Study On Biomimetic Mineralization Of Enamel Induced By Amelotin In Vitro

Objective: To explore the ability of amelotin to induce calcium/phosphorus mineralization and enamel regeneration in situ in vitro.Methods: 1.The recombination and expression of amelotin: The total RNA of tooth germ cells was extracted from the tooth germ of the third molar at the stage of enamel development.The human cDNA fragment was obtained by reverse transcription and amplification.The full-length gene of amelotin was obtained by RT-PCR using cDNA as template.The expression vector of pET-28a(+)-AMTN was constructed by connecting the full-length gene of amelotin with the vector.The pET-28a(+)-AMTN expression vector was transferred into Rosetta(DE3)e.coli receptive cells,and then cultured,the protein expression products were collected.After protein purification,the target protein was obtained and confirmed as amelotin after detection.2.Self-assembly of amelotin: The enamel protein and Tris-HCL / artificial mineralization solution were prepared as an amelotin solution(concentration of 100?g/ml,pH = 7.6).After incubation at room temperature for 1 minute,10 minutes,samples were prepared and the self-assembly of amelotin was observed by transmission electron microscopy,respectively.3.The mineralization ability of amelotin in vitro: Fresh and intact third molars were used to prepare dental slices.After acid etching,ultrasonic cleaning and drying,the amelotin solution was coated.After incubation at room temperature,it was cultured in the newly prepared artificial mineralized solution at 37 ? for 7 days,and the samples were taken for scanning electron microscopy and X-ray diffraction analysis to observe the characteristics of mineralized products.Results: 1.Enamel mature protein was successfully prepared.2.Through transmission electron microscopy,it was found that the self-assembly of amelotin in one minute was nano spheres,and with the extension of time,at 10 minutes and 30 minutes,it could befound that the nano spheres self-assembled by amelotin were connected into beads from the head to the tail.3.Scanning electron microscopy was used to observe the deposit sediment induced by amelotin on enamel surface.The amelotin induced a crystal bundle with the diameter of 700nm-3??m,which was composed of needle like crystals and formed at the angle of each other.The X-ray diffraction analysis showed that the same characteristic peaks of hydroxyapatite appeared at 2 ? = 25.7 °,31.8 °,32.2 ° and 32.9 °,which confirmed that the deposit was hydroxyapatite crystal.Conclusion: amelotin can induce the formation of hydroxyapatite crystals with certain length by self-assembly,and can induce the growth along a and b axes.