| Dental caries, as a common oral disease, seriously impacts on human oral and systemic health. The prevalence rates increase with age. It is always a hot topic of scholars. Dental caries is a disease that tooth hard tissue is chronically destructed under the influence of a variety of factors dominated by bacteria. For many years in the past, people thought that dental caries was a lesion dominated by demineralization. Its occurrence and development processes were irreversible, mineral of the teeth was permanently lost, and a cavity was eventually formed. But the modern caries theory considered that dental caries is an alternative process of demineralization and remineralization. Remineralization is a process that the ions such as calcium, phosphorus in solution redeposit into partially dissolved crystal surface and induce it to regrow. It can not only maintain enamel mineral dynamic equilibrium between losses and gains, but also promotes the natural restoration of caries or makes its healing. Now fluorine is the most effective material in preventing demineralization and promoting the remineralization of teeth. Although the anti-caries efficacy of fluoride has been well-proven, the excessive use of fluoride brings side effects, such as dental fluorosis, skeletal fluorosis etc. Therefore, at the same time of considering improving the use of fluoride, there are still necessary to continue to study alternative, effective non-fluoride anti-caries agents.Hydroxyapatite (HA) is a major inorganic component of tooth hard tissue. Because of its good compatibility and bioactivity, HA becomes one of hot biological materials in recent years. With deeper research of nano materials in adsorption, optics, magnetism, catalysis and chemical activity and so on, the research on HA entered a new stage in exploring and applying the performance of nano-hydroxyapatite (n-HA). Based on the microcrystalline structure of n-HA that is similar to human teeth, preliminary study on the efficacy of n-HA are reported in recent years. It is shown that it has a good effect in remineralizing early enamel caries, relieving dentine hypersensitivity and whitening tooth. Because of its larger specific surface area and more surface atomic particle, n-HA has higher surface energy and electrostatic field, which can increase the Ca2+ migration to promote remineralization of the demineralization area. On the other hand, in the field of environmental function mineral materials, n-HA, as a kind of inorganic material, is an important environmental function material to control the soil and water pollution.With the rapid development of world economy, pollution including water, air and soil becomes major environmental problems that many countries face and urgently solve. Heavy metal is one of the main sources of water pollution. Heavy metal ions can’t be bio-degradable in the natural environment, and they have the biological accumulation and enrichment. When heavy metal ion gets into the human body, it will act with biopolymers to make them lose activity and organ lesions. Because of the bio-accumulation of heavy metals, heavy metal can cause chronic poisoning. In recent years, it is often seen that heavy metal pollution accidents were discovered by the media. Lack of treatment facility for sewage discharge and collection, especially in the vast majority of the urban, leads to a large number of untreated sewage directly discharged into the water body which causes environmental pollution. Therefore, the prevention and control of heavy metal pollution of water are urgent! In recent years, a large number of studies have shown that n-HA, which has a strong adsorption capacity and a good removal efficiency of adsorption for heavy metal ions on the pollution, is an excellent adsorbent.Based on the roles of HA in oral health supplies and adsorption of heavy metal ions, our task is to research adding HA in dentifrice. It can show its oral healthy efficacy in one hand. On the other hand, after the brush, a small amount of toothpaste residue in the mouth, and the vast majority of toothpaste wastewater flows into a sewer, which can play the adsorption capacity of HA for heavy metal ions in living waste water. Thus the "functional toothpaste with environmental protection" concept was first proposed in the world. Its feasibility was verified by the previous studies. Brushing behavior is given the significance of environmental protection, so this research has an important value of environmental protection.Most of the previous studies focused on the remineralization effect of one single particle size of HA, lack of comparative study on various particle sizes of HA. The comparative study of the pure HA and HA dentifrice on the adsorption effect of heavy metal ions has not been reported. With the nanometer particle size decreasing, the adsorption performance is better?ObjectiveUsing citric acid to etch enamel and observing morphological characteristics of enamel surface in different demineralization time with scanning electron microscope, we obtained an efficient, reliable in vitro model for studying the early enamel caries, and provided chemical experimental method closer to natural caries demineralization for further research. Based on establishing the early enamel caries in vitro, through the pH cycle experiment, by simulating oral environment, the effect of HA with various particle sizes on remineralization of artificial enamel lesions was studied in microhardness tester and scanning