CN114668891B

CN114668891B - Phosphate-mediated apatite self-assembly method and application thereof

Info

Publication number: CN114668891B
Application number: CN202111447825.4A
Authority: CN
Inventors: 杜田明; 杨海胜; 乔爱科; 刘有军
Original assignee: Beijing University of Technology
Current assignee: Beijing University of Technology
Priority date: 2021-11-30
Filing date: 2021-11-30
Publication date: 2022-10-25
Anticipated expiration: 2041-11-30
Also published as: CN114668891A

Abstract

The invention discloses a phosphate-mediated apatite self-assembly method and application thereof, and the preparation method comprises the following steps: s1, preparing a polyelectrolyte compound solution, a calcium ion solution, a orthophosphate ion solution, a pyrophosphate ion solution and an alkaline phosphatase solution with proper concentrations; s2, sequentially adding a calcium ion solution, a polyelectrolyte compound solution, a pyrophosphate ion solution, a orthophosphate ion solution and an alkaline phosphatase solution into the type I collagen solution; s3, after the solutions are sequentially added, adjusting the pH value to 8-9, then adding deionized water until the volume of the deionized water is 3-5 times of that of the type I collagen solution, and standing for reaction for 25-35min to obtain a mineralized bone matrix solution; s4, adjusting the pH value of the mineralized bone matrix solution to 7-8, and standing for 24-32 hours to obtain a primary product; s5, removing impurities from the primary product, concentrating to 10% -20% of the original volume to obtain bone matrix mineralized liquid, freezing and forming the bone matrix mineralized liquid at the temperature of below 0 ℃ for 12-24h, taking out the bone matrix mineralized liquid, and drying to obtain the mineralized bone matrix material with the multi-layer flower-like apatite space mineralized structure.

Description

Phosphate-mediated apatite self-assembly method and application thereof

Technical Field

The invention relates to the field of biomedical materials, in particular to a phosphate-mediated apatite self-assembly method and application thereof.

Background

Apatite is the main inorganic phase that makes up bone tissue and constitutes the bone matrix, and is the main source of bone tissue to achieve mechanical strength. Recent studies have shown that apatite has different structural forms in the bone tissue, including filamentous patterns, laced patterns, and nested rosette patterns, on a nanometer scale. On a macroscopic scale, the structure is mainly a lamellar structure. In fact, apatite exists in a multi-scale multi-stage mineralized structure in bone tissues, and apatite exists in different structural forms on different scales, so that the bone can adapt to a complex and changeable external mechanical environment.

However, at present, in the in vitro biomimetic mineralization research, there is a few techniques that can prepare apatite with different pattern structures.

Disclosure of Invention

To this end, embodiments of the present invention provide a phosphate-mediated apatite self-assembly method and an application thereof to solve the above-mentioned problems in the prior art.

In order to achieve the above object, the embodiments of the present invention provide the following technical solutions:

in a first aspect, embodiments of the present invention provide a phosphate-mediated apatite self-assembly method, which includes the following steps:

s1, preparing 0.5-1.5mg/mL polyelectrolyte compound solution, 0.05-0.15M calcium ion solution, 0.05-0.15M orthophosphate ion solution, 2.5-3.5wt% pyrophosphate ion solution and 5-10mg/L alkaline phosphatase solution;

s2, adding 3-5mg/mL of type I collagen solution into a centrifuge tube, and then sequentially adding the calcium ion solution, polyelectrolyte compound solution, pyrophosphate ion solution, orthophosphate ion solution and alkaline phosphatase solution which are prepared in the S1 into the type I collagen solution to form a reaction system, wherein the addition amount of each solution is calculated by the final concentration of each component in the reaction system, the final concentration of the type I collagen is 0.5-2mg/mL, the final concentration of the calcium ion is 10-20mM, the final concentration of the polyelectrolyte compound is 30-100 mu g/mL, the final concentration of the pyrophosphate ion is 0.5-2wt%, the final concentration of the orthophosphate ion is 5-10mM, and the final concentration of the alkaline phosphatase is 30-100 mu g/mL;

s3, after the solutions are sequentially added, adjusting the pH value of the reaction system to 8-9, then adding deionized water into the reaction system until the volume of the deionized water is 3-5 times of that of the type I collagen solution, and then standing for reaction for 25-35min to obtain a mineralized bone matrix solution;

s4, adjusting the pH of the mineralized bone matrix solution to 7-8 by using 1-5wt% acetic acid solution, and then continuously standing for reaction for 24-32h to obtain a primary product;

s5, removing impurities from the primary product, concentrating the primary product after impurity removal to 10% -20% of the original volume according to the total reaction volume to obtain bone matrix mineralized liquid, placing the bone matrix mineralized liquid in a constant temperature environment below 0 ℃ for freezing and forming, taking out after 12-24h, and freeze-drying to obtain the mineralized bone matrix material.

