Alterations in Gene Expression in Pulpal Cells During Expanded Culture

The dentine-pulp complex displays exquisite regenerative potential in response to injury, which depends on the co-ordinated activity of several cell types and the presence of a population of undifferentiated progenitor or stem cells, known as dental pulp stem cells (DPSCs). However, limited information exists regarding the selection and characterisation of these cells. Objectives: Investigate the nature of the cell populations obtained during expanded primary pulpal cell culture. Methods: Cells were isolated from extirpated pulps of young adult male Wistar rat incisor teeth following dissociation in trypsin-EDTA for 30mins. Isolated primary cells were seeded at 1x10⁶ cells/ml and expanded as a monolayer culture in αMEM with 10% FCS, glutamine and antibiotics. Cultures were maintained at 37^oC, in 5% CO₂ in air with media changes every 48 hours until confluent. Once confluent, cells were further passaged for up to 10 times. At each passage, RNA was isolated for downstream sqRT-PCR analysis. Expression levels of markers of cell lineage and genes associated with mineralising tissues were analysed using specific primers for αSM actin, nestin, CD44, collagen1α, osteonectin and alkaline phosphatase. Results: Changes in gene expression were observed for both cell lineage markers and those associated with mineralising tissues. Expression of αSM actin, nestin and CD44 fell sharply during early passages however levels increased in later passages with αSM actin being strongly expressed. Expression of alkaline phosphatase decreased steadily with increasing passage, however expression of osteonectin decreased initially but then increased in the later passages. Conclusions: These data indicate changes in cell population during expansion and provide possible markers for cell selection. Selection will permit further characterisation of these cells to assess their potential in terms of specificity of response during tissue regeneration.