electron microscope. Furthermore, the mechanisms of preventing caries and remineralization on demineralizated enamel were discussed. And the adsorption capacities of HA and dentifrice containing HA with different particle size on aqueous copper ion and cadmium ion were compared, as well as the mechanisms of stability and function of dentifrice containing HA on the heavy metal ion adsorption were researched.Methods(1) Human premolars free of lesions, cracks and fluoric mottle were selected. The crowns were cut into enamel blocks by carborundum needle under continuous water cooling. And the enamel surfaces were polished. After natural drying, baseline surface microhardness (SMH) was measured by a microhardness tester (Knoop head, 10 g,15 s). Enamel blocks with the hardness in the range of 305-355 KHN (Knoop hardness number) were selected. The enamel block surface was marked off a window with 4 mm×4 mm area. The remaining parts of enamel block were coated with nail polish, and embedded in polymethylmethacrylate.The 30 enamel blocks were selected and divided into 3 groups,10 samples in each group. The three groups were placed in the 100 mL solution of 10% citric acid with 90 s,120 s and 150 s, respectively. Removal of citric acid solution, the samples placed in deionized water were cleaned by ultrasonic cleaning machine.5 samples were randomly selected from each group for scanning electron microscope (SEM). All the samples were labeled according to the group, rinsed with deionized water, placed in a drying box for drying, observed (randomly selected 3 different regions) by SEM, and SEM images at varying magnification (×10 000, ×30 000,×60 000) were shot and stored.(2) The prepared enamel blocks samples were placed in 10% citric acid solution with 120 s. After the removal of citric acid solution, artificial caries were formed in enamel blocks. The surface microhardness was performed after demineralization.60 enamel blocks with hardness values range from 155-185 KHN were used for pH cycling experiment. The 60 artificial enamel caries samples were divided randomly into 6 groups (10/group):NaF group (positive control):fluoride solution of 1000 ppm, DDW group (negative control):deionized water,20 nm HA group,30 nm HA group,60 nm HA group,12 μm HA group.Using pH-cycling in vitro, the cycle was repeated for 12 days and temperature maintained at 37℃. Daily pH-cycling includes:the experimental groups were treated 4 times, each time 3 min treatment, soaked in demineralization solution for 2 h, the rest of the time to soak in remineralization solution. All solutions were freshly made daily. The morphologies of normal enamel, demineralization and remineralization were observed by SEM.5 samples were randomly selected from each group and 3 different regions of each samples were randomly selected for SEM, and SEM images at varying magnification (×10 000, ×30 000, ×60 000) were shot and stored.After pH-cycling, another 5 samples of each group were used to measuring microhardness. Surface microhardnesses of the enamel blocks window were remeasured. Then the average of all measured 5 hardness value were compared on three occasions (before demineralization, after demineralization, pH-cycling), and the percentage of the surface hardness recovery (%SMHR) were calculated. Data were analyzed using SPSS 16.0 software. Student’s paired t-test was used to compare Knoop surface microhardness before and after the treatments.%SMHR was analyzed by one-way ANOVA, followed by the LSD test.(3) 20 nm HA,30 nm HA,60 nm HA and 12 μm HA powders were used to prepare for four groups HA suspension with a series of concentrations respectively.1 mL HA suspension of series concentrations was added in 50 mL cadmium or copper ion solution respectively. After standing for 1 d, supernatant were used to determine the concentration of residual ions by inductively coupled plasma emission spectrometer (ICP), and the removal rates were calculated. The dentifrice containing 20 nm HA,30 nm HA,60 nm HA,12 μm HA and blank dentifrice were prepared for five groups dentifrice suspension with a series of concentrations respectively.1 mL dentifrice suspension of series concentrations was added in 50 mL cadmium or copper ion solution respectively. After standing for 1 d, supernatant were used to determine the concentration of residual ions by ICP, and the removal rates were calculated. Repeating the above method of configuration of the reaction system, (standing for 14 d,28 d), the residual ion concentrations were determined and the removal rate were calculated.Results(1) After demineralization, the enamel surface showed an obvious acid corrosion appearance, typical honeycomb and porous defects. When the etching time was 90 s, pore structure of enamel surface was increased and deepened, and the shape was irregular. In high magnification, the enamel surface was rough near pore structure, only a small part of enamel lattices were dissolved by demineralization. Etched for 120 s, at low magnification, the enamel surface showed as typical scaly, dissolved demineralization of head gradually extended to the tail, with distinct boundary, formed a central shallow recessed region. At high magnification, the enamel rods were not tight, loose, and the original enamel rod structure disappeared. The pores and obvious enamel concave ends were visible, the irregular porous structure increased significantly, and honeycomb structure was obvious. With etching enamel surface to 150 s, it was shown that it had obvious corrosion morphologies, dissolution of crystal center led to perforate in crystal center region, crystal dissolution area expanded unceasingly, the perforation area of adjacent crystals mutually fused, which formed crystal structure disintegration with a large area.