Preferably, in S1, the polyelectrolyte compound solution is one of a polyacrylic acid solution and a polyacrylamide solution.

Preferably, in S1, the calcium ion solution is specifically a water-soluble calcium salt solution, and more specifically is one of a calcium chloride solution and a calcium nitrate solution or a mixture thereof.

Preferably, in S1, the orthophosphate ion solution is one of diammonium hydrogen phosphate solution and ammonium dihydrogen phosphate solution or a mixture thereof.

Preferably, in S1, the pyrophosphate ion solution is one of a sodium pyrophosphate solution and a potassium pyrophosphate solution, or a mixture thereof.

Preferably, in S3, the regulator for regulating the pH value of the reaction system is specifically: an alkali solution having a concentration of 1M, an alkali solution having a concentration of 0.1M, and aqueous ammonia having a concentration of 0.1M.

Further, the alkali solution is one of NaOH solution and KOH solution or a mixture of the NaOH solution and the KOH solution.

Preferably, in S5, the specific process of removing impurities from the primary product includes:

a, adding deionized water into the primary product, and cleaning;

procedure B, centrifuging and adding deionized water to obtain a primary product;

c, after centrifugation, filtering out liquid, and adding water again until the volume of the liquid is 2-3 times of that of the mineralized bone matrix solution;

and the process D and the process B-C are repeated for 3 times to obtain an initial product after impurity removal.

In a second aspect, embodiments of the present invention provide a mineralized bone matrix material having a flower-like and multi-layer flower-like apatite spatial mineralization structure obtained by the above method.

In a third aspect, the embodiments of the present invention provide the application of the above method in the preparation of an implantable medical device for bone defect repair.

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

(1) According to the phosphate-mediated apatite self-assembly method provided by the embodiment of the invention, the mineralized apatite, namely the mineralized bone matrix material with different space assembly structures and mineralization degrees is prepared on the surface of the mineralized bone matrix by utilizing apatite self-assembly on a micrometer scale, and the assembly structures are mainly a lamellar structure, a flower-like structure and a multilayer flower-like structure which are consistent with those in a body, so that a powerful technical support is provided for realizing the preparation of the multi-scale high-degree bionic bone material.

(2) Compared with the traditional apatite mineralization method, the phosphate-mediated apatite self-assembly method provided by the embodiment of the invention takes the influences of ion sources, chemical reaction balance and chemical factor addition sequence in mineralization into consideration more fully, constructs a multi-factor cooperative regulation and control system, and better simulates the complex bone matrix mineralization assembly process in vivo; under the action of alkaline phosphatase, pyrophosphate is subjected to enzymolysis to generate a large amount of orthophosphate, which is favorable for the formation of apatite. Orthophosphate is added as a regulating factor at the initial stage of mineralization, so that the enzymolysis speed of pyrophosphate can be effectively controlled, formed apatite is guided to aggregate to form a lamellar structure, the apatite with the lamellar structure is further assembled to form a flower-like and multi-layer flower-like apatite space mineralization structure, and the blank in the technical field at present is filled.

(3) According to the phosphate-mediated apatite self-assembly method provided by the embodiment of the invention, by controlling the addition and the addition sequence of the regulatory factors in the bone matrix, alkaline phosphatase and hydrolyzed pyrophosphate are utilized under the alkalescent condition, meanwhile, the polyelectrolyte compound is utilized to regulate and control the size of nano apatite particles, orthophosphate regulates and controls the hydrolysis of the alkaline phosphatase and the self-assembly mechanism of apatite in a collagen/calcium phosphate biomimetic mineralization system, and therefore apatite space mineralization structures with different shapes can be manufactured.

