阅读说明：本技术 一种磷酸钙前驱体、制备方法及其应用 (Calcium phosphate precursor, preparation method and application thereof ) 是由 曹颖 周庆丽 李全利 郭梦茜 王政 于 2021-02-24 设计创作，主要内容包括：本发明涉及生物材料技术领域,具体涉及一种磷酸钙前驱体、制备方法及其应用。通过将可溶性淀粉与钙试剂混合后加热糊化,得到含钙的淀粉溶液,将可溶性淀粉与磷试剂混合后加热糊化,得到含磷的淀粉溶液,在搅拌下,将含钙的淀粉溶液缓慢滴加入含磷的淀粉溶液或者将含磷的淀粉溶液缓慢滴加入含钙的淀粉溶液,将得到的混合溶液离心,得到含磷酸钙前驱体的上清液,该制备方法简单方便,经济安全,控制性好,环境友好,合成的磷酸钙前驱体尺寸小,大小均一,分布均匀,可长时间稳定存在,不掺杂其它离子。此外,通过添加α-淀粉酶水解淀粉后,前驱体可作为矿物源释放,并在矿化基质上再生釉质样结构,修复牙齿组织缺损。(The invention relates to the technical field of biological materials, in particular to a calcium phosphate precursor, a preparation method and application thereof. The preparation method is simple, convenient, economical and safe, good in controllability and environment-friendly, and the synthesized calcium phosphate precursor is small in size, uniform in size and distribution, can stably exist for a long time and is not doped with other ions. In addition, by hydrolyzing starch with the addition of alpha-amylase, the precursor can be released as a mineral source and regenerate enamel-like structures on a mineralized matrix, repairing tooth tissue defects.)

1. A preparation method of a calcium phosphate precursor is characterized by comprising the following steps:

s1: mixing soluble starch with a calcium reagent, and heating and pasting to obtain a starch solution containing calcium;

s2: mixing soluble starch with a phosphorus reagent, and heating and pasting to obtain a phosphorus-containing starch solution;

s3: slowly dripping the calcium-containing starch solution into the phosphorus-containing starch solution under stirring or slowly dripping the phosphorus-containing starch solution into the calcium-containing starch solution;

s4: and (4) centrifuging the mixed solution obtained in the step S3 at the rotating speed of 14000rpm to obtain the supernatant containing the calcium phosphate precursor.

2. The method of claim 1, wherein the calcium concentration in the calcium reagent of step S1 is greater than 4.5mmol/L, and the starch concentration is 1 wt% to 7 wt%.

3. The method of claim 1, wherein the phosphorus concentration in the phosphorus reagent of step S2 is greater than 3mmol/L, and the starch concentration is 1 wt% to 7 wt%.

4. The method for producing a calcium phosphate precursor according to claim 1, wherein the mixed solution obtained in step S3 has a calcium-phosphorus ion concentration ratio of 3: 2.

5. The method of preparing a calcium phosphate precursor according to claim 1, wherein the pH of the calcium-phosphorus mixed solution obtained in step S3 is 6.

6. The method for producing a calcium phosphate precursor according to claim 1, wherein the calcium phosphate precursor is present in a liquid state during synthesis and is preserved by a freeze-drying technique.

7. A calcium phosphate precursor obtained by the production method according to any one of claims 1 to 6.

8. The calcium phosphate precursor according to claim 7, wherein the size of the calcium phosphate precursor is 0.1nm to 5 nm.

9. Use of a calcium phosphate precursor according to any one of claims 7 to 8 for the preparation of a remineralizing dental article.

Technical Field

The invention relates to the technical field of biological materials, in particular to a calcium phosphate precursor, a preparation method and application thereof.

Background

Over the billions of years, biological systems have optimized their complex organizational and hierarchical structures through evolution and natural selection. Among them, teeth, bones, shells, and the like have attracted attention from scientists because they exhibit significant strength and toughness far exceeding the sum of their constituent components. Therefore, there are a number of biomimetic strategies to replicate this complex hierarchical structure, promoting the development of regenerative medicine and biomimetic materials.

