CN114668891B - A kind of phosphate-mediated apatite self-assembly method and its application - Google Patents

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

A kind of phosphate-mediated apatite self-assembly method and its application

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 technique

Apatite is the main inorganic phase that constitutes bone tissue and bone matrix, and is the main source of mechanical strength of bone tissue. The latest research shows that in bone tissue, apatite includes different structural morphologies such as filamentary patterns, lace patterns, and nested rosette patterns at the nanoscale. On the macro scale, it is mainly a lamellar structure. In fact, apatite has a multi-scale multi-level mineralization structure in bone tissue, and apatite exists in different structural forms at different scales, which enables bone to adapt to the complex and changeable external mechanical environment.

However, at present, in vitro biomimetic mineralization studies, few technologies that can prepare apatite with different patterned structures have emerged.

SUMMARY OF THE INVENTION

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

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

In a first aspect, an embodiment of the present invention provides a phosphate-mediated apatite self-assembly method, comprising the following steps:

S1, configure 0.5-1.5mg/mL polyelectrolyte compound solution, 0.05-0.15M calcium ion solution, 0.05-0.15M orthophosphate ion solution, and 2.5-3.5wt% pyrophosphate ion solution, 5-10mg/L alkali Sex phosphatase solution;

S2. Add 3-5 mg/mL type I collagen solution to the centrifuge tube, and then sequentially add the calcium ion solution, polyelectrolyte compound solution, pyrophosphate ion solution, and orthophosphate ion prepared in S1 to the type I collagen solution. solution and alkaline phosphatase solution to form a reaction system, the addition amount of each solution is calculated from the final concentration of each component in the reaction system, wherein the final concentration of type I collagen is 0.5-2 mg/mL, and the final concentration of calcium ions is 10-20 mM, the final concentration of polyelectrolyte compound is 30-100 μg/mL, the final concentration of pyrophosphate ion is 0.5-2 wt%, the final concentration of orthophosphate ion is 5-10 mM, and the final concentration of alkaline phosphatase is 30-100μg/mL;

S3, after adding each solution in turn, adjust the pH of the reaction system to 8-9, then add deionized water to the reaction system until its volume is 3-5 times the volume of the collagen type I solution, and then let stand for 25-35min to obtain a mineralized bone matrix solution;

S4, adjust the pH of the mineralized bone matrix solution to 7-8 with 1-5wt% acetic acid solution, and then continue to stand for 24-32h to obtain the initial product;

S5. Perform impurity removal on the primary product, and then concentrate the primary product after removal of impurities to 10%-20% of its original volume according to the total reaction volume to obtain a bone matrix mineralized solution, which is placed in a Freeze-molded in a constant temperature environment below 0°C, taken out after 12-24 hours, and freeze-dried to obtain a 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, more specifically 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, ammonium dihydrogen phosphate solution or a mixture thereof.

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

Preferably, in S3, the regulator for adjusting the pH value of the reaction system is specifically: an alkaline solution with a concentration of 1M, an alkaline solution with a concentration of 0.1M, and ammonia water with a concentration of 0.1M.

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

Preferably, in S5, the concrete flow process that carries out impurity removal to initial product comprises:

Process A, adding deionized water to the initial product, cleaning;

Process B, the initial product after adding deionized water by centrifugation;

Process C. After centrifugation, filter out the liquid, and add water again until the volume is 2-3 times that of the mineralized bone matrix solution;

Process D. Repeat process B-C 3 times to obtain the initial product after removal of impurities.

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

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

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

(1) The phosphate-mediated apatite self-assembly method provided in the embodiment of the present invention utilizes apatite self-assembly to prepare the surface of the mineralized bone matrix with different spatial assembly structures and mineralization degrees on the micron scale. Mineralized apatite is a mineralized bone matrix material, and its assembly structure is mainly a lamellar structure, a flower-like structure and a multi-layered flower-like structure consistent with the body, which provides a powerful tool for the preparation of multi-scale highly biomimetic bone materials. Technical Support.

(2) Compared with the traditional apatite mineralization method, the phosphate-mediated apatite self-assembly method provided by the embodiment of the present invention fully considers the source of ions, the balance of chemical reactions and the order of addition of chemical factors in the mineralization process. A multi-factor coordinated regulation system was constructed to better simulate the complex mineralization and assembly process of bone matrix in vivo; under the action of alkaline phosphatase, pyrophosphate was enzymatically hydrolyzed to produce a large amount of orthophosphate , which is conducive to the formation of apatite. Adding orthophosphate as a regulatory factor in the initial stage of mineralization can effectively control the enzymatic hydrolysis rate of pyrophosphate, and at the same time guide the formed apatite to aggregate to form a lamellar structure, and the apatite of the lamellar structure is further assembled to form a flower-like structure And the multi-layer flower-like apatite space mineralization structure makes up for the gap in the current technical field.

