Differentiation of dental pulp cells in a micromass culture system

Objectives: The dental pulp contains progenitor cells which are able to replace irreversible damaged odontoblasts and produce a matrix of reparative dentin. However, the origin of the odontoblast-like cells and their differentiation pathways remain unclear. In vitro differentiation models are mainly based on two dimensional culture systems, require chemicals to induce matrix production, and are considered to be quite far from in vivo conditions. Here we introduce a micromass culture system for promoting differentiation of human dental pulp cells (DPCs). Methods: DPCs were cultured in a dense, 3-dimensional culture format, so that 350,000 cells formed a spheroid pellet. The cell pellets were cultured for 28 days in DMEM supplemented with 10% foetal calf serum without adding any further additives for inducing differentiation. Microarray-based screening of differentially expresses genes, QRT-PCR, histology and TEM were used to identify cell behaviour and fate.

Results: Condensation of DPCs in a 3-dimensional culture format led to a differential regulation of 510 and 375 genes after 3days and 28days of culture respectively. Gene ontology-based clustering showed e.g. a statistically significant underexpression of genes for cell proliferation and M phase and an overexpression of genes related to extracellular matrix components. Collagen type III, metalloproteinases, and different bone morphogenetic proteins were some of the regulated genes detected. QRT-PCR analysis revealed an upregulation of osteocalcin, dentin sialophosphoprotein, and alkaline phosphatase. TEM showed a newly produced collagen-rich matrix and cell-cell interactions (gap-junctions). The results suggest that the 3-dimensional micromass culture format alone induces the production of particular extracellular matrix components, proteases, and growth factors by cells in the local microenvironment. Cell-matrix and cell-cell interactions seem to promote the differentiation of DPCs along osteogenic/odontogenic pathways.

Conclusion: The micromass technology may allow the study of differentiation pathways of DPCs more closely the natural situation and provide important information for tissue engineering strategies.