Comparison of the effects of different pulp capping materials on viability, morphology and ageing of dental pulp cells

A significant amount of research has been directed towards the development of minimally invasive and/or regenerative therapeutic approaches to the maintenance of dental pulp vitality. Direct pulp capping is an example of such a therapeutic approach where the success of the treatment is largely affected by the biological characteristics of pulp capping agent. Tissue engineering approaches also include the use of human dental pulp cells (HDPCs) seeded on biocompatible scaffolds to regenerate lost hard, or soft dental tissues. Such processes may require a large number of cells which can only be produced by expansion of isolated HDPCs in vitro. Under these conditions, biological characteristics of the cells may be altered and their proliferation and differentiation capacity affected as a result of biological or cellular ageing. In this study we have investigated the effects of various pulp capping agents on HDPCs. Additionally we have looked at cell ageing and explored avenues to reduce the effects of this in vitro. HDPCs isolated from extracted human third molars were exposed to pulp capping agents through serial passage in culture. Dycal exhibited highly cytotoxic effects on the HDPCs, whereas, MTA facilitated the maintenance of cell viability and appeared to encourage proliferation of HDPCs. The use of propolis, a natural product of bees that has been reported to have uses in dentistry, resulted in slightly lower cell viability in comparison to the control and MTA groups. MTA and propolis appeared to enhance the osteogenic differentiation potential of HDPCs in comparison to control as indicated by the up-regulation of BMP-2, ALP, RUNX-2 and OCN and the undetectable expression of DSPP and DMP-1.
We also clearly show that the biological characteristics of HDPCs (for example viability, cell proliferation and differentiation) were altered in later passages. A decrease in viability of cells treated with propolis seen in later passages, appeared to coincide
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with the up-regulation of BCN1, a marker for for autophagy. Serially passaging of HDPCs also resulted in telomere shortening as measured by a progressive decrease of telomere restriction fragment (TRF) and this may lead to replicative senescence or cell ageing. Telomere shortening might be expected as expression of the telomerase associated gene (TERT) was undetectable. Contrary to expectations, propolis appeared to cause the greatest shortening of telomere length. Addition of the oligonucleotide “Telome 3”, which was designed to facilitate exogenous priming of telomeric DNA synthesis, to serially passaged HDPCs maintained high cell-viability throughout. Additionally, TRF measurements suggested that “Telome 3” may indeed aid in the maintenance of telomere length in serially passaged HDPCs. These data suggest that within the constraints of this in vitro model, the viability, proliferation and differentiation of HDPCs are largely affected by the cell passage number and also the type and concentration of the pulp capping agent used.