(3) The phosphate-mediated apatite self-assembly method provided in the embodiment of the present invention, by controlling the addition and addition sequence of regulatory factors in the bone matrix, under weak alkaline conditions, using alkaline phosphatase to hydrolyze pyrophosphate At the same time, using polyelectrolyte compounds to control the size of nano-apatite particles, orthophosphate to control the hydrolysis of alkaline phosphatase, and the self-assembly mechanism of apatite in the collagen/calcium phosphate biomimetic mineralization system, can be fabricated with Spatially mineralized structures of apatite with different shapes.

(4) The phosphate-mediated apatite self-assembly method provided by the embodiment of the present invention adjusts the pH value by adding ammonia water, and the ammonia water can form a buffer system, so that the pH of the reaction system can be adjusted autonomously in the reaction process, and the operation is more efficient. Simple, while making the response more stable and controllable.

(5) The mineralized bone matrix material provided in the embodiment of the present invention has a flower-like and multi-layered flower-like apatite space mineralization structure, and at the same time, it has good biocompatibility and is compatible with human bone tissue. The mechanical properties that match the inner bone matrix have a very broad application space.

Description of drawings

In order to illustrate the technical solutions of the present invention more clearly, the following briefly introduces the accompanying drawings that are used in the description of the embodiments of the present invention. Obviously, the drawings in the following description are only exemplary, and for those of ordinary skill in the art, other drawings can also be derived from the provided drawings without creative effort.

The following drawings are only used to cooperate with the content disclosed in the description for the understanding and reading of those who are familiar with the technology, and are not used to limit the conditions for the implementation of the present invention. Any form of adjustment will not affect the On the premise of the effect and the achievable purpose, it should still fall within the scope that the technical content disclosed in the present invention can cover.

Fig. 1 is the operation flow schematic diagram of Embodiment 1 of the present invention;

Fig. 2 is the reaction curve diagram of the enzymatic hydrolysis of pyrophosphate by alkaline phosphatase to form orthophosphate under the control of orthophosphate in Example 1, Example 3 and Comparative Example 1 of the present invention; wherein, Fig. 2-A shows different orthophosphates. The effect curve of alkaline phosphatase addition on pyrophosphate ion concentration under the addition of phosphate, Figure 2-B is the effect curve of alkaline phosphatase addition on orthophosphate ion concentration under different orthophosphate additions picture;

Fig. 3 is the SEM images of Example 1, Comparative Example 1 and Comparative Example 2 of the present invention; wherein Fig. 3-A1 is the SEM image of Comparative Example 2, and Fig. 3-A2 is an enlarged topography of the frame selection area in Fig. 3-A1 Image; Figure 3-B1 is the SEM image of Comparative Example 1, Figure 3-B2 is an enlarged image of the topography of the framed area in Figure 3-B1; Figure 3-C1 is the SEM image of Example 1, Figure 3-C2 is Topographic magnified image of the box-selected area in Figure 3-C1.

Detailed ways

In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention, but not to limit the present invention.

In the description of the present invention, unless otherwise specified, "plurality" means two or more. The terms "first", "second", "third", "fourth", etc. in the description and claims of the present invention are intended to distinguish what is referred to. For a solution with a sequential flow, this term expression does not need to be construed as describing a specific sequence or sequence, and for a device structure solution, this term expression does not have any distinction of importance, positional relationship, or the like.

Furthermore, the terms "comprising", "having" and any variations thereof are intended to cover non-exclusive inclusion, for example, a process, method, system, product or device comprising a series of steps or units is not necessarily limited to those expressly listed Instead, those steps or units may also include other steps or units that are not explicitly listed but are inherent to these processes, methods, products or devices, or added steps or units based on further optimized solutions of the present inventive concept.

