Polyanion hybridized amorphous bone meal and preparation method thereof

Abstract

The invention provides multi-anion hybridized amorphous bone meal and a preparation method thereof. The bone meal is structurally characterized in that the bone meal is in an amorphous state, cations in chemical composition of the bone meal are Ca < 2 + >, anions are PO4 < 3->, C6H5O7 < 3-> and SiO3 < 2->, the composition general formula of the bone meal is Ca5 [(PO4) 3-x-y (C6H5O7) x (SiO3) 1.5 y (OH)], x is larger than or equal to 0.25 and smaller than or equal to 0.50, and y is larger than or equal to 0.05 and smaller than or equal to 0.75. The method for preparing the bone meal comprises the following steps: by taking soluble calcium salt, citrate, a phosphate solution and silicate as raw materials, co-precipitating, washing and freeze-drying by controlling process conditions such as an alcohol-water solution environment, a dropwise adding mode, a reaction temperature, a pH value and the like. The bone meal is convenient to store, has relatively high mineralization activity, good biocompatibility, degradability and angiogenesis promoting function, has a good bone regeneration promoting effect, and is an excellent bone repair material.

Description

Technical Field

[0001] The invention provides a polyanion hybridized amorphous bone powder and a preparation method thereof. The material belongs to the field of medical bone repair materials. Background Art

[0002] Human skeleton is an important part of the human body. It not only supports the weight of the body, protects the internal organs, but also participates in various movements of the human body. From the perspective of chemical composition, inorganic matter in bones accounts for about 70% of its total weight, mainly in the form of hydroxyapatite, which gives human bones hard and stable physical properties; organic matter accounts for about 30%, mainly including protein and collagen fibers, which give it a certain toughness and elasticity; bones also contain water and a small amount of minerals in the form of ions. The three work together to maintain the normal structure and function of bones. When humans face problems such as traumatic fractures, bone tissue necrosis, bone defects after bone tumor resection, the need for craniofacial plastic surgery, and dental implant treatment, calcium phosphate biomaterials, as common bone replacement repair materials and tooth filling repair materials, can not only fill damaged or missing bone parts, but also promote the regeneration of new bone tissue and effectively repair it.

[0003] The chemical composition of calcium phosphate biomaterials is similar to the inorganic composition of natural bones, and can release Ca and P elements through the biodegradation of the material itself, which can promote the biomineralization of bone cells. It has good biocompatibility and osteoconductivity, and helps to accelerate the repair and regeneration of bone tissue. Among them, amorphous calcium phosphate has attracted widespread attention and application because it has better biological activity and biodegradability than crystalline calcium phosphate. Amorphous calcium phosphate is an intermediate in the synthesis of hydroxyapatite. During the synthesis process, it has disadvantages such as harsh experimental environment, difficult process, unstable powder, and easy conversion to crystalline hydroxyapatite. Due to the difficulty in synthesizing amorphous calcium phosphate, the current synthesis method is mainly based on the additive method. The additive method is to add some additives in the synthesis process of amorphous calcium phosphate to reduce its crystallization ability, such as Mg 2+ 、Zn 2+ , Sr 2 + 、P2O7 4- 、CO3 2- 、F - 、C6H5O7 3- Patent US 9108860B2 provides a composite powder of calcium carbonate and calcium phosphate, but the powder is unstable in an amorphous state and difficult to store; Patent US 9795543B1 provides an ACFP material, which is doped with F -The material is endowed with functions such as strengthening tooth enamel and preventing tooth decay, but its application scope is also limited; Patent CN108653804A provides a Si-α-TCP, the mechanical properties of silicon calcium phosphate are generally improved compared with calcium phosphate, and it has a certain angiogenic function, but TCP has a stable crystal structure, so the degradability and biomineralization activity of TCP are not as good as amorphous calcium phosphate; WANG ZQ et al. selected citrate to be doped into calcium phosphate (WANG ZQ, XU ZJ, ZHAO WL, et al. Isoexergonic Conformations of Surface-Bound Citrate Regulated Bioinspired Apatite Nanocrystal Growth [J]. ACS Appl Mater Interfaces, 2016, 8(41): 28116-23.), inhibits the crystallization of hydroxyapatite, and calcium citrate also has good biocompatibility and degradability, as well as excellent biomineralization activity, and is a good bone repair material. However, the amorphous calcium phosphate obtained by doping only with citrate is not stable enough and has no angiogenic function. Therefore, it is currently necessary to obtain a multifunctional amorphous calcium phosphate bone powder that is stable at room temperature.