To replicate the unique complex hierarchical structure, calcium phosphate precursors including pre-nucleation clusters (PNCs) and Amorphous Calcium Phosphate (ACP) nanoparticles are synthesized in vitro to serve as reservoirs of calcium and phosphorus ions, providing a mineral source for biomineralization. However, these PNCs or ACP nanoparticles are very unstable and can spontaneously polymerize and nucleate even within seconds. Thus, additives are commonly used to stabilize PNCs or ACP nanoparticles, where the use of polyelectrolytes to stabilize polymer-induced liquid precursors (pipps) of PNCs has attracted much attention in recent years. However, the content of calcium ions in PILPs is usually limited (typically not more than 5mM) and the conversion of PNCs to HA is hindered by organic templates that are not removable in PILPs. Thus, if there is no effective way to remove the organic template from the precursor to release the PNCs, the mineralization rate may be significantly reduced.

In view of the above, the organic molecule triethylamine is used as a template to stabilize calcium phosphate ion clusters and during application, triethylamine is removed by ethanol volatilization to initiate the formation of calcium phosphate crystals. However, although studies have shown that triethylamine eventually volatilises together with ethanol, the safety of triethylamine remains questionable. More importantly, the stability of the PNCs synthesized by triethylamine is reduced along with the volatilization of the ethanol. The volatilization of ethanol is environment-dependent, and can not accurately regulate the stability of the precursor or promote the nucleation and mineralization of the precursor. Therefore, it is difficult to convert to clinical use.

In view of the above-mentioned drawbacks, the inventors of the present invention have finally obtained the present invention through a long period of research and practice.

Disclosure of Invention

The invention aims to solve the problem that the temperature of a calcium phosphate precursor cannot be accurately adjusted or nucleation and mineralization cannot be promoted in the prior art, so that the calcium phosphate precursor is difficult to apply to clinic, and provides a calcium phosphate precursor, a preparation method and application thereof.

In order to achieve the above object, the present invention provides a method for preparing a calcium phosphate precursor, comprising the steps of:

s1: mixing soluble starch with a calcium reagent, and heating and pasting to obtain a starch solution containing calcium;

s2: mixing soluble starch with a phosphorus reagent, and heating and pasting to obtain a phosphorus-containing starch solution;

s3: slowly dripping the calcium-containing starch solution into the phosphorus-containing starch solution under the condition of vigorous stirring or slowly dripping the phosphorus-containing starch solution into the calcium-containing starch solution;

s4: and (4) centrifuging the mixed solution obtained in the step S3 at the rotating speed of 14000rpm to obtain the supernatant containing the calcium phosphate precursor.

In the step S1, the concentration of calcium in the calcium reagent is more than 4.5mmol/L, and the concentration of starch is 1 wt% -7 wt%.

In the step S2, the concentration of phosphorus in the phosphorus reagent is more than 3mmol/L, and the concentration of starch is 1 wt% -7 wt%.

The concentration ratio of calcium ions to phosphorus ions in the mixed solution obtained in the step S3 is 3: 2. .

The pH of the calcium-phosphorus mixed solution obtained in step S3 is 6.

The calcium phosphate precursor exists in a liquid state during synthesis, and is preserved by a freeze-drying technology.

The invention also discloses a calcium phosphate precursor prepared by the method, and the size of the calcium phosphate precursor is 0.1-5 nm.

The invention also discloses application of the calcium phosphate precursor in preparation of remineralization dental products.

In view of the chemical action that abundant hydroxyl groups in the soluble starch are competitively combined with calcium ions and the local space limitation effect formed after starch gelatinization, the rapid combination of calcium and phosphorus can be inhibited, and then the stable calcium phosphate precursor is synthesized. After the alpha-amylase hydrolyzes the starch template, the local space restriction effect of gelatinized starch disappears, and the precursor is released from the starch template so as to promote the remineralization of the precursor on the mineralized matrix.

Compared with the prior art, the invention has the beneficial effects that: (1) the calcium phosphate precursor prepared by the method is economic and safe, simple to operate, good in controllability, environment-friendly and good in biocompatibility; (2) the prepared calcium phosphate precursor has small size, uniform distribution and high mineralization activity; (3) the soluble starch is used as a template, and can be removed accurately by adding amylase manually or degraded continuously by the amylase rich in saliva, so that the application of the calcium phosphate precursor is facilitated; (4) the calcium phosphate precursor is processed by freeze drying technology, and can be stably stored for a long time.

