Objectives: The aim of the current study was to use atomic force microscopy (AFM) and dynamic force spectroscopy to characterise amelogenin-mineral crystal interactions at the molecular level and under in vivo like conditions.
Methods: Individual crystals were isolated from rat developing enamel using a selective extraction procedure to remove all native matrix protein. Crystals were imaged in the AFM in tapping mode under simulated enamel fluid at pH 7.4 in the presence of full-length amleogenin (rM179). rM179 was also covalently attached to chemically functionalised silicon-nitride AFM tips for dynamic force microscopy against individual crystal targets under similar conditions. Measurements of amelogenin-mineral interactions were calculated from the resulting force-distance curves.
Results: Amelogenin nanospheres were seen to align along crystal surfaces as described previously. Amelogenin-mineral associations were stable when exposed to increasing concentrations of phosphate buffer of up to 200 mM. The amelogenin-mineral dissociation constant (K0off) was found to be 0.08 s-1 at low loading rates when determined using dynamic force spectroscopy.
Conclusions: These results suggest a relatively stable parent amelogenin-mineral association. Post-secretory proteolytic processing of parent amelogenin is known to occur very rapidly in vivo and may be a necessary pre-requisite to permit normal crystal growth. Failure to remove protein may then result in hypomineralised enamel.
Funding: Supported by Wellcome programme grant no. 075945/Z/04/Z and BBSRC Grant no. 24/ABY08147