[0004] Since the amorphous bone powder of the present invention cannot be obtained by simply mixing amorphous calcium phosphate doped with citrate and calcium silicophosphate doped with silicate, and impurities are easily generated during the synthesis process, it is necessary to explore the preparation method and the anion doping ratio. Therefore, in the present invention, citrate and silicate are doped at the same time, and Ca5[(PO4) 3-x-y (C6H5O7) x (SiO3) 1.5y The composition and proportion of (OH)] have been optimized to ensure that the material maintains a stable amorphous state, has high mineralization activity, can promote bone regeneration, and has good biocompatibility, degradability and angiogenesis-promoting function, making it suitable for use as a medical bone repair material. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a polyanion hybridized amorphous bone powder and a preparation method thereof. The bone powder is amorphous and has a general composition formula of Ca5[(PO4) 3-x-y (C6H5O7) x (SiO3) 1.5y (OH)], where 0.25≤x≤0.50, 0.05≤y≤0.75, and the molar ratio of calcium ion to acid anion is Ca 2+ / (PO4 3- +C6H5O7 3-+SiO3 2- )=1.67.

[0006] The steps of the method of the present invention are as follows:

[0007] (1) The molar ratio of calcium ions to acid radical ions is Ca / (PO4 3- +C6H5O7 3- +SiO3 2- )=1.67, weigh calcium nitrate, trisodium citrate dihydrate, diammonium hydrogen phosphate and sodium silicate respectively, and prepare the following at 0℃~40℃ with deionized water as solvent: 0.50mol / L calcium nitrate solution (solution A); 0.05mol / L sodium citrate solution (solution B); 0.25mol / L diammonium hydrogen phosphate solution (solution C); 0.03mol / L diammonium phosphate solution (solution D);

[0008] (2) adding 15 mL of ethanol aqueous solution having an alcohol-water ratio of 2:1 to 20 mL of solution A at a temperature of 0° C. to 40° C. to obtain solution E;

[0009] (3) at a temperature of 0°C to 40°C, 6 to 12 mL of trisodium citrate dihydrate solution, 18 to 23 mL of diammonium hydrogen phosphate solution, and 3 to 45 mL of sodium silicate solution are mixed uniformly to obtain a mixed solution F;

[0010] (4) at a temperature of 0°C to 40°C, the prepared solution F is added dropwise to the solution E at a rate of 7 to 10 mL / min and stirred at a rate of 5 to 60 r / min; the pH value of the reaction solution is adjusted to 8 to 10 with 10 to 25 wt% ammonia water or 10 to 25 wt% sodium hydroxide solution, the suspension is stirred for 20 to 40 min to obtain a suspension, the suspension is allowed to stand for 2 to 12 h, the suspension is washed by centrifugation for 3 to 4 times with deionized water, and then washed by centrifugation for 1 to 2 times with anhydrous ethanol to obtain a precipitate;

[0011] (5) The precipitate is freeze-dried under vacuum at a cold trap temperature of -60°C and a vacuum degree of ≤30.0 Pa for 24 hours to obtain bone powder.

[0012] The beneficial effects of the present invention are as follows:

[0013] (1) The present invention provides a polyanion hybridized amorphous bone powder and a method for preparing the same in an aqueous phase. The preparation method is simple in process and easy to obtain, overcoming the harsh conditions for preparing amorphous calcium phosphate in the past;

[0014] (2) The present invention obtains multifunctional bone powder for bone repair by hybridizing citrate and silicate with biological advantages, thereby improving the stability of amorphous calcium phosphate in amorphous state, which is beneficial to its storage and use;

[0015] (3) The multifunctional bone powder for bone repair provided by the present invention provides calcium ions and phosphate ions during the bone repair process to promote self bone regeneration, making the bone powder more biologically active and having the function of promoting angiogenesis, excellent biocompatibility and degradability, and more advantageous in bone repair applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is the XRD spectrum of the bone powder prepared in Example 1 of the present invention.

