Objective: To characterise pulpal response to EMD and to identify any EMD components that are bioactive against human dental pulp cells (HDPCs).
Methods: EMD (10mg/ml) was fractionated by size exclusion chromatography in 0.125M formic acid (90cm x 1.6cm column; flow rate 0.33ml/min; fraction volume 2ml). Fractions were characterized by SDS-PAGE and western blotting using dentin sialophosphoprotein (DSPP) antibodies. HDPCs were cultured in αMEM supplemented with 5% FBS for 24 hours. Cultures were treated with EMD or EMD fractions containing proteins at the same concentration present in unfractionated EMD. Cells incubated in basal medium alone served as controls. Alkaline phosphatase (ALP) activity was determined after 10 days culture using colourimetric assays.
Results: SDS-PAGE showed that chromatography resolved EMD into fractions containing proteins ranging from 5-200KDa. Western blotting revealed a protein at around 200kDa immunoreactive with anti-DSPP antibodies in fractions 1-5. These early eluting fractions were able to stimulate ALP activity in cultured HDPCs (70% increase in ALP activity for fraction 1(p<0.01)). In contrast, later fractions containing proteins at around 8kDa had a negative effect on ALP activity (30% reduction in ALP activity (P<0.01)).
Conclusion: We believe this is the first report indicating the presence of DSPP in EMD. DSPP-containing fractions stimulated ALP activity in cultured HDPCs suggesting that DSPP may contribute to EMD's bioactivity. However, the DSPP-containing fractions also contained 20-25kDa amelogenins which may also exhibit bioactivity or act synergistically with DSPP in this model system.