| Under normal circumstances, the enamel demineralization and remineralization maintain a dynamic equilibrium. The use of fixed appliance leads this equilibrium to the enamel demineralization. So the patients received the orthodontic treatment are often of high risk to enamel demineralization. Early demineralization of the enamel mostly confined to the surface layer. If the appliance was removed in time, it would impossible to be restored by saliva remineralization. However, at most of the time, when the appliance couldn't be removed immediately, the demineralization of surface layer gradually develops to the subsurface layer, forming the visible white spots. At this time, the lesion is only the loss of the minerals of local enamel, which hasn't caused the destructure and has the possibility of restoring the demineralized enamel. However, the remineralization capacity of saliva is very limited at this time, and needs other measures to remineralize. The key of remineralization is the supplement of losted calcium phosphate ions, but because of the low dissolubility of the calcium phosphate, it is difficult to maintain high concentration bioavailable ions at the surface of the enamel to diffuse into the subsurface enamel. So in traditional methods, calcium and phosphate could only be use at very low concentration. Methods of remineralization are also limited to the enamel surface laeyer by minerals depositing. But there's little effect of restoration in the enamel inner lesion. Therefore, seeking the ways of restoration of the demineralization subsurface layer rather than masked by modification is always the ideal goal of the scholars. The finding of casein phospho-peptides has brought a new breakthrough in this research field. Casein phosphopeptides (CPPs), a bioactive peptide, which is rich of phosphoserine, is obtained from milk casein by hydrolization, segregation and purification. It has the significant capacity of stablizing the calcium and phosphate ions in solution, and can combine with a big amount of calcium, phosphate and OH- ions before spontaneous precipitation under neutral or alkalinity circumstances. So in CPPs aqueous solution, the stablized calcium and phosphate is 100 times more than usual, and forming the nanocomplex, casein phosphopeptide-amorphous calcium phosphate (CPP-ACP). CPP-ACP, acts as the good vector for calcium and phosphate ions, not only supply ions, but also prevent them from spontaneous pricipitation at the enamel surface. Often it promotes the ions diffuse into the subsurface enamel in an appropriate molar ratio. The unique nature of CPP-ACP makes it have a broad application prospects in remineralization of demineralized enamel.Some Scholars had proposed that the CPP combinated with the forming ACP, forming a metastable solution. The researches of CPP-ACP nano micro particle have also confirmed its status of non-crystal. But as the micro-particles of the nano level easily tend to gather together in the solution, there's no relative report of domestic or alien about whether the CPP-ACP particles form together in the solution and the state of existing, as well as the effect of concentration or fluoride. In order to investigate the feature of the particle size distribution and the existant form of nanoparticles groups, we used the laser particle size analysis in experiment 1. By measuring the size and distribution of the CPP-ACP, its aggregation and stability is reflected. By increasing the concentration of 0.1%1% in usual experiment to 5%, as the result, we got stable 5% CPP-ACP colloidal solution, and with the concentration of CPP increases, the particle size of CPP-ACP gradually decreased and the distribution became narrow. When appropriate concentration of fluoride ions added, the particel size was further reduced, showing that in solution of high concentration, the nanoclusters dispersed better, gather together less, and the size and distribution was relatively uniform. The remeasurement carried out one month later showed that the size and distribution of particle had no significantly change, which confirmed that the CPP-ACP solution in the current experiment was relatively stable which laid the foundation for the followed-up experiments.The remineralization effect of CPP-ACP is achieved by stablizing high concentration of calcium ions. The relatively high calcium concentration in plaque inhibits the cariogenic bacteria adhesion to the acquired films and their mutual adhesion, as well as influenced the bacteria metabolism by invaliding the calcium pump in bacteria membrane. In order to verify the remineralization effect of CPP-ACP, we compared the prepared 5% CPP-ACP and the traditional 2% NaCl solution in experiment 2, as well as the quantity and depth of demineralization by microphotography technology. The results showed that 5% CPP-ACP had significant anticariogenic effect compared with the deionized water, which is equivalent to 2% NaF. CPP-ACP is safe, effective, non-toxic or side effects, and can avoid the side effect by local high concentration of fluoride. CPP-ACP has a more wide range of applicable crowd and age than fluorine and it's the ideal succedaneum of the latter.Researches had shown that the calcium and phosphorus ions of CPP-ACP diffused into the subsurface enamel relatively easier by transforming into the neutral ions and ion-pairs. In the early demineralized enamel lesions and lesion surface, the balance between the charged ions and neutral ions maintained the activity gradient of neutral-ion diffused into the lesion. Therefore, CPP-ACP had the obvious advantage in the remineralization of subsurface enamel lesions, but there were few reports about what level of demineralization could be remineralized or the effect after the fluorine was added. Aiming at this question, we used a series CPP-ACP solution of three concentrations in experiment 3 and the fluoride was added into the solution to form CPP-ACFP. By microradiation quantitative study, the results showed that the remineralization throughout the body of subsurface enamel demineralizated lesions was dose-dependent with the CPP-ACP; the added F significantly improved the remineralization ability of CPP-ACP which acts as the vector of the F, which solved the problem of poor permeability of fluorde.In traditional studies, the mineralization solution containing of calcium phosphate and fluoride formed a layer of mineral deposition in the enamel surface, covering the original enamel surface, reaching a so-called "remineralization effect" rather than the recovery of the original enamel structure. In order to verify whether there was any advantage of stablized calcium, phosphate and fluoride ions of CPP in this aspect, we used scanning electron microscopy to observe the