[0017] Figure 2 This is the FTIR spectrum of the bone powder prepared in Example 1 of the present invention.

[0018] Figure 3 This is a SEM photograph of the bone powder prepared in Example 1 of the present invention.

[0019] Figure 4 This is the XRD spectrum of the bone powder prepared in Example 1 of the present invention after mineralization in simulated body fluid. Specific implementation methods

[0020] The present invention is further described below in conjunction with the accompanying drawings and embodiments. The above description is only an embodiment of the present invention, and does not limit the patent scope of the present invention. Any equivalent transformation using the contents of the present invention description and drawings, or directly or indirectly applied to other related technical fields, are also included in the patent protection scope of the present invention.

[0021] Example 1

[0022] Ca5[(PO4) 2.5 (C6H5O7) 0.25 (SiO3) 0.375 (OH)] bone meal

[0023] At a temperature of 0℃~40℃, 20.0mL of 0.50M Ca(NO3)2·4H2O solution was added to 15.0mL of ethanol water solution with an alcohol-water ratio of 2:1 and mixed evenly to obtain solution E; then 6.0mL of 0.05M C6H5Na3O7·2H2O solution, 21.6mL of 0.25M (NH4)2HPO4 solution, 15.0mL of 0.03M Na2SiO3·9H2O solution, uniformly mixed to obtain solution F, and drop solution F into solution E at a speed of 7-10 mL / min and maintain stirring, and the stirring rate is 5-60 r / min; 10-25 wt% ammonia water or 10-25 wt% sodium hydroxide solution is used to control the pH value of the entire reaction solution to 8-10, and the stirring reaction is maintained for 20-40 minutes to obtain a suspension, and the suspension is placed for aging for 2 hours, centrifuged and washed 3-4 times with deionized water, and then centrifuged and washed 1-2 times with anhydrous ethanol to obtain a precipitate; the precipitate is placed in a cold trap at a temperature of -60 DEG C and a vacuum degree of 25.0 Pa for vacuum freeze drying to obtain bone powder.

[0024] Taking Example 1 as an example, Figure 1 The XRD spectrum of the sample shows that the material has no characteristic peaks, indicating that the Ca5[(PO4) 2.5 (C6H5O7) 0.25 (SiO3) 0.375 (OH)] bone powder presents amorphous structural characteristics.

[0025] Figure 2 The FTIR spectrum of the sample shows that the material has -1 、869cm -1 、774cm -1 The characteristic peak at the position of Ca5[(PO4) 2.5 (C6H5O7) 0.25 (SiO3) 0.375 (OH)] bone powder was prepared successfully.

[0026] Figure 3 This is the SEM photo of the sample. It can be seen that the morphology is granular, with a size between 200-600nm. Several or dozens of particles are aggregated into particle clusters, and most of the particles are elliptical.

[0027] Figure 4 This is the XRD spectrum of the sample after mineralization in simulated body fluid. After 6 hours of mineralization, the characteristic peak of hydroxyapatite appeared in the XRD, indicating that the material has transformed from an amorphous state to a crystalline state. It can be seen that the biomineralization activity of the material is very high.

