阅读说明：本技术 一种牙齿矿化液及其矿化方法 (Tooth mineralizing liquid and mineralizing method thereof ) 是由 傅柏平 周子淮 吴志芳 张雷青 赵维家 于 2019-11-05 设计创作，主要内容包括：本发明公开了一种牙齿矿化液及其矿化方法,牙齿矿化液可用于矿化胶原和牙齿。本发明的矿化液成分是分开放置的两部分,即包括含非胶原蛋白类似物与钙盐的复合物的试剂A、含有磷酸盐的试剂B。本发明的牙齿矿化液的制备时需要调节试剂A和试剂B的pH值为5-12。本发明的牙齿矿化液矿化方法为先用试剂A在牙齿表面涂抹静置,再用试剂B在牙齿表面涂抹静置后,根据需要可重复涂布数次,以实现牙齿矿化。此外,该发明的矿化液也可用相同的方法实现胶原的仿生矿化,包括单层重组胶原纤维、胶原凝胶和胶原海绵,浸泡或漂浮于试剂A中,然后取出用滤纸吸干,再浸泡或漂浮于试剂B中,实现仿生矿化,同样也可重复数次。(The invention discloses a tooth mineralization liquid and a mineralization method thereof. The mineralizing liquid component of the invention is divided into two parts, namely an agent A containing a non-collagen analogue and calcium salt complex and an agent B containing phosphate. The preparation of the tooth mineralization liquid of the invention requires adjusting the pH values of the reagent A and the reagent B to 5-12. The tooth mineralization liquid mineralization method comprises the steps of firstly coating and standing the reagent A on the surface of the tooth, then coating and standing the reagent B on the surface of the tooth, and then repeatedly coating for several times according to needs to realize tooth mineralization. In addition, the mineralization liquid can also realize the biomimetic mineralization of the collagen by the same method, comprises a single-layer recombinant collagen fiber, a collagen gel and a collagen sponge, and is soaked or floated in the reagent A, then taken out and sucked dry by filter paper, and then soaked or floated in the reagent B to realize the biomimetic mineralization, and the biomimetic mineralization can also be repeated for a plurality of times.)

1. A tooth mineralization liquid is characterized by comprising a reagent A and a reagent B;

the reagent A is a solution containing non-collagen analogues and calcium salt; the non-collagen analogue is one or more of polyelectrolytes such as polyaspartic acid, polyacrylic acid, polyvinyl phosphonic acid, polyglutamic acid, carboxymethyl chitosan, sodium trimetaphosphate and sodium tripolyphosphate; the calcium salt is one or more of calcium chloride and/or hydrate thereof, calcium fluoride and/or hydrate thereof, calcium carbonate and/or hydrate thereof, calcium nitrate and/or hydrate thereof, and calcium acetate and/or hydrate thereof;

the reagent B is phosphate solution; the phosphate is one or more of trisodium phosphate, tripotassium phosphate, disodium hydrogen phosphate, dipotassium hydrogen phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, triammonium phosphate, ammonium dihydrogen phosphate and diammonium hydrogen phosphate;

the volume ratio of the used dosage of the reagent A to the reagent B is 1 (0.1-10), and the molar ratio of the calcium ion concentration in the reagent A to the total concentration of the phosphate radical, the monohydrogen phosphate radical and the dihydrogen phosphate radical in the reagent B is (0.1-10): 1; reagent a and reagent B must be stored separately.

2. The tooth-mineralizing solution according to claim 1, wherein the amount of the non-collagen analog contained in the agent A is 0.001 to 20g/L, and the concentration of the calcium ion is 0.001 to 10 mol/L.

3. The tooth-mineralizing solution according to claim 1, wherein the total concentration of phosphate, monohydrogen phosphate and dihydrogen phosphate in the phosphate solution in the reagent B is 0.001-10 mol/L.

4. A dental mineralization fluid according to claim 1, preferably a mineralization fluid characterized by: in the reagent A, the content of non-collagen analogues is 3-10g/L, and the concentration of calcium ions is 5.2-10 mol/L; in the reagent B, the total concentration of phosphate radical, phosphoric acid monobasic radical and phosphoric acid dibasic radical in phosphate solution is 5-10 mol/L.

