This technique is suitable to analyze medium complexity samples as secretome. 2. The chondrogenesis differentiation designed through spheroid formation have revealed be an excelent method to obtain chondrocytes-like cells from mesenchymal stem cells of human umbilical cord stromal tissue. 3. Pathways related with differentiation process are been activated during the chondrogenesis process. 175 EFFECT OF HUMAN PLATELET RICH PLASMA RELEASATES ON PROLIFERATION AND MATRIX SYNTHESIS OF HUMAN ARTICULAR CHONDROCYTES GROWN ON A POLYGLYCOLIC ACID-BASED BIOMATERIAL (PGA): MOLECULAR, PROTEOMICAL AND IMMUNOFLUORESCENCE ANALYSIS A. Spreafico 1,2 , F. Chellini 1,2 , B. Frediani 1,2 , S. Niccolini 1 , G. Bernardini 3 , A. Rocchi 3 , R. Marcolongo 2 , M. Galeazzi 1 , A. Santucci 3,2 1 Dept. of Clinical Med. and Immunological Sci., Rheumatology division, Univ. of Siena, Siena, Italy; 2 Centro Interdipartimentale per lo Studio Biochimico e Clinico delle Patologie Osteoarticolari, Università di Siena, Siena, Italy; 3 Dept. of Molecular Biology, Univ. of Siena, Siena, Italy Purpose: Recently proposed cell-based cartilage repair procedures require that autologous articular chondrocytes are isolated from a small biopsy, expanded in vitro, and then either directly injected into the defect or used to engineer implantable grafts. Generally, several technical problems have limited the wide clinical application of these techniques, in particular: (a) the low yield of chondrocytes that can be obtained from a biopsy; (b) the chondrocyte trend to lose their chondrogenic potential during the expan- sion phase in conventional culture systems based on foetal calf serum (FCS) addition to the culture medium; (c) the low capacity of chondrocytes to re-differentiate towards cartilage-forming cells after the expansion phase. In the last years, many of these aspects have been ameliorated thanks to the development of cultural conditions based on the introduction of mixtures of recombinant growth factors (GFs) and the employment of a variety of biocompatible matrices supporting an appropriate tissue formation in vitro and in vivo. However, some disadvantages of recombined GFs have become evident, such as short shelf life and high price. In recent years, a number of clinical studies have suggested that platelet (PLT) derivatives, such as PLT-rich plasma (PRP), have the potential for a substantial therapeutic role in cartilage tissue regeneration. In a previous work, we have demonstrated the high efficacy of a human PRP releasate (PRPr) supplementation in delivering a broad range of GFs and maintaining a chondrocyte-like phenotype in mono- and three-dimensional cultures of human chondrocytes. The aim of the present study was to investigate the anabolic properties of PRPr supplementation on cartilage constructs based on PGA (polyglycolic acid). PGA is a synthetic polymer successfully used in cartilage tissue engineering for its biocompatibility and mechanical properties. In order to evaluate if PRPr, in comparison with the conventional FCS cul- tural supplementation, is really effective in improving the quality of PGA- based cartilage constructs, we investigated, on different chondrocyte/PGA 3D-cultures, the genomic and proteomic expression of several biomarkers of chondrocyte differentiation. Methods: Time-dependent analyses were performed by means of Proteo miner 2D electrophoresis, Real-time PCR and immunofluorecence (IF) mi- croscopy, on several chondrocyte/PGA cultures independently grown with PRPr and FCS at the same concentration. Results: The analysis of protein repertoires of cellular supernatants ob- tained by 3D chondrocyte/PGA cultures grown with PRPr and FCS, after 10 days, shown significant differences in the expression of proteins involved in chondrocytic differentiation and anabolic activity. PRPr-supplemented PGA constructs shown the exclusive expression of at least 7 markers of chondrocytes differentiation, and the increase of about 4 protein involved in cartilagenous anabolism. Real-time PCR gene expression analysis