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

Abstract

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 β-glycerophosphate (DAG) seems promising. The present study evaluated the effects of DAG and MAPK, ERK and PI3K/Akt-pathway inhibitors on growth and mineralization of USSC micromasses. 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 μM dexamethasone in DMEM, 50 mM ascorbic acid in PBS and 1 M β-glycerolphosphate in PBS. Microtome slices were dyed with OsteoImage™ and analyzed under fluorescence microscopy. 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 ERK-pathway inhibitor, FR180204, significantly reduced micromass growth and mineralization in non-DAG treated cells (p<0.001) but showed increased mineralization in DAG-treated cells (p=0.014). The PI3K/Akt-pathway inhibitor, LY294002, did not significantly affect micromass growth but significantly decreased mineralization (p<0.001). The MAP-kinase inhibitor, U0126, significantly reduced micromass growth (p=0.001) and mineralization (p=0.001) of DAG-treated cells. DAG is a strong initiator of osteogenic differentiation. The PI3K/Akt-pathway inhibitor and the ERK-pathway inhibitor, FR180204, control osteogenic differentiation of 3D-micromasses. These results may facilitate preconditioning of cell cultures in guided tissue regeneration.