Analysis of Spontaneous and Induced Osteogenic Differentiation in 3D-micromasses of Human Multipotent Stem Cells

Background/Aim: Craniofacial reconstruction of extensive bone defects causes high morbidity to patients. Contemporary reconstructive surgery aims at restoring lost bone with either autogenous bone or substitutes. Multipotent unrestricted somatic stem cells (USSC) show a potential for osteoblast differentiation and are increasingly used in tissue engineering. The osteogenic potential of USSC micromasses influenced by dexamethasone, ascorbic acid and beta glycerophosphate (DAG) seems promising. The present study pathway inhibitors on growth and mineralization of USSC micromasses. Materials and Methods: Cells: i) USSC-18 (female, Passage 8), ii) USSC-8 (female Passage 9), and iii) USSC- 8/17 (male, Passage 8), all cultured in 350 ml DMEM, with 150 ml fetal bovine serum, 5 ml penicillin/streptomycin and 5 ml L-glutamine. Differentiation was induced using 50 mu M dexamethasone in DMEM, 50 mM ascorbic acid in PBS and 1 M beta-glycerolphosphate in PBS. Microtome slices were dyed with OsteoImageTM and analyzed under fluorescence microscopy. Results: Significant increase in size and mineralization of DAG-treated micromasses was found on days 3 (p<0.001), 6 (p<0.001) and 7 (p<0.001). The ERKpathway inhibitor, FR180204, significantly reduced micromass growth and mineralization in non-DAG treated cells (p<0.001) but showed increased mineralization in DAGtreated cells (p=0.014). The PI3K/Akt-pathway inhibitor, LY294002, did not significantly affect micromass growth but