(4) According to the phosphate-mediated apatite self-assembly method provided by the embodiment of the invention, the pH value is adjusted by adding ammonia water, the ammonia water can form a buffer system, the pH value of the reaction system can be automatically adjusted in the reaction process, the operation is simpler and more convenient, and the reaction is more stable and controllable.

(5) The mineralized bone matrix material provided by the embodiment of the invention has a flower-like and multi-layer flower-like apatite space mineralized structure, and meanwhile, has good biocompatibility, also has mechanical characteristics matched with a bone matrix in human bone tissues, and has a wide application space.

Drawings

In order to more clearly illustrate the technical solution of the present invention, the drawings used in the description of the embodiments of the present invention will be briefly introduced below. It should be apparent that the drawings in the following description are merely exemplary, and that other drawings may be derived from the provided drawings by those of ordinary skill in the art without inventive effort.

The drawings are only for purposes of illustration and are not intended to limit the scope of the invention, which is defined by the claims, which will be obvious to those skilled in the art, and the appended drawings are intended to cover all such modifications, enhancements, and other embodiments which fall within the true scope of the invention.

FIG. 1 is a schematic flow chart showing the operation of embodiment 1 of the present invention;

FIG. 2 is a graph showing the reaction profile of orthophosphate formed by enzymatic hydrolysis of pyrophosphate with alkaline phosphatase under the control of orthophosphate in example 1, example 3 and comparative example 1 of the present invention; wherein, FIG. 2-A is a graph showing the effect of the amount of alkaline phosphatase on the concentration of orthophosphate ions at different amounts of orthophosphate added, and FIG. 2-B is a graph showing the effect of the amount of alkaline phosphatase on the concentration of orthophosphate ions at different amounts of orthophosphate added;

FIG. 3 is SEM images of example 1, comparative example 1 and comparative example 2; wherein FIG. 3-A1 is an SEM image of comparative example 2, and FIG. 3-A2 is a magnified image of the topography of the boxed area in FIG. 3-A1; FIG. 3-B1 is an SEM image of comparative example 1, and FIG. 3-B2 is a magnified image of the features of the boxed area in FIG. 3-B1; FIG. 3-C1 is an SEM image of example 1, and FIG. 3-C2 is a magnified image of the topography of the boxed area of FIG. 3-C1.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

In the description of the present invention, "a plurality" means two or more unless otherwise specified. The terms "first," "second," "third," "fourth," and the like in the description and in the claims of the present invention are intended to distinguish between the referenced items. For a scheme with a time sequence flow, the term expression does not need to be understood as describing a specific sequence or a sequence order, and for a scheme of a device structure, the term expression does not have distinction of importance degree, position relation and the like.

Furthermore, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements specifically listed, but may include other steps or elements not expressly listed that are inherent to such process, method, article, or apparatus or that are added to such process, method, article, or apparatus based on the optimization concepts of the present invention.

Example 1

This example provides a phosphate-mediated apatite self-assembly process by which mineralized bone matrix materials having flower-like and multi-layered flower-like apatite spatial mineralization structures can be prepared. The operation flow of this embodiment is shown in fig. 1, and the specific steps and related parameters are as follows:

step 1, the purchased finished polyacrylic acid (i.e. polyelectrolyte compound in fig. 1), calcium chloride (i.e. calcium ions in fig. 1), diammonium hydrogen phosphate (i.e. orthophosphate in fig. 1) and sodium pyrophosphate (i.e. pyrophosphate in fig. 1), reagents for alkaline phosphatase were first prepared as a reaction stock solution using sterile deionized water. The concentrations of the reaction stock solutions are respectively 1mg/mL of polyacrylic acid, 0.1M of calcium chloride, 0.1M of diammonium hydrogen phosphate and 3 percent of sodium pyrophosphate by mass. Additional pH adjustment requires the use of sodium hydroxide, ammonia and acetic acid. Two concentrations of NaOH solution were prepared for coarse and fine adjustment of pH, 1M and 0.1M respectively. 0.1M of ammonia water, wherein the ammonia water is added to form a buffer system, so that the pH value of the reaction system can be automatically adjusted in the reaction process. The concentration of acetic acid is 2% by mass. Rat tail type I collagen purchased from BD biocoat was the collagen reaction stock (i.e., type I collagen solution in FIG. 1) at a concentration range of 3-5mg/mL. The total activity value of the purchased alkaline phosphatase is more than 10KU, namely more than 10mg, and is prepared into stock solution of 10mg/mL according to the minimum value of 10 mg.