con- firmed, in PRPr-treated constructs, after 20 days of culture, an increase in expression of markers related to the synthesis of mature cartilagenous extracellular matrix (Sox9 and Collagen II respectively +7.7 and +14.5 folds vs starting de-differentiated chondrocytes and +1.8 and +4.8 folds vs FCS-treated constructs). These results were confirmed by IF microscopic analyses. Conclusions: Our results strongly support the efficacy of PRPr supple- mentation to ameliorate the differentiation status of human articular chondrocytes. Moreover, the whole of the differentiating PRPr activity in addition to its stronger effect on cell proliferation, should allow to consider this preparation very useful to the safe and economic engineering of cartilage constructs. 176 LONG-TERM RESULTS OF CARTILAGE REGENERATION AFTER ALLOGENEIC TRANSPLANTATION OF CARTILAGINOUS AGGREGATES FORMED FROM BONE MARROW-DERIVED MESENCHYMAL CELLS WITH CHONDROGENESIS FOR LARGE OSTEOCHONDRAL DEFECTS IN IMMATURE RABBITS T. Yoshioka 1,2 , H. Mishima 2 , S. Sakai 2 , Y. Ohyabu 3 , N. Ochiai 2 , T. Uemura 3 1 Ibaraki Southwest Med. Ctr., Sashima, Japan; 2 Univ. of Tsukuba, Tsukuba, Japan; 3 Natl. Inst. od Advanced Industrial Sci. and Technology, Tsukuba, Japan Purpose: The purpose of this study was to allograft a three-dimensional (3D) cartilaginous aggregates derived from bone marrow mesenchymal cells into large osteochondral defects in rabbit knees, with the aim of elucidating its long-term outcome for cartilage repair. Methods: Bone marrow cells were harvested from long bones of 12-day-old Japanese white rabbits. The cells were subjected to monolayer culture and the spindle-shaped cells attached to the flask surface were defined as bone marrow mesenchymal cells. After approximately 20 days of monolayer culture, a 3D cartilage-like tissue was created by inducing cartilage differ- entiation under rotary culture (1.0×10 7 cells) for one week with serum-free medium containing TGF-β3. We created a full-thickness cartilage defect, 5 mm in diameter and 4 mm in depth, at the femoral trochlea of 10- week-old Japanese white rabbits (average weight, 2.0 kg). Two groups were set up, the graft group (n=19) in which the 3D cartilage-like tissue was transplanted into the defect, and the control group (n=18) in which the defect was untreated. Twenty six and 52 weeks after grafting, rabbits were sacrificed and their tissue repair status was evaluated macroscopically (ICRS macroscore) and histologically (O’Driscoll score). Scores from the two groups were compared, with statistical significance set at p<0.05. Results: ICRS macroscores were: at week-26, 7.2±0.5 (score ± standard error; n=12) and 7.6±0.8 (n=7) for graft and control groups, respectively; at week-52, 7.6±1.1 (n=7) and 9.7±0.7 (n=11) for graft and control groups, respectively. O’Driscoll scores were: at week-26, 12.6±1.9 and 10.1±1.9; at week-52, 9.6±3.0 and 14.0±1.4, each for graft and control groups, respectively. No significant differences were found between the groups. Conclusions: It has been reported that cartilage repair by transplanting bone marrow mesenchymal cells into a full-thickness defect in rabbit articular cartilage results in some short-term repair at hyaline cartilage-like tissue; however, the cartilage layer gradually becomes thinner with time and is eventually replaced by bone tissue. In this study, rather than transplanting mesenchymal cells directly, we induced the differentiation of mesenchymal cells into cartilaginous aggregate and grafted the resulting tissue into the defect. These experiments were conducted based on the hy- pothesis that transplanting the 3D tissue, as opposed to mesenchymal cells themselves, might result in satisfactory repair at the hyaline cartilage-like tissue even in the long term. However, we found no significant differences between graft and control groups, either macroscopically or histologically. Within the graft group, tissues that did not show satisfactory repair were not only ossified but also accompanied by scarring. These results suggest the