075 OSTEOARTHRITIS PATIENTS' PERCEPTIONS REGARDING APPROPRIATENESS FOR TOTAL JOINT REPLACEMENT SURGERY

Abstract

stem cell line (GFP-hES2) was cultured in DMEM/F-12 containing 20% serum replacement, 10% mouse embryonic fibroblast conditioned media and β-Mercaptoethanol on gelatin-coated plates seeded with irradiated mouse feeder cells. Chondrogenic differentiation: We developed a novel two step protocol for chondrogenic differentiation of GFP-hES2. 1. Mesoderm differentiation: Embryoid bodies were developed in lo-cluster tissue culture plates in the presence of BMP4, bFGF, and Act-A. 2. Chondrogenesis: The second step was co-culture of the mesodermal cells with primary bovine chondrocytes (bP0). bP0 and GFP-hES2 were mixed at a ratio of 1:4 and co-cultured on MillicellTM membrane inserts, which allow for 3D growth, in DMEM supplemented with 20%FBS. After 4 weeks the newly formed tissue was characterized and compared with tissue formed by bP0 and hES2 alone. Analysis: FACS analysis of the GFP-hES2 cells was carried out using selected antibodies. Histological qualitative assessment of the newly formed tissue was done by using light microscopy and toluidine blue staining. Proteoglycan accumulation was semi-quantitatively determined by dimethylmethylene blue dye-binding assay. Values were normalized to total DNA content determined by Hoechst 33258 dye binding assay. Gene expression was quantified by qPCR. Statistics: Results were expressed as mean ± SD of 3 independent experiments and significance determined by one-way ANOVA. Results: FACS analysis after the first stage of culture at day 5 showed that only 5-7% of cells remained positive for ckit, an embryonic stem cell marker, indicating a shift towards more differentiated state. Furthermore, 50-60% of the cells were KDR and PDGF double positive, indicating that these cells attained mesodermal features. At this stage cells were co-cultured with bP0. After 4 weeks GFP-hES2 appeared to be present and surrounded by newly deposited cartilaginous matrix when visualized under fluorescent microscopy. Toluidine blue staining and dimethylmethylene blue assay showed that the proteoglycan content of tissue formed by co-cultured embryonic stem cells at 4 weeks was significantly higher than tissue formed by the bovine chondrocytes alone. qPCR showed higher gene expression of type II collagen, aggrecan, and SOX9. GFP-hES2 cultured alone failed to accumulate any cartilaginous matrix. Conclusions: We conclude that bovine primary chondrocytes produce factors which promote chondrogenic differentiation of partially differentiated embryonic stem cells. Understanding the mechanism regulating this differentiation may aid in developing a source of cells suitable to use for cartilage repair. Acknowledgement: Supported by CIHR.