Preliminary Study Of The Mechanisms By Which Amelogenin Bioactive Synthetic Peptide Promote Biomimetic Remineralization Of Early Enamel Caries

Dental caries is a disease that damages the hard tissues of teeth.Because of the lack of cellular repair mechanisms,the development and reversal of early enamel caries depends on the physical and chemical processes?remineralization?around the dental hard tissue.Our previous in vitro study have found that amelogenin bioactive synthetic peptide has the role of promoting biomimetic remineralization of early enamel caries.Objectives:This study is based on previous research to study the biomimetic remineralization mechanism of the amelogenin bioactive synthetic peptide in early enamel caries by isothermal titration calorimetry,transmission electron microscopy,fourier transform infrared spectroscopy and immunofluorescence microscope.Our aim is to provide experimental evidence for the development of biomimetic remineralization agents for the regulation of early enamel caries.Methods:The peptide,comprising the amino acid residues of the N-terminus and the C-terminus of porcine amelogenin,was synthesized by Synpeptide Co Ltd using standard solid-phase peptide synthesis.1.Calcium-binding experiment of amelogenin bioactive synthetic peptide:Isothermal titration calorimetry is used to analyze the binding properties between Ca²⁺and the synthetic peptide.1.25mM Ca²⁺was injected into 0.12mM synthetic peptide solution and carry out at PH=7,T=37?while stirring at 400 rpm.Titration data is analyzed with Nano Analyze Software,and the“independent”model is used for data fitting to obtain the binding constant?KD?,stoichiometric ratio?N?,reaction enthalpy??H?and reaction entropy??S?.2.Study on the effects of amelogenin bioactive synthetic peptide on calcium phosphate compound:Experiments are divided into four groups:absence of peptide?control group?;50ug/ml;200ug/ml and 500ug/ml synthetic peptide groups.Aliquots of CaCl₂ and pH-adjusted KH₂PO₄ is sequentially added to each group.TEM and SAED are used to characterize the products at 15min,45min,4h and 24h during the experiment.FT-IR is used to confirm the final mineral phase at 24h.3.Experimental study on the penetration of amelogenin bioactive synthetic peptide into demineralized enamel:Three days after demineralization in the demineralized solution,the enamel blocks were remineralized for 12 days in a remineralization solution containing 50?g/ml of synthetic peptide.After that indirect immunofluorescence staining and photography were performed on the enamel blocks to observe the distribution of the synthetic peptide in the enamel.Results:1.Amelogenin bioactive synthetic peptide can bind calcium through ionic interactions and the binding constant is up to 9.915×10⁴M^-1,and stoichiometry of binding is about 1 mole of Ca²⁺per mole peptide.2.There is a significant inhibitory function of premature formation of calcium phosphate crystals in all peptide groups by combining and forming stabilized amorphous calcium phosphate.However,in the control group,rapidly occur calcium phosphate spontaneous precipitation reaction.The ability of the peptide groups to stabilize amorphous calcium phosphate to inhibit the premature formation of crystals are shown as follow:50ug/ml<200ug/ml<500ug/ml.What's more,the final mineral products at 24h in the 50ug/ml and 200ug/ml peptide groups are highly ordered hydroxyapatite but random hydroxyapatite in the control group.At the 500ug/ml peptide group is still an ordered amorphous calcium phosphate nanoparticles.3.The distribution of amelogenin bioactive synthetic peptide can be observed both on the surface and subsurface of enamel blocks.Conclusion:1.Amelogenin bioactive synthetic peptide has the ability to bind to calcium and possess the function of ion carrier.2.Amelogenin bioactive synthetic peptide has the ability to maintain bioavailable calcium and phosphorus by combining and forming stabilized amorphous calcium phosphate and this effect varies with the concentration of peptides.It also has the ability to modulate the formation of highly ordered hydroxyapatite.3.Amelogenin bioactive synthetic peptide can penetrate into the deep demineralized area of carious lesions.