In this step:

the polyacrylic acid used may be replaced with other polyelectrolyte compounds, such as polyacrylamide, etc.;

the calcium chloride can be replaced by other water-soluble calcium salts capable of providing calcium ions, such as calcium nitrate;

the diammonium hydrogen phosphate used can be replaced by other water-soluble phosphates which can provide orthophosphate ions, such as ammonium dihydrogen phosphate and the like;

the sodium pyrophosphate used may be replaced by other water-soluble pyrophosphate salts capable of providing pyrophosphate ions, such as potassium pyrophosphate and the like;

the rat tail type I collagen solution used can be replaced by other type I collagen solutions which do not react with substances in the reaction system and have negative effects on the final effect.

And 2, carrying out mineralization reaction in a centrifugal tube. The total amount of the solution in the reaction system was 5mL. In the reaction system, the components in the reaction system were added in the order of addition shown in FIG. 1. And calculating the addition amount of the stock solution required by each component according to the final concentration of each component in the reaction system. Wherein the final concentration of collagen is 1mg/mL, the final concentration of calcium ions is 10mM, the final concentration of polyacrylic acid is 50 μ g/mL, the final concentration of pyrophosphate ions is 1 mass percent, the final concentration of orthophosphate ions is 5mM, and the final concentration of alkaline phosphatase is 60 μ g/mL.

And 3, after adding all reaction substances according to the sequence 1-5 shown in the figure 1, adjusting the pH value of the reaction system to 8-9 by using sodium hydroxide and ammonia water, supplementing sterile deionized water to enable the reaction system to reach 5mL, and maintaining the reaction for 30 min.

In this step:

the sodium hydroxide can be replaced by other alkali which can be completely ionized and can not react with other substances in the reaction system to cause negative influence on the reaction effect, such as KOH and the like;

the ammonia water can be replaced by other alkali liquor which is not completely ionized and can not react with other substances in the reaction system to cause negative influence on the reaction effect.

And 4, after 30 minutes, adjusting the pH value of the reaction system to 7.4 by using acetic acid with the mass fraction of 2%, and reacting for 24 hours to obtain a primary product.

Step 5, flow A: after the reaction is finished, adding sterile deionized water into the primary product, and cleaning; and (B) a process: centrifuging and adding sterile deionized water to obtain a primary product; and (C) a process C: after centrifugation, filtering out liquid, and adding 500 mu L of sterile deionized water again; and (D) a process: repeating the process B-C for 3 times to obtain an initial product after impurity removal; procedure E: repeating for 3 times, removing impurities to 15% of the initial volume (namely the volume of the initial product obtained in the step 4) to obtain a high-concentration bone matrix mineralized liquid, placing the bone matrix mineralized liquid in a refrigerator at-20 ℃ for freezing and forming, transferring the bone matrix mineralized liquid into a vacuum freeze dryer after 12h, and performing freeze drying to obtain the mineralized bone matrix material.

Example 2

This example provides a phosphate-mediated apatite self-assembly process by which mineralized bone matrix materials having flower-like and multi-layered flower-like apatite spatial mineralization structures can be prepared. The operation flow and the specific steps of the embodiment are basically the same as those of the embodiment 1, and the differences are as follows:

in the step 1, the solution of the polyelectrolyte compound is 0.5mg/L polyacrylic acid solution; the calcium ion solution is 0.05M calcium nitrate solution; the pyrophosphate ion solution used was 2.5wt% potassium pyrophosphate solution; the orthophosphate solution used was 0.05M ammonium dihydrogen phosphate solution; the concentration of the alkaline phosphatase solution is 8mg/L;

in the step 2, the final concentration of collagen is 0.5mg/mL, the final concentration of calcium ions is 15mM, the final concentration of polyacrylic acid is 30 mu g/mL, the final concentration of pyrophosphate ions is 0.5wt%, the final concentration of orthophosphate ions is 7mM, and the final concentration of alkaline phosphatase is 30 mu g/mL;

in the step 3, the reaction time is 25min;

in step 4, adjusting the pH value to 7; the reaction time is 28h;

and 5, placing the mixture into a refrigerator at the temperature of-10 ℃ for freezing and molding, transferring the mixture into a vacuum freeze dryer after 18 hours, and performing freeze drying to obtain the mineralized bone matrix material.