[0028] Example 2

[0029] Ca5[(PO4) 2.7 (C6H5O7) 0.25 (SiO3) 0.075 (OH)] bone meal

[0030] At a temperature of 0℃~40℃, 20.0mL of 0.50M Ca(NO3)2·4H2O solution was added to 15.0mL of ethanol water solution with an alcohol-water ratio of 2:1 and mixed evenly to obtain solution E; then 6.0mL of 0.05M C6H5Na3O7·2H2O solution, 22.6mL of 0.25M (NH4)2HPO4 solution, 3.0mL of 0.03M Na2SiO3·9H2O solution, uniformly mixed to obtain solution F, and drop solution F into solution E at a speed of 7-10 mL / min and maintain stirring, and the stirring rate is 5-60 r / min; 10-25 wt% ammonia water or 10-25 wt% sodium hydroxide solution is used to control the pH value of the entire reaction solution to 8-10, and the stirring reaction is maintained for 20-40 minutes to obtain a suspension, and the suspension is placed for aging for 2 hours, centrifuged and washed 3-4 times with deionized water, and then centrifuged and washed 1-2 times with anhydrous ethanol to obtain a precipitate; the precipitate is placed in a cold trap at a temperature of -60 DEG C and a vacuum degree of 25.0 Pa for vacuum freeze drying to obtain bone powder.

[0031] Example 3

[0032] Ca5[(PO4) 2.25 (C6H5O7) 0.25 (SiO3) 0.75 (OH)] bone meal

[0033] At a temperature of 0℃~40℃, 20.0mL of 0.50M Ca(NO3)2·4H2O solution was added to 15.0mL of ethanol water with a ratio of 2:1 and mixed evenly to obtain solution E; then 6.0mL of 0.05M C6H5Na3O7·2H2O solution, 20.4mL of 0.25M (NH4)2HPO4 solution, 30.0mL of 0.03M Na2SiO3·9H2O solution, uniformly mixed to obtain solution F, and drop solution F into solution E at a speed of 7-10 mL / min and maintain stirring, and the stirring rate is 5-60 r / min; 10-25 wt% ammonia water or 10-25 wt% sodium hydroxide solution is used to control the pH value of the entire reaction solution to 8-10, and the stirring reaction is maintained for 20-40 minutes to obtain a suspension, and the suspension is placed for aging for 2 hours, centrifuged and washed 3-4 times with deionized water, and then centrifuged and washed 1-2 times with anhydrous ethanol to obtain a precipitate; the precipitate is placed in a cold trap at a temperature of -60 DEG C and a vacuum degree of 25.0 Pa for vacuum freeze drying to obtain bone powder.

[0034] Example 4

[0035] Ca5[(PO4)2(C6H5O7) 0.25 (SiO3) 1.125 (OH)] bone meal

[0036] At a temperature of 0℃~40℃, 20.0mL of 0.50M Ca(NO3)2·4H2O solution was added to 15.0mL of ethanol water solution with an alcohol-water ratio of 2:1 and mixed evenly to obtain solution E; then 6.0mL of 0.05M C6H5Na3O7·2H2O solution, 19.2mL0.25M (NH4)2HPO4 solution, 45.0mL 0.03M Na2SiO3·9H2O solution, uniformly mixed to obtain solution F, and drop solution F into solution E at a speed of 7-10 mL / min and maintain stirring, and the stirring rate is 5-60 r / min; 10-25 wt% ammonia water or 10-25 wt% sodium hydroxide solution is used to control the pH value of the entire reaction solution to 8-10, and the stirring reaction is maintained for 20-40 minutes to obtain a suspension, and the suspension is placed for aging for 2 hours, centrifuged and washed 3-4 times with deionized water, and then centrifuged and washed 1-2 times with anhydrous ethanol to obtain a precipitate; the precipitate is placed in a cold trap at a temperature of -60 DEG C and a vacuum degree of 25.0 Pa for vacuum freeze drying to obtain bone powder.

[0037] Example 5

[0038] Ca5[(PO4) 2.45 (C6H5O7) 0.5 (SiO3) 0.075 (OH)] bone meal

[0039] At a temperature of 0℃~40℃, 20.0mL of 0.50M Ca(NO3)2·4H2O solution was added to 15.0mL of ethanol water solution with an alcohol-water ratio of 2:1 and mixed evenly to obtain solution E; then 12.0mL of 0.05M C6H5Na3O7·2H2O solution, 21.4mL of 0.25M (NH4)2HPO4 solution, 3.0mL of 0.03M Na2SiO3·9H2O solution, uniformly mixed to obtain solution F, and drop solution F into solution E at a speed of 7-10 mL / min and maintain stirring, and the stirring rate is 5-60 r / min; 10-25 wt% ammonia water or 10-25 wt% sodium hydroxide solution is used to control the pH value of the entire reaction solution to 8-10, and the stirring reaction is maintained for 20-40 minutes to obtain a suspension, and the suspension is placed for aging for 2 hours, centrifuged and washed 3-4 times with deionized water, and then centrifuged and washed 1-2 times with anhydrous ethanol to obtain a precipitate; the precipitate is placed in a cold trap at a temperature of -60 DEG C and a vacuum degree of 25.0 Pa for vacuum freeze drying to obtain bone powder.

