| Objective: To investigate the self-assembly steps of AMEL,AMBN-C and AMTN and its ability to induce calcium and phosphorus ions mineralization at different concentrations in vitro.Methods: 1.Self-assembly of AMEL,AMBN-C and AMTN at different concentrations: Take an appropriate amount of enamel matrix protein stock solution(AMEL,AMBN-C and AMTN)to prepare final concentrations of 0.01mg/ml,0.05mg/ml,0.1mg/ml,0.15mg/ml,0.2mg/ml of 100 ul protein solution(p H 7.6).Drop 20μl of protein solution onto the porous carbon side of the copper mesh,and incubate at room temperature for 1?min and 10min.The self-assembly was observed by JEOL 1400 Plus transmission electron microscope.2.Mineralization capacity of AMEL,AMBN-C and AMTN at different concentrations in vitro: Prepare the concentrations of AMEL,AMBN-C and AMTN solution with 0.01mg/m L,0.05mg/m L,0.1mg/m L,0.15mg/m L and 0.2mg/m L,respectively.Each protein is divided into 11 groups.Experimental groups 1-5: Take 10μl of 0.01mg/ml,0.05mg/ml,0.1mg/ml,0.15mg/ml,and 0.2mg/ml protein solution and spread them on the cell slides surface.After incubation at room temperature,the samples were transferred to 2 ml of artificial saliva for culture at 37 °C.After 7 days and 14 days of incubation,the samples were taken out at each concentration and analyzed by SEM and XRD to observe the characterization of mineralized products.In experimental groups 6-11,10μl of 0.01 mg/ml,0.1 mg/ml,and 0.2 mg/ml protein solutions were coated on the surface of the cell slides.The samples were transferred to 2 ml of artificial saliva for culture at 37 °C.After 7 days of incubation,the samples were taken out and air-dried,respectively coated with 10μl of the other two proteins at 0.1 mg/ml,and incubated for 7 days.The samples were taken out and analyzed by SEM and XRD to observe the characterization of mineralized products.Results: 1.At low concentrations(<0.1mg/ml),AMEL,AMBN-C and AMTN did not form nanospheres although the protein monomers tended to aggregate.When the concentration of AMEL and AMTN ≥ 0.1mg/ml,the aggregation tendency is significant,and a stable nanosphere structure can be formed with a typical selfassembly process.When the concentration of AMBN-C was higher than 0.1mg/ml,only the nanosphere structure was observed,and the higher-order self-assembled structure was not formed.2.At low concentrations(<0.1 mg/ml)of AMEL,AMBN-C and AMTN induced crystals scattered and distributed after 7 days of artificial saliva mineralization at 37°C.At high concentration(≥0.1 mg/ml)protein solutions induce typical needle-like crystal bundles and "cluster-like" crystals with a tendency to further aggregate.When the mineralization time was prolonged to 14 days,the induced crystals increased in length and diameter,and the degree of aggregation was enhanced.0.1mg/ml AMEL,0.1mg/ml AMBN-C,and 0.1mg/ml AMTN can induce hydroxyapatite similar in structure to enamel nanocrystals to micrometers after 14 days of artificial saliva mineralization at 37°C,respectively.The interaction of AMEL,AMBN-C and AMTN promotes the growth of mineralized crystals.Under the interaction of 0.1mg/ml AMEL,0.1mg/ml AMBN-C,and 0.1mg/ml AMTN,hydroxyapatite crystals with the structure of nanometer to micron can be induced.Conclusion: 1.The process of self-assembly and mineralization of enamel matrix proteins is affected by protein concentration.At high concentrations(≥0.1mg/ml)of AMEL and AMTN,a stable nanosphere structure can be formed with a typical selfassembly process.When the concentration of AMBN-C is from 0.1 to 0.2mg/ml,only nanosphere structure is formed,which is an incomplete self-assembly process.2.High concentrations of enamel matrix protein solutions(≥0.1mg/ml)induced typical needle-like crystal bundles.The interaction of AMEL,AMBN-C and AMTN has the synergistic effect of combining calcium and phosphorus ions to nucleate and promote the growth of mineralized crystals. |
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