Example 3

in the step 1, the solution of the polyelectrolyte compound is 1.5mg/L polyacrylamide solution; the calcium ion solution is 0.15M calcium nitrate solution; the pyrophosphate ion solution used was 3.5wt% potassium pyrophosphate solution; the orthophosphate solution used was 0.15M ammonium dihydrogen phosphate solution; the concentration of the alkaline phosphatase solution is 5mg/L;

in the step 2, the final concentration of collagen is 2mg/mL, the final concentration of calcium ions is 20mM, the final concentration of polyacrylamide is 100 mu g/mL, the final concentration of pyrophosphate ions is 2wt%, the final concentration of orthophosphate ions is 10mM, and the final concentration of alkaline phosphatase is 100 mu g/mL;

in the step 3, the reaction time is 35min;

in step 4, adjusting the pH value to 8; the reaction time is 32h;

and 5, placing the mixture in a refrigerator at the temperature of-8 ℃ for freezing and molding, transferring the mixture to a vacuum freeze dryer after 24 hours, and freeze-drying to obtain the mineralized bone matrix material.

Comparative example 1

This comparative example was performed in substantially the same manner as in example 1, except that alkaline phosphatase and orthophosphate were not added, and the addition order of the reactants was random and was not performed in the order of addition in example 1 of the present application.

Comparative example 2

The procedure of this comparative example was substantially the same as that of example 1 except that no orthophosphate was added and the order of addition of the reactants was random and not in the order of addition of example 1 of this application.

A series of experiments are next conducted on examples 1-3 to help illustrate the beneficial effects of the present invention.

Experiment 1

The reaction curves of alkaline phosphatase-catalyzed formation of orthophosphate from pyrophosphate under the control of orthophosphate in example 1, example 3 and comparative example 2 were measured, respectively, and the results are shown in FIG. 2. Wherein, FIG. 2-A is a graph showing the effect of the amount of alkaline phosphatase added on the concentration of pyrophosphate ions at different amounts of orthophosphate added; FIG. 2B is a graph showing the effect of the amount of alkaline phosphatase added on the concentration of orthophosphate ions at different amounts of orthophosphate added.

As can be seen from fig. 2-a, the content of pyrophosphate ions, i.e., pyrophosphate, decreases with the enzymatic hydrolysis of alkaline phosphatase, and the rate of enzymatic hydrolysis of pyrophosphate by alkaline phosphatase decreases with the increase of the amount of orthophosphate added;

as can be seen from fig. 2-B, the content of orthophosphate ions, i.e., orthophosphate, increases with the enzymatic hydrolysis of alkaline phosphatase, and at the same time, the formation rate of orthophosphate ions, i.e., orthophosphate, slows down with the increase in the amount of orthophosphate added;

as can be seen from FIG. 2, pyrophosphate can be enzymatically hydrolyzed by alkaline phosphatase to form orthophosphate, and when the amount of orthophosphate ions, i.e., orthophosphate, initially added is large, the enzymatic reaction becomes slow, indicating that there is a chemical reaction equilibrium between orthophosphate and pyrophosphate. The embodiment of the invention considers the influence of ion sources, chemical reaction balance and chemical factor adding sequence in mineralization more fully, constructs a multi-factor synergistic regulation and control system through the multi-factor synergistic effect of specific reactant adding sequence, adding amount, specific reaction conditions and the like, better simulates the complex bone matrix mineralization assembling process in vivo, and obtains the mineralized bone matrix material with excellent performance and a flower-like and multi-layer flower-like apatite space mineralization structure.

In this experiment, the same measurement was performed as in example 2, and the results were substantially the same as in examples 1 and 3, and the technical effects intended by the present invention were all achieved.

Experiment 2

The microscopic morphology of the mineralized bone matrix materials obtained in comparative example 1, comparative example 2, and example 1 were observed by SEM, and the observed SEM images are shown in fig. 3. Wherein, FIG. 3-A1 is an SEM image of comparative example 1, and FIG. 3-A2 is a morphology-enlarged image of a framed area in FIG. 3-A1; FIG. 3-B1 is an SEM image of comparative example 2, and FIG. 3-B2 is a magnified image of the topography of the boxed area of FIG. 3-B1; FIG. 3-C1 is an SEM image of example 1, and FIG. 3-C2 is a magnified image of the features of the boxed area in FIG. 3-C1.

