Abstracts / Osteoarthritis and Cartilage 20 (2012) S54–S296S274

543THE EFFECTS OF ARTICULAR CARTILAGE FRAGMENTS AND SYNOVIALMEMBRANES ON CHONDROGENESIS OF BONE MARROW- DERIVEDMESENCHYMAL STEM CELLS

C.-H. Chang 1, J.-S. Hu 2, C.-C. Chen 1,2, H.-W. Fang 2,3, Y.-M. Hsu 1. 1Div. ofOrthopedics, Dept. of Surgery, Far Eastern Mem. Hosp., New Taipei City,Taiwan; 2Dept. of Chemical Engineering and Biotechnology & Inst. ofBiotechnology, Natl. Taipei Univ. of Technology, Taipei, Taiwan; 3Div. ofMed. Engineering Res., Natl. Hlth.Res. Inst.s, Miao-Li, Taiwan

Purpose: There are many approaches have been developed to manage andtreat articular cartilage injuries. For example, bone marrow stimulatingtechniques, mosaicplasty and autologous chondrocyte implantation.However, current clinical therapies still have some limitations regarding tothe formation of fibrocartilage. On the other hand, tissue engineering ofcartilage has been recognized as a new treatment for cartilage damage, butit still has some issues need to be verified. In this study, wewanted to createan articular mimetic environment for better hyaline cartilage formation.Therefore, the components of the articular such as cartilage fragments andsynovialmembraneswere added and the effects on chondrogenesis of bonemarrow-derived mesenchymal stem cells (BM-MSCs) which were used forcartilage tissue engineering were investigated (schemed as figure 1).Methods: Cartilage fragments, synovial membranes and BM-MSCs weretested along or in different combinations (as table 1) for chondrogenesis.The components of different groups were wrapped by type I collagen geland treated with induction medium. The matrix were analyzed at 0, 14,and 28 days after induction. Scanning electronic microscopy and histo-logical staining by H&E and alcian blue were employed. Glycosaminogly-cans (GAGs) content were measured and gene expression of Col I, Col II, ColX, SOX9, and aggrecan of matrix were analyzed as well.Results: Type II collagen gene expression of groups containing cartilagefragments were increased. When BM-MSCs were co-cultured with carti-lage fragments and synovial membranes, histological and GAGs analysisshowed that GAGs secretion from BM-MSCs was increased and theproliferation of BM-MSCs which closed to cartilage fragments waspromoted. Finally, aggrecan and type II collagen expression of BM-MSCswere also increased while chondrocyte hypertrophy was inhibited afterincubation for 2 weeks.Conclusions: We demonstrated that the articular cartilage fragments andsynovial membranes in combination could promote chondrogenic differ-entiation of BM-MSCs. We hope these preliminary results could helpdevelop an optimal condition for hyaline cartilage repair and be able to beapplied along with bone marrow stimulating techniques in clinical treat-ment in the future.

Experiment groups

Group Cartilage fragments Synovial membranes BM-MSCs

1 + - -2 - + -3 + + -4 - - +5 + - +6 - + +7 + + +

Ă544ROLE OF POLYAMINES IN CHONDROGENESIS OF ADIPOSE DERIVEDSTEM CELLS

S. Guidotti 1, A. Facchini 2, D. Platano 1, E. Olivotto 1, G. Trisolino 3,E. Martucci 3, S. Cetrullo 2, F. Flamigni 2, A. Facchini 1,4, R. Borzì 1. 1 Lab.Immunoreumatologia e rigenerazione tissutale/RAMSES, Istituto OrtopedicoRizzoli, Bologna, Italy; 2Dipartimento di Biochimica, Università degli Studidi Bologna, Bologna, Italy; 3Chirurgia Ricostruttiva Articolare dell'Anca edel Ginocchio, Istituto Ortopedico Rizzoli, Bologna, Italy; 4Dipartimento diMed. Clinica, Università degli Studi di Bologna, Bologna, Italy

Purpose: Adipose derived stem cells (ASC) are an attractive cell source forregenerative purposes in orthopedics, because of their accessibility andhigh differentiation potential, upon delivery of appropriate stimuli whichmimick the organogenetic natural microenvironment.Polyamines are naturally occurring, positively charged polycations able tointeract with negatively charged compounds and structures within theliving cell thus controlling several cellular processes including cell differ-entiation and already implicated in bone growth and development. Hence,we investigated the effects of exogenously added spermine in chondro-genesis of ASC recapitulated in 3D micromass cultures, to tease out theeffects on gene and protein expression of key chondrogenesis regulatorytranscription factors, markers and effectors.Methods: Adipose tissue was obtained from the surgical subcutaneousarea of 20 OA patients (age 29-72) undergoing hip arthroplasty. ASC wereobtained from the stromal vascular fraction with conventional proceduresand used within p2-p3 passage. To monitor phenotypic homogeneity, flowcytometric analysis of the cells at p1 passage was carried out to evaluatethe expression of CD 31, 34, 45, 271, 44, 73, 90 and 105. Micromasses wereseeded in control (D-MEM 10% FCS and 50 mg/ml ascorbic acid) or chon-drogenic medium, with or without the addition of 5 mM spermine toevaluate its chondrogenic ability across 1, 2 and 3 weeks chondrogenicmaturation. Osteogenic medium was used as control. We evaluated theeffects of spermine addition on molecular markers of chondrocyte differ-entiation at the level of gene (real time PCR) and protein (western blot andimmunohistochemistry) expression as well as the effects on extracellularmatrix deposition and mineralization.Results: The number of ASC per unit (g) of adipose tissue was inverselycorrelated with the age of both male and female subjects and positivelycorrelated with the body mass index. ASC samples were highly positive forCD44, CD73 and CD90, being the expression of the latter inversely corre-lated with the age of the subjects. In micromasses, spermine was able toincrease the gene expression of Sox-9 and Runx-2 which was particularlyconsistent at 2 weeks in chondrogenic medium, and alkaline phosphatasewhich was increased upon spermine addition already at early time pointsboth in control and chondrogenic medium. Western blot confirmed anincreased Runx-2 expression upon spermine addition. Mineralizationevaluated in micromass lysates indicated an increased calcium contentupon spermine addition particularly at 2 weeks in chondrogenic medium.Conclusions: Polyamine can represent a potential tool to improve thedifferentiation of ASC for bone tissue engineering via recapitulation ofendochondral ossification.Acknowledgement: This work was supported by FIRB (MIUR, Italy) andFondi cinque per mille (Ministero della Salute, Italy). The authors wish tothank Prof C. Ventura for helpful discussion during the early phase of thework.

545IMPLANTATION OF BILAYERED PLLA SCAFFOLDS LOADED WITHMESENCHYMAL STEM CELLS (MSCS) IN A SHEEP MODEL OFOSTEOCHONDRAL LESIONS

P. Sanz 1, G. Mora, Sr. 2, P. Ripalda 1, G. Gallego 3, H. Deplaine 3, J. Gómez-Ribelles 3, F. Prósper 3, I. Izal 1. 1Univ. of Navarra, Pamplona, Spain; 2Univ.Clinic of Navarra, Pamplona, Spain; 3Univ. of Valencia, Valencia, Spain

Purpose: Cartilage injuries are poorly repaired after surgery, since carti-lage is a tissue with a very limited capacity to regenerate. Apart from other