5. The dental mineralization fluid of claim 1, wherein the non-collagen analogs comprise polyaspartic acid, polyacrylic acid, polyvinylphosphonic acid, sodium trimetaphosphate, and sodium tripolyphosphate.

6. A method of preparing the tooth mineralization fluid of claim 1, comprising the steps of:

dissolving calcium salt and non-collagen analogues in deionized water, and adjusting the pH value to 5-12 to obtain a reagent A; and (3) dissolving phosphate in deionized water, and adjusting the pH value to 5-12 to obtain a reagent B.

7. A method of mineralizing teeth based on the tooth mineralizing fluid of claim 1, comprising the steps of:

1) smearing the reagent A on the surface of the tooth for 3s-30min, and then standing for 1-60 min; then smearing the reagent B on the tooth surface for 3s-30min, and standing for 1-60 min;

2) repeating the step 1) for a plurality of times or not repeating the step 1), and then putting the teeth into artificial saliva or deionized water for 1 day to realize tooth mineralization, wherein the pH value of the artificial saliva or the deionized water is 5-9.

8. A method of mineralizing collagen based on the tooth mineralizing fluid of claim 1, wherein:

1) soaking or floating the single-layer recombinant collagen fiber, collagen gel or collagen sponge in the reagent A for 3s-30min, taking out, sucking with filter paper, and soaking or floating in the reagent B for 3s-30 min;

2) repeating step 1) several times or not repeating step 1); then placing the collagen in artificial saliva or deionized water at 37 ℃, standing for 1min-24h to realize biomimetic mineralization and generate mineralized collagen.

9. Use of the dental mineralization fluid of claim 1, for the preparation of a dental care product comprising: oral care products for mineralization of teeth, prevention of tooth decay, and dental and periodontal care.

10. The mineralized hydrogel prepared from the tooth mineralized liquid according to claim 1, wherein the mineralized hydrogel is prepared by mixing a reagent A, a reagent B, deionized water and a gelling agent, and the mineralized hydrogel can be used for 3D printing to prepare bone powder, bone cement or dental restoration.

Technical Field

The invention belongs to the field of biological materials, and particularly relates to a tooth mineralization liquid and a method for applying the tooth mineralization liquid to tooth and collagen mineralization.

Background

It is well known that the structure of a tooth comprises an inner dentin layer and an outer enamel shell. Dentin is composed of a multi-stage, ordered arrangement of mineralized collagen. Hydroxyapatite is orderly deposited on the surface and the inner gap area of the collagen template. The minerals within these fibers are particularly important for the mechanical and biological properties of teeth and bone. The complexity of the structure, however, has made dentin mineralization a difficult problem for biomimetic mineralization. Dentin tubules are also present in dentin, communicating with the pulp. If the loss of enamel or cementum and demineralization of dentin are caused by factors such as erosion, abrasion or gingival recession, dentin tubules are exposed, and the sensitive aching pain of dentin is caused by cold or mechanical stimulation.

Methods that have been used to date for tooth mineralization or dentinal tubule occlusion include fluoride, bioactive glass, extremely fast-acting desensitizers, adhesives, and the like. But its tooth mineralization and dentin tubule blocking effects are unstable.

The existing mainstream dentin remineralization mode is that demineralized dentin is soaked in Amorphous Calcium Phosphate (ACP) mineralized liquid with stable collagen analogues, and the ACP is converted into hydroxyapatite after entering collagen, so that intrafibrous mineralization is realized. However, ACP has a low saturation level, a slow calcium and phosphorus supply rate, a long mineralization time and an unstable effect. The bioactive glass can also be used for mineralizing teeth, but the bioactive glass can release calcium and phosphorus ions in a large amount at the same time, and is not stable without non-collagen analogues, and the tooth mineralizing effect is also unstable. The existing tooth mineralization products such as mouthwash, tooth protecting elements, toothpaste and the like can not provide a source of calcium and phosphorus for a long time due to short contact time with teeth.

Patent CN105267046 also mentions a method for rapid mineralization of dentin, which requires that demineralized dentin is immersed in a mineralizing solution for 2 days by synthesizing a non-collagen analogue stabilized amorphous calcium phosphate solution. Calcium phosphate precipitates rapidly as calcium phosphate ion concentration increases, due to the solubility saturation of calcium phosphate. In the existing amorphous calcium phosphate mineralizing liquid for mineralizing, the ion of calcium is usually not more than 50mmol, so the speed of providing the calcium phosphate required by mineralizing is still slow.

