Rapid Regeneration Of Enamel-like Inorganic Crystals Based On Biomineralization

Objectives The excellent mechanical properties of enamel come from its complex multi-hierarchy structure.In recent years,how to regenerate enamel-like structures has become a research hotspot in oral biomimetic materials.Because there are many disadvantages,such as high price,complicated method,wide differences from natural enamel and limited thickness in similar studies.In this study,we aim to develop a simple,economical and efficient method to regenerate enamel-like hydroxyapatite crystals on different substrates in vitro,and try to expand this strategy to regenerate other inorganic crystals,such as zinc oxide(ZnO)crystals.Methods Natural enamel,dentin,titanium and polyethylene(PE)were selected as substrates.Before experiment,the enamel and dentin slices were etched with 37% phosphoric acid,and the titanium and PE slices were immersed in dopamine solution for activation firstly.The substrates were fixed on the inner surface of a rotating flask containing a certain amount of remineralizing solution(Ca-and P-supersaturated solution containing silk fibroin).Then the rotating flask was immersed into a 70 °C water bath and mounted onto a rotary evaporator with 20 rpm.After experiment,the substrates were taken out,cleaned and dried for characterization.The morphology and structure of regenerated minerals on the substrates surface were observed by scanning electron microscopy(SEM)and transmission electron microscopy(TEM).The crystal lattice structures were observed by high resolution transmission electron microscopy(HRTEM).The properties of regenerated minerals were characterized by X-ray diffraction(XRD).The interaction between silk fibroin protein(SF)and HA crystals was characterized by Fourier transform infrared spectrometer(FTIR).Thermo gravimetric analyzer(TGA)was used to analyze the organic and inorganic contents in regenerated minerals.Nano-indentation analyzer was used to analyze the nano hardness and elastic modulus of regenerated minerals and to characterize their mechanical properties.In order to investigate whether this method can be extended to regenerate other enamel-like inorganic crystals(such as ZnO),This kind of rotary evaporation experiment was carried out using silicon wafers as substrates.First,zinc acetate solution was dropped on the surface of the wafer and allowed to dry naturally.After repeated twice,annealing operation was carried out to form a layer of ZnO seed layer on the surface of the silicon wafer.Then,the silicon wafer was fixed on the inner surface of the rotating flask,which was filled with 100 m L of regenerated solution(the same amount of mixed solution of hexamethylenetetramine and zinc nitrate),and then rotary evaporation experiment was carried out at 90 ℃ and 20 rpm for 4 h.After the experiment,the regenerated layer on the surface of the silicon wafer was cleaned and dried,and then characterized by XRD and SEM.Results The minerals regenerated on the surface of enamel,dentin,titanium and PE slices were HA crystals with obvious oriented growth along the c-axis.SEM and TEM results showed that the regenerated mineral crystals were hexagonal crystals with a width of 20-30 nm and were arranged parallel to each other along the c-axis.Their structure was highly similar to the natural enamel structure in crystal size,morphology and arrangement.HRTEM showed that the crystal lattice was well developed,which was mutually supported by SEM and TEM results.FTIR results showed that SF existed in minerals and was connected with HA by chemical bond and hydrogen bond.The results of TGA showed that the inorganic content of the regenerated minerals ranged from 92 Wt% to 93 Wt%,which was very close to that of natural enamel from 96 Wt% to 97 Wt%.The results of nano-indentation showed that the hardness and elasticity modulus of minerals were 1.86 ± 0.27 GPa and 50.01 ± 3.49 GPa,respectively,slightly lower than that of natural enamel(4.61 ± 0.286 GPa and 80.43 ± 4.02 GPa).The XRD results showed that the regenerated minerals on the surface of silicon wafer were ZnO crystals with oriented growth along the c-axis.SEM results showed that the regenerated ZnO crystals had a hexagonal structure with a width of about 60 nm and a thickness of about 1.2 μm;The crystals were highly oriented along c-axis,which was very similar to the structure of natural enamel.Conclusion Though the strategy of rotary evaporation,enamel-like hydroxyapatite crystals have been successfully regenerated on the surface of natural enamel,dentin,titanium and PE slices.The morphology,composition,structure and mechanical properties of these crystals were highly similar to those of natural enamel.It was fast(1-2 μm thickness could be regenerated on the enamel surface in 20 min),and the thickness was controllable(up to 70 μm in 48 h)and was not restricted by the substrate.In addition,this method is not limited to regenerate HA crystals,but also can successfully regenerate enamel-like ZnO crystals.Therefore,this method has potential application values in the field of coating,and also opens a new idea for the construction of other highly oriented inorganic crystal materials.