(2> All experimental groups could raise the surface microhardness of demineralized enamel (P<0.05). The increases of enamel surface microhardness of different groups before and after pH-cycling were significantly different. By one-way ANOVA and LSD test, n-HA is obviously better than that of micro HA. The percentage surface microhardness recovery of 20 nm group HA was 70.5%, higher than that of 30 nm HA group (64.3%), but the difference was not statistically significant (P>0.05), the percentage surface microhardness recovery between the other groups had statistical significance (P<0.05). The percentage surface microhardness recovery of NaF group was up to 81.8%, and that of DDW group was 16.9% as the minimum. The enamel surface appearance after remineralization of n-HA group more regular and more uniform than fluoride group. At high magnification, many uniformly dense crystal particles with acicular or short bar deposited and closed in demineralized surface, which embedded compactly with normal enamel structure. Honey comb structure disappeared, rare or no glazed demineralization pore was seen.20 nm HA group and 30 nm HA group had no obvious difference. But that of 60 nm HA group was less smooth, and a few demineralization pore area was seen. In the 12 μm HA group, only a small part of the sediment were formed on enamel surface. At low magnification, large particles suspended on the surface were visible. At high magnification, the appearances were similar with DDW group, and the crystal size is too large to enter the deep layer of demineralized enamel, but some part honeycomb structures were visible.<3> HA with various particle sizes had a strong ability to absorb cadmium ion from simulated wastewater, the adsorption rate increased with the increase of HA suspension concentration, and the average adsorption rate was about 80%. The adsorption capacity of n-HA was better than that of micro HA. Dentifrice containing HA with various particle sizes on the cadmium ion from simulated wastewater had a strong adsorption capacity, and the adsorption rate increased with the increase of HA suspension concentration, the average adsorption rate compared with that of HA was decreased by about 19%, and the adsorption capacity of HA toothpaste group were higher than that of blank toothpaste. The effects of 20 nm HA and 30 nm HA toothpaste groups on Cd2+ adsorption were relatively stable, and the rate of adsorption did not appear desorption phenomena with the passage of time. The possible mechanism of HA on cadmium ion adsorption were chemical reaction processes such as ion exchange, dissolution-precipitation, surface complexation and so on.HA and dentifrice containing HA with various particle sizes on copper ion from the simulated wastewater had good adsorption capacity, which increased with the increase of their concentrations, and the incorporation of toothpaste enhanced adsorption capacity, with the adsorption capacity of 20 nm HA as the maximum. Pure HA and dentifrice containing HA on copper ion adsorption were relatively stable, and desorption phenomena did not appear. The mechanism of HA on removal of copper ion might mainly be ion exchange adsorption and physical adsorption. The removal of copper ion with dentifrice containing HA might be the cooperative effect of ion exchange adsorption and physical adsorption.This study concluded that, with increasing of HA suspension or HA toothpaste suspension concentration, the adsorption capacity of copper, cadmium ion gradually declined. Through linear approximation on adsorption capacity multiples and concentration reciprocal multiples of HA suspension and dentifrice suspension containing HA, we first discovered a good linear correlation between the adsorption capacity multiple and the concentration reciprocal multiple of HA and dentifrice suspension. Therefore the concentration of HA, as adsorbents for the adsorption of heavy metal ions in domestic wastewater, is not the bigger the better. And we hypothesized that increase of the concentration of HA, may affect the surface binding sites.Conclusions<1> Citric acid solution is firstly used to etch the enamel. The surface morphology after demineralization is observed at different time by SEM. And the results shows that 10% citric acid solution etching enamel with 120 s, is the best etching conditions to obtain reliable early enamel caries model.(2) n-HA has better remineralization of early enamel caries, and can significantly improve the microhardness of artificial enamel caries. The effect of 20 nm HA is the most prominent.(3) HA and dentifrice containing HA on cadmium ions adsorption have strong effects. After mixed with dentifrice the adsorption rate decreases. HA and dentifrice containing HA on copper ions have a strong adsorption, and the adsorption rate increases significantly after mixed with toothpaste. Their adsorption capacities are more stable. The adsorption capacities of 20 nm HA and dentifrice containing 20 nm HA are the best.We first discovered that adsorption capacity multiples on copper, cadmium ion and concentration reciprocal multiples of HA and dentifrice suspension had a good linear correlation. |
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