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90 2 st INTERNATIONAL CONFERENCE ON REGENERATIVE ORTHOPAEDICS COMPARISON BETWEEN TWO ICRS HISTOLOGY SCORING SYSTEMS FOR CARTILAGE REPAIR Andreja Vukasovic, Department of Histology and Embryology, School of Medicine, University of Zagreb, Zagreb, Croatia Davor Jezek, Department of Histology and Embryology, School of Medicine, University of Zagreb, Zagreb, Croatia Petar Kostesic, Clinic for Surgery, Orthopedics and Ophthalmology, Faculty of Veterinary Medicine, University of Zagreb, Zagreb, Croatia Damir Hudetz, Department of Orthopedics, University Hospital Sveti Duh, Zagreb, Croatia Ivan Cerovecki, School of Medicine, University of Zagreb, Zagreb, Croatia Marin Kosovic, Department of Physics and Biophysics, School of Medicine, University of Zagreb, Zagreb, Croatia Tadija Petrovic, Clinic for Traumatology, Clinical Hospital Center “Sestre Milosrdnice”, Zagreb, Croatia Drazen Maticic, Clinic for Surgery, Orthopedics and Ophthalmology, Faculty of Veterinary Medicine, University of Zagreb, Zagreb, Croatia Alan Ivkovic, Department of Orthopedics, University Hospital Sveti Duh, Zagreb, Croatia Marko Pecina, School of Medicine, University of Zagreb, Zagreb, Croatia The histological quality of cartilage is considered to be one of the most important outcome tools to objectify success of different treatment modalities in regenerative orthopedics and tissue engineering. The aim of this pilot study was to compare the applicability of two International Cartilage Research Society (ICRS) visual histological assessment scales, ICRS I and ICRS II in the analysis of cartilage restoration after treatment with genetically modified autologous bone marrow clots. Chondral defects in 28 skeletally mature sheep which were randomly assigned to 4 different groups; TGF group (TGF-β1 gene transduced bone marrow clot), GFP group (Green fluorescent protein transduced bone marrow clot), BMC group (untransduced bone marrow clot) and NC group (negative control, no treatment) were treated accordingly. After 6 months animals were sacrificed and osteochondral explants were analyzed. For histological assessment, slides were stained with hematoxylin - eosin and Safranin O. Two independent evaluators examined 112 slides blinded, 4 slides for each specimen using assessment scale ICRS I then ICRS II. Significant period of time passed between first and second examination, so the possibility of a bias with the respect to the first examination has been avoided entirely. ICRS I assesses 6 features that are graded with grades from 0 to 3. With ICRS II 14 features are evaluated on a visual analogue scale. Each parameter is scored on a scale from 0 to 100%. The ICRS I demonstrated lower inter-reader variability compared with ICRS II. Results yielded with ICRS I assessment scale were 98.7% coincident between two observers while those obtained with ICRS II assessment scale varied greatly. Subchondral bone and overall assessment scores had the best correlation coefficients for inter-reader variability (r = 0.82 and 0.76, respectively). ICRS I score revealed statistical significant difference in cell distribution between TGF and NC group (p=0.002). ICRS II showed statistical significant difference in surface and matrix staining score between TGF and NC group (p=0.009 and 0.007, respectively). ICRS II enabled better discrimination of scoring parameters but it is more suited for evaluators with strong background in cartilage histopathology. ICRS I remains easy to use for less experienced evaluators. Poster
20 - 22 September 2012, Opatija, Croatia OSTEOGENIC POTENTIAL AND STEMNESS OF MESENCHYMAL STEM CELLS AFTER DEXAMETHASONE TREATMENT Andreja Vukasovic, Department of Histology and Embryology, School of Medicine, University of Zagreb, Zagreb, Croatia Davor Jezek, Department of Histology and Embryology, School of Medicine, University of Zagreb, Zagreb, Croatia Petar Kostesic, Clinic for Surgery, Orthopedics and Ophthalmology, Faculty of Veterinary Medicine, University of Zagreb, Zagreb, Croatia Damir Hudetz, Department of Orthopedics, University Hospital Sveti Duh, Zagreb, Croatia Ivan Cerovecki, School of Medicine, University of Zagreb, Zagreb, Croatia Marin Kosovic, Department of Physics and Biophysics, School of Medicine, University of Zagreb, Zagreb, Croatia Tadija Petrovic, Clinic for Traumatology, Clinical Hospital Center “Sestre Milosrdnice”, Zagreb, Croatia Drazen Maticic, Clinic for Surgery, Orthopedics and Ophthalmology, Faculty of Veterinary Medicine, University of Zagreb, Zagreb, Croatia Alan Ivkovic, Department of Orthopedics, University Hospital Sveti Duh, Zagreb, Croatia Marko Pecina, School of Medicine, University of Zagreb, Zagreb, Croatia The purpose of the research was to improve osteogenic differentiation of human bone marrow derived-mesenchymal stem cells (hMSCs) and to evaluate the level of remaining undifferentiated stem cells in culture. Standard cell culture protocol includes continuous exposure to the dexamethasone which is not easy to reproduce in human body. Therefore, our experiment was designed to investigate the possibility of using short-term dexamethasone exposure in order to induce osteogenic differentiation. Human bone marrow-derived MSCs were expanded with FGF-2 supplemented media. Afterwards cells were cultured in osteogenic media (DMEM low glucose, 10% FBS, 10mM β-glycerophosphate, 5µg/ml ascorbic acid, 1mM piruvat) and treated with different concentrations of dexamethasone, both short-term and continuous. The differentiation status of the cells was estimated using several osteogenic markers - alkaline phosphatase (AP) activity, the bone sialoprotein (BSP) and dentin matrix protein 1 (DMP-1) mRNAs. The stemness of the cells was estimated by the presence of pluripotency markers - SRY (sex determining region Y)-box 2 (SOX2) and octamer-binding transcription factor 4 (OCT4). The measurements were conducted on the days 7 and 14 after osteogenic conditions had been introduced. Our results show that continuous treatment induces the highest level of osteogenesis, but 2 hour exposure also induced differentiation in comparison with control. Overall, 10-5 M concentration of dexamethasone was the most powerful in the short-term induction of osteogenesis. SOX2 has not been detected in hMSCs. OCT4 was expressed in undifferentiated pluripotent hMSCs and its expression subsequently went down, indicating that the differentiation process is proceeding. Poster 91
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FINAL PROGRAM BOOK OF ABSTRACTS ORG
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CONTENTS 20 - 22 September 2012, Op
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Organized by: Croatian Academy of S
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CONFERENCE PROGRAMME: September 21
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17:15 - 17:25 Discussion 20:00 Soci
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20 - 22 September 2012, Opatija, Cr
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BOOK OF ABSTRACTS 20 - 22 September
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STEM CELLS FOR THE CURIOUS Stanimir
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