And the prepared calcium-phosphorus mixed mineralized liquid or amorphous calcium phosphate is easy to change phase, forms hydroxyapatite and other crystals in the solution, and cannot be effectively stored for a long time.

It is an object of at least one aspect of the present invention to solve or eliminate at least one or more of the aforementioned problems.

It is an object of at least one aspect of the present invention to provide a tooth mineralization fluid that can be stored for extended periods of time.

It is an object of at least one aspect of the present invention to provide a method of using such a tooth mineralization fluid for tooth and collagen mineralization.

Disclosure of Invention

The invention aims to solve the problems of long treatment time, unstable mineralization effect, continuous supply of calcium and phosphorus sources, difficult long-term storage of mineralized liquid and the like in tooth and collagen mineralization technologies, and provides a tooth mineralized liquid and a method for applying the same to tooth and collagen mineralization.

The invention provides a tooth mineralization liquid, which comprises a reagent A and a reagent B;

the agent A comprises a non-collagen analogue and a calcium salt solution; the non-collagen analogue is one or more of polyelectrolytes such as polyaspartic acid, polyacrylic acid, polyvinyl phosphonic acid, polyglutamic acid, carboxymethyl chitosan, sodium trimetaphosphate and sodium tripolyphosphate; the average molecular weight is about 300-100000. Preferably, the non-collagen analog has an average molecular weight of about 300-40000. The calcium salt is one or more of calcium chloride and/or hydrate thereof, calcium fluoride and/or hydrate thereof, calcium carbonate and/or hydrate thereof, calcium nitrate and/or hydrate thereof, and calcium acetate and/or hydrate thereof;

the reagent B is phosphate solution; the phosphate is one or more of trisodium phosphate, tripotassium phosphate, disodium hydrogen phosphate, dipotassium hydrogen phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, triammonium phosphate, ammonium dihydrogen phosphate and diammonium hydrogen phosphate; preferably, potassium phosphate is used, and potassium ions are also provided, so that nerve depolarization of exposed dentinal tubules is more effectively realized.

In the tooth mineralization liquid, a proper amount of fluoride can be added into the agent A and/or the agent B to improve the caries prevention performance of the material, such as one or any combination of sodium fluoride, potassium fluoride, calcium fluoride, zinc fluoride, stannous fluoride, zinc ammonium fluoride, laurylamine hydrofluoride and diethylaminoethyl hydrofluoride caprylamide, and the addition amount accounts for 0.1-10% of the mass fraction of the tooth mineralization liquid.

The tooth mineralization liquid can also be added with a proper amount of sweetener, such as one or any combination of saccharin, cyclamate, sucrose, glucose, acesulfame potassium and the like, wherein the adding amount accounts for 0.1-10% of the mass fraction of the tooth mineralization liquid.

The tooth mineralization liquid can be added with a proper amount of humectant, such as one or any mixture of polyethylene glycol (with different molecular weights), propylene glycol, glycerol (glycerin) erythritol, xylitol, sorbitol, mannitol and lactitol, wherein the addition amount accounts for 0.1-10% of the mass fraction of the tooth mineralization liquid.

The tooth mineralization liquid can also be added with a proper amount of antibacterial agent to increase the antibacterial and bactericidal performance of materials, such as one or any mixture of benzoic acid, sodium benzoate, potassium benzoate, boric acid, phenolic compounds such as beta naphthol, chlorothymol, thymol, anethole, eucalyptol, carvacrol, menthol, phenol, amylphenol, hexylphenol, heptylphenol, octylphenol, hexylresorcinol, lauryl pyridinium chloride, myristylpyridinium chloride, cetylpyridinium fluoride, cetylpyridinium chloride and cetylpyridinium bromide, wherein the addition amount accounts for 0.1-10% of the mass fraction of the tooth mineralization liquid.

The tooth mineralization liquid can also be added with a proper amount of collagen crosslinking agent to help stabilize the collagen fiber net, such as one or any mixture of carbodiimide such as 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, D-ribose and cyclodextrin polyaldehyde, wherein the addition amount accounts for 0.1-5% of the mass fraction of the tooth mineralization liquid.

