Biomimetic Mineralization Mechanism And Application Of Calcium-containing Minerals Regulated By Amyogenins’ N-terminal Peptide

Crystal formation and dissolution are opposite processes.Some situations crystal formation are required,such as the treatment of osteoporosis and loss of tooth enamel;sometimes crystal dissolution is required,such as crystals such as stones and gout.Understanding the mechanism of biomineralization,including crystal formation and crystal dissolution regulation,as well as the interaction between cells and biomimetic materials,has significant importance for the design of new medical biomaterials.In this paper,using the N-terminal peptide of amelogenin as model macromolecule,we studied the effect of the N-terminal peptide on calcium phosphate and calcium carbonate crystals formation,the effect of the self-assembled N-terminal peptide layer on cell adhesion,and revealing the relationship between peptide molecules,the biological influence of peptide assembly structure on cells,and the effect of peptide conformational changes on crystal growth.In addition,taking calcite crystal as a model crystal,the phenomenon of its dissolution and transformation adjustment in Cu Cl₂ solution was studied,which provided an idea for the subsequent design of new materials by mild means.The specific contents are as follows:（1）Adsorption of the N-terminal peptides of amelogenin on hydroxyapatite（HAp）surface and its dynamic regulation mechanism on calcium phosphate formation.Human body fluid is in constant motion,so studying the dynamic mineralization process of calcium phosphate can better understand the mineralization mechanism.The N-terminal phosphorylated and non-phosphorylated peptide of amelogenin adsorbed layers were constructed by quartz crystal microbalance（QCM）,and the effect of the peptide adsorption layer on calcium phosphate formation under flow dynamics was studied.The results showed that the phosphorylated N-terminal peptide adsorbed on the HAp surface showed a random coil conformation,while the non-phosphorylated N-terminal peptide adsorbed on the HAp surface showed a reverse parallel conformationβfolded conformation,this reverse parallelβfolded conformation will expose more carboxyl groups on the surface,providing more nucleation sites,so the mineralization curve shows a faster crystal growth rate.（2）Study on the formation of amelogenins’N-terminal peptide/HAp bone-like apatite and its osteogenic properties.Since the N-terminus and C-terminus of amelogenin are the key functional domains,on the basis of the research on the regulation of the N-terminus on calcium phosphate,the joint regulation of the N-terminus and the C-terminus on apatite was studied.The results show that the N-terminal peptide can inhibit the phase transformation process of amorphous calcium phosphate（ACP）to HAp,the C-terminal peptide has an inhibitory effect on the phase transformation at the initial stage,and the inhibitory effect is not obvious in the subsequent reaction.Compared to the N-terminus group,the N-terminal+C-terminal group will promote the conversion of ACP to HAp.As time goes on,the N-terminal peptide group makes the crystals oriented along the C-axis,and eventually the N-terminal peptide can form an organic/inorganic bone-like apatite composite material together with apatite.The results of cell differentiation showed that the bone like apatite composite could better promote the expression of osteogenic related genes（ALP,OCN,OPN,Runx2,BMP-2,BSP）than the apatite without N-terminal peptide regulation,and had good osteogenic activity.（3）Regulation of the N-terminal peptide of the amelogenin on calcite crystal by changing its molecular conformation.Because N-terminal peptide is hydrophobic chain segment,it is easy to form spherical structure through hydrophobic interaction under water,which makes it difficult to study its effect on biomineralization.By adding organic solvents DMSO and Acetonitrile（Ace）,the effect of peptide secondary structure change on calcite crystal formation was studied.The results showed that the phosphorylated N-terminal peptide was in random coil conformation in water system,andα-helix conformation formed by adding30%Ace,which mediates the transformation of calcite crystal from porous structure to cluster structure.Adding of DMSO mediate the formation ofα-helical andβ-folded conformation for the phosphorylated peptides.Same to Ace group,α-helix mediated crystals form clusters,theβfolded conformation mediates the formation of"snow"-like calcite crystals.In addition,the organic solvent system with less water regulates the final crystal formation by changing the nucleation rate and phase transition pathway.（4）Construction of the N-terminal peptides of amelogenin self-assembled layer and its adhesion to cells.First,the assembly layers of peptides at different p H values were constructed,and then the adhesion and proliferation behaviors of peptide layers with different assembly morphologies to cells were studied.The results showed that the conformation and surface roughness of peptides could affect cell adhesion and proliferation.The antiparallelβ-folded structure of the non-phosphorylated N-terminal polypeptide makes the surface have better function of promoting cell adhesion and proliferation.The random coil structure of phosphorylated N-terminal peptide reduces cell adhesion behavior but does not affect cell proliferation.However,the phosphorylated N-terminal peptide layer formed under acidic conditions significantly increases its surface roughness,which is benefit to cell adhesion,but will inhibit cell proliferation.It can be seen that the regulation of amelogenins’N-terminal peptide on cells is a process of joint action of molecular structure and aggregation state.（5）Study on the preparation of basic copper chloride by dissolution and transformation of calcite crystal and its antibacterial and angiogenic properties.Based on the idea of crystal dissolution regulation,calcite crystals were dissolved and precipitated in copper chloride solution,and the effects of copper chloride concentration,reaction time and reaction temperature on the morphology of the final crystal were investigated.Finally,the antibacterial and angiogenic properties of the formed basic copper chloride crystals were studied.The results show that nano square and nano plate structures can be formed by changing temperature and time.Antibacterial and angiogenic experiments showed that the material had certain antibacterial and angiogenic abilities.In conclusion,the N-terminal peptide of amelogenin was used as a model molecule to reveal the effect of the conformational change of the N-terminal peptide of amelogenin on calcium containing minerals formation and the influence of related biomaterials on the biological behavior of cells;In addition,based on the idea of crystal dissolution regulation,the preparation of basic copper chloride crystal has certain application prospects.