As can be seen from FIG. 3, the mineralized bone matrix material prepared by the method provided in example 1 of the present invention has a flower-like and multi-layer flower-like apatite spatial mineralization structure, which is not present in comparative example 1 and comparative example 2.

The same measurements as in examples 2 and 3 were carried out in this experiment, and the results were almost the same as in example 1, and the technical effects of the present invention as a whole were achieved.

All technical features of the above embodiments may be combined arbitrarily, and for simplicity of description, all possible combinations of the technical features in the above embodiments are not described; such non-explicitly written embodiments should be considered as being within the scope of the present description.

The present invention has been described in considerable detail by the general description and the specific examples given above. It should be noted that numerous variations and modifications could be made to the specific embodiments described without departing from the inventive concept, and such are intended to be included within the scope of the appended claims. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims

1. A method of phosphate-mediated self-assembly of apatite, comprising the steps of:

s2, adding 3-5mg/mL of a type I collagen solution into a centrifuge tube, and then sequentially adding the calcium ion solution, the polyelectrolyte compound solution, the pyrophosphate ion solution, the orthophosphate ion solution and the alkaline phosphatase solution which are prepared in the S1 into the type I collagen solution to form a reaction system, wherein the addition amount of each solution is calculated by the final concentration of each component in the reaction system, the final concentration of the type I collagen is 0.5-2mg/mL, the final concentration of calcium ions is 10-20mM, the final concentration of polyelectrolyte compound is 30-100 mu g/mL, the final concentration of pyrophosphate ions is 0.5-2wt%, the final concentration of orthophosphate ions is 5-10mM, and the final concentration of alkaline phosphatase is 30-100 mu g/mL;

s4, adjusting the pH value of the mineralized bone matrix solution to 7-8 by using 1-5wt% acetic acid solution, and then continuously standing for reaction for 24-32 hours to obtain a primary product;

s5, removing impurities from the primary product, concentrating the primary product after impurity removal to 10% -20% of the original volume of the primary product according to the total reaction volume to obtain bone matrix mineralized liquid, placing the bone matrix mineralized liquid in a constant-temperature environment below 0 ℃ for freezing and forming, taking out after 12-24 hours, and carrying out freeze drying to obtain the mineralized bone matrix material.

2. The phosphate-mediated apatite self-assembly method as claimed in claim 1, wherein in S1, the polyelectrolyte compound solution is one of a polyacrylic acid solution and a polyacrylamide solution.

3. The method of claim 1, wherein the calcium ion solution in S1 is a water-soluble calcium salt solution.

4. A phosphate-mediated apatite self-assembly method according to claim 3, wherein said calcium ion solution is one of calcium chloride solution, calcium nitrate solution or a mixture thereof.

5. The method of claim 1, wherein in S1 the solution of orthophosphate ions is one of diammonium phosphate solution, ammonium dihydrogen phosphate solution, and a mixture thereof.

6. The method of claim 1, wherein in S1, the pyrophosphate ion solution is one of sodium pyrophosphate solution, potassium pyrophosphate solution or their mixture.

7. The phosphate-mediated apatite self-assembly method as claimed in claim 1, wherein, in S3, the adjusting agent for adjusting the pH value of the reaction system is specifically: an alkali solution having a concentration of 1M, an alkali solution having a concentration of 0.1M, and aqueous ammonia having a concentration of 0.1M.

8. The method of claim 7, wherein the alkali solution is one of NaOH solution and KOH solution or a mixture thereof.

9. The method of claim 1, wherein the step of removing impurities from the initial product in step S5 comprises:

a, adding deionized water into the primary product, and cleaning;

step B, centrifuging the initial product after adding deionized water;

c, after centrifugation, filtering liquid, and adding deionized water again until the volume of the deionized water is 2-3 times of that of the mineralized bone matrix solution;

and D, repeating the process B-C for 3 times to obtain the initial product after impurity removal.

10. A mineralized bone matrix material obtained by the phosphate-mediated apatite self-assembly process according to claim 1, and having a flower-like and multi-layer flower-like apatite spatial mineralization structure.

11. Use of a phosphate-mediated apatite self-assembly method according to claim 1 for the preparation of an implantable medical device for bone defect repair.