Paper 264: Local Administration of Synovium-derived Fibroblasts Cultured in TGF-beta1-supplemented Medium Inhibits Natural Reduction of the Structural Properties of the Tendon Autograft after ACL Reconstruction: A Sheep Model Study

expansion with proliferation medium. Osteogenic assay Cells (n1.0x105) were cultured in 6-well plates in osteogenic medium suppremented with BMP4 (100ng/ ml) and stained for alkaline phosphatase (ALP) at day 7. As well, cells were also cultured as pellets for 21 days in osteogenic medium and evaluated with a micro-CT and stained with von Kossa for assessment of mineralization. Chondrogenic assay Cells (n2.5x105) were placed in a 15ml conical tube, centrifuged at 600g, and cultured in chondrogenic medium (Lonza) supplementted with TGF-3 (10ng/ml). Pellets were assessed macroscopically at day 14, and stained with Alcian blue/nuclear fast red. Adipogenic assay Cells (n1.0x105) were cultured in 6-well plates for 14 days in adipogenic medium and stained for Oil Red O. Reverse Transcription Polymerase Chain Reaction (RT-PCR) Differentiation potential of these cells were confirmed by RT-PCR using osteogenic (osteocalcin and type 1 collagen), chondrogenic (Sox9 and type 2 collegen), and adipogenic (lipoprotein lipase (LPL) and Peroxisome proliferator activated receptor (PPAR))-related markers. Results: Immunohistochemical staining Tissues showed more positive staining for CD34 and CD146 in the ruptured site than in the mid-substance. Flow cytometry analysis The percentage of CD34CD45 and CD146 CD45 cells were significantly higher in the ruptured site compared to the mid-substance (CD34CD45 cells: reptured site, 44.3%, mid-substance, 8.3%; CD146CD45 cells: reptured site, 6.5%, mid-substance, 0.5%) (p0.01). Proliferation potential CD34 and CD146 cells from the ruptured site showed high expansion potential, however, those from the mid-substance did not expand well. Based on these results, we used those from the ruptured site for multilineage differentiation assay. Osteogenesis Cultured CD34 and