Drawings

FIG. 1 is a Transmission Electron Microscope (TEM) image of a calcium phosphate precursor prepared in example 1;

FIG. 2 shows the Dynamic Light Scattering (DLS) results of the calcium phosphate precursor prepared in example 1;

FIG. 3 is a TEM image of the calcium phosphate precursor after enzymatic hydrolysis of starch in example 1;

FIG. 4 is the DLS results of the calcium phosphate precursor after enzymatic hydrolysis of starch in example 1;

figure 5 is a Scanning Electron Microscope (SEM) image of the enamel surface of the control group of enamel remineralization in example 1:

FIG. 6 is an SEM image of enamel surfaces of an enamel remineralization experiment group of example 1;

FIG. 7 is an SEM image of an enamel section of a control group of enamel remineralized by enamel in example 1;

FIG. 8 is an SEM photograph of an enamel section of an enamel remineralization experiment group of example 1;

FIG. 9 is a TEM image of the calcium phosphate precursor used in example 2 after enzymatic hydrolysis;

FIG. 10 is a graph of DLS after enzymatic hydrolysis of a calcium phosphate precursor used in example 2;

FIG. 11 is an SEM image of the surfaces of the remineralized dentin control group (left) and the experimental group (right) in example 2;

FIG. 12 is a high magnification SEM image of the surface of the remineralized dentin control group (left) and the experimental group (right) in example 2;

FIG. 13 is an SEM image of a cross section of a remineralized dentin control group (left) and an experimental group (right) in example 2;

FIG. 14 is a high magnification view of a cross section of an experimental group of remineralized dentin in example 2;

FIG. 15 is an inductively coupled plasma optical emission spectrometer (ICP-OES) plot of different starch concentrations for the synthesized calcium phosphate precursor supernatant;

FIG. 16 is a TEM image of the calcium phosphate precursor preserved for 5 months in example 3;

fig. 17 shows DLS results of the calcium phosphate precursor system stored for 5 months in example 3 after enzymolysis and centrifugation.

Detailed Description

The above and further features and advantages of the present invention are described in more detail below with reference to the accompanying drawings.

Example 1

Mixing 2.5 wt% of soluble starch with 9mmol/L calcium oxide suspension, heating and gelatinizing to obtain calcium-containing starch solution; mixing 2.5 wt% of soluble starch with 6mmol/L phosphoric acid solution, and heating for gelatinization to obtain phosphorus-containing starch solution; stirring vigorously at the rotating speed of 500rpm, and slowly dripping the calcium-containing starch solution cooled to the normal temperature into the phosphorus-containing starch solution; and centrifuging the obtained mixed solution to obtain the supernatant containing the calcium phosphate precursor. A small amount of the sample was subjected to TEM and DLS characterization, and the results are shown in FIGS. 1 and 2. Figure 1 shows the synthesis of uniform sized calcium phosphate precursors in a starch template. The calcium concentration in the supernatant was determined to be about 4mmol/L by inductively coupled plasma emission spectroscopy (ICP-OES). After the supernatant is subjected to enzymolysis of starch by alpha-amylase, a small amount of the supernatant is taken for TEM and DLS characterization, and the results are shown in figures 3 and 4, and the supernatant after enzymolysis is directly used as a mineral source for remineralization of enamel. Figure 3 shows that the starch template has disappeared, leaving only a few nanometers of calcium phosphate precursor. Figure 4 shows calcium phosphate precursors around 1 nm.

(1) Preparing enamel samples, polishing, ultrasonically cleaning and acid etching for later use. One part of the enamel pieces was coated with nail polish as a control group, and the other part was used as an experimental group.

(2) And centrifuging to obtain the calcium phosphate precursor after enzymolysis.

(3) The prepared enamel piece is soaked in the calcium phosphate precursor after enzymolysis, 1ppm of fluorine is added, the supernatant of the calcium phosphate precursor is replaced every day, and the enamel piece is soaked for two weeks.

