59-05-032 Proceeding

197 Proceedings of the Princess Maha Chakri Sirindhorn Congress MESENCHYMAL STEM CELLS IN HUMAN SPINAL LIGAMENTS Baldur Kristjánsson 1 , Sittisak Honsawek 2 , Worawat Limthongkul 3 and Weerasak Singhatanadgige 3 I. Introduction Mesenchymal stem cells (MSCs) capable of osteogenic, chondrogenic and adipogenic differentiation have been gaining much interest for therapeutic applications [1]. Residing predominantly within the bone marrow and adipose tissue they are also found most connective tissues of the human bodywhere they are involved in themaintenance and regeneration [2]. Lumbar spinal stenosis and ossification of the posterior longitudinal ligament (OPLL) are pathological conditions characterized by the hypertrophy and calcifications of connective tissues within the spinal column. The precise etiologies of these conditions are poorly understood. Compression of the spinal canal often leads to surgery where calcified and hypertrophied structures are removed. Heretofore, few have addressed MSCs roles in these conditions; they have been isolated and described from the ligamentum flavum and posterior longitudinal ligaments. MSCs from OPLL patients have been evinced to exhibit significantly stronger osteogenic potentials compared to MSCs from healthy controls [3]. Accordingly, the objective of this study was to identify whether MSCs could be isolated from ligamentum flavum, interspinous ligaments, and facet joints of lumbar spinal stenosis patients. II. Methods Spinal ligaments samples were harvested from patients during posterior decompression surgery. Cells were isolated from ligamentum flavum, interspinous ligaments, and facet joints via direct tissue explants and grown to confluence in culturemedium ( α -minimumessential medium supplementedwith 10% fetal bovine serumand 200U/mL penicillin/streptomycin). Clonogenicity was measured by colony forming unit-fibroblast (CFU-F) assay where 10 cells were seeded into each well of 6-well plates, incubated for 14 days and colonies counted afterwards. To measure cell proliferation, cells were seeded at lowdensity (1,250 cells/cm 2 ), incubated for 14 days, counted and population doublings calculated. Expressions of the MSCs surface markers CD29, CD44, CD90, CD105 and the hematopoietic markers CD34 and CD45 were established using flow cytometry. Tri-lineage differentiation was initiated by growing passage 2-3 cells in ostegenic, chondrogenic and adipogenic medium for 21 days. For osteogenic and adipogenic differentiation 1.5 x 105 cells were seeded in 6-well plates. Osteogenic medium consisted of culture medium supplemented 10 1 Department of Biochemistry, Faculty of Medicine, Chulalongkorn University 2 Department of Biochemistry, Faculty of Medicine, Chulalongkorn University, King ChulalongkornMemorial Hospital, Thai Red Cross Society 3 Department of Orthopaedics, Faculty of Medicine, Chulalongkorn University