Human Embryonic Palatal Mesenchymal Cells Express Osteoblast Features, in vitro

Human embryonic palatal mesenchymal stem cell culture models (HEPM) have been used in evaluating craniofacial palatal closure and effects of teratogenic agents. Objective: We performed studies using a non-transformed undifferentiated freely available female-derived HEPM culture model to characterize migration, proliferation and potential for osteogenic differentiation. Methods: Cells (HEPM 1486; ATCC, Manassas, VA) were grown in EMEM+10% FCS, L-Glut (2mM), neAA (0.1mM), NaPyruvate (1mM) and plated on tissue culture plastic at high cell density (micromass conditions of 5000 cells/mm2) for periods through three weeks. Cultures were grown in the presence or absence of ascorbate (50µg/mL) and/or 5mM Beta-Glycerophosphate. Results: HEPM cells plated at high density on tissue culture plastic have a rapid doubling time of 22h. These conditions create a vertically stacked cell culture of 10-15 cell layers thick. When compared to MC3T3-E1 cells, HEPM cells migrated 4 fold faster/cell (p<0.0001) by 96 hours from the edge of the micromass culture and demonstrate contact guidance with grooved substrate topographies. Cells on grit blasted and etched surface topographies demonstrated a random orientation. HEPM cells developed normal patterns of stress fibers, focal adhesion (FA) formation and subsequent expressed of RUNX-2/cbfa-1, Osteocalcin (OC) and BSP as determined by real time PCR. Significantly (p<0.05) elevated levels of OC, BSP and mineralization were observed between week 2 and week 3. A nodule-like pattern of mineralization was described with alizarin red-S staining and calcium content (atomic absorption) was increased 600 fold between week 2 and 3. Aqueous Dexamethasone (2-hydroxypropyl beta-cyclodextrin at 10-7M) reduced OC message levels and mineral accumulation by 80% at 3 weeks. Conclusion: The human palatal mesenchymal cell line, HEPM 1486, demonstrates hormone sensitive osteoblast-like features with expression of key osteoblast-related phenotypic factors in vitro. Supported by NIH/NIDCR P60 DE13076 and the ITI Foundation for Oral Implantology, Basil, Switzerland.