Amelotin (AMTN) is a recently discovered protein that is specifically expressed during the maturation stage of dental enamel formation. We have recently shown that AMTN is a potent promoter of calcium phosphate mineralisation in vitro, in cell culture, and in vivo using transgenic mice that overexpress AMTN in enamel matrix. The objective of this study is to investigate whether AMTN is also able to accelerate mineralisation in collagen based systems. First, we studied the effect of immobilised AMTN on collagen mineralisation in vitro. Recombinant human (rh) AMTN was expressed in E.coli and affinity purified to near homogeneity. Collagen membranes were immersed in 1 mg/ml rh-AMTN solution in deionized distilled water overnight at RT. Control membranes were immersed in water only overnight. The membranes were then removed from the solution and dried. Each piece was then immersed in SBF mineralisation buffer and incubated at 37 °C for 6 days. Scanning electron microscopy of the membranes coated with AMTN revealed numerous calcium phosphate particles associated with the collagen fibres whereas, control membranes with no AMTN coating contained no mineral deposits. Next, we examined the effect of AMTN on mineralization in a collagen-based cell culture system. AMTN expression vector or empty vector (control) was transfected into the mouse osteogenic cell line MC3T3-E1 by electroporation. 24 hours after transfection, culture medium was changed to α MEM containing 4 mM inorganic phosphate and 50 μg/ml ascorbic acid. The mineral nodules were characterised by Alizarin Red staining and transmission electron microscopy. Overexpression of AMTN in mouse calvaria cells also increased the formation of calcium deposits in the culture medium. These findings open up potential applications for the AMTN protein as a regulator of mineralisation in bone and dentin.
Disclosure: The authors declared no competing interests. This work was supported by the National Sciences and Engineering Research Council of Canada (NSERC, Operating grant #490975) and the Canadian Institutes of Health Research (CIHR, Operating Grant MOP-492418).