In Vitro Study Of Intrafibrillar Mineralization Induced By Biomimetic Peptides With Different Charges

Objective The purpose of this study was to design two kinds of non-collagenous protein biomimetic peptides with different charges,and explore their interaction with collagen and effects on inducing the mineralization of collagen fibers.In addition,the molecular dynamics simulation was used to further explain the mechanism of its interaction with collagen and minerals ions,so as to provide a new idea and reference for the design and manufacture of regenerated mineralized materials.Methods Two kinds of peptides were designed according to the amino acid sequences of Dentin matrix protein 1（DMP1）and Cementum protein 1（CEMP1）.The peptides with different charges were designed to bind to collagen and induce the mineralization in collagen fibers.The model of collagen fiber was established,and two types of peptides were selected for molecular dynamics docking simulation and mineralization simulation.Peptides designed and synthesized:Two kinds of peptides were designed according to the amino acid sequences of DMP1 and CEMP1.The binding constants and reaction heats of peptides with calcium/phosphate ions were determined by isothermal titration calorimetry（ITC）to evaluate their ability to attract mineral ions.The interaction between peptides and collagen fibers:ATR-FTIR was used to measure the infrared spectra of naked collagen and collagen co-assembled with two different polypeptides within the wavelength range of 4000～500cm^-1,and to evaluate the molecular interaction between two different polypeptides and collagen.Circular dichroism spectroscopy（CDS）was used to measure the changes of secondary structure of bare collagen and collagen bound with peptides.Finally,the above experimental results will be combined with molecular dynamics simulation to clarify the interaction mechanism between different peptides and collagen and their differences（binding sites and binding energy）.Peptides induced the intrafibrillar mineralization of collagen:The complex of collagen and peptides was mineralized in two different mineralization systems.Morphology of mineralized collagen:The morphologies of monolayer collagen and collagen membrane were observed by transmission electron microscopy（TEM）and scanning electron microscopy（SEM）,and the differences of mineralization in fibers were compared.Physical and chemical properties:Selective crystal diffraction（SEAD）and elemental analysis（EDS,Mapping）were used to further analyze mineral crystals.Infrared spectroscopy（ATR-FTIR）and X-ray diffraction（XRD）were used to compare the effects of different peptides on the formation and transformation of hydroxyapatite crystals and their differences.Molecular dynamics simulation:e1 and e2 bands were selected as representatives of gap zone to establish 3D models of collagen.The docking simulations of collagen and peptides were carried out under boundary conditions to evaluate their binding energy.The subsequent mineralization simulations were carried out to evaluate the dynamic relationship between polypeptides and collagen and mineral ions.Combined with experimental phenomena,the mechanism of collagen mineralization induced by polypeptides was further explored.Results Both peptides bind to the gap zone of collagen fiber by electrostatic attraction.The results showed that the combination of peptide and collagen could further attract mineralized precursors to aggregate on the surface of collagen,and accelerate the mineralization of collagen fibers.The positively charged peptides showed stronger attraction to mineral ions than that of negatively charged one.However,negatively charged peptides with longer chains can parallel arrange collagen fibers through the non-covalent cross-linking of the peptide chains,increasing the diameter of collagen fibers.Conclusion Peptides with different charges can both bind to the gap zone of collagen fibers.The peptides bounded collagen can significantly improve the efficiency of collagen mineralization.The charged peptides can enhance the driving force of mineral ions into collagen through electrostatic binding force and promote intrafibrillar mineralization.It suggested that electrostatic attraction may be one of the main driving forces to induce mineral ions into collagen fibers,and that proteins rich in basic amino acids may also play an important role in collagen mineralization.