[0040] Example 6

[0041] Ca5[(PO4) 2.25 (C6H5O7) 0.5 (SiO3) 0.375 (OH)] bone meal

[0042] At a temperature of 0℃~40℃, 20.0mL of 0.50M Ca(NO3)2·4H2O solution was added to 15.0mL of ethanol water solution with an alcohol-water ratio of 2:1 and mixed evenly to obtain solution E; then 12.0mL of 0.05M C6H5Na3O7·2H2O solution, 20.4mL of 0.25M (NH4)2HPO4 solution, 15.0mL of 0.03M Na2SiO3·9H2O solution, uniformly mixed to obtain solution F, and drop solution F into solution E at a speed of 7-10 mL / min and maintain stirring, and the stirring rate is 5-60 r / min; 10-25 wt% ammonia water or 10-25 wt% sodium hydroxide solution is used to control the pH value of the entire reaction solution to 8-10, and the stirring reaction is maintained for 20-40 minutes to obtain a suspension, and the suspension is placed for aging for 2 hours, centrifuged and washed 3-4 times with deionized water, and then centrifuged and washed 1-2 times with anhydrous ethanol to obtain a precipitate; the precipitate is placed in a cold trap at a temperature of -60 DEG C and a vacuum degree of 25.0 Pa for vacuum freeze drying to obtain bone powder.

[0043] Example 7

[0044] Ca5[(PO4)2(C6H5O7) 0.5 (SiO3) 0.75 (OH)] bone meal

[0045] At a temperature of 0℃~40℃, 20.0mL of 0.50M Ca(NO3)2·4H2O solution was added to 15.0mL of ethanol water solution with an alcohol-water ratio of 2:1 and mixed evenly to obtain solution E; then 12.0mL of 0.05M C6H5Na3O7·2H2O solution, 19.2mL of 0.25M (NH4)2HPO4 solution, 30.0mL of 0.03M Na2SiO3·9H2O solution, uniformly mixed to obtain solution F, and drop solution F into solution E at a speed of 7-10 mL / min and maintain stirring, and the stirring rate is 5-60 r / min; 10-25 wt% ammonia water or 10-25 wt% sodium hydroxide solution is used to control the pH value of the entire reaction solution to 8-10, and the stirring reaction is maintained for 20-40 minutes to obtain a suspension, and the suspension is placed for aging for 2 hours, centrifuged and washed 3-4 times with deionized water, and then centrifuged and washed 1-2 times with anhydrous ethanol to obtain a precipitate; the precipitate is placed in a cold trap at a temperature of -60 DEG C and a vacuum degree of 25.0 Pa for vacuum freeze drying to obtain bone powder.

[0046] Example 8

[0047] Ca5[(PO4) 1.75 (C6H5O7) 0.5 (SiO3) 1.125 (OH)] bone meal

[0048] At a temperature of 0℃~40℃, 20.0mL of 0.50M Ca(NO3)2·4H2O solution was added to 15.0mL of ethanol water solution with an alcohol-water ratio of 2:1 and mixed evenly to obtain solution E; then 12.0mL of 0.05M C6H5Na3O7·2H2O solution, 18.0mL of 0.25M (NH4)2HPO4 solution, 45.0mL of 0.03M Na2SiO3·9H2O solution, uniformly mixed to obtain solution F, and drop solution F into solution E at a speed of 7-10 mL / min and maintain stirring, and the stirring rate is 5-60 r / min; 10-25 wt% ammonia water or 10-25 wt% sodium hydroxide solution is used to control the pH value of the entire reaction solution to 8-10, and the stirring reaction is maintained for 20-40 minutes to obtain a suspension, and the suspension is placed for aging for 2 hours, centrifuged and washed 3-4 times with deionized water, and then centrifuged and washed 1-2 times with anhydrous ethanol to obtain a precipitate; the precipitate is placed in a cold trap at a temperature of -60 DEG C and a vacuum degree of 25.0 Pa for vacuum freeze drying to obtain bone powder.