The mineralized enamel pieces were ultrasonically cleaned, dried and examined by Scanning Electron Microscopy (SEM), and the results are shown in fig. 6 and 8. Figure 6 shows the mineralization results which are quite different from the control of figure 5. Figure 6 shows the enamel surface covered with remineralized rod-like crystals, which are uniformly densely packed. The enamel profile of fig. 8 is also different from that of the control of fig. 7. The vertical section of figure 8 shows that the boundary between remineralized crystals and natural enamel is not sharp, indicating that enamel-like crystals are regenerated in situ at the enamel surface.

Example 2

Mixing 2.5 wt% of soluble starch with 36mmol/L calcium oxide suspension, heating and gelatinizing to obtain a calcium-containing starch solution; mixing 2.5 wt% of soluble starch with 24mmol/L phosphoric acid solution, and heating for gelatinization to obtain phosphorus-containing starch solution; stirring vigorously at the rotating speed of 500rpm, and slowly dripping the calcium-containing starch solution cooled to the normal temperature into the phosphorus-containing starch solution; and centrifuging the obtained mixed solution to obtain the supernatant containing the calcium phosphate precursor. A small amount of the enzyme was digested and then subjected to TEM and DLS characterization, and the results are shown in FIGS. 9 and 10. The calcium concentration in the supernatant was determined to be about 7mmol by inductively coupled plasma emission spectroscopy (ICP-OES). Fig. 9 and 10 show that although the calcium phosphate precursor concentration (calcium phosphorus concentration) was increased, the morphology of the calcium phosphate precursor was not significantly different from that of fig. 3 and 4, and the size was still around 1 nm. The supernatant can be directly used as mineral source for remineralization of dentin after enzymolysis.

(1) Preparing a dentin sample, polishing, ultrasonically cleaning, performing acid etching for later use, coating nail polish on one part of a dentin sheet to be used as a control group, and coating nail polish on the other part of the dentin sheet to be used as an experimental group.

(2) And centrifuging to obtain the calcium phosphate precursor after enzymolysis.

(3) Soaking the prepared dentin tablets of the control group and the experimental group in the calcium phosphate precursor after enzymolysis, adding 1ppm of fluorine, replacing the supernatant of the calcium phosphate precursor every day, and soaking for two weeks.

The mineralized enamel pieces were ultrasonically cleaned, dried and examined by Scanning Electron Microscopy (SEM), and the results are shown in fig. 11, 12, 13 and 14. Fig. 11 shows that the dentinal tubules of the control group (left side of the figure) are clearly visible, and the dentinal tubules of the experimental group, i.e., the mineralized group (right side of the figure), are covered with a layer of regenerated crystals. Fig. 12 shows, in enlargement, the dentinal tubules of the mineralized group (right panel) completely covered by rod-shaped crystals. The longitudinal section of fig. 13 shows that the dentinal tubules of the control group (left of the figure) have no crystal regrowth, and the dentinal tubules of the mineralized group are filled with regrown crystals by ten-odd microns. The vertical section enlargement of fig. 14 shows no distinct boundary between the regenerated crystals and the natural dentin, the regenerated crystals being densely regenerated in the dentinal tubules.

Example 3

Effect of starch concentration on the synthesis of calcium phosphate precursor system:

mixing soluble starch with the concentration of 2.5 wt%, 5 wt% and 7 wt% with a calcium oxide suspension of 45mmol/L, heating and gelatinizing to obtain a starch solution containing calcium; mixing soluble starch with the concentration of 2.5 wt%, 5 wt% and 7 wt% with a phosphoric acid solution of 30mmol/L, and heating for gelatinization to obtain a starch solution containing phosphorus; stirring vigorously at the rotating speed of 500rpm, and slowly dripping the calcium-containing starch solution cooled to the normal temperature into the phosphorus-containing starch solution; and centrifuging the obtained mixed solution to obtain a supernatant containing the calcium phosphate precursor. Since the supernatant was obtained, the content of calcium and phosphorus ions in the supernatant was definitely different from the theoretical value of the initial charge, and the content of calcium and phosphorus ions in the supernatant was measured by inductively coupled plasma emission spectrometry (ICP-OES), which substantially reflects the concentration of the calcium phosphate precursor, as shown in fig. 15. The results show that as the starch concentration increases, the calcium and phosphorus concentrations increase accordingly.