Example 1

This example provides a phosphate-mediated apatite self-assembly method, through which a mineralized bone matrix material with a flower-like and multi-layered flower-like apatite space mineralization structure can be prepared. The operation flow of the present embodiment is shown in Figure 1, and the specific steps and related parameters are as follows:

Step 1. Combine the purchased finished polyacrylic acid (ie the polyelectrolyte compound in Figure 1), calcium chloride (ie the calcium ion in Figure 1), diammonium hydrogen phosphate (ie the orthophosphate in Figure 1) and pyrophosphate Sodium (ie, pyrophosphate in Figure 1 ) and alkaline phosphatase reagents were first prepared into reaction stock solutions using sterile deionized water. The concentrations of the reaction stock solutions were, respectively, polyacrylic acid 1 mg/mL, calcium chloride 0.1 M, diammonium hydrogen phosphate 0.1 M, and sodium pyrophosphate 3% by mass. In addition, sodium hydroxide, ammonia and acetic acid are required to adjust the pH. Two concentrations of NaOH solution were prepared for coarse and fine adjustment of pH, 1M and 0.1M, respectively. Ammonia water is 0.1M, and the purpose of adding ammonia water is that the ammonia water can form a buffer system, so as to realize the independent adjustment of the pH of the reaction system during the reaction process. The acetic acid concentration was 2% by mass. Rat tail type I collagen purchased from BD biocoat is the reaction stock solution of collagen (ie, the type I collagen solution in Figure 1), with a concentration range of 3-5 mg/mL. The total activity value of the purchased alkaline phosphatase is greater than 10KU, that is, greater than 10mg, calculated according to the minimum value of 10mg, and configured into a stock solution of 10mg/mL.

In this step:

The polyacrylic acid used can be replaced by other polyelectrolyte compounds, such as polyacrylamide, etc.;

The calcium chloride used can be replaced by other water-soluble calcium salts that can provide calcium ions, such as calcium nitrate, etc.;

The diammonium hydrogen phosphate used can be replaced by other water-soluble phosphates that can provide orthophosphate ions, such as ammonium dihydrogen phosphate, etc.;

The sodium pyrophosphate used can be replaced by other water-soluble pyrophosphates that can provide pyrophosphate ions, such as potassium pyrophosphate, etc.;

The rat tail type I collagen solution used can be replaced with other type I collagen solutions that do not react with the substances in the reaction system to negatively affect the final effect.

Step 2. Carry out the mineralization reaction in a centrifuge tube. The total amount of the solution in the reaction system was 5 mL. In the reaction system, each component in the reaction system was added according to the addition sequence shown in FIG. 1 . According to the final concentration of each component in the reaction system, the added amount of the stock solution required by each component is calculated. The final concentration of collagen was 1 mg/mL, the final concentration of calcium ions was 10 mM, the final concentration of polyacrylic acid was 50 μg/mL, the final concentration of pyrophosphate ions was 1% by mass, and the final concentration of orthophosphate ions was 5 mM , the final concentration of alkaline phosphatase was 60 μg/mL.

Step 3. After adding the reaction substances according to the sequence 1-5 shown in Figure 1, adjust the pH of the reaction system to 8-9 with sodium hydroxide and ammonia water, supplement the sterile deionized water to make the reaction system reach 5mL, and maintain it for 30min reaction.

In this step:

The sodium hydroxide used can be replaced by other alkalis that can be completely ionized and will not react with other substances in the reaction system to negatively affect the reaction effect, such as KOH, etc.;

The ammonia water used can be replaced by other alkaline solutions that are not completely ionized and will not react with other substances in the reaction system to negatively affect the reaction effect.

Step 4. After 30 minutes, the pH of the reaction system was adjusted to 7.4 with 2% acetic acid by mass, and the reaction was carried out for 24 hours to obtain the initial product.

Step 5. Process A: after the reaction is completed, add sterile deionized water to the primary product, and wash; Process B: the primary product after centrifugation and adding sterile deionized water; Process C: after centrifugation, filter out the liquid, Add 500 μL of sterile deionized water again; process D: repeat process B-C 3 times to obtain the initial product after removal of impurities; process E: after repeating 3 times, the initial product after removal of impurities to its initial volume (that is, step 4 15% of the volume of the initial product obtained in Mineralized bone matrix material.

Example 2

This example provides a phosphate-mediated apatite self-assembly method, through which a mineralized bone matrix material with a flower-like and multi-layered flower-like apatite space mineralization structure can be prepared. The operation flow and the specific steps of the present embodiment are basically the same as those of the embodiment 1, and the difference is:

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

In step 2, the final concentration of collagen was 0.5 mg/mL, the final concentration of calcium ions was 15 mM, the final concentration of polyacrylic acid was 30 μg/mL, the final concentration of pyrophosphate ions was 0.5 wt%, and the final concentration of orthophosphate ions was 0.5 wt%. was 7 mM, and the final concentration of alkaline phosphatase was 30 μg/mL;

In step 3, the reaction times is 25min;

In step 4, adjust the pH to 7; the reaction time is 28h;

In step 5, it is placed in a refrigerator at -10° C. for freezing and molding, and after about 18 hours overnight, it is transferred to a vacuum freeze dryer and freeze-dried to obtain a mineralized bone matrix material.