Claims

1. A polyanion hybridized amorphous bone powder, characterized in that: The bone powder is amorphous, and the cation in its chemical composition is Ca 2+ , anion is PO4 3- 、C6H5O7 3- and SiO3 2- The general formula is Ca5[(PO4) 3-x-y (C6H5O7) x (SiO3) 1.5y (OH)], where 0.25≤x≤0.50, 0.05≤y≤0.75, and the molar ratio of calcium ion to acid anion is Ca 2+ / (PO4 3- +C6H5O7 3- +SiO3 2- )=1.

67.

2. The polyanion hybridized amorphous bone powder according to claim 1, characterized in that: The bone powder is easy to preserve, has high mineralization activity, good biocompatibility, degradability and angiogenesis-promoting function, and has a good effect in promoting bone regeneration.

3. The polyanion hybridized amorphous bone powder according to claim 1, characterized in that: The preparation method is as follows: (1) The molar ratio of calcium ions to acid radical ions is Ca / (PO4 3- +C6H5O7 3- +SiO3 2- )=1.67, weigh calcium nitrate, trisodium citrate dihydrate, diammonium hydrogen phosphate and sodium silicate respectively, and prepare the following at 0℃~40℃ with deionized water as solvent: 0.50mol / L calcium nitrate solution (solution A); 0.05mol / L sodium citrate solution (solution B); 0.25mol / L diammonium hydrogen phosphate solution (solution C); 0.03mol / L diammonium phosphate solution (solution D); (2) adding 15 mL of ethanol aqueous solution having an alcohol-water ratio of 2:1 to 20 mL of solution A at a temperature of 0° C. to 40° C. to obtain solution E; (3) at a temperature of 0°C to 40°C, 6 to 12 mL of trisodium citrate dihydrate solution, 18 to 23 mL of diammonium hydrogen phosphate solution, and 3 to 45 mL of sodium silicate solution are mixed uniformly to obtain a mixed solution F; (4) at a temperature of 0°C to 40°C, the prepared solution F is added dropwise to the solution E at a rate of 7 to 10 mL / min and stirred at a rate of 5 to 60 r / min; the pH value of the reaction solution is adjusted to 8 to 10 with 10 to 25 wt% ammonia water or 10 to 25 wt% sodium hydroxide solution, the suspension is stirred for 20 to 40 min to obtain a suspension, the suspension is allowed to stand for 2 to 12 h, the suspension is washed by centrifugation for 3 to 4 times with deionized water, and then washed by centrifugation for 1 to 2 times with anhydrous ethanol to obtain a precipitate; (5) The precipitate is freeze-dried under vacuum at a cold trap temperature of -60°C and a vacuum degree of ≤30.0 Pa for 24 hours to obtain bone powder.

4. According to the method for preparing the polyanion hybridized amorphous bone powder of claim 3, the calcium nitrate described in step (1) can be replaced by calcium chloride, the sodium citrate can be replaced by citric acid or ammonium citrate, and the diammonium hydrogen phosphate can be replaced by disodium hydrogen phosphate, diammonium phosphate or phosphoric acid.

5. The doping element range of the polyanion hybrid amorphous bone powder according to claim 1, in particular, the preferred ratio is x=0.25, y=0.25 in the general composition formula, that is, the chemical formula is Ca5[(PO4) 2.5 (C6H5O7) 0.25 (SiO3) 0.375 (OH)], characterized in that: The polyanion hybridized amorphous bone powder has high mineralization activity, good biocompatibility, degradability and angiogenesis promoting function.

6. The polyanion hybridized amorphous bone powder according to claim 1, characterized in that: The bone powder can be used alone for bone repair, or used as an active additive component of a composite bone repair material.

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