surface morphology changes of the enamel (experiment4), the results showed that the newly formed minerals by remineralization of CPP-ACP had a dense structure, forming an organic combination with the surviving structure of the enamel; With the CPP-ACP concentration increased, the surface of the enamel gradually became smoother, the damage of the surface structure caused by acid was gradually restored, the overall smooth appearance was recovered, which was similar to the normal enamel. While NaF and saturated solution of calcium phosphate depositing in the enamel surface in the form of a large number of non-uniform and loose granular, which had loose structure and rough surface. Because of uneven deposition, the pits contour of corrosion was still faintly visible. The morphology observation of the current experiment confirmed the obvious advantages of CPP-ACP in remineralization once again.In recent years, with the increasing consumption of acidic beverages such as fruit juice, great concerns have been taken on the demineralization of the enamel caused by the acid in food. This kind of acid is relatively low in pH (2.04.0), and differs in the mode of enamel corrosion and subsurface demineralizion; a 14μm softened layer formed in the surface of enamel. This softened enamel layer is easily removed by outside force such as teeth brushing. So it is important to protect the destroyed surface by remineralization on the softened surface of enamel. In order to further detect the nano-structure of the softened enamel surface, we used citric acid of pH 3.25 to etch the enamel surface for a short time (120s) in experiment 5. After the softened surface formed, we used CPP-ACP for remineralization, observed the role of remineralization using Atomic Force Microscope (AFM) for the first time and measured the roughness of the enamel surface. The results show that, by remineralization of CPP-ACP, extensing the original crystal structure on the etched enamel surface orderly arranged a lot of micro-crystal fiber (enamel structure of the third level) which was in same size and directions.These micro-fiber lens were parallel to each other and linked closely to further reunion assembled into crystal fiber bundle (the fourth-class structure of enamel), which repaired the destruction part of the enamel surface and reduced the surface roughness caused by etching. This experiment provided the theoretical evidences to verify that the CPP-ACP could restore the nano-structure of the destroyed enamel.The incidence of white spot during the application of fixed appliance is significantly higher than others, and the white spot remains to be chalk colored many years postorthodontic treatment, which affects the patients'appearance. Clinically there is no effective way to cure the already suffered white spot and the traditional fluoride curing the white spot has effect only in a small range, there is no report about completely curing the white spot. In experiment 6, we first used commercialized CPP-ACP (Tooth Mousse) to treat the long existent enamel white spot and quantitative the decrease of white spot. By intraoral colored photos, clinical observation and Image-Pro Plus image analyze software; the white spot and the ratio of the white spot were measured. The paired t test was used to compare among pretreatment, 1 month and 2 months posttreatment. The results showed the surface ot the white spots decreased by 5.456±2.358% 1 month posttreatment and 8.359±3.472 2 months later compared with that of pr-treatment, which was significantly decreased (P<0.01). Part of the patients received treatment as long as 10 months, and the white spots was fully remineralized. The appearance and color returned to normal, which increased the aesthetic effect of the teeth. The quantitative study of the white spots surface by the intraoral photos which was also called the computer assistant image analyzing is reliability and repeatability.By the vitro and vivo study, we proposed that CPP-ACP could stablize the high concentration of calcium and phosphate in aqueous solution without deposition. With the concentration of CPP increased the level of agglomeration of CPP-ACP nano-structure decreases and uniform in distribution. When appropriate concentration of fluorine was added, the diameter of nano-structure was further decreased; 5%CPP-ACP could inhibit the enamel demineralization in vitro, the effect of which was equal to 2% NaCl; CPP-ACP in vitro could make the already demineralized subsurface enamel remineralized, which was dependent on the concentration, and at the same time restored the destroyed enamel surface to normal and had synergy effect with fluoride; The further study by AFM confirmed that the remineralization effect of CPP-ACP was not simply the deposition of minerals, but by the remineralization of enamel crystal and gradually assembling, restored the nano-structure of enamel; The CPP-ACP used to treat white spot after the orthodontic treatment had satisfactory clinical effect, which further confirmed the remineralization effect of CPP-ACP. The innovation of the experiment compared with the relative study of domestic and alien are as follows:①Laser particle size analyzer was used to study the particle diameter and the distribution of different concentration of CPP-ACP and the results supplied the new evidences to the remineralization effect of CPP-ACP and its concentration relevance;②For the first time , Atomic Force Microscope was used to study the remineralization effect of CPP-ACP and observe the enamel remineralizaiton in the nano level; which showed that CPP-ACP could restore the enamel nano-structure by remineralization, providing the evidences for the CPP-ACP clinical using for remineralization treatment.③Commercialized CPP-ACP was used to treat the orthodontic white spots, which broke the situation of having nothing to do with orthodontic white spots and made a new way for treatment. In addition, in alien studies, CPP-ACP was usually used at a low concentrate (0.1%～1%), while this current experiment we increased the concentration to 5% and added 500mg·L-1 NaCl into the CPP-ACP aqueous solution, which formed stable CPP-ACP and CPP-ACFP solution characteristic of good anti-acid and remineralization effect; But in domestic researches of the feature of anti-acid and remineralization of CPP-ACP were limited and mainly focused on the surface of the enamel; this current experiment detected the changes of all layers of the demineralized legion caused by CPP-AC(F)P by Radiographic microscopy techniques, and the demineralization depth and quantity were quantitatively analyzed as well, which was the first time in our country and also one of our research features...
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