Screening And Prelimary Research Of Biomineralization Of Bio-apatite Binding Peptides For Tooth Hard Tissue

Genetically engineered peptides for inorganics (GEPIs) are a group of petides derived from peptide display system. Normally the peptides have the ability of recognizing a group of inorganic surfaces and acting as functional biomolecular for drug&nucleic acid delivery, medical imaging and tissue engineering. According to its binding substrate, GEPIs can be generally divided into three major groups. And mineral binding peptides have affinity to certain mineral surfaces and may act as an active part in biomineralized process. The present research is mainly concerned with screening bio-apatite binding peptides using a piece of well prepared tooth hard tissue. And the binding-peptide relatived activity in biomineralization was evaluated.In the first part, longitude and cross sections were made by cutting parallel or vertical to crown axis. Microstructure chataters of the sample surfaces were observed under a scanning electronic microscopy. Alkaline hydrosis process was used in mild condition to reduce the organic components. High-resolution X-ray microdiffraction technology in combined with scanning electronic microscopy was used to analysis microstructure and crystallographic properties of the sample surface. Then a standard procedure for producing a proper substrate for screening the binding peptides was established. Young permanent premolars were used to minimize the individual variations among differenct teeth. Samples were prepared by cutting in the middle of buccal surface to minimize the variations within a single tooth. Dentin and inner enamel layer should be excluded from sample preparation. Samples were chemically treated with alkaline to reduce organic compontent and damaged enamel crystals. In the second part, the substrates were introduced to a phage based cyclic peptides display system. After a standard screening procedure, two different elute methods were used to collect binding peptides. In each method, thirty clones were identified from the third round and the forth round. Binding affinity of individual clone was evaluated by calculating output/input ratio and strong binders were sequenced. Statistic analysis revealed that no significant different between the two elute methods for screening strong binders (P<0.05). In total, fourteen over fifteen clones were identified with different peptide sequences.In the third part, mineralized activity of identified clones was evaluated in microtiter plate gel system. Most of the identified clones shared the same inhibitive effect with WM13(P>0.05) on calcium phosphate precipitation expect MR414and MR401. Thus, the cyclic peptides of MR414(-CMPQVMPMC-) and MR401(-CMPQVMPMC-) were synthetized. The enamel binding affinity of synthetized cyclic peptides were first verified by observing FTIR-linked synthetized cyclic peptides binding to enamel surface under a fluorescence microscopy. Then mineralized activity of synthetized cyclic peptides was evaluated in microtiter plate gel system and solution system. Calcium phosphate precipitation in gel was inhibited by synthetized cyclic peptide MR414(-CMPQVMPMC-) in a dosage dependant manner. While crystals were well organized along c axis when MR401(-CMPQVMPMC-) presented in solution.In conclusion, a piece of re-treated enamel was served as substrate for screening bio-apatite binding peptides. After a standard procedure, sixty clones were isolated and identified from3rd and4th round. Fourteen clones (out of sixty) were verified to be strong binders. Two of which were reveal significant mineralized activity in vitro experiments.