Example 4

Mixing 2.5 wt% of soluble starch with 18mmol/L calcium oxide suspension, heating and gelatinizing to obtain a calcium-containing starch solution; mixing 2.5 wt% of soluble starch with 12mmol/L phosphoric acid solution, and heating for gelatinization to obtain phosphorus-containing starch solution; stirring vigorously at the rotating speed of 500rpm, and slowly dripping the calcium-containing starch solution cooled to the normal temperature into the phosphorus-containing starch solution; the obtained mixed solution is converted into solid by freeze drying technology and stored at normal temperature. And after 5 months, redissolving the calcium phosphate precursor solid by using deionized water and performing enzymolysis, and then performing characterization, wherein a TEM (transverse electric field) characterization result of the calcium phosphate precursor is shown in figure 16, and a DLS (digital Living system) result obtained by performing enzymolysis and centrifugation on a calcium phosphate precursor system stored for 5 months is shown in figure 17. Figure 16 shows that the morphology of the calcium phosphate precursor did not change significantly after 5 months of storage of the calcium phosphate in a freeze-drying technique. Fig. 17 shows that the size of the calcium phosphate precursor increases slightly after 5 months of storage of calcium phosphate by freeze-drying technique, but remains several nanometers in size, indicating that the calcium phosphate precursor can be stored for a long time by freeze-drying technique and can be applied by re-dissolution.

Example 5

A dental collutory containing calcium phosphate precursor for cleaning oral cavity and preventing dental caries and acid etching comprises calcium phosphate precursor, essence, surfactant, fluoride, strontium chloride, alcohol and water. Wherein the concentration of the calcium phosphate precursor is 4-18 mmol/L. Because saliva is rich in amylase, the saliva can be used for enzymolysis of starch, and calcium phosphate precursors in the starch are released to promote mineralization.

The using method comprises the following steps: the product is used after brushing teeth in the morning and evening or after meal, 20mL is used each time, and the product is spit after gargling for 30 seconds.

Example 6

A dental toothpaste containing calcium phosphate precursor for cleaning and polishing tooth surface, refreshing oral cavity and remineralizing mainly comprises calcium phosphate precursor, abrasive, humectant, surfactant, fluoride, thickener, sweetener, antiseptic, pigment, essence, etc. Wherein the concentration of the calcium phosphate precursor is 4-18 mmol/L. Because saliva is rich in amylase, the saliva can be used for enzymolysis of starch, and calcium phosphate precursors in the starch are released to promote mineralization.

The using method comprises the following steps: an appropriate amount of toothpaste was taken every morning and evening, and the teeth were cleaned with a toothbrush for 3 minutes.

Example 7

A dental chewing gum containing calcium phosphate precursor component for removing food residue on tooth surface, increasing salivary secretion and remineralizing comprises calcium phosphate precursor, colloid, syrup, herba Menthae, sweetener, etc. Wherein the concentration of the calcium phosphate precursor is 4-18 mmol/L. Because saliva is rich in amylase, the saliva can be used for enzymolysis of starch, and calcium phosphate precursors in the starch are released to promote mineralization.

The using method comprises the following steps: the chewing gum is chewed for 5 minutes after meals or when needed and then spit out.

Example 8

A dental spray containing calcium phosphate precursor for removing oral odor, refreshing oral cavity and remineralizing comprises calcium phosphate precursor, water, glycerol, essence, menthol, thymol, etc. Wherein the concentration of the calcium phosphate precursor is 4-18 mmol/L. Because saliva is rich in amylase, the saliva can be used for enzymolysis of starch, and calcium phosphate precursors in the starch are released to promote mineralization.

The using method comprises the following steps: the spray is pressed 2-3 times after meals or when needed to be directed to the oral cavity.

The foregoing is merely a preferred embodiment of the invention, which is intended to be illustrative and not limiting. It will be understood by those skilled in the art that various changes, modifications and equivalents may be made therein without departing from the spirit and scope of the invention as defined in the appended claims.