Example 3

This example provides a phosphate-mediated apatite self-assembly method, through which a mineralized bone matrix material with a flower-like and multi-layered flower-like apatite space mineralization structure can be prepared. The operation flow and the specific steps of the present embodiment are basically the same as those of the embodiment 1, and the difference is:

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

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

In step 3, the reaction times is 35min;

In step 4, the pH is adjusted to 8; the reaction time is 32h;

In step 5, it is placed in a refrigerator at -8° C. for freezing and molding, and after about 24 hours overnight, it is transferred to a vacuum freeze-drying machine, and freeze-dried to obtain a mineralized bone matrix material.

Comparative Example 1

The operation steps of this control example are basically the same as those of Example 1, except that alkaline phosphatase and orthophosphate are not added, and the addition order of each reactant is random, and is not carried out according to the addition order of Example 1 of the present application.

Comparative Example 2

The operation steps of this comparative example are basically the same as those of Example 1, except that no orthophosphate is added, and the addition order of each reactant is random, and is not carried out in accordance with the addition order of Example 1 of the present application.

Next, a series of experiments are carried out on Examples 1-3 to help illustrate the beneficial effects of the present invention.

Experiment 1

The reaction curves of the enzymatic hydrolysis of pyrophosphate by alkaline phosphatase to form orthophosphate 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 . Among them, Figure 2-A is the effect curve of the pyrophosphate ion concentration of alkaline phosphatase addition under different orthophosphate additions; Figure 2-B is the alkaline phosphatase addition under different orthophosphate additions Graph of the effect on orthophosphate ion concentration.

It can be seen from Figure 2-A that with the enzymatic hydrolysis of alkaline phosphatase, the content of pyrophosphate ions, that is, pyrophosphate, decreases. The rate of phosphate is slowed down;

It can be seen from Figure 2-B that with the enzymatic hydrolysis of alkaline phosphatase, the content of orthophosphate ion, that is, orthophosphate, increases. The rate of salt formation is slowed down;

As can be seen from Figure 2 as a whole, pyrophosphate can be enzymatically hydrolyzed to form orthophosphate under the action of alkaline phosphatase. At the same time, when the amount of orthophosphate ions added initially is large, the enzymatic hydrolysis reaction changes. Slow, indicating that there is a chemical reaction equilibrium between orthophosphate and pyrophosphate. In the embodiment of the present invention, the influence of ion source, chemical reaction balance and addition sequence of chemical factors in mineralization is more fully considered, and the specific reactant addition sequence, addition amount and specific reaction conditions and other factors synergistic effect , a multi-factor synergistic regulation system was constructed, which better simulated the complex bone matrix mineralization and assembly process in vivo, and obtained a mineralized bone matrix with excellent performance and a flower-like and multi-layer flower-like apatite spatial mineralization structure. Material.

In this experiment, the same measurement was also carried out for Example 2, and the results were basically the same as those in Examples 1 and 3, and the technical effect to be achieved by the present invention could be achieved on the whole.

Experiment 2

The microscopic morphology of the mineralized bone matrix materials obtained in Comparative Example 1, Comparative Example 2 and Example 1 was observed by SEM, and the observed SEM images were shown in FIG. 3 . Among them, Figure 3-A1 is the SEM image of Comparative Example 1, Figure 3-A2 is an enlarged image of the topography of the framed area in Figure 3-A1; Figure 3-B1 is the SEM image of Comparative Example 2, and Figure 3-B2 is Figure 3-B1 is an enlarged image of the topography of the box-selected region; Figure 3-C1 is the SEM image of Example 1, and Figure 3-C2 is a topographically enlarged image of the box-selected region in Figure 3-C1.

It can be seen from FIG. 3 that the mineralized bone matrix material prepared by the method provided in Example 1 of the present invention has a flower-like and multi-layered flower-like apatite space mineralization structure, which is not available in both Comparative Example 1 and Comparative Example 2. of.

In this experiment, the same measurement was also carried out on Examples 2 and 3, and the results were basically the same as those in Example 1, and the technical effect to be achieved by the present invention could be achieved as a whole.

The technical features of the above embodiments can be combined arbitrarily. In order to simplify the description, all possible combinations of the technical features in the above embodiments are not described; these embodiments that are not explicitly written should also be considered as range described in this manual.

The present invention has been described in more detail and in detail above through the general description and specific embodiments. It should be noted that, without departing from the concept of the present invention, it is obvious that some modifications and improvements can be made to these specific embodiments, which all belong to the protection scope of the present application. Therefore, the scope of protection of the patent of the present application shall be subject to the appended claims.

Claims (11)

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

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 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;

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 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.

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