Effects of solubilised dentine matrix components on pulp cell growth

The dentine extracellular matrix provides a reservoir of bioactive molecules, which when released by bacterial acids during caries, can stimulate the formation of reparative dentine. Previous work has shown that EDTA solutions are effective in solubilising these bioactive dentine matrix proteins (DMPs). Objectives: To investigate the effects of DMPs on the growth characteristics of odontoblast-like cells in vitro. Methods: Dentine was dissected from healthy human teeth and powdered in a percussion mill prior to extraction with 10% EDTA pH 7 for 14 days. Solubilized material was dialysed, lyophilised and its effects (dose range 1-1000 µg/ml) on cell growth examined in immortalised MDPC-23 odontoblast-like cells grown in DMEM without foetal calf serum over a 10-day period. Growth characteristics were analysed by cell counts and trypan blue staining. In addition, the effects on apoptosis induction were determined by DNA laddering assays in cultures exposed to untreated, heat-denatured and micro-filtered DMPs, at a concentration of 1000 µg/ml, for 48 hours. Results: Low concentrations of DMPs (1µg/ml) stimulated increased cellular growth as compared to control cultures and this effect was most pronounced over the first two days of culture. Higher concentrations of DMPs (100+ µg/ml), however, reduced the cellular growth rate and adversely affected cell viability. Cultures exposed to high concentrations of DMPs exhibited characteristics of apoptosis including cellular morphological changes and DNA laddering. To better characterise the DMP component responsible for this effect, DMPs were heat-denatured or size fractionated using 3kDa and 30kDa micro-filtration devices. Both micro-filtered and heat-denatured DMPs retained their ability to induce apoptosis. Conclusion: Low concentrations of DMPs stimulated the growth of MDPC-23 odontoblast-like cells, whilst high concentrations adversely affected cell growth and induced apoptosis. The component(s) of the DMPs responsible for inducing apoptosis were retained by 30kDa cut-off membranes and appeared to be heat stable.