The volume ratio of the used dosage of the reagent A and the reagent B is (1:0.1) to 10, the molar ratio of the calcium ion concentration in the reagent A to the total concentration of the phosphate radical, the monohydrogen phosphate radical and the dihydrogen phosphate radical in the reagent B is (1-10): 1; reagent a and reagent B were stored separately.

The content of the non-collagen analogues in the reagent A is 0.001-20g/L, and the concentration of calcium ions is 0.001-10 mol/L.

In the reagent B, the total concentration of phosphate radical, phosphoric acid monobasic radical and phosphoric acid dibasic radical in phosphate solution is 0.001-10 mol/L.

As a preferable scheme of the invention, the content of the non-collagen analogue in the reagent A is 5-10g/L, and the calcium ion concentration is 5.2-10 mol/L; in the reagent B, the total concentration of phosphate radical, phosphoric acid monobasic radical and phosphoric acid dibasic radical in phosphate solution is 5-10 mol/L.

The tooth mineralized liquid system A liquid of the invention must firstly react with collagen, and form polyelectrolyte calcium compound in the presence of trace polyelectrolyte, so as to quickly convey calcium ions to firstly enter the collagen and then attract phosphate ions to enter the collagen. This is different from the use of synthetic amorphous calcium phosphate because the need to stabilize amorphous calcium phosphate at high concentrations requires the addition of large amounts of non-collagenous leucorrhea analogs. According to the published patent document (CN1488574), since ACP is prepared by mixing it with a phosphorus solution at a maximum calcium concentration of 5M, the actual final calcium concentration is less than 5M, and the effect of mineralizing collagen fibers is unstable. This is an essential difference from the present invention, which uses a polyelectrolyte-calcium complex-guided calcium-first-then-phosphorus biomimetic mineralization pattern. The existing mineralization mode is to adopt amorphous calcium phosphate stabilized by non-collagen analogues as a mineralization precursor to induce biomimetic mineralization.

In a preferred embodiment of the present invention, the non-collagen analogs include sodium trimetaphosphate and sodium tripolyphosphate. Generally in the field of mineralization, the purpose of using oligomers of small molecules such as sodium trimetaphosphate and sodium tripolyphosphate is to dephosphorylate the modified collagen first and then mineralize the collagen with a solution of amorphous calcium phosphate stabilized with a high polymer. These oligomers have not been used to date to stabilize amorphous calcium phosphate. According to the invention, a compound is formed by the micromolecule oligomer and calcium, and a good mineralization effect can be obtained without adding other polyelectrolytes.

The invention also discloses a preparation method of the tooth mineralization liquid, which comprises the following steps:

dissolving calcium salt and non-collagen analogues in deionized water, and adjusting the pH value to 5-12 to obtain a reagent A; and (3) dissolving phosphate in deionized water, and adjusting the pH value to 5-12 to obtain a reagent B.

The invention also discloses a tooth mineralization method of the tooth mineralization liquid, which comprises the following steps:

1) smearing the reagent A on the surface of the tooth for 3s-30min, and then standing for 1-60 min; then smearing the reagent B on the tooth surface for 3s-30min, and standing for 1-60 min;

2) repeating the step 1) for a plurality of times or not repeating the step 1), and then putting the teeth into artificial saliva or deionized water for 1 day to realize tooth mineralization, wherein the pH value of the artificial saliva or the deionized water is 5-9.

Further preferably, the surface application time of the two reagents is about 5 to 10min, and the standing time of the two reagents is about 3 to 5 min. The dentin tubules can be rapidly sealed by the treatment of the tooth mineralization liquid, and the formed hydroxyapatite can reach 200-.

The invention also discloses a collagen mineralization method of the tooth mineralization liquid, which comprises the following steps: 1) soaking or floating the single-layer recombinant collagen fiber, collagen gel or collagen sponge in the reagent A for 3s-30min, taking out, sucking with filter paper, and soaking or floating in the reagent B for 3s-30 min; 2) repeating step 1) several times or not repeating step 1); then placing the collagen in artificial saliva or deionized water at 37 ℃, standing for 1min-24h to realize biomimetic mineralization and generate mineralized collagen. Namely, after the collagen fiber is added into the tooth mineralized liquid reagent A, the tooth mineralized liquid reagent A is mixed with the reagent B, and the tooth mineralized liquid reagent A can also be used for preparing mineralized collagen fiber materials. Type I collagen, as the major organic component of dentin, can also be mineralized with tooth mineralization fluid. Wherein the artificial saliva is used to simulate the environment in the oral cavity, and collagen mineralization can be obtained even if the artificial saliva is placed in deionized water.

The invention also discloses application of the tooth mineralization liquid in preparing a tooth protection product, wherein the tooth protection product comprises: medicaments for dentinal tubule occlusion and tooth desensitization, medicaments for preventing or treating tooth decay, and oral care products for dental care and periodontal scaling.

The mineralized hydrogel is prepared by mixing a reagent A, a reagent B, a gelling agent and deionized water, and can be used for preparing bone powder, bone cement or dental restoration bodies through 3D printing. According to the requirement, a curing agent, a foaming agent and polyelectrolyte can be added in the mixing process of the mineralized hydrogel; the gel is one or more of methylcellulose, sodium alginate, sodium carboxymethylcellulose, carboxypropylmethylcellulose, hydroxypropyl methylcellulose, chitosan, poly (propylene glutarate), and polycaprolactone.

Compared with the prior art, the invention has the following beneficial effects:

the tooth mineralization liquid is different from the original calcium phosphate mineralization liquid with low concentration, and the penetration of the tooth mineralization liquid in dentin tubules and collagen fibers can be enhanced by increasing the concentration and separating calcium and phosphorus. By adding non-collagen analogues such as polyaspartic acid and the like into the tooth mineralized liquid reagent A, calcium ions are stabilized, the speed of forming hydroxyapatite crystals when contacting phosphate ions is slowed down, and the penetration of the reagent B is facilitated to reach deep dentinal tubules. In addition, under the condition of high concentration, a large amount of hydroxyapatite can be generated, dentin tubules are tightly sealed, and the hydroxyapatite is close to hydroxyapatite components in natural dentin and has better biocompatibility. Overcomes the defects of shallow sealing depth, easy falling and abrasion, high preparation cost and repeated long-term use in the prior art, effectively isolates the stimulation of the external to dentinal tubules, is beneficial to long-term storage and has wide market prospect. In addition, the rapid treatment of the tooth mineralized liquid can rapidly provide a large amount of calcium and phosphorus ions for demineralized dentin collagen to be used as a mineral source for mineralized dentin collagen, and the demineralized dentin collagen can be mineralized in a full layer manner after being placed in artificial saliva simulating an oral environment for about 1 day, so that the problems that the existing demineralized dentin mineralized liquid is long in time, unstable in mineralized effect and needs to provide a calcium and phosphorus source continuously are solved.

The tooth mineralization liquid of the invention is extremely useful, not only can be used in oral cavity, but also can realize collagen fiber internal mineralization within 1 day by processing for several minutes, the used synthetic raw materials do not contain toxic materials, the biological safety is good, and the invention also has the prospect of synthesizing materials related to bone transplantation.

Drawings

FIG. 1a is a Scanning Electron Microscope (SEM) image of the surface of demineralized dentin; (ii) a

FIG. 1b is a Scanning Electron Microscope (SEM) image of a demineralized dentinal tubule longitudinal section;

FIG. 2 is an SEM photograph of a dentin sample after treatment with the tooth mineralized liquid according to example 1;

wherein, FIG. 2a shows the surface topography of a dentin sample after treatment with a tooth mineralized liquid; FIG. 2b is a profile of a dentin sample after treatment with a tooth mineralized liquid; fig. 2c is an enlarged view of the surface-proximal dentinal tubules of fig. 2 b.

FIG. 3 is a TEM image of the recombinant type I collagen after mineralization treatment in example 1;

wherein, FIG. 3a is a TEM image under a 5000-fold microscope of the mineralized recombinant type I collagen in example 1; FIG. 3b is a TEM image of 10000 times lower than the mineralized recombinant type I collagen in example 1; FIG. 3c is a SAED plot of recombinant type I collagen after mineralization treatment in example 1 b.

Detailed Description

The invention will be further elucidated and described with reference to the drawings and the detailed description. The technical features of the embodiments of the present invention can be combined correspondingly without mutual conflict.